First morphological description of the larval stages of three Microtendipes species (Diptera, Chironomidae) from South China with molecular confirmation

27 First morphological description of the larval stages of three Microtendipes species (Diptera, Chironomidae) from South China with molecular confirmation Haobo Jin1, Yiyi Wang1, Chao Song1, Xin Qi1 1 College of Life Sciences, Taizhou University, Taizhou, Zhejiang 318000, China Corresponding authors: Chao Song ([email protected]); Xin Qi ([email protected]) Copyright: © Haobo Jin et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract This study presents the first integrated morphological and molecular characterization of larvae from three Microtendipes species, Microtendipes baishanzuensis Song & Qi, 2023, Microtendipes robustus Song & Qi, 2023 and Microtendipes tuberosus Qi & Wang, 2006, collected from subtropical streams in China, providing important insights for advancing Chironomidae taxonomy. Using detailed morphometric analysis (head capsule ratios, mandibular pecten length, and striae counts) in conjunction with mitochondrial COI barcoding, we established larval–adult associations and differentiated these species from their congeners. Microtendipes baishanzuensis is characterized by a brownish head capsule with distinctly paler median teeth compared to the lateral teeth, the longest mandibular pecten within the genus, the highest number of body striae, and an exceptionally anteriorly positioned ring organ. Microtendipes robustus is distinguished by a uniformly dark brown mentum, remarkably wide ventromental plates, the most variable striae count, and a mediumsized mandibular pecten. Microtendipes tuberosus exhibits a uniformly dark brown mentum with median teeth conspicuously shorter than the second lateral teeth, the smallest body size in the genus, the shortest mandibular pecten, the fewest striae, and the most posteriorly located ring organ. A revised larval key for Chinese Microtendipes is presented, improving freshwater biomonitoring and addressing challenges associated with cryptic diversity. Key words: DNA barcodes, freshwater biomonitoring, identification key, larvae, Microtendipes, taxonomy Introduction The genus Microtendipes Kieffer was established by Kieffer in 1915 and currently comprises over 60 described species (Freeman and Cranston 1980; Cranston and Martin 1989; Ashe and Cranston 1990; Oliver et al. 1990; Zorina 2001; Qi and Wang 2006; Qi et al. 2014; Yamamoto and Yamamoto 2014; Hazra et al. 2016; Tang and Hiromi 2017; Song et al. 2023). In China, 18 recorded species have been described (Qi and Wang 2006; Qi et al. 2012, 2014; Song et al. 2023). Taxonomic research on Microtendipes has primarily focused on adult morphology, and larval descriptions are scarce. This gap is largely due to the high morphological homogeneity of larvae and the lack of reliable diagnostic traits, Academic editor: Fabio Laurindo da Silva Received: 12 June 2025 Accepted: 28 August 2025 Published: 9 October 2025 ZooBank: https://zoobank.org/ B39A2C88-5FB6-4B19-A7B699BC9DB78BCD Citation: Jin H, Wang Y, Song C, Qi X (2025) First morphological description of the larval stages of three Microtendipes species (Diptera, Chironomidae) from South China with molecular confirmation. ZooKeys 1255: 27–40. https://doi.org/10.3897/ zookeys.1255.161652 ZooKeys 1255: 27–40 (2025) DOI: 10.3897/zookeys.1255.161652 28 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species particularly distinct autapomorphies. These limitations collectively hinder species differentiation, precise taxonomic classification, and the establishment of a robust larval classification system. Furthermore, significant technical challenges persist in specimen collection and analysis, requiring specialized apparatus and microscopic