molecular characterisation of microorganisms within the
TRANSCRIPT
Molecular characterisation of microorganisms within the
stump-tailed lizard tick, Amblyomma albolimbatum,
in Western Australia
Samuel Elliot
Bachelor of Science
Submitted in fulfilment of the requirements for the degree of
Bachelor of Sciences with Honours.
School of Veterinary and Life Sciences
Murdoch University, 2021
II
Declaration
I declare this thesis is my own account of my research and contains as its main content, work
which has not been previously submitted for a degree at any tertiary education institution.
Samuel Elliot
III
Abstract
Ticks pose a major threat to the health and wellbeing of humans, domesticated animals and wildlife.
Recently, a bacterial community study (16S rRNA) on the stump-tailed lizard tick, Amblyomma
albolimbatum, removed from bobtails, Tiliqua rugosa, in Western Australia (WA), revealed the
presence of Coxiella, Francisella and Rickettsia DNA sequences. However, resolution to species
level could not be achieved due to the short sequences generated through next generation
sequencing. In addition, oocytes proposed to be Hemolivia sp. have been identified within the gut
epithelium of A. albolimbatum; however molecular data confirming the species is lacking. Here we
present for the first time, molecular data supporting the presence of Coxiella burnetii, Francisella sp.
nov., Rickettsia spp. and Hemolivia mariae within A. albolimbatum in WA. Three hundred and six
ticks, morphologically identified as A. albolimbatum, were removed from 88 T. rugosa lizards at the
Kanyana Wildlife Rehabilitation Centre, Perth, WA from 2013 to 2015. A subset of 142 ticks were
subjected to genomic DNA extraction and screened for Coxiella, Francisella, Rickettsia using genus-
specific assays and haemoparasites using a nested 18S rRNA assay followed by Sanger
sequencing. Coxiella burnetii was found in a single A. albolimbatum based on 16S and GroEL genes.
Coxiella burnetii is the causative agent of Q fever and coxiellosis in people and animals, respectively,
and therefore, its presence is of major concern. Secondly, a Francisella sp. nov. based on a
concatenated alignment of 4,230 bp (16S, tpiA, prfB, rpoA, dnaA) was generated with a 3% genetic
distance to F. persica, a known soft tick endosymbiont. Interestingly, there was evidence of three
Rickettsia spp. which warrants further investigation as two were closely related to the Spotted Fever
Rickettsia spp. group. Lastly, two samples had a 100% genetic identity to H. mariae at the 18S
gene, which is consistent with the previous report of Hemolivia within A. albolimbatum and that ticks
have an important role for the sylvatic lifecycle of this parasite. Further research is required to
determine the prevalence of these microbes within questing A. albolimbatum ticks and the role they
play in the health of T. rugosa and wildlife rehabilitation workers.
IV
Acknowledgments
Firstly, I would like to thank my supervisors. My primary supervisor Dr. Charlotte Oskam, I struggle
with words to express the depth of gratitude I have for you taking me on as an honours student. You
have been so supportive, approachable, and generous with your time. My Co-supervisors Dr. Jill
Austen and Dr. Amanda Barbosa, you have both been so helpful and approachable when I have
needed help with anything over the last year. All of you have been so engaging and inspiring. Thank
you to the Vector and Waterborne Pathogens Research Group, I am constantly blown away by how
intelligent and motivated you are. Siobhon Egan, thank you for your patience and willingness to help
towards the bombardment of questions I had when I was beginning my lab work. Professor Peter
Irwin, thank you for allowing us the space to use your desk for the duration of this project while you
were not using it. A special thanks to Xavier Barton, A special thanks to Xavier Barton, who I shared
a lot of troubleshooting and other moments with on this learning journey. Thank you to Ruby
Mckenna, for lending me your lab manual that gave insight into this project, a representation of the
collaborative nature of science. I would also like to thank Dr Shane Tobe, although not an official
supervisor, has been encouraging and willing to give thought provoking advice regarding this project.
I express thanks to Dr Paola Magni for the pictures of the bobtails and the team at the Australian
Rickettsial Reference Laboratory for the use of their Rickettsia culture for my positive control.
I would like to thank my father Derek, who provided me with technical computer related support
through my bachelor’s degree; my mother Susan; and to my brother Rory for lending me money to
purchase a laptop when I started university.
Thanks to Tim for letting me work one day week and supplying me with coffee. Trent and Zoe, thank
you for being amazing over the last year. Finally, A special thanks to Jack Elwin, for your patience
and allowing me to live in your home during the course of this project. You’re a bloody legend, thank
you!
V
Table of Contents
Declaration II
Abstract III
Acknowledgements IV
Table of Contents V
List of Figures VII
List of Tables VIII
List of Abbreviations IX
CHAPTER ONE – INTRODUCTION 1
1.1 Bobtails (Tiliqua rugosa) 1
1.2 Ticks 1
1.2.1 Tick life cycle 2
1.2.2 Ticks of Australia 3
1.2.3 Bobtail ticks 5
1.3 Tick associated microbes and diseases 6
1.3.1 Australian tick-borne diseases 7
1.3.1.1 Coxiella 8
1.3.1.2 Francisella 10
1.3.1.3 Rickettsia 11
1.3.1.4 Hemolivia 12
1.4 Molecular detection of tick associated microbes 13
1.4.1 Polymerase chain reaction (PCR) 13
1.4.1.1 16S rRNA gene 14
1.4.1.2 Additional genes 15
1.5 Aims and Hypotheses 15
CHAPTER TWO – MATERIALS AND METHODS 17
2.1 Sample acquisition 17
2.2 DNA extraction 17
2.3 PCR assays 18
2.3.1 Coxiella detection using conventional PCR 18
2.3.2 Francisella detection using conventional PCR 20
2.3.3 Rickettsia detection using conventional PCR 22
2.3.4 Piroplasm detection using nested PCR 24
2.4 Agarose gel electrophoresis and Sanger sequencing 24
2.5 Phylogenetic analysis 25
CHAPTER THREE – RESULTS 27
3.1 Molecular characterisation of Coxiella 27
3.2 Molecular characterisation of Francisella 35
3.3 Molecular characterisation of Rickettsia 47
3.4 Molecular characterisation of haemoprotozoa 69
VI
CHAPTER FOUR – DISCUSSION 72
4.1 Microbial species of interest 72
4.1.1 Coxiella burnetii confirmed within A. albolimbatum 72
4.1.2 Francisella sp. nov. within A. albolimbatum 75
4.1.3 Rickettsia spp. identified within A. albolimbatum 78
4.1.4 Hemolivia mariae confirmed within A. albolimbatum 81
4.2 Limitations and Future directions 83
4.3 Conclusions 84
References 85
Appendix 97
VII
List of Figures
Figure 1.1 Three host life cycle of a female ixodid tick 3
Figure 1.2 A. albolimbatum parasitising ear canal of a bobtail 5
Figure 1.3 Bobtail parasitised by A. albolimbatum 5
Figure 3.1 Agarose gel electrophoresis of Coxiella 16S (short) PCR products 27
Figure 3.2 Agarose gel electrophoresis of Coxiella 16S (short) PCR products 27
Figure 3.3 Agarose gel electrophoresis of Coxiella 16S (long) PCR products 28
Figure 3.4 Agarose gel electrophoresis of Coxiella GroEL PCR products 28
Figure 3.5 Phylogenetic analysis of 1,140 bp 16S rRNA of Coxiella 31
Figure 3.6 Phylogenetic analysis of 558 bp GroEL of Coxiella 32
Figure 3.7 Phylogenetic analysis of 1,694 bp concatenated 16S and GroEL of Coxiella 34
Figure 3.8 Agarose gel electrophoresis of Francisella 16S PCR products 36
Figure 3.9 Agarose gel electrophoresis of Francisella tpiA PCR products 36
Figure 3.10 Agarose gel electrophoresis of Francisella rpoA PCR products 37
Figure 3.11 Agarose gel electrophoresis of Francisella prfB PCR products 37
Figure 3.12 Agarose gel electrophoresis of Francisella dnaA PCR products 37
Figure 3.13 Agarose gel electrophoresis of Francisella dnaA PCR products 38
Figure 3.14 Phylogenetic analysis of 1,022 bp of 16S rRNA of Francisella 40
Figure 3.15 Phylogenetic analysis of 521 bp of tpiA of Francisella 41
Figure 3.16 Phylogenetic analysis of 900 bp of prfB of Francisella 42
Figure 3.17 Phylogenetic analysis of 723 bp of rpoA of Francisella 43
Figure 3.18 Phylogenetic analysis of 954 bp of dnaA of Francisella 45
Figure 3.19 Phylogenetic analysis of 4,230 bp of concatenated 16S, tpiA, prfB, rpoA, and dnaA of
Francisella 46
Figure 3.20 Agarose Gel Electrophoresis of Rickettsia gltA (long 650bp) PCR products 48
Figure 3.21 Agarose Gel Electrophoresis of Rickettsia gltA (short 381bp) PCR products 48
Figure 3.22 Agarose Gel Electrophoresis of Rickettsia ompA and ompB PCR products 49
Figure 3.23 Agarose Gel Electrophoresis of Rickettsia ompA PCR products 49
Figure 3.24 Agarose Gel Electrophoresis of Rickettsia ompB PCR products 50
Figure 3.25 Agarose Gel Electrophoresis of Rickettsia 17 kDa PCR products 50
Figure 3.26 Agarose Gel Electrophoresis of Rickettsia sca4 PCR products 50
Figure 3.27 Phylogenetic analysis of 344 bp of gltA of Rickettsia 55
Figure 3.28 Phylogenetic analysis of 634 bp of gltA of Rickettsia 56
Figure 3.29 Phylogenetic analysis of 517 bp of ompA of Rickettsia 58
Figure 3.30 Phylogenetic analysis of 251 bp of ompB of Rickettsia 59
Figure 3.31 Phylogenetic analysis of 394 bp of 17 kDa of Rickettsia 61
Figure 3.32 Phylogenetic analysis of 775 bp of sca4 of Rickettsia 63
Figure 3.33 Phylogenetic analysis of 644 bp of concatenated 17 kDa and gltA of Rickettsia 64
Figure 3.34 Phylogenetic analysis of 1,435 bp of concatenated ompA, ompB and gltA of
Rickettsia 65
Figure 3.35 Phylogenetic analysis of 1,639 bp of concatenated ompA, ompB, and sca4 of
Rickettsia 66
Figure 3.36 Phylogenetic analysis of 1,482 bp of concatenated ompB, 17 kDa, and sca4 of
Rickettsia 67
Figure 3.37 Phylogenetic analysis of 633 bp of concatenated ompB and, 17 kDa of Rickettsia 68
Figure 3.38 Agarose Gel Electrophoresis of Haemaprotozoa 18S 70
Figure 3.39 Phylogenetic analysis of 723 bp of 18S of Haemaprotozoa 71
VIII
List of Tables
Table 2.1 Primers and PCR cycling conditions for Coxiella detection 20
Table 2.2 Primers and PCR cycling conditions for Francisella detection 21
Table 2.3 Primers and PCR cycling conditions for Rickettsia detection 23
Table 2.4 Primers and PCR cycling conditions for haemoprotozoa detection 24
Table 2.5 Best-fit nucleotide substitution model for each gene locus 26
Table 3.1 Summary of positive samples from Coxiella assays 29
Table 3.2 BLAST results from 16S and GroEL Coxiella positive samples 29
Table 3.3 Summary of positive samples from Francisella assays 35
Table 3.4 BLAST results from 16S, tpiA, dnaA prfB and rpoA Francisella positive samples 39
Table 3.5 Summary of positive samples from Rickettsia assays 47
Table 3.6 BLAST results from gltA, ompA, ompB, 17 kDa, and sca4 Rickettsia sequences 53
Table 3.7 Summary of positive samples from haemoprotozoa assays 69
Table 3.8 BLAST results from 18S haemoprotozoa sequences 69
IX
List of Abbreviations
17 kDa 17 kDa lipoprotein precursor antigen gene AGRF Australian Genome Research Facility BLAST Basic Local Alignment Sequencing Tool bp base pairs DNA Deoxyribonucleic acid dnaA chromosomal replication initiator protein alpha subunit dNTP deoxyribonucleotide triphosphate EBC Extraction blank control EtOH Ethanol gltA citrate synthase gene GroEL GTR HKY K2
Chaperonin protein gene General Time Reversible Hasegawa-Kishino-Yano Kimura 2-parameter
MEGA Molecular Evolutionary Genetic Analysis software MgCl Magnesium chloride NCBI National Centre for Biotechnology Information nt Nucleotide NTC No-template control ompA Outer membrane protein A ompB Outer membrane protein B PBS Phosphate-buffered saline PCR Polymerase Chain Reaction pgm phosphoglucomutase prfB peptide chain release factor 2 beta subunit rpm Revolutions per minute rpoA DNA-directed RNA polymerase alpha subunit rRNA Ribosomal ribonucleic acid sca4 T92 TN93
120 kDa antigen gene Tamura 3-parameter Tamura-Nei
taq Thermus aquaticus DNA polymerase tpiA Triose-phosphate isomerase alpha subunit TBD Tick-borne disease TBP Tick-borne pathogen ZOTU Zero Operational Taxonomic Unit
1
CHAPTER ONE
INTRODUCTION
In 2019, bacterial community profiling of the 16S rRNA gene was conducted on the stump-tailed
lizard tick, Amblyomma albolimbatum, as part of an ongoing research to characterise and understand
the microbial diversity within Australian ticks (Mckenna, 2019). Several microbial taxa of interest,
those that were related to known tick borne pathogens including those of zoonotic potential, were
identified. However, due to relatively short sequences, taxonomic resolution could not be achieved
to determine the identity of the microbes present within this tick species. Herein this literature review,
a brief description of the vertebrate host Tiliqua rugosa (referred to throughout as ‘bobtail’), followed
by a background of ticks (with a focus on hard ticks), associated microbes (taxa of interest), and
technology used to characterise these microbes will be outlined to provide context for this thesis.
1.1 Bobtails (Tiliqua rugosa)
First described in 1825 by John Edward Gray as Trachydosaurus rugosus, Tiliqua rugosa is a
scincidae lizard; a short tailed, slow moving species with a heavily armoured body found in varying
colours from dark brown to cream (Norval et al, 2019; Pianka and Vitt, 2003). Tiliqua rugosa has four
recognised subspecies, three of which are only found in Western Australia, collectively known as
bobtail, although also known as the stump tailed lizard and the sleepy lizard. The fourth subspecies
of bobtail, Tiliqua rugosa asper, is the only species native to eastern Australia, is also known as the
shingleback (Norval and Gardner, 2019). Tiliqua rugosa rugosa, the subspecies relative to this
project, is distributed in south-west Australia; found throughout and surrounding bushland of Perth.
1.2 Ticks
Ticks are obligate haematophagous arthropods and the most important vector of infectious
microorganisms concerning companion animals and livestock (Parola and Raoult, 2001). Ticks are
members of the phylum Arthropoda. Within this phylum, ticks are assigned to a subphylum
2
Chelierata and then class Arachnida shared with scorpions, spiders, and mites (Guglielmone et al.,
2010). Ticks and mites share a subclass Acari though ticks are classified again into the order
Ixodidae. All ticks are categorised in three families: Ixodidae (hard ticks), Argasidae (soft ticks) and
Nuttalliellidae. Presently there are approximately 900 species of ticks classified globally; over 700
species of hard ticks are currently recognised in Ixodidae and around 200 species of soft ticks are
currently recognised in Argasidae (Guglielmone et al., 2010). Nuttalliellidae, is a third and the
smallest family consisting of a single species, Nuttalliella namaqua, specific and endemic to South
Africa (Jongejan and Uilenberg, 2004).
1.2.1 Tick life cycle
The life cycle of most ixodid ticks consist of four stages spanning one–three hosts: egg, larvae,
nymph, and either female or male adult (Bull and King, 1981; Barker and Walker, 2014). Transition
from one life stage to the next is dependent on a blood meal acquired from a host prior to moulting.
Each life stage requires only one blood meal and full engorgement can take between three days and
three weeks, though duration of feeding varies depending on species, instar, and host (Chilton and
Bull, 1991; Barker and Walker, 2014). This is contrasted by male ticks not completely engorging,
though they require smaller blood meals for sustenance (Barker and Walker, 2014).
Three-host ticks life cycle is as follows: When finished developing in eggs, larvae will hatch around
several weeks and then begin questing for a host, usually a host sharing the environment (Kerr and
Bull, 2006). Once a host is found, larvae will engorge from their blood meal and detach, usually when
host are in their refuge sites, before moulting to a nymph (Petney et al., 1983; Petney and Bull,
1981). This process is repeated by the nymph, moulting to an adult male or female (Smyth, 1973;
Barker and Walker, 2014). Male ticks remain on the host attempting to mate with multiple females.
The male will transfer the sperm sac to the female. Female ticks will only mate once prior to
engorgement. Once engorged to repletion, females will detach from their host and have enough
sperm to fertilize all their eggs before they die (Parola and Raoult, 2001). Eggs are always laid in the
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environment and not on the host (Barker et al., 2014). Life cycle of a three-host tick can vary from
six months to several years depending on environmental conditions and host availability (Barker and
Walker, 2014).
Figure 1.1. Three host life cycle of a female ixodid tick (Apanaskevich and Oliver, 2014).
1.2.2 Ticks of Australia
Australia is home to a range of native and unique ticks. At least 74 valid species are currently known
to exist in Australia; 69 of those species are native to Australia, and five were introduced following
European arrival over 230 years (Barker et al., 2014; Ash et al., 2017; Kwak et al., 2018). The five
introduced tick species are comprised of three hard tick species: Haemaphysalis longicornis (bush
tick), Rhipicephalus australis (Australian cattle tick), and Rhipicephalus linnaei (brown dog tick; syn
Rh. sanguineus sensu lato, tropical lineage; Šlapeta et al. 2021); and two soft ticks: Argas persicus
(poultry tick) and Otobius megnini (spinose ear tick) (Graves and Stenos, 2017).
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Within Australia, there are 57 native hard ticks belonging to the genera Amblyomma, Bothriocroton,
Haemaphysalis and Ixodes, and 12 native soft ticks belonging to two genera, Argas and
Ornithodoros (Barker and Walker, 2014). Since 2017, three new Ixodes species have been
described. Morphological and molecular characterisation by Ash et al. (2017) confirmed Ixodes
woyliei to be the first new species of Ixodes tick species described in over 50 years. Ixodes woyliei
has a high predilection for the endangered marsupial Bettongia penicillate, otherwise known as the
woylie (Ash et al., 2017). Ixodes heathi, an endangered tick found on a critically endangered
mountain pygmy possum (Burramys parvus), was discovered shortly afterwards in 2018 (Kwak et
al., 2018). More recently, Barker et al. (2019) described the species Ixodes barkeri removed from
the short-beaked echidna, Tachyglossus aculeatus (Barker, 2019). The description of these novel
species of tick reveals the importance of accurate species descriptions and the addition of molecular
data.
Companion or domesticated animals are frequently parasitised by 17 ticks in Australia,
approximately 54 species of tick are known to parasitise reptiles, birds and wild mammals, and only
a few species are known to bite humans (Ash et al., 2017; Barker et al., 2014; Graves and Stenos,
2017). The species known to bite humans are the following: 11 hard ticks, Amblyomma triguttatum
(ornate kangaroo tick), Bothriocroton auruginans (wombat tick), B. hydrosauri (southern reptile tick),
Haemaphysalis bancrofti (wallaby tick), Haemaphysalis longicornis (bush tick), Ixodes cornuatus
(southern paralysis tick), Ixodes hirsti (Hirst's marsupial tick), I. holocyclus (paralysis tick), I. tasmani
(common marsupial tick), Rhipicephalus (Boophilus) australis (Australian cattle tick) and R. linnaei
(brown dog tick); and five soft ticks: A. persicus (poultry tick), Argas robertsi (Robert’s bird tick),
Ornithodoros capensis (seabird soft tick), O. gurneyi (kangaroo soft tick), O. megnini (spinose ear
tick). Though these species of tick have been documented to bite humans, it is most likely due to
opportunity rather than the ticks specifically questing for humans (Barker and Walker, 2014).
Similarly ticks not recorded to bite humans may still parasitise people if given the opportunity, though
there are no specific records of this occurring.
5
1.2.3 Bobtail ticks
Australia has more reptile-specific ticks than any other region, which is most likely due to the
abundance and species richness of reptiles (Barker and Walker, 2014; Natusch, 2018). Among the
current 74 tick species documented in Australia, 13 are known to parasitise reptiles. All ticks
historically found on the bobtail are three reptile ticks: A. albolimbatum (the stump-tail lizard tick); A.
limbatum (the reptile tick); and B. hydrosauri (the southern reptile tick).
Ticks commonly parasitise the bobtail under scales and in the external auditory meatus (Figure 1.1
and 1.2) (Smyth, 1973; Barker et al., 2014; Barker and Walker, 2014). Although the bobtail is the
primary host for these three ticks, these ticks have been reported to parasitise, albeit less frequently,
other vertebrate species. For example, B. hydrosauri has been found on other reptile hosts including
lizards, snakes, a terrestrial turtle, and has been reported to parasitise mammals such as cattle,
horses and humans (Barker and Walker, 2014).
Figure 1.2. Amblyomma albolimbatum parasitising ear canal of a bobtail. Source: Dr Paola Magni.
Figure 1.3. Bobtail parasitised with A. albolimbatum. Source: Dr Paola Magni.
6
Smyth (1973) identified and detailed a geographic and host distribution of three species of reptile
tick widespread in Australia. Further research by Professor C. Michael Bull revealed valuable insights
into the biology and ecology of ticks from his long-term study into the parapatric boundaries of hard
ticks A. limbatum and B. hydrosauri (formerly Aponoma hydrosauri) associated with the bobtail,
showing that when any pair of ticks met there is at most 2kms of overlap and clear boundaries
between species (Belan and Bull, 1995). All three tick species occur in Western Australia; however,
Amblyomma limbatum has a northerly distribution, occurring in dryer, more tropical parts of the state;
contrasted by B. hydrosauri and A. albolimbatum having more southern and south-western
distributions, respectively (Sharrad and King, 1981; Godfrey and Gardner, 2017). Interest currently
surrounds ticks known to parasitise reptiles and the putative link to tick-borne disease in humans.
For example, B. hydrosauri has been attributed to causing Flinders Island spotted fever due to
transmission of the bacterium Rickettsia honei (Graves and Stenos, 2017; Whiley et al., 2016).
1.3 Tick associated microbes and diseases
Globally, ticks are established as an important arthropod vector for transmitting a range of microbes
detrimental to the health of its vertebrate host (Colwell et al., 2011). Every tick species has
environment and biotope requirements that determine their geographic distribution, and
subsequently, areas associated with tick-borne diseases (TBD) (Parola and Raoult, 2001). While
identifying the presence of a pathogenic organism within a tick warrants caution, it should be noted
that the presence does not necessarily lead to transmission or result in disease. For transmission of
a pathogenic species to a vertebrate host to occur, the microbe must be present within the salivary
glands of a tick during feeding (Hooper and Gordon, 2001; Parola and Raoult, 2001). Some of the
most important zoonotic TBDs include anaplasmosis (Anaplasma spp.), babesiosis (Babesia spp.),
Lyme borreliosis (Borrelia burgdorferi sensu lato complex), tick-borne encephalitis (Flavivirus),
tularaemia (Francisella tularensis), Crimean-Congo haemorrhagic fever (Nairovirus), and spotted
fever (Rickettsia spp.) (Springer et al. 2021).
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In addition to pathogenic microbes, ticks also harbour a full suite of microorganisms, referred to as
the ‘microbiome’, including bacteria, fungi, protozoa, and viruses, that can be commensal, symbiotic
or pathogenic/parasitic (Narasimhan and Fikrig, 2015). Of note, tick symbionts share a close
phylogenetic relationship to many tick borne pathogens (TBP) (Bonnet et al., 2017). Ticks depend
on symbiotic relationships with endosymbionts of the genera Coxiella, Francisella, Rickettsia, and
Wolbachia, to provide nutrients missing from their diets, aid in reproduction, assist in moulting and
may also promote or inhibit the acquisition or transmission of some TBPs (Gerhart et al., 2016;
Bonnet et al., 2017).
Ticks can also cause non-infectious illness directly to their hosts, such as tick paralysis, mammalian
meat allergy, autoimmune disorders and post-infection fatigue are also reported to occur following a
tick bite in Australia (van Nunen, 2015; Graves and Stenos, 2017). However, non-infectious
diseases, along with fungi and viruses, will not be discussed further as they are beyond the scope
of this thesis.
1.3.1 Australian tick-borne diseases
Compared to overseas, very few Australian tick species are attributed to transmitting tick-borne
pathogens which cause disease (Stenos and Graves, 2017). At present, only three TBD are
recognised; two Rickettsia species, Rickettsia australis and Rickettsia honei which cause
Queensland Tick Typhus (QTT) and Flinders Island spotted fever (FISF), respectively; and Coxiella
burnetii, the causative agent of Q fever. Bothriocroton hydrosauri and I. holocyclus are both known
to harbour and transmit R. honei and R. australis, respectively (Panetta et al., 2017). Both I. tasmani
(common marsupial tick) and I. cornuatus (southern paralysis tick) also have been associated with
R. australis and known to bite humans (Barker and Walker, 2014; Graves and Stenos, 2017). First
discovered on Flinders Island, Tasmania, FISF is an Australian TBD that has been detected in
patients from other parts of Australia (Unsworth et al., 2005). The paralysis tick, I. holocyclus, is
endemic to east coast Australia and causes QTT and Q Fever due to R. australis and C. burnetii
8
respectively (Stenos and Graves, 2017). Withal, the Ornate kangaroo tick, A. triguttatum, a human
biting tick, can be located through central, northern and Western Australia (WA) and has been linked
with the transmission of C. burnetii and a proposed novel SFG pathogen, R. gravesii (Li et al., 2010;
Graves and Stenos, 2017).
1.3.1.1 Coxiella
Coxiella is a genus of gram-negative, obligate intracellular bacteria of the phlyum Proteobacteria;
order Legionellales; family Coxiellaceae (Maurin and Raoult, 1999). The genus Coxiella currently
comprised of three identified species: Coxiella cheraxi, a pathogen of the redclaw crayfish (Cherax
quadricarinatus); Coxiella massilliensis, an etiological agent to humans; and Coxiella burnetii, the
causative agent of Q fever (Angelakis et al., 2016; Duron et al., 2015; Tan and Owens, 2000).
Originally classified as a Rickettsia species ‘Rickettsia burnetii’, C. burnetii was reclassified from
alphaproetobacteria to gammaproteobacteria after phylogenetic analyses of the 16S rRNA gene
locus revealed a genetic relationship closer to Legionellales than Rickettsiales (Leclerque and
Kleespies, 2008; Marrie et al., 2015).
Coxiella burnetii is a zoonotic pathogen that causes the highly infectious disease Query fever (Q
fever) in humans, and coxiellosis in animals (Derrick, 1937; Duron et al., 2015). With the exception
of New Zealand, C. burnetii has been reported to occur worldwide and has a sylvatic life cycle
associated with wildlife, domestic animals, and ticks (Maurin and Raoult, 1999). Human acquisition
of C. burnetii is predominantly through inhalation of aerosols, though Q fever can occur through
contact with infectious tissues and excretions from domestic ruminants and companion animals
(Gregory et al., 2019; Maurin and Raoult, 1999). Most animals infected with C. burnetii are
asymptomatic, however bronchopneumonia, fever, and inflammation of the placenta post-abortion
have been known to occur (Babudieri , 1959; Palmer et al., 1983; Waldman, 1978). In addition,
female animals infected with C. burnetii are known to shed high amounts of the infectious bacteria
in urine, faeces, milk and parturient material (Tan and Owens, 2000). Q fever is considered a
9
notifiable disease across Australia by the Department of Health and is the only endemic tick
associated notifiable disease impacting human health in Australia.
Currently more than 40 tick species have been associated with C. burnetii and Coxiella species
(Parola and Raoult, 2001; Duron et al., 2015). Phylogenetic analyses of the Coxiella genus
emphasised a high genetic diversity and showed the genus can be split into four main clades (Duron
et al., 2015). The Coxiella endosymbionts have been identified within all clades, however C. burnetii
clusters into a unique subclade indicating the species is likely to have evolved from a Coxiella tick
endosymbiont; the closest species to C. burnetii are Coxiella strains associated with soft ticks from
Ornithodoros and Argas. Additionally, Coxiella species genetically related to C. burnetii identified as
endosymbionts in soft and hard ticks, lack virulent characteristics (Duron et al., 2015).
Although more commonly associated with mammals, evidence of C. burnetii has been reported in
water snakes (Natrix natrix); rat snakes (Ptyas korros); Indian python (python molurus); and tortoise
(Kachuga sp.) (Yadav and Sethi, 1979). In addition, overseas reptile ticks have previously been
associated with C. burnetii. The tortoise tick Hyalomma aegyptium (Blanc 1961; Široký, 2007), is
primarily found on Testudo genus tortoises in the Mediterranean region, and has a three host life
cycle alternating between tortoises, other reptiles, and mammals (Široký et al., 2007). The monitor
lizard tick, Amblyomma exornatum, harboured natural infection of C. burnetii in Guinea-Bissau
(Arthur, 1962). Additionally, Kim et al. (1978) has described outbreak of Q fever in New York among
humans removing the African tick, Amblyomma nutalli, from ball python (Python regius). Similarly, a
Coxiella endosymbiont has also been reported in A. nuttalli from Africa (Nardi et al., 2021). Most
recently, two separate studies on Australian reptile ticks, A. albolimbatum and B. undatum, removed
from bobtail and goanna respectively, observed Coxiella sp. reads (proposed C. burnetii) in next
generation sequence (NGS) data (Panetta et al., 2017; Mckenna, 2019). However, both studies were
unsuccessful in confirming the presence of C. burnetii via conventional PCR.
10
1.3.1.2 Francisella
The family Francisellaceae consists of a single genus Francisella which are small coccobacillary,
aerobic, gram-negative, obligate intracellular, non-sporulating bacteria and belong to the phylum
Proteobacteria; class Gammaproteobacteria; order Thiotrichales (Olsufiev et al., 1959; Gerhart et
al., 2016). First identified in 1912 as a plague-like disease of rodents, tularaemia is a highly infectious
zoonotic disease caused by F. tularensis, affecting humans and animals, and considered endemic
in the Northern Hemisphere (Eden et al., 2017). Francisella tularensis can be divided into several
subspecies ranging in pathogenicity: F. tularensis tularensis, which is life threatening; F. tularensis
holartica, causes less severe disease; F. tularensis mediasiatca shows similar virulence to F. t.
holartica; and F. t. novicida, regarded as least virulent (Larson et al., 2020). Acquisition of tularaemia
usually occur through direct contact with infected animals, infected water, inhalation of contaminated
aerosols and tick vectors (Eden et al., 2017). Importantly, while tularaemia is considered a notifiable
disease in Australia, there are currently no reports of ticks transmitting the disease to humans within
Australia. However, tularaemia was reported in Tasmania, Australia in 2011, when an adult human
was bitten by a ringtail possum. The causative agent was proposed to be F. t. holartica, based upon
16S rRNA sequence and serology (Eden et al., 2017; Jackson et al., 2012). Francisella tularensis
has a much broader host range than most other zoonotic pathogens, and has been reported in
species of mammals, birds, amphibians and invertebrates (Mörner and Addison, 2001). In addition
to Francisella causing human disease, closely related Francisella endosymbionts have previously
been described in ticks overseas, for example: Argas, Ornithodoros, Dermacentor, and Amblyomma
ticks.
In 2003, Francisella hispaniensis was isolated from a human in the Northern Territory following a
foot cut received in brackish water (Whipp et al., 2003). Francisella DNA has also been sequenced
from reptile ticks, A. helvoum, and A. varanese, removed from snakes in Thailand, and in a tick
commonly found on reptiles, A. fimbriatum, in the Northern Territory, Australia (Vilcins et al., 2009;
Sumrandee et al., 2014). Following this, Aravena- Román et al. (2015) reported a Francisella sp.
with a 99.8% genetic similarity to F. hispaniensis at the 16S rRNA locus, becoming the first
documented case of bacteraemia attributed to F. hispaniensis in WA; the route of transmission
11
however remains unknown. Furthermore, 16S sequences generated by NGS identified a Francisella
sp. 99% genetically similar to F. hispaniensis was observed in A. alblolimbatum parasitising the
sleepy lizard T. rugosa, was recently reported in Perth, WA (Mckenna, 2019). Polymerase chain
reaction (PCR) developed by Forsman et al, (1994) targeting the 16S rRNA gene of Francisella is
considered to be efficient and cost effective, however is limited by how confidently the assay can
differentiate between species of Francisella (Roux and Raoult, 2000); therefore, additonal genes
need to be considered to identify species more confidently.
While not associated with reptiles, a recent study reported a high prevalence and abundance of
Francisella sequences in the red kangaroo tick, A. triguttatum, a common human biting tick, from
Western Australia (Egan et al., 2020).
1.3.1.3 Rickettsia
The genus Rickettsia are small, gram-negative, pleomorphic coccobalilli bacteria from the phylum
Proteobacteria; class Alphaproteobacteria; order Rickettsiales; family Rickettsiaceae (Roux and
Raoult, 2000). Rickettsia are associated with causing spotted fever and tick-associated typhus
diseases, and are commonly transmitted via hard ticks (Parola et al., 2005). Based on genotypic and
phenotypic differences, the genus can be divided into three groups of disease causing species: The
spotted fever group (SFG), comprised of species mostly associated with ticks; the typhus group
(TG), includes R. typhi and R. prowazekii usually transmitted to humans by fleas and lice; and the
ancestral group, comprised of a single species, R. bellii, the earliest diverging species of Rickettsia
(Ogata et al., 2006; Vilcins et al., 2009; Ogrzewalska et al., 2016; Parola et al., 2016; Abdad et al.,
2017). Several Spotted fever Group (SFG) and Typhus Group (TG) Rickettsia have been recognised
as causing disease in humans (Ogata et al., 2006).
Despite its use for microbiome studies, the 16S rRNA gene is highly conserved within the genus
Rickettsia, and phylogenetic analysis of the 16S cannot be used to confidently differentiate between
12
species, warranting more suitable gene targets (Sekeyova et al., 2001). For example, a recent
microbiome investigation into A. albolimbatum ticks collected from T. rugosa, found several zero
operation taxonomic units (ZOTU) related to rickettsial DNA, suggesting the presence of multiple
Rickettsia spp. Independently, researchers at the Australia Rickettsial Reference Laboratory, have
recently sequenced a single Rickettsia sp. from the same tick species and concluded that sequences
clustered in a distinct subclade to R. gravesii (Tadepalli et al., 2021). However, because each group
utilised different gene assays, it is currently unknown how related the Rickettsia species from both
groups are.
1.3.1.4 Hemolivia
A variety of protistan parasites are present in the blood of vertebrates and are transmitted by
haematophagous vectors, such as ticks. The genus Hemolivia are haemogregarine parasites in the
family Karyolsidae from the sub-order Adeleiorina of the phylum Apicomplexans, and infect
exothermic vertebrates (Carreno and Barta, 1999). There are currently three known species of
Hemolivia: Hemolivia stellata; Hemolivia mariae; and Hemolivia mauratanica. Hemolivia stellata and
H. maratanica are known to infect the cane toad and tortoises respectively. The parasitic protist
Hemolivia mariae infects the erythrocytes of lizards, such as Tiliqua rugosa (Smallridge and
Paperna, 1997; Barker and Walker, 2014). Hemolivia mariae is transmitted by at least two ixodid tick
species known to be a definite host and vector, Amblyomma limbatum and Bothriocroton hydrosauri;
though A. limbatum has been reported to be more susceptible to H. mariae infection (O’Donoghue,
2017; C. Smallridge and Bull, 2001; Spratt and Beveridge, 2018). Amblyomma albolimbatum has
also been implicated as a host for H. mariae based on morphological characteristics and differences
between their host range and geographical distribution; thus, ruling out H. stellata (Moller, 2014).
However, molecular testing is still required to confirm the identity of the Hemolivia species present.
It has been proposed that ticks become infected with H. mariae when taking a blood meal from an
infected bobtail. Similarly, bobtails can become infected with H. mariae after ingesting infected ticks
13
(Barker and Walker, 2014; Smallridge and Bull, 2001). However, studies show that tick load on
infected and non-infected lizards are not significantly different; there is also no evidence of infection-
induced mortality in ticks (Smallridge and Bull, 2001). Furthermore, T. rugosa infected with H. mariae
do not have the energy expenditure of those that do not, resulting in a decrease in their physical
activity and therefore a decrease in their territory. Moreover, B. hydrosauri are also identified to
negatively impact the physical capability of their reptilian hosts (Bouma et al., 2007; Barker and
Walker, 2014). While the presence of H. mariae has been documented in hard ticks, the zoonotic
potential is unknown.
1.4 Molecular detection of tick associated microbes
While serology and culture are the gold standard for the detection of infectious diseases, serology is
limited by lag between initial infection and the development of antibodies (Stenos et al., 2005).
Moreover, culture-based methods can take up to 60 days to produce a positive result with some
pathogens unable to be detected by traditional culture base techniques (Stenos et al., 2005;
Unsworth et al., 2005). For the purpose of this study, the following section will discuss how
polymerase chain reaction (PCR) has been used for the molecular characterisation of microbial taxa
of interest within ticks.
1.4.1 Polymerase chain reaction (PCR)
Polymerase chain reaction is an effective molecular technique allowing DNA fragments to be
amplified within a few hours. This is particularly useful when there is limited quantity of the original
DNA; in some cases, a single molecule (Pierce, 2017). Substantially quicker than tradition methods
such as culture and serological methods, PCR can detect low quantity organisms from a variety of
sample types, due to higher sensitivity, making it a valuable molecular tool (Mullis and Faloona,
1987; Stewart et al., 2017).
14
PCR (coupled with Sanger sequencing) can also be designed to be highly sensitive and specific
making it a valuable diagnostic method. Sequences acquired via PCR and Sanger Sequencing can
be compared with databases composed of reference sequences; this provides a method to draw
inference from phylogenetic relationships of an amplified sequence with prior categorised taxa.
Furthermore, these molecular techniques have been used for detection of bacteria such as Coxiella
and Rickettsia in Australian ticks (Cooper et al., 2013; Izzard et al., 2009). This makes PCR a
particularly useful tool for differentiation of closely related species such as Spotted Fever group
(SFG) and typhus group Rickettsia which cannot be set apart when employing serology (Tadepalli
et al., 2021). Due to the overlap of geographical location known for B. hydrosauri, and confirmed
cases of R. honei isolated from patients, Whiley et al. (2016) conducted a study using real-time PCR
(qPCR) to perform assays on B. hydrosauri collected from T. rugosa from mainland South Australia,
discovering Rickettsia species in all tick samples. While R. honei was identified within ticks, the study
also suggested that B. hydrosauri could be a vector for multiple Rickettsia species.
1.4.1.1 16S rRNA gene
The ubiquitous 16S ribosomal RNA gene (16S) in prokaryotic organisms is fundamental, and
responsible for encoding the small subunit ribosomal RNA molecules of ribosomes (Woese and Fox,
1977; Větrovský and Baldrian, 2013). The gene is universally distributed among the bacterial
domain, making identification and characterisation among different taxa from a variety of sources,
including ticks possible to analyse using phylogenetics (Clarridge, 2004; Větrovský and Baldrian,
2013). Occurring between a single copy and up to 15 copies depending on the taxa, the 16S rRNA
is approximately 1,500 bp long and features both conserved regions and nine hypervariable regions
(V1 – V9) (Klappenbach et al., 2001; Dias et al., 2020) . The conserved regions allow for appropriate
PCR primers target for various taxa due to slow evolution rate of this region (Woese and Fox, 1977).
The nine hypervariable domains of 16S exhibit varying degrees of sequence diversity; among some
bacteria genera the hypervariable regions are highly conserved between species, limiting species-
level identification (Greay et al., 2018; Dias et al., 2020). Although, this limits the 16S rRNA
15
sequencing method due to potential sequence similarity between closely related species, such as
endosymbionts and pathogens (Větrovský and Baldrian, 2013). Thus, further characterisation of
alternative genes is sometimes necessary to more confidently determine a species.
1.4.1.2 Additional genes
Targeting the 16S rRNA has proved a valuable tool and is still widely used, particularly for
microbiome analyses, however it does not suffice for certain taxa, such as Rickettsia, as previously
outlined (Mckenna, 2019). This is due to the conserved nature of the gene, rendering phylogenetic
analysis based on the 16S gene more unreliable, especially concerning members of the SFG
(Fournier et al., 2003; Roux et al., 1997). Additionally, the 16S gene is not adequate for determining
virulence; as the region does not usually encode virulence factors (Clarridge, 2004). In 1997, a study
on enteric bacteria investigating the RNA polymerase b-subunit (rpoB) gene as an alternative to the
16S RNA, it was reported that the rpoB gene is more reliable between some genera (Mollet et al.,
1997). Similarly the citrate synthase gene (gltA) of Rickettsia provides more accuracy between
species differentiation in the genus than 16S (Roux et al., 1997); Furthermore, this is supported by
phylogenetic studies of Rickettsia, prompting a reorganisation of the genus after investigating the
16S rRNA, citrate synthase (gltA), 17 kDa antigen (17 kDa), outer membrane protein A (ompA), outer
membrane protein B (ompB) and surface cell antigen (sca4) (Roux et al., 1997; Roux and Raoult,
2000; Sekeyova et al., 2001; Fournier et al., 2003). Therefore, highlighting the requirement for
sequencing alternative genes when considering phylogenetic analysis.
1.5 Aims and hypotheses
Investigation is still needed to identify unknown microbes within the microbiome of Australian ticks.
