Published by the International Society of Protistologists
The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
SYMPOSIUM ARTICLE
Ciliates and the Rare Biosphere: A Review de ric Mahe a Micah Dunthorna, Thorsten Stoecka, John Clampb, Alan Warrenc & Fre a Department of Ecology, University of Kaiserslautern, D-67663, Kaiserslautern, Germany b Department of Biology, North Carolina Central University, Durham, North Carolina, 27707, USA c Department of Life Sciences Department, Natural History Museum, London, SW7 5BD, United Kingdom
Keywords Abundance; biogeography; Ciliophora; diversity; environmental diversity; rarity. Correspondence M. Dunthorn, Department of Ecology, Uni€dinger versity of Kaiserslautern, Erwin-Schro Street Building 14, D-67663 Kaiserslautern, Germany Telephone number: +49 (0)631 205 3253; FAX number: +49 (0)631 205 2496; e-mail: [email protected]
ABSTRACT Here we provide a brief review of the rare biosphere from the perspective of ciliates and other microbial eukaryotes. We trace research on rarity from its lack of much in-depth focus in morphological and Sanger sequencing projects, to its central importance in analyses using high throughput sequencing strategies. The problem that the rare biosphere is potentially comprised of mostly errors is then discussed in the light of asking community-comparative, noveldiversity, and ecosystem-functioning questions.
Received: 23 January 2014; revised 14 March 2014; accepted March 14, 2014. doi:10.1111/jeu.12121
WHILE issues of rarity have been examined for a long time in animals and plants (Kunin and Gaston 1997; Magurran 2004; Magurran and Henderson 2003), rarity has only recently been a focus in the study of microorganisms. In particular, the “rare biosphere” of Sogin et al. (2006)—in which the majority of microbial species are only detected with substantial sampling effort—has been a driving force in the study of environmental microbial diversity. The rare biosphere in microbes in general has been the subject of recent reviews (Caron and Countway 2009; Caron et al. 2012; Dawson and Hagen 2009; Massana and Logares s-Alio 2006, 2012; Stoeck and Epstein 2009), 2013; Pedro as well as in ciliates in specific in this volume (Edgcomb and Pachiadaki 2014; Hu 2014; Weisse 2014). Here we provide a brief background on the rare biosphere from the perspective of ciliates, although other microbial eukaryotes are mentioned. The review takes a broad perspective of how rarity was viewed as methodologies changed: from rarity not being a focus in morphological and Sanger sequencing studies, to rarity being a focus with high throughput sequencing (HTS), to rarity then being ignored when much of the rarity detected in HTS studies was found to be composed of errors. For each of these methodological changes, only a few representative studies will be cited.
RARITY NOT A FOCUS Morphological investigations of ciliates have largely ignored all but the most common species, except for a few studies (Esteban and Finlay 2007). This was due to the amount of time needed for morphological observations and the tremendous amount of diversity in nature. The focus in most of these studies was on identifying known species and describing new abundant isolates. There was little time left for observing rare ciliates, let alone the difficulties to detect these low-abundant taxa in just a few microliters of water examined under the microscope (Dolan and Stoeck 2011). Nevertheless, in morphological studies the term “rare” is used all the time—even in the paper’s title (Foissner and Oertel 2009; Foissner et al. 2004; Lin et al. 2004; Shen et al. 2009). This is not new; for example, over a century ago Moody (1912) described Spathidium spathula as rare, but it has since been found frequently worldwide (Foissner et al. 2002). Loxophyllum perihoplophorum is another example of a species once thought to be rare; it was originally described by Buddenbrock (1920) and redrawn by Kahl (1931), but was recently found in a mangrove wetland in southern China by Wu et al. (2013).
