Molecular Phylogeny of Noctilucoid Dinoflagellates (Noctilucales, Dinophyceae)

The order Noctilucales or class Noctiluciphyceae encompasses three families of aberrant dinoflagellates (Noctilucaceae, Leptodiscaceae and Kofoidiniaceae) that, at least in some life stages, lack typical dinoflagellate characters such as the ribbon-like transversal flagellum or condensed chromosomes. Noctiluca scintillans, the first dinoflagellate to be described, has been intensively investigated. However, its phylogenetic position based on the small subunit ribosomal DNA (SSU rDNA) sequence is unstable and controversial. Noctiluca has been placed either as an early diverging lineage that diverged after Oxyrrhis and before the dinokaryotes -core dinoflagellates- or as a recent lineage branching from unarmoured dinoflagellates in the order Gymnodiniales. So far, the lack of other noctilucoid sequences has hampered the elucidation of their phylogenetic relationships to other dinoflagellates. Furthermore, even the monophyly of the noctilucoids remained uncertain. We have determined SSU rRNA gene sequences for Kofoidiniaceae, those of the type Spatulodinium (=Gymnodinium) pseudonoctiluca and another Spatulodinium species, as well as of two species of Kofoidinium, and the first gene sequence of Leptodiscaceae, that of Abedinium (=Leptophyllus) dasypus. These taxa were collected from their type localities, the English Channel and the NW Mediterranean Sea, respectively. Phylogenetic analyses place the Noctilucales as a monophyletic group at a basal position close to parasites of the Marine Alveolate Group I (MAGI) and the Syndiniales (MAGII), before the core of dinokaryotic dinoflagellates, although with moderate support.

Introduction

Noctiluca scintillans (Macartney) Kofoid is the first known dinoflagellate (Baker 1753; Slabber 1771). Initially classified as a jelly fish, Haeckel (1873) proposed that Noctiluca Suriray ex Lamarck should be included in the Cystoflagellata within the dinoflagellates. Based on detailed observations of the trophonts, Kofoid (1920) homologized the structures recognised in Noctiluca with the sulcus, cingulum, longitudinal and transverse flagella of the dinoflagellates, and since then, Noctiluca was classified within dinoflagellates as the type of the order Noctilucales. In current taxonomic schemes, dinoflagellates (Dinoflagellata) are divided into two subdivisions: Dinokaryota and Syndinea (Fensome et al. 1993). Syndinea comprises one single order, Syndiniales, of exclusively non-photosynthetic parasitic species that, among other features, lack most characters defining the typical nucleus of the Dinokaryota. The syndinean nucleus contains a low number (usually four to ten) of non-fibrillar and V-shaped chromosomes (Cachon 1964; Hollande 1974). The chromatin of Dinokaryota has fibrillar chromosomes (∼20-300) condensed throughout the cell cycle (Pfiester 1984; Rizzo 2003). Likewise, the diploid Noctiluca trophonts do not retain characters shared by typical dinoflagellates, such as a transverse flagellum and condensed chromosomes. In contrast, the haploid zoospores maintain primitive dinoflagellate-like characteristics including two grooves, slightly differentiated flagella with different lengths and paraxial rod, and condensed chromosomes (Afzelius 1963; Fukuda and Endoh 2006; Höhfeld and Melkonian 1995; Melkonian and Höhfeld 1988; Soyer, 1969, Soyer, 1970, Soyer, 1972; Zingmark 1970). Zingmark (1970) considered Noctiluca not to be a true dinoflagellate because its vegetative nucleus is of conventional eukaryotic type and not dinokaryotic. However, Fensome et al. (1993), based on the dinokaryotic nature of the gamete nucleus and the intergradational nature with other Noctilucales (such as Kofoidinium Pavillard), placed the noctilucoids in the class Noctiluciphyceae within the subdivision Dinokaryota (Fensome et al. 1993).

