THE CHOANOFLAGELLATES Daniel J. Richter1,2,†, Martin Carr3,†, Helge Abildhauge Thomsen4, Parinaz Fozouni2, Timothy J. Smith3, Alexandra Jeuck5, Barry S. C. Leadbeater6 & Frank Nitsche5

IchthyosporeaMetazoa

Microstomoeca  roanoka (ATCC  50931)  Salpingoeca  rosetta (ATCC  50818)

Salpingoeca  infusionum (ATCC  50559)

Hartaetosiga  balthica  (ATCC  50964)Hartaetosiga  gracilis (ATCC  50454)

Monosiga  brevicollis  (ATCC  50154)Choanoeca  perplexa (ATCC  50453)

Salpingoeca  dolichothecata (ATCC  50959)Salpingoeca  tuba

Salpingoeca  qvevrii (ATCC  50929)

Salpingoeca  macrocollata (ATCC  50938)Salpingoeca  punica (ATCC  50788)

Mylnosiga  fluctuans  (ATCC  50635)

Salpingoeca  helianthica (ATCC  50153)Desmarella  sp.

Sphaeroeca  volvox

Codosiga  sp.  M3/MvidCodosiga  botrytis

Codosiga  sp.  M5/Iceland

Codosiga  sp.  M2/MoroccoCodosiga  sp.  M1/pIIp

Acanthoeca  spectabilis (ATCC  PRA-­103)  Savillea  parva  (ATCC  PRA-­391)

Polyoeca  dichotoma

Helgoeca  nana (ATCC  50073)

Didymoeca  costata (ATCC  PRA-­389)

Stephanoeca  aphelesAcanthocorbis  unguiculata

Parvicobicula  pedunculata

Diaphanoeca sp.Diaphanoeca  grandis  (ATCC  50111)

Stephanoeca  cauliculataStephanoeca  paucicostata

Stephanoeca  norrisii

Stephanoeca  diplocostata  (ATCC  PRA-­392)

Salpingoeca  calixa  

Stagondoeca  pyriformis

Salpingoeca  oahui

Codosiga  hollandica (ATCC  PRA-­388)

Salpingoeca  urceolata  (ATCC  50560)

Acanthoecidae

Acanthoecida

Stephanoecidae

Clade  2

Craspedida

Choanoflagellatea

Clade  3

0.06

Hartaetosiga  minima

Clade  1

97/0.95

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*

Salpingoeca  limnea

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77/-­

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99

91

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9799

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9593

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97 94

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Class Choanoflagellatea Cavalier-Smith, 1998 emend. Also at various times known as: Craspedmonadina Stein, 1878; Choanoflagellata Kent, 1880–1882; Craspedomonadaceae Senn, 1900; Craspedophyceae Chadefaud, 1960; Craspédomonadophycidées Bourrelly, 1968; Craspedomonadophyceae Hibberd, 1976 Choanomonadea Krylov et al., 1980; Choanoflagellida Lee, Hutner, and Bovee, 1985, Choanoflagellea Cavalier-Smith, 1997; Choanomonada Adl et al., 2005. Diagnosis. Small, uninucleate protists with a single, centrally positioned, anterior flagellum, which is surrounded by collar of long, actin-supported microvilli. Phagotrophic. Flagellar basal body associated with ring or multiple arcs of cytoskeletal (cortical) microtubules. Second “dormant” basal body located at an angle to flagellar basal body. Fibrillar root if present minor and without obvious banding. Mitochondrial cristae flattened.

Order Craspedida Cavalier-Smith, 1997 emend. Diagnosis. Choanoflagellates with extracellular investment that is entirely organic and does not project above the anterior end of the extended feeding cell. Vegetative stage usually sedentary and stalked. Brief motile stage for dispersal.

