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The original publication is available at: Environmental Pollution 203, 165 174 (2015)
http://dx.doi.org/10.1016/j.envpol.2015.03.047
Metabarcoding of benthic eukaryote communities predicts the ecological condition of estuaries
Anthony A. Charitona, *, Sarah Stephensona, Matthew J. Morganb, Andrew D.L. Stevenc, Matthew J.
Colloffb, Leon N. Courtb, Christopher M. Hardyb
a CSIRO Oceans and Atmosphere, Locked Bag 2007, Kirrawee, NSW 2232, Australia
b CSIRO Land and Water, GPO Box 1700, Canberra, ACT 2601, Australia
c CSIRO Oceans and Atmosphere, GPO Box 2583, Brisbane, QLD 4001, Australia
Abstract
DNA-derived measurements of biological composition have the potential to produce data covering all of
life, and provide a tantalizing proposition for researchers and managers. We used metabarcoding to
compare benthic eukaryote composition from five estuaries of varying condition. In contrast to traditional
studies, we found biotic richness was greatest in the most disturbed estuary, with this being due to the
large volume of extraneous material (i.e. run-off from aquaculture, agriculture and other catchment
activities) being deposited in the system. In addition, we found strong correlations between composition
and a number of environmental variables, including nutrients, pH and turbidity. A wide range of taxa
responded to these environmental gradients, providing new insights into their sensitivities to natural and
anthropogenic stressors. Metabarcoding has the capacity to bolster current monitoring techniques,
enabling the decisions regarding ecological condition to be based on a more holistic view of biodiversity.
Keywords: biomonitoring, metabarcoding, sediments, DNA, eukaryotes, high-throughput sequencing,
18S rRNA, indicator taxa, threshold analysis
Introduction
The increasing human population and its activities are having pronounced deleterious effects on the
ecological condition of the world's estuaries (Rabalais et al., 2009). These activities degrade the physical
environment and modify the chemical composition of the water column and sediments and their
associated biota (Davis and Koop, 2006). Ultimately, such activities are expressed as distinct changes in
ecological composition and function (Dauer et al., 2000; Hooper et al., 2012). In Australia, more than
85% of the population of 22 million live within 50 km of the coast (ABS, 2003). With a population
increase of 82% projected by 2056 (ABS, 2003), the pressures on estuarine environments in rapidly
developing coastal regions such as southeast Queensland are likely to increase markedly.
In order to mitigate the pace of environmental degradation, fundamental information on the chemical,
physical and ecological characteristics and components of estuaries is required. However, many of the
variables which drive the ecology of estuaries are difficult to define and vary greatly across space and
time (Morrisey et al., 1992; Wiens, 1989; Ysebaert and Herman, 2002). The most commonly monitored
ecological component of estuaries is the macrobenthos, with many studies demonstrating its
responsiveness to a range of natural and anthropogenic variables (Johnston and Roberts, 2009). This
approach can lead to management decisions being made on the assumption that the macrobenthos
accurately represents overall ecological condition (Chariton et al., 2010a), despite the knowledge that the
meio- and microbiota are far more species-rich, have a greater diversity of life-histories and ecological
niches, and are often more responsive to environmental change (Austen and Warwick, 1989; Kennedy
and Jacoby, 1999). The inclusion of ecological data derived from these elements of the biota would
provide a more representative, informative ecological picture.
In recent years, there have been considerable advances in applying DNA-based diversity methods
using high-throughput sequencing (Baird and Hajibabaei, 2012; Taberlet et al., 2012a), commonly
referred to as metabarcoding (Taberlet et al., 2012b). Metabarcoding provides previously unattainable
insights into communities and ecosystems, aiding our understanding of them. The approach has proven
especially useful in deriving compositional data from samples containing organisms that are difficult to
identify because of small size, cryptic habits, their occurrence in the form of propagules (e.g. spores and
zoocysts) or a lack of traditional identification keys (Medinger et al., 2010; Valentini et al., 2009).
In this study, we used high-throughput sequencing of the 18S rRNA gene to examine sub-macro
benthic biotic composition of five estuaries in eastern Australia. These estuaries have been routinely
sampled since 2000 as part of a larger monitoring program, but due to time and cost constraints the
ecological health of these estuaries is determined using abiotic surrogates rather than ecological data. Our
initial aim was to examine whether DNA-based eukaryotic composition could differentiate between
estuaries. Secondly, we explored the relationships between the eukaryotic communities and
environmental gradients observed among the estuaries. Finally, we examined whether metabarcoded
eukaryotic data has the potential to produce relevant ecological information which can be used to further
develop DNA-based approaches for the routine monitoring of estuarine sedimentary environments.
Methods
Study region
In February 2010, we sampled five estuaries (Noosa, Maroochydore, Pine, Logan and Currumbin) in
south-east Queensland, Australia (Fig. 1). All estuaries are monitored monthly by the Queensland
Government as part of the Ecosystem Health Monitoring Program (EHMP) (http://www.health-e-
waterways.org). The EHMP includes an Ecological Health Index based on algal productivity derived
from measurement of chlorophyll a, concentrations of dissolved oxygen, major nutrients and turbidity.
The conformation of these variables with national guideline values, together with estimates of seagrass
and riparian vegetation cover is used to develop an annual report card for each estuary (Table S1 in
Supplementary material).
The five estuaries were located no more than 190 km apart (Fig. 1) and represent a range of ecological
conditions (Table 1). There were large differences in morphology between the five estuaries, with the
Currumbin Creek considerably smaller than the others (Table 1). Within each estuary, five sites were
sampled, and in all but two cases (one in each the Noosa and Currumbin) the sites have been routinely
monitored under the EHMP.
Collection and analysis of environmental variables
Sediment collection was confined to non-sandy substrates. Five sediment samples were collected at each
site from ca. 2 m below low water using a Van Veen grab. Sub-samples were taken from the surficial
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layer (1.5-2 cm) of each sample for DNA, grain size and total organic carbon analysis. All samples for
DNA analysis were transferred into clean 50 mL Greiner tubes and placed on ice immediately, then
frozen within 6 h of collection and thawed only just prior to DNA extraction. All materials used for the
collection and storage of DNA samples were pre-rinsed for at least 24 h in 5% sodium hypochlorite, and
rinsed thoroughly five times with Milli-Q water (Millipore, Academic Water Systems, Australia). To
minimise cross contamination, sediments were only sub-sampled from the centre of each grab sample.
The physico-chemical properties of the water column were measured at each sampling site
approximately 0.5 m above the sediment surface using a calibrated YSI 6920 multi-sonde. At all EHMP
sites (excluding one site in both the Noosa and Pine), water samples were collected for nutrient and
chlorophyll a analyses. Water samples for nutrient analysis were filtered upon collection, with the filtrate
stored in clean foil-wrapped containers stored on ice. Total phosphorus, filterable reactive phosphorus,
total nitrogen, organic nitrogen, inorganic nitrates, ammonia and chlorophyll a analyses was performed
using standard methods (Clesceri et al., 1998). Total organic carbon (TOC) and grain size analysis for the
following grain size classes: <63 μm (fines), 0.63 μm- mm (sand), >1 mm (coarse) were performed as
previously described (Chariton et al., 2010c).
