Yeti Royal Society Journal Proceedings B University of Buffalo College of Arts and Sciences

1. Introduction

The Tibetan Plateau, the almost extensive and highest plateau in the globe with an boilerplate altitude of 4500 yard above bounding main level, is partly surrounded by the Himalayan range and many of Earth's highest mountains. Dramatic environmental changes caused by the uplift of the plateau and climatic oscillations during the Fourth glaciations substantially impacted the evolution, diversification, and distribution of local institute and beast species [1]. Because of its heterogeneous habitat and topography, the region sustains a distinct biome with rich biological diversity and high level of endemism [2]. Extant plants and animals on the plateau are likely either descendants of relict colonists that migrated from other areas or recently derived owned species [3–10]. However, the colonization and population expansion history of many species remains poorly understood, despite current and hereafter impacts of climate modify and anthropogenic threats to multifariousness loss.

Two brown deport subspecies, the Himalayan (Ursus arctos isabellinus) and the Tibetan (U. a. pruinosus) brown bear, inhabit the northwestern Himalayan region and southeastern Tibetan Plateau, respectively [11–14] (figure one). These 2 subspecies have singled-out skull features and the Himalayan brown bear is characterized by its paler and crimson-brown fur, while the Tibetan dark-brown bear has generally darker fur with a adult, white 'collar' around the neck [eleven]. Every bit the most widely distributed carry in the world, phylogeography of the chocolate-brown behave has been well studied in North America, Europe and Japan [10,xvi–24]. However, due to limited sampling, very few studies have been conducted on these enigmatic subspecies. Two complete mitochondrial genomes (mitogenomes) from captive Tibetan brown bears are available, while only two brusque fragments of mitochondrial DNA (mtDNA) sequences from the Himalayan brown behave have been published [10,xv]. Phylogenetic analyses based on these sequences suggested that the Tibetan dark-brown acquit might be a relict population of the Eurasian brown behave [ten], and that the Himalayan brown bear, which is genetically distinct from the Tibetan dark-brown bear, may correspond a more ancient lineage [xv]. However, phylogenetic relationships deduced from express genetic data and number of individuals take put these preliminary findings into question. For example, the phylogenetic placement of a Gobi brownish bear (U. a. gobiensis) sequence [25] was inconsistent with a later study also including sequences from Himalayan brown bear [fifteen], and phylogenetic trees based on mtDNA control region and cytochrome b sequences, respectively, of the Tibetan brown deport were incongruent [26]. The other deport species found to inhabit the Tibetan Plateau–Himalaya region is the Asian blackness behave (U. thibetanus), which historically had a continuous distribution from southeastern Iran through Afghanistan and Pakistan to India, Nepal, Communist china, Korea, Nippon, and south into Myanmar and the Malayan peninsula [12,27,28]. Today it occupies a patchy distribution throughout its celebrated range, including across a narrow band from Pakistan, Kashmir and to Kingdom of bhutan, the home range of the Himalayan black carry (U. t. laniger) [27,29], which was described every bit distinguished from other black deport populations past its longer, thicker fur and smaller, whiter chest mark [11]. Although the range of Asian black acquit overlaps with dark-brown bear in the Tibetan Plateau–Himalaya region, information technology is mostly found at lower altitudes in forested hills ranging from 1200 to 3300 m [12,29]. So far, little is known nigh the evolutionary history of black deport in the region and no sequence data are available from the Himalayan black bear. To elucidate the evolutionary and migration history of the Himalayan and Tibetan bears, more genetic data from additional individuals are critically needed.

Figure 1. Distribution of Himalayan and Tibetan brown bear and localities of samples studied. Red and blueish lines outline the judge historical range of the Himalayan brownish comport and the Tibetan chocolate-brown behave, respectively (redrawn from Galbreath *et al.* [xv]). The triangles, diamonds and circles, respectively, betoken the judge collecting localities of the studied samples associated with Asian black bear, Tibetan chocolate-brown bear and Himalayan chocolate-brown acquit.