In 2019, sequences matching Francisella, Rickettsia, and Coxiella were acquired via next-generation
sequencing (NGS) in Amblyomma albolimbatum ticks parasitising the bobtail lizard (T. rugosa) in
Western Australia. This research project will contribute to the molecular characterisation of bacterial
species within A. albolimbatum. This present study will investigate tick DNA samples previously
16
extracted in the aforementioned study and further extractions on ticks identified as being from the
same host; all of which were collected by wildlife health centres in WA. The continued molecular
characterisation of the microbial taxa associated with reptile ticks in Australia will increase our
understanding of the bacterial species these ticks harbour and assess if there is any associated
zoonotic risk.
Based on the previous bacterial community study in A. albolimbatum ticks, the first hypothesis is
additional molecular characterisation will determine how genetically related species of Francisella,
Rickettsia and Coxiella are to known pathogens and endosymbionts. Secondly, it is hypothesised
that haemoprotozoa will be identified as Hemolivia mariae within A. albolimbatum confirming Moller
(2014) morphological observations.
The aims of this thesis are two-fold: first, additional genetic information will be generated and
phylogenetic analyses will be conducted to further characterise Coxiella, Rickettsia, and Francisella
species previously detected by NGS in the tick, A. albolimbatum; and second, to investigate the
presence of haemoprotozoan parasites in A. albolimbatum.
17
CHAPTER TWO
MATERIALS AND METHODS
2.1 Sample acquisition
A total of 142 reptile ticks were collected from 67 bobtail (T. rugosa) by collaborators at Kanyana
Wildlife Rehabilitation Centre, Armadale Reptile Centre, Geovet Busselton and members of the
public, and submitted to the Vector and Waterborne Pathogen Research Group at Murdoch
University. All ticks collected directly and opportunistically from bobtails within Perth metropolitan
area in WA. Ticks were all preserved in 70% ethanol and stored at 4°C until further identification and
molecular analyses. Prior to this current project, Mckenna (2019) morphologically and molecularly
identified all ticks as Amblyomma albolimbatum. This was carried out using the morphologically and
molecular identification keys for Australian ticks to determine species, instar and sex (Roberts, 1970;
Barker and Walker, 2014); and molecular barcoding of the cytochrome C oxidase 1 (CO1) gene
(Hebert et al., 2003).
2.2 DNA extractions
Genomic DNA (gDNA) from 116 ticks were previously extracted using Qiagen DNeasy Blood and
Tissue Kit (Qiagen, Germany) following recommendations provided by manufacturers (Qiagen
Supplementary Protocol: Purification of total DNA from insects) with modifications as described by
Mckenna (2019). In addition to the DNA extracts provided by Mckenna, a further 26 ticks were
extracted during this study, using the same DNA protocols as describe by Mckenna (2019)(DNA
extraction tick instar in Table A1.1). Prior to DNA extraction, 26 ticks were individually surface-
sterilised with a 10% sodium hypochlorite, then 70% ethanol, and washed with sterile DNA-free
Phosphate-Buffered (PBS) and air dried. Each tick was then sliced into quarters using a sterile
scalpel blade and petri dish and placed into a 1.5mL safe lock tube (Eppendorf TM). Equipment was
further decontaminated with DNA AWAY (Molecular Bio-products Inc., San Diego, USA) to further
reduce contamination between samples. Buffer ATL and Proteinase K were aliquoted into the 1.5mL
18
tubes containing the ticks in the following amounts: 200μL buffer ATL and 22μL Proteinase K for
unengorged adults or nymph; 450μL buffer ATL and 50μL Proteinase K enzyme for engorged ticks.
Samples were incubated at 56°C in a headblock overnight (approximately 16 hr) shaking at 700rpm.
Following incubation, samples were spun at 15,0000 rpm for 3 minutes. 200μL of the supernatant
was transferred into a sterile 1.5mL safe lock tube (Eppendorf TM) containing a 1:1 solution of 200μL
of 96% alcohol and 200μL of buffer AL before a thorough vortex. Succeeding 500μL washes of buffer
AW1 and buffer AW2, elution buffer AE was directly added to the membrane in the following
amounts: 100μL for unengorged adults or nymph; 200μL for engorged ticks. Samples were then
incubated at room temperature for 4 minutes, and spun at 8,000rpm for 1 minute. Extracted gDNA
was put into 1.5mL safe lock tube (Eppendorf TM) with DNA ID, internal accession code: PI number,
date and initials for storage at -20°C. All 26 samples were analysed on a nanodrop to determine
nucleic acid yield and purity (Appendix Table A1.2). Extraction reagent blank was carried out to
mirror extraction process.
2.3 PCR assays
Subsets of samples relative to each taxa were analysed due to time and money constraint with
respect to the amount of assays performed. However, due to previous NGS study producing
sequences with a 100% genetic match to C. burnetii, the aetiological agent for the zoonotic disease
Q fever, all 142 samples were screened.
2.3.1 Coxiella detection using conventional PCR
Conventional PCR was used to investigate samples (n=142) for the presence of Coxiella species
within A. albolimbatum, targeting 524 bp of 16S rRNA gene (Short 16S) using primers Cox-sp434F
and Cox-sp1004R (Lalzar et al., 2012) (Table 2.1). Once sequences were obtained, samples that
tested positive using the short 16S assay, were re-tested to obtain a larger product size targeting
1.45kb of the 16S rRNA gene (Long 16S) using primers Cox-16S-1457F and Cox-16S-1457R
(Masuzawa et al., 1997) (Table 2.1). Each PCR reaction was performed in 25μl volumes containing
19
the following: 1 X KAPA buffer with dye (KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA
MgCl2, 0.25mM dNTPs (FisherBiotech, Australia), 0.4μM of each primer, 0.01mg BSA (Fisher
Biotech, Australia), 0.5 U KAPA taq (KAPA Biosystems, Massachusetts, USA) and 2μL of gDNA
template. Extraction blank, no-template and positive control was used in each assay. Thermal cycling
was performed using Applied Biosystems thermal cycler under the following conditions for the short
16S assay: 94°C for 4 minutes, then 40 cycles of 94°C for 30s, 60°C for 30s and 72°C for 45s,
followed by a final extension of 72°C for 5 minutes. In an attempt to amplify better quality bands from
samples that produced weak bands, a re-amplification step was included. The re-amplficiation step
included 1μL of purified amplicon product and an additional 10 cycles of amplification with the Cox-
sp434F and Cox-sp1004R primers. Thermal cycling conditions for the long 16S assay were 94°C for
5 minutes, then 40 cycles of 94°C for 60s, 48°C for 60s and 72°C for 120s, followed by a final
extension of 72°C for 5 minutes.
In addition to the 16S rRNA assay, a Coxiella-specific PCR assay targeting a 659 bp fragment of the
GroEL gene was performed on samples using primers Cox-660f and Cox-1320r in Table 2.1. Each
PCR reaction was performed in 25μl volumes containing the following: 1 X KAPA buffer with dye
(KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA MgCl2, 0.25mM dNTPs (FisherBiotech,
Australia), 0.4μM of each primer, 0.5 U KAPA taq (KAPA Biosystems, Massachusetts, USA) and
2μL of undiluted gDNA template. An extraction blank, no DNA-template and a positive control
(BAB33M, R. sanguineus positive for Coxiella endosymbiont) was used in each assay. Thermal
cycling was performed under the conditions shown in Table 2.1 : 95°C for 5 minutes, then 40 cycles
of 95°C for 30s, 52°C for 30s and 72°C for 1min, followed by a final extension of 72°C for 5 minutes.
20
Table 2.1. Primers used for Coxiella PCR assays.
Gene Primer Sequence (5’-3’) Size (bp) Reference
Short
16S
Cox-sp434F CCTTTTGAGCGTTGACGTTA ~524 bp Lalzar et al., 2012
Cox-sp1004R CCAAAGGCACCAAGTCATTT
Long
16S
Cox-16s-1457F ATTGAAGAGTTTGATTCTGG ~1.45 kb
Masuzawa et al.,
1997 Cox-16s-1457R CGGCTTCCCGAAGGTTAG
GroEL Cox-660f GGCGCICARATGGTTAARGA
~659 bp Angelakis et al.,
2016 Cox-1320r AACATCGCTTTACGACGA
2.3.2 Francisella detection using conventional PCR
Conventional PCR was used to investigate samples (n=21) for the presence of Francisella species
within A. albolimbatum, targeting partial regions of different genes using six different primer sets
(Ramírez-Paredes et al., 2017; Forsman et al., 1994) under the thermal cycling conditions provided
in Table 2.2. The tpiA assay was initially used for screening, with PCR amplified products confirmed
as Francisella DNA using Sanger sequencing, before retesting the samples with the other primer
sets. Each PCR reaction was performed in 25μl volumes containing the following: 1 X KAPA buffer
with dye (KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA MgCl2, 0.25mM dNTPs
(FisherBiotech, Australia), 0.4μM of each primer, 0.5 U KAPA taq (KAPA Biosystems,
Massachusetts, USA) and 2μL of undiluted gDNA template. Extraction blank, and no-DNA template
were used in each assay. No positive control was available, requiring optimisation.
21
Table 2.2. Primers and PCR cycling conditions used for Francisella PCR assays.
Gene Primer Sequence (5’ – 3’) Size (bp) PCR conditions (Temperature; °C/Time in seconds)
Reference Denaturartion Annealing Extension Cycles
tpiA
tpiA-F1 GCAAGTATTGGTGAGGATGTAATC
~674 95/60 63/30 72/60 40
Ramírez-Paredes et al., 2017
tpiA-R1 CTGCGGCACGTGGGTCATAG
rpoA
rpoA-F1 GCGACATCGAGGATATGACAT
~913 95/60 47/30 72/60 35 rpoA-R1 GGAATATTCTAGAACTACCG
prfB
prfB-F1 TTSAATCTTTACGGGACTAT
~1,009 95/60 47/30 72/60 35 prfB-R1 AACTTATCTAAATCACCATCTA
dnaA
dnaA-F1 AAAACCTTTCTACGTTTGAWT
~1,415 95/60 49/30 72/120 40 dnaA-R1 GCAACTCATAATCATCWGAT
pgm
pgm-F1 TGGCTATTCAGACTGTATCWAC
~1,613 95/60 57/30 72/120 50 pgm-R1 CAGTCATTCCTGTSASAGAT
16S
F5 CCTTTTTGAGTTTCGCTCC
~1,140 95/60 55/30 72/120 40 Forsman, et al., 1994 F11 TACCAGTTGGAAACGACTGT
22
2.3.3 Rickettsia detection using conventional and nested PCR
Conventional and nested PCR assays were used to investigate DNA samples from A. albolimbatum
(n=25) for the presence of Rickettsia using assays targeting the gltA, ompA, ompB, 17 kDa and sca4
genes using the primers under the thermal cycling conditions provided in Table 2.3. The gltA nested
assay was initially used for screening due gltA having greater phylogenetic resolution than the 16S.
with PCR amplified products confirmed as Rickettsial DNA using Sanger sequencing, before
retesting the samples with the other primer sets. Samples suggesting positive PCR products were
present were confirmed via Sanger sequencing. Each PCR reaction was performed in 25μl volumes
containing the following: 1 X KAPA buffer with dye (KAPA Biosystems, Cape Town, South Africa),
2.5mM KAPA MgCl2, 0.25mM dNTPs (FisherBiotech, Australia), 0.4μM of each primer, 0.5 U KAPA
taq (KAPA Biosystems, Massachusetts, USA) and 2μL of undiluted gDNA template. Extraction, no-
DNA template and positive control (1/1000 SFG culture from Australian Rickettsial Reference
Laboratory, Geelong) was used in each assay.
23
Table 2.3. Primers and PCR cycling conditions used for Rickettsia PCR assays.
Gene Primer Sequence (5’ – 3’) Size (bp)
PCR conditions (Temperature; °C/Time in seconds) Reference
Denaturartion Annealing Extension Cycles
gltA (1st)
gltA-F1 GCAAGTATTGGTGAGGATGTAATC ~900-1,000
95/60 63/30 72/60 40
Hii et al., 2011 gltA-R1 CTGCGGCACGTGGGTCATAG
gltA (2nd)
gltA-F2 GCGACATCGAGGATATGACAT ~650 95/30 59/30 72/60 35
gltA-R2 GGAATATTCTAGAACTACCG
gltA
RpCS.877p GGGGGCCTGCTCACGGCGG ~381 95/30 48/30 72/60 35 Roux et al., 2011
RpCS. 1258n ATTGCAAAAAGTACAGTGAACA
ompA
RR 190-70 ATGGCGAATATTTCT CCAAAA ~590 95/60 56/30 72/60 40
RR 190-701 GTTCCGTTAATGGCAGCATCT
ompB
omp B-F CGACGTTAACGGTTTCTCATTCT ~252 95/30 52/30 72/60 50 Hii et al., 2011
omp B–R ACCGGTTTCTTTGTAGTTTTCGTC
17 kDa Rr17k.1p TTTACAAAATTCTAAAAACCAT
~520 95/30 50/30 72/60 40 Ishikura et al., 2003 Rr17k.539n TCAATTCACAACTTGCCATT
Sca4 D1f ATGAGTAAAGACGGTAACCT
~910 95/60 50/60 72/60 40 Sekeyova et al., 2001 D928r AAGCTATTGCGTCATCTCCG
24
2.3.4 Piroplasm detection using nested PCR
To evaluate the presence of Piroplasms in samples, a nested PCR was used to screen tick samples
(n=45) targeting a partial region of the 18S rRNA gene of both Babesia and Theileria species using
external and internal primers (Jefferies et al., 2007) (Table 2.4). Each reaction was prepared to a
25µL volume containing: 1X KAPA buffer with dye and MgCl2 1.5 mM, an additional 1.5mM of MgCl2,
100mM dNTPs, 0.4µM of forward and reverse primers (Table 2.4). Reactions were performed under
the following thermal cycling conditions: The primary round of amplification had an initial activation
step of 94°C for 2 minutes, 58°C for 1 minute, and 72°C for 2 minutes, followed by 40 cycles of 94°C
for 30s, 58°C for 20s and 72°C for 1 minute, and a final extension of 72°C for 7 minutes. The
secondary round of amplification followed the same conditions as the primary round with following
exceptions: 1µL of primary PCR product instead of 2µL of gDNA template and the annealing
temperature was increased to 62°C. Extraction, no-DNA template and positive control (Babesia
vogeli from canine) were included with each set of PCR reactions.
Table 2.4. Primers used for Piroplasma PCR assays.
Gene Primer Sequence (5’ – 3’) Size (bp) Reference
18S (External) BTF1
GGCTCATTACAACAGTTATAG ~930 Jefferies et al., 2007
BTR1 CCCAAAGACTTTGATTTCTCTC
18S (Internal) BTF2 CCGTGCTAATTGTAGGGCTAATAC ~800
BTR2 GGACTACGACGGTATCTGATCG
2.4 Agarose Gel Electrophoresis and Sanger sequencing
All nested and conventional PCR products were separated on 1-2% agarose gel using 1 X TAE
buffer and stained using SYBR Safe (Invitrogen, Australia) to visualise under UV light. All PCR
products were visualised against a 100 bp DNA ladder (AXYGEN and Promega, Madison USA).
Amplified PCR products of the appropriate size were removed via a sterile filter tip and spun into a
25
sterilized 2.0 ml Eppendorf tube. After centrifugation the filter tip was removed using a sterile pipette
double wrapped in parafilm and decontaminated between samples (Yang et al., 2016). The purified
DNA was then used for sequencing.
The sequencing preparation of the amplified PCR products consisted of 7μl of Milli-Q water, 1μl of
either forward or reverse primer, and 4μl of PCR product. All PCR amplicons were submitted to the
Australian Genome Research Facility (AGRF) (Perth, Western Australia) for unidirectional
sequencing.
2.5 Phylogenetic analysis
Phylogenetic analyses were conducted by importing Sanger sequences obtained by AGRF into
Geneious v10 (Kearse et al., 2012) for assessment of quality and primer trimming. Sequences were
then subject to BLAST analysis using BLASTN 2.10.0 + (Altschul et al 1990) against nucleotide
collection (nt) database on the National Center for Biotechnology Information (NCBI) wesbite
(Morgulis et al., 2008), to identify most similar species and genotypes. Alignments with reference
sequences acquired from GenBank were built using MAFFT (Katoh, et al. 2002), trimmed to the
same nucldeotide length and realigned using MUSCLE alignment (Edgar, 2004). Aligned sequences
were then imported into MEGAX to determine the most suitable nucleotide substitution model based
on the Bayesian Information Score (BIC) (Kumar, et al., 2020). The best-fit substitution model for
each alignment is shown in Table 2.5. Genetic distance based on percent identity between
sequences were calculated using MEGAX (full genetic distance matrix for each alignment can be
found in appendix). A maximum-likelihood phylogenetic tree for each alignment was generated
based on 1000 bootstrap replications using MEGAX.
26
Table 2.5. Best-fit nucelotide substitution model used for each gene alignment.
Genus Gene Alignment
length BIC
score Model
Coxiella 16S 1,140 bp 10250 K2+G+I
GroEL 558 bp 7829 GTR+G+I
Concatenated 16S and GroEL 1,694 bp 13197 K2+G
Francisella 16S 1,028 bp 6801 K2+G+I
tpiA 521 bp 6093 HKY+G
prfB 900 bp 7255 T92+G
rpoA 723 bp 7265 T92+G+I
dnaA 954 bp 8508 T92+G
Concatenated (16S, tpiA, prfB, rpoA, dnaA) 4,230 bp 40774 GTR+G
Rickettsia gltA (short) 344 bp 3024 T92+G
gltA (long) 634 bp 5966 T92+G
ompA 517 bp 8651 T92+G
ompB 251 bp 3254 T92+G
17 kDa 331 bp 4400 K2+G
sca4 775 bp 8594 GTR+G+I
Concatenated (17 kDa and gltA(short)) 644 bp 5850 T92+G+I
Concatenated (ompA, ompB, and gltA(long)) 1,435 bp 16573 GTR+G+I
Concatenated (ompA, ompB, and sca4) 1,595 bp 20210 GTR+G+I
Concatenated (ompB, 17 kDa, and sca4) 1,482 bp 13722 GTR+G+I
Concatenated (ompB, and 17 kDa) 582 bp 5387 T92+G
Hemolivia 18S 791 bp 5706 T92+G
27
CHAPTER THREE
RESULTS
3.1. Molecular Characterisation of Coxiella
Amblyomma albolimbatum tick samples (n=142) were screened for the presence of Coxiella using
PCR assays targeting a short and long region of the 16S rRNA and GroEL genes (Table 3.1). All A.
albolimbatum samples failed to amplify with the short 16S PCR assay, with the exception of two
samples (C21 and C23) producing a faint 524 bp band and the Coxiella-positive control producing a
strong 524 bp band as visualised by gel electrophoresis (Figure 3.1). The faint products prompted
reamplification using an increase of cycles and from the of samples C21 and C23 which resulted in
stronger bands as shown in Figure 3.2. The no-template and extraction blank controls produced
appropriate results.
Figure 3.1. Agarose Gel Electrophoresis amplification of Coxiella 16S rRNA (short 526 bp). M:
DNA ladder Axygen 100 bp; Lane 1: SR17; Lane 2: SR18; Lane 3: SR19; Lane 4: SR20; Lane 5:
C21; Lane6: C22; Lane7: C23; Lane 8: C24; Lane 9: C25; Lane 10: C26; Lane 11: Empty well; Lane
12: extraction blank control; Lane13: Empty well; Lane 14: No-template control; Lane 15: Empty well;
Lane 16: C. burnetii positive control; Lane 17 - 18: Empty well; M: DNA ladder Axygen 100 bp.
Figure 3.2. Agarose Gel Electrophoresis reamplification of Coxiella 16S (short 526 bp). Lane
1: F6 Lane 2: C21 Lane 3: C23 Lane 4: Empty well; Lane 5: No-template control; Lane 6: Extraction
blank control; Lane 7: C. burnetii positive control; M: DNA ladder Axygen 100 bp.
28
Following successful amplification of the short 16S (529 bp), assays targeting a longer 16S (1,415
bp) and GroEL (659 bp) gene regions were performed on sample C23. Successful amplification
shown of expected product size were produced for the long 16S and GroEL, as visualised by gel
electrophoresis (Figure 3.3 and Figure 3.4 respectively). The no-template and extraction blank
controls produced appropriate results.
Figure 3.3. Agarose Gel Electrophoresis amplification of Coxiella 16S (long 1,375 bp). Lane 1:
Empty Well; Lane 2: C23 (4 µL of DNA template); Lane 3 - 5: Empty well; Lane 6: C23 (2 µL of DNA
template); Lane 7 - 8: Empty well; Lane 9: No-template Control; Lane 10 - 14: Empty well; Lane 15:
C. burnetii positive control; 16 – 18: Empty Well; M: DNA ladder Axygen 100 bp.
Figure 3.4. Agarose Gel Electrophoresis amplification of Coxiella GroEL gene. M: DNA ladder
Axygen 100 bp; Lane 1: C23; Lane 2-5: Empty well; Lane 6: Extraction blank control; Lane 7-9:
Empty well; Lane 10: C. burnetii positive control; Lane 11-12: Empty well; Lane 13: C23 (2 µL of
DNA template); Lane 14-15: Empty well; Lane 16: No-template control; Lane 17-18: Empty well; M:
DNA ladder Axygen 100 bp.
29
Table 3.1. Summary of Coxiella PCR assays.
The short and long 16S, and GroEL nucleotide sequences were compared against the GenBank
nucleotide database using nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide
collection (nt) was used to determine most similar identity. BLAST results of both the short and long
16S and GroEL nucleotide sequences identified in this tick sample presented a 100% nucleotide
identity to the following strains of Coxiella burnetii: C. burnetii Schperling chromosome
(CP014563.1); C. burnetii ‘MSU Goat Q177’ (CP018150.1); C. burnetii str. Namibia genome
(CP007555.1); C. burnetii str. Ammassalik (FJ87329.1); C. burnetii CbuK Q154 (CP001020.1).
Table 3.2. BLAST results for 16S and GroEL Coxiella sequences.
Sample Internal archive code 16S Short (524 bp)
16S Long (1.45 kb)
GroEL (659 bp)
C23 PI1715 + + +
C21 PI1715 +
Sample Internal archive code
Gene Size (bp)
NCBI description E- value
Percent identity
GenBank Accession code
C23 PI1715 16S 524 Coxiella burnetii str. Schperling chromosome
0.0 100% CP014563.1
C23 PI1715 16S 1,391 Coxiella burnetii str. Schperling chromosome
0.0 100% CP014563.1
C23 PI1715 GroEL 621 Coxiella burnetii str. Schperling chromosome
0.0 100% CP014563.1
30
Phylogenetic analysis was peformed on 1,140 bp 16S sequence obtained from sample C23,
including reference sequences from GenBank via NCBI (Figure 3.5). The topology of the
phylogenetic tree shows the 16S nucleotide sequence obtained in this project clustered with strains
of Coxiella burnetii, separated from the Coxiella endosymbionts of other clades, and was
supported by 63% bootstrap confidence. The 16S sequence acquired from C23 was 100%
identical to ten strains of Coxiella burnetii. The 16S sequence generated in this project was 1-3%
distinct from endosymbionts of Ornithodoros spp. (full genetic distance matrix in Table A1.3).
31
Figure 3.5. Maximum likelihood (ML) analysis of the relationships between Coxiella at the 16S
rRNA locus (1,140 bp). Percentage support (>50%) from 1000 replicates is indicated at the left
of the supported node. The sequence generated in the present study is in bold.
32
Phylogenetic analysis was performed on 558 bp GroEL sequence from sample C23, including
refence sequences from GenBank via NCBI (Figure 3.6). The topology of the phylogenetic tree
constructed shows the GroEL sequence from this project clustered with strains of Coxiella burnetii,
seperated from the Coxiella endosymbionts of other clades and was supported by 99% bootstrap
confidence. The GroEL sequence acquired from C23 was 100% identical to ten strains of Coxiella
burnetii. The GroEL sequence generated in this project was 3-4 % distinct from endosymbitons of
Ornithodoros spp. (full genetic distance matrix in Table A1.5).
Figure 3.6. Maximum likelihood (ML) analysis of the relationships between Coxiella at the
GroEL gene locus (558 bp). Percentage support (>50%) from 1000 replicates is indicated at
the left of the supported node. The sequence generated in the present study is in bold.
33
Phylogenetic analysis based on concatenated 1,694 bp 16S and GroEL nucleotide sequences
obtained from sample C23, including reference sequences from GenBank via NCBI (Figure 3.7).
The topology of the phylogenetic tree constructed shows the concatenated sequence from this study
clustering witnah Coxiella burnetii, seperated from the Coxiella endosymbionts of other clades and
was supported by 86% bootstrap confidence. The concatenated sequences acquired from C23 were
100% identical to Coxiella burnetii. The concatenated sequence generated in this project was 2-3
% distinct from endosymbitons of Ornithodoros spp. (full genetic distance matrix in Table A1.7).
34
Figure 3.7. Maximum likelihood (ML) analysis of the relationships between Coxiella using concatenated 16S and GroEL gene loci (1,694
bp). Percentage support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence generated in the present
study is in bold.
35
3.2. Molecular characterisation of Francisella
Conventional PCR assays were conducted on gDNA extracted from A. albolimbatum ticks (n=21) to
amplify Francisella (Table 3.3). The following Francisella genes were targeted: 16S rRNA;
chromosomal replication initiator protein alpha subunit (dnaA); phosphoglucomutase (pgm); peptide
chain release factor 2 beta subunit (prfB); DNA-directed RNA polymerase alpha subunit (rpoA); and
triose-phosphate isomerase alpha subunit (tpiA). No positive control was available for the following
assays. The no-template and extraction blank control of all Francisella PCR assays produced
appropriate results.
Table 3.3. Summary of PCR products tested positive results from Francisella assays.
sample Internal archive code
16S tpiA pgm prfB dnaA rpoA
F6 PI1744 +
F7 PI 767 +
F8 PI 1324 +
F9 PI 2743 +
R100 PI 1297 + +
R108 PI 1736 +
R20 PI 1872 + + + + +
R42 PI 982 + +
R53 PI 680 +
R55 PI 1295 + +
R64 PI 1298 + +
R67 PI 1320 + +
R68 PI 1299 + + +
R73 PI 1322 + +
Successful amplification of approximately 1,140 bp of the 16S rRNA gene was achieved in 14
samples (Figure 3.8) (Table 3.4). PCR products of expected size were obtained for rpoA (n=4), prfB
(n=6), dnaA (n=1) and tpiA (n=2) in following successful amplification and visualised under UV light
on a 1% (Table 3.4) (Figure 3.7 - 3.13 respectively). Amplification of a 1,613 bp region of pgm gene
was abandoned following an unsuccessful attempt at optimisation.
36
Figure 3.8. Agarose Gel Electrophoresis amplification of Francisella 16S rRNA. (A) 16S (1,140
bp). Lane 1-14: No sample; Lane 15: extraction blank control; Lane 16: No sample; Lane 17: no
template control; Lane 18: extra extraction blank; M: DNA ladder Axygen 100 bp. (B) 16S (1,140 bp)
M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: R42; Lane 4: No sample; Lane
5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane 9: R108; Lane 10: R53; Lane 11: R67; Lane
12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane 16: C23; Lane 17: F8 (1:10 dilution); 18: no
sample; M: DNA ladder Axygen 100 bp.
Figure 3.9. Agarose Gel Electrophoresis amplification of Francisella tpiA (674 bp) PCR
products dilutions. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: F1; Lane 4:
F2; Lane 5- 6: no sample; Lane 7: R20 (1:10 dilution); Lane 8: R68 (1:10 dilution); Lane 9: F1 (1:10
dilution); Lane 10: F2 (1:10 dilution); Lane 11: no sample; Lane 12: R20 (1:100 dilution); Lane 13;
R68 (1:100 dilution); Lane 14: F1 (1:100 dilution); Lane 15: F2 (1:100 dilution); Lane 16-18: no
sample; M: DNA ladder Axygen 100 bp.
37
Figure 3.10. Agarose Gel Electrophoresis amplification of Francisella rpoA (674 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R108; Lane 3: R42; Lane 4: R53;
Lane 5: R67; Lane 6: F6; Lane 7: F7; Lane 8: F8; Lane 9: F9; Lane 10: NTC; Lane 11: extraction
blank; Lane 12-13: no sample; M: DNA ladder Axygen 100 bp.
Figure 3.11. Agarose Gel Electrophoresis amplification of Francisella prfB (1,009 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: R42; Lane 4: no
sample; Lane 5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane 9: R108; Lane 10: R53; Lane
11: R67; Lane 12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane 16: C23; Lane 17: F8 (1:10
dilution); Lane 18: no sample; M: DNA ladder Axygen 100 bp.
Figure 3.12. Agarose Gel Electrophoresis amplification of Francisella dnaA (1,415 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R108; Lane 3: R42; Lane 4: R53:
Lane 5: R67; Lane 6: F6; Lane 7: F7; Lane 8: F8; Lane 9: F9; Lane 10: no template control; Lane
11: extraction blank control; M: DNA ladder Axygen 100 bp.
38
Figure 3.13. Agarose Gel Electrophoresis reamplification of Francisella dnaA PCR products.
M: DNA ladder Axygen 100 bp; Lane 1-2: No sample; Lane 3: R20: lane 4: R20; Lane 5: R20; Lane
6: extraction blank control; Lane 7: no sample; Lane 8: no template control; Lane 9-10; no sample;
M: DNA ladder Axygen 100 bp.
Sample R20 tested positive for all genes targeted (except pgm), producing clean chromatograms
used for phylogenetic analyses of individual nucleotide sequences, and a concatenated sequence.
The 16S sequences were compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity (Table 3.4). BLAST results of the 16S Francisella sequences display a 98.85%
nucldeotide similarity to Francisella endosymbionts previously observed in the following species of
tick: Amblyomma paulopunctatum (MN998649.1), Haemaphysalis asiatica (KC170752.1), and
Dermacentor auratus (KC170749.1).
The tpiA sequence was compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the tpiA nucleotide sequence identified in this tick sample
presented a 95.37% nucleotide identity to Francisella persica ATRR VR-331 (CP013022.1).
The prfB sequence was compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the prfB nucleotide sequence identified in this tick sample
presented a 96.28% nucleotide identity to Francisella persica ATRR VR-331 (CP013022.1).
39
The rpoA sequence was compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the rpoA nucleotide sequence identified in this tick sample
presented a 96.15% nucleotide identity to Francisella persica ATRR VR-331 (CP013022.1).
The dnaA sequence was compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the dnaA nucleotide sequence identified in this tick sample
presented a 98.06% nucleotide identity to Francisella persica ATRR VR-331 (CP013022.1).
Table 3.4. BLAST results for 16S, tpiA, dnaA prfB and rpoA Francisella sequences.
Phylogenetic analysis was performed on 954 bp of the 16S sequences obtained from tick samples
in this study, and reference sequences retrieved from GenBank (Figure 3.14). The topology of the
phylogenetic tree shows the 16S sequences from project formed a monophyletic clade, seperated
from the Francisella spp. of other clades, and was supported by 100% boostrap confidence. The
Sample Internal archive code
Gene Size (bp)
NCBI description Query cover
E-value
Percent identity
Accession
F8 PI1324 16S 1,027 F. endoymbiont of A. paulopunctatum
100% 0.0 98.64% CP018093.1
F9 PI 2743 16S 1,038 F. endoymbiont of A. paulopunctatum
100% 0.0 98.36% CP018093.1
R108 PI 1736 16S 1,057 F. endoymbiont of A. paulopunctatum
100% 0.0 98.67% CP018093.1
R20 PI 1872 16S 1,042 F. endoymbiont of A. paulopunctatum
100% 0.0 98.56% CP018093.1
R53 PI 680 16S 1,046 F. endoymbiont of A. paulopunctatum
100% 0.0 98.28% CP018093.1
R55 PI 1295 16S 1,044 F. endoymbiont of A. paulopunctatum
100% 0.0 98.47% CP018093.1
R64 PI 1298 16S 1,043 F. endoymbiont of A. paulopunctatum
100% 0.0 98.39 CP018093.1
R67 PI 1320 16S 1,049 F. endoymbiont of A. paulopunctatum
100% 0.0 98.48% CP018093.1
R68 PI 1299 16S 1,024 F. endoymbiont of A. paulopunctatum
100% 0.0 98.93% CP018093.1
R20 PI 1872 tpiA 626 F. persica ATCC VR-331 100% 0.0 95.37% CP013022.1
R20 PI 1872 dnaA 1,032 F. persica ATCC VR-331 100% 0.0 98.06% CP013022.1
R20 PI 1872 prfB 968 F. persica ATCC VR-331 100% 0.0 96.28% CP013022.1
R20 PI 1872 rpoA 884 F. persica ATCC VR-331 100% 0.0 96.15% CP013022.1
40
16S sequences acquired from A. albolimbatum ticks were 1-2% genetically distinct to known
Francisella endoysmbionts (full genetic distance matrix in Table A1.9).
Figure 3.14. Maximum likelihood (ML) analysis of the relationships between Francisella at the
16S rRNA gene locus (1,022 bp). Percentage support (>50%) from 1000 replicates is indicated
at the left of the supported node. The sequence generated in the present study is in bold.
41
Phylogenetic analysis was performed on 521 bp of the tpiA sequence obtained from sample R20,
and reference sequences retrieved from GenBank (Figure 3.15). The topology of the phylogenetic
tree shows the tpiA sequence from this project formed a monophyletic clade, seperated from the
Francisella spp. of other clades, and was supported by 100% boostrap confidence. The tpiA
sequence acquired from R20 was 5% distinct from Francisella persica ATRR VR-331 (CP013022.1);
11-12 % distinct from F. tularensis and F. hispaniensis; and 14-37% distinct from other Francisella
endosymbionts (full genetic distance matrix in Table A1.11).
Figure 3.15. Maximum likelihood (ML) analysis of the relationships between Francisella at the
tpiA gene locus (521bp). Percentage support (>50%) from 1000 replicates is indicated at the
left of the supported node. The sequence generated in the present study is highlighted in
bold.
42
Phylogenetic analysis was performed on 900 bp of the prfB sequence obtained from sample R20,
and reference sequences retrieved from GenBank (Figure 3.16). The topology of the phylogenetic
tree shows the prfB sequence from this project formed a monophyletic clade, seperated from the
Francisella spp. of other clades, and was supported by 98% boostrap confidence. The prfB sequence
acquired from R20 was 4% distinct from Francisella persica ATRR VR-331 (CP013022.1); 12-17 %
distinct from F. tularensis and F. hispaniensis; and 9-28% distinct from the F. philomiriga clade (full
genetic distance matrix in Table A1.13).
Figure 3.16. Maximum likelihood (ML) analysis of the relationships between Francisella at the
prfB gene locus (900 bp). Percentage support (>50%) from 1000 replicates is indicated at the
left of the supported node. The sequence generated in the present study is highlighted in
bold.
43
Phylogenetic analysis was performed on 723 bp of the rpoA sequence obtained from sample R20,
and reference sequences retrieved from GenBank (Figure 3.17). The topology of the phylogenetic
tree shows the rpoA sequence from this project formed a monophyletic clade, seperated from the
Francisella spp. of other clades, and was supported by 99% boostrap confidence. The rpoA
sequence acquired from R20 was 4% distinct from Francisella persica ATRR VR-331 (CP013022.1);
8-10 % distinct from F. tularensis and F. hispaniensis; and 8-24% distinct from the F. philomiriga
clade (full genetic distance matrix in Table A1.15).
Figure 3.17. Maximum likelihood (ML) analysis of the relationships between Francisella at the
rpoA gene locus (723 bp). Percentage support (>50%) from 1000 replicates is indicated at the
left of the supported node. The sequence generated in the present study is highlighted in
bold.
44
Phylogenetic analysis was performed on 954 bp of the dnaA sequence obtained from sample R20,
and reference sequences retrieved from GenBank (Figure 3.18). The topology of the phylogenetic
tree shows the dnaA sequence from this project formed a monophyletic clade, seperated from the
Francisella spp. of other clades, and was supported by 95% boostrap confidence. The dnA sequence
acquired from R20 was 2% distinct from Francisella persica ATRR VR-331 (CP013022.1); 7-9 %
distinct from F. tularensis and F. hispaniensis; and 13-14% distinct from the F. philomiriga clade (full
genetic distance matrix in Table A1.17).
Phylogenetic analysis was performed on a 4,230 bp concatenated sequence using 16S, tpiA, prfB,
rpoA, and dnaA sequences obtained from sample R20, including reference sequences from
GenBank via NCBI (Figure 3.19). The topology of the phylogenetic tree constructed shows the
concatenated sequence from this study forming a monophyletic clade, seperated from the
Francisella endosymbionts of other clades and supported by bootstrap value of 100%. The
concatenated sequence acquired from R20 was 3% distinct from Francisella persica ATRR VR-331
(CP013022.1) (full genetic distance matrix in Table A1.19).
45
Figure 3.18. Maximum likelihood (ML) analysis of the relationships between Francisella at the dnaA gene locus (954 bp). Percentage
support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence generated in the present study is
highlighted in bold.
46
Figure 3.19. Maximum likelihood (ML) analysis of the relationships between Francisella using concatenated 16S, tpiA, prfB, rpoA, and
dnaA gene loci (4,230 bp). Percentage support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence
generated in the present study is in bold.
47
3.3 Molecular characterisation of Rickettsia
Conventional and nested PCR assays were conducted on gDNA extracted from A. albolimbatum
ticks (n=26) to amplify Rickettsia. The following Rickettsia genes were targeted: citrate synthase
(gltA); outer membrane protein A (ompA); outer membrane protein B (ompB); 17-kDa protein (17
kDa); and surface cell antigen (sca4).
Table 3.5. Summary of samples producing positive results from Rickettsia assays.
Sample Internal archive code gltA ompA ompB 17 kDa sca4
SR12 PI 3329 + + + +
SR16 PI 2745 +
R106 PI 1460 + +
R52 PI 680 + +
R53 PI 680 + + + + +
R92 PI 3323 + + +
R94 PI 3329 +
R66 PI 1320 + +
R8 PI 1725 +
R12 PI 2742 +
SR15 PI 1723 +
SR14 PI 1316 +
Nested PCR on tick gDNA extractions (n=20) were unsuccessful in 19 samples. Successful
amplification of approximately of gltA gene (long 650 bp) using nested PCR assay was achieved in
one sample (SR12) (Table 3.5), and the Rickettsia-positive control. PCR product of expected size
was obtained following successful amplification and visualised under UV light on a 1% agarose gel
(Figure 3.20). Successful amplification using conventional PCR was achieved in gltA (short 380 bp),
ompA, ommB, 17 kDa, and sca4. PCR products of expected size were obtained for gltA (n=2), ompA
(n=2), ompB (n=7), 17 kDa (n=10) and sca4 (n=3) following successful amplification and visualised
under UV light on a 1% agarose gel displayed in Figure 3.21 - 3.26. The no-template and extraction
blank control of all nested and conventional Rickettsia PCR assays produced appropriate results.
48
Figure 3.20. Agarose Gel Electrophoresis amplification of Rickettsia gltA (long 650 bp) PCR
products. (A) M: no sample; Lane 1: SR11; Lane 2: SR12; Lane 3: SR13; Lane 4: SR14; Lane 5:
SR15; Lane 6: SR16; Lane 7: SR17; Lane 8: SR18; Lane 9: SR19; Lane 10: SR20; Lane 11: no
sample; Lane 12: no template control; Lane 13: no sample; Lane 14: extraction blank control; Lane
15: no sample; Lane 16: postive control; Lane 17-18: no sample; M: DNA ladder Axygen 100 bp. (B)
M: no sample; Lane 1: R69; Lane 2: R94; Lane 3: R22; Lane 4: R59; Lane 5: R10; Lane 6: R54;
Lane 7: R56; Lane 8: R82; Lane 9: R68; Lane 10: R88; Lane 11: no sample; Lane 12: DNA ladder
Axygen 100 bp; Lane 13-18: no sample; M: no sample.
Figure 3.21. Agarose Gel Electrophoresis amplification of Rickettsia gltA (short 381 bp) PCR
products. (A) M: DNA ladder Axygen 100 bp; Lane 1: no sample; Lane 2: R108; Lane 3: R42; Lane
4: R53; Lane 5: R67; Lane 6: F6; Lane 7-14 no sample; Lane 15: positive control; Lane 16-18: no
sample; M: DNA ladder Axygen 100 bp. (B) M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2:
R68; Lane 3: R42; Lane 4: no sample; Lane 5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane
9: R108; Lane 10: R53; Lane 11: R67; Lane 12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane
16: C23; Lane 17: F8 (1:10 dilution); Lane 18: no sample; M: DNA ladder Axygen 100 bp.
49
Figure 3.22. Agarose Gel Electrophoresis amplification of ompA (590 bp) and ompB (252 bp)
Rickettsia PCR products. M: DNA ladder Axygen 100 bp; Lane 1: no sample; Lane 2: SR12; Lane
3: no sample; Lane 4: SR12 (1:10 dilution); Lane 5: no template control; Lane 6-7: no sample; Lane
8: extraction blank control; Lane 9: no sample; Lane 10: positive control (1:1000 dilution); Lane 11:
no sample; Lane 12: SR12; Lane 13: R94; Lane 14: extraction blank control; Lane 15: no template
contorl ; Lane 16: SR12 (1:10 dilution); Lane 17: no sample; Lane 18: positive control (1:1000
dilution); M: DNA ladder Axygen 100 bp.
Figure 3.23. Agarose Gel Electrophoresis amplification of ompA (590 bp) Rickettsia PCR
products. Lane 1: R53; Lane 2: no sample; M: DNA ladder Axygen 100 bp; Lane 3: no sample;
Lane 4: R53 (1:10 dilution); Lane 5:; Lane 6: R73; Lane 7: R100; Lane 8-9: no sample; Lane 10:
positive control (1:1000 dilution); Lane 11-13: no sample M: DNA ladder Axygen 100 bp.
