J. Craig Bailey

A PHYLOGENETIC STUDY OF THE CORALLINALES (RHODOPHYTA) BASED ON NUCLEAR SMALL‐SUBUNIT rRNA GENE SEQUENCES

Conflicting classifications for the Corallinales were tested by analyzing partial sequences for the nuclear small‐subunit ribosomal RNA (SSU) gene of 35 species of coralline algae. Parsimony and likelihood analyses of these data yielded congruent hypotheses that are inconsistent with classifications for the group that include as many as eight subfamilies. Four major clades are resolved within the order, including the early‐diverging Sporolithaceae as well as the Melobesioideae and Corallinoideae. The fourth clade, which is supported robustly, includes both nongeniculate and geniculate species classified in the subfamilies Mastophoroideae, Metagoniolithoideae, Lithophylloideae, and Amphiroideae. Molecular and morphological data support the proposal that the latter two subfamilies are sister taxa. Although relationships among some genera are not resolved clearly, the order of branching of taxa among and within the four principal lineages is concordant with paleontological evidence for the group. Relationships inferred among genera within each of the clades is discussed. Seven morphological characters delimiting higher taxonomic groups within the order were combined with the sequence data, analyzed, and optimized onto the resulting tree(s). Except for the presence or absence of genicula, all other characters were found to be phylogenetically informative. Genicula are nonhomologous structures that evolved independently in the Amphiroideae, Corallinoideae, and Metagoniolithoideae. The phenetic practice of separating coralline algae into two categories solely on the basis of the presence or absence of genicula does not accurately reflect the evolutionary history of the group.

The Pinguiophyceae classis nova, a new class of photosynthetic stramenopiles whose members produce large amounts of omega‐3 fatty acids

The Pinguiophyceae class. nov., a new class of photo‐synthetic stramenopiles (chromophytes), is described. The class includes five monotypic genera, Glossomastix, Phaeomonas, Pinguiochrysis (type genus), Pinguio‐coccus and Polypodochrysis. These algae have an unusually high percentage of polyunsaturated fatty acids, especially 20:5 (n‐3)(EPA, eicosapentaenoic acid). These fatty acids are the basis for choosing the Latin noun ‘Pingue’ (= fat, grease) as the root for the class name. Analyses of nuclear‐encoded 18S rRNA and chloroplast‐encoded rbcL gene sequence data showed that these algae formed a monophyletic group that could not be placed in any other class. Morphologically, the species are all single‐celled microalgae from picoplanktonic size to over 40 urn in length. Each cell has one (or two) typical chloroplast(s) with a girdle lamella and a surrounding chloroplast endoplasmic reticulum. Pyrenoids occur within the chloroplast, varying from embedded to stalked, and membranes penetrate into the pyrenoid in all five genera. Phaeomonas has motile cells with two flagella, and the forward‐directed flagellum bears mastigonemes (tripartite flagellar hairs). Two other genera (Glossomastix, Polypodochrysis) produce zoospores that possess only one smooth flagellum (no mastigonemes), and this flagellum apparently is the mature flagellum, a feature previously unknown in the photosynthetic stramenopiles. The major carotenoid pigments in the pinguiophytes are fucoxanthin, violaxanthin, zeaxanthin and P‐carotene, as well as chlorophyll a and chlorophyll c‐related pigment(s). These features support recognition of the Pinguiophyceae class. nov. as a unique group of algae.

Phaeothamniophyceae Classis Nova: A New Lineage of Chromophytes Based upon Photosynthetic Pigments, rbcL Sequence Analysis and Ultrastructure.

A new algal class, the Phaeothamniophyceae classis nova, is established from genera formerly classified in the Chrysophyceae (e.g., Chrysapion, Chrysoclonium, Chrysodictyon, Phaeobotrys, Phaeogloea, Phaeoschizochlamys, Phaeothamnion, Selenophaea, Sphaeridiothrix, Stichogloea, Tetrachrysis, Tetrapion and Tetrasporopsis) as well as one genus previously assigned to the Xanthophyceae (Pleurochloridella). HPLC analysis revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, β-carotene and heteroxanthin, in addition to chlorophylls a and c, in four genera (Phaeoschizochlamys, Phaeothamnion, Stichogloea, Pleurochloridella). The combination of fucoxanthin and heteroxanthin is known only for these organisms. The rbcL sequences of the same four genera, along with representatives of other chromophyte classes, were analyzed phylogenetically and provided independent support for recognition of the Phaeothamniophyceae as a distinct taxon. These data indicate that the Phaeothamniophyceae are more closely related to the classes Xanthophyceae and Phaeophyceae than to the Chrysophyceae. Electron microscopy revealed that Phaeoschizochlamys, Phaeothamnion and Stichogloea possess electron opaque vesicles at the cell periphery, have a cell wall that often appears laminate, form new daughter cell walls via eleutheroschisis, and have plastids with girdle lamellae and a ring-shaped genophore. The flagellar apparatus of Phaeothamnion zoospores (described in a previous study) is chosen as representative of the new class. The flagella are inserted laterally, basal bodies form an angle of ca. 145° or more, a multi-gyred flagellar transitional helix is present and tripartite flagellar hairs lack lateral filaments. Genera placed in the Phaeothamniophyceae are assigned to the orders Phaeothamniales and Pleurochloridellales, each with a single family.

