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The 18S and 28S rDNA identity and phylogeny of thecommon lotic chrysophyte Hydrurus foetidusDag Klaveness a , Jon Bråte a , Vishwanath Patil b , Kamran Shalchian-Tabrizi a , RagnhildKluge c , Hans Ragnar Gislerød c & Kjetill S. Jakobsen a da Microbial Evolution Research Group, Department of Biology, University of Oslo, P.O. Box1066 Blindern, 0316 Oslo, Norwayb Biorefinery R & D, Borregaard, P.O. Box 162, N-1701 Sarpsborg, Norwayc Department of Plant & Environmental Sciences, P.O. Box 5003, Norwegian University ofLife Sciences, N-1432 Ås, Norwayd Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, P.O.Box 1066, University of Oslo, NO-0316 Oslo, Norway
Available online: 10 Aug 2011
To cite this article: Dag Klaveness, Jon Bråte, Vishwanath Patil, Kamran Shalchian-Tabrizi, Ragnhild Kluge, Hans RagnarGislerød & Kjetill S. Jakobsen (2011): The 18S and 28S rDNA identity and phylogeny of the common lotic chrysophyteHydrurus foetidus , European Journal of Phycology, 46:3, 282-291
To link to this article: http://dx.doi.org/10.1080/09670262.2011.598950
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Eur. J. Phycol. (2011) 46(3): 282–291
The 18S and 28S rDNA identity and phylogeny of the common
lotic chrysophyte Hydrurus foetidus
DAG KLAVENESS1, JON BRATE1, VISHWANATH PATIL2, KAMRAN SHALCHIAN-TABRIZI1,
RAGNHILD KLUGE3, HANS RAGNAR GISLERØD3 AND KJETILL S. JAKOBSEN1,4
1Microbial Evolution Research Group, Department of Biology, University of Oslo, P.O. Box 1066 Blindern, 0316 Oslo, Norway2Biorefinery R & D, Borregaard, P.O. Box 162, N-1701 Sarpsborg, Norway3Department of Plant & Environmental Sciences, P.O. Box 5003, Norwegian University of Life Sciences, N-1432 As, Norway4Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, P.O. Box 1066, University of Oslo,
NO-0316 Oslo, Norway
(Received 2 February 2011; revised 22 May 2011; accepted 6 June 2011)
Hydrurus foetidus is a geographically widespread alga commonly detected as 2–10 cm thalli in mountain streams in early
spring, and also in lowland rivers at latitudes where seasonal conditions are appropriate for this cold-water species. Reaching
macroscopic dimensions but with a pronounced phenotypic plasticity, this species is not typical for the golden algae
(Chrysophyceae) – they are almost exclusively microscopic organisms, best known as micro- or nanoplankton in fresh water.
Other sessile multicellular members are less conspicuous in nature, and rarely detected and sampled for further investigation.
Therefore, the phylogenetic position of Hydrurus within the Chrysophyceae is not clear and has been disputed. We determined
the 18S and 28S rDNA subunit sequences from a typical H. foetidus sampled at Finse, Hardangervidda mountain plateau, in
south Norway. Phylogenetic trees inferred from concatenated 18S and 28S sequences, including representatives of all known
groups of heterokonts, safely confirmed Hydrurus as a chrysophyte. Extending the taxon sampling to include nearly all
available 18S chrysophyte sequences from cultured species and environmental DNA, our analysis placed H. foetidus within a
separate, well-defined clade (here named the Hydrurus-clade) dominated by environmental sequences and poorly defined
strains of chrysophytes in culture, mostly from cold environments. The environmental sequences derived from other moun-
tainous regions showed high similarity and may represent homologous or closely related species. However, the phylogenetic
relationship to the closest morphologically described chrysophyte clades remains unresolved. Genetic tools for investigating
theHydrurus complex are now available. An increased sampling ofHydrurus-like heterokont species as well as chrysophytes in
general is crucial for understanding the evolution of this lineage and its relations to other chrysophyte clades.
