1 Host and environmental determinants of microbial community structure in the marine 2 phyllosphere 3 4 Margaret A. Vogel1, Olivia U. Mason2, and Thomas E. Miller1 5 6 7 Author Affiliations: 1Department of Biological Science, Florida State University, Tallahassee, 8 FL, 2Department of Earth, Ocean, and Atmospheric Science, Florida State University, 9 Tallahassee, FL 10 11 Corresponding Author: Margaret A. Vogel, Address 319 Stadium Drive, Tallahassee, FL 12 32301, Phone (850) 644-9823, Fax (850) 645-8447, Email [email protected] 13 14 15 16 17 18 19 20 Abstract 21 Although seagrasses are economically and ecologically critical foundation species, little 22 is known about their blade surface microbial communities and how those communities relate to 23 overall seagrass health. 16S rRNA gene sequencing (iTag) was used to examine the microbial 24 community composition and diversity on blade surfaces at five sites along a gradient of 25 freshwater input in the northern Gulf of Mexico. Additionally, seagrass surveys were performed 26 and environmental parameters were measured to characterize host characteristics and the in situ 27 conditions at each site. Results show that Thalassia testudinum (turtle grass) blades host unique 28 microbial communities that are distinct in composition and diversity from the water column. In 29 addition, compositional changes within these blade surface communities correlated with both 30 environmental conditions, including water depth, salinity, and temperature, and host 31 characteristics, including seagrass growth rates and blade nutrient composition. These 32 correlations may indicate that blade surface community composition changes with stressful 33 conditions either as a direct or indirect effect. Additionally, 15 microorganisms from five phyla 34 (Cyanobacteria, Proteobacteria, Bacteroidetes, Planctomycetes, and Chloroflexi) were present in 35 all blade surface samples, even after a large disturbance event (Hurricane Irma), and may 36 represent a core community for T. testudinum. Members of this core community may have 37 ecological importance for determining community structure or in performing key community 38 functions. Studies such as this are the first step to understanding what processes influence the 39 structure of marine phyllosphere communities in order to determine how these blade surface 40 communities relate to their host and to seagrass health as a whole. 41 Key Words 42 Seagrass; Bacteria; Core Communities; Phyllosphere; Microbial Ecology 43 Introduction 44 In recent years, there has been an increasing number of studies on the microbial 45 communities associated with plant hosts, especially of those communities associated with the 46 phyllosphere, or leaf surfaces (Lindow and Leveau 2002, Lindow and Brandl 2003, Vorholt 47 2012, Vandenkoornhuyse et al. 2015). We now know that the phyllosphere is a rich habitat that 48 can host up to 107 microbial cells per cm2 of leaf tissue and that these epiphytic microbial 49 communities can have a variety of relationships with their host plants ranging from beneficial to 50 pathogenic (Vorholt 2012). However, leaf associated microbial communities of aquatic plants, 51 including seagrasses, remain largely unexplored when compared to those of terrestrial plants. 52 Seagrasses are a polyphyletic group of angiosperms that colonized the marine 53 environment ~100 million years ago and currently have about 72 species distributed worldwide 54 (Hemminga and Duarte 2000, Short et al. 2011). Seagrasses can form dense monospecific or 55 mixed species meadows that serve as nurseries, feeding grounds, and habitats for a wide variety 56 of marine species from invertebrates to sea turtles and manatees. These foundation species also 57 support bacteria, algal epiphytes, and their grazers, creating highly productive ecosystems 58 (Zieman and Zieman 1989, Hemminga and Duarte 2000). In addition, seagrasses provide 59 valuable ecosystem services, such as stabilizing sediments and trapping and cycling nutrients 60 (Costanza et al. 1997, Duarte 2002, Barbier et al. 2011) and are important sites for blue carbon 61 sequestration (Fourqurean et al. 2012, Duarte et al. 2013). However, with rising anthropogenic 62 influence, eutrophication and degraded water quality are increasingly becoming threats to these 63 important habitats (Duarte 2002, Orth et al. 2006, Short et al. 2011) with seagrass coverage 64 declining at a rate of 110 km2 yr-1 worldwide since 1980 (Waycott et al. 2009). 65 Although there have been a few recent studies on the microbial communities that occur 66 on seagrass blade surfaces (Meija et al. 2016, Fahimipour et al. 2017, Crump et al. 2018, Ugarelli 67 et al. 2019), the interactions between these communities and the seagrass host remain poorly 68 understood. This is especially true for the tropical seagrass species Thalassia testudinum Banks 69 ex Kӧnig (turtle grass), for which there is only one previously published study that contains 70 information about its blade surface microbial communities (Ugarelli et al. 2019). Thalassia 71 testudinum is an important climax species and can be a dominant component of shallow waters 72 in the Caribbean, Western Atlantic, and Gulf of Mexico. In these areas, it can act as an 73 ecosystem engineer creating dense meadows which likely provide more ecosystem services than 74 other smaller seagrass species (Nordlund et al. 2016). Adding to their importance, tropical 75 seagrass meadows often occur adjacent to other critical habitats for biodiversity, such as coral 76 reefs and mangrove forests, and their presence has been correlated with a two-fold reduction of 77 disease levels in nearby corals (Lamb et al. 2017). 