Identification of glacial refugia in south-eastern North America by phylogeographical analyses of a forest understorey plant, Trillium cuneatum

Aim We examine several hypotheses emerging from biogeographical and fossil records regarding glacial refugia of a southern thermophilic plant species. Specifically, we investigated the glacial history and post‐glacial colonization of a forest understorey species, Trillium cuneatum. We focused on the following questions: (1) Did T. cuneatum survive the Last Glacial Maximum (LGM) in multiple refugia, and (if so) where were they located, and is the modern genetic structure congruent with the fossil record‐based reconstruction of refugia for mesic deciduous forests? (2) What are the post‐glacial colonization patterns in the present geographical range? Location South‐eastern North America. Methods We sampled 45 populations of T. cuneatum throughout its current range. We conducted phylogeographical analyses based on maternally inherited chloroplast DNA (cpDNA haplotypes) and used TCS software to reconstruct intraspecific phylogeny. Results We detected six cpDNA haplotypes, geographically highly structured into non‐overlapping areas. With one exception, none of the populations had mixed haplotype composition. TCS analysis resulted in two intraspecific cpDNA lineages, with one clade subdivided further by shallower diversification. Main conclusions Our investigation revealed that T. cuneatum survived the LGM in multiple refugia, belonging to two (western, eastern) genealogical lineages geographically structured across south‐eastern North America. The western clade is confined to the south‐western corner of T. cuneatum’s modern range along the Lower Mississippi Valley, where fossil records document a major refugium of mesic deciduous forest. For the eastern clade, modern patterns of cpDNA haplotype distribution suggest cryptic vicariance, in the form of forest contractions and subsequent expansions associated with Pleistocene glacial cycles, rather than simple southern survival and subsequent northward colonization. The north–south partitioning of cpDNA haplotypes was unexpected, suggesting that populations of this rather southern thermophilic species may have survived in more northern locations than initially expected based on LGM climate reconstruction, and that the Appalachian Mountains functioned as a barrier to the dispersal of propagules originating in more southern refugia. Furthermore, our results reveal south‐west to north‐east directionality in historical migration through the Valley and Ridge region of north‐west Georgia.     

Genetically diverse but with surprisingly little geographical structure: the complex history of the widespread herb Carex nigra (Cyperaceae)

Aim Based on extensive range‐wide sampling, we address the phylogeographical history of one of the most widespread and taxonomically complex sedges in Europe, Carex nigra s. lat. We compare the genetic structure of the recently colonized northern areas (front edge) and the long‐standing southern areas (rear edge), and assess the potential genetic basis of suggested taxonomic divisions at the rank of species and below. Location Amphi‐Atlantic, central and northern Europe, circum‐Mediterranean mountain ranges, central Siberia, Himalayas. Methods A total of 469 individuals sampled from 83 populations, covering most of the species’ range, were analysed with amplified fragment length polymorphism (AFLP) and chloroplast DNA (cpDNA) markers. Bayesian clustering, principal coordinates analysis, and estimates of diversity and differentiation were used for the analysis of AFLP data. CpDNA data were analysed with statistical parsimony networks and maximum parsimony and Bayesian inference of phylogenetic trees. Results Overall genetic diversity was high, but differentiation among populations was limited. Major glacial refugia were inferred in the Mediterranean Basin and in western Russia; in addition, there may have been minor refugia in the North Atlantic region. In the southern part of the range, we found high levels, but geographically quite poorly structured genetic diversity, whereas the levels of genetic diversity varied among different areas in the north. North American populations were genetically very similar to the European populations. Main conclusions The data are consistent with extensive gene flow, which has obscured the recent history of the taxon. The limited differentiation in the south probably results from the mixing of lineages expanding from several local refugia. Northward post‐glacial colonization resulted in a leading‐edge pattern of low diversity in the Netherlands, Belgium, Scotland and Iceland, whereas the observed high diversity levels in Fennoscandia suggest broad‐fronted colonization from the south as well as from the east. The patterns found in the American populations are consistent with post‐glacial colonization, possibly even with anthropogenic introduction from Europe. Our data also suggest that the tussock‐forming populations of C. nigra, often referred to as a distinct species (Carex juncella), represent an ecotype that has originated repeatedly from different populations with creeping rhizomes.     

