Post-glacial colonization of Western Europe brown bears from a cryptic Atlantic refugium out of the Iberian Peninsula

The European brown bear (Ursus arctos) shows a particular phylogeography that has been used to illustrate the model for contraction-expansion dynamics related to glacial refugia in Southern European peninsulas. Recent studies, however, have nuanced the once generally accepted paradigm, indicating the existence of cryptic refugia for some species further north. In this paper we collected available data on chronology and mitochondrial haplotypes from Western European brown bears, adding new sequences from present day individuals from the Cantabrian (North Iberia) area, in order to reconstruct the dynamics of the species in the region. Both genetics and chronology show that the Iberian Pleistocene lineages were not the direct ancestors of the Holocene ones, the latter entering the Peninsula belatedly (around 10,000 years BP) with respect to other areas such as the British Isles. We therefore propose the existence of a cryptic refugium in continental Atlantic Europe, from where the bears would expand as the ice receded. The delay in the recolonization of the Iberian Peninsula could be due to the orographic characteristics of the Pyrenean-Cantabrian region and to the abundant presence of humans in the natural entrance to the Peninsula.     

Genetic turnovers and northern survival during the last glacial maximum in European brown bears

The current phylogeographic pattern of European brown bears (Ursus arctos) has commonly been explained by postglacial recolonization out of geographically distinct refugia in southern Europe, a pattern well in accordance with the expansion/contraction model. Studies of ancient DNA from brown bear remains have questioned this pattern, but have failed to explain the glacial distribution of mitochondrial brown bear clades and their subsequent expansion across the European continent. We here present 136 new mitochondrial sequences generated from 346 remains from Europe, ranging in age between the Late Pleistocene and historical times. The genetic data show a high Late Pleistocene diversity across the continent and challenge the strict confinement of bears to traditional southern refugia during the last glacial maximum (LGM). The mitochondrial data further suggest a genetic turnover just before this time, as well as a steep demographic decline starting in the mid‐Holocene. Levels of stable nitrogen isotopes from the remains confirm a previously proposed shift toward increasing herbivory around the LGM in Europe. Overall, these results suggest that in addition to climate, anthropogenic impact and inter‐specific competition may have had more important effects on the brown bear's ecology, demography, and genetic structure than previously thought.     

Extra-Mediterranean refugia: The rule and not the exception?

ABSTRACT: Some decades ago, biogeographers distinguished three major faunal types of high importance for Europe: (i) Mediterranean elements with exclusive glacial survival in the Mediterranean refugia, (ii) Siberian elements with glacial refugia in the eastern Palearctic and only postglacial expansion to Europe and (iii) arctic and/or alpine elements with large zonal distributions in the periglacial areas and postglacial retreat to the North and/or into the high mountain systems. Genetic analyses have unravelled numerous additional refugia both of continental and Mediterranean species, thus strongly modifying the biogeographical view of Europe. This modified notion is particularly true for the so-called Siberian species, which in many cases have not immigrated into Europe during the postglacial period, but most likely have survived the last, or even several glacial phases, in extra-Mediterranean refugia in some climatically favourable but geographically limited areas of southern Central and Eastern Europe. Recently, genetic analyses revealed that typical Mediterranean species have also survived the Last Glacial Maximum in cryptic northern refugia (e.g. in the Carpathians or even north of the Alps) in addition to their Mediterranean refuge areas.     

The crested newt Triturus cristatus recolonized temperate Eurasia from an extra‐Mediterranean glacial refugium

We assess the role of the Carpathians as an extra‐Mediterranean glacial refugium for the crested newt Triturus cristatus. We combine a multilocus phylogeography (one mitochondrial protein‐coding gene, three nuclear introns, and one major histocompatibility complex gene) with species distribution modelling (projected on current and Last Glacial Maximum climate layers). All genetic markers consistently show extensive genetic variation within and genetic depletion outside the Carpathians. The species distribution model suggests that most of the current range was unsuitable at the Last Glacial Maximum, but a small suitable area remained in the Carpathians. Triturus cristatus dramatically expanded its postglacial range, colonizing much of temperate Eurasia from a glacial refugium in the Carpathians. Within the Carpathians, T. cristatus persisted in multiple geographically discrete regions, providing further support for a Carpathian ‘refugia within refugia’ scenario. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 574–587.     

