ORIGINAL ARTICLE

doi:10.1111/evo.12133

GENETIC DIFFERENTIATION AND SELECTIONAGAINST MIGRANTS IN EVOLUTIONARILYREPLICATED EXTREME ENVIRONMENTSMartin Plath,1,2 Markus Pfenninger,3 Hannes Lerp,1 Rudiger Riesch,4,5 Christoph Eschenbrenner,1 Patrick A.

Slattery,1 David Bierbach,1 Nina Herrmann,6,7 Matthias Schulte,6,7 Lenin Arias–Rodriguez,6 Jeane Rimber

Indy,6 Courtney Passow,8 and Michael Tobler8

1J. W. Goethe-University Frankfurt/M., Evolutionary Ecology Group, Max-von-Laue Str. 13, 60438, Frankfurt,

a. M., Germany2E-mail: mplath@bio.uni-frankfurt.de

3J.W. Goethe-University Frankfurt/M., Biodiversity & Climate Research Centre, Molecular Ecology Group, Biocampus

Siesmayerstraße 60323, Frankfurt, a. M., Germany4Department of Biology & W. M. Keck Center for Behavioral Biology, North Carolina State University, 127 David Clark

Labs, Raleigh, North Carolina 276955Department of Animal & Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom6Division Academica de Ciencias Biologicas, Universidad Juarez Autonoma de Tabasco (UJAT), C.P. 86150, Villahermosa,

Tabasco, Mexico7University of Potsdam, Institute of Biochemistry & Biology, Unit of Animal Ecology, Maulbeerallee, 1, 14469 Potsdam,

Germany8Department of Zoology, Oklahoma State University, 501 Life Sciences West, Stillwater, Oklahoma 74078

Received October 11, 2012

Accepted April 2, 2013

Data Archived: Dryad doi:10.5061/dryad.7cn23

We investigated mechanisms of reproductive isolation in livebearing fishes (genus Poecilia) inhabiting sulfidic and nonsulfidic

habitats in three replicate river drainages. Although sulfide spring fish convergently evolved divergent phenotypes, it was unclear

if mechanisms of reproductive isolation also evolved convergently. Using microsatellites, we found strongly reduced gene flow

between adjacent populations from different habitat types, suggesting that local adaptation to sulfidic habitats repeatedly caused

the emergence of reproductive isolation. Reciprocal translocation experiments indicate strong selection against immigrants into

sulfidic waters, but also variation among drainages in the strength of selection against immigrants into nonsulfidic waters.

Mate choice experiments revealed the evolution of assortative mating preferences in females from nonsulfidic but not from

sulfidic habitats. The inferred strength of sexual selection against immigrants (RIs) was negatively correlated with the strength

of natural selection (RIm), a pattern that could be attributed to reinforcement, whereby natural selection strengthens behavioral

isolation due to reduced hybrid fitness. Overall, reproductive isolation and genetic differentiation appear to be replicated and

direct consequences of local adaptation to sulfide spring environments, but the relative contributions of different mechanisms of

reproductive isolation vary across these evolutionarily independent replicates, highlighting both convergent and nonconvergent

evolutionary trajectories of populations in each drainage.

KEY WORDS: Ecological speciation, isolation-by-adaptation, local adaptation, Poecilia mexicana, reinforcement, sexual isolation.

1C© 2013 The Author(s).Evolution

MARTIN PLATH ET AL.

The question of how reproductive isolation between diverg-

ing populations evolves remains essential in evolutionary biol-

ogy. Theory distinguishes between pre- and postzygotic isolat-

ing mechanisms, which can act independently or in concert to

create varying degrees of reproductive isolation (Coyne and Orr

2004). Ecological gradients are particularly well suited to study

the potential role of local adaptation in facilitating reproductive

isolation, because they can influence population genetic struc-

ture via divergent natural selection. If divergent selection across

such gradients is sufficiently strong, it may result in ecologically

based reproductive isolation through reduced fitness of dispersers

between selective environments (Hendry 2004; Nosil 2004; Nosil

et al. 2005) and/or reduced fitness of hybrids between individuals

adapted to alternate ecological conditions (Hatfield and Schluter

1999; Via et al. 2000; Rundle 2002). In addition, local adaptation

is often intertwined with the processes of reinforcement during

which selection promotes assortative mating between individuals

adapted to different environmental conditions (Boughman et al.

2005; Rundle et al. 2005; Snowberg and Benkman 2009; Lenor-

mand 2012). Together, these mechanisms are among the central

components of ecological speciation (Schluter 2000, 2001; Run-

dle and Nosil 2005; Nosil 2012; but see Langerhans and Riesch

2013).

Convergent differentiation along replicated selective gradi-

ents provides strong evidence for a central role of natural selection

in driving adaptive trait divergence (Clarke 1975; Endler 1986;

Wood et al. 2005; Elmer and Meyer 2011). In addition, systems in

which populations of the same species repeatedly evolved repro-

ductively isolated ecotypes with similar phenotypes are valuable

to study the evolution of reproductive isolation (Schluter and

Nagel 1995; Johannesson 2001). Examples of convergent eco-

logical speciation have been documented in organisms inhabiting

different habitat types, exploiting different resources, and experi-

encing different predator regimes (reviewed by Langerhans and

Riesch 2013). Nevertheless, it is yet unclear (1) whether repro-

ductive isolation barriers evolve convergently across replicated

selective gradients in the same way as adaptive phenotypic traits,

and (2)—as gene flow between populations can constrain diver-

gence (Slatkin 1987; Hendry et al. 2001)—how convergent or

divergent isolation barriers affect gene flow patterns (Rasanen

and Hendry 2008). This is of special interest because speciation

can be thought of as a continuum, and pairs of populations facing

the same divergent ecological selection may be at different stages

along the continuum between panmixis and complete reproduc-

tive isolation (Hendry 2009; Nosil et al. 2009a; Langerhans and

Riesch 2013).

One ecological gradient that fundamentally alters the

evolutionary trajectories of populations arises due to naturally

occurring, toxic hydrogen sulfide (H2S). H2S-toxicity results

predominantly from its interference with mitochondrial bioen-

ergetics and blood oxygen transport, which inhibits aerobic

respiration (Bagarinao 1992; Grieshaber and Volkel 1998). Sup-

pression of aerobic respiration is aggravated by the reactivity of

H2S leading to extreme hypoxia in aquatic environments. Adverse

effects notwithstanding a number of livebearing fishes (family

Poeciliidae) thrive in sulfide springs exhibiting continuously

high concentrations of H2S (Tobler and Hastings 2011; Tobler

and Plath 2011). Adaptation to the toxic conditions in these

springs is mediated by complex phenotypic changes, including

physiological, morphological, and life-history traits (Riesch et al.

