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Multigene Evidence for the Placement of a Heterotrophic Amoeboid Lineage Leukarachnion sp. among Photosynthetic Stramenopiles
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Protist, Vol. 160, 376—385, August 2009 http://www.elsevier.de/protis Published online date 11 March 2009
ORIGINAL PAPER
Multigene Evidence for the Placement of a Heterotrophic Amoeboid Lineage Leukarachnion sp. among Photosynthetic Stramenopiles Jessica Granta,1, Yonas I. Teklea,1, O. Roger Andersonb, David J. Pattersonc, and Laura A. Katza,2 a
Department of Biological Sciences, Smith College, Northampton, MA 01063, USA Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA c Marine Biological Laboratory, Woods Hole, MA 02543, USA b
Submitted August 8, 2008; Accepted January 3, 2009 Monitoring Editor: Robert A. Andersen
The colorless amoeboid eukaryote genus Leukarachnion represents one of a long list of microbial lineages for which there have been few taxonomic studies. In this study, we analyze molecular data to assess the placement of a species of Leukarachnion on the eukaryotic tree of life and we report fine structural data to provide additional information on the identity of this taxon. Our multigene analyses indicate that Leukarachnion sp. (ATCCs PRA-24) is a member of the stramenopiles, sister to the Chrysophyceae/Synurophyceae clade. It also forms a sister group relationship to the clade containing Chlamydomyxa labyrinthuloides and Synchroma grande, both of which are characterized by net-like amoeboid phases. Leukarachnion sp. and Chlamydomyxa labyrinthuloides also share fine structural cyst morphology such as bilayered structure of the cyst wall. The amoeboid form and heterotrophic habit of Leukarachnion sp. highlight the multiple origins of diverse body forms and multiple plastid losses within the stramenopiles. & 2009 Elsevier GmbH. All rights reserved. Key words: stramenopiles; Leukarachnion; multigene analysis; heterotrophic; SSU-rDNA; phylogeny; Heterokontophyta; Chrysophyceae; Synurophyceae; amoeboid.
Introduction The colorless amoeboid eukaryote Leukarachnion Geitler is a large multinucleate amoeba with branching ‘net-like’ pseudopodia (Geitler 1942). Leukarachnion grows to several millimeters in size, forming anastomosing networks (Geitler 1942). The genus Leukarachnion has one described species, Leukarachnion batrachospermi, which e-mail [email protected] (L.A. Katz). 1 The authors contributed equally to this work. 2 Corresponding author; fax +11 413 585 3786.
& 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2009.01.001
was originally found in close association with the freshwater red alga, Batrachospermum boryanum (Geitler 1942). The taxonomic affinity of Leukarachnion is uncertain partly due to lack of detailed morphological data (Geitler 1942; Patterson 2002). Leukarachnion has been superficially compared to other large plasmodial amoebae such as Thallasomyxa, Stereomyxa, and Cenetidomyxa (Geitler 1942; Patterson 2002) as well as to lineages that create net-like structures such as Chrysarachnion
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Phylogenetic Position of Leukarachnion
insidians (Geitler 1942). In this study we characterize multiple gene sequences from the Leukarachnion sp. obtained from American Type Culture Collection (ATCCs PRA-24) and present fine structural analyses to enhance the identification of this strain. To assess the phylogenetic position of Leukarachnion sp., we analyze three different data sets that include up to four nuclear encoded genes: SSU-rDNA, actin, alpha- and beta-tubulin. Our preliminary multigene analysis, which included a broad sampling of representative eukaryotes, placed Leukarachnion sp. ATCCs PRA-24 within the stramenopiles, a diverse assemblage of organisms that include a wide array of forms, many of which are photosynthetic. We assess the support for the placement of Leukarachnion sp. within the stramenopiles and investigate its relationship with other members of this clade. Stramenopiles form one of the robust eukaryotic groups that have been recovered consistently in a number of molecular studies (Ben Ali et al. 2002; Bhattacharya et al. 1992; Cavalier-Smith et al. 1994; Cavalier-Smith and Chao 2006; Leipe et al. 1994; Van de Peer and De Wachter 1997; Van der Auwera and De Wachter 1997; Yoon et al. 2008) and share an ultrastructural synapomorphy, tripartite tubular hairs attached to one of their flagella (Patterson 1989). Inclusion of the colorless amoeboid Leukarachnion sp. within this clade enhances the diversity of body forms among stramenopiles and suggests that additional amoeboid lineages of stramenopiles await further study.
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Results Light Microscopic Observations Leukarachnion sp. ATCCs PRA-24 formed a large delicate network with enlarged cell bodies (2—10 mm) and fine cytoplasmic extensions (Fig. 1A). The network was dynamic as ingested bacteria and other cytoplasmic contents moved through the thin strands of the network to the core cell bodies (Fig. 1A). When the network was disturbed it rolled back to form blobby cells, reminiscent of how cysts form during unfavorable conditions. Younger cultures contained cysts of varying sizes, ranging from 5 to 15 mm (Fig. 1B), and as it aged the cysts became smaller in size until the culture was filled with uniform 5 mm cysts. A flagellate was observed in the first culture worked on (used to generate tube 1 DNA) but not in any subsequent cultures (used to generate tubes 2 and 3 DNA). We did not have an opportunity to observe the flagellate in close detail within the first culture so the morphology of the flagella was not determined. Future examination is required to elucidate additional details of the life cycle of Leukarachnion sp. ATCCs PRA-24.
TEM Observations The overall fine structure of Leukarachnion cells (Fig. 2A, B) exhibited a finely granular, relatively hyaline, cytoplasm surrounded by an uncoated plasma membrane. Slender pseudopodial projections were commonly observed projecting from
Figure 1. Light micrograph of Leukarachnion sp. ATCCs-PRA-24. A. Image of a living organism showing an extensively branched network. Scale bar ¼ 50 mm. B. Cysts at different maturation stage. Mature cysts are smaller in size. Scale bar ¼ 25 mm.
