Revisiting the host use and phylogeny of Colastomion Baker (Hymenoptera, Braconidae, Rogadinae), with a new host record from Japan

Kota Sakagami; So Shimizu; Shunpei Fujie; Kaoru Maeto

doi:10.3897/jhr.77.52774

Abstract

We report the solitary parasitism by Colastomion formosanum (Watanabe) (Hymenoptera, Braconidae, Rogadinae) on the larva of Nevrina procopia (Stoll) (Lepidoptera, Crambidae) feeding on Turpinia ternata Nakai (Staphyleaceae) in Amami Ôshima Is., Japan. This is the first host record for the genus Colastomion Baker outside of Papua New Guinea. We have also inferred the phylogenetic relationships of Colastomion species using Bayesian and maximum likelihood approaches, based on the mitochondrial cytochrome oxidase 1 gene. The results indicate two major clades–solitary and gregarious parasitoids–within Colastomion. Colastomion formosanum belongs to the clade of solitary parasitoids that specifically parasitize the crambid subfamily Spilomelinae. Plant-host-parasitoid associations and the evolutionary scenario of the host use of Colastomion are discussed.

Keywords

Colastomion formosanum, Crambidae, endoparasitoid, mtCO1, mummy, Nevrina procopia

Introduction

The family Braconidae is one of the largest lineages in Hymenoptera, containing 21,221 valid species worldwide (Yu et al. 2016). Braconid wasps are parasitoids of various insects (e.g. Lepidoptera, Coleoptera, and Hymenoptera), including important agricultural pests (Wharton 1993; Quicke 2015; Maeto 2018). Their amazing diversity has resulted from the complicated biological interactions with host insects as well as contrasting lifestyles, e.g. solitary vs. gregarious, ecto- vs. endoparasitic, or idio- vs. koinobiont (Shaw 1988). Understanding their biology and phylogeny lends us a greater appreciation of the evolutionary pattern of host-parasitoid systems. Further, from a practical standpoint, the study of Braconidae can also be useful to leverage their parasitic abilities for biological pest control (Quicke 2015).

The subfamily Rogadinae Förster sensu stricto contains diverse koinobiont endoparasitoids of Lepidoptera and comprises 63 genera and 1,243 species that are distributed across all zoogeographical regions, except for polar regions (Quicke 2015; Yu et al. 2016). One of the marked biological features of the rogadine parasitoids is the mummification of their host larvae (Chen and He 1997; Zaldívar-Riverón et al. 2008).

Colastomion Baker is an uncommon genus of Rogadinae and comprises 15 species that occur throughout Papua New Guinea, southern East Asia, and Africa (Quicke et al. 2012; Yu et al. 2016). Crambid larvae have been reported to be hosts of Colastomion only in Papua New Guinea (Quicke et al. 2012). While the host larvae of Colastomion are typically mummified like other rogadine parasitoids, the mummy has been illustrated for only one species of Colastomion to date (Zaldívar-Riverón et al. 2008). Also, although the phylogenetic placement of Colastomion within Rogadinae has been largely established (Zaldívar-Riverón et al. 2008), the phylogenetic relationships within the genus has not been firmly estimated (Quicke et al. 2012).

Recently, the first and second authors (KS and SS) conducted field studies in Amami Ôshima Is., Kagoshima Pref., Japan, and obtained Colastomion specimens from host caterpillars and in a light trap. Here, we identified the Colastomion specimens as a species and conducted phylogenetic analyses based on the mitochondrial cytochrome oxidase 1 (CO1). These provide the first host record of Colastomion outside of Papua New Guinea, detailed mummy morphology, and the evolutionary scenario of host use within Colastomion.

Materials and methods

Field collection and rearing

A field study was conducted at Naze-Ôaza-Chinase (28°21'N, 129°26'E, 16 m alt.), Amami Ôshima Is., Kagoshima Pref., southwest Japan on 11 April 2019. The study site was the evergreen broad-leaved forest dominated by Ficus spp., Ligustrum japonicum Thunb. and Pittosporum tobira (Thunb.) W.T.Aiton. KS collected larvae of Nevrina procopia (Stoll) (Lepidoptera, Crambidae, Spilomelinae) as they were feeding on Turpinia ternata Nakai (Staphyleaceae). The larvae rolled young leaves roughly and hid themselves with the rolled leaves (Fig. 1). Collected larvae were reared in plastic cases under laboratory conditions (25 °C, 16:8 h light:dark). Emerged insects were killed in a freezer.