History and evolution of alpine plants endemic to the Qinghai-Tibetan Plateau: Aconitum gymnandrum (Ranunculaceae)

How Quaternary climatic oscillations affected range distributions and intraspecific divergence of alpine plants on the Qinghai‐Tibetan Plateau (QTP) remains largely unknown. Here, we report a survey of chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) DNA variation aimed at exploring the phylogeographical history of the QTP alpine endemic Aconitum gymnandrum. We sequenced three cpDNA fragments (rpl20–rps12 intergenic spacer, the trnV intron and psbA‐trnH spacer) and also the nuclear (ITS) region in 245 individuals from 23 populations sampled throughout the species’ range. Two distinct lineages, with eastern and western geographical distributions respectively, were identified from a phylogenetic analysis of ITS sequence variation. Based on a fast substitution rate, these were estimated to have diverged from each other in the early Pleistocene approximately 1.45 Ma. The analysis of cpDNA variation identified nine chlorotypes that clustered into two major clades that were broadly congruent in geographical distribution with the two ITS lineages. The east–west split of cpDNA divergence was supported by an amova which partitioned approximately half of the total variance between these two groups of populations. Analysis of the spatial distribution of chlorotypes showed that each clade was subdivided into two groups of populations such that a total of four population groups existed in the species. It is suggested that these different groups derive from four independent glacial refugia that existed during the Last Glacial Maximum (LGM), and that three of these refugia were located at high altitude on the QTP platform itself at that time. Coalescent simulation of chlorotype genealogies supported both an early Pleistocene origin of the two main cpDNA clades and also the ‘four‐refugia’ hypothesis during the LGM. Two previous phylogeographical studies of QTP alpine plants indicated that such plants retreated to refugia at the eastern/south‐eastern plateau edge during the LGM and/or previous glacial maxima. However, the results for A. gymnandrum suggest that at least some of these cold‐tolerant species may have also survived centrally on the QTP platform throughout the Quaternary.     

Reconstructing the demise of Tethyan plants: climate‐driven range dynamics of Laurus since the Pliocene

Aim Climate changes are thought to be responsible for the retreat and eventual extinction of subtropical lauroid species that covered much of Europe and North Africa during the Palaeogene and early Neogene; little is known, however, of the spatial and temporal patterns of this demise. Herein we calibrate ecological niche models to assess the climatic requirements of Laurus L. (Lauraceae), an emblematic relic from the Tethyan subtropical flora, subsequently using these models to infer how the range dynamics of Laurus were affected by Plio‐Pleistocene climate changes. We also provide predictions of likely range changes resulting from future climatic scenarios. Location The Mediterranean Basin and Macaronesian islands (Canaries, Madeira, Azores). Methods We used a maximum‐entropy algorithm (Maxent) to model the relationship between climate and Laurus distribution over time. The models were fitted both to the present and to the middle Pliocene, based on fossil records. We employed climatic reconstructions for the mid‐Pliocene (3 Ma), the Last Glacial Maximum (21 ka) and a CO₂‐doubling future scenario to project putative species distribution in each period. We validated the model projections with Laurus fossil and present occurrences. Results Laurus preferentially occupied warm and moist areas with low seasonality, showing a marked stasis of its climatic niche. Models fitted to Pliocene conditions successfully predicted the current species distribution. Large suitable areas existed during the Pliocene, which were strongly reduced during the Pleistocene, but humid refugia within the Mediterranean Basin and Macaronesian islands enabled long‐term persistence. Future climate conditions are likely to re‐open areas suitable for colonization north of the current range. Main conclusions The climatic requirements of Laurus remained virtually unchanged over the last 3 Myr. This marked niche conservatism imposed largely deterministic range dynamics driven by climate conditions. This species's relatively high drought tolerance might account for the survival of Laurus in continental Europe throughout the Quaternary whilst other Lauraceae became extinct. Climatic scenarios for the end of this century would favour an expansion of the species's range towards northern latitudes, while severely limiting southern populations due to increased water stress.     

Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

Aim The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post‐glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. Methods As our model system we used Pulsatilla vernalis, a widely distributed European herbaceous plant occurring both in the high‐mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution‐wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment‐length polymorphisms (PCR–RFLPs) (trnH–trnK, trnK–trnK, trnC–trnD, psbC–trnS, psaA–trnS, trnL–trnF) and further sequencing of trnL–trnF and trnH–psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus‐scale cpDNA variation. Results Eleven PCR–RFLP polymorphisms were detected in P. vernalis, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section Pulsatilla was low, but P. vernalis did not share haplotypes with other species. Main conclusions The genetic distinctiveness of P. vernalis populations from the south‐western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high‐mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post‐glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post‐glacial colonization of these two areas, with possible founder effects.     

Phylogeography and palaeodistribution modelling of Nassauvia subgenus Strongyloma (Asteraceae): exploring phylogeographical scenarios in the Patagonian steppe

The Patagonian steppe is an immense, cold, arid region, yet phylogeographically understudied. Nassauvia subgen. Strongyloma is a characteristic element of the steppe, exhibiting a continuum of morphological variation. This taxon provides a relevant phylogeographical model not only to understand how past environmental changes shaped the genetic structure of its populations, but also to explore phylogeographical scenarios at the large geographical scale of the Patagonian steppe. Here, we (1) assess demographic processes and historical events that shaped current geographic patterns of haplotypic diversity; (2) analyze hypotheses of isolation in refugia, fragmentation of populations, and/or colonization of available areas during Pleistocene glaciations; and (3) model extant and palaeoclimatic distributions to support inferred phylogeographical patterns. Chloroplast intergenic spacers, rpl32–trnL and trnQ–5′rps16, were sequenced for 372 individuals from 63 populations. Nested clade analysis, analyses of molecular variance, and neutrality tests were performed to assess genetic structure and range expansion. The present potential distribution was modelled and projected onto a last glacial maximum (LGM) model. Of 41 haplotypes observed, ten were shared among populations associated with different morphological variants. Populations with highest haplotype diversity and private haplotypes were found in central‐western and south‐eastern Patagonia, consistent with long‐term persistence in refugia during Pleistocene. Palaeomodelling suggested a shift toward the palaeoseashore during LGM; new available areas over the exposed Atlantic submarine platform were colonized during glaciations with postglacial retraction of populations. A scenario of fragmentation and posterior range expansion may explain the observed patterns in the center of the steppe, which is supported by palaeomodelling. Northern Patagonian populations were isolated from southern populations by the Chubut and the Deseado river basins during glaciations. Pleistocene glaciations indirectly impacted the distribution, demography, and diversification of subgen. Strongyloma through decreased winter temperatures and water availability in different areas of its range.     

Multiple refugia and barriers explain the phylogeography of the Valais shrew,Sorex antinorii (Mammalia: Soricomorpha)

The aim of the present study was to investigate the genetic structure of the Valais shrew (Sorex antinorii) by a combined phylogeographical and landscape genetic approach, and thereby to infer the locations of glacial refugia and establish the influence of geographical barriers. We sequenced part of the mitochondrial cytochrome b (cyt b) gene of 179 individuals of S. antinorii sampled across the entire species' range. Six specimens attributed to S. arunchi were included in the analysis. The phylogeographical pattern was assessed by Bayesian molecular phylogenetic reconstruction, population genetic analyses, and a species distribution modelling (SDM)‐based hindcasting approach. We also used landscape genetics (including isolation‐by‐resistance) to infer the determinants of current intra‐specific genetic structure. The phylogeographical analysis revealed shallow divergence among haplotypes and no clear substructure within S. antinorii. The starlike structure of the median‐joining network is consistent with population expansion from a single refugium, probably located in the Apennines. Long branches observed on the same network also suggest that another refugium may have existed in the north‐eastern part of Italy. This result is consistent with SDM, which also suggests several habitable areas for S. antinorii in the Italian peninsula during the LGM. Therefore S. antinorii appears to have occupied disconnected glacial refugia in the Italian peninsula, supporting previous data for other species showing multiple refugia within southern refugial areas. By coupling genetic analyses and SDM, we were able to infer how past climatic suitability contributed to genetic divergence of populations. The genetic differentiation shown in the present study does not support the specific status of S. arunchi. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 864–880.     