2010, 2011a,c; Tobler et al. 2011). In particular, sulfide spring

fish—as compared to close relatives in nonsulfidic habitats—are

characterized by increased head size and a correlated increase in

gill surface area (Tobler et al. 2008a, 2011; Riesch et al. 2011c;

Tobler and Hastings 2011), which facilitates efficient oxygen ac-

quisition in the hypoxic environment and directly affects survival

(Plath et al. 2007a, 2010a). Intriguingly, adaptation to H2S gave

rise to striking patterns of phenotypic convergence among evo-

lutionarily independent lineages both within and among species

(Riesch et al. 2010; Tobler et al. 2011; Tobler and Hastings 2011).

Adaptation to H2S is best studied in fish of the genus Poe-

cilia in southern Mexico, where three evolutionarily independent

lineages—two within Poecilia mexicana and a more divergent

one described as Poecilia sulphuraria—have colonized sulfidic

springs in three river drainages. Phylogenetic analyses suggest

that colonization of H2S-springs occurred first in the Pichucalco

drainage by P. sulphuraria, which show strong phylogenetic

affinity to present Northern Mexican Poecilia mexicana liman-

touri (Tobler et al. 2011). Invasion of H2S-springs in two other

drainages was more recent, and those sulfide spring ecotypes each

are closely related to P. mexicana in adjacent nonsulfidic habitats

in their respective drainage (Tobler et al. 2011). Still, introgres-

sion of nuclear genes cannot be ruled out, as previous phylogenetic

analyses were solely based on mitochondrial markers (Tobler et al.

2011).

Potential consequences of local adaptation on gene flow and

reproductive isolation between populations from different habi-

tat types have only been investigated in depth in the Tacotalpa

river drainage. There, P. mexicana inhabits both nonsulfidic and

H2S-containing surface habitats and subterranean ecosystems, in

which they are under additional selection from permanent dark-

ness (Tobler et al. 2008a; Riesch et al. 2011a,b). Population ge-

netic analyses indicated reduced gene flow and strong genetic

differentiation among populations residing in ecologically differ-

ent habitat types (Plath et al. 2007a, 2010b), and reproductive

isolation is at least in part mediated by a combination of natural

and sexual selection against immigrants from other habitat types

(Tobler 2009; Tobler et al. 2009).

To date, the degree of reproductive isolation between H2S-

adapted lineages of P. mexicana-like fish and adjacent populations

2 EVOLUTION 2013

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS

from nonsulfidic habitats in other river drainages remains to be

investigated. It is yet unclear whether the consequences of local

adaptation for gene flow are similar across replicated selective gra-

dients and if similar patterns of natural and sexual selection pro-

moting reproductive isolation are found. Specifically, the present

study poses the following questions: (1) Are there any reductions

in gene flow between population pairs inhabiting nonsulfidic and

H2S-containing habitats within different drainages? To address

this, we used 17 unlinked nuclear microsatellites to quantify pat-

terns of gene flow between ecotypes within drainages. Based on

previous findings from the Tacotalpa river drainage (Plath et al.

2007a, 2010b), we predicted reduced gene flow between popu-

lations inhabiting divergent habitat types over short geographi-

cal distances and in the absence of physical barriers; a pattern

that would be indicative of “isolation-by-adaptation” (Nosil et al.

2009b). (2) How strong is the contribution of natural selection

against migrants (RIm) in reducing gene flow among ecotypes?

No barriers other than water chemistry prevent fish movement

across habitat types in our study system; hence, we used recip-

rocal translocation experiments to quantify mortality of migrant

individuals. Previous results from the Tacotalpa river drainage

indicate that viability of fish is low when transferred between

nonsulfidic and H2S-containing habitats (Tobler et al. 2009). (3)

What is the role of sexual selection against immigrants (RIs) in

reducing gene flow among ecotypes? Both male and female mate

choice experiments were conducted, for which focal fish were pre-

sented with a potential resident and immigrant mate. Morpholog-

ical differentiation among ecotypes (Tobler et al. 2011) provides

potential cues for assortative mating. (4) Does total prezygotic

isolation (T), calculated from empirically established values for

RIs and RIm, predict the degree of population genetic differenti-

ation in the three drainages? If selection against immigrants—as

determined in this study—accounts for the majority of reproduc-

tive isolation, total prezygotic isolation should correlate with the

extent of gene flow within each of the three individual drainages.

Alternatively, other reproductive isolation barriers not addressed

here will need to be considered in future studies.

Material and MethodsSTUDY SITES AND SAMPLE COLLECTION

Poecilia spp. were collected in the vicinity of the city of Teapa.

Here, the mountains of the Sierra Madre de Chiapas meet the

wide floodplains of northern Tabasco. Sulfide spring complexes

inhabited by Poecilia spp. are located in the foothills of the Sierra

Madre and are distributed across three major tributaries of the

Rıo Grijalva (from east to west: Rıos Tacotalpa, Puyacatengo,

and Pichucalco). Nonsulfidic and H2S-containing habitats within

each drainage are interconnected and not separated by physical

barriers that would prevent fish migration. All three rivers even-

Figure 1. Overview of the study area in Mexico with reference

cities in gray. Drainages are underlined, numbers indicate sample

sites, black arrows indicate sulfidic sites, and white arrows non-

sulfidic sites. 1, Arroyo Caraco; 2, La Gloria; 3, Puente El Azufre

II; 4, Banos del Azufre; 5, Puyacatengo road crossing; 6, La Lluvia;

7, Arroyo Cristal; 8, El Azufre I; 9, Arroyo Bonita; 10, El Azufre II.

Sulfidic waters usually extend from the point of the black arrows

downstream to the nearest confluence with nonsulfidic waters;

the sole exception being La Gloria, which is sulfidic for only a few

hundred meters.

tually join the Rıo Grijalva and are widely interconnected in the

lowlands during the wet season; however, the sulfide spring com-

plexes are permanently separated by mountains (Miller 1966).

H2S in this region is likely associated with volcanic activity of

the Chichon Volcano (Rosales Lagarde et al. 2006), and average

sulfide concentrations in the spring systems range between 23

and 190 μM (Tobler et al. 2006, 2011). In the Rıo Pichucalco

drainage, the sulfide ecotype has been described as a distinct

species, P. sulphuraria (Alvarez 1948), which is endemic to sul-

fide spring complexes at the Banos del Azufre and Rancho La

Gloria.

