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Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): Evidence from nuclear SSU rDNA and plastidial rbcL genes
Abstract
Xanthophyceae are a group of heterokontophyte algae. Few molecular studies have investigated the evolutionary history and phylogenetic relationships of this class. We sequenced the nuclear-encoded SSU rDNA and chloroplast-encoded rbcL genes of several xanthophycean species from different orders, families, and genera. Neither SSU rDNA nor rbcL genes show intraspecific sequence variation and are good diagnostic markers for characterization of problematic species. New sequences, combined with those previously available, were used to create different multiple alignments. Analyses included sequences from 26 species of Xanthophyceae plus three Phaeothamniophyceae and two Phaeophyceae taxa used as outgroups. Phylogenetic analyses were performed according to Bayesian inference, maximum likelihood, and maximum parsimony methods. We explored effects produced on the phylogenetic outcomes by both taxon sampling as well as selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on a data set including both SSU rDNA and rbcL sequences. Trees obtained in this study show that several currently recognized xanthophycean taxa do not form monophyletic groups. The order Mischococcales is paraphyletic, while Tribonematales and Botrydiales are polyphyletic even if evidence for the second order is not conclusive. Botrydiales and Vaucheriales, both including siphonous taxa, do not form a clade. The families Botrydiopsidaceae, Botryochloridaceae, and Pleurochloridaceae as well as the genera Botrydiopsis and Chlorellidium are polyphyletic. The Centritractaceae and the genus Bumilleriopsis also appear to be polyphyletic but their monophyly cannot be completely rejected with current evidence. Our results support morphological convergence at any taxonomic rank in the evolution of the Xanthophyceae. Finally, our phylogenetic analyses exclude an origin of the Xanthophyceae from a Vaucheria-like ancestor and favor a single early origin of the coccoid cell form.
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... Species of filamentous Tribonema are common among floating mats in still water, particularly in late winter. Coccoid and filamentous species have been isolated from terrestrial habitats in Antarctica (Andreoli et al. 1999;Broady 1976;Maistro et al. 2007;Negrisolo et al. 2004;Rybalka et al. 2009and other citations therein). The most conspicuous forms in nature are species of the siphonous genera Botrydium and Vaucheria. ...
... Current formal classification of Xanthophyceae does not reflect results obtained by modern molecular phylogenetic studies. Indeed, the latter approach has revealed that many orders, families, and genera currently defined by morphological characters (Ettl 1978) are paraphyletic or even polyphyletic and do not form clades Bailey and Andersen 1998;Maistro et al. 2007Maistro et al. , 2009Negrisolo et al. 2004;Potter et al. 1997;Rybalka et al. 2009;Zuccarello and Lokhorst 2005). A modern biological classification must be based on a rigorous phylogenetic approach and every taxonomic unit should be a monophyletic group. ...
... Adl et al. (2005) revised the classification of Xanthophyceae and assigned all taxa to Tribonematales except for Vaucheria that was assigned to Vaucheriales. This classification is also strongly contradicted by phylogenetic analyses (Maistro et al. 2007(Maistro et al. , 2009Negrisolo et al. 2004). ...
The Xanthophyceae is a clade of stramenopilan photoautotrophs containing about 118 genera and 600 species. Morphology ranges from free-living or attached unicells to colonies and unbranched or branched filaments and siphons. A large majority are found in freshwater and soil, while some occur in brackish and marine habitats. Although abundant growth of a few species can occur in nature, none are known to be of practical importance. They are characterized by possession of chlorophylls a, c
1, and c
2 and a range of xanthophylls, but not fucoxanthin, in generally yellowish-green, discoidal, parietal chloroplasts. Thylakoids are in groups of three, and most species investigated have a single thylakoid forming a girdle band around the periphery of the chloroplast. Chloroplasts are surrounded by chloroplast endoplasmic reticulum. Pyrenoids, when present, are typically semi-immersed and are not associated with granules of storage products. A cell wall consisting of two overlapping parts occurs in some coccoid and filamentous species. Reproduction is generally asexual but some, e.g., Vaucheria, exhibit sexual reproduction. The taxonomic status of a significant number of species is uncertain, especially those that are rarely observed, e.g., species of Chloramoebales, Heterogloeales, and Rhizochloridales. Transfer of species to the Eustigmatophyceae and other groups is likely. There are molecular phylogenetic data for fewer than 20% of species. Four major clades are recognized. Two of these contain both coccoid and filamentous species. Many traditional orders, families, and genera are paraphyletic or polyphyletic. It is presently convenient to retain the traditional classification of seven orders based on morphology until these difficulties are resolved following the inclusion of more species in phylogenetic analyses.
... Species of filamentous Tribonema are common among floating mats in still water, particularly in late winter. Coccoid and filamentous species have been isolated from terrestrial habitats in Antarctica (Andreoli et al. 1999;Broady 1976;Maistro et al. 2007;Negrisolo et al. 2004;Rybalka et al. 2009and other citations therein). The most conspicuous forms in nature are species of the siphonous genera Botrydium and Vaucheria. ...
... Current formal classification of Xanthophyceae does not reflect results obtained by modern molecular phylogenetic studies. Indeed, the latter approach has revealed that many orders, families, and genera currently defined by morphological characters (Ettl 1978) are paraphyletic or even polyphyletic and do not form clades Bailey and Andersen 1998;Maistro et al. 2007Maistro et al. , 2009Negrisolo et al. 2004;Potter et al. 1997;Rybalka et al. 2009;Zuccarello and Lokhorst 2005). A modern biological classification must be based on a rigorous phylogenetic approach and every taxonomic unit should be a monophyletic group. ...
... Adl et al. (2005) revised the classification of Xanthophyceae and assigned all taxa to Tribonematales except for Vaucheria that was assigned to Vaucheriales. This classification is also strongly contradicted by phylogenetic analyses (Maistro et al. 2007(Maistro et al. , 2009Negrisolo et al. 2004). ...
The Xanthophyceae is a clade of stramenopilan photoautotrophs containing about 118 genera and 600 species. Morphology ranges from free-living or attached unicells to colonies and unbranched or branched filaments and siphons. A large majority are found in freshwater and soil, while some occur in brackish and marine habitats. Although abundant growth of a few species can occur in nature, none are known to be of practical importance. They are characterized by possession of chlorophylls a, c
1, and c
2 and a range of xanthophylls, but not fucoxanthin, in generally yellowish-green, discoidal, parietal chloroplasts. Thylakoids are in groups of three, and most species investigated have a single thylakoid forming a girdle band around the periphery of the chloroplast. Chloroplasts are surrounded by chloroplast endoplasmic reticulum. Pyrenoids, when present, are typically semi-immersed and are not associated with granules of storage products. A cell wall consisting of two overlapping parts occurs in some coccoid and filamentous species. Reproduction is generally asexual but some, e.g., Vaucheria, exhibit sexual reproduction. The taxonomic status of a significant number of species is uncertain, especially those that are rarely observed, e.g., species of Chloramoebales, Heterogloeales, and Rhizochloridales. Transfer of species to the Eustigmatophyceae and other groups is likely. There is molecular phylogenetic data for fewer than 20 % of species. Four major clades are recognized. Two of these contain both coccoid and filamentous species. Many traditional orders, families, and genera are paraphyletic or polyphyletic. It is presently convenient to retain the traditional classification of seven orders based on morphology until these difficulties are resolved following the inclusion of more species in phylogenetic analyses.
... Molecular studies have also revealed the sister group relationships of the Phaeophyceae, the Phaeothamniophyceae , and the Xanthophyceae (the yellow-green algae) (Bailey et al. 1998; Daugbjerg & Guillou 2001; Daugbjerg & Henriksen 2001) within the stramenopiles. Studies have also investigated generic relationships with the Xanthophyceae (Negrisolo et al. 2004) showing that some genera are not monophyletic. At lower levels, molecular data have elucidated the species relationships within the xanthophycean alga Vaucheria (Andersen & Bailey 2002) confirming intrageneric sectional classification. ...
... A nomenclatural history of the genus is summarized in Ioriya (1986) and Lokhorst (2003). In earlier molecular studies of the heterokont algae some aspects of the phylogenetic position of Tribonema relative to other xanthophytes have been revealed using 18SrRNA and/ or plastid-encoded rbcL gene sequences (Daugbjerg & Andersen 1997; Bailey & Andersen 1998; Negrisolo et al. 2004). It was shown that there are conflicts between the traditional xanthophycean ordinal classification (based on thallus-type) and molecular phylogenies (Bailey & Andersen 1998; Negrisolo et al. 2004), for example molecular data do not support the monophyly of the traditional order Tribonematales containing Tribonema, Xanthonema and Heterococcus. ...
... In earlier molecular studies of the heterokont algae some aspects of the phylogenetic position of Tribonema relative to other xanthophytes have been revealed using 18SrRNA and/ or plastid-encoded rbcL gene sequences (Daugbjerg & Andersen 1997; Bailey & Andersen 1998; Negrisolo et al. 2004). It was shown that there are conflicts between the traditional xanthophycean ordinal classification (based on thallus-type) and molecular phylogenies (Bailey & Andersen 1998; Negrisolo et al. 2004), for example molecular data do not support the monophyly of the traditional order Tribonematales containing Tribonema, Xanthonema and Heterococcus. In these studies only one or two specimens of Tribonema were inves- tigated. ...
2005. Molecular phylogeny of the genus Tribonema (Xanthophyceae) using rbcL gene sequence data: monophyly of morphologically simple algal species. Phycologia 44: 384–392. The genus Tribonema (Xanthophyceae) is a common component of many freshwater ecosystems. It is distinguished by its H-shaped bipartite walls and filamentous habit. Species identification has relied on few morphological characters, especially filament size and plastid numbers. We approached the question of interspecific relationships within the genus by sequencing the plastid-encoded rbcL gene of 10 species of Tribonema. We sequenced multiple samples of seven of these species from throughout Europe. Our data suggest that T. hormidioides (Vischer) Lokhorst is closely related to the genus Xanthonema from which it has recently been removed, and should be returned to that genus. Most other samples of Tribonema belong to a single clade, which includes Bumilleriopsis filiformis Vischer. None of the repeatedly sampled species of Tribonema, except T. microchloron Ettl (two samples), are monophyletic. Trends in character divergence within clades were seen with a clade containing mostly species with wide filaments and another with mostly thin filaments. The question of gene trees vs organismal phylogeny is discussed. Our data suggest that presently identified morpho-species of Tribonema may not be useful as evolutionary units or in biodiversity assessment of freshwater ecosystems.
