DNA barcoding and metabarcoding of standardized
samples reveal patterns of marine benthic diversity
Matthieu Leray and Nancy Knowlton1
National Museum of Natural History, Smithsonian Institution, Washington, DC 20013
Contributed by Nancy Knowlton, December 31, 2014 (sent for review October 29, 2014; reviewed by Naiara Rodriguez-Ezpeleta and Robert Toonen)
Documenting the diversity of marine life is challenging because
many species are cryptic, small, and rare, and belong to poorly
known groups. New sequencing technologies, especially when
combined with standardized sampling, promise to make compre-
hensive biodiversity assessments and monitoring feasible on a
large scale. We used this approach to characterize patterns of
diversity on oyster reefs across a range of geographic scales
comprising a temperate location [Virginia (VA)] and a subtropical
location [Florida (FL)]. Eukaryotic organisms that colonized multi-
layered settlement surfaces (autonomous reef monitoring struc-
tures) over a 6-mo period were identified by cytochrome c oxidase
subunit I barcoding (>2-mm mobile organisms) and metabarcod-
ing (sessile and smaller mobile organisms). In a total area of
∼15.64 m2 and volume of ∼0.09 m3, 2,179 operational taxonomic
units (OTUs) were recorded from 983,056 sequences. However,
only 10.9% could be matched to reference barcodes in public data-
bases, with only 8.2% matching barcodes with both genus and
species names. Taxonomic coverage was broad, particularly for
animals (22 phyla recorded), but 35.6% of OTUs detected via meta-
barcoding could not be confidently assigned to a taxonomic
group. The smallest size fraction (500 to 106 μm) was the most
diverse (more than two-thirds of OTUs). There was little taxonomic
overlap between VA and FL, and samples separated by ∼2 m were
significantly more similar than samples separated by ∼100 m.
Ground-truthing with independent assessments of taxonomic
composition indicated that both presence–absence information
and relative abundance information are captured by metabarcod-
ing data, suggesting considerable potential for ecological studies
and environmental monitoring.
|
oyster reefs operational taxonomic units | meiofauna | ARMS |
cryptic species
U nderstanding the diversity of life in the sea continues to
challenge marine scientists because samples typically con-
tain many rare species, most of them small and difficult to
identify (1). Moreover, recent estimates suggest that between
33% and 91% of all marine species have never been named (2,
3). These constraints have limited our ability to investigate pat-
terns of diversity beyond a few indicator groups (4), most often
conspicuous macroinvertebrates and fish. For this reason, mo-
lecular methods, particularly high-throughput sequencing (HTS)
approaches, hold considerable promise not only for fundamental
understanding of diversity but also for biodiversity monitoring in
the context of global change (5).
Molecular methods are particularly powerful when combined
with standardized sampling, allowing for direct comparisons
across space and through time. In the ocean, analyzing standard
volumes of readily sampled material (e.g., seawater, sediments)
has a long tradition, and, increasingly, HTS approaches are being
applied to these samples (6). Complex hard substrates provide
greater challenges for consistent sampling, which can be met ei-
ther by collecting approximately standard volumes (e.g., of rubble)
or by deploying settlement structures (e.g., ref. 7).
Here, we combine standardized sampling with molecular di-
versity assessments for samples from oyster reefs from one tem-
perate location and one subtropical location on the US Atlantic
2076–2081 | PNAS | February 17, 2015 | vol. 112 | no. 7
Coast. In addition to their commercial value and their role in
maintaining water quality, oyster beds shelter considerable diversity
because of their 3D complexity, essentially the nontropical equiva-
lent of coral reefs. They are also, like coral reefs, highly threatened,
with up to 85% having been lost due to anthropogenic impacts (8).
We report analyses of a nested set of autonomous reef
monitoring structures (ARMS), which provide surfaces and
spaces for mobile and sessile organisms to settle on or shelter
within (SI Text, section I and Fig. S1). ARMS were deployed
for about 6 mo on the ocean side of the Eastern Shore of
Virginia (VA) and in the Indian River Lagoon in Florida (FL).
At each location, there were three replicates ∼2 m apart at
each of three sites ∼100 m apart (total of 18 ARMS; Fig. S1A).
Four fractions were analyzed separately: sessile organisms
growing on the plates and three fractions of organisms retained
by 2-mm, 500-μm, and 106-μm sieves. We sequenced the cy-
tochrome c oxidase subunit I (COI) gene for each specimen of
the >2-mm animals (barcoding). The remaining fractions were
homogenized, and COI amplicons were analyzed from bulk
samples using HTS (metabarcoding). Sequences were clustered
in operational taxonomic units (OTUs) and identified to the
lowest possible taxonomic level using nucleotide BLAST
(BLASTn) searches against public databases or by phylogenetic
assignment when no direct match could be found. The effec-
tiveness of the metabarcoding approach was assessed for
the sessile and 2-mm to 500-μm fractions by comparing num-
bers of sequences with point counts and estimates of total
Significance
High-throughput DNA sequencing methods are revolutionizing
our ability to census communities, but most analyses have fo-
cused on microbes. Using an environmental DNA sequencing
approach based on cytochrome c oxidase subunit 1 primers, we
document the enormous diversity and fine-scale geographic
structuring of the cryptic animals living on oyster reefs, many
of which are rare and very small. Sequence data reflected both
the presence and relative abundance of organisms, but only
10.9% of the sequences could be matched to reference bar-
codes in public databases. These results highlight the enormous
numbers of marine animal species that remain genetically un-
anchored to conventional taxonomy and the importance of
standardized, genetically based biodiversity surveys to monitor
global change.
Author contributions: M.L. and N.K. designed research; M.L. performed research; M.L.
contributed new reagents/analytic tools; M.L. analyzed data; and M.L. and N.K. wrote
the paper.
Reviewers: N.R.-E., AZTI-Tecnalia; and R.T., Hawaii Institute of Marine Biology.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The sequences reported in this paper have been deposited in the Gen-
Bank database (accession nos. KP253982–KP255345), the Barcode Of Life Data Systems
(doi: dx.doi.org/10.5883/DS-ARMS), and the Dryad Digital Repository (doi: doi.org/10.
5061/dryad.d0r79).
1
To whom correspondence should be addressed. Email: knowlton@si.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1424997112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1424997112
DNA per OTU, respectively. Noneukaryotic sequences were
not analyzed.
Results
Patterns of Diversity and Abundance. Organisms >2 mm had rel-
atively low diversity, with FL samples having about 1.7-fold more
OTUs in total than VA samples, and also more phyla repre-
sented. Barcoding revealed a total of 38 OTUs in VA (498
individuals) and 64 OTUs in FL (655 individuals). In both loca-
tions, the most abundant and species-rich higher taxon was the
Arthropoda (16 OTUs in 385 individuals in VA and 30 OTUs in
583 individuals in FL; Fig. 1). Samples from both locations ad-
ditionally contained representatives of the Annelida, Chordata,
and Mollusca; FL samples also contained Platyhelminthes and
Echinodermata. Over half of the sequences at both locations
matched reference sequences (>97% similarity) in the GenBank
sequence database (GenBank) or Barcode of Life Data Systems
(BOLD) (Table 1 and Table S1).
In contrast, the sessile and two smaller sieved fractions had
much higher diversity at the OTU and phylum levels, and a much
lower proportion of OTUs matching sequences in public data-
bases. In total, HTS detected 1,204 OTUs from 572,290
sequences and 1,391 OTUs from 409,613 sequences from VA
and FL, respectively (Table 1). Matches to GenBank or BOLD
sequences were low (<12%) and comparable at both locations.
Several additional OTUs could be identified because they
matched reference barcodes obtained from the >2-mm and 2-mm
to 500-μm fractions that were characterized morphologically
(3.2% in VA and 3.9% in FL). Many more of the remaining
VA FL
Fig. 1. OTU diversity and abundance in VA and FL. The category “Other ani-
mals” comprises Acoelomorpha, Chaetognatha, Ctenophora, Echiura, Ento-
procta, Gastrotricha, Hemichordata, Nematoda, Nemertea, Platyhelminthes,
Rotifera, Sipuncula, Tardigrada, and Xenacoelomorpha.
Leray and Knowlton
OTUs could be assigned to a higher taxonomic group using the
Bayesian phylogenetic approach (45.7% in VA and 55.9% in
FL). However, 40.9% (VA) and 28.3% (FL) of OTUs could not
be confidently assigned to any taxonomic group using this ap-
proach. Taxonomic coverage was very broad, with 22 phyla of
animals, five major groups of protists, two major groups of Fungi,
and three major groups of Plantae. Among the identified OTUs,
Arthropoda were again the most OTU-rich (21.5% in VA and
29% in FL; Fig. 1). In FL, Arthropoda also had the highest
percentage of sequences (28.5%), but in VA, the largest number
of sequences belonged to Cnidaria (38.7%).
As is common in many samples of diversity, there were a few
common taxa and many rare taxa in all samples. The percentage
of singletons was surprisingly uniform, ranging from 31.6–46.9%
in the barcoded samples and from 30.3–39.8% in the metabarcoded
samples (Table 1). The groups with the highest percentage of sin-
gletons were “unidentified” OTUs in both VA and FL (53.2% and
36% of singletons, respectively), followed by Arthropoda (14.6%
and 23.3% of singletons, respectively). The most common OTUs, as
measured by either the number of sequences (Fig. 2) or the number
of samples where they occurred (Fig. S2), were more likely to match
reference barcodes (>97% similarity).
Overall, total diversity was surprisingly high for such small
samples, and statistical analysis revealed that sampling was far
from exhaustive. The mean (±SD) number of taxa per ARMS
was 439.3 ± 54.9 for VA (11.2 ± 4.1 barcoded and 434.2 ± 55.7
metabarcoded) and 545.8 ± 29.6 for FL (15.8 ± 4.9 barcoded and
536.7 ± 30.8 metabarcoded) (Table 1). Chao1, Chao2, and rar-
efaction estimates for increasing numbers of ARMS (Fig. 3) or
sequences (Fig. S3) show numbers continuing to climb with in-
creased sampling effort.
Patterns of Community Composition. For the >2-mm organisms, FL
and VA were highly distinct, with just four of 98 OTUs in
common. Principal component analysis (PCoA) shows FL and
VA samples well separated in 2D space along an axis explaining
32.6% and 62.6% of the variation in community composition
using Jaccard (presence–absence) and Bray–Curtis (relative
abundance) indices, respectively (Fig. S4 A and C). VA and FL
samples also partition on an unweighted pair group method with
arithmetic mean (UPGMA) tree, with differences between
locations well supported by jackknife subsampling (Fig. S4 B and
D). Differences in community composition were also significant
π
between locations (Jaccard: F1,16 = 7.32, P < 0.001; Bray–Curtis:
π
F1,16 = 27.3, P < 0.001).
In contrast, the sessile and smaller sieved fractions from FL
and VA were more similar, with 457 (21%) of the 2,138 OTUs
detected via HTS shared. Nevertheless, VA and FL samples are
consistently separated on PCoA based on incidence or abun-
dance along a first axis explaining 21.0% and 30.4% of the var-
iation in OTU composition, respectively (Fig. 4 A and C), and
they partition into two distinct well-supported groups on a
UPGMA tree (Fig. 4 B and D). With all three fractions aggre-
gated, community composition was significantly different be-
π
tween ARMS from VA and FL (Jaccard: F1,16 = 13.21, P < 0.001;
π
Bray–Curtis: F1,16 = 26.72, P < 0.001) with 15 OTUs contributing
50% of the difference between VA and FL based on relative
abundance (Table S2).
ECOLOGY
The sessile and smaller sieved fractions had distinct OTU
composition in both VA and FL. Their separation is clearly seen
in PCoA and jackknifed UPGMA trees in both FL and VA (Fig.
4). Fractions were significantly different at both locations based on
π
either OTU presence–absence (VA: F2,24 = 6.56, P < 0.001; FL:
π π
F2,24 = 8.08, P < 0.001) or relative abundance (VA: F2,24 = 14.82,
π
P < 0.001; FL: F2,24 = 13.78, P < 0.001). Although samples of the
2-mm to 500-μm and 500- to 106-μm fractions overlay on the 2D
PCoA constructed using the Bray–Curtis metric (Fig. 4C), differ-
ences in OTU composition were significant at both locations (VA:
π π
F1,15 = 13.05, P < 0.001; FL: F1,15 = 7.82, P < 0.001). As expected,
based on the plate appearances (Fig. S1C), Porifera and Chordata
are primarily responsible for the distinctiveness of the sessile
PNAS | February 17, 2015 | vol. 112 | no. 7 | 2077
Table 1. OTU diversity and abundance in VA and FL
VA FL

