Pre-mandibular pharyngeal pouches in early non-teleost fish embryos.

The vertebrate pharynx is a key embryonic structure with crucial importance for the metameric organization of the head and face. The pharynx is primarily built upon progressive formation of paired pharyngeal pouches that typically develop in post-oral (mandibular, hyoid and branchial) domains. However, in the early embryos of non-teleost fishes, we have previously identified pharyngeal pouch-like outpocketings also in the pre-oral domain of the cranial endoderm. This pre-oral gut (POG) forms by early pouching of the primitive gut cavity, followed by the sequential formation of typical (post-oral) pharyngeal pouches. Here, we tested the pharyngeal nature of the POG by analysing expression patterns of selected core pharyngeal regulatory network genes in bichir and sturgeon embryos. Our comparison revealed generally shared expression patterns, including Shh, Pax9, Tbx1, Eya1, Six1, Ripply3 or Fgf8, between early POG and post-oral pharyngeal pouches. POG thus shares pharyngeal pouch-like morphogenesis and a gene expression profile with pharyngeal pouches and can be regarded as a pre-mandibular pharyngeal pouch. We further suggest that pre-mandibular pharyngeal pouches represent a plesiomorphic vertebrate trait inherited from our ancestor's pharyngeal metameric organization, which is incorporated in the early formation of the pre-chordal plate of vertebrate embryos.

A lamprey neural cell type atlas illuminates the origins of the vertebrate brain.

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey-a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits-based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

Cartilage diversification and modularity drove the evolution of the ancestral vertebrate head skeleton.

The vertebrate head skeleton has evolved a myriad of forms since their divergence from invertebrate chordates. The connection between novel gene expression and cell types is therefore of importance in this process. The transformation of the jawed vertebrate (gnathostome) head skeleton from oral cirri to jointed jaw elements required a diversity of cartilages as well as changes in the patterning of these tissues. Although lampreys are a sister clade to gnathostomes, they display skeletal diversity with distinct gene expression and histologies, a useful model for addressing joint evolution. Specifically, the lamprey tissue known as mucocartilage has noted similarities with the jointed elements of the mandibular arch in jawed vertebrates. We thus asked whether the cells in lamprey mucocartilage and gnathostome joint tissue could be considered homologous. To do this, we characterized new genes that are involved in gnathostome joint formation and characterized the histochemical properties of lamprey skeletal types. We find that most of these genes are minimally found in mucocartilage and are likely later innovations, but we do identify new activity for gdf5/6/7b in both hyaline and mucocartilage, supporting its role as a chondrogenic regulator. Contrary to previous works, our histological assays do not find any perichondrial fibroblasts surrounding mucocartilage, suggesting that mucocartilage is non-skeletogenic tissue that is partially chondrified. Interestingly, we also identify new histochemical features of the lamprey otic capsule that diverge from normal hyaline. Paired with our new insights into lamprey mucocartilage, we propose a broader framework for skeletal evolution in which an ancestral soxD/E and gdf5/6/7 network directs mesenchyme along a spectrum of cartilage-like features.

Phylogenomics of Anguis and Pseudopus (Squamata, Anguidae) indicates Balkan-Apennine mitochondrial capture associated with the Messinian event.

A dated phylogenetic hypothesis on the evolutionary history of the extant taxa of the Western Palearctic lizards Anguis and Pseudopus is revised using genome-wide nuclear DNA and mitogenomes. We found overall concordance between nuclear and mitochondrial DNA phylogenies, with one significant exception - the Apennine A. veronensis. In mitochondrial DNA, this species forms a common clade with the earliest diverging lineage, the southern Balkan endemic A. cephallonica, while it clusters together with A. fragilis in nuclear DNA. The nuclear phylogeny conforms to the morphology, which is relatively similar between A. veronensis and A. fragilis. The most plausible explanation for the mitonuclear discordance is ancient mitochondrial capture from the Balkan ancestor of A. cephallonica to the Apennine population of the A. fragilis-veronensis ancestor. We hypothesize that this capture occurred only in a geographically restricted population. The dating of this presumed mitochondrial introgression and capture coincides with the Messinian event, when the Balkan and Apennine Peninsulas were presumably largely connected. The dated nuclear phylogenomic reconstruction estimated the divergence of A. cephallonica around 12 Mya, while the sister clade representing the A. fragilis species complex consisting of the sister species A. fragilis-A. veronensis and A. colchica-A. graeca further diversified around 7 Mya. The depth of nuclear divergence among the evolutionary lineages of Pseudopus (0.5-1.2 Mya) supports their subspecies status.

An Fgf-Shh positive feedback loop drives growth in developing unpaired fins.

