Comparative transcriptomics reveals divergent paths of chitinase evolution 1 underlying dietary convergence in ant-eating mammals

37 Ant-eating mammals represent a textbook example of convergent morphological evolution. 38 Among them, anteaters and pangolins exhibit the most extreme convergent phenotypes with 39 complete tooth loss, elongated skulls, protrusive tongues, and powerful claws to rip open ant 40 and termite nests. Despite this remarkable convergence, comparative genomic analyses have 41 shown that anteaters and pangolins differ in their chitinase gene ( CHIA ) repertoires, which 42 potentially degrade the chitinous exoskeletons of ingested ants and termites. While the 43 southern tamandua ( Tamandua tetradactyla ) harbors four functional CHIA paralogs ( CHIA1 , 44 CHIA2 , CHIA3 , and CHIA4 ), Asian pangolins ( Manis spp.) have only one functional paralog 45 ( CHIA5 ). These two placental mammal lineages also possess hypertrophied salivary glands 46 producing large quantities of saliva to capture and potentially digest their social insect prey. 47 We performed a comparative transcriptomic analysis of salivary glands in 23 representative 48 species of placental mammals, including new ant-eating species and close relatives. Our 49 results on chitinase gene expression suggest that salivary glands play a major role in adapting 50 to an insect-based diet with myrmecophagous and insectivorous species highly expressing 51 CHIA paralogs. Moreover, convergently-evolved pangolins and anteaters express different 52 chitinases in their hypertrophied salivary glands and other additional digestive organs. CHIA5 53 is overexpressed in Malayan pangolin, whereas the southern tamandua exhibits high levels of 54 CHIA3 and CHIA4 expression. Overall, our results demonstrate that divergent molecular 55 mechanisms underlie convergent adaptation to the ant-eating diet in pangolins and anteaters. 56 This work highlights the role of historical contingency and molecular tinkering of the chitin- 57 digestive enzyme toolkit in this classical example of convergent evolution. 58


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The phenomenon of evolutionary convergence is a fascinating process in which distantly 60 related species independently acquire similar characteristics in response to the same selection 61 pressures. A fundamental question famously illustrated by the debate between Stephen Jay 62 Gould (Gould 2002) and Simon Conway Morris (Conway Morris 1999) resides in the relative 63 contribution of historical contingency and evolutionary convergence in the evolution of 64 biodiversity. While Gould (Gould 1990; argued that the evolution of species strongly (Pilosa), aardvarks (Tubulidentata), pangolins (Pholidota), and aardwolves (Carnivora). As a

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Mammalian chitinase gene family evolution 160 The reconciled maximum likelihood tree of mammalian chitinase genes is presented in Figure   161 1A. The evolution of this gene family constituted by nine paralogs is characterized by the 162 presence of numerous inferred gene losses with 384 speciation events followed by gene loss 163 and 48 gene duplications as estimated by the gene tree/species tree reconciliation algorithm 164 of GeneRax. At the base of the reconciled gene tree, we found the clade CHIA1-2/OVGP1 165 (optimal root inferred by the reconciliation performed with TreeRecs) followed by a 166 duplication separating the CHIT1/CHI3L1-2 and CHIA3-5 groups of paralogs. Within the 167 CHIT1/CHI3L clade, two consecutive duplications gave rise to CHIT1, then CHI3L1 and 168 CHI3L2. In the CHIA3-5 clade, a first duplication separated CHIA3 from CHIA4 and CHIA5, 169 which were duplicated subsequently. Marsupial CHIA4 sequences were located at the base of 170 the CHIA4-5 clade suggesting that this duplication might be specific to placentals. The 171 CHIA5 sequences of chiropterans were found at the base of the CHIA5 clade. The duplication 172 that gave rise to the CHIA4 and CHIA5 genes appears recent and specific to eutherians 173 (marsupials and placentals) since no other taxon was found within these clades. This scenario 174 of chitinase gene evolution is consistent with synteny analysis showing physical proximity of 175 CHIA1-2 and OVGP1 on one hand, and CHIA3-5 on the other hand (Fig. 1B), which implies 176 that chitinase genes evolved by successive tandem duplications. However, evidence of gene 177 conversion between the two more recent duplicates (CHIA4 and CHIA5) at least in some taxa 178 suggests that further data are necessary to fully disentangle the origins of these two paralogs The ancestral amino acid sequences of the nine chitinase paralogs have been reconstructed 204 from the reconciled mammalian gene tree and compared to gain further insight into the 205 potential function of the enzymes they encode (Fig. 2). The alignment of predicted amino   To test the hypothesis that salivary glands play an important functional role for the digestion 234 of ants and termites in ant-eating mammals, we analyzed gene expression profiles of the nine 235 chitinase paralogs revealed by the gene family tree reconstruction in 28 salivary gland 236 transcriptomes (Fig. 3)   Expression level is represented as log10 (Normalized Counts + 1).

