New descriptions of the larval and pupal stages of Orthocladiusnitidoscutellatus and Psectrocladiusnevalis from Xizang, China (Diptera, Chironomidae)

Abstract Background Tibetan Plateau is one of the most typical areas of biodiversity in the world because of its unique environmental and regional units, which breed unique biological communities and concentrate on many unique and rare wild animals and plants. Research on Chironomidae in the Tibetan Plateau is relatively weak. At present, the identification of Chironomidae species mainly depends on male adults, while identification of larvae and pupae is relatively difficult and there is less research on them. New information During the investigations of insect diversity in the Tibetan Plateau, larval and pupal stages of Orthocladiusnitidoscutellatus Lundström, 1915 and Psectrocladiusnevalis Akhrorov, 1977 were described and illustrated. Matching and identification of larval and pupal stages were based on DNA barcodes. Neighbour-joining trees were reconstructed, based on known Orthocladius and Psectrocladius COI DNA barcodes, respectively.


New information
During the investigations of insect diversity in the Tibetan Plateau, larval and pupal stages of Orthocladius nitidoscutellatus Lundström, 1915 and Psectrocladius nevalis Akhrorov, 1977 were described and illustrated.Matching and identification of larval and pupal stages ‡ ‡ ‡ ‡ ‡ ‡

Introduction
Chironomidae is one of the most diverse and abundant groups of insects found in various habitats in global aquatic ecosystems, widely used for the impact of pollutants in the aquatic system (Liu et al. 2022).Even for the experienced observer, the aquatic larval level and pupal stages are quite similar and almost impossible to be separated, based purely on morphological criteria (Armitage et al. 1995, Rossaro et al. 2022).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 bioidentifcation system at the species level and proved to be useful in the delimitation of non-biting midge species (Ashe and O'Connor 2012, Gilka et al. 2018, Liu et al. 2021, Liu et al. 2023, Hebert et al. 2003a, Hebert et al. 2003b).
The genus Orthocladius Wulp, 1874, includes 144 species worldwide and is one of the richest in species within the Chironomidae subfamily Orthocladiinae (Ashe and O'Connor 2012, Moubayed et al. 2022, Rossaro et al. 2022).Orthocladius larvae and pupae are rather similar in morphology (Langton andVisser 2003,Epler et al. 2013).Most species live in running waters like rivers and streams, while a few can be found in standing waters, such as ditches and lakes (Cuppen and Tempelman 2022).Orthocladius nitidoscutellatus Lundström, 1915 was reported in east Siberia and described, based on adult males (Lundström 1915).It widely distributed in some countries of the Palearctic and Nearctic Regions, but there is currently no detailed description of larvae and pupae (Ashe andO'Connor 2012, Rossaro et al. 2022).Kieffer (1906) erected the genus Psectrocladius with Orthocladius psilopterus Kieffer, 1906 as the type species.This genus is divided into four subgenera, with a current world record of 61 valid species (Ashe and O'Connor 2012).Since the first report of the male adults of Psectrocladius nevalis Akhrorov, 1977 in Lake Zorkul on the Pamir Plateau in Tajikistan, there has been little research related to this species (Akhrorov 1977,Ashe andO'Connor 2012).To date, only simple descriptions of male adults of this species and public data about its barcode by Chinese scholars are available (Han et al. 2023).
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 (Liu et al. 2023)

Materials and methods
The examined specimens were caught using sweepnets and light traps, 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 (1980).Morphological terminology follows Saether (1969).
The colour 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 deionised H 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 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 Neighbour-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.Fourth instar larva (n = 1).
The larval and pupal specimens were used to extract the COI sequences and compared, all being identified as Orthocladius nitidoscutellatus Lundström, 1915 and these two specimens were well matched.Based on the Neighbour-joining tree of 55 known species in genus Orthocladius, the results showed that this species was close to Orthocladius wetterensis Brundin, 1956 in barcode and the two specimens we collected were clustered into one clade with the existing sequences of O. nitidoscutellatus Lundström, 1915 (Fig. 5).The molecular identification and morphological taxonomy results align, suggesting that DNA barcodes and traditional morphological taxonomy complement each other, with the former serving as a straightforward means to enhance the latter's effectiveness.
Biogeographically, the two species studied are confined to the Tibetan Plateau, thriving at altitudes exceeding 3,000 m.As altitude rises, the climate becomes increasingly harsh, yet Chironomidae inhabiting these high-altitude regions exhibit remarkable cold tolerance.Simultaneously, this underscores the rich biodiversity of the Tibetan Plateau, indicating a broader distribution range for this genus than previously documented.
In summary, this study not only enhances the Chironomidae database in China, but also contributes vital data towards safeguarding the ecological environment and biodiversity of the Tibetan Plateau.

Figure 5 .
Figure 5. Neighbour-joining tree for known species of the genus Orthocladius, based on K2P distances in DNA barcodes.Numbers on branches represent bootstrap support (> 70%) based on 1000 replicates; scale equals the K2P genetic distance.
. Here, we described and illustrated larval and pupal stages of Orthocladius nitidoscutellatus Lundström, 1915 and Psectrocladius nevalis Akhrorov, 1977 from the Tibetan Plateau.Matching and identification of larval and pupal stages are based on DNA barcodes.Neighbour-joining trees were reconstructed, based on known Orthocladius and Psectrocladius COI DNA barcodes, respectively.