A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) in Japan

Abstract Background A gall midge species (Diptera: Cecidomyiidae) inducing leaf bud galls on Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) was found in Hokkaido and northern Honshu, Japan. New information Based on its morphology, the species is regarded as an undescribed species of the genus Pseudasphondylia Monzen (Cecidomyiinae, Cecidomyiidi, Asphondyliini). The species is herein described as Pseudasphondylia saohimea Matsuda, Elsayed and Tokuda sp. n. The new species is easily distinguishable from its congeners by the number of adult palpal segments and the shape of the male terminalia and larval spatula.


Introduction
The genus Pseudasphondylia (Diptera: Cecidomyiidae: Asphondyliini) includes 11 species associated with various plant families (Gagné and Jaschhof 2021). Nine of them are distributed in the eastern Palearctic Region (e.g. Elsayed et al. 2019, Tokuda and, one in India and one in New Caledonia (Gagné and Jaschhof 2021). Amongst the nine eastern Palaearctic species, the following six species have been recorded from Japan: P. elaeocarpi Tokuda Tokuda and Yukawa 2002, Yukawa 1974, Yukawa and Masuda 1996, Elsayed et al. 2019, Yukawa 1983. They have type IIA or IIB life history strategy, in which mature (type IIA) or immature (type IIB) larvae overwinter in galls on their host plants (Yukawa 1974, Yukawa 1987). In addition, some species exhibit polymodal emergence patterns caused by long-term diapause (Takasu and Yukawa 1984) and other species are suspected to alternate host plants (Yukawa and Masuda 1996, Elsayed et al. 2019a).
In recent years, we found an undescribed species of asphondyliine gall midge that induces leaf bud galls on Magnolia kobus DC. var. borealis Sarg. (Magnoliaceae) in Tomakomai, Hokkaido and Aomori Prefecture, Honshu, Japan. Based on morphological comparison, we concluded that the gall midge is a member of Pseudasphondylia. We describe the species as new to science and discuss the phylogenetic relationships amongst Japanese congeners on the basis of molecular analysis. We also compare its life history strategy with those of the Japanese congeners.

Collecting and Rearing
Leaf-bud galls on M. kobus var. borealis (Fig. 1) were collected from Tomakomai City, Hokkaido and Mutsu City, Aomori Prefecture, Honshu, Japan. Some galls were dissected under a stereoscopic microscope to obtain mature larvae and others were kept in plastic bags until emergence of adults. Most specimens were preserved either in 75% ethanol for morphological examinations or in 99.5% ethanol for molecular analyses.

Morphological Examination and Terminology
Gall midge specimens were mounted on slides in Canada balsam, following the technique outlined in Gagné (1994), except for the clearing step for 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 following Elsayed et al. (2020) under a bright-field and phase-contrast microscope (CX43, Olympus, Tokyo). Photomicrographs were taken with a digital camera (DP22, Olympus, Tokyo) attached to a semi-motorised fluorescence microscope (BX53, Olympus, Tokyo).
Morphological terminology basically follows Gagné (2018), except the term "antennal papillae" in pupa follows Elsayed et al. (2020a). Larval and pupal terminology follow Gagné (1994). The holotype and paratypes are deposited in the collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Japan (ELKU). A generic synopsis of Pseudasphondylia is presented in . A new species of Pseudasphondylia (Diptera: Cecidomyiidae) associated with ...

