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ORIGINALARTICLE

Global phylogeography and geographicalvariation in warning coloration ofthe wood tiger moth (Parasemiaplantaginis)Robert H. Hegna1,2*, Juan A. Galarza1 and Johanna Mappes1

1University of Jyvaskyla, Centre of Excellence

in Biological Interactions, Department of

Biological and Environmental Science, 40014

Jyv€askyl€a, Finland, 2Department of Biology,

Palm Beach Atlantic University, West Palm

Beach, FL 33401, USA

*Correspondence: Robert H. Hegna,

Department of Biology, Palm Beach Atlantic

University, 901 S. Flagler Dr., West Palm

Beach, FL 33401, USA.

E-mail: [email protected]

ABSTRACT

Aim To investigate the phylogeography of the aposematic wood tiger moth

(Parasemia plantaginis) across its Holarctic distribution and to explore how its

genetic structure relates to geographical differences in hindwing warning color-

ation of males and females. Males have polymorphic hindwing coloration,

while female hindwing coloration varies continuously, but no geographical

analyses of coloration or genetic structure exist.

Location The Holarctic.

Methods We sequenced a fragment of the mitochondrial cytochrome c

oxidase subunit I gene (COI) from 587 specimens. We also examined more

current population structure by genotyping 569 specimens at 10 nuclear micro-

satellite loci. Species distribution modelling for present conditions and the Last

Glacial Maximum (LGM) was performed to help understand genetic structure.

Geographical patterns in hindwing warning coloration were described from

1428 specimens and compared to the genetic analyses.

Results We found only two instances of genetic divergence that coincided

with distinct, yet imperfect, shifts in male hindwing coloration in the Caucasus

region and Japan. A shift in female hindwing colour did not appear to be asso-

ciated with genetic structure. A change from sexual monomorphism to sexual

dimorphism was also observed. Mitogenetic (mtDNA) structure does not show

the influence of glacial refugia during the LGM. Climate shifts following the

LGM appear to have isolated the red Caucasus populations and other southerly

populations. Populations at opposite ends of the moth’s distribution showed

high levels of differentiation in the microsatellite data analysis compared to the

shallow mitogenetic structure, supporting a more recent divergence.

Main conclusions Parasemia plantaginis populations appeared to have been

historically well connected, but current populations are much more differenti-

ated. This raises the possibility that incipient speciation may be occurring in

portions of the species’ distribution. Some changes in colour align to genetic

differences, but others do not, which suggests a role for selective and non-

selection based influences on warning signal variation.

Keywords

Aposematism, Arctiidae, Arctiinae, colour polymorphism, Erebidae, Holarctic,

Lepidoptera, sexual dimorphism, species distribution model.

INTRODUCTION

Studies of phenotypic differences across species’ distributions

provide valuable insight into the processes of natural

selection and speciation (Endler, 1977). While many species

can exhibit varying degrees of differentiation across their

distributions (Endler, 1977), aposematic organisms with

warning signals that advertise anti-predator defences offer a

ª 2015 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1doi:10.1111/jbi.12513

Journal of Biogeography (J. Biogeogr.) (2015)

unique perspective on such variation because they are pre-

dicted to be under stabilizing selection (Joron & Mallet,

1998). Geographical variation in warning signals is often

found to be the result of co-evolution between the apose-

matic prey and local predators (Kapan, 2001; Chouteau &

Angers, 2012). Thus, studying geographical variation in

warning signals in relation to genetic variation increases our

understanding about warning signal evolution and diversifi-

cation (H€arlin & H€arlin, 2003; Wang & Shaffer, 2008; Hines

et al., 2011). However, many species with geographically

varying warning signals studied to date occur in tropical eco-

systems, where predator/prey relationships are thought to be

more tightly intertwined (Schemske et al., 2009). Therefore,

it is also necessary to study aposematic species distributed in

the north that have experienced different climatic histories

and ecological conditions to fully understand how intraspe-

cific warning signal variation evolves.

We investigated the phylogeography, population structure,

and hindwing warning signal polymorphism in the apose-

matic wood tiger moth, Parasemia plantaginis (Linnaeus,

1758), across its Holarctic distribution to explore evolution-

ary influences on warning coloration. Hindwing colour func-

tions as a warning signal in males and females (Nokelainen

et al., 2012), and is heritable (Nokelainen et al., 2013).

Variable melanized markings on the hindwings can obscure

the colour completely in some areas and limit warning signal

efficacy (Hegna et al., 2013). Male hindwing colour is known

to be polymorphic over much of its distribution, but no

study has systematically examined the distribution or fre-

quency of colours across the entire distribution.

In the Caucasus region, male hindwing coloration is

described as reddish (Koc�ak, 1989; De Freina, 1993; Dubato-lov & Zahiri, 2005). This stands in contrast to neighbouring

populations that have polymorphic males with either yellow

or white hindwings. Such divergence in coloration raises

questions as to how closely connected populations in this

region are to adjacent European and south-western Russian

populations. Given the large species distribution (c.

17,000 km), additional changes in hindwing colour may

occur that are not yet described. For instance, some work

has suggested that Japanese populations mostly comprise

males with white hindwings (Okano & Katayama, 1976;

Kishida, 2011), and variation in coloration is also reported

to occur within North America (Coolidge, 1910). However,

none of these descriptions follow from an examination of

large numbers of specimens and almost nothing is known

about geographical variation in female warning coloration.

