Phylogeographic structuring within recently diverged scorpion species, Euscorpius borovaglavaensis Tropea, 2015 (Scorpiones: Euscorpiidae) in Croatia, with the description of a new subspecies

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({type:entrez-nucleotide,attrs:{text:AF184619,term_id:296940320,term_text:AF184619}}AF184619, {type:entrez-nucleotide,attrs:{text:AF181889,term_id:285026682,term_text:AF181889}}AF181889, {type:entrez-nucleotide,attrs:{text:AF188122,term_id:285809842,term_text:AF188122}}AF188122), Metagonimus yokogawai ({type:entrez-nucleotide,attrs:{text:AF096230,term_id:297039733,term_text:AF096230}}AF096230), Metagonimus takahashii ({type:entrez-nucleotide,attrs:{text:AF096231,term_id:297039734,term_text:AF096231}}AF096231), Metagonimus miyatai ({type:entrez-nucleotide,attrs:{text:AF096232,term_id:297039735,term_text:AF096232}}AF096232), Pygidiopsis summa ({type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884), and Stellantchasmus falcatus ({type:entrez-nucleotide,attrs:{text:AF181887,term_id:285804435,term_text:AF181887}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({type:entrez-nucleotide,attrs:{text:AF096234,term_id:297039736,term_text:AF096234}}AF096234) and Neodiplostomum seoulense ({type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({type:entrez-nucleotide,attrs:{text:EF055885,term_id:119855482,term_text:EF055885}}EF055885) [4] and Paragonimus bangkokensis ({type:entrez-nucleotide,attrs:{text:AB354227,term_id:155369203,term_text:AB354227}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({type:entrez-nucleotide,attrs:{text:AJ628024,term_id:88319716,term_text:AJ628024}}AJ628024, {type:entrez-nucleotide,attrs:{text:AJ628039,term_id:88319746,term_text:AJ628039}}AJ628039, {type:entrez-nucleotide,attrs:{text:FJ469984,term_id:238631966,term_text:FJ469984}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884, {type:entrez-nucleotide,attrs:{text:AY055380,term_id:22203992,term_text:AY055380}}AY055380, and {type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

Papilio chikae and P. hermeli are two endemic swallowtail butterflies in the Philippines found primarily in the Cordillera region and Mindoro Island, respectively. Their species status is still subject to debate due to opposing interpretations of their genitalia and morphological traits. This study aims to delineate these taxa through the use of mitochondrial cytochrome C oxidase subunit I(COI) gene marker, with additional information on their cytochrome C oxidase subunit II(COII) gene. The COI nucleotide sequences from our Philippine P. chikae samples were surprisingly identical to P. hermeliEF514465.1 on the GenBank, whereas our P. hermelisamples showed a 100% similarity with P. hermeliEF514464.1.Based on our phylogenetic analysis, P. chikae and P. hermeli exhibit distinct COI barcodes supported by a high bootstrap value (85%). The P. hermeli voucher specimen with GenBank Acc. No. EF514465.1 clustered with our P. chikae samples, separate from the cluster containing our P. hermelispecimens. Nonetheless, the 3.1–3.4% genetic distance observed in the COI region supported by the formation of two separate clusters suggests species status for these taxa. Moreover, species-specific molecular markers obtained from the COII region displayed a 2.4% distance between these two taxa. These molecular markers could therefore be utilized to distinguish P. chikae from P. hermeli. Nevertheless, a thorough examination of their morphology, behavior, and ecology is still imperative to firmly establish their taxonomic status.

