A taxonomic revision and molecular phylogeny of the eastern Palearctic species of the genera Schizomyia Kieffer and Asteralobia Kovalev (Diptera, Cecidomyiidae, Asphondyliini), with descriptions of five new species of Schizomyia from Japan

Abstract The genus Asteralobia (Diptera, Cecidomyiidae, Asphondyliini, Schizomyiina) was erected by Kovalev (1964) based on the presence of constrictions on the cylindrical male flagellomeres. In the present study, we examine the morphological features of Asteralobia and Schizomyia and found that the male flagellomeres are constricted also in Schizomyiagaliorum, the type species of Schizomyia. Because no further characters clearly separating Asteralobia from Schizomyia were observed, we synonymize Asteralobia under Schizomyia. Molecular phylogenetic analysis strongly supports our taxonomic treatment. We describe five new species of Schizomyia from Japan, S.achyranthesae Elsayed & Tokuda, sp. n., S.diplocyclosae Elsayed & Tokuda, sp. n., S.castanopsisae Elsayed & Tokuda, sp. n., S.usubai Elsayed & Tokuda, sp. n., and S.paederiae Elsayed & Tokuda, sp. n., and redescribe three species, S.galiorum Kieffer, S.patriniae Shinji, and S.asteris Kovalev. A taxonomic key to the Japanese Schizomyia species is provided.


Introduction
Schizomyia Kieffer is a cosmopolitan genus of the subtribe Schizomyiina (Diptera, Cecidomyiidae, Cecidomyiinae, Asphondyliini) with 53 species associated with diverse plant families (Gagné and Jaschhof 2017, Elsayed et al. in press). The genus includes species with needle-like ovipositors, four-segmented palpi, and larval terminal segments with four or fewer pairs of terminal papillae (Gagné and Menjivar 2008). Most of these species induce bud galls, but some induce leaf galls (Gagné 1989). Some Schizomyia species are agricultural pests, e.g. S. loroco Gagné, which induces flower galls on loroco, Fernaldia pandurata (A. DC.) Woodson (Apocynaceae), in El Salvador (Gagné and Menjivar 2008). A few species have been used as potential biological control agents, e.g., S. macrofila (Felt), which induces flower galls on Amsinckia spp. weeds in California (Pantone and Brown 1985). Kovalev (1964), in his revision of the Russian Far East gall midges of the tribe Asphondyliini, erected the genus Asteralobia based on the presence of shallow or deep constrictions on the cylindrical male flagellomeres. Although Asteralobia has been treated as an independent genus of Schizomyiina and presently contains 12 species (Gagné and Jaschhof 2017), some studies have indicated that Asteralobia could be subsumed under Schizomyia because of the lack of known synapomorphic differences between them (e.g. Tokuda et al. 2003;Gagné and Jaschhof 2017). In the present study, we re-examined morphological features of Asteralobia and Schizomyia and analyzed molecular phylogenetic relationships between them, which have led us to the conclusion that Asteralobia should be synonymized under Schizomyia. In addition, we describe five new species of Schizomyia from Japan and provide a taxonomic key to the Schizomyia species from Japan.

Collecting and rearing methods
Galls induced by six gall midge species were collected from various localities in Japan (Table 1). Some galls were dissected to obtain larval specimens, while the remaining galls were kept in plastic bags until the departure of mature larvae from the galls. Thereafter, larvae were transferred to plastic cups (120 mm in diameter, 110 mm in depth) containing a mixture of wet peat moss and sand (1:1), covered with fine mesh, and fixed with a rubber band. Cups containing larvae obtained from fruit galls on Achyranthes bidentata Blume (Amaranthaceae) and Trachelospermum asiaticum (Sieb. et Zucc.) Nakai (Apocynaceae), and flower bud galls on Paederia foetida L. (Rubiaceae) and Patrinia villosa (Thunb.) (Valerianaceae) were transferred to the field on the Saga University campus, Saga Prefecture (about 5.5 m a.s.l.), and half-buried in the soil to allow the mature larvae to overwinter under natural conditions. These cups were brought back to the laboratory in April to rear adults and were kept at room temperature. In cases of larvae obtained from flower bud galls on Diplocyclos palmatus (L.) Jeffrey (Cucurbitaceae) and Castanopsis sieboldii (Makino) Hatus. (Fagaceae), the cups containing larvae were maintained at room temperature in the laboratory until adult emergence. The pupal exuviae protruding from the surface of the soil in the plastic cups were collected at the same time carefully. Reared specimens were preserved in 75% ethanol for morphological study and 99.5% ethanol for the molecular phylogenetic study.

