Cross-amplification of Y chromosome-specific markers isolated from horse (Equus caballus) in Malayan tapir (Tapirus indicus)

This study was conducted to evaluate the cross-amplification of six Y chromosome-specific markers that were isolated from horse (Equus caballus) in Malayan tapir (Tapirus indicus). All the six markers failed to yield positive results in Malayan tapirs with no amplification (Eca.YM2), and with amplification of single band (Eca.YP9) or multiple bands (Eca.YJ10, Eca.YH12, Eca.YE1 and Eca.YA16) in both male and female individuals. Twelve faecal samples were collected from five male individuals of Malayan tapir and stored in three different storage methods (frozen, DET and 80% ethanol). DNA was extracted using a modified protocol of Zhang et al. (2015), yielded DNA concentrations that were not statistically different among the three storage methods based on one-way ANOVA test. Scoring of molecular markers was not carried out because none of the six markers gave a positive result and therefore this step was discontinued

A review of the application of Next Generation Sequencing (NGS) in wild terrestrial vertebrate research

In era of sequencing revolution, scientists seek for knowledge about the ever-expanding field of technology, Next Generation Sequence (NGS) to be applied in their research due to its high reliability and rate of discovery. What is NGS? To obtain a detailed understanding about NGS, it is required to look back the history of sequencing and how the NGS stepped into life science. This review paper gives an overview of NGS projects in wild terrestrial vertebrate including applications such as whole genome sequencing and metagenomics

Lip licking behavior in captive Malayan tapirs (Tapirus indicus): manifestation of a stereotypic or stress related response?

Malayan tapirs are highly endangered and wild populations are fast declining. Thus, captive breeding programs in zoos and governmental breeding centers are the most promising conservation strategy for this species. Despite being common, lip licking, a type of oral behavior, has received little attention in the past, and impacts on the welfare of captive Malayan tapirs have not been quantified. Here, we videoed the behavior of seven captive tapirs for eight hours per diem (0900 - 1700) using instantaneous sampling for six months to investigate which stressors in captivity (enclosure type, enclosure size, humidity, visitors) cause increased lip licking behavior. We show that lip licking is induced by unsuitable humidity whereby dry humidity below 65% caused a significant increase in this behavior. We found lip licking behavior in tapirs is not a stereotypic behavior, but it may indicate a stress response towards heat. Hence, we suggest that breeding centers re-evaluate their exhibit design and behavioral enrichments, implementing simple design changes that would help to reduce lip licking and consequently increase the welfare of captive Malayan tapirs

Cross-species amplification of microsatellite markers in Malayan tapir (Tapirus indicus)

Malayan tapir (Tapirus indicus) is listed as endangered species by IUCN due to population decline caused by habitat loss and fragmentation, and increasing hunting pressure throughout its ranges. Malayan tapir is the only one species of genus Tapiridae, distributed in the tropical rain forest of Southeast Asia. Tapirs are known as the key actors in forest dynamics as they are functionally important seed dispersers and seed predators. Despite their endangered status and functional role in ecosystems, little information is known about their social structure, mating system, population structure and dispersal pattern which are required for better conservation and management. Studies through traditional field methods are extremely difficult because tapirs are shy, cryptic, nocturnal, and prefer to inhabit deep tropical forest. Molecular genetics techniques now provide an alternative way to resolve ecological questions related to tapir. In this study, we tested cross-species amplification of 12 microsatellite markers designed for Lowland tapir (N = 9) and Bairdii tapir (N = 3) in Malayan tapir using fecal DNA. Result showed that all microsatellite markers failed to amplify in Malayan tapir. Poor quality and quantity of DNA, co-purification of PCR inhibitors, unsuitability of DNA extraction and preservation method and high genetic divergence are some of the factors contributing to this result

Optimal method to introduce faeces sample for olfactory-cues studies in Malayan tapir (Tapirus indicus)

