Monday 26 October 2020

Evidence for recent mitochondrial lineage extinction in the critically endangered Javan Rhinoceros.

The Javan Rhinoceros, Rhinoceros sondaicus, is the rarest of the five extant Rhinoceros species, today consisting of a single population in Ujung Kulon National Park on the western tip of the Indonesian island of Java. This critically endangered species is estimated to have only about 60 Animals remaining in the wild, and it is thus one of the rarest mammals in the world. Before the end of the 19th century, its range included much of Southeast Asia, from India and China to the islands of Java and Sumatra, putatively comprising three subspecies: Rhinoceros sondaicus inermis in the northern part of its range, Rhinoceros sondaicus annamiticus mainly in Vietnam, extinct since 2010, and the nominal subspecies Rhinoceros sondaicus sondaicus on Java and Sumatra. However, the distribution and numbers of Rhinoceros sondaicus since the end of the 19th century have decreased dramatically as a result of poaching, loss of habitat and diseases transmitted by Cattle. With the  last continental Asian Javan Rhinoceros killed in Vietnam in 2010, subspecies Rhinoceros sondaicus annamiticus was declared extinct, leaving only the Javan population (Rhinoceros sondaicus sondaicus) in the wild. With the entire population of the world today restricted to the Ujung Kulon National Park, the urgency of its conservation cannot be overstated.

Both fossil and genetic analyses indicate that the Indian Rhinoceros, Rhinoceros unicornis, is the closest extant relative of the Javan Rhinoceros, although there is disagreement on when they diverged. Fossil evidence suggests a divergence in Asia about 3 million years ago, whereas molecular estimates suggest a much earlier split, roughly 12–13 million years ago. Similar phylogenetic relationships between the Indian and Javan Rhinoceros have been observed based on protein sequences.

Previous genetic studies of Rhinoceros unicornis have been restricted to either a whole mitochondrial genome sequence of a single historical specimen, thus representing only a single point in its geographical range, or at the population scale restricted to short mitochondrial fragments, such as 12S rRNA gene and D-loop and 12S rRNA and cytochrome b genes. Thus, there is a need to improve the dataset of complete mitochondrial DNA genome sequences to be more representative of the geographical range that the species once covered. This can be resolved only by analysis of historical samples, such as those held in natural history collections. 

In a paper published in the Zoological Journal of the Linnean Society on 10 March 2020, Ashot Margaryan of the Section for Evolutionary Genomics at the Natural History Museum of Denmark, and the Institute of Molecular Biology of the National Academy of Sciences of Armenia, Mikkel-Holger Sinding, also of the Section for Evolutionary Genomics at the Natural History Museum of Denmark, and of the Greenland Institute of Natural Resources, Shanlin Liu, again of the Section for Evolutionary Genomics at the Natural History Museum of Denmark, and of BGI-Shenzhen, Filipe Garret Vieira, again of the Section for Evolutionary Genomics at the Natural History Museum of Denmark, Yvonne Chan of the Department of Bioinformatics and Genetics at the Swedish Museum of Natural History, Senthivel Nathan of the Sabah Wildlife Department, Yoshan Moodley of the Department of Zoology at the University of Venda, Michael Bruford of the School of Biosciences and Sustainable Places Institute at Cardiff University, and Thomas Gilbert, once again of the Section for Evolutionary Genomics at the Natural History Museum of Denmark, and of the University Museum at the Norwegian University of Science and Technology, present the results of a study which aimed to contribute to this, by using Illumina sequencing technology to generate mitochondrial DNA genomescale data from eight historical (100- to 200-year-old) specimens that span its historical geographical range. These data were also combined with new and existing Rhinoceros mitochondrial DNA sequences, in order to: (1) estimate the divergence times among Javan Rhinoceros; and (2) reveal the loss of mitochondrial DNA diversity in Javan Rhinoceros over the last century. Ultimately, although restricted to a relatively small sample size, Magaryan et al. hope that these mitochondrial DNA sequences from this rare and understudied species will be valuable both for providing insights into its former diversity and for future studies, in which new mitochondrial DNA-based assays might be designed for its monitoring.

Map of Javan Rhinoceros distribution in Southeast Asia, showing their approximate historical (yellow) and current (red) distribution. The green dots indicate the approximate locations of historical samples used in Magaryan et al.'s study. Two samples with unknown locations are not mentioned. Magaryan et al. (2020).

Eight historical Rhinoceros sondaicus specimens, kept in the collections of the Natural History Museum at the University of Oslo or the Natural History Museum of Denmark, were sampled for DNA extraction. Most of the samples were collected or registered in the 19th century and originated from Java, Sumatra and Bhutan (only one sample) according to the museum records. All laboratory work was performed in the ancient DNA laboratories at the Centre for GeoGenetics at the Natural History Museum of Denmark, following standard clean laboratory procedures.

