DNA Barcoding Supports “Color-Pattern’’-Based Species of Stictochironomus from China (Diptera: Chironomidae)

Simple Summary The Chironomidae family stands out as the most widely dispersed and often the most abundant insect group in freshwater habitats. The significance of color patterns is well-recognized, as they fulfill multiple roles such as communication, camouflage, mimicking, and defense. However, the taxonomy of species within this family, relying on color patterns, as well as the shape and distribution of thoracic pigmentation, wing markings, and leg pigmentation, remains controversial and unstable. Here, we conduct a comprehensive review on the taxonomy of a Chiromidae genus, Stictochironomus from China, which is characterized by a combination of distinctive wing and leg markings. Using DNA barcode data and morphological data, two new species to science from China are well supported. Species delimitation analyses performed with distance-based approach and coalescent tree-based approaches also support them as distinct species. Therefore, color patterns should be a good diagnostic characteristic for species delimitation in Stictochironomus. Furthermore, we provided an up-to-date taxonomic key for male adults of Stictochironomus from China. Abstract The genus Stictochironomus (Diptera: Chironomidae) has an almost worldwide distribution, with more than 30 species. However, species delimitation and identification based on the markings on the wings and legs are controversial and uncertain. In this study, we focused on color patterns to review the adults of the genus from China, and two new species (S. trifuscipes sp. nov. and S. quadrimaculatus sp. nov.) are described and figured. DNA barcodes can accurately separate the two new species with specific color patterns. However, heterospecific individuals form a monophyletic cluster in the phylogeny tree. For example, S. maculipennis (Meigen) and S. pictulus (Meigen), which have a lower interspecific genetic divergence, form a single clade. Sequences with the same species name but with high intraspecific distance form more than one phylogenetic clade, such as S. sticticus (Fabricius) of three clades, S. pictulus of four clades, S. akizukii (Tokunaga) and S. juncaii Qi, Shi, and Wang of two clades, might have potential cryptic species diversity. Species delimitation analysis using ASAP, PTP, and GMYC clearly delineated them as separate species. Consequently, color patterns are a good diagnostic characteristic for species delimitation in Stictochironomus. The distance-based analysis shows that a threshold of 4.5–7.7% is appropriate for species delimitation in Stictochironomus. Additionally, an updated key including color pattern variation for male adults of known Stictochironomus species from China is provided.


Introduction
Color patterns play a significant role in the behavior of insects and are recognized as significant characteristics for taxonomic studies [1].Among insects, for instance, the wing color pattern has been important in the evolution of beetles, flies, and moths and butterflies, evolving various functions such as attracting mates and warning predators.One well-studied pattern is butterfly wings, which focuses on the scale-covered wings, pigmentation, and spatial pattering systems [2].Phenotypic variation significantly conceals the cryptic species diversity that is largely hidden within species based on morphology alone.Theoretically, pigmentation intensity in the body, legs, and wings varies widely, often distinguishing sexes, populations, and species [3].
DNA barcoding provides new insights for exploring species diversity and delimitation, and extensive sampling for meta-barcodes has revealed high cryptic diversity [4][5][6][7][8][9][10].In 2004, Hebert et al. discovered ten cryptic species in a neotropical skipper butterfly named Astraptes fulgerator (Walch) using DNA barcoding and morphological traits, including caterpilal color patterns and subtle differences in adult patterns and size [7].In 2017, Janzen et al. employed complete nuclear genome sequencing to obtain DNA barcoding data, the ecological distribution, the natural history, and the subtleties of adult color pattern and size, to reveal the cryptic diversity among the species of Udranomia kikkawai (Weeks) [8].
Chironomidae have evolved a diverse range of pigmentation patterns on different body parts, including the abdomen, thorax, wings, and legs [11][12][13].The genus Stictochironomus is widely distributed, occurring in all biogeographical regions except Antarctica, and is characterized by a combination of distinctive wing and leg markings, lack of frontal setae, conical scutal tubercle, fused tibial combs, usually with only one spur, and a distally flattened gonostylus with mobile inferior volsella bent laterally [13].The larvae of this genus can be found in profundal soft sediments or littoral sand of oligotrophic lakes, as well as in sediments of streams and slowly flowing rivers [14].There are more than thirty species worldwide, with five species recorded in China [15][16][17][18].
In this study, we review the genus Stictochironomus and describe two new species from China.Illustrations based on adult males, diagnoses and descriptions based on morphology, and DNA barcodes are also included.A key to the known adult males of this genus is provided.

