Subspecies assignment

in Chapter 4, mtDNA haplotypes of bears with unknown genetic origin were assigned maternal lineage ancestry based on its phylogenetic relationship to the voucher bear subspecies first. Then, the Bayesian clustering analysis in STRUCTURE based on 8 microsatellite loci was used to assign the biparental genetic ancestry.

Bear samples were considered to have the purebred Formosan black bear ancestry if they were consistently supported by both mitochondrial lineage and microsatellite genotype assignment results. Bear individuals with genetic origin of U. thibetanus formosanus in mtDNA, but admixed origins in microsatellite analysis were also

identified. Others were categorized as bears with admixed origins or with purebred origin of other subspecies in both mtDNA and microsatellite analyses. Bear specimens with only mitochondrial data were considered to have incomplete evidence in maternal lineage only.

5.4 Results

The mtDNA control region and its 5’-flanking region sequences (703 to 706 bp in length) of all samples of 30 captive Asiatic black bears and two paws were successfully amplified and sequenced. In these 32 bear samples, three bear specimens and a paw specimen could not be genotyped for more than seven in ten tetranucleotide microsatellite loci and were not used in further microsatellite analyses.

In the phylogenetic relationship analyses of mtDNA haplotypes which included the source-unknown samples collected from various facilities in Taiwan, the neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) phylogenetic trees showed identical topologies (Fig. 5-1, Fig. 5-2, and Fig. 5-3). Seven source-unknown bears including TWC25, TWC26, TWC27, TWC21, TWC22, TWC23, and TWC29 show the unique haplotypes of the Formosan black bears (Fig. 5-1, Fig. 5-2, and Fig. 5-3). Others are not in the Taiwan clade and commingled with haplotypes of southeastern Asia subspecies (U. thibetanus thibetanus) and southwestern China subspecies (U. thibetanus mupinensis). None of source-unknown samples are in the clades of the northeastern Asian continent subspecies (U. thibetanus ussuricus) or the Japan black bears (U. thibetanus japonicas) (Fig. 5-1, Fig. 5-2, and Fig. 5-3).

In the Bayesian clustering analysis of STRUCTURE with microsatellite loci including source-unknown samples, three of seven bears assigned in the mtDNA Taiwan clade showed a single verified subspecies ancestry of the Formosan black bear, including TWC25, TWC26, and TWC27 (Fig. 5-4). Except for TWC16, TWC17, TWC18, and TWC19 (which were genetically assigned to U. thibetanus mupinensis), most of others were of admixed origin (Fig. 5-4).

We assigned three bear samples, TWC25, TWC26, and TWC27 to Asiatic black

bears with purebred origin of U. thibetanus formosanus, TWC21, TWC22, TWC23, and TWC29 to bears with admixed subspecies origins including U. thibetanus formosanus, TWC16, TWC17, TWC18, and TWC19 to bears with purebred origin of U. thibetanus mupinensis, and determined 17 had admixed subspecies origins of U. thibetanus

thibetanus and U. thibetanus mupinensis. TWC9, TWC10, TWC30, and TWQP5 were

bear samples with admixed origins in mtDNA analyses but without enough microsatellite data for analysis (Table 5-1).

5.5 Discussions

In the 30 origin-unknown captive Asiatic black bears tested in this study, only three bears were with purebred origin of U. thibetanus formosanus, four were U.

thibetanus formosanus in maternal lineage ancestry but with admixed origins of U.

thibetanus thibetanus or U. thibetanus mupinensis in biparental microsatellite analysis.

Namely, more than two thirds of the captive Asiatic black bears in Taiwan were not of native U. thibetanus formosanus origin. Due to the geographical adjacency among Taiwan, China, and southeastern Asia and the frequently legal and illegal trade among these countries, it may be reasonable that most bears of admixed origins or non-native origins are with southeastern Asia subspecies (U. thibetanus thibetanus) and southwestern China subspecies (U. thibetanus mupinensis) ancestries. In addition,

according to the information provided by the owners of these bear specimens, TWC4, TWC16, TWC17 (TWC18 and TWC19 are offspring of TWC16 and TWC17), and TWC28 were bought from indigenous people or mountain areas in Taiwan. However, genetic analysis revealed that these six captive Asiatic black bears were not native bear subspecies. This discordance may demonstrate the necessity of genetic analyses in subspecies identification.

