Report Phylogenomics Reveals Convergent Evolution of Lifestyles in Close Relatives of Animals and Fungi Graphical Abstract Authors Guifre´ Torruella, Alex de Mendoza, Xavier Grau-Bove´ , ..., Ariadna Sitja` -Bobadilla, Stuart Donachie, In˜ aki Ruiz-Trillo Correspondence [email protected] In Brief Torruella et al. provide new molecular data from several protists and infer a novel phylogenomic framework for the opisthokonts that suggests rampant convergent evolution of several characters. Using comparative genomics, the authors show independent losses of the flagellum and delineate the evolutionary history of chitin synthases in this lineage. Highlights d Taxon-rich phylogenomics provides an evolutionary framework for the opisthokonts d Specialized osmotrophy evolved independently in fungi and animal relatives d Opisthokonts underwent independent secondary losses of the flagellum d The last opisthokont common ancestor had a complex repertoire of chitin synthases Torruella et al., 2015, Current Biology 25, 2404–2410 September 21, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.07.053 Current Biology Report Phylogenomics Reveals Convergent Evolution of Lifestyles in Close Relatives of Animals and Fungi Guifre´ Torruella,1,2,12 Alex de Mendoza,1,2,12 Xavier Grau-Bove´ ,1,2 Meritxell Anto´ ,1 Mark A. Chaplin,3 Javier del Campo,1,4 Laura Eme,5 Gregorio Pe´ rez-Cordo´ n,6 Christopher M. Whipps,7 Krista M. Nichols,8,9 Richard Paley,10 Andrew J. Roger,5 Ariadna Sitja` -Bobadilla,6 Stuart Donachie,3 and In˜ aki Ruiz-Trillo1,2,11,* 1Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Marı´tim de la Barceloneta 37-49, Barcelona 08003, Catalonia, Spain 2Departament de Gene` tica, Universitat de Barcelona, Avinguda Diagonal 645, Barcelona 08028, Catalonia, Spain 3Department of Microbiology, University of Hawaii at Manoa, Snyder Hall, 2538 McCarthy Mall, Honolulu, HI 96822, USA 4Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada 5Department of Biochemistry and Molecular Biology, Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, NS B3H 4R2, Canada 6Institute of Aquaculture Torre de la Sal, IATS-CSIC, Ribera de Cabanes s/n, Castello´ 12595, Spain 7Environmental and Forest Biology, State University of New York College of Environmental Science and Forestry (SUNY-ESF), Syracuse, NY 13210, USA 8Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA 9Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, WA 98112, USA 10Centre for Environment Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK 11Institucio´ Catalana de Recerca i Estudis Avanc¸ ats (ICREA), Passeig Lluı´s Companys 23, Barcelona 08010, Catalonia, Spain 12Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.07.053 SUMMARY secondarily underwent flagellar simplification. Anal- ysis of the evolutionary history of chitin synthases The Opisthokonta are a eukaryotic supergroup revealed significant expansions in both animals divided in two main lineages: animals and related and fungi, and also in the Ichthyosporea and protistan taxa, and fungi and their allies [1, 2]. There C. limacisporum, a group of cell-walled animal rela- is a great diversity of lifestyles and morphologies tives. This indicates that the last opisthokont among unicellular opisthokonts, from free-living common ancestor had a complex toolkit of chitin phagotrophic flagellated bacterivores and filopodi- synthases that was differentially retained in extant ated amoebas to cell-walled osmotrophic parasites lineages. Thus, our data provide evidence for conver- and saprotrophs. However, these characteristics do gent evolution of specialized lifestyles in close rela- not group into monophyletic assemblages, suggest- tives of animals and fungi from a generalist ancestor. ing rampant convergent evolution within Opistho- konta. To test this hypothesis, we assembled a new RESULTS AND DISCUSSION phylogenomic dataset via sequencing 12 new strains of protists. Phylogenetic relationships among opis- Broad Taxonomic Sampling Provides New Phylogenetic thokonts revealed independent origins of filopodi- Insights into the Evolution of the Opisthokonta ated amoebas in two lineages, one related to fungi Previous attempts to solve opisthokont phylogeny swayed be- and the other to animals. Moreover, we observed tween species-rich datasets with poor deep-node resolution that specialized osmotrophic lifestyles evolved inde- based on small ribosomal subunit [1–3] and multigene superma- pendently in fungi and protistan relatives of animals, trices that included few taxa [4–6]. To improve upon our previ- indicating convergent evolution. We therefore