BACKGROUND
a central goal of evolutionary ecology is to understand processes that determine the distribution of genetic diversity and population connectivity through gene flow.
the current population genetic structure of a taxon is the result of many factors such as the dispersal capacity of the organism, the degree to which individuals of different populations recognize each other as potential mates, the connectivity of suitable habitats, historical events that impose geographical isolation  and interactions with ecologically similar species or predators  <cit> .
isolation by distance  <cit>  occurs when gene flow declines with increasing distance between pairs of populations, and is characteristic of the overall genetic population structure of many amphibians species  <cit> .
amphibians often exhibit strong site fidelity , have patchy geographical distributions due to specific and complex habitat requirements and low dispersal capacity  <cit> .
most studies of genetic population structure of amphibian show little or no genetic differentiation with moderate to high gene flow and dispersal rates at the local scale , but stronger genetic differentiation at larger geographical scales   <cit> .
less common are pattern of panmixia among distant localities  or strong genetic differentiation across very small geographic distances .
gene flow among populations is fundamentally related to genetic variability within populations.
in areas where gene flow among populations is high, genetic diversity within populations is likely to be higher  than in areas with reduced gene flow .
the most likely explanation for this phenomenon is reduced genetic drift and introduction of rare or new alleles in less isolated populations.
also, colonization events and bottlenecks are expected to have impacts on genetic diversity: generally older lineages are genetically more diverse than populations that have been founded by more recent colonization  <cit> .
multiple landscape features have recently been shown to be important for the magnitude of gene flow and thus for explaining population genetic structure in amphibians.
roads, mountain ridges and open or dry habitats apparently reduce gene flow between populations  <cit>  while forests, wetlands and rivers are associated with increased gene flow  <cit> .
for example, in two neotropical species of the genus craugastor, mountains and dry forests present ecologically unsuitable habitat and act as barriers to gene flow resulting in diverged genetic lineages  <cit> .
both genetic and ecological divergence can lead to reproductive isolation and speciation.
ecological speciation is the evolution of reproductive isolation as a result of divergent natural selection in different environments and can proceed in the presence or absence of gene flow  <cit> .
several general scenarios for speciation have been identified including genetic differentiation, geographic distribution and shift in ecological niches  <cit> .
these scenarios associate speciation processes occurring in sympatry, parapatry or allopatry with presence or absence of ecological divergence.
a combination of environmental niche models with phylogeographic analyses offers the possibility to explore the role of geographic and ecological separation for speciation between diverging lineages.
although phylogenetic and phylogeographic information has been increasing recently, few studies have been conducted simultaneously on both fine and large scale population genetics in tropical anurans  <cit> .
in the neotropics, phylo-geographic studies have been mainly limited to craugastorid  <cit> , strabomantid  <cit>  and dendrobatid frogs  <cit> .
these studies often revealed genetic lineages within species  <cit> , rectified the taxonomic relationship between species or species groups  <cit> , or uncovered previously unknown species diversity  <cit> .
examining ecological divergence in a phylogenetic context has been applied to only a very limited number of neotropical frogs.
these studies demonstrated climatic specialization along temperature and seasonality axes and identified important speciation mechanism for some clades  <cit> .
for most neotropical frogs, however, there is no information about their genetic population structure, genetic diversity within the species, presence of genetic lineages, or even species status and variability in ecological requirements.
still less is known about the relationship between genetic divergence, reproductive isolation and ecological divergence, and the relative importance of all these aspects for speciation.
to our knowledge the spatial genetic organization  <cit>  and reproductive behavior  of the túngara frog physalaemus  pustulosus is more thoroughly documented than any other neotropical frog species.
the distribution of the túngara frog ranges from northern mexico to the caribbean coast of northern south america where these frogs are abundant in dry and wet lowland forests.
allozymes analysis by ryan et al.
<cit>  found two genetic lineages of túngara frogs: a northern lineage containing populations from mexico to northern costa rica and a southern lineage ranging from western panama to northern south america .
this was confirmed by weigt et al.
<cit>  who added an analysis of mtdna  to the previous data set.
the time of the separation between both lineages  was estimated to have occurred prior to the final closing of the panamanian land bridge .
these results were further confirmed by a fine-scaled molecular study in an area of costa rica and panama that encompassed both genetic lineages  <cit>  and a phylogenetic study of the p. pustulosus species group  <cit> .
the northern and southern lineages are separated by a distribution gap of about  <dig> km in central costa rica  <cit> .
the aim of this study was threefold:  to analyze the potential for ecological divergence between the northern and southern lineages,  to test the persistence of the geographic barrier between the northern and southern lineages during the last glaciation and explore the hypothesis of allopatric divergence, and  to provide a more detailed population genetic analysis across  <dig> localities for the aforementioned area  and compare the population genetic structure to that of other amphibian species.
we first conduct a bayesian analysis to detect population subdivision and zones of admixture based on highly polymorphic nuclear markers.
we further calculate population differentiation  between inferred populations and assign individuals to their most probable population of origin for estimating migration between populations.
our analyses include calculation of genetic diversity across the investigated area.
we also estimate divergence time between the northern and southern lineage and between population clusters based on sequence divergence in the mitochondrial cyt b gene.
then we integrate our parameter estimates obtained from classical population differentiation analyses  and recently developed bayesian statistics.
thus we link classical measures of population structure  to population structure inferred from the clustering method, migration patterns deduced from assignment tests and divergence time estimates.
furthermore we conduct environmental niche modeling based on climatic and altitude parameters to estimate the potential for ecological niche divergence between genetic lineages, to measure ecological landscape connectivity across the distribution gap and to predict the distribution of past and current suitable habitat.
the allopatric distribution of the northern and the southern lineage suggests that genetic divergence is a result of geographic isolation.
this evidence, however, is insufficient to support an allopatric model of divergence because distribution ranges could have been in contact in the past.
to explore this possibility, we project niche models to the last glacial maximum.
a result showing equal or higher habitat unsuitability across the distribution gap during the last glacial would provide further support for the scenario of allopatric divergence.
we discuss the possibility that speciation has occurred between the northern and southern lineage based on the combined evidence of genetic and ecological data together with previously collected behavioral data.
moreover, this thorough population genetic and ecological analysis on a species for which the communication system, mating system and mate choice strategies are well studied will offer the opportunity to detect important areas  for studying new aspects of behavior in relation to the ecology and genetic composition of the population.