Fish were caught with a seine (4 m long, 4 mm mesh-width).

Different sites were sampled for each experiment; an overview

is provided in Fig. 1. To ensure that only resident ecotypes were

sampled (and not migrating nonresidents), fish were not collected

in the mixing zone of sulfidic and nonsulfidic waters. All speci-

mens were individually screened to match their respective ecotype

using external morphological features directly upon capture, and

all fish collected exhibited the predicted phenotype (see Tobler

et al. 2011 for ecotype-specific morphological differences), but

it is possible that hybrids might go undetected when using this

method. Several other studies that tried to detect ecological speci-

ation used a design in which the divergence between populations

from comparable environments was compared to the divergence

between populations inhabiting dissimilar environments (e.g., Lu

and Bernatchez 1999; Ogden and Thorpe 2002), and ecological

EVOLUTION 2013 3

MARTIN PLATH ET AL.

speciation was inferred when divergence between populations

from dissimilar environments was greater than between popula-

tions from similar environments. Because phylogenetically in-

dependent invasion of sulfide springs and convergent phenotypic

divergence between sulfidic and nonsulfidic ecotypes was already

established in this system (Riesch et al. 2010; Tobler et al. 2011)

and previous population genetic investigations in the Tacotalpa

system included repeated samples within the different habitat

types (Plath et al. 2007a, 2010b), our present study concentrated

on genetic differentiation and potential gene flow at the interface

between populations from sulfidic and nonsulfidic habitats.

POPULATION GENETIC ANALYSIS

We used 17 nuclear microsatellite loci to genotype N = 180 fish

from six sites (Fig. 1). In the Tacotalpa drainage, samples were

obtained from El Azufre I (sulfidic, N = 25) and Arroyo Bonita

(N = 25); in the Puyacatengo drainage from La Lluvia (sulfidic,

N = 25) and Puyacatengo road crossing (N = 25); and in the

Pichucalco drainage from the Banos del Azufre (sulfidic, N = 25)

and the western branch of the Rıo El Azufre at “Puente El Azufre

II” (N = 55).

We amplified 12 tri-, tetra-, and penta-nucleotide microsatel-

lites specifically designed for P. mexicana and P. sulphuraria

(Slattery et al. 2012), as well as five di-nucleotide microsatel-

lites previously designed for the closely related Amazon molly

(P. formosa; Tiedemann et al. 2005). DNA was extracted from

ethanol-preserved tissue samples (fin clips) using the DNeasy

Blood and Tissue Kit (Qiagen, Hilden, Germany) according to

the manufacturer’s recommendations. Primer pairs for the 17 loci

were arranged in three separate multiplex reactions (primer mix

1: EAI 2566, EAI 343, EAI 916, EAI 1039, EAI 808, EAI 568,

EAI 1426; primer mix 2: GAI29A, GAV18, GTII33, GAI29B,

GTI13B; primer mix 3: AB 195, AB 231, EAI 475, EAI 999,

EAI 96) and amplified using the Type-it Microsatellite poly-

merase chain reaction (PCR) kit (Qiagen). PCR included an initial

denaturation step for 5:00 min at 95◦C, 30 cycles of 1:30 min at

60◦C, and 0:30 min at 72◦C, followed by a final extension step

for 30:00 min at 60◦C. The 5 μL reaction mix included 2.5 μL

Type-it master mix, 0.4 μL primer mix, 0.4 μL Q-solution, 0.9 μL

RNase-free water, and 0.8 μL template DNA.

ARLEQUIN version 3.5 (Excoffier and Lischer 2010) was

used to calculate expected (HE) and observed heterozygosity

(HO), to test for deviations from Hardy–Weinberg equilibrium and

to calculate pairwise FST-values between populations within each

drainage. Allelic richness (A) was calculated using FSTAT version

2.9.3.2 (http://www2.unil.ch/popgen/softwares/fstat.htm). All de-

scriptive statistics are presented in Table S1. We tested for null

alleles at each locus using Micro-checker version 2.2.3 (van Oost-

erhout et al. 2004) while pooling all P. mexicana from nonsulfidic

sites. STRUCTURE version 2.3.3 (Pritchard et al. 2000) was em-

ployed to identify the number of genetically distinct clusters (K) in

each drainage with the method presented by Evanno et al. (2005)

using the web-based tool STRUCTURE HARVESTER version

0.6.8 (Earl and von Holdt 2012). For each value of K = 1–4,

10 iterations were run using the admixture model with a burn-in

period of 100,000 generations, followed by 1,000,000 iterations.

Each simulation was performed using an ancestry model incor-

porating admixture, a model of correlated allele frequencies, and

no prior information on locations.

SELECTION AGAINST MIGRANTS

Natural selection against migrantsWe conducted reciprocal translocation experiments between rep-

resentative pairs of sulfidic and nonsulfidic habitats in all three

drainages using 20-L plastic buckets as experimental containers.

Two holes (18 × 32 cm) were cut on opposite sides of the buckets

and then sealed with 1.5-mm plastic mesh to maintain constant

exchange of water with the environment. Bucket lids were per-

forated with ∼50 small holes to facilitate air exchange. Experi-

mental containers were placed directly into a shallow area of the

natural habitats and equipped with a 3.5-cm layer of natural sub-

strate. Previous studies have shown that water conditions within

these mesocosms closely match the conditions in the adjacent

environment (Tobler et al. 2009).

Upon collection, fish were kept in insulated and aerated cool-

ers. Six haphazardly chosen individuals from a given site were

then introduced into an experimental bucket. Half of the buckets

at each site were set up with resident fish, half with fish from the

other habitat type. Transportation and handling times on average

were 66 min (range 45–95 min) and were balanced for resident

and translocated fish, so differential stress is unlikely to have af-

fected the results, particularly within replicates. Fish were sexed

and measured for standard length (SL) prior to introduction. Ex-

periments were terminated after 24 h to quantify mortality, and

surviving individuals were released at their original collection site.

A subset of data was reanalyzed from Tobler et al. (2009; one set

of N = 27 runs including the sulfidic El Azufre I and the nonsul-

fidic Arroyo Cristal in the Rıo Tacotalpa drainage) and Plath et al.