Staying out in the cold: glacial refugia and mitochondrial DNA phylogeography in ancient European brown bears

Models for the development of species distribution in Europe typically invoke restriction in three temperate Mediterranean refugia during glaciations, from where recolonization of central and northern Europe occurred. The brown bear, Ursus arctos, is one of the taxa from which this model is derived. Sequence data generated from brown bear fossils show a complex phylogeographical history for western European populations. Long-term isolation in separate refugia is not required to explain our data when considering the palaeontological distribution of brown bears. We propose continuous gene flow across southern Europe, from which brown bear populations expanded after the last glaciation.     

Allopatric divergence and secondary contacts in <Emphasis Type="Italic">Euphorbia spinosa</Emphasis> L: Influence of climatic changes on the split of the species

Euphorbia spinosa, a perennial xerophilous shrub naturally distributed across the Italian peninsula, was selected for examination of the role of the Ligurian Alps and Apennines in glacial survival. The Italian Peninsula is considered to be one of the principal glacial refugia in Europe, but few plant population genetic and phylogeography studies have been undertaken in this region. The combined analysis of chloroplast and nuclear loci (ITS, cpSSR and ISSR) enabled us to detect extensive DNA variation and proved to be a very powerful tool for the reconstruction of the phylogeography. Molecular data support the hypothesis of a long-term separation of the Northwestern (Maritime Alps, Sardinia, Corsica, Northern Apennines) and Southeastern (Southern Apennines and Balkan area) lineages in glacial refugia. The existence of allopatrically fragmented lineages is most probably the result of isolation in different glacial refugia, possibly due to the Last Glacial Maximum cooling and the topographic complexity of the Italian peninsula. The most plausible hypothesis assumes the formation of two migration paths during more recent periods: the first one starting with southward migration and the second one moving northwards. The Central Apennines should be considered the confluence of migration routes radiating from separate refugia according to this hypothesis.     

Glacial refugia of mammals in Europe: evidence from fossil records

• 1 Glacial refugia were core areas for the survival of temperate species during unfavourable environmental conditions and were the sources of postglacial recolonizations. Unfortunately, the locations of glacial refugia of animals and plants are usually described by models, without reference to facts about real geographical ranges at that time.
• 2 Careful consideration of the faunal assemblages of archaeological sites from the Younger Palaeolithic, which are precisely dated to the Last Glacial Maximum (LGM), gives indications about the distribution of species during the LGM (23 000–16 000 bp ) and provides evidence for the locations of glacial refugia for mammalian species in Europe.
• 3 In Europe, 47 LGM sites, dating from 23 000 to 16 000 bp and containing typical temperate mammal species, have been described. The geographical range of these archaeological sites clearly shows a distribution which differs from the hypothesized traditional refuge areas of the temperate fauna. A considerable number of sites situated in the Dordogne in south‐western France and the Carpathian region contain records of red deer Cervus elaphus, roe deer Capreolus capreolus, wild boar Sus scrofa and red fox Vulpes vulpes.
• 4 The faunal composition of the majority of the evaluated Palaeolithic sites in the southern European peninsulas (with the exception of Greece), as well as France and the Carpathian region, indicates the co‐occurrence of these temperate species with cold‐adapted faunal elements such as mammoth Mammuthus primigenius and/or reindeer Rangifer tarandus.
• 5 The survival of species in Central European refugia would have significant consequences for phylogeography and would be revealed by the dominant distribution of haplotypes, originating from this region. A Carpathian refuge could also be the reason for the very early records of small mammals or mustelids from the Late‐Glacial or Interstadials before the LGM in regions like southern Germany.

     

Late- and Post-Glacial history of the Mustelidae in Europe

1. Analyses of the subfossil records of mustelid species in Europe indicate specific differences in the pattern of temporal and spatial recolonization of central Europe after the maximum glaciation of the last glacial period. 2. For Meles meles, Martes martes and (with some reservations) Mustela putorius it can be seen that the populations were separated in several glacial refugia during the maximum glaciation of the Weichselian. In contrast, the European population of Lutra lutra was restricted to a single glacial refuge, which had not been clearly localized until now. 3. Besides the known glacial refugia of the Iberian Peninsula, Italian Peninsula and the Balkans, there is evidence of possible additional glacial refugia for mustelids near the Carpathians, in western Moldova and in the northern Pontic region. 4. Gulo gulo, Mustela nivalis, and Mustela erminea show adaptations for survival in Pleistocene conditions, but they were historically also distributed in the warmer areas of southern Europe. 5. Among the more thermophilic mustelid species, Mustela putorius is likely to have been the earliest immigrant following the maximum glaciation. Meles meles has been recorded in comparably early times and also seems to be relatively tolerant of climatic extremes. It is clear that Martes martes had already arrived in central Europe during the Allerød, in connection with the recolonization by birch and pine woods. Lutra lutra, by contrast, seems to have been an absolute Holocene immigrant.     

Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.     

Increased genetic structuring of isolated Salamandra salamandra populations (Caudata: Salamandridae) at the margins of the Carpathian Mountains

The traditional southern Pleistocene refugia hypothesis in Europe has lately been challenged for several animal and plant species. The Carpathian Basin, especially at the marginal regions, is one of the recently recognized biodiversity hotspots in Europe. Marginal populations are prone to have lower genetic diversity and higher genetic differentiation than central populations. Here, we examined one mitochondrial DNA fragment (D‐loop) and nine nuclear (microsatellite) loci to describe the genetic diversity and phylogeographical pattern of fire salamander (Salamandra salamandra) populations in the Carpathian Basin with focusing on the southern margins of the Western Carpathians, where isolated populations of this species are present. Analyses of microsatellites indicated reduced genetic diversity for most of the isolated populations. Based on the mitochondrial DNA, only two haplotypes were found, whereas the analyses with the nuclear markers revealed a more recent genetic split between Western (Alpine) and Eastern (Carpathian) populations, and separated the Apuseni Mountains population (part of the Western Carpathians). Using approximate Bayesian computation analyses, we identified the most probable colonization scenario for the isolated North Hungarian Carpathian Basin populations. The split between isolated salamander populations from the central populations in the Carpathian Mountains dates back to the beginning of the Late Pleistocene, while the split between most of the Hungarian populations can be associated with the Last Glacial Maximum. We found evidence for long‐time isolation between the marginal Carpathian Basin and central populations. Our results also show that S. salamandra survived glacial periods in the temperate forests of north‐east Pannonia (North Hungarian Mountains), confirming that the Carpathian Basin served as important northerly refugia during the Pleistocene climatic oscillations.     

Quaternary history of the European roe deer Capreolus capreolus

• 1 The European roe deer Capreolus capreolus is a typical faunal element of the Holocene. It was already present in Europe at least 600 000 years ago and it has been known from both glacial and interglacial phases since then. With nearly 3000 fossil and subfossil records, it is one of the most frequent mammals in the Late Quaternary.
• 2 During the Middle and Late Weichselian Pleniglacial, the distribution of the roe deer was not restricted to the Mediterranean peninsulas but repeatedly reached regions of central Europe. In contrast to that, roe deer records from the Last Glacial Maximum (LGM, 21.0–14.5 ka ¹⁴C BP) are largely confined to the Mediterranean peninsulas – with the exception of south‐western France and the surroundings of the Carpathians where several records attest to its occurrence during the LGM.
• 3 During the Greenland Interstadial 1 (12.5–10.8 ka ¹⁴C BP), the species' distribution extended further north and the roe deer appeared north of the Alps and reached regions of central Germany. This seems to be correlated with the abrupt change to more favourable environmental conditions during this period. It is very likely that the roe deer disappeared north of the Alps during the Younger Dryas cooling (10.8–10.0 ka¹⁴C BP). The northern regions of the central European lowlands were recolonized by roe deer during the late Preboreal 9.7–9.5 ka ¹⁴C BP for the first time since the Weichselian Glacial.
• 4 The combined pattern of genetic data and fossil records of European roe deer suggests several regions in the Iberian peninsula, southern France, Italy and the Balkans as well as in the Carpathians and/or eastern Europe as glacial refugia. It further suggests that C. capreolus might have recolonized most parts of central‐northern Europe out of one or more eastern European (not Balkan) and/or Carpathian refugia. This recolonization wave might have blocked immigration from the traditional Mediterranean areas.

     

Could brown bears (Ursus arctos) have survived in Ireland during the Last Glacial Maximum?

Brown bears are recorded from Ireland during both the Late Pleistocene and early–mid Holocene. Although most of the Irish landmass was covered by an ice sheet during the Last Glacial Maximum (LGM), Irish brown bears are known to have hybridized with polar bears during the Late Pleistocene, and it is suggested that the Irish brown bear population did not become extinct but instead persisted in situ through the LGM in a southwestern ice-free refugium. We use historical population modelling to demonstrate that brown bears are highly unlikely to have survived through the LGM in Ireland under any combination of life-history parameters shown by living bear populations, but instead would have rapidly become extinct following advance of the British–Irish ice sheet, and probably recolonized Ireland during the end-Pleistocene Woodgrange Interstadial from a closely related nearby source population. The time available for brown bear–polar bear hybridization was therefore restricted to narrow periods at the beginning or end of the LGM. Brown bears would have been extremely vulnerable to extinction in Quaternary habitat refugia and required areas substantially larger than southwestern Ireland to survive adverse glacial conditions.     

