Václav Gvoždík

Slow worm, Anguis fragilis (Reptilia: Anguidae) as a species complex: Genetic structure reveals deep divergences

Phylogenetic relationships of the Western Palearctic legless lizard genus Anguis were inferred based on a fragment of mitochondrial DNA and two nuclear protein-coding loci, C-mos and PRLR. A. cephallonica from the Peloponnese was confirmed as a valid species. It is the sister taxon to a clade comprising all other evolutionary lineages, which were shown to represent three distinct species: (1) A. fragilis sensu stricto occurring in Western and Central Europe, the north-western Balkans, with possibly isolated populations in the eastern Balkans, and presumably also in western Scandinavia and Italy; (2) A. colchica distributed from the eastern Czech Republic and the Baltic region eastward to northern Iran, presumably also in eastern Scandinavia, and the north-eastern Balkans; (3) A. graeca restricted to the southern Balkans, and partially sympatric with A. cephallonica. According to the more variable mitochondrial marker, A. graeca appears to be the sister species to A. colchica, and these taxa together form a sister clade to A. fragilis, whereas the less variable nuclear markers show A. colchica to be closer to A. fragilis. The C-mos gene has not provided substantial variation within this species complex, while the PRLR gene, which was used for the first time in phylogeographic study in a reptile, distinguished all species successfully. Intra-specific differentiation of A. colchica is discussed, and subspecific status of the Caucasian and Caspian populations is proposed. The uncovered genetic differences should be taken into account in all future biogeographical, morphological and ecological studies, as well as in conservation.

Out of Arabia: A Complex Biogeographic History of Multiple Vicariance and Dispersal Events in the Gecko Genus Hemidactylus (Reptilia: Gekkonidae)

The geological history of the Arabian Peninsula has played a crucial role in shaping current diversity and distribution patterns of many Arabian and African faunal elements. The gecko genus Hemidactylus is not an exception. In this study, we provide an insight into the phylogeny and systematics of 45 recognized species of the so-called Arid clade of the genus Hemidactylus from Arabia, the Horn of Africa, the Levant and Iran. The material comprises 358 specimens sequenced for up to two mitochondrial (12S rRNA, cytochrome b) and four nuclear (mc1r, cmos, rag1, rag2) genes with 4766 bp of the concatenated alignment length. A robust calibrated phylogeny and reconstruction of historical biogeography are inferred. We link the history of this genus with major geological events that occurred in the region within the last 30 million years. Two basal divergences correspond with the break-ups of the Arabian and African landmasses and subsequent separation of Socotra from the Arabian mainland, respectively, segregating the genus by means of vicariance. Formation of the Red Sea led to isolation and subsequent radiation in the Arabian Peninsula, which was followed by multiple independent expansions: 13.1 Ma to Iran; 9.8 Ma to NE Africa; 8.2 to Socotra Archipelago; 7–7.3 Ma two colonizations to the Near East; 5.9 Ma to NE Africa; and 4.1 to Socotra. Moreover, using multiple genetic markers we detected cryptic diversity within the genus, particularly in south-western Arabia and the Ethiopian highlands, and confirmed the existence of at least seven new species in the area. These findings highlight the role of Arabia and the Horn of Africa as an important Hemidactylus diversity hotspot.

Systematics of spiny-backed treefrogs (Hylidae: Osteocephalus): An Amazonian puzzle

Spiny‐backed tree frogs of the genus Osteocephalus are conspicuous components of the tropical wet forests of the Amazon and the Guiana Shield. Here, we revise the phylogenetic relationships of Osteocephalus and its sister group Tepuihyla, using up to 6134 bp of DNA sequences of nine mitochondrial and one nuclear gene for 338 specimens from eight countries and 218 localities, representing 89% of the 28 currently recognized nominal species. Our phylogenetic analyses reveal (i) the paraphyly of Osteocephalus with respect to Tepuihyla, (ii) the placement of ‘Hyla’ warreni as sister to Tepuihyla, (iii) the non‐monophyly of several currently recognized species within Osteocephalus and (iv) the presence of low (<1%) and overlapping genetic distances among phenotypically well‐characterized nominal species (e.g. O. taurinus and O. oophagus) for the 16S gene fragment used in amphibian DNA barcoding. We propose a new taxonomy, securing the monophyly of Osteocephalus and Tepuihyla by rearranging and redefining the content of both genera and also erect a new genus for the sister group of Osteocephalus. The colouration of newly metamorphosed individuals is proposed as a morphological synapomorphy for Osteocephalus. We recognize and define five monophyletic species groups within Osteocephalus, synonymize three species of Osteocephalus (O. germani, O. phasmatus and O. vilmae) and three species of Tepuihyla (T. celsae, T. galani and T. talbergae) and reallocate three species (Hyla helenae to Osteocephalus, O. exophthalmus to Tepuihyla and O. pearsoni to Dryaderces gen. n.). Furthermore, we flag nine putative new species (an increase to 138% of the current diversity). We conclude that species numbers are largely underestimated, with most hidden diversity centred on widespread and polymorphic nominal species. The evolutionary origin of breeding strategies within Osteocephalus is discussed in the light of this new phylogenetic hypothesis, and a novel type of amplexus (gular amplexus) is described.

Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. megalocephala)

Grass snakes (Natrix natrix) represent one of the most widely distributed snake species of the Palaearctic region, ranging from the North African Maghreb region and the Iberian Peninsula through most of Europe and western Asia eastward to the region of Lake Baikal in Central Asia. Within N. natrix, up to 14 distinct subspecies are regarded as valid. In addition, some authors recognize big‐headed grass snakes from western Transcaucasia as a distinct species, N. megalocephala. Based on phylogenetic analyses of a 1984‐bp‐long alignment of mtDNA sequences (ND4+tRNAs, cyt b) of 410 grass snakes, a nearly range‐wide phylogeography is presented for both species. Within N. natrix, 16 terminal mitochondrial clades were identified, most of which conflict with morphologically defined subspecies. These 16 clades correspond to three more inclusive clades from (i) the Iberian Peninsula plus North Africa, (ii) East Europe and Asia and (iii) West Europe including Corso‐Sardinia, the Apennine Peninsula and Sicily. Hypotheses regarding glacial refugia and postglacial range expansions are presented. Refugia were most likely located in each of the southern European peninsulas, Corso‐Sardinia, North Africa, Anatolia and the neighbouring Near and Middle East, where the greatest extant genetic diversity occurs. Multiple distinct microrefugia are inferred for continental Italy plus Sicily, the Balkan Peninsula, Anatolia and the Near and Middle East. Holocene range expansions led to the colonization of more northerly regions and the formation of secondary contact zones. Western Europe was invaded from a refuge within southern France, while Central Europe was reached by two distinct range expansions from the Balkan Peninsula. In Central Europe, there are two contact zones of three distinct mitochondrial clades, and one of these contact zones was theretofore completely unknown. Another contact zone is hypothesized for Eastern Europe, which was colonized, like north‐western Asia, from the Caucasus region. Further contact zones were identified for southern Italy, the Balkans and Transcaucasia. In agreement with previous studies using morphological characters and allozymes, there is no evidence for the distinctiveness of N. megalocephala. Therefore, N. megalocephala is synonymized with N. natrix.

Pan‐African phylogeography of a model organism, the African clawed frog ‘Xenopus laevis’

The African clawed frog Xenopus laevis has a large native distribution over much of sub‐Saharan Africa and is a model organism for research, a proposed disease vector, and an invasive species. Despite its prominent role in research and abundance in nature, surprisingly little is known about the phylogeography and evolutionary history of this group. Here, we report an analysis of molecular variation of this clade based on 17 loci (one mitochondrial, 16 nuclear) in up to 159 individuals sampled throughout its native distribution. Phylogenetic relationships among mitochondrial DNA haplotypes were incongruent with those among alleles of the putatively female‐specific sex‐determining gene DM‐W, in contrast to the expectation of strict matrilineal inheritance of both loci. Population structure and evolutionarily diverged lineages were evidenced by analyses of molecular variation in these data. These results further contextualize the chronology, and evolutionary relationships within this group, support the recognition of X. laevis sensu stricto, X. petersii, X. victorianus and herein revalidated X. poweri as separate species. We also propose that portions of the currently recognized distributions of X. laevis (north of the Congo Basin) and X. petersii (south of the Congo Basin) be reassigned to X. poweri.

Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog ( Xenopus , Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).

An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula

Four species of legless anguid lizard genus Anguis have been currently recognized: A. fragilis from western and central Europe, A. colchica from eastern Europe and western Asia, A. graeca from southern Balkans, and A. cephallonica from the Peloponnese. Slow worms from the Italian Peninsula have been considered conspecific with A. fragilis, despite the fact that the region served as an important speciation center for European flora and fauna, and included some Pleistocene glacial refugia. We used mitochondrial and nuclear DNA sequences to investigate the systematic and phylogenetic position of the Italian slow-worm populations and morphological analyses to test for phenotypic differentiation from A. fragilis from other parts of Europe. Our phylogenetic analyses revealed that Italian slow worms form a distinct deeply differentiated mtDNA clade, which presumably diverged during or shortly after the basal radiation within the genus Anguis. In addition, the specimens assigned to this clade bear distinct haplotypes in nuclear PRLR gene and show morphological differentiation from A. fragilis. Based on the differentiation in all three independent markers, we propose to assign the Italian clade species level under the name Anguis veronensisPollini, 1818. The newly recognized species is distributed throughout the Italian Peninsula to the Southern Alps and south-eastern France. We hypothesize that the Tertiary Alpine orogeny with subsequent vicariance might have played a role in differentiation of this species. The current genetic variability was later presumably shaped in multiple glacial refugia within the Italian Peninsula, with the first splitting event separating populations from the region of the Dolomite Mountains.

