Cryptic diversity and phylogeographic patterns of Mediodactylus species in the Eastern Mediterranean region.

Cryptic diversity poses a great obstacle in our attempts to assess the current biodiversity crisis and may hamper conservation efforts. The gekkonid genus Mediodactylus, a well-known case of hidden species and genetic diversity, has been taxonomically reclassified several times during the last decade. Focusing on the Mediterranean populations, a recent study within the M. kotschyi species complex using classic mtDNA/nuDNA markers suggested the existence of five distinct species, some being endemic and some possibly threatened, yet their relationships have not been fully resolved. Here, we generated genome-wide SNPs (using ddRADseq) and applied molecular species delimitation approaches and population genomic analyses to further disentangle these relationships. Τhe most extensive nuclear dataset, so far, encompassing 2,360 loci and âˆ¼ 699,000 bp from across the genome of Mediodactylus gecko, enabled us to resolve previously obscure phylogenetic relationships among the five, recently elevated, Mediodactylus species and to support the hypothesis that the taxon includes several new, undescribed species. Population genomic analyses within each of the proposed species showed strong genetic structure and high levels of genetic differentiation among populations.

Whole-genome SNP genotyping unveils ancestral and recent introgression in wild and domestic goats.

After the domestication of goats around 10,000 years before the present (BP), humans transported goats far beyond the range of their wild ancestor, the bezoar goat. This brought domestic goats into contact with many wild goat species such as ibex and markhor, enabling introgression between domestic and wild goats. To investigate this, while shedding light on the taxonomic status of wild and domestic goats, we analysed genome-wide SNP data of 613 specimens from 14 taxonomic units, including Capra hircus, C. pyrenaica, C. ibex (from Switzerland, Austria, Germany and Slovenia), C. aegagrus aegagrus, C. a. cretica, C. h. dorcas, C. caucasica caucasica, C. c. severtzovi, C. c. cylindricornis, C. falconeri, C. sibirica sibirica, C. s. alaiana and C. nubiana, as well as Oreamnos americanus (mountain goat) as an outgroup. To trace gene flow between domestic and wild goats, we integrated genotype data of local goat breeds from the Alps as well as from countries such as Spain, Greece, Türkiye, Egypt, Sudan, Iran, Russia (Caucasus and Altai) and Pakistan. Our phylogenetic analyses displayed a clear separation between bezoar-type and ibex-type clades with wild goats from the Greek islands of Crete and Youra clustered within domestic goats, confirming their feral origin. Our analyses also revealed gene flow between the lineages of Caucasian tur and domestic goats that most likely occurred before or during early domestication. Within the clade of domestic goats, analyses inferred gene flow between African and Iberian goats. The detected events of introgression were consistent with previous reports and offered interesting insights into the historical relationships among domestic and wild goats.

Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

Infectious disease threats to amphibians in Greece: new localities positive for Batrachochytrium dendrobatidis.

In the early 2000s, numerous cases of European amphibian population declines and mass die-offs started to emerge. Investigating those events led to the discovery that wild European amphibians were confronted with grave disease threats caused by introduced pathogens, namely the amphibian and the salamander chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) and ranaviruses. In Greece, Bd was previously documented among wild amphibian populations in 2 different locations and 3 different species. However, no disease-related mass declines or mortality events have been reported. In this work, we build upon previous findings with new, subsequently obtained data, resulting in a 225-sample dataset of 14 species from 17 different locations throughout Greece, in order to examine the occurrence status of all 3 pathogens responsible for emerging infectious diseases in European amphibians. No positive samples for Bsal or ranavirus were recorded in any location. We confirmed the presence of Bd in 4 more localities and in 4 more species, including 1 urodelan (Macedonian crested newt Triturus macedonicus) and 1 introduced anuran (American bullfrog Lithobates catesbeianus). All insular localities were negative for Bd, except for Crete, where Bd was identified in 2 different locations. Again, no mass declines or die-offs were recorded in any Bd-positive area or elsewhere. However, given the persistence of Bd across Greece over the past ~20 yr, monitoring efforts should continue, and ideally be further expanded.

Endogenous Viral Elements in Shrew Genomes Provide Insights into Pestivirus Ancient History.