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 0, 1–6
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All of these morphological cases of rarity in ciliates, we propose, are merely a case of rarity in ciliate morphological taxonomists. That is, the chance of a rare species being found depends on who is looking for what and how many people are looking. Presently there are far too few morphological taxonomists to investigate the issue of rarity in any depth. And even if we cloned all the living ciliate morphological taxonomists, we would still question if there would be enough of them to get at the diversity of rare ciliates because of the time required for observing both live and silver-stained specimens. Likewise, rarity has not been a focus in studies of ciliates in molecular studies based on cloning and Sanger sequencing methodologies. These methodologies are too time consuming as well, and resulted in too few sequences, to detect any but the most common ciliates pez-Garcıa 2008; Pedro s(Christen 2008; Epstein and Lo 2006). Consequently, when the numbers of ciliate Alio (and other microbial eukaryotic) molecular operational taxonomic units (OTUs) from environmental samples were plotted against the numbers of cloned libraries examined, saturation was not reached (Doherty et al. 2007, 2010; Stock et al. 2009; Stoeck et al. 2007; Tamura et al. 2011). Even the very large Sanger sequencing studies were limited in the number of resulting reads; for example, one of the biggest was when Behnke et al. (2006) analyzed anoxic layers in the Framvaren Fjord, Norway, and were only able to sequence 1,100 clones. RARITY A FOCUS The focus on rarity in environmental diversity surveys began with the implementation of HTS strategies, such as Roche/454 (Margulies et al. 2005) and Illumina sequencing (Bentley et al. 2008). The seminal study in this new focus came when Sogin et al. (2006) examined North Atlantic marine bacteria using Roche/454. They showed with their HTS data that not only is the diversity of bacteria orders of magnitude higher than previously realized with Sanger sequencing, but also that the rare biosphere harbors most of the microbial diversity, at low abundances, within any one environmental sample. And the deeper the HTS is done, the more of this rare biosphere will be uncovered. One hypothesis is that this rare biosphere harbors species that can then take over the role of dominance when environmental conditions change and the need occurs for changes in ecology and metabolisms (Caron and Countway 2009; Sogin et al. 2006; Stoeck and Epstein 2009). In this view of the rare biosphere, community dynamics over time are largely affected by relative changes in abuns-Alio 2006), and there dances (Gibbons et al. 2013; Pedro is a massive seed bank from which species can be recruited in response to ecological changes (Caron and s-Alio 2006; Countway 2009; Caron et al. 2012; Pedro Sogin et al. 2006). Given a large enough time scale, presumably these changes in abundances will cycle through all available species in any one environment, although some species may be recalcitrant to an increase in their
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population sizes (Garland et al. 2009; Massana and Logares 2013; Weisse 2014). Following HTS studies to uncover the extent of the rare biosphere in bacteria and archaea (Garland et al. 2009; Huber et al. 2007; Sogin et al. 2006), the same methods were used to uncover the extent of the rare biosphere in microbial eukaryotes (Amaral-Zettler et al. 2009; Bittner et al. 2013; Logares et al. 2012; Nolte et al. 2010; Pawlowski et al. 2011; Stoeck et al. 2009, 2010). For example, Stoeck et al. (2009) went back to the same DNA samples that Behnke et al. (2006) collected in the Framvaren Fjord, and re-analyzed them with Roche/454. When they grouped the resulting reads at 99% similarity, or less, they were much closer to saturation than what they previously found with Sanger sequencing. They also found that > 90% of reads derived from rare species, which they described as OTUs consisting of fewer than 10 reads— although rarity has been defined differently elsewhere (De Barba et al. 2014; Kunin et al. 2010; Weisse 2014). These studies found, as did Sogin et al. (2006) and others with bacteria and archaea, a rare biosphere in microbial eukaryotes in general and ciliates in particular. This uncovering of the rare biosphere in the ciliates inhabiting the Norwegian fjord by Stoeck et al. (2009) also allowed for the uncovering of a much more complex community, composed of more taxa than just the common spirotrichean and oligohymenophorean species. The aspect of the rare biosphere where community dynamics is largely a reflection of changes in abundances was, for example, shown in microbial eukaryotes when Nolte et al. (2010) sampled lake Fuschlsee, Austria every 3 wk over a 7-mo period; they found that abundances drastically changed, with some very abundant OTUs disappearing within a month. Within the rare biosphere hypothesis there are a number of corollaries about what is occurring in the abundant taxa and what is occurring in the rare taxa. The first corollary is that abundant species are mostly affected by replication as well as predation and parasitism (Caron et al. s-Alio 2006). Predation and parasitism is 2012; Pedro assumed to be only important for the most abundant taxa, as predators and parasites would have a foraging problem when trying to find the rare species; e.g., in the cases in which the predators are actively