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Eastern Michigan UniversityDigitalCommons@EMU
Master's Theses and Doctoral Dissertations Master's Theses, and Doctoral Dissertations, andGraduate Capstone Projects
2016
Phylogeographic and environmental DNA analysisof the mudpuppy (necturus maculosus maculosus)Amber Stedman
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Recommended CitationStedman, Amber, "Phylogeographic and environmental DNA analysis of the mudpuppy (necturus maculosus maculosus)" (2016).Master's Theses and Doctoral Dissertations. 784.http://commons.emich.edu/theses/784
Phylogeographic and Environmental DNA Analysis of the Mudpuppy (Necturus
maculosus maculosus)
by
Amber Stedman
Thesis
Submitted to the Department of Biology
Eastern Michigan University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
in
Biology
Thesis Committee:
Katherine Greenwald, Ph.D, Chair
Steven Francoeur, Ph.D
Margaret Hanes, Ph.D
November 15, 2016
Ypsilanti, MI
ii
Acknowledgments
I would like to thank my advisor, Katy Greenwald, and my committee members,
Dr. Hanes and Dr. Francoeur, for their guidance and support throughout the research and
writing process. I would also like to thank Herpetological Resource and Management,
with a special thanks to Dave Mifsud and Maegan Stapleton, for their admirable
dedication to this project, especially during cold Michigan winters. I also owe a great
deal to the many groups and individuals that have contributed tissue samples to this
study. It was humbling to see so many passionate people willing to take the extra time to
contribute to this study. Thank you to Tom Goniea, Jaquie Craig, Andrew Briggs, Kiley
Briggs, Krista Larson, Mason Murphy, Jeff Briggler, Rich King, Jenny Sutherland, Jean-
Francois Desroches, Jeff LeClere, Tim Matson, Richard Kik, MDNR, USGS, USFWS,
MODNR, Cleveland Museum of Natural History, and Environment and Climate Change
Canada. Finally, thank you to my family, friends, and fellow graduate students, whose
support and encouragement proved invaluable.
iii
Abstract
Understanding species’ genetic diversity allows for informed management that preserves
evolutionary potential. Informed management becomes crucial as genetic diversity
declines and species become susceptible to threats such as climate change. For some
species, it can be difficult to determine whether extant populations persist in an area.
Genetic tools allow us to address both of these issues. Phylogeographic analyses allow
managers to understand a population’s genetic makeup with respect to other populations,
facilitating decisions that preserve local adaptation and evolutionary potential.
Environmental DNA analysis complements traditional survey techniques and increases
confidence when determining species’ range. Here, we explore the phylogeography of
the mudpuppy’s western range and examine the feasibility of using eDNA to complement
traditional surveys. Results show a deep ancestral split between the eastern and western
portions of the mudpuppy’s range. Environmental DNA results show differential
amplification between eDNA samples and negative controls, although the methods
require further optimization.
iv
Table of Contents
Acknowledgments ............................................................................................................. ii
Abstract ............................................................................................................................. iii
List of Tables .................................................................................................................... v
List of Figures ................................................................................................................... vi
Chapter 1:
Introduction .................................................................................................... 1
Methods.......................................................................................................... 7
Results ............................................................................................................ 9
Discussion ...................................................................................................... 15
Chapter 2:
Introduction .................................................................................................... 19
Methods.......................................................................................................... 23
Results ............................................................................................................ 25
Discussion ...................................................................................................... 31
References ......................................................................................................................... 33
v
List of Tables
Table Page
1 Sample sizes for each watershed included in this study ..............................................9
2 Pairwise Fst comparisons of cyt b sequences ..............................................................13
3 Primer sequences used for cyt b gene ..........................................................................14
4 Primer sequences used for eDNA analysis ..................................................................26
vi
List of Figures
Figure Page
1 Range of Necturus spp. .................................................................................................. 5
2 Cyt b haplotypes across the entire geographic range assessed in this study ................. 11
3 Maximum likelihood tree of haplotypes ........................................................................12
4 Haplotype network of cyt b haplotypes .........................................................................13
5 Isolation by distance graph ............................................................................................14
6 Regression line relating log quantity of tissue DNA to C(t) cycle ................................26
7 Aquarium eDNA sample (A1) .......................................................................................27
8 Aquarium eDNA sample (A2) .......................................................................................27
9 Aquarium eDNA sample (A3) .......................................................................................28
10 Aquarium eDNA sample (B1) .....................................................................................28
11 Aquarium eDNA sample (B2) .....................................................................................29
12 Aquarium eDNA sample (B3) .....................................................................................29
13 Distilled H2O Control 1 ................................................................................................30
14 Distilled H2O Control 2 ................................................................................................30
15 Distilled H2O Control 3 ................................................................................................31
1
Chapter 1: Phylogeography of the Mudpuppy (Necturus maculosus maculosus) and the
Implications for Future Conservation and Management Efforts
Introduction
Without baseline genetic information to guide management practices, species may
be managed inefficiently or in a way that limits adaptive potential. To preserve adaptive
potential in populations, it is important to practice evolutionarily enlightened
management (Ashley et al. 2003). Decisions regarding management of species
frequently ignore the evolutionary implications. Sometimes this is simply a result of lack
of genetic data. It is also often a result of the misconception that evolution occurs too
slowly to be relevant to conservation (Avise 1995).
