Diptera: Rhagionidae) with Descriptions of New Species from Japan

Zootaxa 4097 (1): 041–058 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2016 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.4097.1.2 http://zoobank.org/urn:lsid:zoobank.org:pub:6189C0A9-0BDA-4A8E-83B4-717C7A6EDA2B

Bryophyte-feeding of Litoleptis (Diptera: Rhagionidae) with descriptions of new species from Japan

YUME IMADA1 & MAKOTO KATO Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501, Japan 1Corresponding author. E-mail: [email protected]

Abstract

Here we report the larval phytophagous habit of Litoleptis for the first time, and describe six new species of Litoleptis in Japan; L. japonica n. sp., L. kiiensis n. sp., L. niyodoensis n. sp., L. himukaensis n. sp., L. izuensis n. sp., and L. asterellaphile n. sp. All the species described here are thallus-miners of liverworts belonging to Aytoniaceae and Conocephal- aceae (Marchantiopsida: Marchantiophyta). Each fly species mined thalli of only one of the following genera: Conocephalum, Reboulia, and Asterella. The descriptions of the Japanese Litoleptis species here expand the concept of this genus. The female genital morphology of Litoleptis strengthened the current placement of Litoleptis as a member of Spaniinae.

Key words: herbivory, Brachycera, Hilarimorpha, bryophytivore, leaf-mining, keys

Introduction

Diptera accounts for one of the most speciose clades of organisms and displays extremely broad ecological breadth (Grimaldi & Engel 2005; Wiegmann et al. 2011). A major expansion of dipteran families took place in mid- Cretaceous (Yeates 2002), which coincides with the age of angiosperm radiation (Grimaldi 1999). Association of Brachycera and flowering plants date back at least to the mid-Cretaceous (Ren 1998). About half of 13 major clades of brachycera are known to contain important pollinators (Friis et al. 2011) and some brachyceran families have diversified as angiosperm-feeders (Winkler et al. 2009; Novotny et al. 2005; Labandeira 2005), even though the number of species associating with plants is inferred to rather be underestimated (Friis et al. 2001). Lower Brachycera became rich in some assemblages of the Middle and Late Triassic (Krzemiński & Krzemińska 2003), although the origin and phylogenetic relationships of this group are scarcely understood. In particular, Rhagionidae sensu lato (i.e. Rhagionidae, Austroleptidae, and Bolbomyiidae) have experienced the greatest diversification during Middle and Late Jurassic (Mostovski 2000) and have lessened their impact in the modern fauna. The natural history of Rhagionidae s. l. has historically attracted much attention, although the life histories of most species are largely unknown (Oldroyd 1964). As adults, at least some rhagionids are zoophages. Spaniopsis and Symphoromyia have blood-sucking species (Colless & McAlpine 1991; Ferguson 1915; Turner & William 1979). Female flies of Rhagio are believed to be insect predators (Kellogg 1908; Leonard 1930; Narchuk 1988; Paramonov 1962), albeit not proven (Kerr 2010). Two genera, Arthroteles and Austroleptis are reported as flower- visitors (Colless & McAlpine 1991; Stuckenberg 1956). Larval feeding habits of many of rhagionid flies are poorly known. Rhagio and Chrysopilus are mostly terrestrial and are known as predatory (Paramonov 1962; Roberts 1969). Chrysopilus contains terrestrial, rotten wood feeders, and some aquatic species (Roberts 1969; Tsacas 1962; Nagatomi 1958). Some species of Austroleptis are thought to feed on rotten wood or detritus (Colless & McAlpine 1991). Some Symphoromyia species are inferred to be detritivores or partly associated with decayed plants (Sommerman 1962). Spania nigra and Ptiolina obscura are reported as herbivores of bryophytes (Mik 1896; Brindle 1959; Brauer 1883; Lane & Anderson 1982; Nartshuk 1995).

Accepted by C. Lamas: 12 Feb. 2016; published: 30 Mar. 2016 41 Considering the variety of natural history of Rhagionidae s. l., revealing species diversity, systematics and feeding ecology of this group is important in understanding the evolutionary trajectory of flies. Nevertheless, the systematic position and arrangement of Rhagionidae s. l. have been unsettled throughout the history of study since Latreille (1802). Litoleptis is a small genus, which comprises at least four species. It was erected as a monotypic genus, which proposed for L. alaskensis Chillcott, 1963. Hennig (1972) added a new species L. chilensis Hennig and transferred Hilarimorpha orientalis Frey to Litoleptis. Lastly, Arillo et al. (2009) described L. fossilis, which is the first fossil species of this genus preserved in the Lower Cretaceous amber. In addition, three undescribed species of Litoleptis were recorded: two species from Japan and Nepal by Nagatomi (1982), and one from Yunnan, China by Yang et al. (1997). Collection records of Litoleptis are thus sparse and its biology has not been reported so far. The systematic position of Litoleptis has not yet been clarified due to the anomalous morphology and the scarcity of the specimens. This genus was originally included in Rhagionidae by Chillcott (1963). Nevertheless Litoleptis resembles Hilarimorpha Schiner (Hilarimorphidae) in wing venation and in lacking tibial spurs, it is differentiated by the short-styled antenna and the presence of a pulvilliform empodium (Chillcott 1963). Grimaldi & Cumming (1999) noted that Litoleptis is in close proximity to Bolbomyia and Austroleptis based on the lack of wing vein M3 (i.e. the lack of dm cell). It was placed in ‘Spania group’ by Hennig (1973) and subsequently was included in the subfamily Spaniinae (Rhagionidae) by Nagatomi (1982), along with Ptiolina, Spania, and Spaniopsis. Spaniinae was defined primarily based on the characters in the female genitalia besides the antennal form: cerci widely separated each other, tergum 10 absent or short (Nagatomi & Iwata 1976; Nagatomi 1982). Rhagionidae was a heterogenous group, of which sufficient autapomorphies were not identified (Stuckenberg 2001), with subfamilies separated by the shape of female genitalia (Nagatomi 1982). Each subfamily of Rhagionidae (i.e. Rhagioninae, Spaniinae, Austroleptinae) was raised to family rank so as to constitute the respective monophyly (Stuckenberg 2001). The concept of Spaniidae as a family has been rejected by Kerr (2010) because it was incompatible with the concept of Rhagionidae in the result of the combined analyses of morphological and molecular data. This taxonomic group has been instead treated as a subfamily of Rhagionidae, Spaniinae, adding Omphalophora and Symphoromyia (Kerr 2010). Litoleptis has been consistently placed in Spaniinae since Nagatomi (1982), nevertheless its female genital morphology, the most important character for separating the subfamilies of Rhagionidae (Nagatomi 1982), has never been examined. Examining the female genital morphology of Litoleptis is therefore necessary to understand the relationship between Litoleptis and the peripheral genera. From our recent survey of liverwort-dwelling insects, we have found that the larvae of Litoleptis mining within thalli (i.e. leaf-like organ of thallose liverworts) of liverworts are prevalent in Japan. To access the regional species diversity and to reveal the larval diet of Litoleptis, we conducted field observation and larval rearing. All the species obtained were new to science, which were found to be thallus-miners that feed on only single genus of liverwort species. Here we describe six new species of Litoleptis, and discuss the systematic position and concept of Litoleptis with special reference to female genitalia. This study provides a unique example of bryophyte-mining flies, which has long been overlooked despite widespread in Japan.

