Clarifying the Relationships Between Microsporidia and Cryptomycota

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Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE Clarifying the Relationships between Microsporidia and Cryptomycota

David Bassa,b, Lucas Czechc, Bryony A. P. Williamsd,Cedric Berneye, Micah Dunthornf, Frederic Maheg, Guifre Torruellah, Grant D. Stentiforda & Tom A. Williamsi

a Pathology and Microbial Systematics Theme, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, The Nothe, Weymouth DT4 8UB, UK b Department of Life Sciences, The Natural History Museum, London SW7 5BD, UK c Heidelberg Institute for Theoretical Studies, Schloß-Wolfsbrunnenweg, Heidelberg 69118, Germany d Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK e Sorbonne Universite & CNRS, UMR 7144 (AD2M), Station Biologique de Roscoff, Place Georges Teissier, Roscoff 29680, France f Department of Ecology, University of Kaiserslautern, Kaiserslautern, Germany g CIRAD, UMR LSTM, Montpellier, France h Ecologie Systematique Evolution, CNRS, Universite Paris-Sud, AgroParisTech, Universite Paris-Saclay, Orsay, France i School of Biological Sciences, University of Bristol, 24 Tyndall Avenue, Bristol BS8 1TQ, UK

Keywords ABSTRACT Mitosporidium; Nucleophaga; Parami- crosporidium; Rozella; Rozellida. Some protists with microsporidian-like cell biological characters, including Rozellomycota. Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We Correspondence analysed the phylogenetic placement and environmental diversity of David Bass, Pathology and Microbial microsporidian-like lineages that group near the base of the fungal radiation Systematics Theme, Centre for and show that they group in a clade with metchnikovellids and canonical Environment, Fisheries and Aquaculture microsporidians, to the exclusion of the clade including Rozella, in line with Science (Cefas), Barrack Road, The Nothe, what is currently known of their morphology and cell biology. These results Weymouth, Dorset DT4 8UB, UK show that the phylogenetic scope of Microsporidia has been greatly underesti- Telephone number: 01305 276000; mated. We propose that much of the lineage diversity previously thought to e-mail: [email protected] be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. Received: 10 January 2018; revised 2 March This insight has important implications for our understanding of opisthokont 2018; accepted March 20, 2018. evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from Neotropical forest soils doi:10.1111/jeu.12519 group with the short-branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of Microspori- dia provides a unique opportunity to investigate the evolutionary origins of a highly specialized clade of major animal parasites.

MICROSPORIDIA are conventionally considered as highly stages, and multiwalled spores produced by merogony or derived parasitic protists sister to Rozella or diverging as other forms of proliferation (Vavra and Larsson 1999). the next branch below the fungi (James et al. 2013). Microsporidia lack canonical Golgi apparatus (Bez- Microsporidia, Balbiani 1882 display a suite of distinctive noussenko et al. 2007; Vavra and Larsson 1999; Vavra and cell biological characters related to their obligate parasitic Lukes 2013) and their mitochondria have been highly lifestyle, including a characteristic spore-extrusion appara- reduced to mitosomes (reviewed in Dean et al. 2016). tus (represented most conspicuously by the polar ﬁlament These mitosomes are unable to generate their own ATP and its terminal anchoring disc) (Franzen 2004; Vavra and through oxidative phosphorylation, requiring energy to be Lukes 2013), unwalled intracellular trophic (meront) imported from the host via nucleotide transporters.

© 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 0, 1–10 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. An Expanded Microsporidia Bass et al.