50
Figure 3.24. Agarose Gel Electrophoresis amplification of Rickettsia ompB (252 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: SR12; Lane 2: SR16; Lane 3: SR20; Lane 4:
R94; Lane 5: R106; Lane 6: R52; Lane 7: R110; Lane 8: R92; Lane 9: R53; Lane 10: extraction blank
control; Lane 11: no template control; Lane 12: no sample; Lane 13: SR12 (reamplification); Lane
14: no sample; Lane 15: R94 (reamplification): Lane 16: no sample; Lane 17: positive control (1:1000
dilution); Lane 18: no sample M: DNA ladder Axygen 100 bp.
Figure 3.25. Agarose Gel Electrophoresis amplification of Rickettsia 17 kDa (520 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: R52; Lane 2: R106; Lane 3: R110; Lane 4: R12;
Lane 5: R53; Lane 6: R60; Lane 7: R66 ; Lane 8: R8; Lane 9: R95; Lane 10: R92 ; Lane 11: SR12;
Lane 12: SR14; Lane 13: SR15; Lane 14: SR16; Lane 15: SR20; Lane 16: no template control; Lane
17: extraction blank control; Lane 18: positive control (1:1000 dilution); M: DNA ladder Axygen 100
bp.
Figure 3.26. Agarose Gel Electrophoresis amplification of Rickettsia sca4 (910 bp) PCR
products. M: DNA ladder Axygen 100 bp; Lane 1: R52; Lane 2: R106; Lane 3: R110; Lane 4: R12;
Lane 5: R53; Lane 6: R60; Lane 7: R66 ; Lane 8: R8; Lane 9: R95; Lane 10: R92 ; Lane 11: SR12;
Lane 12: SR14; Lane 13: SR15; Lane 14: SR16; Lane 15: SR20; Lane 16: no template control; Lane
17: extraction blank control; Lane 18: positive control (1:1000 dilution); M: DNA ladder Axygen 100
bp.
51
The short gltA sequences were compared against the GenBank nucleotide database using
nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to
determine most similar identity. BLAST results of the short gltA nucleotide sequences obtained from
R53 and SR12 presented a 98.92% nucleotide identity to Rickettsia bellii (MK697586.1). The long
gltA sequence was compared against the GenBank nucleotide database using nucleotide BLAST.
BLAST results of the long gltA nucleotide sequence obtained from SR12 presented a 99.35%
nucleotide identity to Rickettsia raoultii strain Khabarovsk (CP010969.1). The BLAST results of the
short and long gltA sequences showed a nucleotide identity of two different species of Rickettsia.
Closer inspection of the two shorter sequences showed the shorter sequences aligning closer to R.
bellii, and the longer sequence aligning closer with SFG Rickettsia. Additionally, concatenating the
short and long sequences from SR12 formed a chimera, indicating that sample SR12 had possible
mixed infection of at least 2 species of Rickettsia. Furthermore, while the short gltA sequences of
R53 and SR12 from this present study were 100% identical, closer inspection of the R53 forward
and reverse chromatograms showed signs of a mixed chromatogram at over 30 peaks throughout
the sequence suggesting dual infection; therefore, the R53 gltA sequence was left out of further
analyses.
The ompA sequences were compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the ompA nucleotide sequences obtained from R53 and SR12
presented a 97.56% and 97.96% nucleotide identity respectively to the following strains of Rickettsia
raoultii: R. raoultii strain Khabrovsk (CP010969.1); R. raoultii strain IM16 (CP019435.1); R. raoultii
isolate Tomsk (MK304548.1).
The ompB sequences were compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the ompB nucleotide sequences obtained from R52 and R106
presented a 99.20% nucleotide identity to R. monacensis strain IrR/Munich (LN794217.1), and R.
monacensis strain MT34 (JX625150.1); and ompB nucleotide sequences obtained from R53, R92,
and SR12 presented a 99.20% nucleotide identity to Rickettsia endosymbiont of Amblyomma
52
coelebs (MT009186.1), followed by 98.80% nucleotide identity to three R. heilongjiangensis Sendai-
58 strain MT34 (AP019865.1); R. montanensis strain OSU 85-930 (CP003340.1); R. massiliae strain
AZT80 (CP003319.1).
The 17 kDa sequences were compared against the GenBank nucleotide database using nucleotide
BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine
most similar identity. BLAST results of the identical 17 kDa nucleotide sequences obtained from R52
and R106 presented a 99% nucleotide identity to R. monacensis (LN794217), followed by 98.80%
nucleotide identity to R. tamurae (AB812550). BLAST results of the identical 17 kDa nucleotide
sequences obtained from R92 and R8 presented a 99% nucleotide identity R. raoultii strain IM16
(CP019435.1) and R. raoultii strain Khabarovsk (CP010969.1). BLAST results of the identical 17
kDa nucleotide sequences obtained from BLAST results of the R53, SR12, SR14, SR15, and SR16
presented a 99.18% nucleotide identity to Rickettsia endosymbiont of Ornithodoros (MH780988.1).
The identical sca4 sequences were compared against the GenBank nucleotide database using
nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to
determine most similar identity. BLAST results of the sca4 nucleotide sequence identified in this tick
sample presented a 99.75% nucleotide identity to Uncultured Rickettsia sp. clone ARRL2016-159
(MN431845.1) , followed by a 96.81% nucleotide identity to the closest confirmed Rickettsia species,
R. heilongjiangensis (AY331396.1).
53
Table 3.6. BLAST results for gltA, ompA, ompB, 17 kDa, and sca4 Rickettsia sequences.
Sample Internal archive code
Gene Size (bp)
NCBI description Query cover
E-value Percent identity
Accession
SR12 PI 3329 gltA(short) 342 Rickettsia bellii 100% 8e-136 98.92% MK697586.1
R53 PI 680 gltA(short) 342 Rickettsia bellii 100% 8e-136 98.92% MK697586.1
SR12 PI 3329 gltA(long) 612 Rickettsia raoultii strain Khabarovsk 100% 0.0 99.35% CP010969.1
R53 PI 680 ompA 578 Rickettsia raoultii strain Khabarovsk 99% 0.0 97.56% CP010969.1
SR12 PI 3329 ompA 587 Rickettsia raoultii strain Khabarovsk 100% 0.0 97.96% CP010969.1
R52 PI 680 ompB 251 Rickettsia monacensis strain IrR/Munich 100% 3.00e-123 99.20% LN794217.1
R53 PI 680 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1
R92 PI 3323 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1
R106 PI 1460 ompB 251 Rickettsia monacensis strain IrR/Munich 100% 3.00e-123 99.20% LN794217.1
SR12 PI 3329 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1
R8 PI 680 17 kDa 498 Rickettsia raoultii strain Khabarovsk 100% 0.0 99% CP010969.1
R52 PI 680 17 kDa 498 Rickettsia monacensis strain IrR/Munich 100% 0.0 99% LN794217.1
R53 PI 680 17 kDa 544 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1
R92 PI 3323 17 kDa 494 Rickettsia raoultii strain Khabarovsk 100% 0.0 99% CP010969.1
R106 PI 1460 17 kDa 498 Rickettsia monacensis strain IrR/Munich 100% 0.0 99% LN794217.1
SR12 PI 3329 17 kDa 500 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1
SR14 PI 1316 17 kDa 500 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1
SR15 PI 1723 17 kDa 500 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1
SR16 PI 3323 17 kDa 500 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1
R53 PI 680 sca4 889 Uncultured R. sp. ARRL2015-159 91% 0.0 99.75% MN431845.1
R66 PI 1320 sca4 857 Uncultured R. sp. ARRL2015-159 94% 0.0 99.75% MN431845.1
R92 PI 3323 sca4 876 Uncultured R. sp. ARRL2015-159 90% 0.0 99.50% MN431845.1
54
Phylogenetic analysis was peformed on 344 bp gltA sequence obtained from sample SR12, including
reference sequences from GenBank via NCBI (Figure 3.29). The topology of the phylogenetic tree
shows the gltA sequence from this project formed a monophyletic clade, seperated from the
Rickettsia species of other clades, and was supported by 99% boostrap confidence. The shorter gltA
sequence generated in this project was 5% distinct from R. bellii RML369-C (NC007940), followed
by SFG (Genetic distance = 13-17%), TG (Genetic distance = 16-17%). The gltA sequence acquired
from SR12 was 14-17% distinct to transitional group species of Rickettsia (full genetic distance matrix
in Table A1.21).
55
Figure 3.27. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the gltA gene locus (344 bp). Percentage support
(>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are highlighted
in bold.
56
Phylogenetic analysis was peformed on 634 bp gltA sequence obtained from sample SR12, including
reference sequences from GenBank via NCBI (Figure 3.28). The topology of the phylogenetic tree
shows the gltA nucleotide sequence from this project clustered with R. gravesii BWI-1 (DQ269435)
among the SFG, seperated from the Rickettsia species of other clades, however shows low
bootstrap confidence. The gltA sequence generated in this project was 2% distinct from R. gravesii
BWI-1 (DQ269435), followed by SFG (Genetic distance = 1-3%), TG (Genetic distance = 8-9%).
These sequences were 7-8% distinct to transitional group species of Rickettsia (full genetic distance
matrix in Table A1.23).
Figure 3.28. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the
gltA gene locus (long 634 bp). Percentage support (>50%) from 1000 replicates is indicated
at the left of the supported node. The sequences generated in the present study are
highlighted in bold.
57
Phylogenetic analysis was peformed on 517 bp ompA sequence obtained from sample SR12,
including reference sequences from GenBank via NCBI (Figure 3.29). The topology of the
phylogenetic tree shows the ompA nucleotide sequence from this project clustered with R. gravesii
BWI-1 (DQ269437) and Uncultured Rickettsia sp. clone ARRL2016-159 (MN431847), seperated
from the Rickettsia species of other clades, however shows low bootstrap confidence. The ompA
sequence generated in this project was 1-% distinct from R. gravesii BWI-1 (DQ269437) and
Uncultured Rickettsia sp. clone ARRL2016-159 (MN431847), followed by SFG (Genetic distance =
3-15%), TG (Genetic distance = 47-78%). These sequences were 5-10% distinct to transitional group
species of Rickettsia (full genetic distance matrix in Table A1.25).
58
Figure 3.29. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the
ompA gene locus (517 bp). Percentage support (>50%) from 1000 replicates is indicated at
the left of the supported node. The sequences generated in the present study are highlighted
in bold.
Phylogenetic analysis was peformed on 251 bp ompB sequences obtained from this study, including
reference sequences from GenBank via NCBI (Figure 3.30). The topology of the phylogenetic tree
shows the ompB nucleotide sequences from this project clustered in two individual clades, seperated
by a genetic distance of 3%. The R53, R92, and SR12 ompB sequences were identical to each other
and formed a monophyletic clade, seperated from the Rickettsia species of other clades, and was
supported by 86% bootstrap confidence. The ompB sequences acquired from R53, R92, and SR12
were closest to related to R. gravesii BWI-1 (DQ269438) and R. amblyommatis (CP003334) (Genetic
distance = 1-2%), followed by SFG (Genetic distance = 1-4%), and TG (Genetic distance = 13-15%).
59
The sequences were 4-10% distinct to transitional Group species of Rickettsia. The R52 and R106
ompB sequences were identical to each other and formed a monophyletic clade, supported by a
high boostrap value (91%), seperated from other species of Rickettsia. The sequences were
genetically distinct but closest related to R. monacensis (LN794217) and R. tamurae (DQ113911)
(Genetic distance = 1-2%), followed by SFG (Genetic distance = 1-2%), TG (Genetic distance = 12-
13%). These sequences were 5-10% distinct to Transitional Group species of Rickettsia (full genetic
distance matrix in Table A1.27).
Figure 3.30. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the
ompB gene locus (251 bp). Percentage support (>50%) from 1000 replicates is indicated at
the left of the supported node. The sequences generated in the present study are highlighted
in bold.
60
Phylogenetic analysis was peformed on 394 bp 17 kDa sequences obtained from this study,
including reference sequences from GenBank via NCBI (Figure 3.31). The topology of the
phylogenetic tree shows the 17 kDa nucleotide sequences from this project clustered in three
separated clades. The identical R52 and R106 sequences, and the identical R92 and R8 sequences
were 6% distinct from each other; and the R53, SR12, SR14, SR15, and SR16 sequences were 33%
and 36% distinct from the R92 and R8 sequences, and the R52 and R106 sequences respectively.
The R52 and R106 17 kDa sequences were identical and formed a monophyletic clade, supported
by a high boostrap value (96%), seperated from other species of Rickettsia. The sequences were
genetically distinct but closest related to R. monacensis (LN794217) and R. tamurae (AB812550)
(Genetic distance = 1%), followed by SFG (Genetic distance = 6-8%), TG (Genetic distance = 15-
37%). These sequences were 4-9% distinct to transitional Group species of Rickettsia. The R92
AND R8 17 kDa sequences clustered with Uncultured R.sp. clone ARRL2016-149 (MN431847);
Uncultured R.sp. clone ARRL2016-156 (MN431848); Uncultured R.sp. clone ARRL2016-159
(MN431849); and R. raoultii strain Khabrovsk (CP010969.1), supported by a high boostrap value
(94%), however the branches at this are short and phylogenetic resolution is weak. The sequences
were 100% nudelotide identity to Uncultured R.sp. clone ARRL2016-149 (MN431847); Uncultured
R.sp. clone ARRL2016-156 (MN431848); Uncultured R.sp. clone ARRL2016-159 (MN431849); and
R. raoultii strain Khabrovsk (CP010969.1). The R92 and R8 sequences were 1-5% distinct from
SFG, 16-33% distinct from TG, and 4-8% distinct from transitional group Rickettsia. The R53, SR12,
SR14, SR15, and SR16 17 kDa sequences were identical and formed a monophyletic clade,
supported by a high boostrap value (96%), seperated from other species of Rickettsia. The
sequences were 1% distinct from R. endosymbiont of O. rietorrecai (MH780988.1), followed by SFG
(genetic distance = 33-35%), TG (genetic distance = 40-43%). These sequences were 35-37%
distinct to Transitional Group species (full genetic distance matrix in Table A1.29).
61
Figure 3.31. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the 17 kDa gene locus (394bp). Percentage
support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are
highlighted in bold.
62
Phylogenetic analysis was performed on 775 bp sca4 sequence obtained from sample R53, R66,
and R92, including reference sequences from GenBank via NCBI (Figure 3.32). The topology of the
phylogenetic tree shows the sca4 nucleotide sequence from this project clustered with Uncultured
Rickettsia sp. clone ARRL2016-149 (MN431843); Uncultured Rickettsia sp. clone ARRL2016-156
(MN431844); Uncultured Rickettsia sp. clone ARRL2016-159 (MN431845), seperated from the
Rickettsia species of other clades, and was supported by 99% bootstrap confidence. The sca4
sequences generated in this project were 100% idenitcal to ARRL2016-149 (MN431843); Uncultured
Rickettsia sp. clone ARRL2016-156 (MN431844); Uncultured Rickettsia sp. clone ARRL2016-159
(MN431845), and 2% distinct from R. gravesii BWI-1 (DQ269437), followed by and 2-6% distinct
from SFG, 28% distinct from TG, and 11-15% distinct from transitional group Rickettsia (full genetic
distance matrix in Table A1.31).
63
Figure 3.32. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the sca4 gene locus (775 bp). Percentage support
(>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are highlighted
in bold.
64
Phylogenetic analysis was performed on a 644 bp concatenated sequence using 17 kDa and gltA
(short) sequences obtained from sample SR12, including reference sequences from GenBank via
NCBI (Figure 3.33). The topology of the phylogenetic tree constructed shows the concatenated
sequence from this study forming a monophyletic clade, seperated from the Rickettsia species of
other clades and supported by bootstrap value of 100%. The concatenated 17-kda and gltA
sequence acquired from SR12 was 6% distinct from R. bellii RML369-C (NC 007940), followed by
24-26% distinct from SFG, 27-28% distinct from TG, and 23-27% distinct from transitional group
Rickettsia (full genetic distance matrix in Table A1.33).
Figure 3.33. Maximum likelihood (ML) analysis of the relationships between Rickettsia using
concatenated 17 kDa and gltA gene loci (644 bp). Percentage support (>50%) from 1000
replicates is indicated at the left of the supported node. The sequence generated in the
present study is in bold.
65
Phylogenetic analysis was performed on a 1,435 bp concatenated sequence using ompA, ompB,
and gltA (long) sequences obtained from sample SR12, including reference sequences from
GenBank via NCBI (Figure 3.34). The topology of the phylogenetic tree constructed shows the
concatenated sequence from this study forming a monophyletic clade, seperated from the Rickettsia
species of other clades and supported by bootstrap value of 90%. The concatenated ompA, ompB,
and gltA sequence acquired from SR12 was 2-9% distinct from R. gravesii BWI-1 (DQ269438),
followed by 2% distinct from SFG, 18-21% distinct from TG, and 13-18% distinct from transitional
group Rickettsia (full genetic distance matrix in Table A1.35).
Figure 3.34. Maximum likelihood (ML) analysis of the relationships between Rickettsia using
concatenated ompA, ompB, and gltA gene loci (1,435 bp). Percentage support (>50%) from
1000 replicates is indicated at the left of the supported node. The sequence generated in the
present study is in bold.
66
Phylogenetic analysis was performed on a 1,649 bp concatenated sequence using ompA, ompB,
and sca4 sequences obtained from sample R53, including reference sequences from GenBank via
NCBI (Figure 3.35). The topology of the phylogenetic tree constructed shows the concatenated
sequence from this study forming a monophyletic clade, seperated from the Rickettsia species of
other clades and supported by bootstrap value of 99%. The concatenated ompA, ompB, and sca4
sequences acquired from R53 was 2% distinct from R. gravesii BWI-1 (DQ269438), followed by 2-
12% distinct from SFG, 29-33% distinct from TG, and 17-21% distinct from transitional group
Rickettsia (full genetic distance matrix in Table A1.37).
Figure 3.35. Maximum likelihood (ML) analysis of the relationships between Rickettsia using
concatenated ompA, ompB, and sca4 gene loci (1,649 bp). Percentage support (>50%) from
1000 replicates is indicated at the left of the supported node. The sequence generated in the
present study is in bold.
67
Phylogenetic analysis was performed on a 1,482 bp concatenated sequence using ompB, 17 kDa,
and sca4 sequences obtained from sample R92, including reference sequences from GenBank via
NCBI (Figure 3.36). The topology of the phylogenetic tree constructed shows the concatenated
sequence from this study forming a monophyletic clade, seperated from the Rickettsia species of
other clades and supported by bootstrap value of 99%. The concatenated ompB, 17 kDa, and sca4
sequences acquired from R92 was 1% distinct from R. gravesii BWI-1 (DQ269438), followed by 2-
6% distinct from SFG, 20-21% distinct from TG, and 8-9% distinct from transitional group Rickettsia
(full genetic distance matrix in Table A1.39).
Figure 3.36. Maximum likelihood (ML) analysis of the relationships between Rickettsia using
concatenated ompB, 17 kDa, and sca4 gene loci (1,482 bp). Percentage support (>50%) from
1000 replicates is indicated at the left of the supported node. The sequence generated in the
present study is in bold.
68
Phylogenetic analysis was performed on a 633 bp concatenated sequence using 17 kDa, and ompB
sequences obtained from samples R106 and R52, including reference sequences from GenBank
via NCBI (Figure 3.37). The topology of the phylogenetic tree constructed shows the concatenated
sequence from this study forming a monophyletic clade, seperated from the Rickettsia species of
other clades and supported by bootstrap value of 98%. The concatenated 17 kDa and ompB
sequences acquired from R106 and R52 were 1% distinct from R. monacensis (LN794217) and R.
tamurae (AF394896), followed by 4-6% distinct from SFG, 12-14% distinct from TG, and 5-7%
distinct from transitional group Rickettsia (full genetic distance matrix in Table A1.41).
Figure 3.37. Maximum likelihood (ML) analysis of the relationships between Rickettsia using
concatenated ompB and 17 kDa (582 bp). Percentage support (>50%) from 1000 replicates is
indicated at the left of the supported node. The sequence generated in the present study is
in bold.
69
3.4. Molecular characterisation of Hemaprotozoa
Nested PCR assays were conducted on gDNA extracted from A. albolimbatum ticks (n=45) targeting
the 18S rRNA of piroplasms. PCR products of expected size were obtained for 18S (n=13) following
successful amplification and visualised under UV light on a 1% agarose gel displayed in Figure 3.38
and Table 3.7. The no-template and extraction blank controls produced appropriate results.
Table 3.7. Summary of positive samples from haemoprotozoa assays.
The forward and reverse sequences obtained from the A. albolimbatum tick samples produced clean
chromatograms and were aligned in Geneious to produce a consensus sequence. The 18S
nucleotide sequences were compared against the GenBank nucleotide database using nucleotide
BLAST (BLASTN 2.11.0+ (Zhang et al. 2000, Morgulis et al. 2008)). An initial BLAST analysis against
NCBI nucleotide collection (nt) was used to determine most similar identity. BLAST results of the
18S nucleotide sequence identified in this tick sample presented a 100% nucleotide identity to
Hemolivia mariae isolate 4903 (KF992711.1), and Hemolivia mariae isolate 4955 (KF992712.1)
(Table 3.8).
Table 3.8. BLAST results 18S haemoprotozoa sequences.
sample Internal archive code 18S (850bp)
SR14 PI1316 +
R6 PI1735 +
Sample Internal archive code
Gene Size (bp)
NCBI description Query cover
E value
Percent identity
Accession
SR14 PI1316 18S 782 Hemolivia mariae isolate 4903
100% 0.0 100% KF992711.1
R6 PI1735 18S 782 Hemolivia mariae isolate 4955
100% 0.0 100% KF992712.1
70
Figure 3.38. Agarose Gel Electrophoresis amplification of haemoprotozoa 18S rRNA PCR
products. (A) M: DNA ladder Axygen 100 bp; Lane 1: R1; Lane 2: R2; Lane 3: R3; Lane 4: R4; Lane
5: R5; Lane 6: R6; Lane7: R7; Lane 8: R8; Lane 9: R9; Lane 10: R10; Lane 11: R11; Lane 12: R12;
Lane 13: R13; Lane 14: R14; Lane 15: R15; Lane 16: R16; Lane 17: R17; Lane 18: R18; M: DNA
ladder Axygen 100 bp. (B) M: DNA ladder Axygen 100 bp; Lane 1: R19; Lane 2: R20; Lane 3: R21;
Lane 4: R22; Lane 5: R23; Lane 6: R24; Lane7: R25; Lane 8: R26; Lane 9: R27; Lane 10: R28; Lane
11: R29; Lane 12: R30; Lane 13: R32; Lane 14: R33; Lane 15: R34 ; Lane 16: R35; Lane 17: R36;
Lane 18: R37; M: DNA ladder Axygen 100 bp. (C) M: DNA ladder Axygen 100 bp; Lane 1: R38; Lane
2: no sample; Lane 3: R39; Lane 4: R40; Lane 5: no sample; Lane 6: R41; Lane7: no sample; Lane
8: 42; Lane 9: 443; Lane 10: R44; Lane 11: no template control; Lane 12: no sample; Lane 13:
extraction blank control; Lane 14: no sample; Lane 15: positive control; Lane 16-18: no sample; M:
DNA ladder Axygen 100 bp.
71
Phylogenetic analysis was performed on 723 bp of the 18S nucleotide sequence obtained from
sample SR14, including reference sequences from GenBank via NCBI (Figure 3.29). The topology
of the phylogenetic tree constructed shows the 18S sequence from this study clustering with other
Hemolivia species, closest to H. mariae isolate 4903 (KF992711.1), and H. mariae isolate 4955
(KF992712.1), seperated from other clades, supported by 84% bootstrap confidence. The 18S
sequences acquired from SR14, and R6 were 100% identical to Hemolivia mariae (KF992711.1);
99.87% nucleotide to Hemolivia mariae (KF992712.1) and Hemolivia stellata (KP881349.1); and
98% similar to Hemolivia mauritanica isolate TR-8-08 (KF992698.1) (full genetic distance matrix in
Table A1.43).
Figure 3.39. Maximum likelihood (ML) analysis of the relationships between haemoprotozoa
at the 18S rRNA gene locus (723 bp). Percentage support (>50%) from 1000 replicates is
indicated at the left of the supported node. The sequence generated in the present study is
highlighted in bold
72
CHAPTER FOUR
DISCUSSION
4.1 Microbial Species of Interest
This study was prompted by a preliminary study into the microbiome of A. albolimbatum removed
from T. rugosa in 2019 (Mckenna, 2019). Previous results observed a diverse range of
microorganisms, including tick-associated genera Coxiella, Francisella and Rickettsia. However, one
limitation of microbiome analysis is the sequences generated rarely provide valuable taxonomic
resolution to a species level (Gofton et al. 2015). Here in the current study, the main aim was to
provide greater insight into the species of tick-associated microorganisms within A. albolimbatum by
generating more informative gene fragments to determine the genetic relatedness to known tick-
borne pathogens.
4.1.1 Coxiella burnetii confirmed within Amblyomma albolimbatum
Coxiella burnetii is the aetiological agent for the zoonotic disease Q fever, recognised as a global
health concern and a notifiable TBD in Australia (Australian Government Department of Health). Q
fever, or Coxiellosis in animals, is highly infectious to both humans and livestock (Maurin and Raoult,
1999). This study screened 142 A. albolimbatum ticks for the presence and molecular
characterisation of Coxiella at the 16S and GroEL gene loci. An initial 16S (short 524 bp) gene
sequence from Coxiella, genetically similar to C. burnetii, was successfully generated from one A.
albolimbatum tick gDNA sample, warranting further investigation; this fragment was longer than the
original NGS sequence generated by Mckenna (2019). Consistent with the shorter 16S, the longer
16S (long 1,391 bp) and GroEL (621 bp) gene sequences were produced from the tick sample
producing a 100% nucleotide identity of C. burnetii, confirming the presence of this pathogenic
bacterium within A. albolimbatum ticks. The 16S and GroEL sequences produced in this project were
consistent with the 16S (429 bp) sequence generated from NGS by Mckenna (2019). In addition,
further similarities exist between the results here and those reported by Mckenna; the A.
73
albolimbatum ticks gDNA was extracted from during both studies testing positive for C. burnetii,
supported by BLAST analysis, were acquired from the same T. rugosa host (ID 1715).
Phylogenetic analyses were performed on the 16S and GroEL sequences obtained from this study,
which presented 99-100% nucleotide similarity to several strains of C. burnetii. The high sequence
homology between the C. burnetii sequences from this study and available reference C. burnetii
sequences suggests a low genetic diversity of C. burnetii strains within the tick. This is contrasted
by higher genetic diversity among Coxiella endosymbionts of Ornithodoros, Argas, Rhipicephalus
and Dermacentor tick genera. The sequences from this study consistently clustered in a subclade
with previously sequenced C. burnetii strains (Figure 3.3 and 3.4), in a clade that showed the closest
relatives to be Coxiella endosymbionts of soft tick genera Ornithodoros and Argas. This topology is
congruous with earlier observations by Duron et al. (2015), suggesting that the common ancestor of
C. burnetii emanated from a Coxiella species within soft ticks. This is consistent with the evolutionary
history of Coxiella outlined by Duron et al. (2015), reporting a higher genetic diversity among Coxiella
strains of ticks and a lower genetic diversity of C. burnetii.
The bobtail host (ID 1715) ticks implicated to harbour C. burnetii was documented as being admitted
to the Kanyana Wildlife Centre due to an upper respiratory tract infection, infested with nine ticks
and was pregnant. Coxiellosis can lead to abortions, premature delivery and stillbirth in mammals
(Agerholm, 2013). Interestingly, C. burnetii transmission through shedding is at its highest during
birth and causing abortions in infected wildlife (Rousset et al., 2009). As the pregnancy resulted in a
healthy birth, the presence of C. burnetii positive ticks attached to the pregnant bobtail may not have
had any influence on this animal. However, this finding is intriguing. Furthermore, no further samples
associated with the bobtail host were available to determine if C. burnetii shedding occurred. The
potential shedding of C. burnetii in this animal is of a one health concern because other case studies
have shown veterinary staff have acquired Q fever following exposure to infected parturient material
(Kopecny et al. 2013).
74
Predominantly, Q fever is acquired through the inhalation of contaminated aerosols and/or contact
with tissue from infected domestic ruminants and domestic/companion animals (Mathews., et al
2020). While ticks are rarely implicated in the transmission of C. burnetii to humans, the presence of
C. burnetii within this bobtail tick is of great concern, because the first report of A. albolimbatum
parasitising a human was recently observed in WA (S. Egan, Pers. Comm.). Without any substantial
evidence that the bobtail was infected with C. burnetii, these findings alone cannot confirm the origin
of this bacterium within A. albolimbatum ticks, i.e., obtained from parasitising an infected bobtail.
Nevertheless, the presence of C. burnetii within A. albolimbatum ticks in Australia has potential
repercussions for the health of bobtails and their wildlife rehabilitators, necessitating further
investigation. Over the past decade, Q fever notifications without documented exposure sources
have increased. A recent study identified Australian wildlife rehabilitators were two times more likely
to acquiring Q fever than the general public (Mathews et al., 2020). The presence of C burnetii within
the bobtail tick highlights the bobtail as a potential exposure source for humans, placing wildlife
rehabilitators of these lizards at increased risk of exposure to C. burnetii and of Q fever.
While large mammals, mainly cows, were traditionally considered the vertebrate reservoir for C.
burnetii with ticks aiding in the sylvatic lifecycle, there is growing evidence for the role of companion
animals and rodents as smaller vertebrate maintenance hosts. Despite the expansion of research
into the ecology of C. burnetii, there has been a dearth of research into the presence of C. burnetii
within reptiles and their ticks. To date, C. burnetii has only been reported in turtles (Kachuga sp.);
tortoises (Gopherus flavomarginatus); snakes: rat snakes (Ptyas mucosus and Ptyas korros),
Indian/Southeast Asian rock python (Python molurus), water snakes (Natrix natrix); and skinks
(Mabuya carinata) (Stephan and Rao, 1979; Široký, 2010; Medoza-Roldan, 2021). While C. burnetii
has not been identified in Australian reptiles, the NGS study by Mckenna (2019) observed Coxiella
in 13.79% of ticks, while a separated NGS study into Bothriocroton undatum ticks from goannas,
observed C. burnetii in 1.2% of the total reads (Panetta et al. 2017). Importantly, both NGS studies
were unsuccessful in attempts to amplify C. burnetii.
75
4.1.2 Francisella sp. nov. identified within Amblyomma albolimbatum
Ticks are known to contain pathogenic and endosymbiont Francisella. Close genetic relatives of the
pathogenic species are Francisella endosymbionts, which are known to aid in blood meal digestion
(Narasimhan and Fikrig, 2015). Francisella tularensis ssp. tularensis, is the causative agent of
Tularaemia which is a highly infectious zoonosis affecting mammals, birds, and arthropods in the
Northern Hemisphere (Ellis et al., 2002; Sjödin et al., 2012). Tularaemia is a notifiable disease in
Australia1, and to date, no cases of the virulent F. tularensis ssp. tularensis or Tularaemia
transmission by ticks has been documented in Australia. In 2011, a pathogen closely resembling
the Northern hemisphere tick borne pathogen F. tularensis ssp. holartica, was reported in a woman
following a bite from the ringtail possum (Pseudocheirus peregrinus) in Tasmania, Australia (Jackson
et al., 2012). While the bacterium was not associated with a tick in the Tasmanian case, it does
highlight the importance of monitoring ticks and potential wildlife reservoirs, such as reptiles, for
potential human disease causing Francisella. A follow up study identify the presence of F. tularensis
ssp. holartica within ringtail possums (Eden et al. 2017).
Fourteen A. Albolimbatum ticks examined in this study successfully amplified a 1,120 bp region of
Francisella 16S in all samples (100%). The high sequence homology among 16S sequences from
this study (99.50%-100%), in addition to rpoA, tpiA, prfB and dnaA sequences, indicates a single
Francisella species was present among the tick samples. Consistent with Ramírez-Paredes et al.
(2017), the pgm gene failed to amplify despite attempts at optimisation. The 16S, rpoA, tpiA, prfB
and dnaA gene loci were successfully amplified arbitrarily before and after attempts to amplify pgm;
suggesting failure to amplify the gene is unlikely to be attributed to inadequate gene quantity in the
sample. Unsuccessful amplification of pgm assay was likely due to insufficient optimisation of the
1,613 bp pgm assay; analysis of the primers in situ revealed primers should bind to the pgm gene
(data not shown).
1 https://www.health.nsw.gov.au/Infectious/factsheets/Pages/tularaemia.aspx retrieved May 2021.
76
Phylogenetic analyses of the Francisella sequences generated in this study consistently exhibit a
monophyletic clade, supported by high bootstrap support. Furthermore, the sequences generated
through this study did not cluster with known pathogenic or endosymbiont Francisella species;
however they consistently show a close, but genetically distant relationship of 2-5% to F. persica
ATCC 331 (CP013022.1), an endosymbiont of Argus (Persicargas) arboreus, first isolated from the
malpighian soft tubules of the tick species removed from a buff-backed heron (Bubulcus ibis) in
Egypt (Larson et al., 2016). Phylogenetic analysis of the concatenated 16S, tpiA, prfB, rpoA, and
dnaA sequence, clustered in a monophyletic clade, supported by a high bootstrap consensus value
(100%). In addition, phylogenetic analysis of the 16S sequences obtained in this study are separated
from endosymbionts of Amblyomma, Ixodes, Dermacentor, Hyalomma, Ornithodoros,
Haemaphysalis, supported by high bootstrap confidence (100%). The topology among Francisella
trees is consistent, and supports that observed by Ramírez-Paredes et al. (2017) and Gerhart et al.
(2018).
The present study identified a single Francisella species within A. albolimbatum ticks which was
most closely related (3%) to F. persica ATCC 331 (CP013022.1). Francisella persica which is a tick
endosymbiont of Argus (Persicargas) arboreus was recently reclassified from the genus Wolbachia;
a group of bacteria commonly associated with being an insect endosymbiont. The present study was
based on the presence of a 16S Francisella sequence generated from NGS by Mckenna, which had
a genetic identity of 99% to F. hispaniensis (KT281843) (Mckenna, 2019). However, further
investigation of the longer 16S and the additonal housekeeping gene sequences generated in this
study, reveal the species is not related to F. hispaniensis. Instead, this is a novel species most
closely related to F. persica VR 331(CP013022).
The genetically distinct relationship presented among all sequences generated from this study
indicate the singular Francisella species present is a novel Francisella species; and importantly, not
the aforementioned pathogen strain, F. hispaniensis. In accordance with the current criteria for
77
classifying a new bacterial species: A threshold of 98.7% of 16S rRNA gene sequence similarity with
the phylogenetically closest species with standing in nomenclature, to classify a new bacterial
species (Lagier et al., 2018). The 16S sequence generated from this study from, A. albolimbatum, is
outside the threshold at 98.56% nucleotide identity, with 100% query coverage, to F. persica ATCC
331 (CP013022.1).
In Australia, a recent report of a Francisella related bacteraemia during 2015, which F. hispaniensis
was the causative agent (Aravena-Román et al., 2015). The bacterium was isolated from blood
culture from an immunocompromised human following hospital admission, becoming the first
instance of a Francisella-caused bacteraemia reported in Western Australia. hospitalised in Perth,
with the route of Francisella transmission relating to this case remaining unknown (Aravena-Román
et al., 2015). However, it was suggested infection occurred from a Francisella contaminated air
conditioning unit, and no presence of a tick was reported (Aravena-Román et al., 2015).
The results from this study were not the first to identify potential Francisella endosymbionts within
Australia. Francisella DNA has previously been identified within Amblyomma ticks. Previously,
Vilcins et al. (2009) identified Francisella DNA in A. fimbriatum (the tropical reptile tick) ticks removed
from reptiles in the Northern Territory of Australia, reporting the first detection of Francisella DNA
associated with a tick within Australia. Furthermore, a recent study by Egan et al. (2020), reported a
Francisella endosymbiont of high prevalence and abundance in A. triguttatum (the ornate kangaroo
tick) ticks found on red kangaroos from Western Australia. While this study is not the first to identify
potential Francisella endosymbionts within Australian ticks, it is the first to provide sufficient
molecular characterisation of Francisella to establish it is a novel species. Importantly, Francisella
related illness following a tick bite has not currently been documented in Australia. Furthermore, this
study provides additional molecular information towards the Francisella genus within Australian
Amblyomma ticks. Importantly, Francisella related illness following a tick bite has not currently been
documented in Australia.
78
4.1.3 Rickettsia spp. identified within Amblyomma albolimbatum
To provide greater understanding of the genetic composition of rickettsial sequences observed within
the previous NGS study, tick samples (n=26) were screened with five phylogenetically informative
gene assays. Ten (38.5%) samples successfully amplified and were identified as Rickettsia,
providing molecular data for regions of the gltA, ompA, ompB, 17 kDa and sca4 genes. While regions
of all genes were successfully amplified and sequenced, not all sequences were produced from any
single sample (Table 2.3). Rickettsia sequences in this present study indicate the presence of at
least 3 Rickettsia species among the samples producing amplicons. This is consistent with the
observation of 3 ZOTUs identified as Rickettsia spp. from sequences generated from NGS by
Mckenna (2019). In addition, due to expected co-infection of Rickettsia within some ticks, resulting
in either co-amplification with mixed chromatograms, and/or preferential and stochastic amplification
of one Rickettsia sp. over another, thus making interpretation of the results challenging. This was
observed with samples R53 and SR12, whereby for the longer gltA, ompB, sca4 and ompA genes
the generated sequences clustered more with Uncultured Rickettsia sp. ARRL2016, while for shorter
gltA and 17 kDa genes, the generated sequences clustered with Rickettsia endosymbiont of
Ornithodors rietcorreai.
Two separate but overlapping gltA sequences were obtained from the same tick sample (SR12).
Initially, the two sequences were thought to come from the same species. However, sequence
analysis showed amplification of two distinct Rickettsia species. One with genetic similarity close to
SFG R. raoultii observed in ompA; and another more basal species, more closely related to R. bellii
and Rickettsia endosymbiont of O. rietcorreai (Figure 3.27 and 3.28). This observation is similar to
that in 17 kDa, which shows five identical sequences clustering in a similar phylogenetic position to
the gltA sequences, clustering with Rickettsia endosymbiont of O. rietcorreai.
100% sequence homology was present between the shorter gltA sequences from samples SR12
and R53. However, further analysis of the R53 chromatogram revealed the presence of dual peaks
79
at over 30 positions throughout the sequence. The R53 gltA sequence with a mixed chromatogram
from this study, suggesting a co-infected tick, is supported by evidence of previous studies that
observed mixed Rickettsia spp. in ticks. Co-infection of R. bellii and R. rhipicephali was reported in
a D. variabilis tick removed from vegetation in Arkansas, USA (Wikswo et al., 2008); R. bellii, R.
montanensis, and R. rickettsii were found in a D. variabilis tick collected from nature in Ohio, USA
(Carmichael and Fuerst, 2010); and R. bellii and R. parkeri was observed in A. ovale removed from
dogs in Brazil (Szabó et al., 2013). All three cases intially identified co-infection by observing dual
peaks at multiple positions in sequencing electropherograms produced from PCR products of genus-
specific assays i.e. gltA. Furthermore, Abreu et al., (2019), reported two cases of co-infection in
Amblyomma ticks: R. bellii and R. amblyommatis in A. calcaratum ticks; and with R. bellii and a R.
parkeri-like bacterium wihin Amblyomma sp. haplotype Nazaré ticks.
Phylogenetic analyses of the Rickettsia sequences obtained in this study show 3 different groupings
of Rickettsia species. One group was clustered with Uncultured Rickettsia sp. ARRL2016
sequences, and are consistent with the recent findings reported by Tadepalli et al. (2021), however
potentially new strains. Importantly, the grouping identified in the present study is closely related,
however genetically distinct to R. gravesii in A. triguttatum. This was supported by the topology of
the phylogenetic trees built with concatenated sequences (Figure 3.34 - 3.36), which the ARRL
sequences were not included; based on different regions of the ompB gene sequenced.
Another group showed a topology and genetic distance between 1-2% close to SFG Rickettsia, R.
monacensis and R. tamurae. Rickettsia monacensis was first isolated from I. ricinus in Germany
(Simser et al., 2002), and subsequently in the same tick species in Hungary, Portugal, Slovakia,
Czech Republic, and Poland (Thu et al., 2019). Additionally, I, nipponensis and I senensis, are
considered the primary vector of R monacensis in China and Korea respectively; and I. nipponensis
has been documented to contain R. monacensis in Japan (Thu et al., 2019). This Rickettsia has
caused symptoms resembling those of Mediterranean spotted fever in in Italy, and Spain (Jado et
80
al., 2007); and documented in Ixodes genus ticks in a variety of European and Asian countries.
Interestingly, I. ricinus has been reported to parasitise several Lacertidae genera lizards
(Tomassone et al., 2017); however not present in Australia. Vectored by Amblyomma testudinarium,
SFG R. tamurae was identified to cause disease in humans in Japan in 1993 (Imaoka et al., 2011).
Recently in Australia, a Rickettsia sp. resembling R. tamurae , Rickettsia cf. tamurae, was identified
in the goanna tick, B. undatum from Sydney, Australia (Panetta et al., 2017). Intriguingly, 17 kDa
and ompB sequences from this present study were 99% and 98% genetically similar to R. tamurae
(AT-1), and demonstrate a topology similar to that presented by Panetta et al. (2017) at the gltA
gene. However, gltA sequences for the Rickettsia grouping close to R tamurae, and R. monacensis,
in this present study were not obtained. Lastly, another grouping was basal and presented a 1%
genetic distance to Rickettisa endosymbiont of O. rietocorreai, and 9% distinct from R. bellii.
Rickettsia bellii, the most basal diverging species of Rickettsia, is likely to be non-pathogenic to
humans.
