Ashley P. G. Dowling

Evolution of the hymenopteran megaradiation

The Hymenoptera--ants, bees and wasps--represent one of the most successful but least understood insect radiations. We present the first comprehensive molecular study spanning the entire order Hymenoptera. It is based on approximately 7 kb of DNA sequence from 4 gene regions (18S, 28S, COI and EF-1α) for 116 species representing all superfamilies and 23 outgroup taxa from eight orders of Holometabola. Results are drawn from both parsimony and statistical (Bayesian and likelihood) analyses, and from both by-eye and secondary-structure alignments. Our analyses provide the first firm molecular evidence for monophyly of the Vespina (Orussoidea+Apocrita). Within Vespina, our results indicate a sister-group relationship between Ichneumonoidea and Proctotrupomorpha, while the stinging wasps (Aculeata) are monophyletic and nested inside Evaniomorpha. In Proctotrupomorpha, our results provide evidence for a novel core clade of proctotrupoids, and support for the recently proposed Diaprioidea. An unexpected result is the support for monophyly of a clade of wood-boring sawflies (Xiphydrioidea+Siricoidea). As in previous molecular studies, Orussidae remain difficult to place and are either sister group to a monophyletic Apocrita, or the sister group of Stephanidae within Apocrita. Both results support a single origin of parasitism, but the latter would propose a controversial reversal in the evolution of the wasp-waist. Generally our results support earlier hypotheses, primarily based on morphology, for a basal grade of phytophagous families giving rise to a single clade of parasitic Hymenoptera, the Vespina, from which predatory, pollen-feeding, gall-forming and eusocial forms evolved.

Phylogenetic relationships among superfamilies of Hymenoptera

The first comprehensive analysis of higher‐level phylogeny of the order Hymenoptera is presented. The analysis includes representatives of all extant superfamilies, scored for 392 morphological characters, and sequence data for four loci (18S, 28S, COI and EF‐1α). Including three outgroup taxa, 111 terminals were analyzed. Relationships within symphytans (sawflies) and Apocrita are mostly resolved. Well supported relationships include: Xyeloidea is monophyletic, Cephoidea is the sister group of Siricoidea + [Xiphydrioidea + (Orussoidea + Apocrita)]; Anaxyelidae is included in the Siricoidea, and together they are the sister group of Xiphydrioidea + (Orussoidea + Apocrita); Orussoidea is the sister group of Apocrita, Apocrita is monophyletic; Evanioidea is monophyletic; Aculeata is the sister group of Evanioidea; Proctotrupomorpha is monophyletic; Ichneumonoidea is the sister group of Proctotrupomorpha; Platygastroidea is sister group to Cynipoidea, and together they are sister group to the remaining Proctotrupomorpha; Proctotrupoidea s. str. is monophyletic; Mymarommatoidea is the sister group of Chalcidoidea; Mymarommatoidea + Chalcidoidea + Diaprioidea is monophyletic. Weakly supported relationships include: Stephanoidea is the sister group of the remaining Apocrita; Diaprioidea is monophyletic; Ceraphronoidea is the sister group of Megalyroidea, which together form the sister group of [Trigonaloidea (Aculeata + Evanioidea)]. Aside from paraphyly of Vespoidea within Aculeata, all currently recognized superfamilies are supported as monophyletic. The diapriid subfamily Ismarinae is raised to family status, Ismaridae stat. nov.

Molecular phylogenetics of Braconidae (Hymenoptera: Ichneumonoidea), based on multiple nuclear genes, and implications for classification

