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.

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.

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.

The organization of exteroceptive sensory inputs to interneurons of the flight neuropil in locusts.

1. Thirty-seven pairs of mesothoracic interneurons respond selectively to visual or ocellar stimuli corresponding to deviations from course in flight, expressed as angular rotation around the three spatial axes. 2. Sensitivities to roll and yaw are very strongly associated. All interneurons showing a directional preference for yaw rotations showed the same preference for roll rotations. A few roll-sensitive cells were not directionally sensitive to yaw. Some interneurons respond exclusively to pitch rotations, most to both pitch and roll/yaw. 3. Approximately equal numbers of interneurons prefer pitch up, pitch down, roll/yaw to the ipsilateral side and roll/yaw to the contralateral side. All four possible combinations of pitch (up or down) with roll/yaw (ipsilateral or contralateral) preferences occur with equal probability. 4. No relationship between neuronal structure and directional properties could be discerned. 5. The average latency of the ocellar EPSPs recorded in the interneurons is not significantly different from the average latency of the ocellar spike in the descending neurons (at the same temperature and in the same ganglion). The average ocellar IPSP latency is 8.5 ms longer. The data support the hypothesis that most EPSPs are derived from monosynaptic inputs from the DNs, and most IPSPs from polysynaptic inputs. A few EPSPs are also derived from polysynaptic inputs. 6. Most of these neurons are sensitive to wind, at least some directionally so, in a manner functionally compatible with their visual or ocellar directionality, and most are excited. Two neurons respond to movement of small objects in the visual field, and 5 to high frequency sound.

The taxonomy of invertebrate neurons: a plea for a new field.

It is generally accepted that the 'identified cell' concept and its practical application are responsible for the appeal of invertebrate preparations (and perhaps in the near future of certain vertebrate preparations too) for general neurobiology. The considerable number of neurons contained in many such preparations, and the number of investigations to which they are subjected, has led to a minor crisis: it is increasingly hard to determine whether a given neuron has been previously described, and if so, under what name. The taxonomy of identified interneurons requires a far more serious and rigorous approach than has so far been the rule. The main proposals made in this article are for the application of the normal procedural rules of classical organism taxonomy, which are highly applicable to the neural situation, and for a standardized nomenclature. Also recommended is the establishment of networked computer data bases for each of the popular invertebrate preparations (e.g. locusts, leeches) and of international committees for their supervision, and the increased use of confocal fluorescence microscopy to increase the amount of anatomical data which can be gathered from a normal physiological preparation.

Three descending interneurons reporting deviation from course in the locust. I. Anatomy.

Three descending brain interneurons (DNI, DNM, DNC) are described from Locusta migratoria. All are paired, dorsally situated neurons, with soma in the protocerebrum, input dendrites in the proto- and deuterocerebrum, and a single axon running to the metathoracic ganglion and sometimes further. In DNI the soma and all cerebral arborizations lie ipsilateral to the axon. Discrete regions of arborization lie in the ipsilateral and medial ocellar tracts, the midprotocerebrum and the deuterocerebrum. In the other ganglia the axon branches only ipsilaterally, principally laterally in the flight motor neuropil but also towards the midline. DNC is similarly organized to DNI, but the cell crosses the midline in the brain. Soma, the single projection into a lateral ocellar tract, and the midprotocerebral arborization all lie contralateral to the axon. The deuterocerebral arborization is, however, ipsilateral to the axon. The pattern of projections in the remaining ganglia resembles that of DNI. The soma and all cerebral arborizations of DNM lie ipsilateral to the axon. The arborization is only weakly subdivided into protocerebral, deuterocerebral and medial ocellar tract regions. In the remaining ganglia the arborization extends bilaterally to similar areas of both left and right flight motor neuropil. A table of synonymy is given, equating the various names used for these neurons by previous authors. The morphology correlates well with the known input and output connections. They respond physiologically to deviations from the normal flight posture mediated by ocelli, eyes and wind hairs and connect to the thoracic flight apparatus.

Three descending interneurons reporting deviation from course in the locust. II. Physiology.

