Conserv Genet (2018) 19:397–408 DOI 10.1007/s10592-017-1011-1 RESEARCH ARTICLE Genetic variation of complete mitochondrial genome sequences of the Sumatran rhinoceros (Dicerorhinus sumatrensis) Cynthia C. Steiner1 · Marlys L. Houck1 · Oliver A. Ryder1 Received: 9 December 2016 / Accepted: 24 August 2017 / Published online: 12 September 2017 © Springer Science+Business Media B.V. 2017 Abstract The Sumatran rhinoceros (Dicerorhinus sumat- Sumatran rhinoceros subspecies as different conservation rensis) is the smallest and one of the most endangered rhi- units. However, the management of subspecies as part of a noceros species, with less than 100 individuals estimated to metapopulation may appear as the last resource to save this live in the wild. It was originally divided into three subspe- species from extinction, imposing a conservation dilemma. cies but only two have survived, D. sumatrensis sumatren- sis (Sumatran subspecies), and D. s. harrissoni (Bornean). Keywords Mitogenomes · Phylogenetics · Genetic Questions regarding whether populations of the Sumatran structure · Divergence time · Critically endangered species rhinoceros should be treated as different management units to preserve genetic diversity have been raised, particularly in light of its severe decline in the wild and low breeding Introduction success in captivity. This work aims to characterize genetic differentiation between Sumatran rhinoceros subspecies The Sumatran rhinoceros (Dicerorhinus sumatrensis) is using complete mitochondrial genomes, in order to unravel the smallest of the five extant rhinoceros species belong- their maternal evolutionary history and evaluate their status ing to the family Rhinocerotidae. Also known as the hairy as separate management units. We identified three major rhinoceros, it is the closest relative of the extinct woolly phylogenetic groups with moderate genetic differentiation: rhinoceros Coelodonta antiquitatis (Orlando et al. 2003; two distinct haplogroups comprising individuals from both Willerslev et al. 2009). Members of this species once inhab- the Malay Peninsula and Sumatra, and a third group from ited rainforests, swamps, and cloud forests in Bangladesh, Borneo. Estimates of divergence time indicate that the Bhutan, China, India, Indonesia, Laos, Malaysia, Myanmar, most recent common ancestor of the Sumatran rhinoceros and Thailand (van Strien et al. 2008). Currently, fewer than occurred approximately 360,000 years ago. The three mito- 100 individuals are estimated to remain primarily due to chondrial haplogroups showed a common divergence time deforestation and illegal poaching in both protected and about 80,000 years ago corresponding with a major biogeo- non-protected areas. The Sumatran rhinoceros is consid- graphic event in the Sundaland region. Patterns of mito- ered critically endangered and thought to be confined to a chondrial genetic differentiation may suggest considering few remnant upland forest areas on the islands of Sumatra (Barisan Selatan National Park, Gunung Leuser National Park, Gunung Patah, Kerinci-Seblat National Park and Tor- Electronic supplementary material The online version of this gamba Forest) and Borneo (Danum Valley Conservation article (doi:10.1007/s10592-017-1011-1) contains supplementary Area, Tabin Wildlife Reserve, and East Kalimantan Prov- material, which is available to authorized users. ince; Miller et al. 2015). * Cynthia C. Steiner In addition to the critical situation of the Sumatran rhi- [email protected] noceros in the wild, conventional captive breeding has also proven difficult. In the early 1980s, the governments of Indo- 1 San Diego Zoo Institute for Conservation Research, San Diego Zoo Global, 15600 San Pasqual Valley Road, nesia and Malaysia, and international conservation organiza- Escondido, CA 92027, USA tions, supported a captive breeding program by transporting Vol.:(0123456789)1 3 398 Conserv Genet (2018) 19:397–408 40 Sumatran rhinoceroses from their native habitats to zoos to characterize genetic variation in Sumatran rhinoceroses and reserves across the world. By the late 1990s, not a sin- using the complete mt genome, or mitogenome, to unravel gle rhinoceros had been born in the program, and mortality their maternal evolutionary history and evaluate the conser- reached 60% (Foose and van Strien 1997). After years of vation status of subspecies as separate management units. failed reproductive attempts, the first healthy captive calf Results from this study contribute to the discussion about was born at the Cincinnati Zoo in September 2001 (Roth the definition of conservation units of critically endangered 2003). Two other births at the same location followed, in species, and provide practical applications regarding the 2004 and 2007 (Christman 2010). The first captive calf in management of the Sumatran rhinoceros. Asia was born in 2012, followed by another birth recently reported in May 2016 in western Sumatra (http://jakart- aglobe.id/archive/rare-baby-sumatran-rhinoceros-named-a- Materials and methods gift-from-god/; http://phys.org/news/2016-05-rare-sumatran- rhino-calf-born.htm). DNA samples and karyotypes The Sumatran rhinoceros has been divided into three sub- species by Groves (1967) based on measurements of eight We examined a total of 14 wild-born and one captive Suma- morphological characters: D. sumatrensis sumatrensis dis- tran rhinoceroses, three individuals from Sabah–Borneo (D. tributed originally on the island of Sumatra and on the Malay s. harrissoni), one individual from Malaysia (D. s. harris- Peninsula, D. s. harrissoni inhabiting the island of Borneo, soni, likely corresponding to SB26, also caught in Sabah), and D. s. lasiotis, currently extinct, formerly ranging across and 10 from the Malay Peninsula and Sumatra (D. s. sumat- the rest of Southeast Asia and the Indian subcontinent. Ques- rensis; Table 1). DNA, tissue, and cell lines were obtained tions regarding whether the populations in the Malay Pen- from the San Diego Zoo Global Frozen Zoo®. Utilization insula, Sumatra and Borneo should be treated as different of samples was compliant with applicable regulatory pro- management units to preserve genetic diversity have been cedures for CITES and the US Endangered Species Act. raised (Foose and van Strien 1997; Goossens et al. 2013). DNA was extracted using the DNeasy tissue and cell line Molecular studies using mitochondrial (mt) DNA have led to kits (Qiagen, CA, USA) according to the manufacturer’s different interpretations of the conservation status of Suma- instructions. For karyotyping, chromosomes were derived tran rhinoceros subspecies. For instance, Amato et al. (1995) from lymphocytes and/or fibroblast cell cultures. Cell cul- suggested that an average genetic divergence of 1% between turing, harvesting, and chromosome banding followed the the rhinoceros populations of Sumatra and Borneo was not techniques described by Houck et al. (1995). enough evidence to support more than one conservation unit. Morales et al. (1997) in contrast, analyzing the phylogeo- Next generation sequencing data graphic structure of D. s. harrissoni and D. s. sumatrensis, identified a maximum of three management units among the Whole mitogenomes were amplified in two overlapping remaining populations of the Sumatran rhinoceros: Malay amplicons [approximately 10 kilobase pairs (kbp) each] Peninsula–East Sumatra, West Sumatra, and Borneo. using polymerase chain reaction (PCR) and specific primers Although genetic studies have played an important role designed for this study (Supplementary Table 1). Forward in identifying conservation priorities, the Sumatran rhinoc- and reverse primers were defined in conserved regions of eros crisis requires finding effective strategies to manage a multiple-species alignment including all rhinoceros mt the remaining populations in the wild and captivity (Goos- sequences obtained from Genbank. PCRs were performed in sens et al. 2013). The consensus among managers and sci- a 25 µl volume using Eppendorf Mastercycler Gradient ther- entists indicates that both subspecies should be managed mal cyclers. Each reaction included 2 µl of extracted DNA as a metapopulation, disregarding taxonomic distinction (60 ng), 2.5 µl of 10× HiFi PCR buffer, 1.25 µl of MgSO4 (Nardelli 2014). This means interbreeding populations of the 50 mM, 2.0 µl of 2.5 mM deoxynucleoside triphosphates, Sumatran rhinoceros to avoid the extinction of the species 1.0 µl of 10 µM of each primer, and 0.2 µl of High Fidelity as a whole. However, concerns about outbreeding depres- Platinum Taq (Invitrogen Corporation, Carlsbad, CA, USA). sion still exists (Amato et al. 1995; Morales et al. 1997), The PCR cycling conditions were 94 °C for 2 min, followed but this may be outweighed by the risk of extinction due to by 34 cycles of denaturation at 94 °C for 30 s, annealing for inbreeding, demographic stochasticity, disease and poaching 30 s, and 68 °C for 10 min, with a final extension at 68 °C (Miller et al. 2015). for 7 min. Since 1986, the San Diego Zoo has banked in its Fro- The construction of genomic libraries involved three zen Zoo® Sumatran rhinoceros genetic material that can be steps as described by Parson et al. (2013): enzymatic shear- utilized to understand genetic differentiation between liv- ing, ligation of the adapters and size selection. The quan- ing Sumatran rhinoceros subspecies. In this study, we aim tity of amplified DNA was determined with a Nanodrop 1 3 Conserv Genet (2018) 19:397–408 399 Table 1 List of Sumatran rhinoceros