Megaloptera: Corydalidae)

Systematic Entomology (2021), 46, 124–139 DOI: 10.1111/syen.12452

Origin and spatio-temporal diversification of a fishfly lineage endemic to the islands of East Asia (Megaloptera: Corydalidae)

, , , YUNLAN JIANG1 *, FAN YANG1 2 *, LU YUE1, FUMIO HAYASHI3, DING YANG1 andXINGYUE LIU1

1Department of Entomology, China Agricultural University, Beijing, China, 2Capital Airport Customs, Beijing Customs, Beijing, China and 3Department of Biology, Tokyo Metropolitan University, Tokyo, Japan

Abstract. Chauliodinae (fishflies), with their low capacity of long-distance dispersal represent a suitable model insect group to investigate the biogeographical history. The genus Parachauliodes van der Weele, including the herein synonymized genus Sinochauliodes Liu & Yang, is endemic to East Asia. Here, we reconstruct the interspecific phylogeny of Parachauliodes based on mitochondrial DNA sequence data. Sinochauliodes syn.n. was recovered with a group of Parachauliodes species and not the sister group; we therefore treat it as the junior synonym of Parachauliodes. Species delimitation was performed combining the molecular identification with morphological evidence, with Parachauliodes inopinatus syn.n. treated as the junior synonym of Parachauliodes asahinai. The spatio-temporal divergence pattern of Parachauliodes indicates that the genus might have originated from Eurasian continent no later than the early Miocene and the initial divergence within genus was likely to be correlated to the split of southwestern Japanese Islands from the continent. There likely was a southward dispersal in the Parachauliodes japonicus clade from southwestern Japan via the Ryukyus to Taiwan by the end of the Miocene. The present species diversity of the insular lineage of Parachauliodes was possibly shaped by island isolations and sympatric distribution.

Introduction Afrotropical and Neotropical realms. Fishfly genera are largely endemic to the particular zoogeographic regions and most Fishflies (Corydalidae: Chauliodinae) represent one of the major species are in turn distributed in a much narrower area. Fish- groups of the holometabolous order Megaloptera. They are fly larvae are obligate aquatics, generally living in streams and considered to be an archaic insect group with many extant ponds, and they have limited flight capacity for a long-distance species considered ‘living fossils’ since they originated no later dispersal though the adults can fly, therefore their speciation than the Middle Jurassic based on the fossil evidence and dis- as well as population structure have relatively strong depen- play remarkably conserved adult and larval characters between dence on geographical isolation (Heilveil & Berlocher, 2006; Mesozoic-aged and recent species (Liu et al., 2012). Currently, Liu et al., 2010, 2012, 2013). Nevertheless, there are some pre- there are ca. 140 species in 18 genera of extant fishflies world- dicted ancient long-distance dispersal events in ancestral fish- wide. Although fishflies occur in all zoogeographical regions, flies of certain modern lineages from Gondwana to Laurasia, they show a remarkably discontinuous distribution pattern, which might be caused by the northward rafting of the Indian being absent in the western Palaearctic realm and much of the subcontinent (Liu et al., 2012). Thus, standing as a model insect Correspondence: Xingyue Liu, Department of Entomology, group to investigate the correlation between the distribution pat- China Agricultural University, Beijing 100193, China. E-mail: tern and major geological events, the historical biogeography of [email protected] fishflies is of considerable interest. In a proposed scenario on the historical biogeography of ∗These authors are co-first author. Chauliodinae (Liu et al., 2012), the entire subfamily was

C D

Fig. 1. Living adults of Parachauliodes spp. (A) Parachauliodes fujianensis (Yang & Yang), male, from Fujian, China (photograph by Yuchen Zheng); (B) Parachauliodes squalidus (Liu & Yang), male, from Fujian, China (photograph by Yuchen Zheng); (C) Parachauliodes buchi Navás, female, from Anhui, China (photograph by Yuchen Zheng); (D) Parachauliodes japonicus (McLachlan), female, from Kanagawa, Japan (photograph by Hisanori Okamiya); (E) Parachauliodes continentalis van der Weele, female, from Fukui, Japan (photograph by Fumio Hayashi); (F) Parachauliodes rastellus Shimonoya, male, from Fukuoka, Japan (photograph by Fumio Hayashi). Species in panels A, B and C formerly belonged to the presently synonymized Sinochauliodes. [Colour figure can be viewed at wileyonlinelibrary.com]. supposed with a pangaean distribution before the Middle Juras- as the sister of another East Asian endemic Sinochauliodes sic and diversification of some fishfly genera with a gondwanan Liu & Yang (Figs 1, 4; Fig S1) in a morphology-based phy- origin might have be affected by the subsequent breakup of Pan- logeny of Chauliodinae (Liu & Yang, 2006; Liu et al., 2012, gaea. Geographical isolation as a mode of speciation is consid- 2016). These two genera share a similarly shaped male ter- ered to be frequent in fishflies, such as through island forma- gum 9, although Sinochauliodes can be distinguished from tion, fragmentation of favourable habitats, etc. (Liu et al., 2008, Parachauliodes by the sexually dimorphic antennae (pecti- 2010, 2013). However, although the aforementioned hypotheses nate in male but serrate in female) and the male ectoprocts on the historical biogeography of fishflies were proposed based not being bilobed. Still, many species of these two gen- on reconstructed phylogenies, most results were solely obtained era are very similar in wing marking patterns and genitalic from the morphological data. Further testing of the previous morphology. Parachauliodes has an insular distribution while hypotheses is essential with a DNA sequence-based phylogeny Sinochauliodes is distributed on the mainland. Most species of and estimates on the time-line of divergence and ancestral Parachauliodes are confined to the island arc at the western edge distributions. of the Pacific Ocean, including Taiwan, Ryukyus and Japanese Parachauliodes van der Weele is endemic to East Asia, with archipelago, although Parachauliodes asahinai Liu, Hayashi & nine species described (Liu et al., 2008; Hayashi, 2018; Shi- Yang also occurs from the Korean peninsula. Conversely, all monoya, 2019). The adults are characterized by serrate anten- species of Sinochauliodes are distributed in Mainland China. nae in both sexes (except Parachauliodes rastellus Shimonoya Parachauliodes buchi Navás from Zhejiang Province, eastern with pectinate male antennae), posteroventrally expanded male China actually belongs to Sinochauliodes and is a senior syn- tergum 9 enveloping the gonocoxites 10, and bilobed male ecto- onym of Sinochauliodes griseus Yang & Yang, based on mor- procts (Figs 1, 2, 3; Fig S2). Parachauliodes was recovered phological evidence.

