Short Communication
Identifying coral reef fish larvae through DNA barcoding: A test case with the families Acanthuridae and Holocentridae

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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.

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, 2000).

Here, we explore the efficacy of the barcoding 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 assemblages or correspond to single-species schools? Second, without additional knowledge, larvae collected 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 families, 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.

Section snippets

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 surface 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

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

Acknowledgments

This study was conducted during the Master 2 degree from PARIS VI of Erwan Delrieu-Trottin on the ecology of larval assemblages in the “Centre de Biologie et d’Ecologie Tropicale et Méditéranéenne” in Perpignan 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

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