Nian-Hong Jang-Liaw

DNA barcoding using mitochondrial COI is widely applied for animal species identification, but its accuracy in mammals can be affected by uneven sampling, sequence artefacts, and nuclear mitochondrial pseudogenes (numts). In this study, we established a voucher-linked COI DNA barcode reference library for non-volant terrestrial mammals of Taiwan and evaluated its identification performance and potential limitations using the BOLD workbench, Barcode Index Number (BIN) assignments, barcode gap analyses, and comparisons with public sequence data. A total of 157 newly generated COI sequences were obtained, representing seven orders, 16 families, 37 genera and 48 species. Among the 48 sampled taxa, 15 (31.25%) had no COI barcode record available in BOLD before this study; therefore, the deposition of the present library increasing BOLD coverage for the sampled taxa from 68.8% to 100%. In the original BOLD project dataset, 145 of the 157 sequences were assigned to 49 BINs. To evaluate whether the observed barcode patterns were robust for broader sampling, 75 public Taiwanese mammalian COI sequences were incorporated into an expanded Taiwan dataset of 232 sequences. Barcode-gap and Neighbor-Joining analyses showed that 45 of the 48 taxa retained positive barcode gaps after public sequences were added, supporting the utility of COI barcodes for identifying most non-volant terrestrial mammals in Taiwan. Three taxa, Mogera insularis , M. kanoana , and Petaurista grandis , showed negative barcode gaps and/or split clustering patterns and were therefore highlighted for further investigation. In addition, all three newly generated Rattus tanezumi sequences were flagged as putative numts based on anomalous placement and full-length sequence inspection. Our results further emphasize the importance of integrating specimen traceability, functional sequence checks, comparisons with public data, distance-based analyses, and cautious interpretation of BIN results in mammalian DNA barcoding research.

Among the five species of the Mustelidae found in Taiwan, four terrestrial species are potentially sympatric. These species include yellow‐throated marten Martes flavigula , Siberian weasel Mustela sibirica , Taiwan least weasel Mustela nivalis formosana , and Chinese ferret‐badger Melogale moschata . Scat from the first three mustelids are similar in morphology. Therefore, differentiating the scats of these three species is challenging, and without accurate species identification, scatology‐related techniques such as dietary analysis are problematic. Here, we developed a novel set of primers capable of effectively identifying scat samples of these three species through a simple PCR amplification process. This allows for collection of more accurate field data on these species, enabling a better understanding of their ecological niche and basic biological information for future conservation strategies.

Molecular data from 35 of the 50 Acrossocheilinae species suggest that the species‐level diversity in the subfamily has been overestimated, likely due to inadequate taxon and geographic sampling and reliance on morphological characters that vary intraspecifically. Three new genera, one resurrected genus, two resurrected species, and one new species are diagnosed and described herein. Nine synonyms of three valid species of Acrossocheilinae are recognized from the Yangtze, Xijiang, Song Hong, Annamite, and Mekong ecoregions in East and Southeast Asia. Thirty‐two valid and six putative new species are indicated by molecular data and a key to the genera is provided. As more molecular and morphological data become available, additional taxonomic changes in this widespread and generally poorly known subfamily are likely.

The endemic cyprinid, Pararasbora moltrechti Regan, 1908 is briefly redescribed from central Taiwan. A more detailed description was first provided in Mandarin by Chen & Fang, 1999. A second, rather doubtful description error created a new cyprinid species, Aphyocypris amnis Liao et al., 2011, which is almost identical to the original species. The new species was found in a small tributary of the Jushuei River basin, just 0.5 km from Lake Candidius—Sun Moon Lake, the same location where P. moltrechti was originally found. Chen et al. (2012) clearly noted this synonym in the remarks of the Red Data Fish Book in AGC. The incorrect scientific name was added to the Governmental protection list in AGC, causing further issues for conservation management. Solid evidence, not only from morphological data but also from increasing mtDNA documentation, supports the fact that the later published name is redundant. Mito-genetic results also suggest that they are the same species with no distinct genetic differentiation. Herein we clearly state that the Pararasbora moltrechti Regan, 1908 is the senior synonym of Aphocypris amnis Liao et al., 2011 (Teleostei: Cyprinidae) in Taiwan.

