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.     

Range-wide phylogeography of the European temperate-montane herbaceous plant Meum athamanticum Jacq.: evidence for periglacial persistence

Aim  The aim of this study is to analyse the genetic population structure of Meum athamanticum Jacq. in order to explore the alternative hypotheses (1) that the central and northern highland populations are the result of post-glacial recolonization from southern refugia, and the disjunct distribution of M. athamanticum can be explained by modern ecological conditions, or (2) that extant populations north of the Alps and Pyrenees persisted in situ during glacial periods.
Location  Europe.
Methods  Variation of amplified fragment length polymorphisms (AFLPs) was analysed for 23 populations from the entire range of the species. We used band-based approaches and methods based on allele frequencies to measure genetic diversity and to identify population structure.
Results  Our analyses reveal a north–south differentiation within M. athamanticum . High levels of genetic diversity, as well as private fragments, are found in populations both north and south of the Alps. Differentiation among populations is lower in the northern than in the southern population group, and significant isolation-by-distance is found only in the latter group.
Main conclusions  Our results indicate that M. athamanticum survived the last ice age in multiple refugia throughout its contemporary range and did not expand into areas north of the Alps from southern refugia. We found evidence that regional-scale migration in northern, formerly periglacial, parts of the species range has resulted in the intermingling of populations. In contrast, southern populations are characterized by long-term isolation. The south-west Alps represent an area where immigration and mixing of populations from northern and southern refugia appears to have taken place.     

Variable responses of skinks to a common history of rainforest fluctuation: concordance between phylogeography and palaeo-distribution models

There is a growing appreciation of impacts of late-Quaternary climate fluctuations on spatial patterns of species and genetic diversity. A major challenge is to understand how and why species respond individualistically to a common history of climate-induced habitat fluctuation. Here, we combine modelling of palaeo-distributions and mitochondrial-DNA phylogeographies to compare spatial patterns of population persistence and isolation across three species of rainforest skinks ( Saproscincus spp.) with varying climatic preferences. Using Akaike Information Criterion model-averaged projections, all three species are predicted to have maintained one or more small populations in the northern Wet Tropics, multiple or larger populations in the central region, and few if any in the south. For the high-elevation species, Saproscincus czechurai , the warm–wet climate of the mid Holocene was most restrictive, whereas for the generalist S. basiliscus and lower-elevation S. tetradactyla, the cool–dry last glacial maximum was most restrictive. As expected, S. czechurai was the most genetically structured species, although relative to modelled distributions, S. basiliscus had surprisingly deep phylogeographical structure among southern rainforest isolates, implying long-term isolation and persistence. For both S. basiliscus and S. tetradactyla, there was high genetic diversity and complex phylogeographical patterns in the central Wet Tropics, reflecting persistence of large, structured populations. A previously identified vicariant barrier separating northern and central regions is supported, and results from these species also emphasize a historical persistence of populations south of another biogeographical break, the Tully Gorge. Overall, the results support the contention that in a topographically heterogeneous landscape, species with broader climatic niches may maintain higher and more structured genetic diversity due to persistence through varying climates.     

A re-appraisal of nunatak survival in arctic-alpine phylogeography

A long standing and at times fervid debate in biogeography revolves around the question whether arctic and high alpine organisms survived Pleistocene ice ages on small island-like areas protruding above the ice-sheet, socalled nunataks, or whether they did so in peripheral nonglaciated refugial areas. A common picture emerging from a plethora of molecular phylogeographic studies in the last decade is that both in the Arctic and in temperate mountain ranges such as the European Alps nunatak survival needs to be only rarely invoked to explain observed genetic patterns (for a rare example see Stehlik et al. 2002). As two studies in this issue show, depreciation of the nunatak hypothesis is, however, not warranted. In this issue of Molecular Ecology Westergaard et al. (2011) investigate genetic patterns of two arctic-alpine plant species distributed on both sides of the Atlantic exclusively in areas that were mostly covered by ice-sheets during Pleistocene glacial advances. In both species, amplified fragment length polymorphism (AFLP) data identified divergent and partly genetically diverse groups east and west of the Atlantic. This suggests, for the first time in Arctic plants, in situ survival on nunataks. In an entirely different geographic setting and on a different geographic scale, Lohse et al. (2011, this issue) study the colonization of high alpine areas in the Orobian Alps, situated within and adjacent to a prominent peripheral refugial area (massif de refuge) in the Southern Alps of northern Italy, by dispersal-limited carabid ground beetles. Using explicit hypothesis testing and inference of ancestral locations in a Bayesian framework, stepwise colonization from two separate southern refugia is found to shape the genetic pattern of these beetles, but at the northern edge, populations survived at least parts of the last glaciation in situ on nunataks.     

