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Genetic analysis of cave bear specimens from Niedźwiedzia Cave, Sudetes, Poland

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

Plain Language Abstract

We have performed genetic and biometrical analyses of cave bear specimens from Niedźwiedzia Cave in Kletno, Sudetes, Poland. Fragment of mitochondrial D-loop sequence were obtained from two specimens radiocarbon dated to 41,500 ± 1100 yr BP and >49,000 yr BP. Phylogenetic analyses including numerous cave bear sequences from Eurasia showed their close relationship with Ursus ingressus group especially with samples from Ural Mountains and Slovenia. Biometrical analyses of molar teeth and skulls also indicated that Niedźwiedzia cave were inhabited by U. ingressus. Our results represent the first record of U. ingressus north to the Carpathian Arch and some of the oldest specimens of this cave bear taxon known so far. Close relationship with Ural and Slovenian cave bears and internal placement of Ural samples among European specimens in phylogenetic trees may suggest that eastward expansion of U. ingressus may have started from Central Europe.

Resumen en Español

Análisis genético de los ejemplares de oso de las cavernas de la cueva Niedźwiedzia, Sudetes, Polonia

La gran mayoría de los restos fósiles de los depósitos del Pleistoceno superior de la cueva Niedźwiedzia de Kletno (Sudetes, Polonia) pertenece al oso de las cavernas. Los análisis filogenéticos llevados a cabo sobre un fragmento de la región del lazo D mitocondrial extraído de dos muestras del oso de las cavernas muestra claramente su cercana relación con el haplogrupo de Ursus ingressus. Esta filiación taxonómica de los restos de oso de las cavernas de la cueva Niedźwiedzia ha sido confirmada además mediante análisis biométricos de molares y cráneos. Los resultados obtenidos representan el primer registro de U. ingressus al norte del arco carpático, al tiempo que la datación mediante radiocarbono de las muestras (> 49000 años) indica que se trata de unos de los ejemplares más antiguos de esta especie de oso de las cavernas conocidos hasta ahora. Los análisis filogenéticos multimétodo, que incluyen numerosas secuencias del oso de las cavernas disponibles públicamente, permiten examinar en detalle las relaciones entre estas muestras, incluyendo el significado de los distintos clados, y discutir algunos aspectos de la filogeografía de los osos de las cavernas. Las secuencias de U. ingressus de Polonia muestran sus mayores afinidades con las de los ejemplares de los Urales y son también próximas a los de Eslovenia, lo que podría indicar la existencia de migraciones entre poblaciones del centro y el este de Europa. La localización de las muestras de los Urales entre los ejemplares europeos en los árboles filogenéticos y la mayor edad de las muestras polacas que de las de los Urales sugiere que la expansión hacia el este de U. ingressus pudo haberse originado en Europa central.

Palabras clave: ADN antiguo; oso de las cavernas; filogenia; Ursus ingressus; Ursus spelaeus

Traducción: Miguel Company

Résumé en Français

Analyse génétique de spécimens d'ours des cavernes de la grotte de Niedźwiedzia, Sudètes, Pologne

La grande majorité des restes fossiles des dépôts de Pléistocène supérieur de la grotte de Niedźwiedzia à Kletno dans les Sudètes en Pologne appartient à l'ours des cavernes. Les analyses phylogénétiques fondées sur un fragment de la région mitonchondriale D-loop extrait de deux échantillons d'ours des cavernes ont montré sans ambiguité leurs relations proches avec l'haplogroupe Ursus ingressus. Cette affiliation taxonomique des restes d'ours des cavernes de la grotte de Niedźwiedzia a également été confirmée par les analyses biométriques des molaires et des crânes. Nos résultats repésentent le premier enregistrement d'U. ingressus au Nord des Carpathes, tandis qu'une datation radiocarbone (> 49,000 yr BP) des échantillons indique qu'ils représentent certains des plus anciens spécimens de ce taxon d'ours des cavernes connus jusqu'à présent. Les analyses phylogénétiques multi-méthodes incluant de nombreuses séquences publiques d'ours des cavernes ont permis d'analyser les relations entre les échantillons en détail et ont permis de tester la significativité de certains clades ainsi que de discuter certains aspects de la phylogéographie de l'ours des cavernes. Les séquences d'U. ingressus de Pologne sont les plus proches de spécimens de l'Oural puis de Slovénie, ce qui peut mettre en évidence des migrations entre les populations du centre et de l'Est de l'Europe. Le placement interne des spécimens de l'Oural sur les arbres phylogénétiques ainsi que l'âge plus ancien des échantillons polonais par rapport à ceux de l'Oural suggèrent que l'expansion vers l'Est d'U. ingressus ait pu commencer à partir de l'Europe centrale.

