T. Backeljau

18S RRNA SUGGESTS THAT ENTOPROCTA ARE PROTOSTOMES, UNRELATED TO ECTOPROCTA

The Ento- and Ectoprocta are sometimes placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes. However, Entoprocta have also been allied to the pseudocoelomates, while Ectoprocta are often united with the Brachiopoda and Phoronida in the (super)phylum Lophophorata. Hence, the phylogenetic relationships of these taxa are still much debated. We determined complete 18S rRNA sequences of two entoprocts, an ectoproct, an inarticulate brachiopod, a phoronid, two annelids, and a platyhelminth. Phylogenetic analyses of these data show that (1) entoprocts and lophophorates have spiralian, protostomous affinities, (2) Ento- and Ectoprocta are not sister taxa, (3) phoronids and brachiopods form a monophyletic clade, and (4) neither Ectoprocta or Annelida appear to be monophyletic. Both deuterostomous and pseudocoelomate features may have arisen at least two times in evolutionary history. These results advocate a Spiralia-Radialia-based classification rather than one based on the Protostomia-Deuterostomia concept.

Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex

Correct species identification is the starting point for studying the epidemiological role of vectors. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved. The problem with the identification of Anopheles minimus s.l., one of the most widespread malaria vectors in South‐East Asia, is twofold: it is a complex of at least two isomorphic species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was developed for An. minimus species A and C, and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. PCR‐amplified internal transcribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested with restriction endonuclease BsiZI. Clear diagnostic banding patterns for the six species were obtained on agarose gels. Testing field‐collected specimens from different regions in South‐East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear that molecular identification has to focus not only on the species of complexes, but also on related species if they hamper the morphological identification of the ‘sensu lato species’.

Random amplified polymorphic DNA (RAPD) and parsimony methods

Abstract Random amplified polymorphic DNA (RAPD) data possess a number of undesirable features for parsimony analysis. These features include their non-codominant inheritance, their anonymous nature, their different (a)symmetrical transformation probabilities, and their possible GC priming bias. As a consequence, no single parsimony method seems appropriate for RAPD data. Moreover, the presence/absence coding of RAPDs is equivalent to the invalid independent allele model for allozymes. These issues are discussed and the way in which parsimony analysis of RAPDs can be misleading is illustrated.

GENETIC POPULATION STRUCTURE OF THE WINTER MOTH (OPEROPHTERA BRUMATA L.) (LEPIDOPTERA, GEOMETRIDAE) IN A FRAGMENTED LANDSCAPE

As a result of human activities natural environments have been altered in many different ways. One important effect of human disturbance is the fragmentation of natural habitats. As a consequence, genetic differentiation among habitat islands is expected to increase, whereas within-area genetic diversity is expected to decrease. Indirect estimates from allozyme polymorphisms are used to investigate the effects of habitat fragmentation in the winter moth on a very small geographical scale. We demonstrated that genetic differentiation increased whereas genetic diversity decreased with fragmentation, with habitat patches isolated by only a few hundred metres up to 3 km. These results were even more striking considering that no genetic differentiation was detected at a larger scale (10–40 km). This pattern of distribution of genetic variation is in agreement with temporarily variable densities and gene flow levels which prevent an equilibrium being reached between genetic drift and gene flow. Consequently the effects of fragmentation probably remain limited.

Confirmation of Anopheles varuna in vietnam, previously misidentified and mistargeted as the malaria vector Anopheles minimus.

Malaria control programs in Southeast Asia are faced with several questions concerning vector behavior and species identification, which need to be answered to consolidate and further improve the results of control practices. The vector system in Southeast Asia is complex because of the number of species potentially involved in malaria transmission. Additionally, the follow-up and evaluation of preventive control measures are hampered by the misidentification of vectors due to overlapping morphological characters of the female mosquitoes. In central Vietnam, control practices are aimed at 2 main species, Anopheles dirus s.l. and Anopheles minimus s.l. These reputed vectors were studied in an area of Binh Thuan Province of south-central Vietnam. Different collection methods were used to capture mosquitoes quarterly during a 1-year period. Mosquitoes were identified in the field and later subjected to detailed morphological examination and polymerase chain reaction-restriction fragment length polymorphism analysis. What was thought to be an unusual morphotype of An. minimus was shown to be Anopheles varuna, and most specimens identified as the former species in the field proved to be the latter species. Very few An. minimus individuals were found during the study period. The population of An. varuna was found to be highly zoophilic, and based on this behavior, it cannot be considered a vector in Vietnam. Because this species was previously being misidentified as An. minimus, a nonvector was mistargeted as a malaria vector in Binh Thuan Province. Anopheles dirus, which was found positive for Plasmodium falciparum circumsporozoite via enzyme-linked immunosorbent assay, is clearly the main vector in this area. Despite the fact that several potential secondary vectors were found during the study, the primary target for vector control in the region should be An. dirus.

