For Review Only 377 Algomyces Stechlinensis Clustered Together with Environmental Clones from a Eutrophic 378 Lake in France (Jobard Et Al

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1 Running head: Parasitic chytrids of volvocacean algae.

3 Title: Diversity and Hidden Host Specificity of Chytrids infecting Colonial

4 Volvocacean Algae.

5 Authors: Silke Van den Wyngaerta, Keilor RojasJimeneza,b, Kensuke Setoc, Maiko Kagamic,

6 HansPeter Grossarta,d

7 a Department of ExperimentalFor Limnology, Review LeibnizInstitute Only of Freshwater Ecology and Inland

8 Fisheries, Alte Fischerhuette 2, D16775 Stechlin, Germany

9 b Universidad Latina de Costa Rica, Campus San Pedro, Apdo. 101381000, San Jose, Costa Rica

10 c Department of Environmental Sciences, Faculty of Science, Toho University, Funabashi, Chiba,

11 Japan

12 d Institute of Biochemistry and Biology, Potsdam University, Maulbeerallee 2, 14476 Potsdam,

13 Germany

15 Corresponding Author:

16 Silke Van den Wyngaert, Department of Experimental Limnology, LeibnizInstitute of

17 Freshwater Ecology and Inland Fisheries, Alte Fischerhuette 2, D16775 Stechlin, Germany

18 Telephone number: +49 33082 69972; Fax number: +49 33082 69917; email: [email protected],

19 wyngaert@igbberlin.de

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24 ABSTRACT

25 Chytrids are zoosporic fungi that play an important, but yet understudied, ecological role in 26 aquatic ecosystems. Many chytrid species have been morphologically described as parasites on 27 phytoplankton. However, the majority of them have rarely been isolated and lack DNA sequence 28 data. In this study we isolated and cultivated three parasitic chytrids, infecting a common 29 volvocacean host species, Yamagishiella unicocca. In order to identify the chytrids, we 30 characterized morphology and life cycle, and analyzed phylogenetic relationships based on 18S 31 and 28S rDNA genes. Host range and specificity of the chytrids was determined by cross 32 infection assays with host strains, characterized by rbcL and ITS markers. We were able to 33 confirm the identity of two chytrid strains as Endocoenobium eudorinae Ingold and Dangeardia 34 mamillata Schröder and described the third chytrid strain as Algomyces stechlinensis gen. et sp. 35 nov. The three chytrids were assigned to novel and phylogenetically distant clades within the 36 phylum Chytridiomycota,For each Review exhibiting different Only host specificities. By integrating 37 morphological and molecular data of both the parasitic chytrids and their respective host species, 38 we unveiled cryptic hostparasite associations. This study highlights that a high prevalence of 39 (pseudo)cryptic diversity requires molecular characterization of both phytoplankton host and 40 parasitic chytrid to accurately identify and compare host range and specificity, and to study 41 phytoplanktonchytrid interactions in general. 42 43 Keywords: Chytridiomycota, Dangeardia mamillata; Endocoenobium eudorinae; fungal 44 parasites; host specificity; life cycle; phytoplankton

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59 Chytrids are zoosporic true fungi that inhabit a variety of aquatic ecosystems, both as 60 saprophytes and parasites (Sparrow 1960). The parasitic chytrid Chytridium olla on the green 61 algae Oedogonium spp., was identified in the early 19th century by the botanist Alexander Braun 62 (Braun 1851), and was the first chytrid recognized as distinct from other fungi in the later 63 established phylum Chytridiomycota (Hibbett et al. 2007). Currently, more than 400 chytrid 64 species have been morphologically described as parasites on all major groups of phytoplankton 65 (Canter 1954; Sparrow 1960). There is accumulating evidence that parasitic chytrids possess 66 important ecological functions in the aquatic food web such as controlling phytoplankton bloom 67 dynamics (Canter and Lund 1951; Gsell et al. 2013), and serving as trophic link between inedible 68 phytoplankton and zooplankton via the mycoloop (Kagami et al. 2007). However, we still know 69 relatively little about their distribution, phylogeny and host specificity (Frenken et al. 2017). 70 Host specificity, defined as the extent to which parasites can exploit different host 71 species, is a fundamentalFor property Review of parasites both Only from an ecological and evolutionary 72 perspective (Poulin et al. 2011). Host specificity reflects the diversity of resources a parasite is 73 able to use, i.e. the rangewidth of its ecological niche (Agosta et al. 2010). Host specialization 74 often underlies hostparasite coevolutionary processes, potentially leading to local adaptation and 75 speciation (Thompson 2016). Specificity of hostparasite interactions has therefore important 76 implications for the dynamics of parasite extinction, persistence or invasion in spatially and 77 temporally changing ecosystems (Poulin et al. 2011). To measure host specificity of parasites, it 78 is imperative to accurately identify both the host and the parasite. This can be challenging, in 79 particular for microbial hostparasite systems which often display a limited set of morphological 80 features and exhibit a high level of cryptic (identical morphology) or pseudocryptic (fine scale 81 morphological differences) diversity (Le Gac et al. 2007). Most of our knowledge on host 82 specificity of phytoplanktonchytrid parasites up to date has been derived from in situ 83 morphological based identification of phytoplanktonchytrid species pairs (Sparrow 1960). 84 However, morphological traits of chytrid sporangia are scarce and pose limitations for the 85 identification of chytrids at the species level (Letcher et al. 2008). A combination of several traits 86 related to sporangium development, zoospore discharge, sexual reproduction and resting spore 87 formation is needed for chytrid identification (Sparrow 1960). In most cases this information can 88 only be obtained by bringing the chytrid into culture (Longcore 2005). Moreover, chytrid 89 morphology has been shown to be variable depending on host substrate (Barr 1973). 90 Furthermore, identifying the phytoplankton host at the species level, based on morphology alone, 91 is not trivial, especially for nonphytoplankton taxonomists. Currently, an increasing number of 92 cryptic and/or pseudocryptic phytoplankton species are being discovered within traditionally 93 described cosmopolitan morphospecies (Krienitz et al. 2015; Smayda 2011; Van den Wyngaert et 94 al. 2015). The use of molecular tools for species identification could overcome such limitations 95 of traditional microscopic approaches to infer host specificity of chytrids. However, the current 96 bottleneck is the very limited number (e.g. 16 taxa, summarized in Frenken et al. 2017) of 97 obligate and facultative, parasitic chytrid species of phytoplankton in the public sequence 98 databases. Currently, we face an increasing disconnection between contemporary environmental 99 DNA (eDNA) and described chytrid morphospecies. Bridging this gap will allow us to 100 synergistically advance ecological and evolutionary studies on phytoplanktonchytrid 101 interactions. 102 In this study we isolated and cultivated three parasitic chytrids of colonial volvocacean

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103 algae. The first objective was to identify these chytrids with previously described morphospecies, 104 provide reference sequences, and to analyze their molecular phylogenetic relationships. The 105 second objective was to define host range and specificity of the chytrid parasites. By integrating 106 morphological and molecular data of both the parasitic chytrids and their respective host species, 107 we unveiled cryptic hostparasite associations, and were able to better clarify and refine host 108 specificity of these chytrids. The results of our study emphasize the importance and necessity of 109 using molecular tools, also for phytoplankton host identification, in order to infer host specificity 110 of chytrids and to study phytoplanktonchytrid interactions in general.

