Consideration of genetic variation and evolutionary history in future conservation of Indian one-horned rhinoceros (Rhinoceros unicornis)

The extant members of the Eurasian rhino species have experienced severe population and range declines through a combination of natural and anthropogenic factors since the Pleistocene. The one-horned rhino is the only Asian species recovered from such strong population decline but most of their fragmented populations in India and Nepal are reaching carrying capacity. Implementation of any future reintroduction-based conservation efforts would greatly benefit from currently unavailable detailed genetic assessments and the evolutionary history of these populations. We sequenced wild one-horned rhino mitogenomes from all the extant populations (n=16 individuals) for the first time, identified the polymorphic sites and assessed genetic variation (2531bp mtDNA, n=111 individuals) across India. Results showed 30 unique rhino haplotypes distributed as three distinct genetic clades (Fst value 0.68-1) corresponding to the states of Assam (n=28 haplotypes), West Bengal and Uttar Pradesh (both monomorphic). Phylogenetic analyses suggest earlier coalescence of Assam (~0.5 Mya) followed by parallel divergence of West Bengal and Uttar Pradesh/Nepal (~0.06-0.05Mya), supported by the paleobiogeographic history of the Indian subcontinent. Combined together, we propose recognising three Evolutionary Significant Units (ESUs) of the Indian rhino. As recent assessments suggest further genetic isolations of the Indian rhinos at local scales, future management efforts should focus on identifying genetically variable founder animals and consider periodic supplementation events while planning future rhino reintroduction programs in India. Such well-informed, multidisciplinary approach is the only way to ensure evolutionary, ecological and demographic stability of the species across its range.

Identifying and prioritising climate change adaptation actions for greater one-horned rhinoceros (Rhinoceros unicornis) conservation in Nepal

Climate change has started impacting species, ecosystems, genetic diversity within species, and ecological interactions and is thus a serious threat to conserving biodiversity globally. In the absence of adequate adaptation measures, biodiversity may continue to decline, and many species will possibly become extinct. Given that global temperature continues to increase, climate change adaptation has emerged as an overarching framework for conservation planning. We identified both ongoing and probable climate change adaptation actions for greater one-horned rhinoceros conservation in Nepal through a combination of literature review, key informant surveys (n = 53), focus group discussions (n = 37) and expert consultation (n = 9), and prioritised the identified adaptation actions through stakeholder consultation (n = 17). The majority of key informants (>80%) reported that climate change has been impacting rhinoceros, and more than 65% of them believe that rhinoceros habitat suitability in Nepal has been shifting westwards. Despite these perceived risks, climate change impacts have not been incorporated well into formal conservation planning for rhinoceros. Out of 20 identified adaptation actions under nine adaptation strategies, identifying and protecting climate refugia, restoring the existing habitats through wetland and grassland management, creating artificial highlands in floodplains to provide rhinoceros with refuge during severe floods, and translocating them to other suitable habitats received higher priority. These adaptation actions may contribute to reducing the vulnerability of rhinoceros to the likely impacts of climate change. This study is the first of its kind in Nepal and is expected to provide a guideline to align ongoing conservation measures into climate change adaptation planning for rhinoceros. Further, we emphasise the need to integrating likely climate change impacts while planning for rhinoceros conservation and initiating experimental research and monitoring programs to better inform adaptation planning in the future.

Abundance of Multiple-Antibiotic-Resistant Salmonella Strains in Fecal Samples of Rhinoceros unicornis of the Kaziranga National Park, India