In Australia, Rickettsia spp. are one of two recognised tick-borne pathogens, along with Coxiella,
currently acknowledged as causing disease in humans. There have been concerns surrounding
Rickettsia spp. in Australian ticks since Rickettsia honei, the SFG pathogen that causes Flinders
Island spotted fever (FISF), was identified in reptile tick B. hydrosauri, documented to parasitise
humans (Stenos et al., 2003). Furthermore, Rickettsia species were identified in B. hydrosauri ticks
parasitising T. rugosa from South Australia, however T. rugosa hosts tested negative to serological
testing for Rickettsia. Therefore, it cannot currently be determined whether T. rugosa is a reservoir,
though further research investigating presence of the bacteria in the tissues or organs (Whiley et al.,
2016). Close monitoring of these pathogens and hosts is paramount to assess the potential risks to
human and animal health (Tadepalli et al., 2021; Whiley et al., 2016).
In addition, Rickettsia australis causing Queensland tick typhus. More recently, an increase of
screening using molecular techniques has led to recent discovery of infectious bacteria. For
81
example: R. gravesii, removed from A. triguttatum (the ornate kangroo tick), on Barrow Island in
Western Australia; similarly, a closely related bacteria to R honei, R. honei subsp. marmionii causing
Australian spotted fever. Intriguing that Rickettsia spp. identified in this study are related to known
SFG Rickettsia spp., however the pathogenicity of those identified in this study are unknown.
4.1.4 Hemolivia mariae confirmed within Amblyomma albolimbatum
In 2014, oocysts proposed to be Hemolivia were identified for the first time from gut smears of A.
albolimbatum (Moller, 2014). The observation was made using light microscopy, based on a unique
morphological characteristic shared by H. mariae or H. stellata, and unlikley to be H. setllata based
on a difference in host specificity and differing geographical distribution. Moreover, no molecular
tests were available at the time for the identification of the hypothesised Hemolivia. Here in this
present study, we have molecularly confirmed the presence of Hemolivia mariae within two A.
albolimbatum ticks from separate bobtail hosts.
Forty-five tick samples collected previously from T. rugosa at Kanyana Wildlife Centre were screened
for the presence of piroplasm, targeting the 18S gene of these organisms. Amplification was
successful in 13 samples, of which two generated 18S sequences of Hemolivia mariae. Phylogenetic
analysis of the 18S sequences acquired from reptile ticks, revealed the 18S sequences clustered
with sequences associated with known Hemolivia spp. and were genetically identical to H. mariae
sequences (Genbank accession numbers KF992711.1, and KF992712.1). Importantly, the 18S
sequences obtained from this study were genetically identical to H. mariae, while showing a genetic
distance of 2.00% to H. mauritanica and H. stellata. These two species of Hemolivia are known to
infect tortoises and cane toads respectively.
While this is the first molecular identification of Hemolivia mariae within A. albolimbatum, these
haemogregarines have previously been associated with T. rugosa and other ticks that parasitise this
host, A. limbatum and B. hydrosauri (Smallridge and Paperna, 1997; Barker and Walker, 2014). In
82
1997, observations were made using electron microscopy and transmission electron microscopy of
smears from a tick gut of an engorged A. limbatum tick; the engorged tick was removed from T.
rugosa with haemogregarines present in the circulating erythrocytes. Lifecycle of the
haemogregarine oocysts and sporogonic changes resembling those H. stellata were reported. In
addition to the sporokinetes which spread into the gut cells before transforming to sporocysts, were
sporokinetes found spreading throughout the tick tissue and extra-intestinal locations (Smallridge
and Paperna, 1997).
Development of H. mariae occurs by sexual and asexual reproduction phases in the gut epithelium
and lumen of the tick host (Smallridge and Paperna, 1997). Primary sexual reproduction occurs
producing oocysts; progeny of the oocysts then undergo secondary asexual production, to produce
parasite stages contained in oval cysts (Smallridge and Paperna, 1997; Smallridge and Bull (2001).
Lizards are infected following ingestion of infected ticks or transmission by tick during a blood meal,
ticks are infected from parasitising on infectious lizards (Smallridge and Paperna, 1997). In addition,
both tick hosts are susceptible to H. mariae, however susceptibility to infection and burden once
infected, is greater in A. limbatum than B. hydrosauri (Smallridge and Bull, 2001).
Ticks are essential in maintaining the life cycle of H. mariae, where parasite development within the
tick is required before it can be transmitted to the vertebrate host; an indirect life cycle involving
definitive invertebrate hosts; either A. limbatum or B. hydrosauri; and an intermediate host, T. tugosa.
Regarding the geographical distributions of T. rugosa and those of A. albolimbatum, A limbatum,
and B. hydrosauri (Smyth, 1973), when considering the greater susceptibility in A. limbatum ticks,
there is likely a correlation to the distribution with H. mariae. Moreover, the susceptibility to infection
and any burden for A albolimbatum potentially affecting its contribution to H. mariae distribution
remains unexplored.
83
4.2 Limitations and future directions
The results from this study obtained molecular data for genes of Coxiella burnetii, a novel Francisella
sp., two SFG Rickettsia, a Rickettsia endosymbiont, and the blood parasite Hemolivia mariae. The
16S and GroEL gene sequences obtained which showed a 100% genetic match to Coxiella burnetii
warrants further investigation. Future research into the surveillance of C. burnetii within the stump
tailed lizard tick (T. rugosa) comparing blood and tissue to determine if the lizards are a potential
reservoir for the bacterium; moreover, the presence of C. burnetii in the stump-tailed lizard tick, A.
albolimbatum, provides information for surveillance of Q fever in Australia. Previous unsuccessful
amplification attempts may have been attributed to nucleated reptile blood contributing to inhibition
of the PCR assay (Panetta et al. 2017; Mckenna, 2019).
The presence of three Rickettsia was highlighted across five phylogenetically informative gene
regions. The mixed chromatogram of the gltA gene sequence suggesting two species of Rickettsia
in one sample highlights the sensitivity of PCR, while drawing attention to the limitation regarding
differentiation between a potential dual amplification. In addition, two species with genetic similarity
to spotted fever group Rickettsia were present, however the extent of pathogenicity, if any, cannot
be determined by the results of this study. Future research involving cloning strategies of Rickettsia
genes amplified from ticks would be beneficial to make more confident assumptions about Rickettsia
spp. present in A. Albolimbatum ticks (Ishikura et al., 2003). In addition, NGS investigation using the
Rickettsia genes targeted in this study, with emphasis on the gltA and ompA genes, to assess the
degree of similarity between the Rickettsia species within A. albolimbatum in this current study that
was genetically similiar to R. tamurae, and the R. cf. tamurae documented in the goanna tick B.
undatum (Panetta et al., 2017) and to differentiate between SFG and TG Rickettsia.
This study generated sufficient molecular data to establish a novel Francisella species. Further
investigation into Francisella sp. within Australian tick species such as A. triguttatum, and A.
fimbriatum targeting 16S and additional genes, such as those sequenced in this study should be
considered to more accurately identity the Francisella species within Australian ticks.
84
Lastly, future research is necessary to determine the presence of H. mariae within questing A.
albolimbatum ticks to understand the prevalence of this haemogregarine.
4.3 Conclusion
This study successfully achieved all aims. The primary aim of the current study was to generate
genetic information for Coxiella, Rickettsia, and Francisella taxa that were initially identified via NGS.
This study provided vital new sequences confirming the presence of Coxiella burnetii, a novel
Francisella sp. related but genetically distinct to a Francisella endosymbiont, and evidence for two
SFG Rickettsia spp. and a Rickettsia sp. endosymbiont. In addition, this study achieved its secondary
aim of investigating the presence of haemoprotozoan parasites in A. albolimbatum confirming, for
the first time, the presence of Hemolivia mariae within A. albolimbatum ticks in WA. Despite these
novel findings, further research is required to determine the medical and veterinary significance of
these microorganisms within A. albolimbatum.
85
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97
Appendix
Table A1.1. Nanodrop results of 26 samples and extraction blank.
Sample ID ng/uL A260 260/280 260/230 Constant 2ul 3ul 4ul
F1 2.12 0.042 1.11 0.41 50 4.24 6.36 8.48
F2 4.39 0.088 1.24 0.25 50 8.78 13.17 17.56
F3 29.92 0.598 1.52 1.14 50 59.84 89.76 119.68
F4 5.21 0.104 1.37 0.2 50 10.42 15.63 20.84
F5 8.43 0.169 1.32 0.86 50 16.86 25.29 33.72
F6 161.06 3.221 1.98 1.83 50 322.12 483.18 644.24
F7 236.9 4.738 2.13 1.96 50 473.8 710.7 947.6
F8 271.58 5.432 2.01 2.13 50 543.16 814.74 1086.32
F9 312.18 6.244 1.91 1.8 50 624.36 936.54 1248.72
F10 30.89 0.618 2.03 1.84 50 61.78 92.67 123.56
SR11 297.27 5.945 2.13 2.25 50 594.54 891.81 1189.08
SR12 122.74 2.455 2.04 1.92 50 245.48 368.22 490.96
SR13 344.54 6.891 2.04 2.03 50 689.08 1033.62 1378.16
SR14 111.18 2.224 1.99 1.57 50 222.36 333.54 444.72
SR15 110.16 2.203 2.05 1.95 50 220.32 330.48 440.64
SR16 115.4 2.308 2.06 1.87 50 230.8 346.2 461.6
SR17 240.05 4.801 1.97 2.1 50 480.1 720.15 960.2
SR18 76.69 1.534 1.77 0.78 50 153.38 230.07 306.76
SR19 281.18 5.624 2.09 2.27 50 562.36 843.54 1124.72
SR20 136.28 2.726 2 2.1 50 272.56 408.84 545.12
C21 108.83 2.177 2.03 1.81 50 217.66 326.49 435.32
C22 85.54 1.711 1.82 1.55 50 171.08 256.62 342.16
C23 131.61 2.632 2.05 2.06 50 263.22 394.83 526.44
C24 209.9 4.198 2.07 2.15 50 419.8 629.7 839.6
C25 127.13 2.543 2.09 2.16 50 254.26 381.39 508.52
C26 34.91 0.698 1.97 1.7 50 69.82 104.73 139.64
E 0.79 0.016 -26.55 0.1 50 1.58 2.37 3.16
98
Table A1.2. 1,140 bp Coxiella 16S Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
K2+G+I 94 10250 9416 -4614 0.478 0.408 4.537 0.25 0.25 0.25 0.25 0.02 0.02 0.2 0.02 0.2 0.02 0.02 0.2 0.02 0.2 0.02 0.02
HKY+G+I 97 10263 9402 -4604 0.486 0.407 4.825 0.26 0.212 0.22 0.308 0.02 0.02 0.25 0.02 0.18 0.03 0.02 0.17 0.03 0.21 0.02 0.02
T92+G+I 95 10268 9425 -4617 0.482 0.41 4.588 0.236 0.236 0.264 0.264 0.02 0.02 0.22 0.02 0.22 0.02 0.02 0.19 0.02 0.19 0.02 0.02
TN93+G+I 98 10270 9400 -4602 0.478 0.405 4.646 0.26 0.212 0.22 0.308 0.02 0.02 0.22 0.02 0.21 0.03 0.02 0.2 0.03 0.19 0.02 0.02
GTR+G+I 101 10275 9379 -4588 0.493 0.421 4.477 0.26 0.212 0.22 0.308 0.03 0.02 0.22 0.04 0.21 0.03 0.03 0.2 0 0.19 0.02 0
K2+G 93 10337 9511 -4662 n/a 0.374 3.634 0.25 0.25 0.25 0.25 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.2 0.03 0.2 0.03 0.03
HKY+G 96 10356 9504 -4656 n/a 0.37 3.776 0.26 0.212 0.22 0.308 0.02 0.02 0.24 0.03 0.17 0.03 0.03 0.17 0.03 0.2 0.02 0.02
T92+G 94 10357 9522 -4667 n/a 0.374 3.65 0.236 0.236 0.264 0.264 0.03 0.03 0.21 0.03 0.21 0.03 0.03 0.19 0.03 0.19 0.03 0.03
TN93+G 97 10358 9497 -4651 n/a 0.378 3.655 0.26 0.212 0.22 0.308 0.02 0.02 0.2 0.03 0.22 0.03 0.03 0.21 0.03 0.17 0.02 0.02
GTR+G 100 10367 9479 -4639 n/a 0.374 3.598 0.26 0.212 0.22 0.308 0.04 0.03 0.19 0.04 0.21 0.04 0.03 0.21 0.01 0.16 0.03 0.01
K2+I 93 10592 9766 -4790 0.373 n/a 3.298 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
TN93+I 97 10610 9749 -4777 0.373 n/a 3.312 0.26 0.212 0.22 0.308 0.02 0.03 0.19 0.03 0.22 0.04 0.03 0.21 0.04 0.16 0.02 0.03
T92+I 94 10613 9779 -4795 0.373 n/a 3.307 0.236 0.236 0.264 0.264 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.18 0.03 0.18 0.03 0.03
HKY+I 96 10616 9764 -4786 0.373 n/a 3.373 0.26 0.212 0.22 0.308 0.02 0.03 0.24 0.03 0.17 0.04 0.03 0.16 0.04 0.2 0.02 0.03
GTR+I 100 10638 9750 -4775 0.373 n/a 2.823 0.26 0.212 0.22 0.308 0.04 0.03 0.19 0.04 0.2 0.04 0.03 0.19 0.03 0.16 0.03 0.02
JC+G+I 93 10739 9914 -4864 0.462 0.513 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G 92 10801 9984 -4900 n/a 0.409 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
K2 92 10862 10045 -4930 n/a n/a 3.121 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
TN93 96 10879 10026 -4917 n/a n/a 3.122 0.26 0.212 0.22 0.308 0.03 0.03 0.18 0.03 0.21 0.04 0.03 0.21 0.04 0.16 0.03 0.03
T92 93 10884 10058 -4936 n/a n/a 3.122 0.236 0.236 0.264 0.264 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.18 0.03 0.18 0.03 0.03
HKY 95 10892 10049 -4929 n/a n/a 3.12 0.26 0.212 0.22 0.308 0.03 0.03 0.23 0.03 0.17 0.04 0.03 0.16 0.04 0.2 0.03 0.03
GTR 99 10907 10029 -4915 n/a n/a 2.686 0.26 0.212 0.22 0.308 0.03 0.03 0.18 0.04 0.2 0.04 0.03 0.19 0.04 0.16 0.03 0.03
JC+I 92 11023 10206 -5011 0.373 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 91 11280 10472 -5145 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
99
Table A2.3. 1,140 bp Coxiella 16S genetic distance matrix.
Sample C23
ex.
A. A
lbolim
batum
(PI 1
715)
C. b
urne
tii str. N
amibia gen
ome (C
P0075
55.1 )
C. b
urne
tii Dug
way
5J1
08-1
11 (C
P0007
33.1 )
C. b
urne
tii Cb1
75 G
uyan
a (H
G82
5990
.3)
C. b
urne
tii RSA 3
31 (C
P0008
90.1)
C. b
urne
tii Cbu
K Q15
4 (C
P0010
20.1 )
C. b
urne
tii RSA 4
93 (A
E0168
28.3)
C. b
urne
tii M
SU G
oat Q
177 (C
P0181
50.1 )
C. b
urne
tii str. nine
mile
pha
se II (C
P0351
12.1)
C. b
urne
tii str. Hen
zerlin
g (C
P0145
59.1)
C. b
urne
tii str. Sch
perling
(CP01
4563
)
Legion
ella pne
umop
hila
(EU22
1243
)
Can
dida
tus C. m
udro
wiae
(CP02
4961
.1)
C. e
ndos
ymbion
t ex.
R. tur
anicu
s (J
Q48
0822
.1)
C. e
ndos
ymbion
t ex.
R. s
angu
ineu
s (K
U89
2220
)
C. en
dosy
mbion
t ex.
R. g
eigy
i (KP99
4837
)
C. e
ndos
ymbion
t ex.
R. e
verts
i (KP99
4835
)
C. e
ndos
ymbion
t ex.
R. d
ecolor
atus
(KP99
4833
)
C. en
dosy
mbion
t ex. R. b
ursa
(KP99
4831
)
C. en
dosy
mbion
t ex. R. a
ustra
lis (K
P9948
29)
C. en
dosy
mbion
t ex. R. a
nnulatus
(KP99
4827
)
C. en
dosy
mbion
t ex. O. s
phen
iscus
(KP99
4799
)
C. en
dosy
mbion
t ex. O. s
onra
i (K
P9947
97)
C. en
dosy
mbion
t ex. O. ros
tratus
(KP99
4791
)
C. en
dosy
mbion
t ex. O. p
eruv
ianu
s (K
P9947
89)
C. e
ndos
ymbion
t ex.
O. o
cciden
talis
(KP99
4787
)
C. en
dosy
mbion
t ex. O. m
aritim
us (K
P9947
83)
C. e
ndos
ymbion
t ex.
O. k
airo
uane
nsis
(KP99
4779
)
C. e
ndos
ymbion
t ex.
O. e
rratic
us (K
P9947
77)
C. e
ndos
ymbion
t ex. O. d
enm
arki
(KP99
4776
)
C. en
dosy
mbion
t ex. O. c
apen
sis (K
P9947
74)
C. e
ndos
ymbion
t ex.
O. b
rasil
iens
is (K
P9947
72)
C. en
dosy
mbion
t ex. O. a
mblus
(KP99
4770
)
C. e
ndos
ymbion
t ex.
I. ricin
us (K
P9948
25)
C. en
dosy
mbion
t ex. H. s
him
oga
(KC17
0760
.1)
C. en
dosy
mbion
t ex. H. o
besa
(KC17
0759
.1)
C. e
ndos
ymbion
t ex.
H. la
gran
gei (K
C17
0756
.1)
C. e
ndos
ymbion
t ex. H. lo
ngico
rnis
(AY34
2035
.1)
C. e
ndos
ymbion
t e
x. A
rg. m
onac
hus
(KP98
5446
)
C. e
ndos
ymbion
t ex.
Am. tho
lloni
(MN99
5410
)
C. en
dosy
mbion
t ex. Am
. splen
didu
m (M
N99
5413
)
C. en
dosy
mbion
t ex. Am
. nap
onen
se (M
N99
5401
)
C. en
dosy
mbion
t ex. Am
. latep
unctatum
(MN99
5399
)
C. en
dosy
mbion
t ex. Am
. coe
lebs
(MN99
5392
)
C. en
dosy
mbion
t ex.
A.C
ajen
nens
e (K
P9854
82.1)
C. e
ndos
ymbion
t ex.
Am. a
mer
icanu
m (A
Y9398
24)
C. c
hera
xi (N
R 116
014)
Sample C23 ex. A. Albolimbatum (PI 1715)
C. burnetii str. Namibia genome (CP007555.1 ) 0.00
C. burnetii Dugway 5J108-111 (CP000733.1 ) 0.00 0.00
C. burnetii Cb175 Guyana (HG825990.3) 0.00 0.00 0.00
C. burnetii RSA 331 (CP000890.1) 0.00 0.00 0.00 0.00
C. burnetii CbuK Q154 (CP001020.1 ) 0.00 0.00 0.00 0.00 0.00
C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii MSU Goat Q177 (CP018150.1 ) 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii str. Schperling (CP014563) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Legionella pneumophila (EU221243) 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15
Candidatus C. mudrowiae (CP024961.1) 0.04 0.04 0.03 0.03 0.03 0.04 0.03 0.04 0.03 0.03 0.04 0.17
C. endosymbiont ex. R. turanicus (JQ480822.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.00
C. endosymbiont ex. R. sanguineus (KU892220) 0.04 0.04 0.04 0.04 0.03 0.04 0.04 0.04 0.04 0.03 0.04 0.17 0.00 0.00
C. endosymbiont ex. R. geigyi (KP994837) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03
C. endosymbiont ex. R. evertsi (KP994835) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.01
C. endosymbiont ex. R. decoloratus (KP994833) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.01 0.00
C. endosymbiont ex. R. bursa (KP994831) 0.05 0.05 0.04 0.05 0.04 0.05 0.04 0.05 0.04 0.04 0.05 0.17 0.04 0.04 0.04 0.02 0.02 0.02
C. endosymbiont ex. R. australis (KP994829) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 0.04 0.04 0.04 0.01 0.01 0.02 0.03
C. endosymbiont ex. R. annulatus (KP994827) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.00 0.01 0.01 0.02 0.01
C. endosymbiont ex. O. spheniscus (KP994799) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05
C. endosymbiont ex. O. sonrai (KP994797) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04
C. endosymbiont ex. O. rostratus (KP994791) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.05 0.04 0.02 0.03
C. endosymbiont ex. O. peruvianus (KP994789) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.05 0.04 0.04 0.04 0.05 0.05 0.04 0.00 0.03 0.02
C. endosymbiont ex. O. occidentalis (KP994787) 0.03 0.03 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.00 0.03 0.03
C. endosymbiont ex. O. maritimus (KP994783) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.01 0.04 0.02 0.01 0.04
C. endosymbiont ex. O. kairouanensis (KP994779) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.04 0.04 0.04 0.05 0.05 0.05 0.03 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.04
C. endosymbiont ex. O. erraticus (KP994777) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.00 0.03 0.03 0.00 0.03 0.04
C. endosymbiont ex. O. denmarki (KP994776) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.01 0.03 0.02 0.01 0.03 0.01 0.04 0.03
C. endosymbiont ex. O. capensis (KP994774) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.01 0.03 0.02 0.01 0.03 0.01 0.04 0.03 ?
C. endosymbiont ex. O. brasiliensis (KP994772) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.05 0.04 0.05 0.05 0.05 0.05 0.02 0.03 0.02 0.02 0.03 0.02 0.04 0.03 0.02 0.02
C. endosymbiont ex. O. amblus (KP994770) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.01 0.03 0.02 0.01 0.04 0.01 0.04 0.03 0.01 0.01 0.02
C. endosymbiont ex. I. ricinus (KP994825) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.16 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.03 0.03 0.03 0.03 0.04 0.02 0.03 0.03 0.03 0.03 0.03
C. endosymbiont ex. H. shimoga (KC170760.1) 0.05 0.05 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.03 0.05 0.05 0.05 0.05 0.05 0.03
C. endosymbiont ex. H. obesa (KC170759.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.04 0.04 0.04 0.02 0.02
C. endosymbiont ex. H. lagrangei (KC170756.1) 0.05 0.05 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.18 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.05 0.05 0.04 0.05 0.04 0.04 0.05 0.05 0.05 0.05 0.03 0.02 0.02
C. endosymbiont ex. H. longicornis (AY342035.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.04 0.05 0.05 0.02 0.02 0.01 0.00
C. endosymbiont ex. Arg. monachus (KP985446) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.02 0.03 0.02 0.01 0.03 0.02 0.04 0.03 0.02 0.02 0.02 0.02 0.03 0.04 0.04 0.05 0.04
C. endosymbiont ex. Am. tholloni (MN995410) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.03 0.04 0.04 0.03 0.04 0.03 0.03 0.04 0.04 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.04
C. endosymbiont ex. Am. splendidum (MN995413) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 0.06 0.06 0.06 0.07 0.06 0.07 0.06 0.07 0.07 0.06 0.05 0.06 0.06 0.05 0.06 0.04 0.05 0.06 0.06 0.06 0.06 0.04 0.05 0.05 0.05 0.05 0.05 0.04
C. endosymbiont ex. Am. naponense (MN995401) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.04 0.04 0.04 0.06
C. endosymbiont ex. Am. latepunctatum (MN995399) 0.04 0.04 0.03 0.03 0.03 0.04 0.03 0.04 0.03 0.03 0.04 0.16 0.03 0.03 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.05 0.02 0.04 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.05 0.04 0.04 0.06 0.03
C. endosymbiont ex. Am. coelebs (MN995392) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.15 0.04 0.04 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.03 0.05 0.04 0.03 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.03 0.03
C. endosymbiont ex. A.Cajennense (KP985482.1) 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.17 0.07 0.07 0.07 0.06 0.06 0.06 0.07 0.06 0.06 0.07 0.06 0.07 0.07 0.06 0.07 0.06 0.06 0.06 0.06 0.07 0.07 0.06 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.07 0.06 0.05
C. endosymbiont ex. Am. americanum (AY939824) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.18 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.08 0.06 0.07 0.07 0.06 0.08 0.07 0.06 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.06 0.06 0.03
C. cheraxi (NR 116014) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.14 0.05 0.05 0.05 0.06 0.06 0.06 0.07 0.06 0.06 0.05 0.04 0.05 0.05 0.04 0.05 0.06 0.04 0.04 0.04 0.05 0.05 0.05 0.06 0.05 0.06 0.05 0.04 0.05 0.07 0.05 0.05 0.05 0.08 0.08
100
Table A3.4. Coxiella GroEL Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 64 7785 7287 -3579 n/a 0.327 3.164 0.286 0.286 0.214 0.214 0.03 0.03 0.16 0.03 0.16 0.03 0.03 0.22 0.03 0.22 0.03 0.03
T92+G+I 65 7791 7285 -3577 0.248 0.573 3.142 0.286 0.286 0.214 0.214 0.03 0.03 0.16 0.03 0.16 0.03 0.03 0.22 0.03 0.22 0.03 0.03
HKY+G 66 7794 7280 -3574 n/a 0.327 3.238 0.299 0.273 0.171 0.258 0.03 0.02 0.2 0.04 0.13 0.03 0.04 0.21 0.03 0.23 0.03 0.02
HKY+G+I 67 7799 7278 -3572 0.262 0.598 3.223 0.299 0.273 0.171 0.258 0.03 0.02 0.2 0.04 0.13 0.03 0.04 0.21 0.03 0.23 0.03 0.02
TN93+G 67 7800 7279 -3572 n/a 0.328 3.184 0.299 0.273 0.171 0.258 0.03 0.02 0.17 0.04 0.15 0.03 0.04 0.24 0.03 0.2 0.03 0.02
TN93+G+I 68 7806 7277 -3570 0.243 0.566 3.168 0.299 0.273 0.171 0.258 0.03 0.02 0.17 0.04 0.15 0.03 0.04 0.24 0.03 0.2 0.03 0.02
K2+G 63 7814 7324 -3599 n/a 0.332 3.073 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
K2+G+I 64 7821 7323 -3597 0.239 0.563 3.053 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
GTR+G 70 7822 7278 -3569 n/a 0.327 3.197 0.299 0.273 0.171 0.258 0.02 0.03 0.17 0.02 0.15 0.03 0.05 0.24 0.03 0.2 0.04 0.02
GTR+G+I 71 7829 7276 -3567 0.242 0.565 3.178 0.299 0.273 0.171 0.258 0.02 0.03 0.17 0.02 0.15 0.03 0.05 0.24 0.03 0.2 0.04 0.02
T92+I 64 7857 7359 -3615 0.497 n/a 2.684 0.286 0.286 0.214 0.214 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.21 0.03 0.21 0.04 0.03
HKY+I 66 7865 7351 -3609 0.497 n/a 2.763 0.299 0.273 0.171 0.258 0.04 0.02 0.19 0.04 0.13 0.03 0.04 0.2 0.03 0.22 0.04 0.02
TN93+I 67 7873 7352 -3609 0.497 n/a 2.737 0.299 0.273 0.171 0.258 0.04 0.02 0.18 0.04 0.13 0.03 0.04 0.21 0.03 0.21 0.04 0.02
K2+I 63 7888 7398 -3636 0.497 n/a 2.598 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
GTR+I 70 7897 7352 -3606 0.496 n/a 2.734 0.299 0.273 0.171 0.258 0.02 0.03 0.18 0.03 0.13 0.04 0.05 0.21 0.03 0.21 0.05 0.02
JC+G 62 8167 7684 -3780 n/a 0.389 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 63 8172 7682 -3778 0.279 0.782 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 62 8219 7737 -3806 0.491 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
T92 63 8253 7763 -3818 n/a n/a 2.207 0.286 0.286 0.214 0.214 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.2 0.03 0.2 0.04 0.03
HKY 65 8265 7760 -3815 n/a n/a 2.216 0.299 0.273 0.171 0.258 0.04 0.03 0.18 0.05 0.12 0.04 0.05 0.19 0.04 0.21 0.04 0.03
TN93 66 8266 7752 -3810 n/a n/a 2.215 0.299 0.273 0.171 0.258 0.04 0.03 0.15 0.05 0.14 0.04 0.05 0.22 0.04 0.18 0.04 0.03
K2 62 8272 7789 -3833 n/a n/a 2.181 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
GTR 69 8292 7755 -3808 n/a n/a 2.216 0.299 0.273 0.171 0.258 0.04 0.03 0.15 0.04 0.14 0.05 0.06 0.22 0.03 0.18 0.05 0.02
JC 61 8573 8099 -3988 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
101
Table A4.5. Coxiella GroEL genetic distance matrix.
C. b
urne
tii Cbu
K Q15
4 (C
P0010
20.1 )
C. b
urne
tii str. N
amibia gen
ome (C
P0075
55.1 )
C. b
urne
tii M
SU G
oat Q
177 (C
P0181
50.1 )
C. b
urne
tii str. Sch
perling
(CP01
4563
)
C. b
urne
tii str. nine
mile
pha
se II (C
P0351
12.1)
C. b
urne
tii str. Hen
zerlin
g (C
P0145
59.1)
C. b
urne
tii RSA 3
31 (C
P0008
90.1)
C. b
urne
tii RSA 4
93 (A
E0168
28.3)
C. b
urne
tii Dug
way
5J1
08-1
11 (C
P0007
33.1 )
C. b
urne
tii Cb1
75 G
uyan
a (H
G82
5990
.3)
C. en
dosy
mbion
t ex.
A.C
ajen
nens
e (K
P9854
82.1)
C. e
ndos
ymbion
t e
x. A
rg. m
onac
hus
(KP98
5446
)
C. en
dosy
mbion
t ex.
D. m
argina
tus
(KP98
5488
.1)
C. en
dosy
mbion
t ex
. D. s
ilvar
um
(KP98
5490
)
C. en
dosy
mbion
t ex.
H. p
unctata
(KP98
5492
)
C. en
dosy
mbion
t ex.
O. a
mblus
(KP98
5447
)
C. en
dosy
mbion
t ex. O. b
rasil
iens
is (K
P9854
49)
C. en
dosy
mbion
t ex. O.C
apen
sis
(KP98
5451
)
C. en
dosy
mbion
t ex.
O. e
rratic
us
(KP98
5454
)
C. e
ndos
ymbion
t e
x. O
. kairo
uane
nsis
(KP98
5457
)
C. en
dosy
mbion
t ex.
O. m
aritim
us (K
P9854
59)
C. en
dosy
mbion
t ex.
O. m
aroc
anus
(KP98
5462
)
C. e
ndos
ymbion
t ex.
O. o
cciden
talis
(KP98
5464
)
C. en
dosy
mbion
t ex. O. p
eruv
ianu
s (K
P9854
66)
C. e
ndos
ymbion
t ex.
O. ros
tratus
(KP98
5468
)
C. e
ndos
ymbion
t ex. O. rup
estris
(KP98
5472
)
C. en
dosy
mbion
t ex. O. s
onra
i (K
P9854
74)
C. en
dosy
mbion
t ex.
R. p
usillu
s (K
P9855
18)
C. en
dosy
mbion
t ex.
R. s
angu
ineu
s (M
K1192
08)
C. en
dosy
mbion
t ex. R. tur
anicu
s (K
P9855
22)
Sample C23
ex.
A. A
lbolim
batum
(PI 1
715)
Legion
ella pne
umop
hila
(EU22
1243
)
C. burnetii CbuK Q154 (CP001020.1 )
C. burnetii str. Namibia genome (CP007555.1 ) 0.00
C. burnetii MSU Goat Q177 (CP018150.1 ) 0.00 0.00
C. burnetii str. Schperling (CP014563) 0.00 0.00 0.00
C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00
C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00
C. burnetii RSA 331 (CP000890.1) 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii Dugway 5J108-111 (CP000733.1 ) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii Cb175 Guyana (HG825990.3) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. endosymbiont ex. A.Cajennense (KP985482.1)0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39
C. endosymbiont ex. Arg. monachus (KP985446) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.39
C. endosymbiont ex. D. marginatus (KP985488.1)0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.24 0.32
C. endosymbiont ex. D. silvarum (KP985490) 0.29 0.29 0.29 0.29 0.30 0.29 0.29 0.30 0.30 0.30 0.27 0.31 0.04
C. endosymbiont ex. H. punctata (KP985492) 0.32 0.32 0.32 0.32 0.33 0.33 0.33 0.33 0.33 0.33 0.28 0.34 0.20 0.22
C. endosymbiont ex. O. amblus (KP985447) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.38 0.03 0.32 0.32 0.33
C. endosymbiont ex. O. brasiliensis (KP985449)0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.42 0.08 0.32 0.32 0.35 0.07
C. endosymbiont ex. O.Capensis (KP985451) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.39 0.04 0.33 0.33 0.34 0.02 0.07
C. endosymbiont ex. O. erraticus (KP985454) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.29 0.17 0.18 0.18 0.28 0.30 0.29
C. endosymbiont ex. O. kairouanensis (KP985457)0.29 0.29 0.29 0.29 0.28 0.29 0.29 0.28 0.28 0.28 0.38 0.30 0.32 0.35 0.31 0.30 0.31 0.31 0.28
C. endosymbiont ex. O. maritimus (KP985459) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.37 0.03 0.32 0.32 0.31 0.03 0.07 0.02 0.28 0.30
C. endosymbiont ex. O. marocanus (KP985462) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.30 0.17 0.18 0.18 0.28 0.30 0.29 0.00 0.27 0.28
C. endosymbiont ex. O. occidentalis (KP985464)0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.37 0.29 0.21 0.22 0.23 0.29 0.30 0.29 0.08 0.28 0.28 0.09
C. endosymbiont ex. O. peruvianus (KP985466) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.38 0.04 0.31 0.32 0.32 0.03 0.07 0.03 0.28 0.29 0.02 0.28 0.28
C. endosymbiont ex. O. rostratus (KP985468) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.38 0.04 0.33 0.31 0.31 0.04 0.07 0.03 0.28 0.30 0.02 0.28 0.28 0.03
C. endosymbiont ex. O. rupestris (KP985472) 0.26 0.26 0.26 0.26 0.25 0.26 0.26 0.25 0.25 0.25 0.40 0.27 0.33 0.36 0.30 0.25 0.28 0.26 0.28 0.06 0.25 0.28 0.29 0.26 0.26
C. endosymbiont ex. O. sonrai (KP985474) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.30 0.18 0.18 0.18 0.28 0.30 0.29 0.01 0.27 0.28 0.00 0.09 0.28 0.28 0.29
C. endosymbiont ex. R. pusillus (KP985518) 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.29 0.32 0.17 0.19 0.17 0.30 0.32 0.32 0.09 0.29 0.30 0.09 0.15 0.31 0.32 0.30 0.08
C. endosymbiont ex. R. sanguineus (MK119208) 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.29 0.31 0.18 0.19 0.17 0.28 0.31 0.30 0.07 0.30 0.28 0.07 0.14 0.30 0.30 0.30 0.07 0.02
C. endosymbiont ex. R. turanicus (KP985522) 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.29 0.31 0.17 0.18 0.18 0.29 0.32 0.31 0.07 0.30 0.29 0.07 0.14 0.30 0.31 0.29 0.07 0.02 0.01
Sample C23 ex. A. Albolimbatum (PI 1715) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.04 0.30 0.29 0.32 0.04 0.07 0.03 0.27 0.29 0.03 0.27 0.28 0.03 0.03 0.26 0.27 0.30 0.28 0.28
Legionella pneumophila (EU221243) 0.39 0.39 0.39 0.39 0.38 0.39 0.39 0.38 0.38 0.38 0.46 0.40 0.38 0.41 0.43 0.38 0.40 0.39 0.37 0.36 0.38 0.37 0.37 0.39 0.39 0.37 0.37 0.35 0.36 0.36 0.39
102
Table A1.6. Coxiella concatenated 16S and GroEL Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
K2+G 49 13197 12773 -6337 n/a 0.203 2.603 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
K2+G+I 50 13197 12765 -6332 0.384 0.446 2.651 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
HKY+G 52 13201 12752 -6324 n/a 0.201 2.708 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.15 0.04 0.04 0.17 0.04 0.2 0.03 0.03
HKY+G+I 53 13202 12743 -6319 0.39 0.449 2.753 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.15 0.04 0.04 0.17 0.04 0.2 0.03 0.03
TN93+G+I 54 13202 12735 -6314 0.381 0.444 2.67 0.27 0.231 0.205 0.294 0.03 0.03 0.17 0.04 0.18 0.04 0.04 0.21 0.04 0.16 0.03 0.03
TN93+G 53 13203 12745 -6319 n/a 0.206 2.62 0.27 0.231 0.205 0.294 0.03 0.03 0.18 0.04 0.18 0.04 0.04 0.2 0.04 0.16 0.03 0.03
T92+G 50 13207 12774 -6337 n/a 0.203 2.604 0.251 0.251 0.249 0.249 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
T92+G+I 51 13207 12766 -6332 0.384 0.446 2.651 0.251 0.251 0.249 0.249 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
GTR+G+I 57 13216 12723 -6304 0.379 0.446 2.64 0.27 0.231 0.205 0.294 0.03 0.04 0.18 0.04 0.18 0.05 0.05 0.21 0.02 0.16 0.04 0.01
GTR+G 56 13237 12752 -6320 n/a 0.213 2.22 0.27 0.231 0.205 0.294 0.04 0.04 0.18 0.04 0.16 0.05 0.05 0.18 0.04 0.17 0.04 0.03
K2+I 49 13250 12827 -6364 0.661 n/a 2.398 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
HKY+I 52 13254 12804 -6350 0.663 n/a 2.511 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.14 0.04 0.04 0.16 0.04 0.19 0.03 0.03
TN93+I 53 13260 12802 -6348 0.66 n/a 2.437 0.27 0.231 0.205 0.294 0.03 0.03 0.19 0.04 0.16 0.04 0.04 0.19 0.04 0.17 0.03 0.03
T92+I 50 13260 12828 -6364 0.661 n/a 2.399 0.251 0.251 0.249 0.249 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
GTR+I 56 13298 12814 -6351 0.658 n/a 2.048 0.27 0.231 0.205 0.294 0.04 0.04 0.19 0.04 0.14 0.05 0.05 0.16 0.05 0.17 0.04 0.03
JC+G 48 13570 13155 -6529 n/a 0.242 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 49 13574 13150 -6526 0.351 0.505 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 48 13613 13197 -6551 0.648 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
TN93 52 13777 13327 -6612 n/a n/a 1.805 0.27 0.231 0.205 0.294 0.04 0.04 0.15 0.05 0.17 0.05 0.05 0.19 0.05 0.14 0.04 0.04
K2 48 13779 13364 -6634 n/a n/a 1.802 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
T92 49 13789 13366 -6634 n/a n/a 1.802 0.251 0.251 0.249 0.249 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
HKY 51 13791 13350 -6624 n/a n/a 1.803 0.27 0.231 0.205 0.294 0.04 0.04 0.19 0.05 0.13 0.05 0.05 0.15 0.05 0.17 0.04 0.04
GTR 55 13809 13334 -6612 n/a n/a 1.555 0.27 0.231 0.205 0.294 0.05 0.04 0.15 0.06 0.15 0.06 0.05 0.17 0.05 0.14 0.05 0.04
JC 47 14078 13672 -6789 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
103
Table A1.7. Coxiella concatenated 16S and GroEL genetic distance matrix.
C. b
urne
tii M
SU G
oat Q
177 (C
P0181
50.1)
C. b
urne
tii C
b175
Guy
ana (H
G82
5990
.3)
C. b
urne
tii Cbu
K Q15
4 (C
P0010
20.1)
C. b
urne
tii D
ugway
5J1
08-1
11 (C
P0007
33.1)
C. b
urne
tii RSA 4
93 (A
E0168
28.3)
C. b
urne
tii str. N
amibia gen
ome (C
P0075
55.1)
C. b
urne
tii str. H
eizb
erg (C
P0145
61.1)
C. b
urne
tii str. H
enze
rling
(CP01
4559
.1)
C. b
urne
tii str. nine
mile
pha
se II (C
P0351
12.1)
C. en
dosy
mbion
t ex.
Am. c
ajen
nens
e(K
P9854
82.1)
C. e
ndos
ymbion
t ex.
Argas
mon
achu
s
(KP98
5446
)
C. en
dosy
mbion
t ex.
O. a
mblus
(KP99
4770
)
C. en
dosy
mbion
t ex.
O. b
rasil
iens
is (K
P9947
72)
C. en
dosy
mbion
t ex.
O. c
apen
sis (K
P9947
74)
C. e
ndos
ymbion
t ex.
O. e
rratic
us (K
P9947
77)
C. en
dosy
mbion
t ex.
O. k
airo
uane
nsis
(KP99
4779
)
C. e
ndos
ymbion
t ex.
O. m
aritim
us (K
P9854
59)
C. e
ndos
ymbion
t ex.
O. o
cciden
talis
(KP98
5464
)
C. en
dosy
mbion
t ex.
O. p
eruv
ianu
s (K
P9854
66)
C. e
ndos
ymbion
t ex.
O. ros
tratus
(KP99
4791
)
C. en
dosy
mbion
t ex.
O. s
onra
i (KP99
4797
)
C. e
ndos
ymbion
t ex.
R. s
angu
ineu
s (K
U89
2220
)
C. e
ndos
ymbion
t ex.
R. tur
anicus
(JQ48
0822
.1)
Legion
ella pne
umop
hila
230
0/99
(EU22
1243
)
Sample C23
ex.
