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. 2023 May 26;62:e27. doi: 10.6620/ZS.2023.62-27

A New Distinctive Darwin Wasp Represents the First Record of the Ophion minutus Species-group (Hymenoptera: Ichneumonidae: Ophioninae) from Japan and the Far East, with an Analysis of DNA Barcode-based Species Delimitation in Ophion

So Shimizu 1,2,*, Kaoru Maeto 2
PMCID: PMC10390328  PMID: 37533555

Abstract

A new Darwin wasp species, Ophion kobensis Shimizu sp. nov. (Hymenoptera: Ichneumonidae: Ophioninae), is described using the integrated morphological and molecular species delimitation approaches. Our results indicate that the new species is closely related to European O. ventricosus Gravenhorst, 1829 of the O. minutus species-group but can be distinguished using morphological characters, such as entirely black body colour with some light-yellow marks and not inclined epicnemial carina in lateral view. This record of the new species represents the first record of O. minutus species-group from Japan and the Far East. Phylogenetic analysis indicate that the O. minutus species-group is weakly recovered as monophyletic and sister to Ophion s. str. The analysis also indicated that two clades within the O. minutus species-group (O. minutus and O. ventricosus) diverged significantly. This suggests that the species-group, as well as the two included clades, could potentially be treated as separate species-groups or genera. The present study supports previous integrative taxonomic and phylogenetic studies of Ophion and represents a first fundamental step for studies focused on the challenging Japanese and Far Eastern Ophion.

Keywords: bPTP, Integrative taxonomy, MtCO1, Parasitoid wasps, Phylogeny

BACKGROUND

Darwin wasps of the predominantly temperate genus Ophion Fabricius, 1798 (Hymenoptera, Ichneumonidae, Ophioninae) are renowned as some of the most common mid-to large-sized insects that frequently visit lights at night, together with many other nocturnal insects (Townes 1971; Gauld and Mitchell 1981; Gauld 1985; Schwarzfeld et al. 2016). They are solitary koinobiont endoparasitoids of mid-to large-sized lepidopteran larvae (Uchida 1928 1954; Townes 1971; Gauld and Mitchell 1981). Adult wasps of Ophion exhibit the convergent morphology of “ophionoid facies”, characterised by, among others, an orange-brown body, large ocelli, and long antennae, associated with their nocturnal behaviour (Gauld and Huddleston 1976).

Although over 150 valid species of Ophion have been recognised worldwide (Yu et al. 2016; Johansson and Cederberg 2019; Johansson et al. 2021), their taxonomy does not reflect their true species richness, and most species, like other Darwin wasps (Klopfstein et al. 2019), still need to be described. Although many studies have attempted to perform morphology-based taxonomy of Ophion (Gauld and Mitchell 1981; Brock 1982), it is very difficult to delimit species of Ophion based solely on morphological characters due to a wide range of intraspecific morphological variations (Townes 1971; Brock 1982). Integrative taxonomy aims to correctly delimit and describe taxa by incorporating multiple perspectives (e.g., morphology, ecology, and genetics). It is one of the best solutions to resolve taxonomic problems in morphologically challenging and poorly known diverse groups and to understand their biodiversity (Dayrat 2005; Schlick-Steiner et al. 2010; Padial et al. 2010; Ito et al. 2015; Shimizu et al. 2019 2020). Therefore, recent studies on Ophion have employed integrative taxonomic frameworks (at least integrating morphology and DNA barcodes) (Schwarzfeld and Sperling 2014 2015; Johansson and Cederberg 2019).

The Ophion minutus species-group consists of small-sized wasps with typical ophionoid facies, which parasitise geometrid moths. This species-group is a well-recovered monophyletic basal lineage within the genus Ophion and is considered to be a potential candidate for a separate genus (Schwarzfeld et al. 2016; Johansson and Cederberg 2019). It is distinguishable from all other Ophion species by the proximally thickened and slightly angled fore wing vein 2r&RS (Gauld 1985; Schwarzfeld et al. 2016), similar to related non-Ophion genera, such as Eremotylus Förster, 1869 and Stauropoctonus Brauns, 1889. After the phylogenetic reconstruction of the species-groups of Ophion by Schwarzfeld et al. (2016), the number of species in the species-group increased by the great taxonomic studies of a Swedish Entomologist, Mr. Niklas Johansson (Johansson and Cederberg 2019; Johansson et al. 2021). Gauld (1985) suggested that O. minutus could belongs to the O. bicarinatus species-group, previously consisting of Australian species, but reliable evidence has not been presented and further phylogenetic studies are required (Schwarzfeld et al. 2016). Most records of the O. minutus species-group are from Europe, with a few from the western portion of the Eastern Palaearctic region (Yu et al. 2016; Johansson and Cederberg 2019; Johansson et al. 2021), and no Darwin wasps of the species-group have been recorded from the Far East.

