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Restriction site-Associated DNA sequencing (RADseq) has great potential for genome-wide systematics studies of non-model organisms. However, accurately assembling RADseq reads into orthologous loci remains a major challenge in the absence of a reference genome. Traditional assembly pipelines cluster putative orthologous sequences based on a user-defined clustering threshold. Because improper clustering of orthologs is expected to affect results in downstream analyses, it is crucial to design pipelines for empirically optimizing the clustering threshold. While this issue has been largely discussed from a population genomics perspective, it remains understudied in the context of phylogenomics and coalescent species delimitation. To address this issue, we generated RADseq assemblies of representatives of the amphibian generaDiscoglossus, Rana, LissotritonandTriturususing a wide range of clustering thresholds. Particularly, we studied the effects of the intra-sample Clustering Threshold (iCT) and between-sample Clustering Threshold (bCT) separately, as both are expected to differ in multi-species data sets. The obtained assemblies were used for downstream inference of concatenation-based phylogenies, and multi-species coalescent species trees and species delimitation. The results were evaluated in the light of a reference genome-wide phylogeny calculated from newly generated Hybrid-Enrichment markers, as well as extensive background knowledge on the species’ systematics. Overall, our analyses show that the inferred topologies and their resolution are resilient to changes of the iCT and bCT, regardless of the analytical method employed. Except for some extreme clustering thresholds, all assemblies yielded identical, well-supported inter-species relationships that were mostly congruent with those inferred from the reference Hybrid-Enrichment data set. Similarly, coalescent species delimitation was consistent among similarity threshold values. However, we identified a strong effect of the bCT on the branch lengths of concatenation and species trees, with higher bCTs yielding trees with shorter branches, which might be a pitfall for downstream inferences of evolutionary rates. Our results suggest that the choice of assembly parameters for RADseq data in the context of shallow phylogenomics might be less challenging than previously thought. Finally, we propose a pipeline for empirical optimization of the iCT and bCT, implemented in optiRADCT, a series of scripts readily usable for future RADseq studies.

Hybridization between the European smooth and palmate newts has recurrently been mentioned in the literature. The only two studies that attempted to quantify the frequency of hybridization and gene admixture between these two species came to strikingly opposite conclusions. According to Arntzen et al (1998, 42 allozymes), hybrids are rare in nature and introgression negligible, while according to Johanet et al (2011, 6 microsatellites), introgressive hybridization is significant and widespread across the shared distribution range. To clarify this question, we implemented high-throughput SNP genotyping with diagnostic biallelic SNPs on 965 specimens sampled across Europe. Our results are in line with Arntzen et al, since only two F1 hybrids were identified in two distinct French localities, and no further hybrid generations or backcrosses. Moreover, reanalysis of 78 of the samples previously studied by Johanet et al. (2011) using our SNPs panel could not reproduce their results, suggesting that microsatellite-based inference overestimated the hybridization frequency between these two species. Since we did not detect methodological issues with the analyses of Johanet et al., our results suggest that SNP approaches outperform microsatellite-based assessments of hybridization frequency, and that conclusions previously published on this topic with a small number of microsatellite loci should be taken with caution, and ideally be repeated with an increased genomic coverage.

Ecologically distinct species may respond to landscape changes in different ways. In addition to basic ecological data, the extent of the geographic range has been successfully used as an indicator of species sensitivity to anthropogenic landscapes, with widespread species usually found to be less sensitive compared to range-restricted species. In this study, we investigate connectivity patterns of two closely related but ecologically distinct newt species - the range-restricted, Lissotriton montandoni and the widespread, L. vulgaris - using genomic data, a highly replicated setting (six geographic regions per species), and tools from landscape genetics. Our results show the importance of forest for connectivity in both species, but at the same time suggest differential use of forested habitat, with L. montandoni and L. vulgaris showing the highest connectivity at forest-core and forest-edges, respectively. Anthropogenic landscapes (i.e., higher crop- or urban-cover) increased resistance in both species, but the effect was one to three orders of magnitude stronger in L. montandoni than in L. vulgaris. This result is consistent with a view of L. vulgaris as an ecological generalist. Even so, currently, the negative impact of anthropogenic landscapes is mainly seen in connectivity among L. vulgaris populations, which show significantly stronger isolation and lower effective sizes relative to L. montandoni. Overall, this study emphasizes how habitat destruction is compromising genetic connectivity not only in endemic, range-restricted species of conservation concern but also in widespread generalist species, despite their comparatively lower sensitivity to anthropogenic landscape changes.

The slow-worm lizards (Anguis) comprise five species occurring throughout most of the Western Palearctic. Although these species are relatively uniform morphologically – with the exception of A. cephallonica, which exhibits a quite unique morphology – they are genetically deeply divergent. Here, we provide detailed distribution maps for each species and discuss their biogeography and conservation based on updated genetic data and a robust distribution database. We pay particular attention to the so called ‘grey zone’, which typically represents secondary contact zones and in some cases confirmed or presumed hybrid zones. Four of the five species live in parapatry, while only two species, A. cephallonica and A. graeca from the southern Balkans occur in partial sympatry. Further research should focus on the eco-evolutionary interactions between species in contact, including their hybridization rates, to reveal deeper details of the slow-worm evolutionary and natural history.

