Genetic Determination of a Cryptic Species in the Littoraria Genus With Whole-Genome Molecular Resolution

Abstract

Recognizing cryptic species is crucial for understanding global biodiversity. The intertidal snail Littoraria flammea is potentially a cryptic species of L. melanostoma widely distributed in the Northwest Pacific. However, the evidence from traditional morphology and single genetic markers is inconsistent. Our study combined quantitative morphological and whole-genome molecular data to clarify the phylogenetic relationship of three species (L. flammea, L. aff. melanostoma, and L. melanostoma). Three-dimensional models of shells revealed significant differences in morphology between L. flammea and L. melanostoma. Neutral SNPs indicated that individuals of L. flammea and L. melanostoma were in different clusters. The ratio of interspecific F _ST to intraspecific F _ST between L. flammea and L. melanostoma (16) was much larger than the lowest ratio (2.31) in six published genera with cryptic species in gastropods. Non-neutral SNPs disclosed divergence in functional genes related to reproduction and protein binding. The morphological and phylogenetic analyses corroborated the transitional status of L. aff. melanostoma. These results confirmed that the L. flammea snails north of the Yangtze River Estuary is a cryptic species of L. melanostoma, and allopatric speciation occurs in the L. melanostoma complex.

Keywords: Littoraria; Northwest Pacific; cryptic species; morphological differences; whole‐genome molecular data.

FIGURE 1

(a) The sampling locations of Littoraria flammea, L. aff. Melanostoma, and L. melanostoma in Rudong (RD), Yueqing (YQ), and Xiamen (XM), respectively. (b) The number of occurrences of each temperature exceeding the 99th percentile temperatures (T ₉₉) in summer (June–September) from 2011 to 2020 in each locality.

FIGURE 2

A 2D nonmetric multidimensional scaling (nMDS) scatter plot relating to shell morphology for three Littoraria species. Ellipses with different colors indicate 95% confidence ellipses for centroids of morphological parameters. The stress value, indicating the goodness of fit, is remarkably low at 0.014. The density profiles on the right and top of the figure highlight areas of maximum concentration of shell parameters along the different NMDS axes. Notably, shell parameters along the NMDS1 axis play a key role in distinguishing L. flammea from the other two snail species.

FIGURE 3

Phylogenetic relationships of three snail species. (a) The phylogenetic maximum likelihood (ML) tree depicts the relationships among three species: L. flammea (RD), L. aff. melanostoma (YQ), and L. melanostoma (XM). The support values on the branches are based on 1000 bootstrap replicates. (b) Pairwise values of F _ST based on neutral SNPs. (c) Discriminant analysis of principal components (DAPC) for the neutral SNPs. The axes are the first two linear discriminants or DA eigenvalues (901.920 and 3.274, respectively). Each point on the plot represents an individual snail. The analysis revealed two clusters: Cluster 1 consists of L. flammea individuals and cluster 2 includes L. aff. melanostoma and L. melanostoma individuals. (d) Bar graph of ADMIXTURE analysis. The bars represent the proportions of each individual's genetic ancestry from the two ancestral populations (i.e., blue and orange colors represent different ancestral populations).

FIGURE 4

The percentage of genotypes among 4898 outlier SNPs under diversifying selection in three Littoraria species. Heterozygote is defined by the presence of one reference allele and one alternative allele, while homozygote encompasses either two reference alleles or two alternative alleles.

FIGURE 5

Top 30 Gene Ontology (GO) terms annotated for 34 genes containing outlier SNPs in three Littoraria species.