(2010b; one set of N = 40 runs including the sulfidic Banos del

Azufre and an adjacent nonsulfidic tributary in the Rıo Pichucalco

drainage). We completed additional replicates with different sites

for the Rıo Tacotalpa (N = 60 runs between the sulfidic El Azufre

II and the nonsulfidic Arroyo Bonita), the Rıo Pichucalco (N =60 runs between the sulfidic La Gloria springs and the adjacent

nonsulfidic Arroyo Caracol), and the Rıo Puyacatengo drainage

(N = 60 runs between the sulfidic La Lluvia springs and an adja-

cent nonsulfidic tributary). Essentially, this provided us with two

independent sets of replicates for evaluating the performance of

fish translocated between sulfidic and nonsulfidic sites for the

Tacotalpa and the Pichucalco drainages.

4 EVOLUTION 2013

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS

To analyze overall differences in survivability of potential

migrants across drainages and sites, we used a generalized linear

mixed model (GLMM) with a binomial error distribution and a

logit link function; survival (binary data: 1 = survived; 0 = not

survived) was used as the dependent variable. We included “pop-

ulation of origin” (sulfide spring or nonsulfidic water), “testing

environment” (sulfidic or nonsulfidic), “sex” of the test fish, as

well as “drainage” as fixed factors. All possible second- and third-

order interactions of the fixed factors were included in the model.

Body size of the test fish could have affected survivability (Tobler

et al. 2011), so we included “standard length” as a covariate. In ad-

dition, “replicate” (i.e., site combination nested within drainage)

as well as “Bucket ID” (nested within drainage × replicate × ori-

gin × environment) were included as random factors. Removal

of nonsignificant interaction terms did not decrease Akaike’s in-

formation criterion (AIC) for the model; thus, the full model was

analyzed. All survival rates are presented as estimated marginal

means.

Reproductive isolation through mate choiceRecent studies acknowledge both the role played by female and

male mate choice in facilitating reproductive isolation among di-

verging lineages (e.g., Boul et al. 2007; Gregorio et al. 2012),

even though mostly monogamous species were considered so far

(Pierotti et al. 2009; Puebla et al. 2011). Thus, we examined both

female and male mating preferences for resident versus immi-

grant phenotypes using simultaneous, binary association prefer-

ence tests. Female association preferences in another poeciliid

fish, the green swordtail (Xiphophorus hellerii), were good pre-

dictors of male reproductive success when females are allowed to

mate after the association tests (Walling et al. 2010). Furthermore,

male association preferences in P. mexicana predict the number

of copulations with the preferred female (Plath et al. 2006).

Tests were conducted using wild-caught fish at the aqua-

culture facilities of the Academic Division for Biological Sci-

ences, Universidad Juarez Autonoma de Tabasco (DACBIOL-

UJAT), Villahermosa. Fish were caught from proximate sulfidic

and nonsulfidic sites in each of the three drainages (Table S2;

Fig. 1), transferred into closed and aerated Sterilite R© containers,

and immediately brought to the laboratory, where they were kept

separated by sex in aerated 70-L tanks for at least 24 h to al-

low acclimation to laboratory conditions. Fish were kept under

a natural, approximately 12 h:12 h light:dark cycle at ambient

temperatures and were fed ad libitum with commercially avail-

able flake food daily. All tests were conducted in nonsulfidic

water, as fish from sulfide-free sites cannot acclimate to sulfide

conditions.

Tests were conducted in three identical portable test tanks

(42.6 × 30 × 16.5 cm) built with UV-transparent Plexiglas. Each

tank was visually divided into three equally sized zones by black

marks on the outside. The central zone was designated as a neutral

zone, the two lateral zones as preference zones. Two stimulus

fish were presented in two smaller auxiliary tanks (19.5 × 30 ×14.5 cm) on either side of the test tank. To avoid disturbance from

the outside, we set up all test tanks in large oval tubs that were

filled with water to the level inside the test tanks (see Fig. 1 in

Bierbach et al. 2011). The entire set-up was placed on a shelf of

about 1 m height, and the observer was standing approximately

1.5–2 m away from the test apparatus and observed the fish from

diagonally above.

Focal fish were tested with stimulus fish of the opposite

sex, one from the same population and one from the respective

opposite ecotype within a given drainage. Before each trial, the

two stimulus fish were each placed into the auxiliary tanks (see

Table S2). Once the stimulus fish were swimming freely, a focal

fish was introduced into the center of the test tank. Test fish

typically froze on the bottom of the test tank up to a few minutes

after they were introduced, so a trial began only after the focal

individual resumed swimming freely in the water column. We

subsequently measured the time the focal individual spent in each

preference zone during a 5-min observation period. To detect side

biases, the stimuli were switched between sides immediately after

the first 5-min observation period and measurement was repeated.

Upon termination of the trials, SL was measured in all fish. Focal

and stimulus individuals were used only once.

We summed association times near each stimulus from the

two trials (before and after switching of side assignments) and

tested for preferences within each test situation (i.e., for each

population and sex separately) by comparing individual strength

of preference (SOP)-values against a “no choice” expectation of

SOP = 0 using one-sample t-tests. The SOP was calculated as:

SOP = time near resident − time near immigrant

time near resident + time near immigrant.

Hence, SOP-values could range from +1 (complete prefer-

ence for the resident ecotype) to −1 (complete avoidance of the

resident ecotype). SOP-values were used as the dependent vari-

able in a fully factorial general linear model (GLM) to test for

differences among drainages, ecotypes, and sexes. Even though

an attempt was made to use size-matched stimulus pairs, it was

not always possible to assign equal-sized stimulus pairs from our

catches, and we included difference in body size (SL) between the

two stimulus fish (resident–immigrant) as a covariate in the anal-

ysis. Poecilia mexicana females (Tobler et al. 2008b; Bierbach

et al. 2011) and males (Plath et al. 2006) prefer larger mating

partners irrespective of their own body size.

EVOLUTION 2013 5

MARTIN PLATH ET AL.

Figure 2. Population assignment using STRUCTURE version 2.3.2. K = 2 was recovered as the most likely number of genetic clusters

in every drainage separately; given are individual relative assignment values in black (sulfide springs; left side) and white (sulfide-free

waters; right side), divided by a black line and sorted by the relative assignment score for each population separately.