Pond ecosystems are hotspots of freshwater biodiversity, often containing many rare and protected species that are not commonly found elsewhere (Harper et al. 2018;Harper et al. 2019). However, even if they constitute c.a. 30% of freshwaters by area, still not enough effort has been put into pond monitoring and management and pond ecosystems are hence relatively poorly understood. Results of ECOPOND project will lead to add valuable knowledge upon pond diversity in geographic gradient taking for consideration human impact by comparing rural and urban areas. The sample design in ECOPOND includes six geographic regions, spanning from the south of Poland to the middle of Norway, where we will sample five replicates of urban and rural ponds in close geographic proximity, making it possible to test the impact of urbanization on biodiversity and biotic homogenization across latitude. We will sample all ponds at spring and late summer, making it possible to assess also seasonality in biodiversity. ECOPOND will utilize environmental DNA and RNA to perform biodiversity screening. The extracted eDNA and eRNA fragments will be amplified with the use of several selected markers for vertebrates, invertebrates, fungi and bacteria. Comparisons between eDNA and eRNA metabarcoding are hypothesized to allow inference between present and past diversity, as eRNA is thought to be only available from live organisms in the community. Moreover, ECOPOND aims at testing the effects of selected invasives species that can have on whole ecosystems. By sampling a range of biotic and abiotic parameters describing studied ponds, we will incorporate the available data for the ponds and employ occupancy modelling methods to assess the habitat preferences of selected invasive alien species. Then we will develop a method that can contribute towards an earlywarning system of evaluating threats to ecosystem status. One of the focus species will be the parasitic fungus Batrachochytrium dendrobatidis (Bd), an infectious fungal pathogen that has caused a number of amphibian declines and extinctions. The European amphibians seem less affected by the parasite at present. However, the fungi could be a direct driver of reduced genetic variation due to selection, or directly reduce the infected amphibian’s overall fitness by reducing the microbiotic diversity on their skin, which in many cases acts as a second immune system. ECOPOND will therefore provide data on genomic variation (using RADseq) for two amphibian species: the smooth newt (Lissotriton vulgaris) and the common toad (Bufo bufo). We will investigate populations of these species inhabiting ponds that are infected and not infected by Bd as well as collect data on their skin microbes (identified using metabarcoding). We will also contrast the genomic diversity between the replicated urban/rural setup and look for repeatable genomic changes. This setup will also be compared for the genomic variation for a potential native prey, the blue-tailed dragonfly, as will ponds with and without fish and/or amphibians (possibly also comparing between native and IAS top-predators) in order to look for predatory selective sweeps in the genome and transcriptome (experimental setup). All ponds will also be analyzed for over 20 water quality parameters and include data on a range of site characteristics that will be used as explanatory variables in all models. ECOPOND will compare large datasets across large geographic regions and will provide detailed knowledge of biodiversity patterns in vertebrates, invertebrates, fungal and microbial species, as well as genomic composition and skin biodiversity for animals inhabiting the same ponds set in an urban context. As a total, ECOPOND will obtain data on the location and status of biodiversity interests, gather data that can help in preventing the establishment of invasive alien species, and eradicating or controlling species that have already become established. And finally, ECOPOND will work closely with stakeholders and develop statistical techniques that can be used for monitoring, detection and protection of biodiversity.

Płazy to jedna z najbardziej zagrożonych grup zwierząt na świecie, głównie ze względu na niszczenie ich siedlisk przez człowieka. W dobie koronawirusowej pandemii warto wspomnieć również o chorobach, z którymi ta grupa kręgowców mierzy się już od wielu lat. Choroby zakaźne wywołane przez mikroskopijne grzyby pasożytnicze Batrachochytrium dendrobatidis i B. salamandrivorans, a także różne formy ranawirusów (Ranavirus) stanowią ogromne zagrożenie dla płazów, dziesiątkując ich populacje na całym świecie, także w Europie. Przedstawiamy najnowsze doniesienia na temat rozmieszczenia najważniejszych patogenów płazów, w tym także z Polski, oraz perspektywy zachowania płazów w obliczu trwających pandemii. Podsumowujemy również najistotniejsze działania prewencyjne, ograniczające rozprzestrzenianie się patogenów, a także przytaczamy przykłady działań mających eliminować chorobotwórcze organizmy ze środowiska. Artykuł ma na celu zwiększenie świadomości społecznej dotyczącej patogenów, mogącej wpłynąć na podjęcie skutecznych działań zmniejszających negatywne skutki pandemii wśród płazów.

Emerging infectious diseases are a threat to biodiversity and have taken a large toll on amphibian populations worldwide. The chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and the iridovirus Ranavirus (Rv), are of concern as all have contributed to amphibian declines. In central and eastern Europe, their geographical and host distributions and main environmental drivers determining prevalence are poorly known. We screened over 1000 amphibians from natural and captive populations in Poland for the presence of Bd, Bsal and Rv. In wild amphibian populations, we found that Bd is widespread, present in 46 out of 115 sampled localities as well as 2 captive colonies, and relatively common with overall prevalence at 14.4% in 9 species. We found lower prevalence of Rv at 2.4%, present in 11 out of 92 sampling sites, with a taxonomic breadth of 8 different amphibian species. Bsal infection was not detected in any individuals. In natural populations, Pelophylax esculentus and Bombina variegata accounted for 75% of all Bd infections, suggesting a major role for these 2 species as pathogen reservoirs in Central European freshwater habitats. General linear models showed that climatic as well as landscape features are associated with Bd infection in Poland. We found that higher average annual temperature constrains Bd infection, while landscapes with numerous water bodies or artificial elements (a surrogate for urbanization) increase the chances of infection. Our results show that a combination of climatic and landscape variables may drive regional and local pathogen emergence.

Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.

Despite evidence from anatomy, behavior and genomics indicating that the sense of smell in turtles is important, our understanding of chemical communication in this group is still rudimentary. Our aim was to describe the microanatomy of mental glands (MGs) in a freshwater turtle,Mauremys leprosa(Geoemydidae), and to assess the chemical composition of their secretions with respect to variation among individuals and between sexes. MGs are paired sac-like organs on the gular region of the neck and are dimorphic in this species with males having fully functional holocrine glands while those of females appear non-secretory and vestigial. In adult males, the glandular epithelium of the inner portion of the gland provides exocytotic products as well as cellular debris into the lumen of the gland. The contents of the lumen can be secreted through the narrow duct portion of the gland ending in an orifice on the surface of the skin. Females have invaginated structures similar in general outline to male glands, but lack a glandular epithelium. Using gas chromatography coupled to mass spectrometry, we identified a total of 61 compounds in mental gland secretions, the most numerous being carboxylic acids, carbohydrates, alkanes, steroids and alcohols. The number of compounds per individual varied widely (mean (median) ± SD = 14.54 (13) ± 8.44; min = 3; max = 40), but only cholesterol was found in all samples. We found that the relative abundances of only six chemicals were different between the sexes, although males tended to have larger amounts of particular compounds. Although the lipid fraction of mental gland secretions is rich in chemical compounds, most occur in both sexes suggesting that they are metabolic byproducts with no role in chemical signaling. However, the relative amounts of some compounds tended to be higher in males, with significantly larger amounts of two carboxylic acids and one steroid, suggesting their putative involvement in chemical communication.