RESULTS
microsatellites
population structure: most population structure was captured by dividing all sample localities in two genetic lineages  the southern lineage, however, was further subdivided in two or three sub-clusters , while the northern lineage remained undivided even by increasing k to  <dig>  we decided that the most probable population structure is given for k =  <dig>  we base our decision on the following arguments: the likelihood of the data increased considerable when raising k =  <dig> to k =  <dig> and k =  <dig> .
afterwards pr  increased only insignificantly.
on the other hand, there was still a substantial Δk for k =  <dig>  but for higher k values Δk remained close to zero .
also, the assignment of individuals to a population cluster became less clear for k =  <dig> , and this was increasingly the case for higher values of k .
conducting the analysis separately for the northern and southern lineage resulted in the same geographic-genetic pattern, i.e.
by analyzing the southern lineage alone Δksouth was highest for ksouth =  <dig>  in the following we name the identified population clusters north , south_ <dig> , south_ <dig>  and south_ <dig> .
the northern lineage  was located in north costa rica, i.e.
in the north of the gap.
within the southern lineage structure found an admixture zone  in piedras blancas which was evident for k =  <dig> and k =  <dig> .
all individuals in this population possessed genetic material from the two  or three  southern clusters.
the cluster adjacent in the north-west of the admixture zone ranged from the southern edge of the gap  through the lowland areas along the coast and the lowland areas in the valle de general  and valle de cotobrus .
the cluster adjacent towards the south of the admixture zone ranged until el forastero in western panama.
the sample locality on the peninsula osa was included in this cluster.
sixty kilometers to the east, the third sub-cluster of the southern genetic group ranged from galique to gamboa, the eastern most locality .
in order to determine the degree of genetic differentiation among clusters we calculated fst und rst values between each pair of clusters and the admixture zone in piedras blancas .
all pairs of clusters were significantly different in estimates of both fst or rst.
as expected, the northern lineage was most diverged from all other clusters.
comparing the northern cluster with the three southern clusters it is notable that the fst was highest between north and south_ <dig>  while the highest rst values were found between north and the admixture zone.
rst values between the admixture zone and both adjacent clusters were non-significant.
the proportion of membership inferred by structure for each sample locality to the four clusters was for piedras blancas 37% to south_ <dig>  48% to south_ <dig>  13% to south_ <dig> and 2% to north.
this was not in full accordance with the fst and rst values: fst values between piedras blancas and south_ <dig> or south_ <dig> were nearly equal and the rst value was smallest for south_ <dig>  for pairwise rst values see .
fst  and rst  values between population clusters previously identified by structure.
for each comparison p <  <dig> , except *p =  <dig> , ○p =  <dig> , and ∇p =  <dig>
gene flow and migration: in the population cluster north between 17% and 77% of individuals reached the highest assignment score for their home population .
52% of individuals were assigned to another sample locality within the same population cluster  .
in general, in the southern clusters a higher percentage of individuals was assigned to their home population: in south_ <dig> 60-95%  of individuals, in south_ <dig> 41-100% of individuals , in south_ <dig> 58-90% of individuals  per population .
only in the cluster south_ <dig> for several individuals  the highest assignment score was for a sample locality in another population cluster .
overall, the results point to higher gene flow among populations in the cluster north than in the southern clusters.
in the admixture zone  the highest score in  <dig> individuals was for the home population, in  <dig> individuals for population palmar norte , and in one individual for bugaba .
classical migration rates based on fst and rst values were small between north and the southern clusters and much higher among the southern clusters .
migration rates per generation nm  <cit>  among population clusters previously identified by structure, based on fst  and rst  values.
at the sample locality level the software geneclass <dig> detected  <dig> individuals for which the probability that the individual is a resident was < <dig>  .
these individuals are assumed first generation migrants.
no migrants were detected between the northern and southern lineages.
most migrants  were found in the northern cluster.
here several individuals from liberia  seem to have migrated to other sample localities.
within the cluster south_ <dig> five individuals seemed to have migrated between populations.
for south_ <dig> this was true for four individuals.
in the cluster south_ <dig> five individuals were recognized as migrants from very distant source populations either in south_ <dig> or south_ <dig>
genetic diversity: mean allelic richness was higher in the population clusters south of the gap than in the cluster north of the gap  .
allelic richness differed significantly among population clusters .
in general, allelic richness per population or cluster increased from the northern most localities towards south america.
one fact that needs to be emphasized is that populations located somewhat aside from the main route along the pan american highway , possessed lower allelic richness than their neighbor populations.
also the two populations flanking the gap  retained lower allelic richness.
isolation by distance: for the total range, genetic distance  and geographic distance were highly correlated .
62% of the variation in genetic differences were explained by geography .
for the northern and southern lineages separately the correlation coefficients were lower but still significant .
figure  <dig> shows the correlation between geographic and genetic distance within the northern and southern lineage.
inspection of the graph also reveals the effect of geographic and temporal separation between the northern and southern túngara frogs: genetic distances between pairs of localities from both the northern and the southern lineages were considerable higher in comparison with localities within the same lineage separated by a similar geographic distance.
cytochrome b
genetic distance and time of divergence: the mean p distance  between the northern and southern lineage was  <dig>  ±  <dig>  , while the tn distance was calculated to be  <dig>  ±  <dig>  .
distances  between genetic clusters are presented in table  <dig>  whereas all distances between the northern and the southern lineages were between  <dig>  and  <dig> , the distances among groups of the southern lineage were substantially lower.
interestingly the distance of the northern lineage was lowest to south_ <dig>  estimated divergence times between north and south were between  <dig>  to  <dig>  myrs , while southern clusters diverged ~ <dig>  to  <dig>  myrs ago.
within-cluster divergence was lowest for north and increased towards the south .
uncorrected p distances/tn distances  and estimated divergence times  based on uncorrected p distances in myr assuming a pair-wise mutation rate of  <dig> % per myr.
rst  and tn distances  between sample localities within population clusters.
the haplotype network  reveals three main genetic clades, one with two subclades, which correspond nearly perfectly with the population cluster detected by the bayesian analysis based on microsatellites:  <dig>  north ,  <dig>  south_ <dig>  and south_ <dig>  as two subclades, and  <dig>  south_ <dig> .
the two sequences from piedras blancas  fall into south_ <dig>  and south_ <dig> .
one sequence from the group south_ <dig>  clustered together with one sequence from south_ <dig> .
the geographical divide between these two clusters was somewhere between el forastero and galique which are ~ <dig> km apart .
the haplotype network confirms the closer relationship between north and south_ <dig> than between north and south_ <dig>
diversity indices: molecular diversity as measured by number of haplotypes , polymorphic sites and nucleotide diversity was lowest in the northern lineage .
in fact, only one haplotype was found in the north.
all diversity indices increased from north towards the southern populations and were highest in population cluster south_ <dig>
cyt b diversities over all populations, the northern and southern lineage and population clusters.
number of individual sequenced , number of haplotypes , number of polymorphic sites , haplotype diversity  and nucleotide diversity .
ecological niches
the niche models generated with maxent predicted the occurrence of suitable habitat better than random models .
high model performance was also demonstrated by high values of the area under the curve  for the roc analysis  indicating better than random predictions .
the models generated by maxent  consisted of maps with logistic values  ranging from  <dig>  to  <dig> .
the model for the northern lineage predicted regions with high suitability in the dry forests and pine-oak forests of the pacific basin from northern costa rica to southern mexico .
the model for the southern lineage predicted regions with high suitability in the pacific moist forests of provincia puntarenas  and western panama.
high suitability was also present in the atlantic moist forest of central panama .
suitable habitat of the northern lineage showed a lower annual precipitation than for the southern lineage ; also the mean annual temperate was on average colder in the north than in the south .
there were significant differences in precipitation between the localities for the northern lineage and the southern lineage .
differences were not significant for mean annual temperature .
the paleoclimate models suggested significant changes in the location of suitable habitat with opposing trends in range size between both genetic groups .
under a logistic value  threshold of  <dig>  for presence-absence, the suitable range for the southern lineage in middle america, east from the gap, increased from  <dig>   to  <dig>  km <dig> .
for the northern lineage, the suitable range in middle america, west from the gap, decreased from  <dig> , <dig> km <dig>  to  <dig>  km <dig> .
connectivity and suitability across the distribution gap in costa rica: the analyses of suitability and connectivity suggested that the distribution gap in costa rica persisted during the last glacial maximum.
suitability for the northern lineage across the gap was not significantly different between the present and the last glacial maximum .
