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How does molecular taxonomy for deriving river health indices correlate with traditional morphological taxonomy?


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Macroinvertebrate surveys are commonly used for assessing the health of freshwater systems around the world. Traditionally, surveying involves morphologically identifying the families, and sometimes genera, present in samples. Biological indices, derived from taxonomic lists, provide convenient ways to summarise community data and may be fairly insensitive to species-level changes in community compositions. In recent years, molecular techniques for identifying taxa have become increasingly popular and metabarcoding approaches that offer the ability to identify species from mixtures of whole animals (bulk-samples) or from environmental samples have gained much attention. However, generating accurate species lists from metabarcode data is challenging and can be impacted by sample type, choice of primers, community composition within samples, and the availability of reference sequences. This study compares the performance of molecular data extracted from bulk-samples against morphological data in calculating two biological indices (the Stream Invertebrate Grade Number Average Level 2 (SIGNAL2), which is calculated from family-level data, and a genus-level equivalent of this index, SIGNAL_SG) and one biological metric (taxon richness). Further, molecular indices and metrics derived from global, local or mixed reference DNA libraries and with varying degrees of filtering processes applied to them, are compared with respect to the strength of their relationships with morphological indices and metrics. Molecularly derived SIGNAL2 and SIGNAL_SG scores correlated strongly with morphologically derived scores, and were strongest when using a reference library containing a mix of local and global data. Molecularly derived richness metrics were moderately correlated with morphological taxa richness; however, the strongest correlations were observed when taxa that could not be assigned SIGNAL grades were omitted from analyses. This study highlights the utility of using molecular data as an objective and sensitive alternative to traditional freshwater biological assessment using macroinvertebrates.
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Ecological Indicators 125 (2021) 107537
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How does molecular taxonomy for deriving river health indices correlate
with traditional morphological taxonomy?
M.E. Shackleton
, K.A. Dafforn
, N.P. Murphy
, P. Greeneld
, M. Cassidy
, C.H. Besley
Centre for Freshwater Ecosystems, La Trobe University, Australia
Department of Earth and Environmental Sciences, Macquarie University, Sydney, Australia
CSIRO, Energy, Sydney, Australia
Sydney Water, Parramatta, Australia
Biological assessment
Macroinvertebrate surveys are commonly used for assessing the health of freshwater systems around the world.
Traditionally, surveying involves morphologically identifying the families, and sometimes genera, present in
samples. Biological indices, derived from taxonomic lists, provide convenient ways to summarise community
data and may be fairly insensitive to species-level changes in community compositions. In recent years, mo-
lecular techniques for identifying taxa have become increasingly popular and metabarcoding approaches that
offer the ability to identify species from mixtures of whole animals (bulk-samples) or from environmental
samples have gained much attention. However, generating accurate species lists from metabarcode data is
challenging and can be impacted by sample type, choice of primers, community composition within samples, and
the availability of reference sequences. This study compares the performance of molecular data extracted from
bulk-samples against morphological data in calculating two biological indices (the Stream Invertebrate Grade
Number Average Level 2 (SIGNAL2), which is calculated from family-level data, and a genus-level equivalent of
this index, SIGNAL_SG) and one biological metric (taxon richness). Further, molecular indices and metrics
derived from global, local or mixed reference DNA libraries and with varying degrees of ltering processes
applied to them, are compared with respect to the strength of their relationships with morphological indices and
metrics. Molecularly derived SIGNAL2 and SIGNAL_SG scores correlated strongly with morphologically derived
scores, and were strongest when using a reference library containing a mix of local and global data. Molecularly
derived richness metrics were moderately correlated with morphological taxa richness; however, the strongest
correlations were observed when taxa that could not be assigned SIGNAL grades were omitted from analyses.
This study highlights the utility of using molecular data as an objective and sensitive alternative to traditional
freshwater biological assessment using macroinvertebrates.
1. Introduction
Biological assessment of freshwater systems is used globally for
deriving information on stream conditions and aids in tracking the
impact of management actions (Buss et al., 2015; Carew et al., 2017).
Measures of river health are typically evaluated through deriving indices
or metrics based on the presences, or absences, of macroinvertebrate
taxa within freshwater systems. Various metrics and indices have been
developed and tailored to specic regions or countries. Examples
include the Australian River Assessment System (AusRivAS) (Smith
et al., 1999) and Stream Invertebrate Grade Number Average Level
(SIGNAL) (Chessman, 1995) in Australia, the River Invertebrate
Prediction and Classication System (RIVPACS, (Wright et al., 1998) in
the United Kingdom, the Empirical Biotic Index (EBI) (Chutter, 1972) in
South Africa and the Family-level Biotic Index (FBI) (Hilsenhoff, 1988)
in the United States of America. In most cases, these involve identifying
macroinvertebrate specimens to the family level. However, in some
cases genus-level abundance data may be used (Chessman et al., 2007;
Besley and Chessman, 2008). The collection and identication of mac-
roinvertebrates can be costly, with costs increasing as the resolution of
taxonomic identication increases (Marshall et al., 2006).
DNA barcodes are fragments of DNA, typically unique to individual
species, that can be used to delineate and identify taxa (Hebert et al.,
2003; Goldstein and DeSalle, 2011; Carew et al., 2017). There is now a
* Corresponding author.
E-mail address: (M.E. Shackleton).
Contents lists available at ScienceDirect
Ecological Indicators
journal homepage:
Received 5 August 2020; Received in revised form 30 January 2021; Accepted 15 February 2021
Ecological Indicators 125 (2021) 107537
large body of evidence that shows the utility of DNA barcodes for species
identication. Coupled with High-Throughput Sequencing (HTS) tech-
nologies, DNA barcodes can be extracted from multiple target organisms
simultaneously; an approach termed DNA metabarcoding (Taberlet
et al., 2012; Ruppert et al., 2019). The presence of species can be
determined from sample types ranging from bulkmixtures of whole
animals (bulk-samples) to environmental samples, such as water, sedi-
ments or air (Ruppert et al., 2019). Metabarcoding, thus, offers an op-
portunity to collect species-level presence/absence data, for a range of
organisms simultaneously and possibly at lower cost than traditional
Metabarcoding for generating accurate species lists offers some
challenges and can be impacted by sample type, choice of primers,
compositions of the communities within samples, and the availability of
reference sequences. High detectability rates have been achieved from
samples containing whole macroinvertebrate communities (Carew et al.,
2018), whereas many target taxa are missed when analysing water-
based environmental DNA (eDNA) samples using standard Cyto-
chrome Oxidase I (COI) primers (Hajibabaei et al., 2019a); however, see
Leese et al. (2020) for recent improvements in this area. So-called uni-
versal primers do not capture all taxa, for instance commonly used
invertebrate primers fail to detect atworms, and the afnity of primers
can vary among taxa, leading to primer bias (Kanagawa, 2003; Elbrecht
and Leese, 2015). For this reason, multiple primers may need to be used
to comprehensively survey fauna (Hajibabaei et al., 2019b). Ultimately,
assigning correct species names to sequence data requires a compre-
hensive reference database with barcodes that represent species from
the sampling area (Weigand et al., 2019). Where this is lacking, taxon-
omy will be either assigned at higher taxonomic levels or to the next best
matching species, which can often be a species not from the sampled
region (Shackleton and Rees, 2016).
While the above-mentioned issues may have effects on the detect-
ability of taxa or the ability to compile comprehensive species lists, it is
probable that they will have a lesser effect on being able to derive
indices commonly used for inferring river health. Many such indices
require identication to genus or, more commonly, family level only.
Moreover, for indices that use systems to grade macroinvertebrate taxa,
such as The Stream Invertebrate Grade Number Average Level
(SIGNAL, Chessman (1995)), identications incorrectly made to closely
related taxa are not likely to have a signicant impact, as closely related
taxa are more likely to have similar grades due to phylogenetic niche
conservatism. SIGNAL is the index commonly used in Australia to
characterise river health from the presence of macroinvertebrate taxa.