111 MATERIALS AND METHODS 112 Isolation and cultivation 113 A total of 25 colonial volvocacean algae and three parasitic chytrids were isolated from the 114 oligomesotrophic Lake Stechlin (53°09'3.35'' N. 13°01'20.53'' E. Germany). Two algal strains, 115 named PAN1 and PAN4,For were obtained Review from a 010 mOnly integrated sample on 15th October 2014. 116 The rest of the algal strains were isolated on 7th July 2016. A single cell derived culture was 117 obtained as described in Van den Wyngaert et al. (2017). The three chytrid strains (SVdW 118 EUD1, SVdWEUD2, SVdWEUD3) were isolated on 9th June 2015, 15th July 2015, and 1st 119 December 2015 respectively, as described in Van den Wyngaert et al. (2017). PAN4 was used as 120 host strain for the isolation of all three chytrids. Both host cultures and hostchytrid cocultures 121 are cultivated in CHU10 medium (modified after (Stein 1973), under controlled conditions with 122 a light intensity of 40 µE s1 m2, provided by coolwhite fluorescent lamps, a light:dark cycle of 123 16:8 hours and a temperature of 17 °C. Hostparasite cocultures are maintained by transferring 124 0.51 ml of the infected culture to 60 ml of live host cells at 1012 day intervals.

125 Light and epifluorescence microscopy 126 Cell suspensions of chytridinfected host cultures were mounted on glass slides. Different 127 development stages were examined at magnifications of 400x and 1000x with an Olympus BX51 128 microscope and pictures were taken with a ColorView digital camera (Soft Imaging System 129 GmbH). Additional sample preparations were stained with calcofluor white (Müller and 130 Sengbusch 1983) and observed under an epifluorescence microscope (Leitz DMRB, Leica).

131 Molecular and phylogenetic analysis of the chytrid strains 132 Zoospores of strains SVdWEUD2 and SVdWEUD3 were separated from the host cells by 133 filtering the coculture through a 10µm nylon mesh and then concentrated by centrifugation at 134 6000 g for 15 min. DNA was extracted from the zoospore concentrate using the peqGOLD 135 Tissue DNA Mini Kit (Peqlab, Germany) following the manufacturer's instructions. For the 136 chytrid SVdWEUD1, 2 ml of a highly infected hostchytrid coculture was centrifuged and 137 DNA was extracted from the resulting pellet using the same extraction kit. The 18S genes of 138 SVdWEUD1 and SVdWEUD2 were amplified with primers EF4EF3 (Smit et al. 1999) and 139 the 18S gene of SVdWEUD3 with primers NS1NS4 (White et al. 1990). The 28S rDNA genes 140 were amplified with primers LRORLR5 (Rehner and Samuels 1994; Vilgalys and Hester 1990). 141 For PCR reaction, MyTaq Red DNA Polymerase (BIOLINE, Germany) was used with the 142 following conditions: 94 oC for 2 min, 32 cycles at 94 oC for 15 s, 53 oC for 15 s, 72 oC for 30 s, 143 and a final extension at 72 oC for 5 min. PCR products were sequenced at Macrogen Europe 144 using Sanger technology. Sequences were assembled using BioEdit (Hall 1999) and deposited in

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145 GenBank (accession numbers MG605050 MG605055, see also Table S1). 146 For the phylogenetic analysis, representative sequences from all orders of 147 Chytridiomycota were retrieved from GenBank. Sequences of each marker were aligned with 148 MAFFT (Katoh and Standley 2013), refined manually using BioEdit (Hall 1999), and 149 concatenated with SeaView (Gouy et al. 2010). The concatenated alignment used for subsequent 150 phylogenetic analyses included 70 taxa and 941 positions of SSU and 870 positions of LSU. The 151 bestfit nucleotide substitution model was calculated with JModelTest2 (Darriba et al. 2012). 152 This analysis suggested GTR+G+I as the best model according to the Bayesian Information 153 Criterion. A Bayesian estimation of the phylogeny was performed with MrBayes 3.2.6 (Ronquist 154 et al. 2012) using the GTR+G+I model. Monte Carlo Markov Chain analysis simulations were 155 run with two sets of four chains for 2,000,000 generations. Convergence of the chain length was 156 confirmed from the standard deviation of split frequencies (<0.01). For the analysis, 25% of 157 generations were discarded as burnin. The remaining postburnin trees were used to generate a 158 consensus tree and to calculate the posterior probabilities. In addition, a maximum likelihood 159 phylogenetic tree was constructedFor Review using the same modelOnly of evolution with RAxML v 7.7.1 160 (Stamatakis et al. 2008). The resulting trees were visualized with FigTree v1.4.3 and further 161 edited with Inkscape.

162 Molecular and phylogenetic analysis of algal host strains 163 We extracted DNA from 2 ml of the algal strains, concentrated at 6000 g for 15 min, using the 164 peqGOLD Tissue DNA Mini Kit (Peqlab, Germany). The rbcL gene was amplified with primers 165 Fw_rbcL_192 and Rv_rbcL_657 (Hadi et al. 2016). The ITS15.8SITS2 rDNA region was 166 amplified with primers ITS1 and ITS4 (White et al. 1990). Amplifications and sequence 167 processing was performed as described for chytrids (accession numbers MG596989MG597008, 168 MG655241 MG655262, see also Table S1). For the phylogenetic analysis we also included 169 sequences of representative genera of Volvocaceae, retrieved from GenBank. The sequences of 170 the rbcL and ITS markers were aligned separately using MAFFT (Katoh and Standley 2013), 171 refined manually using BioEdit (Hall 1999), and concatenated with MEGA7 (Kumar et al. 2016). 172 A maximum likelihood phylogenetic tree was constructed with FastTree 2.1.9 (Price et al. 2010), 173 using a GTR+G+I model of evolution.

174 Cross infection assays 175 To examine the host specificity of all three chytrid strains, cross infection assays with a total of 176 25 host strains were performed. Strains included the reference strain PAN4, on which all chytrid 177 strains have been isolated, a strain of the desmid Staurastrum sp. (STAU1), and an additional set 178 of 23 colonial volvocacean strains. Each assay was performed in triplicate in 48 well plates 179 containing 400 µl of exponentially growing host and 350 µl of zoospore suspension, which was 180 obtained by filtering a 710 days old infected culture through a 10µm plankton mesh. In the case 181 of SVDWEUD2, no viable zoospore suspension could be obtained and instead 50 µl of a highly 182 infected culture was added. Visual inspection of the infection was performed by light microscopy 183 after 4, 7 and 12 days and were categorized in 4 infection classes; no (0) = no infection, low (+) 184 = <5%, medium (++) = 550%, high (+++) = >50% infected cells.

