It has been suggested that ecosystem fragmentation has important consequences for animal populations by reducing and dividing the distribution areas and by limiting connections between the fragments [1,2]. As a result, this natural or anthropological phenomenon is described as a process promoting the genetic differentiation of isolated populations and could be involved in speciation. Insects are interesting models for studying the effects of fragmentation owing to their abundance, limited dispersal, short generation time and sensitivity to disturbances. In tropical ecosystems, termites (Isoptera) may represent up to 95% of soil insect biomass; humivorous termites contribute to tropical soil fertilization [3-6]. Since soil-feeders are very sensitive to changes in their environment, they constitute interesting bio-indicators in landscape fragmentation studies, when the evolution of their specific richness is surveyed [7-9].
Studying the link between genetics and fragmentation requires evaluation of breeding structure, gene flow and genetic differentiation among populations. This implies unambiguous identification of species. For a long time, termite systematics was based on morphological and\or morphometrical character sets for individuals belonging to various castes (alates, soldiers or workers). During the past decade, an increasing number of taxonomic studies have shown that molecular methods constitute fast and reliable diagnostic systems, which complement morphological identification. Indeed, several studies based on mitochondrial genome sequences such as the cytochrome oxidase genes, the AT-rich region and the 16S rDNA have thrown a great deal of light on termite taxonomy and on phylogenetic and phylogeographic analyses of the Reticulitermes [10-15], Nasutitermes [16-18] and Heterotermes [19] genera. Because mitochondrial gene trees do not necessarily reflect species trees owing to their maternal inheritance, the addition of nuclear markers in molecular studies is useful for confirming the organismal phylogeny [20]. Among the nuclear sequences, the internal transcribed spacers (ITS) of rDNA are more polymorphic between than within species. It is generally assumed than concerted evolution homogenizes individual rDNA repeats and produces a mostly uniform sequence in all repeats in a given species. Although there is intra-individual variation in some taxa, the ITS2 region sequences are considered phylogenetically informative and able to distinguish closely-related species [21]. Such an evolutionary pattern has allowed sibling or cryptic species in the Reticulitermes genus to be discriminated [22-24]. Microsatellites are also very useful nuclear polymorphic markers and have contributed widely to the resolution of colony and population genetic structures in Isoptera [25-27]. Many surveys have concerned the xylophageous genus,Reticulitermes, in non-natural fragmented areas such as urban ecosystems [28,29]. To date, however, microsatellite markers have not been used to investigate termite phylogeny or species discrimination, as has been done for other groups e.g. vertebrates [30], ticks [31], wasps [32] and ants [33].
In the present survey, we studied the humivorous afro-tropical Cubitermes genus (Termitidae, Termitinae), which shows remarkable ecological plasticity, colonizing savannahs or forests according to species. Among the soil-feeding Termitidae, the Cubitermes genus is one of the main builders; its nest can shelter up to 10 genera of true inquiline or optional builder termites [34]. In spite of its undeniable ecological interest, the taxonomy of this genus is poorly resolved and a complete revision appears necessary [35]. Indeed, at least 64 species of Cubitermes have been described on the basis of morphometrical characters, but it is strongly suspected that some of these are synonymic. It is important to note that extensive taxonomic work is necessary for the West African species since the sole currently available key concerns the East African species [36]. Molecular data are also lacking for the Cubitermes genus, as only one sequence from cytochrome oxidase I [37] and two from 12S mtDNA [GenBank: AF475037, AF475001] are registered.
The aim of this work is to clarify the taxonomic status of the Cubitermes colonies from the Lopé Reserve region (Gabon), in order to obtain a better understanding of tropical termite diversity in fragmented areas. The Lopé Reserve is typically characterized by a mosaic of forests and savannahs and constitutes an ideal setting for studies of natural fragmentation.
On the basis of morphological comparisons with type specimens in the collections of the British Museum (London) and the Royal Museum for Central Africa (Tervuren), and because they showed no diagnostic morphometrical variation or molecular divergence in mitochondrial 12S and 16S rDNA (Harry, unpublished data), the Cubitermes colonies were affiliated to a single species, Cubitermes sp. affinis subarquatus (Sjöstedt). Here we attempt to reconstruct a phylogeny based on three types of polymorphic and independent molecular markers. We sequenced a portion of the mitochondrial COII gene and the ITS2 region, and determined the genotypes at five microsatellite loci isolated from Cubitermes subarquatus [38], for 19 Cubitermes colonies from four different sites (Figure 1). Three of these sites were in the same geographical scale within the Lopé Reserve and corresponded to forest patches of different ages, including small savannah patches: Okoumé (75 years old), Rocher (800 years old) and Chameau (800 years old). A fourth site, Doda, was an isolated gallery-forest outside the Reserve within a savannah landscape.
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