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Figure 2. Transmission electron microscopic images of Leukarachnion sp. A. Cross-section of a cell with nucleus (N), tubulo-cristate mitochondria (M) and several food vacuoles (FV) distributed peripherally. Fine pseudopodial projections (arrow) are commonly observed. Scale bar ¼ 1 mm. B. Cells frequently contain more than one nucleus (N) characterized by a central nucleolus and a dense deposit in the nuclear envelope exhibiting a dark halo effect. Occasional elongated sacculi (arrow), possibly smooth endoplasmic reticulum, are observed near the nucleus. The plasma membrane surrounding the cell is consistently uncoated. Scale bar ¼ 1 mm. C. An enlarged view of a tubulo-cristate mitochondrion exhibiting the characteristic osmiophilic matrix and abundant tubular cristae. Scale bar ¼ 0.25 mm. D—F. Fine structure of the cyst and surrounding wall (arrows) including the mature wall with an outer fibrous layer (OW) and an inner more dense layer (IW). D. The cytoplasm is characteristically electron dense and the wall is deposited initially as a fine, fibrillar layer immediately adjacent to the plasmalemma. Scale bar ¼ 0.5 mm. E. Subsequently, an inner layer of the wall is deposited that is initially loosely organized and less dense. Scale bar ¼ 0.5 mm. F. The mature bilayered wall, directly adjacent to the plasmalemma, contains a thicker, electron dense inner layer. Scale bar ¼ 0.1 mm.
the surface of the cells (arrow, Fig. 2A). In some planes of section through the nucleus (about 1—1.5 mm diameter) there was a pronounced central nucleolus (0.5 mm), while in other sections the nucleoplasm appeared to be more uniformly electron dense. The nuclear envelope was somewhat undulating and contained an electron dense deposit producing the effect of a dark halo surrounding the nucleus (Fig. 2B). In general, the
mitochondria (approximately 0.8 mm long and 0.25 mm in diameter) had abundant, closely spaced (about 40 nm diameter) tubular cristae and a dense matrix (Fig. 2C). In many sections, at least two nuclei were observed (e.g., Fig. 2B). The endoplasmic reticulum was sparse and no clearly defined Golgi bodies were observed. However, occasional solitary or scattered flattened, elongated sacculi, possibly smooth endoplasmic
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Phylogenetic Position of Leukarachnion
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reticulum, were observed in the vicinity of the nucleus (Fig. 2B). Cysts were commonly observed in the preparations and exhibited successive stages of maturation and wall deposition (Fig. 2D—F). The cysts (about 5 mm in diameter) presented a circular or nearly circular profile in cross-section and contained densely staining cytoplasm. The cyst wall was initially deposited as a thin (0.1—0.2 mm thick) fibrous envelope followed by secretion of a more electron dense inner layer of approximately equal thickness, yielding a bi-layered mature wall (Fig. 2F). Based on the electron microscopic observation, the wall of the cyst exhibited features that indicate it is largely organic. Walls that are mineralized show evidence of fracture planes during sectioning that were not observed here, but we did not analyze the chemical constituents of the cyst walls using analytical techniques. The wall closely enclosed the cyst cytoplasm and no evidence of a pore was observed.
produced an alpha-tubulin sequence that was 98% similar to one of the haplotypes from tube-1 (GenBank accession number FJ602385). We did not sequence alpha-tubulin from Tube-3. The five beta-tubulin sequences generated from tube-1 were identical to one another (GenBank accession number FJ356263), while tube-2 produced three haplotypes that were 94-98% similar to tube-1 (GenBank accession numbers FJ602388, FJ602389, FJ602390). Tube-3 produced one beta-tubulin sequence that was identical to one of the tube-2 haplotypes. The variable gene sequences found in alpha- and beta-tubulin clustered together in single gene analyses (data not shown) and hence one sequence from each gene was chosen for use in the concatenated alignment. There were no introns found in any of the protein coding genes and the entire sequence was used in the subsequent analyses.
Molecular Evolution
Initial analysis of our newly characterized genes combined with sequences from diverse eukaryotes placed Leukarachnion sp. within the stramenopiles (data not shown). To further examine this placement, we built a concatenated alignment including taxa that span the diversity of the stramenopiles, as well as outgroup taxa from the Alveolata, ‘Rhizaria’, Haptophyta, and Opisthokonta. The resulting topologies of the phylogenetic trees built from our three datasets using two algorithms (RaxML and MrBayes) were generally concordant, with no well-supported nodes in disagreement. Furthermore, the topology of the major clades within the stramenopiles were generally congruent with previously published phylogenies (Andersen 2004; Cavalier-Smith and Chao 2006), although support at deeper nodes is generally poor (Fig. 3). In all of our analyses, Leukarachnion sp. ATCCs PRA-24 consistently fell sister to the amoeboid stramenopiles Chlamydomyxa labyrinthuloides and Synchroma grande with strong support in analyses that include these taxa (Table 1, Fig. 3) (i.e. Bootstrap support (BS) ¼ 100%, Bayesian Posterior Probability (BPP) ¼ 1.0 in the analyses of SSU rDNA only). Together, these three lineages were sister to a clade containing Chrysophyceae and Synurophyceae (see Table 1).
Because of concerns about possible contaminants, particularly in light of the flagellates observed in our first culture, we characterized multiple clones of our target genes from DNA extractions from three separate cultures. We sequenced SSU rDNA as well as actin, and alphaand beta-tubulin from two tubes of DNA (tube-1 and tube-2) extracted at different times at ATCCs, as well as DNA extracted from cultures kept at Smith College (tube-3). The SSU genes from all three samples were identical (GenBank accession number FJ356265). The actin sequences, obtained by sequencing eight clones of tube-1, were identical to one another (GenBank accession number FJ356266) and we were able to amplify an identical 300 base pair segment of this gene from tubes 2 and 3 using taxon-specific primers. There was variation among alpha- and betatubulin sequences from the three DNA extractions, though resulting sequences were consistent with either allelic diversity or recent paralogs within a single taxon. Three unique alphatubulin sequences were obtained from 7 clones from tube-1 DNA (GenBank accession numbers FJ356264, FJ602386, FJ602387). These sequences vary from 3.4% to 6.4% at the nucleotide level, and one sequence (GenBank accession number FJ602385) contained a 7 base pair deletion. We believe this clone may be a PCR artifact, as the nucleotides were conserved both before and after the deletion. Analysis of 1 clone from tube-2
Concatenated Four Genes Analyses
Taxonomic Sampling and Missing Data To explore the robustness of the position of Leukarachnion sp. on our gene trees, we assessed
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Figure 3. Maximum likelihood tree of dataset-A (114 taxa that have data for at least the SSU rDNA and a subset of the protein genes, see supplemental material) inferred using RaxML of four concatenated genes (SSU rDNA plus actin, alpha- and beta-tubulin as nucleotides, ambiguous regions and third positions of protein coding genes excluded). BS/BPP support greater than 50% are shown at the nodes (*indicates o50% support). Solid dots are on nodes with full support under both models. C_Ectocarpus* indicates that the sequences used in the multigene analysis are from two different species. The SSU rDNA is from Ectocarpus siliculosus and the Btub is from Ectocarpus verabilis.
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Support values are RaxML bootstrap support/Bayesian posterior probabilities. (L+(C+S)) ¼ The Leukarachnion sp.+(C. labyrinthuloides+ S. grande) clade or Leukarachnion alone in taxon sets (designated by *) that do not include the other two taxa. Dataset A (114 taxa, 41% missing data) includes all taxa for which at least the SSU sequence was available. Dataset B (57 taxa, 21% missing data) includes all taxa for which at least the SSU and one of the three protein coding genes were available. Dataset C (46 taxa, 12% missing data) includes all taxa for which at least the SSU and two of the three protein coding genes were available. Nm ¼ not monophyletic.