Key words: Chrysophyceae, environmental DNA, evolution, Heterokonta, Hydrurus, phylogeny, 18S and 28S rDNA
Introduction
Few members of the class Chrysophyceae(Chromista, Heterokonta) are large and visiblewithout optical magnification. Most specieswithin the class are planktonic, either unicellularor colonial, consisting of a limited number ofcells and still requiring a microscope to beidentified – examples are the unicellularChromulina or the colonial Dinobryon. There arealso a number of sessile or benthic species – eitherunicellular, like Lepochromulina, or colonial, as inAnthophysa – and a few multicellular species whoseinconspicuous life-form and rarity may have pre-vented a closer acquaintance, such asPhaeodermatium (see Kristiansen & Preisig, 2001,
or Nicholls & Wujek, 2003, for more information).The golden algae are also diverse ecologically,encompassing a few marine and many freshwaterspecies with varying lifestyles, including heterotro-phy. The planktonic Dinobryon was the first photo-trophic chrysophyte also observed to engulfbacteria under natural conditions (Bird & Kalff,1986). A life cycle has been described forDinobryon that involves the formation of a zygotein the shape of a silicified binucleate statospore afterfusion of cells from þ and � strains (cf. Sandgren,1988). The silicified resting stages of chrysophytes(stomatocysts) are characteristic and serve as valu-able indicators for palaeolimnologists (e.g. Smolet al., 2005).Hydrurus foetidus (Villars) Trevisan (1848) is a
large chrysophyte algae, and prominently visibleduring the late winter as ‘an exclusive inhabitantCorrespondence to: Kjetill S. Jakobsen. E-mail:
ISSN 0967-0262 print/ISSN 1469-4433 online/11/030282–291 � 2011 British Phycological Society
DOI: 10.1080/09670262.2011.598950
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of cold mountain streams. . . [that] is distributedworldwide’ (Wehr & Sheath, 2003, p. 12). Thethalli are firmly attached to hard rock or largestones in turbulent water, and easy to recognizedue to their colour and the scarcity of other algaeunder such cold conditions. They may reach a fewcm in unpolluted, soft-water rivers, but can reachlengths of 30 cm or more under more favourableconditions (Bursa, 1934). In fast-running and tur-bulent rivers, the thalli are arbuscular, with moreor less well-developed central axes and branchesmade up of elongated, ellipsoid to ovoid cells in afirm polysaccharide matrix, carrying numerousminor branches of spherical and compressed cells;each axis or branch terminates in a larger triangu-lar to conical apical cell (cf. Klaveness &Lindstrøm, 2011). The entire thallus is embeddedwithin a gel, which gives it a smooth and slipperysurface. In slowly seeping water films, Hydrurusmay appear as a slimy coating upon rock andpebble surfaces (e.g. Klaveness, 1992, fig. 7). Theapparent phenotypic plasticity within this morphos-pecies may reflect ecotypes or different species –a question to be solved by wider sampling anddiscussion of relevant species concepts, and byobtaining genetic sequences from several naturalstrains. A wide range of ‘species’ are also availablefrom herbaria in Europe. Here, we follow the rec-ommendations by recognized authorities, fromPascher & Lemmermann (1913) to Kristiansen &Preisig (2001), treating the genus as monotypicuntil modern evidence points to differentconclusions.Due to its size and prominent morphology and
its rather spectacular occurrence under seasonalclimate regimes, Hydrurus has been the focus formany investigations. The occurrence of Hydrurusin central European alpine regions and the Tatramountains has inspired a number of benchmarkpapers (e.g. Rostafinski, 1882; Klebs, 1893, 1896;Lagerheim, 1888; Geitler, 1927; Avel & Avel, 1932;Bursa, 1934; Kann, 1978), where further referencesmay be found. Early records from the UK arelisted by Whitton et al. (1978), and the distributionthere outlined by Kristiansen (2002). Records inNorth America go back to 1862 (Setchell &Gardner, 1903). In Scandinavia, Hydrurus is wellknown from mountain streams, sometimesfavoured in watersheds of sedimentary origin(e.g. Wille, 1885) or in regions influenced by settle-ments (e.g. Strom, 1926) and at times present alsoduring summer in environments where water tem-peratures remain low. Massive development of thisalga is known (e.g. Wille, 1885; Geitler, 1927;Bursa, 1934; Skuja, 1964; Skulberg &Lillehammer, 1984). Early blooms of Hydrurus inaffected rivers have ecological significance as food
for water insects (Ward, 1994; Milner et al., 2001)and microbial life (e.g. Rott et al., 2006).Hydrurus foetidus has been carefully investigated