There are, however, numerous instances in which evolution is relevant to
conservation (Ashley et al. 2003). In our efforts to conserve biodiversity, we must
remember that genetic diversity is considered a component of biodiversity (Holland and
Hadfield 2002). Thus, knowledge of the distribution of genetic diversity over space and
the preservation of genes is a way of preserving biodiversity. When conservation
resources are limited, knowledge of where increased genetic diversity lies can assist in
making informed management decisions. In practice, this can mean prioritizing a
population that is genetically distinct relative to other populations. As a way of
recognizing distinct evolutionary lineages it is helpful to designate Evolutionary
Significant Units (ESUs). Although the definition of an ESU varies (Allendorf and
Luikart 2007) it can be broadly defined as a unique evolutionary lineage or lineages,
which have diverged from other lineages for a significant period of time. The
organization of populations into ESUs can aid management decisions and facilitate the
2
preservation of genetic diversity (Holland and Hadfield 2002). Furthermore, if captive
breeding or translocation should be required, ESUs can assist in deciding which
individuals ought to be bred and where individuals may be effectively relocated or re-
introduced to avoid outbreeding depression (Soltis and Gitzendanner 1999).
Genetic studies have also identified cryptic species, which may go unrecognized
until genetic studies can establish species status. While they remain unrecognized,
cryptic species may not be fully protected since only species, subspecies, or distinct
population segments can receive official designation under the U. S. Endangered Species
Act (Allendorf and Luikart 2007). This lack of resources for cryptic species may result in
continual decline until their status is elucidated. In the case of the red-legged frog (Rana
aurora) the range of the protected subspecies R. a. draytonii was not fully known until
mtDNA analysis of the full range of R. aurora. The discovery of this cryptic species in
new areas may lead to allocation of conservation protection to new areas where R. a.
draytonii is now known to occur (Shaffer et al. 2004).
Phylogeographic studies may also determine the extent of hybridization occurring
between lineages. If hybridization is detected, it may warrant the implementation of new
management strategies or a revised species designation. For the mottled duck (Anas
fulvigula) hybridization with mallard ducks (Anas platyrhynchos) threatens the
preservation of pure mottled duck lineages (McCracken et al. 2001).
Evolutionary history may also be clarified through phylogeographic methods
using measures of genetic diversity. Assuming an even rate of mitochondrial substitution
across areas, those populations with greater diversity represent older populations and are
likely sources from which individuals migrated to other areas (Nersting and Arctander
3
2001). Overall, the characterization of a species’ genetic diversity and evolutionary
history allow for the implementation of better management practices that work to
maximize evolutionary potential. The preservation of adaptive potential in animals
becomes especially relevant when considering the potential effects of climate change
where few practical management interventions exist (Scoble and Lowe 2010).
The global decline of amphibians was initially recognized at the First World
Congress of Herpetology in 1989, but data indicate that declines had begun as early as the
1970s. Since then, accumulated data show that amphibians comprise one of the most
imperiled animal taxa, experiencing higher extinction rates than both birds and mammals
(Stuart et al. 2004). Many population declines were reported in areas that were otherwise
considered pristine (Blaustein and Wake 1990). Depending on the specific environment,
causes of these declines are likely to vary, and many declines may be a result of complex
interactions among multiple causative agents that exacerbate the decline (Rohr and
Palmer 2013).
Though a single unifying cause cannot be identified, studies suggest that the
disproportionately high rates of declines and extinctions among amphibians are largely
due to habitat destruction and modification (Barrett and Guyer 2008), disease (Kilpatrick
et al. 2009), climate change (Li et al. 2013), pollution (Ficken and Byrne 2013), and
overexploitation (Natusch and Lyons 2012). Although we know that amphibian
populations are disproportionately declining worldwide, many species are still
understudied and categorized as data deficient according to the International Union for
the Conservation of Nature (IUCN). The proportion of amphibians considered “Data
Deficient” also rivals that of birds and mammals and studies suggest these species may be
4
at a higher risk of extinction than their more formally assessed counterparts (Stuart et al.
2004; Howard and Bickford 2014). With increasing alteration of natural habitat
threatening the persistence of amphibian populations, information regarding current
population status is greatly needed to inform best management practices.
The mudpuppy is unique among most amphibians since it is a strictly aquatic,
neotenic species that retains larval characteristics into adulthood. They are also the
second largest North American salamander after the hellbender (Cryptobranchus
alleghaniensis). Mudpuppies belong to family Proteidae, which contains two extant
genera: Necturus and Proteus. Of these genera, only Necturus can be found in North
America. Within Necturus, there are five extant species, all residing in eastern North
America (N. maculosus, N. alabamensis, N. beyeri, N. lewisi, and N. punctatus) (Gendron
2000; Harding 1997). The term mudpuppy can be used to describe two subspecies: N. m.
maculosus and N. m. louisianensis. In this study, we focused only on N. m. maculosus.
