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Chaetophora lobata is the type species of the polyphyletic genus Chaetophora. Clarification of its species identification, phylogenetic relationships, and plastome characteristics is helpful for ta...

One green algal specimen from China was identified as Gongrosira fluminensis Fritsch, due to its unique morphology that pseudoparenchymal basal stratum with rounded or polygonal cells grew into dense little-branched upright threads that reached approximately the same height with specific akinete formation and germination and formed strong cushions without calcification. Examination of the ultrastructural characteristics of plasmodesmata and pyrenoid confirmed that Gongrosira fluminensis Fritsch should be excluded from the order Chaetophorales. The phylogenetic evidence based on DNA sequence data from the nucleus (18S rDNA, ITS rDNA) and chloroplast (tufA) sequences clearly revealed that the Gongrosira fluminensis Fritsch should be classified in the Ulvales (Ulvophyceae) as the new combination species of the genus Lithotrichon Darienko et Proschold, instead of the Chaetophorales (Chlorophyceae). More specimens in conjunction with natural morphological investigation and molecular analyses are required to reevaluate the microfilamentous genus Gongrosira Kutzing and reveal hidden diversities among the Ulvophyceae.

The Chaetophorales (Chlorophyceae) consist of filamentous green algae that were mostly described directly from natural samples by light microscopy. However, the descriptions were often based on mor...

It is generally accepted that land plants evolved from streptophyte algae. However, there are also many chlorophytes (a sister group of streptophyte algae and land plants) that moved to terrestrial habitats and even resemble mosses. This raises the question of why no land plants evolved from chlorophytes. In order to better understand what enabled streptophyte algae to conquer the land, it is necessary to study the chlorophytes as well. This review will introduce the freshwater filamentous chlorophyte alga Draparnaldia sp. (Chaetophorales, Chlorophyceae) as a model for comparative analyses between these two lineages. It will also focus on current knowledge about the evolution of morphological complexity in chlorophytes versus streptophytes and their respective morphological/behavioural adaptations to semi-terrestrial habitats, and will show why Draparnaldia is needed as a new model system.

Background Green plants comprise two lineages: (1) the streptophytes that colonised land and (2) the chlorophytes that have adaptations to land but remained mostly aquatic. To better understand what made streptophytes so successful, we are currently establishing the chlorophyte alga Draparnaldia sp. (Chaetophorales, Chlorophyceae) as a model for comparative analyses between these two lineages. However, establishing Draparnaldia as a valuable model requires that it can be transformed. Thus, our goal is to develop a transformation protocol for this alga. Results We have established the first transformation protocol for Draparnaldia. This protocol is based on protoplast transformation by electroporation. It includes instructions on protoplast isolation, regeneration and transient transfection. It also provides a list of the effective selective agents for future Draparnaldia transformations. Conclusions Our protocol opens a way for Draparnaldia functional genomics analyses. Moreover, it also provides an important base for establishment of stable transformation. Electronic supplementary material The online version of this article (10.1186/s13007-019-0460-6) contains supplementary material, which is available to authorized users.

The broadly defined genus Chaetophora consisted of species with minute, uniseriate branching filaments enveloped in soft or firm mucilage forming macroscopic growths that are spherical, hemispherical, and tubercular or arbuscular, growing epiphytically on freshwater aquatic plants and other submerged surfaces in standing or fast-flowing water. Recent molecular analyses clearly showed that this genus was polyphyletic. In this study, eight strains of Chaetophora and three strains of Stigeoclonium were identified and successfully cultured. In combination with the morphological data, a concatenated data set of four markers (18S + 5.8S + ITS2+ partial 28S rDNA) was also used to determine their taxonomic relationships and phylogenetic positions. The molecular analysis resolved the broadly defined Chaetophora to at least two genera. Species with a globose thallus of genus Chaetophora formed a separate monophyletic clade, which clearly separated from, a type of lobe-form Chaetophora species. Therefore, we propose to erect a new genus, Chaetophoropsis, which includes all globose species of the Chaetophora. Chaetophoropsis aershanensis was determined to be a new species, based on its special characteristic of profuse long rhizoids. Stigeoclonium polyrhizum, as the closest relative to Chaetophoropsis, revealed its distant relationships to other species of Stigeoclonium. A globose thallus with a thick, soft mucilage matrix, and special rhizoidal branches lent further support to the placement of S. polyrhizum in the genus Chaetophoropsis and had the closest relationship to C. aershanensis. Taxonomic diversity was proven by distinctive morphological differences and by phylogenetic divergence in the broadly defined Chaetophora identified herein.

Premise of the study To enhance our knowledge of the diversity of microalgae, a phycological survey of the Canary Islands (Spain) was undertaken. Here we report the discovery of a (semi)terrestrial green filamentous alga isolated from a steep volcanic canyon on La Palma. This alga is continually exposed to changing weather conditions (floods vs. droughts) and thus provides a good opportunity to investigate possible adaptations to a semiterrestrial habitat with large fluctuations of environmental parameters. Methods We used axenic cultures, simulated flood and drought stresses and studied their effect on the life history of the alga using light, confocal laser scanning and scanning electron microscopy including fluorescent staining. Furthermore, phylogenetic analyses using rDNA sequence comparisons were performed. Key results Three specific life-history traits that likely represent adaptations to the fluctuating environment of the canyon were observed: (1) fragmentation through "filament splitting", a unique branching mechanism not reported before in algae and initiated by formation of oblique cross walls, (2) aplanospore formation, and (3) reproduction by multiflagellate zoospores with 4-24 flagella arranged in groups of four. Phylogenetic analyses identified the alga as Barranca multiflagellata gen. et sp. nov. (Barrancaceae fam. nov., Chaetophorales, Chlorophyceae). Moreover, the Chaetophoraceae Greville, 1824 was emended and a new family, Uronemataceae (fam. nov.) erected. Conclusions The discovery of Barrancaceae fam. nov. highlights the importance of investigating nonconventional habitats to explore microalgal diversity. The reproductive versatility demonstrated by Barranca suggests adaptation to a semiterrestrial habitat with large fluctuations in water availability.

