Save

Male terminalia of Ceraphronoidea: morphological diversity in an otherwise monotonous taxon

In: Insect Systematics & Evolution
View More View Less
  • 1 Department of Entomology, Pennsylvania State University, 501 ASI Building, University Park, PA 16802, USA
  • | 2 Agriculture and Agri-Food Canada, Ottawa, ON, Canada K1A 0C6
  • | 3 NCSU-NSCORT, Department of Plant Biology, 2115 Gardner Hall Box 7612, North Carolina State University Raleigh, NC 27695, USA
  • | 4 Illinois Natural History Survey, 1816 South Oak Street, MC 652, Champaign, IL 61820, USA
Open Access

The skeletomuscular system of male terminalia in Evaniomorpha (Hymenoptera) is described and the functional morphology of male genitalia is discussed. Confocal laser scanning microscopy is the primary method used for illustrating anatomical phenotypes, and a domain-specific anatomy ontology is employed to more explicitly describe anatomical structures. A comprehensive data set of ceraphronoid male genitalia is analyzed, yielding the first phylogeny of the superfamily. One hundred and one taxa, including three outgroups, are scored for 48 characters. Ceraphronoidea are recovered as sister to the remaining Evaniomorpha in the implied weighting analyses. Numerous character states suggest that Ceraphronoidea is a relatively basal apocritan lineage. Ceraphronoidea, Ceraphronidae, and Megaspilinae are each retrieved as monophyletic in all analyses. Megaspilidae is not recovered as monophyletic. Lagynodinae is monophyletic in the implied weighting analyses with strong support and is a polytomy in the equal weighting analysis. Lagynodinae shares numerous plesiomorphies with both Megaspilinae and Ceraphronidae. Relationships among genera are weakly corroborated. Masner is sister of Ceraphronidae. Trassedia is nested within Ceraphronidae based on the present analysis. Because of this and numerous features shared between it and Ceraphron we transfer Trassedia from Megaspilidae to Ceraphronidae. Dendrocerus forms a single monophyletic clade, with modest support, together with some Conostigmus species. This result challenges the utility of such traditional diagnostic characters as ocellar arrangement and shape of the male flagellomeres. Aphanogmus is monophyletic in the implied weighting, but remains a polytomy with Ceraphron in the equal weighting analysis. Gnathoceraphron is always nested within a well-supported Aphanogmus clade. Cyoceraphron and Elysoceraphron are nested within Ceraphron and Aphanogmus, respectively. The male genitalia prove to be a substantial source of phylogenetically relevant information. Our results indicate that a reclassification of Ceraphronoidea both at the family and generic level is necessary but that more data are required.

Abstract

The skeletomuscular system of male terminalia in Evaniomorpha (Hymenoptera) is described and the functional morphology of male genitalia is discussed. Confocal laser scanning microscopy is the primary method used for illustrating anatomical phenotypes, and a domain-specific anatomy ontology is employed to more explicitly describe anatomical structures. A comprehensive data set of ceraphronoid male genitalia is analyzed, yielding the first phylogeny of the superfamily. One hundred and one taxa, including three outgroups, are scored for 48 characters. Ceraphronoidea are recovered as sister to the remaining Evaniomorpha in the implied weighting analyses. Numerous character states suggest that Ceraphronoidea is a relatively basal apocritan lineage. Ceraphronoidea, Ceraphronidae, and Megaspilinae are each retrieved as monophyletic in all analyses. Megaspilidae is not recovered as monophyletic. Lagynodinae is monophyletic in the implied weighting analyses with strong support and is a polytomy in the equal weighting analysis. Lagynodinae shares numerous plesiomorphies with both Megaspilinae and Ceraphronidae. Relationships among genera are weakly corroborated. Masner is sister of Ceraphronidae. Trassedia is nested within Ceraphronidae based on the present analysis. Because of this and numerous features shared between it and Ceraphron we transfer Trassedia from Megaspilidae to Ceraphronidae. Dendrocerus forms a single monophyletic clade, with modest support, together with some Conostigmus species. This result challenges the utility of such traditional diagnostic characters as ocellar arrangement and shape of the male flagellomeres. Aphanogmus is monophyletic in the implied weighting, but remains a polytomy with Ceraphron in the equal weighting analysis. Gnathoceraphron is always nested within a well-supported Aphanogmus clade. Cyoceraphron and Elysoceraphron are nested within Ceraphron and Aphanogmus, respectively. The male genitalia prove to be a substantial source of phylogenetically relevant information. Our results indicate that a reclassification of Ceraphronoidea both at the family and generic level is necessary but that more data are required.

