Export of specimens was approved by the DEF under permit: 052N-EA02/MG08 dated 28 February 2008. Sampling for this study was conducted in January, February, and July 2008. No Elbasvir web species protected by the Convention of the International Trade in Endangered Species CITES were concerned by this research. Voucher specimens were euthanized using approved methods (e.g., anaesthesia with MS222, followed by overdose of the same substance and 95 ethanol fixation). These are standard methods included in the permits above that do not require approval by an ethics committee. Killings were recorded and reported to Madagascan authorities as requested in the respective authorisations (included in the permits stated above).Statistical analysesWe assessed species richness (SR) of tadpole assemblages in dry and wet season based on molecular identification of tadpoles sampled. We first confirmed the expectation that species richness (SR) of tadpole assemblages differs between wet and dry season by paired t-test (all software details are stated and RG7800 molecular weight referenced at the end of this section). Before testing whether changes in FD or PD are similar to changes in SR or whether species loss and turnover dependPLOS ONE | DOI:10.1371/journal.pone.0151744 March 25,4 /Seasons Affect Functional and Phylogenetic DiversityFig 2. Scheme illustrating process of setting null assemblages. Scheme illustrating process of setting null assemblages based on observations in the wet and the dry season. (A) ll species found are pooled. (B) his species pool is randomly ordered a thousand times. (C) ull assemblages for wet season are retrieved: the first nine species if there have been nine species observed in the wet season. (D) or the null assemblages of the dry season, first the remaining (here two) species that have not SART.S23506 been used for the respective wet season assemblage are taken and (E) illed up by randomly chosen species from the respective wet season assemblage (here four) until the original species richness of the dry season assemblage is reached. doi:10.1371/journal.pone.0151744.gon the ecological function of species, we developed a null model. For this null model, we remodelled random assemblages (“null assemblages”) of wet and dry season. Modelling was based on observed SR of assemblages in both seasons and therefore the observed changes in SR and the observed species turnover, as well as the observed species pool available for each stream. In detail, for each stream we pooled all species found in wet and/or dry season in this stream (= species pool available). For example, if nine species were found in the wet season and six in the dry season of which two were not sampled in the wet season, the species pool consisted of 9+2 (11) species (Fig 2A). We reordered them randomly 1000 times (Fig 2B). Out of these 1000 species pools, we each picked the first x species (with x being the number of species observed for this stream in the wet season; nine in our example) and thus achieved null assemblages for the wet season (Fig 2C). To achieve null assemblages for the dry season, we first picked each the remaining y species that were not already included in wet season assemblages (with y being the number of species that were found in the dry but not in the wet season in this stream; two in our example; Fig 2D) and then restocked with randomly chosen species from the respective random wet season assemblage (four in our example; Fig 2E) to reach the final number of species for the random.Export of specimens was approved by the DEF under permit: 052N-EA02/MG08 dated 28 February 2008. Sampling for this study was conducted in January, February, and July 2008. No species protected by the Convention of the International Trade in Endangered Species CITES were concerned by this research. Voucher specimens were euthanized using approved methods (e.g., anaesthesia with MS222, followed by overdose of the same substance and 95 ethanol fixation). These are standard methods included in the permits above that do not require approval by an ethics committee. Killings were recorded and reported to Madagascan authorities as requested in the respective authorisations (included in the permits stated above).Statistical analysesWe assessed species richness (SR) of tadpole assemblages in dry and wet season based on molecular identification of tadpoles sampled. We first confirmed the expectation that species richness (SR) of tadpole assemblages differs between wet and dry season by paired t-test (all software details are stated and referenced at the end of this section). Before testing whether changes in FD or PD are similar to changes in SR or whether species loss and turnover dependPLOS ONE | DOI:10.1371/journal.pone.0151744 March 25,4 /Seasons Affect Functional and Phylogenetic DiversityFig 2. Scheme illustrating process of setting null assemblages. Scheme illustrating process of setting null assemblages based on observations in the wet and the dry season. (A) ll species found are pooled. (B) his species pool is randomly ordered a thousand times. (C) ull assemblages for wet season are retrieved: the first nine species if there have been nine species observed in the wet season. (D) or the null assemblages of the dry season, first the remaining (here two) species that have not SART.S23506 been used for the respective wet season assemblage are taken and (E) illed up by randomly chosen species from the respective wet season assemblage (here four) until the original species richness of the dry season assemblage is reached. doi:10.1371/journal.pone.0151744.gon the ecological function of species, we developed a null model. For this null model, we remodelled random assemblages (“null assemblages”) of wet and dry season. Modelling was based on observed SR of assemblages in both seasons and therefore the observed changes in SR and the observed species turnover, as well as the observed species pool available for each stream. In detail, for each stream we pooled all species found in wet and/or dry season in this stream (= species pool available). For example, if nine species were found in the wet season and six in the dry season of which two were not sampled in the wet season, the species pool consisted of 9+2 (11) species (Fig 2A). We reordered them randomly 1000 times (Fig 2B). Out of these 1000 species pools, we each picked the first x species (with x being the number of species observed for this stream in the wet season; nine in our example) and thus achieved null assemblages for the wet season (Fig 2C). To achieve null assemblages for the dry season, we first picked each the remaining y species that were not already included in wet season assemblages (with y being the number of species that were found in the dry but not in the wet season in this stream; two in our example; Fig 2D) and then restocked with randomly chosen species from the respective random wet season assemblage (four in our example; Fig 2E) to reach the final number of species for the random.