for the southern lineage, however, conditions deteriorated significantly .
the analysis of habitat connectivity suggested similar trends.
for the northern lineage, habitat resistance to reach the southern range was almost equal for current conditions and the last glacial maximum .
for the southern lineage, resistance was much higher during the last glacial  than under current conditions .
taken together, these results suggest that geographic separation between genetic lineages persisted during glacial events.
discussion
our analyses show that the two genetic lineages of túngara frogs in middle america differ genetically and reside in ecologically different habitats.
the northern lineage is genetically homogenous while the southern lineage consists of several population clusters: one in south costa rica close to the distribution gap between the lineages , one ranging from south costa rica to western panama , and the last one ranging from western panama towards east panama .
one sample locality in south costa rica  was identified as a contact or admixture zone between south_ <dig> and south_ <dig>  in general, the genetic pattern revealed by microsatellites is confirmed by sequences of the mitochondrial gene cyt b. genetic diversity is lowest in the northern lineage and increased from south costa rica towards east panama.
in contrast, gene flow between sample localities is higher in northern costa rican than in the southern population clusters.
isolation by distance explains a large amount of the genetic variation in both lineages; however, time of divergence between population clusters also plays a significant role for genetic differentiation.
environmental niche modeling revealed that the northern and the southern lineage differ significantly in habitat type.
suitable habitat for the northern lineage is drier and occurs in pine-oak forests, while the southern lineage is associated with moist forests.
annual precipitation is significantly higher for the habitat of the southern lineage.
since the last glacial maximum suitable habitat decreased for the northern lineage but increased for the southern lineage.
the habitat connectivity across the distribution gap in central costa rica was lower during the last glacial maximum, suggesting that ecological factors have prevented secondary contact from at least the pleistocene to the present.
this result is consistent with the large divergence time and the absence of migration between lineages recovered by two independent analyses .
comparison of microsatellite and cyt b analyses
despite two different types of molecular markers  and different statistical methods , both molecular approaches resulted in the same genetic population structure of costa rican and panamanian túngara frogs.
this indicates that the revealed population structure is fairly static given that mtdna indicates ancient, historical events while microsatellites indicate more recent processes.
moreover, the different genetic distance measurements between population clusters  are highly correlated .
pattern of population structure in amphibians and isolation by distance
in general, amphibian populations display a high level of spatial genetic structure, mainly when interpopulation distances exceed several kilometers  <cit> .
túngara frogs also show this pattern as populations are genetically distinguishable at a very local level.
lampert et al.
<cit>  found significant fst values  between populations that were on average three to four km apart , a similar pattern was found in comparing island populations  <cit> .
all studies, including this one, found a significant effect of isolation by distance, i.e.
local migration takes place between neighboring populations, but over longer distances gene flow decreases.
our bayesian structure analysis did not further subdivide the northern lineage into subpopulations and the assignment and migration analyses points to high levels of gene flow between sample localities.
pairwise rst values ranged from  <dig>  to  <dig>  in the northern lineage and  <dig> of the  <dig> population comparisons are significant, i.e.
although rst values ≥  <dig>  -  <dig>  indicate significant genetic differences structure still assigns them to the same genetic cluster.
our results suggest that genetic drift causes genetic differentiation while subpopulations are genetically connected by migrating animals.
marsh et al.
<cit>  showed that túngara frogs have a breeding neighborhood of less than  <dig> m; that is, at smaller distances males tend to move among breeding sites but not so at distances of  <dig> m or more.
on the other hand, marsh et al.
<cit>  showed that these frogs did routinely move among ponds of distances of  <dig> m. this pair of studies suggests that túngara frogs are relatively philopatric but do possess significant dispersal abilities.
nevertheless, we seriously doubt that an individual túngara frog is able to cross distances of  <dig> km or more without the aid of human transport as the analysis of first migrant detection might suggest.
instead, we suggest that túngara frog subpopulations are well connected through ponds and puddles available for breeding during the rainy season as suggested by continuous suitable habitat between liberia and santa rosa .
as the túngara frog is well known to breed in areas of human disturbance, habitat disruption might not hinder and might even facilitate dispersion rather than impede it.
since the distance between our sample localities is on average  <dig> km, the apparent gene flow at such distances is probably established by substantial movements among breeding ponds that are much closer to each other.
our data indicate that the túngara frog is ecologically similar to the natterjack toad  that is known as a pioneering species with a high dispersal capacity, quickly colonizes early-successional habitats and displays low population structure at the local level, increasing population structure at larger distances and exhibits significant isolation by distance  <cit> .
in the north, the assignment of individuals to their population was lower than it was in southern populations , where more individuals were assigned to their true locality.
this indicates either higher gene flow in the north than among southern populations, or a population bottleneck followed by population expansion.
the habitat topography of south costa rica is more complex than north costa rica.
in north costa rica no mountains separate different sample localities.
in south costa rica, buenos aires  and potrero grande  are isolated from other sample localities by the fila costeña mountain range which parallels the coast.
nevertheless, gene flow between palmar norte, cortez and potrero grande seems to be high while buenos aires appears to be more isolated.
it is possible that gene flow between these three sample localities is conveyed by rivers.
the rio coto brus passes potrero grande, downriver it joins the river rio general to form the rio terraba which connects to palmar norte and cortez.
also, osa  and golfito  seem to be genetically more isolated within the southern populations then are the rest of the southern populations.
osa is more remote from other sample localities on the peninsula osa, while golfito is located close to the coast and is separated from the lowland areas with the other sample localities by smaller mountains.
genetic assignment to the other sample localities is also lower in populations close to the distribution gap between la junta  and ojochal  indicating that gene flow and genetic diversity are reduced in peripheral populations.
there is gene flow across the admixture zone of piedras blancas, especially towards south_ <dig>  but the high assignment to the home population indicates that gene flow is reduced as compared to genetically more homogenous areas.
another lowland area of high gene flow is between gloria , gariché  and bugaba .
six individuals found in gloria were genetically assigned with the highest probability to gariché and eight individuals from gariché were assigned with the highest probability to gloria.
in the third southern population cluster some individuals are assigned to south_ <dig>  and some "first generation migrants" stem from very distant home populations  an observation that suggests ancient and current migration between both clusters.
divergences times
our data are consistent with the proposition that túngara frogs invaded middle america from south america at least twice  <cit> .
the calculation of divergence times based on coi and a mitochondrial mutation rate of  <dig> % per myr between northern and southern túngara frogs in the study of weigt et al.
<cit>  resulted in an estimated divergence time of  <dig>  mya  while the parametric bayesian mcmc method calibrated by the rise of the ecuadorian andes estimated the time of divergence as  <dig>  mya .
our calculations resulted in a divergence time of approximately  <dig> myr between the northern and southern lineage which falls into the lower end of the confidence intervals calculated by weigt et al.
