Two new species of Amphinemura (Plecoptera, Nemouridae) from the southern Qinling Mountains of China, based on male, female and larvae

Abstract Two new species of the genus Amphinemura, A.albicaudasp. n. and A.dingoideasp. n. from the southern Qinling Mountains, Foping County of Shaanxi Province, western China, are described based on both sexes and the larval stage. The new species are compared with related taxa, and the A.sinensis species group is defined for an Oriental lineage of the genus. Amphinemurasinensis (Wu, 1926) and A.unihamata (Wu, 1973) are reported from Shaanxi for the first time, and the hitherto unknown female of A.unihamata is described. A distribution map of the Amphinemurinae known from Qinling Mountains is given.

Recent research has highlighted the high diversity of stonefly fauna of the poorly investigated Qinling Mountains, which range from the Qinghai-Tibet Plateau to the North China Plain and separate northern and southern China (Ji et al. 2014;Li and Murányi 2015;Li et al. 2016aLi and Mo 2018). In April 2018, we made a short collecting trip to the southern Qinling Mountains, in the vicinity of Foping, Shaanxi. In the present paper, we describe two new species of Amphinemura collected during that trip, along with some further contributions to the Amphinemurinae, including the definition of a new Oriental species group where one of the new species belongs. We also present a distribution map of the Amphinemurinae hitherto known from the Qinling Mountains.

Material and methods
The material studied was collected along stream banks by hand, or by using a beating sheet or an aquatic net. All material is deposited in the Entomological Museum of China Agricultural University, Beijing (CAUC), the Henan Institute of Science and Technology, Xinxiang (HIST), and the Collection of Smaller Insect Orders, Department of Zoology, Hungarian Natural History Museum, Budapest (HNHM), as indicated in the text. The specimens are preserved in 75% ethanol. The morphological terminology follows that of Baumann (1975) and Zwick (2010). Specimens were examined with the aid of a Leica M420 dissecting microscope and the color images and illustrations were made with the aid of a Leica S8APO and a Keyence LHX5000 digital microscope.
Description. Adult habitus ( Fig. 1): General color light brown to brown. Head and antennae brown, palpi light brown. Thorax brownish, pronotum with distinct rugosities. Legs light brown. Wing membranes grayish, veins brown. Abdomen brown with darker terminalia.
Female inner genitalia ( Fig. 6): Inner sclerite is medially separated into triangular sclerites located anteriorly to the subgenital plate; anterior portion of the sclerite halves with a small, ear-shaped projection, easily observed in oblique or caudal view. Between the sclerites is a membranous tunnel with a tubular median sclerite leading to spermathecal ductus.
Mature larva (Fig. 7): Body relatively slender and small, body length without antennae and cerci 5.5-6.0 mm. General color brown, with some indistinct pattern on pronotum and abdomen, legs and antennae light brown but cerci contrasting white and hairy. Setation long and distinct. Legs moderately long, width of hind femora ca ¼ of their length. The pronotum is trapezoidal, wider than long, as wide as head. Cervical gills long, inner gills with 7, outer with 8 branches. Wing pads more than twice as long as the corresponding segments. Abdomen relatively slender and uniformly brown, integument light matt brown, first 2 abdominal segments divided by pleura. Posterior margin of sternum IX of the male larva short triangular, sternum VIII of female larva slightly incised; paraprocts blunt. Cerci long, with 24-26 cylindrical segments; length of the 15 th segment is about 3 times of its width.
