﻿New species, a new combination, and DNA barcodes of Parachironomus Lenz, 1921 (Diptera, Chironomidae)

﻿Abstract The genus Parachironomus has a cosmopolitan distribution including 85 valid described species worldwide. Species records and studies of the genus in the Tibetan Plateau are scarce. In this study, the genus Parachironomus from China is revised and two new species, Parachironomuswangi Liu & Lin, sp. nov. and Parachironomusnankaiensis Liu & Lin, sp. nov., are described based on adult morphology and molecular data. Paracladopelmademissum Yan, Wang & Bu is placed in the genus Parachironomus as a new combination. A neighbor-joining tree was reconstructed based on all known ParachironomusCOI DNA barcodes. A key to adult males of the genus Parachironomus from China is also provided.


Introduction
The genus Parachironomus was erected by Lenz (1921) based on the characters of larvae and pupae with Chironomus cryptotomus Kieffer, 1915 as type species. Subsequently, the genus was studied by a number of authors in different life stages and geographical areas (Edwards 1929;Townes 1945;Cranston et al. 1989;Saether and Spies 2013). Larvae of Parachironomus can be found in a variety of habitats, such as standing and flowing waters, soft sediments, or within aquatic macrophytes, while others are endoor ectoparasites on snails (Orel 2017). Among the Harnischia generic group, members of Parachironomus can separated from the similar genus Demicryptochironomus Lenz by the extended superior volsella with several distal setae always arising from distinct pits, and the inferior volsella with a pointed or blunt caudal projection (Yan et al. 2015). To date, 85 valid species have been reported worldwide (Lehmann 1970;Ashe and Cranston 1990;Kikuchi and Sasa 1990;Oliver et al. 1990;Saether et al. 2000;Wang 2000;Sasa and Tanaka 2001;Makarchenko et al. 2005;Spies 2008;Trivinho-Strixino et al. 2010;Yan et al. 2015;Orel 2017).
The DNA barcodes corresponding to the 658-bp fragment of the mitochondrial gene cytochrome c oxidase I (COI) has been identified as the core of a global bioidentification system at the species level (Hebert et al. 2003a, b). DNA barcodes also 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. 2015Lin et al. , 2021Giłka et al. 2018;Liu et al. 2021).
The Tibetan Plateau is located in southwest China, with a vast territory and diverse terrain. The Tibetan Plateau is one of the most important areas of biodiversity in the world because of its unique environmental and regional units, which breed unique biological communities and many unique and rare wild animals and plants. Some interesting species were discovered during the investigations of insect diversity in the Tibetan Plateau. In this paper, one new combination and two new species are proposed and described. The partial COI sequences of species in Parachironomus with DNA barcode analysis is conducted. A key to the known Chinese adult males of the genus is presented.

Materials and methods
The examined specimens were caught using sweep net and light trap, stored in the dark at 4 °C, and preserved in 85% ethanol before molecular and morphological analyses. Genomic DNA was extracted from the thorax and leg using a Qiagen DNA Blood and Tissue Kit at Tianjin Normal University, Tianjin, China (TJNU), 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 antennae, legs, wing, and abdomen, following the procedures outlined by Saether (1969). Morphological terminology follows Saether (1980).
The color pattern of all species is described based on the specimen preserved in ethanol. Digital photographs of slide-mounted specimens were taken with a 300-dpi resolution using Nikon Eclipse 80i with Nikon Digital Sight DS-Fil camera at TJNU.
The universal primers LCO1490 and HCO2198 (Folmer et al. 1994) were adopted 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, 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 (Edgar 2004) algorithm on amino acids in MEGA v. 7.0 (Kumar et al. 2016). Some published DNA barcodes of Parachironomus were downloaded from the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2013). Before phylogenetic analysis, nucleotide substitution saturation analysis of gene sequences was performed by DAMBE version 6 (Xia 2017). The pairwise distances were calculated using the Kimura 2-Parameter (K2P) substitution model in MEGA. The neighbor-joining (NJ) tree was constructed using the K2P substitution model, 1000 bootstrap replicates, and the "pairwise deletion" option for missing data in MEGA. Novel sequences, trace-files, and metadata of the new species were uploaded to the BOLD platform.
In this study, the partial COI sequences of Parachironomus were submitted to online ABGD web interface (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html). We used the K2P nucleotide substitution model. The prior intraspecific divergence was set at between 0.001 and 0.1. The minimum relative gap width was 1.0 and other parameters were defaulted.
The holotype of two new species is deposited at the College of Fisheries and Life Science, Shanghai Ocean University (SHOU) and College of Life Sciences, Tianjin Normal University, Tianjin, China (TJNU).

