Female genitalia of Petaluridae provide evidence on the evolution of the reproductive biology in Odonata

RESEARCH Zoomorphology (2025) 144:37 https://doi.org/10.1007/s00435-025-00725-3 Konstantin Nadein [email protected] 1 Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Hlushkova Avenue, 2, Kyiv 03127, Ukraine 2 Senckenberg Natural History Collections Dresden, Museum für Tierkunde, Königsbrücker Landstraße 159, 01109 Dresden, Germany 3 Department of Functional Morphology and Biomechanics, Zoological Institute, Kiel University, Am Botanischen Garten 1–9, 24118 Kiel, Germany 4 Chemin du Salastre, Lagorce 07150, France Abstract The small Pangean dragonfly family Petaluridae poses intriguing questions both on its own evolution and on the evolution of Odonata as a whole. Molecular studies suggest that it began its generic diversification in the Mesozoic and eventually formed two clades, one Laurasian (Tachopteryx and Tanypteryx) and one Gondwanan (Phenes, Uropetala and Petalura). We describe the female genitalia of three species of Petaluridae by light microscopy and scanning electron microscopy and micro-CT for the first time. An evolution from a plesiomorphic “cutting” (endophytic) ovipositor to an apomorphic “pushing” (endosubstratic) ovipositor is indicated in the Laurasian members, whereas a “pushing” ovipositor is found in all Gondwanan members. The “cutting” ovipositor morphology most fully preserved in Tachopteryx is interpreted as a plesiomorphic morphological trait that has been retained after the functional shift in the egg-laying strategy took place. The possible functions of the studied structures are discussed and the present findings are compared with those of other Odonata. Received: 21 March 2025 / Revised: 6 May 2025 / Accepted: 9 May 2025 / Published online: 29 May 2025 © The Author(s) 2025 Female genitalia of Petaluridae provide evidence on the evolution of the reproductive biology in Odonata NataliaMatushkina1· Klaus-DieterKlass2· Stanislav N.Gorb3· KonstantinNadein3· GüntherFleck4 1 3 Zoomorphology (2025) 144:37 Introduction Odonata is an insect lineage of particular scientific interest because of their ancient fossil history, unusual behaviour, crucial ecological roles in freshwater communities, and the interesting morphology of both larvae and imagoes. Petaluridae, with 11 species in 5 genera (Nel et al. 1998), is one of the smallest families within Odonata, yet one posing intriguing questions both on its own evolution and on the evolution of Odonata as a whole, reproductive biology and larval biology being prominent aspects. The evolutionary history of the Petaluridae has thus been a subject of continuing debate for decades (Fleck 2011; Blanke et al. 2013; Kohli et al. 2021). Petaluridae share a unique, and probably derived, semiterrestrial larval lifestyle in which they inhabit boggy ecosystems. Three of its genera encompass species with groundwater-dependent bog-dwelling larvae, which either construct permanent, complex water-filled tunnels or labyrinths in boggy areas (Petalura and Uropetala) or reside in relatively simple burrows (Tanypteryx) (Baird 2019). While the terrestrial larvae of the remaining monotypic genera, Phenes and Tachopteryx, do not construct burrows, they probably also depend on the availability of suitably damp conditions (Baird 2019). Both the burrowing and non-burrowing larvae of Petaluridae are particularly longlived, with a development period of at least four or five years (see Baird 2019 and references therein). The specific lifestyle of the petalurid larvae is reflected in their morphology, which thus may provide numerous characters of putative phylogenetic value (Fleck 2011). Females of Petaluridae deposit their eggs by inserting the ovipositor and, on occasion, the distal abdominal segments, into a moist, organicrich soil substrate, fissures in the substrate, or amongst or under live or decomposing plant material overlying the substrate (for a review, see Baird 2012). Ovipositing females select lightly vegetated places amongst the low fen plants to access the substrate, typically associated with small water tracks or seepage lines. The internal phylogenetic and biogeographic relationships of the Petaluridae were the subject of ongoing debate (Nel et al. 1998; Fleck 2011; Ware et al. 2014). Character systems previously involved in intuitive morphology-based phylogenetic reasoning refer to larval morphology, the proventriculus, mouthparts, and anal appendages (Fleck 2011), and most commonly in the wing venation of both extant and fossil taxa (Nel et al. 1998). The more recent molecular studies suggest that monophyletic Petaluridae had a Pangaean origin in the Mesozoic (Suvorov et al. 2022; Tolman et al. 2024) and split into a Laurasian clade (with Phenes + Graphical Abstract Short description and graphical abstract. We describe for the