The SIGNAL method applies a grade from 1 to 10 to each taxon, with 1
indicating taxa that are highly tolerant and 10 highly sensitive to
pollution (Chessman, 2003). SIGNAL has been used extensively for
biological assessment of freshwater systems and for investigating a va-
riety of environmental impacts (Chessman et al., 2006; Lester et al.,
2007; Besley and Chessman, 2008; Rose et al., 2008; Davies et al., 2010;
Nichols et al., 2010; Tippler et al., 2012). The mean, or weighted mean,
of all taxa within a sample provide a SIGNAL score for a reach, with
scores indicating levels of impact as severe (<4), moderate (45), mild
(56) and healthy (>6). The most commonly used form, SIGNAL2
(Chessman, 2003), uses a system that places grades on families. How-
ever, the SIGNAL_SG system, developed specically for the region
around Sydney, Australia, derives scores based on genus-level grades
(Chessman et al., 2007). SIGNAL-SG was developed in response to
suggestions that region-specic models are more suitable than those
derived for the broad scale as was the case for the original version of
SIGNAL (Bunn, 1995; Bunn and Davies, 2000) and later Australia-wide
objectively derived SIGNAL2 (Chessman, 2003).
Taxonomic richness is also commonly used in routine biological
monitoring programs. However, it often provides a poorer indicator of
possible impacts to a system than SIGNAL. Growns et al. (1997) found
taxa (family) richness to be a weaker measure of the effects of pollution
by municipal sewage efuent and urban stormwater than SIGNAL for 12
streams of outer suburban Sydney and the lower Blue Mountains,
Australia. Walsh (2006) in a study of 16 streams subject to urban
disturbance in eastern Melbourne, Australia, found SIGNAL to be a more
sensitive indicator than taxa (family) richness.
Very few studies have investigated the use of metabarcoding for
deriving water quality indices. Carew et al. (2018) dissected tissues from
macroinvertebrates in river bioassessment samples, metabarcoded COI
fragments, and compared molecularly and morphologically derived
indices and metrics. They found little difference between the two
methods for SIGNAL2 scores, the Number of Families, Key Families and
Australian Rivers Assessment System (AusRivAS) bands. Marshall and
Stepien (2020) found that eDNA metabarcoding of 16S fragments
revealed similar trends in multiple alpha and beta diversity metrics to
those seen in morphological data.
The present study used a short, 313 base-pair, fragment of the COI
barcoding region to determine whether SIGNAL2 and SIGNAL_SG biotic
indices derived from DNA data are comparable to those derived from
traditional, morphological data. However, to differentiate this study
from other studies in this area key differences were made, some of which
aimed to reduce the cost and time involved with sample preparation.
Firstly, DNA was extracted from whole bulk-samples rather than dis-
secting tissue from individual animals as was done in Carew et al.
(2018). Secondly, only a single set of primers was used in this study,
compared to three sets in Carew et al. (2018) and Marshall and Stepien
(2020). While this may reduce taxonomic coverage it also reduces the
sample preparation time and increases the sequencing read depth
available per sample. Thirdly, past studies have investigated family level
metrics, whereas the present study includes a genus level metric (SIG-
NAL_SG). Because taxonomic assignment of Operational Taxonomic
Units (OTUs) can be affected by the taxonomic composition of reference
DNA databases, metrics were derived using three molecular datasets
containing the same OTUs but with differing taxonomic identications
applied from different reference databases in order to investigate how
incomplete barcode libraries effect metric outcomes. The effect that
ltering OTUs, based on their percent contribution to samples, has on
index and metric outcomes was investigated at varying thresholds.
Lastly, comparisons were made between morphological and molecular
taxonomic richness, including for molecular analyses richness of OTUs
as well as taxa richness (i.e. genus, family), taxa lists were compared,
and the detectability of taxa investigated.
2. Methods
2.1. Site selection and macroinvertebrate sampling
Macroinvertebrates were collected from 7 sites in 3 freshwater creeks
(Tributary of Devlins, Lalor and Vineyard) in Sydney, Australia (Fig. 1)
on multiple occasions between December 2016 and July 2017 (see
Supplementary Table S1). On each sampling occasion, three replicate
samples were taken from the edges of pools. Edges were sampled with
hand-held nets (320 ×250-mm opening; 250-µm mesh) and sweep
sampling over transects of approximately 10 m. Macroinvertebrates
were subsampled selectively by the unaided eye for 30 to 60 min, with
the goals of picking approximately 100 specimens per sample and a wide
range of species rather than large numbers of the same species. Further
details are given by Chessman (1995).
Samples were originally collected into 70% ethanol. However, this
was removed for transport between laboratories (approx. 2 days) and
topped up with 100% ethanol and placed in a freezer at 20 C until
DNA extraction. Macroinvertebrates were morphologically identied to
genus level where possible, with the aid of microscopes and published
keys and identication guides. Details of published keys and identi-
cation guides for identication of Australian invertebrates has been
consolidated by the Centre for Freshwater Ecosystems (Hawking et al.,
2013; Keys and
guides used in morphological identication included Arachnida (Cook,
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
1974, 1986; Harvey, 1996; Harvey and Growns, 1998), Annelida
(Pinder, 2010), Diptera (Cranston, 2019; Debenham, 1987;
Elson-Harris, 1990; Madden, 2009), Coleoptera (Davis, 1998; Glaister,
1999; Porch and Perkins, 2010; Watts, 2002), Gastropoda (Ponder,
2013), Hemiptera (Andersen and Weir, 2004; Porch and Perkins, 2010),
Hirudinida (Govedich, 2001), Odonata (Theischinger and Hawking,
1999; Theischinger, 2000, 2001; Theischinger and Endersby, 2009),
Trichoptera (Dean et al., 2004).
2.2. DNA extraction and amplication
Extraction of DNA was undertaken on bulk samples (i.e. all macro-
invertebrates collected in a sample were processed together). DNA
extraction was undertaken in a laminar ow cabinet, with UV serial-
isation, to avoid contamination issues. Prior to extraction, ethanol was
drained from and then evaporated off the samples by placing the open
samples on a heating block at 70 C. Genetic material was extracted
using a DNeasy blood and tissue kit (Qiagen, USA) with the following
modications to the manufacturers guidelines: samples were placed in
a solution of 20 µl Proteinase K (20 mg/mL) and 200400 µl of buffer
and bead beaten for 5 min using a Mini-bead beater (Daintree scientic),
followed by manual crushing using a pestle. Tubes and beads used for
bead beating were from the MoBio Power Water DNA extraction kit.
From each sample, 200 µl of material was taken and used as the sample
from which genetic material would be extracted and the remaining steps
in the DNeasy blood and tissue protocol were adhered to. A blank sample
was prepared, alongside the samples, as a control and underwent all
steps that non-blank samples underwent.
A 313 base-pair (bp) internal region of the COI barcode region was
amplied using the primer pair mlCOIintF (GGWACWGGWT-
CRAARAAYCA) (Leray et al., 2013) with 8 bp index barcodes attached.
Amplication and sequencing were done by the sequence provider Mr
DNA (, Shallowater, TX, USA), during June 2018,
using the providers standard protocols. Duplicate, one-step PCRs were
undertaken using the HotStarTaq Plus Master Mix Kit (Qiagen, USA)
with a protocol of 94 C for 3 min; 30 cycles of 94 C for 30 s, 53 C for
40 s and 72 C for 1 min; and a nal elongation step at 72 C for 5 min.
Successful amplication was assessed through checking on a 2% agarose
gel. Multiple samples were pooled in equal proportions, based on mo-
lecular weight, and puried using Ampure XP beads. An Illumina DNA
library was prepared from the pooled samples. Sequencing was per-
formed on an Illumina MiSeq sequencer using V2 300 cycle kit.
2.3. Taxonomic assignment
Sequence data were demultiplexed using a custom built script pro-
vided by Mr DNA; FASTq Processor (http://www.mrdnafreesoftware.
com/). Processing and cleaning of data, and creation of OTU tables
was performed using the Greeneld Hybrid Analysis Pipeline (GHAP)
(Greeneld, 2017). In summary, sequences with a minimum overlap of
25 bp and homology of at least 80% were merged. Sequences were
quality ltered using a Maximum Expected Error (max_EE) threshold of
1. Only sequences that were between 304 and 350 bp long were retained
for further analyses. Sequences were clustered into operational taxo-
nomic units (OTUs) using a 97% clustering threshold and OTUs that
occurred over less than three samples or consisted of fewer than three
reads were ltered out. To test the effect that ltering OTUs based on
read numbers has on downstream analyses, OTUs were ltered at 6
thresholds where read abundance was greater or equal to 0%, 0.025%,
0.075%, 0.01%, 0.05% and 0.1%.
