The Asiatic black bear (Ursus thibetanus) has a wide distribution in southern and eastern Asia spanning from Pakistan to Russian Far East of Asian continent, and the surrounding islands, including Japan and Taiwan (Servheen et al. 1999; Wozencraft 2005; Garshelis and Steinmetz 2016). This medium-sized bears occupy a variety of forested habitats from near sea level to an elevation of 4,300 m (Garshelis and Steinmetz 2016).
Like many other bear species, the Asiatic black bear has been threatened by habitat loss and poaching, and is listed as “Vulnerable” by the IUCN Red List of Threatened Species since 1990 (Garshelis and Steinmetz 2016). It is also an Appendix I species of the Convention of International Trade on Endangered Species of Wild Fauna and Flora (CITES) (CITES 2017).
Seven subspecies of U. thibetanus have been recognized, including U. thibetanus ussuricus inhabiting southern Siberia, northeastern China, and Korean peninsula, U.
thibetanus japonicus inhabiting Japan, U. thibetanus formosanus inhabiting Taiwan, U.
thibetanus mupinensis inhabiting southwestern China, U. thibetanus laniger inhabiting
Himalaya area, U. thibetanus gedrosianus inhabiting Pakistan, and the nominate
subspecies U. thibetanus thibetanus (Hou and Hu 1997; Ma et al. 1998; Wozencraft 2005).
Traditionally taxonomic differences are based on diagnostic morphological characters, and combinations of measurements, particularly from skulls (Kitchener 2010). However, the subspecies of Asiatic black bears can be recognized only in accordance with their geographic distribution (Wozencraft 2005). The morphological differences among these subspecies reported were from few specimens and description of these differences are vague (Hwang et al. 2008; Kitchener 2010). For examples, Heptner et al. (1998) distinguished U. thibetanus ussuricus from other subspecies by its largest skull measurements, pure black hair, and long fur. Hu (1995) compared the differences among U. thibetanus thibetanus, U. thibetanus mupinensis, and U.
thibetanus laniger by vague descriptions of body sizes, length of hair, and the pattern of
chest mark. Therefore, it is difficult to determine the origin of individual bear specimen based on its morphological characteristics.
Taxonomy is essential for conservation and the implementation of protective legislation (O'Brien and Mayr 1991; Kitchener 2010). Lack of taxonomic delimitation in the wild may result in loss of unique populations, or the recognition of too many subspecies may prevent mixing of depleted gene pools owing to local population
bottlenecks. According to the subspecies concept defined by O'Brien and Mayr (1991), subspecies is defined to include populations below the species level that share a distinct geographic distribution, a group of phylogenetically concordant characters, and a unique natural history relative to other subdivisions of the species. And if a population of a species is genetically distinctive by strong phylogenetic structuring of mitochondrial DNA (mtDNA) variation and nuclear alleles from the others due to long-term evolutionary isolation, it should be treated as an ‘evolutionarily significant unit’ (Ryder 1986; Moritz 1994).
In recent years, the development of molecular techniques allows us to examine genetic variation of animal species distributed over wide geographical areas regardless of sex, age, and local phenotypic responses to the environment which have greatly benefited taxonomy and systematics (Frankham et al. 2002; Van Dike 2008a; Kitchener 2010). Due to the wide distribution and the controversially morphological traits of subspecies identification of Asiatic black bears, the information about the genetic status and genetic partitions is important for the conservation of these subspecies or populations. After all, the conservation strategy for this species will be bound to knowledge of its taxonomy.
The Formosan black bear (U. thibetanus formosanus) is considered an endemic
subspecies of Asiatic black bears inhabiting Taiwan (Wozencraft 2005). Again, its subspecies status was based on geographic distribution and limited information on morphological differences. Similar to other Asiatic black bear subspecies, habitat degradation and fragmentation, as well as poaching, have caused a decline in its population (Wang 1990, 1999; Hwang and Wang 2006). In the conservation of endangered Formosan black bears, molecular genetic techniques could help conservation biologists to define and identify its subspecies status and the management units for conservation more clearly by their genetic constituency.
A few studies have been conducted to examine the genetic status of Formosan black bears. Chu et al. (2000) analyzed the mtDNA control region and cytochrome b of the Asiatic black bears in Taipei Zoo. Chen and Yang (2002) compared partial gene sequences of mitochondrial 12S rRNA and 16S rRNA among 11 captive Asian black bears in Taiwan. Wu et al. (2015) tentatively indicated the black bear from Taiwan was highly nested within the southern East Asian continental population with only one individual in their analysis. However, few bear specimens from Taiwan had been analyzed in all these three studies and the geographical information of captive bear specimens may also be controversial. Therefore, the phylogenetic relationship and genetic status of Formosan black bears remain unclear.
Moreover, the ex situ conservation for possible reintroduction of the Formosan black bears in the future may also suffer from lack of knowledge about the taxonomy of subspecies. In captivity, hybridization may occur due to wrong taxonomy of subspecies.
Descendants of such captive populations would be unavailable for reintroduction to avoid genetically introgression in the wild populations, ultimately wasting resources for breeding program in ex situ conservation (Frankham et al. 2002; Van Dike 2008b;
Kitchener 2010). Thus, the subspecies taxonomy of Formosan black bears should be clarified in genetics for both in situ and ex situ conservation.