Figure 1.

Leaves of Turpinia ternata Nakai rolled by a caterpillar of Nevrina procopia (Stoll).

Further specimen was collected in a field study at Nishinakama (28°15'47.3"N, 129°24'56.1"E, 120 m alt.), Sumiyô Town, Amami Ôshima Is. on 12 July 2019, using High Intensity Discharge light traps by SS.

Specimens examined, repositories and identification

The examined specimens of Colastomion from Japan are deposited in the Institute for Agro-Environmental Sciences, NARO, Tsukuba, Japan (NIAES) and Osaka Museum of Natural History, Osaka, Japan (OMNH): 1♀, Naze-Ôaza-Chinase, Amami City, Amami Ôshima Is., Kagoshima Pref. (28°21'N, 129°26'E, 16 m alt.), 11.IV.2019, K. Sakagami leg. (by rearing host) (NIAES) [DDBJ–LC485659]; 1♀, Nishinakama, Sumiyô Town, Amami Ôshima Is., Kagoshima Pref. (28°15'47.3"N, 129°24'56.1"E, 120 m alt.), 12.VII.2019, S. Shimizu leg. (light trap) (NIAES) [DDBJ–LC499982]; 1♀, Mt. Yui-dake, Setouchi Town, Amami Ôshima Is., Kagoshima Pref., 24.VIII.2004, H. Makihara leg. (sweeping net) (OMNH); 1♂, Yona, Kunigami Village, Okinawa-hontô Is., Okinawa Pref., 29.VI.2013, S. Fujie leg. (sweeping net) (OMNH).

We identified Colastomion specimens based on Watanabe (1932, 1934), Tenma (2002), and Quicke et al. (2012): the specimens were morphologically similar to C. formosanum. To make sure that Colastomion specimens are C. formosanum, all the specimens were compared to photos of the Taiwanese specimens of C. formosanum deposited in the Senckenberg Deutsches Entomologisches Institut, Müncheberg, Germany (SDEI), which include the holotype of the species as follows: holotype ♂, Kankau, Changkou (Koshun), 22.IV.1912, H. Stauter leg.; 1♂, Kankau, Changkou (Koshun), VIII.1912, H. Stauter leg.; 1♀, Kankau, Changkou (Koshun), IV.1912, H. Stauter leg. The photos were provided by Taeger (2020), published online (https://doi.org/10.6084/m9.figshare.11984355.v1).

Morphological observation, photo technique and terms

Morphological observation was conducted using a stereoscopic microscope (SZ61, OLYMPUS, Tôkyô, Japan). Multi-focus photographs were taken using a single lens reflex camera (α7II, Sony, Tôkyô, Japan) with micro-lens (LAOWA 25 mm F2.8 2.5–5X ULTRA MACRO, Anhui Changgeng Optics Technology Co., Ltd, Hefei, China and A FE 50mm F2.8 Macro SEL50M28, Sony, Tôkyô, Japan). The photos were captured in RAW format and developed using Adobe Lightroom Creative Cloud. Then, they were stacked using Zerene Stacker and edited in Adobe Illustrator 2019. Morphological terms follow those of Quicke et al. (2012).

Molecular technique

Part of the mitochondrial protein-coding cytochrome c oxidase 1 (CO1) gene, often referred to as “barcoding gene”, of two individuals of C. formosanum was sequenced for phylogenetic analysis. DNA was extracted from the right middle leg using the DNeasy Blood and Tissue Kit (Qiagen, Düsseldorf, Germany). For amplification, the following primers were used: LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994). Polymerase chain reactions (PCR) were conducted using KOD FX NEO kit (Toyobo, Ōsaka, Japan), and PCR conditions were 94 °C for 2 min as initial denaturation, followed by 35 cycles of denaturation (10 sec at 98 °C), annealing (30 sec at 48 °C), and extension (30 sec at 68 °C), and then a final extension at 72 °C for 10 min. PCR product was purified using Illustra GFX kit (GE Healthcare Life Sciences, Marlborough, USA). The purified product was amplified with the same primers using the BigDyeTM Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, USA). Cycle sequencing products were purified using the 3.0 M sodium acetate, 95% ethanol, 70% ethanol, and Hi-Di formamide. Cycle sequencing reactions were run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Waltham, USA), and the forward and reverse sequences were assembled using the DNA Dynamo Sequence Analyze Software (Blue Tractor Software, North Wales, UK). Finally, we deposited the obtained sequence to DNA Data Bank of Japan (DDBJ).

