Recent Divergence of Neolitsea-associated Pseudasphondylia Gall Midges (Diptera: Cecidomyiidae) with Description of a New Species from Taiwan

© 2025 Academia Sinica, Taiwan Open Access Recent Divergence of Neolitsea-associated Pseudasphondylia Gall Midges (Diptera: Cecidomyiidae) with Description of a New Species from Taiwan Sheng-Feng Lin1,2,* , Makoto Tokuda3, Man-Miao Yang2, Gene-Sheng Tung4, and Liang-Yu Pan4 1Department of Biology, National Museum of Natural Science, Taichung 404, Taiwan. *Correspondence: E-mail: [email protected] (Lin) 2Department of Entomology, National Chung Hsing University, Taichung 402, Taiwan. E-mail: mmy[email protected] (Yang) 3Department of Biological Resource Science, Faculty of Agriculture, Saga University, Japan. E-mail: [email protected] (Tokuda) 4Forest Ecology Division, Taiwan Forestry Research Institute, Taipei 100, Taiwan. E-mail: [email protected].tw (Tung); [email protected] (Pan) urn:lsid:zoobank.org:pub:1716D744-2B26-4B49-89A5-B60391131593 Received 14 May 2024 / Accepted 14 February 2025 / Published 13 May 2025 Communicated by Y. Miles Zhang A Taiwanese Pseudasphondylia species inducing spherical leaf galls on six Neolitsea hosts, viz, N. acuminatissima, N. daibuensis, N. konishii, N. parvigemma, N. sericea and N. variabillima, was regarded as a species allied to Japanese P. neolitseae Yukawa due to similarity of gall structure and host. The Taiwanese species is morphologically different from P. neolitseae in adult palpus segmental number, shapes of pupal antennal horn, pupal prothoracic spiracle, and larval sternal spatula, resulting in its description as a new species to science, Pseudasphondylia hooki sp. n. The species delimitation (Assemble Species by Automatic Partitioning method) supported that P. hooki sp. n. is distinct from P. neolitseae. The sistership of P. hooki sp. n. and P. neolitseae was also supported in the Maximum likelihood tree based on the 1st codon of COI region. The speciation event of them is assumed to be geographical isolation because the divergence corresponded to the separation of Taiwan and Japan in the Pleistocene. The patterns of ecological features (host species) on the phylogeny revealed that galling on N. sericea is primitive of Neolitsea-associated Pseudasphondylia lineage. Accordingly, the divergence pathway from north to south in Taiwan is suggested by the distributions of primitive (N. sericea) and most derived hosts (N. daibuensis). Key words: Asphondyliina, Biogeography, East Asia, Phylogeny, Speciation Citation: Lin SF, Tokuda M, Yang MM, Tung GS, Pan LY. 2025. Recent divergence of Neolitsea-associated Pseudasphondylia gall midges (Diptera: Cecidomyiidae) with description of a new species from Taiwan. Zool Stud 64:09. doi:10.6620/ZS.2025.64-09. BACKGROUND Gall midges (Diptera: Cecidomyidiae) are the largest gall-inducing insect group and use host of various plant families worldwide, although some species are saprophagous, mycophagous or predaceous (Gagné and Jaschhof 2021). Some galling cecidomyiids display unique phenotypes in their gall characteristics, such as shape, galling part/position, and host plant spectrum, which are useful cues to explore the species identity comparing with known cecidomyiid congeners (Tokuda et al. 2004; Lin et al. 2020b 2022). Besides, evolution of these ecological features assists us to know the eco-speciation and biogeography of cecidomyiids, in particular to similar gall sorts occurring on related plants or in adjacent localities, as has been done in Schizomyia species (= Asteralobia syn. in Elsayed et al. 2018) on Ilex (Aquifoliaceae) (Tokuda et al. 2004), Bruggmannilla species on Cinnamomum (Lauraceae) (Lin et al. 2020b), and Pseudasphondylia species on Zoological Studies 64: 9 (2025) doi:10.6620/ZS.2025.64-09 1 © 2025 Academia Sinica, Taiwan Actinidia (Actinidiaceae) (Lin et al. 2022) in the East Asian Arc. Pseudasphondylia neolitseae Yukawa, 1974 induces spherical leaf galls on Neolitsea sericea var. sericea (Lauraceae) and widely