BackgroundThe chloroplast genome sustained extensive changes in architecture during the evolution of the Chlorophyceae, a morphologically and ecologically diverse class of green algae belonging to the Chlorophyta; however, the forces driving these changes are poorly understood. The five orders recognized in the Chlorophyceae form two major clades: the CS clade consisting of the Chlamydomonadales and Sphaeropleales, and the OCC clade consisting of the Oedogoniales, Chaetophorales, and Chaetopeltidales. In the OCC clade, considerable variations in chloroplast DNA (cpDNA) structure, size, gene order, and intron content have been observed. The large inverted repeat (IR), an ancestral feature characteristic of most green plants, is present inOedogonium cardiacum(Oedogoniales) but is lacking in the examined members of the Chaetophorales and Chaetopeltidales. Remarkably, theOedogonium35.5-kb IR houses genes that were putatively acquired through horizontal DNA transfer. To better understand the dynamics of chloroplast genome evolution in the Oedogoniales, we analyzed the cpDNA of a second representative of this order,Oedocladium carolinianum.MethodsTheOedocladiumcpDNA was sequenced and annotated. The evolutionary distances separatingOedocladiumandOedogoniumcpDNAs and two other pairs of chlorophycean cpDNAs were estimated using a 61-gene data set. Phylogenetic analysis of an alignment of group IIA introns from members of the OCC clade was performed. Secondary structures and insertion sites of oedogonialean group IIA introns were analyzed.ResultsThe 204,438-bpOedocladiumgenome is 7.9 kb larger than theOedogoniumgenome, but its repertoire of conserved genes is remarkably similar and gene order differs by only one reversal. Although the 23.7-kb IR is missing the putative foreign genes found inOedogonium, it contains sequences coding for a putative phage or bacterial DNA primase and a hypothetical protein. Intergenic sequences are 1.5-fold longer and dispersed repeats are more abundant, but a smaller fraction of theOedocladiumgenome is occupied by introns. Six additional group II introns are present, five of which lack ORFs and carry highly similar sequences to that of the ORF-less IIA intron shared withOedogonium. Secondary structure analysis of the group IIA introns disclosed marked differences in the exon-binding sites; however, each intron showed perfect or nearly perfect base pairing interactions with its target site.DiscussionOur results suggest that chloroplast genes rearrange more slowly in the Oedogoniales than in the Chaetophorales and raise questions as to what was the nature of the foreign coding sequences in the IR of the common ancestor of the Oedogoniales. They provide the first evidence for intragenomic proliferation of group IIA introns in the Viridiplantae, revealing that intron spread in theOedocladiumlineage likely occurred by retrohoming after sequence divergence of the exon-binding sites.

The green algal genus Cloniophora has been classified in the Chaetophorales (Chlorophyceae) based on morphological characters. This study uses DNA sequence data from the nucleus (SSU) and the chloroplast (tufA) from collections in the Hawaiian Islands and a culture originating from Portugal to test this classification. Taxonomic identities of contemporary collections were confirmed by sequencing small fragments of DNA (rbcL and SSU) from type specimens, including the generitype, Cloniophora willei L. H. Tiffany. These molecular data show that Cloniophora does not have close affinities to the Chaetophorales and belongs instead to the Ulvales (Ulvophyceae). The morphological features of eight or more reproductive products per cell and a pyrenoid with a traversing thylakoid membrane support the molecular data and confirm the placement of this group in the Ulvales. As this genus does not belong to any recognized family in the Ulvales, the family Cloniophoraceae, containing the single genus Cloniophora, is proposed.

The green alga Printzina (Trentepohlia) lagenifera was found in southern Finland in the sori of the leaves of the indigenous fern Polypodium vulgare. The find is noteworthy because P. lagenifera occurred epiphyllous in a boreal, natural habitat. The subcosmopolitan P. lagenifera is a collective species; it was described from a hothouse in Germany. Two earlier finds from Finland are likewise from hothouses. The present alga is hardly conspecific with these lignicolous hothouse algae that most probably are introductions from the tropics. Another green alga, the common Desmococcus olivaceus (Chaetophorales) is reported to occur in Finland on the upper surfaces of the leaves of Polypodium vulgare.

Previously published molecular phylogenetic analyses of the Chaetophorales (Chlorophyceae) suffered from limited taxon sampling (six genera with only a single species per genus). To test the monophyly of species-rich genera, and to analyze the phylogenetic relationships among families and genera in the Chaetophorales, we determined nuclear-encoded SSU rDNA sequences from 30 strains of Chaetophorales, performed phylogenetic analyses using various methods, and screened clades for support by unique molecular synapomorphies in the SSU rRNA secondary structure. The Schizomeridaceae and the weakly supported Aphanochaetaceae were recovered as basal lineages. The derived family Chaetophoraceae diverged into two clades: the "Uronema clade" containing unbranched filaments, and a sister clade designated as "branched Chaetophoraceae" comprising Chaetophora, Stigeoclonium, Draparnaldia, Caespitella, and Fritschiella. Although some terminal clades corresponded to genera described (e.g., Caespitella and Draparnaldia), other clades were in conflict with traditional taxonomic designations. Especially, the genera Stigeoclonium and Chaetophora were shown to be polyphyletic. The globose species Chaetophora elegans was unrelated to lobate Chaetophora spp. (e.g., Chaetophora lobata). Since the original description of Chaetophora referred to a lobate thallus organization, the latter clade represented Chaetophora sensu stricto. In consequence, C. lobata was designated as lectotype of Chaetophora. Two Stigeoclonium species, Stigeoclonium farctum Berthold and Stigeoclonium'Longipilus', diverged independently from the type species of Stigeoclonium, Stigeoclonium tenue (C. Agardh) Kutz. These results indicated that some commonly used taxonomic characters are either homoplasious or plesiomorphic and call for a reevaluation of the systematics of the Chaetophorales using novel morphological and molecular approaches.