by light (e.g. Klebs, 1893, Mack, 1953; Fukushima,1962; Joyon, 1963; Klaveness & Lindstrøm, 2011)and electron microscopy (e.g. Hovasse & Joyon,1960; Vesk et al., 1984; Hoffman et al., 1986;Andersen, 1991), and the details of its peculiarsilicified stomatocyst have been outlined (Hovasse& Joyon, 1960). The motile zoospores haveattracted attention, since their tetrahedral shapeappears to be unique to Hydrurus and genera iden-tified as related, partly due to the morphology ofmotile cells (e.g. Phaeodermatium). A full life cycleinvolving phase transitions has not been outlined,since efforts to culture Hydrurus have failed untilrecently (Klaveness & Lindstrøm, 2011).The few studies where molecular phylogenies
have been inferred for a considerable number ofchrysophyte species show little resolution of thegroup (see for example Lavau et al., 1997; Caronet al., 1999; Andersen, 2007; Patil et al., 2009), withHydrurus yet to be investigated with 18S and/or28S rDNA. Only 5S rDNA gene sequences wereavailable forHydrurus, and the corresponding phy-logeny did not resolve the position of the genus(Lim et al., 1986; Hori & Osawa, 1987). It wasthus uncertain as to where the genus would beplaced in a phylogenetic analysis inferred frommore sequence characters.The goal of this study was to shed light upon the
phylogenetic position of H. foetidus using molecu-lar phylogenetic techniques. We performed phylo-genetic analyses of 18S and 28S rDNA genes toinvestigate the identification and classification ofthe species as well as the more general problemof the evolution and relationships of Hydrurus ina wider context.
Materials and methods
Materials
Hydrurus foetidus was collected at the outlet of LakeFinsevatn (‘Garpefossen’) in the vicinity of FinseAlpine Research Center, at 60� 360 N, 7� 300 E and1215m altitude (for map of locality, see Klaveness &Lindstrøm, 2011). Microscopy confirming identity andabsence of foreign organisms (epiphytes etc.), and pho-tographic documentation were done immediately orwithin hours of sampling; samples were stored at 0�C.The first collection (February 2006) included 20–30mmthalli, which stuck easily to white card left to dry in anupright position. Sampling was repeated in March 2007,specimens being dried on card and also used forsequencing rDNA genes. The sample sheet from whichsequenced material was cut is deposited in the herbariumof the Natural History Museum, University of Oslo,under the species name (‘Hydrurus foetidus (Villars)Trevisan 1848’) and dated 7 March 2007. Stock cultures
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of this strain (named G 070301) are also kept in algalculture collections in Oslo and Gottingen.
DNA isolation and PCR amplification
Material on the cardboard paper was cut into smallpieces and collected in a microfuge tube. 600 ml ofPOWERlyse buffer (Nordiag ASA, Norway) wasadded and incubated at 65�C for 1min. The lysate wasthen centrifuged briefly, supernatant transferred to anew tube and DNeasy Plant mini kit (Qiagen GmbH,Hilden, Germany) used following the manufacturer’sprotocol. The Hydrurus 18S rDNA gene was amplifiedusing universal 18S primers (Medlin et al., 1988). The28S rDNA gene was amplified using the 28S primers;5.8S F, LSU 4256R and LSU 15R (Riisberg et al., 2009).PCR amplification was performed using FinnzymesPhusion (Finnzyme Oy, Finland) high-fidelity DNApolymerase. Cycling conditions for 18S amplificationwere: (1) 98�C for 30 s, (2) 98�C for 10 s, (3) 50�C for20 s, (4) 72�C for 35 s, (5) repeat steps (2)–(4) 34 times,and (6) 72�C for 7min. For the 28S amplification, theconditions were: (1) 98�C for 30 s, (2) 98�C for 10 s,(3) 67�C for 30 s, (4) 72�C for 2.5min, (5) repeat steps(2)–(4) 34 times, and (6) 72�C for 7min. Negative PCRcontrols absent for DNA were run to exclude any pos-sible contamination.
Cloning and sequencing
PCR products were cloned into pCR4 Blunt-TOPOvector using a Zero Blunt cloning kit (Invitrogen,Carlsbad, CA) according to manufacturer’s instructions.Plasmid DNA was extracted from E. coli TOPO10chemically competent cells using Promega PlasmidWizard kit. Plasmids were analysed confirming positiveinsertion using EcoRI. A minimum of five positiveclones was sequenced with a big dye terminator system(v 3.1 Applied Biosystems) using the primers M13F,M13R for 18S sequences. All five clones contained thesame insert sequence. The 28S sequencing primers usedwere LSU4F, LSU4R, LSU5F and LSU5R (Riisberget al., 2009). The sequences generated here are depositedat and available from NCBI Entrez with accessionnumbers FM955256 (18S rDNA) and FM955257(28S rDNA).