searching for specific prey species (although species-specific predation may not be that common in nature). The second corollary is that rare species are mostly affected by immigration and local extinction (Caron et al. s-Alio 2006). Although there is disagreement 2012; Pedro of the extent of dispersal in microbial eukaryotes (Fenchel and Finlay 2006; Finlay 2002; Foissner 1999; Foissner et al. 2008), there is agreement that a large proportion of ciliates are able to immigrate to any community given a long enough time scale, resulting in massive seed banks. Local extinction is thought to be low, because predators and parasites are, as stated above, though to be unimpors-Alio 2006; Sogin et al. 2006). Massana and tant (Pedro Logares (2013) recently argued that local extinction for microbial eukaryotes is higher than bacteria and archaea, since many species lack the protective cysts and very
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small cell sizes that might otherwise prevent predation, although many ciliates are able to encyst (Weisse 2014). Massana and Logares (2013) also posited that becoming too rare would be problematic for microbial eukaryotes that depend on sex as a cohesive evolutionary force. We add to this view that if sex comes into play here for ciliates it would be needed to reduce deleterious mutations (Bell 1988; Kondrashov 1993; Maynard Smith 1978), a process that is especially important in the small populations that would be found in the rare biosphere (but see Weisse 2014). The need for sex to shuffle alleles around to allow for adaption to changing environments (Hamilton 2001; Hamilton et al. 1990; Morran et al. 2011), though, would be reduced as ciliates and other microbial eukaryotes could just lay low and brace themselves until conditions fare better for their current allelic combinations. RARITY IGNORED Rarity began to move from a focus to being ignored, when the rare biosphere was shown to be largely composed of €nemann a long queue of errors (Behnke et al. 2011; Ju et al. 2013; Kunin et al. 2010). For example, Behnke et al. (2011) established a mock community composed of cloned DNA from 24 different microbial eukaryotes, nine of which were ciliates. When these clones were sequenced with Roche/454, they uncovered not only their 24 references in their resulting reads, but also they found over 2,000 OTUs grouped at 100% similarity. Even when grouping the reads at 97% similarity they found 44 OTUs, nearly doubling the number of true morphotypes in the mock community. And when Behnke et al. (2011) removed the singletons, they still found 462 OTUs at 100% similarity. Most of the errors producing these spurious OTUs were due to homopolymers. They concluded that in the absence of using methods that attempt to remove these errors, HTS studies aimed at uncovering environmental microbial eukaryotic diversity should ignore the single singletons (or the rare species). If errors are removed with various error-correcting algorithms, though, you can obtain variable results that can change the taxonomic identities of the OTUs, and greatly limit biodiversity estimates (Coissac et al. 2012; Gaspar and Thomas 2013; Santoferrara et al. 2014). What to do with the rare reads or OTUs that could potentially be composed of errors? That depends on which type of question you want to ask. If you want to ask community-comparative questions, then including rare amplicons OTUs or not can have little effect on some of the comparative statistics that are largely driven by the most abundant species (Caporaso et al. 2011; Gobet et al. 2010). However, it may be better to lose “informative” reads than to introduce potential errors by including only the abundant amplicons—although some diversity will be missed. If you decided to analyze only the abundant OTUs, then you would not be able to use some of the standard statistics like Chao1 (Chao 1984) and ACE (Chao and Lee 1992) since these depend on the number of single singletons (Gihring et al. 2012).
Our ability to measure in environmental samples which species are actually abundant, and which ones should be retained for community-comparative questions, is questionable. This was demonstrated in a study by Stoeck et al. (2014), who compared ciliate abundances in the mountain lake Piburgersee, Austria, with morphological (live and silver-stained) and Roche/454 sequencing approaches. They found not much agreement between morphological and molecular abundances; e.g., Urotricha sp. accounted for 24.5% of the morphological data, but accounted for merely 0.09% of the molecular data; while Pseudomonilicaryon and Orthoamphisiella, which were most abundant in the molecular data, were not recovered with morphology. A similar situation in which abundance estimates drastically differed was found for ciliates and dinoflagellates when Medinger et al. (2010) sampled Lake Fuschlsee, Austria, and when Egge et al. (2013) evaluated a mock community of haptophytes. The incongruence between morphological- and molecular-based abundance estimates may, in part, be due to high variability in SSU-rDNA copy number among species. As pointed out by Prescott (1994) and Riley and Katz (2001), differential processing of macronuclear chromosomes can lead to hundreds of DNA copies in some taxa (e.g., Colpodea, Litostomatea, Prostomatea, and Oligohymenophorea), while leading to 1,000s of copies in other taxa (e.g., Phyllopharyngea and Spirotrichea). Such that, if you sample a freshwater pond morphologically you might find one Halteria grandinella (Spirotrichea) for every 10 Coleps hirtus (Prostomatea), but molecular estimates could tilt toward a 1:1 abundance pattern. Highly variable SSUrDNA copy number can also be due to variable in cell size among species (Zhu et al. 2005). Even within ciliates, the size spectrum of cells spans several orders of magnitude (small scuticocilates compared to the large Stentor), and macronuclei themselves may likewise be highly variable in size and number. Incongruence among abundance estimates may also be due to limitations of the molecular methods, primer ambiguities (Stoeck et al. 2006) and preferential PCR-amplifications of individual DNA templates (Wintzingerode et al. 1997). These purious abundance estimates of ciliates, and other microbial eukaryotes, from environmental molecular methods will affect abundancebased statistics of alpha- and beta-diversity (Haegeman et al. 2013); a preliminary solution to this problem may be the use of incidence-based statistics (presence/absence data) for more robust analyses of environmental HTS data. If you are interested in asking novel-diversity questions, then keeping rare OTUs is important as they are not all the result of sequencing errors (Berney et al. 2013; Br ate et al. 2010; Dunthorn et al. 2014; Lecroq et al. 2011; Orsi et al. 2011). Since most of the common species have already been detected and described in morphological and Sanger sequencing studies, most of the undiscovered diversity is likely to be rare. For example, in a study of six European off-shore marine sites using Roche/454 sequencing of planktonic and sediment samples, Dunthorn et al. (2014) found 87,724 ciliate reads. Of these, 523 reads blasted to the Colpodea, and grouped into 22 OTUs
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at 98% similarity. Nine of these OTUs contained just one read, and 10 contained two to four reads. These rare OTUs could, as suggested by Behnke et al. (2011) and other studies, be treated as mere errors and ignored. But when these rare OTUs were placed into an alignment of full-length Sanger sequences, most of their inferred relationships were very close to already known taxa. These rare marine OTUs, which would otherwise have been discarded in community-comparative studies, provide an alternative view that rather than being primarily terrestrial and freshwater as commonly thought (Foissner 1993; Lynn 2008), the Colpodea also includes multiple marine clades (Dunthorn et al. 2014). Keeping rare OTUs is also important if you want to ask ecosystem-functioning questions. Specifically, are the functional ecological traits hidden in the rare biosphere redundant from what is already known and present in the abundant taxa? If so, the rare biosphere would be responsible for resilience and stability of ecosystems under changing global conditions (Caron and Countway 2009; Dawson and Hagen 2009; Sogin et al. 2006; Stoeck and Epstein 2009); that is, the system can pivot among taxa with no change in the ecosystem. If there is no or only little functional trait redundancy in the rare biosphere, then ecosystem functioning would change drastically with community reassembly due to changes in abundances. Such a knowledge is crucial if we are to understand reactions of ecosystems to disturbances and environmental change. CONCLUSION The massive extent of rarity in ciliates, and other microbial eukaryotes, was largely shown with HTS methodologies, and results point to much diversity that was missed in previous morphological and Sanger sequencing studies. However, molecules by themselves may not be the best approach to get at rarity in these microbes, since they can lead to spurious abundance estimates. A counter-balance with morphology is therefore needed when possible to more accurately estimate which ciliates are abundant and which are rare in environmental samples. ACKNOWLEDGMENTS We thank George McManus, Roberto Docampo, and an anonymous reviewer for helpful suggestions and comments. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, grant # DU1319/1-1) to M.D., DFG (grant # STO414/3-1) to T.S., and U.S. National Science Foundation (DEB grant # 1136580) to J.C. LITERATURE CITED Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W. & Huse, S. M. 2009. A method for studying protistian diversity using massively parallel sequencing of V9 hypervariable region of small-subunit ribosomal RNA genes. PLoS ONE, 4:7. Behnke, A., Engel, M., Christen, R., Nebel, M., Kleln, R. R. & Stoeck, T. 2011. Depicting more accurate pictures of protistan
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morphogenesis and SS rRNA gene sequence. J. Eukaryot. Microbiol., 56:296–304. Sogin, M. L., Morrison, H. G., Huber, J. A., Mark Welch, D., Huse, S. M., Neal, P. R. & Arrieta, J. M. 2006. Microbial diversity in the deep sea and the underexplored “rare biospshere”. Proc. Natl Acad. Sci. USA, 103:12115–12120. €rgen, K., Bunge, J. & Stoeck, T. 2009. Protistan Stock, A., Ju diversity in the suboxic and anoxic waters of the Gotland Deep (Baltic Sea) as revealed by 18S rRNA clone libraries. Aquat. Microb. Ecol., 55:267–284. Stoeck, T. & Epstein, S. S. 2009. Crystal Ball-2009: protists and the rare biosphere. Environ. Microbiol. Rep., 1:20–22. Stoeck, T., Hayward, B., Taylor, G. T., Varela, R. & Epstein, S. S. 2006. A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples. Protist, 157:31–43. Stoeck, T., Breiner, H.