# Morphological Differentiation Among Three Mitochondrial Lineages of Hydrobioides nassa Theobald, 1865 (Gastropoda: Bithyniidae) from Thailand

## TL;DR

This study shows that a Thai freshwater snail species, Hydrobioides nassa, actually consists of three genetically distinct groups with subtle physical differences.

## Contribution

The study reveals hidden genetic diversity within Hydrobioides nassa using combined molecular and morphological approaches.

## Key findings

- Hydrobioides nassa consists of three genetically distinct mitochondrial lineages.
- Subtle but consistent morphological differences support the genetic separation of the lineages.
- Combining DNA and morphology improves species identification in morphologically similar snails.

## Abstract

Freshwater snails are often difficult to identify because many species look very similar, even when they are genetically distinct. In this study, we examined the freshwater snail Hydrobioides nassa from Thailand to better understand its species boundaries. By combining DNA sequence data with detailed observations of shell and anatomical features, we found that H. nassa consists of three genetically distinct lineages. Although these lineages show only subtle morphological differences, these differences are consistent and support their genetic separation. Our findings demonstrate that relying on morphology alone may overlook hidden diversity and highlight the value of integrating molecular and morphological approaches to improve species identification and our understanding of freshwater snail biodiversity.

The identification of species complexes in freshwater snails remains challenging due to limited diagnostic morphological characters and incomplete taxonomic knowledge in many taxa. Within the family Bithyniidae, species have traditionally been classified using shell morphology and genital anatomy to distinguish intraspecific variation from interspecific differences. However, extensive morphological plasticity has hindered reliable species delimitation, and the presence of cryptic diversity further complicates taxonomy. Recent DNA barcoding studies of Hydrobioides have provided evidence of such cryptic diversity, highlighting the need for taxonomic reassessment within the genus. In the present study, we examined morphological variation in Hydrobioides nassa from Thailand in conjunction with mitochondrial DNA sequence data. Molecular phylogenetic analyses based on cytochrome c oxidase subunit I (cox1) sequences revealed three well-supported genetic lineages within H. nassa, accompanied by high levels of pairwise genetic divergence. Morphological comparisons of shell, operculum, and radular characters further supported differentiation among these lineages, although some characters showed overlap. While Hydrobioides has previously been regarded as comprising a single morphologically defined species, our results demonstrate that H. nassa represents a complex of genetically distinct lineages with subtle but consistent morphological differences. This study highlights the importance of integrating molecular approaches with traditional morphological analyses to improve taxonomic resolution and to better understand biodiversity within freshwater snail groups exhibiting cryptic diversity.