TAXONOMIC DESCRIPTION Naseeb et al., Int J Syst Evol Microbiol 2017;67:2046–2052 DOI 10.1099/ijsem.0.002013 Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur Samina Naseeb,1† Stephen A. James,2† Haya Alsammar,1 Christopher J. Michaels,1 Beatrice Gini,1 Carmen Nueno-Palop,2 Christopher J. Bond,2 Henry McGhie,3 Ian N. Roberts2 and Daniela Delneri1,* Abstract Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain. INTRODUCTION of 16 chromosomes [18], and they can be differentiated from one another based on the sequences of their internal The Saccharomyces sensu stricto group is composed of eight transcribed spacer (ITS) and 26S rRNA D1/D2 regions [19, biologically distinct yeast species, namely Saccharomyces 20]. It has been shown that the majority of yeast species can cerevisiae, S. paradoxus, S. cariocanus, S. uvarum, S. mika- be identified from sequence divergence of the D1/D2 tae, S. kudriavzevii, S. arboricola and S. eubayanus [1–6], domain [21]. Sequencing of the ITS1 and D1/D2 regions is and two natural hybrids, namely S. pastorianus [7, 8] and S. therefore routinely used for identifying yeast strains [3, 22– bayanus [9]. S. cariocanus was initially included in the 25]. genus based on karyotyping and reproductive isolation [3]. However, subsequent genome sequence analysis of the only Saccharomyces yeasts have been isolated from a wide variety two known strains (of S. cariocanus) showed them to belong of different substrates including deciduous tree bark, sur- to one of three geographically well-defined populations of S. rounding soil, tree exudates (sap), fruits, insects and vine- paradoxus (i.e. American population) [10, 11]. The most yard grapes [2, 26–28]. S. paradoxus is the most commonly recent phylogenetic analyses of the genus excluded both S. isolated species in nature and has been found globally from cariocanus and S. bayanus, the latter due to it being of natural resources, and most notably from oak trees (Quercus hybrid origin [12, 13]. The cryotolerant yeast S. eubayanus spp.) and surrounding soil [11, 29]. Moreover, S. cerevisae is the latest addition to the genus. This species was first iso- and S. paradoxus have been isolated from the same loca- lated in Nothofagus (southern beech) forests in Patagonia, tions, indicating that populations of the two species coexist Argentina [6], but has since been found in North America, in nature [30, 31]. Saccharomyces hybrids have been often on the Tibetan Plateau and most recently on the North isolated from domesticated environments such as vineyards – Island of New Zealand [14 16]. [32] and breweries, and are known to be associated with fer- The Saccharomyces species are defined by the biological spe- mentation processes for the production of wine and beer. cies concept since they are reproductively isolated via post- The best example of this is the lager yeast S. pastorianus zygotic barriers [3, 10, 17]. All of these species possess (syn. S. carlsbergensis), a cold-adapted S. cerevisiae  S. typical budding shape morphology, have the same number eubayanus alloploid hybrid [6]. Author affiliations: 1Manchester Institute of Biotechnology, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M1 7DN, UK; 2Institute of Food Research, Norwich, Norfolk, UK; 3The Manchester Museum, The University of Manchester, Manchester M13 9PL, UK. *Correspondence: Daniela Delneri, [email protected] Keywords: Saccharomyces; yeast; new species; Saccharomyces jurei; isolation protocol. Abbreviations: ITS, internal transcribed spacer; NJ, neighbour-joining. †These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the 26S rRNA D1/D2 and ITS sequences of D5088T are HG764813 and HG764814, respectively. The MycoBank number for Saccharomyces jurei sp. nov. is MB 819910. Three supplementary tables and one supplementary figure are available with the online Supplementary Material. 002013 ã 2017 IUMS This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. 