Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

Eric D Stein,Peter E Miller,Erik M Pilgrim,Raphael D Mazor,Bryan P White,Dirk Steinke

doi:10.1371/journal.pone.0051273

Abstract

Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93–99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.

Highlights

Molecular methods such as DNA barcoding are emerging as a new tool for species identification [1] and, more recently, environmental biomonitoring [2,3,4]
There was no significant differences between treatments, replicate nested within treatment, or related interaction according to the nested Analysis of Variance (ANOVA) using either the partial barcode or full-barcode criteria (Table 5)
Sample preservation methods typically used in routine bioassessment do not appear to adversely affect the ability to obtain successful barcodes for most benthic macroinvertebrates

Summary

Introduction

Molecular methods such as DNA barcoding are emerging as a new tool for species identification [1] and, more recently, environmental biomonitoring [2,3,4]. Routine application of molecular methods to support species identification for biomonitoring will require that each step of sample collection, handling and processing be considered in terms of its potential to affect DNA integrity. Advantages of molecular methods in biomonitoring include the potential to increase the speed, accuracy and resolution of species identification and thereby support effective biomonitoring programs [5,6,7,8,9]. DNA is extracted from specimen tissue and sequenced for the 650-bp barcoding region of COI [1]. DNA from unknown specimens collected in benthic samples can be identified by comparing their barcode sequences to a reference library, such as the Barcode of Life Data Systems (BOLD) [10]

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Conclusion