The diplobiontic–haplodiplontic life cycle with alternating isomorphic generations in Stigeoclonium tenue (C. Agardh) Kutz. is described for the first time. Sporophytes (2n = 10) arise from tetraflagellate zoospores that are produced by meiosis. Sporic meiosis might be inferred from the cruciform divisions formed during zoosporogenesis and is confirmed through observations of prophase I substages. Zoospores do not germinate directly but produce a haploid cyst that germinates to give rise to a gametophyte (n = 5). Gametophytes produce biflagellate isogametes, which fuse to produce zygotes that germinate by mitosis into the sporophytic stage. Gametophytes and sporophytes reproduce asexually both via mitotic tetraflagellate zoospores and by thallus fragmentation. Results from this study indicate that both the cosmopolitan distribution and dominance of S. tenue in many periphytic communities might be due to its multiple reproductive strategies.

Four specimens of Stigeoclonium spp., sampled in China between 2015 and 2016, were identified as the species Stigeoclonium polyrhizum (Chaetophoraceae, Chaetophorales) due to their unique morphology. A large part of the main filament and branches was tightly enclosed by numerous rhizoidal branches, which extended downward to the base of the plant to form an expanded holdfast. S. polyrhizum has previously been regarded as a synonym of S. longipilum or other related species by most phycologists. Therefore, a reassessment of S. polyrhizum based on morphological and molecular data was conducted. Rhizoidal branch development of S. polyrhizum was also described, showing the abundant rhizoidal branches present from the juvenile stage to the mature stage under controlled culture conditions. Phylogenetic evidence, using nuclear - encoded SSU rDNA data, clearly revealed that the Chaetophoraceae diverged into two well - supported sister clades: the Chaetophora- clade and the Fritschiella- clade. S. polyrhizum was included in the Fritschiella- clade instead of the Chaetophora- clade where Chaetophora draparnaldioides ( S. longipilum ) was found. The morphological and molecular data unambiguously show that S. polyrhizum is a valid species rather than a synonym of S. longipilum or other related species. Thylakoid bands appressed to the periphery of the pyrenoid matrix of S. polyrhizum was also described.

SUMMARY Four species of Stigeoclonium from Argentina were studied by means of transmission electron microscopy and light microscopy. For species identification, we collected data related to the prostrate system and zoospore germination. We also determined the chromosome number for each species. Stigeoclonium aestivale showed a more developed erect system than the prostrate one, zoospore germination was predominantly of erect type and the chromosome number was 8. Stigeoclonium tenue presented well developed, erect and prostrate systems, zoospore germination was initially of the prostrate type and the chromosome number was 5. In Stigeoclonium variabile the prostrate system predominated over the erect one, zoospore germination was strictly of the prostrate type and the chromosome number was 3. Stigeoclonium farctum presented a more developed prostrate system than the erect one, zoospore germination was strictly of the prostrate type and the chromosome number was 8. The ontogeny of the zoospore germination was related to the final relative development of the prostrate and erect portions of adult thalli.

The Chlorophyceae (sensu Mattox and Stewart) is a morphologically diverse class of the Chlorophyta displaying biflagellate and quadriflagellate motile cells with varying configurations of the flagellar apparatus. Phylogenetic analyses of 18S rDNA data and combined 18S and 26S rDNA data from a broad range of chlorophycean taxa uncovered five major monophyletic groups (Chlamydomonadales, Sphaeropleales, Oedogoniales, Chaetophorales, and Chaetopeltidales) but could not resolve their branching order. To gain insight into the interrelationships of these groups, we analyzed multiple genes encoded by the chloroplast genomes of Chlamydomonas reinhardtii P. A. Dang. and Chlamydomonas moewusii Gerloff (Chlamydomonadales), Scenedesmus obliquus (Turpin) Kutz. (Sphaeropleales), Oedogonium cardiacum Wittr. (Oedogoniales), Stigeoclonium helveticum Vischer (Chaetophorales), and Floydiella terrestris (Groover et Hofstetter) Friedl et O'Kelly (Chaetopeltidales). The C. moewusii, Oedogonium, and Floydiella chloroplast DNAs were partly sequenced using a random strategy. Trees were reconstructed from nucleotide and amino acid data sets derived from 44 protein-coding genes of 11 chlorophytes and nine streptophytes as well as from 57 protein-coding genes of the six chlorophycean taxa. All best trees identified two robustly supported major lineages within the Chlorophyceae: a clade uniting the Chlamydomonadales and Sphaeropleales, and a clade uniting the Oedogoniales, Chaetophorales, and Chaetopeltidales (OCC clade). This dichotomy is independently supported by molecular signatures in chloroplast genes, such as insertions/deletions and the distribution of trans-spliced group II introns. Within the OCC clade, the sister relationship observed for the Chaetophorales and Chaetopeltidales is also strengthened by independent data. Character state reconstruction of basal body orientation allowed us to refine hypotheses regarding the evolution of the flagellar apparatus.

The evolutionary affinities of Heterochlamydomonas Cox and Deason and Dictyochloris Vischer ex Sterr were investigated using phylogenetic analyses of a combined data set of 18S and 28S rDNA sequences with those from 38 additional green algae. Previous ultrastructural studies have shown that motile cells of Heterochlamydomonas and Duictyochloris have an unusual flagellar apparatus organization in that the two flagella are of unequal length and the basal bodies are persistently parallel. Because of this similarity these taxa, along with Bracteacoccus Tereg, a third taxon with this same flagellar apparatus arrangement, are hypothesized to be closely related. We show, with maximum parsimony and Bayesian analyses, that the parallel basal bodies are not homologous in the three genera. Rather, Heterochlamydomonas is most closely related to Chlamydomonas baca in the clockwise flagellar apparatus clade, and Dictyochloris and Bracteacooccus are nested within the Sphaeropleales, which has the directly opposite flagellar absolute orientation. Surprisingly, Dictyochloris and Bracteacoccus are not supported as closest relatives. These relationships are supported by morphological features such as the presence or absence of a walled motile cell but not by the orientation of the basal bodies. In addition, our data are derived from multiple isolates of each study genera, and the analyses show that Heterochlamydomonas and Dictyochloris are each monophyletic.