Before studying the genetic status of Formosan black bears and other Asiatic black bears, it is critical to develop suitable genetic markers for better application of genetic methods in assessing genetic partitions, defining the evolutionary significant units for conservation management, and improving the taxonomic designations (Moritz 1994;
Beebee and Rowe 2008). The mitochondrial DNA fragments are useful in addressing questions about species identification, population structure and phylogenetic research (Waits et al. 1999; Murphy et al. 2002; Roon et al. 2003), whereas the microsatellites of nuclear DNA have utility in individual identification (Murphy et al. 2002), kinship analysis, gene flow, and demographic studies (Roon et al. 2003; DeMay et al. 2013).
Thus, these two kinds of molecular markers would be used in the genetic analyses of
Asiatic black bears in this dissertation.
Some microsatellite genetic markers have been developed and used in the genetic studies of Ursid. Most of these markers are dinucleotide loci (Paetkau et al. 1995;
Taberlet et al. 1997; Paetkau et al. 1998; Kitahara et al. 2000; Wu et al. 2010). Two studies reported tetranucleotide loci, which are considered better due to fewer stutter bands and less scoring ambiguity (Hung et al. 2004), from American black bears (Ursus americanus) (Meredith et al. 2009; Sanderlin et al. 2009). There is no report on the
tetranucleotide microsatellite loci for Asiatic black bears. Therefore, the development of tetranucleotide microsatellites should provide an ideal genetic tool kit to study the population genetics of the endangered Formosan black bears and other Asiatic black bear subspecies.
In addition, noninvasive methods have been recommended for collecting samples of wide-ranging and illusive rare carnivores such as the Formosan black bears. For effective application of noninvasive genetic analysis in subtropical Taiwan, it is important to identify the variables which may affect the DNA quality of noninvasive samples, such as faeces or hair. Most studies evaluating the quality and DNA amplification success of noninvasive faeces or hair samples were conducted on brown bears (Ursus arctos) in temperate regions (Murphy et al. 2002; Murphy et al. 2007;
Stenglein et al. 2010). However, few were on bears in regions with different climatic conditions, for instance, tropics and subtropics. Genetic studies using faecal and hair samples of wild populations have been carried out initially in the Formosan black bear.
Therefore, a pilot study is recommended to determine DNA degradation rates in this system and to develop the appropriate noninvasive protocol (Taberlet et al. 1999; Renan et al. 2012; DeMay et al. 2013).
Therefore, the aims of this dissertation were to develop appropriate tools for Asiatic black bear genetic studies and to clarify genetic status of the Formosan black bear. The dissertation ws organized into the next four chapters.
In Chapter 2, ten novel easy-scored polymorphic tetranucleotide repeat (GAAA) microsatellite markers were developed and evaluated for their polymorphism in the Formosan black bears. These microsatellite loci could be applied as molecular tools for genetic analyses of the Formosan black bears and other Asiatic black bears.
To reinforce the optimization of noninvasive sampling approaches in the Asiatic black bear research in subtropical Taiwan, in Chapter 3, the effects of multiple variables on amplification success rate of mitochondrial DNA (mtDNA) extracted from the Asiatic black bear faeces and hair were quantitatively evaluated. The results showed that the amplification success rates decreased with sample age and amplicon size in both
hair and faecal DNA, but did not show differences between two faecal preservation methods, i.e. storage in ethanol then frozen or kept at room temperature, in shorter fragments, and among different sampling locations of faeces. It suggests that careful selection of primers for suitable PCR product sizes depending on sample conditions could optimize success rates of genetic analysis in noninvasive genetic research.
In Chapter 4, mitochondrial phylogeny of bear specimens collected from Taiwan, mainland China, Russia, Vietnam, and Thailand were conducted based on partial mitochondrial DNA control region and its 5’-flanking region to assess the genetic status of the Asiatic black bear populations, and elucidate the unclear genetic taxonomy of the Formosan black bear. The mitochondrial DNA analyses supported the Formosan black bears formed a unique monophyletic group. In addition, the population structure analysis of tetramicrosatellite loci was employed to indicate a clear subdivision scenario of these four subspecies, U. thibetanus formosanus, U. thibetanus mupinensis, U.
thibetanus ussuricus, and U. thibetanus thibetanus.
Finally, in Chapter 5, a pilot study of genetic analysis on both mitochondrial DNA and microsatellite loci from captive bear specimens was conducted to reveal the genetic ancestry of captive Asiatic black bears in Taiwan. In this study, seven captive bears of unknown origin showed the unique mtDNA haplotypes of the Formosan black bear. And
three of them had a single verified subspecies ancestry of the Formosan black bear based on microsatellite data. Given the fact that the size of the wild population is critically small and that the bears of native origin are kept in different zoos, institutes, and rescue centers in Taiwan, these institutions are highly encouraged to cooperate with each other in implementing an ex situ breeding plan for the conservation of this subspecies.
In summary, these studies enhanced genetic tool for conservation genetic studies of the Formosan black bear. They also revealed the level of genetic variation among different populations of Asiatic black bears and provided an explicit basis for subspecies identification of the Formosan black bear. Such information will be important and beneficial for both in situ and ex situ conservation of this Asiatic bear species in the future.