Evolution and phylogeography of Halimeda section Halimeda (Bryopsidales, Chlorophyta)
Introduction
In the tropical marine realm, patterns and processes of speciation are seldom obvious. A striking contradiction in this context is that while marine populations are presumed to be more open than their terrestrial counterparts as a consequence of genetic remixing brought about by ocean currents, many species show large genetic differences between geographically separated populations (e.g., Duke et al., 1998, Lessios et al., 2003, McMillan and Palumbi, 1995), sometimes to such a degree that geographic entities deserve species status (e.g., De Clerck et al., 2005, Muss et al., 2001, Pakker et al., 1996). Additionally, several marine species have been shown to contain cryptic or pseudo-cryptic diversity unlinked with geography (Knowlton, 1993).
Marine macroalgae abound in almost all coastal habitats. Despite their high diversity and abundance, the patterns of their evolution and processes involved in their speciation have not yet been intensively studied. The green algal genus Halimeda, the focus of this paper, is among the better studied (Kooistra et al., 2002, Kooistra and Verbruggen, 2005, Verbruggen et al., 2005c, Verbruggen and Kooistra, 2004).
Halimeda is a common inhabitant of tropical and warm-temperate marine environments and a prominent primary producer, source of food and habitat, and carbonate sand producer (Hillis-Colinvaux, 1980). The algal body of Halimeda is composed of flattened, green, calcified segments interconnected by uncalcified nodes (Hillis-Colinvaux, 1980, Lamouroux, 1812). From the anatomical point of view, the entire algal body consists of a single, tubular cell that branches to form an organized network of siphons (Barton, 1901, Hillis-Colinvaux, 1980). In the medulla, siphons run in the axial direction and ramify sparsely. In the cortex, siphon ramification is denser. The short, cortical siphon branches are inflated and called utricles. Sexual reproduction occurs periodically (Drew and Abel, 1988) and gametes are released into the water column during mass spawning events (Clifton, 1997). Sympatric species have been shown to spawn in slightly different timeframes (Clifton, 1997, Clifton and Clifton, 1999). Reproduction is followed by death of the alga, after which the calcified segments disconnect and contribute to the sediment (Freile et al., 1995, Meinesz, 1980). These segments endure in the fossil record and often make up the bulk of the carbonate structure of tropical reefs (Bassoullet et al., 1983, Hillis-Colinvaux, 1986).
Kooistra et al. (2002) examined the phylogeny, biogeography and historical ecology of the genus on the basis of partial nuclear ribosomal cistron sequences (partial 18S, ITS1, 5.8S and ITS2) of 28 out of the 33 species recognized at that time. Sequences grouped in five clear-cut clades, which subsequently formed the foundation of a new sectional classification (Verbruggen and Kooistra, 2004). Kooistra et al. (2002) also showed that certain clades within the genus were characterized by ecological properties such as growth on unconsolidated substrates and in sheltered localities. Finally, it was shown that each of the five lineages featured distinct Atlantic and Indo-Pacific subgroups, indicating that vicariance has been at play.
The focus of this study is on section Halimeda of the genus, within which eleven morphological species are currently recognized. The majority of these species are epilithic and occur in wave-affected habitats (Verbruggen and Kooistra, 2004). Section Halimeda is of special interest within the genus for a variety of reasons. Firstly, former studies (Kooistra et al., 2002, Verbruggen and Kooistra, 2004) indicated that H. tuna and H. discoidea were non-monophyletic, raising taxonomic interest in the section. Secondly, the section was sparsely sampled in these former studies: most species were represented by only one or a few individuals, and especially the subtropical regions in which the genus occurs were undersampled. Thirdly, the marker used in the studies of Kooistra et al. (2002) and Verbruggen and Kooistra (2004) was unable to completely resolve the relationships between species. Fourthly, the fact that section Halimeda is the most widely distributed section of the genus, occurring also in the Mediterranean Sea, Eastern Atlantic Islands, the Pacific coasts of tropical America, and ranging to higher latitudes than other sections, makes it of special biogeographic interest. Lastly, the section features two species whose phylogeographic patterns could provide insight in the evolution of Indian Ocean algae. It concerns H. cuneata, which has a disjunct distribution in the subtropical basins of the Indian Ocean (SE Africa, SW Australia, Arabian Sea) and Indo-Pacific H. discoidea (sensu Kooistra et al., 2002), which is present in tropical waters of the Indo-Pacific and has a small population in Oman (Jupp, 2002).