Materials and Methods
The examined material was caught using light traps and then preserved in 75% ethanol at 4 • C in a refrigerator before morphological and molecular procedures.Genomic DNA was extracted from the thorax and head following Frohlich et al. (1999) [19] at Taizhou University, Zhejiang, China.The COI sequences were amplified using the universal primers [20].For the PCR, 12.5 µL of 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 deionized H 2 O were used.PCR amplification was performed with the following program: 95 • C for the initial denaturation for 5 min; 34 cycles of 94 • C for 30 s, 51 • C for 30 s, and 72 • C for 1 min; and a final extension for 3 min at 72 • C [21].After the PCRs, 2.5 µL of PCR products were run on a 1.0% agarose gel, purified using ExoProStar 1-Step, and sequenced at the Beijing Genomics Institute Co., Ltd., Hangzhou, China.
After genomic extraction, the exoskeletons were slide-mounted in Euparal together with corresponding body parts following the procedure described by Saether (1969) [22].
The type materials, including holotype and paratypes of the newly described species, were deposited in the collection of the College of Life Sciences, Taizhou University, Zhejiang, China (TZU).The morphological terminology employed in this study follows that of Saether (1980) [23].Images of the habitus of each specimen were captured using a DV500 5MP Digital Camera attached to a stereo microscope (Chongqing Optec Instrument Co., Ltd., Chongqing, China).Images of the mounted specimens were captured using a compound microscope from Leica DMLS (Leica Camera AG, Wetzlar, Germany).Photograph postprocessing was carried out in Adobe Photoshop and Illustrator version 8 (Adobe Inc., San Jose, CA, USA).
Raw sequences were assembled in BioEdit 7.2.5 [25].The sequences were aligned using the ClustalW algorithm in MEGA 7. The pairwise distances were calculated using the Kimura 2-Parameter (K2P) substitution model in MEGA 7. The neighbor-joining tree was constructed using the K2P substitution model, and 1000 bootstrap replicates and the "pairwise deletion" option for missing data were utilized [26].Partitions and model selection were performed in PartitonFinder using greedy search and selected according to the BIC scheme [27].The results were as follows: SYM + G for the first site, F81 + I for the second site, and GTR + I + G for the third site.
For ML analysis, RAxML allows for only a single model of rate heterogeneity in partitioned analyses; we used the GTR + G + I substitution model with 1000 bootstrap replicates in a rapid bootstrap analysis [28].Bayesian inference analysis was carried out using Markov Chain Monte Carlo (MCMC) randomization in MrBayes v3.2.7 [29] with five million generations, and it was stopped when the standard deviation of the split frequencies was below 0.01.A consensus tree was summarized with the first 25% of sampled trees being discarded as burn-in.Trace files of the BI analysis were also inspected in Tracer 1.7 [30], and then the tree was visualized in FigTree v.1.4.2.
ASAP (assemble species by automatic partitioning) analysis was performed on the website https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html(accessed on 19 April 2023) using the K2P model with other parameters the default setting [31].The bPTP analyses were run on online servers (http://species.h-its.org/ptp,accessed on 24 January 2024) and the input ML tree generated in RAxML.We ran 500,000 MCMC generations, used a burn-in of 0.1, and set the other parameters to their defaults [32].The general mixed Yule-coalescent (GMYC) method was applied using the splits package [33] in R. The input ultrametric tree for GMYC was generated in BEAST 1.7 [34] (strict clock, MCMC chain using 10 million generations, TN93 substitution model, and Yule speciation model).Ther convergence and estimated effective sample sizes (ESS > 200) for all the parameters were checked using Tracer v1.7.1 [30]
Stictochironomus species exhibit great phenotypic plasticity, especially in terms of the color patterns (leg, wing, and thorax) and the numbers of setae on the thorax (dorsocentrals, prealars, and scutellars), while the permutation and combination of markings or patterns are sometimes easily overlooked by taxonomists who are more concerned with the structural characteristics of the hypopygium.In our study, we did not observe interspecific overlap of color pattern morphotypes, which could support the idea that color patterns are a good diagnostic characteristic for species delimitation in Stictochironomus.However, species with unique color morphotypes could form several separated clades based on genetic analyses.Four species with unique color patterns formed several phylogenetic clades (Figure 1), which matches the results in the thesis of Reistad (2023) [40].These four species were the same species with high intraspecific divergence and included S. pictulus (four clades), S. sticticus (three clades), S. akizukii (two clades), and S. juncaii (two clades).There are also cases of molecular discordance in which morphospecies have commonly been found in insect groups because of incomplete lineage sorting, introgression, and hybridization, but these rarely pose problems in relation to chironomids [41].Therefore, inaccurate reference taxonomy mainly explains the non-monophyletic phenomena of Chironomidae.Reistad (2023) argued that one of the most notable differences was the number of setae on the thorax for differentiating S. pictulus clusters as well in some other species, which would support undescribed taxa.

