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

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

Phylogeographic structuring and volant mammals: the case of the pallid bat (Antrozous pallidus)

Aim To examine the phylogeographic pattern of a volant mammal at the continental scale. The pallid bat (Antrozous pallidus) was chosen because it ranges across a zone of well‐studied biotic assemblages, namely the warm deserts of North America. Location The western half of North America, with sites in Mexico, the United States, and Canada. Methods PCR amplification and sequencing of the mitochondrial control region was performed on 194 pallid bats from 36 localities. Additional sequences at the cytochrome‐b locus were generated for representatives of each control‐region haplotype. modeltest was used to determine the best set of parameters to describe each data set, which were incorporated into analyses using paup *. Statistical parsimony and measurements of population differentiation (amova , F_ST) were also used to examine patterns of genetic diversity in pallid bats. Results We detected three major lineages in the mitochondrial DNA of pallid bats collected across the species range. These three major clades have completely non‐overlapping geographic ranges. Only 6 of 80 control‐region haplotypes were found at more than a single locality, and sequences at the more conserved cytochrome‐b locus revealed 37 haplotypes. Statistical parsimony generated three unlinked networks that correspond exactly to clades defined by the distance‐based analysis. On average there was c. 2% divergence for the combined mitochondrial sequences within each of the three major clades and c. 7% divergence between each pair of clades. Molecular clocks date divergence between the major clades at more than one million years, on average, using the faster rates, and at more than three million years using more conservative rates of evolution. Main conclusions Divergent haplotypic lineages with allopatric distributions suggest that the pallid bat has responded to evolutionary pressures in a manner consistent with other taxa of the American southwest. These results extend the conclusions of earlier studies that found the genetic structuring of populations of some bat species to show that a widespread volant species may comprise a set of geographically replacing monophyletic lineages. Haplotypes were usually restricted to single localities, and the clade showing geographic affinities to the Sonoran Desert contained greater diversity than did clades to the east and west. While faster molecular clocks would allow for glacial cycles of the Pleistocene as plausible agents of diversification of pallid bats, evidence from co‐distributed taxa suggests support for older events being responsible for the initial divergence among clades.     

Phylogeography and the origins of range disjunctions in a north temperate fish,the pygmy whitefish (Prosopium coulterii), inferred from mitochondrial and nuclear DNA sequence analysis

Aim To investigate the degree of phylogeographical divergence within pygmy whitefish (Prosopium coulterii) and to test hypotheses concerning the origin of disjunct populations within North America. Location North America from western Alaska to Lake Superior. Methods Mitochondrial (ATPase subunit VI) and nuclear (ITS‐1, ITS‐2) DNA sequence variation was assessed across the species’ North American range to test for the existence of distinct phylogeographical groupings of pygmy whitefish associated with known glacial refugia. Coalescent simulations of the mitochondrial DNA (mtDNA) data were used to test hypotheses of population structure. Results This species is composed of two monophyletic mitochondrial clades across its North American range. The two mtDNA clades differed by an average 3.3% nucleotide sequence divergence. These clades were also distinguished by ITS‐2, but the relationships among lineages were not resolved by the ITS‐1 analysis. Coalescent analyses rejected the null hypothesis of the current disjunct distributions being a result of fragmentation of a single widespread ancestral lineage across a variety of effective population sizes and divergence times. Main conclusions The current range disjunctions of pygmy whitefish in North America probably resulted from isolation, genetic divergence, and selective dispersal from at least two major Pleistocene glacial refugia: Beringia and Cascadia. More recent isolation and dispersal from an upper Mississippi refugium is suggested by relationships within one of the clades and by distributional evidence from co‐distributed species. The Beringian and Cascadian refugia have played major roles in the zoogeography of Nearctic temperate aquatics, but the roles of smaller refugia appear more variable among other species.     

