A new genus of anaxyelid wood wasps from the mid-Cretaceous and the phylogeny of Anaxyelidae (Hymenoptera)

Two new species of wood wasps (Anaxyelidae), Orthosyntexis elegans gen. et sp. nov. and Orthosyntexis thanti sp. nov., are described from mid-Cretaceous Kachin amber. Orthosyntexis gen. nov. exhibits characters and character combinations unique to the family, such as the combination of two mesotibial spurs, a length ratio of forewing 1Rs to 1M<1, a length to width ratio of forewing cell 1mcu ≥ 2, and the presence of 2M+Cu in the hind wing. The new species and morphological characters allow for an exploration of anaxyelid phylogeny. The phylogenetic results indicate that Anaxyelidae are monophyletic and it is suggested to classify the genera in two subfamilies, Syntexinae and Anaxyelinae, the latter including Kempendajinae and Dolichostigmatinae.


Taxonomy
All type specimens described herein are housed in the Key Laboratory of Insect Evolution and Environmental Changes, College of Life Sciences and Academy for Multidisciplinary Studies, Capital Normal University, Beijing, China (CNUB; Curator, Dong Ren). Specimens were examined under a Leica M205C dissecting microscope, and photographed using a Nikon SMZ 25 microscope with an attached Nikon DS-Ri2 digital camera system. Line drawings were prepared in Adobe Illustrator CC and Adobe Photoshop CC . Wing venation nomenclature was modified after Rasnitsyn (1969Rasnitsyn ( , 1980, with the following abbreviations: A, anal vein; C, costal vein; Cu, cubital vein; M, median vein; R, radial vein; R1, first radial; Rs, radial sector; 1cu-a, crossvein between 1Cu and A; 2rs-m and 3rs-m, crossveins between Rs and M; 1m-cu and 2m-cu, crossveins between M and 1Cu.

Phylogenetic analyses
A phylogenetic analysis was undertaken using morphological characters to determine the position of the new genus Orthosyntexis and to clarify relationships among the subfamilies Syntexinae, Anaxyelinae, Dolichostigmatinae and Kempendajinae. Seven extant species and 19 fossil species were used in these analyses. The phylogenetic analyses include 27 taxa, with 20 ingroups and seven outgroups -Macroxyela ferruginea (Say 1824) (Xyelidae), Acantholyda erythrocephala (Linnaeus 1758) (Pamphiliidae), Cephus nigrinus (Thomson 1871) (Cephidae), Sirex nigricornis (Fabricius 1781) and Urocerus gigas (Linnaeus 1758) (both Siricidae), Xiphydria camelus (Linnaeus 1758) (Xiphydriidae), and Orussus abietinus (Scopoli, 1763) (Orussidae). Some of the body characters are attributable to Vilhelmsen (2001), while a subset of the wing characters is referenced in Wang et al. (2016). In addition, we made some changes to the character states based on published data and new findings, and we added new characters from the antennae and wing venation. For instance, compared with Wang et al. (2016), we designated the characters of the length ratios of forewing 1Rs to 1M as '0' for '<1', '1' for '≥ 1, < 3' and '2' for '≥ 3'; the length ratios of forewing 1m-cu to 3Cu as '0' for '≤ 1', '1' for '>1'; and the length ratios of forewing 1Cu to 1M as '0' for 'apparently<1', '1' for '≥ 1'. To make the characters of ingroups as broad as possible, we also defined forewing 1r-rs as '0' for 'always present', '1' for 'partly reduced', and '2' for 'completely reduced', and hind wing cell r as '0' for 'closed' and '1' for 'open'. A total of 63 morphological characters and their character states are presented in the Suppl. material 2. Some are clearly present in the two new species, and others are important features of representative ingroups. A character-state data matrix consisting of 27 taxa and 63 morphological characters is provided in the Suppl. material 3.