Microsporidia also lack flagella and an apparent capacity Rozella is sister to a highly diverse clade comprising LB- for phagocytosis. The known diversity of Microsporidia is Microsporidia, the microsporidian-like protists described large, comprising approximately 1,300 described species above, and a large diversity of uncharacterized environ- (Vavra and Lukes 2013), forming a long-branched clade mental sequences. In this study we investigate these rela- (hereafter referred to as LB-Microsporidia) in SSU rRNA tionships further, integrating morphological, phylogenetic, and multigene phylogenies (James et al. 2013). Metch- and sequence diversity data, to determine the phyloge- nikovellids (e.g. Amphiamblys, Amphiacantha) have tradi- netic and taxonomic boundaries of microsporidia and their tionally been referred to as atypical, “primitive” immediate relatives. microsporidians, but share many characters with LB- Microsporidia, and were recently shown to branch as sis- MATERIALS AND METHODS ters to them (Mikhailov et al. 2017). Microsporidia are known primarily as parasites of inver- The nr nucleotide GenBank database was blastn searched tebrates and vertebrates (including humans), but are also using seed SSU rRNA gene sequences of characterized known as endosymbionts of ciliates (Fokin 2012; Fokin microsporidia-like protists, metchnikovellids, deeply branch- et al. 2008), and hyperparasites in protists: metchnikovel- ing LB-Microsporidia (clades 2 and 3 Stentiford et al. lids are parasites of gregarines, protistan gut symbionts of 2017), and the phylogenetic diversity of “Cryptomycota” many invertebrates, and Hyperspora aquatica is a hyper- and aphelids in Karpov et al. (2014). The top 50 matches parasite of the paramyxid Marteilia cochillia, a serious for each sequence were downloaded, aligned with mafft pathogen of European cockles (Stentiford et al. 2017). e-ins-i (Katoh and Standley 2013), deduplicated, and a pre- Rozella species are zoosporic biotrophic parasites of liminary tree constructed on the basis of which the num- oomycetes, chytrids, and Blastocladiomycota (Spatafora ber of closely related sequences was reduced, retaining et al. 2017). Increased attention has recently been given the longest possible sequences. The shorted branched to a large diversity of lineages shown by phylogenetic LB-Microsporidia Janacekia, Trichonosema, and Bacillidium analyses including environmental sequences to be related were selected to represent LB-Microsporidia in order to to Microsporidia, rozellids, and aphelids. The first to high- reduce the possibility of phylogenetic artefacts caused by light this diversity were Lara et al. (2010) and Jones et al. LBA. Published phylogenies (e.g. Stentiford et al. 2017) (2011), who showed a large diversity of environmental show that these form a very robust monophyletic group sequences which, in the absence of microsporidian with all other LB-Microsporidia, so it is reasonable to use sequences, group with Rozella in phylogenetic trees. them as a proxy for the whole group in this study. A Baye- These have been referred to as Rozellida (Fig. 1 in Lara sian phylogeny was inferred under the CAT+GTR+Gamma et al. 2010), Rozellomycota (Corsaro et al. 2014a,b), and (4) model in PhyloBayes-MPI 1.7 (Lartillot et al. 2013). Cryptomycota (Fig. 1 in Jones et al. 2011). Convergence among four MCMC chains was assessed by A few other sequences branching between rozellids and comparing the discrepancies in bipartition frequencies and LB-microsporidia represent microsporidia-like protists in a range of continuous model parameters, along with which have been morphologically characterized: Nucle- the effective sample sizes of the continuous parameters. ophaga (Corsaro et al. 2014a, 2016), Paramicrosporidium A consensus tree was built once all discrepancies were (originally described as a microsporidian; Michel et al. < 0.1, with sample sizes > 100. A maximum likelihood 2000, 2009), and Mitosporidium (Haag et al. 2014). phylogeny was estimated under the GTR+Gamma(4)+F These three genera clearly share some features with model in IQ-Tree (Nguyen et al. 2015), with 200 traditional classical LB-Microsporidia, including forms of polar fila- nonparametric bootstraps. The distribution of microsporid- ments (not necessarily functional as extrusion apparatus), ian characters (Table 1) was constructed from the litera- unwalled intracellular meront stages, and nonflagellated ture. OTUs assigned to any of “Opisthosporidia” (Karpov spores; but in other respects, they are dissimilar, Nucle- et al. 2014); “Cryptomycota” (Jones et al. 2011), ophaga and Paramicrosporidium being the least struc- “Holomycota” (Liu et al. 2009), and “Microsporidia” and turally similar to LB-Microsporidia. Mitosporidium, otherwise unassigned “fungi” by the taxonomic assign- sometimes referred to as the earliest branching ment algorithms of each study were extracted from envi- microsporidian (Mikhailov et al. 2017; Quandt et al. 2017), ronmental amplicon sequencing data of tropical forest has a mitochondrion, albeit lacking Complex I of the oxida- soils clustered into OTUs by Swarm v2.1.5 (Mahe et al. tive phosphorylation pathway. Paramicrosporidium has a 2015) and European coastal water and sediment samples canonical fungal mitochondrial genome, and shares more (Logares et al. 2014). We inferred a maximum likelihood gene content with distantly related fungi than with its tree from our reference database using RaxML v8.2.8 closest relatives (Quandt et al. 2017). Mitosporidium and (Stamatakis 2014). The OTUs were then aligned to the ref- Nucleophaga have possibly nonhomologous finger-like erence database using PaPaRa (Berger and Stamatakis extensions in naked intranuclear trophic stages similar to 2011) and placed on the tree by RaxML-EPA (Berger et al. those of Rozella (Corsaro et al. 2014b; Haag et al. 2014). 2011). The distribution of placements (Fig. 2, 3) was cre- Most published phylogenetic analyses that include ated with Genesis (http://genesis-lib.org/) and visualized Crypto/Rozellomycota/rozellid environmental sequences do with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). not also include LB-Microsporidia. Those that do (Corsaro Based on the 49 flagellar toolkit proteins assembled for a et al. 2016 (Fig. 3); Tedersoo et al. 2017) suggest that previous study (Torruella et al. 2015), we searched by

© 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists 2 Journal of Eukaryotic Microbiology 2018, 0, 1–10 Bass et al. An Expanded Microsporidia

