Molecular Phylogeny Revealing the Single Origin of Cinnamomum-associated Bruggmanniella (Diptera: Cecidomyiidae) in Asia, with Descriptions of Three New and One Newly Recorded Species from Taiwan

© 2020 Academia Sinica, Taiwan Open Access Molecular Phylogeny Revealing the Single Origin of Cinnamomum-associated Bruggmanniella (Diptera: Cecidomyiidae) in Asia, with Descriptions of Three New and One Newly Recorded Species from Taiwan Sheng-Feng Lin1,*, Man-Miao Yang1, and Makoto Tokuda2,* 1Department of Entomology, National Chung Hsing University, Taiwan. *Correspondence: E-mail: [email protected] (Lin) E-mail: mmy[email protected] (Yang) 2Department of Biological Resource Science, Faculty of Agriculture, Saga University, Japan. *Correspondence: E-mail [email protected] (Tokuda) Received 31 July 2020 / Accepted 12 October 2020 / Published 30 November 2020 Communicated by Jen-Pan Huang Cecidomyiid genus Bruggmanniella contains four Lauraceae-associated species in Asia and 13 species associated with various plant families in Latin American. In this article, three new species, B. sanlianensis sp. nov., B. turoguei sp. nov. and B. shianguei sp. nov., and one newly recorded species, B. cinnamomi, are reported on Cinnamomum plant species (Lauraceae) from Taiwan. Molecular phylogenetic analysis was conducted for the four Cinnamomum-associated Bruggmanniella, together with B. brevipes Lin, Yang and Tokuda, B. actinodaphnes Tokuda and Yukawa, three Pseudasphondylia and two Daphnephila species. The Asian Bruggmanniella and the Cinnamomum-associated Bruggmanniella were monophyletic on the Neighbor-joining, Maximum-likelihood, and Bayesian inference trees. In addition, Cinnamomumassociated Bruggmanniella species had the closest sistership with B. brevipes, which are associated with the plant genus Neolitsea (Lauraceae). These results suggest that B. brevipes, B. actinodaphnes and B. cinnamomi are members of genus Bruggmanniella, a finding that is not consistent with another recent morphology-based phylogenetic study. Among the Cinnamomum-associated lineages, the leaf galler B. sanlianensis sp. nov. is a sister to the clade of stem gallers, suggesting that host organ shift from leaf to stem occurred prior to host shift. Additionally, the paraphyly of the Taiwanese stem galler with respect to Japanese B. cinnamomi suggests that the distributional range of B. cinnamomi be expanded from Taiwan to Japan. Key words: Bruggmanniella, Taiwan, Japan, Divergence, Biogeography. BACKGROUND The gall midge genus Bruggmanniella Tavares comprises 17 species, which exhibit amphi-Pacific distribution and special patterns of host spectrum. Thirteen New World species are associated with various host families, while four East Asian species with Lauraceae: B. cinnamomi Tokuda & Yukawa, 2006 with Cinnamomum yabunikkei H. Ohba (= C. japonicum Sebold ex. Nakai), B. actinodaphnes Tokuda & Yukawa, 2006 and B. litseae Lin, Yang & Tokuda, 2020 with Litsea species (including Actinodaphne) and B. brevipes Lin, Yang & Tokuda, 2019 with Neolitsea parvigemma (Hayata) Kanehira & Sasaki (Tokuda and Citation: Lin SF, Yang MM, Tokuda M. 2020. Molecular phylogeny revealing the single origin of Cinnamomum-associated Bruggmanniella (Diptera: Cecidomyiidae) in Asia, with descriptions of three new and one newly recorded species from Taiwan. Zool Stud 59:66. doi:10.6620/ZS.2020.5966. Zoological Studies 59:66 (2020) doi:10.6620/ZS.2020.59-66 1 © 2020 Academia Sinica, Taiwan Yukawa 2006; Maia et al. 2010; Tokuda 2012; Gagné and Joffhorn 2017; Lin et al. 2019 2020; Garcia et al. 2020; Rodrigues et al. 2020). Furthermore, several undescribed species have been found on Lauraceae in Asia. Tokuda and Yukawa (2006) noted one unnamed species on L. acuminata (Blume) Sh. Kurata (= Actinodaphne acuminata (Blume) Meissn.) in Okinawa, Japan. Many other galls that may be induced by Bruggmanniella are known on Cinnamomum and Litsea