Barcoding Metabarcoding Barcoding Metabarcoding

2 mm to 500 to 2 mm to 500 to
Diversity descriptors >2 mm 500 μm 106 μm Sessile Total >2 mm 500 μm 106 μm Sessile Total

No. of sequences 498 256,147 97,439 218,704 572,290 655 155,232 86,350 168,031 409,613
Total no. of OTUs 38 651 828 436 1,204 64 821 976 591 1,391
Mean no. of OTUs 11.2 203.3 290.3 146.6 434.2 15.8 277.2 360.1 222.9 536.7
Mean rarefied no. of OTUs 8.2 117.1 229.7 85.7 333.5 9.6 202.6 312.1 157.4 484.4
Chao1 46.0 1,075.6 1,204.9 638.7 1,711.4 104.8 1,183.0 1,486.0 858.0 1,945.7
Chao1 (rarefied) 49.2 552.7 917.8 451.5 1,174.0 144.7 866.5 1,213.9 562.1 1,483.7
ACE 47.6 1,062.8 1,223.0 628.3 1,743.0 108.6 1,197.8 1,483.1 819.38 1,982.0
ACE (rarefied) 57.6 515.4 975.7 459.1 1,217.8 91.6 837.7 1,213.2 556.02 1,521.3
OTUs with match,* % 60.5 14.1 10.6 16.2 10.2 57.8 15.7 11.8 16.9 11.9
Unidentified OTUs, % NA 35.6 38.8 31.2 40.9 NA 26.8 27.1 23.8 28.3
Singletons, % 31.6 39.8 36.5 34.9 34.8 46.9 32.3 34.6 30.3 31.1

Additional data and calculation methods are provided in Table S1. NA, not applicable.
*Greater than 97% similarity to GenBank or BOLD sequences.

