A New Species of Limnephilus (Insecta: Trichoptera: Limnephilidae) from China, with Revision of the Genus Limnephilus on the Chinese Mainland

Simple Summary As the most diverse genus in the family Limnephilidae, Limnephilus Leach, 1815 (194 extant species and 9 fossil species), has a wide distribution and a broad range of ecological tolerances among its species. Although it has a high species diversity in the Holarctic region, only 16 species have been recognized in China until now, of which 7 are endemic to China. In this study, a new species of the genus Limnephilus was found. The adult females, larvae, and pupae of this new species were associated with the adult males based on COI sequencing, and all the life stages except for the egg stage are described and illustrated. In addition, the biological and habitat information for this new species is provided. Furthermore, all of the Limnephilus species from the Chinese mainland are revised at the species group level, and diagnoses for species groups and species (keys of groups and species) are given, along with a distribution map. Abstract Fifty individuals of Limnephilus from the Qinghai Province, China, were examined, and their COI barcode sequences were extracted and analyzed. Forty individuals of Limnephilus from the Insect Collection of Nanjing Agricultural University (ICNAU), China, were examined, and photos of the male genitalia of four Limnephilus species are here presented. The males, females, larvae, and pupae of a new species, Limnephilus deqianensis n. sp., associated via COI barcode sequences, are described and illustrated. Ecological photos of the male, pupal case, and the habitat of the new species L. deqianensis n. sp. are also provided. Five species groups containing all seventeen Chinese Limnephilus species are revised. Diagnoses, keys, and a distribution map of them are provided. All of the sequences have been uploaded to GenBank. All specimens are deposited in the ICNAU, Nanjing, Jiangsu Province, P. R. China.


Introduction
Limnephilidae Kolenati, 1848, is one of the most diverse caddis families of the Northern Hemisphere, containing 4 subfamilies and 101 genera [1].
Limnephilus Leach, 1815, is the most diverse genus in the family Limnephilidae [2], containing 194 extant species and 9 fossil species [1,3,4]. Out of all known species, 82 are endemic to the Nearctic region; 44 to the West Palearctic region; 20 to the East Palearctic region; 9 to the Oriental region; 3 to the Neotropical region; and 29 of them are distributed in 2 regions, with 16 of them distributed in 3 or more regions.

Materials and Methods
Specimens of the new species studied in this research were collected in Qinghai Province, China. A total of 50 specimens, including 20 adult males and 20 females, 2 larvae, and 8 pupae, were examined in this research. Additional specimens studied in this research were from ICNAU.

Sample Collection
Larvae and pupae were collected by handpicking specimens off of stones in a highaltitude stream ( Figure 1C). Only one male adult ( Figure 1A) was captured by hand during collection; the other 40 adults were reared from pupae collected at the site. The adult male, larvae and some pupae were preserved in 95% ethanol in the field directly after collection. Other collected pupae were returned to the temporary residence for rearing and the adults that emerged from them were stored in 95% ethanol after they were completely sclerotized about 3-5 days later. Two larvae were found in their pupal cases. We determined that the 2 larvae are the final instar when comparing the metamorphotype larval frontoclypeal apotome remaining in the pupal cases to our reared pupae. Ecological photos of the habitat, the collected male, and pupal case ( Figure 1B) were taken with a VIVO X60Pro smartphone. Types and voucher specimens were deposited at the Insect Collection of Nanjing Agricultural University (ICNAU), Nanjing, Jiangsu Province, P. R. China. domen was stored in a microvial together with the remainder of the specimen in 75% ethanol.
The terminology for the male genitalia follows that presented by Schmid [32]; the terminology for the female genitalia was adapted from that of Schmid [32], and for the female spermatheca, the terminology was adapted from that of Nielson [33]. The terminology for the larva corresponds with that of Wiggins [34], while for the pupae, it conforms to that of Denning [35].