Introduction

Hymenoptera, which includes sawflies, wasps, bees, and ants, is one of the four most species-rich insect orders, with more than 145 000 known species (Huber 2009) and perhaps more than 1 million species remaining to be described (Sharkey 2007). The evolutionary history of Hymenoptera has yet to be resolved, though recent efforts have edged closer towards a robust estimate (Vilhelmsen et al. 2010; Heraty et al. 2011). The emerging evolutionary topology reveals a highly supported basal grade of herbivorous hymenopterans (sawflies and woodwasps, known as “Symphyta”), leading to an extraordinarily diverse and notorious rapid radiation of Apocrita (Whitfield & Kjer 2008). Most apocritan superfamilies are robustly monophyletic, but the relationships between them are weakly resolved. Perhaps the most uncertain is the position of the small hyperparasitoid lineage, Ceraphronoidea (Sharanowski et al. 2010, Sharkey et al. 2012), a putatively basal apocritan (Vilhelmsen et al. 2010).

Ceraphronoidea was recently cataloged by Johnson & Musetti (2004) and currently includes four families: Ceraphronidae, Megaspilidae, and the fossil groups Stigmaphronidae and Radiophronidae. These four families include 27 valid genera (plus 28 generic concepts now considered junior synonyms) and 613 valid species (plus 235 species-level concepts now considered junior synonyms). Ceraphronoidea is one of the smallest of the major apocritan clades, yet they are the fourth most commonly collected hymenopterans (Martínez de Murgía et al. 2001; Schmitt 2004). Most ceraphronoids are parasitoids of entomophagous insects that develop in weakly concealed environments, inside cocoons or puparia or hosts that are prepupae (Haviland 1920; Withycombe 1924; Kamal 1939), a lifestyle that is probably the ground plan biology for the clade. Many of their hosts are primary parasitoids or predators of economically important insects (e.g., predators of the coffee berry borer (Evans et al. 2005), spider mite predators (Diptera: Cecidomyiidae) (Oatman 1985), and parasitoids of lepidopteran pests on oil palm (Polaszek & Dessart 1996). Despite their abundance and economic importance, only one systematist, Paul Dessart (active 1962–2001, deceased 2001), has worked on the group in modern times, and his core revisionary hypotheses have never been tested phylogenetically.

It is widely accepted that Apocrita is monophyletic, with Orussoidea as its sister lineage (Rasnitsyn 1988, 2002; Ronquist et al. 1999; Vilhelmsen 2003; Schulmeister 2003a; Sharkey 2007; Heraty et al. 2011). However, our knowledge of the phylogeny of the suborder remains incomplete, and the relationships between putatively basal apocritans (Ceraphronoidea, Evanioidea, Trigonalidae and Megalyroidea, together referred to as Evaniomorpha) remain elusive (Heraty et al. 2011). The importance of Evaniomorpha for resolving the phylogeny of Hymenoptera is broadly recognized, and exemplars are usually involved in analyses attempting to resolve higher-level phylogenies (Shcherbakov 1981; Gibson 1985, 1999; Heraty et al. 1994; Vilhelmsen 1996; 2000a,b, 2003; Schulmeister 2003b). Unfortunately, Ceraphronoidea has been excluded from most of these analyses and in some morphology-based analyses that did include ceraphronoid exemplars, character states were misinterpreted, probably due to their minute size. Numerous observations, however, indicate, that unlike other apocritans some Ceraphronoidea share putatively plesiomorphic character states with “Symphyta”, including: (1) presence of a propleural arm-postoccipital muscle, shared with some Tenthredinoidea (Vilhelmsen et al. 2010), (2) presence of a metanoto-metacoxal muscle, shared with most non-apocritan Hymenoptera (Vilhelmsen et al. 2010), (3) presence of a posterior apical spur on the protibia, shared with most non-apocritan Hymenoptera (Rasnitsyn 1988), (4) presence of a median mesoscutal sulcus that corresponds to an internal ridge, shared with most non-apocritan Hymenoptera and with basal Apocritan lineages (Megalyroidea, Stephanoidea) (Gibson 1985), (5) presence of a mesonotal and mesofurcal depressor of the mesotrochanter with this muscle inserting distinctly ventrally of the site of insertion of the mesonotal depressor on the mesotrochanter (pers. obs.). This latter state of the mesotrochanteral extracoxal depressor complex was considered by Gibson (1999) to represent the hypothetical ancestor of Apocrita, though he did not observe the ceraphronoid furcal muscle in his studies.

