| Woolly rhinoceros (Coelodonta antiquitatis Blummenbach,1799), an improvement species of Coelodonta(Coelodonta Bronn,1831), was highly adapted to the environment on cold grassland, periglacial tundra and permafrost tundra, which was gradually developed into a typical periglacial animal in the influence of Pleistocene glacial climate. During the Pleistocene, woolly rhinoceros was widely distributed in the Northern Hemisphere from72°N to33°N. The extensive woolly rhinoceros fossil records were found in northern Eurasia, but most of them belong to late Pleistocene. The early fossil records of the genus have been founded in several sites from China and the amount of early fossil materials is very limited. Up to now, the earliest representative of the genus, Coelodonta thibetana (3.7Ma), was found in the Zanda Basin in southwestern Tibet. Another early fossil record, Coelodonta nihowanensis (2.5Ma), was found in the Linxia Basin in Gansu Province, China. Comparably, the earliest fossil species outside China appeared later than that from China. A typical representative, Coelodonta tologoijensis (0.75Ma), has been found around the early middle Pleistocene in Buryatia, Russian Federation. In Europe, Coelodonta first appeared around400-460ka according to several early fossil records (such as Romania). Therefore, some researchers suggested that the woolly rhinoceros (Coelodonta) may have originated in China. After the origination of this genus in Asia, it diffused to eastern and central Europe in the middle Pleistocene for the first time, and gradually evolved to adapt the life on the cold tundra and steppe in the influence of Quaternary glacial climate, and it finally evolved into the most famous ice age animal——the true woolly rhinoceros (Coelodonta antiquitatis). After the origination of this species in Europe, it dispersed to north Asia and China in the following cold and dry period. Due to the incompleteness of the fossil record, especially the lack of early fossil records and very limited sampling sites, our understanding on its origin, migration, evolution and other issues is very ambiguous.In1986, with the invention of polymerase chain reaction (PCR) technology, especially the rapid development of molecular biology technology (multiple PCR, large scale sequencing, high throughput sequencing, direct multiplex sequencing, array-based sequence and so on), the extraction and amplification of DNA from ancient biological remains come true. Ancient DNA, as the carrier of genetic information, contains all the information of biological genetic variation and growth. It could provide important research data for people to explore the occurrence of biological system and evolution during the long history. At the same time, it also provides us a new research method to rebuild the origin and evolution process of life. So ancient DNA technology is playing more and more important role in the the study of origin, evolution, migration and genetic diversity of life.At the molecular level, the study on woolly rhinoceros is very limited. Until now, there are only several cases reported in the world. Orlando et al.(2003) extracted complete12S rRNA gene and partial cytochrome b gene of woolly rhinocers collected from Belgium. Based on the sequences, they performed the phylogenetic analyses and the results indicated that woolly rhinoceros is more closely related to extant Dicerorhinus sumatrensis. Binladen et al.(2006) investigated the frequency and types of miscoding lesions in mtDNA and nuDNA marks using two woolly rhinoceros permafrost bone samples, suggesting that there was no significant evidence for nuDNA sequences being more prone to miscoding lesions than mtDNA sequences, the conversion ratio from cytosine into thymine and from guanine into adenine occured more rates than that from Thymine into cytosine and from adenine into guanine. This study indicated that the deamination from cytosine into uracil is the main mismatch in ancient DNA ((both mitochondrial DNA and nuclear DNA). Willerslev et al.(2009) first obtained the whole mitochondrial genome sequences of woolly rhinoceros collected from permafrost inYakutia, Russian. Using the obtained mitochondrial DNA of woolly rhinoceros together with five extant rhino mitochondrial genome sequences, phylogenetic analysis was performed and the results indicated that the six species clustered into three sister groups:Diceros bicornis ICeratotherium simum, Rhinoceros sondaicus/Rhinoceros unicornis, Coelodonta antiquitatis I Dicerorhinus sumatrensis. However, the rhinoceros phylogenetic trees are highly diffuse, the relationship between the three branches will change when using different genes or different out groups. So, the phylogenetic issue between Coelodonta antiquitatis/Dicerorhinus sumatrensis, Rhinoceros sondaicus/Rhinoceros unicornis, Diceros bicornis ICeratotherium simum has not been resolved.Orlando et al.