Using the multiple alignment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html/) and based on 98% gene similarity as a phylotype cutoff [17, 34], clones were grouped together and considered members of the same phylotype. All sequences were compared with the BLAST function (http://www.ncbi.nlm.nih.gov/BLAST/) add to your list for the detection of closest relatives. Sequence data were compiled using the MEGA4 software [35] and aligned with sequences obtained from the GenBank (http://www.ncbi.nlm.nih.gov/) database, using the ClustalX aligning utility. Phylogenetic analyses were performed using the MEGA version 4 software [35] and the topology of the tree was based on neighbour-joining according to Jukes-Cantor. Bootstrapping under parsimony criteria was performed with 1,000 replicates.

Sequences of unique phylotypes found in this study have GenBank accession numbers “type”:”entrez-nucleotide-range”,”attrs”:”text”:”JN090861-JN090912″,”start_term”:”JN090861″,”end_term”:”JN090912″,”start_term_id”:”353255949″,”end_term_id”:”353256000″JN090861-JN090912 and “type”:”entrez-nucleotide-range”,”attrs”:”text”:”JN090913-JN090923″,”start_term”:”JN090913″,”end_term”:”JN090923″,”start_term_id”:”353256001″,”end_term_id”:”353256011″JN090913-JN090923 for the eukaryotes and Cyanobacteria, respectively.Library clone coverage was calculated by the formula of the Good’s C estimator [1 ? (ni/N)] [36], where ni is the number of phylotypes represented by only one clone and N is the total number of clones examined in each library.

The number of predicted phylotypes for each clone library was estimated after the abundance-based richness formula SChao1 [37, 38].3. Results and DiscussionWe investigated the composition of plankton Cyanobacteria and unicellular eukaryotes by combing molecular, 18S/16S rRNA gene diversity, and microscopic analysis in Lake Karla during two fish kill events which happened within the first year of the lake’s partial reconstruction. The prevailing abiotic factors (Table 1) indicated that dissolved oxygen (5.6�C5.8mgL?1) was not limited, while the elevated salinity (7.6�C8.1psu) was possibly attributed to the drainage of the previous lake as well as the result of intensive agricultural and livestock use for four decades. Irrigation in the absence of leaching can increase soil salinity [39] and continued application of livestock manure to agricultural land may result in an accumulation of salt in soil [40].

Table 1Prevailing physical and chemical parameters in L. Karla.The two eukaryotic clone libraries revealed that 45 phylotypes AV-951 occurred in March and only seven in April 2010. However, in both cases, rarefaction curves (Figure 2) reached saturation levels for both clone libraries according to the Good’s C estimator, indicating that the majority of the existing phylotypes were revealed.