Phylogenetics and Species Delimitation
The neighbor-joining (S5), maximum likelihood (S6), and Bayesian inference methods of constructing phylogenetic tress almost share the same topology (S7).The high interspecific variation in COI means that the relationships between species are not well constructed when it is used [42].However, the large interspecific variation aids in the diagnosis of species clusters, resulting in high support values at the ends of the branches.Based on the NJ tree, ML tree, and BI tree, 218 DNA barcodes of 16 initial morphospecies were clustered into 22 clades (Figure 1).The undescribed taxa well form separated clades in all the phylogenic trees, which supports them as new to science.
Species delimitation analyses performed with the distance-based approach and coalescent tree-based approaches almost produced the same results.In the Assemble Species by Automatic Partitioning analysis, dataset A gave the lowest score of 2.5, with the best partition of 26 species (threshold at 4.9%, S8); dataset B gave a score of 4.0, with the best partition of 21 species (threshold at 6.9%, S9); and dataset C gave a score of 3.0, with the best partition of 20 or 24 species (threshold at 7.7% or 4.5%, S10).Using the Poisson Tree Processes (PTP) model to infer putative species, under the maximum likelihood solution, the highest Bayesian-supported solution returned 26 species, while the confidence interval was 26-97 (mean: 40) OTUs (S11).The single-threshold general mixed Yule-coalescent calculations (ST-GMYC) yielded 30 entities, ranging from 30 to 30 (S12).In summary, in the results for ASAP, GMYC, PTP, and phylogeny clades, the species numbers varied from 20 to 30, more than the pre-morphospecies number.The two undescribed species (Stictochironomus trifuscipes and S. trifuscipes) are always regarded as separated species, which supports them as new to science.
Most barcoding research on insect species proposes a threshold of 2-3% for delimiting intra-or interspecific species [42][43][44]; however, genus-specific thresholds have been suggested for some Chironomidae including 4-5% for Tanytarsus [36] and 5-8% for Polypedilum [21].In this study, a barcode gap was explored in Stictochironomus species using three datasets (dataset A: sequences with duplicates; dataset B: sequences without duplicates; dataset C: haplotype sequences) (Figure 2).Barcode gaps appeared at 1-7%, 2-7%, and 3-4%, respectively, which marks the limit between smaller intraspecific distances and larger interspecific distances.From the gap, a distance threshold is estimated and used to partition the samples into putative species.The threshold values generated from ASAP ranged from 4.5 to 7.7%, with species numbers ranging from 20 to 26, which approximately align with the phylogenetic clusters.Therefore, a threshold of 4.5-7.7% is more appropriate for species delimitation in Stictochironomus species.
Insects 2024, 15, 179 6 of 18 and 3-4%, respectively, which marks the limit between smaller intraspecific distances and larger interspecific distances.From the gap, a distance threshold is estimated and used to partition the samples into putative species.The threshold values generated from ASAP ranged from 4.5 to 7.7%, with species numbers ranging from 20 to 26, which approximately align with the phylogenetic clusters.Therefore, a threshold of 4.5-7.7% is more appropriate for species delimitation in Stictochironomus species.Consequently, for future research, more genetic markers are needed to test whether the phylogenetic units are congruent with biological units.On the other hand, the morphological differences between closely related species are often subtle, and information from more than one life stage and data on behavior and ecology tend to be very helpful for species delimitation.Diagnostic characteristics.The male imago can be separated from the known species of Stictochironomus based on the following combination of features: antenna and antennal plumage mostly yellowish brown; wing with six separate markings, two spots in Cell m1+2; thorax yellowish brown or brownish; legs pigmented, especially four dark rings on all tibia; superior volsella sickle-shaped, with three basal long inner setae and microtrichiae at the base; and a distal seta arising at the apical 1/3.
Etymology.The new species is named based on the characteristics of the color patterns of tibia.In Latin, it consists of "quadri" and "maculatus".The Latin word "quadri" means "four", while "maculatus" means "spotted" or "banded".This term is used to describe the features of species that have four dark bands on the tibia.