.
the difference between both studies in average divergence time estimates very likely rests upon the different geographical dimensions, i.e.
in this study sampled populations next to the gap are geographically closer to each other  and the sampled area covers a smaller geographic range.
ecological niches
besides being genetically diverged, the northern and southern lineages of túngara frogs reside in different habitats in panama and costa rica.
while the northern lineage occupies dry forests, the southern lineage occurs in moist forests with higher annual precipitation.
the actual distribution of northern and southern túngara frogs in middle america  <cit>  generally corresponds to the predicted suitable habitat of niche models in costa rica and panama.
the low predicted suitability for the southern lineage across the distribution gap in costa rica is consistent with the known species distribution.
suitability across the gap for the northern lineage, however, was intermediate and within the range of known localities.
thus, the model suggests that conditions in a portion of the gap might be suitable for the northern lineage.
this incorrect prediction of the model could be the result of  the exclusion of an environmental variable that limits the species distribution range,  ecological interactions with other species across the gap , or  an unknown dispersal barrier.
because there are not conspicuous dispersal barriers in the region, we suspect that explanations  or  are the most likely.
niche modeling suggests that the absence of each lineage in the distribution gap correlates with ecological variables and therefore suggests that not only do the two lineages inhabit different habitats but there might be ecological divergence between the lineages.
niche conservatism is the tendency of species to retain aspects of their fundamental niche over longer time periods and limits the adaptation to ecological conditions at geographic barriers  <cit> .
our results show that the northern and southern lineages occupy different environmental envelopes and this suggests that their niches have been evolutionarily labile.
there are two possibilities to explain genetic and ecological divergence in túngara frogs.
the first possibility is that túngara frogs were split in two groups by a vicariance event.
the geographic separation was then maintained by niche conservatism and followed by niche specialization.
otherwise the genetic divergence between lineages in túngara frogs in allopatry could be the result from a combination of niche specialization followed by niche conservatism at the edges of distribution ranges in costa rica.
the absence of the southern lineage in the gap suggests that niche conservatism can promote allopatric speciation as proposed by wiens and graham   <cit> .
at the moment our data cannot discriminate between both possibilities for lineage separation.
in comparison with the present, suitable habitat during the last glacial maximum covered larger areas for the northern lineage while it was more restricted in the southern lineage.
we assume that climatic changes from colder and drier climate to warmer and moister climate are responsible for this development.
drier climate during the last glacial maximum also seems to explain the higher habitat resistance of the distribution gap for the southern lineage.
since habitat resistance persists today it seems improbable that both groups will come into contact in the near future unless the current climate change will result in more favorable conditions across the gap.
speciation in túngara frogs
this and several preceding molecular studies revealed that the túngara frog physalaemus pustulosus consists of two genetically different lineages  <cit> .
in this study we found that the lineages inhabit different habitats and thus suggests the possibility that they might be ecologically divergent.
the geographic separation and the large genetic divergence and ecological dissimilarity between lineages suggest vicariance events together with ecologically mediated divergent selection as potential speciation mechanisms in túngara frogs.
for ecuadorian frogs of the genus epipedobates, sister species with allopatric or parapatric distribution replace each other altitudinally or latitudinally emphasizing the role of environmental niche shift in speciation in neotropical frogs  <cit> .
the ecological separation principally occurs along a temperature/elevational or a seasonality axis.
for divergence between túngara frog lineages a shift in seasonality and precipitation might be more important than differences in temperature.
ecological prezygotic isolation could arise as a result of adaptation to contrasting habitats or temporal isolation  <cit> .
adaptation to different habitats could entail behavioral reproductive isolation because signals involved in mate choice could be under ecologically based divergent selection.
examples include sexual preferences for ecologically selected body size and color pattern in fish or beak size in birds  <cit> .
this possibility needs further exploration in our study species.
ron et al.
<cit>  suggested that each lineage of túngara frogs might deserve species status based on the large molecular divergence , size differences, and large divergence time .
the observed habitat differences are consistent with the recognition of each lineage as a separate species.
species level divergence is also suggested by some evidence of prezygotic isolation.
pröhl et al.
<cit>  conducted experiments showing that captive p. pustulosus females from the south did not produce nests with males from the northern lineage.
however this was not true for northern females which produced nests and tadpoles with males from both lineages.
the pattern of call divergence in the gap region in costa rica shows that several call variables tend to be more divergent near the gap.
this pattern is consistent with ancient reproductive character displacement , however, as discussed below, these differences in calls have no influence on female species recognition.
the results of phonotactic mate choice experiments with female túngara frogs, on the other hand, do not suggest the two lineages should each be given species status.
pröhl et al.
<cit>  showed that calls differ between lineages and populations, and that in about two thirds of the comparisons females showed a significant preference for the local mating call over the foreign call.
this preference for the local call was stronger than that found in females from gamboa, which is well within the southern group  <cit> .
neither study, however, suggested that females exhibited stronger preferences for calls of their own lineage versus the other lineage .
at this point we feel that the data are not conclusive in determining whether the two lineages of túngara frogs represent distinct species and we propose to collect more data on pre- or post-zygotic reproductive isolation  <cit> .
population genetics, ecology and behavior
the results of this study offer the opportunity for new insights on the complex relationship between genetic distance, ecological divergence, call divergence and mate preferences.
the areas between two genetic population clusters and areas adjacent to admixture zones are especially interesting for further behavioral observations.
such studies might determine whether there are abrupt changes in certain call parameters across genetically different populations, or how the female preference pattern varies across admixture zones.
therefore it would be necessary to conduct geographically fine scaled genetic and behavioral analyses in several localities, for example between palmar norte and caracol in south costa rica or between galique and el forastero in western panama .
also, in the context of reproductive character displacement and reinforcement  <cit>  coupled with measurement of gene flow, such areas provide exciting possibilities for future research prospects.
the pattern of call divergence in the gap region in costa rica mentioned above indicates the opportunity for the evolution of character displacement in túngara frogs.
moreover the detected differences in ecology between the two main lineages should influence behavior.
the northern frogs inhabit drier forests than the southern frogs.
we would assume that temperature and precipitation pattern influence the time of the breeding period and the reproductive behavior.
in addition the question whether there is ecological divergence and if it has caused reproductive isolation needs to be examined.
finally more intensive population sampling in the southern genetic clusters could reveal ecological differences between them and help to discover local adaptations that might be involved in divergence between lineages.
CONCLUSIONS
in this study we found population genetic structure and unexpected ecological diversity between lineages of a neotropical frog species, the túngara frog physalaemus pustulosus.
population genetic analyses of middle american populations  revealed two allopatric divergent lineages  and a further sub-division of the southern lineage into three population clusters.
the genetic divergence into two main lineages goes along with ecological differences in habitat type.
these results highlight the importance of intensive population sampling across large ranges of a species distribution.
overall genetic diversity within populations is high across the sampled range but diversity is higher in the south than in the north.
the diversity within populations is maintained by high gene flow between neighbor sample localities.
altogether the genetic data support the idea that the túngara frog is an abundant species whose populations within population clusters are well connected via gene flow and little affected by habitat fragmentation.
the allopatric distribution north and south of the gap in costa rica seems to be maintained by unsuitable climatic conditions across the gap which likely persisted during the last glaciation and probably during previous glacial and interglacial cycles.
since the northern and southern lineages are geographically separated, genetically divergent, and inhabit different habitats there is potential for speciation by ecological divergence in allopatry.
we discuss the possibility that both lineages should receive species status and conclude that additional pre- and postzygotic isolation experiments are needed.
in addition, further ecological and behavioral analyses among and across southern population clusters could shed light on evolutionary divergence and speciation.

