ORIGINAL RESEARCH ARTICLE published: 12 November 2014 doi: 10.3389/fmicb.2014.00605 Benthic protists and fungi of Mediterranean deep hypsersaline anoxic basin redoxcline sediments Joan M. Bernhard 1 *, Konstantinos Kormas 2 , Maria G. Pachiadaki 1 , Emma Rocke 3† , David J. Beaudoin 4 , Colin Morrison 5 , Pieter T. Visscher 6 , Alec Cobban 1 , Victoria R. Starczak 4 and Virginia P. Edgcomb 1 1 Geology and Geophysics Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA 2 Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University ofThessaly,Volos, Greece 3 Division of Life Sciences, Hong Kong University of Science andTechnology, ClearWater Bay, Kowloon, Hong Kong 4 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA 5 Biology Department, University of Nevada Reno, Reno, NV, USA 6 Department of Marine Sciences, University of Connecticut, Groton, CT, USA Edited by: Mark Alexander Lever, ETH Zürich, Switzerland Reviewed by: Gaëtan Burgaud, European University of Brittany, France Aharon Oren, The Hebrew University of Jerusalem, Israel *Correspondence: Joan M. Bernhard, Geology and Geophysics Department, Woods Hole Oceanographic Institution, MS #52, Woods Hole, MA 02543, USA e-mail: jbernhard@whoi.edu † Present address: Emma Rocke,The Marine Research Institute, University of CapeTown, Rondebosch, CapeTown, South Africa Some of the most extreme marine habitats known are the Mediterranean deep hypersaline anoxic basins (DHABs; water depth ∼3500 m). Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB water-column haloclines and brines, the existence and activity of benthic DHAB protists have rarely been explored. Here, we report findings regarding protists and fungi recovered from sediments of three DHAB (Discovery, Urania, L’Atalante) haloclines, and compare these to communities from sediments underlying normoxic waters of typical Mediterranean salinity. Halocline sediments, where the redoxcline impinges the seafloor, were studied from all three DHABs. Microscopic cell counts suggested that halocline sediments supported denser protist populations than those in adjacent control sediments. Pyrosequencing analysis based on ribosomal RNA detected eukaryotic ribotypes in the halocline sediments from each of the three DHABs, most of which were fungi. Sequences affiliated with Usti- laginomycotina Basidiomycota were the most abundant eukaryotic signatures detected. Benthic communities in these DHABs appeared to differ, as expected, due to differing brine chemistries. Microscopy indicated that only a low proportion of protists appeared to bear associated putative symbionts. In a considerable number of cases, when prokaryotes were associated with a protist, DAPI staining did not reveal presence of any nuclei, suggesting that at least some protists were carcasses inhabited by prokaryotic scavengers. Keywords: eukaryote, DHABs, discovery, Urania, L’ Atalante, diversity, rRNA INTRODUCTION Redox boundaries in marine sediments can have significant geo- chemical gradients, transitioning from fully aerated to anoxic (lack of detectable dissolved oxygen) conditions within short ver- tical distances (e.g., Cai and Sayles, 1996; Yucel, 2013). These chemoclines are zones of intense biogeochemical cycling, involv- ing all major elements including carbon, oxygen, nitrogen, sulfur, and hydrogen as well as iron and manganese. While consider- able effort has been dedicated to studying the biogeochemistry of redox boundaries of marine sediments in neritic zones (e.g., Yucel, 2013), silled basins (e.g., Reimers et al., 1996; Bernhard et al., 2003), and hydrocarbon seeps (e.g., Orcutt et al., 2005), less is known regarding chemoclines in the deep bathyal to hadal zones. Microbial eukaryotes inhabiting marine chemoclines can be numerous compared to those from nearby more aerated sites (e.g., Bernhard et al., 2000; Edgcomb et al., 2011c). Addition- ally, marine chemocline microbial eukaryotes typically have associated prokaryotes existing as endobionts and/or ectobionts (e.g., Esteban et al., 1995; Fenchel and Finlay, 1995; Bernhard et al., 2000; Bernhard, 2003). Generally, in the cases receiving dedicated study, evidence suggests these associations are mutu- alistic or commensal. Investigations into the systematics and physiologies of the partners in these putative symbioses often yield surprising results, with multiple structured associations (Camerlenghi, 1990; Edgcomb et al., 2011c) and novel cellular adaptations (e.g., Bernhard and Bowser, 2008; Bernhard et al., 2010a). The deep bathyal Mediterranean has numerous deep hyper- saline anoxic basins (DHABs; Figure 1 and also see Figure 1 in Stock et al., 2013b), which are brine-filled bathymetric depres- sions formed from the dissolution of subterranean Miocene salt deposits exposed to seawater after tectonic activity (Camerlenghi, 1990). Due to the sequence of different chemical ions precipitat- ing from seawater as it evaporates, different layers of those salt deposits are characterized by differing chemistries. It follows that DHABs differ in brine chemistry. In the three DHABs studied here, Urania brine is the highest in free sulfide and methane, Discovery brine is highest in chlorine and magnesium, and L’ Atalante brine is highest in sodium, potassium, and sulfate (Table 1). Further details on the brine chemistries are available in van der Wielen et al. (2005). www.frontiersin.org November 2014 | Volume 5 | Article 605 | 1