As viral genomic imprints in host genomes, endogenous viral elements (EVEs) shed light on the deep evolutionary history of viruses, ancestral host ranges, and ancient viral-host interactions. In addition, they may provide crucial information for calibrating viral evolutionary timescales. In this study, we conducted a comprehensive in silico screening of a large data set of available mammalian genomes for EVEs deriving from members of the viral family Flaviviridae, an important group of viruses including well-known human pathogens, such as Zika, dengue, or hepatitis C viruses. We identified two novel pestivirus-like EVEs in the reference genome of the Indochinese shrew (Crocidura indochinensis). Homologs of these novel EVEs were subsequently detected in vivo by molecular detection and sequencing in 27 shrew species, including 26 species representing a wide distribution within the Crocidurinae subfamily and one in the Soricinae subfamily on different continents. Based on this wide distribution, we estimate that the integration event occurred before the last common ancestor of the subfamily, about 10.8 million years ago, attesting to an ancient origin of pestiviruses and Flaviviridae in general. Moreover, we provide the first description of Flaviviridae-derived EVEs in mammals even though the family encompasses numerous mammal-infecting members. This also suggests that shrews were past and perhaps also current natural reservoirs of pestiviruses. Taken together, our results expand the current known Pestivirus host range and provide novel insight into the ancient evolutionary history of pestiviruses and the Flaviviridae family in general.

Genomic consequences of colonisation, migration and genetic drift in barn owl insular populations of the eastern Mediterranean.

The study of insular populations was key in the development of evolutionary theory. The successful colonisation of an island depends on the geographic context, and specific characteristics of the organism and the island, but also on stochastic processes. As a result, apparently identical islands may harbour populations with contrasting histories. Here, we use whole genome sequences of 65 barn owls to investigate the patterns of inbreeding and genetic diversity of insular populations in the eastern Mediterranean Sea. We focus on Crete and Cyprus, islands with similar size, climate and distance to mainland, that provide natural replicates for a comparative analysis of the impacts of microevolutionary processes on isolated populations. We show that barn owl populations from each island have a separate origin, Crete being genetically more similar to other Greek islands and mainland Greece, and Cyprus more similar to the Levant. Further, our data show that their respective demographic histories following colonisation were also distinct. On the one hand, Crete harbours a small population and maintains very low levels of gene flow with neighbouring populations. This has resulted in low genetic diversity, strong genetic drift, increased relatedness in the population and remote inbreeding. Cyprus, on the other hand, appears to maintain enough gene flow with the mainland to avoid such an outcome. Our study provides a comparative population genomic analysis of the effects of neutral processes on a classical island-mainland model system. It provides empirical evidence for the role of stochastic processes in determining the fate of diverging isolated populations.

Population Genomics of Wall Lizards Reflects the Dynamic History of the Mediterranean Basin.

The Mediterranean Basin has experienced extensive change in geology and climate over the past six million years. Yet, the relative importance of key geological events for the distribution and genetic structure of the Mediterranean fauna remains poorly understood. Here, we use population genomic and phylogenomic analyses to establish the evolutionary history and genetic structure of common wall lizards (Podarcis muralis). This species is particularly informative because, in contrast to other Mediterranean lizards, it is widespread across the Iberian, Italian, and Balkan Peninsulas, and in extra-Mediterranean regions. We found strong support for six major lineages within P. muralis, which were largely discordant with the phylogenetic relationship of mitochondrial DNA. The most recent common ancestor of extant P. muralis was likely distributed in the Italian Peninsula, and experienced an "Out-of-Italy" expansion following the Messinian salinity crisis (∼5 Mya), resulting in the differentiation into the extant lineages on the Iberian, Italian, and Balkan Peninsulas. Introgression analysis revealed that both inter- and intraspecific gene flows have been pervasive throughout the evolutionary history of P. muralis. For example, the Southern Italy lineage has a hybrid origin, formed through admixture between the Central Italy lineage and an ancient lineage that was the sister to all other P. muralis. More recent genetic differentiation is associated with the onset of the Quaternary glaciations, which influenced population dynamics and genetic diversity of contemporary lineages. These results demonstrate the pervasive role of Mediterranean geology and climate for the evolutionary history and population genetic structure of extant species.

Endoparasites of European Wildcats (Felis silvestris) in Greece.