first time the female genitalia of three Petaluridae species using light microscopy, scanning electron microscopy, and micro-CT. A comparative study reveals an ancestral cutting ovipositor transitioning to a pushing type in Laurasian members, whereas Gondwanan members exhibit a pushing ovipositor. Given that all Petaluridae share a similar endosubstratic oviposition, the most fully preserved plesiomorphic ovipositor morphology in Tachopteryx is interpreted as an ancestral morphological trait that was retained after the functional shift in oviposition strategy. Keywords Ovipositor · Spermatheca · Vagina · Evolution · Biogeography · SEM · micro-CT. 1 3 37 Page 2 of 21 Zoomorphology (2025) 144:37 (Uropetala + Petalura) and a Gondwanan clade (with Tanypteryx and Tachopteryx) between 140 and 180 mya in conjunction with the break-up of Pangaea (ca. 160 million years ago). All crown-group genera had separated apparently about 65 million years ago and the ages of individual species range from approximately 100 to 6 million years (Ware et al. 2014; Tolman et al. 2024), leading to petalurids being regarded as iconic “living fossils” in Ware et al. (2014). This evaluation leads to the intuitive suggestion that a specific egg-laying strategy and a unique larval lifestyle are ancient traits of Petaluridae that have persisted for millions of years, and thus raises the question about how these findings may affect our understanding of the evolution of the egg laying strategies in Odonata. Exploring this topic requires a wellfounded hypothesis on the phylogeny of Odonata. Family-level phylogenetic relationships within Odonata, including the position of Petaluridae relative to other Anisoptera, have long been disputed, both among morphological and early molecular phylogenetic studies (Fig. 1). However, phylogenetic hypotheses on Odonata have been largely stabilised by recent molecular-based studies (mainly Kohli et al. 2021; Bybee et al. 2021; Suvorov et al. 2022): The three principal odonatan lineages show the relationships Zygoptera + (Anisozygoptera + Anisoptera), with Epiophlebia being the only extant genus of Anisozygoptera. In the Anisoptera, the basalmost (crown-group) dichotomy separates Aeshnidae (or likely Aeshnidae + Austropetaliidae) from the remaining Anisoptera. For the latter, a trichotomy between Petaluridae, Gomphidae, and the remaining families (= clade Cavilabiata) is currently the consensus assumption, as results vary between Gomphidae + (Petaluridae + remaining families) (Fig. 1F) and (Gomphidae + Petaluridae) + remaining families (Fig. 1H). Thus, the Petaluridae, which have a complete ovipositor, are deeply nested within that clade of Anisoptera, which is characterised by a reduction of the ovipositor. The female genitalia of Petaluridae have rarely been discussed. The shape of the female postabdomen has briefly been described by St. Quentin (1962) in Uropetala carovei, Petalura gigantea, and Phenes, and by Fleck (2011) in Phenes, Tachopteryx, and Uropetala chiltoni. Pfau (1991) provided a semi-diagrammatic representation of the ovipositor musculature of Petalura. So far, Phenes raptor is the only petalurid species for which the female external Fig. 1 Phylogenetic position of Petaluridae and reduction of the endophytic ovipositor following different exemplary tree topologies of Odonata. (A) Bechly 1996 (morphology). (B) Rehn 2003 (morphology). (C) Bybee et al. 2008 (Bayes, morphology + molecules). (D) Fleck et al. 2008 (MR, molecules). (E) Bybee et al. 2008 (DO, morphology + molecules). (F) Carle et al. 2008 (molecules), Kohli et al. 2021 (molecules, fastest genes). (G) Fleck 2011 (morphology). (H) Bybee et al. 2021; Kohli et al. 2021 (molecules, slowest genes), Suvorov et al. 2022 (molecules). Austr. = Austropetaliidae. Check mark at tree base represents presence of a complete unmodified ovipositor in ground plan of Odonata; x upon a tree branch means an instance of strong reduction or modification of ovipositor in the most parsimonious positioning according to the phylogenetic hypothesis 1 3 Page 3 of 21 37 Zoomorphology (2025) 144:37 Tanypteryx pryeri was first dehydrated, dried at critical point, and visualised in microCT (see below), then dissected, macerated, and examined using light microscopy. Light microscopy (LM) The cuticle parts were examined in glycerine under an Olympus SZX12 stereo microscope equipped with camera Olympus U-TVO 5XC3 (CAU, Kiel, Germany) in transmitted and reflected light. The cuticle of internal genitalia of Petaluridae was additionally examined under a Zeiss Axioplan microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at CAU (Kiel, Germany) using combinations of different filters, polarized light, UV, phase contrast, and DIC tools to reveal more details of highly transparent membranous cuticle. Since the biomechanical features of the cuticle were beyond