Palaeontologia Electronica · PALAEO-ELECTRONICA.ORG 3 spelaeus around 28,000 yr BP (32,000 cal....

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Genetic analysis of cave bear specimens fromNiedźwiedzia Cave, Sudetes, Poland

Mateusz Baca, Anna Stankovic, Krzysztof Stefaniak, Adrian Marciszak, Michael Hofreiter, Adam Nadachowski, Piotr Węgleński, and Paweł Mackiewicz

ABSTRACT

The vast majority of fossil remains in Late Pleistocene deposits from NiedźwiedziaCave in Kletno, Sudetes, Poland, belong to the cave bear. Phylogenetic analysesbased on a fragment of the mitochondrial D-loop region extracted from two cave bearsamples unambiguously showed their close relationship with the Ursus ingressus hap-logroup. This taxonomic affiliation of the cave bear remains from Niedźwiedzia Cavewas further confirmed by biometrical analyses of molar teeth and skulls. Our resultsrepresent the first record of U. ingressus north of the Carpathian Arch, while radiocar-bon dating (> 49,000 yr BP) of the samples indicates that they represent some of theoldest specimens of this cave bear taxon known so far. Multi-method phylogeneticanalyses including numerous publicly available cave bear sequences allowed analys-ing the relationships among these samples in details, including the significance of par-ticular clades, and discussing some aspects of cave bear phylogeography. Thesequences of U. ingressus from Poland are most closely related to specimens from theUral Mountains and next to Slovenia, which may indicate migrations between Centraland Eastern European populations. The internal placement of Ural samples amongEuropean specimens in phylogenetic trees and the older age of Polish samples thanthose from Urals suggest that the eastward expansion of U. ingressus may havestarted from Central Europe.

Mateusz Baca (corresponding author). Center for Precolumbian Studies, University of Warsaw, Krakowskie Przedmieście 26/28, 00-927 Warsaw, Poland. [email protected] Stankovic. Institute of Biochemistry and Biophysics, Polish Academy of Science, Pawińskiego 5a, 02-106 Warsaw, Poland. [email protected] Stefaniak. Department of Palaeozoology, Zoological Institute, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland. [email protected] Marciszak. Department of Palaeozoology, Zoological Institute, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland. [email protected] Hofreiter. Department of Biology (Area 2), The University of York, Wentworth Way, Heslington York, YO10 5DD, United Kingdom. [email protected] Nadachowski. Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Cracow, Poland. [email protected] Węgleński. Institute of Biochemistry and Biophysics, Polish Academy of Science, Pawińskiego 5a,

PE Article Number: 15.2.21ACopyright: Palaeontological Association July 2012Submission: 8 September 2011. Acceptance: 5 June 2012

Baca, Mateusz, Stankovic, Anna, Stefaniak, Krzysztof, Marciszak, Adrian, Hofreiter, Michael, Nadachowski, Adam, Węgleński, Piotr, and Mackiewicz, Paweł. 2012. Genetic analysis of cave bear specimens from Niedźwiedzia Cave, Sudetes, Poland. Palaeontologia Electronica Vol. 15, Issue 2;21A,16p; palaeo-electronica.org/content/2012-issue-2-articles/263-cave-bears-from-poland

BACA ET AL.: CAVE BEARS FROM POLAND

02-106 Warsaw, Poland. [email protected]ł Mackiewicz (corresponding author). Department of Genomics, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland. [email protected]

Keywords: ancient DNA; cave bear; phylogeny; Ursus ingressus; Ursus spelaeus

INTRODUCTION

Out of the big mammal species that wentextinct during the Pleistocene, the cave bear isprobably the most thoroughly genetically studiedone. Its fossil material is quite abundant in cavesites across Europe and Asia, and in many casespreserved sufficiently well to allow ancient DNA(aDNA) extraction and analysis (e.g., Hänni et al.,1994; Loreille et al., 2001; Hofreiter et al., 2002,2004a, 2004b, 2007; Orlando et al., 2002; Noonanet al., 2005; Valdiosera et al., 2006; Bon et al.,2008; Krause et al., 2008; Knapp et al., 2009;Stiller et al., 2010). Cave bears diverged from theirsister lineage leading to the brown bear (Ursus arc-tos), approximately 1.2-1.7 million years agoaccording to palaeontological (Kurtén, 1968, 1976;Rustioni and Mazza, 1992; Rabeder and Withalm,2006) and molecular data (Loreille et al., 2001; Bonet al., 2008), although some calculations indicatean earlier split about 2.8 million years ago (Krauseet al., 2008). It is usually assumed that Late Pleis-tocene cave bears (Ursus ex gr. spelaeus) evolvedgradually from the Middle Pleistocene cave bearsfrom the deningeri-group still before the Last Inter-glacial (Mazza and Rustioni, 1994; Rabeder et al.,2000, 2010).

The results of both morphological and geneticstudies have shown that during the Late Pleis-tocene, Europe was inhabited by two main cavebear haplogroups described as separate species,U. spelaeus and U. ingressus (Rabeder, 1995;Hofreiter et al., 2004b; Rabeder and Hofreiter,2004; Rabeder et al., 2004a, 2004b). They sepa-rated between 173,000-414,000 yr BP according tomolecular dating (Knapp et al., 2009). Additionally,among U. spelaeus several high-alpine forms withreduced body size, named U. s. eremus and U. s.ladinicus, were discerned whereas the ‘classic’Western Europe cave bear was called U. s. spe-laeus (Rabeder et al., 2000, 2004a, 2004b, 2008;Hofreiter et al., 2004b; Rabeder and Hofreiter,2004). For a recent review of most described cavebear taxa and a somewhat different conception oftheir classification based on craniometrical fea-tures see Baryshnikov and Puzachenko (2011).According to this study, the morphological differ-

ences between U. spelaeus and U. ingressus donot exceed subspecies level and are too small toconsider them as different species.