Here, we describe variation in hindwing colour across the

distribution of P. plantaginis. We then evaluate the historical

and recent genetic structure of the species across its Holarc-

tic distribution using two independent genetic markers and

determine whether genetic structuring matches any shifts in

male or female hindwing colour. Alignment between hind-

wing colour and genetic differences in neutral markers could

suggest that geographical isolation and drift are primary

drivers of hindwing colour divergence, rather than prezygotic

isolation. Genetic and coloration differences could be either

the product of isolation as a result of climate and/or geo-

graphical factors, or the result of local selection by predators

as observed in other aposematic species. We employed cli-

mate-based species distribution modelling to explore whether

climatic isolation could be a factor in genetic and coloration

differences. In examining hindwing and genetic data concur-

rently we aimed to develop a hypothesis about P. plantaginis

origins that would help in understanding hindwing colour

divergence in this species. Because hindwing coloration in

the Caucasus region clearly differs from that in the sur-

rounding areas (De Freina, 1993; Dubatolov & Zahiri, 2005),

we hypothesized that the Caucasus region would be the most

genetically diverged. We also hypothesized that changes in

coloration would be imperfectly matched to genetic differ-

ences in neutral markers, suggesting that processes other

than drift and mutation (i.e. selective pressures) could be

involved in hindwing colour evolution.

MATERIALS AND METHODS

Specimen collection for genetic analyses

We obtained adult specimens of Parasemia plantaginis from

across the entire Holarctic distribution (see Appendix S1a)

through fieldwork, private collectors and museum collec-

tions. Moths were captured in the field with butterfly nets

and pheromone traps baited with calling females, which

assisted in catching males. Methods for DNA extraction fol-

lowed those of Galarza et al. (2010), with the exception that

we extracted DNA from two legs rather than from the brain.

Mitochondrial DNA genotyping and analysis

We amplified a 630 bp fragment of the mitochondrial cyto-

chrome c oxidase subunit I gene (COI) from 587 specimens

(GenBank accession numbers: KP217220–KP217806; seeAppendix S1b). We used the COI sequences to reconstruct a

maximum parsimony haplotype network to depict relation-

ships among haplotypes of all 587 individuals using tcs

1.2.1, setting a 95% connection limit (Clement et al., 2000).

To further investigate relationships among haplotypes, we

employed a Bayesian approach using MrBayes 3.2.1

(Ronquist & Huelsenbeck, 2003). Our analysis included rep-

resentatives of each haplotype and a representative from each

geographical region bearing each haplotype. In total, we used

82 specimens in the MrBayes analysis. The HKY+G modelwas the most appropriate substitution model indicated by

jModelTest2 (Darriba et al., 2012) using both Akaike infor-

mation criterion corrected for small sample size (AICc) and

Bayesian information criterion (BIC). In the Bayesian

analysis, two runs of four Markov chain Monte Carlo

(MCMC) chains were run simultaneously (one cold and

three hot) for 20 million generations. We set the tree

Journal of Biogeographyª 2015 John Wiley & Sons Ltd

2

R. H. Hegna et al.

http://www.ncbi.nlm.nih.gov/nuccore/KP217220http://www.ncbi.nlm.nih.gov/nuccore/KP217806https://www.researchgate.net/publication/12275236_Clement_MD_Posada_D_Crandall_KA_TCS_a_computer_program_to_estimate_gene_genealogies_Mol_Ecol_9_1657-1659?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/27371926_Western_Lepidoptera-III-Notes_on_Leptarctia_Californiae_Walker?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/225896388_First_microsatellite_panel_for_the_Wood_Tiger_Moth_Parasemia_plantaginis?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/235384852_To_quiver_or_to_shiver_Increased_melanization_benefits_thermoregulation_but_reduces_warning_signal_efficacy_in_the_wood_tiger_moth?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/235378173_Environment-mediated_morph-linked_immune_and_life-history_responses_in_the_aposematic_wood_tiger_moth?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/10618138_MRBAYES_3_Bayesian_Phylogenetic_inference_under_mixed_models?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==

sampling rate to be once every 2000 generations. Following

the completion of the analysis the relative burn-in rate was

set to 0.25. Convergence was checked using Tracer 1.6

(Rambaut & Drummond, 2003). The remaining trees were

used to determine a majority-rule consensus tree.

In order to infer demographic history we used DnaSP

5.10 (Librado & Rozas, 2009) to calculate haplotype diversity

(h), nucleotide diversity (p), Tajima’s D, Fu’s FS and averagenucleotide differences.

Genotyping and analysis of nuclear microsatellite

markers

We amplified a total of 10 nuclear microsatellite loci as iden-

tified by Galarza et al. (2010): Ppla107, Ppla109, Ppla279,

Ppla313, Ppla317, Ppla323, Ppla363, Ppla382, Ppla414 and

Ppla439. A total of 569 specimens were included in the final

analyses because some older specimens failed to amplify for

a majority of loci (see Appendices S1a, S1c & S2a).

We used structure 2.3.4 (Pritchard et al., 2000) to esti-

mate the number of populations (K) in our dataset that are

relevant for studying evolutionary processes (see Appendix

S1d). From the populations identified with structure, we

constructed a neighbour-joining tree with 1000 bootstrap rep-

licates using the DA measure of genetic distance (Nei et al.,

1983) in poptree2 (Takezaki et al., 2010). We conducted a

hierarchical analysis of molecular variance (AMOVA) using

genalex 6.5 (Peakall & Smouse, 2006) to examine variation

within populations, among six regions, and among popula-

tions within the regions. The six regions were based on the

relationships among populations in the poptree2 analysis

and included the Caucasus, Europe, Russia, Japan, Alaska and

the rest of North America (Canada–Utah). Alaskan popula-tions were included as a separate region in the AMOVA

because the populations formed their own well-supported

group in the poptree2 analysis. Russia comprised its own

region in the AMOVA based on geography and no evidence

of connectedness with Japanese populations, rather than by

the results of the poptree2 analysis alone. We estimated FST(Weir, 1996) values among populations using the ENA

method implemented in the program freena (Chapuis &

Estoup, 2007) to account for any effects of null alleles, which

can inflate FST values. We also calculated DEST (Jost, 2008)

among all populations to supplement FST and DA measures of

differentiation using genalex (see Appendix S2b).

To investigate evidence of recent migration among popu-

lations determined from our structure analysis we used the

program BayesAss 3.0 (Wilson & Rannala, 2003). BayesAss

implements a Bayesian method to estimate recent migration

in the past three to four generations using MCMC sampling

techniques (see Appendix S1e).