Taxa were assigned to OTUs using three reference libraries of COI
barcodes, resulting in three sequence datasets. The rst was a library of
curated barcodes obtained from GenBank (Benson et al., 2012)
( accessed May 2018), which
contains data from species across the world. The second was the Aquatic
Invertebrates of Australia reference library (AIA), housed on the Barcode
of Life Database (BOLD) (Ratnasingham and Hebert, 2007)
( accessed July 2018), which contains
only data from Australian macroinvertebrate species and many
Fig. 1. Map of sample locations.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
sequences from species that were not within the GenBank library. The
third was a combination of the two libraries, herein referred to as the
Best of Both Worlds (BoBW) library. Taxonomic assignment for BoBW
was achieved by taking the highest percent identity match from either
the GenBank or AIA datasets. Taxonomic assignment was further ltered
using the arbitrary default thresholds in the GHAP pipeline which assign
OTUs at various taxonomic levels depending on percent homology: 97%
or greater for species, 95 - <97% for genus, 90- <95% for family, 85-
<90% for order and <85% are unassigned. For analyses involving genus
or family level data, OTUs with identical taxonomic assignments were
merged and their read numbers summed. Non-target taxa, such as sh,
fungi and microinvertebrates were removed from the data.
2.4. Analyses
SIGNAL grades provide an indication of how tolerant taxa are to
pollution and SIGNAL scores, calculated as the un-weighted (presence
absence) or weighted (with square root transformed abundance data)
average of grades within a sample, are often used to determine the
health of river systems. Two methods were used to assign SIGNAL grades
to taxa. The rst applied a genus-level grade (SIGNAL_SG) based on
Chessman et al. (2007) regional version of SIGNAL for Sydney,
Australia. For the genus-level analyses OTUs that could not be identied
to genus were removed. The second method applied SIGNAL2 grades as
per Chessman (2003), which were developed from Australia wide
sampling and as such are the most commonly used in Australia. Each
family was assigned a family-level SIGNAL2 grade except organisms
belonging to Oligochaeta and Acarina were assigned a single grade,
respectively, and members of the Chironomidae were assigned grades at
the sub-family level. OTUs that could not be identied to the level
required for their respective SIGNAL2 grade were removed. Both indices
were created with the same approach of setting sensitivity grades of the
taxa objectively (Chessman, 2003; Chessman et al., 2007). For each
reference library, the total number of taxa that could be assigned
SIGNAL grades and the average SIGNAL grades of those taxa were
calculated to investigate possible biases due to reference library
For each sample an un-weighted SIGNAL score was calculated from
both the morphological and molecular datasets. An un-weighted
SIGNAL score was chosen because treating abundances of DNA reads
as abundances of organisms suffers from numerous problems that have
not yet been adequately resolved, such as primer biases (Elbrecht and
Leese, 2015) and differential mitochondrial or cell numbers (Elbrecht
et al., 2017a). Pawlowski et al. (2018) state that there is no simple so-
lution to address the abundance issue and advocated the most conser-
vative approach is to use only presence-absence data. Moreover,
weighted SIGNAL2 scores are generally calculated using predened bins
of taxa counts (e.g. 12, 35 organisms) and it is not clear how these
would correspond with read numbers. Moreover, other studies suggest
that when abundance data are swapped for presence/absence data dif-
ferences in biotic indices are generally low (Beentjes et al., 2018;
Buchner et al., 2019). Correlations between morphologically and
genetically determined SIGNAL scores were assessed using Pearsons
Correlation Coefcient (PCC) tests. SIGNAL scores are often used to
classify river reaches in terms of severity of pollution. Classications
were applied using those provided in Chessman (1995). Confusion
matrices of paired morphological and molecular classications were
created at the generic and family level.
Numbers of taxa were similarly treated, with comparisons made
between the numbers of unique taxa in the morphological data and the
numbers of OTUs and numbers of unique taxa in the molecular data. The
set of unique OTUs within the molecular data contained taxa not
traditionally targeted in macroinvertebrate monitoring (e.g. sh,
microcrustacea and fungi). A comparison using all OTUs was undertaken
to investigate whether richness metrics can be reasonably used without
identifying OTUs to taxa. Two further comparisons of the number of taxa
between morphological and molecular datasets were undertaken using
1) all unique taxa with taxonomic identication taken to genus level
where possible, and 2) only those taxa to which SIGNAL scores could be
applied. The reasoning for the latter being that this better represents
what would be collected in a traditional survey.
Differences in taxonomic assignment between the two methods were
assessed by examining taxa lists. Metrics were calculated to investigate
the detectability of taxa, including accuracy, precision, prevalence, and
true positive, true negative, false positive and misidentication rates. An
score, which provides a harmonic mean between the precision and
true positive rate, was also calculated for each family and genus to
further aid in assessing how well the molecular methods performed at
detecting the presence or absence of taxa. How these metrics change in
response to ltering thresholds was also investigated using a single well-
performing dataset. It should be noted here that the assumption is that
the morphological identication is correct. However, in reality, a false
positive does not necessarily mean a taxon was not present in the sam-
ple, as it is possible taxa may have been missed or mis-identied in the
original morphological assessment. For instance, Chessman et al. (2007)
measured genus-level taxonomic disagreement of 4.2% and difference in
enumeration of 0.05%, for 94 samples selected randomly and reproc-
essed by a person who had not done the original identication and
counting. These 94 samples were drawn from 2740 samples that were
the basis of the Sydney regional version of SIGNAL_SG (Chessman et al.,
2007). Moreover, during the time at which the current samples were
identied the identication error for the laboratory processing the
samples was 3.9%, following similar methods as those of Chessman et al.
3. Results
3.1. Data processing
An r-markdown le of the R script and details of analyses is provided
as a supplementary html le (S2) and r-markdown script (S3) along with
the raw data used in the script (S4.1-S4.8). DNA sequencing resulted in
over 3 million reads. The minimum number of reads within samples was
42,072 and the maximum was 141,085. Filtering samples across read
number thresholds and by removing non-target taxa, had little effect on
reducing the number of reads per sample across the three datasets
(Fig. 2). Analyses at taxonomic levels (i.e. genus and family) required
ltering out OTUs that could not be identied to the required level. For
the AIA dataset there were no reads removed between ltering on OTU
read number and ltering non-target taxa, as the reference library
contained only target taxa, thus OTUs that represent non-target taxa
remained in the dataset until genus or family level ltering was applied.
On average, this ltering resulted in a greater loss in sequences for the
GenBank dataset than either the AIA or BoBW datasets (Fig. 2). An
exception occurred in the AIA dataset for one sample that, in the generic
level analyses, ranged from 50255318 depending on the read threshold
applied, which was similar to samples with lowest read numbers in the
GenBank database. Mean read numbers for family level analyses ranged
from 75,77476,393 for the BoBW dataset, 67,08967,577 in the AIA
dataset and 42,41542,756 in the GenBank dataset. Mean read numbers
for generic level analyses ranged from 67,82368,213 for the BoBW
dataset, 60,41360,701 in the AIA dataset and 37,85638,067 in the
GenBank dataset.
3.2. SIGNAL grades and unique taxa
The numbers of OTUs that could be assigned SIGNAL_SG and
SIGNAL2 grades and that contributed to the total number of unique taxa
(at family or generic levels) varied between datasets (Fig. 3). The
average SIGNAL grades of taxa within the morphological dataset best
matched the average grades of taxa in the BoBW dataset. At the family
level, the average SIGNAL2 grades of taxa within all datasets fell within
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
the 34 band. At the genus level, the average SIGNAL_SG grade of taxa in
both the BoBW and AIA datasets fell within the same band as the
morphological dataset (between 5 and 6), with AIA average grades
marginally higher than BoBW. In contrast, taxa in the GenBank dataset
had an average SIGNAL_SG grade in the 45 band.
