Computational protocol: Eleven nominal species of Burmoniscus are junior synonyms of B.kathmandius (Schmalfuss, 1983) (Crustacea, Isopoda, Oniscidea)

Similar protocols

Protocol publication

[…] A single topotypic (or near-topotypic) material of the eleven species was used for molecular analysis, but a single specimen for Burmoniscus aokii and Burmoniscus boninensis was used. Total DNA was extracted from leg muscle using the Qiagen DNeasy Blood and Tissue Kit, according to the manufacturer’s protocol (Qiagen, Germany). Parts of the mitochondrial cytochrome c oxidase subunit I (COI) and mitochondrial 12S and 16S ribosomal RNA (rRNA) genes were amplified by polymerase chain reaction (PCR) using the following primers: LCO1490 and HCO2198 () for the COI region, 12Sai and 12Sbi () for the 12S rRNA region, and 16Sar and 16Sbr () for the 16S rRNA region. If the 16S rRNA region could not be amplified using these primers, a different primer, 16Sar-int-sf (), was used instead. PCR was carried out in 20-µl reaction volumes with Ex Taq (Takara Bio, Japan). The cycle program comprised an initial denaturation step at 94°C for 3 min followed by 30 cycles of 1 min at 94°C, 1 min at 44–48°C, and 1 min at 72°C, and finally a 7-min extension at 72°C. PCR products were purified using a illustra ExoProStar (GE Healthcare Japan Corp., Japan) and directly sequenced by Macrogen Japan (Japan) using the same primer sets used for PCR. Burmoniscus sp., Burmoniscus meeusei (Holthuis, 1947), Burmoniscus dasystylus Nunomura, 2003, Burmoniscus ocellatus (Verhoeff, 1928), and Ligidium ryukyuensis Nunomura, 1983 from Japan were also added to the molecular analysis, the last as an outgroup. No material Burmoniscus kathmandius from Nepal was available. Sample details and accession numbers are given in Suppl. material .cytochrome c oxidase subunit I ribosomal RNA polymerase chain reaction The sequences were aligned using the default settings in MUSCLE 3.5 () at SeaView 4 (). Gaps were excluded from subsequent analyses. Maximum Likelihood (ML) analysis was performed using RAxML Version 8 (). The best-fit models of sequence evolution for both gene and codon, as determined by the Akaike Information Criterion correction (AICc) in the program KAKUSAN 4 (), were partitioned equal-mean-rate models. Bootstrap support was assessed using 1000 replicates. Genetic distances were calculated as p-distances using MEGA 6 ().Maximum Likelihood Akaike Information Criterion correction […]

Pipeline specifications

Software tools MUSCLE, SeaView, RAxML, MEGA
Application Nucleotide sequence alignment