fraction in FL, whereas unidentified OTUs characterize the 500-
to 106-μm fraction (Fig. S5).
We found no evidence for fine-scale geographic structuring of
the >2-mm samples based on PCoA (Fig. S4 A and C), UPGMA
trees (Fig. S4 B and D), and permutational multivariate analysis
π
of variance (PERMANOVA; Jaccard: F5,12 = 2.33, P = 0.360;
π
Bray–Curtis: F5,12 = 6.40, P = 0.397). In contrast, fine-scale
structuring in FL was apparent for all three fractions analyzed via
metabarcoding on the jackknifed UPGMA tree (Fig. 4 B and D),
with strong branch support for triplets representing adjacent
ARMS. Although most adjacent sites in VA did not cluster as
triplets on the UPGMA trees, differences between sites were
π conspicuous taxa were always present in the overall metabarcoding
highly significant in VA (Jaccard: F2,24 = 1.07, P < 0.001; Bray–
π π
Curtis: F2,24 = 1.41, P = 0.002) as well as in FL (Jaccard: F2,24 = 1.77,
P < 0.001; Bray–Curtis: F2,24π
= 2.11, P < 0.001).
The differences observed between locations, sites within
locations, and fractions cannot be broadly attributed to differences
in multivariate dispersion, because tests were insignificant except
between fractions in FL (Jaccard: F2,24 π
= 14.14, P < 0.001; Bray–
π
Curtis: F2,24 = 14.10, P < 0.001). Moreover, all differences in
community composition remained significant after removing sin-
gletons from the metabarcoding dataset.
Validation of the Metabarcoding Approach. Comparing HTS data
against more direct assessments of community composition re-
vealed that HTS is effective at detecting OTUs. A total of 91.2%
OTUs in VA only OTUs in FL only OTUs at both localities
Fig. 2. Proportion of identified OTUs in a metabarcoding dataset according
to the number of sequences. A match to the reference barcode was defined
as >97% similarity.
(VA) and 77.1% (FL) of OTUs detected via barcoding of in-
dividual specimens isolated from the 2-mm to 500-μm fractions
matched OTUs in the metabarcoding dataset from the same lo-
cation. The proportion of matches between barcoding and meta-
barcoding datasets for individual ARMS ranged from 65.3–88.2%
(i.e., 71.4% match between barcodes and metabarcodes of ARMS
5 in FL). A majority (50–100%) of undetected OTUs were rep-
resented by a single specimen, suggesting that they were rare or
perhaps absent from the subsample (half of the total) crushed for
DNA metabarcoding. Undetected OTUs belonged to several
phyla, suggesting no particular taxonomic bias in OTU detection.
Similarly, for sessile taxa, individual barcodes from subsamples of
dataset (n = 5 in VA and n = 13 in FL). The proportion of matches
between barcoding and metabarcoding datasets for individual
ARMS was 100% in VA and ranged from 90.9–91.7% in FL.
The relative abundance of sequences also showed good
agreement with independent measures of relative abundance. In
the 2-mm to 500-μm fractions, there was a significant positive
relationship between the amount of DNA per OTU and the
number of sequences per OTU for all ARMS from VA (ARMS
1: t22 = 3.95, P < 0.001; ARMS 4: t16 = 3.58, P < 0.001; ARMS 9:
t18 = 6.64, P < 0.001; Fig. 5A) and FL (ARMS 1: t41 = 6.49, P <
0.001; ARMS 5: t34 = 4.54, P < 0.001; ARMS 9: t48 = 8.91, P <
0.001; Fig. 5B). At the phylum level (DNA and sequences per
OTU pooled by phylum), there were too few points to compute
statistics for ARMS individually, but there was an overall sig-
nificant relationship between the amount of DNA and the
number of sequences in VA (t10 = 6.14, P < 0.001; Fig. 5C) and
FL (t20 = 3.72, P = 0.001; Fig. 5D). For sessile taxa in FL,
measures of abundance based on point counts were significantly
correlated with numbers of sequences in the metabarcoding
dataset at both the OTU (ARMS 1: t10 = 3.70, P = 0.005; ARMS
5: t10 = 5.26, P < 0.001; ARMS 9: t12 = 3.60, P = 0.005; Fig. 5F)
and phylum (t13 = 4.02, P = 0.002; Fig. 5H) levels. No statistics
were calculated for the sessile OTUs from VA because of the
limited number of data points (Fig. 5 E and G), but the pattern is
similar to the pattern exhibited by the FL samples.
Discussion
Our intensive survey of the marine diversity of a small area (a
total of 7.82 m2 and 0.05 m3 per locality) yielded a surprising
amount of diversity: 1,218 OTUs in VA and 1,421 OTUs in FL.
Although more than half of the barcode-based OTUs from
invertebrates and fish >2 mm matched barcodes in public li-
braries, only 10–12% (VA/FL) of the metabarcode-based OTUs
in the sessile and smaller sieved fractions matched GenBank or