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185 RESULTS 186 Morphological identification and life cycle of chytrids 187 SVdW-EUD1. Strain SVdWEUD1 was identified as Endocoenobium eudorinae Ingold (1940). 188 Spherical uniflagellate zoospores, which were hard to observe (no picture available), become 189 quickly immotile after their release and encyst, lose or retract their flagellum (Fig. 1A), and 190 produce several rhizoidlike structures which can be up to 1520 µm long (Fig. 1B, C). Encysted 191 zoospores (56 µm) attach with their rhizoidlike structures to motile host cells (Fig. 1D). After 192 attachment, rhizoidlike structures are expulsed or retracted, and a fine germ tube penetrates the 193 mucilage of the host colony (Fig. 1E). Upon contact with a host cell the germ tube enlarges 194 within the host colony and forms a prosporangium (Fig. 1F, G). Sporangial development is 195 exogenous, i.e. the nucleus moves out of the encysted zoospore into the germ tube. The encysted 196 zoospore persists on the surface of the host colony (Fig. 1F, G, I, J). The prosporangium 197 produces multiple simple or branched rhizoids that can infect multiple cells simultaneously 198 within a colony (Fig. 1G,For H). The sporangiumReview is spherical Only to ovoid (6.219 x 819.6 µm) (Fig. 1I, 199 J) and emerges close to the empty encysted zoospore. Infected host colonies can retain their 200 swimming activity, provided that at least a few host cells in the colony remain uninfected. Spiny 201 thickwalled resting spores are produced (10.816.9 x 10.817 µm) (Fig. 1L), probably after 202 sexual reproduction by fusion of two thalli (Fig. 1K). 203 204 SVdW-EUD2. Strain SVdWEUD2 was identified as Dangeardia mamillata Schröder (1898). 205 Two types of uniflagellate zoospores; spherical and more ovoid, are observed (Fig 2A). 206 Zoospores encyst on the surface of the host colony and produce a fine germ tube that penetrates 207 through the mucilage to infect a host cell (Fig 2B). The germ tube enlarges (Fig 2C, D) and the 208 base, which is sessile on the host cell, continues to swell (Fig 2E). Sporangial development is 209 exogenous. In the mature stage, the sporangium still embedded within the mucus of the host 210 colony becomes flask shaped (6.712.4 x 8.315.7 µm) (Fig 2F). Rhizoids could not be 211 observed. The apical region of the mature sporangium protrudes from the mucilage sheath of the 212 host colony (Fig 2E, F), it dissolves and zoospores emerge one by one through the apex opening 213 (Fig. 2G, H). Host colonies retain their swimming activity, provided that at least a few host cells 214 in the colony remain uninfected. Sexual reproduction and resting spores have not been observed. 215 216 SVdW-EUD3. Strain SVdWEUD3 is described for the first time. The zoospores are spherical 217 and uniflagellate (2.53.5 µm) (Fig 3E). The initial steps of the infection process are similar to 218 SVDWEUD2, i.e. zoospores encyst on the surface of the host colony and produce a fine germ 219 tube that penetrates through the mucilage to infect a host cell (Fig. 3A). The host colony can be 220 massively attacked by numerous zoospores simultaneously (Fig. 3C). The development of the 221 encysted zoospore to a sporangium is endogenous (e.g. nucleus remains in the encysted 222 zoospore) and sporangium development occurs entirely external of the host colony (Fig. 3BD). 223 Rhizoids could not be observed. Sporangia morphology is spherical to oval in the early stage 224 (Fig. 3A, B) and becomes obpyriform upon maturation (411.4 x 5.512.5) (Fig. 3D). Zoospores 225 are discharged by the dehiscence of an operculum (Fig. 3F) in a gelatinous matrix from which 226 they eventually escape (Fig. 3E). Sexual reproduction and resting spores have not been observed. 227

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228 Phylogeny of the three chytrid strains 229 The phylogenetic analyses, based on the 18S and 28S rDNA sequences, consistently show that 230 the three parasitic fungal strains belong to distantlyrelated taxa within Chytridiomycetes (Fig. 231 4). Strain SVdWEUD1 clusters together with Polyphagus parasiticus, and forms a clade sister 232 to the orders Gromochytriales and Mesochytriales. Strain SVdWEUD2 clusters together with 233 uncultured chytrids from a lake (Lake Lurleen; Lefèvre et al. 2012) and semiarid soil (Lipson et 234 al. 2014), and is sister to the clade containing several strains of Zygorhizidium spp. This clade 235 was previously recognized as an orderlevel novel clade (Jobard et al. 2012; Seto et al. 2017). 236 Strain SVdWEUD3 forms a cluster with other uncultured chytrids from aquatic environments, 237 e.g. from the Baltic Sea (Majaneva et al. 2012), Lake Adyat (Jobard et al. 2012), the Artic and 238 Subartic Sea (Hassett et al. 2017). This cluster, constitutes a novel clade (SWI) within 239 Lobulomycetales. 240 Phylogeny of colonial volvocaceanFor Review host strains Only 241 Based on morphology and earlier reports of algal species occurrence in Lake Stechlin (Padisák et 242 al. 2003), the isolated algal host strains PAN1 and PAN4 were tentatively identified as Eudorina 243 elegans Ehrenberg and Pandorina morum (O.F. Müller) Bory, respectively. However, a 244 considerable degree of phenotypic plasticity, resulting in the presence of indistinguishable 245 morphotypes in both strains, can be observed in cultures (Supplementary material, Fig. S1). The 246 molecular identification, however, contributed to elucidate the morphologybased identification. 247 According to phylogenetic analysis of the rbcL + ITS sequences, PAN1 and EUDB6 cluster, 248 with high support, together with Eu. elegans, whereas PAN4 is not related to P. morum, but 249 belongs to Yamagishiella unicocca (W.R. Rayburn & R.C. Starr) H. Nozaki (Figure 5A). All 250 other strains also belong to Y. unicocca and together they split up in two clearly distinct 251 subclusters, associated with two different strains of Y. unicocca (Figure 5A). The sequence 252 similarity of both rbcL and ITS sequences between these two subclusters is 9798% and 88% 253 92%, respectively. 254 Host range and specificity 255 Not all host strains are equally susceptible to all chytrid species and both intra and interspecific 256 differences in host susceptibility can be observed (Figure 5A&B, Table 1). Strains from 257 Yamagishiella subcluster 1 are highly susceptible to all three chytrids, whereas strains from 258 Yamagishiella subcluster 2, although infected by all chytrids, show a lower susceptibility to the 259 chytrid strains SVdWEUD1 and SVdWEUD2 (Figs. 5A&B). Conversely, Eu. elegans strains 260 are highly susceptible to SVdWEUD3, weakly susceptible to SVdWEUD1 and nonsusceptible 261 to SVdWEUD2 (Figure 5A&B). Overall, SVdWEUD3 displays the broadest host range and is 262 even able to establish infections on the desmid Staurastrum sp. (Fig. 5B).

263 DISCUSSION 264 In this study we isolated and cultivated three parasitic chytrids all infecting a common 265 volvocacean host species, Y. unicocca (summary Table 1). However, they belong to 266 phylogenetically distant clades within the phylum Chytridiomycota, and exhibit clearly distinct 267 infection patterns and host specificities. 268 Identification of chytrid morphospecies 269 Bringing the parasitic chytrids into culture allowed us to observe the entire life cycle, including

