Phylogeographic analysis reveals a deep lineage split within North Atlantic Littorina saxatilis

Phylogeographic studies provide critical insight into the evolutionary histories of model organisms; yet, to date, range-wide data are lacking for the rough periwinkle Littorina saxatilis, a classic example of marine sympatric speciation. Here, we use mitochondrial DNA (mtDNA) sequence data to demonstrate that L. saxatilis is not monophyletic for this marker, but is composed of two distinct mtDNA lineages (I and II) that are shared with sister species Littorina arcana and Littorina compressa. Bayesian coalescent dating and phylogeographic patterns indicate that both L. saxatilis lineages originated in the eastern North Atlantic, around the British Isles, at approximately 0.64 Ma. Both lineages are now distributed broadly across the eastern, central and western North Atlantic, and show strong phylogeographic structure among regions. The Iberian Peninsula is genetically distinct, suggesting prolonged isolation from northeastern North Atlantic populations. Western North Atlantic populations of L. saxatilis lineages I and II predate the last glacial maximum and have been isolated from eastern North Atlantic populations since that time. This identification of two distinct, broadly distributed mtDNA lineages further complicates observed patterns of repeated incipient ecological speciation in L. saxatilis, because the sympatric origins of distinct ecotype pairs on eastern North Atlantic shores may be confounded by admixture of divergent lineages.     

Phylogeography of the montane caddisfly Drusus discolor: evidence for multiple refugia and periglacial survival

We studied the genetic population structure and phylogeography of the montane caddisfly Drusus discolor across its entire range in central and southern Europe. The species is restricted to mountain regions and exhibits an insular distribution across the major mountain ranges. Mitochondrial sequence data (COI) of 254 individuals from the entire species range is analysed to reveal population genetic structure. The data show little molecular variation within populations and regions, but distinct genetic differentiation between mountain ranges. Most populations are significantly differentiated based on F(ST) and exact tests of population differentiation and most haplotypes are unique to a single mountain range. Phylogenetic analyses reveal deep divergence between geographically isolated lineages. Combined, these results suggest that past fragmentation is the prominent process structuring the populations across Europe. We use tests of selective neutrality and mismatch distributions, to study the demographic population history of regions with haplotype overlap. The high level of genetic differentiation between mountain ranges and estimates of demographic history provide evidence for the existence of multiple glacial refugia, including several in central Europe. The study shows that these aquatic organisms reacted differently to Pleistocene cooling than many terrestrial species. They persisted in numerous refugia over multiple glacial cycles, allowing many local endemic clades to form.     

Phylogeographic structure and past history of the circum-Mediterranean species Tomicus destruens Woll. (Coleoptera: Scolytinae)

Phylogeographic studies are often focused on temperate European species with relict footholds in the Mediterranean region. Past climatic oscillations usually induced range contractions and expansions from refugial areas located in southern Europe, and spatial distribution of genetic diversity show that northward expansions were usually pioneer-like. Actually, few studies have focused on circum-Mediterranean species, which probably were not influenced in the same way by climatic oscillations. We present the phylogeography of the bark beetle Tomicus destruens, which is restricted to the whole Mediterranean basin and the Atlantic coasts of North Africa and Portugal. We systematically sequenced 617 bp of the mitochondrial genes COI and COII for 42 populations (N = 219). Analysis revealed 53 haplotypes geographically structured in two clades, namely eastern and western clades, that diverged during the Pleistocene. A contact zone was identified along the Adriatic coast of Italy. Interestingly, we found contrasting levels of genetic structure within each clade. The eastern group was characterized by a significant phylogeographic pattern and low levels of gene flow, whereas the western group barely showed a spatial structure in haplotype distribution. Moreover, the main pine hosts were different between groups, with the Aleppo-brutia complex in the east and the maritime pine in the west. Potential roles of host species, climatic parameters and geographical barriers are discussed and the phylogeographic patterns are compared to classical models of postglacial recolonization in Europe.     

