Next-generation phylogeography of the banded newts (Ommatotriton): A phylogenetic hypothesis for three ancient species with geographically restricted interspecific gene flow and deep intraspecific genetic structure.

Technological developments now make it possible to employ many markers for many individuals in a phylogeographic setting, even for taxa with large and complex genomes such as salamanders. The banded newt (genus Ommatotriton) from the Near East has been proposed to contain three species (O. nesterovi, O. ophryticus and O. vittatus) with unclear phylogenetic relationships, apparently limited interspecific gene flow and deep intraspecific geographic mtDNA structure. We use parallel tagged amplicon sequencing to obtain 177 nuclear DNA markers for 35 banded newts sampled throughout the range. We determine population structure (with Bayesian clustering and principal component analysis), interspecific gene flow (by determining the distribution of species-diagnostic alleles) and phylogenetic relationships (by maximum likelihood inference of concatenated sequence data and based on a summary-coalescent approach). We confirm that the three proposed species are genetically distinct. A sister relationship between O. nesterovi and O. ophryticus is suggested. We find evidence for introgression between O. nesterovi and O. ophryticus, but this is geographically limited. Intraspecific structuring is extensive, with the only recognized banded newt subspecies, O. vittatus cilicensis, representing the most distinct lineage below the species level. While mtDNA mostly mirrors the pattern observed in nuclear DNA, all banded newt species show mito-nuclear discordance as well.

The effect of short-term fasting on the oxidative status of larvae of crested newt species and their hybrids.

In nature, animals often face periods without food caused by seasonal fluctuations and/or prey scarcity. An organism's physiological response to imposed energetic limitations is followed by changes in mitochondrial functioning (adjustment of energy metabolism) and a reduction of non-essential processes. However, this energy-saving strategy can have its costs. In this study, we examined oxidative stress as one of the possible physiological costs of short-term, two-week-long food deprivation on developing amphibian larvae of the crested newts Triturus macedonicus and Triturus ivanbureschi and their hybrids. We investigated whether this exogenous factor additionally affected the oxidative status (fitness-related trait) of hybrid individuals. The fasting treatment led to lower growth and a lower body mass and body condition index of individuals. The results revealed that the antioxidant system (AOS) of food-deprived larvae could not cope in a proper manner with reactive oxygen species production under limited energy availability, leading to higher lipid oxidative damage. The lowest AOS response was observed for H2O2 scavenging parameters (catalase, glutathione peroxidase, and total glutathione), which together with the elevated activity of superoxide dismutase suggested increased H2O2 concentrations. Comparison between parental species and their hybrids showed that hybrid individuals suffered greater oxidative damage (as demonstrated by higher concentrations of lipid peroxides), indicating that they were more susceptible to fasting-induced oxidative stress. Overall, this study illustrates that: (i) an oxidative event is one of the costs amphibian larvae face during short-term periods of fasting, (ii) hybrids are less capable of dealing with this stressful condition, which can lower their chances of survival in a changing environment.

Discordant patterns of introgression across a narrow hybrid zone between two cryptic lineages of an Iberian endemic newt.

The study of natural hybrid zones can illuminate aspects of lineage divergence and speciation in morphologically cryptic taxa. We studied a hybrid zone between two highly divergent but morphologically similar lineages (south-western and south-eastern) of the Iberian endemic Bosca's newt (Lissotriton boscai) in SW Iberia with a multilocus dataset (microsatellites, nuclear and mitochondrial genes). STRUCTURE and NEWHYBRIDS analyses retrieved few admixed individuals, which classified as backcrosses involving parental individuals of the south-western lineage. Our results show asymmetric introgression of mtDNA beyond the contact from this lineage into the south-eastern lineage. Analysis of nongeographic introgression patterns revealed asymmetries in the direction of introgression, but except for mtDNA, we did not find evidence for nonconcordant introgression patterns across nuclear loci. Analysis of a 150-km transect across the hybrid zone showed broadly coincident cline widths (ca. 3.2-27.9 km), and concordant cline centres across all markers, except for mtDNA that is displaced ca. 60 km northward. Results from ecological niche modelling show that the hybrid zone is in a climatically homogenous area with suitable habitat for the species, suggesting that contact between the two lineages is unlikely to occur further south as their distributions are currently separated by an extensive area of unfavourable habitat. Taken together, our findings suggest the genetic structure of this hybrid zone results from the interplay of historical (biogeographic) and population-level processes. The narrowness and coincidence of genetic clines can be explained by weak selection against hybrids and reflect a degree of reproductive isolation that is consistent with cryptic speciation.

