# Identifying coral reef fish larvae through DNA barcoding: a test case with the families Acanthuridae and Holocentridae

Nicolas Hubert, Erwan Delrieu-Trottin, Jean-Olivier Irisson, Christopher

Meyer, Serge Planes

► To cite this version:

Nicolas Hubert, Erwan Delrieu-Trottin, Jean-Olivier Irisson, Christopher Meyer, Serge Planes. *Identifying coral reef fish larvae through DNA barcoding: a test case with the families Acanthuridae and Holocentridae*. Molecular Phylogenetics and Evolution, 2010, 55 (3), pp.1195-1203. 10.1016/j.ympev.2010.02.023. [hal-00609382](https://hal.science/hal-00609382v1)

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https://hal.science/hal-00609382v1

Submitted on 13 Feb 2025

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# Identifying coral reef fish larvae through DNA barcoding: A test case with the families Acanthuridae and Holocentridae¹

Nicolas HUBERT<sup>a,∗</sup>, Erwan DELRIEU-TROTTIN<sup>b</sup>, Jean-Olivier IRISSON<sup>b</sup>, Christopher MEYER<sup>c</sup>, and Serge PLANES<sup>b,d</sup>

## Abstract

A reference collection of COI barcode (650 bp) for the Pacific Society Islands has been constituted for 22 species of Acanthuridae and 16 species of Holocentridae. Divergence between congeneric species was on average 20-fold to 87-fold higher than divergence between conspecific sequences and this set of DNA-identifiers was used to identify 40 larvae of both families. All larvae sequenced could be identified to species using DNA-barcodes. Pools of larvae constitute multi-specific assemblages and no additional species compared to adult reef communities were sampled in larval pools, suggesting that the larval assemblages originated from adult communities on neighboring reefs.

**Keywords:** Coral reef, Barcoding, Larvae, Identification, Mitochondrial DNA

## 1. Introduction

DNA barcoding seeks to develop automated DNA-based identifications using molecular species tags based on short, standardized gene regions (Hebert et al., 2003; Hebert and Gregory, 2005). The primary goal of DNA barcoding is to create reference DNA-barcode libraries for known species used as DNA-identifiers (e.g. Kerr et al., 2007; Hubert et al., 2008). Mitochondrial DNA (mtDNA) has been widely used in evolutionary studies owing to its higher mutation rate and lower effective population size than nuclear DNA (Brown et al., 1979; Birky et al., 1989), and efforts have converged on a 650-bp portion of the mitochondrial cytochrome c oxidase I gene (COI) that can be readily recovered from a vast array of lineages with a limited set of primers. For a barcoding approach to succeed, within species DNA sequences need to be more similar to one another than those between species and recent studies confirmed that the majority of species examined are well delineated by a tight cluster of very similar sequences (Ward et al., 2005; Clare et al., 2006; Robins et al., 2007; Kerr et al., 2007; Hubert et al., 2008; Foottit et al., 2009; Sheffield et al., 2009). Nevertheless, some pitfalls have been identified due to the presence of pseudogenes, introgressive hybridization, and retention of ancestral polymorphism (Zhang and Hewitt, 1996;

Funk and Omland, 2003; Meyer and Paulay, 2005). The occurrence of mixed genealogies among closely related species were estimated to reach 20% (Funk and Omland, 2003), although recent barcoding surveys suggest that it may not exceed 5–10% (Kerr et al., 2007; Hubert et al., 2008). Coral reefs are among the most diverse ecosystems and the Indo-Pacific region alone hosts 10,490 fish species, nearly 32 percent of Earth’s ichthyofauna (Froese and Pauly, 2000). In ecosystems with no obvious physical barriers, assessing the determinants of connectivity is a priority for conservation practices (Mora et al., 2006; Claudet et al., 2008). In marine systems, connectivity is widely assessed through analysis of gene flow (e.g. Doherty et al., 1995; Jones et al., 1999; Almany et al., 2007). However, community level processes such as competitive exclusion, assortative settlement and habitat selection may strongly influence species distribution and thereby, communities connectivity (Loreau and Mouquet, 1999; Mouquet and Loreau, 2002; Webb et al., 2002; Leibold et al., 2004). Given their high diversity and dramatic phenotypic changes during development, coral reef fish species identification is no easy task and only feasible up to the genera at best for early ontogenetic stages based on diagnostic morphological characters (e.g. Leis and Carson-Ewart, 2004). Species interactions, however, are likely to vary largely depending on ontogenetic stages through which interactions occur (e.g. Webb,

<footnote><sup>a</sup>Laboratoire ECOMAR, Université de la Réunion, 15 Avenue René Cassin, BP 7151, 97715 Saint-Denis Messag Cedex 9, Réunion, France</footnote>

<footnote><sup>b</sup>Équipe récifs coralliens, UMR 5244 CNRS-UPVD-EPHE, Centre de Biologie et Écologie Tropicale et Méditéranéenne, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France</footnote>

<footnote><sup>c</sup>Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, MRC-163, Washington, DC 20013, USA</footnote>

<footnote><sup>d</sup>Centre de Recherches Insulaires et Observatoire de l'Environnement (CRIOBE) UMS 2978 EPHE-CNRS, BP 1013 Papetoai, 98729 Moorea, Polynésie française, France</footnote>

<footnote>*Corresponding authors: myloplus@excite.com</footnote>
2000). Here, we explore the efficacy of the barcod-
ing approach in the identification of coral reef fish
larvae to the species level in order to address the
following questions. First, larvae are aggregated in
patches and schools (Doherty, 1987) and are often
collected in pools of several phylogenetically-related
individuals, e.g. several individuals from one family
or one genus. Do these pools host multi-specific as-
semblages or correspond to single-species schools?
Second, without additional knowledge, larvae col-
lected are assumed to come from the neighboring
reefs. With a more precise species-level identification,
one can ask whether larvae samples contain species
that are not present in adult communities? In such
context, we first assessed the genetic variability at
COI for two of the most abundant coral reef fish fam-
ilies, namely Acanthuridae and Holocentridae, and
further explored its use in a species-level tagging for
the identification of early ontogenetic stages up to
the species level.

