Revision of Japanese species of Nipponomyia Alexander, 1924 (Diptera, Pediciidae)

Abstract Japanese species of the genus Nipponomyia Alexander, 1924 are revised. Two new species, Nipponomyia okinawensis Kolcsár & Kato, sp. nov. and N. yakushimensis Kolcsár & Kato, sp. nov. are described from the Ryukyu Islands. Images of habitus and wings, illustrations of male and female terminalia, and distribution maps are provided for the Japanese species. A key to the world species of Nipponomyia is added. DNA barcodes of three Japanese Nipponomyia are provided, representing the first barcodes from the genus.


Introduction
Nipponomyia Alexander, 1924 is a small crane fly genus belonging to the Pediciidae (Diptera: Tipuloidea). The genus was established based on the Japanese species Tricyphona kuwanai Alexander, 1913 and named after Japan (Nippon in Japanese). Another three species were included in the genus in the original designation, Tricyphona novempunctata (Senior-White, 1922) (originally described as Amalopis) from India, T. symphyletes Alexander, 1923 from Taiwan, and T. trispinosa Alexander, 1920 from Japan.
Nipponomyia is morphologically characterized by the combination of the following characters: compound eye appearing bare, wing with a conspicuous yellow longitudinal stripe along the posterior margin, and gonostylus bearing 2-14 black chitinized spines (Alexander 1924(Alexander , 1935(Alexander , 1958. The phylogenetic position of Nipponomyia within the Pediciidae has not yet been investigated.
The genus includes 15 species known from the Eastern Palearctic and Oriental regions so far ( Fig. 1) (Oosterbroek 2020). The only key to the group was published by Alexander (1935), who included seven species and a partial key to the three Japanese species (Alexander 1958). The biology and immature stages of the species are unknown. The only bionomic note about N. trispinosa is that they swarm in the air, close to the ground at dusk (Alexander 1927a(Alexander , 1958.
In the present paper we review the genus and describe two new species, N. okinawensis Kolcsár & Kato, sp. nov. and N. yakushimensis Kolcsár & Kato, sp. nov. from Ryukyu Islands, Japan. A descriptive note of the genus, images of wings and habitus, and illustrations of male and female terminalia are presented. Additional faunistic records and distribution maps are reported for Japanese species. A key to the world species of the genus is provided based on information from the literature. Finally, we present DNA barcodes for N. kuwanai, N. trispinosa, and N. pentacantha Alexander, 1958 with GenBank accession numbers.

Materials and methods
Fresh materials were collected using sweep nets and stored in 90% ethanol or were dry pinned. In total, 76 specimens of Nipponomyia belonging to five species were examined. Male and female terminalia were described and illustrated from observations in glycerol, after maceration in 10-15% KOH and neutralization with 3% acetic acid, both at room temperature. The cleared terminalia were preserved in terminalia tubes with glycerol. Illustrations were made in Adobe Photoshop CC 2019. Photographs of wing and body were taken using a Zeiss Stemi 508 stereomicroscope equipped with a Canon Kiss M digital camera. Those of terminalia were taken using a Leica M165C stereomicroscope equipped with a Leica MC170HD camera. Stack photos were combined using Zerene Stacker software. Scanning electron microscope photos were taken with a Topcon Electron Microscope SM-200. Morphological terminology in this study follows Cumming and Wood (2017), in the case of the wing venation we follow the venation system, based on McAlpine (1981) and Merz and Haenni (2000), this system is referred to as the traditional venation system in Cumming and Wood (2017: fig. 43b), with modifications based on Starý (2008) as CuA considered as Cu here. For literature collection data an approximate spatial coordinate was selected using Google Earth Pro and distribution maps were made using QGIS3 software.