Mots-clefs:ADN ancien, ours des cavernes, phylogénie, Ursus ingressus, Ursus spelaeu

Translator: Loïc Costeur

Deutsche Zusammenfassung

Genetische Untersuchungen an Höhlenbären aus der Niedźwiedzia Höhle, Sudeten, Polen

Höhlenbären stellen den Großteil der Fossilien aus den spätpleistozänen Ablagerungen der Niedźwiedzia Höhle von Kletno (Sudeten/Polen) dar. Phylogenetische Analysen, die auf einem Fragment der mitochondrischen D-loop Region basieren und die zwei Proben mit Höhlenbär-Material entnommen worden waren, zeigten eindeutig eine nahe Verwandtschaft mit der Ursus ingressus Haploguppe. Diese taxonomische Zuordnung der Höhlenbären aus der Niedźwiedzia Höhle wurde zudem durch biometrische Analysen von Molaren und Schädeln bestätigt. Unsere Ergebnisse stellen den ersten Nachweis von U. ingressus nördlich des Karpatischen Bogens dar, während eine C-14 Datierung (> 49,000 vor heute) der Proben darauf hin deuten, dass es sich um die bislang ältesten Stücke dieses Höhlenbär-Taxons handelt. Mehrfache phylogenetische Analysen mit mehreren öffentlich verfügbaren Höhlenbär-Sequenzen erlaubten eine detaillierte Untersuchung der Verwandtschaftsbeziehungen dieser Proben. Diese Untersuchungen beinhalteten auch die Signifikanz von bestimmten Kladen und einige Aspekte der Höhlenbar-Phylogeographie. Die Sequenzen von U. ingressus aus Polen sind am nächsten verwandt mit Stücken aus dem Ural Gebirge und Slowenien, was möglicherweise auf eine Migration zwischen zentral – und osteuropäischen Populationen hinweist. Die innere Platzierung von Proben aus dem Ural innerhalb der Europäischen Stücke in phylogenetischen Stammbäumen und die polnischen Proben, die älter sind als die aus dem Ural, weisen darauf hin, dass die östliche Ausbreitung von U. ingressus möglicherweise von Zentraleuropa ausging.

SCHLÜSSELWÖRTER: alte DNA; Höhlenbär; Phylogenie; Ursus ingressus; Ursus spelaeus

Translators: Eva Gebauer

Arabic

301 arab

Translator: Ashraf M.T. Elewa

Polski Abstrakt

Analiza genetyczna okazów niedźwiedzia jaskiniowego z Jaskini Niedźwiedziej, Sudety, Polska

Przeważająca część szczątków kopalnych w późnoplejstoceńskich osadach Jaskini Niedźwiedziej w Kletnie, Polska, należy do niedźwiedzia jaskiniowego. Analiza filogenetyczna w oparciu o fragment mitochondrialnego regionu pętli D wyizolowany z dwóch próbek niedźwiedzia jaskiniowego bezsprzecznie wskazała na bliskie pokrewieństwo z haplotypem Ursus ingressus. To taksonomiczne powinowactwo niedźwiedzia jaskiniowego z Jaskini Niedźwiedziej zostało następnie potwierdzone przez analizę biometryczną trzonowców i czasek. Nasz rezultat stanowi pierwsze wystąpienie U. ingressus na północ od łuku Karpat, podczas gdy datowanie radiowęglowe (> 49.000 lat temu) próbek wskazuje, że reprezentują one jeden z najstarszych z dotychczas znanych okazów tego taksonu niedźwiedzia jaskiniowego. Analiza filogenetyczna oparta o wiele metod, wykorzystująca również liczne dostępne publicznie sekwencje niedźwiedzi jaskiniowych, umożliwiła szczegółową analizę pokrewieństw pomiędzy próbkami, w tym istotność konkretnych kladów, oraz dyskusję pewnych aspektów filogeografii niedźwiedzia jaskiniowego. Sekwencje U. ingressus z Polski są najbliższe okazom z Uralu oraz Słowenii, co może wskazywać na migrację między populacjami z centralnej i wschodniej Europy. Centralna pozycja próbek uralskich wśród okazów europejskich na drzewach filogenetycznych i starszy od uralskich wiek polskich próbek, sugeruje że ekspansja na wschód U. ingressus mogła się rozpocząć w Europie Centralnej.