Parsimony analysis was performed using WinClada v.1.00.08 (Nixon 2002) and NONA v.2.0 (Goloboff 1997). Tree search implemented a heuristic search method, and the options were set to hold 10,000 trees, 1000 replications, 100 starting tree replications, and a multiple TBR+TBR search strategy. All characters were treated as unordered and weighted equally. Bootstrap support values were determined in NONA with 1000 replications and are represented as numbers under the branches (in blue). Etymology. The new genus-group name is a combination of the Ancient Greek orthós (ο'ρθός, meaning, "upright" or "erect", and geometrically "right angle"), and the genus Syntexis (itself from Ancient Greek súntēxis (σύντηξις, meaning, "colliquescence", "emaciating", or "wasting away")), type genus of the subfamily Syntexinae. Gender feminine.

Remarks.
Orthosyntexis may be differentiated from Curiosyntexis, in which the forewing pterostigma is desclerotized and 1r-rs is partly reduced (Kopylov 2019), and from Dolichosyntexis, which has an enlarged pterostigma and forewing 1Cu not obviously shorter than 2Cu (Kopylov 2019). The new genus can be separated from Parasyntexis by the latter's narrower pterostigma, narrower than the length of 2r-rs, and 1M longer than 2M in the hind wing (Kopylov 2019). Orthosyntexis can be distinguished from Sclerosyntexis by the latter's elongate scape, which is 3× longer than wide, and the open cell r in the hind wing (Wang et al. 2020). Orthosyntexis differs from the extant genus Syntexis, which has 3Cu longer than 4Cu and 2m-cu 1.5× longer than 1m-cu in the forewing (Rohwer 1915), and from Daosyntexis by 1Rs 3× being longer than 1M and 3rs-m 2× longer than 4M in the forewing . The new genus differs from Cretosyntexis by 2r-rs meeting the pterostigma beyond midlength and Rs+M longer than 2M (Rasnitsyn and Martínez-Delclòs 2000), and from Eosyntexis in which 1RS is not shorter than 1M (Rasnitsyn and Martínez-Delclòs 2000). Etymology. The specific epithet is derived from the Latin word elegans, meaning elegant.

Orthosyntexis elegans
Diagnosis. Antennal scape length to width ratio slightly less than 3. Forewing 1Rs subvertical to R. Meso-and metafemur shorter than associated tibiae; mesotibial apical spurs elongate and distinctly narrowed apically.
Type locality and horizon. The amber specimen was collected from Kachin (Hukawng Valley) in northern Myanmar, and is dated at 98.79 ± 0.62 Mya (Cruickshank and Ko 2003;Shi et al. 2012). Head moderately large, narrower than thorax. Head 1.85 mm wide and 1.23 mm long, nearly quadrate. Compound eyes large and hemispherical; mandible straight, orthogonal with apical margin vertical (parallel to mandibular base) and with lowest tooth not that elongate (Fig. 2C); labial palpus with three palpomeres; maxillary palpus with at least five palpomeres; antenna with 16 flagellomeres, scape 0.40 mm long, maximum width 0.14 mm; pedicel 0.39 mm long, maximum width 0.12 mm; flagellomere I shorter than scape, 0.11 mm wide, 0.22 mm long, flagellomere II 0.10 mm wide, 0.19 mm long, as long as individual lengths of remaining flagellomeres (Fig. 2B). Thorax wide, width across tegulae 1.56 mm; pronotum short, having prominent anterior notch and hind margin, with median longitudinal furrow dorsally. Mesoscutum with median longitudinal sulcus and notauli strongly impressed; mesoscutellum tapering to acute apex; ratio of lengths of prescutum, median longitudinal sulcus between notauli, and mesoscuto-mesoscutellar sulcus and mesoscutellum 2.5/1/4.5, notauli terminating close to mesoscutellum (Fig. 1A). Legs spindly, meso-and metafemora shorter than associated tibiae. Metafemur length 1.30 mm, metatibia thick subapically, length 1.65 mm. Protibia and metatibia with only one apical spur visible, mesotibia with two apical spurs (Fig. 3C, D and E); all tarsomeres with stiff apical setae; basitarsi long but shorter than remaining tarsomeres combined; tarsomere V elongate but shorter than basitarsus; pretarsal claws long, with curved apices and each with a single preapical tooth.
