Biodiversity and evolutionary dynamics of gall midges hosting on Japanese beech trees

Biodiversity and evolutionary dynamics of gall midges hosting on Japanese beech trees | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Ecology and Evolution This is a preprint and has not been peer reviewed. Data may be preliminary. 28 January 2025 V1 Latest version Share on Biodiversity and evolutionary dynamics of gall midges hosting on Japanese beech trees Authors : Shinnosuke Mori 0000-0003-0073-8876 [email protected] , Yugo Dhakhwa , Makoto Tokuda , and Yoko Saikawa Authors Info & Affiliations https://doi.org/10.22541/au.173809505.59741312/v1 656 views 244 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The Japanese Archipelago harbors unique beech flora (Fagus L.; Fagaceae), with a sympatric distribution of two endemic species, F. crenata and F. japonica, upon which a diverse array of 34 types of leaf galls induced by gall midges (Diptera: Cecidomyiidae) have been documented. The inducers of most of these galls remain undescribed, and their phylogenetic relationships with known taxa are still poorly understood. In this study, we collected 29 types of leaf galls from the two Japanese Fagus species, including 6 previously unreported types, and sequenced the cytochrome c oxidase subunit I mitochondrial gene of the inducers to infer maximum-likelihood and Bayesian time-calibrated phylogenies. Our phylogenetic analyses revealed that the Fagus-hosting guild forms a monophyletic clade within the tribe Dasineurini, with F. crenata-hosting taxa occupying a basal position within the lineage. These taxa are closely related to the genera Hartigiola and Mikiola and have likely undergone adaptive radiation on the leaves of F. crenata and F. japonica in the ecologically segregated Japanese Archipelago since the Miocene period, accompanied by multiple host shifts between the two Fagus species and location shifts within their leaves. Manuscript type: Research Article Title Biodiversity and evolutionary dynamics of gall midges hosting on Japanese beech trees Authors (*corresponding authors) Shinnosuke Mori 1,* ( [email protected] ) Yugo Dhakhwa 1 Makoto Tokuda 2,3 ( [email protected] ) Yoko Saikawa 1,* ( [email protected] ) Affiliations 1 Faculty of Science and Technology, Keio University, Kanagawa, Japan 2 Faculty of Agriculture, Saga University, Saga, Japan 3 The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan Abstract The Japanese Archipelago harbors unique beech flora ( Fagus L.; Fagaceae), with a sympatric distribution of two endemic species, F. crenata and F. japonica , upon which a diverse array of 34 types of leaf galls induced by gall midges (Diptera: Cecidomyiidae) have been documented. The inducers of most of these galls remain undescribed, and their phylogenetic relationships with known taxa are still poorly understood. In this study, we collected 29 types of leaf galls from the two Japanese Fagus species, including 6 previously unreported types, and sequenced the cytochrome c oxidase subunit I mitochondrial gene of the inducers to infer maximum-likelihood and Bayesian time-calibrated phylogenies. Our phylogenetic analyses revealed that the Fagus -hosting guild forms a monophyletic clade within the tribe Dasineurini, with F. crenata- hosting taxa occupying a basal position within the lineage. These taxa are closely related to the genera Hartigiola and Mikiola and have likely undergone adaptive radiation on the leaves of F. crenata and F. japonica in the ecologically segregated Japanese Archipelago since the Miocene period, accompanied by multiple host shifts between the two Fagus species and location shifts within their leaves. gall midge, molecular phylogeny, adaptive radiation, co-evolution, biogeography Introduction Insects are one of the most diverse groups in terrestrial ecosystems, and almost half of all extant species are phytophagous (Price et al., 2011). Understanding the processes of speciation and adaptive radiation of phytophagous insects is thus important in order to elucidate the mechanism of biodiversity generation. Insect galls are malformed plant structures induced by insects such as gall midges and gall wasps, which use the galls as their own microhabitats. Galls can form on leaves, stems, floral buds, flowers, fruits, or roots and often exhibit distinctive form and color. Gall midges (Diptera: Cecidomyiidae) represent the most speciose group of galling arthropods in the world, comprising 6,651 known species and thousands of undescribed species (Gagné and Jaschhof, 2021). The currently accepted systematic division of Cecidomyiidae into subfamilies and tribes, as outlined in ‘A Catalog of the Cecidomyiidae (Diptera) of the World’, is based on comparative studies of morphological characteristics (Gagné and Jaschhof, 2021) and corroborated by recent phylogenetic studies (Sikora et al., 2019; Dorchin et al., 2019). The Cecidomyiidae family includes six subfamilies, as follows: five basal subfamilies (Catotrichinae, Lestremiinae, Micromyinae, Winnertziinae, and Porricondylinae) that feed on fungi, and the Cecidomyiinae, the largest and youngest subfamily, which includes fungivorous, herbivorous, and predatory species. Approximately 75% of species in the Cecidomyiinae are herbivorous and diversified significantly during the Tertiary period, coevolving with angiosperms (Gagné and Jaschhof, 2021). Most herbivorous gall midges are host-specific and develop on one or a few closely related host plants, and many genera and even tribes have evolved and diversified on plants of particular families (Gagné and Jaschhof, 2021). Beech ( Fagus L.; Fagaceae), a representative genus of tree species of deciduous forests in temperate areas of the Northern Hemisphere (Peters, 1997), serves as a host for cecidomyiids of the subfamily Cecidomyiinae. The genus Fagus includes two distinct subgenera, Fagus and Engleriana , which include 7–10 and 3 species, respectively (Denk et al., 2005), although the number and rank of several taxa remain controversial (e.g., Gömöry et al., 2018). Regarding trees of the subgen. Fagus , gall midges have been found to infest Siebold’s beech ( F. crenata Blume) in Japan, Oriental beech ( F. orientalis Lipsky) in areas around the Caspian Sea and Black Sea, and European beech ( F. sylvatica L.) in Europe (Yukawa et al., 2021). The Fagus -hosting gallers comprise six genera across three tribes, as follows: Contarinia (Cecidomyiini), Hartigiola , Janetiella , Macrolabis , Mikiola (Dasineurini), and Phegomyia (Trotteriini) (Yukawa et al., 2021). By contrast, field surveys in North America reported the absence of cecidomyiid galls on American beech ( F. grandifolia Ehrhart) and Mexican beech ( F. mexicana Martinez) (Sato and Yukawa, 2001). In subgen. Engleriana , Japanese beech ( F. japonica Maximowicz) is the only species infested by cecidomyiids, with Hartigiola annulipes (Hartig) as a described species. The Japanese Archipelago is home to unique beech flora due to the sympatric distribution of two endemic species, each of which belongs to a different subgenus: F. crenata (subgen. Fagus ) and F. japonica (subgen. Engleriana ). At least 26 types of leaf galls have been documented on F. crenata , with 8 types documented on F. japonica (summarized in Sato et al., 2010). As gall morphology is essentially species-specific (Yukawa and Masuda, 1996), formation of the respective types of galls is thought to be induced by different cecidomyiid species. Fagus crenata harbors the greatest abundance of gall-inducing cecidomyiids in the Japanese Archipelago (Yukawa and Masuda, 1996), and elucidating their speciation processes will be necessary in order to understand the diversification of gall inducers in relation to specific host plants. Among cecidomyiids associated with F. crenata and F. japonica , six species have been described and identified to date: Mikiola bicornis Sato & Yukawa, Mikiola glandaria Sato & Yukawa, Hartigiola faggalli (Monzen), Hartigiola annulipes (Hartig), Janetiella infrafoli Monzen, and Phegomyia tokunagai Sasakawa & Koyama (Table 1; Sato et al., 2010), all of which belong to the supertribe Lasiopteridi of subfamily Cecidomyiinae (Gagné and Jaschhof, 2021). Although the nucleotide sequences of cytochrome oxidase c subunit I ( COI ), a ‘DNA barcode’, are publicly available for Hartigiola faggalli and Hartigiola annulipes , sequence data for cecidomyiids that induce other types of galls are lacking; thus, the molecular phylogenetic relationships between these species remain unknown. This study therefore addressed this knowledge gap by collecting COI sequences from Fagus -hosting cecidomyiids that induce 29 types of galls, including previously unreported types, in order to infer the molecular phylogeny within the Cecidomyiinae. The study also investigated the taxonomy, evolutionary context, and larval feeding modes of these cecidomyiids and explored the relationship between the morphological diversity of galls and genetic variations in gall inducers within a phylogenetic framework. \received DD MMMM YYYY \acceptedDD MMMM YYYY Materials and Methods Gall midge collection A total of 29 types of galls (designated A – AC ) were collected from leaves of F. crenata and F. japonica specimens across five mountainous areas of the Kanto and Chubu regions of Japan (Figs. 1 and 2; Table 1): Mt. Amagi (elevation, 1,406 m; 34°51’30.6”N, 139°0’27.6”E) in Shizuoka, Mt. Gozen (elevation, 1,405 m; 35°46’18.4”N, 139°4’51.0”E) in Tokyo, Mt. Kanyudo (elevation, 1,418 m; 35°30’40.1”N, 139°0’49.0”E) and Mt. Nabewari (elevation, 1,272 m; 35°26’39.5”N, 139°8’31.6”E) in Kanagawa, and Mt. Mito (elevation, 1,531 m; 35°43’53.0”N, 138°58’37.4”E) in Yamanashi. Galls were collected during the periods April–June and August–October of 2022 to 2024. On Mt. Gozen and Mt. Mito, F. crenata and F. japonica grow sympatrically, whereas no F. japonica were observed on Mt. Amagi, Mt. Kanyudo, or Mt. Nabewari. All types of galls found on F. crenata and F. japonica were classified as shown in Table 1 according to Yukawa (1991), Yukawa and Masuda (1996), and Sato and Yukawa (2004). Six types of galls ( Q , R , and Z – AC ) exhibited previously unreported morphologies, for which Japanese names were assigned based on the characteristics (Table 1) . Gall specimens were photographed either at the collection site or in the laboratory before collection of gall midges. To collect midges, galls were gently excised using a scalpel blade, and larvae or pupae ( a – ac ) within the galls were collected under a stereomicroscope. Gall midge specimens were preserved in 99.5% ethanol at 4°C until use. DNA extraction, amplification, sequencing, and editing Larval and pupal specimens were homogenized using a BioMasher II (Takara Bio, Tokyo, Japan), and gDNA was extracted from the homogenized samples using NucleoSpin DNA Insect (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The extracted DNA was subjected to PCR to amplify approx. 700 bp within the mitochondrial COI gene using the primer set LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) (Folmer et al., 1994). The primers were synthesized by Eurofins Genomics (Tokyo, Japan). Amplification was performed using KOD FX DNA polymerase (Toyobo, Osaka, Japan) and a MiniAmp Thermal Cycler (Thermo Fischer Scientific, MA, USA) under the following conditions: initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 sec, annealing at 40–60°C for 30 sec, and extension at 68°C for 15–30 sec. PCR amplification products were cleaned up enzymatically using ExoSAP-IT PCR Product Cleanup (Thermo Fischer Scientific) or purified using either NucleoSpin Gel and PCR Clean-up XS (Macherey-Nagel) or the E-Gel Power Snap Electrophoresis System with CloneWell II 0.8% agarose gels (Thermo Fischer Scientific). The amplicons were then bidirectionally Sanger-sequenced by Eurofins Genomics using the same primers described above. Forward and reverse sequences were manually edited with reference to the chromatograms and assembled into consensus sequences using 4Peaks ver. 1.8 (https://nucleobytes.com/4peaks/index.html). Sequence ends exhibiting ambiguity were trimmed. The nucleotide sequence data generated in this study were deposited in the NCBI GenBank nucleotide sequence database under the accession numbers listed in Table S1 (accession, PQ838091–PQ838157). Phylogenetic analysis A phylogenetic analysis was conducted on the collected cecidomyiids within the subfamily Cecidomyiinae, which includes previously reported species that infest Fagus species ( Contarinia , Janetiella , Macrolabis , Hartigiola , Mikiola , and Phegomyia ). Sequence data for the COI , 16S rRNA ( 16S ), 28S rRNA ( 28S ), internal transcribed spacer 1 ( ITS1 ), and carbamoyl-phosphate synthetase 2 , aspartate transcarbamylase , and dihydroorotase ( CAD ) genes of Cecidomyiinae species used in Dorchin et al. (2019) were retrieved from GenBank. Sequences for cecidomyiids known to induce galls on Fagus were also included, if available: Contarinia