Phylogenomic insights into the first multicellular streptophyte

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants)1–4. Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments—from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic); Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats5,6. Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using phylogenomic analysis built on 420 loci and sophisticated models, we establish a novel phylogenetic structure, dividing the six distinct genera of Klebsormidiophyceae in a novel four-order-system, with deep divergences more than 898, 765, and 734 million years ago. The reconstruction of ancestral states for habitat suggests an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with Klebsormidiales having conquered land earlier than embryophytes. Focusing on the body plan of the last common ancestor of Klebsormidiophyceae, we postulate it was likely filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. Our data reveal that the first multicellular streptophytes likely lived more than 900 million years ago.

A phylogenomic framework and four-order-system for Klebsormidiophyceae Using the Illumina NovaSeq6000 platform, we sequenced the transcriptomes of 24 isolates of Klebsormidiophyceae, including 15 strains of Klebsormidium, 4 Interfilum, and one of each Hormidiella and Streptofilum as well as three isolates of Streptosarcina collected from five continents and various habitats from all climate zones.In total, we sequenced 1.407 billion paired-end transcriptomic reads, providing more than 423 gigabases of raw sequence information.To complement our dataset, we integrated these data with 14 previously published transcriptomes and 24 additional samples of algae and land plants (see Methods).
With a focus on 420 densely sampled loci, we used maximum likelihood with the complex LG+F+I+Γ4+C60 mixture model to construct a robust phylogenomic tree (Figure 2).The maximum likelihood tree was fully resolved and accurately represented the accepted phylogeny of the green lineage (Chloroplastida).The tree confirmed the positioning of the Klebsormidiophyceae as the sister group to all Phragmoplastophyta 1,30 with full nonparametric bootstrap support (Figure 2).Within Klebsormidiophyceae, two major clades emerged: (i) a clade consisting of Entransia, Hormidiella, and a monophylum of the three Streptosarcina strains that branched sister to (ii) the clade of the other Klebsormidiophyceae, consisting of clades A to G (formed by the genera Interfilum and Klebsormidium) and Streptofilum.Importantly, we recovered a monophyletic genus Klebsormidium (25 strains, full support) and a monophyletic genus Interfilum (4 strains, full support; clade A).Klebsormidium flaccidum (SAG 2307) and Klebsormidium flaccidum (A1-1a) formed an assemblage (a non-supported monophylum) next to the other Klebsormidium spp., yielding a clades B and C 21 .Indeed, while the genus Klebsormidium was monophyletic, some species were recovered as paraphyletic and will require taxonomic revision.Thus, Klebsormidium and Interfilum form a monophylum, to which Streptofilum capillatum branches as sister.
Our phylogenomic analyses recover a topology that features a deep split in the Klebsormidiophyceae.According to our molecular clock analyses, this split happened 898.46    (702.56-1147.595% HPD age estimates) million years ago (Suppl.Figure 2).The resulting two clades again split 734.2 (565.56-945.55 95% HPD age estimates; Streptofilum-Interfilum/Klebsormidium) and 764.97 (591.72-977.4595% HPD age estimates; Entransia-Hormidiella/Streptosarcina) million years ago.We recover that even these two more shallow splits are deeper than the deepest divergence in Embryophyta (for timing on embryophytes, see Morris et al. 31 ).We therefore propose to account for this deep genetic structure by diving Klebsormidiophyceae into a four-order system (Table 1): Klebsormidiales with the genera Interfilum and Klebsormidium (forming a fully-supported monophylum that encompasses the clades and grades A to G), Hormidiellales ord.nov., Streptofilales ord.nov., and Entransiales ord.nov.(Figure 2).
The enigmatic Entransia was first described from Nova Scotia 32 and for a long time tentatively placed in the Zygnematophyceae.We here describe it as Entransiales ord.nov., consisting of a fully-supported clade and the family Entransiaceae fam.nov., forming a clade with the Hormidiellales ord.nov.Previous molecular phylogenetic analyses based on several genes demonstrated that Entransia is a member of Klebsormidiophyceae [33][34][35] .Cook 36 conducted a detailed study of the two available isolates of Entransia and firmly anchored its Overall, while there is a set of shared and distinct traits to all orders in Klebsormidiophyceae, our phylogenomic data establish a robust and unequivocal backbone of Klebsormidiophyceae evolution consisting of two deep dichotomies nested within an even deeper dichotomy in Klebsormidiophyceae-each of these splits being more distant in the past than the split of all land plants.We next used this new phylogenomic framework and four-order-system to understand the evolutionary history of key traits.
The first multicellular streptophyte emerged more than 900 million years ago Land plants are among those photosynthetic eukaryotes with the most complex true multicellularity.The evolutionary emergence of streptophyte multicellularity is thus one of the general interest topics.Here, streptophyte algae hold important information and surprises.Among those streptophyte algae most closely related to land plants, the Zygnematophyceae, we find unicells and (at times branched) filaments.This stands in stark contrast to other (phragmoplastophytic) streptophyte algae, which have parenchymatous growth (Coleochaetophyceae) or even erect growth with organs and thus 3D growth 37 (Charophyceae).It was inferred that the common ancestor of Zygnematophyceae likely underwent reduction and might have even ancestrally transitioning to a unicellular body 38 .
To understand the propensity for multicellularity among Phragmoplastophyta, one must turn to its sister group: the Klebsormidiophyceae.