the main scope of this research, the choice of microscope tools was determined by the clarity and informativeness of the image obtained. Scanning electron microscopy (SEM) The cuticular parts of macerated female postabdomina were rinsed in distilled water, dehydrated in a graded ethanol series, and then processed in different ways dependent on the species studied: cuticular parts of Phenes raptor were air-dried, coated with gold-palladium, and examined with the scanning electron microscopes LEO 1530VP, Hitachi S-4800 (in 2011 at MPI-MF, Stuttgart, Germany), and Zeiss EVO-50 SEM (SNSD, Dresden, Germany); the dehydrated cuticle of the rest of Petaluridae species were critical point dried (Leica EM CPD300), coated with gold-palladium (Leica BalTec SCD 500), and examined with a table top TM3000 SEM (CAU, Kiel, Germany); the ovipositors of Epallage fatime, Epiophlebia superstes, and Aeshna cyanea were rinsed in acetone, critical point dried (OM CPD 7501), coated with gold-palladium (OM-SC7640) and examined with a Zeiss EVO 50 SEM scanning electron microscope (SNSD, Dresden, Germany). X-ray micro-computed tomography (micro-CT) and three-dimensional (3D) reconstruction The dried female postabdomen of Tanypteryx pryeri was examined with a SkyScan 1172 (Bruker Corp., Billerica, USA.) (CAU, Kiel, Germany) at 40 kV and 250 µA, with a camera pixel size of 8.95 μm, image pixel size 4.62 μm; 2454 projections were recorded over the 360° rotation. For 3D reconstruction, the open source software Drishti was used (Hu et al. 2020a, b). As resulting images revealed underdeveloped musculature, the specimen was considered to be either a teneral imago (more likely) or an adult imago genitalia have been intensively studied with light and scanning electron microscopy (Matushkina and Klass 2011). The female internal genitalia have apparently never been studied in Petaluridae, including their cuticle-bearing ectodermal parts. The present study was conducted to describe the exoskeleton of the external and cuticle-bearing internal female genitalia in Petaluridae through light microscopy, scanning electron microscopy, and micro-CT. The potential functions of the studied structures are discussed, and the findings are compared with other Odonata. The evolution of the egg-laying strategy in Petaluridae and Odonata in general is discussed, and the arguments in support of a specific biogeographic origin of the Petaluridae are critically analysed based on original and published data. Materials and methods Materials studied and initial sample preparation The postabdomen was examined in six females of Petaluridae: two dried adult females of Phenes raptor Rambur, 1842 (specimens described in Matushkina and Klass 2011), one adult ethanol-preserved female each of Ph. raptor (November 1998, Chili, Vilches, VII Region, GF leg.), Tachopteryx thoreyi (Hagen 1858) (Oakmulgee Rd, Talladega NF, Bibb County, Alabama, USA; 5/5/2019; Tennessen), and Tanypteryx hageni (Selys 1879) (Toddlake, Deschutes County, Oregon, USA; 8/7/2019; S. Valley), and one presumably teneral ethanol-preserved female of Tanypteryx pryeri (Selys 1889) (30.VI.1998, [Kyuhsu], Inakadai, Tsuiku-machi, Fukuoka Pref., Y. Shinkai leg.). To compare ovipositor morphology of Petaluridae with other Odonata, we examined ethanol-preserved adult females from five taxa apart from Petaluridae: Epallage fatime (Charpentier 1840) (Zygoptera: Euphaeidae), Epiophlebia superstes (Selys 1889) (Anisozygoptera: Epiophlebiidae) (Japan), Aeshna cyanea (Latrelle 1805) (Anisoptera: Aeshnidae), Cordulegaster insignis Schneider, 1845 (Anisoptera: Cordulegastridae), and Gomphus vulgatissimus (Linnaeus 1758) (Anisoptera: Gomphidae). General features of the external morphology of the ovipositor of Uropetala were reconstructed from photographs retrieved from GBIF.org (Fig. S4). Prior to maceration, dried postabdomina of Ph. raptor were soaked in Bouin’s fluid for several months to soften the cuticle (Matushkina and Klass 2011). Postabdomina of Ph. raptor and ethanol-preserved postabdomina of other insects except Tanypteryx pryeri were then washed in distilled water, dissected in a paramedian plane and macerated in 10% KOH solution in water. The postabdomen of 1 3 37 Page 4 of 21 Zoomorphology (2025) 144:37 corresponds to that of Phenes (Matushkina and Klass 2011): the gonapophyses 8 and 9 (= ventral and dorsal valves, respectively) form together the ovipositor shaft, partly covered ventrolaterally by the gonoplacs 9 (= third, or sheathing valves; = coxal lobes 9) with styli 9 (= styli) (Figs. 2, 3 and 4). In all studied species of Petaluridae, the external surface of gonapophysis 8 lacks projecting cutting sculpture, such as ridges or teeth, but bears numerous sensilla scattered over the entire surface (more densely apically) (Fig. 3). The ventral basal (= anterior) margin of gonapophyseal sclerite 