## 2. Material and Methods

**2.1. Sampling reef fishes larvae in the pelagic realm**

Fish larvae were sampled during an oceanographic
campaign aboard the N.O. Alis in May 2006 all
around the atoll of Tetiaroa in the Society archipelago
(17°S, 149°55W). Owing to its relative isolation by sur-
face currents, the atoll of Tetiaroa is likely to be an
autonomous system where self-recruitment sustains
most of the local populations. In order to describe the
spatial distribution of larvae, thirteen stations laid in
a radiating pattern around the atoll were sampled
twice. Samples were obtained by trawling with a
Multiple Opening-Closing Net and Environmental
Sampling System (MOCNESS) with an 800 µm mesh
and 4 m² opening. Trawling was conducted between
the surface and 100 m where the majority of coral
reef fish larvae are found (Boehlert et al., 1992). Sam-
pling was stratified vertically: net M0 sampled from
0 down to 100 m and nets M1 to M4 sampled back up
to the surface, in 25 m layers. M0 samples were pre-
served in 90% ethanol to allow the use of DNA-based
identification. M1–M4 samples were preserved in a
4% buffered formaldehyde-sea water solution, which
preserves pigmentation, for morphological identifi-
cation and further oceanographic analysis. In the
M0 net, larvae were identified to the lowest taxo-
nomic level possible using a stereo-microscope to
look for morphological criteria described in previ-
ous systematic surveys (e.g. Moser, 1996; Leis and
Carson-Ewart, 2004; Miller and Tsukamoto, 2004)
such as general shape, fin rays and spines count,
head spine location, and pigmentation. Equivocal
identifications were confirmed by using an online
photography database developed for this purpose

(http://cbetm.univ-perp.fr/larvae). Over all dataset,
morphological identification discriminated 82 fam-
ilies, with a number of larvae ranking from 4 to
231 individuals per station. The families Acanthuri-
dae and Holocentridae were the most abundant in
the formaldehyde-preserved samples, particularly in
stations 9 and 10 during the first round of sampling
since 87 out of the 158 Acanthuridae and 78 out of the
120 Holocentridae were sampled there (Irisson, 2008).
Likewise, Acanthuridae and Holocentridae were the
most abundant families in ethanol-preserved sam-
ples and stations 9 and 10 of the first sampling round
also contained high concentrations of larvae, with
15 Acanthuridae and 20 Holocentridae at sta-
tions 9 and 11 Acanthuridae at station 10. Finally, a total
of 46 larvae (sum of Holocentridae and Acanthuri-
dae of stations 9 and 10) were used for DNA-based
identifications.

**2.2. Adult sampling, reference library and barcoding** Fish adults used as DNA-identifiers for larvae in the reference library were sampled apart in the context of the Moorea biocode project and FISH-BOL campaign (Ward et al., 2009) in 2006 all around the atoll of Moorea in the Society Archipelago. The reference library of adult sequences was built from ethanol-preserved fin clips on specimens identified by experts using morphological criteria (Randall, 2005). For each species, reference specimens were deposited as vouchers in publicly available collections, namely the ‘Muséum National d’Histoire Naturelle’ (MNHN) in Paris and the Moorea Biocode collection (MBIO) in Berkeley. In order to fit with the BARCODE criteria in GenBank (Hubert et al., 2008), sequences were bi-directionally sequenced for at least 500 bp and electropherogram trace files were made accessible in the NCBI Trace Archive as well as forward and reverse PCR amplification primers. In addition, a link with the Barcode of Life Data System (BOLD) has been created to provide access to detailed voucher data including collection record and photographs. These data are also publicly available in the Moorea Biocode databases (http://biocode.berkeley.edu) (Table 1).

Genomic DNA was extracted using the Gentra
System Puregene DNA Purification Kit accord-
ing to manufacturer specifications. A 650-bp
segment was amplified from the 5' region of the
mitochondrial COI gene using the primers FF2d-
5'TTCTCCACCAACCACAARGAYATYGG3' and
FR1d-5'CACCTCAGGGTGTCCGAARAAYCARAA3'
(Ivanova et al., 2007). PCR amplifications were
performed in 27 µl including 10.7 µl of ultrapure
water, 2.5 µl of 10 PCR buffer, 3 µl of MgCl2 (25 mM),
2.5 µl of each primers (10 mM), 3 µl of each dNTP (2
mM), 0.3 µl of Taq DNA polymerase (5U/µl), and 4
µl of template DNA. The PCR conditions consisted of 94°C (5 min), 10 cycles of 94°C (1 min), 60–50°C decreasing 1°C per cycle (1 min), 72°C (1 min 30 s) followed by 25 cycles of 94°C (1 min), 50°C (1 min), 72°C (1 min 30 s), with a final extension at 72°C (5 min). All sequences have been deposited in GenBank (Accession Numbers XX–XX). Accession Numbers for the barcodes, specimen and collection data, sequences, trace files, and primers details are available within the project "in progress" in BOLD (http://www.barcodinglife.org). Sequence divergence was calculated using the JC69 model (Jukes and Cantor, 1969) and ultrametric trees were computed using the UPGMA algorithm as implemented in *PAUP* 4.0b10 (Swofford, 2002) to provide a graphic representation of species divergence. Finally, several metrics were computed from the pairwise distance matrix using the package APE for R (Paradis et al., 2004), namely the mean, minimum and maximum of the distance within species ($D_{within species}$), the distance to the nearest neighbor ($D_{NN}$) and the distance between species ($D_{between species}$).

## 3. Results and discussion

All adult specimens were successfully amplified using the primers FF2d and FR1d. Four genera and 22 species were discriminated among the 53 adults of Acanthuridae. Another four genera and 16 species were characterized from 53 adults of Holocentridae (Table 1). Those 106 sequences constituted the reference library of DNA-identifiers used to assign larvae to known species. Among the 46 larvae collected in this study, only three Acanthuridae and three Holocentridae failed to amplify using FF2d and FR1d. The 40 remaining larvae provided COI consensus established through bi-directional sequencing. A total of 146 COI barcodes of 650-bp were thus obtained for 38 species and eight genera. No insertions, deletions or codon stops were found, supporting that all amplified sequences constitute functional mitochondrial COI. All the amplified sequences were larger than 600-bp, the maximum size limit observed for non-functional nuclear copies of mtDNA genes (Zhang and Hewitt, 1996).

The distribution of pairwise differences among COI barcodes of adults revealed little to no overlap in the distribution of divergence within and between species (Figure 1). The majority of COI sequences were identical within species, while reaching up to four differences in Acanthuridae and two differences in Holocentridae. The minimum number of inter-specific differences was eight for Acanthuridae and two for Holocentridae, hence only slightly overlapping with intra-specific differences for the

latter. The divergence of sequences within species ($D_{within species}$) was relatively homogeneous between genera, ranging from 0.001 to 0.004 in both families (Table 2). By contrast, divergence between species ($D_{between species}$) greatly varied among genera from an average of 0.063 in *Myripristis* to 0.11 on average in *Acanthurus*. Overall, the divergence was 20-fold to 87-fold higher among congeneric species than among conspecific sequences. The average distance to the nearest neighbor ($D_{NN}$), i.e. the minimum genetic distance between a species and its closest congeneric relative, was lower than the average distance between species while remaining 13-fold to 85-fold higher than the mean divergence within species. Unexpectedly, $D_{NN}$ was more homogeneous between genera than the average congeneric distance between species, averaging 0.05 in *Naso*, *Zebrasoma*, *Myripristis*, and *Sargocentron* while reaching 0.08 in *Acanthurus* and *Ctenochaetus*.

