Systematic and Applied Microbiology 41 (2018) 139–150 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ournal homepage: www.elsevier.de/syapm Biogeographical patterns of bacterial and archaeal communities from distant hypersaline environments a,∗ a a b c M.del R. Mora-Ruiz , A. Cifuentes , F. Font-Verdera , C. Pérez-Fernández , M.E. Farias , d e a B. González , A. Orfila , R. Rosselló-Móra a Department of Ecology and Marine Resources, Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Spain b Environmental Microbiology Laboratory, Puerto Rico University, Rio Piedras campus, Puerto Rico c Laboratorio de Investigaciones Microbiológicas de Lagunas Andinas (LIMLA), Planta Piloto de Procesos Industriales Microbiológicos (PROIMI), CCT, CONICET, San Miguel de Tucumán, Tucumán, Argentina d Facultad de Ingeniería y Ciencias, Universidad Adolfo Ibá˜nez — Center of Applied Ecology and Sustainability, Santiago, Chile e Marine Technology and Operational Oceanography Department, IMEDEA (CSIC-UIB), Esporles, Spain a r t a b i c l e i n f o s t r a c t Article history: Microorganisms are globally distributed but new evidence shows that the microbial structure of their Received 10 July 2017 communities can vary due to geographical location and environmental parameters. In this study, 50 sam- Received in revised form 22 October 2017 ples including brines and sediments from Europe, Spanish-Atlantic and South America were analysed by Accepted 23 October 2017 applying the operational phylogenetic unit (OPU) approach in order to understand whether microbial community structures in hypersaline environments exhibited biogeographical patterns. The fine-tuned Keywords: identification of approximately 1000 OPUs (almost equivalent to “species”) using multivariate analysis Archaea revealed regionally distinct taxa compositions. This segregation was more diffuse at the genus level and Bacteria Brines pointed to a phylogenetic and metabolic redundancy at the higher taxa level, where their different species acquired distinct advantages related to the regional physicochemical idiosyncrasies. The presence of pre- Hypersaline sediments Operational phylogenetic units viously undescribed groups was also shown in these environments, such as Parcubacteria, or members of Salterns Nanohaloarchaeota in anaerobic hypersaline sediments. Finally, an important OPU overlap was observed between anoxic sediments and their overlaying brines, indicating versatile metabolism for the pelagic organisms. © 2017 Elsevier GmbH. All rights reserved. Introduction formed on the global distribution of microorganisms, but they have mostly focused on their possible pathogenic effects [24,30,52,101]. In recent decades, the paradigm of the global distribution of Nonetheless, attention has drifted away from clinical microbiology microorganisms (“everything is everywhere”) has been constantly towards global environmental studies, including oceanic sediments questioned [71,98]. Traditional explanations for cosmopolitan dis- [47], ocean waters [13] and hypersaline environments such as tributions are large microbial population sizes, high probability salterns [8,36,96]. of dispersion and low probability of extinction [28]. The actual Extreme environments, due to their isolated nature, that are concept of microbial biogeography and the possibility of non- often scattered between different geographical points without random distribution of microorganisms are currently considered direct connexions are excellent systems to evaluate biogeo- to be hot topics [31,45,83]. The analysis of microbial commu- graphical patterns and allopatric speciation [84,95]. Hypersaline nities sampled at different distant locations can facilitate the environments are globally distributed in different climatic regions understanding of the underlying drivers causing the differentiation and include some of the most extreme environments, such as the of populations/communities [38]. Some studies have been per- Atacama desert, or the Arctic and Antarctic regions [27,29,69,97]. Therefore, they offer an excellent opportunity to compare com- plex halophilic communities in very distant yet similar hypersaline environments around the world. Depending on the origin of the ∗ ionic composition of their brines, salterns can be divided into Corresponding author at: Marine Microbiology Group, Department of Ecology and Marine Resources, Mediterranean Institute for Advanced Studies, CSIC-UIB, thalassohaline and athalassohaline. Briefly, thalassohaline salterns C/Miquel Marqués 21, 07190 Esporles, Spain. present a similar ionic composition to seawater and they ultimately E-mail address: [email protected] (M.delR. Mora-Ruiz). https://doi.org/10.1016/j.syapm.2017.10.006 0723-2020/© 2017 Elsevier GmbH. All rights reserved. 