Setation of the larva (Figs 8-13): Head, antennae and palpi with dense short setae. Pronotum covered with very short setae; marginal setae distinct and blunt, row interrupted in anteromedial and posteromedial third, corners have setae as long as one 15 th of pronotum width (Fig. 12). Setae on meso-and metanotum as long as longest marginal setae on pronotum; wing pads with long and acute, whitish setae. Legs with dense setation, all tibiae bear distinct swimming hairs as long as femur width (Fig.  13). Longest acute setae of all femora are longer than half of the corresponding femur width, long setae on fore femur arranged in an incomplete transversal line. Tarsi and claws relatively long. Tergal segments covered with thin setae, all segments bear a pair of thin, erect and irregularly curved hairs that are distinct in lateral view and reach nearly the segment length ( Fig. 9); posterior margin with row of 14-16 acute setae, of various length, longest nearly reaches half of segment length (Figs 8, 10). Cercal segments with dense and long, white setation; setae sparser and shorter on basal and apical segments (Figs 10, 11). Cercomeres 14-16 with intercalary setae-like fine hairs, longer than the segment width, and an apical whorl of 14-17 acute setae that are as long as segment length (Fig. 11).
Type material. Holotype male ( Affinities. The new species belongs to a lineage of Amphinemura that is distributed in Oriental areas of China and Vietnam. This lineage can be characterized by the horn-shaped lateral processes of the dorsal sclerite, and pointed ventral sclerite of the male epiproct. It is defined here as the A. sinensis species group. The species belonging to this group are A. ancistroidea Li & Yang, 2007a, A. caoae Stark & Sivec, 2010, A. cestroidea Li & Yang, 2005, A. chui Wu, 1935, A. divergens Stark & Sivec, 2010, A. elongata Li, Yang & Sivec, 2005 fleurdelia (Wu, 1949), A. furcostyla (Wu, 1973), A. giay Stark & Sivec, 2010, A. guangdongensis Yang, Li & Zhu, 2004, A. hamiornata Li & Yang, 2008b, A. leigong Wang & Du in Wang et al. 2006 (Wu, 1938), A. malleicapitata Li & Yang, 2006, A. maoi (Wu, 1938), A. nanlingensis , A. nigritubulata Li & Yang, 2008d (Wu, 1926), A. tianmushana Li & Yang, 2011, A. viet Stark & Sivec, 2010, and A. yao Mo, Yang, Wang & Li, 2017 Males of the new species can be distinguished from other members of the group by their armed outer lobe of the paraproct, which is unique within the group, and also by the distinctive shape of the processes of epiproct. The female outer genital sclerites are less distinctive, but the inner sclerite is unique because of the ear-shaped projections. The larva is distinctive by its conspicuous white and rather hairy cerci. Distribution and ecology. The new species was found at a single locality at the lower section of a large stream at moderate elevation (Figs 31,35). The stream runs between forests, less-used agricultural areas, and ruderal bush. Its width varies between 3 and 8 m, and its maximum depth is less than 1 m. Rocky rapids are mixed with nearly stagnant pools and slow, stony sections. The substrate is mostly stony or sandy, with a moderate amount of debris (Fig. 32). Both last instar larvae and fully mature adults were present, suggesting April is the peak season of its emergence. Accompanying stoneflies were A. sinensis, a Rhopalopsole sp. collected as females, a few larvae of a Neoperla sp., and larvae of a Kamimuria sp. that occurred in high numbers.
Etymology. The specific name is composed of the Latin words albus (white) and cauda (tail), and refers to the distinctive white cerci of the larva.
Remarks. The adults and larvae were associated on the basis of pharate male and female adult terminalia dissected from matured larvae. dian lobe long, curved and with 5 or 6 apical spines, outer lobe short and lacks spine. Female: subgenital plate strongly bilobed with dome-like median notch, paragenital plate with two branches, inner genitalia simple. Larva: general color light brown with specific, distinct dark brown dorsal pattern, setation long.
Description. Adult habitus (Fig. 16): Head dark brown without pattern; compound eyes dark brown; antennae dark brown; palpi light brown. Pronotum lighter than head, arrangement of brown pattern similar to larvae. Legs generally brown, coxae and venter of femora lighter. Abdominal segments reddish brown, terminalia brown.