fagi Rübsaamen ( COI , JQ684875.1; host, F. sylvatica ), Hartigiola faggalli (Monzen) (host, F. crenata ) of adaxial side-type isolate M01hf ( COI , AB753796.1), and abaxial side-type isolate M23hf ( COI , AB753817.1). Additionally, four taxa representing the basal subfamilies Catotrichinae ( Catotricha subobsoleta ), Micromyinae ( Catocha angulata ), and Porricondylinae ( Asynapta sp.; Porricondyla nigripennis ) were employed as outgroups, as per Dorchin et al. (2019). Sequences were aligned using MAFFT ver. 7 (Kuraku et al., 2013). COI sequences were aligned using the FFT-NS-1 progressive algorithm (Katoh et al., 2002), and sequences of other loci were aligned independently using the E-INS-i algorithm (Katoh et al., 2005). Poorly aligned regions of CAD were trimmed using trimAl ver. 1.50 (Capella-Gutiérrez et al., 2009), with gap and similarity thresholds of 0.5 and 0.001, respectively. The final lengths of aligned sequences, including gaps, were as follows: 16S , 670 bp (108 taxa); ITS1 , 1,606 bp (120 taxa); 28S , 754 bp (115 taxa); COI , 711 bp (203 taxa); and CAD , 752 bp (132 taxa). All loci were then concatenated into a single dataset using MEGA ver. 11.0.11 (Tamura et al., 2021), resulting in a complete dataset containing 216 taxa with a maximum length of 4,491 bp. The dataset was then partitioned by locus and further divided according to first, second, and third codon positions for protein-coding regions. Phylogenetic analyses were conducted using the maximum-likelihood (ML) algorithm in IQ-TREE2 ver. 2.3.4 (Minh et al., 2020). The following substitution models were determined using Modelfinder (Kalyaanamoorthy et al. 2017): 16S , GTR + F + I + G4; ITS1 , GTR + F + I + G4; 28S , TVM + F + I + G4; COI _1st, GTR + F + I + R5; CAD _1st, GTR + F + I + G4; CAD _2nd + COI _2nd, GTR + F + R3; COI _3rd, TPM2u + F + I + R5; and CAD _3rd, TIM3 + F + I + R4. Nodal support was evaluated using the Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) with 1,000 replicates (Guindon et al., 2010) and ultrafast bootstrap (UFBoot) analysis with 1,000 replicates (Hoang et al., 2018). Default settings were used for other parameters. Finally, the phylogenetic tree was visualized using iTOL ver. 6.8.1 (Letunic and Bork, 2024). Ancestral state reconstruction The Fagus -hosting clade was pruned from the ML IQ-TREE and subjected to ancestral state reconstruction using Mesquite 3.81 (Maddison and Maddison, 2023). The Markov k-state 1 (Mk1) parameter model (Lewis, 2001) was used for ML reconstructions, assuming equal probability for any character change. Host plant species ( F. crenata , F. japonica , F. orientalis , or F. sylvatica ), gall shape (globoid, horn, discoid, bivalve, or pocket), galling location (lamina, edge, vein, or midrib), and galling side of the leaf (adaxial or abaxial) were coded and reconstructed. One taxon per gall type was included in the analysis. Divergence time estimation Divergence time was estimated using Bayesian Evolutionary Analysis Sampling Trees (BEAST2) ver. 2.7.7 (Bouckaert et al., 2019). The concatenated dataset was partitioned by locus and codon position, as described above. The bModelTest (Bouckaert and Drummond, 2017) with transition-transversion split was used to identify the most appropriate substitution model for each partition. A Birth-Death model (Gernhard, 2008) was used as the tree prior, with estimation of the relative death rate (μ/λ) and birth difference rate (λ − μ). An optimized relaxed clock model (Douglas et al., 2021) under a log-normal prior distribution was used with an exponentially distributed mean (ORCucldMean.c = 10) and gamma-distributed standard deviation. Six priors exhibiting log-normal distribution (M = 1.0, S = 1.25) were applied to the estimated time to the most recent common ancestor (TMRCA), referring to the Paleobiology Database ver. 1.3 (McClennen et al., 2024): genera Contarinia , Lestodiplosis , and Clinodiplosis (amber fossils collected in Mexico, 16.0 Ma; Gagné, 1973), tribe Dasineurini, based on Jaapiella acinaciformis (amber fossil collected in Ukraine, 33.9 Ma; Fedotova and Perkovsky, 2005), subfamily Micromyinae, based on Eltxo cretaceus (amber fossil collected in Northern Spain, 99.6 Ma; Arillo and Nel, 2000), and subfamily Catotrichinae, based on Mesotrichoca mesozoica (sediment fossil collected in Russia, 145.0 Ma; Kovalev, 1990; Jaschhof and Jaschhof, 2008). Bayesian Markov chain Monte Carlo (MCMC) simulations were repeated for 200 million generations, with sampling every 1,000 generations. Log files were analyzed in Tracer ver. 1.7.2 (Rambaut et al., 2018) to examine convergence with reference to the effective sample size (ESS) of each inferred parameter. An ESS > 200 was ensured for all parameters. The first 10% of the generations were discarded as “burn-in”, leaving a total of 180,002 trees remaining. A maximum clade credibility (MCC) tree with 95% highest posterior density (95% HPD) intervals was compiled using TreeAnnotator ver. 2.7.7. The Bayesian inference (BI) time-calibrated tree was visualized using iTOL ver. 6.8.1 (Letunic and Bork, 2024). \received DD MMMM YYYY \acceptedDD MMMM YYYY Rate through time analysis Using the R package ‘ape’ ver. 5.8 (Paradis et al., 2004), the Fagus -hosting clade was pruned from the time-calibrated BI tree to include single representatives of each taxon for each type, resulting in a tree with 28 taxa. This subtree was subjected to lineage-through-time (LTT) analysis (Nee et al., 1992) using the R package ‘phytools’ ver. 2.3.0 (Revell et al., 2014; R Core Team, 2024). The γ-test was used to infer historical changes in the diversification rate of a clade (Pybus & Harvey, 2000), in which γ 0 indicated a decrease in the rate of diversification over time. \received DD MMMM YYYY \acceptedDD MMMM YYYY Results Collection of galls from Fagus forests A total of 18 types of galls were observed on the leaves of F. crenata (8 on Mt. Amagi, 7 on Mt. Gozen, 7 on Mt. Kanyudo, 5 on Mt. Mito, and 11 on Mt. Nabewari), and 11 types were found on the leaves of F. japonica (9 on Mt. Gozen and 3 on Mt. Mito), as summarized in Fig. 2 and Table 1. Among these, galls Q and R on F. crenata and Z – AC on F. japonica exhibited distinctive morphologies compared to the known types (Yukawa, 1991); thereby, they were distinguished from the latter. Galls of type Q were horn-shaped with a rounded tip and measured approx. 