A large number of radiocarbon dates revealedthat cave bears became extinct just before the LastGlacial Maximum (LGM), ca. 24,000 yr BP (27,800cal. yr BP; Pacher and Stuart, 2009). Comparisonof effective female population sizes of cave bearsand their sister species, the brown bear, showedthat the genetic decline of cave bears precededtheir extinction by about 25,000 years, while brownbear population size remained constant (Stiller etal., 2010). The reason(s) for the extinction of cavebears are still unclear but it seems that not onlyone, but probably several causes played a role inits disappearance, possibly including climaticchanges, human impact (Stiller et al., 2010), andpredator stress caused by cave lion and cavehyena (Diedrich, 2010).

The phylogeographic picture of cave bears inEurasia does not offer clear indications of themigration routes of cave bears during the Pleis-tocene glacials and interglacials. It has been sug-gested that U. ingressus dominated mainly inEastern and Central Europe while U. spelaeus didso in Western Europe (Hofreiter et al., 2004a;Knapp et al., 2009). In addition to this picture inEurope, remains of both species were also discov-ered in Asia (Knapp et al., 2009). Whereas U. spe-laeus inhabited mainly low and medium elevationareas, U. ingressus has mostly been found inmedium and high elevated regions (Baryshnikovand Puzachenko, 2011). Recent isotopic analysesshowed also some dietary differentiation betweenthese cave bear haplogroups (Bocherens et al.,2011; Dotsika et al., 2011). Ursus ingressus waslikely better adapted to continental environmentsand, thus, might have outperformed U. spelaeusduring cold and arid climate conditions (Barysh-nikov and Puzachenko, 2011). In fact, it was shownthat U. ingressus immigrated into the Alps around50,000 years ago where it replaced two formerpopulations of U. s. eremus and U. s. ladinicus(Rabeder and Hofreiter, 2004; Rabeder et al.,2008). Similarly, the stratigraphically younger U.ingressus replaced quite suddenly the older U. s.

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PALAEO-ELECTRONICA.ORG

spelaeus around 28,000 yr BP (32,000 cal. BP) inthree geographically close caves in the SwabianJura of Germany (Münzel et al., 2011). However,despite the numerous samples that have beengenetically analysed so far, there are a number ofregions from which no genetic data have beenobtained so far and which could contribute to a bet-ter understanding of the complex picture of recentcave bear evolution.

Although fossil remains of cave bears arequite common in Poland, i.e., north of the Car-pathian and Sudetes Mountain ridges, no DNAanalyses were so far performed on cave bearremains from this region of Europe. Such analyseswould be an important step towards reconstructinga more complete picture of phylogeographic struc-ture and genetic variation of cave bears sensu lato.In this paper we present the results of the analysisof cave bear specimens discovered inNiedźwiedzia Cave (Wiszniowska, 1976; Wisznio-wska et al., 1996; Bieroński et al., 2009) located inthe eastern Sudetes Mountains. Phylogenetic anal-yses were performed with a variety of methods toensure that the obtained tree topology was notdependent on a specific method.

MATERIALS AND METHODS

Characterization of Niedźwiedzia Cave, Its Deposits and Palaeoenvironment

Niedźwiedzia Cave (Polish for Bear Cave)(50° 14' 03'' N; 016° 50' 03'' E) is located in the vil-lage of Kletno, Stronie Śląskie commune, in theŚnieżnik Massif of the Sudetes (Figure 1). It wasdiscovered in 1967 during exploitation of crystalinelimestone (marble) in a small quarry at the Kleśnicastream valley. Artificial entrances to the cave aresituated at 800 and 807 m above sea level. Thelength of the known passages is more than 3,000m. A systematic, detailed palaeontological explora-tion of Niedźwiedza Cave from 1967 until todayyielded a huge collection of a few hundred thou-sand vertebrate remains and snail shells (Wisznio-wska, 1976, 1978, 1989; Wiszniowska et al., 1996;Bieroński et al., 2009). The age of the depositscontaining bone debris of Pleistocene animals wasestimated as upper Vistula glaciation (> 50,000 –12,000 yr BP), with Holocene deposits only in thetop parts of some profiles (Wiszniowska, 1989;Wiszniowska et al., 1996; Bieroński et al., 2009).

The radiocarbon dating of cave bear remainsfrom different profiles gave ages from32,100±1,300 to > 49,000 yr BP (Bieroński et al.,2009). All dates correspond to the period MIS 3,

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FIGURE 1. Location of Niedźwiedzia Cave (1 and 2) and position of profiles in the middle horizon of NiedźwiedziaCave (3). Two fossil cave bear specimens used for DNA extraction were excavated from profiles located in Lion’s Halland Primitive Man’s Corridor, respectively.

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from the Grudziądz Interstadial to the main stadialof the Vistula glaciation.

All cave bear remains from diverse localitiesin Poland date to a period before the LGM as in therest of Europe. However, the youngest dates fromPoland (ca. 26,000 yr BP, cal. 31,000 yr BP) areolder than those of cave bears from the Alps, sug-gesting that in areas north of the Sudetes and theCarpathian ranges cave bears became extinct a bitearlier (Nadachowski et al., 2010).

