From the Past to the Present: Wolf Phylogeography and ... - Core

ORIGINAL RESEARCH published: 02 December 2016 doi: 10.3389/fevo.2016.00134

From the Past to the Present: Wolf Phylogeography and Demographic History Based on the Mitochondrial Control Region Erik Ersmark 1, 2*, Cornelya F. C. Klütsch 3 , Yvonne L. Chan 1 , Mikkel-Holger S. Sinding 4 , Steven R. Fain 5 , Natalia A. Illarionova 6 , Mattias Oskarsson 7 , Mathias Uhlén 7 , Ya-ping Zhang 8 , Love Dalén 1 † and Peter Savolainen 7 †

Edited by: Badri Padhukasahasram, Illumina, USA Reviewed by: Klaus-Peter Koepfli, Smithsonian Conservation Biology Institute, USA Jouni Aspi, University of Oulu, Finland *Correspondence: Erik Ersmark [email protected] †

These authors have contributed equally to this work.

Specialty section: This article was submitted to Evolutionary and Population Genetics, a section of the journal Frontiers in Ecology and Evolution Received: 22 July 2016 Accepted: 09 November 2016 Published: 02 December 2016 Citation: Ersmark E, Klütsch CFC, Chan YL, Sinding M-HS, Fain SR, Illarionova NA, Oskarsson M, Uhlén M, Zhang Y-P, Dalén L and Savolainen P (2016) From the Past to the Present: Wolf Phylogeography and Demographic History Based on the Mitochondrial Control Region. Front. Ecol. Evol. 4:134. doi: 10.3389/fevo.2016.00134

1 Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Stockholm, Sweden, 2 Department of Zoology, Stockholm University, Stockholm, Sweden, 3 Biology Department, Trent University, Peterborough, ON, Canada, 4 Centre for GeoGenetics, Natural History Museum of Denmark, Copenhagen University, Copenhagen, Denmark, 5 National Forensics Laboratory, U. S. Fish and Wildlife Service, Ashland, OR, USA, 6 Laboratory of Microevolution and Domestication of Mammals, A. N. Severtsov Institute of Ecology and Evolution, Moscow, Russia, 7 Science for Life Laboratory, Department of Gene Technology, KTH Royal Institute of Technology, Solna, Sweden, 8 Laboratory of Cellular and Molecular Evolution, and Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China

The global distribution of the gray wolf (Canis lupus) is a complex assembly consisting of a large number of populations and described subspecies. How these lineages are related to one another is still not fully resolved, largely due to the fact that large geographical regions remain poorly sampled both at the core and periphery of the species’ range. Analyses of ancient wolves have also suffered from uneven sampling, but have shown indications of a major turnover at some point during the Pleistocene-Holocene boundary in northern North America. Here we analyze variation in the mitochondrial control region in 122 contemporary wolves from some of the less studied populations, as well as six samples from the previously unstudied Greenland subspecies (Canis l. orion) and two Late Pleistocene samples from Siberia. Together with the publicly available control region sequences of both modern and ancient wolves, this study examines genetic diversity on a wide geographical and temporal scale that includes both Eurasia and North America. We identify 13 new haplotypes, of which the majority is found in northern and eastern Asia. The results show that the Greenland samples are all represented by one haplotype, previously identified in North American wolves, among which this population seems to trace its maternal lineage. The phylogeny and network analyses show a wide spatial distribution of several lineages, but also some clusters with more distinct geographical affiliation. In North America, we find support for an end-Pleistocene population bottleneck through coalescent simulations under an approximate Bayesian framework in contrast to previous studies that suggested an extinction-replacement event. However, we find no support for a similar bottleneck in Eurasia. Overall, this global analysis helps to clarify our understanding of the complex history for wolves in Eurasia and North America. Keywords: Canis lupus, phylogeography, mtDNA, turnover, control region