Phenotypic data

We used 1187 male and 241 female individuals to determine

the distribution of hindwing warning signal colour across the

Holarctic distribution of P. plantaginis (Table 1; also see

Appendix S1a for detailed coordinates). Because the speci-

mens were gathered from a total of 351 sampling sites (with

some sites only having 1–5 moths) it was necessary to poolsamples collected from different sites in looking at the distri-

bution of male and female hindwing colour across the Hol-

arctic (Fig. 1c, see Appendix S1f for details). We visually

determined the hindwing colour of males (white and yellow)

because it is a polymorphic trait (white or yellow) without

intermediate phenotypes (Galarza et al., 2014). Males from

the Caucasus region with reddish hindwings were classified

as red for simplicity on a large spatial scale and because we

found no red individuals outside this area. Female hindwing

colour varies continuously between red and yellow (Fig. 1),

but can be matched to six standard colour tiles that were

previously tested against the wing colour using spectropho-

tometer data (see Lindstedt et al., 2011 for additional

method detail). This visual classification method for female

colour allowed us to reliably analyse geographical patterns

without touching the fragile museum specimens with a spec-

trophotometer. The number of specimens of each hindwing

colour was counted for each region and analysed in ArcGIS

10 (ESRI, Redlands, CA, USA).

Table 1 Number of specimens obtained from differentcountries used to examine geographical variation in colorationacross the distribution of Parasemia plantaginis.

Country Males Females Collector Field Museum

Armenia 6 0 1 0 5

Austria 96 8 4 100 0

Azerbaijan 1 0 0 0 1

Bulgaria 14 11 0 0 25

Canada 110 11 3 108 10

China 6 1 0 0 8

Czech Republic 3 1 4 0 0

Denmark 11 0 0 0 11

Estonia 39 12 0 51 0

Finland 246 41 0 287 0

Georgia 14 6 0 0 20

Italy 6 0 0 6 0

Japan 92 11 0 103 0

Kazakhstan 32 3 0 0 35

Latvia 2 0 0 0 2

Macedonia 1 1 2 0 0

Mongolia 17 8 0 0 25

North Korea 2 0 0 0 2

Norway 29 0 0 0 29

Poland 6 1 0 0 7

Romania 2 0 2 0 0

Russia 213 88 35 0 266

Spain 1 0 0 0 1

Sweden 5 1 0 0 6

Switzerland 17 0 0 17 0

Turkey 4 1 1 0 4

Ukrane 4 0 0 0 4

UK 56 2 0 58 0

USA 152 34 110 64 12

Total 1187 241 162 794 473


3

Geographical variation and phylogeography of an aposematic moth

https://www.researchgate.net/publication/24036324_Jost_L_GST_and_its_relatives_do_not_measure_differentiation_Mol_Ecol_17_4015-4026?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/24257674_DnaSP_v5_A_Software_for_Comprehensive_Analysis_of_DNA_Polymorphism_Data?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/227464332_Direction_and_strength_of_selection_by_predators_for_the_color_of_the_aposematic_wood_tiger_moth?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/223995688_Peakall_ROD_Smouse_PE_GENALEX_6_genetic_analysis_in_Excel_Population_genetic_software_for_teaching_and_research_Mol_Ecol_Notes_6_288-295?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==https://www.researchgate.net/publication/12483622_STRUCTURE_version_233_inference_of_population_structure_using_multilocus_genotype_data?el=1_x_8&enrichId=rgreq-7779eccf-9f53-4ea6-aee0-6afb3e55ac67&enrichSource=Y292ZXJQYWdlOzI3NTA1NzIzNDtBUzoyMjAxNDA3NTE4NTU2MjJAMTQyOTQ5NzA1NDAyMQ==

Species distribution modelling

Combining climate-based distribution modelling with phylo-

geographical data allows for development of spatially explicit

hypotheses of past distribution patterns (Waltari et al., 2007).

To perform the climatic species distribution model (SDM) of

P. plantaginis we used Maxent 3.3.3 (Phillips et al., 2006).

Climate data for the present and for the Last Glacial Maxi-

mum (LGM) were downloaded from the WorldClim 1.4 data-

base (Hijmans et al., 2005), using a grid resolution of 2.5

arcmin. Simulations used to create the climate data for the

LGM used the Community Climate System Model (CCSM).

Modelling was replicated 20 times to explore the reliability

and robustness of the results using 75% of the 211 locality

records for model training and 25% for model testing (see

Appendix S1g). Only collection sites with precise locality data

were used, although such analyses are somewhat robust to

imprecise locality data (Graham et al., 2008).

RESULTS

Hindwing colour distribution

Hindwing colour varied across the distribution of Parasemia

plantaginis for both males and females (Fig. 1). Males with

red hindwings were only found in the Caucasus area. Much

of the western Palaearctic areas appeared to have a high

proportion of yellow males, and white males became increas-

ingly predominant east of Europe, except for interior China.

We found males with completely melanized hindwings in

central Siberia and also in the Nearctic.

Female hindwing colour also exhibited geographical varia-

tion, but not concordant with male variation. Females with

deep-red hindwings were most common in the Caucasus.

However, unlike males, deep-red females can be found in

much of the western Palaearctic. Eastwards from the Cauca-

sus, female coloration became predominantly yellow across

the rest of the Palaearctic and Nearctic. Females with highly

melanized hindwings could be found occasionally in the

Nearctic (Fig. 1).

Phylogenetic relationships and haplotype structure

Our phylogeny from the COI gene fragment showed shallow

mitogenetic (mtDNA) structure across most of the Holarctic

distribution (Fig. 2). Samples from the Caucasus region

formed a highly supported group (1.0 posterior probability,

PP). However, one Georgian individual shared a haplotype

and clustered with moths from central and eastern Russia.