-W., Filker, S., Ostermaier, V., Kammerlander, B. & Sonntag, B. 2014. A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology. Environ. Microbiol., 16:430–444. Stoeck, T., Kasper, J., Bunge, J., Leslin, C., Ilyin, V. & Epstein, S. 2007. Protistan diversity in the Arctic: a case of paleoclimate shaping modern biodiversity? PLoS ONE, 2:e728. Stoeck, T., Bass, D., Nebel, M., Christen, R., Jones, M. D. M., Breiner, H. W. & Richards, T. A. 2010. Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water. Mol. Ecol., 19:21–31. Stoeck, T., Anke, B., Christen, R., Amaral-Zettler, L., RodriguezMora, M. J., Chistoserdov, A., Orsi, W. & Edgcomb, V. P. 2009. Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities. BMC Biol., 7:72. Tamura, M., Katz, L. A. & McManus, G. B. 2011. Distribution and diversity of oligotrich and choreotrich ciliates across an environmental gradient in a large temperate estuary. Aquat. Microb. Ecol., 64:51–67. Weisse, T. 2014. Ciliates and the rare biosphere – community ecology and population dynamics. J. Eukaryot. Microbiol., doi:10.1111/jeu.12123. € bel, U. B. & Stackebrandt, E. 1997. Wintzingerode, F. V., Go Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev., 21:213–229. Wu, L., Chen, R., Yi, Z., Li, J., Warren, A. & Lin, X. 2013. The morphology of three Loxophyllum species (Ciliophora, Pleurostomatida) from Southern China, L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with notes on the molecular phylogeny of Loxophyllum. J. Eukaryot. Microbiol., doi:10.1111/jeu.12089. Zhu, F., Massana, R., Not, F., Marie, D. & Vaulot, D. 2005. Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene. FEMS Microbiol. Ecol., 52:79–92.
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The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
SYMPOSIUM ARTICLE
Ciliates and the Rare Biosphere: A Review de ric Mahe a Micah Dunthorna, Thorsten Stoecka, John Clampb, Alan Warrenc & Fre a Department of Ecology, University of Kaiserslautern, D-67663, Kaiserslautern, Germany b Department of Biology, North Carolina Central University, Durham, North Carolina, 27707, USA c Department of Life Sciences Department, Natural History Museum, London, SW7 5BD, United Kingdom
Keywords Abundance; biogeography; Ciliophora; diversity; environmental diversity; rarity. Correspondence M. Dunthorn, Department of Ecology, Uni€dinger versity of Kaiserslautern, Erwin-Schro Street Building 14, D-67663 Kaiserslautern, Germany Telephone number: +49 (0)631 205 3253; FAX number: +49 (0)631 205 2496; e-mail: [email protected]
ABSTRACT Here we provide a brief review of the rare biosphere from the perspective of ciliates and other microbial eukaryotes. We trace research on rarity from its lack of much in-depth focus in morphological and Sanger sequencing projects, to its central importance in analyses using high throughput sequencing strategies. The problem that the rare biosphere is potentially comprised of mostly errors is then discussed in the light of asking community-comparative, noveldiversity, and ecosystem-functioning questions.
Received: 23 January 2014; revised 14 March 2014; accepted March 14, 2014. doi:10.1111/jeu.12121
WHILE issues of rarity have been examined for a long time in animals and plants (Kunin and Gaston 1997; Magurran 2004; Magurran and Henderson 2003), rarity has only recently been a focus in the study of microorganisms. In particular, the “rare biosphere” of Sogin et al. (2006)—in which the majority of microbial species are only detected with substantial sampling effort—has been a driving force in the study of environmental microbial diversity. The rare biosphere in microbes in general has been the subject of recent reviews (Caron and Countway 2009; Caron et al. 2012; Dawson and Hagen 2009; Massana and Logares s-Alio 2006, 2012; Stoeck and Epstein 2009), 2013; Pedro as well as in ciliates in specific in this volume (Edgcomb and Pachiadaki 2014; Hu 2014; Weisse 2014). Here we provide a brief background on the rare biosphere from the perspective of ciliates, although other microbial eukaryotes are mentioned. The review takes a broad perspective of how rarity was viewed as methodologies changed: from rarity not being a focus in morphological and Sanger sequencing studies, to rarity being a focus with high throughput sequencing (HTS), to rarity then being ignored when much of the rarity detected in HTS studies was found to be composed of errors. For each of these methodological changes, only a few representative studies will be cited.
RARITY NOT A FOCUS Morphological investigations of ciliates have largely ignored all but the most common species, except for a few studies (Esteban and Finlay 2007). This was due to the amount of time needed for morphological observations and the tremendous amount of diversity in nature. The focus in most of these studies was on identifying known species and describing new abundant isolates. There was little time left for observing rare ciliates, let alone the difficulties to detect these low-abundant taxa in just a few microliters of water examined under the microscope (Dolan and Stoeck 2011). Nevertheless, in morphological studies the term “rare” is used all the time—even in the paper’s title (Foissner and Oertel 2009; Foissner et al. 2004; Lin et al. 2004; Shen et al. 2009). This is not new; for example, over a century ago Moody (1912) described Spathidium spathula as rare, but it has since been found frequently worldwide (Foissner et al. 2002). Loxophyllum perihoplophorum is another example of a species once thought to be rare; it was originally described by Buddenbrock (1920) and redrawn by Kahl (1931), but was recently found in a mangrove wetland in southern China by Wu et al. (2013).
