Donald L. J. Quicke

Early tetrapod relationships revisited

In an attempt to investigate differences between the most widely discussed hypotheses of early tetrapod relationships, we assembled a new data matrix including 90 taxa coded for 319 cranial and postcranial characters. We have incorporated, where possible, original observations of numerous taxa spread throughout the major tetrapod clades. A stem‐based (total‐group) definition of Tetrapoda is preferred over apomorphy‐ and node‐based (crown‐group) definitions. This definition is operational, since it is based on a formal character analysis. A PAUP* search using a recently implemented version of the parsimony ratchet method yields 64 shortest trees. Differences between these trees concern: (1) the internal relationships of aistopods, the three selected species of which form a trichotomy; (2) the internal relationships of embolomeres, with Archeria crassidisca and Pholiderpeton scutigerum collapsed in a trichotomy with a clade formed by Anthracosaurus russelli and Pholiderpeton attheyi; (3) the internal relationships of derived dissorophoids, with four amphibamid species forming an unresolved node with a clade consisting of micromelerpetontids and branchiosaurids and a clade consisting of albanerpetontids plus basal crown‐group lissamphibians; (4) the position of albenerpetontids and Eocaecilia micropoda, which form an unresolved node with a trichotomy subtending Karaurus sharovi, Valdotriton gracilis and Triadobatrachus massinoti;(5) the branching pattern of derived diplocaulid nectrideans, with Batrachiderpeton reticulatum and Diceratosaurus brevirostris collapsed in a trichotomy with a clade formed by Diplocaulus magnicornis and Diploceraspis burkei. The results of the original parsimony run ‐ as well as those retrieved from several other treatments of the data set (e.g. exclusion of postcranial and lower jaw data;character reweighting; reverse weighting) ‐ indicate a deep split of early tetrapods between lissamphibian‐ and amniote‐related taxa. Colosteids, Crassigyrinus, Whatcheeria and baphetids are progressively more crownward stemtetrapods. Caerorhachis, embolomeres, gephyrostegids, Solenodonsaurus and seymouriamorphs are progressively more crownward stem‐amniotes. Eucritta is basal to temnospondyls, with crown‐lissamphibians nested within dissorophoids. Westlothiana is basal to Lepospondyli, but evidence for the monophyletic status of the latter is weak. Westlothiana and Lepospondyli form the sister group to diadectomorphs and crown‐group amniotes. Tuditanomorph and microbrachomorph microsaurs are successively more closely related to a clade including proximodistally: (1) lysorophids; (2) Acherontiscus as sister taxon to adelospondyls; (3) scincosaurids plus diplocaulids; (4) urocordylids plus aïstopods. A data set employing cranial characters only places microsaurs on the amniote stem, but forces remaining lepospondyls to appear as sister group to colosteids on the tetrapod stem in several trees. This arrangement is not significantly worse than the tree topology obtained from the analysis of the complete data set. The pattern of sister group relationships in the crownward part of the temnospondyl‐lissamphibian tree re‐emphasizes the important role of dissorophoids in the lissamphibian origin debate. However, no specific dissorophoid can be identiffed as the immediate sister taxon to crown‐group lissamphibians. The branching sequence of various stem‐group amniotes reveals a coherent set of internested character‐state changes related to the acquisition of progressively more terrestrial habits in several Permo‐Carboniferous forms.

A phylogenetic reconstruction of the Ichneumonoidea (Hymenoptera) based on the D2 variable region of 28S ribosomal RNA

The D2 variable region of 28S rRNA was sequenced in a wide range of Ichneumonoidea to provide the first comprehensive phylogenetic reconstruction of this superfamily. The two constituent families (Braconidae and Ichneumonidae) were each found to contain a single well‐supported clade dominated by the more plesiomorphic life history strategies (idiobiosis, ectoparasitism and attacking endoephytic hosts). In the Braconidae this clade corresponds to the morphologically‐defined group called the cyclostomes. In the Ichneumonidae the clade unites for the first time the pimpliformes (sensu Wahl) with most of the phygadeuontoid subfamilies and several small taxa including Adelognathus and Euceros. Relationships among the remaining, more biologically‐derived, subfamilies were less well resolved, but included among the Braconidae a well‐supported microgastroid clade and strong evidence for a sister group relationship between the Agathidinae and Sigalphinae.

Phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with a reclassification of Eulophinae and the recognition that Elasmidae are derived eulophids

Eulophidae is a large and biologically varied family of parasitoid wasps, traditionally split into four subfamilies; Elasmidae is a uniform (single genus) and morphologically distinct family of wasps that are thought to be related to Eulophidae. The D2 region of the 28S rDNA gene (≈ 560 bp) of eighty‐seven species of eulophid, three species of elasmid and sixteen outgroup species in five families was sequenced. Cladograms were constructed, and the results compared with conclusions drawn from morphological studies. The gene was most informative at the level of subfamily and tribe. The monophyly of both Eulophinae and Tetrastichinae is supported; that of Entedoninae and Euderinae is less clear. Results indicate that Eulophinae is a derived group within Eulophidae, rather than an ancestral group as previously thought, and that Elasmus, the sole genus of Elasmidae, belongs within this subfamily. The tribes of Eulophinae are reassessed and only three accepted: Eulophini (including Euplectrini and Elachertini), Elasmini and Cirrospilini LaSalle trib.n. for Boučeks Ophelimini with Ophelimus and Australsecodes excluded. Three small Australian tribes, Anselmellini, Ophelimini and Platytetracampini, are removed from Eulophinae and Entedoninae, respectively, but their exact relationships and subfamily status cannot as yet be decided. Another tribe, Keryini, known from a single Australian genus, is excluded from both Eulophinae and Eulophidae.

Robustness of Ancestral State Estimates: Evolution of Life History Strategy in Ichneumonoid Parasitoids

We test hypotheses for the evolution of a life history trait among a group of parasitoid wasps (Hymenoptera: Ichneumonoidea), namely, the transition among koinobiont parasitoids (parasitoids whose hosts continue development after oviposition) between attacking exposed hosts and attacking hosts that are concealed within plant tissue. Using a range of phylogeny estimates based on 28S rDNA sequences, we use maximum parsimony (MP) and maximum likelihood (ML) methods to estimate the ancestral life history traits for the main clades in which both traits occur (using the programs MacClade and Discrete, respectively). We also assess the robustness of these estimates; for MP, we use step matrices in PAUP* to find the minimum weight necessary to reverse estimates or make them ambiguous, and for ML, we measure the differences in likelihood after fixing the ancestral nodes at the alternative states. We also measure the robustness of the MP ancestral state estimate against uncertainties in the phylogeny estimate, manipulating the most-parsimonious tree in MacClade to find the shortest suboptimal tree in which the ancestral state estimate is reversed or made ambiguous. Using these methods, we find strong evidence supporting two transitions among koinobiont Ichneumonoidea: (1) to attacking exposed hosts in a clade consisting of the Helconinae and related subfamilies, and (2) the reverse transition in a clade consisting of the Euphorinae and related subfamilies. In exploring different methods of analyzing variable-length DNA sequences, we found that direct optimization with POY gave some clearly erroneous results that had a profound effect on the overall phylogeny estimate. We also discuss relationships within the superfamily and expand the Mesostoinae to include all the gall-associated braconids that form the sister group of the Aphidiinae.

Host location and oviposition in a basal group of parasitic wasps: the subgenual organ, ovipositor apparatus and associated structures in the Orussidae (Hymenoptera, Insecta)

