Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes—the 22 autosomes and the X and Y sex chromosomes—can be stained or “painted” with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Background: Molecular characters have been added in integrative taxonomic approaches in recent years. Nevertheless, taxon diagnoses are still widely restricted to morphological characters. The inclusion of molecular characters into taxon diagnoses is not only hampered by problems, such as their definition and the designation of their positions in a reference alignment, but also by the technical effort. Results: DeSignate is a tool for character-based taxon diagnoses that includes a novel ranking scheme. It detects and classifies individual and combined signature characters (diagnostic molecular characters) based on so-called character state vectors. An intuitive web application guides the user through the analysis process and provides the results at a glance. Further, formal definitions and a uniform terminology of characters are introduced. Conclusions: DeSignate facilitates the inclusion of diagnostic molecular characters and their positions to complement taxon diagnoses. Compared to previous solutions, the tool simplifies the workflow and improves reproducibility and traceability of the results.

Tintinnid ciliates have traditionally been described and classified exclusively based on their lorica features. Although information on the cell characters is urgently needed for a natural classification, more molecular than cytological data has been accumulated over recent years. Apparently, the tintinnids developed in the marine environment and entered freshwater several times independently. Typical freshwater tintinnids belong to the genera Tintinnidium and Membranicola. The species are comparatively well‐known regarding their morphology and characterised by two unusual de novo originating ciliary rows, the ventral organelles. In contrast, the cell features in the marine/brackish Tintinnidium species, specifically their somatic ciliary patterns, are insufficiently known or not known at all. Therefore, the morphology of a common marine/brackish representative, Tintinnidium mucicola, is redescribed based on live observation and protargol‐stained material. Furthermore, biogeographical and autecological data of the species are compiled from literature and own records. The phylogenetic relationships of T. mucicola are inferred and the diversity of the family Tintinnidiidae is assessed from 18S rDNA sequences. The study shows that T. mucicola is not only molecularly distinct, but also characterised by many plesiomorphic features, for instance, it does not possess a verifiable homologue to the ventral organelles. Hence, a new genus, Antetintinnidium nov. gen., is established for T. mucicola. The new insights into the diversity of Tintinnidiidae shed light on the early evolution of tintinnids and might provide clues on their adaptions to freshwater.

Unraveling geographic distribution patterns of planktonic protists is a central goal in marine microbial ecology. Using a novel combination of recently developed phylogenetic and network analyses on a V4 18S rDNA metabarcoding dataset, we here analyzed the genetic diversity of marine planktonic ciliate communities in Chinese and European coastal waters. Thereby, our approach provided an unprecedented perspective on geographic patterns inferred from ciliate genetic diversity and accomplished a very fine resolution down to single nucleotides within operational taxonomic units (OTUs). While most OTUs (87%) exclusively contained sequences of either Chinese or European origin, those OTUs detected in both regions comprised the vast majority of reads (84%). Phylogenetic analyses of OTUs belonging to the same taxon revealed genetically distinct clades that were geographically restricted to either Chinese or European coastal waters. The detection of signature nucleotides emphasized this genetic distinction of Chinese and European clades. Second-level clustering of OTUs and reference sequences in two selected taxa (the oligotrichid Spirotontonia and the tintinnid Tintinnidium) revealed the presence of several potentially new species or ones lacking genetic reference data. Geographic patterns were also discovered by network analyses within 700 widespread and abundant OTUs; in 77 of these OTUs, European and Chinese sequences formed significantly assortative groups. These assortative groupings indicated a higher genetic similarity among sequences from the same region than between sequences from different regions. Our results demonstrate that detailed analyses of metabarcoding data down to single nucleotide differences expand our perception of geographical distribution patterns and provide insights into historic and ongoing effective dispersal in protists. The congruent discovery of geographic patterns at different levels of resolution (between and within OTUs) suggests that cosmopolitan distribution in marine planktonic ciliates is less common than previously postulated.

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