The photic-aphotic divide is a strong ecological and evolutionary force determining the distribution of ciliates (Alveolata, Ciliophora) in the ocean.

The bulk of knowledge on marine ciliates is from shallow and/or sunlit waters. We studied ciliate diversity and distribution across epi- and mesopelagic oceanic waters, using DNA metabarcoding and phylogeny-based metrics. We analyzed sequences of the 18S rRNA gene (V4 region) from 369 samples collected at 12 depths (0-1000 m) at the Bermuda Atlantic Time-series Study site of the Sargasso Sea (North Atlantic) monthly for 3 years. The comprehensive depth and temporal resolutions analyzed led to three main findings. First, there was a gradual but significant decrease in alpha-diversity (based on Faith's phylogenetic diversity index) from surface to 1000-m waters. Second, multivariate analyses of beta-diversity (based on UniFrac distances) indicate that ciliate assemblages change significantly from photic to aphotic waters, with a switch from Oligotrichea to Oligohymenophorea prevalence. Third, phylogenetic placement of sequence variants and clade-level correlations (EPA-ng and GAPPA algorithms) show Oligotrichea, Litostomatea, Prostomatea, and Phyllopharyngea as anti-correlated with depth, while Oligohymenophorea (especially Apostomatia) have a direct relationship with depth. Two enigmatic environmental clades include either prevalent variants widely distributed in aphotic layers (the Oligohymenophorea OLIGO5) or subclades differentially distributed in photic versus aphotic waters (the Discotrichidae NASSO1). These results settle contradictory relationships between ciliate alpha-diversity and depth reported before, suggest functional changes in ciliate assemblages from photic to aphotic waters (with the prevalence of algivory and mixotrophy vs. omnivory and parasitism, respectively), and indicate that contemporary taxon distributions in the vertical profile have been strongly influenced by evolutionary processes. Integration of DNA sequences with organismal data (microscopy, functional experiments) and development of databases that link these sources of information remain as major tasks to better understand ciliate diversity, ecological roles, and evolution in the ocean.

The Mixoplankton Database (MDB): Diversity of photo-phago-trophic plankton in form, function, and distribution across the global ocean.

Protist plankton are major members of open-water marine food webs. Traditionally divided between phototrophic phytoplankton and phagotrophic zooplankton, recent research shows many actually combine phototrophy and phagotrophy in the one cell; these protists are the "mixoplankton." Under the mixoplankton paradigm, "phytoplankton" are incapable of phagotrophy (diatoms being exemplars), while "zooplankton" are incapable of phototrophy. This revision restructures marine food webs, from regional to global levels. Here, we present the first comprehensive database of marine mixoplankton, bringing together extant knowledge of the identity, allometry, physiology, and trophic interactivity of these organisms. This mixoplankton database (MDB) will aid researchers that confront difficulties in characterizing life traits of protist plankton, and it will benefit modelers needing to better appreciate ecology of these organisms with their complex functional and allometric predator-prey interactions. The MDB also identifies knowledge gaps, including the need to better understand, for different mixoplankton functional types, sources of nutrition (use of nitrate, prey types, and nutritional states), and to obtain vital rates (e.g. growth, photosynthesis, ingestion, factors affecting photo' vs. phago' -trophy). It is now possible to revisit and re-classify protistan "phytoplankton" and "zooplankton" in extant databases of plankton life forms so as to clarify their roles in marine ecosystems.

Genome architecture used to supplement species delineation in two cryptic marine ciliates.

The purpose of this study is to determine which taxonomic methods can elucidate clear and quantifiable differences between two cryptic ciliate species, and to test the utility of genome architecture as a new diagnostic character in the discrimination of otherwise indistinguishable taxa. Two cryptic tintinnid ciliates, Schmidingerella arcuata and Schmidingerella meunieri, are compared via traditional taxonomic characters including lorica morphometrics, ribosomal RNA (rRNA) gene barcodes and ecophysiological traits. In addition, single-cell 'omics analyses (single-cell transcriptomics and genomics) are used to elucidate and compare patterns of micronuclear genome architecture between the congeners. The results include a highly similar lorica that is larger in S. meunieri, a 0%-0.5% difference in rRNA gene barcodes, two different and nine indistinguishable growth responses among 11 prey treatments, and distinct patterns of micronuclear genomic architecture for genes detected in both ciliates. Together, these results indicate that while minor differences exist between S. arcuata and S. meunieri in common indices of taxonomic identification (i.e., lorica morphology, DNA barcode sequences and ecophysiology), differences exist in their genomic architecture, which suggests potential genetic incompatibility. Different patterns of micronuclear architecture in genes shared by both isolates also enable the design of species-specific primers, which are used in this study as unique "architectural barcodes" to demonstrate the co-occurrence of both ciliates in samples collected from a NW Atlantic estuary. These results support the utility of genomic architecture as a tool in species delineation, especially in ciliates that are cryptic or otherwise difficult to differentiate using traditional methods of identification.

