Convergence in the ovipositor system of platygastroid wasps ( Hymenoptera )

It is widely accepted that there are two ovipositor system types in Platygastroidea. The Scelio-type ovipositor system features telescoping conjunctiva between metasomal tergites 6 and 7 and operates via internal changes in hydrostatic pressure alone, whereas muscles are involved in ovipositor extension and retraction in the Ceratobaeus-type. We here provide analyses of ovipositor systems in two platygastrine genera that cannot be classified as either Scelioor Ceratobaeus-type, but exhibit telescoping conjunctivae and extend the ovipositor system without musculature. These represent unique derivations that are clearly identifiable by the location of the telescoping conjunctiva. In Gastrotrypes Brues, the telescoping conjunctiva occurs between T5 and T6, and in Platygaster tubulosa Brues it occurs between each segment from T3 to T6. If there are convergent derivations of a Scelio-type ovipositor system within Scelionidae (sensu Masner 1976), their identification will require examination of new characters, one of which we provide here, the presence and form of a ring of acanthae on the conjunctiva between T6 and T7.


Introduction
The mechanism of ovipositor extension in Platygastroidea is unique among hymenopterans with retracted ovipositors.Outside of Platygastroidea the ovipositor is protracted by the contraction of the 2nd valvifer-2nd valvula muscles and the anterior pivot of the bulb is often accompanied by posterior rotation of the ovipositor (Figure 1C, Ernst et al. 2013).In Platygastroidea, the mid portion of T9 (tA9: Figures 6, 11 Austin and Field 1997) is elongate and weakly sclerotized, allowing the rest of the ovipositor assembly (first and second valvifers, first, second and third valvulae and proximal (lateral) region of abdominal tergite 9) to protrude telescopically.The biological machinery (e.g.muscles, apodemes) underlying the ovipositor protraction and retraction system of Platygastroidea has not been aligned to that of other Hymenoptera and thus its evolutionary origin has yet to be elucidated.The morphology of platygastroid ovipositor systems has been used for generic diagnoses (Masner 1976, Talamas et al. 2011) and was the sole character source for a morphology-based phylogeny of the superfamily (Austin and Field 1997).Austin and Field (1997) classified the platygastroid ovipositors into two types: Scelio-and Ceratobaeus-types.In the Ceratobaeus-type, telescopic movement occurs only at the elongate and weakly sclerotized mid portion of T9 and the proximal portion of the ovipositor remains within the apical part of the metasoma, with the exception of Teleasinae (Austin and Field 1997) and Anteris Förster (Talamas et al. 2011).An additional telescoping region can be found between metasomal tergites 6 and 7 in the Scelio-type ovipositor system, causing metasomal tergites 7-9 to be extended with the ovipositor away from the preceding metasomal segments.In this system, the proximal portion of the ovipositor substantially protrudes from T7+8.Although muscles and apodemes on T7+8 and corresponding sternites are present in the Ceratobaeus-type system, internal changes in hydrostatic pressure likely contribute to ovipositor protraction (Austin 1983, Field andAustin 1994) and thus facilitated the evolution of ovipositor systems that operate via hydrostatic pressure alone.The Scelio-type system was documented in numerous scelionine genera, including Archaeoteleia Masner (Austin and Field 1997, Masner 1976, Masner 1968) and was used to define a putatively monophyletic group, Scelionini sensu lato (Austin and Field 1997).Austin and Field (1997) provided diagnoses and discussed differences in major platygastroid clades, including Platygastridae s.s., and acknowledged that their observations in this family were preliminary.They listed the reduction and lack of sensory structures on T7+8, and the lack of additional telescoping structures (conjunctiva not extended between T6 and T7) among the diagnostic characters for Platygastridae s.s.. Brues (1922) and Huggert (1980) reported the presence of additional telescoping structures in the ovipositor system of Gastrotrypes and Platygaster tubulosa.Comparison between these ovipositor systems and the telescoping ovipositor system of Scelionidae (sensu Masner 1976) were not mentioned in Austin and Field (1997).We here use scanning electron microscopy and confocal laser scanning microscopy to further document remarkable evolutionary convergence in telescoping ovipositor systems of platygastroid wasps.Gastrotrypes is cosmopolitan in distribution with seven valid species and numerous species that remain undescribed.Individuals are typically small, often less than 1 mm in length, and are known to be associated with gall midges that feed in the vascular tissue of freshly cut wood (Brues 1922).Females can be separated from other platygastrine genera by an absence of venation in the fore wing, presence of five externally visible tergites, and by the cylindrical "nozzle" formed by T5+S5 at the apex of the metasoma.Gastrotrypes is very similar to Metanopedias Brues, which also has a reduced number of visible tergites (4 or 5), but has the apical segments of the metasoma distinctly spatulate (Figures 1-5).Males of both genera have 7 visible tergites and reliable characters to separate male Gastrotrypes from male Metanopedias are not known to us.
Platygaster tubulosa is recognizable by the presence of only two external tergites when the ovipositor system is retracted.Like Gastrotrypes, it is associated with Cecidomyiidae that feed in wood vascular tissue (Brues 1922).Huggert (1980) considered the lumber trade to be the means by which P. tubulosa became a "tramp" species, now known from Asia, Europe, Africa and the Americas (Huggert 1980, Notton 2006, Buhl 2011).