DNA barcode analysis
In this study, five COI sequences were obtained, and 19 COI sequences of Parachironomus were downloaded from BOLD, totaling 24 COI sequences. All sequences could be translated successfully into amino acids without indels and stop codons. MEGA analysis showed that the average total length of the sequence was 658 bp, and that there were 427 conserved sites, 231 variable sites, 196 parsimony informative sites, and 35 singleton sites. The mean nucleotide base compositions were 27.1% A, 17.9% C, 16.2% G, and 38.8% T for COI genes ( Table 1). The ratio of A + T was 65.9%, which was significantly higher than that of G + C (34.1%), showing obvious AT bias, which was consistent with the bias of base composition of mitochondrial genes in most insects.
The neighbor joining tree based on available COI DNA barcodes of the Parachironomus revealed two species new to science (Fig. 1). Parachironomus wangi Liu & Lin, sp. nov. is closer to Parachironomus biannulatus Staeger, 1839; and Parachironomus nankaiensis Liu & Lin, sp. nov. is closer to Parachironomus cayapo Spies, Fittkau & Reiss, 1994. The new species separate from the other sequenced species by more than 11% divergence in the COI barcode sequences (Table 2).
When the interspecific genetic distance is greater than the intraspecific genetic distance, barcode gaps will appear through the frequency histogram of genetic data. There is an obvious barcode gap in the genetic distance of all Parachironomus COI sequences, which fully confirms the feasibility of COI as a DNA barcode (Fig. 2).   0.13 0.09 0.14 0.14 P. gracilior|MZ624787 0.13 0.12 0.14 0.13 0.10 P. siljanensis|KC250820 0.14 0.10 0.13 0.15 0.11 0.11
Coloration. Thorax yellowish brown with pale brown spots. Femora and tibiae of front legs yellowish green with distal parts brown, anterior 1/2 of tarsi I yellowish green, remainder of front legs dark brown; femora, tibiae and basal 1/2 of tarsi I of mid and hind legs yellowish green, remaining dark brown. Abdomen yellowish green to dark brown, with tergites I-V yellowish green, tergites VII, VIII, and hypopygium dark brown.
Coloration. Thorax yellowish brown with pale brown spots. Front legs dark brown; femora and basal 1/3 of tarsi I of mid and hind legs yellowish brown, remaining dark brown. Abdomen pale yellow to yellowish brown, with tergites I-VI pale yellow, tergites VII, VIII, and hypopygium yellowish brown.
Etymology. Name after Prof. Xin-Hua Wang, for his outstanding contribution towards increasing our knowledge of aquatic insect taxonomy; noun in nominative case.
Distribution. China (Xizang). Diagnostic characters. The species can be distinguished from known species of Parachironomus by the following combination of characters: frontal tubercles small; squama with seven setae; anal tergite bands V-shaped, separated; superior volsella narrower at base, curved and expanded in the distal part, with a bare lamellar projection as wide as apex of volsella; inferior volsella reaching slightly beyond anal tergite margin; gonostylus slender, slightly curved in the middle, tapered to the apex.
Hypopygium (Figs 4C,F,I,7B,C). Tergite IX with 14 setae at base of anal point and shoulder-like posterior margin of anal tergite. Laterosternite IX with three setae. Anal point originating from caudal margin of anal tergite, almost parallel-sided, moderately expanded at apex, 50 µm long, 9 µm wide at base, 10 µm wide apically. Anal tergite bands V-shaped, separated (Fig. 4F). Phallapodeme 80 µm long. Transverse sternapodeme 59 µm long. Superior volsella narrower at base, curved and expanded in the distal part, with a bare lamellar projection as wide as apex of volsella, 80 µm long, 22 µm wide at apex; and bearing six long setae at apex, free microtrichia. Inferior volsella with fairly blunt caudal projection, reaching slightly beyond anal tergite margin,

Discussion
In this study, the holotype of Paracladopelma demissum were examined, and the original description has been modified. The distinguishing feature of Parachironomus are that the superior volsella usually has a distinct preapical tooth as well as setae arising from distinct pits (Yan et al. 2012;pers. comm. Martin SpiesJuly. 2022). We re-checked the holotype, and the characters of tergite IX with shoulder-like posterior margin, superior volsella slender and with 2 distal setae arising from distinct pits, inferior volsella with blunt or pointed caudal projection conform to the characters of Parachironomus; therefore, Paracladopelma demissum should be placed in Parachironomus. Parachironomus demissum comb. nov. resembles Parachironomus digitalis Edwards, 1929 in having similarly shaped tergite IX, anal point and superior volsella, but the antenna ratio and some other measurements are different. Parachironomus wangi Liu & Lin, sp. nov. resembles Parachironomus biannulatus Staeger, 1839 in having similar shapes of the superior volsella and posterior margin of tergite IX, but can be separated by the following combination characters: AR 1.54-1.55, anal point parallel-sided and gonostylus expanded apically in P. wangi Liu & Lin,sp. nov,, the anal point is constricted in the middle, and the gonostylus is tapered to the apex in P. biannulatus.
Parachironomus nankaiensis Liu & Lin, sp. nov. resembles Parachironomus cayapo Spies, Fittkau & Reiss, 1994 in having similar shapes of anal point, inferior volsella, and anal tergite bands, but can be separated from the latter by the following combination characters: squama seven setae, the superior volsella expanded in the distal part and with a bare lamellar projection, plus with the gonostylus tapered to the apex. In contrast, the squama of P. cayapo is bare, the superior volsella is not widened in the distal part and has no projection, and the gonostylus has a protruding hump.
The results of molecular identification and morphological taxonomy are consistent, indicating that DNA barcodes and traditional morphological taxonomy are complementary in this case; therefore, the DNA barcode can be used as a simple method to supplement traditional morphological taxonomy for Parachironomus.
Biogeographically, the three species examined in this study are all distributed in the Tibetan Plateau at an altitude of more than 3,000 meters (Fig. 8). With the gradual increase of altitude, the climate gradually deteriorates, and Parachironomus living in high altitude areas have strong cold tolerance. The high biodiversity in Tibetan Plateau is demonstrated, as well as the distribution range of this genus extended.
In conclusion, this study not only enriches the database of Chironomidae in China, but also provides baseline data for the protection of the environment and biodiversity in the Tibetan Plateau.