Lachancea lanzarotensis sp. nov., an ascomycetous yeast isolated from grapes and wine fermentation in Lanzarote, Canary Islands

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1 International Journal of Systematic and Evolutionary Microbiology (2013), 63, DOI /ijs Lachancea lanzarotensis sp. nov., an ascomycetous yeast isolated from grapes and wine fermentation in Lanzarote, Canary Islands Sara S. González, 1 Julia Alcoba-Flórez 2 and Federico Laich 3 Correspondence Federico Laich flaich@icia.es 1 Bodegas Insulares, S. A. Vereda del Medio, 8, acoronte, Santa Cruz de enerife, Spain 2 Servicio de Microbiología, Hospital Universitario Ntra. Sra de Candelaria, Santa Cruz de enerife, Spain 3 Instituto Canario de Investigaciones Agrarias, Ctra. Boquerón s/n, Valle de Guerra, Santa Cruz de enerife, Spain During the characterization of the microbiota biodiversity associated with grapes and wineries in different bioclimatic conditions of the Canary Islands (Spain), a novel yeast species was isolated from Lanzarote, the driest wine-producing region of the archipelago. Seven strains isolated from grapes, microvinifications and wineries are described. Sequence analysis of the D1/D2 domain of the LSU rdna gene and 5.8S-IS regions revealed that the isolates were phylogenetically a member of the genus Lachancea and are closely related to Lachancea meyersii NRRL Y and Lachancea nothofagi NRRL Y On the basis of morphological, biochemical and physiological characterization and phylogenetic analysis, a novel ascosporogenous yeast species, Lachancea lanzarotensis sp. nov., is proposed. he type strain is L2C-15 (5CBS CEC ) which was isolated from grape berries of Vitis vinifera L. cv. Listán Negro red grape variety in inajo, Lanzarote. he MycoBank no. is MB Introduction Yeasts are part of the natural microbial communities associated with fresh grapes and are the most important micro-organisms in wine production (Fleet et al., 2002).he yeast community on grapes is influenced by biotic and abiotic factors, including climatic conditions (mainly temperature and rainfall), geographical location and vineyard factors (age, size, grape variety and vintage year), vineyard treatments, physical grape damage, microbial vectors, microbial interactions and enzymic activities (Barata et al., 2012). Spontaneous alcoholic fermentation of grape must is characterized by the presence of a high number of yeast genera and species. In grape must and during the early phase of wine fermentation, apiculate species are the most frequent yeasts. As fermentations continue and ethanol concentration increases, Saccharomyces cerevisiae and related Abbreviations: IS, Internal ranscribed Spacer; LSU, Large Subunit; m.a.s.l., metres above sea level; PCR-IS-RFLP, PCR restriction fragment length polymorphism analysis of the rdna 5.8S-internal transcribed spacer region. he GenBank/EMBL/DDBJ accession numbers for the D1/D2 domain of the LSU rdna sequences of Lachancea lanzarotensis L2C-15, FLN2-4, 12BA-11, 5BA-1, CMV1-9, GMV1-4 and GMV1-7 are JX515589, JX515590, JX515591, JX515592, JX515593, JX and JX515595, respectively. he GenBank/EMBL/DDBJ accession number for the 5.8S-IS rdna region of Lachancea lanzarotensis L2C-15 is JX species become the dominant yeasts and complete the process (Fleet & Heard, 1993). Indigenous yeast species from grapes and wine have been described in many wineproducing regions of the world. he Canary Islands, located in the Atlantic Ocean, near the ropic of Cancer, off the African coast of the Western Sahara, have a long tradition of viticulture. he Canarian archipelago comprises seven main islands together with a number of smaller islets. Most Canarian wines are produced in Lanzarote, enerife and La Palma. hese three islands have