Dtsch. Entomol. Z. 70 (2) 2023, 387-401 | DOI 10.3897/dez.70.108096 yee ee BERLIN Two new species of Deuterophlebia Edwards, 1922 from Southwestern China (Diptera, Deuterophlebiidae) Xuhongyi Zheng!, Changfa Zhou? 1 The Key Laboratory of Jiangsu Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China https://zoobank. org/95 E6EB75-94FA-4DA 7-9DE4-D3324C9EDICE Corresponding author: Changfa Zhou (zhouchangfa@njnu.edu.cn) Academic editor: Matthias Seidel # Received 16 June 2023 @ Accepted 9 October 2023 Published 23 Ocotber 2023 Abstract The family Deuterophlebiidae 1s considered the most plesiomorphic Diptera family, with striking morphology and life history. In this study, we provide detailed descriptions and figures of two new species from Southwestern China: Deuterophlebia pseudopoda sp. nov. and Deuterophlebia pachychaeta sp. nov., along with genetic distances of COI sequences between the seven known Chinese species of Deuterophlebia Edwards, 1922. The two new species can be identified by the adults’ male terminalia, head structures, male and female antennae and mesothoracic spines or abdominal projections of the pupae. As a result, China is currently the country with the highest Deuterophlebia species diversity. Key Words aquatic insects, barcoding, COI, genetic identification, mountain midge Introduction Deuterophlebiidae (or mountain midge) is a monotypic family in the order Diptera. Members of this family show unique morphology and living habits. In contrast to other dipteran groups, the larvae of Deuterophlebia Edwards, 1922 have paired pseudopodia and live in torrent streams; adults mate at sunrise, male adults die immediately after mating, while females shed their wings, go back to the wa- ter and lay eggs (Courtney 1989; Zheng et al. 2022). This kind of specialized taxon and its living history has attract- ed more and more attention and studies in recent years. The biogeographic and phylogenetic status of the fam- ily is still enigmatic (Courtney 1994; Zheng et al. 2022). In the latest phylogenetic topology based on mitochon- drial sequences, Deuterophlebiidae was considered the earliest branching lineage of Diptera, a sister group of all other dipterans (Zhang et al. 2022). This result differs from previous inferences, which usually consider Bleph- ariceridae and Deuterophlebiidae as sister groups (Court- ney 1991). Obviously, many questions are still waiting for answers in the systematic study of this family. Up to 2022, there were 14 named species tn this fami- ly, eight species reported from the Palearctic Region (Ki- takami 1938; Jedli¢ka and HalgoS 1981; Courtney 1994; Sofi et al. 2020) and six from the Nearctic realm (Pennak 1945, 1951; Shewell 1954; Kennedy 1958, 1960; Court- ney 1989, 1990). China was a blank area in the distribu- tion maps (Courtney 1994). In 2022, five species were reported from Southwestern and Eastern China, bringing some new insights into the diversity and biogeography of Deuterophlebiidae (Zheng et al. 2022). Encouraged by that, extensive investigations were carried out, and it was hypothesized that China is possibly the center of the diversity of Deuterophlebiidae. More studies on these Chinese species can provide more information on their evolution and dispersal. During a trip in Yunnan Province of southwestern Chi- na, some Deuterophlebia adults and pupae were collected. After careful examination, they were recognized as two new species. We presented detailed images of their adults and pupae, described them in detail and compare them with the known species using COI and morphological charac- ters, including some that have not been mentioned before. Copyright Xuhongyi Zheng & Changfa Zhou. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 388 Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China Materials and methods Pupae were hand-picked from the surface of stones un- derwater. Adults were found floating nearby the banks of creeks. Specimens were examined under a stereomicro- scope (Nikon SMZ 745T). Habitus of pupae and adults were photographed using a SONY a7R II camera with a LAOWA 25 mm 5x macro lens. Details of heads and legs were studied by dissection and treatment in 10% NaOH (30 °C, 30 min), observed and photographed with a cam- era (Nikon 501) coupled on a microscope. Terminology mainly follows that of Courtney (1994). All specimens were preserved in 85% ethanol and deposited in the Dip- tera collection of College of Life Sciences, Nanjing Nor- mal University and School of Grain Science and Technol- ogy, Jiangsu University of Science and Technology. To associate the pupae and winged stages, total genom- ic DNA was extracted from the abdomen of specimens using Animal Genomic DNA Kit (TsingKe Biotech Co., Bejing, China). The mitochondrial gene cytochrome c oxidase subunit I fragment was PCR-amplified using the Premix Tag (Takara Bio Inc., Beijing, China) with forward primer LCO1490 (5’°-GGTCAACAAATCATAAAGA- TATTGG-3’) and reverse primer HCO2198 (5’-TA- AACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). PCR conditions included initial denaturation at 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for 40 s, with a final extension at 72 °C for 10 min (Zheng et al. 2022). Sequences were aligned using Muscle, and the K2P genetic distances were estimated in MEGA11 (Ta- mura et al. 2021). The intraspecific genetic distances were lower than 0.01. The sequence of each species has been uploaded to GenBank (Table 1) with the same specimen information as for the holotype of each species. Table 1. GenBank accession numbers of COI sequences used in this research. Species GenBank Accession Number D. sinensis Zheng et al., 2022 ON637906 D. yunnanensis Zheng et al., 2022 ON637909 D. alatus Zheng et al., 2022 ON637916 D. acutirhina Zheng et al., 2022 ON637914 D. wuyiensis Zheng et al., 2022 ON637910 D. pseudopoda sp. nov. OR064529 (this study) D. pachychaeta sp. nov. OR064528 (this study) Results Deuterophlebia pseudopoda sp. nov. https://zoobank.org/E1B5D9F5-9797-4350-8 1 5F-197CEBE47826 Description. Male adults. Body length ca. 2.2-2.6 mm (n=5), uniformly brownish black (Fig. 1). Head brownish black, flattened, nearly trapezoidal, width ca. 0.50 mm, folded backward under thorax, hidden in dorsal view (Fig. 1). Head densely covered with microtrichia. Median dez.pensoft.net wf — Figure 1. Male adult of D. pseudopoda sp. nov. Scale bar: 1.0 mm. clypeal lobe slightly convex, semicircle shaped, with around 20 sharp setae (Fig. 2A). Mouthparts in form of an invaginated tubule, oral region depressed (Fig. 2A, B). Edges of oral region (or mouth opening) ridged, convex medially on ventral ridge, forming a blunt mental tooth (Fig. 2A, B). Postgena and oral region with sparser mi- crotrichia than other regions, a pair of tentorial pits pres- ent on each side of oral region (Fig. 2A, B). Compound eyes glabrous, width ca. 0.18 mm, distance between eyes ca. 0.30 mm (Fig. 2A). Antennae 8.5—10.0 mm (n=5) (Fig. 2A, C). Scape oval shaped, pedicel globular, both covered with microtrich- ia (Fig. 2A, C). Flagella four segmented, flagellomeres I-III slender cylindrical, each with a subapical tubercle on front margin, bearing 9-12, 6-9 and 4-6 digitiform setae respectively (Fig. 2C). Flagellomere IV flattened and elongated, broader than flagellomeres I-HI, narrowed gradually, with curved hair-like setae on the anterior side of basal half, apical half generally glabrous but bear- ing 4—5 clusters of curved hair-like setae, apex slightly expanded with some curved hair-like setae (Fig. 2C). Antennal ratio: 4.0: 2.0: 5.0: 3.0: 3.0: 238.0 (Figs 1, 2C). Flagellomere IV about 14 combined length of five basal antennal articles or about 4x body length (Fig. 1). Thorax uniformly brownish black, densely covered with microtrichia (Fig. 1). Pronotum almost hidden, me- sonotum strongly expanded (Fig. 1). Wings ca. 4.0 mm, uniformly