Extinction Threat to a Previously Undescribed Species of Gall Wasp (Hymenoptera: Cynipidae) and Two Associated Parasitoid Species (Hymenoptera: Braconidae and Eulophidae) on a Threatened Rose

Abstract Diplolepis ogawai Abe and Ide sp. nov. (Hymenoptera: Cynipidae) induces galls on Rosa hirtula (Regel) Nakai (Rosales: Rosaceae), which is endemic to a restricted area of Honshu, the main island of Japan. The gall is induced mainly on the leaf of R. hirtula in spring and the mature gall falls to the ground in early summer. The gall-inducing wasp emerges from the gall on the ground in the following spring, suggesting that D. ogawai is univoltine. From spring to summer, the braconid Syntomernus flavus Samartsev and Ku and the eulophid Aprostocetus sp. are parasitic on the larva of D. ogawai in the gall, and the adult wasp of both parasitoid species emerges from the gall on the ground in summer. For S. flavus, this is the first distribution record in Japan and the first host record. Since R. hirtula is threatened with extinction by succession and deforestation, D. ogawai and its two parasitoid wasp species are considered to be at risk of coextinction with the threatened rose. In the event that the population size of this rose species is further reduced, D. ogawai and its parasitoids may become extinct prior to the extinction of R. hirtula. To conserve these three wasp species associated with R. hirtula, protection of remnant vegetation where individuals of this threatened rose species grow is necessary.

To protect endangered and vulnerable species of wild fauna and flora, conservation of their habitats is important (Noss et al. 1997). A better understanding and increased interest among citizens regarding the biodiversity crisis are necessary in order to promote measures to conserve threatened species. For this reason, the Ministry of the Environment in Japan published the 'Red Data Book 2014-Threatened Wildlife of Japan-'. Among the vulnerable plant species designated as 'threatened class II', Rosa hirtula (Regel) Nakai (Rosales: Rosaceae) is endemic to a limited area of Kanagawa, Shizuoka, and Yamanashi Prefectures on the main island of Honshu, Japan (Ministry of the Environment of Japan 2015). This rose species is found in thickets and their margins, and also on marshy slopes at 800-1,800 m above sea level (Ohba 2016). The decrease in the population size over the last 10 years and the probability of extinction within 100 years were estimated to be approximately 31% and 82%, respectively (Ministry of the Environment of Japan 2015). The main threats facing the continued survival of R. hirtula are succession and deforestation (Ministry of the Environment of Japan 2015). Host plant species sometimes harbor diverse insect communities on them (Strong et al. 1984), and in many cases herbivorous insects are quite host specific and hence reliant upon the host plant's viability (Cardoso et al. 2011). If the host plant as a keystone species is at risk of extinction, then there is a risk of a cascade of extinctions of the associated herbivores and their natural enemies (Begon et al. 1996).
Rose gall wasps (Hymenoptera: Cynipidae: Diplolepidini), which consist of the genera Diplolepis Geoffroy and Liebelia Kieffer, are gall inducers associated with Rosa spp. (Csóka et al. 2005, Ronquist et al. 2015. The global phylogenetic relationships among members of the genus Diplolepis have been inferred, suggesting that this genus is composed of a Holarctic leaf-galler clade and a Nearctic stemgaller clade (Zhang et al. 2019. However, the biogeographic origin of this genus has not yet been determined, mainly due to the lack of information about eastern Palearctic species . Based on the substantial flora of Rosa spp. in China (Ohba 1997), the existence of a potentially diverse fauna of rose gall wasps in the eastern Palearctic was pointed out (Abe et al. 2007). In recent years, taxonomic studies on Diplolepis in this region were conducted and several new species were discovered (Wang et al. 2013, Pujade-Villar et al. 2020, Zhu et al. 2021. During a field survey of insect galls, Haruo Ogawa discovered galls on R. hirtula shrubs. He observed that gall wasps and their parasitoids emerged from the galls he reared. Preliminarily, morphological examinations of these wasps revealed that the gall wasp was a member of Diplolepis, and the parasitoids consisted of one braconid and one eulophid species. Many rare and undescribed species of insects become extinct before description (Pimm et al. 2014). In terms of biodiversity conservation, it is important to give a scientific name to a threatened and undescribed species to facilitate communication about it among scientists (Braby and Williams 2016). Because the galls induced by an undescribed species of Diplolepis are only found in a few places, describing the new gall wasp species and identifying both parasitoid species are urgently required. In the present study, in addition to revealing a previously unknown insect fauna on the vulnerable plant, formal description or identification of that Hymenopteran fauna and partial sequences of the mtDNA COI region of these three species are provided. Furthermore, the conservation of the wasps associated with the threatened rose species is discussed.