In the 1990s, the phylogenetic position of Noctiluca was re-evaluated using molecular information. The first phylogenetic analyses based on the large subunit (LSU) and small subunit (SSU) rDNA sequences suggested that Noctiluca represented a basal lineage within the dinoflagellates (Lenaers et al. 1991; Saunders et al. 1997). Recent studies based on other markers (light-emitting enzyme luciferase gene, editing density of mitochondrial cox1 mRNA and Spliced-Leader RNA trans-splicing groups, β-tubulin and heat shock protein 90 -Hsp90-) also suggested that Noctiluca might belong to an early diverging dinoflagellate lineage that diverged after that leading to Oxyrrhis Dujardin and before the core dinoflagellates, although the corresponding phylogenies were most often poorly supported (Fukuda and Endoh 2008; Liu and Hastings 2007; Zhang and Lin 2008). Similarly, in the SSU rDNA phylogeny, the most extended marker for protist phylogenetic and diversity studies, the placement of Noctiluca was unstable, its position likely influenced by the number of nucleotides used in the alignment (Saldarriaga et al. 2004). An analysis using diverse alveolate SSU rDNA sequences as outgroup suggested that Noctiluca occupies a basal position among dinoflagellates, while another analysis rooted using sequences only from Perkinsus Levine and Syndiniales placed Noctiluca within one of the clades of the order Gymnodiniales (Saldarriaga et al. 2004). The SSU rDNA phylogeny turned out to be too weak to place Noctiluca with confidence. Since the enrichment of taxonomic sampling is well known to improve the resolution of phylogenetic analyses, the sequencing of SSU rDNA from other noctilucoids might help to resolve the phylogenetic position of this group within the dinoflagellates.

In addition to Noctiluca, which is easily accessible from cultures and widespread in eutrophic coastal waters, other noctilucoids have a predominantly tropical to warm-temperate oceanic distribution (Cachon and Cachon, 1967, Cachon and Cachon, 1969; Gómez and Furuya, 2005, Gómez and Furuya, 2007. Most of the Noctilucales have been described from the NW Mediterranean and knowledge on their morphology and life cycle is almost completely restricted to the studies by Cachon and Cachon, 1967, Cachon and Cachon, 1969. More than 100 nucleotide sequences are publicly available for N. scintillans, but sequences from other noctilucoid protists are lacking. Spatulodinium pseudonoctiluca (Pouchet) Cachon et Cachon, the type of the family Kofoidiniaceae, was first named Gymnodinium pseudonoctiluca due to the intermediate morphological characters between Gymnodinium Stein and Noctiluca. Pouchet (1885) already noted that members of this taxon undergo a complex ontogenetic process from gymnodinioid or amphidinioid immature stages to tentacle-bearing noctilucoid trophonts and he showed that G. pseudonoctiluca is a single species with two different morphotypes. The immature morphotype has two differentiated flagella, including the ribbon-like transverse flagellum, and the cingulum and the sulcus, whereas the mature morphotype, which also keeps these dinoflagellate morphological features, has radiating contractile fibrils from the perinuclear region and a tentacle that resembles the one of Noctiluca. Other authors described the immature stages of Spatulodinium J. Cachon et M. Cachon as separate species (i.e., Gymnodinium conicum Kofoid et Swezy, G. fulgens Kofoid et Swezy, and G. lebouriae Pavillard). Pouchet’s observations were confirmed by Cachon and Cachon (1967) and more recent studies (Gómez and Souissi 2007; Konovalova and Selina 2002). Nevertheless, G. lebouriae and S. pseudonoctiluca often appear as a separate species in the literature (Hansen and Larsen 1992; Hoppenrath 2004). The family Leptodiscaceae is the least known among the Noctilucales due to their delicacy and warm-water oceanic distribution. Their cell bodies are strongly antero-posteriorly flattened with a bilateral symmetry or with equatorial wing-like expansions, being able to contract suddenly (Cachon and Cachon 1967; Cachon and Cachon-Enjumet, 1964, Cachon and Cachon-Enjumet, 1966).

The present study provides the first SSU rRNA gene sequences for representatives of the families Kofoidiniaceae and Leptodiscaceae. This allows us to test the monophyly of the noctilucoid dinoflagellates and to study the placement of Noctiluca in the SSU rDNA phylogeny, namely, to test whether the noctilucoid protists are an early diverging lineage or a recent lineage derived from typical dinokaryotes phylogenetically related to Gymnodiniales.