Absence of Co-Phylogeny Indicates Repeated Diatom Capture in Dinophytes Hosting a Tertiary Endosymbiont

Org Divers Evol (2018) 18:29–38 https://doi.org/10.1007/s13127-017-0348-0

ORIGINAL ARTICLE

Absence of co-phylogeny indicates repeated diatom capture in dinophytes hosting a tertiary endosymbiont

Anže Žerdoner Čalasan1 & Juliane Kretschmann1 & Marc Gottschling1

Received: 28 July 2017 /Accepted: 26 October 2017 /Published online: 18 November 2017 # Gesellschaft für Biologische Systematik 2017

Abstract Tertiary endosymbiosis is proven through Keywords Chloroplast . Dinoflagellates . Dinotoms . dinophytes, some of which (i.e. Kryptoperidiniaceae) have Endosymbiosis . Evolution . Mutualism engulfed diatom algae containing a secondary plastid. Chloroplasts are usually inherited together permanently with the host cell, leading to co-phylogeny. We compiled a diatom Introduction sequence data matrix of two nuclear and two chloroplast loci. Almost all endosymbionts of Kryptoperidiniaceae found their As one of the important groups of unicellular eukaryotic life closest relatives in free-living diatoms and not in other forms, Dinophyceae (or Dinoflagellata under zoological no- harboured algae, rejecting co-phylogeny and indicating that menclature) are nothing less but diverse from every single resident diatoms were taken up by dinophytes multiple times point of view (Morden and Sherwood 2002; Pochon et al. independently. Almost intact ultrastructure and insignificant 2012; Gottschling and McLean 2013; Burki 2014). Together genome reduction are supportive for young, if not recent with Ciliata and Apicomplexa (= Sporozoa), Dinophyceae events of diatom capture. With their selective specificity on belong to Alveolata and are a well-supported monophyletic the one hand and extraordinary degree of endosymbiotic flex- group based on molecular data and numerous unique anatom- ibility on the other hand, dinophytes hosting diatoms share ical characteristics (Harper et al. 2005; Medlin and Fensome more traits with lichens or facultatively phototrophic ciliates 2013; Keeling et al. 2014; Janouškovec et al. 2017). Compared than with green algae and land plants. Time estimates indicate to all other eukaryotes, the genome of dinophytes is highly the dinophyte lineages as consistently older than the hosted unusual with respect to structure and regulation (Moreno Díaz diatom lineages, thus also favouring a repeated uptake of en- de la Espina et al. 2005; Wisecaver and Hackett 2011). The dosymbionts. The complex ecological role of dinophytes nucleus contains chromosomes that are permanently condensed employing a variety of organismic interactions may explain throughout cell development (displaying a liquid crystalline their high potential and plasticity in acquiring a great diversity state: Rill et al. 1989) except in the DNA replication stage of plastids. (Dodge 1966; Rizzo 2003). Dinophyte morphology also ex- hibits unique traits such as the coiled transverse flagellum asso- ciated with a transverse groove termed the ‘cingulum’ (Taylor 1980; Fensome et al. 1999; Leander and Keeling 2004; Electronic supplementary material The online version of this article Okamoto and Keeling 2014). (https://doi.org/10.1007/s13127-017-0348-0) contains supplementary Despite the fact that they represent only about a half of all material, which is available to authorized users. dinophyte species (Taylor et al. 2008), photosynthetically active taxa have gained chloroplasts in numerous advanced * Marc Gottschling [email protected] ways (Gagat et al. 2014; Dorrell and Howe 2015), excluding the most basic primary engulfment (primary endosymbiosis). 1 Department Biologie, Systematische Botanik und Mykologie, The most common and widespread chloroplast type found in GeoBio-Center, Ludwig-Maximilians-Universität München, dinophytes indicates a secondary engulfment event (second- Menzinger Str. 67,, 80 638 Munich, Germany ary endosymbiosis) of a red algae (Dodge 1989; Jeffrey 1989; 30 Žerdoner Čalasan A. et al.