Morphological Study
The following methods were used for specimen preparation: abdomens of the adults were cut from their bodies, transferred into 10% NaOH solution, and heated to 90 • C for 2 h to remove all of the non-chitinous tissues. Then, the cleaned abdomens were rinsed in distilled water and mounted on a depression slide with 75% ethanol for examination. Photographs of the male and female genitalia, larvae, and pupae were taken by using a stereomicroscope (Nikon SMZ18), and photos of the female spermathecal structure were taken by using a compound microscope (Olympus BX41). Each of these pieces of equipment had a built-in camera and Nis-Element D ® software (Version 3.22.14, Nikon, Shanghai, China). A series of photos at different focal depths were taken and stitched together with Zerene Stacker ® software (Zerene Systems LLC, Richland, WA, USA) and then arranged using Adobe Photoshop ® (version 2017.0.0, Adobe, San Jose, CA, USA). Subsequently, each abdomen was stored in a microvial together with the remainder of the specimen in 75% ethanol.
The terminology for the male genitalia follows that presented by Schmid [32]; the terminology for the female genitalia was adapted from that of Schmid [32], and for the female spermatheca, the terminology was adapted from that of Nielson [33]. The terminology for the larva corresponds with that of Wiggins [34], while for the pupae, it conforms to that of Denning [35].