Taxonomy
Gall midge specimens were mounted on slides in Canada balsam using the technique outlined in Gagné (1994), except for the clearing step following Elsayed et al. (2018). The slide-mounted specimens were examined under a bright-field and phase-contrast microscope (H550L, Nikon, Tokyo) and illustrations were made with the aid of a drawing tube. A semi-motorized fluorescence microscope (BX53, Olympus, Tokyo) was used to photograph some of the characters from mounted specimens with the aid of a microscope-attached camera (DP22, Olympus, Tokyo). Morphological terminology basically follows McAlpine et al. (1981) for adults, but the thoracic sclerite "mesanepisternum" follows Gagné (1968) and the wing venation follows Yukawa (1971). Larval and pupal terminology follows Gagné (1994). All the types of the newly described species are deposited in the collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (KUEC).
Adult and immature specimens of Asteralobia humuli Shinji, A. patriniae Shinji, A. sasakii Monzen and A. soyogo Kikuti, and larvae of A. doellingeriae Kovalev were examined in KUEC. Adults of A. asteris Kovalev, A. calathidiphaga Kovalev, A. doellingeriae, and A. solidaginis Kovalev, as well as four females and two males of S. galiorum were examined in the B. M. Mamaev Collection in the Museum of Nature and Human Activities, Hyogo, Japan. A female and a pupa of Schizomyia galiorum Kieffer were examined in the National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (USNM).
The sequence data were analyzed with the maximum likelihood (ML) method using MEGA (ver. 6.0) (Tamura et al. 2013). Sequences of Asphondylia gennadii Marchal (AB198012 and AB115580) and Pseudasphondylia neolitseae Yukawa (AB334237 and LC422092) were used as outgroup species. The best model was identified with the jModelTest 2 software (Darriba et al. 2012, Guindon andGascuel 2003) on the basis of hierarchical likelihood ratio tests (hLRT) was GTR+G+I.
Female abdomen (Figs 10, 11): Tergites with anterior pair of trichoid sensilla; tergites I-VII rectangular and evenly covered with scales, tergites I-VI with a posterior row of setae and some scattered setae laterally at midlength; tergite VII with 1-2 posterior rows of setae and some scattered setae laterally at midlength; tergite VIII bare, notched laterally, posterior margin with a pair of well-developed dorsal lobes. Sternites II-VI rectangular, bare and less pigmented medially, with the lateral pair of trichoid sensilla situated anterior to the sclerotized sternite, several scattered setae on the anterior half, and 1-2 rows of setae posteriorly; sternite VII about 2.6 times as long as preceding sternites, with anterior pair of trichoid sensilla laterally situated on the sternite and setae covering posterior two-thirds. Ovipositor: protrusible needlelike portion about 4 times as long as sternite VII (Fig. 10); cerci fused, each lobe with few setae (Fig. 11).
Male abdomen: Tergites I-VII as in female; tergite VIII weakly sclerotized medially, with anterior pair of trichoid sensilla. Sternites II-VI as in female; sternite VII with lateral pair of trichoid sensilla situated anterior to the sclerotized sternite, several setae scattered anteriorly, median membranous bare area and 1-2 posterior row of setae; sternite VIII setose, with lateral pair of trichoid sensilla situated intersegmentally between sterna VII and VIII. Terminalia ( Fig. 12): gonocoxite extending ventrally as convex lobe beyond of gonostylus; gonostylus stout, with unfused and compressed denticles, dorsally with setae on distal third, ventrally with a cluster of setae on the basal half. Cerci notched, each cercus with 4 strong setae and a few fine setae. Hypoproct shorter than cerci and aedeagus, bilobed, with a seta on each lobe. Parameres about half length of hypoproct. Aedeagus gradually tapering, acute apex, longer than cerci.