While the total numbers of families or genera that could be assigned
SIGNAL grades was lower in the molecular datasets than the morpho-
logical dataset, the BoBW had the greatest number, followed by AIA and
then GenBank. The morphological dataset had around 30 more genera
that the BoBW dataset. Applying increasingly more stringent read
number threshold lters had little effect on average SIGNAL grades or
numbers of unique taxa.
3.3. Correlations of molecular and morphological SIGNAL_SG and
SIGNAL2 scores
Molecularly derived SIGNAL_SG and SIGNAL2 scores of site samples
across collection events were generally signicantly and strongly
correlated with morphologically derived scores as long as some read
Fig. 2. Effect on total read number within samples from ltering out OTUs below a read number threshold (dark blue), non-target taxa (red), OTUs that could not be
identied to the appropriate level for family (green) and generic (light blue) level analyses. Note the read number threshold of 0 represents no ltering having been
applied and is thus the original total number of reads. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of
this article.)
Fig. 3. Four metrics of mean SIGNAL_SG grade, mean SIGNAL2 grade, total number of unique taxa at family or generic levels in three eDNA datasets and a
morphological dataset (colours), and the effect of applying read number threshold lters (x axis) on these four metrics.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
number ltering had been applied; although, generic level GenBank
scores were only moderately correlated (Fig. 4). Correlations in unl-
tered datasets were weak and in one case non-signicant. At the generic
level the AIA dataset marginally outperformed the BoBW, with the
strongest correlations seen in the comparisons that used 0.05percent
threshold for the AIA dataset (PCC =78.5 p-value <0.001). At the
family level, correlations with the GenBank database improved sub-
stantially from the generic level analyses and also with increased
threshold ltering. While at the family level the BoBW dataset per-
formed best across most thresholds, except at 0.1 percent threshold the
GenBank database outperformed the BoBW and provided the highest
correlation. In contrast the correlations for the AIA dataset grew
markedly weaker with increasing threshold ltering when analysed at
the family level.
Comparisons between morphological and molecular SIGNAL_SG and
SIGNAL2 scores at each threshold are graphically illustrated in the
supplementary r-markdown le, Figs. 2.3 and 2.11, respectively. When
threshold ltering was applied, molecular-based scores mostly fell
within the same unit of morphologically-based scores. Scores that fell
outside the same unit tended to be only 1 unit either side of the
morphological score, with GenBank tending to skew towards lower
scores at both the generic and family level and BoBW and AIA tending to
skew towards higher scores in the family level analyses (see supple-
mentary r-markdown Figs. 2.4 and 2.10).
Confusion matrices of the classications into which each dataset
places river reaches showed a greater spread of discrepancy between
morphological and molecular classications at the generic level than the
family level (see supplementary r-markdown le Figs. 2.5 and 2.13
respectively). At the generic level, the GenBank dataset was more likely
to overestimate the severity of pollution, for instance 15 cases assessed
by the morphological analyses as moderately polluted were classied as
severely polluted by the GenBank analyses at 0.1% read number
threshold. In contrast, the AIA and BoBW most often ascribed the same
classication as in the morphological analyses, with few cases in
disagreement. For instance, the AIA analysis with a 0.05% read number
lter had only 6 samples that disagreed with the morphological classi-
cation and four of these either ascribing mild pollution to moderately
polluted reaches or vice versa. The percentage of cases where the mo-
lecular classication agreed with the morphological classication at the
generic level, when read number ltering greater than 0 was applied,
was between 67.6 and 86.5% in the AIA analyses, 67.678.4 in the
BoBW analysis and 35.148.6% in the GenBank analysis (Table 1). The
family level analyses were in much greater agreement. However, all but
one sample was morphologically categorised as severely polluted. The
percentage of cases where the molecular classication agreed with the
morphological classication at the family level was between 89.2 and
97.3% in the AIA analyses, 94.6100 in the BoBW analysis and 97.3%
across all read number thresholds in the GenBank analysis (Table 1).
3.4. Correlations of molecular and morphological taxonomic richness
Correlations between taxa richness of morphological data and OTU
or taxa richness of molecular data were investigated for each dataset and
across all read number lters. Analyses were conducted using three
levels of the molecular data 1) all OTUs, 2) all unique taxa with genus as
the lowest taxonomic level, and 3) only those taxa for which SIGNAL
grades could be applied.
In general, morphological taxa richness was weak to moderately
correlated with molecular OTU and unique taxa richness measures. OTU
richness returned signicant PCCs between 0.34 and 0.44 when some
level of read number thresholding was applied (Fig. 5). Molecular
unique taxa richness correlated best with morphological taxa richness
when using the AIA dataset, with PCCs over 0.5 when read numbers that
contributed at least <0.025% were removed. At the 0.025 read number
threshold the PCC reached 0.59 (p-value <0.01). The BoBW and Gen-
Bank datasets correlated relatively poorly with morphological taxa
richness, with PCCs increasing as read number thresholds increased.
However, at the 0.1% read number threshold PCCs were 0.51 and 0.49
respectively, with p-values <0.01. Including only those taxa for which
SIGNAL grades could be applied (i.e. SIGNAL taxa richness) increased
the performance of the BoBW dataset, and this dataset returned com-
parable although slightly lower PCC values than the AIA dataset (Fig. 5).
In contrast, the GenBank PCC values increased and became signicant at
the 0% read number threshold, but became non-signicant for thresh-
olds between 0.01 and 0.075%. The greatest correlation over all ana-
lyses was between the morphological taxa richness and the AIA SIGNAL
taxa richness at 0.01% read number threshold (PCC =0.63, p-value <
Fig. 4. Pearson correlation coefcient scores among three datasets (colour) and thresholds (x axis). Shapes of points indicate the degree of signicance.
Table 1
Percent agreement between morphological and molecular water classications.
Dataset Threshold Generic level Family level
AIA 0 37.84 97.30
0.01 67.57 94.59
0.025 70.27 97.30
0.05 83.78 89.19
0.075 81.08 89.19
0.1 86.49 89.19
BoBW 0 45.95 97.30
0.01 67.57 97.30
0.025 75.68 100
0.05 72.97 97.30
0.075 78.38 94.60
0.1 78.38 91.90
GenBank 0 59.46 97.30
0.01 48.65 97.30
0.025 43.24 97.30
0.05 45.95 97.30
0.075 40.54 97.30
0.1 35.14 97.30
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
0.01) (Fig. 5). Fig. 6 shows the scatter of data points between the
morphological and AIA dataset at the 0.01% threshold. For brevity only
these scatterplots are presented here. However, further scatterplots,
including for analyses at all thresholds, are provided in the r-markdown
supplementary material Figs. 2.25, 2.26 and 2.27. Similarly, Fig. 7
shows the relationship between morphological and molecular richness
at sites over time for the AIA dataset with a read number threshold of
0.1%. Here, OTU richness is generally higher, taxa richness higher or
lower and SIGNAL taxa richness generally lower than morphological
3.5. Detectability
The ability of the molecular approach illustrated by the BoBW
dataset to detect taxa, varied among genera (Fig. 8) and families (Fig. 9).
Increasing the read number threshold reduced the number of taxa that
occurred exclusively in the molecular data but also reduced the per-
centage of genera and families that were shared between the molecular
(AIA, BoBW and GenBank) and morphological datasets (Fig. 10).
Overall, the morphological dataset shared more taxa with the BoBW
dataset than the AIA or GenBank datasets. Whereas the BoBW and AIA
datasets had at least some samples with 100% of the families present in
the morphological dataset, the GenBank dataset only included 100% of
families when a 0% read number threshold was applied and never
included 100% of the genera (Fig. 10). The BoBW dataset was the only
dataset to have samples that contained 100% of the genera present in the
morphological dataset (Fig. 10).
Metrics of detectability and prevalence were calculated across all
thresholds and are provided as supplementary material (S5 for genera
and S6 for families). These calculations were based on the assumption
that the morphological identication was correct. For brevity, only
those for the BoBW dataset using a read number threshold of 0.1% are
provided here for genera (Fig. 11) and families (Fig. 12). Prevalence was
highly variable among families and genera, with the majority of taxa
having a prevalence below 50%. Prevalence below 10% occurred in 28
of the 59 families and 64 of 101 genera.