Refugia within refugia: the case study of a canopy tree (Eurycorymbus cavaleriei) in subtropical China

Aim Eurycorymbus cavaleriei (Lévl.) Rehd. et Hand.‐Mazz. (Sapindaceae) is a Tertiary relict tree endemic to subtropical China. This area is a centre for speciation and evolution within the East Asia biome and one of its most important refugial locations. In this study we aim to elucidate the phylogeographical patterning in E. cavaleriei, in order to identify the locations of the species’ main refugia and the predominant patterns of migration that have led to the contemporary spatial genetic structure of chloroplast variation. Location Subtropical China. Methods We sampled 18 populations of E. cavaleriei throughout its geographical range. Chloroplast DNA (cpDNA) sequence data from two non‐coding regions ((trnS/trnG and pl20/5′‐rps12) were obtained from 170 individuals for phylogeographical analyses. Relationships among cpDNA haplotypes were determined using median‐joining networks. Genetic structure was examined by spatial analysis of molecular variance (SAMOVA). Population differentiation was estimated by G_ST and N_ST statistics. Results Ten distinct haplotypes were identified. The level of differentiation among populations was relatively high (G_ST = 0.817), and N_ST was significantly higher than G_ST (P < 0.05), indicating that strong phylogeographical structure is exhibited by this species. The SAMOVA revealed five diverging groups of related haplotypes, which coincide with major landscape features in this region. Main conclusions The high differentiation among populations of E. cavaleriei may be a combined effect of historical and contemporary processes, such as the low effective population size for the chloroplast genome of a dioecious species, long‐term range fragmentation and limited seed dispersal for the species. Clear‐cut geographical distributions of ancestral haplotypes of the species suggest multiple potential refugia across subtropical China. The identified refugial regions have long been recognized as centres of plant diversity and endemism for China and have also been suggested as glacial refugia for many other plant species. The combination of these factors means that these locations should be considered as the highest priority for inclusion in conservation policies and sustainable forest management strategies for subtropical China.     

Mitochondrial DNA of pre‐last glacial maximum red deer from NW Spain suggests a more complex phylogeographical history for the species

The major climatic oscillations that characterized the Quaternary had a great influence on the evolution and distribution of several species. During cold periods, the distribution of temperate‐adapted species became fragmented with many surviving in southern refugia (Iberian, Italian, and Balkan Peninsulas). Red deer was one of the species that contracted its original range to southern refugia. Currently, two main lineages have been described for the species: western and eastern. We have analyzed fossils pre‐dating the last glacial maximum (LGM) from Liñares cave (NW Spain) that belongs to the peripheral range of the western clade, and fossils from the Danish Holocene belonging to the central part of the same clade. Phylogenetic analyses place our samples in the western clade. However, some specimens from Liñares represent an early split in the tree along with other pre‐LGM western samples from previous studies. Despite low bootstrap values in the Bayesian phylogenies, haplotype networks connect these foreign haplotypes to the eastern clade. We suggest a mixed phylogeographical model to explain this pattern with range expansions from the east during the expansion phase after the cold periods in marine isotope stage 3. We find slight isolation by distance in post‐LGM populations that could be a consequence of the recolonization from southern refugia after the LGM.     