In Central Europe, water frog species coexist in assemblages consisting of Pelophylax lessonae, P. ridibundus and their hybridogenetic hybrid, P. esculentus. Population compositions are poorly understood, partly because of difficulties in distinguishing hybrids from parentals by means of morphology alone. Environmental change and human-mediated, cryptic introductions of non-native water frog species have modified local assemblages. In this contribution we examined the structure of nine water frog populations inhabiting mostly large fish ponds in the Upper Vistula river valley of southern Poland using morphology and PCR-based genotyping of single loci in the nuclear and mitochondrial genomes. We found four different water frog population compositions: two with only P. ridibundus, one composed of P. lessonae and P. esculentus, three with P. ridibundus and P. esculentus, and three containing all three water frog taxa. Historical data show that the area was previously inhabited by lessonae-esculentus populations. We propose that both ecological and genetic replacement by expanding P. ridibundus may have contributed to the decline of the former. Overall, 18% of P. ridibundus frogs contained introgressed P. lessonae mtDNA, however, the frequency of introgressants was most pronounced in populations with a high proportion of P. esculentus. Exotic water frogs were not detected in the study area. Our results bear significance for water frog population dynamics and conservation in Central Europe and highlight the importance of long-term monitoring for the detection of changes in population composition in these amphibians.

The European corn borer (Ostrinia nubilalis) infests a wide array of host plants and is considered one of the most serious pests of maize in Europe. Recent studies suggest that individuals feeding on maize in Europe should be referred to O. nubilalis (sensu nov.), while those infesting dicots as Ostrinia scapulalis (sensu nov.). We test if the clear genetic distinctiveness among individuals of O. nubilalis living on maize vs. dicots is tracked by mitochondrial DNA (mtDNA). We used fragments of COI and COII genes of 32 individuals traditionally recognized as O. nubilalis collected on three host plants, maize, mugwort and hop, growing in different parts of Poland. In addition, we reconstructed the mtDNA phylogeny of Ostrinia species based on our data and sequences retrieved from GenBank to assess host and/or biogeographic patterns. We also compared haplotype variation found in Poland (east-central Europe) with other regions (Anatolia, Eastern Europe, Balkans, Far East, North America). Our study showed high mtDNA diversity of O. nubilalis in Poland in comparison with other regions and revealed rare haplotypes likely of Asian origin. We did not find distinct mtDNA haplotypes in larvae feeding on maize vs. dicotyledonous plants. Phylogenetic analyses showed an apparent lack of mtDNA divergence among putatively distinct lineages belonging to the O. nubilalis group as identical haplotypes are shared by Asian and European individuals. We argue that human-mediated dispersal, hybridization and sporadic host jumps are likely responsible for the lack of a geographic pattern in mtDNA variation.

Speciation processes have long been inferred from phylogenetic, phylogeographic, and biogeographic pattern-driven perspectives. Now much current speciation research is attempting to more directly describe the underlying processes and mechanisms of divergence leading to speciation. Ideally, researchers should integrate both process- and pattern-based approaches for a more comprehensive understanding of speciation. To this end, a symposium was organized during the 7th World Congress of Herpetology in Canada with the goal of bringing leading experts together to share successful examples of these perspectives and to promote a more cohesive understanding of reptile and amphibian speciation. Here we present a joint paper of short and updated summaries of each of these contributions with the aim of providing a reference source and launching pad for students and researchers interested in speciation in amphibians and reptiles.

The ‘smooth newt’, the taxon traditionally referred to as Lissotriton vulgaris, consists of multiple morphologically distinct taxa. Given the uncertainty concerning the validity and rank of these taxa, L. vulgaris sensu lato has often been treated as a single, polytypic species. A recent study, driven by genetic data, proposed to recognize five species, L. graecus, L. kosswigi, L. lantzi, L. schmidtleri and a more restricted L. vulgaris. The Carpathian newt L. montandoni was confirmed to be a closely related sister species. We propose to refer to this collective of six Lissotriton species as the smooth newt or Lissotriton vulgaris species complex. Guided by comprehensive genomic data from throughout the range of the smooth newt species complex we 1) delineate the distribution ranges, 2) provide a distribution database, and 3) produce distribution maps according to the format of the New Atlas of Amphibians and Reptiles of Europe, for the six constituent species. This allows us to 4) highlight regions where more research is needed to determine the position of contact zones.