Molecular signature characters complement taxonomic diagnoses: A bioinformatic approach exemplified by ciliated protists (Ciliophora, Oligotrichea).

Traditionally, taxa following the botanical or zoological codes of nomenclature are diagnosed mainly by morphological characters, although integrative taxonomy advocates including additional features. While many taxonomic studies include DNA sequence analyses, a systematic integration of diagnostic molecular characters (signature characters) is still rare. Here, we suggest a practical guideline for the detection and evaluation of signature characters that provides the means necessary to complement diagnoses and facilitates identifications. The guideline comprises generally applicable criteria exemplified by a case study on an ecologically important group of planktonic protists, the Oligotrichea. The detection of signature characters and their discrete states in multiple sequence alignments is facilitated by the recently developed tool DeSignate. Moreover, we introduce a novel bioinformatic approach to test the influence of different alignment programs on the consistency of signature characters. Our workflow enabled detection of consensus signature characters for most tested taxa and inclusion of such characters in the diagnoses of three orders, eight families, and two genera in the Oligotrichea. The suggested approach is a step towards an integrative taxonomy linking reliable molecular sequence data to organisms' traits.

The importance of type species and their correct identification: A key example from tintinnid ciliates (Alveolata, Ciliophora, Spirotricha).

Types and the corresponding rules in the International Code of Zoological Nomenclature are crucial for taxonomy and are meant to provide nomenclatural stability. In the case of neotypification, especially diligent taxonomic work is required to retain continuity. In the present communication, we first outline the main principles of typification and neotypification. We then discuss a critical case, using a current example from the marine planktonic tintinnid genus Tintinnopsis Stein, 1867 (Alveolata, Ciliophora). This diverse and ubiquitous genus is nonmonophyletic, but its revision and the erection of new related genera is currently prevented by the uncertain affiliation of its type species.

Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes.

BACKGROUND: The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods. RESULTS: Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa. CONCLUSION: The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.

Combined Genome and Transcriptome Analyses of the Ciliate Schmidingerella arcuata (Spirotrichea) Reveal Patterns of DNA Elimination, Scrambling, and Inversion.

Schmidingerella arcuata is an ecologically important tintinnid ciliate that has long served as a model species in plankton trophic ecology. We present a partial micronuclear genome and macronuclear transcriptome resource for S. arcuata, acquired using single-cell techniques, and we report on pilot analyses including functional annotation and genome architecture. Our analysis shows major fragmentation, elimination, and scrambling in the micronuclear genome of S. arcuata. This work introduces a new nonmodel genome resource for the study of ciliate ecology and genomic biology and provides a detailed functional counterpart to ecological research on S. arcuata.

EukRef: Phylogenetic curation of ribosomal RNA to enhance understanding of eukaryotic diversity and distribution.

Environmental sequencing has greatly expanded our knowledge of micro-eukaryotic diversity and ecology by revealing previously unknown lineages and their distribution. However, the value of these data is critically dependent on the quality of the reference databases used to assign an identity to environmental sequences. Existing databases contain errors and struggle to keep pace with rapidly changing eukaryotic taxonomy, the influx of novel diversity, and computational challenges related to assembling the high-quality alignments and trees needed for accurate characterization of lineage diversity. EukRef (eukref.org) is an ongoing community-driven initiative that addresses these challenges by bringing together taxonomists with expertise spanning the eukaryotic tree of life and microbial ecologists, who use environmental sequence data to develop reliable reference databases across the diversity of microbial eukaryotes. EukRef organizes and facilitates rigorous mining and annotation of sequence data by providing protocols, guidelines, and tools. The EukRef pipeline and tools allow users interested in a particular group of microbial eukaryotes to retrieve all sequences belonging to that group from International Nucleotide Sequence Database Collaboration (INSDC) (GenBank, the European Nucleotide Archive [ENA], or the DNA DataBank of Japan [DDBJ]), to place those sequences in a phylogenetic tree, and to curate taxonomic and environmental information for the group. We provide guidelines to facilitate the process and to standardize taxonomic annotations. The final outputs of this process are (1) a reference tree and alignment, (2) a reference sequence database, including taxonomic and environmental information, and (3) a list of putative chimeras and other artifactual sequences. These products will be useful for the broad community as they become publicly available (at eukref.org) and are shared with existing reference databases.

EukRef-Ciliophora: a manually curated, phylogeny-based database of small subunit rRNA gene sequences of ciliates.

