Peter Schlup Stefan T. Hertwig (Eds.) Of Bears and Men
Peter Schlup, Stefan T. Hertwig (Eds.)
Of Bears and Men Conclusions of the 1st International Bear Meeting IBEAR
Haupt Verlag
We are grateful to the insurance Die Mobiliar and the Bugergemeinde Bern for funding this publication:
1st edition: 2019 This publication is listed in the Deutsche Nationalbibliografie; detailed bibliographic data are available at: http://dnb.dnb.de The publisher Haupt Verlag was awarded a grant from the Federal Office of Culture for the period 2016 to 2020. ISBN 978-3-258-08145-8 All Rights reserved. Copyright Š 2019 Haupt Berne Any kind of reproduction without permission of the owner of copyright is not allowed. Copyright photos: unless otherwise stated the photos were taken by the respective authors. The photos between each article were all taken by Reno Sommerhalder, Banff, Canada. Editor: Helen Johnson, UK, www.brownfoxlazydog.co.uk Cover design: pooldesign.ch Typesetting: Die Werkstatt Medien-Produktion GmbH, D-GÜttingen Printed in Germany www.haupt.ch
Contents Introduction By Stefan T. Hertwig A Summary of Brown Bear Genetic Studies: Phylogeny, Phylogeography and Evolutionary Relationships with Polar Bears By Frank Hailer, Verena E. Kutschera, Frank E. Zachos Five hundred years of Bern and bears By Bernd Schildger Keeping bears and wolves together: what effect does this have on the social structure of wolf packs? By Udo Ganslosser and Theresa Ettner Mixed species exhibit of Andean Bears (Tremarctos ornatus) and South-American Coatis (Nasua nasua) in the “Sangay mountainous cloud forest” at Zoo Zurich: a review of 21 years of the enclosure’s operation. By Cordula Galeffi and Dr. Alex Rübel, Zoo Zurich Possibilities for the diagnosis and treatment of fractures in bears By J. Thielebein, Ch. Lischer, Ch. Klaus, R. Georgi, S. Troll, D. Wujciak, U. Fischer, A. Filz, M. Biller, T. Theuß, E. Ludewig, M. Gerwing und I. Kiefer The use of minimally invasive surgery on bears: worthwhile or a bit of fun? By Dr. Dipl. mult. Uwe Ziemann Bear necessities: a study on the rehabilitation of bears, based on the examples of the sub-standard bear parks in Worbis and Schwarzwald, Germany By Rüdiger Schmiedel
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Project Ursina: measures for a low-conflict co-existence with bears in an alpine setting By Joanna Schoenenberger, WWF Switzerland
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Living among bears: possibilities of peaceful coexistence between man and bear By Reno Sommerhalder
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Introduction Bears are among the most fascinating animals on our planet. They long since have played an important role in the mysticism of ancient cultures and even today bears arouse strong emotions – both positive and negative. Their survival and gradual return to their previous range in several parts of Europe has caused concerns, conflicts and legal issues. On other continents, too, bears have come into conflict with the interests of men, and, as a result, the long-term survival of bears is threatened by fear, hunting, poaching and habitat loss. In light of this critical global situation for bears, two Bernese institutions, the Tierpark Bern and the Natural History Museum, organised the International Bear Meeting (IBEAR) in 2014. The aim was to bring together people interested in different aspects of the evolution, conservation and husbandry of bears. The lectures covered various issues, ranging from the phylogeny of bears to the history of bear keeping in Bern. In this conference proceedings, the vast majority of the IBEAR speakers summarise their presentations. This compilation addresses numerous fascinating aspects of bear-related information. Readers will find, for example, an excellent overview of the current knowledge on the complex phylogeny and biogeography of brown bears and their white cousins, the polar bears. Three chapters deal with the life of bears in captivity: as well as discussing historical, ethological, and ethical viewpoints, experiences regarding cohabitation of bears and other mammals are presented alongside ideas regarding the rehabilitation of bears having previously been kept poor conditions. The veterinary care of bears is covered within two articles, which detail diagnostics and surgical care of fractures and discuss the value of minimal invasive surgery. Another chapter describes the problems and perspectives regarding the return of bears in the Swiss Alps. Of particular note are the excellent and emotional illustrative photos provided by Reno Sommerhalder, which show bears in the wild and their relationships to men. In publishing these conference proceedings, we – the editors – aim to make this fascinating melange of expert contributions on bear research available to the public. It is also our intention, and hope, to contribute to an improved understanding of bears and their specific needs in our anthropogenic world. Dr. Stefan T. Hertwig Head Curator Vertebrate Animals Department Natural History Museum Bern
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A Summary of Brown Bear Genetic Studies: Phylogeny, Phylogeography and Evolutionary Relationships with Polar Bears By Frank Hailer1,*, Verena E. Kutschera2,3, Frank E. Zachos4,5, 6
1
School of Biosciences, Cardiff University, CF10 3AX, Wales, UK Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, 752 36 Uppsala, Sweden 3 Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, 171 21 Solna, Sweden 4 Natural History Museum Vienna, Mammal Collection, 1010 Vienna, Austria 5 Department of Integrative Zoology, University of Vienna, 1090 Vienna, Austria 6 Department of Genetics, University of the Free State, Bloemfontein 9300, South Africa * Correspondence to: F.H., HailerF@cardiff.ac.uk 2
Abstract Brown bears (Ursus arctos) are among the most extensively studied mammal species in terms of genetics. This work has contributed profoundly to our understanding of various aspects of evolutionary biology: (i) Brown bears have essentially become a model organism in phylogeography, exhibiting at least seven different mitochondrial DNA haplogroups, one of which is widely distributed in Eurasia and North America. (ii) Their evolutionary and phylogenetic relationship with their closest relative, the polar bear (Ursus maritimus), has recently been addressed in detail based on different molecular markers, uncovering a complex speciation process involving (probably several) hybridization and introgression events. (iii) The phylogenetic relationships among the extant bears, Ursidae, have also recently been studied in greater detail than before, and analyses have yielded a textbook example of discordance between mitochondrial and nuclear markers due to hybridization and incomplete lineage sorting. In this chapter, we summarise the current thinking in these three areas of research.