Janouškovec et al. 2017). Some dinophyte predators capture Takano et al. 2008) surrounded by a triple-membrane structure additional plastids of their prey and keep them up to several (Horiguchi and Pienaar 1994). weeks in an evolutionary phenomenon known as All Kryptoperidiniaceae constitute a monophyletic group kleptoplastidy (Schnepf and Elbrächter 1999; Stoecker 1999; as a part of the Peridiniales (Pienaar et al. 2007; Takano et al. Gast et al. 2007; Takano et al. 2014). Other dinophytes have 2008; Gottschling and McLean 2013; Janouškovec et al. taken up a different endosymbiont in a more permanent way 2017; Price and Bhattacharya 2017; Kretschmann et al. in and have brought the endosymbiosis even further to a tertiary press), but their endosymbionts originate from at least three level by engulfing organisms such as diatoms, which already different diatom lineages. The currently accepted scenario de- possessed secondarily gained chloroplasts (Keeling 2004, scribes the single acquisition of a Nitzschia Hassall-related 2010; Hehenberger et al. 2014). organism (present today in Durinskia Carty & El.R.Cox, An example for a chloroplast replacement at a second- Galeidinium Tam. & T.Horig. and Kryptoperidinium ary level of endosymbiosis is Lepidodinium M.Watan., Er.Lindem.: Tamura et al. 2005; Pienaar et al. 2007; Zhang S.Suda, I.Inouye et al. (Gymnodiniaceae s.str.) enclosing et al. 2011a) that has been replaced by either Cyclotella a green alga (Kamikawa et al. 2015). Tertiary endosym- (Kütz.) Bréb.-related (in freshwater Unruhdinium biosis, primarily found in dinophytes, is even more com- Gottschling: Takano et al. 2008; Zhang et al. 2011b;You plex from the symbiont’s point of view. Tertiary plastids et al. 2015)orbyChaetoceros Ehrenb.-related symbionts (in are present in, for example, species of Dinophysis Ehrenb. Blixaea Gottschling: Horiguchi and Takano 2006)intwofur- (Dinophysaceae) possessing cryptomonad-derived sym- ther evolutionary steps. The phenomenon of monophyletic bionts separated from the host by two membranes hosts and polyphyletic symbionts has thus been explained (Schnepf and Elbrächter 1988; Garcia-Cuetos et al. by ‘serial replacement’ (Horiguchi and Takano 2006; 2010). Taxa such as Karenia Gert Hansen & Moestrup Takano et al. 2008) of an already engulfed diatom. However, and Karlodinium J.Larsen (Brachidiniaceae) possess chlo- this idea has been developed based on single-locus analyses roplasts that have originated from haptophyte algae with a rather limited diatom taxon sample. (Hansen et al. 2003;Gastetal.2007). Endosymbiont While preparing a similar study of our own, we took inter- reduction is severe in this group, and the three- est in the study of Yamada et al. (2017). The study advocates membrane layer as part of the chloroplast is the only serial replacement, considering a few diatom acquisition structure left of the engulfed organism. An ancestral events, after which endosymbionts are established, maintained peridinin chloroplast may have co-existed with the newly and inherited. Subsequently, endosymbionts are in permanent gained chloroplast of haptophyte origin but has been lost association with the host cell and should thus find their closest eventually (Nosenko et al. 2006;Patronetal.2006; relatives among other endosymbionts. Co-phylogenetic struc- Takano et al. 2014). However, there is no ultrastructural tures should therefore be present between separately derived evidence that Brachidiniaceae have ever possessed two tress of diatoms and their hosting dinophytes. This hypothesis different functional chloroplasts. is not explicitly phrased in Yamada et al. (2017), although it is Based on ultrastructural analyses of those dinophytes the necessary conceptual basis to understand the origin of harbouring diatoms (Kryptoperidiniaceae, commonly referred dinophytes harbouring tertiary endosymbionts and the dy- to as ‘dinotoms’: Imanian et al. 2010), the endosymbiont namics of this (endo)symbiont/host association. Our data pro- keeps not only its photosynthesis machinery but also the func- vide evidence for a repeated acquisition of endosymbiotic tional nucleus with protein encoding genes, a substantial diatoms through their evolutionary history and for the rejec- amount of cytosol and mitochondria (McEwan and Keeling tion of overall co-phylogeny. Tertiary endosymbiosis present 2004; Imanian and Keeling 2007; Imanian et al. 2012; Takano in Kryptoperidiniaceae may have happened recently in com- et al. 2008). However, numerous studies also show a loss of parison to many other algal groups, in which severe reduction motility, cell wall and mitotic division (Dodge 1971; Tomas has taken place leaving behind only the chloroplasts and their and Cox 1973). Despite this major autonomy and minimal membranes. reduction, the resident diatom is present in the host cell in all stages of development, which indicates synchrony of life history between both host and endosymbiont (Figueroa et al. Materials and methods 2009). Moreover, diatom mitochondria seem to be almost un- affected by endosymbiosis, retaining nearly all of their char- From the dinophyte endosymbionts of Durinskia oculata acteristics and functions (Imanian and Keeling 2007;Imanian (F.Stein) Gert Hansen & Flaim and Kryptoperidinium sp., et al. 2012). Engulfed diatoms due to tertiary endosymbiosis we sequenced the nucleomorph small subunit (SSU) and replace the ancestral peridinin chloroplast, whose remnants Internal Transcribed Spacers (ITS) and plastid rbcLlocias are considered to be a unique form of an eyespot (Dodge previously described (Kretschmann et al. in press). In order 1983; Horiguchi et al. 1999; Moestrup and Daugbjerg 2007; to place dinophyte endosymbionts on diatom reference trees, we Absence of co-phylogeny indicates repeated diatom capture in dinophytes hosting a tertiary endosymbiont 31 gathered extensive sequence data (Tab. S1, mainly provided by et al. 1996). For the GTR+Γ substitution model with four Alverson et al. 2007; Lee et al. 2013;Lietal.2015;Medlin discrete categories, we applied an uncorrelated relaxed 2015; Theriot et al. 2015). For a global diatom analysis, we molecular clock with a lognormal distribution of rate changes. compiled all diatom operational taxonomic units (OTUs) Starting tree was constructed at random, and the final topology possessing at least two nuclear (SSU and large subunit: LSU) was estimated by combining five independent chains each of and two chloroplast loci (rbcLandeitherpsbAorpsbC), along 50 million generations, sampling every 10,000th iteration. with sequences from the Kryptoperidiniaceae possessing at least TRACER v1.6 (http://tree.bio.ed.ac.uk/software/tracer/)was one nuclear (SSU) and one chloroplast (rbcL) locus. In a con- used to evaluate effective sample size values and to confirm siderable number of cases, further plastid loci such as atpB, adequate combination of the Markov chain Monte Carlo psaA, psaBandpsbA were added, when they were available chains with an appropriate burn-in (10%). from the same strains. For two additional analyses at taxonomically subordinate levels (i.e. Bacillariaceae and Stephanodiscaceae), we performed BLAST searches (Altschul Results and discussion et al. 1997) using the endosymbiont sequences as query to ob- tain all available sequences of closely related diatoms. The To hinder evolutionary artefacts due to limited and biased alignments included then all diatom and endosymbiont se- taxon sample, we first carried out a phylogenetic analysis, quences (excluding ITS due to the high divergence), regardless including all diatom taxa whose data corresponded with our of the locus coverage. criteria. Accordingly, we constructed a concatenated phyloge- Separate matrices were constructed, aligned using netic analysis with a wide range of endosymbiotic and free- ‘MAFFT’ v6.502a (Katoh and Standley 2013)and living diatom grades and clades. Our multi-locus and well- concatenated afterwards with SeaView v4.6.1 (Gouy et al. resolved phylogenetic tree of diatoms (Fig. S1) confirmed 2010). Phylogenetic analyses were carried out using both a the existence of three lineages presenting dinophyte endosym- ML (Stamatakis 2014) as well as a Bayesian approach bionts that are only distantly related (Chesnick et al. 1997; (Ronquist et al. 2012), as described in detail previously Horiguchi and Takano 2006; Pienaar et al. 2007;Takano (Gottschling et al. 2012). The aligned matrices are available et al. 2008; Yamada et al. 2017). We interpret that tertiary as *.nex files upon request. All new DNA sequences were endosymbiosis evolved either early, with many extant free- deposited in GenBank. living diatoms deriving from captured cells, or more likely, In order to estimate divergence times in resident diatoms were taken up by dinophytes multiple times Kryptoperidiniaceae, we constituted a concatenated alignment independently (not necessarily in a form of serial replace- (defined by the nuclear rRNA loci SSU, ITS and LSU plus ment). Thus, we reject the hypothesis on overall homology mitochondrial MT-CO1 and MT-CYB) that was shown to be of functional chloroplasts present in Kryptoperidiniaceae. In suitable for phylogenetic inference (Gottschling et al. 2012; this respect, Kryptoperidiniaceae differ from green algae and Gu et al. 2013;Tillmannetal.2014). It included all land plants, in which plastids have been engulfed only once Peridiniales, of which sequence information of all three nucle- and have since been inherited making them homologous. ar regions was available, along with a representative set of Two endosymbiotic lineages were recovered as rRNA sequences covering the known diversity of paraphyletic with regard to free-living diatoms, indicating Kryptoperidiniaceae (irrespectively whether they were com- multiple independent endosymbiotic events within each sym- plete or not). The phylogeny was dated using BEAST v1.8.3 biont clade. Therefore, two further phylogenetic analyses were (Drummond et al. 2012), with settings recommended for in- carried out, including all available sequences (regardless of terspecific data that might or might not satisfy the molecular the coverage) within each lineage to elucidate the actual clock. A Yule branching process with lognormal priors and host-endosymbiont dynamics within each group. As inferred empirically defined base frequencies was adopted using the from the trees with a focus on distinctive diatom lineages following four calibration points (minimal ages for crown (Figs. 1 and 2), almost every endosymbiont DNA sequence groups, see also Gottschling et al. 2008): Peridiniales were had its closest relative among free-living algae. Even the res- estimated at 200 ± 1 Mya (Fensome et al. 1996), ident diatoms within particular dinophyte taxa such as Peridiniales excluding Heterocapsaceae were estimated at Durinskia and Kryptoperidinium did not constitute monophy- 160 ± 4 Mya based on the first fossil occurrence of letic groups. Notably, the ITS sequence of our (calcareous) coccoid cells with tabulation (Keupp 1984; Kryptoperidinium strain GeoB 459 was almost identical Keupp and Ilg 1989), Scrippsiella Balech s.l. was estimated (> 99% similarity) to an ITS sequence (AY574381) derived at 70 ± 0.5 Mya based on the combination archaeopyle (Streng from a free-living Nitzschia pusilla Grunow (data not shown). et al. 2004) as apomorphic trait and the T/Pf clade was esti- This observation rejects any higher-level co-phylogenetic matedat70±0.5Myabasedon the first occurrence of structure and requires a new interpretation of the origin of Thoracosphaera Kamptner in the fossil record (Fensome the dinotoms’ chloroplasts. 32 Žerdoner Čalasan A. et al.

