View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Crossref Hindawi Publishing Corporation Archaea Volume 2015, Article ID 646820, 11 pages http://dx.doi.org/10.1155/2015/646820 Research Article Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil Yendi E. Navarro-Noya,1 César Valenzuela-Encinas,2 Alonso Sandoval-Yuriar,2 Norma G. Jiménez-Bueno,2 Rodolfo Marsch,2 and Luc Dendooven2 1 Catedras´ CONACYT, Centro de Investigacion´ en Ciencias Biologicas,´ Universidad Autonoma´ de Tlaxcala, Km. 10.5 Autopista Tlaxcala-Texmelucan, 90120 San Felipe Ixtacuixtla, TLAX, Mexico 2LaboratoryofSoilEcology,ABACUS,Cinvestav,AvenidaInstitutoPolitecnico´ Nacional 2508, 07360 Mexico City, DF, Mexico Correspondence should be addressed to Luc Dendooven; [email protected] Received 1 February 2015; Accepted 15 April 2015 Academic Editor: Antonio Ventosa Copyright © 2015 Yendi E. Navarro-Noya et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC) ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5), indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena,andNatronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances) clearly clustered the communities by pH. 1. Introduction soda lakes bettering our understanding of the physiology, ecology, and distribution of polyextremophiles, such as the Hypersaline environments are found in natural or anthro- haloalkaliphiles [13]. Their terrestrial counterparts, for exam- pogenic aquatic or terrestrial habitats in most parts of ple, soda desert, however, remain largely unexplored and we the world [1]. Athalassohaline and thalassohaline aquatic still have an inadequate understanding of terrestrial Archaea systems, saline deserts, solonchaks, solar salterns, and former [7, 14]. Previous studies on Archaea in hypersaline soil lakebeds are examples of them. Cultivation- and molecular- focusedoftenononesalinitylevel[1, 13, 15]. Consequently, based studies have been used to reveal the diversity of bacte- our understanding of how a salinity gradient affects archaeal rial and archaeal communities in hypersaline environments diversity and their functionality is limited. [2–6].Insuchhypersalinehabitats,thearchaealcommunity The soil of the former lake Texcoco (Mexico) is a unique dominates the microbial population [1, 7]. The majority of extreme-haloalkaline terrestrial ecosystem formed from vol- theArchaeaidentifiedinsuchenvironmentsbelongtothe canic ash deposited in situ in a lacustrine environment Halobacteriales [7]. coveredrecentlybycolluvialmaterials.ThelakeTexcoco Soda lakes are exceptional hypersaline (up to saturation) covered 50% of the original lakes around the Aztec city aquatic habitats that have simultaneously an extreme alkaline of Tenochtitlan (current day Mexico City). The lakes were pH. Several soda lakes from around the world have been drained since the 17th century, and, nowadays, it is a large studied [8–12]. Novel Archaea have been isolated from these area of lacustrine bed exposed to desertification [16]. Since 2 Archaea Table 1: Some physical-chemical characteristics of soils from the former lake Texcoco. Particle size distribution a WHCb Total N Total C Soil sampling site EC pH (dS/m) Clay Silt Sand (g/kg soil) TX 1 0.7 8.5 851 1.9 32.8 127 247 623 TX 2 9.0 10.3 1046 1.3 23.2 270 341 181 TX 3 84.8 10.5 1120 0.9 16.7 653 270 77 TX 4 139.1 10.3 850 0.9 22.6 332 216 462 TX 5 143.7 10.0 923 0.9 30.9 332 92 576 TX 6 157.2 10.2 747 1.4 46.7 478 131 391 aEC: electrolytic conductivity; bWHC: water holding capacity. thebeginningofthe1970s,ahydraulicdrainagesystemhas different EC and pH. At each location, the 0–10 cm soil layer 2 been installed and the soil irrigated with wastewater effluent was sampled five times from three 20 m areas with a small to reduce the salt content and pH so that the former lakebed hand spade in August 2013. The soil collected from each can be vegetated. The artificial drainage together with the area was pooled separately so that eighteen soil samples were variability inherent of the former lakebed has generated a obtained. The soil samples were taken to the laboratory in a heterogenic soil [16]. According to the FAO soil classification black polyethylene bag kept on ice. The eighteen samples were [17], a very strong saline soil has an electrolytic conductivity 5 mm sieved separately under aseptic conditions. The soil was ∘ (EC) > 16 dS/m. Soils with