Fig. 2. Male genitalia of insular species of Parachauliodes in ventral view. e: ectoproct, gx: gonocoxite, T: tergum. Scale bar: 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

A preliminary hypothesis on the historical biogeography of Material and methods Parachauliodes and Sinochauliodes was proposed by Liu & Yang (2006) and Liu et al. (2008). The assumed scenario of Taxon sampling divergence was outlined as follows. Firstly, the ancestral species of Parachauliodes might have migrated via a land bridge from All species of Parachauliodes and Sinochauliodes hitherto the eastern Chinese mainland to Taiwan, Ryukyus, and Japanese described were included. As Sinochauliodes is synonymized archipelago during the late Miocene and Pliocene (∼10–5 with Parachauliodes based on the present result, to avoid Ma) after divergence from Sinochauliodes. Second, the deep ambiguity, the name Sinochauliodes is placed in quotation divergence between two major clades of Parachauliodes (i.e. a marks in the Results and Discussion where necessary. Eight clade of P. asahinai + Parachauliodes continentalis and a clade species of Megaloptera were selected as outgroups, including including the remaining Parachauliodes species) might be due Sialis hamata Ross (Sialidae), Protohermes concolorus Yang & to the formation of the Tokara Strait. Finally, dispersal from Yang (Corydalidae: Corydalinae), Dysmicohermes ingens Chan- western Japan to the Korean peninsula might have taken place in dler (Corydalidae: Chauliodinae), Protochauliodes biconicus P. asahinai via a land connection during the Pleistocene glacial Kimmins (Corydalidae: Chauliodinae), Archichauliodes decep- period. tor Kimmins (Corydalidae: Chauliodinae), Chauliodes pectini- Here, we present the first DNA-based phylogeny of cornis (Linnaeus) (Corydalidae: Chauliodinae), Neochauliodes Parachauliodes and Sinochauliodes reconstructed from the bowringi (McLachlan) (Corydalidae: Chauliodinae) and Nigro- mitochondrial DNA sequence data. We used an integrative nia serricornis (Say) (Corydalidae: Chauliodinae). Among approach to identify species, including morphology and mul- these outgroup taxa, Nigronia and Neochauliodes were previ- tiple methods for molecular identification, which clarified the ously recovered to be closely related to Parachauliodes and present taxonomic problems on these two genera, especially Sinochauliodes (Liu & Yang, 2006; Liu et al., 2012), while the synonymy of Sinochauliodes with Parachauliodes. We esti- the other taxa belong to various lineages of the monophyletic mated the temporal and spatial divergence of the Parachauliodes group comprising the above four fishfly genera (Liu et al., 2016). species and present a historical framework for the phylogeny The morphological identifications prior to DNA extraction were and biogeography of these East Asian endemic fishflies. made by XL and FH. In total, there were 90 specimens of

Fig. 3. Male genitalia of insular species of Parachauliodes in ventral view. e: ectoproct, gx: gonocoxite, T: tergum. Scale bar: 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com].

Parachauliodes and 17 specimens of Sinochauliodes.Speci- sampling is the most comprehensive one for the phylogenetic mens of Parachauliodes were collected from islands or areas analysis in terms of both taxonomy and distribution. Moreover, in Taiwan, Ryukyus, the Japanese archipelago and the Korean we examined a large number of additional specimens of these peninsula, covering almost the entire distribution of the genus. two genera for better understanding the morphological varia- Specimens of Sinochauliodes were collected from six provinces tions of species, to aid in accurate species determination. in central and southern China, including Zhejiang, Fujian, The specimens used in this study are deposited in 19 institu- Guangdong, Guangxi, Henan and Shaanxi (Sinochauliodes is tions (see Table S1 for the information of voucher specimens first recorded from the latter two provinces). Thus, the present used in the molecular phylogenetic study, and Table S4 for the

Fig. 4. Male genitalia of continental species of Parachauliodes in ventral view. e: ectoproct, gx: gonocoxite, T: tergum. Scale bar: 1.0 mm. [Colour figure can be viewed at wileyonlinelibrary.com]. deposition of other specimens herein examined). Male genitalia in this study are presented in Table S2. The amplification are used as the key character for morphological identification of conditions of PCRs are: 95∘C for 30s, 40 cycles of denaturation species. Genitalia preparations were made by clearing the apex at 95∘C for 10s, annealing at 43–60∘C (depending on the primer of the abdomen in a cold, saturated potassium hydroxide (KOH) pair used) for 50s, elongation at 65∘C for 1 kb/min (depending solution for 8–10 h. After rinsing with acetic acid and water, the on the size of amplicon), and the final elongation step at apex of the abdomen was transferred to glycerin for further dis- 65∘C for 10 min. The quality of PCR products was assessed section and examination. The genitalic terminology follows Liu by electrophoresis in agarose gel that is stained with Gold et al. (2016). View. All PCR products were sequenced in both directions using the BigDye Terminator Sequencing Kit (Applied Bio Systems, San DNA extraction, amplification and sequencing Francisco, CA, U.S.A.) and the ABI 3730XL Genetic Analyser (PE Applied Biosystems) with two vector-specific primers and Genomic DNA was extracted from the mesothoracic muscle internal primers for primer walking. of adults or larvae using TIANamp Genomic Kit (TIANGEN, Beijing, China). Complete mitochondrial (mt) genome or partial sequences of mitochondrial genes (COI, ND2 and 16S rRNA) were amplified and sequenced. Two fragments of COI termed Sequence alignment and phylogenetic analysis as COI1 and COI2 were sequenced, the latter of which is used as the standard DNA barcode of insects (Folmer et al., 1994; Sequence assembly was completed using ContigExpress. Hebert et al., 2003). The mt genomes of eight ingroup species, Mitochondrial genome annotation followed Cameron (2014). that is, P. asahinai Liu, Hayashi & Yang, P. continentalis van The complete mt genomes and gene fragments herein sequenced der Weele, P. rastellus Shimonoya, Parachauliodes yanbaru are deposited in GenBank (Table S1). The ribosomal RNA genes Asahina, Parachauliodes japonicus (McLachlan), S. griseus were adjusted using G-Blocks Server (http://molevol.cmima Yang & Yang, and Sinochauliodes squalidus (Liu & Yang) were .csic.es/castresaana/Gblocks_server.html). The protein coding generated by amplification of overlapping PCR fragments. Most genes were aligned based on the amino acid translation using gene fragments were amplified using universal primers (Folmer Clustal X (Thompson et al., 1997) incorporated in MEGA 7.0 et al., 1994; Simon et al., 1994, 2006; Yu et al., 2015), while the (Kumar et al., 2016). Individual gene regions were concatenated remaining fragments were amplified using specifically designed excluding the stop codons using SequenceMatrix v1.7.8 (Vaidya primers based on previous sequences. Primer sequences used et al., 2010).

Fig. 5. Phylogenetic tree of Parachauliodes based on the concatenated matrix. Numbers on nodes are Bayesian posterior probabilities (PB)/Bayesian posterior probabilities (BI)/maximum likelihood bootstrap values (ML). Nodes where the posterior probabilities are equal or greater than 0.95 and the likelihood bootstrap values no less than 95 are marked by asterisk, while nodes without posterior probabilities or likelihood bootstrap values are inconsistent in the topologies from the three analyses. Results of molecular species delimitation based on Automatic Barcoding Gap Discovery (ABGD), Poisson tree process maximum likelihood (PTP_ML) and Bayesian (PTP_sh) methods are annotated on the right side of the tree. The ingroup names are given as the former names before the present taxonomic treatments. [Colour figure can be viewed at wileyonlinelibrary.com].