Table 1. Support for various taxonomic hypotheses with datasets of varying taxon sampling and missing data.
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relationships under varying taxon samples and with varying levels of missing data. The placement of Leukarachnion sp. within the stramenopiles was not substantially affected when data matrices with varying percentages of missing data (4—41%) and taxon numbers (46—114 taxa) were analyzed (Table 1). In general, the support for the grouping of Leukarachnion sp. with the Chrysophyceae+ Synurophyceae clade was shown to increase as the percentage of missing data and taxon sampling decreased. For example, in the 114 taxon matrix with 41% missing data, the support for the clade containing Leukarachnion sp.+(Synchroma grande+Chlamydomyxa labyrinthuloides) with the Chrysophyceae/Synurophyceae clade was 0.97 BPP (70% BS). The support increased to 1.0 BPP (100% BS) when the 57 taxon matrix with 21% missing data was analyzed (Table 1).
Discussion ATCCs PRA-24 Isolate is Leukarachnion sp. The ATCCs PRA-24 isolate shares a number of characters with the one published description of Leukarachnion batrachospermi, including network and cyst formation as well as plasmodia. Leukarachnion batrachospermi was described in 1942 with beautiful line drawings; there are neither electron micrographs nor molecular data for this taxon. Our isolate is much smaller than L. batrachospermi, which could grow to a few millimeters in size (Geitler 1942). Further comparison of our isolate with L. batrachospermi cannot be made due to lack of a culture of L. batrachospermi. The light and TEM data provided here will contribute to a formal description of our isolate, which awaits more detailed data for L. batrachospermi. In the meantime, the ATCCs PRA-24 isolate is identified as a Leukarachnion sp.
Phylogenetic Placement of Leukarachnion sp. ATCCs PRA-24 Molecular analyses show Leukarachnion sp. ATCCs PRA-24 grouping with the photosynthetic stramenopiles Chlamydomyxa labyrinthuloides and Synchroma grande (Fig. 3, Table 1). These lineages are similar in gross morphology including amoeboid body form, plasmodia and network/ branching pseudopodia. This sister relationship is further corroborated by ultrastructural characters, such as the similar fine structure of the cyst wall with Chlamydomyxa labyrinthuloides (Pearlmutter
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and Timpano 1984); cysts are not reported for Synchroma grande. Both Leukarachnion sp. and Chlamydomyxa labyrinthuloides cysts possess a similar bilayered wall, an inner dense layer and an outer more fibrous layer, each layer of similar thickness (Pearlmutter and Timpano 1984). The recently described Synchroma grande was placed in a monotypic class, Synchromophyceae Horn et Wilhelm based primarily on unique chloroplast structures and distinctive 18S rRNA and rbcL sequences (Horn et al. 2007). The analyses presented here with the additional taxon sampling group S. grande with Leukarachnion sp. ATCCs PRA-24, an organism with no chloroplasts, and C. labyrinthuloides, an organism with more traditional chloroplasts. As neither of these taxa matches the class description of Synchromophyceae, this class designation must be formally reevaluated. This might best be done following characterization of several other amoeboid lineages described in earlier literature that are morphologically similar to Leukarachnion sp. but for which no molecular data are yet available (see below). In a recent classification, Cavalier-Smith and Chao (2006) placed Chlamydomyxa and Picophagus in a new class, Picophagea, based on an analysis of SSU-rDNA. None of our analyses, including SSU rDNA only or concatenated SSU rDNA plus three protein-coding genes, recover the Chlamydomyxa+Picophagus clade (Fig. 3). On the contrary, the Leukarachnion sp.+(Chlamydomyxa labyrinthuloides+Synchroma grande) clade leans more to the Chrysophyceae/Synurophyceae clade to the exclusion of Picophagus flagellatus (Table 1; Fig. 3). Although an AU-test (p-value ¼ 0.43) could not reject the grouping of P. flagellatus at the base of Leukarachnion sp.+Chlamydomyxa labyrinthuloides+Synchroma grande clade, three alternative hypotheses (see Methods) that place Chlamydomyxa and Picophagus together were significantly worse (p-value o0.0001 (for AU test) and p-values o0.0001 (for SH/KH tests)). These findings indicate that the class Picophagea sensu CavalierSmith and Chao 2006 requires further scrutiny.
Evolution of Stramenopiles Despite its heterotrophic habit and reticulate amoeboid form, Leukarachnion sp. falls with robust support within a clade of predominantly photosynthetic, flagella-bearing lineages (Fig. 3; see also Ben Ali et al. 2002). This indicates that Leukarachnion sp. ATCCs PRA-24 is secondarily heterotrophic, though ultrastructural analysis of
our isolate shows no evidence of a leukoplast or any plastid-like structure (Fig. 2). We attempted to amplify the rbcL gene from Leukarachnion sp. using degenerate primers but we were not successful (data not shown). Within the autotrophic heterokonts there are other lineages that have lost photosynthetic ability, though many of these lineages, such as Spumella and Paraphysomonas in the Chrysophyceae and Pteridomonas in the Dictyochophyceae, are reported to possess vestigial colorless plastids known as leukoplasts (Belcher and Swale 1976; Mignot 1977). Intriguingly, Leukarachnion may represent just one of a diverse clade of colorless amoeboid lineages described by light microscopy in earlier literature. Examples of such taxa include lineages that form net-like structures similar to Leukarachnion such as Chrysarachnion insidians (Pascher 1917), Leukapsis vorax (Pascher 1940), Chrysidiastrum catenatum (Lauterborn 1913), and Heliapsis mutabilis (Pascher 1940). As taxonomic sampling of diverse eukaryotes continues, we will be able to assess the hypothesis that these morphologically similar taxa represent a monophyletic and diverse clade within the stramenopiles. The placement of Leukarachnion sp. ATCCs PRA-24 within the stramenopiles adds to the known diversity within this clade. Stramenopiles contain unicellular and multicellular forms; lineages with hetrotrophic, mixotrophic and autotrophic modes of nutrition; lineages with flagellate and amoeboid forms, free-living species and parasites (Andersen 2004). Moreover, Leukarachnion is just one of about 200 described eukaryotic lineages whose taxonomic position is undetermined (Adl et al. 2005; Patterson 1999). The findings reported here further reinforce the power of multigene analyses in placing lineages of uncertain origin (Tekle et al. 2007; Yoon et al. 2008). Despite the great morphological diversity among stramenopiles, their monophyly has not been questioned. However, their relationship to other eukaryotic groups is controversial: a number of studies suggest that stramenopiles are sister to alveolates (Baldauf et al. 2000) while some multigene and phylogenomic analyses suggest that stramenopiles plus alveolates are sister to the putative supergroup ‘Rhizaria’ (Burki et al. 2007; Hackett et al. 2007). Though our analyses were not structured to test these hypotheses fully, it is noteworthy that stramenopiles group with alveolates with strong support (Table 1). In contrast, the monophyly of ‘Rhizaria’ plus alveolate plus stramenopile is not supported in our taxon-rich SSU
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Phylogenetic Position of Leukarachnion
rDNA only analysis, and receives mixed support with inclusion of protein genes (Table 1).