The collective phenome of ceraphronoid wasps is not only relatively monotonous (i.e., lacking distinct apomorphies) compared to other microhymenoptera (e.g., Chalcidoidea, Platygastroidea), but the few possibly suitable morphological variables (e.g., body shape, sculpture) are often affected by allometry (Fig. 1). Male genitalia, however, provide a source of discrete and size-independent characters. The utility of male genitalia in species delimitation was recognized relatively early and from the mid 20th century became the key element in species diagnoses (majority of Paul Dessart’s publications since 1963; Teodorescu 1967; Takada 1973).

Fig. 1.
Fig. 1.

Bright field images of undescribed species of Ceraphron showing the body size variability and related allometric changes. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1876312x.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

Male genitalia characters are certainly informative for higher level classification of basal Hymenoptera (Schulmeister 2003b) and have been successfully applied in generic level phylogenetic studies within Apocrita (Rozen 1951; Andena et al. 2007; Owen et al. 2007; Brajković et al. 2010; Žikić et al. 2011). While ceraphronoid male genitalia serve as an important source of species diagnostic characters their phylogenetic signal has never been tested. Here we provide the first phylogenetic analysis of Ceraphronoidea, based exclusively on 48 morphological characters related to the skeletomuscular system of the male terminalia.

Materials and Methods

Depositories and locality data of specimens examined in the present study are documented in Appendix C.

tab3

Resulting anatomical phenotype descriptions were based on observations made during dissections under stereo (Olympus SZX16 with SDFPL APO 26PF objective, 2306) and compound (Olympus BX51 with LMPLFLN506 objective, 5006) microscopes. Wet specimens in a glycerin droplet and critical point dried specimens were both dissected on Blu-Tack (Bostik Findley, Wauwatosa, WI, USA) using Super Personna razor blades (American Safety Razor, Cedar Knolls, NJ, USA) and #2 insect pins. For the better visualization of skeletal structures some specimens were macerated in 20% KOH for one week (Figs 10, 11, 25, 57).

Confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and bright field digital imaging were used to visualize anatomical structures. Bright field images were made using an Olympus CX41 compound microscope and DP71 digital camera. SEM micrographs were made using a Hitachi S-3200 Scanning Electron Microscope (wd = 23.5, av = 5kV). Specimens were critical point dried and coated with palladium prior to examination. CLSM images were made on glycerin-stored specimens between 1.5 mm thick, 24 × 50 mm cover glasses with a Leica LSM 710 laser scanning confocal microscope using 488 nm laser for excitation of the sample. We collected the autofluorescence of insect anatomical structures between 500 and 700 nm with two channels (500–580; pseudocolor green and 580–700 pseudocolor red) using 106 and 206 Plan Achromat objectives. Volume rendered images and media files were generated by Imaris Bitplane (Bitplane, Zürich, Switzerland) software. Media files, SEM micrographs and bright field images are available from figshare.com (Appendix A).

tab1

We applied a dissection-based morphological approach for the description of complex anatomical systems because section based techniques (micro-CT or histological sections) would have limited the breadth of our taxon sampling. The appropriate visualization of very small anatomical structures (the male genitalia are 100–400 μm in most Ceraphronoidea) and the textualization of the highly complex and diverse skeletal systems were the two most challenging components of our study. Imaging dissected specimens with bright field digital imaging techniques results in the loss of the ability to see anatomical structures in three dimensions and a very low tissue contrast (i.e., the ability to differentiate muscles and skeletal structures). By applying CLSM we were able to eliminate these complications. The 3D reconstructions of CLSM data have very high tissue specific contrast due to differences in the wavelength of emitted fluorescence light by soft and skeletal structures (Deans et al. 2012) and CLSM volume rendered media files let us to share our observations in 3D with a reader.