Molecular Analysis
The left hind legs of 12 individuals (males, females, and larvae) and left forelegs of 5 individuals (pupae) were taken from the bodies for DNA extractions. Extraction followed the animal tissue protocols of the Ezup Column Animal Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). The PCR amplification, fragment sequencing, and analyses followed the procedures of Xu [36]. The primers [37] (LCO1490/HCO2198) are listed in Table 2. Neighbor-joining (NJ) trees were constructed using Mega v10.2.6 [38]. Calculation parameters were set as follows: Kimura 2-parameter substitution model, pairwise gap deletion and others as defaults. The NJ tree was plugged into ITOL v5 [39]. Female, larval, and pupal specimens were associated with adult males for L. deqianensis n. sp. according to the criteria outlined by Zhou [40]. Their COI sequences were uploaded to GenBank and their accession numbers are included in Table 3.
Etymology: The specific epithet is from the Chinese name of the type locality, De-qian Town, Qinghai Province.
Male genitalia. Tergum VIII in dorsal view ( Figure 3A) with basal portion subrectangular and distal portion triangular and produced into a bifid apicodorsal lobe; in lateral view ( Figure 3B), basal portion subrectangular, apicodorsal lobe with apex beak-shaped. Segment IX in dorsal view ( Figure 3C Figures 3D and 4A), each somewhat triangular, with lower portion of each inner margin produced into small membranous process, in caudal view ( Figures 3F and 4C), each somewhat semicircular; apical lobes in lateral view ( Figures 3D and 4A), each tapered to apex and with apex slightly curved upwards; in dorsal view ( Figure 3C), each digitate. Inferior appendages fused with segment IX in lower portion of posterior margins; in lateral view ( Figures 3D and 4A), higher than long, each with upper portion thicker than lower portion, upper portion with posterior margins sinuate, and produced into the free portion. Phallic apparatus slender, phallocrypt cylindrical tube ( Figure 3C,D), endotheca membranous. Aedeagus ( Figure 3C-E) slender, tube-like, tapering from base to apex, slightly curved upwards. Parameres (Figrues 3D,G and 4D) each slightly longer than aedeagus, in lateral view tubular with flattened setose apex, and having a small setose lobe on inner side.     Figures 5G and 6E), and vase-like in dorsal view ( Figure 5E); posterior section membranous, elliptic in lateral view ( Figure 5G) and somewhat circular in dorsal view ( Figure 5G).         side ( Figure 10D) bearing 4 black short setae and posterior side ( Figure 10C) 2 setae. Each hind femur ( Figure 10E,F) with upper margin bearing 5 brown spike-like setae and with anterior side ( Figure 10E) having 6 short black short setae and posterior side ( Figure 10F) having 5. Light brown major femoral setae present in all legs. All femora with lower margins bearing inconspicuous setal buds. Mid-and hind-tibiae ( Figure 10C-F) are same shape as foretibiae, but slightly more elongate; tibial spurs present same as foretibiae. Light brown spur-like basal setae present in all legs.  Tracheal gills trifid, bifid or unbranched. Gill forms and their distributions from segments II-VII as in Table 4. Lateral fringe present on each side from segment II to segment VIII.
Dorsal sclerite of segment IX ( Figure 11C) semicircular and yellowish, with about 10 long black spike-like setae arranged in arc, intermingled with 5 short fine black setae. Lateral sclerites ( Figure 11C,D) each with 7 long, dark spike-like setae apically, 1 long black seta subapically, and 1 long seta and about 3 short black setae mesally. Ventral sole plate ( Figure 11D) triangular with anterior margin black in lateral view. Anal proleg claws (   posterior margin having 17-19 curved hair-like setae ( Figure 12C) overlying eye surface. Labrum ( Figure 12B) wide, brown with 14 strong and slender setae on surface. Mandibles ( Figure 12C) blackish brown and crossing each other apically, distal parts relatively slender; median edges of blades slightly serrated; basal parts yellowish and each with 2 strong black hair-like setae. Wing sheaths reaching end of abdominal segment IV. Mid-and hindlegs natatorial; tarsal segments I-IV ( Figure 12E,G) each with dark fringe. Abdominal segments V-VIII fringed ( Figure 12G).  G and 6E,F) somewhat elliptic, spermathecal sclerites slightly sclerotized, each with lateral margin arc-shaped, with apodeme straight and strongly scerotized, somewhat calvate; processus spermatheca obviously divided into 2 sections, anterior section slightly sclerotized, saccular in lateral view ( Figures 5G and 6E), and vase-like in dorsal view ( Figure 5E); posterior section membranous, elliptic in lateral view ( Figure 5G) and somewhat circular in dorsal view ( Figure 5G). and fine teeth more strongly sclerotized than supporting hook plate.
Posterior pair of dorsal hook plates on abdominal terga I and V. Ip ( Figure 13A) with each anterior margin indiscernible, with stubby teeth arranged mesally. Vp ( Figure 13D) subrectangular with 21-23 strong and short hooks more strongly sclerotized than supporting hook plate. Hook numbers on each hook plate as follows: Ip 13-15; IIIa 4; IVa 3-4; Va 4-5; Vp 21-23; VIa 4-5, VIIa 4-5. Gill forms and their distributions from segments II-VII are list as in Table 5. Nineteen mitochondrial COI sequences, including seventeen from Limnephilus deqianensis n. sp., and two downloaded from GenBank were pruned to 658bp to construct the NJ tree ( Figure 14).
The genetic distances are displayed at the node and rounded to four decimal places. Larvae, pupae, and female adults of Limnephilus deqianensis n. sp. were clustered with the male adults of L. deqianensis n. sp. The maximum genetic distances of the L. deqianensis n. sp. cluster were lower than 2.43%. Therefore, we concluded that the association is successful. Mandibles ( Figure 8A,C) shaped for scraping, black, asymmetrical in shape and arrangement of hairs; only left mandible with stiff hairs on subbasomesal edge of dorsal margin. Cardo ( Figure 7B) subpentagonal, with inner and outer ends black, and intermediate portion yellow to brown.
Mesonotum ( Figure 9B) subrectangular dorsally, subdivided longitudinally by mesal suture, each part yellowish brown, with scattered long spike-like setae and random muscle scars; each anterior margin with convex black spot mesal of anterolateral corner, dark spots forming transverse W-shaped mark across middle of pair of nota, posterolateral margins each black and produced ventrolaterad as small triangular projection, posterior margins black except for pair of narrow yellowish brown transverse bands mesal of posterolateral corners.
Mid-and hind-coxae ( Figure 10C-F) longer and slenderer, each bearing a row of black spike-like setae on upper margins. Mid-and hind-trochanters ( Figure 10C-F) each 2-segmented with basal joint subtriangular and shorter than apical joint; apical joint trapezoidal with anterior side (Figure 10D,E) bearing 3 light brown spur-like major setae, with ventral margin having trochanteral brush. Each mid-femur ( Figure 10C,D) with anterior side ( Figure 10D) bearing 4 black short setae and posterior side ( Figure 10C Light brown major femoral setae present in all legs. All femora with lower margins bearing inconspicuous setal buds. Mid-and hind-tibiae ( Figure 10C-F) are same shape as foretibiae, but slightly more elongate; tibial spurs present same as foretibiae. Light brown spur-like basal setae present in all legs.
Abdomen. Abdominal segment I ( Figure 11A,B) with 1 dorsal and 2 lateral humps. About 60 black setae arranged in two rows on tergum I ( Figure 11A); anterior row with about 40 setae and most of them each with basal sclerite; posterior row with about 20 setae and most with basal sclerites smaller than in anterior row, only 2 of them same size as anterior sclerites. Sternum I ( Figure 11B) with 2 triangular median sa2 sclerites and 2 irregular lateral sa3 sclerites; each median sclerite with about 20 setae and lateral sclerites each with about 9 setae.
Pupa: Length 13.7-14.6 mm (n = 10). Antennae slightly shorter than body, each straight at the end; scape longer and thicker than remaining joints. Each eye with entire posterior margin having 17-19 curved hair-like setae ( Figure 12C) overlying eye surface. Labrum ( Figure 12B) wide, brown with 14 strong and slender setae on surface. Mandibles ( Figure 12C) blackish brown and crossing each other apically, distal parts relatively slender; median edges of blades slightly serrated; basal parts yellowish and each with 2 strong black hair-like setae. Wing sheaths reaching end of abdominal segment IV. Mid-and hind-legs natatorial; tarsal segments I-IV ( Figure 12E,G) each with dark fringe. Abdominal segments V-VIII fringed ( Figure 12G).
Anterior pair of dorsal hook plates on abdominal terga III-VII, each with 4-6 strong and curved hooks. IIIa ( Figure 13B) relatively small, suboval; IVa ( Figure 13C), Va ( Figure 13D), VIa ( Figure 13E) and VIIa ( Figure 13F) round and subequal in size, each with strong and fine teeth more strongly sclerotized than supporting hook plate.

