First record of the genus Fagineura Vikberg & Zinovjev (Hymenoptera, Tenthredinidae) with descriptions of two new species from China

Abstract Fagineura Vikberg & Zinovjev, 2000 is recorded from China for the first time. Two species of Fagineura are described as new, F.flactoserrulasp. n. and F.xanthosomasp. n. A key to the species of Fagineura worldwide is provided, now including four species. In addition, a simple phylogenetic analysis of Fagineura species is provided, based on sequences of the COI and NaK genes.


Introduction
Fagineura Vikberg & Zinovjev, 2000(Shinohara et al. 2000 is a very small genus of the subfamily Nematinae (Tenthredinidae). Until now, there are only two known species in the world (Taeger et al. 2010), namely F. crenativora Vikberg & Zinovjev, 2000 (type species) and F. quercivora Togashi, 2006, both of which are distributed in Japan. In a study of Nematinae from China, two species of Fagineura were found that are different from the two known species in Japan, and they are described herein as new species. Additionally, the genus Fagineura is recorded as a new genus in China. The two species are described and illustrated, a key to the known species of Fagineura worldwide is provided, and a simple phylogenetic analysis based on DNA sequence data from two genes (COI and NaK) is provided.

Imaging, terminology, deposition of material
The specimens were examined with a Motic-SMZ-171 stereomicroscope. Images of the imagines were taken with a Nikon D700 digital camera and a Leica Z16APO separately. The genitalia were examined with a Motic BA410E microscope, and images of the genitalia were taken with Motic Moticam Pro 285A. The series of images produced were montaged using Helicon Focus (HeliconSoft, Kharkiv, Ukraine) and further processed with Adobe Photoshop CS 11.0.
Morphological descriptions of the new species are based on the holotypes. The terminology of genitalia follows Ross (1945) and that of general morphology follows Viitasaari (2002). For a few terms, including middle fovea, lateral fovea, and lateral walls, we follow Takeuchi (1952).
Specimens examined in this study are deposited in the Central South University of Forestry and Technology, Changsha (CSCS), China, including all holotypes and paratypes of the two new species.

Phylogenetic analyses
DNA was extracted from adult samples stored in 99.5% ethanol at -20 °C by using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Sequence data were obtained from the mitochondrial gene cytochrome oxidase I (COI; 810 bp) and the nuclear gene sodium-potassium adenosine triphosphatase (NaK; 952 bp). PCR amplification of COI and NaK were performed as described previously (Normark et al. 1999;Nyman et al. 2006;Leppänen et al. 2012). New sequences have been deposited in GenBank under accession numbers MH544099-MH544102. COI and NaK sequences of Nematinae species used in previous phylogenetic analyses are available in GenBank, and their accession numbers and references are shown in Table 1.
The data of each newly sequenced sample are as follows: The final two-gene alignment is 1762 base pairs long and contains 42 specimens from 13 genera. The genetic distances among species were calculated based on Kimura 2-parameter model of the two genes in Mega 7 (Kumar et al. 2016). Bayesian phylogenetic analyses were performed in MrBayes 3.2.6 (Ronquist et al. 2012). The dataset was not partitioned, and the best-fitting DNA substitution model for the twogene alignment was selected using jModelTest 2.1.7 (Darriba et al. 2012), which uses PhyML (Guindon and Gascuel 2003) for likelihood calculations. Model selection was done by selecting among 11 substitution schemes (including 88 different models) on the basis of the Akaike Information Criterion (AIC).
Abbreviations used in the text and illustrations are as follows: OCL The distance between a lateral ocellus and the occipital carina, or the hind margin of the head where this carina would be if it were developed (Benson 1954). OOL The distance between an eye and a lateral ocellus. POL The distance between the mesal margins of the 2 lateral ocelli.