(2003) and Willerslev et al.(2009) carried out the molecular clock calculations based on the calibration time points of out groups, and they obstained the divergent timescales21-26Ma and17.5-22.8Ma for woolly rhinoceros from Sumatran rhinoceros, there is a big gap between these timescales and the known earlist fossil record of woolly rhinoceros. Moreover, Orlando et al.(2003) also pointed out the evolutionary timescale for woolly rhinoceros from Sumatran rhinoceros should be taken as preliminary because the same calibration failed to recover the date of emergence of the Equus genus. So, people need to do further researches to explore the divergence time between woolly rhinoceros and its relatives. In addition, to investigate phylogenetic status of Coelodonta thibetana (3.7Ma), Deng et al.(2011) reconstructed phylogenetic trees based on the morphological data, using seventeen rhinoceros genera which contained all the five extant rhinoceros and twelve extinct rhinoceros (including all the known extinct Coelodonta:Coelodonta thibetana, Coelodonta nihowanensis, Coelodonta tologoijensis and Coelodonta antiquitatis). The results show that Dicerorhinus sumatrensis clustered together with two extant one-horned rhinoceros (Rhinoceros sondaicus/Rhinoceros unicornis) to form a sister group, it also formed a sister group with the branch of Coelodonta, or two extant one-horned rhinoceros first clustered together with the branch of Coelodonta, and then Dicerorhinus sumatrensis formed a sister group with the larger branch. However, at the molecular level, the the phylogenetic trees showed that woolly rhinoceros is most closely related to one of the extant rhinoceros species, Sumatran rhinoceros, and this result was inconsistent with the study based on morphological data.Which extant rhinoceros is more closely related to woolly rhinoceros? When did woolly rhinoceros separate from its relatives? When did Coelodonta diffuse from Asia to Europe? What routes did it experience during the process of migration? And other issues such as its extinction also need further exploration.Woolly rhinoceros remains from China occupy a very important role in the evolution history of woolly rhinoceros. The fossils found in China last a long period, there are a large number of late Pleistocene fossils in northeastern China and Salawusu region, the sample source is very rich, and the temperature in these places is low all year round, all these are very conducive to the preservation of ancient DNA. Our samples were collected from the late Pleistocene stratigraphy, and the age of the samples does not exceed the theoretical retention period of ancient DNA, these provide very good research materials for our ancient DNA work. Up to now, ancient DNA studies to the woolly rhinoceros fossil materials collected from China are very limited though there are a lot of late Pleistocene fossils.In this study, we obtained complete or partial cytochrome b sequences from eight late Pleistocene Coelodonta antiquitatis bones excavated from Salawusu (Inner Mongolia,42000YBP), Zhaodong (Heilongjiang Province,39000YBP) and Qinggang (Heilongjiang Province,35000YBP). The sequences obtained in this study enriched the GenBank data of woolly rhinoceros and they also make people better understand the genetic diversity of woolly rhinoceros samples, which were collected from different locations and belonged to different ages. At the molecular level, we reconstructed the phylogenetic trees of woolly rhinoceros, and this work improved us better understanding the phylogenetic status of Coelodonta antiquitatis from China. In addition, we also derived the divergence time between woolly rhinoceros and Sumatran rhinoceros using molecular clock. Firstly, we estimate the divergence time using the software MEGA4.0based on the split time between the equids and ceratomorpha (56Ma) or the split time between Artiodactyla and Cetacea (60Ma). We also carried out Bayesian analyse using the software of BEAST1.6.1and Markov chain Monte Carlo (MCMC) algorithm to estimate the divergence time based on the split time between the equids and ceratomorpha (56Ma). Secondly, we estimated the divergence time between woolly rhinoceros and Sumatran rhinoceros using the software Network4610based on the evolution rate of the cytochrome b gene as2%per million years. In addition, we tried to reveal the Eurasian migration of woolly rhinoceros.We mainly drew the following conclusions:(1) We obtained complete or partial cytochrome b gene sequences from eight woolly rhinoceros fossil materials collected from northeastern China and Salawusu region, the length of the sequences ranges from84bp to1140bp. Among them, we obtained the complete cytochrome b gene from the sample C.α._HS14, which was collected from Zhaodong, and to another Zhaodong sample C.