Geodesics to characterize the phylogenetic landscape.

Phylogenetic trees are fundamental for understanding evolutionary history. However, finding maximum likelihood trees is challenging due to the complexity of the likelihood landscape and the size of tree space. Based on the Billera-Holmes-Vogtmann (BHV) distance between trees, we describe a method to generate intermediate trees on the shortest path between two trees, called pathtrees. These pathtrees give a structured way to generate and visualize part of treespace. They allow investigating intermediate regions between trees of interest, exploring locally optimal trees in topological clusters of treespace, and potentially finding trees of high likelihood unexplored by tree search algorithms. We compared our approach against other tree search tools (Paup*, RAxML, and RevBayes) using the highest likelihood trees and number of new topologies found, and validated the accuracy of the generated treespace. We assess our method using two datasets. The first consists of 23 primate species (CytB, 1141 bp), leading to well-resolved relationships. The second is a dataset of 182 milksnakes (CytB, 1117 bp), containing many similar sequences and complex relationships among individuals. Our method visualizes the treespace using log likelihood as a fitness function. It finds similarly optimal trees as heuristic methods and presents the likelihood landscape at different scales. It found relevant trees that were not found with MCMC methods. The validation measures indicated that our method performed well mapping treespace into lower dimensions. Our method complements heuristic search analyses, and the visualization allows the inspection of likelihood terraces and exploration of treespace areas not visited by heuristic searches.

The population genetics of speciation by cascade reinforcement.

Species interactions drive diverse evolutionary outcomes. Speciation by cascade reinforcement represents one example of how species interactions can contribute to the proliferation of species. This process occurs when the divergence of mating traits in response to selection against interspecific hybridization incidentally leads to reproductive isolation among populations of the same species. Here, we investigated the population genetic outcomes of cascade reinforcement in North American chorus frogs (Hylidae: Pseudacris). Specifically, we estimated the frequency of hybridization among three taxa, assessed genetic structure within the focal species, P. feriarum, and ascertained the directionality of gene flow within P. feriarum across replicated contact zones via coalescent modeling. Through field observations and preliminary experimental crosses, we assessed whether hybridization is possible under natural and laboratory conditions. We found that hybridization occurs among P. feriarum and two conspecifics at a low rate in multiple contact zones, and that gene flow within the former species is unidirectional from allopatry into sympatry with these other species in three of four contact zones studied. We found evidence of substantial genetic structuring within P. feriarum including a divergent western allopatric cluster, a behaviorally-distinct sympatric South Carolina cluster, and several genetically-overlapping clusters from the remainder of the distribution. Furthermore, we found sub-structuring between reinforced and nonreinforced populations in the two most intensely-sampled contact zones. Our literature review indicated that P. feriarum hybridizes with at least five heterospecifics at the periphery of its range providing a mechanism for further intraspecific diversification. This work strengthens the evidence for cascade reinforcement in this clade, revealing the geographic and genetic landscape upon which this process can contribute to the proliferation of species.

Genetic diversity and population structure of Leishmania (Viannia) braziliensis in the Peruvian jungle.

BACKGROUND: Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is highly prevalent in the Peruvian jungle, where it affects military forces deployed to fight against drug trafficking and civilian people that migrate from the highland to the lowland jungle for economic activities such as mining, agriculture, construction, and chestnut harvest. We explored the genetic diversity and population structure of 124 L. (V.) braziliensis isolates collected from the highland (Junín, Cusco, and Ayacucho) and lowland Peruvian jungle (Loreto, Ucayali, and Madre de Dios). All samples were genotyped using Multilocus Microsatellite Typing (MLMT) of ten highly polymorphic markers. PRINCIPAL FINDINGS: High polymorphism and genetic diversity were found in Peruvian isolates of L. (V.) braziliensis. Most markers are not in Hardy-Weinberg equilibrium; this deviation is most likely caused by local inbreeding, as shown by the positive FIS values. Linkage Disequilibrium in subpopulations was not strong, suggesting the reproduction was not strictly clonal. Likewise, for the first time, two genetic clusters of this parasite were determined, distributed in both areas of the Peruvian jungle, which suggested a possible recent colonization event of the highland jungle from the lowland jungle. CONCLUSIONS: L. (V.) braziliensis exhibits considerable genetic diversity with two different clusters in the Peruvian jungle. Migration analysis suggested a colonization event between geographical areas of distribution. Although no human migration was observed at the time of sampling, earlier displacement of humans, reservoirs, or vectors could have been responsible for the parasite spread in both regions.

