PHYLOGENETIC SYSTEMATICS OF THE ULVACEAE (ULVALES,ULVOPHYCEAE) USING CHLOROPLAST AND NUCLEAR DNA SEQUENCES1

Systematic hypotheses for the Ulvaceae were tested using phylogenetic analysis of sequences for the gene encoding the large subunit of RUBISCO, small subunit rDNA and a combined data matrix. Representatives of eight putative ulvaceous genera and twelve additional taxa from the Ulvophyceae and Trebouxiophyceae were included in analyses using maximum parsimony and maximum likelihood criteria. Molecular data supported hypotheses for the Ulvaceae that are based on the early development of vegetative thalli and motile cell ultrastructure. Ulvaceae sensu Floyd and O'Kelly, including Percursaria Bory de Saint‐Vincent, Ulvaria Ruprecht and a complex of closely related species of Chloropelta Tanner, Enteromorpha Link and Ulva L. was supported; however, monophyly of Enteromorpha and Ulva was not supported. The Ulvales and Ulotrichales sensu Floyd and O'Kelly were monophyletic. Blidingia Kylin and Kornmannia Bliding were allied with the former and Capsosiphon Gobi with the latter, although relationships among these and other taxa in these orders remain uncertain. The Ulvales are characterized by an isomorphic life history pattern, gametangia and sporangia that are identical in structure and development, motile cells with bilobed terminal caps and proximal sheaths consisting of two equal subunits. Method of motile cell release and the gross morphology of vegetative thalli are not systematically reliable characters.     

178 Molecular Systematics of the Ulvellaceae (Ulvales, Ulvophyceae) Inferred from Nuclear and Chloroplast DNA Sequences

The traditional use of thallus morphology to define lineages within the Ulvophyceae proved problematic because parallelism and convergence obscured natural relationships. The advent of transmission electron microscopy led to a re-evaluation of groups on the basis of zoid characters. Within TEM-defined lineages, the Ulotrichales and Ulvales were considered closely related orders, and molecular evidence has substantiated this hypothesis. In our molecular systematic studies of ulvophyceous green algae, we have broadened taxon sampling among these two orders. We show that the Ulvales and Ulotrichales are indeed monophyletic orders. However, the phylogenetic placement of species in the genera Bolbocoleon, Ctenocladus, Phaeophila, Pseudendoclonium and Acroblaste indicates that one or more additional lineages, placed between Ulvales and Ulotrichales in gene sequence trees, must be recognized. These species may constitute a separate order (Ctenocladales) consisting of multiple families. Our findings will be compared with taxonomic concepts based on morphological criteria.     

Taxonomic study of a new green alga,Annulotesta cochlephila gen. et sp. nov. (Kornmanniaceae,Ulvales, Ulvophyceae), growing on the shells of door snails

Some algae are known to grow on shellfish shells. Most of these have been reported in aquatic environments. The species specificity for substrate shells varies, and some algae grow only on the shells of a certain species of shellfish, such as Pseudocladophora conchopheria (Cladophorales, Ulvophyceae) on Lunella coreensis (Trochida, Gastropoda). There are very few reports of algae that grow on land snails. In this study, we discovered green algae growing on the shells of six species of door snails (Clausiliidae) from nine localities in Japan. These green algae formed a green mat composed of thalli embedded in the extracellular matrix. The thallus was composed of aggregated oval cells and peripheral branched filaments. The cells possessed a single parietal chloroplast with a pyrenoid surrounded by two starch sheaths and transversed by a thylakoid. Oil droplets in the cell and ring-like structures on the cell wall surface were frequently observed. The 18S rDNA sequences of all shell-attached algae on different clausiliid species from different localities were almost identical and formed a new clade in the family Kornmanniaceae (Ulvales, Ulvophyceae). No other algae forming visible colonies on the clausiliid shell were found. These findings indicate the presence of specificity between the alga and clausiliid shells. Based on the results of morphological observation and molecular phylogenetic analysis, we propose a new genus and new species of shell-attached green alga, Annulotesta cochlephila.

