Sergey gen. n., a new doryctine genus from temperate forests of Mexico and Cuba (Hymenoptera, Braconidae)

Abstract The new doryctine genus Sergey gen. n. is described with four new species (Sergey cubaensis Zaldívar-Riverón & Martínez, sp. n., Sergey coahuilensis Zaldívar-Riverón & Martínez, sp. n., Sergey tzeltal Martínez & Zalídivar-Riverón, sp. n., Sergey tzotzil Martínez & Zalídivar-Riverón, sp. n.) from temperate forests of Mexico and Cuba. Similar to many other doryctine taxa, the new genus has a considerably elongated, petiolate basal sternal plate of the first metasomal tergite, although it can be distinguished from these by having the mesoscutum sharply declivous anteriorly with sharp anterolateral edges. The described species have been characterised molecularly based on two mitochondrial (COI, cyt b) and one nuclear (28S) gene markers. Based on the mitochondrial gene genealogies reconstructed, the evidence suggests the existence of incomplete lineage sorting or hybridization in the populations from Chiapas and Oaxaca assigned to Sergey tzeltal sp. n.


Introduction
The braconid wasp subfamily Doryctinae is a highly diverse, cosmopolitan group that currently comprises 198 genera and about 1,700 species (Braet 2016;Yu et al. 2012). This group gathers a wide array of genera with distinct morphologies and biologies, though most of its species appear to be idiobiont ectoparasitoids of bark-boring or xylophagous beetle larvae (Belokobylskij 1992;Marsh 1997). Previous attempts trying to elucidate the phylogenetic relationships among doryctine genera based on morphological evidence yielded poorly resolved hypotheses Belokobylskij et al. 2004). Subsequent molecular phylogenetic studies carried out for the subfamily (Zaldívar-Riverón et al. 2007, 2008 refuted most of the previously proposed tribes and subtribes Fischer 1981). These molecular phylogenies have served as a base to start building a stable higher-level classification for the group (Zaldívar-Riverón et al. 2007, 2008Samacá-Sáenz et al. 2016).
One of the main external morphological features that was traditionally used to group genera within the Doryctinae is the relative length of the basal sternal plate of the first metasomal tergite (acrosternite sensu Belokobylskij 1995). This structure can be petiolate, tubular and long or sessile and short (Belokobylskij 1995;Marsh 1997). Within the Doryctinae, a long and tubular basal sternal plate has been shown to have independently evolved in various unrelated genera. Two of these genera are among the most speciose within the subfamily, the cosmopolitan, mainly Old World Spathius Nees, and the exclusively Neotropical Notiospathius Mathews & Marsh.
In a recent molecular phylogenetic study of Notiospathius, various species originally assigned to this genus were nested in two distantly related clades (Ceccarelli and Zaldívar-Riverón 2013). Members of these two clades have consistent external morphological features that distinguish them from each other and from the remaining doryctine genera. Species of one of these clades were placed in the newly described genus Bolivar Zaldívar-Riverón & Rodríguez-Jimenez (Zaldívar-Riverón et al. 2013).
In this work, a new doryctine genus, Sergey gen. n., is erected to include the species of the second clade, and four new species are described. Three of these species were collected in cloud forests from México and Cuba, whereas the remaining one was collected in a submontane forest in Coahuila, northeast Mexico. Members of the new genus are morphologically distinct from other doryctine genera with petiolate first metasomal tergite by having the anterolateral corners of mesoscutum sharply pointed and a different pattern of ornamentation in the propodeum, with two divergent carinae that sometimes enclose a more or less distinguishable areola. The phylogenetic relationships within the new genus have been assessed based on separate analyses of one nuclear and two mitochondrial (mt) markers, and provide evidence that suggests the existence of incomplete lineage sorting between two populations of one of the described species.

Specimens and terminology
Specimens were collected in four different localities in Mexico and Cuba, preserved in 100% ethanol, kept at 20°C until they were processed for DNA sequencing, and subsequently dried, labelled and mounted. The examined specimens are deposited in the Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Autónoma de México (IB-UNAM), Mexico City, Mexico, and the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia" (MACN), Buenos Aires, Argentina.
The morphological terminology follows Sharkey and Wharton (1997), except for the sculpture characters, which follow Harris (1979), and the term precoxal sulcus, which replaces the term sternaulus according to Wharton (2006). Digital colour images were taken with a Leica® Z16 APO-A stereoscopic microscope, a Leica® DFC295/ DFC290 HD camera, and the Leica Application Suite® program. Digital SEM images were taken with a FEI® INSPECT (Oregon, USA) and a Hitachi® SU1510 SEM microscopes in low vacuum at the Museo Nacional de Ciencias Naturales (CSIC, Madrid, Spain) and the IB-UNAM, respectively.