DNA extraction, amplification and sequencing
DNA was extracted from 1.5 g of sediment and purified using UltraCleanSoil DNA extraction kits
(MO BIO, Carlsbad, CA) following the manufacturer's protocols. In addition to the sediment samples,
three internal reference samples containing sixteen clones from a range of eukaryotic taxa were also
processed, as previously described (Morgan et al., 2013). Polymerase chain reaction (PCR)
amplification of a 200e500-bp fragment of the 18S rRNA gene was carried out with the universal
primers All18SF-TGGTGCATGGCCGTTCTTAGT and All18SR-CATCTAAGGGCATCACAGACC
(Hardy et al., 2010), and sample preparation was conducted as previously described (Baldwin et al.,
2013). Sequencing was performed by the Australian Genome Research Facility (St Lucia, Queensland)
using a single plate of Roche 454 GS FLX Titanium. Demultiplexing and the removal of potential PCR
artefacts, sequencing errors and chimeric sequences were performed using the Amplicon Pyrosequence
Denoising Program (APDP) (Morgan et al., 2013). Taxon identification of each unique sequence,
herein referred to as a Molecular Operational Taxonomic Unit (MOTU) was inferred using the RDP
classifier with the SILVA 18S rRNA database (release 113) (www.arb-silva.de).
Statistical analysis
As there is a weak statistical relationship between the number of sequence reads and organism biomass
or abundance (Egge et al., 2013), all MOTU data were converted to presence or absence prior to
computation (Chariton et al., 2014). Ordination of MOTU data was performed by non-metric
multidimensional scaling (nMDS) using the Jaccard similarity coefficient in the Primer 6 þ statistical
package (Plymouth Marine Laboratory, UK). Statistical differences between estuaries were tested by a
two-factor permutational multivariate analysis of variance (PERMANOVA), with sitesnested within
estuary. Differences between treatments were identified by pairwise a posteriori tests based on 9999
random permutations. The proportions of explained variation at spatial scales of estuary, site and
residual were calculated using the procedure described by Quinn and Keough (2002). Differences in
richness of total MOTU and dominant taxonomic groups were examined using a two-factor nested
ANOVA. Residuals were assessed for skewness, kurtosis, and omnibus normality using D'Agostino's
tests (D'Agostino et al.,1990) with homogeneity of variances examined using a modified Levene equal
variance test (Levene, 1960). When assumptions of homogeneity were violated, appropriate
transformations were performed (Sokal and Rolf, 1995). In cases in which the data remained
heteroscedastic, the level of statistical significance was set at P < 0.01. All ANOVAs were performed
in NCSS v8 (NCSS, Kaysville, UT). MOTUs indicative of each estuary and combinations of estuaries
were identified using the R package Indispecies, with Indictor Values (IV) reflecting both the
conditional probability of the MOTU as an indicator of a particular estuary and the probability of
finding the MOTU in samples associated with the estuary. In addition to the package's multipatt
function, the signassoc function was used to determine whether the occurrences of each potential
indicator MOTU identified by the multipatt analysis were random and to correct for multiple testing.
The relationships between community composition and environmental variables were examined
using distance-based linear models (DISTLM) (Legendre and Anderson,1999). In order to match the
number of biological and physico-chemical samples, i.e. one sample per site, the similarity matrix for
the biological data was recalculated using the distance between centroids for each site. The two sites
where nutrient data were unavailable were discarded from the analyses. Following the procedure of
Bellchambers et al. (2011), an initial analysis was performed using forward selection of all
environmental variables with the goodness-of-fit examined using Akaike's information criterion. The
most parsimonious model was re-run using only the variables selected for this model and distance-
based redundancy analysis (dbRDA) was performed to visualise the influence of predictor variables
identified by the DISTLM. Threshold Indicator Taxa ANalysis (TITAN) in R was used to estimate
MOTUs whose occurrences declined (z scores) or increased (þz scores) and to examine community
thresholds along the significant environmental gradients identified from the DISTLM's sequential tests
(total phosphorus, mono-nitrogen oxides, turbidity and pH). TITAN is an extension of indicator
analysis which partitions the biological data into two groups at the value of a predictor variable that
maximizes the association of each taxon (or MOTU) with each side of the partition. Using standardized
z-scores, TITAN can distinguish those taxa whose occurrences declined (z scores) or increased (þz
scores) along the environmental variable. Prior to running TITAN, all MOTUs which were observed in
fewer than three samples or and in more than 95% of samples were removed. Peak values in z and þz
scores were used to respectively determine negative and positive community responses to the
environmental variable. Bootstrapping was used to estimate the confidence limits of the change points
(King and Baker, 2010).
Results
Sequencing
The pyrosequencing run produced >1.3 million sequences. The dataset for the sample estuaries contained
2937 MOTUs after all potentially erroneous sequences had been removed and sample rarefaction to 2093
reads. The data are accessible via the CSIRO data portal http://dx.doi.org/10.4225/08/52DF4D8008B99.
For all three internal reference samples, the APDP bioinformatics pipeline correctly identified only the
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16 clone sequences as valid. As such, we consider our measurement of sample richness to reflect the
truevariant richness of the targeted region. The largest proportion of MOTUs that could be confidently
assigned to a kingdom (59%) belonged to the Metazoa (16%). The eukaryote supergroups Chromista and
Chromalvelolata each contributed 10% to the total taxon richness and fungi, Rhizaria and Viridiplantae
contributing 6e8%.
Physico-chemical attributes of the estuaries
A summary of the physico-chemical attributes of the estuaries and relevant Australian water quality
guideline values are presented in Supplementary Material Tables S2 and S3. All estuaries had total
nitrogen (TN) concentrations that exceeded the Australian guideline value (ANZECC/ARMCANZ,
2000), with all sites within the Logan Estuary exceeding this value by at least four-fold. With the
exception of the Noosa Estuary, guideline values for mono-nitrogen oxides (NOx), total reactive
phosphorus (TRP) and total phosphorus (TP) were exceeded in almost all samples. On average, the
Logan Estuary had TRP and TP concentrations 32 and 10 times greater than the guideline values,
respectively. Chlorophyll a concentrations exceeded guideline values in both the Maroochydore and Pine
estuaries. It is emphasised that the physico-chemical attributes of the estuaries are highly variable, with
measurements taken at the time of sampling occasionally deviating from the mean values observed over
of the previous six months (Figs. S1 and S2 in Supplementary material). Most notably, in the Pine and
Currumbin, pH and concentrations of total nitrogen and dissolved organic nitrogen at several sites were
considerably elevated, with conductivity also being substantially below the long term mean in the some
of the Pine Estuary sites. These deviations reflect the influence of a substantial rainfall event which
occurred during sampling. There were some inter-estuarine variation in physicochemical properties
(Tables S2 and S3 in Supplementary material), but the Noosa, Maroochydore, Pine and Currumbin
mostly had similar profiles while those of the Logan Estuary were markedly different: the overlying
waters were more turbid (Secchi depth) and hypoxic. Furthermore, the low conductivity and pH of the
Logan water column indicates that the estuary was being driven by freshwater inputs at the time of
sampling. The environmental data indicates that sites from the Noosa Estuary were relatively
homogeneous and contained the lowest concentrations of nutrients. At the time of sampling, the Pine,
Maroochydore and Currumbin estuaries contained environmentally significant concentrations of
nutrients, i.e. exceeded regional trigger values, with the relative contribution of each nutrient and its
inorganic and organic components varying between estuaries. The water of the Logan Estuary was highly
eutrophic and turbid.
