Mitochondrial DNA sequence analysis reveals multiple Pleistocene glacial refugia for the Yellow‐spotted mountain newt,Neurergus derjugini (Caudata: Salamandridae) in the mid‐Zagros range in Iran and Iraq

Phylogeography is often used to investigate the effects of glacial cycles on current genetic structure of various plant and animal species. This approach can also identify the number and location of glacial refugia as well as the recolonization routes from those refugia to the current locations. To identify the location of glacial refugia of the Yellow‐spotted mountain newt, Neurergus derjugini, we employed phylogeography patterns and genetic variability of this species by analyzing partial ND4 sequences (867 bp) of 67 specimens from 15 sampling localities from the whole species range in Iran and Iraq. Phylogenetic trees concordant with haplotype networks showed a clear genetic structure among populations as three groups corresponding to the populations in the north, center, and south. Evolutionary ages of clades north and south ranging from 0.15 to 0.17 Myr, while the oldest clade is the central clade, corresponding to 0.32 Myr. Bayesian skyline plots of population size change through time show a relatively slight increase until about 25 kyr (around the last glacial maximum) and a decline of population size about 2.5 kyr. The presence of geographically structured clades in north, center, and south sections of the species range signifies the disjunct populations that have emerged in three different refugium. This study illustrates the importance of the effect of previous glacial cycles in shaping the genetic structure of mountain species in the Zagros range. These areas are important in terms of long‐term species persistence and therefore valuable areas for conservation of biodiversity.     

Structure Organization of Urinary System in the Yellow Spotted Mountain Newts （Salamandridae： Neurergus microspilotus）

This study deals with the histomorphology of the mesonephros in male and female Neurergus microspilotus. The slender and narrow kidneys are positioned in the retro peritoneal position up against the ventral aspect of vertebral column and may extend the length from the esophagus-stomach junction to cloaca. The kidney in both sexes is composed of sexual（anterior） and pelvic（posterior） parts. The duct of sexual kidney is a narrow duct which is lying alongside its lateral edge. In the female, it is connected to the ureters and then the duct of defi nitive kidney. Before entering the cloaca, two ureters are joined together and open to the apex of the cloaca. In the male, after entering the sexual kidney, the sperm leave the testis through efferent ducts, then these ducts join together and eventually form Bidder＇s duct. The Bidder＇s duct joins the Bowman＇s capsule of the nephrons in the sexual kidney and the nephrons make collecting ducts which are fi lled with both sperm and urine. After leaving the kidney, all the collecting ducts are connected to the Wolffi an duct. Wolffi an duct joins the ureters（merge from defi nitive kidney） just before entering the cloaca. Based on serial paraffi n sections, nephrons consist of a fi ltration unit, the Malpighian corpuscle, and a renal tubule, which can be divided into 4 morphologically distinct segments： proximal tubule（first and second segment）, distal tubule, and collecting tubule. Collecting tubules merge and form a branch system that opens into collecting ducts.     

River islands,refugia and genetic structuring in the endemic brown frog Rana kukunoris (Anura,Ranidae) of the Qinghai‐Tibetan Plateau

Frequently, Pleistocene climatic cycling has been found to be the diver of genetic structuring in populations, even in areas that did not have continental ice sheets, such as on the Qinghai‐Tibetan Plateau (QTP). Typically, species distributed on the plateau have been hypothesized to re‐treat to south‐eastern refugia, especially during the Last Glacial Maximum (LGM). We evaluated sequence variation in the mitochondrial DNA gene Cytb and the nuclear DNA gene RAG‐1 in Rana kukunoris, a species endemic to the QTP. Two major lineages, N and S, were identified, and lineage N was further subdivided into N1 and N2. The geographical distribution and genealogical divergences supported the hypothesis of multiple refugia. However, major lineages and sublineages diverged prior to the LGM. Demographical expansion was detected only in lineage S and sublineage N2. Sublineage N1 might have survived several glacial cycles in situ and did not expand after the LGM because of the absence of suitable habitat; it survived in river islands. Genetic analysis and environment modelling suggested that the north‐eastern edge of QTP contained a major refugium for R. kukunoris. From here, lineage S dispersed southwards after the LGM. Two microrefugia in northern Qilian Mountains greatly contributed to current level of intraspecific genetic diversity. These results were found to have important implications for the habitat conservation in Northwest China.     