The rainforest biome of eastern Madagascar is renowned for its extraordinary biodiversity and restricted distribution ranges of many species, whereas the arid western region of the island is relatively species poor. We provide insight into the biogeography of western Madagascar by analyzing a multilocus phylogeographic dataset assembled for an amphibian, the widespread Malagasy bullfrog, Laliostoma labrosum. We find no cryptic species in L. labrosum (maximum 1.1% pairwise genetic distance between individuals in the 16S rRNA gene) attributable to considerable gene flow at the regional level as shown by genetic admixture in both mtDNA and three nuclear loci, especially in central Madagascar. Low breeding site fidelity, viewed as an adaptation to the unreliability of standing pools of freshwater in dry and seasonal environments, and a ubiquitous distribution within its range may underlie overall low genetic differentiation. Moreover, reductions in population size associated with periods of high aridity in western Madagascar may have purged DNA variation in this species. The mtDNA gene tree revealed seven major phylogroups within this species, five of which show mostly non-overlapping distributions. The nested positions of the northern and central mtDNA phylogroups imply a southwestern origin for all extant mtDNA lineages in L. labrosum. The current phylogeography of this species and paleo-distributions of major mtDNA lineages suggest five potential refugia in northern, western and southwestern Madagascar, likely the result of Pleistocene range fragmentation during drier and cooler climates. Lineage sorting in mtDNA and nuclear loci highlighted a main phylogeographic break between populations north and south of the Sambirano region, suggesting a role of the coastal Sambirano rainforest as a barrier to gene flow. Paleo-species distribution models and dispersal networks suggest that the persistence of some refugial populations was mainly determined by high population connectivity through space and time.

Acute die-offs of amphibian populations worldwide have been linked to the emergence of viral and fungal diseases. Inter and intraspecific immunogenetic differences may influence the outcome of infection. Toll-like receptors (TLRs) are an essential component of innate immunity and also prime acquired defenses. We report the first comprehensive assessment of TLR gene variation for urodele amphibians. The Lissotriton newt TLR repertoire includes representatives of 13 families and is compositionally most similar to that of the anuran Xenopus. Both ancient and recent gene duplications have occurred in urodeles, bringing the total number of TLR genes to at least 21. Purifying selection has predominated the evolution of newt TLRs in both long (∼70 Ma) and medium (∼18 Ma) timescales. However, we find evidence for both purifying and positive selection acting on TLRs in two recently diverged (2–5 Ma) allopatric evolutionary lineages (Lissotriton montandoni and L. vulgaris graecus). Overall, both forms of selection have been stronger in L. v. graecus, while constraint on most TLR genes in L. montandoni appears relaxed. The differences in selection regimes are unlikely to be biased by demographic effects because these were controlled by means of a historical demographic model derived from an independent data set of 62 loci. We infer that TLR genes undergo distinct trajectories of adaptive evolution in closely related amphibian lineages, highlight the potential of TLRs to capture the signatures of different assemblages of pathogenic microorganisms, and suggest differences between lineages in the relative roles of innate and acquired immunity.

Aim To assess the role of the Balkan Peninsula as a Pleistocene refugium for the smooth newt, Lissotriton vulgaris, and to test whether its genetic differentiation is temporally compatible with the southern refugia model. Location The Balkan Peninsula. Methods Phylogeographical analyses were conducted using mitochondrial DNA (mtDNA) sequences sampled from 49 populations of L. vulgaris. A fossil-calibrated estimate of divergence times among major mtDNA clades was obtained. Results We detected seven parapatrically distributed mtDNA clades with very little admixture among populations. Whereas most clades diverged in the Pleistocene, the earliest splits between Caucasian, Anatolian and Balkan clades occurred in the Pliocene. Clades C, D, K and M have local distributions within the Balkans and have evolved in isolation from other groups. Clade L originated in the Pannonian Basin and northern margin of the Balkan Peninsula and recently expanded across central and western Europe. Clades H and E have recently arrived in the Balkans from source populations in the Apennine Peninsula and Anatolia, respectively. Main conclusions The history of L. vulgaris involves multiple, independent refugial populations in the Balkans. Only one of them, located at the northern periphery of the peninsula, showed evidence of post-glacial expansion into western and northern Europe. The Balkans have therefore served as a reservoir of old diversity for L. vulgaris. By contrast, populations at the northern and eastern frontiers of the peninsula have experienced non-equilibrium dynamics. Our dating revealed that very little, if any, pre-Pliocene genetic diversity has survived in Europe, despite an extensive fossil record for this species in the Miocene and Pliocene. Differentiation of the European mtDNA clades thus seems to have been primarily moulded by Pleistocene climate change. None of the currently recognized subspecies present in the Balkans are reciprocally monophyletic in their mtDNA. We hypothesize that incomplete lineage sorting and mtDNA introgression account for the observed discrepancies.

A highly resolved and time-calibrated phylogeny based on nucleotide variation in 18 complete mitochondrial genomes is presented for all extant species and major lineages of fire-bellied toads of the genus Bombina ( Bombinatoridae ). Two sets of divergence time estimates are inferred by applying alternative fossil constraints as minima. Divergence time estimates from both analyses differed for the two oldest nodes. The earliest phylogenetic split occurred between small- and large-bodied Bombina (subgenera Bombina and Grobina , respectively) either in the Middle Oligocene or the Early Miocene. East Asian B. orientalis and European B. bombina + B. variegata diverged in the early or Middle Miocene. Divergence times inferred using the alternative fossil calibration strategies converged for the younger nodes, with broadly overlapping HPD intervals. The split between Bombina bombina and B. variegata occurred in the Late Miocene of Europe and somewhat preceded another deep mtDNA division between the Balkan B. v. scabra and B. v. variegata inhabiting the Carpathian Mts. Concurrently, the genetically distinct B. maxima diverged from other Grobina in southeast Asia in the Late Miocene or Pliocene. Our mtDNA phylogeny and a new species-tree analysis of published data (nuclear and mtDNA) suggest that B. fortinuptialis , B. lichuanensis and B. microdeladigitora may be conspecific geographic forms that separated due to Pleistocene climatic fluctuations in southeastern Asia. In the western Palearctic, the Late Pliocene to Pleistocene climatic vagaries most probably induced vicariant events in the evolutionary history of B. variegata that led to the formation of the two Balkan B. v. scabra lineages and the allopatric B. v. pachypus in the Apennine Peninsula. Divergence among B. bombina mtDNA lineages is low, with an Anatolian Turkey lineage as the sister group to the European mtDNA clades. In sum, Miocene diversification in the genus Bombina established six allopatrically distributed major mtDNA lineages that diversified during the Pliocene and Pleistocene and have survived until the present. The narrow habitat requirements of fire-bellied toads and extensive environmental changes throughout the Palearctic in the Neogene may have contributed to a putatively high extinction rate in these anurans resulting in the current east/west disjunction of their ranges.