Despite only moderate haplotype diversity, nucleotide diver-

sity was greatest in the Caucasus group (Table 2).

The Bayesian analysis identified two groups in the area

extending from Europe across to eastern Russia. Two Czech

moths from a mountainous area formed the first well-sup-

(a)

(b)

(c)

Figure 1 Hindwing coloration of males andfemales of Parasemia plantaginis across theirgeographical distribution. Colours in circles

of (a) represent polymorphic male hindwingcolour and (b) represent female hindwing

colour that varies continuously. Circles in

(c) enclose sampling sites pooled togetherfor each pie chart based on genetic

populations (letters A, G, F, E, K, O, P, Q,S, T, U, V, W, X), samples that spatially

cluster without genetic basis for grouping toraise the sample size above 10 (B, H, I, J,

M, L, N, R, Y, Z), and a large geneticpopulation split to show greater detail in an

area adjacent to the Caucasus region (C, D).


4

R. H. Hegna et al.

ported group (0.96 PP). The second group, though not sta-

tistically supported, consisted of samples from central Russia

to the Kamchatka peninsula on the east coast (0.94 PP).

However, several specimens within this region had haplo-

types that grouped among European samples.

Japanese specimens formed three additional groups rang-

ing from no support to strong support (0.54–0.99 PP). Oneindividual from the Hokkaido population grouped with a

population from Honshu and one specimen from Honshu

shared a haplotype with the European group (Fig. 2).

North American specimens grouped together, but did not

form a statistically supported group (0.54 PP, Fig. 2).

Despite this, no haplotypes were shared with Palaearctic

moths, or vice versa (Figs 3 & 4). Haplotype diversity within

the North American group was the highest compared to

other groups of samples in the analysis (Table 2).

Our mtDNA haplotype network paralleled results from the

phylogenetic analysis and also suggested surprisingly little

differentiation across the Holarctic (Figs 3 & 4). The most

differentiated haplotypes belonged to individuals from the

Caucasus. However, one moth from the Caucasus had a hap-

lotype in common with specimens from Siberia. With the

exception of a single Japanese and Georgian specimen, all

other haplotypes were broadly clustered in a geographical

sense (i.e. Caucasus, Europe–Russia–Japan, and North Amer-ica). No haplotypes were shared between Palaearctic and

Nearctic specimens.

Coalescent theory predicts older haplotypes will dominate

the interior of a network, occur with high frequency, and

encompass a greater diversity of individuals (Posada & Cran-

dall, 2001), while younger descendant haplotypes surround

them and occur at lower frequencies (Crandall & Templeton,

1993). Consistent with these predictions, our haplotype net-

work shows one central high-frequency haplotype shared

across European, Russian, and one Japanese specimen.

Central haplotypes of relative high frequency also appeared

in the Caucasus, Russia and North America. Japanese

moths had several unique haplotypes in addition to the one

Table 2 Number of samples (n), Tajima’s D and significance level, Fu’s FS value and significance, nucleotide diversity (p), haplotypediversity (h), number of haplotypes (N), and average nucleotide differences within groups (K) calculated from the COI gene fragmentfrom samples of Parasemia plantaginis.

Group n Tajima’s D P-value FS P-value p h N (K)

Europe 229 �2.2683 < 0.001 �23.819 < 0.001 0.00093 0.45 20 0.584Caucasus 37 �1.5919 0.033 �0.661 0.379 0.00268 0.428 7 1.686Russia 52 �1.4011 0.037 �2.650 0.053 0.00094 0.501 6 0.591Japan HOK 26 �1.3463 0.052 �1.240 0.130 0.00083 0.348 4 0.520Japan ABO 30 1.0115 0.835 0.605 0.676 0.00172 0.634 4 1.083

Japan SAJ 23 �1.1533 0.168 0.905 0.693 0.0013 0.53 3 0.822North America 178 �1.3234 0.067 �9.204 0.002 0.00153 0.673 15 0.966Dalton 12 �1.1405 0.133 �0.476 0.333 0.00026 0.167 2 0.167

Figure 2 Phylogeny showing relationshipsamong Parasemia plantaginis specimensfrom different regions including all 56

haplotypes of the COI gene fragment.Numbers at nodes are posterior

probabilities larger than 0.5; values 0.95 andabove are well supported. Red branches

denote samples from the Caucasus region,black branches are European and Russian

samples, green branches denote Japanese,and blue branches represent North

American specimens. Two underlinedspecimens are the haplotypes from the

Caucasus and Japan that did not group withother haplotypes from those areas. The scale

bar unit is substitutions per site.


5


individual that shared the most common European haplo-

type. The same patterns in global haplotype structure are evi-

dent when haplotype frequencies are mapped across

geographical space (Fig. 3). North American specimens

exhibited two predominant haplotypes. One haplotype was

common above the southern Canadian–American border anda second haplotype was predominant below. However, we

also found each of these two haplotypes to be dominant in

two different portions of Alaska. Haplotypes were shared by

males irrespective of hindwing colours (Fig. 4). This lack of

Figure 3 COI gene fragment haplotypefrequencies of Parasemia plantaginis across

the entire geographical range of the species.Each colour represents a separate haplotype.

(a)

(b)

Figure 4 Haplotype networks showing eachof the 56 COI haplotypes of Parasemiaplantaginis represented as circles sized in

proportion to the number of specimenshaving each haplotype. The first network (a)

shows the geographical origin of sampleswith each haplotype. The second network

(b) illustrates the proportion of males ofeach haplotype with the four hindwing

colours observed across their distribution.


6

R. H. Hegna et al.

association between hindwing colour and COI haplotypes

found only in particular geographical regions suggests that

coloration did not differentiate under a scenario of

geographical isolation.

