Phylogeography of European roe deer (Capreolus capreolus) in Central and Eastern Europe

The roe deer (Capreolus sp.) are one of the most numerous and widespread mammals of Cervidae in Eurasia. They inhabit diverse environments including forests and open areas, lowlands and mountains. They are able to adapt to various climatic conditions and play an important role as a game species. Nowadays genus Capreolus consists of two sister species: European roe deer (C. capreolus Linnaeus, 1758) and Siberian roe deer (C. pygargus Pallas, 1771). In general, the contemporary ranges of the two species are distinct geographically. The Siberian roe deer is distributed throughout the continental Asia, while the European species occurs throughout Europe with the exception of northernmost Scandinavia, Ireland and the Mediterranean Sea islands. The two species are co-occurring only in the region of Volga-Don rivers but until the early 20th century, their ranges were also overlapping in the northern Caucasus. Siberian roe deer is larger in size and better adapted to colder climatic conditions than European roe deer. Climate fluctuations during the Last Glacial Period between 115,000 and 11,500 years ago played an important role in shaping the current distribution and genetic diversity of European mammals, including the roe deer. During glaciations, the ranges of temperate and thermophile species were limited to southern refugial areas. The contributions by given refugia into postglacial recolonization process varied a lot among species. Those that have very broad biogeographic occurrence (from the Mediterranean to the boreal zones) usually dispersed from several glacial refugia located at both lower and higher latitudes, where isolated populations could have developed adaptations to different climate, habitat and food-related conditions. Large scale phylogenetic studies on European roe deer consistently indicated the division of the contemporary population into three main mitochondrial DNA (mtDNA) lineages – Central, Eastern and Western. The Central lineage has been most common throughout Europe, the Eastern one is restricted mainly to the Balkans, and the Western lineage has been recorded in the Iberian Peninsula, only. On the basis of internal structuring within lineages, only one subspecies (C. c. italicus) was undoubtedly confirmed. In contrast to the European roe deer, the Siberian species has so far been subject of very few studies on phylogeography and the genetic structuring of population. Genetic analyses revealed several haplogroups of this species, but the mapping of their spatial distribution varied among studies. Recent studies revealed the introgression of the Siberian roe deer mtDNA genes in some populations of the European roe deer. This indicated an additional level of the genetic complexity of the species and opened a debate on the sources of introgression. However, the limited sampling allowed neither to define the whole spatial range at which the introgression has occurred nor the relation of mtDNA populations of the introgressed deer to the contemporary populations of the Siberian roe deer. The main aims of my thesis were to: - reveal the roe deer phylogeny, identify the spatial patterns of mtDNA clade distribution and demographic processes at the level of lineages, clades and haplogroups of the species at the European scale (Paper 1); - describe the mtDNA diversity and its spatial pattern in roe deer throughout the species range in Europe and determine the population genetic structure of roe deer including factors affecting it (Paper 2); - identify the general phylogenetic and phylogeographic patterns of the Siberian roe deer in its entire range of occurrence and elucidate the phylogeny and origin of the Siberian roe deer haplotypes found within the European range of roe deer as well as their relation to the entire population of the Siberian deer (Paper 3). I hypothesized that: (1) The mtDNA haplotype diversity of roe deer in Europe is high and the most numerous mtDNA haplotypes will be clustering with the Central and Eastern clades (Paper 1); (2) It is highly probable that there are (not recorded earlier) clades of mtDNA with distribution restricted to Eastern Europe (Paper 1); (3) There had been one or more Last Glacial Maximum (LGM) refugial areas for European roe deer in the eastern part of the continent and populations from those refugia colonized the eastern and northern regions of Europe in the postglacial times (Paper 1); (4) Genetic diversity hotspots are effects of the Siberian roe deer (C. pygargus) mtDNA introgression and the secondary contact of mtDNA clades (Paper 2); (5) We will find different proportion of Siberian roe deer haplogroups in European and Asian populations (Paper 3); (6) Introgression of the Siberian roe deer haplotypes in the European roe deer range is an effect of both human activities (including translocations) and natural processes, which occurred in the past (Paper 3). To investigate the haplotype diversity and distribution of clades, I sequenced a fragment of the mtDNA control region (610 bp) from 1469 European roe deer samples collected in 21 countries in central and eastern