Downloaded from http://mbe.oxfordjournals.org/ at Centro de Información y Documentación Científica on March 17, 2015 ), ). and .Our Carr et al. del Campo figure 1 Corallochytrium , FRESCHO 3, and a table 1). Our tree also Open Access Marshall and Berbee 2010 elieved. Our results provide chthyosporea: one freshwater equences used in this analysis, onts have focused exclusively t diversity among the filaster- Cavalier-Smith and Allsopp 1996 ), with some internal clades being clade, for which so far only one single Weber et al. 2012 isthokonts, except for the filastereans. ). We also recover three environmental Corallochytrium Nitsche et al. 2011 lineage of marine . It is worth mentioning Our final phylogenetic tree, based on complete 18S ribo- icellular lineages, such as choanoflagellates, filastereans, that the species has been described ( is fairly wellrepresents populated with an environmentalopisthokonts sequences important in and their percentagerecovers entirety all of (5%, previously describedthe the Ichthyosporea. clades There are and unicellular two familiesvironmental well-defined clades exclusively within among en- the I (FRESHIP 1) and(Marine Ichthyosporean the 1) is other fairlymental abundant marine clone libraries, in with (MAIP the 34 environ- s 1). The latter These two marinenovel clades may well represent a completely somal DNA (rDNA) sequences, is shown in topology confirms the divisiontwo of main the choanoflagellates clades: into Acanthoecida2008; and Craspedida ( formed exclusively of environmental sequences ( and Massana 2011 clades (Clade L from singleton) of mainly freshwater choanoflagellates thatas branch a sister group toFurthermore, the branching Acanthoecida and as Craspedida a clades. flagellates, sister there is group a group to constituted all of the choano- two clades ofMAOP marine environmental sequences: 1(Uncultured the Opisthokonts novel 1 and in the previously described MAOP 2 tionary diversity of opisthok ns represent a significant percentage of overall unicellular l use, distribution, and reproduction in any nmental sequences, especially among ichthyosporeans and e environments than previously b Thus,verylittleisknownabou org, [email protected]. ution, choanoflagellates, ichthyosporeans. ), the Carr et al. Creative Commons Attribution Non-Commercial License (http:// ). Although ¸ats (ICREA), Barcelona, Catalonia, half of the Society for Molecular Biology and Evolution. ,1,2,3 Brown et al. ( ensive diversity among all unicellular op at Pompeu Fabra), Barcelona, Catalonia, Spain 30(4):802–805 doi:10.1093/molbev/mst006 Advance Access publication January 16, 2013 eral unicellular lineages nt understanding of the evo- alba ), which permits unrestricted non-commercia aki Ruiz-Trillo* Corallochytrium limacisporum ˜ and abundance) of all nonfungal del Campo and Massana). 2011 ), ), ; ). These include the nucleariids Mol. Biol. Evol. Shalchian-Tabrizi et al. 2008 E-mail: inaki.ruiz@multicellgenome. Paps and Ruiz-Trillo 2010 and In tica, Universitat de Barcelona, Barcelona, Catalonia, Spain ), and therefore, it is the ideal candidate ` 1 Charles Delwiche unicellular opisthokonts, diversity, distrib Catalana per a la Recerca i Estudis Avanc Ragan et al. 1996

´