techniques, and database limitations hinder reliable larva–adult matching. Despite these challenges, DNA barcoding has emerged as an effective identification tool (Carew et al. 2005, 2007; Gadawski et al. 2022). DNA barcodes are included in most new species descriptions as a standard method for delimiting non-biting midges (Song et al. 2016, 2022; Yan et al. 2017; Lin et al. 2019). DNA barcoding has also proven effective for associating specimens across different life stages (Song et al. 2018). However, the larval stages of these species have remained undiscovered. To address this gap in the literature, we conducted extensive larval collections and performed DNA barcode analyses. Consequently, we successfully collected and identified the larvae of three Microtendipes species: M. baishanzuensis Song & Qi, 2023, M. robustus Song & Qi, 2023, and M. tuberosus Qi & Wang, 2006, providing the first descriptions of their larval stages. Material and methods Larval specimens were collected from multiple rivers and streams using D-shaped nets. All specimens were preserved in 75% ethanol and stored at −20 °C in the laboratory until subsequent morphological and molecular analyses. For morphological examination, specimens were slide-mounted in Euparal and examined under a microscope. Measurements followed standardized protocols, and morphological terminology and abbreviations were adopted from Sæther (1980) and Maschwitz and Cook (2000). Values are reported as ranges accompanied by mean values, with the number of observed specimens (N) indicated in parentheses. All specimens are deposited in the College of Life Sciences, Taizhou University. Genomic DNA was extracted using the protocol outlined by Song et al. (2018). The standard barcode region of the 5′ portion of the mitochondrial gene cytochrome c oxidase I (COI-5P) was amplified using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR amplifications were performed as described by Song et al. (2018). PCR products were electrophoresed on a 1.0% agarose gel, purified, and sequenced using an ABI 3730XL capillary sequencer (Beijing Genomics Institute Co., Ltd., Hangzhou, China). Raw sequences were edited using BioEdit v. 7.2.5 (Hall 1999). Publicly available Microtendipes sequences (≥500 bp in length) were retrieved from the Barcode of Life Data System (BOLD; http://www.boldsystems. org/) on June 20, 2024 (see Suppl. material 1 for details). Sequence alignment was conducted in MEGA v. 12 (Kumar et al. 2024) using the ClustalW algorithm. Pairwise genetic distances were calculated using the K2P model in MEGA v. 12. A neighbor-joining (NJ) tree was constructed based on the Kimura 2-Parameter (K2P) substitution model with 1000 bootstrap replicates, using the “pairwise deletion” option to handle missing data. A maximum-likelihood (ML) tree was generated using IQ-TREE v. 2.1.3 (Nguyen et al. 2015), with node support assessed through ultrafast bootstrapping with 1000 replicates. Bayesian inference (BI) analysis was conducted using MrBayes v. 3.2.7 (Ronquist et al. 2012), employing Markov chain Monte Carlo (MCMC) randomization for 10 million 29 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species generations, with the first 25% of trees discarded as burn-in. Trace files from the BI analysis were examined using Tracer v. 1.7 (Rambaut et al. 2018), and the final phylogenetic tree was visualized in FigTree v. 1.4.2. Abbreviations Morphological abbreviations of larval characters are used as follow: Ant 1–6, length of antennal segments 1–6 in µm; A1R, first antennal segment ratio, length of segment 1: width of segment 1 through the ring organ; AR, antennal ratio, length of basal segment: combined lengths of segments 2 to apex; B. l., total body length in mm; Bl, antennal blade length; BlR, blade