The Japanese fauna of Ophion has been poorly studied (Smith 1874; Uchida 1928 1954), with only 10 valid species records (Yu et al. 2016), although over 50 morphospecies have been recognised by the first author (Shimizu unpublished data). Therefore, revisional studies are necessary but are difficult at present due to the enormous species richness, taxonomic difficulties, and incomplete sampling. The Ophion minutus species-group is not known from Japanese fauna, but the first author of this paper recently recognised specimens of the species-group from Japan for the first time. Therefore, as a first step in the taxonomy of the Japanese Ophion, the present study aims to report the O. minutus species-group from Japan for the first time and to classify it using integrated morphology and molecular species delimitation methods, with molecular phylogenetic reconstruction for the genus Ophion to test the phylogenetic position of the Japanese species.

MATERIALS AND METHODS

Terms and indices

Morphological terms and indices followed those of Broad et al. (2018), Johansson and Cederberg (2019), and Shimizu et al. (2019 2020). The density of punctures was indicated as follows: “densely” = punctures separated by approximately their own diameter or less, “moderately” = punctures separated by approximately twice their own diameter, “sparsely” = punctures separated by over triple their own diameter. The “mandibular gape” was used for the angle between mandibular teeth (Brock 1982; Johansson and Cederberg 2019). The following abbreviations were used: FL = flagellomere (e.g., FL1 = 1st and FL2 = 2nd flagellomeres), IOD (inter-ocellar distance) = shortest distance between inner margin of lateral ocelli, LOD (lateral-ocellar diameter) = maximum diameter of lateral ocelli, OOD (orbito-ocellar distance) = shortest distance between outer margin of lateral ocellus and orbit of eye, POD (post-ocellar distance) = shortest distance between posterior margin of lateral ocellus and anterior margin of occipital carina, S = metasomal sternite (e.g., S1 = 1st and S2 = 2nd metasomal sternites), and T = metasomal tergite (e.g., T1 = 1st and T2 = 2nd metasomal tergites).

Examined samples

The materials were deposited at the Institute for Agro-Environmental Sciences, NARO (= National Institute for Agro-Environmental Sciences), Tsukuba, Japan (NIAES).

Morphological observation

Morphology of the examined adult wasps was observed under a stereoscopic microscope (SMZ1500, Nikon, Tokyo, Japan). For accurate measurement, left wings were removed from the body, and the wing wrinkles were pressed out by putting them between two microscope slides in 80% ethanol. Then, wing photos were taken (cf. below for photo technique). Finally, the wing measurements were conducted using Adobe Photoshop CC v.20.0.4 (Adobe Systems Inc., San Jose, CA, USA). Body length was measured between the wasp head and the terminus of the metasoma, not including antennae and ovipositor.

Multi-focus photographs were taken using a single lens reflex camera (α7II, Sony, Tokyo, Japan) with a micro-lens (LAOWA 25 mm F2.8 2.5–5× ULTRA MACRO, Anhui Changgeng Optics Technology Co., Ltd, Hefei, China and A FE 50mm F2.8 Macro SEL50M28, Sony, Tokyo, Japan), captured in RAW format, developed using Adobe Lightroom CC v.2.2.1 (Adobe Systems Inc., San Jose, CA, USA), and stacked using Zerene Stacker v.1.04 (Zerene Systems LLC., Richland, WA, USA).

Molecular species delimitation

Partial sequences of a mitochondrial protein-encoding gene, cytochrome c oxidase 1 (CO1), frequently referred to as the DNA barcoding gene for arthropods, were sequenced from the Japanese species of the O. minutus species-group for the delimitation of molecular species. DNA was extracted from the right fore or mid-leg, and DNA extraction and sequencing protocols followed those described by Shimizu et al. (2020).

Sequences of the global Ophion species were compiled from BOLD systems (Ratnasingham and Hebert 2007) (available at: http://v3.boldsystems.org/) (accessed on 14 Dec. 2022), and together with the sequences of the Japanese species of the O. minutus species-group, a multiple sequence alignment (MSA) was conducted using MEGA v.10.0.5 (Kumar et al. 2018) based on both nucleotides and amino acids (see Fujie et al. 2021 for detailed protocols). Poorly aligned regions and identical haplotypes were removed from the MSA using trimAl v.1.2 (Capella-Gutiérrez et al. 2009) and the web server of ALTER (Glez-Peña et al. 2010) (available at: http://sing.ei.uvigo.es/ALTER/) respectively.

Uncorrected pairwise nucleotide genetic distances (p-distances) were calculated using MEGA.