... In their scheme, Tribonematales contains all species except for those of siphonous Vaucheria which are placed in the Vaucheriales. Our previous molecular data (Negrisolo et al., 2004) suggested that Vaucheria is not sister taxon of all other Xanthophyceans. ...
... The traditional Tribonematales has been found to be para/polyphyletic in all molecular analyses performed to date Negrisolo et al., 2004;Zuccarello and Lokhorst, 2005). A close phylogenetic relationship was identified between some uniseriate filamentous species of Tribonema and Xanthonema and members of the coccoid genus Bumilleriopsis (Negrisolo et al., 2004;Zuccarello and Lokhorst, 2005). ...
... The traditional Tribonematales has been found to be para/polyphyletic in all molecular analyses performed to date Negrisolo et al., 2004;Zuccarello and Lokhorst, 2005). A close phylogenetic relationship was identified between some uniseriate filamentous species of Tribonema and Xanthonema and members of the coccoid genus Bumilleriopsis (Negrisolo et al., 2004;Zuccarello and Lokhorst, 2005). In contrast, species of Heterococcus were closely related to coccoid forms of Mischococcus or Chlorellidium (Negrisolo et al., 2004). ...
Tribonematales is an order of filamentous algae in the class Xanthophyceae (Heterokonta). Few molecular studies, all with a limited taxon sampling, have previously investigated its evolutionary history and phylogenetic relationships. We sequenced the chloroplast-encoded rbcL and psaA genes of several tribonematalean species and of several coccoid and siphonous forms that previous studies revealed to be strictly related to Tribonematales. Multiple alignments included mostly new sequences obtained from 42 taxa. Phylogenetic reconstructions were performed using the maximum likelihood method. The rbcL and psaA data sets were analyzed independently and combined in a single multiple alignment. Neither rbcL nor psaA genes showed intraspecific sequence variation. The former proved to be a better diagnostic marker than the latter for characterization of species. We explored effects produced on phylogenetic outcomes by selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on the combined data set. There is strong statistical support for trees that show several currently recognized taxonomic groups to be polyphyletic. The siphonous orders Botrydiales and Vaucheriales do not form a clade. Botrydiales and Tribonematales are polyphyletic as are the families Botrydiaceae, Centritractaceae and Tribonemataceae and the genera Xanthonema and Bumilleriopsis. We tentatively define new boundaries of the Tribonematales to include both coccoid and filamentous species having a bipartite cell wall and also the siphonous members of the genus Botrydium. Also, our results support morphological convergence at all taxonomic ranks in the evolution of the Xanthophyceae.
... that the filamentous species with H-shaped cell walls constitute one lineage of the Tribonematales and those with entire cell walls (e.g., Heterococcus) constitute a second lineage, but this finding has not yet been put forward in a classification (Bailey et al., 1998;Adl et al., 2005). Also the monophyly of Vaucheriales is questionable as well as the appropriateness of Botrydiales (Negrisolo et al., 2004;Maistro et al., 2009). Many investigated morphologically defined genera, for example, Bumilleriopsis, Botrydiopsis, Chlorellidium, Xanthonema, and Ophiocytium, were found not to be monophyletic (Negrisolo et al., 2004;Maistro et al., 2007Maistro et al., , 2009). ...
... Also the monophyly of Vaucheriales is questionable as well as the appropriateness of Botrydiales (Negrisolo et al., 2004;Maistro et al., 2009). Many investigated morphologically defined genera, for example, Bumilleriopsis, Botrydiopsis, Chlorellidium, Xanthonema, and Ophiocytium, were found not to be monophyletic (Negrisolo et al., 2004;Maistro et al., 2007Maistro et al., , 2009). Using DNA sequence data, it has also been shown that criteria for species delimitation and species borders should be re-evaluated within many other genera of Xanthophyceae (Zuccarello and Lokhorst, 2005;Rybalka et al., 2009). ...
The Xanthophyceae, Eustigmatophyceae, and Raphidophyceae are three independent classes of stramenopile algae (Heterokontophyta or Ochrophyta); they are not closer related with each other. Most Xanthophytes are unicellular or colonial coccoid algae, others from multicellular filaments and or exhibit thalli composed of multinucleate siphons. The Eustigmatophyceae comprises only coccoid members which are very difficult to distinguish from the coccoid Xanthophytes. Freshwater Raphidophytes are rather distinct, because they form flagellated vegetative stages. The color of Xanthophytes and Eustigmatophytes is yellowish green due to the absence of the brown fucoxanthin, present in Raphidophytes and other stramenopile algae. Only the Eustigmatophytes lack chlorophyll c. Many Xanthophytes and Eustigmatophytes share that they predominately occur in terrestrial habitats, e.g. soil, representing a small group of terrestrial algae with their plastids obtained from an ancestral red alga by secondary symbiosis. For Raphidophytes only three genera are recognized in freshwater yet and they are observed within the plankton.
... Recent genetic investigations indicate that the genus Botrydiopsis is polyphyletic and therefore should be divided (neGriSolo et al. 2004;MaiStro et al. 2009). For B. pyrenoidosa it is even uncertain if it should be placed at the basis of the Xanthophyceae or if it belongs to a different group of algae because it is genetically so different (Fig. 8,9). ...
... In current investigations already a new name is proposed: Polykaryon pyrenoidosum (MiSner 2004; J.C. Bailey, personal comment, in press). For B. constricta it was shown that it certainly belongs to the Xanthophyceae but it groups at a distinct position far from the other species of the genus Botrydiopsis (neGriSolo et al. 2004;MaiStro et al. 2009). According to our investigations B. constricta together with the new isolates from New Zealand (in concordance with noViS et al. 2008) and E. fraunhoferiana lay close together. ...
A new aerophytic species of the xanthophycean genus Excentrochloris – Excentrochloris fraunhoferi-ana Hofbauer, Gärtner, rennebartH, Sedlbauer, Mayer et breuer from a building surface is described. Light microscopically investigation and sequence analyses showed a clear relationship of the new alga to Botrydiopsis constricta broady. Cytomorphology and reproduction of the new species was investigated on cultures. E. fraun-hoferiana differs from the type species E. gigas by bigger size of adult vegetative cells with obligate thickening of wall, and none amoeboid zoospores with one chloroplast. Adult zoospores are peripherically arranged in the spo-rangia. The relation to Botrydiopsis constricta is discussed in comparison with characters of E. fraunhoferiana.
... 21 Among the most promising commercial applications for algae are the production of lipids for biodiesel production 1,2,22-24 and high value lipids for human nutrition. 25,26 H. sp. DN1 produces large amounts of intracellular lipids, so it is a candidate for commercial lipid production. ...
... consequences such as overfishing the oceans and risk of contaminants. 25,27 Even in cases where companies purify their fish oil another high-cost process is added to the manufacture of a EPA-supplying product. Companies V-pure (Bedford, UK) and Deva (Chelsea, AL) have already been established to supply EPA from algae as people are demanding a commercial alternative to fish oil. ...
A new strain of yellow-green algae (Xanthophyceae, Heterokonta), tentatively named Heterococcus sp. DN1 (UTEX accession number UTEX ZZ885), was discovered among snow fields in the Rocky Mountains. Axenic cultures of H. sp. DN1 were isolated and their cellular morphology, growth, and composition of lipids were characterized. H. sp. DN1 was found to grow at temperatures approaching freezing to accumulate large intracellular stores of lipids. H. sp. DN1 produces the highest quantity of lipids when grown undisturbed with high light in low temperatures. Of particular interest was the accumulation of eicosapentaenoic acid, known to be important for human nutrition, and palmitoleic acid, known to improve biodiesel feedstock properties. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
... chrysophyceans: the synurophyceans Synura curtispina, S. sphagnicola, S. uvella, and Mallomonas asmundae (based on Andersen (2004), who showed a sister group relationship between synurophyceans and chrysophyceans). Xanthophyceans: the brown algae Stichogloea globosa, Phaeoschizochlamys mucosa, and Phaeothamnion confervicola (based on Negrisolo et al. (2004), who also used brown algae). chlamydomonads: Carteria obtusa and Pseudocarteria mucosa (based on Nozaki et al. 2003). ...
... The genus Botrydiopsis is polyphyletic, occurring in at least four separate lineages within the Xanthophyceae ( fig. 5) (Negrisolo et al. 2004). Two of these lineages have austral representatives. ...
Chrysocapsa flavescens (Chrysophyceae), Botrydiopsis constricta (Xanthophyceae), and Chlamydomonas pseudogloeogama (Chlorophyceae) were recovered from the alpine zone and constitute new records for New Zealand. Pseudococcomyxa simplex and Stichococcus sp. were also found and subjected to molecular phylogenetic analysis for the first time. Klebsormidium dissectum was isolated for the first time from the alpine zone. RbcL sequences suggest a mix of range restrictions in these taxa, from very little (C. pseudogloeogama, highly similar to a Czech strain) to a well‐supported clade of New Zealand strains (K. dissectum). This mix of biogeographic patterns in these “soft algae” reflects those of the diatom flora, although evidence for widespread range restriction is presently less compelling. Despite the challenges posed by the difficulty of undertaking surveys of microbes, and a paucity of molecular data for many taxa, we suggest Baas Becking's widely quoted biogeographical tenet can be reconciled with both biogeographic structure and long‐distance dispersal under a simple probabilistic framework.
... The siphonous yellow-green alga Vaucheria de Candolle frequently occurs in freshwater, marine, and terrestrial habitats forming greenish mats, often appearing as velvety, felt-like coatings on various substrata. The genus consists of nearly 70 species worldwide; in Europe about 40 species are known, which are distributed mainly in freshwaters and terrestrial habitats (Rieth 1980 ). Within the Xanthophyceae , siphonous taxa are found in the orders Vaucheriales and Botrydiales, which however are not placed in a single clade (Negrisolo et al. 2004; Potter et al. 1997 ). Initially, it was supposed that the Xanthophyceae originated from a siphonous Vaucheria-like ancestor (Bailey and Anderson 1998 ). ...