Methods Sampling and molecular methods: DNA samples of Leukarachnion sp. ATCCs PRA-24TM, originally isolated from a salt marsh in Virginia, using DNA Stat60TM (Tel-Test, Inc., Friendswood, Texas; Cat. No. TL-4220) per manufacturer’s instructions and with the addition of a phenol-chloroform-isoamyl step using Phase Lock Gel Heavy tubes (Eppendorf AG, Hamburg, Germany; Cat. No. 955154070). Additional genomic DNA from the same isolate cultured at Smith College was extracted with a DNeasy Tissue kit (Qiagen, Valencia, CA, USA 28106; Cat. No. 69504). Data analysis: Primers for SSU rDNA genes are from Medlin et al. (1988) with three additional primers used to generate overlapping sequences from each clone as described in Snoeyenbos-West et al. (2002). Primers for actin, alpha-tubulin and beta-tubulin are from Tekle et al. (2007). Vent DNA Polymerase (NEB Cat. No. M0254L) and Phusion DNA Polymerase (NEB Cat. No. F-540L), strict proofreading enzymes, were used to amplify our genes of interest and we have used Lucigen PCRSmart (Lucigen Inc., Middleton, WI 53562, Cat. No. 41120-4), Novagen Perfectly Blunt (Novagen Inc., Madison WI 53719, Cat. No. 70191-4), and Invitrogen Zero Blunt Topo (Invitrogen, Inc., Carlsbad, CA 92008, Cat. No. K2800) cloning kits. Sequencing of cloned plasmid DNA was accomplished using vector-or gene-specific primers and the BigDye terminator kit (Perkin Elmer, Waltham, MA). Sequences were run on an ABI 3100 automated sequencer. We have fully sequenced 2—6 clones of each gene and surveyed up to 10 clones per gene in order to detect paralogs. The SSU rRNA sequences were aligned using ClustalW (Thompson et al. 1994) as implemented in DNAstar’s Lasergene software. The resulting alignment was further edited manually in MacClade v4.08 (Maddison and Maddison 2005). Protein coding genes were aligned as amino acids using Clustal W (Thompson et al. 1994) as implemented in DNAstar’s Lasergene software and adjusted by eye in MacClade (Maddison and Maddison 2005). Our genealogies excluded ambiguously aligned regions. In addition, the nucleotide genealogies excluded 3rd positions. The SSU rDNA alignment contained a total of 1600 characters, and the concatenated alignments contained 3766 characters (SSU rDNA plus protein coding genes as nucleotides) and 2683 (SSU rDNA plus protein coding genes as amino acids). Alignments are available at http://www.science.smith.edu/ departments/Biology/lkatz/data/Leukarachnion. We analyzed three datasets to investigate the impact of taxonomic sampling and missing data on our results. Dataset A (114 taxa) includes a broad sampling of taxa for which at least the ssu rDNA gene sequence data was available. Dataset B (57 taxa) includes those taxa from set A for which at least one of the three protein coding genes in our study was available and dataset C (46 taxa) includes those taxa from set A for which at least two of the three protein coding genes was available. As the taxa for which there were fewer data available were removed to form the smaller datasets, the number of missing characters decreased as well. Dataset A has 41% missing data, while datasets B and C have 21% and 12%, respectively. We also analyzed the SSU rRNA gene alone for the taxa in dataset A, for an analysis with the least amount of missing data (4%).
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Phylogenies were constructed in MrBayes (Huelsenbeck and Ronquist 2001) and RaxML (Stamatakis et al. 2005a, 2005b). Bayesian analyses were performed with the parallel version of MrBayes 3.1.2 using the GTR+I+G (for nucleotide) and rtREV (for amino acid) models of sequence evolution (Ronquist and Huelsenbeck 2003). Four simultaneous MCMCMC chains were run for 4,000,000 generations sampling every 100 generations. The 50% majority-rule consensus tree was determined to calculate the posterior probabilities for each node. RaxML (Stamatakis et al. 2005a, 2005b) was run for 100 iterations using the GTRGAMMA model for nucleotide data and PROTGAMMA with matrix rtREV for amino acid data. The datasets were partitioned to allow for different parameters for each gene. A total of 100 independent bootstrap analyses were run in RaxML and a 50% majority rule consensus was calculated to determine the support values for each node. MrModeltest (Nylander 2004) and ProtTest (Abascal et al. 2005) were used to select the appropriate model of sequences evolution for the nucleotides and amino acid data, respectively. To compare competing hypotheses on the placement of Leukarachnion and the monophyly of Chlamydomyxa and Picophagus (Class Picophagea) to the exclusion of other taxa, we performed the approximately unbiased (AU) test (Shimodaira 2002) as well as the more conventional Kishino—Hasegawa and Shimodaira—Hasegawa tests, as implemented in Consel 0.1j (Shimodaira and Hasegawa 2001). We tested for the following alternative hypotheses (a) the monophyly of ((Leukarachnion sp.+(C. labyrinthuloides, S. grande))+ P. flagellatus) (b) the monophyly of (C. labyrinthuloides+ P. flagellatus) (c) the monophyly of ((C. labyrinthuloides+ P. flagellatus)+(C. labyrinthuloides+S. grande)) (d) the monophyly of ((C. labyrinthuloides+P. flagellatus)+(Chrysophyceae/ Synurophyceae)).
Acknowledgements The authors thank Jeff Cole for technical assistance. This work is supported by the National Science Foundation Assembling the Tree of Life grant (043115) to LAK and DJP. This is LamontDoherty Earth Observatory Contribution Number 7176.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/ j.protis.2009.01.001.