As Schwarzfeld and Sperling (2015) concluded in the comparison of the species delimitation methods using Ophion, tree-based General Mixed Yule Coalescent (GMYC) (Pons et al. 2006; Fontaneto et al. 2007; Fujisawa and Barraclough 2013) and Poisson Tree Processes (PTP) (Zhang et al. 2013) models were less reliant on arbitrary parameters than the distance-based threshold and Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012) methods. They also suggested that PTP was less successful than GMYC in delimiting test species. However, PTP is fast and easy to run without ultrametricised trees. This is a significant advantage over GMYC, which requires ultrametricised trees. Therefore, bPTP (Zhang et al. 2013), an updated version of the maximum likelihood PTP with Bayesian support values for delimited species, was selected to delimit the species in the present study.

The bPTP input tree was generated with IQ-TREE v.2.1.2 (Minh et al. 2020) under the maximum likelihood framework. The optimal substitution models and partitioning schemes were determined using PartitionFinder v.2.1.1 (Lanfear et al. 2017) with the greedy search algorithm under the corrected Akaike information criterion (AIC). A Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010) and ultrafast likelihood bootstrap replicates (UFBoot2) (Minh et al. 2013; Hoang et al. 2018) were performed with 10,000 replicates. To reduce the risk of overestimating nodal supports, the ”-bnni” option was also employed.

A standalone version of bPTP was run with the following MCMC parameters: iterations = 5,000,000, sampling interval = 200, burn-in = 0.50, and seed = 7777. The Bayesian support values of posterior probabilities (PP) for molecular operational taxonomic units (MOTUs, considered to be approximately equivalent to species) were interpreted as follows: strongly supported: 0.95 ≤ PP ≤ 1.00, moderately supported: 0.90 ≤ PP < 0.95, weakly supported: 0.80 ≤ PP < 0.90, and not supported: PP < 0.80.

Molecular phylogeny

A phylogenetic analysis was conducted to determine the phylogenetic position of the Japanese species of the Ophion minutus species-group. One sequence was selected per MOTU delimited by bPTP analysis to reduce sampling bias per species. Thirteen sequences from other ophionine genera, i.e., Afrophion hynnis (Gauld & Mitchell, 1978) (ASQIC051-09), Barytatocephalus mocsaryi (Brauns, 1895) (ASQIC059-09), Dicamptus sp. (ASQAS285-11), Dictyonotus sp. (GBMIN25686-13), Enicospilus ramidulus (Linnaeus, 1758) (COLHH305-18), Eremotylus sp. (BBHYG815-10), Hellwigiella dichromoptera (Costa, 1886) (ASQIC060-09), Leptophion sp. (GBMND19516-21), Riekophion sp. (GBAH21297-19), Skiapus sp. (ASQIC197-09), Stauropoctonus bicarinatus (Cushman, 1947) (STAT1173-07), Thyreodon sp. (ASINB1539-12), and Xylophion sevrapek Villemant, 2012 (ICHSA002-12), were selected as outgroups. Two non-ophionines, i.e., Habronyx (Camposcopus) nigricornis (Wesmael, 1849) (Anomaloninae) (COLHH348-18) and Cremastus incompletus (Provancher, 1875) (Cremastinae) (HYMBB978-10), were also selected to define a distinct root of Ophioninae. The phylogenetic tree was reconstructed in IQ-TREE using the same process as described above for the input tree preparation for bPTP (cf. above “Molecular species delimitation”). As recommended by the manual of IQ-TREE, nodal support values were interpreted as follows: supported = SH-aLRT ≥ 0.80 and UFBoot2 ≥ 0.95, weakly supported = SH-aLRT ≥ 0.80 or UFBoot2 ≥ 0.95, and not supported = SH-aLRT < 0.80 and UFBoot2 < 0.95.

Figure editing

All figures were edited in Adobe Illustrator CC v.23.0.2 or Photoshop CC v.20.0.4 (Adobe Systems Inc., San Jose, CA, USA). Phylogenetic trees were edited with the interactive Tree of Life (iTOL) (available at: https://itol.embl.de) (Letunic and Bork 2021).

RESULTS

Morphological species delimitation

Based on two female specimens from the Kinki region, the western portion of Honshu Island in the Japanese Archipelago, only a single morphospecies of the O. minutus species-group was recognised in the Japanese fauna, as described as a new species below (O. kobensis Shimizu sp. nov.). The species displayed the most distinctive morphology (i.e., entirely black body with yellowish wings, and proximally thickened and slightly angled fore wing vein 2r&RS) among the Japanese species of Ophion but resembled the European O. ventricosus Gravenhorst, 1829. However, the body colour of O. kobensis sp. nov. was entirely black with some small light-yellow marks, and that of the European O. ventricosus was entirely testaceous with some small black marks; therefore, they could easily be distinguished from each other by their colouration. Additionally, they can be distinguished from each other by the characters summarized in table 1.

Table 1.