different bioclimatic and topographic characteristics. he relief is the main factor that affects the local rainfall distribution. In general, precipitation increases across the archipelago from east to west. enerife is situated near the centre of the archipelago and has the largest number of bioclimatic belts (26). Maximum rainfall is recorded on the northern side (up to 500 mm per year), while the southern side is drier (200 mm per year) (del-arco et al., 2006). La Palma, more oceanic and humid (rainfall exceeds 700 mm in most of the area), is the north-westernmost of the Canary Islands and has a wide variety of habitats (23 bioclimatics belts) (del-arco et al., 1999). Lanzarote is the northeasternmost and closest island to Africa coast (100 km). he low altitude does not allow the development of trade wind clouds. herefore, annual precipitation is below 200 mm and a limited number of bioclimatic belts (8) are defined (Reyes-Betancort et al., 2001) G 2013 IUMS Printed in Great Britain On: hu, 23 Nov :01:59

2 Lachancea lanzarotensis sp. nov. During a study of yeast communities associated with grape berries in vineyards and wineries in Lanzarote, enerife and La Palma (Canary Islands, Spain), conventional taxonomic tests and routine PCR restriction fragment length polymorphism analysis of the rdna 5.8S-internal transcribed spacer region (PCR-IS-RFLP) were used for identification. A group of novel yeast strains with identical PCR-IS-RFLP patterns was detected in Lanzarote. Analysis of D1/D2 domains of the large subunit (LSU) rdna and the 5.8-IS sequences of seven isolates indicated that these strains represented a distinct species closely related to Lachancea meyersii and Lachancea nothofagi. On the basis of phenotypic and phylogenetic analysis, a novel species of Lachancea, Lachancea lanzarotensis sp. nov., is proposed. Methods Sample collection and yeasts isolation. Collections were conducted in vineyards and wineries located in different bioclimatic conditions of the Canary Islands. Samples of healthy grapes and wineries were obtained from Lanzarote, enerife and La Palma. Grapes from 34 vineyards and samples from 23 wineries were collected during the 2009 harvest season, taking into account the bioclimatic factors of each island. In enerife, bunches were harvested from 18 vineyards belonging to three different zones and seven different bioclimatic belts ranging from 60 to 1175 m above sea level (m.a.s.l.). Vineyards sampled from this island are included in three wine-growing areas with Denominación de Origen. In La Palma, bunches were harvested from eight vineyards corresponding to three winemaking regions and four different bioclimatic belts ranging from 365 to 1180 m.a.s.l. Samples from Lanzarote were obtained from eight vineyards belonging to three winemaking regions and two different bioclimatic belts ranging from 188 to 390 m.a.s.l. en grape bunches each from red (Listán Negro) and white grape varieties (Listán Blanco, in enerife and La Palma or Malvasía, in Lanzarote) were randomly and aseptically collected from each vineyard and stored in sterile plastic bags. All the samples were kept cool during transport to the laboratory and were then directly processed. Each grape bunch was homogenized in a Stomacher for 2 min and 100 ml of the resulting must was plated on Dichloran Rose Bengal Chloramphenicol agar (DRBC) plates and incubated at 25 uc for 48 h. A total of ten yeast colonies from each plate (corresponding to each bunch) were randomly isolated. In addition a pool of musts elaborated with ten bunches from each variety and vineyard was made. From each pool, aseptic microvinifications were performed in 450 ml sterile bottles with 200 ml must. At the end of the fermentation process, 40 random