On the genus Polypedilum, subgenus Collartomyia, with description of P. (Col.) baishanzuensis sp. nov. from Baishanzu Nature Reserve, China (Diptera, Chironomidae)

Abstract A new species of the genus Polypedilum Kieffer, 1912 is described from Baishanzu Nature Reserve, China, based on molecular and morphological data. Molecular phylogenetic analysis based on standard barcode sequences confirmed a new clade of Polypedilum (Collartomyia) species. The new species is easily distinguished from its congeners by a combination of the following morphological characters: membrane of wing with a large spot occupying 70% of the proximal area; tergite without dark brown band pigmentation; tarsi I–V dark brown; superior volsella with three outer lateral setae and six long setae along inner base; inferior volsella with setose tubercules. An updated key to adult males of the subgenus Collartomyia is also provided.

DNA barcoding provides an effective and quick tool for species identification and delimitation, and has been proven successful in many different kinds of animals (Herbert et al. 2003). Chironomid researchers around the world have uploaded 3,310 species including 599,223 sequences in the Barcode of Life Database (BOLD) before 16 June, 2021. Barcode sequences are becoming a necessary character for chironomid species identification and new species descriptions (Song et al. 2016(Song et al. , 2018Lin et al. 2018Lin et al. , 2020Makarchenko et al. 2020;Qi et al. 2020).
Baishanzu National Nature Reserve is located in the south Zhejiang and north Fujian provinces of China; this region is well known for its high level of biodiversity and hot spots in Asia. It belongs to the tropical to warm temperate transitional zone. During field surveys in Baishanzu Nature Reserve, an unknown species of the genus Polypedilum were collected. Molecular data and morphological comparisons supported it as an undescribed taxon that we describe herein as a new species.

Material and methods
The examined material was collected by light trap and then preserved in 75% ethanol at 4 °C in a refrigerator before final slide mounting. Tissues for total genomic DNA extraction were removed from the thorax and head of the adults. The extraction procedure followed the Qiagen DNeasy Blood and Tissue kit guide except for the use of an elusion buffer quantity of 120 µl. After extraction, the exoskeletons were cleared and mounted on corresponding slides following the procedure described by Saether (1969). Morphological terminology follows that of Saether (1980). The photograph of the dorsal habitus was obtained with a DV500 5MP Digital Camera attached to a stereo microscope (Chongqing Optec SZ680). The photograph of the body parts was obtained using a Leica DMLS compound microscope. Photograph post-processing was done in Adobe photoshop and Illustrator (Adobe Inc., California, USA). The standard barcode region of COI-5P was amplified using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR amplifications were carried out 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 deionized H 2 O following Song et al. (2018). PCR products were electrophoresed in 1.0% agarose gel, purified, and sequenced in both directions using an ABI 3730XL capillary sequencer (Beijing Genomics Institute Co., Ltd., Hangzhou, China). Raw sequences were assembled into contigs and edited in BioEdit 7.2.5 (Hall 1999). The pairwise distances were calculated using the Kimura 2-Parameter (K2P) substitution model in MEGA 7 (Kumar et al. 2016). The neighbor joining tree was constructed using the K2P substitution model, 1,000 bootstrap replicates and the "complete deletion" option for missing data. Sequences, trace-files, and metadata of the new species were uploaded to the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2013).

Barcode analysis
The species was primarily blasted in GenBank and molecularly confirmed as a species of Polypedilum. Morphological characters support it belonging to the subgenus Collartomyia. All ten species with public COI sequences of P. (Collartomyia) species were used to construct the neighbor-joining tree based on COI barcode sequences and a distinct genetic branch suggests that our specimen belongs to a species new to science (Fig. 1). The minimum interspecific genetic distance within the subgenus Collartomyia is up to 14.8% divergence in partial COI sequences (Table 1), larger than the 5-8% threshold suggested by Song et al. (2016Song et al. ( , 2018 The holotype is deposited in the collection of the College of Life Sciences, Taizhou University, Taizhou, China (TZU).
Diagnostic characters. The male adult can be distinguished from other P. (Collartomyia) species by the following combination of characters: most of the body yellowish; wing with distinct spots on 70% of the proximal part; tarsomeres dark brown; tergite without dark brown band pigmentation; superior volsella with six inner basal setae and three outer lateral setae; dorsal side of inferior volsella with three distinct setiferous tubercles.
Etymology. The specific name refers to the Baishanzu National Nature Reserve, where the holotype was collected.
Immatures and female unknown. Ecology. Material composed of male adults was light-trapped from stones and boulders in a flowing mountain stream (Fig. 6), located at an altitude of 1,450 m.
Distribution. Only known from the type locality in Zhejiang province, China.

Discussion
The morphological characters of the well-developed gonocoxite bulb of the new species clearly fit the subgeneric definition by Tang et al. (2021) and Saether and Sundal (1998