Ultrastructural analyses of the flagellar apparatus suggested that Sphaeroplea, Atractomorpha, the Hydrodictyaceae, and the Neochloridaceae, all of which produce biflagellate motile cells with directly opposed (DO) basal bodies, are allied in an order Sphaeropleales. Recent studies of 18S rDNA sequence data supported an alliance of the DO group, but no data from Sphaeroplea and its allies were included. This investigation presented a test of the phylogenetic hypothesis suggested by the flagellar apparatus evidence using sequence data from the nuclear-encoded small-subunit rDNA (18S) and large subunit rDNA (26S) genes, combined with additional taxon sampling. Results from phylogenetic analyses weakly supported monophyly of biflagellate DO taxa and indicated that pyrenoids with cytoplasmic invaginations are present in numerous distinct lineages. Analysis of both molecular data sets supported a class Chlorophyceae comprised of at least six major groups that generally correspond to currently recognized orders or families: Chaetophorales, Chae- topeltidales, Chlamydomonadales, Sphaeropleales, Sphaeropleaceae, and Oedogoniales. In addition, Cylindrocapsa, Elakatothrix, Treubaria, and Trochiscia formed a seventh chlorophycean clade that is new to science. This investigation demonstrated that the 26S rDNA gene provides more phylogenetic signal, per unit sequence, than the 18S rDNA gene and that combined analysis yields topologies with more robust support than independent analysis of either data set.

SUMMARY Two populations of Chaetophora elegans (Roth) C, Agardh and two of Stigeoclonium helveticum Vischer were investigated for microhabitat characteristics and morphological variation in streams of Sao Paulo State, southeastern Brazil. Different patterns of microhabitat distribution were found between species investigated. Populations of C, elegans were distributed under relatively narrow microhabitat conditions (high irradiance, low depth, moderate to high current velocity, rocky substrata and lower values of niche width) and showing little morphometric variation (colony diameter, main axis cell size, and apical branch number), Stigeoclonium helveticum occurred under more diverse microhabitat conditions, revealed by lack of significant difference between sampling units with and without the alga and wider niche width, but also exhibited relatively narrow morphometric variation (plant length, main axis cell and lateral branch cell sizes). The narrow microhabitat conditions and smaller niche width of C. elegans can explain its low abundance (percentage cover) in streams from the area studied as well as in other regions of Sao Paulo State, In contrast, the wider variation of microhabitat conditions and the higher niche widths of S. helveticum suggest that this green alga is able to grow in a high number of stream ecosystems in the region investigated, ranging from undisturbed to highly disturbed habitats. Thus, the results suggest that S. helveticum is a generalist species.

The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other chlorophycean orders is uncertain. Chaetophoralean taxa include filamentous species which are both branched and unbranched. Ultrastructural studies of zoospores have revealed similar flagellar apparatuses in a number of genera, including Uronema, Stigeoclonium, and Fritschiella, suggesting a close phylogenetic relationship among these taxa. The order Oedogoniales represents a second group of branched and unbranched filamentous green algae whose relationships to other chlorophycean orders also has been unclear. A possible close relationship between the Chaetophorales and Oedogoniales has been suggested. Using DNA sequences from the small-subunit ribosomal RNA gene (SSU rRNA) of several members of each order, we have examined the monophyly of the Chaetophorales and Oedogoniales, as well as the nature of their relationship to other chlorophycean orders. Our results show that chaetophoralean and oedogonialean taxa form separate monophyletic groups. Results also suggest that the two orders are not closely related to each other.

SUMMARY To elucidate the phylogeny of the Dunaliellales sensu Ettl. the taxon often thought to be intermediate between primitive green flagellates such as the prasinophytes and the advanced chlorophycean algae, the sequences of the nuclear-encoded small subunit ribosomal RNA gene (18SrDNA) were determined and analyzed for five green algae, including three dunaliellalean algae. Phylogenetic trees based on 18SrDNA suggest that Oltmannsiellopsis viridis (Margraves et Steele) Chihara et Inouye represents an early divergence in the Ulvophyceae/Trebouxio-phyceae/Chlorophyceae clade and has no close relationship to any other green algae, as also suggested from ultrastructural characters. We propose Oltmannsiellopsidates ord. nov. for this genus. Hafniomonas and Polytomella are included in the clade which is characterized by clockwise basal bodies (CW group). The 18SrDNA trees suggest that multiple losses of the cell wall of the flagellate cell occurred in the CW group, and that the Dunaliellales sensu Ettl has a polyphyletic nature. This study also suggests that Planophita terrestns Groover et Hof-stetter (Chaetopeltidales) and Chaetophora incras-sata (Hudson) Hazen (Chaetophorales) are distinct lineages in the Chlorophyceae.

Two microfilamentous green algal specimens from Tibet were identified as Ctenocladus circinnatus Borzi, due to their unique morphology and saline habitat. The phylogenetic evidence based on DNA sequence data from the nucleus (SSU) and chloroplast elongation factor TU (tufA) sequences clearly revealed that the genus Ctenocladus, which has been classified in the Chaetophorales (Chlorophyceae) or Trebouxiophyceae incertae sedis by most phycologists, should be included in the Ulvales (Ulvophyceae) instead, and has a close relationship with the genera Pseudendoclonium and Phaeophila. We speculate that there may be undescribed or cryptic species especially in freshwater and other non-marine habitats. A phylogenetic re-evaluation based on large samples of microfilamentous ulvophycean algae especially freshwater specimens is needed.

Clones of the filamentous green alga Ctenocladus circinnatus Borzi were isolated from algae collected at Abert Lake (Oregon) and Mono Lake (California). Stock cultures were exposed to varied salinities of natural lake water to examine the effects on growth rate, cell form, chlorophyll a, and water content. Growth rates were reduced in both clones with increased salinity over the range 25–100 g·L−1 and were almost completely inhibited at 150 g·L−1. Chlorophyll a increased between salinities of 25 and 100 g·L−1, reflecting slower growth, higher proportions of akinetes, and smaller cell sizes as salinity increased. Tissue water content remained essentially constant from 25 to 100 g·L−1 salinity. Shorter cell dimensions with increased salinity suggest that a lower surface-to-volume ratio may reduce the potential for passive loss of cell water. Prior acclimation of stock cultures to elevated salinity provided no enhancement of growth response at any salinity. The results indicate that environmental salinity can limit the productivity and distribution of Ctenocladus in nature.

