fmicb-10-01018 May 31, 2019 Time: 15:59 # 1 ORIGINAL RESEARCH published: 31 May 2019 doi: 10.3389/fmicb.2019.01018 Actinobacteria and Cyanobacteria Diversity in Terrestrial Antarctic Microenvironments Evaluated by Culture-Dependent and Independent Methods Adriana Rego1,2, Francisco Raio1, Teresa P. Martins1, Hugo Ribeiro1,2, António G. G. Sousa1, Joana Séneca1, Mafalda S. Baptista1,3, Charles K. Lee3,4, S. Craig Cary3,4, Vitor Ramos1†, Maria F. Carvalho1, Pedro N. Leão1 and Catarina Magalhães1,5* Edited by: 1 Peter Convey, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Porto, Portugal, 2 3 British Antarctic Survey (BAS), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal, International Centre 4 United Kingdom for Terrestrial Antarctic Research, University of Waikato, Hamilton, New Zealand, School of Science, University of Waikato, Hamilton, New Zealand, 5 Faculty of Sciences, University of Porto, Porto, Portugal Reviewed by: Kimberley Warren-Rhodes, Ames Research Center, United States Bacterial diversity from McMurdo Dry Valleys in Antarctica, the coldest desert on Geok Yuan Annie Tan, earth, has become more easily assessed with the development of High Throughput University of Malaya, Malaysia Sequencing (HTS) techniques. However, some of the diversity remains inaccessible *Correspondence: Catarina Magalhães by the power of sequencing. In this study, we combine cultivation and HTS [email protected]; techniques to survey actinobacteria and cyanobacteria diversity along different soil [email protected] and endolithic micro-environments of Victoria Valley in McMurdo Dry Valleys. Our † Present address: Vitor Ramos, results demonstrate that the Dry Valleys actinobacteria and cyanobacteria distribution Instituto Politécnico de Bragança is driven by environmental forces, in particular the effect of water availability and (IPB-CIMO), Campus de Santa endolithic environments clearly conditioned the distribution of those communities. Apolónia, Bragança, Portugal Data derived from HTS show that the percentage of cyanobacteria decreases from Specialty section: about 20% in the sample closest to the water source to negligible values on the This article was submitted to last three samples of the transect with less water availability. Inversely, actinobacteria Extreme Microbiology, a section of the journal relative abundance increases from about 20% in wet soils to over 50% in the driest Frontiers in Microbiology samples. Over 30% of the total HTS data set was composed of actinobacterial strains, Received: 23 August 2018 mainly distributed by 5 families: Sporichthyaceae, Euzebyaceae, Patulibacteraceae, Accepted: 24 April 2019 Published: 31 May 2019 Nocardioidaceae, and Rubrobacteraceae. However, the 11 actinobacterial strains Citation: isolated in this study, belonged to Micrococcaceae and Dermacoccaceae families Rego A, Raio F, Martins TP, that were underrepresented in the HTS data set. A total of 10 cyanobacterial strains Ribeiro H, Sousa AGG, Séneca J, from the order Synechococcales were also isolated, distributed by 4 different genera Baptista MS, Lee CK, Cary SC, Ramos V, Carvalho MF, Leão PN and (Nodosilinea, Leptolyngbya, Pectolyngbya, and Acaryochloris-like). In agreement with Magalhães C (2019) Actinobacteria the cultivation results, Leptolyngbya was identified as dominant genus in the HTS data and Cyanobacteria Diversity in Terrestrial Antarctic set. Acaryochloris-like cyanobacteria were found exclusively in the endolithic sample Microenvironments Evaluated by and represented 44% of the total 16S rRNA sequences, although despite our efforts Culture-Dependent and Independent we were not able to properly isolate any strain from this Acaryochloris-related group. Methods. Front. Microbiol. 10:1018. doi: 10.3389/fmicb.2019.01018 The importance of combining cultivation and sequencing techniques is highlighted, as Frontiers in Microbiology| www.frontiersin.org 1 May 2019| Volume 10| Article 1018 fmicb-10-01018 May 31, 2019 Time: 15:59 # 2 Rego et al. Bacterial Diversity in Antarctic Microenvironments we have shown that culture-dependent methods employed in this study were able to retrieve actinobacteria and cyanobacteria taxa that were not detected in HTS data set, suggesting that the combination of both strategies can be usefull to recover both abundant and rare members of the communities. Keywords: actinobacteria, McMurdo Dry Valleys, Antarctic soil, bacteria diversity, bacterial cultivability, endolitic microbiota, Antarctic microenvironments, cyanobacteria INTRODUCTION other microorganisms (Vincent, 2000). They are well adapted to the stress of desiccation and despite most of the Dry Valleys The continent of Antarctica comprises about 0.34% ice-free areas soils