Hybridization among Arctic white-headed gulls (Larus spp.) obscures the genetic legacy of the Pleistocene

We studied the influence of glacial oscillations on the genetic structure of seven species of white-headed gull that breed at high latitudes (Larus argentatus, L. canus, L. glaucescens, L. glaucoides, L. hyperboreus, L. schistisagus, and L. thayeri). We evaluated localities hypothesized as ice-free areas or glacial refugia in other Arctic vertebrates using molecular data from 11 microsatellite loci, mitochondrial DNA (mtDNA) control region, and six nuclear introns for 32 populations across the Holarctic. Moderate levels of genetic structure were observed for microsatellites (F(ST)= 0.129), introns (Φ(ST)= 0.185), and mtDNA control region (Φ(ST)= 0.461), with among-group variation maximized when populations were grouped based on subspecific classification. Two haplotype and at least two allele groups were observed across all loci. However, no haplotype/allele group was composed solely of individuals of a single species, a pattern consistent with recent divergence. Furthermore, northernmost populations were not well differentiated and among-group variation was maximized when L. argentatus and L. hyberboreus populations were grouped by locality rather than species, indicating recent hybridization. Four populations are located in putative Pleistocene glacial refugia and had larger τ estimates than the other 28 populations. However, we were unable to substantiate these putative refugia using coalescent theory, as all populations had genetic signatures of stability based on mtDNA. The extent of haplotype and allele sharing among Arctic white-headed gull species is noteworthy. Studies of other Arctic taxa have generally revealed species-specific clusters as well as genetic structure within species, usually correlated with geography. Aspects of white-headed gull behavioral biology, such as colonization ability and propensity to hybridize, as well as their recent evolutionary history, have likely played a large role in the limited genetic structure observed.     

Pleistocene climatic cycling and diversification of the Andean treefrog, Hypsiboas andinus

Our understanding of the causes of diversification of Neotropical organisms lags behind that of Northern Hemisphere biota, especially for montane and temperate regions of southern South America. We investigated the mitochondrial DNA genealogical patterns in 262 individuals of the frog Hypsiboas andinus from 26 sites across the eastern ranges of the Andes Mountains in Argentina and Bolivia. Our phylogenetic analyses indicate at least three distinct lineages: one representing H. andinus from Northwestern Argentina and southern Bolivia, at least one H. andinus lineage from northern Bolivia, and one clade containing both H. andinus (from the southern portion of the species range) and its putative sister taxon Hypsiboas riojanus. Hypsiboas andinus samples from northern Bolivia are well differentiated and may represent distinct species. The northern Argentine H. andinus lineage and southern H. andinus/H. riojanus lineage likely diverged between 2 and 6 million years ago; their current sympatry may be the result of secondary contact due to range expansion after isolation during Andean uplift or may reflect cryptic species. Within the geographically extensive northern H. andinus clade, we found significant geographical structuring consistent with historical fragmentation and subsequent range expansion. The timing of this fragmentation and range expansion coincide with the Pleistocene, a time of extensive climatic cycling and vegetational shifts. Average divergence among clades is lower than those found for other Neotropical taxa, highlighting the potential importance of recent climatic history in diversification in the southern Andes.     