Abstract. Lubis K, Sudibyo M, Farajallah A, Hanim N. 2020. Short Communication: The mitochondrial cytochrome c oxidase subunit I (COI) for identification of batoids collected from landing sites in Medan, Indonesia. Biodiversitas 21: 5414-5421. Batoids are member of Elsamoranch subclass which consist of many species. Most of batoids species are overexploited, especially in Medan Indonesia. Up to presents, the information about diversity of rays on the east coast of North Sumatra, Indonesia was very limited. Therefore, this research aimed to investigate the diversity of rays on the east coast of North Sumatra. We examined the morphological trait of 82 individuals of batoid from three landing sites on the east coast of North Sumatra, namely: Tanjung Balai, Belawan, and Percut, then identify its species based on determination key. After that, we collected pectoral muscle tissue from an individual in each species which successfully identified to extract its genomic DNA. Molecular based identification was carried out by using DNA fragment form COI gene. The successfully amplificated COI gene DNA fragment then was sequenced and analyzed. Based on morphological trait, we successfully identifying nine species of batoid, which is Maculabatis gerrardi, Gymnura poecilura, Dasyatis zugei, Brevitrygon heterura, Neotrygon kuhlii, Hemitrygon bennettii, Rhinobatos jimbaranensis, Rhinoptera javanica, and Taeniura lymma. The result of identification based on COI gene DNA fragment was in congruent with morphological-based identification based on data BLAST-N and genetic distance value within same species. The nucleotide diversity within same species ranged from 0-15 nucleotide variants.

DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

To report a case of imported furuncular cutaneous myiasis, and to analyze the sequence of the mitochondrial cytochrome C oxidase subunit Ⅰ (COⅠ) gene of the pathogenic Cordylobia anthropophaga. A 33-year-old female patient had a travel history to Ghana and Cameroon in Africa 1 month prior to the presentation. No anti-mosquito measures were taken during her stay, and she hung up the laundries outside to dry for several times. Skin examination showed furuncular protuberances with diameters of 1 - 2 cm on the inner side of the left upper arm as well as on the outer side of the left chest, which were bright red and hard on palpation with irregular borders and a small hole on their central surface. Morphological identification revealed that the larva squeezed from the lesion was suspected as myiasis. After PCR amplification of the COⅠ gene of the larva, an about 650-bp PCR product was acquired. Sequencing and BLAST analysis showed that this product was most closely related to the COⅠ gene (GenBank accession number: FR719158.1) of Cordylobia anthropophaga isolated in Cameroon in 2010 with the sequence similarity being 99.84%, and they were grouped together on the phylogenetic tree. According to the clinical features and travel history of the patient and the sequencing results of the pathogenic Cordylobia anthropophaga, this case was confirmed as imported furuncular cutaneous myiasis caused by Cordylobia anthropophaga. Key words: Hypodermyiasis; Muscidae; Electron transport complex Ⅳ; Furuncular cutaneous myiasis; Cordylobia anthropophaga

Sphaeroma terebrans, a wood-boring isopoda, is distributed worldwide in tropical and subtropical mangroves. The taxonomy of S. terebrans is usually based on morphological characteristics, with its molecular identification still poorly understood. The number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod are considered as the major morphological characteristics in S. terebrans, which can cause difficulty in regards to accurate identification. In this study, we identified S. terebrans via molecular and morphological data. Furthermore, the validity of the mitochondrial cytochrome c oxidase subunit I (COI) gene as a DNA barcode for the identification of genus Sphaeroma, including species S. terebrans, S. retrolaeve, and S. serratum, was examined. The mitochondrial COI gene sequences of all specimens were sequenced and analysed. The interspecific Kimura 2-parameter distances were higher than intraspecific distances and no intraspecific-interspecific distance overlaps were observed. In addition, genetic distance and nucleotide diversity (π) exhibited no differences within S. terebrans. Our results revealed that the mitochondrial COI gene can serve as a valid DNA barcode for the identification of S. terebrans. Furthermore, the number of teeth on the uropodal exopod and the length of the propodus of the seventh pereopod were found to be unreliable taxonomic characteristics for S. terebrans.