It has been reported that the brown comport populations in the Tibetan Plateau–Himalaya region have declined by more one-half in the by century because of habitat loss, fragmentation, poaching and intense hunting by humans [12,29–31]. Facing the same threats as brown bears, Asian black conduct populations accept also decreased in the past few decades [29,32,33]. The Himalayan brown bear is listed in the IUCN (International Wedlock for the Conservation of Nature) red list of threatened species as critically endangered [34], while the Asian black bear is listed as vulnerable [27]. Hence, clarifying population construction and genetic variety for conservation management purposes is also urgently needed for these endangered bear species.

The Tibetan Plateau–Himalaya region is also known for the legend of purported 'hominid'-like creatures, referred to every bit the 'yeti', 'chemo', 'mheti' or 'bharmando', amid other regional monikers (for simplicity they are referred to in this paper every bit yeti). Despite decades of enquiry and anecdotal association with bears and other mammals in the region [35,36], the species identity of the mysterious yeti is still debated, given the lack of conclusive evidence. A survey of hair samples attributed to yeti and other anomalous, supposed primates, was recently conducted to identify their genetic affinities [37]. Based on a short fragment of the mtDNA 12S rRNA factor from 2 samples collected in Ladakh, India and Kingdom of bhutan, respectively, and a 100% match to a sequence recovered from a subfossil polar bear [38], Sykes et al. [37] speculated that an unclassified bear species or hybrid of polar carry and dark-brown acquit might exist present in the Tibetan Plateau–Himalaya region. Nonetheless, this speculation was critiqued by others [39,40], and their phylogenetic analyses using the sequences from Sykes et al. and other available Ursidae sequences did not rule out the possibility that the samples belonged to brown bear. Thus, to get accurate species identification, comprehensive phylogenetic analyses using genetic data from more variable and informative loci are needed.

Here, we report on new analyses of 24 field-collected and museum specimens, including hair, bone, skin and faecal samples, collected from bears or purported yetis in the Tibetan Plateau–Himalaya region. Based on both amplified mtDNA loci equally well equally consummate mitogenomes, we reconstructed maternal phylogenies to increase cognition well-nigh the phylogenetic relationships and evolutionary history of Himalayan and Tibetan bears.

ii. Material and methods

(a) Samples

A total of 24 samples, including hair, tissue, os and faeces, were analysed in this report (electronic supplementary material, table S1). Of these, 12 samples had been nerveless for a previous analysis of Himalayan chocolate-brown conduct in the Khunjerab National Park, Northern Pakistan [xxx], two samples were from purported Himalayan brown bears housed in the Lahore and Islamabad Zoos, one bone sample (M-70448) recorded equally U. a. pruinosus was obtained from the American Museum of Natural History, and ix samples were provided to united states of america by the Reinhold Messner Museum and the Icon Film Visitor.