The distribution of pairwise differences within and among species slightly overlapped in Holocentridae, but all species from both families were monophyletic and formed clusters of tightly related sequences (Table 2, Figure 2). It has been recently proposed that the efficacy of the barcoding approach relies on the existence of a 'barcoding gap', between intra- and inter-specific divergence (Meyer and Paulay, 2005). This gap is created by the fact that mutation is more frequent than speciation, therefore lineages diversify more quickly between species than within species (Pons et al., 2006). Provided that speciation is stochastic, sampling large pools of species increases the probability of sampling species pairs that diverged recently. Nevertheless, overlap in the distribution of divergence will only lead to erroneous molecular identification if sister species remain in the lineage sorting period and still share polymorphism through common ancestry (Funk and Omland, 2003). This was not the case here since the sister species examined were all reciprocally monophyletic, even the pair *Sargocentron caudimaculatum* and *S. spiniferum* that diverged only by 0.007 on average. This result reinforces the view that no canonical threshold applies to the frontier separating populations and species in fishes (e.g. Hubert et al., 2008).

The pattern of divergence together with the topology of species' genealogies support that barcoding with COI is effective for the Acanthuridae and Holocentridae communities from the Society Islands. Thus, this set of DNA-identifiers was used for the molecular identification of the 40 larvae sequenced for both families (Figure 2). The 40 larval sequences all branched unambiguously within a species' genealogy. At station 9 (MOCNESS M9/1), three *Acanthurus* and one *Naso* species were identified (*A. glaucopareius*,
<table><caption>**Table 1:** Details of the capture and registration of specimens. The columns are: voucher catalog number either in the Muséum National d'Histoire Naturelle (MParis) or in Moorea Biode Databases (MBIO), MBIO specimen numbers, Barcode of Life Database (BOLD) specimen and sequence numbers, GenBank Accession Numbers, and geographic location. Stars ( ) denote species names assigned to an early stage specimen by the present molecular analysis.</caption><thead><tr><th rowspan="2">Family</th><th rowspan="2">Species</th><th rowspan="2">Voucher</th><th rowspan="2">Biocode ref</th><th colspan="2">BOLD</th><th rowspan="2">Genbank</th><th rowspan="2">Island</th><th rowspan="2">Island group</th><th rowspan="2">Latitude/Longitude</th></tr><tr><th>Specimen ref</th><th>Sequence ref</th></tr></thead><tbody><tr><td>Acanthuridae</td><td>Acanthurus acanthus</td><td>MHNH2008-953</td><td>MB101277</td><td>MB101277</td><td>MBF4764-07</td><td>HM034179</td><td>Moorea</td><td>Society Islands</td><td>17.48440/-149.91580</td></tr><tr><td>Acanthuridae</td><td>Acanthurus acanthus</td><td>MHNH2008-954</td><td>MB101278</td><td>MB101278</td><td>MBF4765-07</td><td>HM034178</td><td>Moorea</td><td>Society Islands</td><td>17.48440/-149.91580</td></tr><tr><td>Acanthuridae</td><td>Acanthurus acanthus</td><td>MBIO18242</td><td>MO1101</td><td>MB1018242</td><td>MBF4008-10</td><td>HM034177</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Acanthurus blochii</td><td>MHNH2008-891</td><td>MBIO1159</td><td>MB1011594</td><td>MBF4681-07</td><td>HM034180</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Acanthuridae</td><td>Acanthurus glaucopareius</td><td>MBIO18213</td><td>MO772</td><td>MB1018213</td><td>MBF4003-10</td><td>HM034184</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.96/-138.838</td></tr><tr><td>Acanthuridae</td><td>Acanthurus glaucopareius</td><td>MHNH2009-1628</td><td>MOCNESS M9/1-MO-2</td><td>MOCNESS M9/1-MO-2</td><td>FPFL001-09</td><td>HM034137</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus lineatus</td><td>MHNH2008-762</td><td>MBIO918</td><td>MBIO918.4</td><td>MBF4565-07</td><td>HM034183</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus lineatus</td><td>MBIO919</td><td>MBIO919</td><td>MBIO919.4</td><td>MBF4566-07</td><td>HM034182</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricans</td><td>MHNH2008-239</td><td>MBIO136</td><td>MBIO136.4</td><td>MBF4780-07</td><td>HM034186</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.85630</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricans</td><td>MHNH2008-240</td><td>MBIO137</td><td>MBIO137.4</td><td>MBF4708-07</td><td>HM034185</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricauda</td><td>MHNH2008-888</td><td>MBIN1154</td><td>MB101154.4</td><td>MBF4679-07</td><td>HM034187</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricauda</td><td>MHNH2008-454</td><td>MBIO441</td><td>MB10441.4</td><td>MBF4263-07</td><td>HM034189</td><td>Moorea</td><td>Society Islands</td><td>17.50270/-149.92500</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricauda</td><td>MHNH2008-761</td><td>MBIO917</td><td>MBIO917.4</td><td>MBF4564-07</td><td>HM034188</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2008-230</td><td>MBIO126</td><td>MB10126.4</td><td>MBF4068-07</td><td>HM034190</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2008-168</td><td>MBIO41</td><td>MB1041.4</td><td>MBF4013-07</td><td>HM034192</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2008-169</td><td>MBIO41</td><td>MB1041.4</td><td>MBF4042-07</td><td>HM034191</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1629</td><td>MOCNESS M9/1-MO-1</td><td>MOCNESS M9/1-MO-1</td><td>FPFL002-09</td><td>HM034138</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1630</td><td>MOCNESS M9/1-MO-3</td><td>MOCNESS M9/1-MO-3</td><td>FPFL003-09</td><td>HM034139</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1631</td><td>MOCNESS M9/1-MO-4</td><td>MOCNESS M9/1-MO-4</td><td>FPFL004-09</td><td>HM034140</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1632</td><td>MOCNESS M9/1-MO-6</td><td>MOCNESS M9/1-MO-6</td><td>FPFL005-09</td><td>HM034141</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1633</td><td>MOCNESS M9/1-MO-7</td><td>MOCNESS M9/1-MO-7</td><td>FPFL006-09</td><td>HM034142</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1634</td><td>MOCNESS M9/1-MO-11</td><td>MOCNESS M9/1-MO-11</td><td>FPFL007-09</td><td>HM034143</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1635</td><td>MOCNESS M9/1-MO-12</td><td>MOCNESS M9/1-MO-12</td><td>FPFL008-09</td><td>HM034144</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2009-1636</td><td>MOCNESS M9/1-MO-13</td><td>MOCNESS M9/1-MO-13</td><td>FPFL009-09</td><td>HM034145</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus nigricoccus</td><td>MHNH2008-788</td><td>MBIO953</td><td>MBIO953.4</td><td>MBF4569-07</td><td>HM034193</td><td>Moorea</td><td>Society Islands</td><td>17.48430/-149.89404</td></tr><tr><td>Acanthuridae</td><td>Acanthurus olivaceus</td><td>MBIO1233</td><td>MB101233</td><td>MB101233.4</td><td>MBF4725-07</td><td>HM034197</td><td>Moorea</td><td>Society Islands</td><td>17.48230/-149.89330</td></tr><tr><td>Acanthuridae</td><td>Acanthurus olivaceus</td><td>MHNH2008-763</td><td>MBIO920</td><td>MBIO920.4</td><td>MBF4567-07</td><td>HM034198</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus olivaceus</td><td>MBIO18257</td><td>MO116</td><td>MB1018257</td><td>MBF4011-10</td><td>HM034196</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Acanthurus olivaceus</td><td>MBIO18261</td><td>MO1120</td><td>MB1018261</td><td>MBF4012-10</td><td>HM034195</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Acanthurus olivaceus</td><td>MBIO18328</td><td>MO1187</td><td>MB1018328</td><td>MBF4014-10</td><td>HM034194</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.972/-138.842</td></tr><tr><td>Acanthuridae</td><td>Acanthurus pyroferus</td><td>MBIO1316</td><td>MBIO1316</td><td>MB101316.4</td><td>MBF4793-07</td><td>HM034200</td><td>Moorea</td><td>Society Islands</td><td>17.47140/-149.77280</td></tr><tr><td>Acanthuridae</td><td>Acanthurus pyroferus</td><td>MHNH2008-757</td><td>MBIO909</td><td>MBIO909.4</td><td>MBF4559-07</td><td>HM034200</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus pyroferus</td><td>MHNH2008-758</td><td>MBIO910</td><td>MBIO910.4</td><td>MBF4041-07</td><td>HM034201</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Acanthuridae</td><td>Acanthurus pyroferus</td><td>MBIO18263</td><td>MO1122</td><td>MB1018263</td><td>MBF4013-10</td><td>HM034199</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Acanthurus thomsoni</td><td>MBIO1620</td><td>MB101620</td><td>MB101620.4</td><td>MBF4396-07</td><td>HM034203</td><td>Moorea</td><td>Society Islands</td><td>17.48530/-149.85860</td></tr><tr><td>Acanthuridae</td><td>Acanthurus thomsoni</td><td>MHNH2008-808</td><td>MBIO987</td><td>MBIO987.4</td><td>MBF4612-07</td><td>HM034204</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Acanthuridae</td><td>Acanthurus thomsoni</td><td>MHNH2008-161</td><td>MBIO33</td><td>MB1033.4</td><td>MBF4007-07</td><td>HM034205</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2008-774</td><td>MBIO934</td><td>MB103934.4</td><td>MBF4578-07</td><td>HM034207</td><td>Moorea</td><td>Society Islands</td><td>17.48430/-149.86940</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MBIO935</td><td>MBIO935</td><td>MB103935.4</td><td>MBF4579-07</td><td>HM034206</td><td>Moorea</td><td>Society