Cytotaxonomy, morphology and molecular systematics of the Bioko form of Simulium yahense (Diptera: Simuliidae).

Cytotaxonomic analysis of the polytene chromosomes from larvae of the Simulium damnosum Theobald complex from the island of Bioko in Equatorial Guinea is reported, and a new endemic cytoform is described. Chromosomally this cytoform is close to both S. squamosum (Enderlein) and S. yahense Vajime & Dunbar, but is not identical to either. However, it is morphologically and enzymatically identical to S. yahense. The Bioko form was also found to differ from other cytoforms of the S. damnosum complex in West Africa in the copy number or RFLP pattern of several different repetitive DNA sequences. It is clear that the Bioko form is genetically distinct from other populations of the S. damnosum complex, and whilst it is closest to S. yahense, it shows features that suggest a high degree of geographical and genetic isolation. Such isolation is an important consideration in the assessment of the potential for onchocerciasis vector eradication on Bioko.

Secondary structure and conserved motifs of the frequently sequenced domains IV and V of the insect mitochondrial large subunit rRNA gene.

We have analysed over 400 partial insect mitochondrial large subunit (mit LSU) sequences in order to identify conserved motifs and secondary structures for domains IV and V of this gene. Most of the secondary structure elements described by R. R. Gutell et al. (unpublished) for the LSU were identified. However, we present structures for helices 84 and 91 that are not recognized in previous universal models. The portion of the 16S gene containing domains IV and V is frequently sequenced in insect molecular systematic studies so we have many more sequences than previous studies which focused on the complete mitochondrial LSU molecule. In addition, we have the advantage of investigating several sets of closely related taxa. Aligned sequences from thirteen insect orders and nine secondary structure diagrams are presented. These conserved sequence motifs and their associated secondary structure elements can now be used to facilitate the alignment of other insect mit LSU sequences.

Molecular phylogenetic analysis of the Pneumoroidea (Orthoptera, Caelifera): molecular data resolve morphological character conflicts in the basal acridomorpha.

A key transition in the evolution of the insect suborder Caelifera (Orthoptera; Insecta) was from predominantly non-angiosperm-feeding basal lineages to the modern acridomorph fauna (grasshoppers and related insects). However, because of conflicts in the distribution of several complex morphological characters, the relationships of the presumed intermediates, and in particular of the superfamily Pneumoroidea, are presently unclear. We undertook a phylogenetic study of representatives of all of the transitional acridomorph families using mitochondrial and nuclear DNA sequences. No support for pneumoroid monophyly was obtained from nonparametric bootstrap analysis. Furthermore, adopting a maximum-likelihood approach, specific hypotheses of relationships within the Pneumoroidea were firmly rejected using parametric bootstrapping and Kishino-Hasegawa tests. The results indicate that the Pneumoroidea are at best a grade. This distinction implies that the evolution of the proposed pneumoroid synapomorphies, femoro-abdominal stridulation and simple male genital structure, might previously have been misinterpreted as cases of single character gains or losses within lineages. Reconstructions of character states for the femoro-abdominal stridulation indicate that, in fact, multiple losses or gains are equally likely. An important implication of our findings is that, in grasshoppers, auditory tympana may have evolved before stridulation, supporting the argument that the original function of tympana may have been related not to conspecific communication but to predator detection. Overall, the results of this study emphasize the high information content of these minor groups (in this case, the four intermediate families under consideration contain only 0.2% of extant orthopteran species diversity). Our analyses also demonstrate the advantages of model-based methods in analyzing systematic problems and, in particular, of the importance of testing specific phylogenetic hypotheses when a priori support for groupings (e.g., from nonparametric bootstrapping) is marginal.

Combined molecular phylogenetic analysis of the Orthoptera (Arthropoda, Insecta) and implications for their higher systematics.

A phylogenetic analysis of mitochondrial and nuclear rDNA sequences from species of all the superfamilies of the insect order Orthoptera (grasshoppers, crickets, and relatives) confirmed that although mitochondrial sequences provided good resolution of the youngest superfamilies, nuclear rDNA sequences were necessary to separate the basal groups. To try to reconcile these data sets into a single, fully resolved orthopteran phylogeny, we adopted consensus and combined data strategies. The consensus analysis produced a partially resolved tree that lacked several well-supported features of the individual analyses. However, this lack of resolution was explained by an examination of resampled data sets, which identified the likely source of error as the relatively short length of the individual mitochondrial data partitions. In a subsequent comparison in which the mitochondrial sequences were initially combined, we observed less conflict. We then used two approaches to examine the validity of combining all of the data in a single analysis: comparative analysis of trees recovered from resampled data sets, and the application of a randomization test. Because the results did not point to significant levels of heterogeneity in phylogenetic signal between the mitochondrial and nuclear data sets, we therefore proceeded with a combined analysis. Reconstructing phylogenies under the minimum evolution and maximum likelihood optimality criteria, we examined monophyly of the major orthopteran groups, using nonparametric and parametric bootstrap analysis and Kishino-Hasegawa tests. Our analysis suggests that phylogeny reconstruction under the maximum likelihood criteria is the most discriminating approach for the combined sequences. The results indicate, moreover, that the caeliferan Pneumoroidea and Pamphagoidea, as previously suggested, are polyphyletic. The Acridoidea is redefined to include all pamphagoid families other than the Pyrgomorphidae, which we propose should be accorded superfamily status.