 a central goal of evolutionary ecology is to understand processes that determine the distribution of genetic diversity and population connectivity through gene flow. the current population genetic structure of a taxon is the result of many factors such as the dispersal capacity of the organism, the degree to which individuals of different populations recognize each other as potential mates, the connectivity of suitable habitats, historical events that impose geographical isolation and interactions with ecologically similar species or predators. isolation by distance occurs when gene flow declines with increasing distance between pairs of populations, and is characteristic of the overall genetic population structure of many amphibians species. amphibians often exhibit strong site fidelity, have patchy geographical distributions due to specific and complex habitat requirements and low dispersal capacity. most studies of genetic population structure of amphibian show little or no genetic differentiation with moderate to high gene flow and dispersal rates at the local scale, but stronger genetic differentiation at larger geographical scales. less common are pattern of panmixia among distant localities or strong genetic differentiation across very small geographic distances. gene flow among populations is fundamentally related to genetic variability within populations. in areas where gene flow among populations is high, genetic diversity within populations is likely to be higher than in areas with reduced gene flow. the most likely explanation for this phenomenon is reduced genetic drift and introduction of rare or new alleles in less isolated populations. also, colonization events and bottlenecks are expected to have impacts on genetic diversity: generally older lineages are genetically more diverse than populations that have been founded by more recent colonization. multiple landscape features have recently been shown to be important for the magnitude of gene flow and thus for explaining population genetic structure in amphibians. roads, mountain ridges and open or dry habitats apparently reduce gene flow between populations while forests, wetlands and rivers are associated with increased gene flow. for example, in two neotropical species of the genus craugastor, mountains and dry forests present ecologically unsuitable habitat and act as barriers to gene flow resulting in diverged genetic lineages. both genetic and ecological divergence can lead to reproductive isolation and speciation. ecological speciation is the evolution of reproductive isolation as a result of divergent natural selection in different environments and can proceed in the presence or absence of gene flow. several general scenarios for speciation have been identified including genetic differentiation, geographic distribution and shift in ecological niches. these scenarios associate speciation processes occurring in sympatry, parapatry or allopatry with presence or absence of ecological divergence. a combination of environmental niche models with phylogeographic analyses offers the possibility to explore the role of geographic and ecological separation for speciation between diverging lineages. although phylogenetic and phylogeographic information has been increasing recently, few studies have been conducted simultaneously on both fine and large scale population genetics in tropical anurans. in the neotropics, phylo-geographic studies have been mainly limited to craugastorid, strabomantid and dendrobatid frogs. these studies often revealed genetic lineages within species, rectified the taxonomic relationship between species or species groups, or uncovered previously unknown species diversity. examining ecological divergence in a phylogenetic context has been applied to only a very limited number of neotropical frogs. these studies demonstrated climatic specialization along temperature and seasonality axes and identified important speciation mechanism for some clades. for most neotropical frogs, however, there is no information about their genetic population structure, genetic diversity within the species, presence of genetic lineages, or even species status and variability in ecological requirements. still less is known about the relationship between genetic divergence, reproductive isolation and ecological divergence, and the relative importance of all these aspects for speciation. to our knowledge the spatial genetic organization and reproductive behavior of the túngara frog physalaemus pustulosus is more thoroughly documented than any other neotropical frog species. the distribution of the túngara frog ranges from northern mexico to the caribbean coast of northern south america where these frogs are abundant in dry and wet lowland forests. allozymes analysis by ryan et al. found two genetic lineages of túngara frogs: a northern lineage containing populations from mexico to northern costa rica and a southern lineage ranging from western panama to northern south america. this was confirmed by weigt et al. who added an analysis of mtdna to the previous data set. the time of the separation between both lineages was estimated to have occurred prior to the final closing of the panamanian land bridge. these results were further confirmed by a fine-scaled molecular study in an area of costa rica and panama that encompassed both genetic lineages and a phylogenetic study of the p. pustulosus species group. the northern and southern lineages are separated by a distribution gap of about km in central costa rica. the aim of this study was threefold: to analyze the potential for ecological divergence between the northern and southern lineages, to test the persistence of the geographic barrier between the northern and southern lineages during the last glaciation and explore the hypothesis of allopatric divergence, and to provide a more detailed population genetic analysis across localities for the aforementioned area and compare the population genetic structure to that of other amphibian species. we first conduct a bayesian analysis to detect population subdivision and zones of admixture based on highly polymorphic nuclear markers. we further calculate population differentiation between inferred populations and assign individuals to their most probable population of origin for estimating migration between populations. our analyses include calculation of genetic diversity across the investigated area. we also estimate divergence time between the northern and southern lineage and between population clusters based on sequence divergence in the mitochondrial cyt b gene. then we integrate our parameter estimates obtained from classical population differentiation analyses and recently developed bayesian statistics. thus we link classical measures of population structure to population structure inferred from the clustering method, migration patterns deduced from assignment tests and divergence time estimates. furthermore we conduct environmental niche modeling based on climatic and altitude parameters to estimate the potential for ecological niche divergence between genetic lineages, to measure ecological landscape connectivity across the distribution gap and to predict the distribution of past and current suitable habitat. the allopatric distribution of the northern and the southern lineage suggests that genetic divergence is a result of geographic isolation. this evidence, however, is insufficient to support an allopatric model of divergence because distribution ranges could have been in contact in the past. to explore this possibility, we project niche models to the last glacial maximum. a result showing equal or higher habitat unsuitability across the distribution gap during the last glacial would provide further support for the scenario of allopatric divergence. we discuss the possibility that speciation has occurred between the northern and southern lineage based on the combined evidence of genetic and ecological data together with previously collected behavioral data. moreover, this thorough population genetic and ecological analysis on a species for which the communication system, mating system and mate choice strategies are well studied will offer the opportunity to detect important areas for studying new aspects of behavior in relation to the ecology and genetic composition of the population.  microsatellites population structure: most population structure was captured by dividing all sample localities in two genetic lineages the southern lineage, however, was further subdivided in two or three sub-clusters, while the northern lineage remained undivided even by increasing k to we decided that the most probable population structure is given for k = we base our decision on the following arguments: the likelihood of the data increased considerable when raising k = to k = and k =. afterwards pr increased only insignificantly. on the other hand, there was still a substantial Δk for k = but for higher k values Δk remained close to zero. also, the assignment of individuals to a population cluster became less clear for k =, and this was increasingly the case for higher values of k. conducting the analysis separately for the northern and southern lineage resulted in the same geographic-genetic pattern, i.e. by analyzing the southern lineage alone Δksouth was highest for ksouth = in the following we name the identified population clusters north, south_, south_ and south_. the northern lineage was located in north costa rica, i.e. in the north of the gap. within the southern lineage structure found an admixture zone in piedras blancas which was evident for k = and k =. all individuals in this population possessed genetic material from the two or three southern clusters. the cluster adjacent in the north-west of the admixture zone ranged from the southern edge of the gap through the lowland areas along the coast and the lowland areas in the valle de general and valle de cotobrus. the cluster adjacent towards the south of the admixture zone ranged until el forastero in western panama. the sample locality on the peninsula osa was included in this cluster. sixty kilometers to the east, the third sub-cluster of the southern genetic group ranged from galique to gamboa, the eastern most locality. in order to determine the degree of genetic differentiation among clusters we calculated fst und rst values between each pair of clusters and the admixture zone in piedras blancas. all pairs of clusters were significantly different in estimates of both fst or rst. as expected, the northern lineage was most diverged from all other clusters. comparing the northern cluster with the three southern clusters it is notable that the fst was highest between north and south_ while the highest rst values were found between north and the admixture zone. rst values between the admixture zone and both adjacent clusters were non-significant. the proportion of membership inferred by structure for each sample locality to the four clusters was for piedras blancas 37% to south_ 48% to south_ 13% to south_ and 2% to north. this was not in full accordance with the fst and rst values: fst values between piedras blancas and south_ or south_ were nearly equal and the rst value was smallest for south_ for pairwise rst values see. fst and rst values between population clusters previously identified by structure. for each comparison p <, except *p =, ○p =, and ∇p = gene flow and migration: in the population cluster north between 17% and 77% of individuals reached the highest assignment score for their home population. 