Deep mitochondrial divergence within a Heliconius butterfly species is not explained by cryptic speciation or endosymbiotic bacteria.

BACKGROUND: Cryptic population structure can be an indicator of incipient speciation or historical processes. We investigated a previously documented deep break in the mitochondrial haplotypes of Heliconius erato chestertonii to explore the possibility of cryptic speciation, and also the possible presence of endosymbiont bacteria that might drive mitochondrial population structure. RESULTS: Among a sample of 315 individuals from 16 populations of western Colombia, two principal mtDNA clades were detected with 2.15% divergence and we confirmed this structure was weakly associated with geography. The first mtDNA clade included 87% of individuals from northern populations and was the sister group of H. erato members of Andes western, while the second clade contained most individuals from southern populations (78%), which shared haplotypes with an Ecuadorian race of H. erato. In contrast, analysis using AFLP markers showed H. e. chestertonii to be a genetically homogeneous species with no association between mitochondrial divergence and AFLP structure. The lack of congruence between molecular markers suggests that cryptic speciation is not a plausible explanation for the deep mitochondrial divergence in H. e chestertonii. We also carried out the first tests for the presence of endosymbiontic bacteria in Heliconius, and identified two distinct lineages of Wolbachia within H. e. chestertonii. However, neither of the principal mitochondrial clades of H. e. chestertonii was directly associated with the patterns of infection. CONCLUSIONS: We conclude that historical demographic processes are the most likely explanation for the high mitochondrial differentiation in H. e. chestertonii, perhaps due to gene flow between Cauca valley H. e. chestertonii and west Pacific slope populations of H. erato.

Multiple sources of reproductive isolation in a bimodal butterfly hybrid zone.

An important evolutionary question concerns whether one or many barriers are involved in the early stages of speciation. We examine pre- and post-zygotic reproductive barriers between two species of butterflies (Heliconius erato chestertonii and H. e. venus) separated by a bimodal hybrid zone in the Cauca Valley, Colombia. We show that there is both strong pre- and post-mating reproductive isolation, together leading to a 98% reduction in gene flow between the species. Pre-mating isolation plays a primary role, contributing strongly to this isolation (87%), similar to previous examples in Heliconius. Post-mating isolation was also strong, with absence of Haldane's rule, but an asymmetric reduction in fertility (< 11%) in inter-specific crosses depending on maternal genotype. In summary, this is one of the first examples of post-zygotic reproductive isolation playing a significant role in early stages of parapatric speciation in Heliconius and demonstrates the importance of multiple barriers to gene flow in the speciation process.

A hybrid zone provides evidence for incipient ecological speciation in Heliconius butterflies.

In Heliconius butterflies, it has been proposed that speciation occurs through a combination of divergence in ecological habitat preferences and mimetic colour patterns. Here we test this hypothesis by investigating a parapatric form of the widespread species Heliconius erato. Mendelian (colour patterns) and molecular genetic data permit us to address hypotheses about introgression and genetic differentiation between different populations. Combined analysis of colour pattern, microsatellite loci and mitochondrial DNA showed that Heliconius erato venus and Heliconius erato chestertonii form a bimodal hybrid zone implying partial reproductive isolation. In a sample of 121 individuals collected in sympatry, 25% were hybrids representing a significant deficit of heterozygotes compared to the Hardy-Weinberg expectation. Seven microsatellite loci, analysed for a subset of these individuals, showed marked differentiation between the parental taxa, and unambiguously identified two genotypic clusters concordant with our phenotypic classification of individuals. Mitochondrial DNA analysis showed H. erato venus as a monophyletic group well differentiated from H. erato chestertonii, implying a lack of historical introgression between the populations. Heliconius erato chestertonii is therefore an incipient species that maintains its integrity despite high levels of hybridization. Moreover, H. erato chestertonii is found at higher altitudes than other races of H. erato and has a distinct colour pattern and mimetic relationship. Hence, there are now two examples of parapatric incipient species related to H. erato, H. himera and H. erato chestertonii, both of which are associated with higher altitudes, more arid habitats and distinct mimetic relationships. This implies that parapatric habitat adaptation is a likely cause of speciation in this group.