(b) Dna extraction

Genomic Deoxyribonucleic acid from 12 faecal samples nerveless in the Khunjerab National Park, Northern Pakistan [41], were previously extracted using the QIAmp DNA Stool Kit (Qiagen, USA) in a room dedicated to processing hairs and faeces [xxx]. DNA from two ethanol-preserved hair samples from Lahore and Islamabad Zoos were isolated in a room defended to nucleic acid extraction from modern samples. A DNeasy Blood & Tissue DNA Kit (Qiagen, USA) was used according to the manufacturer'due south protocol, except for the following modifications to optimize extraction of DNA from hair: 10 strands of pilus from each sample were cut into fragments of approximately 0.5 cm with a sterile razor blade. Ethanol was allowed to evaporate (approx. 1 h), and hair fragments were transferred to a microcentrifuge tube. Three hundred microlitres of ATL buffer, 20 µl proteinase K, 20 µl 1M DTT (dithiothreitol) and four µl RNase A were added, and samples were incubated at 56°C overnight until completely lysed. A negative control was prepared alongside each hair sample. Following lysis, 300 µl AL buffer and 300 µl 100% EtOH were added to each sample, and the mixture was pipetted into the DNeasy Mini Spin Column and centrifuged for ii min. Dna was eluted twice with 50 µl AE buffer for a total elution book of 100 µl. The remaining ten samples, which had not been intentionally preserved for later extraction of Deoxyribonucleic acid, were regarded as not-modern (ancient) samples, and thus DNA extractions and pre-amplifications were performed in a dedicated state-of-the-art cleanroom facility, physically separated from whatsoever modernistic DNA laboratory and appropriate for aboriginal Dna inquiry. The following protocols designed for aboriginal Deoxyribonucleic acid extraction were used: for bone samples, 50–100 mg fine bone powder was obtained from each sample past using a dental drill (HKM Surgical Handpiece, Pearson Dental, USA), and l–100 mg skin samples were sliced into approximately ane mm pieces with a sterile razor blade. DNA from the bone powder and the sliced skin samples was extracted using the protocol in Dabney et al. [42]. DNA from the hair samples were extracted using the protocol provided past Gilbert et al. [43] with the following modifications: one ml digestion buffer was used for each pilus extraction. After purification with phenol and chloroform, boosted purification was performed using Qiagen MinElute PCR Purification Kit (Qiagen, USA). Finally, a 12.5 µl EB buffer elution footstep was performed twice to obtain a total elution volume of 25 µl. Dna from approximately 100 mg faecal samples was extracted using the QIAmp Deoxyribonucleic acid Stool Kit (Qiagen, USA). The final elution footstep was also performed twice to obtain a total book of 100 µl. Negative controls were prepared alongside all extractions.

(c) PCR amplification

PCR amplifications from mod DNA were performed in a 25 µl reaction book each containing two.v µl of 10 × PCR buffer (Applied Biosystems, U.s.), 1.0 µl of dNTP mixture (two.5 mM each dNTP; Applied Biosystems), 2.v µl of MgCl₂ (25 mM, Applied Biosystems), 0.one µl of Taq DNA polymerase (5–10 U µl⁻¹; Applied Biosystems, AmpliTaq Gold), i µl each of the frontward and reverse primers (x µM), 2 µl of the genomic DNA and 17.4 µl of H₂O. The PCR reaction mix for ancient DNAs was prepared in the cleanroom past adding 21 µl H₂O, i µl of each frontward and reverse primer, and 2 µl genomic Deoxyribonucleic acid to each GE illustra PuReTaq Fix-To-Go PCR dewdrop (GE Healthcare, USA). A touchdown thermal cycling protocol was used as follows: 10 min at 94°C, ten cycles of 30 s at 94°C, 30 s annealing with the temperature decreasing every cycle by 0.v°C from 55°C to 50°C, and xxx s extension at 72°C, followed past 25 cycles the annealing temperature set to 50°C and denaturation and extension phases as above. For samples of unknown identity, two sets of mtDNA 12S rRNA primers [44,45] were used to determine their approximate taxonomic analogousness. Bear-specific primers targeting the mtDNA control region and cytochrome b ([46] and primers designed for this written report; run across electronic supplementary material, table S2) were used for samples identified as ursid bears. PCR products were Sanger sequenced direct using the same primers equally in the PCR.

