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.

Chironominae, Collartomyia, DNA barcode, key, morphology, new species, Polypedilum, taxonomy

Polypedilum Kieffer, 1912 is the largest chironomid genus, with more than 520 known species worldwide. Its subgeneric divisions and phylogeny have always been disputable and intractable (Sæther et al. 2010; Cranston et al. 2016; Yamamoto et al. 2016; Pinho and Silva 2020; Tang et al. 2021). Only two subgenera, Collartomyia Goetghebuer, 1936 and Tripodura Townes, 1945, form certain monophyletic groups. The subgenus Collartomyia was recently recognized by Tang et al. (2021) for the species having wing with a brownish band or dark spots, a well-developed gonocite bulb, split setae usually present on inner margin of gonostylus, including the previous subgenus Cerobregma Sæther & Sundal, 1998 and the monotypic genus Yaethauma Yamamoto, Yamamoto & Tang, 2018. The subgenus now includes 21 valid species recorded in the Afrotropical, Holarctic, and Oriental regions (Sæther and Sundal 1999; Kobayashi et al. 2003; Zhang and Wang 2005; Zhang et al. 2006; Moubayed-Breil 2007; Tang and Niitsuma 2017; Yamamoto et al. 2018; Lin et al. 2019; Qi et al. 2020; Liu et al. 2021).

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, 2018; Lin et al. 2018, 2020; Makarchenko 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.

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 Sæther (1969). Morphological terminology follows that of Sæther (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).

Abbreviations used are as follows:

AR antennal ratio;

BR bristle ratio;

BV beinverhältnisse;

Cu cubitus;

Dc dorsocentrals;

Fe femur;

HR hypopygium ratio;

HV hypopygium value;

IV inner verticals;

LR leg ratio;

M media;

MCu crossvein between media and cubitus;

OV outer verticals;

Pa prealars;

Po post orbitals;

R radius;

RM crossvein between radius and media;

Ta tarsomere;

Ti tibia;

VR venarum ratio.

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 H₂O 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).

The morphological characters of the well-developed gonocoxite bulb of the new species clearly fit the subgeneric definition by Tang et al. (2021) and Sæther and Sundal (1998). The new species shows close morphological similarity with other P. (Collartomyia) species on the basis of the spotted wings, including those of P. (C.) heberti Lin & Wang, 2018 and P. (C.) huapingensis Liu & Lin, 2021, but can be clearly distinguished by tergite IX without a dark brown band or spots, tarsomeres of P1 dark brown, inferior volsella present, with three dorsal setiferous tubercles. Other differences are listed in Table 3.

The authors are grateful to financial support from the National Natural Science Foundation of China (NSFC, Grant No. 32100353, 32070481), the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY17C040001), the Science & Technology Project of Taizhou (Grant No. 1902gy23), and the Project of Biodiversity Survey in Lishui Municipality, Zhejiang Province of China.