bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.160382; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 2 3 4 Symbiotic cyanobacteria communities in hornworts across time, space, 5 and host species 6 7 Jessica M. Nelson1, Duncan A. Hauser1, and Fay-Wei Li1,2,* 8 9 1Boyce Thompson Institute, Ithaca, NY, USA 10 2Plant Biology Section, Cornell University, Ithaca, NY, USA 11 *Corresponding author ([email protected]) 12 13 Word counts 14 • Total: 5952 15 • Introduction: 1140 16 • Materials and Methods: 1603 17 • Results: 1505 18 • Discussions: 1704 19 Display items 20 • 5 Figures (all color) 21 Supporting Information 22 • 6 SI Figures 23 • 5 SI Tables 24 25 26 27 28 29 30 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.160382; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 Summary 33 Rationale. While plant-microbe interactions have been intensively studied in mycorrhizal and 34 rhizobial symbioses, much less is known about plant symbioses with nitrogen-fixing 35 cyanobacteria. Here we focused on hornworts (a bryophyte lineage), and investigated the 36 diversity of their cyanobionts and how these communities are shaped by spatial, temporal, and 37 host factors. 38 Method. We carried out repeated samplings of hornwort and soil samples in upstate New York 39 throughout the growing season. Three sympatric hornwort species were included, allowing us to 40 directly compare partner specificity and selectivity. To profile cyanobacteria communities, we 41 established a new metabarcoding protocol targeting rbcL-X with PacBio long reads. 42 Results. Hornwort cyanobionts have a high phylogenetic diversity, including clades that do not 43 contain other known plant or lichen symbionts. While the sympatric hornwort species have 44 similarly low specificity, they exhibit different preferences toward cyanobionts, although this 45 depended on what cyanobacteria were present in the soil. Cyanobacterial communities varied 46 spatially, even at small scales, but time did not play a major organizing role. 47 Conclusion. This study highlights the importance of sampling soil and sympatric species to infer 48 partner compatibility and preference, and marks a critical step toward better understanding the 49 ecology and evolution of plant-cyanobacteria symbiosis. 50 51 Keywords: Anthoceros, cyanobacteria, hornworts, Nostoc, Notothylas, PacBio, Phaeoceros, 52 symbiosis 53 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.160382; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 54 Introduction 55 Symbiotic interactions with microbes are key drivers in plant evolution and ecology. They 56 not only facilitated plants’ initial transition to land (Bidartondo et al., 2011), but also played a 57 pivotal role in subsequent species diversification and ecosystem functioning (Lugtenberg & 58 Kamilova, 2009; Van Der Heijden et al., 2015; Lutzoni et al., 2018). The most well-studied 59 plant-microbe mutualisms are those with arbuscular mycorrhizal fungi (AMF) and rhizobia. In 60 both cases the symbiosis is endophytic and plants provide carbon sources to the symbionts in 61 exchange for phosphate (AMF) or nitrogen (rhizobia and AMF). The natural histories and 62 genetic mechanisms for these two types of symbiosis have been intensively studied (Oldroyd et 63 al., 2011; Van Der Heijden et al., 2015; Andrews & Andrews, 2017; MacLean et al., 2017). 64 Cyanobacteria also form specialized nutritional symbioses with plants in which they 65 exchange fixed nitrogen for carbon supplements (Rai et al., 2000), but these relationships have 66 been much less studied than those with AMF and rhizobia. Five disparate plant lineages have 67 independently evolved the ability to engage in this specialized symbiosis with cyanobacteria: the 68 angiosperm genus Gunnera, all cycads (gymnosperm), the water fern genus Azolla, a small 69 family of liverworts (Blasiaceae), and all hornworts (Meeks, 1998; Rai et al., 2000). In all these 70 lineages, the symbiotic cyanobacteria (hereafter cyanobionts) are hosted inside specialized plant 71 structures that develop independent of contact with cyanobacteria, for example slime cavities in 72 hornworts and coralloid roots in cycads (Rai et al., 2000; Santi et al., 2013). The cyanobionts are 73 all extracellular endophytes except in Gunnera where they are intracellular in the cells of the 74 stem glands (Rai et al., 2000). Research on plant-cyanobacteria symbiosis mechanisms thus far 75 has determined the sequence of events for establishment of the interaction and some of the 76 necessary cyanobacterial genes (Santi et al., 2013). The symbioses have been experimentally 77 reconstituted in hornworts, liverworts, Gunnera, and cycads (Enderlin & Meeks, 1983; Ow et al., 78 1999; Chiu et al., 2005; Santi et al., 2013). 