Introduction
Benthic macrofauna are abundant in marine and freshwater ecosystems and
play vital roles in nutrient cycling and secondary production
(Snelgrove, 1997,
1998). For example, macrofauna decompose organic inputs by feeding
directly on organic matter, and they promote metabolism of pollutants by
bacteria through biological transport and by increasing the oxygen
supply in sediments
(Pant, Negi, &
Kumar, 2017; Sanz-Lázaro & Marín, 2011; Snelgrove, 1998). Because
macrofauna are important to ecosystem processes, can be identified by
microscopy, and have differing tolerances to enrichment gradients, they
have been used widely to assess environmental impacts associated with
human activities, including marine finfish farming
(Keeley, Forrest,
Crawford, & Macleod, 2012; Sanz-Lázaro & Marín, 2011), oil-drilling
(Denoyelle,
Jorissen, Martin, Galgani, & Mine, 2010), and mining
(Josefson, Hansen,
Asmund, & Johansen, 2008). Macrofauna assessments have also been used
to monitor environmental recovery from these activities
(Daan, Booij,
Mulder, & Weerlee, 1996; Machado et al., 2018; Zhulay, Reiss, & Reiss,
2015).
To date, characterization of macrofauna communities has been done by
traditional morpho-taxonomic identification, which is very time- and
labour-consuming and relies heavily on highly specialized taxonomists.
These factors intrinsically limit the speed and magnitude of sample
throughput that can be achieved by traditional taxonomic approaches
(Bourlat et al.,
2013; Danovaro et al., 2016; Goodwin et al., 2017; Ji et al., 2013;
Pawlowski et al., 2018). Furthermore, individuals that are damaged
during mechanical collection and sieving processes as well as with
poorly described life stages may be misidentified by visual
identification
(Danovaro et al.,
2016; Goodwin et al., 2017). Exacerbating these challenges is that
species that are difficult to distinguish by morpho-taxonomy are
commonly present in these communities
(Knowlton, 1993),
and if improperly identified could lead to underestimation of
biodiversity and/or misleading interpretations about indicator species
used for environmental assessment
(Dean, 2008;
Nygren, 2014).
DNA barcoding alleviates the reliance on morphological characters for
taxonomic identification and can offer several benefits including higher
taxonomic resolution of closely-related or cryptic species and the
ability to verify morphologically-identified taxa
(Nygren, 2014;
Westheide & Schmidt, 2003; Witt, Threloff, & Hebert, 2006). It can
also be used to characterize the taxonomic composition of opportunistic
indicator complexes
(Silva et al.,
2017). However, it is still relatively time- and cost-intensive due to
the one-by-one nature of the workflow: samples need to be manually
sorted into individual specimens and the cost of Sanger DNA sequencing.
Environmental DNA (eDNA) metabarcoding is a high-throughput, rapid,
cost-effective, and relatively new method of characterizing species
composition in complex environmental samples
(Shokralla, Spall,
Gibson, & Hajibabaei, 2012; Taberlet, Bonin, Zinger, & Coissac, 2018;
Taberlet, Coissac, Hajibabaei, & Rieseberg, 2012). It has been proven
to be useful for detecting invasive
(Klymus, Marshall,
& Stepien, 2017) and rare species
(Pikitch, 2018),
assessing biodiversity for conservation
(Lacoursière‐Roussel
et al., 2018; Stat et al., 2019), and monitoring environmental health
status (Laroche et
al., 2016; Pawlowski, Esling, Lejzerowicz, Cedhagen, & Wilding, 2014).
Environmental DNA metabarcoding has been used to characterize benthic
macrofauna responses to human activities associated with marine finfish
aquaculture
(Lejzerowicz et
al., 2015), offshore oil-drilling
(Lanzén, Lekang,
Jonassen, Thompson, & Troedsson, 2016), oil spills
(Xie et al.,
2018), and chemical contamination
(Xie et al.,
2017). It has the potential to supplement traditional morpho-taxonomic
analysis of macrofauna from sediments for environmental assessment and
biomonitoring
(Aylagas, Borja,
& Rodríguez-Ezpeleta, 2014; Pawlowski et al., 2018), and to increase
the spatial and temporal scales at which biomonitoring can realistically
be done (Gibson et
al., 2015). Available studies comparing eDNA metabarcoding with
traditional morpho-taxonomy have only used visual morpho-taxonomy of
macrofaunal individuals without the support of DNA barcoding for
resolving morphologically similar (or cryptic) species. This makes it
difficult to compare macrofauna inventories among studies, particularly
for taxonomic groups prone to cryptic speciation including Capitellidae,
a family that is especially relevant to benthic biomonitoring. In the
absence of an objective means of classifying species based on
morphology, there will inevitably be variation among studies as to how
taxa were classified when morpho-taxonomy alone is used.
Benthic organic enrichment is a significant environmental issue
associated with marine finfish aquaculture
(Forrest et al.,
2007; Wilson, Magill, & Black, 2009). It can influence faunal diversity
and abundance, causing population shifts towards opportunistic taxa
(Pearson &
Rosenberg, 1978) thereby eliminating a diverse community and the
functions originally provided by it within the ecosystem
(Snelgrove, 1997,
1998). As such, benthic impacts of finfish farming are subject to
regulatory control and routine monitoring in many countries
(Wilson et al.,
2009). In New Zealand, Norway, and Scotland, biological indices based on
morpho-taxonomic macrofauna data are implemented for regulatory purposes
(Keeley et al.,
2012; Maurer, Nguyen, Robertson, & Gerlinger, 1999). To date, a
regulatory biological index for evaluating benthic impacts has not been
implemented in Canada. Geochemical indicators (e.g. sediment pore-water
sulphide concentration) are used for routine regulatory benthic soft
sediment monitoring
(Hargrave, 2010;
Hargrave, Holmer, & Newcombe, 2008), and macrofauna are analyzed only
under certain benthic conditions
(AAR, 2016;
Wildish, Pohle, Hargrave, Sutherland, & Anderson, 2005). The addition
of a biotic measure to routine assessments of soft sediments to
complement information obtained by geochemistry would advance the state
of the science of benthic monitoring in Canada, providing regulators
additional information for decision-making. This could come in the form
of eDNA-based data, either by metabarcoding or assays targeting
particular taxa once eDNA indicators are identified.
Here we advance existing knowledge on the responses of benthic metazoans
to organic enrichment with the goal of testing the potential application
of eDNA metabarcoding to benthic impact assessments of fish farms. At
seven stations along a distance gradient at each of six salmon farms in
British Columbia, Canada, we collected sediment samples for eDNA
metabarcoding analysis of metazoans, geochemistry measurements, and
morpho-taxonomy combined with DNA barcoding analyses. For
morpho-taxonomy and DNA barcoding, we focused on Polychaeta as this was
the most abundant group of species observed and taxa therein are known
to be good indicators of benthic impacts
(Dean, 2008;
Tomassetti & Porrello, 2005), especially the Capitella capitatacomplex (Dean,
2008; Wildish et al., 2005). The specific objectives of this study were
to: (1) compare biotic signals associated with benthic impacts of salmon
farming between morpho-taxonomic polychaete and eDNA metabarcoding data;
and (2) identify potential eDNA indicators that could form the basis for
development of eDNA-based monitoring methods in Canada. We analyzed
biotic signals associated with benthic impacts of fish farms in eDNA
metabarcoding data and macrofaunal polychaetes data, and used multiple
methods to analyze the relationship between operational taxonomic units
(OTUs) and organic enrichment.