DNA isolation, amplification, and sequencing
Genomic DNA was individually extracted using DNease blood tissue kits (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions. Extracted DNA was amplified using primers LCO-1490and HCO-2198(Folmer et al. 1994) on a 658 bp region of the mitochondrial cytochrome oxidase I (COI, cox1) gene, with an annealing temperature of 48 °C and 40 PCR cycles. We purified the PCR products using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and sequenced by Eurofins Operon (Tokyo, Japan) in both directions using the same primers as mentioned above. Forwards and reverse reads were assembled and edited using CodonCode Aligner v 3.5 (Codon Code Corporation, Dedham, USA). The sequences from N. kuwanai, N. trispinosa, and N. pentacantha were submitted to GenBank (accession number: MT874511-MT874514). The sequences were aligned using ClustalW (Larkin et al. 2007). We calculated the pairwise genetic distance (i.e., between species) and overall mean genetic distance with the Kimura 2-parameter model on the aligned sequences using DnaSp v5.10 (Librado and Rozas 2009 Descriptive notes on Nipponomyia Alexander, 1924 based on Japanese species. General coloration yellow to black, with or without conspicuous marking on thorax. Markings of body not differing significantly between sexes. Head: Rostrum short. Eye appearing bare; however, a few small setae present between ommatidia, near to border of compound eye ( Fig. 2A, B). Eyes widely separated. Antenna short in both sexes, only little longer than head. Scape 1.2-1.4 × longer and wider than pedicel. Pedicel 1.8-2.2 × wider than first flagellomere. Flagellum 11-13-segmented, evenly narrow toward apical segment. Flagellomeres oval to cylindrical, first 9 or 10 flagellomeres with 1 or 2 long, erected verticils dorsally (Fig. 2C, D). Last 3 or 4 flagellar segments with 3 or 4 verticils arranged irregularly. Last flagellar segment with 3 or 4 dark apical verticils, slightly curved upward, differing in shape to other verticils. Ventral part of flagellomeres densely covered with whitish sensilla, shorter than diameter of basal segment (Fig. 2E). Additional microtrichia on flagellomere (Fig. 2E). Palpi 5-segmented, length varying among species.
Thorax: Elongated in dorso-ventral direction (Fig. 3A, C). Cervical sclerite elongated fusiform. Pronotum well developed, medial part of antepronotum with hump and long setae; antepronotal lobe well developed, dorsal part slipping under medial part of antepronotum; postpronotum relative narrow. Prescutum with anterior part rounded, greatly protruding anteriorly, above to the pronotum in lateral view. Scutum usually with conspicuous spots. Presutural area of scutum without longitudinal suture, just with solid line of some long hairs (Fig. 3D); area under line of hairs before transverse suture bare in SEM photo (N. trispinosa) (Fig. 3D); not evident under stereomicroscopes. Transverse suture deep, V-shaped, generally with dark patch in middle. Mediotergite elongated, dorsal margin almost straight in lateral view (Fig. 3A, C). Episternum, epimeron, and laterotergite each virtually not divided. Pit between episternum and epimeron deep (Fig.  3C). Meron relatively small, narrow in middle, forming two triangular parts, ventral one bigger. Metepisternum angular, additional divisions indistinct.
Larva: Unknown. Pupa: Unknown. Distribution. Eastern Palearctic and Oriental (Fig. 1). Biology. Adults swarm in the air close to the ground or above the vegetation, in shadow and windless conditions. They rest on ventral surfaces of substrates like leaves, spreading their wings horizontally, even during copulation. Nipponomyia kuwanai and N. trispinosa males walk fast on the vegetation and fly short distances to find females. Nipponomyia kuwanai females were observed ovipositing in muddy, wet soil, near mosses on a mountain lakeshore. A N. trispinosa female was observed searching for oviposition sites around wet soil, rich of organic matter next to a waterfall, but the oviposition has not yet been observed. Sometimes N. kuwanai, N. trispinosa, and N. pentacantha inhabit the same habitat.

Species groups
Japanese species of the genus can be classified into two morphological species groups. The kuwanai species group is characterized by the presence of black transverse lines (dashes) in costal cell (
Thorax: In dry specimens general coloration yellow (Fig. 8C) to fulvous (Fig. 8A, B); 4 dark spots on presutural area of scutum and 7 spots on postsutural area, sizes of spots variable, especially lateral pair of spots on presutural area (Fig. 8B, C).
Legs: General coloration yellow, covered with yellowish setae (Fig. 8A). Femora without apical darkened area, apical part of tibiae brownish, with darker setae. Tarsomeres 1-3 each with narrow brown ring at tip. Tarsomeres 4 and 5 brown. Spurs on tarsomeres (2 in each segment), small but relatively easy to recognize for their darker coloration than setae.
Wing: As in Fig. 4D. Wing with transverse dark lines in costal cell. Crossvein m-m present. Dark band from base of R 2+3 extending to crossvein m-m. Dark band along crossveins r-m and m-cu pale.
