﻿A new species of Andricus Hartig, 1840 (Hymenoptera, Cynipidae) from China, with references to DNA taxonomy and Wolbachia infection

﻿Abstract In the present paper, a new species of cynipid gall wasp, Andricuselodeoides Liu & Pang, is described from several provinces in southern China. The new species is closely related to the recently redescribed A.mairei (Kieffer, 1906). In addition to differences in adult and gall morphology, the new species is also readily separated by COI sequences, with a 6.2–8.9% genetic distance between populations of the new species and those of A.mairei. A contrasting difference in sex ratios was also observed between the two species, with A.elodeoides extremely female-biased (95.5–97.8% female) while A.mairei male-biased to more balanced (5.4–43.5% female). PCR screening for Wolbachia infection further revealed contrasting infection rates between populations of A.elodeoides and A.mairei: the Wolbachia infection rate was 0% in A.elodeoides and 100% in A.mairei. Cytoplasmic incompatibility induced by Wolbachia is proposed as a potential mechanism of speciation of the sympatric A.elodeoides and A.mairei.


Introduction
The genus Andricus Hartig, 1840 (Hymenoptera, Cynipoidea, Cynipidae, Cynipini) is the largest genus of the oak-gall wasp tribe Cynipini, currently comprising approximately 400 known species (Melika 2006) and making up 40% of the known species diversity of the tribe (Wachi et al. 2011). The genus is predominantly Holarctic, with the highest recorded species diversity from the Nearctic and Western Palearctic (Wang et al. 2013). However, a number of new species of the genus have also been described in the last decade or so from Mesoamerica in the Neotropical realm (Melika et al. 2009a, b;Pujade-Villar et al. 2016) and the Oriental realm (Tang et al. 2009(Tang et al. , 2012Wang et al. 2013;Pujade-Villar et al. 2014;Ide et al. 2018). In Eastern Asia, which stretches from the Palearctic to the Oriental, 19 Andricus species are known (Ide et al. 2018;Penzes et al. 2018;Pujade-Villar et al. 2020).
The unusually high diversity of Andricus species among all the genera of the tribe Cynipini may be an artifact, as the genus is not well defined and often has been treated as a "trash can" genus in Cynipini (Melika 2006). In their taxonomic review of the world genera of cynipine wasps, Melika and Abrahamson (2002) treated several previously recognized genera as junior synonyms of the genus because of the lack of reliable diagnostic characteristics, rather than because of the existence of defining synapomorphies (Melika and Abrahamson 2002). One of the synonymized genera, Druon Kinsey, 1937 has since been re-established as a valid genus (Cuesta-Porta et al. 2022). Although multiple phylogenetic studies involving Cynipini have invariably shown Andricus to be paraphyletic or polyphyletic (Stone and Cook 1998;Cook et al. 2002;Rokas et al. 2003;Stone and Schönrogge 2003;Liljeblad et al. 2008;Ronquist et al. 2015), the current concept of the genus is still largely based on that of Melika and Abrahamson (2002).
One of the genera synonymized with Andricus Hartig, 1840 by Melika and Abrahamson (2002) is Parandricus Kieffer, 1906, which is known from China and includes a single species, P. mairei Kieffer, 1906. A detailed redescription of the species was done based on specimens collected from Zhejiang Province of China because the original type of P. mairei Kieffer, 1906 was lost and the original description was inadequate by today's standards (Pujade-Villar et al. 2020). In the last few years, we have reared a large series of specimens that apparently belong to multiple, known or unknown, species of Andricus, including A. mairei (Kieffer, 1906)  ). In the present paper, we describe a new species from that series of Andricus specimens and provide a detailed comparison between it and the apparently closely related A. mairei (Kieffer 1906). We also sequenced the mitochondrial COI gene for both species for DNA barcoding as well as the nuclear 28S D2 region to place the new species within the current phylogenetic framework of all Andricus species that had both COI and 28S sequences available.

Specimen collection
The galls of gall wasps were collected from 12 locations in six provinces in southern China in late spring to early summer from 2012 to 2019 (Table 1). The collected galls were cage-reared at room temperature in the laboratory of the College of Life Science and Technology, Central South University of Forestry and Technology (CSUFT) and checked daily for emergence. Adult wasps were directly preserved in 100% ethanol within 2 days after emergence and stored in freezer at −80 °C until being retrieved for morphological and molecular studies.