Islands</td><td>17.48430/-149.86940</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1637</td><td>MOCNESS M10/1-MO-37</td><td>MOCNESS M10/1-MO-37</td><td>FPFL010-09</td><td>HM034146</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1638</td><td>MOCNESS M10/1-MO-38</td><td>MOCNESS M10/1-MO-38</td><td>FPFL011-09</td><td>HM034147</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1639</td><td>MOCNESS M10/1-MO-39</td><td>MOCNESS M10/1-MO-39</td><td>FPFL012-09</td><td>HM034148</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1640</td><td>MOCNESS M9/1-MO-5</td><td>MOCNESS M9/1-MO-5</td><td>FPFL013-09</td><td>HM034149</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1641</td><td>MOCNESS M9/1-MO-8</td><td>MOCNESS M9/1-MO-8</td><td>FPFL014-09</td><td>HM034150</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1642</td><td>MOCNESS M9/1-MO-9</td><td>MOCNESS M9/1-MO-9</td><td>FPFL015-09</td><td>HM034151</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1643</td><td>MOCNESS M9/1-MO-10</td><td>MOCNESS M9/1-MO-10</td><td>FPFL016-09</td><td>HM034152</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus triostegus</td><td>MHNH2009-1644</td><td>MOCNESS M9/1-MO-14</td><td>MOCNESS M9/1-MO-14</td><td>FPFL017-09</td><td>HM034153</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Acanthurus xanthopterus</td><td>MHNH2008-887</td><td>MBIO1153</td><td>MB101153.4</td><td>MBF4678-07</td><td>HM034208</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Acanthuridae</td><td>Acanthurus xanthopterus</td><td>MHNH2008-805</td><td>MBIO982</td><td>MB10982</td><td>MBF4608-07</td><td>HM034212</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Acanthuridae</td><td>Ctenoaeetus flavicuda</td><td>MHNH2008-806</td><td>MBIO983</td><td>MBIO983.4</td><td>MBF4609-07</td><td>HM034211</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Acanthuridae</td><td>Ctenoaeetus flavicuda</td><td>MBIO18256</td><td>MO115</td><td>MB1018256</td><td>MBF4010-10</td><td>HM034209</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Ctenoaeetus striatus</td><td>MHNH2008-242</td><td>MBIO140</td><td>MB10140.4</td><td>MBF4079-07</td><td>HM034214</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Ctenoaeetus striatus</td><td>MHNH2008-243</td><td>MBIO141</td><td>MB10141.4</td><td>MBF4080-07</td><td>HM034213</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Naso anulatus</td><td>MHNH2008-1130</td><td>MBIO1798</td><td>MB101798</td><td>MBF4998-07</td><td>HM034241</td><td>Moorea</td><td>Society Islands</td><td>17.53500/-149.77110</td></tr><tr><td>Acanthuridae</td><td>Naso anulatus</td><td>MHNH2009-1645</td><td>MOCNESS M10/1-MO-41</td><td>MOCNESS M10/1-MO-41</td><td>FPFL024-09</td><td>HM034154</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Naso anulatus</td><td>MHNH2009-1646</td><td>MOCNESS M10/1-MO-46</td><td>MOCNESS M10/1-MO-46</td><td>FPFL025-09</td><td>HM034155</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-820</td><td>MBIO1002.4</td><td>MB101002.4</td><td>MBF4624-07</td><td>HM034242</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-271</td><td>MBIO177</td><td>MB10177.4</td><td>MBF4105-07</td><td>HM034246</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-272</td><td>MBIO178</td><td>MB10178.4</td><td>MBF4106-07</td><td>HM034245</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-158</td><td>MBIO29</td><td>MBIO29.4</td><td>MBF4005-07</td><td>HM034248</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-159</td><td>MBIO30</td><td>MBIO30.4</td><td>MBF4006-07</td><td>HM034247</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-725</td><td>MBIO861</td><td>MBIO861.4</td><td>MBF4525-07</td><td>HM034244</td><td>Moorea</td><td>Society Islands</td><td>17.57840/-149.87250</td></tr><tr><td>Acanthuridae</td><td>Naso lituratus</td><td>MHNH2008-726</td><td>MBIO862</td><td>MBIO862.4</td><td>MBF4526-07</td><td>HM034243</td><td>Moorea</td><td>Society Islands</td><td>17.57840/-149.87250</td></tr><tr><td>Acanthuridae</td><td>Naso unicorns</td><td>MHNH2008-292</td><td>MBIO206</td><td>MB10206.4</td><td>MBF4124-07</td><td>HM034249</td><td>Moorea</td><td>Society Islands</td><td>17.57007/-149.82240</td></tr><tr><td>Acanthuridae</td><td>Naso unicorns</td><td>MHNH2009-1647</td><td>MOCNESS M9/1-MO-15</td><td>MOCNESS M9/1-MO-15</td><td>FPFL026-09</td><td>HM034156</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Acanthuridae</td><td>Naso unicorns</td><td>MHNH2009-1648</td><td>MOCNESS M10/1-MO-42</td><td>MOCNESS M10/1-MO-42</td><td>FPFL027-09</td><td>HM034157</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Naso unicorns</td><td>MHNH2009-1649</td><td>MOCNESS M10/1-MO-44</td><td>MOCNESS M10/1-MO-44</td><td>FPFL018-09</td><td>HM034158</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Naso unicorns</td><td>MHNH2009-1650</td><td>MOCNESS M10/1-MO-45</td><td>MOCNESS M10/1-MO-45</td><td>FPFL019-09</td><td>HM034159</td><td>Tetiaroa</td><td>Society Islands</td><td>16.95403/-149.56125</td></tr><tr><td>Acanthuridae</td><td>Naso vlamingii</td><td>MHNH2008-921</td><td>MBIO1214</td><td>MB101214.4</td><td>MBF4711-07</td><td>HM034251</td><td>Moorea</td><td>Society Islands</td><td>17.48230/-149.89330</td></tr><tr><td>Acanthuridae</td><td>Naso vlamingii</td><td>MBIO1215</td><td>MBIO1215</td><td>MB101215</td><td>MBF4569-07</td><td>HM034250</td><td>Moorea</td><td>Society Islands</td><td>17.48230/-149.89330</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MBIO18243</td><td>MO1102</td><td>MB101824</td><td>MBF4009-10</td><td>HM034282</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.95/-138.832</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MHNH2008-257</td><td>MBIO160</td><td>MB10160.4</td><td>MBF4094-07</td><td>HM034286</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MHNH2008-258</td><td>MBIO161</td><td>MB10161.4</td><td>MBF4095-07</td><td>HM034285</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MBIO442</td><td>MBIO442</td><td>MB10442.4</td><td>MBF4264-07</td><td>HM034284</td><td>Moorea</td><td>Society Islands</td><td>17.50270/-149.92500</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MBIO18386</td><td>MOOP30</td><td>MB1018386</td><td>MBF4015-10</td><td>HM034283</td><td>Moorea</td><td>Society Islands</td><td>17.481/-149.903</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MHNH2008-839</td><td>MBO1033</td><td>MBO1033.4</td><td>MBF4641-07</td><td>HM034288</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Acanthuridae</td><td>Zenoscaenus traepturus</td><td>MBIO1034</td><td>MBO1034</td><td>MBO1034.4</td><td>MBF4642-07</td><td>HM034287</td><td>Moorea</td><td>Society Islands</td><td>17.48260/-149.89990</td></tr><tr><td>Holocentridae</td><td>Myripristis adusta</td><td>MHNH2008-903</td><td>MBIO1181</td><td>MB10118.4</td><td>MBF4691-07</td><td>HM034216</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Myripristis adusta</td><td>MBIO1182</td><td>MBIO1182</td><td>MB101182.4</td><td>MBF4692-07</td><td>HM034215</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Myripristis amaena</td><td>MHNH2008-1075</td><td>MBIO1598</td><td>MB101598.4</td><td>MBF4291-07</td><td>HM034220</td><td>Moorea</td><td>Society Islands</td><td>17.59220/-149.84030</td></tr><tr><td>Holocentridae</td><td>Myripristis amaena</td><td>MHNH2008-1094</td><td>MBIO1634</td><td>MB101634.4</td><td>MBF4946-07</td><td>HM034218</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis amaena</td><td>MHNH2008-518</td><td>MBIO535</td><td>MB10535.4</td><td>MBF4947-07</td><td>HM034217</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MBIO18218</td><td>MO77</td><td>MB18218</td><td>MBF4005-10</td><td>HM034224</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.96/-138.838</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MHNH2008-918</td><td>MBIO1210</td><td>MB1210.4</td><td>MBF4709-07</td><td>HM034227</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MHNH2008-919</td><td>MBIO1211</td><td>MB1211.4</td><td>MBF4710-07</td><td>HM034226</td><td>Moorea</td><td>Society Islands</td><td>17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MHNH2009-1652</td><td>MOCNESS M9/1-MO-32</td><td>MOCNESS M9/1-MO-32</td><td>FPFL021-09</td><td>HM034161</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MHNH2009-1653</td><td>MOCNESS M9/1-MO-35</td><td>MOCNESS M9/1-MO-35</td><td>FPFL022-09</td><td>HM034162</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Myripristis berndti</td><td>MHNH2008-1093</td><td>MBIO1632</td><td>MB101632.4</td><td>MBF4494-07</td><td>HM034231</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis kuntee</td><td>MBIO1633</td><td>MBIO1633</td><td>MB101633.4</td><td>MBF4495-07</td><td>HM034230</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis kuntee</td><td>MBIO18235</td><td>MOH94</td><td>MB18235</td><td>MBF4007-10</td><td>HM034232</td><td>Mohotani</td><td>Marquesas Islands</td><td>9.96/-138.838</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-976</td><td>MBIO1321</td><td>MB101321</td><td>MBF4797-07</td><td>HM034236</td><td>Moorea</td><td>Society Islands</td><td>17.47140/-149.77280</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-977</td><td>MBIO1322</td><td>MB101322.4</td><td>MBF4798-07</td><td>HM034235</td><td>Moorea</td><td>Society