Periodic Reporting for period 4 - ConvergeAnt (An Integrative Approach to Understanding Convergent Evolution in Ant-eating Mammals)
Reporting period: 2021-03-01 to 2022-08-31
Understanding how different species have repeatedly adapted to similar environmental conditions is one of the fundamental questions in evolutionary biology. Despite its widespread occurrence at many levels of the tree of life, fundamental questions about the fascinating phenomenon of convergent evolution remain unanswered. By providing natural evolutionary replay experiments, convergently evolved taxa have the potential to shed light on the predictability of evolution. The convergent evolution of species living in similar environments illustrates the power of natural selection to produce phenotypic similarity despite different evolutionary histories. The large scale of convergent morphological evolution, the importance of molecular convergence, and the potential role of the host microbiome are becoming increasingly recognised. The ConvergeAnt project aimed to address these questions by studying ant-eating mammals, which provide a textbook example of morphological convergence with at least five independent origins in placentals (armadillos, anteaters, aardvarks, pangolins and aardwolves). Taking advantage of the unique set of convergently evolved characters associated with the ant-eating diet, we are investigating the molecular mechanisms underlying phenotypic adaptation through an integrative approach combining morphometric, genomic and metagenomic approaches. The ultimate goal of the ConvergeAnt project is to provide fundamental insights into the complex interplay between morphology, genome and microbiome in a classic case of adaptive convergence driven by a highly specialised diet. Our results show that historical contingency has played a major role, as the major convergent ant-eating mammalian lineages have taken different evolutionary paths to adapt to the myrmecophagous diet at the morphological, genomic, and gut microbiome levels.
For all three tasks of the project, we have provided a wealth of data that has been key to the project and will be an excellent resource for the research community. For the morphological task, 3D digital data of skulls were collected to characterise the evolutionary processes by which convergent tooth reduction occurs and how it has affected skull shape in the different ant-eating lineages. We show that convergent tooth loss in anteaters and pangolins resulted in a different fate for the mandibular canal, with anteaters retaining a neurovascular system (dorsal canaliculi) in the mandible, whereas pangolins did not. This suggests that the external similarities between anteater and pangolin mandibles have overshadowed the complex evolution of their internal morphology. The geometric morphometric data were used to examine cranial covariance patterns in convergently evolved myrmecophagous placentals. The results confirmed that morphological integration of the mammalian skull is highly constrained and may therefore rely on conserved developmental modules and underlying gene networks. For the genomic task, we overcame a major challenge of the project by demonstrating that a hybrid sequencing and assembly strategy could be successfully used to generate high quality genomes from roadkill. This resulted in nine new complete genomes of elusive myrmecophagous species and relatives, which we used to unravel the genomic mechanisms underlying convergent ant-eating phenotypes. An important result of this task concerns the evolution of chitinase genes (CHIAs), which encode enzymes capable of digesting the chitin of insect exoskeletons. By conducting a detailed comparative genomic survey of these genes, we were able to infer that the ancestor of placental mammals likely possessed five functional CHIA genes, which have undergone divergent evolutionary fates among myrmecophagous species. Indeed, anteaters, armadillos and aardvarks retain 4-5 functional CHIA genes, whereas pangolins and aardwolves have only one. Using comparative transcriptomics, we found that convergently evolved pangolins and anteaters express different chitinases in their hypertrophied salivary glands and other additional digestive organs. These results show that divergent molecular mechanisms underlie the convergent adaptation to myrmecophagy in pangolins and anteaters, highlighting the role of historical contingency and molecular tinkering of their chitin digestive enzyme toolkit. Finally, for the microbiome task, we use long-read metagenomics from field-collected faecal samples of nine myrmecophagous species to reconstruct more than 300 high-quality bacterial genomes. We show that more than a hundred of these carry chitinase genes, suggesting a potential role for the gut microbiota in insect prey digestion in myrmecophagous mammals. Furthermore, while some of the chitinolytic bacteria were recruited convergently across myrmecophagous species, others appeared to be host species specific. This sheds further light on the potential role of the holobiont in convergent adaptation to myrmecophagy.
The project has allowed us to begin to understand how convergent lineages of ant-eating mammals evolved. Our results have already shown that convergent evolution in anteaters and pangolins proceeded through different underlying mechanisms, both at the phenotypic level with the example of the mandibular canal, and at the genomic and transcriptomic level with the discovery of different chitinase genes involved in the digestion of social insects and expressed in different organs in the two most ancient ant-eating lineages. The new phenotypic and genomic data provide a deeper understanding of the mechanisms involved in convergent evolution. On the morphological front, detailed digital dissections of the masticatory muscles have allowed fine-scale characterisation of the processes associated with tooth reduction/loss and snout elongation in the different ant-eating lineages. The 3D digitised specimens were used to test levels of skull shape integration in convergent lineages using modularity methods. Despite drastic functional changes in the skull, such as rostral elongation, tooth and masticatory loss, we did not detect a convergent shift in cranial modularity and integration in myrmecophagous placentals, which have conserved the typical mammalian scheme. On the genomic front, the high quality genomes generated for nine ant-eating species are still being used to unravel the genomic mechanisms underlying the previously characterised convergent ant-eating phenotypes. The high quality of our genomes, in terms of both contiguity and completeness, allows us to apply state-of-the-art statistical methods to detect convergent molecular evolution in both protein-coding and non-coding regions, as well as to confidently assess patterns of gene loss and pseudogenisation. Finally, on the microbiome front, we have used the hundreds of samples already collected to assess the diversity and patterns of phylosymbiosis of gut microbes in ant-feeding lineages and closely related species. This was complemented by shotgun metagenomic analyses to characterise microbial functional diversity in convergent myrmecophagous species. The characterisation of hundreds of bacterial genomes containing chitinase genes highlighted the potential role of the gut microbiome in convergent adaptation to this highly specialised diet. The combination of these three approaches has greatly improved our understanding of the mechanisms underlying this textbook example of convergent evolution.