in Taiwan (Tung et al. 2018), which suggests a high diversification of Bruggmanniella on Asian Lauraceae. From a systematics viewpoint, the identity of Bruggmanniella is controversial due to its heterogeneity on ecological aspects and morphological features. Tokuda and Yukawa (2006) described two Bruggmanniella species from Japan and mentioned that Asian Bruggmanniella species are similar to the Palaearctic genus Pseudasphondylia Monzen, 1955 in male morphological features such as the presence of parameres and separate teeth on the gonostylus. However, Bruggmanniella can be distinguished from Pseudasphondylia by the presence of setae on larval cervical papillae and on all ventral papillae. Recently, Garcia et al. (2020) provided a cladistics hypothesis based on 57 characters and 27 species belonging to genera Asphondylia Loew, Bruggmannia Tavares, Bruggmanniella Tavares, Illiciomyia Tokuda, Parazalepidota Maia, Pseudasphondylia Monzen, Schizomyia Kieffer and Lopesia Rübsaamen. Most Bruggmanniella species are included in this analysis except B. litseae and B. byrsonimae. Based on this analysis by Garcia et al. (2020), B. brevipes is situated in the basal clade of Pseudasphondylia and Bruggmanniella, and two Japanese Bruggmanniella species were subsumed in the clade of Pseudasphondylia. Thus, Garcia et al. (2020) erected the new genus Odontokeros Garcia, Lamas and Urso-Guimarães, 2020 for B. brevipes and combined two Japanese Bruggmanniella with Pseudasphondylia. However, bootstraps of key nodes on the topology are poorly supported (less than 30%), especially on the node of B. brevipes + (Pseudasphondylia + Bruggmanniella), and among Pseudasphondylia and two Asian Bruggmanniella species (B. cinnamomi and B. actinodaphnes). In addition, their morphological definition of the new genus is obscure, consisting of only a slight difference in the larval sternal spatula and “shorter adult legs.” In this study, we show that the hypothetical system proposed by Garcia et al. (2020) is not supported. We described three new Bruggmanniella species from Taiwan that induce stem and leaf galls on Cinnamomum (Fig. 1), two on C. osmophloeum and one on C. subavenium; additionally, we record B. cinnamomi, which induces stem galls on C. insularimontanum, in Taiwan for the first time. Furthermore, we conducted molecular phylogenetic analyses of Cinnamomum-associated Bruggmmanniella to infer its possible divergence processes. MATERIALS AND METHODS Collecting galls and gall midges Galls were collected from various localities of Taiwan from 2014 to 2020. Larval and pupal specimens were obtained from galls and dissected under a Fig. 1. Plant galls induced by Taiwanese Bruggmanniella species on Cinnamomum species. (A) Stem galls on C. insularimontanum. (B) Stem galls on C. subavenium. (C) Stem galls on C. osmophloeum. (D) Leaf galls on C. osmophloeum. page 2 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan stereoscopic microscope. Adults were reared in the laboratory and pupal exuviae were saved. Specimens were preserved in 70% ethanol for morphological studies or in 99.5% ethanol for molecular work. Slide preparation Specimens were mounted on slides following Gagné (1994). Specimens were examined under a microscope (Leica DM 750, Germany) and illustrated with a drawing tube. Morphological terminology of adult thoracic plates follows Tokuda (2004) and the morphology of other parts follows McAlpine (1981). Larval and pupal morphological terminology follows Gagné (1994). Type specimens were deposited in the Laboratory of Insect Systematics and Evolution, National Cheng-Hsing University, Taichung, Taiwan (NCHU). Type materials of B. cinnamomi were examined (ELKU: Collection of the Entomological Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan). Molecular analysis A partial region of mitochondrial DNA cytochrome oxidase subunit I (COI) was sequenced following Pan et al. (2015) and