Materials and methods

We collected and reared the bryophyte-mining larvae of Litoleptis in Japan, thereby obtaining adult flies. We collected liverworts from 24 localities in Japan (Fig. 1) and kept the plant material until the larvae exited the plant and emerged as adults. For larval rearing, the plant material was packed in small plastic cases and occasionally moistened, and kept under laboratory conditions. Emerged adults were completely dried in a refrigerator. We examined 223 adult specimens of Litoleptis for this study. For specimen preparation, we removed the abdomen, placed it in 10% potassium hydroxide warmed in a hot water bath (70~75°C) for approximately 30 minutes to remove adipose tissue, and then washed it with distilled water and subsequently 50% ethanol to clean out debris. After dissection of the genitalia by separating the epandrium from the gonocoxites, the specimen was mounted in glycerine on a glass slide and observed under a microscope with transillumination.

FIGURE 1. Map showing the sampling localities. Locality numbers correspond to those in the text.

All the specimens examined in this study are deposited in the following collections: National Museum of Nature and Science (NMNS), Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan (KUHE). All specimens studied have a serial number label added in the following format: “Rh XXXX”. The terminology follows Kerr (2010) with minor modifications. The abbreviation “Pref” using below stands for “prefecture”. The author’s names are abbreviated: MK and YI stand for Makoto Kato and Yume Imada, respectively.

Taxonomy

Litoleptis Chillcott

Type species: Litoleptis alaskensis Chillcott 1963: 1187, by original designation. L. chilensis Hennig 1972: 6. L. orientalis as Hilarimorpha; Frey 1954: 25. L. fossilis Arillo et al. 2009.

Description. Head (Fig. 2b, c) dark brown, glossy. Vertex, occiput, postgena with sparse setae, gena bare. Eyes inconspicuously setulose; slightly dichoptic or holoptic in male, dichoptic in female; in male, upper part orange, lower part black. Mandibles absent. Antenna black or pale grey. Pedicel clearly larger than scape. Scape minute, pedicel as broad as long and weakly bristled around distal edge. Clypeus slightly bulbous. Palpus one-segmented, with black setae. Labellum developed, without pseudotracheae. Thorax (Fig. 2a) brownish black with pruinosity and with three dark brown vittae. Postpronotal lobe, proepimeron, bare. Scutellum with sparse setae on posterior margin. Mesonotum without vittae. Proscutellum absent. Subscutellum inconspicuous. Proepimeron developed. Postscutellum bare. Anatergite and katatergite indistinguishable. Wing (Fig. 4) infuscate, without any dark markings, despite having intraspecific variation in color darkness. Pterostigma inconspicuous. Lower calypter reduced. Upper calypter triangular in form, underdeveloped; with

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 43 broad curvature. Costa ending before wing tip. Humeral crossvein weakly developed. Crossvein sc-r present, positioned proximal of humeral crossvein. R1 dorsal and ventral surface bare. R2+3 sinuous, apical third of R2+3 ultimately bends anteriorly slightly, toward leading edge of wing margin; longer than R5, but less than twice as long. Fork of R4+5 distal of distal end of fork of M1+2. R4 at base mostly straight, ending at wing tip entire length. R5 ending at wing tip. M3 absent. Medial crossvein absent. CuA2 generally join with A1. CuA2+A1 reaching or sometimes not reaching wing margin. Anal cell closed usually before and sometimes at wing margin. Alula reduced with narrow curvature; rounded evenly. Legs dark brown to black; coxae and femora more or less pollinose with weak setae. Empodium pulvilliform. Tibial spur formula 0:0:0. Macrotracheae absent. Postmetacoxal bridge absent. Hind coxae bare behind.

FIGURE 2. Morphological features of Litoleptis applied in this study. (a) General appearance of adult of L. japonica sp. n. in lateral view, (b) head of adult male of L. japonica sp. n. in anterior view, (c) head of adult male of L. kiiensis sp. n. in anterior view, (d) female genitalia in dorsal view. (e) ventrolateral process of female of L. kiiensis sp. n. in lateral view. Abbreviations: anatg, anatergite; anepm, anepimeron; anepst, anepisternum; fc, face; gn, gena; hlt, halter; kepst, katepisternum; ktg, katatergite; lbl, labellum; mr, meron; oc, ocellar; pgn, postgena; plp, palpus; pprn lb, postpronotal lobe; prepm, proepimeron; prepst, proepisternum; sctl, scutellum; vl proc, ventrolateral process.