Diagnostic characters between O. kobensis sp. nov. and O. ventricosus

graphic file with name zoolstud-62-027-t001.jpg

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Molecular species delimitation

Dataset: A total of 1,606 public records with CO1 sequences of Ophion, forming 148 BINs, were compiled from BOLD systems. Among them, the deposited photograph of a single record of NBINS376-15 was not ophionine Darwin wasp; seven sequences (i.e., GBMIN71038-17 = GenBank KU753332 of O. obscuratus obscuratus, GBMIN77568-17 = KU753331 of O. luteus, GBAH23440-19 = KU753333 of O. obscuratus obscuratus, GBAH23448-19 = KU753334 of O. takaozanus, SSEIB12923-13, DBFCI467-15, and ACGBA1775-12 of Ophion spp.) were enigmatic with non-triplet indels or suspicious regions. Therefore, these eight sequences were removed from the dataset (1,598 sequences, forming 140 BINs). Additionally, two sequences of O. kobensis sp. nov. were obtained (DDBJ: LC756991 and LC756992). The aligned and trimmed dataset contained 1,600 sequences of 567 bp in length. This represented 616 haplotypes. Therefore, the final dataset without identical haplotypes consisted of 616 sequences (Supplementary file S1). It included 243 conserved and 324 variable sites, of which 268 were parsimony informative.

Tree and phylogeny: The TVM+I+G model was selected as the optimal substitution model for each codon position under AIC. All branches of the BOLD systems project code of “HYSAF” (South African Hymenoptera) and “HYSA” (South Australian Hymenoptera) were significantly longer than those of the other Ophion (Fig. S1). However, most of the reconstructed topologies were consistent with those reported by Schwarzfeld et al. (2016) and Johansson and Cederberg (2019).

For the O. minutus species-group (Fig. 1), the monophyly of the O. ventricosus clade (O. kobensis sp. nov. + O. ventricosus) was highly supported (SH-aLRT = 1.00 and UFBoot2 = 0.99), and O. minutus was also strongly recovered as a monophyletic clade (the O. minutus clade; SH-aLRT = 0.98 and UFBoot2 = 1.00). However, the monophyly of the O. minutus species-group was not recovered (SH-aLRT = 0.66 and UFBoot2 = 0.69). The two clades in this species-group were more or less divergent. Divergences in p-distances were 0.2–2.6% among the O. minutus clade, 2.7–4.2% among the O. ventricosus clade (0.2–0.7% among O. ventricosus and 0.7% among O. kobensis sp. nov.), and 12.2–14.3% between the two clades (Table 2).

bPTP: The bPTP analysis estimated between 168 and 265 species (mean = 213.11), and the most supported result indicated that 199 species, consisting of 80 supported (i.e., 57 strongly, 7 moderately, and 16 weakly supported species) and 119 unsupported species, were delimited (Figs. 1, S1, Supplementary file S2). Ophion kobensis sp. nov. of the O. minutus species-group was recovered as a weakly supported single species (PP = 0.866).

Fig. 1.

Fig. 1.

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Result of the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion, based on the maximum likelihood CO1 gene topology reconstructed by IQ-TREE2. This figure only shows the O. minutus species-group but whole result is available at figure S1. Stripes indicate delimited molecular operational taxonomic units (MOTUs), and stripe colours and numbers on them indicate a degree of the Bayesian supports of posterior probabilities (PP) to delimited species. Pie charts on each node indicate nodal support of SH-aLRT ≥ 0.80 and/or UFBoot2 ≥ 0.95. Ophion kobensis Shimizu sp. nov. was recovered as a single MOTU (PP = 0.866).

Table 2.

Divergences of p-distances among the Ophion minutus species-group

graphic file with name zoolstud-62-027-t002.jpg

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Phylogeny

Dataset: The final dataset consisted of 214 sequences of the CO1 gene (199 from the bPTP MOTUs of Ophion, 13 of the other ophionine genera, and two of the non-ophionines) and 555 bp in total length (Supplementary file S3), comprising 224 conserved and 331 variable sites, with 264 parsimony informative sites.

Phylogeny: The TIM+I+G model for the 1st and 2nd codon positions and the GTR+I+G for the 3rd codon were selected as the optimal substitution model under AIC. Terminals with the BOLD systems project code of “HYSAF” and “HYSA” were strongly nested into ophionine outgroups (Figs. 2, S2). Our phylogeny result was mostly consistent with that reported by Schwarzfeld et al. (2016) and Johansson and Cederberg (2019). Except for the O. minutus species-group, Ophion species were weakly supported as a monophyletic clade (referred to as Ophion s. str. here; Figs. 2, S2), with SH-aLRT = 0.93 and UFBoot2 = 0.72. However, Hellwigiella dichromaptera (ASQIC060-09) was nested in the Ophion s. str. Among the Ophion s. str., the monophyly of the following species-groups were strongly recovered: the O. scutellaris species-group (SH-aLRT = 0.98 and UFBoot2 = 0.99) and the O. slossonae species-group (SH-aLRT = 0.99 and UFBoot2 = 1.00).

Fig. 2.

Fig. 2.