Fig. 1 Polyphyletic endosymbionts of freshwater Unruhdinium with closest relatives among free-ranging diatoms. Maximum likelihood (ML) tree (−ln = 21,700) of all 37 Stephanodiscaceae operational taxonomic units available, derived from the comparison of concatenated rRNA and plastid sequences. Endosymbiont taxa are highlighted. Note that we did not observe any contradictory tree topologies between separated analyses of nuclear and plastid loci (not shown). Endosymbionts of Unruhdinium segregated into two distinct lineages. Four identical sequences of different U. penardii (Lemmerm.) Gottschling strains clustered together (94LBS, .98BPP) and built a well-supported (99LBS, 1.00BPP) sister group relationship with free-living Discostella nipponica (Skvortzov) A.Tuji & D.M.Williams (strain TNS:AL-5776). A second lineage (100LBS, 1.00BPP) did not include endosymbiont sequences of U. cf. kevei (G.X.Liu & Z.Y.Hu) Gottschling and U. jiulongense (H. Gu) Gottschling only, but also those of the free-living strains L435 and LO4-2. All sequences of this lineage were nearly identical. Branch lengths are drawn to scale, with the scale bar indicating the number of nt substitutions per site. The numbers on the branches are statistical support values (ML bootstrap values, values < 50 are not shown, posterior probabilities below branches, values < 90 are not shown; asterisks indicate maximal support)

