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