     

177 Molecular Systematics of the Green Algal Orders Ulotrichales and Ulvales Based on 18S rDNA Sequences

The traditional use of thallus morphology to define lineages within the Ulvophyceae proved problematic because parallelism and convergence obscured natural relationships. The advent of transmission electron microscopy led to a re‐evaluation of groups on the basis of zoid characters. Within TEM‐defined lineages, the Ulotrichales and Ulvales were considered closely related orders, and molecular evidence has substantiated this hypothesis. In our molecular systematic studies of ulvophyceous green algae, we have broadened taxon sampling among these two orders. We show that the Ulvales and Ulotrichales are indeed monophyletic orders. However, the phylogenetic placement of species in the genera Bolbocoleon, Ctenocladus, Phaeophila, Pseudendoclonium and Acroblaste indicates that one or more additional lineages, placed between Ulvales and Ulotrichales in gene sequence trees, must be recognized. These species may constitute a separate order (Ctenocladales) consisting of multiple families. Our findings will be compared with taxonomic concepts based on morphological criteria.     

PHYLOGENY OF THE ULVOPHYCEAE (CHLOROPHYTA): CLADISTIC ANALYSIS OF NUCLEAR-ENCODED rRNA SEQUENCE DATA1

Cladistic analysis of nuclear-encoded rRNA sequence data provided us with the basis for some new hypotheses of relationships within the green algal class Ulvophyceae. The orders Ulotrichales and Ulvales are separated from the clade formed by the remaining orders of siphonous and siphonocladous Ulvophyceae (Caulerpales, Siphonocladales /Cladophorales [S/C] complex, and the Dasycladales), by the Chlorophyceae and Pleurastrophyceae. Our results suggest that the Ulvophyceae is not a monophyletic group. Examination of inter- and intra-ordinal relationships within the siphonous and siphonocladous ulvophycean algae revealed that Cladophora, Chaetomorpha, Anadyomene, Microdictyon, Cladophoropsis and Dictyosphaeria form a clade. Thus the hypothesis, based on ultrastructural features, that the Siphonocladales and Cladophorales are closely related is supported. Also, the Caulerpales is a monophyletic group with two lineages; Caulerpa, Halimeda, and Udotea comprise one, and Bryopsis and Codium comprise the other. The Dasycladales (Cymopolia and Batophora) also forms a clade, but this clade is not inferred to be the sister group to the S/C complex as has been proposed. Instead, it is either the sister taxon to the Caulerpales or basal to the Caulerpales and S/C clade The Trentepohliales is also included at the base of the siphonous and siphonocladous ulvophycean clade. The Pleurastrophyceae, which, like the Ulvophyceae, posses a counter-clockwise arrangement of flagellar basal bodies, are more closely related to the Chlorophyceae than to the Ulvophyceae based on rRNA sequences. Thus, the arrangement of basal bodies does not diagnose a monophyletic group. Previously reported hypotheses of phylogenetic relationships of ulvophycean algae were tested. In each case, additional evolutionary steps were required to obtain the proposed relationships. Relationships of ulvophycean algae to other classes of green algae are discussed.     

MOTILE CELL ULTRASTRUCTURE AND THE CIRCUMSCRIPTION OF THE ORDERS ULOTRICHALES AND ULVALES (ULVOPHYCEAE,CHLOROPHYTA)

We have examined the motile cell ultrastructural features of several green algal species having filamentous or foliose thallus morphology and probable affinities with the Ulvophyceae, and compared them with the structural, reproductive, and life history features known for these taxa. We separate the algae studied into the orders Ulotrichales and Ulvales on the basis of consistent variations in terminal cap and proximal sheath structure that correlate well with life history patterns and certain features of sporangial and gametangial structure and development. Body scales are present only in certain members of the Ulotrichales. Both orders encompass a variety of thallus forms, demonstrating parallel evolution of thallus morphology. Flagellar apparatus features common to all the motile cells examined include 180° rotational symmetry, counterclockwise absolute orientation, the positioning of the basal bodies in an apical papilla, and the presence of one or more sets of striated bands associated with the X rootlets. Additional features that are usually present include basal body overlap and orientation roughly perpendicular to the long axis of the cell during forward swimming, striated distal fibers, and a single, striated, microtuble-associated component underlying each two-membered rootlet. These similarities indicate to us that the two groups are closely related members of the Ulvophyceae. We suggest that the Ulotrichales is the most primitive ulvophyceous assemblage known, but that all groups studied have advanced features relative to those supposed to have been present in the ancestral members of the Ulvophyceae.     