Population divergence time estimation using individual lineage label switching.

Divergence time estimation from multilocus genetic data has become common in population genetics and phylogenetics. We present a new Bayesian inference method that treats the divergence time as a random variable. The divergence time is calculated from an assembly of splitting events on individual lineages in a genealogy. The time for such a splitting event is drawn from a hazard function of the truncated normal distribution. This allows easy integration into the standard coalescence framework used in programs such as Migrate. We explore the accuracy of the new inference method with simulated population splittings over a wide range of divergence time values and with a reanalysis of a dataset of 5 populations consisting of 3 present-day populations (Africans, Europeans, Asian) and 2 archaic samples (Altai and Ust'Isthim). Evaluations of simple divergence models without subsequent geneflow show high accuracy, whereas the accuracy of the results of isolation with migration models depends on the magnitude of the immigration rate. High immigration rates lead to a time of the most recent common ancestor of the sample that, looking backward in time, predates the divergence time. Even with many independent loci, accurate estimation of the divergence time with high immigration rates becomes problematic. Our comparison to other software tools reveals that our lineage-switching method, implemented in Migrate, is comparable to IMa2p. The software Migrate can run large numbers of sequence loci (>1,000) on computer clusters in parallel.

Population Genetic Inference With MIGRATE.

Many evolutionary biologists collect genetic data from natural populations and then need to investigate the relationship among these populations to compare different biogeographic hypotheses. MIGRATE, a useful tool for exploring relationships between populations and comparing hypotheses, has existed since 1998. Throughout the years, it has steadily improved in both the quality of algorithms used and in the efficiency of carrying out those calculations, thus allowing for a larger number of loci to be evaluated. This efficiency has been enhanced, as MIGRATE has been developed to perform many of its calculations concurrently when running on a computer cluster. The program is based on the coalescence theory and uses Bayesian inference to estimate posterior probability densities of all the parameters of a user-specified population model. Complex models, which include migration and colonization parameters, can be specified. These models can be evaluated using marginal likelihoods, thus allowing a user to compare the merits of different hypotheses. The three presented protocols will help novice users to develop sophisticated analysis techniques useful for their research projects. © 2019 The Authors. Basic Protocol 1: First steps with MIGRATE Basic Protocol 2: Population model specification Basic Protocol 3: Prior distribution specification Basic Protocol 4: Model selection Support Protocol 1: Installing the program MIGRATE Support Protocol 2: Installation of parallel MIGRATE.

Fractional coalescent.

An approach to the coalescent, the fractional coalescent (f-coalescent), is introduced. The derivation is based on the discrete-time Cannings population model in which the variance of the number of offspring depends on the parameter α. This additional parameter α affects the variability of the patterns of the waiting times; values of [Formula: see text] lead to an increase of short time intervals, but occasionally allow for very long time intervals. When [Formula: see text], the f-coalescent and the Kingman's n-coalescent are equivalent. The distribution of the time to the most recent common ancestor and the probability that n genes descend from m ancestral genes in a time interval of length T for the f-coalescent are derived. The f-coalescent has been implemented in the population genetic model inference software Migrate Simulation studies suggest that it is possible to accurately estimate α values from data that were generated with known α values and that the f-coalescent can detect potential environmental heterogeneity within a population. Bayes factor comparisons of simulated data with [Formula: see text] and real data (H1N1 influenza and malaria parasites) showed an improved model fit of the f-coalescent over the n-coalescent. The development of the f-coalescent and its inclusion into the inference program Migrate facilitates testing for deviations from the n-coalescent.

A synthesis of genetic connectivity in deep-sea fauna and implications for marine reserve design.