Population structure from microsatellite markers

Violations of Hardy–Weinberg equilibrium (HWE) or link-age equilibrium were not found across a majority of the

inferred populations for any loci. The initial analysis of

HWE and linkage equilibrium with samples grouped by

country, state, or province also found no violations in any

loci across a majority of groups. Previous studies indicate the

influence of null alleles to be negligible at low frequencies

(< 0.2) (Dakin & Avise, 2004). Our analysis with freenafound that only 23/170 loci by population combinations had

null alleles with frequencies above 0.2. The influence of null

alleles on FST values calculated within freena was minimal

when we compared estimates adjusted for the influence of

null alleles alongside non-adjusted estimates. The average dif-

ference was 0.017 and the maximum was 0.06, but none of

the differences affected the interpretation of FST values (i.e.

the difference of 0.06 reduced an FST value from 0.48 to

0.41). Population assignment and delimitation tests, such as

structure, are also robust to the effects of null alleles, even

at frequencies as high as 0.9 (Carlsson, 2008). Therefore, our

ability to infer current populations and examine differences

among them was not affected.

Analysis of population structure using structure inferred

17 populations, with Q > 0.8 across all specimens for theirdetermined population. Populations inferred by structure

corresponded to some of the strong and weakly supported

groups in the analysis of the COI gene fragment. These pop-

ulations included Scotland, central Europe (Europe_C),�Aland, Finland, Turkey, Caucasus (i.e. the northern Caucasus

Mountains), central Russia (Rus_Central), south-eastern

Russia (Rus_SE), north-eastern Russia (Rus_NE), the island

of Hokkaido, Japan (HOK), two populations on Honshu,

Japan (ABO and SAJ), Fairbanks, Alaska (Alaska_F), south-

central Alaska (Alaska_K), Canada, Utah and south-eastern

Utah (Dalton). Populations also tended to correspond to

large geographical areas, sometimes spanning more than

200 km wide or more (see Appendix S2c). For instance,

individuals across Finland grouped together in a population

spanning 800 km. Specimens from across central Europe

(Switzerland, Italy, Austria, Czech Republic, Poland, Roma-

nia, Macedonia and Bulgaria) also formed a single popula-

tion. Specimens from Estonia indicated a heavily admixed

ancestry between central Europe and Finland populations

and did not form their own unique population.

In contrast to our COI results, analysis of relationships

among populations using Nei’s genetic distance (DA) in pop-

tree2 showed a high degree of structure and support for

many groups only weakly supported in the COI tree (Fig. 5).

However, none of the structure among populations

contradicted broad sample groups inferred using the COI

gene fragment (see Appendix S2d).

Our analysis of molecular variance attributed 61% of

molecular variance to within-populations (P < 0.001) and10% to among populations (P < 0.001; Table 3). Among-region variance comprised 29% of molecular variance

Figure 5 Neighbour-joining tree representing differentiationamong the 17 populations of Parasemia plantaginis, which wederived from analysis of microsatellite markers in structure,

constructed from genetic distances between populations (DA).Numbers at nodes indicate bootstrap values (1000 bootstrap

replicates); values above 0.7 indicate strong support for groups.Branches are colour-coded to represent regions as in Fig. 2, with

Caucasus populations in red, European and Russianpopulations in black, Japanese in green, and North American in

blue.

Table 3 Results of a hierarchical analysis of molecular variance (AMOVA) showing the division of genetic variance from themicrosatellite marker data for Parasemia plantaginis across its Holarctic distribution. Regions and populations are defined in the maintext.

Source d.f. SS MS Est. var. % P-value

Among regions 5 1072.404 214.481 1.099 29% 0.001

Among Pops within regions 11 275.938 25.085 0.375 10% 0.001

Within Pops 1071 2457.633 2.295 2.295 61% 0.001

Total 1087 3805.974 3.769 1

d.f., degrees of freedom; SS, sum of squares; MS, mean squares; Est. var., estimated variance.


7


(P < 0.001). Regions included Europe, Caucasus, Russia,Japan, Alaska and the southern North American populations

grouped as regions. All FST values calculated with freena to

account for null alleles were well supported, having confi-

dence intervals that did not include zero after 5000 bootstrap

replicates (Table 4). Our pairwise population FST values indi-

cate a high degree of differentiation between populations

separated by 10,000 km (0.48–0.77 when comparing NorthAmerican, Japanese and Caucasus populations), but also sug-

gest that many neighbouring populations are more moder-

ately differentiated. Specimens from Caucasus and Turkish

populations were more strongly differentiated compared with�Aland and Scottish populations (FST = 0.15–0.25) than whencompared with central European or Finland populations

(FST = 0.12–0.19). While significant, the degree of differenti-ation indicated between the Scottish and central European

populations was low to moderate (FST = 0.027–0.169).Among Europe and central and eastern Russia, FST values

indicated moderate differentiation (0.086–0.164). BetweenAlaska and eastern Russia differentiation was high (0.327–0.479). Estimates of DEST showed similar patterns compared

to FST estimates, except a slightly greater degree of differenti-

ation among European populations and somewhat less dif-

ferentiation among North American populations (see

Appendix S2b).

Our analysis of gene flow using BayesAss found that

migration between most population pairs was low enough

that estimates of recent migration could not be detected

above background noise. The BayesAss results were consis-

tent with the populations inferred from structure. Recent

migration was only detected above background noise

between two populations in Japan (from ABO to SAJ:

m = 0.0916, CI95 = 0.043–0.140), and both populations inthe Caucasus region (from Caucasus to Turkey: m = 0.20,CI95 = 0.106–0.290). No evidence of recent migration existedbetween any of the Japanese populations and either south-

eastern or north-eastern Russia (see Appendix S2e).

Demographic history and distribution modelling

Within the Palaearctic, values of Fu’s FS statistic were both

significant and negative for Europe and Russia, indicative of

population expansion, but values for other locations were

not significant (Table 2). In North America, Fu’s FS statistic

was significant and indicated a negative deviation, except for

Dalton in southern Utah. Tajima’s D statistic was signifi-

cantly negative for Europe and the Caucasus, which can sug-

gest demographic expansion or selection, but was non-

significantly negative for Hokkaido and North America

(Table 2).

