﻿Corynoneura Winnertz species from Hunan Province, Oriental China, delineated with morphological and 16S rDNA data (Diptera, Chironomidae)

﻿Abstract The genus Corynoneura Winnertz, 1846 from Hunan Province in Oriental China is reviewed. Four new species, C.enormis Fu sp. nov., C.gibbera Fu sp. nov., C.incuria Fu sp. nov., and C.longshanensis Fu sp. nov. are described and illustrated based on adult males. Sequence data from the 16S rDNA gene were used to infer relationships between these species and complement morphological delineation. Sequences from the mitochondrial large ribosomal subunit (16S rDNA) from these species are uploaded to the National Center for Biotechnology Information (NCBI). Relationships were inferred using the Neighbor-Joining method based on 16S rDNA.


Introduction
Corynoneura was erected by Winnertz (1846) with Corynoneura scutellata Winnertz, 1846 as the type species. Fu et al. (2009) and Fu and Saether (2012) reviewed the East Asia and Nearctic members of this genus. In addition, the different life stages of species

Materials and methods
Adults were mainly collected in the habitats of small streams and lakes next to mountain forests. Adults were collected by light traps near the water body or swept from marginal vegetation beside natal aquatic sites. The specimens were preserved in 85% ethanol, and stored in the dark at 4 °C before molecular analyses. Total genomic DNA of specimens was extracted from the thorax and legs using Qiagen DNA Blood & Tissue Kit. The standard protocol of the Qiagen DNeasy Blood & Tissue Kit was used, except that the final elution volume was 100 L due to the small specimen size. PCR amplification of the mitochondrial 16S ribosomal RNA gene was carried out with the primers and temperature regimes given in Ekrem et al. (2010). After DNA extraction, the clear exoskeleton was washed with 96% ethanol and mounted in Euparal on microscope slides together with the corresponding antennae, head, wings, and legs following the procedure outlined by Saether (1969). Morphological nomenclature follows Saether (1980). Measurements are given as ranges followed by the mean, when three or more specimens were measured. The specimens examined in this study are deposited at the College of Biology and Agricultural Resources, Huanggang Normal University (HNU), Huanggang, China.
Abbreviations used in text as follows:
Measurements and ratios of hind tibia follow Schlee (1968) as follows: Length of ventral elongation; c 1 Length of strong broad part, measured from apex; c 2 Total length of broadening; d Width of tibia basally to the apical broadening. Diagnosis. The male imago is characterized by having an antenna with eight flagellomeres, AR 0.51; anterior margin of cibarial pump distinctly concave; hind tibia with hooked spur; superior volsella small rounded and undeveloped; inferior volsella narrow, with dented edge, along the inner margin of gonocoxite; phallapodeme apically curved, placed in lateral position of sternapodeme; sternapodeme curved into a U-shape, and lateral sternapodeme with large caudal attachment point.
Remarks. This species is similar to Corynoneura ascensa Fu & Saether, 2012 and Corynoneura sesquipedalis Fu & Fang, 2018 by having a large attachment point on the lateral sternapodeme. The new species can be separated from C. ascensa by having antenna with 8 flagellomeres, narrow and undeveloped inferior volsella; and differs from C. sesquipedalis by having a narrow inferior volsella, transverse sternapodeme present and with an oral projection (broad inferior volsella, transverse sternapodeme V-shaped,without transverse part in C. sesquipedalis). The sequence of 16S rDNA from this species is highly similar to Corynoneura tumula Fu & Fang, 2018, but there are distinct morphological differences between them: in C. enormis the antenna has 8 flagellomeres, AR 0.51; inferior volsella narrow, lateral sternapodeme with large caudal attachment point, while C. tumula has an antenna with 9 flagellomeres, AR 0.46; inferior volsella relatively broad, lateral sternapodeme with small caudal attachment point.  Etymology. From Latin, gibbera, protuberant, referring to the prominent inferior volsella. Diagnostic characters. The male imago is characterized by having an antenna with nine or ten flagellomeres, AR 0.43-0.57, 0.52; superior volsella triangular; inferior volsella prominent, like a small rectangle, and placed caudally of gonocoxite; transverse sternapodeme inverted U-shaped; phallapodeme scalpel-like, in caudal position of sternapodeme.
Remarks. This species is closely related to Corynoneura macula Fu & Saether, 2012 by having similarly shaped inferior volsella and an inverted U-shaped sternapodeme. The new species can be separated from the latter by having AR 0.43-0.57, 0.52, gonostylus relatively long and slender, apically curved, while C. macula has a yellowish antenna Diagnostic characters. The male imago is characterized by having antenna with eleven flagellomeres, AR 0.31; anterior margin of cibarial pump strongly concave; superior volsella developed and with right-angled corner; inferior volsella almost absent, fused with the inner margin of gonocoxite; transverse sternapodeme curved into U-shaped; phallapodeme scalpel-like, apical slightly curved, placed caudal position of sternapodeme. The female imago is characterized by coxosternapodeme with a single transparent, well-developed lamella.
Coloration. Head and thorax brown, eyes dark brown. Legs pale yellow. Abdominal tergites I-V yellowish, VI-IX yellow-brown.
Remarks. This species is closely related to Corynoneura tokarapequea Sasa & Suzuki, 1995 by having antenna with eleven flagellomeres, the same shaped sternapodeme and phallapodeme, and a similar gonostylus. The new species can be separated from the latter by having AR 0.31, the inferior volsella almost absent and fused with the inner margin of the gonocoxite, while C. tokarapequea has AR 0.62-0.70, the inferior volsella obvious and near rectangular. The new species is also similar to Corynoneura floridaensis Fu & Saether, 2012 by the antenna with eleven flagellomeres, AR 0.36, same shaped sternapodeme and phallapodeme, but differs from the latter by having a thick transverse sternapodeme, and the gonostylus is strongly curved in C. floridaensis.  Etymology. Named after the type locality. Diagnostic characters. The male imago is characterized by having an antenna with seven flagellomeres, AR 0.55; superior volsella undeveloped, and inferior volsella with right-angular corner, fused with the inner margin of gonocoxite; sternapodeme inverted U-shaped; phallapodeme scalpel-like, apical curved, in caudal position of sternapodeme.
Remarks. This new species is similar to Corynoneura hortonensis Fu & Saether, 2012 by having the same shaped sternapodeme and phallapodeme. The new species can be separated from the latter by the broad and thick transverse sternapodeme, and the median part of the gonostylus expanded with a rugged inner edge.