Collection, Rearing, and Morphological Examination
In 2020 and 2021, fresh galls that had fallen to the ground from R. hirtula shrubs were collected from a few localities in Kanagawa and Shizuoka Prefectures, Japan, by Haruo Ogawa (Table 1). After keeping these galls in shade under field conditions in Susono City, Shizuoka Prefecture, the adults of the gall wasps and their parasitoids emerged from the galls. The wasps were collected and stored in 99% ethanol for morphological observation and DNA analysis. Some specimens of the new species of rose gall wasp, Diplolepis ogawai, and its parasitoids were dried and examined under stereomicroscopes (models S8APO and MZ APO, Leica Microsystems KK, Tokyo, Japan) fitted with digital single-lens reflex camera (model E-30, Olympus, Tokyo, Japan), and under a scanning electron microscope (model JSM-5600LV, JEOL, Tokyo, Japan) at 1.5 kV. The length of body parts was measured with an ocular micrometer. Focus stacking was performed using CombineZP software (available from: https://combinezp.software.informer.com/) for the microscopic images. All images were processed and assembled using the GNU Image Manipulation Program (GIMP 2.10.20; available from https://www.gimp.org/).
In the description of D. ogawai, the following morphological abbreviations are used: POL, postocellar line (the distance between the inner edges of the two lateral ocelli); OOL, ocular-ocellar line (the distance from the outer edge of the lateral ocellus to the compound eye); and LOL, lateral-ocellar line (the distance between the median and lateral ocelli). The morphological terminology for gall wasps follows Ronquist and Nordlander (1989), Melika (2006), and Liljeblad et al. (2008); the description of surface sculptures follows Harris (1979).

Examination of Galls
The shape of nine galls from which wasps of D. ogawai and its parasitoids emerged was observed and their diameters were measured with digital calipers to clarify the effects of parasitism on gall shape and size. The galls were then dissected under a binocular stereomicroscope to determine their contents. To compare the structure of the galls on R. hirtula by D. ogawai with that of the galls induced by Diplolepis japonica (Walker), galls of the latter species were collected from leaves of Rosa multiflora Thunb. by Yoriko Abe in Kyonan-cho, Musashino City, Tokyo on 25 May 2021 and were reared under field conditions on the Ito Campus of Kyushu University until 26 March 2022 when the female wasps emerged. These wasps were identified as D. japonica based on Yasumatsu and Taketani (1967).

DNA Analysis
Partial sequences (658 bp) of the COI gene of mtDNA were determined for one wasp of D. ogawai and that of the braconid using previously described methods (Ide and Abe 2019). DNA of two eulophid specimens were destructively extracted after morphological identification. The primers used in the analysis of the eulophid were: forward; COI_pF2 5ʹ-ACC WGT AAT RAT AGG DGG DTT TGG DAA-3ʹ and reverse; COI_2437d 5ʹ-GCT ART CAT CTA AAW AYT TTA ATW CCW G-3ʹ (Kaartinen et al. 2010). PCR conditions were as follows: 94°C for 2 min, followed by four repeated cycles of 94°C for 30 s, 45°C for 1 min, and 72°C for 1 min. These steps were followed by 34 repeated cycles of 94°C for 30 s, 50°C for 1 min, and 72°C for 1 min, before storage at 4°C. The determined sequences were deposited in GenBank (https://www.ncbi.nlm. nih.gov/genbank/) under the accession numbers OP281689 for D. ogawai, OP281690 for the braconid and OP281691, OP281692 for the eulophid. The COI sequence of D. ogawai was compared with those of other congeners in GenBank using BLAST (https://blast. ncbi.nlm.nih.gov/Blast.cgi; accessed 31 January 2022).