The Author 2013. Published by Oxford University Press on be Departament de Gene Institucio Institut de Biologia Evolutiva (CSIC-Universit Steenkamp etAmaral-Zettler al. et 2006 al. 2001 Raghu-Kumar 1987), and the choanoflagellates ( Marshall and Berbee 2010 Pawlowski et al. 2012 Associate editor: *Corresponding author: medium, provided the original work is properly cited. 802 ß on metazoans, fungi, and, very recently, choanoflagellates. ichthyosporeans, and nucleariids. To date, studies of the evolu eans, ichthyosporeans, and nucleariids. Tohere better understand we the analyze evolutionary published diversity environmentalphylogenetic and data ecology analyses. from of Our the nonfungal data opisthokonts, reveal unicellular ext opisthokonts and report 18S ribosomal DNA The Opisthokonta clade includes Metazoa, Fungi, and several un Abstract 2 3 among Unicellular Opisthokonts Javier del Campo 1 choanoflagellates. Moreover, we show that the ichthyosporea Environmental Survey Meta-analysis Reveals Hidden Diversity We identify several clades that consist exclusivelyopisthokont of diversity, enviro with a greater ecological role in marin a useful phylogenetic framework for futureKey ecological words: and evolutionary studies of these poorly known lineages. This is an Opencreativecommons.org/licenses/by-nc/3.0/ Access article distributed under the terms of the The opisthokonts include two ofdoms the of most well-studied life: king- thenetic metazoans and and the fungi.opisthokonts phylogenomic Recent analyses phyloge- also( have include shown( that sev the ichthyosporeans (alsozoeans) known ( as DRIPs or mesomyceto- 2009), the filastereans ( ( 2008)(forareviewsee for searching for environmental diversity. lutionary diversity of thepoor. To other date, most phylogenetic lineages studieshave is of focused the very on opisthokonts dataences from as cultured organisms above). (samesequences A refer- into handful account, ofand/or but studies use they take a focus environmental ( limited on number a ofTo single environmental fill clade sequences this gapdiversity (i.e., and species obtain richness anunicellular exhaustive description opisthokonts, of we the somal screened gene clone published libraries andyses. 18S performed In phylogenetic ribo- terms anal- ofribosomal the environmental gene data( provided, is, the 18S by far, the most sequenced gene we have some knowledge of theand diversity choanoflagellates, of our metazoans, fungi, curre

Letter Evolutionary Diversity of Unicellular Opisthokonts . doi:10.1093/molbev/mst006 MBE Downloaded from http://mbe.oxfordjournals.org/ at Centro de Información y Documentación Científica on March 17, 2015

FIG.1. Maximum likelihood (ML) phylogenetic tree of the opisthokonts constructed with 227 complete 18S rDNA sequences (1,436 informative positions). Exclusively environmental clades are indicated with *. The last bar on the right indicates the main environmental origin of the differentclades: blue (marine), green (freshwater), and yellow (symbiont). Clades indicated as symbiont are those in which the species are known to be symbiont and that had almost no environmental sequences (maximum one sequence). Environmental clades consisting of a single sequence have not been named. Because of the increasing number of new clades described and for the sake of clarity, the names have been changed from del Campo and Massana (2011) (supplementary information S1, Supplementary Material online). 1,000 pseudoreplicate ML bootstrap values over 50% are indicated with a black dot for the defined clades. The scale bar indicates 0.09 substitutions per position. The complete tree is shown in the supplementary information S2, Supplementary Material online. The order and class names given are based on Cavalier-Smith and Chao (2003), Shalchian-Tabrizi et al. (2008),and Cavalier-Smith (2009). MACHO, marine choanoflagellates; FRESCHO, freshwater choanoflagellates; MAOP, marine opisthokonts; FRESHIP, freshwater ichthyosporeans; MAIP, marine ichthyosporeans; FRESHOP, freshwater opisthokonts. 803 del Campo and Ruiz-Trillo . doi:10.1093/molbev/mst006 MBE