Family Salpingoecidae Kent (1880–1882), emend. sensu Nitsche et al. (2011). Diagnosis. Vegetative cells with extracellular investment that is entirely organic. Investment may be either a flexible, nonrestrictive fibrous matrix or a rigid, microfibril-based theca. Sedentary cells adhere to a surface by a peduncle extending from the base of the investment. Cell division when investment is nonrestricting is longitudinal in situ. Cell division when investment is restricting (theca) is emergent and involves cell becoming amoeboid and dividing outside the theca. Juvenile daughter cells can disperse as naked, flagellated cells. Under certain conditions colonies of flagellated cells may develop. Omnipresent in freshwater, marine, and brackish water habitats. Type genus: Salpingoeca James-Clark (1867). Recognized genera: Astrosiga Kent, 1880, Aulomonas Lackey, 1942, Choanoeca Ellis, 1929, CladospongiaIyengar and Ramanathan, 1940, Codonocladium Stein, 1878, Codonosigopsis Senn, 1900, Codosiga (junior synonym Codonosiga) James-Clark, 1867, Desmarella Kent, 1878 (junior synonyms Codonodesmus and Kentrosiga), Dicraspedella Ellis, 1929, Diploeca Ellis, 1929, Diplosiga Frenzel, 1891, Diplosigopsis Francé, 1897, Kentia Schiller, 1953, Lagenoeca Kent, 1880, Monosiga Kent, 1880*, Pachysoeca Ellis, 1929, Proterospongia Kent 1880*, Salpingoeca James-Clark, 1867*, Salpingorhiza Klug, 1936, Sphaeroeca Lauterborn, 1894, Stelexomonas Lackey, 1942, Stylochromonas Lackey, 1940.

Order Acanthoecida Cavalier-Smith, 1997 emend. Diagnosis. Cells surrounded by a basket-like lorica of siliceous costae comprising rod-shaped costal strips and a partial or entire organic investment on inner surface.

Family Acanthoecidae Norris, 1965, emend. sensu Nitsche et al. 2011 Diagnosis. Lorica with costae arranged in two layers, outer longitudinal and inner helical. Occasionally only one layer around cell body, in which case costae are helical. Adult loricate cells sedentary, cell cycle, and lorica production accord to nudiform condition. Cell division is diagonal resulting in both daughter cells facing forwards. Upper daughter cell (juvenile) is naked with flagellum that serves for dispersal. Juvenile attaches to surface, withdraws flagellum, and deposits costal strips in correct orientation. Strips are accumulated in vertical bundles on the surface of the spindle-shaped cell body. Costal strips destined for longitudinal costae are deposited first followed by those for the inner layer of costae. Lorica assembly is achieved by a forwards and clockwise rotation which extends costal strips to form mature pattern of costae. Type genus: Acanthoeca Ellis 1929. Recognized genera: Acanthoeca Ellis, 1929, Helgoeca Leadbeater, 2008, Polyoeca Kent, 1880, Savillea Loeblich, 1967.

Family Stephanoecidae fam. n. Leadbeater Diagnosis. Costae arranged in two layers with longitudinal costae outermost; internal costal layer is helical in posterior chamber of some species, but usually comprises transverse costae (rings) in the anterior chamber of lorica. Some species only possess transverse rings. Cell cycle and lorica production accords to tectiform condition. Costal strips are deposited in inverted orientation; strips destined for the inner layer of costae are deposited first followed by those for the outer layer of longitudinal costae. Costal strips are exocytosed through anterior end of cell and accumulated in bundles at the top of the inner surface of the collar. Cell division is inverted with juvenile daughter cell being turned upside down and pushed into accumulated strips and emerging from parent lorica backwards. Costal strips destined for longitudinal and helical costae are located vertically on juvenile cell, costal strips destined for transverse costae are located horizontally. Once free of parent lorica, juvenile cell constructs lorica immediately and there is no swimming dispersal stage. Type genus: Stephanoeca Ellis 1929. Recognized genera: Acanthocorbis Hara & Takahashi, 1984, Amoenoscopa Hara & Takahashi, 1987, Apheloecion Thomsen, 1983 in Thomsen and Boonruang (1983b), Bicosta Leadbeater, 1978, Calliacantha Leadbeater, 1978, Calotheca Thomsen & Moestrup, 1983, Campanoeca, Throndsen, 1974, Campyloacantha Hara & Takahashi, 1987, Conion Thomsen, 1982, Cosmoeca Thomsen, 1984, in Thomsen and Boonruang (1984), Crinolina Thomsen, 1976, Crucispina Espeland, 1986 in Espeland and Throndsen (1986), Diaphanoeca Ellis, 1929, Didymoeca Doweld, 2003, Kakoeca Buck & Marchant 1990 in Buck et al. (1990), Monocosta Thomsen, 1979, Nannoeca Thomsen, 1988, Parvicorbicula Deflandre, 1960, Platypleura Thomsen, 1983 in Thomsen and Boonruang (1983a), Pleurasiga Schiller, 1925, Polyfibula Manton, 1981 in Manton and Bremer (1981), Saepicula Leadbeater, 1980, Saroeca Thomsen, 1979, Spinoeca Thomsen, 1995 in Thomsen, Østergaard and Hansen (1995), Spiraloecion Marchant & Perrin, 1986, Stephanacantha Thomsen, 1983 in Thomsen and Boonruang (1983a), Stephanoeca Ellis, 1930*, Syndetophyllum Thomsen & Moestrup, 1984. *Denotes genera known to require taxonomic revision. From: Higher Level Taxonomy and Molecular Phylogenetics of the Choanoflagellatea, Frank Nitsche†, Martin Carr†, Hartmut Arndt and Barry S. C. Leadbeater, Journal of Eukaryotic Microbiology, Volume 58, Issue 5, pages 452–462, September/October 2011