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270 the different development stages of the chytrids on their host. Using this information, we were 271 able to confirm the identity of the chytrid strains SVdWEUD1 as En. eudorinae and SVdW 272 EUD2 as D. mamillata. 273 Endocoenobium eudorinae (SVdWEUD1), was for the first time described by Ingold 274 (1940) as a parasite on Eu. elegans in Swithland Reservoir, UK. The thallus and formation of 275 nonmotile spores with radiating rhizoidlike structures in En. eudorinae shows morphological 276 similarities to Polyphagus, a genus that contains several parasitic species of single celled, 277 filamentous and colonial green algae (Sparrow 1960) including Eu. elegans (Johns 1964). 278 However, in Polyphagus the encysted zoospore enlarges directly into the thallus, whereas in 279 Endocoenobium the encysted zoospore remains as a knob on the surface of the coenobium, and 280 the thallus forms within the coenobium as an outgrowth from this knob (Fig. I, J) (Ingold 1940). 281 Dangeardia mamillata (SVdWEUD2) was first described by Schröder (1898) as a 282 parasite on P. morum in a small pond of the botanical garden Wroclaw, Poland. Few reports have 283 been made on this chytrid since its first description. Canter (1946) provided a detailed 284 description of the developingFor stages Review of D. mamillata onOnly Eu. elegans from Barn Elms Reservoir, 285 UK. In contrast to the original description of an endobiotic resting spore by Schröder, Canter 286 observed epibiotic and sexually formed resting spores. Although in this study no resting spores 287 were observed, we still consider the chytrid to be identical to D. mamillata since all other 288 morphological characteristics of the zoospores, sporangia development stages and zoospore 289 discharge behavior are in perfect agreement with the descriptions made by Schröder and Canter. 290 Although we observed the life cycle and many relevant morphological features of the 291 chytrid strain SVdWEUD3 (e.g. zoospore discharge), we were still unable to identify it with any 292 of the previously described chytrid species on Eudorina spp. or Pandorina spp. (Sparrow 1960). 293 The morphology of SVdWEUD3 displayed a high similarity with Chytridium oocystidis 294 (HuberPestalozzi 1944), i.e. ovalobpyriform sporangium with operculate dehiscence, absence 295 of resting spores, and gregarious infection behavior. The sporangia of C. oocystidis, however, are 296 slightly narrower. HuberPestalozzi described this chytrid as a parasite on Oocystis lacustris in 297 Walensee, Switzerland, and Pongratz (1966) observed a similar looking chytrid on O. lacustris, 298 O. planctonica and O. solitaria in Lake Geneva, Switzerland. Although strain SVdWEUD3 was 299 isolated from Eu. elegans, cross infection experiments revealed that this chytrid has a generalist 300 infection behavior, suggesting that it can infect a broad range of green algae. The broad host 301 range of SVdWEUD3 was further evidenced by the observation of a similar looking chytrid, 302 infecting a spherical, nonflagellated, colonial green algal species in Lake Stechlin (September 303 2016). Multiple displacement amplification (MDA), followed by PCR and sequencing of a single 304 infected colony confirmed that the 28S rDNA sequence of this chytrid was identical to strain 305 SVdWEUD3. The ITS sequence of the green algal host was 94% similar to Oocystis sp. 306 FG2/8.5E (Supplementary material, Fig. S2). Our results highlight that even when a parasitic 307 chytrid is brought in culture, identification of chytrid morphospecies can remain problematic, in 308 particular for those that display a broad host range. For highly host specific chytrids, host 309 identity can serve as an important guideline for chytrid identification. However, when dealing 310 with generalists, host identity becomes less useful for identification purposes, especially when 311 the chytrid has been found in a hostassociation that has previously not been described yet. 312 Moreover, since it is known that chytrid phenotype can be variable (Barr 1973), depending on 313 the host species it infects, morphological identification becomes practically impossible. Because 314 of subtle differences in sporangium morphology, the broad host range, and phylogenetic position 315 within the order Lobulomycetales, we decided to describe strain SVdWEUD3 as a new genus

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316 and species Algomyces stechlinensis. 317 318 Taxonomy. Fungi; Chytridiomycota; Chytridiomycetes; Lobulomycetales; Lobulomycetaceae 319 Algomyces S. Van den Wyngaert, K. Rojas & K. Seto, gen. nov. 320 321 Diagnosis. Fungus parasitic on colonial green algae, with the capability to infect, albeit with low 322 preference, desmids. Zoospores spherical and uniflagellate, containing single lipid globule. 323 Thalli eucarpic, monocentric, having endogenous development. Sporangia occurring entirely 324 external of the host colony, with apical operculate pore. Zoospore discharge by the dehiscence of 325 an operculum, as a mass in a gelatinous matrix from which zoospores eventually escape. 326 Type species. Algomyces stechlinensis (HOLOTYPE) 327 Etymology. The genus name refers to the Latin alga for algae. 328 Mycobank. MB825142 329 330 Algomyces stechlinensis S.For Van den Review Wyngaert, K. Rojas Only & K. Seto, sp. nov. 331 332 Diagnosis. Fungus parasitic on colonial green algae, with the capability to infect, albeit with low 333 preference, desmids, and having a gregarious infection behavior. Zoospores spherical, 2.53.5 334 µm in diameter, uniflagellate, containing single lipid globule. The development of the encysted 335 zoospore to a sporangium endogenous. Sporangium occurring entirely external of the host 336 colony, spherical to oval, becoming obpyriform upon maturation, 411.4 µm in width, 5.512.5 337 µm in height, with apical operculate pore. Zoospore discharge by the dehiscence of an 338 operculum, as a mass in a gelatinous matrix from which zoospores eventually escape. Sexual 339 reproduction and resting spores not observed. 340 Type material. ICBN: Fig. 3 in this publication (HOLOTYPE), strain SVdWEUD3 (extype 341 culture). 342 Type host. Eudorina elegans Ehrenberg. 343 Other hosts. Yamagishiella unicocca (W.R.Rayburn & R.C.Starr) H.Nozaki and Staurastrum sp. 344 (strain STAU1). 345 Type locality. GERMANY, 53°9´5.59´´N 13°1´34.22´´E, from Lake Stechlin, collected on 1st 346 December 2015 by Silke Van den Wyngaert. 347 Etymology. Latin; stechlinensis, referring to the name of type location Lake Stechlin. 348 Gene sequence. GenBank Accession Number MG605055 (18S), MG605052 (28S). 349 Mycobank. MB825143 350 351 352 Phylogenetic diversity 353 This study is the first to obtain cultures and DNA sequences of the morphospecies En. eudorinae 354 and D. mamillata, enabling the analysis of their phylogenetic position within Chytridiomycota. 355 The taxonomic position of the morphogenus Endocoenobium has been regarded as problematic 356 (Canter 1985). Because of morphological similarities with Polyphagus spp., it was originally 357 placed within the order Chytridiales, family Rhizidiaceae, subfamily Polyphagoideae (Ingold 358 1940, Sparrow 1960). Barr´s reclassification of the order Chytridiales replaced En. eudorinae in 359 the family Endochytriaceae Sparrow ex D.J.S. Barr (1980) and molecular phylogenetic studies 360 placed the family Endochytriaceae into the newly described order Cladochytriales (Mozley 361 Standridge et al. 2009). Recently, the first SSU based molecular phylogeny of a Polyphagus sp.,