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Introduction The brown bear (Ursus arctos) is one of the most widespread and most iconic large carnivore species worldwide. As such, brown bears have stimulated a great deal of human interest both within and outside the scientific community. Indeed, the genetics of few – if any – non-model mammal species has been studied in as much detail. Early phylogeographic studies in the 1990s uncovered a surprising genetic pattern throughout Europe, which has since become paradigmatic as Hewitt’s “bear pattern” (see below). More recently, the brown bear’s evolutionary relationships with its closest relative, the polar bear (Ursus maritimus), have come under scrutiny once again since nuclear genetic analyses called into question the mitochondrial (mt) DNA-based conclusion that the two separated only very recently, and that polar bears were phylogenetically nested within brown bears, rendering the latter paraphyletic. In this chapter, we summarise the present knowledge on the evolutionary genetics of brown bears: we begin with an overall phylogeny of the extant Ursidae, then focus on the latest insights on brown/polar bear speciation and conclude the chapter with a synthesis of the intraspecific range-wide phylogeography of Ursus arctos.
Phylogenetic relationships among extant bears The closest extant relatives of brown and polar bears are the American black bear (Ursus americanus), the Asiatic black bear (Ursus thibetanus), the sun bear (Helarctos malayanus), and the sloth bear (Melursus ursinus), constituting the monophyletic taxon Ursinae. Together with the spectacled bear (Tremarctos ornatus), the giant panda (Ailuropoda melanoleuca), and a number of extinct taxa they are included into the Ursidae. Observations of hybridization between different bear species in zoos and in the wild (Gray, 1972; Kelly et al., 2010) indicate that gene flow between bear species might have occurred since their initial divergence. Indeed, the brown bear genome not only shows signals of introgressive hybridization with the polar bear (Miller et al., 2012; Cahill et al., 2013; Liu et al., 2014), but nuclear intron (Kutschera et al., 2014) and genome-wide single nucleotide polymorphism (SNP) data (Miller et al., 2012) also indicate a history of admixture with the American black bear lineage. Moreover, Barlow et al. (2018) found that ancient hybridization with extinct cave bears (Ursus spelaeus complex) contributed ca. 0.9-2.4 % to the genomes of the analysed brown bears. Similarly, Kumar et al. (2017) found widespread gene flow among ursine bears and across the ursine phylogenetic tree. This increasing evidence for gene flow between different bear species provides an intriguing explanation for remaining uncertainties and apparent contradictions in pre-
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
vious phylogenetic analyses of bears (Yu et al., 2004; Pagès et al., 2008; Nakagome et al., 2008). Gene flow causes some loci in the genome to show a different phylogeny than the original branching pattern of the species, which complicates inferences of the species tree (Nichols, 2001). Another evolutionary process that can cause gene tree discordance (and thereby complicate phylogenetic inferences) is incomplete lineage sorting (ILS) (Nichols, 2001). After the divergence of two populations, ancestral polymorphisms remain for a certain time period in both descendant populations (that time depends upon the effective population size and if and how the loci are under selection). If the ancestral population had a high diversity and/or if the time between divergence events was short, ancestral polymorphisms may not have been sorted completely according to the true species tree at the time of the following divergence event. In that case, a proportion of the genome remains incongruent with the species tree with a certain probability (Tajima, 1983; Pamilo & Nei, 1988). The lineage sorting process is stochastic and can be modelled using coalescence theory (Kingman, 1982; Hudson, 1991). Several studies suggested that the genomes of bears show signals of widespread ILS, reflecting their relatively recent evolutionary divergence and, thus, a short amount of time for complete sorting of polymorphisms at individual loci on their autosomes into reciprocally monophyletic clades (Miller et al., 2012; Hailer et al., 2013; Liu et al., 2014; Kutschera et al., 2014). In contrast, the mitochondrial (mt) DNA and Y-chromosomal lineages, which sort more rapidly (due to lower effective population sizes), show reciprocal monophyly for most species (but note the case of polar/brown bears; see below) (Waits et al., 1999; Yu et al., 2004; Nakagome et al., 2008; Pagès et al., 2008; Kutschera et al., 2014). Based on analyses of genome-wide data from all extant ursid species, however, Kumar et al. (2017) suggested that gene flow better explained the phylogenetic conflict among bears than ILS. The fossil record and divergence time estimates from mitochondrial genomes suggest a rapid radiation of Ursinae (KurtÊn & Anderson, 1980; Krause et al., 2008); a time frame that may not have provided enough time for complete lineage sorting among bear species. However, the phylogenetic methods that were available at the time of the first phylogenetic studies in bears did not model ILS. Moreover, only complementing gene flow estimates would have made further interpretations of branches with low statistical support possible. In a phylogenetic study of ursids, Kutschera et al. (2014) analysed nuclear sequence data of several individuals from each extant bear species. To increase the phylogenetic resolution compared to earlier studies, nuclear introns were analysed. Compared to exons, introns are less affected by purifying selection and thus typically evolve at a faster rate (Friesen, 2000). In addition, the authors analysed Y-chromosomal markers to specifically study the evolutionary history of male lineages. Analyses of whole mitochondrial genome sequences complemented the picture obtained from nuclear data. Coalescence theory-based multi-locus approaches were used to model the lin-