Abdomen dark brown to black with pruinosity. Male Cercus displaced away from epandrium, widely displaced from one another, separation distance about 3/ 4 width of cercus; held vertically over abdomen; flat dorso-ventrally. Tergite 10 absent. Hypandrium fused entirely to gonocoxites. Gonocoxal ridge absent. Gonocoxal apodeme absent. Gonostylus with developed extension apically from apex of main component. Aedeagus slender and conical-shaped; apical surface covered with tiny pustules; connected with paramere at base. Lateral ejaculatory process either present or absent. Aedeagal tines absent. Endoaedeagal process absent. Sperm sac bulbous, generally without distinct lobes, envelope aedeagus ventrally. Female (for L. japonica, kiiensis, niyodoensis, asterellaphile; Fig. 2d) Tergite 7 wider than long. Tergite 9 bare, inconspicuous and largely retracted within tergite 8; having ventrolateral projections, extending posteriorly, surrounding and fusing sternite 9 laterally. Intersegmental membrane clearly separating tergites 7 and 8. Spermathecae three, membranous. Spermathecal duct short, less than length of sternite 9. Spermathecal duct accessory glands with short, sclerotized columnar duct. Circular ridge of ejection apparatus absent. Ring at base of spermathecal ducts lightly or not sclerotized. Spermathecal duct ejection apparatus absent. Common spermathecal duct absent or inconspicuous. Surrounding area of genital chamber membranous, contained sclerotization of sternite 9 laterally. Diagnosis. Litoleptis can immediately be distinguished from the other genera of Rhagionidae. Major

44 · Zootaxa 4097 (1) © 2016 Magnolia Press IMADA & KATO autapomorphies are as follows: medial crossvein (and discal cell) absent; all tibiae without spurs; gonocoxal apodeme absent. Litoleptis specifically mostly resembles Spania in general appearance and stylus-shaped antenna, although they can easily be distinguished by the following character states besides the above-mentioned traits; male gonostylus with an extension from apex of main component expanding into an irregular form (as opposed to constantly slender or club-like form in Spania); gonocoxal apodeme absent. Also, the Japanese Litoleptis shows inconsistency in having the following traits unlike other congeners (e.g. L. alaskensis): male eyes dichoptic; lateral ejaculatory process present (except L. japonica); ejaculatory apodeme present. The following traits are uniquely found in the Japanese Litoleptis: apex of aedeagus covered with numerous tiny pustules; spermathecal duct accessory glands connected by short, sclerotized columnar duct arising from spermathecal duct.

Litoleptis japonica Imada & Kato sp. n. [Japanese name: jagoke-shitone-abu] (Figs. 3a, 3b, 4a, 5)

Description. Body length: 3.0 mm (n=3) in male, 3.0 mm (n=12) in female. Wing length: 3.2 mm (n=3) in male, 3.4 mm (n=9) in female. Head (Fig. 3a, b) Background color dark brown, clypeus in male generally darker than face. Frons bare in male, generally covered with sparse setae in female. Antennal length 0.5 mm (n=3). Antenna 0.75 times as long as diameter of eye, covered with dense, appressed microsetae, consistently except distal half of first flagellomere (Fig. 5a). First flagellomere laterally compressed, oval and enlarged at basal half, sharply tapering toward apex, sharpened to be needle-like at tip and covered with sparse and rough microsetae. Male (Fig. 5b) Gonostylus wide and thick at basal main component and tapering steeply toward tip (apex approximately 3 times narrower than width at base), with forceps-like extension hollowed dorsally and bifid unevenly at apex. Sperm sac barely visible. Lateral ejaculatory process absent. Ejaculatory apodeme relatively long, reaching anterior margin of gonocoxite. Dorsal surface of gonocoxite with clear medial chevron in anterior margin. Posteromedial margin in ventral surface of gonocoxite without bristles. Aedeagus deeply notched at tip, smoothly connected with paramere at base. Paramere widest at connection with aedeagus, approximately three times wider than narrowest point. Female (Fig. 5c) Spermatheca spherical, not sclerotized. Spermathecal duct accessory gland with short, sclerotized columnar duct, curved at middle to make a right angle. Spermathecal duct accessory glands arising at base of spermatheca. Ring at base of spermathecal duct lightly sclerotized. Common spermathecal duct nearly absent. Genital chamber moderately sized, oval. Type material. Holotype. JAPAN [HONSHU] 1♂, emerged on 20.IV.2011 from larva collected by MK on 2.IV.2011 at Haruno-cho, Shizuoka Pref (Fig. 1:20), “Rh 0001”, NMNS. Paratype. 2♂, 5♀(Rh 0002–0008), emerged 20–22.IV.2011 from larvae collected by MK on 2.IV.2011, same locality as holotype, Shizuoka Pref (Fig. 1:20), KUHE. Additional materials. In total, 53 specimens were collected. All following materials were obtained as larvae. All specimens are stored in Kyoto University (KUHE). JAPAN [HOKKAIDO] 1♀(Rh 0009), emerged 1.V.2010 from larva collected by MK on 19.X.2009 at Aizan-kei, Hokkaido Pref (Fig. 1:1); 2♀(Rh 0010, 0011), emerged 16.VI.2012 from larvae collected by MK on 11.VI.2012 at Mt. Daisengen, Hokkaido Pref (Fig. 1:2). [HONSHU] 1♀(Rh 0012), emerged 29.IV.2010 from larva collected by MK on 24.XI.2009 at Sekikawa-mura, Niigata Pref (Fig. 1:3); 1♀(Rh 0013), emerged 18.V.2010 from larva collected by MK on 26.IV.2010 at Hakusan, Fukui Pref (Fig. 1:5). 25♀(Rh 0014–0038), emerged 21.IV–4.V.2010 from larvae collected by MK on 6.IV.2010 at Kibune, Kyoto Pref (Fig. 1:6). 5♀(Rh 0039–0043), emerged 30.III–2.IV.2011 from larvae collected by MK on 20.II..2011 at Akame-no-taki, Mie Pref (Fig. 1:7). 2♂, 3♀(Rh 0044–0048), emerged 26.III–1.IV.2012 from larvae collected by MK on 4.III.2012 at Koto-no-taki, Wakayama Pref (Fig. 1:14). 2♀(Rh 0049, 0050), emerged 14.IV.2009 from larvae collected by MK on 1.IV.2009 at Kuki, Wakayama Pref (Fig. 1:17). 1♀(Rh 0051), emerged 20.IV.2011 from larva collected by MK on 2.IV.2011 at Wayama-touge, Shizuoka Pref (Fig. 1:20). 1♀(Rh 0052), emerged 26.IV.2008 from larva collected by MK on 20.IV.2008 at Shirokura-kyo, Shizuoka Pref (Fig. 1:22). [SHIKOKU] 3♀(Rh 0053–0055), emerged 1–8.IV.2011 from larvae collected by MK on 27.II.2011 at Yasui-keikoku, Kouchi Pref (Fig. 1:11). [KYUSHU] 2♀ (Rh 0056, 0057), emerged 20–25.IV.2010 from larvae collected by MK on 12.IV.2010 at Gokanosho, Kumamoto Pref (Fig. 1:10). 4♀(Rh 0058–0061), emerged 18–22.IV.2010 from larvae collected by MK on 11.IV.2010 at Mt. Kosho, Fukuoka Pref (Fig. 1:8).