Cladistic analyses were carried out with TNT 1.1 (Goloboff et al. 2008). Space for 1000 trees was reserved in memory. Traditional searches in equal-weighting analyses and implied-weighting analyses (Goloboff 1993) with the concavity constant k set in turn to 3, 5, 10 and 25 were run to test the stability of clades under different weighting conditions. Analyses were run with collapsing rules set to maximum length = 0. One thousand replications with 1000 trees saved per replication were run, followed by a round of branch breaking on the optimal trees. Jackknife support values were calculated with 10000 pseudoreplications.

Morphological terminology follows Schulmeister (2001, 2003b), Snodgrass (1941) and Bohart and Menke (1976). The integumentary part of the external male genitalia, similar to other areas of the insect cuticle, is composed of sclerites (more rigid, usually well tanned areas with thick exocuticle) and conjunctivae (less flexible, usually less tanned areas with thin exocuticle) whose number and spatial distribution change during the course of evolution. The statements “sclerites fused” and “sclerite subdivided” are widely used to refer to processes during which the usually narrow and elongate conjunctivae separating sclerites disappear or appear during the course of evolution. Unfortunately this phrasing presents two major problems:

  1. 1.The phenotypic concepts “fused” and “separated” are relational, based on comparisons with the phenotypes of other taxa and therefore can be decoded only by comparisons with the phenotypes of other taxa. This predicament limits the accessibility of character descriptions to the non-expert community.
  2. 2.If the above mentioned phenotypic concepts are applied in descriptions supported by anatomical ontologies, i.e., semantic phenotype annotations (Deans et al. 2012; Mullins et al. 2012), where concepts are explicitly defined by structural relationships (e.g., by their bordering sclerites), “fused” and “separated” actually refer to anatomical structures that do not exist.

For example, the descriptors that “the parossiculus is delimited medially from the other parossiculus and laterally from the gonostipes by conjunctivae” are necessary components of its differentia (what distinguishes this sclerite from other sclerites). In the statement “the parossiculi are fused laterally with the gonostipites in Ceraphronidae and fused medially with each other and laterally with the gonostipes in most Dendrocerus species” qualities are provided for anatomical structures that lack the necessary conditions and therefore do not exist in these wasps. We therefore chose a more objective approach to describe what was observed rather than what is hypothesized—“medioventral conjunctiva of the male genitalia (separating parossiculi) present or absent”. We have also mapped all anatomical terms used to anatomical concepts in the Hymenoptera Anatomy Ontology (Appendix B).

tab2

Character list

The scorings of the characters defined here are listed in a character matrix in Appendix D. This matrix is available in .tnt format at http://dx.doi.org/10.684/mg.figshare.790672

  1. 1.Cercal plate: 0, present (cp: Fig. 2B); 1, absent (Fig. 18).
tab4

A cercal plate is present in Xyela, Orthogonalys, Pristaulacus, Megalyroidea and Megischus. The cercus is located on a posterior lobe of T9 in Gasteruptidae (crc: Fig. 18) and Ceraphronoidea (Fig. 2A) and no cercus is present in Evaniidae.

Fig. 18.
Fig. 18.

SEM micrographs showing the male terminalia of Gasteruption sp., distal view.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

Fig. 2.
Fig. 2.

SEM micrographs and CLSM volume rendered images of the apical abdominal sclerites of Evaniomorpha. A, Megaspilus armatus, T9 and T10, dorsal view; B, Orthogonalys pulchella, S9, ventral view; C, Ceraphron sp. 5, ventral view (see also http://dx.doi.org/10.6084/m9.figshare.100557); D, Trassedia luapi, lateral view (see also http://dx.doi.org/10.6084/m9.figshare.95699). This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1876312x.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

This character is modified after character 353 of Schulmeister (2003b).