Am
. albolim
batum
(PI 1
715)
C. burnetii MSU Goat Q177 (CP018150.1)
C. burnetii Cb175 Guyana (HG825990.3) 0.00
C. burnetii CbuK Q154 (CP001020.1) 0.00 0.00
C. burnetii Dugway 5J108-111 (CP000733.1) 0.00 0.00 0.00
C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00
C. burnetii str. Namibia genome (CP007555.1) 0.00 0.00 0.00 0.00 0.00
C. burnetii str. Heizberg (CP014561.1) 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C. endosymbiont ex. Am. cajennense (KP985482.1) 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15
C. endosymbiont ex. Argas monachus (KP985446) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.16
C. endosymbiont ex. O. amblus (KP994770) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02
C. endosymbiont ex. O. brasiliensis (KP994772) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.17 0.04 0.04
C. endosymbiont ex. O. capensis (KP994774) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.01 0.04
C. endosymbiont ex. O. erraticus (KP994777) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.13 0.10 0.10 0.10 0.10
C. endosymbiont ex. O. kairouanensis (KP994779) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.15 0.11 0.11 0.11 0.11 0.10
C. endosymbiont ex. O. maritimus (KP985459) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.04 0.02 0.10 0.11
C. endosymbiont ex. O. occidentalis (KP985464) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.15 0.10 0.10 0.10 0.10 0.03 0.10 0.10
C. endosymbiont ex. O. peruvianus (KP985466) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.01 0.04 0.01 0.10 0.11 0.01 0.10
C. endosymbiont ex. O. rostratus (KP994791) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.03 0.04 0.03 0.10 0.11 0.02 0.10 0.02
C. endosymbiont ex. O. sonrai (KP994797) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.13 0.10 0.10 0.10 0.10 0.00 0.10 0.10 0.03 0.10 0.10
C. endosymbiont ex. R. sanguineus (KU892220) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.13 0.11 0.11 0.11 0.11 0.04 0.11 0.11 0.05 0.11 0.11 0.03
C. endosymbiont ex. R. turanicus (JQ480822.1) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.13 0.11 0.11 0.11 0.12 0.04 0.11 0.11 0.06 0.12 0.11 0.04 0.00
Legionella pneumophila 2300/99 (EU221243) 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.25 0.23 0.22 0.23 0.22 0.21 0.22 0.22 0.21 0.22 0.22 0.21 0.22 0.22
Sample C23 ex. Am. albolimbatum (PI 1715) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.15 0.02 0.02 0.03 0.02 0.09 0.10 0.02 0.09 0.02 0.02 0.09 0.10 0.10 0.22
104
Table A1.8. Francisella 16S Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
K2+G 103 6791 5877 -2835 n/a 0.35 2.41 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
K2+G+I 104 6801 5879 -2835 0.28 0.64 2.45 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
HKY+G 106 6802 5862 -2825 n/a 0.34 2.47 0.268 0.217 0.207 0.309 0.03 0.03 0.22 0.04 0.15 0.05 0.04 0.15 0.05 0.19 0.03 0.03
T92+G 104 6803 5880 -2836 n/a 0.35 2.42 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04
TN93+G 107 6803 5855 -2820 n/a 0.35 2.44 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.2 0.04 0.04 0.21 0.04 0.14 0.03 0.03
K2+I 103 6806 5893 -2843 0.4 n/a 2.22 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
HKY+G+I 107 6813 5864 -2825 0.29 0.63 2.51 0.268 0.217 0.207 0.309 0.03 0.03 0.22 0.04 0.15 0.05 0.04 0.15 0.05 0.19 0.03 0.03
T92+G+I 105 6813 5882 -2836 0.28 0.64 2.45 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04
TN93+G+I 108 6814 5856 -2820 0.27 0.64 2.44 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.2 0.04 0.04 0.21 0.04 0.14 0.03 0.03
T92+I 104 6818 5896 -2844 0.4 n/a 2.22 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04
HKY+I 106 6819 5879 -2833 0.4 n/a 2.23 0.268 0.217 0.207 0.309 0.03 0.03 0.21 0.04 0.14 0.05 0.04 0.15 0.05 0.18 0.03 0.03
TN93+I 107 6819 5870 -2828 0.4 n/a 2.22 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.19 0.05 0.04 0.2 0.05 0.14 0.03 0.03
K2 102 6826 5922 -2859 n/a n/a 2.1 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
GTR+G 110 6832 5856 -2818 n/a 0.36 2.37 0.268 0.217 0.207 0.309 0.05 0.02 0.16 0.06 0.2 0.05 0.02 0.21 0.04 0.14 0.04 0.03
T92 103 6838 5925 -2859 n/a n/a 2.11 0.242 0.242 0.258 0.258 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.16 0.04 0.16 0.04 0.04
TN93 106 6839 5899 -2843 n/a n/a 2.1 0.268 0.217 0.207 0.309 0.04 0.03 0.16 0.04 0.18 0.05 0.04 0.19 0.05 0.14 0.04 0.03
HKY 105 6839 5908 -2849 n/a n/a 2.1 0.268 0.217 0.207 0.309 0.04 0.03 0.21 0.04 0.14 0.05 0.04 0.14 0.05 0.18 0.04 0.03
GTR+G+I 111 6841 5856 -2817 0.27 0.65 2.38 0.268 0.217 0.207 0.309 0.05 0.02 0.16 0.06 0.2 0.05 0.02 0.21 0.04 0.14 0.04 0.03
GTR+I 110 6848 5873 -2826 0.4 n/a 1.98 0.268 0.217 0.207 0.309 0.05 0.03 0.16 0.06 0.17 0.06 0.04 0.18 0.04 0.14 0.04 0.03
GTR 109 6868 5902 -2842 n/a n/a 1.87 0.268 0.217 0.207 0.309 0.05 0.03 0.16 0.06 0.17 0.06 0.04 0.18 0.05 0.14 0.04 0.03
JC+G 102 6902 5997 -2896 n/a 0.42 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 103 6912 5999 -2896 0.23 0.71 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 102 6914 6010 -2903 0.4 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 101 6931 6036 -2917 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
105
Table A1.9. Francisella 16S genetic distance matrix.
Sample R68 ex.
A albolimbatum
(PI 1
299)
Sample R55 ex.
A albolimbatum
(PI 1
295)
Sample R64 ex.
A albolimbatum
(PI 1
298)
Sample R108 ex.
A albolimbatum
(PI 1
736)
Sample R53 ex.
A albolimbatum
(PI 6
80)
Sample R67 ex.
A albolimbatum
(PI 1
320)
Sample F8 ex. A albolim
batum (P
I 1324)
Sample F9 ex. A albolim
batum (P
I 2743)
Sample R20 ex.
A. albolim
batum (PI 1
872)
F. cf. n
ovicida
3523 (CP002558)
F. cf. n
ovicida
Fx1 (C
P002557)
F. endosymbiont ex.
Am. geoemyd
ae (K
T382871.1)
F. endosy
mbiont ex.
Am. goeldii (
MW287918.1)
F. endosy
mbiont ex.
Am. humerale (M
W287921.1)
F. endosy
mbiont ex.
Am. latum
(MW287937.1)
F. endosy
mbiont ex.
Am. oblongogutta
tum (M
W287912.1)
F. endosymbiont e
x. Am. p
acae (M
W287930.1)
F. endosy
mbiont ex.
Am. longiro
stre
(MW287926.1)
F. endosy
mbiont ex.
Am. paulopuncta
tum (M
W287939.1)
F. endosy
mbiont ex.
Am. scu
lptum (M
W287940.1)
F. endosy
mbiont ex.
D. atro
signatus
(KC170748.1)
F. endosy
mbiont ex.
Am. variu
m (M
W287934.1)
F. endosy
mbiont ex. D. o
ccidentalis
(MW287945.1)
F. endosy
mbiont ex.
D. varia
bilis st
r. MV (A
Y805307.1)
F. endosy
mbiont ex.
D. anderso
ni (MT712191.1)
F. endosy
mbiont ex.
D. a
uratus (KC170749.1)
F. endosy
mbiont ex.
D. c
ompactus
(KC170750.1)
F. endosy
mbiont ex.
D. retic
ulatus (M
G859281.1)
F. endosy
mbiont ex.
Hyl. Aegyp
tium
(MH645203.1)
F. endosy
mbiont ex.
Hyl.
Asiatic
um (K
X852464.1)
F. endosymbiont e
x. Hyl.
trunca
tum (J
F290387.1)
F. endosy
mbiont ex.
Hyl.
excava
tum (M
W287952.1)
F. endosy
mbiont ex.
Hyl. Lusit
anicum
(MW287950.1)
F. endosy
mbiont ex.
Hae. fl
ava (K
Y797658)
F. endosy
mbiont ex.
Hae. hys
tricis
(KC170753.1)
F. endosy
mbiont ex.
Hae. shim
oga (K
C170755.1)
F. endosy
mbiont ex.
Hae. papuana (K
C170754.1)
F. endosy
mbiont ex.
R. h
aemaphysaloides
(KC170751.1)
F. endosy
mbiont ex.
I. ric
inus (MW287984.1)
F. endosy
mbiont ex.
I. sca
pularis (M
W287955.1)
F. endosy
mbiont ex.
O. p
orcinus
(MW287943.1)
F. endosy
mbiont ex.
R. sanguineus
(MH645201.1)
F. hisp
aniensis FSC454 (C
P018093)
F. hisp
aniensis st
r. 2008651S (K
T281843)
F. noatunensis
subsp
. noatunensis
FSC774 (NZ C
P053850)
F. noatunensis
subsp
. orie
ntalis st
r. Toba 04 (C
P003402)
F. orie
ntalis FNO12 (C
P011921)
F. persi
ca ATCC VR-331 (C
P012505)
F. philo
miragia str
. 1951 (A
Y496933)
F. philo
miragia
str. G
A01-2794 (CP009440)
F. philo
miragia
subsp
. noatunensis
str. 2
006/09/130 (EF490217)
F. philo
miragia
subsp
. philo
miragia ATCC 25015 (C
P010019)
F. opportu
nistica
str. 1
4-2155 (CP022375)
F. sp. M
A067296 (CP016930)
F. philo
miragia
subsp
. philo
miragia
ATCC 25017 (CP000937.1)
F. salin
a (F7308 R0007 - 1
0861471)
F. tularensis
str. 3
523 (AY243028.1)
F. tularensis
str. S
chu4 (C
P013853)
F. tularensis
subsp
. holarct
ica FSC200 (F
TS 1142 - 14009524)
F. halio
ticida
str. S
himane-1 (J
F290376)
F. tularensis
subsp
. holarct
ica LVS (A
M233362.1)
F. tularensis
subsp
. mediasia
tica
FSC147 (CP000915)
F. tularensis
subsp
. novic
ida F6168 (CP000915)
F. tularensis
subsp
. novic
ida U112 (FTN 0472 - 4
548231)
F. tularensis
subsp
. tularensis
(NE061598)
F. tularensis
subsp
. tularensis
str. W
Y96 (CP012037)
P. salm
onis str
. LF-89 (C
P011849)
Sample R68 ex. A albolimbatum (PI 1299)
Sample R55 ex. A albolimbatum (PI 1295) 0.00
Sample R64 ex. A albolimbatum (PI 1298) 0.00 0.00
Sample R108 ex. A albolimbatum (PI 1736) 0.00 0.00 0.00
Sample R53 ex. A albolimbatum (PI 680) 0.00 0.00 0.00 0.00
Sample R67 ex. A albolimbatum (PI 1320) 0.00 0.00 0.00 0.00 0.00
Sample F8 ex. A albolimbatum (PI 1324) 0.00 0.00 0.00 0.00 0.00 0.00
Sample F9 ex. A albolimbatum (PI 2743) 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Sample R20 ex. A. albolimbatum (PI 1872) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
F. cf. novicida 3523 (CP002558) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02
F. cf. novicida Fx1 (CP002557) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00
F. endosymbiont ex. Am. geoemydae (KT382871.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01
F. endosymbiont ex. Am. goeldii (MW287918.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00
F. endosymbiont ex. Am. humerale (MW287921.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00
F. endosymbiont ex. Am. latum (MW287937.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
F. endosymbiont ex. Am. oblongoguttatum (MW287912.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00
F. endosymbiont ex. Am. pacae (MW287930.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00
F. endosymbiont ex. Am. longirostre (MW287926.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.00
F. endosymbiont ex. Am. paulopunctatum (MW287939.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00
F. endosymbiont ex. Am. sculptum (MW287940.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.00
F. endosymbiont ex. D. atrosignatus (KC170748.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01
F. endosymbiont ex. Am. varium (MW287934.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
F. endosymbiont ex. D. occidentalis (MW287945.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01
F. endosymbiont ex. D. variabilis str. MV (AY805307.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.02 0.01 0.00
F. endosymbiont ex. D. andersoni (MT712191.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.01 0.00 0.00
F. endosymbiont ex. D. auratus (KC170749.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.00 0.01 0.02 0.02 0.02
F. endosymbiont ex. D. compactus (KC170750.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00
F. endosymbiont ex. D. reticulatus (MG859281.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02
F. endosymbiont ex. Hyl. Aegyptium (MH645203.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01
F. endosymbiont ex. Hyl. Asiaticum (KX852464.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.02 0.01 0.00
F. endosymbiont ex. Hyl. truncatum (JF290387.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.00
F. endosymbiont ex. Hyl. excavatum (MW287952.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.00 0.00
F. endosymbiont ex. Hyl. Lusitanicum (MW287950.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.00 0.00 0.00 0.00
F. endosymbiont ex. Hae. flava (KY797658) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.00 0.01 0.01 0.01
F. endosymbiont ex. Hae. hystricis (KC170753.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02
F. endosymbiont ex. Hae. shimoga (KC170755.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00
F. endosymbiont ex. Hae. papuana (KC170754.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01
F. endosymbiont ex. R. haemaphysaloides (KC170751.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01
F. endosymbiont ex. I. ricinus (MW287984.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02
F. endosymbiont ex. I. scapularis (MW287955.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.02 0.02 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.02 0.02 0.02 0.02 0.01
F. endosymbiont ex. O. porcinus (MW287943.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.02 0.02 0.02 0.01 0.00
F. endosymbiont ex. R. sanguineus (MH645201.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0 0.00 0.00 0.00 0.00 0.01 0.02 0.02 0.02 0.02 0.01 0.00 0.00
F. hispaniensis FSC454 (CP018093) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01
F. hispaniensis str. 2008651S (KT281843) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00
F. noatunensis subsp. noatunensis FSC774 (NZ CP053850) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02
F. noatunensis subsp. orientalis str. Toba 04 (CP003402) 0.03 0.04 0.04 0.03 0.04 0.04 0.03 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.02 0.03 0.01
F. orientalis FNO12 (CP011921) 0.03 0.04 0.04 0.03 0.04 0.04 0.03 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.02 0.03 0.01 0.00
F. persica ATCC VR-331 (CP012505) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02
F. philomiragia str. 1951 (AY496933) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02
F. philomiragia str. GA01-2794 (CP009440) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00
F. philomiragia subsp. noatunensis str. 2006/09/130 (EF490217)0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00
F. philomiragia subsp. philomiragia ATCC 25015 (CP010019) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00 0.00
F. opportunistica str. 14-2155 (CP022375) 0.03 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03
F. sp. MA067296 (CP016930) 0.03 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.00
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1)0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00 0.00 0.00 0.02 0.02
F. salina (F7308 R0007 - 10861471) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.03 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.03 0.03 0.01
F. tularensis str. 3523 (AY243028.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.00 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
F. tularensis str. Schu4 (CP013853) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00
F. tularensis subsp. holarctica FSC200 (FTS 1142 - 14009524)0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.03 0.03 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00
F. halioticida str. Shimane-1 (JF290376) 0.03 0.04 0.03 0.03 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.01 0.02 0.02 0.03 0.01 0.01 0.01 0.01 0.04 0.04 0.01 0.01 0.03 0.03 0.03
F. tularensis subsp. holarctica LVS (AM233362.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.03 0.03 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00 0.00 0.03
F. tularensis subsp. mediasiatica FSC147 (CP000915) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00
F. tularensis subsp. novicida F6168 (CP000915) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0.00
F. tularensis subsp. novicida U112 (FTN 0472 - 4548231) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0.00 0.00
F. tularensis subsp. tularensis (NE061598) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0 0 0
F. tularensis subsp. tularensis str. WY96 (CP012037) 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00 0.00 0.03 0.00 0.00 0.00 0.00 0.00
P. salmonis str. LF-89 (CP011849) 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.17 0.17 0.16 0.15 0.16 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.16 0.16 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.16
106
Table A1.10. Francisella tpiA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
HKY+G 46 6093 5757 -2832 n/a 0.49 2.2 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.23 0.03 0.23 0.05 0.02
HKY+G+I 47 6100 5756 -2831 0.24 1.08 2.31 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.1 0.03 0.05 0.23 0.03 0.23 0.05 0.02
TN93+G 47 6102 5758 -2832 n/a 0.49 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.17 0.05 0.09 0.03 0.05 0.21 0.03 0.25 0.05 0.02
T92+G 44 6106 5784 -2848 n/a 0.5 2.19 0.323 0.323 0.177 0.177 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
TN93+G+I 48 6108 5757 -2830 0.24 1.05 2.33 0.325 0.321 0.134 0.22 0.05 0.02 0.17 0.05 0.09 0.03 0.05 0.21 0.03 0.25 0.05 0.02
HKY+I 46 6111 5774 -2841 0.35 n/a 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.23 0.03 0.23 0.05 0.02
T92+G+I 45 6112 5783 -2846 0.24 1.1 2.3 0.323 0.323 0.177 0.177 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.23 0.03 0.23 0.05 0.03
TN93+I 47 6119 5776 -2841 0.35 n/a 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.21 0.03 0.24 0.05 0.02
T92+I 44 6122 5800 -2856 0.35 n/a 2.24 0.323 0.323 0.177 0.177 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.23 0.03 0.23 0.05 0.03
GTR+G 50 6125 5759 -2829 n/a 0.51 2.17 0.325 0.321 0.134 0.22 0.06 0.02 0.16 0.06 0.09 0.02 0.04 0.21 0.05 0.24 0.03 0.03
GTR+G+I 51 6132 5759 -2828 0.23 1.07 2.27 0.325 0.321 0.134 0.22 0.06 0.02 0.17 0.06 0.09 0.02 0.04 0.21 0.04 0.25 0.03 0.03
GTR+I 50 6142 5776 -2838 0.35 n/a 2.23 0.325 0.321 0.134 0.22 0.06 0.01 0.16 0.06 0.09 0.02 0.04 0.22 0.04 0.24 0.03 0.02
K2+G 43 6161 5846 -2880 n/a 0.56 1.94 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+G+I 44 6168 5846 -2879 0.25 1.31 2.04 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
K2+I 43 6173 5858 -2886 0.35 n/a 2.07 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
HKY 45 6235 5906 -2908 n/a n/a 1.45 0.325 0.321 0.134 0.22 0.06 0.03 0.13 0.06 0.08 0.04 0.06 0.2 0.04 0.2 0.06 0.03
TN93 46 6243 5907 -2907 n/a n/a 1.46 0.325 0.321 0.134 0.22 0.06 0.03 0.14 0.06 0.07 0.04 0.06 0.18 0.04 0.21 0.06 0.03
T92 43 6248 5933 -2924 n/a n/a 1.45 0.323 0.323 0.177 0.177 0.06 0.03 0.11 0.06 0.11 0.03 0.06 0.2 0.03 0.2 0.06 0.03
GTR 49 6264 5906 -2904 n/a n/a 1.46 0.325 0.321 0.134 0.22 0.08 0.02 0.14 0.08 0.07 0.03 0.06 0.18 0.05 0.21 0.04 0.03
K2 42 6287 5979 -2948 n/a n/a 1.43 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC+G 42 6291 5984 -2950 n/a 0.69 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 43 6299 5985 -2949 0.02 0.73 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 42 6307 5999 -2958 0.33 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 41 6389 6090 -3004 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
107
Table A1.11. Francisella tpiA genetic distance matrix.
F. philo
miragia subsp. p
hilomira
gia str.
1951 (EU683025)
F. philo
miragia
subsp. noatunensis str.
DSM 18777 (EU683027)
Sample R20 ex.
A. albolim
batum (P
I 1872)
F. halio
ticida str.
Shimane-1 (J
F290385)
F. philo
miragia subsp. p
hilomira
gia ATCC 25015 (C
P010019.1)
F. philo
miragia
str. GA01-2794 (C
P009440.1)
P. salm
onis (CP011849)
F. tularensis
subsp. mediasiatic
a FSC147 (CP000915.1)
F. noatunensis
subsp. orie
ntalis str.
Toba 04 (CP003402.1)
F. orie
ntalis FNO12 (C
P011921.2)
F. tularensis
subsp. tularensis W
Y96-3418 (CP000608.1)
F. opportu
nistica str.
14-2155 (CP022375.1)
F. noatunensis
subsp. noatunensis FSC774 (C
P053850.1)
F. cf. n
ovicida Fx1 (CP002557.1)
F. tularensis
str. Schu4 (C
P013853.1)
F. cf. n
ovicida 3523 (CP002558.1)
F. philo
miragia
subsp. philo
miragia
ATCC 25017 (CP000937.1)
F. persica ATCC VR-331 (C
P012505.1)
F. hispaniensis FSC454 (C
P018093.1)
F. sp. MA067296 (C
P016930.1)
F. tularensis
subsp. holarctic
a LVS (AM233362.1 )
F. tularensis
subsp. novicida F6168 (C
P009353.1)
F. philomiragia subsp. philomiragia str. 1951 (EU683025)
F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.06
Sample R20 ex. A. albolimbatum (PI 1872) 0.33 0.34
F. halioticida str. Shimane-1 (JF290385) 0.33 0.35 0.36
F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.05 0.04 0.35 0.34
F. philomiragia str. GA01-2794 (CP009440.1) 0.06 0.02 0.37 0.35 0.06
P. salmonis (CP011849) 1.09 1.08 1.04 1.07 1.13 1.07
F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.31 0.31 0.11 0.35 0.33 0.34 1.06
F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.07 0.02 0.36 0.35 0.05 0.03 1.09 0.34
F. orientalis FNO12 (CP011921.2) 0.07 0.02 0.36 0.35 0.05 0.03 1.09 0.34 0.00
F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.32 0.31 0.11 0.36 0.33 0.35 1.06 0.00 0.34 0.34
F. opportunistica str. 14-2155 (CP022375.1) 0.30 0.34 0.14 0.37 0.37 0.35 1.02 0.16 0.38 0.38 0.16
F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.06 0.00 0.34 0.35 0.04 0.02 1.08 0.31 0.02 0.02 0.31 0.34
F. cf. novicida Fx1 (CP002557.1) 0.32 0.32 0.11 0.36 0.34 0.35 1.07 0.01 0.35 0.35 0.01 0.16 0.32
F. tularensis str. Schu4 (CP013853.1) 0.32 0.31 0.11 0.36 0.33 0.35 1.06 0.00 0.34 0.34 0.00 0.16 0.31 0.01
F. cf. novicida 3523 (CP002558.1) 0.31 0.34 0.11 0.35 0.37 0.37 1.02 0.05 0.38 0.38 0.05 0.16 0.34 0.05 0.05
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.01 0.05 0.34 0.34 0.05 0.06 1.10 0.32 0.07 0.07 0.33 0.32 0.05 0.33 0.33 0.33
F. persica ATCC VR-331 (CP012505.1) 0.32 0.31 0.05 0.35 0.33 0.34 1.06 0.11 0.34 0.34 0.11 0.16 0.31 0.11 0.11 0.13 0.33
F. hispaniensis FSC454 (CP018093.1) 0.32 0.34 0.12 0.36 0.37 0.37 1.03 0.05 0.38 0.38 0.05 0.16 0.34 0.05 0.05 0.00 0.34 0.13
F. sp. MA067296 (CP016930.1) 0.30 0.34 0.14 0.37 0.37 0.35 1.02 0.16 0.38 0.38 0.16 0.00 0.34 0.16 0.16 0.16 0.32 0.16 0.16
F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.31 0.30 0.11 0.36 0.32 0.34 1.08 0.01 0.33 0.33 0.01 0.17 0.30 0.02 0.01 0.05 0.32 0.12 0.05 0.17
F. tularensis subsp. novicida F6168 (CP009353.1) 0.31 0.32 0.11 0.35 0.34 0.34 1.06 0.01 0.35 0.35 0.01 0.16 0.32 0.01 0.01 0.06 0.32 0.12 0.06 0.16 0.02
108
Table A1.12. Francisella prfB Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 44 7255 6920 -3416 n/a 0.58 2.03 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
T92+G+I 45 7265 6922 -3416 0.09 0.73 2.05 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
HKY+G 46 7271 6921 -3414 n/a 0.58 2.03 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
T92+I 44 7275 6940 -3426 0.33 n/a 2.02 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
TN93+G 47 7277 6920 -3413 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.2 0.05 0.02
HKY+G+I 47 7281 6923 -3414 0.08 0.71 2.05 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
TN93+G+I 48 7287 6922 -3413 0.05 0.67 2.04 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.21 0.05 0.02
HKY+I 46 7291 6941 -3425 0.32 n/a 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
TN93+I 47 7299 6941 -3423 0.32 n/a 2 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.25 0.03 0.21 0.05 0.02
GTR+G 50 7299 6919 -3409 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02
GTR+G+I 51 7308 6920 -3409 0.1 0.77 2.05 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02
GTR+I 50 7317 6936 -3418 0.33 n/a 2.07 0.346 0.317 0.136 0.202 0.07 0.02 0.13 0.07 0.11 0.02 0.05 0.25 0.02 0.22 0.03 0.02
K2+G 43 7345 7018 -3466 n/a 0.67 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+G+I 44 7355 7020 -3466 0 0.67 1.81 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+I 43 7361 7033 -3474 0.33 n/a 1.82 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
T92 43 7361 7034 -3474 n/a n/a 1.46 0.331 0.331 0.169 0.169 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03
HKY 45 7377 7034 -3472 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.21 0.06 0.03
TN93 46 7383 7033 -3470 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.11 0.07 0.1 0.04 0.07 0.22 0.04 0.19 0.06 0.03
GTR 49 7403 7030 -3466 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.07 0.02 0.11 0.08 0.1 0.02 0.06 0.22 0.05 0.19 0.04 0.03
K2 42 7430 7111 -3513 n/a n/a 1.42 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC+G 42 7480 7160 -3538 n/a 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 43 7490 7162 -3538 0 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 42 7494 7175 -3545 0.3 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 41 7545 7233 -3575 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
109
Table A1.13. Francisella prfB genetic distance matrix.
Sample R20
ex. A. a
lbolim
batum
(PI 1
872)
F. tu
lare
nsis
sub
sp.
holarctic
a LVS (A
M23
3362
.1 )
F. tu
lare
nsis
sub
sp.
med
iasiatica
FSC
147 (C
P0009
15.1)
F. philom
iragia
sub
sp.
philo
mira
gia
ATC
C 250
17 (C
P0009
37.1)
F. cf. no
vicida
Fx1
(CP00
2557
.1)
F. cf. no
vicida
352
3 (C
P0025
58.1)
F. noa
tune
nsis
sub
sp. or
ientalis
str. Tob
a 04
(CP00
3402
.1)
F. tu
lare
nsis
sub
sp.
novic
ida
F61
68 (C
P0093
53.1)
F. philom
iragia
str. G
A01-2
794 (C
P0094
40.1)
F. orie
ntalis
FNO12
(CP01
1921
.2)
F. tu
lare
nsis
sub
sp. tu
lare
nsis
WY96
(CP01
2037
.1)
F. per
sica
ATCC V
R-3
31 (C
P0125
05.1)
F. tu
lare
nsis
str. S
chu4
(CP01
3853
.1)
F. sp
. MA06
7296
(CP01
6930
.1)
F. hispa
nien
sis
FSC45
4 (C
P0180
93.1)
F. noa
tune
nsis
sub
sp.
noatun
ensis
FSC
774 (C
P0538
50.1)
F. philom
iragia
subs
p. ph
ilom
iragia
ATC
C 250
15 (CP01
0019
.1)
F. philom
iragia
subs
p. ph
ilom
iragia
str. 195
1 (E
U68
3025
)
F. philom
iragia
sub
sp.
noatun
ensis
str. DSM
187
77 (E
U68
3027
)
F. haliotic
ida
str. S
him
ane-
1 (JF2
9038
5)
F. opp
ortunistica
str. 14-
2155
(CP02
2375
.1)
P. salm
onis
(C
P0118
49)
Sample R20 ex. A. albolimbatum (PI 1872)
F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.15
F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.16 0.01
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.22 0.18 0.18
F. cf. novicida Fx1 (CP002557.1) 0.17 0.04 0.04 0.15
F. cf. novicida 3523 (CP002558.1) 0.14 0.08 0.09 0.19 0.10
F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.23 0.19 0.19 0.06 0.17 0.20
F. tularensis subsp. novicida F6168 (CP009353.1) 0.17 0.05 0.05 0.15 0.01 0.11 0.17
F. philomiragia str. GA01-2794 (CP009440.1) 0.22 0.16 0.16 0.05 0.15 0.18 0.05 0.16
F. orientalis FNO12 (CP011921.2) 0.23 0.19 0.19 0.06 0.17 0.20 0.00 0.17 0.05
F. tularensis subsp. tularensis WY96 (CP012037.1) 0.16 0.00 0.00 0.18 0.04 0.09 0.19 0.05 0.16 0.19
F. persica ATCC VR-331 (CP012505.1) 0.04 0.13 0.14 0.22 0.15 0.12 0.24 0.15 0.22 0.24 0.14
F. tularensis str. Schu4 (CP013853.1) 0.16 0.00 0.00 0.18 0.04 0.09 0.19 0.05 0.16 0.19 0.00 0.14
F. sp. MA067296 (CP016930.1) 0.09 0.14 0.14 0.21 0.16 0.12 0.23 0.16 0.20 0.23 0.14 0.09 0.14
F. hispaniensis FSC454 (CP018093.1) 0.12 0.09 0.10 0.19 0.11 0.03 0.20 0.12 0.18 0.20 0.10 0.11 0.10 0.13
F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.24 0.17 0.17 0.07 0.16 0.20 0.06 0.16 0.06 0.06 0.17 0.24 0.17 0.22 0.20
F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.22 0.18 0.18 0.01 0.15 0.19 0.06 0.15 0.06 0.06 0.18 0.22 0.18 0.21 0.18 0.07
F. philomiragia subsp. philomiragia str. 1951 (EU683025) 0.22 0.17 0.18 0.02 0.14 0.19 0.07 0.15 0.07 0.07 0.17 0.22 0.17 0.21 0.19 0.07 0.02
F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.24 0.17 0.17 0.07 0.16 0.20 0.06 0.16 0.06 0.06 0.17 0.24 0.17 0.22 0.20 0.00 0.07 0.07
F. halioticida str. Shimane-1 (JF290385) 0.28 0.28 0.29 0.23 0.28 0.25 0.25 0.28 0.25 0.25 0.29 0.26 0.29 0.25 0.24 0.23 0.23 0.24 0.23
F. opportunistica str. 14-2155 (CP022375.1) 0.09 0.14 0.14 0.21 0.16 0.12 0.23 0.16 0.20 0.23 0.14 0.09 0.14 0.00 0.13 0.22 0.21 0.21 0.22 0.25
P. salmonis (CP011849) 0.59 0.55 0.55 0.56 0.54 0.57 0.56 0.53 0.56 0.56 0.54 0.60 0.54 0.54 0.58 0.56 0.57 0.56 0.56 0.53 0.54
110
Table A1.14. Francisella rpoA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 44 7255 6920 -3416 n/a 0.58 2.03 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
T92+G+I 45 7265 6922 -3416 0.09 0.73 2.05 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
HKY+G 46 7271 6921 -3414 n/a 0.58 2.03 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
T92+I 44 7275 6940 -3426 0.33 n/a 2.02 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03
TN93+G 47 7277 6920 -3413 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.2 0.05 0.02
HKY+G+I 47 7281 6923 -3414 0.08 0.71 2.05 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
TN93+G+I 48 7287 6922 -3413 0.05 0.67 2.04 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.21 0.05 0.02
HKY+I 46 7291 6941 -3425 0.32 n/a 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02
TN93+I 47 7299 6941 -3423 0.32 n/a 2 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.25 0.03 0.21 0.05 0.02
GTR+G 50 7299 6919 -3409 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02
GTR+G+I 51 7308 6920 -3409 0.1 0.77 2.05 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02
GTR+I 50 7317 6936 -3418 0.33 n/a 2.07 0.346 0.317 0.136 0.202 0.07 0.02 0.13 0.07 0.11 0.02 0.05 0.25 0.02 0.22 0.03 0.02
K2+G 43 7345 7018 -3466 n/a 0.67 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+G+I 44 7355 7020 -3466 0 0.67 1.81 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+I 43 7361 7033 -3474 0.33 n/a 1.82 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
T92 43 7361 7034 -3474 n/a n/a 1.46 0.331 0.331 0.169 0.169 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03
HKY 45 7377 7034 -3472 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.21 0.06 0.03
TN93 46 7383 7033 -3470 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.11 0.07 0.1 0.04 0.07 0.22 0.04 0.19 0.06 0.03
GTR 49 7403 7030 -3466 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.07 0.02 0.11 0.08 0.1 0.02 0.06 0.22 0.05 0.19 0.04 0.03
K2 42 7430 7111 -3513 n/a n/a 1.42 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC+G 42 7480 7160 -3538 n/a 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 43 7490 7162 -3538 0 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 42 7494 7175 -3545 0.3 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 41 7545 7233 -3575 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
111
Table A1.15. Francisella rpoA genetic distance matrix.
F. per
sica
ATCC V
R-3
31 (C
P0125
05.1)
Sample R20
ex. A. a
lbolim
batum
(PI 1
872)
F. cf. no
vicida
352
3 (C
P0025
58.1)
F. cf. no
vicida
Fx1
(CP00
2557
.1)
F. haliotic
ida
str. S
him
ane-
1 (JF2
9038
5)
F. hispa
nien
sis
FSC45
4 (C
P0180
93.1)
F. noa
tune
nsis
sub
sp.
noatun
ensis
FSC
774 (C
P0538
50.1)
F. noa
tune
nsis
sub
sp. or
ientalis
str. Tob
a 04
(CP00
3402
.1)
F. opp
ortunistica
str. 14-
2155
(CP02
2375
.1)
F. orie
ntalis
FNO12
(CP01
1921
.2)
F. philom
iragia
str. G
A01-2
794 (C
P0094
40.1)
F. philom
iragia
sub
sp.
noatun
ensis
str. DSM
187
77 (E
U68
3027
)
F. p
hilom
iragia
sub
sp. p
hilom
iragia ATC
C 250
15 (C
P0100
19.1)
F. philom
iragia
sub
sp.
philo
mira
gia
ATC
C 250
17 (C
P0009
37.1)
F. philom
iragia su
bsp.
philo
mira
gia
str. 195
1 (E
U68
3001
)
F. sp
. MA06
7296
(CP01
6930
.1)
F. tu
lare
nsis
str. S
chu4
(CP01
3853
.1)
F. tu
lare
nsis
sub
sp.
holarctic
a LVS (A
M23
3362
.1 )
F. tu
lare
nsis
sub
sp.
med
iasiatica
FSC
147 (C
P0009
15.1)
F. tu
lare
nsis
sub
sp.
novic
ida
F61
68 (C
P0093
53.1)
F. tu
lare
nsis
sub
sp. tu
lare
nsis
WY96
-341
8 (C
P0006
08.1)
P. salm
onis
(CP01
1849
)
F. persica ATCC VR-331 (CP012505.1)
Sample R20 ex. A. albolimbatum (PI 1872) 0.04
F. cf. novicida 3523 (CP002558.1) 0.10 0.11
F. cf. novicida Fx1 (CP002557.1) 0.08 0.09 0.07
F. halioticida str. Shimane-1 (JF290385) 0.24 0.25 0.24 0.21
F. hispaniensis FSC454 (CP018093.1) 0.10 0.11 0.00 0.08 0.25
F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.23 0.23 0.23 0.20 0.26 0.23
F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.24 0.25 0.23 0.21 0.26 0.23 0.01
F. opportunistica str. 14-2155 (CP022375.1) 0.09 0.10 0.10 0.09 0.25 0.10 0.25 0.26
F. orientalis FNO12 (CP011921.2) 0.24 0.25 0.23 0.21 0.26 0.23 0.01 0.00 0.26
F. philomiragia str. GA01-2794 (CP009440.1) 0.23 0.23 0.22 0.20 0.27 0.23 0.02 0.02 0.26 0.02
F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.23 0.23 0.23 0.20 0.26 0.23 0 0.01 0.25 0.01 0.02
F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.24 0.24 0.25 0.22 0.28 0.25 0.08 0.09 0.27 0.09 0.07 0.08
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.24 0.24 0.24 0.21 0.27 0.24 0.08 0.09 0.27 0.09 0.07 0.08 0.01
F. philomiragia subsp. philomiragia str. 1951 (EU683001) 0.24 0.24 0.25 0.22 0.28 0.25 0.08 0.09 0.27 0.09 0.07 0.08 0.01 0.01
F. sp. MA067296 (CP016930.1) 0.09 0.10 0.10 0.09 0.25 0.10 0.25 0.26 0.00 0.26 0.26 0.25 0.27 0.27 0.27
F. tularensis str. Schu4 (CP013853.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09
F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00
F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00 0.00
F. tularensis subsp. novicida F6168 (CP009353.1) 0.08 0.09 0.08 0.00 0.22 0.08 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.01 0.01 0.01
F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00 0.00 0.00 0.01
P. salmonis (CP011849) 1.02 1.01 1.00 0.91 1.12 1.01 1.04 1.03 1.03 1.03 1.05 1.04 1.07 1.03 1.03 1.03 0.92 0.92 0.92 0.90 0.92
112
Table A1.16. Francisella dnaA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 44 8508 8159 -4035 n/a 0.17 2.93 0.335 0.335 0.165 0.165 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02
T92+G+I 45 8518 8161 -4035 0 0.17 2.93 0.335 0.335 0.165 0.165 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02
TN93+G 47 8528 8155 -4031 n/a 0.19 2.91 0.367 0.304 0.138 0.191 0.03 0.02 0.1 0.04 0.15 0.02 0.04 0.33 0.02 0.19 0.03 0.02
GTR+G 50 8531 8135 -4017 n/a 0.19 2.92 0.367 0.304 0.138 0.191 0.06 0.01 0.11 0.07 0.15 0.01 0.03 0.34 0 0.2 0.02 0
HKY+G 46 8534 8169 -4038 n/a 0.17 3.02 0.367 0.304 0.138 0.191 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.04 0.02
TN93+G+I 48 8536 8155 -4030 0.34 0.44 3.1 0.367 0.304 0.138 0.191 0.03 0.01 0.1 0.04 0.16 0.02 0.04 0.34 0.02 0.19 0.03 0.01
GTR+G+I 51 8537 8132 -4015 0.36 0.47 3.15 0.367 0.304 0.138 0.191 0.06 0.01 0.1 0.07 0.16 0.01 0.02 0.35 0 0.2 0.02 0
HKY+G+I 47 8544 8171 -4038 0 0.17 3.03 0.367 0.304 0.138 0.191 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.04 0.02
T92+I 44 8582 8233 -4073 0.47 n/a 2.52 0.335 0.335 0.165 0.165 0.04 0.02 0.12 0.04 0.12 0.02 0.04 0.25 0.02 0.25 0.04 0.02
TN93+I 47 8591 8218 -4062 0.47 n/a 2.55 0.367 0.304 0.138 0.191 0.04 0.02 0.09 0.05 0.15 0.02 0.05 0.32 0.02 0.18 0.04 0.02
GTR+I 50 8600 8204 -4052 0.47 n/a 2.06 0.367 0.304 0.138 0.191 0.06 0.01 0.1 0.08 0.14 0.04 0.03 0.3 0 0.19 0.06 0
HKY+I 46 8611 8246 -4077 0.47 n/a 2.51 0.367 0.304 0.138 0.191 0.04 0.02 0.14 0.05 0.1 0.03 0.05 0.22 0.03 0.27 0.04 0.02
K2+G 43 8738 8397 -4156 n/a 0.23 2.2 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
K2+G+I 44 8748 8399 -4155 0.14 0.31 2.22 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
K2+I 43 8790 8449 -4182 0.48 n/a 2.17 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
T92 43 8824 8483 -4198 n/a n/a 1.42 0.335 0.335 0.165 0.165 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03
GTR 49 8829 8441 -4171 n/a n/a 1.44 0.367 0.304 0.138 0.191 0.08 0.03 0.09 0.1 0.12 0.02 0.07 0.26 0.01 0.18 0.04 0
TN93 46 8838 8473 -4190 n/a n/a 1.43 0.367 0.304 0.138 0.191 0.06 0.03 0.09 0.07 0.11 0.04 0.07 0.25 0.04 0.17 0.06 0.03
HKY 45 8849 8493 -4201 n/a n/a 1.42 0.367 0.304 0.138 0.191 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.22 0.06 0.03
JC+G 42 8912 8579 -4247 n/a 0.32 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 43 8922 8581 -4247 0 0.32 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 42 8962 8629 -4272 0.45 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
K2 42 8972 8639 -4277 n/a n/a 1.39 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC 41 9102 8777 -4347 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
113
Table A1.17. Francisella dnaA genetic distance matrix.