8 is slightly concave (as in Matushkina and Klass 2011; Fig. 2a). The basal part of gonapophysis 8 is expanded. The mesal surface of the expanded part is dorsally folded and smooth, i.e. there are no oblique ridges. Laterally to the mesal fold, the internal surface of gonapophysis 8 has a notch of spinose cuticle. The rest of the internal surface of gonapophysis 8 is smooth. The olistheter (sliding interlock between gonapophyses 8 and 9) reaches from the base of gonapophysis 8 up to about 3/4 of the whole ovipositor length, whereby an apical interlock between gonapophyses 8 and 9 is absent (Fig. 5). As gonapophyses of the left and right sides are entirely separated from each other (i.e. neither fused nor interlocked), they can likely be pushed aside during oviposition. Microstructures on the internal surface of the gonapophyses consist of bud-like clusters of elongated, apparently moveable microtrichia approximately 10 to 20 μm in length, which are surrounded by rigid solitary leaf-like microtrichia and spinules (Fig. 5C-K). There are minor differences between species in the shape and number of the microtrichia in a cluster. Solitary microtrichia and spinules are present on both pairs of gonapophyses. Bud-like clusters of microtrichia are found on gonapophyses 9 in all species and very few clusters were additionally found on basal dorsal regions of gonapophyses 8 in Tanypteryx pryeri and Phenes. This egg channel microsculpture is well developed on the ventral half of the internal surface of the gonapophysis 9. There is thus altogether a pair of continuous ribbons of semi-erect, posteriorly directed microstructures on the internal walls of the gonapophyses. This ribbon starts on each side basally behind the vaginal opening to run upon the internal walls of gonapophyses 8 at the base of the ovipositor, then bends upward to the walls of gonapophyses 9 to end upon these subapically (Fig. 5, dashed line). The shape and length of the ovipositor, the sculpture of the external surface of gonapophysis 9, and the sensory array of both pairs of gonapophyses differ in the species studied as described below. Tachopteryx thoreyi has a strongly curved ovipositor, which hardly reaches the middle region of segment 10, this being the shortest among all the species studied; distally all that had been partially macerated during conservation procedures (less likely). Image processing Resulting photographs were corrected using HDR toning in Adobe Photoshop 26.0.0 to visualize the cuticle of different sclerotisation levels on one image and using brightness/ contrast and levels tool to contrast the images. In the figures, unless otherwise stated, the orientation of the structures is with the posterior end on the left and the dorsal side on the top. Terminology Morphological terms and abbreviations follow the insect-wide approach from Klass (2008), Matushkina and Klass (2011), and Matushkina and Stetsun (2025) (see also Klass and Matushkina 2012). The terminology traditionally used in odonatology (e.g. Pfau 1991; Fleck 2011) is given as synonyms. The classification of sensilla to a particular morphotype is based on the external morphology of the cuticular components and does not imply the same function (see Rebora et al. 2013a, b). The following abbreviations are used both in the illustrations and in the text (“segment” = abdominal segment): cr, collar-shaped ridge or fold of vagina; CX8, (gono)coxal sclerite of segment 8; CX9, (gono)coxal sclerite of segment 9 (including sclerotisation of gl9); gl9, gonoplac (= coxal lobe) of segment 9 (third, or lateral valve of ovipositor); gp8, gonapophysis of segment 8 (first, or ventral valve of ovipositor); GP8, sclerotisation associated with gonapophysis gp8; gp9, gonapophysis of segment 9 (second, or dorsal valve of ovipositor); GP9, sclerotisation associated with gonapophysis gp9; LC9, laterocoxal sclerite of segment 9 (gonangulum); oc, common oviduct (cuticlebearing part); rh, rhachis, ridge on gonapophysis of segment 9 (dorsal part of tongue-and-groove interlocking mechanism on gonapophyses of segments 8 and 9 called the olistheter); S8, S9, S10, segment 8, 9, and 10, respectively; sl9, stylus of segment 9; vt, tendon ventromedially on vagina. The inner side of the cuticle facing the epidermis is called its epidermal side; the outer side facing the outer world is called the non-epidermal side. Regarding the non-epidermal surfaces of the gonapophyses, those facing the egg channel enclosed by the gonapophyses are specified as the internal surfaces, while those facing the surroundings are specified as the external surfaces. Results Ovipositor The overall morphology of the ovipositors of Tachopteryx thoreyi, Tanypteryx hageni, and Tanypteryx pryeri 1 3 Page 5 of 21 37 Zoomorphology (2025) 144:37 Fig. 2 Brightfield images of the macerated female postabdomen (A, C, E) and