Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

PHYLOGENY OF ZYGNEMOPHYCEAE BASED ON COXIII GENE SEQUENCE DATA

Molecular phylogenetic analysis of the conjugating green algae (Class Zygnemophyceae) using nuclear (SSU rDNA) and chloroplast (rbcL) gene sequences has resolved hypotheses of relationship at the class, order, and family levels, but several key questions will require data from additional genes. Based on SSU and rbcL sequences, the Zygnemophyceae and Desmidiales are monophyletic, and families of placoderm desmids are distinct clades (Desmidiaceae, Peniaceae, Closteriaceae, and Gonatozygaceae). In contrast, the Zygnemataceae and Mesotaeniaceae are paraphyletic, although whether these two traditional families constitute a clade is uncertain. In addition, relationships of genera within families have proven resistant to resolution with these two oft‐used genes. We have sequenced the coxIII gene from the mitochondrial genome to address some of these ambiguous portions of the phylogeny of conjugating green algae. The coxIII gene is more variable than rbcL or SSU rDNA and offers greater resolving power for relationships of genera. We present preliminary analyses of coxIII sequences from each of the traditional families of Zygnemophyceae and contrast the resulting topologies with those derived from nuclear and chloroplast genes.     

PHYLOGENETIC AFFINITIES OF “CHANTRANSIA” STAGES IN MEMBERS OF THE BATRACHOSPERMALES AND THOREALES (RHODOPHYTA)1

This study evaluated the phylogenetic relationship among samples of “Chantransia” stage of the Batrachospermales and Thoreales from several regions of the world based on sequences of two genes—the plastid‐encoded RUBISCO LSU gene (rbcL) and the nuclear SSU ribosomal DNA gene (SSU rDNA). All sequences of “Chantransia macrospora” were shown to belong to Batrachospermum macrosporum based on both molecular markers, confirming evidence from previous studies. In contrast, nine species are now associated with “Chantransia pygmaea,” including seven species of the Batrachospermales and two of the Thoreales. Therefore, the presence of “C. macrospora” in a stream can be considered reliable evidence that it belongs to B. macrosporum, whereas the occurrence of “C. pygmaea” does not allow the recognition of any particular species, since it is associated with at least nine species. Affinities of “Chantransia” stages to particular taxa were congruent for 70.5% of the samples comparing the rbcL and SSU analyses, which were associated with the same or closely related species for both markers. Sequence divergences have been reported in the “Chantransia” stage in comparison to the respective gametophyte, and this matter deserves further attention.     

DNA sequence data reveal polyphyly of Brexioideae (Brexiaceae; Saxifragaceae sensu lato)

Previous molecular phylogenetic analyses have demonstrated that Saxifragaceae sensu lato are polyphyletic, with component lineages scattered throughout the eudicots. As part of our effort to elucidate the relationships of members of Engler and Prantl's Saxifragaceae s. l., we undertook a molecular systematic study of subfamily Brexioideae, which comprises three genera:Brexia, Ixerba, andRoussea. Not all taxonomic treatments have concurred, however, in placing these genera together. To elucidate relationships among these three genera as well as their relationships to other angiosperms we constructed large data sets ofrbcL, 18S rDNA, andrbcL + 18S rDNA sequences. Our phylogenetic analyses indicate clearly that Brexioideae are polyphyletic.Brexia is part of a celastroid clade that also includesParnassia, Lepuropetalon, and Celastraceae.Ixerba appears as sister to a large eurosid I clade;Roussea appears as part of Asterales. Molecular data, therefore, indicate that Brexioideae are a polyphyletic assemblage and component genera should ultimately be incorporated into other groups. Our studies continue to demonstrate the polyphyly not only Saxifragaceae s. l., but also of its constituent subfamilies.The first author would like to dedicate this paper to Kurt Schuchart, a good friend who passed away during this research.     

Investigation of a freshwater acrochaetioid alga (Rhodophyta) with molecular and morphological methods

An enigmatic acrochaetioid alga was collected from Niangziguan spring in Shanxi Province, northern China. Morphological data indicated that this alga reproduces exclusively asexually by monosporangia and its morphological characteristics suggested that it might be referred to Audouinella heterospora. To ascertain its phylogenetic position, phylogenetic trees were reconstructed using partial sequences of the plastid‐encoded gene (rbcL) and the nuclear‐encoded gene (SSU rDNA) applying Bayesian inference (BI), maximum parsimony (MP) and maximum likelihood (ML). However, phylogenetic reconstructions showed that this acrochaetioid alga does not belong in a clade with the genus Audouinella, but forms a clade with Thorea hispida (Thoreales). Based on this analysis it is concluded that A. heterospora represents the ‘chantransia’ stage of T. hispida.     