Distribution of the Simulium damnosum complex on Bioko island, Equatorial Guinea, and the potential for onchocerciasis elimination by vector eradication.

Onchocerciasis is endemic on the island of Bioko, Equatorial Guinea, where it is transmitted by the 'Bioko form' of the Simulium damnosum complex, a cytospecies unique to the island. To determine the distribution of vector breeding, three dry season and two wet season expeditions were made in 1989, 1996 and 1997, and 226 of the island's 247 rivers (91.5%) were visited. Of these 226 rivers, 130 (58%) were flowing during the dry season, forty-five (20%) supported aquatic stages of Simuliidae of any species and twenty-five (11%) contained larvae or pupae of the S. damnosum complex. The twenty-one rivers not prospected were in the mountainous south of the island, where an additional seventeen rivers were reached but not satisfactorily prospected. Of these thirty-eight rivers, twenty-nine were considered highly likely to support vector breeding, bringing the total number of rivers which could harbour the vector during the dry season to fifty-four (21.9% of the island's total). Breeding was believed to be limited to river stretches below 1000 m altitude, and during the dry season the total length of those stretches which could support breeding on Bioko was estimated to be 1020 km. A combination of factors, including low river discharges during the dry season, the relatively low water temperature on Bioko, the suitability of limited stretches of most rivers as vector breeding sites and the close proximity of many rivers within a small geographical area, render the vector vulnerable to eradication by aerial treatment of rivers with insecticide. The isolation of the Bioko form of the S. damnosum complex suggests that reinvasion following treatment would be unlikely, and eradication of the vector might be achieved by a dry season larviciding programme in one or two years.

Inferences about orthopteroid phylogeny and molecular evolution from small subunit nuclear ribosomal DNA sequences.

We determined DNA sequences of SSU rRNA genes in twenty-nine polyneopteran insect species and aligned these with homologues from eight other insects. In a phylogenetic analysis we recovered the classic divisions of Palaeoptera and Neoptera, with the latter divided into monophyletic Paraneoptera and Polyneoptera. The polyneopterans divided into three lineages: one includes the Grylloblattodea, Dermaptera and Plecoptera, the second contains the Blattodea, and the third (Orthopteroidea sensu Hennig) contains the Embiidina, Phasmida, and Orthoptera, in that order. The monophyly of the Orthoptera is supported by the analyses, as is the separation between taxa from its suborders Caelifera and Ensifera. The Caelifera are not always supported as a monophyletic group; the basal Tridactyloidea are separated from the rest of the Caelifera in some analyses. Inside of Tridactyloidea, the Acridoidea, Pamphagoidea, Pneumoroidea and Trigonopterygoidea are always recovered as a monophyletic group. We also examined the basal orthopteran relationships, with the specific aim of assessing the antiquity of the Ensifera. Character state reconstructions indicated that the ancestral ensiferan sequence is very similar to the ancestral orthopteran sequence. However, likelihood ratio tests rejected the null hypothesis of a molecular clock and we conclude that a change in substitution rate has occurred within the Orthoptera and several of the other polyneopteran orders. Similar observations have been made in holometabolous insects, suggesting that variation in substitution rate is a general feature of insect nuclear rRNA evolution.

Molecular population studies of Simulium damnosum s.l. (Diptera: Simuliidae) using a novel interspersed repetitive DNA marker.

Simulium damnosum s.l., the vector of West African onchocerciasis, has been the target of a major insect control initiative for the past 20 years. However, attempts to study the migration patterns of reinvading infective flies into controlled areas have been restricted by the lack of suitable genetic markers. Here, the results of the first population-level study of S. damnosum s.l. using a repetitive DNA marker, pSO11, are presented. Sequence analysis of pSO11 revealed a complex internal pattern of repetition and an open reading frame with 56.8 per cent similarity to a mouse retrotransposon protein. Combined with an interspersed genomic distribution, the internal structure of pSO11 suggested that it represents the 5' half of a transposable element. The genomic diversity of the sequence was analysed using Southern blot analysis of genomic DNA. Data were collected from 475 individuals from the island of Bioko (in Equatorial Guinea), Cameroon, Côte d'Ivoire and Sierra Leone. The results indicate that pSO11 is useful as an indicator of genomic diversity and that the experimental design used here permits comparison of an approximately random sample of chromosomal loci. Making this assumption, estimates of homozygosity based on pSO11 diversity were used to study inter- and intraspecific variability. The results indicate that pSO11 is potentially useful for studying population-level processes in S. damnosum s.l.