close-up of the gonapophyses (B, D, F) of Petaluridae. (A, B) Tachopteryx thoreyi. (C, D) Tanypteryx hageni. (E, F) Tanypteryx pryeri. As a result of the maceration, the cuticlular regions are deformed to some extent and the gonapophyses are separated. Abbreviations: gl9, gonoplac 9 = coxal lobe 9; gp8, gonapophysis 8; gp9, gonapophysis 9; sl9, stylus 9; S8-S10, 8th, 9th and 10th abdominal segments, respectively 1 3 37 Page 6 of 21 Zoomorphology (2025) 144:37 basal half, where it bears setae of variable length (longer basally) and campaniform sensilla (Fig. 4C). The apical half of gonapophysis 9 bears a distinct, regular sculpture (Fig. 4C): In the dorsolateral region there are ten transverse dorsolateral ridges, their steep slopes facing the gonapophyseal base, and the two distal ridges being quite indistinct. In the dorsolateral region there are seven short longitudinal ridges placed in one line, but with the basal end of each ridge bending ventrally (Fig. 4C, asterisks) towards near the lateral gonapophyseal edge; the ventrally bending parts are gonapophyses 8 and 9 narrow strongly, quite gradually, and evenly and have a pointed apex (Figs. 2A, B and 4C). — Gonapophysis 8 is strongly and quite abruptly expanded in its basal third, where its external side bears numerous setae of different length (Fig. 3C-E). Elongated obliquely directed campaniform sensilla and coeloconic sensilla occur sparsely all over the external wall of gonapophysis 8, but campaniform sensilla occur in greater density in the expanded basal part (between the setae), along the ventral edge, and near the apex. — Gonapophysis 9 is slightly expanded in its Fig. 3 SEM images of external surfaces of the gonapophyses 8 (A–E) and gonoplacs 9 (third valves) with styli 9 (F–H) of Petaluridae. (A, B, F) Tanypteryx pryeri: isolated gonapophysis 8 (A) and its distal half (B), gonoplac 9 with laterally deflected stylus 9 (G). (C–E, H) Tachopteryx thoreyi: isolated gonapophysis 8 (С), its basis (D) and middle part (E), gonoplac 9 (H). (G) Tanypteryx hageni: gonoplac 9. Abbreviations: LC9, laterocoxa 9 (gonangulum); gp8, gonapophysis 8; CX8, (gono)coxa 8; gl9, gonoplac 9 = coxal lobe 9; sl9, stylus 9 1 3 Page 7 of 21 37 Zoomorphology (2025) 144:37 apical part of gonapophysis 9 (about the area of the three distal dorsolateral ridges). — Gonoplac 9 and styli 9 are densely and uniformly covered with long setae (Figs. 3H and 6A-D). Gonoplac 9 has a strongly convex ventral edge, the ventral marginal part is mesolaterally compressed, and further dorsally the lateral gonoplac wall is abruptly bulged (Fig. 3H). Stylus 9 is short and club-shaped (i.e. proximal part slightly narrower). Dispersed campaniform sensilla are present on the gonoplac wall near the lateral base of stylus 9 mostly in alignment with the transverse dorsal ridges. The series of lateral ridges has the effect that the gonapophyseal wall dorsal to them forms a shallow longitudinal depression. The unsculptured depressed longitudinal area between dorsolateral and lateral ridges bears several obliquely directed campaniform sensilla and less frequent coeloconic sensilla, whereas the areas of the dorsolateral and lateral ridges bear mainly coeloconic sensilla and few campaniform sensilla. The coeloconic sensilla are extremely dense in the most Fig. 4 SEM images of the lateral surface of the ovipositor in some Anisoptera. (A) Aeshna cyanea (Aeshnidae). (B) Phenes raptor (Petaluridae). (C) Lateral view on the isolated gonapophysis 9 (second valve) of Tachopteryx thoreyi (Petaluridae) with close-up of the apical region in insets. (D) External view on the isolated gonapophysis 9 (second valve) of Tanypteryx hageni (Petaluridae) with close-up of the basal and apical regions in insets. (E) Lateral view on the isolated gonapophysis 9 (second valve) of Tanypteryx pryeri (Petaluridae) with close-up of the apex and subapical lateral region in insets. Asterisks mark short lateral ridges of gonapophysis 9. Abbreviations: gp8, gonapophysis 8; gp9, gonapophysis 9 1 3 37 Page 8 of 21 Zoomorphology (2025) 144:37 Fig. 5 SEM images of mesal surfaces of the ovipositor, showing egg channel microsculpture putatively used for egg transport (dashed arrow). (A) Epallage fatime (Zygoptera: Euphaeidae). (B) Epiophlebia superstes (Anisozygoptera: Epiophlebiidae). (C–E) Phenes raptor (Anisoptera: Petaluridae): apical region of the ovipositor in C and microsculpture of gonapophyses 9 with bud-like clusters of microtrichia at different magnifications in D and E. (F–H) Tachopteryx thoreyi (Anisoptera: Petaluridae): apical region of the ovipositor collaged from isolated gonapophyses 8 and 9 in F and macrosculpture on gonapophyses 9 with bud-like clusters of microtrichia at different magnifications in G and H. (I–K) Tanypteryx hageni (Anisoptera: Petaluridae): apical region of the ovipositor collaged from isolated