Table 1. Assignation of Sequences from Both the Cultures and the which represent 6% of the total of unicellular opisthokonts. Environment to the Different Defined Clades (fig. 1). Unexpectedly, we did not retrieve any filasterean environ- Clade #seq %env %env Environments mental sequences, either from the symbiont Capsaspora or opisto MSLF SB from the free-living marine Ministeria, which both appear as Acanthoeca 48 79 6 94 6 — — singletons. Among the , we retrieved , Stephanoeca 17 71 2 100 — — — Fonticula, and the environmental clade FRESHOP (previously Diaphanoeca 55 91 9 100 — — — described by Marshall and Berbee 2010) as a monophyletic MACHO 1 16 88 2 100 — — — group but with no statistical support. This clade was previ- Acanthocorbis 71 90 11 100 — — — ously described as class Discicristoidea (Cavalier-Smith 2009), Monosiga 35 71 4 100 — — — and our data show that it is a mainly a freshwater group. Langenoeca 17 88 3 82 6 12 — Besides finding new environmental clades within the Downloaded from FRESCHO 1 22 100 4 — 5 95 — opisthokonts, our data suggest that the ichthyosporeans Urceolata 12 75 2 100 — — — could play a more significant ecological role in both marine Salpingoeca 31 87 5 — 6 94 — and freshwater environments than previously thought. To Napiformis 15 93 2 — — 100 — date, all the described ichthyosporeans have been isolated FRESCHO2 5 100 1 — — 100 — from metazoans. Therefore, it was assumed that all ichthyos- Pyxidium 250NA——100— poreans were either parasites or commensals of vertebrates http://mbe.oxfordjournals.org/ FRESCHO 4a 18 100 3 — — 100 — and invertebrates (Ragan et al. 1996; Mendoza et al. 2002). FRESCHO 3 33 100 6 — — 100 — However, our data show that some ichthyosporean groups, Clade L 18 100 3 44 — 56 —— such as the Abeoforma, Pseudoperkinsus, and Anurofeca Other MACHOa 9 100 2 100 — — — clades, contain an important proportion (up to 82%) of Other FRESCHOa 5 100 1 — 20 80 — environmental sequences. These are similar percentages of Choanoflagellatea 429 88 66 66 2 32 — environmental sequences as some choanoflagellate clades MAOP 2 15 100 3 100 — — — (table 1). In contrast, clearly parasitic ichthyosporean clades, MAOP 1 9 100 2 100 — — — such as Ichthyophonus and Dermocystidium,havemuchsmal- at Centro de Información y Documentación Científica on March 17, 2015 Corallochytrium 29 97 5 100 — — — ler percentages of environmental sequences (0–7%). Marine Opisthokonts 42 98 9 100 — — — Moreover, we identified two ichthyosporean clades, MAIP 1 Eccrinales 15 — — — — — 100 and FRESHIP 1, which are exclusively environmental. Ichthyophonus 38 — — — — — 100 Moreover, the ichthyosporeans represent an important Amoebidium 7571——5743 percentage (24%) of the overall unicellular opisthokont Psorospermium 1— ————100 sequences, with some clades (such as MAIP1 and FRESHIP 1 4 100 1 — — 100 — Pseudoperkinsus) representing 6% and 11% of the total of Abeoforma 48 33 3 31 2 — 67 unicellular opisthokont sequences, respectively; similar MAIP 1 34 100 6 94 3 3 — values to those of some choanoflagellate groups (table 1). FRESHIP 2a 31001 ——100— A possible explanation for this large percentage of environ- Anurofeca 11 82 2 — — 82 18 mental sequences among the ichthyosporeans is that some Pseudoperkinsus 82 82 11 82 — — 18 clades may have representatives with a free-living stage, at Dermocystidium 29 7 NA — 3 3 93 least. However, we cannot rule out the possibility that they Other MAIPa 5 100 1 100 — — — are infecting small organisms (such as Syndiniales infecting Ichthyosporea 277 52 24 43 1 8 48 Dinoflagellates, see Guillou et al. 2008) that can be recovered Capsaspora 5— ————100 in the 0.2–3 mmfractionusedinmostofthestudies(Massana Ministeria 2— —100——— and Pedro´s-Alio´ 2008). 