The fossil faunal assemblage fromNiedźwiedza Cave includes 30 mammal species(Wiszniowska, 1989; Bieroński et al., 2009), manyof which live today in the temperate zone and areassociated with meadows, tree stands, and forests,such as field vole Microtus arvalis, European watervole Arvicola terrestris, bank vole Myodes glareo-lus, badger Meles meles, pine marten Martesmartes, brown bear Ursus arctos, wild boar Susscrofa, red deer Cervus elaphus, and roe deerCapreolus capreolus. The remaining species, suchas red fox Vulpes vulpes and wolf Canis lupus, areregarded as euryoecious, i.e., adapted to an arrayof habitats. Extinct mammals are represented bycave bear Ursus ex gr. spelaeus, cave hyena Cro-cuta crocuta spelaea, cave lion Panthera spelaea,and bison Bison priscus. The remains of the cavebear were the most abundant and constituted from70 to 90% of the bone material in all profiles. Theextinct species were components of the faunalassociation of the so-called mammoth steppe,characteristic of cold periods of the Pleistocenewhile the presence of chamois Rupicapra rupic-apra remains indicates open areas or pine forest,or alternatively mountain forests on a rocky sub-stratum.

Fossil Material Used in a DNA Extraction

Two fossil specimens of cave bear used in thisstudy, i.e., a small fragment of the crown of onecave bear’s canine and a proximal epiphysis of aleft ulna bone, were excavated from profileslocated in Lion`s Hall and Primitive Man’s Corridorin Niedźwiedzia Cave (Figure 1.3). They werefound during the 1967-1972 excavations in caveloam deposit, which for the most part filled the

cave’s corridors. They do not provide any morpho-metric diagnostic features to classify them to one ofthe two Late Pleistocene cave bear taxa U. ingres-sus or U. spelaeus. It can only be concluded thatthe relatively small size of the ulna bone (anterio-posterior length ca. 71.0 mm) allows assigning it toa female. The specimens are hosted in the collec-tion of the Palaeozoology Department, ZoologicalInstitute, Wrocław University (ZPALUWr/JN/K).Samples that yielded reliable mtDNA sequenceswere AMS C14 dated in the Poznan RadiocarbonLaboratory, Poznań, Poland (Table 1). The radio-carbon dates were calibrated using CalPal (http://www.calpal.de/).

Contamination Precautions

DNA extraction and PCR setup were per-formed in a laboratory dedicated especially toancient DNA work with positive air pressure. Allstaff wore lab coats, face masks and gloves. Work-ing areas and benches were frequently cleanedwith bleach and DNA ExitusPlus (Applichem). Allthe reactions were carried out in laminar flow cabi-nets with HEPA filters. Only filter tips and steriledisposables were used.

DNA Extraction, Amplification and Sequencing

Prior to DNA extraction each tooth or bonefragment was cleaned with bleach, rinsed withddH2O, UV irradiated for at least 20 minutes oneach side and pulverized in a cryogenic mill (SpexCentriPrep). About 100 mg of powder were incu-bated overnight at 40°C in 1.6 ml of extraction buf-fer (0.5 M EDTA, 0.7 mg of proteinase K (Bioline),0.1 M DTT, 50 mM PTB, 0.5% N-Lauryl sarcosinesalt) with constant agitation. The supernatant wassubjected to phenol:chloroform:isoamyl alcohol(25:24:1, v:v:v) extraction, followed by two chloro-form extractions and isopropanol precipitation(Sambrook et al., 1989). After precipitation, theDNA was resuspended in 60 μl of ddH2O (Fluka).At least two independent extractions were per-formed from each sample. A 285 bp fragment ofmtDNA control region was amplified with threeoverlapping primer pairs (1F: 5’-CTA TTT AAA CTA

TABLE 1. List of samples used in aDNA extraction and associated 14C dates.

DNA sample Number in collection Description Age BP (Laboratory no.) Calibrated age

CB4 KIII/220 Fragment of upper canine 41500±1100(Poz-35024)

45034±1160

CB5 KV Left ulna > 49000(Poz-35025)

NA

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TTC CCT GGT ACA TAC-3’; 1R: 5’-ATG GGGGCA CGC CAT TAA TGC-3’; 2F: 5’-CAT CTC ATGTAC TGT ACC ATC ATA GT-3’; 2R: 5’-TAA ACTTTC GAA ATG TAG GTC CTC ATG-3’; 3F: 5’-GCCCCA TGC ATA TAA GCA TG-3’; 3R: 5’-GGA GCGAGA AGA GGT ACA CGT-3’). All primers weretaken from Hofreiter et al. (2002) with exception ofthe primer 3R, which was redesigned. PCR reac-tions were carried out in 25 μl of reaction mix con-taining 0.2 μM of each primer, 1X AmpliTaq GoldMaster Mix (ABI), 0.1 M BSA and 1.5 μl ofextracted DNA. Initial denaturation of 12 min. at95°C was followed by 40 cycles of 30 sec. at 95 °C,45 sec. at 41°C, 1 min. at 72°C followed by a finalextension of 7 min. at 72°C. PCR products werepurified with SureClean Kit (Bioline) andsequenced. To ensure that PCR products werehomogenous and the obtained sequences werenot erroneous due to post-mortem DNA modifica-tions, each product was cloned with the PCR Clon-ing Kit (Qiagen) according to the manufacturer’sinstruction. Several clones were sequenced fromeach isolation and primer pair and checked for con-sistency.