Phylogeographic diversification of antelope squirrels (Ammospermophilus) across North American deserts

We investigated the biogeographic history of antelope squirrels, genus Ammospermophilus, which are widely distributed across the deserts and other arid lands of western North America. We combined range‐wide sampling of all currently recognized species of Ammospermophilus with a multilocus data set to infer phylogenetic relationships. We then estimated divergence times within identified clades of Ammospermophilus using fossil‐calibrated and rate‐calibrated molecular clocks. Lastly, we explored generalized distributional changes of Ammospermophilus since the last glacial maximum using species distribution models, and assessed responses to Quaternary climate change by generating demographic parameter estimates for the three wide‐ranging clades of A. leucurus. From our phylogenetic estimates we inferred strong phylogeographic structure within Ammospermophilus and the presence of three well‐supported major clades. Initial patterns of historical divergence were coincident with dynamic alterations in the landscape of western North America, and the formation of regional deserts during the Late Miocene and Pliocene. Species distribution models and demographic parameter estimates revealed patterns of recent population expansion in response to glacial retreat. When combined with evidence from co‐distributed taxa, the historical biogeography of Ammospermophilus provides additional insight into the mechanisms that impacted diversification of arid‐adapted taxa across the arid lands of western North America. We propose species recognition of populations of the southern Baja California peninsula to best represent our current understanding of evolutionary relationships among genetic units of Ammospermophilus. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 949–967.     

Climate change and evolution of the New World pitviper genus Agkistrodon (Viperidae)

Aim We derived phylogenies, phylogeographies, and population demographies for two North American pitvipers, Agkistrodon contortrix (Linnaeus, 1766) and A. piscivorus (Lacépède, 1789) (Viperidae: Crotalinae), as a mechanism to evaluate the impact of rapid climatic change on these taxa. Location Midwestern and eastern North America. Methods We reconstructed maximum parsimony (MP) and maximum likelihood (ML) relationships based on 846 base pairs of mitochondrial DNA (mtDNA) ATPase 8 and ATPase 6 genes sequenced over 178 individuals. We quantified range expansions, demographic histories, divergence dates and potential size differences among clades since their last period of rapid expansion. We used the Shimodaira–Hasegawa (SH) test to compare our ML tree against three biogeographical hypotheses. Results A significant SH test supported diversification of A. contortrix from northeastern Mexico into midwestern–eastern North America, where its trajectory was sundered by two vicariant events. The first (c. 5.1 Ma) segregated clades at 3.1% sequence divergence (SD) along a continental east–west moisture gradient. The second (c. 1.4 Ma) segregated clades at 2.4% SD along the Mississippi River, coincident with the formation of the modern Ohio River as a major meltwater tributary. A single glacial refugium was detected within the Apalachicola region of southeastern North America. Significant support was also found for a hypothesis of trans‐Gulf rafting by the common ancestor of A. piscivorus from eastern Mexico (possibly the Yucatan Peninsula) to northern Florida. There, a Mid–Late Pliocene marine transgression separated it at 4.8% SD from mainland North America. Significant range expansions followed compressive glacial effects in three (of four) A. contortrix clades and in two (of three) A. piscivorus clades, with the Florida A. piscivorus clade exhibiting significant distributional stasis. Main conclusions Pliocene glaciations, rapidly developing western aridity, and Pleistocene glacial meltwaters seemingly led to the diversification of A. contortrix and A. piscivorus in North America. Both species were pushed southwards by Pleistocene climate change, with subsequent northward expansions uninhibited topographically. The subspecific taxonomy used for A. contortrix and A. piscivorus today, however, appear non‐representative. The monophyletic Florida subspecies of A. piscivorus may be a distinct species (at 4.8% SD), whereas two western subspecies of A. contortrix also appear to constitute a single distinct species, pending additional analyses. We conclude that both species of Agkistrodon can be used as suitable ectothermic models to gauge impacts of future climate change.     