This study examined subfamilial relationships within Braconidae, using 4 kb of sequence data for 139 taxa. Genetic sampling included previously used markers for phylogenetic studies of Braconidae (28S and 18S rDNA) as well as new nuclear protein‐coding genes (CAD and ACC). Maximum likelihood and Bayesian inference of the concatenated dataset recovered a robust phylogeny, particularly for early divergences within the family. This study focused primarily on non‐cyclostome subfamilies, but the monophyly of the cyclostome complex was strongly supported. There was evidence supporting an independent clade, termed the aphidioid complex, as sister to the cyclostome complex of subfamilies. Maxfischeria was removed from Helconinae and placed within its own subfamily within the aphidioid complex. Most relationships within the cyclostome complex were poorly supported, probably because of lower taxonomic sampling within this group. Similar to other studies, there was strong support for the alysioid subcomplex containing Gnamptodontinae, Alysiinae, Opiinae and Exothecinae. Cenocoeliinae was recovered as sister to all other subfamilies within the euphoroid complex. Planitorus and Mannokeraia, previously placed in Betylobraconinae and Masoninae, respectively, were moved to the Euphorinae, and may share a close affiliation with Neoneurinae. Neoneurinae and Ecnomiinae were placed as tribes within Euphorinae. A sister relationship between the microgastroid and sigalphoid complexes was also recovered. The helconoid complex included a well‐supported lineage that is parasitic on lepidopteran larvae (macrocentroid subcomplex). Helconini was raised to subfamily status, and was recovered as sister to the macrocentroid subcomplex. Blacinae was demoted to tribal status and placed within the newly circumscribed subfamily Brachistinae, which also contains the tribes Diospilini, Brulleiini and Brachistini, all formerly in Helconinae.

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.

Characterization and tissue‐specific expression of two lepidopteran farnesyl diphosphate synthase homologs: Implications for the biosynthesis of ethyl‐substituted juvenile hormones

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl‐substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl‐branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type‐I FPPSs display unique active site substitutions, including several in and near the first aspartate‐rich motif, whereas type‐II proteins have a more “conventional” catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type‐I enzymes are better suited than type‐II for generating ethyl‐substituted products, a quantitative real‐time PCR assessment of their relative abundance in insect tissues indicated that type‐I expression is ubiquitous whereas that of type‐II is essentially confined to the JH‐producing glands, where its transcripts are ∼20 times more abundant than those of type‐I. These results suggest that type‐II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity. Proteins 2006.

Multivariate Analysis of Morphological Variation in Two Cryptic Species of Sancassania (Acari: Acaridae) from Costa Rica

Abstract Populations of Sancassania mites were collected in Costa Rica from scarabaeid and passalid beetles and cultured. The populations proved to be reproductively incompatible due to postzygotic isolation and show 1.4% difference in domains 2 and 3 of the 28S nuclear rDNA gene, indicating the populations represent distinct species. Because the mites were virtually indistinguishable morphologically, 61 morphological characters of 50 females and 101 characters of 60 deutonymphs of the two species were analyzed. Traditional univariate morphometrics could not separate them. Multivariate analyses of variance (principal component and discriminant function) were used to interpret morphological differences between the two species in relation to factors that influence their morphology in a laboratory and field setting. Principal component analyses were done on size and shape as well as shape variables alone. The discriminant function analysis was done on a reduced subset of shape variables. In both cases, the shape analyses resulted in complete separation of the two species and the characters contributing strongly to the discrimination were used in formal description of the two species, Sancassania salasi sp. nov. and S. ochoai sp. nov. Although deutonymphs of S. salasi taken from field-collected beetles show a significantly smaller magnitude of size variation, they show significant deviation in shape compared with cultured deutonymphs of the same species, a potential problem for correlation of specimens of other Sancassania species from culture and nature. Characters that provide the strongest contribution to these intraspecific shape changes are, therefore, taxonomically unreliable.

Functional characterization of PAS and HES family bHLH transcription factors during the metamorphosis of the red flour beetle, Tribolium castaneum.

The basic helix-loop-helix transcription factors are present in animals, plants and fungi and play important roles in the control of cellular proliferation, tissue differentiation, development and detoxification. Although insect genomes contain more than 50 helix-loop-helix transcription factors, the functions of only a few are known. RNAi has become a widely used tool to knock-down the expression to analyze the function of genes. As RNAi works well in Tribolium castaneum, we utilized this insect and RNAi to determine functions of 19 bHLH transcription factors belonging to PAS and HES families during the larval stages of the red flour beetle, T. castaneum. We searched the genome sequence of T. castaneum and identified 53 bHLH genes. Phylogenetic analyses classified these 53 genes into ten families; PAS, HES, Myc/USF, Hand, Mesp, Shout, p48, NeuroD/Neurogenin, Atonal and AS-C. In RNAi studies, knocking-down the expression of seven members of the PAS and HES families affected the growth and development of T. castaneum. An inability to grow to reach critical weight to undergo metamorphosis, failure to complete larval-pupal or pupal-adult ecdysis and abnormal wing development are among the most common phenotypes observed in RNAi insects. Among the bHLH transcription factors studied, the steroid receptor coactivator (SRC) showed the most severe phenotypes. Knock-down in the expression of the gene coding for SRC caused growth arrest by affecting the regulation of lipid metabolism. These studies demonstrate the power of RNAi for functional characterization of members of the multigene families in this model insect.

