Molecular Phylogenetic Analysis of the Main Lineages of Nymphalinae(Nymphalidae: Lepidoptera) Based on the Partial Mitochondrial COI Gene

The phylogenetic relationships of the subfamily Nymphalinae （sensu Chou 1994） were analyzed based on 1 488 bp of mtDNA cytochrome oxidase subunit I （COI） gene sequence data obtained from 24 individuals, along with those of eight species obtained from GenBank. The base compositions of this COI fragment varied among the individuals as follows： T 39.9%, C 14.6%, A 32.2%, and G 13.4%, with a strong AT bias （72.1%）, as usually found in insect mitochondrial genomes. The A ＋T contents of the third, second, and first codon positions of the COI fragments in this study was 92.4, 62.2, and 61.4%, respectively. The phylogenetic trees were reconstructed by neighbor-joining （NJ）, maximum likelihood （ML）, and Bayesian methods by using Byblia anvatara as outgroup. Phylogenetic analyses based on the COI gene sequence data created very similar topologies, which were producing trees with two main clades A and B, and five subclades. The data indicated that the tribes Nymphalini and Hypolimni （sensu Chou 1994） are not monophyletic groups, and the genus Junonia should be removed from Nymphalini to Hypolimni （=Junoniini）. On the basis of the data, the Symbrenthia and Araschnia had a relative distant relationship with the rest of Nymphalini. The relationships of species in the Nymphalini were confirmed via the NJ, ML, and Bayesian methods, namely （（（（Nymphalis ＋ Kaniska） ＋ Polygonia） ＋Aglais） ＋ Vanessa） ＋ （Symbrenthia ＋Araschnia）. This investigation provides a little novel information for Chinese researches of butterflies.

Genetic difference of Chinese horseshoe crab(Tachypleus tridentatus) in southeast coast of China based on mitochondrial COI gene analysis

Population genetic structure and historical demography of Chinese horseshoe crab (T.tridentatus)along southeast coast of China were inferred from the sequence data of mitochondrial cytochrome c oxidase subunit Ⅰ (COI) fragment.The sequence analysis for 964 bp COI fragment was conducted in 28 individuals collected from five localities:Ninghai in Zhejiang Province,Meizhou and Zhangpu in Fujian Province,Beihai of Guangxi Zhuang Autonomous Region and Danzhou of Hainan Province.Sequence variation was relatively low with a total of seven transitions observed.In all localities,Haplotype H3 was the dominant type observed among eight haplotypes defined previously,and was at the center of radiation in Median-Joining network.The prolonged star-like network suggests a signature of population expansions.The level of diversity was low in total,with haplotype diversity ( Hd) being equal to 0.765 and nucleotide diversity (π) being equal to 0.00118,respectively.The genetic structure analysis revealed the significant genetic difference between Ninghai and Danzhou populations.Both mismatch distribution analysis and Fu's Fs test provided consistent inference of historic population expansion.The low genetic diversity of horseshoe crab observed along China coast indicated that urgent measures should be taken to protect this rare marine animal.

Zeylanicobdella arugamensis,the marine leech from cultured crimson snapper (Lutjanus erythropterus),Jerejak Island,Penang,Malaysia

Objective:To investigate the prevalence,phylogenetics and DNA barcoding of Zeylanicobdella arugamensis(Z.arugamensis) from crimson snapper(Lutjanus erythropterus),Jerejak Island,Penang,Malaysia.Methods:Experiment was conducted with 200 fish specimens of cultured Lutjanus erythropterus from Jerejak Island,Penang,Peninsular Malaysia.The water temperature and length for each fish were measured prior to parasites examination.Next,the morphological identification of parasites was performed.Genomic DNA from parasites was extracted for further molecular analysis.After PCR amplification,phylogenetic tree was constructed.The lowest Bayesian information criterion scores showed that the most compatible model is Tajima and Nei.Finally,data sets of cytochrome oxidase subunit I gene sequence and trace file have been submitted to Barcode of Life Data System.Results:The prevalence rate of Z.arugamensis was recorded to be 11.5%,and the intensity was 1.48.The low intensity was due to the water temperature recorded in this study(32.9–33.2 C).All the individuals of Z.arugamensis recorded in this study showed a close relationship with species that were recorded in NCBI database(Z.arugamensis DQ414344,Aestabdella leiostomi DQ414305,Pterobdella amara DQ414334 and Cystobranchus meyeri DQ414315) but less relationship with Aestabdella abditovesiculata DQ414300.Finally,the DNA sequences submitted to Barcode of Life Data System in accordance to species have already obtained Barcode Index Number as BOLD:ACM3477.Conclusions:This study has provided an overview of sequence divergence at cytochrome oxidase subunit I gene,DNA barcodes and parasite prevalence of Z.arugamensis.

A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.