With anthropogenic impacts rapidly advancing into deeper waters, there is growing interest in establishing deep-sea marine protected areas (MPAs) or reserves. Reserve design depends on estimates of connectivity and scales of dispersal for the taxa of interest. Deep-sea taxa are hypothesized to disperse greater distances than shallow-water taxa, which implies that reserves would need to be larger in size and networks could be more widely spaced; however, this paradigm has not been tested. We compiled population genetic studies of deep-sea fauna and estimated dispersal distances for 51 studies using a method based on isolation-by-distance slopes. Estimates of dispersal distance ranged from 0.24 km to 2028 km with a geometric mean of 33.2 km and differed in relation to taxonomic and life-history factors as well as several study parameters. Dispersal distances were generally greater for fishes than invertebrates with the Mollusca being the least dispersive sampled phylum. Species that are pelagic as adults were more dispersive than those with sessile or sedentary lifestyles. Benthic species from soft-substrate habitats were generally less dispersive than species from hard substrate, demersal or pelagic habitats. As expected, species with pelagic and/or feeding (planktotrophic) larvae were more dispersive than other larval types. Many of these comparisons were confounded by taxonomic or other life-history differences (e.g. fishes being more dispersive than invertebrates) making any simple interpretation difficult. Our results provide the first rough estimate of the range of dispersal distances in the deep sea and allow comparisons to shallow-water assemblages. Overall, dispersal distances were greater for deeper taxa, although the differences were not large (0.3-0.6 orders of magnitude between means), and imbalanced sampling of shallow and deep taxa complicates any simple interpretation. Our analyses suggest the scales of dispersal and connectivity for reserve design in the deep sea might be comparable to or slightly larger than those in shallow water. Deep-sea reserve design will need to consider the enormous variety of taxa, life histories, hydrodynamics, spatial configuration of habitats and patterns of species distributions. The many caveats of our analyses provide a strong impetus for substantial future efforts to assess connectivity of deep-sea species from a variety of habitats, taxonomic groups and depth zones.

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region.

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.

Assortative mating drives linkage disequilibrium between sperm and egg recognition protein loci in the sea urchin Strongylocentrotus purpuratus.

Sperm and eggs have interacting proteins on their surfaces that influence their compatibility during fertilization. These proteins are often polymorphic within species, producing variation in gamete affinities. We first demonstrate the fitness consequences of various sperm bindin protein (Bindin) variants in the sea urchin Strongylocentrotus purpuratus, and assortative mating between males and females based on their sperm Bindin genotype. This empirical finding of assortative mating based on sperm Bindin genotype could arise by linkage disequilibrium (LD) between interacting sperm and egg recognition loci. We then examine sequence variation in eight exons of the sea urchin egg receptor for sperm Bindin (EBR1). We find little evidence of LD among the eight exons of EBR1, yet strong evidence for LD between sperm Bindin and EBR1 overall, and varying degrees of LD between sperm Bindin among the eight exons. We reject the alternate hypotheses of LD driven by shared evolutionary histories, population structure, or close physical linkage between these interacting loci on the genome. The most parsimonious explanation for this pattern of LD is that it represents selection driven by assortative mating based on interactions among these sperm and egg loci. These findings indicate the importance of ongoing sexual selection in the maintenance of protein polymorphisms and LD, and more generally highlight how LD can be used as an indication of current mate choice, as opposed to historic selection.

A continuous method for gene flow.

Most modern population genetics inference methods are based on the coalescence framework. Methods that allow estimating parameters of structured populations commonly insert migration events into the genealogies. For these methods the calculation of the coalescence probability density of a genealogy requires a product over all time periods between events. Data sets that contain populations with high rates of gene flow among them require an enormous number of calculations. A new method, transition probability-structured coalescence (TPSC), replaces the discrete migration events with probability statements. Because the speed of calculation is independent of the amount of gene flow, this method allows calculating the coalescence densities efficiently. The current implementation of TPSC uses an approximation simplifying the interaction among lineages. Simulations and coverage comparisons of TPSC vs. MIGRATE show that TPSC allows estimation of high migration rates more precisely, but because of the approximation the estimation of low migration rates is biased. The implementation of TPSC into programs that calculate quantities on phylogenetic tree structures is straightforward, so the TPSC approach will facilitate more general inferences in many computer programs.