Mitochondrial differentiation in a polymorphic land snail: evidence for Pleistocene survival within the boundaries of permafrost

The genetic differentiation of populations having colonized formerly unsuitable habitats after the Pleistocene glaciations depends to a great extent on the speed of expansion. Slow dispersers maintain their refugial diversity whereas fast dispersal leads to a reduction of diversity in the newly colonized areas. During the Pleistocene, almost the entire current range of the land snail Arianta arbustorum has repeatedly been covered with ice or been subjected to permafrost. Owing to the low potential for dispersal of land snails, slow (re)colonization of the wide range from southern refugia can be excluded. Alternatively, fast, passive dispersal from southern refugia or survival in and expansion from multiple refugia within the area subjected to permafrost may account for the current distribution. To distinguish between these scenarios we reconstructed a phylogeography based on the sequences of a fragment of the cytochrome oxidase I from 133 individuals collected at 45 localities and analysed the molecular variance. Seventy-five haplotypes were found that diverged on average at 7.52% of positions. This high degree of diversity suggests that A. arbustorum is an old species in which the population structure, isolation and the hermaphroditic nature have reduced the probability of lineage extinction. The genetic structure was highly significant with the highest variance partition found among regions. Geographic distance and mitochondrial differentiation were not congruent. Lineages had overlapping ranges. The clear genetic differentiation and the patchy pattern of haplotype distribution suggest that colonization of formerly unsuitable habitats was mainly achieved from multiple populations from within the permafrost area.     

Phylogeography of the rare Balkan endemic Martino's vole, Dinaromys bogdanovi, reveals strong differentiation within the western Balkan Peninsula

The spatial genetic structure of Martino's vole, a rare palaeoendemic species of the western Balkans, was investigated using DNA isolated from archived museum samples. The study was based on partial sequencing (555 bp) of the mitochondrial cytochrome b gene for 63 specimens from 20 different localities throughout the species' range. Three highly divergent allopatric phylogenetic lineages (Northwestern, Central and Southeastern) were recognized among 47 haplotypes, suggesting three independent glacial differentiation centres within the western Balkans. The Northwestern lineage, which showed the highest divergence from all other samples (mean sequence divergence of 6.64% +/- 1.10), comprised samples collected from northwest of the Neretva River in Croatia, western Bosnia and Herzegovina. The Central and Southeastern lineages were separated by an average sequence divergence of 2.95% +/- 0.66 and were geographically divided by the Drim River (the Kosovo Basin in Serbia). Overall, haplotype diversity decreased from the Northwestern lineage to the Central and subsequently the Southeastern lineage, in a geographical pattern consistent with a stepping stone colonization. The observed distribution indicates a gradual southerly expansion with subsequent allopatry across the Neretva River and Drim River approximately 1 and 0.3 million years ago, respectively. Such a scenario is concordant with palaeontological evidence. Several highly divergent sublineages within the Northwestern and Central lineages showed no significant geographical structuring, suggesting secondary contact of allopatrically evolved lineages. We hypothesize that the topographical complexity of the Balkans promoted allopatry and isolation on a small geographical scale during interglacial periods, with secondary contact during glacial maxima. Furthermore, the three main lineages should be regarded as evolutionary significant units with important implications for conservation. Ecological data show that the Northwestern lineage in particular fulfils all criteria for a highly endangered, evolutionarily significant unit.     

Isolation and characterization of microsatellite markers in the caddisfly Drusus discolor (Trichoptera: Limnephilidae)

We describe the development of and amplification conditions for microsatellite primers isolated from the caddisfly Drusus discolor. Eight polymorphic microsatellite loci were developed and screened for variability using 37 individuals from two populations from central Europe. The primers yielded an average of 8.6 alleles per loci. No linkage disequilibrium between loci was detected, while three loci showed deviations from Hardy–Weinberg equilibrium in one of the two tested populations.     

Phylogeography and historical demography of the Italian treefrog, Hyla intermedia, reveals multiple refugia, population expansions and secondary contacts within peninsular Italy