The species distribution model suggests that movement

across the Palaearctic region during the LGM was possible to

some extent (Fig. 6). The Caucasus region was more con-

nected to Europe in the past by climatically suitable areas,

compared to its more isolated present condition. The poten-

tial for connectivity across large geographical areas appearsTable

4DifferentiationbetweenParasem

iaplantaginispopulationsestimated

bypairw

iseFSTvalues

(Weir,1996)adjusted

fornullallelesusingtheprogram

freena(below

thediagonal).

AllFSTvalues

had

confidence

intervalsnotincludingzero

afterbootstrapping,

indicatingthey

werewellsupported.Genetic

distance

betweenpopulations(D

A)isgivenabove

thediagonal.

Population

Scotland

Europe_C

� Aland

Finland

Turkey

Caucasus

Rus_Central

Rus_SE

Rus_NE

Jap_ABO

Jap_HOK

Jap_SA

JAlaska_F

Alaska_K

Canada

Utah

Utah_D

Scotland

00.250

0.570

0.379

0.538

0.543

0.426

0.460

0.397

0.522

0.495

0.547

0.614

0.631

0.544

0.563

0.565

Europe_C

0.027

00.429

0.207

0.496

0.478

0.330

0.429

0.355

0.466

0.424

0.476

0.518

0.546

0.477

0.528

0.524

� Aland

0.169

0.143

00.291

0.733

0.690

0.647

0.656

0.662

0.686

0.696

0.650

0.747

0.762

0.702

0.767

0.756

Finland

0.068

0.042

0.102

00.551

0.531

0.402

0.407

0.406

0.505

0.445

0.502

0.575

0.629

0.530

0.583

0.570

Turkey

0.151

0.116

0.211

0.122

00.338

0.467

0.521

0.461

0.629

0.543

0.622

0.650

0.702

0.655

0.648

0.655

Caucasus

0.220

0.186

0.252

0.177

0.106

00.549

0.609

0.540

0.724

0.650

0.693

0.714

0.700

0.662

0.690

0.703

Rus_Central

0.120

0.087

0.223

0.109

0.165

0.259

00.264

0.290

0.466

0.311

0.481

0.509

0.530

0.507

0.614

0.584

Rus_SE

0.175

0.150

0.240

0.135

0.242

0.337

0.126

00.303

0.444

0.340

0.459

0.443

0.460

0.418

0.489

0.458

Rus_NE

0.179

0.135

0.259

0.153

0.261

0.332

0.141

0.164

00.304

0.244

0.294

0.511

0.465

0.465

0.481

0.447

Jap_ABO

0.393

0.307

0.409

0.302

0.562

0.532

0.394

0.373

0.341

00.230

0.104

0.665

0.675

0.662

0.684

0.706

Jap_HOK

0.339

0.251

0.375

0.247

0.494

0.475

0.288

0.351

0.274

0.262

00.209

0.621

0.608

0.569

0.581

0.611

Jap_SA

J0.457

0.345

0.443

0.336

0.676

0.581

0.470

0.485

0.400

0.264

0.280

00.676

0.670

0.666

0.702

0.707

Alaska_F

0.418

0.329

0.417

0.332

0.525

0.528

0.446

0.371

0.479

0.620

0.595

0.670

00.073

0.102

0.195

0.174

Alaska_K

0.361

0.280

0.361

0.292

0.487

0.480

0.390

0.327

0.397

0.586

0.560

0.641

0.084

00.135

0.241

0.189

Canada

0.410

0.325

0.445

0.333

0.563

0.559

0.453

0.355

0.471

0.627

0.596

0.672

0.135

0.159

00.121

0.139

Utah

0.491

0.374

0.489

0.380

0.711

0.621

0.558

0.506

0.610

0.711

0.687

0.765

0.333

0.353

0.207

00.146

Utah_D

0.445

0.341

0.444

0.341

0.702

0.582

0.514

0.463

0.576

0.712

0.675

0.772

0.268

0.281

0.175

0.418

0


8

R. H. Hegna et al.

to remain high under current conditions except for the

Caucasus. The average test area under the curve (AUC) of a

receiver operating characteristic (ROC) across all replicate

runs was 0.925 (SD = 0.014), which indicated the model out-put was of good quality. The parameters with the highest

explanatory power included annual mean temperature

(43.6%), the maximum temperature of the warmest period

(18.4%), minimum temperature of the coolest period (7%),

isothermality (6.4%), and annual precipitation (4.4%).

DISCUSSION

The wood tiger moth (Parasemia plantaginis) has only two

main lineages and surprisingly shallow levels of mitogenetic

structure, given its roughly 17,000 km geographical distribu-

tion. The first lineage is composed entirely of specimens

from the Caucasus region and Middle East. The second line-

age includes specimens from across Europe, Russia, Japan

and North America, but one individual from the Caucasus

also has a haplotype in common with this group. Therefore,

genetic divergence does not completely follow expectations

based on geographical location, and suggests historical gene

flow. Hindwing warning colour differences are most strongly

linked to the split between these two lineages, but such dif-

ferences are also not exclusive. Males with reddish hindwings

that vary in the degree of redness occur only in the Caucasus

region, but yellow males can also be found there on occasion

(Fig. 1). Female hindwing warning colour is also reddish in

the Caucasus, but can be red elsewhere in Europe and is pre-

dominantly yellow across the rest of the distribution east of

the Ural Mountains. A change in female hindwing colour to

be predominantly yellow does not appear to correspond with

any clear genetic differentiation. Therefore, while phenotypic

differentiation coincides with the greatest mitogenetic diver-

gence across the distribution in the Caucasus, hindwing col-

our differences do not appear to be completely exclusive

between the two groups. Japanese specimens also formed

their own groups and a shift to predominantly white

hindwings in males appears to have occurred there. However,

we observed one yellow individual in Japan and their exis-

tence is also reported in previous literature (Okano & Katay-

ama, 1976; Kishida, 2011). Female coloration in Japan is

similar compared to neighbouring areas (Fig. 1). Therefore,

genetic structure across the distribution of P. plantaginis

appears to be somewhat imperfectly matched to the two

shifts in male coloration in the Caucasus and Japan and not

associated with female hindwing colour changes. This sug-

gests that divergence in male and female coloration could be

the result of selection rather than solely a product of drift

and long term isolation.