occurs in Japan (Honshu, Shikoku, Kyushu, and Southwest Islands) and southern Korea (Jeju Island) (Paik et al. 2004; Tokuda and Yukawa 2005). In Taiwan, undescribed Pseudasphondylia species are assumed to have an allied relationship with P. neolitseae due to the similar gall features and host affinity. Namely, the spherical leaf galls induced by unclarified Pseudasphondylia species have been noted from six Neolitsea species consisting of two native species, N. konishii and N. sericea var. sericea, and four endemic species, N. acuminatissima, N. daibuensis, N. parvigemma, and N. variabillima (Liang et al. 1999; Yang et al. 1999; Tung et al. 2018; Fig. 1). These provide an opportunity to explore the ecological divergence and island biogeography of Neolitseaeassociated Pseudasphondylia species. In this article, the species identity of the Taiwanese Pseudasphondylia species on six Neolitsea host species was first clarified based on both morphological and molecular evidence. Then, we conducted phylogenetic analysis to address the following questions for Neolitsea-associated Pseudasphondylia species: (1) What is their possible divergence pathway in the East Asian Arc? (2) How did they diverse on Neolitsea plants? Clarifications of these two questions will contribute to the understanding of the divergence modes of cecidomyiids in this region. Fig. 1. Leaf galls of Pseudasphondylia hooki sp. n. on Neolitseae species (A) N. acuminatissima, (B) N. daibuensis, (C) N. konishii, (D) N. sericea (E) N. parvigemma, and (F) N. variabillima. page 2 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan MATERIALS AND METHODS Collecting and preparation of specimens Leaf galls induced by Neolitsea-associated Pseudasphondylia species were collected from various localities in Taiwan and Japan between 2009 to 2024. Larval, pupal, and adult specimens were mostly preserved in 70% ethanol after dissecting the galls or adult emergence. A part of the fresh specimens was kept in 95% ethanol for molecular studies. Slide-mounted specimens were prepared following Gagné (1994). Specimens were observed using an optical microscope (Leica® DM 750, Germany) and deposited in National Chung Hsing University, Taiwan (NCHU), National Museum of Natural Science, Taiwan (NMNS), and Taiwan Forestry Research Institute, Taiwan (TFRI). The morphological terminology of adult followed Gagné (2018) and Tokuda (2004), and that of larva and pupa followed Gagné (1994). Molecular analysis Three individuals of P. neolitseae and 17 individuals of Taiwanese Neolitsea-associated Pseudasphondylia species were used in the present molecular work (Table 1). DNA extraction and PCR procedures followed Lin et al. (2019). The COI maker primers were referenced Cameron et al. (2007): Forward primer: Diptera-49F (5'-AATCATAAAGATATTGGAAC-3') and reverse primer: Diptera-734R (5'-CAACATTTATTTTGATTT TTTGG-3'). The 20 COI sequences were successfully analyzed and deposited in the DNA Data Bank of Japan (DDBJ) with accession numbers shown in table 1. The COI sequences of Illiciomyia yukawai (LC348719), P. kiwiphila (LC710532), and P. rokuharensis (LC538357) were used as outgroups. The sequences were aligned using ClustalW (Thompson et al. 1994) with BioEdit (Hall 1999) and the species delimitation methods using ASAP (Assemble Species by Automatic Partitioning) online service (Puillandre et al. 2021) with Jukes-Cantor model. The genetic distances among Pseudasphondylia species were calculated by p-distance with MEGA XI (Tamura et al. 2021) and the divergence time was assessed with the divergent rates of 2.0 and 2.3% pairwise sequence divergence per million years according to DeSalle et al. (1987) and Brower (1994). For the phylogeny, we evaluate the saturation of codon substitutions in order to obtain greater phylogenetic signal. The analyzed COI dataset were evaluated for transition and transversion substitutions by DAMBE 7 (Xia 2018). As result, the first codon and first plus second codons dataset were analyzed. The best model (T93+G) was selected for phylogenetic analysis using MEGA XI (Tamura et al. 2021) with Maximum likelihood method. Bootstrap replication was set as 1000. RESULTS TAXONOMY Order Diptera Linnaeus, 1758 Family Cecidomyiidae Newman, 1834 Tribe Asphondyliini Enderlein, 1914 Subtribe Asphondyliina Enderlein, 1914 Genus Pseudasphondylia Monzen, 1995 Pseudasphondylia Monzen 1955: 41. Type species: Pseudasphondylia rokuharensis Monzen, 1955. Philadelphella Kovalev 1964: 440. Type species. Philadelphella philadelphi Kovalev, 1964. The genus is known from China, Far-East Russia, India, Japan, New Caledonia, and Taiwan, and until now comprised 14 species (Gagné and Jaschhof 2021; Matsuda et al. 2021; Elsayed et al. 2023). A fifteenth Table 1. Pseudasphondylia specimens used in the molecular phylogenetic analysis, with information on host plant, locality, and DNA accession numbers Gall midge Host plant Locality* Taxon No. [Accession No.] P. neolitseae Neolitsea sericea Fukuoka, JP A348-350 [PV446537-39] P. hooki sp. n. N. acuminatissima Mt. Dahan, Pingtung, TW A74,A77 [PV446529-30] N. daibuensis Jinshuiying Trail 0.7K, Pingtung, TW A354-356 [PV446540-42] N. konishii Donggua Trial, Miaoli, TW A180,A360 [PV446531,PV446543] N. parvigemma Mt. Dahan forest Trial, Pingtung, TW A257-258 [PV446532-33] N. sericea Erziping trail, Taipei, TW A247-249,A375-259 [PV446534-36,PV446544-46] N. variabillima Mt. Beidongyan-Nantungyen 3.4K, Nantou, TW A362-363 [PV446547-48] *TW and JP indicate Taiwan and Japan, respectively. page 3 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan species is described below from Taiwan on Neolitseae species. The description, DNA, and host spectrum of the new species were provided below. Pseudasphondylia hooki sp. n. Lin, Tokuda & Yang (Table 2; Figs. 3–4) urn:lsid:zoobank.org:act:716CE8F9-AF4D-42B5-81FF882AF8940986 Type materials: Holotype: ♂ (on slide, NCHU), TAIWAN: Taipei City, Mt. Yang-Ming National Park, gall collected on 17.ii.2011 and adult emerged on 23.ii.2011, ex. Neolitsea sericea, leg. SF Lin. Paratypes: TAIWAN: Ex. Neolitsea acuminatissima: [Taipei City] 4♀, 4 pupal exuviae (on slides, NCHU), gall collected on 22.i.2013 and adult emerged on 26.i.2013, Mt. Yang-Ming National Park, leg. SF Lin; [Nantou Co.] 4♂, 1♀, 10 pupal exuviae (on slides, NCHU), gall collected on 23.iii.2013 and adult emerged on 25–28.iii.2013, Mt. Liying, leg. SF Lin; [Yilan Co.] 2 pupae (on slides, NCHU), 12.ii.2009, Taiwan Beech National Trail, Mt. Taiping, leg. TC Tang. Ex. Neolitsea konishii: [Taipei City] 4♂, 9♀, 3 pupae, 26 pupal exuviae (on slides, NCHU), gall collected on 17.ii.2011 and adult emerged on 24–26. ii.2011, Mt. Yang-Ming National Park, leg. SF Lin; 5♂, 8♀, 2 pupae, 6 pupal exuviae (on slide, NCHU), 11.iii.2011, Mt. Yang-Ming National Park, leg. SF Lin; 8♀, 12 pupal exuviae (on slides, NCHU), gall collected on 22.i.2013, adult emerged on 28.i.2013, Mt. YangMing National Park, leg. SF Lin. Ex. Neolitsea sericea [Taipei City] 2♂, 6♀, 7 pupae, 50 pupal exuviae (on slides, NCHU), same data as holotype; 1♀, 2 pupae, 9 pupal exuviae (on slides, NCHU), gall collected on 18.iii.2011 and adult emerged on 20.iii.2011, Mt. YangMing National Park, leg. SF Lin; 2 mature larvae (on slides, NCHU), 30.xii.2013, Mt. Yang-Ming National Park, leg. SF Lin; 5♂, 1 pupa (in EtOH, NCHU), 11.ii.2001, Mt. Yang-Ming National Park, leg. LH Liao, MM Yang; 10♂ (in EtOH, NMNS), gall collected on 29.i.2024 and adult emerged on 4~5.ii.2024, Mt. Yang-Ming National Park, leg. SF Lin. Ex. Neolitsea parvigemma: [Pingtung Co.] 8 mature larvae (six in EtOH, two on slide, NCHU), 17.ii.2017, Dahan forest rd. leg. SF Lin. Ex. Neolitsea variabillima: [Nantou Co.] 1 mature larva (in EtOH, TFRI), 18.i.2010, Mt. Peitungyen-Nantungyen 3.4K, leg. GS Tung. Adult: Head. Eye bridge 3–4 facets long. Frontoclypeal setal count as in table 2. Labella hemispherical in lateral view, with setae. Palpus threesegmented (Fig. 2A), first segment globose, 25–30 μm long, with 2–4 setae; second 2.0 times as