Phylogenetic analysis guided by intragenomic SSU rDNA polymorphism refines classification of 'Alexandrium tamarense' species complex

摘要

We analyzed small subunit ribosomal rRNA gene (SSU rDNA) in single-cell isolated strains of "Alexandrium tamarense", "Alexandrium fundyense" and "Alexandrium catenella" (Atama complex) by both direct and clone-based sequencing, and found 42-50 intragenomic SSU rDNA polymorphic sites (High IRP) in some of the strains but none or one (No/Low IRP) in others. Clone sequencing of the High-1RP amplicons revealed numerous variants with 0-3.5% nudeotide differences. Phylogenetic analyses including reported and new Atama complex SSU rDNA data divided this complex into two major well-separated clades. All the High-IRP sequences obtained in this study were grouped in Clade I (High-IRP clade), which also contained strains of Atama complex previously reported from northern Asia. Clade Ⅱ (No/Low-IRP clade), with no evidence of IRP except for the one polymorphic site found in one of the strains, contained a subclade (ⅡC) exclusively of "A catenella" from various geographic locations and several other subclades (HA, ⅡB) predominantly of "A tamarense". Clade I corresponded to Group I in the large subunit (LSU) rDNA-based phylogenetic tree (Lilly et al., 2007), and subclades in our Clade Ⅱ corresponded to the LSD-based Groups Ⅱ-V. Our !RP information further unites the many seemingly different genotypes into a coherent group (Clade I) and provides delineating boundary between this and other genotypes (Clade Ⅱ) in Atama complex. Based on the currently available data, we propose that Clade I and subclade ⅡC represent two distinct species, while the rest of Clade Ⅱ represents another one or more species. Clade I should be considered one species because (1) intragenomic rDNA variants dispersed across strains in the phylogenetic tree uniting the many different genotypes to same or cfoseiy related populations, (2) their expressed SSU rDNAs (i.e. rRNAs) are almost identical (1 out of 1700 nt, 0.06% difference), and (3) the corresponding Group I in the LSU-based tree also appears to be a coherent group. Subclade ⅡC should be considered another species because it has a long distance from the rest of CladeⅡ as well as Clade I, Whether the rest of CladeⅡ (mainly European "A tamarense" subclades) represents another one or more species requires further study. Our results show that the three original morphospecies designations are invalid, and the strains do not group based on geographic locations or whether they are toxic in general, although some subclades are predominated by part of a morphotype from a region. Applying the IRP-guided analysis to a toxic Alexandrium bloom in Long Island Sound revealed that although some undocumented Atexandrium-related and other dinoflagellate lineages co-existed, the majority of the "diverse" SSU sequences detected belonged to one single population identical to "A fundyense" in Gulf of Maine. Our result suggests that negligence of IRP could lead to incorrect recognition of the intragenomic SSU rDNA variants as distinct genotypes, thus overestimating strain diversity of a bloom of this species complex and possibly other HAB lineages.