Abdomen: Abdomen covered with comparatively long pale setae. Tergites 2-6 in both sexes, each with longitudinal narrow black line on lateral side, situated on basal 1/3-1/2 of each tergite in male (Fig. 8A, B) and 1/2-2/3 of each tergite in females. Sternite 2 with short black line positioned on lateral side in the middle between membranous area and posterior end of sternite 2. Sternites 3-6 with a little, wider than line on tergite (Fig. 8A, B). Sometimes line on sternite 6 indistinct or absent. Tergites and sternites 7 and 8 slightly darker than previous segments, dark yellow to brown.
Wing length: male 9-12 mm, female 9.5-11.5 mm. Head: Brown with grayish pruinosity (Fig. 10B), reddish in some dry specimens, grayish pruinosity not visible in specimens stored in ethanol. Palpi brown, 5-segmented, segments 2-4 subequal in length, last segment elongated, ca. 1.5 × longer than palpomere 4 in male, maximum at most 1.3-1.4 × longer in female, measurable clearly only in specimens stored in ethanol. Tip of palpomere 5 darker than other part of palpus. Antenna short, just a little longer than head. Antenna yellow to brown, gradually lightening to apical end. Scape darker than pedicel, often color difference very contrasting. Flagellum 13-segmented, flagellomeres gradually narrowing to apical end.
Thorax: General coloration yellow for specimens in alcohol, dark yellow, with reddish shade in dry specimens, dorsal parts light brown (Fig. 10). Decayed specimens more reddish; 4 spots on presutural area of scutum, lateral spots on presutural area very variable in size, and almost lacking in specimens collected in Ishikari Mountains (Asahidake, Hokkaido) and 7 or 9 spots on postsutural area of scutum. Pair of diffused spots in middle on postsutural area of scutum variable in size and shape, sometimes spots divided, forming 4 diffuse spots as in Fig. 10C.
Legs: General coloration yellow, covered with yellowish setae. Femora without apical darkened area, apical part of tibia slightly brownish, with darker setae. Apical ends of tarsomeres 1-3 each with narrow brown to dark brown ring, tarsomeres 4 and 5 light brown to brown (Fig. 10A). Tarsomeres each with two spurs, small but relatively easy to recognize for their darker coloration than setae.  Fig. 4B, C. Wing with transverse dark lines in costal cell. Dark band from R 2+3 not extending to crossvein m-m, shorter in specimens from Honshu (Aomori prefecture) (Fig. 4B) than those from Hokkaido (Fig. 4C)

. Cell d closed in specimens collected by us (crossvein m-m present), open in type specimens.
Abdomen: Abdomen covered with relatively long pale setae, dorsal setae darker than ventral ones. Tergites 2-6 (male) and 2-7 (female) each with a longitudinal narrow black line on lateral side, its length 1/2 of tergite in male (Fig. 10A, B) and 1/2-1 in female. Sternite 2 with short black line at corner of membranous area. Sternites 3-5, sometimes also sternite 6 with a brown line, a little wider than line on tergite (Fig. 10A, B). Sometimes line on sternite 6 less distinct or absent. Tergite and sternites 7 and 8 dark yellow to brown, darker than previous segments.
Male terminalia: Dark yellow to brown (Fig. 10A). Tergite 9 with median projection almost straight at posterior margin (Fig. 11A, B). Gonocoxite without apical lobe 1.6-1.7 × longer than wide (at middle), and 1.7-1.8 × longer than tergite 9 in lateral view (Fig. 11E, F). Apical lobe of gonocoxite slightly separated from gonocoxite, more prominent in inner lateral view, as long as 2/3 of width of gonocoxite in lateral view (Fig. 11G, H). Gonostylus with 11-14 black spines, but generally with 12. Interbase dilated apically, with two pointed parts; interbase with apical part twice as wide as basal part in dorsal view (Fig. 11A, B). In inner lateral view interbase variable in shape in different angle, tip pointed and directing posterodorsally (Fig. 11G, H). Aedeagus short, as long as wide in lateral view, tip rounded (Fig. 11I, J).
Flying period. The species flies from April to early August.   1/6-1/7 of length of genital fork (1/3 of length of genital fork in N. pentacantha and less than 1/5 of length of genital fork in N. kuwanai). Genital fork cross-shaped, lateral branch curved caudally (spoon-shaped in N. kuwanai and cross-shaped in N. pentacantha but lateral branch almost straight).