Gene genealogies
Sequences of three gene markers have been examined for specimens belonging to the new genus. These included 34 and 26 sequences that were previously published in Ceccarelli and Zaldívar-Riverón's (2013) phylogenetic study of Notiospathius belonging to the cytochrome oxidase I (COI; 531 bp) mt and the second and third domain regions of the 28S nuclear ribosomal (r) (~617 bp;) DNA genes, respectively. Moreover, COI and 28S sequences were generated of additional specimens of this genus, as well as sequences of a 371 bp fragment of the cytochrome b mt DNA gene for a subset of the examined specimens. Sequences of Heterospilus tauricus Telenga (DNA voucher number CNIN884; GenBank accession nos. KC822008, 36, 72 for COI, 28S and cyt b, respectively) were also included to root the trees. Heterospilus was closely related to the newly described genus in the above molecular phylogenetic study. The sequenced ingroup specimens, their localities and GenBank accession numbers are provided in the description section.
Corrected pairwise genetic distances for the three gene markers were calculated using the K2P model with MEGA version 6 (Kimura 1980;Tamura et al. 2013). Separate gene genealogies were carried out with the program MrBayes version 3.2.6 (Ronquist et al. 2012) in the Cipres Science Gateway (Miller et al. 2010). Each analysis consisted of two independent runs of 20 million generations each, used uniform priors and sampled trees every 1000 generations. The following evolutionary models selected for each partition were obtained using the Bayesian criterion with JMOD-ELTEST2 (Darriba et al. 2012): 28S.-K2; CytB.-1 st pos, TrN + G, 2 nd pos, HKY + I, 3 rd pos, GTR + G; COI.-1 st pos, F81, 2 nd pos F81, 3 rd pos HKY. Burn-in was determined assessing convergence between runs verifying the potential scale reduction factors (PSRF) and the estimated sample size (ESS) for all tested parameters. The burnin fraction was set to 0.25, which corresponded to 5,000 trees (5 × 10 6 generations) in all analyses. The remaining trees from the two independent runs were employed to reconstruct a majority rule consensus tree using the 'halfcompat' option implemented in MrBayes. Clades were regarded as significantly supported if they had a posterior probability 0.95 (Ronquist et al. 2012).  5-segmented, apical segment longer than fourth segment; labial palpi short, 4-segmented, third segment not shortened. Scape of antenna wide and rather short, without flange apically and ventroapical lobe, without basal constriction; ventral margin of scape shorter than dorsal margin in lateral view. First flagellar segment about the same length as second segment, usually several apical or subapical segments whithish. Apical segment more or less pointed apically, without "spine".
Wings: veins RS and r-m present, thus first and second submarginal cells entirely closed. Second submarginal cell rather long and narrow. First subdiscal cell open postero-apically, vein 2cu-a absent. Veins 1a and 2a absent. Hind wing with vein C+Sc+R longer than vein SC+R. Vein RS arising from vein R far from vein r-m. Marginal cell more or less distinctly narrowed towards apex, without vein r. Vein cu-a present. Vein M+CU about 0.6-0.7 times as long as 1M; vein m-cu straight. Male hind wing without stigma-like swelling of basal veins.
Legs: Fore tibia on inner surface with several long and slender spines arranged along its anterior margin in almost single vertical line. Hind coxa long and narrow, with basoventral tubercle. Claws simple.
Metasoma: first tergite petiolate, long and narrow, usually striate-coriaceous, with some transverse carinae basally, these carinae sometimes reduced. Basal sternal plate (acrosternite) of first tergite long, 0.6-0.7 times as long as first tergite, extended distinctly beyond level of spiracles. Dorsope of first tergite small and shallow; spiracular tubercles indistinct, situated in basal 0.3 of tergite. Second tergite without distinct furrows and areas. Second suture considerably shallow, complete, almost straight in females and distinctly curved in males. Third tergite without transverse furrow and basal area. Tergites behind second with a single transverse line of sparse long erect setae. Ovipositor distinctly darkened apically, with two distinct subapical nodes. Ovipositor sheaths long, about as long as metasoma or slightly longer.
Etymology. We are very pleased to name this genus after our dear friend and colleague Dr. Sergey A. Belokobylskij, for his great contribution to the taxonomic knowledge of the braconid subfamily Doryctinae. Gender is to be considered masculine.