RIm, RIs, and total isolation (T)The individual contribution of selection against migrants (RIm)

was calculated as 1 − (migrant survival/resident survival) and the

individual contribution of sexual isolation (RIs) as 1 − (immigrant

mate choice/resident mate choice). This formula was originally

developed to contrast heterotypic and homotypic mating (Nosil

2004), and we adjusted it for the binary choice tests employed in

this study by using association times as a proxy of individuals’

likelihood to mate. To uncover a potential signature of reinforce-

ment, we tested for a negative correlation between RIs and RIm,

which is predicted if low-migrant mortality increases the likeli-

hood of heterotypic mating allowing for direct selection on RIs

(see Howard 1993; Higgie et al. 2000; Hobel and Gerhardt 2003;

Nosil et al. 2007). We used RIs-values as dependent variable

in a GLM, while including RIm as a covariate and “sex” as a

factor.

Following Nosil (2004), we estimated the relative contribu-

tions of reduced migrant survival and sexual isolation to total pre-

mating isolation (T; ranging from 0 to 1; see Ramsey et al. 2003).

In these sulfidic/nonsulfidic systems, natural selection against

migrants has an immediate impact on fitness (i.e., survival) prior

to sexual selection. Thus, the absolute contribution of selection

against migrants is RIm, whereas the absolute contribution of sex-

ual isolation is RIs(1 − RIm), and total prezygotic isolation is T =RIm + RIs(1 − RIm). We tested for a correlation between total

isolation (average T for males and females of each population)

and mean genetic assignment to the nonresident genetic cluster

(obtained from a population genetic assignment test and averaged

for the ten runs for K = 2) using a nonparametric Spearman rank

correlation.

ResultsPOPULATION GENETIC STRUCTURE

The population assignment test identified K = 2 as the most likely

number of genetically distinct clusters in all three drainages with

all runs separating the sulfide-spring populations from the pop-

ulations from surrounding nonsulfidic waters (Fig. 2). Although

this separation is relatively clear in the Tacotalpa and Pichucalco

drainages, it is considerably weaker in the Puyacatengo drainage,

where some degree of gene flow across habitats was uncovered,

even though first-generation migrants were not detected in any

drainage (Fig. 2). The strong differentiation between ecotypes

was corroborated by high pairwise FST-values of 0.296 (Pichu-

calco) and 0.203 (Tacotalpa drainage). Differentiation in the Puy-

acatengo drainage was lower, but still significant (FST = 0.103).

6 EVOLUTION 2013

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS

Table 1. Results from a generalized linear mixed model (with binomial error distribution and logit link function) on survival rates during

the reciprocal translocation experiment. As random factors we included “site nested within drainage” as well as “bucket ID nested within

site and drainage.” The model included “population of origin,” “testing environment,” “drainage,” and “sex” as fixed factors, and “SL”

as a covariate. Significant P-values are in bold face. AIC = 7,969.859.

Random effects Estimate SE z P

Replicate (drainage) 0.551 0.621 0.887 0.375Bucket ID (drainage × replicate × origin × environment) 0.563 0.213 2.624 0.008Fixed effects F df1 df2 POrigin 1.934 1 1459 0.165Environment 77.895 1 1459 <0.001Drainage 1.312 2 1459 0.269Sex 34.298 1 1459 <0.001SL 101.807 1 1459 <0.001Origin × Environment 196.564 1 1459 <0.001Origin × Drainage 8.982 2 1459 <0.001Origin × Sex 2.558 1 1459 0.110Environment × Drainage 5.028 2 1459 0.007Environment × Sex 1.158 1 1459 0.282Drainage × Sex 6.202 2 1459 0.002Origin × Environment × Drainage 11.672 2 1459 <0.001Origin × Environment × Sex 4.048 1 1459 0.044Location × Environment × Sex 3.851 2 1459 0.021Origin × Drainage × Sex 0.256 2 1459 0.774

RECIPROCAL TRANSLOCATION EXPERIMENT

The interaction term between “origin of fish” (sulfidic vs. nonsul-

fidic habitat) and “test environment” (sulfidic vs. nonsulfidic habi-

tat) had a highly significant effect explaining survival of fish, indi-

cating habitat-specific performance-differences between ecotypes

(Table 1). Resident fish consistently had higher survival rates than

immigrant fish, and mortality was highest for fish from nonsulfidic

waters being transferred into sulfidic water (Fig. 3). Nevertheless,

a significant three-way interaction between “origin × test envi-

ronment × drainage” indicates that performance differences were

not uniform across drainages. Survivability of H2S ecotypes in

nonsulfidic sites varied across drainages (Fig. 3A), whereas eco-

types from nonsulfidic waters consistently had low survival rates

in sulfidic environments (Fig. 3B). In the Tacotalpa drainage,

survivability of H2S ecotypes in nonsulfidic environments was

almost as low as survivability of the ecotype from nonsulfidic wa-

ter in the sulfidic environment. In contrast, survivability of H2S

ecotypes in nonsulfidic habitats of the Puyacatengo and the Pichu-

calco drainages was comparatively high. No differences were un-

covered among replicate site pairs within drainages, suggesting

that survivability estimates for each drainage were not caused

by the specific sites we chose for this experiment. Our model

also revealed a strong overall effect of the factor “sex” (Table 1),

and males generally exhibited lower survival rates than females

(Fig. 3C). This pattern varied to some extent across drainages and

between sulfidic and nonsulfidic locations (significant three-way

interaction “drainage × environment × sex”; Table 1). Our model

also identified a significant effect of body size (SL) on survivabil-

ity, and a post hoc Pearson correlation with standardized residuals

for survivability detected a significant negative correlation with

SL (rp = −0.220, P < 0.001, N = 1,482; Fig. 3D).

FEMALE AND MALE MATE CHOICE

In the GLM comparing the SOP of individual focal fish for mating

partners from the same population of origin, a significant main

effect of “ecotype” and a significant interaction term of “sex by

ecotype” were uncovered (Table 2). Closer inspection of SOP

values indicates that only fish from nonsulfidic, but not from sul-

fidic habitats, showed a preference for resident over immigrant

mating partners, and females generally expressed stronger pref-

erences than males (Fig. 4). Qualitatively, fish from the Tacotalpa

drainage showed weaker association preferences than fish from

the other two drainages (Fig. 4A,B); however, neither the main

effect nor any interaction involving “drainage” were statistically

significant (Table 2).

RIm AND RIs

A GLM using RIs as the dependent variable uncovered a signif-

icant effect of the covariate RIm (F1,8 = 10.00, P = 0.013, η2p =

0.555). Closer investigation of the relationship between both vari-

ables revealed a negative correlation (Fig. 5). There was also a

significant difference between sexes (F1,8 = 5.46, P = 0.048,

EVOLUTION 2013 7

MARTIN PLATH ET AL.