Differential regional response of the bushy‐tailed woodrat (Neotoma cinerea) to late Quaternary climate change

Aim To reconstruct the regional biogeographical history of the bushy‐tailed woodrat, Neotoma cinerea (Rodentia: Cricetidae), across its distribution using multiple sources of information, including genetic data, ecological niche models and the palaeorecord. Location Western North America. Methods We analysed complete cytochrome b gene (1143 bp) sequences from 182 specimens of N. cinerea using Bayesian and coalescent methods to infer phylogenetic relationships, time of major divergences, and recent demographic trends. For comparison, we developed clade‐specific ecological niche models for groups of interest and analysed spatial trends of N. cinerea in the palaeorecord as well as temporal frequency trends across strata of individual palaeomiddens. Results We found two largely allopatric clades within N. cinerea, with several regionally distinct subclades showing contrasting recent population dynamics. Niche models showed consistent habitat at the Last Glacial Maximum (LGM) and modern times in the Rocky Mountains and northern United States, while the Great Basin may have been markedly less suitable at the LGM than today. The palaeorecord showed great spatio‐temporal variability in the presence of N. cinerea, but documents broad‐scale patterns of occupancy and regional population trends. Main conclusions The Quaternary dynamics and evolutionary history of N. cinerea appear to have been shaped by both vicariant events associated with geographical barriers and the availability of suitable habitat through time. Divergence of the two major clades dates to the Pliocene–Pleistocene transition, with clades separated by the Green and Colorado rivers and northern Rocky Mountain Pleistocene glaciations. We found largely concordant genetic, niche model and palaeorecord patterns suggesting long‐term population stability in the Rocky Mountains, while extant clades in the Great Basin and far north appear to have expanded or re‐expanded into these regions relatively recently. Furthermore, disjunct haplotype distributions, regional demographic history, and historical distribution of suitable habitat suggest that the Great Basin has been a particularly dynamic region.     

Inter‐basin exchange and repeated headwater capture across the Sierra Madre Occidental inferred from the phylogeography of Mexican stonerollers

Aim Geomorphic evolution of river basins can shape the structure and diversity of aquatic communities, but understanding the biological significance of basin evolution can be challenging in semi‐arid regions with ephemeral or endorheic conditions and complex drainage configurations such as the Sierra Madre Occidental (SMO) in North America. In this study, we characterized range‐wide patterns of genetic variation in the Mexican stoneroller (Campostoma ornatum) to infer how orogenic and erosional influences on river basin connectivity have given rise to the diverse and largely endemic freshwater communities across the SMO region. Location Twelve drainage basins across northern Mexico and the south‐western United States, centred on the SMO. Methods  We collected 202 specimens from 98 localities across the range of C. ornatum. We performed phylogenetic analyses of DNA sequences from one mitochondrial (cytochrome b) and one nuclear (intron S7) gene. Phylogenetic trees were estimated for each data set using maximum likelihood and Bayesian inference. Results Phylogenetic analyses consistently resolved a monophyletic C. ornatum composed of multiple evolutionary lineages within two markedly divergent clades that differentiate northern drainages from southern drainages in the SMO region. Within‐clade patterns of divergence corresponded to fine‐scale geographic structure within and among SMO drainage basins. However, the geographic distribution of evolutionary lineages within the northern and southern clades did not always correspond to the geographic configuration of drainage basins. Some subclades encompassed multiple drainages, and individuals from a single drainage were sometimes recovered in multiple subclades. Main conclusions Our findings indicate that a common ancestor of Mexican Campostoma is likely to have entered north‐west Mexico through an ancient Rio Grande system that extended as far south as the Rio Nazas and Rio Aguanaval. The geographic orientation of the two strongly divergent clades recovered within C. ornatum provides evidence of long‐standing isolation of southern basins from northern basins within the ancestral Rio Grande system, possibly due to the combined influence of tectonic events and increasing regional aridity. Geographic patterns of genetic variation also provide evidence of range expansion from Atlantic to Pacific drainages due to drainage evolution and river capture events, as well as further inter‐basin exchange via more recent headwater capture events, hydrological connections and possible anthropogenic introductions.     