Overall, the accuracy of the molecular method was relatively high,
with the notable exception of the atworm Dugesiidae which had a true
positive rate of 0% and very high misclassication rate (81.1%)
(Fig. 11). However, the primers used in this project are known to not
Fig. 5. Pearsons correlation coefcients between morphological taxa richness and richness of molecularly derived OTUs, taxa, and taxa for which SIGNAL grades
could be applied. Colour represents datasets and shape of points provide the signicance of the correlations. Note that at the OTU level all datasets were the same so
here only the GenBank values are supplied.
Fig. 6. Scatterplots of correlations between morphological taxa richness and the molecular dataset and threshold with the highest correlated richness measure for A)
OTUs, B) unique taxa and C) SIGNAL taxa, at the 0.1% read number threshold. Blue line is a linear model of best t with 95% condence intervals. (For interpretation
of the references to colour in this gure legend, the reader is referred to the web version of this article.)
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
amplify atworm DNA. The high accuracy values were, in most cases,
largely driven by high true negative rates. To gain a better under-
standing of how the molecular methods performed at determining the
presences or absences of taxa an F
score was used, which provides a
balance between precision and true positive rate. The molecular ana-
lyses correctly determined the presences/absences for ve families and
six genera (i.e. F
scores =1): Pionidae Piona (mite), Scirtidae (beetle),
Psychodidae Psychoda (true y), Simuliidae Simulium (true y), and
Culicidae culex (mosquito), and Philopotamidae Chimarra (Caddisy)
(Fig. 11). Generally, most families returned F
scores greater than 0.5.
However, one or two families within most orders returned null F
These were often families that occurred at low prevalence within the
samples. However, some occurred at a similar prevalence to other
families of their order; for instance, Synthemistidae and Gomphidae
both had a prevalence of 0.08, but where the former returned a null F
score, the latter returned a score of 0.8 (Fig. 12).
Values of the detectability metrics changed with increasing read
number threshold (Fig. 13). As read number thresholds are increased,
there is a trade-off between decreasing the mean and increasing the
spread of values in the true positive rate and increasing the mean and
reducing the range of values in the true negative rate. However, all
metrics improve with at least some degree of read number ltering while
ltering beyond the 0.05% threshold only marginally improved these
4. Discussion
This study demonstrates that the river health SIGNAL biotic indices
can be derived from bulk sample DNA data with results that are com-
parable to those derived through traditional morphological analyses.
Strong and signicant correlations between morphologically and
molecularly derived SIGNAL scores were observed for both family
(SIGNAL2) and genus-level (SIGNAL_SG) analyses (Fig. 4). However, the
choice of DNA reference library and data pre-processing inuences the
signicance and strength of correlations. In both generic and family
level analyses, correlations greatly improved when at least some
ltering of low contribution OTUs was performed (e.g. ltering those
that contributed<0.01%). At the generic level, datasets with taxonomic
identications made using a reference database of local taxa (i.e. AIA)
performed better than using the GenBank reference library, with the AIA
dataset returning the highest Pearson Coefcient Correlation (PCC) of
0.785 (p-value <0.001) when using a 0.05% read number threshold. At
the family level, the correlation of the GenBank with the morphological
SIGNAL2 scores was greatly improved and when applying a read num-
ber lter at the 0.1% threshold the GenBank dataset had the highest
correlation of the molecular datasets (PCC =0.795, p-value =<0.001).
However, at lower thresholds the BoBW dataset performed marginally
better. In contrast the performance of the AIA dataset decreased.
In practice, SIGNAL scores are interpreted as bands (water quality
status classes) indicating gradients of pollution. Chessman (1995), who
introduced the rst signal score classied bands as greater than 6 =
clean water, 56 possible mild pollution, 45 moderate pollution and <4
severe pollution. In the present study, when applying at least some de-
gree of read number ltering, most molecularly derived scores were in
agreement with the morphologically derived scores in terms of water
quality classication. When molecular classications deviated from
morphological classications they predominately classied to the next
lower or higher classication; the notable exception being in the genus
level analyses using the GenBank dataset which classied a few mildly
polluted samples as severely polluted.
While application of the SIGNAL biotic indices can be used to assign
water quality status classes, Besley and Chessman (2008) demonstrated
graphical and statistical assessment of SIGNAL_SG scores based on
morphological data collected from paired sites situated upstream and
Fig. 7. Per site comparisons between number of taxa in the morphological (Morph) data and in the AIA molecular dataset with a read number threshold of 0.1% for
the number of A) OTUs, B) unique taxa and C) taxa for which SIGNAL grades could be placed.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
Fig. 8. Comparison of genera detected in samples within the morphological (morph) and BoBW datasets across read number thresholds (panels). Sample names have
been removed for ease of plotting; however, grey divisions (x-axis) indicate different samples, which are arranged alphabetically and by date.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
downstream of point source discharge of treated sewage wastewater.
That graphical assessment illustrated SIGNAL_SG scores do not neatly
fall into a band, and often occur across two water quality status classes
(bands). Addition of an overall upstream mean of SIGNAL_SG scores
with error bars of ±one standard deviation for a temporal period allows
presentation in a process control chart for ecological monitoring as
advocated by Burgman et al. (2012). An example of this control chart
approach is provided by the 25-year long-term study (1995 to 2020) of
the Nepean River near the West Camden sewage treatment plant in the
Sydney region, Australia, which illustrates the SIGNAL_SG range of
morphologically derived scores of about a unit uctuation as typical
variation (see Fig. S1 in Supplementary material). In adopting meta-
barcoding data as the basis for assessment with biotic indices such as
SIGNAL, our study suggests the underlying barcode library will inu-
ence slight differences in SIGNAL scores and a period where both
morphological and metabarcoding data are obtained would provide an
understanding of the potential site specic ranges. This conservative
approach would consider Buchner et al. (2019) advocation of the
importance of properly evaluating the potential to link metabarcoding
data to established indices and relating them to existing data. This
approach seems prudent as management decisions can be expensive, for
example the Blue Mountains Sewage Transfer Scheme in the Sydney
region, Australia, was established to upgrade the sewerage system at a
cost of $AU 360 million, by progressively closing small, local plants and
diverting sewage to a larger, more efcient plant (Besley and Chessman,
Fig. 9. Comparison of families detected in samples within the morphological (morph) and BoBW datasets across read number thresholds (panels). Sample names
have been removed for ease of plotting; however, grey divisions (x-axis) indicate different samples, which are arranged alphabetically and by date.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
Overall, molecular classication was relatively accurate with accu-
racies over 80% at the generic level and over 95% at the family level
obtainable. At these levels, managers of river health could apply the
molecular techniques described here with some condence that their
results will be relatively consistent with those of traditional methods.
However, a limitation to the current study is that it lacked samples
classied as mildly polluted and had only one sample classied as
moderately polluted at the family level. Further experimentation on a
wider variety of streams with less surrounding urbanisation would
provide greater insight into how family level classications perform
outside the category of severely polluted.
The morphologically derived taxa richness metric was generally
signicantly, but only weak to moderately, correlated with the molec-
ularly derived metrics of richness. At the OTU level, correlations were
signicant and exceeded PCC values of 0.3 only when some degree of
read number thresholding was applied. Correlations improved for
measures of unique taxa but only when using the AIA dataset. However,
correlations for the GenBank and BoBW datasets increased with
increasing read number threshold, with the highest and signicant
correlations occurring at the 0.1% read number threshold. When only
taxa for which SIGNAL grades could be applied were included in ana-
lyses, correlations became moderately strong in both the AIA and BoBW
datasets, with PCC values ranging from 0.44 at the 0% and 0.63 at the
0.1% read number threshold for the AIA dataset and 0.44 at the 0% and
0.59 at the 0.1% read number threshold for the BoBW dataset. In
contrast, the GenBank analyses were only signicant at the 0% and 0.1%
read number thresholds. The improvement in correlations from the OTU
level analyses to the unique taxa and SIGNAL taxa analyses, is likely
partly due to the removal of sequences of non-target taxa such as sh,
microinvertebrates or terrestrial organisms, thus better representing the
suite of taxa collected in a traditional sample. Moreover, the OTU
clustering method used a 97% threshold which perhaps better represents
delineations of species rather than genera, as in the morphological data
(Hebert et al., 2003). Fig. 7 shows a trend where, compared with
morphological richness, OTU richness is generally higher, taxa richness
is either higher or lower and SIGNAL taxa richness is generally lower.
