﻿Xenosyangi sp. nov.: A new twisted-wing parasite species (Strepsiptera, Xenidae) from Gaoligong Mountains, Southwest China

﻿Abstract Here we report a new twisted-wing parasite species of the family Xenidae based on both morphological and molecular evidence. By using nearly complete mitogenomes, we confirmed the twisted-wing parasites on two wasps (Vespavelutina and Vespabicolor) (China: Yunnan) as the same species, and associated its neotenic females and alate males. Combining the mitogenomic data (COI) and morphological traits, this species was identified to be a new species of the genus Xenos, namely Xenosyangi Dong, Liu & Li, sp. nov. Detailed descriptions and illustrations are provided for the new species.


Morphological description
Images of the living adults were taken using a Canon 70D camera in conjunction with a Canon EF 100 mm f/2.8L IS USM. The habitus images were taken using a stereomicroscope Nikon, SMZ18 equipped with NIS-Elements (Nikon, Japan). Scanning electron microscopes (SEM) images were taken using TM4000 II (Hitachi, Japan). The specimens used for SEM were directly fixed in 70% ethanol, and then dried at the room temperature. Morphological terminology follows those of Kinzelbach (1971), Kifune and Maeta (1985), Kathirithamby and Hughes (2006) and Koeth et al. (2012).
DNA extraction, library construction, sequencing, mitogenome assembling and sequence comparison Total genomic DNA of one male collected from V. velutina nest and one neotenic female collected from V. bicolor nest was extracted using a TIANamp Genomic DNA Kit (TIANGEN, China) based on manual instruction. Library construction, sequencing, mitogenome assembly follows those in our previous work (Zhang et al. 2021), in which the mitogenome of one neotenic female collected from V. velutina was sequenced. We assembled the nearly complete mitogenomes of both male and neotenic female individuals, and compared them with that in our previous work (Zhang et al. 2021). Then the mitogenome sequences of the three individuals were compared in pairs using BLAST in NCBI website.

Phylogenetic analyses
COI is an useful molecular marker for species identification in many insects, including twisted-wing parasites (Nakase and Kato 2013;Jůzová et al. 2015;Benda et al. 2021Benda et al. , 2019. Here, we used the COI sequences from the nearly complete mitogenomes of one male and one neotenic female of Xenos yangi sp. nov. and another Xenos neotenic female individual in our previous work (Zhang et al. 2021) for the association between neotenic female and male adults.