Ecological comparisons between estuaries
As illustrated by the accumulation curve (Fig. S3), 125 replicate samples were sufficient to account for
>99.8% of the 2941 MOTUs estimated (Chao 2) that occur in the five estuaries. The 25 replicates
collected in each estuary accounted for between 79% (Pine) and 90% (Logan) of the estimated eukaryote
richness. Total MOTU richness varied significantly within the estuaries (ANOVA: F = 4.43, P < 0.001).
The Logan Estuary (mean 391 ± 8 SE) had higher MOTU richness (ANOVA: F = 7.69, P < 0.001) than
the other four estuaries, all of which had similar richness (Noosa 294 ± 11; Maroochydore 259 ± 9; Pine
301 ± 6; Currumbin 256 ± 13). The high MOTU richness of the Logan Estuary was due to richer fungal
(ANOVA: F = 20.78, P < 0.001) and protozoan (ANOVA: F = 10.2, P < 0.001) communities.
Chromoalveolata communities were richer in the Logan Estuary (ANOVA: F = 8.0, P < 0.001).
Metazoan richness was lower in the Noosa and Maroochydore estuaries, with the Pine and Currumbin
estuaries containing the richest metazoan communities (ANOVA: F = 9.6, P < 0.001).
The composition of the benthic biota varied within (PERMANOVA: F = 2.90, P = 0.001) and between
the estuaries (PERMANOVA: F = 5.71, P = 0.001), with post-hoc analysis identifying that all five
estuaries contained significantly different assemblages (P < 0.01). Spatial scale was shown to influence
variability, with approximately half of the variation occurring from sample to sample (residuals =
0.52%). Variation in composition at the largest scale of estuary(28%) was more important than that at
the intermediate scale of site(20%).
As illustrated by the nMDS ordination plot (Fig. 2), the greatest disparity in benthic eukaryote
composition was between the largely unmodified Noosa Estuary and highly eutrophic Logan Estuary
(similarity 5.6%), with both containing markedly different compositions to the other estuaries. While the
compositions of the other three intermediately modified estuaries did differ, their compositions were
more similar to each other (similarity 15.1-15.9%) than to either the Noosa (similarity 7.0-10.8%) or
Logan (similarity 8.1-8.8%).
Indicator analysis identified 426 MOTUs which were characteristic of the estuaries at the time of
sampling (Fig. 3). The largest proportions of these were in the Logan (38%) and Noosa (28%) estuaries.
MOTUs with the ten highest Indicator Values (IV) for each estuary are provided in the Supplementary
material (Table S4).
Relationships between benthic communities and environmental variables
The most parsimonious distance-based linear regression model which used 15 of the original 16 variables
(excluding percentage silt) explained 71.3% of the total variation in benthic community structure. The
first dbRDA coordinate axis explained 19.6% of the total variation in the benthos (Fig. 4), with high
concentrations of nutrients (total phosphorus, total nitrogen, organic nitrogen) and high turbidity clearly
separating communities of the Logan Estuary from the others. The second dbRDA coordinate axis
explained 14.4% of the total variation and indicated that compositional changes within Noosa and
Maroochydore estuaries were correlated with variation in conductivity, pH and temperature. Oxygen
saturation, concentrations of NOx and ammonia, and primary production assisted in distinguishing the
benthic communities from the Pine and Currumbin from the Noosa Estuary. Benthic community
composition was significantly correlated with TP (18.6%, P = 0.01), NOx (7.9%, P = 0.01), turbidity
(8.1%, P = 0.004) and pH (6.12%, P = 0.009). The first three of these variables were symptomatic of
eutrophication and clearly separated the Logan Estuary's benthos from the rest (Fig. 4).
Threshold analysis (TITAN) identified 330 MOTUs whose occurrence (i.e. number of times identified
as present) responded negatively (z scores) to increasing TP concentration (Table S5 in the
Supplementary material), with 40% of those which could be taxonomically assigned belonging to
Bacillariophyta (mostly Bacillariophyceae). The most sensitive indicators of elevated total phosphorus
(i.e. largest z scores) observed across the five estuaries were all bacillariophytes and foraminiferans (Fig.
5). As indicated by their synchrony in change points and consistently small percentile ranges, declines in
the presence (i.e. occurrence) of phosphorus sensitive taxa were generally abrupt, with the most
pronounced loss of sensitive taxa occurring at a TP concentration of 24 mg L-1 (22-34 mg L-1, 5-95
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percentile; Fig. 5). In contrast, 465 MOTUs had significant þ z scores in response to increases in TP
(Table S5). Many of these were identified as potential indicator MOTUs for the Logan Estuary. The
MOTUs with the largest þ z scores included Dinophyceae, Coscinodiscophyceae, Annelida, Gastrotricha,
Rotaliida and Micronuclearia (Fig. 5). The confidence limits for these were broad indicating occurrence
over a wide range of TP concentrations. The largest peak in TP tolerant MOTUs was at 100 mg L-1 (100-
290 mg L-1), with smaller peaks also occurring at 180 and 260 mg P L-1. The community threshold (or
change point), i.e. the point where a maximum change in composition occurred was at TP = P 185 mg L-1
(26-269 mg L-1).
There were some strong similarities between the responses of MOTUs to TP and to NOx and turbidity
(Table S5 in the Supplementary material), with 127 MOTUs responding negatively to all three variables
which are generally elevated in eutrophic estuaries. These MOTUs all shared small tolerance ranges (5-
95 percentiles) and a high representation of MOTUs from Bacillariophyceae, Arthropoda (notably
Crustacea) and Rotaliida (Table S5 in the Supplementary material). There was peak decline in MOTUs
when NOx concentrations reached 5 μg N/L (4-12 μg N/L). No such clear synchronous decline was
observed for turbidity-sensitive MOTUs, with the change point occurring 16 NTU with 9-95 percentiles
encompassing a wide turbidity range (15-68 NTU). Some 265 MOTUs that responded positively to
increases in TP also responded positively to increases in NOx and turbidity (Table S5). They were from
Coscinodiscophyceae, Heliozoa (Heterophryidae), Ciliophora, Ascomycota and Chytridiomycota,
Apusozoa (Rigifilida), Choanozoa and Kinorhyncha (Echinoderes spp). The sum (z+) change points for
NOx and turbidity occurred at 100 μg N/L (79-130 μg N/L) and 134 NTU (49-145 NTU), respectively.