Among the Ixodid ticks, Hyalomma anatolicum is a well-known vector that transmits various pathogens to terrestrial and semi-terrestrial vertebrates including humans, and its population differ in ecology and vector competence. Expansion of this tick to new areas changes the genetic structure, and lead to affect the vector-pathogen interaction and disease outcomes. The present study was designed to infer the haplotype diversity, demographic dynamics, gene flow and genetic differentiation, and phylogeny of H. anatolicum from different countries based on the cytochrome oxidase I (COI) and 16S rDNA sequences. A total of 320 ticks were collected from cattle, buffaloes, and sheep in five districts of Khyber Pakhtunkhwa, Pakistan, morphologically identified as H. anatolicum, and subjected to genetic analysis. A total 85 and 138 sequences for COI and 16S rDNA, including 11 and 2 sequences generated in this study, respectively, were analyzed to assess haplotype network, population structure and divergence, demographic changes, and phylogenetic analysis. Analysis based on COI sequences yielded 29 haplotypes in which haplotype 1 and 15 were the predominant consisting of 35 and 20 sequences, respectively, from Pakistan, India, China, Bangladesh, Iraq, Saudi Arabia, Kazakhstan and Egypt. The 16S rRNA yielded 30 haplotypes in which haplotype 1 was predominant consisting of total 86 sequences from Pakistan, India, China, United Arab Emirates, Tajikistan, Kazakhstan, Turkey, Egypt, and Iraq. Complete haplotype network based on COI and 16S rRNA confirmed stellate structure, together with high haplotype diversity (COI 0.77899, 16S rRNA 0.60774) and low nucleotide diversity (COI 0.00445, 16S rRNA 0.00431), which support recent population expansion. Similarly, neutrality indices for the whole dataset, Tajima’s D (COI − 2.36363**, 16S rRNA − 2.54127***), Fu and Li’s D (COI − 5.72992, 16S rRNA − 6.31313*), and Fu and Li’s F (COI − 5.04435*, 16S rRNA − 5.56085*) were negative, indicating deviation from neutrality and recent population dispersal. In the phylogenetic tree based on the COI and 16S rDNA sequences, with exception of one sequence for a single haplotypes, which appeared independently, there is a single main clade that includes the largest number of sequences for all other haplotype. Based on COI and 16S rDNA sequences, the present study provided first detail information about the population genetics and haplotype networks of H. anatolicum.

Background and Aim:Illegal wildlife trafficking is a critical threat to biodiversity, particularly in megadiverse countries such as Colombia. Birds, notably psittacines, are among the most targeted taxa. Morphological identification is often insufficient, especially when dealing with cryptic species or degraded samples. This study aimed to assess the utility of mitochondrial markers cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) as molecular tools for species-level identification of psittacines housed at the Conservation Park of Medellín.Materials and Methods:Six adult psittacines from the genera Ara and Pionus were selected based on availability. Blood samples were collected and genomic DNA was extracted using a commercial kit. Polymerase chain reaction amplification of partial COI and 16S rRNA gene fragments was performed, followed by Sanger sequencing. Sequence identity was confirmed using BLASTn and the Barcode of Life Data System (BOLD). Phylogenetic relationships were analyzed using Neighbor-Joining, Maximum Likelihood, and Bayesian Inference approaches.Results:Molecular results showed 100% concordance with prior morphological identification for all six individuals. COI and 16S rRNA sequences allowed clear species-level identification with similarity values >98%. Phylogenetic analyses for both markers yielded congruent tree topologies, with high branch support (>90%), further validating species identification. Maximum interspecific divergence for COI was observed between Ara macao and Pionus fuscus (0.15980), while 16S rRNA showed lower divergence values. All generated sequences were submitted to GenBank and BOLD in accordance with findable, accessible, interoperable, reusable principles.Conclusion:This study confirms the robustness of COI and 16S rRNA mitochondrial markers in accurately identifying psittacine species. The integration of molecular and morphological approaches enhances forensic investigations, facilitates biodiversity conservation, and contributes to efforts against wildlife trafficking. Expanding genetic databases for Neotropical avifauna, especially for commonly trafficked species, is imperative. Future research should adopt integrative genomic approaches involving nuclear markers to overcome the maternal inheritance limitation of mitochondrial DNA.