1 mm in diameter and 3 mm in height (Fig. 2Q). The morphology resembled that of P (Fig. 2P) but differed in terms of galling location; Q formed on the edge on the abaxial side of the leaf (Fig. 2Q), whereas P formed exclusively on the adaxial side of the lamina. Galls of type R were nodular structures that formed at the inward-curling edge on the abaxial side of leaves and measured 1.5–2 mm in diameter (Fig. 2R). Unlike C (Fig. 2C), which also formed at the leaf margin, R exhibited flattened swelling rather than a spherical shape. Galls of type Z were fang-shaped structures found on the lateral veins (Fig. 2Z). These galls developed on the adaxial side of leaves and measured 1–1.5 mm in diameter and 5–6 mm in height, with a smooth surface, pointed tip, and brown color. The morphology was similar to that of I on F. crenata leaves (Fig. 2I). Galls of type AA formed along the lateral veins of leaves, with narrow gaps on the adaxial side (Fig. 2AA). Although the swollen morphology resembled that of A (Fig. 2A) and T (Fig. 2T), which formed on a section of the lateral vein (6–15 mm long; Yukawa, 1991), AA extended from the midrib to near the leaf margin. Galls of type AB were onion-shaped structures on the edge of the abaxial side of the leaf and exhibited a smooth green surface (Fig. 2AB). AB were similar to the globular S on the leaf edge (Fig. 2S) but differed by having a pointed tip. AB measured approx. 5 mm in diameter and 4 mm in height (Fig. 2AB), slightly smaller than S . Galls of type AC exhibited a hairy surface and formed on the adaxial side of the leaf (Fig. 2AC). These galls were characterized by standing upright above the concave leaf and appeared to be pointed when viewed from the abaxial side. The galls measured 1.2–1.5 mm in diameter and 1.5–2 mm in height. The surface was reddish-brown, akin to U , which were globular with red hair , although the hairs of AC were shorter than those of U (Fig. 2U). Molecular phylogeny and divergence time A total of 67 COI sequences (654–687 bp) were obtained from cecidomyiids associated with 28 types of galls ( a – ac , excluding f due to insufficient PCR amplification). The sequences of most cecidomyiids within the same gall type ( b , c–e , g–k , o–s , u , w , y , and aa–ac ; n = 2–7) exhibited high similarity (> 99.0%), regardless of collection site. Nevertheless, lower similarity was observed for m (97.5%; n = 2), n (93.0%; n = 4), and z (97.5%; n = 2). The COI sequence from e and h , which induce bivalve-shaped galls, showed high homology to H. faggalli , the reported inducer of E (98.6% identity to isolate M01hf; GenBank accession, AB753796.1) and H (98.4% identity to isolate M23hf; GenBank accession, AB753817.1), respectively. By contrast, the sequence from s exhibited only 90.0% identity with that of Hartigiola annulipes (GenBank accession, MN191300.1), with a sequence difference of 60 of 607 bp. Although the inducer of S is believed to be Hartigiola annulipes (Yukawa et al., 2021), the species also reportedly induces galls on F. sylvatica (Pilichowski and Giertych, 2020), and this apparent discrepancy raises questions regarding the taxonomic assignment of the S inducer. Phylogenetic analyses of 67 OTUs and 149 known species produced congruent topologies in both the ML and BI trees (Figs. 3 and 4). The supertribe Lasiopteridi was clearly divided into three tribes: Dasineurini, Lasiopterini, and Alycaulini, which were fully supported (SH-aLRT = 99.8 and UFBoot = 100 in ML; PP = 1 in BI). With the exception of F. japonica -hosting t , which was classified into tribe Cecidomyiini in Cecidomyiidi, all leaf gallers hosting on F. crenata and F. japonica trees were assigned to the Lasiopteridi. Within the Lasiopteridi clade, all Japanese Fagus -hosting taxa other than l were assigned to Dasineurini, alongside previously described Fagus -hosting taxa ( Hartigiola annulipes , Hartigiola faggalli , Mikiola fagi , and Mikiola bicornis ). Only l occupied a basal position relative to tribe Lasiopterini, which was assumed to be ‘ambrosia gallers’ associated with symbiotic fungi (Gagné and Jaschhof, 2021). Within the Fagus -hosting clade of tribe Dasineurini (Figs. 3b and 4b), most nodes were well supported (SH-aLRT ≥ 80%, UFBoot ≥ 80%, PP ≥ 0.9). The phylogenetic positions of Hartigiola faggalli and Hartigiola annulipes indicate that the genus Hartigiola is paraphyletic. Although support for the monophyly of Mikiola fagi and b (described as Mikiola bicornis ) was relatively low (SH-aLRT = 77.5, UFBoot = 78, PP = 0.4), these appeared to be sister species. Some relationships among taxa remained unresolved. The monophyly of F. crenata -hosting k and m was highly supported (SH-aLRT = 98.4, UFBoot = 100, PP = 1), suggesting a possible sister relationship; however, they were intermixed within a single clade. The relationships among F. japonica -hosting u , v , and ac also could not be clearly resolved. Within the tribe Dasineurini (Fig. 4a), the Fagus -hosting lineage and Janetia , gall inducers hosting on Quercus (Fagaceae; Gagné and Jaschhof, 2021) were estimated to have diverged in the late Oligocene period (ca. 25.4 Ma; 95% HPD, 30.14–20.71 Ma). The crown age of the Fagus -hosing taxa was inferred at the beginning of the Miocene period (Fig. 4b; ca. 21.4 Ma; 95% HPD, 26.07–16.77 Ma). Along with the divergences of West Eurasian Fagus -hosting species, Hartigiola annulipes (ca. 15.6 Ma; 95% HPD, 19.42–11.73 Ma) and Mikiola fagi (ca. 10.3 Ma; 95% HPD, 14.95–5.86 Ma), the Japanese Fagus -hosting taxa rapidly radiated during the Miocene and beyond. Ongoing diversification is further supported by the LTT plot and the negative γ statistic (Fig. 4c; γ = −2.11, p = 0.035). Evolution of gall characteristics Likelihood-based ancestral character states for host plant species, gall shape, galling location, and leaf side (adaxial or abaxial) are presented in Fig. 5. Reconstruction of the evolutionary history of host plant use indicated that the root-ancestral host was of the F. crenata lineage (76.6%; Fig. 5a). Other potential ancestral hosts were identified with significantly lower probabilities (10.6% for F. japonica and approx. 