(d) Mitochondrial genome target enrichment and sequencing

50 microlitres of DNA extracts from iv samples were sent to MYcroarray (http://world wide web.mycroarray.com) for training of Ion Torrent sequencing libraries and mtDNA target enrichment and sequencing, using the following protocol. Sample libraries were quantified using spectrofluorometry, which indicated betwixt 5 and 255 total nanograms (0.2–8.5 ng µl⁻¹) of double-stranded Dna. Each library was then individually target enriched using a custom-designed ursid mitogenome bait set manufactured by MYcroarray. The standard MYbaits five. 3.0 protocol was applied with hybridization for 21 h at lx°C at all relevant steps. Following clean up, half of each dewdrop-bound library was amplified in a fifty µl reaction with universal Ion Torrent adapter-primers for 10 cycles using a KAPA HiFi premix (KAPA Biosystems) and the manufacturer's recommended thermal contour coupled with 62°C annealing temperature. After amplification, the beads were pelleted and the supernatant was purified using SPRI beads and eluted in Tris-HCl buffer containing 0.05% Tween-20. The enriched libraries were quantified with spectrofluorometry, which indicated between ane.12 and iv.21 total nanograms dsDNA per library (0.03–0.12 ng µl⁻¹). Equal masses of each library were pooled, bead-templated and sequenced alongside other project libraries on the Ion Proton platform using the Ion PI Chip Kit v2 chemistry. Post-obit sequencing, reads were de-multiplexed, quality trimmed and filtered using the default settings on the Ion Torrent Suite v. 4.4.three.

(e) Mitochondrial genome associates

Assembly of mitochondrial genomes was performed using the following strategy: species-specific mitochondrial reference genomes were selected from initial species identification based on phylogenetic analyses of amplicon sequences (results not shown). All Ion Torrent reads were first aligned against the reference genome using BWA aln (v. 0.7.thirteen) [47] using the default parameters, except for the parameter '-fifty 1024' to disable the seed and increment high-quality hits for the damaged ancient DNA reads [48]. The remaining unmapped reads were then aligned against the aforementioned reference using BWA mem with default parameters (run across electronic supplementary material, tabular array S3, for assembly statistics). Nosotros filtered for human being contamination past applying an edit-altitude based strategy [48]. All reads were mapped to a human mitochondrial genome reference (NCBI accession J01415.ii) using the aforementioned BWA mapping method described in a higher place. Reads with a higher mapping edit-distance to human mtDNA than to carry mitochondrial genomes were considered of likely human origin and were removed from the bear mitogenome mapping results. PCR duplicates were removed with the MarkDuplicates tool in the Picard software suite v. 1.112 using lenient validation stringency (http://broadinstitute.github.io/picard/). Consensus calling was carried out using Samtools mpileup [49] with default settings.

(f) Phylogenetic analyses

Complete mitochondrial genomes, partial command region sequences, and cytochrome b sequences for 11 Asian black bears, 76 American black bears, two cave bears (U. spelaeus), 200 brown bears, and 52 polar bears were obtained from GenBank (electronic supplementary material, tabular array S4). Ii GenBank datasets were created: one dataset included but complete mitogenomes for the non-Tibetan/Himalayan bears and both partial (amplicon sequences) and complete mitogenomes for Tibetan and Himalayan bear lineages, while the other dataset included both amplicon sequences and consummate mitogenomes for non-Tibetan/Himalayan bears. All new sequences produced in this written report were added to these two GenBank datasets and used in the phylogenetic analyses. Sloth bear (U. ursinus) and sun deport (U. malayanus) sequences were included to root the trees (electronic supplementary material, tabular array S4). Alignments were generated using MAFFT [fifty] followed by manual adjustment in BioEdit [51] to exclude the variable number tandem repeats of the D-loop. The total length of the concluding alignment was 16 412 bp. Maximum-likelihood (ML) phylogenetic analyses were performed using RAxML-HPC BlackBox v. 8.2.eight [52] in the CIPRES Scientific discipline Gateway under the GTR substitution model, which was identified as the best-supported model by jmodeltest2 [53,54]. A full of 1000 bootstrap replicates were conducted to evaluate co-operative support. Bayesian inference (BI) phylogenetic analyses were carried out using MrBayes v. 3.ii.6 [55] in two runs of 5 000 000 Markov chain Monte Carlo (MCMC) generations, with trees for estimation of the posterior probability distribution sampled every 100 generations. The best-fit substitution model was determined by the program by setting Nst=mixed; 500 000 trees were discarded as burn-in.