STUDIES ON ULTRASTRUCTURE AND THREE‐GENE PHYLOGENY OF THE GENUS MALLOMONAS (SYNUROPHYCEAE)1

The genus Mallomonas, a common and often abundant member of the planktic community in many freshwater habitats worldwide, consists of 180 species divided into 19 sections and 23 series. Classification of species is based largely on ultrastructural characteristics of the siliceous scales and bristles that collectively form a highly organized covering over the cell. However, the relative importance of the different siliceous features of the scales, such as the dome, V rib, and secondary structures, as well as the different types of scales, in understanding the evolution and phylogeny of the genus is little known. In this study, we investigated the scale and bristle ultrastructure, along with sequences of three genes, for 19 isolates (18 species) of Mallomonas (18 isolates were from Korean habitats). The isolates represented nine of the 19 sections. Sequences for both the nuclear SSU and LSU rDNA and plastid LSU of RUBISCO (rbcL) genes for each of the 19 Mallomonas isolates and four outgroups were determined. Bayesian and maximum‐likelihood (ML) analyses of the data revealed that Mallomonas consists of two strongly supported clades. Mallomonas bangladeshica (E. Takah. et T. Hayak.) Siver et A. P. Wolfe was at the base of the first clade that included taxa from the sections Planae and Heterospinae, both of which lack a V rib on the shield of the scales. Our results indicated that the sections Planae and Heterospinae should be combined. The second clade, with Mallomonas insignis Penard and Mallomonas punctifera Korshikov at the base, contained taxa from the sections Mallomonas, Striatae, Akrokomae, Annulatae, Torquatae, Punctiferae, and Insignes, all of which have V ribs or well‐developed marginal ribs on the scales. Sister relationships between Mallomonas and Striatae were strongly supported, but interrelations among the remaining sections were not resolved, probably due to inclusion of too few species. Our results suggest that the current classification of the genus Mallomonas at the section level will require some revision. Additional species will need to be added in future analyses.     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Characterization of the crustose red alga Peyssonnelia japonica (Rhodophyta,Gigartinales) and its taxonomic relationship with Peyssonnelia boudouresquei based on morphological and molecular data

The vegetative and reproductive morphology of the crustose red alga Peyssonnelia japonica (Segawa) Yoneshigue was re‐examined based on the holotype specimen and recent collections from various localities in Japan, including the type locality, and Hawaii. This species is characterized by the following features: thallus with appressed margins, perithallial filaments arising from the entire upper surface of each hypothallial cell (the Peyssonnelia rubra‐type), easily separable perithallial filaments in a gelatinous matrix, hypothallial filaments arranged in parallel rows, unicellular rhizoids, hypobasal calcification, gonimoblasts derived mainly from connecting filaments, and spermatangia produced in a series of whorls comprised of one to four paired spermatangia surrounding each central cell (the Peyssonnelia dubyi‐type). In addition to these features, the dimensions of the vegetative and reproductive structures of Peyssonnelia boudouresquei Yoneshigue described from Brazil were consistent with those of P. japonica. Molecular phylogenetic analyses using partial 26S rDNA, rbcL, and cox2‐3 spacer DNA sequences also supported the monophyly of P. japonica (from 16 localities in Japan and one locality in Hawaii) and P. boudouresquei (from two localities in Brazil). Therefore, P. boudouresquei may be a taxonomic synonym of P. japonica. However, considering the relatively high sequence divergences between the two taxa (2.1–2.5% in partial 26S rDNA, 5.9–6.7% in rbcL, and 5.8–6.7% in cox2‐3 spacer), and the relatively limited geographic sampling ranges, we reserve the taxonomic conclusion until further morphological and genetic data of the specimens from other geographic areas connecting Japan and Brazil become available.     

Elucidating the evolution and diversity of Uroglena-like colonial flagellates (Chrysophyceae): polyphyletic origin of the morphotype

ABSTRACT

The Uroglena-like morphotype represents a prototype of a colonial naked chrysophyte, comprising plastid-bearing cells that are arranged as the surface monolayer of the spherical colony. So far, insufficient molecular characterization appears to be the most significant brake on the modern taxonomic revision of this ecologically and morphologically coherent group of organisms. The general aim of this work was to conduct a modern taxonomic revision of Uroglena-like flagellates by using combined molecular, morphological and ultrastructural methodology, complemented by exploring type localities of Uroglena volvox and Uroglenopsis americana in Europe and North America, respectively. On the basis of phylogenetic analysis of concatenated nuclear SSU rDNA and plastid rbcL sequences we show that Uroglena-like colonial flagellates form three genetically and morphologically distinct lineages within the Ochromonadales (Chrysophyceae), distinguished here as Uroglena, Uroglenopsis and Urostipulosphaera gen. nov. The taxonomic status of the other chrysophyte genera with spherical colonies is discussed in light of our findings.     