Fig trees at the northern limit of their range: the distributions of cryptic pollinators indicate multiple glacial refugia

Climatic oscillations during the last few million years had well‐documented effects on the distributions and genomes of temperate plants and animals, but much less is known of their impacts on tropical and subtropical species. In contrast to Europe and North America, ice‐sheets did not cover most of China during glacial periods, and the effects of glacial cycles were less dramatic. Fig trees are a predominantly tropical group pollinated by host‐specific fig wasps. We employed partial mitochondrial COI (918 bp) and nuclear ITS2 (462 bp) gene sequences to investigate the genetic structure and demographic histories of the wasps that pollinate the subtropical Ficus pumila var. pumila in Southeastern China. Deep genetic divergence in both mitochondrial (7.2–11.6%) and nuclear genes (1.6–2.9%) indicates that three pollinator species are present and that they diverged about 4.72 and 6.00 Myr bp . This predates the Quaternary ice ages, but corresponds with the formation of the Taiwan Strait and uplifting of the Wuyi–Xianxia Mountains. The three pollinators have largely allopatric distribution patterns in China and display different postglacial demographic histories. Wiebesia spp. 1 and 2 occupy, respectively, the northern and southern regions of the mainland host range. Their populations both underwent significant postglacial spatial expansions, but at different times and at different rates. Wiebesia sp. 3 is largely restricted to northern islands and shows less evidence of recent population expansion. Their mainly allopatric distributions and different demographic histories are consistent with host plant postglacial expansion from three distinct refugia and suggest one mechanism whereby fig trees gain multiple pollinators.     

Molecular phylogeny of the Eremias persica complex of the Iranian plateau (Reptilia: Lacertidae), based on mtDNA sequences

The Persian racerunner Eremias persica Blanford, 1875 is confined to the Iranian plateau, and forms one of the most widespread but rarely studied species of the family Lacertidae. With many local populations inhabiting a variety of habitats, and exhibiting considerable morphological, genetic, and ecological variations, it represents a species complex. We analysed sequences of mitochondrial cytochrome b and 12S ribosomal RNA (rRNA) genes derived from 13 geographically distant populations belonging to the E. persica complex. Using our knowledge of palaeogeographical events, a molecular clock was calibrated to assess the major events in fragmentation, radiation, and intraspecific variation. The sequence data strongly support a basal separation of the highland populations of western Iran from those of the open steppes and deserts, occurring in the east. The subsequent radiation, fragmentation, and evolution of these major assemblages have led to several discernable geographical lineages across the wide area of the Iranian plateau. The results indicate a middle‐Miocene origin for the clade as a whole. The first split, isolating the western and eastern clades, appears to have occurred 11–10 Mya. Further fragmentations and divergence within the major clades began about 8 Mya, with an evolutionary rate of 1.6% sequence divergence per million years among the lineages in the genes studied (combined data set). Molecular and morphological data strongly support a taxonomic revision of this species complex. At least four of the discovered clades should be raised to species, and two to subspecies, rank. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 641–660.     

Population genomic evidence for multiple Pliocene refugia in a montane‐restricted harvestman (Arachnida,Opiliones, Sclerobunus robustus) from the southwestern United States

The integration of ecological niche modelling into phylogeographic analyses has allowed for the identification and testing of potential refugia under a hypothesis‐based framework, where the expected patterns of higher genetic diversity in refugial populations and evidence of range expansion of nonrefugial populations are corroborated with empirical data. In this study, we focus on a montane‐restricted cryophilic harvestman, Sclerobunus robustus, distributed throughout the heterogeneous Southern Rocky Mountains and Intermontane Plateau of southwestern North America. We identified hypothetical refugia using ecological niche models (ENMs) across three time periods, corroborated these refugia with population genetic methods using double‐digest RAD‐seq data and conducted population‐level phylogenetic and divergence dating analyses. ENMs identify two large temporally persistent regions in the mid‐latitude highlands. Genetic patterns support these two hypothesized refugia with higher genetic diversity within refugial populations and evidence for range expansion in populations found outside hypothesized refugia. Phylogenetic analyses identify five to six genetically divergent, geographically cohesive clades of S. robustus. Divergence dating analyses suggest that these separate refugia date to the Pliocene and that divergence between clades pre‐dates the late Pleistocene glacial cycles, while diversification within clades was likely driven by these cycles. Population genetic analyses reveal effects of both isolation by distance (IBD) and isolation by environment (IBE), with IBD more important in the continuous mountainous portion of the distribution, while IBE was stronger in the populations inhabiting the isolated sky islands of the south. Using model‐based coalescent approaches, we find support for postdivergence migration between clades from separate refugia.     