7 — — 29 — 71 Overall, our data show a large and hidden diversity of Simplex 11 64 1 — 9 91 — nonfungal unicellular opisthokonts, except for the filaster- Termophila 8 38 1 — 13 88 — eans. This hidden diversity, unveiled only after analyzing the Fonticula 250NA50—50— complete set of 18S rDNA sequences from all published en- FRESHOP 2 100 NA — — 100 — vironmental studies based on clone libraries, indicates an im- Discicristoidea 23 57 2 4 9 87 — portant bias in the representation previously assigned to Total 789 75 100 58 2 23 17 cultures, which constitute only a very small fraction of the real diversity. As expected, choanoflagellates are the most NOTE.—#seq, total number of sequences retrieved from GenBank; %env, percentage of environmental sequences; %env opisto, percentage of environmental sequences abundant and important unicellular opisthokont group in within the unicellular opisthokonts; M, marine; SL, salt lake; F, freshwater; SB, sym- the environment, but both the ichthyosporeans and biont. NA, not appicable. Sequences were assigned using two different maximum Corallochytrium clade represent as well an important fraction likelihood phylogenetic trees: one with only complete sequences (fig. 1)andone inferred from both complete and partial 18S rDNA sequences (442 sequences in of the unicellular opisthokont diversity and have the potential total, 1,286 informative positions, shown in supplementary information S3, to be prominent ecological players. Thus, it is crucial to Supplementary Material online). increase our efforts in culturing the little-known unicellular a Clades that appear as an unnamed singletons in figure 1 or that are only recovered opisthokonts to better understand their ecological role. from partial sequences. Our phylogenetic tree provides an important phylogenetic

804 Evolutionary Diversity of Unicellular Opisthokonts . doi:10.1093/molbev/mst006 MBE framework for future evolutionary studies of the ichthyospor- Carr M, Leadbeater BSC, Hassan R, Nelson M, Baldauf SL. 2008. eans, nucleariids, Corallochytrium, filastereans, and choano- Molecular phylogeny of choanoflagellates, the sister group to Metazoa. Proc Natl Acad Sci U S A. 105:16641–16646. flagellates. Moreover, new environmental data, single-cell Cavalier-Smith T. 2009. Megaphylogeny, cell body plans, adaptive zones: genomics, and the application of fluorescence in situ hybrid- causes and timing of basal radiations. J Eukaryot Microbiol. ization to environmental samples are also needed to further 56:26–33. increase our knowledge of the real unicellular opisthokont Cavalier-Smith T, Chao EEY. 2003. Phylogeny of , , diversity. and other protozoa and early eukaryote megaevolution. J Mol Evol. 56:540–563. Materials and Methods Cavalier-Smith T, Paula Allsopp MTE. 1996. Corallochytrium,anenig- matic non-flagellate protozoan related to choanoflagellates. Eur J Environmental 18S rDNA sequences of opisthokonts were Protistol. 32:306–310. obtained from GenBank following a two-step screening del Campo J, Massana R. 2011. Emerging diversity within chrysophytes, process. First, we retrieved sequences using the NCBI taxon- choanoflagellates and bicosoecids based on molecular surveys. Downloaded from omy tool. The sequences were further checked by blastn to 162:435–448. confirm their phylogenetic assignment. Second, we used Guillou L, Bachar D, Audic S, et al. (32 co-authors). 2013. The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukar- these and other published sequences from cultures or envi- yote Small Sub-Unit rRNA sequences with curated . ronmental surveys that belong to the target groups (but are Nucleic Acids Res. 41:597–604. not labeled as such in GenBank) to retrieve additional se- Guillou L, Viprey M, Chambouvet A, Welsh RM, Kirkham AR, Massana http://mbe.oxfordjournals.org/ quences using blastn. Putative chimeric sequences were fur- R, Scanlan DJ, Worden AZ. 2008. Widespread occurrence and ther checked with KeyDNATools (Guillou et al. 2013)as genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environ Microbiol. 