Phylogenetic analyses

In order to exclude the taxon sampling bias, a single sequence for each species was selected from 42 sequences of CO1 of the nine Colastomion species from Papua New Guinea and Benin deposited in the DNA databases (Quicke et al. 2012). Megarhogas maculipennis Chen & He and Myocron sp., closely related genera to Colastomion (Zaldívar-Riverón et al. 2008), were selected as outgroups. A total of 10 sequences from ingroup and two from outgroups were used for analyses (see Table 1).

Table 1.

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GenBank and DNA Data Bank of Japan accession numbers and information for specimens used for the phylogenetic analyses.

Species	Identifer	Locality	Latitude / longitude	Date	Collector	Accession number
Colastomion crambidiphagus	DLJ Quicke	PNG: Madang, Wanang	5.23088S, 145.182E	16.II.2007	local collector	JF963127
Colastomion formosanum	K Maeto	JPN: Amami-Oshima, Kagoshima	28.3500N, 129.433E	11.IV.2019	K. Sakagami	LC485659
Colastomion gregarius	DLJ Quicke	PNG: Madang, Wanang	5.23088S, 145.182E	24.V.2007	local collector	JF963128
Colastomion maclayi	DLJ Quicke	PNG: East Sepik, Yapsiei	4.62825S, 141.097E	27.I.2004	local collector	JF271312
Colastomion madangensis	DLJ Quicke	PNG: Madang, Wanang	5.23088S, 145.182E	24.V.2007	local collector	JX034716
Colastomion masalaii	DLJ Quicke	PNG: West Sepik, Sandaun, Utai	3.38405S, 141.586E	28.VII.2004	local collector	JF271307
Colastomion parotiphagus	DLJ Quicke	PNG: Madang, Wanang	5.23088S, 145.182E	30.V.2007	local collector	JX034711
Colastomion pukpuk	DLJ Quicke	PNG: East Sepik, Wamangu	3.78713S, 143.652E	3.XI.2005	local collector	JF271303
Colastomion wanang	DLJ Quicke	PNG: Madang, Wanang	5.23088S, 145.182E	29.IV.2005	local collector	JF271302
Colastomion sp.	–	BEN	–	–	–	AY935370
Myocron sp.	–	KEN	–	5.V.2005	R Copeland	JN278218
Megarhogas maculipennis	–	THA: Chanta Bari, Pong Nani Ron	–	–	–	EU979615

Multiple sequence alignment was conducted in MAFFT v7.409 (Katoh and Toh 2008) using the L-INS-I algorithm. The aligned sequences were checked visually. Subsequently, they were manually optimized for phylogenetic analysis.

Using Bayesian Inference (BI) and maximum likelihood (ML) approaches, phylogenetic analyses were performed. Evolutionary models were determined using Kakusan4 v4.0 (Tanabe 2011). The best-fit models were selected based on the lowest corrected Akaike information criterion (AICc) for ML and the lowest Bayesian Information Criterion (BIC) for BI.

For the ML analysis, we used RAxML v8.2.10 (Stamatakis 2014) with 1,000 bootstrap replications, the codon separate model, and GTRGAMMA as a substitution model.

For the BI analysis, we used MrBayes v3.2.2 (Ronquist et al. 2012) with two independent runs of a Bayesian Markov chain Monte Carlo (MCMC) analyses of eight chains each, heating at 0.1, as well as random starting trees with trees sampled every 1,000^th generations for 10,000,000 generations. If the average standard deviation of split frequencies was below 0.01, chain stationarity was checked with Tracer v1.6 (Rambaut and Drummond 2007) and two converged MCMC runs were considered adequate (Ronquist and Huelsenbeck 2003). The anterior half of the generations were discarded as a conservative burn-in and estimates were obtained for the harmonic means of the likelihood scores from the remaining half generations using the sump command. A final check of the convergence of the runs by the value of the potential scale reduction factor was conducted and a majority-rule consensus tree was obtained using the sumt command. The phylogenetic tree was edited using FigTree v1.4.3 (Rambaut 2006–2016) and Adobe Illustrator 2019.