Mudpuppies can be found throughout the Great Lakes region, parts of Southern
Canada, as far east as Vermont, south to Louisiana, and west to Oklahoma and Minnesota
(Fig. 1). Recent geographical analyses suggest that mudpuppies may have migrated from
west to east during the Nipissing Phase when water levels were sufficiently high to
connect the Mississippi River to Lake Michigan and allow colonization of previously
glaciated regions (Mills and Hill 2016). Though they are geographically widespread,
mudpuppies are a behaviorally secretive species that tends to be most plentiful in habitats
containing cover objects such as flat rocks, logs, and planks or retreats such as crayfish
burrows, undercut banks, and tree roots (Lannoo 2005). Throughout their range
mudpuppies are known to occupy a variety of aquatic habitats including lakes, reservoirs,
5
canals, and streams. Unlike many other amphibians, mudpuppies are also quite cold-
tolerant and remain active even when the water’s surface is covered in ice. Mudpuppies
feed mostly on insects, crustaceans, and earthworms (Lagler and Goellner 1941) but have
also been known to feed on small fish, amphibians (including other mudpuppies) and
carrion (Holman 2012). Mudpuppies may live for more than 20 years and reach sexual
maturity between 4-6 years of age (>200 mm total length). Adults will move to shallow
water to breed beginning in October or November and females will begin laying eggs as
early as April. Females will attach eggs to the underside of rocks or other solid substrate
and will guard the nest from fish and crayfish (Holman 2012).
Figure 1: Range of Necturus spp. (Gendron 2000).
6
When they were first described, mudpuppies were reported as being common
(Eycleshymer 1906) but have since been reported as declining in parts of their range
(Harding 1997; King et al. 1997). Instances of direct, human-caused mortality have
contributed to these declines. Historically, biological supply companies would harvest
mudpuppies by the millions to be used in comparative anatomy classes. At the time,
mudpuppies were an ideal specimen for their relative abundance and minimal cost to
universities. Furthermore, there are reports of some fishermen mistaking mudpuppies for
predators of game fish or a danger to humans. These misconceptions have led some to
leave mudpuppies to die on ice or be cut from hooks rather than be returned humanely to
the water (Holman 2012). Another direct threat facing mudpuppy populations includes
mortality due to lampricide application (Gilderhus and Johnson 1980), which was
initiated around 1960 as an additional method of controlling invasive sea lamprey
populations. Both adult and juvenile mudpuppy mortality has been linked to 3-
trifluoromethyl-4-nitrophenol (TFM) application, but whether TFM disproportionately
affects juveniles is still unclear (Matson 1990; Boogaard et al. 2003).
Some of the indirect threats to mudpuppies include habitat degradation through
siltation, pollution, and climate change (Lanoo 2005; Holman 2012). Together, these
direct and indirect threats to survival are likely to act synergistically, exacerbating the
current decline. In Michigan, mudpuppies are currently considered a Species of Special
Concern (Michigan Department of Natural Resources). Due to their sensitivity to
environmental pollutants, mudpuppies are also thought to serve as an indicator species,
and their decline suggests a concurrent decline in overall ecosystem health (Welsh and
Ollivier 1998; Barrett and Guyer 2008). Given this alarming trend, this species warrants
7
further study and implementation of evolutionarily enlightened conservation practices.
An understanding of the population history of the mudpuppy and current genetic diversity
can aide in more effective management of this species.
Little information exists regarding the genetic diversity of the mudpuppy.
However, a study by Chellman (2011) analyzed the genetic variation of mudpuppy
populations in nine river drainages across Ohio, New York, Massachusetts, and Vermont.
The author concatenated cyt b and d-loop sequences and found nine unique haplotypes
and significant genetic structure among populations. To investigate the genetic diversity
in the unexamined western portion of the mudpuppy’s range, I conducted a
phylogeographic analysis in five watersheds spanning six U.S. states and two Canadian
provinces. A phylogeographic analysis may detect historical population structure.
Additionally, it may help identify populations that are genetically diverse. Such
genetically distinct populations may warrant separate management as a way to avoid the
effects of outbreeding depression and maximize evolutionary potential (Allendorf and
Luikart 2007; Sabatino and Routman 2009). Based on historic population structure,
populations can be organized into evolutionary significant units (ESUs) or management
units (MUs), which may be used to guide future management (Pan et al. 2014; Palsboll et
al. 2006).
Methods
The phylogeographic analysis included mudpuppies from six states in the U. S.
(Iowa, Kentucky, Ohio, Michigan, Minnesota, and Missouri) and two Canadian provinces
(Ontario and Quebec). Most tissue samples were collected from tail tips of mudpuppies.