10:3349–3365. described elsewhere (Guillou et al. 2008). A total of 789 Marshall WL, Berbee ML. 2010. Facing unknowns: living cultures (Pirum sequences were finally retrieved from GenBank. Maximum gemmata gen. nov., sp. nov., and Abeoforma whisleri,gen.nov.,sp. likelihood phylogenetic trees were constructed using nov.) from invertebrate digestive tracts represent an undescribed RAxML with the GTRCATI model of evolution and with clade within the unicellular Opisthokont lineage Ichthyosporea wide taxon coverage, to establish whether ambiguous diver- (). Protist 162:33–57. Massana R, Pedro´s-Alio´ C. 2008. Unveiling new microbial in gent sequences belonged to a given group. Repeated runs on at Centro de Información y Documentación Científica on March 17, 2015 the surface ocean. Curr Opin Microbiol. 11:213–218. distinct starting trees were carried out to select the tree with Mendoza L, Taylor JW, Ajello L. 2002. The class mesomycetozoea: a the best topology (the most likely of 1,000 alternative trees). heterogeneous group of microorganisms at the animal-fungal Details on the phylogenetic methods are explained elsewhere boundary. Ann Rev Microbiol. 56:315–344. (del Campo and Massana 2011). Nitsche F, Carr M, Arndt H, Leadbeater BS. 2011. Higher level taxonomy and molecular phylogenetics of the choanoflagellatea. JEukaryot Supplementary Material Microbiol. 58:452–462. Paps J, Ruiz-Trillo I. 2010. Animals and their unicellular ancestors. In: Supplementary information S1–S3 are available at Molecular Encyclopedia of life sciences (ELS). John Wiley & Sons, Ltd: Biology and Evolution online (http://www.mbe.oxfordjournals. Chichester, UK. doi:10.1002/9780470015902.a0022853. org/). Pawlowski J, Audic S, Adl SM, et al. (33 co-authors). 2012. CBOL protist working group: barcoding eukaryotic richness beyond the animal, Acknowledgments plant, and fungal kingdoms. PLoS Biol. 10:e1001419. Ragan MA, Goggin CL, Cawthorn RJ, Cerenius L, Jamieson AV, Plourde The authors are grateful to Diego Mallo for bioinformatic SM, Rand TG, So¨derhall K, Gutell RR. 1996. A novel clade of protis- support and to Ramon Massana for helpful discussions. tan parasites near the animal-fungal divergence. Proc Natl Acad Sci This work was supported by an Institucio´ Catalana per a USA.93:11907–11912. la Recerca i Estudis Avanc¸ats contract, a European Research Raghu-kumar S. 1987. Occurrence of the traustochytrid Council Starting Grant (ERC-2007-StG-206883), and a Corallochytrium limacisporum gen. et sp. nov. in the coral reef la- goons of the Lakshadweep Islands in the Arabian Sea. Bot Mar. 30: grant (BFU2011-23434) from Ministerio de Economı´ay 83–89. Competitividad (MINECO) to I.R.-T. Shalchian-Tabrizi K, Minge MA, Espelund M, Orr RJS, Ruden T, Jakobsen KS, Cavalier-Smith T. 2008. Multigene phylogeny of choanozoa and References the origin of animals. PLoS One 3:e2098. Amaral-Zettler LA, Nerad TA, O’Kelly CJ, Sogin ML. 2001. The nucleariid Steenkamp ET, Wright J, Baldauf SL. 2006. The protistan origins of amoebae: more at the animal-fungal boundary. JEukaryot animals and fungi. MolBiolEvol.23:93–106. Microbiol. 48:293–297. WeberF,delCampoJ,WylezichC,MassanaR,Ju¨rgens K. 2012. Brown MW, Spiegel FW, Silberman JD. 2009. Phylogeny of the “forgot- Unveiling trophic functions of uncultured protist taxa by ten” cellular , Fonticula alba, reveals a key evolutionary incubation experiments in the brackish Baltic Sea. PLoS One 7: branch within Opisthokonta. Mol Biol Evol. 26:2699–2709. e41970.

805