2046 Naseeb et al., Int J Syst Evol Microbiol 2017;67:2046–2052 To date, most of the Saccharomyces strains held in interna- Morphological and physiological characterization of tional yeast collections (e.g. the Westerdijk Fungal Biodiver- yeasts sity Institute, CBS) have been isolated from substrates The two strains were characterized biochemically, morpho- collected and sampled at low altitudes [26, 30, 33, 34]. Some logically and physiologically according to standard methods species found from higher altitude include S. eubayanus iso- described previously [35]. Growth temperature was deter- lated from the Tibetan Plateau [14] and S. arboricola from mined by cultivation on YM (yeast extract-malt extract) the Qinling Mountains [2]. Consequently, very little is agar. Sporulation tests were performed on cornmeal agar, known about the ecology and geographical distribution of Gorodkowa agar, potassium acetate agar and YM agar, and Saccharomyces yeasts found at higher altitudes and cooler plates were incubated at 25 C for 3–4 weeks in individual conditions. Thus, sampling substrates such as soil and trees and mixed cultures. at higher altitudes may lead to the discovery of new cryoto- lerant yeast strains and species. In this study, we sampled Images of the asci were taken using an Olympus model BH- 2 light microscope and a scanning electron microscope. The oak tree bark and surrounding soil at an altitude of 1000 m above sea level in Saint Auban, France. The yeast commu- asci formed on acetate agar after 5 days at 25 C, and sponta- nity was isolated and the species identities were determined neously broke as result of the general fixing process. by standard ITS and D1/D2 sequencing. Whilst the majority DNA extraction of isolated Saccharomyces were identified as S. paradoxus, two strains (D5088T and D5095) were recovered and found For ITS1 and 26S rRNA D1/D2 sequencing, genomic DNA to represent a novel species belonging to the genus Saccha- was isolated from cultures freshly grown on plates using the romyces. The novel species is named Saccharomyces jurei sp. Masterpure Yeast DNA extraction kit (catalogue no. nov., in memory of the yeast researcher Professor Jure Piš- MPY80200) and following the manufacturer’s protocol. kur. We show here that S. jurei is reproductively isolated DNA yields and A260/A280 ratios were measured using a from other Saccharomyces species by performing genetic Nanodrop spectrophotometer (ND-1000), while DNA crosses and testing for hybrid sterility. Both strains formed purity and integrity were checked by 0.8 % agarose gel viable hybrids with all other Saccharomyces species and electrophoresis. were, as expected from crosses between different biological species, predominantly sterile (with a spore viability ranging DNA sequencing from 0 to 3 %). The variable D1 and D2 domains of the 26S rRNA gene were amplified and sequenced using primers NL1 and NL4 [36]. The ribosomal ITS region was amplified using primers METHODS ITS4 and ITS5, and sequenced using these primers as well Yeast isolation, media and maintenance as internal primers ITS2 and ITS3 [20, 37]. Translation EF- 1aA(TEF1) and RPB2 genes were amplified and sequenced Samples of bark and soil were obtained in July 2013 from as described previously [23]. Other nuclear genes (CAT8, oak trees (Quercus) growing at an altitude of 1000m CYR1, GSY1, MET6 and OPY1) were amplified and above sea level in the Saint Auban region of south-eastern sequenced using previously published primers [32]. The ¢ ¢ France (43 5.2 N 006 44 E). The samples were collected PCR fragments were analysed by standard 1 % agarose gel aseptically and stored in sterile bags or Petri dishes. Equal electrophoresis, purified and concentrated using QIAquick amounts of each bark and soil sample were independently PCR purification spin columns (Qiagen) following the man- placed into one of two 50ml sterile Falcon tubes contain- ufacturer’s instructions. The purified products were ing Sniegowski enrichment medium consisting of 3 g sequenced using the BigDye Terminator Ready Reaction kit, yeast extract, 3 g malt extract, 5 g peptone, 10 g sucrose, version 3.1 (Applied Biosystems) following the manufac- 76ml EtOH, 1mg chloramphenicol and 1ml of 1M HCl turer’s instructions. Sequence traces were edited manually, per litre [31]. The Falcon tubes were tightly capped and and consensus sequences were generated using the program one set was incubated (without agitation) at 30 C, while SEQMAN, version 11 (DNASTAR). The sequences were com- – the other was incubated at 20 C for 20 25days. The tubes pared pairwise