Phylogeny and species delimitation of near Eastern Neurergus newts (Salamandridae) based on genome-wide RADseq data analysis.

We reconstruct the molecular phylogeny of Near Eastern mountain brook newts of the genus Neurergus (family Salamandridae) based on newly determined RADseq data, and compare the outcomes of concatenation-based phylogenetic reconstruction with species-tree inference. Furthermore, we test the current taxonomy of Neurergus (with four species: Neurergus strauchii, N. crocatus, N. kaiseri, and N. derjugini) against coalescent-based species-delimitation approaches of our genome-wide genetic data set. While the position of N. strauchii as sister species to all other Neurergus species was consistent in all of our analyses, the phylogenetic relationships between the three remaining species changed depending on the applied method. The concatenation approach, as well as quartet-based species-tree inference, supported a topology with N. kaiseri as the closest relative to N. derjugini, while full-coalescent species-tree inference approaches supported N. crocatus as sister species of N. derjugini. Investigating the individual signal of gene trees highlighted an extensive variation among gene histories, most likely resulting from incomplete lineage sorting. Coalescent-based species-delimitation models suggest that the current taxonomy might underestimate the species richness within Neurergus and supports seven species. Based on the current sampling, our analysis suggests that N. strauchii, N. derjugini and N. kaiseri might each be subdivided into further species. However, as amphibian species are known to be composed of deep conspecific lineages that do not always warrant species status, these results need to be cautiously interpreted in an integrative taxonomic framework. We hypothesize that the rather shallow divergences detected within N. kaiseri and N. derjugini likely reflect an ongoing speciation process and thus require further investigation. On the contrary, the much deeper genetic divergence found between the two morphologically and geographically differentiated subspecies of N. strauchii leads us to propose that N. s. barani should be considered a distinct species, Neurergus barani Öz, 1994.

Comparative Anatomy of Trunk Musculature in Salamandridae with Different Habitats.

The diversity of trunk muscle morphology of Salamandridae occupying different habitats (aquatic: Pachytriton labiatus; terrestrial: Tylototriton kweichowensis and Salamandra salamandra salamandra) was examined. Trunk muscles were dissected, and muscle weight ratios were quantified. The terrestrial species have larger abdominal trunk muscles than the aquatic species do. In contrast, the lateral hypaxial muscles are larger in the aquatic species. The M. rectus abdominis profundus is located subjacent to the M. rectus abdominis in the terrestrial species. In the aquatic species, the ventral muscle is composed of the M. rectus abdominis alone. The lateral hypaxial muscles in the aquatic species are suited to lateral bending during underwater locomotion in the denser aquatic medium. Larger abdominal muscles may be used in supporting body weight against gravity in the terrestrial species. The function of the M. rectus abdominis profundus may be to support the M. rectus abdominis in the terrestrial species. These findings suggest a divergent evolution of trunk muscle characteristics within the Salamandridae, which correlate with both habitats and modes of locomotion.

A biogeographic and ecological perspective to the evolution of reproductive behaviour in the family Salamandridae.