The origin and diversification of appendage types is a central question in vertebrate evolution. Understanding the genetic mechanisms that underlie fin and limb development can reveal relationships between different appendages. Here we demonstrate, using chemical genetics, a mutually agonistic interaction between Fgf and Shh genes in the developing dorsal fin of the channel catfish, Ictalurus punctatus. We also find that Fgf8 and Shh orthologs are expressed in the apical ectodermal ridge and zone of polarizing activity, respectively, in the median fins of representatives from other major vertebrate lineages. These findings demonstrate the importance of this feedback loop in median fins and offer developmental evidence for a median fin-first scenario for vertebrate paired appendage origins.

A Comprehensive Analysis of Fibrillar Collagens in Lamprey Suggests a Conserved Role in Vertebrate Musculoskeletal Evolution.

Vertebrates have distinct tissues which are not present in invertebrate chordates nor other metazoans. The rise of these tissues also coincided with at least one round of whole-genome duplication as well as a suite of lineage-specific segmental duplications. Understanding whether novel genes lead to the origin and diversification of novel cell types, therefore, is of great importance in vertebrate evolution. Here we were particularly interested in the evolution of the vertebrate musculoskeletal system, the muscles and connective tissues that support a diversity of body plans. A major component of the musculoskeletal extracellular matrix (ECM) is fibrillar collagens, a gene family which has been greatly expanded upon in vertebrates. We thus asked whether the repertoire of fibrillar collagens in vertebrates reflects differences in the musculoskeletal system. To test this, we explored the diversity of fibrillar collagens in lamprey, a jawless vertebrate which diverged from jawed vertebrates (gnathostomes) more than five hundred million years ago and has undergone its own gene duplications. Some of the principal components of vertebrate hyaline cartilage are the fibrillar collagens type II and XI, but their presence in cartilage development across all vertebrate taxa has been disputed. We particularly emphasized the characterization of genes in the lamprey hyaline cartilage, testing if its collagen repertoire was similar to that in gnathostomes. Overall, we discovered thirteen fibrillar collagens from all known gene subfamilies in lamprey and were able to identify several lineage-specific duplications. We found that, while the collagen loci have undergone rearrangement, the Clade A genes have remained linked with the hox clusters, a phenomenon also seen in gnathostomes. While the lamprey muscular tissue was largely similar to that seen in gnathostomes, we saw considerable differences in the larval lamprey skeletal tissue, with distinct collagen combinations pertaining to different cartilage types. Our gene expression analyses were unable to identify type II collagen in the sea lamprey hyaline cartilage nor any other fibrillar collagen during chondrogenesis at the stages observed, meaning that sea lamprey likely no longer require these genes during early cartilage development. Our findings suggest that fibrillar collagens were multifunctional across the musculoskeletal system in the last common ancestor of vertebrates and have been largely conserved, but these genes alone cannot explain the origin of novel cell types.

Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates.

In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or "basal" vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants' various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.

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.

Lamprey lecticans link new vertebrate genes to the origin and elaboration of vertebrate tissues.

The evolution of vertebrates from an invertebrate chordate ancestor involved the evolution of new organs, tissues, and cell types. It was also marked by the origin and duplication of new gene families. If, and how, these morphological and genetic innovations are related is an unresolved question in vertebrate evolution. Hyaluronan is an extracellular matrix (ECM) polysaccharide important for water homeostasis and tissue structure. Vertebrates possess a novel family of hyaluronan binding proteins called Lecticans, and studies in jawed vertebrates (gnathostomes) have shown they function in many of the cells and tissues that are unique to vertebrates. This raises the possibility that the origin and/or expansion of this gene family helped drive the evolution of these vertebrate novelties. In order to better understand the evolution of the lectican gene family, and its role in the evolution of vertebrate morphological novelties, we investigated the phylogeny, genomic arrangement, and expression patterns of all lecticans in the sea lamprey (Petromyzon marinus), a jawless vertebrate. Though both P. marinus and gnathostomes each have four lecticans, our phylogenetic and syntenic analyses are most consistent with the independent duplication of one of more lecticans in the lamprey lineage. Despite the likely independent expansion of the lamprey and gnathostome lectican families, we find highly conserved expression of lecticans in vertebrate-specific and mesenchyme-derived tissues. We also find that, unlike gnathostomes, lamprey expresses its lectican paralogs in distinct subpopulations of head skeleton precursors, potentially reflecting an ancestral diversity of skeletal tissue types. Together, these observations suggest that the ancestral pre-duplication lectican had a complex expression pattern, functioned to support mesenchymal histology, and likely played a role in the evolution of vertebrate-specific cell and tissue types.