Phylogenetic position of Jaoa,a green algal genus endemic to China

Jaoa prasina, a freshwater green alga endemic to China, was collected from a stream in Hubei province, China. Unialgal cultivation, morphological observation, and phylogenetic analyses of small subunit ribosomal DNA and RuBisCO large subunit sequences were performed. When cultured on agar medium, the alga was irregularly filamentous, similar to marine species of Acrochaete. Aplanospores were observed on solid medium. A vesicular‐like thallus without rhizoids developed in liquid medium, similar to specimen development in natural habitats. Molecular phylogenetic analyses revealed that Jaoa was closely related to the marine genera Acrochaete Pringsheim and Ulvella Crouan & Crouan. The results suggested the genus Jaoa is a member of the family Ulvellaceae (Ulvophyceae), which contains mostly marine algae. The family name Jaoaceae should be abandoned. We speculate that Jaoa may have evolved from a marine Ulvellaceae ancestor.     

GENE SEQUENCE DIVERSITY AND THE PHYLOGENETIC POSITION OF ALGAE ASSIGNED TO THE GENERA PHAEOPHILA AND OCHLOCHAETE (ULVOPHYCEAE,CHLOROPHYTA)1

The phylogenetic position of microfilamentous marine green algae assigned to the species Phaeophila dendroides, Entocladia tenuis (Phaeophila tenuis, and Ochlochaete hystrix was examined through phylogenetic analyses of nuclear‐encoded small subunit rDNA and chloroplast‐encoded tufA gene sequences. These analyses placed the P. dendroides strains within the Ulvophyceae, at the base of a clade that contains representatives of the families Ulvaceae, Ulvellaceae, and the species Bolbocoleon piliferum, supporting an earlier hypothesis that P. dendroides constitutes a distinct lineage. Substantial divergence in both nuclear and plastid DNA sequences exists among strains of P. dendroides from different geographic localities, but these isolated strains are morphologically indistinguishable. The lineage may have an accelerated rate of gene sequence evolution relative to other microfilamentous marine green algae. Entocladia tenuis and O. hystrix are placed neither in the P. dendroides clade nor in the Ulvellaceae as previous taxonomic schemes predicted but instead form a new clade or clades at the base of the Ulvaceae. Ruthnielsenia gen. nov. is proposed to accommodate Kylin's species, which cannot be placed in Entocladia (=Acrochaete), Phaeophila, or Ochlochaete. Ruthnielsenia tenuis (Kylin) comb. nov., previously known only from Atlantic coasts, is reported for the first time from the Pacific coast of North America (San Juan Island, WA, USA). Isolates of R. tenuis from the Atlantic and Pacific coasts of North America have identical small subunit rDNA and tufA gene sequences.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure

Phylogenetic analyses, based upon nuclear small-subunit ribosomal RNA gene sequences, of four ‘chlorosarcinoid’ species referred to Planophila Gerneck show that the genus is polyphyletic. The type species, P. laetevirens Gerneck, is closely related to species in the Ulotrichales, Ulvophyceae. The monotypic sarcinoid genus Pseudendocloniopsis is the closest relative of Planophila; the two genera represent the addition of a new morphological type to the Ulotrichales. Planophila microcystis (Dangeard) Kornmann & Sahling forms a clade at the base of the Ulvophyceae with Oltmannsiellopsis, and thus belongs to the Oltmannsiellopsidales. This result is also supported by the Oltmannsiellopsis-like ultrastructure of P. microcystis zoospores. Planophila sp. B from Antarctica, which has Trebouxia-like pyrenoid structure, is a trebouxiophyte closely related to Chlorella-like unicellular coccoids, Stichococcus bacillaris and Prasiola species. This is the first robustly supported molecular phylogenetic analysis that places Prasiola in the Trebouxiophyceae. As shown previously, P. terrestris Groover & Hofstetter belongs to the Chaetopeltidales, Chlorophyceae. Dangemannia gen. nov. (type species : D. microcystis (Dangeard) comb. nov.), Floydiella gen. nov. (type species : F. terrestris (Groover & Hofstetter) comb. nov.) and Pabia gen. nov. (type: P. signiensis sp. nov.) are proposed.     