Tilapia (Oreochromis mossambicus) was first introduced to the Philippines in 1950 for aquaculture. Since then, other species of tilapia have been introduced to the country and some of them (mainly Oreochromis niloticus) have become established in lakes and other water bodies. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase subunit I (COI) gene was done to assess the reliability of morphological identification and the degree of introgression among feral tilapias (Oreochromis spp.) in seven major Philippine lakes, namely Laguna de Bay, Lake Lanao, Taal Lake, Lake Mainit, Lake Naujan, Lake Bato, and Lake Buhi. Specimens were also collected from a private hatchery in Sual, Pangasinan to serve as reference. Morphological traits, Nucleotide BLAST (BLASTn), and Translated BLAST (BLASTx) analyses were used to classify the specimens. A Neighbor-Joining tree was constructed using the Kimura 2-Parameter method, incorporating 66 COI sequences generated from the study and 20 additional reference sequences obtained from GenBank. Three Oreochromis clusters were obtained and were classified as the O. niloticus group, O. mossambicus group, and O. aureus group, with bootstrap support values of 99%, 74%, and 99%, respectively. The mean K2P genetic distances within each group were 0.008%, 0.959%, and 0.086%, respectively. The clustering of COI sequences generated from this study corresponded with the results of the BLASTn analysis. Oreochromis hybrids were also found in all the lakes. The study highlights the usefulness of DNA barcoding for molecular identification and detection of introgressed individuals, with potential applications in management of feral stocks.

The striped false limpet Siphonaria pectinata (Linnaeus, 1758) has traditionally been considered a pulmonate gastropod (but see current phylogenetic work for the placement of Siphonariidae; e.g. Grande, Templado & Zardoya, 2008; Jorger et al., 2010) with a limpet-like shell adapted to life on wave-swept rocky intertidal shores. With its 12 synonyms, S. pectinata is known from the Atlantic coast of Portugal, European and African Mediterranean and West African coast to Angola, Macaronesian archipelagos in the eastern Atlantic and from the Caribbean and nearby Atlantic, including eastern Florida, Texas and Mexico (Abbott, 1974; Rolan, 2005). Although its presence in the western Atlantic has been attributed to a Nineteenth Century invasion from the Mediterranean Sea (Morrison, 1963, 1972), this view was questioned by Carlton (1992) based on habitat and broad Western Atlantic distribution, and it is thus considered by some a cryptogenic species (Carlton, 1992; Baker, Baker & Fajans, 2004) – a species that may be either native or introduced. The potential invasive capacity of Mediterranean Siphonaria pectinata has recently been demonstrated by a considerable range extension to the Gulf of Tunis (northeastern Tunisia), where it was established after 2005 (Antit, Gofas & Azzouna, 2007) and Croatia (unpublished sequence data available in GenBank, accessioned October 2010). The dietary specialization of the species on soft microalgae that may serve to reduce competition with other grazers that consume harder encrusting material (Ocana & Fa, 2003) has been interpreted as one of the advantages of this species for colonizing new localities. In order to test the potential invasion of Siphonaria pectinata into (or from) the western Atlantic, we collected samples from near the extreme of the species distribution in localities in the province of Cadiz, southeastern Spain (La Caleta and el Puerto de Santa Maria), southern Cameroon (Kribi) and the Atlantic coast of Florida (Fort Pierce) (Table 1), totalling 23 individuals, collected between 1993 and 2010 (see Fig. 1 for some of the sampled specimens). The oldest specimens were preserved in 70% ethanol and transferred to 96% ethanol a few years after collection; newer specimens were preserved in 96% ethanol and stored at –808C until processed for DNA extraction. Specimens were retained as vouchers and, together with their DNA extractions, are deposited in the Museum of Comparative Zoology, Department of Invertebrate Zoology DNA collection under numbers MCZ DNA100660, DNA104633, DNA104886 and DNA105664. Total DNA was extracted from a small tissue sample from the foot using the DNeasy Tissue Kit from QIAGEN# and the protocol provided by the manufacturer. The purified DNA was used as a template for PCR amplification of fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI), and 16S rRNA. A 658-bp fragment of the COI gene was amplified and sequenced using the primer pair LCO1490–HCO2198 (Folmer et al., 1994), and part of the 16S rRNA gene, between 436 and 437 bp, was amplified and sequenced using primer pair 16Sa–16Sb (Xiong & Kocher, 1991; Edgecombe, Giribet & Wheeler, 2002). PCRs (25 ml) included 1 ml of the template DNA, 1 ml of each primer, 2.5 ml EconoTaq 10X PCR buffer containing 15 mM MgCl2 (Lucigen), 0.25 ml of dNTPs 100 mM, 1.25 U of EconoTaq DNA polymerase (Lucigen). The PCRs were carried out using an Eppendorf Mastercycler epgradient thermal cycler, and involved, for COI, an initial denaturation at 958C for 2 min, followed by 36 cycles of denaturation step at 958C (45 s), annealing at 41–448C (COI) or 48–538C (16S rRNA) (1 min) and elongation at 728C (90 s). The final elongation step at 728C (4 min) and a rapid thermal ramp for 48C were applied to finalize the process. The double-stranded PCR products were visualized by agarose-gel electrophoresis (1.5% agarose), cleaned up with 2 ml of diluted (1:3) ExoSAP-IT (USB Corp., Cleveland, OH, USA) in a volume of 22 ml PCR product and performed at 378C for 30 min followed by enzyme inactivation at 808C for 15 min. Sequencing reactions were performed in a 10-ml reaction volume using 3.2 ml primer (1 mM), a 1 ml of ABI BigDye Terminator v. 3.0 (Applied Biosystems), 0.5 ml BigDye 5 Sequencing Buffer (Applied Biosystems) and 3.3 ml of cleaned PCR product. The sequencing reaction, performed by using the thermal cycler described above, involved an initial denaturation step for 3 min at 958C, 25 cycles (958C for 10 s, 508C for 5 s and 608C for 4 min) and a rapid thermal ramp to 48C. The BigDye-labelled PCR products were cleaned using Performa DTR Plates (Edge Biosystems, Gaithersburg, MD, USA). The chromatograms were visualized, edited and assembled using Sequencher (Gene Codes Corporation#1991–2006). External primers were cropped and discarded from the edited sequences. Subsequently, the sequences were edited using the sequence alignment editor Se-Al v. 2.0a11. The same program was used to translate COI sequences to ensure that stop codons were not present. The sequences generated in this study have been deposited in GenBank under accession numbers HQ386632–HQ386679. Using Siphonaria concinna G.B. Sowerby I, 1824 (GenBank accession numbers EF489378.1 and EF489300.1 for COI and 16S rRNA, respectively), S. serrata (Fischer, 1807) (GenBank accession numbers EF489380.1 and EF489302.1) and the western Atlantic species S. alternata Say, 1826 (new data for voucher MCZ DNA105833; GenBank accession numbers HQ386678 and HQ386679) as outgroups we performed a maximum-likelihood (ML) phylogenetic tree search. Phylogenetic analyses were conducted in RAxML 7.0.4 (Stamatakis, 2006). Nodal support was estimated via bootstrapping (1,000 replicates) (Stamatakis, Hoover & Rougemont, 2008). Because COI and 16S rRNA can be considered part of the same locus, all analyses were conducted for both genes independently and as a concatenated dataset. The