A possible explanation for the population size discrepancy in tuna (genus Thunnus) estimated from mitochondrial DNA and microsatellite data.

A recent study using both mitochondrial DNA (mtDNA) and microsatellite data reported on a population size discrepancy in the eastern tiger salamander where the effective population size (N(e)) estimate of the former exceeded that of the latter. That study suggested, among other hypotheses, that homoplasy of microsatellite alleles is responsible for the discrepancy. In this investigation, we report 10 new cases of a similar discrepancy in five species of tuna. These cases derive from our Bayesian inferences using data from Pacific Bluefin Tuna (Thunnus orientalis) and Yellowfin Tuna (Thunnus albacares), as well as from published estimates of genetic diversity for additional populations of Yellowfin Tuna and three other tuna species. Phylogenetic character analyses of inferred genealogies of Pacific Bluefin and Yellowfin Tuna reveal similar reduced levels of mtDNA and microsatellite homoplasy. Thus, the discrepancy between inferred population sizes from mtDNA and microsatellite data in tuna is most likely not an artifact of the chosen mutation models used in the microsatellite analyses, but may reflect behavioral differences between the sexes such as female-biased philopatry and male-biased dispersal. This explanation now warrants critical testing with more local populations of tuna and with other animal and plant groups that have different life histories.

Balancing a cline by influx of migrants: a genetic transition in water frogs of eastern Greece.

Variation patterns of allozymes and of ND3 haplotypes of mitochondrial DNA reveal a zone of genetic transition among western Palearctic water frogs extending across northeastern Greece and European Turkey. At the western end of the zone, allozymes characteristic of Central European frogs known as Pelophylax ridibundus predominate, whereas at the eastern end, alleles characteristic of western Anatolian water frogs (P. cf. bedriagae) prevail. The ND3 haplotypes reveal 2 major clades, 1 characteristic of Anatolian frogs, the other of European; the European clade itself has distinct eastern and western subclades. Both the 2 major clades and the 2 subclades overlap within the transition zone. Using Bayesian model selection methods, allozyme data suggest considerable immigration into the Nestos River area from eastern and western populations. In contrast, the ND3 data suggest that migration rates are so high among all locations that they form a single panmictic unit; the best model for allozymes is second best for mitochondrial DNA (mtDNA). Nuclear markers (allozymes), which have roughly 4 times as deep a coalescent history as mtDNA data and thus may reflect patterns over a longer time, indicate that eastern and western refugial populations have expanded since deglaciation (in the last 10,000 years) and have met near the Nestos River, whereas the mtDNA with its smaller effective population size has already lost the signal of partitioning into refugia.

Biogeographic models of gene flow in two waterfowl of the Australo-Papuan tropics.

There are many large, easy-to-observe anseriform birds (ducks, geese, and swans) in northern Australia and New Guinea and they often gather in large numbers. Yet, the structure of their populations and their regional movements are poorly understood. Lack of understanding of population structure limits our capacity to understand source-sink dynamics relevant to their conservation or assess risks associated with avian-borne pathogens, in particular, avian influenza for which waterfowl are the main reservoir species. We set out to assess present-day genetic connectivity between populations of two widely distributed waterfowl in the Australo-Papuan tropics, magpie goose Anseranas semipalmata (Latham, 1798) and wandering whistling-duck Dendrocygna arcuata (Horsfield, 1824). Microsatellite data were obtained from 237 magpie geese and 64 wandering whistling-duck. Samples were collected across northern Australia, and at one site each in New Guinea and Timor Leste. In the wandering whistling-duck, genetic diversity was significantly apportioned by region and sampling location. For this species, the best model of population structure was New Guinea as the source population for all other populations. One remarkable result for this species was genetic separation of two flocks sampled contemporaneously on Cape York Peninsula only a few kilometers apart. In contrast, evidence for population structure was much weaker in the magpie goose, and Cape York as the source population provided the best fit to the observed structure. The fine scale genetic structure observed in wandering whistling-duck and magpie goose is consistent with earlier suggestions that the west-coast of Cape York Peninsula is a flyway for Australo-Papuan anseriforms between Australia and New Guinea across Torres Strait.