Characteristics and utility of nuclear-encoded large-subunit ribosomal gene sequences in phylogenetic studies of red algae

Primer sequences are described for amplifying and sequencing a large fragment (approximately 2500 b.p.) of the nuclear‐encoded large‐subunit ribosomal RNA gene (LSU) from red algae. In comparison to RuBisCo large‐subunit gene (rbcL) and nuclear‐encoded small‐subunit ribosomal RNA gene (SSU) sequence data, LSU sequence data was intermediate in the number of phylogenetically informative positions and sequence divergence. Parsimony analysis of LSU sequences for 16 Gelidiales species resolved some nodes unresolved in rbcL and SSU parsimony trees. An analysis of LSU sequences from 13 species of red algae classified in 11 orders suggests that this gene may be useful in studies of higher‐level relationships of red algae.

Nuclear 18S rRNA gene sequence analyses indicate that the Mastophoroideae (Corallinaceae, Rhodophyta) is a polyphyletic taxon

Abstract Nuclear small subunit ribosomal RNA (18S rRNA) gene sequences were determined for six species representing three genera classified within the Mastophoroideae, and also for two species of Amphiroa (Lithophylloideae). These data were combined with previously published 18S rRNA sequences for 38 other coralline species and analysed (1) to determine the phylogenetic position of the Mastophoroideae within the Corallinaceae and (2) to examine relationships among Hydrolithon, Metamastophora, Neogoniolithon and Spongites. Trees derived from parsimony and maximum likelihood analyses of these data indicate that the Mastophoroideae is not monophyletic. Instead, our data suggest that the group is polyphyletic and includes species belonging to four distinct evolutionary lineages. For example, Neogoniolithon spp. were associated with geniculate members of the Corallinoideae, and Hydrolithon was resolved as sister to the geniculate genus Metagoniolithon. We demonstrate that each of the four ‘mastophoroid’ lineages share a number of vegetative and reproductive features with their respective sister taxa. Together the molecular and morphological data imply that the Mastophoroideae is not monophyletic and that genera placed in this taxon might more naturally be classified in other subfamilies. Also, on the basis of our findings, Porolithon pachydermum is transferred to Hydrolithon as H. pachydermum (Foslie) Bailey, Gabel & Freshwater.

ANALYSIS OF CLONAL CULTURES OF THE BROWN TIDE ALGAE AUREOCOCCUS AND AUREOUMBRA (PELAGOPHYCEAE) USING 18S rRNA, rbcL, AND RUBISCO SPACER SEQUENCES

Variability among clonal cultures of the brown tide algae Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson was examined by DNA sequence comparisons. Nuclear‐encoded 18S rRNA and plastid‐encoded rbcL gene sequences were determined for six Aureococcus strains. RUBISCO spacer sequences were determined for 14 strains of Aureococcus. No differences among Aureococcus strains were found in the DNA regions examined. The rbcL and RUBISCO spacer sequences for three Aureoumbra strains were identical but differed from those of Aureococcus. These data indicate that blooms of these species are comprised of cells that are very similar and also imply that Aureococcus and Aureoumbra do not contain varieties or cryptic species. Separate and combined phylogenetic analyses of the 18S rRNA and rbcL gene sequences were performed. Results confirm that the brown‐tide‐causing algae of Long Island Sound, New York (Aureococcus), and Laguna Madre, Texas (Aureoumbra), are best classified in separate genera within the Pelagophyceae. Phylogenetic trees place Aureococcus and Aureoumbra within the Pelagomonadales and Sarcinochrysidales, respectively.

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.

Pinguiococcus pyrenoidosus gen. et sp. nov. (Pinguiophyceae), a new marine coccoid alga

A coccoid marine alga, collected from an aquaculture tank and maintained in culture as CCMP1144, was examined using light and electron microscopy. Young, rapidly growing cells were mostly spherical in shape, approximately 4–6 μm in diameter. Older cells often produced protrusions and pseudopodia‐like extensions, giving cells an amoeboid‐like appearance, but no amoeboid movement was observed and the pseudopodia‐like extensions exhibited no active movement. The single chloroplast had a typical photosynthetic stramenopile ultrastructure. A large stalked pyrenoid was easily observed by light microscopy. Ultrastructurally, the granular portion of the pyrenoid was divided into sections by a penetrating chloroplast envelope. A mitochondrion was often, but not always, adjacent to the pyrenoid, and in some cases the mitochondrion formed a ‘cap’ over the protruding pyrenoid. The Golgi cisternae were (when viewed in cross‐section) curved toward the nucleus. A peripheral network of anastomosing tube‐like membranes was located immediately beneath the plasmalemma. Two centrioles were located adjacent to the nuclear envelope. Lipid‐like and electron transparent vacuoles were present. Based on this investigation and data published elsewhere (large percentage of eicosapentaenoic acid, 18S rRNA and rbcL genes), this alga was described as Pinguiococcus pyrenoidosus gen. et sp. nov.

MOLECULAR SYSTEMATIC ANALYSES INDICATE THAT THE ENIGMATIC APOPHLAEA IS A MEMBER OF THE HILDENBRANDIALES (RHODOPHYTA, FLORIDEOPHYCIDAE)

Nuclear small‐subunit (SSU) ribosomal DNA sequences were determined for Apophlaea lyallii Hooker f. et Harvey from New Zealand and an isolate of Hildenbrandia rubra (Sommerfelt) Meneghini from France. These data, in addition to SSU sequences representative of all the major florideophyte lineages, were used in molecular systematic investigations to confirm inclusion of Apophlaea in the Hildenbrandiales. Anatomical features that serve to unite Apophlaea and Hildenbrandia are discussed to buttress this result. Furthermore, our analyses support recognition of the Hildenbrandiales as sister to all other extant florideophyte lineages studied to date. An anomalous result positioning the SSU sequence generated herein for Hildenbrandia rubra closer to Apophlaea lyallii than to a previously published sequence for Hildenbrandia rubra requires further investigation.