However, some Canadian samples were derived from liver tissue. To collect tail tips,
8
mudpuppies were restrained in clear plastic tubes sufficiently wide to accommodate body
width and reduce discomfort, while also restricting movement. The tip of the tail was
placed on a surface sterilized with ethanol and a small section, measuring a few
millimeters, was removed from the end of the tail using a razor blade sterilized with
ethanol. Since different people collected our samples from numerous locations, there
may have been slight alterations to the methods just described. All samples were stored
at -20°C in 95% ethanol until DNA extraction. A Qiagen Dneasy Blood and Tissue Kit®
was used to extract genomic DNA following the manufacturer’s protocol. We then
amplified a 746 bp segment of the cytochrome b (cyt b) region of the mitochondrial
genome with primers (Table 3) developed by Moritz et al. (1992). Each reaction
contained 4µl of MyTaqTM Red Mix, 1 µM of each primer, 50–100 ng of genomic DNA,
and distilled H2O for a total volume of 10 µl. The PCR program consisted of an initial
denaturation of 2 min at 95 °C followed by 35 cycles of denaturation at 95 °C for 30s,
annealing at 50 °C for 1 min, and extension at 72 °C for 2 min. A final extension cycle
followed for 5 min at 72 °C. Successful amplification was confirmed using a 1% agarose
gel. Those reactions that were successful were sequenced at the University of Michigan
DNA Sequencing Core (Ann Arbor, MI). Forward and reverse sequences were edited
using Geneious ver. 6.1.8. A total of 272 mudpuppies were successfully sequenced at cyt
b. A haplotype tree using a minimal spanning network was generated using TCS in
Popart ver 1.7. A phylogenetic tree was created in Mega ver. 7.0.16 using maximum
likelihood. The tree followed the Hasegawa-Kishino-Yano model of evolution, which
was deemed most appropriate by Mega given our data. A cyt b sequence from Necturus
beyeri was included as an outgroup. Molecular diversity was measured using DNAsp
9
ver. 5 and Arlequin ver. 3.5 was used to measure population-level diversity. For
Arlequin analysis, populations were grouped by watershed. We had samples from five
different watersheds including: Great Lakes, Mississippi, Red River, Missouri, and Ohio
(Table 1). To determine whether increasing geographic distance correlates with
increasing genetic distance, I used a Mantel test. Genetic distances were supplied by
pairwise Fst values and geographic distances were obtained by measuring the pairwise
distances in kilometers between the midpoints of sampling areas within each watershed.
Table 1: Sample sizes for each watershed included in this study.
Watershed Sample Size
Great Lakes 128
Mississippi 113
Red River 24
Missouri 4
Ohio 3
Results
A total of 272 cyt b sequences, measuring 746 bp, were analyzed. Cyt b
sequences represented six U.S. states (OH, IA, MI, MN, MO, and KY) and two Canadian
provinces (ON and QC). The cyt b sequences contained 21 unique haplotypes. The most
prevalent haplotype of the cyt b sequences was haplotype D, which was found
exclusively in Iowa and Minnesota. Haplotype B was the second most prevalent
haplotype and was found exclusively in eastern states and provinces (OH, MI, KY, ON,
10
and QC; Fig. 2). Our phylogenetic tree of cyt b sequences shows a distinct separation
between eastern and western haplotypes (Fig. 3). Haplotype I is shown to be the
intermediate haplotype linking the two most common haplotypes (B and D; Fig. 4).
Haplotype B is one of the most ancestral haplotypes and gave rise to many eastern and
western haplotypes. The tree also demonstrates that many haplotypes are separated by
short genetic distances, which is the result of many haplotypes being separated by only
one or two mutations.
Measures of sequence diversity revealed that cyt b has a haplotype gene diversity
(Hd) of 0.612 ± 0.017 (SD). Nucleotide diversity (π) was 0.00715, and the number of
mutations per site (θ) was 0.12286. The average number of nucleotide differences
between samples (k) was 5.320.
There was a high degree of population differentiation. The Fst value across all
populations for cyt b analysis was 0.82. Variation among populations at this locus
accounted for 81.95% of the total variation while within population variation accounted
for 18.05% of the total variation (Table 2). Likewise, our Mantel test supports a
correlation between increasing geographic distance and increased genetic distance
(R2=0.63, p<0.05; Fig 5).
11
Figure 2: Cyt b haplotypes across the entire geographic range assessed in this study. The genetic divide between eastern and western groups is clearly visible.
12
Figure 3: Maximum likelihood tree of haplotypes.
13
Figure 4: Haplotype network of cyt b haplotypes.
Table 2: Pairwise Fst comparisons of cyt b sequences. Asterisks indicate significant values. Great Lakes Mississippi Red River Ohio Missouri
Great Lakes -
Mississippi 0.83998* -
Red River 0.87513* 0.00769 -
Ohio -0.13158 0.81540* 0.89921* -
Missouri 0.79202* 0.70969* 0.77570* 0.66667* -
A
B
C
D
E
F
G
H
I
J
KL
M
N
O
P
QR
ST
U
10 samples
1 sample
cluster1
cluster2
cluster3
cluster4
cluster5
cluster6
cluster7
cluster8
cluster9
cluster10
cluster11
cluster12
cluster13
cluster14
cluster15
14
Figure 5: Isolation by distance graph. This figure shows that genetic distance increases
as geographic distance increases (R2=0.63, p<0.05).