The community threshold for NOx occurred at the same concentration (5 μg N/L, 4-15 μg N/L) as the
sum (z+) change point. The most pronounced shifts in MOTU composition occurred when the overlying
waters reached a turbidity of 101 NTU (49-145 NTU).
The observed pH gradient reflects the transition from fresh to marine waters. Some 433 MOTUs had
significant z scores and occurred less frequently as the water became more marine (Table S5 in the
Supplementary material). Declines began at pH 7 and were maximal at pH 7.06 (6.96-7.31, 5-95
percentile), coinciding with the mean community change-point (pH = 7.06, 6.96-7.32). Some 9% of the
Bacillariophyta MOTUs showed pronounced declines in occurrence as pH increased (Table S5). The
most sensitive taxa were the protistan Micronuclearia, Dinophyceae and Thalassiosirophycidae
(Coscinodiscophyceae) (Fig. 5). The relatively broad percentile ranges of many of the MOTUs indicated
they could persist at a pH 0.5 greater than their mean change points. Some 279 MOTUs responded
positively to an increase in pH (þz scores), with a change point occurring at pH 8.04 (7.91-9.08). MOTUs
that showed increased occurrence with increased pH of waters were generally observed within a
relatively small pH range. Some 33% of bacillariphytes responded positively to increased pH, as did
Apicomplexa, Ciliophora, Cnidaria, Foraminifera and Labyrinthista (Table S5). The most responsive
MOTUs to the increase in pH were Rotaliida, Bacillariophyceae and Arthropoda (Fig. 5).
Discussion
Comparisons between estuaries
Taxonomic richness is a commonly used index for comparing benthic communities, with the assumption
that endemic taxon richness will be lower in disturbed environments (Dauer, 1993). Previously, we have
cautioned against the use of MOTU richness in DNA-derived ecological studies, as bioinformatic
pipelines generally overly inflate true sequence richness (Chariton et al., 2014). As shown in the present
study, and that by Morgan et al. (2013), an accurate measure of sequence richness (i.e. the number of true
variants of the targeted region) can be obtained using the bioinformatics pipeline APDP, and it is timely
to examine the potential usefulness of MOTU richness as an ecological metric. The underpinning trend
from estuarine macrobenthic studies is that eutrophication leads to a pronounced reduction in richness,
generally in the order of 30% (Johnston and Roberts, 2009). The Logan Estuary is significantly modified,
with the adjacent catchment (3076 km2) now 12% urbanized and the remainder under a variety of
agricultural uses including beef, chicken and lawn farms in the mid reaches; prawn farms, sugar cane
farms and rural residential in the estuarine reaches. There are also two major sewage treatment plants
(STPs) in this system: The Loganholme and Beenleigh STPs, which collectively discharge an average
920 ML/ day into the Logan Estuary. As a consequence the Logan estuary is considered highly
eutrophic. Nevertheless, we observed relatively high biotic richness in the Logan Estuary, with richness
being 25-35 percent greater than that of the other four less disturbed estuaries. We have observed a
similar trend when comparing a heavily contaminated and moderately disturbed estuary (Chariton et al.,
2010b). Protozoan and fungal richness in particular was greater in the Logan than the other estuaries. We
observed no difference in MOTU richness between the other four estuaries. This was surprising given the
marked differences in catchment size, land use, salinity gradients, nutrient concentrations and other
variables commonly shown to alter composition (Archambault and Bourget, 1996; Dauer et al., 2000;
Remane and Schlieper, 1971; Witman et al., 2004).
Many MOTUs specific to the Logan Estuary were associated with the breakdown of detritus material
(Sandgren et al., 1995). These included Oomycetes which have been shown to proliferate in
environments subjected to high organic inputs, aggressively outcompeting fungal species (Newell and
Fell, 1995, 1997) and Order Pythiales, parasites known to affect humans, fish, plants, fungi and insects
(Yu, 2001). The Logan Estuary also contained large volumes of catchment-derived material, as sampling
was performed during an unseasonal high rainfall event (255 mm in February, 2010; median since 1960
¼ 107 mm) (http://www.bom.gov.au/climate/data/; sampling station 040312). Consequently, the high
richness in the Logan estuary was due to MOTUs derived from extraneous sources (e.g. sewage
treatment plants and agriculture) as well as from organisms which were inherently part of the estuary's
biocoenosis. By contrast, in the Noosa Estuary, a large proportion of the richness was associated with
diatoms and a less diverse metazoan fauna. Collectively, our findings suggest that total MOTU richness
is not a sensitive indicator of ecological condition. While a greater breadth of studies is required to
support or refute these patterns, the initial signs suggest that the richness of key taxonomic groups
appears more sensitive than total MOTU richness to environmental conditions.
Diatoms along with protozoans, Chromoalveolata and fungal phyla exhibit characteristics which
potentially make them informative indicators of ecological condition. These include their trophic
positioning and responsiveness to a range of environmental contaminants (Harding et al., 2005;
McCormick and Cairns,1994). Although there is a wealth of information describing broad univariate
responses in diatoms to eutrophication, there is currently a paucity of information regarding the structural
attributes of naturally functioning diatom communities and how these may change in response to
eutrophication and other environmental stressors. Pronounced changes in diatom composition have
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frequently been shown to correlate with environmental degradation (Desrosiers et al., 2013; Snoeijs,
2013). In our study, diatom composition (derived from presence/absence) was a strong feature of the
largely unmodified Noosa Estuary, with more than 40% of the indicator MOTUs associated with families
within Bacillariophyceae. Palaeoecological evidence capturing 2500 years of history of the Great Lakes
further supports strong correlations between eutrophication and land-use and a decline in diatom richness
(Cooper and Brush, 1993). By contrast, only 7-12 % of the indicator MOTUs for the other estuaries were
diatoms, with these more commonly associated with Coscinodiscophyceae.
While still containing different compositions, the three estuaries (Maroochydore, Pine and Currumbin)
which have been historically shown to be in similar ecological condition (EHMP scores C to C-), were
more similar to each other than either the Noosa or Logan estuaries. As one of the metrics used to
calculate indicator scores is a MOTU's specificity to a particular estuary (De Caceres et al., 2010), the
indicator scores for the MOTUs from these three estuaries were generally lower than those from the
Logan and Noosa estuaries. MOTUs whose increased occurrences aided discrimination between the
Maroochydore, Pine and Currumbin estuaries were derived from a wide breadth of taxonomic groups. In
particular, the Pine estuary had a number of metazoan indicator MOTUs, including nematodes, rotifers
and turbellarians. Indicator MOTUs associated elsewhere for bacillariophytes and ciliophorans were
common to all three estuaries.
