A trio of tiny snails collected along China’s coasts and waterways has provided scientists with new genetic clues to one of evolution’s most remarkable ecological stories: how members of the same gastropod family repeatedly moved between the sea, brackish estuaries and freshwater streams. By sequencing the complete mitochondrial genomes of three neritid species, researchers have expanded the molecular record for a family whose members are found clinging to tropical rocks, grazing on mangrove mudflats and crawling across seagrass blades. The study also exposes a persistent problem in modern biodiversity science: public DNA databases can contain mislabeled specimens, and those errors may quietly distort evolutionary trees.
Neritidae, commonly known as nerites, includes roughly 300 living species. Their shells vary from smooth and emerald-green to boldly striped or limpet-like, but the family’s most striking feature is its ecological breadth. About two-thirds of neritid species live in freshwater or brackish environments, while the rest are marine. Some species spend their adult lives in rivers but produce larvae that develop in the ocean, a life cycle known as amphidromy. Others are tied to intertidal habitats, mangrove forests or seagrass meadows. This repeated movement across environmental boundaries makes neritids useful natural experiments for studying adaptation, dispersal and the evolutionary transition from marine to freshwater life.
The new study focuses on three species from China: the seagrass-dwelling marine snail Smaragdia rangiana, the brackish Neripteron pileolus and the brackish-to-freshwater Vittina cumingiana. S. rangiana was collected from seagrass in a lagoon in Lingshui, Hainan Province, in June 2024. Its living shell is bright green, marked by white blotches or bars edged with black, an appearance that helps explain its association with the genus Smaragdia. N. pileolus was found on intertidal rocks near Shanghai in October 2024. V. cumingiana came from the Lingshui River in Hainan in April 2024 and is notable not only for its striking black shell with fine yellow zigzag stripes, but also for its popularity in the aquarium trade.
The researchers confirmed the snails’ identities using a combination of shell morphology and molecular evidence. They examined diagnostic features such as the shape of the spire, the structure of the shell opening and the number and arrangement of teeth along the columella, the central pillar inside the aperture. They then compared mitochondrial sequences with records in the National Center for Biotechnology Information database using BLAST, a tool that identifies similar DNA sequences, and reconstructed phylogenetic trees from the mitochondrial COI and 16S ribosomal RNA genes. This two-stage approach is important because shell characters can overlap among closely related species, while a DNA sequence can reveal whether an apparent match is biologically plausible.
To obtain the genomes, the team extracted DNA from foot tissue, which reduces contamination from material in the digestive tract, and used genome skimming on an Illumina NovaSeq X Plus platform. Genome skimming generates enormous numbers of short DNA fragments from all genetic material in a sample; because mitochondrial DNA occurs in many more copies per cell than most nuclear genes, mitochondrial genomes can often be assembled from this mixture without first isolating the organelle. The sequencing runs produced between 3.86 and 4.44 gigabytes of raw data per specimen. After filtering low-quality reads, the scientists assembled the mitochondrial chromosomes and manually checked automated annotations.
The resulting genomes were compact but information-rich: 15,824 base pairs for S. rangiana, 15,794 for N. pileolus and 15,681 for V. cumingiana. Each contained the standard set of 37 mitochondrial genes found in most animal mitochondria: 13 protein-coding genes involved primarily in energy production, two ribosomal RNA genes and 22 transfer RNA genes, together with a large non-coding region. The gene order was identical across all three species and consistent with previously studied neritids. That conservation is biologically useful for identifying broad similarities among species, but it also limits the amount of evolutionary information that gene arrangement itself can provide.
The nucleotide composition of the genomes showed a familiar mitochondrial bias. Between 62.8 and 66.0 percent of the bases were adenine or thymine, with N. pileolus having the highest A+T content. The researchers found negative AT-skew values, meaning thymine slightly exceeded adenine on the strand analyzed, and positive GC-skew values, indicating more guanine than cytosine. These asymmetries can arise from strand-specific mutation and DNA-replication processes. The protein-coding genes also showed unequal base composition: ATP8 was particularly rich in A and T, reaching 72.1 percent in N. pileolus, whereas COX3 was comparatively more balanced. All protein-coding genes began with the conventional ATG start codon, while their stop signals included TAA, TAG and the abbreviated stop codon T.
The genetic analyses revealed that not all mitochondrial genes evolve at the same speed. Nucleotide diversity, a measure of sequence variation among sampled neritids, was lowest in COX1 and COX2 and highest in ND6, ND2 and ND4. The ratio of nonsynonymous to synonymous substitutions, known as Ka/Ks, was below one for every protein-coding gene. This pattern indicates purifying selection: mutations that change the amino-acid sequence of an essential mitochondrial protein are generally removed by natural selection more often than mutations that do not alter the protein. COX1, CYTB, ND1 and ATP6 were especially conserved, while ATP8, ND2 and ND6 showed comparatively faster evolution and may offer more resolution for distinguishing closely related lineages.