Klebsormidiophyceae includes sarcinoid (a thallus comprised of cellular colonies organized in a three-dimensional, packet-like structure), uniserial unbranched filaments, and filaments that easily disintegrate into unicells 26,28 .According to phylogenetic reconstruction based on single (or few) gene(s) or multigene approaches 21,25,26 , the position of the sarcinoid morphotype is spread among different clades and probably is derived from filamentous type.Interestingly, both sarcinoid and filamentous morphotypes are present sometimes within the same species or genus, for example in Streptosarcina costaricana or Interfilum 25,26 .This could represent an advantage for colonizing different terrestrial substrates, due to the lower surface-to-volume relationship of large cell assemblages or the possibility of crust formation by filaments.To understand the evolution of growth types in Klebormidiophyceae and streptophytes in general, we conducted Ancestral Character State Reconstructions (ACSR) by maximum likelihood.Multiple data coding strategies were employed, particularly focused on the type of cellular growth (Figure 3; Supp. Figure 3).In the simplest coding pattern, we recovered full support for a multicellular ancestor of Klebsormidiphyceae (posterior probability [PP] of 0.992) and for a multicellular ancestor of Klebsormidophyceae and Phragmoplastophyta (PP of 0.988).If we employ a three-character coding, distinguishing between unicells, sarcinoid cell packages, and filamentous or more complex body plans, we also recover a multicellular ancestor of Klebsormidophyceae and Phragmoplastophyta, with a likely filamentous (PP of 0.620 and 0.613) and less likely sarcinoid (PP of 0.359 and 0.366) body plan.Thus, the first multicellular streptophyte likely lived around 900 million years ago.
What does this mean for the ability to grow on land?While some streptophyte lineages, such as Mesostigma or Chlorokybus, are rare algae with very narrow ecological niches, one of the most striking successes in colonizing terrestrial habitats can be found in Klebsormidium.Klebsormidium spp.are one of the few eukaryotes that are capable of forming biological soil crusts on their own or together with cyanobacteria, mosses, or lichens 39 .We coded habitat occurrence and, using our newly established phylogenetic backbone, performed ancestral character state reconstruction to identify the history of habitat shifts in Klebsormidiophyceae.No clear pattern support was recovered for the deep ancestors, but a full support (PP of 0.994) for ancestrally terrestrial Klebsormidiales (Figure 3B, node 519.84 mya [384.37-692.68 95% HPD age estimates]).This aligns with physiological traits.Interfilum and Klebsormidium are known to produce special mycosporine-like amino acids acting as UV-protectors 40 .Another source of protectants could be phenylpropanoidderived compounds.The first enzyme in the pathway, phenylalanine-ammonia lyase (PAL), was thought to be a land plant innovation that was proposed to have emerged via horizontal gene transfer from soil-associated bacteria 41 .However, when the genome of K. nitens was published 42 , it was found to have a PAL homolog 43,44 , raising the question of when the streptophyte PAL emerged.In our dataset, we found candidates for PAL for other Klebsormidiales (Klebsormidium and Interfilum), which formed a fully-supported clade with bacterial PALs (Suppl.Figure 4).No homologs outside of Klebsormidiales-neither other Klebsormidiophyceae nor other algae-were found.Overall, this suggests, that the ancestor of Klebsormidium and Interfilum may have acquired a bacterial PAL from soil-associated bacteria independent from the origin in land plants 41 .
Our data suggest that the ability for filamentous growth is ancient in streptophytes -it emerged at least 900 million years ago.Recently, Hess et al. 38 found that the Zygnematophyceae (the algal sister lineage of land plants) might have (re-)gained a filamentous body plan multiple times independently.Our ACSR on Klebsormidiophyceae might help explain this: The molecular machinery for filamentous growth might be a set of homologous genes shared since the last common ancestor of Klebsormidiophyceae and Phragmoplastophyta.Hence, there is an ancient 900-million-year-old genetic potential for multicellularity among streptophytes, explaining the propensity to become multicellular.This propensity, building on this genetic potential, was realized multiple times throughout streptophyte evolution, sometimes resulting in very complex multicellular organisms like Chara and land plants, other times in manifesting in mere filamentous growth.That there is a smooth transition between unicellular and multicellular growth is palpable when taking a closer look at, for example, Interfilum sp.SAG2147 whose cells are often organized in clumps (Supplementary Figure 5).However, these clumps easily fall apart, resulting mainly in groups of two or four cells.Groups with four cells can be arranged longitudinally, suggesting that cytokinesis was transverse only.However, in packs of four, it appears that the cell division plane has changed by 90 degrees since the previous division.This indicates rotations in the cell division plane.Additionally, cell wall remodeling after cell division seems to be extensive and fast.
Filamentous Klebsormidiophyceae exhibit, like filamentous Zygnematophyceae, one of the simplest forms of multicellularity: non-branching ("1D") filaments 37,45 .A classical vegetative centripetal cleavage gives rise to these filaments 46,47 .That said, some intricate molecular mechanisms known from land plants might be at play, foremost the ancient phytohormone auxin 48 .Ohtaka et al. 49 found that Klebsormidium nitens NIES-2285 alters its cell division and cell elongation upon auxin treatment and it was later confirmed that K. flaccidum has a functional auxin efflux carrier 50 -which in land plants is key for polar auxin transportmediated morphogenesis 51 .Thus, some key morphogenic processes likely had a deep evolutionary origin in the first filamentous streptophyte.
The frequent loss and gain of filamentous growth suggest that this habit involves several independent genes, each with additional functions.Indeed, also unicellular green algae have most of the genes needed for multicellularity 52,53 .This implies that the complete loss of these genes, even when the lineage reverts to a simpler growth type (likely unicells), is unlikely.This enables both forward and backward evolutionary transitions in body plans across many clades and millennia, as only one or a few genes need to undergo slight changes in their activity.