2078 | www.pnas.org/cgi/doi/10.1073/pnas.1424997112 Leray and Knowlton

 VA FL
Fig. 3. Sample-based rarefaction curves and Chao1 estimates of total OTU
diversity.
BOLD barcodes. As a result, identification of OTUs detected via
metabarcoding relied mostly on phylogenetic assignments to
taxonomic groups represented in GenBank, a database still lack-
ing COI references for numerous families of marine inverte-
brates. Moreover, numerous OTUs remained unidentified, which
may be due to the scarcity of representative COI sequences for
A B
C D
Leray and Knowlton
entire branches of the tree of life, COI barcode misidentifications
in GenBank, or limitations in using the hypervariable COI region
for phylogenetic assignments. Methodological artifacts (e.g., PCR
and sequencing errors or amplification of pseudogenes) are also
a possibility, but they likely account for a minor proportion of
unidentified OTUs, given our stringent quality filtering based on
amino acid translation.
Regardless of taxonomy, our data provide a unique opportu-
nity to investigate local and regional patterns of diversity across
size fractions of mobile and sessile taxa. As expected, the >2-mm
fraction is much less diverse than smaller mobile fractions (over
an order of magnitude in VA and FL), a difference partly in-
herent to the sequencing approach used. Although barcoding
provides specimen level data, metabarcoding captures not only
“free-living” forms but also parasites, gut contents, and other
forms of environmental DNA. We found the smallest sized
organisms (500 to 106 μm) to have a 1.96- to 1.54-fold greater
rarefied diversity than the 2-mm to 500-μm organisms in VA and
FL, respectively, and the sessile fraction had the lowest rarefied
OTU richness of the three metabarcoded fractions at both sites
(1.37- to 1.29-fold smaller than 2-mm to 500-μm organisms, re-
spectively). A higher local diversity in smaller sized organisms is
consistent with the literature (9). However, the small sieve (106
μm) is likely accumulating debris and body parts shedding from
sessile and larger motile animals (retained by coarser sieves)
during field processing, thereby “artificially” increasing diversity
in the 500- to 106-μm fraction [e.g., some sessile OTUs (i.e.,
Porifera, Bryozoa) are major contributors to differences between
sieved fractions in both VA and FL; Table S2].
Also, as expected [(10) and ubiquity hypothesis (11)], the larger
organisms showed a greater difference in estimated diversity be-
tween the temperate (37.6°N) and subtropical (27.4°N) locations
(2.3-fold greater diversity in FL than VA) than did organisms be-
longing to the smaller sized fractions (1.1- to 1.4-fold greater in FL
than VA for the two smaller mobile fractions). (The sessile fraction
is composed of large and small organisms, and so cannot be sep-
arated in this fashion.) The latitudinal inflation in diversity of the
>2-mm fraction is comparable to the latitudinal inflation in di-
versity observed for Western Atlantic coastal fishes from these
latitudes (a two- to threefold difference) and somewhat less than
the latitudinal inflation in diversity observed for decapod crusta-
ceans (five- to sixfold difference) (figure 2 of ref. 12).
VA
FL
VA
FL
ECOLOGY
Fig. 4. Clustering analyses [PCoA (A and C) and jackknifed
UPGMA trees (B and D)] depicting similarity in community
composition based on OTU incidence (Jaccard; A and B)
and relative abundance (Bray–Curtis; C and D). PC, principal
component.
PNAS | February 17, 2015 | vol. 112 | no. 7 | 2079
A VA B FL
C D
Fig. 5. Relationship between the number of sequences per OTU (A, B, E, and F) or per phylum (C, D, G, and H) obtained via metabarcoding and the total
amount of DNA (2-mm to 500-μm samples; A–D) or plate coverage (sessile taxa; E–H).
Our data showed no evidence for fine-scale spatial structuring
in larger animal communities but demonstrated community par-
titioning at the 100-m scale for assemblages of sessile and mi-
croscopic taxa. More limited postsettlement dispersal abilities
make these communities sensitive to local scale differences in
environmental factors (13, 14). Moreover, because numerous
microscopic animals may have specific associations with sessile
taxa, spatial structuring in sessile assemblages may amplify dif-
ferences between communities of small mobile taxa.
Finally, the assessment of the robustness of the metabarcoding
approach targeting the COI gene suggests this method can be
used reliably to detect OTU presence–absence, and it provides
useful information on OTU relative abundance as well. Notably,
the reliability improves as one moves to coarser groupings, be-
cause we have shown a remarkable fidelity at the level of the
phylum, which suggests limited PCR bias among distant taxo-
nomic groups. This finding is noteworthy because many ecological
assessments work at the level of functional groups rather than
at the level of species. Alternatively, PCR-free shotgun meta-
genomic approaches will be less prone to bias but require much
higher sequencing depth (15), therefore increasing the cost for
sufficient replication. Taxonomic coverage among animals will
also increase with sequencing multiple independent markers
[i.e., 18S, 16S (16)], but targeting nonprotein-coding genes may
increase the probability of including sequencing artifacts. More-
over, alternative barcode genes would provide a more compre-
hensive survey of fungi [i.e., internal transcribed spacer (17)] and
protists [i.e., 18S (18)]. As marine monitoring moves from the
use of indicator groups to more comprehensive community-level
analysis of alpha and beta diversity, this study provides support to
encourage more routine use of a metabarcoding approach.
2080 | www.pnas.org/cgi/doi/10.1073/pnas.1424997112
E VA F FL
G H
Materials and Methods
ARMS Deployment, Collection, and Sampling. ARMS were deployed subtidally
adjacent to natural oyster reefs in VA and FL for ∼6 mo (Fig. S1 and SI Text,
section II). Upon retrieval, each plate was kept submerged in seawater. Plates
were photographed on both sides, and representative sessile taxa were in-
dividually tissue-sampled for DNA barcoding. Sessile organisms growing on
the plates were then scraped into a tray and homogenized using a kitchen
blender, and ∼45 g of tissue was preserved in DMSO buffer (SI Text, sections
II.D and III). Water holding ARMS was filtered through 2-mm, 500-μm, and
106-μm sieves. Mobile specimens retained by the 2-mm sieve were photo-
graphed alive, identified to the lowest taxon level possible based on mor-
phology, and individually preserved in 95% ethanol (EtOH). The two smaller
sieved fractions were initially bulk-preserved in 95% EtOH, and the organic
fraction was later separated from sediments by decantation (SI Text, section
IV). Each organic fraction was split in half by weight; the first half was crushed
using a mortar and pestle to be analyzed via DNA metabarcoding, and the
second half was archived in 95% EtOH (a summary of the protocol is provided
in Fig. S1D). The biomass and amount of sediment are provided in Table S3.
DNA Barcoding and Metabarcoding. For barcoding, tissue was subsampled
from each specimen and placed individually in 96-well Costar plates (Corning)
for phenol DNA extraction. DNA amplification and Sanger sequencing used
standard protocols (SI Text, section V.A) and previously published primers
(19, 20). For metabarcoding, DNA was extracted from 10 g of homogenized
sessile tissue and the crushed half of the 2-mm to 500-μm and 500- to 106-μm
samples using the MO-BIO Powermax Soil DNA Isolation Kit (SI Text, section
V.B). Three replicate PCR assays were performed to amplify an ∼313-bp COI
fragment for each of the 54 bulk samples (SI Text, section V.B). We used
a hierarchical tagging approach for sample multiplexing [combination of
tailed PCR primers and Ion Xpress (Life Technologies) barcode adapters;
SI Text, section V.B and Table S4]. Amplicons were sequenced on the Ion
Torrent platform (Life Technologies) following the manufacturer’s instructions
(SI Text, section V.B). Barcode sequences were deposited in GenBank (accession
Leray and Knowlton
nos. KP253982–KP255345) and BOLD (doi: dx.doi.org/10.5883/DS-ARMS), and
the metabarcode datasets were deposited in the Dryad Digital Repository (doi:
doi.org/10.5061/dryad.d0r79).
Sequence Analysis of Barcodes and Metabarcodes. For barcodes, forward and
reverse sequences were assembled, checked for stop codons or frame shifts,
and edited in Geneious (Biomatters). We used the Bayesian clustering al-
gorithm implemented in clustering 16S rRNA for OTU prediction (CROP)
(21) to delineate OTUs based on the natural distribution of sequence
dissimilarity in the dataset (SI Text, section VI.A). CROP outputs a repre-
sentative sequence per OTU that was used for taxonomic identification.
For metabarcodes, higher quality reads prefiltered by Torrent Suite Soft-
ware version 4.0.2 (Life Technologies) were assigned to samples based on
the combination primer tail-Ion Xpress barcode. Additional sequences
were removed if they did not meet several criteria (SI Text, section VI.B).
We then took advantage of the coding property of the COI gene to im-
prove the quality and reliability of our dataset further by discarding reads
with any anomaly in their amino acid translation using Multiple Alignment
of Coding Sequences (MACSE) (22) (SI Text, section VI.B). Finally, reads were
screened for chimeras using UCHIME (23). Remaining reads were clustered in
OTUs using CROP following the parameters detailed in SI Text, section VI.A.
For taxonomic assignments, we performed BLASTn searches of OTU repre-
sentative sequences of the barcoding and metabarcoding datasets against full
GenBank and BOLD databases. We accepted a species level match when sim-
ilarity to the reference barcode was >97%. In the absence of a direct match,
we used a phylogenetic approach implemented in the Statistical Assignment
Package (24) to assign a higher taxonomic level (SI Text, section VI.C).
Statistical Analyses. We summarized barcoding and metabarcoding data in
separate OTU tables. Sample-based rarefaction curves and nonparametric
species richness estimators were computed in EstimateS (25). Each OTU table
was rarefied to the lowest number of sequences using Quantitative Insights
into Microbial Ecology (QIIME) (26) to account for differences in abundance.
We used Jaccard (presence–absence) and Bray–Curtis (relative abundance)
metrics to calculate pairwise community distance matrices and examine
differences in beta diversity. Patterns of sample dissimilarity were visualized
using PCoA. Hierarchical cluster trees were also constructed using UPGMA
with jackknife support to examine the robustness of sample clustering. We
examined differences in community composition between locations and sites
using PERMANOVA (27). We also tested whether the average distance to
the group centroid is equivalent among groups [multivariate dispersion:
PERMDISP (28)]. To account for the stratified structure of the design, we
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Leray and Knowlton
constrained permutations within factors when necessary (i.e., within locality
to test for differences among sites). Metabarcoding data were initially ag-
gregated per ARMS to test for overall differences in OTU composition and
dispersion between locations and sites. We then partitioned the OTU table
between locations to test for differences between fractions and sites. We
repeated all statistical analysis after removing singletons from the meta-
barcoding OTU table to test for the robustness of the patterns. Finally, we
conducted a similarity of percentage analysis to determine which OTUs were
major contributors to differences between locations and fractions based on
relative abundance. All tests were computed in the R package Vegan (29)
and significance-tested using 1,000 permutations.
Assessment of Reliability of Metabarcoding Approach. One ARMS from each of