Our alternative scenario proposed here describes the re- (Inagaki et al. 2000)—is based on isozyme analysis of two peated capture of free-living diatoms and their transitory diatom-harbouring dinophytes. There are extensive enzymatic maintenance, but not necessarily the inheritance by descen- differences between Durinskia baltica (Levander) Carty & dants over geological times. Evolutionary dynamics in El.R.Cox and Kryptoperidinium foliaceum (F.Stein) Kryptoperidiniaceae thus appears higher than assumed so Er.Lindem., indicating rather divergent adaptations of a host far. An almost intact ultrastructure and insignificant genome to its endosymbiont (Whitten and Hoyhome 1986). Similar reduction (Imanian et al. 2010, 2012; Hehenberger et al. 2014) conclusions supporting the independent acquisition of endo- favour our scenario, indicating a rather young and not yet symbionts have been made based on histone analysis of en- optimal mutualism. Independent acquisition of diatoms was dosymbiotic nuclei (Morris et al. 1993;Chesnicketal.1997). postulated for dinophytes already in the previous millennium. Such older publications are not cited in the era of molecular This idea of ‘separate endosymbiosis’—that dinophytes have phylogenetics anymore (and also not in Yamada et al. 2017), captured the same or similar diatom species independently although the studies might have elucidated the accurate Absence of co-phylogeny indicates repeated diatom capture in dinophytes hosting a tertiary endosymbiont 33