(yard) Divergence time interpretation

Bayesian MCMC-based divergence time estimation was carried out using Animal version 1.8.0 under the GTR substitution model. The dataset used for molecular dating analysis included only consummate mitogenome, since shorter mtDNA regions (e.g. command region and cytochrome b) are by and large associated with considerable dubiety and may bias molecular dating analyses due to homoplasy [x,17]. The uncorrelated lognormal relaxed clock and the abiding size coalescent prior were used. Radiocarbon dates and stratigraphically estimated dates for four ancient sequences were used to calibrate ages for terminal nodes, including three sequences from extinct comport species (U. spelaeus and U. deningeri) dated to 31.8 thousand years (ka) BP [56], 44.1 ka BP [57], and 409 ka BP [42], an approximately 120 ka BP polar bear subfossil [38], and vii European brown bears dated to approximately 4.1–37 ka BP [58]. Trees were sampled every 1000 generations from a total of i 000 000 000 generations. The maximum clade credibility tree was generated using TreeAnnotator, implemented in the Beast bundle [59], with 10% burn-in. Effective sampling size value greater than 200 for all parameters sampled from the MCMC and the posterior distributions were examined using Tracer v. 1.half dozen [threescore].

3. Results

(a) Identity and phylogenetic placement of the Tibetan Plateau–Himalayan samples

Except for one tooth sample nerveless from a stuffed exhibit at the Reinhold Messner Mountain Museum, which Blast-matched domestic dog (Canis lupus familiaris), all other samples were identified as ursid bears. ML tree reconstruction based on amplicon and mitogenome sequences (electronic supplementary fabric, figure S1) grouped the 23 samples inside four bear lineages: Himalayan brownish bear, Tibetan brown bear, Continental Eurasian brown bear and Asian blackness bear. Complete mitogenomes were assembled from one private in each of the four identified bear lineages (electronic supplementary material, table S3). ML and BI phylogenetic trees were reconstructed using the newly obtained amplicon sequences, consummate mitogenome sequences, and previously published deport mtDNA sequences, using sloth comport (U. ursinus) equally an outgroup (figure ii and electronic supplementary textile, figures S2 and S3). In general, the ML and BI tree topologies are consistent and in agreement with previous studies [10,17,61], with all major polar, brownish and blackness behave clades well-resolved and strongly supported. The 2 Tibetan–Himalayan black acquit samples formed a well-supported sis lineage to all other Asian blackness acquit subspecies. The polar and brown bears grouped into ix clades (clades 1, 2a, 2b, 3a1, 3a2, 3b, 4, five and a Himalayan clade, with numerical clade nomenclature following [x,17]). Fourteen samples collected in Islamic republic of pakistan and the Himalayas grouped with a previously reported Gobi brown bear (GOBI-1) and two Himalayan brown bears (DQ914409 and DQ914410), and formed a sister lineage to all other brown and polar behave clades with stiff bootstrap support. 6 samples collected from the Tibetan Plateau grouped with previously sequenced Tibetan dark-brown bears, which together formed a sister clade to several other Due north American and Eurasian chocolate-brown bear lineages (clade 3a1, 3a2, 3b and 4). I specimen (Chiliad-70448), which was sampled from the American Museum of Natural History'south mammal collection and identified every bit a Tibetan brownish comport, maybe of 'mixed breed', grouped in clade 3a with brownish bears from Syria, Turkey, and animals held at Zoos in Europe [24] (electronic supplementary material, effigy S1 and table S4).