Mature larva (Figs 13-15): Cylindrical, yellow. Number and position of thoracic and abdominal spiracles as in other Asphondyliini (see Möhn 1961); 6 dorsal papillae present on all thoracic segments and abdominal segments I-VII, each with a seta; abdominal segment VIII with 2 dorsal lobes, each with a setose dorsal papilla. Sternal spatula bilobed (Fig. 13) with posterior portion about 3.6 times as wide as the base of the anterior free portion. Two groups of lateral papillae on all thoracic segments, the inner group of 2 setose papillae and the outer group of 2 setose and 1 asetose papillae. Two sternal papillae on each thoracic segment and 4 sternal papillae on abdominal segments I-VIII, all without setae and situated on slight swellings. Ventral papillae each with a seta on meso-and metathoracic segments and on abdominal segments I-VII. Anus situated ventrally, with simple opening and 4 asetose anal papillae (Fig. 14). Two pairs of pleural papillae present on all thoracic segments and abdominal segment VIII, and 3 pairs on abdominal segments I-VII. Terminal segment (Fig. 15) with 2 setose and 2 corniform terminal papillae.
Pupa (Figs 16,17): Exuviae not pigmented except prothoracic spiracles and dorsal spines of abdomen. Antennal horns short; 2 pairs of cephalic papillae present, a pair with short seta; 2 pairs of lower facial papillae, a pair with seta; 3 lateral facial papillae present on each side, 1 with short seta, 2 without setae. Prothoracic spiracle, slightly curved, 23-29 μm long (n = 6), connected with trachea to the tip. Spiracles present on abdominal segments II-VI. Abdominal terga I-VIII each with anterior pair of trichoid sensilla and 2 pleural papillae; terga I-VII each with 3 pairs of dorsal papillae, only outermost pair with a seta; tergum VIII with a pair of dorsal papillae, each with a seta. Abdominal terga II-VIII each with 3-4 rows of spines on median third.
Gall and life history. Schizomyia achyranthesae induces subglobular fruit galls on A. bidentata, 5.07-5.17 mm in diameter (n = 5) ( Fig. 1) [Gall No. C-246 in Yukawa and Masuda (1996)]. Based on Yukawa and Masuda (1996) and present observations, each gall contains 1-13 chambers and each chamber contains a single larva. The galls appear in September. The mature larvae leave the galls between October and November and overwinter in the soil. The adults of S. achyranthesae emerge during the flowering season of the host plant in August and September.
Remarks. Schizomyia achyranthesae is distinguishable from the known Schizomyia species, except S. asteris and S. solidaginis, by its shallowly constricted male flagellomeres, lateral position of anterior pair of trichoid sensilla and presence of four larval terminal papillae, as well as two setose papillae in inner group of lateral papillae. S. achyranthesae can be separated from S. solidaginis based on the larval characters as follows: S. achyranthesae possesses a more elongated sternal spatula than S. solidaginis; the inner group of lateral papillae consists of two setose papillae in S. achyranthesae, but one setose and one asetose papillae in S. solidaginis; the anal opening is simple in S. achyranthesae, while branched in S. solidaginis. Then, S. achyranthesae can be separated from S. asteris by the following features: female cerci is less divided in S. achyranthesae; dorsal setae are present on the gonostylus in S. achyranthesae, but absent in A. asteris; and the gonocoxite is only slightly extends ventrally beyond the gonostylus in S. achyranthesae, and the larval anal opening is simple in S. achyranthesae while branched in S. asteris.  Table S1 Characters given in S. achyranthesae except for the following:

Schizomyia diplocyclosae
Etymology. The species name, diplocyclosae, is based on the generic name of the host plant, Diplocyclos palmatus (Cucurbitaceae). Description. Head (Fig. 18): Fronto-clypeus with 17-24 setae (n = 6). Palpus: first segment ca 38.3 μm, second 1.4 times as long as the first, third 1.3 as long as the second, fourth 1.5 as long as the third.
Female abdomen (Figs 23, 24): Posterior margin of tergite VIII with a pair of slightly developed dorsal lobes. Sternite VII about 2.5 times as long as preceding sternites. Ovipositor: distal protrusible needle-like portion about 3 times as long as sternite VII.
Mature larva: Sternal spatula with posterior portion about 2.8 times as wide as the base of the anterior free portion (Fig. 26). Larval anus with 2 asetose anal papillae.