Table 3: Primer sequences used for cyt b gene. MVZ15 5’-GAACTAATGGCCCACACWWTACGNAA-3’
MVZ16 5’-AAATAGGAARTATCAYTCTGGTTTRAT-3’
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400 1600
GeneticDistance(Fst)
GeographicDistance(km)
15
Discussion
The results of this study are the most geographically widespread analysis of
mudpuppy genetics to date. A similar study of mudpuppy populations in the northeastern
United States found nine unique mitochondrial haplotypes (Chellman 2011). Our study,
which analyzed mudpuppies from a broader geographic region found 21 haplotypes for
cyt b sequences. Similar to the study by Chellman (2011) our data also indicate
significant population structure across the region we analyzed, as demonstrated by high
Fst values. High Fst values are typical of population comparisons over large distances
where gene flow becomes increasingly unlikely (Allendorf and Luikart 2007). Given that
some of the groups compared in this study were separated by hundreds of miles, such a
high Fst value would be expected.
The results of the Mantel test support the idea that increasing geographic
separation between populations correlates with increased genetic distance between
populations, possibly as a result of reduced gene flow between populations or varying
selective forces in different environments. .Eight of ten pairwise Fst comparisons showed
significant genetic differentiation between watersheds (Fst>0.25), a result likely
influenced by reduced gene flow and different selective pressures. One of the two
exceptions was a comparison between the Mississippi and Red River watersheds.
Samples from these two locations were separated by the least distance (<400 km) and
thus gene flow was likely to have occurred much more recently between these two
watersheds than in other pairwise comparisons. .The second exception occurred when
comparing the Great Lakes watershed to the Ohio watershed. In this case the geographic
16
distance between the waterhsheds was greater (~600 km). However, the lack of a
significant Fst result may be due to low sample size in the Ohio watershed.
For both cyt b and concatenated sequence analysis, most of the genetic variation
was found among rather than between populations. This is not surprising given that some
of the populations in this study have been separated for hundreds of miles and thousands
of years. Additionally, overall sequence diversity was low, a result that may be partially
influenced by the relatively large body size of mudpuppies compared to other
salamanders (Mezhzherin 2002).
The phylogenetic tree of cyt b sequences indicates a deep evolutionary split
between eastern and western haplotypes. The divergence of eastern and western
haplotypes suggests that there is significant genetic separation between these two broad
regions. During the Wisconsinan glaciation (80,000–10,000 BP) the Great Lakes region,
where many mudpuppies currently reside, would have been inhospitable, as this region
was covered with glacial ice. During this time, mudpuppies would have to have persisted
in refugia either east or west of the Great Lakes region. For the purposes of determining
likely colonization routes, the mudpuppy may be assessed similarly to fish species since
the mudpuppy’s migratory route would have also proceeded solely along aquatic routes
(Hecht and Walters 1955). A study by Mandrak and Crossman (1992) examines the
likely colonization routes of fish with present-day distributions in Ontario, Canada. They
identified three possible refugia: Missourian, Mississippian, and Atlantic Coastal. Of the
three refugia, most fish were believed to have originated from the Mississippian
refugium. Another study comparing current distributions to ancient lake maxima in
Southern Ontario suggests the most probable time of colonization to have occurred
17
during the Nippissing phase (4,000-5,000 BP) (Mills and Hill 2016). During this time,
Lake Michigan would have had a connection to the Mississippi river via the Sag Channel,
which then connected to Illinois River (Hansel et al. 1985). A third study suggests a
Mississippian origin and subsequent northward colonization from Minnesota via a
drainage reversal that caused the Red River of the North to flow into ancient Lake
Agassiz and into the Hudson Bay (Hecht and Walters 1955). Although it is plausible that
mudpuppies colonized the Great lakes region from western refugia following the retreat
of the Wisconsinan glaciation, definitively resolving the migration route may require
additional genetic data. One interesting exception to the overall pattern of east-west
separation is the grouping of Iowa haplotype T with the eastern group. This may be a
result of homoplasy or human introduction since descent from eastern animals is
unlikely.
These data provide insight into the genetic evolution of mudpuppy populations
and may help elucidate patterns of migration across the United States. These data can
also be used to identify genetically unique populations and determine how these
populations may be best managed in the future (Ashley et al. 2003; Pearse and Crandall
2004). Given the deep separation between eastern and western groups, based on the
phylogenetic tree and Fst analyses, all watersheds with significantly high Fst values
should be managed as separate entities to avoid outbreeding depression. Separate
management of these watersheds will help preserve local adaptations needed for survival.