GLOBAL SYSTEMATIC AND PHYLOGENETIC ANALYSIS OF SARGASSUM IN THE GULF OF MEXICO,CARIBBEAN AND PACIFIC BASIN

Sargassum is one of the most species‐rich genera in the brown algae with over 400 described species worldwide. The bulk of these species occurs in Pacific‐Indian ocean waters with only a small portion found on the Atlantic side of the Isthmus of Panama. Sargassum also has one of the most subdivided and complex taxonomic systems used within the algae. Systematic distinctions within the genus are further complicated by high rates of phenotypic variability in several key morphological characters. Molecular analyses in such systems should allow testing of systematic concepts while providing insights into speciation and evolutionary patterns. Global molecular phylogenetic analyses using both conserved and variable regions of the Rubisco operon (rbcL and rbcL‐IGS‐rbcS) were performed with species from the Gulf of Mexico, Caribbean, and Pacific basin. Results confirm earlier analyses based on rbcL‐IGS‐rbcS from Pacific species at the subgeneric and sectional level while providing additional insights into the systematics and phylogenetics on a global scale. For example, species east of the Isthmus of Panama form a distinct well‐resolved clade within the tropical subgenus. This result in sharp contrast to traditional systematic treatments but provides a window into the evolutionary history of this genus in the Pacific and Atlantic Ocean basins and a possible means to time speciation events.     

GRACILARIOPSIS MCLACHLANII SP. NOV. AND GRACILARIOPSIS PERSICA SP. NOV. OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYCEAE) FROM THE INDIAN OCEAN1

Two new species of Gracilariopsis from the Indian Ocean are proposed—Gracilariopsis (Gp.) mclachlanii Buriyo, Bellorin et M. C. Oliveira sp. nov. from Tanzania and Gracilariopsis persica Bellorin, Sohrabipour et E. C. Oliveira sp. nov. from Iran—based on morphology and DNA sequence data (rbcL gene and SSU rDNA). Both species fit the typical features of Gracilariopsis: axes cylindrical throughout, freely and loosely ramified up to four orders, with an abrupt transition in cell size from medulla to cortex, cystocarps lacking tubular nutritive cells and superficial spermatangia. Nucleotide sequence comparisons of rbcL and SSU rDNA placed both species into the Gracilariopsis clade as distinct species from all the accepted species for this genus, forming a deeply divergent lineage together with some species from the Pacific. The new species are very difficult to distinguish on morphological grounds from other species of Gracilariopsis, stressing the importance of homologous molecular marker comparisons for the species recognition in this character‐poor genus.     

Multigene phylogeny of Synura (Synurophyceae) and descriptions of four new species based on morphological and DNA evidence

We used phylogenetic analyses based on multiple gene sequences (partial nr SSU and LSU rDNA, partial pt LSU rDNA, psaA and rbcL) from 148 strains (including three outgroups) and scale ultrastructure to examine phylogenetic relationships among species of the colonial genera Synura and Tessellaria. The phylogenetic tree based on the combined dataset was congruent with ultrastructural characteristics of the scales. Synura was divided into three major clades, two including species in section Synura, and one representing section Peterseniae. One clade, consisting of seven strains of S. uvella (section Synura), diverged at the base of the genus. The second clade consisted of the remaining species belonging to the section Synura. The third clade, containing organisms in the section Peterseniae and characterized by scales possessing a keel, was monophyletic with strong support values. Based on our findings, S. uvella needs to be in a separate section from other spine-bearing species, and we therefore propose new sectional ranks; Synura, Peterseniae, Curtispinae (presence of body scales with slender spines, tubular scales and caudal scales). We further propose four new species based on phylogenetic analyses and unique scale characters: S. longitubularis sp. nov., S. sungminbooi sp. nov., S. soroconopea sp. nov. and S. lanceolata sp. nov. Lastly, we propose a new genus name, Neotessella, to replace the invalid use of the name Tessellaria.     