ratio, length of blade: total length of Ant 2–6; Pmd, premandible length; H. l., head capsule length, length from anterior labrum to posterior margin of capsule; L, larva; Md, mandible length; M. w, mentum width; Mmw, median mental tooth width; PM, postmentum length; ROR, ring organ ratio, distance from basal to location of ring organ: length of basal antennal segment; SAS, length of supraanal seta; SSm–SSm: distance between setae submenti; V. w, ventromentum width; V. l, ventromentum length; IPD, inter-ventromentum plates distance; Str, striae (Sæther 1980; Maschwitz and Cook 2000). Results and discussion DNA barcodes analysis This study analyzed a total of 161 sequences, including eight newly obtained larval sequences. A clear barcode gap of 4–6% was observed (Fig. 1), confirming the effectiveness of DNA barcoding for species delimitation within this genus. Based on three known male specimens, the maximum intraspecific genetic distance was 0.7% (mean: 0.5%) for M. baishanzuensis, 5.8% (mean: 2.9%) for M. robustus, and 3.2% (mean: 1.4%) for M. tuberosus. After including larval DNA sequences, these values changed slightly: M. baishanzuensis exhibited a maximum of 0.9% (mean: 0.5%), M. robustus a maximum of 5.8% (mean: 2.5%), and M. tuberosus a maximum of 6.1% (mean: 2.6%) (Suppl. material 2). Notably, the maximum intraspecific distance observed in M. tuberosus (6.1%) remained below the minimum interspecific distance (8.0%) between M. baishanzuensis and M. robustus. Figure 1. Histogram of pairwise K2P distances between morphological species of Microtendipes. The horizontal axis represents the pairwise K2P distance, the vertical axis represents the number of pairwise sequence comparisons. 30 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Phylogenetic analyses (Neighbor-joining, maximum likelihood, and Bayesian inference) revealed strong concordance between larval and adult sequences (Fig. 2). For M. tuberosus, although minor topological variations occurred across trees, all three methods provided robust support for the same clade structure. In M. robustus, consistent results across all three methods (NJ/ML/BI) confirmed larval–adult conspecificity via DNA barcoding. For M. baishanzuensis, all three phylogenetic reconstructions yielded well-resolved, congruent clades. This high consistency in branching patterns, combined with the low maximum pairwise genetic distance (0.9%), conclusively identifies the larvae as M. baishanzuensis using DNA barcoding. Figure 2. Phylogeny tree for Microtendipes based on DNA barcode sequences. The tree was based on partial COI sequences and the generalized time-reversible substitution model. Omisus caledonicus (Edwards) was used as an outgroup. The phylogenetic analysis incorporated data from maximum-likelihood (ML), Bayesian inference (BI), and neighbor-joining (NJ) methods for all three identified species; the data are presented as ML/BI/NJ analyses. Only nodes with Ultra-BS (ML) > 80%, PP > 0.80, and BS(NJ) > 0.75 are shown; different colors within each area represent larvae. 31 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Taxonomy Microtendipes baishanzuensis Song & Qi, 2023 Figs 3B–E, 4 Microtendipes baishanzuensis Song & Qi, 2023: 8. Material examined (N = 2): 2 larvae, China, Zhejiang Province, Lishui City, Qingyuan County, Baishanzu Town, 1600 m a.s.l., 27.750°N, 119.198°E, 14.VIII.2020, C. Song. Diagnosis (Fig. 3B, C). Mentum dark brown, with median teeth distinctly paler than lateral teeth; cephalic capsule brown. Total length 7.1–7.8, 7.5 mm; head capsule length 490.0–520.0, 505.0 μm; head capsule width 450.0–460.0, 455.0 μm. Mentum (Fig. 3C). Width 152.5 μm; median tooth width 37.5 μm; ventromental plate width 125.0–132.5, 128.8 μm; distance between ventromental plates 83.0–90.0, 86.5 μm; striae