... Initially, it was supposed that the Xanthophyceae originated from a siphonous Vaucheria-like ancestor (Bailey and Anderson 1998 ). This hypothesis was rejected by Negrisolo et al. (2004), who inferred from their results obtained by data of the large subunit of the ribulosebisphosphate carboxylase M. Schagerl (&) Á M. Kerschbaumer Department of Marine Biology, University of Vienna, Althanstraße 14, 1090 Vienna, Austria e-mail: Michael.schagerl@univie.ac.at gene (rbcL) and sequence comparisons of the smallsubunit (SSU) rDNA, that a basic unicellular organism represents the primary Xanthophyceae. Early reports of Vaucheria mainly dealt with its morphology, taxonomy, and the development of sexual organs (Götz 1897; Heering 1921; Heidinger 1908; Oltmanns 1895; Walz 1866). ...
In this study, the yellow-green alga Vaucheria was collected from 86 sites with different environmental characteristics. A principal component analysis of the sites revealed
a model with four principal components explaining 64.7% of the total variance in the data set. The principal components were
interpreted as nutrient enrichment, ions, current, and buffer capacity. In total, seven Vaucheria species were found: V. taylorii, V. debaryana, V. sessilis, V. geminata, V. terrestris, V. walzii, and V. woroniniana. V. taylorii was noted only three times, V. terrestris occured exclusively at terrestrial sites, the remaining taxa were frequent. Vaucheria seems to have its optimal growth at lower temperatures; in the cold season thick covers were observed, which exceeded the
abundance in summer. A discriminant analysis revealed that growth types are highly explained by current; high flow velocity
favors the development of pads. Species occurrence along artificial gradients consisting of the obtained principal components
were analyzed simultaneously by means of a canonical correspondence analysis. V. sessilis occurred widely with no preferences, but three species showed a distinct pattern within the gradient analysis: V. woroniniana mainly occurred at elevated pH and total alkalinity, V. walzii was favored by increased ion contents and slow current, and V. geminata clearly preferred unpolluted sites with decreased pH and alkalinity values. This species may be used as an indicator species
for unpolluted locations, however some taxonomic uncertainties remain to be first resolved.
... Due to the diversity in Heterokonta it was later raised to infrakingdom (Cavalier-Smith 1997) with two main groups; Ochrophyta (Cavalier-Smith 1986 ) consisting mainly of autotrophic heterokonts, and a purely heterotrophic group, which was again further subdivided in two phyla: Bigyra and Pseudofungi (Cavalier-Smith and Chao 2006). Due to their diversity and ecological significance , a number of molecular phylogenetic studies have been performed on separate heterokonts groups; predominantly single gene inferences (Andersen et al. 1999; Guillou et al. 1999; Moriya et al. 2000; Negrisolo et al. 2004; Potter et al. 1997). Investigations on the global phylogeny of heterokonts are limited to relatively few studies of nuclear-encoded ribosomal RNAs (rDNA) or chloroplast-encoded rbcL (ribulose-1,5- bisphosphate carboxylase/oxygenase) (Ben Ali et al. 2001; Daugbjerg and Guillou 2001; Edvardsen et al. 2007). ...
... This topology is contradictory to Figures 1 and 2 as well as Smith and Chao (2006), where the Bacillariophyceae (together with Bolidophyceae ) formed a basal group within Ochro- phyta.Figure 3 shows Phaeothamniophyceae cluster strongly with Xanthophyceae. These two clades may belong to a single class as earlier suggested (Goertzen and Theriot 2003; Negrisolo et al. 2004). However, more data from deeply diverging xanthophytes are needed to test this scenario. ...
Nucleotide ssu and lsu rDNA sequences of all major lineages of autotrophic (Ochrophyta) and heterotrophic (Bigyra and Pseudofungi) heterokonts were combined with amino acid sequences from four protein-coding genes (actin, beta-tubulin, cox1 and hsp90) in a multigene approach for resolving the relationship between heterokont lineages. Applying these multigene data in Bayesian and maximum likelihood analyses improved the heterokont tree compared to previous rDNA analyses by placing all plastid-lacking heterotrophic heterokonts sister to Ochrophyta with robust support, and divided the heterotrophic heterokonts into the previously recognized phyla, Bigyra and Pseudofungi. Our trees identified the heterotrophic heterokonts Bicosoecida, Blastocystis and Labyrinthulida (Bigyra) as the earliest diverging lineages. A separate analysis of the phototrophic lineages, by adding the rbcL gene, further resolved the Ochrophyta lineages by increased support for several important nodes. Except for the positioning of Chrysophyceae, Eustigmatophyceae, Raphidophyceae and Pinguiophyceae, all main branches of Ochrophyta were resolved. Our results support the transfer of classes Dictyochophyceae and Pelagophyceae from subphylum Phaeista to Khakista. Based on all our trees, in combination with current knowledge about ultrastructure of heterokonts we suggest that a more advanced flagellar apparatus originated at one occasion in the ancestor of Phaeista whereas, Khakista independently reduced their flagellar apparatus and gained chlorophyll c(3).
... Benthic diatom Cylindrotheca closterium identified through both traditional morphological method and molecular phylogenetic method. For the molecular phylogenetic analyses, the 18S rRNA and the ribulose bisphosphate carboxylase large subunit coding gene (rbcL) were sequenced (Negrisolo et al., 2004). PCR has been used to effectively determine the presence or absence of an organism in a sample. ...
... Botrydiopsis callosa (AJ579340.1), Heterococcus pleurococcoides/fuornensis/chodatii (Xanthophyceae; Broady 1976;Negrisolo et al. 2004), Chloroidium saccharophilum (KX024691.1; Darienko et al. 2010), Lobosphaera sp. ...
Melting snow fields are an extremophilic habitat dominated by closely related Chlamydomonadaceae (Chlorophyta). Microscopy-based classification of these cryophilic microalgae is challenging and may not reveal the true diversity. High-throughput sequencing (HTS) allows for a more comprehensive evaluation of the community. However, HTS approaches have been rarely used in such ecosystems and the output of their application has not been evaluated. Furthermore, there is no consensus on the choice for a suitable DNA marker or data processing workflow. We found that the correct placement of taxonomic strings onto OTUs strongly depends on the quality of the reference databases. We improved the assignments of the HST data by generating additional reference sequences of the locally abundant taxa, guided by light microscopy. Furthermore, a manual inspection of all automated OTU assignments, oligotyping of the most abundant 18S OTUs, as well as ITS2 secondary structure analyses were necessary for accurate species assignments. Moreover, the sole use of one marker can cause misleading results, either because of insufficient variability within the locus (18S) or the scarcity of reference sequences (ITS2). Our evaluation reveals that HTS output needs to be thoroughly checked when the studied habitats or organisms are poorly represented in publicly available databases. We recommend an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two- molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended.
... There are many cases of incongruences between phylogenies from nuclear-and plastid (organellar) encoded markers, most likely due to different evolutionary dynamics of nuclear and organellar genomes, e.g. in the green algae (e.g. De Cambiaire et al., 2007;Lemieux et al., 2014;Turmel et al., 2015), Stramenopiles (Xanthophyceae; Negrisolo et al., 2004) andhigher plants (e.g. Small et al., 2004;Yu et al., 2013). ...
There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.
... There are many cases of incongruences between phylogenies from nuclear-and plastid (organellar) encoded markers, most likely due to different evolutionary dynamics of nuclear and organellar genomes, e.g. in the green algae (e.g. De Cambiaire et al., 2007;Lemieux et al., 2014;Turmel et al., 2015), Stramenopiles (Xanthophyceae; Negrisolo et al., 2004) andhigher plants (e.g. Small et al., 2004;Yu et al., 2013). ...
... The traditional ordinal classification of xanthophyceans, while somewhat unsatisfactory, is based on these levels of structural organisation. Molecular genetic evidence now suggests that the traditional classification is artificial and displays polyphyly at the ranks of order, family, and genus (Negrisolo et al. 2004). ...
... Recent investigations (e.g. Bailey & Andersen, 1998;Negrisolo et al., 2004;Zuccarello & Lokhorst, 2005) have shown that the traditional, morphologically-based, classification of the Xanthophyceae, is often incongruent with molecular data and that a new classification system is needed. For example, molecular data do not support the monophyly of the traditional order Tribonematales, containing Tribonema Derbe`s et Solier, Xanthonema Silva and Heterococcus Chodat but ultrastructural investigations have been important for determining the taxonomic and phylogenetic relationships in the algae. ...
The ultrastructure of a Xanthonema strain featuring multinucleate cells was investigated by transmission electron microscopy. An important specific feature of the organisation of the photosynthetic apparatus in this strain is its association with mitochondrial profiles. The chloroplast girdle is composed of two different U-shaped lamellae, one peripheral and one subcentral. Multinuclearity is observed as often as the uninucleate state. The transition from the uninucleate to the multinucleate stage is connected to disturbances in the normal division pattern of the parietal chloroplast-mitochondria complex during interphase. As a result mitosis is not coordinated with cytokinesis. The return to the uninucleate stage occurs as a result of asynchronous cytokinesis or by aplanospore formation. Mitosis is of the semi-closed type, as in Tribonema. Centrioles replicate in early interphase, after the end of karyokinesis and progeny nuclei separate with the aid of CER invagination. Filament fragmentation takes place between neighbouring cells where two U-shaped segments adjoin, resulting in fragment ends being rounded rather than ‘zweispitzig’. The taxonomic significance of various ultrastructural features for the classification of filamentous Xanthophyta is discussed.
... Clones from an Arctic (S-M2) and an Antarctic (A-S1) community were sequenced and revealed distinct communities. Clones from the Svalbard Midtre Lovénbreen community were all closely related to Stramenopiles Xanthophyceae organisms, with the majority of the clones (82.5 %) having 98-99 % identity to Botrydiopsis constricta rbcL sequences (accession number: AJ579566, Negrisolo et al. 2004). Clones from the Antarctic Signy Island community contained sequences related to a greater range of taxa, including those from Stramenopiles and Haptophyceae (Fig. 5). ...
