Short Communication
Molecular phylogenetics and evolution of Holacanthus angelfishes (Pomacanthidae)

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Abstract

The angelfish genus Holacanthus includes seven species in the tropical Eastern Pacific and Atlantic. In this study we performed an analysis on all species, the closely related regal angelfish, and the Cortez angelfish, using four mitochondrial and one nuclear marker. Our results support a monophyletic Holacanthus. The Indo-Pacific regal angelfish, Pygoplytes diacanthus, was found to be the closest relative to Holacanthus. We found a split into two clades with divergences that were consistent with the rise of the Isthmus of Panama. An internally calibrated molecular clock thus placed the origin of Holacanthus to ∼10.2–7.6 million years ago.

Introduction

The angelfish genus Holacanthus (family Pomacanthidae) is relatively small with only seven recognized species. Holacanthus resides in the tropical portion of three discrete biogeographic regions: The Eastern Atlantic, the Western Atlantic, and the tropical Eastern Pacific (Fig. 1). The West African angelfish, Holacanthus africanus, is the only species found along the tropical Eastern Atlantic coastline, in the Gulf of Guinea (São Tomé) and the Cape Verde Islands. Within the Western Atlantic, three species occur on coral and rocky reefs, H. tricolor, H. bermudensis, and H. ciliaris. The rock beauty, H. tricolor, and the queen angelfish, H. ciliaris have the widest distributions, from Bermuda to Brazil, where different color morphs of H. ciliaris can be found on the NE coast and at isolated offshore islands (Luiz, 2003, Feeley et al., 2009). The blue angelfish, H. bermudensis has a more restricted distribution, from Bermuda, along the entire Atlantic coast of the US from North Carolina south, and throughout the Gulf of Mexico (Smith-Vaniz et al., 1999). Atlantic Holacanthus have traditionally been divided into two separate subgenera, Angelichthys, which comprises H. africanus, H. bermudensis, and H. ciliaris, and Holacanthus which comprises only H. tricolor (Allen et al., 1998, Debelius et al., 2003).

Within the tropical Eastern Pacific, H. passer, H. clarionensis, and H. limbaughi occur on rocky and coral reefs, (Robertson and Allen, 2008) (Fig. 1). The king angelfish, H. passer, has the widest distribution, from central Baja California, Mexico, through the Gulf of California south to northern Peru, plus the Revillagigedo (its abundance there is unclear), Cocos, Malpelo, and the Galapagos Islands. Two insular endemic species, the Clipperton angelfish H. limbaughi, and the Clarion angelfish, H. clarionensis, are found at Clipperton Atoll and the Revillagigedo Islands, respectively. Small numbers, most likely vagrants, of H. clarionensis are found at the southern tip of Baja California, while there is a single record of that species at Clipperton Island (Allen and Robertson, 1997). There are no records of H. limbaughi from any site other than Clipperton, or of H. passer at that island. Traditionally, these three species have been grouped in a single subgenus, Plitops (Allen et al., 1998, Debelius et al., 2003).

The rise of the Central American Isthmus separated many populations of marine organisms, with the final closure of the Isthmus of Panama producing geminate pairs of similar-looking species (Jordan, 1908). Holacanthus has been described as a potential candidate, with H. passer and H. bermudensis being the likely geminates (Thomson et al., 2000). In a molecular (mtDNA) study of angelfishes that included four Holacanthus species, Bellwood et al. (2004) proposed a phylogenetic hypothesis for the group. That analysis indicated that H. bermudensis and H. passer were sister species (thus potentially breaking up the subgenera Plitops and Angelichthys). In addition, that study, which included a thorough representation of most angelfish genera, indicated that the regal angelfish, Pygoplites diacanthus, the sole member of its genus, is the closest relative of the genus Holacanthus, raising the possibility of it being a basal Holacanthus (Bellwood et al., 2004), or potentially well within Holacanthus, thus disrupting its monophyletic status. Although Pygoplites diacanthus was originally described as Holacanthus diacanthus (Bleeker, 1857), an early morphological phylogenetic analysis by Shen and Liu (1978) indicated that Pygoplites is sister to Apolemichthys. A later allozyme comparison done by Chung and Woo (1998) then placed Pygoplites closer to Pomacanthus than to Holacanthus (see Fig. 1 from Bellwood et al., 2004). However, neither of the two molecular studies incorporated sampling of all seven members of Holacanthus.

Pygoplites diacanthus is widely distributed over the entire Indo-central Pacific. This distribution does not indicate whether Holacanthus originated either from the ancestor of Pygoplites in the TEP after migrating from the central Pacific, or in West Africa, after colonization from the Indian Ocean. Thus, a complete phylogeny of the genus Holacanthus is necessary to elucidate the evolutionary history of this genus.

To achieve this goal, we sampled all seven known Holacanthus species (including different color morphs), P. diacanthus and the Cortez angelfish, Pomacanthus zonipectus, a TEP species, as an outgroup, and used four mitochondrial and one nuclear molecular markers. We first established a complete phylogeny of the genus, and then further investigated the status of geminate groups using the more variable mitochondrial control region marker.

Section snippets

Collections and DNA samples

Sampling sizes and locations are listed in Table 1. Preservation and DNA extraction protocols followed Bernardi et al. (2008).

PCR amplification and sequencing

Amplifications of 12S rRNA, 16S rRNA, cytochrome b, and control region segments were performed using the universal primers 12SAL-12SBH, 16SAR-16SBR, GLUDGL-CB3H, and CRA-CRE, respectively (Palumbi et al., 1991, Lee et al., 1995) with 35 cycles at a denaturation temperature of 94 °C for 30 s, an annealing temperature of 52–54 °C, and an extension of 30 s at 72 °C.

Sequences

We used two sets of data, one broad set for a phylogenetic analysis and one more restricted set for a detailed analysis of Trans-Isthmian species. The dataset for the broad analysis comprised 2283 aligned base pairs (bp), which included 346, 547, 699 and 692 bp for 12S rRNA, 16S rRNA, cytochrome b, and 1st intron of the ribosomal protein S7 fragments, respectively. The dataset for the restricted analysis, based on the mitochondrial control region, was a set of 21 individuals that were

Phylogenetic relationships, evolutionary history and biogeographic patterns

The genus Holacanthus comprises only seven species, yet it offers unique possibilities in testing biogeographic, ecological and evolutionary hypotheses. The overall monophyly of the genus is confirmed here. Considering the thorough investigation presented by Bellwood et al. (2004), it is unlikely that other unsampled angelfish species would fall within Holacanthus. Pygoplites diacanthus was found to be very close and basal to Holacanthus. Keeping Pygoplites in its own monospecific genus or

Conclusion

Holacanthus, although a small and geographically confined genus (compared to other angelfish genera), joins other angelfishes in providing a very useful model for the study of marine reef fish evolution. The results of our phylogenetic analyses indicate when and how the genus originated, that its seven species include Trans-Isthmian geminate species complexes, and that speciation likely occurred well after the closure of the Central American Isthmus in both the Caribbean and the tropical

Acknowledgments

Several people generously helped in the field and or suggestions to earlier versions: B. Campbell, V. Domingues, M. Ramon, T. Skelton, H. Crizanto, J.L. Gasparini, L.A. Rocha and R. Beldade. We also thank Forrest Young for information on Holacanthus bermudensis at Florida and the Gulf of Mexico. The sample (non-lethal removal of fin clip) of Holacanthus clarionensis was donated by the Aquarium of the Pacific, Long Beach, California, courtesy of Derek Smith. Finally we would like to acknowledge

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