RESEARCH ARTICLE Open Access Origin of land plants: Do conjugating green algae hold the key? Sabina Wodniok1���, Henner Brinkmann2���, Gernot Gl��ckner3, Andrew J Heidel4, Herv�� Philippe2, Michael Melkonian1 and Burkhard Becker1* Abstract Background: The terrestrial habitat was colonized by the ancestors of modern land plants about 500 to 470 million years ago. Today it is widely accepted that land plants (embryophytes) evolved from streptophyte algae, also referred to as charophycean algae. The streptophyte algae are a paraphyletic group of green algae, ranging from unicellular flagellates to morphologically complex forms such as the stoneworts (Charales). For a better understanding of the evolution of land plants, it is of prime importance to identify the streptophyte algae that are the sister-group to the embryophytes. The Charales, the Coleochaetales or more recently the Zygnematales have been considered to be the sister group of the embryophytes However, despite many years of phylogenetic studies, this question has not been resolved and remains controversial. Results: Here, we use a large data set of nuclear-encoded genes (129 proteins) from 40 green plant taxa (Viridiplantae) including 21 embryophytes and six streptophyte algae, representing all major streptophyte algal lineages, to investigate the phylogenetic relationships of streptophyte algae and embryophytes. Our phylogenetic analyses indicate that either the Zygnematales or a clade consisting of the Zygnematales and the Coleochaetales are the sister group to embryophytes. Conclusions: Our analyses support the notion that the Charales are not the closest living relatives of embryophytes. Instead, the Zygnematales or a clade consisting of Zygnematales and Coleochaetales are most likely the sister group of embryophytes. Although this result is in agreement with a previously published phylogenetic study of chloroplast genomes, additional data are needed to confirm this conclusion. A Zygnematales/ embryophyte sister group relationship has important implications for early land plant evolution. If substantiated, it should allow us to address important questions regarding the primary adaptations of viridiplants during the conquest of land. Clearly, the biology of the Zygnematales will receive renewed interest in the future. Background The ancestors of modern land plants (embryophytes) colonized the terrestrial habitat about 500 to 470 million years ago (Ordovician period [1-3]). This event was undoubtedly one of the most important steps in the evolution of life on earth [4-6], thereby establishing the path to our current terrestrial ecosystems [7] and signifi- cantly changing the atmospheric oxygen concentration [8,9]. Since this time three major groups of land plants evolved: bryophytes (liverworts, hornworts and mosses), pteridophytes (lycophytes and monilophytes) and sper- matophytes with the latter dominating most habitats today. It is widely accepted that embryophytes evolved from green algae, or more specifically, from a small but diverse group of green algae known as the streptophyte algae (char- ophycean algae). Streptophyte algae and embryophytes together constitute the division Streptophyta, which likely split from the Chlorophyta (all other green algae) about 725-1200 MY ago [10-12]. Streptophyta and Chlorophyta comprise the Viridiplantae, one of the three evolutionary lineages derived from the single primary endosymbiosis of a cyanobacterium and a eukaryotic host cell [13]. The Streptophyta are characterized by several mor- phological (e.g., structure of flagellate reproductive cells, * Correspondence: b.becker@uni-koeln.de ��� Contributed equally 1Biozentrum K��ln, Botanik, Universit��t zu K��ln, Z��lpicher Stra��e 47b, 50674 K��ln, Germany Full list of author information is available at the end of the article Wodniok et al. BMC Evolutionary Biology 2011, 11:104 http://www.biomedcentral.com/1471-2148/11/104 �� 2011 Wodniok et al licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

if present [14]), and physiological characters (e.g., occur- rence of glyceraldehyde-3-phosphate dehydrogenase iso- form B, GAPDH B [15], leaf peroxisome type of photorespiration [16,17]). Furthermore, several typical embryophyte traits have evolved within the streptophyte algae (e.g., cell division using a phragmoplast, structure of the cellulose synthase complex [4]). However, the streptophyte algae differ greatly in cellular organization and reproduction. Molecular phylogenies indicate that the Mesostigmatales and Chlorokybales form a clade that is a sister-group to all other streptophytes, currently containing only two genera: the biflagellate Mesostigma and the sarcinoid (non-motile cells occurring in packages of four) Chlorokybus [18,19]. The Klebsormi- diales, which is comprised of