6% for other Fagus species). Among 28 taxa studied, F. crenata remained the host for 16 taxa. The phylogenetic positions of the F. japonica -hosting taxa aa , x and z , and s , u–w , y , ab , and ac suggest that at least three independent host shifts from F. crenata to F. japonica occurred. Additionally, the positions of d and q suggest that their common ancestor shifted back to F. crenata . Host shifts to the West Eurasian beech species F. sylvatica and F. orientalis may have also occurred once or twice. The reconstruction of gall shapes suggested that the globular shape is the most probable ancestral state (Fig. 5b), although it was not highly favored at Node A (globoid, 48.1%, with other shapes ranging from 10–15%). The vein pocket shape ( a , aa ), which occupies a basal position, likely evolved once from an uncertain ancestral state. At Node B, globoid was more strongly favored (82.0%), followed by independent evolution of the horn shape five times and single occurrences of the bivalve ( e , h ; Hartigiola faggalli ) and discoid ( x ) shapes. Regarding the evolution of galling location (Fig. 5c), Node A showed nearly 50% probability for lamina (49.5%), 20.0% for vein, 18.9% for leaf margin, and < 6% for others. Although the ancestral location at the basal node was not clearly attributed, lamina was the most probable (70.7%) at Node B, with leaf margin (22.2%) and others (≥ 4%) being less likely. Of the 26 descendant taxa, the lamina is still the galling site of 17. Location shifts from the lamina to leaf margin likely occurred once or twice. The basal taxa a and aa exclusively form galls at the side vein, and midrib and/or side vein galling evolved independently several times in more recent groups. At the root node, the probability of adaxial versus abaxial galling was approximately equal (50%); however, several basal nodes exhibited a higher probability of adaxial galling (Fig. 5d). The probability of galling on the adaxial side was 63.2% at Node B and 95.6% at Node C, suggesting that adaxial gall formation was the ancestral state in Clades B and C. Multiple independent shifts to the abaxial side occurred across the phylogeny, often accompanied by a shift to the leaf margin. \received DD MMMM YYYY \acceptedDD MMMM YYYY Discussion Phylogenetic positions of Fagus -hosting cecidomyiids The ML and BI phylogenetic analyses illustrated the presence of robust divisions within the subfamily Cecidomyiinae and provided insights into the phylogenetic relationships between cecidomyiids associated with Fagus trees. The taxonomic assignment of leaf gallers collected from both F. crenata and F. japonica into tribes Dasineurini and Lasiopterini was consistent with Yukawa (1991), who speculated that the majority of cecidomyiids hosting on Japanese Fagus trees likely belong to the supertribe Lasiopteridi. Cecidomyiids a – e , g – k , m – s , and u – ac belong to the same lineage, along with the described species of Hartigiola and Mikiola hosting on F. orientalis and F. sylvatica . Although the generic level cannot be determined solely based on molecular phylogeny, leaf gallers a – e , g – k , m – s , and u – ac most likely belong to or are related to either Hartigiola or Mikiola . These putative congeners may have radiated on Fagus leaves, as discussed in the following section. The pocket vein gall-inducer t , classified within tribe Cecidomyiini, may be related to Contarinia fagi , a bud galler hosting on F. sylvatica , in addition to several other Contarinia species. Contarinia is a highly diverse genus within Cecidomyiini and currently includes over 300 described species worldwide and serves as a catch-all genus for the tribe (Gagné and Jaschhof, 2021). Although Contarinia species are associated with a wide range of plant families, including flower buds and leaves (Yukawa et al., 2005), they have not been previously reported on F. crenata or F. japonica . \received DD MMMM YYYY \acceptedDD MMMM YYYY Gall morphology in relation to the phylogenetic positions of inducers Gall morphology is often regarded as an extended phenotype of galling insects (reviewed in Stone and Schönrogge, 2003). Previous studies on aphids (Stern, 1995), thrips (Crespi and Worobey, 1998), wasps (Stone and Cook, 1998), and sawflies (Nyman et al., 2000) suggested that galling insects exert control over the key aspects of gall morphology. The diverse array of gall morphologies observed on the single host plants F. crenata and F. japonica can be attributed to factors originating from the cecidomyiids, rather than to constraints imposed by the host plant. Although galls induced by closely related taxa do not always display similar morphologies (e.g., N and O ), sister taxa often exhibit similar gall morphologies in analogous foliar locations (i.e., C and R on F. crenata ; I and P on F. crenata ; D and Q on F. crenata ; S and AB on F. japonica ; U , V , and AC on F. japonica ). In some cases, this pattern persists even after a host shift occurs, as seen with the similarity between gall morphologies on F. crenata - A and F. japonica - AA and the galls induced by Mikiola fagi on F. orientalis and F. sylvatica and Mi. bicornis -induced B on F. crenata (Ellis, 2001–2024). These similar or analogous morphologies may arise from shared behavioral characteristics, such as oviposition and/or larval feeding activity (Ferreira et al., 2019). As inferred from ancestral state reconstruction analyses, the horn-shaped morphology has evolved repeatedly from ancestral taxa that produce globular galls. Convergent evolution may involve adaptive significance such as enhancement of nutritive supply and/or defense against predators and pathogens (Joy and Crespi, 2007; Stireman et al., 2012). Feeding modes of Fagus -hosting cecidomyiids Larval feeding modes in the subfamily Cecidomyiinae are considered to have evolved from ancestral fungivory to herbivory, ambrosia, and predation. The basal supertribes Stomatosematidi and Brachyneuridi, along with the tribe Mycodiplosini within Cecidomyiidi, retain the fungivorous habit (Dorchin et al., 2019). Although some taxa in the Clinodiplosini have reverted from herbivory to fungivory, plant-feeders represent the most species-rich group, having undergone explosive diversification between 25–50 Ma (Dorchin et al., 2019). Tribes Dasineurini and Cecidomyiini are exclusively herbivorous (Gagné and Jaschhof, 2021), suggesting that a – ac (except for f , l , and t ) are herbivorous. Notably, no cecidomyiids with COI sequences identical to those of inducers found on F. crenata were found on F. japonica , even in areas in which these Fagus trees exist sympatrically. This discrepancy suggests that cecidomyiid species found on Mt. Gozen and Mt. Mito are monophagous and associate exclusively with either F. crenata or F. japonica . Biogeographical and evolutionary history of Fagus and associated gall inducers The divergence of the West Eurasian Fagus -hosting species Hartigiola annulipes (ca. 15.6 Ma) and Mikiola