We found that the largest proportion of variance occurred at the smallest spatial scale, that is, between
samples. This suggests that benthic community composition is driven by small-scale localised processes,
e.g. competitive interactions, habitat heterogeneity and disturbance events (Archambault and Bourget,
1996; Thrush et al., 1996; Whitlatch and Zajac, 1985). While Anderson et al. (2005) proposed that
similar patterns are likely to occur in other biological assemblages, the present study is the first to
demonstrate the preservation of this pattern using presence/absence compositional data simultaneously
derived from numerous phyla. The low level of variation which occurred at the site scale (20%) reflects
the lack of true independence between sampling sites due to the ebb and flow of the overlying waters.
Variation at the estuary scale was more pronounced than at the site scale. While all five estuaries were
located within the southeast Queensland biogeographical zone and shared the same oceanic input (Pacific
Ocean) e although the Pine and Logan estuaries are buffered by Moreton Bay e marked differences in
catchment size, channel morphology, land use, salinity profiles and nutrient concentrations undoubtedly
contributed to biological variation at the estuary scale. Biological connectivity within estuaries can occur
via active migration, passive movement via the water column, or adherence to other organisms, sediment
and organic material. Unique to DNA surveys is that patterns in intra-estuary connectivity are
undoubtedly distorted by the inadvertent sequencing of DNA which is either adhered or retained within
the gut contents of the targeted biota (Chariton et al., 2010b).
Relationships between biological composition and environmental variables
Whilst nutrient inputs are critical for estuarine functioning, extensive changes in the land use of south-
east Australia have profoundly altered the timing of riverine flows and their nutrient loadings into the
region's estuaries, altering the ecological compositions of environments and potentially creating
conditions ideal for cyanobacterial blooms (Davis and Koop, 2006; Quigg et al., 2010; Roy et al., 2001).
In the present study, elevated concentrations of total nitrogen were reported in all five estuaries, however,
elevated concentrations of nitrogen (total, organic, ammonia) and phosphorus (total and filterable
reactive) were clearly greater in the Logan Estuary, reflecting its current land uses. A majority of the
variables which explained a significant proportion of the variation in benthic community data were either
directly (e.g. total phosphorus and mono-nitrogen oxides) or indirectly (turbidity) associated with
excessive nutrient inputs (Fig. 4). Consequently, it was unsurprising that the constrained analysis clearly
separated the Logan Estuary's benthic communities from those of the other four estuaries (Fig. 4). To a
lesser degree, differences in NOx concentrations differentiated the Currumbin and the Pine estuaries
from the Maroochydore and Noosa estuaries.
Threshold analysis (TITAN) indicated that there was a pronounced decline in TP sensitive taxa
(change point in z scores) when the mean overlying water reached 24 μg P/L (23-34 μg P/L, 5 and 95
percentiles), which is close to the Australian default water quality guideline for south-eastern Australian
estuaries of 30 mg P/L (ANZECC/ARMCANZ, 2000). As measurements of TP and other environmental
variables, including their co-variates, can vary dramatically across a range of temporal scales (e.g. tides,
seasons and run-off events) it is highly unlikely that there is absolute synchrony between water column
concentrations of nutrients and the turnover rates of benthic communities. As such, in the current study
the derived threshold values are considered to be notional, with the additional information regarding the
tolerances of MOTUs being obtained from the width of the percentiles created from the threshold
analysis.
Diatoms were the most responsive group to TP, with the occurrences of a large proportion of MOTUs
declining with increasing concentrations of TP. With the exception of Thalassiosira, a common attribute
of the most sensitive MOTUs was their relatively small tolerance ranges (Fig. 5), with the occurrences of
the taxa declining rapidly as indicated by their narrow percentile range. In contrast, the MOTUs which
responded favourably to TP, NOx and turbidity were generally present across a wide range of
concentrations (Fig. 5). While nutrient addition has been shown to stimulate algal biomass (Anderson et
al., 2002; Hecky and Kilham, 1988), our findings emphasize that compositional change also occurs, with
diatom richness being substantially reduced by elevated concentrations of TP. The ecological
ramifications of a compositional shift in diatoms is difficult to define, however, because of their rapid
generation time (days to hours), evidence of a shift in diatom composition may be a precursor for
subsequent changes in ecological integrity, e.g. trophic bottle necks and cyanobacteria blooms
(Desrosiers et al., 2013; Logan and Taffs, 2013; Snoeijs, 2013). The latter can be determined by the
inclusion of the chloroplast 16S rRNA gene in subsequent studies.
There were many commonalities between those MOTUs which responded positively to TP, NOx and
turbidity (Table S4 in the Supplementary material). In addition to the Apusozoa and dinoflagellate
MOTUs, these included relatively large proportions of the MOTUs representative of Heliozoa,
Ciliophora and Choanozoa.
Highly diverse protozoan communities are commonly associated with sewage treatment plants
(Madoni, 1994), and consequently their observed increase in occurrence is most likely to be associated
with the sewage treatment plants within the Logan Estuary catchment, a primary source of the estuary's
nutrient enrichment. Further evidence of this was the increase in the occurrence of fungal MOTUs from
the Phyla Ascomycota and Chytridiomycota. Increases in the abundance of chytrid fungi have been
shown to occur in organic materials derived from sewage plants (Novinscak et al., 2009). Interestingly,
11
some chytrids are parasitic and can cause dramatic shifts in diatom populations, with this phenomenon
potentially contributing to the observed decline in diatoms (Bruning et al., 1992). Collectively, the
findings from the current study show a strong correlation between anthropogenic induced changes to the
estuaries and benthic composition, with the communities shifting from primary producing taxa (e.g.
diatoms) to ones dominated by protozoans and fungi, and other taxa associated with the consumption of
bacteria and the breakdown of organic material.
In those estuaries that were less influenced by nutrients, e.g. Noosa and Maroochydore, compositional
changes were more strongly correlated with pH, with an increase in pH reflecting an increase in the
influence of marine waters (Bianchi, 2012). In general, taxa which preferred low pH waters were found
at a pH below 7.06, with most taxa persisting across a relatively broad pH range. It should be noted that
the Logan Estuary had the lowest pH, and as such, other variables which contributed to the composition
of this estuary (i.e. nutrients and turbidity) may also be contributing to the perceived composition of
lower pH environments. The pH levels can vary dramatically within estuaries, however, as indicated by
the synchrony between the mean z scores and community change point (from TITAN analysis), and the
relatively narrow tolerances of the taxa associated with the higher pH waters, even relatively small
changes can substantially alter biotic composition.