The European wildcat (Felis silvestris) is the only wild felid living in Greece. Wildcat populations are declining due to anthropogenic and phenological unfavourable conditions, and parasites may have an additional negative impact. In the present study, the occurrence of endoparasites in wildcats in Greece and the potential threats posed to wildcats, domestic animals, and humans in the study areas has been investigated. In a six-year period, 23 road-killed wildcats and 62 wildcat faecal samples were collected from different areas of the country. Necropsy for the detection of endoparasites and standard parasitological examinations of faecal samples were performed. Parasites were morphologically identified and, in selected cases, molecularly analysed. All necropsied wildcats (100%) were infected by three to 10 different parasite taxa, with the most prevalent being Taenia taeniaeformis (73.9%), Toxocara cati (60.9%), Angiostrongylus chabaudi (56.5%), Ancylostoma tubaeforme (39.1%), Cylicospirura spp. (34.8%), Troglostrongylus brevior (34.8%), and Capillaria aerophila (33.8%). Of the 62 faecal samples examined, 53 (85.5%) were positive for one or more parasite elements (larvae, eggs, or oocysts). The most frequent were T. cati (45.2%), A. chabaudi (29%), C. aerophila (24.2%), and Ancylostomatidae (17.7%). This is the first survey on endoparasites affecting wildcats in Greece. Some of the parasites here found are frequent in domestic and wild felids, while others, i.e., Oslerus rostratus and Cylicospirura petrowi, were described for the first time in the European wildcat. Most of them have a significant pathogenic potential, causing severe to hazardous diseases to infected felids and some, under specific circumstances, can also threaten human health.

Long-term stability of sex chromosome gene content allows accurate qPCR-based molecular sexing across birds.

Embryos, juveniles, and even adults of many bird species lack pronounced external sexually dimorphic characteristics. Accurate identification of sex is crucial for research (e.g., developmental, population, and evolutionary studies), management of wildlife species, and captive breeding programmes for both conservation and poultry. An accurate molecular sexing method applicable across the entire bird radiation is theoretically possible thanks to the long-term stability of their ZZ/ZW sex chromosomes, but current methods are not applicable in a wide range of bird lineages. Here, we developed a novel molecular sexing method based on the comparison of gene copy number variation by quantitative real-time PCR (qPCR) in conserved Z-specific genes (CHRNA6, DDX4, LPAR1, TMEM161B, VPS13A), i.e. genes linked to Z but absent from W chromosomes. We tested the method across three paleognath and 70 neognath species covering the avian phylogeny. In addition, we designed primers for four Z-specific genes (DOCK8, FUT10, PIGG and PSD3) for qPCR-based molecular sexing in three paleognath species. We have demonstrated that the genes DOCK8, FUT10, PIGG and PSD3 can identify sex in paleognath birds and the genes CHRNA6, DDX4, TMEM161B, and VPS13A can reveal sex in neognath birds. The gene LPAR1 can be used to accurately identify sex in both paleognath and neognath species. Along with outlining a novel method of practical importance for molecular sexing in birds, our study also documents in detail the conservation of sex chromosomes across the avian phylogeny.

The wall lizards of the Balkan peninsula: Tackling questions at the interface of phylogenomics and population genomics.

Wall lizards of the genus Podarcis (Sauria, Lacertidae) are the predominant reptile group in southern Europe, including 24 recognized species. Mitochondrial DNA data have shown that, with the exception of P. muralis, the Podarcis species distributed in the Balkan peninsula form a species group that is further sub-divided into two subgroups: the one of "P. tauricus" consisting of P. tauricus, P. milensis, P. gaigeae, and P. melisellensis, and the other of "P. erhardii" comprising P. erhardii, P. levendis, P. cretensis, and P. peloponnesiacus. In an attempt to explore the Balkan Podarcis phylogenomic relationships, assess the levels of genetic structure and to re-evaluate the number of extant species, we employed phylogenomic and admixture approaches on ddRADseq (double digested Restriction site Associated DNA sequencing) genomic data. With this efficient Next Generation Sequencing approach, we were able to obtain a large number of genomic loci randomly distributed throughout the genome and use them to resolve the previously obscure phylogenetic relationships among the different Podarcis species distributed in the Balkans. The obtained phylogenomic relationships support the monophyly of both aforementioned subgroups and revealed several divergent lineages within each subgroup, stressing the need for taxonomic re-evaluation of Podarcis' species in Balkans. The phylogenomic trees and the species delimitation analyses confirmed all recently recognized species (P. levendis, P. cretensis, and P. ionicus) and showed the presence of at least two more species, one in P. erhardii and the other in P. peloponnesiacus.

Landscape Connectivity Limits the Predicted Impact of Fungal Pathogen Invasion.