long as first, with 4–8 setae and third 2.2 times as long as first, with 7–10 setae. Twelve flagellomeres, the last three equal in length in male (Fig. 2B). Female distal flagellomeres gradually shortened, flagellomere 11 subglobular and terminal one globular (Fig. 2C). Thorax: Thoracic setal and scale counts as in table 2. Legs with dense blackish scales; first tarsomeres of all legs with apicoventral spur; tarsal claws bent after midlength on all legs; pulvilli shorter than claws; empodia slightly shorter than claws on all legs (Fig. 2D–E). Wing 3.1–4.0 mm long, 2.0–2.4 times as long as wide in male; 3.2–3.4 mm, 2.3 times as long as wide in female. R5 joining costa posterior to wing apex (Fig. 2F–G). Male abdomen: Tergites I–VII rectangular, covered with scales on medium part, with a few lateral setae; tergites I–VI with 1–2 posterior row of setae; tergite VII with few setae on posterior part; sternites II–VII with anterior pair of lateral setae, cover with scattered setae and scales, sternites I–VI with three to four posterior row of setae; sternites VII with three to four posterior row of setae; tergite VIII covered with scattered setae and few scales. Terminalia (Fig. 2H): cerci forming a pair of lobes, each lobe with a few apical setae; hypoproct bilobed, deeply separated, each lobe with two apical setae; gonostylus suboval, with two separate sclerotized teeth cover with setae; gonocoxite elongate, cover with setae dorsally and ventrally; mediobasal lobe of gonocoxite present; aedeagus laterally sclerotized in basal part, distally tapering. Table 2. Frontoclypeal and thoracic setal counts of Pseudasphondylia hooki sp. n. Specimens Male Female nMean ± SD Range nMean ± SD Range Frontoclypeal setae 3 29.3 ± 4.7 24–33 2 41.5 ± 0.7 41–42 Anterior dorsolateral setae 4 66.5 ± 6.4 63–76 4 56.3 ± 5.7 49–63 Posterior dorsolateral setae 3 65.3 ± 7.0 58–72 4 64.3 ± 5.6 59–72 Mesopleural scales 5 14.6 ± 1.8 12–17 4 12.0 ± 1.8 10–14 Mesepimeral setae 6 46.5 ± 9.4 30–55 4 35.5 ± 4.1 32–40 page 4 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan Fig. 2. Pseudasphondylia hooki sp. n. (A) Male head (ventral view) (B) Male antenna (8–12 segment). (C) Female antenna (8–12 fragellomeres). (D) Male 5th tarsomere. (E) Female 5th tarsomere. (F) Male wing. (G) Female wing. (H) Male genitalia. (I) Female abdomen. (J) Pupal abdomen. Scale bars: A–C = 0.03 mm; D–E = 0.2 mm; F–G = 1 mm; H = 0.2 mm; I–J = 0.5 mm. page 5 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan Female abdomen: First through seventh tergites and second through sixth sternites as in male. Seventh sternite 0.5–0.6 mm long, protrusible part of ovipositor 1.3–1.7 mm long (Fig. 2I), 2.5–3.1 times as long as the length of seventh sternite. Pupa: Body length 2.7–3.0 mm, pupal skin not pigmented except for antennal horns. Antennal horn 280–320 µm long, triangular, lateral margin without irregular serration (Fig. 3); cephalic papilla with seta, 40–65 µm long; frons without horns; two pairs of facial and three pairs lateral facial papillae present; prothoracic spiracle (Fig. 3) 180–210 µm long; second to sixth abdominal spiracles 50–70 µm long; second to seventh abdominal segments with 4 to 5 transverse rows of spines (Fig. 2J) ; eighth abdominal segments with 3 to 4 transverse rows of spines; eight dorsal papillae on first to the seventh abdominal segments, most outer and second inner pair papillae with seta; two dorsal papillae on eighth abdominal segment, each with seta; pleural papilla present on each side, with seta. Mature Larva: Body color in life yellow, length 2.0–2.3 mm. Second antennal segment short, conical; cervical papillae without seta. Sternal spatula 330–370 µm long, anteriorly with two lobes (Fig. 3); Fig. 3. Phylogenetic tree based on the 1st codon positions of COI (T93+G) and morphological differences of Neolitsea-associated Pseudasphondylia gall midge, P. hooki sp. n. (left) and P. neolitseae (right). The bootstraps (%) of Maximum-Likelihood inferences were shown beside nodes (1st