Special attention goes to the species H. cuneata, H. discoidea, and H. tuna. The former species (Figs. 1A–N) is defined morphologically by a stalk zone between subsequent segments and peripheral utricles adhering laterally over approximately half their length (Hillis-Colinvaux, 1980). The stalk zone can take the form of a stretch of uncorticated medullar filaments (Figs. 1B, F, H, and N) or a corticated cushion segment (Fig. 1D). Segments are wedge-shaped (Figs. 1A, C, E, I, and M) or discoid (Figs. 1G and K). Two forms of Halimeda cuneata (forma undulata and forma digitata) described by Barton (1901) do not possess a stalk zone (Figs. 1J and L) and are recognized as forms within H. cuneata by some (e.g., Littler and Littler, 2003) or synonymized with other species by other taxonomists (e.g., Hillis-Colinvaux, 1980). Halimeda discoidea (Figs. 1O–Q) is characterized by discoid to kidney-shaped segments and large subperipheral utricles bearing multiple peripheral utricles (Hillis-Colinvaux, 1980). Halimeda tuna (Figs. 1R and S) can be recognized by relatively small wedge-shaped to discoid segments and rather small subperipheral utricles typically bearing three peripheral utricles (Hillis-Colinvaux, 1980). Hillis-Colinvaux (1980) lists H. discoidea and H. tuna as pantropical; the latter also occurs in the Mediterranean Sea. Halimeda cuneata has a disjunct distribution in the subtropical parts of the Indian Ocean, with populations in Australia, SE Africa, SW Madagascar and the Arabian Sea (Hillis-Colinvaux, 1980) and has recently been reported from Brazil (Bandeira-Pedrosa et al., 2004). The H. cuneata forms undulata and digitata occur in the tropical Indo-Pacific.
The goals of this study are (1) to elucidate the phylogenetic history of Halimeda section Halimeda using plastid DNA markers (tufA, rps19–rps3, and rpl5–rps8–infA) and nuclear ribosomal sequences (partial 18S, ITS1, 5.8S, and ITS2), (2) to evaluate the phylogenetic status of Halimeda cuneata, H. discoidea, and H. tuna using molecular tools and specimens covering more of the morphological variability and distribution ranges than was the case in former studies, and (3) to examine biogeographic patterns within the section as a whole and phylogeographic patterns within H. discoidea and H. cuneata.
Section snippets
Materials and methods
Taxa of Halimeda section Halimeda were collected throughout most of their distribution ranges. Vouchers were deposited in the Ghent University Herbarium (GENT). Identifications followed Hillis-Colinvaux, 1980, Ballantine, 1982 and Noble (1986) and, for H. cuneata forms, Barton (1901) and Littler and Littler (2003). We were unable to obtain specimens suitable for DNA analysis of three species of section Halimeda (H. gigas, H. scabra, and H. xishaensis). Halimeda gigas was represented in the
DNA sequence data
Information on length, variability and base composition of the molecular markers can be found in Table 1. Different species exhibited markedly divergent ITS1–5.8S–ITS2 and partial rps19–rps3 sequences. The species H. cuneata, H. discoidea, and H. tuna each comprised two or more genotypic groups. Sequences within such genotypic groups differed in only one or a few positions (or not at all) while sequences among genotypic groups differed more substantially (Fig. 2). Modeltest suggested different
Discussion
The obtained molecular phylogenetic data broach several issues about the evolutionary history of Halimeda section Halimeda. First, the rps19–rps3 and ITS1–5.8S–ITS2 phylograms show that the group under scrutiny contains many more genetically delineable species than those recognized by classical taxonomy. Second, the topological discordances between nuclear and plastid phylograms are suggestive of reticulate speciation. Third, the fact that some cryptic species are restricted to the margins of
Acknowledgments
We are grateful to E. Cocquyt, C. De maire, C. VanKerckhove and A. Vierstraete for laboratory and administrative assistance. We thank K. Arano, C. Battelli, P. Colinvaux, Y. De Jong, R. Diaz, C. Galanza, S. Guimaraes, O. Gussmann, R. Haroun, I. Hendriks, L. Hillis, L. Kirkendale, T. & C. Leigh, F. Leliaert, J. Maté, A. Maypo, A. N’Yeurt, D. Olandesca, F. Parrish, K. Pauly, C. Payri, G. Procaccini, W. Prud’homme van Reine, E. Tronchin, S. Wilson, and B. Wysor for providing specimens and/or
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