As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex‐situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex‐specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty‐five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.

Populations of Eurasian otters Lutra lutra, one of the most widely distributed apex predators in Eurasia, have been depleted mainly since the 1950s. However, a lack of information about their genomic diversity and how they are organized geographically in East Asia severely impedes our ability to monitor and conserve them in particular management units. Here, we re‐sequenced and analyzed 20 otter genomes spanning continental East Asia, including a population at Kinmen, a small island off the Fujian coast, China. The otters form three genetic clusters (one of L. l. lutra in the north and two of L. l. chinensis in the south), which have diverged in the Holocene. These three clusters should be recognized as three conservation management units to monitor and manage independently. The heterozygosity of the East Asian otters is as low as that of the threatened carnivores sequenced. Historical effective population size trajectories inferred from genomic variations suggest that their low genomic diversity could be partially attributed to changes in the climate since the mid‐Pleistocene and anthropogenic intervention since the Holocene. However, no evidence of genetic erosion, mutation load, or high level of inbreeding was detected in the presumably isolated Kinmen Island population. Any future in situ conservation efforts should consider this information for the conservation management units.

We investigated the genetic structure of the freshwater fish Aphyocypris normalis, in 33 populations around Hainan Island and southern mainland China. Sequencing of the mitochondrial DNA (mtDNA) cytochrome b from 127 specimens yielded 47 haplotypes, from which we inferred a Bayesian tree. This revealed three major divergences: a principal clade of specimens with widespread geographic distribution, plus two clades with limited distribution. We estimated that these diverged between 1.05–0.16 Ma. Additionally, based on molecular data and comparing with the climate patterns of Hainan Island, eight phylogeographic ranges (populations) of A. normalis were constructed: the eastern plain (E), northeastern hills and plain (NE), northwestern hills and lowlands (NW), central mountains (C), southeastern hills and plain (SE), southern mountains and hills (S), southwestern mountains and lowlands (SW), and western lowlands (W). The patterns of geographical divergence in this species do not reflect the isolation caused by the Qiongzhou (Hainan) Strait, which would generally be experienced by terrestrial animals on isolated islands. The present results indicate that the major clades within A. normalis have diverged before the temporary land bridge existed across the strait during the Last Glacial Maximum.

The Chinese pangolin Manis pentadactyla is critically endangered because of over-exploitation and illegal trafficking and includes three subspecies. However, the taxonomic status of the three subspecies of the Chinese pangolin has not been well resolved, which impedes regional conservation and illegal trade traces. In this study, the complete mitogenome sequence of M. p. pentadactyla, an endemic subspecies of the Chinese pangolin in Taiwan, was determined. The complete mitogenome of M. p. pentadactyla is 16,570 base pairs (bp) in length with 13 protein-coding genes (PCG), 23 transfer RNAs (tRNAs), two ribosomal RNAs and a 1164 bp control region. The overall base composition of the genome showed a slight A + T bias (59.9%), positive AT skew (0.1515) and negative GC skew (-0.3406), which is similar to that of other pangolins. All PCGs started with a typical ATN codon and all tRNAs were typical cloverleaf-shaped secondary structures, except for tRNA-Ser^(GCU). Phylogenetic analysis indicated a monophyletic relationship for M. p. pentadactyla and M. p. aurita and was monophyletic for M. p. pentadactyla, but paraphyletic for M. p. aurita. The paraphyly of M. p. aurita resulted from an incomplete lineage sorting. This study enriched the mitogenome database of the Chinese pangolin and the molecular information obtained should be very useful for future research on mitogenome evolution and genetic diversification in M. pentadactyla.