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eage sorting process in phylogenetic and gene flow analyses. Several haplotypes and variants were shared among species at nuclear loci, likely reflecting ILS in bears. Divergence time estimates obtained from phylogenetic analyses of multiple nuclear loci confirmed a rapid radiation of Ursinae (Table 1) – further indication of insufficient time for complete lineage sorting between divergence events in bears. Figure 1 gives an overview of the phylogenetic results. In trees based on multiple nuclear loci, the six Ursinae clustered together (Figure 1). The spectacled bear was the sister taxon to all Ursinae, and the giant panda was sister to all other extant bears. Within Ursinae, the American black bear was placed as sister taxon to a brown/polar bear clade, and the three Asian bear species (Asiatic black bear, sun bear and sloth bear) formed a monophylum with high statistical support. Phylogenetic relationships within the Asian clade remained unresolved, which was further reflected in phylogenetic conflict among gene trees (Figure 1, network). This was consistent with gene flow estimated from the Asiatic black bear into the sloth bear lineage at nuclear loci. Notably, the Asian bears are currently classified in three different genera (Ursus, Melursus, Helarctos), but these new results support a re-classification of all extant Ursine bears in one monophyletic genus Ursus. Kumar et al. (2017), in a phylogenomic analysis based on 869 mega base pairs and modelling ILS, confirmed the species tree topology found by Kutschera et al. (2014), but found significantly older divergence times (Table 1), more similar to previous estimates from mitochondrial genomes (Krause et al., 2008). A network analysis of genome-wide data (Kumar et al., 2017) found similarly strong signals of phylogenetic conflict as in the analysis of nuclear introns shown in Figure 1 (network from Kutschera et al., 2014), but with the American black bear placed between polar/brown bears and the Asian bear clade. Table 1. Divergence time estimates obtained from starBEAST based on multiple nuclear loci (14 autosomal introns and Y-chromosomal sequence; Kutschera et al. 2014). A minimum root height of 11.6 million years (Abella et al. 2012) was used to calibrate the tree. The older estimates found by Kumar et al. (2017) are given in grey.
*
Split in the nuclear phylogeny
Estimated divergence time, in million years before present (95 % HPD* interval)
Giant panda / (spectacled bear + Ursinae)
12.46 (11.6–14.48)
–––
Spectacled bear / Ursinae
5.88 (4.67–7.18)
10.6 (6.7–13.0)
(Polar + brown + American black bear) / (Asiatic black + sun + sloth bear) 1.78 (1.42–2.2)
5.0 (4.5–6)
Asiatic black bear / (sun + sloth bear)
1.56 (1.2–1.96)
4.4 (3.6–5.8)
Sun / sloth bear
1.42 (1.04–1.81)
3.6 (2.4–5.6)
American black bear / (polar + brown bear)
0.94 (0.67–1.25)
3.4 (2.0–4.7)
Polar / brown bear
0.62 (0.38–0.89)
0.9 (0.6–1.1)
HPD: highest posterior density
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
In the analysis by Kutschera et al. (2014), nuclear and mtDNA topologies differed from each other with high statistical support (Figure 1): the placement of the American black bear as sister taxon to polar bears and brown bears at nuclear loci, but with the Asiatic black bear in the mtDNA gene tree was consistent with nuclear gene flow from the brown bear into the American black bear lineage. Kutschera et al. (2014) introduced two hybridization scenarios that could explain the incongruent placement of the American black bear in nuclear and mtDNA phylogenetic trees: 1) the replacement of the original American black bear mitochondrial genome by an Asiatic black bear-like lineage through introgressive hybridization (mitochondrial capture), leading to a matrilineal sister relationship of the two species for the mitochondrial genome. 2) alternatively, nuclear swamping of the American black bear genome by genetic material from brown bears through male-mediated gene flow, causing the placement of the American black bear close to brown/polar bears in the nuclear tree. Hence, discrepancies between nuclear phylogenies and the mtDNA gene tree of bears (Yu et al., 2007; Krause et al., 2008) can be reconciled when introgressive hybridization during the ursid evolutionary history is considered (Kutschera et al., 2014). Indeed, Kumar et al. (2017) found signals of gene flow between the American and Asiatic black bears, supporting hybridization scenario 1. We anticipate that additional genome-wide studies complementing phylogenomic with population genomic approaches will be necessary to further understand the processes leading to the observed gene tree discordance in bears. We hypothesise that analysis of ancient DNA will play a prominent role in this process, following up on the recent study of cave and brown bears by Barlow et al. (2018). In conclusion, the bear family emerges as a textbook example of reticulate evolution (e.g. Allendorf et al., 2012), emphasising the utility of coalescence theory-based approaches (which allow modelling and thus incorporating ILS in the inference) to study the evolutionary processes of divergence and gene flow among closely related species. Introgressive hybridization may have provided the receiving species with alleles already adapted from the donor species (adaptive introgression) (Hedrick, 2013; for a potential example in hares, Lepus spp., see Alves et al., 2008). Such pre-adapted variants may have been especially advantageous during the strong and recurrent climatic and habitat changes of the Pliocene (5.3–2.6 million years before present [YBP]) and Pleistocene (2.6 million–12 thousand YBP; http://quaternary.stratigraphy.org), the time period during which most splits within the extant Ursidae species appear to have occurred.