Shallow mitogenetic structure in lepidopterans across large

species ranges similar to P. plantaginis has only been

reported for two other species: the Apollo butterfly, Parnas-

sius phoebus, which occurs across the Holarctic (Todisco

et al., 2012), and the pea blue butterfly, Lampides boeticus,

which occurs between Africa and Australia (Lohman et al.,

2008). A greater degree of mitogenetic structure has been

reported for the butterfly Hesperia comma, which is also dis-

tributed across the Holarctic (Forister et al., 2004). On a

smaller geographical scale, some studies of other temperate

and northern lepidopterans have found greater degrees of

genetic structure and phylogeographical signal (Wahlberg &

Saccheri, 2007; Gratton et al., 2008; Von Reumont et al.,

2012).

Climate oscillations during the Pleistocene that caused

species’ distributions to contract southwards before the recol-

onization of northern areas are commonly thought to be a

predominant driver of mitogenetic structure (Ibrahim et al.,

1996; Schmitt, 2007; Provan & Bennett, 2008). The influence

of glacial refugia has been described for many lepidopterans

(Schmitt, 2007; Kerdelhue et al., 2009). One way of inter-

preting the deeper split between most of the P. plantaginis

Caucasus haplotypes and those from the rest of the distribu-

tion is that the Caucasus served as a glacial refugium, similar

to what has been hypothesized for other species (Schmitt,

2007; Kerdelhue et al., 2009). However, our species

(a)

(b)

Figure 6 Species distribution models.Modelled geographical distribution for

Parasemia plantaginis based on (a) climateunder present conditions showing localities

used in the model and (b) the modelprojected onto past conditions during the

Last Glacial Maximum (LGM). Layersprojecting the glacial distribution during the

LGM in ArcGIS were obtained from Ray &Adams (2001). Areas coloured grey-black

represent increasing probabilities of climatefavouring the presence of P. plantaginis

above 10%.


9


distribution model (SDM) suggests that suitable climate for

P. plantaginis existed between southern Europe and the

Caucasus during the LGM, rather than a disconnected and

fragmented set of small refugia. Additionally, across Europe

and Russia we did not find any Caucasus haplotypes, unlike

patterns of genetic structure in organisms that migrated

northwards from southern refugia following glacial retreat in

Europe (Schmitt, 2007; Provan & Bennett, 2008). Less evi-

dence of population expansion in Russia and in Japan also

suggests demographic stability during the LGM. Our SDM

for present conditions, together with molecular data, suggests

that one explanation for the mitogenetic differentiation

between the Caucasus and other areas is isolation after post-

glacial climate change. Exactly when connectivity was lost

and to what extent gene flow may occur across patches of

habitable areas in Turkey is unknown. Recent work is reveal-

ing additional lepidopteran species that show mitogenetic

structuring not congruent with classic refugia hypotheses

(Schmitt et al., 2003; Schmitt, 2007; Todisco et al., 2012),

along with other invertebrates (Crease et al., 2012; Porretta

et al., 2013). Such patterns appear to be linked to greater

dispersal abilities of the species in question (Wahlberg &

Saccheri, 2007). Climate isolations during the Pleistocene

may have driven selection for greater dispersal abilities in

northerly taxa (Dynesius & Jansson, 2000), which might help

explain shallow mitogenetic structure in some Holarctic

species. Greater potential for past population connectivity

was also thought to explain the lack of deeply diverged

clades in the Apollo butterfly across its Holarctic distribution

(Todisco et al., 2012).

The deep split between most Caucasus haplotypes and

other locations, combined with the geographically wide-

spread predominant European haplotype, suggests that the

origin of P. plantaginis is based in either south-eastern

Europe or the Caucasus. Our finding supports previous

hypotheses suggesting that P. plantaginis has a Eurasian

origin (Dubatolov, 2008). The question of whether the

originating populations of P. plantaginis were within the

Caucasus, or in south-eastern Europe poses fundamental

questions concerning the evolution of polymorphism and

sexual dimorphism. Within the European–North Americangroup, males have polymorphic hindwings (yellow or

white) and females can vary gradually between yellow and

dark red. However, within the Caucasus group both males

and females are reddish to varying degrees and very few

males are yellow. It is interesting that P. plantaginis

became highly sexually dimorphic over a large portion of

its distribution because sexual dimorphism does not appear

in other closely related members of the subfamily Arctiinae

(Dubatolov, 2008). Given that sexual monomorphism

appears to be ancestral in arctiines (using phylogenetic

relationships described in Dubatolov, 2008), we hypothesize

that the Caucasus population and the red coloration of P.

plantaginis represents the ancestral form. A more thorough

understanding of phylogenetic relationships within the Arc-

tiinae is needed before more detailed inference methods

can be applied to explore hindwing colour evolution with

a high degree of confidence.

Suitable climatic conditions still exist across much of the

distribution of P. plantaginis according to our SDM and

should allow for gene flow at some level. This differs

from the distribution model for the Holarctic-occurring

Parnassius phoebus (Todisco et al., 2012), which appears to

be more dependent on cold climate for dispersal than

P. plantaginis. Despite the possibility for gene flow across

large areas of suitable climate, we found that recent gene

flow has not occurred between most populations. High

levels of current population structure suggest that migration

among many populations may be rare, but not impossible

because we detected recent migration between two Japanese

populations despite the high degree of population differen-

tiation (FST = 0.26–0.28). Evidence of recent migration inJapan and higher rates of migration into Estonia from both

central Europe and Finland (but not between central

Europe and Finland) suggests that some form of selection

by predators, mates or environment may limit gene

flow between some populations. The large spatial area

of genetic populations probably makes intra-population

connectivity an important influence of male hindwing

colour polymorphism and female variation. Detailed future

analyses will be important to understanding how intra-

population dynamics may influence the hindwing warning

signal colour.