Molecular Phylogenetic Analysis
Genome DNA was extracted from 99.5% ethanol-preserved female gall midges reared from leaf galls of M. kobus var. borealis and of P. tominagai reared from flower bud galls of E. spinosus (Elsayed et al. 2019) and from adults of P. elaeocarpi obtained from leaf galls on E. sylvestris var. ellipticus (Elaeocarpaceae). A 472 bp fragment of the mitochondrial gene cytochrome oxidase subunit I (COI) of the species from leaf galls of M. kobus var. borealis and P. tominagai was sequenced and aligned following Elsayed et al. (2017), using the following primer set: Forward: J-1718 (5'-GGA GGA TTT GGA AAT TGA TTA GTT CC-3') (Simon et al. 1994) and reverse: COIA (5'-CCC GGT AAA ATT AAA ATA TAA ACT TC-3') (Funk et al. 1995), while a 439 bp fragment of the same gene of P. elaeocarpi was sequenced using the primer set: COIS (5'-GGA TCA CCT GAT ATA GCA TTC CCA TAT TGG-3') and COIA (5'-CCC GGT AAA ATT AAA ATA TAA ACT TC-3') (Funk et al. 1995). The sequences obtained were compared using MEGA (version X) (Kumar et al. 2018) and deposited in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL) and GenBank nucleotide sequence databases as accession numbers LC621302-621303, LC621304-621305 and LC621306 for P. tominagai, the gall midge of M. kobus var. borealis and P. elaeocarpi, respectively.
Female abdomen (Fig. 3a). Tergites I-VII rectangular, evenly covered with scales, with lateral setae and without anterior pair of trichoid sensilla; tergites I-VI with 1-2 posterior rows of setae, but tergite VII with 2-3 rows; tergite VIII bare. Sternites II-VII with anterior pair of trichoid sensilla situated laterally; sternites II-VI rectangular, anteriorly with scattered setae and scales, posteriorly with one row of setae usually mixed with some scales; sternite VII about two times longer than VI, covered with scattered setae and scales. Ovipositor: protrusible needle-like portion about 3.5 (3.4-3.6; n = 3) times longer than sternite VII; cerci undifferentiated.
Mature larva (Fig. 4). Body colour in life orange. Spatula: anteriorly with four lobes, outer two longer than inner two. Three lateral papillae present on each side of midline, two with setae. Three pairs of asetose pleural papillae present anteriorly on prothorax. Two pairs of asetose pleural papillae on meso-and metathorax. One pair of setose 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 dorsal papillae present, without setae on thoracic segments and only outer pair with setae on abdominal segments I-VII; a pair of setose dorsal papillae on abdominal segment VIII. Terminal abdominal segment with two setose terminal papillae and two asetose anal papillae present. Pupa (Fig. 5). Two setose and two asetose cephalic papillae on tubercles. Antennal horns greatly enlarged, tapered and dorsoventrally flattened, serrate along anterior margin. Antennal papillae absent. Lower and lateral facial papillae not visible. Prothoracic spiracle elongated, slightly curved, about 160 μm long, with tracheae extending to tip. Abdominal spiracles present on segments II-VI, each spiracle about 0.3 times as long as prothoracic spiracle. Abdominal terga I-VII with anterior pair of trichoid sensilla, 5-6 rows of spines and three pairs of setose dorsal papillae; tergum VIII with 5-6 rows of spines and two setose dorsal papillae.

Etymology
The specific name, saohimea, is derived from "Saohime", a Japanese goddess of spring, because blooming of the host plant Magnolia kobus var. borealis is a symbolic event announcing the beginning of spring in northern Japan. Galls of P. saohimea become conspicuous on the host also in early spring.

Biology
Pseudasphondylia saohimea is univoltine. Third instars and pupae were found in the galls in mid-May and adults emerged directly from the galls in mid-to late May. All mature galls collected in July were empty, indicating that no individuals had entered th long-term diapause. In rearing conditions, adults emerged in the morning and mated around 11:00 h, suggesting that the gall midge is a diurnal species. The adults are supposed to oviposit into host buds. First instars were found in undeveloped bud galls in late September. They possibly overwinter in the undeveloped bud galls and develop to the second and third instars in the following spring.
Gall: Pseudasphondylia saohimea induces hairy leaf bud galls on Magnolia kobus var. borealis (Magnoliaceae). The galled buds remain closed and indistinguishable in appearance from ungalled buds until the following spring and rapidly grow with bud burst. Mature galls are 2.7-6.0 mm in diameter and 5.1-13.7 mm in length (n = 45). Galls are multi-chambered and each chamber contains a single gall midge larva.

Notes
The new species is distinguishable from most of its other congeners in Japan by the number of palpal segments: three in the new species, but two in P. neolitseae and four in P. rokuharensis, P. kiritanii and P. tominagai. Although P. matatabi and P. elaeocarpi have three-segmented palpi, they are easily distinguished from the new species by their cerci which are shorter than the hypoproct. The larval spatula of the new species has four lobes of which the outer two are longer than the inner two. However, the larval spatula of P. neolitseae has only two lobes anteriorly and the other species have four lobes of which the inner two are longer than the outer two. In the pupa, five pairs of long abdominal spiracles are present in the new species, while only three pairs are present in P. rokuharensis, P. kiritanii, P. elaeocarpi and P. tominagai and four pairs in P. matatabi. The pupa of P. neolitseae, which has five pairs of abdominal spiracles, is otherwise similar to the new species, but it can be distinguished by dorsal abdominal spines that are markedly shorter than in P. saohimea.
In the key to the males of world Pseudasphondylia species in Elsayed et al. (2019), P. saohimea will run to couplet 5 that separates P. matatabi and P. elaeocarpi. In order to update the key and include P. saohimea, couplet 5 is amended and a sub-couplet is added as in Table 1.