As a general rule, organisms removed from their environment should be returned as
closely as possible to their original location especially since much remains unknown
about the fine-scale genetic structure within watersheds. Within a single watershed,
18
microsatellite markers may be useful for revealing fine-scale molecular structure. If
more molecular markers are analyzed, we may find that the Great Lakes and Ohio
watershed should also be managed separately. Similarly, additional markers may reveal
significant genetic differentiation between the Red River watershed and the Mississippi
watershed.
19
Chapter 2: Evaluating the Utility of Environmental DNA (eDNA) Analysis to Detect the
Presence of Mudpuppies (Necturus maculosus maculosus)
Introduction
Environmental DNA (eDNA) consists of extra-organismal DNA deposited in the
air, water, or soil (Díaz-Ferguson and Moyer 2014). This type of DNA can originate
from several sources including: sloughed skin, gametes, mucous secretions, feces, and
urine (Barnes et al. 2014; Furlan et al. 2015). Once in the environment, the lifespan of
eDNA is determined by several forces including: temperature, pH, conductivity, salinity,
and microbial community composition (Dejean et al. 2011; Barnes et al. 2014; Strickler
et al. 2015). The lifespan of eDNA also depends on whether it is extracellular,
intracellular, free-floating, or adsorbed to particles (Barnes et al. 2014). Together, these
forces limit the stability and lifespan of eDNA. Due to its short lifespan, from hours to
weeks, in the environment, eDNA has been used as an indicator of the recent presence of
organisms. Scientists began analyzing eDNA in the mid-1980s to describe microbial
communities in marine sediments (Díaz-Ferguson and Moyer 2014). Recently, eDNA
has also been used to monitor invasive species (Ficetola et al. 2008; Jerde et al. 2011),
estimate biodiversity (Thomsen et al. 2012), and survey for threatened and endangered
species (Goldberg et al. 2011).
The mudpuppy (Necturus maculosus maculosus) is an ideal candidate for the
implementation of eDNA surveys in future monitoring efforts. Since mudpuppies are
both aquatic and behaviorally secretive, it can be difficult to assess presence using
traditional monitoring methods (Ficetola et al. 2008). Compared to traditional survey
20
methods eDNA surveys in other taxa have saved time (Jerde et al. 2011; Sisgaard et al.
2015), money (Biggs et al. 2015; Sisgaard et al. 2015), and have detected the presence of
species in sites where a species was declared absent in traditional surveys (Dejean et al.
2011; Biggs et al. 2015). Furthermore, eDNA surveys do not require physical handling
of animals and result in a lesser degree of habitat disturbance compared to traditional
surveys (Jones 1992; Thomsen and Willerslev 2015). This can be a great advantage
when surveying threatened and endangered species as habitat degradation is cited as a
cause of decline for many, and human handling of species risks disease transfer, undue
stress, and injury for the animals involved.
Freshwater environments are the most common habitat for studies isolating
macro-organismal DNA with dozens of studies published to date. Taxa surveyed in
freshwater environments include mammals, insects, crustaceans, mollusks, reptiles, fish,
and amphibians. The most common taxa surveyed in freshwater are amphibians and fish
(Thomsen and Willerslev 2015). The first amphibian freshwater eDNA study focused on
the detection of invasive bullfrog species in France (Ficetola et al. 2008). Other
amphibian studies have focused on using eDNA as a tool for detecting the presence of
behaviorally secretive species (Goldberg et al. 2011) and species of conservation concern
(Thomsen et al. 2012; Olssen et al. 2013; Santas et al. 2013; Biggs et al. 2015) where low
densities make these species particularly difficult to monitor using traditional survey
methods alone.
In addition to investigating the relative success of eDNA amphibian surveys,
several studies have also begun to elucidate the caveats involved with this type of
research. Although it has been demonstrated that eDNA exhibits a general pattern of
21
exponential decay following the removal of an organism (Barnes et al. 2014), estimates
of how long eDNA may persist in the environment vary. For example, Thomsen et al.
(2012) showed that eDNA was able to persist for two weeks following the removal of
two amphibian species (Pelobates fuscus and Triturus cristatus) from mesocosms.
However, in a similar experiment using Lithobates catesbeianus housed in aquaria,
Dejean et al. (2011) were able to detect eDNA 25 days after the removal of tadpoles from
aquaria. Given that both studies were conducted in laboratory settings, it is possible that
eDNA would have an increased rate of degradation in a natural setting where a multitude
of environmental variables would affect degradation rate. This is demonstrated by Pilliod
et al. (2014) who found that following a 24-hour introduction and subsequent removal of
Idaho Giant Salamanders from a previously unoccupied stream, no eDNA was detectable
after only one hour following the removal of the salamanders.
Since environmental factors will influence the rate of eDNA degradation, other
studies have begun to describe some of the environmental effects on the rate of eDNA
degradation. Some studies on amphibian eDNA have found higher rates of decay with
increased exposure to UV radiation (Pilliod et al. 2014; Strickler et al. 2015).
Furthermore, Strickler et al. (2015) suggest that higher temperatures combined with an
acidic (pH= 4) or neutral pH (pH= 7) will also increase the rate of eDNA degradation.