Morphological observations
Specimens for conventional morphological examination were air dried at room temperature before mounting. Specimens mounted to pinned triangle-card paper were studied under a stereomicroscope (SZX7, Olympus, Japan) and automatically stacked photographs were taken with Leica M205C microscope system (Leica, Germany) equipped with Leica DMC6200 digital camera connected to a computer. Additional specimens were dissected out and transferred to diluted ammonia (5%) and kept overnight to remove debris that might interfere with observation. Cleansed parts were then rinsed in distilled water and dehydrated gradually through 25%, 50%, 75%, and 100% ethanol solutions, and finally stored in 100% ethanol. Dehydrated specimen parts were air-dried before being mounted onto aluminum stub (Ted Pella, Redding, CA, USA) with copper conductive tape (3M). Gold-coated specimens were examined with JEOL JSM-6380Lv SEM (JEOL, Japan) at CSUFT with 15 KV voltage, and selected frames were saved as digitized high-resolution TIFF images.
We follow Ronquist and Nordlander (1989) and Ronquist (1995) for structural terminology, Melika (2006) for measurement definitions, and Harris (1979) for surface sculpture descriptions. Abbreviations: F1 and F2 = the first and second flagellomeres, respectively; POL (post-ocellar distance) = the distance between the inner margins of the posterior ocelli; OOL (ocellar-ocular distance) = the distance from the outer margin of a posterior ocellus to the inner margin of the compound eye; LOL (lateral-frontal ocelli distance) = the distance between anterior and lateral ocelli. Type specimens are deposited in Insect Collection, Central South University of Forestry and Technology (CSUFT), Changsha, Hunan.

DNA extraction and sequencing
Three individuals from each population of two gall wasp species were used for DNA extraction. The insects were washed in sterile water before DNA extraction to avoid surface contamination. Total DNA was extracted from each individual using SDS/ proteinase K digestion and a phenol-chloroform extraction. Extracted DNA pellets were air dried, resuspended in 50 µl sterile water, and then stored at 4 °C before being processed for PCR and sequencing.
For phylogenetic analysis, we chose a specific region of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the nuclear large ribosomal subunit gene (28S), which were amplified with the primes HCO-2198 (5′-TAAACTTCAGGGTGAC-CAAAAAATCA-3′) and LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) (Folmer et al. 1994), and D2F (5′-CGTGTTGCTTGATAGTGCAGC-3′) and D2R (5′ TCAAGACGGGTCCTGAAAGT 3′) (Dowton and Austin 2001), respectively. This gene fragment was chosen because of its suitability for recovering inter-and intrageneric phylogenies within the Hymenoptera in general and Cynipidae in particular (Rokas et al. 2002) as well as sequence availability for a reasonable number of congeneric species from public depositories. The PCR mixture was composed of 1 µl of PrimeSTAR HS DNA Polymerase (Takara Biomedical Technology Co., Dalian, China), 10 µl of buffer, 4 µl of dNTPs, 1 µl of each primer, and 2 µl of DNA with water added to achieve a total volume of 50 µl. The amplification was conducted using a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, United States). The cycling conditions were 98 °C for 3 min, 35 cycles of 98 °C for 10 s, 50-57 °C for 30 s, and 72 °C for 1 min. Amplified PCR products were sequenced in both directions using an ABI 3730XLDNA sequencer (Applied Biosystems, Foster City, CA, USA) with M13F/R at Wuhan Icongene Co., Ltd. The sequences have been deposited in GenBank under the following accession numbers: COI ON803612 to ON803631 and 28S ON911591 to ON911610 (Table 2).

Phylogenetic analysis
The COI and 28S gene sequences of 11 species of Andricus (including eight populations of A. mairei) and Dryocosmus liui and Melikaiella bicolor (as outgroups) were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) ( Table 2). The final dataset consists of 14 species including the new species and outgroup. Multiple sequence alignment was performed using ClustalW (Thompson et al. 1994) implemented in MEGA 11.0 (Kumar et al. 2016) using default parameters. ClustalW aligned sequences were then visually edited in MEGA 11.0 and trimmed, resulting a final aligned length of 1154 bp nucleotides for COI and 1053 bp nucleotides for 28S. The final dataset was subjected to MEGA 11.0 for evaluation of best fit nucleotide substitution model (Nei and Kumar 2000) using the maximum likelihood (ML) method with default settings except that we used "very strong" branch swap filter. Phylogenetic analysis was conducted using MrBayes 3.2.6 x64 for Windows (Ronquist et al. 2012) (Bayesian inference method, BI), assuming a generalized Time-reversible (GTR) model with gamma distributed rate variation across sites (+G) based on best fit nucleotide substitution model evaluation performed earlier. For Bayesian analysis, two independent runs were performed with the default priors and MCMC parameters except the following: nst = 6, rates = gamma, MCMC runs comprised 10 million generations sampled at every 1,000 generations with 30% burn-in time. Convergence was achieved as being diagnosed by the average standard deviation of split frequencies between the two independent runs (<0.01) and PSRF values (1 with < 1% deviation). The final tree from both analyses was rooted with D. liui and M. bicolor based on published phylogeny of Cynipidae (Ronquist et al. 2015).
To compare directly with a recent study on A. mairei and related species based solely on COI (Pujade-Villar et al. 2020), we also performed a phylogenetic analysis based on COI only to include the sequences of A. mairei from various populations published in that study.
Finally, the pair-wise genetic distance in the COI sequences from all populations of A. elodeoides and A. mairei, and other two Andricus species were calculated, using the MEGA 11.0 (Kumar et al. 2016).

