Biodiversity Data Journal :
Research Article
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Corresponding author: Yongde Cui (ydcui@ihb.ac.cn)
Academic editor: Wagner Magalhães
Received: 27 Aug 2021 | Accepted: 08 Dec 2021 | Published: 14 Dec 2021
© 2021 Tingting Zhou, Wei Jiang, Hongzhu Wang, Yongde Cui
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhou T, Jiang W, Wang H, Cui Y (2021) DNA barcoding of Naididae (Annelida, Oligochaeta), based on cytochrome C oxidase gene and ITS2 region in China. Biodiversity Data Journal 9: e73556. https://doi.org/10.3897/BDJ.9.e73556
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Exploring the effectiveness of DNA barcoding in species identification is a prerequisite for biodiversity conservation and environmental monitoring. Aquatic oligochaetes could serve as excellent indicators in aquatic monitoring programmes. However, few studies have examined the effectiveness of DNA barcoding in these specific organisms. The mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to 40 species of 18 genera were sequenced in this study. The results showed that there was a barcode gap between species of Naididae and the intraspecific genetic distances of each species were smaller than interspecific genetic distances. The classification results of ABGD (Automatic Barcode Gap Discovery) were consistent with those of morphological identification, except for Tubifex tubifex and Lumbriculus variegatus. All species were successfully distinguished in the phylogenetic tree, based on the ITS2 region, which was coincident with the morphological result. Our results provided evidence that DNA barcoding can be used as an effective and convenient tool for species identification of the family Naididae and even for other aquatic oligochaetes.
aquatic oligochaete, Naididae, DNA barcoding, COI, ITS2
The family Naididae, as the most diverse family within the class Oligochaeta, includes more than 1,100 valid species (
Rapid and unambiguous identification of species is an essential prerequisite for environmental monitoring and biodiversity (
DNA-based identification methods can not only quickly and accurately identify morphologically damaged specimens (
Recent advances in developed countries have established a national barcode database of aquatic oligochaete (
In this study, we sequenced the COI sequences of 83 specimens under 40 species belonging to 18 genera to study the barcoding of Naididae. A total of 75 sequences of the ITS2 region, including 28 GenBank sequences and 47 new sequences, were used as additional evidence. We aim to explore the accuracy of DNA barcoding technology for the species identification and to construct a bio-identification system for Naididae.
The specimens were collected between 2017 and 2020 in China (Suppl. material
Total genomic DNA was extracted using the TIANGEN blood tissue kit, following the manufacturer’s protocol strictly (TIANGEN Blood and Tissue Handbook). Approximately 658 bp were amplified of the COI gene using universal primers, LCO1490-GGTCAACAAATCATAAAGATATTGG and HCO2198-TAAACTTCAGGGTGACCAAAAAATCA (
Raw sequences were calibrated in BioEdit and assembled in SeqMan (DNASTAR). All sequences were aligned by ClustalW using MEGA5. The newly-acquired COI sequences in our study were compared to Genbank (NCBI) sequences using BLAST.
All of COI sequences were imported into MEGA5 for multi-sequence alignment and base composition, conserved sites, variable sites, parsimony informative sites and transitions/transversions were calculated, respectively.
The uncorrected pairwise genetic distances between sequences were obtained with MEGA5 (
The genetic distance matrix of all Naididae specimens was calculated using the Kimura two parameter (K2P) model (
The Bayesian trees were generated using the software Phylosuite1.2.1 (
A total of 40 species belonging to 18 genera were analysed, giving altogether 83 COI sequences. Ten of them were acquired for the first time, including Tubifex laxus Peng, Wang & Cui, 2017; T. conicus He, Wang & Cui, 2012; Isochaetides palmatus He, Cui & Wang, 2012; Dero dorsalis Ferronière, 1899; Haemonais waldvogeli Bretscher, 1900; Nais simplex Piguet, 1906; N. inflata Liang, 1963; N. badia Peng, Wang & Cui, 2014; N. longidentata Cui, He, Peng & Wang, 2015 and Rhyacodrilus sinicus (Chen 1940).
The aligned length for COI gene used in the study was 658 bp and the sequences were AT-biased (60.8%). There were 337 conserved sites and 321 variable sites, of which 319 were parsimony informative. The value of transitions/transversions was 1.35.
A total of 75 ITS sequences were obtained in this study, of which 47 were successfully amplified and the remaining 28 were from GenBank. Compared with the COI gene, the ITS2 region is often difficult to sequence.
The average genetic distances between species range from 7.6% to 26.5%. Amongst them, N. longidentata and N. communis had the smallest inter-specific distances, while Limnodrilus grandisetosus and Lumbriculus variegatus had the largest.
The mean intra-specific genetic distance was 0.0-3.9% and Aulophorus furcatus was maximum. The phylogenetic tree of COI sequences, based on the neighbour-joining method (NJ) was established to visualise the genetic distances (Fig.
Overall, our results showed that there was more variation amongst species than intraspecific differences.
The partition results, based on the ABGD method, included recursive partition and initial partition and the latter was relatively stable. The COI sequences were grouped into 40 taxa (Fig.
The phylogenetic analysis of family Naididae, based on the ITS2 region was constructed by Bayesian Inference (Fig.
The core principle of DNA barcoding is that inter-specific genetic distance is greater than intra-species differences (
Sequences can be divided quickly and effectively by the ABGD method according to the principle of "DNA barcode gap" (
The phylogeny revealed that Naidinae and Tubificinae were monophyletic, whereas Rhyacodrilinae was not. This was supported by recent research (
The study represents the first DNA barcoding study of the aquatic oligochaete in China, including endemic species and common species. Our findings detected that DNA barcoding, based on the COI gene is practicable and effective in identifying aquatic oligochaetes; however, combining ITS2 would provide more information. The ITS2 region can be used to help build a barcode database (for delimitation of species), but not for biomonitoring. The taxonomic status of some species needs to be confirmed through comprehensive taxonomic research, by expanding the sampling range and increasing the number of specimens. The barcodes of oligochaete species need to be continuously studied to establish a native database so as to lay a solid foundation for the biological research of aquatic oligochaete.
We are very grateful to Junyan Wu, Yajing He, Yongjing Zhao and Tingfeng Xie (IHB, CAS), Wenqiang Ren and Xuhao Zhao (Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences) for assistance in collecting all the specimens. We also thank Zhenyuan Liu (IHB, CAS) for the help with the drawing.
This study was supported by Major Science and Technology Program for Water Pollution Control and Treatment (Grant Nos. 2017ZX07302-002) and National Natural Science Foundation of China (No. 31471962).
TTZ conceived the idea and wrote the first draft of the manuscript. TTZ and WJ performed the data analyses. YDC and HZW revised the manuscript and provided valuable suggestions. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Collection information of specimens of Naididae. The sequences from China are shown in bold. Missing data are marked with “-”. The remaining sequences are downloaded from Genbank.
Uncorrected pairwise genetic distance of COI gene for species of the family Naididae. Intra-specific distances are given as maximum distance, and intra-specific as minimum distance. Missing data are marked with “-”.