Eun Hwa Choi

Mitochondrial genome phylogeny among Asiatic black bear Ursus thibetanus subspecies and comprehensive analysis of their control regions

The complete mitochondrial genome (16,824 bp) of an Asiatic black bear Ursus thibetanus ussuricus (Mammalia, Carnivora, Ursidae) was newly sequenced and characterized in detail. It is the second mitochondrial genome from this subspecies which has been completely sequenced. The two U. t. ussuricus individuals were compared with each other and then with individuals from the other four U. thibetanus subspecies and the other nine ursid species, focusing especially on the control regions in the 14 mitochondrial genomes. Within these control regions, tandem repeats of basically 10 bp (5′-ACGCACGTGT-3′ or its derivatives) were found in Domain II. Plausible secondary structures of the repeat region were compared between the North and South Korean individuals of U. t. ussuricus. According to the maximum likelihood and Bayesian inference trees inferred from the nucleotide sequences of 13 protein-coding and two rRNA genes, the ursine members within the monophyletic ursid clade can be divided into at least three groups: A, B, and C. According to this analysis, U. thibetanus subspecies were found with Ursus americanus and Ursus malayanus within Group A, showing the following relationships with nodal bootstrap values above 91% and Bayesian posterior probabilities of 1.00: ([(U. t. thibetanus, U. t. formosanus), U. t. spp.], U. t. ussuricus), U. t. mupinensis. In addition, we present a hypothetical scenario of the evolution of the major repeat motifs in the control region.

Complete mitochondrial genome of the Hodgson's bat Myotis formosus (Mammalia, Chiroptera, Vespertilionidae)

The first complete mitochondrial genome (17,159 bp) of the Hodgsons bat Myotis formosus, which is an endangered species in South Korea, was sequenced and characterized. The genome included 13 protein-coding, 22 tRNA, and 2 rRNA genes, and 1 control region. It has high AT content and the same gene arrangement pattern as those of typical vertebrate mitochondrial genome. Within the control region, a 80 bp tandem repeat unit was iterated five times which was found in Domain I. It has been observed only in the vespertilionid bat group, and could contribute to identifying the species or genus, and also distinguishing it from other bat families.

Mitochondrial genome of the Korean colubrid snake Elaphe schrenckii (Reptilia; Squamata; Colubridae)

In the present study, we determined the complete mitochondrial genome of a colubrid snake from Korea, Elaphe schrenckii (Reptilia, Squamata, Colubridae). Six universal primer pairs suggested by Kumazawa and Endo (2004) were used for long polymerase chain reactions. All methods were carried out following standard laboratory procedures (Woo et al. 2007). The complete mitochondrial genome (17,164 bp in length) consists of 13 protein-coding, 22 tRNA, and 2 rRNA genes, 2 identical, large control regions (CR1 and CR2), and a small CR (OL; Table I). Except for the seven tRNA genes and Nd6, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand is 35.21% A, 25.93% T, 26.33% C, and 12.53% G, with an AT content of 61.14%. The AT content was higher than the GC content, as generally shown in other vertebrate mitochondrial genomes (Lee et al. 2008). All 21 tRNA genes can fold into a typical cloverleaf secondary structure except for tRNA, which lacks a dihydrouridine arm (D-arm). A pseudotRNA exists in the 50-upstream region of CR1 that can be folded into a potentially imperfect tRNA having a distinct anticodon arm and a D-arm with four base pairings, but lacks a TCC. The 12S and 16S rRNA genes are located between tRNA and tRNA (926 bp), and between tRNA and Nd1 (1483 bp), respectively. Except for Nd2 (ATA start codon) and Cox1 and Nd4 l (GTG start codon), the remaining protein-coding genes initiate with ATG. Five genes end with an incomplete stop codon T__ (Nd1, Cox2, Cox3, Nd3, and Cytb) that may be completed by polyadenylation after transcript processing (Ojala et al. 1981). As observed in the mitochondrial genomes of other snake taxa—such as the viperids himehabu (Trimeresurus okinavensis), the western rattlesnake (Crotalus oreganus), the colubrid akamata (Dinodon semicarinatus), and the boid boa constrictor—that of the Korean E. schrenckii has loci CR1 and CR2 (1017 bp in length, high AT content of 60.7%) surrounded by tRNA and tRNA, and by tRNA and tRNA, respectively. CR1 and CR2 have putative terminationassociated sequences (TASs) and conserved sequence blocks (CSBs) in common. TASs exhibit a conserved motif that can form a stable hairpin-loop structure for the regulation of mitochondrial gene replication (Saccone et al. 2002). Each CR contains TAS1 and TAS2, and three CSBs (CSB1, CSB2, and CSB3), which are thought to be involved in positioning RNA polymerase for both transcription and priming replication (Clayton 1991; Shadel and Clayton 1997). E. schrenckii is categorized as an endangered species in Korea. We expect the present study will contribute to the preservation of endangered species’ genetic resources and will assist species identification and research in colubroid systematics.