Conclusion
Significant efforts have been made in the past decade to understand the phylogenetic relationships within streptophytes, particularly in relation to embryophytes, and the deep evolutionary origin of land plant traits 30,38,42,[54][55][56][57][58][59][60] .The evolutionary history of one of the defining traits of land plants however remains debated: multicellularity and complex body plans.We investigate Klebsormidiophyceae using a phylotranscriptomic approach building on isolates from around the world to establish the deep genetic structure within Klebsormidiophyceae and their relation to Phragmoplastophyta.Through ancestral character state reconstruction, we demonstrate that the common ancestor of Phragmoplastophyta and Klebsormidiophyceae was already a multicellular alga; this alga lived almost a billion years ago.across 900 million years of klebsormidiophyceaen evolution.A: To examine the ancestral character states of growth types in unicellular or multicellular organisms, coding schemes represented varying levels of complexity and hypotheses regarding the homology of growth types.The shown color-coded character state distributions represents yellow for unicellular growth, purple for multicellular growth sensu lato (including sarcinoid, filamentous, and parenchymatous growth); a different coding scheme is shown in Supp. Figure S3.Note ancestor nodes for (i) Klebsormidiophyceae and Phragmoplastophyta 988.5 mya and (ii) Klebsormidiophyceae 898.5 mya.B: To examine the ancestral habitats of the Klebsormidiophyceae, we coded the habitat occurrence of the species as blue for aquatic and green for terrestrial.Note the terrestrial ancestor of Klebsormidiales, which lived 290.4 mya; a different coding scheme is shown in Supp. Figure S3.Divergence dating is based on the molecular clock analyses shown in Suppl.Figure S2.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jan de Vries (devries.jan@uni-goettingen.de).