Key to species of Sergey
1 Eyes small, their height about as long as malar space (Fig. 4); first metasomal tergite 1.5 times longer than its apical width ( Fig. 8)  Antenna with a white apical or subapical band composed of 3-7 (rarely two) flagellomeres in females (Figs 24,26); males either with two apical flagellomeres whithish (Fig. 25), or with antenna entirely brown ( Diagnosis. This is the most distinctive species of the genus. It can be distinguished from the remaining members of Sergey by having the eyes considerably smaller, their height about as long as malar space (distinctly longer than malar space in the remaining species); and the first metasomal tergite broad, 1.5 times longer than its apical width (slender, at least 2.1 times longer than its apical width in the remaining species). Description. Body length 2.1mm (Fig. 3), fore wing 1.5mm; ovipositor sheaths 1.1mm. Colour: head excluding antennae, mesoscutum and mesopleuron brown, otherwise uniformly honey yellow. Head: about as high as wide (anterior view) (Fig. 4), 0.7 times as long as wide (dorsal view). Clypeus, face, frons and vertex largely smooth and shining (Fig. 5), with a few shallow rugae near the mandible insertion and antennal sockets; temple smooth. Eye small 1.5 times higher than wide. Malar space height/eye height ratio 1.1 (Fig. 4). Temple/eye length ratio (dorsal view) 0.6. Antenna incomplete, only with nine basal flagellomeres; first flagellomere about four times longer than wide and as long as second.
Mesosoma: 2.0 times longer than wide and 1.9 times longer than high (Fig. 6). Pronotal groove wide, and scrobiculate, pronotal carina distinct. Propleuron rugose on median third. Mesoscutum transverse, 0.6 times as long as wide. Mesoscutal lobes smooth, notauli deep and scrobiculate, obscured in an irregularly rugose median area before reaching the scuto-scutellar suture (Fig. 9). Prescutellar sulcus with three carinae, the median one straight, and the lateral ones irregular. Scutellar disc smooth and triangular. Mesopleuron smooth. Precoxal sulcus, deep, wide and scrobiculate-rugose, running along the entire length of mesopleuron. Subalar sulcus deep and rugose. Metanotum with a median carina but without a distinct projection. Metapleuron entirely areolate rugose. Propodeum with two divergent carinae running from median anterior edge delimiting two smooth dorsolateral areas, beyond these carinae it is almost uniformly areolate rugose.
Legs: fore tibia with a row of spines. Hind coxa slightly striate dorsally, smooth ventrally, with a small but distinct basoventral tubercule.
Male. Unknown. Distribution. Known only from a submontane forest at the type locality in Coahuila, Mexico. Diagnosis. This distinctive species can be distinguished from the remaining species of Sergey by having: 1) a mostly yellow body colour (brown to black in the remaining species); 2) head and mesoscutum distinctly sculptured, transversally striate (entirely smooth and polished in the remaining species); and 3) fore wing with vein m-cu reaching vein RS+M basally to 2RS, thus vein (RS+M)b present and distinct (m-cu reaching vein RS+M interstitial with respect to vein 2RS, thus vein (RS+M)b absent in the remaining species).
Description. Body length 3.1mm (Fig. 11), fore wing 2,5 mm; ovipositor sheaths 3.5 mm. Colour: most part of the body yellow; apical third of propodeum and first metasomal tergite dark brown, second metasomal tergite yellow with lateral areas brown; antennae honey yellow, gradually darkening toward apex, subapical 20 th to 23 rd segments white (Fig. 15), apical three segments dark brown; fore and middle coxae pale yellow; fore and middle tibiae brown to dark brown; trochanter and trochantellus pale yellow; tarsi brown to dark brown; hind coxa pale yellow basally, dark brown apically; hind femur and tibia with four alternate yellow and dark brown transversal bands. Wings hyaline; pterostigma and veins brown. Ovipositor sheaths yellow to honey yellow.
Legs: fore tibia with a row of spines. Hind coxa transversally striate-rugose, with a small but distinct basoventral tubercle..
Males. Unknown. Distribution. Known only from the type locality in southern Cuba.

Biology. Unknown.
Etymology. This species is named after the Caribbean country where it occurs, Cuba. Diagnosis. This species is similar to S. tzotzil, but it can be distinguished from the latter species by the colour pattern of the white band on the female antenna. In S. tzeltal, the white band is either apical or subapical and is composed of at least two entire whitish flagellomeres, usually more, with at most three apical flagellomeres brown. In S. tzotzil, the white band is subapical and consists only of the lighter color on the articulation between the 19 th and 20 th flagellomeres, and with the five apical flagellomeres brown.
Description. Body length 3.1 mm (Fig. 20), fore wing 2.7 mm; ovipositor sheaths 1.5mm. Colour: head uniformly brown, antenna brown, gradually darkening towards apex, except for a subapical white band composed of 2-4 flagellomeres (apical in Oaxaca population composed of 6-8 flagellomeres, see below) (Figs 24,26). Mesosoma uniformly dark brown, except for a slightly lighter area on the median area of mesoscutum. Metasoma brown. Legs light brown, except fore and middle coxae, trochanters and trochantelli and hind trochantellus, which are pale yellow; hind coxa and apical three fourths of hind femur dark brown. Wings hyaline; pterostigma and veins brown. Ovipositor sheaths brown.
