Exploring confocal laser scanning microscopy (CLSM) and fluorescence staining as a tool for imaging and quantifying traces of marine microbioerosion and their trace-making microendoliths.

Microscopic organisms that penetrate calcareous structures by actively dissolving the carbonate matrix, namely microendoliths, have an important influence on the breakdown of marine carbonates. The study of these microorganisms and the bioerosion traces they produce is crucial for understanding the impact of their bioeroding activity on the carbonate recycling in environments under global climate change. Traditionally, either the extracted microendoliths were studied by conventional microscopy or their traces were investigated using scanning electron microscopy (SEM) of epoxy resin casts. A visualisation of the microendoliths in situ, that is within their complex microbioerosion structures, was previously limited to the laborious and time-consuming double-inclusion cast-embedding technique. Here, we assess the applicability of various fluorescence staining methods in combination with confocal laser scanning microscopy (CLSM) for the study of fungal microendoliths in situ in partly translucent mollusc shells. Among the tested methods, specific staining with dyes against the DNA (nuclei) of the trace making organisms turned out to be a useful and reproducible approach. Bright and clearly delineated fluorescence signals of microendolithic nuclei allow, for instance, a differentiation between abandoned and still populated microborings. Furthermore, infiltrating the microborings with fluorescently stained resin seems to be of great capability for the visualisation and quantification of microbioerosion structures in their original spatial orientation. Potential fields of application are rapid assessments of endolithic bio- and ichnodiversity and the quantification of the impact of microendoliths on the overall calcium carbonate turnover. The method can be applied after CLSM of the stained microendoliths and retains the opportunity for a subsequent investigation of epoxy casts with SEM. This allows a three-fold approach in studying microendoliths in the context of their microborings, thereby fostering the integration of biological and ichnological aspects of microbial bioerosion.

Bioerosion describes the process of active erosion of hard substrates induced by the activity of living organisms. Beside numerous marine macroscopic bioeroding organisms such as sponges, annelids or bivalves, there is an astonishing 'hidden diversity' of microscopic bioeroding organisms which produce minute tunnels and chambers, for example in calcareous shells and skeletons of other marine organisms. These so-called microendoliths belong to bacteria, microalgae, foraminiferans, or fungi. Due to their lifestyle hidden inside the hard substrate, scientific investigation is often laborious and involves complex preparation techniques, electron microscopy, or even nano-computed tomography. Photo-autotrophic microendoliths (eg cyanobacteria and algae) have been studied with fluorescence microscopy using autofluorescence properties, for example of their chloroplasts. However, microendoliths of aphotic depths, mostly of fungal origin, do not show autofluorescence. With the present study we test different fluorescent dyes staining the microbioeroders 'in situ', that is, inside their microscopic tunnels, and visualise them using three-dimensional confocal laser scanning microscopy (CLSM). Very good results have been obtained with the dye Sybr Green I that stains DNA molecules and thereby the cell nuclei of the microendoliths. This method can be used, for instance, to measure the infestation rate of a given substrate by discriminating between abandoned microborings and those still inhabited by microendoliths. Another approach that was successfully tested in the course of the present study was the infiltration of the cleaned microborings with resin that was previously mixed with the fluorescent dye Safranin-O. The datasets obtained with the CLSM were used to reconstruct 3D-surface models of the microborings of three different microendoliths. Such models can be used to analyse the original spatial arrangement inside the hard substrate and to measure exact volumes. The resulting possibility to make exact quantifications is of high value for future investigations that focus on the role and proportion of microbioerosion in the (re)cycling of marine carbonates.

A new species of Cephalodasys (Gastrotricha, Macrodasyida) from the Caribbean Sea with a determination key to species of the genus.

A new marine gastrotrich species of the genus Cephalodasys is described from shallow sublittoral coralline sand sampled between Lee Stocking Island and Norman's Pond Cay (Exuma Cays), Bahamas. Cephalodasys interinsularis n. sp. reaches a body length of 471 µm and is characterized by a new combination of characters including six total anterior adhesive tubes and five pairs of ventrolateral adhesive tubes. The new species is morphologically similar to C. swedmarki but can be distinguished by the different number of anterior adhesive tubes, the spatial arrangement of the ventrolateral adhesive tubes, and a shorter pharynx. We provide an updated diagnosis of the genus and a determination key to all known species of Cephalodasys. C. interinsularis n. sp. is the third known species of Cephalodasys from the Caribbean marine province.

The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.

Spatially structured populations with a low level of cryptic diversity in European marine Gastrotricha.

Species of the marine meiofauna such as Gastrotricha are known to lack dispersal stages and are thus assumed to have low dispersal ability and low levels of gene flow between populations. Yet, most species are widely distributed, and this creates a paradox. To shed light on this apparent paradox, we test (i) whether such wide distribution may be due to misidentification and lumping of cryptic species with restricted distributions and (ii) whether spatial structures exist for the phylogeography of gastrotrichs. As a model, we used the genus Turbanella in NW Europe. DNA taxonomy using a mitochondrial and a nuclear marker supports distinctness of four traditional species (Turbanella ambronensis, T. bocqueti, T. mustela and T. cornuta) and provides evidence for two cryptic species within T. hyalina. An effect of geography on the within-species genetic structure is indeed present, with the potential for understanding colonization processes and for performing phylogeographic inference from microscopic animals. On the other hand, the occurrence of widely distributed haplotypes indicates long-distance dispersal as well, despite the assumed low dispersal ability of gastrotrichs.

Ultrastructure of the protonephridial system in Neodasys chaetonotoideus (Gastrotricha: Chaetonotida) and in the ground pattern of Gastrotricha.

The taxon Neodasys has a basal position within Gastrotricha. This makes it very interesting for phylogenetic considerations in this group. To complete the reconstruction of the nephridial system in the stem species of Gastrotricha started earlier, we have studied the whole protonephridial system of Neodasys chaetonotoideus by means of complete sets of ultrathin sections and TEM. In many characters, protonephridia of N. chaetonotoideus resemble those of macrodasyidan gastrotrich species. For example, each of the six protonephridia, arranged in three pairs, consists of three distinct cells that constitute the continuous protonephridial lumen. Especially, the terminal cell of the protonephridia of N. chaetonotoideus shows a striking pattern: The perforation of the filter region is a meandering cleft that is continuous with the seam of the enfolded lumen of that cell. With the results presented here and that of former TEM studies, we give a comprehensive idea of the excretory organs in the ground pattern of Gastrotricha. Moreover, we can elaborate on the hypothesized protonephridial system in the stem species of Bilateria. We suggest that a meandering filtration cleft is a feature of the ground pattern of the Bilateria.