Despite having a rapid degradation rate, eDNA surveys have been demonstrated to
outperform traditional surveys, particularly when a species occurs in low densities
(Pilliod et al. 2013; Biggs et al. 2015).
Even though eDNA surveys present numerous advantages compared to traditional
surveys, there are also several drawbacks to these types of surveys. One of the main
22
concerns when conducting eDNA surveys is contamination (Thomsen and Willerslev
2015). Given that there is no way to reliably assess contamination issues, it is crucial to
prevent contamination of samples both during the collection of samples, when
contamination may occur between sites or through the use of common equipment, and
during sample processing when it is possible to contaminate samples with genomic DNA
stored in labs. Therefore, vigilance in the collection and processing of eDNA samples is
key to avoiding false positives. To help determine whether contamination occurred, it is
useful to have negative controls in the field and in the lab by bringing containers filled
with sterile water to collection sites and subsequently filtering that water using the same
equipment and methods used to process eDNA samples. Field controls can aid in
assessing whether any contamination occurred while collecting samples as a result of
contaminated equipment, improper handling, or contaminated storage during transport.
Lab controls can help assess the degree to which lab equipment or workspace may be
contaminated, which is especially important if one is working in a lab containing
genomic DNA of the organism of interest. Including multiple replicates and a threshold
value of positive replicates before indicating presence of a species can also aid in
reducing error rate (Schmidt et al. 2013; Rees et al. 2014).
Although it is known that eDNA has a relatively short lifespan, estimates on its
lifespan vary from less than a day (Foote et al. 2012) to approximately one month
(Dejean et al. 2011), and it is unclear just how long ago an animal was present in a given
location since studies measuring the lifespan of eDNA vary widely. Furthermore,
although eDNA is often correlated with species presence, such a correlation may not
always be accurate since the DNA may be from deceased individuals, feces of an animal
23
that consumed the species of interest, resuspended sediments, or introduced ballast water
(Rothlisberger and Lodge 2013; Roussell et al. 2015; Turner et al. 2015). In aquatic
habitats, eDNA may also be transported far from where the animal is located (Deiner and
Altermatt 2014). Species abundance is also difficult to discern with eDNA analysis.
Quantitative PCR (qPCR) may help correlate population size with signal strength, but
whether reliable inferences may be made remains to be seen (Thomsen and Willerslev
2015). Ultimately, the design of protocol to limit contamination, the inclusion of
multiple replicates, and the concurrent use of traditional surveys will ensure the most
accurate assessment of population status.
Our goals for this preliminary experiment were to determine if eDNA is a feasible
method for detecting the presence of mudpuppies. At this stage, we may be able to detect
issues with filtration methods, contamination, primer specificity, primer binding, and
PCR conditions. Although mudpuppies may still occur at high densities in some areas,
their tendency to remain hidden throughout the day makes them a difficult species to
survey using traditional methods. Mudpuppies tend to have limited movements with
male and female home ranges measured at 75.6 m2 and 17.5 m2 respectively, which would
tend to support assumptions of recent presence in an area (Gendron 2000). Given that
mudpuppies inhabit a variety of permanent bodies of water, including lakes and rivers,
we may find differential success of eDNA studies in these habitats, which is an area of
research that may be investigated with field sampling.
Methods
Prior to conducting an eDNA field study, it is necessary to ensure that DNA can
be successfully amplified from animals in a controlled setting. To this end, two one-liter
24
water samples were collected from an aquarium housing two juvenile mudpuppies.
Water samples were vacuum-filtered on site using a hand-powered vacuum pump kit with
Thermo Scientific™ Nalgene™ Analytical Test Filter Funnels connected to a one-liter
Erlenmeyer flask. Each filter funnel setup was individually packaged with a 0.45 μm
cellulose nitrate filter installed on each setup. One liter of distilled water was also filtered
as a negative control. After the water was filtered, forceps treated with DNA-away were
used to transfer each filter into 1.5 ml tubes filled with 95% ethanol. Tubes were stored
in a -20°C freezer until DNA extraction. DNA was extracted using a Qiagen Dneasy
Blood and Tissue Kit®. Following extraction samples were amplified using primers
developed by Stephen Spear (Table 4; unpublished). The PCR program consisted of an
initial denaturation of 95 °C for 15 min followed by 50 cycles of denaturation at 94 °C
for 60 s and annealing/extension at 60 °C for 60 s. Each reaction contained 7.5μl of
MyTaqTM Red Mix, 15 nm of each primer, 5 nm of fluorescent probe, 3 µl DNA and
distilled H2O for a total volume of 15µl. Reactions were amplified using a Chromo 4TM
qPCR machine (BIO-RAD). Since eDNA analysis is prone to false type I and type II
errors, three replicates of each aquarium sample and three replicates of the distilled H2O
control were included to reduce erroneous conclusions. Following amplification, the
threshold fluorescence value indicating successful amplification in eDNA samples was
set by finding the best-fit line relating log quantity DNA to C(t) value using tissue
standards. The C(t) value indicates the cycle at which the DNA has achieved a threshold
level of amplification. Tissue standards were determined to have reached a C(t) value
when the DNA amplification curve moved beyond the baseline region and into the
exponential region.
