Sex identification of birds in Taipei Zoo.

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.

Genomic targets for high-resolution inference of kinship, ancestry and disease susceptibility in orang-utans (genus: Pongo).

BACKGROUND: Orang-utans comprise three critically endangered species endemic to the islands of Borneo and Sumatra. Though whole-genome sequencing has recently accelerated our understanding of their evolutionary history, the costs of implementing routine genome screening and diagnostics remain prohibitive. Capitalizing on a tri-fold locus discovery approach, combining data from published whole-genome sequences, novel whole-exome sequencing, and microarray-derived genotype data, we aimed to develop a highly informative gene-focused panel of targets that can be used to address a broad range of research questions. RESULTS: We identified and present genomic co-ordinates for 175,186 SNPs and 2315 Y-chromosomal targets, plus 185 genes either known or presumed to be pathogenic in cardiovascular (N = 109) or respiratory (N = 43) diseases in humans - the primary and secondary causes of captive orang-utan mortality - or a majority of other human diseases (N = 33). As proof of concept, we designed and synthesized 'SeqCap' hybrid capture probes for these targets, demonstrating cost-effective target enrichment and reduced-representation sequencing. CONCLUSIONS: Our targets are of broad utility in studies of orang-utan ancestry, admixture and disease susceptibility and aetiology, and thus are of value in addressing questions key to the survival of these species. To facilitate comparative analyses, these targets could now be standardized for future orang-utan population genomic studies. The targets are broadly compatible with commercial target enrichment platforms and can be utilized as published here to synthesize applicable probes.

DNA barcoding reveals CITES-listed species among Taiwanese government-seized chelonian specimens.

Compared to traditional morphological identification, DNA barcoding-molecular identification based on sequencing of a segment of mitochondrial cytochrome c oxidase subunit I (COI)-provides a shortcut to authenticating chelonian identifications. Here, we selected 63 government-seized chelonian specimens deposited at Taipei Zoo for DNA barcoding analysis. DNA barcoding and subsequent phylogenetic analysis successfully authenticated 36 chelonian species, including five that are listed in CITES Appendix I. Approximately 90% (57/63) of the specimens were successfully authenticated by our molecular approach, but lack or error of BOLD reference sequences, biological processes such as hybridization, and uncertain species delimitation all reduced the accuracy of DNA barcoding. To increase the accuracy of DNA barcoding, Taipei Zoo will continue to enrich the BOLD database and also establish a genetic database, to include additional genetic markers, by using government-seized chelonian specimens. A fast and accurate method to authenticate seized samples could assist law enforcement agencies to prosecute criminals and restrict illegal exploitation of wild chelonian resources.

Spatiotemporal Diversification of the True Frogs (Genus Rana): A Historical Framework for a Widely Studied Group of Model Organisms.

True frogs of the genus Rana are widely used as model organisms in studies of development, genetics, physiology, ecology, behavior, and evolution. Comparative studies among the more than 100 species of Rana rely on an understanding of the evolutionary history and patterns of diversification of the group. We estimate a well-resolved, time-calibrated phylogeny from sequences of six nuclear and three mitochondrial loci sampled from most species of Rana, and use that phylogeny to clarify the group's diversification and global biogeography. Our analyses consistently support an "Out of Asia" pattern with two independent dispersals of Rana from East Asia to North America via Beringian land bridges. The more species-rich lineage of New World Rana appears to have experienced a rapid radiation following its colonization of the New World, especially with its expansion into montane and tropical areas of Mexico, Central America, and South America. In contrast, Old World Rana exhibit different trajectories of diversification; diversification in the Old World began very slowly and later underwent a distinct increase in speciation rate around 29-18 Ma. Net diversification is associated with environmental changes and especially intensive tectonic movements along the Asian margin from the Oligocene to early Miocene. Our phylogeny further suggests that previous classifications were misled by morphological homoplasy and plesiomorphic color patterns, as well as a reliance primarily on mitochondrial genes. We provide a phylogenetic taxonomy based on analyses of multiple nuclear and mitochondrial gene loci. [Amphibians; biogeography; diversification rate; Holarctic; transcontinental dispersal.