Hind Etymology. The specific epithet honours 3 rd Secretary General of the United Nations and Burmese diplomat U Thant  and his dedication to seeking peace within and between nations. Diagnosis. Antennal scape length to width ratio more than 3. Forewing 1Rs proclival to R. Mesofemur longer than mesotibia, metafemur nearly as long as metatibia; mesotibial apical spurs somewhat shortened and not narrowed apically.
Forewing with dense microtrichia but no coloration pattern, except for slightly darkened costal area. C and R thick, costal area narrower than C and R widths. Pterostigma completely sclerotized; Sc absent; 1r-rs and 2rs-m absent; 2r-rs issuing from pterostigma at its basal 1/3; 1Rs short and slightly proclival to R, about 0.7× as long as 1M, meeting 1M at right angle (Fig. 7E). 2Rs+M distinct but short, 0.1× as long as 1Rs+M (Fig. 7E)  with an angle at 2Rs and shorter than 1Rs. 1M straight; m-cu absent; 2M+Cu present (about 0.40 [0.33] mm in length), free abscissa of Cu and cu-a (about 0.40 [0.43] mm in length) developed, 1Cu and cu-a straight.
Regarding the intrafamiliar relationships of Anaxyelidae, two principle branches are supported in the consensus topology (Fig. 8), one equivalent to the subfamily Syntexinae, and the other composed of Dolichostigmatinae, Kempendajinae, and Anaxyelinae, and with the dolichostigmatines and kempendajines rendering anaxyelines paraphyletic. While support values of the two main branches are not great, there are convincing apomorphies supporting these clades. For example, the monophyly of Syntexinae is supported by the ratio of the maximum width of the pterostigma to the length of 2r-rs of forewing nearly = 1 (character 24 length of 2Cu to the length of 1Cu in the hind wing ≥ 5 (character 55), and crossvein m-cu of hind wing absent (character 57). At the same time, the monophyly of Anaxyelinae + Dolichostigmatinae + Kempendajinae is supported by length of 1r-rs to 2Rs of forewing < 1 (character 38), length of Rs+M to 2M of forewing < 1 (character 39), and hind wing crossvein 1rs-m at or quite close to Rs midlength (character 52).

Discussion
The placement of Orthosyntexis in Anaxyelidae is well-supported, most notably by the fore-and hind wings each with a single rs-m and the pedicel length to width ratio ≥ 2. Moreover, the genus can be attributed to Syntexinae mainly based on the following combination of characters: forewing with the maximum width of pterostigma not shorter than 2r-rs, 1r-rs absent and hind wing m-cu absent. Given that most of the available fossil anaxyelids are preserved as compressions, often with body structures poorly discernible, there is a natural reliance on wing traits and so most characters currently supporting nodes are derived from the venation. Naturally, as more amber fossils become available, it is hoped that a finer comparison of body structures can be made across living and fossil taxa in the future.