52% of individuals were assigned to another sample locality within the same population cluster. in general, in the southern clusters a higher percentage of individuals was assigned to their home population: in south_ 60-95% of individuals, in south_ 41-100% of individuals, in south_ 58-90% of individuals per population. only in the cluster south_ for several individuals the highest assignment score was for a sample locality in another population cluster. overall, the results point to higher gene flow among populations in the cluster north than in the southern clusters. in the admixture zone the highest score in individuals was for the home population, in individuals for population palmar norte, and in one individual for bugaba. classical migration rates based on fst and rst values were small between north and the southern clusters and much higher among the southern clusters. migration rates per generation nm among population clusters previously identified by structure, based on fst and rst values. at the sample locality level the software geneclass detected individuals for which the probability that the individual is a resident was <. these individuals are assumed first generation migrants. no migrants were detected between the northern and southern lineages. most migrants were found in the northern cluster. here several individuals from liberia seem to have migrated to other sample localities. within the cluster south_ five individuals seemed to have migrated between populations. for south_ this was true for four individuals. in the cluster south_ five individuals were recognized as migrants from very distant source populations either in south_ or south_ genetic diversity: mean allelic richness was higher in the population clusters south of the gap than in the cluster north of the gap. allelic richness differed significantly among population clusters. in general, allelic richness per population or cluster increased from the northern most localities towards south america. one fact that needs to be emphasized is that populations located somewhat aside from the main route along the pan american highway, possessed lower allelic richness than their neighbor populations. also the two populations flanking the gap retained lower allelic richness. isolation by distance: for the total range, genetic distance and geographic distance were highly correlated. 62% of the variation in genetic differences were explained by geography. for the northern and southern lineages separately the correlation coefficients were lower but still significant. figure shows the correlation between geographic and genetic distance within the northern and southern lineage. inspection of the graph also reveals the effect of geographic and temporal separation between the northern and southern túngara frogs: genetic distances between pairs of localities from both the northern and the southern lineages were considerable higher in comparison with localities within the same lineage separated by a similar geographic distance. cytochrome b genetic distance and time of divergence: the mean p distance between the northern and southern lineage was ±, while the tn distance was calculated to be ±. distances between genetic clusters are presented in table whereas all distances between the northern and the southern lineages were between and, the distances among groups of the southern lineage were substantially lower. interestingly the distance of the northern lineage was lowest to south_ estimated divergence times between north and south were between to myrs, while southern clusters diverged ~ to myrs ago. within-cluster divergence was lowest for north and increased towards the south. uncorrected p distances/tn distances and estimated divergence times based on uncorrected p distances in myr assuming a pair-wise mutation rate of % per myr. rst and tn distances between sample localities within population clusters. the haplotype network reveals three main genetic clades, one with two subclades, which correspond nearly perfectly with the population cluster detected by the bayesian analysis based on microsatellites: north, south_ and south_ as two subclades, and south_. the two sequences from piedras blancas fall into south_ and south_. one sequence from the group south_ clustered together with one sequence from south_. the geographical divide between these two clusters was somewhere between el forastero and galique which are ~ km apart. the haplotype network confirms the closer relationship between north and south_ than between north and south_ diversity indices: molecular diversity as measured by number of haplotypes, polymorphic sites and nucleotide diversity was lowest in the northern lineage. in fact, only one haplotype was found in the north. all diversity indices increased from north towards the southern populations and were highest in population cluster south_ cyt b diversities over all populations, the northern and southern lineage and population clusters. number of individual sequenced, number of haplotypes, number of polymorphic sites, haplotype diversity and nucleotide diversity. ecological niches the niche models generated with maxent predicted the occurrence of suitable habitat better than random models. high model performance was also demonstrated by high values of the area under the curve for the roc analysis indicating better than random predictions. the models generated by maxent consisted of maps with logistic values ranging from to. the model for the northern lineage predicted regions with high suitability in the dry forests and pine-oak forests of the pacific basin from northern costa rica to southern mexico. the model for the southern lineage predicted regions with high suitability in the pacific moist forests of provincia puntarenas and western panama. high suitability was also present in the atlantic moist forest of central panama. suitable habitat of the northern lineage showed a lower annual precipitation than for the southern lineage; also the mean annual temperate was on average colder in the north than in the south. there were significant differences in precipitation between the localities for the northern lineage and the southern lineage. differences were not significant for mean annual temperature. the paleoclimate models suggested significant changes in the location of suitable habitat with opposing trends in range size between both genetic groups. under a logistic value threshold of for presence-absence, the suitable range for the southern lineage in middle america, east from the gap, increased from to km. for the northern lineage, the suitable range in middle america, west from the gap, decreased from, km to km. connectivity and suitability across the distribution gap in costa rica: the analyses of suitability and connectivity suggested that the distribution gap in costa rica persisted during the last glacial maximum. suitability for the northern lineage across the gap was not significantly different between the present and the last glacial maximum. for the southern lineage, however, conditions deteriorated significantly. the analysis of habitat connectivity suggested similar trends. for the northern lineage, habitat resistance to reach the southern range was almost equal for current conditions and the last glacial maximum. for the southern lineage, resistance was much higher during the last glacial than under current conditions. taken together, these results suggest that geographic separation between genetic lineages persisted during glacial events. discussion our analyses show that the two genetic lineages of túngara frogs in middle america differ genetically and reside in ecologically different habitats. the northern lineage is genetically homogenous while the southern lineage consists of several population clusters: one in south costa rica close to the distribution gap between the lineages, one ranging from south costa rica to western panama, and the last one ranging from western panama towards east panama. one sample locality in south costa rica was identified as a contact or admixture zone between south_ and south_ in general, the genetic pattern revealed by microsatellites is confirmed by sequences of the mitochondrial gene cyt b. genetic diversity is lowest in the northern lineage and increased from south costa rica towards east panama. in contrast, gene flow between sample localities is higher in northern costa rican than in the southern population clusters. isolation by distance explains a large amount of the genetic variation in both lineages; however, time of divergence between population clusters also plays a significant role for genetic differentiation. environmental niche modeling revealed that the northern and the southern lineage differ significantly in habitat type. suitable habitat for the northern lineage is drier and occurs in pine-oak forests, while the southern lineage is associated with moist forests. annual precipitation is significantly higher for the habitat of the southern lineage. since the last glacial maximum suitable habitat decreased for the northern lineage but increased for the southern lineage. the habitat connectivity across the distribution gap in central costa rica was lower during the last glacial maximum, suggesting that ecological factors have prevented secondary contact from at least the pleistocene to the present. this result is consistent with the large divergence time and the absence of migration between lineages recovered by two independent analyses. comparison of microsatellite and cyt b analyses despite two different types of molecular markers and different statistical methods, both molecular approaches resulted in the same genetic population structure of costa rican and panamanian túngara frogs. this indicates