Testing the accuracy of TreeMap and Brooks parsimony analyses of coevolutionary patterns using artificial associations

Abstract Brooks parsimony analysis and TreeMap are the two most commonly used methods and are tested using artificially evolved host–parasite associations with varying amounts of coevolutionary events occurring between the two “phylogenies.” The purpose is to test the precision with which each method recovers the true coevolutionary history. The reconstructions recovered by each method are compared against the original test case to determine how closely the reconstruction resembles the artificially created coevolutionary history. Brooks parsimony analysis is found to be consistently less prone to gross overestimation of coevolutionary events and misleading results. Brooks parsimony analysis performs better overall because it is more adept at dealing with host-switching events, both between and within lineages leading to widespread parasite taxa, which provides enough evidence for implementing Brooks parsimony analysis instead of TreeMap in coevolutionary studies.

External mouthpart morphology in the Tenuipalpidae (Tetranychoidea): Raoiella a case study

The use of low-temperature scanning electron microscopy (LTSEM) to study external mouthpart morphology in the Tenuipalpidae, in particular the genus Raoiella, has brought some aspects of the mechanics of feeding in this group into question. In addition, an LTSEM study on the specialized feeding behaviour of Raoiellaindica Hirst (Tetranychoidea: Tenuipalpidae) revealed host plant use in this species could be affected by stomatal complex morphology.

A priori and a posteriori methods in comparative evolutionary studies of host–parasite associations

Brooks parsimony analysis (BPA) and reconciliation methods in studies of host–parasite associations differ fundamentally, despite using the same null hypothesis. Reconciliation methods may eliminate or modify input data to maximize fit of single parasite clades to a null hypothesis of cospeciation, by invoking different a priori assumptions, including a known host phylogeny. By examining the degree of phylogenetic congruence among multiple parasite clades, using hosts as analogs of taxa but not presuming a host phylogeny or any degree of cospeciation a priori, BPA modifies the null hypothesis of cospeciation if necessary to maintain the integrity of the input data. Two exemplars illustrate critical empirical differences between reconciliation methods and BPA: (1) reconciliation methods rather than BPA may select the incorrect general host cladogram for a set of data from different clades of parasites, (2) BPA rather than reconciliation methods provides the most parsimonious interpretation of all available data, and (3) secondary BPA, proposed in 1990, when applied to data sets in which host‐switching produces hosts with reticulate histories, provides the most parsimonious and biologically realistic interpretations of general host cladograms. The extent to which these general host cladograms, based on cospeciation among different parasite clades inhabiting the same hosts, correspond to host phylogeny can be tested, a posteriori, by comparison with a host phylogeny generated from nonparasite data. These observations lead to the conclusion that BPA and reconciliation methods are designed to implement different research programs based on different epistemologies. BPA is an a posteriori method that is designed to assess the host context of parasite speciation events, whereas reconciliation methods are a priori methods that are designed to fit parasite phylogenies to a host phylogeny. Host‐switching events are essential for explaining complex histories of host–parasite associations. BPA assumes coevolutionary complexity (historical contingency), relying on parsimony as an a posteriori explanatory tool to summarize complex results, whereas reconciliation methods, which embody formalized assumptions of maximum cospeciation, are based on a priori conceptual parsimony. Modifications of basic reconciliation methods, embodied in TreeMap 1.0 and TreeMap 2.02, represent the addition of weighting schemes in which the researcher specifies allowed departures from cospeciation a priori, with the result that TreeMap results more closely agree with BPA results than do reconciled tree analysis results.