Depository of Voucher Specimens
The data of all the sample details are provided in Table 1.

Nomenclature
This paper and the nomenclatural act(s) it contains have been registered in Zoobank (www.zoobank.org), the official register of the International Commission on Zoological Nomenclature. The LSID (Life Science Identifier) number of the publication is: urn:lsid:zoobank. org:pub:89AAB7FF-3A3B-49B1-9493-D67A0FB72B4A.

Species Identification
Morphological differences between D. ogawai and other congeners were detected and this new species is described below. The parasitoids reared from the new Diplolepis were identified as Syntomernus flavus Samartsev and Ku (Hymenoptera: Braconidae) and Aprostocetus sp. (Hymenoptera: Eulophidae) based on Samartsev and Ku (2020) and Graham (1987), respectively.

Specimens Examined
See Table 1.
Wing surface and margin closely ciliated. Marginal cell of forewing closed with pale anterior margin, 2.3 times as long as wide, 2r curved without median prolongation into the marginal cell. Areolet indistinct. Hind femur without flange. Apex of metatarsal claw bent; base not expanded to lobe. Lengths of forewing and hind tibia 2.75 and 0.96 mm, respectively. Metasoma (Fig. 2E) smooth, longer than mesosoma in lateral view. Metasomal tergum II large (more than half of metasoma) with sparse setae laterally.

Male
Not known.

Remarks
The genus Diplolepis contains 46 species worldwide (Zhu et al. 2021). Of these, D. japonica has been recorded in Japan (Yasumatsu and Taketani 1967). This species is also native to Korea and China (Wang et al. 2013, Ide andAbe 2020), but the Chinese population could be an undescribed species (Pujade-Villar et al. 2020). The Diplolepis ogawai female can be distinguished from the D. japonica female by the following four morphological characteristics: 1) metasoma is dark brown and partially reddish-brown in D. japonica, but black in D. ogawai; 2) mesoscutal impression is present in D. japonica, but absent in D. ogawai; 3) areolet is distinct in D. japonica, but indistinct or absent in D. ogawai; 4) forewing is infuscate along all the veins of the marginal cell in D. japonica, but not infuscate in D. ogawai (Fig. 1B).
D. ogawai is most similar to the Chinese species, Diplolepis valtonyci Zhu, Wang, and Pujade-Villar. The D. ogawai female can be distinguished from the D. valtonyci female by the following three morphological characteristics: 1) metasoma is chestnut brown in D. valtonyci, but black in D. ogawai; 2) mesoscutal impression is present in D. valtonyci, but absent in D. ogawai; 3) forewing is infuscate along all the veins of the marginal cell in D. valtonyci, but not infuscate in D. ogawai.

Etymology
The new species is named in honor of Haruo Ogawa, who collected the specimens.

Gall
According to personal observations of Haruo Ogawa, the galls of D. ogawai can be characterized as follows. Galls are induced on both the upper and lower surfaces of the R. hirtula leaf (Figs. 3A and B), and also on the sepal (Fig. 3C) and petiolule (Fig. 3D). They are spherical and smooth without spines. Galls are white, pink, or red when on the host plant, but turn brown after falling on the ground. Each gall has one larval chamber (Fig. 4A). The diameter of the galls from which the gall-inducing wasps emerged was 3.3-3.4 mm (n = 4). In contrast, the gall induced by D. japonica bears sharppointed spines (Yasumatsu and Taketani 1967) and the maximum diameter of galls from which D. japonica females emerged was 6.8-9.8 mm (n = 5). The gall wall is thin (ca. 0.2 mm) in D. ogawai, but thick (2.0-2.7 mm) in D. japonica (Fig. 4). According to Yasumatsu and Taketani (1967), the diameter and wall of D. japonica gall are typically 8-9 mm and 1.5-2.0 mm, respectively.