한국산 붉바리 집단에서 유전적 구조와 계통 유연관계를 mtDNA COI 유전자 서열의 다형성을 이용하여 조사하였다. COI 유전자 서열을 결정하였고 기존에 보고된 서열들과 비교하였다. 본 연구를 통해 결정된 COI 서열들은 기존에 보고된 EF607565에 대하여 99.1-99.8%의 동일성을 나타내었다. 전체 20가지의 haplotype들이 발견되었고, 한국산 붉바리 집단은 19가지의 haplotype을 나타내었다. 이들 중 Hap_03과 Hap_08은 각각 제주도와 중국-특이적인 COI 서열들을 보였다. 반면, Hap_07은 한국에서 채집된 시료들과 홍콩과 대만에서 보고된 기록 등 여러 COI 서열들을 포함하였다. COI haplotype들의 다형성에 근거한 계통 유전학적 분석을 통해 작성된 NJ tree는 Epinephelus 속 내에서 단계통적인 분지양상을 나타내었고, 이는 붉바리 집단들이 공통의 모계 선조에서 진화한 것임을 나타내었다. 또한 중국해에서 보고된 COI 서열만을 포함하였던 Hap_08은 NJ tree의 중앙부에서 위치하였고, Hap_07의 서열들과도 근연의 관계임을 보여주었다. 이 결과는 중국산 붉바리 역시 동아시아의 다른 집단들과 모계적으로 연관되어있음을 보여주었다. 결과적으로, 동아시아 붉바리 집단들은 모계적으로 연관되어있을 뿐만 아니라 공통의 진화 역사를 공유하고 있으며 여전히 동아시아 해류(Kuroshio 해류)에 의해 영향을 받는 집단이라고 할 수 있다. 본 연구는 붉바리의 유전적 구조와 계통 유연관계를 이해하는 데 도움을 줄 수 있으며, 인공증식과 산업화에 관련된 연구에 있어 중요한 역할을 담당할 것으로 기대된다. The genetic structure and phylogenetic relationship were investigated in Korean red spotted grouper populations using the nucleotide sequence polymorphisms of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) gene. The COI gene was sequenced showed 99.1-99.8% identity with the EF607565 sequence previously reported. A total of twenty haplotypes were found, and the Korean population showed nineteen haplotypes. Among those, Hap_03 and Hap_08 showed Jeju-do and China-specific COI sequences, respectively. However, Hap_07 had twelve COI sequences from South Korea and records from Hong Kong and Taiwan. Neighbor-joining (NJ) trees constructed from the phylogenetic analyses based on the polymorphisms of the COI haplotypes showed a monophyletic branching pattern within the genus Epinephelus. This indicated that the red spotted grouper populations had evolved from common maternal ancestors. In addition, the Hap_08, which had the COI sequence recorded only from China Sea, was found in the middle of the NJ tree nearby Hap_07 and showed a close relationship with Hap_07. This indicates that Chinese red spotted grouper is also maternally related to other populations in East Asia. Consequently, East Asian red spotted grouper populations are maternally related, as well as sharing the same evolutionary history, and are still affected by the East Asian ocean current (Kuroshio). These findings help to explain the genetic structure and phylogenetic relationship of red spotted grouper and also contribute to research on artificial breeding and industrialization.