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362 Polyphagus parasiticus, showed that P. parasiticus belongs to a deep branching clade of 363 chytridiomycetes sister to the orders Gromochytriales and Mesochytriales. The results of our 364 DNA based phylogenetic analysis shows that En. eudorinae clusters together with the 365 morphological similar P. parasiticus and as such confirms the previously suggested relationship 366 between the two chytrids. This result strengthens the taxonomic position of both chytrids and 367 provides additional justification for the new order Polyphagales, erected by Karpov et al. 2016. 368 The morphogenus Dangeardia is currently placed within the order Chytridiales, family 369 Chytridiaceae. As was found for En. eudorinae, our molecular phylogenetic analysis did not 370 support the current taxonomic position of D. mamillata within Chytridiales and Dangeardia 371 could not be assigned to any known order within Chytridiomycota. According to our analyses, D. 372 mamillata clusters together with environmental clones from a mesoeutrophic lake (Lefèvre et al. 373 2012) and semiarid soil (Lipson et al. 2014), as a sister group to the orderlevel novel clade II 374 including Zygorhizidium spp. (Jobard et al. 2012, Seto et al. 2017). Interestingly, all currently 375 described species within the Zygorhizidium spp. novel clade contain parasites of diatoms (Seto et 376 al. 2017). For Review Only 377 Algomyces stechlinensis clustered together with environmental clones from a eutrophic 378 lake in France (Jobard et al. 2012) and from the Baltic (Majaneva et al. 2011) and Arctic sea ice 379 (Hassett et al. 2017) in a separate, novel clade, sister to the order Lobulomycetales (Simmons et 380 al. 2009). Interestingly, the arctic sea ice clones most likely represent parasitic chytrids of 381 diatoms, since microscopic observations by Hassett and coworkers (2017) indicated that a 382 substantial portion of large pennate diatoms in the arctic sea ice were infected by chytrids during 383 the sampling period. Morphologically these arctic chytrids also shared the feature of an apical 384 operculum with A. stechlinensis. Lobulomycetales represent a small order, currently containing a 385 single family and five genera. Most of the described species within this order are isolated from 386 soil. Algomyces stechlinensis represents the first freshwater parasitic taxa within the 387 Lobulomycetales but could not be placed in any of the five known genera. The morphological 388 characteristics of A. stechlinensis (i.e. endogenous development, eucarpic, monocentric, 389 operculate) are in agreement with those described for the family Lobulomycetaceae (Simmons et 390 al. 2009). Thus, at this moment, we consider the placement of all recognized members of the 391 order into the same family Lobulomycetaceae. This placement should be further verified by 392 investigations on the affiliation of A. stechlinensis to the family Lobulomycetaceae, including 393 additional strains and zoospore ultrastructure analyses.

394 Cryptic host range and specificity 395 Although the three chytrids share a common host, they differ in host range and specificity. The 396 generalist strain SVdWEUD3 was even able to establish infections, albeit with low prevalence, 397 on a nonvolvocacean, and phylogenetically distant desmid species. Further evidence of its broad 398 host range, including other colonial green algal species, has been discussed above. En. eudorinae 399 and D. mamillata displayed a narrower host range. En. eudorinae is able to infect Y. unicocca 400 and, to a smaller extent, Eu. elegans, whereas D. mamillata was only able to infect Y. unicocca. 401 Previous microscopybased studies, have defined the host range of En. eudorinae and D. 402 mamillata to include Eu. elegans, Eu. unicocca, P. morum (Canter 1985, Pongratz 1966) and Eu. 403 elegans, P. morum, respectively (Blinn and Button 1973; Canter 1946; Ingold 1940; Schröder 404 1898). However, Schröder (1898) and Pongratz (1966) already pointed out the difficulties to 405 morphologically distinguish Pandorina and Eudorina and addressed the related problem for 406 defining host range and specificity of parasitic chytrids. Here, we resolved this issue by

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407 providing a molecular characterization of the host strains used in our crossinfection 408 experiments, which revealed the presence of two distinct host species Eu. elegans (PAN1) and Y. 409 unicocca (PAN4). Yamagishiella H. Nozaki (Nozaki and Kuroiwa 1992) is a monotypic genus 410 containing Y. unicocca, which was originally described as Pandorina unicocca (Rayburn and 411 Starr 1974). As such this genus was still unknown when studies on En. eudorinae and D. 412 mamillata were published. The vegetative morphology of the three genera Eudorina, Pandorina 413 and Yamagishiella is very similar (Herron 2016) and often indistinguishable, especially between 414 Y. unicocca and Eu. unicocca G. M. Smith (Yamada et al. 2008). Moreover, molecular 415 phylogenetic studies have indicated that many morphospecies within the Volvocaceae, including 416 Eu. elegans and P. morum form polyphyletic groups (Nozaki et al. 2000). Our study identified Y. 417 unicocca as the most susceptible host species for all three chytrids. Moreover, D. mamillata was 418 not able to infect the strains of Eu. elegans from this study. The observed host specificity might 419 well reflect local adaptation to the most abundant host species in Lake Stechlin (Lively and 420 Dybdahl 2000). When we assume that Y. unicocca has been falsely identified as P. morum, then, 421 in Lake Stechlin, the mostFor susceptible Review host, Y. unicocca Only, is occurring more frequent and regularly 422 than the suboptimal host Eu. elegans (Padisák et al. 2003). The dominance of Y. unicocca in 423 Lake Stechlin is also suggested by the high proportion of Yamagishiella colonies that were 424 isolated in this study compared to Eudorina. It could, however, well be that in other 425 geographical locations where Eu. elegans is the more dominant species, it will represent the most 426 susceptible host. Serial transfers on the suboptimal host Eu. elegans could clarify the 427 evolutionary potential of En. eudorinae for rapid host adaptation. The fact that we also found 428 differences in host susceptibility on the intraspecific level suggests the potential of rapid 429 antagonistic coevolution between Y. unicocca and both chytrids En. eudorinae and D. mamillata 430 (De Bruin et al. 2004; Kyle et al. 2015). Overall, these results emphasize that i) a high 431 prevalence of cryptic diversity requires molecular characterization of both phytoplankton host 432 and parasitic chytrid to accurately identify and compare host range and specificity of chytrids; ii) 433 extending the geographical scale might reveal additional phytoplankton hosts of chytrid species 434 and reveal patterns of local specialist versus regional/global generalists which are important for 435 predicting parasite spread, invasion and extinction dynamics.

436 Conclusion 437 By integrating morphological, molecular and host specificity data of three chytrid species, 438 infecting colonial volvocacean algae, we have contributed with high quality reference sequences 439 of parasitic chytrids, which are ecologically important and diverse, but poorly represented in 440 current sequence databases. The fact that previously described morphospecies did not have any 441 close relatives in reference databases, emphasizes the large incompleteness of our current 442 knowledge of chytrid diversity. Moreover, our results highlight that a high prevalence of 443 (pseudo)cryptic diversity requires molecular characterization of both phytoplankton host and 444 parasitic chytrid to accurately identify and compare host range and specificity of chytrids. 445 Ignoring this hidden diversity may lead to false interpretations on infection dynamics and 446 mechanisms, as well as species distribution patterns of phytoplanktonchytrid interactions. We 447 encourage the scientific community to apply both cultivation dependent approaches and single 448 infected cell/colony PCR (Ishida et al. 2015) to increase the number of DNA sequences of 449 parasitic chytrids and their phytoplankton hosts. These efforts will not only improve the 450 phylogenetic resolution of Chytridiomycota but will also facilitate interpretation of 451 environmental sequence data and the use of molecular methods to study the distribution and

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452 ecological role of phytoplanktonchytrid interactions in aquatic ecosystems. 453 454 ACKNOWLEDGEMENTS 455 456 SVdW was supported by a IGB postdoc fellowship and the German Research Foundation (DFG) 457 Grant number 347469280 and HPG by the Leibniz SAW project “Mycolink”. This study was 458 supported by JSPS KAKENHI Grant Numbers JP15KK0026 and JP16H02943. 459