In this study, we developed a method to assess subspecies ancestry of the Asiatic black bears with uncertain background, especially to identify native Formosan black bear from other subspecies. It should be a powerful tool for ex situ conservation of the Formosan black bear to increase the number of purebred bears suitable for conservation breeding. However, our method could not clarify the difference between southeastern Asia subspecies (U. thibetanus thibetanus) and southwestern China subspecies (U.

thibetanus mupinensis). According to the results described in Chapter 4, it may be due

to the gene flow between U. thibetanus mupinensis and U. thibetanus thibetanus. It will be worth of conducting further studies with more effective genetic markers to increase the accuracy of subspecies identification between these subspecies in the future.

For ex situ conservation breeding program of the Formosan black bear, TWC25, TWC26, and TWC27 in this study and three voucher bears in captivity described in

Chapter 4 (TW14, TW17, and TW18) should be the core founders because of their purebred origin of U. thibetanus formosanus. Four bears (TWC21, TWC22, TWC23, and TWC29) with maternal lineage ancestry of U. thibetanus formosanus but admixed origins in biparental nuclear genealogy may result from asymmetric breeding between two subspecies in captivity. These four bears with partial U. thibetanus formosanus origin may be included as of second priority for conservation breeding if there are not enough young breeders in the breeding program. Other bears of admixed origins are suggested to avoid breeding unless they are used for developing animal husbandry techniques.

Genetic analysis conducted in our study is important in helping relevant organizations to cooperate and better plan for ex situ conservation of the Formosan black bears. Given the fact that the critically small size of wild population and that the bears of native origin are kept in different organizations, these institutions are highly encouraged to cooperate with each other in implementing an ex situ breeding plan for the conservation of this subspecies.

5.6 References

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Frankham R, Briscoe DA, Ballou JD (2002) Introduction to conservation genetics.

Cambridge University Press, Cambridge, UK

Hwang M-H, Wang Y (2006) The status and management of Asiatic black bears in Taiwan. In: Yamazaki K (ed) Understanding Asian bears to secure their future.

Japan Bear Network, Japan, pp 107-110

Ishibashi Y, Saitoh T (2004) Phylogenetic relationships among fragmented Asian black bear (Ursus Thibetanus) populations in western Japan. Conservation Genetics 5:311-323

IUCN (1987) The IUCN policy statement on captive breeding. IUCN (World Conservation Union) Gland, Switzerland

Kitchener AC (2010) Taxonomic issues in bears: impacts on conservation in zoos and the wild, and gaps in current knowledge. International Zoo Yearbook 44:33-46 Matsuhashi T, Masuda R, Mano T, Yoshida MC (1999) Microevolution of the

mitochondrial DNA control region in the Japanese brown bear (Ursus arctos)

O'Brien SJ, Joslin P, Smith GL, Wolfe R, Schaffer N, Heath E, Ott‐Joslin J, Rawal PP, Bhattacharjee KK, Martenson JS (1987) Evidence for African origins of founders of the Asiatic lion species survival plan. Zoo Biology 6:99-116

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Shih C-C, Huang C-C, Li S-H, Hwang M-H, Lee L-L (2009) Ten novel tetranucleotide microsatellite DNA markers from Asiatic black bear, Ursus thibetanus.

Conservation Genetics 10:1845-1847

Shih C-C, Wu S-L, Hwang M-H, Lee L-L (2017) Evaluation on the effects of ageing factor, sampling and preservation methods on Asiatic black bear (Ursus thibetanus) noninvasive DNA amplification. Taiwania 62:363-370

Swofford DL (2002) Paup*: Phylogenetic analysis using parsimony (*and other methods) verson 4.0.b10. Sinauer Associates, Sunderland, Massachusetts

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Figures

Fig. 5-1 Phylogenetic relationships of source-unknown bear samples and voucher samples based on neighbor-joining analysis.

Bootstrap supports were provided above branch at node for the divergence of lineages.

The bear sample sizes with each voucher haplotype were showed in parentheses. The source-unknown bear samples were highlighted with dark red color.

Fig. 5-2 Phylogenetic relationships of source-unknown bear samples and voucher samples based on Maximum Likelihood analysis.

Bootstrap supports were provided above branch at node for the divergence of lineages.

The bear sample sizes with each voucher haplotype were showed in parentheses. The source-unknown bear samples were highlighted with dark red color.