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Figure 1. Phylogenies of ursids. Left corner: Consensus network of 14 autosomal gene trees obtained from a starBEAST (Drummond et al. 2012) analysis. All splits found in at least two gene trees are shown. Number of individuals analysed per species is shown in brackets. Left phylogenetic tree: Maximum credibility tree of multiple nuclear loci estimated in starBEAST (14 autosomal introns and 5.9 kb of Y-chromosomal sequence). Right phylogenetic tree: Maximum credibility tree of mitochondrial (mt) DNA (concatenated protein-coding genes, excl. ND6) estimated in BEAST. Black dots at nodes in both phylogenetic trees indicate posterior support >0.95. Modified from Kutschera et al. (2014). Bear illustrations: © Fauna, http://www.fauna.is.
Brown and polar bears: hybridization, introgression and speciation Further indications for the role of introgressive hybridization – or in other words, reticulate evolution – are found in the relatively recent evolutionary history of brown and polar bears. Classical taxonomists, such as Kurtén (1964), had already suspected that these two species were closely related. Kurtén analysed morphometric data and concluded that the two species likely diverged in the Middle Pleistocene, an epoch between 781,000–126,000 YBP (http://quaternary.stratigraphy.org). Indeed, the oldest known polar bear fossil known at the time (found near Kew Bridge, London) only dated back to the Late Pleistocene, a period which began 126,000 YBP. Kurtén therefore suggested a scenario of polar bear evolution in which a northern brown bear population would have been isolated in northerly areas, ultimately adapting to novel food resources and the Arctic climate.
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
Early results obtained from mtDNA showed an intriguing pattern: while, as expected, polar and brown bears were each other’s closest relatives, polar bears were nested within brown bear diversity rather than clustering separately (Figure 2A). This finding, originally reported by Cronin et al. (1991) and consistently confirmed by later work on mtDNA (e.g. Talbot & Shields, 1996; Waits et al., 1999; Leonard et al., 2000), rendered brown bears paraphyletic, because not all descendants of the ancestor of all brown bears were brown bears. Some brown bears, in particular those from the Admiralty, Baranof, and Chichagof (ABC) Islands off the Alaskan Pacific coast, appeared to be more closely related to polar bears than to other conspecific brown bears. The next crucial step towards our understanding of bear evolutionary history was made by the analysis of nuclear, biparentally inherited DNA. Work by Yu et al. (2004), Pagès et al. (2008) and Nakagome et al. (2008) yielded phylogenies that again showed polar and brown bears as each other’s most closely related extant bear species. However, these studies were based on only a single representation per species, precluding inferences about paraphyly versus a sister lineage relationship: based on analysis of one sequence per species, it was not possible to assess whether one lineage was nested within the diversity of another species. In 2009, Ingólfsson and Wiig reported the discovery of a subfossil polar bear jawbone in coastal sediment on Svalbard (Barents Sea). Infrared stimulated luminescence dating suggested that the jawbone was ca. 80,000–150,000 years old. Further stratigraphic information indicated an age of 110,000–130,000 YBP, and stable isotope analysis (Lindqvist et al., 2010) was consistent with a marine diet, as expected for polar bears rather than the terrestrial brown bear. The picture became even more surprising, because Lindqvist et al. (2010) were able to recover and sequence the whole mtDNA genome of the jawbone individual – the oldest mtDNA genome sequenced at the time. Phylogenetic analysis placed the jawbone’s mtDNA adjacent to, but outside, current polar bear variation. Utilising the estimated jawbone age to date the inferred mtDNA branching events, Lindqvist et al. (2010) found that this individual had lived just ca. 20–30 thousand years after the brown/polar bear mtDNA split, which was dated to ca. 150,000 YBP. These findings indicated that the animal’s morphology and feeding behaviour were already similar to that of today’s polar bears. Such a short time scale for the emergence of a distinct polar bear phenotype was striking, making polar bears a textbook example of unusually rapid mammalian speciation and adaptation. Several subsequent studies have provided independent evidence that mtDNA in brown and polar bears shows a phylogenetic pattern that deviates from their original species tree, i.e. that their original evolutionary divergence is earlier than mtDNA phylogenies would indicate. Edwards et al. (2011) retrieved partial mtDNA control region sequences from Late Pleistocene and Holocene brown bears from Ireland, showing the latter to be closely related to the extant polar bear lineage. The authors suggested three scenarios to explain this result: aside from the (by then) classical ‘very recent