Sequences and phylogenetic analyses
We assembled nearly complete mitogenomes of one male adult collected from a V. velutina nest (15324 bp) (GenBank accession number: OK329871) and one neotenic female collected from a V. bicolor nest (14670 bp) (GenBank accession number: OK32987). The mitogenomes of these two individuals in this study and one neotenic female in our previous work (Zhang et al. 2021) contain the same sequence except for the A+T-rich region and a gap between trnaM and trnaI, suggesting the nature of the same species for these three individuals with different sexes and different host. In this study, the mitogenome of male adult was annotated as 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs) and an A+T-rich region, while only 36 mitogenomic genes (excl. trnM ) were annotated in the incomplete mitogenome sequence of the neotenic female.
We further extracted a major fragment (1518 bp) of COI sequences from three Chinese Xenos individuals (one male and two neotenic females), and combined 45 COI sequences of identified species of Xenos reported by others to make the dataset for the phylogenetic analyses using ML and MP methods. All phylogenetic trees show that the three Chinese Xenos individuals (one male individual from the V. velutina nest and two female individuals from the V. velutina and V. bicolor nests) cluster together with high bootstrap values (Fig. 6). The genetic divergence among three Chinese Xenos individuals varies from 0 to 0.014 (Table 2, Fig. 6), which is equal to that among X. moutoni individuals and less than that among X. oxyodontes individuals. Especially, one male and one neotenic female from the same host nests (V. velutina) showed no genetic divergence, suggesting their conspecific identity. These findings confirm that these male and neotenic female individuals collected from different host populations are the same species. This species can be differentiated from all the other Eurasian species of Xenos based on the genetic analyses and further morphological examination, and thus stands as a new species described below.  2B) four-segmented, 1 st with distal lateral extension and wider than 2 nd , 3 rd and 4 th flabellate with subequal length. Palpus twice as long as maxilla (Fig. 2C). Mandible (Fig. 2D) slender, widened at base, tapering at tip. Prescutum pentagonal. Scutellum longitudinally elongated, triangular.
Hind wing sector with nine veins (Fig. 2A). C and Sc fused, half length of costal margin. R1 and R2 veins almost glued together, R2 vein extending from middle to wing apex; R3 vein from middle to outer margin of wing; R4 vein terminated at distal 1/4 of the wing and approximating R5 vein. MA, CuA1, CuA2 and CuP veins present and uninterrupted.
Comparative notes. Considering the geographic distance and host association of those species of Africa and Americas, we mainly compared the male adult, the cephalotheca of the male papurium, and the neotenic female of this new species with ten described known species distributed in Asia (nine species) and Europe (one species) (Table 1). These species were originally described based on the male adult, the cephalotheca of the male Table 1. Distribution and described stages of 11 Xenos species from Asia and Europe. Literature in which the species was originally described is highlighted in bold. papurium, and/or the neotenic female (Table 1). The new species can be distinguished from X. moutoni (China: Yunnan, Anhui, Taiwan), X. dianshuiwengi (China: Fujian), X. formosanus (China: Taiwan), X. provesparum (Indonesia) and X. oxyodontes (Japan) based on the external characters of male adult. The male adult of X. moutoni maxilla as long as palpus and the postlumbium is straight anteriorly and posteriorly (Kifune and Maeta 1985). The male adult of X. oxyodontes (Japan) has the postlumbium rounded anteriorly and posteriorly (Nakase and Kato 2013). The proventrite is not concaved in X. dianshuiwengi (China: Fujian), X. formosanus (China: Taiwan) and X. provesparum (Indonesia) (Kifune and Maeta 1985;Kifune 1986;Yang 1999).
The new species can be distinguished from X. circularis (China: Taiwan), X. yamaneorum (China: Taiwan), X. vespularum (Japan), X. hebraei (India) and X. vesparum (Europe; Northern Africa) by the female cephalothorax. It is almost circular or ovoid in X. yamaneorum, X. circularis, X. vespularum and X. vesparum (Kifune and Maeta 1975;Kifune and Maeta 1985). The female cephalothorax is slightly wider than long in X. hebraei. Besides that, this new species can be also separated from X. yamaneorum and X. vespularum by the oval maxillae of the male cephalotheca (the two compared species lack the oval maxillae of the male cephalotheca).
Distribution. China (Yunnan). Biology. The hosts of this new species are Vespa velutina (Fig. 5A) and Vespa bicolor (Fig. 5B). It parasitizes in the host abdomen. Its body partly protrudes from the portion between the two abdominal segments of the hosts. One wasp can usually carry 1-4 parasite individuals (Fig. 5C). After emergence, male adults fly away from their hosts (Fig. 5D). Neotenic females remain in the host's abdomen with their anterior cephalothorax protruding. When neotenic females are removed from their host abdomen, they can be seen to be covered with larval exuviae.
Etymology. The specific epithet is dedicated to the late famous Chinese entomologist Chi-Kun Yang, who made significant contributions to the studies on Strepsiptera in China.