For two specimens, CB4 and CB5, a 285 bpfragment of the D-Loop sequence was obtained.The sequences were deposited in GenBank underaccession numbers JN112374 and JN112375.

Comparative Biometrical Analysis of Cave Bear Remains

For comparison of the cave bear remains fromNiedźwiedzia Cave with other samples, whose tax-onomic affiliation was indicated by genetic or mor-phological studies, we used length and width of allmolars published by Baryshnikov (2006), Barysh-nikov et al. (2003), Nagel et al. (2005), Rabeder(1995), Rabeder (2004b), Sabol (2005), Tsoukalaet al. (2006) and Wiszniowska (1976). Apart fromNiedźwiedzia Cave (Poland) this data consideredthe following localities: Arcy-sur-Cure (France),Conturines Cave (Italy), Cova Eiros (Spain),Gamssulzen Cave (Austria), Goyet (Belgium),Loutra Arideas Bear Cave (Greece), MedvezhiyaCave (Urals, Russia), Medvedia Cave (Slovakia),Nerubajskoe (Ukraine), Ramesch Cave (Austria),Rübeland (Germany), and Zoolithen Cave (Ger-many). Hierarchical clustering analysis was carriedout on mean values assuming UPGMA(Unweighted Pair-Group Method Arithmetic Aver-ages), agglomerative method and squared Euclid-ean distance measure using the R package (RDevelopment Core Team, 2010). To evaluate thereliability of specific clades in the obtained dendro-

grams we applied approximately unbiased testsand bootstrap resampling assuming 1000 replica-tions.

Skull measurements for six cave bears fromNiedźwiedzia Cave were taken from Wiszniowska(1976) and compared to corresponding measure-ments of cave bear taxa by Baryshnikov andPuzachenko (2011).

Phylogenetic Analyses

To determine the phylogenetic relationships ofthe DNA sequences from Niedźwiedzia Cave, wecompared them to cave bear sequences that weregathered by thorough BLAST searches of the Gen-Bank database. Three U. arctos sequences werechosen as outgroup according to Knapp et al.(2009). After exclusion of incomplete sequenceswe obtained a set of 70 sequences covering thepart of the mitochondrial D-loop region analysed inthis study. Next, we eliminated redundantsequences from this set leaving only one represen-tative for each haplotype. However, all locations ofeach sequence were included in the final tree. Afterremoval of an ambiguously alignable long pyrimi-dine stretch, a final set of 39 aligned sequenceswith 254 bp length was used in all subsequentanalyses.

Phylogenetic trees were inferred using 10approaches. Three Bayesian analyses were doneusing PhyloBayes 3.2f (Lartillot and Philippe, 2004)and one using MrBayes 3.1.2 (Ronquist andHuelsenbeck, 2003). Maximum likelihood (ML)trees were constructed with PhyMl 3.0 (Guindonand Gascuel, 2003) and PAUP* 4.0b (Swofford,1998). Additionally, we applied maximum parsi-mony (MP) and three distance methods: neighborjoining (NJ), minimum evolution (ME) and weightedleast squares (WLS). All trees were reconstructedwith PAUP. The best-fit substitution model HKY+Ias estimated in jModeltest 0.1.1 (Posada, 2008)according to AIC, AICc and BIC criteria, was usedwith MrBayes, ML, NJ, ME and WLS.

For the PhyloBayes analyses, we appliedthree of the available models: Poisson (i.e., F81),GTR and the CAT-Poisson model with the numberof components, weights and profiles inferred fromthe data. Two independent Markov chains were runfor 1,000,000 generations in each of these analy-ses. The last 250,000 trees from each chain werecollected to compute posterior consensus treesafter reaching convergence (i.e., the largest dis-crepancy across all bipartitions was lower than0.015, much below the proposed threshold of 0.1).For the MrBayes analyses, two independent runs

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starting from random trees using four Markovchains each were carried out. Trees were sampledevery 100 generations for 30,000,000 generations.In the final analysis we selected trees from the last6,650,000 generations that reached the stationaryphase and convergence (i.e., the standard devia-tion of split frequencies stabilized and was lowerthan 0.0014, much below the proposed thresholdof 0.01).

We used two heuristic search algorithms inPhyMl, i.e., NNI and SPR. Node support wasassessed by non-parametric bootstrap analysiswith 1000 replicates and by the approximate likeli-hood ratio test (aLRT) based on the 2 and the Shi-modaira-Hasegawa-like procedures (Guindon andGascuel, 2003). The minimum of the support val-ues from the latter two procedures was shown atnodes in the presented tree. In the case of ML, ME,WLS and MP methods used with PAUP, final treeswere searched from 10 starting trees obtained bystepwise addition with random-addition sequence.Non-parametric bootstrap analyses were per-formed with 1000 replicates for each reconstructiondone with PAUP. The tree-bisection-reconnection(TBR) branch-swapping algorithm was applied inthe final tree search and bootstrap procedures forPAUP ML, ME, WLS and MP methods.

The reliability of clades in the resulting treewas also evaluated by the weighted least-squareslikelihood ratio test (WLS-LRT) and F-test asimplemented in the WeightLESS 3.05 program(Sanjuan and Wrobel, 2005). The distance matrixfor the original sequences and 1000 bootstrap dis-tance matrices used in this program were calcu-lated with PAUP assuming the HKY+I model.