Abstract Anatomical studies and behavioural observations indicate that representatives of the Orussidae use vibrational sounding to detect suitable oviposition sites. During host location, vibrations generated by tapping the tips of the antennae against the wood are picked up by the fore legs through the basitarsal spurs, transmitted along the basitarsi to thin-walled areas on the tibiae and through haemolymph to the subgenual organs, where they are transduced into nerve impulses. The apical antennomeres are distinctly shaped and have the cuticle thickened distally. The fore basitarsi have weakly sclerotised basitarsal lines proximally and membranous basitarsal spurs distally. The external wall of the fore tibiae have thin-walled areas distally on their posterior parts. Internally, large subgenual organs are situated opposite the thin-walled areas and each organ consists of 300–400 scolopidial units suspended between a lateral cuticular spine, a ventral sheet and a median ridge. The ovipositor is several times the length of the body of the wasp. When at rest, it extends all the way into the prothorax, where it is coiled before extending posteriorly to lie between the third valvulae distally. The ovipositor lies in a membranous ovipositor sac attached posteriorly to the proximal parts of the ovipositor apparatus and the posterior margin of sternum 7. In the ovipositor apparatus, the anterior parts of the second valvifers are displaced and expanded anterodorsally, inverting the first valvifers and the base of the ovipositor. When in use, the ovipositor is extended and retracted by median apodemes situated on the anterior margins of abdominal sterna 3–7. Longitudinal muscles between the apodemes allow the latter to grip the ovipositor in troughs between them. The ovipositor extends from the abdomen at the tip of sternum 7, and an internal trough on sternum 7 serves to guide the ovipositor into the wood. Despite the alterations observed in the ovipositor apparatus in the Orussidae, the musculature is almost complete and the mode of operation presumably not much different from that of other representatives of the Hymenoptera. The different ways parasitic wasps with very long ovipositors handle and accommodate these and the implications for the evolutionary history of Hymenoptera are discussed.

Wolbachia and DNA barcoding insects: Patterns, potential, and problems

Wolbachia is a genus of bacterial endosymbionts that impacts the breeding systems of their hosts. Wolbachia can confuse the patterns of mitochondrial variation, including DNA barcodes, because it influences the pathways through which mitochondria are inherited. We examined the extent to which these endosymbionts are detected in routine DNA barcoding, assessed their impact upon the insect sequence divergence and identification accuracy, and considered the variation present in Wolbachia COI. Using both standard PCR assays (Wolbachia surface coding protein – wsp), and bacterial COI fragments we found evidence of Wolbachia in insect total genomic extracts created for DNA barcoding library construction. When >2 million insect COI trace files were examined on the Barcode of Life Datasystem (BOLD) Wolbachia COI was present in 0.16% of the cases. It is possible to generate Wolbachia COI using standard insect primers; however, that amplicon was never confused with the COI of the host. Wolbachia alleles recovered were predominantly Supergroup A and were broadly distributed geographically and phylogenetically. We conclude that the presence of the Wolbachia DNA in total genomic extracts made from insects is unlikely to compromise the accuracy of the DNA barcode library; in fact, the ability to query this DNA library (the database and the extracts) for endosymbionts is one of the ancillary benefits of such a large scale endeavor – for which we provide several examples. It is our conclusion that regular assays for Wolbachia presence and type can, and should, be adopted by large scale insect barcoding initiatives. While COI is one of the five multi-locus sequence typing (MLST) genes used for categorizing Wolbachia, there is limited overlap with the eukaryotic DNA barcode region.

Molecular detection of trophic links in a complex insect host-parasitoid food web

Previously, host–parasitoid links have been unveiled almost exclusively by time‐intensive rearing, while molecular methods were used only in simple agricultural host–parasitoid systems in the form of species‐specific primers. Here, we present a general method for the molecular detection of these links applied to a complex caterpillar–parasitoid food web from tropical rainforest of Papua New Guinea. We DNA barcoded hosts, parasitoids and their tissue remnants and matched the sequences to our extensive library of local species. We were thus able to match 87% of host sequences and 36% of parasitoid sequences to species and infer subfamily or family in almost all cases. Our analysis affirmed 93 hitherto unknown trophic links between 37 host species from a wide range of Lepidoptera families and 46 parasitoid species from Hymenoptera and Diptera by identifying DNA sequences for both the host and the parasitoid involved in the interaction. Molecular detection proved especially useful in cases where distinguishing host species in caterpillar stage was difficult morphologically, or when the caterpillar died during rearing. We have even detected a case of extreme parasitoid specialization in a pair of Choreutis species that do not differ in caterpillar morphology and ecology. Using the molecular approach outlined here leads to better understanding of parasitoid host specificity, opens new possibilities for rapid surveys of food web structure and allows inference of species associations not already anticipated.