Male : Forewing length 6.3-6.5 mm. Tergum IX lightly but evenly sclerotized, two paramedial groups of 6-8 long setae present along posterior margin (Fig. 17). Vesicle of sternum IX very long, constricted medially, about 5 times longer than wide (Figs 18, 19). Hypoproct basal half rectangular, medial portion rounded, apex short and up-curved (Fig. 18). Tergum X widely sclerotized, medial light area beneath epiproct very small and narrow, with 3 or 4 very small lateral spinules (Fig. 17). Cercus lightly sclerotized, stout and short. Epiproct (Figs 16, 17, 19) weakly modified, nearly rectangular with lightly sinuous margin, less than 3 times longer than wide, apically scaled and with a dark medioapical notch. Dorsal sclerite mostly membranous, basal sclerite narrow, lateral sclerite evenly thin and S-curved, basal portion hidden beneath the large membrane, appearing as weak stripe in dorsal aspect, apical portion black in dorsal and lateral aspects and ending subapically (Figs 17, 19); ventral sclerite with weak ridge fringed by a row of short ventral teeth (Fig. 19). Paraproct trilobed (Figs 17-20): inner lobe slightly bilobed, relatively elongate and reach end of terminalia, basally connected to median lobe and membranous portion between the lobes bear minute apical setae; median lobe sclerotized, up-curved at apical half, apical portion in dorsal aspect with 5 or 6 medium-sized spines: 2 or 3 upper spines and 3 lateroapical spines (Figs 17, 19, 20); outer lobe sclerotized and medially curved along cerci, apex up-curved, without spines (Figs 18, 20). Female (Fig. 15): Forewing length 7.5-7.8 mm. Sternum VII posteriorly greatly produced in a large semicircular pregenital plate, the plate mainly being pale but posterior margin brown. Sternum VIII forms bilobed subgenital plate with a deep medial indentation at inner margin of the lobes, the median notch between subgenital plate dome-like. Paragenital plate with two branches, lower plate large, rounded and lightly pigmented lobe with hairs in ventral surface, upper plate forming a dark brown sloping sclerite seemingly like the pod brim. Sternum IX with anterior margin slightly protruded medially. Paraproct and cerci brownish.
Female inner genitalia (Fig. 21): Inner structure under pregenital plate is simple and membranous, lightly sclerotized, ovum-shaped anterior shield attached to the spermathecal ductus; attached muscles linking with margin of paragenital plate easily observed.
Mature larva (Fig. 22): Body relatively stout and small, body length without antennae and cerci 5.5 mm. General color light brown with distinct and characteristic dark brown dorsal pattern: head mostly dark brown with well delimited light brown pattern on occiput; scape and pedicel dark brown, rest of the antennae and palpi light brown; pronotum mostly pale, dark brown pattern similar to adults; meso-, metanotum and wing pads mostly pale but with distinct, paired Z-shaped dark pattern; legs light brown, apex of femora and base of tibiae darker; abdominal terga I-II entirely pale, terga III-V entire dark, terga VI-X laterally dark with medial pale area gradually widened towards the apex; cerci pale brown. Ventral aspect of the body entirely pale. Setation long and distinct. Legs moderately short, width of hind femora about one-third of their length. The pronotum is rounded trapezoidal, wider than long, slightly wider than head. Cervical gills long, inner gills with 6, outer with 7 branches. Wing pads more than twice as long as the corresponding segments. Abdomen relatively stout, integument light matt brown, first 3 abdominal segments fully, further 3 partly divided by pleura. Posterior margin of sternum IX of the male larva short and blunt triangular. Cerci long, with 25 slightly clubbed segments; length of the 15 th segment is more than 3 times of its width.