Tree topologies obtained by all 10 approacheswere compared according to the approximatelyunbiased test (AU) with the Consel v0.1k program(Shimodaira and Hasegawa, 2001) with10,000,000 replicates. Site-wise log-likelihoods forthe analysed trees were calculated in PAUP underthe HKY+I model.

In addition to the phylogenetic analyses, amedian-joining network (Bandelt et al., 1999)based on the 254 bp alignment of all 70 cave bearsequences was constructed using the Network 4.6software (fluxus-engineering.com). The MaximumParsimony algorithm was used to resolve reticula-tions in the final network (Polzin and Dane-schmand, 2003).

RESULTS AND DISCUSSION

Taxonomic Affiliation of Cave Bear Remains from Niedźwiedzia Cave Based on Biometrical Studies

Although it is possible to discriminate U.ingressus from high-alpine forms of U. spelaeus bydental morphometry and long bones measure-ments (Rabeder, 2004; Rabeder and Hofreiter,2004: Rabeder et al., 2004b, 2008), the morpho-logical differences between U. ingressus and U. s.spelaeus are much less pronounced (Münzel et al.,2009, 2010; Baryshnikov and Puzachenko, 2011).Nevertheless, we compared length and width ofmolars (Figure 2) as well as main skull measure-ments (Figure 3) of cave bear specimens fromNiedźwiedzia Cave with other remains whose taxo-nomic affiliation was confirmed by genetic or mor-phological studies.

Molars from Niedźwiedzia Cave are muchlarger than those from Conturines and Rameschcaves, and in a clustering analysis, they group withspecimens assigned to U. ingressus with statisti-cally significant support (Figure 2). Interestingly,this analysis also showed a clear separation ofmost analysed representatives of the two largecave bear forms U. ingressus and U. s. spelaeusinto two clades. Only teeth from Zoolithen Cave, alocality typical of U. s. spelaeus, were usually clus-tered with U. ingressus. However, these resultsmay be biased by an unbalanced sex ratio of theteeth sets used and a mixture of teeth coming fromdifferent cave bear taxa. For example, geneticanalyses of Zoolithen Cave samples revealed thepresence of mtDNA typical of not only U. s. spe-laeus but also U. ingressus in this cave (Stiller etal., 2009).

The comparison of available skull measure-ments including sexual dimorphism also revealedcloser similarity of the remains from NiedźwiedziaCave to U. ingressus (Figure 3). The medians ofskull measurements calculated by Baryshnikovand Puzachenko (2011) showed an increasingtrend from U. s. eremus, through U. s. spelaeus toU. ingressus. The skull measurements of theNiedzwiedzia Cave specimens fit well into thistrend and generally resemble the ones of U.ingressus skulls.

Dating of Cave Bear Remains from Niedźwiedzia Cave

The sample CB4 was AMS C14 dated to41,500±1,100 yr BP, while sample CB5 was datedto > 49,000 yr BP (Table 1). The most recent AMS

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FIGURE 2. UPGMA cluster dendrogram for all molar teeth (1) and lower carnassial m1 (2) from cave bear speci-mens with confirmed or the most probable taxonomic affiliation. Numbers at nodes correspond to p-valuesexpressed as percentages calculated using approximately unbiased test (AU) and bootstrap resampling (BP),respectively. For full names of localities see Material and Methods.


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FIGURE 3. Comparison of skull measurements from Niedźwiedzia Cave (Nie) specimens with medians calculatedby Baryshnikov and Puzachenko (2011) for Ursus ingressus (Ing), U. s. spelaeus (Spe), and U. s. eremus (Ere): (1)total length, (2) basal length, (3) neurocranium length, (4) viscerocranium length, (5) palatal length, and (6) tooth-rowlength. Triangles denote males, circles females, and squares juveniles.


radiocarbon dating of other cave bear remainsfrom Niedźwiedzia Cave also indicated similarages from 41,300±1,300 to > 49,000 yr BP. Thus,both samples analysed in this work belong to theoldest U. ingressus specimens described so far(Table 2).

Phylogenetic Analyses of mtDNA from Cave Bears

For construction of phylogenetic trees a 254bp long D-loop alignment of 39 cave bearsequences was used. Trees obtained with 10approaches gave almost identical or very similartopologies (Figure 4). The AU test did not showstatistically significant differences between them.

Similarly to the results obtained by Knapp etal., (2009), several main well-supported clades ofcave bears can be recognized in our phylogenies(Figure 4). One clade includes sequencesassigned to U. deningeri kudarensis from the Cau-casus and Siberia (Yana River) (Knapp et al.,2009). The Siberian sample occupies a positionbasal to a strongly supported subclade of cavebear sequences originating from the Caucasus. Asa monophyletic clade, the U. deningeri kudarensisgroup is, however, strongly and moderately sup-ported only by the two PhyloBayes approaches.Thus, the Caucasus clade alone is strongly sup-ported, but the position of the Siberian sequence israther unstable.

Statistical significance in all applied tests,strong support with Bayesian methods (> 0.95) andmoderate support (72-81%) in all other analyseswere obtained for the second large clade that

groups sequences from ‘advanced’ cave bears, U.spelaeus and U. ingressus. Early branches in thisclade are created by two sequences representingalpine forms with reduced body size (named U. s.ladinicus) that were discovered in Merveilleuse andConturines caves. However, their position in thetrees is very poorly supported. Although thesequence from Conturines Cave is grouped in alltrees with U. spelaeus sequences, mostapproaches show an affiliation of the sequencefrom Merveilleuse Cave with U. ingressus and onlyin distance trees (LS, ME and NJ) it is placed basalto the rest U. spelaeus sequences. The latter groupof sequences is well-supported by Bayesian meth-ods (0.75-0.99) and F-test at the 0.001 level. Thisgroup can be further divided into two lineages. Thefirst contains samples from Southwestern andWestern Europe whereas the second consists ofsequences from the Altai, which cluster with speci-mens from Austrian medium-sized cave bearsclassified as U. s. eremus.