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.

Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single-gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms.

Over the past two decades, molecular phylogenetic data have allowed evaluations of hypotheses on the evolution of green algae based on vegetative morphological and ultrastructural characters. Higher taxa are now generally recognized on the basis of ultrastructural characters. Molecular analyses have mostly employed primarily nuclear small subunit rDNA (18S) and plastid rbcL data, as well as data on intron gain, complete genome sequencing, and mitochondrial sequences. Molecular-based revisions of classification at nearly all levels have occurred, from dismemberment of long-established genera and families into multiple classes, to the circumscription of two major lineages within the green algae. One lineage, the chlorophyte algae or Chlorophyta sensu stricto, comprises most of what are commonly called green algae and includes most members of the grade of putatively ancestral scaly flagellates in Prasinophyceae plus members of Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The other lineage (charophyte algae and embryophyte land plants), comprises at least five monophyletic groups of green algae, plus embryophytes. A recent multigene analysis corroborates a close relationship between Mesostigma (formerly in the Prasinophyceae) and the charophyte algae, although sequence data of the Mesostigma mitochondrial genome analysis places the genus as sister to charophyte and chlorophyte algae. These studies also support Charales as sister to land plants. The reorganization of taxa stimulated by molecular analyses is expected to continue as more data accumulate and new taxa and habitats are sampled. Twenty years ago, a relatively slim volume with chapters by leading chlorophycologists celebrated the systematics of green algae (Irvine and John, 1984), a field that was undergoing rapid and fascinating changes, both in content and theory. ‘‘The present period may be termed the ‘Age of Ultrastructure’ in green algal systematics,’’ wrote Frank Round (1984, p. 7) in the introductory chapter, which summarized the history and state of the art. Round (1984) argued that light microscopy had laid the foundation in the preceding two centuries, but that the foundation was largely descriptive—alpha taxonomy in the most restricted sense. Ultrastructure, he asserted, had enlarged and presumably would continue to expand our horizons to unify systematics of green algae and overcome the fragmented alpha taxonomy that had dominated the field. Little did Round know that this golden age of green algal systematics was about to go platinum. Molecular systematics, in concert with a rigorous theoretical approach to data analysis and hypothesis testing (Theriot, 1992; Swofford et al., 1996), would at first complement and then transform the age of ultrastructure and usher in the ‘‘Age of Molecules.’’ In this article, we review the major advances in green algal systematics in the past 20 years, with a focus on well-supported, monophyletic taxa and the larger picture of phylogeny and evolution of green algae. We will review the types of data that have fueled these advances. As will become obvious, this perspective entails discussion of some embryophytes as well as their closest green algal relatives. In addition, we will point

Three species of Gongrosira were isolated from freshwater habitats in England and Denmark. Developmental morphology and reproduction of the isolated strains were studied over a wide range of light and temperature regimes by means of a cross-gradient apparatus. Some of the traits traditionally used for defining infraspecific taxa (e.g. comparative development of prostrate and erect branches, position of zoosporangia) were found to be of questionable diagnostic value. New information is provided on previously described species and the isolates compared with cultures of related taxa. Gongrosira scourfieldii was collected from its type locality and culture-based observations have enabled an improved description of this little-known alga. A new species of Gongrosira is described, G. pseudoprostrata L. Johnson nov. sp., characterized by the presence of a well-defined primary prostrate axis and secondary axes. Problems of defining some of the main character traits and the possible ecological implications of the ...

Clonal algal cultures isolated from a stream in south-west England and a lake in northern Denmark correspond to the little-known genus Dilabifilum. The developmental morphology and reproduction of these isolates were compared with cultures (often type cultures) of the three species of Dilabifilum species recognized by Tschermak-Woess. All our isolates closely corresponded with a previously described species, Dilabifilum printzii. Some of the vegetative traits traditionally used for defining species were found to be of questionable diagnostic value. The most useful traits proved to be the branching habit, degree of compactness of the filaments, length to breadth ratio of the cells, form of the cells, and physiological features (e.g. growth rate, readiness to become coccoidal). The genus remains ill-defined, with earlier observations concerning the production of quadriflagellate zoospores by the type culture of D. arthopyreniae still requiring reconfirmation.

Recent studies of the Chlorophyceae using 18S and 26S rDNA data in meta-analysis have demonstrated the power of combining these two sets of rDNA data. Furthermore, the 26S rDNA data complement the more conserved 18S gene for many chlorophycean lineages. Consequently, this data approach was pursued in an expanded taxon-sampling scheme for the Chlorophyta, with special reference to the classes Chlorophyceae and Trebouxiophyceae. Results of these new phylogenetic analyses identify Microspora sp. (UTEX LB 472) and Radiofilum transversale (UTEX LB 1252) as sister taxa which, in turn, form a basal clade in the Cylindrocapsa alliance (Treubaria, Trochiscia, Elakatothrix). The relative position of the “Cylindrocapsa” clade within the Chlorophyceae remains uncertain. The enhanced taxon-sampling has not resolved the relative positions of the Oedogoniales, Chaetophorales or Chaetopeltidales. Furthermore, the Sphaeropleaceae are supported as members of the Sphaeropleales in only some analyses, raising concerns about the status of the order. Although based on a limited set of taxa (currently

Pure (axenic) strains of an intercellular endophyte identified as Chlorochytrium lemnae Cohn (= Chlorosphaeropsis lemnae Moewus) were isolated from infected duckweed (Lemna spp.). Pure cultures of the host were also obtained. Both grew well in mineral media, requiring no organic growth factors. In suitably dilute media, the algae could be induced to infect dead leaves of several different Lemna clones. Since its cells divide vegetatively by the formation of common cross walls (“desmoschisis”), Chlorochytrium lemnae should be transferred from the Chlorococcales to the Chaetophorales. The taxonomic status of other algae identified as Chlorochytrium—some of them demonstrated to be stages in the life-cycle of filamentous algae—should be re-evaluated.