We investigated the geographical patterns of genetic diversity in the Italian treefrog through sequence analysis of a mitochondrial cytochrome b gene fragment. Three main mitochondrial lineages were identified, distributed in northern, central and southern Italy, respectively. Their divergence appears indicative of a split time largely predating Late Pleistocene climatic oscillations, and syntopy between them was only observed in the geographically intermediate populations. The historical demographic reconstructions suggest that in both northern and central Italy, an expansion occurred during the last major glacial phase, when a vast widening of the lowland habitats followed the glaciation-induced fall of the sea level. Instead, in southern Italy an expansion event likely followed the end of the last glaciation, although the inference of expansion appears less reliable for the southern clade than for the others. Within this geographical area, a sharp phylogeographic discontinuity separated peninsular from Sicilian populations, and the overall pattern of diversity suggests that the latter derived from a recent colonization of the island, probably through a Late Pleistocene land bridge. Phylogenetic, phylogeographic and historical demographic analyses thus concur in delineating a scenario of multiple refugia, with four groups of populations which survived the last glacial-interglacial cycles in at least three distinct refugia arranged along peninsular Italy, and have recently come into contact following range expansions. Therefore, these results support the hypothesis that a plethora of microevolutionary processes, rather than the prolonged stability of populations, were mainly responsible for shaping the patterns of diversity within this major biodiversity hotspot.     

Contrasting phylogeographic patterns in Sphagnum fimbriatum and Sphagnum squarrosum (Bryophyta, Sphagnopsida) in Europe

The chloroplast phylogeography of two peat mosses (Sphagnum fimbriatum and Sphagnum squarrosum) with similar distributions but different life history characteristics was investigated in Europe. Our main aim was to test whether similar distributions reflect similar phylogeographic and phylodemographic processes. Accessions covering the European distributions of the species were collected and approx. 2000 bp of the chloroplast genome of each species was sequenced. Maximum parsimony, statistical parsimony and phylodemographic analyses were used to address the question of whether these species with similar distributions show evidence of similar phylogeographic and phylodemographic processes. The chloroplast haplotypes of the currently spreading species S. fimbriatum showed strong geographic structure, whereas those of S. squarrosum, which has stable historical population sizes, showed only very weak geographic affinity and were widely distributed. We hypothesize that S. fimbriatum survived the last glaciations along the Atlantic coast of Europe, whereas S. squarrosum had numerous, scattered refugia in Europe. The dominance of one haplotype of S. fimbriatum across almost all of Europe suggests rapid colonization after the last glacial maximum. We hypothesize that high colonizing ability is an inherent characteristic of the species and its recent expansion in Europe is a response to climate change.     

Phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum: contrasting results from mitochondrial and microsatellite data

Phylogeographical studies are typically based on haplotype data, occasionally on nuclear markers such as microsatellites, but rarely combine both. This is unfortunate because the use of markers with contrasting modes of inheritance and rates of evolution might provide a more accurate and comprehensive understanding of a species' history. Here we present a detailed study of the phylogeography of the greater horseshoe bat, Rhinolophus ferrumequinum , using 1098 bp of the mitochondrial ND2 gene from 45 localities from across its Palaearctic range to infer population history. In addition, we re-analysed a large microsatellite data set available for this species and compared the results of both markers to infer population relationships and the historical processes influencing them. We show that mtDNA, the most popular marker in phylogeography studies, yielded a misleading result, and would have led us to conclude erroneously that a single expansion had taken place in Europe. Only by combining the mitochondrial and microsatellite data sets are we able to reconstruct the species' history and show two colonization events in Europe, one before the Last Glacial Maximum (LGM) and one after it. Combining markers also revealed the importance of Asia Minor as an ancient refugium for this species and a source population for the expansion of the greater horseshoe bat into Europe before the LGM.     

The rise and fall of the mountain hare (Lepus timidus) during Pleistocene glaciations: expansion and retreat with hybridization in the Iberian Peninsula