Postglacial northward expansion and genetic differentiation between migratory and sedentary populations of the broad‐tailed hummingbird (Selasphorus platycercus)

Unlike other migratory hummingbirds in North America, the broad‐tailed hummingbird (Selasphorus platycercus) exhibits both long‐distance migratory behaviour in the USA and sedentary behaviour in Mexico and Guatemala. We examined the evolution of migration linked to its northward expansion using a multiperspective approach. We analysed variation in morphology, mitochondrial and nuclear DNA, estimated migration rates between migratory and sedentary populations, compared divergence times with the occurrence of Quaternary climate events and constructed species distribution models to predict where migratory and sedentary populations resided during the Last Glacial Maximum (LGM) and Last Interglacial (LIG) events. Our results are consistent with a recent northward population expansion driven by migration from southern sedentary populations. Phylogeographical analyses and population genetics methods revealed that migratory populations in the USA and sedentary populations in Mexico of the platycercus subspecies form one admixed population, and that sedentary populations from southern Mexico and Guatemala (guatemalae) undertook independent evolutionary trajectories. Species distribution modelling revealed that the species is a niche tracker and that the climate conditions associated with modern obligate migrants in the USA were not present during the LIG, which provides indirect evidence for recent migratory behaviour in broad‐tailed hummingbirds on the temporal scale of glacial cycles. The finding that platycercus hummingbirds form one genetic population and that suitable habitat for migratory populations was observed in eastern Mexico during the LIG also suggests that the conservation of overwintering sites is crucial for obligate migratory populations currently facing climate change effects.     

A skeletochronological analysis of a population of the Anatolia Newt,Neurergus strauchii (Steindachner, 1887) (Caudata: Salamandridae), in Eastern Anatolia,Turkey

This study presents data on age, growth and longevity of a population of Neurergus strauchii in Eastern Anatolia (Bingöl, Turkey) based on skeletochronological data. The phalangeal diaphyseal cross-sections of 54 individuals studied (12♂, 42♀) showed that females are on average older than males: the ages ranged from 6 to 14 years, with an average age of 8.8 years in males and 10.9 years in females. Sexual maturity is reached at an age of 4-5 years in both sexes. The slow growth and the longevity make the species vulnerable.     

Inferring the roles of vicariance,climate and topography in population differentiation in Salamandra algira (Caudata,Salamandridae)

Integrating information from species occurrence data, environmental variables and molecular markers can provide valuable insights about the processes of population persistence and differentiation. In this study, we present the most comprehensive overview of the evolutionary history of the North African salamander Salamandra algira (Caudata, Salamandridae) to date, including analyses of climatic and topographical variables, and sequences of two mitochondrial and two nuclear DNA fragments, with a special focus on Algerian populations, under‐represented in previous studies. Coalescent‐based phylogenetic analyses of mtDNA data recover four well‐supported population groups corresponding to described subspecies, with a western clade including populations in north‐western Morocco (with two subclades corresponding to the subspecies tingitana and splendens), and an eastern clade including populations from north‐eastern Morocco (subspecies spelaea) and Algeria (subspecies algira). Inferred split times between major clades date back to the Miocene, with additional splits within each major clade in the Plio‐Pleistocene. Present climatic (aridity) and topographical factors account for geographical discontinuities across population groups and help identify potential areas of secondary contact between clades corresponding to the subspecies tingitana and splendens in the Rif mountains in Morocco. Niche analysis indicates the absence of phylogenetic signal in the use of environmental space in this species.     