gonapophyses 8 and 9 in I and microsculpture on gonapophyses 9 with bud-like clusters of microtrichia at different magnifications in J and K. Empty arrowheads in A and B show the apical lock on gonapophyses 8 (absent in Petaluridae). Abbreviations: gp8, gonapophysis 8; gp9, gonapophysis 9 1 3 Page 9 of 21 37 Zoomorphology (2025) 144:37 the presence in Phenes of stiff setae as anchoring modifications in both contact regions of the postabdomen (like in e.g. Epiophlebia, see above). In the case of Tachopteryx thoreyi, for which both endosubstratic oviposition and the structural features of an ‘endophytic ovipositor’ are now clearly documented, the only explanation we can offer is that the ancestral ovipositor oblique ridges on the distal part of gonapophysis 9 (reduced F2). Tanypteryx pryeri shows no features of the endophytic ovipositor. Like in the latter species, in extant members of the Gondwanan clade, the ‘cutting’ adaptations (F1)-(F5) are absent throughout, i.e. in Phenes raptor (Matushkina and Klass 2011), and probably in Uropetala (Fig S4) and Petalura (Pfau 1991: Fig. 22). The only exception to this is Fig. 8 Phylogenetic tree of extant Petaluridae (Tolman et al. 2024) showing the presence of the plesiomorphic cutting design of the ovipositor in Laurasian clade and the living of larvae in burrows independently originated in two clades (A), and distribution of extant Petaluridae and fossil records of their close relatives (B). Question marks indicate that character states were reconstructed from public data (see Material and methods and Fig. S4). Symbols used for fossil records: grey triangles, Aktassiidae; black squares, Cretapetaluridae; white squares, Protolindeniidae; grey circle, fossil Petaluridae (details of fossil records are given in Table S1) 1 3 37 Page 16 of 21 Zoomorphology (2025) 144:37 and Stetsun 2025). If this prediction, which has not yet been experimentally confirmed, is correct, then the distribution of microtrichia could be used to determine at which point exactly the egg leaves the ovipositor at its apex. In Zygoptera and Aeshnidae (Anisoptera), this point lies mainly between gonapophyses 8 (Gorb 1996; Matushkina and Lambret 2011; NM unpubl. observ.), and in Epiophlebia (Anisozygoptera) and Petaluridae (Anisoptera) between gonapophyses 9 (Matushkina 2008 – for Epiophlebia, Matushkina and Klass 2011 and this study – for Petaluridae). Bud-like clusters of microtrichia of the egg channel have yet not been found in other Odonata lineages. What should be re-examined in the light of current findings on female genital morphology in Petaluridae? Life history of larvae of Petaluridae The evolution of the burrowing behaviour of Petaluridae larvae is an intriguing topic for further investigation. All larvae of extant Petaluridae are classified as semi-terrestrial, and, in particular, are not able either to propel themselves by expelling water from the rectal chamber or to swim with leg movements (Winstanley 1982; Fleck 2011). They can be divided into three morphotypes (Fleck 2011): (1) Flattened larvae with well-developed lateral spines mimicking dead leaves (Tachopteryx); (2) Elongated subcylindrical larvae with lateral protuberances and body excrescences mimicking rotting sticks (Phenes); (3) Cylindrical larvae lacking lateral abdominal and body protuberances and living in relatively simple but variable burrows (Tanypteryx) or complex, water-filled tunnels (Petalura and Uropetala). Tachopteryx with non-fossorial larvae is considered the sister group of Tanypteryx with fossorial larvae and Phenes with non-fossorial larvae is considered most likely the sister group of Petalura and Uropetala with larvae constructing complex tunnels (Tolman et al. 2024). Other Odonata have non-fossorial larval lifestyle. Thus, the burrowing behaviour seems, by simple parsimony, to have evolved independently in the Laurasian and Gondwanan clades of Petaluridae (Fig. 8A) (2 steps of acquisition) rather than having evolved in the stem of Petaluridae and then lost in Tachopteryx and Phenes (3 steps: 1 acquisition and 2 losses) (see Fleck 2011 for an alternative hypothesis of the evolution of burrowing larvae in Petaluridae). However, Tachopteryx larvae were found to inhabit small cavities (the author calls them ‘crypts’), pits, or shallow depressions under or among fallen wet litter and sphagnum in wet hillside bogs or fens with at most minimal surface flow; they are able to modify their environment by constructing these shallow pits (Baird 2019). If taken as a plesiomorphic behaviour, this morphology was preserved after functional changes in oviposition strategy had occurred. It is also reasonable to surmise that the plesiomorphic ovipositor morphology preserved in Tachopteryx indicates that the relationship between vegetation and dragonflies was closer and more specialised in the past than it is