Limits...
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

Molecular phylogeny of the Elliptochloris-clade (Trebouxiophyceae) based on SSU and ITS rDNA sequence comparisons.The phylogenetic tree shown was inferred using the maximum likelihood method based on a concatenated data set of 2671 aligned positions of 43 taxa using PAUP 4.0b10. For the analysis, the GTR+I+G model (base frequencies: A 0.2372, C 0.2575, G 0.2781, T 0.2272; rate matrix: A-C 1.3929, A-G 1.8871, A-T 1.0278, C-G 0.6574, C-T 3.7034, G-T 1.0000) with the proportion of invariable sites (I = 0.5745) and gamma distribution shape parameter (G = 0.3685) was chosen, which was calculated as the best model by Modeltest 3.7. Bayesian and bootstrap values of the branches 1–23 were calculated from the concatenated data set as well as from both genes separately using different methods, as described in Material and Methods, and these values are shown in S2 Table. The branches in bold are highly supported (Bayesian values > 0.95; bootstrap values > 70%) in all analyses. The sister group of Coccomyxa (Elliptochloris bilobata and Hemichloris antarctica) was chosen as an outgroup. Names at the branch are the original designation of the strains; names in the boxes were assigned after revision using the integrative approach. The circles in the boxes showed the results of different species delimitation approaches (distance-based methods: ABGD and K/θ; phylogeny-based methods: GMYC (ST = single threshold, MT = multi threshold) and PTP (ML = maximum likelihood, MB = Bayesian); see details in Material and Methods) compared to the ITS2/CBC approach presented in this study (black circles = species delimitation supported, grey circles = single taxa needed to be excluded before analyzing with ABGD, white circles = species delimitation not resolved). Authentic strains (‘type cultures’) are marked with an asterisk. S highlighted the photobionts of lichens.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4469705&req=5

pone.0127838.g002: Molecular phylogeny of the Elliptochloris-clade (Trebouxiophyceae) based on SSU and ITS rDNA sequence comparisons.The phylogenetic tree shown was inferred using the maximum likelihood method based on a concatenated data set of 2671 aligned positions of 43 taxa using PAUP 4.0b10. For the analysis, the GTR+I+G model (base frequencies: A 0.2372, C 0.2575, G 0.2781, T 0.2272; rate matrix: A-C 1.3929, A-G 1.8871, A-T 1.0278, C-G 0.6574, C-T 3.7034, G-T 1.0000) with the proportion of invariable sites (I = 0.5745) and gamma distribution shape parameter (G = 0.3685) was chosen, which was calculated as the best model by Modeltest 3.7. Bayesian and bootstrap values of the branches 1–23 were calculated from the concatenated data set as well as from both genes separately using different methods, as described in Material and Methods, and these values are shown in S2 Table. The branches in bold are highly supported (Bayesian values > 0.95; bootstrap values > 70%) in all analyses. The sister group of Coccomyxa (Elliptochloris bilobata and Hemichloris antarctica) was chosen as an outgroup. Names at the branch are the original designation of the strains; names in the boxes were assigned after revision using the integrative approach. The circles in the boxes showed the results of different species delimitation approaches (distance-based methods: ABGD and K/θ; phylogeny-based methods: GMYC (ST = single threshold, MT = multi threshold) and PTP (ML = maximum likelihood, MB = Bayesian); see details in Material and Methods) compared to the ITS2/CBC approach presented in this study (black circles = species delimitation supported, grey circles = single taxa needed to be excluded before analyzing with ABGD, white circles = species delimitation not resolved). Authentic strains (‘type cultures’) are marked with an asterisk. S highlighted the photobionts of lichens.

Mentions: To determine the evolutionary model that fit the dataset best (concatenated and separated according to their genes) the program Modeltest 3.7 [49] was used. Considering the results of these tests, the best models were selected by the Akaike Information Criterion [50]. For datasets, concatenated or partitioned according to their genes, the GTR model with a proportion of invariable sites (I) and a gamma shape parameter (G) was used for the phylogenetic analyses. The phylogenetic tree (Fig 2) was inferred by distance (neighbor-joining [NJ] using the GTR+I+G model), maximum parsimony (MP), and maximum likelihood (ML; using GTR+I+G) criteria using PAUP version 4.0b10 [51], by randomized accelerated maximum likelihood using RAxML version 7.0.3 [52], and by Bayesian inference (BI) using MrBayes version 3.1 [53, 54] and the PHASE package 2.0 [55–59]. The RAxML analyses of the concatenated dataset were performed partitioned according to their genes. For PHASE analyses, four models were used according to their secondary structures and to their genes; the REV model [60] was used for the unpaired regions and the RNA7D model [61] was used for the paired regions. The Bayesian and bootstrap support for each branch in Fig 2 are summarized in S2 Table. The tree topology presented in Fig 2 was compared with user-defined trees that were manually generated with the program TreeView 1.6.6 [62]. The log-likelihood values of these tree topologies was calculated using PAUP and compared via approximately unbiased (AU), Kishino-Hasegawa (KH), and Shimodaira-Hasegawa (SH) tests using CONSEL version 0.1i [63–64]. The results of these tests are summarized in S3 Table.