Amphibians have a complex reproductive behaviour, which shows the highest diversity among tetrapodes. The family Salamandridae, distributed across the entire Holarctic, is one of the most diverse groups of extant salamanders comprising 114 species in 21 genera. The family has a remarkable diversity of courtship modes, amplexus and sperm transfer. It is often hypothesised that this diversity has evolved in adaptation to a specific mating and/or breeding habitat. We test this hypothesis based upon a phylogenetic reconstruction using the complete mitochondrial genome sequences of 45 Salamandridae species, representing all existing genera. We used ancestral character state reconstruction methods and geographic range models and applied relaxed Bayesian molecular clock models to discuss the results in a temporal framework of Salamandridae evolution. Our results show that the family Salamandridae started to diversify in the Late Cretaceous (ca. 87 mya) and is of Western Palearctic origin. Ancestral character state reconstruction predicts that its common ancestor was oviparous, mated on land without amplexus and probably showed a pin wheel spermatophore transfer, which is still found in the Italian endemic Salamandrina terdigidata. Our results suggest that several colonization of continents with subsequent radiations took place, once to the Nearctic and twice into Eastern Asian realms. However, these events were only in one case associated with a change in mating behaviour (dorsal amplexus in Nearctic newts). Around the Cretaceous-Paleogene boundary (K-Pg boundary) several Salamandridae lineages further diverged, again with no obvious changes in mating behaviour. Overall, there is no significant signal for mating character evolution being caused by changes in habitat type, with only a slight tendency that changes in mating habitat might have led to changes in the type of sperm transfer which in turn was associated with changes in the presence or absence of amplexus.

Isolation and gene flow in a speciation continuum in newts.

Because reproductive isolation often evolves gradually, differentiating lineages may retain the potential for genetic exchange for prolonged periods, providing an opportunity to quantify and to understand the fundamental role of gene flow during speciation. Here we delimit evolutionary lineages, reconstruct the phylogeny and infer gene flow in newts of the Lissotriton vulgaris species 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 evolutionary lineages and show that two principal 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 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 following secondary contact, previously suggested by discordance between mtDNA phylogeny and morphology. Our study reveals long-term evolutionary persistence of evolutionary lineages that may periodically exchange genes with one another: although some of these lineages may become extinct or fuse, others will acquire complete reproductive isolation and will carry signatures of this complex history in their genomes.

Integrative inference of population history in the Ibero-Maghrebian endemic Pleurodeles waltl (Salamandridae).

Inference of population histories from the molecular signatures of past demographic processes is challenging, but recent methodological advances in species distribution models and their integration in time-calibrated phylogeographic studies allow detailed reconstruction of complex biogeographic scenarios. We apply an integrative approach to infer the evolutionary history of the Iberian ribbed newt (Pleurodeles waltl), an Ibero-Maghrebian endemic with populations north and south of the Strait of Gibraltar. We analyzed an extensive multilocus dataset (mitochondrial and nuclear DNA sequences and ten polymorphic microsatellite loci) and found a deep east-west phylogeographic break in Iberian populations dating back to the Plio-Pleistocene. This break is inferred to result from vicariance associated with the formation of the Guadalquivir river basin. In contrast with previous studies, North African populations showed exclusive mtDNA haplotypes, and formed a monophyletic clade within the Eastern Iberian lineage in the mtDNA genealogy. On the other hand, microsatellites failed to recover Moroccan populations as a differentiated genetic cluster. This is interpreted to result from post-divergence gene flow based on the results of IMA2 and Migrate analyses. Thus, Moroccan populations would have originated after overseas dispersal from the Iberian Peninsula in the Pleistocene, with subsequent gene flow in more recent times, implying at least two trans-marine dispersal events. We modeled the distribution of the species and of each lineage, and projected these models back in time to infer climatically favourable areas during the mid-Holocene, the last glacial maximum (LGM) and the last interglacial (LIG), to reconstruct more recent population dynamics. We found minor differences in climatic favourability across lineages, suggesting intraspecific niche conservatism. Genetic diversity was significantly correlated with the intersection of environmental favourability in the LIG and LGM, indicating that populations of P. waltl are genetically more diverse in regions that have remained environmentally favourable through the last glacial cycle, particularly southern Iberia and northern Morocco. This study provides novel insights into the relative roles of geology and climate on the biogeography of a biodiversity hotspot.

The Near East as a cradle of biodiversity: A phylogeography of banded newts (genus Ommatotriton) reveals extensive inter- and intraspecific genetic differentiation.