Historical biogeography of lowland species of toads (Bufo) across the Trans-Mexican Neovolcanic Belt and the Isthmus of Tehuantepec

Aim In this study, we investigate phylogeographic structure in two different species groups of lowland toads. First, we further investigate strict parapatry of the Pliocene‐vicariant Bufo valliceps/B. nebulifer species pair. Secondly, we test for similar phylogeographic structure in the distantly related toad B. marinus, a species we hypothesize will show a Pleistocene dispersal across the same area. Location The eastern extension of the Trans‐Mexican Neovolcanic Belt (TMNB) contacts the Atlantic Coast in central Veracruz, Mexico. Although it is not a massive structure at this eastern terminus, the TMNB has nonetheless effected vicariance and subsequent speciation in several groups of animals. The Isthmus of Tehuantepec unites the North American continent with Nuclear Central America and is also known to be a biogeographic barrier for many taxa. Methods We use sequence data from two mitochondrial DNA genes (c. 550 base‐pairs (bp) of 16S and c. 420 bp of cyt b) from 58 individuals of the B. valliceps/nebulifer complex, collected from 24 localities. We also present homologous sequence data from 23 individuals of B. marinus, collected from 12 localities. We conduct maximum‐parsimony, maximum‐likelihood and Bayesian analyses to investigate phylogeographic structure. We then use parsimony‐ and likelihood‐based topology tests to assess alternative phylogenetic hypotheses and use a previously calibrated molecular rate of evolution to estimate dates of divergence. Results Our results further define the parapatric contact zone across the TMNB between the Pliocene‐vicariant sister species B. valliceps and B. nebulifer. In contrast, phylogenetic structure among populations of B. marinus across the TMNB is much shallower, suggesting a more recent Pleistocene dispersal in this species. In addition, we found phylogeographic structure associated with the Isthmus of Tehuantepec in both species groups. Main conclusions The existence of a Pliocene–Pleistocene seaway across the Isthmus of Tehuantepec has been controversial. Our data depict clades on either side of the isthmus within two distinct species (B. valliceps and B. marinus), although none of the clades associated with the isthmus, for either species, are reciprocally monophyletic. In the B. valliceps/B. nebulifer complex, the TMNB separation appears to predate the isthmian break, whereas in B. marinus dispersal across the TMNB has occurred subsequent to the presence of a barrier at the Isthmus of Tehuantepec.     

Mitochondrial phylogeography and subspecies of the wide‐ranging sub‐Saharan leopard tortoise Stigmochelys pardalis (Testudines: Testudinidae) – a case study for the pitfalls of pseudogenes and GenBank sequences