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All of these morphological cases of rarity in ciliates, we propose, are merely a case of rarity in ciliate morphological taxonomists. That is, the chance of a rare species being found depends on who is looking for what and how many people are looking. Presently there are far too few morphological taxonomists to investigate the issue of rarity in any depth. And even if we cloned all the living ciliate morphological taxonomists, we would still question if there would be enough of them to get at the diversity of rare ciliates because of the time required for observing both live and silver-stained specimens. Likewise, rarity has not been a focus in studies of ciliates in molecular studies based on cloning and Sanger sequencing methodologies. These methodologies are too time consuming as well, and resulted in too few sequences, to detect any but the most common ciliates pez-Garcıa 2008; Pedro s(Christen 2008; Epstein and Lo 2006). Consequently, when the numbers of ciliate Alio (and other microbial eukaryotic) molecular operational taxonomic units (OTUs) from environmental samples were plotted against the numbers of cloned libraries examined, saturation was not reached (Doherty et al. 2007, 2010; Stock et al. 2009; Stoeck et al. 2007; Tamura et al. 2011). Even the very large Sanger sequencing studies were limited in the number of resulting reads; for example, one of the biggest was when Behnke et al. (2006) analyzed anoxic layers in the Framvaren Fjord, Norway, and were only able to sequence 1,100 clones. RARITY A FOCUS The focus on rarity in environmental diversity surveys began with the implementation of HTS strategies, such as Roche/454 (Margulies et al. 2005) and Illumina sequencing (Bentley et al. 2008). The seminal study in this new focus came when Sogin et al. (2006) examined North Atlantic marine bacteria using Roche/454. They showed with their HTS data that not only is the diversity of bacteria orders of magnitude higher than previously realized with Sanger sequencing, but also that the rare biosphere harbors most of the microbial diversity, at low abundances, within any one environmental sample. And the deeper the HTS is done, the more of this rare biosphere will be uncovered. One hypothesis is that this rare biosphere harbors species that can then take over the role of dominance when environmental conditions change and the need occurs for changes in ecology and metabolisms (Caron and Countway 2009; Sogin et al. 2006; Stoeck and Epstein 2009). In this view of the rare biosphere, community dynamics over time are largely affected by relative changes in abuns-Alio 2006), and there dances (Gibbons et al. 2013; Pedro is a massive seed bank from which species can be recruited in response to ecological changes (Caron and s-Alio 2006; Countway 2009; Caron et al. 2012; Pedro Sogin et al. 2006). Given a large enough time scale, presumably these changes in abundances will cycle through all available species in any one environment, although some species may be recalcitrant to an increase in their
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population sizes (Garland et al. 2009; Massana and Logares 2013; Weisse 2014). Following HTS studies to uncover the extent of the rare biosphere in bacteria and archaea (Garland et al. 2009; Huber et al. 2007; Sogin et al. 2006), the same methods were used to uncover the extent of the rare biosphere in microbial eukaryotes (Amaral-Zettler et al. 2009; Bittner et al. 2013; Logares et al. 2012; Nolte et al. 2010; Pawlowski et al. 2011; Stoeck et al. 2009, 2010). For example, Stoeck et al. (2009) went back to the same DNA samples that Behnke et al. (2006) collected in the Framvaren Fjord, and re-analyzed them with Roche/454. When they grouped the resulting reads at 99% similarity, or less, they were much closer to saturation than what they previously found with Sanger sequencing. They also found that > 90% of reads derived from rare species, which they described as OTUs consisting of fewer than 10 reads— although rarity has been defined differently elsewhere (De Barba et al. 2014; Kunin et al. 2010; Weisse 2014). These studies found, as did Sogin et al. (2006) and others with bacteria and archaea, a rare biosphere in microbial eukaryotes in general and ciliates in particular. This uncovering of the rare biosphere in the ciliates inhabiting the Norwegian fjord by Stoeck et al. (2009) also allowed for the uncovering of a much more complex community, composed of more taxa than just the common spirotrichean and oligohymenophorean species. The aspect of the rare biosphere where community dynamics is largely a reflection of changes in abundances was, for example, shown in microbial eukaryotes