F. tu
lare
nsis
sub
sp.
holarctic
a LVS (A
M23
3362
.1 )
F. tu
lare
nsis
sub
sp.
med
iasiatica
FSC
147 (C
P0009
15.1)
F. philom
iragia
sub
sp.
philo
mira
gia
ATC
C 250
17 (C
P0009
37.1)
F. cf. no
vicida
Fx1
(CP00
2557
.1)
F. cf. no
vicida
352
3 (C
P0025
58.1)
F. noa
tune
nsis
sub
sp. or
ientalis
str. Tob
a 04
(CP00
3402
.1)
F. tu
lare
nsis
sub
sp.
novic
ida
F61
68 (C
P0093
53.1)
F. philom
iragia
str. G
A01-2
794 (C
P0094
40.1)
F. philom
iragia
subs
p. ph
ilom
iragia
ATC
C 250
15
F. orie
ntalis
FNO12
(CP01
1921
.2)
F. tu
lare
nsis
sub
sp. tu
lare
nsis
WY96
-341
8 (C
P0006
08.1)
F. per
sica
ATCC V
R-3
31 (C
P0125
05.1)
F. tu
lare
nsis
str. S
chu4
(CP01
3853
.1)
F. sp
. MA06
7296
(CP01
6930
.1)
F. hispa
nien
sis
FSC45
4 (C
P0180
93.1)
F. opp
ortunistica
str. 14-
2155
(CP02
2375
.1)
F. noa
tune
nsis
sub
sp.
noatun
ensis
FSC
774 (C
P0538
50.1)
P. salm
onis
LF-
89 A
TCC V
R-1
361 (2
9882
744)
F. philom
iragia
subs
p. ph
ilom
iragia
str. 195
1 (E
U68
3025
)
F. philom
iragia
sub
sp.
noatun
ensis
str. DSM
187
77 (E
U68
3027
)
F. haliotic
ida
str. S
him
ane-
1 (JF2
9038
5)
Sample R20
ex. A. a
lbolim
batum
(PI 1
872)
F. tularensis subsp. holarctica LVS (AM233362.1 )
F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.00
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.14 0.14
F. cf. novicida Fx1 (CP002557.1) 0.01 0.01 0.13
F. cf. novicida 3523 (CP002558.1) 0.07 0.06 0.14 0.06
F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.14 0.14 0.03 0.13 0.14
F. tularensis subsp. novicida F6168 (CP009353.1) 0.01 0.01 0.13 0.01 0.07 0.14
F. philomiragia str. GA01-2794 (CP009440.1) 0.15 0.15 0.05 0.14 0.15 0.03 0.15
F. philomiragia subsp. philomiragia ATCC 25015 0.13 0.13 0.02 0.12 0.12 0.04 0.13 0.06
F. orientalis FNO12 (CP011921.2) 0.14 0.14 0.03 0.13 0.14 0.00 0.14 0.03 0.04
F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.00 0.00 0.14 0.01 0.06 0.14 0.01 0.15 0.13 0.14
F. persica ATCC VR-331 (CP012505.1) 0.06 0.07 0.15 0.07 0.09 0.15 0.06 0.16 0.14 0.15 0.06
F. tularensis str. Schu4 (CP013853.1) 0.00 0.00 0.14 0.01 0.06 0.14 0.01 0.15 0.13 0.14 0.00 0.06
F. sp. MA067296 (CP016930.1) 0.08 0.08 0.15 0.08 0.10 0.15 0.08 0.17 0.14 0.15 0.08 0.06 0.08
F. hispaniensis FSC454 (CP018093.1) 0.07 0.06 0.13 0.06 0.00 0.14 0.07 0.16 0.12 0.14 0.06 0.09 0.06 0.09
F. opportunistica str. 14-2155 (CP022375.1) 0.08 0.08 0.15 0.08 0.10 0.15 0.08 0.17 0.14 0.15 0.08 0.06 0.08 0.00 0.09
F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.14 0.13 0.04 0.13 0.14 0.01 0.14 0.03 0.04 0.01 0.13 0.15 0.14 0.15 0.14 0.15
P. salmonis LF-89 ATCC VR-1361 (29882744) 0.68 0.69 0.71 0.68 0.72 0.72 0.68 0.69 0.71 0.72 0.68 0.66 0.68 0.71 0.71 0.71 0.70
F. philomiragia subsp. philomiragia str. 1951 (EU683025) 0.13 0.13 0.02 0.12 0.12 0.03 0.13 0.05 0.00 0.03 0.13 0.14 0.13 0.14 0.12 0.14 0.04 0.71
F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.14 0.14 0.04 0.13 0.15 0.01 0.14 0.03 0.04 0.01 0.14 0.15 0.14 0.15 0.15 0.15 0.00 0.71 0.04
F. halioticida str. Shimane-1 (JF290385) 0.16 0.15 0.15 0.16 0.16 0.15 0.16 0.15 0.15 0.15 0.15 0.17 0.15 0.17 0.16 0.17 0.15 0.68 0.15 0.15
Sample R20 ex. A. albolimbatum (PI 1872) 0.07 0.07 0.14 0.07 0.09 0.15 0.07 0.17 0.14 0.15 0.07 0.02 0.07 0.06 0.09 0.06 0.15 0.68 0.14 0.15 0.17
114
Table A1.18. Francisella concatenated 16S, tpiA, prfB, rpoA and dnaA Maximum Likelihood fits of 24 different nucleotide substitution
models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
GTR+G 48 40774 40325 -20114 n/a 0.295 1.948 0.323 0.28 0.158 0.239 0.06 0.03 0.13 0.07 0.14 0.02 0.06 0.24 0.03 0.17 0.03 0.02
GTR+G+I 49 40779 40321 -20111 0.181 0.425 1.981 0.323 0.28 0.158 0.239 0.06 0.03 0.13 0.07 0.14 0.02 0.06 0.24 0.03 0.17 0.03 0.02
TN93+G 45 40804 40384 -20147 n/a 0.294 1.947 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.13 0.04 0.05 0.24 0.04 0.17 0.05 0.03
TN93+G+I 46 40810 40380 -20144 0.175 0.418 1.979 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.14 0.04 0.05 0.24 0.04 0.17 0.05 0.03
HKY+G 44 40836 40424 -20168 n/a 0.291 1.965 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.11 0.04 0.05 0.19 0.04 0.21 0.05 0.03
HKY+G+I 45 40841 40421 -20165 0.171 0.41 1.997 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.11 0.04 0.05 0.19 0.04 0.22 0.05 0.03
T92+G 42 40854 40461 -20189 n/a 0.296 1.945 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03
T92+G+I 43 40860 40458 -20186 0.167 0.412 1.975 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03
GTR+I 48 41097 40648 -20276 0.491 n/a 1.928 0.323 0.28 0.158 0.239 0.07 0.03 0.13 0.08 0.13 0.03 0.05 0.23 0.03 0.18 0.03 0.02
TN93+I 45 41133 40712 -20311 0.49 n/a 1.9 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.13 0.04 0.05 0.23 0.04 0.18 0.05 0.03
HKY+I 44 41164 40752 -20332 0.491 n/a 1.908 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.1 0.04 0.05 0.18 0.04 0.21 0.05 0.03
T92+I 42 41181 40788 -20352 0.491 n/a 1.898 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03
K2+G 41 41230 40847 -20382 n/a 0.317 1.824 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+G+I 42 41237 40845 -20380 0.146 0.423 1.842 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
K2+I 41 41535 41151 -20535 0.488 n/a 1.782 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
JC+G 40 42072 41698 -20809 n/a 0.343 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 41 42083 41699 -20809 0.051 0.378 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 40 42367 41993 -20956 0.479 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
GTR 47 42418 41978 -20942 n/a n/a 1.401 0.323 0.28 0.158 0.239 0.08 0.03 0.12 0.09 0.11 0.03 0.07 0.2 0.04 0.17 0.04 0.02
TN93 44 42475 42064 -20988 n/a n/a 1.399 0.323 0.28 0.158 0.239 0.06 0.03 0.12 0.07 0.11 0.05 0.07 0.2 0.05 0.16 0.06 0.03
HKY 43 42505 42103 -21008 n/a n/a 1.398 0.323 0.28 0.158 0.239 0.06 0.03 0.14 0.07 0.09 0.05 0.07 0.16 0.05 0.19 0.06 0.03
T92 41 42519 42135 -21027 n/a n/a 1.4 0.301 0.301 0.199 0.199 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04
K2 40 42757 42383 -21152 n/a n/a 1.395 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC 39 43469 43104 -21513 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
115
Table A1.19. Francisella concatenated 16S, tpiA, prfB, rpoA and dnaA genetic distance matrix.
P. s
almon
is
str.
LF-8
9 (C
P0118
49)
F. haliotic
ida
str.
Shim
ane-
1 (J
F290
376)
F. hisp
aniens
is
FSC45
4 (C
P01
8093
)
F. cf. no
vicida
352
3 (C
P00
2558
)
F. tular
ensis
sub
sp. ho
larc
tica
LVS (A
M23
3362
.1)
F. tular
ensis
sub
sp. m
ediasiat
ica F
SC14
7 (C
P00
0915
)
F. tular
ensis
str.
Sch
u4 (C
P01
3853
)
F. tular
ensis
sub
sp. tu
lare
nsis
str.
WY96
(CP01
2037
)
F. cf. no
vicida
Fx1
(CP00
2557
)
F. tular
ensis
sub
sp. no
vicida
F61
68 (C
P00
0915
)
F. sp
. MA06
7296
(CP01
6930
)
F. opp
ortunistica
str.
14-
2155
(CP02
2375
)
Sam
ple
R20
ex. A
. albolim
batum
(PI 1
872)
F. per
sica
ATC
C V
R-3
31 (C
P01
2505
)
F. noa
tune
nsis
subs
p. n
oatune
nsis
FSC77
4 (N
Z CP05
3850
)
F. philom
iragia
sub
sp. ph
ilom
iragia
ATC
C 250
15 (C
P01
0019
)
F. philom
iragia
str.
1951
(AY49
6933
)
F. philom
iragia
sub
sp. ph
ilom
iragia
ATC
C 250
17 (C
P00
0937
.1)
F. noa
tune
nsis
subs
p. o
rient
alis
str.
Tob
a 04
(CP00
3402
)
F. orie
ntalis
FNO12
(CP01
1921
)
F. philom
iragia
str.
GA01
-279
4 (C
P00
9440
)
P. salmonis str. LF-89 (CP011849)
F. halioticida str. Shimane-1 (JF290376) 0.58
F. hispaniensis FSC454 (CP018093) 0.60 0.17
F. cf. novicida 3523 (CP002558) 0.60 0.17 0.01
F. tularensis subsp. holarctica LVS (AM233362.1) 0.58 0.17 0.05 0.05
F. tularensis subsp. mediasiatica FSC147 (CP000915) 0.58 0.17 0.05 0.05 0.00
F. tularensis str. Schu4 (CP013853) 0.58 0.17 0.05 0.05 0.00 0.00
F. tularensis subsp. tularensis str. WY96 (CP012037) 0.58 0.17 0.05 0.05 0.00 0.00 0.00
F. cf. novicida Fx1 (CP002557) 0.58 0.17 0.06 0.05 0.02 0.02 0.01 0.01
F. tularensis subsp. novicida F6168 (CP000915) 0.58 0.17 0.06 0.06 0.02 0.01 0.01 0.01 0.01
F. sp. MA067296 (CP016930) 0.58 0.18 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09
F. opportunistica str. 14-2155 (CP022375) 0.58 0.18 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.00
Sample R20 ex. A. albolimbatum (PI 1872) 0.59 0.18 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.07
F. persica ATCC VR-331 (CP012505) 0.59 0.17 0.08 0.08 0.07 0.07 0.07 0.07 0.08 0.08 0.07 0.07 0.03
F. noatunensis subsp. noatunensis FSC774 (NZ CP053850) 0.64 0.21 0.21 0.21 0.19 0.19 0.19 0.19 0.19 0.19 0.22 0.22 0.22 0.21
F. philomiragia subsp. philomiragia ATCC 25015 (CP010019) 0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.13 0.14 0.16 0.16 0.16 0.16 0.10
F. philomiragia str. 1951 (AY496933) 0.59 0.16 0.14 0.14 0.14 0.14 0.14 0.14 0.13 0.13 0.16 0.16 0.16 0.15 0.10 0.01
F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1)0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.13 0.14 0.16 0.16 0.16 0.16 0.10 0.01 0.01
F. noatunensis subsp. orientalis str. Toba 04 (CP003402) 0.59 0.17 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.11 0.11 0.14 0.13 0.12 0.07 0.07 0.07
F. orientalis FNO12 (CP011921) 0.59 0.17 0.15 0.16 0.15 0.15 0.15 0.15 0.14 0.14 0.17 0.17 0.17 0.16 0.09 0.04 0.05 0.04 0.04
F. philomiragia str. GA01-2794 (CP009440) 0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.14 0.14 0.16 0.16 0.17 0.16 0.09 0.05 0.05 0.04 0.05 0.03
116
Table A1.20. 344 bp Rickettsia gltA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 62 3024 2574 -1224 n/a 0.267 3.221 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02
T92+I 62 3029 2579 -1227 0.644 n/a 3.148 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02
T92+G+I 63 3031 2574 -1224 0.353 0.592 3.237 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02
HKY+G 64 3037 2573 -1222 n/a 0.262 3.284 0.366 0.291 0.143 0.2 0.03 0.02 0.16 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.03 0.02
TN93+G 65 3041 2569 -1219 n/a 0.272 3.212 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.16 0.02 0.04 0.32 0.02 0.2 0.03 0.02
HKY+I 64 3044 2579 -1225 0.646 n/a 3.187 0.366 0.291 0.143 0.2 0.03 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.03 0.02
HKY+G+I 65 3046 2574 -1222 0.351 0.574 3.309 0.366 0.291 0.143 0.2 0.03 0.02 0.16 0.04 0.11 0.02 0.04 0.23 0.02 0.29 0.03 0.02
TN93+I 65 3046 2574 -1222 0.639 n/a 3.128 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.15 0.02 0.04 0.31 0.02 0.2 0.03 0.02
TN93+G+I 66 3048 2569 -1218 0.376 0.656 3.237 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.16 0.02 0.04 0.32 0.02 0.2 0.03 0.02
K2+G 61 3060 2617 -1247 n/a 0.24 3.283 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
GTR+G 68 3064 2570 -1217 n/a 0.281 2.504 0.366 0.291 0.143 0.2 0.03 0.03 0.12 0.03 0.13 0.03 0.07 0.27 0.02 0.21 0.05 0.01
K2+I 61 3065 2622 -1250 0.667 n/a 3.223 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
GTR+I 68 3067 2573 -1218 0.631 n/a 2.872 0.366 0.291 0.143 0.2 0.01 0.03 0.11 0.02 0.14 0.03 0.07 0.29 0.02 0.21 0.05 0.02
K2+G+I 62 3067 2617 -1246 0.388 0.575 3.319 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
GTR+G+I 69 3070 2569 -1215 0.355 0.637 2.652 0.366 0.291 0.143 0.2 0.02 0.03 0.12 0.03 0.14 0.03 0.07 0.28 0.02 0.21 0.05 0.01
T92 61 3092 2649 -1263 n/a n/a 2.899 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.25 0.02 0.25 0.04 0.02
TN93 64 3108 2643 -1257 n/a n/a 2.894 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.15 0.02 0.04 0.3 0.02 0.2 0.03 0.02
HKY 63 3108 2651 -1262 n/a n/a 2.89 0.366 0.291 0.143 0.2 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.22 0.02 0.28 0.04 0.02
GTR 67 3132 2645 -1255 n/a n/a 2.141 0.366 0.291 0.143 0.2 0.03 0.03 0.11 0.04 0.12 0.04 0.07 0.25 0.02 0.21 0.05 0.02
K2 60 3135 2699 -1289 n/a n/a 2.865 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03
JC+G 60 3151 2716 -1298 n/a 0.259 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 60 3157 2721 -1300 0.659 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 61 3160 2717 -1297 0.366 0.6 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 59 3222 2793 -1337 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
117
Table A1.21. 344 bp Rickettsia gltA genetic distance matrix.
Sample SR
12 ex.
A. a
lbolim
batum
(P
I 332
9)
Unc
ulture
d R. sp
. clone
ARRL2
016-
156 (M
N43
1835
)
Unc
ulture
d R. s
p. clone
ARRL2
015-
159 (M
N43
1836
)
R. tam
urae
str. A
T 1 (A
F394
896 )
R. s
lova
ca is
olate Xin
jiang
(MF0
0252
8)
R. s
ibirica
246
(AABW
0100
0001
)
R. rick
ettsii
str. A
rizon
a (C
P0033
07)
R. rhipice
phali
str. H
J 5 (C
P0131
33)
R. rao
ultii
str. K
haba
rovs
k (C
P0109
69)
R. p
arke
ri str. P
ortsm
outh (NC 017
044)
R. m
ontane
nsis
str. O
SU 85 93
0 (C
P0033
40)
R. m
onac
ensis
str. IrR/M
unich (L
N79
4217
)
R. m
assil
iae
str. A
ZT80
(CP00
3319
)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. h
onei
RB (N
Z AJT
T010
0000
4)
R. h
eilong
jiang
ensis
Sen
dai 5
8 DNA (A
P0198
65)
R. g
rave
sii B
WI 1
(DQ26
9435
)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. c
onor
ii str. M
alish 7 (A
E0069
14)
R. a
mblyo
mm
atis
str. GAT
30V (
CP00
3334
)
R. a
frica
e E
SF 5 (C
P0016
12)
Unc
ulture
d R. s
p. clone
ARRL2
016-
149 (M
N43
1834
)
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. a
kari
str. H
artfo
rd (NC 009
881)
R. felis
URRW
XCal2 (C
P0000
53)
R. a
sem
bone
nsis
str. N
MRCii (
NZ
JWSW
0100
0078
)
R. typ
hi str. W
ilmington
(NC 006
142)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample SR12 ex. A. albolimbatum (PI 3329)
Uncultured R. sp. clone ARRL2016-156 (MN431835) 0.14
Uncultured R. sp. clone ARRL2015-159 (MN431836) 0.14 0.00
R. tamurae str. AT 1 (AF394896 ) 0.15 0.04 0.04
R. slovaca isolate Xinjiang (MF002528) 0.15 0.01 0.01 0.04
R. sibirica 246 (AABW01000001) 0.14 0.00 0.00 0.04 0.00
R. rickettsii str. Arizona (CP003307) 0.14 0.01 0.01 0.04 0.01 0.01
R. rhipicephali str. HJ 5 (CP013133) 0.14 0.01 0.01 0.05 0.01 0.01 0.02
R. raoultii str. Khabarovsk (CP010969) 0.14 0.00 0.00 0.04 0.01 0.00 0.01 0.01
R. parkeri str. Portsmouth (NC 017044) 0.14 0.01 0.01 0.04 0.01 0.00 0.01 0.01 0.01
R. montanensis str. OSU 85 930 (CP003340) 0.13 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01
R. monacensis str. IrR/Munich (LN794217) 0.15 0.03 0.03 0.01 0.04 0.04 0.04 0.04 0.03 0.04 0.03
R. massiliae str. AZT80 (CP003319) 0.14 0.01 0.01 0.05 0.01 0.01 0.02 0.00 0.01 0.01 0.01 0.04
R. japonica str. M14012 (NZ AP017596) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01
R. honei RB (NZ AJTT01000004) 0.15 0.01 0.01 0.04 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.04 0.01 0.01
R. heilongjiangensis Sendai 58 DNA (AP019865) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.00 0.01
R. gravesii BWI 1 (DQ269435) 0.15 0.01 0.01 0.05 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.05 0.02 0.02 0.02 0.02
R. helvetica C9P9 (CM001467) 0.17 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.05
R. conorii str. Malish 7 (AE006914) 0.14 0.01 0.01 0.05 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.04 0.02 0.01 0.01 0.01 0.02 0.06
R. amblyommatis str. GAT 30V (CP003334) 0.14 0.00 0.00 0.04 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.01 0.01 0.01 0.02 0.05 0.01
R. africae ESF 5 (CP001612) 0.14 0.00 0.00 0.04 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.04 0.01 0.01 0.00 0.01 0.02 0.05 0.01 0.01
Uncultured R. sp. clone ARRL2016-149 (MN431834) 0.14 0.00 0.00 0.04 0.01 0.00 0.01 0.01 0.00 0.01 0.00 0.03 0.01 0.00 0.01 0.00 0.01 0.05 0.01 0.00 0.00
R. aeschlimannii str. RH15 (HM050289) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.01 0.01 0.01 0.02 0.05 0.01 0.01 0.01 0.00
R. australis str. Cutlack (NC 017058) 0.14 0.03 0.03 0.04 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.04 0.04 0.03 0.04
R. akari str. Hartford (NC 009881) 0.16 0.05 0.05 0.03 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.03 0.06 0.05 0.05 0.05 0.06 0.06 0.06 0.05 0.05 0.05 0.05 0.03
R. felis URRWXCal2 (CP000053) 0.15 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.03 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.04
R. asembonensis str. NMRCii (NZ JWSW01000078) 0.17 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05 0.06 0.07 0.04
R. typhi str. Wilmington (NC 006142) 0.17 0.08 0.08 0.09 0.08 0.09 0.09 0.09 0.08 0.09 0.08 0.08 0.09 0.09 0.09 0.09 0.09 0.11 0.09 0.08 0.09 0.08 0.09 0.10 0.09 0.09 0.12
R. prowazekii str. Madrid E (NC 000963) 0.16 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.10 0.09 0.08 0.08 0.08 0.08 0.10 0.10 0.09 0.11 0.04
R. bellii RML369-C (NC 007940) 0.05 0.16 0.16 0.17 0.17 0.16 0.16 0.16 0.16 0.16 0.15 0.17 0.16 0.16 0.16 0.16 0.17 0.17 0.16 0.16 0.16 0.16 0.16 0.17 0.18 0.18 0.17 0.19 0.18
118
Table A1.22. 634 bp Rickettsia gltA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq AFreq TFreq CFreq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 56 5966 5533 -2710 n/a 0.37 2.58 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.23 0.02 0.23 0.04 0.02
K2+G 55 5973 5547 -2718 n/a 0.31 2.6 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
T92+G+I 57 5976 5534 -2710 0.12 0.47 2.66 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.23 0.02 0.23 0.04 0.02
K2+G+I 56 5982 5548 -2718 0.15 0.43 2.67 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
HKY+G 58 5986 5537 -2710 n/a 0.37 2.56 0.3 0.33 0.19 0.18 0.04 0.02 0.13 0.04 0.14 0.02 0.04 0.24 0.02 0.22 0.04 0.02
T92+I 56 5988 5554 -2721 0.41 n/a 2.91 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.24 0.02 0.24 0.04 0.02
TN93+G 59 5994 5538 -2710 n/a 0.36 2.6 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.13 0.02 0.04 0.23 0.02 0.23 0.04 0.02
HKY+G+I 59 5994 5538 -2710 0.13 0.49 2.64 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.22 0.04 0.02
K2+I 55 5999 5573 -2731 0.41 n/a 2.78 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
TN93+G+I 60 6004 5539 -2709 0.13 0.49 2.64 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.13 0.02 0.04 0.23 0.02 0.23 0.04 0.02
HKY+I 58 6006 5557 -2720 0.41 n/a 2.9 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.23 0.04 0.02
TN93+I 59 6016 5559 -2720 0.41 n/a 2.91 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.23 0.04 0.02
GTR+G 62 6016 5536 -2706 n/a 0.38 2.05 0.3 0.33 0.19 0.18 0.05 0.04 0.15 0.04 0.11 0.04 0.06 0.19 0.01 0.24 0.07 0.01
GTR+G+I 63 6021 5533 -2703 0.09 0.46 2.21 0.3 0.33 0.19 0.18 0.04 0.04 0.15 0.03 0.11 0.04 0.06 0.21 0 0.24 0.07 0
GTR+I 62 6025 5545 -2710 0.41 n/a 2.87 0.3 0.33 0.19 0.18 0.02 0.04 0.14 0.02 0.14 0.04 0.06 0.24 0 0.23 0.07 0
T92 55 6074 5648 -2769 n/a n/a 1.66 0.32 0.32 0.18 0.18 0.06 0.03 0.12 0.06 0.12 0.03 0.06 0.2 0.03 0.2 0.06 0.03
K2 54 6082 5664 -2778 n/a n/a 1.68 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
HKY 57 6092 5651 -2768 n/a n/a 1.66 0.3 0.33 0.19 0.18 0.06 0.03 0.12 0.05 0.12 0.03 0.05 0.21 0.03 0.19 0.06 0.03
TN93 58 6102 5653 -2768 n/a n/a 1.66 0.3 0.33 0.19 0.18 0.06 0.03 0.12 0.05 0.12 0.03 0.05 0.22 0.03 0.19 0.06 0.03
JC+G 54 6108 5690 -2791 n/a 0.39 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 55 6118 5692 -2791 0 0.39 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
GTR 61 6118 5646 -2762 n/a n/a 1.55 0.3 0.33 0.19 0.18 0.04 0.04 0.11 0.03 0.11 0.05 0.07 0.2 0.03 0.19 0.09 0.03
JC+I 54 6137 5719 -2805 0.39 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 53 6193 5783 -2838 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
119
Table A1.23. 634 bp Rickettsia gltA genetic distance matrix.
Sample SR
12 ex.
A. a
lbolim
batum
(P
I 332
9)
R. a
esch
liman
nii s
tr. R
H15
(HM
0502
89)
R. a
frica
e E
SF 5 (C
P0016
12)
R. a
mblyo
mm
atis
str. GAT
30V (
CP00
3334
)
R. c
onor
ii str. M
alish 7 (A
E0069
14)
R. h
eilong
jiang
ensis
Sen
dai 5
8 DNA (A
P0198
65)
R. h
onei
RB (N
Z AJT
T010
0000
4)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. m
assil
iae
str. A
ZT80
(CP00
3319
)
R. m
ontane
nsis
str. O
SU 85 93
0 (C
P0033
40)
R. p
arke
ri str. P
ortsm
outh (NC 017
044)
R. p
eaco
ckii
str. Rus
tic (NC 012
730)
R. rao
ultii
str. Kha
baro
vsk (C
P0109
69)
R. rhipice
phali s
tr. H
J 5 (C
P0131
33)
R rick
ettsii
str. A
rizon
a (C
P0033
07)
R. s
ibirica
246
(AABW
0100
0001
)
R. s
lova
ca is
olate Xin
jiang
(MF0
0252
8)
R. g
rave
sii B
WI 1
(DQ26
9435
)
R. m
onac
ensis
str. IrR/M
unich
(LN79
4217
)
R. tam
urae
str. A
T 1 (A
F394
896 )
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. a
sem
bone
nsis
str. N
MRCii (
NZ
JWSW
0100
0078
)
R. felis
URRW
XCal2 (C
P0000
53)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. a
kari
str. H
artfo
rd (NC 009
881)
R. typ
hi str. W
ilmington
(NC 006
142)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample SR12 ex. A. albolimbatum (PI 3329)
R. aeschlimannii str. RH15 (HM050289) 0.01
R. africae ESF 5 (CP001612) 0.01 0.01
R. amblyommatis str. GAT 30V (CP003334) 0.01 0.01 0.01
R. conorii str. Malish 7 (AE006914) 0.01 0.01 0.01 0.01
R. heilongjiangensis Sendai 58 DNA (AP019865) 0.01 0.01 0.00 0.01 0.01
R. honei RB (NZ AJTT01000004) 0.01 0.01 0.00 0.01 0.00 0.01
R. japonica str. M14012 (NZ AP017596) 0.01 0.01 0.00 0.01 0.01 0 0.01
R. massiliae str. AZT80 (CP003319) 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01
R. montanensis str. OSU 85 930 (CP003340) 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01
R. parkeri str. Portsmouth (NC 017044) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01
R. peacockii str. Rustic (NC 012730) 0.01 0.01 0.00 0.01 0.00 0.01 0.00 0.01 0.01 0.01 0.00
R. raoultii str. Khabarovsk (CP010969) 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01
R. rhipicephali str. HJ 5 (CP013133) 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.00 0.01 0.01 0.01 0.01
R rickettsii str. Arizona (CP003307) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.02
R. sibirica 246 (AABW01000001) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.00
R. slovaca isolate Xinjiang (MF002528) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.00
R. gravesii BWI 1 (DQ269435) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
R. monacensis str. IrR/Munich (LN794217) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04
R. tamurae str. AT 1 (AF394896 ) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.01
R. australis str. Cutlack (NC 017058) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.07
R. asembonensis str. NMRCii (NZ JWSW01000078) 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.07 0.08 0.07 0.07 0.05
R. felis URRWXCal2 (CP000053) 0.08 0.07 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.08 0.07 0.07 0.08 0.07 0.08 0.08 0.08 0.07 0.06 0.01
R. prowazekii str. Madrid E (NC 000963) 0.08 0.08 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.09 0.08 0.08 0.10 0.11 0.12
R. akari str. Hartford (NC 009881) 0.08 0.08 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.07 0.08 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.03 0.06 0.07 0.10
R. typhi str. Wilmington (NC 006142) 0.09 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.09 0.08 0.08 0.09 0.08 0.08 0.11 0.12 0.13 0.03 0.10
R. bellii RML369-C (NC 007940) 0.17 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.17 0.16 0.16 0.18 0.16 0.16 0.17 0.15 0.16 0.17 0.17 0.16
120
Table A1.24. Rickettsia ompA Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 58 8651 8237 -4060 n/a 3.25 1.21 0.2949 0.2949 0.2051 0.2051 0.07 0.05 0.11 0.07 0.11 0.05 0.07 0.16 0.05 0.16 0.07 0.05
TN93+G 61 8656 8220 -4049 n/a 3.46 0.92 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.13 0.07 0.07 0.06 0.07 0.12 0.06 0.16 0.08 0.05
T92 57 8657 8250 -4068 n/a n/a 0.89 0.2949 0.2949 0.2051 0.2051 0.08 0.05 0.1 0.08 0.1 0.05 0.08 0.14 0.05 0.14 0.08 0.05
HKY+G 60 8666 8238 -4059 n/a 3.28 1.21 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.13 0.06 0.1 0.05 0.06 0.17 0.05 0.16 0.07 0.04
HKY 59 8669 8248 -4065 n/a n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.11 0.08 0.09 0.06 0.08 0.14 0.06 0.14 0.08 0.05
T92+I 58 8671 8257 -4070 0.05 n/a 1.19 0.2949 0.2949 0.2051 0.2051 0.07 0.05 0.11 0.07 0.11 0.05 0.07 0.16 0.05 0.16 0.07 0.05
T92+G+I 59 8671 8250 -4066 0 3.08 1.35 0.2949 0.2949 0.2051 0.2051 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.17 0.04 0.17 0.06 0.04
TN93 60 8674 8246 -4063 n/a n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.12 0.08 0.08 0.06 0.08 0.13 0.06 0.15 0.08 0.05
TN93+I 61 8675 8240 -4059 0.05 n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.12 0.07 0.08 0.06 0.07 0.12 0.06 0.16 0.08 0.05
GTR+G 64 8677 8220 -4046 n/a 3.53 0.92 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.13 0.07 0.07 0.05 0.09 0.12 0.06 0.16 0.07 0.05
TN93+G+I 62 8679 8237 -4056 0 3.35 1.15 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.12 0.07 0.1 0.05 0.07 0.16 0.05 0.16 0.07 0.04
HKY+I 60 8684 8256 -4067 0.05 n/a 1.19 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.12 0.06 0.1 0.05 0.06 0.17 0.05 0.16 0.07 0.04
HKY+G+I 61 8684 8249 -4063 0 3.11 1.35 0.2877 0.3021 0.1835 0.2267 0.06 0.04 0.13 0.06 0.11 0.05 0.06 0.18 0.05 0.17 0.06 0.04
GTR+G+I 65 8686 8222 -4046 0 3.52 0.92 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.13 0.07 0.07 0.05 0.09 0.12 0.06 0.16 0.07 0.05
GTR 63 8693 8244 -4058 n/a n/a 0.88 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.12 0.06 0.08 0.05 0.09 0.13 0.07 0.15 0.07 0.05
GTR+I 64 8696 8239 -4055 0.05 n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.12 0.07 0.08 0.05 0.09 0.12 0.07 0.15 0.07 0.05
K2+G 57 8756 8349 -4117 n/a 2.68 1.25 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
K2+G+I 58 8776 8362 -4123 0 2.53 1.39 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC+G 56 8776 8376 -4132 n/a 3.29 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
K2 56 8777 8377 -4132 n/a n/a 1.12 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06
K2+I 57 8782 8375 -4130 0.06 n/a 1.23 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
JC+G+I 57 8785 8378 -4132 0 3.29 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 56 8799 8399 -4143 0.06 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 55 8799 8407 -4148 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
121
Table A1.25. Rickettsia ompA genetic distance matrix.
R. m
ontane
sis OSU
85-
390
(CP00
3340
)
R. p
arke
ri str. Por
tsm
outh (N
C 017
044)
R. s
ibirica
246
(AABW
0100
0001
)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P0198
65)
R. a
frica
e E
SF-5 (C
P0016
12)
R. rhipice
phali
HJ 5 (C
P0131
33)
R. g
rave
sii
BWI-1
(DQ26
9437
)
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. m
assil
iae
str. AZT
80 (C
P0033
19)
R. m
onac
ensis
str. IrR
/Mun
ich (L
N79
4217
)
R. s
lova
ca is
olate Xin
jiang
(MF0
0253
5)
R. rao
ultii
str. Kha
baro
vsk (C
P1096
9)
Unc
ulture
d R. s
p. clone
ARRL2
016-
159 (M
N43
1847
)
R. p
eaco
cki
(NC 012
730)
R. c
onor
ii str. M
alish
7 (A
E0069
14)
R. rick
ettsii
str. A
rizon
a (C
P0033
07)
R. h
onei
RB (N
Z AJT
T000
0000
0.1)
Sample SR
12 ex. Am
. albolim
batum
(PI 3
329)
R. b
ellii
RM
L369
-C (N
C 007
940)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. tam
urae
str. AT-
1 (A
F394
896)
R. a
mblyo
mm
atis
str. G
AT-30
V (CP00
3334
)
R. typ
hi st
r. W
ilmington
(NC-0
0614
2)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. felis
URRW
XCal2 (C
P0000
53)
R. a
kari
(NC 009
881)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. a
sem
bone
nsis
str. NM
RCii (
NZ JW
SW00
0000
00.1)
R. montanesis OSU 85-390 (CP003340)
R. parkeri str. Portsmouth (NC 017044) 0.10
R. sibirica 246 (AABW01000001) 0.12 0.04
R. heilongjiangensis Sendai-58 (AP019865) 0.10 0.07 0.08
R. africae ESF-5 (CP001612) 0.12 0.03 0.02 0.07
R. rhipicephali HJ 5 (CP013133) 0.08 0.08 0.10 0.09 0.10
R. gravesii BWI-1 (DQ269437) 0.05 0.06 0.09 0.09 0.09 0.04
R. aeschlimannii str. RH15 (HM050289) 0.07 0.08 0.10 0.10 0.10 0.04 0.04
R. massiliae str. AZT80 (CP003319) 0.07 0.07 0.09 0.09 0.09 0.02 0.03 0.03
R. monacensis str. IrR/Munich (LN794217) 0.12 0.20 0.22 0.20 0.22 0.17 0.14 0.16 0.16
R. slovaca isolate Xinjiang (MF002535) 0.10 0.03 0.02 0.07 0.01 0.09 0.07 0.09 0.08 0.20
R. raoultii str. Khabarovsk (CP10969) 0.06 0.07 0.08 0.08 0.09 0.04 0.04 0.04 0.03 0.16 0.07
Uncultured R. sp. clone ARRL2016-159 (MN431847) 0.08 0.08 0.11 0.11 0.10 0.06 0.03 0.06 0.05 0.18 0.09 0.05
R. peacocki (NC 012730) 0.15 0.10 0.09 0.13 0.08 0.15 0.13 0.14 0.14 0.24 0.07 0.15 0.15
R. conorii str. Malish 7 (AE006914) 0.11 0.05 0.04 0.07 0.04 0.09 0.07 0.09 0.08 0.18 0.03 0.07 0.09 0.07
R. rickettsii str. Arizona (CP003307) 0.11 0.05 0.03 0.07 0.03 0.10 0.09 0.10 0.09 0.23 0.03 0.09 0.10 0.10 0.04
R. honei RB (NZ AJTT00000000.1) 0.14 0.04 0.03 0.09 0.02 0.12 0.10 0.12 0.11 0.24 0.03 0.10 0.12 0.10 0.05 0.04
Sample SR12 ex. Am. albolimbatum (PI 3329) 0.05 0.06 0.09 0.09 0.09 0.03 0.01 0.04 0.03 0.15 0.07 0.03 0.02 0.13 0.07 0.09 0.10
R. bellii RML369-C (NC 007940) 0.74 0.81 0.89 0.86 0.88 0.73 0.70 0.76 0.70 0.94 0.86 0.75 0.78 0.90 0.88 0.91 0.97 0.73
R. japonica str. M14012 (NZ AP017596) 0.12 0.09 0.11 0.04 0.10 0.11 0.11 0.11 0.11 0.20 0.09 0.09 0.14 0.16 0.08 0.10 0.12 0.12 0.90
R. tamurae str. AT-1 (AF394896) 0.10 0.16 0.16 0.16 0.15 0.12 0.09 0.12 0.11 0.08 0.14 0.11 0.13 0.18 0.13 0.15 0.16 0.10 0.91 0.18
R. amblyommatis str. GAT-30V (CP003334) 0.07 0.07 0.08 0.10 0.08 0.04 0.04 0.05 0.04 0.15 0.06 0.04 0.06 0.11 0.08 0.07 0.10 0.03 0.77 0.12 0.09
R. typhi str. Wilmington (NC-006142) 0.53 0.61 0.60 0.65 0.56 0.54 0.51 0.45 0.49 0.67 0.54 0.51 0.50 0.56 0.56 0.56 0.64 0.47 1.45 0.67 0.63 0.41
R. prowazekii str. Madrid E (NC 000963) 0.81 0.86 0.88 0.87 0.86 0.74 0.79 0.75 0.72 0.95 0.86 0.79 0.82 0.97 0.83 0.86 0.91 0.78 1.62 0.92 0.93 0.85 1.02
R. australis str. Cutlack (NC 017058) 0.77 0.84 0.84 0.87 0.82 0.72 0.73 0.75 0.72 0.87 0.84 0.80 0.73 0.90 0.79 0.85 0.86 0.74 1.80 0.86 0.88 0.67 1.31 1.53
R. felis URRWXCal2 (CP000053) 0.66 0.66 0.72 0.74 0.71 0.65 0.66 0.63 0.61 0.80 0.68 0.68 0.63 0.77 0.67 0.72 0.78 0.64 1.13 0.82 0.74 0.60 1.10 1.42 0.98
R. akari (NC 009881) 0.78 0.76 0.76 0.89 0.79 0.76 0.75 0.74 0.71 0.98 0.77 0.77 0.73 0.82 0.78 0.82 0.86 0.74 1.42 0.99 0.86 0.66 1.12 1.65 1.06 0.12
R. helvetica C9P9 (CM001467) 0.55 0.52 0.50 0.53 0.51 0.53 0.51 0.51 0.53 0.59 0.51 0.49 0.55 0.50 0.47 0.46 0.56 0.50 1.69 0.57 0.55 0.50 1.29 1.16 1.71 1.36 1.75
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.57 0.57 0.61 0.65 0.62 0.58 0.60 0.57 0.59 0.64 0.62 0.54 0.67 0.69 0.61 0.57 0.68 0.59 1.77 0.69 0.65 0.56 1.59 1.69 1.70 1.52 1.57 0.29
122
Table A1.26. Rickettsia ompB Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 66 3254 2791 -1329 n/a 1.24 1.71 0.304 0.304 0.196 0.196 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.19 0.04 0.19 0.05 0.04
T92+G+I 67 3261 2792 -1328 0 1.22 1.71 0.304 0.304 0.196 0.196 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.2 0.04 0.2 0.05 0.04
T92 65 3265 2809 -1339 n/a n/a 1.42 0.304 0.304 0.196 0.196 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04
T92+I 66 3266 2803 -1335 0.1789 n/a 1.58 0.304 0.304 0.196 0.196 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.19 0.04 0.19 0.06 0.04
HKY+G 68 3272 2795 -1329 n/a 1.24 1.71 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.19 0.04 0.2 0.05 0.03
K2+G 65 3275 2819 -1344 n/a 1.17 1.64 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
TN93+G 69 3279 2796 -1328 n/a 1.26 1.7 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.2 0.04 0.19 0.05 0.03
HKY+G+I 69 3280 2796 -1328 1E-05 1.24 1.71 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.19 0.04 0.2 0.05 0.03
HKY 67 3282 2812 -1339 n/a n/a 1.42 0.305 0.303 0.183 0.21 0.06 0.04 0.13 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04
HKY+I 68 3283 2806 -1335 0.1777 n/a 1.59 0.305 0.303 0.183 0.21 0.06 0.03 0.13 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.03
K2+G+I 66 3283 2821 -1344 0 1.15 1.65 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
K2+I 65 3287 2831 -1350 0.1962 n/a 1.54 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
K2 64 3288 2839 -1355 n/a n/a 1.38 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05
TN93+G+I 70 3288 2798 -1328 1E-05 1.23 1.72 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.21 0.04 0.19 0.05 0.03
GTR+G 72 3290 2785 -1320 n/a 1.22 1.81 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.21 0.02 0.19 0.09 0.02
TN93 68 3291 2815 -1339 n/a n/a 1.42 0.305 0.303 0.183 0.21 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.2 0.04 0.17 0.06 0.04
TN93+I 69 3292 2808 -1335 0.1765 n/a 1.59 0.305 0.303 0.183 0.21 0.06 0.03 0.13 0.06 0.12 0.04 0.06 0.2 0.04 0.18 0.06 0.03
GTR+G+I 73 3299 2787 -1320 0 1.18 1.83 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.21 0.02 0.19 0.09 0.02
GTR+I 72 3301 2796 -1326 0.1924 n/a 1.72 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.19 0.02 0.19 0.1 0.02
GTR 71 3314 2816 -1336 n/a n/a 1.13 0.305 0.303 0.183 0.21 0.05 0.05 0.12 0.05 0.09 0.06 0.09 0.16 0.04 0.17 0.09 0.03
JC+G 64 3317 2868 -1370 n/a 1.52 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 63 3324 2882 -1378 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 65 3325 2869 -1369 1E-05 1.52 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 64 3327 2879 -1375 0.1573 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
123
Table A1.27. Rickettsia ompB genetic distance matrix.