The leopard tortoise (Stigmochelys pardalis) is the most widely distributed sub‐Saharan tortoise species, with a range extending from the Horn of Africa all over eastern Africa to the Republic of South Africa, Namibia and southernmost Angola. Using 1938 bp of mitochondrial DNA (cyt b gene, partial ND4 gene plus adjacent tRNA genes) from a nearly range‐wide sampling, we examined its phylogeographic structure and compared our findings with previously published GenBank sequences. We identified seven major clades that are largely parapatrically distributed. A few records of distinct haplotypes at the same locality or in close proximity could be the result of translocation of tortoises by man. The greatest diversity occurs in the south of the species’ range, with five out of the seven clades. Testing for isolation‐by‐distance suggests that the observed phylogeographic structure is the result of restricted geographical gene flow and not of historical vicariance. This is in sharp contrast to wide‐ranging thermophilic reptiles from the western Palaearctic, whose phylogeographic structure was significantly shaped by Pleistocene range interruptions, but also by earlier dispersal and vicariant events. Most cyt b sequences of S. pardalis from GenBank turned out to be nuclear pseudogenes, or to be of chimerical origin from such pseudogenes and authentic mitochondrial sequences, which argues for caution regarding uncritical usage of GenBank sequences. The recent revalidation of the two subspecies of S. pardalis was based on such a chimerical sequence that was erroneously identified with the subspecies S. p. babcocki. Furthermore, according to our data, the distribution of mitochondrial clades does match neither the traditional subspecies ranges nor the pronounced geographical size variation of leopard tortoises. We conclude that there is no rationale for recognizing subspecies within S. pardalis.     

Complete taxon sampling of the avian genus Pica (magpies) reveals ancient relictual populations and synchronous Late‐Pleistocene demographic expansion across the Northern Hemisphere

Previous studies have suggested that bird populations in east Asia were less affected by Pleistocene climatic fluctuations than those in Europe and North America. However, this is mainly based on comparisons among species. It would be more relevant to analyse geographical populations of widespread species or species complexes. We analyzed two mitochondrial genes and two nuclear introns for all taxa of Pica to investigate 1) which Earth history factors have shaped the lineage divergence, and 2) whether different geographical populations were differently affected by the Pleistocene climatic changes. Our mitochondrial tree recovered three widespread lineages, 1) in east Asia, 2) across north Eurasia, and 3) in North America, respectively, with three isolated lineages in northwest Africa, Arabia and the Qinghai‐Tibet Plateau, respectively. Divergences among lineages took place 1.4–3.1 million yr ago. The northwest African population was sister to the others, which formed two main clades. In one of these, Arabia was sister to Qinghai‐Tibet, and these formed the sister clade to the east Asia clade. The other main clade comprised the North American and north Eurasian clades. There was no or very slight structure within these six geographical clades, including a lack of differentiation between the two North American species black‐billed magpie P. hudsonia and yellow‐billed magpie P. nutalli. Demographic expansion was recorded in the three most widespread lineages after 0.06 Ma. Asymmetric gene flow was recorded in the north Eurasian clade from southwestern Europe eastward, whereas the east Asian clade was rooted in south central China. Our results indicate that the fragmentation of the six clades of Pica was related to climatic cooling and aridification during periods of the Pliocene–Pleistocene. Populations on both sides of the Eurasian continent were similarly influenced by the Pleistocene climate changes and expanded concomitantly with the expansion of steppes. Based on results we also propose a revised taxonomy recognising seven species of Pica.     

The geography of speciation in narrow‐range endemics of the ‘Haenydra’ lineage (Coleoptera,Hydraenidae, Hydraena)

Aim We test whether species of western Mediterranean aquatic Coleoptera of the ‘Haenydra’ lineage (Hydraenidae, Hydraena) originated through: (1) successive periods of dispersal and speciation, (2) range fragmentation by random vicariance, or (3) range fragmentation by geographic isolation owing to a general reduction of population density. Location Europe. Methods To discriminate between scenarios we use contrasting predictions of the relationship between phylogenetic and geographic distance. The phylogeny was based on 3 kb of four mitochondrial and two nuclear gene fragments of about half of the known species of ‘Haenydra’, including most western Mediterranean taxa. Divergences were estimated using a molecular clock. The relationship between phylogenetic and geographic distance was tested using bivariate plots, Mantel tests and comparison of the observed phylogeny with the one minimizing geographic distances between species, as measured using Euclidean minimum spanning trees (EMSTs). Results The monophyly of ‘Haenydra’ was strongly supported, although its phylogenetic placement was not resolved. ‘Haenydra’ was estimated to be of late Miocene age, with most species originating during the Pleistocene. In two clades (Hydraena tatii and Hydraena emarginata clades) there was a significant association between geographic and phylogenetic distance, and the reconstructed phylogeny was identical to that obtained through the EMST, demonstrating a strong non‐randomness of the geographic distribution of the species. In two other clades (Hydraena iberica and Hydraena bitruncata clades) there was no association between geographic and phylogenetic distance, and the observed phylogeny was not the one minimizing geographic distances. In one of the clades this seems to be due to a secondary, recent range expansion of one species (H. iberica), which erased the geographic signal of their distributions. Main conclusions We show that it is possible to obtain strong evidence of stasis of the geographic ranges of narrow‐range endemic species through the study of their phylogenetic relationships and current distributions. In at least two of the studied clades, current species seem to have originated through the fragmentation of a more widely distributed species, without further range movements. A process of range expansion and fragmentation may have occurred repeatedly within the ‘Haenydra’ lineage, contributing to the accumulation of narrow‐range endemics in Mediterranean Pleistocene refugia.     

