A new species of Megastigmus (Hymenoptera, Megastigmidae) from China

Abstract Background Most species of Megastigmus are considered important economic pests that grow in seeds, especially of conifers. Accurate identification of species is a crucial step for the biological research of parasitic pests and the further application of biological control. However, their large variety, small size, similar morphology and different growth and development stages have brought great challenges to taxonomic research. Traditional morphological identification often takes a long time and this requires us to seek a new method for rapid and accurate identification. Therefore, the better identification of Megastigmus urgently needs to be combined with molecular methods to help taxonomic development. New information Here, Megastigmusdaduheensis sp. n. (Chalcidoidea: Megastigmidae) was identified, based on morphology and molecular markers, such as COI and Cytb. M.daduheensis sp. n. is distinct from other known species of the same genus in the morphology. The results of the molecular phylogenetic tree, similarity alignment and genetic distance indicate that the COI and Cytb sequences of M.daduheensis sp. n. are highly similar to M.sobinae and M.duclouxiana, but there are some genetic differences. The genetic distances of M.daduheensis sp. nov. with M.duclouxiana and M.sabinae were 0.34 and 0.33 and the percentages of shared base pairs were 76.3% and 76.8%, respectively. Both morphological and molecular data classified M.daduheensis sp. n. as a new species. The obtained COI and Cytb sequences of M.daduheensis sp. n. can be used as DNA barcodes, providing molecular data for rapid and accurate identification of this species in the future.


Introduction
Megastigmidae comprises over 200 species currently classified into 12 valid genera which are generally considered to have a worldwide distribution and Megastigmus is the only cosmopolitan genus of the family (Böhmová et al. 2022).Megastigmus was described by Dalman (1820) as the subgenus of Torymus Dalman, with Pteromalus bipunctatus Swederus, 1795 as its type species.Later, Megastigmus was recorded as a valid genus by several scholars and Crosby (1913) designated its type species as P. bipunctatus (Curtis 1829, Dalla 1898, Ashmead 1904).
Megastigmus is a diverse group with 144 valid species described in the Universal Chalcidoidea Database (Noyes 2019).Of these, most species have been described from the Holarctic and Australasian Regions (Bouček 1988, Grissel 1999).So far, 18 species in Megastigmus have been reported from China.The species of Megastigmus display a great variety of feeding strategies in which many species are seed feeders and others are mostly solely carnivorous (Roques andSkrzypczynska 2003, Böhmová et al. 2022).Their habit leads to a decrease in seed yield which seriously endangers the reproduction of trees.We need to quickly identify and control the species to reduce damage.However, their small size and similar appearance makes it difficult to identify.The identification of closely-related species can no longer be met by using morphological characters alone.Therefore, rapid and accurate identification of Megastigmus insects and monitoring of their distribution and growth are one of the main measures for the prevention and control of these insects.
DNA barcoding is a method for species identification, based on a relatively short DNA gene fragment with sufficient variation and easy amplification (Folmer et al. 1994, Nuriye et al. 2021).Currently, sequence fragments of mitochondrial DNA (mtDNA: COI, Cytb, COII, 16S etc.) have been widely used in this method, which can quickly and accurately identify insects (Janzen et al. 2017).In mitochondrial DNA, molecular markers, such as cytochrome c oxidase subunit I (COI) and Cytochrome b (Cytb), are commonly used to study intra-species, inter-species and even inter-family phylogenetic relationships.They have the characteristics of high evolutionary rate, obvious interspecific variation, but relative conservation within species, good versatility of primers and easy amplification, which can effectively solve some long-standing controversies in morphological taxonomy ( Meyer and Wilson 1990, Koch 2010, Cheng et al. 2015, Chen et al. 2020).Therefore, the morphological characteristics combined with two molecular markers (COI and Cytb) can more effectively solve the problem of classification and identification of Megastigmus.