fagi (ca. 10.3 Ma) from the F. crenata -hosting lineage suggests that these host trees coexisted in the same geographical region during the middle Miocene period (Fig. 4, 5a). These host shifts may be closely associated with the biogeographic history of Fagus species, as indicated by the following palaeobotanical and molecular phylogenetic studies. The genus Fagus originated in the North Pacific region during the early Eocene period (ca. 50 Ma; Manchester and Dillhoff, 2004), and by the Pliocene period (5.3–2.6 Ma), Fagus had disappeared from western North America (Smiley, 1963), whereas extant species, F. grandifolia and F. mexicana , persisted in the southern and eastern regions without any association with gall midges (Fig. 6). By contrast, the derivative species migrated to the Eurasian continent across Beringia (Fig. 6; Denk and Grimm, 2009). The subgen. Engleriana diverged from the subgen. Fagus in the North Pacific region ca. 63–43 Ma (Renner et al., 2016). Unlike subgen. Fagus , the range of subgen. Engleriana seems to have remained confined to Northeast Asia (Uemura, 2002). After closure of the Turgai Strait ca. 37 Ma, the range of Fagus expanded westward during the early Oligocene period (Denk and Grimm, 2009). By the early/middle Miocene, a single species, F. castaneifolia Unger, was prevalent throughout the Northern Hemisphere, forming a homogenous biota (Denk, 2004). Coinciding with expansion of the range of F. castaneifolia , the common ancestor of the Fagus -hosting lineage in tribe Dasineurini diverged from other Fagaceae-hosting taxa (e.g., Janetia ) approximately 30.1–20.7 Ma (Fig. 4), presumably originating in Eurasia. The most ancestral host of the Fagus -hosting lineage in tribe Dasineurini might be F. castaneifolia . After the middle Miocene period, global cooling and uplift of the Himalaya-Tibetan Plateau led to aridification in Central Asia (An et al., 2001; Miao et al., 2012; Ding et al., 2022). This climatic shift likely disrupted the homogeneous distribution of Fagus and led to its retreat into East Asia and West Eurasia (Jiang et al., 2022). Such geographic fragmentation may have driven the divergence of subgen. Fagus into the Northeast Asian, Central European, and Turkish lineages. Indeed, previous molecular phylogenetic studies inferred a sister relationship between F. crenata and the West European lineages ( F. orientalis + F. sylvatica ; Denk et al., 2005; Renner et al., 2016; Cardoni et al., 2022). The ecological segregation of Fagus hosts would have isolated their associated cecidomyiids into corresponding regions, thereby driving their speciation. Notably, the earliest fossil record of a Fagus gall, found in Miocene strata in Spain, indicated the gall was formed by the extinct Mikiola pontiensis Villalta on F. castaneifolia (Diéguez et al., 1996). The Fagus -hosting lineage in tribe Dasineurini likely underwent adaptive radiation by the middle Miocene period, as indicated by the LTT plot (Fig. 4c). During the early Miocene period, Japan was separated from the Eurasian continent and transformed into an archipelago through continental uplift ca. 25–13 Ma (Jolivet et al., 1994). The opening of the Sea of Japan led to ecological segregation between the Eurasian continent and the Japanese Archipelago, where distinct niches fostered the development of a unique assemblage of Fagus flora and associated cecidomyiid fauna. In this period, Japanese beech flora included several species, such as F. stuxbergi (Nathorst) Tanai (subgen. Fagus ), F. palaeocrenata Okutsu (subgen. Fagus ), and F. palaeojaponica Suzuki (subgen. Engleriana ), which likely led to the modern F. hayatae , F. crenata , and F. japonica , respectively (Okutsu, 1955; Tanai, 1974). Although details regarding the evolutionary processes that led to modern Fagus species remain elusive (Momohara and Ito, 2023; Hara, 2024), Japanese Fagus -hosting leaf gallers likely coevolved with these relict Fagus hosts in the eastern Palearctic realm, particularly in the segregated Japanese Archipelago during the Neogene period. Possible mechanisms of speciation with/without host shifts The diversification of Fagus -hosting gall inducers within tribe Dasineurini appears to have been driven by host and location shifts on the leaves, including both the adaxial and abaxial surfaces. The lineage of cecidomyiids associated with Fagus seems to have originally hosted on F. castaneifolia . During radiation, these gall inducers likely shifted hosts between the F. crenata and F. japonica lineages in Japan, where these two lineages coexisted sympatrically during the Miocene period. Adaptive radiation on the single host species F. crenata gave rise to taxa such as b , g , i – k , and m – p . The common ancestor of Node C shown in Fig. 4a likely originated through a host shift to a non-natal congener F. japonica and subsequently radiated on this host, giving rise to taxa s , u – w , y , ab , and ac . Some taxa, such as d and q , appear to have shifted back to F. crenata from F. japonica . Additionally, several nested taxa, such as x and z , which are associated with F. japonica , are included in a clade predominantly hosted by F. crenata . The occurrence of these nested taxa may also have been accompanied by host plant shifts in regions in which both species coexist sympatrically. Host shifts to non-natal species can result from opportunistic ovipositional mistakes, a phenomenon observed in various taxa of herbivorous insects, including many Cecidomyiidae (Price, 2005). Through host exchange experiments, Yukawa et al. (2021) demonstrated that the monophagous gall midge Daphnephila machilicola , which natively induces leaf galls on Machilus thunbergii , lays its eggs on the leaves of a congener, Machilus japonica , resulting in the formation of galls with different morphology. Host plant shifts typically entail adaptations to distinct plant characteristics related to morphology, chemistry, and phenology (Jaenike, 1989, 1990; Becerra and Venable, 1999; Cook et al., 2002). Given that congeners often share various traits, such as chemical profiles (e.g., leaf volatiles; Boddum et al., 2018; Molnár et al., 2018) and morphological characteristics (e.g., leaf thickness) that potentially affect host-choice behavior, host shifts are expected to occur readily. Speciation can occur even in the absence of host plant shifts; within-host speciation may instead arise from shifts in the feeding niche, such as a shift from leaf to stem (Joy and Crespi, 2007). Developmental