Molecular phylogeny and historical biogeography of the cosmopolitan parasitic wasp subfamily Doryctinae (Hymenoptera : Braconidae)

The phylogenetic relationships among representatives of 64 genera of the cosmopolitan parasitic wasps of the subfamily Doryctinae were investigated based on nuclear 28S ribosomal (r) DNA (~650 bp of the D2–3 region) and cytochrome c oxidase I (COI) mitochondrial (mt) DNA (603 bp) sequence data. The molecular dating of selected clades and the biogeography of the subfamily were also inferred. The partitioned Bayesian analyses did not recover a monophyletic Doryctinae, though the relationships involved were only weakly supported. Strong evidence was found for rejecting the monophylies of both Doryctes Haliday, 1836 and Spathius Nees, 1818. Our results also support the recognition of the Rhaconotini as a valid tribe. A dispersal–vicariance analysis showed a strong geographical signal for the taxa included, with molecular dating estimates for the origin of Doryctinae and its subsequent radiation both occurring during the late Paleocene–early Eocene. The divergence time estimates suggest that diversification in the subfamily could have in part occurred as a result of continental break-up events that took place in the southern hemisphere, though more recent dispersal events account for the current distribution of several widespread taxa.

A thousand and one wasps: a 28S rDNA and morphological phylogeny of the Ichneumonidae (Insecta: Hymenoptera) with an investigation into alignment parameter space and elision

Abstract The internal phylogeny of the Ichneumonidae is investigated using parsimony analysis of a large data set including 1001 partial 28S ribosomal DNA sequences, 621 of which are newly reported, and a morphological data set of 162 characters scored variously at subfamily, tribe, genus group and genus levels and including only informative characters. The data set includes members of 630 named genera, representing all currently recognized subfamilies, all but four tribes and all but one of the taxa noted by Townes as being of uncertain placement. Sequences were aligned using clustal X, and a sensitivity analysis was performed varying gap-opening and gap-extension parameters. Alignments were appraised by reference to their ability to recover a range of traditional and morphologically recognized groups. Each alignment was analysed both independently and simultaneously with the morphological data set, and also with gap characters treated as both missing data and as informative. No single set of alignment parameters was found to be markedly better by this criterion, and different ranges of parameters led to the recovery of different recognized groups of taxa. Elision (combining all alignments into a single analysis) was therefore used, both with and without morphology and with both gap character treatments, to summarize the overall molecular signal. Analysis of the morphological matrix alone produced a number of results that are undoubtedly a consequence of convergence of morphological characters as the result of parallel evolution of similar life histories. Simultaneous analysis of the morphological data set with each of the 120 DNA alignments recovered most accepted subfamilies as monophyletic. Several currently recognized subfamilies are supported by most of the molecular analyses but some appear to be paraphyletic or polyphyletic. The Ctenopelmatinae are paraphyletic with respect to the Metopiinae. Robustly recovered results lead us to resurrect the Brachyscleromatinae to include Brachyschleroma and the Erythrodolius group of Phrudinae. The Neorhacodinae and the Phrudus group of Phrudinae are transferred to the Tersilochinae. Nonnus is transferred to the Nesomesochorinae. Hyperacmus is transferred to the Cylloceriinae. The major groupings of subfamilies that have recently been proposed (i.e. ichneumoniformes, pimpliformes and ophioniformes) were recovered as monophyletic, but their exact limits remain in question.

MOLECULAR MARKERS INDICATE RARE SEX IN A PREDOMINANTLY ASEXUAL PARASITOID WASP

The parasitoid wasp genus Lysiphlebus (Hymenoptera: Braconidae: Aphidiinae) contains a taxonomically poorly resolved group of both sexual (arrhenotokous) species and asexual (thelytokous) clones. Maximum‐parsimony and maximum‐likelihood analyses of mitochondrial DNA sequence data from specimens collected across Western Europe showed that asexuality, which does not appear to be caused by the bacterium Wolbachia, is concentrated in two geographically widespread lineages, the older of which diverged from the closest extant sexual taxa approximately 0.5 million years ago. However, the DNA sequences of a nuclear intron (elongation factor—1α) showed no congruence with this pattern, and a much higher frequency of heterozygotes with very high allelic diversity was observed among the asexual females compared to that among females from the sexual species. This pattern is consistent with maternally inherited asexuality coupled with a history of rare sex with members of several closely related sexual populations or species. Our observations reinforce recent arguments that rare sex may be more important for the persistence of otherwise asexual lineages than hitherto appreciated.