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divergence and paraphyly’ hypothesis (see Figure 2A), two hypotheses proposing a Late and Middle Pleistocene divergence between the two bear species were put forward (see Edwards et al., 2011 for details). Indeed, when analysing independently inherited intron loci from the nuclear genomes of brown, polar and American black bears, Hailer et al. (2012) found that brown and polar bears form distinct, reciprocally monophyletic sister lineages (Figure 2B). The authors estimated the lineage divergence to have occurred ca. 600,000 YBP (95 % confidence intervals ranging from 338,000– 940,000 YBP). While their analyses only found tentative evidence of gene flow from brown into polar bears at one locus, the genomes of the analysed individuals appeared largely pure, at least with regard to recent admixture. However, since numerous nuclear loci also showed patterns of paraphyly when analysed independently from each other (Nakagome et al., 2013) – especially when including American black bears in the analysis (Hailer et al., 2013) – both gene flow and ILS appear to have an impact on topologies at individual nuclear loci (Hailer et al., 2013). A similar result was later obtained from numerous additional individuals analysed for microsatellites, amplified fragment length polymorphism (AFLP) and single nucleotide polymorphism (SNP) markers (Cronin and MacNeil, 2012; Cronin et al., 2013, 2014). Hailer et al. (2012) suggested that brown bear mtDNA paraphyly could have resulted from Late Pleistocene introgression of brown bear mtDNA into polar bears, later replacing the original polar bear lineage. This view was consistent with their finding of polar bears having only ca. 20 % of the nuclear variation found in brown bears, indicative of past bottlenecks (and/or long-term low effective population size) that would accompany a replacement of mtDNA in polar bears. Taking another large methodological step forward for bear genetics, Miller et al. (2012) sequenced the entire genomes of several polar, brown and black bears, investigating numerous aspects of their evolutionary history and adaptations (see Liu et al., 2014 and Hailer & Welsh, 2016 for reviews of recent findings about the adaptations in brown and polar bears). Miller et al. (2012) also recovered an overall (genome-wide) phylogeny of polar and brown bears as separately clustering lineages, but analysis of two Alaskan ABC-Island individuals suggested that some brown bears carry up to ca. 10 % introgressed genetic material from polar bears. Cahill et al. (2013, 2015) further investigated the genetics of ABC-Island bears, and elegantly suggested a complex scenario of ‘island conversion’ in this population: after initially being colonised by polar bears that got stranded in the area in a phase of Late Pleistocene sea ice retreat, a prolonged and likely ongoing (Paetkau, 1998) phase of immigration of mainland brown bear males led to the gradual replacement of most of the original polar bear genome in this archipelago population. Unlocking the highly informative, paternally inherited Y chromosome for evolutionary analyses in bears, Bidon et al. (2013, 2014, 2015) found that Y-chromosome SNPs and microsatellites show brown and polar bears to constitute sister lineages (or,
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
since this pattern is due to paternal rather than maternal inheritance, ‘brother lineages’). This was consistent with both the genome-wide data suggesting a sister lineage relationship of brown and polar bears, and with the ABC-Island conversion scenario proposed by Cahill and colleagues: with male brown bears seemingly responsible for the influx of genetic material onto the ABC-Islands, an increasing amount of introgression is found for increasingly ‘male’ parts of the genome (mtDNA < X chromosome < autosomes < Y chromosome; Cahill et al., 2013, 2015; Bidon et al., 2014). Despite this work, several important aspects of brown and polar bear evolutionary history remain somewhat unclear. As is common to any phylogenetic dating, uncertainty remains regarding the precise divergence time of brown and polar bears. Liu et al. (2014) used genome sequences and relatively complex demographic models to obtain a time estimate that was towards the lower end of the confidence intervals reported by Hailer et al. (2012), suggesting a divergence at ca. 343,000–479,000 YBP. Kumar et al. (2017) reported older divergence time estimates at ca. 600,000–1,100,000 YBP based on entire genomes, but still using a phylogenetic model that does not explicitly take demography into account. It is possible that additional genome sequencing and improved demographic modelling will provide more precise estimates of the time since speciation. However, progression of the field is currently also hampered by the absence of any polar bear remains that date back to a time shortly after the speciation. Discovery and analysis of early polar bear remains would likely provide much-needed information. Furthermore, we are still lacking a clear explanation for the seemingly aberrant phylogenetic pattern for mtDNA compared to many other parts of bear genomes (Figure 2) (Hailer, 2015). While introgression of mtDNA could have occurred from brown into polar bears (Hailer et al., 2012; Miller et al., 2012), the opposite direction is also conceivable (Hassanin, 2015). Moreover, ILS could provide an explanation for the observed phylogenetic pattern, without the requirement of introgression. Finally, the presence of brown bear remains found in the fossil record in ca. 1.1 million yearold deposits in Europe (Wagner, 2010), along with a speciation of polar bears a few 100,000 years ago (e.g. Hailer et al., 2012; Liu et al., 2014; Hassanin, 2015) would suggest that the species tree of polar and brown bears might in fact be expected to show a paraphyly pattern – but only if ancient brown bear lineages predating the brown/polar bear split have indeed survived to this day.
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Figure 2. Phylogenetic relationships of brown and polar bears, obtained from differently inherited parts of the genome. A. Paraphyly, based on partial mtDNA control region sequences. Black circles denote possible instances of mtDNA introgression that could explain the apparent incongruence of mtDNA and nuclear phylogenies. B. Reciprocally distinct sister lineages in a species tree based on 14 autosomal intron markers. C. Sister (or rather ‘brother’) lineages in a phylogeny of Y chromosome sequences. Data from Hailer et al. (2012) (A, B) and Bidon et al. (2014) (C). Modified and reproduced with permission from Hailer & Welsh (2016). Bear illustrations: © Fauna, http:// www.fauna.is.