Surviving glacial ages within the Biotic Gap: phylogeography of the New Zealand cicada Maoricicada campbelli

Aim New Zealand is an ideal location in which to investigate the roles of landscape and climate change on speciation and biogeography. An earlier study of the widespread endemic cicada Maoricicada campbelli (Myers) found two phylogeographically distinguishable major clades – northern South Island plus North Island (northern‐SI + NI) and Otago. These two clades appeared to have diverged on either side of an area of the South Island known as the Biotic Gap. We sampled more intensively to test competing theories for this divergence. We aimed to discover if M. campbelli had survived within the Biotic Gap during recent glacial maxima, and if predicted areas of secondary contact between the two major clades existed. Location New Zealand. Methods We analysed mitochondrial DNA sequences (1520 bp; 212 individuals; 91 populations) using phylogenetic (maximum likelihood, Bayesian), population genetic (analysis of molecular variance) and molecular dating methods (Bayesian relaxed clock with improved priors). Results We found strong geographical structuring of genetic variation. Our dating analyses suggest that M. campbelli originated 1.83–2.58 Ma, and split into the two major clades 1.45–2.09 Ma. The main subclades in the northern‐SI + NI clade arose almost simultaneously at 0.69–1.03 Ma. Most subclades are supported by long internal branches and began to diversify 0.40–0.78 Ma. We found four narrow areas of secondary contact between the two major clades. We also found a difference between calling songs of the Otago vs. northern‐SI + NI clades. Main conclusions Phylogeographical patterns within M. campbelli indicate an early Pleistocene split into two major clades, followed by late Pleistocene range expansion and in situ population differentiation of subclades. The northern‐SI + NI clade diversified so rapidly that the main subclade relationships cannot be resolved, and we now have little evidence for a disjunction across the Biotic Gap. Structure within the main subclades indicates rapid divergence after a common bottlenecking event, perhaps attributable to an extremely cold glacial maximum at c. 0.43 Ma. Clade structure and dating analyses indicate that M. campbelli survived in many refugia during recent glacial maxima, including within the Biotic Gap. The narrow overlap between the two major clades is attributed to recent contact during the current interglacial and slow gene diffusion. The two major clades appear to be in the early stages of speciation based on genetic and behavioural differences.     

INFERRING THE PAST AND PRESENT CONNECTIVITY ACROSS THE RANGE OF A NORTH AMERICAN LEAF BEETLE: COMBINING ECOLOGICAL NICHE MODELING AND A GEOGRAPHICALLY EXPLICIT MODEL OF COALESCENCE

The leaf beetle Chrysomela aeneicollis occurs across Western North America, either at high elevation or in small, isolated populations along the coast, and thus has a highly fragmented distribution. DNA sequence data (three loci) were collected from five regions across the species range. Population connectivity was examined using traditional ecological niche modeling, which suggested that gene flow could occur among regions now and in the past. We developed geographically explicit coalescence models of sequence evolution that incorporated a two‐dimensional representation of the hypothesized ranges suggested by the niche‐modeling estimates. We simulated sequence data according to these models and compared them to observed sequences to identify most probable scenarios regarding the migration history of C. aeneicollis. Our results disagreed with initial niche‐modeling estimates by clearly rejecting recent connectivity among regions, and were instead most consistent with a long period of range fragmentation, extending well beyond the last glacial maximum. This application of geographically explicit models of coalescence has highlighted some limitations of the use of climatic variables for predicting the present and past range of a species and has explained aspects of the Pleistocene evolutionary history of a cold‐adapted organism in Western North America.     