Effects of mating, breeding system and parasites on reproduction in hermaphrodites: pulmonate gastropods (Mollusca)

There are approximately 20 000 pulmonate gastropod species that are all hermaphroditic and (with a few exceptions) can act in both (i.e., male and female) sexual roles. Life history traits such as growth (rate), age at first reproduction, fecundity, fertility, future survival and offspring survival are highly variable within pulmonate species, even among individuals of the same population. Here, we review some aspects of reproduction (availability of partners, size-dependent sex allocation, courtship, (multiple) mating, sperm longevity/viability, social facilitation), breeding system (self-fertilisation, outcrossing or mixed) and parasitism that may influence an individuals reproductive success and therefore account for part of the intraspecific variation in life history traits. A literature study showed that fecundity, fertility and/or growth are significantly affected by: i) the mating group size through changes in interference competition (e.g., crowding), breeding system and sex allocation; ii) individual body size with larger individuals producing more eggs than smaller individuals; iii) mating whereby female fitness may be positively or negatively affected; iv) social facilitation whereby female fitness is positively affected by the presence of conspecifics; v) the breeding system including the phenomena of inbreeding and outbreeding depression; and vi) parasites that may suppress or stimulate reproduction, especially egg-laying, in parasitised individuals. Moreover, multiple mating and multiple paternity seem very common in pulmonates. Interestingly, several of the above-mentioned aspects seem to interact or even act synergetically. Although many aspects of life history variation in pulmonate gastropods are still poorly understood which makes it difficult to draw general conclusions, pulmonates offer ample opportunities to study the evolution of major topics in evolution and life history strategies. Indeed, there is a growing number of basommatophoran and stylommatophoran model species, experimental setups and molecular, histological and histochemical techniques that are used to test current hypothesis on sex allocation, sexual selection (sexual conflict, sperm competition or cryptic female choice), the evolution of breeding systems and host-parasite interactions which will yield much information for the study of life history strategies as well.

Age and size of European saltmarshes and the population genetic consequences for ground beetles

Abstract Enzyme and dispersal polymorphisms of the saltmarsh carabid beetles Pogonus chalceus and Dicheirotrichus gustavii were studied in European populations varying in size and in isolation in space and time. D. gustavii, a constantly fullwinged species, has a larger genetic diversity and a smaller genetic differentiation between populations than the wing-polymorphic P. chalceus. Clear relationships between population or site characteristics and genetic structure were not observed, except for the special position taken by some small populations in both species. The dispersal power of P. chalceus in small populations is larger than in large populations, suggesting that these populations are unstable and/or young. Small populations, however, do not always show a lower genetic diversity than large populations, as would be expected from genetic drift. Dispersal power in P. chalceus declines with increasing age of the saltmarsh, probably due to continuous emigration of winged individuals. Age and size of saltmarshes, although difficult to study independently, both appear to be important in determining the genetic structure of saltmarsh beetles. Maximum diversity in both parameters is therefore recommended as an optimal nature conservation strategy.

Review of the ultrastructure of the nematode body cuticle and its phylogenetic interpretation

The phylogenetic interpretation of the nematode cuticle ultrastructure is reviewed within the framework of recent DNA‐sequence data. In particular, the structure of the median and basal zones is discussed. Several structural elements of the cuticle seem to have arisen independently several times within the Nematoda and thus are highly homoplasious (e.g. the cortical or basal radial striae, spiral fibre layers and a fluid matrix with struts). Moreover, identifying the homology of the nematode cuticle ultrastructures is often very difficult at deep taxonomic levels. Hence, the cuticle appears to be unreliable regarding resolution of deep‐level relationships in the Nematoda. However, at less inclusive taxonomic levels (e.g. families, genera, …) the cuticle seems to be a more reliable phylogenetic marker.

Cryptic diversity and gene flow among three African agricultural pests: Ceratitis rosa, Ceratitis fasciventris and Ceratitis anonae (Diptera, Tephritidae).

The ‘Ceratitis FAR complex’ is a species complex of African fruit flies (Diptera, Tephritidae) including the major agricultural pest Ceratitis rosa and the morphologically similar Ceratitis fasciventris and Ceratitis anonae. To resolve their intra‐ and interspecific genetic relationships and to estimate gene flow within this complex, we surveyed allelic variation at 16 microsatellite loci in 27 African populations of the three morphospecies. Interpopulation genetic distances and individual Bayesian assignments distinguished five genotypic clusters: two involving C. rosa (R1, R2; that may occur in sympatry), two involving C. fasciventris (F1, F2; with parapatric distributions) and one involving C. anonae (A). Intra‐ and interspecific patterns of genetic differentiation were not hierarchically structured and genetic differentiation between conspecific clusters (F1–F2 and R1–R2) was higher or comparable with differentiation between heterospecific clusters (e.g. F1‐A or R2‐A). In some cases, gene flow estimates among morphospecies or among heterospecific genotypic clusters were significantly different from zero, showing the lack of reproductive isolation. Genetic differentiation between genotypic clusters was partly supported by morphological differences observed a posteriori in male secondary sexual characters. These results suggest important revisions to current models of ecological niche requirements and invasion risk of the major agricultural pest C. rosa and provide a basis for a taxonomic re‐interpretation of the FAR complex.

Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa

The complete nucleotide sequence of the small ribosomal subunit RNA of the gastropod, Limicolaria kambeul, was determined and used to infer a secondary structure model. In order to clarify the phylogenetic position of the Mollusca among the Metazoa, an evolutionary tree was constructed by neighbor‐joining, starting from an alignment of small ribosomal subunit RNA sequences. The Mollusca appear to be a monophyletic group, related to Arthropoda and Chordata in an unresolved trichotomy.