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Pascher A (1917) Flagellaten und Rhizopoden in ihren gegenseitigen Beziehungen. Arch Protistenkd 38: 1—88 Patterson DJ (1989) Stramenopiles: Chromophytes from a Protistological Perspective. In Green JC, Leadbeater BSC, Diver WL (eds) The Chromophyte Algae: Problems and Perspectives. Clarendon Press, Oxford, pp 357—379 Patterson DJ (1999) The diversity of eukaryotes. Am Nat 154: S96—S124 Patterson DJ (2002) Changing Views of Protistan Systematics: The Taxonomy of Protozoa — An Overview. In Lee JJ (ed) An Illustrated Guide to the Protozoa. 2nd Ed. Society of Protozoologists, Lawrence, KS Pearlmutter NL, Timpano P (1984) The biology of Chlamydomyxa montana — ultrastructure of the cyst. Protoplasma 122: 68—74 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572—1574 Shimodaira H (2002) An approximately unbiased test of phylogenetic tree selection. Syst Biol 51: 492—508 Shimodaira H, Hasegawa M (2001) CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17: 1246—1247 Snoeyenbos-West OLO, Salcedo T, McManus GB, Katz LA (2002) Insights into the diversity of choreotrich and oligotrich ciliates (Class: Spirotrichea) based on genealogical analyses of multiple loci. Int J Syst Evol Microbiol 52: 1901—1913 Stamatakis A, Ludwig T, Meier H (2005a) RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics 21: 456—463 Stamatakis A, Ott M, Ludwig T (2005b) RAxML-OMP: an efficient program for phylogenetic inference on SMPs. Parall Comput Technol 3606: 288—302 Tekle YI, Grant J, Cole JC, Nerad TA, Anderson OR, Patterson DJ, Katz LA (2007) A multigene analysis of Corallomyxa tenera sp. nov. suggests its membership in a clade that includes Gromia, Haplosporidia and Foraminifera. Protist 158: 457—472 Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673—4680
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Protist, Vol. 160, 376—385, August 2009 http://www.elsevier.de/protis Published online date 11 March 2009
ORIGINAL PAPER
Multigene Evidence for the Placement of a Heterotrophic Amoeboid Lineage Leukarachnion sp. among Photosynthetic Stramenopiles Jessica Granta,1, Yonas I. Teklea,1, O. Roger Andersonb, David J. Pattersonc, and Laura A. Katza,2 a
Department of Biological Sciences, Smith College, Northampton, MA 01063, USA Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA c Marine Biological Laboratory, Woods Hole, MA 02543, USA b
Submitted August 8, 2008; Accepted January 3, 2009 Monitoring Editor: Robert A. Andersen
The colorless amoeboid eukaryote genus Leukarachnion represents one of a long list of microbial lineages for which there have been few taxonomic studies. In this study, we analyze molecular data to assess the placement of a species of Leukarachnion on the eukaryotic tree of life and we report fine structural data to provide additional information on the identity of this taxon. Our multigene analyses indicate that Leukarachnion sp. (ATCCs PRA-24) is a member of the stramenopiles, sister to the Chrysophyceae/Synurophyceae clade. It also forms a sister group relationship to the clade containing Chlamydomyxa labyrinthuloides and Synchroma grande, both of which are characterized by net-like amoeboid phases. Leukarachnion sp. and Chlamydomyxa labyrinthuloides also share fine structural cyst morphology such as bilayered structure of the cyst wall. The amoeboid form and heterotrophic habit of Leukarachnion sp. highlight the multiple origins of diverse body forms and multiple plastid losses within the stramenopiles. & 2009 Elsevier GmbH. All rights reserved. Key words: stramenopiles; Leukarachnion; multigene analysis; heterotrophic; SSU-rDNA; phylogeny; Heterokontophyta; Chrysophyceae; Synurophyceae; amoeboid.
Introduction The colorless amoeboid eukaryote Leukarachnion Geitler is a large multinucleate amoeba with branching ‘net-like’ pseudopodia (Geitler 1942). Leukarachnion grows to several millimeters in size, forming anastomosing networks (Geitler 1942). The genus Leukarachnion has one described species, Leukarachnion batrachospermi, which e-mail [email protected] (L.A. Katz). 1 The authors contributed equally to this work. 2 Corresponding author; fax +11 413 585 3786.
& 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2009.01.001
was originally found in close association with the freshwater red alga, Batrachospermum boryanum (Geitler 1942). The taxonomic affinity of Leukarachnion is uncertain partly due to lack of detailed morphological data (Geitler 1942; Patterson 2002). Leukarachnion has been superficially compared to other large plasmodial amoebae such as Thallasomyxa, Stereomyxa, and Cenetidomyxa (Geitler 1942; Patterson 2002) as well as to lineages that create net-like structures such as Chrysarachnion
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Phylogenetic Position of Leukarachnion
insidians (Geitler 1942). In this study we characterize multiple gene sequences from the Leukarachnion sp. obtained from American Type Culture Collection (ATCCs PRA-24) and present fine structural analyses to enhance the identification of this strain. To assess the phylogenetic position of Leukarachnion sp., we analyze three different data sets that include up to four nuclear encoded genes: SSU-rDNA, actin, alpha- and beta-tubulin. Our preliminary multigene analysis, which included a broad sampling of representative eukaryotes, placed Leukarachnion sp. ATCCs PRA-24 within the stramenopiles, a diverse assemblage of organisms that include a wide array of forms, many of which are photosynthetic. We assess the support for the placement of Leukarachnion sp. within the stramenopiles and investigate its relationship with other members of this clade. Stramenopiles form one of the robust eukaryotic groups that have been recovered consistently in a number of molecular studies (Ben Ali et al. 2002; Bhattacharya et al. 1992; Cavalier-Smith et al. 1994; Cavalier-Smith and Chao 2006; Leipe et al. 1994; Van de Peer and De Wachter 1997; Van der Auwera and De Wachter 1997; Yoon et al. 2008) and share an ultrastructural synapomorphy, tripartite tubular hairs attached to one of their flagella (Patterson 1989). Inclusion of the colorless amoeboid Leukarachnion sp. within this clade enhances the diversity of body forms among stramenopiles and suggests that additional amoeboid lineages of stramenopiles await further study.
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Results Light Microscopic Observations Leukarachnion sp. ATCCs PRA-24 formed a large delicate network with enlarged cell bodies (2—10 mm) and fine cytoplasmic extensions (Fig. 1A). The network was dynamic as ingested bacteria and other cytoplasmic contents moved through the thin strands of the network to the core cell bodies (Fig. 1A). When the network was disturbed it rolled back to form blobby cells, reminiscent of how cysts form during unfavorable conditions. Younger cultures contained cysts of varying sizes, ranging from 5 to 15 mm (Fig. 1B), and as it aged the cysts became smaller in size until the culture was filled with uniform 5 mm cysts. A flagellate was observed in the first culture worked on (used to generate tube 1 DNA) but not in any subsequent cultures (used to generate tubes 2 and 3 DNA). We did not have an opportunity to observe the flagellate in close detail within the first culture so the morphology of the flagella was not determined. Future examination is required to elucidate additional details of the life cycle of Leukarachnion sp. ATCCs PRA-24.
TEM Observations The overall fine structure of Leukarachnion cells (Fig. 2A, B) exhibited a finely granular, relatively hyaline, cytoplasm surrounded by an uncoated plasma membrane. Slender pseudopodial projections were commonly observed projecting from
Figure 1. Light micrograph of Leukarachnion sp. ATCCs-PRA-24. A. Image of a living organism showing an extensively branched network. Scale bar ¼ 50 mm. B. Cysts at different maturation stage. Mature cysts are smaller in size. Scale bar ¼ 25 mm.