BLAST in available early-branching microsporidian pre- This “expanded” Microsporidia concept is consistent dicted protein sets from genomic data, in Mitosporidium with the original descriptions of Paramicrosporidium (Michel daphniae (Haag et al. 2014), Paramicrosporidium (Quandt et al. 2000, 2009) as “microsporidian” or “microsporidian- et al. 2017), and Amphiamblys sp. (Mikhailov et al. 2017), like”, and Mitosporidium, which exhibits merogony and a using an e-value threshold of 1e-10 and manual scrutiny. coiled polar filament, the latter exclusive to Microsporidia, being “profoundly morphologically similar to Microsporidia” (Haag et al. 2014). This phylogenetically broader circum- RESULTS AND DISCUSSION scription of Microsporidia is morphologically distinct because all characterized lineages in the microsporidian An expanded Microsporidia clade possess the key morphological features of Microspor- We constructed an SSU rRNA gene sequence alignment idia: spores with multilayered cell walls containing polar fila- including a comprehensive selection of the microsporidian- ment apparatus (Richards et al. 2017; Vavra and Lukes like protists and their related environmental sequences, 2013), and merogony, whereas Rozella, aphelids, and fungi metchnikovellids, and representatives of short-branch LB- do not possess these characters. Microsporidia (see Methods), aphelids, rozellids, and related unclassified groups. Maximum Likelihood and Morphological vs. genomic evolution in Microsporidia Bayesian phylogenetic analyses (Fig. 1) showed that the expected metchnikovellid+LB-Microsporidia clade emerges Corresponding morphological and genomic datasets are from within a diverse and well-supported (Bayesian poste- available for only a very small proportion of lineages repre- rior probability (BPP) = 0.98; Maximum Likelihood boot- senting SB-Microsporidia and metchnikovellids. Until very strap 96%) clade including Paramicrosporidium, recently this applied to only Mitosporidium (Haag et al. Nucleophaga, Mitosporidium, LKM-11, and many other 2014) and Amphiamblys (Mikhailov et al. 2017), but the environmental sequences referred to as rozello-/cryptomy- addition of nuclear and mitochondrial genomes of Parami- cotans. Rozella formed a clade with high support with a crosporidium saccamoebae (Quandt et al. 2017) have pro- relatively small number of environmental sequences. vided several intriguing new perspectives on microsporidian It is well recognized that SSU rRNA gene data alone are evolution. It is increasingly apparent that, although all SB- unable to resolve deep phylogenetic relationships. How- microsporidia exhibit microsporidian-defining morphological ever, although not a substitute for multigene data, the characters, their genomic evolution appears far more high taxon sampling afforded by SSU provides phyloge- mosaic (Quandt et al. 2017); Table 1. Rozella and LB-Micro- netic information currently inaccessible for phylogenomic sporidia both have horizontally acquired Rickettsia-like NTT analyses (Berney et al. 2004; Cavalier-Smith 2004). We ATP/ADP transporters, but metchnikovellids, Mitosporidium calculated a range of trees of varying taxon samplings and Paramicrosporidium do not (Table 1). LB-Microsporidia (e.g. Fig. 2; other data not shown), and although the lack mitochondrial genomes, which are present in branching order within the clade was not consistent Mitosporidium and Paramicrosporidium. The mitochondrial between them (although many subclades were consis- genomes of Mitosporidium and Rozella lack Complex I of tently recovered), the microsporidian clade as marked on the oxidative phosphorylation pathway, are degenerate, and Fig. 1 was invariably and strongly recovered. AT-rich, but that of Paramicrosporidium includes all genes These analyses provide additional evidence for an of that pathway typically found in fungi; in fact, the total expanded and strongly supported microsporidian clade, gene complement of both Paramicrosporidium and including all of the LB-Microsporidia, (metchnikovellids, Mitosporidium have more in common with fungi than with the “microsporidian-like” protists discussed above, and its closest relatives (Haag et al. 2014; Quandt et al. 2017). almost all of the environmental “crypto/rozellomycotan” The very fast rates of sequence evolution (and so long diversity indicated in Lara et al. 2010; Jones et al. 2011; branches in the phylogeny) observed for LB-Microsporidia Corsaro et al. 2014a,b, 2016; Karpov et al. 2014; Lazarus correlate with extensive cellular and genomic reduction, and James 2015; Tedersoo et al. 2017), but excluding including strong mutational bias to AT and the loss of some rozellids (=Rozella), NAMAKO-37, and NCLC1 (Basal Clone DNA replication and repair genes that, in other eukaryotes, Group 1). We therefore propose that this clade including help to promote genome stability (Williams et al. 2016). the large diversity of environmental sequences, are all While genome data are currently very sparse, the slower actually microsporidia, and we refer to them (excluding evolutionary rates observed for the 18S genes of SB-Micro- LB-Microsporidia) here as short-branched Microsporidia sporidia suggest that, while these organisms are also para- (SB-Microsporidia). For the purposes of this study we also sites, reductive evolution has not proceeded to the same exclude metchnikovellids from the definition of SB-Micro- extreme degree as in the LB clade. Another instance of sporidia as sequenced metchnikovellids have significantly potentially horizontally acquired genes (the distribution of longer branches than the SB-Microsporidia shown on which may understandably not correlate with phylogeny) Fig. 2. However, it is likely that as more related lineages are thymidine kinases found in Rozella and LB-Microspori- are discovered a more gradual continuum of branch dia, but not Paramicrosporidium (Alexander et al. 2016; lengths between SB-Microsporidia such as Nucleophaga Quandt et al. 2017). Paramicrosporidium possesses a full (Fig. 2), metchnikovellids, and LB-Microsporidia will be set of meiosis genes and significant evidence for diploidy, revealed. more similar to LB-Microsporidia than is known to be the

Table 1. Molecular and genomic characteristics of Microsporidia and their relatives. Two long-branch microsporidia infecting nematodes (Nematocida parisii) and vertebrates (Encephalitozoon cuniculi) and Rozella (Rozellida)) are shown in comparison. Dashes represent unavailable data. ✓/x indicate presence/absence of a character; where uncertain these are shown in parentheses. Note that, following ancestral reduction, some LB-Microsporidia have experienced independent secondary expansions in genome size (e.g. up to 51.3 Mb in Edhazardia aedis Desjardins et al. ©

08TeAtosJunlo uaytcMcoilg ulse yWlyProias n.o eafo nentoa oit fProtistologists of Society International of behalf on Inc. Periodicals, Wiley by published Microbiology Eukaryotic of Journal Authors The 2018 2015)), but the additional material largely comprises noncoding and repetitive sequences, without a concomitant increase in cellular or metabolic complexity. The polar ﬁlament was reported as absent in Nucleophaga amoebae, although a form of it is present in the very closely related N. terricolae. It is possible that it has been lost in. N. amoebae or was simply not seen in the cells investigated by Corsaro et al. (2014b)