Discussion
Due to the discovery of X. yangi sp. nov., the number of Chinese Xenos species increases to six (Fig. 7) while the Asian species add up to ten. In general, the Asian Xenos species are endoparasites of Vespinae (yellow jackets and hornets) and Polistinae (paperwasps) (Suppl. material 1: Table S1). Vespa (Vespinae) and Polistes (Polistinae) are common hosts for most Xenos species (Cook 2019). Except two Taiwanese species parasiting on Polistes, all other eight Asian species parasite on Vespa. Considering the species diversity of Vespinae and Polistinae in China (Carpenter 2011), we confirm the rich Xenos species diversity in China.
Among the 10 Asian Xenos species, six species (including the new species here) are described based on both males and neotenic females, one species solely based on males, two species are based on neotenic females and the cepholotheca of the male puparium, and one species is solely based on neotenic females (Table 1). This situation Figure 6. Phylogeny tree of Xenos species inferred from mitochondrial cytochrome c oxidase subunit 1 (COI) using Maximum parsimony method. In total, 48 COI sequences of different Xenos species were used to investigate their phylogenetic relationships. two sequences (str6-b-female and Xenos-male) were sequenced in this study, and that of Xenos cf. moutoni (MW222190.2) was sequenced in Zhang et al. (2021). Other 45 sequences were published by the following studies (Benda et al. 2021;McMahon et al. 2011;Nakase and Kato 2013;Jůzová et al. 2015;Carapelli et al. 2006). Stylops ater Reichert, 1914, Melittostylops hesperapium Kinzelbach, 1971, Halictoxenos tumulorum Perkins, 1918 and Crawfordia warnckei Kinzelbach, 1970 (outgroup) were used as outgroups. The phylogenetic trees were constructed using Maximum Parsimony (MP), and Maximum Likelihood (ML). Branch support values are described as Maximum Parsimony (MP)/Maximum Likelihood (ML) in MP tree.   in describing new species based only on neotenic females is also common in the taxonomy of Xenos from Africa and America (Suppl. material 1: Table S1). Considering the sexual dimorphism in twisted-wing parasites it is feasible to describe a new Xenos species when both male and female specimens are available. Thus, the association of both sexes and different stages of development in the same species of Xenos is crucial for future studies. This study provide an example of associating both sexes using combined biological, morphological and molecular evidence.
Xenos moutoni was originally described by Buysson (1903) based on only neotenic female specimens collected in Anhui (Ngan-hoei = Anhui Prov., Yng-chan = Xuanchen?宣城) and Yunnan (Yun-nam = Yunnan Prov., Tsé-kou = Cigu茨古 ( Xu and Qiu 2020). Then, Buysson (1904) recorded its male puparium cephalotheca based on the specimens collected from the type locality. Kifune (1985) redescribed the male adult and cephalotheca of the male puparium of this species from Taiwan. In Buysson's work, the cephalotheca of the male puparium might be the main diagnostic trait to identify Taiwan X. moutoni. However, the author did not give a detailed description of the male puparium cephalotheca. According to available male specimens, cephalotheca of the male puparium and the neotenic females of the new species in the present study, we compared the different stages of the new species with the description of a male adult (Taiwan), cephalotheca of the male paparium (type locality), or the neotenic female (type locality) of X. moutoni, facilitating the delimitation of these two species both recorded from Yunnan. In addition, our study affirms again that molecular data, e.g., the DNA barcodes, are essential for the association of dimorphic sexes and different developmental stages in twisted-wing parasites taxonomy.
In the molecular data analysis, we noticed that different populations of five monophylic species (X. moutoni, X. oxyodontes, X. yangi, X. pecki, and X. vespularum) show genetic divergence of less than 0.036 (Table 2). Especially for X. moutoni, the genetic divergence among their different populations from Laos, China, Japan is less than 0.014 (Table 2). For the other lineages including specimens identified as X. minor, X. vesparum and X. ropalidiae, we noticed that X. minor and X. vesparum form a clade including four groups (group 6, 7, 8 and 9) (Fig. 6) and their genetic divergences are less than 0.007 (Table 2), suggesting these specimens may be the same species (Benda et al. 2021). On the other hand, different populations (Laos, Nepal, Malaysia) of X. ropalidiae form two separate groups (5 and 10) with a genetic divergence of 0.368-0.421 (Table 2), which may include different species (Benda et al. 2021). These findings suggest that an integrated methodology of molecular, biological, and morphological evidence should be adopted in taxonomy of such endoparasites as twisted-wing insects.