Notes on 16S rDNA analysis
The primary structure of the mitochondrial large ribosomal subunit (16S rDNA) gene is conservative, while the secondary structure shows spiral differences, which are more suitable for systematic studies of species and genera (Simon et al.1994;Harrison 2004). This gene has been successful in identification of chironomids (Cranston et al. 2002;Ekrem et al. 2010). A neighbor-joining tree (Fig. 6) based on 16S rDNA sequences has been proven effective for quickly delimiting and identifying specimens, and supports differentiation of the new species. This study is the first to use 16S rDNA for auxiliary delimitation and identification of specimens in the genus Corynoneura.

Discussion
The four new species referred in this study share the same morphological features: a transverse sternapodeme inverted U-shape, and the attachment point for the phallapodeme is placed in a caudal position of the sternapodeme. According to Fu et al. (2009), these new species belong to the celeripes species group. Additionally, short DNA fragments have been shown to play an important role in the definition of morphospecies (Hebert et al. 2004;Sharkey et al. 2021). In this study, 16S rDNA was used to match male and female individuals from different collections. A similarity of 100% was considered as the same species: thus, the female of C. incuria sp. nov. was successfully matched with the male by 16S rDNA. Figure 6. Neighbor-joining Kimura 2 parameter tree based on 16S rDNA of five Corynoneura species and Thienemanniella convexa Fu in Fang et al. (2021). Numbers on branches refer to the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates). Taxa names include scientific names and GenBank accession numbers of corresponding 16S rDNA.