Zhi-Teng Chen. 2019. A campaign to DNA barcode Chinese stoneflies (Insecta: Plecoptera). Illiesia, 15(08):111-117. https://doi.org/10.25031/2019/15.08 http://zoobank.org/urn:lsid:zoobank.org:pub:005BE947-9CA3-4B00-B7BD-C8F0047B32E2 Illiesia – http://illiesia.speciesfile.org Volume 15 – Number 8 – Page 111 A CAMPAIGN TO DNA BARCODE CHINESE STONEFLIES (INSECTA: PLECOPTERA) Zhi-Teng Chen School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang 212004, China. E-mail: 741208116@qq.com ABSTRACT A DNA barcode library for Chinese stoneflies would increase the speed and accuracy of species identification, help associate life stages, improve our basic understanding of the phylogeny of the group, and build a roadmap for future systematic, conservation, biodiversity, and population genetic work in the region. However, no such library exists in China. This study synthesizes all published Cytochrome Oxidase Subunit 1 (COI) sequence data available from online databases and published literature. This contribution outlines a proposal for establishing a DNA barcode library for Chinese stoneflies. Keywords: biodiversity, rapid identification, molecular database, COI gene INTRODUCTION Plecoptera is an order of hemimetabolous insects whose nymphs inhabit clean water and are the most environmentally sensitive of aquatic insects (DeWalt & Ower 2019). The accelerating urbanization of China is rapidly degrading water quality across the country. Consequently, stoneflies are also rapidly disappearing, some being lost before we find and describe them. Accurate and rapid identification of the ubiquitous aquatic insects, including stoneflies, is a vital precondition for water quality monitoring and biodiversity surveys (Macher et al. 2016). However, the traditional way based on morphology is time-consuming, requiring professional taxonomic skill (Zhou et al. 2009). DNA barcoding is a well-developed technique that uses a short fragment of the Cytochrome Oxidase Subunit 1(COI) gene for species delimitation (Hebert et al. 2003). Three main functions of DNA barcodes have been used in Plecoptera, including rapid identification, association of different life stages, and population genetics (DeWalt 2011, Mynott 2015, Boumans et al. 2016). Many entities have conducted studies of DNA barcodes of stoneflies and established DNA barcode libraries (Zhou et al. 2009, Gattolliat et al. 2016, Morinière et al. 2017). However, in the Barcode of Life Data System (BOLD), a global library for DNA barcode sequences, most sequences originate from Europe, North America, Japan and New Zealand; very few sequences are from China (Fig. 1). This data gap hinders the ability of Chinese scholars to advance the study of stoneflies in a number of scientific endeavors. Chinese researchers must begin to erase this deficit if we are to document and protect our fauna and the habitats on which they depend. In this study, I have summarized and accumulated all available COI sequences from Chinese Zhi-Teng Chen. 2019. A campaign to DNA barcode Chinese stoneflies (Insecta: Plecoptera). Illiesia, 15(08):111-117. https://doi.org/10.25031/2019/15.08 Illiesia – http://illiesia.speciesfile.org Volume 15 – Number 2 – Page 112 Fig. 1. Barcoding data sources in Genbank and BOLD databases. stoneflies from Genbank, BOLD, and published literature. I herein propose a collaborative campaign to DNA barcode all stonefly species in China. MATERIALS AND METHODS Public COI sequences were downloaded from Genbank (https://www.ncbi.nlm.nih.gov/) and the BOLD System (http://www.boldsystems.org/). All obtained sequences were temporarily stored on a server in the Insect Collection of Jiangsu University of Science and Technology (ICJUST), Jiangsu Province, China. In next steps, these data and newly obtained sequences will be submitted to the project entitled “DNA barcodes of Chinese Plecoptera” in the BOLD system, along with all relevant information and images of sequenced specimens. RESULTS AND DISCUSSION The currently available COI sequences of Chinese stoneflies are mostly obtained from partial or whole mitochondrial genomes. Only a few studies from China have combined the molecular data with morphological characters (Li et al. 2017, Chen 2019). Approximately 658 stonefly species representing 67 genera and 10 families are known from China (DeWalt & Ower 2019, Yang et al. 2015, Yang & Li 2018). Only 36 species of 26 genera have COI sequences (Table 1, Fig. 2). Perlidae, Nemouridae and Leuctridae have relatively more available COI sequences, whereas Pteronarcyidae and Taeniopterygidae have not been sequenced (Fig. 3). The number of COI sequences for each family is found to have a positive correlation with the number of species in each family, suggesting that availability of specimens has more to do with which species are sequenced rather than the existence of a larger objective. Previous molecular contributions to Chinese stoneflies, especially the mitochondrial genomic contributions made mainly by Drs. Yu-Zhou Du, Wei-Hai Li and Ding Yang and students, have contributed a lot to the taxonomy of the Plecoptera of China (Table 1). However, no comprehensive DNA barcode study has been considered or conducted for Chinese stoneflies, the reason for the campaign proposed in this study. The limited DNA barcode sequences and metadata are all deposited in Genbank and BOLD, but only some of them are available for public use. A new BOLD campaign to build a Zhi-Teng Chen. 2019. A campaign to DNA barcode Chinese stoneflies (Insecta: Plecoptera). Illiesia, 