Setation of the larva (Figs 23-28): Head, antennae and palpi with short setae. Pronotum covered with short setae; marginal setae distinct and blunt, row continuous but setae shorter in anteromedial and posteromedial third, corners have setae as long as 1/15 th of pronotum width (Fig. 27). Setae on meso-and metanotum with marginal setae as long as longest marginal setae on pronotum; wing pads with short setae besides marginal ones. Legs with relatively sparse but diverse setae, all tibiae bear sparse and indistinct swimming hairs as long as tibia width (Fig. 28). Longest acute setae of all femora are about as long as half of the corresponding femur width, not arranged in line but restricted to apical half. Tarsi and claws normal. Tergal segments covered with thin setae and a few short hairs; posterior margin with row of 14-16 acute and erect setae, of various length, longest reaches more than half of segment length (Figs 23, 24). Cercal segments with relatively sparse and moderately long setation, apical whorl of setae consist of both dark and whitish setae; setae sparser and shorter on the basal segments (Figs 25, 26). Cercomeres 14-16 with intercalary setae-like indistinct fine hairs, as long as the segment width, and an apical whorl of 8 or 9 acute setae that are much shorter than segment length (Fig. 26).  Affinities. On the basis of the simple epiproct and rather elongated median lobe of the paraproct, the male of A. dingoidea is similar to several other Chinese species, e.g. A. curvispina (Wu, 1973), A. filarmia Li & Yang, 2000, A. microhamita Li, Dong & Yang, 2018, and A. ovalis Li & Yang, 2005. However, the combination of the long and slightly bilobed inner lobe and short, spine-less outer lobe of paraproct, together with simple but apically notched epiproct, distinguish the new species from all congeners. The female can be easily distinguished from females of the hitherto known Asian Amphinemura species on the basis of the distinctive shape of their subgenital plate combined with rather simple inner genitalia. The larva is distinctive by its rather conspicuous pale and dark brown color pattern.
Distribution and ecology. Most specimens were found along a small forest brook (Fig. 33), but a single male was collected by the upper, faster flowing section of the same large stream where A. albicauda was collected at the lower section (Fig. 35). Both localities are at moderate elevations, have fast current, and the littoral vegetation consists of deciduous forest, willow bush, and dense littoral grasses and sedges. The width of the brook at the type locality is less than 1 m and usually less than 10 cm in depth, but deeper, nearly stagnant pools also occur. The substrate is bedrock with small stones and variable debris (Fig. 34). Only a single mature larva was found among several other Nemouridae larvae, whereas most adults were fully mature and abundant among the adult stoneflies collected at this stream, suggesting late April is after the peak emergence. At the type locality, accompanying stoneflies were Nemouridae: Amphinemura sinensis, A. unihamata (Wu, 1973), Sphaeronemoura grandicauda (Wu, 1973), an unidentified Indonemoura sp. found only as premature larvae, and a yet undetermined Nemoura sp. of the ovocercia species group. In the Dizhuang valley, the paratype male was caught together with the recently described Cryptoperla nangongshana Huo & Du, 2018. Etymology. The specific name refers to the shape of the pod-like subgenital plate and the pot-like anterolateral branches, which overall resembles the Chinese "ding", an ancient pot unique in Chinese culture.
Remarks. The adults were associated with the single mature larva on the basis of the distinct pronotal pattern and similar, contrasting body color. (Wu, 1926)  Distribution and ecology. This species was described from Jiangsu Province, later reported also from Beijing and Henan , and is the only member of the sinensis group that occurs in Palaearctic areas of China. We collected this species at the type localities of the new species above (Figs 31-34). Our new records represent the first records from Shaanxi.  (Wu, 1973), terminalia of adult female. 29 Terminalia in ventral view 30 Inner genitalia, dorsal view. Scale bar: 0.5 mm.

Remarks.
From both streams where we found this species, we also collected mature larvae that are probably belong to this species. These are very similar to the larva of A. albicercia in color pattern and setation, but lacks the distinctive, dense white hairs on the cerci.
Amphinemura unihamata (Wu, 1973) Figs 29, 30, 35 Description of the female. (Fig. 29) Forewing length 9.0-9.6 mm. Sternum VII posteriorly produced in a wide but short, semicircular pregenital plate, the plate pale. Sternum VIII forms trapezoid subgenital plate that is about half as wide as the segment, posterior lobes distinctly divided into rounded sublobes, nearly equal in size; posteromedial indentation narrow and triangular, with white medial area widened towards anterior edge. Paragenital plate large, rounded, but pale and rather indistinct. Sternum IX with anterior margin slightly protruded medially. Paraproct and cerci brown.
