The phylum Ciliophora plays important roles in a wide range of biological studies. However, the evolutionary relationships of many groups remain unclear due to a lack of sufficient molecular data. In this study, molecular dataset was expanded with representatives from 55 orders and all major lineages. The main findings are: (1) 14 classes were recovered including one new class, Protocruziea n. cl.; (2) in addition to the two main branches, Postciliodesmatophora and Intramacronucleata, a third branch, the Mesodiniea, is identified as being basal to the other two subphyla; (3) the newly defined order Discocephalida is revealed to be a sister clade to the euplotids, strongly suggesting the separation of discocephalids from the hypotrichs; (4) the separation of mobilids from the peritrichs is not supported; (5) Loxocephalida is basal to the main scuticociliate assemblage, whereas the thigmotrichs are placed within the order Pleuronematida; (6) the monophyly of classes Phyllopharyngea, Karyorelictea, Armophorea, Prostomatea, Plagiopylea, Colpodea and Heterotrichea are confirmed; (7) ambiguous genera Askenasia, CyclotrichiumParaspathidium and Plagiocampa show close affiliation to the well known plagiopyleans; (8) validity of the subclass Rhynchostomatia is supported, and (9) the systematic positions of Halteriida and Linconophoria remain unresolved and are thus regarded as incertae sedis within Spirotrichea.

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN‐BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN‐BC proposes to populate The Ciliate Guide, an online database, with biodiversity‐related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: , and We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

Sequence-based approaches, such as analyses of ribosome DNA (rDNA) clone libraries and high-throughput amplicon sequencing, have been used extensively to infer evolutionary relationships and elucidate the biodiversity in microbial communities. However, recent studies demonstrate both rDNA copy number variation and intra-individual (intra-genomic) sequence variation in many organisms, which challenges the application of the rDNA-based surveys. In ciliates, an ecologically important clade of microbial eukaryotes, rDNA copy number and sequence variation are rarely studied. In the present study, we estimate the intraindividual small subunit rDNA (SSU rDNA) copy number and sequence variation in a wide range of taxa covering nine classes and 18 orders of the phylum Ciliophora. Our studies reveal that: (i) intra-individual sequence variation of SSU rDNA is ubiquitous in all groups of ciliates detected and the polymorphic level varies among taxa; (ii) there is a most common version of SSU rDNA sequence in each cell that is highly predominant and may represent the germline micronuclear template; (iii) compared with the most common version, other variant sequences differ in only 1-3 nucleotides, likely generated during macronuclear (somatic) amplification; (iv) the intra-cell sequence variation is unlikely to impact phylogenetic analyses; (v) the rDNA copy number in ciliates is highly variable, ranging from 10 3 to 10 6 , with the highest record in Stentor roeselii. Overall, these analyses indicate the need for careful consideration of SSU rDNA variation in analyses of the role of ciliates in ecosystems.

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In variational data assimilation, optimal ingestion of the observational data and optimal use of prior physical and statistical information involve the choice of numerous weighting, smoothing and tuning parameters which control the ltering and merging of divers sources of information. Generally these weights must be obtained from a partial and imperfect understanding of various sources of errors, and are frequently chosen by a combination of historical information, physical reasoning and trial and error. Generalized Cross Validation (GCV) has long been one of the methods of choice for choosing certain tuning, smoothing, regularization parameters in ill-posed inverse problems, smoothing and ltering problems. In theory, it is well suited for the adaptive choice of certain parameters that occur in variational objective analysis, and data assimilation problems that are mathematically equivalent to variational problems. The main drawback of the use of GCV in data assimilation problems was that matrix decompositions were apparently needed to compute the GCV estimates. This limited the application of GCV to data sets of the order of less than about 1000. Recently the randomized trace technique for computing the GCV estimates has been developed, and this makes the use of GCV feasible in essentially any variational problem which has an operating algorithm to produce estimates, given data. In this paper we demonstrate that the answers given by the randomized trace estimate are indistinguishable in a practical sense from those computed more exactly by traditional methods. Then we carry out an experiment to choose one of the main smoothing parameters () in the context of a variational objective analysis problem which is approximately solved by k iterations of a conjugate gradient algorithm. We show how the randomized trace technique can be used to obtain good values of both and k in this context. Finally, we describe how the method can be applied in operational-sized three and four dimensional variational data assimilation schemes, as well as in conjuction with a Kalman lter.

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Chromosome rearrangements occur in a variety of eukaryotic life cycles, including during the development of the somatic macronuclear genome in ciliates. Previous work on the phyllopharyngean ciliate Chilodonella uncinata revealed that macronuclear β-tubulin and protein kinase gene families share alternatively processed germ line segments nested within divergent regions. To study genome evolution in this ciliate further, we characterized two additional alternatively processed gene families from two cryptic species of the ciliate morphospecies C. uncinata: those encoding histidine acid phosphatase protein (Hap) and leishmanolysin family protein (Lei). Analyses of the macronuclear Hap and Lei sequences reveal that each gene family consists of three members in the macronucleus that are marked by identical regions nested among highly divergent regions. Investigation of the micronuclear Hap sequences revealed a complex pattern in which the three macronuclear sequences are derived either from a single micronuclear region or from a combination of this shared region recombined with additional duplicate micronuclear copies of Hap. We propose a model whereby gene scrambling evolves by gene duplication followed by partial and reciprocal degradation of the duplicate sequences. In this model, alternative processing represents an intermediate step in the evolution of scrambled genes. Finally, we speculate on the possible role of genome architecture in speciation in ciliates by describing what might happen if changes in alternatively processed loci occur in subdivided populations.