Data mining and phylogenetic analyses
The 18S and 28S rDNA alignment of Heterokonta andChrysophyceae was the same as in Riisberg et al. (2009)with sequences from Hydrurus generated in this studyadded to it. Publicly available 18S rDNA sequences ofChrysophyceae for the generation of the 18S rDNAChrysophyceae alignment were acquired fromGenBank via BLAST searches using Hydrurus querysequences. Sequence alignments were constructed withMAFFT (Katoh et al., 2002) and subsequently editedmanually. After removing ambiguously aligned sites, the18S alignment consisted of 94 taxa and 1627 nucleotidecharacters, whereas the 28S had 2367 nucleotide charac-ters and 48 taxa. The concatenated (18Sþ 28S)
alignment contained 48 taxa and 3904 nucleotide char-acters. The alignments are available in the supplemen-tary material, accessible via the Supplementary Contenttab on the article’s online page at http://dx.doi.org.10.1080/09670262.2011.598950.
The alignments were subjected to maximum-likeli-hood (ML) and Bayesian analyses. ML analyses wereperformed with RaxML v7.2.6 (Stamatakis et al.,2005) using the General Time Reversible (GTR) modelconsidering the proportion of invariable sites (I) andgamma (G) distributed site rates, as suggested by theprogram Modeltest (Posada & Buckley, 2004). Thegamma distribution was approximated with four ratecategories and the analyses were run from a randomstarting tree. The topology with the highest likelihoodscore out of 100 heuristic searches with a randomlyselected starting tree for each search was chosen. Non-parametric bootstrap scores were calculated from 500pseudo-replicates using the best topology as startingtree. The Bayesian inferences were performed usingMrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003), apply-ing the GTRþGþ I model as selected by MrModeltest(Nylander, 2004). Ten independent analyses were run,each from a random starting tree and with four MonteCarlo Markov Chain (MCMC) chains that lasted for4 000 000 generations. The MCMC chains includedthree heated and one cold chain. The harmonic meanlikelihood values, posterior probability and tree topol-ogy were calculated from the sampled trees after burn-in. Burn-in was set at 25% after visual inspection of themarginal likelihood scores of the sampled trees.Harmonic mean values and posterior probabilityvalues for the internal nodes were almost identical ineach run, suggesting convergence of the MCMC chains.
Fast-evolving sites were identified using PAMLimplemented in the AIR package (Kumar et al., 2009)on the 18S rDNA dataset. From 10% to 90% (with 10%intervals) of the fastest evolving sites were removed andML analyses on each reduced alignment were run asbefore.
To test for possible outgroup artifacts we analysed the18S dataset with different sets of closely related out-groups [Picophagus, Chlamydomyxa, Synchroma,Leukarachnion and a closely related environmentalsequence (AB534476); for details see Andersen (2007),Patil et al. (2009) and Grant et al. (2009)], as well aswithout any outgroup. For the final analysesLeukarachnion and sequence AB534476 were chosen asoutgroup, because they had the shortest branches of theoutgroup taxa.
All phylogenetic analyses were performed onUniversity of Oslo’s Bioportal Platform (http://www.bioportal.uio.no cf. Kumar et al., 2009).
Results
Morphology
The strain from the outlet of Lake Finsevatn isdocumented in Figs 1–5. Specific charactersinclude the arbuscular thalli, consisting of a firmcentral axis and peripheral branches (Figs 1–3)
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containing characteristic cells within a viscousgelatinous coat (Fig. 4). By March thalli averaged3–5 cm in length, with a maximum of 7 cm.Zoospores were released from the periphery ofthe thallus; they were rounded at first, beforeattaining a tetrahedral shape (Fig. 5) with asingle visible flagellum within minutes of release.The thallus, and the cells in the periphery in par-ticular, were sensitive to temperature and quicklychanged morphology (eventually decomposing)as they warmed to room temperature.Comparable morphologies and details have beendocumented and discussed by Rostafinski (1882),Lagerheim (1888), Klebs (1893), Mack (1953),Hovasse & Joyon (1960), Hoffmann et al. (1986),Canter-Lund & Lund (1995) and Graham &Wilcox (2000).