Studying the hidden meaning behind the behaviours of wildlife can provide plenty of information for the betterment of their conservation. In this research, a preliminary study had been conducted to find out whether Malayan tapir (Tapirus indicus) (1) sniffs on the faeces or not and (2) to identify the optimal method of presenting the faeces samples. Five individuals of Malayan tapirs were contributed to this research as the samples donors and/or the subject. Basically, for this experiment several replicates of faeces samples were collected from the donors and frozen at -20˚C, and the thawed samples were introduced to the subjects at different time slots (morning, afternoon and night). The results showed that tapirs exhibited both investigation and identification behaviours when sniff on faeces samples of other individuals. However, the sniffing was observed only during early in the morning and night when they were active due to their nocturnal characteristic. We found that, the location and the sample nature (i.e., thawed at sufficient period of time) had influenced the attractiveness of presented faeces sample to tapirs. Presenting the samples at feeding and sleeping areas increased the chance for the tapirs to sniff on samples and well thawed samples which emits strong odour drawn tapir’s attention. Thus, it is recommended to take into account the above factors for further investigation related to the individual recognition through olfactory cues in Malayan tapir. The findings of this study could be incorporated in ex-situ conservation mainly during the selection of mating partners prior to physical introduction of the animals (strategy to avoid any injuries to tapir due to fighting and inbreeding in captivity)

Non-invasive measurement of progesterone and cortisol metabolites in the faeces of captive female Rusa unicolor at Zoo Negara, Malaysia and its reproductive and stress behaviour

Sambar deer are listed as vulnerable and are bred in captivity under governmental management. The success of captive breeding programs varies, and the underlying causes are unclear. The advantage of using non-invasive faecal samples to obtain hormonal profiles without the animal being sedated or restrained has not been tested in sambar deer. This experiment was aimed to study the reproductive and stress behaviours of sambar deer and to measure the levels of reproductive and stress hormones in captive female sambar deer via a non-invasive procedure using faeces samples. Data on reproductive and stress behaviour were collected from six sambar deer for six months. Behaviours were recorded by instantaneous sampling method using direct observation. The reproductive and stress hormones in faecal samples were analysed using ELISA procedures. There are differences in frequency of certain reproductive behaviours recorded within different sessions of data collections while stress behaviour was in the low count and no huge difference in frequency between different sessions. Progesterone metabolites showed some trend of high concentrations in July and started to drop at the end of July till the end of December with constantly negative concentrations. Sambar deer in Zoo Negara can be considered not in stress due to low reading of cortisol concentration even though there was a presence of visitors. In future, it is important to make sure the faecal samples for hormonal analysis are collected daily to look for the pattern of the oestrus cycle in sambar deer

Genetic diversity and phylogenetic relationships of Malayan tapir (Tapirus indicus) populations in the Malay Peninsula based on mitochondrial DNA control region

The Malayan tapir (Tapirus indicus) is an endangered species in Southeast Asia (SEA). Over the years, there has only been a few reports on its population genetic structure and evolutionary history in SEA. In particular, while the captive Malayan tapir population in Thailand has received fairly more research in recent years, there has not been any widescale population genetic study in the Malay Peninsula since the last decade. In this study, we report on the genetic diversity and attempt to make inferences on the phylogeography of the Malayan tapir in the Malay Peninsula, using the mitochondrial DNA control region, including also data from previous studies. We identifed 12 novel haplotypes and two distinct Malayan tapir clades with a divergence time of 14.6 million years ago (mya) in the Peninsular Malaysia (i.e. southern Malay Peninsula) samples, as opposed to only one clade found in the Thai (i.e. Thailand western border and northern Malay Peninsula) captive individuals, with an almost two-fold higher nucleotide diversity compared to the latter. The co-occurrence of the two lineages in southern Malay Peninsula suggested past historical events of population isolation, migration and admixture as a result of episodic glacial periods and paleoenvironmental changes in SEA. Only one haplotype shared between the populations of the two regions suggested further gene fow restriction at the narrow corridor of the Malay Peninsula as of present day

Population genetic structure of wild Malayan tapirs (Tapirus indicus) in Peninsular Malaysia revealed by nine cross-species microsatellite markers

