Using eDNA to biomonitor the fish community in a tropical oligotrophic lake

Abstract

Environmental DNA (eDNA) is an effective approach for detecting vertebrates and plants, especially in aquatic ecosystems, but prior studies have largely examined eDNA in cool temperate settings. By contrast, this study employs eDNA to survey the fish fauna in tropical Lake Bacalar (Mexico) with the additional goal of assessing the possible presence of invasive fishes, such as Amazon sailfin catfish and tilapia. Sediment and water samples were collected from eight stations in Lake Bacalar on three occasions over a 4-month interval. Each sample was stored in the presence or absence of lysis buffer to compare eDNA recovery. Short fragments (184-187 bp) of the cytochrome c oxidase I (COI) gene were amplified using fusion primers and then sequenced on Ion Torrent PGM or S5 before their source species were determined using a custom reference sequence database constructed on BOLD. In total, eDNA sequences were recovered from 75 species of vertebrates including 47 fishes, 15 birds, 7 mammals, 5 reptiles, and 1 amphibian. Although all species are known from this region, six fish species represent new records for the study area, while two require verification. Sequences for five species (2 birds, 2 mammals, 1 reptile) were only detected from sediments, while sequences from 52 species were only recovered from water. Because DNA from the Amazon sailfin catfish was not detected, we used a mock eDNA experiment to confirm our methods would enable its detection. In summary, we developed protocols that recovered eDNA from tropical oligotrophic aquatic ecosystems and confirmed their effectiveness in detecting fishes and diverse species of vertebrates.

Fig 1. Sampling locations.

The map was prepared using QGIS v 2.8.2 under the INEGI (Instituto Nacional de Geografía Estadística e Informática) free documentation license from topographic chart E16A66 (Scale 1:50,000) (www.inegi.org.mx/app/areasgeograficas/?ag=23).

Fig 2. Experimental design for the water and sediment samples.

Fig 3. Number of species detected versus the number of samples analyzed for water, sediments, and combined.

Species accumulation curves were calculated in EstimateS software with 1000 randomizations and classic formula for Chao 1 and Chao 2.

Fig 4. Number of vertebrate species recovered in eDNA from each sampling event at each collection site.

Fig 5. Species recovery and DNA concentration for each sampling event and treatment.

A–Number of vertebrate species recovered for each sampling event and treatment (only PGM data for fishes are shown, circle size indicates average read count per sampling event). B–DNA concentration of sediment and water samples (one replicate per sampling event) measured on Qubit. Whiskers correspond to data within 1.5x of interquartile range (IQR).

Fig 6. Percentage of total read coverage for each species recovered in water samples from April 2016 and mock experiment, analyzed with Ion Torrent PGM and S5 instruments.

Fig 7. Top hit process IDs mapped on the Neighbor-Joining tree for fish taxa visualized in iTOL.

Blue–species previously reported from the Lake Bacalar region; red–species new to Lake Bacalar); orange–species complexes lacking resolution with COI: 1 –Bramocharax-Astyanax complex, 2 –Cyprinodon simus/beltrani; green–Pterygoplichthys from mock eDNA experiment.