Mitochondrial genome of the cockscomb pearl mussel Cristaria plicata (Bivalvia, Unionoida, Unionidae).

The complete mitochondrial genome sequence of the cockscomb pearl mussel Cristaria plicata, which is an endangered species in South Korea, was sequenced. The circle genome (15,708 bp in size) consists of 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. There were 26 noncoding regions (NCs) found throughout the mitogenome of C. plicata, ranging in size from 2 to 327 bp, and the two largest NC regions, NC286 and NC326, were found between ND5 and tRNAGln (286 bp) and between tRNAGlu and ND2 (326 bp), respectively. The 13 mitochondrial protein-coding genes of a female individual of C. plicata collected from Korea (15,708 bp) were compared to those of the Chinese individual (15,712 bp) published before. The result showed that ND3 is the most conserved with 100% nucleotide similarity, and each of the other protein-coding genes has ca. 99%, respectively. The two largest NCs among 26 NCs have totally 98% nucleotide similarity between Korean and Chinese ones.

Complete mitochondrial genome of a Chinese scorpion Mesobuthus martensii (Chelicerata, Scorpiones, Buthidae)

The complete mitochondrial genome (15,034 bp) of a Chinese scorpion Mesobuthus martensii (Buthidae) was sequenced and characterized in detail. The genome contains 13 protein-coding genes, 21 transfer RNA genes, two ribosomal RNA genes and a large non-coding region ( = CR). Its gene arrangement pattern is identical to that of Limulus polyphemus (Chelicerata, Xiphosura), with the exceptions of the tRNAGlu–tRNAIle–tRNAMet (Q–I–M) arrangement and tRNAAsp-loss. Additional interesting features are found and discussed: high frequency of Leu(UUG) codon use, low A+T content of the genome (66.75%), and six repeat units (five 60-nt-long and one 58-nt-long repeats) in the 998-nt CR. Bayesian analysis based on amino acid sequences of the 12 proteincoding genes (excluding ATP8) reveals that the family Buthidae (Order Scorpiones) and the class Arachnida form strong monophyletic groups within Chelicerata, respectively. It indicated that the scorpions are the most ancestral arachnids.

Complete Mitochondrial Genomes of Carcinoscorpius rotundicauda and Tachypleus tridentatus (Xiphosura, Arthropoda) and Implications for Chelicerate Phylogenetic Studies

Horseshoe crabs (order Xiphosura) are often referred to as an ancient order of marine chelicerates and have been considered as keystone taxa for the understanding of chelicerate evolution. However, the mitochondrial genome of this order is only available from a single species, Limulus polyphemus. In the present study, we analyzed the complete mitochondrial genomes from two Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus tridentatus to offer novel data for the evolutionary relationship within Xiphosura and their position in the chelicerate phylogeny. The mitochondrial genomes of C. rotundicauda (15,033 bp) and T. tridentatus (15,006 bp) encode 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. Overall sequences and genome structure of two Asian species were highly similar to that of Limulus polyphemus, though clear differences among three were found in the stem-loop structure of the putative control region. In the phylogenetic analysis with complete mitochondrial genomes of 43 chelicerate species, C. rotundicauda and T. tridentatus were recovered as a monophyly, while L. polyphemus solely formed an independent clade. Xiphosuran species were placed at the basal root of the tree, and major other chelicerate taxa were clustered in a single monophyly, clearly confirming that horseshoe crabs composed an ancestral taxon among chelicerates. By contrast, the phylogenetic tree without the information of Asian horseshoe crabs did not support monophyletic clustering of other chelicerates. In conclusion, our analyses may provide more robust and reliable perspective on the study of evolutionary history for chelicerates than earlier analyses with a single Atlantic species.