Wolbachia screening
Wolbachia infections were screened by PCR with the Wolbachia-specific primers wsp-81F and wsp-691R that amplify a 575-625 bp fragment of the wsp gene encoding Wolbachia surface protein (Zhou et al. 1998). To verify the presence of Wolbachia infection in A. elodeoides, gatB, coax, ftsZ, and hcpA genes were amplified for various populations using the respective primers reported by Baldo et al. (2006). Amplification methods and conditions were as previously described (Hou et al. 2020).  Diagnosis. The new species is similar to A. mairei (Kieffer 1906), but differs from the latter in having: 1) vertex and frons glabrate with long setae evenly-spaced on vertex and scatted on frons in the new species (Fig. 3), whereas vertex coriaceous and  fig.  1b, d); 2) male antenna F1 strongly curved medially in the new species (Fig. 10), but straight in A. mairei (Pujade-Villar et al. 2020: fig. 2b); 3) mesopleuron glabrous in the new species (Fig. 6), whereas with weak longitudinal striation medially in A. mairei (Pujade-Villar et al. 2020: fig. 3c, d, but compare with fig. 3e); 4) mature galls of A. elodeoides are straight and cylindrical, fully covered with dense resinous white hairs (Fig. 14), whereas the galls of A. mairei are curved or strongly tapering in distal half, mostly shining smooth with an apical cluster of white hairs (Fig. 15).
Coloration. Head area of compound eyes and frons black and gena yellow. Antenna uniformly dark brown to black, except for scape, pedicel and F1 brownish yellow. Mandible, maxillar and labial palpi dark brown. Legs uniformly brownish yellow. Mesosoma black; metasoma mostly reddish brown and posteriorly black. Hypopygial spine reddish brown.
Head coriaceous, 1.2 times as wide as high in anterior view, nearly oval, broader than mesosoma in front view and 2.2 times as broad as long in dorsal view. Gena not broadened behind eyes in dorsal view. Height of eye about 3.4 times the length of malar space. Frons glabrate with evenly spaced long setae, with ocellar triangle indistinctly rugose; lower face and malar space glabrate and distinctly setose. Clypeus distinct and impressed; epistomal sulcus distinct; anterior tentorial pits small, but distinct; clypeo-pleurostomal line distinct. Transfacial distance slightly bigger than height of eye; distance between inner margin of eye and outer rim of antennal torulus slightly wider than distance between antennal toruli, but as wide as diameter of torulus (Fig. 1). Posterior ocelli widely separated from each other, ratios of POL/OOL, POL/LOL, and LOL/OOL 2.1, 2.7 and 0.9, respectively. In dorsal view, posterior margin of anterior ocellus nearly aligned with anterior margin of posterior ocelli (Fig. 3). Vertex glabrate, covered with scattered long setae. Gena coriarious, posteriorly with sparce long setae; postgena mostly glabrate with dense setae in outer edge. Occiput very finely imbricate and setose except medially; posterior tentorial pits distinct. Gular sulci absent; area around occipital foramen glabrous (Fig. 2).
Mesosoma longer than high in lateral view. Pronotum median length two ninth of length of outer lateral margin. Anterior plate of pronotum areolate to rugose and densely setose laterally (Fig. 6); Mesoscutum nearly as long as width measured at anterior tip of tegulae, with some small foveae and setae along outer edge. Notauli distinct and glabrous, lined with setae along sides, and slightly broadened posteriorly.
Metasoma 1.2 times as long as high in lateral view; abdominal tergite II 1.5 times as high as long in lateral view, laterally with anterior patch of short setae; tergite VII dorsally and VIII with long setae. Prominent part of hypopygium slender, distally not pointed; and ventrally with a row of short setae (Fig. 4).
Gall. Galls are monolocular and form clusters of 50-60 galls on twigs of host plant. Galls are covered with very dense resinous white hairs, which become brown at the terminal of the galls as galls mature. Individual galls straight and cylindrical (Fig. 14), but not curved or strongly tapering in distal half as in A. mairei (Fig. 15).
Biology. All specimens emerged from galls collected from Quercus serrata. The adults of the new species appeared in early to mid-May (which overlaps with the emergence period of A. mairei). Populations were extremely female-biased at 95.5-97.8% (while that of A. mairei were 5.4-43.5%) ( Table 1).
Distribution. The new species is currently known from China in several provinces in the middle to lower reaches of the Yangtze River, including Henan (Xinyang), Anhui (Jinzhai, Shucheng, and Taihu), Hubei (Wuhan), Hunan (Changsha and Shaoyang), and Zhejiang (Suichang and Qingyuan).