α._HS12, we got the total length of1130bp cytochrome b gene fragment. To the Qinggang sample C. α._Qgl3, we got the length of490bp cytochrome b gene fragment. To the Salawusu sample C. α.SL1, we got the total length of651bp cytochrome b gene fragment, and to the other one Salawusu sample C. α._SL4, we got the total length of1100bp cytochrome b gene fragment. We have submitted the above gene sequences to GenBank respectively, and the GenBank accession numbers are GU371439(C.α._HS14), GU371440(C.α._HS12)、JQ974919(C.α._Qg13)、JQ974920(C.α.SL1) and JQ974921(C.α._SL4). The sequence length obtained from the other three samples is very short. To the sample C. a._Qg13collected from Qinggang, we only got84bp cytochrome b gene fragment. Another Qinggang sample C.α._Qg13, we only got140bp cytochrome b gene fragment. And to the sample C.α._SL5collected from Salawusu, we just got204bp cytochrome b gene fragment. The repeated experiments were carried out at the centre for ancient genetics, university of Copenhagen, and identical results were obtained, these all proved the sequences obtained in this study are true and reliable.(2) In this study, we obtained ancient DNA sequences from late Pleistocene Coelodonta antiquitatis remains excavated from northeastern China and Salawusu region. Our studies revealed that the soil, climate and environment conditions of northern and northeastern China are suitable for the preservation of ancient DNA. But the preservation of our samples is not as good as the samples excavated from Belgium Scladina cave and Siberia permafrost. Moreover, the preservation of our samples is quite different even them from the same location and the same stratigraphy.(3) Phylogenetic analyses show that all ancient Coelodonta antiquitatis samples analyzed are clustered together using both MEGA and Bayesian methods, this reveals that the sequences analyzed are homology. At the same time phylogenetic trees also show that Coelodonta antiquitatis group shared the closest relationship with the extant Sumatran rhinoceros. In addition, our results also reveal that the Coelodonta antiquitatis samples from Salawusu (C. α._SL1、C. α._SL4、C. α._SL5) together with one sample from Zhaodong County (C.α._HS14) appear at one sub-clade of the Coelodonta antiquitatis clade, the other samples from Zhaodong County and Qinggang County, Heilongjiang Province (C.α._HS12and C.α._Qg13) are grouped with the sample from Yakut, Russia (C.a._Willerslev). This may reflect the genetic exchange of woolly rhinoceros among the regions of northeastern China, Salawusu region and northern Asia during the late Pleistocene. Our study may reveal the migration of woolly rhinoceros, the typical periglacial animal. It dispersed from north to south or from south to north under the influence of climate change during the alternation of Quaternary glacial/interglacial periods.(4) We calculated the divergence time between woolly rhinoceros and Sumatran rhinoceroses using two molecular clock methods. Firstly, based on the calibration standard of outgroup fossil records, we obtained woolly rhinoceros and Dicerorhinus sumatrensis diverged at about24.5-27.6Ma by using MEGA phylogenetic analyses and22.5Ma by using BEAST phylogenetic analyses. Secondly, we derived another estimate of the divergence time considering the evolution rate of the cytochrome b gene as2%per million years, the separation between woolly rhinoceros and Sumatran rhinoceros occurred at about3.8-4.7Ma. Compared the two different timescales derived from different molecular dating methods, We considered that the older timescale approach of using the fossil calibration of the outgroup may have overestimated the divergent event. We suggested that the younger timescale obtained by using the evolution rate of the cytochrome b gene as2%per million years is more likely, because this timescale is more consistent with the earliest known fossil record.(5) We have analyzed the woolly rhinoceros cytochrome b sequences obtained in this study compared with previously published data from GenBank, the results indicated that woolly rhinoceros might be lack of genetic variation among analyzed samples in the late Pleistocene. Perhaps it is one of the important factor which led to woolly rhinoceros extinction at the beginning of Holocene. Considering the samples within differents regions, genetic diversity of woolly rhinoceros from Salawusu region is the highest, the genetic diversity of woolly rhinoceros from northeastern China is the second. Comparison sample gene diversity between regions, the results showed that gene diversity is the lowest between northeastern China samples and Yakut samples, and the gene diversity is relatively high between northeastern China samples and Salawusu samples. |
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