ARTICLES https://doi.org/10.1038/s41477-019-0560-3 Genomes of early-diverging streptophyte algae shed light on plant terrestrialization Sibo Wang1,2,3,12, Linzhou Li1,4,5,12, Haoyuan Li1,2, Sunil Kumar Sahu 1,4, Hongli Wang1,2, Yan Xu1,6, Wenfei Xian1,2, Bo Song1,2, Hongping Liang1,6, Shifeng Cheng1,2, Yue Chang 1,2, Yue Song1,2, Zehra Çebi7, Sebastian Wittek7, Tanja Reder7, Morten Peterson3, Huanming Yang1,2, Jian Wang1,2, Barbara Melkonian7,11, Yves Van de Peer8,9, Xun Xu1,2, Gane Ka-Shu Wong 1,10*, Michael Melkonian7,11*, Huan Liu 1,3,4* and Xin Liu 1,2,4* Mounting evidence suggests that terrestrialization of plants started in streptophyte green algae, favoured by their dual exis- tence in freshwater and subaerial/terrestrial environments. Here, we present the genomes of Mesostigma viride and Chlorokybus atmophyticus, two sister taxa in the earliest-diverging clade of streptophyte algae dwelling in freshwater and subaerial/terres- trial environments, respectively. We provide evidence that the common ancestor of M. viride and C. atmophyticus (and thus of streptophytes) had already developed traits associated with a subaerial/terrestrial environment, such as embryophyte-type photorespiration, canonical plant phytochrome, several phytohormones and transcription factors involved in responses to envi- ronmental stresses, and evolution of cellulose synthase and cellulose synthase-like genes characteristic of embryophytes. Both genomes differed markedly in genome size and structure, and in gene family composition, revealing their dynamic nature, pre- sumably in response to adaptations to their contrasting environments. The ancestor of M. viride possibly lost several genomic traits associated with a subaerial/terrestrial environment following transition to a freshwater habitat. ransition to a terrestrial environment, termed terrestrializa- but differ in their cellular organization, life history and type of habi- tion, is generally regarded as a pivotal event in the evolution tat18. Mesostigma viride is a scale-covered flagellate with an eyespot Tand diversification of land plant flora1. Extant green plants that reproduces by binary division at the flagellate stage (Fig. 1b). (Viridiplantae) can be subdivided into two lineages, Chlorophyta Chlorokybus atmophyticus consists of sarcinoid cell packets where (most of the green algae) and Streptophyta (embryophytes and each cell has its own cell wall, occasionally producing biflagellate, their closest algal relatives, a grade collectively known as strepto- scaly zoospores (Fig. 1b). Importantly, M. viride is found in the ben- phyte algae2). There is now compelling evidence that adaptation thos of small, shallow ponds whereas C. atmophyticus is a subaerial/ to subaerial/terrestrial habitats is a feature of streptophyte algae, terrestrial alga that occurs among bryophytes, on soil and on stones. arising from their dual existence in freshwater and subaerial/ter- Previous analyses of two streptophyte algal genomes, restrial environments throughout their evolutionary history. Recent Klebsormidium nitens6 and Chara braunii7, have not only revealed transcriptomic and genomic studies have shown that the molecu- embryophyte-like genomic traits with gains and expansions lar toolkit for life in a terrestrial environment was already present of respective genes/gene families in both taxa, but also loss of in streptophyte algae3. Homologues of genes once thought to be genes involved in responses to abiotic stresses that prevail in a restricted to embryophytes are now being detected in streptophyte terrestrial environment, in the aquatic (freshwater) C. braunii algae. Examples include those involved in symbiotic or pathogenic (K. nitens thrives in a subaerial/terrestrial environment). The draft interactions with soil microbes4, phytohormone signalling5–8, des- genomes of M. viride and C. atmophyticus reported here allowed iccation/stress9, plastid/nucleus retrograde signalling10,11 and cell us to address two questions important for plant terrestrialization: wall metabolism12. Importantly, many transcription factors (TFs) (1) did the common ancestor of streptophytes already display thought to be specific to embryophytes originated in streptophyte embryophyte-like genomic traits that would be indicative for adap- algae and also substantially expanded there13. These findings raise tation to a terrestrial environment; and (2) how do the genomes exciting questions about the functional role of embryophyte-like of M. viride and C. atmophyticus differ from each other in light genes in streptophyte algae. of previous genome studies on two streptophyte algae that occur In phylogenomic analyses, the earliest-diverging streptophyte in contrasting environments (subaerial/terrestrial and aquatic) algae are represented by a clade comprising two monospecific gen- but belong to different streptophyte classes (Klebsormidiophyceae era, Mesostigma and Chlorokybus14–17. Both are structurally simple and Charophyceae)? 