Materials and methods
Dissections for scanning electron microscopy were performed with a minuten probe and forceps and body parts were mounted to a 12 mm slotted aluminum mounting stub (EMS Cat.#75220) using a carbon adhesive tab (EMS Cat.#77825-12) and sputter coated with approximately 70 nm of gold/palladium using a Cressington®™ 108 sputter coater.Micrographs were captured using a Hitachi®™ TM3000 Tabletop Microscope at 15 keV.Sample preparation for CLSM followed Mikó et al. 2016: specimens were temporarily mounted between two coverslips (#1.5) in a glycerin droplet.Blu-tack (Bostik, Wauwatosa, WI, USA) was used as spacer because this material does not interact with glycerol.Specimens were examined with an Olympus FV10i desktop CLSM using the 60X objective.Brightfield images were captured with a Z16 Leica®™ lens with a JVC KY-F75U digital camera using Cartograph®™ software.In some cases, multiple SEM micrographs or photographs were stitched together in Adobe Photoshop CS4®™ to produce larger images at high resolution and magnification.Full resolution images are archived at the image database at The Ohio State University (specimage.osu.edu).
The numbers prefixed with "USNMENT" or "OSUC " are unique identifiers for the individual specimens (note the blank space after some acronyms).Details on the data associated with these specimens may be accessed at the following link: purl.oclc.org/NET/hymenoptera/hol, by entering the identifier in the form (e.g."OSUC 123123") We apply the terminology from Austin and Field 1997.Morphological terms were matched to concepts in the Hymenoptera Anatomy Ontology (Yoder et al. 2010) using the text analyzer function.A table of morphological terms URI links is provided in Suppl.material 1.

Gastrotrypes (Figures 6-14)
The ovipositor system in Gastrotrypes is functionally identical to the Scelio-type in that it possesses elongate, telescoping conjunctiva and distal metasomal segments are protracted with the ovipositor.It differs in that elongation of the conjunctiva occurs between T5 and T6 , versus between T6 and T7 in scelionids, and in the longitudinally striate surface sculpture of the conjunctiva (Figures 10-11).T6 is immovably connected to and extruded with T7+8 during ovipositor extension (Figures 10-11).We interpret the apex of the tergal portion of the ovipositor system to be the fusion of T7 and T8 based on the presence of cerci (Figure 11).The conjunctiva between T5 and T6 in Gastrotrypes features acanthae where it attaches to the posterior margin of T6 and a second ring of acanthae anterior to its distal inflection site (Figures 11,14).Similar structures exist on the telescoping conjunctiva of at least two scelionine genera, Paridris Kieffer and Calliscelio Ashmead.The location of the acanthae in these scelionines is strikingly analogous with Gastrotrypes, as they are located anterior to the distal inflection site of the conjunctiva between T6 and T7.However, in Paridris and Calliscelio the free ends of the acanthae are directed anteriorly (Figures 23-24) and in Gastrotrypes they are directed posteriorly or outward (Figures 11,14).The form of the acanthae varies between the scelionine genera: the apices are rounded in Paridris coorgensis (Sharma) (Figure 23) and pointed in Calliscelio rubriclavus (Ashmead) (Figure 24).We did not find this ring of acanthae in Platygaster tubulosa, Probaryconus Kieffer (Figure 27) or Archaeoteleia (Figure 28).In the latter two cases the ring of acanthae may simply remain inside the portion of the conjunctiva that is not fully extended.We consider this character to have potential for informing relationships between genera of Scelionini sensu lato (Austin and Field 1997) but can draw no further conclusion without examination of more genera.
Similar acanthae types were found on the internal surface of the third valvula in other Hymenoptera (Vilhelmsen 2003, Shah et al. 2013), inside the egg canal (Le Relac
As in Gastrotrypes, the conjunctiva is striate and T6 is immovably attached to T7+8 (Figure 21).Unlike in Gastrotrypes, Paridris or Calliscelio, we found no indication of acanthae on any part of the conjunctiva.

Discussion
We treat the telescoping ovipositor systems in Gastrotrypes and Platygaster tubulosa as independent derivations based on multiple lines of evidence: the plesiomorphic condition for Platygastridae s.s. is a Ceratabaeous-type ovipositor (Murphy et al. 2007); Platygastrinae is phylogenetically distant from scelionids with a Scelio-type ovipositor (Murphy et al. 2007); and elongation of the conjunctiva occurred on different places on the metasoma: between T5 and T6 in Gastrotrypes and between multiple segments in P. tubulosa.These results suggest a degree of plasticity in the ovipositor systems of Platygastroidea that is consistent with the findings of Murphy et al. (2007), in which phylogenetic analysis retrieved multiple reversals from Scelio-to Ceratobaeus-type ovipositors in Scelionidae, and two derivations of the Scelio-type ovipositor.One derivation of the Scelio-type ovipositor occurred in the main scelionid clade (= Scelionidae sensu Masner (1976) excluding Archaeoteleia and Sparasionini), and the other in Archaeoteleia.
Identification of convergence will ultimately be determined by morphological analysis and the phylogenetic placement of Archaeoteleia relative to the main scelionid clade, a task challenged by what appears to be a significant age difference between these lineages.Archaeoteleia has existed for at least 99 Mya based on a specimen in Burmese amber (Talamas et al. 2017), whereas the oldest reliable fossil of a Scelio-type ovipositor from the main scelionid clade has an age of ~45 Mya (Talamas and Buffington 2015).Morphological evidence that elongation of conjunctiva between T6 and T7 occurred independently in Archaeoteleia and the main scelionid clade exists in two characters, albeit tenuously.Within Scelionidae, the presence of longitudinal striation on the conjunctiva is known only from Archaeoteleia (Austin and Field 1997), and the ring of acanthae at inflection points on the conjunctiva is known from two scelionid genera, but not Archaeoteleia.Examination of the latter character in specimens from a broad range of genera, and from specimens with fully extended ovipositor systems, is needed to determine the degree to which it is informative.

CNCI
Canadian National Collection of Insects, Ottawa, Canada OSUC C. A. Triplehorn Collection, The Ohio State University, Columbus, OH, USA