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Figure 2. Transmission electron microscopic images of Leukarachnion sp. A. Cross-section of a cell with nucleus (N), tubulo-cristate mitochondria (M) and several food vacuoles (FV) distributed peripherally. Fine pseudopodial projections (arrow) are commonly observed. Scale bar ¼ 1 mm. B. Cells frequently contain more than one nucleus (N) characterized by a central nucleolus and a dense deposit in the nuclear envelope exhibiting a dark halo effect. Occasional elongated sacculi (arrow), possibly smooth endoplasmic reticulum, are observed near the nucleus. The plasma membrane surrounding the cell is consistently uncoated. Scale bar ¼ 1 mm. C. An enlarged view of a tubulo-cristate mitochondrion exhibiting the characteristic osmiophilic matrix and abundant tubular cristae. Scale bar ¼ 0.25 mm. D—F. Fine structure of the cyst and surrounding wall (arrows) including the mature wall with an outer fibrous layer (OW) and an inner more dense layer (IW). D. The cytoplasm is characteristically electron dense and the wall is deposited initially as a fine, fibrillar layer immediately adjacent to the plasmalemma. Scale bar ¼ 0.5 mm. E. Subsequently, an inner layer of the wall is deposited that is initially loosely organized and less dense. Scale bar ¼ 0.5 mm. F. The mature bilayered wall, directly adjacent to the plasmalemma, contains a thicker, electron dense inner layer. Scale bar ¼ 0.1 mm.
the surface of the cells (arrow, Fig. 2A). In some planes of section through the nucleus (about 1—1.5 mm diameter) there was a pronounced central nucleolus (0.5 mm), while in other sections the nucleoplasm appeared to be more uniformly electron dense. The nuclear envelope was somewhat undulating and contained an electron dense deposit producing the effect of a dark halo surrounding the nucleus (Fig. 2B). In general, the
mitochondria (approximately 0.8 mm long and 0.25 mm in diameter) had abundant, closely spaced (about 40 nm diameter) tubular cristae and a dense matrix (Fig. 2C). In many sections, at least two nuclei were observed (e.g., Fig. 2B). The endoplasmic reticulum was sparse and no clearly defined Golgi bodies were observed. However, occasional solitary or scattered flattened, elongated sacculi, possibly smooth endoplasmic
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reticulum, were observed in the vicinity of the nucleus (Fig. 2B). Cysts were commonly observed in the preparations and exhibited successive stages of maturation and wall deposition (Fig. 2D—F). The cysts (about 5 mm in diameter) presented a circular or nearly circular profile in cross-section and contained densely staining cytoplasm. The cyst wall was initially deposited as a thin (0.1—0.2 mm thick) fibrous envelope followed by secretion of a more electron dense inner layer of approximately equal thickness, yielding a bi-layered mature wall (Fig. 2F). Based on the electron microscopic observation, the wall of the cyst exhibited features that indicate it is largely organic. Walls that are mineralized show evidence of fracture planes during sectioning that were not observed here, but we did not analyze the chemical constituents of the cyst walls using analytical techniques. The wall closely enclosed the cyst cytoplasm and no evidence of a pore was observed.
produced an alpha-tubulin sequence that was 98% similar to one of the haplotypes from tube-1 (GenBank accession number FJ602385). We did not sequence alpha-tubulin from Tube-3. The five beta-tubulin sequences generated from tube-1 were identical to one another (GenBank accession number FJ356263), while tube-2 produced three haplotypes that were 94-98% similar to tube-1 (GenBank accession numbers FJ602388, FJ602389, FJ602390). Tube-3 produced one beta-tubulin sequence that was identical to one of the tube-2 haplotypes. The variable gene sequences found in alpha- and beta-tubulin clustered together in single gene analyses (data not shown) and hence one sequence from each gene was chosen for use in the concatenated alignment. There were no introns found in any of the protein coding genes and the entire sequence was used in the subsequent analyses.
Molecular Evolution
Initial analysis of our newly characterized genes combined with sequences from diverse eukaryotes placed Leukarachnion sp. within the stramenopiles (data not shown). To further examine this placement, we built a concatenated alignment including taxa that span the diversity of the stramenopiles, as well as outgroup taxa from the Alveolata, ‘Rhizaria’, Haptophyta, and Opisthokonta. The resulting topologies of the phylogenetic trees built from our three datasets using two algorithms (RaxML and MrBayes) were generally concordant, with no well-supported nodes in disagreement. Furthermore, the topology of the major clades within the stramenopiles were generally congruent with previously published phylogenies (Andersen 2004; Cavalier-Smith and Chao 2006), although support at deeper nodes is generally poor (Fig. 3). In all of our analyses, Leukarachnion sp. ATCCs PRA-24 consistently fell sister to the amoeboid stramenopiles Chlamydomyxa labyrinthuloides and Synchroma grande with strong support in analyses that include these taxa (Table 1, Fig. 3) (i.e. Bootstrap support (BS) ¼ 100%, Bayesian Posterior Probability (BPP) ¼ 1.0 in the analyses of SSU rDNA only). Together, these three lineages were sister to a clade containing Chrysophyceae and Synurophyceae (see Table 1).
Because of concerns about possible contaminants, particularly in light of the flagellates observed in our first culture, we characterized multiple clones of our target genes from DNA extractions from three separate cultures. We sequenced SSU rDNA as well as actin, and alphaand beta-tubulin from two tubes of DNA (tube-1 and tube-2) extracted at different times at ATCCs, as well as DNA extracted from cultures kept at Smith College (tube-3). The SSU genes from all three samples were identical (GenBank accession number FJ356265). The actin sequences, obtained by sequencing eight clones of tube-1, were identical to one another (GenBank accession number FJ356266) and we were able to amplify an identical 300 base pair segment of this gene from tubes 2 and 3 using taxon-specific primers. There was variation among alpha- and betatubulin sequences from the three DNA extractions, though resulting sequences were consistent with either allelic diversity or recent paralogs within a single taxon. Three unique alphatubulin sequences were obtained from 7 clones from tube-1 DNA (GenBank accession numbers FJ356264, FJ602386, FJ602387). These sequences vary from 3.4% to 6.4% at the nucleotide level, and one sequence (GenBank accession number FJ602385) contained a 7 base pair deletion. We believe this clone may be a PCR artifact, as the nucleotides were conserved both before and after the deletion. Analysis of 1 clone from tube-2
Concatenated Four Genes Analyses
Taxonomic Sampling and Missing Data To explore the robustness of the position of Leukarachnion sp. on our gene trees, we assessed
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Figure 3. Maximum likelihood tree of dataset-A (114 taxa that have data for at least the SSU rDNA and a subset of the protein genes, see supplemental material) inferred using RaxML of four concatenated genes (SSU rDNA plus actin, alpha- and beta-tubulin as nucleotides, ambiguous regions and third positions of protein coding genes excluded). BS/BPP support greater than 50% are shown at the nodes (*indicates o50% support). Solid dots are on nodes with full support under both models. C_Ectocarpus* indicates that the sequences used in the multigene analysis are from two different species. The SSU rDNA is from Ectocarpus siliculosus and the Btub is from Ectocarpus verabilis.