Microorganisms have a crucial role to play in the cycling of nutrients within glacial environments. These systems are often nutrient-limited, and so biogeochemical reactions, which ensure the availability of nutrients for microbial communities, are critical for the maintenance of these systems. This study uses molecular biology to characterise the supraglacial cryoconite microbial communities that are capable of cycling carbon and nitrogen in a range of glacial environments. Organisms with the potential to photosynthesise were identified, including Cyanobacteria, Actinobacteria, Betaproteobacteria, Stramenopiles and Haptophyceae. Organisms with the potential to perform nitrification and denitrification processes were also identified and featured Betaproteobacteria, Alphaproteobacteria, Thaumarchaeota and Cyanobacteria. While it is unlikely that the chemical and physical parameters of the supraglacial environment will facilitate optimal rates of all of the nitrogen-related biogeochemical processes, the transport of these cryoconite communities to downstream locations, where more favourable conditions may prevail, will perhaps provide a valuable inoculation of microorganisms with the genetic potential to catalyse these reactions elsewhere.
... The sequences revealed the probable monophyletic origin of the genus and its basal position within the Xanthophyceae, which was distinct from other filamentous members (e.g. Tribonema, Vaucheria)12131415 . We used molecular phylogenetics, especially within the framework of the monophyletic species concept161718, to evaluate 33 culture strains identified as Heterococcus (Figure 2). ...
Background
Heterococcus is a microalgal genus of Xanthophyceae (Stramenopiles) that is common and widespread in soils, especially from cold regions. Species are characterized by extensively branched filaments produced when grown on agarized culture medium. Despite the large number of species described exclusively using light microscopic morphology, the assessment of species diversity is hampered by extensive morphological plasticity.
Results
Two independent types of molecular data, the chloroplast-encoded psbA/rbcL spacer complemented by rbcL gene and the internal transcribed spacer 2 of the nuclear rDNA cistron (ITS2), congruently recovered a robust phylogenetic structure. With ITS2 considerable sequence and secondary structure divergence existed among the eight species, but a combined sequence and secondary structure phylogenetic analysis confined to helix II of ITS2 corroborated relationships as inferred from the rbcL gene phylogeny. Intra-genomic divergence of ITS2 sequences was revealed in many strains. The ‘monophyletic species concept’, appropriate for microalgae without known sexual reproduction, revealed eight different species. Species boundaries established using the molecular-based monophyletic species concept were more conservative than the traditional morphological species concept. Within a species, almost identical chloroplast marker sequences (genotypes) were repeatedly recovered from strains of different origins. At least two species had widespread geographical distributions; however, within a given species, genotypes recovered from Antarctic strains were distinct from those in temperate habitats. Furthermore, the sequence diversity may correspond to adaptation to different types of habitats or climates.
Conclusions
We established a method and a reference data base for the unambiguous identification of species of the common soil microalgal genus Heterococcus which uses DNA sequence variation in markers from plastid and nuclear genomes. The molecular data were more reliable and more conservative than morphological data.
... observed in these soils need further characterization. Molecular genetical studies (NEGRISOLO et al. 2004), incorporating Antarctic isolates of Botrydiopsis morphotypes, suggest the genus is polyphyletic. ...
A description is provided of the distribution of algae amongst terrestrial and hydro-terrestrial habitats at Victoria Valley, Garwood Valley and Cape Geology (77-78°S, 161-164°E), southern Victoria Land, Antarctica. 358 samples were taken from 12 distinct terrestrial and hydro-terrestrial habitats, with an emphasis on the former. More than 82 species were recorded including eight new records for southern Victoria Land. Data for presence or absence of each species within each sample was subjected to Two Way Indicator Species Analysis (TWINSPAN) which resulted in the recognition of eight groups of samples with different species associations. Particularly distinctive were associations of terrestrial chasmoendolithic and epilithic algae and those in dry soils of Victoria Valley which either lacked visible algal growths or had thin algal crusts. Four TWINSPAN groups each accommodated samples mostly from just one of these habitats. In contrast, a single TWINSPAN group accommodated the majority of samples from several hydro-terrestrial habitats, water-flushed soils and moss cushions. Restriction in duration of liquid water availability results in development of distinct species associations.
... It was suggested that chloroplast DNA, being only maternally inherited, may not provide the ideal molecular tool and analysis of nuclear DNA and more detailed morphological studies will be needed to systematically annotate the genus Vaucheria (Andersen and Bailey, 2002). However, rbcL sequences together with sequences for small ribosomal subunits (SSU rDNA) proved to be useful as molecular markers to distinguish Vaucheriales as a distinct order in a class Xantophyceae, including mostly unicellular and colonial algae, but also several multinucleate siphonous forms and multicellular filamentous species (Negrisolo et al., 2004). Vaucheria litorea was first described and appointed as a specific species by a Swedish botanist Carl Adolph Agardh in 1821. ...
Sea slugs of the genus Elysia (e.g. E. chlorotica) are known for their ability to incorporate chloroplasts from the yellow-green alga Vaucheria litorea. These “kleptoplasts” stay active in the digestive tract of the sea slug for several months. Chloroplasts from Vaucheria litorea are also reported to be significantly more stable after in vitro isolation than chloroplasts of other algae or of higher plants. In organello assays with isolated chloroplasts are used in studies on photosynthetical and biochemical processes in these organelles, chloroplast-nucleus communication, plant development and plant responses to environmental changes. The major limiting factor of the chloroplast in organello systems is the rapid decline in function and intactness of the plastids. This study is investigating the stability and longevity of chloroplasts isolated from V. litorea and its relative V. bursata in comparison to an angiosperm Pisum sativum. The structural intactness of the chloroplasts was investigated by phase contrast microscopy and the overall transcriptional activities were analyzed by run-on transcription assays. The recently completed sequence of the plastid genome from V.litorea has made it possible to investigate also the specific changes in isolated chloroplasts on the transcriptional level. The expression patterns of chloroplast-encoded genes trnE, rrn23, rrn16, rbcL, psbD, psbA and psaA directly after isolation and 4 hours post-isolation time were analyzed using dot blot hybridization. The ability of isolated chloroplasts to incorporate 35S-methionine into de novo synthesized proteins at 0, 4 and 24 hours after isolation was tested by translational assays. The chloroplasts of Vaucheria litorea were both transcriptionally and translationally stable over an extended period of time. However, the culturing limitations and low plastid yields diminish the potential of this alga as a chloroplast donor for in organello assays. Chloroplasts of Pisum sativum, previously suspected to be more unstable, did not show a decline in transcription rate until 4 hours after isolation. The decrease in incorporation of 35S-methionine between time 0 and 4 hours after isolation was only minor, between 4 and 24 hours more significant but the radioactive signal was still readily detectable at 24 hours. Pisum sativum has simple growth requirements and offers high yields of isolated chloroplasts. Chloroplasts from this plant should therefore be in the centre of further investigations into their suitability to express foreign genes and their potential for in organello assays.
... relationship (with posterior probability 1.0) with Chrysomerophyceae representative Giraudyopsis stellifera, a result that has independently been inferred from 18S rDNA [48]. Additionally, our analyses reject the clustering of Botrydiopsis pyrenoidosa with Xanthophyceae [49], or indeed with any of the other classes, suggesting that additional major evolutionary lineages may exist among the photosynthetic stramenopiles. Finally, our results indicate that Chrysophyceae and Synurophyceae are paraphyletic taxa (for discussion, see [50]), due to the Chrysosphaerella lineage (Synurophyceae) forming a sister-relationship with Chrysamoeba mikrokonta (Chrysophyceae) with posterior probability 1.0 in all analyses. ...
Stramenopiles constitute a large and diverse eukaryotic clade that is currently poorly characterized from both phylogenetic and temporal perspectives at deeper taxonomic levels. To better understand this group, and in particular the photosynthetic stramenopiles (Ochrophyta), we analyzed sequence data from 135 taxa representing most major lineages. Our analytical approach utilized several recently developed methods that more realistically model the temporal evolutionary process.
Phylogenetic reconstruction employed a Bayesian joint rate- and pattern-heterogeneity model to reconstruct the evolutionary history of these taxa. Inferred phylogenetic resolution was generally high at all taxonomic levels, sister-class relationships in particular receiving good statistical support. A signal for heterotachy was detected in clustered portions of the tree, although this does not seem to have had a major influence on topological inference. Divergence time estimates, assuming a lognormally-distributed relaxed molecular clock while accommodating topological uncertainty, were broadly congruent over alternative temporal prior distributions. These data suggest that Ochrophyta originated near the Proterozoic-Phanerozoic boundary, diverging from their sister-taxon Oomycota. The evolution of the major ochrophyte lineages appears to have proceeded gradually thereafter, with most lineages coming into existence by ∼200 million years ago.
The evolutionary timescale of the autotrophic stramenopiles reconstructed here is generally older than previously inferred from molecular clocks. However, this more ancient timescale nevertheless casts serious doubt on the taxonomic validity of putative xanthophyte/phaeophyte fossils from the Proterozoic, which predate by as much as a half billion years or more the age suggested by our molecular genetic data. If these fossils truly represent crown stramenopile lineages, then this would imply that molecular rate evolution in this group proceeds in a fashion that is fundamentally incompatible with the relaxed molecular clock model employed here. A more likely scenario is that there is considerable convergent morphological evolution within Heterokonta, and that these fossils have been taxonomically misdiagnosed.
... This gene was chosen because rbcL sequences are conserved enough to design consensus primers (e.g. Manhart 1994, McCourt et al. 2000, but distinct enough between even the most closely related species to distinguish them (Chase et al. 2005, Negrisolo et al. 2004). ...