1BGI-Shenzhen, Shenzhen, China. 2China National GeneBank, BGI-Shenzhen, Shenzhen, China. 3Department of Biology, University of Copenhagen, Copenhagen, Denmark. 4State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen, China. 5Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark. 6BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China. 7Botanical Institute, Cologne Biocenter, University of Cologne, Cologne, Germany. 8Department of Plant Biotechnology and Bioinformatics, Ghent University and VIB/UGent Center for Plant Systems Biology, Ghent, Belgium. 9Centre for Microbial Ecology and Genomics, Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, South Africa. 10Department of Biological Sciences and Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. 11Present address: University of Duisburg-Essen, Campus Essen, Faculty of Biology, Essen, Germany. 12These authors contributed equally: Sibo Wang, Linzhou Li. *e-mail: [email protected]; [email protected]; [email protected]; [email protected] NATURE PLAnts | VOL 6 | FEBRuaRY 2020 | 95–106 | www.nature.com/natureplants 95 ARTICLES NATURE PLANTS a 100 Oryza sativa japonica 41 Arabidopsis thaliana 100 Selaginella moellendorffii 100 Physcomitrella patens 100 Chara braunii 100 Klebsormidium nitens 100 Chlorokybus atmophyticus Mesostigma viride 100 100 Volvox carteri 01234567 100 Chlamydomonas reinhardtii log2(gene number + 1) 100 100 Chlorella variabilis 100 Ostreococcus tauri Micromonas commoda Cyanophora paradoxa Chondrus crispus Cyanidioschyzon merolae b e a O. sativa A. thaliana S. moellendorffii P. patens Embryophyt M. polymorpha K. nitens C. atmophyticus M. viride V. carteri C. reinhardtii 10 µm 10 µm Algae C. variabilis Mesostigma viride Chlorokybus atmophyticus O. tauri M. pusilla C. merolae 0 50% 100% Algae and embryophytaEAlgae mbryophyta c C. atmophyticus K. nitens f 0.3 Cso Mco 1,893 Chlorophyta Gpe 2,470 8,196 Czo Rhodophyta Ceu Vca 3,683 Streptophyta 0.2 333 700 Cre 3,639 Mne Mpo Cva 0.1 M. viride Csu Ppa Kni Mco 0 Cbr M. viride C. merolae C. atmophyticusC. merolae Smo Mvi Mpu PC2 (8% variance) Csc Cat Apr 275 210 −0.1 Afi 3,896 1,767 4,624 1,888 Scu Ota Men Olu 1,817 2,296 Osp 1,842 215 1,534 575 Bpr −0.2 4,958 5,114 Ccr Ath M. commoda M. commoda Osa −0.3 Pum Cme −0.4 −0.20 0.2 PC1 (34% variance) d + Significant increases of gene families – Significant decreases of gene families Expansion Contraction Plant hormone signal transduction Alpha-linolenic acid metabolism RNA polymerase Streptophyte Starch and sucrose metabolism algae Photosynthesis:antenna proteins Pentose and glucuronate interconversions Plant–pathogen interaction Purine metabolism Carbon fixation in photosynthetic organisms Endocytosis Plant hormone signal transduction Other types of O-glycan biosynthesis +225 –74 +89 Glycosphingolipid biosynthesis Purine metabolism –41 Vitamin metabolism DNA replication Plant–pathogen interaction Pyrimidine metabolism Basal transcription factors Arachidonic acid metabolism Photosynthesis:antenna proteins Purine metabolism Plant hormone signal transduction Pentose and glucuronate interconversions +188 Rhodophyta Chlorophyta Plant–pathogen interaction Endocytosis –44 Starch and sucrose metabolism Pyrimidine metabolism Carbon fixation in photosynthetic organisms Spliceosome Fig. 1 | Comparative genome profile of M. viride and C. atmophyticus. a, This phylogenetic tree was constructed by maximum likelihood based on the concatenated sequences of single-copy genes, while species-specific gene duplicates were excluded from the analysis. A k-means clustering of gene families based on the gene abundance of each species is shown in the right-hand panel; each column represents a family and each row represents one species. b, Differential interference contrast micrographs showing M. viride (left) and C. atmophyticus (right). c, Venn diagrams showing the number of gene families shared among M. viride, C. atmophyticus and a representative rhodophyte, streptophyte or chlorophyte. d, Significant increases and decreases in gene families; filled red circles, triangles and rhombi denote function enrichment of significant increased gene families in the KEGG pathway, while empty symbols denote function enrichment of significant decreased gene families in the KEGG pathway. Details of these functions are shown in the right- hand panel. e, Percentages of total proteins found in both algae and embryophytes (red), proteins shared