The Malayan tapir (Tapirus indicus) is an endangered species endemic to Southeast Asia. Previous research have used maternally inherited, mitochondrial DNA genetic markers to investigate the population genetics of the species but not the biparentally inherited, nuclear genetic markers. However, the increasing pressure of habitat fragmentation and roadkill on the Malayan tapirs has called for an urgent need to assess the species’ genetic status. In this study, the genetic diversity, population genetic structure, and sex-biased dispersal patterns of the tapir population in Peninsular Malaysia were investigated using nine cross-species microsatellite markers, using sixty-seven tapir samples (39 wild, 11 captive-born, and 17 of unknown origin) provided by the wildlife department, zoo and conservation centre. Low genetic diversity of the wild population was found based on heterozygosity and allelic richness, and a cryptic pattern of population genetic structure was inferred. One (K = 3) to two (K = 4) genetic clusters in proximity to the south-western part of the major forest complexes seemed to be experiencing restricted gene flows compared to the other more widespread clusters or clines. Effective to census population size was estimated at 0.39-0.46. Sex-biased dispersal was not found in the current dataset. Even though these results may need cautious interpretation due to possible sampling bias, this study is the first study that investigated the genetic diversity distribution for the species using nuclear markers, and therefore will have conservation implications for the implementation of the Central Forest Spine Master Plan in Peninsular Malaysia to connect major forest complexes

A revisit to a low-cost method for the isolation of microsatellite markers: the case of the endangered Malayan tapir (Tapirus indicus)

There are many approaches to develop microsatellite markers. Despite the availability of the more advanced technology in the market, due to budget constraints, we revisited an easy and rapid polymerase chain reaction (PCR) cloning-sequencing method to design microsatellite markers for Tapirus indicus. Using six random amplified microsatellite markers, this study had rapidly generated 45 unique genomic sequences containing microsatellites. After screening 15 terminal and seven intermediate microsatellite loci, we shortlisted five and seven which were amplified either by single- or multiplex PCR using the economical three-primer PCR method. Genotyping attempts were made with ten T. indicus individuals using three of the terminal microsatellite loci and all seven intermediate loci. However, none of the terminal microsatellite loci were considered useful for population genotyping studies, while the seven intermediate loci showed good amplification but were monomorphic in the ten samples and the subsequent 51 tapir samples. Despite successful detection of amplified loci, we would like to highlight that, researchers who are interested in this alternative method for isolation of microsatellite loci to be cautious and be aware of the limitations and downfalls reported herein that could render these loci unsuitable for population genotyping

Genetic, socio-ecological and fitness correlates of extra-group paternity in the European badger (Meles meles)

The evolution of extra-group paternity (EGP) is a contentious issue in evolutionary biology. This thesis examines the factors and adaptive benefits driving EGP in a high-density, group-living population of European badgers (Meles meles). To improve power to assign parentage, I isolated and characterised 21 new polymorphic microsatellite markers. I genotyped 83% of 1410 badger trapped 1987‒2010 using 35 autosomal microsatellite markers. Maternity and paternity were assigned at 80% confidence ca. 82% of individuals. 48% of paternities were extra-group, where 85% were attributable to neighbouring-group males and EGP was detected in 47% of litters; thus badger social group do not correspond with a breeding unit. I tested whether indirect genetic benefits explain these high EGP rates. (1) ‘Good-gene-as-heterozygosity Hypothesis’: Paternal heterozygosity, but not maternal or an individual’s own heterozygosity, associated positively with first-year survival probability. Under benign environmental conditions, cubs fathered by more heterozygous males had a higher first year survival probability. Despite this correlation, the EGP rate per litter correlated with neither average nor maximum within-group heterozygosity of candidate fathers. (2) Fitness benefit Hypothesis: Extra-group offspring (EGO) had lower first-year survival probability and lived 1.3 years less than within-group offspring (WGO). Female WGO produced more litters and offspring over their lifetime than female EGO, whereas male EGO produced more offspring than male WGO. (3) Inbreeding avoidance hypothesis: The EGP rate within a litter increased with greater average pair-wise relatedness between mothers and within-group candidate fathers. No inbreeding depression on first-year survival probability was detected, but small sample sizes limited statistical power. Socio-ecologically, at the litter level, EGP correlated negatively with the number of within-group candidate fathers, and positively with neighbouring-group candidate fathers. In conclusion, EGP in badgers may reduce inbreeding and be maintained in the population through a sex-specific antagonistic selection and indirect genetic benefits may occur when the total fitness benefits of producing extra-group sons outweigh the costs of producing extra-group daughters. These indirect genetic benefits only partially explain the evolution of promiscuity in European badgers, highlighting that evolutionary factors underlying promiscuity remain unclear. </p