Diagnosis
Male: The male genitalia of this new species are similar to those of Limnephilus kaumarajiva Schmid, 1961, from Pakistan, but differ from those of the latter in that (1)  Female: The female genitalia of this new species are similar to those of Limnephilus primoryensis Nimmo, 1995, from Russia (Primorye). This female was described by Nimmo, 1995 [41], but L. deqianensis differs from the latter in that (1) segment IX is subtriangular and broad in the middle in the lateral view (the upper portion of the posterior margin is concave anteriorly, narrow in the upper portion in L. primoryensis); (2) segment IX has paired button-like processus (this processus is absent in L. primoryensis); (3) segment X is narrow in the lateral view (segment X is broad and triangular laterally in L. primoryensis).
Larva: The larva of this new species is similar to that of Limnephilus ademus Ross, 1941, from America; this larva was described by Ruiter [42], but differs from the latter in that (1) the head capsule is without blotches outside the anterior constriction of the frontoclypeal suture (with two blotches primarily outside the anterior constriction of the frontoclypeal suture in L. ademus); (2) the meso and metafemora are without accessory setae on the lateral margins in the new species (accessory setae are present on the lateral margins of the meso and metafemora in L. ademus); (3) 5 small brown sclerites are arranged randomly between the metanotal sa2 sclerites (without such 5 small sclerites in this position in L. ademus).
Pupa: The pupa of the new species is far from any known described pupa and can be distinguished from other species by the numbers of hooks on each hook plate.