The basal positions of polyphyletic lineages ofsmall bears to the large ones suggest that bodymass increased independently in several lineagesduring the evolution of cave bears (Hofreiter et al.,2002). Further supporting this view, recent analy-ses of mtDNA from another small cave bear form,U. rossicus found in Kizel Cave in the Urals, placesthis species close to U. ingressus (Münzel et al.,2010: Pacher et al., 2009). These results togetherwith morphological analyses by Baryshnikov andPuzachenko (2011) that demonstrated substantialsimilarity of U. ingressus to cave bears describedas U. kanivetz from the Volga River region, suggest

TABLE 2. Radiocarbon dates for the oldest known cave bear remains from different localities, in which Ursus ingressus

was reported.

Locality Date (yr BP) Reference

Niedźwiedzia Cave, Poland (CB4) 41,500 This study

Niedźwiedzia Cave, Poland (CB5) > 49,000 This study

Nerubajskoe, Ukraine > 52,450 Pacher and Stuart (2009)

Peştera cu Oase Cave, Romania 51,453 Pacher and Stuart (2009)

Medvedia Cave, Slovakia 51,100 Sabol et al. (2008)

Divje babe I Cave, Slovenia 50,400 Pacher and Stuart (2009)

Vindija Cave, Croatia 48,990 Pacher and Stuart (2009)

Tayn, Secrets Cave, Russia 47,600 Pacher and Stuart (2009)

Gamssulzen Cave, Austria 47,300 Pacher and Stuart (2009)

Križna jama Cave, Slovenia 45,400 Pacher and Stuart (2009)

Serpievskaya Cave, Russia 44,050 Stiller et al. (2010)

9


10

Ramesch Austria( )

Winden Austria( )

Abruzzo Italy( )0.86/0.99/0.99/0.90.67 9

1/ 7/92/87/76/78/100

Oskhordokh Siberia Russia( . )

Kudaro Georgia3 ( )

Hovk Armenia Kudaro Georgia1 ( ); ( )

Ossenyaga Caucasus Russia( , )0.75/0.96/0.91/0.70.77 90

5/ /66/71/-/93/660.99/1/1/1

0.94 9/ 8/95/98/98/99/100

Balme a Collomb France( )

Gailenreuth Germany( )

Hohlefels Germany( )

Scladina Belgium Chauvet Arcy sur Cure France Eiros Spain( ); , ( ); ( )

Cova Linares Spain( )

Cova Linares Spain( )

A Ceza Spain( )

Arcy sur Cure France( )0.53/0.5/0.52/0.86 4

0.47/ 2/-/52/-/-/-

0.45 6-/ /48/55/51/46/-

/0.69/0.62/0.551

- - - -/-/-/54/52/58/-

/ / /0.83

- - - -/ /-/66/64/65/-

/ / /- -

0.53/0.93/0.83/0.0.66 64

53/ /48/61/57/72/58

0.99/1/1/11 98/ /98/99/96/99/100

0.91/0.99/0.98/0.0.88 5

88/ 3/53/45/47/-/58

- 44 42 42-/-/-/-/-/-/-

/0. /0. /0.

Conturines Italy( )

0.75/0.99/0.97/0.80.89 4

0/ 7/49/40/-/-/46

- - - --/-/-/47/-/-/-

/ / /

Merveilleuse France( )

0.71/0.8/0.8/0.87 49

0.68/ /52/50/49/47/-

Pe tera cu Oase Romaniaş ( )

Gamssulzen Herdengel Austria Pe tera cu Oase Romania, ( ); ş ( )

Herdengel Austria( )

Nerubajskoe Ukraine( )

Pe tera cu Oase Romania Medvedia Slovakiaş ( ); ( )


Gamssulzen Austria Vindjia Croatia( ); ( )

Vindija Croatia( )

Geissenkloesterle Germany( )

Gamssulzen Herdengel Nixloch AustriaPe tera cu Oase Romania Potocka Zijalka Slovenia

, , ( );ş ( ); ( )

Divje babe Slovenia( )

Bolshoi Glukhoi MedvezhiyaSerpievskaya Ural Russia

, ,( , )

CB5 Nied wiedzia Polandź ( )

CB4 Nied wiedzia Polandź ( )0.99/1/1/10.92 9/ 1/87/87/92/92/88

0.47 0.41/-/-/-/-/-/-

/0.52/0.52/0.79

0.65/0.95/0.88/0.0.84 4

50/ 4/42/40/46/49/43

0.57/0.94/0.85/0.85

0.46/-/-/-/-/-/-

- 0.49/-/-/-/-/-/-

/0.5/ /-0.87

0.44 - --/-/-/-/44/-/-

/ / /-

0.95/0.99/0.99/10.88 7/ 4/76/69/69/69/59

0.40/-/-/-/-/44/42/0.56/0.52/-

0.77

0.97/1/1/0.90.96 63

5/ /57/68/57/60/64

- 0.47 -/ /-/-/-/-/-

/0.55/ /- -

0. /99 1/1/10.98/99/99/99/100/99/99

0.98/1/0.99/0.90.96 8

6/ 1/74/77/72/80/79

0.98/1/0.99/0.90.96 8

6/ 1/74/77/72/80/79

Ramesch Austria( )

