eDNA metabarcoding as a means to assess distribution of subterranean fish communities: Iranian blind cave fishes as a case study

Abstract

One of the most important steps in conservation of the subterranean life forms is to decipher their distribution and ecology, which is challenging using traditional approaches. Development of an environmental DNA (eDNA) assay provides an efficient means for discovering and monitoring subterranean life forms. In this study, the distribution of three Iranian blind cave fish species (blind Iran cave barb Garra typhlops, blind Lorestan cave barb Garra lorestanensis, and blind cave loach Eidinemacheilus smithi) was assessed using 12S rRNA eDNA metabarcoding performed using MiFish‐U PCR primers and preliminary species distribution modeling (SDM) using bioclimatic data. The majority of sampling localities with positive detection of cave barb eDNA fall within suitable habitats in the Zagros Mountains of Iran. Our results revealed that Lorestan and Iran cave barbs have differential distribution patterns, with some extent of habitat overlap in the vicinity of the originally discovered cave barb locality. According to the observed distribution patterns, the blind Lorestan cave barb and cave loach are mostly distributed in habitats close to the Sezar River (Dez River drainage, Iran), and the blind Iran cave barb is distributed towards the west and probably in a few springs in the Karkheh River drainage. Our data support the previously proposed distribution pattern for the cave barbs, in which the species show partial niche separation and reproductive isolation, with the Lorestan cave barb being a water flow‐dependent species and the Iran cave barb being a generalist species preferring variable flow rates. We showed eDNA metabarcoding to be a useful approach for ecological surveys of subterranean fish biodiversity with implications for conservation.

eDNA metabarcoding as a means to track distributions of different fish species in a protected area

“…While genetic studies on fish tissues are routinely performed in other sections of this laboratory, the eDNA facilities are isolated and carefully disinfected. DNA extraction was performed using the DNeasy® Blood & Tissue Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany), with some modifications (Hashemzadeh Segherloo, Tabatabaei, Abdolahi‐Mousavi, et al., 2022). All instruments used in eDNA extraction were sterilized with bleach and UV light to avoid contamination.…”

“…Among its advantages, eDNA is very sensitive, nondestructive, and noninvasive compared to conventional methods. In particular, the eDNA approach has been successfully applied to detect low‐abundance and introduced species and to assess the relative abundance of different fish species (Hashemzadeh Segherloo, Tabatabaei, Abdolahi‐Mousavi, et al., 2022; Jensen et al., 2022; Thomas et al., 2020; Xia et al., 2021) in ecosystems where application of conventional approaches such as nets and electrofishing are difficult, impossible, or less efficient (Casselman et al., 1990).…”

Rapid detection of subterranean fauna from passive sampling of groundwater eDNA

“…Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach in terrestrial, marine, and aquatic biodiversity assessments with applications including, biosecurity (Kim et al., 2018), conservation (Harper et al., 2019), and cryptic taxa detection (Nester et al., 2020), with recent studies effectively using this method to assess subterranean biodiversity (Hashemzadeh Segherloo et al., 2022; van der Heyde et al., 2023; West et al., 2020). This tool uses whole and fragmented DNA shed into the environment, though scales, skin cells, saliva, feces, mucous, or other material (reviewed by Thomsen & Willerslev, 2015), which can then be extracted from a substrate (e.g.…”

“…Environmental DNA (eDNA) metabarcoding is a sensitive and widely used approach in terrestrial, marine, and aquatic biodiversity assessments with applications including, biosecurity (Kim et al, 2018), conservation (Harper et al, 2019), and cryptic taxa detection (Nester et al, 2020), with recent studies effectively using this method to assess subterranean biodiversity (Hashemzadeh Segherloo et al, 2022;van der Heyde et al, 2023;West et al, 2020).…”

Two different mental disc forms in the Tashan caveGarra:different species or ecotypes?

“…Based on mental disc characters, two phylogenetic groups had been considered for Garra spp., which include the Garra variabilis and Garra rufa clades (Hashemzadeh Segherloo et al 2017); the members of G. rufa clade usually have a mental disc and the members of G. variabilis group lack the disc. In the clade with a mental disc ( G. ruffa ), the disc character has been used for description of the Lorestan cave barb Garra lorestanensis , which is sympatric with the blind Iran cave barb Garra typhlops in the Zagros Mountains of Iran (Hashemzadeh Segherloo et al 2021; Musavi-Sabet and Eagderi 2016). This difference in mental disc of these species was also supported by mtDNA (Hashemzadeh Segherloo et al 2012, 2017; Farashi et al 2014) and genomic data (Hashemzadeh Segherloo et al 2018).…”