set with dark micro-tubercles, grayish translu- cent, cubital area greatly enlarged, costal margin slightly thickened (Fig. 1). Outer margin fringed with soft hair- like setae, denser and longer on cubital margin (Fig. 1). Veins radially arranged, pale and inconspicuous (Fig. 1). Halteres transparent, ca. 0.35 mm (Fig. 1). Legs brownish black, slender, sharing similar chaeto- taxy with four types of setae: (1) microtrichia, densely covered on all segments; (2) sharp macrotrichia, sparsely on dorsal margin of femora and tibiae; (3) long capitate setae, ventrally on tarsomeres I-IV of each leg, distal half of ventral edge of all tibiae, surrounding the top of fore- and midtibiae, and also densely arranged radially on each empodium; (4) digitiform setae, 1-3 pairs for each tar- somere (Fig. 2D-—F). In all legs, coxae much broader than trochanters, coxae about twice the length of trochanters (Fig. 2D-F). In foreleg, femur: tibia: tarsus = 9.0: 14.0: 14.0; femur slightly flattened, tibia slender, cylindrical, Dtsch. Entomol. Z. 70 (2) 2023, 387-401 389 ae Figure 2. Male adult of D. pseudopoda sp. nov.: A. Head (ventral view); B. Oral region (ventral view); C. Flagellomeres; D. Foreleg; E. Midleg; F. hindleg; G. Terminalia (dorsal view). Abbreviations: cp, median clypeal lobe; pd, pedicel; fl, flagellomere; or, oral region; mt, mental tooth; pg, postgena; co, coxa; tr, trochanter; fe, femur; tb, tibia; ts, tarsomere; em, empodium; dp, dorsal plate; gc, gonocoxite; gs, gonostylus; ad, aedeagus. Scale bars: 0.1 mm (A, C, G); 0.05 mm (B); 0.5 mm (D-F). dez.pensoft.net 390 and gradually broader apically; tarsomere I: II: III: IV: V = 64: 2.3: 1.4: 1.4: 1.0, tarsomeres I-IV cylindrical, tarsomere V conical; empodium shell-shaped, length sub- equal to tarsomere V; claw slender, tapered, shorter than empodium (Fig. 2D). Midleg shortest among all legs, sim- ilar to foreleg, femur: tibia: tarsus = 8.0: 10.5: 12.0, tar- somere I: IT: If: IV: V=6.0: 1.9: 1.1: 1.1: 1.0 (Fig. 2E). In hindleg, femur: tibia: tarsus of hindleg = 10.0: 13.5: 10.0, tarsomere T: Il: II: TV: V = 3.5: 1.5:.1.2: 1.2: 1.0 Fig. 2F). Abdomen brownish black, densely covered with mi- crotrichia, nine segmented, tapering posteriorly (Fig. 1). First two segments strongly fused with each other, paler and shorter than others (Fig. 1). Segment VII in form ofa Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China short chitin ring (Fig. 1). Sternite IX almost glabrous, fused with dorsal plate, connected with gonocoxite (Fig. 2G). Gonocoxite with posterior projection which length sub- equal to gonostylus (Fig. 2G). Gonostylus subequal to the dorsal plate in length, flattened, oval-shaped, flexor sur- face with numerous curved sharp setae (Fig. 2G). Dorsal plate parallel-sided, posterior margin slightly depressed without cleft with some stout curved setae on margin (Fig. 2G). Aedeagus in form of a smooth tube, length sub- equal to gonostylus and dorsal plate (Fig. 2G). Female adult. Body length ca. 2.0 mm (n= 1). Besides sexual differences, generally similar to the males except following features (Fig. 3A—D). B C D ~ Figure 3. Female adult of D. pseudopoda sp. nov.: A. Head (ventral view); B. Foreleg; C. Midleg; D. Hindleg. Scale bars: 0.1 mm. dez.pensoft.net Dtsch. Entomol. Z. 70 (2) 2023, 387-401 Head width ca. 0.36 mm (Fig. 3A). Median clypeal lobe strongly protruded medially with ca. 20 setae (Fig. 3A). Oral region located near anterior margin of head (Fig. 3A). Compound eyes more prominent than males (Fig. 3A). Antenna ca. 0.3 mm. Scape slender oval shaped, pedicel globular, both scape and pedicel covered with microtri- chia and bearing several sharp setae (Fig. 3A). Flagel- lomere I slender cylindrical, flagellomeres II-III slender oval shaped, flagellomere IV dripping shaped, strongly narrowed basally (Fig. 3A). Each of flagellomeres J-III bearing ca. 5 digitiform setae apically, flagellomere IV with 4 sharp setae. Antennal ratio ca. 7.0: 3.0: 10.0: 4.0: 5.0: 4.0 (Fig. 3A). 