Hitherto, no attempt has been made to explore relationships among anaxyelids in a cladistic framework, likely owing to the fact that there is only one extant species and that many of the known fossils are quite incomplete. Recent phylogenetic treatments of the families of Hymenoptera (e.g., Vilhelmsen 2000Vilhelmsen , 2001Sharkey et al. 2012), identified a single putative apomorphy for the family, specifically the longitudinal subdivision of the pronotum, easily observed in our amber fossils. Our analysis recovered a monophyletic Anaxyelidae, even when including the considerable fossil diversity and the lack of critical information from many compression fossils. In addition, our analysis indicated the pedicel length to width ratio ≥ 2 and the loss of forewing 2rs-m and hind wing 3rs-m to be additional putative apomorphies for the family (Fig. 8). Zhang and Rasnitsyn (2006) and Kopylov (2019) organized the diversity of Anaxyelidae into four subfamilies. The establishment of Dolichostigmatinae and Kempendajinae was based on four fossil specimens from three species: Dolichostigma tenuipes Rasnitsyn 1968, Kempendaja jacutensis Rasnitsyn 1968and Mangus magnus Kopylov 2019. The diagnosis of the subfamily Kempendajinae (Rasnitsyn 1980) focused generally on characters of the body and hind wing, which are plesiomorphies; for example, desclerotized pterostigma in the forewing and rs-m located in the distal third of hind wing cell r. Desclerotized pterostigma is also observed in two anaxyelids, Anaxyela gracilis Martynov 1925 and Sphenosyntexis pallicornis Rasnitsyn 1968. In addition, rs-m in the hind wing is also located in the distal third of cell r in U. undosa. Thus, the features used to justify the subfamily Kempendajinae are unreliable and our analysis robustly placed the group within Anaxyelinae. Similarly, the feature used to differentiate Dolichostigmatinae, i.e., large forewing pterostigma also exists in Dolichosyntexis transbaikalicus Kopylov 2019. Again, our analysis indicated that the subfamily Dolichostigmatinae is merely a subgroup of Anaxyelinae. Accordingly, we recognize only two monophyletic subfamilies in Anaxyelidae: Syntexinae and Anaxyelinae. Of course, this is only an analysis based on our phylogenetic results, and many fossil specimens provide few useful features due to preservation reasons. Therefore, it is hoped that more fossils of Anaxyelidae will provide more favorable evidence for our analysis in the future.
Recently, the anaxyelid Sclerosyntexis hirsuta was described from a single specimen in Kachin amber (Wang et al. 2020). Here, we add two new species, O. elegans and O. thanti, from the same deposit, based on three specimens. They have thick legs, particularly inflated metatibiae, and a pattern of forewing venation similar to Sclerosyntexis, such as 2r-rs joining the pterostigma proximal to pterostigmal midlength, 1r-rs absent, and 2Rs+M present. Furthermore, Sclerosyntexis + Parasyntexis was recovered as the sister group to Orthosyntexis in our phylogenetic estimate for the family.
Although Orthosyntexis belongs to the same clade as the modern Syntexis, these genera are relatively distantly related within the subfamily, and many features differ significantly between them, e.g., flagellomere I length to width ratio < 3, mesotibia with two apical spurs, 1r-rs absent, 2Rs+M present, hind wing cell r closed, and hind wing 2M+Cu present. However, they do share some similar morphological traits, such as notauli close to the mesoscutellum and 1rs-m at the base of Rs in the hind wing. In addition, the new genus, like extant Anaxyelidae, has the ovipositor apically modified with marginal serrations. Extant Siricoidea use their ovipositor to insert their eggs and spores of a symbiotic fungus into dead or dying trees (Goulet 1993;Grimaldi and Engel 2005). However, there has been no evidence of fungal spores in extinct Anaxyelidae, so a symbiotic relationship between extinct Anaxyelidae and fungi has not been conclusively established, although the distribution of this trait across wood wasps tends to suggest that the extinct diversity had a similar association (e.g., Morgan 1968;Kajimura 2000;Grimaldi and Engel 2005). Nonetheless, conclusive evidence of a fungal symbiosis in the fossils is lacking and requires further study as more fossil Anaxyelidae are discovered. Indeed, µCT scans of amber-preserved anaxyelids would be critical to look for potential fungal spores carried on adult females or the presence of distinct mycangia. To date, only seven symphytan species have been described in Kachin amber, of which five are syspastoxyelids, one is an anaxyelid, and one is an orussid (Engel et al. 2016;Zhang et al. 2018Zhang et al. , 2020Zheng et al. 2019;Wang et al. 2020Wang et al. , 2021. And eight symphytan specimens have not been described in Kachin amber, of which seven are anaxyelids and one is an orussid (Zhang et al. 2018). Compared with Mesozoic fossil deposits of China and Russia, the number of Symphyta in Kachin amber is quite small. The reason for such a low diversity and abundance may be that the mid-Cretaceous fauna of Kachin amber is characterized by high levels of endemism indicative of an insular biota (Grimaldi et al. 2002;Rasnitsyn and Öhm-Kühnle 2018;Lin et al. 2019;Zhao et al. 2020), as well as its high termophilic (tropical) nature (Grimaldi et al. 2002;Zhang et al. 2018).