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https://learn-us-east-1-prod-fleet01-xythos.s3.amazonaws.com/5ddee2fc8b3b3/29148723?response-cache-control=private%2C%20max-age%3D21600&response-content-disposition=inline%3B%20filename%2A%3DUTF-8%27%27BIOL3408MovesHW%25282%2529.docx&response-content-type=application%2Fvnd.openxmlformats-officedocument.wordprocessingml.document&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Date=20201011T210000Z&X-Amz-SignedHeaders=host&X-Amz-Expires=21600&X-Amz-Credential=AKIAZH6WM4PL5SJBSTP6%2F20201011%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Signature=51f9707c890dbcc9f368865e09b3a77d7288656b1cb6cb6a8b44a758c30d1ab3Overlapping Patterns of Gene Expression Between Gametophyte and Sporophyte Phases in the Fern Polypodium amorphum (Polypodiales) fpls-09-01450 October 9, 2018 Time: 15:32 # 1 ORIGINAL RESEARCH published: 09 October 2018 doi: 10.3389/fpls.2018.01450 Edited by: Stefan A. Rensing, Philipps-Universität Marburg, Germany Reviewed by: Chao Cai, Purdue University, United States Caspar Christian Cedric Chater, University of Sheffield, United Kingdom Jan De Vries, Dalhousie University, Canada *Correspondence: Erin M. Sigel erinsigel@louisiana.edu Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 05 May 2018 Accepted: 12 September 2018 Published: 09 October 2018 Citation: Sigel EM, Schuettpelz E, Pryer KM and Der JP (2018) Overlapping Patterns of Gene Expression Between Gametophyte and Sporophyte Phases in the Fern Polypodium amorphum (Polypodiales). Front. Plant Sci. 9:1450. doi: 10.3389/fpls.2018.01450 Overlapping Patterns of Gene Expression Between Gametophyte and Sporophyte Phases in the Fern Polypodium amorphum (Polypodiales) Erin M. Sigel1* , Eric Schuettpelz2, Kathleen M. Pryer3 and Joshua P. Der4 1 Department of Biology, University of Louisiana at Lafayette, Lafayette, LA, United States, 2 Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC, United States, 3 Department of Biology, Duke University, Durham, NC, United States, 4 Department of Biological Science, California State University Fullerton, Fullerton, CA, United States Ferns are unique among land plants in having sporophyte and gametophyte phases that are both free living and fully independent. Here, we examine patterns of sporophytic and gametophytic gene expression in the fern Polypodium amorphum, a member of the homosporous polypod lineage that comprises 80% of extant fern diversity, to assess how expression of a common genome is partitioned between two morphologically, ecologically, and nutritionally independent phases. Using RNA- sequencing, we generated transcriptome profiles for three replicates of paired samples of sporophyte leaf tissue and whole gametophytes to identify genes with significant differences in expression between the two phases. We found a nearly 90% overlap in the identity and expression levels of the genes expressed in both sporophytes and gametophytes, with less than 3% of genes uniquely expressed in either phase. We compare our results to those from similar studies to establish how phase-specific gene expression varies among major land plant lineages. Notably, despite having greater similarity in the identity of gene families shared between P. amorphum and angiosperms, P. amorphum has phase-specific gene expression profiles that are more like bryophytes and lycophytes than seed plants. Our findings suggest that shared patterns of phase- specific gene expression among seed-free plants likely reflect having relatively large, photosynthetic gametophytes (compared to the gametophytes of seed plants that are highly reduced). Phylogenetic analyses were used to further investigate the evolution of phase-specific expression for the phototropin, terpene synthase, and MADS-box gene families. Keywords: gametophyte, life cycle, MADS-box, phototropins, sporophyte, terpene synthases, transcriptomics INTRODUCTION All land plants share a life cycle that alternates between a multicellular spore-producing phase (sporophyte) and a multicellular gamete-producing phase (gametophyte). However, it is in ferns that the manifestation of these two phases (also referred to as generations) as independent entities is most extreme (Niklas and Kutschera, 2010; Haufler et al., 2016). Fern sporophytes are typically Frontiers in Plant Science | www.frontiersin.org 