460 LITERATURE CITED

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588 M. A. Suchard, and J. P. Huelsenbeck. 2012. MrBayes 3.2: Efficient Bayesian 589 Phylogenetic Inference and Model Choice Across a Large Model Space. Systematic 590 Biology 61:539542. 591 Schröder, B. 1898. Dangeardia, ein neues Chytridineen Genus auf Pandorina morum Bory. . Ber. 592 dtsch. bot. Ges. XVI:314321. 593 Seto, K., M. Kagami, and Y. Degawa. 2017. Phylogenetic Position of Parasitic Chytrids on 594 Diatoms: Characterization of a Novel Clade in Chytridiomycota. J. Eukaryot. Microbiol. 595 64:383393. 596 Simmons, D. R., T. Y. James, A. F. Meyer, and J. E. Longcore. 2009. Lobulomycetales, a new 597 order in the Chytridiomycota. Mycol. Res. 113:450460. 598 Smayda, T. J. 2011. Cryptic planktonic diatom challenges phytoplankton ecologists. PNAS 599 108:42694270. 600 Smit, E., P. Leeflang, B. Glandorf, J. Dirk van Elsas, and K. Wernars. 1999. Analysis of Fungal 601 Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCRAmplified Genes 602 Encoding 18S rRNAFor and Review Temperature Gradient Only Gel Electrophoresis. Appl. Environ. 603 Microbiol. 65:26142621. 604 Sparrow, F. 1960. Aquatic phycomycetes. 2nd edn edition. University of Michigan Press, Ann 605 Arbor. 606 Stamatakis, A., P. Hoover, and J. Rougemont. 2008. A Rapid Bootstrap Algorithm for the 607 RAxML Web Servers. Syst. Biol. 57:758771. 608 Stein, J. 1973. Culture methods and growth measurements. Handbook of phycological methods. . 609 Cambridge University Press, UK. 610 Thompson, J. N. 2016. Coevolution, local adaptation and ecological speciation. Mol. Ecol. 611 5:56085610. 612 Van den Wyngaert, S., M. Möst, R. Freimann, B. W. Ibelings, and P. Spaak. 2015. Hidden 613 diversity in the freshwater planktonic diatom Asterionella formosa. Mol. Ecol. 24:2955 614 2972. 615 Van den Wyngaert, S., K. Seto, K. RojasJimenez, M. Kagami, and H.P. Grossart. 2017. A New 616 Parasitic Chytrid, Staurastromyces oculus (Rhizophydiales, Staurastromycetaceae fam. 617 nov.), Infecting the Freshwater Desmid Staurastrum sp. Protist 168:392407. 618 Vilgalys, R., and M. Hester. 1990. Rapid genetic identification and mapping of enzymatically 619 amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172:4238 620 4246. 621 White, T., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal 622 ribosomal RNA genes for phylogenetics. in In: Innis M, Gelfand D, Shinsky J, White T 623 (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, pp 315322. 624 Yamada, T. K., K. Miyaji, and H. Nozaki. 2008. A taxonomic study of Eudorina unicocca 625 (Volvocaceae, Chlorophyceae) and related species, based on morphology and molecular 626 phylogeny. Eur. J. Phycol. 43:317326. 627 628 629 FIGURE LEGEND 630 631 Fig. 1 Light and fluorescence microscopy images of the infection and development stages of 632 Endocoenobium eudorinae (SVdWEUD1) on the host Yamagishiella unicocca (PAN4) (A) en 633 cysted zoospores, (B) encysted zoospore initiating the development of rhizoidlike structures, (C)

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634 encysted zoospore with complete development of rhizoidlike structures, (D) encysted zoospore 635 (blue color = calcofluor white) attached to a host colony (red color = autofluorescence), (E) ini 636 tial infection stage (FH) development of prosporangium , (I, J) maturing sporangium, (K) fusion 637 of two thalli, potentially indicative for sexual reproduction, (L) resting spore. ez = encysted zoo 638 spore, l = lipid globule, h = host, gt = germ tube, rh = rhizoids, ps = prosporangium, sp = sporan 639 gium, rs = resting spore, black arrows = rhizoidlike structures, white arrows = empty encysted 640 zoospore. Scale bars = 10 µm. 641 642 Fig. 2 Light microscopy images of the infection and development stages of Dangeardia mamilla- 643 ta (SVdWEUD2) on the host Yamagishiella unicocca (PAN4). (A) zoospores, (B) initial infec 644 tion stage, (CE) developing sporangia, (F) mature sporangium, (GH) zoospore discharge and 645 empty sporangium. sz = spherical shaped zoospore, oz = oval shaped zoospore, l = lipid globule, 646 gt = germ tube, dsp = developing sporangial stages, msp = mature sporangium, black arrows = 647 zoospore discharge through the apex opening of the sporangium. Scale bars = 10 µm. 648 For Review Only 649 Fig. 3 Light microscopy images of the infection and development stages of Lobulomycetales sp. 650 (SVdWEUD3) on the host Eudorina elegans (PAN1). (A) initial infection stage, (B) developing 651 sporangia, (C) gregarious infection, (D) mature sporangium, (E) zoospore discharge, (F) empty 652 sporangium. gt = germ tube, oc = operculum. Scale bars = 10 µm. 653 654 Fig. 4 Molecular phylogenetic tree of Chytridiomycota. The analysis was performed on a 655 concatenated alignment of the 18SrDNA and 28 rDNA regions (1811 bp) using both Maximum 656 Likelihood and Bayesian methods. The evolutionary history was inferred based on the General 657 Time Reversible model with gammashaped distribution and the proportion of invariable sites. 658 On the nodes are indicated the maximum likelihood boostrap values of 1000 repetitions (first 659 number), calculated with FastTree, and the Bayesian posterior probabilities (second number) 660 estimated with Mrbayes. 661 662 Fig. 5 (A) Molecular phylogenetic tree, including 25 colonial volvocacean strains and 663 representative genera of Volvocaceae. The analysis was performed on a concatenated alignment 664 of the rbcL gene and ITS15.8SITS2 rDNA region (912 bp). using both Maximum Likelihood 665 and Bayesian methods. The evolutionary history was inferred based on the General Time 666 Reversible model with gammashaped distribution and the proportion of invariable sites. On the 667 nodes are indicated the maximum likelihood boostrap values of 1000 repetitions, calculated with 668 B)FastTree. ( Cross infection assays with a total of 22 colonial volvocacean strains, including the 669 reference strain PAN4, on which all chytrid strains have been isolated, strain PAN1 and the 670 desmid Staurastrum sp (STAU1). Infections were evaluated by light microscopy after 4, 7 and 12 671 days and were categorized in 4 infection classes; no (0) = no infection, low (+) = <5%, medium 672 (++) = 550%, high (+++) = >50% infected cells. 673 674 SUPPORTING INFORMATION 675 676 Supplemental Fig. S1 Phenotypic variability in the algal culture strains PAN4 (Yamagishiella 677 unicocca) and PAN1 (Eudorina elegans). 678 679 Supplemental Fig. S2 (A) infected green algal colony “moC1, that was picked up manually with

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680 a micropipette, from a water sample from Lake Stechlin (September 2016). B) a similar looking 681 infected colony, from the same water sample, with multiple chytrid infections. 682 683 Supplemental Table 1 GenBank accession numbers for chytrid and host strains.

For Review Only

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For Review Only

Fig. 1 Light and fluorescence microscopy images of the infection and development stages of Endocoenobium eudorinae (SVdWEUD1) on the host Yamagishiella unicocca (PAN4) (A) encysted zoospores, (B) encysted zoospore initiating the development of rhizoidlike structures, (C) encysted zoospore with complete development of rhizoidlike structures, (D) encysted zoospore (blue color = calcofluor white) attached to a host colony (red color = autofluorescence), (E) initial infection stage (FH) development of prosporangium , (I, J) maturing sporangium, (K) fusion of two thalli, potentially indicative for sexual reproduction, (L) resting spore. ez = encysted zoospore, l = lipid globule, h = host, gt = germ tube, rh = rhizoids, ps = prosporangi um, sp = sporangium, rs = resting spore, black arrows = rhizoidlike structures, white arrows = empty encysted zoospore. Scale bars = 10 µm.