Materials availability statement
This study did not generate new unique reagents.
Data and code availability statement

Light microscopy
High-resolution images of the studied strains were done with Olympus BX-60 microscope (Olympus, Japan) with DIC equipped with a ProgRes C14plus camera and the ProgRes® CapturePro Software (version 2.9.01) (JENOPTIK AG, Jena, Germany).All investigated strains examined at the 21st day of cultivation.

RNA isolation
For the RNA extraction of 24 different strains, 50 ml of 21-day old liquid culture were centrifuged for 5 min at 20 °C and 11000 rpm and the supernatant were removed.The pellet was transferred into the Tenbroek tissue homogenizer and each sample was manually disrupted during 10 min on ice.Further extraction was done using RNA the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich Chemie GmbH, Germany) according to the manufacturer's instructions.DNAse I treatment (Thermo Fisher, Waltham, MA, USA) was applied to the RNA samples, and their quality and quantity were assessed using a 1% agarose gel with a SDS stain, and nanodrop (Thermo Fisher), respectively.The RNA samples were shipped on dry ice to Novogene (Cambridge, UK).

RNAseq and transcriptome assembly
At Novogene (Cambridge, UK), the samples underwent quality checks using a Bionanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and library preparation was performed based on polyA enrichment and using directional mRNA library preparation.
The resultant trimmed alignments were then combined into a matrix consisting of 62 taxa and 420 loci.This matrix comprised a total of 141,000 aligned amino acid positions.To perform maximum-likelihood inference, we employed IQ-Tree2 108 , specifically utilizing the multicore version 2.2.2.7.6.Our analysis consisted of rapid searches, the selection of best-fit nuclear models based on the Bayesian Information Criterion (BIC), and SH-like aLRT branch support (-fast -st AA -m TEST -msub nuclear -alrt 1000).The final tree was then reconstructed using the concatenated file and its partition file using IQ-Tree2 v2.2.2.7 (-m TEST -msub nuclear -s concatenated.fas-p partition.txt-bb 1000 -alrt 1000)