The effectiveness of mitochondrial rRNA gene sequences for the reconstruction of the phylogeny of an insect order (Orthoptera).

We investigated the value of mitochondrial rRNA sequences for analyzing pre-Cainozoic divergence events in insects. Using small subunit and large subunit rRNA sequences from 38 orthopteroid species, we examined several aspects of sequence evolution including secondary structure, substitution rate, and base composition. Substitution matrices calculated from the two genes were very similar, though differences were detected in rates of C-T transitions between paired and unpaired positions in secondary structures. By contrast, extreme disparities between substitution frequencies at different phylogenetic levels make character-transition weighting essential in parsimony reconstruction. The analysis of base composition indicated that branch attraction of at least two important lineages was due to shared base composition biases and to long branch attraction. The importance of taxonomic sampling and sequence length for the effectiveness of phylogenetic recovery using the rRNA fragments was also assessed. Significantly, combining the two sequences seemed both justifiable and necessary for this taxonomic sample. However, for reconstructing deep branches of phylogeny, it seems that increasing either or both the number of taxa or nucleotide positions will not necessarily solve all problems. Instead, the resolution of ancient branching events using mtDNA sequences probably depends upon the development and application of better specified reconstruction methods.

The phylogeny of the Caelifera (Insecta, Orthoptera) as deduced from mtrRNA gene sequences.

Fragments of both mitochondrial ribosomal RNA genes of 32 caeliferan taxa (representing six of the seven superfamilies) and six outgroup Orthopteroids were sequenced. The combined alignment length was 630 bp after removal of all ambiguously aligned positions. Separation for the basal taxa was problematic and analysis using the LogDet transformation indicated that shared base composition biases were a confounding factor. The suborder Caelifera and all traditional caeliferan superfamilies except the Pamphagoidea are retrieved as monophyletic groups, though the Eumastacoidea lack significant bootstrap support. Of the traditional pamphagoid taxa, the Pamphagidae is embedded between classically acridid subfamilies, whereas Pyrgomorphidae is placed close to the Pneumoroidea. The morphological similarities of the Pyrgomorphidae and Pamphagidae may thus be homoplasic. A consensus tree based on five different methods of analysis indicated the following order: (Tridactyloidea, Tetrigoidea (Eumastacidae, Proscopiidae (Pneumoridae, Pyrgomorphidae (Acrididae + Pamphagidae)))).

The sequence, organization, and evolution of the Locusta migratoria mitochondrial genome.

The sequencing of the cloned Locusta migratoria mitochondrial genome has been completed. The sequence is 15,722 bp in length and contains 75.3% A+T, the lowest value in any of the five insect mitochondrial sequences so far determined. The protein coding genes have a similar A+T content (74.1%) but are distinguished by a high cytosine content at the third codon position. The gene content and organization are the same as in Drosophila yakuba except for a rearrangement of the two tRNA genes tRNAlys and tRNAasp. The A+T-rich region has a lower A+T nucleotide content than in other insects, and this is largely due to the presence of two G+C-rich 155-bp repetitive sequences at the 5'end of this section and the beginning of the adjacent small rRNA gene. The sizes of the large and small rRNA genes are 1,314 and 827 bp, respectively, and both sequences can be folded to form secondary structures similar to those previously predicted for Drosophila. The tRNA genes have also been modeled and these show a strong resemblance to the dipteran tRNAs, all anticodons apparently being conserved between the two species. A comparison of the protein coding nucleotide sequences of the locust DNA with the homologous sequences of five other arthropods (Drosophila yakuba, Anopheles quadrimaculatus, Anopheles gambiae, Apis mellifera, and Artemia franciscana) was performed. The amino acid composition of the encoded proteins in Locusta is similar to that of Drosophila, with a Dayhoff distance twice that of the distance between the fruit fly and the mosquitoes. A phylogenetic analysis revealed the locust genes to be more similar to those of the Dipterans than to those of the honeybee at both the nucleotide and amino acid levels. A comparative analysis of tRNA orders, using crustacean mtDNAs as outgroups, supported this. This high level of divergence in the Apis genome has been noted elsewhere and is possibly an effect of directional mutation pressure having resulted in an accelerated pattern of sequence evolution. If the general assumption that the Holometabola are monophyletic holds, then these results emphasize the difficulties of reconstructing phylogenies that include lineages with variable substitution rates and base composition biases. The need to exercise caution in using information about tRNA gene orders in phylogenetic analysis is also illustrated. However, if the honeybee sequence is excluded, the correspondence between the other five arthropod sequences supports the findings of previous studies which have endorsed the use of mtDNA sequences for studies of phylogeny at deep levels of taxonomy when mutation rates are equivalent.