Lin et al. (2020). Primers were designed based on Cameron et al. (2007); forward: Diptera-49F (5’-AAT CAT AAA GAT ATT GGA AC-3’) and reverse: Diptera-734R (5’-CAA CAT TTA TTT TGA TTT TTT GG-3’). DNA sequences were aligned by ClustalW (Thompson et al. 1994) via software BioEdit (Hall 1999). Then 19 new and two prepared sequences (B. cinnamomi and P. kiritanii) were uploaded to the DNA Data Bank of Japan (DDBJ) (Table 1). Sequences of two Daphnephila species, D. urnicola Chiang, Yang & Tokuda, 2015 (in Pan et al. 2015) (AB857360) and D. truncicola Tokuda, Yang & Yukawa, 2008 (AB334228), and one Pseudasphondylia species, P. matatabi Yuasa & Kumazawa, 1938 (AB085873), were downloaded from the National Center for Biotechnology Information (NCBI) (Table 1). Sequences of D. truncicola was selected as the root taxon in phylogenetic analyses according to the results of the morphology-based phylogeny in Tokuda and Yukawa (2007) and Tokuda et al. (2008). Phylogenetic clustering was performed by Maximum Likelihood (ML) and Neighbor-joining (NJ) methods with MEGA7 (Kumar et al. 2016), and Bayesian inference (BI) via MrBayes 3.2 (Ronquist et al. 2012). For ML inference, the GTR+I+G model was determined based on jModelTest 2.1.10 (Darriba et al. 2012) and pairwise deletion in proportional distance was selected with 1000 bootstrap replicates. The NJ method was performed using the same settings and in the ML method, except that the model used was Kimura’s twoparameter divergence (Kimura 1980). As for BI, the best-fit substitution model TIM2+I was determined via jModelTest 2.1.10 (Darriba et al. 2012) using Bayesian Information Criterion (BIC). Markov chain Monte Carlo (MCMC) chain length was 5 × 105 generations with sampling every 1000 generations with the first 25% of steps discarded as burn‐in by TreeAnnotator. Posterior clade probabilities were summarized on a maximum clade credibility tree. Divergence distances between Bruggmanniella species were calculated via P-distance with MEGA7 (Kumar et al. 2016). The LSID (Life Science Identifier) numbers of the new species were registered in ZooBank (www. zoobank.org) and are given in the taxonomy section below. Table 1. DNA information on Bruggmanniella species and relatives Scientific name Host Gall organ Locality Collector Accession Number B. cinnamomi Cinnamomum yabunikkei Stem Okinawa , Japan M Tokuda LC441006*, LC517103** B. cinnamomi C. insularimontanum Stem Pingtung, Taiwan SF Lin LC516718-20 B. shianguei sp. nov. C. subavenium Stem Nantou, Taiwan SF Lin LC516726-28 B. turoguei sp. nov. C. osmophloeum Stem Taichung, Taiwan SF Lin LC516722-25 B. sanlianensis sp. nov. C. osmophloeum Leaf Taichung, Taiwan SF Lin LC516721 B. brevipes Neolitsea parvigemma Bud Pingtung, Taiwan SF Lin LC516729-34 B. actindaphnes Litsea coreana Stem Mt. Mukabaki, Japan M Tokuda AB334238* Pseudasphondylia matatabi Actinidia polygama Flower bud Hisayama, Fukuoka, Japan J Yukawa, N Uechi, M Tuda AB085873* P. rokuharensis Viburnum dilalatum Fruit Hanayama, Honshu, Japan M Tokuda, J Yukawa LC538357 P. kiritanii Cornus controversa Flower bud Futo, Honshu, Japan J Yukawa, K Kiritani LC538356** Daphnephila urnicola Machilus zuihonensis Leaf Nantou, Taiwan TC Chiang AB857360* Daphnephila truncicola Machilus thunbergii Stem Taipei, Taiwan TC Chiang AB334228* “*” indicates DNA sequences downloaded from the National Center for Biotechnology Information; “**” indicates DNA sequences prepared by M Tokuda and used in the present study. page 3 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan RESULTS TAXONOMY Order Diptera Linnaeus, 1758 Family Cecidomyiidae Newman, 1834 Tribe Asphondyliini Gagné, 1994 Genus Bruggmanniella Tavares, 1909 Bruggmanniella Tavares, 1909: 19. Type species: Bruggmanniella braziliensis Tavares, 1909. Hemibruggmanniella Möhn, 1961b: 6. Type species: Bruggmanniella obita Tavares, 1920. Odontokeros Garcia, Lamas and Urso-Guimarães, 