the three sites at each location was randomly chosen. Archived bulk samples
were resuspended in a graduated beaker containing 100 mL of 95% EtOH
and homogenized with a spatula, and 20 mL was immediately collected
using a Hensel–Stempel pipette. All specimens were isolated and identified
to the lowest taxonomic level; the entire specimen was used for phenol
DNA extraction, and the mitochondrial COI gene was sequenced for OTU
delineation. The amount of DNA in each individual extract was measured
with a Qubit fluorometer (dsDNA HS Assay kit; Invitrogen), enabling the
calculation of the total amount of DNA represented by each OTU and
subsequent comparison with the number of reads obtained for the same
OTU via metabarcoding (SI Text, section VII.A). For the sessile organisms, we
individually sampled and barcoded morphologically distinctive taxa to
identify matching OTUs in the metabarcoding dataset. The number of reads
per OTU was then compared with their estimated cover on each ARMS as
measured by a point count approach implemented in Coral Point Count
with Excel extensions (CPCe) (30) (SI Text, section VII.B).
The relationship between the number of reads in metabarcoding dataset
and the amount of DNA or number of point counts (for 2-mm to 500-μm and
sessile specimens, respectively) was tested using a generalized linear model.
Given the nature and significant overdispersion of the data, we fitted a quasi-
Poisson model with log link function.
ACKNOWLEDGMENTS. We thank Natalia Agudelo, Sherry Reed, Woody Lee,
and Sean Fate for help in the field; the Smithsonian Laboratory of Analytical
Biology staff for assistance; and Daryl Hurley II for allowing research to be con-
ducted on his oyster reef in VA. Research Permit SAJ-2012-02893(NW-SLR) was
provided by the US Army Corps of Engineers in FL. Financial support was pro-
vided by the Sant Chair and Smithsonian Tennenbaum Marine Observatories
Network, for which this paper is Contribution 1.
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a universal DNA barcode marker for Fungi. Proc Natl Acad Sci USA 109(16):6241–6246.
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cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa
biotic surveys. Mol Ecol Resour 13(5):851–861.
20. Leray M, et al. (2013) A new versatile primer set targeting a short fragment of the
mitochondrial COI region for metabarcoding metazoan diversity: Application for
characterizing coral reef fish gut contents. Front Zool 10:34.
21. Hao X, Jiang R, Chen T (2011) Clustering 16S rRNA for OTU prediction: A method of
unsupervised Bayesian clustering. Bioinformatics 27(5):611–618.
22. Ranwez V, Harispe S, Delsuc F, Douzery EJP (2011) MACSE: Multiple Alignment of
Coding SEquences accounting for frameshifts and stop codons. PLoS ONE 6(9):e22594.
23. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensi-
tivity and speed of chimera detection. Bioinformatics 27(16):2194–2200.
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signment of DNA sequences using Bayesian phylogenetics. Syst Biol 57(5):750–757.
25. Colwell RK (2006) EstimateS: Statistical Estimation of Species Richness and Shared
ECOLOGY
Species from Samples. Version 9.1. Available at purl.oclc.org/estimates. Accessed
August 15, 2014.
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quencing data. Nat Methods 7(5):335–336.
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iance. Austral Ecol 26:32–46.
28. Anderson MJ, Ellingsen KE, McArdle BH (2006) Multivariate dispersion as a measure
of beta diversity. Ecol Lett 9(6):683–693.
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PNAS | February 17, 2015 | vol. 112 | no. 7 | 2081
Supporting Information
Leray and Knowlton 10.1073/pnas.1424997112
SI Text
I. ARMS Specifications
ARMS consist of ten 22.5 × 22.5-cm PVC plates separated by
1.27-cm spacers, anchored to a baseplate (Fig. S1). In alternate
layers, water flow through the spaces was obstructed by bars
running from the corners to the center of the plate. The total
surface area sampled was 0.869 m2 per ARMS, and the total
volume between plates was 0.005 m3 per ARMS.
II. Field Sampling Protocols
A. Deployment and Recovery. ARMS were deployed subtidally
adjacent to natural oyster reefs on September 19, 2013 in VA and
on November 6, 2013, in FL. We used stainless-steel stakes to
anchor the structures to the reef at a level guaranteeing that they
remained submerged at low tide. ARMS were collected on May
3–4, 2014, and May 26–29, 2014, in VA and FL, respectively, for
a soak time of ∼6 mo. To prevent loss of community members,
a 100-μm Nitex-lined crate was placed over the ARMS structure
and fastened with two or three hooked elastic cords (bungies)
before removal of the ARMS from the bottom. Lined crates are
designed to cover the central structure made of 10 PVC plates
only, which means that mobile specimens occurring on the base
plate are able to escape during ARMS handling in the field.
Stakes used for anchoring ARMS to the substrate were then
removed, and a small cable tie was placed at the northern corner
of the baseplate to keep track of orientation. ARMS were then
placed in a large plastic container with seawater and at least two
aeration stones and transported to the wet laboratory.
B. Disassembly. The lined crate was removed, rinsed over the
plastic container in which the structure was transported with
seawater from the plastic container, and examined for any hiding
organisms. The ARMS were positioned upside-down to unscrew
nuts and bolts at each corner (long bolts are left in place to allow
the removal of each plate one by one). The baseplate was re-
moved first, brushed minimally inside the plastic container to
remove any mobile animals, and placed aside (not analyzed).
Each of the 10 plates was then removed one by one and lightly
brushed, a small cable tie was placed at the northern corner of the
plate, and the plate was photographed on both sides and finally
placed in labeled (with ARMS and plate number) 5-gallon
buckets containing 45-μm filtered seawater and an air stone.
C. Processing Sieved Fractions. Water from the large plastic con-
tainer was filtered through three sets of sieves [2 mm (no. 10),
500 μm (no. 32), and 106 μm (no. 140)], and each fraction was
placed in an individual tray with an air stone. Mobile specimens
retained by the 2-mm sieve were sorted to morphospecies; photo-
graphed alive to document color patterns; and anesthetized using
clove oil, magnesium chloride, or chilling before preservation in
95% EtOH. Pieces of algae and other sessile organisms retained by
the 2-mm sieve were not processed. The two smaller sieved frac-
tions (2 mm to 500 μm and 500 to 106 μm) were washed with
seawater into a 45-μm Nitex net and preserved in falcon tubes (or
larger jars depending on the volume of the sample) containing 95%
EtOH. Both individual mobile animals larger than 2 mm and
smaller sieved fractions were kept at −20 °C until DNA extraction.
D. Processing the Sessile Fraction. The most common and conspic-
uous sessile taxa found on the plates were photographed, and
a small tissue sample was preserved in salt-saturated 25% (vol/vol)
Leray and Knowlton www.pnas.org/cgi/content/short/1424997112
DMSO buffer [0.25 M EDTA (pH 7.5), DMSO, NaCl-saturated]
for DNA barcoding. The surface and sides of all PVC plates were
then scraped into a tray of seawater or EtOH, and the total content
was poured into a kitchen blender with 45-μm filtered seawater
(roughly 1:1 in volume) for homogenization for 30 s at maximum
speed. Blended material was then immediately poured into
a collection net (45-μm Nitex mesh) and rinsed with seawater or
95% EtOH, squeezing out the liquid through the mesh at least
twice. On-site homogenization followed by the washing step was
found to give high-molecular-weight DNA as detailed below.
After the last wash and squeezing out of liquid, ∼15 g of material
was placed inside each of three falcon tubes that were then filled
with DMSO buffer. Falcon tubes were placed at −20 °C, along
with any remaining tissue that was frozen, in plastic bags.
E. Avoiding Contamination. Because the PCR-based approach to
characterize communities is very sensitive to contamination, each
piece of equipment was soaked in 10% bleach (sodium hypo-
chlorite) for a minimum of 5 min before first use and between
samples for sterilization. Nitrile gloves were used to manipulate
equipment at all times.
III. Tests of DNA Preservation of Sessile Fraction
Preliminary tests were conducted to determine the best approach
to obtain high-molecular-weight DNA from the sessile fraction.
An initial protocol was designed in which plates were first sub-
merged in 95% EtOH for several hours to reduce the amount of
water in animal tissues. Then, tissues were scraped into a large
container filled with EtOH (ratio of ∼1:10) and preserved at
−20 °C for several days or weeks. Finally, tissues were homog-
enized (using a blender) in a small amount of EtOH, and ∼10 g
of material was immediately collected for DNA extraction in the
laboratory. That approach provided very low DNA quality
[100% of DNA fragments shorter than 300 bp as measured
by a TapeStation (Agilent Technologies)] for 90% of samples
tested. After ruling out the potential effect of mechanical
shearing during tissue homogenization, the protocol was modi-
fied to minimize storage time by conducting tissue homogeni-
zation and DNA extraction in the field. Nevertheless, DNA was
still degraded, which suggested that chemical denaturation po-
tentially caused by substances released by sessile animals oc-
curred quickly following tissue homogenization. We were able to
obtain very high-quality DNA (75% of DNA fragments longer
than 10 kb as measured by the Agilent TapeStation) across all
samples by homogenizing samples shortly after scraping (plates
are kept in aerated seawater) and immediately rinsing the ho-
mogenate in a 45-μm mesh collection net using seawater or
EtOH. We used DMSO buffer for tissue preservation because it
was shown to be more effective than EtOH for preserving DNA
of several sessile taxonomic groups (1).
IV. Decantation of Sieved Fractions
Small sieved fractions (2 mm to 500 μm and 500 to 106 μm)
contain sediments that should be separated from the organic
fraction before DNA extraction. Each sample was therefore
transferred into a 2-L cylinder and filled up to the 1.5-L level
with deionized water. The cylinder was sealed with parafilm and
shaken vigorously to resuspend animals and other organic mat-
ter, and the water was poured quickly into a 45-μm sieve. Sample
resuspension was repeated five times (or until no organic par-
ticulates could be observed after shaking). The material retained
by the sieve was weighed and homogenized with a spatula. Half
1 of 11
of the sample was then crushed using a mortar and pestle for
2 min and preserved in a falcon tube with 95% EtOH for DNA
extraction. The other half was archived (in 95% EtOH) or used
for morphological analysis, as in the present study. Sediments
collected in the bottom of the cylinder were also archived. All
equipment used for decantation was bleached and UV-sterilized
between samples.
We examined the 2-mm to 500-μm sediments from VA and FL
to quantify the abundance and diversity of mollusks and other
organisms. There was a mean (±SD) of 4.1 (±4.3) and 5 (±4.7)
specimens in the 2-mm to 500-μm sediments from VA and FL,
respectively, with a majority being mollusks [2 (±2.3) and 3.7
(±3.7) specimens per ARMS, respectively]. We also found a few
amphipods and isopods. We obtained COI sequences from 18
specimens found in sediments from VA. They belonged to nine
OTUS, all of them (including two mollusk OTUs) matching
reference OTUs in the metabarcoding dataset, which shows the
effectiveness of the decantation process in retaining organisms
for metabarcoding.
V. Laboratory Protocols
A. DNA Barcoding. A small piece of tissue was collected from each