ABSTRACT: A comparison of Elysia (=Tridachia) crispata (Mörch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki. Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters the chloroplasts, which actively photosynthesize for up to 4 months. We have determined the algal source of the chloroplasts in adult E. clarki from 2 populations in the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that adult E. clarki sequester chloroplasts from 7 different species of algae, representing two genera, of which 5 were identified; Penicillus lamourouxii, P. capitatus, Halimeda incrassata, H. monile, and Bryopsis pennata. In addition, chloroplasts from more than 1 species of algae are sequestered in the same digestive cell simultaneously. Phylogenetic analysis of rbcL sequences from the order Bryopsidales showed that E. clarki feeding was restricted to calcareous members of the family Udoteaceae and the family Bryopsidaceae. Feeding experiments were conducted, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae. For the first 14 d post-metamorphosis, juveniles ate only the thin filamentous coenocytes, Bryopsis plumosa or Derbesia tenuissima. Electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other macroalga, including the four calcareous species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ~ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae. Dissertation (Ph.D.)--University of South Florida, 2006. Includes bibliographical references. Text (Electronic dissertation or thesis) in PDF format. System requirements: World Wide Web browser and PDF reader. Mode of access: World Wide Web. Title from PDF of title page. Document formatted into pages; contains 115 pages. Includes vita.
... So far, there has been only one study for inferring phylogenetic relationships of Antarctic Xanthophyceae, and it was based on the chloroplast-encoded rbcL and psaA genes and culture strains (Maistro et al., 2007). The nuclear-encoded SSU rRNA gene, widely used for prokaryotes and some eukaryotes, has been too conservative for xanthophycean species level phylogenies (Bailey and Andersen, 1998;Negrisolo et al., 2004). Because of its limited use, only six 18S rDNA sequences for Tribonemataceae are in GenBank, whereas 26 rbcL sequences available (Maistro et al., 2007). ...
The genetic diversity of all available culture strains of the Tribonemataceae (Stramenopiles, Xanthophyceae) from Antarctica was assessed using the chloroplast-encoded psbA /rbcL spacer region sequences, a highly variable molecular marker, to test for endemism when compared with their closest temperate relatives. There was no species endemic for Antarctica, and no phylogenetic clade corresponded to a limited geographical region. However, species of the Tribonemataceae may have Antarctic populations that are distinct from those of other regions because the Antarctic strain spacer sequences were not identical to sequences from temperate regions. Spacer sequences from five new Antarctic isolates were identical to one or more previously available Antarctic strains, indicating that the Tribonemataceae diversity in Antarctic may be rather limited. Direct comparisons of the spacer sequences and phylogenetic analyses of the more conserved rbcL gene revealed that current morphospecies were inadequate to describe the actual biodiversity of the group. For example, the genus Xanthonema, as currently circumscribed, was paraphyletic. Fortunately, the presence of distinctive sequence regions within the psbA/rbcL spacer, together with differences in the rbcL phylogeny, provided significant autoapomorphic criteria to re-define the Tribonemataceae species.
The photosynthetic world is not only dominated by the green color of trees and plants.
Eustigmatophyceae is one of the ∼17 classes of the vast algal phylum Ochrophyta. Over the last decade, the eustigmatophytes emerged as an expansive group that has grown from the initially recognized handful of species to well over 200 genetically distinct entities (potential species). Yet the majority of eustigs, remain represented by unidentified strains, or even only metabarcode sequences obtained from environmental samples. Moreover, the formal classification of the group has not yet been harmonized with the recently uncovered diversity and phylogenetic relationships within the class. Here we make a major step towards resolving this issue by addressing the diversity, phylogeny and classification of one of the most prominent eustigmatophyte clades previously informally called the “Eustigmataceae group”. We obtained 18S rDNA and rbcL gene sequences from four new strains from the “Eustigmataceae group”, and from several additional eustig strains, and performed the most comprehensive phylogenetic analyses of Eustigmatophyceae to date. Our results of these analyses confirm the monophyly of the “Eustigmataceae group” and define its major subclades. We also sequenced plastid genomes of five “Eustigmataceae group” strains to not only improve our understanding of the plastid gene content evolution in eustigs, but also to obtain a robustly resolved eustigmatophyte phylogeny. With this new genomic data, we have solidified the view of the “Eustigmataceae group” as a well-defined family level clade. Crucially, we also have firmly established the genus Chlorobotrys as a member of the “Eustigmataceae group”. This new molecular evidence, together with a critical analysis of the literature going back to the 19th century, provided the basis to radically redefine the historical concept of the family Chlorobotryaceae as the formal taxonomic rubric corresponding to the “Eustigmataceae group”. With this change, the family names Eustigmataceae and Characiopsidaceae are reduced to synonymy with the Chlorobotryaceae, with the latter having taxonomic priority. We additionally studied in detail the morphology and ultrastructure of two Chlorobotryaceae members, which we describe as Neustupella aerophytica gen. et sp. nov. and Lietzensia polymorpha gen. et sp. nov. Finally, our analyses of partial genomic data from several Chlorobotryaceae representatives identified genes for hallmark flagellar proteins in all of these strains. The presence of the flagellar proteins strongly suggests that zoosporogenesis is a common trait of the family and also occurs in the members never observed to produce flagellated stages. Altogether, our work paints a rich picture of one of the most diverse principal lineages of eustigmatophyte algae.
Eustigmatophyceae is one of the ∼17 classes of the vast algal phylum Ochrophyta. Over the last decade, the eustigmatophytes emerged as an expansive group that has grown from the initially recognized handful of species to well over 200 genetically distinct entities (potential species). Yet the majority of eustigs, remain represented by unidentified strains, or even only metabarcode sequences obtained from environmental samples. Moreover, the formal classification of the group has not yet been harmonized with the recently uncovered diversity and phylogenetic relationships within the class. Here we make a major step towards resolving this issue by addressing the diversity, phylogeny and classification of one of the most prominent eustigmatophyte clades previously informally called the “Eustigmataceae group”. We obtained 18S rDNA and rbcL gene sequences from four new strains from the “Eustigmataceae group”, and from several additional eustig strains, and performed the most comprehensive phylogenetic analyses of Eustigmatophyceae to date. Our results of these analyses confirm the monophyly of the “Eustigmataceae group” and define its major subclades. We also sequenced plastid genomes of five “Eustigmataceae group” strains to not only improve our understanding of the plastid gene content evolution in eustigs, but also to obtain a robustly resolved eustigmatophyte phylogeny. With this new genomic data, we have solidified the view of the “Eustigmataceae group” as a well-defined family level clade. Crucially, we also have firmly established the genus Chlorobotrys as a member of the “Eustigmataceae group”. This new molecular evidence, together with a critical analysis of the literature going back to the 19th century, provided the basis to radically redefine the historical concept of the family Chlorobotryaceae as the formal taxonomic rubric corresponding to the “Eustigmataceae group”. With this change, the family names Eustigmataceae and Characiopsidaceae are reduced to synonymy with the Chlorobotryaceae, with the latter having taxonomic priority. We additionally studied in detail the morphology and ultrastructure of two Chlorobotryaceae members, which we describe as Neustupella aerophytica gen. et sp. nov. and Lietzensia polymorpha gen. et sp. nov. Finally, our analyses of partial genomic data from several Chlorobotryaceae representatives identified genes for hallmark flagellar proteins in all of these strains. The presence of the flagellar proteins strongly suggests that zoosporogenesis is a common trait of the family and also occurs in the members never observed to produce flagellated stages. Altogether, our work paints a rich picture of one of the most diverse principal lineages of eustigmatophyte algae.
Vaucheria species have previously been restricted to upper shore habitats in the Wadden Sea (eastern North Sea, European Atlantic). In contrast to these previous observations, we have now found Vaucheria longicaulis and a distinct plastid lineage of V. velutina spreading along the lower shores, forming extensive turfs at and below low tide level under fully marine conditions near the Island of Sylt. Species and populations were identified by morphological features and reconfirmed by sequencing the plastid-encoding gene rbcL and the psbA-rbcL spacer region. We needed to modify primer sequences to successfully amplify the rbcL region of marine and brackish Vaucheria species. Due to distinct phylogenetic grouping, we can reject the possibility of niche expansions from the upper shore and propose recent introductions as the cause of the newly formed populations at the lower shore.
Taxonomic identification of algae is widely used in algal genetics, physiology, ecology, applied phycology, and particularly bioassessment. Errors in identification have been caused by the inherent problems of atypical or absent taxonomic features, research-grade microscopes, and inadequate taxonomic expertise. In recent years, as longer, cheaper, and faster DNA sequencing technologies have become more accessible, molecular markers have increasingly become universal tools in algae identification. The selection of molecular markers is critical for the molecular identification of algae because there are obvious differences among various phyla. We reviewed the progress in selecting and applying molecular markers in Cyanophyta, Bacillariophyta, Chlorophyta, Pyrrophyta, Euglenophyta, Cryptophyta, Chrysophyta, Xanthophyta, Rhodophyta, and Phaeophyta, and examined the principles governing selection, common molecular markers, improvements in the sequence database, and the advantages and disadvantages of the application in different phyla. Identifying species using nucleic, mitochondrial, and chloroplast DNA has become commonplace in recent years. To date, it has been considered that combining multiple markers of ribosomal DNA (rDNA), internal transcribed spacers (ITS), and conserved protein coding genes is much more effective than using a single marker for algae molecular identification. In addition, the mitochondrial, chloroplast, and nuclear genome, transcriptome, and metagenome contain large amounts of phylogenetic information, and their use is becoming significant for phylogenetic analysis as well as in the classification of algae. Potentially, they represent giant molecular markers for use in future research into algae systematics. Phylogenetic analysis on the basis of molecular markers, together with morphological features, is insufficient for exact identification. A polyphasic approach-combining features from molecular biology, morphology, physiology, and biochemistry-has been applied in algae classification and will become the strategy of choice for accurate and systematic algae classification and identification. Databases are essential for the application of algae molecular identification, and much work needs to be done to perfect the databases for the molecular marker for each alga. The rapid identification approach on the basis of molecular markers will be widely applied in future.
A new culture strain of Navicula veneta Kützing (Bacillariophyceae) was isolated from brown mats covering thermal muds in the SPA town of Abano Terme, Padova, Italy. The morphology, 18S rDNA and rbcL gene sequences of this strain were analysed. The results do not support an existing taxonomic proposal to lower the taxonomic rank of Navicula veneta to a variety of Navicula cryptocephala Kützing. Therefore, we consider Navicula veneta as a good species in its own right. We also hypothesize that the species Navicula veneta, as defined morphologically, contains a number of cryptic species yet to be discovered and analysed genetically.