3911 Legs sharing similar chaetotaxy and exhibiting three types of setae, chaetotaxy similar to males but without capitate setae (Fig. 3B—D). In foreleg and midleg, femur: tibia: tarsus = 1.0: 2.0: 1.3, tarsomere I: II: HI: ['V: V = 1.0: 0.8: 0.8: 0.8: 2.4. (Fig. 3B, C) In hindleg, femur: tibia: tarsus = 1.0: 1.7: 1.1, tarsomere I: II: HW: 1'V: V = 1.0: 0.9: 0.9: 0.9: 2.7 (Fig. 3D). Claws of all legs similar, paired, stout and curved, with a blunt protrusion in the middle (Fig. 3B—D). Empodium in form of a long and hairy spine, subequal to the length of claw (Fig. 3B—D). Male pupae. Pupae flattened oval shaped, length 2.3 mm (n = 2), width 1.6 mm. Dorsal integument dark brown, divided into 11 segments (Fig. 4A, B). D Figure 4. Pupae of D. pseudopoda sp. nov.: A. Male pupa (dorsal view); B. Male pupa (ventral view); C. Female pupa (dorsal view); D. Female pupa (ventral view). Scale bars: 1.0 mm. dez.pensoft.net 392 Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China C Figure 5. Pupa of D. pseudopoda sp. nov.: A. Thoracic spine (dorsal view); B. Gill (ventral view); C. Posterior end (ventral view). Abbreviation: pp, posterolateral projections. Scale bars: 0.1 mm. Prothorax fused with mesothorax, forming a conical segment with a median suture (Fig. 4A, B). Mesotho- racic lateral margins each with a sharp spine and a gill (Figs 4A, B, 5A, B). Spines ca. 0.4 mm, slightly curved, dark brown, originated from a round base (Fig. 5A, B). Ventral gills light to dark brown, length subequal to the dor- sal spines, hand-shaped and consisting of three filaments: posterior filament shorter, pointing backward; anterior two filaments similar in shape, twisted and light in color apically (Fig. 5A, B). Metathorax completely surround- ed by mesothorax and first abdominal segment (Fig. 4A, B). Abdominal segments J-II with a pair of anterolateral projections, each projection pointing forward and bearing ca. 13 spines (Fig. 4A, B). Segments VI—VII with a pair of posterolateral projections, projections foot-shaped and each bearing ca. 8 spines (Fig. SC). Segment VIII shield- shaped, surrounded by segments VII and IX (Fig. 4A, B). Adult structures visible on ventral side (Fig. 4A, B). Head present directly below mesothorax; anten- nal sheaths in form of a large elliptic ring, surrounding body 2.0 times (Fig. 4A, B). Leg sacs extended to pos- terior end of antennal ring, strongly expanded apically. dez.pensoft.net Abdominal segments IN—V with a pair of black adhesive discs (Fig. 4A, B). Female pupae. Length ca. 2.2 mm (n = 2), width ca. 1.5 mm (Fig. 4C, D). Dorsal morphology similar to male except for smaller mesothorax (Fig. 4C, D). Gender can be identified through the absence of antennal ring, apex of female leg sheaths not expanded (Fig. 4C, D). Material examined. Holotype: male adult, China: Yunnan Province, Gongshan County, Dulongjiang Town- ship, Dulongjiang River, 27°50'14.16"N, 98°19'54.2"E, 1470 ma.s.1., 4.11.2023, Xuhongyi Zheng leg. Paratypes: 6 male adults, 1 female adult, 2 male pupae, 2 female pupae, same locality and data as holotype. Diagnosis. Male adults of Deuterophlebia pseudopo- da sp. nov. can be identified by their terminalia: gonosty- lus short, length of gonostylus subequal to the gonocoxite and dorsal plate; posterior margin of dorsal plate slightly depressed but without a median cleft (Fig. 2G). Such a terminalia differs from the 19 named Deuterophlebia spe- cies (Courtney 1990, 1994; Zheng et al. 2022). The shape of their heads is also distinct among known species: me- dian clypeal lobe slightly convex, inner side of compound Dtsch. Entomol. Z. 70 (2) 2023, 387-401 eye without a protruded corner (Fig. 2A) (Courtney 1990, 1994; Zheng et al. 2022). Female adults of D. pseudopoda sp. nov. can be recog- nized through a combination of the pronounced median clypeal lobe, chaetotaxy of antennae, and shape of flagel- la (Fig. 