Number of Polar ﬁlament Spore protein ATP/DP Electron Flagellate Genome Gene density coding translocases Mitochondrial transport Posterior stage size (Mb) genes/kbp genes (HGT) genome chain Regular Atypical Absent Exo+endospore vacuole

Encephalitozoon cuniculi x 2.9 0.81 1,997 ✓ xx✓✓✓ (LB-Microsporidia)1,2 Nematocida parisii ERTm1 x 4.1 0.65 2,661 ✓ xx✓✓✓ (LB-Microsporidia)3,4 Amphiamblys sp. WSBS x5–7 0.66 2,529 (nr) x* xx---- - 2006 (metchnikovellid)5 Amphiamblys capitellides x------✓✓x (metchnikovellid)6 Mitosporidium daphniae x 5.64 0.585 3,300 x ✓ Partial ✓✓x (SB-Microsporidia)7 Paramicrosporidium x 7.3 - 3,750 x ✓✓ ✓ ✓ x saccamoebae (SB-Microsporidia)8,9 LKM11-01; CM1.1 ✓ ------(SB-Microsporidia)10 Nucleophaga terricolae x------✓✓x (SB-Microsporidia)11,12 †‡ ora fEkroi Microbiology Eukaryotic of Journal Nucleophaga amoebae x------✓? x (SB-Microsporidia)13 Rozella allomycis (rozellid)14 ✓ 11.9 0.535 6,350 ✓✓Partial ✓ xx

nr, nonredundant. 1. Katinka et al. (2001), 2. Karpov et al. (2014), 3. Cuomo et al. (2012), 4. Troemel et al. (2008), 5. Mikhailov et al. (2017), 6. Larsson and Koie (2006), 7. Haag et al. (2014), 8. Corsaro et al. (2014a), 9. Quandt et al. (2017), 10. Jones et al. (2011), 11. Corsaro et al. (2016), 12. Michel et al. (2012), 13. Corsaro et al. (2014b), 14. James et al. (2013), 15. Letcher and Powell (2005), and 16. Ajello (1948). *Incomplete genome (estimated 90% complete). †Undetected or potential loss. ‡Two-layered cell wall. 2018, ase al. et Bass 0 1–10 , Bass et al. An Expanded Microsporidia