ously published phylogenomic dataset [6], we therefore sampled analyzed the evolution of two key fungal characters representative species in all described opisthokont lineages (see Table S1 and Supplemental Experimental Procedures). This in Opisthokonta, the flagellum and chitin synthases. included representatives of nucleariids, choanoflagellates, filas- Comparative analyses of the flagellar toolkit showed tereans, and the two main lineages of Ichthyosporea (Dermocys- a previously unnoticed flagellar apparatus in two tidia and Ichthyophonida). In addition, we included two different close relatives of animals, the filasterean Ministeria strains of the enigmatic Corallochytrium limacisporum, a spher- vibrans and Corallochytrium limacisporum. This im- ical free-living walled saprotroph found in coral reefs [7]. Origi- plies that at least four different opisthokont lineages nally classified as a thraustochytrid based on its morphology, 2404 Current Biology 25, 2404–2410, September 21, 2015 ª2015 Elsevier Ltd All rights reserved C. limacisporum has been unstably placed within the Opistho- mensals [18], despite being frequently found in environmental konta in all molecular phylogenies to date because of the scarce surveys [3]. The life cycles of C. limacisporum and Ichthyospor- molecular data available [8–11]. In order to improve the opistho- eans [7, 18] are strikingly similar: both start as a single cell that kont outgroup, we also sampled the ancyromonad Nutomonas grows as a coenocyte until it reaches maturation, when it un- longa CCAP 1958/5 [12], which is putatively related to Apusomo- dergoes schizogony. The dispersive amoeboid or flagellated nadida [11]. Overall, we generated new transcriptomic data for progeny (merozoites) settle and close the cycle [18]. Chytrid fungi 10 protistan taxa (11 strains in total, highlighted in bold in Fig- show a similar developmental mode, with both coenocytic growth ure 1), plus new genomic data from another strain (Ichthyopho- and amoeboid or flagellated stages [19]. Similarly, fungi also nus hoferi). This represents the broadest taxon sampling to evolved from phagotrophic ancestors (Discicristoidea, Rozella, date to infer the opisthokont phylogeny. and Aphelida [20]) to become saprotrophs and parasites. More- To investigate the phylogenetic relationships, we assembled over, some Ichthyosporea species (A. parasiticum and I. hoferi) two datasets comprising a total of 93 single-copy protein do- present a mode of polar growth that clearly resembles fungal hy- mains: one with 83 taxa and 18,218 aligned amino acid positions phae [21]. Thus, teretosporeans and fungi present tantalizing sim- (S83), and the other with 70 taxa and 22,313 amino acid positions ilarities regarding life style adaptations and morphologies. (S70). The latter dataset was constructed to maximize alignment The resulting opisthokont tree also confirms the convergent length and to minimize topological artifacts by excluding putative evolution of filose amoebas, Filasterea within the Holozoa and problematic taxa with long branches (e.g., Microsporidia, Exca- Discicristoidea within the Holomycota. Both lineages have vata) and high percentages of missing data (e.g., taxa with only evolved a similar cell morphology comprising long, actin-based expressed sequence tag data) (see Table S1). Both datasets filopodia [22], with some taxa going through an aggregative were consistent in recovering the backbone of the eukaryotic multicellular cell stage in their life cycles [23]. phylogeny using both Bayesian inference (BI) (Figures 1 and S1C) and maximum likelihood (ML) (Figures S1A and S1B; see Independent Loss of the Flagellum within the Supplemental Experimental Procedures for details). Opisthokonta As sister groups to Opisthokonta, we recovered Apusomona- A single posterior motile flagellum is a defining character of opis- dida and Breviatea as recently reported [13], branching as inde- thokonts [2]. Our observation that both filose amoebas and pendent lineages and not forming a monophyletic group or fungal-like lineages evolved in independent branches within clustering with amoebozoans. Interestingly, the topology of opisthokonts therefore predicts independent loss of the flagel- the S83 dataset placed Nutomonas longa (Ancyromonadida) lum. To address this hypothesis, we analyzed the evolution of branching closer to the Excavata and not closely related to the the flagellar toolkit [24, 25]. The molecules that comprise the fla- Apusomonadida and Opisthokonta. This contrasts with previous gellum include specialized tubulins (epsilon, delta)[26],