Five Stigeoclonium tenue and five S. aestivale strains (both species having wide ecological ranges) and five S. farctum strains (a species with relatively narrow ranges) were examined for chlorine tolerance and morphological plasticity under laboratory conditions. Hair formation, cell dimensions and thallus form were studied. Differentiation in chlorine tolerance was most pronounced in S. aestivale, next in S. tenue and less in S. farctum. For morphological differentiation the sequence was S. tenue, S. farctum and S. aestivale, and for morphological plasticity S. farctum, S. tenue and S. aestivale. It is concluded that in a widespread algal genus such as Stigeoclonium, the ecological ranges are predominantly determined by the degree of ecotypic differentiation, rather than by morphological plasticity. The present study indicates that the separation of S. farctum and S. variabile by Cox & Bold (1966) is not justified and it is suggested that S. variabile may be an ecotype of S. farctum, but to some extent ...

Comparative investigations on the morphology, life-history, and reproduction of 3 cultivated strains of the atmophytic green algaApatococcus lobatus are presented. Some ultrastructural features are added to the results of earlier investigations, and comments on the systematics and ecology are given. Morphology and cytology of each strain is variable and shows considerable overlapping. The results support the view thatApatococcus consists of a single variable species (A. lobatus) only. For the epiphytic algal associationPleurococcetum vulgaris the more significant termApatococcetum lobati is proposed.

SUMMARY Pseudendoclonium basiliense and Trichosarcina polymorpha are essentially identical with regard to the fine structural details of cell division even though one was previously classified in the Chaetophorales and the other in the Ulvales. Cell division in the 2 genera is also shown to be like that in Ulva, as previously suggested might be the case. The combination of mitotic and cytokinetic characteristics common to the 3 genera is distinctive: (1) precocious development of a thick cleavage furrow, (2) centrioles distinctly lateral to polar fenestrae, (3) collapse of the interzonal spindle at telophase, and. (4) a cleavage furrow not associated with microtubules. It is suggested that features of vegetative cell division presently provide the best, characteristics for defining the Ulvaceae and that the use of growth habit should be abandoned. Despite the fact that a phycoplast is not present, in these algae, it is concluded that their affinities lie with genera that do possess a phycoplast.

The fine structure of the quadriflagellate zoospores of Draparnaldia glomerata (Vauch.) Agardh is described with emphasis on the flagellar root system and compared with the flagellar apparatus of related green algae. It is demonstrated that the flagellar root system in Draparnaldia is similar to that of the zoospore of Uronema belkae. Common features include presence of a cruciate root system (formula 2–5–2–5), prominent striated distal fibre connecting opposite basal bodies, a system I striated root component associated with the 2–stranded root, association of electron dense material with the 5–stranded root, mode of arrangement of the basal bodies in the absolute configuration model, and presence of four striated peripheral fibres interconnecting adjacent basal bodies. Differences exist in the shape of the striated peripheral fibres, the origin of the 2– and 5– stranded roots in the proximal part of the flagellar apparatus, and the architecture and striation pattern of the proximal part of the system I fibre that detaches from the 2–stranded root between adjacent basal bodies. Both the 2– and 5–stranded roots originate near the basal bodies and descend deeply into the zoospore. One of the 5–stranded roots passes near the eyespot of the chloroplast. The implications of these findings for the taxonomic position of the genus Draparnaldia are discussed. In addition, an evaluation is given of the present status of the order Chaetophorales. Suggestions are given to standardize some aspects of the current terminology of the cruciate flagellar root system in green algae.

Helicodictyon planctonicum (Whitford) Whitford & Schumacher (Chlorophyta) is a rare freshwater alga usually assigned to the order Chaetophorales but also reported to show features resembling members of the order Chlorococcales. In order to help clarify its phylogenetic position, an ultra-structural study was performed. The filamentous branching pattern and absence of a perinuclear envelope of endoplasmic reticulum during mitosis in H. planctonicum resemble features found among members of the Chaetophorales. However, the first and second cell divisions are perpendicular to each other as is common among chlorococcalean algae (Biebel, 1968). Additional chlorococcalean features exhibited by H. planctonicum include pyrenoids traversed by thylakoids, centrioles at the division plane following mitosis, and cytokinesis by furrowing. Therefore, it appears that H. planctonicum may be more closely allied with the Chlorococcales. However, failure to observe a phycoplast suggests Helicodictyon should be exami...

SUMMARY In vivo growth rates of Gongrosira [probably G. incrustans (Reinsch) Schmidle], a cushion-forming fresh-water green alga (Chaetophorales) are reported for the first time. The annual radial growth rate averaged 0-42 mm in a mountain stream from northern England and was positively correlated with water temperature (r = + 0-919, P < 0 05). The colonies were heavily incrusted with calcium carbonate (calcite). Photosynthetic activity probably promotes calcification between the closely packed basal filaments, but most of the precipitation appears to be abiogenic from supersaturated stream waters. Calcified Gongrosira bioherms are also described from a subtropical site in Mexico. The survival period for Gongrosira at the English site was approximately one year, and at both sites Gongrosira was a permanent and significant component of the epilithtc algal flora.

Extracts prepared from 10 bacteria-free algal cultures and 4 naturally occurring seaweeds were examined for urease and ATP-urea amidolyase (UAL-ase) activities. UAL-ase activity is confined to members of the classes Volvocales, Chlorococcales and Chaetophorales in the Chlorophyceae. Members of the Ulotrichales may possess either urease or UAL-ase. Ulva contains urease. All other algae, so far examined, which can grow with urea as nitrogen source contain urease but not UAL-ase.