Comparative phylogeography in a genus of coral reef fishes: biogeographic and genetic concordance in the Caribbean

Geographic barriers that limit the movement of individuals between populations may create or maintain phylogenetically discrete lineages. Such barriers are often inferred from geographic surveys of a single mitochondrial marker to identify phylogenetic splits. Mitochondrial DNA, however, has an effective population size one-fourth that of nuclear DNA, which can facilitate the rapid evolution of monophyletic mtDNA lineages in the absence of geographic barriers. The identification of geographic barriers will thus be more robust if barriers are proposed a priori, and tested with multiple independent genetic markers in multiple species. Here, we tested two proposed marine biogeographic breaks located at the Mona Passage in the Caribbean Sea and at the southern end of Exuma Sound in the Bahamas. We sequenced mitochondrial cytochrome b (400 bp) and nuclear rag1 (573 bp) for nine species and colour forms (183 individuals total) within the teleost genus Elacatinus (Gobiidae) that span the proposed breaks. Our results showed that Mona Passage separated mtcyb and rag1 lineages, with no genetic exchange between populations separated by just 23 km. However, the Central Bahamas barrier was only weakly supported by our data. Importantly, neither barrier coincided with deep genetic splits. This suggests that these two barriers did not initially isolate regional populations, but instead disrupt ongoing gene flow between regions. Our inferred relationships further suggested a division of the Caribbean region into northwestern and southeastern regions, a pattern reflected by some freshwater and terrestrial vertebrates. Our results, coupled with genetic and demographic data from other reef fishes and corals, provide robust support for the Mona Passage as a long-term biogeographic barrier for Caribbean animals.     

Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn.

Traditionally, information on the postglacial history of plant species has been gained from the analysis of fossil pollen data. More recently, surveys of present patterns of genetic variation have given valuable insights into species phylogeography. The genus Alnus , based on fossil data, is known to have had at least four glacial refugia. A survey of chloroplast DNA (cpDNA) diversity in populations of black alder ( A. glutinosa ) was undertaken in order to gain more insight into its postglacial history. This revealed a high degree of structuring of 13 cpDNA haplotypes on a European scale which indicated that most of northern and central Europe was colonized from a refuge in the Carpathian Mountains. Based on the distribution of two common cpDNA haplotypes, colonization routes from this refuge can be determined. The locations of other previously identified refugia are confirmed and two formerly unconfirmed refugial areas for alder (southern Spain and Turkey) are proposed.     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

Three times out of Asia Minor: the phylogeography of Arabis alpina L. (Brassicaceae)

Arabis alpina is a characteristic plant in arctic-alpine habitats and serves as a classical example to demonstrate biology, ecology and biogeography of arctic-alpine disjuncts. It has a wider distribution than most other arctic-alpine plants, covering all European mountain systems, the Canary Islands, North Africa, the high mountains of East Africa and Ethiopia, the Arabian Peninsula and mountain ranges of Central Asia in Iran and Iraq. Additionally it is found in the northern amphi-Atlantic area including northeastern North America, Greenland, Iceland, Svalbard and northwestern Europe. We used markers from the nuclear (internal transcribed spacer of ribosomal DNA) and chloroplast genome (trnL-F region) to reconstruct its phylogeographic history. Both markers revealed clear phylogeographic structure. We suggest that A. alpina originated in Asia Minor less than 2 million years ago based on synonymous mutation rates of different genes (plastidic matK, nuclear adh and chs). From the Asian ancestral stock one group migrated via the Arabian Peninsula to the East African high mountains. A second group gave rise to all European and northern populations, and also served as source for the northwest African populations. A third group, which is still centred in Asia, migrated independently southwards and came into secondary contact with the East African lineage in Ethiopia, resulting in high genetic diversity in this area. In the Mediterranean regions, the genetic diversity was relatively high with numerous unique haplotypes, but almost without geographic structure. In contrast, the populations in the northern amphi-Atlantic area were extremely depauperate, suggesting very recent (postglacial) expansion into this vast area from the south.     