Linking Choanoflagellate Morphological and Sequence Data by Sampling and Single-Cell PCR

Choanoflagellate Phylogeny Based on 42 Species

(a) - (c) Phase contrast light microscopy images of choanoflagellates. (a) Codosiga hollandica, a freshwater stalked craspedid without a theca. (b) An undescribed freshwater flask-shaped thecate craspedid from the River Rhine, Germany. (c) Didymoeca elongata, a marine tectiform loricate, (d) Scanning electron microscopy image of an empty Didymoeca elongata lorica. b = bacterium, c = collar, f = flagellum, p = pedicel, t = theca, v = food vacuole; lc = longitudinal costae of the lorica, tc = transverse costae of the lorica. All scale bars = 10 µm.

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1e−051e−040.0010.010.10.89

Rel. Abund in %

Google Maps

Calliacantha simplex Crinolina isefiordensis

Pleurasiga reynoldsii

Calliacantha longicaudata

Calliacantha natans Cosmoeca ventricosa

Diaphanoeca undulata

Pleurasiga minima

Parvicorbicula circularis

Stéphane Audic, Nicolas Henry

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1e−051e−040.0010.010.10.59Rel. Abund in %

1 Station Biologique de Roscoff, France, 2 Department of Molecular and Cell Biology, University of California, Berkeley, United States, 3 School of Applied Sciences, University of Huddersfield, United Kingdom, 4 Institute for Aquatic Resources, Denmark Technical University, Denmark,5 General Ecology and Limnology, University of Cologne, Germany, 6 School of Biosciences, University of Birmingham, United Kingdom

Choanoflagellates in Tara Oceans

The phylogeny is based upon 9,463 aligned nucleotides positions from partial sequences of the genes SSU, LSU, tubA, hsp90, EFL and EF-1A. Branches are drawn proportional to the number of nucleotide substitutions per site as indicated by the scale bar at the lower left. Values of 100% mlBP and 1.00 biPP support are denoted by an *, and mlBP and biPP values reflecting partial support are otherwise given above and below branches, respectively. Values are omitted from weakly supported branches (i.e. mlBP<50%, biPP<0.70). Newly described species are written in bold font. Numerous undescribed environmental lineages (del Campo and Ruiz-Trillo, Mol. Biol. Evol., 2013) are not depicted on the tree.

Over the course of two separate one-week field trips in Denmark, we identified loricate choanoflagellates by light microscopy, performed single-cell PCR using universal 18S and 28S ribosomal primers and Sanger sequenced the resulting PCR products. We established a link between morphology and sequence data for 13 species and 5 genera that had never previously been sequenced. The ribosomal sequences we provided can now be used to search for choanoflagellate sequences in Tara Oceans and other barcoding data sets, and as a mechanism to provide species identification for single-cell amplified genomes, to cite two examples. We propose short field trips such as this one, involving experts in group-specific morphology and in single-cell isolation, as a time- and cost-effective means to link morphology with molecular data for all protist groups where morphology can be used for species identification.

Phylogenetic Tree Including Newly Sequenced Species

Sampling Locations and “Traveling Laboratory”

in Denmark

Selected Images of Newly Sequenced Choanoflagellate Species

Relative Abundance of Newly Identified Tara V9 Barcodes

Diaphanoeca sphaerica

Parvicorbicula superpositus

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Higher-Order Choanoflagellate Taxonomy

All Choanoflagellates Loricate Choanoflagellates

Craspedid Choanoflagellates

Relative Abundance of V9 Barcodes (0.8 - 5 um, surface)

Ichthyosporea

Polyoeca dichotoma

Diaphanoeca spiralifurca Crinolina isefiordensis

Didymoeca costata Didymoeca elongata