This trend is driven by the distinct clustering of OTUs into taxa and then
the ltering out of taxa to which SIGNAL grades could not be applied.
However, it should be noted that SIGNAL taxa richness was generally
lower than morphological taxa richness because not all the taxa in the
morphological dataset have been barcoded, and thus are missing from
the SIGNAL taxa richness analysis.
The performance of the genetic data to determine the presence or
absence of genera was assessed using F
scores derived from the BoBW
dataset with 0.1% read number threshold. Around 63% of families and
43% of genera had F
scores above 0.5; and 42% of families and 28% of
genera had F
scores over 0.7. Within orders there were usually one or
two families that returned null F
scores, with these predominantly
being at low prevalence among samples. The Odonata were the most
family diverse taxa and performed relatively well in terms of F
with scores ranging from 0.67 to 0.92. The notable exception in the
Odonata was the family Synthemistidae, which returned a null F
despite being as prevalent as Gomphidae. The family Planorbidae was a
similar notable exception among the snails, having a prevalence of 0.16
but a null F
score. For the Dugesiidae, the null F
score can be explained
by the choice of primers used for this study, as they are known not to
amplify this taxon (Vanhove et al., 2013; Carew et al., 2018). For other
families with related taxa that did return F
scores it is less clear as to
why they did not. It is possible that genetic variation could have been
inadequate to distinguish between some taxa, and that taxa were mis-
identied to sister taxa, due to the small size of the barcodes used. This
occurs usually when large reference libraries are used for identication
(Hajibabaei et al., 2006). However, mini-barcodes are frequently used
for metabarcoding analyses and lengths of 100 and 250 bp have been
shown to distinguish around 90 and 95% of species, respectively
(Meusnier et al., 2008; Yeo et al., 2020). The use of references from local
species and a barcode length of 313 bp in this study should also have
improved performance. Moreover, a cursory investigation into the
variation in the gene fragment used among genera suggests that all
genera should be reasonably distinct.
In one case, the null F
score can be attributed to an error in the
reference library. The two mite genera Oxus and Frontipoda were
morphologically recorded among the samples but were never found
within the same sample. Frontipoda did not occur in the molecular data;
however, suspiciously, Oxus was recorded in all of the samples that
contained Frontipoda. A review of the Oxus voucher specimens that
contributed to the AIA barcode library, revealed, incorrect identica-
tions placed onto some of the Oxus specimens, specically those that
genetically matched the misidentied Frontipoda, and that the vouchers
were in fact Frontipoda. These voucher specimens were the same that
genetically matched to the misidentied Frontipoda in the molecular
analyses. This highlights the issue of incorrectly assigned taxonomy in
DNA databases and the need for adequately curated reference libraries,
as has been emphasized by other authors (Nilsson et al., 2006; Tixier
et al., 2012; Shen et al., 2013; Shackleton and Rees, 2016; Carew et al.,
Fig. 10. Boxplots of the percent of genera and families shared between the morphological and molecular datasets and how these percentages change with read
number threshold.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
In general, this study attributed discrepancies between morpholog-
ical and molecular taxonomic assignments to be errors in molecular
assignment. Because the samples used were destroyed during the genetic
extraction process, morphological identications could not be double
checked. However, errors in morphological identication are probable
and not unexpected. An error rate of about 4% was documented for the
laboratory that performed morphological identication and this corre-
sponds with the rate found by Chessman et al. (2007) for genus level
analyses on taxa in the same region.
One possibility for unexpected positive results is that trace or envi-
ronmental DNA (eDNA) may have been present in the samples (Beer-
mann et al., 2020), including for species that may have been present as
dietary components of the collected specimens (Zaidi et al., 1999;
Sheppard et al., 2005; Hosseini et al., 2008). A further possibility, as
recorded for Oxus above, is that specimens have been miss-identied in
the reference databases used and thus the best matches for sequence
data are to incorrectly assigned species. Pawlowski et al. (2018) sug-
gested the most cited explanation for discrepancies between molecular
and morphological datasets is the incompleteness and lack of accuracy
of the molecular reference databases that impedes the correct taxonomic
assignment of DNA sequences. Elbrecht et al. (2017b) describes data-
bases like BOLD (where AIA is hosted) as containing misidentied taxa
or conicting taxonomic assignments for the same Barcode Index
Number. Some of the cases of false negatives were due to taxa missing
from the reference databases, but these were limited to Physolimnesia
(mite), Illebdella, Vivabdella (leeches), Synthemis (Dragony) and Pyg-
manisus (snail).
Despite the differences between morphologically and genetically
detected taxa, SIGNAL scores (for both index versions tested) were
generally comparable between data types. This is likely to be primarily
driven by phylogenetic signal to pollution tolerance occurring within
taxa, with closely related taxa likely to share similar SIGNAL grades. For
instance, Carew et al. (2011) showed phylogenetic signal within the
Ephemeroptera and Chironomidae to tolerance to organic pollution and
zinc concentration, respectively. In the presence of phylogenetic signal,
it follows that if sequences are assigned to incorrect but closely related
taxa the effect on SIGNAL grade assignment would be minimal. This
suggests that while the molecular data used in this study may not have
provided 100% detection rate for certain taxa, reliable SIGNAL scores
can still be derived.
Our ndings support those of Carew et al. (2018) who found little
difference between DNA derived and morphologically derived family
level indices: SIGNAL2, AusRivAS (Reynoldson et al., 1997) and a
Chironomidae-based pollution index developed as part of their study.
However, unlike Carew et al. (2018), the present study used a single
primer pair, reducing the time and costs involved in processing samples
and increasing the available sequencing depth per sample. This does,
however, come at the cost of increasing the number of undetected taxa
due to primer bias and primer-template mismatches. While Carew et al.
(2018) were able to recover 85% of families known to be in their sam-
ples, in the present study the average number of families recovered
ranged from 70.3% to 84.3% in the BoBW dataset with a range from
38.1% to 100% of families known to occur in the samples were recov-
ered. It should be noted that many of the species used in the present
study have since been added to GenBank, and it is thus possible that
these percentages will increase if analyses are performed on updated
libraries. However, our work has also shown that databases with local
taxa maybe more important, thus GenBank identications are unlikely
to improve in regions where local data is depauperate.
While the full value of using DNA as a way to track river health lies in
its ability to monitor species-level changes in communities, species-level
metrics are yet to be developed (Nichols et al., 2020). Buchner et al.
Fig. 13. Changes in detectability metrics over read number thresholds for family and generic level analyses.
M.E. Shackleton et al.
Ecological Indicators 125 (2021) 107537
(2019) suggest the central incentive for including genetic data in
assessment of ecological status should be the fundamental improvement
of resolution down to species or even population level that can be ob-
tained in a standardised fashion. For biotic indices, such as SIGNAL, this
will require compiling species responses to environmental stressors.
Additionally, biotic index grade values could be inferred through ma-
chine learning predictive models trained on metabarcoding data linked
to associated pressure data (Pawlowski et al., 2018). Buchner et al.
(2019) suggests using supervised machine learning based on direct
comparisons of metabarcoding data and traditional morphological taxa.
Hence, suitable data for this development could be obtained from bulk-
sample DNA methods as they are applied to routine biomonitoring,
especially as detectability of individual species improves. In the mean-
time, our results show how bulk-sample DNA derived data can be used as
an alternative way to calculate family- and genus-level river health
metrics with similar results to current practices. Buchner et al. (2019)
indicated DNA metabarcoding provided high-resolution taxonomic data
for the data sets required by any of the currently used EU Water
Framework Directive assessment methods including those at the genus
level. While still in its infancy, DNA metabarcoding shows promise as a
cheaper, yet robust, alternative to traditional morphological methods
for biological monitoring.