now, and that dragonfly habitats may have been associated with locations where certain plant species suitable for egg-laying were available. The case of Tachopteryx thoreyi also demonstrates that the presence of pointed gonapophyses and of distal teeth/ridges on gonapophysis 9 cannot be taken as clear evidence for endophytic oviposition. This is of particular relevance with regard to fossils, where oviposition can no longer be observed and functional interpretations can only be concluded from morphology. Unfortunately, the fossil record of Petaluridae and related extinct families (together the Petalurida, see below) does not bear any evidence on the morphology of the ovipositor. It is noteworthy in this context that even petalurids lacking ‘cutting’ adaptations have some limited ability to penetrate into some coherent soft substrate: “The Tanypteryx [pryeri] ovipositor is surely functionable to insert eggs into wet paper but very superficially… just under the thin uppermost paper of the wet paper pile, one by one apart from each other, at least 12 mm in distance” (Miyakawa 1990: P. 459). Kozo Miyakawa noted then that “This oviposition pattern differs from that of the true endophytic Aeshna [juncea] females who lay eggs more deeply and closely” (Miyakawa 1990: P. 459–460) and concluded that “Petaluridae show a transitional state between Aeshnidae and Gomphidae having a disintegrating endophytic ovipositor and exophytic ellipsoid egg” (Miyakawa 1990: P. 460). John W.H. Trueman also observed Petalura gigantea inserting eggs into wet paper tissue under laboratory conditions (Baird 2012: P. 30). Female Odonata practising endophytic oviposition can prefer specific plant species, plant anatomy characteristics, and tissue conditions, like live vs. dry conditions, for egg deposition (Martens 2001; Matushkina and Gorb 2002a, 2007; Lambret et al. 2015; Matushkina et al. 2016). Gustatory and mechanosensory sensilla of the ovipositor may evaluate the properties of the oviposition substrate (Rebora et al. 2013a, b). Bilateral fields of campaniform sensillae on the base of stylus 9 control a specific pattern of egg deposition in the substrate (Matushkina 2007; Matushkina and Gorb 2000, 2002b; Matushkina and Lambret 2011; Matushkina et al. 2015). It is unclear to what extent these features of the sensillary equipment also apply to ovipositors used for endosubstratic oviposition. The inner surface of the ovipositor in Odonata is covered with posteriorly directed microtrichia, which was predicted to ensure unidirectional egg transport as gonapophyses move (Austin and Browning 1981; Gorb 1996; Matushkina 1 3 Page 17 of 21 37 Zoomorphology (2025) 144:37 archangelskyi, has been found from the Atlantic side of Southern Patagonia (Petrulevičius and Nel 2003), which belonged to Gondwana. It has been recovered as the sister group to Phenes and is thus subordinate in the Gondwanan clade of Petaluridae (Tolman et al. 2024) and bears no evidence on the geographic origin of Petaluridae. Petaluridae is the only surviving clade of the ancient group Petalurida, which also included fossil families Aktassiidae (considered the sister group of Petaluridae), Cretapetaluridae (sister to Aktassiidae + Petaluridae), and Protolindeniidae (sister to Cretapetaluridae + (Aktassiidae + Petaluridae) (Nel et al. 1998; Fleck 2011). Systematic analysis of the fossil record of Petalurida revealed a considerable prevalence in areas of Laurasian origin (Table S1). Indeed, ten of eleven currently undoubtedly recognised species of Aktassiidae were reported from Eurasia (Spain, Germany, UK, Kazakhstan, Mongolia, and China), i.e. from Laurasia region. Conclusion and limitations of the study Although this work was carried out on only three of the five genera of the Petaluridae, it represents the most comprehensive morphological study of the female genitalia of the Petaluridae to date. We find ovipositors of different morphotypes in three representatives of the Laurasian clade of Petaluridae, which use the same egg-laying methods, so that the observed morphological differences cannot yet be explained by different function. Thus, the plesiomorphic ovipositor morphology most fully preserved in Tachopteryx is interpreted as an ancestral trait that persisted after the functional shift in oviposition strategy. Unfortunately, the fossil records of the stem group Petaluridae provide no data on the ancient morphology of the female genitalia. The distribution of fossil records in Petalurida, primarily in the Laurasian region, combined with the presence of some signs of the plesiomorphic “cutting” ovipositor and a plesiomorphic non-burrowing lifestyle of the leaf-mimicking larva in the member of the Laurasian clade, Tachopteryx, has been interpreted with caution as indirect evidence for the biogeographic origin of Petaluridae in the northern part of Pangea. This