The DNI, DNM and DNC descending interneurons all have very similar properties and are each at the convergence of visual, ocellar, wind-hair and other mechanoreceptor inputs. The 3 neurons respond almost exclusively to movement of the animal in space about its three axes of rotation. All are spatially and directionally selective. Movements in the preferred sense produce increasingly strong responses with amplitude and absolute position, while movements in the antipreferred sense usually elicit no response at all. Movements in the preferred sense, but towards, rather than away from, the normal flying position start to produce responses only as the animal approaches the normal flight position. The neurons function as feature detectors, responding only to specific sorts of deviation from course. DNI, DNM and DNC differ from one another principally in their directionality. DNI responds optimally to a diving banked turn to the ipsilateral side, DNM to downwards pitch, and the DNC to a diving banked turn to the contralateral side. The DN neurons contribute to the production of steering manoeuvres. They appear to be representatives of a larger class of descending interneurons bringing exteroceptive sensory input to the thoracic locomotory neuropil. The occurrence of this class of units in locusts and other insects is discussed.

Integration of nonphaselocked exteroceptive information in the control of rhythmic flight in the locust.

The integration of exteroceptive information in the flight control system of the locust was studied by determining the cellular basis of ocellar- (simple eye) mediated control of flight. Neural interactions that transform phase-independent sensory input into phase-specific motor output were characterized. Ocellar information about course deviations during flight was conveyed to the segmental thoracic ganglia by three pairs of large fast multimodal descending neurons. These made connections with thoracic motoneurons directly, via short-latency mono-or disynaptic pathways, and indirectly, via a population of intercalated thoracic interneurons. The synaptic potentials caused in the motoneurons by the direct pathway occurred at short latency and were adequate for summation with other types of sensory input. However, the strength of the synaptic effects of this pathway was weak compared with the central flight oscillator drive to the same motoneurons. In contrast, synaptic potentials evoked by the descending neurons in the thoracic interneurons were often large and brought these cells close to threshold. In turn, these interneurons always had stronger synaptic effects on postsynaptic flight motoneurons than did the descending neurons alone. We conclude that the indirect interneuronal pathway is more powerful in its effects on motoneurons than the direct pathway. Premotor thoracic interneurons, which received ocellar input appropriate for a role in correctional steering, were also rhythmically modulated during flight motor activity in phase with either depressor or elevator motoneurons. This phasic modulatory drive occurred in deafferented preparations, indicating that its source is the central oscillator for flight. Presentation of ocellar stimulation during flight motor activity showed that the central oscillatory modulation of the thoracic interneurons gated the transmission of sensory information through these interneurons. Ocellar-mediated postsynaptic potentials influenced the firing of thoracic interneurons only if they arrived during the proper phase of rhythmic drive. Thus the transmission of ocellar information from interneuron to motor neuron is possible only during appropriate phases of the flight cycle.

The feeding biology of a species-rich genus of rainforest grasshoppers (Rhachicreagra: Orthoptera, Acrididae) : I. Foodplant use and foodplant acceptance.

1. 11 Costa Rican species of the forest light-gap grasshopper genus Rhachicreagra were shown by direct observation and by faecal analysis to be each narrow-range disjunct oligophages, eating typically only 3-6 species from the hundreds present in their habitat. The diet of the different species varies considerably, some pairs showing no overlap, others having several species in common. 2. All Rhachicreagra spp. accepted in captivity any plant found in the diet of any other congeneric species, but refused almost all other plants. There is thus a generic spectrum of acceptable plants. It includes one to several members of each of 7 families: Compositae, Urticaceae, Umbellifereae, Amaranthaceae, Phytolaccaceae, Gramineae and an unidentified monocotyledenous family. Within this range, the diet of any given species or population appears to be determined primarily by availability within the habitat. There is no evidence for the hypothesis that food-plant shifts are directly associated with speciation within the genus. 3. Compositae are a component of the diet of most species, and include the principal (=most used) foodplant of many, including the morphologically most primitive species. In lowland habitats, Urticaceae supplement or replace the Compositae in the diet, in montane habitats Hydrocotyle (Umbelliferae) is important. Phytolacca and Iresine (Amaranth.) are usually minor constituents of the diet of lowland species, but Iresine is the principal foodplant of Rh. obsidian. The monocotyledenous families appear to be eaten only when other footplants are in short supply. The literature suggests that the dicotyledenous families favoured are listinguished by their relatively high content of nitrogen and cations. They are also variously rich in saponins, flavonoids, alkaloids, polyacetylenes and sesquiterpene lactones. 4. The present ecological distribution of the genus is wider han that of any single known foodplant, and appears to be made possible by the diversity of the plants accepted; these include species typical of lowland and montane habitats and of drier and wetter climates.

The feeding biology of a species-rich genus of rainforest grasshoppers (Rhachicreagra, Orthoptera, Acrididae) : II. Foodplant preference and its relation to speciation.

