Cultivation-Independent Species Identification of Dinobryon Species (Chrysophyceae) by Means of Multiplex Single-Cell Pcr1

Abstract

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.

Comparing Morphological and Molecular Estimates of Species Diversity in the Freshwater Genus Synura (Stramenopiles): A Model for Understanding Diversity of Eukaryotic Microorganisms

“…This molecular locus has been shown to represent an ideal DNA barcode to distinguish among Chrysophycean species, including those belonging to the genus Synura (Jost et al. , Škaloud et al. , Bock et al.…”

Assessing morphological congruence in Dinobryon species and their stomatocysts, including a newly established Dinobryon pediforme–stomatocyst connection

“…Other No relationships in size between loricas and stomatocysts belonging to the same taxa analysed in the present study may result from different amounts of elements which are used by individual taxa to build stomatocysts. So far, we do not have much data on environmental conditions causing the encystment process 5 , as many chrysophyte taxa are usually not willing to grow in cultures and finding the encysting specimens in natural and laboratory conditions is problematic 22 . The fact that the diameter of stomatocysts in analysed Dinobryon species was similar or bigger (in D. sertularia) than the lorica width resulted from the encystment process which in all Dinobryon species occurs within the special chamber at the mouth of the lorica 1 .…”

A Resurgence in Field Research is Essential to Better Understand the Diversity, Ecology, and Evolution of Microbial Eukaryotes

“…; Chantangsi and Leander ; Jost et al. ). Culture‐independent assessments of diversity based on environmental PCR surveys or fluorescent in situ hybridization have contributed greatly to our overall understanding of heterotrophic uncultured protistan biodiversity in aquatic and terrestrial environments (López‐García et al.…”

“…Most protists have yet to be discovered, especially freeliving heterotrophic species that occur in low abundance and have patchy distributions (Foissner 1999;Lef evre et al 2008;Lin et al 2012;Orsi et al 2011;Takishita et al 2007). The discovery, characterization, and identification of free-living heterotrophic protists that might play a crucial role in ecosystems are also complicated by the inherent difficulties of cultivating them, their small cell sizes, and the lack of conspicuous morphological traits (Boenigk 2008;Boenigk et al 2005;Chantangsi and Leander 2010;Jost et al 2010). Culture-independent assessments of diversity based on environmental PCR surveys or fluorescent in situ hybridization have contributed greatly to our overall understanding of heterotrophic uncultured protistan biodiversity in aquatic and terrestrial environments (L opez-Garc ıa et al 2001; Stoeck et al 2009).…”