Europe. Additionally, I included 1541 roe deer sequences available in GenBank and their frequencies in different populations reported in scientific publications. I assigned the obtained sequences to haplotypes and revealed the internal structure of haplogroups according to the results of modelling of the genetic trees and networks. Summary statistics were calculated for three classification levels: lineage, clade and haplogroup. Neutrality tests were performed to evaluate possible models of demographic fluctuations (expansion or bottleneck). The reconstruction of the potential LGM refugial range of European roe deer was based on results of mtDNA analyses supported by fossil and subfossil records of the species provided in the literature (Paper 1). To analyze population genetic structure and define hotspot regions I used sequences obtained in the first part of the study (Paper 1) and clustered them into the geographically defined demes. For each deme I calculated diversity indices and extrapolated them on the area of whole Europe. The population genetic structure of roe deer in Europe was investigated also by using several clustering methods and the results were analyzed to evaluate if the identified genetic populations are in spatial or demographic expansion (Paper 2). To describe genetic diversity of the Siberian roe deer and detect the potential sources of hybridization, I analyzed mtDNA control region fragment of 352 Siberian roe deer samples combined with 139 sequences available in GenBank. I assigned the obtained sequences to haplotypes and revealed internal structure of subclades based on haplotype genealogy of phylogenetic trees and networks. I presented the spatial distribution of subclades on maps to detect phylogenetic structure of species. The genetic structure of the Siberian roe deer population in Eurasia was then determined using the Genaland program. (Paper 3). In Europe, I revealed the presence of two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined towards central regions. Additionally, I confirmed that the European lineage contained three clades (Central, Eastern and Western) but also found that they were composed of several haplogroups, many of which have been separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia (Paper 1). I showed that most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian Pleniglacial period before the Last Glacial Maximum, while the expansion time of the Western clade overlapped with the Eemian interglacial. I also showed that the high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large contiguous range spanning from the Iberian Peninsula to the Caucasus Mts. and in two northern refugia (Paper 1). In the next step (Paper 2), I found two major diversity hotspots in European roe deer. I proposed that they result from the Siberian roe deer (C. pygargus) mtDNA introgression and the secondary contact of the European roe deer mtDNA clades. Significantly lower values of genetic diversity (nucleotide and haplotype diversity) were recorded in the peripheral areas of the species’ range, including the southernmost parts of the LGM refugial areas. I found that, in the eastern parts of the continent, the introgression of mtDNA of C. pygargus has been the main driver of roe deer population subdivision. Spatial genetic analyses showed a complex structure of deer population on a pan-European scale (Paper 2). Analyses of the Siberian roe deer samples (Paper 3) revealed that the shares of different haplogroups in the regional populations varied longitudinally, with a major shift in Central Siberia. Three haplogroups were the most frequent in Europe, while their shares declined or vanished in Asia. I detected the presence of seven genetic populations of the Siberian roe deer, including two populations in the range of the European roe deer with the Siberian mtDNA introgression. I showed that genetic diversity of mtDNA in the Siberian roe deer was greater than documented earlier. Two ancient haplogroups, occurring predominantly in Europe, have been a remnant of the past natural interbreeding between the Siberian and the European roe deer that most probably had occurred hundred(s) kyr BP. Translocations of the Siberian roe deer to Eastern Europe performed in the 19th and 20th centuries have also left signals in the genetic diversity of the European roe deer (Paper 3). To sum up, on the basis of the analysis of mitochondrial DNA fragment of roe deer across Eurasia, I have demonstrated that phylogenetic studies conducted on a wide geographic scale expose signs of species history, which can be missed in regional studies. I confirmed the presence of three, previously known clades, and I described their novel spatial distribution. I discovered some new subclades in the newly studied areas. I proposed that the European roe deer survived LGM in a vast refugial area. The postglacial recolonization have created the genetic diversity hotspots of roe deer in Central and Eastern Europe. I detected many new regions of the past introgression of C. pygargus into C. capreolus, especially in Central and Eastern Europe, and revealed two sources (natural and human-related) of this process.