AF372713 Uncult BAQA254 AY916571 Uncult Zeuk2 AB971034 Uncult Cryptomycota Jp13Cm04S CM1.1 FISH probe 0.98/100 AB971033 Uncult Cryptomycota Jp13Cm03S (Jones et al 2011; flagellated cells) Laz I (& II); ‘LKM-11’ (Corsaro et al 2016) AJ130849Uncult LKM11 0.66/49 FJ687268 Uncult WS CM1 3701 KY687824 Uncult Branch 2, Rozellomycota (Tedersoo et al 2017) JN054676 Uncult C10 0.76/- FJ976649 Uncult PRS2 4E 06 JQ796369 Paramicrosporidium saccamoebae (KSL-3) 0.53/42 FJ577832 Uncult Joinv23 1968 JQ796368 Paramicrosporidium vannellae 0.9/99 AB901672 Uncult AOox H 2012Dec 37 Laz III; ‘Paramicrosporidium’ (Corsaro et al 2016) KP096167 Uncult Cryptomycota CES3204-27 AB695512 Uncult MPE217 0.66/66 AY821991 Uncult CH1 S2 50 Laz IV 1/100 DQ244017 Uncult PFD5AU2004 LKM11-01 FISH probe AJ130857 Uncult LKM46 (Jones et al 2011; 0.98/65 DQ244016 Uncult PFB12AU2004 ‘LKM-46’ (Corsaro et al 2016) AB695465 Uncult MPE1-22 flagellated cells) 0.86/27 EU091850 Uncult P2 3m3 Laz VIII (part) 0.58/18 KY687737 Uncult GS11 ‘wweuk’ (Corsaro et al 2016) 0.99/98 GU568155 Uncult soil fungus clone 564 EU091829 Uncult P1 3m3 Laz VII AJ506016 Uncult RSC CHU 42 Laz VI 1/99 KY687783 Uncult GS10 0.65/40 EF023474 Uncult Amb 18S 1059 Laz V 0.96/90 AF372716 Uncult LEM108 EU162634 Uncult PFB5SP2005 AY64270 Uncult P34-42 Laz VIII (part); P34.42 (Corsaro et al 2016) GS01 0.5/13 KY687512 Uncult x4 KX214677 Amphiacantha ex Lecudina x4 x4 1/100 KX214676 Amphiacantha ex Lecudina x4 1/100 KX214678 Uncult Metchnikovellidae p1 44 1/100 x4 KX214674 Amphiamblys WSBS2011 0.99/88 KX214672 Amphiamblys WSBS2006 1/100 0.91/- x4 AJ252950 Janacekia debaisieuxi Canonical AF484695 Trichonosema pectinatellae 0.97/87 1/100 AJ581995 Bacillidium vesiculoformis Microsporidia KY687850 Uncult GS03 1/99 KX017226 Nucleophaga terricolae JQ288099 Nucleophaga amoebae ‘Nucleophaga’ (Corsaro et al 2016) KY687857 Uncult GS07 KM067412 Uncult DH134F06 Microsporidia KY687859 Uncult GS08 0.51/9 FM178232 Uncult hww6 0.94/67 KY687716 Uncult GS06 (expanded) AB252776 Uncult NAMAKO-36 Unaffiliated 0.68/94 EF023360 Uncult Amb 18S 686 AB901732 Uncult AS N 2012Sep10 Laz IX 0.99/100 DQ244011 Uncult PFA12AU2004 AM114814 Uncult WIM27 AB971036 Uncult Cryptomycota Jp13Cm06E ‘WIM27’ (Corsaro et al 2016) KY687580 Uncult 0.98/96 AJ130850 Uncult LKM15 1/96 AB901699 Uncult AOox H 2012Dec 71 AF372718 Uncult BAQA64 HM487046 Uncult HA001 KY687629 Uncult 0.86/- AB695464 MPE1-21 Laz X; ‘Mitosporidium’ (Corsaro et al 2016) Mitosporidium daphniae Branch 1, Rozellomycota (Tedersoo et al 2017) 0.99/88 EF024492 Uncult Elev 18S 791 KY687565 Uncult GS04 AB534337 Uncult A 3 61 AB191431 Uncult TAGIRI23 0.94/52 AB275064 Uncult DSGM64 Laz XI; ‘Basal Marine Group’ (Corsaro et al 2016); NAMAKO-37 AB252777 Uncult NAMAKO-37 AB971038 Uncult Cryptomycota Jp13Cm08S 1/100 AB971037 Uncult Cryptomycota Jp13Cm07E JQ689418 Uncult Pa2007A1 Laz XII; ‘Rozella’ (Corsaro et al 2016) 0.95/73 NG 017174 Rozella allomycis Rozellida KY687657 Uncult GS02 GU067982 Uncult ESS270706 1/98 FJ976648 Uncult PR5 4E 71 EU050974 Uncult SS1 E 01 42 0.98/100 EU050973 Uncult SS1 E 01 10 AB468614 Uncult S04B23 EU154992 Uncult KD14-BASS NCLC1 (Basal Clone Group 1) AB275063 Uncult DSGM63 AY180024 Uncult CCW48 aphelid AM114806 Uncult WIM48 1/100 GQ995419 Uncult T4P1AeE08 AB252775 Uncult NAMAKO35 GQ995418 Uncult T6P2AeG07 FJ157332 Uncult 110904-24 HM487005 Uncult GA089 JX507298 Amoeboaphelidium protococcarum GQ995416 Uncult T3P1AeG08 JX967274 Amoeboaphelidium PML2014-FD01 Aphelidea HQ191302 Uncult PA2009E13 GU067917 Uncult ESS270706-024 EF100212 Uncult D1P02G09 DQ244010 Uncult PFF3AU2004 KX576682 Paraphelidium tribonemae 0.99/97 AY230211 CCW24 aphelid GU067817 Uncult ESS220206 FJ976650 Uncult PRS2 4E 31 1/100 Nuclearia pattersoni Nuclearia simplex Outgroup (Nucleariida)

2.0

Figure 1 Phylogenetic relationships among canonical, long-branching (LB) Microsporidia, metchnikovellids, Rozellida, and a diversity of related short-branching lineages. LB-Microsporidia form a clade with a diversity of short-branching lineages (SB-Microsporidia) that share key cell biological characters deﬁning the microsporidian clade. Lineages that have been labelled in other studies are labelled: GSxx from Tedersoo et al. (2017), Laz x from Lazarus and James (2015); others as marked. The extent of the expanded Microsporidia is shown by the bracket on the far right. The tree was inferred under the CAT+GTR model in PhyloBayes-MPI, on a ﬁnal alignment of 1729 sites from the SSU rRNA gene. Black blobs indicate support values of > 0.96 BPP and > 95% ML bootstrap (actual values also shown). Branch lengths are proportional to the expected number of substitutions per site, as denoted by the scale bar.

Paraphelidium s earlier-diverging Mitosporidium (or the metchnikovellid Amoeboaphelidium GU067817 e FJ976650

DQ24401 AY230211 Amoeboa GU067917 HQ191302 Amphiamblys sp.), but were unable to ﬁnd any signiﬁcant 3

0212 ium FJ350301 pamyc ophydium551 hits (E < 1e-10). We were also unable to find significant ap 6 GQ9954168phelid hytr EF10 Y642742 K Rhizophydium yc A Rhiz 313 AB69 hits to most of the proteins associated with flagellar struc- FJ592325 ium Pol 44 learia ria GQ995419 Chytridiumc EF Nu ture and function detected in Paramicrosporidium by AB252775 Nuclea GQ995418 Homo Caenorhabditis Quandt et al. (2017). These analyses suggest that none of FJ157332 Trichoplax HM4870 5 Monosiga these lineages has a cryptic flagellum that might have AM11480605 Ichthyophonus Amoebidium been missed by microscopy. However, the branching posi- AY180024 4 Corallochytrium EU154992 6 Dermocystidium tion of the lineages targeted by FISH in Jones et al. (2011) 3 Ministeria AB275063 Capsaspora is unresolved within the microsporidian clade. If these lin- AB468614 Paramicrosporidium EU050973 FJ577832 eages are actually more deeply branching than other char- 2 Paramicrosporidium EU050974 AB90 1 1672 acterized SB-Microsporidia then it is possible that the AB275064 KP096 252777 AB 167 AB AB971971 flagellar apparatus in microsporidia was lost early in the 033 AB191431 AJ130 F 03 diversification of the clade. If this is the case, it would be 976648 J68 84 4 FJ la AJ506 8 GU5 7268 9 AB5121774 AY6427 very interesting to know whether this pre-dated or over- Rozel DQ24 6815503 A 0 AB97103 232 AB90J130850 lapped with the evolution of the polar filament apparatus, AF AB971037 HM48 4 00 EF Mi 016 EF024492 3