Fagineura Vikberg & Zinovjev, 2000
Diagnosis. Medium-sized; clypeus and labrum yellowish-white to yellow; clypeus with broad and moderately deep (0.4-0.5) emargination apically; mandibles symmetrical; malar space shorter than diameter of median ocellus, and in most species not exceeding 0.5 × of diameter of median ocellus; postocellar area short, more than 2.0 × as wide as long; antenna usually shorter than thorax and abdomen together; posterior part of mesopleural katepimeron covered with hairs; distance between cenchri almost as long as breadth of a cenchrus; forewing without radial cross-vein; the costa of forewing less dilated than in Pristiphora; hindwing with anal cell petiolate; claws bifid, inner tooth large; sawsheath short; annular suture 1 with setae band; the longest setae bands of lancet is at least 0.5 × length of annulus (Figs 1i, 2h); cypsella of basal serrulae almost absent, apically short and with somewhat deep emargination; tangium of lancet with campaniform sensilla in most species; radix at least 0.5 × as long as lamnium, in most species radix not shorter than lamnium.
Remarks. The genus resembles Pristiphora, Mesoneura, Euura and Nematus, but Fagineura can be distinguished from Pristiphora by having an emarginate clypeus; less dilated costa of the forewing; claws with a large inner tooth; in males, the posterior end of tergum 8 with distinct apical projection; distinguished from Mesoneura by the lack of radial cross-veins; apex of vein C in forewing slightly enlarged; abdomen longer than the head and thorax together; ovipositor sheath longer than fore tibia; distinguished from Euura and Nematus by an annular suture 1 with setae band; malar space narrower than the diameter of the median ocellus; katepimeron of the mesopleuron with hairs; having campaniform sensilla on the tangium in most cases.  . 1e); orbit black in female (Fig. 1b-c); clypeus emarginated for about 0.3 of its length (Fig. 1c); postocellar area 3.0 × as wide as long (Fig. 1b); ovipositor sheath without emargination apically, cerci almost as long as sheath (   . 1b-c); most parts of mesepisternum yellowish-brown (Fig. 1e); most of stigma pale yellowish-brown but margins black brown, veins in most part black brown; labrum and clypeus smooth and shiny, with few faint setigerous punctures, without microsculpture; frons slightly shiny, with hair warts and few wrinkles, punctures minute and sparse (Fig. 1b-c); vertex and postocellar area shiny, punctures faint and sparse, without microsculpture; malar space 0.5 × as long as diameter of median ocellus; postocellar area slightly convex, without mesosulcus, approx. 3.0 × as wide as long; relative length of antennomere 3 : antennomere 4 : antennomere 5 = 1.0 : 1.5 : 1.2 (Fig. 1d); forewings with cross-vein cu-a joining cell 1M at basal 0.5, cell 2Rs 1.2 × as long as wide, petiole of anal cell of hindwing 1.6 × as long as cu-a; lancet with 14 serrulae (Fig. 1i); annular suture 1 oblique and slightly curved, sutures 1-10 with setae bands, longest setae band about 0.7× length of annulus; tangium 3.4 × as long as annulus 1, radix 1.1 × as long as lamnium (Fig.1j).