DNA barcoding provides support for a cryptic species complex within the globally distributed and fishery important opah (Lampris guttatus)

The cornerstone of fisheries management relies on a solid taxonomic base and an understanding of how animals can be grouped into coherent management units. Surprisingly, little is known about the basic biology and ecology of opah (Lampris guttatus), a globally distributed species that is commercially exploited and regionally common in the North Pacific. Recent efforts to collect life history data on this species uncovered evidence of two North Pacific morphotypes. Sequencing of the mitochondrial cytochrome c oxidase I gene (655 bp) for these morphotypes and other specimens collected worldwide (n = 480) produced five strongly diverged and well‐supported clades. Additional sequence data from the cytochrome b gene (1141 bp) as well as the nuclear recombination activating gene 1 (1323 bp) corroborated these results, suggesting these five clades probably represent separate species. Our conclusion that opah is a complex of five separate species has implications for management and indicates a need to gather additional data on these poorly understood fishes.     

Phylogeny and biogeography of the hollies (Ilex L., Aquifoliaceae)

The holly genus, Ilex L., in the monogeneric Aquifoliaceae, is the largest woody dioecious genus (>664 spp.), with a near‐cosmopolitan distribution in mesic environments. We constructed a phylogeny based on two nuclear genes, representing 177 species spread across the geographical range, and dated using macrofossil records. The five main clades had a common ancestor in the early Eocene, much earlier than previously suggested. Ilex originated in subtropical Asia and extant clades colonized South America by 30 Ma, North America by 23 Ma, Australia by 8 Ma, Europe by 6 Ma, and Africa by 4 Ma. South and North America were colonized multiple times. Ilex also reached Hawaii (10 Ma) and other oceanic islands. Macrofossil and pollen records show the genus has tracked mesic climates through time and space, and had a wider distribution before late Miocene global cooling. Our phylogeny provides a framework for studies in comparative ecology and evolution.     

Ephemeral Pleistocene woodlands connect the dots for highland rattlesnakes of the Crotalus intermedius group

Aim To test how Pleistocene climatic changes affected diversification of the Crotalus intermedius species complex. Location Highlands of Mexico and the south‐western United States (Arizona). Methods We synthesize the matrilineal genealogy based on 2406 base pairs of mitochondrial DNA sequences, fossil‐calibrated molecular dating, reconstruction of ancestral geographic ranges, and climate‐based modelling of species distributions to evaluate the history of female dispersion. Results The presently fragmented distribution of the C. intermedius group is the result of both Neogene vicariance and Pleistocene pine–oak habitat fragmentation. Most lineages appear to have a Quaternary origin. The Sierra Madre del Sur and northern Sierra Madre Oriental are likely to have been colonized during this time. Species distribution models for the Last Glacial Maximum predict expansions of suitable habitat for taxa in the southern Sierra Madre Occidental and northern Sierra Madre Oriental. Main conclusions Lineage diversification in the C. intermedius group is a consequence of Pleistocene climate cycling. Distribution models for two sister taxa in the northern and southern Sierra Madre Occidental and northern Sierra Madre Oriental during the Last Glacial Maximum provide evidence for the expansion of pine–oak habitat across the Central Mexican Plateau. Downward displacement and subsequent expansions of highland vegetation across Mexico during cooler glacial cycles may have allowed dispersal between highlands, which resulted in contact between previously isolated taxa and the colonization of new habitats.