Nucleophaga FM178 AB534337 7 1699 and whether the latter arose at the origin of the 1 tosp 2718 KM067412 0 KP685328 0 7 04 132 AB252776 35 microsporidian clade. Alternatively, it is possible that only oridium 6 100 AM114814 3 AB971036 DQ244011 AB901732 some SB-Microsporidia lost their ﬂagella apparatus (and EF023360

Nucleophaga associated genes) and that those lineages represented in

Amphiamblys 10 Jones et al. (2011) are exceptions to the generality sug- 1 Expanded Microsporidia 4 Aphelidea gested by the other characterized lineages in this part of 2 Rozellida & NAMAKO-37 5 Chytridiomycota the tree. In that case the use of FISH enabled detection of ≤1 3 NCLC1 6 Holozoa life-stages and lineages that have so far eluded cell isola- Figure 2 Evolutionary placement of tropical soil microsporidian OTUs tion-based methods of investigation. Screening genomic on the opisthosporidian reference tree. The branch colours correspond data alone may not provide all such information: even to the distribution of OTU placement: The darker a branch, the more though Paramicrosporidium has an obvious polar filament, OTUs are placed on it. The OTU frequency scaling is logarithmic due polar filament proteins (PFPs) known from LB-Microspori- to the large range of placement density. The branch labels for Micro- dia were not found in the Paramicrosporidium or sporidia are red (characterized taxa in bold), rozellids orange, NCLC1 Mitosporidium genomes (Haag et al. 2014; Quandt et al. purple, aphelids blue, chytrids green, and the holozoan outgroup grey. 2017), suggesting rapid evolution of PFPs in the Nucleariids are the sister clade to Fungi+Opisthosporidia. microsporidian clade, earlier forms of these proteins being too dissimilar to their highly derived homologs to be detectable by gene similarity searches. This observation is case for other SB-Microsporidia. In summary, as Quandt supported by the low number (589–664; 24–27%) of et al. (2017) note, “shared gene content is clearly not corre- orthologous genes shared between Paramicrosporidium lated with evolutionary relationships”; instead, gene con- and all sequenced LB-Microsporidia. Haag et al. (2014) tent evolution within the microsporidian clade depicted in found only four orthologs shared between Mitosporidium Fig. 1 appears to be characterized by repeated, lineage- and LB-Microsporidia, but not with other fungi. specific gene losses rather than a stepwise trend towards The small number of genomic comparisons currently genome reduction in LB-Microsporidia. possible between members of Opisthosporidia shows a mosaic evolution, at least partly mediated by horizontal gene transfer, independent gene losses, and perhaps mul- Flagella and polar filament evolution in Microsporidia tiple transitions to parasitism. This is a fascinating situa- While rozellids have a lifecycle of alternating zoosporic and tion, worthy of intense study, but does not detract from a nonflagellate trophic stages, flagellate (zoosporic) stages simple and robust classification as proposed here. As are unknown for any lineages in the expanded microsporid- more lineages are detected and characterized, this hetero- ian clade, with two intriguing exceptions. Jones et al. geneity is set to increase. By adopting a phylogenetically (2011) showed that members of two SB-microsporidia lin- driven, character-based classification structure based on eages (Laz I and LKM-46, indicated on Fig. 1) have zoospo- the monophyly of an expanded microsporidian clade, our ric stages without chitin in their cell walls. Assuming the rationalization provides a clearer set of hypotheses on cells reconstructed from those FISH experiments repre- which to base future studies by pinpointing the origin of sent the branches indicated on Fig. 1 and were not the microsporidian radiation, the relation to which genomic false-positive FISH signals, this shows that some SB- and cellular characters can be ascertained. Microsporidia do possess flagella at some stage of their life cycle. In this study, we used BLASTP to search for Classification of Microsporidia and their relatives homologs of the specialized epsilon and delta tubulins, intraflagellar transport system, or flagellar-specific motor Taxonomic circumscriptions of Rozellomycota and, particu- molecules in the genomic datasets for the putatively larly, Cryptomycota, vary significantly (see Berbee et al.

© 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists 6 Journal of Eukaryotic Microbiology 2018, 0, 1–10 Bass et al. An Expanded Microsporidia