Differences in developmental potential predict the contrasting patterns of dental diversification in characiform and cypriniform fishes.

Morphological diversification during adaptive radiation may depend on factors external or internal to the lineage. We provide evidence for the latter in characiform fishes (tetras and piranhas), which exhibit extensive dental diversity. Phylogenetic character mapping supported regain of lost teeth as contributing to this diversity. To test for latent potential for dentition that would facilitate its evolutionary expansion, we overexpressed a tooth initiation signal, the tumour necrosis factor pathway ligand ectodysplasin, in a model characiform, the Mexican tetra (Astyanax mexicanus). This manipulation resulted in extensive ectopic dentition, in contrast with its previously reported limited effect in the zebrafish (Danio rerio). Tooth location in the order Cypriniformes, to which the zebrafish belongs, is much more restricted than in characiforms, a pattern that may be explained by differences in the retention of ancestral developmental potential. Our results suggest that differences in evolvability between lineages may lead to contrasting patterns of diversification.

Phenotypic differentiation of the slow worm lizards (Squamata: Anguis) across their contact zone in Central Europe.

BACKGROUND: The application of molecular-phylogenetic approaches to taxonomy has had a dramatic effect on our understanding of the diversity of reptiles. These approaches have allowed researchers to reveal previously hidden lineages as well as taxonomic overestimation in morphologically plastic taxa. Slow worms, legless lizards of the genus Anguis (Squamata: Anguidae), were previously considered to comprise either one or two species, and morphology-based intraspecific taxonomy of Anguis fragilis remained controversial throughout the 20th century. After the discovery of deep genetic divergences within the genus, its taxonomy was reconsidered, and as a result, five extant species have been recognized. In order to better understand the patterns of their interspecific differentiation, here we studied phenotypic differences between the two most widespread of them-A. fragilis and A. colchica, and their putative hybrids across the contact zone of both species in Central Europe. METHODS: We used multivariate and univariate statistics and analyzed ten metric, eleven meristic, and six categorical phenotypic variables in material comprising a total of 326 individuals. We also genotyped individuals from the contact zone for one mitochondrial and two nuclear DNA fragments in order to delineate the distribution of individuals of hybrid and non-hybrid origin. The clines in morphological traits were studied using HZAR. RESULTS: We show that the two species are morphologically differentiated. Anguis fragilis has a less robust head, fewer scales covering the body, lower frequency of the external ear opening presence, lower frequency of separated prefrontal scales, higher frequency of prefrontal scales in contact with each other, and body coloration more similar to the juvenile coloration than A. colchica. Slow worms from the contact/hybrid zone are characterized by an intermediate morphology, with more similarities to A. fragilis than to A. colchica. DISCUSSION: None of the analyzed characters alone proved to be fully diagnostic, although more than 90% of all individuals could be successfully assigned to one or another species based on numbers of scales around the body. Our results indicate concordant, coincident, and steep clines in character states change. We present several hypotheses on the origin and evolutionary maintenance of the morphological divergence between both species and suggest that different evolutionary histories of the taxa rather than recently acting selection explain the observed morphological variation.

Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer.

Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.

An inventory of online reptile images.

No central online repository exists for the collection of animal images; hence it remains unclear how extensively species have been illustrated in the published literature or online. Here we compiled a list of more than 8000 reptile species (out of 11,341) that have photos in one of six popular online repositories, namely iNaturalist (6,349 species), the Reptile Database (5,144), Flickr (4,386), CalPhotos (3,071), Wikimedia (2,952), and Herpmapper (2,571). These sites have compiled over one million reptile photos, with some species represented by tens of thousands of images. Despite the number of images, many species have only one or a few images. This suggests that a considerable fraction of morphological and geographic variation is under documented or difficult to access. We highlight prominent gaps in amphisbaenians, lizards, and snakes, with geographic hotspots for species without images in Central Africa, Pacific Islands, and the Andes Mountains. We present a list of ~3,000 species without photos in any of the six databases and ask the community to fill the gaps by depositing images on one of these sites (preferably with minimal copyright restrictions).

An inventory of online reptile images

No central online repository exists for the collection of animal images; hence it remains unclear how extensively species have been illustrated in the published literature or online. Here we compiled a list of more than 8000 reptile species (out of 11,341) that have photos in one of six popular online repositories, namely iNaturalist (6,349 species), the Reptile Database (5,144), Flickr (4,386), CalPhotos (3,071), Wikimedia (2,952), and Herpmapper (2,571). These sites have compiled over one million reptile photos, with some species represented by tens of thousands of images. Despite the number of images, many species have only one or a few images. This suggests that a considerable fraction of morphological and geographic variation is under documented or difficult to access. We highlight prominent gaps in amphisbaenians, lizards, and snakes, with geographic hotspots for species without images in Central Africa, Pacific Islands, and the Andes Mountains. We present a list of ~3,000 species without photos in any of the six databases and ask the community to fill the gaps by depositing images on one of these sites (preferably with minimal copyright restrictions).