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 45 FIGURE 3. Habitus of adults of Japanese Litoleptis species. L. japonica sp. n. (♂: a [holotype], ♀: b [Rh 0017]), L. kiiensis sp. n. (♂: c [Rh 0221], ♀: d [Rh 0114]), L. niyodoensis sp. n. (♂: e [holotype], ♀: f [Rh 0186]), L. himukaensis sp. n. (♂: g [Rh 0201], ♀: h [Rh 0196]), L. izuensis sp. n. (♂: i [holotype]), L. asterellaphile sp. n. (♂: j [Rh 0207], ♀: k [Rh 0206]). Scales = 0.1 mm.

FIGURE 4. Wings of Japanese Litoleptis species. (a) L. japonica sp. n. [Rh 0017], (b) L. kiiensis sp. n. [Rh 0221], (c) L. niyodoensis sp. n. [Rh 0186], (d) L. himukaensis sp. n. [Rh 0197], (e) L. izuensis sp. n. [holotype], (f) L. asterellaphile sp. n [Rh 0211]. Abbreviation: H, humeral crossvein.

46 · Zootaxa 4097 (1) © 2016 Magnolia Press IMADA & KATO FIGURE 5. Litoleptis japonica sp. n. (a) Antenna of male in lateral view [holotype], (b) female genitalia in ventral view [Rh 0014], (c) male gonocoxite in dorsal (left) and ventral (right) view [Rh 0008]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm. Abbreviations: aedg, aedeagus; ejc apd, ejaculatory apodeme; gns, gonostylus; gnt chm, genital chamber; prm, paramere; sp sac, sperm sac; spmtc, spermatheca; spr dct, spermathecal duct; spr dct acc g, spermathecal duct accessory gland; vl proc, ventrolateral process.

Etymology. The specific epithet is a noun in apposition, derived from the distribution of this species. Distribution. Japan (Hokkaido, Honshu, Shikoku, Kyushu) (Fig. 1). Natural history. Adults of this species emerged from thalli of the Conocephalum conicum species complex (Marchantiales: Conocephalaceae) growing on moist rocky or clay slopes along streams in both evergreen and deciduous forests (Fig. 11a). Larvae of this species are thallus miners of C. conicum species complex (Fig. 11d). Adults emerged in spring (March–May, in laboratory conditions). Diagnosis. Litolepis japonica is easily separated from all other congeners by having a long stout setae at the tip of first flagellomere. This species can also be easily distinguished from the other species by the form of apical extension of gonostylus that is unevenly bifurcated in males, and spermathecal duct accessory glands arising at base of spermatheca in females. Remarks. It is noteworthy that Litoleptis japonica has extremely female-biased sex ratios: only seven males of 61 adult flies of L. japonica were obtained, even though the collecting method and timing (collecting as larvae mining in the liverwort mats) did not appear to produce the sampling biases.

Litoleptis kiiensis Imada & Kato sp. n. [Japanese name: kii-jingasa-shitone-abu] (Figs. 3c, 3d, 4b, 6)

Description. Body length: 2.6 mm (n=6) in male, 3.0 mm (n=7) in female. Wing length: 2.6 mm (n=7) in male, 2.8 mm (n=7) in female. Head Frons bare in male, bare or having a pair of setae in female. Antenna 0.55 mm (n=3). Antenna 1.0 times in male and 1.3 times in female as long as diameter of eye. First flagellomere with enlarged base; with distinctive depression at middle part (at basal 1/3–2/3) of anterior margin; apex slightly enlarged (wider than midpoint width) (Fig. 6a). Antenna covered with short setae consistently. Male (Fig. 6b) Gonostylus with semicircle-shaped extension apically, pointed inwardly at apex; smooth in posterior edge of basal major part. Sperm sac bulbous hardly visible. Lateral ejaculatory process integrated into sperm sac basally, but freely elongate apically. Ejaculatory apodeme short, round at anterior margin of gonocoxite. Less than four bristles at inner corner

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 47 of ventral surface of gonocoxite. Base of aedeagus connected with paramere at thick and wide joint. Paramere widest at connection with aedeagus, approximately three times as wide as narrowest point. Apex of aedeagus flat with shallow incision at apex. Female (Fig. 6c) Spermatheca membranous, cone-shaped. Spermathecal duct accessory gland with short, straight sclerotized columnar duct, arising at less than two-thirds distal length from genital chamber to spermatheca. Common spermathecal duct absent. Genital chamber small.

