New or little-known Boreoheptagyia (Diptera, Chironomidae) in China inferred from morphology and DNA barcodes

Abstract The male adult of Boreoheptagyia zhengi Lin & Liu, sp. nov. is described and illustrated based on material collected in China. Associated morphological characteristics and reference to its DNA barcode are provided. Boreoheptagyia kurobebrevis (Sasa & Okazawa, 1992) is newly recorded from China based on both a male and female, with additional associated data on the DNA barcode of the male adult. A neighbor-joining tree based on available Boreoheptagyia DNA barcodes and a key to the adults of Boreoheptagyia from China are given.


Introduction
erected the genus Boreoheptagyia with Heptagyia rugosa Saunders, 1930 as type species by original designation. Larval populations of this genus live in cool, fast-flowing streams and other harsh environments (Thienemann 1954;Oliver 1989).
The DNA barcode corresponding to the 658-bp fragment of the mitochondrial gene cytochrome c oxidase I (COI) has been identified as the core of a global bio-identification system at the species level (Hebert et al. 2003a, b) and has proved to be useful in the delimitation of non-biting midge species and has provided important evidence to confirm new species (Anderson et al. 2013;Lin et al. 2015;Giłka et al. 2018;Song et al. 2018;Lin et al. 2019;Liu et al. 2021).
In the present study, morphology and the DNA barcode of B. zhengi Lin & Liu, sp. nov. are provided based on material collected in Yunnan Province, China. Boreoheptagyia kurobebrevis (Sasa & Okazawa, 1992) is newly recorded from China based on a male and female, the latter was associated with the male by standard DNA barcodes. DNA barcode analysis including the partial COI sequences of species of genus Boreoheptagyia is conducted. A key to the known adults of Boreoheptagyia from China is also given.

Materials and methods
The examined adults were preserved in 85% ethanol and stored in the dark at 4 °C before morphological and molecular analyses. Genomic DNA was extracted from the thorax and head using a Qiagen DNA Blood and Tissue Kit at Nankai University, Tianjin, China (NKU), following the standard protocol ) except for the final elution volume of 100 µl. After DNA extraction, the exoskeleton of each specimen was mounted in Euparal on a microscope slide together with the corresponding wings, legs, antennae and abdomen, following the procedures outlined by Saether (1969). Morphological terminology follows Saether (1980). Digital photographs of the mounted specimens were taken at 300-dpi resolution using a Nikon Digital Sight DS-Fil camera mounted on Nikon Eclipse 80i compound microscope using the software NIS-Elements F v.4.60.00. at the College of Life Sciences, Nankai University, Tianjin, China (NKU).
The universal primers LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify the standard 658-bp mitochondrial COI barcode region. Polymerase chain reaction (PCR) amplifications followed Song et al. (2018) and were conducted in a 25 µl volume including 12.5 µl 2× Es Taq MasterMix (CoWin Biotech Co., Beijing, China), 0.625 µl of each primer, 2 µl of template DNA and 9.25 µl of deionized H 2 O. PCR products were electrophoresed in 1.0% agarose gel, and purified and sequenced in both directions at Beijing Genomics Institute Co., Ltd., Beijing, China.
Raw sequences were assembled and edited in Geneious Prime 2020 (Biomatters Ltd., Auckland, New Zealand). Alignment of the sequences was carried out using the MUSCLE algorithm (Edgar 2004) on amino acids in MEGA X (Kumar et al. 2018). The pairwise distances using the Kimura 2-Parameter (K2P) substitution model of ten species within the genus Boreoheptagyia were calculated in MEGA. The neighbor-joining tree was constructed using the K2P substitution model, 1000 bootstrap replicates and the "pairwise deletion" option for missing data in MEGA. Novel sequence, trace-files, and metadata of the new species are uploaded to the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2013). GenBank accessions of the Chinese specimens are list in Table 1. The holotype of the new species and other examined specimens are deposited in the collection of the College of Life Sciences, Nankai University, Tianjin, China.
Female and immature stages unknown. Etymology. The species is named 'zhengi' after Prof. Le-Yi Zheng, for his outstanding contribution to the knowledge of insect taxonomy in China; noun in nominative case.

Discussion
Morphological characters of B. kurobebrevis from China fit well with the original description by Sasa and Okazawa (1992) and Makarchenko et al. (2008), but there are a few differences in numeric measurements: total length (2.95 mm), AR 0.82 and scutellum with 46 setae in Chinese specimen, as compared with total length 3.34 mm, AR 0.64 and scutellum with 50 setae in Japanese specimen (Makarchenko et al. 2008). The new species can be easily separated from other related members of the genus by the following combination of morphological characters found in the male adult: antenna with seven flagellomeres; wing membrane covered with macrotrichia on almost the entire surface except a bare spot near the anal lobe. Boreoheptagyia zhengi sp. nov. keys out close to B. tibetica from which it can be separated in having: 1) antenna with seven flagellomeres in B. zhengi Lin & Liu, sp. nov., whereas the latter has six flagellomeres; 2) well-developed anal lobe in the new species and the wing membrane with microtrichiae on almost the entire surface except for a bare area near the anal lobe, whereas B. tibetica has a reduced anal lobe and wing membrane with macrotrichia on the entire surface; 3) differing number of chaetae on thorax: (acrostichals 27, dorsocentrals 14 in two rows, prealars 5 in the new species) compared with (acrostichals 14, dorsocentrals 6, prealars 15-16 in B. tibetica).
The neighbor-joining tree based on COI DNA barcodes of Boreoheptagyia revealed nine distinct genetic clades (Fig. 7). The new species B. zhengi sp. nov. separates from B. brevitarsis by more than 11% divergence in COI barcodes ( Fig. 7; Table 1). In addition, there are two genetic clades of Boreoheptagyia brevitarsis (Fig. 7), indicating that a potential cryptic species or misidentification. A further integrative taxonomic study on Boreoheptagyia brevitarsis is needed when the more public vouchers are available to access.