A new species of Polypedilum (Cerobregma) (Diptera, Chironomidae) from Oriental China

Abstract Polypedilum (Cerobregma) huapingensis Liu & Lin, sp. nov. is described and illustrated based on an adult male from Huaping National Nature Reserve, Guangxi, China. A DNA barcode analysis, including the known partial COI sequences of species in the Cerobregma subgenus, was conducted. An updated key to adult males of the subgenus Cerobregma is provided.


Introduction
The genus Polypedilum Kieffer is one of the largest chironomid genera, containing eight subgenera and more than 520 described species (Saether et al. 2010;Cranston et al. 2016;Yamamoto et al. 2016;P. Ashe, pers. comm.). The larvae mostly occur in sediments, but some species are associated with mines of aquatic plants or co-inhabit pupal retreats of caddisflies (Cranston et al. 1989). Adult males of the subgenus Cerobregma Saether & Sundal, 1999 are characterized by having extremely long and strong, split setae along the inner margin of the gonostylus and gonocoxite, with an apicolateral bulb-like extension with deep lateral incision between the bulb and the gonostylus. The subgenus Cerobregma includes 15 valid species recorded in the Afrotropical, Holarctic and Oriental regions (Tokunaga 1940;Saether and Sundal 1999;Kobayashi et al. 2003;Zhang and Wang 2005;Zhang et al. 2006;Moubayed-Breil 2007;Tang and Niitsuma 2017;Lin et al. 2019).
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 non-biting midge species delimitation (Anderson et al. 2013;Silva et al. 2014;Lin et al. 2015;Montagna et al. 2016;Giłka et al. 2018;Lin et al. 2018;Qi et al. 2017). COI barcodes have provided important evidence to confirm new species descriptions within Polypedilum species (Song et al. 2016(Song et al. , 2018Lin et al. 2019).
The Nanling-Mountain region, located in the middle subtropical zone of China, rich in biological resources and with a warm and moist climate, is a typical natural ecosystem and one of the most biologically diverse areas in the world. Recently, during investigations of insect diversity in the Nanling Mountains, we discovered an unknown species of the subgenus Cerobregma from Huaping National Nature Reserve. In the present study, Polypedilum (Cerobregma) huapingensis Liu & Lin sp. nov. is described and delimited by its morphology and DNA barcode. An updated key to adult males of the subgenus is provided.

Materials and methods
The single specimen of the new species, collected by a Malaise trap, was 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, 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).
The color pattern of new species is described based on the specimen preserved in ethanol. Digital photographs of slide-mounted specimens were taken with a 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.
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 6 (Tamura et al. 2013). The pairwise distances using the Kimura 2-Parameter (K2P) substitution model of six species within the subgenus Cerobregma were calculated in MEGA. The neighborjoining 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 Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2013). The GenBank accession number for the new species is MW472357.
The holotype of the new species is deposited at the College of Life Sciences, Nankai University, Tianjin, China (NKU).

DNA barcode analysis
The neighbor joining tree based on COI DNA barcodes of the six sequenced species within the subgenus Cerobregma revealed six distinct genetic clusters, suggesting one species new to science (Fig. 1). In some barcode studies in Chironomidae, the average threshold intraspecific divergence is 4-5% in Tanytarsus van der Wulp (Lin et al. 2015) and 5%-8% in Polypedilum Kieffer (Song et al. 2016(Song et al. , 2018. Polypedilum (Cerobregma) huapingensis sp. nov. can be differentiated from the other sequenced species by a more than 13% divergence in the COI barcode sequence (Tab. 1; Fig. 1).  Diagnostic characters. According to the morphological characters of the adult male, the new Polypedilum species keys to the subgenus Cerobregma, and can be distinguished from other known species of the subgenus by the following combination of characters: tergites III-VI brown with dark brown spots at middle; wing pale brown with a large black spot on entire basal area of wing; superior volsella with basal microtrichia and two inner setae; anal point strong, contracted in middle, a large inflated globe apically with candle-like spine.
Adult male (n = 1). Total length 4.29 mm. Wing length 2.71 mm. Total length/ wing length 1.58. Wing length/length of profemur 1.57. Coloration (Fig. 2). Head brown. Antenna yellow. Thorax ground color brown with dark brown stripes on scutum, laterally under parapsidal suture, postnotum and on preepisternum. Tergites III-VI brown with dark brown spots at middle; tergites I, II, VII, and VIII and hypopygium largely dark brown. Most of femora and tibiae dark brown, all tarsomeres yellow. Wing pale brown with a large black spot on entire basal area.
Hypopygium (Fig. 4). Anal tergite with 46 median setae. Laterosternite IX with seven setae. Anal point as in Fig. 4D, strong, contracted in middle, as a large inflated globe apically with a single candle-like spine, tapering, 143 µm long. Transverse sternapodeme 93 µm long; phallapodeme 168 µm long. Gonocoxite 320 µm long. Superior volsella 143 µm long, with basal microtrichia and two inner setae (Fig. 4E). Inferior Discussion. The characters of the anal point and superior volsella of the new species place it within the subgenus Cerobregma. The morphology of the new species resembles that of Polypedilum heberti Lin &Wang, 2019, but it can be separated from it on the basis of the following: 1) tergites III-VI brown with dark brown spots at middle in the new species, versus tergites III-VI with dark brown bands at middle in P. heberti; 2) thorax of the new species (acrostichals 31; humerals 38; dorsocentrals 104) with much more setae than in P. heberti (acrostichals 8; humerals 5; dorsocentrals 20); 3) anal point strong and tapering in P. heberti, versus constricted in middle, with a large inflated globe apically with candle-like spine in the new species.  Updated key to known adult males of Polypedilum (Cerobregma) The following key replaces couplet 5 in Lin et al. (2019) and adds a couplet 5a to include the male of the newly described species.