Draparnaldia iyengarii sp. nov. is described from two desert localities in India. The new species has been compared with D. ravenellii Wolle, D. jaoii Cook and D. champlainesis Cook. D. iyengarii differs from all known species in having well defined and two types of short laterals composed of characteristically ellipsoidal cells. Each short lateral possesses only one multicellular, long hair.

Karyological investigations on six species of Stigeoclonium and three of Chaetophora (Chaetophorales, Chlorophyceae) are reported. While Stigeoclonium spp. were grown in BBMPTB 12 medium, the species of Chaetophora were cultivated in modified Chu-10 medium under standard culture conditions. The range of chromosome numbers was found to vary from n=5 to n= 14. Chromosome numbers of three taxa of Stigeoclonium, namely S. helveticum (n=5), S. aestivale (n = 10) and S. subsecundum (n= 14) are new records and the chromosome number obtained for Chaetophora pisiformis (n=8) is a first report.

The seta-bearing cells of Coleochaete scutata have been studied with the light and electron microscopes. These cells differentiated from vegetative cells and represent 3–5% of the cells in a typical thallus. They have the following modifications: (1) a tube-like extension of the cell wall (the seta collar); (2) the seta, which is a thin membrane-bound structure originating from the cytoplasm but exuded through the seta collar; (3) the setasome, a granular body found in the cytoplasm near the base of the seta collar; and (4) a crescent-shaped chloroplast that revolves around the base of the seta collar and extends a diverticulum of its outer membrane into the seta. Functional and evolutionary implications of seta-bearing cells are discussed.

The chlorophycean genus Schizomeris is usually considered to be a member of the Ulvales, often in the family Schizomeridaceae. However, Schizomeris differs from Ulva in several important cytological aspects. In Ulva cytokinesis is by furrowing; there are no microtubules associated with the cleavage furrow and there are no plasmodesmata. In Schizomeris cytokinesis is by cell plate formation with associated phycoplast microtubules, and plasmodesmata are present in the transverse walls. Schizomeris is similar to the investigated chaetophoralean algae in these and other respects. At present Schizomeris should be considered a distinct genus, apparently related to the Chaetophorales.

In the first paper of this series it was shown that mitosis and cytokinesis in Schizomeris are more like those events in Stigeoclonium rather than Ulva. It was concluded that Schizomeris should be included in the Chaetophorales. The present work shows that the zoospores of Schizomeris are somewhat different from those of both Stigeoclonium and Ulva. It is concluded that Schizomeris should be retained in a separate family of the Chaetophorales. Specifically, quadriflagellate zoospores of Schizomeris lack a two-stranded microtubular root, but rather have four, wide, cruciately arranged microtubular roots which probably contain an equal number of microtubules. Evolution in green algal motile cells is briefly discussed, and it is suggested that a flagellar ring such as occurs in the Oedogoniales may have evolved from quadriflagellate zoospores similar to those of Schizomeris.

Behaviour of Draparnaldiopsis indica Bharadwaja (Chlorophyta) was studied in a newly synthesized nutrient medium. The medium induced the development of normal morphological features in the alga. Branches of unlimited growth also showed differentiation into nodal and internodal cells. The typical morphology of the alga found in nature could only be obtained in this new medium.

Six different isolates of Ochlochaete Thwaites ex Harvey have been studied under identical culture conditions. All the isolates show open branching, a character previously ascribed specifically to O. hystrix Thwaites ex Harv. sensu stricto, and all form hairs on rounded cells in the central part of the thallus, a character hitherto attributed only to O. ferox Huber. Consequently, separation of these two entities on the species level is untenable. The plant described by Huber is referred to O. hystrix var. ferox (Huber) var. nov. The type material of Chaetobolus gibbus Rosenvinger is similar to one of the isolates studied and is included in O. hystrix. Quadriflagellate zoospores have been observed in all the isolates, and in one of them also biflagellate swarmers.

Quadriflagellate zoospores ofChaetophora incrassata andPseudoschizomeris caudata have similar features including an appressed membrane between the pyrenoid matrix and the starch sheath, and identical flagellar apparatuses. Components of the flagellar apparatus include: directly opposed upper basal bodies, lower basal bodies in the clockwise absolute orientation, a grooved distal fiber, peripheral and terminal fibers between adjacent basal bodies, proximal fibers connecting the lower basal bodies to the X-membered rootlets two- and X-membered rootlets associated with electron-dense components, and at least one rhizoplast. The X-membered rootlets, are comprised of five microtubules inC. incrassata and four or five inP. caudata. These features of the flagellar apparatus suggest that the two algae are closely related, and together withStigeoclonium, Uronema, Draparnaldia andFritschiella, form a natural group, the Chaetophoraceae, Chaetophorales (sensu Mattox and Stewart).

The influence of various element deficiencies was tested on 13 strains of Chaetophorales (1 Chaetophora, 2 Draparnaldia, 10 Stigeoclonium) isolated from sites in Belgium, N.E. England and Germany. Judged by the yield in batch culture, the relative response to each deficiency was quite similar in all strains: control > –P > –Fe > –Ca = –S > –Mg > –N. Judged by the extent of hair formation, the relative response was quite different: –P > –N > –Fe = –S > –Ca > –Mg > control. P- and N-limited cultures were the only ones to have typical hairs, and hair development was much greater in the former. It proved possible to maintain continued growth of a culture in a hairy condition by developing a continuous culture technique and using a medium with a high N:P ratio. Five strains were selected for more detailed study. N and P compositions, percentage hairiness and surface phosphatase activity were followed during growth in batch culture in medium leading to P-limitation. Although the maximum P composition r...