Coloration (Figure 3).A mature male adult is mostly pale yellowish to light brown; the antenna is yellowish brown and antenna plumage yellowish; the ground of th thorax is yellowish brown with dark lateral stripes, anepisternum II, and the postnotum is dark brown; the abdomen is yellowish; the wings are without markings; the legs have poorly Consequently, for future research, more genetic markers are needed to test whether the phylogenetic units are congruent with biological units.On the other hand, the morphological differences between closely related species are often subtle, and information from more than one life stage and data on behavior and ecology tend to be very helpful for species delimitation.Diagnostic characteristics.The male imago can be separated from the known species of Stictochironomus based on the following combination of features: antenna and antennal plumage mostly yellowish brown; wing with six separate markings, two spots in Cell m 1+2 ; thorax yellowish brown or brownish; legs pigmented, especially four dark rings on all tibia; superior volsella sickle-shaped, with three basal long inner setae and microtrichiae at the base; and a distal seta arising at the apical 1/3.
Etymology.The new species is named based on the characteristics of the color patterns of tibia.In Latin, it consists of "quadri" and "maculatus".The Latin word "quadri" means "four", while "maculatus" means "spotted" or "banded".This term is used to describe the features of species that have four dark bands on the tibia.
Coloration (Figure 3).A mature male adult is mostly pale yellowish to light brown; the antenna is yellowish brown and antenna plumage yellowish; the ground of th thorax is yellowish brown with dark lateral stripes, anepisternum II, and the postnotum is dark brown; the abdomen is yellowish; the wings are without markings; the legs have poorly defined pigmentation.P1: mostly pale, the anterior part of the femur has a pale brown ring, the tibia is slightly pale brown, tarsus pale.P2 and P3: pale, except the knees are slightly brown.defined pigmentation.P1: mostly pale, the anterior part of the femur has a pale brown ring, the tibia is slightly pale brown, tarsus pale.P2 and P3: pale, except the knees are slightly brown.Head.AR 1.44.The head was destroyed for genome exception except for the antenna.Thorax (destroyed).Acrostichals more than 3; dorsocentrals 7; prealars 6; scutellars 5. Scutral tubercle present.
Legs (Figure 5A).The spur on the median tibiae is 27 µm long, including an 18 µm long comb and the un-spurred comb is 18 µm long; the spur on the hind tibia is 32 µm long including a 20 µm long comb and the un-spurred comb is 15 µm long.The width at the apex of the front tibia is 53 µm; the width of the middle tibia is 53 µm; and the width Head.AR 1.44.The head was destroyed for genome exception except for the antenna.Thorax (destroyed).Acrostichals more than 3; dorsocentrals 7; prealars 6; scutellars 5. Scutral tubercle present.
Legs (Figure 5A).The spur on the median tibiae is 27 µm long, including an 18 µm long comb and the un-spurred comb is 18 µm long; the spur on the hind tibia is 32 µm long including a 20 µm long comb and the un-spurred comb is 15 µm long.The width at the apex of the front tibia is 53 µm; the width of the middle tibia is 53 µm; and the width of the hind tibia is 57 µm.The lengths (in µm) and proportions of the legs can be found in Table 3. of the hind tibia is 57 µm.The lengths (in µm) and proportions of the legs can be found in Table 3.    Hypopygium (Figure 4B,C).The anal point is straight and parallel in the dorsal view, 88 µm long and 37 µm wide at the base and 6 µm wide at the apex.Tergite IX with 9 setae medially; Laterosternite IX with 2-3 setae.The transverse sternapodeme is 55 µm long and the phallapodeme is 77 µm long.The gonocoxite is 192 µm long and the gonostylus is 130 µm long.The superior volsella is sickle-shaped, 77 µm long, 30 µm wide, with a 3basal long inner seta and microtrichiae in the base and 1-distal seta arising at apical 1/3.Inferior volsella with 12 setae, is 100 µm long, and extends beyond the apex of anal point.HR 1.48; HV 2.88.