Phylogenetic placement of H. foetidus within theheterokont group from 18Sþ 28S rDNA phylogeny
In an attempt to place H. foetidus within the phy-logeny of heterokonts, we amplified both 18S and
28S rDNA sequences from the species and per-formed phylogenetic inferences of the concatenated
sequences from the entire heterokont clade (Fig. 6).
The analysis recovered the photosynthetic classes
of heterokonts as a monophyletic clade with high
ML bootstrap support and Bayesian posterior
probability values [95% bootstrap support (BS)
and 1.00 Bayesian posterior probability (PP)].
The heterotrophic lineages comprised two clades,
though these were without significant support
(OomycetesþHyphochytridiomycetesþDevelop-
ayella and ThraustochytridaeþBlastocystisþ
Bicosoecida). In accordance with other reports
on the rDNA phylogeny of heterokonts (Cava-
lier-Smith & Chao, 2006; Riisberg et al.,
2009), the heterotrophic clades were not sup-
ported as monophyletic. The Chrysophyceae,
however, was recovered as monophyletic
with maximum support. Within this, H. foetidus
was the closest relative to Ochromonas sp.
and Chrysolepidomonas dendrolepidota (73% BS/
1.00 PP) (Fig. 6).
Figs 1–5. Morphology of Hydrurus foetidus collected at Finse, Norway. 1. Specimen dried on cardboard paper and depicted
by optical scanning at 600 dpi showing a small, richly branched specimen, collected late February 2006. 2. Individual thallicollected March 2007, in good growth. 3. Specimen sampled in March 2007, apex of two individual branches in good growth.4. Detail of branch apex in good growth, showing the dominant apical cell and adjacent vegetative cells. 5. Zoospore released
from cells a few hours after collection. The zoospore is released as a spherical cell but develops rapidly (within minutes) intothe tetrahedal zoospore (shown here) characteristic for this species. Scale bars: 10mm (Figs 1, 2: bar in Fig. 2), 100 mm (Fig. 3)or 10mm (Figs 4, 5: bar in Fig. 5).
Molecular phylogeny of Hydrurus 285
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Fig. 6. Bayesian phylogenetic tree based on the concatenated alignments of 18S and 28S rDNA sequences (3904 bp and 48
taxa) for Heterokonta, rooted with Haptophyceae, Cryptophyceae, Apicomplexa, Dinophyceae and Cercozoa. Maximum-likelihood bootstrap (BS, in %) and posterior probability values (PP) at the branches are separated by slashes. Support valuesare only shown for branches that received maximum-likelihood BS support >50% and Bayesian support >0.80 PP. Thick
lines indicate maximum support values (100% BS/1.00 PP) An asterisk (�) indicates that the branch length is divided by two.The Hydrurus 18S and 28S rDNA sequences are deposited in Genbank under the accession numbers FM955256 (18S) andFM955257 (28S). For the other accession numbers see table in Riisberg et al. (2009).
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Phylogeny of H. foetidus related to Chrysophyceae:18S rDNA phylogeny
The 18Sþ 28S rDNA phylogeny did not unequiv-ocally reveal the placement of Hydrurus within theChrysophyceae clade, due to the low taxon sam-pling in the concatenated alignment (only fivechrysophyte species including Hydrurus).Therefore, we inferred an 18S phylogeny with anextended sampling, based on the sequences alreadyavailable, including those originating from envi-ronmental samples. The tree was rooted withLeukarachnion sp. and a closely related environ-mental clone (AB534476) (Fig. 7) and H. foetiduswas recovered among the Chrysophyceae with highsupport (100% BS/1.00 PP). The nearest sister toH. foetidus was the environmental sequenceAJ867745 (97% BS/1.00 PP). These two taxa clus-tered together with the undescribed strainCCMP1899 and several other environmentalsequences in a highly supported clade (85% BS/1.00 PP), which is referred to here as theHydrurus-clade (Fig. 7). The Hydrurus-clade wasin turn contained within a larger group thatincluded several sequences from environmentalsamples, as well as the species Phaeoplaca thallosaand the culture collection strains CCCM41 andCCMP2296 (88% BP/1.00 PP). Additional infor-mation about the related sequences in theHydrurusclade is summarized in Table 1. No further resolu-tion of the position of Hydrurus and its closest rel-atives was given by the 18S rDNA phylogeny asthere was no support for the backbone nodes in thetree. Removing fast-evolving sites and testing dif-ferent outgroups (not shown) did not result in adifferent topology or improved support valuesthan in the phylogeny presented in Fig. 7.