Słowa kluczowe: pradawne DNA; niedźwiedź jaskiniowy; filogeneza; Ursus ingressus; Ursus spelaeus

Translators: Dawid Mazurek, Robert Bronowicz, and Daniel Madzia

 

 

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bacaCenter for Precolumbian Studies
University of Warsaw
Krakowskie Przedmieście 26/28
00-927 Warsaw
Poland

Mateusz Baca is a research assistant in Centre for Precolumbian Studies at University of Warsaw. His main research field is ancient DNA. His Master’s thesis (2007) concerned genetic characterization of pre-Columbian populations from Peruvian Andes. He is also interested in genetic analyses of Pleistocene mammals. Now he is preparing his PhD thesis concerning impact of late Pleistocene climatic oscillations on population dynamics of Dicrostonyx gulielmi from Biśnik Cave (Poland). 

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stankovicInstitute of Biochemistry and Biophysics
Polish Academy of Science
Pawińskiego 5a
02-106 Warsaw
Poland

Anna Stankovic (born in 1971) She is a member of the group of geneticists from the Institute of Genetics and Biotechnology (University of Warsaw) and she is working on Molecular Ecology and Conservation Genetics of marine and freshwater fish for restitution and management projects in Poland including sturgeon, salmon and few others. Recently her work is also focused on several Pleistocene animal species like red deer, wolverine, lemming, bear, and some others. 

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stefaniak Department of Palaeozoology
Zoological Institute
University of Wrocław
Sienkiewicza 21
50-335 Wrocław
Poland

Krzysztof Stefaniak graduated from the Wroclaw University, Poland in 1986 and received his PhD in 2001. He has been working at the Department of Palaeozoology, Zoological Institute, University of Wroclaw, Poland since 1984. His interests are focused on fossil mammals, especially ungulates, including Neogene and Quaternary deer. His research activity includes also archaeozoology, caves and fossil DNA. He has participated in excavations in karst areas of Poland and the Ukraine for 30 years. He lectures palaeontology, archaeozoology and the history of the formation of the modern fauna and flora during the Quaternary at the University of Wroclaw.
 

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marciszakDepartment of Palaeozoology
Zoological Institute
University of Wroc?aw
Sienkiewicza 21
50-335 Wrocław
Poland

Adrian Marciszak, born in 1983, is married, has one child. In 2007 completed master thesis entitled “Carnivore mammals (Carnivora, Mammalia) from Żabia Cave". Recently focused on Tertiary carnivore mammals, mostly from territory of Poland, but also from Germany and Ukraine. Recently he prepared doctoral thesis entitled: “Mustelids (Mustelidae, Carnivora, Mammalia) from Pleistocene of Poland”. 

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hofreiterDepartment of Biology (Area 2)
The University of York
Wentworth Way
Heslington York
YO10 5DD
United Kingdom

Michael Hofreiter’s research is centred on the use of ancient DNA data for addressing questions in evolutionary genetics. Michael’s main interest lies in mammalian evolution ranging from phylogenetic questions, often investigating the phylogenetic relationships of extinct species to their living relatives, over population genetics to functional analyses of phenotypically or physiologically relevant genes. Because of the limited temporal survival of DNA in the fossil record he mostly investigates late Pleistocene and Holocene remains. Main areas of research are: 1) The evolutionary history of Ursidae, with a focus on the late Pleistocene cave bear complex; 2) the evolution of elephants and mammoths; 3) the domestication history of the domestic horse.  