The climatic fluctuations during glaciations have affected differently arctic and temperate species. In the northern hemisphere, cooling periods induced the expansion of many arctic species to the south, while temperate species were forced to retract in southern refugia. Consequently, in some areas the alternation of these species set the conditions for competition and eventually hybridization. Hares in the Iberian Peninsula appear to illustrate this phenomenon. Populations of Iberian hare (Lepus granatensis), brown hare (Lepus europaeus) and broom hare (Lepus castroviejoi) in Northern Iberia harbour mitochondrial haplotypes from the mountain hare (Lepus timidus), a mainly boreal and arctic species presently absent from the peninsula. To understand the history of this past introgression we analysed sequence variation and geographical distribution of mitochondrial control region and cytochrome b haplotypes of L. timidus origin found in 378 specimens of these four species. Among 124 L. timidus from the Northern Palaearctic and the Alps we found substantial nucleotide diversity (2.3%) but little differentiation between populations. Based on the mismatch distribution of the L. timidus sequences, this could result from an expansion at a time of temperature decrease favourable to this arctic species. The nucleotide diversity of L. timidus mtDNA found in Iberian L. granatensis, L. europaeus and L. castroviejoi (183, 70 and 1 specimens, respectively) was of the same order as that in L. timidus over its range (1.9%), suggesting repeated introgression of multiple lineages. The structure of the coalescent of L. granatensis sequences indicates that hybridization with L. timidus was followed by expansion of the introgressed haplotypes, as expected during a replacement with competition, and occurred when temperatures started to rise, favouring the temperate species. Whether a similar scenario explains the introgression into Iberian L. europaeus remains unclear but it is possible that it hybridized with already introgressed L. granatensis.     

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     

Phylogenetic analysis and differentiation of Veronica subgenus Stenocarpon in the Balkan Peninsula

The Balkan Peninsula is considered the most important refugium for species during the Pleistocene glaciations and today harbours c. 2000 endemic species, but we know surprisingly little about the evolution of taxa in this region. Veronica saturejoides, V. thessalica and V. erinoides are a group of closely related alpine taxa endemic to the Balkan Peninsula. Here, we analyse four DNA regions [the nuclear chalcone synthase intron (CHSi) and ribosomal internal transcribed spacer (ITS) region and the plastid rpoB‐trnC spacer and trnL‐trnL‐trnF region] and amplified fragment length polymorphism (AFLP) fingerprints to provide a phylogenetic hypothesis for the relationships among these taxa. Additionally, we analyse leaf morphological characters used to distinguish the three subspecies of V. saturejoides. The analyses support the distinction of the three subspecies based on previously intuitively suggested characters. Nuclear chalcone synthase intron data indicate that the southern taxa are genetically much more diverse than the more northern V. saturejoides subsp. saturejoides. Phylogenetic relationships inferred from this region and AFLP fingerprints support the monophyly of V. saturejoides. In contrast, plastid DNA regions suggest a closer relationship of V. saturejoides subsp. saturejoides to V. thessalica. The most likely scenario involves introgression into V. saturejoides subsp. saturejoides from V. thessalica. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 616–636.     

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.     

Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability

Climatic oscillations throughout the Pleistocene combined with geological and topographic complexity resulted in extreme habitat heterogeneity along the Atlantic coast of Brazil. Inferring how these historic landscape patterns have structured the current diversity of the region's biota is important both for our understanding of the factors promoting diversification, as well as the conservation of this biodiversity hotspot. Here we evaluate potential historical scenarios of diversification in the Atlantic Coastal Forest of Brazil by investigating the population genetic structure of a frog endemic to the region. Using mitochondrial and nuclear sequences, we generated a Bayesian population-level phylogeny of the Thoropa miliaris species complex. We found deep genetic divergences among five geographically distinct clades. Southern clades were monophyletic and nested within paraphyletic northern clades. Analyses of historical demographic patterns suggest an overall north to south population expansion, likely associated with regional differences in habitat stability during the Pliocene and early Pleistocene. However, genetic structure among southern populations is less pronounced and likely represents more recent vicariant events resulting from Holocenic sea-level oscillations. Our analyses corroborate that the Atlantic Coastal Forest has been a biogeographically dynamic landscape and suggest that the high diversity of its fauna and flora resulted from a combination of climatic and geologic events from the Pliocene to the present.     

Glacial vicariance in the Pacific Northwest: evidence from a lodgepole pine mitochondrial DNA minisatellite for multiple genetically distinct and widely separated refugia