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)

Molecular phylogeny of the Elliptochloris-clade (Trebouxiophyceae) based on SSU and ITS rDNA sequence comparisons.The phylogenetic tree shown was inferred using the maximum likelihood method based on a concatenated data set of 2671 aligned positions of 43 taxa using PAUP 4.0b10. For the analysis, the GTR+I+G model (base frequencies: A 0.2372, C 0.2575, G 0.2781, T 0.2272; rate matrix: A-C 1.3929, A-G 1.8871, A-T 1.0278, C-G 0.6574, C-T 3.7034, G-T 1.0000) with the proportion of invariable sites (I = 0.5745) and gamma distribution shape parameter (G = 0.3685) was chosen, which was calculated as the best model by Modeltest 3.7. Bayesian and bootstrap values of the branches 1–23 were calculated from the concatenated data set as well as from both genes separately using different methods, as described in Material and Methods, and these values are shown in S2 Table. The branches in bold are highly supported (Bayesian values > 0.95; bootstrap values > 70%) in all analyses. The sister group of Coccomyxa (Elliptochloris bilobata and Hemichloris antarctica) was chosen as an outgroup. Names at the branch are the original designation of the strains; names in the boxes were assigned after revision using the integrative approach. The circles in the boxes showed the results of different species delimitation approaches (distance-based methods: ABGD and K/θ; phylogeny-based methods: GMYC (ST = single threshold, MT = multi threshold) and PTP (ML = maximum likelihood, MB = Bayesian); see details in Material and Methods) compared to the ITS2/CBC approach presented in this study (black circles = species delimitation supported, grey circles = single taxa needed to be excluded before analyzing with ABGD, white circles = species delimitation not resolved). Authentic strains (‘type cultures’) are marked with an asterisk. S highlighted the photobionts of lichens.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127838.g002: Molecular phylogeny of the Elliptochloris-clade (Trebouxiophyceae) based on SSU and ITS rDNA sequence comparisons.The phylogenetic tree shown was inferred using the maximum likelihood method based on a concatenated data set of 2671 aligned positions of 43 taxa using PAUP 4.0b10. For the analysis, the GTR+I+G model (base frequencies: A 0.2372, C 0.2575, G 0.2781, T 0.2272; rate matrix: A-C 1.3929, A-G 1.8871, A-T 1.0278, C-G 0.6574, C-T 3.7034, G-T 1.0000) with the proportion of invariable sites (I = 0.5745) and gamma distribution shape parameter (G = 0.3685) was chosen, which was calculated as the best model by Modeltest 3.7. Bayesian and bootstrap values of the branches 1–23 were calculated from the concatenated data set as well as from both genes separately using different methods, as described in Material and Methods, and these values are shown in S2 Table. The branches in bold are highly supported (Bayesian values > 0.95; bootstrap values > 70%) in all analyses. The sister group of Coccomyxa (Elliptochloris bilobata and Hemichloris antarctica) was chosen as an outgroup. Names at the branch are the original designation of the strains; names in the boxes were assigned after revision using the integrative approach. The circles in the boxes showed the results of different species delimitation approaches (distance-based methods: ABGD and K/θ; phylogeny-based methods: GMYC (ST = single threshold, MT = multi threshold) and PTP (ML = maximum likelihood, MB = Bayesian); see details in Material and Methods) compared to the ITS2/CBC approach presented in this study (black circles = species delimitation supported, grey circles = single taxa needed to be excluded before analyzing with ABGD, white circles = species delimitation not resolved). Authentic strains (‘type cultures’) are marked with an asterisk. S highlighted the photobionts of lichens.
Mentions: To determine the evolutionary model that fit the dataset best (concatenated and separated according to their genes) the program Modeltest 3.7 [49] was used. Considering the results of these tests, the best models were selected by the Akaike Information Criterion [50]. For datasets, concatenated or partitioned according to their genes, the GTR model with a proportion of invariable sites (I) and a gamma shape parameter (G) was used for the phylogenetic analyses. The phylogenetic tree (Fig 2) was inferred by distance (neighbor-joining [NJ] using the GTR+I+G model), maximum parsimony (MP), and maximum likelihood (ML; using GTR+I+G) criteria using PAUP version 4.0b10 [51], by randomized accelerated maximum likelihood using RAxML version 7.0.3 [52], and by Bayesian inference (BI) using MrBayes version 3.1 [53, 54] and the PHASE package 2.0 [55–59]. The RAxML analyses of the concatenated dataset were performed partitioned according to their genes. For PHASE analyses, four models were used according to their secondary structures and to their genes; the REV model [60] was used for the unpaired regions and the RNA7D model [61] was used for the paired regions. The Bayesian and bootstrap support for each branch in Fig 2 are summarized in S2 Table. The tree topology presented in Fig 2 was compared with user-defined trees that were manually generated with the program TreeView 1.6.6 [62]. The log-likelihood values of these tree topologies was calculated using PAUP and compared via approximately unbiased (AU), Kishino-Hasegawa (KH), and Shimodaira-Hasegawa (SH) tests using CONSEL version 0.1i [63–64]. The results of these tests are summarized in S3 Table.

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