2020: 10. Syn. nov. Genus Bruggmanniella belongs to the subtribe Asphondyliina and comprises 17 known species worldwide (Gagné and Jaschhof 2017; Lin et al. 2019 2020; Garcia et al. 2020; Rodrigues et al. 2020). In this article, three new species are described and one known species is recorded from Taiwan for the first time; all the species are associated with Cinnamomum species. Morphological features of Bruggmanniella are summarized in Gagné (1994) and Gagné et al. (2004), and they were compared to related Palearctic genera in Tokuda and Yukawa (2006). Briefly, Bruggmanniella species are separated from Pseudasphondylia Monzen, 1955 and Probruggmanniella Möhn, 1961 by the presence of setae on the larval cervical papillae and on all papillae of the ventral surface, less constricted male flagellomeres, convolute male circumfila, presense of an apical spur on the first tarsomeres, and the absense of setae on lateral papillae on larval thoracic segments (Tokuda 2004; Möhn 1961a; Tokuda and Yukawa 2006). In addition, two separate teeth on the gonostylus are regarded as a common feature of Bruggmanniella (Tokuda and Yukawa 2006). However, in this article we describe a new species based on its bidentate tooth of the gonostylus and other morphological features that fit well with the genus; in addition, our molecular phylogenetic analysis clearly indicates that the species is in the clade of Bruggmanniella (see below). Bruggmanniella turoguei sp. nov. Lin, Yang and Tokuda (Figs. 2, 5A, 6A, D; Table 2) urn:lsid:zoobank.org:act:369831A8-5701-4D23-A7A1B6403FCCEA8F Type materials: Holotype. Male (on slide, NCHU), TAIWAN: Taichung City, Shalian Ln, adult reared from galls that collected on 28.iv.2016 and emerged on 2.v.2016, S.F. Lin leg. Paratypes. TAIWAN: 7♂7♀ (on slides, NCHU) collected data same as holotype; 2 pupal exuviae (on slides, NCHU) Taichung City, Shalian Ln, 28.iv.2018, S.F. Lin leg.; 1 pupa (in ethanol, NCHU), 2 pupal exuviae (on slide, NCHU) Taichung City, Shalian Ln, 1.iv.2018, S.F. Lin leg.; 10 larvae (6 on slides, 4 in ethanol, NCHU), Nantou Co., Lianhuachi Research Center, 20.iii.2020, S.F. Lin leg. Other material examined: TAIWAN: 1 larval skin (on slide, NCHU), Nantou, Lianhuachih Research Center, 2.iv.2019, S.F. Lin leg. Etymology: The species name is derived from transliteration of the Chinese common name of the host species. Host: Cinnamomum osmophloeum Kaneh is endemic to Taiwan (Lu et al. 2000). Gall: Multi-chambered stem galls with irregular swellings; approximately 1 cm in diameter and 1 to 12 cm long. Distribution: Mountain areas of central Taiwan Life history: Immature larvae are found in the galls from January to early April; larvae mature and pupate during April; adults emerge from galls in early May. One or two years are required to complete one generation. Adult: Head. Eye bridge three to four facets long; frontoclypeal setal count as in table 2; palpus foursegmented, first segment globose, second to fourth successively elongated; twelve flagellomeres all with flexuous circumfila, gradually becoming shorter from first to tenth, distal three subequal in length in male, gradually shortened and the terminal one subglobular in female (Fig. 2B, C). Thorax. Thoracic setal and scale counts as in table 2; legs with dense blackish scales; first tarsomeres of all legs with apical spur (Fig. 2D); female fifth tarsomere shorter than male; claws strongly curved on all legs; pulvilli shorter than claws; empodia as long as claws (Fig. 2E); Wing densely covered with dark grayish hairs; wing length 2.5–2.8 mm in male (n = 8) and 3.1–3.4 mm in female (n = 7), 2.6 times as long as wide in male and 2.2–2.5 times as long as wide in female, R5 joining costa posterior to wing apex (Fig. 2G, H). Male abdomen. First through seventh abdominal tergites rectangular, with one or two rows of posterior setae; both tergites and sternites without anterior pair of trichoid