Materials and methods
The infested cones of C. chengiana were collected from Ri'er Township (102°35′ E, 30°59′ N), Xiaojin County, Aba Autonomous Prefecture of Tibetan and Qiang (Ngawa), Sichuan Province, China.The cones were kept in mesh bags, where the spawning adult insects were collected and immediately frozen in liquid nitrogen and preserved at -80°C until DNA extraction.All type specimens are deposited in the Natural History Museum of Sichuan University, Chengdu (NHMSU) with an accession number NHMSU-20201015-XJ.
We used Mitoz 3.5.0software (Meng et al. 2019) to assemble two mitochondrial gene fragments (COI and Cytb) from genomic sequencing data of M. daduheensis sp.n. which was performed with Illumina HiSeq 2000 at Novogene, Beijing, China.Subsequently, a total of 27 COI and Cytb gene sequences of Megastigmus species were found in the National Center for Biotechnology Information (NCBI).Hence, the phylogenetic analysis used genetic data from the above species and one outgroup of Torymus geranii Walker, 1833 (Table 1).The sequences of different species were manually aligned with clustalW in MEGA 7.0 (Kumar et al. 2016).The trimmed alignment was used to estimate the phylogeny by Maximum Likelihood (ML) using IQ-TREE 2.0.4 and an ultrafast bootstrap approximation approach with 1000 replicates (Fu et al. 2014) was used to calculate bootstrap scores for each node (BP) (Minh et al. 2020).MEGA 7.0 software was used to calculate the interspecific genetic distance, based on p-distance and the percentage of base pairs are shared between the species (Kimura 1980, Kumar et al. 2016).
Head.Head about 1.6× as wide as long in dorsal view; whole face with pale, dense setae; frons with many obvious striated sculptures; ocellus arranged in a blunt triangle, POL about twice as long as OOL and OCL; several short setae between ocelli, the rest on other parts of head (Fig. 1B-E 1F).
Mesosoma.Pronotum and mesoscutum with irregular sculpture and orientation of rugae, notauli brown and sparse setae scattered on both sides (Fig. 1H); axillae with oblique longitudinal ridge; scultellum with transverse costula and white setae on both sides; propodeum with oriaceous, callus with long white setae and irregular reticulate sculpture; callus densely covered with white long setae.Basal cell of fore wing with more than ten setae and the lower part of the basal cell closed by a row of setae; marginal vein shorter than postmarginal vein; stigma large and 2.2× as long as width, stigma vein short and 0.11× as long as stigma (Fig. 1G).Hind coxa with white long setae on both sides, femora and tibia densely covered with white setae (Fig. 1A).

Metasoma.
Gaster not compressed laterally, 1.21 mm long, with sparse bristles scattered with four black horizontal bands and the spacing between the horizontal bands decreasing successively (Fig. 1A).Ovipositor densely covered with brown long setae and 4.5× as long as hind-leg tibia.
Male.Body black, 2.4 ~ 2.6 mm long (Fig. 1I).Gaster black, 1.0 mm long, 1.5× as long as hind tibia.Antennae brown.Femora wider than female femora.Marginal vein tan, stigma dark brown.Antennae flagellum about the same length as gaster, funicle length greater than width, anellus about 1/3 of pedicel length.Stigma 1.6× as long as width, stigmal vein short and 0.18× as long as stigma.Other morphology is similar to female.

Diagnosis
The key from China distinguished the females of Megastigmus daduheensis sp.n. by their body length less than 5 mm, ovipositor exceeding body length, stigma neck less than 0.5 times the width of the stigma and scultellum dark yellow.(Fig. 1A, G, E).Megastigmus daduheensis sp.n. is similar to Megastigmus duclouxianae Xu & He, 1995 (Fig. 2A, B) and Megastigmus sabinae Xu & He, 1989 (Fig. 3A,B), but can be distinguished from the latter two species by: 1) stigma light brown, 2.2× as long as width, stigma neck short and 0.11× as long as stigma; 2) ovipositor 4.5× as long as metatibia; and 3) mesosctum black anteriorly to brownish-yellow posteriorly.A new species of Megastigmus (Hymenoptera, Megastigmidae) from China

Etymology
The new species is named after its type locality, Daduhe, Sichuan Province.

Biology
The eggs are laid in the cones of Cupressus chengiana S.Y.Hu, feeding on the nuts and the adults fly out of the cones from October to November every year.

Identification keys
Key to species of Megastigmus from China (females)

Analysis Phylogenetic relationship
Megastigmus is sometimes difficult to classify accurately because of its small size and similar morphological characteristics.Therefore, the phylogenetic relationships are important to resolve species boundaries.Here, the existing mitochondrial data (COI + Cytb) was used to construct a molecular phylogenetic tree of Megastigmus, so as to verify the taxonomic status of the new species described above (Fig. 4).In terms of phylogenetic relationships, M. daduheensis sp.nov.and M. sabinae were sister species and then they constituted one clade with M. duclouxiana.This result is consistent with morphological identification.
Pairwise genetic distances of COI and Cytb amongst 28 species of Megastigmus.