that the revealed population structure is fairly static given that mtdna indicates ancient, historical events while microsatellites indicate more recent processes. moreover, the different genetic distance measurements between population clusters are highly correlated. pattern of population structure in amphibians and isolation by distance in general, amphibian populations display a high level of spatial genetic structure, mainly when interpopulation distances exceed several kilometers. túngara frogs also show this pattern as populations are genetically distinguishable at a very local level. lampert et al. found significant fst values between populations that were on average three to four km apart, a similar pattern was found in comparing island populations. all studies, including this one, found a significant effect of isolation by distance, i.e. local migration takes place between neighboring populations, but over longer distances gene flow decreases. our bayesian structure analysis did not further subdivide the northern lineage into subpopulations and the assignment and migration analyses points to high levels of gene flow between sample localities. pairwise rst values ranged from to in the northern lineage and of the population comparisons are significant, i.e. although rst values ≥ - indicate significant genetic differences structure still assigns them to the same genetic cluster. our results suggest that genetic drift causes genetic differentiation while subpopulations are genetically connected by migrating animals. marsh et al. showed that túngara frogs have a breeding neighborhood of less than m; that is, at smaller distances males tend to move among breeding sites but not so at distances of m or more. on the other hand, marsh et al. showed that these frogs did routinely move among ponds of distances of m. this pair of studies suggests that túngara frogs are relatively philopatric but do possess significant dispersal abilities. nevertheless, we seriously doubt that an individual túngara frog is able to cross distances of km or more without the aid of human transport as the analysis of first migrant detection might suggest. instead, we suggest that túngara frog subpopulations are well connected through ponds and puddles available for breeding during the rainy season as suggested by continuous suitable habitat between liberia and santa rosa. as the túngara frog is well known to breed in areas of human disturbance, habitat disruption might not hinder and might even facilitate dispersion rather than impede it. since the distance between our sample localities is on average km, the apparent gene flow at such distances is probably established by substantial movements among breeding ponds that are much closer to each other. our data indicate that the túngara frog is ecologically similar to the natterjack toad that is known as a pioneering species with a high dispersal capacity, quickly colonizes early-successional habitats and displays low population structure at the local level, increasing population structure at larger distances and exhibits significant isolation by distance. in the north, the assignment of individuals to their population was lower than it was in southern populations, where more individuals were assigned to their true locality. this indicates either higher gene flow in the north than among southern populations, or a population bottleneck followed by population expansion. the habitat topography of south costa rica is more complex than north costa rica. in north costa rica no mountains separate different sample localities. in south costa rica, buenos aires and potrero grande are isolated from other sample localities by the fila costeña mountain range which parallels the coast. nevertheless, gene flow between palmar norte, cortez and potrero grande seems to be high while buenos aires appears to be more isolated. it is possible that gene flow between these three sample localities is conveyed by rivers. the rio coto brus passes potrero grande, downriver it joins the river rio general to form the rio terraba which connects to palmar norte and cortez. also, osa and golfito seem to be genetically more isolated within the southern populations then are the rest of the southern populations. osa is more remote from other sample localities on the peninsula osa, while golfito is located close to the coast and is separated from the lowland areas with the other sample localities by smaller mountains. genetic assignment to the other sample localities is also lower in populations close to the distribution gap between la junta and ojochal indicating that gene flow and genetic diversity are reduced in peripheral populations. there is gene flow across the admixture zone of piedras blancas, especially towards south_ but the high assignment to the home population indicates that gene flow is reduced as compared to genetically more homogenous areas. another lowland area of high gene flow is between gloria, gariché and bugaba. six individuals found in gloria were genetically assigned with the highest probability to gariché and eight individuals from gariché were assigned with the highest probability to gloria. in the third southern population cluster some individuals are assigned to south_ and some "first generation migrants" stem from very distant home populations an observation that suggests ancient and current migration between both clusters. divergences times our data are consistent with the proposition that túngara frogs invaded middle america from south america at least twice. the calculation of divergence times based on coi and a mitochondrial mutation rate of % per myr between northern and southern túngara frogs in the study of weigt et al. resulted in an estimated divergence time of mya while the parametric bayesian mcmc method calibrated by the rise of the ecuadorian andes estimated the time of divergence as mya. our calculations resulted in a divergence time of approximately myr between the northern and southern lineage which falls into the lower end of the confidence intervals calculated by weigt et al.. the difference between both studies in average divergence time estimates very likely rests upon the different geographical dimensions, i.e. in this study sampled populations next to the gap are geographically closer to each other and the sampled area covers a smaller geographic range. ecological niches besides being genetically diverged, the northern and southern lineages of túngara frogs reside in different habitats in panama and costa rica. while the northern lineage occupies dry forests, the southern lineage occurs in moist forests with higher annual precipitation. the actual distribution of northern and southern túngara frogs in middle america generally corresponds to the predicted suitable habitat of niche models in costa rica and panama. the low predicted suitability for the southern lineage across the distribution gap in costa rica is consistent with the known species distribution. suitability across the gap for the northern lineage, however, was intermediate and within the range of known localities. thus, the model suggests that conditions in a portion of the gap might be suitable for the northern lineage. this incorrect prediction of the model could be the result of the exclusion of an environmental variable that limits the species distribution range, ecological interactions with other species across the gap, or an unknown dispersal barrier. because there are not conspicuous dispersal barriers in the region, we suspect that explanations or are the most likely. niche modeling suggests that the absence of each lineage in the distribution gap correlates with ecological variables and therefore suggests that not only do the two lineages inhabit different habitats but there might be ecological divergence between the lineages. niche conservatism is the tendency of species to retain aspects of their fundamental niche over longer time periods and limits the adaptation to ecological conditions at geographic barriers. our results show that the northern and southern lineages occupy different environmental envelopes and this suggests that their niches have been evolutionarily labile. there are two possibilities to explain genetic and ecological divergence in túngara frogs. the first possibility is that túngara frogs were split in two groups by a vicariance event. the geographic separation was then maintained by niche conservatism and followed by niche specialization. otherwise the genetic divergence between lineages in túngara frogs in allopatry could be the result from a combination of niche specialization followed by niche conservatism at the edges of distribution ranges in costa rica. the absence of the southern lineage in the gap suggests that niche conservatism can promote allopatric speciation as proposed by wiens and graham. at the moment our data cannot discriminate between both possibilities for lineage separation. in comparison with the present, suitable habitat during the last glacial maximum covered larger areas for the northern lineage while it was more restricted in the southern lineage. we assume that climatic changes from colder and drier climate to warmer and moister climate are responsible for this development. drier climate during the last glacial maximum also seems to explain the higher habitat resistance of the distribution gap for the southern lineage. since habitat resistance persists today it seems improbable that both groups will come into contact in the near future unless the current climate change will result in more favorable conditions across the gap. speciation in túngara frogs this and several preceding molecular studies revealed that the túngara frog physalaemus pustulosus consists of two genetically different lineages. in this study we found that the lineages inhabit different habitats and thus suggests the possibility that they might be ecologically divergent. the geographic separation and the large genetic divergence and ecological dissimilarity between lineages suggest vicariance events together with ecologically mediated divergent selection as potential speciation mechanisms in túngara frogs. for ecuadorian frogs of the genus epipedobates, sister species with allopatric or parapatric distribution replace each other altitudinally or latitudinally emphasizing the role of environmental niche shift in speciation in neotropical frogs. the ecological separation principally occurs along a temperature/elevational or a seasonality axis. for divergence between túngara frog lineages a shift in seasonality and precipitation might be more important than differences in temperature. ecological prezygotic isolation