yeast colonies obtained in DRBC were isolated from each microvinification. Wineries samples were obtained at the beginning, middle and end of the fermentation process, from the same regions described above. From each sample 40 random yeast colonies were isolated from DRBC medium. Subcultivations were performed on YPD agar at 25 uc for 3 days and were subsequently preserved in 30 % (w/v) glycerol at 280 uc. Yeast identification and characterization. Conventional taxonomic tests and routine PCR-IS-RFLP (Esteve-Zarzoso et al., 1999) were used for identification. A group of novel yeast strains with identical PCR-IS-RFLP patterns were detected in samples from Lanzarote and seven strains were selected for phenotypic characterization and sequence analysis (able 1). Physiological, biochemical and ascospore examination of the yeasts were carried out as described by Yarrow (1998). For Gorodkowa and V8 agar media, used to induce the sporulation, two different formulations of each were used: Gorodkowa A agar (0.1 % glucose, 0.5 % NaCl, 1 % peptone, 2 % agar), Gorodkowa B agar (0.25 % glucose, 0.5 % NaCl, 1 % malt extract, 2 % agar), Vegetable juice agar 1 (50 % V8 vegetable juice, 0.7 % yeast extract, 2 % agar) and V8 juice agar (Samson et al., 2010) (17.5 % V8 vegetable juice, 0.3 % CaCO 3, % ZnSO 4 7H 2 O, % CuSO 4 5H 2 O, 2 % agar). All assimilation and fermentation able 1. Origin of strains of Lachancea lanzarotensis sp. nov. isolated from grape berries, microvinifications and wineries of Lanzarote, Canary Islands FGM, Fermented grape must; Mv, Malvasía; LB, Listán Blanco; LN, Listán Negro; LSa-Xeric-UI, Lower-Semiarid Xeric Upper-Inframediterranean; UA-Desertic-UI, Upper-Arid Desertic Upper-Inframediterranean; m.a.s.l., metres above sea level; Lat., Latitude; Long., Longitude. he sampling areas are arranged from SW to NE. Strain Source Sampling area Bioclimatic characteristic 5BA-1 (5CBS CEC 13067) L2C-15 (5CBS CEC ) CMV1-9 (5CBS CEC 13072) 12BA-11 (5CBS CEC 13068) FLN2-4 (5CBS CEC 13069) GMV1-7 (5CBS CEC 13071) GMV1-4 (5CBS CEC 13070) FGM (Mv/LB) from wine cellar Vineyards (LN grapes) FGM (Mv) FGM (LN) from wine cellar FGM (LN) FGM (Mv) FGM (Mv) Altitude (m.a.s.l.) Coordinates (Lat., Long.) La Geria LSa-Xeric-UI u, u inajo UA-Desertic-UI u, u inajo UA-Desertic-UI u, u inajo UA-Desertic-UI u, u Máguez UA-Desertic-UI u, u Ye LSa-Xeric-UI u, u Ye LSa-Xeric-UI u, u On: hu, 23 Nov :01:59

3 S. S. González, J. Alcoba-Flórez and F. Laich tests were performed at 25 uc in duplicate and results were recorded after 1, 2 and 3 weeks. DNA sequence analysis. Nucleotide sequences of the D1/D2 domain of the LSU rdna gene and 5.8S-IS region of the strains L2C-15, CMV1-7, GMV1-4, GMV1-7, FLN2-4, 5BA-1 and 12BA-11 were determined using the methods described by Kurtzman & Robnett (1998). Sequences were deposited in the GenBank database and the strains were deposited in the CBS and CEC culture collections as shown in able 1. Comparisons with sequences from GenBank were performed using BLASN (Altschul et al., 1997). he sequences were initially aligned using the multiple alignment program CLUSAL W version 2.0 (Larkin et al., 2007). he phylogenetic tree was constructed using the neighbour-joining method (based on 1000 bootstrap iterations) in MEGA version 5 software (amura et al., 2011). Results and Discussion All strains tested exhibited similar physiological and morphological characteristics. However some differences were observed between strains in fermentation and assimilation tests for certain compounds. Strain GMV1-4 fermented D-galactose and a,a,-trehalose, but strains L2C- 15, GMV1-7, 12BA-11, 5BA-1, FLN2-4 and CMV1-9 were not fermentative. Strain L2C-15 did not assimilate xylitol, but the other strains did. Strain GMV1-7 