Infectious diseases are major drivers of biodiversity loss. The risk of fungal diseases to the survival of threatened animals in nature is determined by a complex interplay between host, pathogen and environment. We here predict the risk of invasion of populations of threatened Mediterranean salamanders of the genus Lyciasalamandra by the pathogenic chytrid fungus Batrachochytrium salamandrivorans by combining field sampling and lab trials. In 494 samples across all seven species of Lyciasalamandra, B. salamandrivorans was found to be absent. Single exposure to a low (1000) number of fungal zoospores resulted in fast buildup of lethal infections in three L. helverseni. Thermal preference of the salamanders was well within the thermal envelope of the pathogen and body temperatures never exceeded the fungus' thermal critical maximum, limiting the salamanders' defense opportunities. The relatively low thermal host preference largely invalidates macroclimatic based habitat suitability predictions and, combined with current pathogen absence and high host densities, suggests a high probability of local salamander population declines upon invasion by B. salamandrivorans. However, the unfavorable landscape that shaped intraspecific host genetic diversity, lack of known alternative hosts and rapid host mortality after infection present barriers to further, natural pathogen dispersal between populations and thus species extinction. The risk of anthropogenic spread stresses the importance of biosecurity in amphibian habitats.

Genomic and mitochondrial evidence of ancient isolations and extreme introgression in the four-lined snake.

Comparing mitochondrial and genomic phylogenies is an essential tool for investigating speciation processes, because each genome carries different inheritance properties and evolutionary characteristics. Furthermore, mitonuclear discordance may arise from ecological adaptation, historic isolation, population size changes, and sex-biased dispersal. Closely related taxa are expected to experience gene flow; however, this may not be true for insular populations or populations isolated in refugia. The four-lined snake Elaphe quatuorlineata has a fragmented distribution, separating populations of the Italian and Balkan Peninsulas, whereas several insular Aegean populations of significantly smaller body size (Cyclades island group and Skyros Island, Greece) are currently considered distinct subspecies. We constructed the species-tree phylogeny of this species utilizing genome-wide single nucleotide polymorphisms and a gene-tree based on complete cytochrome b sequences, aiming to detect convergence and discrepancies between biparentally and maternally inherited genomes. Population structuring, phylogenetic patterns and migration events among geographically defined lineages supported our hypothesis of isolation in multiple sub-refugia. Where biogeographical barriers did not restrict subsequent dispersal, extensive genetic exchange occurred between mainland Balkan populations. This process has led to the mitochondrial sweep of an ancestral mitolineage that survived only in peripheral (East Greece) and insular populations (North Cyclades and Skyros). The Central Cyclades represent an ancient lineage for both molecular markers that emerged almost 3.3 Mya. Considering their distinct morphology, insular E. quatuorlineata populations should be the future focus of an extensive sampling, especially since the mitonuclear discordance observed in this species could be related to ecological adaptations, such as the island-dwarfism phenomenon.

Impacts of the synthetic androgen Trenbolone on gonad differentiation and development - comparisons between three deeply diverged anuran families.

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17ß-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in three environmentally and/or physiologically relevant concentrations (0.027 µg/L (10-10 M), 0.27 µg/L (10-9 M), 2.7 µg/L (10-8 M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10-9 M) and 2.7 µg/L (10-8 M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2).

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates).

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species-level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near-East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.

Little evidence for switches to environmental sex determination and turnover of sex chromosomes in lacertid lizards.

Amniotes possess variability in sex determination, from environmental sex determination (ESD), where no sex chromosomes are present, to genotypic sex determination (GSD) with highly differentiated sex chromosomes. Some evolutionary scenarios postulate high stability of differentiated sex chromosomes and rare transitions from GSD to ESD. However, sex chromosome turnovers and two independent transitions from highly differentiated ZZ/ZW sex chromosomes to ESD were previously reported in the lacertid lizards. Here, we examined the homology of sex chromosomes in the wide phylogenetic spectrum of lacertids and their outgroups by comparing gene copy numbers between sexes in genes previously found to be Z-specific in some lacertids. Our current sampling covers 45 species from 26 genera including lineages supposed to possess a derived sex determining systems. We found that all tested lacertids share homologous differentiated ZZ/ZW sex chromosomes, which were present already in their common ancestor living around 85 million years ago. These differentiated sex chromosomes are not present in amphisbaenians and teiid lizards, the close relatives of lacertids. Our study demonstrates how inaccuracies in data can influence the outcome of phylogenetic reconstructions of evolution of sex determination, in this case they overestimated the number of shifts from GSD to ESD and the rate in turnovers of sex chromosomes.

Multigene phylogeny, phylogeography and population structure of Podarcis cretensis species group in south Balkans.