Gene flow and population structure in the Mexican blind cavefish complex (Astyanax mexicanus).

BACKGROUND: Cave animals converge evolutionarily on a suite of troglomorphic traits, the best known of which are eyelessness and depigmentation. We studied 11 cave and 10 surface populations of Astyanax mexicanus in order to better understand the evolutionary origins of the cave forms, the basic genetic structuring of both cave and surface populations, and the degree to which present day migration among them affects their genetic divergence. RESULTS: To assess the genetic structure within populations and the relationships among them we genotyped individuals at 26 microsatellite loci. We found that surface populations are similar to one another, despite their relatively large geographic separation, whereas the cave populations are better differentiated. The cave populations we studied span the full range of the cave forms in three separate geographic regions and have at least five separate evolutionary origins. Cave populations had lower genetic diversity than surface populations, correlated with their smaller effective population sizes, probably the result of food and space limitations. Some of the cave populations receive migrants from the surface and exchange migrants with one another, especially when geographically close. This admixture results in significant heterozygote deficiencies at numerous loci due to Wahlund effects. Cave populations receiving migrants from the surface contain small numbers of individuals that are intermediate in both phenotype and genotype, affirming at least limited gene flow from the surface. CONCLUSIONS: Cave populations of this species are derived from two different surface stocks denoted "old" and "new." The old stock colonized caves at least three times independently while the new stock colonized caves at least twice independently. Thus, the similar cave phenotypes found in these caves are the result of repeated convergences. These phenotypic convergences have occurred in spite of gene flow from surface populations suggesting either strong natural or sexual selection for alleles responsible for the cave phenotype in the cave environment.

BEAGLE: an application programming interface and high-performance computing library for statistical phylogenetics.

Phylogenetic inference is fundamental to our understanding of most aspects of the origin and evolution of life, and in recent years, there has been a concentration of interest in statistical approaches such as Bayesian inference and maximum likelihood estimation. Yet, for large data sets and realistic or interesting models of evolution, these approaches remain computationally demanding. High-throughput sequencing can yield data for thousands of taxa, but scaling to such problems using serial computing often necessitates the use of nonstatistical or approximate approaches. The recent emergence of graphics processing units (GPUs) provides an opportunity to leverage their excellent floating-point computational performance to accelerate statistical phylogenetic inference. A specialized library for phylogenetic calculation would allow existing software packages to make more effective use of available computer hardware, including GPUs. Adoption of a common library would also make it easier for other emerging computing architectures, such as field programmable gate arrays, to be used in the future. We present BEAGLE, an application programming interface (API) and library for high-performance statistical phylogenetic inference. The API provides a uniform interface for performing phylogenetic likelihood calculations on a variety of compute hardware platforms. The library includes a set of efficient implementations and can currently exploit hardware including GPUs using NVIDIA CUDA, central processing units (CPUs) with Streaming SIMD Extensions and related processor supplementary instruction sets, and multicore CPUs via OpenMP. To demonstrate the advantages of a common API, we have incorporated the library into several popular phylogenetic software packages. The BEAGLE library is free open source software licensed under the Lesser GPL and available from http://beagle-lib.googlecode.com. An example client program is available as public domain software.

Global migration dynamics underlie evolution and persistence of human influenza A (H3N2).

The global migration patterns of influenza viruses have profound implications for the evolutionary and epidemiological dynamics of the disease. We developed a novel approach to reconstruct the genetic history of human influenza A (H3N2) collected worldwide over 1998 to 2009 and used it to infer the global network of influenza transmission. Consistent with previous models, we find that China and Southeast Asia lie at the center of this global network. However, we also find that strains of influenza circulate outside of Asia for multiple seasons, persisting through dynamic migration between northern and southern regions. The USA acts as the primary hub of temperate transmission and, together with China and Southeast Asia, forms the trunk of influenza's evolutionary tree. These findings suggest that antiviral use outside of China and Southeast Asia may lead to the evolution of long-term local and potentially global antiviral resistance. Our results might also aid the design of surveillance efforts and of vaccines better tailored to different geographic regions.