FIGURE 6. Litoleptis kiiensis sp. n. (a) Antenna of male in lateral view [Rh 0214], (b) female genitalia in ventral view [Rh 0135], (c) male gonocoxite in dorsal (left) and ventral (right) view [Rh 0133]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm.

Type material. Holotype. JAPAN [HONSHU] 1♂, emerged on 28.III.2011 from larva collected by MK on 22.XI.2011 at Akabanegawa, Mie Pref (Fig. 1:18), “Rh 0109”, NMNS. Paratypes. 2♂, 9♀ (Rh 0110–0132) emerged from 27.III–30.IV.2011, from the larvae collected by MK on 22.XI.2011 at same locality as holotype, KUHE. Additional materials. In total, 58 specimens were collected. All following materials were obtained as larvae. All specimens are stored in Kyoto University (KUHE). JAPAN [HONSHU] 13♂, 15♀ (Rh 0133–0161) emerged on 30.III.2014 from larvae collected by YI on 9.III.2014 at Nanataki, Wakayama Pref (Fig. 1:12). 7♂, 7♀ (Rh 0162–0175) emerged during 3.IV.2014 from larvae collected by YI on 9.III.2014 at Hyakkennzannkeikoku, Wakayama Pref (Fig. 1:13). 4♂, 2♀ (Rh 0176–0181) emerged on 30.III.2014 from larvae collected by YI on 10.III.2014 at Takinohai, Wakayama Pref (Fig. 1:15). 2♂ (Rh 0182, 0183) emerged during 7–9.IV.2009 from larvae collected by MK on 1.IV.2009 at Youjigawa, Wakayama Pref (Fig. 1:16). 4♂, 4♀ (Rh 0212–0223) emerged during 9–11.IV.2010 from larvae collected by MK on 3. IV 2010 at Shimajigawa, Mie Pref (Fig. 1:19). Etymology. The specific epithet is a noun in apposition, taken from the Kii peninsula where this species exists. Distribution. Japan (Honshu: Wakayama and Mie Prefectures) in Japan (Fig. 1). Natural history. Larvae of this species are thallus miners of Reboulia hemisphaerica (Marchantiales: Aytoniaceae) growing on rocky or clayey slopes in both evergreen and deciduous forests (Fig. 11f). This species prefers sunny and relatively arid microhabitats compared to the habitat of L. japonica, depending on the microhabitat preference of its host-plant. Adults emerged in spring (March-July, in laboratory condition). Diagnosis. Litoleptis kiiensis resembles L. niyodoensis and himukaensis, and can be distinguished from them by the following characters: first flagellomere with distinctive depression at basal one-third to two-thirds of dorsal margin and swollen at apex; gonostylus with semicircle-shaped extension at apex (as opposed to square-shaped in the other two species), and smooth posterior edge of basal part (as opposed to undulate dorsal edge in the other two species); less than four bristles at posteromedial margin of ventral surface of gonocoxite; spermatheca conical- shaped in female (as opposed to round-shaped in L. niyodoensis).

Description. Body length: 2.9 mm (n=7) in male, 3.1 mm (n=3) in female. Wing length: 2.7 mm (n=7) in male, 3.0 mm (n=3) in female. Head Frons bare in male, with sparse setae in female. Antenna 0.47 mm (n=3). Antenna 1.1 times as long as diameter of eye, densely covered with short setae consistently. First flagellomere elongate as long tapering segment or oval and enlarged near base, concave ventrally at basal one-third to half; distal end not enlarged (Fig. 7a). Male (Fig. 7b) Gonostylus with flat, oblong-shaped extension apically, bluntly pointed inwardly at apex; undulating in posterior edge of basal part. Sperm sac bulbous hardly visible. Lateral ejaculatory process integrated into sperm sac, elongate transversely accompanying with distal end of sperm sac. Ejaculatory apodeme short, round at anterior margin of gonocoxite. Posteromedial margin in ventral surface of gonocoxite bear 5–7 bristles. Aedeagus tapering toward apex, connected with gonocoxite at paramere at thick and wide joint. Paramere swollen dorsally near distal end, widest at connection with aedeagus, approximately three times as wide as distal end. Female (Fig. 7c) Spermatheca membranous, rounded at end. Spermathecal duct accessory gland prominent, with short and sclerotized columnar duct, arising at approximately two-thirds the distal length between genital chamber and spermatheca, with saccate structure visible. Common spermathecal duct absent. Genital chamber small.

FIGURE 7. Litoleptis niyodoensis sp. n. (a) Antenna of male in lateral view [Rh 0193], (b) female genitalia in ventral view [Rh 0194], (c) male gonocoxite in dorsal (left) and ventral (right) view [Rh 0187]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm.

Type material. Holotype. JAPAN [HONSHU] 1♂, emerged 29.III.2011 from larva collected by MK on 27.II.2011 at Yasuikeikoku, Kouchi Pref (Fig. 1:11), “Rh 0184”, NMNS. Paratypes. 7♂, 3♀ (Rh 0185–0194) emerged during 27–29.III.2011 from larvae collected by MK on 27.II.2011, same locality as holotype, KUHE. Etymology. The specific epithet is a noun in apposition, taken from Niyodo river, the main stream of Yasui river flowing near the type locality. Distribution. Japan (Honshu: Kouchi Prefecture) (Fig. 1).

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 49 Natural history. Habitat of this species is similar to that of L. kiiensis. Larvae of this species are miners of thallus of Reboulia hemisphaerica. Adults emerged in spring (March, in laboratory condition). Diagnosis. Litoleptis niyodoensis resembles L. kiiensis and himukaensis, and be distinguished from them by the following characters: first flagellomere tapering toward apex with a slight depression at basal one-third to half without enlarged apex; gonostylus with oblong-shaped extension, undulating in posterior edge of basal part; posteromedial margin in ventral surface of gonocoxite bear 5–7 bristles; paramere swollen posteriorly near distal end (without swollen in other two species); spermatheca rounded at end in female (as opposed to conical-shaped in L. kiiensis).