Complete mitochondrial genome of the Korean spotted seal Phoca largha (Mammalia, Pinnipedia, Phocidae): Genetic differences between P. largha and Phoca vitulina

The spotted seal Phoca largha is an endangered marine mammal whose range covers wide regions of the continental shelves of the North Pacific and Arctic Oceans (Shaughnessy and Fay 1977; Mizuno et al. 2003). Very little is known about the genetic diversity of the species, with only a single complete mitochondrial genome from an Alaskan individual so far available (Arnason et al. 2006). Here, we report the complete mitochondrial genome (16,728 bp; GenBank accession number FJ895151) of a Korean specimen of P. largha sampled at the exact opposite end of Alaska within the distribution area of this species. This mitochondrial genome, slightly longer than the Alaskan individual’s one (16,701 bp; Arnason et al. 2006; AM181031), exhibits the classic features of mammalian mitochondrial genomes (Figure 1a; Lee et al. 2008). The nucleotide sequence similarity between large control regions (OH, 1200–1300 bp) of the Korean P. largha (KPL) and Alaskan P. largha (APL) is 92.4%. Except for OH, Nd5 exhibits 99% similarity and is the most variable among 13 protein-coding and two rRNAcoding genes between the two species. In the remaining genes, nucleotide similarity ranges from 99.4% (Atp6) to 99.9% (12S rDNA). Similarly, comparison of mitochondrial genomes of an Icelandic individual (IPV; X63726) and a Baltic individual of a closely related species, the harbor seal Phoca vitulina (BPV; AM181032), showed that OH (92.9%) and Nd5 (99.5%) have the lowest levels of similarity, as in P. largha. The OH of KPL is not identical to any one of 57 haplotypes of P. largha reported from Mizuno et al. (2003). Within the OH, the lengths of the repeat regions of KPL, APL, IPV, and BPV are 156, 143, 199, and 163 bp, respectively. The similarity between the repeat regions of KPL and APL is 62.7% and that of IPV and BPV is 66.8%. As shown in Figure 1b, the ACGC and GC repeat motifs are more abundant in KPL (19 occurrences) than in APL (three occurrences), IPV (nine occurrences), and BPV (nine occurrences). In APL, the AC-rich motif consisting of AC plus ACGTAC appears repetitively (up to 19 times), which is not found in KPL. According to Stanley et al. (1996), IPV and BPV belong to the east Atlantic populations that are very closely related each to other. Nevertheless, we were able to distinguish them by examining OH variation. A variety of characteristics such as sequence diversity, repeat motifs, and repeat patterns of OH (potentially also Nd5) may be helpful to distinguish individuals of P. largha and P. vitulina.

Complete mitochondrial genome of the Korean stumpy bullhead Pseudobagrus brevicorpus (Siluriformes, Bagridae)

We describe the complete mitochondrial genome sequence of the Korean stumpy bullhead Pseudobagrus brevicorpus, which is an endangered species in Korea. The circle genome (16,526 bp) consists of 13 protein coding, 22 tRNA, 2 rRNA genes and 1 control region. It has the typical vertebrate mitochondrial gene arrangement.

DNA Barcoding of Metazoan Zooplankton Copepods from South Korea.