Ontogenetic changes in spot configuration (numbers,circularity, size and asymmetry) and lateral line in Neurergus microspilotus (Caudata: Salamandridae)

Coloration in three of four species of the genus Neurergus including N. microspilotus is characterized by the presence of yellow spots on a dark skin, but there is no available information about changes in spot configuration, speed of development and degree of association between melanophore‐free region and the lateral line. In this study, spot numbers, spot circularity, spot size and spot asymmetry were studied during larval to adult growth in N. microspilotus during July 2012 to June 2015. The mean numbers of spots increased during the late larval stage till postmetamorphic period from 13.33 ± 3.77 to 22.53 ± 4.09 and reached 42.62 ± 4.06 in adults. At the same time, the extent of spots gradually decreased in size from 5.80 ± 1.00 to 3.57 ± 0.97 mm² and reached 3.55 ± 1.42 mm² in adults, but the spot circularity increased from 0.48 ± 0.23 to 0.78 ± 0.49 and reached 0.80 ± 0.15 in adults. In adults, the numbers, circularity, size and asymmetry of spots remain stable with little but non‐significant changes during the study period. Histological study shows that formation of a melanophore‐free region correlates with the development of the lateral line receptors. This study demonstrates that the effects of lateral line on chromatophores persist through middle larval stages but diminish as metamorphosis completes.     

Geological habitat template overrides late Quaternary climate change as a determinant of range dynamics and phylogeography in some habitat‐specialist water beetles

Aim We investigated the roles of lithology and climate in constraining the ranges of four co‐distributed species of Iberian saline‐habitat specialist water beetles (Ochthebius glaber, Ochthebius notabilis, Enochrus falcarius and Nebrioporus baeticus) across the late Quaternary and in shaping their geographical genetic structure. The aim was to improve our understanding of the effects of past climate changes on the biota of arid Mediterranean environments and of the relative importance of history and landscape on phylogeographical patterns. Location Iberian Peninsula, Mediterranean. Methods We combined species distribution modelling (SDM) and comparative phylogeography. We used a multi‐model inference and model‐averaging approach both for assessment of range determinants (climate and lithology) and for provision of spatially explicit estimates of the species current and Last Glacial Maximum (LGM) potential ranges. Potential LGM distributions were then contrasted with the phylogeographical and population expansion patterns as assessed using mitochondrial DNA sequence data. We also evaluated the relative importance of geographical distance, habitat resistance and historical isolation for genetic structure in a causal modelling framework. Results Lithology poses a strong constraint on the distribution of Iberian saline‐habitat specialist water beetles, with a variable, but generally moderate, additional influence by climate. The degree to which potential LGM distributions were reduced and fragmented decreased with increasing importance of lithology. These SDM‐based suitability predictions were mostly congruent with phylogeographical and population genetic patterns across the study species, with stronger geographical structure in the genetic diversity of the more temperature‐sensitive species (O. glaber and E. falcarius). Furthermore, while historical isolation was the only factor explaining genetic structure in the more temperature‐sensitive species, lithology‐controlled landscape configuration also played an important role for those species with more lithology‐determined ranges (O. notabilis and N. baeticus). Main conclusions Our data show that lithology is an important constraint on the distribution and range dynamics of endemic Iberian saline‐habitat water beetles, in interaction with climate and long‐term climate change, and overrides the latter in importance for some species. Hence, geological landscape structure and long‐term history may codetermine the overall range and the distribution of genetic lineages in endemic species with specialized edaphic requirements.     

Phylogeography and genetic structure of the red-legged partridge (Alectoris rufa): more evidence for refugia within the Iberian glacial refugium

The Pleistocene climatic oscillations promoted the diversification in avian species during the last glacial period. The red‐legged partridge (Alectoris rufa, Family Phasianidae) has a large natural distribution extending from the Mediterranean to humid temperate zones. However, the genetic structure for this species is unknown. The present study investigates the phylogeography, genetic structure and demographic history of A. rufa across its distribution, employing both mitochondrial DNA control region sequences and nuclear microsatellite loci. Our results propose that this species was greatly affected by Pleistocene glaciations. The mismatch analyses suggest that the current populations resulted from post‐glacial expansion and subsequent differentiation resulting in five diagnosable genetic clusters: Southwestern, Central‐eastern, Northwestern, Balearic and French and Italian. Further, we found evidence of three glacial refugia within the currently recognized Iberian glacial refugium. The intraspecific structure revealed by both maternal and biparental phylogeographic analyses was not resolved in the phylogenetic analyses. Based on all considerations, we recommended that five management units be recognized.     