Figure 2. Phylogenetic copse based on (a) ML and (b) BI analyses of new mtDNA sequence information produced in this study and sequence data obtained from GenBank. New sequences are marked with triangles, diamonds, circles and a square, indicating the Asian black comport, Tibetan brown bear, Himalayan brown bear and the brown behave from the AMNH, respectively. GenBank data include complete mitogenomes of non-Tibetan–Himalayan bears, as well as amplicon and complete mitochondrial sequences of Tibetan and Himalayan bears. Major maternal clades and their geographic range are labelled following [10,17]. Come across electronic supplementary material, figures S2 and S3, for complete versions of the copse, shown with posterior probability and bootstrap values.

(b) Divergence fourth dimension estimations

MCMC-based divergence times discussed in the text are shown in effigy 3 (see electronic supplementary material, figure S4, for divergence times estimated for all nodes). For the brownish bear clades, the difference time betwixt the Himalayan lineage and all other brown deport lineages was estimated to be 658 ka BP (95% HPD: 336–1258 ka BP). The divergence time between the Tibetan lineage and its sis North American and Eurasian lineages (clade 3 and four) was estimated at 342 ka BP (95% HPD: 99–618 ka BP), and the separate of the Continental Eurasian lineage (clade 3a) was estimated to exist 146 ka BP (95% HPD: fourteen–799 ka BP). For the blackness bear clades, the ancestor of the Himalayan black bear lineage diverged from other Asian blackness bear lineages at approximately 475 ka BP (95% HPD: xv–831 ka BP).

Figure 3. — Figure iii. Maximum clade brownie tree from a Creature analysis based on complete mitogenomes. The numbers at nodes betoken the median estimated divergence time in ka BP (HPD values are shown in brackets and the lower scale indicates time in ka BP). The coloured vertical confined indicate, from left to correct, time spans of four Pleistocene glaciations: the Xixabangma, Nyanyaxungla, Guxiang and Baiyu. New mitogenomes sequenced in this written report are indicated with symbols as in figure 2. Meet electronic supplementary material, figure S4, for a consummate version of the tree and difference times estimated for all nodes.

4. Discussion

(a) Phylogenetic placement and evolutionary history of Himalayan and Tibetan brownish bears

Few genetic studies have been conducted of bears in the Tibetan Plateau and surrounding Himalaya region, and their evolutionary history remains enigmatic. Specially little is known about the Himalayan dark-brown comport (U. a. isabellinus). First, Masuda et al. [25] reported a 269 bp mtDNA command region sequence from a Gobi comport nerveless from the Great Gobi National Park in Mongolia, and suggested information technology was more closely related to Western European brown bears based on a neighbour-joining phylogenetic analysis. Later on, Galbreath et al. [15] investigated homologous DNA fragments from 2 brownish bears nerveless from the Deosai Plains of the western Himalayas. Their analyses demonstrated that the two Himalayan brownish bears grouped together with the Gobi bear, confirming a close relationship between these 2 populations and a clear separation from European and Tibetan brown bears. Our results, providing more data and meliorate resolution, demonstrate that the Himalayan dark-brown bears, including the previously reported Gobi bear and Deosai bears, form a well-supported, sis lineage to all other extant brown behave clades included here. This result strongly supports Himalayan dark-brown bears as a relict population that diverged early from other brown bear populations.

The phylogenetic position of Tibetan brown bears (U. a. pruinosus), which form a sis clade to Due north American and Eurasian brown bears consistent with previous reports [10,17–nineteen,25], indicates that the Tibetan and other Eurasian brown bears, every bit well as North American dark-brown bears, are all descendants of a common ancestral lineage. It was proposed that the Tibetan brownish bears migrated to the Tibetan Plateau from its source population—ancestral Eurasian brownish bears—approximately 343 ka BP, and that they remained geographically isolated from this source population thereafter [10]. Our phylogenetic analyses strongly back up this migration scenario.