Combining mitochondrial and nuclear evidences: a refined evolutionary history of Erebia medusa (Lepidoptera: Nymphalidae: Satyrinae) in Central Europe based on the COI gene

We aim to infer a combined scenario for the evolution of the Woodland Ringlet, Erebia medusa, in Central Europe based on analyses of part of the mitochondrial cytochrome oxydase subunit I (COI) and a published allozyme data set. We sequenced 529 bp of COI for 158 butterflies from 32 populations from almost the entire western distribution range of the species. We applied population genetic [spatial analysis of molecular variance (samova )] and phylogeographical analyses as well as coalescence simulation to test if the published allozyme scenario supports or contradicts the observed haplotype distribution. We recorded 16 haplotypes of which four represent a total of 82%. samova grouped the 32 populations into four geographically coherent groups: (1) western Central Europe, (2) Central Europe, (3) eastern Central Europe and (iv) western Pannonia. Mismatch distribution analyses and haplotype networks are in coincidence with constant population growth and reveal a relatively shallow phylogeographical structure. We evaluated the level of discordance between population histories and gene trees using Slatkins s and the deep coalescence statistics based on our mtDNA data. These estimators decline the previously published allozyme scenario of survival in different extra‐Mediterranean refugia in Europe with an onset of differentiation at the beginning of the last ice age some 70 000 years ago. However, it supports a refined scenario if we assume an onset of vicariance driven differentiation in these refugia after the end of the middle Würm interstadials some 30 000 years ago. Therefore, the general evolutionary history of this species in Europe apparently is very recent. Most probably, areas adjoining the high mountain regions as Alps and northern Carpathians were of great importance for the late Würm glacial survival of species like Erebia medusa.     

From Mediterranean shores to central Saharan mountains: key phylogeographical insights from the genus Myrtus

Aim The Mediterranean region is often regarded as a crossroads where species of various origins meet. However, the biogeographical relationships between this region and contiguous Saharan, Macaronesian and Irano‐Turanian regions have not been investigated in detail. The aim of this study was to characterize the phylogeography of the circum‐Mediterranean species Myrtus communis and to investigate the origin of isolated central Saharan populations of Myrtus nivellei. Location The distribution ranges of M. communis from Macaronesia to the Irano‐Turanian region (173 sampled populations) and of M. nivellei in the mountains (Hoggar, Tassili n’Ajjer, Immidir, Tibesti) of the central Sahara (23 populations). Methods Nuclear ribosomal DNA (nrDNA) sequences of Myrtaceae were used to root the phylogeny of Myrtus, and to date its crown node, according to a detailed review of the palaeobotanical records used for multiple fossil calibration. Chloroplast DNA (cpDNA) sequences were analysed through the determination of genetic diversity indices and by statistical phylogeography. Results Both cpDNA and nrDNA markers indicated east–west genetic differentiation within M. communis. During the late Miocene, a key vicariance event affected the previous circum‐Mediterranean distribution of Myrtus, leading to the isolation of eastern populations. During the late Miocene or early Pliocene, two clades diverged: one is now scattered in the Mediterranean Basin and adjacent regions, whereas the other evolved in the western Mediterranean region. The differentiation of lineages during the Plio‐Pleistocene occurred mainly in the western part of the Mediterranean Basin, which has been at the origin of migrations towards Macaronesian islands and Saharan mountains. This is one of the first plant phylogeographical studies to report migrations from the Mediterranean to the Sahara. Main conclusions The genus Myrtus has persisted in the Mediterranean region since at least the Neogene and its biogeography reflects the cumulation of the species’ responses to successive palaeoenvironmental changes. The current distribution of the genus Myrtus in the Mediterranean Basin and in isolated areas, such as the Macaronesian islands and Saharan mountains, can be explained by the striking ability of this plant not only to persist locally in various refugia, but also to migrate.     