sensilla. Terminalia (Fig. 5A): cerci setose, each rounded at apex; hypoproct deeply incised V-shaped emargination, each lobe with an apical seta; gonostylus suboval, distally with 2 very closely situated solid teeth; gonocoxite massive, with mediobasal lobe; aedeagus longer than hypoproct, distally tapering, basal part laterally sclerotized. Female abdomen. Seventh sternite 448–680 μm long (n = 7), 2.1 times as long as sixth sternite; ovipositor protractile, slender, aciculate, basally with a bilobed cerci-like structure; needle part of ovipositor 0.88–0.96 mm long (n = 7), 1.4–2.1 times as long as seventh sternite. Otherwise as in male. page 4 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan Table 2. Frontoclypeal and thoracic setal counts of Bruggmanniella turoguei sp. nov. Specimens Male Female n Mean ± SD Range n Mean ± SD Range Frontoclypeal setae 3 18.0 ± 2.0 16–21 5 18.6 ± 1.3 17–20 Anterior dorsolateral setae 8 31.4 ± 4.5 21–35 7 38.9 ± 5.2 29–45 Posterior dorsolateral setae 4 25.5 ± 1.3 24–27 4 36.5 ± 1.0 36–38 Mesopleural scales 6 26.0 ± 3.7 21–31 7 26.7 ± 6.4 16–35 Mesepimeral setae 8 31.9 ± 3.8 27–37 7 36.6 ± 6.2 27–44 Fig. 2. Bruggmanniella turoguei sp. nov. (A) Male head (ventral view) (B) Male antenna (7–12 segment). (C) Female antenna (7–12 segment). (D) Male 1st tarsomere. (E) Male 5th tarsomere. (F) Female 5th tarsomere. (G) Male wing. (H) Female wing. Scale bars: A–C = 0.03 mm; D–F = 0.1 mm; G–H = 1 mm. page 5 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan Pupa: Body length 3.2–3.4 mm, pupal skin not pigmented except for antennal horn. Antennal horn 450– 500 µm long, dorsoventrally flattened, anterior margin narrowed, posterolaterally with small lobe (Fig. 6A); cephalic seta 80–90 µm long; frons without horns; facial papillae not visible; prothoracic spiracle 280–320 µm long; spiracles on second to sixth abdominal segments 25–30 µm long; second to seventh abdominal segments with 9 to 10 transverse rows of spines; eight dorsal papillae on first to seventh abdominal segments, most outer and second inner pairs with seta; two dorsal papillae on eighth abdominal segment, each with seta; each segment with pleural papilla, each with seta. Full growth larva: Body color yellow, body length 2.8–3.0 mm. Second antennal segment short, conical; cervical papillae with seta. Sternal spatula approximately 300 µm long (Fig. 6D), anteriorly with two lobes; four lateral papillae and a sternal papillae present on each side of all thoracic segments, each with seta; four dorsal papillae on all thoracic and first through seventh abdominal segments, 2 dorsal papillae on eighth abdominal segment, each with seta; one pleural papilla present on each side, each with seta; terminal papillae not apparent. Each abdominal segment, except terminal one, ventrally with many transverse rows of minute spines and covered with small triangular spines. Bruggmanniella shianguei sp. nov. Lin, Yang and Tokuda (Figs. 3 and 5B, 6B, E; Table 3) urn:lsid:zoobank.org:act:151A1E27-B237-44CC-9EEC41BD4CA08E66 Type material: Holotype. Male (on slide, NCHU), TAIWAN: Nantou Co., Huisun Forest Area, adult emerged on 27.iv.2016 reared from stem gall collected on 21.iv.2016, S.F. Lin leg. Paratypes. TAIWAN: 5♂4♀ (on slides, NCHU), same data as holotype; 2♂4♀ (on slides, NCHU) same data as holotype except emergence date on 2.v.2016, S.F. Lin leg.; 2♀ (on slides, NCHU) same data as holotype except emergence date on 23.iv.2016, S.F. Lin leg.; 1 larva, 6 pupal exuviae (on slides, NCHU), same collection data as holotype, S.F. Lin leg.; 2 larvae (on slides, NCHU), 13 pupae (4 on slides and 9 in ethanol, NCHU), Taichung City, Mt. Tungma, 25.iv.2017, S.F. Lin leg. Etymology: The species name is derived from Chinese pronunciation of host plant species. Host: Cinnamomum subavenium Miq. is distributed in Borneo, Cambodia, China South-Central, China Southeast, Malaya, Myanmar, Sulawesi, Taiwan, and