We consider the node to be supported by either the Baysian posterior probabilities (PP) > 0.95 or the bootstrap (BT) > 80%.

Results

Rearing and mummy morphology

One adult female of Colastomion emerged from a mummified final instar larva of N. procopia on 30 April 2019 (Figs 2, 3). The mummy remained unfixed within rolled leaves. It was mildly hardened, having a posterolateral and irregular, noncircular emergence hole (Fig. 4). Five adults of N. procopia emerged from unparasitized pupae, of which two emerged on 30 April, two on 8 May, and one on 9 May 2019.

Figure 2.

Female adult wasp of Colastomion formosanum (Watanabe) from Amami Ōshima Is., Japan A habitus B head, frontal view C head and mesosoma, lateral view D head and mesosoma, dorsal view E propodeum, dorsal view F metasoma, dorsal view.

Identification and adult morphology

All Japanese Colastomion specimens were identified as C. formosanum because of yellow face, antennae (although apical segments were brown) and legs (although middle and hind coxae and telotarsi were brown), sharply contrasting with brown mesosoma and metasoma (Fig. 2A, B); epicnemial area finely strigose, precoxal sulcus shallowly impressed and with some rugae, and pleural sulcus crenulate (Fig. 2C); notauli deep and weakly crenulate (Fig. 2D); propodeum rugose and with complete midlongitudinal carina (Fig. 2E); 1^st metasomal tergite 1.7–1.8× longer than posteriorly wide; 2^nd tergite as long as maximally wide (Fig. 2F); pterostigma entirely dark brown; fore wing cu-a postfurcal to 1-M (Fig. 3); and subquadrate vein 2-SC+R of hind wing (Fig. 3).

Figure 3.

Wings of Colastomion formosanum (Watanabe).

A female specimen from Taiwan shows a distinct protuberance on the base of the first metasomal tergite (Taeger 2020), whereas it is absent in males (including holotype) from Taiwan (Taeger 2020) and females and a male from Japan. The protuberance will probably be due to ontogenetic deformation in the individual. On the other hand, the sculpture of epicnemial area and precoxal sulcus tends to be stronger in females than in males, which is most likely a sexual variation.

Figure 4.

A larva of Nevrina procopia (Stoll) mummified by Colastomion formosanum (Watanabe), lateral view.

Phylogeny

The Bayesian majority-rule consensus tree of Colastomion obtained from the CO1 sequences is shown in Figure 5. Both the BI and ML topologies were consistent with each other. Colastomion formosanum was identified as a sister group of the well-supported clade (C. crambidiphagus and C. parotiphagus) by both the BT and PP. Monophyly of the clade of solitary parasitoids (C. crambidiphagus, C. formosanum, C. maclayi, C. parotiphagus, and C. wanang) plus C. pukpuk was good supported only by the PP. On the other hand, the clade of gregarious parasitoids (C. masalaii, C. gregarius, and C. madangensis) was not fully supported.

Figure 5.

Bayesian majority-rule consensus tree of Colastomion species based on CO1. Posterior probabilities (> 0.80) and bootstrap values (> 50%) are indicated at below of each node (PP / BT). Lifestyles are indicated as shown in Quicke et al. (2012), except for C. formosanum.

Discussion

We have found that N. procopia feeding on rolled leaves of T. ternata are the host species of C. formosanum in Japan. This supports the hypothesis of the host specificity of Colastomion to the crambids subfamily Spilomelinae as mentioned by Quicke et al. (2012), whereas the genus Nevrina of the tribe Udeini is a new host genus and the Staphyleaceae is a new host plant family for Colastomion (Table 2). Interestingly, Quicke et al. (2012) has reported that two solitary species of Colastomion use some species of crambid moths on a certain plant: C. crambidiphagus parasitizes several species of different tribes of Spilomelinae feeding only on the Convolvulaceae, and C. parotiphagus uses various host tribes of moth while mostly feeding on the Rubiaceae. Moreover, all gregarius species (C. gregarious, C. madangensis, and C. masalaii) consistently use Margaroniini on the Moraceae (Table 2). These may indicate that adult wasps search the host larvae by plant cues, such as herbivory-induced plant volatiles (Arimura et al. 2009) or oviposition-induced plant volatiles (Hilker and Fatoaros 2015). It is therefore important to understand the specificity of parasitoids not only in host insects but also in host plants.