Magaryan et al. complemented this dataset with the rhinoceros mitogenome sequences available in GenBank and through reconstruction of an additional four full mitochondrial genomes from currently unpublished data from the Rhinoceros genome sequencing consortium. This unpublished dataset includes three modern Rhinoceros species (one each for the Black Rhinoceros, Diceros bicornis, the Indian Rhinoceros, Rhinoceros unicornis, and the Sumatran Rhinoceros, Dicerorhinus sumatrensis, and
the extinct Woolly Rhinoceros, Coelodonta antiquitatis.

In total, 362 066 719 sequence reads were produced from the eight historical Javan Rhinoceroses. After removal of adapters and trimming for stretches of Ns and low-quality bases, 315 609 438 sequences remained, with an average read length ranging from 48.4 to 63.2 base pairs.

The number of reads mapping to the mitochondrial DNA genome of Rhinoceros sondaicus varied greatly among the samples and ranged from as few as 494 (JR524) to 558 067 reads (JR734), which probably reflected different preservation states of these historical samples. For five out of eight historical samples, the depth of coverage of mitochondrial DNA was more than 24X, which allowed Magaryan et al. to reconstruct the whole mitochondrial DNA sequences of these samples reliably, with close to 100% mitochondrial DNA coverage. For the remaining three samples with low depth of coverage (1.64X–1.96X), only partial mitochondrial DNA sequences were generated, spanning about 50% of the genome. The shotgun sequencing reads from all historical samples showed increased C→T deamination rates at the 5′ ends of the sequences when compared with the R. sondaicus reference mitochondrial sequence. Together, these elevated C→T damage profiles and the short average length of mapped reads are consistent with the notion that the DNA molecules were of ancient origin. 

The phylogenetic relationship between the Javan and other Rhinoceros species based on whole mitochondrial DNA genomes showed that Elasmotherium was a sister group to all modern Rhinoceros (the Rhinocerotinae group), with 89% posterior probability with the split time of about 31 million years. The most recent common ancestor of the group including all three extant Rhinoceros species was estimated to have lived about 18 million years ago. According to the Bayesian phylogeny, the Javan Rhinoceros and the Indian Rhinoceros were closest to the African species, although the node support was low, with about 70% posterior probability. Magaryan et al. also note that the maximum likelihood tree based on all Rhinoceros whole mitochondrial DNA sequences showed an identical topology, with a lack of high resolution in the Rhinocerotinae group.

Bayesian phylogeny of all available (36) Rhinoceros whole mitochondrial DNA sequences (published + new sequences from Magaryan et al.'s study). Two Equus mitochondrial DNA sequences were included as the outgroup. The blue bars represent the 95% highest posterior density intervals of the divergence times. All branches within a species level have been collapsed to improve readability. Node labels in bold show the Bayesian posterior probability values for two nodes; the rest of the nodes had PP values of one. Geological time scale abbreviations: Q, Quaternary; Ple, Pleistocene; Pli, Pliocene. Extinct Rhinoceros lineages are shown with daggers after species names. Magaryan et al. (2020).

Magaryan et al. expanded dataset of Javan sequences derived principally from Indonesian samples did enable them to show, for the first time, that the most recent common ancestor of this group lived at least 400 000 years ago, with the oldest branches represented by two historical samples, JR3851 and JR734. A Bayesian skyline plot based on the seven Javan rhinos with complete mitochondrial DNA sequences showed a largely constant female population size of the Javan Rhinoceros over the past 300 000 years until about 150 years ago (when most of the samples were collected), when the effective female population size was likely to have been about 9000 individuals.

Magaryan et al. subsequent analyses, which included the lower coverage samples, provided further insights. First, although two of the low-coverage samples (JR28 and JR29) were closely related to most other samples, sample JR524 fell basal to all other specimens, diverging in the Pleistocene at about 540 000 years before present. Identical tree topologies with similar time splits were observed when using more stringent criteria (using at least three reads for calling a base) for constructing consensus mitochondrial DNA sequences of the three low-coverage samples, although this reduced the number of informative sites further. This indicated that: (1) the method for constructing the consensus mitochondrial DNA sequences was relatively robust; and (2) relatively few informative sites were enough to assess the phylogenetic relationship within the Rhinoceros sondaicus lineage.

Bayesian phylogeny of the historical Javan Rhinoceros mitochondrial DNA lineages, using the Indian Rhinoceros (Rhinoceros unicornis; GenBank ID: X97336) as the outgroup and including the partial (7606-base pair-long) mitogenome of the Javan Rhinoceros sample collected in Bhutan (JR524). The blue bars represent the 95% highest posterior density intervals of the divergence times. Node labels in bold show the Bayesian posterior probability values for the major clades. All specimens in the blue clade originated from Indonesia (apart from JR27, which has largely unknown ‘Calcutta?’ label) and are clearly divergent from the Bhutanese sample. Magaryan et al. (2020).

Given that no whole mitochondrial DNA genomes have been published for modern Javan Rhinoceros, Magaryan et al. restricted their analysis to the few tRNAPro gene and partial D-loop sequences available for modern samples. Despite the small number of modern sequences, the analysis showed that most of the genetic diversity within the species was represented by some of the newly analysed historical sequences (e.g. JR734, JR3851 and JR26), in addition to the now extinct (since 2010) Vietnamese subspecies of the Javan rhino, Rhinoceros sondaicus annamiticus.