Copepods, small aquatic crustaceans, are the most abundant metazoan zooplankton and outnumber every other group of multicellular animals on earth. In spite of ecological and biological importance in aquatic environment, their morphological plasticity, originated from their various lifestyles and their incomparable capacity to adapt to a variety of environments, has made the identification of species challenging, even for expert taxonomists. Molecular approaches to species identification have allowed rapid detection, discrimination, and identification of cryptic or sibling species based on DNA sequence data. We examined sequence variation of a partial mitochondrial cytochrome C oxidase I gene (COI) from 133 copepod individuals collected from the Korean Peninsula, in order to identify and discriminate 94 copepod species covering six copepod orders of Calanoida, Cyclopoida, Harpacticoida, Monstrilloida, Poecilostomatoida and Siphonostomatoida. The results showed that there exists a clear gap with ca. 20 fold difference between the averages of within-specific sequence divergence (2.42%) and that of between-specific sequence divergence (42.79%) in COI, suggesting the plausible utility of this gene in delimitating copepod species. The results showed, with the COI barcoding data among 94 copepod species, that a copepod species could be distinguished from the others very clearly, only with four exceptions as followings: Mesocyclops dissimilis–Mesocyclops pehpeiensis (0.26% K2P distance in percent) and Oithona davisae–Oithona similis (1.1%) in Cyclopoida, Ostrincola japonica–Pseudomyicola spinosus (1.5%) in Poecilostomatoida, and Hatschekia japonica–Caligus quadratus (5.2%) in Siphonostomatoida. Thus, it strongly indicated that COI may be a useful tool in identifying various copepod species and make an initial progress toward the construction of a comprehensive DNA barcode database for copepods inhabiting the Korean Peninsula.

Complete mitochondrial genome of a Steller sea lion Eumetopias jubatus (Carnivora, Otariidae)

The Steller sea lion (Eumetopias jubatus Schreber, 1776; Mammalia, Carnivora, Otariidae) is one of the most endangered species (IUCN 2009; Ministry of Environment of Korea 2004). The population of the Steller sea lion has dramatically declined during the past five decades worldwide (Trites and Donnelly 2003). Thus, it is very important for the systematic conservation of the endangered species to characterize the mitochondrial genomes, a technique that has been usefully employed in the fields of population genetics, phylogeography, species identification, and DNA barcoding (Tunez et al. 2006; Yoo et al. 2006; Koyama et al. 2008; Phillips et al. 2009). In the present study, the nearly complete mitochondrial genome (16,310 bp; GenBank accession number GU475464) of E. jubatus collected from Pohang, South Korea, was sequenced except for the ca. 330 bp downstream region of the control region not sequenced possibly due to the poly-G track. Its characteristics are presented in Table I. It is the second (nearly) complete mitochondrial genome sequenced from this species (Arnason et al. 2002). Although eight nearly complete mitochondrial genome sequences had been deposited directly in GenBank (AB300601–AB3006018), ca. 500 bp of the control region had not been sequenced. Overall characteristics including the mitochondrial genome organization and the gene arrangement pattern were identical to the typical vertebrate mitochondrial genome. The complete mitochondrial genome consists of 13 protein-coding, 22 tRNA, and 2 rRNA genes and one control region. Except for the seven tRNA genes and ND6, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand is 33.51% A, 25.78% T, 26.96% C, and 13.75% G, with an AT content of 59.29%. The AT content was higher than the GC content, as generally shown in other vertebrate mitochondrial genomes (e.g. Bahn et al. 2009; Jang et al. 2009; Woo et al. 2009). Compared with that of Arnason et al. (2002), all 13 protein-coding and two rRNA genes have identical gene lengths and high nucleotide sequence similarities, ranging from 99 to 100%, and the control region is most variable with at least 3% sequence differences. All 21 tRNA genes can fold into a typical cloverleaf secondary structure except for tRNA, which lacks a dihydrouridine arm. The 12S and 16S rRNA genes are located between tRNA and tRNA (963 bp), and between tRNA and tRNA (UUR) (1577bp), respectively. Except for ND2 and ND3 (ATA start codon) and ND5 (ATC start codon), all protein-coding genes initiate with ATG. CO3, ND3, and ND4 end with an incomplete stop codon only with a single “T”. We expect that the present result will contribute to the preservation of genetic resources of endangered