An assessment of the AFLP method for investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae)

The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR, these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Amino acid sequence of a ferredoxin from Rhodymenia palmata, a red alga in the florideophyceae

The amino acid sequence of a [2Fe-2S] ferredoxin from a red alga, Rhodymenia palmata in the family Florideophyceae, was determined by conventional methods. The ferredoxin is composed of 97 amino acid residues having five cysteines, but lacking methionine and tryptophan. It possesses a number of structural features of particular interest. The amino acid sequence is compared with those previously determined for ferredoxins from two red algae in the family Bangiophyceae. Conclusions from a comparison of the structures, by noting features such as the presence of gaps in the sequences and by constructing a phylogenetic tree, were consistent with the proposed taxonomic relationship among these algae.     

MOLECULAR SYSTEMATICS OF THE FLORIDEOPHYCEAE (RHODOPHYTA) USING NUCLEAR LARGE AND SMALL SUBUNIT rDNA SEQUENCE DATA

Sequence data are presented for approximately 85% of the nuclear large subunit (LSU) rDNA gene for one member of the Bangiophyceae and 47 members of the Florideophyceae, the latter representing all but one of the currently recognized florideophyte orders. Distance, parsimony, and maximum likelihood analyses of these data were used to generate phylogenetic trees, and bootstrap resampling was implemented to infer robustness for distance and parsimony results. LSU phylogenies were congruent with published nuclear small subunit (SSU) rDNA results in that four higher level florideophyte lineages were resolved: lineage 1, containing the order Hildenbrandiales; lineage 2, recovered only under distance analysis, composed of the orders Acrochaetiales, Balliales, Batrachospermales, Corallinales, Nemaliales, Palmariales, and Rhodogorgonales; lineage 3, containing the Ahnfeltiales; and lineage 4, composed of the orders Bonnemaisoniales, Ceramiales, Gelidiales, Gigartinales, Gracilariales, Halymeniales, Plocamiales, and Rhodymeniales. Analyses were also performed on a combined LSU–SSU data set and an SSU-only data set to account for differences in taxon sampling relative to published studies using this latter gene. Combined LSU–SSU analyses resulted in phylogenetic trees of similar topology and support to those obtained from LSU-only analyses. Phylogenetic trees produced from SSU-only analyses differed somewhat in particulars of branching within lineages 2 and 4 but overall were congruent with the LSU-only and combined LSU–SSU results. We close with a discussion of the phylogenetic potential that the LSU has displayed thus far for resolving relationships within the Florideophyceae.     

DEFINING THE MAJOR LINEAGES OF RED ALGAE (RHODOPHYTA)1

Previous phylogenetic studies of the Rhodophyta have provided a framework for understanding red algal phylogeny, but there still exists the need for a comprehensive analysis using a broad sampling of taxa and sufficient phylogenetic information to clearly define the major lineages. In this study, we determined 48 sequences of the PSI P700 chl a apoprotein A1 (psaA) and rbcL coding regions and established a robust red algal phylogeny to identify the major clades. The tree included most of the lineages of the Bangiophyceae (25 genera, 48 taxa). Seven well‐supported lineages were identified with this analysis with the Cyanidiales having the earliest divergence and being distinct from the remaining taxa; i.e. the Porphyridiales 1–3, Bangiales, Florideophyceae, and Compsopogonales. We also analyzed data sets with fewer taxa but using seven proteins or the DNA sequence from nine genes to resolve inter‐clade relationships. Based on all of these analyses, we propose that the Rhodophyta contains two new subphyla, the Cyanidiophytina with a single class, the Cyanidiophyceae, and the Rhodophytina with six classes, the Bangiophyceae, Compsopogonophyceae, Florideophyceae, Porphyridiophyceae classis nov. (which contains Porphyridium, Flintiella, and Erythrolobus), Rhodellophyceae, and Stylonematophyceae classis nov. (which contains Stylonema, Bangiopsis, Chroodactylon, Chroothece, Purpureofilum, Rhodosorus, Rhodospora, and Rufusia). We also describe a new order, Rhodellales, and a new family, Rhodellaceae (with Rhodella, Dixoniella, and Glaucosphaera).     

Actin Phylogeny and Intron Distribution in Bangiophyte Red Algae(Rhodoplantae)

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic “Bangiophyceae,” was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.[Reviewing Editor: Dr. Yves Van de Peer]     

Egg release in gametophytes of Laminaria saccharina: Induction by darkness and inhibition by blue light and u.v.