A low relief, green turf-forming alga of a heterotrichous habit was discovered in the coral reef microcosm, Museum of Natural History, Smithsonian Institution. Erect filaments bore lateral, specialized sporangia and together with basal filaments possessed septal plugs between adjacent cells, grossly similar to the "pit connections" of red algae. Data are presented which: 1) establish the identity of our plant with a plant recently described as Pilinia earleae Gallagher et Humm from the Florida Gulf coast; 2) support our establishment of the new genus Smithsoniella and our transfer of P. earleae to this new taxon. Additional data on pigmentation and cytology are related to the fine structure of other selected green algae to develop and test three hypotheses, viz. Smithsoniella earleae represents either: 1) a symbiotic association between a green and a red alga; 2) an alga which belongs to either the Ulotrichales, Chaetophorales or the Chroolepidales; or 3) an alga representing an evolutionary link between filamentous forms of the Ulvophyceae and members of the coenocytic siphonalean complex (e.g., Codiales or Caulerpales) of the Chlorophyta. Data refute hypotheses I and 2 but do lend support to the third hypothesis. RECENT FINE STRUCTURAL STUDIES of the green algae (Roberts et al., 1980; Sluiman et al., 1980; Stewart and Mattox, 1975, 1978; Stewart, Mattox and Floyd, 1973) have substantially modified our view of the relationships of members of the Chlorophyta from older systems based on vegetative and reproductive morphology. The origins of the coenocytic green algae have been especially difficult to discern (Stewart, Mattox and Floyd, 1 Received for publication 13 July 1981; revision accepted 3 November 1981. We thank individuals of the Smithsonian Institution who supported the concept of the coral reef microcosm developed by Dr. Walter Adey and colleagues and especially Dr. Adey for use of facilities in the Marine Systems Laboratory at the Smithsonian Institution. We also thank Dr. John Ogden and Nancy Ogden of the West Indies Laboratory, St. Croix, U.S.V.I. for providing space, facilities and field expertise during our visits to St. Croix, and Dr. Kenneth Towe for use of his electron microscopy facilities. Discussions with Drs. David Domozych, Ken Stewart, and Karl Mattox were especially helpful in developing our hypotheses and comments by Drs. Max Hommersand, Paul Silva, and Charles O'Kelly aided in taxonomic and nomenclatural treatments. We thank Dr. H. Humm for providing a culture of Smithsoniella from the Florida Gulf coast and Dr. Hannah Croasdale, Dartmouth College, for providing the Latin diagnosis of Smithsoniella. Support for travel to St. Croix by J. Sears was generously provided by Southeastern Massachusetts University and was greatly appreciated. 2 Present address: Department of Physiology, Health Center, University of Connecticut, Farmington, CT 06032. 1973) but recent studies (Roberts et al., 1980; Sluiman et al., 1980) suggest that they are related to the Ulvophyceae as proposed by Stewart and Mattox (1978). While surveying the green algae within the Smithsonian Institution's 6.5 kl coral reef microcosm (Brawley and Adey, 1981) in 1977, one of us discovered a most unusual heterotrichous green alga growing as a low relief, green turf on the walls and coral rock substratum of the microcosm. The fine, erect filaments bore specialized, lateral sporangia along their axes. These prominent reproductive structures were of special interest; among green algae, specialized sporangia are largely restricted to siphonous, coenocytic forms within the Caulerpales and Codiales or to multicellular forms of the Chaetophorales and Trentepohliales (Bourrelly, 1972). Even more unusual was the presence of "pit connections" between adjacent cells. Subsequent studies of these structures (Brawley and Sears, 1980, 1982) demonstrated that the apparent "pit connections" were true septal plugs occluding perforated end walls and consisted of a core and paired caps. Such structures have never been demonstrated within the Chlorophyta, although they are common in members of the Rhodophyta where they also consist of a perforated end wall occluded by a septal plug (Ramus, 1969). Stewart, Mattox and Floyd (1973) pointed out that "coe

The development and morphology of Draparnaldia mutabilis under various culture conditions is described. The germination of zoospores is strictly erect. In the first stage of development a primary rhizoid or a small prostrate system is formed, which is generally replaced in a later stage by persistent secondary rhizoids originating from basal cells of the main axis. Optimal growth occurs at about 20°C and at relatively high light intensities. The phenotypic appearance found in field material is maintained best in culture in a dilute modified Woods Hole medium at 8°C under short day conditions. In more nutrient-rich media the typical features of Draparnaldia, especially differentiation of main axes and lateral fascicles, disappear and the plants attain a Stigeoclonium-like habit. Morphological variation has been studied in herbarium material and freshly collected specimens from several localities in The Netherlands. Evidence is presented that in The Netherlands only one, rather polymorphic Draparnaldia spec...

The prostrate thallus of Stigeoclonium was studied under defined culture conditions. The development from zoospore to adult stage was followed in detail with the result that no more than three morphological groups (or species) can be discerned. The three groups are designated as Stigeoclonium helveticum, S. tenue, and S. farctum. Considerable intra- and intergenotypic variation exists, especially in the erect system of Stigeoclonium helveticum and in the prostrate system of S. tenue. The prostrate system of Stigeoclonium farctum is not always a closed pseudoparenchymatous disc, but may form an open star-like ‘variabile’ habit.

A study was made of zinc concentrations in field populations of Stigeoclonium and the aqueous chemistry of sites from which the materials were harvested; the sites included streams polluted by old mines. A significant positive relationship between zinc in the alga and that in the water was obtained when data were plotted on log10-log10 axes; its predictive ability was improved further by use of multiple stepwise regression (MSR) procedures to account for the influence of other environmental factors. The relationship between accumulation and toxicity was studied using the present and previously published data for toxicity (measured as tolerance index concentration). In both cases the environmental variables affecting Zn accumulation indicated during the first three MSR steps were (aqueous) Zn, Fe and Mg and/or Ca. Accumulation increased at a faster rate than toxicity with increasing environmental concentrations of zinc.

The unicellular, sessile and setate green alga Dicranochaete reniformis Hieronymus has been referred to the orders Tetrasporales, Chlorococcales and Chaetophorales. Ultrastructural investigation of the alga points to a pseudocilial nature of the seta and reveals the occurrence of a contractile vacuole near the base of the seta. These observations suggest a classification of this taxon within the Tetrasporales. We are, however, of opinion that this order as presently conceived, is insufficiently characterized to be considered a natural group. The pyrenoid of Dicranochaete reniformis possesses cytoplasmic invaginations of a type not previously recorded among tetrasporalean algae. The possible taxonomic significance of this character is discussed.