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nadachowskiInstitute of Systematics and Evolution of Animals
Polish Academy of Sciences
Sławkowska 17
31-016 Cracow
Poland

Zoologist, palaeontologist; professor of the Vertebrate Zoology Department, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, and professor and head of Palaeozoology Department, Wrocław University, Wrocław, Poland. The author of about 150 original contributions, including 5 books, 60 other publications, 40 presentations in 30 international meetings, about 30 national scientific conferences. Research interests: palaeobiology of small mammals, palaeoecology, archaeozoology, biostratigraphy and Quaternary extinctions, biology and systematics of recent mammals. The member of Editorial Boards of several Polish and international journals, e.g. Quaternary International. The member of Committees of the Quaternary Research and of Zoology of the Polish Academy of Sciences.  

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weglenskiInstitute of Biochemistry and Biophysics
Polish Academy of Science
Pawińskiego 5a
02-106 Warsaw
Poland

University of Warsaw professor of genetics. 1980-2009 - Head of the Institute of Genetics and Biotechnology, 2000 -2006 - Rector of the Warsaw University. Post-doc and visiting scientist at the John Innes Institute GB, MIT and Paris XI. Main scientific interests: regulation of gene expression in microorganisms, genomics, ancient DNA analysis.
 

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mackiewiczDepartment of Genomics
Faculty of Biotechnology
University of Wrocław
Przybyszewskiego 63/77
51-148 Wrocław
Poland

Paweł Mackiewicz (born in 1971) is an Assistant Professor in the Faculty of Biotechnology at the University of Wrocław in Poland. His fascination for dinosaurs in childhood sparked his interest in paleontology and in natural sciences in general. Therefore he decided to study biology at the Faculty of Natural Sciences at the University of Wrocław. His first Master’s thesis in general biology (1996) concerned analyses of dental enamel structure and thickness of Carnivora with special attention to fossil bears. During his studies he also got interested in molecular biology and bioinformatics that later became his main field of research activity. They resulted in BSc (1996) and MSc in microbiology (1997), and PhD (2000) as well as habilitation (2004) in biological sciences-genetics. He is involved in many research projects in the field of bioinformatics and genomics, as well as molecular phylogeny and evolution, related to gene recognition, analysis of bacterial genome structure, computer simulations of genome evolution, endosymbiosis, phage genomics, nematode bar-coding, and evolution of various genes and proteins, including ancient DNA samples. He also continues his studies of dental enamel thickness of carnivorans including fossil ursids in relation to their diet and phylogeny. The analysis of aDNA is fulfillment of his past dreams to connect paleontology with molecular biology in one interdisciplinary field. Dr. Mackiewicz is an author and co-author of more than 75 scientific publications and more than 135 conference presentations. For his scientific achievements, he has received numerous awards from the Foundation for Polish Science, the Polish Genetics Society, the Education Minister, and the President of the University of Wrocław.

 

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

fig1

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

fig2

FIGURE 3. Comparison of skull measurements from Niedźwiedzia Cave (Nie) specimens with medians calculated by 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-row length. Triangles denote males, circles females, and squares juveniles.

fig3

FIGURE 4. Bayesian tree for the mitochondrial D-loop region from cave bears obtained in PhyloBayes under the CAT-Poisson model. The two sequences obtained in this study are shown in bold. Numbers at nodes, in the order shown, correspond to posterior probabilities estimated in Phylobayes under CAT-Poisson model (CAT-Poi), Poisson model (Poi), and GTR model (GTR), as well as in MrBayes (MB). Values placed below them correspond to support values calculated in PhyML and PAUP. PhyML's support values were obtained by the approximate likelihood ratio test (aLRT) and bootstrap analysis (boot). Bootstrap values in PAUP were ordered by method used: maximum likelihood (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 indicated 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 tree topologies inferred by the ten applied methods.

fig4

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

fig5

 

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

 

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

 

Locality

Date (yr BP)

Reference

Nied?wiedzia Cave, Poland (CB4)

41,500

This study

Nied?wiedzia Cave, Poland (CB5)

> 49,000

This study

Nerubajskoe, Ukraine

> 52,450

Pacher and Stuart (2009)

Pe?tera 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)

Krina jama Cave, Slovenia

45,400

Pacher and Stuart (2009)

Serpievskaya Cave, Russia

44,050

Stiller et al. (2010)