Remarks.The new species is similar to Stictochironomus han Na and Bae, 2010.Both species share the following characteristics: wing with obvious separated spots; an anal point that is long, slender, nearly parallel-sided, and with a rounded apex; a superior volsella that is broad basally with microtrichiae and three long setae on the inner margin and with the long distal process slightly curved, bare, with a hooked apex, and a long seta on the dorsal surface in distal 1/3 [45].Stictochironomus quadrimaculatus sp.nov.differs from S. han by a lower AR (1.44), shorter wing (1.80 mm), larger foreleg LR (1.88), and having unique pigmentation patterns on the legs and wings, such as spot locations, sizes, and numbers (Figure 1A,D in [43]).In S. han, these measurements are AR 2.48, the wing length is 3.04 mm, and the foreleg LR is 1.12 (Table 4).Moreover, the average species k2p distance to the other Stictochironomus ranges from 15% to 22% (S4), which to some extent, supports it as a new species.Hypopygium (Figure 4B,C).The anal point is straight and parallel in the dorsal view, 88 µm long and 37 µm wide at the base and 6 µm wide at the apex.Tergite IX with 9 setae medially; Laterosternite IX with 2-3 setae.The transverse sternapodeme is 55 µm long and the phallapodeme is 77 µm long.The gonocoxite is 192 µm long and the gonostylus is 130 µm long.The superior volsella is sickle-shaped, 77 µm long, 30 µm wide, with a 3-basal long inner seta and microtrichiae in the base and 1-distal seta arising at apical 1/3.Inferior volsella with 12 setae, is 100 µm long, and extends beyond the apex of anal point.HR 1.48; HV 2.88.
Remarks.The new species is similar to Stictochironomus han Na and Bae, 2010.Both species share the following characteristics: wing with obvious separated spots; an anal point that is long, slender, nearly parallel-sided, and with a rounded apex; a superior volsella that is broad basally with microtrichiae and three long setae on the inner margin and with the long distal process slightly curved, bare, with a hooked apex, and a long seta on the dorsal surface in distal 1/3 [45].Stictochironomus quadrimaculatus sp.nov.differs from S. han by a lower AR (1.44), shorter wing (1.80 mm), larger foreleg LR (1.88), and having unique pigmentation patterns on the legs and wings, such as spot locations, sizes, and numbers (Figure 1A,D in [43]).In S. han, these measurements are AR 2.48, the wing length is 3.04 mm, and the foreleg LR is 1.12 (Table 4).Moreover, the average species k2p distance to the other Stictochironomus ranges from 15% to 22% (S4), which to some extent, supports it as a new species.Diagnostic characteristics.The male imago can be separated from the known species of Stictochironomus based on following combination of features: the antenna and antennal plumage is mostly dark brown; the wing is smokey with markings; the thorax is yellowish Diagnostic characteristics.The male imago can be separated from the known species of Stictochironomus based on following combination of features: the antenna and antennal plumage is mostly dark brown; the wing is smokey with markings; the thorax is yellowish brown or brownish with the postnotum dark brown; the legs are pigmented, especially the fore Ta 1-3 which is dark brown; the superior volsella sickle-shaped, with three basal long inner setae and microtrichiae at the base and one distal seta arising at the apical 1/3.
Etymology.The new species is named based on the characteristics of the patterns of the fore tarsi.In Latin, it consists of "tri" and "fuscipes".The Latin word "tri" means "three", while "fuscipes" means "dark legs".This term is used to describe the feature of species where the first three segments of the fore tarsi are dark in color.
Coloration (Figures 6 and 7).A mature male adult is mostly brownish or yellowish brown.The head is brownish or dark brown; the antenna and antenna plumage are dark brown; the thorax is yellowish brown or brownish with the postnotum dark brown.The Abdomen is yellowish or brownish.Legs.P1: the femur is not clearly defined and is mostly pale with two brown rings; most of the tibia is pale with the anterior 2/5 brownish; ta 1-3 is dark brown and distal 2/5 of ta 4 and distal 1/2 of ta 5 are brown.P2: most of the femur is brownish with three pale rings, most of the tibia is dark brown with two pale parts, and most of the tarsus is pale, with basal 1/4 and apical 1/5 of ta 1 and apical 1/2 of ta 4 , and ta 5 being dark brown.P3: most of the femur is dark brown with three pale rings, the tibia is dark brown except for a pale basal part; the tarsus is pale except for ta1, apical 1/2 of ta4, and ta5, which are dark brown.