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INTRODUCTION

and on Greenland (Canis l. orion; Pocock, 1935; Wozencraft, 2005). By applying ancient DNA techniques to subfossil remains, many studies have sought to reconstruct the wolf ’s phylogeographic history, often specifically in relation to its domestication by humans (Verginelli et al., 2005; Germonpré et al., 2009; Skoglund et al., 2011; Thalmann et al., 2013). Like numerous studies on modern wolves, these have generally lacked coverage in terms of geography and/or genetic material, often to a great extent. Samples of ancient wolf remains have mainly been collected from sites in Europe and Alaska, leaving out much of the historical distribution (Stiller et al., 2006; Leonard et al., 2007; Germonpré et al., 2009), and a majority of these samples have exclusively been targeted for a short but variable fragment of the mitochondrial control region (CR; Verginelli et al., 2005; Stiller et al., 2006). Despite these limitations, ancient DNA studies have provided important insights into the population dynamics in certain regions. In a study on Late Pleistocene wolves from eastern Beringia (Alaska) a population turnover was detected at the Pleistocene-Holocene transition, where a diverse group of haplotypes (haplogroup 2) seemed to have been replaced by a more distinct and monophyletic group (haplogroup 1), which also represents the modern wolves in North America (Leonard et al., 2007). Interestingly, the former group was morphologically described as a robust ecomorph, possibly adapted to large megafaunal prey. The division into these two genetic groups can also be applied to the larger Eurasian population, where both groups are still present in contemporary wolves. However, following the pattern in North America, all Late Pleistocene European specimens have been shown to fall within haplogroup 2 (Pilot et al., 2010; Thalmann et al., 2013), which might suggest that a population turnover occurred in Eurasia as well. Although only present at low frequency today, haplogroup 2 exhibits a great deal of diversity, especially when ancient specimens are included (Pilot et al., 2010). This is also mirrored by indications of past morphological diversity observed both in North America and Europe, which suggests that the wolf has suffered a general decrease, not only in genetic but also morphological diversity across its range (Leonard et al., 2007; Germonpré et al., 2009). In addition to the issue of the wolf ’s relationship to the modern dog, there have been several debated uncertainties concerning specific wolf populations and their status in terms of subspecies, hybrids, or even distinct species. It has for instance recently been suggested that wolves from the Great Lakes region of Canada and the United States (Canis l. lycaon) constitutes a hybrid between gray wolves and coyotes or even a species on its own (Fain et al., 2010; vonHoldt et al., 2011, 2013, 2016). To provide a more comprehensive phylogeograpy of the gray wolf, we have used CR-sequences to study the mtDNA diversity of wolves on a worldwide scale. We specifically aimed to include more modern samples from previously less studied areas, as well as to collate and analyze ancient sequences globally in order to assess the relationships between wolf populations and how they have changed over time. We also sampled wolves from the Great Lakes states Michigan and Minnesota in order to evaluate if this population shows more sharing of haplotypes with

The gray wolf (Canis lupus) exhibits a tremendous ecological flexibility with respect to different environments, ranging from the Arctic tundra to the deserts and dry shrub lands of the Middle East. This iconic canid was one of the most widely distributed large terrestrial mammals in the Late Quaternary with a historical range that covered most of the northern hemisphere (Nowak, 2003), and its range has expanded even further alongside humans as the domestic dog (C. lupus familiaris). In addition to domestication, humans have impacted the wolf considerably by restricting its habitat through active persecution, which has resulted in a dramatic decrease in population sizes, especially over the last two centuries (Boitani, 2003; Leonard et al., 2005). Shrinking habitats overlapping with closely related dogs and coyotes (Canis latrans) have also led to numerous occurrences of hybridization (Andersone et al., 2002; Lucchini et al., 2004; Fain et al., 2010; vonHoldt et al., 2011, 2013). Along with recent turnovers, these characteristics and events make the phylogeographic history of the wolf in many ways difficult to disentangle. The divergence between wolves and coyotes most likely took place in America at some point between 4.5 and 1.8 million years ago (Mya; Nowak, 2003). However, the more recent time point seems more plausible, considering that a sudden expansion of the genus Canis, sometimes referred to as the “wolf event,” took place at the beginning of the Pleistocene (∼2.5–1.8 Mya; Azzaroli, 1983; Wang et al., 2010). This expansion was most likely facilitated by intense continental glaciations, which created open landscapes including the “mammoth steppe biome” (Azzaroli, 1983; Azzaroli et al., 1988). According to the fossil record, the wolf C. lupus ssp. appeared in Europe around 800 thousand years ago (kya) during the Middle Pleistocene and in the mid-latitudes of North America around 100 kya, where its ancestor previously had gone extinct (Wang et al., 2010). The wolf appears to have been wellestablished in Europe from around 400 kya and onwards (Meloro et al., 2007). Older records are known only from Siberia and Alaska (Beringia), leading to the assumption that wolves originated somewhere in this region, whence they spread all across the Holarctic (Wang et al., 2010). Despite the wide geographical distribution and complex evolutionary history of the gray wolf, most genetic analyses to date have concentrated on specific geographical regions (Randi et al., 2000; Aggarwal et al., 2007; Pilot et al., 2010; Weckworth et al., 2010) and/or short fragments of the mitochondrial DNA (mtDNA; Vila et al., 1999; Valiere et al., 2003; Sharma et al., 2004; Jedrzejewksi et al., 2005). Many of these studies have thus suffered from limited geographical coverage and/or insufficient resolution at the genetic level. Further, large areas have remained scarcely sampled, such as Russia/Siberia, China, and the Middle East; regions holding large interconnected populations that potentially contain high genetic diversity. There are also remote regions where wolves have not yet been studied in terms of genetic differentiation and diversity. Among these are the extreme north—home to phenotypically distinct arctic subspecies, which occur in the Canadian arctic (Canis l. arctos)