filamentous algae [14], is the sister group to the remaining streptophyte algae and the embryophytes. The phylogenetic position of the other three groups of streptophyte algae is currently controversial. The conjugating green algae (Zygnema- tales) today represent the most species-rich group of streptophyte algae and are characterized by their unique mode of sexual reproduction. They have completely lost flagellate cells, using instead conjugation for sexual reproduction [20]. The conjugating green algae include both filamentous and unicellular forms. The last two groups of streptophyte algae, the Coleochaetales and Charales, are filamentous with apical growth and an oogamous mode of sexual reproduction. Based on mor- phological complexity, either of the latter two groups have been suggested to be the sister group of the embryophytes [4]. In many illustrations referring to the evolution of streptophyte algae and embryophytes in textbooks [e.g. [21]] or review articles [14,22,23], the Charales (stoneworts) are depicted as the sister group of the embryophytes. The strongest support for a sister group relationship between Charales and embryophytes was obtained in a phylogenetic analysis using four genes (atpB and rbcL [plastid], nad5 [mitochondria], and SSU RNA [nuclear] using 26 streptophyte algae, eight embry- ophytes, and five chlorophytes and one glaucophyte as outgroup [24]). In contrast, analyses using plastid LSU and SSU ribosomal RNAs or whole chloroplast genomes support the Zygnematales or a clade consisting of Zygnematales and Coleochaetales as sister group of the embryophytes [25-27]. Here we use ESTs from six different streptophyte algae for a phylogenomic analysis including 21 embryo- phytes. We show that the Charales are most likely not the sister group of the embryophytes, instead our ana- lyses indicate that either the conjugating green algae or less likely a sister group formed by Coleochaete and the Zygnematales might be the closest living relatives of embryophytes, in agreement with previous phylogenetic analyses based on chloroplast genomes. Results Zygnematales alone or together with Coleochaetales as the sister group of embryophytes New ESTs were sequenced from the streptophyte algae Klebsormidium subtile, Coleochaete scutata, and Chara vulgaris and the chlorophyte alga Pyramimonas parkeae (see Material and Methods for details). We assembled a data set of 129 expressed genes (30,270 unambiguously aligned amino acid positions) for 40 viridiplant taxa including six streptophyte algae (Mesostigma, Klebsormi- dium, Chara, Coleochaete, Closterium and Spirogyra) using the chlorophytes as outgroup to root the trees. The data set was analyzed by maximum likelihood (ML) and Bayesian inference (BI) methods using several evolutionary models. We first evaluated the fits of the models to our data set using cross validation (Table 1). The site-heterogeneous CATGTR model is the best of the four models under study. The site-heterogeneous CAT model, which assumes uniform exchangeability rates among amino acids, has a much better fit than the site-homogeneous LG+F and GTR models, and is just slightly worse than the CATGTR model. Interestingly, the data set is sufficiently large to accurately estimate the amino acid exchangeability rates, since the GTR model has a better fit to the data than the LG+F model, where these parameters were learned from numerous align- ments [28]. The simplifying assumption of equal rates of the CAT model, albeit biologically unsound and rejected by cross validation (in favor of the CATGTR model), has the advantage of allowing a significant increase in computational speed [29], and was therefore used for bootstrap analysis. Despite very different model fits, the same tree topol- ogy (Figure 1) was obtained in all analyses. Bootstrap support values were computed for both methods, using the site homogeneous GTR+��4 model (ML) and the site heterogeneous CAT+��4 model (BI). The posterior prob- abilities of all nodes for both the CAT+��4 and CATGTR +��4 models were 1 except for three nodes (0.99 each, indicated with an asterisk in Figure 1). The molecular phylogeny of embryophytes and chlorophyte algae (out- group) is in agreement with other recently published phylogenies [14,23,30-32] and supports the monophyly of liverworts and mosses which is however still a matter of debate [33]. The phylogeny of the streptophyte algae is Table 1 Cross-validation results for the data set of 40 viridiplant species and 30,270 positio ns (a positive score indicates a better fit) Model compared Likelihood difference (��SD) GTR+��4 vs LG+��4 377.99 �� 28.51 CAT+��4 vs LG+��4 1187.27 �� 78.75 CATGTR+��4 vs LG+��4 1547.08 �� 63.35 Wodniok et al. BMC Evolutionary Biology 2011, 11:104 http://www.biomedcentral.com/1471-2148/11/104 Page 2 of 10