Exploring the effects of acid mine drainage on diatom teratology using geometric morphometry.

Metal pollution of aquatic habitats is a major and persistent environmental problem. Acid mine drainage (AMD) affects lotic systems in numerous and interactive ways. In the present work, a mining area (Roșia Montana) was chosen as study site, and we focused on two aims: (i) to find the set of environmental predictors leading to the appearance of the abnormal diatom individuals in the study area and (ii) to assess the relationship between the degree of valve outline deformation and AMD-derived pollution. In this context, morphological differences between populations of Achnanthidium minutissimum and A. macrocephalum, including normal and abnormal individuals, were evidenced by means of valve shape analysis. Geometric morphometry managed to capture and discriminate normal and abnormal individuals. Multivariate analyses (NMDS, PLS) separated the four populations of the two species mentioned and revealed the main physico-chemical parameters that influenced valve deformation in this context, namely conductivity, Zn, and Cu. ANOSIM test evidenced the presence of statistically significant differences between normal and abnormal individuals within both chosen Achnanthidium taxa. In order to determine the relative contribution of each of the measured physico-chemical parameters in the observed valve outline deformations, a PLS was conducted, confirming the results of the NMDS. The presence of deformed individuals in the study area can be attributed to the fact that the diatom communities were strongly affected by AMD released from old mining works and waste rock deposits.

European river typologies fail to capture diatom, fish, and macrophyte community composition.

Typology systems are frequently used in applied and fundamental ecology and are relevant for environmental monitoring and conservation. They aggregate ecosystems into discrete types based on biotic and abiotic variables, assuming that ecosystems of the same type are more alike than ecosystems of different types with regard to a specific property of interest. We evaluated whether this assumption is met by the Broad River Types (BRT), a recently proposed European river typology system, that classifies river segments based on abiotic variables, when it is used to group biological communities. We compiled data on the community composition of diatoms, fishes, and aquatic macrophytes throughout Europe and evaluated whether the composition is more similar in site groups with the same river type than in site groups of different river types using analysis of similarities, classification strength, typical species analysis, and the area under zeta diversity decline curves. We compared the performance of the BRT with those of four region-based typology systems, namely, Illies Freshwater Ecoregions, the Biogeographic Regions, the Freshwater Ecoregions of the World, and the Environmental Zones, as well as spatial autocorrelation (SA) classifications. All typology systems received low scores from most evaluation methods, relative to predefined thresholds and the SA classifications. The BRT often scored lowest of all typology systems. Within each typology system, community composition overlapped considerably between site groups defined by the types of the systems. The overlap tended to be the lowest for fishes and between Illies Freshwater Ecoregions. In conclusion, we found that existing broad-scale river typology systems fail to delineate site groups with distinct and compositionally homogeneous communities of diatoms, fishes, and macrophytes. A way to improve the fit between typology systems and biological communities might be to combine segment-based and region-based typology systems to simultaneously account for local environmental variation and historical distribution patterns, thus potentially improving the utility of broad-scale typology systems for freshwater biota.

DNA metabarcoding and morphological methods show complementary patterns in the metacommunity organization of lentic epiphytic diatoms.

Diatoms are important organisms in freshwater ecosystems due to their position as primary producers and therefore, analyzing their assemblages provides relevant information on ecosystem functioning. Diatoms have historically been identified based on morphological traits, which is time-consuming and requires well-trained specialists. Nevertheless, DNA barcoding offers an alternative approach to overcome some limitations of the morphological method. Here, we assess if both approaches are comparable methods to study patterns and mechanisms (including environmental filtering and dispersal limitation) of epiphytic diatom metacommunities using a comprehensive dataset from 22 Mediterranean ponds at different taxonomic resolutions. We used a fragment of rbcL barcode gene combined with High-Throughput Sequencing to infer diatom community composition. The overall degree of correspondence between both approaches was assessed by Procrustean rotation analysis and Procrustean randomization tests, whereas the role of local environmental variables and geographical distances was studied using a comprehensive combination of BIOENV, Mantel tests and distance-based redundancy analysis. Our results showed a relatively poor correspondence in the compositional variation of diatom metacommunity between both approaches. We speculate that the incompleteness of the reference database and the bioinformatics processing are the biases most likely affecting the molecular approach, whereas the limited counting effort and the presence of cryptic species are presumably the major biases related with the morphological method. On the other hand, variation in diatom community composition detected with both approaches was strongly related to the environmental template, which may be related with the narrow community-environment relationships in diatoms. Nevertheless, we found no significant relationship between compositional variation and geographical distances. Overall, our work shows the complementary nature of both approaches and highlights the importance of DNA metabarcoding to address empirical research questions of community ecology in freshwaters, especially once the reference databases include most genotypes of occurring taxa and bioinformatics biases are overcome.

Oblique illumination in microscopy: A quantitative evaluation.

Many biological objects are barely distinguished with the brightfield microscope because they appear transparent, translucent and colourless. One simple way to make such specimens visible without compromising contrast and resolution is by controlling the amount and the directionality of the illumination light. Oblique illumination is an old technique described by many scientists and microscopists that however has been largely neglected in favour of other alternative methods. Oblique lighting (OL) is created by illuminating the sample by only a portion of the light coming from the condenser. If properly used it can improve the resolution and contrast of transparent specimens such as diatoms. In this paper a quantitative evaluation of OL in brigthfield microscopy is presented. Several feature descriptors were selected for characterising contrast and sharpness showing that in general OL provides better performance for distinguishing minute details compared to other lighting modalities. Oblique lighting is capable to produce directionally shadowed differential contrast images allowing to observe phase details in a similar way to differential contrast images (DIC) but at lower cost. The main advantage of OL is that the resolution of the light microscope can be increased by effectively doubling the angular aperture. OL appears as a cost-effective technique both for the amateur and professional scientist that can be used as a replacement of DIC or phase contrast when resources are scarce.

Low-cost oblique illumination: an image quality assessment.

We study the effectiveness of several low-cost oblique illumination filters to improve overall image quality, in comparison with standard bright field imaging. For this purpose, a dataset composed of 3360 diatom images belonging to 21 taxa was acquired. Subjective and objective image quality assessments were done. The subjective evaluation was performed by a group of diatom experts by psychophysical test where resolution, focus, and contrast were assessed. Moreover, some objective nonreference image quality metrics were applied to the same image dataset to complete the study, together with the calculation of several texture features to analyze the effect of these filters in terms of textural properties. Both image quality evaluation methods, subjective and objective, showed better results for images acquired using these illumination filters in comparison with the no filtered image. These promising results confirm that this kind of illumination filters can be a practical way to improve the image quality, thanks to the simple and low cost of the design and manufacturing process.

Disentangling diatom species complexes: does morphometry suffice?

Accurate taxonomic resolution in light microscopy analyses of microalgae is essential to achieve high quality, comparable results in both floristic analyses and biomonitoring studies. A number of closely related diatom taxa have been detected to date co-occurring within benthic diatom assemblages, sharing many morphological, morphometrical and ecological characteristics. In this contribution, we analysed the hypothesis that, where a large sample size (number of individuals) is available, common morphometrical parameters (valve length, width and stria density) are sufficient to achieve a correct identification to the species level. We focused on some common diatom taxa belonging to the genus Gomphonema. More than 400 valves and frustules were photographed in valve view and measured using Fiji software. Several statistical tools (mixture and discriminant analysis, k-means clustering, classification trees, etc.) were explored to test whether mere morphometry, independently of other valve features, leads to correct identifications, when compared to identifications made by experts. In view of the results obtained, morphometry-based determination in diatom taxonomy is discouraged.