Signatures of Divergence, Invasiveness, and Terrestrialization Revealed by Four Apple Snail Genomes

Abstract

The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history, and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here, we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, P. maculata, and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquisition of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.

Keywords: Hox genes; gastropod; gene duplication; genomics; interchromosome rearrangement; mollusc.

Fig. 1.

Native ranges of the four ampullariid species included in this study, and pictures of their adults and egg masses. Dashed lines demarcate the distribution overlap of Pomacea spp. The pink-reddish calcareous egg masses of the two Pomacea species are deposited terrestrially, whereas the white gelatinous egg masses of Lanistes nyassanus and Marisa cornuarietis are deposited underwater.

Fig. 2.

Maximum-likelihood phylogenetic tree, constructed using RaxML version 8.2.4 (Stamatakis 2014) with the GTR + Γ model assigned to each partition. The tree shows the relationships and divergence times of 15 molluscan species, with 2 annelids and 1 brachiopod served as outgroups. Blue lines indicate the 95% confidence interval on estimated node depths. The names of four ampullariid species used in this study are written in bold font. Red circles indicate that the nodes are constrained with fossil calibration data (see Supplementary Material for details). All nodes have a bootstrap support of 100.

Fig. 3.

Schematic illustration of Hox and ParaHox gene clusters in four ampullariid species and five other selected molluscans. Data for non-ampullariid genomes were adopted from Wang et al. (2017). Gene names are labeled on top of the colored graphs, and species names are labeled at the left of each row. Genes located on the same scaffold or chromosome are connected with a line but the line length is not proportional to their sequence length. The symbol “//” indicates a break between different scaffolds. Transcription direction, when available, is indicated by a bullet head.

Fig. 4.

Dotplot showing the conserved macrosynteny between the 14 chromosomes of Pomacea canaliculata and the 17 bilaterian ancient linkage groups (ALGs). Pomacea canaliculata has a chromosome conservation index (CI) of 0.58, which is smaller than that of the scallop Patinopecten yessoensis (0.89), but larger than those of all other bilaterians (0.00–0.42) whose chromosome-level assembly has been examined by Wang et al. (2017), including seven protostomes (three lophotrochozoans and four ecdysozoans) and four deuterostomes. The analysis and the resultant plot were made using MCScanX (Wang et al. 2012).

Fig. 5.

Unrooted maximum-likelihood tree showing the massive expansion of the G-protein coupled receptor (GPCR) GRL101 gene family in Ampullariidae. Among the 18 lophotrochozoan genomes examined, there are 299 sequences from the 4 ampullariids. Only 11 sequences are available for other gastropods (1 for Biomphalaria glabrata, 9 for Aplysia californica, 1 for Lottia gigantea), 15 for bivalves (1 from each of Lo. gigantea, Patinopecten yessoensis, and Crassostrea gigas, 3 from each of Pinctada fucata and Bathymodiolus platifrons, 6 from Modiolus philippinarum), and 2 for the annelid Capitella teleta. Each gene is labeled with a prefix of either the genus name for non-ampullariids or the first letter of the genus name and the first two letters of the species name for ampullariids (e.g., Pca for Pomacea canaliculata). Numbers on the nodes are bootstrap values (>50%).

Fig. 6.

Maximum-likelihood phylogenetic tree of calcium binding proteins (CaBP) in 18 lophotrochozoans, with numbers on nodes showing bootstrap values (>50%) (A), and their expression in different early stages and adult tissues of ampullariids (B). Note that in Pomacea canaliculata, many different development stages and different tissues are available, but in the other species, only AG and OT are available. Abbreviations of developmental stages/tissues: E5-12, day 5–12 embryo; J, juvenile; AG, albumen gland; DG, digestive gland; PK, posterior kidney; S, stomach; T, testis; L, lung; G, gill, M, mantle; F, Foot; OT, Other tissues (pooled tissues of DG, F, M, and T for P. maculata, Marisa cornuarietis, and Lanistes nyassanus, except the latter without testis data). Names of non-ampullariids: Aplysia californica, Biomphalaria glabrata, Haliotis discus hannai, Crassostrea gigas, Modiolus philippinarum, Patinopecten yessoensis, Lingula anatina, and Hellobdella robusta.

Fig. 7.

Phylogeny and expression of homologues of PV2 subunits in lophotrochozoans. (A) MACPF-like genes and (B) tachylectin-like genes. Ampullariid genes are colored by species, whereas albumen gland overexpressed genes are highlighted with an orange box. Numbers on nodes are bootstrap values (>50%). Gene expression levels are in logarithmic scale. E5-12, 5- to 12-day embryo; J, juvenile; AG, albumen gland; DG, digestive gland; F, Foot; PK, posterior kidney; L, lung; M, mantle; S, stomach; T, testis; OT, other tissues. Of the 14 non-ampullariid examined, only the following have MACPF-like genes, but none of them have a tachylectin-like gene: Aplysia californica; Biomphalaria glabrata; Radix auricularia; Haliotis discus hannai; Modiolus philippinarum.

Fig. 8.

Schematic illustration of the evolution of the MACPF–tachylectin complex in ampullariids. Based on the genomic arrangements of the MACPF and tachylectin genes, the model proposes that a single copy of MACPF–tachylectin complex was present in the common ancestor of ampullariids. Only in the two species of Pomacea has it become highly diversified, with both MACPF-tachylectin genes that are conserved across Ampullariidae, and multiple Pomacea specific MACPF-tachylectin genes that were generated by tandem duplication. The final 1-MACPF and 2-tachylectin configuration is exclusively expressed in the albumen gland of Pomacea and the proteins detected in their egg PVF. Numbers below and inside gene diagram boxes are scaffold numbers and gene numbers in the scaffold, respectively. For instance, Lanistes nyassanus contains a MACPF gene (Lny_22924_0.25) and a tachylectin gene (Lny_22924_0.27) in scaffold Lny_22924. For P. canaliculata, the chromosome numbers are shown above the gene diagram boxes.