Because reproductive isolation often evolves gradually, differentiating lineages may retain the potential for genetic exchange for prolonged periods, providing an opportunity to quantify and understand the fundamental role of gene flow during speciation. Here we delimit taxa, reconstruct the phylogeny and infer gene flow in newts of theLissotriton vulgarisspecies complex based on 74 nuclear markers sampled from 127 localities. We demonstrate that distinct lineages along the speciation continuum in newts exchange nontrivial amounts of genes, affecting their evolutionary trajectories. By integrating a wide array of methods, we delimit nine taxa and show that two principle factors have driven their genetic differentiation: time since the last common ancestor determining levels of shared ancestral polymorphism, and shifts in geographic distributions determining the extent of secondary contact. Post-divergence gene flow, indicative of evolutionary non-independence, has been most extensive between sister and non-sister taxa in Central Europe, while four southern European lineages have acquired the population genetic hallmarks of independent species (L. graecus,L. kosswigi,L. lantzi,L. schmidtleri). We obtained strong statistical support for widespread mtDNA introgression, previously suggested by discordance between mtDNA phylogeny and morphology. Our study suggests that long-term evolution in structured populations that may periodically exchange genes may be common: although some of these populations may become extinct or fuse, others will acquire complete reproductive isolation and will carry signatures of this complex history in their genomes.

Based on DNA sequences of fragments of the mitochondrial COI and 16S rRNA genes we provide a first assessment of genetic variability of Iberian treefrog populations that have been historically allocated to Hyla arborea, partly as subspecies H. a. molleri. Our data from 147 specimens and 33 populations confirm strong divergence between these frogs and Central European H. arborea but relatively low differentiation across their range, supporting their status as a separate species, H. molleri. Preliminary phylogeographic data indicate a possible weak genetic differentiation of populations from the northern coast of the region of Galicia. We suggest inclusion of nuclear markers and an extension of the sampling into the coastal regions of Asturias and Cantabria, as well as the identification of the contact zone between H. molleri and H. arborea in either the Spanish Basque country or in France, as priorities for future research on this species.

Green frogs of Central Europe consist of three taxa: Pelophylax ridibundus, P. lessonae and their natural hybridogenetic hybrid, P. esculentus, which forms as a rule mixed populations with its parental species. We examined 659 095 eggs from P. ridibundus (48 females), P. lessonae (133 females) and P. esculentus (170 females) originating from 39 populations in Austria, Denmark, Germany and Poland. Some females of each taxon laid eggs that fell into discrete size classes (small, medium and large). Large eggs were mostly diploid gametes from which triploids developed. They were found in P. esculentus (25 233 large eggs in 152 spawns), P. lessonae (81 in 10 spawns) and P. ridibundus (7 in 3 spawns). The main purpose of the paper was to demonstrate that the numbers of large eggs were clearly associated with triploid P. esculentus frogs. In pure hybrid (esculentus) populations large eggs comprised between 2.44-40.96% of all ova, while triploid adult frogs constituted between 13.9-73.2% of all individuals, in mixed ridibundus-esculentus populations the large eggs and triploid frogs ranged between 0.85-36.6% and 9.2-56.2%, respectively. However, in mixed lessonae-esculentus populations large eggs comprised only 1.74% of the spawns, whereas triploid frogs represented 2.1% of the adults in the population.

We describe two new species of semiarboreal frogs from the northern central east coast of Madagascar which occur syntopically, at least on the island of Nosy Boraha. The two species are morphologically closest to Blommersia wittei, but differ in advertisement calls and molecular phylogenetic relationships. One of the new species has a remarkable femoral gland structure, as these are shifted towards the distal part of the thigh, close to the knee joint. The new species are apparently microendemic to a small stretch of Madagascar's east coast and have so far not been found at higher elevations. Since both new species are tolerant of habitat disturbance and occur in anthropogenically altered habitats, such as plantations and villages, we propose an IUCN threat status of Least Concern despite their restricted extent of occurrence.

Major histocompatibility complex (MHC) genes are essential in pathogen recognition and triggering an adaptive immune response. Although they are the most polymorphic genes in vertebrates, very little information on MHC variation and patterns of evolution are available for amphibians, a group known to be declining rapidly worldwide. As infectious diseases are invoked in the declines, information on MHC variation should contribute to devising appropriate conservation strategies. In this study, we examined MHC variation in 149 Alpine newts (Mesotriton alpestris) from three allopatric population groups in Poland at the northeastern margin of the distribution of this species. The genetic distinctiveness of the population groups has previously been shown by studies of skin graft rejection, allozymes and microsatellites. Two putative expressed MHC II loci with contrasting levels of variation and clear evidence of gene conversion/recombination between them were detected. The Meal-DAB locus is highly polymorphic (37 alleles), and shows evidence of historical positive selection for amino acid replacements and substantial geographical differentiation in allelic richness. On the contrary, the Meal-DBB locus exhibits low polymorphism (three alleles differing by up to two synonymous substitutions) and a uniform distribution of three alleles among geographical regions. The uniform frequencies of the presumptively neutral Meal-DBB alleles may be explained by linkage to Meal-DAB. We found differences in allelic richness in Meal-DAB between regions, consistent with the hypothesis that genetic drift prevails with increasing distance from glacial refugia. Pseudogene loci appear to have evolved neutrally. The level of DAB variation correlated with variation in microsatellite loci, implying that selection and drift interplayed to produce the pattern of MHC variation observed in marginal populations of the Alpine newt.

We sequenced the complete mitochondrial genome of the Greek marsh frog Pelophylax cretensis, a water frog species endemic to the island of Crete. The genome sequence was 17,829 bp in size, and the gene order and contents were identical to those of previously reported mitochondrial genomes of other water frog species. This is the first complete mitogenome (i.e. including control region) described for western Palaearctic water frogs.