2017; Spatafora et al. 2017; Richards et al. 2017; and Rozella, and in Fig. 1 of this study [under the best-fitting examples cited throughout this study). Some authors CAT+GTR phylogenetic model], and in Lazarus and James include aphelids within Cryptomycota (e.g. Letcher et al. (2015) is sister to microsporidia), and (2) NCLC1 (Basal 2013), but more normally Crypto/Rozellomycota are used Clone Group I), which in Fig. 1 branches between to encompass Paramicrosporidium, Mitosporidium, Nucle- NAMAKO-37 and aphelids, but whose actual branching ophaga, the lineages detected by FISH in Jones et al. position is unresolved (Richards et al. 2015, 2017). On the (2011), the strongly supported clade containing Rozella, basis of their phylogenetic position and existing prove- and the large diversity of environmental sequences nance data we suggest that the last two groups are zoos- branching around and among these lineages. This is the poric parasites of marine microbial eukaryotes. most frequently used classification, informally described We infer that the “unaffiliated” group (Fig. 1) is by Letcher et al. (2018) and shown in Jones et al. (2011), microsporidian (and therefore not cryptomycotan) based Lazarus and James (2015), and (excluding Mitosporidium, on tree topology and bipartition support. These cells could which is classified as microsporidian) Quandt et al. (2017). provide key insights into early microsporidian evolution. We suggest that the frequently referred to “paraphyly We hypothesize that they possess mitochondria, and pos- of Rozello/cryptomycota” is both misleading and avoid- sibly flagellar structures or/and simpler cell extrusion appa- able. The defining morphological characters of microspori- ratus than in other SB-Microsporidia. The NAMAKO-37 dia are spores with multilayered cell walls containing polar clade cannot be classified until more data are available. filament apparatus homologs (not necessarily functioning There is negligible support for their being microsporidian in extrusion). The phylogenetic distribution of these char- or belonging to any other recognized group, so the label acters is coincident with the clade containing only SB- and “Cryptomycota” could be used for this monophyletic lin- LB-Microsporidia, which is recovered by both SSU and eage, at least for the time being. However, the message multigene phylogenetic analyses. Therefore, all members of this article remains unchanged whether the whole of that clade are in fact Microsporidia, and there is no Rozellida + Microsporidia clade is referred to as Rozello- or need for them to be regarded as belonging to a para- Cryptomycota or whether these names fall out of use, phyletic group. The closest known relatives of microspori- what the relationships between aphelids, rozellids, and dia, again according to SSU (Fig. 1) and multigene microsporidia actually are, and where the boundary phylogenies, are rozellids, which are restricted to a between fungi and other protists is. Crucially, acknowledg- robustly supported clade including the genus Rozella. ing an expanded Microsporidia as an evolutionarily and Rozella shares some microsporidian-like features with the morphologically coherent unit both unambiguously clarifies more distantly related LB-Microsporidia (e.g. horizontally the taxonomy/classification of this very interesting clade in acquired Rickettsia-like NTT ATP/ADP transporters, degen- the eukaryote tree, and provides a clear framework for erate, and AT-rich mitochondrial genomes lacking Complex future research. I of the oxidative phosphorylation pathway, thymidine kinases), but not with all of their shorter-branch relatives, High diversity of SB-Microsporidia in neotropical soils and are highly morphologically distinct. What then are Cryptomycota (Jones et al. 2011) and An important consequence of our analyses is that the tax- Rozellomycota (Corsaro et al. 2014a)? On the basis that onomic affiliations of large-scale SSU rRNA amplicon they refer to the same assemblage of lineages (which is sequencing studies must now be revisited. Most of the usually the case) two names are unnecessary and confus- sequences annotated as “crypto/rozellomycota” in taxo- ing. The purpose of this study is neither to decide nomically curated databases such as SILVA and PR2 (Guil- between them nor suggest an alternative, nor even to lou et al. 2013) are likely to be SB-Microsporidia. The invalidate them. However, to be consistent with recent rozellid annotation should be restricted to the clade indi- usage this label could be applied to an uncharacterized cated on Fig. 1. This is not purely a matter of classifica- monophyletic group branching somewhere in the tion: Microsporidia and rozellids are each monophyletic opisthosporidian clade, excluding aphelids, the expanded and distinct in terms of biology and ecology, but are cur- microsporidia, or the clade including Rozella. The latter rently conflated in the major sequence databases. Clarify- already has order or class status, according to different ing the composition of the two clades will enable much authorities (Lara et al. 2010; Ruggiero et al. 2015). Fig- more accurate and high-resolution analyses, and interpre- ure 1 indicates the diversity revealed by general eukary- tation of microsporidian diversity and function. ote-wide environmental sequencing studies, including To demonstrate the particular relevance of this more those clades coded and labelled by Corsaro et al. (2016), inclusive definition of microsporidia to the annotation and Tedersoo et al. (2017), and the diversity detected by the interpretation of environmental sequencing studies, we targeted PCR approach of Lazarus and James (2015). Of reevaluated a recent SSU rRNA high-throughput sequenc- this very substantial environmental diversity, only two ing study of three Neotropical rainforest soils (Mahe et al. groups within Opisthosporidia do not branch within the 2017), using the Evolutionary Placement Algorithm as expanded microsporidian, rozellid, or aphelid clades: implemented in RAxML (Stamatakis 2014) to place opera- (1) the NAMAKO-37 clade (which in Corsaro et al. (2016) tional taxonomic units (OTUs) previously annotated as branches outside of the microsporidian clade before the Opisthosporidia, crypto/rozellomycota, or unassigned divergence of Rozella, in Jones et al. (2011) is sister to fungi. We similarly analysed an OTU dataset from