EC 16.3 dS/m have been described characterized and a 25 g subsample was stored at −20 Cfor in other studies as “extremely saline soils” [18]; the EC in less than three weeks until extracted for DNA. soil of Texcoco can reach 100 dS/m and the pH can be as Soil pH was measured in 1 : 2.5 soil-H2O suspension using high as 10.5 [16]. Culture-based studies with soil of former aglasselectrode[27]. The EC was measured according to lake Texcoco have yielded a number of novel prokaryotic the saturated paste method [28]. The total carbon (TOC) T species, that is, the Archaea Natronobacterium texcoconens in soil was determined by oxidation with K2Cr2O7 and T and Natronorubrum texcoconens and the Bacteria Texco- trapping the evolved CO2 inNaOH,followedbytitration conibacillus texcoconensiT [19–21]. Valenzuela-Encinas et al. with 0.1 M HCl [29]. Total nitrogen (TN) was measured by [22–24] first studied the bacterial and archaeal communities the Kjeldahl method [30]andsoilparticlesizedistribution using cloned sequences of the 16S rRNA gene. It was found by the hydrometer method as described by Gee and Bauder that in the soil with EC 159 dS/m and pH 10.5 more than [31]. The water holding capacity (WHC) was measured on 95% of the clones were affiliated with members of the family soil samples water-saturated in a funnel and left to stand Halobacteriaceae belonging to phylum Euryarchaeota [22], overnight (Table 1). whileinthedrainedsoilwithEC0.68dS/mandpH7.8 most of the cloned Archaea were related to mesophilic Crenarchaeota and were not-yet-cultured [24]. Few studies 2.2. DNA Isolation and PCR-Amplification of Archaeal 16S described the microbial community in the remaining lake rRNA Genes. Metagenomic DNA was extracted from soil [25, 26]. samples using the Power Soil DNA Isolation Kit (MO It is most likely that the soil of the former lake Texcoco BIO Laboratories, CA, USA) following the manufacturer’s harbors more novel archaeal species with unique characteris- instructions. The V1–V3 region (about 550 bp) of archaeal tics. However, the extent to which new archaeal species can be 16S rRNA gene was chosen for amplification and subsequent found in this environment has not been determined. In this pyrosequencing. The DNA samples were amplified using the study, archaeal-specific primers combined with taxon-based set of archaeal primers 25F 5 -CYG GTT GAT CCT GCC and phylogenetic approaches were used to investigate and RG-3 [32]andA571R5-GCT ACG GNY SCT TTA RGC-3 identify archaeal diversity patterns in soil from the former [33]. Each ribosomal primer set was flanked by a 454-adapter lake Texcoco with different EC (0.7–157.2 dS/m) and pH (8.5– sequence. A 10-nucleotide tag was incorporated between the 10.5). 454-adapter and the forward primer for sample identification among mixed amplicon libraries. PCR products per soil 2. Material and Methods sample were amplified in triplicate with a 30-cycle-based protocol, pooled, and purified using the DNA Clean and 2.1. Site Description and Soil Sampling. The sampling site Concentrator Columns (Zymo Research, Irvine, CA, USA). is located in the former lake Texcoco (northern latitude Each library was quantified using NanoDrop 2000 (Thermo ∘ ∘ 19 29 46 ,westernlongitude9858 01 )intheStateof Fisher Scientific Inc., Suwanee, GA, USA) and mixed in equal Mexico, Mexico, at an altitude of 2240 m.a.s.l., with a mean amount. Sequencing was done unidirectionally by Macrogen ∘ annual temperature of 16 C and mean annual precipitation of Inc. (Seoul, Korea) using the Roche 454 GS-FLX Titanium 705 mm. Soil was sampled from six different locations with (Roche 454 Life Sciences, Branford, CT, USA). Archaea 3 2.3. Pyrosequencing Reads Processing. Sequences were pro- assemblages.TheCCAwasruninR(vegan package (http:// cessed for quality, barcode sorting, and denoising through the cran.r-project.org/web/packages/vegan/index.html)). QIIME pyrosequencing pipeline (http://qiime.org/). Briefly, reads shorter than 250 nt, with quality scores less than 25, 3. Results or containing errors in adaptors and primers were discarded. Onemismatchwasallowedinthebarcodesequence.Denois- The sequencing of eighteen libraries yielded 75,727 V1–V3 ing of the reads was done with the script denoise wrapper.py archaeal 16S rRNA raw sequence reads, with an average using the barcode-sorted libraries and the standard flowgram length of 486 nt.