Molecular phylogeny
The Bayesian and maximum-likelihood phylogenetic trees of various populations of A. elodeoides, A. mairei, and other Andricus species based on the COI and 28S genes had identical topology while showing minor differences in support level for some nodes. According to the Bayesian trees presented here (Fig. 16), the sampled populations of Figure 16. Bayesian phylogenetic tree of A. elodeoides sp. nov. and other Andricus species based on COI and 28S sequences. Bold font refers to the sequence obtained in this study, and others are downloaded from NCBI. The photograph on the right refers to the gall of adult emergence. The letters in parentheses indicate the sampled populations shown in Table 1. The length of the branches is drawn to scale of genetic distance and the number over branches is posterior probability. Melikaiella bicolor and Dryocosmus liui were used as the outgroup.
A. elodeoides and A. mairei form their own monophyletic clades, and the two species are sister to each other. The genetic distance between the two species is similar to other Andricus species pairs, while the distance between this clade and the other including Andricus species is rather distinct (Fig. 16).
Pair-wise comparison of the COI gene segment used in this study showed interspecific genetic distances ranged from 6.2 to 11.7% among Andricus species. In A. elodeoides and A. mairei, the interspecific genetic distance ranged from 6.2 to 8.9%. The level of intraspecific genetic variation in A. mairei was higher than that in A. elodeoides. The intraspecific genetic distances were 0-1.8% in A. elodeoides and 0-2.6% in A. mairei, while the distance between "A. mairei ILV91" and A. elodeoides, "A. mairei ILV91" and A. mairei were 0.2-1.8%, and 6.5-8.2%, respectively (Table 3).

Wolbachia infection
Using PCR screening for Wolbachia infection with wsp gene-specific primers, in all sampled populations of A. elodeoides and A. mairei, we found that all individuals from 12 populations of A. mairei (N = 360) were infected with Wolbachia, whereas no Wolbachia infection was found in the seven studied populations of A. elodeoides (N = 350), including samples collected from Jinzhai and Shucheng populations through four consecutive years (Table 1). The negative results of Wolbachia infection in A. elodeoides adults were further verified by PCR using specific primers for the multilocus sequence type genes (ftsZ, coxA, hcpA, and gatB).