We estimated the best partitioning scheme and model for our prevalent features of mitogenomes (Song et al., 2010; Talavera dataset with PartitionFinder 2 (Lanfear et al., 2016). The con- & Vila, 2011; Li et al., 2015). Therefore, an additional Bayesian catenated matrix was partitioned into two subsets, and GTR+G analysis (PB) was conducted in PhyloBayes under the hetero- was selected as the best-fitting model for the present phylo- geneous model CAT-GTR, which contained four independent genetic analysis (Table S3). Phylogenies were inferred using chains. The run was stopped after the likelihood of the sam- Bayesian inference (BI) and Maximum-likelihood (ML) meth- pled trees had stabilised and chains had satisfactorily converged ods. The BI analysis was conducted using MrBayes 3.2.6 (Ron- (maxdiff <0.3). The ML analysis was performed using RaxML quist et al., 2012) and PhyloBayes (Lartillot et al., 2009) via 8.0.2 (Stamatakis, 2014) via the CIPRES web portal with 200 the CIPRES web portal (http://www.phylo.org). The BI anal- bootstrap replicates using the rapid bootstrap feature. ysis in MrBayes contains four simultaneous Markov chain Monte Carlo (MCMC) runs of 2 million generations. Trees were sampled every 1000 generation and the burn-in frac- Species delimitation tion set to 25%. Analyses terminated when the standard devi- ation of clade frequencies fell below 0.01, which indicates We delimited species using a total evidence approach combin- that stationarity had been reached. A majority-rule consensus ing morphological and molecular evidence. We used two inde- tree was calculated with posterior probabilities (PPs) for each pendent species delimitation approaches based on molecular node. Recent publications on phylogenetic studies of arthro- data, that is, the Automatic Barcoding Gap Discovery (ABGD) pods based on concatenated sequences of mt genomes indicated and the Poisson tree processes (PTP) method, to identify the that heterogeneous models were better than homogenous mod- molecular operational taxonomic unit (MOTU), which has a els at accounting for homoplasy, which is resulted from two similar extent as traditional species (Blaxter, 2004). ABGD is an

© 2020 The Royal Entomological Society, Systematic Entomology, 46, 124–139 130 Y. Jiang et al. iterative method to sort sequences into genetic clusters or can- whether the MCMCs had reached a stationary distribution by didate species based solely on pairwise distances without an a visual inspection of plotted posterior estimates. We further priori species hypothesis (Puillandre et al., 2012). The calcu- checked whether the effective sample size (ESS) was greater lations were conducted on the ABGD web-interface based on than 200 for most important parameters (e.g. posterior and the dataset of COI2. Prior maximum divergence of intraspe- tree likelihood). We resampled the tree files of both runs with cific diversity P was investigated under a Kimura’s 2-parameter a frequency of 10 000 and combined them in LogCombiner model (K2P) (Kimura, 1980), and the parameters were set as (BEAST package), of which the first 25% were removed as follows: Pmin = 0.001, Pmax = 0.1, Steps = 50, X (relative burn-in. We summarized the subsampled trees in a maximum gap width) = 0.5, Nb bins (for distance distribution) = 20. The clade credibility tree with common ancestor heights as node PTP analysis was conducted using the bPTP web server (Zhang heights using TreeAnnotator (BEAST package). The mean et al., 2013) based on the trees generated from RaxML. The anal- heights and 95% highest probability density (95% HPD) values yses ran for 500 000 MCMC generations, thinning set to 100 and were displayed in FigTree v1.4.4 (Rambaut, 2018). burn-in at 25%. The probability of clusters representing putative species was calculated in two ways: (i) the Bayesian solution (PTP_sh), which considers the frequency of the nodes across the Ancestral area reconstruction sampling; and (ii) the maximum likelihood solution (PTP_ML), which considers the most likely solution among the sampling We defined the following eight biogeographical areas of (Zhang et al., 2013). Parachauliodes and Sinochauliodes according to their known distributions: A, central and southern parts of Mainland China; B, Honshu (including Oki Islands) and Shikoku; C, Taiwan; Divergence time estimation D, Ishigaki-jima and Iriomote-jima Islands; E, Okinawa Island; F, Amami Islands (Amami-oshima Island, Tokunoshima Island, We estimated the divergence time using the arthropod mito- Kakeroma Island, etc.); G, Kyushu and some affiliated islands chondrial molecular clock (2.3% Ma−1; Brower, 1994) and fos- (i.e. Tsushima Island, Fukue-jima Island, Amakusa Islands, sil calibrations. Then we compared the fit of our results to Yaku-shima Island, Iki Island, etc.); H, Korean peninsula. the geological record, and evaluated support for feasibility of Ancestral ranges were reconstructed using RASP (Reconstruct either of these temporal frameworks. Divergence times were Ancestral State in Phylogenies) 4.0 beta, build 20 180 508 (Yu estimated using BEAST 2.4.7 (Bouckaert et al., 2014) via the et al., 2015) with the BioGeoBEARS package (Matzke, 2013). CIPRES web portal. The mt genome alignment and the topol- The program implements a parameter describing founder-event ogy obtained from PhyloBayes were used in the divergence time speciation (+ j) likely to be important in an oceanic setting estimation. The partition scheme for the nucleotide alignment (Matzke, 2014) and allows the comparison of different models defined in BEAUti (BEAST package) was estimated using Par- in a statistical framework, where DIVALIKE + j was estimated titionFinder 2, which is same to that used in the above phylo- as the best model. We inputted the specific distribution for each genetic analyses (Table S3). A Yule model was implemented of these ingroup taxa. The maximum number of ancestral areas as the tree prior with fixed rate calibration priors (birth-death was limited to two. To account for phylogenetic uncertainty, process of speciation in fossil calibrations method), and the 5001 post-burn-in trees yielded from the BEAST analyses were substitution rate set to 0.0115, with the time-tree and clock included with outgroup taxa removed. model linked across partitions. An uncorrelated, relaxed molecular clock model (Drummond & Rambaut, 2007), and an expo- nential prior were used in the analysis. For the fossil calibrations Results method, the birth-death process of speciation was implemented and we used three minimum age constraints based on the follow- Species delimitation ing published fossil records as calibrations: (i) the oldest known fossil of Sialidae, Dobbertinia reticulata Handlirsch (from the Results of species delimitation from the ABGD and Lower Jurassic of Dobbertin, Germany) as the stem of Corydali- PTP approaches were summarized in Fig. 5. Considering dae + Sialidae, with a minimum age of 185 Ma (Ansorge, 2001); Parachauliodes, the ABGD analysis clustered the taxa of eight (ii) Jurochauliodes ponomarenkoi Wang & Zhang (from the morphological species of this genus into 17 MOTUs. The Middle Jurassic of Inner Mongolia, China) for the split of PTP analyses, however, resulted in more MOTUs (26 and 24) Chauliodinae and Corydalinae, with a minimum age of 165 under the best-fit ML search (PTP_ML) and simple heuristic Ma (Liu et al., 2012); (iii) Chauliodes carsteni Wichard, 2003 search (PTP_sh). Similarly, in ‘Sinochauliodes’, the ABGD (from the middle Eocene Baltic amber) for the split between analysis clustered the taxa of four morphological species of this Chauliodes and the clade including Neochauliodes, Nigronia, genus into seven MOTUs, while the PTP analyses resulted in Parachauliodes and Sinochauliodes, with a minimum age of 44 10 MOTUs under PTP_ML and PTP_sh. Thus, the molecular Ma (Wichard, 2003). species delimitation in general implied that there may be many Two independent MCMC runs were conducted with 500–600 cryptic species in these two genera. million generations, and trees were sampled every 1000 In Parachauliodes, there are no subdivisions, respectively, generations. We used Tracer 1.7 (Rambaut et al., 2018) to check within P. asahinai, Parachauliodes inopinatus, P. continentalis