Gametophytes of Laminaria saccharina cultivated from zoospores in a light-dark regime (16:8), release eggs exclusively during the dark cycles, 8–10 days after seeding of the zoospores, and mainly during the first 30 min of darkness. The inhibiting effects of light during the light cycle of 16 h per day is also apparent in gametophytes which have experienced only two dark cycles prior to day 8, when egg release begins. Egg release can be shifted to any time during the light cycle by prolonging the irradiation with white fluorescent light and by subsequent darkening for 1 h. In gametophytes cultivated in continuous white fluorescent light eggs are also released from day 8–10, so in this case no inhibiting activity of light is apparent. Egg release is inhibited by blue light and u.v., with peak wavelengths for inhibition at 372, 413, 438 and 481 nm. No inhibition occurs at wavelengths above 513 nm. The light requirement for inhibition is very low. A photon fluence rate of 1·4 μE m^-2s^-1, given for 45 min at 449 nm, inhibits egg release in 50% of the mature gametophytes. There is some evidence that a circadian rhythm is involved, primarily since in gametophytes which are transferred at the beginning of day 8 from 16:8 to constant conditions (darkness, continuous red or green light) the diel rhythm of egg release persists until day 11.     

PHYLOGENETIC ANALYSES OF THE RED ALGAL ORDER RHODYMENIALES SUPPORTS RECOGNITION OF THE HYMENOCLADIACEAE FAM. NOV., FRYEELLACEAE FAM. NOV., AND NEOGASTROCLONIUM GEN. NOV.1

Systematics of the red algal order Rhodymeniales was investigated using combined large‐subunit nuclear ribosomal DNA (LSU) and elongation factor 2 (EF2) analyses. These data were subjected to distance, parsimony, and Bayesian analyses, and the resulting phylogenies were largely congruent with previously published SSU results in that the four currently recognized rhodymenialean families (Champiaceae, Faucheaceae, Lomentariaceae, and Rhodymeniaceae) were resolved as monophyletic lineages (with the exception of Coelothrix, which is here transferred to the Champiaceae from the Rhodymeniaceae). In addition, taxa presently considered as incertae sedis consisted of two lineages (Fryeella lineage and Hymenocladia lineage). Based on these results, two new families are proposed: (i) the Fryeellaceae fam. nov. to accommodate the genera Fryeella, Hymenocladiopsis, and a new taxon from Tasmania, Australia; and (ii) the Hymenocladiaceae fam. nov., to accommodate Asteromenia, Hymenocladia, and Erythrymenia. In addition to resolving familial relationships, these analyses resolved some novel interspecific affinities, and we propose a new genus, Neogastroclonium gen. nov., for Gastroclonium subarticulatum, a species that differs significantly in both morphology and molecular data from genuine species of Gastroclonium. Relationships among additional faucheacean and lomentariacean taxa were investigated using LSU data only, and these results are discussed. The familial classification of the Rhodymeniales proposed herein is discussed in light of vegetative and reproductive anatomy, most notably the ontogeny of the tetrasporangia.     

A nuclear phylogeny of the Florideophyceae (Rhodophyta) inferred from combined EF2, small subunit and large subunit ribosomal DNA: establishing the new red algal subclass Corallinophycidae

Previous studies have indicated that resolution of supraordinal relationships in the red algal class Florideophyceae will require additional characters, improved taxon sampling and optimized methods of phylogenetic analysis. To this end, we have generated data to introduce a novel nuclear marker to red algal systematics, elongation factor 2, as well as expanded ribosomal DNA alignments (SSU and LSU) to include 62 ingroup and 4 outgroup taxa. Both single gene and combined data sets were considered. Our analyses resulted in better resolution of both deep as well as more recent divergences, with higher support realized at many nodes. Distance, parsimony and bayesian analyses of the single gene and combined data sets indicated that the subclasses Hildenbrandiophycidae, Ahnfeltiophycidae and Rhodymeniophycidae were monophyletic, whereas the Nemaliophycidae was polyphyletic: one lineage containing the Rhodogorgonales and Corallinales (CR complex); and the other containing the Acrochaetiales, Balbianiales, Balliales, Batrachospermales, Colaconematales, Nemaliales, Palmariales, and Thoreales (APB complex). Based on these results a new subclass of the Florideophyceae, the Corallinophycidae subclassis nov., is proposed to accommodate the Corallinales and Rhodogorgonales. In addition to resolving supraordinal relationships, the present analyses resolved some novel ordinal affinities within the Nemaliophycidae and Rhodymeniophycidae, which are discussed here.     

Sterols of Mediterranean Florideophyceae

The distribution of sterols in 31 Mediterranean Florideophyceae has been investigated. Cholesterol is present in the greatest majority of the species examined, while the occurrence of other C-27 sterols (desmosterol, 22-dehydrocholesterol, liagosterol and cholest-7-en-3β-ol) is much more restricted. Two species (Rytiphloea tinctoria and Vidalia volubilis) contain, in addition to C-27 sterols, large amounts of C-28 and C-29 compounds.