when Nolte et al. (2010) sampled lake Fuschlsee, Austria every 3 wk over a 7-mo period; they found that abundances drastically changed, with some very abundant OTUs disappearing within a month. Within the rare biosphere hypothesis there are a number of corollaries about what is occurring in the abundant taxa and what is occurring in the rare taxa. The first corollary is that abundant species are mostly affected by replication as well as predation and parasitism (Caron et al. s-Alio 2006). Predation and parasitism is 2012; Pedro assumed to be only important for the most abundant taxa, as predators and parasites would have a foraging problem when trying to find the rare species; e.g., in the cases in which the predators are actively searching for specific prey species (although species-specific predation may not be that common in nature). The second corollary is that rare species are mostly affected by immigration and local extinction (Caron et al. s-Alio 2006). Although there is disagreement 2012; Pedro of the extent of dispersal in microbial eukaryotes (Fenchel and Finlay 2006; Finlay 2002; Foissner 1999; Foissner et al. 2008), there is agreement that a large proportion of ciliates are able to immigrate to any community given a long enough time scale, resulting in massive seed banks. Local extinction is thought to be low, because predators and parasites are, as stated above, though to be unimpors-Alio 2006; Sogin et al. 2006). Massana and tant (Pedro Logares (2013) recently argued that local extinction for microbial eukaryotes is higher than bacteria and archaea, since many species lack the protective cysts and very
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small cell sizes that might otherwise prevent predation, although many ciliates are able to encyst (Weisse 2014). Massana and Logares (2013) also posited that becoming too rare would be problematic for microbial eukaryotes that depend on sex as a cohesive evolutionary force. We add to this view that if sex comes into play here for ciliates it would be needed to reduce deleterious mutations (Bell 1988; Kondrashov 1993; Maynard Smith 1978), a process that is especially important in the small populations that would be found in the rare biosphere (but see Weisse 2014). The need for sex to shuffle alleles around to allow for adaption to changing environments (Hamilton 2001; Hamilton et al. 1990; Morran et al. 2011), though, would be reduced as ciliates and other microbial eukaryotes could just lay low and brace themselves until conditions fare better for their current allelic combinations. RARITY IGNORED Rarity began to move from a focus to being ignored, when the rare biosphere was shown to be largely composed of €nemann a long queue of errors (Behnke et al. 2011; Ju et al. 2013; Kunin et al. 2010). For example, Behnke et al. (2011) established a mock community composed of cloned DNA from 24 different microbial eukaryotes, nine of which were ciliates. When these clones were sequenced with Roche/454, they uncovered not only their 24 references in their resulting reads, but also they found over 2,000 OTUs grouped at 100% similarity. Even when grouping the reads at 97% similarity they found 44 OTUs, nearly doubling the number of true morphotypes in the mock community. And when Behnke et al. (2011) removed the singletons, they still found 462 OTUs at 100% similarity. Most of the errors producing these spurious OTUs were due to homopolymers. They concluded that in the absence of using methods that attempt to remove these errors, HTS studies aimed at uncovering environmental microbial eukaryotic diversity should ignore the single singletons (or the rare species). If errors are removed with various error-correcting algorithms, though, you can obtain variable results that can change the taxonomic identities of the OTUs, and greatly limit biodiversity estimates (Coissac et al. 2012; Gaspar and Thomas 2013; Santoferrara et al. 2014). What to do with the rare reads or OTUs that could potentially be composed of errors? That depends on which type of question you want to ask. If you want to ask community-comparative questions, then including rare amplicons OTUs or not can have little effect on some of the comparative statistics that are largely driven by the most abundant species (Caporaso et al. 2011; Gobet et al. 2010). However, it may be better to lose “informative” reads than to introduce potential errors by including only the abundant amplicons—although some diversity will be missed. If you decided to analyze only the abundant OTUs, then you would not be able to use some of the standard statistics like Chao1 (Chao 1984) and ACE (Chao and Lee 1992) since these depend on the number of single singletons (Gihring et al. 2012).