Herdengel Austria( )

Denisova Altai Russia( , )

Strashnaya Altai Russia( , )





0.02nt substitution/site

0.67/0.95/0.87/0. 70.72 48

6/ /42/-/40/41/41

0.65/0.69/0.69/0.0.85

43/-/-/-/-/-/-

0.53/0.94/0.85 0.-/-

/ 51/-/-/-/-/61

CAT-Poi/Poi/GTR/MBPhyloBayes

PhyMl PAUP�2-SH/boot/ML/LS/ME/NJ/MP

WLS-LRT/F-test

*/***

-/**

-/***

-/***

***/***

***/***

***/***

**/***

-/***

-/***

-/*

-/***

-/***

-/**

***/***

-/***

**/***

**/***

**/***

Ursus arctos

Ursus deningerikudarensis

Ursus spelaeus

U. s. ladinicus

U. s. eremus

Ursus ingressus

Edge support:

0.47 0.49 0.47/ /-/73/62/69/-

/0.51/ /- -

FIGURE 4. Bayesian tree for the mitochondrial D-loop region from cave bears obtained in PhyloBayes under theCAT-Poisson model. The two sequences obtained in this study are shown in bold. Numbers at nodes, in the ordershown, correspond to posterior probabilities estimated in Phylobayes under CAT-Poisson model (CAT-Poi), Poissonmodel (Poi), and GTR model (GTR), as well as in MrBayes (MB). Values placed below them correspond to supportvalues calculated in PhyML and PAUP. PhyML’s support values were obtained by the approximate likelihood ratiotest (aLRT) and bootstrap analysis (boot). Bootstrap values in PAUP were ordered by method used: maximum likeli-hood (ML), weighted least squares (WLS), minimum evolution (ME), neighbor joining (NJ), and maximum parsimony(MP). Values of probabilities and bootstrap percentages lower than 0.40 and 40%, respectively, were omitted or indi-cated by a dash “-”. Asterisks at nodes denote corrected significance of branches at 0.05 (*), 0.01 (**) and 0.001 (***)level, estimated by WLS-LRT and F-test in WeightLESS. Thick branches were present in at least nine of the ten treetopologies inferred by the ten applied methods.


that U. ingressus had likely an East-European ori-gin.

The subclade of the second ‘advanced’ cavebear species, U. ingressus is strongly supported byaLRT (0.96) and Bayesian methods (≥ 0.95) butonly moderately by others (57-64%). However, theWLS-LRT and F-tests unambiguously confirm thestatistical significance of this subclade at the 0.001level. This subclade is further divided into twobranches. The one groups four samples fromPeştera cu Oase Cave, which are supported by ≥0.95 posterior probabilities, 59-76% bootstrap val-ues and the two applied tests. The second branchsupported with generally low bootstrap and supportvalues contains samples from both Peştera cuOase Cave and all other U. ingressus sequences.This tree topology suggests that the Southeasternpart of Europe was the place of origin for U. ingres-sus from which it expanded to other territories inEurope. However, with one exception (see below)the sequences of U. ingressus originating fromother countries do not cluster according to theirgeographic location. Therefore, we cannot inferparticular migration routes from the current data.

Both specimens from Niedźwiedzia Cave evi-dently branch within the U. ingressus subclade andsignificantly cluster together (posterior probabilities≥ 0.99; bootstrap values 88-92%, aLRT = 0.92, p-values < 0.05 and < 0.001). These specimens aremost closely related to samples that originate fromthe Urals (Bolshoi Glukhoi, Medvezhiya and Ser-pievskaya caves). Although the support for thisgrouping is weak, nine of ten reconstructed treesrecovered this relationship. Only the ME treeshowed a close relationship between the samplesfrom Niedźwiedzia Cave and the sequence fromDivje babe (Slovenia) but with non-significant sup-port. In addition, this tree was marginally rejectedby the AU test (with p-value = 0.05). This resultindicates that a grouping of the specimens fromNiedźwiedzia with those from the Ural caves ismore likely than one with the Divje babe (Slovenia)sequence. In agreement with this conclusion, clus-tering analyses of molar measurements also showa close relationship of Niedźwiedzia and Medve-zhiya caves (Figure 2). All sequences from Poland,Urals and Slovenia fall into a distinct cladeobtained in all 10 approaches. Bootstrap values ofthis grouping are slightly lower than 50% butBayesian support reaches 0.95 and F-test branchsignificance is at the 0.01 level.

The median joining network shows a similarposition of the specimens from Niedźwiedzia Cave(Figure 5). There are also clearly separated groups

of U. spelaeus, U. ingressus and U. deningerikudarensis with U. s. ladinicus in the middle of thenetwork. The haplotypes from Niedźwiedzia Caveare situated in the U. ingressus group and are con-nected to haplotypes from the Urals and Divjebabe.