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Support values are RaxML bootstrap support/Bayesian posterior probabilities. (L+(C+S)) ¼ The Leukarachnion sp.+(C. labyrinthuloides+ S. grande) clade or Leukarachnion alone in taxon sets (designated by *) that do not include the other two taxa. Dataset A (114 taxa, 41% missing data) includes all taxa for which at least the SSU sequence was available. Dataset B (57 taxa, 21% missing data) includes all taxa for which at least the SSU and one of the three protein coding genes were available. Dataset C (46 taxa, 12% missing data) includes all taxa for which at least the SSU and two of the three protein coding genes were available. Nm ¼ not monophyletic.
Table 1. Support for various taxonomic hypotheses with datasets of varying taxon sampling and missing data.
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relationships under varying taxon samples and with varying levels of missing data. The placement of Leukarachnion sp. within the stramenopiles was not substantially affected when data matrices with varying percentages of missing data (4—41%) and taxon numbers (46—114 taxa) were analyzed (Table 1). In general, the support for the grouping of Leukarachnion sp. with the Chrysophyceae+ Synurophyceae clade was shown to increase as the percentage of missing data and taxon sampling decreased. For example, in the 114 taxon matrix with 41% missing data, the support for the clade containing Leukarachnion sp.+(Synchroma grande+Chlamydomyxa labyrinthuloides) with the Chrysophyceae/Synurophyceae clade was 0.97 BPP (70% BS). The support increased to 1.0 BPP (100% BS) when the 57 taxon matrix with 21% missing data was analyzed (Table 1).
Discussion ATCCs PRA-24 Isolate is Leukarachnion sp. The ATCCs PRA-24 isolate shares a number of characters with the one published description of Leukarachnion batrachospermi, including network and cyst formation as well as plasmodia. Leukarachnion batrachospermi was described in 1942 with beautiful line drawings; there are neither electron micrographs nor molecular data for this taxon. Our isolate is much smaller than L. batrachospermi, which could grow to a few millimeters in size (Geitler 1942). Further comparison of our isolate with L. batrachospermi cannot be made due to lack of a culture of L. batrachospermi. The light and TEM data provided here will contribute to a formal description of our isolate, which awaits more detailed data for L. batrachospermi. In the meantime, the ATCCs PRA-24 isolate is identified as a Leukarachnion sp.
Phylogenetic Placement of Leukarachnion sp. ATCCs PRA-24 Molecular analyses show Leukarachnion sp. ATCCs PRA-24 grouping with the photosynthetic stramenopiles Chlamydomyxa labyrinthuloides and Synchroma grande (Fig. 3, Table 1). These lineages are similar in gross morphology including amoeboid body form, plasmodia and network/ branching pseudopodia. This sister relationship is further corroborated by ultrastructural characters, such as the similar fine structure of the cyst wall with Chlamydomyxa labyrinthuloides (Pearlmutter
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and Timpano 1984); cysts are not reported for Synchroma grande. Both Leukarachnion sp. and Chlamydomyxa labyrinthuloides cysts possess a similar bilayered wall, an inner dense layer and an outer more fibrous layer, each layer of similar thickness (Pearlmutter and Timpano 1984). The recently described Synchroma grande was placed in a monotypic class, Synchromophyceae Horn et Wilhelm based primarily on unique chloroplast structures and distinctive 18S rRNA and rbcL sequences (Horn et al. 2007). The analyses presented here with the additional taxon sampling group S. grande with Leukarachnion sp. ATCCs PRA-24, an organism with no chloroplasts, and C. labyrinthuloides, an organism with more traditional chloroplasts. As neither of these taxa matches the class description of Synchromophyceae, this class designation must be formally reevaluated. This might best be done following characterization of several other amoeboid lineages described in earlier literature that are morphologically similar to Leukarachnion sp. but for which no molecular data are yet available (see below). In a recent classification, Cavalier-Smith and Chao (2006) placed Chlamydomyxa and Picophagus in a new class, Picophagea, based on an analysis of SSU-rDNA. None of our analyses, including SSU rDNA only or concatenated SSU rDNA plus three protein-coding genes, recover the Chlamydomyxa+Picophagus clade (Fig. 3). On the contrary, the Leukarachnion sp.+(Chlamydomyxa labyrinthuloides+Synchroma grande) clade leans more to the Chrysophyceae/Synurophyceae clade to the exclusion of Picophagus flagellatus (Table 1; Fig. 3). Although an AU-test (p-value ¼ 0.43) could not reject the grouping of P. flagellatus at the base of Leukarachnion sp.+Chlamydomyxa labyrinthuloides+Synchroma grande clade, three alternative hypotheses (see Methods) that place Chlamydomyxa and Picophagus together were significantly worse (p-value o0.0001 (for AU test) and p-values o0.0001 (for SH/KH tests)). These findings indicate that the class Picophagea sensu CavalierSmith and Chao 2006 requires further scrutiny.
Evolution of Stramenopiles Despite its heterotrophic habit and reticulate amoeboid form, Leukarachnion sp. falls with robust support within a clade of predominantly photosynthetic, flagella-bearing lineages (Fig. 3; see also Ben Ali et al. 2002). This indicates that Leukarachnion sp. ATCCs PRA-24 is secondarily heterotrophic, though ultrastructural analysis of
our isolate shows no evidence of a leukoplast or any plastid-like structure (Fig. 2). We attempted to amplify the rbcL gene from Leukarachnion sp. using degenerate primers but we were not successful (data not shown). Within the autotrophic heterokonts there are other lineages that have lost photosynthetic ability, though many of these lineages, such as Spumella and Paraphysomonas in the Chrysophyceae and Pteridomonas in the Dictyochophyceae, are reported to possess vestigial colorless plastids known as leukoplasts (Belcher and Swale 1976; Mignot 1977). Intriguingly, Leukarachnion may represent just one of a diverse clade of colorless amoeboid lineages described by light microscopy in earlier literature. Examples of such taxa include lineages that form net-like structures similar to Leukarachnion such as Chrysarachnion insidians (Pascher 1917), Leukapsis vorax (Pascher 1940), Chrysidiastrum catenatum (Lauterborn 1913), and Heliapsis mutabilis (Pascher 1940). As taxonomic sampling of diverse eukaryotes continues, we will be able to assess the hypothesis that these morphologically similar taxa represent a monophyletic and diverse clade within the stramenopiles. The placement of Leukarachnion sp. ATCCs PRA-24 within the stramenopiles adds to the known diversity within this clade. Stramenopiles contain unicellular and multicellular forms; lineages with hetrotrophic, mixotrophic and autotrophic modes of nutrition; lineages with flagellate and amoeboid forms, free-living species and parasites (Andersen 2004). Moreover, Leukarachnion is just one of about 200 described eukaryotic lineages whose taxonomic position is undetermined (Adl et al. 2005; Patterson 1999). The findings reported here further reinforce the power of multigene analyses in placing lineages of uncertain origin (Tekle et al. 2007; Yoon et al. 2008). Despite the great morphological diversity among stramenopiles, their monophyly has not been questioned. However, their relationship to other eukaryotic groups is controversial: a number of studies suggest that stramenopiles are sister to alveolates (Baldauf et al. 2000) while some multigene and phylogenomic analyses suggest that stramenopiles plus alveolates are sister to the putative supergroup ‘Rhizaria’ (Burki et al. 2007; Hackett et al. 2007). Though our analyses were not structured to test these hypotheses fully, it is noteworthy that stramenopiles group with alveolates with strong support (Table 1). In contrast, the monophyly of ‘Rhizaria’ plus alveolate plus stramenopile is not supported in our taxon-rich SSU
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rDNA only analysis, and receives mixed support with inclusion of protein genes (Table 1).