Ribulose-1, 5-bisphosphate carboxynase/oxygenase large subunit gene (rbcL) of Jiaozhou Bay phytoplankton was amplified from spring, summer and autumn surface seawater DNAs and cloned respectively. About 50 clones were randomly selected from each library and sequenced. If identical amino acid sequences lare considered as the same operational taxonomy unit (OTU), 61 OTUs are identified according to inferred amino acid sequences, among them, 21 from spring seawater, 15 from summer seawater and 25 from autumn seawater. Shannon index calculated from OTU abundances reflects the genetic diversity of a community. The indexes of spring, summer and autumn surface seawater phytoplankton are 2.69, 2.44 and 2.76 respectively, indicating that phytoplankton genetic diversity of autumn seawater is the richest. Seasonal variation of phytoplankton community is significant; the community compositions of three seasons are almost completely different except for the two OTUs shared by summer and autumn. Surface seawater phytoplankton communities are possibly metacommunities different spatially and temporally.
The Verrucariaceae (Ascomycota) is a family of mostly lichenized fungi with a unique diversity of algal symbionts, including some algae that are rarely or never associated with other lichens. The phylogenetic position of most of these algae has not yet been studied and, because morphology-based identifications can often be misleading, molecular data is necessary to revisit their identity and to explore patterns of association between fungal and algal partners. For this reason, the diversity of photobionts in this lichen family was investigated using molecular markers (rbcL and nuSSU) amplified from DNA extracts of lichen thalli and cultured isolates. Although a single algal genus, Diplosphaera (Trebouxiophyceae), was associated with 12 out of the 17 sampled genera of Verrucariaceae, representatives of eight other genera in five orders of the Chlorophyta and one genus in the Xanthophyceae also form lichen associations with members of the family. Fungal genera with simple crustose thalli (e.g. Hydropunctaria, Wahlenbergiella, Bagliettoa) use a high diversity and unusual selection of photobionts. In contrast, fungal genera with more complex thalli (e.g. Placidium, Dermatocarpon) tend to have lower photobiont diversity. Habitat requirements and phylogenetic histories are both partly reflected in the observed patterns of associations between lichenized fungi from the family Verrucariaceae and their photobionts.
The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA (SSU rDNA) and chloroplast-encoded
rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. Lineage I was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. Lineage II contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the
average distance within lineage I was significantly higher than that of other microalgae species (P < 0.01). These results suggested the existence of cryptic species within C. closterium.
Extremophile microalgae show a remarkable ability to cope with harsh environments, yet still relatively little is known about the molecular basis for such tolerance. In this work the susceptibility of a psychrophylic alga isolated from alpine snowbanks, Xanthonema sp., to either water or salt stress conditions was assessed, and mechanisms for osmotic adjustment were investigated. Cultures were treated with increasing concentrations of either salts or non-permeant solutes, as polyethylene glycol, and the resulting effect on growth rate was measured. Both the accumulation of compatible osmolytes and the activity of cation transporters were studied in response to the exposure to hyperosmotic conditions. Xanthonema showed a differential sensitivity to osmotic and ionic stress, with a noteworthy tolerance to NaCl. No evidence was found supporting an osmo-induced intracellular accumulation of the most common osmoprotectants. Salt tolerance seems to rely upon the inducible expression of an amiloride-resistant Na+/H+ antiporter. Since in snow fields osmotic unbalance due to freeze/thaw is more likely to occur than excess salts, results suggest an allochthonous origin of the strain.
To unravel the concept structure and dynamics of the bioinformatics field, we analyze a set of 7401 publications from the Web of Science and MEDLINE databases, publication years 1981–2004. For delineating this complex, interdisciplinary field, a novel bibliometric retrieval strategy is used. Given that the performance of unsupervised clustering and classification of scientific publications is significantly improved by deeply merging textual contents with the structure of the citation graph, we proceed with a hybrid clustering method based on Fisher’s inverse chi-square. The optimal number of clusters is determined by a compound semi-automatic strategy comprising a combination of distance-based and stability-based methods. We also investigate the relationship between number of Latent Semantic Indexing factors, number of clusters, and clustering performance. The HITS and PageRank algorithms are used to determine representative publications in each cluster. Next, we develop a methodology for dynamic hybrid clustering of evolving bibliographic data sets. The same clustering methodology is applied to consecutive periods defined by time windows on the set, and in a subsequent phase chains are formed by matching and tracking clusters through time. Term networks for the eleven resulting cluster chains present the cognitive structure of the field. Finally, we provide a view on how much attention the bioinformatics community has devoted to the different subfields through time.
As a discipline, phylogenetics is becoming transformed by a flood of molecular data. These data allow broad questions to be
asked about the history of life, but also present difficult statistical and computational problems. Bayesian inference of
phylogeny brings a new perspective to a number of outstanding issues in evolutionary biology, including the analysis of large
phylogenetic trees and complex evolutionary models and the detection of the footprint of natural selection in DNA sequences.
A microalga was isolated from the pack-ice of Wood Bay (Antarctica) during austral summer 1993–4. By comparison with an authentic strain obtained from Sammlung von Algenkulturen at the University of Go$ ttingen (SAG 860–3), its ultrastructural details and pigment composition were very similar to those of Pleurochloris meiringensis. However, sequence analysis of the entire 18S rRNA gene revealed substantial genetic divergence (57 nucleotide substitutions and 19 insertions/deletions) between SAG 860–3 and the Antarctic isolate. Phylogenetic analysis of complete 18S rRNA gene sequences from 18 microalgal species, including the two strains studied, rejected the hypothesis that the Antarctic isolate is the sister species to Pleurochloris meiringensis. Molecular evidence indicates that the Antarctic isolate is the sister group of a clade that comprises two other representatives of the Xanthophyceae (synonym Tribophyceae): Pleurochloris meiringensis and Botrydiopsis intercedens. This leads us to conclude that this microalga is a new species belonging to a new genus and the name Pseudopleurochloris antarctica is proposed.
The program MRBAYES performs Bayesian inference of
phylogeny using a variant of Markov chain Monte Carlo.
Availability: MRBAYES, including the source code, documentation,
sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.
Contact: johnh{at}brahms.biology.rochester.edu
Some earlier studies suggested an evolutionary relationship between the Raphidophyceae (chloromonads) and Xanthophyceae (yellow-green algae), whereas other studies suggested relationships with different algal classes or the öomycete fungi. To evaluate the relationships, we determined the complete nucleotide sequences of the 18S ribosomal RNA gene from the raphidophytes Vacuolaria virescens, Chattonella subsalsa, and Heterosigma carterae, and the xanthophytes Vaucheria bursata, Botrydium stoloniferum, Botrydiopsis intercedens, and Xanthonema debile. The results showed that the Xanthophyceae were most closely related to the Phaeophyceae. A cladistic analysis of combined data sets (nucleotide sequences, ultrastructure, and pigments) suggested the Raphidophyceae are the sister taxon to the Phaeophyceae-Xanthophyceae clade, but the bootstrap value was low (40%). The raphidophyte genera were united with high (100%) bootstrap values, supporting a hypothesis based upon ultrastructural features that marine and freshwater raphidophytes form a monophyletic group. We examined the relationship between Vaucheria, a siphoneous xanthophyte alga, and the öomycetes, and we confirmed that Vaucheria is a member of the class Xanthophyceae. Partial nucleotide sequences of the 18S rRNA gene from eight xanthophytes (including Bumillariopsis filiformis, Heterococcus caespitiosus, and Mischococcus sphaerocephalus) produce a phylogeny that is not congruent with the current morphology-based classification scheme.
Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
We introduce a graphical method, likelihood-mapping, to visualize the phylogenetic content of a set of aligned sequences. The method is based on an analysis of the maximum likelihoods for the three fully resolved tree topologies that can be computed for four sequences. The three likelihoods are represented as one point inside an equilateral triangle. The triangle is partitioned in different regions. One region represents star-like evolution, three regions represent a well-resolved phylogeny, and three regions reflect the situation where it is difficult to distinguish between two of the three trees. The location of the likelihoods in the triangle defines the mode of sequence evolution. If n sequences are analyzed, then the likelihoods for each subset of four sequences are mapped onto the triangle. The resulting distribution of points shows whether the data are suitable for a phylogenetic reconstruction or not.
Using the large subunit of RuBisCo (rbcL) sequences from cyanobacteria, proteobacteria, and diverse groups of algae and green plants, we evaluated the plastid relationship between haptophytes and heterokont algae. The rbcL sequences were determined from three taxa of heterokont algae (Bumilleriopsis filiformis, Pelagomonas calceolata, and Pseudopedinella elastica) and added to 25 published sequences to obtain a data set comprising 1,434 unambiguously aligned sites (approximately 98% of the total rbcL gene). Higher levels of mutational saturation in third codon positions were observed by plotting the pairwise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In accordance with this finding phylogeny reconstructions were completed by omitting third codon positions, thus using 956 bp in weighted-parsimony and maximum-likelihood analyses. The midpoint-rooted phylogenies showed two major clusters, one containing cyanobacteria, glaucocystophytes, a phototrophic euglenoid, chlorophytes, and embryophytes (the green lineage), the other containing proteobacteria, haptophytes, red algae, a cryptophyte, and heterokont algae (the non-green lineage). In the nongreen lineage, the haptophytes formed a sister group to the clade containing heterokont algae, red algae, and the cryptophyte Guillardia theta. This branching pattern was well supported in terms of bootstrap values in weighted-parsimony and maximum-likelihood analyses (100% and 92%, respectively). However, the phylogenetic relationship among red algae, heterokonts, and a cryptophyte taxon was not especially well resolved. A four-cluster analysis was performed to further explore the statistical significance of the relationship between proteobacteria, red algae (including and excluding Guillardia theta), haptophytes, and heterokont algae. This test strongly favored the hypothesis that the heterokonts and red algae are more closely related to each other than either is to proteobacteria or haptophytes. Hence, this molecular study based on a plastid-encoded gene provides additional evidence for a distant relationship between haptophytes and the heterokont algae. It suggests an evolutionary scenario in which the ancestor of the haptophyte lineage engulfed a phototrophic eukaryote and, more recently, the heterokont lineage became phototrophic by engulfing a red alga.