Vietnam (Lu et al. 2000; Hassler 2019). Gall: Multi-chambered ellipsoid galls are induced on stem. The size is smaller than 1 cm long and wide. Distribution: Mountain areas of Taiwan. Life history: Immature larvae are found in the gall from October to the following February and mature larvae in March. Pupae are found from late March to early April and adults emerge in early May. This species does not seem to be univoltine, because well-developed galls are not found every year in the same locality. Instead, fresh galls are found biennially, suggesting that the species has a two-year life type of cycle. Adult: Head. Eye bridge two or three facets long; frontoclypeal setal counts as in table 3; palpus 4-segmented (Fig. 3A), first globose, second to fourth elongated and subequal in length; distal three flagellomeres as in figure 3B and 3C. Thorax. First tarsomeres as in figure 3D; empodia equal length to claws in male, shorter than claw in female (Fig. 3E, F). Wing 2.0–2.8 mm long in male (n = 7, Fig. 3G) and 2.8–3.2 mm long in female (n = 4, Fig. 3H); thoracic setal and scale counts as in table 3. Male abdomen. Terminalia (Fig. 6B): hypoproct shorter than cerci, shallowly incised V-shaped emargination; 2 solid teeth of gonostylus rather apart from each other; gonocoxite slightly extended beyond gonostylus. Female abdomen. Seventh sternite 469–538 μm long (n = 8), 2.0 times as long as sixth sterminte; needle part of ovipositor 0.85– 0.92 mm long, 1.8 times as long as seventh sternite. Otherwise as in B. turoguei. Pupa: Body length 1.8–2.4 mm. Antennal horn dorsoventrally flattened, anterior margin narrowed, posterolaterally with small lobe, antennal horn Table 3. Frontoclypeal and thoracic setal counts of Bruggmanniella shianguei sp. nov. Specimens Male Female nMean ± SD Range nMean ± SD Range Frontoclypeal setae 4 19.3 ± 4.2 15–25 9 17.9 ± 1.7 15–20 Anterior dorsolateral setae 8 33.9 ± 4.5 24–38 8 42.5 ± 5.2 37–54 Posterior dorsolateral setae 6 29.0 ± 3.9 23–35 7 37.1 ± 3.6 32–43 Mesopleural scales 7 19.9 ± 3.1 17–26 9 27.4 ± 6.3 19–38 Mesepimeral setae 8 32.4 ± 6.0 21–38 9 35.2 ± 4.4 29–42 page 6 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan 460–480 µm long (Fig. 6B); cephalic papilla with seta, 75–85 µm long; prothoracic horn 280–310 µm long; spiracles on second to sixth abdominal segments 25–30 µm long; second to seventh abdominal segments with nine to 10 transverse rows of spines. Otherwise as in B. turoguei. Full growth larva: Body color yellow, body length 1.6–1.8 mm. Second antennal segment short, conical; cervical papillae with seta. Sternal spatula 260–300 µm long (Fig. 6E), anteriorly with two lobes; four lateral papillae and a sternal papillae present on each side of all thoracic segments (Fig. 6E), each with seta. Otherwise as in B. turoguei sp. nov. Bruggmanniella sanlianensis sp. nov. Lin, Yang and Tokuda (Figs. 4, 5C, 6C, F; Table 4) urn:lsid:zoobank.org:act:9F8D07AA-13AF-4335-BCA9822D12240DF4 Type material: Holotype. Male (on slide, NCHU), TAIWAN: Taichung City, Shalian Ln, adult emerged on 15.v.2017 and reared from collected galls on 4.v.2017, S.F. Lin leg. Paratypes. TAIWAN: 3♂3♀ (on slides, Fig. 3. Bruggmanniella shianguei sp. nov. (A) Male head (ventral view) (B) Male antenna (8–12 segment). (C) Female antenna (8–12 segment). (D) Male 1st tarsomere. (E) Male 5th tarsomere. (F) Female 5th tarsomere. (G) Male wing. (H) Female wing. Scale bars: A–C = 0.03 mm; D–F = 0.1 mm; G–H = 1 mm. page 7 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan Fig. 4. Bruggmanniella sanlianensis sp. nov. (A) Male head (ventral view) (B) Male antenna (8–12 segment). (C) Female antenna (8–12 segment). (D) Male 1st tarsomere. (E) Male 5th tarsomere. (F) Female 5th tarsomere. (G) Male wing. (H) Female wing. Scale bars: A–C = 0.03 mm; D–F = 0.1 mm; G–H = 1 mm. Table 4. Frontoclypeal and thoracic setal counts of Bruggmanniella sanlianensis