Neighbour-Joining network. The analysis of 14 modern and historical Javan and Indian Rhinoceros mitochondrial DNA sequences was conducted using the ‘Integer Neighbor-Joining’ algorithm implemented in POPART. Magaryan et al. used the partial D-loop region of 413 base pairs to maximize the number of available Javan Rhinoceros samples. ‘Javan annamiticus’ represents the recently extinct (since 2010) Vietnamese subspecies Rhinoceros sondaicus annamiticus. Each circle represents a certain haplotype; smaller black circles indicate median vectors. Small black lines connecting branches between the haplotypes denote the number of mutation steps separating the haplotypes. Sample identities and regions of origin for the Javan Rhinoceros samples are indicated next to each circle. Numbers in the legend indicate the number of sequences in each group. Magaryan et al. (2020).

Magaryan et al. reconstructed five complete and three partial mitochondrial DNA sequences of critically endangered Javan Rhinoceros by sequencing eight historical museum specimens, which more than doubled the number of available complete mitochondrial DNA lineages from this species.

The phylogenetic placement of the newly sequenced Javan Rhinoceros samples confirmed the species identity, and the overall tree topology based on all available mitochondrial DNA sequences was in accordance with recent studies, namely showing a lack of resolution among the three main lineages of Rhinoceros species (Indian + Javan, White + Black and Sumatran + Woolly + Stephanorhinus). However, the highest Bayesian probability tree reflected a greater genetic similarity between the two African and two Asian Rhinoceros species, with the clade of the Sumatran Rhinoceros as a sister group. These results differed from those based on phylogenetic analyses of protein (collagen alpha) sequences, which showed higher resolution within Rhinocerotinae and a different tree topology, with the African Rhinoceros forming a sister group to the Asian species. However, we caution that this might reflect the different genetic histories and/or power of resolution between nuclear (coding collagen alpha in this case) and mitochondrial DNA genomes. Ultimately, full nuclear genome-based analyses will be needed to resolve this question satisfactorily.

Although the overall topology of the mitochondrial DNA phylogenetic tree from the present study is similar to those from previous studies, the molecular dating estimates differ significantly. This is attributable to the fact that Magaryan et al. used well-documented fossil data for node calibration of the phylogenetic tree rather than the molecular estimates of previous studies, which are likely to be overestimated by a large margin, as has been shown for the Equus genus based on nuclear data. However, Magaryan et al. feel it is worth mentioning that genomewide data will be required from various Rhinoceros species to assess whether these observed differences do, in fact, reflect different nuclear and mitochondrial DNA evolutionary histories.

The relatively constant effective female population size of the Javan Rhinoceros for the past roughly 300 000 years (until 150 years ago) indicated that the dramatic decline of their numbers in the past two centuries is attributable solely to anthropogenic factors.

As might be expected, the oldest lineage of the Javan Rhinoceros in Magaryan et al.'s dataset (represented by the sample JR524) was from the specimen sampled in Bhutan, in continental Asia. Magaryan et al. therefore hypothesise that it might represent an individual of the now-extinct subspecies Rhinoceros sondaicus inermis, although additional samples and the incorporation of nuclear DNA will be needed to test this further.

The dramatic decline of the population size of the Javan Rhinoceros in recent years is reflected in the network analysis of the mitochondrial DNA sequences, in which Magaryan et al. show that the most diverse lineages are represented by individuals that no longer exist, i.e. the historic samples from their dataset and recently extinct Rhinoceros sondaicus inermis.

This difference in genetic diversity is likely due to the temporal as opposed to geographic differences since most (apart from JR27 which has largely unknown 'Calcutta?' label) of the Rhinoceros sondaicus lineages originated from Indonesia (Java and Sumatra). This result is similar to a recent study comparing museum specimens of now extinct populations of Black Rhinoceros with modern samples, suggesting a general reduction in genetic diversity in modern Rhinoceros populations, a consequence of anthropogenic population collapse. Unfortunately, Magaryan et al. were unable to recover the tRNA-Pro gene and partial D loop region from their three least sequenced samples including JR524, which represented the oldest branch in the Javan Rhinoceros mtDNA lineage.

In summary, although Magaryan et al.'s dataset is relatively small, reflecting the challenge of obtaining genetic data from Rhinoceros sondaicus owing to the rarity of modern specimens and the poor preservation conditions of the historical material, our results clearly show that the genetic diversity of its mitochondrial lineage has contracted significantly during the past two centuries. With two subspecies already extinct, the importance of survival of the last one (Rhinoceros sondaicus sondaicus) cannot be overstressed. Currently, there are no modern complete mtDNA sequences available from this species. Therefore, Magaryan et al. hope that their newly assembled sequences from historical samples might provide a valuable starting dataset, upon which future studies and conservation efforts might be able to build, in order that more insights can be gained into the evolutionary history of this critically endangered species.

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