Detection of genetic changes involved in adaptive evolution by comparing the genomes of Cuban Anolis lizards

概要

Introduction
　Elucidation of the genomic loci and the sequence changes involved in adaptive evolution is important for considering the effects of genetic factors on the evolvability, the evolutionary constraint, and the repeatability of the evolution, and is therefore one of the key issues in evolutionary ecology. Parallel evolution and adaptive radiation provide the opportunity to study replicates of evolution in the field in the taxon and is a particularly very good model of study to consider them. The genomes of many species of finches and cichlids, model organisms for parallel adaptive diaphanous evolution, have been sequenced and analyzed to examine the processes of genetic variation creation and accumulation involved in their adaptive evolution, suggesting that common variation and common mutation creation processes have influenced subsequent repeated evolution.
　Anolis lizards in the West Indies are another model organisms of parallel evolution and adaptive evolution with diversified morphology and ecology. Their morphology and behavior vary depending on the structural site of the tree used by the species, and species with the same structural site of the tree used have similar morphology and behavioral patterns. It is estimated that ecomorphs have evolved many times independently on each of the West Indies islands. Furthermore, because of the diversity of thermal habitat, the range of average body temperatures is relatively wide. It is common to see differences in the elevation, tree cover, sunlight intensity, and amount of shade in habitats between closely related species or populations. Recently, progress has been made in identifying genes involved in the adaptive evolution of Anolis lizards. The genes involved in thermal adaptation and their functions have been recently elucidated. Furthermore, for Anolis cristatellus in Puerto Rico, RNA-seq reads were mapped to the genome of A. carolinensis and the results of genome-wide SNP analysis suggests that particular amino acid sites of rars, which is thought to be involved in heat tolerance in urban areas, were under selection between forest and urban populations. Furthermore, genome assembly of three Anolis lizards from Central America have been reconstructed and comparative analysis of them detected positive selection in several Hox genes involved in limb development. It has also been shown that the expression pattern of hoxd13 during development differs among species. However, while progress has been made in understanding what genes are involved in the adaptive evolution of Anolis lizards, in many species, the genetic variation selected during adaptive evolution has not been detected. Moreover, very little work has been done to detect mutations other than SNPs. genome-wide detection of transposable element insertions has not been performed in most Anolis lizards, and it is not known how transposable element insertions have occurred during species diversification and adaptive evolution. There has also been little progress in detecting gene duplication and non-coding regions that have undergone natural selection. The detection of such mutations will also need to be advanced to elucidate the genetic basis of parallel evolution and adaptive evolution in West Indies Anolis lizards.
　Cuban Anolis lizards are an excellent model for studying the mechanism of adaptive evolution because of the diversity of its ecomorphs and the differentiation among closely related lineages in their thermal habitats.
　A. sagrei, A. allisoni and A. porcatus, which inhabit open areas, are also common in urban areas. In addition, A. carolinensis, closely related species of A. porcatus, and A. sagrei, have been introduced and established in various regions of the world. Therefore, the analysis of genetic variation among Cuban Anolis lizards is expected to lead to elucidation of the molecular and genetic basis of adaptive evolution of morphology, adaptive evolution to thermal habitat, urban tolerance and invasiveness.
　In my thesis, I detected genetic variation occurred during adaptive evolution of Cuban Anolis lizards. In Chapter 1, I conducted a phylogenetic analysis of the transcriptome sequences of nine species (Anolis alutaceus, Anolis garridoi, A. isolepis, A. porcatus, A. allisoni, A. allogus, A. homolechis, A. mestrei and A. sagrei) and detected genes positively selected in the common ancestral branch of A. allisoni and A. porcatus and the A. sagrei branch, which inhabit hot and open areas and currently thrive in urban areas. In Chapter 2, I constructed de novo draft genome assembly of six species (A. isolepis, A. allisoni, A. porcatus, A. allogus, A. sagrei and A. homolechis), compared each feature of their genomes and detected genetic changes that occurred among their genomes during adaptive evolution.

Chapter 1. Detection of genes positively selected in Cuban Anolis lizards that naturally inhabit hot and open areas and currently thrive in urban areas
　By phylogenetic analysis of the transcriptome assemblies of Anolis alutaceus, Anolis garridoi, Anolis isolepis, Anolis allisoni, Anolis porcatus, Anolis allogus, Anolis homolechis, Anolis mestrei, and Anolis sagrei, I detected genes positively selected in the common ancestor of A. allisoni and A. porcatus, and A. sagrei, which inhabit hot and open areas and thrive in urban areas currently. I did not detect any positive selection in genes common to those. However, positive selection was detected for genes involved in the response to stresses such as oxidative stress and DNA damage, and in cardiac function in commonly both of them. These findings suggest that adaptive evolution of the response to stress caused by heat or ultraviolet radiation might have occurred in ancestors of Anolis species inhabiting hot and open areas and might be related to the current thriving in urban areas of them.