Ancestral character state reconstruction
To infer marginal ancestral state estimates for internal nodes in the tree, we conducted ancestral character state reconstruction (ACSR) using Phytools 93 .This software implements Yang's re-rooting method 95 .We performed two separate ACSR analyses, considering different character coding schemes, to examine the impact on the inferred ancestral character states.The first analysis utilized a 2-character state model, distinguishing between position within Klebsormidiophyceae solely based on morphological and cytological features.The morphological characters shared by Entransia and other Klebsormidiophyceae include cylindrical cells, unbranched filaments, parietal laminated chloroplast, H-shaped cross-wall pieces, asexual reproduction by fragmentation as well as zoospores and aplanospores.Hormidiella is here described as part of Hormidiellales ord.nov.and the family Hormidiellaceae fam.nov..Both Entransia to Hormidiella have the tendency for upright growth and differentiation into three types of vegetative cells: cells with adhesive structures (Entransia) or stalks (Hormidiella), normal vegetative cells and upper tapered cell.Both genera also produce asexual zoospores.The sister genus Streptosarcina appears to have lost asexual zoospores during the adaptation to the arid habitats and developed instead 2Ddivision 26 , which may serve as protection from desiccation; the mechanisms and induction of branching in Streptosarcina however remain obscure.Some traits are also shared by Gclade Klebsormidium spp.and Hormidiella.Morphological investigation of mature cultures demonstrated that these organisms possess relative coin-like cells and filaments disintegrating into short filaments 26 with tapered end cells.Representatives of the G-clade have generally smaller cell sizes that could also be adaptative to desiccation.Streptofilum is here described as part of Streptofilales ord.nov.and the family Streptofilaceae fam.nov., which are sister to the clade of Klebsormidium and Interfilum (the Klebsormidiales).In the description of Streptofilum, the authors 26 noted a shared feature with mature vegetative cells of Mesostigma: a scaly cell wall.Other representatives of the Streptophyta, including some mosses and ferns, possess cell-covering scales only in asexual reproductive stages (zoospores).Hence, while Streptofilum and Interfilum might have superficial similarities in morphology and ecology, the ultrastructure of the vegetative cells showed organic scales in Streptofilum (in contrast to Interfilum).This is underscored by the 734-million-year divergence between both genera.

Figure 1 :
Figure 1: Biogeography of Klebsormidiophyceae. A: World map with all the klebsormidiophycean strains used within this study.An interactive map can be accessed under https://tinyurl.com/yph2s4ma.B: Details on the strains of Klebsormidiophyceae used in this study.C: Cladogram of the genera in Klebsormidiophyceae. Dots label their distribution across climate zones, habitats, and body plan diversity.Character information were guided by Mikhailyuk et al.26

Figure 2 :
Figure 2: A new four-order-system of Klebsormidiophyceae based on phylogenomics.A: Maximum likelihood phylogenetic analyses based on 420 loci and the complex LG+F+I+Γ4+C60 mixture model.An SH-like aLRT branch support test was employed and the size of the dots corresponds to the support values (legend).B: A selection of the morphological diversity found across Klebsormidiophyceae.

Figure 3 :
Figure 3: Ancestral character state reconstruction of body plan and habitat characters