Key to species of Fagineura in the world
Description. Holotype, female. Body length approximately 6.5 mm (Fig. 1a).
Color. Body mostly black. Labrum, clypeus, most parts of pronotum, most parts of propleuron, tegula, most parts of all coxae, all trochanters and femora pale yellow; most parts of vertex and temple, triangular spot of median mesoscutal lobe and mesoscutellum, most parts of mesepisternum, speckles on terga, sterna of abdomen, all tibiae and tarsi yellowish-brown; valvifer 2 pale yellow, valvula 3 yellowish-brown to black; cenchrus yellowish-white. Wings hyaline, most parts of stigma pale yellowishbrown with margins black brown, veins in most part black brown.
Thorax. Mesonotum shiny, with fine and slightly dense punctures, without microsculpture; median mesoscutal groove shallow and thin; mesoscutellum shiny, with faint and sparse punctures, and flat, posterior half of middle ridge distinct, about 0.8 × as long as wide; mesoscutellar appendage slightly shiny, with weak and sparse punctures, microsculpture faint, about 0.3 × length of scutellum, middle ridge low and blunt. Distance between cenchri as long as breadth of a cenchrus. Mesepisternum smooth and shiny, setigerous punctures and microsculpture indistinct; anepimeron of mesepimeron slightly shiny, with few wrinkles, punctures faint; katepimeron shiny, most parts with microsculpture and posterior part distinct, punctures weak and very sparse, posterior part covered with few setae; metapleuron shiny and smooth, with few weak punctures, microsculpture indistinct (Fig. 1e). Vein Sc interstitial with origin of vein M from R, and vein M slightly shorter than vein R+M; forewings with cross-vein cu-a joining cell 1M at basal 0.5, cell 2Rs 1.2 × as long as wide, petiole of anal cell of hindwing 1.6 × as long as cu-a.
Remarks. The new species is similar to F. crenativora Vikberg & Zinovjev, 2000, but can be distinguished from the latter by the following characters: metapleuron mostly black; orbit black in the female; postocellar area 3.0 × as wide as long; sheath without emargination apically; cerci almost as long as the sheath; lancet with 14 serrulae, annular suture 1 of lancet oblique and slightly curved, and with 7 marginal sensilla below.
Color. Body pale yellow to pale yellowish-brown. Lateral fovea, around ocelli, dorsal side of scape and pedicel, anterior edge and medial spot of tergum 1 black; cenchrus yellowish-white. Wings hyaline, stigma and most parts of veins pale yellow.
Thorax. Mesonotum slightly shiny, with minute and dense punctures, microsculpture indistinct; median mesoscutal groove shallow and narrow; mesoscutellum shiny, with weak and slightly sparse punctures, and flat, middle ridge indistinct, 0.8 × as long as wide; mesoscutellar appendage shiny, with faint and sparse punctures, without microsculpture, approx. 0.4 × as long as scutellum, middle ridge faint. Distance between cenchri as long as breadth of cenchrus. Mesepisternum shiny, setigerous punctures weak and slightly dense, microsculpture indistinct; mesepimeron shiny, with few faint punctures, with some microsculpture on margins, posterior part of katepimeron extensively covered with setae; metapleuron shiny and smooth, punctures and microsculpture indistinct (Fig. 2d). Vein Sc little basad of origin of vein M from R, vein M about as long as vein R+M; forewings with cross-vein cu-a joining cell 1M at basal 0.6, cell 2Rs 1.4 × as long as wide, petiole of anal cell of hindwing 1.3 × as long as cu-a (Fig. 2a).
Male. Unknown. Distribution. China (Hubei, Hunan). Variation. Body length 6.0-8.0mm; scape and pedicel partly to entirely black; around ocelli more or less black; vein Sc a little basad or interstitial with origin of vein M from R, and vein M as long as or slightly shorter than vein R+M; petiole of anal cell of hindwing 1.2-1.6 × as long as cu-a; in dorsal view, apex of cercus protruding far as or beyond valvula 3.
Remarks. The new species is similar to F. quercivora Togashi, 2006, but can be distinguished from the latter by the following characters: mesepisternum entirely pale yellowish-brown; all coxae and hind tibia pale yellowish; terga 3-10 entirely pale yellowish-brown; ovipositor sheath yellow; malar space 0.8 × as long as diameter of median ocellus; petiole of anal cell of hindwing longer than cu-a; tarsal claw with inner tooth shorter than outer tooth; lancet with 21 serrulae.
Etymology. The specific name is derived from the body color of adults.

Phylogenetic analyses
A Kimura 2-parameter model of COI and NaK distances within Fagineura species is shown in Table 2, and the mean distances within Nematus, Fagineura, Pristiphora, Euura, Mesoneura respectively and distances between these genera are shown in Table 3. The best-fitting model for the two-gene alignment was GTR+I+G (Nei and Kumar 2000). In MrBayes, default priors were used, and two parallel runs having four incrementally heated chains for 1.5 million generations were made, while sampling trees from the current cold chain every 1000 generations. 375 trees sampled were discarded prior to reaching chain stationarity as a burn-in from both runs, and the remaining 1126 trees were used to calculate a 50% majority consensus rule tree, showing all groupings with posterior probability more than 0.5 (Fig. 3). The two new species and Fagineura crenativora are separated by an adequate distance (Tables 2, 3), and all three species form a monophyletic group (Fig. 3). The K2P   distance based on COI and NaK between F. flactoserrula and F. xanthosoma are 6.7% and 0.5%, between F. flactoserrula and F. crenativora 5.2% and 0.5%, and between F. xanthosoma and F. crenativora 5.0% and 0.4%, respectively. The distance, based on COI and NaK, is 11.6% between Fagineura and Mesoneura and 8.9% between Fagineura and Nematus. These results are consistent with the morphological taxonomy described above. Unfortunately, sequences of F. quercivora are not available in GenBank, and we did not have any specimens of this species available. However, the two new species can be easily separated from F. quercivora by morphological characters.

Discussion
In this paper, two new species of Fagineura are described and illustrated. Compared to the generic characters of Fagineura proposed by Shinohara et al. (2000) and Prous et al. (2014), there are two differences in F. flactoserrula, including that the tangium lacks or has only one campaniform sensillum, and that the mesothoracic katepimeron is covered with only few setae. However, the generic characterisation in the earlier publications was based only on the two species known at that time, so that the previous definition of the genus apparently does not encompass the full range of interspecific variability. The phylogenetic analyses support placement of the two new species in Fagineura, and that they are different from F. crenativora. The new species are also different from F. quercivora based on morphological characters.