Discussion
The identification of species based on morphological differences is essential to insect taxonomy.Nevertheless, the traditional classification method has drawbacks when it comes to tiny and similar-looking species, while molecular data can make up for the shortcomings of this method.Currently, many studies have stated explicitly the appropriateness of mtDNA in resolving the relationships amongst subspecies or closelyrelated species (Sperling and Hickey 1995, Davison et al. 2001, Wahlberg et al. 2003) 2017) has calculated intra-group and inter-group distances of Megastigmus, based on the COI dataset and the lower values were observed within groups rather than between groups, with values ranging from 6% to 7.9%.However, in our study, the average pairwise genetic distance (COI/Cytb: 0.16/0.13) of Megastigmus species was higher and the genetic distance of M. daduheensis sp.nov.supported it as a separate species.
This study not only increased the species diversity of Megastigmus, but also provided the genetic information of M. daduheensis sp.nov.and enriched the genetic database.However, our study only obtained COI and Cytb genes of this genus, which contained incomplete genetic information.Therefore, it can be combined with other mitochondrial or nuclear genes for a comprehensive analysis and verification in the future.In addition, there are a large number of species within this genus, but most of them are studied, based on morphology, ecology and physiology, while molecular data is very scarce.Hopefully, we will be able to collect more samples and obtain molecular data to make up for the shortfall.

Figure 1 .
Figure 1.Megastigmus daduheensis sp.n.A-H female holotype; A Body, lateral view; B Head, lateral view; C Head, ventral view; D Head, dorsal view; E Head, frontal view; F Antenna; G Fore wing; H Head and mesosoma, dorsal view; I Male, Body, lateral view.

Table 1 .
List of the mitochondrial genes analysed in the present study.
Table2) and common base pairs between species for 28 species of the Megastigmus, based on COI and Cytb.The results showed that the interspecific genetic distance base on the COI and Cytb were 0.
and the percentages of shared base pairs (COI+Cytb) were 76.3% and 76.8%, respectively.Molecular evidence confirmed M. daduheensis sp.nov.was distantly related to M. duclouxiana and M. sabinae and it was a separate species.
Note: COI fragment before backslash(/)and Cytb fragment after backslash.1 M. Sabinae; 2 M. duclouxiana; 3 M. daduheensis sp.nov.; 4 M. dorsalis; 5 M. aculeatus; 6 M. amicorum; 7 M. bipunctatus; 8 M. pinus; 9 M. spermotrophus; 10 M. tsugae; 11 M. zebrinu; 12 M. schimitscheki; 13 M. strobilobius; 14 M. synophri; 15 M. stigmatizans; 16 M. pinsapinis (Auger-Rozenberg et al. 2010))ermotrophus nigrodorsatus has been considered until now as a subspecies of M. spermotrophus, but molecular results (mean distances of 3.3% in Cytb and 0.3% in 28S) suggested that the species actually could be a distinct species rather than a subspecies of M. spermotrophus( Auger-Rozenberg et al. 2010).Similarly, two specimens from Greece and Kenya were identified as Megastigmus pistaciae, but Roques et al. (2017) study showed that they diverged by 4.5% in the COI data and the two are probably sibling species.Therefore, the combination of the two methods can make insect identification more accurate, which is of great significance for improving the efficiency and accuracy of species identification.In this paper, morphological characters and molecular data (COI and Cytb) were used to classify parasitic wasps from Cupressus chengiana S.Y.Hu.Both methods confirm that M. daduheensis sp.nov. is a separate species, which is clearly different from other species in the genus and has a certain degree of diversification.Although M. daduheensis sp.n. was similar to M. sobinae and M. duclouxiana in morphology, it can be distinguished from other species, based on characters on the mesoscutum, stigma and ovipositor.M. daduheensis sp.nov.andM.sabinaewere sister species, based on the phylogenetic relationship and genetic distance and, together, they constituted one clade with M. duclouxiana.This result is consistent with previous studies and supports Megastigmus on Cupressaceae plants formed a distinct clade(Auger-Rozenberg et al. 2010).Roques et al. (