specimen retained by the 2-mm sieve and placed individually in
96-well Costar plates (Corning) for phenol DNA extraction
performed on an AutoGeneprep 965 (Autogen). DNA from
sessile taxa and whole specimens from the 500-μm to 2-mm
fractions were also extracted using the same procedure. Eluted
DNA was used for PCR amplification of a ∼658-bp fragment of
the mitochondrial COI gene using the following PCR mixture:
19-μL reaction with 10 μL of Promega GoTaq G2 Hot Start
Master Mix, 0.6 μL of 10 μM each forward or reverse primer
[jgLCO/jgHCO (2)] and 0.2 μL of 20 mg/mL BSA. PCR thermal
cycling conditions were as follows: 5 min at 95 °C; four cycles of
30 s at 94 °C, 45 s at 50 °C, and 60 s at 72 °C; 34 cycles at 45 °C
annealing temperature; and a final extension of 8 min at 72 °C.
PCR product was purified using ExoSAP-IT (Affymetrix), and
sequences were generated in both directions with the Sanger
sequencing platform. We then repeated the PCR assay with the
mlCOIintF/jgHCO primer combination (3) whenever the initial
reaction was not successful (∼8% of samples) to increase our
success rate.
B. DNA Metabarcoding.
DNA extractions. DNA was extracted from 10 g of homogenized
sessile tissue, and the crushed half of the 2-mm to 500-μm and
500- to 106-μm samples using the MO-BIO Powermax Soil DNA
Isolation Kit. The initial bead-beating step of the kit was found
to shear DNA. Therefore, we added proteinase K (0.4 mg/mL)
to the powerbead solution (+ C1 solution) instead and incubated
samples in a shaking incubator overnight at 56 °C, which ensured
effective tissue lysis. We followed the manufacturer’s instructions
for the rest of the protocol. However, for sessile samples, we only
used one-third of the homogenized tissue lysate for extraction to
prevent clogging of the silica membrane of the spin column.
Extracted DNA was purified using the MO-BIO Powerclean
DNA Clean-Up Kit and quantified with a Qubit fluorometer
(dsDNA HS Assay kit; Invitrogen) before PCR amplification.
PCR amplification, tagging, and sequencing. We used a hierarchical
tagging approach (as in ref. 3) combining seven tailed PCR
primers (Table S4) and eight Ion Xpress barcode adapters (Life
Technologies) for sample multiplexing. Three replicate PCR
assays were performed to amplify an ∼313-bp COI fragment for
each of the 54 bulk samples using the following PCR mixture:
20-μL reaction with 1 μL of 10 μM each forward or reverse primer
(tailed-mlCOIintF/tailed-jgHCO; Table S4), 1.4 μL of 10 mM
dNTP, 0.4 μL of Clontech Advantage 2 Polymerase Mix, 2 μL of
Clontech Advantage 2 PCR buffer, and 1 μL (10 ng) of purified
DNA. We used the touchdown PCR profile with 16 initial cycles:
Leray and Knowlton www.pnas.org/cgi/content/short/1424997112
denaturation for 10 s at 95 °C, annealing for 30 s at 62 °C (−1 °C
per cycle), and extension for 60 s at 72 °C, followed by 20 cycles at
an annealing temperature of 46 °C. Triplicate PCR products were
pooled and purified using Agencourt AMPure XP beads, and
equimolar amounts of each sample were pooled, with each pool
containing amplicons generated with each of the seven tailed-
primer pairs (total of eight pools). End-repair (Ion Plus Fragment
Library kit) and ligation of Ion Xpress barcode adapters were
conducted following the manufacturer’s instructions (Life Tech-
nologies). Library templates were clonally amplified using the
OT2 400-bp kit on the Ion One Touch 2, and enriched template
ISPs were sequenced on the Ion Torrent platform using the Ion
PGM 400-bp version 2 protocol (all from Life Technologies).
VI. Data Analysis
A. DNA Barcoding. Forward and reverse sequences were assembled,
checked for stop codons or frame shifts, and edited in Geneious
(Biomatters). Our dataset comprised a diversity of taxonomic
groups so that using a fixed sequence dissimilarity cutoff (i.e., 5%)
for clustering OTUs would not result in accurate species delin-
eations. Therefore, we used the Bayesian clustering algorithm
implemented in CROP (4) to delineate OTUs based on the
natural distribution of sequence dissimilarity in the dataset.
Lower and upper bound variance was set to 3 and 4, respectively,
because these settings were shown to provide the best results for
marine invertebrates (3). CROP outputs a representative se-
quence per OTU that was used for taxonomic identification.
B. DNA Metabarcoding. Higher quality reads prefiltered by Torrent
Suite Software version 4.0.2 (Life Technologies) were assigned to
samples based on the combination primer tail-Ion Xpress barcode.
Additional sequences were removed from the prefiltered dataset if
they (i) were shorter than 250 bp, (ii) had more than two mis-
matches in the primer sequence, (iii) had any ambiguous base call,
or (iv) had at least one homopolymer region longer than 8 bp. We
then used the option “enrichAlignment” in Multiple Alignment of
Coding Sequences (MACSE) (5) to align our reads to the high-
quality library of COI barcodes of the Moorea Biocode project
(7,675 sequences from 30 animal phyla represented), an all-taxa
biodiversity inventory of the Moorea Island ecosystem (6), re-
taining sequences that had zero stop codons (using invertebrate
mitochondrial translation table), zero frame shifts, zero insertions,
and no more than three deletions. This latter step maximizes the
reliability of the sequence dataset.
C. Taxonomic Assignments. We performed BLASTn searches (7)
of OTU representative sequences of the barcoding and meta-
barcoding datasets in GenBank and BOLD. Previous papers that
used similar DNA sequencing approaches to look at terrestrial
(8) and marine diversity (3, 9) used 98% assignments. However,
the distribution of sequence similarity in our dataset showed
a significant number of matches between 97% and 98% before
rapidly dropping below 90%, a pattern that may be driven by
matches to specimens collected across the Atlantic Ocean.
Therefore, we accepted species level matches when similarity to
the reference barcode was higher than 97%.
In the absence of a direct match we used a phylogenetic approach
implemented in the Statistical Assignment Package (10) to assign
OTUs to higher taxonomic levels. The program was set to down-
load up to 40 homologs from GenBank with ≥70% sequence
identity. We accepted taxonomic assignments at an 80% posterior
probability cutoff, but we did not consider assignment lower than
order level to minimize misidentifications due to the lack of data
for some taxonomic groups in GenBank. OTUs that matched or
were assigned to bacteria were removed.
2 of 11
VII. Assessment of Reliability of Metabarcoding Approach
A. Fraction Sized 2 mm to 500 μm. One ARMS from each of the three
sites at each location was randomly chosen for analysis. Archived
bulk samples (see SI Text, section IV), which correspond to half of
the sample measured by weight, were resuspended in a graduated
beaker containing 100 mL of 95% EtOH and homogenized with
a spatula, and 20 mL was immediately collected using a Hensel–
Stempel pipette. All specimens were isolated and identified to the
lowest taxonomic level using morphology, the entire specimen was
used for phenol DNA extraction according to the protocol de-
scribed in SI Text (section V.A), and the mitochondrial COI gene
was sequenced for OTU delineation.
The amount of DNA in each individual extract was measured
with a Qubit fluorometer. The total amount of DNA represented
by each OTU was calculated by summing the amount of DNA of
each specimen belonging to that same OTU according to COI
barcodes. To identify OTUs shared between datasets, we ran local
BLAST searches [using Geneious (Biomatters)] of one repre-
sentative sequence per OTU obtained via barcoding against the
full database of OTU representative sequences obtained via
metabarcoding.
To evaluate the efficacy of metabarcoding at detecting di-
versity, we first calculated the overall proportion of OTUs shared
by the barcoding and metabarcoding datasets at each location.
We then conducted similar calculations but between datasets of
corresponding ARMS. For example, we compared the proportion
of shared OTUs between the barcoding and metabarcoding
datasets of ARMS 1 from FL. To evaluate the efficacy of meta-
barcoding at estimating OTU relative abundance, we first tested
the relationship between the amount of DNA per OTU and
1. Gaither MR, Szabó Z, Crepeau MW, Bird CE, Toonen RJ (2010) Preservation of corals
in salt-saturated DMSO buffer is superior to ethanol for PCR experiments. Coral
Reefs 30(2):329–333.
2. Geller J, Meyer C, Parker M, Hawk H (2013) Redesign of PCR primers for mitochondrial
cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa
biotic surveys. Mol Ecol Resour 13(5):851–861.
3. Leray M, et al. (2013) A new versatile primer set targeting a short fragment of the
mitochondrial COI region for metabarcoding metazoan diversity: Application for
characterizing coral reef fish gut contents. Front Zool 10:34.
4. Hao X, Jiang R, Chen T (2011) Clustering 16S rRNA for OTU prediction: A method of
unsupervised Bayesian clustering. Bioinformatics 27(5):611–618.
5. Ranwez V, Harispe S, Delsuc F, Douzery EJP (2011) MACSE: Multiple Alignment of
Coding SEquences accounting for frameshifts and stop codons. PLoS ONE 6(9):
e22594.
Leray and Knowlton www.pnas.org/cgi/content/short/1424997112
number of reads per OTU in the metabarcoding dataset. We
pooled amounts of DNA and read number for OTUs belonging to
the same phylum to test for the same relationship at the level of
functional groups.
We sorted and photographed a total of 251 and 954 animals in
three 2-mm to 500-μm fractions from VA and FL, respectively
(representing one ARMS from each of the three sites at the two
locations). A total of 671 specimens were individually barcoded,
which includes all specimens except Tanaidacea and Ostracoda
from FL, for which only ∼25% of specimens were individually
barcoded because of their high abundance. Based on the subset
of specimens analyzed, abundant Ostracoda and Tanaidacea
belonged to one and three OTUs, respectively. We extrapolated
the amount of DNA for each of these four OTUs for subsequent
analysis.
B. Sessile Fraction. We individually subsampled and barcoded mor-
phologically distinctive sessile taxa to identify matching OTUs in the
metabarcoding dataset using local BLASTn searches (as discussed
above). The number of reads per OTU was then compared with the
estimated cover of each OTU on each ARMS as measured by
a point count approach implemented in Coral Point Count with
Excel extensions (CPCe) (11). A 15 × 15 grid was positioned over
each plate photograph, and the taxon located under each in-
tersection of the grid was recorded. All 10 plates (19 sides) were
scored for each ARMS.
The efficacy of metabarcoding at detecting diversity and relative
abundance of sessile taxa was evaluated using similar calculations
as presented in the previous section (SI Text, section VII.A).
6. Leray M, Boehm JT, Mills SC, Meyer CP (2012) Moorea BIOCODE barcode library as
a tool for understanding predator–prey interactions: Insights into the diet of common
predatory coral reef fishes. Coral Reefs 31(2):383–388.
7. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment
search tool. J Mol Biol 215(3):403–410.
8. Yu DW, et al. (2012) Biodiversity soup: Metabarcoding of arthropods for rapid bio-
diversity assessment and biomonitoring. Methods Ecol Evol 3(4):613–623.
9. Machida RJ, Hashiguchi Y, Nishida M, Nishida S (2009) Zooplankton diversity analysis
through single-gene sequencing of a community sample. BMC Genomics 10:438.
10. Munch K, Boomsma W, Huelsenbeck JP, Willerslev E, Nielsen R (2008) Statistical as-
signment of DNA sequences using Bayesian phylogenetics. Syst Biol 57(5):750–757.
11. Kohler KE, Gill SM (2006) Coral Point Count with Excel extensions (CPCe): A Visual
Basic program for the determination of coral and substrate coverage using random
point count methodology. Comput Geosci 32(9):1259–1269.
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 VA FL
VA