Islands</td><td>17.47140/-149.77280</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-1090</td><td>MBIO1627</td><td>MB101627.4</td><td>MBF4940-07</td><td>HM034234</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-1091</td><td>MBIO1628</td><td>MB101628.4</td><td>MBF4941-07</td><td>HM034233</td><td>Moorea</td><td>Society Islands</td><td>17.49220/-149.92530</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-265</td><td>MBIO170</td><td>MB10170.4</td><td>MBF4100-07</td><td>HM034238</td><td>Moorea</td><td>Society Islands</td><td>17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Myripristis pralinia</td><td>MHNH2008-753</td><td>MBIO903</td><td>MB10903.4</td><td>MBF4555-07</td><td>HM034237</td><td>Moorea</td><td>Society Islands</td><td>17.48900/-149.85800</td></tr><tr><td>Holocentridae</td><td>Myripristis violacea</td><td>MHNH2008-1121</td><td>MBIO1744</td><td>MB101744.4</td><td>MBF4985-07</td><td>HM034240</td><td>Moorea</td><td>Society Islands</td><td>17.48970/-149.89140</td></tr><tr><td>Holocentridae</td><td>Myripristis violacea</td><td>MHNH2008-1122</td><td>MBIO1745</td><td>MB101745.4</td><td>MBF4986-07</td><td>HM034239</td><td>Moorea</td><td>Society Islands</td><td>17.48970/-149.89140</td></tr><tr><td>Holocentridae</td><td>Myripristis violacea</td><td>MHNH2009-1654</td><td>MOCNESS M9/1-MO-34</td><td>MOCNESS M9/1-MO-34</td><td>FPFL023-09</td><td>HM034163</td><td>Tetiaroa</td><td>Society Islands</td><td>16.96977/-149.56289</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><tr><td>Holocentridae</td><td>Neoniphon samara</td><td>MHNH2008-457</td><td>MBIO446</td><td>MBIO446.4</td><td>MBFA268-07</td><td>HM034255</td><td>Moorea</td><td>Society Islands</td><td>-17.50270/-149.92500</td></tr><tr><td>Holocentridae</td><td>Neoniphon samara</td><td>MHNH2008-458</td><td>MBIO447</td><td>MBIO447.4</td><td>MBFA269-07</td><td>HM034254</td><td>Moorea</td><td>Society Islands</td><td>-17.50270/-149.92500</td></tr><tr><td>Holocentridae</td><td>Neoniphon samara</td><td>MHNH2008-1064</td><td>MBIO1574</td><td>MBIO1574.4</td><td>MBFA913-07</td><td>HM034253</td><td>Moorea</td><td>Society Islands</td><td>-17.59220/-149.84030</td></tr><tr><td>Holocentridae</td><td>Neoniphon samara</td><td>MBIO1575</td><td>MBIO1575</td><td>MBIO1575.4</td><td>MBFA914-07</td><td>HM034252</td><td>Moorea</td><td>Society Islands</td><td>-17.59220/-149.84030</td></tr><tr><td>Holocentridae</td><td>Plectrypops lima</td><td>MHNH2008-266</td><td>MBIO171</td><td>MBIO171.4</td><td>MBFA101-07</td><td>HM034256</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron caudimaculatum</td><td>MBIO609</td><td>MBIO609</td><td>MBIO609.4</td><td>MBFA369-07</td><td>HM034272</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron caudimaculatum</td><td>MBIO18217</td><td>MOH76</td><td>MBIO18217</td><td>MBFC004-10</td><td>HM034257</td><td>Mohotani</td><td>Marquesas Islands</td><td>-9.96/-138.838</td></tr><tr><td>Holocentridae</td><td>Sargocentron caudimaculatum</td><td>MHNH2009-1655</td><td>MOCNESS M9.1-MO-21</td><td>MOCNESS M9.1-MO-21</td><td>FPFL028-09</td><td>HM034164</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron caudimaculatum</td><td>MHNH2009-1656</td><td>MOCNESS M9.1-MO-23</td><td>MOCNESS M9.1-MO-23</td><td>FPFL029-09</td><td>HM034165</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron diadema</td><td>MHNH2008-915</td><td>MBIO1206</td><td>MBIO1206.4</td><td>MBFA707-07</td><td>HM034259</td><td>Moorea</td><td>Society Islands</td><td>-17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Sargocentron diadema</td><td>MHNH2008-916</td><td>MBIO1207</td><td>MBIO1207.4</td><td>MBFA708-07</td><td>HM034258</td><td>Moorea</td><td>Society Islands</td><td>-17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Sargocentron melanospilos</td><td>MHNH2008-914</td><td>MBIO1205</td><td>MBIO1205.4</td><td>MBFA706-07</td><td>HM034260</td><td>Moorea</td><td>Society Islands</td><td>-17.51330/-149.84960</td></tr><tr><td>Holocentridae</td><td>Sargocentron melanospilos</td><td>MHNH2008-710</td><td>MBIO839</td><td>MBIO839.4</td><td>MBFA508-07</td><td>HM034261</td><td>Moorea</td><td>Society Islands</td><td>-17.57840/-149.87250</td></tr><tr><td>Holocentridae</td><td>Sargocentron microstoma</td><td>MHNH2008-262</td><td>MBIO166</td><td>MBIO166.4</td><td>MBFA098-07</td><td>HM034265</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron microstoma</td><td>MHNH2008-263</td><td>MBIO167</td><td>MBIO167.4</td><td>MBFA099-07</td><td>HM034264</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron microstoma</td><td>MHNH2008-651</td><td>MBIO751</td><td>MBIO751.4</td><td>MBFA449-07</td><td>HM034263</td><td>Moorea</td><td>Society Islands</td><td>-17.60630/-149.83400</td></tr><tr><td>Holocentridae</td><td>Sargocentron microstoma</td><td>MHNH2008-652</td><td>MBIO752</td><td>MBIO752.4</td><td>MBFA450-07</td><td>HM034262</td><td>Moorea</td><td>Society Islands</td><td>-17.60630/-149.83400</td></tr><tr><td>Holocentridae</td><td>Sargocentron punctatissimum</td><td>MHNH2008-305</td><td>MBIO223</td><td>MBIO223.4</td><td>MBFA137-07</td><td>HM034270</td><td>Moorea</td><td>Society Islands</td><td>-17.48250/-149.88210</td></tr><tr><td>Holocentridae</td><td>Sargocentron punctatissimum</td><td>MHNH2008-306</td><td>MBIO224</td><td>MBIO224.4</td><td>MBFA138-07</td><td>HM034269</td><td>Moorea</td><td>Society Islands</td><td>-17.48250/-149.88210</td></tr><tr><td>Holocentridae</td><td>Sargocentron punctatissimum</td><td>MHNH2008-699</td><td>MBIO824</td><td>MBIO824.4</td><td>MBFA498-07</td><td>HM034268</td><td>Moorea</td><td>Society Islands</td><td>-17.57840/-149.87250</td></tr><tr><td>Holocentridae</td><td>Sargocentron punctatissimum</td><td>MHNH2008-700</td><td>MBIO825</td><td>MBIO825.4</td><td>MBFA499-07</td><td>HM034267</td><td>Moorea</td><td>Society Islands</td><td>-17.57840/-149.87250</td></tr><tr><td>Holocentridae</td><td>Sargocentron microstoma</td><td>MBIO18192</td><td>MOH51</td><td>MBIO18192</td><td>MBFC002-10</td><td>HM034266</td><td>Mohotani</td><td>Society Islands</td><td>-9.96/-138.838</td></tr><tr><td>Holocentridae</td><td>Sargocentron spiniferum</td><td>MHNH2008-455</td><td>MBIO443</td><td>MBIO443.4</td><td>MBFA265-07</td><td>HM034276</td><td>Moorea</td><td>Society Islands</td><td>-17.50270/-149.92500</td></tr><tr><td>Holocentridae</td><td>Sargocentron spiniferum</td><td>MBIO444</td><td>MBIO444</td><td>MBIO444.4</td><td>MBFA266-07</td><td>HM034275</td><td>Moorea</td><td>Society Islands</td><td>-17.50270/-149.92500</td></tr><tr><td>Holocentridae</td><td>Sargocentron spiniferum</td><td>MHNH2008-514</td><td>MBIO530</td><td>MBIO530.4</td><td>MBFA321-07</td><td>HM034274</td><td>Moorea</td><td>Society Islands</td><td>-17.50270/-149.92500</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2008-288</td><td>MBIO200</td><td>MBIO200.4</td><td>MBFA120-07</td><td>HM034279</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2008-289</td><td>MBIO201</td><td>MBIO201.4</td><td>MBFA121-07</td><td>HM034278</td><td>Moorea</td><td>Society Islands</td><td>-17.48240/-149.88300</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2008-752</td><td>MBIO902</td><td>MBIO902.4</td><td>MBFA122-07</td><td>HM034277</td><td>Moorea</td><td>Society Islands</td><td>-17.48900/-149.85800</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1657</td><td>MOCNESS M9.1-MO-17</td><td>MOCNESS M9.1-MO-17</td><td>FPFL030-09</td><td>HM034166</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1658</td><td>MOCNESS M9.1-MO-18</td><td>MOCNESS M9.1-MO-18</td><td>FPFL031-09</td><td>HM034167</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1659</td><td>MOCNESS M9.1-MO-19</td><td>MOCNESS M9.1-MO-19</td><td>FPFL032-09</td><td>HM034168</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1660</td><td>MOCNESS M9.1-MO-22</td><td>MOCNESS M9.1-MO-22</td><td>FPFL033-09</td><td>HM034169</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1661</td><td>MOCNESS M9.1-MO-24</td><td>MOCNESS M9.1-MO-24</td><td>FPFL034-09</td><td>HM034170</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1662</td><td>MOCNESS M9.1-MO-25</td><td>MOCNESS M9.1-MO-25</td><td>FPFL035-09</td><td>HM034171</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1663</td><td>MOCNESS M9.1-MO-26</td><td>MOCNESS M9.1-MO-26</td><td>FPFL036-09</td><td>HM034172</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1664</td><td>MOCNESS M9.1-MO-27</td><td>MOCNESS M9.1-MO-27</td><td>FPFL037-09</td><td>HM034173</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1665</td><td>MOCNESS M9.1-MO-29</td><td>MOCNESS M9.1-MO-29</td><td>FPFL038-09</td><td>HM034174</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiare</td><td>MHNH2009-1666</td><td>MOCNESS M9.1-MO-30</td><td>MOCNESS M9.1-MO-30</td><td>FPFL039-09</td><td>HM034175</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiereoides</td><td>MHNH2008-751</td><td>MBIO901</td><td>MBIO901.4</td><td>MBFA553-07</td><td>HM034280</td><td>Moorea</td><td>Society Islands</td><td>-17.48900/-149.85800</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiereoides</td><td>MBIO658</td><td>MBIO658</td><td>MBIO658.4</td><td>MBFA391-07</td><td>HM034281</td><td>Moorea</td><td>Society Islands</td><td>-17.49460/-149.86190</td></tr><tr><td>Holocentridae</td><td>Sargocentron tiereoides</td><td>MHNH2009-1667</td><td>MOCNESS M9.1-MO-20</td><td>MOCNESS M9.1-MO-20</td><td>FPFL040-09</td><td>HM034176</td><td>Tetiaroa</td><td>Society Islands</td><td>-16.96977/-149.56289</td></tr></table>