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MS_wolf5 (149 bp), and MS_wolf6 (132 bp). Primer sequences are listed in Table S4. Polymerase chain reactions (PCRs) were set up using; 1 mM MgCl2 (Qiagen), 0.2 mM dNTPs, 0.2 µM of each primer, 1X PCR-buffer (Qiagen), 0.1 mg/ml BSA, 2 units of Hotstar Taq (Qiagen) and 2 µl of DNA extract, making a total volume of 25 µl/reaction. Amplifications started with a 10 min denaturation step at 95◦ C, followed by 55 cycles of denaturation at 95◦ C for 30 s, annealing for 30 s at 58–62◦ C, followed by extension at 72◦ C for 30 s. A final extension step at 72◦ C for 7 min was included at the end of the procedure. Confirming successful amplifications was done using gel electrophoresis, by applying 5 µl PCR product on a 1.5% agarose gel prepared with fluorescent GelRed (Biotium Inc.). The gel was run in 0.5X TBE buffer (50 ml) and inspected with UV-light. The PCR-products (20 ul) were further purified with EXO-SAP enzymes (5 ul; Thermo Fisher Scientific). Sequencing reactions were then performed using the BigDye Terminator kit ver.1.1 (Applied Biosystems Inc.), and the products were purified with the DyeEx 96 Kit (Qiagen). An ABI 3130xl Genetic analyzer (Applied Biosystems Inc.) was used for the final sequencing analysis. To easily detect contamination and minimize the risk of cross-contamination, all extractions were made in small series with regular inclusions of blank samples. The same procedure was applied for the PCR preparation, and a minimum of two PCR products were sequenced for every sample to confirm its authenticity and detect possible discrepancies caused by postmortem DNA damage (Hofreiter et al., 2001).

wolf or coyote. Finally, to formally test the proposed population turnover in North America, as well as the possibility of a similar turnover in Eurasia, we analyzed the data using serial coalescent simulations.

MATERIALS AND METHODS Samples Tissue samples (n = 128) from wolves were collected from across the range including Scandinavia, Russia/Siberia, Iran, China, North America, and Greenland (Table S1). With the exception of two captive Mexican wolves, all samples were derived from wild animals. Of the six samples from Greenland, two were dated to the fifth and the early eighteenth century, respectively (Table S1). Two Late Pleistocene wolf remains were also analyzed, both taken from permafrost sites on the Taimyr Peninsula in Siberia. After positive DNA screening, these samples were radiocarbon dated at the Oxford Radiocarbon Accelerator Unit (ORAU), yielding approximate ages of 35 and 42 thousand calibrated radiocarbon years before present (kyr BP). For the sake of consistency, all subsequent radiocarbon dates are presented in this (calibrated) form (Tables S1, S3). In summary, a total of 130 novel samples were analyzed (Table S1). For more information regarding samples included in the current study, see Tables S1, S2.

Laboratory Methods For the modern samples, DNA was extracted from blood and tissue using Proteinase K and organic solvents (Sambrook et al., 1989), or from hairs (Hopgood et al., 1992). PCR amplification of a 582 bp long fragment of the hypervariable domain (HVR1) of the mitochondrial control region was performed with a set of primers used in a previous study (Savolainen et al., 2002). This specific region has commonly been applied in several previous phylogenetic studies on the wolf and was thus targeted to facilitate inclusive comparisons. PCR products were sequenced using the primers above with BigDye Terminator chemistry on ABI 377 and 3700 instruments (Applied Biosystems Inc.). Both extraction and pre-PCR preparation of the historical and ancient samples was performed at a specifically designated laboratory with high standards of sterility at the Swedish Museum of Natural History in Stockholm, and the Centre for Geogenetics at the Natural History Museum of Denmark. DNA was extracted using a silica-based method, where ca. 50 mg bone powder from each sample was incubated overnight under motion at 55◦ C in 715 µl extraction buffer (0.45 M EDTA, 0.1 M UREA, 150 µg proteinase K). The samples were then centrifuged at 2300 rpm for 5 min and the supernatants were collected and concentrated using 30K MWCO Vivaspin filters (Sartorius). Purification and elution was performed following Brace et al. (2012). Taking the fragmented state of ancient DNA into account, six partially overlapping sequences of the mitochondrial control region were amplified to cover a total of 686 bp (including primers), matching the fragment targeted for the modern samples; MS_wolf1- dogdl5 (148 bp; Leonard et al., 2005), MS_wolf2 (205 bp), MS_wolf3 (211 bp), MS_wolf4 (175 bp),


Alignment and Data Set Establishment All sequences obtained from the extracted samples were aligned and edited using the software BioEdit 7.2.3 (Hall, 1999; http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and Geneious 7.1.3 (Kearse et al., 2012). In addition to the samples extracted and sequenced by the authors, currently available sequences from GenBank were downloaded for wolves (C. lupus; Table S2). However, since available and published CR-sequences varied to a great extent both in overlap and length, two alignments were initially constructed; one covering the complete stretch of 582 bp (alignment A, n = 314) and a second where all sequences