Jeffrey (1982) 61 was 64 ,
the first who used the name Klebsormidiophyceae without formal description of the class.Later on, van den Hoek and co-authors (1995) 62 separated this group based on ultrastructural features of flagellar apparatus, presence of VII and VIII types of mitosis and cytokinesis.The authors proposed two orders Klebsormidiales and Coleochaetales within Klebsormidiophyceae. Unfortunately, the class Klebsormidiophyceae was not formally described.The Latin diagnosis was not delivered and type order was not defined.In the later publications as justification for the establishment of the class,Stewart and Mattox (1975)   63 was cited.However, the authors also did not formally describe the order and refer to the Latin diagnosis ofSilva et al. (1972)   who corrected the generic name Klebsormidium within the family, but did not formally describe the family Klebsormidiaceae or order Klebsormidiales (seeSilva 1980   65   ).As results of our findings, we formally describe the class Klebosormidiophyceae according to the ICN and proposed four orders as follows:Class Klebsormidiophyceae class.nov.Description: Cell division by the formation of cleavage furrow (type VII according to van den Hook et al. 1995).Flagellar apparatus associated with MLS.Cell wall bipartite or H-like, cap-like, or with scales (?).Zoospores (if present) unilateral possess two flagella covered with squared scales and hairs and inserted asymmetrically, without stigma and cell wallless.Comprises filaments, packages or unicells with parietal chloroplast.Type order (designated here): Klebsormidiales ordo nov.Order Klebsormidiales ordo nov.Description: With features of the class.Diagnosis: Differ by the absence of heteropolarity in comparison to Entransiales and Hormidiellales (see below).Type family (designated here): Klebsormidiaceae fam.nov.Family Klebsormidiaceae fam.nov.Description: With features of the order.Type genus (designated here): Klebsormidium Silva, Mattox & Blackwell 1972, Taxon 21: 643.Note: Currently, it contains two genera, Klebsormidium and Interfilum Chodat (1922).Order Entransiales ordo nov.Description: comprises unbranched filaments with H-like cell wall, cells with parietal chloroplast containing several pyrenoids.Asexual reproduction via zoospores (if present).Zoospore germination often with formation amorphous adhesive holdfast and tendency to heteropolarity (forming tapering spines at the tip of germinating filaments).Currently contains only one genus (Entransia).Diagnosis: Differ by the presence of heteropolarity in comparison to Klebsormidiales and Streptofilales (see above).Type family (designated here): Entransiaceae fam.nov.Family Entransiaceae fam.nov.Diagnosis: with characteristic of order Entransiales.Type genus (designated here): Entransia E.O.Hughes 1948, Amer.J. Bot.35: 427.Note: Currently it contains only two species: E. fimbriata and a doubtful E. dichloroplastes Prescott (1967).Order Streptofilales ordo nov.Description (according Mikhailyuk et al. 2018 for Streptofilum): comprises unbranched, short filaments often disintegrating into unicells.Cells naked, surrounded by dense layers of piliform scales possibly organic nature (visible with TEM) and layer of homogenous mucilage.Vegetative cells ellipsoid to ovoid and hemispherical.Chloroplast parietal with pyrenoid.Asexual reproduction by fragmentation.Zoospore formation and sexual reproduction unknown.Diagnosis: Differ by the presence of piliform scales on vegetative cells and genetically in comparison to the other orders.Type family (designated here): Streptofilaceae fam.nov.Family Streptofilaceae fam.nov.Diagnosis: with characteristic of order Streptofilales.Type genus (designated here): Streptofilum Mikhailyuk & Lukešová 2018 in Mikhailyuk et al., Protist 169: 422-423.Currently it contains one species, S. capillatum Mikhailyuk & Lukešová 2018 (Mikhailyuk et al. 2018).Order Hormidiellales ordo nov.Description: comprises branched or unbranched filaments and packets with H-like cell wall, cells with parietal chloroplast containing one pyrenoid.Asexual reproduction via unilateral zoospores without stigma.Zoospore germination often with formation of stalk in Hormidiella and tendency to heteropolarity or without adhesive structure (Streptosarcina).Diagnosis: Differ genetically in comparison to the other orders.Type family (designated here): Hormidiellaceae fam.nov.

Table 1 :
Revision of the class Klebsormidiophyceae and its orders 346Taxonomical and nomenclatural problems of the class Klebsormidiophyceae and justification for emendation.
: with characteristic of order Hormidiellaceae.Type genus (designated here): Hormidiella Iyengar & Kanthamma 1940, J. Indian Bot.Soc.19: 165.Note: Currently, it contains two genera Hormidiella and Streptosarcina Mikhailyuk & Lukešová 2018 (Mikhailyuk et al. Diagnosis The source code for the novel tools discussed in the paper (See RESOURCE TABLE - orthogroups, Phylopypruner result, Prequal; ginsi; clipkit result, the concatenated alignment file and the tree file can be found on Zenodo doi: 10.5281/zenodo.10058795• Six authentic strains representing the recently described species of Klebsormidium 28 were received from one of the authors (AL) and officially deposited in at the Culture collection at Institute of Soil Biology (BCCO), Ceske Budejovice, Czech Republic.All strains were cultivated in 3NBBM (medium 26a 101 ) at 18°C under full-spectrum fluorescent lamps (25-35 µmol photons m -2 s -1 ; 14:10h light-dark cycle).
Table -Software and Algorithms) only sequences that showed the best match to predicted Klebsormidiophyceae nuclear proteins for phylogenetic analysis.For each type of See Resource Table -Software and Algorithms).This selection criterion required the presence of at least one sequence from each of the 10 different taxonomic groups out of the total 14.Additional information regarding the taxonomic group ordering can be found on Zenodo.Homologous sets were aligned using MAFFT 92 v7.304 with default settings, and maximumlikelihood inference was performed using IQ-Tree2 91,107 multicore version 2.2.2.7.The analysis involved fast searches, BIC-selected best-fit nuclear models, and SH-like aLRT