Life Cycle
A univoltine life cycle was indicated by rearing the wasps (Table 1).

Specimens Examined
See Table 1.

Geographic Distribution
South Korea (Samartsev and Ku 2020), Japan (Kanagawa and Shizuoka Prefectures in Honshu). New to Japan.

Remarks
S. flavus was previously only known from Korea and its host was unknown (Samartsev and Ku 2020). For S. flavus, Japan is a new distributional record. Japanese specimens were identical to the original description of the South Korean specimens (Samartsev and Ku 2020), except that the pterostigma was entirely brown (without a basal yellow patch) in some males (Fig. 5A).
Integumentary fragments of the gall wasp larva were found in two galls from which S. flavus wasps emerged (Fig. 5B). Because only a single braconid wasp emerged from any particular host gall and no dead specimens of S. flavus were found inside, this braconid species is considered to be a solitary parasitoid. The diameters of the two host galls were 3.4 and 3.5 mm. No difference in gall shape and size was observed between galls fostering S. flavus and those fostering D. ogawai. The members of Syntomernus include parasitoids of both cecidomiid gall midges and insect larvae inside dipterocarp fruits; some species of this genus have been reared from fig syconia and one species is known to be phytophagous on syconium tissues (Samartsev and Ku 2020 and references therein). Based on the findings of this study, S. flavus is considered to be a solitary parasitoid of D. ogawai larva. This is the first record of the genus Syntomernus associated with a cynipid gall inducer. This braconid parasitoid depends on D. ogawai from spring to summer. Because the galls of D. ogawai fall to the ground in early summer, no fresh galls induced by D. ogawai are present on R. hirtula when the adult wasp of S. flavus emerges from its host gall on the ground in summer. Therefore, the host(s) of S. flavus from summer to the following spring is unknown.

Specimens Examined
See Table 1.

Remarks
The genus Aprostocetus contains more than 800 species worldwide (Noyes 2019). Of these, six species have been recorded in Japan (Matsuo 2020). Detailed morphological comparisons among the Japanese species are required for precise identification of the Aprostocetus sp. that was obtained in this study. However, due to the lack of detailed morphological descriptions and illustrations, substantial taxonomic efforts will be required in order to characterize the known Japanese species. For example, morphological information of A. pallidipes (Ashmead), a Japanese species, has not been amended since its original description (Ashmead 1904). However, since the taxonomic revision of the Japanese Aprostocetus is beyond  the scope of this paper, the specimens obtained in this study have not been identified to the species level at present.
Integumentary fragments of wasp larvae were found in three galls from which Aprostocetus sp. wasps emerged (Fig. 5D). It is not clear whether they are from a cynipid larva or from some other hymenopteran larva, e.g., S. flavus. Because 6-9 wasps emerged from one host gall and no dead specimens of this eulophid were found in the galls, this eulophid species is considered to be a gregarious parasitoid. The diameters of the three host galls were 2.9, 3.5, and 3.5 mm. One gall was somewhat smaller than the others, but no difference in the shape and size was observed between galls fostering Aprostocetus sp. and those fostering D. ogawai.
As in S. flavus, the host(s) of Aprostocetus sp. from summer to the following spring is unknown.
In addition, the BLAST analysis selected Baryscapus pallidae Graham and Aprostocetus cerricola (Erdös) as the two species with COI sequences most similar to Aprostocetus sp. These two species had COI sequence homologies in the range 90-92% compared to Aprostocetus sp.