What defines a species is not always clear (Mallet, 2008;

Petit & Excoffier, 2009; Lumley & Sperling, 2011). Our

results show that populations of P. plantaginis at opposite

ends of the distribution are highly differentiated, but many

adjacent populations exhibit more moderated levels of popu-

lation structure. For instance, Caucasus populations are dif-

ferentiated the most from Japanese and North American

populations, but are less differentiated from neighbouring

European populations (Table 4). Regardless of the level of

genetic divergence between Caucasus and other populations,

individuals from the Caucasus are the most diverged in

hindwing colour. Prezygotic isolation is often held to be a

critical component in ultimately determining whether intra-

specific divergence and speciation will take place (Panhuis

et al., 2001). We know from previous work that the hind-

wing coloration of males can influence mating success in the

polymorphic Finnish population (Nokelainen et al., 2012),

but this has not been tested for the red Caucasus and nearby

non-red populations in the wild. Lepidopteran pheromones

can also provide clues concerning species delimitation (Lum-

ley & Sperling, 2011). During fieldwork in Japan, Canada,

Alaska, and recently in Caucasus, we found that pheromones

of Finnish females consistently attracted only P. plantaginis

males (R.H.H., J.M. & Atsushi Honma, pers. obs.). Further-

more, we observed no obvious signs of mating hesitancy in

laboratory crosses between specimens from distant popula-

tions (n = 59 pairs between Finnish and Japanese individu-als, n = 20 pairs between Caucasus and Finnish individuals;J.M. & Bibiana Rojas, unpublished data). Detailed studies of


10

R. H. Hegna et al.

sexual selection in laboratory and field conditions, along with

better sampling in Turkey and the lower Caucasus range, will

be needed to fully understand the influence of sexual selec-

tion on population divergence.

Overall, our study suggests that recent climatic isolation,

rather than past isolation during the LGM, is an ongoing

major influence in the genetic structure of P. plantaginis at

continental scales, along with sexual selection (Nokelainen

et al., 2012) and predation (Nokelainen et al., 2012; Hegna

et al., 2013; Hegna & Mappes, 2014) at other spatial scales.

Indications of more recent genetic differentiation, probably

due to the combination of these factors, raises the possibility

that incipient speciation is taking place in portions of the

distribution, such as the Caucasus. The apparent shift from

sexual monomorphism to distinct sexual dimorphism in col-

oration outside the Caucasus region raises the possibility that

mate choice, in addition to isolation, may have contributed

to the divergence of male coloration. Prezygotic isolation

may be a factor maintaining phenotypic and genetic

differences in the Caucasus if immigration from Europe does

happen periodically, but only direct experimentation will be

able to confirm mate choice. It is also possible that predator-

driven selection on the hindwing warning signal is more

intensive in the Caucasus, favouring warning signal mono-

morphism, as is expected for aposematic species (Joron &

Mallet, 1998). Future work with P. plantaginis promises to

contribute to our understanding of the evolution of colour

polymorphism, sexual dimorphism, and warning coloration.

ACKNOWLEDGEMENTS

We thank the following people who assisted in the collection

of moths on such a large scale: Ossi Nokelainen, T€onis Ta-

sane, Lauri Mikonranta, Kari Kulmala, Otmar Wanninger,

Felix Sperling, Jordi Maggi, Reza Zahiri, Kyle Johnson, Jona-

than Hegna, Chuck Harp, Evgenij Derzhinski, Sergey Sinev,

Alexej Matov, Venera Tyukmaeva, Ted Pike, Jostein Engdal,

Julien Delisle, Vladamir Gusarov, Leif Aarvik, and Robert

Mower. Sari Viinikainen, Carol Gilsenan, and Roghaieh

Ashrafi assisted in lab work. We thank the following muse-

ums for loaning collections or allowing us to visit: the Zoo-

logical Museum of the Zoological Institute of the Russian

Academy of Sciences, University of Oslo, National Royal

Museum of Sweden, University of Helsinki, the American

Museum of Natural History, University of Alberta, Brigham

Young University, University of Wisconsin-Madison, and the

Finnish Museum of Natural History. We also thank Emily

Knott, Niklas Wahlberg, Kyle Summers, Mathieu Joron, Tho-

mas Jones, and Swanne Gordon who improved the quality of

this manuscript with their comments. Permits included: the

Swiss National Park (permit no. CH-4168), Hohe Tauen

National Park (permit no. 21303-96/8/11-2010, permit no.

SP3-NS-1545/2010), Waterton National Park (WL-2009-

2915), US Fish and Wildlife Service (KN-11-001), and Alaska

Department of Fish and Game (FH11-III-002-SA). Finnish

Centre of Excellence in Biological Interactions 2012–2017

(no. 252411) and BIOINT graduate school provided funding

(R.H.H.).

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 Supplementary methods.

Appendix S2 Supplementary results.

BIOSKETCHES

Robert Hegna is a former PhD student at the University of

Jyv€askyl€a in Finland. His research interests include the evolu-

tionary ecology and behaviour of aposematic species.

Juan Galarza is currently a postdoc at the University of

Jyv€askyl€a working on the evolution of aposematism, and the

ecology of insect social behaviour.

Johanna Mappes is a professor of evolutionary ecology at

the University of Jyv€askyl€a interested in evolution of animal

signalling and predator–prey interactions. She leads the Cen-tre of Excellence in Biological Interactions research group.

Author contributions: J.M., R.H.H. and J.A.G. conceived the

ideas; R.H.H. collected the data; R.H.H. and J.A.G. analysed

the data; and R.H.H. led the writing.

Editor: Luiz Rocha


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