Fig. 6. Chronogram with estimated divergence times based on fixed rate calibration amongst Parachauliodes species, with ancestral areas reconstructed from DIVALIKE + j in BioGeoBEARS. Letters at nodes and colour for squares ahead of taxon name correspond to the areas of endemism of Parachauliodes in the map left: A, central and southern parts of Mainland China; B, Honshu (including Oki Islands) and Shikoku; C, Taiwan; D, Ishigaki-jima and Iriomote-jima Islands; E, Okinawa Island; F, Amami Islands (Amami-oshima Island, Tokunoshima Island, Kakeroma Island, etc.); G, Kyushu and some affiliated islands (i.e. Tsushima Island, Fukue-jima Island, Amakusa Islands, Yaku-shima Island, Iki Island, etc.); H,Korean peninsula. Dispersal and vicariant events are indicated by red stars and green circles, respectively. Numbers on nodes are consistent with those in Table S6. Maps at bottom denote inferred major dispersal (indicated by arrows) and vicariance (indicated by dash lines) of insular Parachauliodes species along geological times. Geological reconstructions based on Otofuji et al. (1985), Ota (1998), and Tojo et al. (2017).[Colour figure can be viewed at wileyonlinelibrary.com]. and Parachauliodes niger. However, despite the distinct genetic samples from Okinawa Island represent a MOTU that is different distance between P. asahinai from Korea and P. inopinatus from from a group of all samples from Amami Islands in all analyses, central Honshu, Japan, these two species possess identical mor- and there are two or three subdivided MOTUs within the sam- phological characters, including the male genitalia. Therefore, ples from Amami Islands; in particular referring to the sample we consider that P. inopinatus represents a geographical popu- from Tokunoshima Island (H856) and the other samples from lation of P. asahinai. The taxonomic treatment of P. inopinatus Amami Island. It is noteworthy that the length and shape of the as the junior synonym of P. asahinai is proposed (see Discus- male fused gonocoxites 10 are slightly different among the spec- sion below). In P. rastellus only PTP_sh assigned one sample imens from different islands (Figs 2, 3). With respect to P.japon- (i.e. F933 from Iki Island, Nagasaki) as a different MOTU from icus, which is the most widespread species of Parachauliodes, that including the other samples of this species. However, there there is also the highest number of MOTUs (eight MOTUs based is no morphological difference between these two MOTUs. In on the ABGD analysis and 13 MOTUs based on the two PTP Parachauliodes nebulosus the sample from Iriomote-jima Island analyses). Indeed, most samples from different prefectures of (H838) and the two samples from Taiwan (A342 and K498) were Japan were assigned to be independent MOTUs. The slight dif- divided into two MOTUs based on ABGD and PTP_ML anal- ferences on the shape of male fused gonocoxites 10 are also yses, while the two samples from Taiwan were further divided reflected among these MOTUs (Figs 2, 3). In light of thegreat into different MOTUs by the PTP_sh analysis. The latter sub- genetic distance among different MOTUs of P. yanbaru and division is certainly due to overestimation because these two P. japonicus, it is possible that some cryptic species formed by specimens were collected from same locality and on same day allopatric speciation are hidden within these two species. How- with identical morphological characters. Moreover, we also did ever, holding the present criteria to distinguish the corydalid not find any different characters between the samples from species based on morphology, such subtle difference aforemen- Iriomote-jima Island and Taiwan (Figs 2, 3). In P. yanbaru all tioned cannot be used as reliable species diagnosis. At present, to

© 2020 The Royal Entomological Society, Systematic Entomology, 46, 124–139 132 Y. Jiang et al. avoid overestimation of species solely based on molecular data, clade, including P. japonicus, P. nebulosus, P. n i ge r , P. ra s t e l - we consider that there are seven valid species of Parachauliodes lus and P. yanbaru. Notably, all above analyses resulted in (all distinguishable based on both morphological and molecular Parachauliodes rendered paraphyletic by ‘Sinochauliodes’. As evidence) but cryptic species may exist pending further investi- the CAT-GTR model performs better when inferring deep phy- gation. logenies based on mitochondrial genome data (Talavera & Vila, All four described species of ‘Sinochauliodes’, that is, ‘S.’ 2011; Li et al., 2015; Song et al., 2016), the PB tree was used griseus,‘S.’ fujianensis,‘S.’ maculosus and ‘S.’ squalidus, for the following analyses of divergence time estimation and are greatly different genetically based on present molecu- ancestral area reconstruction. Moreover, the similar wing and lar species delimitation, individually representing independent male genital characters also support the sister-group relationship species. However, the samples of ‘S.’ squalidus were divided between ‘Sinochauliodes’ and the P. japonicus clade. into two MOTUs, which did not form a monophylum based on P. japonicus was recovered as the sister group of the other the ABGD analysis, and more MOTUs were recovered by the species belonging to the P. japonicus clade. P. nebulosus was PTP analysis respectively within the former two heterogeneous recovered as the sister of P. rastellus + (P. n i ge r + P. yanbaru). groups. We consider these two MOTUs of ‘S.’ squalidus recov- Within ‘Sinochauliodes’, ‘S.’ fujianensis and a potential cryptic ered by the ABGD analysis to be different species because they new species superficially similar to ‘S.’ squalidus were recov- are not closely related as sister group and both include speci- ered to be a pair of sister species, while the remaining species, mens from Mt. Wuyishan, Fujian (i.e. YL062, YL063, H897, that is, ‘S.’ griseus,‘S.’ maculosus,and‘S.’ squalidus formed a H898, HLiu13), suggesting a sympatric distribution of these monophylum. ‘Sinochauliodes’ griseus was assigned to be the two species. Nevertheless, we could not find any morphological sister group of ‘S.’ maculosus + ‘S.’ squalidus, although there is characters for separating these samples into two species (Fig. 4). one taxon identified as S. griseus from Henan that merged into A similar case was found in ‘S.’ griseus, which was divided into the lineage of ‘S.’ maculosus + ‘S.’ squalidus. two heterogeneous MOTUs respectively from Zhejiang (H872, H873) and Henan (YF037), but without any corresponding Divergence time estimation morphological evidence. There were also two MOTUs recovered in ‘S.’ maculosus, respectively from Guangxi (YL057, The chronogram in Fig. 6 presents the divergence time YL058, H899, HLiu12) and Shaanxi (YL059). The male gen- estimation based on the topology recovered from PB analysis. italia between these two MOTUs are almost the same, while A summary of the age estimates (mean and 95% HPD) for all the specimen from Shaanxi has much fewer forewing dark spots nodes from the BEAST are given in Tables S6 and S7. The than those from Guangxi including the types of ‘S.’ maculosus. clade of Parachauliodes + ‘Sinochauliodes’ was inferred to be S maculo- Thus far, we have examined only one specimen of ‘ .’ diverged from Nigronia at 13.0 Ma (95% HPD: 10.4–15.9 Ma). sus from Shaanxi. Clarification of the identity of this species will The P. continentalis clade was found to be diverged from require examination of more specimens from Shaanxi. Based ‘Sinochauliodes’ + the P.japonicus clade at 11.8 Ma (95% HPD: on the above results, there are probably some cryptic species 9.4–14.4 Ma). The P. japonicus clade and ‘Sinochauliodes’ of ‘Sinochauliodes’. However, as we could not find any reliable were estimated to split at 11 Ma (95% HPD: 8.9–13.5 Ma). morphological characters to distinguish these potential cryptic The two Parachauliodes species widespread in the Japanese species based on a very limited number of individuals, we refrain archipelago (i.e., P. continentalis and P. japonicus) respectively to describe any new species in this paper. were found to diverge from their sister groups during a broadly overlapped time interval, that is, 9.2 Ma (95% HPD: 6.0–12.1 Ma) for the former species or 9.5 Ma (95% HPD: 7.4–11.6 Phylogenetic analysis Ma) for the latter species. Within the P. japonicus clade, P. nebulosus from Taiwan and Iriomote Island was estimated to An inclusive matrix including the sequence data of both mt diverge at 3.1 Ma (95% HPD: 1.2–5.5 Ma), while P. rastellus genomes and gene fragments was analysed using BI (GTR), from Kyushu was diverged from P. n i ge r + P. yanbaru from ML (GTR) and PB (CAT-GTR). The best-fitting partition- Amami and Okinawa Islands at 6.7 Ma (95% HPD: 4.9–8.6 ing scheme is summarized in Table S3. The BI, ML and Ma). The divergence between P. n i ge r and P. yanbaru was dated PB analyses recovered phylogenies largely similar in topol- at 5.7 Ma (95% HPD: 3.9–7.5 Ma). The initial divergence ogy and overall high nodal support (Fig. 5). The monophyly among the ‘Sinochauliodes’ species was dated at 7.9 Ma (95% of Parachauliodes + ‘Sinochauliodes’ was always recovered. HPD: 5.6–10.2 Ma). There are two main clades of Parachauliodes recovered in all analyses, namely the P. continentalis clade and the P. japonicus clade. All species of ‘Sinochauliodes’ formed a monophy- Ancestral area reconstruction lum, which, emerged as the sister group of the P. continentalis clade, including P. asahinai and P. continentalis, based on the The results of ancestral area reconstruction from DIVALIKE + BI and ML analyses. The phylogenetic tree inferred from the j in BioGeoBEARS are shown in Fig. 6 (summary in Table S8). PB analysis is mostly similar to the BI and ML trees except The ancestral range for Parachauliodes + ‘Sinochauliodes’cov- for the position concerning ‘Sinochauliodes’. In the PB tree ers central and southern parts of Mainland China (A) and two ‘Sinochauliodes’ emerged as the sister group of the P. japonicus Japanese major islands, that is, Honshu and Shikoku (B). The