Thorax: Apical half of thorax dark brown, almost black, partly due to decay inside, posterior part yellowish brown (Fig. 14). Pattern of thorax hardly recognizable, only 2 lateral large spots on presutural area of scutum distinct. Postsutural area of scutum with 5 spots, 1 triangular black spot at middle of suture, other 2 spots at anterior corners of transverse suture, and 2 small spots at posterior corners of scutum (parascutum) (Fig. 14B, C).
Legs: General coloration yellow, covered with yellowish setae. Femora without apical dark area, tip of tibiae with a narrow darker ring. Apical ends of tarsomeres 1 to 4 each with narrow dark yellow to light brown ring. Tarsomeres 4 and 5 yellowish (Fig. 14A). Tarsomeres each with 2 spurs, black, easily discernible.
Wings: As in Fig. 4E. Wing with transverse dark lines in costal cell. Crossvein m-m present. Narrow band on R 2+3 not extending to crossvein m-m. Small yellowish brown area around connection of m-cu to Cu.
Abdomen: Yellow to light brown, relatively short setae dark on tergites and pale on sternites. Tergites 2-6, each with longitudinal narrow black line on lateral side, 1/4-1/3 length of tergite length, less prominent compared to other species. Sternite 2 without dark mark. Sternites 3-5 each with narrow brown line, not continuous in sternite 3 (Fig. 14A, B). The abdomen removed in specimen for DNA extraction.
Male: Unknown. Larva: Unknown. Pupa: Unknown. Distribution. Japan: Ryukyu Islands: Okinawa Island (Fig. 9). Oriental region. Flying period. Type specimen collected at the end of May. Biogeographic notes. Okinawa Island is the largest island of the Ryukyu Archipelago, located roughly midway between Kyushu and Taiwan. The island was formed by complex process of Paleogene volcanic activities and Neogene-Quaternary sedimentations and reef deposits (Osozawa et al. 2012;Fujita et al. 2018). Okinawa is a continental island, separated and reconnected to the Eurasian mainland by land bridges few times during Neogene-Quaternary sea level fluctuations (Ota 1998). The last separation of Okinawa from mainland occurred 1.552 ± 0.154 million years ago (Osozawa et al. 2012). The island is situated in the Oriental faunal realm. The northern part of the island, the so called Yambaru Forest consists of unique, relatively well-preserved subtropical rainforest, which is home to numerous endemic plant and animal species (Ito et al. 2000). The crane fly fauna of the island very poorly known, with six species known as endemic to the island so far. The new species, Nipponomyia okinawensis Kolcsár & Kato, sp. nov., is most probably more closely related to the Taiwanese N. symphyletes than to other Japanese species; however, to support this hypothesis additional specimens must be collected from both species and both sexes.
Thorax: Specimens stored in ethanol whitish yellow. General coloration yellow, dorsal parts somewhat darker yellow in pinned specimens (Fig. 15A). Sclerites in lateral view as (Fig. 3). Four uniformly dark spots on presutural area of scutum, 7 dark spots on postsutural area of scutum (Fig. 15C).
Legs: General coloration yellow, covered with yellowish setae. Femora without apical dark area, apical part of tibiae brownish, with a few darker setae. Apical ends of tarsomeres light brown. Apical half of tarsomere 4 brown, tarsomere 5 slightly lighter than tarsomere 4 (Fig. 15A). Tarsomeres with very small spurs, hardly discernable. Abdomen: Abdomen covered with relatively long pale setae. Tergites 2-6 in male and 2-7 in female each with a longitudinal narrow black line on lateral side, its length ranging from 1/3-1/2 of tergite length (Fig. 15A). Sternite 2 with a short black line at corner of membranous area, but without other line (Fig. 15B). Membranous area of sternite 2 as in Fig. 3B. Sternites 3-6 each with a brown line, a little wider and shorter than tergite line (Fig. 15A). Sometimes line on sternite 6 indistinct or absent. Tergites and sternites 7 and 8 dark yellow to brown.
Flying period. Usually flying between the end of July and the middle of November, but also collected in May.