Biology and Habitat
Limnephilus deqianensis n. sp. larvae and pupae were found in a small high-altitude stream (2930 m) in open areas. The riparian vegetation is mainly alpine meadow. Stream margins were covered with detritus (mainly grass blades). The water in the stream was clear and transparent at the collection site. The anterior ends of each pupal case was attached under a flat stone at the bottom of the stream. The species has the habit of aggregating to pupate. A newly emerged male was found during collection, and the pupae in the pupal cases were well developed.

Molecular Analysis
Nineteen mitochondrial COI sequences, including seventeen from Limnephilus deqianensis n. sp., and two downloaded from GenBank were pruned to 658bp to construct the NJ tree ( Figure 14).
The genetic distances are displayed at the node and rounded to four decimal places. Larvae, pupae, and female adults of Limnephilus deqianensis n. sp. were clustered with the male adults of L. deqianensis n. sp. The maximum genetic distances of the L. deqianensis n. sp. cluster were lower than 2.43%. Therefore, we concluded that the association is successful.

Revision of Limnephilus Species from the Chinese Mainland
Key to the males of 5 species groups of Limnephilus species occurring on the Chinese mainland: We proposed renaming this species group to the "L. perpusillus Species Group", from the species L. incisus Curtis, 1834, which was used to name the group when it had been transferred to the genus Colpotaulius by Vshivkova et al. [43], and L. perpusillus Walker, 1852, which was the oldest species in the group. Originally, this species group included the following 7 species: L. hyalinus Hagen, 1861; L. acnestus Ross, 1938; L. ademus Ross, 1941; L. janus Ross, 1938; L. major Martynov, 1909; L. perpusillus Walker, 1852; and L. secludens Banks, 1914. The species L. homeros Malicky, 2011, was distributed in China and was not ascribed to any species group by the author [16]. Here, we place L. homeros Malicky, 2011, into this species group based on its male genitalia characteristics. The group can be diagnosed by following characteristics; (1) superior appendages, each with a posterior margin having an incision, as in L. major Martynov, 1909 Ross, 1938; or almost invisible, as in. L. janus Ross, 1938, and L. perpusillus Walker, 1852 (Table 6); (3) aedeagus with a thick extensible extremity.   Schmid, 1955 This species group contains the following two species: L. externus Hagen, 1861, and L. thorus Ross, 1938, of which L. externus Hagen, 1861, was distributed in China. The group can be diagnosed by following characteristics: (1) superior appendages are triangular, each with the upper margin straight and the apical corner acute in the lateral view; (2) inferior appendages, each with the free portion bearing an apicodorsal angle produced upwards;
Diagnosis: (1) The male genitalia of this species are similar to those of L. nipponicus but differ from the latter in that: (1)

Distribution
A total of 17 Limnephilus species were recognized on the Chinese mainland, their distribution pattern is shown in Figure 19.    Figures 1-9, Russia. Previously, Schmid placed this species in the L. asaticus Species Group. However, based on the morphological phylogenetic analysis, Vshivkova and her co-authors showed that the species belonged to the genus incertae sedis, not Limnephilus [43]. Studies conducted after that (e.g., Oláh et al. 2019) still treat it as a member of Limnephilus; therefore, we here also place it in the L. asiaticus Species Group.
Diagnosis: The male genitalia of this species are similar to those of L. alaicus and L. tricalcaratus but differ from L. alaicus and L. tricalcaratus in that the apical hook on the aedeagus is shorter than the extensible extremity.  The male genitalia of this species are similar to those of L. abstrusus but differ from the latter in that: (1) The length of the free portion of each inferior appendage is about 3/4 of the width of segment IX (inferior appendages, each with the free portion almost as long as segment IX in L. abstrusus); (2) posterior margins of segment IX are concave at the insertion of the inferior appendages (straight in L. abstrusus).