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Maximum likelihood CO1 gene phylogeny of the genus Ophion based on bPTP MOTUs. Gray terminals indicate the apparently non-Ophion species although they have been deposited in the database as Ophion species. Ophion s. str. was collapsed (cf. Fig. S2 for whole tree). Pie charts on each node indicate a nodal support of SH-aLRT ≥ 0.80 and/or UFBoot2 ≥ 0.95. The Ophion minutus species-group and two well-recovered clades were represented as coloured clades. Ophion kobensis Shimizu sp. nov. was recovered as a sister to O. ventricosus. (SH-aLRT = 0.99 and UFBoot2 = 1.00).

The O. minutus species-group (Figs. 2, S2) was weakly recovered as a monophyletic clade (SH-aLRT = 0.84 and UFBoot2 = 0.76) and placed as a sister to Ophion s. str., together with Xylophion sevrapek (ICHSA002-12) and Ophion sp. (GBAH21304-19). However, the monophyly of Ophion s. lat. (the O. minutus species-group + Ophion s. str.) was not recovered. Ophion kobensis sp. nov. of the group was strongly recovered as a member of the O. ventricosus clade (SH-aLRT = 1.00 and UFBoot2 = 1.00). The O. minutus clade was also strongly recovered (SH-aLRT = 0.96 and UFBoot2 = 0.99).

TAXONOMY

As mentioned above, O. kobensis sp. nov. was recovered as a single species and was easily distinguishable from other previously described species based both on the morphological and molecular species delimitations. Therefore, it is described as a new species below.

Ophion kobensis Shimizu sp. nov.

[Japanese name: Kobe-amebachi]

(Figs. 3–5)

urn:lsid:zoobank.org:act:AD2F2A64-1324-4B7B-A339-4963585070F9

Materials examined (type series): Holotype ♀ (NIAES) (SHOP24) (Figs. 3–5): 34°43'27"N 135°13'58"E (110 m alt.), Kobe Univ., Kobe City, Hyogo Pref., Japan, 29.IV.2015, Masato Ito leg. Paratype ♀ (NIAES) (SHOP190): N34.281 E135.4077, Kokawa, Kinokawa City, Wakayama Pref., Japan, 1–31. V.2016, Takuto Hirooka leg. by light trap.

Etymology: Named after the type locality, Kobe, Hyogo, Japan.

Description of holotype female (Figs. 3–5): Body entirely more or less shiny (Figs. 3, 4), and length ca. 13.5 mm.

Fig. 3.

Fig. 3.

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Habitus of female holotype of Ophion kobensis Shimizu sp. nov.

Head buccate behind compound eyes in dorsal view (Fig. 4c) with GOI = 1.3 (Fig. 4b). Antenna with 49 flagellomeres; FL1 3.2× longer than wide and 1.5× longer than FL2; FL20 and FL40 1.6× longer than wide. Lower face transverse and 1.2× wider than high, strongly polished, and densely punctate with setae (Fig. 4a); clypeus 2.2× wider than high, slightly convex in profile, and ventral margin evenly rounded in frontal view and weakly impressed and sub-blunt in profile (Fig. 4a, b). Malar space 0.4× longer than basal mandibular width. Mandibular gape slightly acute angle, with internal angle. Posterior ocelli not touching eye (Fig. 4c). OOD /LOD = 0.4; IOD /LOD = 0.5; POD /LOD = 1.0. Occipital carina complete, mediodorsally evenly curved; ventral end joining oral carina.

Mesosoma entirely weakly shagreened to polished with sparse to dense punctures and setae (Fig. 4d–f). Mesoscutum 1.3× longer than width (Fig. 4e); polished (Figs. 4d–f); notauli absent (Fig. 4d–f); and evenly rounded in profile (Fig. 4d). Scutellum moderately convex in profile (Fig. 4d) and narrowed posteriorly in dorsal view with lateral longitudinal carinae along anterior 0.4 of scutellum (Fig. 4e, f). Epicnemial carina, in lateral view, almost straight and not inclined to anterior and upper end evenly curved to anterior (Fig. 4d); and, in antero-ventral view, virtually evenly curved, therefore pleurosternal angles strongly obtuse and sternal angles almost absent. Submetapleural carina broadened anteriorly (Fig. 4d). Propodeum rugose and almost evenly rounded in profile (Fig. 4d, f); anterior transverse carina absent (Fig. 4d, f); posterior transverse carina strongly defined laterally but vestigial and ill-defined centrally; lateromedian longitudinal carina present between about anterior transverse carina and posterior end of propodeum (Fig. 4d, f); lateral longitudinal carina complete between posterior longitudinal carina and posterior end of propodeum (Fig. 4d, f); spiracle elliptical and joining pleural carina (Fig. 4d, f).

Fig. 4.

Fig. 4.

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Detailed photographs of Ophion kobensis Shimizu sp. nov. (a) head, frontal view; (b) head, lateral view; (c) head, dorsal view; (d) mesosoma, lateral view; (e) mesoscutum and scutellum, dorsal view; (f) mesosoma, dorso-lateral view; (g) metasoma, lateral view.