discrepancies among plant organs involve potential phenological separation (Condon and Steck 1997; Després et al., 2002; Ferdy et al., 2002). However, the Fagus -hosting taxa diverged exclusively on the leaves without any organ shifts. Some taxa may have diverged through fine-scale location shifts within leaves. For instance, the bivalve-shaped gall inducers e and h seem to be in the process of speciation through a shift in galling location between the adaxial and abaxial sides of the leaf (Sato and Yukawa, 2004; Mishima et al., 2014). By contrast, no clear differences in galling location were observed in potential sister taxa (i.e., n and o [lamina], i and p [lamina], s and ab [leaf margin], and d and q [leaf margin]). One possible driver of inducer divergence is adaptation to different age classes of a single host plant species. Zhang et al. (2015) suggested that adaptation to different ages (e.g., seedling versus mature tree) of host elm trees ( Ulmus pumila L.) can lead to the divergence of sympatric sister species of the elm leaf beetles Pyrrhalta maculicollis and Pyrrhalta aenescens , likely due to differences in the chemical profiles of the leaf surface at different tree ages. A gradual shift in the allocation of metabolic energy from the roots in seedlings to the shoots in mature trees was demonstrated in F. crenata (Kurosawa et al., 2023). Such ontogenetic changes in host plant leaves could impact the fitness of phytophagous cecidomyiids via age-specific differences in nutritional content of the leaves. Concluding remarks This study provides the first molecular phylogeny of cecidomyiids hosting on F. crenata and F. japonica . With few exceptions, most Fagus -hosting species share a common lineage, likely belonging to or closely related to Hartigiola or Mikiola within the tribe Dasineurini, underscoring their herbivorous nature. These cecidomyiids appear to have undergone adaptive radiation on relict Fagus species in Japan since the Miocene epoch (ca. ~21 Ma), coinciding with the separation of the Japanese Archipelago from the Eurasian continent and the development of the Asia monsoonal climate. 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Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 38, 3022–3027. 87. Tanai T (1974) Evolutionary trend of the genus Fagus around the northern Pacific basin. Birbal Sahni Inst Palaeobot Spec Publ 1, 62–83. 88. Tanai T (1995) Fagacean leaves from the Paleogene of Hokkaido, Japan. Bull Natl Sci Mus C 21, 71–102. 89. Tanaka N, Matsui T (2007) PRDB: Phytosociological Relevé Database, Forestry and Forest Products Research Institute. http://www.ffpri.affrc.go.jp/labs/prdb/index.html 90. Uemura K (2002) “Asiatic elements” in the Cenozoic floras and their phytogeographic implication. Bunrui 2, 1–7. 91. Wen J, Nie ZL, Ickert-Bond SM (2016) Intercontinental disjunctions between eastern Asia and western North America in vascular plants highlight the biogeographic importance of the Bering land bridge from late Cretaceous to Neogene. J Syst Evol 54, 469–490. 92. Yukawa J (1991) Gall midges producing galls on Fagus crenata and Fagus japonica . Forest Pests 40, 198–205. 93. Yukawa J, Masuda H (1996) Insect and Mite Galls of Japan in Colors. Zenkoku Noson Kyoiku Kyokai, Tokyo. 94. Yukawa J, Tokuda M, Sato S, Ganaha-Kikumura T, Uechi N (2021) Speciation. In: Yukawa J, Tokuda M (eds) Biology of Gall Midges, Entomology Monographs. Springer, Singapore. 95. Yukawa J, Uechi N, Tokuda M, Sato S (2005) Radiation of gall midges (Diptera: Cecidomyiidae) in Japan. Basic Appl Ecol 6, 453–461. Table 1 Types of galls on the leaves of Japanese Fagus trees ( F. crenata and F. japonica ), gall inducers, gall sites, and month of collection. F. crenata 1 A Pocket gall along the side vein Buna-hamyaku-kobufushi a undescribed N May, Sep 2 B Conical gall Buna-hasuji-togaritamafushi b Mikiola bicornis Sato & Yukawa† G, N May, Sep 3 - Buna-hasuji-dongurifushi - Mikiola glandaria Sato & Yukawa† - - 4 C Globular gall on the leaf edge Buna-haberi-tamafushi c undescribed G, N May, Sep 5 D Horn-shaped gall on the rolled leaf edge Buna-haberi-hosofushi d undescribed A, G Apr, May 6 E Bivalve-shaped gall (abaxial side) Buna-haura-kaigarafushi e Hartigiola faggalli (Monzen)‡,§ M Oct 7 - Buna-haura-kefushi - undescribed - - 8 - Buna-haura-kobufushi - undescribed - - 9 F Hairy gall at the intersection of veins Buna-haura-kometsubufushi f Janetiella infrafoli Monzen† M, N Sep, Oct 10 - Buna-haura-hishigatafushi - undescribed - - 11 G Globular gall with red hair Buna-ha-akagetamafushi g undescribed A, G, K, N Apr, May 12 - Buna-ha-ootsunofushi - undescribed - - 13 H Bivalve-shaped gall (adaxial side) Buna-ha-kaigarafushi h Hartigiola faggalli (Monzen)‡,§ M, N Sep, Oct 14 I Fang-shaped gall Buna-ha-kibatsunofushi i undescribed M Oct 15 - Buna-ha-ketamafushi - undescribed - - 16 J Small horn-shaped gall Buna-ha-kotsunofushi j undescribed A, K May 17 K Globular gall with smooth surface Buna-ha-tamafushi k undescribed A, K, N May, Sep 18 - Buna-ha-tsunofushi - undescribed - - 19 - Buna-ha-togetsunofushi - undescribed - - 20 L Horn-shaped gall Buna-ha-nagatsunofushi l undescribed N Sep 21 M Nodular gall Buna-ha-fukurefushi m undescribed K, G, N May, Sep 22 N Thick horn-shaped gall Buna-ha-futotsunofushi n undescribed A, G, K May 23 - Buna-ha-hosotogaritamafushi - Phegomyia tokunagai Sasakawa & Koyama† - - 24 O Globular gall with a constricted base Buna-ha-magetamafushi o undescribed A, K, G, N Apr, May 25 - Buna-ha-marutamafushi - undescribed - - 26 P Horn-shaped gall with a rounded tip Buna-ha-marutsunofushi p undescribed M, N Sep, Oct 27 Q Horn-shaped gall on the leaf edge* Buna-haberi-tsunofushi q undescribed A May 28 R Nodular gall on the leaf edge* Buna-haberi-kobufushi r undescribed A, K May F. japonica 29 S Globular gall on the leaf edge Inubuna-haberi-tamafushi s undescribed G, M Jun, Sep 30 - Inubuna-haberi-hosofushi - undescribed - - 31 T Pocket gall along the side vein Inubuna-hamyaku-kobufushi t undescribed G Apr, May, Jun 32 U Globular gall with red hair Inubuna-ha-akagetamafushi u undescribed G May 33 V Cylindrical gall with white hair Inubuna-ha-ketsunofushi v Hartigiola annulipes (Hartig)‡ G May, Jun, Aug, Sep 34 W Horn-shaped gall with a rounded tip Inubuna-ha-tsunofushi w undescribed G May, Jun 35 X Button-shaped gall Inubuna-ha-botanfushi x undescribed G Aug 36 Y Globular gall with smooth surface Inubuna-ha-marutamafushi y undescribed G, M May, Jun, Sep 37 Z Fang-shaped