Table 2.

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Host tribes (Crambidae: Spilomelinae), host plant families, and lifestyles of Colastomion. Sources: Quicke et al. (2012), except for C. formosanum. Systematics of host tribes follows Mally et al. (2019).

Species	Host tribe	Host plant family	Lifestyle
C. crambidiphagus	Hydririni, Udeini	Convolvulaceae	Solitary
C. formosanum	Udeini	Staphyleaceae	Solitary
C. gregarius	Margaroniini	Moraceae	Gregarious
C. maclayi	Udeini	Rubiaceae	Solitary
C. madangensis	Margaroniini	Moraceae	Gregarious
C. masalaii	Margaroniini	Moraceae	Solitary/gregarious
C. parotiphagus	Agroterini	Malvaceae	Solitary
ditto	Margaroniini	Rubiaceae	Solitary
ditto	Unidentified	Lauraceae, Ulmaceae	Solitary
C. pukpuk	Unidentified	Rubiaceae	Unknown
C. wanang	Udeini	Myrtaceae, Vitaceae	Solitary

Colastomion formosanum (Figs 2, 3) is a solitary endoparasitoid that forms a hard mummy (Fig. 4). Solitary parasitism in Colastomion is considered the ancestral relative to gregarious parasitism because Megarhogas, which is closely related to Colastomion (and is included as an outgroup in our phylogenetic analyses), and most species in Rogadini are solitary (Quicke and Shaw 2005; Zaldívar-Riverón et al. 2008), while the phylogenetic placement of species is still unresolved (Fig. 5). The formation of a hard mummy as displayed by C. formosanum remains unusual within Rogadinae, although various types of host mummies appear to act as a protective roll during parasitoid metamorphosing (Zaldívar-Riverón et al. 2008; Maeto 2018). The hosts of most rogadine genera are killed as prepupae within host cocoons, which can protect the parasitoid larvae and pupae, and thus relatively frail mummies are the norm. In contrast, the hosts of genera that form hard mummies are killed in the larval stage, and the hardness of the mummies plays a vitally important protective role. In the case of C. formosanum, the host is killed in the larval stage probably because the immature host caterpillar is large enough for the parasitoid. Host stage of mummifying, which may vary according to relative size of host caterpillars to parasitoids, would therefore be relevant to the hardness of mummies.

Colastomion formosanum was originally described from Taiwan (as Formosa) (Watanabe 1932), later recorded from Hainan Is., China (Chen and He 1997), and was most recently recorded from Okinawa-hontô Is. and Iriomotejima Is., Japan (Tenma 2002). Our collection of C. formosanum from Amami Ôshima Is., Japan, has therefore expanded the northernmost border of the genus Colastomion as well as the species. It is most likely that C. formosanum has advanced northward from the tropics where species of Colastomion have highly diversified (Table 1; Fig. 5), although further investigations are needed to reveal the biogeographical history of Colastomion. C. formosanum is found only in the subtropical islands of China and Japan, whereas its host moth N. procopia is distributed much more broadly, from southern Japan to China, India, Papua New Guinea and western Africa (Swinhoe 1916; Chandra 1994; Nasu et al. 2013; Poltavsky et al. 2018). It would be interesting to know whether C. formosanum is truly an insular species of East Asia or is also further distributed in continental parts.

Acknowledgements

We thank Satoru Matsubara, Masafumi Harada, and Naoto Shimada (Kobe University) for assistance with the field observation of KS. We are grateful to Andreas Taeger (SDEI) for providing photos. This research is partially supported by the Grant-in-Aid for JSPS Fellows Grant Number 18J20333 to SS and the JSPS KAKENHI Grant Number 19H00942 to KM. We also thank Donald L. Quicke for making very helpful comments.

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