Chapter 2. Draft genome of six Cuban Anolis lizards and insights into genetic changes involved in adaptive evolution
　In order to build a genomic information for studying the relationship between repeated adaptive radiation and various genetic variations, including those in non-coding regions, I reconstructed de novo genome assemblies of A. isolepis, A. allisoni, A. porcatus, A. allogus, A. homolechis and A. sagrei. Six new draft genome assemblies with relatively long and high gene completeness were obtained. The features of these genomes, together with those of mainland Anolis lizards already obtained in previous studies, were compared. I detected differences in the composition of transposable elements among lineages or species (Fig. 1A). Furthermore, the accumulation process of transposable elements was estimated to be different in each lineage or species. The analysis of gene copy number showed the association between adaptive evolution to environments and gene duplication. Species inhabiting shaded areas such as forests tended to have lower the proportion of duplicated genes (PD), and the only species native to America, A. carolinensis, had a higher PD than three related species of Cuban Anolis lizards with the same ecomorph (Fig. 1B). However, further studies on gene copy number in a larger number of species and the investigation the biological effects of copy number are needed to verify the association. In addition, the estimation of past effective population size for each species indicated the possibility that population size have been influenced by the formation of the island of Cuba and adaptive evolution to thermal habitats. These results indicate that during the diversification of the Cuban Anolis lizards, many genetic variations that could drive adaptive evolution has occurred in each lineage and species, suggesting that many parts of the repeated adaptive evolution might be caused a variety of separate genetic variations. The research for phenotypic consequences of these genetic variants found and the process by which they were created will help to elucidate in more detail the molecular and genetic basis of adaptive evolution in Anolis lizards.

Discussion
　Anolis lizards of the West Indies have diversified in morphology and ecology, and it is thought that their morphology and ecology have evolved many times on several islands. Clarifying how genetic variation has occurred and accumulated during this repeated evolution, and by what kind of variation adaptive evolution has been achieved, will provide important insights into the mechanisms of evolution. In this thesis, in order to detect genetic variation occurred during adaptive evolution, I conducted transcriptome phylogenetic analysis of Cuban Anolis lizards and detected positive selection in protein-coding regions of genes in species inhabiting hot and open habitats, and reconstructed novel genome assemblies of six species with different ecomorphs and thermal habitats, and compared genome characteristics. Phylogenetic analysis of the transcriptome in chapter 1 did not detect genes positively selected commonly in the common ancestor of A. allisoni and A. porcatus, and A. sagrei, which inhabit hot and open areas. However, in both, positive selection was detected in genes involved in response to DNA damage and oxidative stress, and cardiac function. These results suggest that the protein- coding sequences of genes may have evolved adaptively to the common stresses experienced there, although evolution of the same genes is not common in hot and open areas-dwelling species. In chapter 2, I reconstructed novel genome assemblies of six species of Cuban Anolis lizards with different ecomorphs and thermal habitats and detected differences in their genomic characteristics. As a result, I obtained draft genome assemblies of good quality with relatively long and high gene completeness. Comparisons of transposable element composition and repeat landscapes suggest that there were different transposable element accumulation processes among each lineages or species. Analysis of gene copy numbers showed that forest species tended to have lower proportion of duplicated genes than other species. Thus, I could provide an overview of the activities of transfer factors and gene duplication during the diversification of Cuban Anolis lizards. Furthermore, the results of estimation of past effective population size indicate that all six Cuban Anolis lizards have increased in effective population size between tens of millions of years ago and around one million years ago. The results also suggest the possibility that population size has fluctuated in relation to the formation of the island of Cuba, global temperature, interspecific competition and thermal adaptation. In this thesis, I detected genes and mutation sites involved in adaptive evolution to hot and open areas, and changes in the composition and accumulation process of transposable elements and in gene copy number among lineages or species. Analysis of the transcriptome sequences in Chapter 1 detected no genes where positive selection occurred in both the common ancestor of A. allisoni and A. porcatus and A. sagrei, which inhabit hot and open areas. In Chapter 2, I found that waves of accumulation of transition factors and many gene duplications, the driving forces of adaptive evolution, have occurred separately in each lineage and species. These results indicate the possibility that repeated adaptive evolution in Anolis lizards may have been driven by many different mutations. However, even if the mutations are disparate, it is also possible that there are more fundamental factors in common that give rise to the mutations. Further investigation of the biological effects of these mutations and the factors that lead to their creation will help us to better understand the mechanisms of the diversification. The transcriptome and genome constructed in this thesis, as well as the inter-species genomic variation revealed, will provide an important stepping stone to understanding the genetic basis of the diversification of Anolis lizards in the West Indies.