3A). Compared to other species, its pronounced median clypeal lobe is similar to D. oporina Courtney, 1994 and D. nipponica Kitakami, 1938, but can be dif- ferentiated from them by its antenna: flagellomeres I-III bearing ca. 5 digitiform setae respectively, flagellomere IV with only 4 sharp setae, antennal ratio = 7.0: 3.0: 10.0: 4.0: 5.0: 4.0 (Courtney 1990, 1994; Zheng et al. 2022). Pupae can be easily identified by their foot-shaped posterolateral projections of abdominal segments VI-— VII (Fig. 5C). This feature is absent in the 15 species with clear pupal stage (Courtney 1990, 1994; Zheng et al. 2022). They can also be separated from other spe- cies by their single mesothoracic spines, gills consisting of three filaments, and absence of conspicuous thoracic ridges (Figs 4A—D, 5A, B) (Courtney 1990, 1994; Zheng etal 2022): Etymology. The specific epithet “pseudopoda” means “pseudopodia”, refers to the pseudopodia-like lateral pro- jections of pupal abdominal segments VI—VII. Distribution. China (Yunnan Province). Deuterophlebia pachychaeta sp. nov. https://zoobank. org/99503019-F78C-49B5-B602-8CE1D53BD96A Description. Male adults. Body length ca. 3.0 mm (n = 2), uniformly brownish black. Head brownish black, flattened and nearly trapezoidal, width ca. 0.50 mm (Fig. 6A). Head densely covered with microtrichia. Me- dian clypeal lobe slightly convex, semicircle shaped with around 20 sharp setae (Fig. 6A). Mouthparts in form of an invaginated tubule, oral region depressed (Fig. 6A, B). Edges of oral region (or mouth opening) ridged, forming a protruded angle on inner side of each eye (Fig. 6A, B). Ventral ridge of oral region convex medially and form- ing a blunt mental tooth (Fig. 6A, B). Postgena and oral region with microtrichia but more sparse than other re- gions, a pair of tentorial pits present on each side of oral region (Fig. 6A, B). Compound eyes glabrous, width ca. 0.13 mm, distance between eyes ca. 0.30 mm (Fig. 6A). Antennae ca. 13.0 mm (n=5) (Fig. 6A, C). Scape oval shaped, pedicel globular, both scape and pedicel covered with microtrichia (Fig. 6A, C). Flagella four segmented, flagellomeres I-III slender cylindrical, each with a sub- apical tubercle on front margin and bearing 8-10 digiti- form setae (Fig. 6C). Flagellomere IV flattened and elon- gated, with curved hair-like setae on the anterior side of basal half, apical half generally glabrous but bearing 4—5 clusters of curved hair-like setae, apex slightly expanded with some curved hair-like setae (Fig. 6C). Antennal ratio ca. 4.0: 2.0: 5.0: 3.0: 3.0: 287.0, flagellomere IV about 17x combined length of five basal antennal articles or about 4x body length (Fig. 6C). 393 Thorax brownish black, densely covered with mi- crotrichia. Mesonotum strongly expanded. Wings ca. 5.5 mm, shape, venation and other features similar to D. pseudopoda sp. nov. (Fig. 1). Legs brownish black, slender, three pairs sharing sim- ilar chaetotaxy exhibiting four types of setae: (1) micro- trichia, densely covered on all segments; (2) sharp mac- rotrichia, sparsely on dorsal margin of femora and tibiae; (3) long capitate setae, on ventral side of tarsomere I-IV of each leg, distal half of ventral edge of all tibiae, sur- rounding the top of fore- and midtibiae, and also densely arranged radially on each empodium; (4) digitiform se- tae, 1-3 pairs for each tarsomere (Fig. 6D-F). In fore- leg, femur: tibia: tarsus = 11.0: 17.0: 16.0; femur slightly flattened, tibia slender cylindrical and gradually broader apically; tarsomere I: II: HI: IV: V = 8.5: 2.0: 2.0: 1.5: 0.8, tarsomeres I-IV cylindrical, tarsomere V conical; empodium shell-shaped, length subequal to tarsomere V; claw slender tapered, shorter than empodium (Fig. 2B) (Fig. 6D). Midleg shortest among all legs, features gen- erally similar to foreleg, femur: tibia: tarsus = 10.0: 12.5: 14.0, tarsomere I: I: TI: IV: V = 8.5: 2.0: 2.0: 1.5: 0.8 (Fig. 6E). In hindleg, femur: tibia: tarsus