Phylogenetic relationships in Interfilum and Klebsormidium (Klebsormidiophyceae, Streptophyta)

https://doi.org/10.1016/j.ympev.2010.11.030Get rights and content

Abstract

Members of the genus Klebsormidium have cosmopolitan distribution and occur in a very wide range of freshwater and terrestrial habitats. Due to its simple filamentous morphology, this genus represents a taxonomically and systematically complex taxon in which phylogenetic relationships are still poorly understood. The phylogeny of Klebsormidium and closely related taxa was investigated using new ITS rRNA and rbcL sequences generated from 75 strains (isolated from field samples or obtained from culture collections). These sequences were analyzed both as single-marker datasets and in a concatenated dataset. Seven main superclades were observed in the analyses, which included sixteen well-supported clades. Some species of Klebsormidium, including the type species Klebsormidium flaccidum, were polyphyletic. Interfilum was recovered with high statistical support as sister taxon to a clade of Klebsormidium formed mainly by strains identified as K. flaccidum. Whereas some clades could be easily associated with described species, this was not possible for other clades. A new lineage of Klebsormidium, isolated from arid soils in southern Africa and comprising undescribed species, was discovered. Several morphological characters traditionally used for taxonomic purposes were found to have no phylogenetic significance and in some cases showed intra-clade variation. The capacity to form packet-like aggregates (typical of Interfilum), features of the morphology of the chloroplast and the type of habitat were the main phylogenetically relevant characters. Overall, Klebsormidium and Interfilum formed a more diverse algal group than was previously appreciated, with some lineages apparently undergoing active evolutionary radiation; in these lineages the genetic variation observed did not match the morphological and ecological diversity.

Research highlights

► The genetic diversity of the Klebsormidiales is higher than previously believed. ► A new lineage of Klebsormidium from arid soils of southern Africa is discovered. ► Paraphyly of Klebsormidium caused by Interfilum is demonstrated. ► The habitat occupied is phylogenetically more significant than previously appreciated. ► Morphological homoplasy is demonstrated in several lineages of Klebsormidium.

Introduction

Green algae with a thallus formed by uniseriate filaments are among the most widespread and ecologically versatile photosynthetic eukaryotes. Organisms with this type of morphology occur in almost all aquatic and terrestrial environments and belong to five major green algal lineages (Chlorophyceae, Klebsormidiophyceae, Trebouxiophyceae, Ulvophyceae and Zygnemophyceae; Lewis and McCourt, 2004).

Klebsormidium P.C. Silva, Mattox et Blackwell is a cosmopolitan genus of filamentous green algae to which 20 species are currently ascribed (Guiry and Guiry, 2010). Species of Klebsormidium are common in the microalgal vegetation of many terrestrial and freshwater environments, occurring in habitats as diverse as streams and rivers (Printz, 1964, John, 2002), margin of lakes (Lokhorst, 1996), bogs (John, 2002), soil (Ettl and Gärtner, 1995), natural rocks in plains and mountainous areas (Mikhailyuk et al., 2003), tree bark (Handa et al., 1991), acidic post-mining sites and water bodies (Lukešová, 2001, Sabater et al., 2003), golf courses (Baldwin and Whitton, 1992), sand dunes (Smith et al., 2004), biotic crusts of hot deserts (Lewis, 2007), bases of urban walls (Rindi and Guiry, 2004) and building façades (Barberousse et al., 2006). Species of Klebsormidium consist of uniseriate filaments devoid of differentiated or specialized cells. Each cell has a parietal chloroplast containing a pyrenoid and encircling half to 3/4 of the cell wall; reproduction takes place by biflagellate asymmetrical zoospores devoid of stigma that are produced singly in unspecialized cells, from which they escape through a pore (Silva et al., 1972, Lokhorst, 1991, Sluiman et al., 2008). The distinctness of Klebsormidium from similar genera of green algae was highlighted by TEM observations of cell division and zoospore ultrastructure (Floyd et al., 1972, Marchant et al., 1973, Lokhorst and Star, 1985). Stewart and Mattox (1975) established the placement of Klebsormidium in the Charophyceae, noting the presence of ultrastructural characters typical of this class (a persistent interzonal mitotic spindle, two laterally inserted flagella, a single broad band of microtubules associated with the flagellar basal bodies). These authors also based on Klebsormidium the new family Klebsormidiaceae and the new order Klebsormidiales, using characters specific to this taxon (centripetal cleavage furrow, little change in chromosome to spindle pole distance during anaphase, persistent chromosomal microtubules at telophase, and absence of plasmodesmata). The circumscription of the Klebsormidiales has changed over time as new data became available (Lokhorst, 1991, Van den Hoek et al., 1995, Sluiman et al., 2008) and is not yet fully clarified; it has been established that species of two other filamentous genera, Entransia Hughes and Hormidiella Iyengar et Khantamma, are the closest known relatives of Klebsormidium (Karol et al., 2001, Sluiman et al., 2008). Molecular data have confirmed conclusions based on ultrastructure and have robustly established the position of Klebsormidium and related taxa in the streptophytan lineage (Kranz et al., 1995, Karol et al., 2001, Turmel et al., 2002, Qiu et al., 2006). In more recent treatments this group has been regarded as an independent class, the Klebsormidiophyceae (Van den Hoek et al., 1995, Lewis and McCourt, 2004, Becker and Marin, 2009).