In our study, brownish behave samples collected in the northwestern to western Himalayas were all identified as Himalayan brown acquit, while the ones nerveless in the southeastern Himalayas and Tibetan Plateau were all identified every bit Tibetan brownish carry (figure i). The historical range of the Himalayan chocolate-brown bear extends from the n and west of the Taklimakan Desert to the western Himalayas, while the historical range of the Tibetan chocolate-brown bear lies in the Tibetan Plateau and the southeastern Himalayas [15]. While the Tibetan dark-brown bears share a common ancestry with extant North American and Eurasian brown bears, the Himalayan chocolate-brown bear appears to take originated from an aboriginal lineage that experienced long isolation in the mountains of central Asia, at to the lowest degree over the concluding 658 ka. Although the habitats of the two brown bear subspecies are geographically shut, the high-altitude peaks of the Himalayan Mountains have likely impeded migration between these populations, and subsequently kept them as genetically distinct lineages.

(b) Phylogenetic placement and evolutionary history of the Himalayan black comport

The phylogenetic topology of Asian black bears is in agreement with a previous finding [61], except here nosotros also include the rare Himalayan black bear (U. t. laniger), which forms a sister lineage to all other Asian blackness bears. Although sampling is limited, this effect indicates that the Himalayan black bear originated from an ancient lineage and experienced long isolation in the Himalayan Mountains, a similar scenario to the deviation of the Himalayan brown bear lineage. However, the departure time for the Himalayan blackness bear is younger, estimated at 475 ka BP, suggesting the isolation of Himalayan black bear occurred afterward than the isolation of the higher-altitude Himalayan brown bear. Reportedly, other described subspecies occur in the region, the Tibetan (U. t. thibetanus) and Indochinese (U. t. mupinensis) black comport, simply whether these subspecies overlap is unclear given no modern revisionary piece of work exists. Our phylogenetic relationships point that individuals from the Himalayas are genetically distant from other populations analysed, suggesting that petty if whatsoever gene menstruation has occurred between this and other Asian black behave populations. Similar to the brown deport situation, the high mountains may also have separated the habitats of these black behave subspecies, possibly keeping U. t. laniger to the western Himalayas, and U. t. mupinensis and U. t. thibetanus to the due east. Analyses of more than individuals throughout the region and inclusion of nuclear Dna would be needed, however, to explore if this pattern is restricted to maternal gene flow but.

(c) 4th climatic oscillations and divergence of local carry lineages in the Tibetan Plateau–Himalaya region

The Tibetan Plateau is one of the youngest plateaus on Earth, created by the collision of the Indian subcontinent with the Eurasian continental plate in early Cenozoic times, followed past diachronous and extensive surface uplifts in the Miocene and even into the Pleistocene [62,63]. Although the dates and details of the uplifts take long been debated, many studies indicate they caused dramatic climatic changes and topographic variation, which facilitated the introduction and evolution of new plant and animal clades and greatly influenced the current spatial distribution of local species and their genetic diversity [64]. The Pleistocene glaciations of the Tibetan Plateau, which is closely related to the progressive uplift of the plateau and the surrounding Himalayan Mountains, accept been suggested to have had a highly circuitous pattern, occurring asynchronously with the Northern Hemisphere glaciation events [65]. Four Pleistocene glaciations have been described in several geological and geographical studies [66–68]; the Xixabangma (Early Pleistocene, 1170–800 ka BP), Nyanyaxungla (Middle Pleistocene, 720–500 ka BP), Guxiang (Middle-Late Pleistocene, 300–130 ka BP) and Baiyu (Late Pleistocene, 70–ten ka BP) events. The nearly widespread Nyanyaxungla glaciation [64,69] was initiated by successive Kunlun-Huanghe tectonic movements. Interestingly, the divergence time of the Himalayan brown conduct at effectually 658 ka BP overlaps with the Nyanyaxungla glaciation event, suggesting that this glaciation event may have caused the initial isolation of Himalayan brown behave. Glacial retreat occurred following the Nyanyaxungla glaciation, causing changes in environmental conditions from cold and arid to warm and wet during the great interglacial flow (500–300 ka BP) [68]. Both the divergence of the Himalayan black bear at effectually 475 ka BP and the Tibetan brown deport at effectually 342 ka BP overlap with this interglacial period, indicating that ancestors of these bear lineages migrated from lower altitudes to college distance locales afterwards glaciers retreated. Subsequently, these populations may have diverged from lower distance populations due to isolation in the high mountains and the post-obit Guxiang glaciation effect. Phylogeographic studies of many Tibetan establish and animal species betoken that local extant institute and fauna populations, which mainly derived from colonists migrating from other areas or represent endemic species that diverged recently [3–ten], experienced extensive oscillations and survived through glacial periods in multiple refugia or microrefugia on the plateau [1,65,70–75]. Similarly, we speculate that ancestral bear lineages on the Tibetan Plateau and Himalayan Mountains probable immigrated to the region from nearby Asian locales. These ancestral lineages so likely experienced extensive population oscillations caused by local climatic changes and diverged from other bear populations in refugia during the Pleistocene glaciations.