Discussion
Morphological examination and phylogenetic analysis clearly indicate that P. saohimea is distinct from its congeners in Japan, although phylogenetic relationships amongst Pseudasphondylia species were not revealed in the analyses, except for the sister group relationship of P. kiritanii and P. tominagai. Pseudasphondylia was hypothesised to be an ancient clade within the subtribe Asphondyliina Yukawa 2007, Tokuda 2012), which suggests that it would be more promising to analyse more conservative DNA regions to illuminate the inner generic relationships.
Based on overwintering sites and larval stages, the life-history of gall midges is divided into two main types, of which each can be subdivided into two subtypes regardless of uni-, bior multivoltine (Yukawa 1987, Yukawa andUechi 2021). Adaptive significance of respective life history strategies has been attributed particularly to the avoidance of parasitoid attack (Yukawa 1987, Tokuda et al. 2004, Yukawa and Rohfritsch 2005, Yukawa and Uechi 2021. Most species of Asphondyliini adopt type IIA or IIB life history strategy. Mature larvae overwinter in the galls on host plants in type IIA, while young larvae do so in type IIB. Amongst the six Japanese species of Pseudasphondylia, P. neolitseae and P. rokuharensis are univoltine-type IIA species, P. elaeocarpini is a univoltine-type IIB species and P. kiritanii, P. matatabi and P. tominagai are host-alternating, bivoltine and possibly type IIB species (Yukawa 1974, Yukawa and Masuda 1996, Elsayed et al. 2019a Cerci deeply separated, each cercus oval (Fig. 3b); wing length approximately 2.4 times as long as wide ( Fig. 2g) P. saohimea sp. n. Table 1. Maximum Likelihood phylogenetic tree of Japanese Pseudasphondylia species, based on a fragment of COI. Bootstrap values are indicated for nodes that gained > 50% support in 1,000 replications.

Placement of
As mentioned above, P. saohimea belongs to the univoltine type IIB, as does P. elaeocarpi . However, leaf galls induced by P. elaeocarpi become fullgrown in summer and the first instars spend summer, autumn and winter in fully developed galls Masuda 1996, Tokuda and. This contrasts with the situation in P. saohimea, which passes through these seasons in inconspicuous galls.
Some Asphondyliini, other than Pseudasphondylia, have a life history pattern similar to that of P. saohimea. For example, females of Oxycephalomyia styraci (Shinji) oviposit into axillary overwintering buds of Styrax japonica Siebold and Zucc. (Styracaceae) and its larvae spend summer, autumn and winter as the first instar in the host buds; then, galls and larvae rapidly grow, coinciding with host bud burst (Tokuda et al. 2004). Tokuda et al. (2004) proposed that the life history of O. styraci is adaptive to avoid late parasitoids, which are generally idiobiont ectoparasitoids attacking mature host larvae. Similarly, Asphondylia tojoi Elsayed and Tokuda, inducing unremarkable leaf bud galls on Schoepfia jasminodora Siebold and Zucc. (Schoepfiaceae), overwinters as the first instar in inconspicuous overwintering buds, although this species is multivoltine and repeatedly uses axillary buds throughout the year (Elsayed et al. 2018b).
Some large genera, such as Asphondylia, Contarinia, Dasineura and Lasioptera, are associated with various plant families, while others are restricted to particular plant genera, namely Caryomyia on Carya (Juglandaceae), Procontarinia on Mangifera (Anacardiaceae), Rabdophaga on Salix (Salicaceae) and Semudobia on Betula (Betulaceae) . Pseudasphondylia belongs to the former group, although it, so far, contains only a small number of species. Considering the antiquity of Pseudasphondylia and its wide host range and diverse life history patterns, many more species may remain undiscovered, since generally, in Cecidomyiidae, ecological diversifications are involved in radiation at the species level (Yukawa et al. 2019).