25
Results
A regression line of log quantity tissue standard DNA to C(t) value (Fig. 6)
determined the most appropriate threshold value for the amplified samples. The most
appropriate threshold fluorescence value for the tested samples was -2.6. The two one-
liter samples collected from an aquarium housing juvenile mudpuppies show some
positive eDNA amplification using qPCR since five out of six reactions reached a
threshold value of amplification. However, there was a large amount of background
noise, and though five out of six reactions reached threshold, none of the reactions
remained above the threshold value for the duration of the qPCR run (Figs. 7–15). To
improve this protocol, we may include more tissue DNA standards with concentrations
that would be expected to closely match those found in the environment to more
accurately determine an appropriate threshold value. Additionally, we may consider
including a greater number of eDNA replicates in the qPCR reaction to increase
confidence in our conclusions. Each of the negative controls had threshold values below
-2.6, indicating no amplification (Figs. 22–24). The amplification efficiency measures the
percentage of DNA amplified after each cycle. The average amplification efficiency of
tissue standards was 34.66 ± 20.24% SD. The average amplification efficiency of
aquaria samples was 23.81 ±15.35% SD. There was no difference in amplification
efficiency between tissue standards and aquaria samples (t=1.07, df=11, p=0.30). The
average C(t) value for tissue standards was 37.03 ± 10.48. The average C(t) value for
aquaria samples was 39.90 ± 12.14. There was no difference between the C(t) values of
26
tissue standards and aquarium samples (t=0.46, df=11, p=0.65). The next phase of this
research will focus on amplification of freshwater samples.
Table 4: Primer sequences used for eDNA analysis
NEMA F AGCAACAGCCTTTGTAGGGTA
NEMA R TCGCCTTATCGACGGAGAATC
Biosearch probe (Quasar
670) CGTACTACCATGAGGCCAAATATCCTTC
Figure 6: Regression line relating log quantity of tissue DNA to C(t) cycle.
27
Figure 7: Aquarium eDNA sample (A1). This sample shows sufficient amplification
to cross the threshold value of -2.6.
Figure 8: Aquarium eDNA sample (A2). This sample shows sufficient amplification
to cross the threshold value of -2.6.
28
Figure 9: Aquarium eDNA sample (A3). This sample shows sufficient amplification
to cross the threshold value of -2.6.
Figure 10: Aquarium eDNA sample (B1). This sample shows sufficient amplification
to cross the threshold value of -2.6.
29
Figure 11: Aquarium eDNA sample (B2). This sample shows sufficient amplification
to cross the threshold value of -2.6.
Figure 12: Aquarium eDNA sample (B3). This sample shows sufficient amplification
to cross the threshold value of -2.6.
30
Figure 13: Distilled H2O Control 1. This sample shows no threshold amplification.
Figure 14: Distilled H2O Control 2. This sample shows no threshold amplification.
31
Figure 15: Distilled H2O Control 3. This sample shows no threshold amplification.
Discussion
The successful amplification of mudpuppy eDNA from an aquarium
housing captive animals demonstrates that our methods are able to successfully detect
the DNA of interest in a concentrated and controlled system. Furthermore, our
demonstrated lack of amplification of negative water samples suggests that there were
no major contamination issues. This is a required first step in any eDNA study as it
can help detect issues with filtration methods, contamination, primer specificity,
primer binding, and PCR conditions (Pilliod et al. 2012; Goldberg et al. 2016). The
lack of a statistically significant difference in amplification efficiency and C(t) values
between eDNA samples and tissue samples suggests that eDNA samples amplified
just as effectively as a group compared to the group of tissue samples. When
sampling from natural environments, we may find that eDNA amplification is
hindered compared to tissue DNA extracts as a result of a greater concentration of
32
inhibitors in the environment, environmental degradation, or low copy number
(Dejean et al. 2011; Barnes et al. 2014; Jane et al. 2015).
The next step in this study will be to test whether mudpuppy eDNA can be
amplified in a natural environment where increased environmental degradation will
make detection more difficult than captive environments. Sampling from
environments with known populations of mudpuppies will be a priority to establish a
positive control for our methods in a natural system. After establishing that our
methods can successfully detect mudpuppy eDNA from natural settings, we may be
able to correlate eDNA amplification with demographic data (Kelly et al. 2014). We
may also find that eDNA amplification correlates with habitat quality. As one of the
overarching goals of mudpuppy conservation is the successful restoration of desirable
breeding habitat, it would be useful for conservationists to know whether a given
restoration site is being utilized by mudpuppies. Although traditional survey methods
can assist in answering this question, eDNA analysis of a given site can be completed
more easily with less disturbance to animals and habitat (Hunter et al. 2015; Rees et
al. 2014).
33
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