Mitochondrial DNA variation and the evolutionary history of the Mediterranean species of Cicada L. (Hemiptera,Cicadoidea)

Cicadas are widely distributed in the Mediterranean area and are mainly distinguished by male acoustic signals, which act as specific mate recognition systems. Within the genus Cicada L. several species have diverged in their calling songs without showing external morphological differences, so acoustic recordings and genetic studies are particularly useful for systematic, biogeography and evolutionary studies. This study assesses sequence variation in closely related species of Cicada from the Mediterranean area, using domain III of the 12S rRNA mitochondrial gene in order to determine the phylogenetic relationships and the evolutionary history of this group, as well as the population structure of the two most common species, C. orni and C. barbara. Five distinct haplogroups were identified, C. orni, C. barbara, C. mordoganensis, C. cretensis and C. lodosi, each corresponding to a distinct evolutionary group. C. barbara was the most divergent species within this group, while C. orni and C. mordoganensis were the most similar. The population structure and demographic parameters of the species were not completely resolved. However, there is evidence for the separation of the C. orni Greek populations from the rest of Europe and also for demographic expansions probably related to Pleistocene climate changes. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 266–288.     

The Quaternary evolutionary history,potential distribution dynamics,and conservation implications for a Qinghai–Tibet Plateau endemic herbaceous perennial,Anisodus tanguticus (Solanaceae)

Various hypotheses have been proposed about the Quaternary evolutionary history of plant species on the Qinghai–Tibet Plateau (QTP), yet only a handful of studies have considered both population genetics and ecological niche context. In this study, we proposed and compared climate refugia hypotheses based on the phylogeographic pattern of Anisodus tanguticus (three plastid DNA fragments and nuclear internal transcribed spacer regions from 32 populations) and present and past species distribution models (SDMs). We detected six plastid haplotypes in two well‐differentiated lineages. Although all haplotypes could be found in its western (sampling) area, only haplotypes from one lineage occurred in its eastern area. Meanwhile, most genetic variations existed between populations (F_ST = 0.822). The SDMs during the last glacial maximum and last interglacial periods showed range fragmentation in the western area and significant range contraction in the eastern area, respectively, in comparison with current potential distribution. This species may have undergone intraspecific divergence during the early Quaternary, which may have been caused by survival in different refugia during the earliest known glacial in the QTP, rather than geological isolation due to orogenesis events. Subsequently, climate oscillations during the Quaternary resulted in a dynamic distribution range for this species as well as the distribution pattern of its plastid haplotypes and nuclear genotypes. The interglacial periods may have had a greater effect on A. tanguticus than the glacial periods. Most importantly, neither genetic data nor SDM alone can fully reveal the climate refugia history of this species. We also discuss the conservation implications for this important Tibetan folk medicine plant in light of these findings and SDMs under future climate models. Together, our results underline the necessity to combine phylogeographic and SDM approaches in future investigations of the Quaternary evolutionary history of species in topographically complex areas, such as the QTP.     

A phylogenetic view on skull size and shape variation in the smooth newt (Lissotriton vulgaris,Caudata, Salamandridae)

In this study, we explore skull size and shape variation in the smooth newt, a taxon with substantial morphological differentiation and complex phylogeographic relations. By projecting phylogenies into the morphospace of the skull shape, we explore the variation in and differentiation of this complex morphological structure within a phylogenetic framework. For these analyses, we used a dataset that covers the most southern part of the species’ distribution range, including all conventionally recognized subspecies. The study revealed different patterns of divergence in skull shape between sexes, which is paralleled by intraspecific differentiation. The divergence in dorsal skull shape is concordant with the phylogenetic divergence, as the most diverged clades of the smooth newt (Lissotriton vulgaris kosswigi and Lissotriton vulgaris lantzi) exhibit a skull shape that significantly diverges from the smooth newt’s mean shape configuration. The results of this study also indicate that ventral skull portion, which is more directly related to feeding and foraging, shows higher variation between populations than dorsal skull portion, which appears to be less variable and phylogenetically informative.     