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Distribution of the number of pairwise differences among COI sequences between and within species for Acanthuridae (A) and Holocentridae (B).</figcaption></figure>
<table><caption>**Table 2:** Summary of genetic divergences (JC69 model used for computing distances) at COI for the 38 species of the 8 genera analyzed here. Early stage specimens are not included. N, number of pairwise comparisons; D<sub>within species</sub>, distance between conspecific haplotypes; D<sub>NN</sub>, distance to the nearest neighbor (i.e. the minimum of D<sub>between species</sub>), D<sub>between species</sub>, distance between two heterospecific haplotypes; M, percent of monophyletic species.</caption><thead><tr><th rowspan="2">Family</th><th rowspan="2">Genus</th><th rowspan="2">N</th><th rowspan="2">Species</th><th colspan="3">D<sub>within species</sub></th><th colspan="3">D<sub>NN</sub></th><th colspan="3">D<sub>between species</sub></th><th rowspan="2">M</th></tr><tr><th>min</th><th>mean</th><th>max</th><th>min</th><th>mean</th><th>max</th><th>mean</th><th>max</th></tr></thead><tbody><tr><td rowspan="4">Acanthuridae</td><td>Acanthurus</td><td>465</td><td>13</td><td>0</td><td>0.002 ± 0.002</td><td>0.008</td><td>0.028</td><td>0.075 ± 0.026</td><td>0.104</td><td>0.110 ± 0.018</td><td>0.134</td><td>100</td></tr><tr><td>Ctenochaetus</td><td>6</td><td>2</td><td>0</td><td>0.001 ± 0.001</td><td>0.003</td><td>0.085</td><td>0.085</td><td>0.085</td><td>0.087 ± 0.002</td><td>0.090</td><td>100</td></tr><tr><td>Naso</td><td>55</td><td>4</td><td>0</td><td>0.004 ± 0.004</td><td>0.010</td><td>0.034</td><td>0.052 ± 0.021</td><td>0.072</td><td>0.081 ± 0.014</td><td>0.093</td><td>100</td></tr><tr><td>Zebrasoma</td><td>21</td><td>3</td><td>0</td><td>0.003 ± 0.003</td><td>0.006</td><td>0.017</td><td>0.056 ± 0.067</td><td>0.134</td><td>0.104 ± 0.054</td><td>0.140</td><td>100</td></tr><tr><td rowspan="4">Holocentridae</td><td>Myripristis</td><td>300</td><td>6</td><td>0</td><td>0.001 ± 0.002</td><td>0.009</td><td>0.040</td><td>0.050 ± 0.013</td><td>0.050</td><td>0.063 ± 0.012</td><td>0.080</td><td>100</td></tr><tr><td>Neoniphon</td><td>6</td><td>1</td><td>0</td><td>0.002 ± 0.003</td><td>0.006</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Plectrypops</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Sargocentron</td><td>253</td><td>8</td><td>0</td><td>0.002 ± 0.002</td><td>0.009</td><td>0.007</td><td>0.052 ± 0.047</td><td>0.117</td><td>0.102 ± 0.038</td><td>0.138</td><td>100</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Figure 2:** UPGMA dendrograms based on a JC69 model of sequence evolution for Acanthuridae (A) and Holocentridae (B). Thick branching represent within species genetic variability. COI barcodes obtained from larvae are labeled MOCNESS and printed in bold.</figcaption></figure>
*A. triostegus*, *A. nigrofuscus*, and *N. unicornis*) while three *Myripritis* and three *Sargocentron* species were identified (*M. violacea*, *M. amaena*, *M. berndti*, *S. caudimaculatum*, *S. tiere*, and *S. tiereoides*). By contrast, station 10 (MOCNESS M10/1) was dominated by the genus *Naso* with two species (*N. annulatus*, *N. unicornis*) and a single-species of *Acanthurus* was detected (*A. triostegus*).

