Pyrosequencing of Amphilophus cichlid ESTs
Natural selection and divergence times inferred from transcriptome sequence variation in young species of ecologically distinct cichlid fishes
The current study aimed to compare the EST sequence divergence between two ecologically distinct, sympatric sister taxa of neotropical cichlid from the Nicaraguan crater lake Apoyo: Amphilophus astorquii and Amphilophus zaliosus. EST sequences were generated using massively parallel 454 pyrosequencing.
The species rich cichlid fishes display a surprising breadth of ecological adaptations. Crater lakes provide a natural laboratory in which to study cichlid speciation by ecological divergence. Up to now there is a dearth of transcriptomic and genomic information that would aid in understanding the molecular basis of the phenotypic differentiation between young species. We used next-generation sequencing (Roche 454 pyrosequencing) to characterize the diversity of expressed sequence tags (ESTs) between ecologically divergent, endemic and sympatric species of cichlid fishes from crater lake Apoyo, Nicaragua: benthic Amphilophus astorquii and limnetic A. zaliosus. We obtained 24 174 A. astorquii and 21 382 A. zaliosus "high quality EST" contigs, of which 13 106 pairs are orthologous between them. Based on the ratio of non-synonymous and synonymous substitutions, we identified six sequences exhibiting signals of strong diversifying selection (Ka/Ks > 1). These included genes involved in biosynthesis, metabolic processes and development. Therefore we have identified transcriptome sequence variation that may be reflective of natural selection acting on the genome in these young, sympatric sister species. Based on Ks ratios and p-distances between 3’-untranslated regions (UTRs) calibrated to previously published species divergence times, we estimated a neutral transcriptome-wide substitutional mutation rate of ~1.25 × 10-6/ site / year. We conclude that next-generation sequencing technologies allow us to infer natural selection acting to diversify the genome of young species, such as crater lake cichlids, with greater scope than ever previously possible.