Limnephilus Isolated Species
Limnephilus sibiricus Martynov, 1929 Limnophilus subfuscus sibiricus Martynov, 1929, 305-308, Figures 13-16, female, China. Limnephilus spurisi Grigorenko, 2002 Schmid treated this species as being isolated [5]. Information about its distribution in China was provided by Malicky. So far, we do not have any specimens from the Provinces of Qinghai and Sichuan; therefore, we could not provide a Diagnosis nor any illustrations of the male genitalia of this species.

Distribution
A total of 17 Limnephilus species were recognized on the Chinese mainland, their distribution pattern is shown in Figure 19.

Discussion
Limnephilus larvae live predominantly in lentic habitats, but a few [21,31] have been collected in streams and cold springs, including the new species Limnephilus deqianensis n. sp., which was collected in a cold stream during this study. Unfortunately, the investigation was performed very late, and all the larvae of the new species had pupated, so the only two larvae studied in this research were prepupae, acquired from the pupal cases. For this reason, we had no opportunity to observe the larval biology of the new species, including its feeding style, stadia, case-making behavior, etc., or its phenology and adult biology, such as the number of generations per year, the development speed, the synchronization of emergence, the life span of the adults, or the dispersal ability of the females. Therefore, further investigations are needed to determine these biological characteristics. We believe that the discovery of these biological features will enrich the stream macroinvertebrate traits datasets of China, which will allow them to be used in monitoring water quality and may even be helpful for inferring phylogenetical relationships among species in the genus Limnephilus and its higher taxa.
On the other hand, the taxonomic studies of the genus Limnephilus are mainly based on the structures of the male genitalia; species descriptions based only on female genitalia were rare, and certainly, there are some species known from both the male and female genitalia. For the species diagnoses based on the female genitalia, the abdominal segments and the vulvar scales were the most important diagnostic characteristics. However, the spermatheca is a chitinous organ inside the abdomen, potentially indicating a specificity among the species, as described by Nielsen [33]. Chuluunbat [47] applied his terminology for diagnosing the females of some Apataniidae species (also in the superfamily Limnephiloidea). Combined with the external morphology of the abdominal segments, the descriptions of the vulvar scales and the internal spermatheca can greatly improve our species identification ability and enrich the knowledge of our trichopteran fauna.
Terminology for the female genitalia presented by Schmid [27] has been widely used; however, he did not provide terms for the structure of the internal spermatheca. Nielsen [33] studied the female genital chamber of 26 species in detail and formulated terms for the structure of the internal spermatheca. In this article, we combined the terms of Schmid [32] and Nielson [33].
Finally, the morphological study of the larva and pupa of this species will be helpful in diagnosing Limnephilus species larvae and pupae in China, a common benthic macroinvertebrates group, which is useful for the biomonitoring of water quality. This morphological study of the male, female, larva, and pupa of L. deqianensis n. sp. will also provide morphological data for building hypotheses of phylogenetic relationships among species of this genus and its sister taxa. Perhaps because of the spumaline that covers the eggs of most Trichoptera, very little is known about the morphology of caddisfly eggs. Therefore, it is not likely that information about caddisfly embryos and other chorionic details will be useful for Diagnosis and phylogenetic inference in the near future.

Conclusions
In this study, most of the life stages of a new Limnephilus species, determined by COI sequencing, are described and illustrated. Ecological information regarding this new species is also provided.
Additionally, based on the male genitalia and following Schmid's system, we revised all of the Chinese mainland Limnephilus species at the species group level while emphasizing their morphological characteristics. However, phylogenetic homologues or synapomorphies were not determined and cannot support the grouping of these species. Further phylogenetic studies between the species groups of Limnephilus are needed.