Wings (Fig. 5). Fore wing (Fig. 5a) length ca. 10.5 mm with AI=1.4, CI=0.5, DI=0.6, ICI=0.7, SDI = 1.2, SRI = 0.5; vein 2r&RS weakly thickened and angulated proximally; vein 1m-cu&M between ramellus and bulla as long as 1m-cu&M between bulla and 2m-cu; ramellus short and less longer than bulla of 1m-cu&M; angles between 1m-cu&M and M (between 2rs-m and 2m-cu) 153°; vein 1cu-a postfurcal to M&RS by 0.1× length of 1cu-a. Hind wing (Fig. 5b) with NI = 1.2; vein RA with 8 uniform hamuli.

Fig. 5.

Fig. 5.

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Wings of Ophion kobensis Shimizu sp. nov. (a) fore and (b) hind wing.

Legs. Hind leg with trochantellus shorter than wide in dorsal view; femur 5.1× longer than wide; inner spur 1.6× longer than outer one and 0.5× longer than basitarsus; basitarsus 2.5× longer than 2nd tarsomere; tarsal claw simply pectinate.

Metasoma (Fig. 4g) with DMI = 0.9, PI = 1.6. T1–2 polished; T3 and after that shagreened; posterior margin of anterior sclerotised section of S1 distinctly posterior to spiracle at nearly equal distance to shortest distance between spiracle and ventral margin of T1; T2 2.2× longer than deep; T6 distinctly vertically long and 0.4× longer than deep. Ovipositor sheath not longer than posterior depth of metasoma.

Colour (Figs. 3–5). Entirely blackish. Head with antennae brown; lower face black, and inner orbit and clypeus yellow; mandible yellow and apical teeth black; outer orbit yellow; frons, stemmaticum, vertex, and occiput black. Mesosoma entirely black, except for ventral end of pronotum, anterior margin of mesopleuron, and scutellum yellow. Wing membrane strongly yellowish; veins dark brown to black. Legs with all coxae and hind femur black; fore and mid legs excluding coxae, hind trochanter and trochantellus more or less testaceous; hind legs after tibia testaceous to brownish. Metasoma entirely dark brown to black except for testaceous parts on T1–3 and S1–3 and ovipositor.

Variation in females (n = 2): Body length 13.5–15.5 mm. Head with GOI = 1.3–1.5. Antenna with 49–55 flagellomeres; FL1 3.2–3.3× longer than wide; FL20 1.3–1.6× and FL40 1.5–1.6× longer than wide. Clypeus 2.1–2.2× wider than high. Malar space 0.3–0.4× longer than basal mandibular width. OOD /LOD = 0.3–0.4. Mesoscutum 1.3–1.4× longer than width. Fore wing length ca. 10.5–12.5 mm with AI = 1.3–1.4, ICI = 0.7–0.8, SDI = 1.0–1.2; angles between 1m-cu&M and M (between 2rs-m and 2m-cu) 148–153°; vein 1cu-a postfurcal to M&RS by 0.1–0.2× length of 1cu-a. Hind wing with NI = 1.0–1.2; vein RA with 7–8 uniform hamuli. Hind leg with femur 5.1–5.9× longer than wide. Metasoma with DMI = 0.9–1.0; T2 polished to shagreened; T2 2.2–2.5× longer than deep; T6 0.4–0.5× longer than deep.

DNA barcode: Two CO1 sequences were deposited at DDBJ with the following accession numbers: LC756991 (SHOP24) and LC756992 (SHOP190).

Male: Unknown.

Distribution: Western portion of Honshu Island (Hyogo and Wakayama Prefs.), Japan.

DISCUSSION

Species delimitation

Although the estimated number of molecular species supported by our bPTP analysis was stable, many unsupported clusters were recognised and the number of such clusters was variable, resulting in a wide range of variation in the estimated species number by bPTP. This could be because the low divergence rate between the Ophion species sometimes provides biased input trees and complicates molecular species delimitation analysis. Many species are delimited within narrow divergence ranges, as shown in Figure 1. Such a small divergence of the CO1 sequences has already been reported in Darwin wasps’ studies of the Diplazon (Klopfstein et al. 2016) and Ophion (Johansson and Cederberg 2019), with potential negative effects on CO1 sequences caused by Wolbachia infections. Therefore, CO1 sequences are sometimes insufficient to delimit and/or describe a species, as indicated by many published studies (Schwarzfeld and Sperling 2015; Johansson and Cederberg 2019; Meier et al. 2022; Zamani et al. 2022). As suggested by many researchers (Ahrens et al. 2021; Meier et al. 2022; Zamani et al. 2021 2022), we would like to emphasize that the result of the molecular species delimitation analysis should always be carefully interpreted as a complementary source to other delimitation sources, such as morphological and ecological sources. Furthermore, maximalist integrative approaches, rather than recently spotlighted revolutionary minimalist approaches, should be employed (Meierotto et al. 2019; Sharkey et al. 2021a b; Zamani et al. 2021 2022; Meier et al. 2022), especially for challenging taxa, such as Ophion, as in Schwarzfeld and Sperling (2014). Additional nuclear gene markers, which have a lower risk of introgression or Wolbachia infections than mitochondrial genes (e.g., internal transcribed spacer 2 (ITS2)), have partially resolved issues with CO1 genes (e.g., Klopfstein et al. 2016; Schwarzfeld and Sperling 2014). However, published markers are not universal for a wide taxonomic range and are difficult to use with many indels. Therefore, additional universal gene markers should be explored.