  1. 2.T10: 0, present (Fig. 2A); 1, absent (Figs 3B, 18)

T10 is surrounded laterally by the posteriorly extended lateral part of the T9, which bears the cerci and is connected to it via a pair of rodlike T9-T10 muscles (Figs 2C, D). T10 is present in Xyela among the outgroup taxa. The relative position of T10 in Ceraphronoidea is similar to that of Orussus (T10: fig. 15D in Schulmeister 2003b; XT: plate IV in Snodgrass 1941). Unfortunately we were not able to study the musculature of the male terminalia of Orussus and hence can only suggest that T10 of Ceraphronoidea is structurally equivalent to the abdominal tergum 10 of Orussus.

Fig. 15.
Fig. 15.

CLSM volume rendered images and brightfield image showing the male genitalia and S9 of Ceraphronoidea. A, Aphanogmus sp. 27, aedeagus, median view (see also http://dx.doi.org/10.6084/m9.figshare.95675); B, Lagynodes crassicornis, S9, dorsal view; C, Conostigmus sp. 31, male genitalia, dorsal view (see also http://dx.doi.org/10.6084/m9.figshare.95680); D, Conostigmus sp. 31, male genitalia, ventral view (see also http://dx.doi.org/10.6084/m9.figshare.95684). This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1876312x.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

This character is modified after character 352 of Schulmeister (2003b).

  1. 3.Anterior margin shape of S9: 0, convex (Figs 2B and 3A); 1, concave (Fig. 3C,D)

The anterior margin of S9 is concave in Ceraphronidae and Trassedia and convex in all other taxa examined. The shape of the proximal margin of S9 corresponds to the site of origin of the mediolateral S9-cupulal muscle. The anterior margin is convex if the muscle arises medially from the sternite and concave if it arises from the anterolateral edge. In most taxa, the site of origin of the muscle corresponds with a distinct spiculum (Figs 2B and 3A), but the spiculum is absent from Masner (Fig. 4A).

Fig. 3.
Fig. 3.

CLSM volume rendered images and bright field images of the male terminalia of ceraphronoidea. A, Megaspilus armatus, ventrolateral view (see also http://dx.doi.org/10.6084/m9.figshare.95706); B, Megaspilus armatus, dorsolateral view (see also http://dx.doi.org/10.6084/m9.figshare.95704); C, Ceraphron testaceipes, ventral view; D, Ceraphron testaceipes, lateral view. This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1876312x.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

Fig. 4.
Fig. 4.

CLSM volume rendered images and SEM micrographs of the male and female terminalia of Ceraphronidae. A, Masner lubomirus, S9, dorsal view (see also http://dx.doi.org/10.6084/m9.figshare.95701); B, Trassedia sp., male terminalia, ventral view (see also http://dx.doi.org/10.6084/m9.figshare.95768); C, Cyoceraphron sp., female terminalia, lateral view; D, Masner lubomirus, male genitalia, ventral view (see also http://dx.doi.org/10.6084/m9.figshare.95683). This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/1876312x.

Citation: Insect Systematics & Evolution 44, 3-4 (2013) ; 10.1163/1876312X-04402002

This character is modified after character 347 from Schulmeister (2003b).

  1. 4.Row of short setae delimiting apical, cercus bearing area of abdominal tergum 9: 0, absent (Figs 2A, 3B and 18); 1, present (rss: Figs 2C,D and 4C)

In Megaspilidae the cercus is uniformly setose whereas in Ceraphronidae a row of shorter setae delimits a posterior area from which 1 or 2 longer setae arise.

  1. 5.Proximal lobe of vas deferens: 0, absent (Fig. 5B); 1, present (pvd: Fig. 5A)

A proximal lobe emptying into the lumen of the vas deferens is present in most Ceraphronoidea and numerous outgroup taxa. Based on its shape and location, the lobe might be an accessory gland. In Megalyra, Pseudofoenus, Megaspilus and Conostigmus sp. 23 the lobe is absent. The distal part of the vas deferens, just proximally of the vesicula seminalis, is enlarged relative to the more distal and more proximal areas in Megaspilus and Conostigmus sp. 23 (Fig. 5B). This enlarged area of the duct might also have an accessory gland function. Male accessory gland structures are great sources of species diagnostic and phylogenetic characters in some Hymenoptera (Schulmeister 2003b; Ferreira et al. 2004; Mikheyev 2004). The shape of the vesicula seminalis and the putative accessory glands is variable in different ceraphronoid taxa, and might be useful for species delimitation or even for phylogenetic analyses. Unfortunately, however, the internal male genitalia