Gall and life history. Schizomyia diplocyclosae induces subglobular and pale green flower bud galls on D. palmatus, about 6-10 mm in diameter. Each gall consists of 10-45 chambers and each chamber contains a single larva [Gall No. C-409 in Yukawa and Masuda (1996)]. Galls become mature between December and March and larvae depart from galls to drop to the ground. The adults of S. diplocyclosae emerge in February and March when the larvae were reared under laboratory temperature (Yukawa and Masuda 1996, present data). Similar flower bud galls were found on Melothria liukiuensis Nakai (Cucurbitaceae) and considered to be induced by this species or a closely related one (Yamauchi et al. 1982, Yukawa andMasuda 1996). Remarks. Schizomyia diplocyclosae is morphologically very similar to S. achyranthesae but differs from it by the following characters: S. diplocyclosae has a shorter ovipositor (protrusible needle-like-portion three times as long as sternite VII while four times as long in S. achyranthesae), less developed dorsal lobes on the posterior margin of female tergite VIII, gonocoxite more pointed posteroapically, empodia longer than claws and larva with only two anal papillae (four in S. achyranthesae).
Remarks. Schizomyia castanopsisae is morphologically close to S. asteris, S. achyranthesae and S. diplocyclosae. Schizomyia castanopsisae can be separated from S. asteris by a shorter ovipositor (protrusible needle-like portion 3.3 times as long as sternite VII, while 5.7 times in S. asteris), the presence of dorsal setae on the gonostyli, and the tooth of gonostylus, which extends more dorsally than in that of S. asteris; from S. achyranthesae by a shorter ovipositor (four times as long as sternite VII in S. achyranthesae), more posteroapically pointed gonocoxite, and branched anal opening of larva; and from S. diplocyclosae by shorter empodia than tarsal claws (empodia are as long as claws in S. diplocyclosae) and the number of larval anal papillae (four in S. castanopsisae while two in S. diplocyclosae).
Description. Head (Fig. 43): Compound eyes separated on vertex by a diameter of 0.0-0.5 facets. Fronto-clypeal setae 15-16 setae (n = 4). Palpus: first segment ca 53.5 μm, second about as long as the first, third 1.6 as long as the second, fourth 1.4 as long as the third.
Mature larva: Sternal spatula (Fig. 51) with posterior portion about 3.5 times as wide as the base of the anterior free portion. Anus with branched opening (Fig. 52).
Pupa (Figs 54, 55): Prothoracic spiracle 250-350 μm long (n = 6). Distribution. Japan: The Izu Islands (Tokuda et al. 2012b, Tokuda and Kawauchi 2013 Honshu, and Kyushu (Yukawa and Masuda 1996).  Gall and life history. The normal fruit of Trachelospermum asiaticum (Apocynaceae) is V-shaped, consisting of a pair of very long and thin seed pods. When the fruits are galled by S. usubai, the apical parts of the fruit become fused and swollen, more or less cat-bell shaped (Fig. 3), about 12-18 mm in diameter and 27 mm in length [Gall No. D-033 in Yukawa and Masuda (1996)]. Each gall consists of 10-25 chambers and each chamber contains 10-25 larvae. Galls mature between late September and October and the larvae depart from galls to overwinter in soil. The adults of S. usubai emerge between late April and July (Yukawa 1978;Yukawa and Masuda 1996;present study). Similar galls probably induced by this species were found on Trachelospermum gracilipes Hook. f. var. kiukiuense (Hatus.) Kitam. on Tanegashima Island .
Remarks. Schizomyia usubai is close to S. asteris, S. achyranthesae, S. diplocyclosae and S. castanopsisae. Schizomyia usubai can be distinguished from S. asteris by a shorter ovipositor (protrusible needle-like portion about 4.5 times as long as sternite VII, while 5.5 times in S. asteris), longer empodia, and the presence of dorsal setae on gonostyli; from S. achyranthesae and S. diplocyclosae by a longer ovipositor (four and three times as long as sternite VII in S. achyranthesae and S. diplocyclosae, respectively), longer empodia, and branched opening of the larval anus. In addition, larva of S. usubai has four anal papillae, but two in S. diplocyclosae. Schizomyia castanopsisae is very similar to S. usubai, but can be separated by a shorter ovipositor (protrusible needle-like portion about 3.3 times as long as sternite VII, while 4.5 times in S. usubai), longer empodia, and less compressed circumfila of female flagellomeres. Characters as in S. achyranthesae except for the following:

Schizomyia paederiae
Etymology. The species name, paederiae, is based on the generic name of the host plant, Paederia foetida (Rubiaceae).