This work was supported by Sydney Water, Sydney Australia.
CRediT authorship contribution statement
M.E. Shackleton: Conceptualization, Methodology, Formal anal-
ysis, Investigation, Data curation, Writing - original draft, Writing - re-
view & editing. K.A. Dafforn: Conceptualization, Writing - original
draft, Writing - review & editing. N.P. Murphy: Resources, Writing -
original draft, Writing - review & editing. P. Greeneld: Software,
Writing - original draft, Writing - review & editing. M. Cassidy: Funding
acquisition, Writing - original draft, Writing - review & editing. C.H.
Besley: Conceptualization, Funding acquisition, Resources, Writing -
original draft, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
We thank Sydney Water for nancial assistance and for support in
the collection of macroinvertebrate samples and morphological labo-
ratory work. We also thank Ms. Merran Grifth from Sydney Water for
providing valuable feedback on the manuscript. We thank and are very
grateful to two anonymous reviewers who provided valuable feedback
and enhanced our manuscript greatly.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
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... Sequences with a minimum overlap of 25 bp and homology of at least 80% were merged and only sequences between 301 and 313 bp (i.e. close to the target amplicon length) were retained for further analyses 41 . Sequences were clustered into OTUs, using a 97% clustering threshold as this threshold corresponds reasonably well with COI delimitation between invertebrate species 42 . ...
... gov/ genba nk/, accessed 10th of June 2021). We then refined this taxonomic assignment using percent homology thresholds, arbitrarily assigned as part of the GHAP pipeline: 97% or greater for species, 95 to < 97% for genus, 90 to < 95% for family, 85-< 90% for order, and those with < 85% were not assigned because of taxonomic uncertainty 41 . ...
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Platypuses ( Ornithorhynchus anatinus ) forage for macroinvertebrate prey exclusively in freshwater habitats. Because food material in their faeces is well digested and mostly unidentifiable, previous dietary studies have relied on cheek pouch assessments and stable isotope analysis. Given DNA metabarcoding can identify species composition from only fragments of genetic material, we investigated its effectiveness in analysing the diet of platypuses, and to assess variation across seasons and sexes. Of the 18 orders and 60 families identified, Ephemeroptera and Diptera were the most prevalent orders, detected in 100% of samples, followed by Trichoptera, Pulmonata, and Odonata (86.21% of samples). Caenidae and Chironomidae were the most common families. Diptera had a high average DNA read, suggesting it is an important dietary component that may have been underestimated in previous studies. We found no variation in diet between sexes and only minimal changes between seasons. DNA metabarcoding proved to be a highly useful tool for assessing platypus diet, improving prey identification compared to cheek pouch analysis, which can underestimate soft-bodied organisms, and stable isotope analysis which cannot distinguish all taxa isotopically. This will be a useful tool for investigating how platypus prey diversity is impacted by habitat degradation as a result of anthropogenic stressors.
... Nevertheless, generating accurate species lists from MBC data is challenging [60]. Thus, the availability of reference sequences linked to known species needs to be developed [17,61]. Several researchers have found that MBC was superior to MOI in single-sample comparisons [62,63]. ...
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Background: The water level of Poyang Lake (China) fluctuates seasonally. Shahu Lake, a smaller body of water connected to Poyang Lake during the wet season, is separated in the dry season. Due to a special fishing method termed 'lake enclosed in autumn', the water level is lowered and reaches its lowest point in January, which is <0.5 m deep in the middle of the lake. Our research investigated the effect of water level changes on the zooplankton community composition in Shahu Lake. Methods: We used both DNA metabarcoding method (MBC) (18S rRNA gene V4 region) and morphological method (MOI) to track the zooplankton community structure over four seasons in Shahu Lake (China). Results: Totals of 90 and 98 species of zooplankton were detected by MOI and MBC, respectively, with rotifers being the main zooplankton component. The α-diversity index of both methods increased from spring to summer and decreased from summer to autumn, reaching the lowest value in winter. NMDS and a cluster analysis showed that all zooplankton communities detected by MOI and MBC were significantly separated by season. The zooplankton community in winter was separated from that of the other three seasons, but the summer and autumn communities were more similar. Conclusions: Changes in the water level had significant effects on the zooplankton community composition. We found that MBC was more able to detect the differences in the zooplankton composition than MOI. MBC also had more advantages in copepod recognition. In our study, 37 species of copepods were detected by MBC, but only 11 species were detected by MOI. We concluded that MBC should be used to research the seasonal variations of zooplankton.
... Astonishingly, these rapid advances in eDNA-based technologies are rather timidly implemented in routine biomonitoring (Hering et al., 2018;Shackleton et al., 2021). Although the concept of Biomonitoring 2.0 is widely endorsed, its acceptance in practice is hampered for various reasons. ...
... Astonishingly, these rapid advances in eDNA-based technologies are rather timidly implemented in routine biomonitoring (Hering et al., 2018;Shackleton et al., 2021). Although the concept of Biomonitoring 2.0 is widely endorsed, its acceptance in practice is hampered for various reasons. ...
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Species compositions and diversity levels of aquatic macroinvertebrate communities provide important indicators of ecosystem health. However, such community analyses typically are limited by time and effort of sampling, sorting, and identifications, as well as morphological character uncertainty for some taxa, especially at early life stages. Our objective was to evaluate a suite of targeted metabarcode high‐throughput sequencing assays to characterize the macroinvertebrate communities (specifically targeting Annelida, Bryozoa, Crustacea, Insecta, and Mollusca) from environmental (e)DNA water samples along 160 km of the Maumee River, OH (a major Lake Erie, Laurentian Great Lakes tributary). Multiple alpha (richness, Shannon Index, and Simpson's Index) and beta (Bray–Curtis and Sørensen dissimilarities) diversity metrics from the metabarcode assays were compared with an Invertebrate Community Index (ICI) metric calculated from traditional morphological sampling surveys conducted by the Ohio Environmental Protection Agency. The 15 Maumee River sites varied in their ICI scores (ranging from 4 to 46), with seven sites rated as “poor” or “very poor” and eight scoring “fair” or “good.” Metabarcoding assays yielded greater gamma richness, delineating 181 Operational Taxonomic Units versus 172 taxa from morphology (the latter often limited to family or genus‐level identifications). Both datasets supported similar river‐wide trends, with comparable gamma, alpha, and beta diversity patterns and community compositions across habitat types and habitat quality scores. Metabarcode assays revealed similar detection of important bioindicator Insecta, but missed most Trichoptera (caddisflies). eDNA identified eight aquatic invasive species on the GLANSIS (Great Lakes Aquatic Nonindigenous Species Information System) list, including three missed by the morphological surveys (Branchiura sowerbyi, Potamothrix bedoti, and Skistodiaptomus pallidus; with Lophopodella carteri, Faxonius rusticus, Corbicula fluminea, Dreissena polymorpha, and Valvata piscinalis in both datasets). Findings illustrate the utility of eDNA sampling and targeted metabarcode assays to enhance and complement environmental assessments of aquatic ecosystems. Species compositions and diversity levels of macroinvertebrate communities are analyzed using a suite of targeted metabarcode high‐throughput sequencing assays from environmental (e)DNA water samples along a 106 river km stretch of the Maumee River, a primary Great Lakes tributary. Targeted metabarcoding generated greater gamma richness, delineating 181 Operational Taxonomic Units (OTUs) versus 172 taxa from morphology, with both displaying similar river‐wide trends, with comparable gamma, alpha, and beta diversity patterns and community compositions across habitat types and habitat quality scores.
