Comparative transcriptomics reveals divergent paths of chitinase evolution underlying dietary convergence in ant-eating mammals

<strong>Comparative transcriptomics reveals divergent paths of chitinase evolution underlying dietary convergence in ant-eating mammals</strong><br> Rémi Allio^1,2,§,*, Sophie Teullet^1,§, Dave Lutgen^1,3,4,§, Amandine Magdeleine¹, Rachid Koual¹, Marie-Ka Tilak¹, Benoit de Thoisy^5,6, Christopher A. Emerling^1,7, Tristan Lefébure⁸, and Frédéric Delsuc^1,* <br> ¹ISEM, Univ. Montpellier, CNRS, IRD, Montpellier, France ²CBGP, INRAE, CIRAD, IRD, Montpellier SupAgro, Univ. Montpellier, Montpellier, France ³Institute of Ecology and Evolution, University of Bern, Bern, Switzerland ⁴Swiss ornithological Institute, Sempach, Switzerland ⁵Institut Pasteur de la Guyane, Cayenne, French Guiana, France ⁶Kwata NGO, Cayenne, French Guiana, France ⁷Biology Department, Reedley College, Reedley, CA, USA ⁸Univ. Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France ^§Equal contribution *Correspondence Rémi Allio: [e-mail] Frédéric Delsuc: [e-mail] <strong>Abstract</strong> Ant-eating mammals represent a textbook example of convergent evolution. Among them, anteaters and pangolins exhibit the most extreme convergent phenotypes with complete tooth loss, elongated skulls, protruding tongues, hypertrophied salivary glands producing large amounts of saliva, and powerful claws for ripping open ant and termite nests. However, comparative genomic analyses have shown that anteaters and pangolins differ in their chitinase gene (<em>CHIA</em>) repertoires, which potentially degrade the chitinous exoskeletons of ingested ants and termites. While the southern tamandua (<em>Tamandua tetradactyla</em>) harbors four functional <em>CHIA </em>paralogs (<em>CHIA1</em>-<em>4</em>), Asian pangolins (<em>Manis</em> spp.) have only one functional paralog (<em>CHIA5</em>). Here, we performed a comparative transcriptomic analysis of salivary glands in 33 placental species, including 16 novel transcriptomes from ant-eating species and close relatives. Our results suggest that salivary glands play an important role in adaptation to an insect-based diet, as expression of different <em>CHIA </em>paralogs is observed in insectivorous species. Furthermore, convergently-evolved pangolins and anteaters express different chitinases in their digestive tracts. In the Malayan pangolin, <em>CHIA5</em> is overexpressed in all major digestive organs, whereas in the southern tamandua, all four functional paralogs are expressed, at very high levels for <em>CHIA1</em> and <em>CHIA2 </em>in the pancreas, and for <em>CHIA3</em> and <em>CHIA4</em> in the salivary glands, stomach, liver, and pancreas. Overall, our results demonstrate that divergent molecular mechanisms underlie convergent adaptation to the ant-eating diet in pangolins and anteaters. This study highlights the role of historical contingency and molecular tinkering of the chitin-digestive enzyme toolkit in this classic example of convergent evolution. <strong><em>Figures &amp; Tables</em></strong> <strong>Figure 1: </strong>A. Mammalian chitinase gene family tree reconstructed using a maximum likelihood gene-tree/species-tree reconciliation approach on protein sequences. The nine chitinase paralogs are indicated on the outer circle. Scale bar represents the mean number of amino acid substitutions per site. B. Synteny of the nine chitinase paralogs in humans (<em>Homo sapiens</em>), tarsier (<em>Carlito syrichta</em>), nine-banded armadillo (<em>Dasypus novemcinctus</em>) and the two main focal convergent ant-eating species: the southern tamandua (<em>Tamandua tetradactyla</em>) and the Malayan pangolin (<em>Manis javanica</em>). Assembly names and accession numbers are indicated below species names. Arrows represent genes with scaffold/contig names and BLAST hit positions indicated below. Arrow direction indicates gene transcription direction as inferred in Genomicus v100.01 (Nguyen et al. 2022) for genes located on short contigs. Ψ symbols indicate pseudogenes as determined in Emerling et