Head: 0.8 times as high as wide in anterior view (Fig. 21), and 0.7 times as long as wide in dorsal view (Fig. 22). Clypeus, face, frons and vertex smooth and polished; temple smooth. Eye 1.3 times higher than wide. Malar space height/eye height ratio 0.3. Temple/eye length ratio (dorsal view) 0.6. Antenna with 24 flagellomeres, first flagellomere about 4.0 times longer than wide, as long as second one.
Legs: fore tibia with a row of spines. Hind coxa transversally striate dorsally, smooth ventrally, with a distinct basoventral tubercule.
Distribution. This species is known from cloud forests located in the Reserva el Triunfo, Chiapas, and Santiago Comaltepec, Oaxaca, in southeast Mexico.
Biology. Unknown. Comments. This species has a considerable variation in the antennal color pattern. We had originally grouped the specimens assigned to this taxon in two morphospecies, each represented by the specimens from Chiapas and Oaxaca, respectively. Females from Oaxaca have a distinct apical white band composed of 6-8 flagellomeres (Fig.  24), whereas in males this apical band is smaller (Fig. 25). On the other hand, most females from Chiapas have a white antennal band that is subapical and is only composed of 2-4 flagellomeres (Fig. 26). However, one female that could not be sequenced has an apical band similar to the specimens from Oaxaca. Other external morphological features (e.g. sculpture of propodeum and first metasomal tergite) also varied but we could not find any correlation with the geographical provenance of the specimens.
There was no concordance between the corrected COI distances and the geographic provenance and morphological variation for the above specimens. Some of the specimens from Oaxaca had lower COI distances with those from Chiapas than with the remaining specimens from the same locality (0.38-0.76 and 1.7-1.9%, respectively). This incongruence suggests that the existence of incomplete lineage sorting or hybridization between two recently diverged, sympatric species (see below). We have followed a conservative approach and consider the members of the populations from Chiapas and Oaxaca as a single species. One of the specimens from Oaxaca (DNA voucher number CNIN573) has considerably higher COI distances compared with the remaining conspecific specimens (3.6-4.7%). However, it is morphologically undistinguishable and we thus placed it within S. tzeltal.
Etymology. The name of this species refers to the Tzeltal ethnic group, descendant from the Mayans that inhabits Los Altos, a mountain region located in central Chiapas Description. Body length 3.7mm (Fig. 30), fore wing 3.2mm; ovipositor sheaths 2.3mm. Colour: head uniformly brown, antenna brown, gradually darkening towards apex, except for a light band composed of most of the 19 th and the basal half of the 20 th flagellomeres (Fig. 34). Mesosoma uniformly dark brown, except for a slightly lighter area on median area of mesoscutum. Metasoma brown. Legs light brown, except fore and middle coxae, trochanters and trochantelli and hind trochantellus which are pale yellow; hind coxa and apical three fourths of hind femur dark brown. Wings hyaline; pterostigma and veins brown. Ovipositor sheaths brown.
Head: in anterior view 0.9 times as high as wide (Fig. 31), and 0.6 times as long as wide in dorsal view (Fig. 32). Clypeus, face, frons and vertex smooth and shining; temple smooth (Fig. 33). Eye 1.3 times higher than wide. Malar space height/eye height ratio 0.4. Temple/eye length ratio (dorsal view) 0.6. Antenna with 25 flagellomeres, first flagellomere five times longer than wide and about as long as the second one.
Mesosoma: 2.1 times longer than wide (Fig. 35) and 2.0 times longer than high (Fig. 36). Pronotal groove wide, deep, and scrobiculate, pronotal carina distinct. Propleuron smooth. Mesoscutum slightly transverse, 0.7 times as long as wide. Mesoscutal there were three non-significantly supported, subclades, two of which were composed of specimens from Comaltepec, Oaxaca, but with one of them being more closely related to the subclade containing the specimens from El Triunfo, Chiapas.
The bayesian phylogram derived from the cyt b sequences yielded similar relationships with the COI topology. Again, some of the specimens of S. tzeltal from Comaltepec, Oaxaca were more closely related to the ones from El Triunfo, Chiapas (PP = 0.6) than with the remaining specimens from the same locality. The 28S tree was largely unresolved (phylogram not shown), with the sequenced specimens S. tzeltal and S. tzotzil grouped together (PP = 1.0). The reconstructed mt gene genealogies, together with the geographic provenance and morphological variation found in the specimens of S. tzeltal from Oaxaca and Chiapas suggests that this taxon could consist of two sympatric, recently derived lineages in which there is incomplete lineage sorting or hybridization. Further morphological and genetic studies will help to confirm the taxonomic status of the populations of S. tzeltal from the latter two Mexican regions.