was positive for assimilation of 2-keto-D-gluconate, but the other strains were negative. Strains GMV1-7, CMV1-9, GMV1-4, FLN2-4 were positive for growth in ethanol, but strains L2C-15, 12BA-11 and 5BA-1 had a weak growth. AcomparisonofchemotaxonomicpropertiesofL. lanzarotensis sp. nov. with those of other recognized species of the genus Lachancea are shown in able 2. L. lanzarotensis sp. nov., L. meyersii and L. nothofagi, displayed similar morphological and physiological characteristics. However, L. lanzarotensis sp. nov. could be distinguished from L. meyersii by its ability to assimilate D-galactose and from L. nothofagi by the ability to assimilate ethanol as a sole carbon source. For accurate identification of these species, sequencing of the 5.8S-IS rdna regions is needed. As an alternative to sequencing, a restriction analysis PCR-IS- RFLP can be performed. Restriction of the 5.8S-IS rdna regions with enzymes HinfI or DdeIseparatesL. lanzarotensis sp. nov. from L. meyersii and L. nothofagi. he three species have the same amplified product size (680 bp) with primers IS1 and IS4. With the enzyme HinfI the PCR-IS-RFLP pattern of L. lanzarotensis sp. nov. has five fragments ( bp), while L. meyersii and L. nothofagi have four fragments ( bp). With the enzyme DdeI L. lanzarotensis sp. nov. produces two fragments ( bp), while L. meyersii and L. nothofagi produce three fragments ( bp). he PCR-IS-RFLP technique is reproducible, rapid and easy to use for yeast species identification. his study also demonstrates its potential use in the detection of possible new species during yeast population diversity analysis. he seven strains were sequenced in the D1/D2 domain of the LSU rdna gene. Strains L2C-15, GMV1-4, GMV1-7 and CMV1-9 were found to share identical sequences, able 2. Comparison of phenotypic properties of Lachancea lanzarotensis sp. nov. and other recognized species of the genus Lachancea Species: 1, Lachancea lanzarotensis sp. nov. (data from this study); 2, Lachancea meyersii (data from Fell et al., 2004); 3, Lachancea nothofagi (Mestre et al., 2010); 4, Lachancea dasiensis (Lee et al., 2009); 5, Lachancea thermotolerans (Lachance, 2011); 6, Lachancea waltii (Lachance, 2011); 7, Lachancea kluyveri (Lachance, 2011); 8, Lachancea cidri (Lachance, 2011); 9, Lachancea fermentati (Lachance, 2011); 10, Lachancea mirantina (Pereira et al., 2011). +, Positive; 2, negative; W, weak; V, variable; D, delayed growth; S, slow growth. Characteristic Assimilation of: D-Galactose +/W 2 D + V V + L-Sorbose V + V 2 Maltose V +/W S a,a-rehalose W + 2 V + +/W S Melibiose Melezitose V V 2 Inulin 2 +D/2 2 2 V V S Glycerol V +D V + V + DL-Lactate V V +/W S Succinate V 2 V + V 2 Ethanol +/W +D 2 2 V /S 2 Growth with: 0.01 % Cycloheximide % NaCl + + V /S 2 50 % D-Glucose + V V +++ V V + 2 however one nucleotide insertion was found in strains 5BA-1 and FLN2-4, and two nucleotide insertions in strain 12BA-11. A similar analysis was performed by 5.8S-IS rdna sequencing and in this case all the isolates had identical sequences. he phylogenetic tree constructed using the neighbourjoining method based on D1/D2 sequences showed that the novel species clustered with L. meyersii NRRL Y (GenBank accession no. AY645656) and L. nothofagi NRRL Y (GQ411403) (Fig. 1). Strain L2C-15 showed 98.3 % sequence similarity (8 substitutions+2 gaps) with L. meyersii and 97.3 % sequence similarity (16 substitutions) with L. nothofagi. A similar analysis was performed with the 5.8S-IS rdna sequences and the results demonstrated that 5.8S-IS tree was congruent with D1/ D2 tree. In the case of IS locus, strain L2C-15 showed 22 (96.24 % sequence similarity with 2 gaps) and 29 (95.05 % sequence similarity with 3 gaps) substitutions with L. meyersii and L. nothofagi, respectively. BLASN analysis of the nucleotide sequence of the D1/D2 domain of strain L2C-15 with the closest phylogenetic relatives retrieved from the GenBank database showed an identical sequence with Lachancea sp. GJ3L14 (GenBank accession no. FJ527091) which has been isolated from 360 International Journal of Systematic and Evolutionary Microbiology 63 On: hu, 23 Nov :01:59