FL

VA

Fig. S1. Illustration of study design and diversity encountered. (A) Map of experimental design. (B) Photographs of Virginia location, ARMS, and ARMS re-
covery. (C) Photographs of representative ARMS plates and organisms in the 2-mm to 500-μm fraction. (D) Sample processing workflow. In C, scale bars are
provided for individual organisms, and the square plates are 22.5 cm on each side.

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 OTUs in VA only OTUs in FL only OTUs at both localities

Fig. S2. Proportion of identified OTUs in the metabarcoding dataset according to the number of ARMS where they were detected. (A) Virginia only.
(B) Florida only. (C) Both localities. OTUs were considered to match a reference barcode if they had >97% similarity to a COI sequence in the BOLD or GenBank
or in a reference barcode generated in this study.

VA

FL

VA

FL

Fig. S3. Individual-based rarefaction curves.

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 VA

FL

Fig. S4. Clustering analyses [PCoA (A and C) and UPGMA trees (B and D)] depicting similarity in community composition among >2-mm samples based on OTU
incidence (Jaccard; A and B) and relative abundance (Bray–Curtis; C and D). PC, principal component.

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 VA

FL

VA

FL

FL

VA

FL

VA

FL
VA

VA

FL

Fig. S5. PCoA with coordinates of the 10 most abundant phyla. The size of the sphere is proportional to the mean relative abundance of the taxon across samples.

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 Table S1. OTU diversity and abundance as revealed by DNA barcoding and DNA metabarcoding in ARMS from VA and FL
VA FL

Barcoding Metabarcoding Barcoding Metabarcoding

2 mm to 500 to 2 mm to 500 to
Diversity descriptors >2 mm 500 μm 106 μm Sessile Total >2 mm 500 μm 106 μm Sessile Total

No. of sequences 498 256,147 97,439 218,704 572,290 655 155,232 86,350 168,031 409,613

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Total no. of OTUs 38 651 828 436 1,204 64 821 976 591 1,391
Mean (±SD) no. of OTUs 11.2 ± 4.1 203.3 ± 52.3 290.3 ± 32.1 146.6 ± 28.9 434.2 ± 55.7 15.8 ± 4.9 277.2 ± 37.6 360.1 ± 28.5 222.9 ± 24.3 536.7 ± 30.8
Mean (±SD) rarefied 8.2 ± 2.1 117.1 ± 21.4 229.7 ± 20.5 85.7 ± 15.1 333.5 ± 34.9 9.6 ± 2.2 202.6 ± 16.4 312.1 ± 23.2 157.4 ± 15.5 484.4 ± 30.7
no. of OTUs
Chao1 [95% CI] 46.0 [40.1, 1,075.6 [953.3, 1,204.9 [1,106.4, 638.7 [568.1, 1,711.4 [1,596.5, 104.8 [80.2, 1,183.0 [1,082.1, 1,486.0 [1,356.2, 858.0 [769.8, 1,945.7 [1,821.2,
67.8] 1,247.2] 1,338.2] 746.9] 1,859.9] 166.9] 1,322.8] 1,660.2] 989.7] 2,106.2]
Chao1 [95% CI] 49.2 [39.4, 552.7 [469.3, 917.8 [843.7, 451.5 [380.0, 1,174.0 [1,082.3, 144.7 [70.7, 866.5 [772.2, 1,213.9 [1,108.9, 562.1 [505.6, 1,483.7 [1,384.1,
(rarefied) 80.8] 690.4] 1,021.6] 569.8] 1,298.7] 403.5] 1,007.4] 1,358.7] 653.5] 1,617.3]
Chao2 [95% CI] 50.5 [41.7, 1,126.2 [1,001.7, 1,309.5 [1,191.1, 706.7 [621.0, 1,891.0 [1,746.4, 140.06 [94.3, 1,213.3 [1,117.7, 1,536.0 [1,406.2, 880.2 [796.2, 2,056.3 [1,921.3,
80.3] 1,295.0] 1,466.5] 832.0] 2,074.1] 254.8] 1,339.8] 1,705.1] 998.5] 2,225.7]
Chao2 [95% CI] 49.4 [39.7, 535.6 [468.3, 1,007.3 [913.2, 557.3 [449.4, 1,256.3 [1,149.8, 116.1 892.4 [804.3, 1,280.5 [1,167.8, 596.6 [533.6, 1,562.7 [1,455.1,
(rarefied) 79.4] 638.5] 1,136.2] 730.4] 1,398.0] [64.6, 280] 1,015.4] 1,431.1] 692.6] 1,702.1]
ACE 47.6 1,062.8 1,223.0 628.3 1,743.0 108.6 1,197.8 1,483.1 819.38 1,982.0
ACE (rarefied) 57.6 515.4 975.7 459.1 1,217.8 91.6 837.7 1,213.2 556.02 1,521.3
ICE 56.5 1,205.5 1,345.6 741.2 1,928 139.9 1,326.1 1,537.2 892.0 2,078.5
ICE (rarefied) 70.6 580.0 1,066.3 551.3 1,300.7 105.6 942.8 1,269.0 588.6 1,583.0
OTUs with match to 60.5 14.1 10.6 16.2 10.2 57.8 15.7 11.8 16.9 11.9
BOLD/GenBank, %
Unidentified OTUs, % NA 35.6 38.8 31.2 40.9 NA 26.8 27.1 23.8 28.3
Singletons, % 31.6 39.8 36.5 34.9 34.8 46.9 32.3 34.6 30.3 31.1

Each diversity estimate was calculated using both raw and rarefied OTU tables. ACE, abundance-based coverage estimator; CI, confidence interval; ICE, incidence-based coverage estimator; NA, not applicable.