1 October 2018 | Volume 9 | Article 1450 https://www.frontiersin.org/journals/plant-science/ https://www.frontiersin.org/journals/plant-science#editorial-board https://www.frontiersin.org/journals/plant-science#editorial-board https://doi.org/10.3389/fpls.2018.01450 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.3389/fpls.2018.01450 http://crossmark.crossref.org/dialog/?doi=10.3389/fpls.2018.01450&domain=pdf&date_stamp=2018-10-09 https://www.frontiersin.org/articles/10.3389/fpls.2018.01450/full http://loop.frontiersin.org/people/559055/overview http://loop.frontiersin.org/people/523853/overview http://loop.frontiersin.org/people/247009/overview http://loop.frontiersin.org/people/114584/overview https://www.frontiersin.org/journals/plant-science/ https://www.frontiersin.org/ https://www.frontiersin.org/journals/plant-science#articles fpls-09-01450 October 9, 2018 Time: 15:32 # 2 Sigel et al. Phase-Specific Gene Expression in Ferns long-lived, complex plants composed of roots, stems, and leaves (Figure 1). Fern gametophytes are considerably smaller (usually less than one centimeter long), possess fewer tissue types, and are often ephemeral, but are usually photosynthetic, and can sometimes persist independently for multiple growing seasons (Foster and Gifford, 1974; Sato, 1982; Raghavan, 2005; Watkins et al., 2007; Haufler et al., 2016; Pinson et al., 2017). Thus, fern sporophytes and gametophytes are distinct and separate organisms, varying in morphology, physiology, persistence, ecology, and usually ploidy, while sharing a common genome (Qui et al., 2012). The fern life cycle, with its two free-living phases, occupies a pivotal phylogenetic position in the spectrum of green plant life cycles, which ranges from the charophyte algae (i.e., the closest relative to land plants) life cycle that lacks a multicellular sporophyte phase to the angiosperm life cycle in which gametophytes have been reduced to a few cells (Figure 2; Kenrick and Crane, 1997a,b; Graham et al., 2000). Ferns, with their two free- living phases, represent an ideal but under-utilized resource for addressing questions pertaining to the morphological and functional differences between generations in life cycle evolution (Whittier, 1971; Barker and Wolf, 2010; Der et al., 2011). The phase-specific morphologies and functions of fern gametophytes and sporophytes, like those of all land plants, result largely from different gene expression patterns (Graham et al., 2000), with some genes uniquely expressed FIGURE 1 | The homosporous fern life cycle. Sporophyte tissues, which are usually but not necessarily diploid, are shown in green. Gametophyte tissues and spores, which are often but not necessarily haploid, are shown in brown. Spores are generated by meiosis in sporangia. Gametes, both eggs and sperm, are generated by mitosis in archegonia and antheridia, respectively. For simplicity, fertilization is depicted between an egg and sperm from the same gametophyte, but fertilization is also likely to occur between gametes from different gametophytes that are derived from the same or different sporophytes. an, antheridium; ar, archegonium; e, egg; gam, gametophyte; spa, sporangium; spe, sperm; spo, spore; sporo, sporophyte; z, zygote. Images are not to scale. in each phase. Previous isozyme, microarray, and RNA-Seq transcriptome profiling studies comparing gene expression between sporophytes and gametophytes in angiosperms and bryophytes uncovered a negative relationship between the morphological and functional divergence of the two phases and the similarity of their gene expression profiles (e.g., Tanksley et al., 1981; Gorla et al., 1986; Pedersen et al., 1987; Becker et al., 2003; Honys and Twell, 2003; Pina et al., 2005; Szövényi et al., 2011; Rutley and Twell, 2015). In general, the highly reduced male and female gametophytes of seed plants (the sister lineage to ferns) have reduced transcriptome profiles, expressing fewer and different genes than sporophyte tissues (Chettoor et al., 2014; Rutley and Twell, 2015). For example, in Arabidopsis thaliana, approximately one third of the genes expressed in sporophyte tissue are expressed in pollen, with approximately 10% of pollen-expressed genes being unique to that phase (Becker et al., 2003; Honys and Twell, 2003; Pina et al., 2005). In contrast, in the