Fig. 2 Polyphyletic endosymbionts of Durinskia, Galeidinium and Kryptoperidinium with closest relatives among free-ranging diatoms. Maximum likelihood (ML) tree (−ln = 14,945) of all 77 Bacillariaceae operational taxonomic units available, derived from the comparison of concatenated rRNA and plastid sequences. Highlighted endosymbiont taxa were scattered along up to ten only distantly related lineages over the Bacillariaceae phylogeny. Note that accessions of neither Durinskia nor Kryptoperidinium constituted monophyletic groups. Within such lineages, endosymbionts were usually closer related to free-ranging diatoms than to other endosymbionts (though statistical support was not always high). Some endosymbionts were phylogenetically isolated from free-living diatoms exhibiting long branches (e.g. D. kwazulunatalensis N.Yamada, Sym & T.Horig., D. oculata and G. rugatum Tam. & T.Horig.). Branch lengths are drawn to scale, with the scale bar indicating the number of nt substitutions per site. The numbers on the branches are statistical support values (ML bootstrap values, values < 50 are not shown, posterior probabilities below branches, values < 90 are not shown; asterisks indicate maximal support)

evolutionary scenario. Based on our new phylogenetic in- endosymbiont of marine Blixaea, for example, is a part of a sights, future research should answer the question, well-resolved group within Chaetoceros—probably the larg- whether a particular dinophyte species always hosts a unique est taxon of marine centric diatoms (Rines and Hargraves diatom genotype or if it has the potential to take up different 1988)—and endosymbionts of freshwater Unruhdinium endosymbionts (as it is indicated for Durinskia and cluster within freshwater Cyclotella (Alverson et al. 2011), but Kryptoperidinium in our phylogenetic trees). endosymbionts are neither found within other freshwater species Irrespectively of the endosymbionts’ variety, the diatom- of Nitzschia nor of Stephanodiscus Ehrenb. The situation is not harbouring dinophytes are highly selective towards specific as congruent in the diatom clade including endosymbionts of groups and do not recruit diatoms arbitrarily: The Durinskia, Galeidinium and Kryptoperidinium,inwhich 34 Žerdoner Čalasan A. et al. Absence of co-phylogeny indicates repeated diatom capture in dinophytes hosting a tertiary endosymbiont 35