Lunachloris lukesovae gen. et sp. nov. (Trebouxiophyceae,Chlorophyta), a novel coccoid green alga isolated from soil in South Bohemia,Czech Republic

Culture collections of microorganisms can still hold undiscovered biodiversity; with molecular techniques, considerable progress has been made in characterizing microalgae which were isolated in the past and misidentified due to a lack of morphological features. However, many strains are still awaiting taxonomic reassessment. Here we analysed the phylogenetic position, morphology and ultrastructure of the strain CCALA 307 previously identified as Coccomyxa cf. gloeobotrydiformis Reysigl isolated in 1987 from field soil in South Bohemia, Czech Republic. Molecular phylogenetic analyses based on SSU rDNA and the plastid rbcL gene revealed that the strain CCALA 307 formed a distinct sister lineage to Neocystis and Prasiola clades within the Trebouxiophyceae. We describe this strain as a new genus and species, Lunachloris lukesovae. Multiple conserved nucleotide positions identified in the secondary structures of the highly variable ITS2 rDNA barcoding marker provide further evidence of the phylogenetic position of Lunachloris. Minute vegetative cells of this newly recognized species are spherical or ellipsoid, with a single parietal chloroplast without a pyrenoid. Asexually, it reproduces by the formation of 2–6 autospores. Since the majority of recent attention has been paid to algae from the tropics or extreme habitats, the biodiversity of terrestrial microalgae in temperate regions is still notably unexplored and even a ‘common’ habitat like agricultural soil can contain new, as yet unknown species. Moreover, this study emphasizes the importance of culture collections of microorganisms even in the era of culture-independent biodiversity research, because they may harbour novel and undescribed organisms as well as preserving strains for future studies.     

DISTRIBUTION,MORPHOLOGY, AND PHYLOGENY OF KLEBSORMIDIUM (KLEBSORMIDIALES,CHAROPHYCEAE) IN URBAN ENVIRONMENTS IN EUROPE1

Klebsormidium is a cosmopolitan genus of green algae, widespread in terrestrial and freshwater habitats. The classification of Klebsormidium is entirely based on morphological characters, and very little is understood about its phylogeny at the species level. We investigated the diversity and phylogenetic relationships of Klebsormidium in urban habitats in Europe by a combination of approaches including examination of field‐collected material, culture experiments conducted in many different combinations of factors, and phylogenetic analyses of the rbcL gene. Klebsormidium in European cities mainly occurs at the base of old walls, where it may produce green belts up to several meters in extent. Specimens from different cities showed a great morphological uniformity, consisting of long filaments 6–9 μm in width, with thin‐walled cylindrical cells and smooth wall, devoid of false branches, H‐shaped pieces, and biseriate parts. Conversely, the rbcL phylogeny showed a higher genetic diversity than expected from morphology. The strains were separated in four different clades supported by high bootstrap values and posterior probabilities. In culture, these clades differed in several characters, such as production of a superficial hydro‐repellent layer, tendency to break into short fragments, and inducibility of zoosporulation. On the basis of the taxonomic information available in the literature, most strains could not be identified unambiguously at the species level. The rbcL phylogeny showed no correspondence with classification based on morphology and suggested that the identity of many species, in particular the type species K. flaccidum (kütz.) P.C. Silva, Mattox et W. H. Blackw., needs critical reassessment.     

Morphology and molecular phylogeny of the marine diatom genus Nagumoea (Bacillariophyceae) from Japan

The canal-bearing diatom genus Nagumoea, described based on only morphological evidence, was tentatively assigned to the order Bacillariales, although its phylogenetic position remained unclear. Because three isolates of Nagumoea (SK002, SK024 and SK053) were successfully established from Japanese coasts, we performed their morphological observations and molecular phylogenetic analyses to discuss the phylogeny and taxonomic position of this genus. Strains SK002 and SK024 were identified as Nagumoea africana, whereas SK053 conformed with Nagumoea serrata. There was high interspecific divergence between N. africana and N. serrata in the rbcL sequences (8.03–8.17%), indicating their distinctness. Furthermore, intraspecific variations were detected within N. africana (2.35%) in the rbcL, implying its cryptic diversity. The maximum likelihood and Bayesian phylogenetic trees inferred from the plastid rbcL, psbC and nuclear 18S rDNA genes recovered Nagumoea as monophyletic with strong statistical support and embedded within an unresolved, poorly supported lineage containing Achnanthes, Craspedostauros, Staurotropis and Undatella in the canal-bearing order Bacillariales (= the family Bacillariaceae). Although the constrained tree based on the monophyly of Nagumoea and the other canal-bearing clade (Surirellales and Rhopalodiales) was statistically rejected by the topology tests, the phylogenetic position of Nagumoea with other Bacillarialean members remains equivocal. The possession of two plastids positioned fore and aft, observed in the present study, and lack of keel, typical of the Bacillariales, indicate the possibility of Nagumoea being part of the ingroup of the Bacillariales or its closely related outgroup.