Aims: This study was undertaken to assess the abundance of multiple-antibiotic-resistant (MAR) Salmonella strains in fecal samples of Rhinoceros unicornis of the Kaziranga National Park (KNP), India. Study Design: Antibiotic-resistance profile of the Salmonella isolates from fecal samples of rhinoceros was carried out by replica plating on Muller Hinton Agar (MHA) plates containing antibiotics. The presence of class 1 integrons in metallo-β-lactamase (MBL) producing Salmonella isolates was determined by multiplex PCR. Place and Duration of Study: The study was carried out on rhinos of KNP situated in the Golaghat district of Assam, India in April 2015. Methodology: Fresh rhino fecal samples (designated as 1R, 2R, 3R, 4R and 5R) were dilution plated onto MacConkey agar. Purified bacterial colonies were then streaked separately on bismuth sulphite (BS) agar plates. All black colonies which are characteric growth of Salmonella were selected and used to make master plates on Luria Agar. To determine the antibiotic-resistance profile of the isolates, master-plates of purified single colonies of Salmonella spp. were replicate-printed on plates containing antibiotics from the β-lactam, aminoglycoside, or quinolone groups. To detect the presence of an integron, a conserved segment polymerase chain reaction was used. Results: 97.6% of the Salmonella isolates tested were resistant to >1 antibiotics (multidrug resistant or MAR). A total of 100 isolates from two fecal samples, 4R and 5R, were found to be imipenem resistant; 52 of them tested positive for the presence of MBLs. Five of the twenty MBL producing strains contained class 1 integrons. Conclusion: Because Salmonella is usually spread by drinking contaminated water, it is likely that water bodies in KNP are contaminated with MAR Salmonella strains. In case of Salmonellosis outbreak among Indian one-horned rhinos, our antibiogram will assist the veterinarians to choose the appropriate regimen of antibiotics for the rhinos in the KNP.

Case Report: Ovulation Induction in Greater One-Horned Rhinoceros (Rhinoceros unicornis)

Despite a profound knowledge on reproduction biology in greater one-horned (GOH) rhinoceros, many individuals cope with sub or infertility or an-ovulatory estrous. At the same time, early and regular captive breeding is of high importance in female GOH rhinoceros due to their high prevalence to develop genital tract tumors with consequent cessation of reproduction. Thus, mature, an-ovulatory GOH rhinoceros represent a challenge for captive breeding programs and warrant for means of reliable ovulation induction. Here, we used hCG and GnRH analog histrelin to induce ovulation in an-ovulatory GOH rhinoceros. Upon ultrasound diagnosis of a preovulatory follicle hCG or GnRH were injected to induce ovulation (n = 11). As a result, 75% of the hCG (n = 6/8) and 33% of GnRH (n = 1/3) inductions resulted in ovulation. Ovulation occurred when fecal estrogen concentration increased before and pregnane concentration after induction. Thirty-six percent of all treatments (n = 4/11) failed to induce ovulation. When ovulation induction by hCG/GnRH injection failed, estrogen and pregnane concentrations were significantly lower compared to ovulatory estrous (P < 0.001). Our results suggest that hCG and GnRH analog facilitate an easily applicable treatment to induce ovulation in females with behavioral but at times an-ovulatory estrous. Frequent use of hCG as an ovulation inducer might help to achieve pregnancies in genetically important but an-ovulatory GOH rhinoceroses.

Limb myology and muscle architecture of the Indian rhinoceros Rhinoceros unicornis and the white rhinoceros Ceratotherium simum (Mammalia: Rhinocerotidae)

Land mammals support and move their body using their musculoskeletal system. Their musculature usually presents varying adaptations with body mass or mode of locomotion. Rhinocerotidae is an interesting clade in this regard, as they are heavy animals potentially reaching three tons but are still capable of adopting a galloping gait. However, their musculature has been poorly studied. Here we report the dissection of both forelimb and hindlimb of one neonate and one adult each for two species of rhinoceroses, the Indian rhinoceros (Rhinoceros unicornis) and the white rhinoceros (Ceratotherium simum). We show that their muscular organisation is similar to that of their relatives, equids and tapirs, and that few evolutionary convergences with other heavy mammals (e.g. elephants and hippopotamuses) are present. Nevertheless, they show clear adaptations to their large body mass, such as more distal insertions for the protractor and adductor muscles of the limbs, giving them longer lever arms. The quantitative architecture of rhino muscles is again reminiscent of that of horses and tapirs, although contrary to horses, the forelimb is much stronger than the hindlimb, which is likely due to its great role in body mass support. Muscles involved mainly in counteracting gravity (e.g. serratus ventralis thoracis, infraspinatus, gastrocnemius, flexores digitorum) are usually highly pennate with short fascicles facilitating strong joint extension. Muscles involved in propulsion (e.g. gluteal muscles, gluteobiceps, quadriceps femoris) seem to represent a compromise between a high maximal isometric force and long fascicles, allowing a reasonably fast and wide working range. Neonates present higher normalized maximal isometric force than the adults for almost every muscle, except sometimes for the extensor and propulsor muscles, which presumably acquire their great force-generating capacity during the growth of the animal. Our study clarifies the way the muscles of animals of cursorial ancestry can adapt to support a greater body mass and calls for further investigations in other clades of large body mass.