Discussion
In Pascher’s keystone publications (Pascher &Lemmermann, 1913; Pascher, 1914), Hydruruswas assigned to the class Chrysophyceae, a policycontinued in subsequent classifications and sup-ported by more recent light and transmission elec-tron microscopy (e.g. Preisig, 1995; Kristiansen &Preisig, 2001; Andersen, 2007). The 18Sþ 28SrDNA phylogeny presented here (Fig. 6) confirmsthat Hydrurus has an evolutionary origin amongthe golden algae. When it came to understandingwhether Hydrurus is a primitive or advancedmember among the golden algae, traditional view-points diverged. While Klebs (1893) depicted thecellular organelles in detail, including contractilevacuoles within the vegetative cells, and treatedHydrurus as a member of the flagellates, Fritsch(1935, p. 546) pointed to the fact that Hydrurus‘in its marked division of labour far surpasses
any of the palmelloid forms found in other classesand may in some respect be ranked as high asDraparnaldia’. Although electron microscopyrevealed a wealth of new information and resolvedimportant structural and functional differencesbetween classes of heterokonts (see Andersen,2007, for recent developments), this laboriousmethod contributed little to an understanding ofevolutionary relationships among chrysophytes.A problem with the morphological evidence atthe transmission electron microscopy level is theuneven availability of information concerningcharacters of possible evolutionary significance,like mitosis, meiosis, cytoskeletal constructionand function, and the ultrastructure of the flagellarbases. Mitosis inHydrurus has been investigated byelectron microscopy (Vesk et al., 1984) and exhibitsa variant of orthomitosis (Raikov, 1994), involvinga symmetrical metaphase within the boundariesof a more or less deconstructed nuclear membrane.This is known also from other chrysophytes(Raikov, 1994). The bases of two vestigial flagellaon the vegetative cell of Hydrurus were first shownby Vesk et al. (1984) and in more detail byHoffman et al. (1986; see also Andersen, 1991) –gained by a very laborious (and expensive) methodonly available in dedicated laboratories. We areconfronted with the fact that molecular methodsare the most efficient tools for evaluation of evo-lutionary relationships, across diverging morphol-ogies, incommensurable information, and differentenvironments.The 5S rDNA sequences analysed by Lim et al.
(1986) placed Hydrurus at the base of the chromo-phyte phylogeny. However, the resolution and sup-port provided by the short 5S rDNA sequence waspoor. Generally, the concatenation of 18S and 28SrDNA has improved reconstruction of the hetero-kont phylogeny (Ben Ali et al., 2002; Edvardsenet al., 2007; Riisberg et al., 2009). In our concate-nated analysis, many of the deep branches are wellsupported, but there are few taxa for which data onboth genes are available and this hinders any fur-ther resolution of the placement of Hydrurus, otherthan its allocation to the Chrysophyceae.In our 18S rDNA phylogeny, there is essentially
no support for the backbone nodes and it is there-fore not possible to confidently determine the evo-lutionary position of the Hydrurus-clade and itsallies (see Table 1) among the Chrysophyceae.This is also seen in other 18S phylogenies of theChrysophyceae (Andersen, 2007). The Hydrurussequence does not cluster with any other describedchrysophyte species. Instead, its closest relativesare environmental samples from snow and ice inalpine or arctic areas, or from Baltic Sea ice(Table 1). These form the highly supported‘Hydrurus-clade’ (AY689714 to AJ867745).
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Closely related to this clade are unclassified anduncultured chrysophytes (AY180010 toEU247834), of which at least two are from coldenvironments. Of interest is also the affinity toPhaeoplaca thallosa (AF123296), a pseudoparen-chymatous chrysophyte. In the comparable
phylogenetic tree of Andersen (2007), this speciesgrouped (96% BS) with EF165134 (¼CCCM41)and EF165133 (¼CCMP1899), the latter being amember of the Hydrurus clade here. The linkwith P. thallosa suggests that it will be rewardingto generate new phylogenies that include colonial
Fig. 7. Bayesian phylogenetic tree of Chrysophyceae based on an 18S rDNA alignment of 1627 characters and 97 taxa, rooted
with Leukarachnion sp. and an uncultured eukaryote (AB534476). Maximum likelihood bootstrap values and Bayesianposterior probability values as in Fig. 6.