Brown bear phylogeography Brown bears occur, or occurred in historical times, throughout large parts of the Holarctic, similar to wolves (Canis lupus) or red foxes (Vulpes vulpes). They have been eradicated in north-western Africa and large parts of North America and western Europe (Garshelis, 2009). In Japan, they occur only on Hokkaido, i.e. north of Blakiston’s Line, to the south of which they are replaced by the Asiatic black bear. Throughout this vast range, body size varies considerably, ranging from about 100kg in, for example, small Mediterranean and Near Eastern brown bear populations, to more than 700kg in Kodiak brown bears (Garshelis, 2009).
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
Since the 1990s, brown bears have been studied genetically with respect to intraspecific diversity and population genetic structuring (e.g. Hartl & Hell, 1994; Randi et al., 1994; Taberlet & Bouvet, 1994). The first surprising phylogeographic insight from mitochondrial DNA analyses was the presence of deeply divergent lineages within Europe that were found to form a contact zone in Scandinavia. This pattern was interpreted as having emerged from two distinct glacial refugia and subsequent recolonisation of northern Europe along different routes: one from western Europe north towards southern Scandinavia, and another one from the east, north towards Russia and Finland and then south into the Scandinavian peninsula (Taberlet & Bouvet, 1994; Taberlet et al., 1995; but see Bray et al., 2013 below). The two lineages (1a/b and 3a in the nomenclature below) were found to co-occur in the Romanian Carpathians (Kohn et al., 1995; Zachos et al., 2008). The recolonisation pattern of the brown bear (extensive recolonisation from both a western and an eastern glacial refugium, with a suture zone in Scandinavia1) became one of the three paradigmatic postglacial colonisation routes in Europe, the other two being the grasshopper pattern (recolonisation predominantly from the Balkans, after Chorthippus parallelus) and the hedgehog pattern (recolonisation from three refugia â&#x20AC;&#x201C; Iberia, Italy and the Balkans, after Erinaceus roumanicus (formerly E. concolor) and E. europaeus) (Hewitt, 1999, 2000). European species with similar phylogeographic patterns to that of the brown bear are, amongst others, shrews (Sorex araneus, Crocidura suaveolens) and water voles (Arvicola sp.). Among the European carnivores, the brown bear seems to have been the first to recolonise the northern parts of the continent, and its distribution range was only confined to glacial refugia for a comparatively short time: probably no more than 10,000 years during the Last Glacial Maximum (Sommer & Benecke, 2005). A large amount of research on the phylogeography of brown bears has been published over the last 20 years, including a comprehensive summary and review by Davison et al. (2011; Anijalg et al., 2018 add an in-depth analysis of the wide-spread 3a clade in particular). The main results of lineage distribution are shown in Figure 3 which is taken (and slightly adapted) from Davison et al. (2011). A number of haplogroups (as identified in networks) or clades (monophyletic groups identified in phylogenetic trees) have been found; some localised, some more widespread. Among the latter, clade 3a is the most common and widespread, ranging from eastern Europe (from northern Scandinavia in the north to Romania in the south) to eastern Turkey and the Caucasus and through all of northern and temperate Asia (including Hokkaido, Japan) across the Bering Strait into Alaska (Figure 3).
1
This suture zone only exists with respect to mitochondrial DNA, however. Nuclear genetic analyses based on autosomal microsatellite loci and Y-chromosomal markers have revealed substantial male-mediated gene flow throughout Scandinavia (Waits et al., 2000; Schregel et al., 2015), also apparent on continental and cross-continental geographic, and hence on phylogeographic, time scales (Bidon et al., 2014).
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Apart from the extant haplogroups depicted in Figure 3, additional extinct lineages were recovered from ancient DNA analyses: subclades 2c and 3c from North America and a divergent clade from North Africa (from where clade 1a is also known). The extinct North African clade from Algeria and Morocco seems to have been the sister group to all other brown bear clades and the polar bear clade that is nested within them (Calvignac et al., 2008, 2009). Calibration of divergence dates for the various lineages is difficult and contingent upon many assumptions. Davison et al. (2011, Table 1) recalculated node ages using different molecular clock calibrations and compared them to available estimates from the literature, in particular Korsten et al. (2009). These calculations suggest 263 thousand YBP (95 % posterior density intervals: 162–400 thousand YBP) for the last common ancestor of all sampled extant brown bears worldwide, and 45 (10–91) thousand YBP for the youngest analysed clade (2a in North America). However, based on fewer but much longer sequences (complete mtDNA) and a slightly different coverage of clades (lacking, for example, clade 7), Hirata et al. (2013) found values of 566 (251– 944) thousand YBP for all brown bears, and 31 (9–60) thousand YBP for clade 2a. Also based on complete mitogenomes, Anijalg et al. (2018) found similar results: 514 (406–640) thousand YBP for all brown bears/polar bears and also 31 (19–43) thousand YBP for clade 2a. The most wide-spread clade 3a they date to 53 (38–71) thousand YBP. In any case, all clades are clearly older than the Last Glacial Maximum (LGM, ca. 25–18 thousand YBP), showing that phylogeographic patterns in the wake of the LGM resulted from sorting of existing lineages rather than the emergence of new ones (Taberlet et al., 1998; Hewitt, 2000). This is in line with findings from ancient DNA of brown bears and various other species (cave bears, cave hyenas and Neanderthals), which seem to have exhibited little (or no) phylogeographic structuring at the onset of the LGM (Hofreiter et al., 2004). In brown bears, the western and eastern lineages (1a/b and 3a) found separated in Europe