239x149mm (300 x 300 DPI)

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For Review Only Fig. 2 Light microscopy images of the infection and development stages of Dangeardia mamillata (SVdW EUD2) on the host Yamagishiella unicocca (PAN4). (A) zoospores, (B) initial infection stage, (CE) developing sporangia, (F) mature sporangium, (GH) zoospore discharge and empty sporangium. sz = spherical shaped zoospore, oz = oval shaped zoospore, l = lipid globule, gt = germ tube, dsp = developing sporangial stages, msp = mature sporangium, black arrows = zoospore discharge through the apex opening of the sporangium. Scale bars = 10 µm.

293x122mm (300 x 300 DPI)

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For Review Only

Fig. 3 Light microscopy images of the infection and development stages of Lobulomycetales sp. (SVdW EUD3) on the host Eudorina elegans (PAN1). (A) initial infection stage, (B) developing sporangia, (C) gregarious infection, (D) mature sporangium, (E) zoospore discharge, (F) empty sporangium. gt = germ tube, oc = operculum. Scale bars = 10 µm.

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Zygorhizidium aff. melosirae C1 (LC176287/ LC176292) 100/99 Zygorhizidium aff. melosirae KS94Journal (LC176288/ of Eukaryotic LC176293) Microbiology Page 38 of 43 100/92 Zygorhizidium aff. melosirae KS99 (LC176290/ LC176295) 81/93 Zygorhizidium planktonicum KS98 (LC176289/ LC176294) 100/100 77/85 Uncultured AY2009A5 (HQ219392/-) Novel Clade II (sensu Jobard et al. 2012) Uncultured AY2009B15 (HQ219406/-). 95/99 Uncultured PA2009B1 (HQ191393/-). 100/98 Dangeardia mamillata SVdW EUD2 (MG605054/MG605051) 100/96 Uncultured E4e4731 (-/KF750554) Chytridiomycota sp. LLMB2 (-/JN049538) 98/89 Spizellomycete sp. JEL355 (DQ536477/DQ273821) 100/100 Thoreauomyces humboldtii JEL95 (AF164245/HQ901662) Spizellomycetales 60/89 Spizellomyces punctatus ATCC48900 (NG017173/NG027618) 92/85 Gaertneriomyces semiglobifer BK9110 (AF164247/DQ273778) 87/74 Rhizophlyctis rosea JEL318 (NG017175/NG027649) 100/97 Rhizophlyctis rosea BK47 (AH009028/-) Rhizoplyctiales Rhizophlyctis rosea BK57 (AH009027/-) 100/99 Rhizophydium brooksianum JEL136 (AY601710/AY349086) 92/79 Uebelmesseromyces harderi JEL171 (AF164272/DQ273775) 100/100 Boothiomyces macroporosum PLAUS21 (NG017171/NG027566) Rhizophydiales 100/97 Kappamyces laurelensis AFTOLID690 (DQ536478/DQ273824) Rhizophydium sphaerotheca AFTOLID37 (AY635823/DQ273781) 72/98 Diplophlyctis sp. JEL331 (EU828477/EU82850) 100/97 Cylindrochytridium johnstonii JEL596 (JF796051/JF796052) 60/99 Allochytridium luteum JEL324 (AY635844/DQ273816) 100/98 62/99 Cladochytriales sp. JEL72 (AH009044/AY349083) Cladochytriales 71/98 Endochytrium ramosum JEL402 (EU828484/EU828513) Nowakowskiella sp. JEL127 (AY635835/DQ273798) 98/99 Cladochytrium replicatum JEL180 (NG017169/NG027614) 100/96 Polychytrium aggregatum JEL109 (NG017168/NG027613) 100/92 Lacustromyces hiemalis JEL31 (AH009039/HQ901700) Polychytriales 90/98 For ReviewArkaya lepida JEL93 (AF164278/DQ273814) Only Karlingiomyces asterocystis JEL572 (HQ901769/HQ901708) 100/99 Podochytrium dentatum JEL30 (AH009055/AY439060) 100/85 Obelidium mucronatum JEL57 (AH009056/AY439071) 100/93 Rhizoclosmatium sp. JEL347 (AY601709/DQ273769) 79/98 Chytriomyces hyalinus AFTOLID1537 (DQ536487/DQ273836) 100/100 100/95 Rhizophydium planktonicum AstB5 (LC176286/LC176291) Chytridiales Rhizophydium planktonicum (FMS34600/-) 100/96 Pseudorhizidium endosporangiatum JEL221 (DQ536484/DQ273834) 100/94 Phlyctochytrium planicorne AFTOLID628 (DQ536473/DQ273813) Dendrochytridium crassum JEL354 (AY635827/DQ273785) 100/99 Synchytrium minutum BPI 880898 (HQ324138/HQ324139) 100/88 Synchytrium decipiens AFTOLID 634 (DQ536475/DQ273819) 100/76 Synchytrium macrosporum AFTOLID635 (NG017170/NG027565) Synchytriaceae 100/98 Synchytrium microbalum JEL517 (KU672728/-) 100/99 Uncultured VM3 116 (HG530947/-) 100/95 Uncultured C10 (FN690492/-) 100/98 Uncultured 6c A1 (FN690496/-) 100/93 Uncultured Ay2007E7 (JQ689413/-) Novel Clade SW-I 100/95 Algomyces stechlinensis SVdW EUD3 (MG605055/MG605052) 99/99 Uncultured BTH (-/KU898745) 100/92 Uncultured BTH (-/KU898738) Lobulomycetales 100/100 Uncultured BTH (-/KU898746) 100/98 Lobulomyces angularis JEL45 (AF164253/DQ273815) 100/100 Lobulomyces poculatus JEL343 (EF443134/EF443139) 98/85 Clydeae vesicula PL70 (EF443138/EF443143) 100/99 Maunachytrium keaense AF021 (EF432822.2/EF432822.2) Chytridiales sp. AF021 (EF432822.1/EF432822.1) 100/94 100/99 Uncultured PFF5SP2005 (EU162641/-) 98/98 100/94 Uncultured Pa2007C10 (JQ689425/-) 100/96 Uncultured PFD6SP2005 (EU162637/-) Mesochytrium penetrans CALUX10 (FJ804149/FJ804153) Mesochytriales 89/98 100/97 Uncultured PA2009B6 (HQ191400/-) 88/99 Uncultured PA2009D8 (HQ191406/-) 99/98 100/95 Uncultured Va2007BB6 (JQ689445/-) Uncultured PA2009E7 (HQ191286/-) 100/96 88/99 Gromochytrium mamkaevae CALUX51 (KF586842/KF586842) Uncultured kor 110904 (FJ157331/-) Gromochytriales 100/99 Endocoenobium eudorinae SVdW EUD1 (MG605053/MG605050) Polyphagus parasiticus (KX449337/-) Polyphagales 100/98 Monoblepharella mexicana AFTOLID33 (AF164337/DQ273777) 100/91 Gonapodya sp. JEL183 (AH009066/KJ668088) 100/97 Oedogoniomyces sp. CR84 (AY635839/DQ273804) Monoblepharidiomycetes Hyaloraphidium curvatum SAG235 1 (NG017172/NG027646) 100/100 Rozella allomycis AFTOLID297 (NG017174/NG027561) Rozella sp. JEL347 (AY601707/DQ273766) Outgroup: Cryptomycota