To place the new genomes in an evolutionary context, the researchers compared them with publicly available mitochondrial data and mitochondrial genes extracted from transcriptomes. They began with 62 Neritidae records in GenBank, removed redundant sequences and retained 44 unique mitogenomes. They also assembled the 13 protein-coding genes and two ribosomal RNAs from transcriptomic data, selecting one representative sequence per species. The team tested several data treatments, including all codon positions, only the first and second positions, amino-acid translations and combinations that included ribosomal RNA. Maximum-likelihood and Bayesian analyses produced broadly similar results, although the choice of dataset affected the precise branching order more strongly than the statistical method.
Across the principal tree, Neritidae divided into two major lineages. One consisted solely of the marine genus Nerita and corresponded to the subfamily Neritinae. The second contained the other sampled genera, including Theodoxus, Neritona, Smaragdia, Neritina, Septaria, Neripteron, Vittina, Vitta, Puperita and Clithon. This larger lineage corresponds to Neritininae, whose members are predominantly freshwater or brackish. All three newly sequenced species fell within this second group. S. rangiana clustered with other Smaragdia sequences, V. cumingiana formed a distinct branch near Vitta and Puperita, and N. pileolus grouped closely with sequences assigned to Neripteron or to the historical name Neritina violacea.
That last result points to a taxonomic complication with consequences far beyond the names printed on museum labels. The newly sequenced N. pileolus showed more than 99 percent identity in COI, 16S and mitochondrial-genome comparisons to some records labeled Neritina violacea. Yet another authenticated N. violaceum sequence occupied a different position. The researchers suggest that at least some database records may actually represent N. pileolus, or may have been deposited under an older synonym. The problem is difficult to resolve because one relevant record lacks a locality, voucher information and images. The authors therefore present the proposed correction as provisional rather than definitive, emphasizing that re-examination of original specimens or fresh, well-documented collections will be needed.
A similar warning emerged from a transcriptome record labeled “Clithon parvulum.” That sequence fell inside the Smaragdia group rather than with other Clithon species. Its associated museum metadata said the specimen had been collected from seagrass blades, a habitat characteristic of Smaragdia but unusual for a freshwater Clithon. The shell had been broken during tissue extraction and no photographs were available, making direct confirmation impossible. The sequence may therefore represent a Smaragdia specimen misidentified as Clithon, perhaps reflecting the complicated history of names that have been applied inconsistently to seagrass-associated nerites.
The findings reinforce both the promise and the limits of mitochondrial phylogenetics. Complete mitochondrial genomes offer thousands of aligned characters—far more than a single marker such as COI—and can clarify relationships when taxon sampling is adequate. But mitochondrial DNA is inherited as a single linked unit, so it represents only one genetic history and may not capture hybridization, incomplete lineage sorting or gene flow between populations. In this study, mitochondrial gene order was completely conserved, and relationships among some genera remained sensitive to the dataset used. One outgroup, the hydrocenid Georissa bangueyensis, shifted position among analyses, raising the possibility of long-branch attraction, an artifact in which rapidly evolving lineages appear related because they independently accumulate similar changes.
The broader evolutionary question—how often neritids crossed between marine and freshwater environments—therefore remains open. Smaragdia is unusual because it is the only completely marine genus embedded in the predominantly brackish and freshwater Neritininae lineage, potentially indicating a return to the sea. Earlier work has proposed numerous transitions among marine, brackish and freshwater habitats across the wider group. Testing those scenarios will require complete sampling of all 16 recognized genera, more authenticated specimens and nuclear genomic data capable of revealing evolutionary histories that mitochondrial DNA alone cannot show.
For now, the three new genomes fill important gaps. S. rangiana represents the first complete mitochondrial genome reported for Smaragdia, while V. cumingiana is the first for Vittina. The sequences, deposited in GenBank under PX869896, PX869897 and PX869898, provide reference points for identifying future specimens and checking existing records. They also offer a practical lesson for the age of biodiversity genomics: a DNA sequence is only as reliable as the specimen and information attached to it. Clear photographs, locality data, diagnostic descriptions and preserved vouchers may seem like bureaucratic details, but together they determine whether genetic databases become a dependable map of evolution—or a maze of inherited mistakes.
Subject of Research: Complete mitochondrial genomes and phylogenetic relationships of three neritid gastropods from China
Article Title: Complete mitochondrial genomes of three neritid species and their phylogenetic implications
Article References: Rao, Yiyong, Yuanzheng Meng, Sheng Zeng, and Deyuan Yang. “Complete mitochondrial genomes of three neritid species and their phylogenetic implications.” Original research page not provided in the source material.
Image Credits: AI Generated
DOI: 10.1002/ece3.73888
Keywords: Neritidae, mitochondrial genomes, gastropod phylogeny, freshwater snails, brackish-water snails, Smaragdia rangiana, Neripteron pileolus, Vittina cumingiana, DNA barcoding
Tags: amphidromous life cycle in neritidsbiodiversity database accuracy issuesecological breadth of neritid snail speciesfreshwater and marine gastropodsgastropod dispersal mechanismsgastropod ecological adaptationgastropod evolutionary relationshipsimpact of DNA mislabeling on phylogeneticsmitochondrial DNA in evolutionary studiesmolecular phylogenetics of neritidsneritid species distributionNeritidae mitochondrial genome sequencing