Paratype ♂, same data as holotype (pinned, BLKU). Diagnostic characters. Dark yellow species with 11 large darker spots on thorax (N. trispinosa light yellowish species with 11 smaller dark spots, N. gracilis brownish species without any dark spots on thorax). Wing without transverse dark line on costal cell. Brown band running from base of R 2+3 to crossvein m-m, but not reaching wing margin (reaching the wing margin in N. trispinosa). Brown band along crossveins r-m and m-cu conspicuous. Second sternite with black marking at corner of membranous area, but without other line. Gonostylus with 2 spines (3 spines in N. trispinosa), aedeagus long, rod-shaped and acute at tip (aedeagus short, triangular in N. trispinosa).
Legs: General coloration yellow, covered with yellowish setae. Femora without clear apical dark area, but with some darker setae. Apical part of tibiae light brown, with a few darker setae. Apical ends of tarsomeres narrowly dark yellow to light brown (Fig. 18A). Tarsomeres with spurs very small, hardly discernible.
Wing: As in Fig. 5G. Yellow pattern less intensive compared to other Japanese species. Spots around yellow costal region brown, not blackish as in N. trispinosa. Brown band running from base of R 2+3 to crossvein m-m, but not reaching wing margin. Brown band along crossveins r-m and m-cu conspicuous. In paratype, wing with Rs divided to R 2+3+4 and R 5 (Fig. 5G), in holotype as usual in genus, divided to R 2+3 and R 4+5 .
Abdomen: Abdomen covered with relative long dark setae. Tergites 2-6 each with a longitudinal narrow black line on lateral side, its length ranging from 1/2-3/4 of tergite length. Sternite 2 with a short black line at corner of membranous fold. Sternites 3-7 each with a broad brown patch, covering anterior half of segment (Fig. 18A, B). The abdomen of paratype removed for DNA extraction. Male terminalia: Dark yellow to light brown (Fig. 18A). Median part of tergite 9 with posterior margin convex with two small obtuse peaks laterally (Fig. 19A, B). Gonocoxite with apical lobe 2.2 × longer than wide (in the middle) and 1.6 × longer than tergite 9 (Fig. 19E, F). Apical lobe of gonocoxite squarish in dorsal and ventral views (Fig. 19A-D), as long as width of gonocoxite at middle in lateral view (Fig. 19G, H). Basal lobe of gonocoxite prominent, both in ventral and lateral views, triangular in ventral view (Fig. 19C, D). Outer part of gonostylus slender in inner view (Fig. 19G, H), with 2 black spines, inner part of gonostylus triangular (Fig. 19A-D). Interbase elongated, gradually widening to tip, widest part twice wider than basal part in dorsal view (Fig. 19A, B), interbase curved dorsally in lateral view (Fig. 19G, H). Aedeagus rod-shaped, extending beyond interbase, tip acute, curved dorsally (Fig. 19I, J).  Distribution. Japan: Ryukyu Islands: Yakushima Island (Fig. 9). Flying period. Type specimens were collected at the end of April. Biogeographic notes. Yakushima Island is one of the northmost members of Ryukyu Islands, and also the largest island of the Osumi Archipelago. Yakushima is located approximately 70 km south of Kyushu and formed by a combination of sedimentary and orogenic volcanism processes (Shibasaki 2018). The island is one of the world's wettest locations, with the annual rainfall around 10000 mm in the mountains whose peaks reach 1900 meters. The island is characterized by a unique wet climate, which ranges from subtropical to high alpine climates, and hosts numerous endemic species (Yahara et al. 1987;Smith and Kamiya 2006;Shibasaki 2018). Yakushima is located in the southern boundary of Palearctic faunal realm, and the new biogeographic boundary between the Palearctic and Oriental realm was proposed between Yakushima/Taneshima and Amami Islands (Komaki and Igawa 2017). The crane fly fauna of the island is poorly known, at the moment only six species are known as endemic to the island; however, the second author has an additional 8-10 undescribed species from Yakushima. Based on the male terminalia the new species N. yakushimensis Kolcsár & Kato, sp. nov. is more closely related to N. gracilis, than to N. trispinosa. Both, N. yakushimensis Kolcsár & Kato, sp. nov. and N. gracilis have 2 spines on the gonostylus, aedeagi elongated, and the shapes of their interbases are also similar. Presumably the two species diverged from each other at least 1.706 Ma ago, when the Korean Peninsula & Kyushu and also Yakushima and Kyushu separated (Osozawa et al. 2012).