Mature larva: Abdominal segment VIII with 2 setose dorsal papillae. Posterior portion of sternal spatula about 3.3 times as wide as the base of the anterior free portion (Fig. 64); 2 groups of lateral papillae present on all thoracic segments, each consisting of 2 setose and 1 asetose papillae. Terminal segment with 8 terminal papillae, consisting of 4 setose, 2 asetose and 2 corniform ones (Fig. 65).
Gall and life history. Schizomyia paederiae induces flower bud galls on P. foetida. Basal parts of the galled flower buds are swollen, 3.0-5.6 mm in diameter and 4.0-6.1 mm in length (Fig. 4) [Gall No. D-037 in Yukawa and Masuda (1996)]. Galls are single-chambered and each gall contains 1-10 larvae. The larvae depart from mature galls from late August to September and overwinter in the soil. The adults of S. paederiae emerge in early August when the flower buds are available on the host plant (Yukawa and Masuda 1996).
Remarks. Schizomyia paederiae is distinguishable from other Schizomyia species, except four Russian species, i.e. S. calathidiphaga, S. clematidis, S. spiraeae, and S. veronicastrum, by its deeply constricted male flagellomeres (Kovalev 1964;Fedotova 2002). Firstly, the adults of S. paederiae differs from S. calathidiphaga by a slightly longer ovipositor (protrusible needle-like portion about 4.8 times as long as sternite VII, while 4.5 times in S. calathidiphaga), longer empodia (empodia are as long as claws in S. paederiae, but shorter in S. calathidiphaga), the position of gonostylus tooth (mostly covering only the apical margin in S. paederiae, but on the posteroapical margin in S. calathidiphaga), and the arrangement of papillae on the larval terminal segment (the two asetose terminal papillae are situated more posteriorly in S. paederiae, while more anteriorly in S. clathidiphaga). Then, the adults of S. paederiae can be separated from S. clematidis, S. spiraeae and S. veronicastrum by a longer neck of male flagellomere III, which is about 0.25 as long as node in S. paederiae but about 0.15 as long as node in other species, the position of gonostylus tooth (mostly covering the apical margin in S. paederiae, but on the posteroapical margin in the other species), and a much narrower hypoproct than S. clematidis.
Description. Head: Compound eyes with rounded facets; facets on vertex and eye bridge unobservable because the specimens mounted laterally. Palpus: first segment ca 23.4 μm, second 1.6 times as long as the first, third 1.4 as long as the second, fourth 1.4 as long as the third.
Male abdomen: Sternites with median pair of trichoid sensilla. Sternite VII with two posterior rows of setae. Terminalia (Fig. 74): Gonocoxite with slightly developed apical lobe. Gonostylus with several setae on distal half dorsally, with group of setae on the basal half ventrally, and with distinctive unfused denticles. Hypoproct slightly longer than cerci.
Mature larva: Sternal spatula bilobed, the anterior free portion slightly wider than the posterior portion (Kieffer 1889). Two groups of lateral papillae present on each side of the spatula, each group of 2 setose and 1 asetose papillae. Terminal segment with 6 setose and 2 corniform terminal papillae (Möhn 1955).
Remarks. This species is distinguished from eastern Holarctic congeners by the distinctly short ovipositor, the absence of dorsal lobes on the posterior margin of female tergite VIII, and the conjunction of wing vein C with R 5 before wing apex. Shinji, 1938 comb. rev. Figs 77-85 ; Table S4 Schizomyia patriniae Shinji, 1938: 372. Asphondylia partriniae Shinji, 1944. of patriniae. Asteralobia patriniae (Shinji, 1938) Characters as in S. achyranthesae except for the following:
Distribution. Japan: Hokkaido, Honshu and Shikoku (Yukawa and Masuda 1996). Remarks. This species had been described by Shinji (1938) under the genus Schizomyia. Then, Yukawa (1983) combined the species with Asteralobia because of its shallowly constricted male flagellomeres. Because Asteralobia is synonymized under Schizomyia in this paper, S. patriniae is combined again with Schizomyia.
