Abstract
Macroinvertebrates have been recognized as key ecological indicators of environmental and biodiversity assessment in aquatic ecosystems. However, species identification of macroinvertebrates (especially aquatic insects) proves to be very difficult due to lack of expertise. In this study, we evaluated the feasibility of DNA barcoding for the classification of benthic macroinvertebrates and investigated the genetic differentiation in nine taxonomic groups (Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera, Odonata, Mollusca and Annelida) from four large transboundary rivers of northwest China, and further explored its potential application to environment and biodiversity assessment. A total of 1227 COI sequences, belonging to 189 species, 122 genera and 59 families were obtained. The barcode gap analysis supported species status using the barcode gap approach. Meanwhile, NJ phylogenetic trees showed that all species group into single-species representing clusters whether from the same population or not, except two species (Polypedilum. laetum andPolypedilum. bullum ). The ABGD analysis divided into 190 OTUs (P = 0.0599) and BIN analysis generated 201 different BINs. Phylogenetic diversity (PD) metrics can reflect environmental stress and serve as a metrics of Index of Biotic Integrity (IBI) to reflect the degree of disturbance in river systems.
Keywords Benthic macroinvertebrates; Species identification; Environmental assessment; DNA barcoding;
Introduction
Macroinvertebrates (e.g., aquatic insects) have been considered as one of primary components in aquatic ecosystems and a key food resources for higher trophic levels. They provide unique ecosystem services in nutrient cycling as well as energy flow (Sivaramakrishnan et al., 2014). Macroinvertebrates have been frequently utilized to monitor the water quality worldwide (Chandler et al., 1970; Helson et al., 2013), especially for those water bodies (e.g., rivers and lakes) intervened by anthropogenic activities (Wang et al., 2006; Zhang et al., 2011). However, these ecological functions depend, to a larger extent, on the absolutely accuracy and precision of species identification (Frezal et al., 2008; Macher et al., 2016). Traditional taxonomy always relies on the morphology-based traits to identify a specific species, but this approach proves to be difficult, time-consuming and costly (Wong et al., 2014). Despite their important roles on water quality assessment, morphological delineation of aquatic insects (especially females and immatures) remains a challenging task, as some important traits for reliable identification are only available in a single sex or at a certain stage of development (Zhou, Adamowicz, Jacobus, DeWalt, & Hebert, 2009; Zhou et al., 2011). For example, identification of Hydropsychidae caddisflies at the species level is only possible in adult organisms (Zhou et al., 2007). Unlike those model species such as butterflies (Dinca et al., 2015) and spiders (Blagoev et al., 2016), the information on aquatic insects is quite limited due to the complex life history. Moreover, despite the increasing demand for well-trained taxonomists to support the assessment of aquatic ecosystems, the number of ecologists and researchers with taxonomic expertise are decreasing (Wheeler, 2014). Accordingly, such subjective or objective factors may lead to an inaccurate description of macroinvertebrates in terms of traditional classification.
To circumvent the above-mentioned hurdles associated with morphology-based species identification, DNA barcoding emerged as a new tool that utilizes short standard sequences of the genome for species identification and classification, based on the mitochondrial gene cytochrome c oxidase subunit I (COI) (Hebert et al., 2003; Moriniere et al., 2017). DNA barcoding has the potential to identify cryptic species and highlight the diversity of macroinvertebrates in aquatic ecosystems (Bucklin et al., 2010). This could provide valuable information on the selection of taxa for further analyses (Hajibabaei et al., 2007) and make it possible to associate with all life history stages and genders (Casiraghi et al., 2010). Thus, DNA barcoding offsets some shortcomings in traditionally morphological delineation (Kress et al., 2012). Unknown specimens could be identified by comparing their DNA barcode sequences with a barcode reference library (Moriniere et al., 2017). Previous studies illuminated the great reliability of DNA barcoding in species identification of aquatic insects, e.g., Ephemeroptera (Ball et al., 2005; Curt et al., 2012), Plecoptera and Trichoptera (Gillet et al., 2014) and Diptera (Brodin et al., 2013; Hernandez et al., 2014; Hunter et al., 2008). However, these studies focused primarily on the application of DNA barcoding in 1-3 orders, and few studies considered all the taxa of macroinvertebrates in a specific ecosystem and further applied to the environmental and biodiversity assessment.
China spans a great range from east to central Asia and stretches across Eurasia (e.g., neighboring to Russia), and exhibits very different characteristics of climate and geography. Thus, great differences and high diversity in aquatic biota occur in China. Until now, DNA barcoding has been applied to some organisms, e.g., amphibians (Che et al., 2012), birds (Yang et al., 2014), plants (Huang et al., 2015), Noctuoidea moths (Zheng et al., 2015), molluscs (Andrea et al., 2016), crickets (Hawlitschek et al., 2016), herpetofauna (Hawlitschek et al., 2016), fish (sonnet et al., 2018), spiders (Ivanov et al., 2018), but rarely focused on aquatic invertebrates (Zhou et al., 2009b; Zhou et al., 2009a), especially aquatic insects. In this study, we attempted to test the feasibility of DNA barcoding in the classification of benthic macroinvertebrates in the transboundary rivers and their affiliated water bodies of northwest China. The main objectives of this study were to (1) establish a DNA barcoding reference library for benthic macroinvertebrates in northwest China; (2) apply phylogenetic diversity using community data to reflect the degree of disturbance in these rivers.
Materials and Methods