Complete Mitochondrial Reveals a New Phylogenetic Perspective on the Brackish Water Goby Mugilogobius Group (Teleostei: Gobiidae: Gobionellinae).

The Mugilogobius group consists of brackish water gobionellines widely distributed in the Indo-West Pacific region. Complete mitochondrial genome and morphological evidence was collected to estimate their phylogenetic relationship and taxonomic status. A total of 11 genera were sampled, including Brachygobius, Calamiana, Hemigobius, Mugilogobius, Pandaka, Pseudogobiopsis, Pseudogobius, Redigobius, Rhinogobius, Stigmatogobius, and Wuhanlinigobius, five of which were sequenced for the first time. A morphological phylogenetic tree was also reconstructed based on 35 characters. The molecular phylogenetic trees reveal that the Mugilogobius group contains four major clades. The present study also reveals that the adult male mouth size and forked sensory papillae row d can be considered as synapomorphies, and that the head pores on inter-orbital, anterior oculoscapular, and preopercular regions can be regarded as derived features among the Mugilogobius group. Furthermore, the absence of posterior oculoscapular pores may provide a clue for understanding the evolutionary history of the Mugilogobius group.

Low but highly geographically structured genomic diversity of East Asian Eurasian otters and its conservation implications.

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.

Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data.

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.

Phylogeography of the fanged dicroglossine frog, Limnonectes fujianensis (Anura, Ranidae), in Taiwan.

A phylogenetic analysis of Taiwanese fanged dicroglossine frog, Limnonectes fujianensis (Anura, Ranidae), was conducted to examine its genetic diversification using sequence data from a portion of the mitochondrial DNA (mtDNA) cytochrome b sequences. We collected genetic data from 200 individuals at 23 localities in Taiwan and three localities in China. A neighbor-joining tree of 39 haplotypes revealed two clades in Taiwan and a clade in China, each showing restricted geographical distribution. The pattern of geographical divergence suggests a single invasion into Taiwan. Divergence times between clades were inferred using molecular clock tests. The population relationship of L. fujianensis between Taiwan and mainland China, and the phylogenetic relationships with its congeners, e.g., L. bannaensis, L. fragilis and L. kuhlii, were obtained and discussed.

A barcoding-based scat-analysis assessment of Eurasian otter Lutra lutra diet on Kinmen Island.

While it is well known that Eurasian otters principally feed on fishes and crustaceans, their detailed diet taxonomies are not fully understood. This is partly due to their nocturnal behavior and the limited resolving power of traditional morphological identification from scat. A suitable, reliable molecular method for diet studies is therefore needed.I performed a series of Sanger-sequencing reactions, utilizing nine primer sets for Eurasian otter diet research. These are mainly based on the barcoding concept to determine the taxonomic composition of spraints. The primer sets target different types of animals, amplifying each separately. This procedure was used to detect the prey contents of 64 spraint samples collected from Kinmen Island. Through high-resolution gel electrophoresis and sequencing, it was evident that PCR products could be successfully amplified by the different primer sets and from spraint samples comprising multiple prey species.Extracted DNA from all spraint samples was PCR-amplified with 9 primer sets. In total, 16 prey types were identified across all 64 samples. Fourteen were identified at the species level.The aim of this study was to develop and apply a novel diet research method to Eurasian otters. Eight of the primers are universal primers designed for COI segments of different animal groups, and one primer set was designed specifically for tilapia groups. This method can be applied to study the diets of not only Kinmen Eurasian otter populations, but also other Eurasian otter populations and other small carnivorous animals.

Complete mitochondrial genome of Manispentadactylapentadactyla (Mammalia: Pholidota), an endemic subspecies of Chinese pangolin: mitogenome characterisation and phylogenetic implications.

The Chinese pangolin Manispentadactyla 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.

Intraspecific relationships of populations of the brown frog Rana sauteri (Ranidae) on Taiwan, inferred from mitochondrial cytochrome b sequences.