Figure 3. Survival in reciprocal translocation experiments. Within each drainage, fish were either transferred from (A) sulfidic to

nonsulfidic sites or (B) from nonsulfidic to sulfidic sites with H2S-ecotypes in black and ecotypes inhabiting nonsulfidic waters in white.

R, resident fish; T, transplanted fish. (C) Visualization of the sex-effect from our generalized linear mixed model (GLMM) with females

in light gray and males in dark gray. (A–C) Shown are estimated marginal means (+ SEM; SL was fixed at 34.41 mm) from a GLMM.

(D) Regression of residual survival versus SL.

η2p = 0.406) owing to males showing higher values for RIs than

females. The interaction term was not significant (F1,8 = 2.79,

P = 0.133, η2p = 0.259).

TOTAL ISOLATION AND GENE FLOW

Total prezygotic isolation (T) differed significantly between sexes

(t5 = 3.30, P = 0.022) and was higher for males (mean ± SD:

0.842 ± 0.129) than females (0.564 ± 0.230). Mean values of total

prezygotic isolation did not correlate with population-wise means

of assignment to the nonresident genetic cluster, our estimate of

gene flow (Spearman rank correlation: rs = −0.49, P = 0.33, N =6 . . . ; Fig. 6). The same pattern was uncovered when only male

(rs = −0.77, P = 0.07, N = 6) or only female T-values were used

instead (rs = −0.14, P = 0.66, N = 6).

8 EVOLUTION 2013

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS

Table 2. Comparison of the strength of preference (SOP, see main text) for mating partners from the same population in males and

females across drainages (three levels) and ecotypes (two per drainage). The full factorial general linear model included the difference

in body size between the two stimulus individuals (SL own–SL alien ecotype) as a covariate. The interaction term “drainage × sex ×ecotype” was not significant (mean square = 0.56, F1, 354 = 0.61, P = 0.54, partial η2 = 0.003) and thus, removed from the final model.

Significant effects are highlighted in bold typeface.

Source df Mean square F P Partial η2

Sex 1 0.10 0.75 0.39 0.002Drainage 2 0.34 2.46 0.087 0.014Ecotype 1 0.86 6.34 0.012 0.018Sex × Drainage 2 0.13 0.99 0.37 0.006Drainage × Ecotype 2 0.29 2.14 0.12 0.012Sex × Ecotype 1 0.57 4.21 0.041 0.012Stimulus size difference 1 2.58 18.91 <0.001 0.050Error 356 0.14

Figure 4. Mate discrimination in female (A) and male (B) Poecilia spp. Within each drainage focal fish could choose between two

stimulus fish of the opposite sex, the respective ecotypes from sulfidic and nonsulfidic waters. Positive strength of preference (SOP)

values indicate preference for mating partners from the own population. Focal fish stemmed from sulfidic (•) and nonsulfidic sites (◦).

Results from two-sided one-sample t-tests are shown, testing against the null assumption of SOP = 0, where ** signifies P < 0.01 (from

left to right: t30 = −0.12, P = 0.91; t29 = 3.31, P = 0.002; t29 = −1.63, P = 0.12; t29 = 3.14, P = 0.004; t29 = −1.01, P = 0.32; t34 = 1.79, P =0.083; t29 = −0.86, P = 0.40; t29 = 2.85, P = 0.008; t29 = −0.93, P = 0.36; t29 = 0.73, P = 0.47; t29 = 1.52, P = 0.14; t30 = −0.70, P = 0.49).

Shown are means ± SEM.

DiscussionWe investigated genetic differentiation and patterns of gene flow

in livebearing fishes of the genus Poecilia, inhabiting nonsul-

fidic and H2S-containing habitats in three distinct river drainages.

We found reduced yet variable gene flow resulting in strong ge-

netic differentiation between ecotypes in all river drainages. The

varying degree of genetic differentiation across drainages is in-

dicative of population pairs in contrasting environments being

at different stages along the speciation continuum. Nevertheless,

high pair-wise FST-values in all cases suggest that adaptation

to H2S-containing habitats repeatedly caused the emergence of

reproductive isolation across evolutionarily independent popula-

tion pairs despite the lack of physical barriers that would prevent

fish migration. Translocation and mate choice experiments indi-

cated that both natural and sexual selection against immigrants

contribute to reproductive isolation between ecotypes, but their

relative contributions vary among drainages, reflecting unique

evolutionary trajectories in each replicate drainage. Because the

EVOLUTION 2013 9

MARTIN PLATH ET AL.

Figure 5. Reproductive isolation due to sexual selection (RIs) ver-

sus reproductive isolation due to selection against migrants (RIm)

for males (triangles) and females (diamonds) from three different

drainages. Closed symbols refer to RI-values estimated for fish

from sulfidic sites in sulfide-free habitats, open symbols refer to

RI-values estimated for fish from sulfide-free sites in sulfidic habi-

tats.

Figure 6. Proportion of immigrant assignment based on STRUC-

TURE version 2.3.2 versus total prezygotic isolation. Closed sym-

bols refer to immigration of fish from sulfidic sites into nonsulfidic

habitats, open symbols refer to immigration of fish from nonsul-

fidic sites into sulfidic habitats. Pi, Pichucalco drainage; Pu, Puya-

catengo drainage; T, Tacotalpa drainage.

inferred amount of gene flow did not correlate with total prezy-

gotic isolation estimated by our experiments, additional reproduc-

tive isolation barriers need to be explored to better understand all

factors contributing to speciation in this system.

GENETIC DIFFERENTIATION BETWEEN ECOTYPES

Divergent selection can reduce gene flow in natural populations,

but genetic structuring in relation to ecological variables is not

ubiquitous among systems in which phenotypically divergent

populations experience different selection pressures (reviewed in

Thibert-Plante and Hendry 2010). Potentially, fitness of dispersers

and their hybrids may not be sufficiently reduced among selective

environments to detect a reduction in gene flow in microsatellites

or other neutral loci (see Rasanen and Hendry 2008). Our pop-

ulation genetic analyses revealed that fish from nonsulfidic and

H2S-containing habitats in all three drainages were genetically

divergent and gene flow (estimated from STRUCTURE assign-

ment scores) was virtually absent between population pairs from

the Tacotalpa and Pichucalco drainages, corroborating previous

studies on the Tacotalpa drainage (Plath et al. 2007a, 2010b).

