PHYLOGENETIC ANALYSIS OF MORPHOLOGICAL SPECIES OF CARTERIA (VOLVOCALES,CHLOROPHYTA) BASED ON rbcL GENE SEQUENCES1

Four related species in the unicellular volvocalean genus Carteria [C. crucifera Pascher, C. eugametos Mitra, C. inversa (Korshikov) Bourrelly and C. cerasiformis Nozaki et al.] were delineated on the basis of recent comparative light and electron microscopy of a large number of culture strains. However, the species thus delineated may not represent natural or monophyletic entities. In the present study, 1128 base pairs of the chloroplast protein-coding gene (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene) from 12 Carteria strains representing the four species as well as from related volvocalean species were analyzed to elucidate the phylogenetic status of the taxonomic or morphologic species of Carteria. The sequence data showed that the 12 Carteria strains exhibit four robust monophyletic groups which are strictly consistent with the four taxonomic species. These results are discussed in relation to contrasting results found in other microalgal genera. It is concluded that phylogenetic analysis, based on DMA sequence data and comparative morphologic characterization of species and using a large number of culture strains, is essential to a natural system of microalgal species taxonomy.     

A MOLECULAR PHYLOGENY OF THE HETEROKONT ALGAE BASED ON ANALYSES OF CHLOROPLAST-ENCODED rbcL SEQUENCE DATA1

Nearly complete ribulose-1,5-bisphosphate carboxylase/ oxygenase (rbcL)sequences from 27 taxa of heterokont algae were determined and combined with rbcL sequences obtained from GenBank for four other heterokont algae and three red algae. The phylogeny of the morphologically diverse haterokont algae was inferred from an unambiguously aligned data matrix using the red algae as the root, Significantly higher levels of mutational saturation in third codon positions were found when plotting the pair-wise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In light of this observation, third codon positions were excluded from phylogenetic analyses. Both weighted-parsimony and maximum-likelihood analyses supported with high bootstrap values the monophyly of the nine currently recognized classes of heterokont algae. The Eustigmatophyceae were the most basal group, and the Dictyochophyceae branched off as the second most basal group. The branching pattern for the other classes was well supported in terms of bootstrap values in the weightedparsimony analysis but was weakly supported in the maximum-likelihood analysis (<50%). In the parsimony analysis, the diatoms formed a sister group to the branch containing the Chrysophyceae and Synurophyceae. This clade, charactetized by siliceous structures (frustules, cysts, scales), was the sister group to the Pelagophyceae/Sarcinochrysidales and Phaeo-/Xantho-/ Raphidophyceae clades. In the latter clade, the raphido-phytes were sister to the Phaeophyceae and Xanthophyceae. A relative rate test revealed that the rbcL gene in the Chrysophyceae and Synurophyceae has experienced a significantly different rate of substitutions compared to other classes of heterokont algae. The branch lengths in the maximum-likelihood reconstruction suggest that these two classes have evolved at an accelerated rate. Six major carotenoids were analyzed cladistically to study the usefulness of carotenoid pigmentation as a class-level character in the heterokont algae. In addition, each carotenoid was mapped onto both the rbcL tree and a consensus tree derived from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) sequences. Carotenoid pigmentation does not provide unambiguous phylogenetic information, whether analyzed cladistically by itself or when mapped onto phylogenetic trees based upon molecular sequence data.     

Phylogenetic relationships among actinorhizal plants. The impact of molecular systematics and implications for the evolution of actinorhizal symbioses

A review of recent molecular systematic studies of actinorhizal plants and their Frankia endosymbionts is presented. For comparative purposes, a discussion of recent studies pertaining to the evolution of nodulation in the legume-rhizobium system is included. Molecular systematic studies have revealed that actinorhizal plants are more closely related than current taxonomic schemes imply. Broad-based analyses of the chloroplast gene rbcL indicate that all symbiotic root-nodulating higher plants belong to a single large clade. More focused molecular analyses of both legume and actinorhizal hosts within this large clade indicate that symbioses have probably arisen more than once. By comparing host phylogenies and recently published bacterial phylogenies, we consider the coevolution of bacterial symbionts with their actinorhizal hosts.     