The reconstruction of the phylogenetic relationships in Klebsormidium and closely related taxa is problematic. Until recently the taxonomy of these algae was based on morphological data, which offer only a limited set of characters. Width and length of filaments, shape of cells, texture of the cell wall, presence/absence of a mucilaginous envelope, presence/absence of cell doublets or false branches, formation of H-shaped pieces, shape of the chloroplast and shape and size of the pyrenoid are the characters most commonly used for identification of field-collected specimens of Klebsormidium (Hazen, 1902, Printz, 1964, Ramanathan, 1964, Ettl and Gärtner, 1995, Lokhorst, 1996, Rifón-Lastra and Noguerol-Seoane, 2001, John, 2002). Some authors (Lokhorst, 1996) have attached more taxonomic significance to characters observable in liquid cultures, such as: presence/absence of a superficial hydrorepellent layer; tendency to fragment into short unattached filaments; type of reproduction (zoosporogenesis only or zoosporogenesis in combination with aplanosporogenesis); ease of inducibility of release of zoospores; shape of release aperture in lateral wall of zoosporangial cell (large and distinct or small and indistinct); and germination pattern of zoospores (unipolar and bipolar or unipolar only). It is known, however, that several characters exhibit great phenotypic plasticity and may vary dramatically in relation to environmental conditions and physiological state of the specimens examined (Lokhorst, 1996, Dřímalová and Poulíčková, 2003, Škaloud, 2006, Rindi et al., 2008). It is presently unclear how morphological characters have evolved and which characters must be considered phylogenetically significant. In this regard, unfortunately, our understanding of the evolutionary history of the group is too limited to be of any assistance. No fossil record is available for Klebsormidium (a situation that is general to the green algae, with the exception of a few groups provided with calcified cell walls, e.g. McCourt et al., 1996, Verbruggen et al., 2009). Therefore, it is unknown what was the morphology of the ancestral klebsormidialean alga and what character states should be considered ancestral.

Molecular studies focused on phylogeny at genus and species level have become available recently and have led to several unexpected discoveries (Novis, 2006, Mikhailyuk et al., 2008, Rindi et al., 2008, Sluiman et al., 2008). Sluiman et al. (2008) demonstrated that Entransia fimbriata Hughes and Hormidiella attenuata Lokhorst are the closest relatives to Klebsormidium currently known. The topology of the ITS rRNA tree presented by these authors is in disagreement with the morphological parsimony trees presented by Lokhorst (1996). Rindi et al. (2008), whose analyses included the largest taxon sampling of Klebsormidium strains currently available, found great difficulties in mapping morphological characters with phylogenetic significance on their rbcL trees. Recently, Mikhailyuk et al. (2008) showed that Interfilum Chodat et Topali is closely related to Klebsormidium, but they were not able to clarify the relationships between these genera. Species of Interfilum consist of unicells, packet-like aggregates or filaments of cells united by thin mucilaginous envelopes or strands of cell wall material (Chodat and Topali, 1922, John, 2002, Mikhailyuk et al., 2008). This finding revolutionised the concept of morphological evolution in Klebsormidium and close relatives, revealing that this group encompasses a wider range of morphological forms than previously appreciated. Overall, these studies indicate that our understanding of the genetic diversity and phylogeny of this algal group is still very incomplete and that new data are required.

In the present study, 29 new rbcL sequences and 66 new ITS rRNA sequences of Interfilum and Klebsormidium are added to the body of molecular data previously available. Sequences of these markers are also analyzed for the first time in a concatenated dataset. Our results extend considerably the taxon sampling and provide robust phylogenies that advance substantially our understanding of the relationships of these algae. These data will be of fundamental importance for future reassessments of the classification of the order Klebsormidiales.