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 Table S2. Percent contribution of individual OTUs to differences between localities and fractions (based on similarity of percentage analyses)
VA FL

500 to 500-μm 106-μm 500 to 500-μm 106-μm

OTU no. Kingdom Phylum Class Subclass/order Genus/species VA-FL 106 μm sessile sessile 106 μm sessile sessile

x17 Animalia Annelida Clitellata Haplotaxida Tubificoides 1.9 2.2

parapectinatus
x19 Animalia Annelida Polychaeta Eunicida Marphysa 2.9 5.5 1.6 4.6 3.0 3.0 1.6
sanguinea
x350 Animalia Annelida Polychaeta Sabellida Branchiomma 1.6 1.8
cf. bairdi
x1039 Animalia Annelida Polychaeta Spionida Polydora 1.7 1.7
cornuta
x283 Animalia Annelida Polychaeta Spionida Streblospio 1.2 4.2 4.2
benedicti
x432 Animalia Annelida Polychaeta Terebellida Polycirrus 1.0
x1066 Animalia Annelida Polychaeta Terebellida 1.1 1.0
x26 Animalia Annelida Polychaeta 1.3 1.4
x282 Animalia Annelida Polychaeta 1.9 1.7

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x38 Animalia Annelida 1.0 1.0
x791 Animalia Arthropoda Malacostraca Amphipoda Caprella 1.0 1.1
penantis
x320 Animalia Arthropoda Malacostraca Amphipoda Monocorophium 1.0
acherusicum
x136 Animalia Arthropoda Malacostraca Amphipoda Gammarus 1.4 4.1 5.1
mucronatus
x165 Animalia Arthropoda Malacostraca Amphipoda 1.1 4.1 4.4
x372 Animalia Arthropoda Malacostraca Amphipoda 1.5 1.3
x83 Animalia Arthropoda Malacostraca Amphipoda 2.3 8.3 9.0
x102 Animalia Arthropoda Malacostraca Decapoda 2.8 2.5
x352 Animalia Arthropoda Malacostraca Decapoda Dyspanopeus 1.2 1.3
sayi
x998 Animalia Arthropoda Malacostraca Decapoda Panopeus 2.5 2.3
occidentalis
x418 Animalia Arthropoda Malacostraca Isopoda Cilicaea 2.1 1.8
x37 Animalia Arthropoda Malacostraca Stomatopoda Neogonodactylus 2.3 2.2
bredini
x597 Animalia Arthropoda Maxillopoda Calanoida Centropages 1.2 1.5
hamatus
x574 Animalia Arthropoda Maxillopoda Calanoida 1.9 1.3
x983 Animalia Arthropoda Maxillopoda Calanoida 1.3 1.1
x62 Animalia Arthropoda Maxillopoda Harpacticoida 1.8 1.7
x607 Animalia Arthropoda Maxillopoda Siphonostomatoida 1.5 1.4
x39 Animalia Arthropoda Maxillopoda 2.0 6.1 5.4
x397 Animalia Arthropoda Maxillopoda 1.3 1.2
x492 Animalia Arthropoda Maxillopoda 1.4 1.3
x871 Animalia Arthropoda Maxillopoda 1.9 1.4
x84 Animalia Arthropoda Ostracoda 4.1 6.2 7.1 5.8

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 Table S2. Cont.
VA FL

500 to 500-μm 106-μm 500 to 500-μm 106-μm

OTU no. Kingdom Phylum Class Subclass/order Genus/species VA-FL 106 μm sessile sessile 106 μm sessile sessile

x688 Animalia Arthropoda Pycnogonida Pantopoda 1.0 1.0

x1054 Animalia Arthropoda 2.7 2.7
x2 Animalia Arthropoda 1.0
x651 Animalia Arthropoda 2.7 2.4
x799 Animalia Arthropoda 1.2 1.0
x5 Animalia Bryozoa Gymnolaemata Cheilostomatida Bugula 1.2 4.1 3.9
neritina
x3 Animalia Bryozoa Gymnolaemata Cheilostomatida Biflustra 1.0 2.5 2.9
arborescens
x182 Animalia Bryozoa Gymnolaemata Cheilostomatida Schizoporella 4.0 11.8 10.6 7.7 8.0
errata
x245 Animalia Bryozoa Gymnolaemata Cheilostomatida 1.6 2.4 1.6 2.3
x198 Animalia Chordata Ascidiacea Phlebobranchia Ascidia 2.7 8.5 8.6
virginea

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x13 Animalia Chordata Ascidiacea Stolidobranchia Symplegma 3.7 1.4 9.4 8.9
rubra
x1 Animalia Chordata 1.9 1.9
x437 Animalia Chordata 1.1 3.9 3.5
x841 Animalia Cnidaria Hydrozoa Leptothecata Obelia 11.5 12.4 12.2 11.0
bidentata
x1114 Animalia Cnidaria Hydrozoa 7.3 7.7 9.5 9.0
x139 Animalia Echinodermata Ophiuroidea Ophiurida Amphipholis 1.8 1.6
cf. squamata
x132 Animalia Mollusca Bivalvia Ostreoida Ostrea equestris 1.6 1.6
x544 Animalia Mollusca Gastropoda Littorinimorpha Crepidula plana 1.1 1.1
x575 Animalia Porifera Demospongiae Halichondrida 2.2 2.3
x1061 Animalia Porifera Demospongiae Homosclerophorida Oscarella 3.1 3.0 6.5 6.2
x8 Chromista Ochrophyta Phaeophyceae Ectocarpales Ectocarpus 1.0 1.3 1.4 1.1
siliculosus
x423 Plantae Rhodophyta Florideophyceae Ceramiales Polysiphonia 1.2 1.3
x366 Plantae Rhodophyta Florideophyceae Gracilariales Gracilaria 2.2 2.5
vermiculophylla
x733 Plantae Rhodophyta Florideophyceae 1.1 1.0
x11 Unidentified 1.5 1.4
x112 Unidentified 1.3 1.2
x131 Unidentified 1.8 1.7
x147 Unidentified 2.1 1.9
x158 Unidentified 1.0 3.7 3.7
x21 Unidentified 1.5 1.4
x36 Unidentified 1.0
x581 Unidentified 1.1 1.1

Only OTUs with a contribution superior to 1% are presented.

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 Table S3. Biomass and sediment in samples from VA and FL
Location: fraction Site Mean (±SD) biomass, g Mean (±SD) sediment, g

VA: 2 mm to 500 μm 1 19 (±13) 59 (±87)

2 19 (±7) 82 (±29)
3 17 (±13) 9 (±5)
VA: 500 to 100 μm 1 19 (±2) 791 (±386)
2 18 (±3) 497 (±400)
3 14 (±4) 835 (±216)
VA: Sessile 1 148 (±54) NA
2 249 (±28) NA
3 85 (±9) NA
FL: 2 mm to 500 μm 1 6 (±1) 4 (±2)
2 8 (±1) 25 (±19)
3 11 (±2) 8 (±4)
FL: 500 to 100 μm 1 11 (±2) 110 (±109)
2 12 (±2) 34 (±36)
3 21 (±10) 175 (±139)
FL: Sessile 1 118 (±25) NA
2 127 (±18) NA
3 225 (±8) NA

NA, not applicable, because there was no sediment in the sessile fraction.

Table S4. Tailed PCR primers

Primer label Primer sequence (5′–3′)

mlCOIint_Tag1 AGACGCGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag2 AGTGTAGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag3 ACTAGCGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag4 ACAGTCGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag5 ATCGACGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag6 ATGTCGGGWACWGGWTGAACWGTWTAYCCYCC
mlCOIint_Tag7 ATAGCAGGWACWGGWTGAACWGTWTAYCCYCC
jgHCO_Tag1 AGACGCTAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag2 AGTGTATAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag3 ACTAGCTAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag4 ACAGTCTAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag5 ATCGACTAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag6 ATGTCGTAIACYTCIGGRTGICCRAARAAYCA
jgHCO_Tag7 ATAGCATAIACYTCIGGRTGICCRAARAAYCA

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