Multiple periglacial refugia in the Patagonian steppe and post‐glacial colonization of the Andes: the phylogeography of Calceolaria polyrhiza

Aim We perform a phylogeographical study of an endemic Patagonian herbaceous plant to assess whether geographical patterns of genetic variation correspond to in situ Pleistocene survival or to glacial retreat and post‐glacial expansion. We also seek to determine the locations of potential glacial refugia and post‐glacial colonization routes. Location Southern Andes and Patagonian steppe. Methods We used Calceolaria polyrhiza, a widely distributed Patagonian herbaceous plant that occurs mainly in the understorey of Nothofagus rain forests and in the arid Patagonian steppe, as our model system. The chloroplast intergenic spacer trnH–psbA was sequenced for 590 individuals from 68 populations. Sequence data were analysed using phylogenetic (maximum parsimony, maximum likelihood and Bayesian inference) and population genetic (spatial analyses of molecular variance, mismatch distributions and neutrality tests) methods. Nested clade phylogeographic analyses, and divergence time estimates using a calibrated molecular clock, were also conducted. Results A total of 27 haplotypes identified in the present study clustered into four primary genealogical lineages, revealing three significant latitudinal phylogeographical breaks. The two high Andean lineages probably split first, during the late Miocene, and the Patagonian lineage split around 4 Ma, coincident with the establishment of the Patagonian steppe. Within each haplogroup, major diversification occurred in the Pleistocene. The Patagonian groups show a pattern consistent with a rapid post‐glacial expansion and colonization of the Andean flanks, achieved independently by four lineages. The highest haplotype diversity was found along a longitudinal transect that is remarkably congruent with the limit of the ice‐sheet extension during the Greatest Patagonian Glaciation. A north‐east expansion is evident, which is probably associated with the ‘Arid Diagonal’ fluctuations. Main conclusions Glacial climate fluctuations had a substantial impact on the diversification, distribution and demography of the study species. A scenario of multiple periglacial Pleistocene refugia and subsequent multiple recolonization routes, from eastern Patagonia to the Andean flanks, may explain the phylogeographical patterns observed. However, current genetic structure also preserves the imprints of older events that probably occurred in the Miocene and Pliocene, providing evidence that multiple processes, operating at different spatial and temporal scales, have moulded biodiversity in Patagonia.     

From middens to molecules: phylogeography of the piñon pine,Pinus edulis

Aim Data from packrat middens have established a hypothesized historical biogeography of piñon pine, Pinus edulis, including locations of glacial refugia in the south‐western USA and subsequent migration out of the refugia. In this study, we used molecular techniques to test the glacial refugial hypotheses inferred from packrat (Neotoma) midden data for P. edulis. Location South‐western USA. Methods Two fragments of chloroplast DNA (a portion of the matK gene and a portion of the rbcL gene) for a total of 1045 base pairs were amplified and sequenced for 100 individuals. Thirty‐one populations were sampled throughout the range of P. edulis. Phylogenetic analyses included maximum parsimony and maximum likelihood. Results Very little variation existed among the individuals sampled. Four haplotypes were identified. The inferred ancestral haplotype was the most widespread; it was most common in Texas and New Mexico where, with the exception of one individual, it was the only haplotype found. Arizona and Utah populations were more diverse, with almost half of the populations containing two or more haplotypes. The most derived haplotype was most abundant in Arizona. Main conclusions The distribution of haplotypes is geographically informative. Only one haplotype exists in the south‐eastern portion of the range of P. edulis whereas up to four haplotypes are found in other populations, suggesting one of two hypotheses: either all modern populations are descended from a refugial population in central Arizona, or modern populations are descended from two refugial populations, one in central Arizona and another in Texas–southern New Mexico. Interpreting these data in the light of packrat midden data gives more support for the latter hypothesis.     