Sample R52
ex. A a
lbolim
batum
(PI 6
80)
Sample R92
ex. A a
lbolim
batum
(PI 3
323)
Sample R10
6 ex
. A a
lbolim
batum
(P
I 146
0)
Sample SR
12 ex. A a
lbolim
batum
(PI 3
329)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. a
frica
e E
SF-5 (C
P0016
12)
R. a
mblyo
mm
atis
str. G
AT-30
V (CP00
3334
)
R. a
kari
Har
tford
(NC 009
881)
R. a
ustra
lis str. C
utlack
(NC 017
058)
R. felis
URRW
XCal2 (C
P0000
53)
R. a
sem
bone
nsis
str. NM
RCii (
NZ JW
SW00
0000
00.1)
R. c
onor
ii str. M
alish 7 (A
E0069
14.1)
R. g
rave
sii
BWI-1
(DQ26
9436
)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P0198
65)
R. h
onei R
B (NZ
AJTT0
0000
000.1)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. m
assil
iae
str. A
ZT80
(CP00
3319
)
R. m
onac
ensis
str. IrR
/Mun
ich (L
N79
4217
)
R. m
ontane
nsis
OSU
85-
390 (C
P0033
40)
R. p
arke
ri str. Por
tsm
outh (N
C 017
044)
R. rao
ultii
str. K
haba
rovs
k (C
P1096
9)
R. rhipice
phali
HJ 5 (C
P0131
33)
R. rick
ettsii
str. Ariz
ona (C
P0033
07)
R. s
ibirica
24
6 (A
ABW01
0000
01)
R. s
lova
ca iso
late X
injia
ng (M
F002
535)
R. tam
urae
str. A
T-1
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. typ
hi str. W
ilmington
(NC-0
0614
2)
Sample R53
ex. A. a
lbolim
batum
(PI 6
80)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample R52 ex. A albolimbatum (PI 680)
Sample R92 ex. A albolimbatum (PI 3323) 0.03
Sample R106 ex. A albolimbatum (PI 1460) 0.00 0.03
Sample SR12 ex. A albolimbatum (PI 3329) 0.04 0.00 0.04
R. helvetica C9P9 (CM001467) 0.03 0.04 0.03 0.04
R. africae ESF-5 (CP001612) 0.02 0.02 0.02 0.03 0.02
R. amblyommatis str. GAT-30V (CP003334) 0.04 0.02 0.04 0.03 0.04 0.02
R. akari Hartford (NC 009881) 0.10 0.10 0.10 0.09 0.08 0.08 0.09
R. australis str. Cutlack (NC 017058) 0.07 0.07 0.07 0.07 0.05 0.05 0.07 0.06
R. felis URRWXCal2 (CP000053) 0.05 0.04 0.05 0.04 0.03 0.03 0.03 0.05 0.03
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.05 0.04 0.05 0.05 0.04 0.04 0.05 0.07 0.04 0.01
R. conorii str. Malish 7 (AE006914.1) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04
R. gravesii BWI-1 (DQ269436) 0.03 0.01 0.03 0.02 0.03 0.02 0.02 0.09 0.06 0.03 0.04 0.02
R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01
R. honei RB (NZ AJTT00000000.1) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01
R. japonica str. M14012 (NZ AP017596) 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02
R. massiliae str. AZT80 (CP003319) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01 0.00 0.01 0.01
R. monacensis str. IrR/Munich (LN794217) 0.01 0.02 0.01 0.03 0.03 0.02 0.03 0.10 0.07 0.04 0.04 0.02 0.02 0.01 0.02 0.02 0.01
R. montanensis OSU 85-390 (CP003340) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01 0.00 0.01 0.01 0.00 0.01
R. parkeri str. Portsmouth (NC 017044) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01
R. raoultii str. Khabarovsk (CP10969) 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02
R. rhipicephali HJ 5 (CP013133) 0.03 0.02 0.03 0.02 0.03 0.01 0.02 0.09 0.06 0.03 0.03 0.01 0.01 0.00 0.01 0.01 0.00 0.02 0.00 0.01 0.01
R. rickettsii str. Arizona (CP003307) 0.02 0.02 0.02 0.03 0.03 0.00 0.03 0.09 0.06 0.04 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.02
R. sibirica 246 (AABW01000001) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.01 0.00
R. slovaca isolate Xinjiang (MF002535) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.01 0.00 0.00
R. tamurae str. AT-1 (AF394896) 0.02 0.04 0.02 0.04 0.03 0.03 0.03 0.11 0.07 0.05 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.01 0.03 0.03 0.03 0.03 0.04 0.03 0.03
R. aeschlimannii str. RH15 (HM050289) 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.01 0.01 0.02 0.02 0.03
R. prowazekii str. Madrid E (NC 000963) 0.13 0.15 0.13 0.16 0.14 0.13 0.14 0.20 0.17 0.14 0.16 0.13 0.15 0.14 0.13 0.15 0.14 0.13 0.14 0.13 0.15 0.14 0.13 0.13 0.13 0.14 0.13
R. typhi str. Wilmington (NC-006142) 0.12 0.13 0.12 0.14 0.10 0.11 0.12 0.12 0.11 0.09 0.11 0.11 0.13 0.11 0.11 0.13 0.11 0.12 0.11 0.11 0.13 0.11 0.12 0.11 0.11 0.12 0.12 0.08
Sample R53 ex. A. albolimbatum (PI 680) 0.03 0 0.03 0.00 0.04 0.02 0.02 0.10 0.07 0.04 0.04 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.15 0.13
R. bellii RML369-C (NC 007940) 0.36 0.36 0.36 0.36 0.37 0.37 0.35 0.38 0.36 0.33 0.36 0.37 0.35 0.35 0.37 0.37 0.35 0.36 0.35 0.37 0.37 0.34 0.36 0.37 0.37 0.38 0.33 0.41 0.38 0.36
124
Table A1.28. Rickettsia 17 kDa Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 76 4395 3834 -1840 n/a 0.81 2.24 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.19 0.04 0.19 0.04 0.04
K2+G 75 4400 3846 -1848 n/a 0.81 2.23 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
T92+G+I 77 4405 3836 -1840 0 0.8 2.24 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.19 0.04 0.19 0.04 0.04
K2+G+I 76 4410 3848 -1848 0 0.83 2.21 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
TN93+G 79 4411 3827 -1834 n/a 0.97 2.21 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.18 0.04 0.05 0.24 0.04 0.14 0.04 0.03
HKY+G 78 4414 3838 -1841 n/a 0.78 2.33 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.04 0.05 0.17 0.04 0.2 0.04 0.03
TN93+G+I 80 4421 3830 -1834 0 0.95 2.21 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.18 0.04 0.05 0.24 0.04 0.14 0.04 0.03
HKY+G+I 79 4424 3840 -1841 0 0.79 2.32 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.04 0.05 0.17 0.04 0.2 0.04 0.03
T92+I 76 4427 3866 -1856 0.2 n/a 2.03 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.18 0.04 0.18 0.04 0.04
K2+I 75 4432 3877 -1863 0.19 n/a 2.02 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
T92 75 4434 3880 -1864 n/a n/a 1.8 0.269 0.269 0.231 0.231 0.05 0.04 0.15 0.05 0.15 0.04 0.05 0.17 0.04 0.17 0.05 0.04
GTR+G 82 4434 3828 -1832 n/a 1.02 1.6 0.295 0.243 0.18 0.282 0.05 0.05 0.14 0.06 0.14 0.04 0.08 0.19 0.04 0.15 0.04 0.03
K2 74 4438 3891 -1871 n/a n/a 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04
TN93+I 79 4439 3855 -1848 0.16 n/a 2.01 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.17 0.05 0.05 0.23 0.05 0.14 0.04 0.03
GTR+G+I 83 4440 3827 -1830 0 1 1.66 0.295 0.243 0.18 0.282 0.05 0.05 0.14 0.06 0.14 0.04 0.08 0.19 0.04 0.15 0.03 0.03
TN93 78 4440 3864 -1854 n/a n/a 1.85 0.295 0.243 0.18 0.282 0.04 0.03 0.12 0.05 0.17 0.05 0.05 0.23 0.05 0.13 0.04 0.03
HKY+I 78 4445 3869 -1856 0.2 n/a 2.1 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.05 0.05 0.16 0.05 0.2 0.04 0.03
HKY 77 4453 3884 -1865 n/a n/a 1.81 0.295 0.243 0.18 0.282 0.04 0.03 0.18 0.05 0.11 0.05 0.05 0.15 0.05 0.19 0.04 0.03
GTR+I 82 4460 3854 -1845 0.16 n/a 1.52 0.295 0.243 0.18 0.282 0.05 0.05 0.13 0.06 0.14 0.04 0.08 0.19 0.04 0.14 0.04 0.03
GTR 81 4463 3865 -1851 n/a n/a 1.39 0.295 0.243 0.18 0.282 0.05 0.05 0.13 0.06 0.14 0.05 0.08 0.18 0.05 0.13 0.04 0.03
JC+G 74 4501 3955 -1903 n/a 0.88 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 75 4511 3957 -1903 0 0.89 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 74 4529 3982 -1917 0.17 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 73 4529 3990 -1922 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
125
Table A1.29. Rickettsia 17 kDa genetic distance matrix.
Sample SR
16 ex. A. a
lbolim
batum
(P
I 274
5)
Sample SR
15 ex. A
. albolim
batum
(PI 1
723)
Sample SR
14 ex. A. a
lbolim
batum
(PI 1
316)
Sample SR
12 ex. A. a
lbolim
batum
(PI 3
329)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample R92
ex.
A. a
lbolim
batum
(PI 3
323)
Sample R8 ex
. A. a
lbolim
batum
(PI 1
725)
Sample R10
6 ex
. A. a
lbolim
batum
(PI 1
460)
Sample R52
ex. A. a
lbolim
batum
(PI 6
80)
Unc
ulture
d R. s
p. clone
ARRL2
016-
156 (M
N43
1838
)
Sample R53
ex. A. a
lbolim
batum
(PI 6
80)
R. typ
hi st
r. W
ilmington
(NC-0
0614
2)
R. tam
urae
str. AT-
1 (A
F394
896)
R. s
lova
ca is
olate Xin
jiang
(MF0
0253
5)
R. s
ibirica
246
(AABW
0100
0001
)
R. rick
ettsii
str. A
rizon
a (C
P0033
07)
R. rhipice
phali
HJ 5 (C
P0131
33)
R. rao
ultii
str. Kha
baro
vsk (C
P1096
9)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. p
arke
ri str. Por
tsm
outh (N
C 017
044)
R. m
ontane
sis
OSU
85-
390 (C
P0033
40)
R. m
onac
ensis
str. IrR
/Mun
ich (L
N79
4217
)
R. m
assil
iae
str. AZT
80 (C
P0033
19)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. h
onei
RB (N
Z AJT
T000
0000
0.1)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P0198
65)
R. g
rave
sii
BWI-1
(DQ26
9437
)
R. felis
URRW
XCal2 (C
P0000
53)
R. c
onor
ii str. M
alish
7 (A
E0069
14)
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. a
sem
bone
nsis
str. NM
RCii (
NZ JW
SW00
0000
00.1)
R. a
mblyo
mm
atis
str. G
AT-30
V (CP00
3334
)
R. a
frica
e E
SF-5 (C
P0016
12)
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. a
kari
str.
Har
tford
(NC 009
881)
Unc
ulture
d R. s
p. clone
ARRL2
016-
159 (M
N43
1849
)
Unc
ulture
d R. s
p. clone
ARRL2
016-
149 (M
N43
1837
)
Sample SR16 ex. A. albolimbatum (PI 2745)
Sample SR15 ex. A. albolimbatum (PI 1723) 0.00
Sample SR14 ex. A. albolimbatum (PI 1316) 0.00 0.00
Sample SR12 ex. A. albolimbatum (PI 3329) 0.00 0.00 0.00
R. bellii RML369-C (NC 007940) 0.08 0.08 0.08 0.08
Sample R92 ex. A. albolimbatum (PI 3323) 0.31 0.31 0.31 0.31 0.30
Sample R8 ex. A. albolimbatum (PI 1725) 0.31 0.31 0.31 0.31 0.30 0.00
Sample R106 ex. A. albolimbatum (PI 1460) 0.34 0.34 0.34 0.34 0.31 0.06 0.06
Sample R52 ex. A. albolimbatum (PI 680) 0.34 0.34 0.34 0.34 0.31 0.06 0.06 0.00
Uncultured R. sp. clone ARRL2016-156 (MN431838) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06
Sample R53 ex. A. albolimbatum (PI 680) 0.00 0.00 0.00 0.00 0.08 0.31 0.31 0.34 0.34 0.31
R. typhi str. Wilmington (NC-006142) 0.37 0.37 0.37 0.37 0.32 0.12 0.12 0.11 0.11 0.12 0.37
R. tamurae str. AT-1 (AF394896) 0.33 0.33 0.33 0.33 0.30 0.05 0.05 0.01 0.01 0.05 0.33 0.11
R. slovaca isolate Xinjiang (MF002535) 0.33 0.33 0.33 0.33 0.30 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06
R. sibirica 246 (AABW01000001) 0.33 0.33 0.33 0.33 0.30 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06 0.01
R. rickettsii str. Arizona (CP003307) 0.32 0.32 0.32 0.32 0.28 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01
R. rhipicephali HJ 5 (CP013133) 0.31 0.31 0.31 0.31 0.30 0.01 0.01 0.05 0.05 0.01 0.31 0.12 0.05 0.02 0.02 0.02
R. raoultii str. Khabarovsk (CP10969) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01
R. prowazekii str. Madrid E (NC 000963) 0.40 0.40 0.40 0.40 0.35 0.15 0.15 0.14 0.14 0.15 0.40 0.05 0.13 0.16 0.16 0.16 0.14 0.15
R. parkeri str. Portsmouth (NC 017044) 0.32 0.32 0.32 0.32 0.29 0.01 0.01 0.06 0.06 0.01 0.32 0.13 0.05 0.00 0.00 0.00 0.02 0.01 0.16
R. montanesis OSU 85-390 (CP003340) 0.32 0.32 0.32 0.32 0.30 0.01 0.01 0.06 0.06 0.01 0.32 0.12 0.05 0.01 0.01 0.01 0.02 0.01 0.15 0.01
R. monacensis str. IrR/Munich (LN794217) 0.33 0.33 0.33 0.33 0.30 0.05 0.05 0.01 0.01 0.05 0.33 0.11 0.00 0.06 0.06 0.06 0.05 0.05 0.13 0.05 0.05
R. massiliae str. AZT80 (CP003319) 0.32 0.32 0.32 0.32 0.31 0.01 0.01 0.06 0.06 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.01 0.01 0.16 0.02 0.02 0.06
R. japonica str. M14012 (NZ AP017596) 0.32 0.32 0.32 0.32 0.30 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01 0.01 0.02 0.01 0.16 0.01 0.02 0.06 0.02
R. honei RB (NZ AJTT00000000.1) 0.32 0.32 0.32 0.32 0.30 0.00 0.00 0.06 0.06 0.00 0.32 0.13 0.05 0.01 0.01 0.01 0.02 0.00 0.15 0.00 0.01 0.05 0.02 0.01
R. heilongjiangensis Sendai-58 (AP019865) 0.33 0.33 0.33 0.33 0.31 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06 0.02 0.02 0.02 0.03 0.01 0.17 0.01 0.02 0.06 0.03 0.01 0.01
R. gravesii BWI-1 (DQ269437) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01
R. felis URRWXCal2 (CP000053) 0.34 0.34 0.34 0.34 0.33 0.07 0.07 0.08 0.08 0.07 0.34 0.14 0.07 0.08 0.07 0.08 0.07 0.07 0.16 0.08 0.08 0.07 0.09 0.08 0.07 0.08 0.07
R. conorii str. Malish 7 (AE006914) 0.32 0.32 0.32 0.32 0.28 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01 0.00 0.02 0.01 0.16 0.00 0.01 0.06 0.02 0.01 0.01 0.02 0.01 0.08
R. australis str. Cutlack (NC 017058) 0.32 0.32 0.32 0.32 0.30 0.03 0.03 0.06 0.06 0.03 0.32 0.12 0.06 0.04 0.04 0.04 0.04 0.03 0.15 0.04 0.04 0.06 0.05 0.04 0.04 0.05 0.03 0.06 0.04
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.31 0.31 0.31 0.31 0.29 0.05 0.05 0.03 0.03 0.05 0.31 0.12 0.03 0.06 0.06 0.05 0.05 0.05 0.14 0.05 0.05 0.03 0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.04
R. amblyommatis str. GAT-30V (CP003334) 0.32 0.32 0.32 0.32 0.32 0.01 0.01 0.08 0.08 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.03 0.01 0.16 0.02 0.02 0.06 0.03 0.03 0.02 0.03 0.01 0.08 0.02 0.04 0.06
R. africae ESF-5 (CP001612) 0.32 0.32 0.32 0.32 0.29 0.01 0.01 0.07 0.07 0.01 0.32 0.13 0.06 0.01 0.01 0.01 0.03 0.01 0.16 0.01 0.02 0.06 0.03 0.02 0.01 0.02 0.01 0.09 0.01 0.05 0.06 0.02
R. aeschlimannii str. RH15 (HM050289) 0.32 0.32 0.32 0.32 0.32 0.01 0.01 0.06 0.06 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.02 0.01 0.15 0.02 0.02 0.06 0.02 0.03 0.02 0.03 0.01 0.08 0.02 0.04 0.06 0.03 0.03
R. helvetica C9P9 (CM001467) 0.34 0.34 0.34 0.34 0.30 0.04 0.04 0.06 0.06 0.04 0.34 0.12 0.06 0.05 0.05 0.05 0.05 0.04 0.14 0.05 0.05 0.06 0.06 0.05 0.04 0.05 0.04 0.09 0.05 0.06 0.06 0.06 0.05 0.06
R. akari str. Hartford (NC 009881) 0.34 0.34 0.34 0.34 0.32 0.05 0.05 0.07 0.07 0.05 0.34 0.13 0.07 0.06 0.06 0.06 0.06 0.05 0.16 0.06 0.06 0.07 0.07 0.06 0.05 0.06 0.05 0.08 0.06 0.03 0.06 0.07 0.06 0.06 0.06
Uncultured R. sp. clone ARRL2016-159 (MN431849) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01 0.00 0.07 0.01 0.03 0.05 0.01 0.01 0.01 0.04 0.05
Uncultured R. sp. clone ARRL2016-149 (MN431837) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01 0.00 0.07 0.01 0.03 0.05 0.01 0.01 0.01 0.04 0.05 0.00
126
Table A1.30. Rickettsia sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
GTR+G+I 73 8594 8004 -3929 0.16 1.725 1.192 0.38 0.261 0.18 0.179 0.04 0.07 0.1 0.06 0.11 0.05 0.14 0.15 0.01 0.2 0.07 0.01
GTR+G 72 8604 8022 -3939 n/a 1.001 1.08 0.38 0.261 0.18 0.179 0.04 0.07 0.1 0.06 0.09 0.05 0.14 0.14 0.03 0.2 0.07 0.03
HKY+G 68 8607 8057 -3960 n/a 0.893 1.407 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04
TN93+G 69 8609 8051 -3956 n/a 0.919 1.391 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.18 0.04 0.2 0.05 0.04
GTR+I 72 8610 8027 -3941 0.312 n/a 1.057 0.38 0.261 0.18 0.179 0.04 0.06 0.1 0.06 0.09 0.05 0.14 0.14 0.03 0.2 0.07 0.03
HKY+I 68 8610 8060 -3962 0.33 n/a 1.411 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04
HKY+G+I 69 8613 8055 -3958 0.177 1.719 1.419 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04
TN93+I 69 8614 8056 -3959 0.327 n/a 1.402 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.19 0.04 0.19 0.05 0.04
TN93+G+I 70 8618 8052 -3956 0.2 2.048 1.404 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.18 0.04 0.2 0.05 0.04
T92+G 66 8620 8086 -3977 n/a 0.921 1.389 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04
T92+I 66 8624 8090 -3979 0.326 n/a 1.397 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04
T92+G+I 67 8626 8084 -3975 0.146 1.531 1.398 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04
GTR 71 8659 8085 -3971 n/a n/a 1.106 0.38 0.261 0.18 0.179 0.03 0.07 0.09 0.04 0.11 0.05 0.14 0.16 0.04 0.18 0.07 0.04
HKY 67 8677 8136 -4001 n/a n/a 1.219 0.38 0.261 0.18 0.179 0.06 0.04 0.1 0.08 0.1 0.04 0.08 0.15 0.04 0.22 0.06 0.04
TN93 68 8680 8130 -3997 n/a n/a 1.225 0.38 0.261 0.18 0.179 0.06 0.04 0.09 0.08 0.12 0.04 0.08 0.18 0.04 0.18 0.06 0.04
T92 65 8687 8161 -4015 n/a n/a 1.231 0.32 0.32 0.18 0.18 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.18 0.04 0.18 0.07 0.04
K2+G 65 8738 8213 -4041 n/a 0.846 1.368 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05
K2+G+I 66 8745 8211 -4039 1E-05 0.843 1.369 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05
K2+I 65 8748 8222 -4046 0.339 n/a 1.355 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05
K2 64 8815 8297 -4084 n/a n/a 1.191 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
JC+G 64 8842 8324 -4098 n/a 0.994 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 65 8848 8323 -4096 6E-06 0.991 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 64 8851 8333 -4102 0.313 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 63 8903 8393 -4134 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
127
Table A1.31. Rickettsia sca4 genetic distance matrix.
Sample R53 ex. A
. albolim
batum (PI 6
80)
Sample R66 ex. A
. albolim
batum (PI 1
320)
Sample R92 ex. A
. albolim
batum (PI 3
323)
R. aeschlim
annii str.
RH15 (HM050275)
R. afric
ae ESF-5 (CP001612)
R. akari H
artford (N
C 009881)
R. amblyommatis
str. GAT-30V (C
P003334)
R. asembonensis s
tr. NMRCii (
NZ JWSW00000000.1)
R. austra
lis str. Cutla
ck (NC 017058)
R. prowazekii s
tr. Madrid
E (NC 000963)
R. typhi s
t Wilm
ington (NC 006142)
R. conorii
str. Malish 7 (A
E006914.1)
R. felis U
RRWXCal2 (CP000053)
R. gravesii B
WI-1 (D
Q269439)
R. heilo
ngjiangensis Sendai-5
8 (AP019865)
R. honei R
B (NZ AJTT00000000.1)
R. japonica str.
M14012 (NZ AP017596)
R. massilia
e str. AZT80 (C
P003319)
R. monacensis s
tr. IrR
/Munich (L
N794217)
R. montanensis O
SU 85-930 (CP003340)
R. parkeri s
tr. Ports
mouth (NC 017044)
R. peacockii s
tr. Rustic
(NC_012730)
R. raoultii
str. Khabarovsk (C
P10969)
R. rhipicephali H
J 5 (CP013133)
R. ricketts
ii str.
Arizona (C
P003307)
R. sibiric
a 246 (AABW01000001)
R. slovaca is
olate Xinjiang (M
F002531)
R. tamurae s
tr. AT-1 (D
Q113911)
R. helvetic
a C9P9 (C
M001467)
Uncultured R
. sp. c
lone ARRL2016-149 (MN431843)
Uncultured R
. sp. c
lone ARRL2016-156 (MN431844)
Uncultured R
. sp. c
lone ARRL2016-159 (MN431845)
R. bellii
RML369-C (N
C 007940)
Sample R53 ex. A. albolimbatum (PI 680)
Sample R66 ex. A. albolimbatum (PI 1320) 0.00
Sample R92 ex. A. albolimbatum (PI 3323) 0.00 0.00
R. aeschlimannii str. RH15 (HM050275) 0.03 0.03 0.03
R. africae ESF-5 (CP001612) 0.03 0.03 0.03 0.02
R. akari Hartford (NC 009881) 0.15 0.15 0.15 0.15 0.15
R. amblyommatis str. GAT-30V (CP003334) 0.03 0.03 0.03 0.02 0.02 0.14
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.13 0.13 0.13 0.12 0.12 0.07 0.12
R. australis str. Cutlack (NC 017058) 0.14 0.14 0.14 0.13 0.13 0.05 0.12 0.05
R. prowazekii str. Madrid E (NC 000963) 0.28 0.28 0.28 0.27 0.28 0.32 0.28 0.29 0.30
R. typhi str. Wilmington (NC 006142) 0.29 0.29 0.29 0.28 0.29 0.31 0.28 0.28 0.30 0.07
R. conorii str. Malish 7 (AE006914.1) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28
R. felis URRWXCal2 (CP000053) 0.11 0.11 0.11 0.10 0.10 0.08 0.10 0.04 0.07 0.29 0.29 0.11
R. gravesii BWI-1 (DQ269439) 0.02 0.02 0.02 0.03 0.03 0.15 0.03 0.14 0.14 0.28 0.28 0.03 0.12
R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.02 0.03 0.02 0.01 0.14 0.02 0.12 0.13 0.27 0.27 0.01 0.10 0.02
R. honei RB (NZ AJTT00000000.1) 0.03 0.03 0.03 0.02 0.01 0.14 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01
R. japonica str. M14012 (NZ AP017596) 0.03 0.03 0.03 0.02 0.02 0.15 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.01
R. massiliae str. AZT80 (CP003319) 0.03 0.03 0.03 0.01 0.02 0.15 0.02 0.12 0.13 0.28 0.28 0.02 0.10 0.03 0.01 0.02 0.02
R. monacensis str. IrR/Munich (LN794217) 0.06 0.06 0.06 0.05 0.05 0.12 0.04 0.10 0.10 0.28 0.29 0.05 0.11 0.06 0.04 0.05 0.05 0.05
R. montanensis OSU 85-930 (CP003340) 0.03 0.03 0.03 0.02 0.02 0.15 0.02 0.12 0.13 0.28 0.28 0.02 0.10 0.03 0.02 0.02 0.02 0.02 0.05
R. parkeri str. Portsmouth (NC 017044) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.27 0.28 0.01 0.10 0.03 0.01 0.01 0.02 0.02 0.05 0.02
R. peacockii str. Rustic (NC_012730) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.13 0.14 0.27 0.28 0.01 0.11 0.03 0.01 0.01 0.02 0.02 0.05 0.02 0.01
R. raoultii str. Khabarovsk (CP10969) 0.03 0.03 0.03 0.02 0.02 0.14 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.02 0.02 0.02 0.05 0.02 0.02 0.02
R. rhipicephali HJ 5 (CP013133) 0.03 0.03 0.03 0.01 0.02 0.15 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.02 0.02 0.01 0.05 0.02 0.02 0.02 0.02
R. rickettsii str. Arizona (CP003307) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01 0.01 0.01 0.02 0.05 0.02 0.01 0.01 0.02 0.02
R. sibirica 246 (AABW01000001) 0.03 0.03 0.03 0.02 0.01 0.15 0.03 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.02 0.01 0.02 0.02 0.05 0.03 0.00 0.01 0.02 0.03 0.01
R. slovaca isolate Xinjiang (MF002531) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01 0.00 0.01 0.02 0.05 0.02 0.01 0.00 0.02 0.02 0.01 0.01
R. tamurae str. AT-1 (DQ113911) 0.04 0.04 0.05 0.03 0.02 0.15 0.04 0.13 0.13 0.29 0.30 0.02 0.11 0.04 0.03 0.02 0.03 0.03 0.06 0.04 0.02 0.02 0.03 0.03 0.02 0.02 0.02
R. helvetica C9P9 (CM001467) 0.09 0.09 0.09 0.09 0.09 0.13 0.08 0.10 0.11 0.28 0.28 0.08 0.09 0.09 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.09 0.08 0.09 0.09 0.09 0.08 0.10
Uncultured R. sp. clone ARRL2016-149 (MN431843) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.28 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09
Uncultured R. sp. clone ARRL2016-156 (MN431844) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.29 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09 0.00
Uncultured R. sp. clone ARRL2016-159 (MN431845) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.28 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09 0.00 0.00
R. bellii RML369-C (NC 007940) 0.61 0.61 0.61 0.60 0.62 0.71 0.61 0.69 0.71 0.75 0.71 0.61 0.67 0.63 0.61 0.61 0.61 0.59 0.63 0.60 0.62 0.61 0.60 0.59 0.62 0.62 0.61 0.63 0.66 0.61 0.62 0.61
128
Table A1.32. Rickettsia concatenated 17 kDa and gltA (short) Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 62 5841 5352 -2614 n/a 0.304 2.764 0.304 0.304 0.196 0.196 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.23 0.03 0.23 0.04 0.03
T92+G+I 63 5850 5352 -2613 0.253 0.508 2.778 0.304 0.304 0.196 0.196 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.23 0.03 0.23 0.04 0.03
TN93+G 65 5850 5337 -2603 n/a 0.321 2.744 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.03 0.12 0.02 0.03 0.17 0.02 0.29 0.04 0.03
HKY+G 64 5858 5352 -2612 n/a 0.298 2.883 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.03 0.17 0.02 0.03 0.25 0.02 0.2 0.04 0.03
TN93+G+I 66 5859 5338 -2603 0.247 0.536 2.757 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.03 0.12 0.02 0.03 0.17 0.02 0.29 0.04 0.03
T92+I 62 5865 5375 -2626 0.598 n/a 2.672 0.304 0.304 0.196 0.196 0.04 0.03 0.14 0.04 0.14 0.03 0.04 0.22 0.03 0.22 0.04 0.03
HKY+G+I 65 5866 5353 -2611 0.253 0.497 2.897 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.03 0.17 0.02 0.03 0.25 0.02 0.2 0.04 0.03
GTR+G 68 5871 5334 -2599 n/a 0.325 2.56 0.27 0.338 0.232 0.16 0.03 0.02 0.16 0.03 0.12 0.04 0.03 0.17 0.02 0.27 0.08 0.03
TN93+I 65 5872 5358 -2614 0.587 n/a 2.655 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.04 0.12 0.02 0.04 0.17 0.02 0.28 0.04 0.03
GTR+G+I 69 5876 5332 -2597 0.254 0.551 2.79 0.27 0.338 0.232 0.16 0.03 0.03 0.17 0.03 0.12 0.04 0.03 0.17 0.01 0.29 0.08 0.02
HKY+I 64 5882 5377 -2624 0.601 n/a 2.756 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.04 0.17 0.02 0.04 0.25 0.02 0.2 0.04 0.03
K2+G 61 5887 5406 -2642 n/a 0.296 2.768 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
GTR+I 68 5895 5358 -2611 0.583 n/a 2.385 0.27 0.338 0.232 0.16 0.03 0.03 0.16 0.03 0.12 0.04 0.03 0.17 0.02 0.27 0.08 0.03
K2+G+I 62 5895 5406 -2641 0.265 0.506 2.784 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
K2+I 61 5912 5430 -2654 0.606 n/a 2.681 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03
TN93 64 6012 5507 -2689 n/a n/a 2.338 0.27 0.338 0.232 0.16 0.05 0.03 0.16 0.04 0.11 0.02 0.04 0.16 0.02 0.28 0.05 0.03
T92 61 6012 5531 -2704 n/a n/a 2.329 0.304 0.304 0.196 0.196 0.04 0.03 0.14 0.04 0.14 0.03 0.04 0.22 0.03 0.22 0.04 0.03
HKY 63 6034 5536 -2705 n/a n/a 2.325 0.27 0.338 0.232 0.16 0.05 0.03 0.11 0.04 0.16 0.02 0.04 0.24 0.02 0.19 0.05 0.03
GTR 67 6034 5505 -2685 n/a n/a 2.137 0.27 0.338 0.232 0.16 0.04 0.03 0.15 0.03 0.11 0.04 0.03 0.16 0.02 0.26 0.08 0.04
K2 60 6062 5588 -2734 n/a n/a 2.315 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
JC+G 60 6072 5599 -2739 n/a 0.324 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 61 6081 5599 -2738 0.246 0.533 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 60 6094 5621 -2750 0.597 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 59 6232 5766 -2824 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
129
Table A1.33. Rickettsia concatenated 17 kDa and gltA (short) genetic distance matrix.
Sample
SR12
ex
Am. a
lbolim
batum
(PI 3
329)
Uncult
ured
R.sp. cl
one A
RRL2
016-1
49 (MN43
1847
)
R. aes
chlim
annii
str. R
H15 (H
M05
0289
)
R. afric
ae ESF-
5 (C
P0016
12)
R. aka
ri str.
hartfo
rd (NC 00
9881
)
R. ambly
ommati
s
str. G
AT-30
V (CP00
3334
)
R. ase
mbo
nens
is str
. NMRC
ii (NZ JW
SW00
0000
00.1)
R. aus
tralis
str
. Cutl
ack (
NC 017
058)
R. bell
ii RM
L369
-C (N
C 00
7940
)
R. felis
URRW
XCal2 (
CP0000
53)
R. grav
esii
BWI-1
(DQ26
9439
)
R. helv
etica
C9P
9 (C
M00
1467
)
R. hon
ei R
B (NZ AJ
TT00
0000
00.1)
R. japo
nica
str. M
1401
2 (NZ
AP01
7596
)
R. mas
siliae
str. A
ZT80
(CP00
3319
)
R. mon
acen
sis
str. Ir
R/Mun
ich (L
N7942
17)
R. park
eri
str. P
ortsm
outh
(NC 01
7044
)
R. pea
cock
i (N
C 0127
30)
R. prow
azek
ii str
. Mad
rid E
(NC 00
0963
)
R. raou
ltii
str. K
haba
rovs
k (CP1
0969
)
R. rick
ettsii
str. A
rizon
a (CP0
0330
7)
R. sibi
rica
246 (
AABW01
0000
01)
R. tamurae
str
. AT-
1 (AF3
9489
6)
R. con
orii
str. M
alish
7 (A
E006
914)
R. heil
ongjian
gens
is
Sen
dai-5
8 (AP0
1986
5)
R. typh
i str
. Wilm
ington
(NC-00
6142
)
R. mon
tanes
is OSU 85
-390
(CP0
0334
0)
R. slov
aca
isola
te Xinj
iang (
MF0
0253
5)
Sample SR12 ex Am. albolimbatum (PI 3329)
Uncultured R.sp. clone ARRL2016-149 (MN431847) 0.23
R. aeschlimannii str. RH15 (HM050289) 0.24 0.01
R. africae ESF-5 (CP001612) 0.25 0.01 0.01
R. akari str. hartford (NC 009881) 0.26 0.05 0.06 0.06
R. amblyommatis str. GAT-30V (CP003334) 0.24 0.01 0.02 0.01 0.06
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.25 0.05 0.06 0.06 0.06 0.06
R. australis str. Cutlack (NC 017058) 0.24 0.04 0.04 0.04 0.03 0.04 0.05
R. bellii RML369-C (NC 007940) 0.06 0.23 0.24 0.23 0.26 0.24 0.23 0.24
R. felis URRWXCal2 (CP000053) 0.26 0.05 0.06 0.06 0.06 0.06 0.05 0.05 0.26
R. gravesii BWI-1 (DQ269439) 0.24 0.01 0.01 0.01 0.06 0.02 0.06 0.04 0.24 0.06
R. helvetica C9P9 (CM001467) 0.27 0.05 0.05 0.05 0.06 0.06 0.06 0.06 0.24 0.06 0.05
R. honei RB (NZ AJTT00000000.1) 0.24 0.00 0.01 0.00 0.05 0.01 0.05 0.04 0.24 0.06 0.01 0.05
R. japonica str. M14012 (NZ AP017596) 0.24 0.01 0.01 0.01 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.01
R. massiliae str. AZT80 (CP003319) 0.24 0.01 0.01 0.02 0.06 0.02 0.06 0.05 0.24 0.07 0.02 0.06 0.02 0.02
R. monacensis str. IrR/Munich (LN794217) 0.25 0.04 0.05 0.05 0.05 0.05 0.04 0.05 0.24 0.05 0.05 0.06 0.05 0.05 0.05
R. parkeri str. Portsmouth (NC 017044) 0.24 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05
R. peacock i (NC 012730) 0.25 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05 0.00
R. prowazek ii str. Madrid E (NC 000963) 0.28 0.11 0.12 0.12 0.14 0.12 0.13 0.13 0.27 0.13 0.12 0.13 0.12 0.12 0.12 0.11 0.12 0.12
R. raoultii str. Khabarovsk (CP10969) 0.23 0.00 0.01 0.01 0.05 0.01 0.05 0.04 0.23 0.05 0.01 0.05 0.00 0.01 0.01 0.04 0.01 0.01 0.11
R. rickettsii str. Arizona (CP003307) 0.24 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.00 0.00 0.12 0.01
R. sibirica 246 (AABW01000001) 0.25 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05 0.00 0.00 0.12 0.01 0.00
R. tamurae str. AT-1 (AF394896) 0.25 0.04 0.05 0.05 0.05 0.05 0.04 0.05 0.24 0.05 0.05 0.05 0.05 0.05 0.06 0.00 0.05 0.05 0.11 0.04 0.05 0.05
R. conorii str. Malish 7 (AE006914) 0.24 0.01 0.02 0.01 0.06 0.02 0.06 0.05 0.23 0.06 0.02 0.06 0.01 0.01 0.02 0.05 0.01 0.01 0.13 0.01 0.01 0.01 0.05
R. heilongjiangensis Sendai-58 (AP019865) 0.25 0.01 0.02 0.01 0.05 0.02 0.06 0.04 0.24 0.06 0.02 0.05 0.01 0.00 0.02 0.05 0.01 0.01 0.12 0.01 0.01 0.01 0.05 0.02
R. typhi str. Wilmington (NC-006142) 0.27 0.11 0.11 0.12 0.12 0.11 0.12 0.12 0.26 0.12 0.11 0.12 0.11 0.12 0.12 0.10 0.11 0.12 0.04 0.11 0.12 0.12 0.10 0.12 0.12
R. montanesis OSU 85-390 (CP003340) 0.23 0.01 0.01 0.01 0.05 0.02 0.05 0.04 0.23 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.01 0.01 0.12 0.01 0.01 0.01 0.05 0.02 0.02 0.11
R. slovaca isolate Xinjiang (MF002535) 0.25 0.01 0.01 0.00 0.06 0.01 0.06 0.04 0.24 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.00 0.00 0.12 0.01 0.01 0.00 0.05 0.01 0.01 0.11 0.01
130
Table A1.34. Rickettsia concatenated ompA, ompB, and gltA (long) Maximum Likelihood fits of 24 different nucleotide substitution
models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 56 16534 16063 -7975 n/a 0.36 1.379 0.309 0.309 0.191 0.191 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04
T92+G+I 57 16540 16061 -7973 0.167 0.505 1.38 0.309 0.309 0.191 0.191 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04
TN93+G 59 16547 16051 -7966 n/a 0.359 1.408 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.06 0.08 0.04 0.06 0.15 0.04 0.22 0.06 0.04
HKY+G 58 16552 16065 -7974 n/a 0.361 1.377 0.299 0.32 0.183 0.199 0.06 0.04 0.12 0.06 0.11 0.04 0.06 0.19 0.04 0.18 0.06 0.04
HKY+G+I 59 16559 16062 -7972 0.169 0.507 1.379 0.299 0.32 0.183 0.199 0.06 0.04 0.12 0.06 0.11 0.04 0.06 0.19 0.04 0.18 0.06 0.04
TN93+G+I 60 16565 16060 -7970 0.166 0.486 1.641 0.299 0.32 0.183 0.199 0.06 0.03 0.14 0.05 0.1 0.04 0.05 0.18 0.04 0.21 0.06 0.03
GTR+G 62 16567 16046 -7961 n/a 0.355 1.412 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.05 0.08 0.05 0.07 0.15 0.03 0.22 0.08 0.02
GTR+G+I 63 16573 16043 -7959 0.171 0.502 1.414 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.05 0.08 0.05 0.07 0.15 0.02 0.22 0.08 0.02
T92+I 56 16711 16240 -8064 0.465 n/a 1.237 0.309 0.309 0.191 0.191 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04
TN93+I 59 16726 16230 -8056 0.465 n/a 1.246 0.299 0.32 0.183 0.199 0.07 0.04 0.13 0.06 0.09 0.04 0.06 0.15 0.04 0.2 0.07 0.04
HKY+I 58 16728 16240 -8062 0.465 n/a 1.235 0.299 0.32 0.183 0.199 0.07 0.04 0.11 0.06 0.1 0.04 0.06 0.18 0.04 0.17 0.07 0.04
GTR+I 62 16750 16228 -8052 0.466 n/a 1.244 0.299 0.32 0.183 0.199 0.07 0.05 0.13 0.06 0.09 0.05 0.08 0.15 0.03 0.2 0.07 0.03
K2+G 55 16762 16299 -8095 n/a 0.352 1.381 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05
K2+G+I 56 16768 16297 -8093 0.174 0.499 1.384 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
K2+I 55 16938 16476 -8183 0.472 n/a 1.243 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
JC+G 54 16979 16525 -8208 n/a 0.378 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 55 16986 16524 -8207 0.174 0.543 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 54 17138 16684 -8288 0.47 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
T92 55 17351 16888 -8389 n/a n/a 1.145 0.309 0.309 0.191 0.191 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.17 0.04 0.17 0.07 0.04
HKY 57 17366 16887 -8386 n/a n/a 1.145 0.299 0.32 0.183 0.199 0.07 0.04 0.11 0.07 0.1 0.04 0.07 0.18 0.04 0.16 0.07 0.04
TN93 58 17369 16881 -8382 n/a n/a 1.145 0.299 0.32 0.183 0.199 0.07 0.04 0.12 0.07 0.09 0.04 0.07 0.16 0.04 0.18 0.07 0.04
GTR 61 17394 16881 -8380 n/a n/a 1.146 0.299 0.32 0.183 0.199 0.07 0.05 0.12 0.06 0.09 0.05 0.08 0.16 0.04 0.18 0.08 0.04
K2 54 17606 17152 -8522 n/a n/a 1.122 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06
JC 53 17786 17340 -8617 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
131
Table A1.35. Rickettsia concatenated ompA, ompB, and gltA (long) genetic distance matrix.