bryophyte Physcomitrella patens approximately 85% of genes are expressed by both the sporophyte and gametophyte (Ortiz-Ramírez et al., 2016). Similarly, a transcriptomic survey of Lygodium japonicum suggested that ferns may have more genes expressed in both gametophytes and sporophytes than do angiosperms (Aya et al., 2015). Here, we evaluate the partitioning of gene expression between gametophyte and sporophyte phases in the well- studied, homosporous fern species Polypodium amorphum Sukds. This diploid member of the Polypodium vulgare species complex (Sigel et al., 2014) belongs to the leptosporangiate order Polypodiales (henceforth, polypod ferns) that encompasses approximately 80% of extant fern species (PPG I, 2016). Polypodium amorphum embodies the cytological, morphological, and life history traits typical of the clear majority of ferns. It has a high base chromosome number (x = 37; Haufler et al., 1993), a large genome size (C-value = 11.5 pg; Murray, 1985), and perennial sporophytes whose spores produce cordate, photosynthetic gametophytes. We generated sporophyte-leaf and whole-gametophyte transcriptomes from three individuals of P. amorphum (each collected from independent populations) to characterize expression profiles for this iconic polypod fern and to investigate its phase- specific gene expression in the broader context land plant life-cycle diversity. Despite having greater overlap in gene- family identity with seed plants, we found P. amorphum to have phase-specific expression profiles like those of its more distant relatives—bryophytes and lycophytes. Our study supports the hypothesis that plants with relatively large, photosynthetic gametophytes will exhibit substantially more overlap in the identity and expression levels of genes in their gametophyte and sporophyte phases regardless of their phylogenetic relatedness to each other or whether they have gametophyte-dominant or sporophyte-dominant life cycles. In addition, we investigate the phototropin, terpene synthase, and Type II MADS-box gene families to determine whether phase-specific gene expression can inform our understanding of gene family function and evolution. Frontiers in Plant Science | www.frontiersin.org 2 October 2018 | Volume 9 | Article 1450 https://www.frontiersin.org/journals/plant-science/ https://www.frontiersin.org/ https://www.frontiersin.org/journals/plant-science#articles fpls-09-01450 October 9, 2018 Time: 15:32 # 3 Sigel et al. Phase-Specific Gene Expression in Ferns FIGURE 2 | Simplified phylogeny of the major clades of streptophyte plants, illustrating the gametophyte (colored brown) and sporophyte (colored green) phases for exemplar lineages. Charophyte algae have a multicellular gametophyte and a single celled sporophyte. All embryophytes, or land plants, have multicellular gametophytes and multicellular sporophytes. Synapomorphies are shown for the major clades. MATERIALS AND METHODS Plant Material We sampled sporophyte leaf material and whole gametophytes from three individuals of P. amorphum Suksd. (Supplementary Table S1). Species determination followed Haufler et al. (1993) and Sigel et al. (2014). Living sporophytes were initially collected from wild populations and transported to the glasshouses at Duke University, Durham, NC, United States, where rhizomes were cleaned to remove soil and repotted in Farfard Mix 2 (Sun Gro Horticulture Canada Ltd., United States). Plants were maintained under common glasshouse conditions (photoperiod 18 h: 6 h, light: dark, with luminosity of 200–400 Umol sec−1 cm2; 27– 67% humidity; daytime temperature: 18.3–21.1◦C; nighttime temperature: 17.8–20.6◦C) for a minimum of 18 months prior to sampling for RNA extraction. Material from a single leaf was taken from each individual and flash frozen in liquid nitrogen 21 days after it had fully unfurled but before sporangia had developed. Spores from each sporophyte individual of P. amorphum were collected and cleaned with bleach solution, as described in Hoshizaki (1975). For each individual, four replicated cultures of gametophytes were grown from spores in a modified Knop’s liquid medium to which 0.1% glucose was added (Miller and Miller, 1961; Smith and Robinson, 1969) under controlled conditions (photoperiod 12 h: 12 h, light: dark; light source: Philips 392282 40W Plant and Aquarium linear