Pleistocene divergence of Dinaric Drusus endemics (Trichoptera, Limnephilidae) in multiple microrefugia within the Balkan Peninsula

The Balkan Peninsula is one of three major European refugial areas. It has high biodiversity and endemism, but data on the age and origin of its fauna, especially endemics, are limited. Mitochondrial sequence data (COI and 16S genes) were used to study the population structure and phylogeography of the caddisfly Drusus croaticus and the phylogeny and divergence of seven other Drusus species, mostly range-restricted endemics of the Dinaric region of the Balkan Peninsula. The divergence of D. croaticus populations in Croatia and allopatric Drusus species in Bosnia dated to the Pleistocene, showing the importance of this time period for the origin and diversification of Balkan endemic taxa. The divergence of more distantly related species dated to the Late Miocene/Early Pliocene. Population genetic and phylogeographic analysis of 115 individuals from 11 populations of D. croaticus revealed a high level of genetic differentiation and absence of gene flow between populations separated by more than 10 km. The existence of allopatrically fragmented lineages in D. croaticus and the endemic Bosnian species is most likely the result of long-term isolation in multiple microrefugia, probably due to the specific habitat requirements and life-history traits of Drusinae coupled with the topographic complexity and historical changes in geomorphology of the region. Overall, these findings shed light on the processes generating the high genetic complexity of this refugial region that parallels the 'refugia within refugia' pattern widely reported from the Iberian refugium.     

Multiple Pleistocene refugia and Holocene range expansion of an abundant southwestern American desert plant species (Melampodium leucanthum,Asteraceae)

Pleistocene climatic fluctuations had major impacts on desert biota in southwestern North America. During cooler and wetter periods, drought‐adapted species were isolated into refugia, in contrast to expansion of their ranges during the massive aridification in the Holocene. Here, we use Melampodium leucanthum (Asteraceae), a species of the North American desert and semi‐desert regions, to investigate the impact of major aridification in southwestern North America on phylogeography and evolution in a widespread and abundant drought‐adapted plant species. The evidence for three separate Pleistocene refugia at different time levels suggests that this species responded to the Quaternary climatic oscillations in a cyclic manner. In the Holocene, once differentiated lineages came into secondary contact and intermixed, but these range expansions did not follow the eastwardly progressing aridification, but instead occurred independently out of separate Pleistocene refugia. As found in other desert biota, the Continental Divide has acted as a major migration barrier for M. leucanthum since the Pleistocene. Despite being geographically restricted to the eastern part of the species’ distribution, autotetraploids in M. leucanthum originated multiple times and do not form a genetically cohesive group.     

A phylogeographic, demographic and historical analysis of the short-tailed pit viper (Gloydius brevicaudus): evidence for early divergence and late expansion during the Pleistocene

The impact of quaternary glaciation in eastern China on local fanua and flora has been a topic of considerable interest. We used mitochondrial DNA (mtDNA) sequence data and coalescent simulations to test two general biogeographic hypothesis related to the effects of Pleistocene climatic fluctuations for a widespread ophidian species (Gloydius brevicaudus) in eastern China and Korean Peninsula. The phylogenetic analysis revealed three major lineages, the southeast Coastal, Yangtze and North Lineages. The latter two are closely related and jointly form a continental lineage. Divergence dating and coalescent simulations indicate a Late Pliocene to Early Pleistocene divergence between lineages from the southeast coast and continental interior, followed by a mid-to-late Pleistocene divergence between lineages from the north and the middle-lower Yangtze Valley across East China, suggesting that all these lineages predated the last glacial maximum. An overlapping range between the two lineages within the continental lineage and a secondary contact associated with ecological transition zones on the margins of the North China Plain were also observed. These results show that vicariance patterns dominated the history of G. brevicaudus. Though the climatic events of the Pleistocene have had a marked effect on the historical distribution and intra-specific divergence of reptiles in China, coalescent and non-coalescent demographic analyses indicate that all lineages of G. brevicaudus seem not to have been adversely affected by glacial cycles during the Late Pleistocene, presumably because of an increase in the amount of climatically mild habitat in East Asia due to a decline in elevation and the development of monsoons since the Mid-End Pleistocene.     