Fossil evidence and phylogeography of temperate species: 'glacial refugia' and post-glacial recolonization

We present a short synthesis of the Pleistocene distribution dynamics and phylogeographic recolonization hypotheses for two temperate European mammal species, the red deer ( Cervus elaphus ) and the roe deer ( Capreolus capreolus ), for which high-resolution patterns of fossil evidence and genetic data sets are available. Such data are critical to an understanding of the role of hypothesized glacial refugia. Both species show a similar pattern: a relatively wide distribution in the southern part of Central Europe 60,000–25,000 years ago, and a strong restriction to areas in southern Europe for nearly 10,000 years during the Last Glacial Maximum (LGM) and the early Late Glacial (25,000–14,700 years ago). With the beginning of Greenland Interstadial 1 (Bølling/Allerød warming, c. 14,700–11,600 years ago) a sudden range expansion into Central Europe is visible, but the colonization of most of Central Europe, including the northern European Lowlands, only began in the early Holocene. In a European context, regions where the species were distributed during the LGM and early Late Glacial are most relevant as potential origins of recolonization processes, because during these c. 10,000 years distribution ranges were smaller than at any other time in the Late Quaternary. As far as the present distribution of temperate species and their genetic lineages is concerned, so-called 'cryptic refugia' are important only if the species are actually confirmed there during the LGM, as otherwise they could not possibly have contributed to the recolonization that eventually resulted in the present distribution ranges.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

Exceptional maternal lineage diversity in brown bears (Ursus arctos) from Turkey

The genetic diversity and phylogeography of maternal lineages in Ursus arctos Linnaeus, 1758 (the brown bear) have been studied extensively over the last two decades; however, sampling has largely been limited to the northern Holarctic, and was possibly biased towards lineages that recolonized the vast expanses of the north as the Last Glacial Maximum (LGM) ended. Here we report the genetic diversity and phylogeography of U. arctos from Turkey based on 35 non‐invasive samples, including five from captive individuals. Bayesian phylogenetic analyses based on a 269‐bp fragment of the mitochondrial DNA control region revealed 14 novel haplotypes belonging to three major lineages. The most widespread lineage was found to be the Eastern clade 3a, whereas geographically more restricted Western and Middle Eastern lineages were reported for the first time in Turkey. A specimen from the Taurus mountain range carried a haplotype closely related to the presumably extinct bears in Lebanon. Moreover, we identify a unique new lineage that appears to have split early within the Middle Eastern clade. Despite limited sampling, our study reveals a high level of mitochondrial diversity in Turkish U. arctos, shows that the ranges of both European and Middle Eastern clades extend into Turkey, and identifies a new divergent lineage of possibly wider historical occurrence. Obtaining these results with 35 samples also demonstrates the value of proper sampling from regions that have not been significantly affected by the LGM. © 2015 The Linnean Society of London     

Genetics and conservation of European brown bears Ursus arctos

• 1 We review the genetics research that has been conducted on the European brown bear Ursus arctos, one of the genetically best‐studied mammalian species.
• 2 The first genetics studies on European brown bears were on phylogeography, as a basis for proposed population augmentations. Two major mitochondrial DNA lineages, western and eastern, and two clades within the western lineage were found. This led to a hypothesis that brown bears had contracted to southern refugia during the last glacial maximum. More recent results suggest that gene flow among brown bears blurred this structure and they survived north of these putative refugia. Thus, today's structure might be a result of population fragmentation caused by humans.
• 3 The nuclear diversity of European brown bears is similar in range to that in North American bears: low levels occur in the small populations and high levels in the large populations.
• 4 Many non‐invasive genetic methods, developed during research on brown bears, have been used for individual identification, censusing populations, monitoring migration and gene flow, and testing methods that are easier to use in endangered populations and over large areas.
• 5 Genetics has been used to study many behavioural and population ecological questions that have relevance for the conservation and management of brown bears.
• 6 The European brown bear has served, and will continue to serve, as a model for the development of methods, analyses and hypotheses in conservation genetics.