Both morphological and phylogenetic evidence indicate that O. kobensis sp. nov. is closely related to O. ventricosus. The interspecific divergences of the majority of morphological characters between these species are relatively small. However, body colour and mesosomal characters exhibit considerably large divergences between them and provide useful diagnostic characteristics for morphological species delimitation. In particular, the characteristics of the epicnemial carina are useful for distinguishing them, as suggested in previous taxonomic studies of Ophion (Brock 1982; Broad 2018; Johansson and Cederberg 2019). The mesopleural furrow is also useful for distinguishing O. kobensis sp. nov. from O. ventricosus, although this character has not been mentioned in the most recent revision of the Swedish Ophion (Johansson and Cederberg 2019). The characteristic black and light-yellow colour patterns of O. kobensis sp. nov. provide excellent diagnostic information for this species. It is rare among Ophion for body colour to provide informative diagnostic characters, as in the O. ventricosus clade, because most Ophion species (as well as other genera of Ophioninae) usually exhibit uniform testaceous colour patterns.

Ophion kobensis sp. nov. and O. ventricosus exhibit a disjunct distribution, and intermediate species are currently unknown. However, taxonomic efforts on the temperate Ophioninae are strongly biased toward Europe and its adjacent areas, and further studies of the Asian fauna of Ophion are strongly needed to reveal the biodiversity and biogeography of Ophion species.

Quality of data from databases

Although the doubtful sequences were removed from the dataset in the present study, some terminals with the BOLD systems project code of “HYSAF” and “HYSA” have very long branches in the species delimitation input tree (Figs. 1, S1) and are strongly nested into ophionine outgroups in the phylogenetic tree (Figs. 2, S2), indicating that these wasps are not Ophion species. Examining sequenced specimens using low-quality photographs on BOLD systems revealed that the fore wing vein 2r&RS of HYSA was strongly thickened and angulated in the proximal half, also indicating that these wasps belong to other genera of the tribe Ophionini. These misidentifications of sequenced specimens are likely caused by the extreme difficulties of morphology-based species identification of parasitoid wasps for many non-taxonomists. To improve the data quality of databases and achieve accurate results, confirmation of the species identification of sequenced samples by taxonomists is necessary, and the accessibility of sequenced specimens is, therefore, most important. However, sequence-based databases (i.e., GenBank and DDBJ) sometimes lack information on sequenced specimens, making it impossible to verify the quality of identification. Therefore, the demand for specimen-based DNA sequence databases, like BOLD systems will continue to increase.

Status of the O. minutus species-group

Although the monophyly of the O. minutus species-group is not strongly supported, the sister relationship between the O. minutus and the O. ventricosus clades was suggested in our molecular phylogenetic analysis, as in all recently published molecular phylogenetic trees (Schwarzfeld et al. 2016; Johansson and Cederberg 2019). These wasps can be distinguished from the remaining species of Ophion s. str. by both morphology and molecular phylogeny and should therefore be treated as a separate new genus, as suggested by Johansson and Cederberg (2019). However, high CO1 gene divergences with p-distances of 12.2–14.3% and differences in morphological characteristics (i.e., the typical testaceous ophionoid facies and small-sized wasps with more or less distinctly thickened and angulated fore wing vein 2r&RS proximally in the O. minutus clade versus the unusual blackish ophionoid facies and medium-sized wasps with slightly thickened and angulated 2r&Rs proximally in the O. ventricosus clade) between the two clades among the O. minutus species-group also suggest that these two clades should be treated as separate species-groups or genera. Further global phylogenetic studies of the tribe Ophionini, as well as the subfamily Ophioninae, are needed to assess this because the previous molecular phylogenies mainly focused on taxa from the Nearctic and Western Palaearctic regions, while mostly excluding Afrotropical, Australasian, Neotropical, Eastern Palaearctic and Oriental samples (Schwarzfeld et al. 2016; Johansson and Cederberg 2019; Johansson et al. 2021). In addition, previous phylogenetic analyses of Ophion have not been sufficiently resolved (Schwarzfeld et al. 2016; Johansson and Cederberg 2019; Johansson et al. 2021; the present study), and phylogenomic methods should be employed in future studies of Ophion to revise systematics and reveal their evolutionary histories.