Section snippets

Collections, taxon sampling and vouchering

Samples of Klebsormidium and Interfilum were isolated by the authors or external collaborators or obtained from culture collections (Supplementary Table 1). For the new samples isolated in the course of this study, the following morphological characters were examined in freshly isolated cultures: habit of filaments; tendency to fragmentation; width and length of cells; shape of cells; formation of H-shaped pieces; thickness and texture of the cell wall; formation of a mucilaginous envelope;

Results

Details of the characteristics of the datasets analyzed are presented in Table 1. For each dataset analyzed, analyses performed with different methods of inference provided similar topologies, usually with limited differences in nodal support. The rbcL gene and the ITS rRNA showed congruent phylogenetic signals and identical groups were recovered in the two phylogenies. However, most internal nodes did not receive significant statistical support in any analysis for any dataset.

The results of

Characteristics of the datasets analyzed

The larger taxon sampling of the phylogenies presented here resulted in an increased statistical support and better resolution in comparison with previous investigations. This is also the first study on Klebsormidium and Interfilum in which two different molecular markers are combined and analyzed in a concatenated dataset. Not unexpectedly, in the combined dataset the nodal support is higher than in single-marker analyses; it is well established that an increase in the number of genes or

Acknowledgments

We wish to express sincere thanks to Dr. Alena Lukešová, Dr. Tatyana Darienko, Dr. Opayi Mudimu, Dr. Lira Gaysina and Dr. Mario Salisch for providing some strains of Interfilum and Klebsormidium. The study was funded by the US National Science Foundation (Systematics Program DEB-1036495 and Assembling the Tree of Life Program DEB-0542924 to J.M.L.-B.) and the European Community (INTAS Grant for Young Scientist Fellowship 06-1000014-6216 to T.I.M.). Parts of the research were carried out in the

References (73)

  • D. Dřímalová et al.

    Filament fragmentation of Klebsormidium flaccidum

    Biologia

    (2003)
  • H. Ettl et al.

    Syllabus der Boden-, Luft- und Flechtenalgen

    (1995)
  • A.J. Fazekas et al.

    Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well

    PloS One

    (2008)
  • J. Felsenstein

    Confidence limits on phylogenies: an approach using the bootstrap

    Evolution

    (1985)
  • G.L. Floyd et al.

    Cellular organization, mitosis and cytokinesis in the ulotrichalean alga Klebsormidium

    J. Phycol.

    (1972)
  • W. Foissner

    Protist diversity and distribution: some basic considerations

    Biodivers. Conserv.

    (2008)
  • T. Friedl et al.

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

    Eur. J. Phycol.

    (2002)
  • Guiry, M.D., Guiry, G.M., 2010. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway....
  • S. Handa et al.

    Some corticolous algae from Shibetsu, Hokkaido, northern Japan

    J. Jpn. Bot.

    (1991)
  • H.S. Hayden et al.

    Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera

    Eur. J. Phycol.

    (2003)
  • T.E. Hazen

    The Ulotrichaceae and Chaetophoraceae of the United States

    Mem. Torrey Bot. Club

    (1902)
  • J.P. Huelsenbeck et al.

    MRBAYES: Bayesian inference of phylogeny

    Bioinformatics

    (2001)
  • Jobb, G., 2008. TREEFINDER version October 2008. Munich, Germany....
  • D.M. John

    Orders Chaetophorales, Klebsormidiales, Microsporales, Ulotrichales

  • N. Jürgens et al.

    Biodiversity in southern Africa

    (2010)
  • K.G. Karol et al.

    The closest living relatives of land plants

    Science

    (2001)
  • H.D. Kranz et al.

    The origin of land plants: phylogenetic relationships among charophytes, bryophytes and vascular plants inferred from complete small-subunit ribosomal RNA gene sequences

    J. Mol. Evol.

    (1995)
  • W.J. Kress et al.

    Use of DNA barcodes to identify flowering plants

    Proc. Natl. Acad. Sci. USA

    (2005)
  • Kützing, F.T., 1849. Species algarum. F.A. Brockhaus, Leipzig, 922...
  • R. Lahaye et al.

    DNA barcoding the floras of biodiversity hotspots

    Proc. Natl. Acad. Sci. USA

    (2008)
  • G. Lear et al.

    Biofilm bacterial community structure in streams affected by acid mine drainage

    Appl. Environ. Microbiol.

    (2009)
  • C. Lemieux et al.

    A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies

    BMC Biol.

    (2007)
  • L.A. Lewis

    Chlorophyta on land: independent lineages of green eukaryotes from arid lands

  • L.A. Lewis et al.

    Green algae (Chlorophyta) of desert microbiotic crusts: diversity of North American taxa

    Taxon

    (2002)
  • L.A. Lewis et al.

    Green algae and the origin of land plants

    Am. J. Bot.

    (2004)
  • G.M. Lokhorst

    Synopsis of Genera of Klebsormidiales and Ulotrichales

    Cryptogam. Bot.

    (1991)
  • Cited by (0)

    View full text