In tropical soil-feeding termites, it is impractical to apply a Biological Species Concept [39] because of the difficulties in realizing experimental crosses and observing natural hybridizations. Thus, our purpose here was to apply a Phylogenetic Species Recognition (PSR) based on the Genealogical Concordance Concept [40] to diagnose the Cubitermes species or to detect cryptic species. Such a concept has often been applied in bacteria, fungi and animals. In termites, research involving DNA-based taxonomy of structural and agricultural pest species is important [10]. Comparison of multiple molecular markers has allowed the taxonomic status of new species in Reticulitermes to be assessed [60,61] and has provided evidence for species synonymy in this genus [12,14,23]. Many DNA-based species recognitions are corroborated by evidence of morphological/chemical differences or geographically defined groups. However, PSR can be a powerful tool for diagnosing otherwise undistinguishable species, because genetic changes occurring in recently-isolated species may be observed before morphological or behavioral changes arise [41]. As found recently in other social insects, genetic isolation is not always accompanied by evident morphological differentiation. In fire ants belonging to the Solenopsis genus, genetic analyses based on allozyme and mitochondrial markers demonstrated the occurrence of sympatric and indistinguishable cryptic species [42-44].
The genetic results obtained in our study from the combined mitochondrial, nuclear and microsatellite markers unequivocally show deep separation among four groups of genotypes in the Cubitermes sp. affinis subarquatus colonies from the Lopé Reserve region. The congruence of these three types of unlinked molecular markers strongly supports the existence of differentiated genetic pools in this limited area. The mitochondrial and nuclear sequences show very little variation within the four groups but there is considerable variation among them. Although the allele ranges overlap for all the loci, possibly because of size homoplasy, the microsatellite data confirm the occurrence of four differentiated groups. These unexpected levels of subdivision are unlikely to have arisen under gene flow, so these four newly-detected groups can be seen as good evidence for the existence of cryptic species previously unrecognized by morphological techniques.
The lack of detectable morphological differences among Cubitermes species is not surprising in view of the data in the taxonomic literature, which imply that the genus is highly complex (great number of species, synonymy, missing data, etc.) [35]. Furthermore, the boundaries between nominal Cubitermes species are often concealed by intra-specific morphometrical variability. In his revision of the East African Cubitermes species, Williams [36] mentioned that most of the specific characters vary greatly in size and can also vary markedly in shape and proportion within a particular species.
In addition, our genetic data inform the current debate about the use of comparative phylogenetic methods for studying present-day species distributions. These distributions do not necessarily reflect the geographical range of the ancestral species at the time of speciation, because geographical distributions are often labile owing to climate fluctuations, territory expansion or extinction of competitors. In particular, we have certainly not sampled the entire species range and enabled definite conclusions to be drawn about biogeographic differentiation and speciation. However, the repartition of the cryptic Cubitermes species is quite interesting in relation to the species distributions of poorly dispersive insects.
In our study, we can consider two landscape units: (1) the Lopé Reserve south of the Ogooué River, where Cubitermes spA, C. spB and C. spC live sympatrically in the closely apposed sites of Okoumé, Chameau and Rocher, the only exception being that Cubitermes spA is absent from the Chameau site; (2) the Doda zone to the north, where Cubitermes spD is restricted to the distant and isolated gallery-forest of Doda and represents the only Cubitermes species in the Doda zone across 10 km. The ecological differentiation of the two zones results from paleogeographic events. Indeed, since the last glaciations, the North Ogooué has progressively run dry, resulting in the regression of all forest types except riparian. In the South Ogooué, a wet environment has been maintained by the well-developed hydro-geographical network and this has allowed the forest to be preserved.
The contemporary sympatric distributions of three of the Cubitermes species (Cubitermes spA, C. spB and C. spC) in the Reserve zone could reflect their ability to disperse within a mosaic of forests of variable ages (e.g. differing in biotic and edaphic parameters) and savannah "buffer-zones". Indeed, their dispersion seems not to be affected by fragmentation on this small scale. Little is known about the dispersion modalities and the reproductive strategies of the Cubitermes species. It is very likely that swarming (i.e. alate dispersal flight) is the main mode of dispersal in this genus, since budding (i.e. local secondary reproduction initiated by the differentiation of neotenic reproductives, derived from the nymphs or workers of the colony) is not as common in Termitidae as in lower termites [45]. It has been suggested that although the active flight of winged termites is very limited (a few hundreds of meters), the meteorological conditions accompanying dispersal flights could strongly influence the distance covered by these sexual alates. Actually, a recent genetic study of Macrotermes michaelseni (Termitidae) suggested that some winged termites can travel considerable distances (50 km), most likely by passive drift [46]. Furthermore, the effectiveness of dispersal obviously depends upon the number of alates and the rate of predation [47].
Finally, the particular distribution pattern of Cubitermes spD raises the question of the link between the history of the forest fragmentation and the modalities of speciation. One can indeed wonder whether the history of successive modifications of habitats in this geographical zone has not contributed to the isolation of the termite species populating it. Areas such as isolated gallery forests, like Doda, may constitute refuges for fauna, where some species are led to disappear while others begin to differentiate under the influence of genetic drift.
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