ULTRASTRUCTURE AND PHYLOGENETIC RELATIONSHIPS OF CHAETOPELTIDALES ORD. NOV. (CHLOROPHYTA,CHLOROPHYCEAE)1

Vegetative cells and zoospores of Hormotilopsis gelatinosa Trainor & Bold, H. tetravacuolaris Arce & Bold, Planophila terrestris Groover & Hofstetter, and Phyllogloea fimbriata (Korchikov) Silva were examined by transmission electron microscopy. All cells had pyrenoids traversed by cytoplasmic channels. Zoospores were quadriflagellate and had essentially cruciate flagellar apparatuses. Scales were present on free-swimming zoospores. These features are essentially identical to those of Chaetopeltis sp. and are dissimilar to those of other described green algae. The new order Chaetopeltidales is created to accommodate the genera Chaetopeltis, Hormotilopsis, Planophila sensu Groover & Hofstetter, Phyllogloea, Dicranochaete, and Schizochlamys, organisms previously scattered among the orders Tetrasporales, Chloro-coccales, Chlorosarcinales, and Chaetophorales. Members of the order are closely related to the ancestral chlorophycean flagellate genus Hafniomonas, may be ancestral with respect to other Chlorophyceae, and may also be closely related to the ulvophycean order Ulotrichales.     

The Distribution of Elongation Factor-1 Alpha (EF-1α), Elongation Factor-Like (EFL), and a Non-Canonical Genetic Code in the Ulvophyceae: Discrete Genetic Characters Support a Consistent Phylogenetic Framework

ABSTRACT. The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1α (EF-1α) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1α, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1α of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1α sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis , and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.     

Nuclear behavior and roles indicate that Codiolum phase is a sporophyte in Monostroma angicava (Ulotrichales,Ulvophyceae)

The life‐cycle system of Ulotrichales, a major order of Ulvophyceae, remains controversial because it is unclear whether the Codiolum phase, a characteristic unicellular diploid generation in ulotrichalean algae, is a zygote or a sporophyte. This controversy inhibits the understanding of the diversified life cycles in Ulvophyceae. To distinguish between zygotes and sporophytes, we have to examine not only whether diploid generations function as sporophytes, but also whether mitosis occurs before meiosis in diploid generations. However, the nuclear behavior in the Codiolum phases is largely unknown, probably because no suitable methods are available. Using fluorescent microscopy with ethidium bromide and transmission electron microscopy of cell‐wall‐dissected specimens, we report the nuclear behavior in the Codiolum phases of an ulotrichalean alga with a representative life cycle, Monostroma angicava. Each vegetative Codiolum phase had a single polyploid nucleus due to endoreduplication, a type of mitosis without nuclear division. During zoosporogenesis, the nucleus had a structure that would be a meiosis‐specific complex. We quantitatively showed that Codiolum phases grew extremely large and produced numerous zoospores. Our results suggest that an event comparable to mitosis occurs before meiosis in the Codiolum phase of M. angicava. This nuclear behavior and the functions (growth and zoospore production abilities) correspond to those of sporophytes. Therefore, the life‐cycle system of M. angicava is a heteromorphic haplo‐diplontic cycle. This system appears to be widely adopted among other ulotrichalean algae.     

Ultrastructure and phylogenetic relationships of the unicellular green algae Ignatius tetrasporus and Pseudocharacium americanum (Chlorophyta)

The phylogenetic relationships of two unicellular green algae, Ignatius tetrasporus Bold et MacEntee and Pseudocharacium americanum Lee et Bold were investigated by ultrastructural and molecular methods. The zoospores from both species were covered neither by scales nor cell walls. The flagellar apparatus of the zoospores commonly included these features: the upper basal bodies were displaced counterclockwise in half to two‐thirds of the basal body diameter and did not overlap with each other; the lower basal bodies were directly opposed or slightly displaced clockwise; the distal fiber had gently sigmoid central striations; terminal caps were absent from the ends of the basal bodies; a V‐shaped proximal sheath extended from the upper basal bodies; a posterior fiber lay between the opposite lower basal bodies; and the coarsely striated band linked the sinister rootlet to the lower basal body. The suite of these features was not identical to that of any other quadriflagellate swimming cells, but some features including the lower basal body orientation, the striated distal fiber, and the coarsely striated fiber resemble those of the several organisms of the Siphonocladales sensu Floyd and O’Kelly. Phylogenetic analysis using 18S rDNA sequence data revealed that I. tetrasporus and P. americanum formed a monophyletic clade within the clade of Ulvophyceae sensu López‐Bautista and Chapman, but was not nested within any of the orders of the class that were examined.     