conclusion should be considered as partly speculative, as it contradicts the current molecular hypothesis (Ware et al. 2014). Nevertheless, we think it is necessary to state it here, because this option should be taken into account in future research of the family Petaluridae, especially those addressing fossil and molecular data. Coupled morphological studies of male and female genitalia, and probably of the egg, will provide further more accurate clarification of how the reproduction occurs and probably explain why the ancestral design of the ovipositor was retained in some representatives of Petaluridae and lost in others. optional ability could explain the parallel evolution of the derived burrowing larval stage in Petaluridae. The capacity of the common petalurid ancestor to live freely in wet habitats and excavate shallow depressions could have led, under variable selective pressures, to the capacity to dig deeper excavations, simple and short tunnels as observed in Tanypteryx or longer and more complex tunnels (with lateral extensions) as observed in Uropetala and Petalura. Conversely, the behavioural plasticity of the ancestor could lead to the existence of non-excavating forms as Phenes. Indeed, Phenes larvae appear highly ecologically versatile, occupying microhabitats ranging from shallow, lotic, and lentic aquatic to fully terrestrial (Baird 2019). In this context, the retention of the plesiomorphic design of the ovipositor of Tachopteryx, possibly suitable for oviposition in plant substrate, is co-occurring with the possibly plesiomorphic non-burrowing behaviour and leaf-mimicking shape of the larvae, suggesting a closer association of this dragonfly lineage with the vegetation that existed in the past but is not being observed today. History of Petaluridae Recent molecular studies suggest that the Petaluridae originated somewhere in Pangaea, and its two principal clades, Gondwanan (south) and Laurasian (north), diverged approximately 160 mya (Ware et al. 2014; Tolman et al. 2024). The disintegration of the supercontinent Pangaea, as well as the isolation of populations and extinction of species have been proposed to be the major driving forces behind the speciation events in Petaluridae (Ware et al. 2014; Tolman et al. 2024), resulting in a very specific and disjunctive distribution pattern of the recent species of the family (see Fig. 8B). Despite the detailed and intensive research on the molecular phylogeny of Petaluridae conducted most recently by J. Ware and colleagues, the biogeographic origin of Petaluridae is not yet definitely established. «… Findings [resulting from molecular data] suggest that Petaluridae radiated across the southern and western coasts of Pangaea, expanding further along the eastern and western coasts of Laurasia after the split from Gondwana, and expanding further into Japan» (Ware et al. 2014: P. 7). The later analysis revealed support for “… the hypothesis that the Laurasian taxa migrated to Eastern North America and Japan, while Phenes, Uropetala, and Petalura diverged with the separation of Gondwana, with all Gondwanan remnants as highly plausible ancestral states in this clade” (Tolman et al. 2024: P. 5). However, fossil history is not quite in line with an origin of Petaluridae in the south of Pangaea, but it is in agreement with the hypothesis of Nel et al. (1998) of an origin of the Petaluridae in the north Pacific area. The oldest known representative of Petaluridae, Argentinopetala 1 3 37 Page 18 of 21 Zoomorphology (2025) 144:37 petalurid. 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Schmalhausen Institute of Zoology of NASU, Ukraine) for the contributing materials, Anika Preuss and Helen Gorges (Department of Functional Morphology and Biomechanics, Zoological Institute, Kiel University, Germany) for help in conducting X-ray micro-computed tomography, Ian Baird (Australia) for valuable discussion on the egg-laying habits of Petaluridae, and Albert Orr (Australia) for his editing the manuscript, providing English corrections and helpful suggestions for early version of the manuscript. Author contributions N.M.: Conceptualization; data curation; methodology; investigation; writing – original draft; writing – review and editing; visualization. K.D.K.: Writing – original draft; writing – review & editing; resources; funding acquisition. S.G.: Writing – review & editing; resources; funding acquisition. K.N.: Writing – review & editing; methodology; visualization. G.F.: Conceptualization; writing – original draft; review & editing. Funding Open Access funding enabled and organized by Projekt DEAL. Data availability All data are shown in this study; raw data can be made available upon reasonable request to the authors. Declarations Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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