8 [98], and then translated into distance matrixes (1 minus
Bray-Curtis index value) for UPGMA cluster analyses. Bray-Curtis similarity index is a modified version of the Sørensen index, which considers abundance distribution (also known as the Sørensen abundance Index or the quantitative Sørensen index [99, 100]. To assess an effect of distance on community similarities, Jaccard and Chao-Sørensen indices were plotted against distance data among individual sample sites in a https://www.selleckchem.com/products/Liproxstatin-1.html Pearson-rank correlation using the Statistica software package. A Student’s t-test for paired samples was used for significance testing. A Mantel test between the geographic distance and the Bray Curtis distance matrices was conducted to evaluate the significance of the correlation
coefficient between geographic and genetic distance. The Mantel test was conducted using the software add-in AL3818 in vitro for Microsoft Excel XLSTAT (http://www.xlstat.com) with 10000 permutations. Geographical distances were calculated via the subtraction of different depths on a single geographical position, which resulted in the altitude difference within the same basin. For the calculation of the 2-dimensional great-circle distance between two points on a sphere from their longitudes and latitudes this website (same depth) the haversine formula [101] was implemented in the script as provided by Chris Veness (2002–2011) at http://www.movable-type.co.uk/scripts/latlong.html. A canonical correspondence analysis (CCA) of quantitative amplicon profiles was conducted to describe the relationships between ciliate community composition patterns and underlying environmental gradients, which shape these diversity patterns. Data were log-transformed [102] and unconstrained permutations (n = 499) were run under a reduced model. Monte Carlo significance tests of first ordination axes and of all canonical axes together were performed. Initially, all available environmental variables
(see above) were included in the model. In order to develop a robust model explaining as much variance as possible 6-phosphogluconolactonase while avoiding multi-colinearity, individual variables were removed in a step-wise manner. We used the Canoco software (Microcomputer Power, Ithaca, NY, USA) for the ordination analysis. Scanning electron microscopy (SEM) preparation and enumeration of ciliates We used SEM to visualize ciliate morphotypes and to amend the molecular diversity survey with imaging analyses. We followed the method for SEM described in [25, 103]. In short, fixed samples were filtered onto 0.4-μm polycarbonate Transwell membrane filters (Corning, USA) and washed with 1X PBS (pH 7.4) that were taken through a dehydration series and fixed with 100% hexamethyldisilizane (Electron Microscopy Sciences, Hatfield, Pennsylvania) before air-drying. Transwell filters were not exposed to air at any point during the protocol, until the final step to prevent collapse of fixed protists.