Mitochondrial sequence analyses
Mitochondrial sequence dataset consisted in 558 bp sequenced for one individual from each of the 19 colonies collected in the four sites, and for the outgroup taxa, Apilitermes longiceps and Crenetermes albotarsalis (Termitidae, Termitinae).
In total for the Cubitermes sequences, 42 nucleotidic sites were variable (7.5%) and the overall proportion of A+T reached 66.7%. Height haplotypes were scored (Table 1 and Figure 2) differing at 1–30 nucleotide sites (0.18–5.40% sequence divergence). Maximum Parsimony (MP), Bayesian Inference (BI) and Maximum Likelihood (ML) reconstructions clearly showed four distinct COII haplotype groups (Figure 3). We named the first clade Cubitermes spA, including sequences from the T7, T16, T42 and T14A colonies. The second clade, named Cubitermes spB, included T5, T26, T34, T37, T38 and T14B sequences. The third clade, Cubitermes spC, comprised T17, T24, T31, T33, T46, T45 and TX sequences and the fourth group, Cubitermes spD, only the two sequences from the colonies of the Doda site, TD1 and TD2.
Sequence divergence was quite low within these groups. Indeed, in the
Cubitermes spA group, only one haplotype was detected and sequence divergence was inferior to 1% within
Cubitermes spB and
Cubitermes spC groups (sequences differed by 0–0.18% and 0–0.36% respectively).
On the opposite, the divergence between the four mitochondrial lineages was high. The Cubitermes spA and Cubitermes spB sequences differed from each other by 3.94% (net sequence divergence), Cubitermes spA and Cubitermes spC by 4.22% and Cubitermes spB and Cubitermes spC by 4.99%. Finally, Cubitermes spD diverged by 1.07% from Cubitermes spA.
ITS2 sequence analyses
A total of 298 positions sequenced for one individual from each of the 19 Cubitermes colonies, for Apilitermes longiceps and Crenetermes albotarsalis were aligned for ITS2 sequences. Regarding Cubitermes sequences, we found 262 invariable sites, 25 alignment gaps and 11 polymorphic sites. MP, BI and ML reconstructions confirmed the four lineages found with the COII gene (Figure 4). However, the position of Cubitermes spD was quite different since it was not found univocally clustered with Cubitermes spA. In this ITS2 tree, high BI posterior probabilities, ML and MP bootstrap support values were found for grouping Cubitermes spA, C. spD and C. spC in the same clade (Figure 4, 99/68/100) whereas a lower resolution of this branch appeared in the tree of mtDNA haplotypes (Figure 3, 54/
Sequence data from ITS2 revealed no polymorphism within the putative four species. Since no heterozygote was detected, ITS2 sequences were assigned to four unique haplotypes (a for Cubitermes spA, b for Cubitermes spB, c for Cubitermes spC, and d for Cubitermes spD, Table 1).
Again, divergence between groups was high in comparison with within-group divergence. Haplotypes of Cubitermes spA differed from the Cubitermes spC haplotypes by 2.18% (net sequence divergence). The Cubitermes spA and Cubitermes spB haplotypes differed from each other by 7.61% and Cubitermes spB and Cubitermes spC by 8.36%. Finally, the haplotype of Cubitermes spD differed from the haplotype of Cubitermes spA by 1.43%.
Microsatellite analyses
A total of 447 individuals for the 19 nests were surveyed at five microsatellite loci. The genetic differentiation among colonies within each putative species (FCT = 0.275, 0.258, 0.322 for Cubitermes spA, C. spB and C. spC, respectively) was substantially low. Otherwise, very high genetic differentiation was detected between colonies of different Cubitermes putative species. Cubitermes spA and Cubitermes spD were the less differentiated (FSTAD = 0.12; CI = -0.09–0.38) followed by Cubitermes spA and Cubitermes spC groups (FSTAC = 0.25, CI = 0.11–0.39). Cubitermes spA and Cubitermes spB (FSTAB = 0.49, CI = 0.27–0.67) and Cubitermes spB and Cubitermes spC (FSTBC = 0.48, CI = 0.25–0.68) showed similar high patterns of differentiation.
The NJ trees based on the DAS distance, the minimum genetic distance of Nei and the chord distance (DC) showed a genetic structure with four main clusters (Figure 5).
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