today showed a much more overlapping distribution before the LGM: Valdiosera et al. (2008) identified an eastern haplotype in a Pleistocene brown bear from Iberia. In fact, even shortly after the LGM in the Holocene, clades 1 and 3 were more widely spread than today, suggesting that present-day phylogeographic patterns may be due to female philopatry and anthropogenically-caused decline rather than isolation in peninsular refugia (Valdiosera et al., 2007; but see Bray et al., 2013 for contrary evidence from Scandinavia). Furthermore, analysis of ancient DNA has even documented clade 2 haplotypes in western Europe: Edwards et al. (2011) identified this clade in ancient remains of brown bears from Ireland dating from ca. 38 to 9 thousand YBP. Interestingly, these clade 2 haplotypes clustered as a sister lineage to all extant polar bear sequences. Finally, Calvignac et al. (2009) found clade 1 haplotypes in Lebanese brown bears from the 19th century, while a Syrian sample of similar age produced a haplotype from clade 3a. This again shows that the pattern we see today may have
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
been less complex than before the recent human impact on brown bears. For Europe, Hassanin (2015) provided a synthesis of present and past distributions of brown bear haplogroups in Europe (clades 1a, 1b, 2, 3a), showing substantial geographical overlap in southern France and Ireland of subclades 1a and 1b (cf. the present-day distribution; Figure 3). Clade 1b was also found in a fossil brown bear in Denmark, suggesting that the colonisation of southern Scandinavia may have been more diverse and complex than previously recognised (Bray et al., 2013). In conclusion, phylogeographic patterns in European brown bears appear to have changed over time: mitochondrial lineages started to diverge long before the LGM, but came into contact again around the LGM and shortly thereafter, followed by the recent extinction of brown bear lineages as a result of humans. A general shortcoming of phylogeographic studies in brown bears is that there is a bias towards samples from Europe and North America, while more than half of the global population of brown bears lives in Asia. Davison et al. (2011) concluded that, at the time, 4.3 %, 0.3 % and 1.5 % of all bears in Europe, Asia and North America, respectively, had been sampled. Although there have been studies since, the general picture certainly still holds, which means that sampling of the largest population (Asia) is an order of magnitude lower than in other parts of the world. It may not be surprising, therefore, that recent studies on Asian brown bears have yielded new insights. While a distinct clade (now called clade 7) from Iranian brown bears that seems to have shared an early common ancestor with clades 3 and 4 (Davison et al., 2011) was known from previous studies based on five specimens (including two from zoos) (Miller et al., 2006; Calvignac et al., 2009), more recent studies with larger sample sizes have shown that its range reaches into Turkey and that there is considerable sub-structuring into subclades within it (Ashrafzadeh et al., 2016; Çilingir et al., 2016). North America and Japan were colonised by brown bears multiple times independently, as summarised by Davison et al. (2011) based on the available literature on paleontological evidence and both modern and ancient DNA data. Japanese brown bears, present only on Hokkaido (see above), exhibit mtDNA clades 3a, 3b and 4. The latter two clades may have reached Japan at about the same time as they colonised North America, i.e. earlier during the last glacial period, while clade 3a was introduced to Japan during a later colonisation event, probably between 18 and 12 thousand YBP. A recent study based on complete mtDNA sequences (rather than the usually analysed 200–300bp) with a focus on Japanese brown bears has confirmed the overall picture (Hirata et al., 2013). Hokkaido harbours the clades 3a, 3b and 4 (in the centre and north, in the east, and in the south of the island, respectively). Brown bears on the Kuril Islands off the east coast of Hokkaido showed the same clade as the closest Hokkaido bears (3b), and the 3a clade that is found in Hokkaido’s north is also present across the La Pérouse Strait on the Russian island of Sakhalin (although Hokkaido bears show the subclade 3a2, while Sakhalin bears exhibit subclade 3a1).
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Figure 3. a) Approximate geographic distribution of extant mtDNA haplogroups plotted on the present (dark red shade) and historical (light red shade) brown bear distribution range. Haplogroup 2b refers to polar bears (circumpolar distribution, unshaded). Davison et al. (2011) showed haplogroups 1–6 and the Iranian haplogroup, which is now called haplogroup (or clade) 7 and whose approximate distribution is highlighted by the white oval. The T in the white circle refers to clade 7 haplotypes found in north-eastern Turkey by Çilingir et al. (2016), where they co-occur with clade 3a. Iranian clade 7 haplotypes have recently been confirmed in a more detailed study by Ashrafzadeh et al. (2016). b) Biomes in the Northern hemisphere during the LGM ca 18 thousand YBP. c) Putative glacial refugia (dashed outlines) with their hypothesised haplogroups and postglacial recolonisation routes (arrows). Whether the large eastern European/Asian refuge (CM/CA/UR/ CS) was continuous or comprised several separate refugia is uncertain, as is the exact contribution of the European southern peninsulas to the recolonisation. Also, the PC refuge in the American Northwest may not have been occupied by subclade 2a during the LGM. ‘N Africa’ refers to the extinct divergent clade found there (together with clade 1a). For refugial areas in Asia, see also Anijalg et al. (2018). IB: Iberia, IT: Italy and Balkans, CM: Carpathians, CA: Caucasus, UR: Ural Mountains, CS: central Siberia, NAF: North Africa, ME: Middle East, SA: South Asia, JA: Japan, BE: Beringia, PC: Pacific coastal islands, NA: continental North America. Slightly modified (and reproduced with permission from Elsevier) from Davison et al. (2011), Quaternary Science Reviews 30:418-430, p. 419.
