Pseudasphondylia tominagai, a new gall midge species (Diptera: Cecidomyiidae) inducing flower bud galls on Eleutherococcus spinosus (Araliaceae) in Japan

Abstract Background The genus Pseudasphondylia (Diptera: Cecidomyiidae: Asphondyliini: Asphondyliina) comprises ten Palearctic, Oriental and Australian species associated with various hosts belonging to at least ten plant families. New information A new species, Pseudasphondylia tominagai Elsayed & Tokuda n. sp., that induces flower bud galls on Eleutherococcus spinosus (L.f.) S.Y.Hu (Araliaceae) is described. This species is considered to alternate between host plants seasonally. A key to males of known Pseudasphondylia species is provided.

bags until emergence of adults. Most of the specimens collected were preserved in 75% ethanol for morphological examinations.

Morphological Examination and Terminology
Gall midge specimens were mounted on microscope slides in Canada balsam, following the technique outlined in Gagné (1994), except for the clearing step for some of the larval and adult specimens, following Elsayed et al. (2018). The slide-mounted specimens were examined under a bright-field and phase-contrast microscope (H550L, Nikon, Tokyo) and line illustrations were made with the aid of a drawing tube. Photomicrographs were taken with a digital camera (DP22, Olympus, Tokyo) attached to a semi-motorised fluorescence microscope (BX53, Olympus, Tokyo). Contrasts of some photographs and illustrations were adjusted and the figure plates were arranged using Adobe photoshop (version CS2, Adobe Systems).
Morphological terminology mainly follows McAlpine et al. (1981) for adults. The term "gonocoxal lobes" is used in accordance with Gagné (2018) and wing venation according to Yukawa (1971). Larval and pupal terminology follows Gagné (1994). All types of the newly described species are deposited in the collection of Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (KUEC).
The new species was compared to specimens of five Japanese congeners in KUEC.
Third instar. Pale yellow, body strongly bowed backwards. Spatula (Fig. 6a): quadridentate, inner teeth slightly longer than outer two; posterior portion about 3.7 times as long as width of anterior free portion. Three lateral papillae present, two with setae. Three asetose pleural papillae present anteriorly on each side of prothorax. Two pairs of asetose pleural papillae on meso-and metathorax. One pair of asetose pleural  papillae on abdominal segments I-VIII. Two sternal papillae on each thoracic segment and abdominal segments I-VII, with setae, except on prothorax without setae. Two pairs of asetose elliptical papillae present anterodorsally on all thoracic and abdominal segments I-VIII. Two pairs of dorsal papillae present, without setae on thoracic segments and only outer pair with setae on abdominal segments I-VII. Terminal abdominal segment with two pairs of terminal papillae, outer pair with large setae and inner pair with minute setae (Fig. 6b). Four asetose anal papillae present.
Pupa (Fig. 7a-c). Four cephalic papillae present on tubercle, two with setae. Antennal horns greatly enlarged, tapered and flattened in ventral view, with serrate outer margins. One pair of setose lower facial papillae present. Two pairs of lateral facial papillae present, one pair with minute setae. Prothoracic spiracle elongated, slightly curved, about 320 μm long, with tracheae extending to tip. Abdominal spiracles present on segments II-IV, each spiracle about 0.5 times as long as the prothoracic spiracle.
Abdominal terga I-VII with anterior pair of trichoid sensilla, 4-5 rows of spines and two pairs of dorsal papillae, only outer pair with setae; terga VIII with 3-4 rows of spines and two pairs of setose dorsal papillae. Gall and life history: Pseudasphondylia tominagai induces flower bud galls on E. spinosus. The galled flower bud remains closed and reaches a diameter of 2.06-2.38 mm and length of 3.58-4.27 mm (n = 5) when matured. Larvae grow and pupate in the apical third of the galled bud. Third instars were found in the dissected galls in mid-April and adults emerged in late May.

Diagnosis
Amongst the five known Pseudasphondylia species in Japan, P. tominagai can be separated easily from P. neolitseae, P. matatabi and P. elaeocarpi at least by the following characters: narrower wings (Fig. 3d, e, f), more palpal segments and shape of sternal spatula (Yukawa 1971, Yukawa 1974, Tokuda and Yukawa 2005. It can be distinguished from P. rokuharensis as follows: wings of P. tominagai are slightly wider (Fig. 3c); the male of P. tominagai has the hypoproct with shallower-notch and the cerci more rounded and with deeper emargination in between; the female of P. tominagai has a longer ovipositor (the protrusible needle-like part of ovipositor is 2.7 times as long as sternite VII in P. tominagai compared to 1.8 times in P. rokuharensis) (Tokuda and Yukawa 2005); the pupa of P. tominagai has four dorsal papillae on abdominal segments I-VII, compared to six dorsal papillae in P. rokuharensis (Tokuda and Yukawa 2002); the larva of P. tominagai has four setose papillae on the terminal abdominal segment compared to only two in P. rokuharensis. Pseudasphondylia tominagai is closest to P. kiritanii because both species resemble each other in wing shape (Fig. 3a, b) and larval and pupal morphology; they can be distinguished from each other by the following characters: the male of P. tominagai has the hypoproct narrower than each cercus and with a shallower notch, whereas the hypoproct of P. kiritanii is slightly wider than each cercus and with a deeper notch (Fig. 5b); and the protrusible, needle-like part of the female ovipositor is slightly longer in P. tominagai (about 2.7 times as long as sternite VII) than in P. kiritanii (about 2.5 times as long as sternite VII).

Identification keys
Key to males of known Pseudasphondylia species

Discussion
In Pseudasphondylia tominagai, females of the overwintering generation lay eggs into flower buds of E. spinosus, which appear in March. Adult midges emerge from these flowers in late May when uninfested flower buds have already bloomed. Since we could not find any sign of larval presence in overwintering buds, we consider that P. tominagai possibly uses an alternative host plant from May to the following March.
In Diptera, the host-alternating habit has been known for only a few species of Asphondylia , such as A. gennadii (Marchal), A. yushimai Yukawa and Uechi, A. baca Monzen and A. sphaera Monzen (Harris 1975, Gagné and Orphanides 1992, Uechi et al. 2005, Uechi et al. 2004, Yukawa et al. 2003, Yukawa et al. 2016. Since Asphondylia and Pseudasphondylia belong to the same subtribe (Asphondyliina), they provide a great opportunity to study the evolutionary process of host-alternating habit by gall midges through molecular genetic analyses. Further taxonomic and ecological investigations are needed to clarify the life history of possible host alternating species in Pseudasphondylia.