Evaluating the Species Boundaries of Green Microalgae (Coccomyxa, Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples.

Tatyana Darienko,Lydia Gustavs,Anja Eggert,Wiebke Wolf,Thomas Pröschold,Diego Fontaneto

doi:10.1371/journal.pone.0127838

Abstract

Integrative taxonomy is an approach for defining species and genera by taking phylogenetic, morphological, physiological, and ecological data into account. This approach is appropriate for microalgae, where morphological convergence and high levels of morphological plasticity complicate the application of the traditional classification. Although DNA barcode markers are well-established for animals, fungi, and higher plants, there is an ongoing discussion about suitable markers for microalgae and protists because these organisms are genetically more diverse compared to the former groups. To solve these problems, we assess the usage of a polyphasic approach combining phenotypic and genetic parameters for species and generic characterization. The application of barcode markers for database queries further allows conclusions about the ‘coverage’ of culture-based approaches in biodiversity studies and integrates additional aspects into modern taxonomic concepts. Although the culture-dependent approach revealed three new lineages, which are described as new species in this paper, the culture-independent analyses discovered additional putative new species. We evaluated three barcode markers (V4, V9 and ITS-2 regions, nuclear ribosomal operon) and studied the morphological and physiological plasticity of Coccomyxa, which became a model organism because its whole genome sequence has been published. In addition, several biotechnological patents have been registered for Coccomyxa. Coccomyxa representatives are distributed worldwide, are free-living or in symbioses, and colonize terrestrial and aquatic habitats. We investigated more than 40 strains and reviewed the biodiversity and biogeographical distribution of Coccomyxa species using DNA barcoding. The genus Coccomyxa formed a monophyletic group within the Trebouxiophyceae separated into seven independent phylogenetic lineages representing species. Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.

Highlights

Microalgae and protozoa are traditionally classified according to morphological and cytological characteristics of vegetative stages in their life cycle
We focussed on the following questions: (i) How we can detect species boundaries in those asexual microorganisms? (ii) Can we find a suitable molecular barcode marker to discriminate species? (iii) Do Coccomyxa-like algae form a monophyletic group and how many species can be distinguished within this genus? (iv) Is the missing occurrence in marine habitats caused by highly sensitive physiological response to enhanced salinity? (v) Does the biodiversity of Coccomyxa in culture represent its genetic diversity in nature, and are there any geographical distribution pattern, or ecological and lifestyle preferences?
The strains investigated are subdivided into seven lineages within Coccomyxa, designated as C. subellipsoidea, C. simplex, C. dispar, C. viridis, and three new lineages called C. polymorpha, C. vinatzeri, and C. galuniae

Summary

Introduction

Microalgae and protozoa (protists) are traditionally classified according to morphological and cytological characteristics of vegetative stages in their life cycle. In species that exhibit sexual reproduction, crossing experiments can be conducted to test the biological species concept and identify organisms at the species level, but the situation for asexual species is more complex Organisms such as coccoid green microalgae often lack characteristic morphological features and cannot be classified at a higher taxonomic level without additional data. The latest version of ICN allows the depositing of DNA and cultured material (in a metabolic inactive state) as type material for a species name, but it is not commonly used Taxonomic revision using this approach is impossible for large scale environmental studies and has been recently replaced by DNA barcoding. The internal transcribed spacer region 2 (ITS-2) is often used for phylogenetic studies of microalgae at species level, but it has not been proposed as a barcode marker because of the difficulties with the alignment of sequences and the prediction of secondary structure. The usage of ITS-2 as barcode marker among protists and microalgae, especially in asexual species is still unused at a larger scale and must be validated

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