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or multicellular species, such as Celloniella palensis(Pascher, 1929), the presumably more closelyrelated Chrysonebula holmesii (cf. Hibberd, 1977)and the ‘advanced’, pseudoparenchymatousPhaeodermatium rivulare (see e.g. chapter 12 inWehr & Sheath, 2003, for an overview of chryso-phyte diversity); addressing this goal will requireadditional taxon and gene sampling.In his paper on the systematics of the
Chrysophyceae and Synurophyceae, Andersen(2007) has shown that traditional classificationsbased upon characters like flagellar numbers orthe formation of colonies or thalli are not compat-ible with phylogenetic trees constructed from 18SrDNA and/or rbcL genes. Unicellular genera witheither a single or two flagella (visible in the lightmicroscope) may have appeared several timesduring the evolution of chrysophytes and thereforedo not constitute natural clades in the phylogenetictrees. Similarly, groupings of life forms with cap-soid thalli (with individual non-motile cells embed-ded in mucilage) seem also not to be natural.Since the 18S rDNA phylogenetic tree shown by
us here reveals environmental sequences from high-altitude watersheds with snow and ice, and othercold localities as closest relatives to our Hydrurus,it is likely that a clade of Hydrurus-related speciesexists on a geographically widespread scale.Unfortunately, the winter and early spring seasons
traditionally have had low priority for sampling inlakes and rivers (cf. Salonen et al., 2009). However,since the recent emergence of a Working Group ofWinter Limnology within the InternationalLimnological Society, and because symposia onwinter limnology are arranged biannually, thereis now a realistic hope of more samples and insitu identifications.
Acknowledgements
We are grateful to Ingvild Riisberg and Russell Orr
for providing the heterokont 28S alignment, and the
latter also for improving the language. The Research
Council of Norway by a grant to KSJ funded theproject (Project # 172572/S40). The Bioportal plat-
form (http://www.bioportal.uio.no) is acknowledged
for providing computer resources. KST thank
University of Oslo for a fellowship to JB.
Supplementary material
The following supplementary material is availablefor this article, via the Supplementary Content tabof the article’s online page at http://dx/doi.org/10.1080/09670262.2011.598950:Hydrurus18S-28S-alignment.nxsHydrurus18S-alignment.nxs
Table 1. Accession numbers, origin of samples and sequences clustering within the Hydrurus clade (AY689714 to AJ867745)
and their closest neighbours in the 18S rDNA tree (Fig. 7). The unpublished author information is from the NCBI-Entrezpages for the accession numbers given in the first column.
GenBank
Accession
Taxonomic
information Strain/Sample
Author
information Location/Sampling site
AY689714 Uncultured stramenopile BSR1LC01 Baeseman et al., unpubl. Acidic subalpine stream
sediment
37777777777777777777777775
The Hydrurus
Clade
AY689712 Uncultured stramenopile BSR1LD02 Baeseman et al., unpubl. Acidic subalpine stream
sediment
HQ230104 Uncultured eukaryote CLBe4 Harding et al., unpubl. High arctic snow
EF432525 Chrysophyceae sp. I76 clone A1 Beaudoin et al., unpubl. Laboratory, cold
adaption exp.
EF165133 Ochromonas sp. CCMP 1899 Andersen (2007)
FN690692 Uncultured stramenopile 3c-D7 Majaneva et al., unpubl. Baltic sea ice, wintertime
water
FM955256 Hydrurus foetidus G 070301 This paper Mountain river in early
spring
AJ867745 Uncultured chrysophyte JFJ-ICE-Uni-10 Yuhana, unpubl. High mountain snow
communities
AY180010 Uncultured chrysophyte CCW7 Stoeck & Epstein (2003) Oxygen-depleted marine
salt marsh
FN690663 Uncultured stramenopile 6c-G3 Majaneva et al., unpubl. Baltic sea ice, wintertime
water
EF165134 Unclassified chrysophyte CCCM 41 Andersen (2007)
EU247834 Chrysophyceae sp. CCMP 2296 Hamilton et al. (2008) Cold complex nearshore
marine system
AY919744 Uncultured eukaryote LG18-10 Richards et al. (2005) Oligotrophic lake
AY179989 Uncultured stramenopile CCI40 Stoeck & Epstein (2003) Oxygen-depleted marine
salt marsh
AF123296 Phaeoplaca thallosa CCMP634 Andersen et al. (1999, 2007) Uncertain, N. America
Molecular phylogeny of Hydrurus 289
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