Sample SR
12 ex.
Am
. albolim
batum
(PI 3
329)
R. p
eaco
cki
(NC 012
730)
R. a
kari
(NC 009
881)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. m
ontane
sis OSU
85-
390 (C
P0033
40)
R. tam
urae
str. AT-
1 (A
F394
896)
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. a
frica
e E
SF-5 (C
P0016
12)
R. a
mblyo
mm
atis
str. G
AT-30
V (CP00
3334
)
R. a
sem
bone
nsis
str. NM
RCii (
NZ JW
SW00
0000
00.1)
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. b
ellii
RM
L369
-C (N
C 007
940)
R. c
onor
ii str. M
alish
7 (A
E0069
14)
R. felis
URRW
XCal2 (C
P0000
53)
R. g
rave
sii
BWI-1
(DQ26
9439
)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P0198
65)
R. h
onei
RB (N
Z AJT
T000
0000
0.1)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. m
assil
iae
str. AZT
80 (C
P0033
19)
R. m
onac
ensis
str. IrR
/Mun
ich (L
N79
4217
)
R. p
arke
ri str. Por
tsm
outh (N
C 017
044)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. rao
ultii
str. Kha
baro
vsk (C
P1096
9)
R. rhipice
phali
HJ 5 (C
P0131
33)
R. rick
ettsii
str. A
rizon
a (C
P0033
07)
R. s
ibirica
246
(AABW
0100
0001
)
R. s
lova
ca is
olate Xin
jiang
(MF0
0253
5)
R. typ
hi st
r. W
ilmington
(NC-0
0614
2)
Sample SR12 ex. Am. albolimbatum (PI 3329)
R. peacocki (NC 012730) 0.04
R. akari (NC 009881) 0.16 0.16
R. helvetica C9P9 (CM001467) 0.09 0.08 0.17
R. montanesis OSU 85-390 (CP003340) 0.02 0.05 0.15 0.08
R. tamurae str. AT-1 (AF394896) 0.06 0.07 0.17 0.09 0.04
R. aeschlimannii str. RH15 (HM050289) 0.03 0.04 0.15 0.09 0.03 0.05
R. africae ESF-5 (CP001612) 0.03 0.02 0.16 0.08 0.04 0.07 0.04
R. amblyommatis str. GAT-30V (CP003334) 0.02 0.04 0.15 0.09 0.03 0.05 0.02 0.03
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.13 0.13 0.16 0.11 0.12 0.13 0.12 0.13 0.12
R. australis str. Cutlack (NC 017058) 0.18 0.17 0.19 0.18 0.16 0.17 0.17 0.16 0.17 0.17
R. bellii RML369-C (NC 007940) 0.29 0.29 0.35 0.30 0.29 0.30 0.27 0.28 0.27 0.31 0.39
R. conorii str. Malish 7 (AE006914) 0.03 0.02 0.15 0.09 0.03 0.06 0.03 0.01 0.03 0.12 0.17 0.28
R. felis URRWXCal2 (CP000053) 0.16 0.16 0.10 0.17 0.15 0.16 0.15 0.15 0.14 0.11 0.22 0.33 0.16
R. gravesii BWI-1 (DQ269439) 0.02 0.04 0.16 0.09 0.03 0.06 0.03 0.04 0.02 0.13 0.18 0.29 0.03 0.16
R. heilongjiangensis Sendai-58 (AP019865) 0.03 0.03 0.16 0.09 0.03 0.06 0.03 0.02 0.03 0.13 0.17 0.27 0.02 0.16 0.03
R. honei RB (NZ AJTT00000000.1) 0.03 0.03 0.16 0.09 0.04 0.07 0.04 0.01 0.03 0.12 0.17 0.28 0.01 0.16 0.04 0.02
R. japonica str. M14012 (NZ AP017596) 0.03 0.04 0.17 0.09 0.04 0.07 0.04 0.03 0.04 0.13 0.18 0.28 0.02 0.17 0.04 0.01 0.03
R. massiliae str. AZT80 (CP003319) 0.02 0.04 0.15 0.08 0.03 0.05 0.01 0.03 0.02 0.12 0.17 0.27 0.03 0.15 0.03 0.03 0.03 0.03
R. monacensis str. IrR/Munich (LN794217) 0.05 0.07 0.17 0.09 0.04 0.02 0.06 0.07 0.05 0.13 0.18 0.30 0.06 0.17 0.05 0.06 0.06 0.06 0.05
R. parkeri str. Portsmouth (NC 017044) 0.03 0.03 0.15 0.09 0.04 0.06 0.03 0.01 0.03 0.12 0.18 0.28 0.01 0.16 0.03 0.02 0.01 0.03 0.03 0.06
R. prowazekii str. Madrid E (NC 000963) 0.21 0.20 0.27 0.22 0.20 0.20 0.20 0.19 0.19 0.24 0.35 0.37 0.19 0.29 0.20 0.20 0.20 0.21 0.20 0.21 0.20
R. raoultii str. Khabarovsk (CP10969) 0.02 0.04 0.15 0.08 0.02 0.05 0.02 0.03 0.02 0.13 0.17 0.28 0.03 0.15 0.02 0.02 0.03 0.03 0.02 0.05 0.02 0.20
R. rhipicephali HJ 5 (CP013133) 0.02 0.05 0.16 0.09 0.03 0.06 0.02 0.03 0.03 0.13 0.18 0.29 0.03 0.15 0.03 0.03 0.03 0.03 0.01 0.06 0.03 0.21 0.02
R. rickettsii str. Arizona (CP003307) 0.03 0.04 0.16 0.09 0.03 0.06 0.03 0.02 0.03 0.13 0.18 0.28 0.02 0.17 0.03 0.02 0.02 0.03 0.03 0.06 0.01 0.20 0.02 0.03
R. sibirica 246 (AABW01000001) 0.03 0.03 0.15 0.09 0.03 0.06 0.03 0.01 0.03 0.13 0.17 0.28 0.01 0.16 0.03 0.02 0.01 0.02 0.03 0.06 0.01 0.20 0.02 0.03 0.01
R. slovaca isolate Xinjiang (MF002535) 0.02 0.03 0.16 0.09 0.03 0.06 0.03 0.01 0.03 0.13 0.18 0.28 0.01 0.16 0.03 0.01 0.01 0.02 0.02 0.06 0.01 0.20 0.02 0.03 0.01 0.01
R. typhi str. Wilmington (NC-006142) 0.18 0.16 0.23 0.16 0.17 0.17 0.17 0.16 0.16 0.21 0.24 0.33 0.16 0.24 0.18 0.16 0.17 0.17 0.18 0.18 0.17 0.15 0.18 0.18 0.18 0.17 0.17
132
Table A1.36. Rickettsia concatenated ompA, ompB, and sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
HKY+G 58 20185 19692 -9788 n/a 0.576 1.214 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.16 0.04 0.19 0.06 0.04
TN93+G 59 20194 19692 -9787 n/a 0.575 1.221 0.344 0.278 0.182 0.196 0.06 0.04 0.12 0.08 0.1 0.04 0.08 0.15 0.04 0.2 0.06 0.04
HKY+G+I 59 20195 19694 -9788 0 0.575 1.215 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.16 0.04 0.19 0.06 0.04
T92+G 56 20198 19722 -9805 n/a 0.579 1.211 0.311 0.311 0.189 0.189 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04
GTR+G 62 20200 19672 -9774 n/a 0.566 1.239 0.344 0.278 0.182 0.196 0.05 0.05 0.12 0.06 0.1 0.05 0.1 0.15 0.03 0.2 0.08 0.03
TN93+G+I 60 20205 19694 -9787 1E-05 0.574 1.222 0.344 0.278 0.182 0.196 0.06 0.04 0.12 0.08 0.1 0.04 0.08 0.15 0.04 0.2 0.06 0.04
T92+G+I 57 20209 19724 -9805 1E-05 0.578 1.212 0.311 0.311 0.189 0.189 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04
GTR+G+I 63 20210 19674 -9774 1E-05 0.565 1.24 0.344 0.278 0.182 0.196 0.05 0.05 0.12 0.06 0.1 0.05 0.1 0.15 0.03 0.2 0.08 0.03
HKY+I 58 20406 19913 -9898 0.324 n/a 1.136 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.15 0.04 0.19 0.06 0.04
TN93+I 59 20415 19913 -9898 0.325 n/a 1.14 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.09 0.04 0.08 0.14 0.04 0.2 0.06 0.04
T92+I 56 20419 19943 -9915 0.324 n/a 1.131 0.311 0.311 0.189 0.189 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.17 0.04 0.17 0.07 0.04
GTR+I 62 20428 19901 -9888 0.324 n/a 1.148 0.344 0.278 0.182 0.196 0.05 0.05 0.11 0.06 0.09 0.05 0.1 0.14 0.03 0.2 0.07 0.03
K2+G 55 20465 19998 -9944 n/a 0.552 1.212 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
K2+G+I 56 20476 20000 -9944 0 0.552 1.212 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06
JC+G 54 20679 20220 -10056 n/a 0.591 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 55 20690 20222 -10056 1E-05 0.591 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
K2+I 55 20701 20233 -10061 0.333 n/a 1.126 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06
HKY 57 20777 20292 -10089 n/a n/a 1.054 0.344 0.278 0.182 0.196 0.07 0.04 0.1 0.08 0.1 0.05 0.08 0.15 0.05 0.18 0.07 0.04
T92 55 20787 20319 -10104 n/a n/a 1.057 0.311 0.311 0.189 0.189 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.16 0.04 0.16 0.07 0.04
TN93 58 20788 20294 -10089 n/a n/a 1.054 0.344 0.278 0.182 0.196 0.07 0.04 0.1 0.08 0.1 0.05 0.08 0.15 0.05 0.18 0.07 0.04
GTR 61 20796 20278 -10078 n/a n/a 1.055 0.344 0.278 0.182 0.196 0.05 0.05 0.1 0.07 0.1 0.05 0.1 0.15 0.04 0.18 0.07 0.04
JC+I 54 20902 20442 -10167 0.328 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
K2 54 21094 20635 -10263 n/a n/a 1.032 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06
JC 53 21273 20822 -10358 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
133
Table A1.37. Rickettsia concatenated ompA, ompB, and sca4 genetic distance matrix.
R. pea
cock
i (N
C 012730
)
R. aka
ri (NC 00
9881
)
R. helv
etica
C9P9 (CM
0014
67)
R. mon
tane
sis O
SU 85-3
90 (C
P0033
40)
R. tam
urae
str. A
T-1 (A
F39489
6)
R. aes
chlim
annii
str. R
H15 (H
M050
289)
R. afric
ae E
SF-5 (C
P0016
12)
R. am
blyom
matis
str.
GAT-30V
(CP00
3334)
R. ase
mbon
ensis
str. N
MRCii (
NZ JWSW
000000
00.1)
R. aus
tralis
str. C
utlack
(NC 0
17058
)
R. bell
ii RM
L369-C
(NC 0
07940
)
R. con
orii str
. Mali
sh 7
(AE006
914)
R. felis
URRWXCal2
(CP00
0053)
R. gra
vesii
BWI-1
(DQ269
439)
R. heil
ongji
ange
nsis S
endai-
58 (A
P0198
65)
R. hon
ei R
B (NZ A
JTT00
00000
0.1)
R. japon
ica str
. M14
012 (
NZ AP017
596)
R. mas
siliae
str. A
ZT80 (CP003
319)
R. mon
acensis
str. Ir
R/Mun
ich (L
N79421
7)
R. par
keri
str. P
ortsm
outh (N
C 0170
44)
R. pro
wazekii
str. M
adrid
E (N
C 000963
)
R. rao
ultii str
. Kha
baro
vsk (
CP1096
9)
R. rhip
icepha
li HJ 5
(CP013
133)
R. rick
ettsii
str.
Arizon
a (CP003
307)
R. sibi
rica
246 (A
ABW010
00001)
R. slov
aca is
olate
Xinjiang
(MF00
2535)
R. typhi
str. W
ilming
ton (N
C-006
142)
Sample
R53 ex
Am. a
lbolim
batum
(PI 6
80)
R. peacocki (NC 012730)
R. akari (NC 009881) 0.19
R. helvetica C9P9 (CM001467) 0.12 0.19
R. montanesis OSU 85-390 (CP003340) 0.05 0.19 0.12
R. tamurae str. AT-1 (AF394896) 0.06 0.21 0.12 0.05
R. aeschlimannii str. RH15 (HM050289) 0.05 0.19 0.12 0.03 0.05
R. africae ESF-5 (CP001612) 0.03 0.19 0.12 0.04 0.06 0.04
R. amblyommatis str. GAT-30V (CP003334) 0.05 0.19 0.12 0.03 0.05 0.03 0.04
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.16 0.16 0.12 0.15 0.16 0.15 0.15 0.15
R. australis str. Cutlack (NC 017058) 0.20 0.19 0.19 0.19 0.21 0.20 0.19 0.20 0.16
R. bellii RML369-C (NC 007940) 0.56 0.68 0.62 0.54 0.58 0.54 0.55 0.53 0.66 0.74
R. conorii str. Malish 7 (AE006914) 0.02 0.19 0.12 0.04 0.05 0.04 0.01 0.03 0.15 0.19 0.54
R. felis URRWXCal2 (CP000053) 0.17 0.10 0.17 0.17 0.18 0.16 0.17 0.15 0.12 0.21 0.66 0.17
R. gravesii BWI-1 (DQ269439) 0.05 0.19 0.13 0.03 0.06 0.04 0.04 0.03 0.16 0.20 0.55 0.04 0.18
R. heilongjiangensis Sendai-58 (AP019865) 0.04 0.19 0.12 0.03 0.05 0.03 0.02 0.03 0.15 0.19 0.53 0.02 0.17 0.03
R. honei RB (NZ AJTT00000000.1) 0.03 0.18 0.12 0.04 0.05 0.04 0.01 0.04 0.15 0.19 0.54 0.01 0.17 0.04 0.02
R. japonica str. M14012 (NZ AP017596) 0.04 0.20 0.12 0.04 0.06 0.04 0.03 0.04 0.16 0.20 0.54 0.03 0.18 0.04 0.01 0.03
R. massiliae str. AZT80 (CP003319) 0.05 0.19 0.12 0.03 0.05 0.02 0.03 0.03 0.14 0.20 0.53 0.03 0.16 0.03 0.03 0.03 0.03
R. monacensis str. IrR/Munich (LN794217) 0.07 0.20 0.12 0.05 0.05 0.06 0.07 0.06 0.15 0.19 0.58 0.06 0.18 0.06 0.06 0.07 0.06 0.06
R. parkeri str. Portsmouth (NC 017044) 0.03 0.19 0.12 0.04 0.05 0.04 0.01 0.04 0.15 0.21 0.55 0.01 0.17 0.04 0.03 0.01 0.03 0.03 0.07
R. prowazekii str. Madrid E (NC 000963) 0.31 0.41 0.35 0.32 0.33 0.32 0.32 0.31 0.36 0.48 0.75 0.31 0.40 0.32 0.32 0.32 0.33 0.32 0.34 0.32
R. raoultii str. Khabarovsk (CP10969) 0.05 0.19 0.12 0.03 0.05 0.03 0.03 0.03 0.15 0.20 0.53 0.03 0.16 0.03 0.02 0.03 0.03 0.02 0.06 0.03 0.31
R. rhipicephali HJ 5 (CP013133) 0.05 0.19 0.12 0.03 0.05 0.02 0.04 0.03 0.15 0.20 0.54 0.04 0.17 0.03 0.02 0.03 0.03 0.01 0.06 0.04 0.32 0.02
R. rickettsii str. Arizona (CP003307) 0.04 0.19 0.12 0.04 0.05 0.04 0.02 0.03 0.15 0.20 0.55 0.02 0.18 0.03 0.02 0.01 0.03 0.03 0.06 0.02 0.32 0.03 0.03
R. sibirica 246 (AABW01000001) 0.03 0.19 0.12 0.04 0.05 0.04 0.02 0.04 0.15 0.20 0.55 0.01 0.17 0.04 0.02 0.02 0.03 0.03 0.07 0.01 0.33 0.03 0.04 0.02
R. slovaca isolate Xinjiang (MF002535) 0.03 0.19 0.12 0.03 0.05 0.03 0.01 0.03 0.15 0.20 0.55 0.01 0.17 0.03 0.02 0.01 0.02 0.03 0.06 0.01 0.32 0.02 0.03 0.01 0.01
R. typhi str. Wilmington (NC-006142) 0.27 0.37 0.29 0.29 0.30 0.29 0.28 0.29 0.31 0.37 0.69 0.28 0.35 0.29 0.28 0.28 0.29 0.29 0.31 0.29 0.18 0.29 0.29 0.30 0.30 0.29
Sample R53 ex Am. albolimbatum (PI 680) 0.05 0.19 0.12 0.03 0.06 0.04 0.04 0.03 0.15 0.21 0.55 0.04 0.17 0.02 0.03 0.04 0.04 0.03 0.06 0.04 0.33 0.03 0.03 0.03 0.03 0.03 0.29
134
Table A1.38. Rickettsia concatenated ompB, 17 kDa, and sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
GTR+G+I 63 13722 13183 -6528 0.135 0.748 1.78 0.348 0.258 0.182 0.211 0.02 0.07 0.11 0.03 0.14 0.04 0.12 0.2 0.01 0.18 0.05 0.01
GTR+G 62 13747 13217 -6546 n/a 0.582 1.39 0.348 0.258 0.182 0.211 0.04 0.06 0.12 0.05 0.11 0.04 0.12 0.16 0.03 0.19 0.05 0.03
HKY+G 58 13764 13267 -6576 n/a 0.526 1.772 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03
TN93+G 59 13765 13260 -6571 n/a 0.543 1.739 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03
GTR+I 62 13768 13238 -6557 0.438 n/a 1.535 0.348 0.258 0.182 0.211 0.02 0.06 0.11 0.03 0.13 0.04 0.12 0.18 0.04 0.19 0.05 0.03
T92+G 56 13772 13293 -6590 n/a 0.535 1.744 0.303 0.303 0.197 0.197 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03
HKY+G+I 59 13773 13268 -6575 0.033 0.565 1.773 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03
TN93+G+I 60 13774 13260 -6570 0.069 0.633 1.742 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03
T92+G+I 57 13781 13293 -6589 0.031 0.571 1.745 0.303 0.303 0.197 0.197 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03
TN93+I 59 13796 13291 -6586 0.446 n/a 1.721 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03
HKY+I 58 13797 13301 -6592 0.451 n/a 1.748 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03
T92+I 56 13804 13325 -6607 0.449 n/a 1.724 0.303 0.303 0.197 0.197 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.2 0.04 0.2 0.05 0.04
K2+G 55 13902 13431 -6660 n/a 0.511 1.738 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
K2+G+I 56 13910 13431 -6659 1E-04 0.51 1.739 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
K2+I 55 13940 13469 -6680 0.458 n/a 1.711 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
GTR 61 13971 13449 -6663 n/a n/a 1.313 0.348 0.258 0.182 0.211 0.03 0.06 0.1 0.04 0.12 0.04 0.12 0.17 0.04 0.17 0.05 0.03
TN93 58 13998 13502 -6693 n/a n/a 1.455 0.348 0.258 0.182 0.211 0.05 0.04 0.1 0.07 0.13 0.04 0.07 0.19 0.04 0.17 0.05 0.04
HKY 57 14008 13520 -6703 n/a n/a 1.448 0.348 0.258 0.182 0.211 0.05 0.04 0.13 0.07 0.11 0.04 0.07 0.15 0.04 0.21 0.05 0.04
T92 55 14009 13538 -6714 n/a n/a 1.456 0.303 0.303 0.197 0.197 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04
K2 54 14151 13689 -6790 n/a n/a 1.437 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05
JC+G 54 14160 13698 -6795 n/a 0.59 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 55 14169 13698 -6794 7E-06 0.59 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 54 14196 13734 -6813 0.436 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 53 14373 13920 -6907 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
135
Table A1.39. Rickettsia concatenated ompB, 17 kDa, and sca4 genetic distance matrix.
Sam
ple
R92
ex. Am
. albolim
batu
m
(PI 3
323)
R. g
rave
sii
BW
I-1 (D
Q26
9437
)
R. a
esch
liman
nii
str.
RH15
(HM05
0289
)
R. m
assilia
e str.
AZT80 (C
P00
3319
)
R. rhipice
phali
HJ 5 (C
P01
3133
)
R. m
ontane
sis OSU 85-
390
(CP00
3340
)
R. p
eaco
cki
(NC 012
730)
R. a
frica
e E
SF-
5 (C
P00
1612
)
R. c
onor
ii str.
Malish 7
(AE00
6914
)
R. h
onei
RB (N
Z AJT
T000
0000
0.1)
R. p
arke
ri str.
Portsm
outh (N
C 017
044)
R. rick
ettsii
str.
Ariz
ona
(CP00
3307
)
R. s
ibiric
a 246
(AABW
0100
0001
)
R. s
lova
ca is
olat
e Xinjia
ng (M
F002
535)
R. a
mblyo
mm
atis
str.
GAT-3
0V (C
P00
3334
)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P01
9865
)
R. jap
onica
str.
M14
012 (N
Z AP01
7596
)
R. rao
ultii
str.
Khaba
rovs
k (C
P10
969)
R. m
onac
ensis
str.
IrR/M
unich
(LN79
4217
)
R. tam
urae
str.
AT-1 (A
F3948
96)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. a
kari
(NC 009
881)
R. felis
URRW
XCal2 (C
P00
0053
)
R. a
sem
bone
nsis
str.
NM
RCii (
NZ J
WSW
0000
0000
.1)
R. a
ustra
lis
str.
Cutlack
(NC 017
058)
R. typ
hi
str.
Wilm
ington
(NC-0
0614
2)
R. p
rowaz
ekii
str.
Mad
rid E
(NC 000
963)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample R92 ex. Am. albolimbatum (PI 3323)
R. gravesii BWI-1 (DQ269437) 0.01
R. aeschlimannii str. RH15 (HM050289) 0.02 0.02
R. massiliae str. AZT80 (CP003319) 0.02 0.02 0.01
R. rhipicephali HJ 5 (CP013133) 0.02 0.02 0.01 0.01
R. montanesis OSU 85-390 (CP003340) 0.02 0.02 0.01 0.01 0.02
R. peacocki (NC 012730) 0.02 0.02 0.02 0.02 0.02 0.02
R. africae ESF-5 (CP001612) 0.02 0.02 0.02 0.02 0.02 0.02 0.01
R. conorii str. Malish 7 (AE006914) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01
R. honei RB (NZ AJTT00000000.1) 0.02 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01
R. parkeri str. Portsmouth (NC 017044) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.00 0.01
R. rickettsii str. Arizona (CP003307) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.00 0.01
R. sibirica 246 (AABW01000001) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01
R. slovaca isolate Xinjiang (MF002535) 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.00 0.00 0.00 0.00 0.01
R. amblyommatis str. GAT-30V (CP003334) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02
R. japonica str. M14012 (NZ AP017596) 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.02 0.01 0.02 0.01
R. raoultii str. Khabarovsk (CP10969) 0.02 0.02 0.01 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.02 0.01 0.02 0.01 0.02 0.01 0.01
R. monacensis str. IrR/Munich (LN794217) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04
R. tamurae str. AT-1 (AF394896) 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.04 0.03
R. helvetica C9P9 (CM001467) 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.07
R. akari (NC 009881) 0.08 0.09 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.08
R. felis URRWXCal2 (CP000053) 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.06
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.08 0.07 0.05 0.03
R. australis str. Cutlack (NC 017058) 0.09 0.09 0.09 0.08 0.09 0.09 0.09 0.09 0.09 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.10 0.08 0.04 0.06 0.05
R. typhi str. Wilmington (NC-006142) 0.20 0.19 0.19 0.20 0.19 0.19 0.20 0.20 0.20 0.19 0.19 0.20 0.20 0.19 0.20 0.19 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.19 0.20
R. prowazekii str. Madrid E (NC 000963) 0.21 0.21 0.20 0.21 0.20 0.20 0.21 0.21 0.21 0.20 0.20 0.21 0.21 0.21 0.21 0.20 0.20 0.20 0.20 0.20 0.20 0.22 0.21 0.21 0.23 0.07
R. bellii RML369-C (NC 007940) 0.44 0.45 0.44 0.44 0.43 0.43 0.44 0.44 0.43 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.45 0.45 0.45 0.47 0.46 0.46 0.48 0.48 0.52
136
Table A1.40. Rickettsia concatenated ompB, and 17 kDa Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 58 5387 4939 -2411 n/a 0.415 2.496 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03
T92+I 58 5393 4945 -2414 0.531 n/a 2.475 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03
T92+G+I 59 5396 4941 -2411 0.298 0.911 2.507 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03
HKY+G 60 5404 4940 -2410 n/a 0.409 2.552 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03
K2+G 57 5405 4965 -2425 n/a 0.405 2.502 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
HKY+I 60 5410 4946 -2413 0.533 n/a 2.531 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03
K2+I 57 5410 4970 -2428 0.536 n/a 2.475 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
TN93+G 61 5411 4940 -2409 n/a 0.426 2.494 0.301 0.265 0.183 0.25 0.04 0.03 0.15 0.04 0.16 0.04 0.04 0.23 0.04 0.18 0.04 0.03
HKY+G+I 61 5413 4942 -2410 0.288 0.863 2.561 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03
K2+G+I 58 5414 4966 -2425 0.27 0.796 2.511 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04
GTR+G 64 5415 4920 -2396 n/a 0.424 2.502 0.301 0.265 0.183 0.25 0.02 0.06 0.15 0.02 0.16 0.04 0.1 0.23 0.01 0.18 0.04 0.01
TN93+G+I 62 5419 4940 -2408 0.328 1.066 2.51 0.301 0.265 0.183 0.25 0.04 0.03 0.15 0.04 0.16 0.04 0.04 0.23 0.04 0.18 0.04 0.03
GTR+G+I 65 5423 4921 -2395 0.4 1.567 2.523 0.301 0.265 0.183 0.25 0.02 0.06 0.15 0.02 0.16 0.04 0.1 0.23 0.01 0.18 0.04 0.01
TN93+I 61 5443 4972 -2425 0.32 n/a 2.232 0.301 0.265 0.183 0.25 0.04 0.03 0.14 0.05 0.16 0.04 0.05 0.23 0.04 0.17 0.04 0.03
GTR+I 64 5456 4961 -2416 0.32 n/a 1.952 0.301 0.265 0.183 0.25 0.02 0.06 0.14 0.03 0.14 0.04 0.1 0.2 0.04 0.17 0.04 0.03
T92 57 5482 5041 -2463 n/a n/a 2.042 0.283 0.283 0.217 0.217 0.05 0.04 0.15 0.05 0.15 0.04 0.05 0.19 0.04 0.19 0.05 0.04
HKY 59 5500 5044 -2463 n/a n/a 2.042 0.301 0.265 0.183 0.25 0.04 0.03 0.17 0.05 0.12 0.04 0.05 0.18 0.04 0.2 0.04 0.03
K2 56 5501 5069 -2478 n/a n/a 2.029 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
TN93 60 5504 5040 -2460 n/a n/a 2.051 0.301 0.265 0.183 0.25 0.04 0.03 0.14 0.05 0.15 0.04 0.05 0.22 0.04 0.17 0.04 0.03
GTR 63 5511 5024 -2449 n/a n/a 2.063 0.301 0.265 0.183 0.25 0.03 0.06 0.14 0.03 0.15 0.04 0.1 0.22 0.02 0.17 0.04 0.01
JC+G 56 5550 5117 -2502 n/a 0.461 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 57 5559 5119 -2502 0.109 0.582 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 56 5580 5147 -2517 0.32 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC 55 5634 5209 -2549 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
137
Table A1.41. Rickettsia concatenated ompB, and 17 kDa genetic distance matrix.
Sample R52
ex. Am
. albolim
batum
(PI 6
80)
Sample R10
6 ex
. Am
. albolim
batum
(PI 6
80)
R. tam
urae
str. AT-
1 (A
F394
896)
R. m
onac
ensis
str. IrR
/Mun
ich (L
N79
4217
)
R. h
onei
RB (N
Z AJT
T000
0000
0.1)
R. a
sem
bone
nsis
str. NM
RCii (
NZ JW
SW00
0000
00.1)
R. m
assil
iae
str. AZT
80 (C
P0033
19)
R. rhipice
phali
HJ 5 (C
P0131
33)
R. p
eaco
cki
(NC 012
730)
R. a
kari
(NC 009
881)
R. h
elve
tica
C9P
9 (C
M00
1467
)
R. m
ontane
sis OSU
85-
390
(CP00
3340
)
R. a
esch
liman
nii
str. RH15
(HM
0502
89)
R. a
frica
e E
SF-5 (C
P0016
12)
R. c
onor
ii str. M
alish
7 (A
E0069
14)
R. g
rave
sii
BWI-1
(DQ26
9437
)
R. h
eilong
jiang
ensis
Sen
dai-5
8 (A
P0198
65)
R. ja
ponica
str. M
1401
2 (N
Z AP01
7596
)
R. p
arke
ri str. Por
tsm
outh (N
C 017
044)
R. rao
ultii
str. Kha
baro
vsk (C
P1096
9)
R. rick
ettsii
str. A
rizon
a (C
P0033
07)
R. s
ibirica
246
(AABW
0100
0001
)
R. s
lova
ca is
olate Xin
jiang
(MF0
0253
5)
R. a
mblyo
mm
atis
str. G
AT-30
V (CP00
3334
)
R. a
ustra
lis
str. Cutlack
(NC 017
058)
R. felis
URRW
XCal2 (C
P0000
53)
R. typ
hi st
r. W
ilmington
(NC-0
0614
2)
R. p
rowaz
ekii
str. M
adrid
E (N
C 000
963)
R. b
ellii
RM
L369
-C (N
C 007
940)
Sample R52 ex. Am. albolimbatum (PI 680)
Sample R106 ex. Am. albolimbatum (PI 680) 0.00
R. tamurae str. AT-1 (AF394896) 0.01 0.01
R. monacensis str. IrR/Munich (LN794217) 0.01 0.01 0.01
R. honei RB (NZ AJTT00000000.1) 0.04 0.04 0.04 0.04
R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.04 0.04 0.04 0.04 0.05
R. massiliae str. AZT80 (CP003319) 0.04 0.04 0.05 0.04 0.01 0.05
R. rhipicephali HJ 5 (CP013133) 0.04 0.04 0.05 0.04 0.02 0.05 0.01
R. peacocki (NC 012730) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02
R. akari (NC 009881) 0.05 0.05 0.05 0.04 0.03 0.04 0.04 0.04 0.04
R. helvetica C9P9 (CM001467) 0.05 0.05 0.05 0.05 0.04 0.06 0.04 0.04 0.04 0.04
R. montanesis OSU 85-390 (CP003340) 0.05 0.05 0.04 0.04 0.01 0.05 0.01 0.02 0.02 0.03 0.04
R. aeschlimannii str. RH15 (HM050289) 0.05 0.05 0.05 0.04 0.01 0.05 0.01 0.02 0.02 0.04 0.04 0.01
R. africae ESF-5 (CP001612) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02
R. conorii str. Malish 7 (AE006914) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00
R. gravesii BWI-1 (DQ269437) 0.05 0.05 0.04 0.04 0.01 0.04 0.01 0.02 0.01 0.03 0.04 0.01 0.01 0.01 0.01
R. heilongjiangensis Sendai-58 (AP019865) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02 0.02 0.03 0.04 0.01 0.02 0.01 0.01 0.01
R. japonica str. M14012 (NZ AP017596) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02 0.02 0.04 0.04 0.02 0.02 0.02 0.02 0.01 0.01
R. parkeri str. Portsmouth (NC 017044) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.00 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.01
R. raoultii str. Khabarovsk (CP10969) 0.05 0.05 0.04 0.04 0.01 0.04 0.01 0.02 0.01 0.03 0.04 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
R. rickettsii str. Arizona (CP003307) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.01 0.00 0.00 0.01 0.01 0.02 0.00 0.01
R. sibirica 246 (AABW01000001) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.02 0.00 0.01 0.00
R. slovaca isolate Xinjiang (MF002535) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.02 0.00 0.01 0.00 0.00
R. amblyommatis str. GAT-30V (CP003334) 0.06 0.06 0.05 0.05 0.02 0.05 0.03 0.03 0.03 0.04 0.05 0.02 0.03 0.02 0.03 0.02 0.03 0.03 0.02 0.02 0.03 0.03 0.03
R. australis str. Cutlack (NC 017058) 0.07 0.07 0.07 0.06 0.05 0.04 0.05 0.05 0.05 0.03 0.06 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06
R. felis URRWXCal2 (CP000053) 0.07 0.07 0.07 0.06 0.06 0.03 0.06 0.06 0.06 0.05 0.07 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.05
R. typhi str. Wilmington (NC-006142) 0.12 0.12 0.12 0.12 0.13 0.12 0.14 0.13 0.14 0.12 0.12 0.13 0.13 0.13 0.13 0.13 0.14 0.14 0.13 0.13 0.13 0.13 0.13 0.14 0.12 0.12
R. prowazekii str. Madrid E (NC 000963) 0.14 0.14 0.14 0.13 0.15 0.15 0.15 0.15 0.16 0.15 0.14 0.15 0.14 0.16 0.16 0.15 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.16 0.17 0.16 0.06
R. bellii RML369-C (NC 007940) 0.34 0.34 0.35 0.34 0.34 0.33 0.34 0.33 0.34 0.34 0.34 0.33 0.33 0.34 0.33 0.33 0.34 0.34 0.33 0.34 0.33 0.34 0.34 0.35 0.34 0.34 0.36 0.39
138
Table A1.42. Haemoprotozoa 18S Maximum Likelihood fits of 24 different nucleotide substitution models.
Model #Param BIC AICc lnL (+i) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C
T92+G 40 5706 5400 -2660 n/a 0.21 2.13 0.315 0.315 0.185 0.185 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.22 0.03 0.22 0.05 0.03
T92+G+I 41 5714 5401 -2659 0.361 0.464 2.147 0.315 0.315 0.185 0.185 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.22 0.03 0.22 0.05 0.03
HKY+G 42 5717 5396 -2656 n/a 0.212 2.124 0.321 0.308 0.148 0.223 0.05 0.02 0.15 0.05 0.1 0.03 0.05 0.21 0.03 0.22 0.05 0.02
TN93+G 43 5725 5396 -2655 n/a 0.212 2.114 0.321 0.308 0.148 0.223 0.05 0.02 0.13 0.05 0.12 0.03 0.05 0.25 0.03 0.19 0.05 0.02
HKY+G+I 43 5725 5397 -2655 0.368 0.477 2.142 0.321 0.308 0.148 0.223 0.05 0.02 0.15 0.05 0.1 0.03 0.05 0.21 0.03 0.22 0.05 0.02
TN93+G+I 44 5733 5397 -2654 0.346 0.444 2.131 0.321 0.308 0.148 0.223 0.05 0.02 0.13 0.05 0.12 0.03 0.05 0.25 0.03 0.19 0.05 0.02
GTR+G 46 5752 5401 -2654 n/a 0.216 1.971 0.321 0.308 0.148 0.223 0.06 0.02 0.14 0.06 0.11 0.03 0.05 0.23 0.03 0.2 0.04 0.02
K2+G 39 5756 5458 -2690 n/a 0.22 2.015 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
GTR+G+I 47 5758 5399 -2652 0.346 0.445 2.144 0.321 0.308 0.148 0.223 0.06 0.03 0.14 0.06 0.12 0.03 0.06 0.25 0.02 0.2 0.04 0.01
K2+G+I 40 5765 5459 -2689 0.382 0.529 2.029 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04
T92+I 40 5788 5482 -2701 0.374 n/a 1.788 0.315 0.315 0.185 0.185 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.21 0.03 0.21 0.05 0.03
HKY+I 42 5797 5477 -2696 0.374 n/a 1.792 0.321 0.308 0.148 0.223 0.05 0.03 0.15 0.06 0.1 0.04 0.06 0.2 0.04 0.21 0.05 0.03
TN93+I 43 5806 5477 -2696 0.374 n/a 1.785 0.321 0.308 0.148 0.223 0.05 0.03 0.13 0.05 0.11 0.04 0.05 0.22 0.04 0.19 0.05 0.03
GTR+I 46 5830 5478 -2693 0.374 n/a 1.778 0.321 0.308 0.148 0.223 0.07 0.03 0.13 0.07 0.11 0.03 0.06 0.23 0.02 0.19 0.04 0.01
K2+I 39 5831 5533 -2727 0.374 n/a 1.755 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
JC+G 38 5860 5570 -2747 n/a 0.239 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+I 38 5867 5577 -2750 0.661 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
JC+G+I 39 5869 5571 -2746 0.347 0.527 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
T92 39 5872 5574 -2748 n/a n/a 1.674 0.315 0.315 0.185 0.185 0.06 0.03 0.12 0.06 0.12 0.03 0.06 0.2 0.03 0.2 0.06 0.03
HKY 41 5881 5568 -2743 n/a n/a 1.675 0.321 0.308 0.148 0.223 0.06 0.03 0.14 0.06 0.09 0.04 0.06 0.2 0.04 0.21 0.06 0.03
TN93 42 5890 5569 -2742 n/a n/a 1.673 0.321 0.308 0.148 0.223 0.06 0.03 0.13 0.06 0.1 0.04 0.06 0.22 0.04 0.19 0.06 0.03
K2 38 5910 5620 -2772 n/a n/a 1.679 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05
GTR 45 5912 5568 -2739 n/a n/a 1.66 0.321 0.308 0.148 0.223 0.07 0.03 0.13 0.07 0.11 0.03 0.06 0.22 0.02 0.19 0.04 0.01
JC 37 6002 5720 -2823 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
139
Table A1.43. Haemoprotozoa 18S genetic distance matrix.
Sample
SR14
ex.
A. albo
limba
tum
(PI 1
316)
Hepato
zoon
felis
(AB7
7150
1.1)
Hepato
zoon
felis
AB771
552.1
Hepato
zoon
canis
(AF1
7683
5)
Hepato
zoon
felis
isolat
e La
CONES
/Asia
tic lio
n 01 (
HQ82
9438
.1)
Hepato
zoon
felis
isola
te La
CONES/In
dian L
eopa
rd 02
(HQ82
9444
.1)
Hepato
zoon
felis
isola
te La
CONES/B
enga
l Tiger
01 (H
Q82
9445
.1)
H. mau
ritanic
a
isola
te TR
-8-08
(KF9
9269
8.1)
H. mariae
isolate
490
3 (KF9
9271
1.1)
H. mariae
isolate
495
5 (KF9
9271
2.1)
H. sp
. ex.
R. pulc
herrima
(KF9
9271
3.1)
H. stel
lata
KP88
1349
.1
K. sp
. Bto-20
19a i
solate
B3 (
MK39
6906
.1)
K. cf. l
acaz
ei (M
K497
254.1
)
K. sp
. isolate
CR12
(MK62
1304
.1)
B. gibs
oni
(KC46
1261
)
B. stab
leri
(HQ22
4961
)
D. rana
rum
(HQ22
4957
)
K. heli
cina
(HQ22
4955
)
A. bam
baroo
niae
(AF4
9405
9)
Sample SR14 ex. A. albolimbatum (PI 1316)
Hepatozoon felis (AB771501.1) 0.03
Hepatozoon felis AB771552.1 0.03 0.00
Hepatozoon canis (AF176835) 0.05 0.04 0.04
Hepatozoon felis isolate LaCONES/Asiatic lion 01 (HQ829438.1)0.03 0.00 0.01 0.04
Hepatozoon felis isolate LaCONES/Indian Leopard 02 (HQ829444.1)0.03 0.01 0.01 0.04 0.00
Hepatozoon felis isolate LaCONES/Bengal Tiger 01 (HQ829445.1)0.03 0.02 0.02 0.04 0.02 0.02
H. mauritanica isolate TR-8-08 (KF992698.1) 0.02 0.03 0.04 0.04 0.04 0.04 0.04
H. mariae isolate 4903 (KF992711.1) 0 0.03 0.03 0.05 0.03 0.03 0.03 0.02
H. mariae isolate 4955 (KF992712.1) 0.00 0.03 0.03 0.05 0.03 0.03 0.03 0.02 0.00
H. sp. ex. R. pulcherrima (KF992713.1) 0.02 0.02 0.03 0.04 0.03 0.03 0.03 0.01 0.02 0.02
H. stellata KP881349.1 0.02 0.02 0.02 0.04 0.02 0.03 0.03 0.02 0.02 0.02 0.01
K. sp. Bto-2019a isolate B3 (MK396906.1) 0.04 0.02 0.02 0.05 0.02 0.02 0.03 0.04 0.04 0.04 0.03 0.03
K. cf. lacazei (MK497254.1) 0.04 0.02 0.02 0.05 0.02 0.03 0.03 0.05 0.04 0.04 0.04 0.03 0.01
K. sp. isolate CR12 (MK621304.1) 0.03 0.00 0.01 0.04 0.01 0.01 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02
B. gibsoni (KC461261) 0.16 0.18 0.18 0.19 0.18 0.18 0.18 0.17 0.16 0.16 0.17 0.17 0.17 0.17 0.18
B. stableri (HQ224961) 0.07 0.06 0.06 0.08 0.06 0.06 0.07 0.07 0.07 0.07 0.06 0.06 0.07 0.07 0.06 0.18
D. ranarum (HQ224957) 0.07 0.07 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.06 0.07 0.07 0.07 0.18 0.01
K. helicina (HQ224955) 0.13 0.12 0.12 0.14 0.12 0.13 0.14 0.13 0.13 0.13 0.13 0.13 0.14 0.13 0.12 0.17 0.13 0.14
A. bambarooniae (AF494059) 0.12 0.12 0.12 0.12 0.12 0.12 0.13 0.11 0.12 0.12 0.11 0.11 0.13 0.12 0.12 0.15 0.12 0.12 0.07