Luciana F. Santoferrara

Utility of Genetic Markers and Morphology for Species Discrimination within the Order Tintinnida (Ciliophora, Spirotrichea)

We evaluated the small- and large-subunit rDNA (SSU and LSU, respectively) for their ability to discriminate morphospecies of tintinnid ciliates. Multiple individuals from 29 morphospecies were identified according to microscopically-observed characteristics of the lorica, and then sequenced for both loci (21 new species for SSU and all of them new for LSU). Sequences from public databases were included in our analyses, and two hypervariable SSU regions (V4 and V9) were separately examined. Of the four regions, LSU is the most useful as a potential barcoding tool. It showed a gap in distances within and between species, and discriminated the maximum number of phylotypes (86% at 1% cut-off). SSU and V4 were less consistent, sometimes lumping together very distinctive morphospecies, even at the 1% level of sequence divergence. V9 was the least reliable marker in delimitating morphospecies. The agreement in sequences and morphology suggests that the lorica is useful for species discrimination, even in agglomerated forms. However, the observation of both genetically constant yet polymorphic groups of species, as well as similar morphospecies with divergent sequences, indicates that previous taxonomic schemes are complementary to the emerging molecular database.

Diversity of diversity: conceptual and methodological differences in biodiversity estimates of eukaryotic microbes as compared to bacteria

Recent advances such as high-throughput sequencing (HTS) have changed conceptions about the magnitude of diversity on Earth. This is especially true for microbial lineages, which have seen the discovery of great numbers of rare forms in places such as the human gut as well as diverse environments (e.g., freshwater, marine, and soil). Given the differences in perceptions of diversity for bacterial and eukaryotic microbes, including divergent species concepts, HTS tools used to eliminate errors and population-level variation in bacteria may not be appropriate for microbial eukaryotes and may eliminate valid species from the data. We discuss here how the nature of biodiversity varies among microbial groups and the extent to which HTS tools designed for bacteria are useful for eukaryotes.

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

Our knowledge on microbial biogeography depends on the way we define and study diversity. In contrast to most microbes, some protist lineages have conspicuous structures that allow comparisons of diversity concepts and measures—those based on molecules and those based on morphology. We analyzed a group of shell-bearing planktonic ciliates, the tintinnids, in a coast-to-ocean gradient using high-throughput sequencing and microscopy. First, we compared molecular operational taxonomic units (OTUs) and morphospecies in terms of assemblage composition, distribution and relationships with the environment. OTUs revealed potentially novel and rare taxa, while morphospecies showed clearer correlations with environmental factors, and both approaches coincided in supporting a coastal versus oceanic pattern. Second, we explored which processes influence assembly across the environmental gradient examined. Assemblage fluctuations were associated with significant distance–decay and changes in morphospecies size and prey proxies, thus suggesting niche partitioning as a key structuring mechanism. Our conclusion is that molecules and morphologies generally agreed, but they provided complementary data, the first revealing hidden diversity, and the latter making better connections between distribution patterns and ecological processes. This highlights the importance of linking genotypes and phenotypes (using multidisciplinary analyses and/or reliable databases of barcoded species), to understand the diversity, biogeography and ecological roles of microbes.

Beyond the “Code”: A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora)

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.

Updating Biodiversity Studies in Loricate Protists: The Case of the Tintinnids (Alveolata, Ciliophora, Spirotrichea)

Species determination is crucial in biodiversity research. In tintinnids, identification is based almost exclusively on the lorica, despite its frequent intraspecific variability and interspecific similarity. We suggest updated procedures for identification and, depending on the aim of the study, further steps to obtain morphological, molecular, and ecological data. Our goal is to help improving the collection of information (e.g. species re‐/descriptions and DNA barcodes) that is essential for generating a natural tintinnid classification and a reliable reference for environmental surveys. These suggestions are broadly useful for protistologists because they exemplify data integration, quality/effort compromise, and the need for scientific collaborations.