The Mascarene ridged frog, Ptychadena mascareniensis, is a species complex that includes numerous lineages occurring mostly in humid savannas and open forests of mainland Africa, Madagascar, the Seychelles, and the Mascarene Islands. Sampling across this broad distribution presents an opportunity to examine the genetic differentiation within this complex and to investigate how the evolution of bioclimatic niches may have shaped current biogeographic patterns. Using model-based phylogenetic methods and molecular-clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the group based on mitochondrial 16S rRNA and cytochrome b (cytb) genes and the nuclear RAG1 gene from 173 individuals. Haplotype networks were reconstructed and species boundaries were investigated using three species-delimitation approaches: Bayesian generalized mixed Yule-coalescent model (bGMYC), the Poisson Tree Process model (PTP) and a cluster algorithm (SpeciesIdentifier). Estimates of similarity in bioclimatic niche were calculated from species-distribution models (maxent) and multivariate statistics (Principal Component Analysis, Discriminant Function Analysis). Ancestral-area reconstructions were performed on the phylogeny using probabilistic approaches implemented in BioGeoBEARS. We detected high levels of genetic differentiation yielding ten distinct lineages or operational taxonomic units, and Central Africa was found to be a diversity hotspot for these frogs. Most speciation events took place throughout the Miocene, including "out-of-Africa" overseas dispersal events to Madagascar in the East and to São Tomé in the West. Bioclimatic niche was remarkably well conserved, with most species tolerating similar temperature and rainfall conditions common to the Central African region. The P. mascareniensis complex provides insights into how bioclimatic niche shaped the current biogeographic patterns with niche conservatism being exhibited by the Central African radiation and niche divergence shaping populations in West Africa and Madagascar. Central Africa, including the Albertine Rift region, has been an important center of diversification for this species complex.

Molecular genetic studies have in recent years revealed the presence of hidden species diversity in Madagascar's amphibians, and numerous genera contain candidate species awaiting description. We here revise the taxonomy of one such genus within the endemic Malagasy-Comorian family Mantellidae. The target taxon, Aglyptodactylus, is classified in the subfamily Laliostominae and to date contained three recognized nominal species. We focus in particular on clarifying the status of three candidate species proposed for the genus, and for this purpose integrate evidence from molecular genetics, bioacoustics and morphology. We furthermore summarize the information available for all species in the genus concerning their taxonomic identities, distributions and advertisement calls. Our analyses of advertisement calls revealed significant differences coinciding with major clades revealed by phylogenetic analysis of the mitochondrial 16S rRNA gene. Together with the new molecular dataset this provides clear evidence for evolutionary lineage divergence and supports the three candidate species previously characterized by mtDNA divergence of single specimens only. We also observed morphological differences among some of the lineages but these were in most cases not unambiguously diagnostic. As a taxonomic consequence we describe two new species: Aglyptodactylus australis sp. nov. from Andohahela rainforest in southeastern Madagascar, which so far is the largest species known in the genus with females reaching 82.5 mm snout-vent length; and Aglyptodactylus chorus sp. nov. from northeastern lowland rainforests in the Maroantsetra region. For the third proposed and confirmed candidate species we resurrect the name Rana inguinalis Günther, 1877 as Aglyptodactylus inguinalis (Günther, 1877) from the synonymy of A. madagascariensis. This name is applied to populations distributed in lowlands along most of the east coast of Madagascar, from Marojejy in the north southward to Tolagnaro. Genetic data indicate strong differentiation of A. inguinalis with three sub-lineages receiving support, and we cannot exclude that A. inguinalis as defined herein is a complex of species warranting further partition in the future. As a result of the revised taxonomy, A. madagascariensis remains restricted to rainforests at higher elevations (650‒1500 m a.s.l.) ranging from northern to south-central Madagascar, and showing some further differentiation with respect to a northern and a southern sub-lineage. Difficulties in the assignment of historical type specimens are discussed, as well as the biogeography and conservation status of the recognized species.

Genetic variation in 13 populations of the Alpine newt, Triturus alpestris, was assessed at the northeastern margin of its range (southern Poland). Variation at six microsatellite loci was scored in 354 newts, and two mitochondrial DNA fragments (c. 2000 bp) were sequenced in a subset of 27 individuals. Significant differences in allele frequencies and the presence of private alleles determined genetic units corresponding to three separate mountain ranges, i.e. the Carpathian, Sudetes and Holy Cross Mountains. F(ST)'s were three times greater in among than in within mountain range pairwise comparisons. An assignment test and pairwise F(ST)'s suggested relatively high levels of gene flow at the local level, although the Sudetes populations revealed some subtle structuring. Genetic variation was lower in the Carpathians and Holy Cross Mountains. The geographic pattern of mitochondrial DNA variation indicated that these newt populations originated from a single glacial refugium/founder population, and that the colonization of southern Poland took place in an easterly direction. The data show that substantial neutral variation and between group divergence has accumulated relatively quickly in these low-vagility organisms. The Alpine newt case exemplifies species history as a factor determining patterns of genetic diversity in marginal populations.