Even though genetic drift can significantly affect the frequency

of alleles at neutral loci and its effects cannot be conclusively

separated from those of divergent selection especially in cases of

very low gene flow (Thibert-Plante and Hendry 2010), our results

match previous findings of convergent phenotypic divergence be-

tween different ecotypes across all three drainages (Riesch et al.

2010; Tobler et al. 2011), suggesting “isolation-by-adaptation”

(Nosil et al. 2009a). In contrast to the Tacotalpa and Pichucalco

drainages, a low level of gene flow was detected in the Puya-

catengo drainage, suggesting that this population pair falls onto

a different point along the speciation continuum than the other

two population pairs (Hendry 2009; Nosil et al. 2009a; Langer-

hans and Riesch 2013). More specifically, there are two drainages

(Pichucalco and Tacotalpa) in which the speciation process is

well advanced, whereas the population pair from the Puyacatengo

drainage is still in the process of speciation. This may be due to

a stronger spatial and temporal heterogeneity in the Puyacatengo

drainage compared to the other two drainages (authors, unpubl.

data), or it could be due to a more recent colonization of sulfidic

springs.

NATURAL SELECTION AGAINST IMMIGRANTS

Translocation experiments revealed that resident ecotypes in each

habitat had higher probabilities of survival compared to immi-

grant fish, which is indicative of local adaptation (Kawecki and

Ebert 2004) and highlights the costs of local adaptation in terms of

reduced individual performance in nonnative environments (Nosil

2012). This was particularly evident when fish were moved from

nonsulfidic to H2S-containing environments, and high mortalities

are readily explained by the lack of adaptation to cope with the

extreme environmental conditions in sulfidic habitats, including

H2S-toxicity and hypoxia. H2S is a potent respiratory toxicant that

is usually lethal to nonadapted organisms in micromolar amounts

(Bagarinao 1992; Grieshaber and Volkel 1998). Although the ex-

act physiological mechanisms allowing fish from H2S-containing

1 0 EVOLUTION 2013

GENETIC DIFFERENTIATION AND SELECTION AGAINST MIGRANTS

habitats to withstand continuous exposure to H2S remain un-

clear, eco-toxicological experiments revealed that they exhibit

significantly higher sulfide tolerance than fish from nonsulfidic

environments (Tobler et al. 2011). Furthermore, fish from non-

sulfidic habitats lack the increased respiratory efficiency of fish

from H2S-containing habitats, which—due to their increase in

gill size—can acquire oxygen more efficiently at low ambient

concentrations (Tobler et al. 2011).

We also found high mortality rates in translocations from

H2S-containing into nonsulfidic environments, but the magnitude

of this effect varied across drainages. Survivability was compara-

tively high in the Puyacatengo and the Pichucalco drainages, but

in the Tacotalpa drainage it was almost as low as survivability

of fish transferred from nonsulfidic into H2S-containing habi-

tats. Mortality of fish from H2S-containing habitats in nonsulfidic

environments could be caused by oxidative stress, possibly in

combination with poor body condition and energy limitation of

H2S-ecotypes (Tobler 2008; Riesch et al. 2010, 2011a). Oxygen

can have adverse physiological effects due to its biotransforma-

tion into reactive oxygen species, and organisms have evolved

biochemical pathways with antioxidant activity to mitigate such

effects (Halliwell and Gutteridge 1999). In aquatic organisms, the

expression of antioxidant enzymes is often downregulated dur-

ing periods of hypoxia (Hermes-Lima and Zenteno-Savın 2002;

Olsvik et al. 2006), and subsequent exposure to normoxic condi-

tions can cause substantial oxidative stress with profound fitness

consequences (Sies 1986).

Both laboratory and field experiments further indicate that

survival in adults is dependent on body size (with small individ-

uals having a higher probability of survival than larger ones). We

hypothesize that H2S-detoxification becomes inefficient in larger

individuals, possibly due to their higher absolute demand for read-

ily available oxygen (Hildebrandt and Grieshaber 2008). In addi-

tion, standard length in poeciliid fishes is correlated with age (e.g.,

Snelson 1989), so the positive correlation between mortality and

body size could also reflect a decreased physiological capacity as

a result of senescence (e.g., Kirkwood and Austad 2000; but see

Reznick et al. 2002). Finally, females consistently outperformed

males, which could be caused by different life-history strategies.

Males in these species have consistently lower amounts of stored

body fat than females and a larger proportional investment into

reproduction (measured as gonadosomatic index) in sulfidic com-

pared to nonsulfidic habitats (Riesch et al. 2010, 2011a). In other

words, male poeciliids seem to have a higher total investment into

reproduction (including costly mate searching and mating behav-

ior) than females even at the risk of lowering survival capability.

This interpretation is consistent with a previous study that found

male P. mexicana to perform more aquatic surface respiration than

females in H2S-containing water (Plath et al. 2007b). Clearly, ad-

ditional studies are warranted to understand size- and sex specific

patterns of mortality mechanistically.

SEXUAL SELECTION AGAINST IMMIGRANTS

Our mate choice experiment revealed the evolution of assortative

mating preferences in at least some of the populations investi-

gated. Particularly, fish from nonsulfidic (but not from sulfidic)

habitats showed a preference for resident over immigrant mating

partners. The inferred strength of sexual selection against immi-

grants (RIs) was negatively correlated with the strength of natural

selection (RIm). We propose to attribute this pattern to reinforce-

ment, a process by which natural selection strengthens behavioral

isolation due to reduced hybrid fitness (Kirkpatrick 2001; Serve-

dio and Noor 2003). Direct selection on mating preferences is

more likely to occur when individuals have an increased likeli-

hood of encountering a nonnative mating partner (Coyne and Orr

1997); that is, when RIm is relatively low, like in migrants from sul-

fidic to nonsulfidic environments. It is important to point out that

occasional mating (and thus a role for reinforcement) might still

be possible even for migrants from nonsulfidic to sulfidic environ-

ments, because our experiments indicate that H2S-related death

is not instantaneous upon exposure to sulfidic waters. Further-

more, reinforcement can shape male and female mate preferences

even when reductions in hybrid fitness are small (Kirkpatrick

2001; Svensson et al. 2007; Servedio 2007). Future experiments

will need to establish (potentially drainage-specific) differences

in hybrid offspring fitness both under sulfidic and nonsulfidic

conditions (Servedio 2007).