© 2020 The Royal Entomological Society, Systematic Entomology, 46, 124–139 Parachauliodes phylogeny and biogeography 133 ancestral area of the P. continentalis clade was reconstructed not justified to separate Parachauliodes and Sinochauliodes as to be Honshu and Shikoku (B). Within this clade, the ancestral different genera and we herein propose Sinochauliodes syn.n. area of P. asahinai was reconstructed as Honshu and Shikoku as a junior synonym of Parachauliodes. Accordingly, three new (B), as well as that of P. continentalis.‘Sinochauliodes’and combinations are proposed, that is, Parachauliodes fujianensis the P. japonicus clade together have the reconstructed ancestral (Yang & Yang, 1999) comb.n., Parachauliodes maculosus (Liu area ranging central and southern parts of the Chinese main- & Yang, 2006) comb.n.,andParachauliodes squalidus (Liu land (A). The central and southern parts of Mainland China (A) & Yang, 2006) comb.n. Moreover, after examination of the were reconstructed to be the ancestral area of ‘Sinochauliodes’. primary types of P. bu ch i from Zhejiang, China (see type photos Kyushu and its nearby islands (G) were recovered as the ances- in Shimonoya, 2019: fig. 1), it is clear that S. griseus (type tral area of the P.japonicus clade. Within this clade, the ancestral locality also from Zhejiang) is the same as P. bu ch i based on area of P. japonicus was reconstructed to be Kyushu with some the pectinate male antennae, the entirely greyish wings, and the affiliated islands (G). The ancestral range for the lineage of P. median plate of male fused gonocoxites 10 with nearly truncate yanbaru (including species: P. nebulosus, P. rastellus, P. n i ge r tip. Here we treat Neochauliodes griseus Yang & Yang, 1992 and P.yanbaru) covered Kyushu and some affiliated islands (G). syn.n. to be a junior synonym of P. bu ch i . We also examined Taiwan (C) was reconstructed to be the ancestral area of P. neb- the primary types of Neochauliodes pielinus Navás, 1933 from ulosus. P. rastellus and (P. n i ge r + P. yanbaru) had an ancestral Zhejiang, China (one male herein designated as lectotype and area of Kyushu with some affiliated islands (G). The ancestral one female herein designated as paralectotype), and we found area of P. n i ge r + P. yanbaru was found to be Okinawa Island that this species is also the same as P. bu ch i . Thus, N. pielinus (E). The ancestral area of P. n i ge r was Okinawa Island (E), the syn.n. is also considered to be a junior synonym of P. bu ch i . same as that of P. yanbaru. P. inopinatus was originally described by Shimonoya (2015) based on specimens from Fukui Prefecture, Central Honshu, Japan. Morphologically, this species resembles P. asahinai, Discussion which is known from Korea and Kyushu, Japan. The present molecular data assigned these two species as sister to each other. Taxonomic treatment Shimonoya (2015) proposed several characters to distinguish P.inopinatus from P.asahinai, including the slightly larger body Based on the results from our phylogenetic analyses, size, the posteromedially notched male sternum 9, the ovoid dor- Parachauliodes is rendered paraphyletic by Sinochauliodes sal and posteriorly tapering ventral lobes of male ectoproct, the as the sister group of the P. japonicus clade. Liu & Yang (2006) male gonocoxites 9 (attached laterally to the fused gonocoxites originally described Sinochauliodes as a closely related genus 10 as lateral arms) posteriorly protruding, and the median plate to Parachauliodes but differing based on the sexually dimorphic of male fused gonocoxites 10 posteriorly subtriangular. Based antennae and the male ectoproct not bilobed. Considering on our examination of the specimens of P. inopinatus from its the morphology of male antennae, the recent discovery of P. type locality of Fukui, the above characters are not significantly rastellus with pectinate male antennae (Shimonoya, 2019) different from those in P.asahinai. First, the body size in fishflies shows that both of the pectinate and subserrate male antennae can be greatly varied and in most cases is not used to distinguish are present in Parachauliodes. Such within-genus difference closely related species of Parachauliodes. Second, the postero- on the male antennae is also found in the Nearctic fishfly median notch on male sternum 9 is a common character state in genera Chauliodes and Nigronia (see more information in Parachauliodes formed by the slight inflation of the posterolat- Shimonoya, 2019). Therefore, the morphological modification eral part of sternum 9, which can be distinct in some individuals of male antennae does not always stand as a reliable char- but not distinct in the others, due to the development condition. acter for distinguishing fishfly genera. Considering the male Third, there is no constant difference in the shape of ectoprocts, genital characters, there are a number of important genital gonocoxites 9 and gonocoxites 10 between these two species in sclerites that are very similar between Parachauliodes and our examined specimens, while the aforementioned slight differ- Sinochauliodes, such as the posterolaterally enlarged tergum ences can be found in both species. It should be also noted that 9 and the fused gonocoxites 10 with apex of median plate the population of P. asahinai from Kyushu was not included in enveloped by the tergum 9. The bilobed male ectoproct is the present sampling. Considering the location of this population indeed a good character to distinguish Parachauliodes from between Korea and central Honshu, it may represent an interme- Sinochauliodes because it is present in all species of the former diate clade between P. asahinai and P. inopinatus, and further genus but absent in all species of the latter. However, it can be obscure the genetic gap between these two species. Therefore, seen that the anteroventral portion of male ectoprocts is inflated regardless of a great genetic distance between P. asahinai and in all species of Sinochauliodes (see Appendix, File S1, Figs 2, P. inopinatus, here we treat P. inopinatus Shimonoya syn.n. as 3, 4). This inflation should be homologous with the ventral a junior synonym of P. asahinai because there are no reliable lobe of male ectorpocts in Parachauliodes. It might either be morphological characters to distinguish them. a plesiomorphic condition compared to the well-developed Parachauliodes in the context of this phylogenetic investiga- ventral lobe of male ectorpocts in Parachauliodes or a section, comprises 11 valid species with a broad distribution from ondary reduction from the ventral lobe. Thus, considering the the central and southeastern parts of Mainland China, the Korean present morphological and molecular evidence in total, it is peninsula and the larger islands of the western Pacific (i.e.