Compared to morphological diagnostic characters,
the present barcoding approach provided an unprece-
dented level of resolution in the identification of early
stages of fish. Among the 26 of Acanthuridae larvae,
identifications to the sub-family level were feasible
for all specimens due to the convex profile of the
head in Acanthurinae and a pigmentation spot on
the caudal peduncle in Nasinae. Within the Nasinae,
the eight specimens were assigned unambiguously to
*Naso*, the only genus of Nasinae in the region, while
only two specimens in the Acanthurinae were unam-
biguously identified as *Acanthurus* due to the pres-
ence of nine dorsal fin spines. Likewise, identification
in Holocentridae was possible to the sub-family level
due to the shape of the rostrum (long and simple
in Holocentrinae, short and bifurcated in Myripristi-
nae). A single specimen was large enough to count
anal and dorsal fins rays which allowed to assign it
unambiguously to the genus *Sargocentron*. Species-
level meristic characters are not even available for
those families in the region. Overall, identifications
up to the genus were not feasible for 16 out of 26
Acanthuridae larvae and 19 out of 20 Holocentridae
larvae, due to overlap in meristic characters or to
the small size of the specimens prohibiting unam-
biguous counts of spines and fin rays. Morphologi-
cal identification and molecular assignment were in
agreement, although DNA-barcoding allowed unam-
biguous identifications up to the species level for all
40 specimens.

