Research Article |
Corresponding author: Qian Wang ( wqgt1999@163.com ) Academic editor: Fabio Laurindo da Silva
© 2020 Hai-Jun Yu, Xiao-Long Lin, Rui-Lei Zhang, Qian Wang, Xin-Hua Wang.
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:
Yu H-J, Lin X-L, Zhang R-L, Wang Q, Wang X-H (2020) Species delimitation and life stage association of Propsilocerus Kieffer, 1923 (Diptera, Chironomidae) using DNA barcodes. ZooKeys 975: 79-86. https://doi.org/10.3897/zookeys.957.54668
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The utility of COI DNA barcodes in species delimitation is explored as well as life stage associations of five closely related Propsilocerus species: Propsilocerus akamusi (Tokunaga, 1938), Propsilocerus paradoxus (Lundström, 1915), Propsilocerus saetheri Wang, Liu et Paasivirta, 2007, Propsilocerus sinicus Sæther et Wang, 1996, and Propsilocerus taihuensis (Wen, Zhou et Rong, 1994). Results revealed distinctly larger interspecific than intraspecific divergences and indicated a clear “barcode gap”. In total, 42 COI barcode sequences including 16 newly generated DNA barcodes were applied to seven Barcode Index Numbers (BINs). A neighbor-joining (NJ) tree comprises five well-separated clusters representing five morphospecies. Comments on how to distinguish the larvae of P. akamusi and P. taihuensis are provided.
barcode gap, bioindicator, chironomid, COI, genetic distance, larval association, Propsilocerus taihuensis
The genus Propsilocerus Kieffer, 1923 (Fig.
All four common species, Propsilocerus akamusi (Tokunaga, 1938), Propsilocerus paradoxus (Lundström, 1915), Propsilocerus sinicus Sæther et Wang, 1996, and Propsilocerus taihuensis (Wen, Zhou et Rong, 1994) are present in Yuqiao Reservoir, Jizhou Distinct, Tianjin, China during the spring and autumn. As a result, larvae of these four species usually have been misidentified as Propsilocerus akamusi by ecologists in China.
DNA barcodes (
Here we explore the utility of DNA barcodes in species delimitation and in associating life stage in Propsilocerus. Registering new barcodes of Propsilocerus species will improve the reference library of Chironomidae (
In this study, 42 specimens of five Propsilocerus species (P. akamusi, P. paradoxus, P. saetheri, P. sinicus, and P. taihuensis) from China, Japan, Norway, and South Korea with COI barcodes were included. Twenty-six specimens with public COI barcodes were retrieved from BOLD and GenBank, and an additional 16 individuals of four Propsilocerus species were collected from the eutrophic lakes and reservoirs from Hebei Province, Shanghai and Tianjin, China, using D-nets, sweep nets, and light traps.
Larvae were preserved in 95% ethanol, adults in 85% ethanol, and stored at 4 °C in the dark before morphological and molecular studies. Photographs of all intact specimens were taken before dissection using a ZEISS camera mounted on a ZEISS stereomicroscope using the software AxioVision Rel. 4.8. at the College of Life Sciences, Nankai University, Tianjin, China. Digital photographs of slide specimens were taken at 300-dpi resolution using a Nikon Digital Sight DS-Fi1 camera mounted on a Nikon Eclipse 80i compound microscope.
Extraction of genomic DNA was done following the standard protocol of the Qiagen DNeasy Blood & Tissue Kit, except the volume of DNA template was 110 μl in the final step. Morphological terminology used in this work is according to
DNA amplifications of COI barcode sequences with the universal primers LCO1490 and HCO2198 (
Raw sequences were edited and assembled in SeqMan version 7.1.0 (in the LaserGene package, DNASTAR, Madison, USA), aligned using the Muscle algorithm (
In general, the data showed distinctly larger interspecific than intraspecific divergence, and there was a clear “barcode gap” in the pairwise K2P distances (Fig.
The neighbor-joining tree (Fig.
Although it is feasible to distinguish species of Propsilocerus by referring to the works of
It was also discovered that the undescribed Nearctic larva (
Head capsules of Propsilocerus akamusi (Tokunaga, 1938) and Propsilocerus taihuensis (Wen, Zhou & Rong, 1994) A head capsule of P. akamusi, ventral view B head capsule of P. taihuensis, ventral view C mandible of P. akamusi D mandible of P. taihuensis E antenna of P. akamusi F antenna of P. taihuensis G premento-hypopharyngeal complex of P. taihuensis. Scale bar: 100 µm (A, B), 50 µm (C, D), 25 µm (E, F).
Our study has revealed strong concordance between morphospecies and DNA barcodes of Propsilocerus. Distinct “barcode gaps” were discovered among Propsilocerus species. DNA barcodes have been used to associate different life stages, and the unidentified species (Propsilocerus sp. JC-2015) was confidently assigned to P. taihuensis. Comments on how to distinguish this species from congeners on the larvae of P. taihuensis are given.
We thank the following people who have kindly sent us specimens for our study: Dr Wen-Bin Liu (Tianjin Normal University, Tianjin, China) and Tao Zheng (Tianjin Agricultural University, Tianjin, China). Financial support from the National Natural Science Foundation of China (31672264, 31301908, 31900344) and the China Postdoctoral Science Foundation Grant (2018M640227) are acknowledged with thanks.