Survey of the Efficacy of a Short Fragment of the rbcL Gene as a Supplemental DNA Barcode for Diatoms

Abstract

DNA barcoding is a tool that uses a short, standard segment of DNA to identify organisms. In diatoms, a consensus on an appropriate DNA barcode has not been reached, but several markers show promise. These include the 5.8S gene plus a fragment of the internal transcribed spacer 2 (ITS-2) of nuclear-encoded ribosomal RNA, a 420-bp segment of the 18S rRNA gene, and a 748-bp fragment at the 3'-end of the ribulose bisophosphate carboxylase large subunit (rbcL) gene. Here, we tested a 540-bp fragment 417-bp downstream of the start codon of the rbcL gene for its efficacy in distinguishing diatom species in a wide range of taxa. Overall, 381 sequences representing 66 genera and 245 species from the classes Mediophyceae and Bacillariophyceae were examined. Intra/interspecific thresholds were set at p = 0.01 differences per site (diff./site) for Mediophyceae and p = 0.02 diff./site for Bacillariophyceae and correctly segregated 96% and 93% of morphological congeners, respectively. When testing reproductively isolated or biological species, which are only available from Bacillariophyceae, 80% of species were discriminated. Therefore, we concluded that, alone, the rbcL region tested herein as potential a DNA barcode was not a sufficient discriminator of all diatoms. We suggest that this fragment could be used in a dual-locus barcode with the more variable 5.8S+ITS-2 to discriminate species without sufficient interspecific divergences in the tested rbcL region and to provide insight into species identity from a separately evolved genome.

Molecular evidence of a Didymosphenia geminata (Bacillariophyceae) invasion in Chilean freshwater systems

“…Molecular identification is useful for more accurate species identification and for assessing the biodiversity of environmental samples (Kermarrec et al, 2013(Kermarrec et al, , 2014Leliaert et al, 2014;Zimmermann et al, 2015). The molecular markers most commonly employed for species delimitations in diatoms are: the plastid gene rbcL, the mitochondrial gene cythocrome oxidase I (COI), the small ribosomal subunit (SSU), the large ribosomal (LSU), and the internal transcribed spacer (ITS) of the nuclear rDNA (Amato et al, 2007;Evans et al, 2007;Hamsher et al, 2011;MacGillivary and Kaczmarska, 2011;Zimmermann et al, 2011;Kermarrec et al, 2013). Most recently advances in nextgeneration sequencing (NGS) technologies offer the possibility to use molecular barcoding for fast and reliable diversity surveys.…”

Identification of benthic diatoms isolated from the eastern tidal flats of the Yellow Sea: Comparison between morphological and molecular approaches

“…Furthermore, long branch artefacts were not found among the rbcL phylogeny. In addition, the rbcL gene, a plastid–encoded gene, is advantageous in its use as a genetic marker because of its high PCR success rate (i.e., ease of amplification), simplicity of alignment, and low susceptibility to interference by heterotrophic contaminants [53]. However, the deficiencies in databases must still be addressed.…”

Sedimentary DNA versus morphology in the analysis of diatom-environment relationships

“…Those differences are produced by variations in the specificity of the primerbinding region in taxa not present in the reference database, the efficiency of PCR amplifications and J Paleolimnol dilution effects during library preparation (Pawluczyk et al 2015;Schirmer et al 2015). The diversities gained through metabarcoding studies are mostly reliant on the genetic marker, but so far there is no consensus on an appropriate diatom barcode, although several markers have been tested and used (Hamsher et al 2011;MacGillivary and Kaczmarska 2011). Generally, markers were developed for different genomic regions, i.e.…”