gall* Inubuna-ha-kibatsunofushi z undescribed G Apr, May 38 AA Elongated pocket gall along the side vein* Inubuna-hamyaku-nagakobufushi aa undescribed G Apr 39 AB Onion-shaped gall on the leaf edge* Inubuna-haberi-giboshifushi ab undescribed M Sep 40 AC Ovoid gall with a hairy surface* Inubuna-ha-kekotamafushi ac undescribed M Sep Collection sites are abbreviated as follows: A, Mt. Amagi; G, Mt. Gozen; K, Kanyudo; M, Mt. Mito; N, Mt. Nabewari. *Types newly reported in this study. †No sequence deposited in any public database. ‡ COI sequence available in GenBank (Note: the COI sequence identity between H. annulipes [MN191300.1] collected from a gall on F. sylvatica in Germany and v was 90.1% [547/607 bp]). §The gall midges inducing these two types (morphotypes) are currently regarded as the same species (Sato and Yukawa, 2004). Figure legends Fig. 1 Geographical distribution of F. crenata and F. japonica and collection sites . Reproduced from Phytosociological Relevé Database (Tanaka and Matsui, 2007). The map was generated using the R (R Core Team, 2024) ‘maps’ package version 3.3.0 [Original S code by Richard A. Becker, Allan R. Wilks. R version by Ray Brownrigg. Enhancements by Thomas P Minka and Alex Deckmyn (2018) maps: Draw Geographical Maps. https://CRAN.R-project.org/package=maps]. Fig. 2 Types of galls found on the leaves of F. crenata and F. japonica in this study. Leaf galls (A–R) on F. crenata and (S–AC) on F. japonica (see Table 1 for detailed descriptions of each gall). ( U ) The red gall on the right is U , and the white one on the left is V . View from the adaxial side (left) and abaxial side (right) in each panel. Fig. 3 ML phylogenetic tree of cecidomyiids galling on F. crenata and F. japonica and known taxa of Cecidomyiinae based on concatenated COI , 16S , ITS1 , 28S , and CAD sequences. ( a ) Whole tree and ( b ) pruned subtree (Clade I). Hyphenated values indicate individual specimens collected from different galls. Nodal support indicated by UFBoot values (1,000 replications) and SH-aLRT values (1,000 replications). Tribes: Bra, Brachineuridi; Myc, Mycodiplosini; Sto, Stomatosematidi. Strips around the tree indicate the host Fagus species, in the order F. crenata , F. japonica , F. sylvatica , and F. orientalis from the inside. Fig. 4 BI time-calibrated phylogenetic tree of cecidomyiids galling on F. crenata and F. japonica and known taxa of Cecidomyiinae based on concatenated COI , 16S , ITS1 , 28S , and CAD sequences. Support for branches is indicated by PP values above branches. Yellow circles indicate nodes used as calibrated priors in divergence time estimation in BEAST2. Nodes are at mean divergence times. The time scale is Ma. ( a ) Whole tree and ( b ) pruned subtree (Clade I). ( c ) LTT plot of the phylogeny of the Fagus -hosting lineage (Clade I). The natural logarithm of the number of lineages versus time since the origin of the lineage (Ma) is shown with the γ statistic and corresponding p value. Fig. 5 Ancestral state reconstructions for Fagus -hosting taxa in Dasineurini using ML and stored Mk1 models. Areas of pie charts indicate relative support for ancestral states. *Significant support for ancestral state reconstruction at the node. ( a ) Host plant species. ( b ) Gall shape. ( c ) Galling location on the leaf. ( d ) Galling side of the leaf. Fig. 6 Hypothetical migratory routes of Fagus and associated gall inducers. Species distributions of major accepted Fagus species (Tanaka and Matsui, 2007; Rodríguez-Ramírez et al., 2013; EUFORGEN, 2024; POWO, 2024) are shown by circles. Symbols + and × indicate the distribution of Fagus -hosting gall midges currently reported in Europe (i.e., Hartigiola annulipes and Mikiola fagi , respectively; GBIF Secretariat, 2023). a – ac , Japanese Fagus -hosting gall midges (see Table 1). Solid arrows indicate the migratory direction of ancestral Fagus species. Dashed arrows indicate routes in which migration likely did not occur; or, if migration occurred, it was likely associated with a range reduction due to factors such as the aridification of Central Asia. Mid-Mio., middle Miocene period; Plio., Pliocene period. Author contributions Shinnosuke Mori: conceptualization (supporting); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); software (lead); visualization (lead); writing - original draft preparation (lead); writing - review & editing (equal). Yugo Dhakhwa: investigation (supporting). Makoto Tokuda: methodology (supporting); writing - review & editing (equal). Yoko Saikawa: project administration (lead); conceptualization (lead); funding acquisition; writing - original draft preparation (supporting); writing - review & editing (equal). Acknowledgements The authors would like to thank Dr. Shinsuke Sato (Horticultural Institute, Agricultural Center, Ibaraki, Japan) for his advice on the classification of gall types, and FORTE Science Communications (https://www.forte-science.co.jp/) for English language editing. Funding information This study was supported by Amano Institute of Technology to YS. Conflict of interest statement The authors declare no conflict of interest. Data availability statement Sequence data in this study are available on the NCBI GenBank accession PQ838091–PQ838157. \received DD MMMM YYYY \acceptedDD MMMM YYYY ORCID Shinnosuke Mori: 0000-0003-0073-8876 Makoto Tokuda: 0000-0001-7162-0715 Yoko Saikawa: 0000-0002-4571-1821 Information & Authors Information Version history V1 Version 1 28 January 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Ecology and Evolution Keywords comparative ecosystem evolutionary ecology invertebrate molecular genetics sequencing terrestrial Authors Affiliations Shinnosuke Mori 0000-0003-0073-8876 [email protected] Keio University Faculty of Science and Technology Graduate School of Science and Technology View all articles by this author Yugo Dhakhwa Keio University Faculty of Science and Technology Graduate School of Science and Technology View all articles by this author Makoto Tokuda Saga University View all articles by this author Yoko Saikawa Keio University Faculty of Science and Technology Graduate School of Science and Technology View all articles by this author Metrics & Citations Metrics Article Usage 656 views 244 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shinnosuke Mori, Yugo Dhakhwa, Makoto Tokuda, et al. Biodiversity and evolutionary dynamics of gall midges hosting on Japanese beech trees. Authorea . 28 January 2025. 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