Legs (Figure 5B).The spurs on the median tibiae are 25-30 µm long, including a 25-32 µm long comb and an un-spurred comb that is 32-35 µm long; the spur on the hind tibia is 30-32 µm long including a 30-32 µm long comb and an un-spurred comb that is 32-35 µm long.The width at the apex of the front tibia is 73-78 µm; the middle tibia is 65-78 µm; and the hind tibia is 63-65 µm.Lengths (in µm) and proportions of legs are shown in Table 5. Hypopygium (Figure 7B,C).The anal point is straight and parallel in the dorsal view, 95-115 µm long and 27-27 µm wide at the base and 5-5 µm wide at the apex.Tergite IX has 6-11 setae medially and laterosternite IX has 2-3 setae.The transverse sternapodeme is 55-60 µm long and the phallapodeme is 75-75 µm long.The gonocoxite is 260-275 µm long and the gonostylus is 135-140 µm long.The superior volsella is sickle-shaped, 65-75 µm long, 28-30 µm wide, with three basal long inner setae and microtrichiae at the base and a distal seta arising at the apical 1/3.The inferior volsella has 18-20 setae and is 110-120 µm long, not extending beyond the apex of the anal point.HR 1.92-1.94;HV 3.61-3.71.
Remarks.The new species is similar to Stictochironomus simantomaculatus (Sasa, Suzuki and Sakai, 1998).Both species have similar wing patterns and pigmentation of the fore femur and fore tibia, but they can be differentiated by the morphology of the hypopygium.Specifically, the superior volsella has three inner setae and one lateral outer seta in S. trifuscipes sp.nov.versus two inner setae and zero lateral outer setae in S. simantomaculatus.In addition, the gonostylus is normally developed in S. trifuscipes sp.nov.and abruptly constricted at about the middle in S. simantomaculatus.The wing patterns and superior volsellae of Stictochironomus trifuscipes sp.nov.also resemble Stictochironomus multannulatus (Tokunaga, 1938) (Figures 38 and 39 in [46]), but these species can be distinguished by leg pigmentation and details on the inferior volsella.For example, ta1-3 of P1 is totally dark brown (Figure 5B), while it is mostly pale in the latter species.In addition, the inferior volsella has 18-20 recurved setae versus 16 setae; the gonostylus lacks long strong setae along the inner margin but has strong setae on the ventral surface and several long bristles arising from the basal half (Table 6).In addition, the average species k2p distance to the other Stictochironomus ranged from 16% to 24% (S4), which to some extent, supports it as a new species.Diagnosis.The male imago can be separated from the known species of Stictochironomus based on the following combination of features: wing with only one dark area around the cross vein r-m; superior volsella with four basal setae; gonostylus with twelve long setae along the inner margin; a thorax yellow with scutum, postnotum, and scutellum brown; and the abdominal tergites I-IV pale yellow, with apical brown band, tergites V-IX brown.
Distribution.China (Tianjin, Inner Mongolia, Sichuan, Yunan), Japan.Diagnostic characters.The male imago can be separated from the known species of Stictochironomus based on following combination of features: wing with several dark spots, posterior area somewhat smoky; the anal point slender and parallel-sided; the gonostylus robust, basal gonostylus sturdy, gradually narrowed to apical, with 13 strong setae along the inner margin.
Diagnosis.The male imago can be separated from the known species of Stictochironomus by the following combination of features: wing smoky, with transverse markings; legs with conspicuous dark and pale rings, as the tibia is mostly black with a preapical pale ring, ta 1-3 mostly pale with apical dark; superior volsella hooked, with 3-4 setae at the base and one dorsal seta arising at the apical 1/3.
Distribution.China (Jiangxi, Guizhou), Japan, Russian Far East [53].Diagnosis.The male imago can be separated from the known species of Stictochironomus by the following combination of features: wing with four separated spots; abdominal tergites mostly black but each tergite with a pale band along posterior margin; legs with conspicuous dark and pale rings, as the femora are largely dark and have a short preapical pale ring; tibiae with three dark rings and two pale rings; superior volsella with a narrow base bearing four inner setae; and a narrow, apically hooked and pointed distal horn bearing a long lateral seta arising at near the tip.
Distribution.China (Xinjiang); Holarctic region.Diagnosis.The male imago can be separated from the known species of Stictochironomus based on following combination of features: wing with only one dark area around the cross vein r-m; legs with conspicuous dark and pale rings, as the fore femur is yellowish, with two apical dark brown rings, the mid-and hind femur brown, with one apical pale ring; superior volsella with a narrow base bearing 6-7 inner setae; and a narrow, apically hooked and pointed distal horn bearing a long lateral seta arising at distal 1/3.
Distribution.China (Hebei, Qinghai); Holarctic region.An updated key for the known males of Stictochironomus from China.
The following key is modified from Qi et al. (2008) [18], with color pattern variations seen in S13.