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By Frank Hailer, Verena E. Kutschera, Frank E. Zachos
North America was reached via Beringia, the land bridge that formed several times during the Pleistocene when the Bering Strait disappeared due to lower sea-levels during glaciations (Hoffecker & Elias, 2007). The first colonisation occurred about 70,000 YBP (clades 2c, 3c, 4 and possibly 2a). The second colonisation came around 21,000 YBP, i.e. during the LGM, and introduced clade 3b to North America. This suggests that northeast Eurasia was also inhabited by brown bears at this time. Interestingly, brown bears seem to have been absent from Beringia for 12,000 years between 33 and 21,000 YBP, possibly because of competition with the giant North American short-faced bear (Arctodus simus) (Barnes et al., 2002). The final wave of brown bears, carrying clade 3a haplotypes, entered North America before the Bering Strait reappeared and became an insurmountable barrier to migration sometime around 10,000 YBP. According to Anijalg et al. (2018), this last immigration wave reached Alaska ca. 15,000 YBP, after having been present in Beringia for 6–7,000 years without entering North America. This so-called ‘Beringian Standstill’ is also known from other large mammal species, in particular wapitis (Cervus canadensis) and humans (see references in Anijalg et al., 2018). The switch in recent work to investigate entire mitochondrial genomic sequences (mitogenomes) certainly represents an important methodological improvement, and soon sample sizes in terms of numbers of individuals surveyed will be high enough to allow for a geographical coverage similar to the one based on shorter sequences2. Keis et al. (2013) analysed complete mtDNA genomes in 95 brown bears from north-western Eurasia (Estonia, Finland and European Russia) and compared their results to those of the same dataset trimmed to shorter sequence lengths. The increase in resolution power was highly relevant for the inference of demographic history: while the short alignments yielded a single star-like haplogroup, the complete mitochondrial genomes recovered five distinct geographically confined and partially overlapping haplogroups that allowed for an in-depth analysis of distribution history in the region. Nevertheless, nuclear markers are indispensable for a complete picture of present phylogeographic structuring and the reconstruction of the demographic and biogeographical history of brown bears. The genomic revolution will undoubtedly contribute to this and yield further and deeper insights. Nuclear markers will also contribute to a more complete picture because mtDNA, being maternally inherited, only uncovers female-mediated genetic structure. Important insights into sex-biased gene flow can be provided by analysis of the male-inherited Y chromosome. Bidon et al. (2014) recently sequenced 5.3 kilobases of the bear Y chromosome in 90 brown bears across the Holarctic distribution range. Overall, this recovered few variable sites for the sequenced loci – yielding only 5–7 haplotypes (depending on the length of the alignment), with a single dominant haplotype throughout the entire distribution range (Bidon et al., 2014). Along with the pronounced range-wide structuring for maternally inherited mtDNA, this 2
Previously, common sequences shared among studies were usually very short (between 200 and 300 bp).
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suggests that gene flow in brown bears is predominantly mediated by males, and that estimates of population subdivision based on maternally inherited mtDNA provide a particularly structured view. Using the Y markers of Bidon et al. (2014), this has been confirmed for Japanese brown bears where paternally-mediated patterns differed from, and were generally weaker than, maternally-mediated mitochondrial structuring (Hirata et al. 2017). Bidon et al. (2014) also developed and analysed nine Y-linked microsatellite markers (which have since been successfully tested for forensic applications (Aarnes et al., 2015)), and with this strongly increased resolution also found only little evidence of pronounced Y-chromosomal structuring in brown bears. While brown bears from Kamchatka showed a lineage not encountered in any other of the surveyed regions, all other populations contained haplotypes that had their most closely related haplotypes in geographically distant populations. In summary, it thus seems that range-wide genetic structuring in brown bears is largely shaped by female philopatry and male-biased gene flow. However, some mtDNA lineages such as 3a are geographically widespread, and the mechanisms underlying lineage distribution require more investigation: lineages can be dispersed by wide-ranging movement of individuals, or the spread can be favoured by natural selection, despite relatively short-range movement of the carriers of such beneficial alleles. It is thus clear that research is increasingly focussing on pattern rather than process, and that the genomic era is finally seeing researchers investigate the genetic basis of brown bear adaptations, including the species’ persistence across continents and geological time.
References Aarnes, S.G., Hagen, S.B., Andreassen, R., Schregel, J., Knappskog, P.M., Hailer, F., Stenhouse, G., Janke, A., Eiken, H.G. (2015) Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples. Forensic Sci Int Genet 19:197–204 Abella, J., Alba, D.M., Robles, J.M., Valenciano, A., Rotgers, C., Carmona, R., Montoya, P., Morales, J. (2012). Kretzoiarctos gen. nov., the Oldest Member of the Giant Panda Clade. PLoS ONE 7:e48985 Allendorf, F.W., Luikart, G., Aitken, S.N. (2012) Conservation and the genetics of populations. Wiley-Blackwell. 624 pp. Alves, P.C., Melo-Ferreira, J., Freitas, H., Boursot, P. (2008) The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus. Phil Trans Roy Soc B 363:2831–2839 Anijalg, P., Ho, SYW., Davison, J. (2018) Large-scale migrations of brown bears in Eurasia and to North America during the Late Pleistocene. J Biogeogr 45:394–405 Ashrafzadeh, M.R., Kaboli, M., Naghavi, M.R. (2016) Mitochondrial DNA analysis of Iranian brown bears (Ursus arctos) reveals new phylogeographic lineage. Mamm Biol 81:1–9