High-throughput sequencing (HTS) surveys, among the most common approaches currently used in environmental microbiology, require reliable reference databases to be correctly interpreted. The EukRef Initiative (eukref.org) is a community effort to manually screen available small subunit (SSU) rRNA gene sequences and produce a public, high-quality and informative framework of phylogeny-based taxonomic annotations. In the context of EukRef, we present a database for the monophyletic phylum Ciliophora, one of the most complex, diverse and ubiquitous protist groups. We retrieved more than 11 500 sequences of ciliates present in GenBank (28% from identified isolates and 72% from environmental surveys). Our approach included the inference of phylogenetic trees for every ciliate lineage and produced the largest SSU rRNA tree of the phylum Ciliophora to date. We flagged approximately 750 chimeric or low-quality sequences, improved the classification of 70% of GenBank entries and enriched environmental and literature metadata by 30%. The performance of EukRef-Ciliophora is superior to the current SILVA database in classifying HTS reads from a global marine survey. Comprehensive outputs are publicly available to make the new tool a useful guide for non-specialists and a quick reference for experts.

Dartintinnus alderae n. g., n. sp., a Brackish Water Tintinnid (Ciliophora, Spirotrichea) with Dual-ended Lorica Collapsibility.

A tintinnid ciliate isolated from waters of the Thames River (Connecticut, USA) is described through combined in vivo observation, protargol impregnation, and phylogenetic analysis. The novel genus Dartintinnus and its type species, D. alderae are distinct from established tintinnid taxa by a lorica that collapses on both anterior and posterior ends. Dartintinnus is placed in the family Eutintinnidae based on a hyaline, elongated lorica opened at both ends, a ciliary pattern including a ventral kinety, at least one dorsal kinety, and right, left and lateral fields, and a sister relationship with Eutintinnus in gene trees. Main differences between D. alderae and Eutintinnus species include a 5.5-6.5% divergence in the small subunit rRNA gene, the geometry of the lorica (resembling an isosceles tetrahedron when collapsed vs. a cylinder, respectively), the number of macronuclear nodules (two vs. four), and the number of dorsal kineties (one vs. usually two). Considering the features of the new genus, we improve the diagnosis of the family Eutintinnidae, including the presence of a lateral ciliary field that had been overlooked in some Eutintinnus species. This work exemplifies the potential for novel diversity, even in these relatively well-studied protists, and the importance of an integrated approach for the description of tintinnid taxa.

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.

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.

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.

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.

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.

Discrimination of closely related species in tintinnid ciliates: new insights on crypticity and polymorphism in the genus Helicostomella.

This study focuses on the utility of molecular markers for the discrimination of closely related species in tintinnid ciliates. We analyzed the ecologically important genus Helicostomella by sequencing part of the large-subunit rDNA (LSU rDNA) and the 5.8S rDNA combined with the internally transcribed spacer regions 1 and 2 (5.8S rDNA-ITS) from forty-five individuals collected in NW and SW Atlantic waters and after culturing. Although all described Helicostomella species represent a continuum of morphologies, forms with shorter or longer loricae would correspond to different species according to previous molecular data. Here we observed that long forms show both crypticity (i.e. two almost identical long forms with different DNA sequences) and polymorphism (i.e. some long forms develop significantly shorter loricae after culturing). Reviewing all available tintinnid sequences, we found that 1) three Helicostomella clusters are consistent with different species from a molecular perspective, although these clusters are neither clearly differentiated by their loricae nor unambiguously linked to described species, 2) Helicostomella is closely related (probably to the family or genus level) to four "Tintinnopsis-like" morphospecies, and 3) if considered separately, neither LSU rDNA nor 5.8S rDNA-ITS completely discriminate closely related species, thus supporting the use of multi-gene barcodes for tintinnids.

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.

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.

Pyrosequencing for assessing diversity of eukaryotic microbes: analysis of data on marine planktonic ciliates and comparison with traditional methods.

Assessing microbial diversity requires analysis of all three domains of life, including eukaryotic microbes. We examined the diversity of two ecologically important clades of microbial eukaryotes, ciliates in the subclasses Oligotrichia and Choreotrichia (class Spirotrichea), by comparing pyrosequencing to Sanger-sequenced clone libraries and microscopy. Using samples from a large temperate estuary (Long Island Sound, USA), we gained three major insights. First, richness estimates varied by up to one order of magnitude either using different criteria for pyrosequence processing or among pyrosequencing, cloning and microscopy, while taxon identification was almost always coherent. Error-correcting algorithms for pyrosequences ('denoising') reduced discrepancies in richness but also removed known morphospecies from the data. Second, although most of the pyrosequenced operational taxonomic units (OTUs) were distributed within known orders and families, we found evidence of a previously uncharacterized or unknown clade even in these ciliate lineages that have a rich history of morphological description. Third, pyrosequencing allowed the detection of OTUs that were either dominant or extremely rare in different samples. Our findings confirm the potential of pyrosequencing for quantifying microbial diversity, but also highlight the importance of careful evaluation of pyrosequence processing for using this method to address ecological questions.

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.