We studied the phylogenetic relationships among populations of Rana sauteri using partial sequences of the mitochondrial cytochrome b gene from 244 samples from 29 localities In Taiwan. We detected 77 haplotypes among these sequences. The phylogenetic trees contained five distinct lineages: the northern (NL), eastern (EL), southern hill (SHL), northern mountain (NML), and southern mountain (SML) lineages, defined by geographical distribution. The lineage phylogeny did not support the two-species hypothesis Inferred from larval morphology. To describe the possible colonization history of R. sauteri in Taiwan, we propose hypotheses of within-island differentiation and a multiple-invasion model. Using a molecular clock, we estimated the order of divergence times between lineages in order to test the migration hypothesis. The multiple-invasion model was well supported by the phylogeny and a nested clade network.

Phylogeography of Sylvirana latouchii (Anura, Ranidae) in Taiwan.

Biogeographic studies are important for understanding the natural history of faunas. To comprehend the geographical patterns of genetic variation in anurans in Taiwan, we investigated the genetic structure of Sylvirana latouchii (Anura, Ranidae) from 31 populations by using mitochondrial DNA (mtDNA) cytochrome b sequences. A neighbor-joining tree of 38 haplotypes revealed three major divergences in Taiwanese S. latouchii: the northern, western, and eastern-and-southern clades. Each clade was restricted to a single geographical district and showed obvious differentiation. The patterns of geographical divergence in this species reflect common historical events experienced by other native animals distributed in Taiwan. The order of divergence times between clades was inferred using a molecular clock test. The population relationship of S. latouchii between Taiwan and mainland China is discussed. Further study employing more populations of S. latouchii from mainland China is necessary to clarify the original geographical patterns and migratory history of this species.

Complete mitochondrial genome sequence for the Taiwan Blue Magpie Urocissa caerulea (Passeriformes: Corvidae).

Taiwan Blue Magpie (Urocissa caerulea) is endemic to Taiwan and listed as threatened species protected by law. In this study, we first determined and described the complete mitochondrial genome of Taiwan Blue Magpie. The circle genome is 16,928 bp in length, and contains 13 protein coding, 22 tRNA, two rRNA genes, and one non-coding control region (CR). The overall base composition of the mitochondrial DNA is 30.99% for A, 24.69% for T, 30.07% for C, and 14.25% for G. The percentage of G + C content is 44.32%. This work provides fundamental molecular data which will be useful for evolution and phylogeny studies on Corvidae in the future.

The complete mitochondrial genome of the Kinmen's population of endangered minnow Metzia mesembrinum (Cypriniformes, Cyprinidae) from Taiwan.

Here we describe the complete mitochondrial genome sequence of endangered minnow, Metzia mesembrinum, which collected from Kimnen island, Fujian Province of Taiwan. The circular mitogenome (16,603 bp) consists of 13 protein coding, 22 tRNA, 2 rRNA genes and 1 control region. It has the typical vertebrate mitochondrial gene arrangement. The overall base composition in descending order is A (32.0%), T (27.1%), C (25.2%), G (15.7%) with rather higher AT content as 59.1%. Yuan et al. reported the complete mitogenome (KF997093) of M. mesembrimum from southern China that may belong to other congeneric species, but not exactly M. mesembrinum collected from either southern Fujian or Taiwan. The well-identified species sequence data of Metzia mesembrinum would be rather beneficial for further phlyogenetic approach for congeneric species in East and Southeast Asia.

Complete mitochondrial genome of the longfin mako shark, Isurus paucus (Chondrichthyes, Lamnidae).

Here we describe the complete mitochondrial genome sequence of the longfin mako, Isurus paucus, which is a pelagic shark found in temperate and tropical waters. The circle genome (16,704 bp) consists of 13 protein coding, 22 tRNA, 2 rRNA genes and 1 control region. It has the typical vertebrate mitochondrial gene arrangement.

Phylogeography, Historical Demography, and Genetic Structure of the Rose Bitterling, Rhodeus ocellatus (Kner, 1866) (Cypriniformes: Acheilognathidae), in East Asia.