A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation

Phylogenetic analysis of the chloroplast-encoded rbcL gene in Leguminosae are consistent with previous hypotheses in suggesting that the family as a whole is monophyletic, but that only two of its three subfamilies are natural. The earliest dichotomies in the family appear to have involved tribes Cercideae or Cassieae (subtribe Dialiinae), followed by Detarieae/ Macrolobieae, all of which are members of subfamily Caesalpinioideae. The remainder of the family is divided into two clades: (1) Mimosoideae and the caesalpinioid tribes Caeasalpinieae and Cassieae (subtribes Ceratoniinae and Cassiinae); (2) Papilionoideae. Basal groups within Papilionoideae are, as expected, elements of the grade tribes Sophoreae and Swartzieae. Major clades within Papilionoideae include: (1) a Genistoid Alliance comprising Genisteae, Crotalarieae, Podalyrieae, Thermopsideae, Euchresteae, and also some Sophoreae; (2) a clade marked by the absence of one copy of the chloroplast inverted repeat, with which are associated Robinieae. Loteae, and some Sophoreae; (3) Phaseoleae, Desmodieae. Psoraleeae, and most Millettieae, a group also marked by presence of pseudoracemose inflorescences; and (4) a well-supported clade comprising Aeschynomeneae, Adesmieae, and some Dalbergieae. Nodulation is most parsimoniously optimized on the rbcL strict consensus tree as three parallel gains, occurring in Papilionoideae, the caesalpioioid ancestors of Mimosoideae, and in the genus Chamaecrista (Caesalpinieae: Cassieae).     

TAXONOMY AND PHYLOGENY OF OSMUNDEA (RHODOMELACEAE, RHODOPHYTA) IN ATLANTIC EUROPE

Two species of Osmundea Stackhouse (Rhodomelaceae, Rhodophyta) that occur in Atlantic Europe have been confused under the names Osmundea ramosissima (Oeder) Athanasiadis and Osmundea truncata (Kützing) Nam et Maggs, regarded until now as a synonym of O. ramosissima. An epitype from its type locality (Stavanger, Norway) is selected for Osmundea ramosissima Athanasiadis, recognized here as a valid name for Fucus ramosissimus Oeder, nom. illeg. Details of vegetative and reproductive morphology of O. ramosissima are reported, based on material from France, the British Isles, and Helgoland. Osmundea ramosissima resembles other species of Osmundea in its vegetative axial segments with two pericentral cells and one trichoblast, spermatangial development from apical and epidermal cells (filament type), the formation of five pericentral cells in the procarp‐bearing segment of the female trichoblast, and tetrasporangial production from random epidermal cells. Among the species of Osmundea, O. ramosissima is most similar to O. truncata. Both species have discoid holdfasts, secondary pit connections between epidermal cells, and cup‐shaped spermatangial pits. They differ in that: (a) O. ramosissima lacks lenticular wall thickenings and refractive needle‐like inclusions in medullary cells, both of which are present in O. truncata; (b) O. ramosissima has branched spermatangial filaments that terminate in a cluster of several cells, whereas in O. truncata the unbranched spermatangial filaments have a single large terminal sterile cell; and (c) cystocarps of O. ramosissima lack protuberant ostioles but ostioles are remarkably protuberant in O. truncata. Phylogenetic analyses of rbcL sequences of Laurencia obtusa (Hudson) Lamouroux and all five Atlantic European species of Osmundea, including the type species, strongly support the generic status of Osmundea. Osmundea ramosissima and O. truncata are closely related (5.2% sequence divergence) and form a well‐supported clade sister to a clade consisting of O. pinnatifida (Hudson) Stackhouse, O. osmunda Stackhouse and O. hybrida (A. P. de Candolle) Nam. The formation of secondary pit connections between epidermal cells is a synapomorphy for the O. ramosissima+O. truncata clade. The close relationship between species with cup‐shaped spermatangial pits (Osmundea hybrida) and urn‐shaped pits (Osmundea pinnatifida and Osmundea osmunda) shows that spermatangial pit shape is not an important phylogenetic character. Parsimony analysis of a morphological data set also supports the genus Osmundea but conflicts with the molecular trees in infrageneric relationships, placing O. hybrida basal within the Osmundea clade and grouping O. osmunda and O. pinnatifida but not O. truncata and O. ramosissima. A key to Osmundea species is presented.     

Ultrastructural and molecular characterization of diversity among small araphid diatoms all lacking rimoportulae. I. Five new genera,eight new species

Pennate diatoms are important contributors to primary production in freshwater and marine habitats. But the extent of their diversity, ecology, and evolution is still largely unknown. This is particularly evident among the clades of pennate diatoms without raphe slits, whose diversity is likely underestimated due to their small size and features that can be difficult to discern under light microscopy. In this study, we described five new araphid genera with eight new species based on morphological observations (light and electron microscopy) and molecular data (nuclear‐encoded small subunit ribosomal RNA and chloroplast‐encoded rbcL and psbC): Serratifera varisterna, Hendeyella rhombica, H. dimeregrammopsis, H. lineata, Psammotaenia lanceolata, Castoridens striata, C. hyalina, and Cratericulifera shandongensis. We also transferred Dimeregramma dubium to Hendeyella dubia. Phylogenetic analysis of the molecular data revealed that all the newly established taxa fell into a monophyletic group, with Fragilariforma virescens located at the base. The group was composed by two subclades: one comprising Castoridens, Cratericulifera, and Plagiostriata, and the larger including also the rest of the new genera plus some of the smallest known diatoms, such as Nanofrustulum, Opephora, Pseudostaurosira, Staurosirella, and Staurosira with a high level of support. This study enhances the general knowledge on the phylogeny and biodiversity of a group of small araphid diatoms that have been generally poorly described both by electron microscopy and DNA sequence data.     