1. Two geographically adjoining but allopatric and altitudinally disjunct species (nothra and anchidiphalara) of the forest grasshopper genus Rhachicreagra were compared for food plant preferences in the laboratory. It has been shown that both are oligophagous and that their natural diets are markedly different. Both were offered the same 8 foodplants in a matrix of 2-way choice experiments. These plants included all important constituents of the natural diets of both species. 2. The resultant preference rank order was virtually identical for the two species, suggesting that the proportions of the different plants in the natural diet are determined principally by their relative availability. 3. The only major differences between the rank orders of the two species arise from the fact that both show an increased preference (relative to the other species) for the commonest foodplant in their own habitat. Indirect evidence suggests that this is the result of evolutionary change, not of conditioning during the lifetime of the individual. 4. There is no suggestion that host plant switching has played any significant role in speciation within the genus.

Neuronal basis of a sensory analyser, the acridid movement detector system. III. Control of response amplitude by tonic lateral inhibition.

1. The Lobular Giant Movement Detector neurone (LGMD) of Schistocerca responds with spikes when small areas of the visual field change in luminance. Previous work has shown that changes of +/- 1 log 10 unit are enough to produce maximal ON and OFF responses. 2. Using a 5 degree test area, it is shown that the number of spikes generated by such a stimulus depends on the luminance of the surrounding area. When the surround is dark, the response is maximal; when it is brightly lit, the response is minimal. Intermediate intensities produce intermediate values of response. A X 2 change in response is produced by about 3 log 10 units change in surround intensity. 3. A bright annulus, with diameters of 10-5 degrees and 25-8 degrees, inhibits both ON and OFF responses when concentric with the 5 degree test area, but not when it is 30 degrees eccentric to the test area. The inhibitory effect shows no decrease after 4 min. 4. These results are interpreted to indicate a tonic lateral inhibitory network, sited peripherally in the optic lobe prior to the divergence of the separate ON and OFF channels found in the projection from the medulla to the LGMD. It is probably identical with that described for the lamina by previous workers.

The neuronal basis of a sensory analyser, the acridid movement detector system. II. response decrement, convergence, and the nature of the excitatory afferents to the fan-like dendrites of the LGMD.

No dendritic spikes occur in the input fan of the lobular giant movement detector (LGMD) neurone. The action potentials are initiated at the point of thickening of the axon, which therefore represents the site of convergence of the retinotopic projection in the MD system. Previous work has shown that the site of decrement in response to repetitive visual stimulation is distal to this point. No change in spiking threshold in the LGMD could be demonstrated, and decrement in the number of LGMD action potentials is completely explained by the observed decrement of EPSPs recorded in the LGMD input dendritic fan. Possible postsynaptic mechanisms which might affect EPSP amplitude are excluded experimentally or shown to be improbable. Latency measurements during electrical stimulation in the second chiasma (which produces a decrementing EPSP in the fan) indicate that the pathway from the chiasma afferents to the LGMD fan is probably monosynaptic. By exclusion, the site of decrement appears to be located at the presynaptic terminal of that synapse. Generalization of habituation of the response to ON and OFF stimuli is demonstrated, showing that the presynaptic neurone at the labile synapse is an ON/OFF unit. The greater part of the previously described sensitivity gradient on the retina, relative to the MD response, appears to be explicable by the geometry of the LGMD fan and of the retinotopic projection. We conclude that the LGMD is fed by a homogeneous population of ON/OFF units running in the second optic chiasma, which form labile synapses on the input fan.

The neuronal basis of a sensory analyser, the acridid movement detector system. I. Effects of simple incremental and decremental stimuli in light and dark adapted animals.

1. The response of the movement detector (MD) system to proportionally constant incremental and decremental stimuli has been studied at various degrees of light and dark adaptation. Action potentials in the descending contralateral movement detector neurone were taken as the indicator of response. 2. Over a range of at least six log10 units of adapting luminance, the MD system behaves as an ON/OFF unit, giving responses to both incremental and decremental changes in the illumination of a 5 degrees target. 3. With increasing amplitudes of stimuli, both the ON and OFF responses saturate rapidly. Saturation is reached sooner at higher levels of light adaptation. At all levels of light adaptation, the OFF response is greater than the ON. The ratio for saturating stimuli is approximately constant at around 3:2. 4. At the brightest adapting luminances used (20 000 cd/m2) the ON response is reduced but not lost. At the lowest (0-004 cd/m2) the OFF response to a 5 degrees disc fails, but can be regained by increasing the test area to 10 degrees. 5. From what is known of the retina of locusts and other insects, it is thought that light and dark adaptation in the MD system can be adequately explained by events at the retinula cell.