Taiwan, the Ryukyu Islands, and Japanese archipelago). Here Phylogenetic position of Parachauliodes we term the Parachauliodes species from Mainland China (i.e. those species previously placed in Sinochauliodes) to be conti- Our mitochondrial phylogenomic data recovered the mono- nental Parachauliodes species, while the remaining species to phyletic group comprising Neochauliodes, Nigronia and be insular Parachauliodes species for the following discussion. Parachauliodes (including Sinochauliodes), which is consistent with the result from morphological studies by Liu & Yang (2006) and Liu et al. (2012). Here we recovered Cryptic diversity versus population differentiation Parachauliodes as the sister of Nigronia, in contrast to Liu & Yang (2006), where Parachauliodes + Sinochauliodes was According to the results of species delimitation using ABGD, recovered as the sister-group of Neochauliodes + Nigronia. we calculated the genetic distance among, and within each The sister-group relationship between Neochauliodes and MOTUs. The interspecific distance among the insular species Nigronia was supported by the wings with transversely ranged from 12.6 to 19.9% (Table S5), much greater than 2.2%, band-like markings, the male tergum 9 acutely tapering which has been proposed empirically as an appropriate level of divergence delimiting species across diverse insect taxa (Hebert ventrally in lateral view, and the short gonocoxites 9 (i.e. et al., 2003; Ball et al., 2005; Zhou et al., 2010). The maximum the lateral arms) (see Liu & Yang, 2006: characters 11, value (19.9%) is between P. continentalis and P. yanbaru, while 27, 41). However, the former two characters are shared the minimum value (12.6%) is between P. continentalis and by Ctenochauliodes, and the latter character is not always P. rastellus. The genetic distance between these sister-pairs of present in Neochauliodes (e.g. absent in the early diverged species is as follows: between P. continentalis and P. asahinai Neochauliodes sundaicus group; see Liu et al., 2010). In Liu is about 15% (15.3%: between MOTU1 and MOTU3; 15.2%: et al. (2012) Neochauliodes + Parachauliodes was supported between MOTU2 and MOTU3), while that between P. n i ge r by the presence of brush-like setae on male ectoprocts (char- and P. yanbaru is more than 14%. The intraspecific distance acter 33 in Liu et al., 2012). However, this character, together within each insular species is around 10%, and there is no with the absence of male gonocoxites 11 (characters 27, 47 in significant difference among the values obtained from samples Liu et al., 2016), supported a monophyletic group comprising on the same, or on different islands. In the most widespread P. Parachauliodes, Neochauliodes, Anachauliodes and Chauliodes japonicus, the maximum value is 12.9%, obtained from samples in Liu et al. (2016). distributed in different regions of Kyushu, while the minimum Given the sister group relationship between Nigronia and of 7.4% is observed among samples from Honshu. The same Parachauliodes, two male genital characters might serve as the phenomenon occurs in P. asahinai, as the distance between autapomorphies of this lineage, that is, the male tergum 9 dis- samples distributed from Korea and Ishikawa is 9.1%. High tinctly protruding posteriad and the ventral separation of male levels of intraspecific genetic divergence have been reported in some groups of aquatic insects, such as mayflies and stoneflies, ectoproct. The former character was not used in previous phylo- which is probably caused by the low dispersal capacity of these genetic analyses. However, another character related to this one, species (Hebert et al., 2003; Ball et al., 2005; Ratnasingham alternatively described as the male gonocoxites 10 enveloped by & Hebert, 2007; Zhou et al., 2010; Morinière et al., 2017). tergum 9, was included in all preceding studies and assigned Zhang et al. (2017) advocated that determination of a cryptic as an autapomorphy of Parachauliodes + Sinochauliodes (Liu species should be made based on the minimum interspecific & Yang, 2006; Liu et al., 2012, 2016). The posterior protru- genetic distance of congeneric species rather than a fixed sion is present in Nigronia (see Liu et al., 2016: 6C) but over- empirical threshold. Accordingly, although the results of species looked previously, although it is not developed as well as that delimitation clustered the insular Parachauliodes species into found in Parachauliodes. Nevertheless, this character is also many MOTUs, we consider that there may be few cryptic species present in a few species of Neochauliodes (see Liu et al., 2010: but population differentiation correlated with island formation fig. 12). The latter character refers to the ventrally bilobed male because the intraspecific genetic distance in almost all of these ectoproct in Parachauliodes.InNigronia there is a pair of scle- insular species is smaller than the minimum interspecific genetic rites fused on the ventral side of male ectoprocts. In light of distance (12.6%). The relatively large intraspecific genetic the median fusion between the sclerites beneath the anus, Liu distance is probably caused by the restricted gene flow and et al. (2016) considered this peculiar structure to be homolo- isolation by distance among the allopatric populations, which gous with the fused gonocoxites 11 in Ctenochauliodes, Platy- has also been reported in the Nearctic fishfly N. serricornis chauliodes Esben-Petersen, Puri Cardosa-Costa et al., and other (Heilveil & Berlocher, 2006). As for the continental species, the genera. Unlike Nigronia, the fused male gonocoxites 11 is not interspecific distances are in 7.9–14.0%, while the intraspecific distances are around 10%, being similar to that in the insular amalgamated with the ectoprocts in these other genera. Thus, relatives. However, it is notable that the recovered MOTUs of the homology of this ventral sclerite in Nigronia with a part of the same species (i.e. P. squalidus and P. griseus) were not male ectoproct should be re-considered. Integrating the afore- clustered into one monophyletic lineage. This result suggests mentioned morphological evidence and the present molecular that some cryptic species may exist in these species, although evidence, the sister group relationship between Nigronia and no supportive morphological evidence has yet been found. Parachauliodes should be most plausible.