For many researchers, the ability to obtain compositional data from the amplification of DNA/RNA
provides an exciting prospect, increasing the pool of taxa which can be included in biological surveys
and monitoring; enabling the identification of cryptic or decomposed organisms (e.g. in gut contents); as
well as potentially reducing the costs, latency and identification issues associated with traditional
surveys. DNA-based monitoring is in its infancy and as such considerable research is required to further
develop, refine, and evaluate the utility of the approach. One major limitation is the unreliability of
proportional data derived from PCR-based approaches, and hence our use of presence/absence data. To
address this limitation we have recently resequenced three of the estuaries system using a PCR-free
metagenomic approach (Chariton in prep.). Although we have demonstrated the capacity for
metabarcoding to discriminate between the five estuaries, show correlative patterns between composition
and the major environmental variables, and identify those taxa which responded positively and
negatively to the key environmental variables; it is emphasised that the present study was from a single
sampling event and flows can greatly vary. Consequently, repetitive temporal sampling is required to add
credence or refute the observed trends. To address this knowledge gap we resampled the five estuaries in
2012 using an expanded program (additional sites, particulate metals and organic contaminants,
additional genes, and a large sediment volume for DNA extraction). Although there were differences in
estuary specific MOTU indicators, the broad differences among the estuaries and the relationships
between biotic assemblages and the key environmental variables (e.g. total phosphorus, turbidity and pH)
remained unchanged (Chariton in prep.).
The current approach used by the EHMP to establish the ecological condition of estuaries uses data
solely derived from water quality parameters and habitat condition, and excludes ecological data. The
aim of the presented approach is not to replace traditional sampling programs but rather, to add an
additional line of ecological evidence which encompasses a greater breadth of diversity. As with all
monitoring programs, a considerable wealth of data is required to identify predictable patterns and to
understand the ecological ramifications of any observed changes in community composition. Only when
additional data has been obtained and methodological issues refined can the utility of this approach be
fairly evaluated.
Acknowledgements
Financial support for the project was provided by the CSIRO Oceans and Atmosphere. The authors wish
to thank John Ferris and James Fells (Queensland Environmental Protection Agency) for their assistance
in the collection of samples, Ray Williams (Queensland Department of Environment and Heritage
Protection) for facilitating access to the physico-chemical data, and Chris Moeseneder (CSIRO) for his
map illustration.
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15
Table 1 Morphological characteristics and environmental condition of the five estuaries
Estuary
Distance
upstream
sampled
(km)
Catchment
area (km2)
Condition
EHMP
score
(2010)a
Entrance
Noosa
Maroochydore
18.6
25.3
841
630
Largely
unmodified
Modified
C
Pacific
Ocean
Pacific
Ocean
Pine
Currumbin
10.5
5.3
806
54
Extensively
modified
Modified
C
C
Morton
Bay
Pacific
Ocean
Logan
23.0
3822
Highly modified
F
Morton
Bay
Information compiled from www.ozcoasts.gov.au and www.health-e-waterways.org. a Indicates ecological condition derived
under the Ecosystem Health Monitoring Program (EHMP)
Figure 1. Locations of the five estuaries (Noosa, Maroochydore, Pine, Logan and Currumbin) sampled
within south-east Queensland (Australia). Bold letters represent EMPH Report Card scores for 2010 (see
Table 1 for details). Shaded areas indicate the catchments for the estuaries
17
Figure 2. nMDS plot illustrating the similarities and differences in the compositions of benthic eukaryotic
communities from the five estuaries. The shading of site markers indicates their position from upstream
(light) to downstream (dark)
Figure 3. Summary of the indicator analysis illustrating the relative proportion of MOTUs associated
with each taxonomic group for each estuary. Bracketed values after estuary names
represent the total number of potential indicator MOTUs identified in each estuary
19
Figure 4. A dbRDA ordination plot illustrating the relationships between benthic community structure
and the measured environmental variables. Sites are derived from their distances among centroids
Figure 5. Summary of Threshold Indicator Taxa ANalysis (TITAN) results illustrating the change points
and 95% confidence limits for the top 20 (highestz and þz scores) significant MOTUs for key
environmental gradients: Total phosphorus (TP); Mono-nitrogen oxides (NOx); turbidity; and pH. Black
and orange points indicate MOTUs with z and þz scores, respectively. The size of the points is scaled to
reflect the magnitude of their response (z scores)
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... (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Lawes et al., 2016Lawes et al., , 2017Santi et al., 2019;Stoeck et al., 2018) and eukaryotic (Chariton et al., 2015;Santi et al., 2019) communities to be sensitive indicators of enrichment, with diatom and bacterial communities often responding more strongly than general eukaryotes (Birrer et al., 2018;Minerovic et al., 2020;Pochon et al., 2019). Clear shifts in eukaryotic and bacterial indicator taxa were seen in response to nutrient loading but indicator taxa common to both sites were restricted to bacterial communities. ...
... The use of DNA sequencing methods detects even rare taxa and cryptic life stages and allows diversity comparisons even when only a fraction of the species is described and, thus, often exceeds conventional diversity assessments in terms of taxonomic resolution, precision and sensitivity (Zaiko et al., 2018). Thus, metabarcoding is an excellent tool in ecological monitoring (Chariton et al., 2015;DiBattista et al., 2020). ...
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Tidal marshes are among the most valuable, productive, and vulnerable ecosystems with high biodiversity. Louisiana’s saltmarshes are endangered by natural and man-made stressors, including oil pollution, saltwater intrusion, and land loss due to sea level rise and erosion. Freshwater diversions have been planned to restore sediment input from the Mississippi River to rebuild marsh habitats in South Louisiana. These proposed diversions will undoubtedly change salinity levels, which is a major controlling factor in the distribution of marsh organisms, including those in soil; however, detailed pre-event inventories are lacking. This study describes the diversity of metazoan meiofauna (organisms between 45 and 500 μm) and environmental DNA in marsh soil collected in 2018 from Barataria and Caillou Bay, Louisiana, across three salinity zones and four distances from the marsh edge. Diversity analyses using 18S rRNA gene metabarcodes identified salinity as a factor impacting soil metazoan composition. Nematoda and Mollusca were equally distributed across salinity zones. Gastrotricha, Bryozoa, Rotifera, and Platyhelminthes were more prevalent in low salinity while Kinorhyncha were not detected in low salinity. Annelida and insects were equally common in low and high salinity but less in mid salinity. Five nematodes ( Eumonhystera filiformis , two Prismatolaimus spp., Anoplostoma sp., and Prodorylaimus sp.), two annelids ( Marionina southerni and Dendronereis aestuarina ), two platyhelminthes ( Rhynchoscolex simplex and Olisthanella truncula ), the gastrotrich Chaetonotus novenarius and four collembola and ostracods appear to be low salinity bioindicators and are expected to expand range with freshwater diversions. No frequently detected organisms were unique to mid or high salinity zones, but four Nematoda ( Meleidogyne spartinae , Prochaetosoma sp., Halalaimus sp., and Dichromadora sp.), two Annelida ( Alitta succinea and Namalycastis jaya ), two Platyhelminthes ( Macrostomum kepneri and Mesorhynchus terminostylis ), and one Kinorhyncha ( Echinoderes sp.) were never detected in low salinity zones. None of the frequently detected taxa were unique for a particular distance from the marsh edge or bay. This dataset will be useful as baseline for assessing how soil communities will change in response to salinity changes caused by freshwater diversions and saltwater intrusion as well as measuring the environmental impact of pollution and other stressors.