CONCLUSIONS

The Ophion minutus species-group was recorded for the first time from Japan and the Far East, with the description of the new species (O. kobensis Shimizu sp. nov.) based on integrated morphological and molecular species delimitation methods. Phylogenetic analysis indicated that the O. minutus species-group was sister to Ophion s. str., and the two clades within the O. minutus species-group (O. minutus and O. ventricosus clades) have diverged considerably. These results suggest that the species-group and the two included clades should potentially be treated as separate species-groups or genera, and further global phylogenomic reconstruction is strongly needed to assess and reveal their evolutionary history. Our findings support previous integrative taxonomic and phylogenetic studies of Ophion and represent a first fundamental step for studies focused on the challenging Japenese and Far Eastern Ophion.

Supplementary materials

Fig. S1.

The complete result of the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion, based on the maximum likelihood CO1 gene topology reconstructed by IQ-TREE2. Stripes indicate delimited molecular operational taxonomic units (MOTUs), and stripe colours indicates a degree of the Bayesian supports of posterior probabilities (PP) to delimited species. Pie charts on each node indicate nodal support of SH-aLRT ≥ 0.80 and/or UFBoot2 ≥ 0.95. The O. minutus species-group is indicated as a colored clade.

Fig. S2.

The complete maximum likelihood CO1 gene phylogeny of the genus Ophion based on bPTP MOTUs.

Supplementary file S1.

The final multiple sequence alignment dataset for the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion based on DNA barcodes. The dataset consists of 616 haplotypes (= sequences) and 567 bp in length.

Supplementary file S2.

The most supported result of the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion based on DNA barcodes. A total of 199 MOTUs were delimited.

Supplementary file S3.

The final multiple sequence alignment dataset for the maximum likelihood CO1 gene phylogenetic analysis of the genus Ophion based on bPTP MOTUs. The dataset consists of 214 sequences (199 from the bPTP MOTUs of the Ophion, 13 of the other ophionine genera, and two of the non-ophionines) and 555 bp in length.

zoolstud-62-027-s005.fas (124.3KB, fas)

Acknowledgments

This work and the new species name were registered with ZooBank under urn:lsid:zoobank.org:pub:2BFD9FA4-32E7-44EC-BC8B-3DC69D5E4123. The authors thank Dr Masato Ito, Mr Shunpei Fujie, and Mr Takuto Hirooka for providing valuable samples of the Japanese Ophioninae and supporting the authors’ studies of Darwin wasps. This study was partly supported by the JSPS KAKENHI Grant Number 19H00942 to KM.

Footnotes

Authors’ contributions: SS designed the study, carried out the morphological observations and molecular analysis, and drafted the manuscript; and KM helped draft the manuscript; both the authors read and approved the final manuscript.

Competing interests: The authors have declared that they have no competing interests with the present study.

Availability of data and materials: Type series of the newly described species in the present study are preserved at NIAES, meaning that it is publicly available. DNA sequences are deposited in databases (DDBJ: LC756991 and LC756992). All data are provided within the paper and supplemental materials.

Consent for publication: Both the authors agree to the publication of the present study in Zoological Studies.

Ethics approval consent to participate: Not applicable.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1.

The complete result of the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion, based on the maximum likelihood CO1 gene topology reconstructed by IQ-TREE2. Stripes indicate delimited molecular operational taxonomic units (MOTUs), and stripe colours indicates a degree of the Bayesian supports of posterior probabilities (PP) to delimited species. Pie charts on each node indicate nodal support of SH-aLRT ≥ 0.80 and/or UFBoot2 ≥ 0.95. The O. minutus species-group is indicated as a colored clade.

zoolstud-62-027-s001.png (3MB, png)
Fig. S2.

The complete maximum likelihood CO1 gene phylogeny of the genus Ophion based on bPTP MOTUs.

zoolstud-62-027-s002.png (1.9MB, png)
Supplementary file S1.

The final multiple sequence alignment dataset for the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion based on DNA barcodes. The dataset consists of 616 haplotypes (= sequences) and 567 bp in length.

zoolstud-62-027-s003.txt (25KB, txt)
Supplementary file S2.

The most supported result of the bPTP molecular species delimitation test of Ophion kobensis Shimizu sp. nov. among the genus Ophion based on DNA barcodes. A total of 199 MOTUs were delimited.

zoolstud-62-027-s004.fas (376KB, fas)
Supplementary file S3.

The final multiple sequence alignment dataset for the maximum likelihood CO1 gene phylogenetic analysis of the genus Ophion based on bPTP MOTUs. The dataset consists of 214 sequences (199 from the bPTP MOTUs of the Ophion, 13 of the other ophionine genera, and two of the non-ophionines) and 555 bp in length.

zoolstud-62-027-s005.fas (124.3KB, fas)

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