5. Conclusion

Samples collected in the field and archived in museum or private collections can significantly assistance in our understanding of the genetic variation and phylogeographic patterns of rare and widespread species. To determine accurate species identification and clade affinity, however, phylogenetically informative genetic markers and appropriate phylogenetic analyses are critically needed. Based on a Boom search using a 104 bp fragment of the mitochondrial 12S rRNA locus, which gave a 100% lucifer to a complete mitogenome recovered from a subfossil polar bear [38], Sykes et al. [37] suggested that a previously unrecognized acquit species or possibly a hybrid betwixt brown behave and polar bear exists in the Himalayas. All the same, every bit too demonstrated by others [39,40], the brusque 12S rRNA factor fragment is insufficiently informative to make up one's mind precise taxonomic identity, particularly among closely related species, although it tin can exist a useful screening mark to appraise preliminary species affinities. We isolated Dna and assembled a complete mitogenome from a hair sample (collected in Ladakh, India, and named 'YHB' in this written report), which based on their shared collection locality and other anecdotal bear witness obtained from Icon Films, our sample source, may come from the same specimen that Sykes et al. [37] speculated represents an unknown or hybrid bear. Here, we unambiguously prove that this sample is from a bear that groups with extant Himalayan dark-brown comport. Similarly, we were able to make up one's mind the clade affinities of all other purported yeti samples in this study and infer their well-supported and resolved phylogenetic relationships among extant bears in the Tibetan Plateau and surrounding Himalayan Mountains. This study represents the most rigorous analysis to appointment of samples suspected to derive from anomalous or mythical 'hominid'-like creatures, strongly suggesting that the biological basis of the yeti legend is local brown and black bears.

Information accessibility

The DNA sequences generated in this study are deposited in the NCBI database under accession nos MG066702–MG066705 and MG131869–MG131905.

Authors' contributions

T.L. and C.L. designed the study; T.Fifty. and S.G. generated the sequence information; T.L. and C.L. analysed the data; East.B., R.B. and M.A.North. provided of import samples and Deoxyribonucleic acid; T.L. and C.L. drafted the manuscript and all authors contributed to the writing of the manuscript and gave their final blessing for publication.

Competing interests

The authors take no competing interests.

Funding

We thank the Icon Film Company for fiscal back up. C.L. is funded past the National Science Foundation (DEB #1556565).

Acknowledgements

We thank the Icon Film Company, the Reinhold Messner Museum, and the American Museum of Natural History for providing samples and permissions to undertake destructive sampling. Special thanks to Harry Marshall, Charles Allen, Pemba Tashi and Sonam Norbu for sharing their samples and to MYcroarray for technical assistance.

Footnotes

Electronic supplementary textile is available online at https://dx.doi.org/10.6084/m9.figshare.c.3933163.

Published by the Regal Social club under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted apply, provided the original author and source are credited.

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