140 M.delR. Mora-Ruiz et al. / Systematic and Applied Microbiology 41 (2018) 139–150 occur due to evaporation of seawater or dissolution of evaporite Environmental DNA was extracted from sediment pellets and rocks. On the other hand, athalassohaline hypersaline environ- thawed cut filters from brine samples. Sediment pellets and cut ments show different multiple ionic compositions, distinct from filter pieces were separately vortexed in 2 mL extraction buffer seawater, which depend directly on the composition of the sur- (100 mM Tris–HCl, 100 mM EDTA) in 50 mL polypropylene cen- rounding substrates [82]. trifuge tubes. The supernatant was then transferred to a new tube −1 −1 In general, diversity in hypersaline environments is domi- and 20 ␮L 10 mg mL proteinase K (Roche), 24 ␮L 300 mg mL −1 nated by halophilic microorganisms belonging to the bacterial lysozyme (Roche), and 20 ␮L 1000 U mL mutanolysin (Roche) and archaeal taxa, such as Bacillus [49] and Salinibacter [5], and were added, and the tubes were incubated for 1 h in an orbital ◦ Haloquadratum, Halorubrum [22,70] and the candidate division shaker (Thermo Electron Corp.) at 15,700 × g at 37 C. After the incu- Nanohaloarchaeota [3], respectively. Despite the local descriptions bation period, 10% sodium dodecyl sulphate (Panreac) was added to ◦ of hypersaline environments, a global vision of these environments a final concentration of 1% and the tubes were incubated at 55 C for is still necessary to understand microbial adaptation to different 30 min. Lysates were extracted with phenol-chloroform-isoamyl environmental conditions and its functioning [55]. In addition, the alcohol, as previously described [54]. Then, the DNA was precipi- analysis of physicochemical characteristics is necessary for achiev- tated overnight with 0.7 (v/v) isopropanol, centrifuged for 30 min at ◦ ing a better understanding of habitats, as well as the response of 15,700 × g and 4 C, rinsed with 70% ethanol (v/v) and centrifuged the microbial communities to environmental variations [54,76,78]. again for 15 min. After air-drying, nucleic acids were resuspended ◦ Recently, Filker et al. [32], using some of the same samples in 50 ␮L sterile nuclease-free water (Sigma), and stored at −20 C. also included in the present study, found a high degree of novel genetic diversity and an effect of geographical distance in protis- PCR amplification and pyrosequencing of 16S rRNA tan communities separated by more than 500 km. To complement the biogeographical findings on protist global patterns, this current 16S rRNA gene sequences of environmental samples were study characterized the structure of the bacterial and archaeal com- amplified using the primer pairs GM3 and S for Bacteria, and 21F and ∼ munities in a larger set from hypersaline sediments and brines in 1492R for Archaea [20,66,94] obtaining fragments of 1450 pb. The geographically distant salterns from Europe, Spanish-Atlantic and PCR reactions were performed as previously described by Mora- South America. Coastal seawater-fed (thalassohaline) and inland Ruiz et al. [63]. A secondary PCR reaction was performed to add endorheic (athalassohaline) systems were also compared. barcodes and sequencing linkers to the previously obtained ampli- cons. This was undertaken using 1:10 ␮L of the original PCR product Materials and methods as a template, and the same PCR conditions but only for five cycles. Primers GM3-PS and 907-PS were used for Bacteria [63] and 21F- Sampling sites and sample collection PS for Archaea [64]. This PCR was carried out in triplicate for each sample in order to minimize PCR-bias, and the final products were ® Between January and March 2011, a total of 17 brine and 33 mixed and purified using MSB Spin PCRapace (INVITEK), follow- sediment samples were collected from 27 sites in ten locations in ing the manufacturer’s instructions. The samples were sequenced Spain, Argentina and Chile (Table 1; Fig. 1). The Spanish samples using the 454 GS-FLX+ Titanium technology. All sequences were included six located in the Mediterranean region, one located in the submitted to the European Nucleotide Archive (ENA) under the inland saltpan Penahueca,˜ and two insular salterns from the Canary accession numbers ERR2003672-ERR2003764. Islands. Nine sampling sites were located in the Argentinean Alti- plano. Finally, there was one sampling site on the Chilean Pacific Processing of pyrosequencing data Ocean coast from the Boyeruca salterns. In all cases, sediments were extracted using methacrylate cores, and sterilized bottles were Data were processed following the Mothur pipeline [86]. Briefly, ◦ filled with brines. All samples were stored at 4 C until processing. low-quality sequences were removed (sequences <500