... The various organisms in salt lakes play an important role in increasing the diversity and maintaining the balance of salt lake ecosystems [4]. Benthic eukaryotes can modify sediment habitats [5][6][7], and their participation in the formation of benthic food webs makes an essential contribution to the material cycling and energy transfer of aquatic ecosystems [8,9]. In addition, benthic eukaryotic communities are susceptible to changes in external environmental conditions [10]. ...
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Eukaryotes exist widely in aquatic ecosystems. It is of great importance to study their species composition, diversity, and relationship with environmental factors to protect and maintain ecosystem balance. Salt lakes are essential lakes rich in biological and mineral resources and have significant research value. To understand the characteristics of eukaryotic diversity in salt lake sediments, we conducted a sampling survey of the benthos in Kyêbxang Co, Tibet, in July and August 2020. The sampling area was divided into littoral, sublittoral, and profundal zones. A total of 42 species of Metazoa, 159 species of Protozoa, 63 species of Viridiplantae, and 46 species of Fungi were identified by the high-throughput sequencing of 18S ribosomes. Alpha diversity analysis revealed significant differences in species composition among the three study zones. The littoral zone had the highest Sobs index and Chao index, indicating that the eukaryotic diversity and richness in this zone were significantly higher than those in the profundal and sublittoral zones. Redundancy analysis (RDA) showed that water depth, temperature, and sediment organic matter content significantly affected the community structure of eukaryotes zones, especially the distribution of dominant genera such as Dunaliella, Psilotricha and Brachionus. Cooccurrence network analysis showed that Dunaliella, Aphelidium, temperature, water depth, and organic matter represent essential nodes in the entire network. This study can provide baseline data and new insights for eukaryotic diversity research for salt lakes.
... In the large Elbe River estuary in Germany, eDNA metabarcoding revealed that tidal flows influenced species detection: downstream sampling locations were similar to sites further upstream during low tide, but species composition changed at the downstream sites at high tide (Schwentner et al. 2021). Other eDNA approaches such as indirect detection using sediment eDNA (e.g., Chariton et al. 2015;Lallias et al. 2015) and direct detection of plankton (Abad et al. 2016;Jungbluth et al. 2021) have been used in estuaries. Taken as a whole, the estuarine eDNA literature is limited, but growing. ...
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Environmental DNA (eDNA) approaches enable sensitive detection of rare aquatic species. However, water conditions like turbidity can limit sensitivity, resulting in false negative detections. The dynamics of eDNA detection in turbid conditions are poorly understood, but can be better characterized through experimental work. In this study, 1-L field-collected water samples were spiked with tank-sourced eDNA from a rare, endangered estuarine fish at concentrations similar to eDNA samples collected from the natural environment. Samples using non-turbid water (5 NTU), turbid water (50 NTU), and prefiltered turbid water were filtered using four filter types (pore size range 0.45 μm-10 μm). Detection success using a species-specific Taqman qPCR assay was assessed as both eDNA copy number and detection/non-detection. Glass fiber filters (nominal pore size 1.6 μm) yielded the highest number of eDNA copies and detections in non-turbid water and the highest detection rate in turbid water when used without a prefilter. Detection was a more robust metric for evaluating species presence across turbidity conditions compared with eDNA copy number. Prefiltration improved detection rates for the other filters tested (polycarbonate and cartridge and filters). Filter material and design appear to interact differently with the prefiltration step, and may be more important considerations than pore size for eDNA capture in turbid water. Interactions between eDNA particles, suspended particulate matter, and filters are important to consider for eDNA methods optimization and interpretation of rare species detections in turbid water.
... In the large Elbe River estuary in Germany, eDNA metabarcoding revealed that tidal flows influenced species detection: downstream sampling locations were similar to sites further upstream during low tide, but species composition changed at the downstream sites at high tide (Schwentner et al. 2021). Other eDNA approaches such as indirect detection using sediment eDNA (e.g., Chariton et al. 2015;Lallias et al. 2015) and direct detection of plankton (Abad et al. 2016;Jungbluth et al. 2021) have been used in estuaries. Taken as a whole, the estuarine eDNA literature is limited, but growing. ...
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Environmental DNA (eDNA) detection methods can complement traditional biomonitoring to yield new ecological insights in aquatic systems. However, the conceptual and methodological frameworks for aquatic eDNA detection and interpretation were developed primarily in freshwater environments and have not been well established for estuaries and marine environments that are by nature dynamic, turbid, and hydrologically complex. Environmental context and species life history are critical for successful application of eDNA methods, and the challenges associated with eDNA detection in estuaries were the subject of a symposium held at the University of California Davis on January 29, 2020 ( https://marinescience.ucdavis.edu/engagement/past-events/edna ). Here, we elaborate upon topics addressed in the symposium to evaluate eDNA methods in the context of monitoring and biodiversity studies in estuaries. We first provide a concise overview of eDNA science and methods, and then examine the San Francisco Estuary (SFE) as a case study to illustrate how eDNA detection can complement traditional monitoring programs and provide regional guidance on future potential eDNA applications. Additionally, we offer recommendations for enhancing communication between eDNA scientists and natural resource managers, which is essential for integrating eDNA methods into existing monitoring programs. Our intent is to create a resource that is accessible to those outside the field of eDNA, especially managers, without oversimplifying the challenges or advantages of these methods.
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Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all β-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.
Chapter
To identify and distinguish the biodiversity of the various organisms and plants, the efficient approach is needed to monitor the environment and its associated factors. Environmental DNA (eDNA) extracted from soil and water samples, however, can include taxa represented by both active and dormant tissues, seeds, pollen, and detritus in plant diversity and in animals tissues, liver, muscle like different organs were utilized for the experiment. Analysis of this eDNA through DNA metabarcoding provides a more comprehensive view of biodiversity of marine organisms and plant diversity at a site from a single assessment but it is not clear which DNA markers are best to be used to capture this diversity. Whatever the platform whether plant or marine animal target both the diversity studies require the same methodology such as sequence recovery, annotation, and sequence resolution among taxa were evaluated for four established DNA markers in silico using database sequences and in situ using high throughput sequencing of soil samples from a remote boreal wetland. Here, we describe a new skeleton to evaluate DNA metabarcodes and, contrary to existing statement, based on this by using current DNA barcoding markers rbcL and ITS2 for plant metabarcoding, we can take advantage of existing resources such as the growing DNA barcode database. This book chapter brings the new insights into the applications of eDNA approach to establishes the value of standard DNA barcodes for soil plant eDNA analysis in ecological investigations and biomonitoring programs.