Cranial shape variation and molecular phylogenetic structure of crested newts (Triturus cristatus superspecies: Caudata,Salamandridae) in the Balkans

In the present study, we investigated the degree of congruence between phylogeny, as inferred from mitochondrial (mt)DNA sequences, and cranium shape variation of crested newts (Triturus cristatus superspecies) in the Balkans. These newts belong to four phylogenetic clades defined by mtDNA analysis, and significantly differed in cranial shape. Allometry explained a high percentage of shape variation in crested newts. The clade‐specific allometric slopes significantly diverged for both the ventral cranium and dorsal cranium, indicating that differences in shape between clades could not be a simple consequence of their difference in size. The analysis of hierarchical and spatial variation showed similarity in the patterns of global and spatially localized hierarchical variation of cranial shape. We also found significant congruence between the pattern of cranial shape variation and molecular phylogeny. The differences in morphology of Triturus dobrogicus in comparison to other crested newt clades, including marked differences in cranium shape, is discussed in the context of the evolution and ecology of crested newts. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 348–360.     

Quaternary range dynamics of ecologically divergent species (Edraianthus serpyllifolius and E. tenuifolius,Campanulaceae) within the Balkan refugium

Aim We investigated Quaternary range dynamics of two closely related but ecologically divergent species (cold‐tolerant Edraianthus serpyllifolius and thermophilic Edraianthus tenuifolius) with overlapping distribution ranges endemic to the western Balkan Peninsula, an important yet understudied Pleistocene refugium. Our aims were: to test predictions of the ‘refugia‐within‐refugia’ model of strong genetic subdivisions due to population isolation in separate refugia; to explore whether two ecologically divergent species reacted differently to Pleistocene climatic fluctuations; and to test predictions of the displacement refugia model of stronger differentiation among populations in the thermophilic E. tenuifolius compared with the cold‐tolerant E. serpyllifolius. Location The western Balkan Peninsula. Methods We gathered amplified fragment‐length polymorphism (AFLP) data and plastid DNA sequences from two to five individuals from 10 populations of E. serpyllifolius and 22 populations of E. tenuifolius, spanning their entire respective distribution areas. AFLP data were analysed using a Bayesian clustering approach and a distance‐based network approach. Plastid sequences were used to depict relationships among haplotypes in a statistical parsimony network, and to obtain age estimates in a Bayesian framework. Results In E. serpyllifolius, both AFLP and plastid sequence data showed clear geographic structure. Western populations showed high AFLP diversity and a high number of rare fragments. In E. tenuifolius, both markers congruently identified a major phylogeographic split along the lower Neretva valley in central Dalmatia. The most distinct and earliest diverging chloroplast DNA (cpDNA) haplotypes were found further south in the south‐easternmost populations. North‐western populations, identified as a separate cluster by Bayesian clustering, were characterized by low genetic diversity and a low number of rare AFLP markers. Main conclusions Clear evidence for multiple Pleistocene refugia is found not only in the high‐elevation E. serpyllifolius, but also in the lowland E. tenuifolius, despite the lack of obvious dispersal barriers, in line with the refugia‐within‐refugia model. Genealogical relationships and genetic diversity patterns support the hypothesis that cold‐adapted E. serpyllifolius responded to climatic oscillations mostly by elevational range shifts, whereas thermophilic E. tenuifolius did so mainly by latitudinal range shifts, with different phases (and probably extents) of range expansion. In contrast to the displacement refugia hypothesis, the two elevationally differentiated species do not differ in their genetic diversity.