Placement of larval COI barcodes in the COI tree
provided insights into our first concern, 'Do these
pools host multi-specific assemblages or correspond
to single-species schools?'. The two samples MOC-
NESS M9/1 and MOCNESS M10/1 yielded multi-
specific assemblages of the most specious genera,
namely *Acanthurus*, *Naso*, *Myripristis*, and *Sargocen-
tron*. This result suggests that larvae from several
species may be found concomitantly in a limited vol-
ume of water (i.e. 1000 m³ of water sampled on
average per trawling), and further evidence that con-
specific larvae does not necessarily aggregate in large
school as previously suggested (Doherty, 1987). For
example, samples from MOCNESS M9/1 yielded 32
larvae from 10 species suggesting that conspecific lar-
vae do not necessarily aggregate in large schools but
rather mix in multi-specific assemblages. In addition,

the two samples of Acanthuridae showed consider-
able heterogeneity since *Acanthurus* dominated in
MOCNESS M9/1 while *Naso* was more common in
MOCNESS M10/1. Finally, the question regarding
the potential occurrence of species caught as larvae
but undetected in adult reef communities remains
open since no additional species compared to adult
communities were sampled in larvae at these two
stations.

Acknowledgements

This study was conducted during the Master 2 degree
from PARIS VI of Erwan Delrieu-Trottin on the ecoly-
ogy of larval assemblages in the "Centre de Biologie
et d'Ecologie Tropicale et Méditéranéenne" in Per-
pignan under the supervision of Serge Planes, Jean-
Olivier Irisson and Nicolas Hubert. The authors wish
to thank the crew of the N.O. Alis and the scientific
team (R. Crechriou, C. Paris, C. Guigand, L. Carassou,
D. Lecchini, P. Ung). Many thanks go to R.K. Cowen
for providing facilities with the MOCNESS and J.
Grondin for his help with the sorting of MOCNESS
samples. The authors are also grateful to A. Fukui,
D. Johnson, J.M. Leis, M.J. Miller, D. Richardson, and
A. Suntsov for their advice on the identification of
fish larvae, Elisabeth Rochel for genetic lab support,
Emmanuel Paradis for his help with APE scripts, Ben-
jamin Ciotti for his help in improving the manuscript
and the staff of the CRIOBE, in Moorea, for pre and
post-fieldwork support. Funding for this work came
from the ACI MOOREA, the Agence Nationale de la
Recherche (ANR, project Bioaqua), the "Délégation à
la recherche du Haut Commissariat de la République
en Polynésie", and the DIREN of French Polynesia.
This work was supported by 'Agence Nationale de
la Recherche' (France: BIOTAS, ANR-2006-BDIV002)

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