count 35.0–40.0, 37.5. Mandible (Fig. 3D). Length 168.0–195.0, 181.5 μm. Antenna (Fig. 3E). Segment lengths, 102.5–112.5, 107.5 μm; 25.0–27.5, 26.3 μm; 27.0 μm; 25.0 μm; 17.0–17.5,17.3 μm; 10.0 μm. Antennal ratio (AR) 1.0–1.1, 1.0. Ring organ width 34.0–35.0, 34.5 μm; ring organ positioned at 0.2–0.3, 0.3 of basal segment length, 22.5–35.0, 28.8 μm from segment base; antennal blade 106.0–125.0, 115.5 μm in length, width 2.0–18.0 μm. Labrum. Premandible with two apical teeth; length 97.5–120.0, 108.8 μm. Pecten epipharyngis with three broad marginal teeth apically; labral lamella composed of 15 small teeth. Figure 3. Larva of Microtendipes baishanzuensis Song & Qi, 2023. A. Habitus of larva; B. Head capsule; C. Mentum; D. Mandible; E. Antenna. 32 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Thorax. Anterior claws pale with a little golden posterior claws pale golden, both simple and dense. Abdomen. 8 tail hairs of anal seta, 520.0–611.0, 565.5 μm (Table 1). Ecological environment (Fig. 4). A forest stream, winding through wooded areas, exhibiting high-clarity water flowing over a predominantly rocky substrate. Remarks. A comparative analysis was conducted between our data and the larval morphological measurements reported by Tang (2006). The larvae of M. baishanzuensis resemble Microtendipes pedellus (De Geer, 1929) and Microtendipes chloris (Meigen, 1933) in the shape of the mentum. However, M. baishanzuensis has more striae than M. pedellus (mean 37.5 vs. 30.0). The total length of the postmentum in M. baishanzuensis is shorter than in M. pedellus (mean 201.3 μm vs. 223.0 μm). The ring organ in M. baishanzuensis is located at 0.2–0.3 (mean 0.267) of the basal segment, compared to approximately 0.33. Compared to M. chloris, M. baishanzuensis has a distinctly shorter blade (mean 115.5 μm vs. 145.0 μm), a longer postmentum (mean 201.3 μm vs. 215.0 μm), and more striae (mean 37.5 vs. 28.0). Microtendipes tuberosus Qi & Wang, 2006 Figs 5B–E, 6 Microtendipes tuberosus Qi & Wang, 2006: 43. Material examined (N = 3): 1 larva, China, Zhejiang Province, Lishui City, Suichang County, Jiulong Mountain National Nature Reserve, 370 m a.s.l., 28.408°N, 118.811°E, 29.VIII. 2020, C. Song; 2 larvae, China, Zhejiang Province, Lishui City, Suichang County, Jiulong Mountain National Nature Reserve, 400 m a.s.l., 28.380°N, 118.799°E, 29.VIII. 2020, C. Song. Diagnosis (Fig. 5B, C). Mentum dark brown, with median teeth concolorous with lateral teeth; median tooth slightly shorter than the second lateral tooth. The cephalic capsule is light brown. Total length 2.1–3.7, 2.8 mm; head capsule length 375.0–400.0, 387.5 μm; head capsule width 287.5–300.0, 292.5 μm. Figure 4. Habitat of Microtendipes baishanzuensis larvae. 33 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Mentum (Fig. 5C). Width 105.0–110.0, 108.0 μm; median tooth width 23.0–25.0, 24.3 μm; ventromental plate width 83.0–122.5, 96.8 μm; distance between ventromental plates 59.0–85.0, 68.8 μm; striae count 22–29, 26. Mandible (Fig. 5D). Length 125.0–132.5, 129.8 μm. Antenna (Fig. 5E). Segment lengths: 100.0–102.5, 101.2 μm; 27.0–28.0, 27.5 μm; 23.0–26.3, 24.8 μm; 16.0–21.3, 19.1 μm; 10.0–11.3, 10.4 μm; 7.5–8.8, 7.9 μm). Antennal ratio (AR) 1.1–1.2, 1.1. Ring organ width 25.5 μm; ring organ positioned at 0.15–0.22, 0.20 of basal segment length, 15.0–22.5, 20.0 μm from segment base; antennal blade 53.0–72.0, 59.7 μm in length. Labrum. Premandible bifid; length 62.5–65.0, 63.8 μm. Pecten epipharyngis is composed of 8 teeth. Thorax. Anterior claws pale with a faint golden tint; posterior claws pale golden. Procercus and apical setae are yellowish brown. Abdomen. Anal setae comprising 8 hairs, length 197.4–445.0, 327.5 μm (Table 1). Ecological environment (Fig. 6). The stream flows through