The program MODELTEST uses log likelihood scores to establish the model of DNA evolution that best fits the data.
AVAILABILITY: The MODELTEST package, including the source code and some documentation is available at http://bioag.byu. edu/zoology/crandall_lab/modeltest.html.
As a discipline, phylogenetics is becoming transformed by a flood of molecular data. These data allow broad questions to be asked about the history of life, but also present difficult statistical and computational problems. Bayesian inference of phylogeny brings a new perspective to a number of outstanding issues in evolutionary biology, including the analysis of large phylogenetic trees and complex evolutionary models and the detection of the footprint of natural selection in DNA sequences.
Unlabelled:
CONSEL is a program to assess the confidence of the tree selection by giving the p-values for the trees. The main thrust of the program is to calculate the p-value of the Approximately Unbiased (AU) test using the multi-scale bootstrap technique. This p-value is less biased than the other conventional p-values such as the Bootstrap Probability (BP), the Kishino-Hasegawa (KH) test, the Shimodaira-Hasegawa (SH) test, and the Weighted Shimodaira-Hasegawa (WSH) test. CONSEL calculates all these p-values from the output of the phylogeny program packages such as Molphy, PAML, and PAUP*. Furthermore, CONSEL is applicable to a wide class of problems where the BPs are available.
Availability:
The programs are written in C language. The source code for Unix and the executable binary for DOS are found at http://www.ism.ac.jp/~shimo/
Contact:
shimo@ism.ac.jp
TREE-PUZZLE is a program package for quartet-based maximum-likelihood phylogenetic analysis (formerly PUZZLE, Strimmer and von Haeseler, Mol. Biol. Evol., 13, 964-969, 1996) that provides methods for reconstruction, comparison, and testing of trees and models on DNA as well as protein sequences. To reduce waiting time for larger datasets the tree reconstruction part of the software has been parallelized using message passing that runs on clusters of workstations as well as parallel computers.
Availability:
http://www.tree-puzzle.de. The program is written in ANSI C. TREE-PUZZLE can be run on UNIX, Windows and Mac systems, including Mac OS X. To run the parallel version of PUZZLE, a Message Passing Interface (MPI) library has to be installed on the system. Free MPI implementations are available on the Web (cf. http://www.lam-mpi.org/mpi/implementations/).
An approximately unbiased (AU) test that uses a newly devised multiscale bootstrap technique was developed for general hypothesis
testing of regions in an attempt to reduce test bias. It was applied to maximum-likelihood tree selection for obtaining the
confidence set of trees. The AU test is based on the theory of Efron et al. (Proc. Natl. Acad. Sci. USA 93:13429–13434; 1996),
but the new method provides higher-order accuracy yet simpler implementation. The AU test, like the Shimodaira–Hasegawa (SH)
test, adjusts the selection bias overlooked in the standard use of the bootstrap probability and Kishino–Hasegawa tests. The
selection bias comes from comparing many trees at the same time and often leads to overconfidence in the wrong trees. The
SH test, though safe to use, may exhibit another type of bias such that it appears conservative. Here I show that the AU test
is less biased than other methods in typical cases of tree selection. These points are illustrated in a simulation study as
well as in the analysis of mammalian mitochondrial protein sequences. The theoretical argument provides a simple formula that
covers the bootstrap probability test, the Kishino–Hasegawa test, the AU test, and the Zharkikh–Li test. A practical suggestion
is provided as to which test should be used under particular circumstances.
The Xanthophyta are much less species-diverse than the Chlorophyta, with about 600 species and many of the 100 known genera containing only a few species. However, they show a wide range of form and include biflagellate and non-motile unicells, simple or branched uniseriate filaments, and others are coenocytic or siphonous (large multinucleate cells). Colonial forms may or may not have a well-defined shape. Some species are epiphytes and sessile or attached by a stalk.
Most are non-motile, single-celled or colonial, although there a few more advanced filamentous forms and coenocytic forms such as Vaucheria. If motile, they are biflagellate, and often possess associated photoreceptors. Asexual reproduction is mainly by fragmentation into portions of one or more cells in multicellular species, aplanospores or zoospores that each have two unequal flagella and sometimes an eyespot. Sexual reproduction is apparently comparatively rare, although well-known in Vaucheria, where it is distinctive and provides important taxonomic characters. Resting structures or cysts are known and often have walls impregnated with silica. Heterogamy is uncommon and isogametes known only for a few genera.
The distinction between the Xanthophyta and the Chlorophyta rests largely on chloroplast pigments and food storage products rather than on morphological characters. Traditionally, there are three features that distinguish the xanthophytes: (i) yellow or yellowgreen colour of the chloroplasts; (ii) carbohydrate storage as oil droplets or chrysolaminarin (usually termed leucosin) granules, with starch and pyrenoids rare; (iii) walls of pectin or pectic acid (sometimes in association with cellulose or siliceous substances) and consisting of two spliced and overlapping sections (most conspicuous in Tribonema), which on dissociation of the filaments tend to break into H-shaped sections or pieces. Although a useful taxonomic character, these sections are not present in all genera, and certainly not always readily visible even in genera where they occur.
In practice, the yellow-green colour of these plants is not always easy to distinguish, and in the case of Vaucheria, the colour is indistinguishable from that of green algae (Vaucheria was initially described as a green alga). The xanthophytes differ in containing no chlorophyll b, but in addition to chlorophyll a have chlorophylls c1 and c2; other pigments are carotenoids (especially b-carotene) and at least three xanthophylls. These xanthophylls can give these algae a blue-green colour when treated with hot hydrochloric acid in the laboratory.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
We report six complete 18S ribosomal DNA (rDNA) sequences representing five brown algal orders: Sporochnus comosus C. A. Agardh (Sporochnales), Chorda tomentosa Lyngbye (Chordaceae, Laminariales), Saccorhiza polyschides (Lightfoot) Batters (Phyllariaceae, Laminariales), Desmarestia ligulata (Lightfoot) Lamouroux (Desmarestiales), Ectocarpus siliculosus (Dillwyn) Lyngbye (Ectocarpales), and Scytosiphon lomentaria (Lyngbye) J. G. Agardh (Syctosiphonales). These sequences were compared with published laminarialean (Alaria marginata Postel et Ruprecht [Alariaceae] and Macrocystis integrifolia Bory [Lessoniaceae]) and fucalean (Fucus gardneri Silva) rDNA sequences for phylogeny inference using both the distance-matrix and parsimony methods. The inferred 18S phylogenies clustered Sporochnus, Desmarestia, Chorda, Saccorhiza, Alaria, and Macrocystis in an assemblage. This Sporochnales-Desmarestiales-Laminariales (S-D-L) complex was consistently separated from the Ectocarpales, Scytosiphonales, and Fucales by bootstrap analyses. The inferred phylogenies are consistent with several possible evolutionary processes leading to this S-D-L complex. Members in this assemblage lack eyespots in their sperm, and their sperm have the atypical brown algal flagellation: shorter anterior and longer posterior flagella. In addition, they are oogamous with a heteromorphic alteration of generations between a microscopic gametophyte and a macroscopic sporophyte. Members of the S-D-L complex can be separated into different phylogenetic lines based on the presence/absence of eyespots in their meiospores. Our findings support the contention that the Sporochnales, Desmarestiales, and Laminariales are closely related. In addition, our rDNA tree suggests that the Laminariales is paraphyletic.
This book presents the statistical methods that are useful in the study of molecular evolution and illustrates how to use them in actual data analysis. Molecular evolution has been developing at a great pace over the past decade or so, driven by the huge increase in genetic sequence data from many organisms, the improvement of high-speed microcomputers, and the development of several new methods for phylogenetic analysis. This book for graduate students and researchers, assuming a basic knowledge of evolution, molecular biology, and elementary statistics, should make it possible for many investigators to incorporate refined statistical analysis of large-scale data in their own work. Nei is one of the leading workers in this area. He and Kumar have developed a computer program called MEGA, which has been sold for about $20 to over 1900 users. For the book, the authors are thoroughly revising MEGA and will make it available via FTP. The book also included analysis using the other most popular programs for phylogenetic studies, including PAUP, PHYLIP, MOLPHY, and PAML.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.
Gene sequence data were used to evaluate traditional classifications for the Xanthophyceae based on the level of organization of vegetative cells and to rest the hypothesis that larger morphologically complex species are highly derived members of the class. Sequences for the plastid-encoded large subunit of ribulose-1,5- bisphosphate carboxylase/oxygenase (rbcL) and nuclear-encoded small subunit ribosomal RNA (18S rRNA) genes were aligned for nine xanthophyte species. Separate and combined analyses of these data matrices yielded topologically identical trees. The molecular data do not support the monophyly of the orders Mischococcales or Tribonematales, to which coccoid and filamentous species, respectively, are traditionally assigned. Multiple, independent origins of each of these life forms are inferred from the gene tree. Thus, the level of organization of cells of species may not be a phylogenetically reliable character for ordinal classification. Although placed in the same order by some authorities, evidence is lacking for a close relationship between the siphonous, sexually reproducing genera Botrydium and Vaucheria. Vaucheria, the largest and reproductively most complex of all xanthophytes, is positioned on a long branch at the base of the ingroup and is not closely related to any other species examined. The contention that Vaucheria is the most highly advanced xanthophyte taxon is rejected by the molecular data. Results tentatively suggest that species may be divided between two major lineages characterized by the presence or absence of a bipartite cell wall.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Complete 18S rDNA sequences were determined for 25 strains representing five genera of the Eustigmatophyceae, including re-examination of three strains with previously published sequences. Parsimony analysis of these and 44 published sequences for other heterokont chromophytes (unalignable sites removed) revealed that the Eustigmatophyceae were a monophyletic group. Analysis of eustigmatophyte taxa only (complete gene analyzed) supported the current familial classification scheme. Twenty one strains of Nannochloropsis were also examined using light microscopy. Gross morphology of cells was variable and overlapped among the strains; cell size was consistent within strains but sometimes varied considerably among strains of a species. The 18S rDNA of N. gaditana, N. oculata and N. salina was re-sequenced for strains used in previous publications and one or more nucleotide differences were found. Nucleotide sequences for Nannochloropsis species varied by up to 32 nucleotides. Identical sequences were found for six strains of N. salina, five strains of N. gadifana, four strains of N. granulata, and two strains of N. oculata, respectively. Four strains could not be assigned to described species and may represent two new species. The unique 18S rDNA sequences for each sibling species of Nannochloropsis demonstrates the presence of considerable genetic diversity despite the extremely simple morphology in this genus.