The banded newt (genus Ommatotriton) is widely distributed in the Near East (Anatolia, Caucasus and the Levant) - an understudied region from the perspective of phylogeography. The genus is polytypic, but the number of species included and the phylogenetic relationships between them are not settled. We sequenced two mitochondrial and two nuclear DNA markers throughout the range of Ommatotriton. For mtDNA we constructed phylogenetic trees, estimated divergence times using fossil calibration, and investigated changes in effective population size with Bayesian skyline plots and mismatch analyses. For nuDNA we constructed phylogenetic trees and haplotype networks. Species trees were constructed for all markers and nuDNA only. Species distribution models were projected on current and Last Glacial Maximum climate layers. We confirm the presence of three Ommatotriton species: O. nesterovi, O. ophryticus and O. vittatus. These species are genetically distinct and their most recent common ancestor was dated at ∼25Ma (Oligocene). No evidence of recent gene flow between species was found. The species show deep intraspecific genetic divergence, represented by geographically structured clades, with crown nodes of species dated ∼8-13Ma (Miocene to Early Quaternary); evidence of long-term in situ evolution and survival in multiple glacial refugia. While a species tree based on nuDNA suggested a sister species relationship between O. vittatus and O. ophryticus, when mtDNA was included, phylogenetic relationships were unresolved, and we refrain from accepting a particular phylogenetic hypothesis at this stage. While species distribution models suggest reduced and fragmented ranges during the Last Glacial Maximum, we found no evidence for strong population bottlenecks. We discuss our results in the light of other phylogeographic studies from the Near East. Our study underlines the important role of the Near East in generating and sustaining biodiversity.

Concordant species delimitation from multiple independent evidence: A case study with the Pachytriton brevipes complex (Caudata: Salamandridae).

Mitochondrial DNA (mtDNA) sequence data are widely used to delimit species. However, owing to its strict maternal inheritance in most species, mtDNA tracks female dispersion and dispersal only. The accuracy of mtDNA-derived species delimitation is often not explicitly tested using other independent evidence, such as nuclear DNA (nDNA) data, morphological data, or ecological data. Because species are independent evolutionary lineages that can form testable hypotheses, we present a multi-evidence case study on species delimitation that combines statistical approaches with spatially explicit ecological analysis. Montane salamanders of the Pachytriton brevipes complex (Salamandridae) from southeastern China exhibit conservative morphology and variable color patterning that impede species diagnosis. Recent studies proposed splitting P. brevipes into four species based on deep mtDNA divergence but also found discordance between mtDNA and nDNA trees. In this study, we test evolutionary independence of these hypothesized species lineages using two coalescent-based Bayesian methods (Bayes factor and BP&P). Despite significant conflict between mtDNA gene tree and the species phylogeny, the results reinforce the inference of at least four species in the complex as opposed to the one species recognized for over 130 years. Correlative ecological niche modeling and statistical analysis of environmental data indicate that suitable habitats for each species are isolated by incompatible intervening lowland regions, so the likelihood of gene flow among species is very low, which means species lineages should maintain their evolutionary independence. We demonstrate that concordance among independent evidence confirms species status, which forms the basis for accurate assessment of regional biodiversity.

I Environmental DNA sampling is more sensitive than a traditional survey technique for detecting an aquatic invader.

Effective management of alien species requires detecting populations in the early stages of invasion. Environmental DNA (eDNA) sampling can detect aquatic species at relatively low densities, but few studies have directly compared detection probabilities of eDNA sampling with those of traditional sampling methods. We compare the ability of a traditional sampling technique (bottle trapping) and eDNA to detect a recently established invader, the smooth newt Lissotriton vulgaris vulgaris, at seven field sites in Melbourne, Australia. Over a four-month period, per-trap detection probabilities ranged from 0.01 to 0.26 among sites where L. v. vulgaris was detected, whereas per-sample eDNA estimates were much higher (0.29-1.0). Detection probabilities of both methods varied temporally (across days and months), but temporal variation appeared to be uncorrelated between methods. Only estimates of spatial variation were strongly correlated across the two sampling techniques. Environmental variables (water depth, rainfall, ambient temperature) were not clearly correlated with detection probabilities estimated via trapping, whereas eDNA detection probabilities were negatively correlated with water depth, possibly reflecting higher eDNA concentrations at lower water levels. Our findings demonstrate that eDNA sampling can be an order of magnitude more sensitive than traditional methods, and illustrate that traditional- and eDNA-based surveys can provide independent information on species distributions when occupancy surveys are conducted over short timescales.