Phylogeography of a subalpine tall‐herb Ranunculus platanifolius (Ranunculaceae) reveals two main genetic lineages in the European mountains

A phylogeographical analysis of Ranunculus platanifolius, a typical European subalpine tall‐herb species, indicates the existence of two main genetic lineages based on amplified fragment length polymorphism (AFLP) markers. One group comprises populations from the Balkan Peninsula and the south‐eastern Carpathians and the other includes the remaining part of the range of the species, encompassing the western Carpathians, Sudetes, Alps, Pyrenees and Scandinavia. The main phylogeographical break observed in this species runs across the Carpathians and separates the main parts of this range (western and south‐eastern Carpathians), supporting a distinct glacial history of populations in these areas. The high genetic similarity of the Balkan Peninsula and south‐eastern Carpathian populations could indicate a common glacial refugium for these contemporarily isolated areas of species distribution. The western and northern part of the species range displays an additional weak differentiation into regional phylogeographical groups, which could have been shaped by isolation in glacial refugia or even by a postglacial isolation. The observed weak phylogeographical structure could also be linked with ecological requirements, allowing survival along streams in relatively low, forested mountain ranges. © 2013 The Linnean Society of London     

Phylogeography of red deer (Cervus elaphus) in Europe

Aim To investigate the phylogeographical patterns of red deer (Cervus elaphus) in Europe, and to disentangle the influence of ancient (e.g. Pleistocene ice ages) from more recent processes (e.g. human translocations). Location Europe. Methods In this study we provide by far the most extensive analysis of genetic structure in European red deer, based on analyses of variation at two mitochondrial markers (cyt b and D‐loop) in a large number of individuals from 39 locations. Relationships of mitochondrial DNA haplotypes were determined using minimum spanning networks and phylogenetic analyses. Population structure was examined by analyses of molecular variance. Historical processes shaping the present patterns were inferred from nested clade analysis and nucleotide diversity statistics. Results Within Europe, we detected three deeply divergent mitochondrial DNA lineages. The three lineages displayed a phylogeographical pattern dividing individuals into western European, eastern European and Mediterranean (Sardinia, Spain and Africa) groups, suggesting contraction into three separate refugia during the last glaciation. Few haplotypes were shared among these three groups, a finding also confirmed by F_ST values. Calculations of divergence times suggest that the groups probably split during the Pleistocene. Main conclusions The observed pattern is interpreted to result from isolation in different refugia during the last glaciation. The western and eastern European lineages could be linked to an Iberian and Balkan refugium, respectively. The third lineage might originate from a Sardinian or African refugium. We link local phylogeographical patterns observed in Europe to the post‐glacial recolonization process, shaped by the geographical localization of refugia and barriers to gene flow. Regardless of the importance of red deer as a game species and the tradition of translocating red deer in Europe, we detected few individuals that did not match the trichotomous pattern, suggesting that translocations have occurred mainly at smaller spatial scales.     

Evidence for range stasis during the latter Pleistocene for the Atlantic Coastal Plain endemic genus,Pyxidanthera Michaux

The general phylogeographical paradigm for eastern North America (ENA) is that many plant and animal species retreated into southern refugia during the last glacial period, then expanded northward after the last glacial maximum (LGM). However, some taxa of the Gulf and Atlantic Coastal Plain (GACP) demonstrate complex yet recurrent distributional patterns that cannot be explained by this model. For example, eight co‐occurring endemic plant taxa with ranges from New York to South Carolina exhibit a large disjunction separating northern and southern populations by >300 km. Pyxidanthera (Diapensiaceae), a plant genus that exhibits this pattern, consists of two taxa recognized as either species or varieties. We investigated the taxonomy and phylogeography of Pyxidanthera using morphological data, cpDNA sequences, and amplified fragment length polymorphism markers. Morphological characters thought to be important in distinguishing Pyxidanthera barbulata and P. brevifolia demonstrate substantial overlap with no clear discontinuities. Genetic differentiation is minimal and diversity estimates for northern and southern populations of Pxyidanthera are similar, with no decrease in rare alleles in northern populations. In addition, the northern populations harbour several unique cpDNA haplotypes. Pyxidanthera appears to consist of one morphologically variable species that persisted in or near its present range at least through the latter Pleistocene, while the vicariance of the northern and southern populations may be comparatively recent. This work demonstrates that the refugial paradigm is not always appropriate and GACP endemic plants, in particular, may exhibit phylogeographical patterns qualitatively different from those of other ENA plant species.     

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.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.     