Litoleptis himukaensis Imada & Kato sp. n. [Japanese name: himuka-shitone-abu] (Figs. 3g, 3h, 4d, 8)

Description. Body length: 2.8 mm (n=3) in male, 2.9 mm (n=2) in female. Wing length: 2.4 mm (n=4) in male, 2.6 mm (n=2) in female. Head Frons bare in both sexes. Antenna 0.53 mm (n=3). Antenna 1.0 times as long as diameter of eye. First flagellomere straight and gradually tapering towards apex; covered with comparatively rough and long setae (Fig. 8a). Male (Fig. 8b) Gonostylus with oblong-shaped extension apically; posterior surface of apex hollowed and weakly pointed inwardly; undulating in posterior edge of basal part. Sperm sac bulbous and hardly visible. Lateral ejaculatory process apparently not integrated into sperm sac, freely elongate transversely. Ejaculatory apodeme moderately long but not reaching anterior margin of gonocoxite. Posteromedial margin in ventral surface of gonocoxite bear less than four bristles. Aedeagus smoothly connected with paramere at thick and wide joint. Paramere slightly swollen posteriorly near distal end; become widest at connection with aedeagus, less than two times as wide as distal end. Type material. Holotype. JAPAN [KYUSHU] 1♂, emerged 14. II 2009 from larvae collected by MK on 17.I.2009 at Udo Jingū, Miyazaki Pref, Japan (Fig. 1:9), “Rh 0195”, NMNS. Paratypes. 3♂, 3♀ (Rh 0196–0201), emerged 10–17.II.2009 from larvae collected by MK on 17.I.2009 at same locality as holotype (Fig. 1:9), KUHE. Etymology. The specific epithet is taken from classical name of the type locality, “Himuka”, southern part of Miyazaki Prefecture. Distribution. Japan (Honshu: Miyazaki Prefecture) (Fig. 1). Natural history. Habitat of this species is rocky slope along streams in coastal evergreen Castanopsis forests. Larvae of this species are thallus miner of Reboulia hemisphaerica (Fig. 11g). Adults emerged in spring (March- July, in laboratory condition). Diagnosis. Litoleptis himukaensis resembles L. kiiensis and L. niyodoensis, and can be distinguished from them by the gonostylus with oblong-shaped extension apically (as opposed to L. kiiensis), of which posterior surface of apex is hollowed (as opposed to flat at apex in L. niyodoensis).

Litoleptis izuensis Imada & Kato sp. n. [Japanese name: izu-jingasa-shitone-abu] (Figs. 3i, 4e, 9)

Description. Body length: 3.4 mm (n=2) in male. Wing length: 4.3 mm (n=2) in male. Head Frons bare in male, with sparse hairs in female. Antenna 0.32 mm (n=2). Antenna 1.1 times as long as diameter of eyes. First flagellomere straight and gradually tapering towards apex, slightly bulbous dorsally near apex; densely covered with comparatively long hairs (Fig. 9a). Male (Fig. 9b) Gonostylus with triangle-shaped extension posteriorly, tapering toward apex, widest point at base twice as wide as apex. Sperm sac posteriorly cylindrical in internal aedeagual process, and widen and forked toward anterior end. Lateral ejaculatory process small and round-shaped without elongation, weakly sclerotized. Ejaculatory apodeme short, posterior end broad and tapering toward anterior margin of gonocoxite. Posteromedial margin in ventral surface of gonocoxite bear less than four bristles. Aedeagus tapering toward apex; smoothly connected with paramere. Paramere consistently slender, approximately six times as narrow as width of gonostylus at basal widest point. Female. Unknown.

50 · Zootaxa 4097 (1) © 2016 Magnolia Press IMADA & KATO FIGURE 8. Litoleptis himukaensis sp. n. (a) Antenna of male in lateral view [Rh 0195], (b) male gonocoxite in dorsal (left) and ventral (right) view [Rh 0200]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm.

Type material. Holotype. JAPAN [HONSHU] 1♂, holotype, emerged on 7.IV.2009 from larva collected by MK on 22.III.2009 at Izu Oshima, Tokyo Metropolitan, Japan (Fig. 1:23), “Rh 0202”, NMNS. Paratype. 1♂ (Rh 0203), emerged on 2.IV.2009 from larva collected by MK on 22.III.2009 at same locality as holotype (Fig. 1:23), KUHE. Etymology. The specific epithet is a noun in apposition, taken from the Izu Oshima Island where this species was found. Distribution. Japan (Honshu: Tokyo Metropolitan) (Fig. 1). Natural history. Habitat of this species is shaded clayey slope along streams and roads in evergreen Castanopsis forests. Larvae of this species are thallus miners of Reboulia hemisphaerica. Adults emerged in spring (April, in laboratory condition). Diagnosis. Litoleptis izuensis can be distinguished from the congeners by the following characters: gonostylus greatly wide at base with triangle-shaped extension posteriorly, tapering toward apex, widest point at base twice as wide as apex; paramere narrow, approximately six times as narrow as width of gonostylus at basal widest point; lateral ejaculatory process small and round-shaped without elongation; sperm sac developed and forked at anterior end.

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 51 FIGURE 9. Litoleptis izuensis sp. n. (a) Antenna of male in lateral view [holotype], (b) male gonocoxite in dorsal (left) and ventral (right) view [holotype]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm. Abbreviation: ltr ejc prc, lateral ejaculatory process.