Evolutionary history of Ichthyosaura alpestris (Caudata, Salamandridae) inferred from the combined analysis of nuclear and mitochondrial markers.

Widespread species with morphologically and ecologically differentiated populations are key to understand speciation because they allow investigating the different stages of the continuous process of population divergence. The alpine newt, Ichthyosaura alpestris, with a range that covers a large part of Central Europe as well as isolated regions in all three European Mediterranean peninsulas, and with strong ecological and life-history differences among populations, is an excellent system for such studies. We sampled individuals across most of the range of the species, and analyzed mitochondrial (1442 bp) and nuclear (two nuclear genes -1554 bp- and 35 allozyme loci) markers to produce a time-calibrated phylogeny and reconstruct the historical biogeography of the species. Phylogenetic analyses of mtDNA data produced a fully resolved topology, with an endemic, Balkan clade (Vlasina) which is sister to a clade comprising an eastern and a western group. Within the former, one clade (subspecies I. a. veluchiensis) is sister to a clade containing subspecies I. a. montenegrina and I. a. serdara as well as samples from southern Romania, Bosnia-Herzegovina, Serbia and Bulgaria (subspecies I. a. reiseri and part of I. a. alpestris). Within the western group, populations from the Italian peninsula (subspecies I. a. apuana and I. a. inexpectata) are sister to a clade containing samples from the Iberian Peninsula (subspecies I. a. cyreni) and the remainder of the samples from subspecies I. a. alpestris (populations from Hungary, Austria, Poland, France, Germany and the larger part of Romania). Results of (∗)BEAST analyses on a combined mtDNA and nDNA dataset consistently recovered with high statistical support four lineages with unresolved inter-relationships: (1) subspecies I. a. veluchiensis; (2) subspecies I. a. apuana+I. a. inexpectata; (3) subspecies I. a. cyreni+part of subspecies I. a. alpestris (the westernmost populations, plus most Romanian populations); and (4) the remaining populations, including subspecies I. a. serdara, I. a. reiseri and I. a. montenegrina and part of subspecies I. a. alpestris, plus samples from Vlasina. Our time estimates are consistent with ages based on the fossil record and suggest a widespread distribution for the I. alpestris ancestor, with the split of the major eastern and western lineages during the Miocene, in the Tortonian. Our study provides a solid, comprehensive background on the evolutionary history of the species based on the most complete combined (mtDNA+nDNA+allozymes) dataset to date. The combination of the historical perspective provided by coalescent-based analyses of mitochondrial and nuclear DNA variation with individual-based multilocus assignment methods based on multiple nuclear markers (allozymes) also allowed identification of instances of discordance across markers that highlight the complexity and dynamism of past and ongoing evolutionary processes in the species.

A stable niche assumption-free test of ecological divergence.

Understanding the impact of geological events on diversification processes is central to evolutionary ecology. The recent amalgamation between ecological niche models (ENMs) and phylogenetic analyses has been used to estimate historical ranges of modern lineages by projecting current ecological niches of organisms onto paleoclimatic reconstructions. A critical assumption underlying this approach is that niches are stable over time. Using Notophthalmus viridescens (eastern newt), in which four ecologically diverged subspecies are recognized, we introduce an analytical framework free from the niche stability assumption to examine how refugial retreat and subsequent postglacial expansion have affected intraspecific ecological divergence. We found that the current subspecies designation was not congruent with the phylogenetic lineages. Thus, we examined ecological niche overlap between the refugial and modern populations, in both subspecies and lineage, by creating ENMs independently for modern and estimated last glacial maximum (LGM) newt populations, extracting bioclimate variables by randomly generated points, and conducting principal component analyses. Our analyses consistently showed that when tested as a hypothesis, rather than used as an assumption, the niches of N. viridescens lineages have been unstable since the LGM (both subspecies and lineages). There was greater ecological niche differentiation among the subspecies than the modern phylogenetic lineages, suggesting that the subspecies, rather than the phylogenetic lineages, is the unit of the current ecological divergence. The present study found little evidence that the LGM refugial retreat caused the currently observed ecological divergence and suggests that ecological divergence has occurred during postglacial expansion to the current distribution ranges.