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Indicator species are species that are used as ecological indicators of community or habitat types, environmental conditions, or environmental changes. In order to determine indicator species, the characteristic to be predicted is represented in the form of a classifi cation of the sites, which is compared to the patterns of distribution of the species found at the sites. Indicator species analysis should take into account the fact that species have diff erent niche breadths: if a species is related to the conditions prevailing in two or more groups of sites, an indicator species analysis undertaken on individual groups of sites may fail to reveal this association. In this paper, we suggest improving indicator species analysis by considering all possible combinations of groups of sites and selecting the combination for which the species can be best used as indicator. When using a correlation index, such as the point-biserial correlation, the method yields the combination where the diff erence between the observed and expected abundance/frequency of the species is the largest. When an indicator value index (IndVal) is used, the method provides the set of site-groups that best matches the observed distribution pattern of the species. We illustrate the advantages of the method in three diff erent examples. Consideration of combinations of groups of sites provides an extra fl exibility to qualitatively model the habitat preferences of the species of interest. Th e method also allows users to cross multiple classifi cations of the same sites, increasing the amount of information resulting from the analysis. When applied to community types, it allows one to distinguish those species that characterize individual types from those that characterize the relationships between them. Th is distinction is useful to determine the number of types that maximizes the number of indicator species.
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Competition experiments were performed using precolonized leaves or leaf disks of red mangrove Rhizophora mangle with: (1) disks containing pure cultures of single species of marine true fungi or species of Halophytophthora (the principal genus of marine oomycotes); and (2) leaves bearing bacterial films. Preoccupied leaves were exposed to natural microflorae in mangrove creeks at 2 Gays in the Bahama Islands, or placed in laboratory seawater enclosures wherein pairs of halophytophthoras were given equivalent opportunity to occupy fresh leaf material. The ubiquitous coastal-marine oomycote H. vesicula was found to be an able competitor versus true fungi and versus other halophytophthoras. Against other halophytophthoras, this was true for both primary and secondary resource capture. The one exception among the fungi was a species (Dendryphiella salina) common in decaying drift material in high-intertidal zones. H. spinosa was a weak competitor with true fungi and with H. vesicula, though it was not displaced by H, vesicula, and H. spinosa could depress the frequency of H. vesicula occupation when H, spinosa was well established. H. bahamensis did not routinely form sporangia, preventing identification and firm conclusions regarding competitiveness, other than that it could not block H, vesicula, but could block H. spinosa from entering its occupied arenas. When bacterial films were present on leaves prior to access by halophytophthoras, the occupation frequency of halophytophthoras was sharply depressed (by about 70 to 90% with 48 h bacterial films), including for H, vesicula, implying that in some types or parts of mangrove systems, submerged-leaf decomposition may sustain low levels of participation by halophytophthoras.
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Currently there is no commercial or subsistence fishing for holothurians on the Cocos (Keeling) Islands and, despite a prominent government presence, no reported cases of poaching or illegal fishing. However, because of recent interest in developing a commercial fishery for holothurians there, a survey was initiated to provide baseline data on the previously unfished local holothurian populations. Fourteen species of holothurians were recorded during the survey, with the most abundant species being Holothuria atra; only 4 other species had relatively high abundances. A total of 20 556 holothurians were counted; however, 97% of these were considered to be of low commercial value. The high-and medium-value species found in this survey were all in extremely low abundances, with restricted distributions. The distribution and abundance of holothurians was closely linked with benthic habitats, with 48% of the variation in holothurian populations explained by 13 habitat variables. Several species displayed distinct habitat preferences: H. atra was associated with sand-dominated habitats, Actinopyga mauritiana was associated with relic reefs and soft corals, while Holothuria fuscopunctata and Stichopus herrmanni were both associated with reef flats. The densities recorded in the present study represent the natural abundance and distribution of holothurian populations at this atoll. Given the low numbers of commercially important species, it is highly unlikely that a commercial fishery would be economically viable at the Cocos (Keeling) Islands and it would be more beneficial to maintain the natural holothurian population.
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For testing that an underlying population is normally distributed the skewness and kurtosis statistics, √b1and b2, and the D’Agostino-Pearson K2 statistic that combines these two statistics have been shown to be powerful and informative tests. Their use, however, has not been as prevalent as their usefulness. We review these tests and show how readily available and popular statistical software can be used to implement them. Their relationship to deviations from linearity in normal probability plotting is also presented.
Book
1. Introduction 2. Estimation 3. Hypothesis testing 4. Graphical exploration of data 5. Correlation and regression 6. Multiple regression and correlation 7. Design and power analysis 8. Comparing groups or treatments - analysis of variance 9. Multifactor analysis of variance 10. Randomized blocks and simple repeated measures: unreplicated two-factor designs 11. Split plot and repeated measures designs: partly nested anovas 12. Analysis of covariance 13. Generalized linear models and logistic regression 14. Analyzing frequencies 15. Introduction to multivariate analyses 16. Multivariate analysis of variance and discriminant analysis 17. Principal components and correspondence analysis 18. Multidimensional scaling and cluster analysis 19. Presentation of results.
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An important ecological issue is developing an understanding of how patterns and processes vary with scale. We designed a field experiment to test how differences in the aerial extent of disturbance affected macrofaunal recolonization on a sandflat. Three different plot sizes (0.203 m2, 0.81 m2, and 3.24 m2) were dafaunted, and samples were collected to assess recovery over a 9-mo period. As the sandflat used for the experiment was prone to disturbance by wind-driven waves, we also measured changes in sediment bed height (an indicator of sediment stability) over the course of the experiment. Most common species revealed significant relationships between density and disturbance plot size. Scale-dependent recovery was also demonstrated by differences in species assemblage structure over the course of the experiment. Relative rates of colonization varied by ≈50% between large and small experimental plots. However, these differences were not related to specific species, particular functional groups, or potential modes of colonizations. The results revealed an unusually slow rate of faunal recovery following defaunation. Both increasing numbers of colonists, and density changes in ambient sediments made an important contribution to recovery. The relationship found between changes in sediment bed height and wind velocity indicated that wind-driven wave disturbance was an important factor influencing sediment instability. Sediment instability was higher in all experimental plots than in the ambient sediments, due to the initial removal of a dense spionid polychaete tube mat characteristically found at the study site. Sediment instability also increased with increasing plot size. Thus in this dynamic sandflat habitat, faunal emigration from recovering disturbed patches of sediment may significantly slow rates of recolonization. These results demonstrate that incorporating patch size, emigration, recovery time, and interactions between hydrodynamic conditions and habitat stability (particularly where colonists influence sediment stability) are crucial to generating a general understanding of recovery processes in sof-sediment habitats. While our results demonstrate the need for caution in scaling-up from small-scale studies, they do indicate that larger scale disturbances that destroy organisms with a role in maintaining habitat stability are likely to result in very slow recovery dynamics, particularly in wave-disturbed soft-sediment habitats.