Made available in DSpace on 2018-11-26T16:16:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-09-01 Deutsche Forschungsgemeinschaft Humboldt Foundation Katholischer Akademischer Austauschdienst Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Many tropical organisms show large genetic differences among populations, yet the prevalent drivers of the underlying divergence processes are incompletely understood. We explored the effect of several habitat and natural history features (body size, macrohabitat, microhabitat, reproduction site, climatic heterogeneity, and topography) on population genetic divergence in tropical amphibians, based on a data set of 2680 DNA sequences of the mitochondrial cytochrome b gene in 39 widely distributed frog species from Brazil, Central America, Cuba, and Madagascar. Generalized linear models were implemented in an information-theoretic framework to evaluate the effects of the six predictors on genetic divergence among populations, measured as spatially corrected pairwise distances. Results indicate that topographic complexity and macrohabitat preferences have a strong effect on population divergence with species specialized to forest habitat and/or from topographically complex regions showing higher phylogeographic structure. This relationship changed after accounting for phylogenetic relatedness among taxa rendering macrohabitat preferences as the most important feature shaping genetic divergence. The remaining predictors showed negligible effects on the observed genetic divergence. A similar analysis performed using the population-scaled mutation rate (I similar to) as response variable showed little effect of the predictors. Our results demonstrate greater evolutionary independence among populations of anurans from forested regions versus species from open habitats. This pattern may result from lower vagility and stringency in reproductive requirements of rainforest species. Conversely, open landscapes may offer ephemeral and unstable breeding sites suitable for vagile generalist species, resulting in reduced intraspecific divergence. Our results predict that, for a given period of time, there should be a higher chance of speciation in tropical anurans living in forests than in species adapted to open habitats. Tech Univ Carolo Wilhelmina Braunschweig, Inst Zool, Div Evolutionary Biol, D-38106 Braunschweig, Germany Jagiellonian Univ, Inst Zool, Dept Comparat Anat, PL-30387 Krakow, Poland Jagiellonian Univ, Inst Environm Sci, PL-30387 Krakow, Poland Univ Fed Rio Grande do Norte, DBEZ, Ctr Biociencias, BR-59078900 Natal, RN, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Zool, BR-13506900 Rio Claro, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Zool, BR-13506900 Rio Claro, SP, Brazil Deutsche Forschungsgemeinschaft: VE247/7-1 FAPESP: 2008/50928-1 FAPESP: 2013/50741-7 CNPq: 302518/2013-4

The phylogenetic relationships for rapid species radiations are difficult to disentangle. Here we study one such case, namely the genus Triturus, which is composed of the marbled and crested newts. We analyze data for 38 genetic markers, positioned in 3-prime untranslated regions of protein-coding genes, obtained with 454 sequencing. Our dataset includes twenty Triturus newts and represents all nine species. Bayesian analysis of population structure allocates all individuals to their respective species. The branching patterns obtained by data concatenation, Bayesian concordance analysis and coalescent-based estimations of the species tree differ from one another. The data concatenation based species tree shows high branch support but branching order is considerably affected by allele choice in the case of heterozygotes in the concatenation process. Bayesian concordance analysis expresses the conflict between individual gene trees for part of the Triturus species tree as low concordance factors. The coalescent-based species tree is relatively similar to a previously published species tree based upon morphology and full mtDNA and any conflicting internal branches are not highly supported. Our findings reflect high gene tree discordance due to incomplete lineage sorting (possibly aggravated by hybridization) in combination with low information content of the markers employed (as can be expected for relatively recent species radiations). This case study highlights the complexity of resolving rapid radiations and we acknowledge that to convincingly resolve the Triturus species tree even more genes will have to be consulted.

We reconstruct range–wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA � 3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

We analyzed the influence of large rivers on the phylogeography of endemic widespread amphibians along Madagascar’s east coast, using as models various species and species complexes of Malagasy reed frogs (Heterixalus spp.) that are specialized to either highland or lowland habitats. We assembled a dense sampling across the full species ranges and used mitochondrial (cob) as well as nuclear (Rag-1) DNA sequences to assess their phylogeographies. A multigene mtDNA phylogeny of each species was constructed to establish the relationships among the main phylogroups, in order to understand the geographical regions of clade origins and possible directions of historical range expansions. Distinct intraspecific lineages as diagnosed by mitochondrial haplotype clades were found in highlands and lowlands. Most geographical boundaries among these phylogroups did not coincide with rivers, indicating that the influence of rivers on the primary divergence of phylogroups is probably minor in these frogs. Nevertheless, we found evidence for the influence of one riverine barrier in the lowland species complex, where the most important genetic discontinuity (the border between Heterixalus madagascariensis and H. alboguttatus) coincides with the geographical position of the Mangoro River on Madagascar’s central east coast. Analyses of the highland species H. betsileo revealed the existence of six deep haplotype lineages, separated in two major subpopulations which differ largely in altitudinal distribution and do not overlap with the geographical settings of rivers in the highlands. Furthermore, our analyses indicated that most of the major intraspecific lineages of reed frogs show signs of a rather recent population expansion.

The processes affecting species diversification may also exert an influence on patterns of genetic variability within species. We evaluated the contributions of five variables potentially influencing clade diversification (body size, reproductive mode, range size, microhabitat and skin texture) on mtDNA divergence and polymorphism among populations of 40 species of frogs (Mantellidae) from two rainforest communities in Madagascar. We report an inverse association between body size and nucleotide divergence between populations but find no influence of other variables on genetic variation. Body size explained ca. 11% of the variation in nucleotide divergence between populations and was coupled with high F(ST) levels and an absence of haplotype sharing in small-bodied and medium-sized frogs. Low dispersal ability is likely the proximate mechanism producing higher population differentiation in small mantellids. The lack of genetic cohesion among populations establishes regional genetic fragmentation which in turn has the potential to accelerate rates of allopatric speciation in small frogs relative to large species. However, there is little evidence of increased speciation rates in these or other small-bodied organisms. We reconcile these contradictory observations by suggesting that lower dispersal ability also curbs colonization of new areas, decelerating diversification in weak dispersers. Our results imply that the intermediate dispersal model also applies to amphibians and may explain inconsistent previous results on the correlation of body size and speciation rate.