of hindleg = 14.0: 17.0: 11.0, tarsomere I: II: III: IV: V = 3.0: 2.0: 2.0: 1.5: 0.8 (Fig. 6F). Abdomen brownish black, densely covered with mi- crotrichia, nine segmented, tapering posteriorly. Segment VIII in form of a short chitin ring, sternite [IX almost glabrous, connected with dorsal plate and gonocoxite (Fig. 6G). Gonocoxite with posterior projection ca. 1/2 length of gonostylus (Fig. 6G). Gonostylus subequal dor- sal plate, flattened, slender oval shaped, flexor surface with numerous curved sharp setae (Fig. 6G). Dorsal plate parallel-sided, posterior margin slightly depressed with a median cleft and some stout setae on margin (Fig. 6G). Aedeagus in form of a smooth tube, length subequal to gonostylus, longer than dorsal plate (Fig. 6G). Female adult. Body length ca. 2.3 mm (n= 1). Besides sexual differences, generally similar to the males except following features (Fig. 7A—D). Head width ca. 0.40 mm (Fig. 7A). Median clypeal lobe slightly convex with ca. 20 setae (Fig. 7A). Oral re- gion located near anterior margin of head, postgena and oral region with microtrichia but more sparse than other regions (Fig. 7A). Compound eyes more prominent than males (Fig. 7A). Antenna ca. 0.3 mm. Scape slender oval- shaped, pedicel globular, both scape and pedicel covered with microtrichia with several sharp setae (Fig. 7A). Flagellomere I slender cylindrical, flagellomeres H-III slender oval shaped, flagellomere IV dripping shaped (Fig. 7A). Flagellomeres I-IV bearing 2, 4, 10 and 2 dig- itiform setae apically respectively, flagellomere IJ-I'V with 1 and 4 sharp setae respectively (Fig. 7A). Antennal ratio = 7.0: 4.0: 10.0: 5.0: 5.0: 4.0 (Fig. 7A). Legs sharing similar chaetotaxy and exhibiting three types of setae, chaetotaxy similar to males but without capitate setae (Fig. 7B—D). In all legs, coxae about twice longer and broader than trochanters, femora slightly dez.pensoft.net 394 Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China Figure 6. Male adult of D. pachychaeta sp. nov.: A. Head (ventral view); B. Oral region (ventral view); C. Flagellomeres; D. Foreleg; E. Midleg; F. Hindleg; G. Terminalia (dorsal view). Scale bars: 0.1 mm (A, C, G); 0.05 mm (B); 0.5 mm (D-F). dez.pensoft.net Dtsch. Entomol. Z. 70 (2) 2023, 387-401 395 Figure 7. Female adult of D. pachychaeta sp. nov.: A. Head (ventral view); B. Foreleg; C. Midleg; D. Hindleg. Scale bars: 0.1 mm. dez.pensoft.net 396 Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China flattened, tibiae and tarsomeres slender cylindrical (Fig. 7B—D). In foreleg and midleg, femur: tibia: tarsus = 1.0: 2.0: 1.5, tarsomere I: II: Ill: IV: V = 1.2: 0.8: 0.8: 0.8: 2.4 (Fig. 7B, C). In hindleg, femur: tibia: tarsus = 1.0: 1.7: 1.1, tarsomere I: II: III: 1V: V = 1.0: 0.8: 0.8: 0.8: 2.4 (Fig. 7D). Claws of all legs similar, paired, stout and curved, with a blunt protrusion in the middle (Fig. 7B—D). Empodium in form of a long and hairy spine, subequal to the length of claw (Fig. 7B—D). Male pupae. Pupae flattened oval shaped, length 2.8 mm (n= 2), width 2.0 mm (Fig. 8A, B). Dorsal integ- ument dark brown, divided into 11 segments (Fig. 8A, B). Prothorax fused with mesothorax, forming a conical segment with a median suture (Fig. 8A, B). Mesothoracic lateral margins each with a pair of sharp spines and a gill (Figs 8A, B, 9A, B). Spines ca. 0.4 mm, expanded basally, slightly curved, dark brown, originated from a round base (Fig. 9A). Ventral gills light to dark brown, length subequal C Figure 8. Pupae of D. pachychaeta sp. nov.: A. Male pupa (dorsal view); B. Male pupa (ventral view); C. Female pupa (dorsal view); D. Female pupa (ventral view). Scale bars: 1.0 mm. dez.pensoft.net Dtsch. Entomol. Z. 70 (2) 2023, 387-401 397 Figure 9. Male pupa of D. pachychaeta sp. nov.: A. Thoracic spine (dorsal view); B. Gill (ventral view). Scale bar: 0.1 mm. to the dorsal spines, each including four filaments: poste- rior filament single and short, pointing