Litoleptis asterellaphile Imada & Kato sp. n. [Japanese name: saihai-shitone-abu] (Figs. 3j, 3k, 4f, 10)

Description. Body length: 2.3 mm (n=5) in male, 2.4 mm (n=1) in female. Wing length: 2.3 mm (n=6) in male, 2.6 mm (n=2) in female. Head Frons bare in both sexes. Antenna 0.31 mm (n=2). Antenna 0.8 times in male and 1.2 times in female as long as diameter of eyes. First flagellomere oval and enlarged near base, elongate as long tapering segment, sharpened like a straight long hair at tip; in male, flagellomere sometimes sharply curved at middle (Fig. 10a). Male (Fig. 10b) Gonostylus slightly tapering toward apex (basal width at most 1.8 times as wide as width of narrowest part), posterior surface of apex hollowed and posterior margin projected as truncated, upward apex inwardly. Sperm sac bulbous not visible in ventral view. Lateral ejaculatory process baculiform. Ejaculatory apodeme moderately long but not reaching anterior margin of gonocoxite. Posteromedial margin in ventral surface of gonocoxite bear one bristle. Aedeagus smoothly connected with paramere at wide joint. Female (Fig. 10c)

52 · Zootaxa 4097 (1) © 2016 Magnolia Press IMADA & KATO Spermatheca membranous, long and narrow, less than half as wide as long. Spermathecal duct accessory gland with short, sclerotized columnar duct curves middle at right angle, arising at approximately three-fourth distal length from genital chamber to spermathecal ducts. Common spermathecal duct absent. Genital chamber oval, very narrow, occupying fraction of sternite 9 area.

FIGURE 10. Litoleptis asterellaphile sp. n. (a) Antenna of male in lateral view [Rh 0204], (b) female genitalia in ventral view [Rh 0210], (c) male gonocoxite in dorsal (left) and ventral (right) view [Rh 0205]. Joint of gonocoxite and epandrium is circled and pointed by arrow. Scales = 0.1 mm.

Type material. Holotype. JAPAN [HONSHU] 1♂, emerged on 22.IV.2011 from the larva collected by MK on 14.XI.2010 at Nakatsugawa-keikoku, Saitama Pref (Fig. 1:24), “Rh 0204”, NMNS. Paratype. 5♂, 2♀ (Rh 0205–0211), emerged on 2–22.IV.2011 from the larvae collected by MK on 14.XI.2010 at the same place (Fig. 1:24). Etymology. The specific epithet is a noun in apposition, formed by the combination of ”asterella”, which being named after the genus of their host-plant, and “phile”, a suffix that derived from Latin and means “lover of”. Distribution. Japan (Honshu: Saitama Prefecture) (Fig. 1:24). Natural history. Habitat of this species is rocky cliff along streams and roads in deciduous forests. Larvae of this species are thallus miners of Asterella odora (Marchantiales: Aytoniaceae). Adults emerged in spring (April, in laboratory condition). Diagnosis. Litoleptis asterellaphile is smaller in body size than the other species of Litoleptis known in Japan. This species can be distinguished from the other Litoleptis species in Japan by the following characters: gonostylus slightly tapering toward apex (basal width at most 1.8 times as wide as width of narrowest part), posterior surface of apex hollowed and posterior margin projected as truncated, upward apex inwardly; posteromedial margin in ventral surface of the gonocoxite bear one bristle; lateral ejaculatory process baculiform.

Key to the species of Litoleptis in Japan

1. First flagellomere sharpened to be like a needle at apex (Fig. 5a). Female spermathecal duct accessory gland originates at base of spermatheca (Fig. 5b). Male gonostylus with forcep-like extension hollowed dorsally and bifid unevenly at apex (Fig. 5c). Aedeagus deeply notched at apex (Fig. 5c). Lateral ejaculatory process absent (Fig. 5c)...... L. japonica n. sp.

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 53 - First flagellomere stylate, tapering toward apex. Female spermathecal duct accessory gland originates in middle of genital chamber and spermatheca. Male gonostylus with extension apically, of which tip not bifurcated. Aedea- gus without notch at apex. Lateral ejaculatory process present...... 2 2. Lateral ejaculatory process round-shaped, without elongation (Fig. 9b). Sperm sac widened and forked at anterior end (Fig. 9b) ...... L. izuensis n. sp. - Lateral ejaculatory process elongated transversely (e.g. Fig. 5b). Sperm sac not forked at anterior end (e.g. Fig. 5b) ...... 3 3. Body length shorter than 2.5 mm. Antennal length less than 0.4 mm. Lateral ejaculatory process baculiform without extension at distal end (Fig. 10c) ...... L. asterellaphile n. sp. - Body size longer than 2.5 mm. Antennal length longer than 0.4 mm. Lateral ejaculatory process elongate slightly curved at distal end (e.g. Fig. 6c) ...... 4 4. Ejaculatory apodeme more than one-fourth as long as length of gonocoxite (Fig. 8b) ...... L. himukaensis n. sp. - Ejaculatory apodeme more than one-fourth as long as length of gonocoxite...... 5 5. First flagellomere with distinctive depression at basal one-third to two-thirds of dorsal margin and enlarged at apex. Spermatheca conical-shaped in female (Fig. 6b). Posteromedial margin in ventral surface of gonocoxite bear less than four bristles (Fig. 6c) ...... L. kiiensis n. sp. - First flagellomere with a slight depression at basal one-third to two-thirds of dorsal margin without enlarged apex (Fig. 7a). Spermatheca rounded at end in female (Fig. 7b). Posteromedial margin in ventral surface of gonocoxite bear more than five bristles (Fig. 7c) ...... L. niyodoensis n. sp.