No evidence for nuclear introgression despite complete mtDNA replacement in the Carpathian newt (Lissotriton montandoni).

Patterns of interspecific introgression may vary geographically, and the distribution of introgressed variants can yield insight into the historical dynamics of genetic interactions between hybridizing species. Urodele amphibians, often characterized by limited mobility, deep intraspecific genetic structuring and vulnerability to climatic changes, constitute suitable models for such historical inferences. Here, we combine an extensive survey of the mitochondrial (mtDNA) and nuclear (15 microsatellites) genomes in the Carpathian newt, Lissotriton montandoni (Lm) with species distribution modelling (SDM). Populations of the smooth newt, L. vulgaris (Lv) from the areas surrounding the Lm range were also sampled to test whether gene flow between these hybridizing species extends beyond the area of strict syntopy. The extent of introgression differs dramatically between the mitochondrial genome and the nuclear genome. While multiple, spatially and temporally distinct introgression events from Lv resulted in complete mtDNA replacement in Lm, there was little evidence of recent interspecific nuclear gene flow in the assayed markers. Microsatellite differentiation within Lm defines three units, probably derived from separate glacial refugia, located in the northern, eastern and southern part of the Carpathians. In situ survival and range fragmentation of Lm are supported by SDM, corroborating the role of the Carpathians as a major refugial area. Our results, in combination with previous reports of extensive introgression of the major histocompatibility complex (MHC) genes, emphasize the complexity of historical gene exchange between Lm and Lv.

A geographic mosaic of evolutionary lineages within the insular endemic newt Euproctus montanus.

Islands are hotspots of biodiversity, with a disproportionately high fraction of endemic lineages, often of ancient origin. Nevertheless, intra-island phylogeographies are surprisingly scarce, leading to a scanty knowledge about the microevolutionary processes induced on island populations by Plio-Pleistocene climatic oscillations, and the manner in which these processes contributed to shape their current genetic diversity. We investigated the phylogeography, historical demography and species distribution models of the Corsican endemic newt Euproctus montanus (north-western Mediterranean). As for many island endemics, the continuous distribution of E. montanus throughout its range has hitherto been considered as evidence for a single large population, a belief that also guided the species' categorization for conservation purposes. Instead, we found a geographic mosaic of ancient evolutionary lineages, with five main clades of likely Pliocene origin (2.6-5.8 My), all but one restricted to northern Corsica. Moreover, the copresence between main lineages in the same population was limited to a single case. As also suggested by growing literature on intra-island phylogeographic variation, it seems that the extensive use of simplifying assumption on the population structure and historical demography of island populations--both in theoretical and applicative studies--should be carefully reconsidered, a claim that is well exemplified by the case presented here.

Phylogeny and historical demography of Cynops pyrrhogaster (Amphibia: Urodela): taxonomic relationships and distributional changes associated with climatic oscillations.

We investigated the phylogenetic relationships and estimated the historical demography of the Japanese fire-bellied newt, Cynops pyrrhogaster, from Japanese mainlands using 1407-bp sequences of the mitochondrial DNA (NADH6, tRNAglu, cyt b) and 1208-bp sequences of nuclear DNA (Rag-1) genes. Phylogenetic trees based on mitochondrial DNA revealed four major haplotype clades (NORTHERN, CENTRAL, WESTERN, and SOUTHERN clades) within this species. Degree of genetic differentiation among major haplotype clades was very large for intraspecific variation, suggesting this species to be composed of four species lineages that replace each other geographically. Nuclear genetic variation presented no obvious patterns of geographic structure except for the distinctness of populations diagnosed by NORTHERN clade of mitochondrial haplotypes, suggesting results of incomplete lineage sorting. Current distribution and estimated divergence times for the genus Cynops suggest that the common ancestor of two Japanese species (C. pyrrhogaster and C. ensicauda from the Ryukyu Islands) had diverged at the edge of the continent corresponding to the present East China Sea and Central Ryukyus. Subsequent range expansion to Japanese mainland seems to have occurred in the middle Miocene. Population-genetic analyses indicated that all species lineages, except for the SOUTHERN one, experienced geographic population reductions and expansions associated with glacial and postglacial climatic oscillations.