lack any visible cyanobacterial growth, their presence (Convey, 2011) characterized by extreme cold and dry conditions was detected through HTS even in low moisture soil samples (Wierzchos et al., 2004). In the McMurdo Dry Valleys (henceforth (Wood et al., 2008). Dry Valleys), the largest ice-free region of the Antarctic continent In fact, HTS techniques have revolutionized the traditional (Pointing et al., 2009) and the coldest and driest desert on biodiversity studies based on culturing approaches and opened earth (Wood et al., 2008), the environmental stresses range an array of new opportunities to explore previously hard to access from high variations in temperature (Doran et al., 2002), low environments, as are extreme environments. These approaches nutrient availability and soil moisture (Buelow et al., 2016) to can be used to study both cultured and uncultured diversity, high ultraviolet solar radiation incidence (Perera et al., 2018). being of particular relevance in these types of environments – Under such constraints, embracing the limits of physiological where the unique environmental conditions are hard to mimick adaptability, microorganisms developed specialized strategies in the laboratory. Cultivation-independent studies have revealed to survive, such as the colonization of edaphic and endolithic that in the Dry Valleys, abiotic factors drive the diversity and microenvironments (Walker and Pace, 2007), the entry into structure of microbial communities (Pointing et al., 2009; Lee dormancy states (Goordial et al., 2017) and the biosynthesis et al., 2012; Magalhães et al., 2012). Aridity seem to be the most of secondary metabolites (Wilson and Brimble, 2009; preponderant factor shaping bacterial communites, not only in Tian et al., 2017). Dry Valleys but across multiple deserts (Kastovská et al., 2005; Oligotrophic soils from the Dry Valleys are considered to Pointing et al., 2007; Wood et al., 2008). The type of habitat be microbiologically distinct from all other soils worldwide (lithic vs. soil) also has a preponderant role in shaping the (Fierer et al., 2012b) and High Throughput Sequencing (HTS) bacterial community composition as shown for Antarctic and studies have proved their bacterial diversity is much larger than other hyper-arid deserts (Azúa-Bustos et al., 2011; Stomeo et al., previously thought (Lee et al., 2012; Wei et al., 2016). 2013; Makhalanyane et al., 2015). The Antarctica Dry Valleys soils are usually dominated by Although HTS techniques promised to be able to replace Actinobacteria, the prevalent phylum in cold arid soils (Pointing bacterial culturing (Venter et al., 2004), cultivation techniques et al., 2009; Van Goethem et al., 2016; Goordial et al., 2017). are still necessary to improve taxonomic resolution and even Although the molecular basis behind actinobacteria dominance diversity coverage (Lagier et al., 2015; Choi et al., 2017; Ramos in cryoenvironments is still unknown (Goordial et al., 2015), et al., 2017), as well as to recover whole genomes or allow metabolic activity at subzero temperatures has been detected physiological and metabolic studies (Sørensen et al., 2002; (Soina et al., 2004). The formation of spores allows the survival Ramos et al., 2018; Lambrechts et al., 2019). The information in desert-like habitats (Mohammadipanah and Wink, 2016) and provided by the bacterial isolates can further allow to understand cyst-like resting forms have been described for non-sporulating the cultivation requirements and develop directed cultivation actinobacteria species (Soina et al., 2004). techniques leading to potential novel discoveries (Ramos et al., In addition to soil environments, rocky niches are of 2018; Tahon et al., 2018). Antarctic bacteria, by evolving in particular relevance in the Dry Valleys ecosystems since they such extreme conditions are particularly interesting in terms provide protection to different biota from harsh environmental of biotechnological applications, including for bioremediation conditions such as intense solar radiation exposure, temperature (Gran-Scheuch et al., 2017; Lee et al., 2018), antimicrobials fluctuations, wind, and desiccation (Cockell and Stokes, 2004; (Gesheva and Vasileva-Tonkova, 2012; Tedesco et al., 2016) and Walker and Pace, 2007). These rock-inhabiting organisms are production of anti-freezing molecules (Muñoz et al., 2017). very important in Dry Valleys because of the extent of rock- Combined approaches encompassing culture dependent and exposed surface, thus accounting largely for the productivity and independent techniques to retrieve a broader bacterial diversity biomass in this system (Omelon, 2008; Pointing and Belnap, have been employed for Antarctic studies