Transfer of selected Ahnfeltiopsis (Phyllophoraceae,Rhodophyta) species to the genus Besa and description of Schottera koreana sp. nov.

We investigated species in the family Phyllophoraceae from Korea using nuclear large-subunit ribosomal RNA, mitochondrial COI and plastid rbcL sequences and morphological examination. To understand the taxonomic relationships of Korean Ahnfeltiopsis, we also analysed topotype materials of A. leptophylla and Besa papillaeformis from California. Both individual and combined datasets revealed that Korean Ahnfeltiopsis (except A. flabelliformis) consistently formed a clade with Californian A. leptophylla and Besa papillaeformis with strong support, and this Besa clade was distinct from other genera or genetic groups in the Phyllophoraceae. The form and development of reproductive structures in topotype material of B. papillaeformis as well as those of the four Ahnfeltiopsis species supported the molecular phylogeny and underlined the distinctiveness of the Besa clade. We therefore propose to transfer four Ahnfeltiopsis species to the genus Besa as Besa catenata, B. divaricata, B. leptophylla and B. paradoxa. The concept of the genus Besa is extended to species having a heteromorphic life history and conspicuous erect gametophytes. On the basis of our molecular phylogeny, we restore A. flabelliformis to the genus Gymnogongrus. We describe a new member of the Phyllophoraceae, Schottera koreana sp. nov., a deep-water species occurring along western and southern coastlines of Korea.     

A new blue-pigmented hasleoid diatom,Haslea provincialis,from the Mediterranean Sea

Haslea provincialis Gastineau, Hansen & Mouget, sp. nov., is a new, morphologically semicryptic blue diatom discovered on the French shores of the Mediterranean Sea. Like H. ostrearia and H. karadagensis, H. provincialis shares the capacity to synthesize a marennine-like blue pigment. Sexual reproduction between clones of H. provincialis has been repeatedly observed and resulted in viable initial cells. There were no sexual interactions with sexually competent clones of H. ostrearia or H. karadagensis, as would be expected for a separate biological species. There are strong similarities between the H. provincialis pigment and the marennine produced by H. ostrearia, evidenced by UV-visible spectrophotometry and Raman spectrometry. However, unlike the marennine from H. ostrearia, no differences were found between the extracellular and the intracellular forms of the pigment in H. provincialis. This indicates that the synthesis pathways and excretion mechanisms among the three ‘blue’ Haslea may be species-specific. Molecular taxonomy and phylogeny (based on rbcL, cox1 and SSU V4 DNA sequences) confirmed the distinct position of this species among the blue Haslea species. Haslea provincialis occurs in environments from which H. ostrearia has already been reported (mostly based on the presence of the blue cell vacuoles). Possible species misidentifications and the impact of the complex geological history of the Mediterranean Sea on blue diatom diversification are also discussed.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF AUREOPHYCUS ALEUTICUS GEN. ET SP. NOV. (LAMINARIALES,PHAEOPHYCEAE) FROM THE ALEUTIAN ISLANDS1

A previously unknown species of kelp was collected on Kagamil Island, Aleutian Islands. The species can be easily distinguished from any known laminarialean alga: the erect sporophytic thallus is composed of a thin lanceolate blade attaining ～2 m in height and ～0.50 m in width, without midrib, and the edge of the blade at the transition zone is thickened to form a V‐shape; the stipe is solid and flattened, slightly translucent, attaining ～1 m in length; the holdfast is semidiscoidal and up to 0.15 m in diameter. Anatomically, the blade has the typical trumpet‐shaped hyphae characteristic of the Chordaceae and derived foliose laminarialean species (i.e., Alariaceae/Laminariaceae/Lessoniaceae). No hair pits or mucilaginous structures were observed on the blade or stipe. No fertile sporophytes were collected, but abundant juvenile sporophytes were observed in the field. In the molecular phylogenetic analyses using chloroplast rbcL gene, nuclear ITS1‐5.8S‐ITS2 rDNA, and mitochondria nad6 DNA sequences, the new species (Aureophycus aleuticus gen. et sp. nov.) showed a closer relationship with Alariaceae of conventional taxonomy, or the “Group 1” clade of Lane et al. (2006) including Alaria and related taxa than with other groups, although the species was not clearly included in the group. Aureophycus may be a key species in elucidating the evolution of the Alariaceae within the Laminariales. Because of the lack of information on reproductive organs and insufficient resolution of the molecular analyses, we refrain from assigning the new species to a family, but we place the new species in a new genus in the Laminariales.