The Canadian side of the Pacific Northwest was almost entirely covered by ice during the last glacial maximum, which has induced vicariance and genetic population structure for several plant and animal taxa. Lodgepole pine ( Pinus contorta Dougl. ex. Loud.) has a wide latitudinal and longitudinal distribution in the Pacific Northwest. Our main objective was to identify relictual signatures of glacial vicariance in the population structure of the species and search for evidence of distinct glacial refugia in the Pacific Northwest. A maternally inherited mitochondrial DNA minisatellite-like marker was used to decipher haplotype diversity in 91 populations of lodgepole pine located across the natural range. Overall population differentiation was sizeable ( G _ST = 0.365 and R _ST = 0.568). Four relatively homogeneous groups of populations, possibly representative of as many genetically distinct glacial populations, were identified for the two main subspecies, ssp. latifolia and ssp. contorta . For ssp. contorta , one glacial lineage is suggested to have been located at high latitudes and possibly off the coast of mainland British Columbia (BC), while the other is considered to have been located south of the ice sheet along the Pacific coast. For ssp. latifolia , two genetically distinct glacial populations probably occurred south of the ice sheet: in the area bounded by the Cascades and Rocky Mountains ranges, and on the eastern side of the Rockies. A possible fifth refugium located in the Yukon may have also been present for ssp. latifolia . Zones of contact between these ancestral lineages were also apparent in interior and northern BC. These results indicate the role of the Queen Charlotte Islands and the Alexander Archipelago as a refugial zone for some Pacific Northwest species and the vicariant role played by the Cascades and the American Rocky Mountains during glaciation.     

Persistent genetic signatures of colonization in Brachionus manjavacas rotifers in the Iberian Peninsula

Recent phylogeographical assessments have consistently shown that continental zooplankton display high levels of population subdivision, despite the high dispersal capacity of their diapausing propagules. As such, there is an apparent paradox between observed cosmopolitanism in the zooplankton that is associated with long-distance dispersal, and strong phylogeographical structures at a regional scale. Such population dynamics, far from migration-drift equilibrium, have been shown in the rotifer species complex Brachionus plicatilis, a group of over a dozen species inhabiting salt lakes and coastal lagoons worldwide. Here we present the mitochondrial DNA phylogeography of one of these species, Brachionus manjavacas, in the Iberian Peninsula, where it often co-occurs with the morphologically similar species B. plicatilis sensu stricto. We obtained sequences from 233 individuals from diapausing eggs and clonal cultures from 16 lakes in the Iberian Peninsula, and a Tunisian lake. Two strongly supported deep mitochondrial DNA clades were found (A and B). Phylogenetic and nested clade analysis showed that clade A has a strong phylogeographical structure, with a strong similarity of phylogeographical patterns between B. manjavacas clade A and B. plicatilis s.s. These include (i) signatures of allopatric fragmentation between central and southern populations, and (ii) range expansions in the Iberian Peninsula, both likely to have occurred during the Pleistocene. We find evidence for a glacial refugium in the Guadiana basin. Clades A and B co-occurred in several of these lakes because of range expansion and secondary contact between both clades. The co-occurrence between B. plicatilis s.s. and B. manjavacas is not recent, and both species might have experienced similar environmental challenges during the Pleistocene. The strong correlation of genetic and geographical distance found suggests that historical events can lead to such correlation, mirroring the effects of 'isolation by distance' in equilibrium populations.     

A relict bank vole lineage highlights the biogeographic history of the Pyrenean region in Europe

The Pyrenean region exhibits high levels of endemism suggesting a major contribution to the phylogeography of European species. But, to date, the role of the Pyrenees and surrounding areas as a glacial refugium for temperate species remains poorly explored. In the current study, we investigated the biogeographic role of the Pyrenean region through the analyses of genetic polymorphism and morphology of a typical forest-dwelling small mammal, the bank vole ( Myodes glareolus ). Analyses of the mitochondrial cytochrome b gene and the third upper molar (M³) show a complex phylogeographic structure in the Pyrenean region with at least three distinct lineages: the Western European, Spanish and Basque lineages. The Basque lineage in the northwestern (NW) Pyrenees was identified as a new clearly differentiated and geographically localized bank vole lineage in Europe. The average M³ shape of Basque bank voles suggests morphological differentiation but also restricted genetic exchanges with other populations. Our genetic and morphological results as well as palaeo-environmental and fossils records support the hypothesis of a new glacial refugium in Europe situated in the NW Pyrenees. The permissive microclimatic conditions that prevailed for a long time in this region may have allowed the survival of temperate species, including humans. Moreover, local differentiation around the Pyrenees is favoured by the opportunity for populations to track the shift of the vegetation belt in altitude rather than in latitude. The finding of the Basque lineage is in agreement with the high level of endemic taxa reported in the NW Pyrenees.     

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.