Methods Sampling and molecular methods: DNA samples of Leukarachnion sp. ATCCs PRA-24TM, originally isolated from a salt marsh in Virginia, using DNA Stat60TM (Tel-Test, Inc., Friendswood, Texas; Cat. No. TL-4220) per manufacturer’s instructions and with the addition of a phenol-chloroform-isoamyl step using Phase Lock Gel Heavy tubes (Eppendorf AG, Hamburg, Germany; Cat. No. 955154070). Additional genomic DNA from the same isolate cultured at Smith College was extracted with a DNeasy Tissue kit (Qiagen, Valencia, CA, USA 28106; Cat. No. 69504). Data analysis: Primers for SSU rDNA genes are from Medlin et al. (1988) with three additional primers used to generate overlapping sequences from each clone as described in Snoeyenbos-West et al. (2002). Primers for actin, alpha-tubulin and beta-tubulin are from Tekle et al. (2007). Vent DNA Polymerase (NEB Cat. No. M0254L) and Phusion DNA Polymerase (NEB Cat. No. F-540L), strict proofreading enzymes, were used to amplify our genes of interest and we have used Lucigen PCRSmart (Lucigen Inc., Middleton, WI 53562, Cat. No. 41120-4), Novagen Perfectly Blunt (Novagen Inc., Madison WI 53719, Cat. No. 70191-4), and Invitrogen Zero Blunt Topo (Invitrogen, Inc., Carlsbad, CA 92008, Cat. No. K2800) cloning kits. Sequencing of cloned plasmid DNA was accomplished using vector-or gene-specific primers and the BigDye terminator kit (Perkin Elmer, Waltham, MA). Sequences were run on an ABI 3100 automated sequencer. We have fully sequenced 2—6 clones of each gene and surveyed up to 10 clones per gene in order to detect paralogs. The SSU rRNA sequences were aligned using ClustalW (Thompson et al. 1994) as implemented in DNAstar’s Lasergene software. The resulting alignment was further edited manually in MacClade v4.08 (Maddison and Maddison 2005). Protein coding genes were aligned as amino acids using Clustal W (Thompson et al. 1994) as implemented in DNAstar’s Lasergene software and adjusted by eye in MacClade (Maddison and Maddison 2005). Our genealogies excluded ambiguously aligned regions. In addition, the nucleotide genealogies excluded 3rd positions. The SSU rDNA alignment contained a total of 1600 characters, and the concatenated alignments contained 3766 characters (SSU rDNA plus protein coding genes as nucleotides) and 2683 (SSU rDNA plus protein coding genes as amino acids). Alignments are available at http://www.science.smith.edu/ departments/Biology/lkatz/data/Leukarachnion. We analyzed three datasets to investigate the impact of taxonomic sampling and missing data on our results. Dataset A (114 taxa) includes a broad sampling of taxa for which at least the ssu rDNA gene sequence data was available. Dataset B (57 taxa) includes those taxa from set A for which at least one of the three protein coding genes in our study was available and dataset C (46 taxa) includes those taxa from set A for which at least two of the three protein coding genes was available. As the taxa for which there were fewer data available were removed to form the smaller datasets, the number of missing characters decreased as well. Dataset A has 41% missing data, while datasets B and C have 21% and 12%, respectively. We also analyzed the SSU rRNA gene alone for the taxa in dataset A, for an analysis with the least amount of missing data (4%).
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Phylogenies were constructed in MrBayes (Huelsenbeck and Ronquist 2001) and RaxML (Stamatakis et al. 2005a, 2005b). Bayesian analyses were performed with the parallel version of MrBayes 3.1.2 using the GTR+I+G (for nucleotide) and rtREV (for amino acid) models of sequence evolution (Ronquist and Huelsenbeck 2003). Four simultaneous MCMCMC chains were run for 4,000,000 generations sampling every 100 generations. The 50% majority-rule consensus tree was determined to calculate the posterior probabilities for each node. RaxML (Stamatakis et al. 2005a, 2005b) was run for 100 iterations using the GTRGAMMA model for nucleotide data and PROTGAMMA with matrix rtREV for amino acid data. The datasets were partitioned to allow for different parameters for each gene. A total of 100 independent bootstrap analyses were run in RaxML and a 50% majority rule consensus was calculated to determine the support values for each node. MrModeltest (Nylander 2004) and ProtTest (Abascal et al. 2005) were used to select the appropriate model of sequences evolution for the nucleotides and amino acid data, respectively. To compare competing hypotheses on the placement of Leukarachnion and the monophyly of Chlamydomyxa and Picophagus (Class Picophagea) to the exclusion of other taxa, we performed the approximately unbiased (AU) test (Shimodaira 2002) as well as the more conventional Kishino—Hasegawa and Shimodaira—Hasegawa tests, as implemented in Consel 0.1j (Shimodaira and Hasegawa 2001). We tested for the following alternative hypotheses (a) the monophyly of ((Leukarachnion sp.+(C. labyrinthuloides, S. grande))+ P. flagellatus) (b) the monophyly of (C. labyrinthuloides+ P. flagellatus) (c) the monophyly of ((C. labyrinthuloides+ P. flagellatus)+(C. labyrinthuloides+S. grande)) (d) the monophyly of ((C. labyrinthuloides+P. flagellatus)+(Chrysophyceae/ Synurophyceae)).
Acknowledgements The authors thank Jeff Cole for technical assistance. This work is supported by the National Science Foundation Assembling the Tree of Life grant (043115) to LAK and DJP. This is LamontDoherty Earth Observatory Contribution Number 7176.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/ j.protis.2009.01.001.
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