PSEUDONEOCHLORIS MARINA (CHLOROPHYTA), A NEW COCCOID ULVOPHYCEAN ALGA, AND ITS PHYLOGENETIC POSITION INFERRED FROM MORPHOLOGICAL AND MOLECULAR DATA

Ultrastructural and molecular sequence data were used to assess the phylogenetic position of the coccoid green alga deposited in the culture collection of the University of Texas at Austin under the name of Neochloris sp. (1445). This alga has uninucleate vegetative cells and a parietal chloroplast with pyrenoids; it reproduces by forming naked biflagellate zoospores. Electron microscopy revealed that zoospores have basal bodies displaced in the counterclockwise absolute orientation and overlapped at their proximal ends. Four microtubular rootlets numbering 2 and 2/1 are alternatively arranged in a cruciate pattern. A system I fiber extends beneath each d rootlet and a system II fiber (rhizoplast) originates from each basal body and extends peripherally along each d rootlet. These features differ substantially from those of the three genera, Ettlia (Komárek) Deason et al., Neochloris (Starr) Deason et al., and Parietochloris Watanabe et Floyd, all of which were previously accommodated in the single genus Neochloris Starr. Sequence data from the nuclear small subunit ribosomal RNA gene were obtained and compared with published green algal sequences. Results from the ultrastructural and sequence data support the placement of Neochloris sp. (The Culture Collection of Algae at the University of Texas at Austin [UTEX] no. 1445) in the Ulvophyceae. This isolate is described as Pseudoneochloris marina , gen. et sp. nov. in the Ulotrichales, Ulvophyceae.     

THE FLAGELLAR APPARATUS OF ENTOCLADIA VIRIDIS MOTILE CELLS,AND THE TAXONOMIC POSITION OF THE RESURRECTED FAMILY ULVELLACEAE (ULVALES,CHLOROPHYTA)1

The flagellar apparatuses of the quadriflagellate zoo-spores and biflagellate female gametes of the marine chaetophoracean alga Entocladia viridis Reinke are significantly different from those of algae belonging to Chaetophoraceae sensu stricto, but closely resemble those of ulvacean genera. These differences permit the taxonomic reassignment of certain marine chaetophoracean genera and an evaluation of the flagellar apparatus features used to characterize the class Ulvophyceae. Critical features of the zoospore include arrangement of the four basal bodies into an upper and a lower pair with the proximal ends of the upper basal bodies overlapping, terminal caps, proximal sheaths connected to one another by striated bands, and a cruciate microtubular rootlet system having a 3-2–3-2 alternation pattern and striated microtubule-associated components that accompany the two-membered rootlets. An indistinct distal fiber occurs just anterior to the basal bodies, and is closely associated with the insertion into the flagellar apparatus of the three-membered rootlets. The flagellar apparatus demonstrates 180° rotational symmetry, and its components show counterclockwise absolute orientation when viewed from above. Newly described features include the prominently bilobed structure of the terminal caps on the upper basal body pair, and the presence of both a granular zone and an additional single microtubule anterior to each of the four rootlets, an arrangement termed the “stacked rootlet configuration.” Rhizoplasts were not observed and are presumed to be absent. The gamete is identical, except for the absence of the lower basal body pair and the presence of an electron-dense membrane associated structure that resembles the mating structure found in Ulva gametes. These findings, correlated with life history data, sporangial and gametangial structure and developmental patterns, chloroplast pigment arrays, and vegetative cell ultrastructural features, compel the removal of Entocladia viridis and similar members of the marine Chaetophoraceae to a separate family, the Ulvellaceae. The latter is referred to the order Ulvales of the Ulvophyceae. The counterclockwise absolute orientation of components, and terminal caps, may be the most consistent flagellar apparatus features of ulvophycean green algae, while variations in other features previously considered diagnostic for the Ulvophyceae may serve instead to identify discrete lineages within this class.     

ENDOPHYTIC ALGAE OF CHONDRUS CRISPUS (RHODOHYTA). II. ACROCHAETE HETEROCLADA SP. NOV., A. OPERCULATA SP. NOV., AND PHAEOPHILA DENDROIDES (CHLOROPHYTA)1

The occurrence of three endophytic green algae within Chondrus crispus Stackh. is reported. Two of them are new to science and are described as Acrochaete heteroclada Correa and Nielsen sp. nov. and A. operculata Correa and Nielsen sp. nov. The algae were studied in unialgal culture and in association with the host following infection of C. crispus under laboratory conditions. The experimental infection showed A. heteroclada to be initially epiphytic, with endophytic filaments growing into the cortex of the host during late infection. A. operculata is entirely endophytic when associated with the host. Phaeophila dendroides (Crouan frat.) Batters behaves as an epiphyte at 15 and 24°C but penetrates the host tissues at 20°C. For all three species, a close physical association with the host is established only when zoospores settle and germinate on the surface of C. crispus.