Temporal diversification and historical biogeography The insular species of Parachauliodes display a more sym- of Parachauliodes patric distribution pattern, represented as P. asahinai, P. continentalis, P. japonicus and P. rastellus from the Japanese Liu et al. (2012) postulated that the species of the archipelago, and P. n i ge r and P. yanbaru from Okinawa. How- Neochauliodes subclade (a lineage includes all extant Asian fish- ever, it can be seen that the species distributed from the fly genera plus Chauliodes and Nigronia) had been widespread islands on either side of the Tokara Strait and Kerama Strait in Eurasia and dispersed during the Eocene. According to this are completely different. This pattern generally agrees with scenario, the ancestral species of Chauliodes and Nigronia, Liu et al. (2008) in that the oceanic barriers formed by the managed to disperse from Europe to eastern North America Tokara Strait and the Kerama Strait might be correlated with via the Thulean Bridge, which is considered to be an important divergences among Parachauliodes species from Taiwan and the route for the trans-Atlantic exchange of temperate biota during Ryukyus (e.g. P. nebulosus, P. n i ge r , P. yanbaru, etc.). These the Eocene (McKenna, 1983; Sanmartín et al., 2001). Notably, vicariance events require that dispersal of the common ancestor a recently described Eocene Baltic amber fishfly species, ten- of these species occurred along the island arc via intermittent tatively placed in Nigronia, provides new evidence to support land bridges, because over-sea dispersal could hardly be man- this hypothesis (Liu & Ansorge, ). As the sister group of Nigro- aged by fishflies due to their limited dispersal capacity. Despite nia, the origin of Parachauliodes is likely Eurasia during the different palaeogeographical hypotheses depicting the forma- Eocene (Liu et al., 2008), which is supported by the present tion process of the Ryukyus, and the associated land bridges or results of fossil-calibrated temporal estimation and ancestral insular isolations shaped by plate tectonics or Pleistocene glacia- area reconstruction. Concerning the timing of divergence of tion (Ota, 1998; Kimura, 2002; Su et al., 2016; Tojo et al., 2017), Parachauliodes from Nigronia herein estimated with fossil it is generally accepted that the formation of this island arc may calibrations (30.7 Ma, 95% HPD: 22.5–38.2 Ma; Table S7; have been driven by the opening of the Okinawa Trough in Fig S3), the origin of Parachauliodes is postulated to be early thelateMiocene(Leeet al., 1980; Sibuet et al., 1995; Wang Miocene when Japan was still a part of eastern margin of et al., 2014). Under this circumstance, the predicted dispersal Eurasia, prior to separation (Otofuji, 1996). Notably, estimates of ancestral Parachauliodes species along the island arc and using the priors based on an insect mitochondrial clock suggest the subsequent speciation might not have taken place before the a much younger (13.0 Ma, 95% HPD: 10.4–15.9 Ma), middle late Miocene. Thus, comparing the present divergence time esti- Miocene age for Parachauliodes. When comparing the spatial mates based on two calibration schemes, the fossil-calibrated temporal divergence among the insular Parachauliodes species divergence pattern among the Parachauliodes species with is much earlier than the late Miocene, which appears to be not the time estimates, the result based on fixed rate calibration compatible to the known geological history of the island arc appears to be more compatible with the putative history of the mentioned above. Instead, the result from the fixed substitution East Asian islands harbouring the insular species of this genus rate calibration corresponds to the pattern of island formation. (Otofuji et al., 1985; Kimura, 2002; Sibuet & Hsu, 2004; see The divergence between the P. continentalis clade and the con- the following discussion). tinental Parachauliodes clade + the P. japonicus clade, might The tectonic separation of the Japanese Islands eastward from have taken place during the middle and late Miocene (11.8 Ma, Eurasia occurred around 15 Ma in the middle Miocene (Otofuji 95% HPD: 9.4–14.4 Ma) when the southwestern portion of et al., 1985). Interestingly, the northeastern and the southwest- the Japanese Islands was largely separated from the Eurasian ern portions of these Japanese Islands split independently from continent, but with a land connection that remained between the Eurasian continent, known as the ‘dual origin’ model (Oto- Kyushu and the Korean peninsula up to the late Pleistocene fuji et al., 1985). The timing of divergence of Parachauliodes (Kizaki & Oshiro, 1977; Ota, 1998; Tojo et al., 2017). This con- estimated under fixed substitution rate priors, suggests the south- tradicts the previous hypothesis that the P. continentalis clade western portion of the Japanese Islands were still connected diverged due to the formation of the Tokara Strait about 5 Ma with the nearby continent at the Korean peninsula (see Tojo (Liu et al., 2008). Our results support dispersal events during et al., 2017: fig. 3C). The earliest dispersal of the Parachauliodes the initial divergence within Parachauliodes. Furthermore, Hon- ancestor probably took place along this peninsula-like land- shu was reconstructed as the ancestral range of the P. continen- mass as its reconstructed distribution covers Mainland China talis clade, while the Mainland China was recovered to be the and two Japanese major islands, that is, Honshu and Shikoku. ancestral range of the continental Parachauliodes clade + the Conversely, the northeastern portion of the Japanese Islands, ini- P.japonicus clade. It is notable that the two widespread Japanese tially fragmented from the Russian Far East about 20 Ma (see Parachauliodes species, P. continentalis and P. japonicus,have Tojo et al., 2017: fig. 3C), is less likely to be related to the different habitats of larvae. Larvae of P. continentalis are found early dispersal of Parachauliodes from the Eurasian continent in shallow stony streams while P. japonicus are found from as several subsequent divergence events of species from south- small muddy streams near riverside (Hayashi, 1989a,b). Hence, western Japan occurred prior to the closure of Fossa Magna (an sympatric distribution driven by niche partition could account oceanic barrier that isolated the southwestern portion and the for the initial divergence within Parachauliodes. Unfortunately, northeastern portion of the Japanese Islands; Otofuji et al., 1985) ecological data of other Parachauliodes species remains largely about 5 Ma. unknown.