79 Plant cyanobionts belong to cyanobacterial groups able to produce nitrogen-fixing 80 heterocysts and motile hormogonia (Santi et al., 2013). Most of these plant cyanobionts have 81 been commonly referred to as Nostoc (Rai et al., 2000), but this genus is notoriously polyphyletic 82 and contains multiple distinct genetic clades (Rajaniemi et al., 2005; Shih et al., 2013; Komárek, 83 2016). The taxonomic confusion for Nostoc could conceal wider symbiont diversity and patterns 84 of compatibility and preference between partners. Until recently, research on the diversity of 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.160382; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 plant cyanobionts had relied on culturing the cyanobacteria or limited methods of genetic 86 differentiation such as RFLPs (Leizerovich et al., 1990; West & Adams, 1997; Costa et al., 87 1999; Costa et al., 2001; Guevara et al., 2002; Zheng et al., 2002; Papaefthimiou et al., 2008; 88 Rikkinen & Virtanen, 2008; Thajuddin et al., 2010; Yamada et al., 2012; Liaimer et al., 2016). 89 Metabarcoding and metagenomic methods using next generation sequencing platforms have 90 begun to be applied recently, covering Azolla, cycads, and one hornwort (Dijkhuizen et al., 2018; 91 Zheng et al., 2018; Suárez-Moo et al., 2019; Zheng & Gong, 2019; Bell‐Doyon et al., 2020; 92 Bouchard et al., 2020). 93 These next generation sequencing studies have relied on rRNA regions for identification 94 of bacteria, but this approach poses several challenges in characterizing symbiotic cyanobacteria 95 communities. First, the standard 16S primers are nonspecific and will amplify the entire bacterial 96 community as well as plant organelles, limiting the measurement of cyanobacteria in a given 97 sample. This is a particular challenge when targeting cyanobacteria since they share ancestry 98 with plastids. Second, the short 16S amplicons obtained with Illumina sequencing do not provide 99 much phylogenetic signal. This is especially problematic because the classification of 100 cyanobacteria—from order to species levels—has been largely unresolved, so assigning accurate 101 taxonomy with conventional 16S classifiers is difficult. Third, cyanobacterial genomes typically 102 contain multiple rRNA operons which skews read counts, and in some cases the operons can be 103 highly divergent in the same genome (Johansen et al., 2017), which makes rRNA genes 104 inappropriate species markers in this case. 105 Concomitant with these challenges of defining and surveying cyanobacterial diversity is a 106 lack of information on the environmental factors controlling the composition of cyanobiont 107 communities. Only a few studies have attempted to survey the cyanobacterial diversity of soils 108 near hosts (West & Adams, 1997; Cuddy et al., 2012; Liaimer et al., 2016; Suárez-Moo et al., 109 2019) and have found differing amounts of overlap between soil and symbiotic communities. It 110 is also unclear how geographic distance and host species affect the communities. While some 111 studies have reported very little overlap of cyanobacterial taxa between sites (West & Adams, 112 1997; Bouchard et al., 2020), others have reported the appearance of the same symbiont on 113 distant continents (Rikkinen & Virtanen, 2008; Gehringer et al., 2010). Studies that have 114 compared different host species so far have not detected clear specificity of symbionts to certain 115 hosts (Gehringer et al., 2010; Suárez-Moo et al., 2019), but this may be due in part to host 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.160382; this version posted June 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 116 species and location being confounded. In addition, none of the previous studies have explicitly 117 tested the effects of time, a commonly ignored dimension in plant microbiome studies in general 118 (Peršoh, 2015; Wagner et al., 2016). Plants’ symbiotic structures are usually continuously 119 developed and colonized by cyanobacteria (Adams & Duggan, 2008), which implies that