could arise as a result of adaptation to contrasting habitats or temporal isolation. adaptation to different habitats could entail behavioral reproductive isolation because signals involved in mate choice could be under ecologically based divergent selection. examples include sexual preferences for ecologically selected body size and color pattern in fish or beak size in birds. this possibility needs further exploration in our study species. ron et al. suggested that each lineage of túngara frogs might deserve species status based on the large molecular divergence, size differences, and large divergence time. the observed habitat differences are consistent with the recognition of each lineage as a separate species. species level divergence is also suggested by some evidence of prezygotic isolation. pröhl et al. conducted experiments showing that captive p. pustulosus females from the south did not produce nests with males from the northern lineage. however this was not true for northern females which produced nests and tadpoles with males from both lineages. the pattern of call divergence in the gap region in costa rica shows that several call variables tend to be more divergent near the gap. this pattern is consistent with ancient reproductive character displacement, however, as discussed below, these differences in calls have no influence on female species recognition. the results of phonotactic mate choice experiments with female túngara frogs, on the other hand, do not suggest the two lineages should each be given species status. pröhl et al. showed that calls differ between lineages and populations, and that in about two thirds of the comparisons females showed a significant preference for the local mating call over the foreign call. this preference for the local call was stronger than that found in females from gamboa, which is well within the southern group. neither study, however, suggested that females exhibited stronger preferences for calls of their own lineage versus the other lineage. at this point we feel that the data are not conclusive in determining whether the two lineages of túngara frogs represent distinct species and we propose to collect more data on pre- or post-zygotic reproductive isolation. population genetics, ecology and behavior the results of this study offer the opportunity for new insights on the complex relationship between genetic distance, ecological divergence, call divergence and mate preferences. the areas between two genetic population clusters and areas adjacent to admixture zones are especially interesting for further behavioral observations. such studies might determine whether there are abrupt changes in certain call parameters across genetically different populations, or how the female preference pattern varies across admixture zones. therefore it would be necessary to conduct geographically fine scaled genetic and behavioral analyses in several localities, for example between palmar norte and caracol in south costa rica or between galique and el forastero in western panama. also, in the context of reproductive character displacement and reinforcement coupled with measurement of gene flow, such areas provide exciting possibilities for future research prospects. the pattern of call divergence in the gap region in costa rica mentioned above indicates the opportunity for the evolution of character displacement in túngara frogs. moreover the detected differences in ecology between the two main lineages should influence behavior. the northern frogs inhabit drier forests than the southern frogs. we would assume that temperature and precipitation pattern influence the time of the breeding period and the reproductive behavior. in addition the question whether there is ecological divergence and if it has caused reproductive isolation needs to be examined. finally more intensive population sampling in the southern genetic clusters could reveal ecological differences between them and help to discover local adaptations that might be involved in divergence between lineages.  in this study we found population genetic structure and unexpected ecological diversity between lineages of a neotropical frog species, the túngara frog physalaemus pustulosus. population genetic analyses of middle american populations revealed two allopatric divergent lineages and a further sub-division of the southern lineage into three population clusters. the genetic divergence into two main lineages goes along with ecological differences in habitat type. these results highlight the importance of intensive population sampling across large ranges of a species distribution. overall genetic diversity within populations is high across the sampled range but diversity is higher in the south than in the north. the diversity within populations is maintained by high gene flow between neighbor sample localities. altogether the genetic data support the idea that the túngara frog is an abundant species whose populations within population clusters are well connected via gene flow and little affected by habitat fragmentation. the allopatric distribution north and south of the gap in costa rica seems to be maintained by unsuitable climatic conditions across the gap which likely persisted during the last glaciation and probably during previous glacial and interglacial cycles. since the northern and southern lineages are geographically separated, genetically divergent, and inhabit different habitats there is potential for speciation by ecological divergence in allopatry. we discuss the possibility that both lineages should receive species status and conclude that additional pre- and postzygotic isolation experiments are needed. in addition, further ecological and behavioral analyses among and across southern population clusters could shed light on evolutionary divergence and speciation. 
most population structure is captured by dividing all sample localities into two allopatric genetic lineages. we measure gene flow and migration within and between population clusters and compare genetic diversity between population clusters. gene flow is higher within the northern lineage than within the southern lineage, perhaps due to increased landscape heterogeneity in the south. niche conservatism possibly contributes to preventing movements across the gap and gene flow between both groups. niche modeling reveals differences in suitable habitat between the northern and southern lineages: the northern lineage inhabits dry/pine-oak forests, while the southern lineage is confined to tropical moist forests. in this context we discuss whether the northern and southern lineages should be recognized as separate species, and we conclude that further studies of pre- and post-zygotic isolation are needed for a final assessment. here we combine population genetic methods with phylogenetic information to define genetic population clusters of the wide-spread neotropical túngara frog. the northern genetic lineage is genetically homogenous while the southern lineage is sub-divided into three population clusters by both microsatellite and mtdna analyses. identified population clusters should motivate future behavioral and ecological research regarding within-species biodiversity and speciation mechanisms. both lineages seem to have had little movement across the distribution gap, which persisted during the last glacial maximum. the allopatric distribution is attributed to unsuitable habitat and probably other unknown ecological factors present across the distribution gap. genetic and ecological data indicate that there is the potential for ecological divergence in allopatry between lineages. by applying ecological niche modeling we determine whether the two most divergent genetic groups of the túngara frog inhabit different habitats, and are separated geographically by unsuitable habitat across a gap in the distribution. this study confirms the finding of previous studies that túngara frogs diverged into two allopatric genetic lineages north and south of the gap in the distribution in central costa rica several million years ago. the lack of movement was more pronounced for the southern lineage than for the northern lineage.
 uncovering how populations of a species differ genetically and ecologically is important for understanding evolutionary processes. here we combine population genetic methods with phylogenetic information to define genetic population clusters of the wide-spread neotropical túngara frog. we measure gene flow and migration within and between population clusters and compare genetic diversity between population clusters. by applying ecological niche modeling we determine whether the two most divergent genetic groups of the túngara frog inhabit different habitats, and are separated geographically by unsuitable habitat across a gap in the distribution.  most population structure is captured by dividing all sample localities into two allopatric genetic lineages. the northern genetic lineage is genetically homogenous while the southern lineage is sub-divided into three population clusters by both microsatellite and mtdna analyses. gene flow is higher within the northern lineage than within the southern lineage, perhaps due to increased landscape heterogeneity in the south. niche modeling reveals differences in suitable habitat between the northern and southern lineages: the northern lineage inhabits dry/pine-oak forests, while the southern lineage is confined to tropical moist forests. both lineages seem to have had little movement across the distribution gap, which persisted during the last glacial maximum. the lack of movement was more pronounced for the southern lineage than for the northern lineage.  this study confirms the finding of previous studies that túngara frogs diverged into two allopatric genetic lineages north and south of the gap in the distribution in central costa rica several million years ago. the allopatric distribution is attributed to unsuitable habitat and probably other unknown ecological factors present across the distribution gap. niche conservatism possibly contributes to preventing movements across the gap and gene flow between both groups. genetic and ecological data indicate that there is the potential for ecological divergence in allopatry between lineages. in this context we discuss whether the northern and southern lineages should be recognized as separate species, and we conclude that further studies of pre- and post-zygotic isolation are needed for a final assessment. identified population clusters should motivate future behavioral and ecological research regarding within-species biodiversity and speciation mechanisms. 