backward; anterior three gill filaments on a common base, similar in shape, slender and twisted; second and third filaments fused at base (Fig. 9B). Metathorax completely surrounded by me- sothorax and first abdominal segment (Fig. 8A, B). Abdominal segment I and II similar, each with a pair of anterolateral projections, each projection pointing for- ward and bearing ca. 18 spines (Fig. 8A, B). Lateral mar- gins of segments VI and VII with several spines on both dorsal and ventral sides. Segment VIII shield-shaped, surrounded by segments VII and IX (Fig. 8A, B). Adult structures visible on ventral side (Fig. 8B). Head present below mesothorax; antennal sheaths in form of a large elliptic ring, surrounding body 2.0 times (Fig. 8B). Leg sacs extended to posterior end of antennal ring, strongly expanded apically. Abdominal segments III—V each with a pair of black adhesive discs (Fig. 8B). Female pupae. Length ca. 2.8 mm (n = 1), width ca. 2.0 mm (Fig. 8C, D). Dorsal morphology similar to male except for smaller mesothorax (Fig. 8C, D). Gender can be identified through the absence of antennal ring, apex of female leg sheaths not expanded (Fig. 8C, D). Material examined. Holotype: male adult, China: Yun- nan Province, Nujiang Lisu Autonomous Prefecture, Gong- shan County, Dulongjiang Township, Dulongjiang River, 27°50'14.16"N, 98°19'54.2"E, 1470 m as.l., 411.2023, Xuhongyi Zheng leg. Paratypes: 1 male adult, 2 male pupae, same locality and data as holotype; 1 female adult dissected from a pupa, Yunnan Province, Nujiang Lisu Autonomous Prefecture, Fugong County, 26°57'17.32"N, 98°52'3.04"E, 1265 m a.s.l., 3. VII. 2022, leg. Xuhongyi Zheng, Zhenxing Ma, Zhiteng Chen & Pengxu Mu. Diagnosis. Male adults of Deuterophlebia pachychaeta sp. nov. can be identified by their head shape and chaetotaxy of antennae (Fig. 6A, C). Following the keys of Courtney (1994) and Zheng et al. (2022), males of this species, which are similar to D. mirabilis Edwards, 1922, D. blepharis Courtney, 1994 and D. sinensis Zheng et al., 2022, can be separated from other Asian species by their relatively long antennae (longer than 8.0 mm), and post- gena covered with microtrichia (Fig. 6B). Chaetotaxy on flagellomeres can be used to differentiate those four sim- ilar species: in the new species, there were ca. 8—10 dig- itiform setae on each of flagellomeres I-III (Fig. 6A, C) (Courtney 1990, 1994; Zheng et al. 2022). Female adults of D. pachychaeta sp. nov. can be rec- ognized through a combination of the smooth median clypeal lobe, chaetotaxy of antennae, and shape of flagel- la (Fig. 7A). Its smooth and convex median clypeal lobe is similar to D. mirabilis Edwards, 1922, D. blepharis Courtney, 1994, D. sinensis Zheng et al., 2022, D. yunna- nensis Zheng et al., 2022 and D. wuyiensis Zheng et al., 2022, but can be differentiated from them by its antenna: flagellomeres I-IV bearing ca. 2, 4, 10 and 2 digitiform setae respectively, flagellomere IV with additionally 4 sharp setae; antennal ratio = 7.0: 4.0: 10.0: 5.0: 5.0: 4.0 (Fig. 7A) (Courtney 1990, 1994; Zheng et al. 2022). Pupae can be identified by their mesothoracic spines: there are two spines on each side, expanded basally (Fig. 9A, B). Compared with the 15 species with a clear pupal stage (Courtney 1990, 1994; Zheng et al. 2022), only D. acutirhina Zheng et al., 2022 has similar basally expanded spines, but less expanded than in the new spe- cies. They can also be distinguished by the absence of abdominal dots (Zheng et al. 2022). Etymology. The specific epithet “pachychaeta” is a combination of the words pachy and chaeta, which refers to its thickened spines on pupal mesothorax. Distribution. China (Yunnan Province). dez.pensoft.net 398 Xuhongyi Zheng & Changfa Zhou: Two new Deuterophlebia species from China Keys to Asian Deuterophlebia Key to male adults Modified from the key by Zheng et al. 2022. 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