sp. nov. Specimens Male Female nMean ± SD Range nMean ± SD Range Frontoclypeal setae 4 20.5 ± 1.7 19–23 2 19.0 ± 0.0 19 Anterior dorsolateral setae 1 32.0 32 2 36.0 ± 1.4 35–37 Posterior dorsolateral setae 2 41.5 ± 10.6 34–49 0 NA NA Mesopleural scales 1 14.0 14 1 24.0 24 Mesepimeral setae 2 35.0 ± 2.8 33–37 4 25.8 ± 2.1 23–28 “NA” indicate no data. page 8 of 16Zoological Studies 59:66 (2020) © 2020 Academia Sinica, Taiwan NCHU), data are same as holotype; 1♂1♀ (on slide, NCHU) Taichung City, Shalian Ln, adult emerged on 5.v.2016 and reared from collected galls on 25.iv.2017, S.F. Lin leg. (NCHU); 5 larvae, 2 pupa, 7 pupal exuviae (on slides, NCHU) Taichung City, Shalian Ln, 1.iv.2018, S.F. Lin leg.; 2 larvae (on slides, NCHU), Taichung City, Shalian Ln, 25.iv.2017, S.F. Lin leg. Etymology: The species name is derived from the type locality, Sanlian Ln. ost. Cinnamomum osmophloeum Kaneh is an endemic plant species to Taiwan. Gall: Tear-shaped and single-chambered gall on leaf veins (Fig. 1D), 2–3 mm long and 1–2 mm wide. Possibly 1–20 galls occur on one leaf. Distribution: Mountain areas of Taiwan. Life history: This gall midge is univoltine. Immature larvae are found in the galls from December to the following March. Mature larvae are found in April and pupate from middle to late April and adults emerged in early May. Adult: Head. Frontoclypeal setal counts as in table 4; palpus as in figure 4A, first segment round, second to fourth gradually becoming longer. Thorax. First tarsomere as in figure 4D; thoracic setal and scale counts as in table 4; empodia equal length to claws in male and shorter than claws in female (Fig. 4E, F); wing 2.2–2.4 mm long in male (n = 5) and 2.2–2.7 mm (n = 4) in female (Fig. 4G, H). Otherwise as in B. turoguei. Male abdomen. Terminalia (Fig. 5C): hypoproct entire or slightly emarginated, slightly shorter than cerci; gonostylus distally with a bidentate tooth. Female abdomen. Seventh sternite 365–410 μm long (n = 4), 2.5–3.0 times as long as sixth sternite; needle part of ovipositor 0.64–0.74 mm long, 1.8 times as long as seventh sternite. Otherwise as in B. turoguei. Pupa: Body length 1.5–1.8 mm. Antennal horn 260–290 µm long (Fig. 6C), anteriorly narrowed, posterolaterally with small lobe; cephalic setae 50–60 µm long; frons without horns; prothoracic spiracle 150–160 µm long; spiracles on second to sixth abdominal segments 30–35 µm long; second to seventh cover with six to eight and eighth cover with two to four transverse rows spines. Otherwise as in B. turoguei. Full growth larva: Body length 1.2–1.6 mm. Sternal spatula 200–225 µm long, anteriorly with two angular lobes; four lateral papillae and one sternal papillae of each sides on all thoracic segments, each with seta (Fig. 6F). Otherwise as in B. turoguei sp. nov. Bruggmanniella cinnamomi Tokuda & Yukawa (Fig. 7; Table 5) Description: See Tokuda and Yukawa (2006). Number of palpal segment is four in both sex (Fig. 7A) that was misidentified in Tokuda and Yukawa (2006). Additional descriptions are as follows: female tarsal claws thicker than male (Fig. 7B–C); Wing 2.6–2.9 mm long in male (n = 8, Fig. 7D) and 3.1–3.6 mm long in female (n = 8); Frontoclypeal and thoracic setal counts as in table 5. Specimen examined: Holotype. Male (on slide, ELKU), JAPAN: Nakagusuku, Okinawa, adult reared by M. Tokuda and emerged on 15.iii.2001 from stem galls that collected on 25.ii.2001, leg. J. Yukawa, S. Yamauchi. Paratypes. JAPAN: (Cecid. Nos. C7101– 7114; C7151–C7164; see Tokuda and Yukawa (2006) for detailed information). Other specimens: TAIWAN: 8♂, 8♀, 2 pupal exuviae (on slide, NCHU), Pingtung Co., Dahan forest road, adult emerged on 25–27.iv.2016 from galls that Fig. 5. Male genitalia of Taiwanese Cinnamomum–associated Bruggmanniella. (A) Bruggmanniella turoguei sp. nov., (B) B. shianguei sp. nov., and (C) B. sanlianensis sp. nov. 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