Abstract
This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker.
Highlights
DNA barcoding is a method used for species identification, which identifies specimens based on DNA sequence similarity against a sequence database of a priori defined species[1]
This study aimed to identify neotropic green microalgae specimens isolated from Brazilian inland waters through the use of Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL), nuITS1 and nuITS2 molecular markers as DNA barcodes
The ITS1-5.8S-ITS2 region could be successfully sequenced from DNA samples extracted from 47 strains (92,15% sequencing success rate) by using the universal primers described by White and coworkers (1990) [35] (Table 1)
Summary
DNA barcoding is a method used for species identification, which identifies specimens based on DNA sequence similarity against a sequence database of a priori defined species[1]. This powerful technique has brought significant improvements to applications such as taxonomy [2,3,4], ecology [5, 6], biosecurity [7,8,9] and food product regulation [10,11,12]. PLOS ONE | DOI:10.1371/journal.pone.0149284 February 22, 2016
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