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
Fig 2
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.