Females consistently expressed stronger preferences than

males. Females generally have a lower reproductive potential

(Andersson 1994), and their reproductive fitness may thus de-

crease disproportionally if unfit hybrid offspring were produced

(Servedio and Noor 2003). Hence, females are expected to be

choosier than males and contribute more to premating isolation

between populations (Servedio 2007).

To date, it is unclear on what cues individuals base their

preferences. Our experimental design only allowed for the trans-

mission of visual cues, and general phenotypic differences be-

tween ecotypes—for example, in body shape (Tobler et al. 2011)

and coloration (authors, pers. obs.)—may provide sufficient in-

formation. If mating decisions are indeed based on such broad

phenotypic differences shaped by natural selection, reproductive

isolation through mate choice could evolve rapidly because any

detectable modification of a phenotype essentially could serve as

a “magic trait” (a character shaped by divergent natural selec-

tion that pleiotropically affects mate choice decisions; Servedio

et al. 2011). Alternatively, assortative mating may be based on

condition-dependent signals. Female preferences for condition-

dependent male traits signaling local adaptation through enhanced

EVOLUTION 2013 1 1

MARTIN PLATH ET AL.

performance may promote assortative mating even when female

preferences are unimodal (van Doorn et al. 2009). Males from

H2S-containing habitats are known to have poor overall body

condition even in their native environment (Plath et al. 2005; To-

bler et al. 2006; Riesch et al. 2011a), which may provide females

from nonsulfidic environments with cues to discriminate against

alien males.

Our results provide fruitful ground for future studies illumi-

nating mechanisms of assortative mating in this system. If repro-

ductive isolation evolved as a byproduct of adaptation (as pre-

dicted by ecological speciation theory; Langerhans et al. 2007;

Nosil 2012), across drainage comparisons should uncover sig-

nals of reproductive isolation between different ecotypes, but a

lack thereof between individuals of the same ecotype. Further

cross-drainage experiments are clearly warranted both to make

inferences about convergent and nonconvergent mechanisms un-

derlying assortative mating and the potential role of reinforcement

(Rasanen and Hendry 2008; Schwartz et al. 2010). Future studies

will also need to elucidate the role of nonvisual cues, such as

pheromones (Stacey 2003; Stacey and Sorensen 2005; Rosenthal

et al. 2011), in mate choice decisions. It also remains unclear

whether preferences are innate (reflecting genetically based di-

vergence) or shaped by sexual imprinting (Verzijden et al. 2012),

highlighting the need for future experimentation using common-

garden reared fish (Jennions and Petrie 1997).

TOTAL REPRODUCTIVE ISOLATION AND GENE FLOW

Total reproductive isolation was calculated based on our empiri-

cal estimates of natural and sexual selection against immigrants

and did not correlate significantly with the degree of gene flow.

The virtual absence of gene flow in the Tacotalpa and Pichucalco

drainages points towards nearly complete reproductive isolation

between ecotypes, which in theory should be reflected by T values

around 1 (Ramsey et al. 2003). However, we evaluated T rang-

ing between 0.64 and 0.85 for these drainages, suggesting that

additional reproductive isolation barriers, not investigated in this

study, are present. Considering the toxic effects of H2S, we suspect

that gene flow in this system is constrained by local adaptation.

However, we cannot unequivocally establish a causal relation-

ship, because the reverse—gene flow constraining adaptation—

also receives support from empirical studies (Rasanen and Hendry

2008). An assessment of multiple ecological and evolutionary

factors, including dispersal rates and effective population sizes,

is requisite to determine cause and effect (Rasanen and Hendry

2008).

ConclusionOverall, reproductive isolation and genetic differentiation appear

to be consistent consequences of local adaptation to sulfide spring

environments. Nonetheless, genetic divergence as well as the rela-

tive contributions of different reproductive isolation barriers vary

across the evolutionarily independent replicates investigated here,

supporting the view that both convergent and nonconvergent as-

pects of evolutionary differentiation are common during transi-

tions along the speciation continuum even in response to repli-

cated environmental gradients. This could be due to a variety of

reasons, among them as yet undetected (i.e., cryptic) differences

among seemingly similar environments, genetic covariances of

traits, differences in the amount of gene flow between popula-

tions (i.e., replicated population pairs falling onto different points

along the speciation continuum), or mutation-order speciation

(e.g., Kaeuffer et al. 2012; McGee and Wainwright 2013).

Prezygotic isolation through selection against immigrants

(either through natural selection or sexual selection) is a key in

driving genetic differentiation between sulfidic and nonsulfidic

populations in all three river systems. Observed gene flow pat-

terns, however, are lower than the total reproductive isolation

calculated based on the empirical patterns of natural and sexual

selection against immigrants would predict, which points toward

the existence of additional isolation barriers that need further

evaluation. Thus, current efforts in identifying genes underlying

adaptation to sulfidic spring environments (see Kelley et al. 2012)

in conjunction with experiments on hybrid performance, both in

the laboratory (to test for intrinsic postzygotic isolation) and under

natural conditions (to test for environmentally based postzygotic

isolation), are the logical next steps to further evaluate whether

and how ecological speciation is operating in our system.

ACKNOWLEDGMENTSThe authors thank members of the Tobler lab, G. Mayer, A. P. Hendry,and an anonymous reviewer for their helpful suggestions. Financial sup-port came from the German Science Foundation (DFG, PL 470/1-2, 3-1),the German Academic Exchange Service (DAAD), the initiative Nach-wuchswissenschaftler im Fokus by the presidential office of the Universityof Frankfurt, and the Herrmann Willkomm-Foundation (to M. Pl.), theresearch funding program LOEWE – Landesoffensive zur Entwicklungwissenschaftlich-okonomischer Exzellenz of Hesse’s Ministry of HigherEducation, Research, and the Arts (to M. Pl. and M. Pf.), the Stiftung Poly-technische Gesellschaft Frankfurt am Main (to HL), the Erwin Riesch-Stiftung (to RR), and National Science Foundation (IOS-1121832, toMT). Collection of fish and experimental work in Mexico were con-ducted under authorization by the Municipal de Tacotalpa, Tabasco, andthe federal agency SEMARNAT.

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Associate Editor: G. Mayer

Supporting InformationAdditional Supporting Information may be found in the online version of this article at the publisher’s website:

Table S1. Descriptive statistics of genetic variability for 17 microsatellite loci across six populations of mollies (Poecilia

sulphuraria and Poecilia mexicana).

Table S2. Number and standard length of individuals sampled for the mate choice experiments as well as water chemistry from

the different field sites investigated.

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