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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.

Darienko T, Gustavs L, Eggert A, Wolf W, Pröschold T - PLoS ONE (2015)

Bottom Line: 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.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.Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.

View Article: PubMed Central - PubMed

Affiliation: M. G. Kholodny Institute of Botany, National Academy Science of Ukraine, Kyiv 01601, Ukraine.

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.

No MeSH data available.


Related in: MedlinePlus

ITS-2 DNA Barcode of the Coccomyxa species.The aligned conserved region of ITS-2 was translated into a base-pair alignment using a number coding for each base-pair (1 = A-U, 2 = U-A, 3 = G-C, 4 = C-G, 5 = G•U, 6 = U•G, 7 = mismatch, 8 = deletion, unpaired or single bases) and is given for each strain in S2 Fig. Equal base-pairs within species are marked with a dot. The ITS-2 barcodes were designated by numbers (variants within species were named by lowercase letters) for each species.
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pone.0127838.g004: ITS-2 DNA Barcode of the Coccomyxa species.The aligned conserved region of ITS-2 was translated into a base-pair alignment using a number coding for each base-pair (1 = A-U, 2 = U-A, 3 = G-C, 4 = C-G, 5 = G•U, 6 = U•G, 7 = mismatch, 8 = deletion, unpaired or single bases) and is given for each strain in S2 Fig. Equal base-pairs within species are marked with a dot. The ITS-2 barcodes were designated by numbers (variants within species were named by lowercase letters) for each species.

Mentions: To find molecular signatures that can be used as DNA barcodes for the identification of Coccomyxa species, the conserved region of ITS-2 was extracted manually from the alignment created by MARNA. According to the recommendation of Coleman [15], the conserved region (highlighted in numbered boxes in Fig 3; nos. 1–73), using the 14 bp of the 5.8S-LSU stem, the first 5 bp of Helix I, the first 11 bp of Helix II (including the pyrimidine-pyrimidine mismatch), and all base-pairs of Helix III were selected for the DNA barcode (highlighted in blue for each strain in S2 Fig). The resulting sequences were manually aligned again and proven automatically using MARNA. This alignment was used for finding compensatory base changes (CBCs; see S4 Table) using the program 4SALE version 1.7 [47]. This sequence alignment was translated into the base-pair alignment by replacing each base-pair by a number (A–U = 1; U–A = 2, G–C = 3, C–G = 4, G•U = 5, U•G = 6, mismatch = 7, deletion or unpaired or single bases = 8; Fig 4). In addition, a reduced SSU and ITS dataset (the taxa were represented by only one to four sequences that were removed from the complete dataset) was analyzed using the program ABGD [65] to identify barcoding gaps among the species. For another distance-based analysis (K/θ; [66]), the uncorrected p-distances were calculated by PAUP and compared following the instruction provided in Birky et al. 2010 [66]. The results of these analyses were compared with those of phylogeny-based methods, which were proposed in the guideline of Fontaneto et al. 2015 [67]. For these analyses, the SSU and ITS data set was modified according to the instructions for each method. Identical sequences and sequences of both outgroup taxa were excluded for GMYC and PTP analyses. For GMYC [68], an ultrametric tree was calculated with BEAST 1.8.0 [69] using the HKY model and a strict clock (MCMC: 10,000,000 generations; burnin 1,000,000). The consensus of the sampled trees was built with TreeAnnotator 1.8.0 of the BEAST package. The consensus tree was used as input file for the GMYC analyses (http://species.h-its.org/gmyc/) using both methods (single and multi threshold). For PTP [70], a maximum likelihood of the reduced GMYC data set was calculated by RAxML version 7.0.3 [52] and the resulting best tree was used as input file for analysis (http://species.h-its.org/gmyc/) with 500,000 MCMC generations. The results of all these analyses are summarized in Fig 2.


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.

Darienko T, Gustavs L, Eggert A, Wolf W, Pröschold T - PLoS ONE (2015)

ITS-2 DNA Barcode of the Coccomyxa species.The aligned conserved region of ITS-2 was translated into a base-pair alignment using a number coding for each base-pair (1 = A-U, 2 = U-A, 3 = G-C, 4 = C-G, 5 = G•U, 6 = U•G, 7 = mismatch, 8 = deletion, unpaired or single bases) and is given for each strain in S2 Fig. Equal base-pairs within species are marked with a dot. The ITS-2 barcodes were designated by numbers (variants within species were named by lowercase letters) for each species.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4469705&req=5

pone.0127838.g004: ITS-2 DNA Barcode of the Coccomyxa species.The aligned conserved region of ITS-2 was translated into a base-pair alignment using a number coding for each base-pair (1 = A-U, 2 = U-A, 3 = G-C, 4 = C-G, 5 = G•U, 6 = U•G, 7 = mismatch, 8 = deletion, unpaired or single bases) and is given for each strain in S2 Fig. Equal base-pairs within species are marked with a dot. The ITS-2 barcodes were designated by numbers (variants within species were named by lowercase letters) for each species.
Mentions: To find molecular signatures that can be used as DNA barcodes for the identification of Coccomyxa species, the conserved region of ITS-2 was extracted manually from the alignment created by MARNA. According to the recommendation of Coleman [15], the conserved region (highlighted in numbered boxes in Fig 3; nos. 1–73), using the 14 bp of the 5.8S-LSU stem, the first 5 bp of Helix I, the first 11 bp of Helix II (including the pyrimidine-pyrimidine mismatch), and all base-pairs of Helix III were selected for the DNA barcode (highlighted in blue for each strain in S2 Fig). The resulting sequences were manually aligned again and proven automatically using MARNA. This alignment was used for finding compensatory base changes (CBCs; see S4 Table) using the program 4SALE version 1.7 [47]. This sequence alignment was translated into the base-pair alignment by replacing each base-pair by a number (A–U = 1; U–A = 2, G–C = 3, C–G = 4, G•U = 5, U•G = 6, mismatch = 7, deletion or unpaired or single bases = 8; Fig 4). In addition, a reduced SSU and ITS dataset (the taxa were represented by only one to four sequences that were removed from the complete dataset) was analyzed using the program ABGD [65] to identify barcoding gaps among the species. For another distance-based analysis (K/θ; [66]), the uncorrected p-distances were calculated by PAUP and compared following the instruction provided in Birky et al. 2010 [66]. The results of these analyses were compared with those of phylogeny-based methods, which were proposed in the guideline of Fontaneto et al. 2015 [67]. For these analyses, the SSU and ITS data set was modified according to the instructions for each method. Identical sequences and sequences of both outgroup taxa were excluded for GMYC and PTP analyses. For GMYC [68], an ultrametric tree was calculated with BEAST 1.8.0 [69] using the HKY model and a strict clock (MCMC: 10,000,000 generations; burnin 1,000,000). The consensus of the sampled trees was built with TreeAnnotator 1.8.0 of the BEAST package. The consensus tree was used as input file for the GMYC analyses (http://species.h-its.org/gmyc/) using both methods (single and multi threshold). For PTP [70], a maximum likelihood of the reduced GMYC data set was calculated by RAxML version 7.0.3 [52] and the resulting best tree was used as input file for analysis (http://species.h-its.org/gmyc/) with 500,000 MCMC generations. The results of all these analyses are summarized in Fig 2.

Bottom Line: 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.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.Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.

View Article: PubMed Central - PubMed

Affiliation: M. G. Kholodny Institute of Botany, National Academy Science of Ukraine, Kyiv 01601, Ukraine.

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.

No MeSH data available.


Related in: MedlinePlus