78 In the terrestrial phyllosphere, host-species has been found to be a significant driver of 79 variation in microbial community composition with more variation in leaf-associated 80 communities often occurring across plant-host species rather than within a host species even 81 across large spatial scales (Redford et al. 2010, Finkel et al. 2012, Laforest-Lapointe et al. 82 2016a). For instance, intra-specific variability of the microbial communities on Pinus ponderosa 83 was found to be less than inter-specific variability within and across continents (Redford et al. 84 2010). However, variation also occurs between these leaf-associated communities due to 85 environmental conditions, including precipitation/moisture, temperature, and salt content 86 (Jackson et al. 2006, Finkel et al. 2012, Vorholt et al. 2012, Laforest-Lapointe et al. 2016a, 87 2016b, 2017). In a study of seven tree species, the proportion of Alphaproteobacteria, a dominant 88 class in the natural plant microbiome, was found to decrease along a gradient of urban intensity 89 (Laforest-Lapointe et al. 2017). These compositional shifts can lead to changes in the 90 relationship between these leaf associated microbial communities and their host plant which can 91 ultimately affect host fitness and performance (Lindow and Leveau 2002, Vandenkoornhuyse et 92 al. 2015, Saleem et al. 2017). However, it is unknown how much variation exists within leaf- 93 associated microbial communities in the marine phyllosphere and what roles both biotic and 94 abiotic factors play in determining microbial community structure. 95 Characterizing the variation in blade surface microbial communities on seagrasses is the 96 first step to understanding the relative influence of host and environmental conditions on 97 community structure in the marine phyllosphere, which is essential to elucidating the potential 98 role of these microbial communities as a part of the seagrass holobiont. This study is the first to 99 use 16S rRNA amplicon sequencing (iTag: Illumina platform) to characterize the structure and 100 diversity of the microbial communities associated with T. testudinum blades and to examine 101 whether these microbial communities vary in composition with environmental and host 102 characteristics. 103 104 Methods 105 Sampling Location 106 This study took place in Apalachee Bay in the northern Gulf of Mexico along the Florida 107 Panhandle. The coastline of this area is mostly undeveloped with the St. Marks National Wildlife 108 Refuge occupying the adjacent land area. Five sites (ABT-1 – ABT-5) were established starting 109 near the mouth of the St. Marks River (30.07059°N, 84.16687°W) and extending south in a 110 linear fashinon approximately two miles into the bay (30.04194°N, 84.16634°W). These sites are 111 situated along a gradient of abiotic conditions caused by riverine input with the farthest site from 112 shore located on a shoal. Seagrasses in this region form dense meadows that have mixed species 113 composition. Thalassia testudinum is the dominant species at all study sites, however 114 Syringodium filiforme Kützing (manatee grass) is common along with small amounts of 115 Halodule wrightii Ascherson (shoal grass) and Halophila engelmannii Ascherson (star grass). 116 Microbial Sampling 117 To determine microbial community structure and diversity, samples were taken at all five sites 118 on three separate dates (22-Jul, 20-Aug, and 21-Sep-2016) capturing both spatial and temporal 119 variation. On each date, samples were taken from both the blade surface and water column with 120 all sites visited during a six-hour period. To sample water column communities, 1 liter of 121 seawater was collected from above the seagrass canopy, filtered using a sterile syringe with a 2.7 122 µM pre-filter, and microbial biomass was collected on a 0.22 μM Sterivex™ filter. To capture the 123 blade surface microbial communities, T. testudinum blades from five haphazardly chosen shoots 124 at least one meter apart were removed using sterile forceps at each site. The blade surface 125 microbial communities were then sampled using a sterile swab (PurFlock® Ultra, Puritan 126 Diagnostics, LLC). Microbial sampling was standardized by using the second oldest blade in the 127 shoot and only swabbing healthy tissue free of algal epiphytes. Microbial samples (swabs and 128 Sterivex™ filters) were immediately fixed in RNAlater® and placed on dry ice to be transported 129 to Florida State University where all samples were stored at -80°C until further processing.