Two new species of Tylototriton from Thailand (Amphibia: Urodela: Salamandridae).

Three morphological groups are found in a salamandrid newt Tylototriton shanjing from Thailand. We describe two of them as new species, one from northern and the other from northeastern Thailand, based on molecular and morphological data, however we could not make a taxonomic decision on the remaining one group because of the lack of voucher specimens and sufficient genetic data. The northern species differs morphologically from all known congeners by having the combination of orange to reddish brown markings, narrow and sharply protruding dorsolateral bony ridges on head, weakly segmented vertebral ridge, and long and high tail. The northeastern species is characterized by having the combination of yellow, orange, or reddish brown markings, wide and moderately protruding dorsolateral bony ridges on head, smooth vertebral ridge, black limbs, and black tail except for edges. Validity of taxonomic subdivision of the genus Tylototriton is discussed.

Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes.

Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.

Phylogeny and biogeography of the Anderson's crocodile newt, Echinotriton andersoni (Amphibia: Caudata), as revealed by mitochondrial DNA sequences.

The Anderson's crocodile newt, Echinotriton andersoni, is considered a relic and endangered species distributed in the Central Ryukyus. To elucidate phylogenetic relationships and detailed genetic structures among populations, we analyzed variation in the mitochondrial cytochrome b gene. Results strongly support a primary dichotomy between populations from the Amami and Okinawa Island Groups with substantial genetic divergence, favoring a primary divergence between the two island groups. Within the latter, populations from the southern part of Okinawajima Island are shown to be more closely related to those from Tokashikijima Island than to those from the northern and central parts of Okinawajima. The prominent genetic divergence between the two island groups of the Central Ryukyus seems to have initiated in the Miocene, i.e., prior to formation of the strait that has consistently separated these island groups since the Pleistocene. The ancestor of the southern Okinawajima-Tokashikijima is estimated to have migrated from the northern and central parts of Okinawajima into southern Okinawajima at the Pleistocene, and dispersed into Tokashikijima subsequently.

Evolutionary history of Lissotriton helveticus: multilocus assessment of ancestral vs. recent colonization of the Iberian Peninsula.

The Pleistocene was characterized by climatic changes that greatly altered the distribution of organisms. Population extinctions, bottlenecks, isolation, range expansions and contractions were often associated with glaciations, leaving signatures in the spatial patterns of genetic diversity across species. Lissotriton helveticus belongs to a Pan-European lineage of newts that were strongly affected by glaciations and represent an excellent model to analyse the effect of generalized climatic changes in phylogeographic patterns. We studied the genetic diversity of the species using data from two mitochondrial and three nuclear genes analyzed in a Bayesian phylogenetic framework to investigate the historical processes shaping spatial patterns of genetic diversity. Mitochondrial haplotypes cluster in four different groups present in the Iberian Peninsula and of Pleistocene origin, probably by allopatric fragmentation. Nuclear genes present no obvious geographic structure patterns, suggesting gene flow and generalized incomplete lineage sorting. Populations north of the Pyrenees are closely related to those from northeastern Iberia, suggesting recent range expansion from this region. Historical demographic analyses indicate a demographic expansion starting about 100,000years ago and more recent population declines. Compared to other Lissotriton species, L. helveticus includes only relatively young genetic lineages, suggesting a Central European pre-Pleistocene distribution followed by complete extirpation of the species during glaciations in that area. Historical demographic trends in the Iberian Peninsula are reversed with respect to the more Mediterranean species Lissotriton boscai, indicating different responses of both species to climate changes. Diversity patterns among Lissotriton species seem to be defined by four main factors: ancestral distributions, colonization capabilities, interactions with other species and effective population sizes. Differences in these factors define two types of species, referred to as "R" (refugia) and "S" (sanctuaries) that explain part of the diversity in patterns of genetic diversity created by glaciations in Western Europe.