Sky Islands of the Cameroon Volcanic Line: a diversification hot spot for puddle frogs (Phrynobatrachidae: Phrynobatrachus)

The continental highlands of the Cameroon Volcanic Line (CVL) represent biological ‘sky islands’ with high levels of species richness and endemism, providing the ideal opportunity to understand how orogenesis and historical climate change influenced species diversity and distribution in these isolated African highlands. Relationships of puddle frogs (Phrynobatrachus) endemic to the CVL are reconstructed to examine the patterns and timing of puddle frog diversification. Historical distributions were reconstructed using both elevation and geography data. Puddle frogs diversified in the CVL via several dispersal and vicariance events, with most of the locally endemic species distributed across the northern part of the montane forest area in the Bamenda‐Banso Highlands (Bamboutos Mts., Mt. Lefo, Mt. Mbam, Mt. Oku and medium elevation areas connecting these mountains). Two new species, P. jimzimkusi sp. n. and P. njiomock sp. n., are also described based on molecular analyses and morphological examination. We find that these new species are most closely related to one another and P. steindachneri with the ranges of all three species overlapping at Mt. Oku. Phrynobatrachus jimzimkusi sp. n. is distributed in the southern portion of the continental CVL, P. njiomock sp. n. is endemic to Mt. Oku, and P. steindachneri is present in the northeastern part of the montane forest area. Both new species can be distinguished from all other puddle frogs by a combination of morphological characters, including their large size, ventral coloration and secondary sexual characteristics present in males. These results highlight the Bamenda‐Banso Highlands, and specifically emphasize Mt. Oku, as a centre of diversification for puddle frogs, supporting the conservation importance of this region. Our results also provide new insights into the evolutionary processes shaping the CVL ‘sky islands’, demonstrating that lineage diversification in these montane amphibians is significantly older than expected with most species diverging from their closest relative in the Miocene. Whereas climatic changes during the Pliocene and Pleistocene shaped intraspecific diversification, most speciation events were significantly older and cannot be linked to Africas aridification in response to Pleistocene climate fluctuations.

Contrasting evolutionary histories of the legless lizards slow worms (Anguis) shaped by the topography of the Balkan Peninsula

BackgroundGenetic architecture of a species is a result of historical changes in population size and extent of distribution related to climatic and environmental factors and contemporary processes of dispersal and gene flow. Population-size and range contractions, expansions and shifts have a substantial effect on genetic diversity and intraspecific divergence, which is further shaped by gene-flow limiting barriers. The Balkans, as one of the most important sources of European biodiversity, is a region where many temperate species persisted during the Pleistocene glaciations and where high topographic heterogeneity offers suitable conditions for local adaptations of populations. In this study, we investigated the phylogeographical patterns and demographic histories of four species of semifossorial slow-worm lizards (genus Anguis) present in the Balkan Peninsula, and tested the relationship between genetic diversity and topographic heterogeneity of the inhabited ranges.ResultsWe inferred phylogenetic relationships, compared genetic structure and historical demography of slow worms using nucleotide sequence variation of mitochondrial DNA. Four Anguis species with mostly parapatric distributions occur in the Balkan Peninsula. They show different levels of genetic diversity. A signature of population growth was detected in all four species but with various courses in particular populations. We found a strong correlation between genetic diversity of slow-worm populations and topographic ruggedness of the ranges (mountain systems) they inhabit. Areas with more rugged terrain harbour higher genetic diversity.ConclusionsPhylogeographical pattern of the genus Anguis in the Balkans is concordant with the refugia-within-refugia model previously proposed for both several other taxa in the region and other main European Peninsulas. While slow-worm populations from the southern refugia mostly have restricted distributions and have not dispersed much from their refugial areas, populations from the extra-Mediterranean refugia in northern parts of the Balkans have colonized vast areas of eastern, central, and western Europe. Besides climatic historical events, the heterogeneous topography of the Balkans has also played an important role in shaping genetic diversity of slow worms.

No ecological opportunity signal on a continental scale? Diversification and life-history evolution of African true toads (Anura: Bufonidae).

The niche‐filling process predicted by the “ecological opportunity” (EO) model is an often‐invoked mechanism for generating exceptional diversity in island colonizers. Whether the same process governs lineage accumulation and trait disparity during continental colonization events is less clear. Here, we test this prediction by investigating the rate dynamics and trait evolution of one of Africas most widespread amphibian colonizers, the true toads (Bufonidae). By reconstructing the most complete molecular phylogeny of African Bufonidae to date, we find that the diversification of lineages in Africa best conforms to a constant rate model throughout time and across subclades, with little support for EO. Evolutionary rates of life‐history traits have similarly been constant over time. However, an analysis of generalists and specialists showed a shift toward higher speciation rates associated with habitat specialization. The overall lack of EO signal can be interpreted in a number of ways and we propose several explanations. Firstly, methodological issues might preclude the detection of EO. Secondly, colonizers might not experience true EO conditions and due to the size, ecological heterogeneity and age of landmasses, the diversification processes might be more complex. Thirdly, lower speciation rates of habitat generalists may have affected overall proliferation of lineages.