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DNA metabarcoding of freshwater communities typically relies on PCR amplification of a fragment of the mitochondrial cytochrome c oxidase (COI) gene with degenerate primers. The advantage of COI is its taxonomic resolution and the availability of an extensive reference database. However, when universal primers are used on environmental DNA (eDNA) isolated from stream water, macroinvertebrate read and OTU numbers are typically `watered down', i.e. diluted, compared to whole specimen `bulk samples' due to greater co-amplification of abundant non-target taxa such as algae and bacteria. Because stream macroinvertebrate taxa are of prime importance for regulatory biomonitoring, more effective ways to capture their diversity via eDNA isolated from water are important. In this study, we aimed to improve macroinvertebrate assessment from eDNA by minimizing non-target amplification. Therefore, we generated data using universal primers BF2/BR2 throughout 15 months from a German Long-Term Ecological Research (LTER) site, the River Kinzig, to identify most abundant non-target taxa. Based on these data, we designed a new reverse primer (EPTDr2n) with 3'-specificity towards macrozoobenthic taxa and validated its specificity in silico together with universal forward primer fwhF2 using available data from GenBank and BOLD. We then performed in vitro tests using 20 eDNA samples taken in the Kinzig catchment. We found that the percentage of target reads was much higher for the new primer combination compared to two universal macrozoobenthic primer pairs, BF2/BR2 and fwhF2/fwhR2n (>99 % vs. 21.4 % and 41.25 %, respectively). Likewise, number of detected macroinvertebrate taxa was substantially higher (351 vs. 46 and 170, respectively) and exceeded the number of 257 taxa identified by expert taxonomists at nearby sites across two decades of sampling. While few taxa such as Turbellaria were not detected, we show that the optimized primer avoids the dilution problem and thus significantly improves macroinvertebrate detection for bioassessment and -monitoring.
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New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared to full-length barcodes. This is here tested based on 20 empirical datasets covering ca. 30,000 specimens (5,500 species) and six clade-specific datasets from GenBank covering ca. 98,000 specimens (>20,000 species). All specimens in these datasets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (3 windows: 100-bp, 200-bp, 300-bp) and by excising nine mini-barcodes with established primers (length: 94 - 407-bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular Operational Taxonomic Units (mOTUs) that were obtained using three different species delimitation techniques (PTP, ABGD, objective clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length (>200-bp). Only very short mini-barcodes (<200-bp) perform poorly, especially when they are located near the 5' end of the Folmer region. The mean congruence between morphospecies and mOTUs was ca. 75% for barcodes >200-bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes.
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Benthic invertebrates are the most commonly used organisms used to assess ecological status as required by the EU Water Framework Directive (WFD). For WFD-compliant assessments, benthic invertebrate communities are sampled, identified and counted. Taxa × abundance matrices are used to calculate indices and the resulting scores are compared to reference values to determine the ecological status class. DNA-based tools, such as DNA metabarcoding, provide a new and precise method for species identification but cannot deliver robust abundance data. To evaluate the applicability of DNA-based tools to ecological status assessment, we evaluated whether the results derived from presence/absence data are comparable to those derived from abundance data. We analysed benthic invertebrate community data obtained from 13,312 WFD assessments of German streams. Broken down to 30 official stream types, we compared assessment results based on abundance and presence/absence data for the assessment modules “organic pollution” (i.e., the saprobic index) and “general degradation” (a multimetric index) as well as their underlying metrics. In 76.6% of cases, the ecological status class did not change after transforming abundance data to presence/absence data. In 12% of cases, the status class was reduced by one (e.g., from good to moderate), and in 11.2% of cases, the class increased by one. In only 0.2% of cases, the status shifted by two classes. Systematic stream type-specific deviations were found and differences between abundance and presence/absence data were most prominent for stream types where abundance information contributed directly to one or several metrics of the general degradation module. For a single stream type, these deviations led to a systematic shift in status from ‘good’ to ‘moderate’ (n = 201; with only n = 3 increasing). The systematic decrease in scores was observed, even when considering simulated confidence intervals for abundance data. Our analysis suggests that presence/absence data can yield similar assessment results to those for abundance-based data, despite type-specific deviations. For most metrics, it should be possible to intercalibrate the two data types without substantial efforts. Thus, benthic invertebrate taxon lists generated by standardised DNA-based methods should be further considered as a complementary approach.
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Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub‐sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.
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Biomonitoring programs have evolved beyond the sole use of morphological identification to determine the composition of invertebrate species assemblages in an array of ecosystems. The application of DNA metabarcoding in freshwater systems for assessing benthic invertebrate communities is now being employed to generate biological information for environmental monitoring and assessment. A possible shift from the extraction of DNA from net-collected bulk benthic samples to its extraction directly from water samples for metabarcoding has generated considerable interest based on the assumption that taxon detectability is comparable when using either method. To test this, we studied paired water and benthos samples from a taxon-rich wetland complex, to investigate differences in the detection of arthropod taxa from each sample type. We demonstrate that metabarcoding of DNA extracted directly from water samples is a poor surrogate for DNA extracted from bulk benthic samples, focusing on key bioindicator groups. Our results continue to support the use of bulk benthic samples as a basis for metabarcoding-based biomonitoring, with nearly three times greater total richness in benthic samples compared to water samples. We also demonstrated that few arthropod taxa are shared between collection methods, with a notable lack of key bioindicator EPTO taxa in the water samples. Although species coverage in water could likely be improved through increased sample replication and/or increased sequencing depth, benthic samples remain the most representative, cost-effective method of generating aquatic compositional information via metabarcoding.
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Identification keys are provided for the final (4th) instar larvae of genera of Chironomidae (Diptera), from aquatic habitats in Australia and New Zealand. Morphological features of taxonomic utility are discussed and illustrated by line drawings. Summaries of described species for each genus and their distribution is provided, with reference to means of further identification where available. In the subfamily Podonominae, 5 genera are keyed of which 3 are recorded from New Zealand; the 4 genera of Aphroteniinae are from Australia (absent from New Zealand); in Diamesinae 1 genus is Australian, 2 are from New Zealand; in the Tanypodinae 21 genera are found in Australia and 4 are from New Zealand; in Orthocladiinae 31 genera are reported from Australia, 14 from New Zealand; and in Chironominae 43 genera are keyed from Australia, 9 from New Zealand. Larvae of Axarus Roback, Chernovskiia Sæther and Omisus Townes (Chironomini) are recognised in Australia for the first time. The undescribed larva of Paucispinigera Freeman, endemic to New Zealand, is keyed and several other New Zealand taxa are included based on unpublished records. Genera reported from Australia and New Zealand as adults, but unknown as larvae, are listed.
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Mixed community or environmental DNA marker gene sequencing has become a commonly used technique for biodiversity analyses in freshwater systems. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to test whether COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of target assemblages in the benthos kick-net samples typically used in freshwater biomonitoring. We examine six commonly used COI primer sets on samples collected from six freshwater sites. Biodiversity analyses show that richness is sensitive to primer choice and the combined use of multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. In ordination analyses based on community dissimilarity, samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Chironomidae as well as Arthropoda site indicators were differentially detected by each amplicon tested. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that either maximize taxon-detection or the selection of amplicons for water quality or Arthropoda site indicators.
Worldwide, multiple stressors that often lead to complex and non-linear biological responses affect stream ecosystems. Combined stressor effects on ecologically diverse and functionally important macroinvertebrate communities are often difficult to assess (such as species-specific responses of many species, effects of different stressors and stressor levels in concert). A central limitation is taxonomic resolution applied for specimen identification in such studies. DNA metabarcoding can resolve taxonomy and provide greater insights into multiple stressor effects. This was detailed by results of a recent multiple stressor mesocosm experiment, where only for the dipteran family Chironomidae 183 Operational Taxonomic Units (OTUs) could be distinguished. Numerous OTUs showed very different response patterns to multiple stressors. In this study, we applied DNA metabarcoding to assess multiple stressor effects on all non-chironomid invertebrates from the same experiment. In the experiment, we applied three stressors (increased salinity, fine sediment deposition, reduced flow velocity) in a full-factorial design. Afterwards, 99,498 macroinvertebrates were sampled from two microhabitats, i.e. streambed and leaf litter. We compared stressor responses inferred through DNA metabarcoding of the mitochondrial COI gene to responses based on morphotaxonomic taxa lists. We identified 435 OTUs, of which 122 OTUs were assigned to EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa. The most common 35 OTUs alone showed 15 different response patterns to the experimental manipulation, ranging from insensitivity to any applied stressor to sensitivity to single and multiple stressors. These response patterns even comprised differences within one family. The species-specific taxonomic resolution and the inferred response patterns to stressors highlights the potential of DNA metabarcoding in the context of multiple stressor research, even for well-known taxa such as EPT species.