title: Crustose coralline algal species host distinct bacterial assemblages on their       surfaces
design: In this study, we examined the bacterial communities on four different species of CCA collected in Belize using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP®) of the V1-V3 region of the 16S rDNA. We chose CCA species that had previously been tested for their ability to facilitate coral recruitment.  The CCA Titanoderma prototypum and Hydrolithon boergesenii facilitate larval settlement and post-settlement survival of two important reef-building acroporid species in the Caribbean (Acropora palmata and Acropora cervicornis) (Ritson-Williams et al., 2010; Ritson-Williams et al., 2014). These corals are of special interest because they have declined so drastically that they are now listed as threatened on the endangered species list. Two co-occurring CCA species, Paragoniolithon solubile and Porolithon pachydermum, are common members of the reef community but do not directly facilitate recruitment in these acroporid corals (Ritson-Williams et al., 2010; Ritson-Williams et al., 2014).  We compared the bacterial communities associated with these four CCA species to determine if these algae harbor species specific bacterial communities that may have implications for coral larval settlement preferences.   Individual pieces of CCA were collected in individual sterile Whirl-Pak bags or 50 ml Falcon tubes. Samples were kept in the shade in a 5-gallon bucket with seawater until processing (< 3 hours).  CCA were rinsed under a stream of 30 ml 0.22µm-filtered seawater (FSW) and the surfaces (3-14 cm2) were swabbed vigorously with a sterile swab.  Swabs were placed in 1 ml FSW and vortexed for 1 minute on high.  Swabs were removed with sterile forceps, 200 µl was removed for use in other assays and the remaining 800 µl suspension was centrifuged for 25 minutes at 13 000 rcf.  The supernatant was removed and the resulting pellet was dissolved in 175 µl of lysis buffer (400 mM NaCl, 750 mM sucrose, 20 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl pH 9.0) and stored at -20 °C.    Water samples (n = 5) were taken from each CCA collection site within Belize in 2 ml sterile microcentrifuge tubes by opening the tubes underwater, allowing them to fill, and then closing them before removing them from the water column. Using aseptic technique, 100 µl were removed from each sample for other assays and the remaining volume was centrifuged at 13 000 rcf for 25 minutes. The supernatant was removed and 175 µl of lysis buffer was added. Samples were stored at -20 °C. Samples were sent to Molecular Research LP (MR DNA, Shallowater, TX) for DNA extraction and Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP®) of the V1 – V3 region of the 16S rDNA (Dowd et al., 2008). 16S rDNA was amplified using universal bacterial primers 27Fmod (AGRGTTTGATCMTGGCTCAG) and 519Rmodbio (GWATTACCGCGGCKGCTG). Amplification was performed in a single step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) starting with an initial denaturation step (3 min at 94 °C), followed by 28 cycles (30 sec at 94 °C, 40 sec at 53 °C, 1 min at 72 °C), and a final elongation step (5 min at 72 °C).  Amplicon products were mixed in equal concentrations, purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA), and sequenced with Roche 454 FLX titanium instruments and reagents following the manufacturer’s guidelines. 