European coastal water and sediment samples (BioMarKs: 65 OOTUsTUs Logares et al. 2014; Massana et al. 2015). 26 OTUsOTUs Of the 1,279 candidate tropical soils OTUs, 94% clus- 7 OTUsOTUs LBLB-Microsporidia,-Microsporidia, NNS1405S1405 MMetchnikovellids,etchnikovellids, tered within the microsporidian clade (Fig. 2, 3). Figure 3 NS9695NS9695 + 2 OOTUsTUs shows the branching positions of clades of OTUs relative 2 OTUsOTUs 4 OTUsOTUs to the reference sequences. OTUs clustering with the NS22327NS22327 7 OTUsOTUs metchnikovellid branch are putatively parasites of gre- 1313 NSNS OTUs,OTUs, garines. Others grouping nearer Nucleophaga, at the KX017226KX017226 NucleophagaNucleophaga cladeclade 2 BM OTUsOTUs JQ288099JQ288099 base of the branch leading to metchnikovellids and LB- NNS5987S5987 Microsporidia, may be parasites of amoebae or/and other 2 OTUsOTUs 2 OTUsOTUs protists (perhaps including hyperparasites). Mahe et al. 3 OTUsOTUs 1414 OOTUsTUs (2017) show that both gregarines and Amoebozoa are 5 OTUsOTUs highly diverse in their Neotropical soil samples. However, 1199 OOTUsTUs 2 OTUsOTUs until more lineages are found in nature we will be unable 4411 OOTUsTUs 110404 OOTUsTUs to conclusively determine whether microsporidian diver- NS4619NS4619 sity branching nearer Paramicrosporidium or Mitosporid- 5500 OOTUsTUs PParamicrosporidiumaramicrosporidium ccladelade + 4455 OOTUsTUs ium are parasites of protists and arthropods respectively. 1155 NSNS OTUs,OTUs, 3 BM OOTUsTUs 3 BM Although there were far fewer opisthosporidian OTUs in 2266 OOTUsTUs the BioMarKs data, the majority of these also branched NNS17605S17605 2 NNSS OOTUsTUs within the microsporidian clade (Fig. 3). In both datasets 2 NS OTUs a small proportion of OTUs was placed in the rozellid LLKM11KM11 clade + 94 OTUs 4477 OTUs clade (two from soil), and the NAMAKO-37 clade (four NNS39442S39442 GGU568155,U568155, AJ506030 + 137 OOTUsTUs from BioMarKs), consistent with the apparently limited 3388 OOTUsTUs diversity of these clades compared to Microsporidia. 6644 OOTUsTUs DQDQ244016,244016, AY642700 + 145 OOTUsTUs LB-Microsporidia are predominantly parasites of ani- NS25395NS25395 NS34422NS34422 mals, but Nucleophaga and Paramicrosporidium are para- 2 OOTUsTUs MiMitosporidiumtosporidium ccladelade + 197 OTUsOTUs sites of protists (amoebozoan hosts are only known so 5533 OTUs far, which are relatively scarce in marine habitats). ‘‘Unaffiliated’Unaffiliated’ clade + 78 OOTUsTUs NAMAKO37 (4 BM OTUs) Metchnikovellids are parasites of gregarines (protistan Rozellids (2 OTUs) gut parasites of a wide range of invertebrates; Des- NS3943 Dikarya (0 OTUs) portes and Schrevel 2013). Mitosporidium is a parasite Aphelids (1 NS OTU, 1 BM OTU) DQ536489 Physoderma maculare of Daphnia (Haag et al. 2014), and perhaps other lin- Chytrids (0 OTUs) eages in the Mitosporidium clade are also parasites of NCLC1 (0 OTUs) arthropods. SB-Microsporidia therefore appear to occupy Outgroup a broad and little understood set of niches. The phyloge- 0.6 netic distribution of characters typically associated with LB-Microsporidia may be determined at least as much Figure 3 Maximum Likelihood phylogeny of SSU rRNA gene V4 region OTUs from tropical soil and European marine (Biomarks) sam- by host-specific adaptation as phylogenetic relatedness. ples. The OTUs are aligned to reference sequence dataset of long- For instance, shorter/reduced polar filaments may be and short-branch Microsporidia, metchnikovellids, rozellids, other more characteristic of Microsporidia infecting protists clades of environmental sequences, and representative aphelids and than invertebrates. On the other hand, if at least in fungi with a holozoan outgroup. Light and dark grey shading indicates some cases cell host invasion is mediated by host extent of expanded Microsporidia; dark grey box encloses all mem- phagocytosis, the length/complexity of the polar filament bers of the subclade including long-branch Microsporidia. Green trian- may not always be directly related to the physical barri- gles (collapsed clades) represent OTUs from Neotropical soil samples ers it must cross to invade the host (Franzen 2004). only, ‘BM’ from Biomarks only, and blue from both.

CONCLUSIONS many uncharacterized environmental sequence types. The We analysed the phylogenetic placement and environmen- concept of Rozellomycota/Cryptomycota requires revision tal diversity of microsporidian-like lineages that group near to avoid encompassing lineages that are actually the base of the fungal radiation. These lineages form a microsporidian. Our analyses suggest hypotheses for inves- monophyletic group including canonical Microsporidia and tigations into the relative timings of acquisition of the polar metchnikovellids, but excluding a strongly supported rozel- filament apparatus and loss of flagella, key microsporidian lid clade. This topology is concordant with the phylogenetic characteristics. We show that this revised classification has distribution of defining microsporidian cell characters, but major implications for our understanding of microsporidian not shared gene content across Opisthosporidia and fungi. diversity as inferred from environmental sequencing sur- The genetic diversity of Microsporidia is far higher than pre- veys. The large diversity and abundance of SB-Microspori- viously realized, and includes the SB-microsporidian taxa dia offer unique opportunities to study the evolution of the Mitosporidium, Paramicrosporidium,andNucelophaga,and highly specialized cells and genomes of canonical

© 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists 8 Journal of Eukaryotic Microbiology 2018, 0, 1–10 Bass et al. An Expanded Microsporidia

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