The grouping of the Ural specimens withthose from Poland may reflect more frequent orrecent migrations between Central Europe and theUral Mountains populations across the East Euro-pean Plain. The placement of sequences from Uralbears within European samples (see also Stiller etal., 2009) suggests that long-distance migrationswere probably directed from Central Europe to theEast. Although based on a limited number of sam-ples, this conclusion agrees well with the compara-tively old dates obtained for the Polish specimens,which seem to be older than those from the Uralcaves. The dates for samples from NiedźwiedziaCave are 41,500±1,100 and > 49,000 yr BP,whereas a sample from Serpievskaya Cave analy-sed for DNA has been dated to 44,050 yr BP(Stiller et al., 2010) and the oldest date reported byKosintev (2007) for mammal bones from BolshoiGlukhoi is 38,200±900 yr BP. More generally, theoldest published remains of cave bears (includingthe U. ingressus type) found in the Ural Mountains,from Tayn (Secrets) Cave, do so far not exceed47,600±900 yr BP (Pacher and Stuart, 2009;Pacher et al., 2009).

This dating indicates that the migration ofEuropean cave bears to the Urals may have takenplace during Early or Middle Weichselian/Valdaiwarming periods, e.g., during the Odderade(MIS5a) or Oerel Interstadials (early MIS3). TheOdderade Interstadial correlates with the Gniew,Krastudy or Rudunki Interstadials in Polish stratig-raphy (Krzywicki, 2002) and with the Krugitsa Inter-stadial in East European stratigraphy (Velichko andSpasskaya, 2002), whereas the Oerel Interstadialcorresponds to the beginning of the GrudziądzInterstadial in Polish stratigraphy (Krzywicki, 2002)and the Krasnogorsk warming of the Bryansk orGrazhdanski megainterval in East European stra-tigraphy (Velichko et al., 2010).

However, it cannot be excluded that themigration was driven by the asymmetric develop-ment of Eurasian ice sheets (Svendsen et al.,2004) and the resulting differences in climatic andenvironmental conditions between Central andEast Europe. For example, during the MiddleWeichselian glacial maximum (MIS4) the Barents-Kara Ice Sheet terminated on the northern marginof the Eurasian mainland, whereas the Scandina-

11


vian Ice Sheet covered the whole of Finland,reached Denmark and the north-eastern part ofPoland. In Polish territory it is known as Świecie orVistulian 3 Stadial (Krzywicki, 2002), which corre-sponds to Schalkholz in North Europe and theShestikhino Stadial in Eastern European stratigra-phy (Velichko and Spasskaya, 2002). A moreadvanced expansion of Ice Sheets in CentralEurope could have forced emigrations of cavebears toward the East European Plain, which wasfree of ice sheets. This region was covered bynorthern taiga and mixed forests during the Kru-gitsa and Krasnogorsk Interstadials whereas dur-ing the Shestikhino Stadial, the area was coveredby periglacial forest-steppe (Velichko et al., 2004).However, more dated DNA samples especially

from the East European Plain region are requiredto support this hypothesis. Such analyses may wellreveal more complexity of both the genetic diver-sity of cave bears and their migration patterns, e.g.,in two directions.

Genetic analyses of small cave bears fromRamesch Cave and larger ones from GamssulzenCave showed a lack of gene flow between thesepopulations, which inhabited the same alpineregion during at least 15,000 years (Hofreiter et al.,2004b). On the other hand, it was found that differ-ences between mtDNA correlate neither with geo-graphical distance nor with the age of samples(Hofreiter et al., 2002) and particular cave bearhaplogroups are spread over wide geographicareas (Orlando et al., 2002). Moreover, clustering

FIGURE 5. Median-joining network of 70 cave bear sequences. Haplotypes are colored according to their speciesaffiliation. Inferred missing haplotypes are presented as black dots. The area of the circle is proportional to the haplo-type frequency whereas the length of connecting lines corresponds to the number of substitutions between the haplo-types.

12


in phylogenetic trees was observed between geo-graphically distant samples from U. deningerikudarensis (Caucasus and Yana River) and U.spelaeus (Altai and Austria) (Knapp et al., 2009).Baryshnikov et al., (2003) did not find importantmorphometrical differences in cave bear cheekteeth from different localities, which they explainedby exchange of genetic material between adjacentcave bear populations, whereas they justified thediscrepancy between samples from northern andsouthern sites by different dietary habits. Alto-gether, the data indicate that long distance migra-tions occurred in cave bear evolutionary history.Such a view is also supported by the close relation-ships between the mtDNA sequences from Polandand the Ural, respectively. A specialization anddependence of cave bears on vegetarian diet (seePacher and Stuart, 2009; Mackiewicz et al., 2010;Bocherens et al., 2011; Dotsika et al., 2011; andreferences therein) could have been behind thispattern, by forcing cave bear populations tomigrate into regions rich enough in food.

CONCLUSIONS

Genetic analyses of mtDNA extracted fromcave bear remains found in Niedźwiedzia Cave inPoland clearly indicate its affiliation with the U.ingressus haplogroup. This conclusion is furthersupported by biometrical analyses of molars andskull. For the first time remains of U. ingressus areidentified in the territory of Poland, north of the Car-pathians and Sudetes. The investigated samplesfrom Niedźwiedzia Cave belong to one of the old-est recognized remains of this species in Europe. Aclose relationship of samples from NiedźwiedziaCave with those from Ural caves, and their respec-tive radiocarbon dates may indicate that migrationsor gene flow occurred between Polish cave bearpopulations and those from the Ural Mountains.These results suggest the existence of a migratorycorridor connecting Western and Eastern Europeacross the area where Poland is situated today.

ACKNOWLEDGMENTS

This work was supported by Operational Pro-gramme Innovative Economy POIG.02.02.00-14-024/08-00.

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Palaeontologia Electronica · PALAEO-ELECTRONICA.ORG 3 spelaeus around 28,000 yr BP (32,000 cal....

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