Discussion
In this study, we described the gall wasp, D. ogawai. In addition, two parasitoids, S. flavus and Aprostocetus sp., were reared from D. ogawai. This is a new distributional record for S. flavus from Japan.
Both BLAST searches for D. ogawai and its parasitoid Aprostocetus sp. result in about 90% match. Moreover, the three best hits for D. ogawai are two Nearctic and one European species, and the two best hits for Aprostocetus sp. are European species. Such a situation is ascribable to the lack of appropriate sequence data from most of the Asian representatives of Diplolepis spp. and their parasitoids. Effective DNA barcoding requires a well-sampled, extensive reference database. Reliable DNA-based determination of phylogenetic position of this new species needs nuclear data as well as better Asian sampling.
Because the members of Diplolepis induce galls on plants of the genus Rosa (e.g., Zhang et al. 2020), the distributions of these gall wasps may be associated with those of specific species of Rosa (Sardón-Gutiérrez et al. 2021). However, with the exception of Diplolepis spinosissimae (Giraud) in Rosa pimpinellifolia L., no reliable data indicates that Diplolepis species are host-specific in Europe at the level of host plant species (Stille 1984, Kohnen et al. 2011). In the eastern Palearctic, D. japonica has been recorded from R. multiflora and R. rugosa (Yasumatsu andTaketani 1967, Wang et al. 2013). Since the distribution ranges of both plant species include Japan, Korea, China, and Russia (Nagamitsu 2017, Jeon andKim 2019), D. japonica is widely distributed in East Asia, including Japan, Korea, and China (Yasumatsu and Taketani 1967, Wang et al. 2013, Ide and Abe 2020. However, according to Pujade-Villar et al. (2020), the Chinese population of D. japonica could be an undescribed species. In contrast to the typical Diplolepis species, galls of D. ogawai have been only recorded from R. hirtula. The exceptionally high host specificity of D. ogawai to R. hirtula, which has a very limited natural distribution, most likely means that the distribution range of this gall wasp is restricted to an area on the main island of Japan. The vulnerable rose R. hirtula is considered to be a keystone species within a local ecological community (a network in which species are trophically connected; e.g., Paine 1980), because the previously unknown biodiversity including three hymenopteran species depends on it. As suggested by Dunn et al. (2009), coextinction through cascading effects across trophic levels may be the most common form of biodiversity loss. Because the loss of a keystone species is likely to result in extinction of interdependent species (Koh et al. 2004a), conservation efforts should extend from R. hirtula to the newly discovered fauna associated with it.
Although some species of cynipid gall inducers often cause serious damage to their host plants (Stone et al. 2002), the rarity of D. ogawai means that it likely does not adversely affect the host plant population. Therefore, when R. hirtula suffers anthropogenic habitat degradation, the extinction of D. ogawai due to the decline of the host plant populations is rather likely to come earlier. R. hirtula is distributed in a limited area (Ohba 2016) and is threatened with extinction by a decrease in favorable habitats due to the progress of succession (Ministry of the Environment, Japan 2015). Moreover, loss of rose habitats due to deforestation is another concern (Ministry of the Environment of Japan 2015). In the present study, the previously unknown insect fauna depending on R. hirtula was discovered. The new gall wasp D. ogawai appears to be host-limited to the rare rose, and two parasitoids: S. flavus and Aprostocetus sp. depend on this gall wasp from spring to summer. These three wasp species are also considered to be at risk of coextinction with this vulnerable plant as follows: the primary extinction of R. hirtula causes the secondary extinction of D. ogawai, followed by the tertiary extinction of S. flavus and Aprostocetus sp. Generally, host-limited herbivorous insects may become extinct before their host plants (Koh et al. 2004b, Moir et al. 2010). If the population size of R. hirtula is reduced further, these three wasp species may become extinct before the vulnerable host plant. Therefore, the insect fauna depending on the rare rose is considered vulnerable. As pointed out by Cardoso et al. (2011) and Moir and Brennan (2020), criteria for listing threatened species should be modified to consider coextinction of species that depend on threatened hosts/prey. To conserve the new gall wasp species and its two parasitoid species, protection of areas with remnant populations of this vulnerable rose species is necessary.