Chloroplast DNA phylogeography of Fagus crenata (Fagaceae) in Japan

 CpDNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 45 populations distributed throughout the species' range. Two cpDNA regions were sequenced: the non-coding region between the trnL (UAA) 5′exon and trnF (GAA), and the trnK region (including matK). Thirteen distinct cpDNA haplotypes were recognized and each haplotype was found to be geographically structured. Two major clades (I and II+III) were revealed in phylogenetic analyses among the haplotypes using F. sylvatica as an outgroup. The haplotypes of Clade I were distributed mainly along the Japan Sea side of the Japanese Archipelago, while those of Clade II+III occurred chiefly along the Pacific Ocean side. Consequently, the distribution of the two major cpDNA clades suggests that there were two migration routes in the history of F. crenata; one along the Japan Sea and the other along the Pacific Ocean side of the Japanese Islands. Received March 19, 2001 Accepted November 22, 2001     

Of glaciers and refugia: a decade of study sheds new light on the phylogeography of northwestern North America

Glacial cycles have played a dominant role in shaping the genetic structure and distribution of biota in northwestern North America. The two major ice age refugia of Beringia and the Pacific Northwest were connected by major mountain chains and bordered by the Pacific Ocean. As a result, numerous refugial options were available for the regions taxa during glacial advances. We reviewed the importance of glaciations and refugia in shaping northwestern North America’s phylogeographic history. We also tested whether ecological variables were associated with refugial history. The recurrent phylogeographic patterns that emerged were the following: (i) additional complexity, i.e. refugia within refugia, in both Beringia and the Pacific Northwest; and (ii) strong evidence for cryptic refugia in the Alexander Archipelago and Haida Gwaii, the Canadian Arctic and within the ice‐sheets. Species with contemporary ranges that covered multiple refugia, or those with high dispersal ability, were significantly more likely to have resided in multiple refugia. Most of the shared phylogeographic patterns can be attributed to multiple refugial locales during the last glacial maximum or major physiographic barriers like rivers and glaciers. However, some of the observed patterns are much older and appear connected to the orogeny of the Cascade‐Sierra chain or allopatric differentiation during historic glacial advances. The emergent patterns from this review suggest we should refine the classic Beringian‐southern refugial paradigm for northwestern North American biota and highlight the ecological and evolutionary consequences of colonization from multiple refugia.     

Chloroplast DNA phylogeography of the shrub Cistus ladanifer L. (Cistaceae) in the highly diverse Western Mediterranean region

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity=0.32). High differentiation among populations was estimated (G(ST)=0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.     

Phylogeography of the North American red fox: vicariance in Pleistocene forest refugia

Fossil, archaeological, and morphometric data suggest that indigenous red foxes in North America were derived from vicariance in two disjunct refugia during the last glaciation: one in Beringia and one in the contiguous USA. To test this hypothesis, we conducted a phylogeographical analysis of the North American red fox within its presettlement range. We sequenced portions of the mitochondrial cytochrome b (354 bp) gene and D-loop (342 bp) from 220 historical red fox specimens. Phylogenetic analysis of the cytochrome b gene produced two clades that diverged c . 400 000 years before present ( bp ): a Holarctic and a Nearctic clade. D-loop analyses of the Nearctic clade indicated three distinct subclades (≥ 99% Bayesian posterior probability); two that were more recently derived (rho estimate c . 20 000 bp ) and were restricted to the southwestern mountains and the eastern portion of North America, and one that was older (rho estimate c . 45 000 bp ) and more widespread in North America. Populations that migrated north from the southern refugium following deglaciation were derived from the colonization of North America during or prior to the Illinoian glaciation (300 000–130 000 bp ), whereas populations that migrated south from the northern refugium represent a more recent colonization event during the Wisconsin glaciation (100 000–10 000 bp ). Our findings indicate that Nearctic clade red foxes are phylogenetically distinct from their Holarctic counterparts, and reflect long-term isolation in two disjunct forest refugia during the Pleistocene. The montane lineage, which includes endangered populations, may be ecologically and evolutionarily distinct.