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Chitinase gene expression in other digestive and non-digestive organs 267 The expression level of the nine chitinase paralogs in several organs was compared among 268 three species including an insectivorous xenarthran (the nine-banded armadillo; D. 269 novemcinctus) and the two main convergent myrmecophagous species (the southern anteater;

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T. tetradactyla, and the Malayan pangolin; M. javanica) (Fig. 4). This analysis revealed 271 . CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 30, 2022. ; https://doi.org/10.1101/2022.11.29.518312 doi: bioRxiv preprint marked differences in expression level of these genes among the three species and among 272 their digestive and non-digestive organs. CHIT1 was expressed in all tissues in M. javanica 273 and only in the spleen, testes, tongue and small intestine in T. tetradactyla, and in the 274 cerebellum, liver, lungs and salivary glands in D. novemcinctus. CHI3L1 was found to be 275 expressed in the majority of digestive and non-digestive tissues in all three species. CHI3L2 276 is non-functional or even absent in the genome of these three species and was therefore not   Expression level is represented as log10 (Normalized Counts + 1).

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. CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in In the nine-banded armadillo (D. novemcinctus), although only CHIA1 is pseudogenized and 288 therefore logically not expressed, we did not detect any expression of CHIA2, CHIA3, and 289 CHIA4 in the tissues studied here, and CHIA5 was only weakly expressed in one spleen  and CHIA2 genes. In mammals, OVGP1 has a role in fertilization and embryonic 318 . CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in However, different name aliases for OVGP1 include Mucin 9 and CHIT5 320 (www.genecards.org), which suggests a possible digestive function. This result was further 321 confirmed by synteny analyses suggesting a common origin by tandem duplication for 322 CHIA1-2 and OVGP1 within the conserved chromosomal cluster also including CHIA3-5 and 323 CHI3L2 (Fig. 1B). The comparison of ancestral amino acid sequences of the nine chitinase 324 paralogs revealed differences in their ability to bind and degrade chitin (Fig. 2), suggesting

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. CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 30, 2022. ; https://doi.org/10.1101/2022.11.29.518312 doi: bioRxiv preprint Since the presence of enlarged salivary glands is a hallmark of convergent ant-eating 387 mammals ensuring massive production of saliva to help catch and potentially digest prey, we 388 first investigated chitinase gene expression in mammalian salivary glands. Our comparative 389 transcriptomic study encompassing a diversity of species with different diets revealed that in CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 30, 2022. ; https://doi.org/10.1101/2022.11.29.518312 doi: bioRxiv preprint physiology by confirming its ubiquitous expression in all major digestive tissue types 421 (tongue, salivary glands, stomach, pancreas, liver, and large and small intestines) (Ma et al.  However, the comparison with the two myrmecophagous species seems to fit well with its 446 less specialized insectivorous diet and actually further underlines the contrasted specific use       CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 30, 2022. ; https://doi.org/10.1101/2022.11.29.518312 doi: bioRxiv preprint Transcriptomes from additional organs -Tissue biopsies from eight additional organs (testis, 608 lungs, heart, spleen, tongue, stomach, liver, and small intestine) were sampled during 609 dissections of three individuals of southern tamandua (T. tetradactyla; Table 1). Total RNA 610 extractions from these RNAlater-preserved tissues, RNA-seq library construction, and 611 sequencing were conducted as described above resulting in 12 newly generated  (Table 1).              Yates AD, Achuthan P, Akanni W, Allen James, Allen Jamie, Alvarez-Jarreta J, Amode MR,   ψ ψ ψ . CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 30, 2022. ; https://doi.org/10.1101/2022.11.29.518312 doi: bioRxiv preprint