1.
Wing with only one dark area around cross vein r-m

Figure 1 .
Figure 1.Phylogeny tree for Stictochironomus based on DNA barcode sequences.The numbers on the branches refer to the bootstrap support, varying from 75 to 100/ML; bootstrap values vary from 70 to 100 and posterior probabilities vary from 0.95 to 1 (* means that no support indicated on nodes); different colors mean different species.

Figure 1 .
Figure 1.Phylogeny tree for Stictochironomus based on DNA barcode sequences.The numbers on the branches refer to the bootstrap support, varying from 75 to 100/ML; bootstrap values vary from 70 to 100 and posterior probabilities vary from 0.95 to 1 (* means that no support indicated on nodes); different colors mean different species.

Figure 7 .
Figure 7. Male adult of Stictochironomus trifuscipes sp.nov.: (A) wing; (B) hypopygium in dorsal view; (C) hypopygium in ventral view.Type material.Holotype: male (BOLD and TZU sample ID: ZJCH206; Field ID: BSZ73), China, Zhejiang Province, Lishui City, Qingyuan County, Baishanzu National Nature Reserve, N 27.581944, E 117.154722, 12 August 2020, leg.C. Song, light trap; Paratype: 1 male, China, Fujian Province, Nanping City, Wuyishan County, Wuyi Mountain National Forest Park, N 27.601667, E 117.789167, 16 April 2021, leg.K.H. Zhong, light trap.Diagnostic characteristics.The male imago can be separated from the known species of Stictochironomus based on following combination of features: the antenna and antennal plumage is mostly dark brown; the wing is smokey with markings; the thorax is yellowish

Table 1 .
Variable and informative sites, and average nucleotide composition in the aligned COI gene sequences.

Table 2 .
The highest intraspecific divergence in Stictochironomus species; * means only one sample.

Table 2 .
The highest intraspecific divergence in Stictochironomus species; * means only one sample.

Table 4 .
Comparison of main characteristics that show differences between male S. han and S. quadrimaculatus sp.nov.Stictochironomus trifuscipes Song and Qi, sp.nov.

Table 4 .
Comparison of main characteristics that show differences between male S. han and S. quadrimaculatus sp.nov.Stictochironomus trifuscipes Song and Qi, sp.nov.