A fine-scale phylogenetic and phylogeographic analysis of Peripatopsis lawrencei s.l. was conducted with both mitochondrial and nuclear DNA sequence data, using both external morphology and scanning electron microscopy of taxonomically important characters. A total of 119 sequences were used for the mitochondrial cytochrome c oxidase subunit I (COI ) whereas a single representative specimen from each locality was sequenced for the nuclear 18S rRNA locus. Phylogenetic analyses were conducted on the total COI data set and the combined COI + 18S rRNA data set using a Bayesian analysis and maximum likelihood analyses. For the combined DNA sequence data set, a divergence time estimation was further undertaken in BEAST and specimens placed in a phylogenetic framework including all the described Peripatopsis species from South Africa. In addition, a phylogeographic study was conducted exclusively on P. lawrencei s.s. (clade A) using an analysis of molecular variance and haplotype network. Phylogenetic results indicated that, at the Oubos sample locality, two highly distinct genetic lineages were present (clades A and B), whereas a divergence time estimation suggests a Miocene cladogenesis of the novel Oubos lineage. Marked phylogeographic structure was observed for P. lawrencei s.s. (restricted to clade A) across the distribution range with limited maternal dispersal. Morphologically, the two sympatric lineages at Oubos A and B differed in leg pair number, ventral colour and dorsal scale rank counts, as evident from scanning electron microscopy. Our results support the recognition of a distinct species that occurs in sympatry with P. lawrencei s.s. The new species, P. aereus sp. nov. (clade B) is described and the implication for fine-scale taxonomic studies on saproxylic taxa is discussed. ZooBank: urn:lsid:zoobank.org:pub:AB6E0BDA-7B5F-4FD3-A863-BA7C814E278C.

Metabarcoding extra‐organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci – COI, cytochrome b, 12S and 16S – in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs – three COI and one 12S – for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra‐organismal DNA at low template concentrations, both metazoan‐targeted and fish‐targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non‐specific amplification of prokaryotic and non‐target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome‐skimming existing tissue collections.