Painted frogs (Discoglossus) are an anuran clade that originated in the Upper Miocene. Extant species are morphologically similar and have a circum-Mediterranean distribution. We assembled a multilocus dataset from seven nuclear and four mitochondrial genes for several individuals of all but one of the extant species and reconstructed a robust phylogeny by applying a coalescent-based species-tree method and a concatenation approach, both of which gave congruent results. The earliest phylogenetic split within Discoglossus separates D. montalentii from a clade comprising all other species. Discoglossus montalentii is monophyletic for haplotype variation at all loci and has distinct morphological, bioacoustic and karyotypic characters. We find moderate support for a sister-group relationship between the Iberian taxa and the Moroccan D. scovazzi, and high support for a D. pictus – D. sardus clade distributed around the Tyrrhenian basin. Topological discordance among gene trees during the speciation of D. galganoi, D. scovazzi, D. pictus and D. sardus is interpreted as the consequence of nearly simultaneous, vicariant diversification. The timing of these events is unclear, but possibly coincided with the final geotectonic rearrangement of the Western Mediterranean in the Middle Miocene or later during the Messinian salinity crisis. The Iberian taxa D. galganoi galganoi and D. g. jeanneae are reciprocally monophyletic in mitochondrial DNA but not in nuclear gene trees, and are therefore treated as subspecies of D. galganoi.

We describe a new frog species of the genus Guibemantis in the subgenus Pandanusicola from southeastern Madagascar and provide a redescription of a morphologically similar species, G. pulcher, based on its type series and on newly collected material. The new species, Guibemantis tasifotsy, differs from most Pandanusicola by probably not breeding in leaf axils of Pandanus plants but instead was collected calling from the vegetation above large, open lowland swamps. The green dorsal color of the new species is reminiscent of Guibemantis pulcher but can be distinguished from this Pandanus-breeding species by the presence of prominent white blotches along the flanks and by the structure of femoral glands in males. It shares these character states with G. liber from which the new species differs in details of coloration and advertisement call. Newly determined DNA sequences of the cytochrome b gene confirm that G. tasifotsy is genetically highly divergent from all other species of Guibemantis.

Field surveys in northeastern Madagascar have revealed the existence of a new Blommersia frog species (Anura: Mantellidae), populations of which were recorded within a 30 km radius of the town of Maroantsetra. We combined morphological, bioacoustic and molecular techniques and show that it is indeed a distinct evolutionary lineage which we describe as Blommersia variabilis sp. nov. from the type locality Ambodivoahangy near Maroantsetra. This new species is morphologically most similar to B. wittei by the presence of vomerine teeth and relatively small, well-delimited femoral glands, but differs from that species by advertisement call consisting of only 2–6 notes of comparatively longer duration, and a wider separation of femoral glands in males. It is associated with dense secondary vegetation fringing lentic water bodies. Anecdotal evidence suggests its life history is similar to other congeners. The restricted range of this species implies that it is microendemic, being possibly confined to the Antainambalana watershed. Mitochondrial and nuclear DNA variation show that its closest known relatives are Blommersia galani and B. dejongi, both of which also have restricted ranges on the east coast. A putative hybrid between a B. galani female and B. dejongi male with intermediate morphology was identified based on nuclear and mitochondrial DNA variation.

Depletion of polymorphism at major histocompatibility complex (MHC) genes has been hypothesized to limit the ability of populations to respond to emerging pathogens, thus putting their survival at risk. As pathogens contribute substantially to the global amphibian decline, assessing patterns of MHC variation is important in devising conservation strategies. Here, we directly compare levels of MHC class II and neutral variation between multiple populations of the great crested newt (Triturus cristatus) from refugial (REF: Romania) and postglacial expansion (PGE: Germany, Poland and UK) areas. REF populations harboured high levels of adaptive variation (24 expressed alleles), exhibiting clear signatures of historical positive selection, which points to the overall importance of MHC class II variation in this species. On the other hand, PGE populations were extremely depauperate (two alleles) but nevertheless have survived for c. 10,000 years, since the postglacial expansion. Variation in putative MHC class II pseudogenes, microsatellites and allozymes also showed a significant southern richness-northern purity pattern. The populations in the postglacial expansion area thus provide the clearest example to date of the long-term survival of populations in which MHC variation, historically under positive selection, has been depleted.

The complete mitochondrial genomes of two basal anurans, Bombina bombina and B. variegata (Anura; Bombinatoridae), were sequenced. The gene order of their mitochondrial DNA (mtDNA) is identical to that of canonical vertebrate mtDNA. In contrast, we show that there are structural differences in regulatory regions and protein coding genes between the mtDNA of these two closely related species. Corrected sequence divergence between the mtDNA of B. bombina and B. variegata amounts to 8.7% (2.3% divergence in amino acids). Comparisons with two East Asian congeners show that the control region contains two repeat regions, LV1 and LV2, present in all species except for B. bombina, in which LV2 has been secondarily lost. The rRNAs and tRNAs are characterized by low nucleotide divergence. The protein coding genes are considerably more disparate, although functional constraint is high but variable among genes, as evidenced by dN/dS ratios. A mtDNA phylogeny established the distribution of autapomorphic nonsynonomous substitutions in the mitogenomes of B. bombina and B. variegata. Nine of 98 nonsynonomous substitutions led to radical amino acid replacements that may alter mitochondrial protein function. Most radical substitutions were found in ND2, ND4, or ND5, encoding mitochondrial subunits of complex I of the electron transport system. The extensive divergence between the mitogenomes of B. bombina and B. variegata is discussed in terms of its possible role in impeding gene flow in natural hybrid zones between these two species.

Genetic variation in 19 populations of the alpine newt, Triturus alpestris, was assessed at the north-eastern margin of its range (southern Poland). Allozyme variation at five polymorphic loci was used in order to evaluate the degree of intrapopulational variation and interpopulational differentiation and define conservation units. Three management units were established on the basis of significant differences in allele frequencies. These correspond to three separate mountain ranges, i.e. the Carpathian, Sudetes and Holy Cross Mts. Contemporary gene flow between them is limited. In the extensively sampled Carpathian Mts, genetic structure is less pronounced, although still significant. Among population differentiation may be attributed to genetic drift in accordance with the isolation by distance model. The consequences of recent changes in land use in Central and Eastern Europe for low vagility and genetically structured organisms are discussed.