0.05 Page 39 of 43 Journal of Eukaryotic Microbiology

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Fig. 5 (A) Molecular phylogenetic tree, including 25 colonial volvocacean strains and representative genera of Volvocaceae. The analysis was performed on a concatenated alignment of the rbcL gene and ITS1-5.8S- ITS2 rDNA region (912 bp). using both Maximum Likelihood and Bayesian methods. The evolutionary history was inferred based on the General Time Reversible model with gamma-shaped distribution and the proportion of invariable sites. On the nodes are indicated the maximum likelihood boostrap values of 1000 repetitions, calculated with FastTree. (B) Cross infection assays with a total of 22 colonial volvocacean strains, including the reference strain PAN4, on which all chytrid strains have been isolated, strain PAN1 and the desmid Staurastrum sp (STAU1). Infections were evaluated by light microscopy after 4, 7 and 12 days and were categorized in 4 infection classes; no (0) = no infection, low (+) = <5%, medium (++) = 5-50%, high (+++) = >50% infected cells.

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Page 40 of 43

sp.

gen. gen. operculate

rhizoid infects a infects rhizoid single host cell host single infection gregarious sp. sp.

pyriform, ob - utside host colony host utside Lobulomycetales Lobulomycetales spherical o oval unicocca Yamagishiella (SVdW-EUD3) (SVdW-EUD3) µm) 5.5-12.5 x (4-11.4 elegans Eudorina Staurastrum

rhizoid infects a infects rhizoid single host cell cell host single , inoperculate nside host colony host nside shaped - ompletely i ompletely lask Dangeardia mamillata Dangeardia oval and spherical 2 types: c f elegans Eudorina unicocca Yamagishiella (SVdW-EUD2) (SVdW-EUD2) µm) 8.3-15.7 x (6.7-12.4 morum Pandorina Journal of Eukaryotic Microbiology

For Review Only

rhizoidal system infects infects system rhizoidal simultaneously multiple host cells multiple host , inoperculate oval nside host colony host nside -

artially i artially Endocoenobium eudorinae Endocoenobium spherical p spherical elegans Eudorina unicocca Yamagishiella (SVdW-EUD1) (SVdW-EUD1) µm) 8-19.6 x (6.2-19 unicocca Eudorina morum Pandorina elegans Eudorina

zoospore Infection Sporangia Sporangium Host range Host range Table 1. Summary of the main characteristics of the three parasitic chytrids, infecting colonial volvocacean algae. algae. volvocacean colonial infecting chytrids, parasitic the three of characteristics main the of Summary 1. Table pattern development (previous observations) study) (this Page 41 of 43 Journal of Eukaryotic Microbiology

SUPPORTING INFORMATION

Supplemental Fig. S1 Phenotypic variability in the algal culture strains PAN4 (Yamagishiella unicocca) and PAN1 (Eudorina elegans). Supplemental Material and methods Supplemental Results Supplemental Fig. S2 A) infected colony “moC1” that was picked up manually with a micropipette, from a water sample from Lake Stechlin (September 2016). B) a similar looking infected colony, from the same water sample, with multiple chytrid infections. Supplemental Table 1 GenBank accession numbers for chytrid and host strains. For Review Only Supplemental Fig. S1 Phenotypic variability of the algal culture strains PAN4 (Yamagishiella unicocca) and PAN1 (Eudorina elegans).

PAN4 PAN4 PAN1 PAN1

Supplemental Material and methods Multiple displacement amplification (MDA), a non-PCR based DNA amplification technique (Spits et al. 2006) was performed on a single infected colony (Fig. S2 A), using the illustra single cell genomPhi DNA amplification kit (GE-Healthcare). The 28S rDNA gene of the chytrid was amplified with primers LROR-LR5 (Vilgalys and Hester 1990). The ITS1-5.8S-ITS2 rDNA region of the host was amplified with primers ITS1 and ITS4 (White et al 1990). For PCR reaction, MyTaq Red DNA Polymerase (BIOLINE, Germany) was used with the following conditions: 94oC for 2 min, 32 cycles at 94oC for 15 sec, 53oC for 15 sec, 72oC for 30 sec, and a final extension at 72oC for 5 min. PCR products were sequenced at Macrogen Europe using Sanger technology. Sequences were assembled using BioEdit (Hall 1999). Supplemental results Sequencing results confirmed that the 28S sequence of the chytrid (Fig. S2) was identical to strain SVdW-EUD3. Blast search revealed that the ITS sequence of the green algal host had highest sequence similarity (94%) with Oocystis sp. FG2/8.5E. Journal of Eukaryotic Microbiology Page 42 of 43

Supplemental Fig. S2 A) infected colony “moC1” that was picked up manually with a micropipette, from a water sample from Lake Stechlin (September 2016). B) a similar looking infected colony, from the same water sample, with multiple chytrid infections.

A B

For Review Only

Supplemental Table 1 GenBank accession numbers for chytrid and host strains.

Chytrid strain 18S rRNA 28S rRNA Endocoenobium eudorinae SVdW-EUD1 MG605053 MG605050 Dangeardia mammillata SVdW-EUD2 MG605054 MG605051 Lobulomycetales sp. SVdW-EUD3 MG605055 MG605052 Host strain ITS1-5.8S-ITS2 rbcL Yamagishiella unicocca SVdW-A2 MG596989 MG655241 Yamagishiella unicocca SVdW-A3 MG596990 MG655242 Yamagishiella unicocca SVdW-A4 MG596991 MG655243 Yamagishiella unicocca SVdW-A5 MG596992 MG655244 Yamagishiella unicocca SVdW-A6 MG596993 MG655245 Yamagishiella unicocca SVdW-A7 MG596994 MG655246 Yamagishiella unicocca SVdW-A8 MG596995 MG655247 Yamagishiella unicocca SVdW-B1 MG596996 MG655248 Yamagishiella unicocca SVdW-B3 MG596997 MG655249 Yamagishiella unicocca SVdW-B4 MG596998 MG655250 Yamagishiella unicocca SVdW-B5 MG596999 MG655251 Yamagishiella unicocca SVdW-C2 MG597001 MG655253 Yamagishiella unicocca SVdW-C3 MG597002 MG655254 Yamagishiella unicocca SVdW-C4 MG597003 MG655255 Yamagishiella unicocca SVdW-C5 MG655256 Yamagishiella unicocca SVdW-D1 MG597004 MG655257 Yamagishiella unicocca SVdW-D2 MG655258 Yamagishiella unicocca SVdW-D3 MG597005 MG655259 Yamagishiella unicocca SVdW-D4 MG597006 MG655260 Yamagishiella unicocca SVdW-D6 MG597007 MG655261 Yamagishiella unicocca SVdW-D7 MG597008 MG655262 Page 43 of 43 Journal of Eukaryotic Microbiology

Yamagishiella unicocca SVdW-D8 MG597009 MG655263 Yamagishiella unicocca SVdW-F1 MG597010 MG655264 Yamagishiella unicocca SVdW-PAN4 MG597012 MG655265 Eudorina elegans SVdW-B6 MG597000 Eudorina elegans SVdW-PAN1 MG597011

For Review Only