al. (2018). Genes with negative BLAST results were not represented and are probably not functional or absent. <strong>Figure 2: </strong>Comparison of predicted ancestral sequences of the nine mammalian chitinase paralogs. A. Conserved residues of the canonical chitinolytic domain active site (DXXDXDXE). Arrows indicate paralogs in which changes occurred in the active site. B. Summary of the evolution of chitinase paralogs functionality. C. Conserved cysteine residues of the chitin-binding domain. The arrow indicates OVGP1 in which the last four cysteines have been replaced. <strong>Figure 3:</strong> Comparative expression of the nine chitinase paralogs in 40 mammalian salivary gland transcriptomes. The 33 species are presented in the phylogenetic context covering the four major placental clades: Afrotheria (AFR), Xenarthra (XEN), Euarchontoglires (EUA), and Laurasiatheria (LAU). The chronogram was extracted from www.timetree.org (Kumar et al. 2022). Non-functional pseudogenes of the three focal species (in bold) are represented by the Ψ symbol: nine-banded armadillo (<em>Dasypus novemcinctus</em>), southern tamandua (<em>Tamandua tetradactyla</em>) and Malayan pangolin (<em>Manis javanica</em>). Expression level is represented as log10 (Normalized Counts + 1). Asterisks indicate the 16 new transcriptomes produced in this study. Silhouettes were obtained from www.phylopic.org. <strong>Figure 4:</strong> Comparative expression of the nine chitinase paralogs in 72 transcriptomes from different organs of the three focal species: the nine-banded armadillo (<em>Dasypus novemcinctus</em>), the Malayan pangolin (<em>Manis javanica</em>), and the southern tamandua (<em>Tamandua tetradactyla</em>). Non-functional pseudogenes are represented by the Ψ symbol and hatched background. Boxes indicate organs of the digestive tract. Expression level is represented as log10 (Normalized Counts + 1). Silhouettes were obtained from www.phylopic.org. <em><strong>Supplementary Materials</strong></em> <strong>Table S1: </strong>Detailed information on the tissues sequenced or retrieved from public databases for the project. <strong>Table S2</strong>: BUSCO v5 scores of all transcriptomes based on a dataset of 9,226 single-copy orthologs conserved in over 90% of mammalian species (Manni et al. 2021). <strong><em>Zenodo supplementary files</em></strong> <strong>CHIAs_OG_tree-RAxML_EPA</strong><strong>.zip </strong>contains CHIA sequences (obtained from the OrthoFinder orthogroups and the sequences used to infer the chitinase genes evolution) and the corresponding ML tree. <strong>Chitinases_ancestral_sequences.zip </strong>contains the alignment of the ancestral sequence reconstruction inferred by RAxML-NG. <strong>Chitinases_gene_tree.zip</strong> contains input and output files corresponding to the chitinase gene tree presented in Figure 1: - mammalina_species_tree_input_Generax.newick = species tree used for the reconciliation with Generax - chitinase_gene_alignment_renamed_input_Generax.fasta = chitinase gene alignment with the sequence names renamed for Generax - chitinase_gene_alignment_not_renamed.fasta = chitinase gene alignment with the original sequence names (for information) - chitinases_gene_tree_sequences_renamed_input_Generax.newick = chitinase gene tree inferred with RAxML-NG and reconciled using the TreeRecs algorithm to find the optimal rooting scheme; this tree was used for Generax - reconciled_chitinase_genes_tree_output_Generax.newick = reconciled chitinase gene tree inferred by Generax and presented in Figure 1 <strong>Chitinases_expression.zip </strong>contains all orthogroup gene expressions plus chitinase gene expressions. <strong>Kallisto_abundances.zip</strong> contains the abundances estimated with kallisto for each organ of each species<em>.</em> <strong>Supplementary table figure 1B - BLAST</strong> <strong>results.xlsx</strong> contains BLAST results supporting sentence inferences. <strong>Transcriptome_assemblies.tar.gz</strong> contains the transcriptome assemblies obtain for each organ and species with Trinity.