Yao-Feng Tsao, Wen-Wen Lin, Chia-Hao Chang, Takayoshi Ueda, Nian-Hong Jang-Liaw, Ya-Hui Zhao, and Hsiao-Wei Kao (2016) Rose bitterling, Rhodeus ocellatus, is a small cyprinid fish distributed in East Asia. To infer its phylogeography and genetic structure, specimens from Taiwan, China, and Japan were collected, and complete mitochondrial cytochrome b (cyt b) DNA sequences were amplified and sequenced. Phylogenetic analyses identified seven mitochondrial lineages (A-G). Among them, three lineages (A, B, and C) distributed in mainland China. Lineages D, E, and F distributed in Japan, Korea, and Taiwan, respectively. Lineage G distributed in both China and Japan. The results of the Bayesian Binary MCMC analysis (BBM) suggested that the most recent common ancestor of R. ocellatus was from Lower Yangtze region. Divergence times among lineages inferred by molecular clock ranged from 7.55 to 1.44 million years ago. We propose that topography and climate changes by uplift of the Tibetan Plateau in the Late Miocene-Pliocene and the glacial-interglacial cycles in the Pleistocene might account for population expansion and genetic differentiation. Divergence times among lineages A, B, and C in Yangtze River basin ranged from 7.55 to 2.27 million years ago that might result from changes of flow directions of rivers from westward to eastward driven by the uplift of the Tibetan Plateau. The glacial-interglacial cycles in the Pleistocene might further cause population expansion to the northward of lineage G at about 0.19 million years ago. Lineage D in Japan was dispersed from the mainland China before the opening of the Sea of Japan, and lineage F in Taiwan was dispersed from the mainland China through the land bridge in the Pleistocene. Because of the genetic differentiation is statistically significant among populations, protection of genetic diversity and distinctness of R. ocellatus should be considered in the future conservation management.

The complete mitochondrial genome of the salmon shark, Lamna ditropis (Chondrichthyes, Lamnidae).

The complete mitochondrial genome of the salmon shark consists of 16,699 bp and includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, 1 replication origin region and 1 control region. The mitochondrial gene arrangement of the salmon shark is the same as that of most vertebrates. Base composition of the genome is A (29.6%), T (28.6%), C (27.1%), and G (14.8%).

Complete mitochondrial genome of the megamouth shark Megachasma pelagios (Chondrichthyes, Megachasmidae).

Here we describe the complete mitochondrial genome sequence of the megamouth shark, Megachasma pelagios, which is an extremely rare species of deepwater shark. The circle genome (16,694 bp) consists of 13 protein coding, 22 tRNA, 2 rRNA genes and 1 control region. It has the typical vertebrate mitochondrial gene arrangement.

A molecular forensic method for identifying species composition of processed marine mammal meats.

We used universal primers designed for the cytochrome oxidase I (CO I) sequence of the order Cetacea and the family Phocidae to prove that meat fritters sold in Taiwan contained meat from two seal, six cetacean, and one pig species. The sequence information for CO I obtained in this study was limited and population genetics data for the eight sampled marine mammalian species was insufficient to deduce where these marine mammals were hunted. Regardless of the geographic origins of the marine mammal flesh, sale and consumption of marine mammals in Taiwan violates the Wildlife Conservation Act. This study provides PCR primers that could enable government testing of suspect meats to curtail the illegal trade in marine mammal products.

Complete mitochondrial genome of the Kikuchi's minnow Aphyocypris kikuchii (Teleostei, Cyprinidae).

We have sequenced the complete mitochondrial genome of the Kikuchi's minnow, Aphyocypris kikuchii (Oshima 1919), which is an endemic species to Taiwan. The complete mitochondrial genome is 16,601 bp in size, containing 37 genes coding for 13 proteins, 2 rRNAs, 22 tRNAs, and 1 control region. It has the typical vertebrate mitochondrial gene arrangement. The sequence information could play an important role in resolving the conflict on its current taxonomic position and preservation of genetic resources for helping conservation of the endangered species.