Fig. 5-3 Phylogenetic relationships of source-unknown bear samples and voucher samples based on Bayesian inference analysis.

Bayesian posterior probabilities were provided above branch at node for the divergence of lineages. The bear sample sizes with each voucher haplotype were showed in

parentheses. The source-unknown bear samples were highlighted with dark red color.

Fig. 5-4 Bayesian population genetic structure of source-unknown bear samples and voucher samples of 4 subspecies of Asiatic black bears using STRUCTURE 2.3.4.

Bar plot showed the population structure of clustering result with the highest posterior probability, K=5.

Tables

Table 5-1 Genetic ancestries of captive Asiatic black bear samples/ paw specimens used in this study.

Code Name/Local ID Source/Owner Suspected Origin^a Genetic Origin^a

(Asiatic black bears with purebred origin of U. thibetanus formosanus in both mtDNA and microsatellite analyses, n=3)

TWC25 愛德華 Taipei Zoo U/ formo formo

TWC26 六龜(♂) Pingtung Wildlife Rescue Center U/ formo formo

TWC27 六龜(♀) Pingtung Wildlife Rescue Center U/ formo formo

(Asiatic black bears with origin of U. thibetanus formosanus in mtDNA, but admixed origins in microsatellite analysis, n=4)

TWC21^b 黑皮 Shousan Zoo U formo/thibe/mupi

TWC22 ^b 波比 Shousan Zoo U formo thibe/mupi

TWC23 阿妹 Shousan Zoo U formo/thibe/mupi

TWC29 寶貝 Shousan Zoo U formo/thibe/mupi

(Asiatic black bears with admixed origins or with purebred origin of other subspecies in both mtDNA and microsatellite analyses, n=21)

TWC1 小敏 Taipei Zoo U thibe/mupi

TWC2 嘉女 Taipei Zoo U thibe/mupi

TWC3 黑梅蓓 Taipei Zoo U thibe/mupi

TWC4 小熊 Taipei Zoo U/formo thibe/mupi

TWC5 嘉男 Taipei Zoo U thibe/mupi

TWC6 小黑 Taipei Zoo U thibe/mupi

TWC7 寶貝 Taipei Zoo U thibe/mupi

TWC8 嘟嘟 Taipei Zoo U thibe/mupi

TWC11 卡特 Taiwan Endemic Species Research Institute

U thibe/mupi

TWC12 黑妞 Taiwan Endemic Species Research Institute

U thibe/mupi

TWC13 阿財 Taiwan Endemic Species Research Institute

U thibe/mupi

TWC14 小妞 Taiwan Endemic Species Research U thibe/mupi

Institute

TWC15 元元 Taiwan Endemic Species Research Institute

U thibe/mupi

TWC16 皮皮 Private owner (Teng-Zheng, Li) U/formo mupi

TWC17 乖乖 Private owner (Teng-Zheng, Li) U/formo mupi

TWC18^b 平平 Private owner (Teng-Zheng, Li) U/formo mupi

TWC19 ^b 安安 Private owner (Teng-Zheng, Li) U/formo mupi

TWC20 龍谷 Private farm (Long-Gu) U thibe/mupi

TWC24 乖乖 Shousan Zoo U thibe/mupi

TWC28 梅山-1 Private owner (Jin-Xiu, Lin), U/formo thibe/mupi

TWQP1 六龜山產店腳掌 Pingtung University of Science and Technology (Mei-Hsiu Hwang)

U/formo thibe/mupi

(Asiatic black bears with admixed origins in mtDNA analyses but without enough microsatellite data for analysis, n=4)

TWC9 阿里 Taipei Zoo U thibe/mupi/U

TWC10 日月 Taipei Zoo U thibe/mupi/U

TWC30 阿胖 Shousan Zoo U thibe/mupi/U

TWQP5 蔡 O 松案右後腳

掌

Pingtung University of Science and Technology (Mei-Hsiu Hwang)

U thibe/mupi/U

a Subspecies Code: formo, U.thibetanus formosanus; mupi, U. thibetanus mupinensis; thibe, U. thibetanus thibetanus; U, unknown.

b TWC21 and TWC22 are Offsprings of TWC29; TWC18 and TWC19 are Offsprings of TWC16 and TWC17.

Appendix Publications

A. Shih C-C, Huang C-C, Li S-H, Hwang M-H, Lee L-L (2009) Ten novel