codon/ 1st +2nd codons). Score less than 60 were hidden. page 6 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan four lateral papillae and a sternal papilla present on each side of all thoracic segments, each with seta; four dorsal papillae on all thoracic and first to seventh abdominal segments, two dorsal papillae on eighth abdominal segment, each with seta; pleural papilla present on each side, with seta; 2 asetose terminal papillae present; anal papillae not apparent. Distribution: Taiwan. Gall and Host: Monothalamus, spherical gall on the leaves of six Neolitsea species, N. acuminatissima, N. daibuensis, N. konishii, N. parvigemma, N. sericea, and N. variabillima. Gall development and morphology are shown in Liang et al. (1999) and Tung et al. (2018). A total of 1059 galls of P. hooki were examined, including 225 galls on N. acuminatissima, 62 on N. daibuensis, 98 on N. konishii, 90 on N. parvigemma, and 500 on N. variabillima. All of them occurred on the abaxial side of the host leaf (Table 3). Biological notes: This species is fundamentally univoltine. Adults emerge directly from galls in February and females lay eggs possibly into new buds as in P. neolitseae. Larvae soon hatch and remain inside the host tissue as the first instar until October. Liang et al. (1999) mentioned the gall started to grow from August and reached the maturation phase in October. Subsequently, larvae within the galls undergo maturation in January and pupate during middle midJanuary and early February. While Takasu and Yukawa (1984) reported the occurrence of extended diapause and a semivoltine life cycle in Japanese P. neolitseae, the presence of such individuals in P. hooki remains unconfirmed Remarks: Male terminalia and larval features, sternal spatula with two lobes and cervical and ventral papillae lacking seta, of the new species are similar to those of P. neolitseae. Nevertheless, the new species is distinguishable from P. neolitseae by the deeper insersion of larval sternal spatula (Fig. 3), serrate lateral margin of the antennal horns (Fig. 3) and hookliked prothoracic spiracle (Fig. 3) in pupa, and the three-segmented palpus. In contrast to these features, P. neolitseae possesses sternal spatula with shallower insersion in larva, antennal horns with flat lateral margin and long prothoracic spiracle in pupa, and twosegmented palpus in adult. Species delimitation and divergence A total of 20 COI sequences (~658 bp) of Neolitsea-associated Pseudasphondylia species were obtained. Among them, 17 COI sequences of P. hooki were obtained from six host plants in Taiwan and three from P. neolitseae on N. sericea in Japan (Table 1). Excluding the three outgroups, the ASAP revealed that P. neolitseae formed one entity under all asap-score modes, whereas P. hooki formed one to 11 entities with different asap-score modes. The ASAP branch nodes of the two species did not support their distinction (P > 0.1) (Fig. 4). The COI genetic distance is 0.7% within P. hooki sp. n., 0% within P. neolitseae, while 2.7% (2.5– Table 3. Galled position of Pseudasphondylia hooki sp. n. on Neolitsea leaves Host Locality Abaxial side Adaxial side N. acuminatissima Mt. Beidongyan, Nantou 27 0 Mt. Liying, Nantou 60 0 Cika Cabin, Taichung 90 0 Dasyueshan forest rd. 48 0 N. daibuensis Jinshuiying Trail, Pingtung 62 0 N. konishii Erziping Trail, New Taipei 48 0 Sanyi, Maioli 4 0 Huisun forest area, Nantou 16 0 Mt. Dongmao, Taichung 25 0 Dasyueshan forest rd., Taichung 5 0 N. parvigemma Jinshuiying Trail, Pingtung 90 0 N. variabillima Mt. Beidongyan, Nantou 23 0 Mt. Baimao, Taichung 2 0 Lianhuachi Research Center, Nantou 2 0 Dasyueshan forest rd., Taichung 57 0 N. sericea Erziping Trail, New Taipei 500 0 page 7 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan 3.1%) between the two species. The divergence time between them was estimated at around 1.2–1.3 mya. Molecular phylogeny The comparison of the transition and transversion frequencies of our two dataset revealed that the 1st codon