The biogeography of Elaphe sauromates (Pallas, 1814), with a description of a new rat snake species.

BACKGROUND: The rat snake genus Elaphe once comprised several dozens of species distributed in temperate through tropical zones of the New and Old World. Based on molecular-genetic analyses in early 2000s, the genus was split into several separate genera, leaving only 15 Palearctic and Oriental species as its members. One of the three species also occurring in Europe is Elaphe sauromates, a robust snake from the Balkans, Anatolia, Caucasus, Ponto-Caspian steppes, and Levant that has been suspected to be composed of two or more genetically diverse populations. Here, we studied the genetic structure and morphological variation of E. sauromates, aiming to better understand its inter-population relationships and biogeography, and subsequently revise its taxonomy. METHODS: We reconstructed the phylogeography and analyzed the genetic structure of E. sauromates populations originating from most of its geographic range using both mitochondrial (COI, ND4) and nuclear (C-MOS, MC1R, PRLR, RAG1) DNA gene fragments. We employed Maximum likelihood and Bayesian inference methods for the phylogenetic tree reconstructions, supplemented with species delimitation methods, analysis of haplotype networks, and calculation of uncorrected p-distances. Morphological variation in 15 metric and 18 meristic characters was studied using parametric univariate tests as well as multivariate general linearized models. In total, we analyzed sequences originating from 63 specimens and morphological data from 95 specimens of E. sauromates sensu lato. RESULTS: The molecular phylogeny identified two clearly divergent sister lineages within E. sauromates, with both forming a lineage sister to E. quatuorlineata. The genetic distance between them (5.80-8.24% in mtDNA) is similar to the distances among several other species of the genus Elaphe. Both lineages are also moderately morphologically differentiated and, while none of the characters are exclusively diagnostic, their combination can be used for confident lineage identification. Here, following the criteria of genetic and evolutionary species concepts, we describe the lineage from eastern Anatolia and parts of the Lesser and Great Caucasus as a new species E. urartica sp. nov. DISCUSSION: Elaphe urartica sp. nov. represents a cryptic species whose ancestors presumably diverged from their common ancestor with E. sauromates around the Miocene-Pliocene boundary. The intraspecific genetic structure indicates that the recent diversity of both species has been predominantly shaped by Pleistocene climatic oscillations, with glacial refugia mainly located in the Balkans, Crimea, and/or Anatolia in E. sauromates and Anatolia and/or the Caucasus in E. urartica sp. nov.

Bichir external gills arise via heterochronic shift that accelerates hyoid arch development.

In most vertebrates, pharyngeal arches form in a stereotypic anterior-to-posterior progression. To gain insight into the mechanisms underlying evolutionary changes in pharyngeal arch development, here we investigate embryos and larvae of bichirs. Bichirs represent the earliest diverged living group of ray-finned fishes, and possess intriguing traits otherwise typical for lobe-finned fishes such as ventral paired lungs and larval external gills. In bichir embryos, we find that the anteroposterior way of formation of cranial segments is modified by the unique acceleration of the entire hyoid arch segment, with earlier and orchestrated development of the endodermal, mesodermal, and neural crest tissues. This major heterochronic shift in the anteroposterior developmental sequence enables early appearance of the external gills that represent key breathing organs of bichir free-living embryos and early larvae. Bichirs thus stay as unique models for understanding developmental mechanisms facilitating increased breathing capacity.

Tracing the maternal origin of the common wall lizard (Podarcis muralis) on the northern range margin in Central Europe.

The maternal origin of isolated populations of the common wall lizard (Podracis muralis) in the Czech Republic, representing the north-eastern range border of the species, was addressed. We compared mitochondrial DNA sequences of the cytochrome b gene of samples from these populations with those from within the continuous range in Slovakia, the northern Balkan region, and those available from previous studies. We recorded five main haplogroups in the studied region, with all available Central European samples belonging to the same haplogroup. The star-like structure of this haplogroup suggests a scenario of relatively recent, post-glacial population expansion, which is further supported by a coalescent-based demographic analysis. The presence of unique haplotypes in two of the three isolated Czech populations together with close phylogenetic relationships to adjacent Slovak populations suggests either autochthonous origin or human-mediated introductions from geographically and genetically closest populations. We therefore support conservation programs for all three isolated Czech populations.

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.