Female inner genitalia (Fig. 30): Inner structure consist of a beak-like, dark median sclerite positioned under pregenital plate at the anterior edge of subgenital plate, is simple and a smaller but thick, ring-shaped anterior shield attached to the spermathecal ductus; membranous portion wide and attached to the paragenital plates.
Material examined. CHINA, Shaanxi Province, Hanzhong, Foping County, steep forest brook in Panda valley, 1270Panda valley, m, 33°40.322'N, 107°58.190'E, 2018. Distribution and ecology. Previously, this species was known only from the holotype, collected in Sichuan. It was recently redescribed by Yang et al. (2015). We found it at the type locality of A. dingoidea (Figs 31, 32) and a similar small brook at slightly higher elevation, representing its first records from Shaanxi.
Remarks. Amphinemura annulata Du & Ji in Ji et al., 2014 was described one year earlier before the specific identity of A. unihamata was resolved by Yang et al. (2015). The holotype of A. annulata was designated from Zhejiang Province but the paratype series included specimens also from Shanxi, Shaanxi, and Guizhou provinces. The two species are probably conspecific, but without examination of holotype of A. annulata, we do not propose a synonymy of A. unihamata at this time.

Concluding remarks: the Amphinemurinae known from the Qinling Mountains
Sloping from the Qinghai-Tibet Plateau to the North China Plain, the Qinling Mountains form the boundary between the Palaearctic and Oriental realms and are an important biodiversity hotspot (Chen et al. 2008, Zhang et al. 2014. Even though one of the earliest Chinese publications on stoneflies was published on species from the Qinling (Klapálek 1908), the Amphinemurinae remained unknown until the last ten years.
The first known Amphinemurinae species, Mesonemoura membranosa Du & Zhou in Du et al., 2007a was described from the central part of the mountain range in Shaanxi. Subsequently, four additional species were reported from the eastern extremities of the range in Henan Province: M. tritaenia Li & Yang, 2007b, and Indonemoura auriformis Li & Yang, 2008e were described as new, whereas A. didyma was reported from Baotianman as new for Henan (Li and Yang 2008e). Later, Sphaeronemoura separata Li, Murányi & Yang, 2014 Li, Dong & Yang, 2018, A. longihamita Li, Dong & Yang, 2018, and A. microhamita Li, Dong & Yang, 2018, from the Shaanxi areas of the Qinling, and reported A. multispina (Wu, 1973) and S. grandicauda (Wu, 1973) from the same ranges.
Including the newly described or reported species in the present paper, 16 species are now known from the Qinling Mountains. However, this number must be just a fraction of the possible diversity. Seven of the species described from the range are known only from their Qinling type localities, whereas A. mamillata was later found also in Ningxia , and the type series of A. annulata included specimens from Zhejiang, Shanxi, and Guizhou. The paratypes of A. lingulata were from Sichuan ). Among species subsequently reported from the Qinling, A. didyma is also known from Ningxia and Inner Mongolia, A. multispina, A. unihamata, and S. grandicauda from Sichuan, and A. sinensis from Jiangsu, Beijing, and Henan . These distributions support well the idea that the Qinling Mountains, as a border zone between realms, include species distributed mainly in the Palaearctic as well as other species that are members of the Oriental fauna. However, none of the known species have dispersed far into the both realms, e.g. no species is known to occur in northeastern or southern China.
Amphinemura is a widespread Holarctic and Oriental genus, whereas Indonemoura Baumann, 1975, Mesonemoura Baumann, 1975, and Sphaeronemoura Shimizu & Sivec, 2001 are distributed primarily in the Oriental Region. Among these three genera, Indonemoura is not known to occur on the mainland north to Qinling, further supporting the uniqueness of this Palaearctic and Oriental border region.