15(08):111-117. https://doi.org/10.25031/2019/15.08 Illiesia – http://illiesia.speciesfile.org Volume 15 – Number 2 – Page 113 Fig. 2. Percentage of Chinese species, genera, and species within families that have been barcoded. The percentages reflect only the publicly available data from Genbank and BOLD Systems. DNA barcode library for Chinese stoneflies and other aquatic insects (especially Ephemeroptera and Trichoptera), should be established with the joint effort of researchers from China and worldwide. The remaining stonefly species known from China should be sequenced to facilitate: 1) rapid identification; 2) association of life stages; and 3) studies of evolutionary history and biogeography of Chinese stoneflies. This campaign must build a highly vetted resource that notes where voucher specimens for each sequence may be borrowed for re-examination, allows for submission of multiple taxonomically informative images that aid in verification of species identification, and that will provide open access for other researchers as data are published. All these attributes are already available in BOLD Systems in a global, collaborative platform. In the next stages, the following phases are expected to be realized with our joint efforts: 1) relevant scholars join in the new project in BOLD with full access to the database; 2) develop a jointly maintained database of freshly collected species contained within major stonefly collections in China so that a plan for collecting and sequencing may be advanced; 3) sequence at least five individuals from across the range of each species; 4) submit all DNA sequences and photos of specimens to the new project in BOLD along with detailed geographical and biological information; and 5) analyze and publish these data to support the goals mentioned above. Please join me in this collaborative project that will help us attack taxonomic, ecological, and water quality issues with species-level resolution. The proposed work will require at least 5 years to complete. The development of a joint specimen database and barcode library will demonstrate a level of collaboration heretofore not experienced by stonefly researchers in China. This work will also push Chinese researchers to adopt museum and specimen database standards that will help preserve type and non-type specimens for the future and allow sharing of biodiversity data with the Global Biodiversity Informatics Facility (https://www.GBIF.org), increasing the visibility of Chinese studies of stoneflies and the researchers who conducted Zhi-Teng Chen. 2019. A campaign to DNA barcode Chinese stoneflies (Insecta: Plecoptera). Illiesia, 15(08):111-117. https://doi.org/10.25031/2019/15.08 Illiesia – http://illiesia.speciesfile.org Volume 15 – Number 2 – Page 114 Fig. 3. Percentage of all sequenced Chinese stoneflies relative to families in which they are classified. Table 1. Information of available DNA barcodes for Chinese stoneflies. Family Species Accession No. Reference Capniidae Capnia zijinshana Du & Chen, 2016 NC_034661 Chen & Du 2017a Mesocapnia daxingana Chen & Du, 2016 KY568983 Wang et al. 2017a Chloroperlidae Haploperla ussurica Navas, 1934 XJDQD193-18 BOLD Suwallia teleckojensis (Šámal, 1939) NC_037754 Wang et al. 2018a Sweltsa longistyla (Wu, 1938) KM216826 Chen & Du 2015 Leuctridae Paraleuctra cuihuashana Chen, 2019 MK995183- MK995195 Chen 2019 Paraleuctra orientalis (Chu, 1928) MK995196- MK995198 Chen 2019 Rhopalopsole duyuzhoui Sivec & Harper, 2008 KY304497 Li et al. 2017 Rhopalopsole longispina Yang & Yang, 1991 KY304503 Li et al. 2017 Rhopalopsole sinensis Yang & Yang, 1993 KY304499 Li et al. 2017 Nemouridae Amphinemura yao Mo, Yang, Wang & Li, 2017 NC_044749 Cao et al. 2019a Indonemoura jacobsoni (Klapálek, 1912) NC_044750 Cao et al. 2019a Indonemoura scalprata (Li & Yang, 2007) MK132455 Gamboa et al. 2019 Mesonemoura tritaenia Li & Yang, 2007 NC_044720 Cao et al. 2019a Nemoura nankinensis Wu, 1926 NC_034939 Chen & Du 2017b Nemoura papilla Okamoto, 1922 MK290826 Cao et al. 2019b Nemoura securigera Klapálek, 1907 MK132393 Gamboa et al. 2019 Zhi-Teng Chen. 2019. A campaign to DNA barcode Chinese stoneflies (Insecta: Plecoptera). Illiesia, 15(08):111-117. https://doi.org/10.25031/2019/15.08 Illiesia – http://illiesia.speciesfile.org Volume 15 – Number 2 – Page 115 Sphaeronemoura grandicauda (Wu, 1973) NC_044754 Cao et al. 2019a Sphaeronemoura hamistyla (Wu, 1962) NC_044755 Cao et al. 2019a Perlidae Acroneuria hainana Wu, 1938 KM199685 Huang et al. 2015 Agnetina brevipennis (Navás, 1912) XJDQD106-18 BOLD Caroperla siveci Li & Wang, 2013 MG677942 Cao et al. 2019c Etrocorema hochii (Wu, 1938) MK905888 Liu et al. 2019 Kamimuria chungnanshana Wu, 1938 NC_028076 Wang et al. 2015 Kamimuria klapaleki (Wu & Claassen, 1934) MN400755 Chen et al. 2019 Kamimuria wangi Sun & Du, 2012 KC894944 Qian et al. 2014 Neoperlops gressitti Banks, 1939 MN400756 Zhang et al. 2019 Sinacroneuria dabieshana Li & Murányi, 2014 MK492253 Cao et al. 2019d Perlodidae Isoperla eximia Zapekina-Dulkeit, 1975 NC_038167 Wang et al. 2018b Isoperla lunigera (Klapálek, 1923) XJDQD196-18 BOLD Skwala compacta (McLachlan, 1872) XJDQD135-18 BOLD Peltoperlidae Cryptoperla stilifera Sivec, 1995 KC952026 Wu et al. 2014 Microperla geei Chu, 1928 MN096323 Cao et al. 2019e Soliperla wanglanga Huo & Du, 2017 NC_038189 Chen & Du 2018 Styloperlidae Cerconychia sapa Stark & Sivec, 2007 MF100783 Wang et al. 2018c Styloperla spinicercia Wu, 1935 NC_034809 Wang et al. 2017b them. 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