Within the P. continentalis clade, the late Miocene diver- northern Kyushu, diverged from the central Ryukyu species P. gence between P. asahinai and P. continentalis was recovered niger and P. yanbaru probably due to the vicariance formed although no geological event correlated to the divergence could by the Tokara Strait. Lastly, by the end of Miocene (5.7 Ma, be traced. Nevertheless, the Korean Strait probably acted as a 95% HPD: 3.9–7.5 Ma) P. n i ge r and P. yanbaru had diverged, barrier blocking further dispersal of the widespread P. continen- probably from speciation within the Okinawa Island. talis to the Korean peninsula (P. continentalis occurs only as Insular isolation has also significantly contributed as biogeo- far as Tsushima Island). Our results suggest a dispersal event graphic barriers against gene flow between populations. For from Honshu to Korea for the ancestor of P. asahinai. During instance, the isolation between Taiwan and Yaeyama corre- this period, the formation and submergence of land bridges in sponds to the distinct differentiation of populations of P. neb- western Japan across the Korean Strait (Tojo et al., 2017; Kawa- ulosus from these two islands. Similarly, the isolation between mura, 2014; Taruno, 2010) probably had significant impacts on Okinawa and Amami Islands likely caused the separation of shaping the current distribution of P. asahinai. populations of P. yanbaru. Concerning the Japanese widespread The divergence between the continental Parachauliodes species P. japonicus, the Kyushu populations that are geneti- species and the P. japonicus clade was probably influenced by cally distinct from the other populations in Honshu and Shikoku, the splitting between the southwestern portion of the Japanese may be attributed to the geographical isolation between Kyushu Islands and the continent during the late Miocene. Our results and Honshu + Shikoku. Moreover, three other genetically dis- recover Kyushu as the ancestral range of the P. japonicus clade tinct lineages of P. japonicus are also recovered within Honshu and multiple subsequent dispersals to Ryukyus and Taiwan, and Shikoku. Interestingly, we did not find any genetically dis- suggesting a general southward dispersal of the common ances- tinct lineage within P. continentalis, which is also widespread tor of the P. japonicus clade across the land bridge shortly after in Kyushu, Honshu and Shikoku. Based on this observation, its divergence in the late Miocene. This scenario is contrary the population structure and comparative phylogeography of the to the previous hypothesis in Liu et al. (2008) that common above two species are worthy of further investigation. ancestor of the insular Parachauliodes species might have migrated to the Japanese archipelago along a land bridge from Mainland China via Taiwan and Ryukyus during the Conclusions late Miocene. Following this putative southward dispersal, the speciation within the P. japonicus clade was driven by several Here, we presented the first interspecific phylogeny of two late Miocene (9.5 Ma, 95% HPD: 7.4–11.6 Ma) island isola- East Asian endemic fishfly genera Parachauliodes and tion events. In addition, sympatric distribution after speciation Sinochauliodes based on mitochondrial DNA sequence might also have been a factor shaping the faunal diversity of data using comprehensive sampling. Based on our results, Parachauliodes from Kyushu, Ryukyus, and Taiwan. Firstly, P. Sinochauliodes is a junior synonym of Parachauliodes and japonicus might have split from the common ancestor of the is the sister group of the P. japonicus clade. Integrating both P. japonicus clade locally within a wider ranging distribution morphological and molecular evidence, we revised the clas- because our results do not suggest any vicariance in relation sification of Parachauliodes and highlight the cryptic species to this late Miocene divergence. However, P. japonicus only diversity found within this genus. Moreover, we provided occurs in the area north of the Tokara Strait, while the other new insights into the spatio-temporal divergence pattern of species of this clade, except P. rastellus, are distributed from Parachauliodes, with emphasis on the insular species of this the islands south of the Tokara Strait. Although the formation genus distributed along the major islands of East Asia, based on of the Tokara Strait is thought to have isolated the central and divergence time estimation and ancestral area reconstruction. southern Ryukyus from Kyushu and the northern Ryukyus in We corroborated the Eurasian origin of Parachauliodes during the early Pleistocene (Kimura, 2002), whether there had been the middle Eocene, with the two independent clades of insular any geographical barrier at the similar location of the Tokara Parachauliodes species diverging due to separation of the Strait during the late Miocene (7.2 Ma, 95% HPD: 5.3–9.2 southwestern Japanese Islands and the continent. We suggest a Ma) is not certain. Second, P. nebulosus, the endemic species to southward dispersal of the ancestral species of the P. japonicus Taiwan and Ishigaki-jima and Iriomote-jima Islands, diverged clade from southwestern Japan via land bridges, which enabled through allopatric speciation. Among the Taiwanese Corydali- the colonization of Parachauliodes via Ryukyus to Taiwan by dae fauna, P. nebulosus is the only species that is not distributed the end of Miocene. Finally, a series of island isolations, espe- in Mainland China (Yang & Liu, 2010). This distribution pattern cially formed by the Tokara Strait and Kerama Strait, possibly may provide further evidence to support that colonization of along with distributional expansions after speciation, was found Taiwan by Parachauliodes originated from Japan. The isola- to significantly shape the present species diversity of the insular tion formed by the Kerama Strait probably accounted for this lineage of Parachauliodes. Future studies may focus on (i) divergence although it was documented to be formed much the cryptic diversity and phylogeography of some widespread later in the middle to late Pleistocene (Kimura, 2002). Notably, species of Parachauliodes, (ii) the interspecific phylogeny of examples similar to this pre-Pleistocene divergence pattern the continental species of Parachauliodes and (iii) whether the had been reported from some vertebrate and arthropod groups biogeographical patterns found in the other megalopteran taxa (Maekawa et al., 1999; Matsui et al., 2005; Honda et al., 2014; from the major islands of East Asia are consistent to that of Su et al., 2016). Third, P. rastellus, which only occurs in Parachauliodes.

Supporting Information heights and the 95% high posterior density (HPD) interval. Nodal age unit is in Ma. Additional supporting information may be found online in the Supporting Information section at the end of the article. Table S8. Summary of ancestral area reconstruction from DIVALIKE + j approaches test in BioGeoBEARS for nodes Appendix S1. Species checklist of Parachauliodes with in Fig. 5. present taxonomic treatments.

Figure S1. Habitus photos of continental species Acknowledgements of Parachauliodes (formerly Sinochauliodes). (A) Parachauliodes fujianensis (Yang & Yang, 1999), male, We are much indebted to the following curators or persons for their kind help on getting access to valuable material: U. Aspöck, from Fujian, China; (B) Parachauliodes buchi Navás, D. Burckhardt, P. Chvojka, J. Constant, W. Chou, O.S. Flint Jr., Parachauliodes maculosus male, from Zhejiang, China; (C) D. Goodger, W. Hogenes, S.W. Jung, S. Kim, V. Krivokhatsky, (Liu & Yang), holotype male, from Guangxi, China; (D) R. Kuranishi, J. Legrand, W. Li, R. Matsumoto, M. Ohl, S. Parachauliodes squalidus (Liu & Yang), holotype male, Randolf, A. Saito, C. Sun, A. Taeger, R. de Vries, K. Watanabe, from Guangdong, China. H. Yin, K. Zhang, W. Zhu. We are also grateful to Y. Zheng and H. Okamiya for photographs of some living adults and Figure S2. Habitus photos of insular species of specimens of Parachauliodes. We also thank many persons Parachauliodes.(A)Parachauliodes asahinai Liu, Hayashi who collected useful materials for this study. Finally, we thank & Yang, holotype male, from Busan, Korea; (B) P. asahinai B.W. Price, A. Contreras-Ramos and an anonymous reviewer for Liu, Hayashi & Yang, male, from Ishikawa, Japan; (C) the comments to improve the manuscript. We declare that the Parachauliodes continentalis van der Weele, male, from authors have no conflicts of interest to this work. This research Miyagi, Japan; (D) Parachauliodes japonicus (McLachlan), was supported by the National Natural Science Foundation of male, from Tottori, Japan; (E) Parachauliodes nebulosus China (Nos. 31322051, 31972871, 31672322, 31320103902), (Okamoto), female, from Taiwan, China; (F) Parachauliodes the Changjiang Scholars Programme (No: Q2018035), and the niger Liu, Hayashi & Yang, paratype female, from Okinawa key project of Science-technology basic condition platform from The Ministry of Science and Technology of the People’s Island; (G) Parachauliodes rastellus Shimonoya, male, from Republic of China (Grant No. 2005DKA21402). Fukuoka, Japan; (H) Parachauliodes yanbaru Asahina, male, from Amai-oshima Island, Japan. Data availability statement Figure S3. Chronogram with estimated divergence times with fossil calibrations. Data openly available in a public repository that issues datasets with DOIs. File S1. Taxonomic notes and additional material examined.

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