Unified framework to evaluate panmixia and migration direction among multiple sampling locations.

For many biological investigations, groups of individuals are genetically sampled from several geographic locations. These sampling locations often do not reflect the genetic population structure. We describe a framework using marginal likelihoods to compare and order structured population models, such as testing whether the sampling locations belong to the same randomly mating population or comparing unidirectional and multidirectional gene flow models. In the context of inferences employing Markov chain Monte Carlo methods, the accuracy of the marginal likelihoods depends heavily on the approximation method used to calculate the marginal likelihood. Two methods, modified thermodynamic integration and a stabilized harmonic mean estimator, are compared. With finite Markov chain Monte Carlo run lengths, the harmonic mean estimator may not be consistent. Thermodynamic integration, in contrast, delivers considerably better estimates of the marginal likelihood. The choice of prior distributions does not influence the order and choice of the better models when the marginal likelihood is estimated using thermodynamic integration, whereas with the harmonic mean estimator the influence of the prior is pronounced and the order of the models changes. The approximation of marginal likelihood using thermodynamic integration in MIGRATE allows the evaluation of complex population genetic models, not only of whether sampling locations belong to a single panmictic population, but also of competing complex structured population models.

Genetic variation of Anastrepha suspensa (Diptera: Tephritidae) in Florida and the Caribbean using microsatellite DNA markers.

Anastrepha suspensa (Loew) (Diptera: Tephritidae), the Caribbean fruit fly, is indigenous to Florida and the Greater Antilles where it causes economic losses in fruit crops, including citrus. Because of the geographic separation of many of its native locations and anecdotal descriptions of regional differences in host preferences, there have been questions about the population structure of A. suspensa. Seven DNA microsatellite markers were used to characterize the population genetic structure of A. suspensa, in Florida and the Caribbean from a variety of hosts, including citrus. We genotyped 729 A. suspensa individuals from Florida, Puerto Rico, Cayman Island, Dominican Republic, and Jamaica. The investigated seven loci displayed from 5 to 19 alleles, with expected heterozygosities ranging from 0.05 to 0.83. There were five unique alleles in Florida and three unique alleles in the Caribbean samples; however, no microsatellite alleles were specific to a single host plant. Genetic diversity was analyzed using F(ST) and analysis of molecular variance and revealed low genetic diversity between Florida and Caribbean samples and also between citrus and noncitrus samples. Analyses using migrate revealed there is continuous gene flow between sampling sites in Florida and the Caribbean and among different hosts. These results support previous comparisons based on the mitochondrial cytochrome oxidase I locus indicating there is no genetic differentiation among locations in Florida and the Caribbean and that there is no separation into host races.

Phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs have been determined by geological processes and climate change in the Late Cenozoic.

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.

In defence of model-based inference in phylogeography.

Recent papers have promoted the view that model-based methods in general, and those based on Approximate Bayesian Computation (ABC) in particular, are flawed in a number of ways, and are therefore inappropriate for the analysis of phylogeographic data. These papers further argue that Nested Clade Phylogeographic Analysis (NCPA) offers the best approach in statistical phylogeography. In order to remove the confusion and misconceptions introduced by these papers, we justify and explain the reasoning behind model-based inference. We argue that ABC is a statistically valid approach, alongside other computational statistical techniques that have been successfully used to infer parameters and compare models in population genetics. We also examine the NCPA method and highlight numerous deficiencies, either when used with single or multiple loci. We further show that the ages of clades are carelessly used to infer ages of demographic events, that these ages are estimated under a simple model of panmixia and population stationarity but are then used under different and unspecified models to test hypotheses, a usage the invalidates these testing procedures. We conclude by encouraging researchers to study and use model-based inference in population genetics.