forests and villages, featuring a streambed composed of bedrock and sediment, with excellent water clarity. Remarks. A comparative analysis was conducted between our data and the larval morphological measurements reported by Tang (2006). The larvae of M. tuberosus resemble those of Microtendipes britteni (Edwards, 1983) in mentum morphology. However, M. tuberosus is noticeably smaller in body size (mean 2.8 mm vs. 6.9 mm in M. britteni) and has a shorter mandibular pecten (mean 153.3 μm vs. 195.0 μm in M. britteni). Among congeners, M. tuberosus is notably smaller overall. Both its median and lateral teeth are uniformly dark brown. Figure 5. Larva of Microtendipes tuberosus Qi & Wang, 2006. A. Habitus of larva; B. Head capsule; C. Mentum; D. Mandible; E. Antenna. 34 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Figure 6. Habitat of Microtendipes tuberosus larvae. Table 1. Mensural features of the larvae of Microtendipes baishanzuensis, Microtendipes tuberosus and Microtendipes robustus. Abbreviations follow as in the text. M. baishanzuensis M. tuberosus M. robustus N2 3 3 B.l. /mm 7.1–7.8, 7.5 2.1–3.7, 2.8 2.9–9.6, 5.0 H. w. 450–460, 455 287.5–300, 292.5 287.5–620.0, 480.6 PM 200–202.5, 201.25 150–157.5, 153.3 125–228, 190.8 SSm–SSm 105–100, 102.5 67.5–77.5, 72.1 71–128, 109.8 Ant1 102.5–112.5, 107.5 100–102.5, 101.2 42.5–117.5, 75.8 Ant2 25–27.5, 26.3 27–28, 27.5 15.5–25, 22.6 Ant3 27.0 23–26.3, 24.8 17.5–27.5, 21.9 Ant4 25.0 16–21.3, 19.1 15–18, 17.1 Ant5 17–17.5, 17.3 10–11.3, 10.4 11.0–15.0, 12.9 Ant6 10.0 7.5–8.8, 7.9 5.0–8.0, 6.1 AR 1.0–1.1, 1.0 1.1–1.2, 1.1 0.8–1.3, 0.9 AIR 3.0–3.2, 3.1 3.7–4.4,4.0 1.4–3.2, 2.5 ROR 0.2–0.3, 0.27 0.2–0.2, 0.2 0.3–0.7, 0.4 Bl 106–125, 115.5 53–72, 59.67 87.5–137.5, 114.4 BlR 1.0–1.2, 1.1 0.6–0.8, 0.7 1.3–1.6, 1.4 Pmd 97.5–120, 108.8 62.5–65, 63.8 60–110, 90 Md 168–195, 181.5 125–132.5, 129.8 115–200, 174.4 M. w 152.5–152.5, 152.5 105–110, 108 92.5–185, 151.9 Mmw 37.5 23–25, 24.3 20.5–42.5, 33.3 AS 520–611, 565.5 197.4–445, 327.5 347–602.3, 492.5 V. w 125–132.5, 128.8 83–122.5, 96.8 75–150, 120 V. l 75–80, 77.5 37.5–57.5, 49.3 48–87.5, 72.3 IPD 83–90, 86.5 59–85, 68.8 51–125, 105.9 Str 35–40, 37.5 22–29, 26 28–43, 38.3 SAS 8.0–10.0, 9.0 8.5–9.5, 9.0 8.5–12.5, 10.3 35 ZooKeys 1255: 27–40 (2025), DOI: 10.3897/zookeys.1255.161652 Haobo Jin et al.: First larval description of three Chinese Microtendipes species Microtendipes robustus Song & Qi, 2023 Figs 7B–E, 8 Microtendipes robustus Song & Qi, 2023:16. Material examined (N = 3): 1 larva, China, Zhejiang Province, Lishui City, Qingyuan County, Baishanzu Town, 1370 m, 27.754°N, 119.186°E, 14.VIII.2020, C. Song; 1 larva, China, Zhejiang Province, Lishui City, Qingyuan County, Baishanzu Town, 1650 m a.s.l., 27.751°N, 119.199°E, 14.VIII.2020, C. Song; 1 larva, China, Zhejiang Province, Wenzhou City, Taishun County, Wuyanling National Nature Reserve, 1060 m a.s.l., 27.424°N, 119.404°E, 25.IX.2020, C. Song; 1 larva, China, Zhejiang Province, Wenzhou City, Taishun County, Wuyanling National Nature Reserve, 660 m a.s.l., 27.423°N, 119.404°E, 25.IX.2020, C. Song. Diagnosis (Fig. 7B, C). The larvae possess a light brown cephalic capsule and dark brown mentum, with the pale median teeth forming a distinct contrast against the darker lateral teeth. Total length. 2.9–9.6 mm, 5.0 mm; head capsule length 325.0–650.0, 521.3 μm; head capsule width 287.5–620.0, 480.6 μm. Mentum (Fig. 7C). Width 92.5–185.0, 151.9 μm; median tooth width 20.5–42.5, 33.3 μm; ventromental plate width 75.0–150.0, 120.0 μm; distance between ventromental plates 51.0–125.0, 105.9 μm; number of striae 28–43, 38.3. Mandible (Fig. 7D). Length 115.0–200.0, 174.4 μm. Figure 7. Larva of Microtendipes robustus Song & Qi, 2023. A. Habitus of larva; B. Head capsule; C. Mentum; D. Mandible; E. Antenna.