Fig. 3 Cretaceous diversification of dinophytes hosting a diatom McLean 2013; Gottschling and Söhner 2013;Kretschmann endosymbiont. Dated phylogeny of Peridiniales, with et al. in press). Nevertheless, it is disputable why an organism Kryptoperidiniaceae fully uncollapsed (ultrametric maximum clade credibility tree with node ages from the Bayesian uncorrelated would try to integrate a new endosymbiont, if it has already lognormal analysis). Median rate is given in units of substitutions per established a permanent functional chloroplast of its own. The million years (including 95% confidence intervals), and branches are observations of Kryptoperidinium foliaceum without an endo- coloured according to the estimated evolutionary rates. Absolute ages symbiont (Kempton et al. 2002) also challenge the scenario are in million years, and epochs are indicated. The numbers on the branches are statistical support values (Bayesian posterior probabilities, that mutualism is entirely obligatory. Thus, the facultativity of values < .90 are not shown; asterisks indicate maximal support). Note that heterotrophy may represent the ancestral condition in Kryptoperidiniaceae were monophyletic (.96BPP) and started to diversify Kryptoperidiniaceae, as there is no evidence of co-existence at the Jurassic/Cretaceous boundary. Durinskia (.98BPP) was first of two types of fully functional chloroplasts either. branching, diversifying since the mid Cretaceous and built a sister group relationship to the remaining Kryptoperidiniaceae (.94BPP). Dinophytes hosting a tertiary endosymbiont appear as well- Stem node of Unruhdinium (1.00BPP) is subsequently also old, but the trained taxonomists, which selectively recognise their target extant members did not start to diversify before the late Palaeogene. symbionts (putatively former prey: Zhang et al. 2011b)fortheir Among the remaining lineages of Kryptoperidiniaceae, Galeidinium tight organismal interactions. The evolutionary dynamics is (.99BPP) and Kryptoperidinium appeared closely related (.99BPP), and diversification of their individual lineages did not take place before the more complex than previously assumed, and phylogenetic data K/Pg boundary indicates that diatom acquisition has taken place repeatedly and might be ongoing. The complex ecological role of dinophytes employing a variety of nutrition modes such as phototrophy, freshwater and marine taxa are intermingled and cannot be dif- heterotrophy and mixotrophy may explain their potential and ferentiated. However, they are all assigned to different plasticity in acquiring a great diversity of plastids. The inferred Nitzschia taxa from marine and freshwater habitats recent origin is also corroborated by our time estimates (Fig. 3), underlining the taxonomically selective recognition by in which the age of most host lineages is clearly older than those of the dinophyte hosts. Cell size may matter in this respect the corresponding resident diatoms. It is, for example, impossible (Litchman et al. 2009), as free-living close relatives of for the last common ancestor of Kryptoperidiniaceae, which endosymbionts are rather small diatoms, and they belong appeared in early Cretaceous, to possess a Nitzschia-like diatom to the same taxonomic groups as the endosymbionts from as an endosymbiont, whose stem age has been dated no earlier large foraminifers (Lee et al. 1989, 1995). Furthermore, the than to the late Cretaceous (Sorhannus 2007;Medlin2015). The endosymbiotic cytoplasm of Kryptoperidiniaceae is rich in young and incomplete realisation of mutualism makes ribosomes (Horiguchi and Pienaar 1994;Tamuraetal. Kryptoperidiniaceae an exceptional model for studying the first 2005; Pienaar et al. 2007; Takano et al. 2008), which may steps of chloroplast establishment, as the reduction of the exces- indicate a high metabolic activity. Instead of the well- sive morphological and biochemical components has not yet tak- developed mutualism as present in other photosynthetically en place. This feature makes them different from other dinophytes active algal groups, the dinophyte host may virtually drain its also possessing a tertiary endosymbiont (Gast et al. 2007). symbiont. Overall, the situation rather resembles the domestication of phycobionts in lichens (Lücking et al. 2009), Acknowledgements Financial support was provided by the Deutsche sacoglossan sea slugs (Händeler et al. 2010)orthefacultatively Forschungsgemeinschaft (grant GO 1549 10-1) and the Münchener phototrophic ciliates (Qiu et al. 2016) and foraminifers (Lee Universitätsgesellschaft. We thank Nina Simanovic for improving the English text, and the Scientific Committee of the 11th International et al. 1989, 1995). Similar phenomena are also known from Conference on Modern and Fossil Dinoflagellates for awarding the first other dinophytes exhibiting kleptoplastidy (Schnepf and author of this study with the Best Young Scientist Oral Presentation. Elbrächter 1999; Stoecker 1999; Gast et al. 2007; Takano et al. 2014). The question arises whether the last common ancestor of References Kryptoperidiniaceae was phototrophic and replaced the primary chloroplast (Keeling 2010) or if it was heterotrophic Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., and developed the ability to photosynthesise secondarily. Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI- This question is irrespective of the interpretation that the pri- BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25,3389–3402. mary chloroplast of the Peridiniales may have developed to an Alverson, A. J., Jansen, R. K., & Theriot, E. C. (2007). Bridging the eyespot in Kryptoperidiniaceae (Dodge 1983; Horiguchi et al. Rubicon: phylogenetic analysis reveals repeated colonizations of 1999; Moestrup and Daugbjerg 2007; Takano et al. 2008). marine and fresh waters by thalassiosiroid diatoms. Molecular Outgroup comparison does not help answer the question, as Phylogenetics and Evolution, 45,193–210. phototrophic symbionts (i.e. Zooxanthella K.Brandt) as well Alverson, A. J., Beszteri, B., Julius, M. L., Theriot, E. C. (2011). The model marine diatom Thalassiosira pseudonana likely descended as heterotrophic parasites (i.e. Blastodinium) are found in a from a freshwater ancestor in the genus Cyclotella. BMC close relationship with Kryptoperidiniaceae (Gottschling and Evolutionary Biology, 11,125. 36 Žerdoner Čalasan A. et al.

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