Phylogeny, classification and diversity of Choreotrichia and Oligotrichia (Ciliophora, Spirotrichea)

Ciliated protists in the subclasses Choreotrichia and Oligotrichia are major components of marine plankton. Despite their ecological relevance, there are uncertainties in their systematics and diversity. We retrieved and curated all the GenBank ribosomal DNA (rDNA) sequences from these groups, which were analyzed in two ways. The first approach was based on morphologically-identified sequences (including those of two families and six genera newly studied here by single-cell sequencing), and aimed at improving phylogenetic inferences using concatenated sequences of three rDNA loci. Based on phylogenetic and morphological support, we update the taxonomic classification of these subclasses into 23 families, including the re-established Favellidae. We also propose an informal naming system for incertae sedis taxa, namely Tintinnopsis and five related genera that are spread among eleven lineages. The second approach included unidentified environmental sequences, and was used to explore potentially novel diversity in these subclasses. Our results support high proportions of both synonyms in tintinnids and uncharacterized taxa in choreotrichids and oligotrichs. One previously unidentified, environmental clade is here linked to our new Leegaardiellidae sequences. Our curation of almost 4000 rDNA sequences exemplifies known issues of public repositories, and suggests caution in both the use and contribution to these unique resources for evolutionary and diversity studies.

Phylogeny of the order Tintinnida (Ciliophora, Spirotrichea) inferred from small- and large-subunit rRNA genes.

Concatenated sequences of small‐ and large‐subunit rRNA genes were used to infer the phylogeny of 29 species in six genera of Tintinnida. We confirmed previous results on the positions of major clusters and the grouping of various genera, including Stenosemella, the paraphyletic Tintinnopsis, the newly investigated Helicostomella, and some species of the polyphyletic Favella. Tintinnidium and Eutintinnus were found to be monophyletic. This study contributes to tintinnid phylogenetic reconstruction by increasing both the number of species and the range of genetic markers analyzed.

Unexpected biodiversity of ciliates in marine samples from below the photic zone

Marine microbial eukaryotes play critical roles in planktonic food webs and have been described as most diverse in the photic zone where productivity is high. We used high‐throughput sequencing (HTS) to analyse the spatial distribution of planktonic ciliate diversity from shallow waters (<30 m depth) to beyond the continental shelf (>800 m depth) along a 163 km transect off the coast of New England, USA. We focus on ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), as these taxa are major components of marine food webs. We did not observe the decrease of diversity below the photic zone expected based on productivity and previous analyses. Instead, we saw an increase of diversity with depth. We also observed that the ciliate communities assessed by HTS cluster by depth layer and degree of water column stratification, suggesting that community assembly is driven by environmental factors. Across our samples, abundant OTUs tend to match previously characterized morphospecies while rare OTUs are more often undescribed, consistent with the idea that species in the rare biosphere remain to be characterized by microscopy. Finally, samples taken below the photic zone also reveal the prevalence of two uncharacterized (i.e. lacking sequenced morphospecies) clades – clusters X1 and X2 – that are enriched within the nano‐sized fraction (2–10 μm) and are defined by deletions within the region of the SSU‐rDNA analysed here. Together, these data reinforce that we still have much to learn about microbial diversity in marine ecosystems, especially in deep‐waters that may be a reservoir for rare species and uncharacterized taxa.

De novo transcriptomes of a mixotrophic and a heterotrophic ciliate from marine plankton.

Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich Strombidinopsis sp., and their respective algal food using Illumina RNAseq. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We detected approximately 10,000 and 7,600 amino acid sequences for S. rassoulzadegani and Strombidinopsis sp., respectively. About half of these transcripts had significant BLASTP hits (E-value <10−6) against previously-characterized sequences, mostly from the model ciliate Oxytricha trifallax. Transcriptomes from both the mixotroph and the heterotroph species provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance and functioning in the transcriptome of this ciliate. While chloroplasts are known sources of reactive oxygen species (ROS), we found the same complement of antioxidant pathways in both ciliates, except for one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy.

Integrating dimensions of biodiversity in choreotrichs and oligotrichs of marine plankton

Choreotrichs and oligotrichs are the main ciliate groups in marine plankton, where they play major roles as trophic intermediaries. We have studied these groups with a variety of approaches to combine the three dimensions of biodiversity-taxonomy, genetics and function. Here we revisit our findings with an integrative perspective, and highlight future directions. In our studies, the correspondence between classical taxonomy (mostly based on morphology) and the increasingly available genetic data (DNA sequences) is examined at the individual, population, species, and assemblage levels. We use a combination of single-cell and environmental sequencing to quantify diversity, track distribution patterns, and explain biogeography processes. Comparativelly, we know little about how the morphological and genetic estimates of diversity relate to function, but we expect to better link these aspects by incorporating modern -omics approaches. For example, we have pioneered functional transcriptomic analyses in these groups by contrasting a heterotrophic choreotrich and a mixotrophic oligotrich. These data provide a tremendous resource to start building reference databases needed to measure differential expression of key functional genes, either experimentally or directly in the environment.