Phylogeography of Japanese water crowfoot based on chloroplast DNA haplotypes

Water crowfoot, Ranunculus subgenus Batrachium, are submerged macrophytes in the Ranunculaceae. We aimed to infer the Quaternary history of these aquatic macrophytes in the Japanese archipelago. We studied 212 individuals of three perennial and one annual species from 46 populations covering the entire geographic range and found eight haplotypes based on approximately 1800 bp of four spacers in chloroplast DNA. The relationships among haplotypes were resolved using maximum parsimony and parsimony network analyses. To identify the zones of clear genetic boundaries, Monmonier's algorithm was used. The eight haplotypes were distinguished from adjacent haplotypes by one substitution or indel. Each of the 46 populations was fixed for a single haplotype. Inconsistency between cpDNA haplotypes and Batrachium taxa was found, except in one annual species. The distribution of the haplotypes in perennial species was highly geographically structured. An abrupt genetic change was detected between the Tohoku region and more southerly regions. Since the perennial Batrachium are cold-adapted, this genetic differentiation may be due to historical changes in their distributions caused by Quaternary climatic oscillations: interglacial retreats in colder refugia and glacial range expansions. The single haplotype composition of each population may have been shaped by founder effects during colonization and/or by genetic drift. The interglacial refugial populations must have been small enough to deplete haplotype diversity. Inconsistency between cpDNA haplotypes and Batrachium taxa may be due to incomplete lineage sorting of ancestral polymorphic haplotypes.     

Mitochondrial differentiation and biogeography of Hyla meridionalis (Anura: Hylidae): an unusual phylogeographical pattern

Aim To study the patterns of genetic variation and the historical events and processes that influenced the distribution and intraspecific diversity in Hyla meridionalis Boettger, 1874. Location Hyla meridionalis is restricted to the western part of the Mediterranean region. In northern Africa it is present in Tunisia, Algeria and Morocco. In south‐western Europe it is found in the south of France, north‐western Italy and north‐eastern and south‐western Iberian Peninsula. There are also insular populations, as in the Canaries and Menorca. Methods Sampling included 112 individuals from 36 populations covering the range of the species. We used sequences of mitochondrial DNA Cytochrome Oxidase I (COI) for the phylogeographical analysis (841 bp) and COI plus a fragment including part of tRNA lysine, ATP synthase subunits 6 and 8 and part of Cytochrome Oxidase III for phylogenetic analyses (2441 bp). Phylogenetic analyses were performed with paup *4.0b10 (maximum likelihood, maximum parsimony) and Mr Bayes 3.0 (Bayesian analysis). Nested clade analysis was performed using tcs 1.18 and Geo Dis 2.2. A dispersal‐vicariant analysis was performed with diva 1.0 to generate hypotheses about the geographical distribution of ancestors. Results We found little genetic diversity within samples from Morocco, south‐western Europe and the Canary Islands, with three well‐differentiated clades. One is distributed in south‐western Iberia and the High Atlas, Anti‐Atlas and Massa River in Morocco. The second is restricted to the Medium Atlas Mountains. The third one is present in northern Morocco, north‐eastern Iberia, southern France and the Canaries. These three groups are also represented in the nested clade analysis. Sequences from Tunisian specimens are highly divergent from sequences of all other populations, suggesting that the split between the two lineages is ancient. diva analysis suggests that the ancestral distribution of the different lineages was restricted to Africa, and that an explanation of current distribution of the species requires three different dispersal events. Main conclusions Our results support the idea of a very recent colonization of south‐western Europe and the Canary Islands from Morocco. South‐western Europe has been colonized at least twice: once from northern Morocco probably to the Mediterranean coast of France and once from the western coast of Morocco to southern Iberia. Human transport is a likely explanation for at least one of these events. Within Morocco, the pattern of diversity is consistent with a model of mountain refugia during hyperarid periods within the Pleistocene. Evaluation of the phylogenetic relationships of Tunisian haplotypes will require an approach involving the other related hylid taxa in the area.