A new algal class, the Phaeothamniophyceae classis nova, is established from genera formerly classified in the Chrysophyceae (e.g., Chrysapion, Chrysoclonium, Chrysodictyon, Phaeobotrys, Phaeogloea, Phaeoschizochlamys, Phaeothamnion, Selenophaea, Sphaeridiothrix, Stichogloea, Tetrachrysis, Tetrapion and Tetrasporopsis) as well as one genus previously assigned to the Xanthophyceae (Pleurochloridella). HPLC analysis revealed the presence of fucoxanthin, diadinoxanthin, diatoxanthin, β-carotene and heteroxanthin, in addition to chlorophylls a and c, in four genera (Phaeoschizochlamys, Phaeothamnion, Stichogloea, Pleurochloridella). The combination of fucoxanthin and heteroxanthin is known only for these organisms. The rbcL sequences of the same four genera, along with representatives of other chromophyte classes, were analyzed phylogenetically and provided independent support for recognition of the Phaeothamniophyceae as a distinct taxon. These data indicate that the Phaeothamniophyceae are more closely related to the classes Xanthophyceae and Phaeophyceae than to the Chrysophyceae. Electron microscopy revealed that Phaeoschizochlamys, Phaeothamnion and Stichogloea possess electron opaque vesicles at the cell periphery, have a cell wall that often appears laminate, form new daughter cell walls via eleutheroschisis, and have plastids with girdle lamellae and a ring-shaped genophore. The flagellar apparatus of Phaeothamnion zoospores (described in a previous study) is chosen as representative of the new class. The flagella are inserted laterally, basal bodies form an angle of ca. 145° or more, a multi-gyred flagellar transitional helix is present and tripartite flagellar hairs lack lateral filaments. Genera placed in the Phaeothamniophyceae are assigned to the orders Phaeothamniales and Pleurochloridellales, each with a single family.
Nuclear ribosomal small subunit and chloroplast rbcL sequence data for heterokont algae and potential outgroup taxa were analyzed separately and together using maximum parsimony. A series of taxon sampling and character weighting experiments was performed. Traditional classes (e.g. diatoms, Phaeophyceae, etc.) were monophyletic in most analyses of either data set and in analyses of combined data. Relationships among classes and of heterokont algae to outgroup taxa were sensitive to taxon sampling. Bootstrap (BS) values were not always predictive of stability of nodes in taxon sampling experiments or between analyses of different data sets. Reweighting sites by the rescaled consistency index artificially inflates BS values in the analysis of rbcL data. Inclusion of the third codon position from rbcL enhanced signal despite the superficial appearance of mutational saturation. Incongruence between data sets was largely due to placement of a few problematic taxa, and so data were combined. BS values for the combined analysis were much higher than for analyses of each data set alone, although combining data did not improve support for heterokont monophyly.
Variability among clonal cultures of the brown tide algae Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis Stockwell, DeYoe, Hargraves et Johnson was examined by DNA sequence comparisons. Nuclear-encoded 18S rRNA and plastid-encoded rbcL gene sequences were determined for six Aureococcus strains. RUBISCO spacer sequences were determined for 14 strains of Aureococcus. No differences among Aureococcus strains were found in the DNA regions examined. The rbcL and RUBISCO spacer sequences for three Aureoumbra strains were identical but differed from those of Aureococcus. These data indicate that blooms of these species are comprised of cells that are very similar and also imply that Aureococcus and Aureoumbra do not contain varieties or cryptic species. Separate and combined phylogenetic analyses of the 18S rRNA and rbcL gene sequences were performed. Results confirm that the brown-tide-causing algae of Long Island Sound, New York (Aureococcus), and Laguna Madre, Texas (Aureoumbra), are best classified in separate genera within the Pelagophyceae. Phylogenetic trees place Aureococcus and Aureoumbra within the Pelagomonadales and Sarcinochrysidales, respectively.
The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA-rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA-rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA-rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair-wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.
The 16S-like ribosomal RNA genes from Mallomonas papillosa Harris et Bradley (Synurophyceae) and Tribonema aequale Pascher (Xanthophyceae) were sequenced and compared to those of other eukaryotes. Mallomonas is closely related to Ochromonas (Chrysophyceae) and supports the general hypothesis of a close phylogenetic relationship between the Synurophyceae and Chrysophyceae. Tribonema is specifically related to Costaria costata (C. A. Agardh) Saunders (Phaeophyceae) demonstrating an unexpected phylogenetic relationship between the Xanthophyceae and Phaeophyceae. Distance and parsimony analysis place these four chromophyte genera in a complex evolutionary assemblage that includes the Bacillariophyceae and Oomycetes but excludes the Dinophyceae. The close relationship between the chromophyte algae and the Öomycete fungi supports the hypothesis that protists with tripartite hairs form a natural assemblage.
According to currently accepted theories, rapidly evolving nucleotide sites are phylogenetically less informative than more slowly evolving ones, especially for recognizing more ancient groupings. For this reason third codon positions are often regarded as less reliable than first and second positions as indicators of phylogeny. Analysis of the largest nucleotide matrix treated to date—2538 rbc L sequences covering all major lineages of green plants—shows the opposite: although rapidly evolving and highly homoplastic, third positions contain most of the phylogenetic structure in the data. Frequency of change should thus be used with caution as a criterion for weighting or selecting characters.
Slowly evolving characters, such as amino acids and replacement substitutions, have generally been favored over faster evolving characters for inferring phylogenetic relationships. However, amino acids constitute composite characters and, because of the degenerate genetic code, are subject to convergence. Based on an analysis of atpB and rbcL in 567 seed plants, we show that silent substitutions may be more phylogenetically informative than replacement substitutions and that artifacts caused by composite characters and/or convergence cause clades on amino acid trees to conflict with nucleotide trees and independent evidence. These findings indicate that coding nucleotide sequences only as amino acid characters for phylogenetic analysis provides little benefit and may yield misleading results.
The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas phi. Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.
The nucleotide sequence and the 5' flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria, the P. littoralis rbcL gene is more closely related to that of a beta-purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996-4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599-608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
In phylogenetic inference by maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods, it is customary to conduct extensive heuristic searches of MP, ME, and ML trees, examining a large number of different topologies. However, these extensive searches tend to give incorrect tree topologies. Here we show by extensive computer simulation that when the number of nucleotide sequences (m) is large and the number of nucleotides used (n) is relatively small, the simple MP or ML tree search algorithms such as the stepwise addition (SA) plus nearest neighbor interchange (NNI) search and the SA plus subtree pruning regrafting (SPR) search are as efficient as the extensive search algorithms such as the SA plus tree bisection-reconnection (TBR) search in inferring the true tree. In the case of ME methods, the simple neighbor-joining (NJ) algorithm is as efficient as or more efficient than the extensive NJ+TBR search. We show that when ME methods are used, the simple p distance generally gives better results in phylogenetic inference than more complicated distance measures such as the Hasegawa-Kishino-Yano (HKY) distance, even when nucleotide substitution follows the HKY model. When ML methods are used, the simple Jukes-Cantor (JC) model of phylogenetic inference generally shows a better performance than the HKY model even if the likelihood value for the HKY model is much higher than that for the JC model. This indicates that at least in the present case, selecting of a substitution model by using the likelihood ratio test or the AIC index is not appropriate. When n is small relative to m and the extent of sequence divergence is high, the NJ method with p distance often shows a better performance than ML methods with the JC model. However, when the level of sequence divergence is low, this is not the case.
As the amount of molecular sequence data in the public domain grows, so does the range of biological topics that it influences through evolutionary considerations. In recent years, a number of developments have enabled molecular phylogenetic methodology to keep pace. Likelihood-based inferential techniques, although controversial in the past, lie at the heart of these new methods and are producing the promised advances in the understanding of sequence evolution. They allow both a wide variety of phylogenetic inferences from sequence data and robust statistical assessment of all results. It cannot remain acceptable to use outdated data analysis techniques when superior alternatives exist. Here, we discuss the most important and exciting methods currently available to the molecular phylogeneticist.
Strains of Heterococcus endolithicus sp. nov., H. pleurococcoides Pitschmann, H. caespitosus Vischer, and H. protonematoides Vischer isolated from terrestrial habitats in Antarctica were studied in culture. Morphology of the algae changes with stage in life history. The characteristic branching patterns are not present in very young or old cultures. Filament formation is suppressed when cultures are grown outside their optimal temperature range.
In this study, we used an empirical example based on 100 mitochondrial genomes from higher teleost fishes to compare the accuracy of parsimony-based jackknife values with Bayesian support values. Phylogenetic analyses of 366 partitions, using differential taxon and character sampling from the entire data matrix of 100 taxa and 7,990 characters, were performed for both phylogenetic methods. The tree topology and branch-support values from each partition were compared with the tree inferred from all taxa and characters. Using this approach, we quantified the accuracy of the branch-support values assigned by the jackknife and Bayesian methods, with respect to each of 15 basal clades. In comparing the jackknife and Bayesian methods, we found that (1) both measures of support differ significantly from an ideal support index; (2) the jackknife underestimated support values; (3) the Bayesian method consistently overestimated support; (4) the magnitude by which Bayesian values overestimate support exceeds the magnitude by which the jackknife underestimates support; and (5) both methods performed poorly when taxon sampling was increased and character sampling was not increases. These results indicate that (1) the higher Bayesian support values are inappropriate (in magnitude), and (2) Bayesian support values should not be interpreted as probabilities that clades are correctly resolved. We advocate the continued use of the relatively conservative bootstrap and jackknife approaches to estimating branch support rather than the more extreme overestimates provided by the Markov Chain Monte Carlo-based Bayesian methods.
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The branch swapping applied the tree bisection– reconnection TBR algorithm
- Swofford
2002). The branch swapping applied the tree bisection– reconnection TBR algorithm (Swofford et al., 1996) with