Biology of the Japanese Litoleptis

All Japanese Litoleptis species were found to be thallus-miners of thallose liverworts belonging to Aytoniaceae and Conocephalaceae (Marchantiopsida, Marchantiophyta) growing on moist riverine slopes or cliffs in evergreen or deciduous forests (Fig. 11a). All Litoleptis species in Japan are univoltine, and their larval stages spanned almost eleven months, from spring till late winter. The mines were formed within photosynthetic tissue of the thalli particularly near the upper side but not in the upper epidermis, and sometimes easily were seen through the upper thallus (Fig. 11b). The mines were linear at first, but occasionally became radiate or marked with blotches later. The number of larvae per thallus was usually one. The larvae underwent pupation inside the mine in early spring (Fig. 11c). The pupal stage spanned approximately a couple of weeks and then the adults emerged, which lived for only a few days. The adult flies were inactive and their feeding behavior has not been observed so far, nevertheless their labella were well-developed (Fig. 2a). The adults were seldom found in the field; therefore, it was difficult to observe their behavior. We did however observe in the field a L. japonica female ovipositing at the apical-dorsal part of the thallus of their host-plant later in spring. Each Japanese species of Litoleptis fed on only a single liverwort genus as their host-plant: L. japonica feed on Conocephalum, L. asterellaphile on Asterella, and the other four species on Reboulia. Among the host-plants of the Japanese Litoleptis, Conocephalum conicum species complex and Reboulia hemisphaerica are common in Japan, whereas Asterella odora is extremely rare and whose distribution is restricted in the Kanto Region (Iwatsuki 2001). In general, the Japanese Litoleptis species appeared to be locally distributed in limited area except for L. japonica, nevertheless the mines of Litoleptis spp. were found relatively frequently in each population. In Japan, the association between liverworts and Litoleptis spp. seemed to be as common as it is in Micropterigidae, the most basal moth family (Kristensen 1984). The micropterigid moths in Japan feed on liverworts and collectively have a wide distribution in Japan (Imada et al. 2011). In particular, all the Japan endemic species of Micropterigidae belonging to four endemic genera, feed only on Conocephalum liverworts (Imada et al. 2011). The distribution pattern of these micropterigid species in Japan, which is local and allopatric (Imada et al. 2011), is in contrast to that of Litolepis japonica, which is widespread and covers the main islands of Japan (Hokkaido, Honshu, Shikoku, Kyushu), even though these two lineages share the same host-plant species.

Discussion on taxonomy of Litoleptis

Spaniinae has been defined based on morphology of female genitalia (Nagatomi 1982): cerci widely separated from each other; segment 1 of cercus usually not dilated outward or downward; tergum 10 absent or short; intersegmental membrane between tergum 7 and 8 hardly visible or short; tergum 7 much wider than long. In

54 · Zootaxa 4097 (1) © 2016 Magnolia Press IMADA & KATO addition, Kerr (2010) proposed that the presence of a paired anterior-facing ventrolateral process of tergite 9 in female as defining character of Spaniinae. By and large, the character states of female genitalia of the Japanese Litoleptis satisfy the above-mentioned conditions, therefore, Litoleptis may be confidently placed in Spaniinae. Besides, Litoleptis mostly resemble Spania among all genera of Rhagionidae in general appearance, nevertheless Litoleptis and Spania have few synapomorphies, such as tergite 10 absent in male.

FIGURE 11. Habits of Japanese Litoleptis. (a) Typical habitat of Litoleptis japonica sp. n. at Nanataki in Wakayama Pref., (b) adult female of L. japonica sp. n. at Kakuma-onsen in Nagano Pref., (c) cross section of a thallus of Conocephalum conicum, in which a larva of L. japonica sp. n. mined. (d) larva of Litoleptis japonica sp. n., (e) pupa of Litoleptis japonica sp. n. (white arrow), (f) larva of Litoleptis kiiensis sp. n., (g) mines of L. himukaensis (black arrows) (h) pupa of L. asterellaphile (white arrows).

Due to interspecific variation found in several of the Japanese species, the concept of Litoleptis has been expanded. The following characters are newly found in the Litoleptis species in Japan: male eyes holoptic (in all Japanese species); sperm sac developed into bulbous sac or separated lobes (in all Japanese species); lateral ejaculatory process present (in all but L. japonica); aedeagus developed (in all Japanese species). The character states of the male genitalia of Litoleptis are somewhat similar to those of Spania, and that may blur the identity of this genus among all genera of Spaniinae. Considering this, genus Litoleptis is best defined by the following characters: wing lacks a medial crossvein (dm cell is absent); all tibiae lack spurs; gonocoxal apodemes absent. Furthermore, the female abdomen and genitalia of Litoleptis display several autapomorphic traits of this genus:

BRYOPHYTE-FEEDING OF LITOLEPTIS Zootaxa 4097 (1) © 2016 Magnolia Press · 55 tergite 9 inconspicuous and retracted within tergite 8; spermathecae membranous without sclerotization (e.g. Fig. 5b); spermathecal duct accessary glands connected with sclerotized short duct (e.g. Fig. 5b). The short sclerotized duct at the base of spermathecal duct accessary glands is particularly unique in this genus. A paired anterior-facing ventrolateral process (Fig 2e) of tergite 9 most resembles that in Spania, although careful comparison is necessary taking into account the interspecific variation. Overall, the morphology of female genitalia of Rhagionidae remains an untapped source of taxonomic information, and further investigations on it will greatly develop our understanding of the classification of Rhagionidae. The Japanese Litoleptis may be divided into two groups, Litoleptis japonica and the rest. The former has pronounced difference in the following traits: first flagellomere with a long stout setae at the tip; lateral ejaculatory processes absent; apical extension of gonostylus unevenly bifurcated; common spermathecal duct presents; spermathecal duct accessory glands arising at the base of spermatheca in female. Interestingly, this division is consonant with the phylogenetic division of their host-plants at family level, i.e. L. japonica use Conocephalaceae, and the others Aytoniaceae. It is noteworthy that the bryophyte-feeding habit of Litoleptis is shared with two genera, Spania and Ptiolina, which are reported as herbivores of liverworts and mosses, respectively (Mik 1896; Nartshuk 1995). These findings corroborate the relatedness among Litoleptis, Spania and Ptiolina and imply that the close association with bryophytes may have influenced on the evolution of Spaniinae.

Acknowledgements

We greatly appreciate Dr M Sueyoshi (EEPRI) and Dr P Kerr for providing critical and helpful comments on earlier drafts. This research was partly supported by Ministry of Education, Science, Sports and Culture, Grant-in- Aid for Scientific Research (B) 2010–2014 (22405009) to MK and also by Grant-in-Aid for JSPS Fellows (No. 26- 160) to YI.

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