The evolutionary history of taxa with limited overseas dispersal abilities is considered to be majorly influenced by vicariant events constituting them as model organisms for the interpretation of evolutionary processes. An excellent candidate are the wall lizards of the genus Podarcis exhibiting an impressive level of genetic and morphological diversification and harboring several cases of recently discovered cryptic diversity. In this study, we investigated the effect of palaeogeographic events on the wall lizards' biodiversity patterns in the Aegean (Greece) as well as the evolutionary processes that acted both in space and time. To accomplish that we studied a group of three endemic Podarcis species (i.e., P. cretensis, P. levendis, and P. peloponnesiacus) both at the intra and interspecific levels employing mitochondrial and nuclear DNA sequence data as well as microsatellites. Furthermore, presence information coupled with bioclimatic data (i.e., species distribution modeling, and niche similarity analyses) shed light on the necessary ecological factors for the species' occurrence. These approaches revealed yet another case of cryptic diversity for this group of lizards, with the existence of two slightly overlapping lineages within P. peloponnesiacus and highly structured populations within P. cretensis. Species diversification occurred during the Pliocene with P. peloponnesiacus divergence into the two lineages dating back to 1.86 Mya. Furthermore, temperature and precipitation related environmental parameters were the most important ones regarding the current distribution of the studied species. Based on the results, we propose a more detailed phylogeographic scenario where both the paleogeography of the area and several environmental parameters have shaped the genetic diversity and the current distribution pattern of this species group.

Phylogeography of Aegean green toads (Bufo viridis subgroup): continental hybrid swarm vs. insular diversification with discovery of a new island endemic.

BACKGROUND: Debated aspects in speciation research concern the amount of gene flow between incipient species under secondary contact and the modes by which post-zygotic isolation accumulates. Secondary contact zones of allopatric lineages, involving varying levels of divergence, provide natural settings for comparative studies, for which the Aegean (Eastern Mediterranean) geography offers unique scenarios. In Palearctic green toads (Bufo viridis subgroup or Bufotes), Plio-Pleistocene (~ 2.6 Mya) diverged species show a sharp transition without contemporary gene flow, while younger lineages, diverged in the Lower-Pleistocene (~ 1.9 Mya), admix over tens of kilometers. Here, we conducted a fine-scale multilocus phylogeographic analysis of continental and insular green toads from the Aegean, where a third pair of taxa, involving Mid-Pleistocene diverged (~ 1.5 Mya) mitochondrial lineages, earlier tentatively named viridis and variabilis, (co-)occurs. RESULTS: We discovered a new lineage, endemic to Naxos (Central Cyclades), while coastal islands and Crete feature weak genetic differentiation from the continent. In continental Greece, both lineages, viridis and variabilis, form a hybrid swarm, involving massive mitochondrial and nuclear admixture over hundreds of kilometers, without obvious selection against hybrids. CONCLUSIONS: The genetic signatures of insular Aegean toads appear governed by bathymetry and Quaternary sea level changes, resulting in long-term isolation (Central Cyclades: Naxos) and recent land-bridges (coastal islands). Conversely, Crete has been isolated since the end of the Messinian salinity crisis (5.3 My) and Cretan populations thus likely result from human-mediated colonization, at least since Antiquity, from Peloponnese and Anatolia. Comparisons of green toad hybrid zones support the idea that post-zygotic hybrid incompatibilities accumulate gradually over the genome. In this radiation, only one million years of divergence separate a scenario of complete reproductive isolation, from a secondary contact resulting in near panmixia.

Multilocus phylogeny and coalescent species delimitation in Kotschy's gecko, Mediodactylus kotschyi: Hidden diversity and cryptic species.

Kotschy's Gecko, Mediodactylus kotschyi, is a small gecko native to southeastern Europe and the Levant. It displays great morphological variation with a large number of morphologically recognized subspecies. However, it has been suggested that it constitutes a species complex of several yet unrecognized species. In this study, we used multilocus sequence data (three mitochondrial and three nuclear gene fragments) to estimate the phylogenetic relationships of 174 specimens from 129 sampling localities, covering a substantial part of the distribution range of the species. Our results revealed high genetic diversity of M. kotschyi populations and contributed to our knowledge about the phylogenetic relationships and the estimation of the divergence times between them. Diversification within M. kotschyi began approximately 15 million years ago (Mya) in the Middle Miocene, whereas the diversification within most of the major clades have been occurred in the last 5 Mya. Species delimitation analysis suggests there exists five species within the complex, and we propose to tentatively recognize the following taxa as full species: M. kotschyi (mainland Balkans, most of Aegean islands, and Italy), M. orientalis (Levant, Cyprus, southern Anatolia, and south-eastern Aegean islands), M. danilewskii (Black Sea region and south-western Anatolia), M. bartoni (Crete), and M. oertzeni (southern Dodecanese Islands). This newly recognized diversity underlines the complex biogeographical history of the Eastern Mediterranean region.