and 1st +2nd codons did not reach saturation. In contrast, saturation was observed in other datasets, including those containing the 2nd, 3rd, and all codon positions (Fig. 5). Therefore, a total of 202 positions from the 1st codon and 404 positions from the 1st + 2nd codons were used for further phylogenetic analysis. As a result, phylogenetic trees form both datasets shared similar topologies but some branches weakly supported in the tree reconstructed from the 1st + 2nd codons. In the present study, we showed the tree based on 1st codon dataset as it most clearly illustrated the phylogenetic relationship. The phylogenetic tree supported the monophyly of Neolitsea-associated Pseudasphondylia species (bootstrap value: 90; Fig. 3), as well as of P. hooki lineage (bootstrap value: 79) and P. neolitseae lineage (bootstrap value: 93). In the P. hooki clade, individuals associated with N. sericea situated at the basal part and formed a paraphyletic group with respective a clade of P. hooki individuals on the other hosts (bootstrap value: 64). Then, P. hooki individuals on N. daibuensis formed a subclade (bootstrap value: 63) and situated at the most terminal part. DISCUSSION The gall shapes together with host species of cecidomyiids are useful features for researchers to recognize cecidomyiid congeners in adjacent unexplored areas. In East Asia, P. elaeocarpi associated with Elaeocarpus sylvestris (Elaeocarpaceae) and inducing finger-shaped leaf galls in both Taiwan and Japan (Lin et al. 2022), and Bruggmanniella cinnamomi Tokuda & Yukawa inducing swollen stem galls on Cinnamomum yabunikkei (= C. japonica) in Japan and on Cinnamomum insulari-montanum in Taiwan (Lin et al. 2020b). However, similar ecological features may represent in distinct cecidomyiid species. For example, the shape of swollen stem galls of Taiwanese Bruggmenniella turoguei Lin, Yang, & Tokuda on Cinnamomoum osmophloeum is similar to Japanese B. cinnamomi on C. yabunikkei (Lin et al. 2020b). In the present study, spherical leaf galls on six Taiwanese Neolitsea species were all induced by P. hooki. This suggests the necessity of adopting the integrative taxonomic approach, in particular for galling cecidomyiid inducing similar galls and associated with related hosts in adjacent areas. Besides, gall morphospecies are used to evaluate the richness of galling insects in many studies (Yang et al. 2000; Tung et al. 2006; Pan et al. 2018). The case of P. hooki highlights the asymmetry of cecidomyiid species and gall morphospecies richness. In Taiwan, several Asphondyliina species are oligophagous and/or display Fig. 4. Assemble Species by Automatic Partitioning (ASAP) analyses of Neolitsea-associated Pseudasphondylia gall midges based on mitochondrial partial COI region. Colors represent unique partitions. Gray and Yellow dots of branches refer to P value incalculable and greater than 0.1, respectively. page 8 of 12Zoological Studies 64: 9 (2025) © 2025 Academia Sinica, Taiwan gall polymorphism (Pan et al. 2015; Lin et al. 2020a b; present study). These result in an overestimation of cecidomyiid richness based on host species and gall morphology. The further taxonomic efforts of Taiwanese Asphondyliina are needed to realize the actual cecidomyiid diversity. Additionally, these study systems are suitable materials to explore the divergence mode of galling cecidomyiids at the species level. Species recognition and divergence The mtDNA marker, the partial COI region, is a useful tool for the recognition of cecidomyiid species and the study of their evolutionary history. Tokuda et al. (2004) clarified the host spectrum and biogeography of Ilex-associated S. sasakii and S. soyogo (mentioned as Asteralobia species at that time) in Japan; Lin et al. (2020b) confirmed the species identify and possible evolutionary history of four Cinnamomum-associated Fig. 5. Saturation plots of transition (blue line) and transversion (green line) rates against F84 distance. page 9 of 12Zoological Studies 64: 9 (2025)