Our ability to measure in environmental samples which species are actually abundant, and which ones should be retained for community-comparative questions, is questionable. This was demonstrated in a study by Stoeck et al. (2014), who compared ciliate abundances in the mountain lake Piburgersee, Austria, with morphological (live and silver-stained) and Roche/454 sequencing approaches. They found not much agreement between morphological and molecular abundances; e.g., Urotricha sp. accounted for 24.5% of the morphological data, but accounted for merely 0.09% of the molecular data; while Pseudomonilicaryon and Orthoamphisiella, which were most abundant in the molecular data, were not recovered with morphology. A similar situation in which abundance estimates drastically differed was found for ciliates and dinoflagellates when Medinger et al. (2010) sampled Lake Fuschlsee, Austria, and when Egge et al. (2013) evaluated a mock community of haptophytes. The incongruence between morphological- and molecular-based abundance estimates may, in part, be due to high variability in SSU-rDNA copy number among species. As pointed out by Prescott (1994) and Riley and Katz (2001), differential processing of macronuclear chromosomes can lead to hundreds of DNA copies in some taxa (e.g., Colpodea, Litostomatea, Prostomatea, and Oligohymenophorea), while leading to 1,000s of copies in other taxa (e.g., Phyllopharyngea and Spirotrichea). Such that, if you sample a freshwater pond morphologically you might find one Halteria grandinella (Spirotrichea) for every 10 Coleps hirtus (Prostomatea), but molecular estimates could tilt toward a 1:1 abundance pattern. Highly variable SSUrDNA copy number can also be due to variable in cell size among species (Zhu et al. 2005). Even within ciliates, the size spectrum of cells spans several orders of magnitude (small scuticocilates compared to the large Stentor), and macronuclei themselves may likewise be highly variable in size and number. Incongruence among abundance estimates may also be due to limitations of the molecular methods, primer ambiguities (Stoeck et al. 2006) and preferential PCR-amplifications of individual DNA templates (Wintzingerode et al. 1997). These purious abundance estimates of ciliates, and other microbial eukaryotes, from environmental molecular methods will affect abundancebased statistics of alpha- and beta-diversity (Haegeman et al. 2013); a preliminary solution to this problem may be the use of incidence-based statistics (presence/absence data) for more robust analyses of environmental HTS data. If you are interested in asking novel-diversity questions, then keeping rare OTUs is important as they are not all the result of sequencing errors (Berney et al. 2013; Br ate et al. 2010; Dunthorn et al. 2014; Lecroq et al. 2011; Orsi et al. 2011). Since most of the common species have already been detected and described in morphological and Sanger sequencing studies, most of the undiscovered diversity is likely to be rare. For example, in a study of six European off-shore marine sites using Roche/454 sequencing of planktonic and sediment samples, Dunthorn et al. (2014) found 87,724 ciliate reads. Of these, 523 reads blasted to the Colpodea, and grouped into 22 OTUs
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at 98% similarity. Nine of these OTUs contained just one read, and 10 contained two to four reads. These rare OTUs could, as suggested by Behnke et al. (2011) and other studies, be treated as mere errors and ignored. But when these rare OTUs were placed into an alignment of full-length Sanger sequences, most of their inferred relationships were very close to already known taxa. These rare marine OTUs, which would otherwise have been discarded in community-comparative studies, provide an alternative view that rather than being primarily terrestrial and freshwater as commonly thought (Foissner 1993; Lynn 2008), the Colpodea also includes multiple marine clades (Dunthorn et al. 2014). Keeping rare OTUs is also important if you want to ask ecosystem-functioning questions. Specifically, are the functional ecological traits hidden in the rare biosphere redundant from what is already known and present in the abundant taxa? If so, the rare biosphere would be responsible for resilience and stability of ecosystems under changing global conditions (Caron and Countway 2009; Dawson and Hagen 2009; Sogin et al. 2006; Stoeck and Epstein 2009); that is, the system can pivot among taxa with no change in the ecosystem. If there is no or only little functional trait redundancy in the rare biosphere, then ecosystem functioning would change drastically with community reassembly due to changes in abundances. Such a knowledge is crucial if we are to understand reactions of ecosystems to disturbances and environmental change. CONCLUSION The massive extent of rarity in ciliates, and other microbial eukaryotes, was largely shown with HTS methodologies, and results point to much diversity that was missed in previous morphological and Sanger sequencing studies. However, molecules by themselves may not be the best approach to get at rarity in these microbes, since they can lead to spurious abundance estimates. A counter-balance with morphology is therefore needed when possible to more accurately estimate which ciliates are abundant and which are rare in environmental samples. ACKNOWLEDGMENTS We thank George McManus, Roberto Docampo, and an anonymous reviewer for helpful suggestions and comments. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, grant # DU1319/1-1) to M.D., DFG (grant # STO414/3-1) to T.S., and U.S. National Science Foundation (DEB grant # 1136580) to J.C. LITERATURE CITED Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W. & Huse, S. M. 2009. A method for studying protistian diversity using massively parallel sequencing of V9 hypervariable region of small-subunit ribosomal RNA genes. PLoS ONE, 4:7. Behnke, A., Engel, M., Christen, R., Nebel, M., Kleln, R. R. & Stoeck, T. 2011. Depicting more accurate pictures of protistan
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