Schizomyia patriniae is distinguishable from known Schizomyia species, except three species that were previously treated as Asteralobia and newly combined here under Schizomyia, i.e. S. sasakii, S. soyogo, and S. humuli, by the presence of shallow constrictions on male flagellomeres and the absence of corniform papillae on the terminal larval segment. S. patriniae can be easily separated from S. sasakii, S. soyogo and S. humuli based on the number of papillae on the larval terminal segment: S. patriniae possesses two setose and six asetose terminal papillae, but S. sasakii and S. soyogo have only six setose terminal papillae, and S. humuli has four setose terminal papillae. (Kovalev, 1964)  Description. Head: Compound eyes with rounded facets; facets on the vertex and eye bridge unobservable. Palpus: first segment ca 34.5 μm, second 1.8 times as long as the first, third 1.2 as long as the second, fourth 1.1 as long as the third.
Mature larva: Sternal spatula with posterior portion about 3.3 times as wide as the base of the anterior free portion (Fig. 91). Abdominal segment VIII with three dorsal lobes, the outer two each with 1 setose dorsal papilla (Fig. 92). Anus with branched opening, and 4 asetose anal papillae (Fig. 93).

Molecular phylogenetic study
The complete molecular dataset of COI and 12S consisted of approximately 800 bp. The monophyly of Schizomyia was strongly supported with a 99% bootstrap value, and the genus was divided into two main clades. One clade with a 55% bootstrap support was subdivided into three subclades: one including S. galiorum, S. doellingeriae, S. humuli, S. patriniae and S. kovelavi; another including S. sasakii, S. soyogo, and S. paederia; and the third comprising S. buboniae. The second main clade contains six morphologically-close species: S. diplocyclosae, S. castanopsisae, S. achyranthesae, S. solidaginis, S. asteris and S. usubai, and gained a 95% bootstrap support.

Discussion
In the present study, we showed that constricted male flagellomeres, the only character used to separate Asteralobia from Schizomyia (Kovalev 1964), can be also observed in the type species of Schizomyia, S. galiorum, and hence Asteralobia is synonymized here under Schizomyia. Our molecular phylogenetic analysis strongly supported this conclusion with high bootstrap values. Because of the broad definition of Schizomyia, which depends only on plesiotypic characters (Gagné and Marohasy 1997;Gagné 1994), some other genera of Schizomyiina, e.g., Metasphondylia, Placochela and Schizandrobia, are considered to fit easily into its definition (Gagné and Jaschhof 2017). Comprehensive taxonomic and molecular analyses including these genera are needed for further progress on the taxonomy of Schizomyia.
In the present study, six eastern Palearctic Schizomyia species, namely S. achyranthesae, S. asteris, S. diplocyclosae, S. castanopsisae, S. usubai and S. solidaginis, were shown to be close to each other and (although we have never examined the phylogenetic position of S. solidaginis) constructed a monophyletic clade in the molecular analysis. They differ from all known Schizomyia spp. by the laterally situated anterior pair of trichoid sensilla, which are present anterior to the sclerotized sternite. This character can be considered as derived because in other genera of Schizomyiina, the anterior pair of trichoid sensilla are usually located on the sternites. Future comprehensive taxonomic studies may treat these species as a natural cluster within Schizomyia.
Several important characters need to be re-evaluated in order to meet current taxonomic standards in many Schizomyia species. For example, the ovipositors of most known Schizomyia species were not described in detail, although they can be expected to be variable because of their adaptation for oviposition on different organs of hosts belonging to various, not related, families. Similarly, the pupa, which offers many diagnostic features for taxonomy in Schizomyiina (Möhn 1961), is still unknown and Figure 96. A phylogenetic reconstruction based on partial sequences of cytochrome oxidase subunit I (COI) and 12S small ribosomal subunit genes. The topology and branch length were produced by the maximum likelihood method (note the scale bar). Bootstrap values are indicated at branches gaining more than 50% support (10 3 replications). undescribed in many species, especially in those that develop in the soil and are not easily found. Descriptions of these unknown morphological features of Schizomyia species are essential to clarify the generic concept.