# Morpho‐Molecular Identification of Common Freshwater Loaches Collected From Different Ecosystems of Bangladesh

## TL;DR

This study identifies seven freshwater loach species in Bangladesh using both physical traits and DNA analysis to aid conservation efforts.

## Contribution

The study confirms seven loach species using morphology and molecular markers, revealing differences in genetic diversity between COI and RAG1 genes.

## Key findings

- Morphological and molecular analysis confirmed the existence of seven loach species.
- RAG1 gene showed lower intraspecific diversity compared to COI gene.
- Phylogenetic analysis grouped species into distinct clades with strong support.

## Abstract

The biodiversity of hill stream fishes, especially loaches, in Bangladesh is declining, despite limited exploration. Conservation strategies can flourish through the effective identification of species. Ambiguous loaches were collected to resolve taxonomic confusion. Morphology was analyzed through morphometric and meristic traits, with mitochondrial COI and nuclear RAG1 genes as molecular markers. Sixteen morphotypes are found in 60 specimens, and the molecular study confirmed the existence of seven species. Furthermore, the species delimitation method ASAP suggests seven species according to the best partition. A low level of intraspecific diversity is found in the RAG1 gene analysis compared with the COI gene. The lowest interspecific genetic divergence (3.7%) was observed between
Botia lohachata
and
Botia rostrata
, whereas Canthophrys gongota showed the highest interspecific genetic divergence (24.45%) with
B. lohachata. A potential barcoding gap of 2.52% in the COI gene is typically the threshold for distinguishing intra‐species from inter‐species comparisons. In the maximum likelihood phylogenetic tree of both COI and RAG1 gene sequences, the species are divided into distinct clades with high bootstrap support, and specimens within the same species do not converge. Morphology, along with the molecular data of this study, will strengthen conservation efforts for loaches.

Taxonomic identification of seven morphologically ambiguous freshwater loach species of Bangladesh has been confirmed through morphological study as well as analyzing mitochondrial and nuclear DNA sequences. A relatively low level of intraspecific genetic diversity has been found in the nuclear RAG1 gene compared to the mitochondrial COI gene of the loaches.