Discussion
Andricus elodeoides sp. nov. is considered a distinct from A. mairei (Kieffer) based on differences in adult and gall morphology, and phylogenetic reconstruction based on COI sequence data (Fig. 17), as well as combined dataset of 28S and COI genes (Fig. 16) and pair-wise genetic distance of the COI gene marker (Table 3). However, intraspecific variation of adult morphology exists in A. elodeoides as well as in A. mairei (Pujade-Villar et al. 2020). For example, the median propodeal area is rugose in specimens from Hunan (Changsha and Yueyang), but smooth in specimens from Guizhou (Guiding) and Fujian. The lateral propodeal carinae are parallel to each other in A. elodeoides, as we observed, which appear to be highly variable in A. mairei from being "subparallel to divergent anteriorly and bent outwards in the middle" (Pujade-Villar et al. 2020). Such variations in the morphology of both species, while needing to be further evaluated using large series of specimens from broad regional populations, certainly make it difficult to separate the two species based on adult morphology alone. In such situations, gall morphology and DNA barcoding based on COI sequence is necessary.  Table 1. The length of the branches is drawn to scale and show the genetic distances, and the number over branches is posterior probability. Table 3. Pair-wise COI sequence distances in various geographic populations of A. elodeoides sp. nov. and A. mairei. Pujade-Villar et al. (2020) suspected that one of specimens included in their study as A. mairei (ILV91) was probably a new species based on the COI genetic distance. Our COI tree including this sequence (Fig. 17) and our pairwise genetic distance analysis (Table 3) supported their hypothesis. In addition, galls in one photograph in that paper (Pujade-Villar et al. 2020: fig. 7b) very likely belonged to A. elodeoides, although it is not clear to us whether these galls were the same as those which A. mairei-ILV91 was reared from.
Our phylogenetic analyses of gene sequence data support A. elodeoides and A. mairei as sister species (Figs 16, 17). The two species are sympatric in distribution and share the same host plant species, make galls on the same host plant structure (the stalk of male catkins), and overlap in time of gall formation and the emergence of adults. In addition, the galls of the two species share striking structural similarities despite distinct morphological differences (Figs 14, 15). Given these facts, it is intriguing what speciation mechanisms might have been involved given the lack of barriers in biogeography, host plant use, and phenology between the two species. It is possible that Wolbachiainduced cytoplasmic incompatibility was one of the potential causes for speciation between A. mairei, which is infected with Wolbachia, and its uninfected sister species A. elodeoides. Nonetheless, we did not conduct interspecific mating experiments after curing of Wolbachia due to the difficulties in artificial breeding of gall wasps.
Wolbachia (Anaplasmataceae) are maternally inherited endosymbiotic bacteria that infect arthropods and nematodes and has been shown to be associated with multiple effects on the reproduction of their hosts, such as cytoplasmic incompatibility (CI), induced parthenogenesis, feminization of genetic males, and male killing (Werren et al. 2008). Several studies have revealed Wolbachia infection in diverse cynipid species, involving tribe Aylacini, Diplolepidini, Cynipini, and Synergini (Plantard et al. 1998;Abe and Miura 2002;Zhu et al. 2007;Yang et al. 2013;Hou et al. 2020). In this study, we found that all examined individuals of A. mairei were infected with Wolbachia, whereas individuals of A. elodeoides collected from seven sites were all Wolbachia-free. Reproductive isolation between different populations or incipient species can evolve in both sympatry and allopatry (Turelli and Bierzychudek 2001). In arthropods, sympatry isolation may result from infection by Wolbachia reproductive manipulators (Engelstädter and Hurst 2009;Weinert et al. 2015). Cytoplasmic incompatibility, the most common form of reproductive manipulation induced by Wolbachia to its hosts, is characterized by partial or complete embryonic lethality in crosses between infected males and uninfected females or between hosts carrying incompatible symbiont strains. Thus, Wolbachia-induced CI may create substantial barriers to genetic exchange between individuals with different infection status and act as an agent of speciation (Werren 1998;Wade 2001;Turelli 2010). Bordenstein et al. (2001) reported a preeminent case of symbiont-assisted isolation because of Wolbachia-induced CI in the parasitoid wasp genus Nasonia (Hymenoptera, Chalcidoidea). This study demonstrated that Wolbachia-induced reproductive isolation via CI preceded the evolution of other mating barriers in Nasonia species and was the first major step in the process of speciation.
A contrasting difference in sex ratio was observed between A. elodeoides and A. mairei. Populations of of A. elodeoides were extremely female-biased, with female rates being 95.5-97.8%, while populations of A. mairei were more male biased to nearly balanced, with female rates being 5.4-43.5%. For two A. mairei populations in Jinzhai and Shucheng, which were investigated for six and four consecutive years, the female rates were 17.3% and 24.6%, or lower, respectively. This is consistent with observations made by other studies. Weld (1952) reported that there was only one female among the six adults of A. mairei collected from Hankou. Yang et al. (2012) collected specimens from multiple locations, including Yueyang, Changsha and Shaoyang, in Hunan Province, with a female ratio of less than 20%. The contrasting sex ratio biases of A. elodeoides and A. mairei are an interesting phenomenon that might be associated with Wolbachia infection. Genetic mutation or recombination may result in differences in susceptibility to Wolbachia infection in gall wasps and somehow effectively interrupted the genetic exchange between genotypes by mechanisms mentioned above. Consequently, a sympatric speciation event could take place relatively quickly due to founder effect (Joly 2011). This may explain our observation that the COI genetic distance between A. elodeoides and A. mairei is comparable to the average distance among known Andricus species from Eastern Asian while the two species are very similar in morphology, phenology, and gall morphology (Table 3). Nonetheless, the exact mechanism involved could only be understood by further investigations.