     

Multiple Glacial Refugia of the Low-Dispersal Ground Beetle Carabus irregularis: Molecular Data Support Predictions of Species Distribution Models

Classical glacial refugia such as the southern European peninsulas were important for species survival during glacial periods and acted as sources of post-glacial colonisation processes. Only recently, some studies have provided evidence for glacial refugia north of the southern European peninsulas. In the present study, we combined species distribution models (SDMs) with phylogeographic analyses (using mitochondrial DNA = mtDNA) to investigate if the cold-adapted, stenotopic and flightless ground beetle species, Carabus irregularis, survived the Last Glacial Maximum (LGM) in classical and/or other refugia. SDMs (for both a western European and for a Carpathian subgroup) were calculated with MAXENT on the basis of 645 species records to predict current and past distribution patterns. Two mtDNA loci (CO1 and ND5, concatenated sequence length: 1785 bp) were analyzed from 91 C. irregularis specimens to reconstruct the phylogeography of Central and eastern European populations and to estimate divergence times of the given lineages. Strong intra-specific genetic differentiation (inter-clade Φ_ST values ranged from 0.92 to 0.99) implied long-term isolation of major clades and subsclades. The high divergence between the nominate subspecies and the Carpathian subspecies C. i. montandoni points to two independent species rather than subspecies (K-2P distance 0.042 ± 0.004; supposed divergence of the maternal lineages dated back 1.6 to 2.5 million years BP) differing not only morphologically but also genetically and ecologically from each other. The SDMs also inferred classical as well as other refugia for C. irregularis, especially north of the Alps, in southeastern Europe and in the Carpathians. The coincidences between the results of both methods confirm the assumption of multiple glacial refugia for the studied species and the usefulness of combining methodological approaches for the understanding of the history of low-dispersal insect species.     

Tracing the evolutionary history of the mole,Talpa europaea,through mitochondrial DNA phylogeography and species distribution modelling

Our understanding of the effect of Pleistocene climatic changes on the biodiversity of European mammals mostly comes from phylogeographical studies of non‐subterranean mammals, whereas the influence of glaciation cycles on subterranean mammals has received little attention. The lack of data raises the question of how and to what extent the current amount and distribution of genetic variation in subterranean mammals is the result of Pleistocene range contractions/expansions. The common mole (Talpa europaea) is a strictly subterranean mammal, widespread across Europe, and represents one of the best candidates for studying the influence of Quaternary climatic oscillation on subterranean mammals. Cytochrome b sequences, as obtained from a sampling covering the majority of the distribution area, were used to evaluate whether Pleistocene climate change influenced the evolution of T. europaea and left a trace in the genetic diversity comparable to that observed in non‐subterranean small mammals. Subsequently, we investigated the occurrence of glacial refugia by comparing the results of phylogeographical analysis with species distribution modelling. We found three differentiated mitochondrial DNA lineages: two restricted to Spain and Italy and a third that was widespread across Europe. Phylogenetic inferences and the molecular clock suggest that the Spanish moles represent a highly divergent and ancient lineage, highlighting for the first time the paraphyly of T. europaea. Furthermore, our analyses suggest that the genetic break between the Italian and the European lineages predates the last glacial phase. Historical demography and spatial principal component analysis further suggest that the Last Glacial Maximum left a signature both in the Italian and in the European lineages. Genetic data combined with species distribution models support the presence of at least three putative glacial refugia in southern Europe (France, Balkan Peninsula and Black Sea) during thelast glacial maximum that likely contributed to post‐glacial recolonization of Europe. By contrast, the Italian lineage remained trapped in the Italian peninsula and, according to the pattern observed in other subterranean mammals, did not contribute to the recolonization of northern latitudes. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 495–512.     

Multiple cryptic refugia of forest grass Bromus benekenii in Europe as revealed by ISSR fingerprinting and species distribution modelling

Despite not having been fully recognized, the cryptic northern refugia of temperate forest vegetation in Central and Western Europe are one of the most important in the Holocene history of the vegetation on the subcontinent. We have studied a forest grass Bromus benekenii in 39 populations in Central, Western and Southern Europe with the use of PCR-ISSR fingerprinting. The indices of genetic population diversity, multivariate, and Bayesian analyses, supplemented with species distribution modelling have enabled at least three putative cryptic northern refugial areas to be recognized: in Western Europe—the Central and Rhenish Massifs, in Central Europe—the Bohemia–Moravia region and in the Eastern/Western Carpathians. Central Poland is the regional genetic melting-pot where several migratory routes might have met. Southern Poland had a different postglacial history and was under the influence of an Eastern/Western Carpathian cryptic refugium. More forest species should be checked in a west–east gradient in Europe to corroborate the hypothesis on the Western European glacial refugia.