4 Lachancea lanzarotensis sp. nov Lachancea mirantina CBS (FJ666084) Lachancea sp. GJ3L14 (FJ527091) Lachancea thermotolerans (EU541357) Lachancea sp. CBS 6924 (EF463105) Lachancea meyersii NRRL Y (AY645656) Lachancea nothofagi NRRL Y (GQ411403) Lachancea dasiensis CBS (EU523636) Lachancea thermotolerans NRRL Y-8284 (U69581) Lachancea waltii NRRL Y-8285 (U69582) Lachancea kluyveri NRRL Y (U68552) Lachancea cidri NRRL Y (U84236) Lachancea fermentati NRRL Y-1559 (U84239) Lachancea lanzarotensis L2C-15 (JX515589) Lachancea lanzarotensis FLN2-4 (JX515590) 96 Lachancea lanzarotensis 12BA-11 (JX515591) Lachancea lanzarotensis 5BA-1 (JX515592) Kluyveromyces marxianus NRRL Y-8281 (U94924) Fig. 1. Neighbour-joining phylogenetic tree based on the D1/D2 domain of the LSU rdna sequence of Lachancea lanzarotensis sp. nov. and related species of the Lachancea clade. Kluyveromyces marxianus NRRL Y-8281 (U94924) was used as an outgroup. Bootstrap values (%) were obtained from 1000 replicates. Bar, 0.01 substitutions per site. plants in aiwan; a difference in one position (one insertion) with a group of strains isolated from grapes in Portugal and identified as Lachancea thermotolerans (GenBank accession no. EU541357) by their phenotypic characteristics (Barata et al., 2008); and three positions difference (three substitutions) with Lachancea sp. CBS 6924 (GenBank accession no. EF463105) isolated by van der Walt from garden soil in South Africa (Fig. 1). According to Kurtzman & Robnett (1998), yeast strains with 0 3 nt difference are conspecific or sister species, for this reason and using the proposed phenetic standard these isolates could be considered conspecific. Lachance (2011) discusses in detail the phylogenetic position of strain CBS 6924 in the genus Lachancea, and concludes that this strain deposited in 1975 by van der Walt as a specimen of Kluyveromyces thermotolerans is closely related to L. meyersii, but it is unclear whether it represents a distinct species. According to our phylogenetic tree constructed with the sequences available in GenBank (D1/D2 and 5.8S-IS) this strain could be considered conspecific with the proposed species. However this investigation did not attempt to revise previous taxonomy. Isolation of the strains of L. lanzarotensis sp. nov. only from Lanzarote could probably indicate that the proposed species has better capacity for adaptation or tolerance to desertic bioclimatic conditions. However, in enerife and La Palma samples were obtained from xeric bioclimatic areas on the southern sides of these islands and the proposed species was not detected. For this reason, it is probable that the population of this species is lower in enerife and La Palma, and the sampling protocol employed (10 randomly isolated colonies per bunch) did not allow detection. Also, it must be remembered that this species was isolated by other authors from grapes, plants and soil in different environments (Portugal, aiwan and South Africa), which shows the potential distribution and adaptability in distinct climatic regions. Based on phenotypic and phylogenetic analysis, strains L2C- 15, GMV1-4, GMV1-7, CMV1-9, 5BA-1, FLN2-4 and 12BA-11 represent a novel species of the genus Lachancea, for which the name Lachancea lanzarotensis sp. nov., is proposed. Latin diagnosis of Lachancea lanzarotensis González, Alcoba-Flórez & Laich sp. nov. In medio liquido YM et ME5 % post 2 dies ad 25 uc, cellulae globosae vel subglobosae ( mm), singulae vel binae. Per gemmationem multipolarem reproducentes. Post 1 mensem ad 25 uc sedimentum formatur. Post 7 dies ad 25 uc in agaro YM et ME5 %, colonias cremeas, butyrosa et margine integrae. Pseudohyphae et hyphae non formantur. Asci formantur ex conjugatione inter cellulae et gemmae. Asci continentes 1 2 ascosporas globosae ( mm). Glucosum, maltosum, et sucrosum fermentantur. Galactosum, a,a-trehalosum, lactosum, inulinum et amylum non fermentantur. Assimilantur glucosum, galactosum (infirme), sucrosum, maltosum, a,atrehalosum, methyl a-d-glucosidum, raffinosum, melezitosum, glycerolum (variabile), D-glucitolum (infirme), D-mannitolum, acidum D-gluconicum (infirme), ethanolum (infirme) et palatinosum. Non assimilantur L-sorbosum, D-glucosaminum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L- rhamnosum, cellobiosum, salicinum, arbutinum, melibiosum, lactosum, inulinum, amylum, erythritolum, xylitolum, ribitolum, galactitolum, myo-inositolum, acidum 2-keto-D-gluconicum, acidum 5-keto-D-gluconicum, acidum D-glucuronicum, acidum D-galacturonicum, acidum DL-Iacticum, acidum On: hu, 23 Nov :01:59

5 S. S. González, J. Alcoba-Flórez and F. Laich succinicum, acidum citricum, methanolum, acidum levulinicum, acidum malicum et N-acetyl-D-glucosaminum. Non assimilantur nitratum, nitritum, L-lysinum, glucosaminum et D-prolinum. Amylum non formatur. Ureum non hydrolysatur. Vitaminum externum ad crescentiam necessarium est. Non crescit in medio cum 0.1 % cycloheximido addito. Non crescit in substrato 16 % sal/5 % glucosum continente. Non crescit in 60 % glucosum addito. Maxima temperature crescentiae: 32 uc. ypus stirpis L2C-15 (5CBS CEC ) isolatus ex uva (Vitis vinifera L. cv. Listán Negro) in insula Lanzarote (Hispania). Colección Española de Cultivos ipo (CEC), Valencia, Spain, et Centraalbureau voor Schimmelcultures (CBS), Utrecht, he Netherlands, deposita est. Description of Lachancea lanzarotensis González, Alcoba-Flórez & Laich sp. nov. Lachancea lanzarotensis (lan.za.ro.ten9sis. N.L. fem. adj. lanzarotensis of or belonging to Lanzarote, Spain). After 7 days on 5 % Malt extract (ME5 %) and YM agar at 25 uc, colonies ( mm in diameter) are creamy, butyrous, convex, centrally heaped up (especially on YM) with circular and entire margins, and smooth and glistening surfaces (on ME5 % some isolates are matt) (Fig. 2a). After growth in ME5 % and YM broth at 25 uc for 2 days, cells are usually spherical to subspheroidal ( mm in diameter) and occur singly or in pairs (Fig. 2b). Budding is multilateral. After 1 month at 25 uc, sediment formation is observed. Pseudohyphae and hyphae are not observed on any of the culture media tested. Abundant ascosporulation was observed after 8 days at 25 uc on Gorodkowa B agar, V8 juice agar, ME5 % agar and Acetate agar 1. A minor amount of ascospores were observed on Glucose-Peptone-Yeast extract agar, Restricted growth agar, Gorodkowa A agar and Yeast extract-2 % Glucose agar. Ascospores were not observed on Vegetable juice agar 1, Acetate agar 2 and YM-2 % sodium chloride agar. he asci are formed by conjugation between independent cells or autogamously by bud-mother cell conjugation. Ascospores (1 2 per ascus) are spheroidal ( mm in diameter) with smooth walls and are quickly liberated from the ascus (Fig. 2c & d). Ferments glucose, galactose (variable), maltose, sucrose and a,atrehalose (variable), but not lactose, inulin or starch. Assimilates glucose, galactose (weak), sucrose, maltose, a,a-trehalose, methyl a-d-glucoside, raffinose, melezitose, glycerol (variable), xylitol (variable), D-glucitol (weak), D- mannitol, 2-keto-D-gluconate (variable), D-gluconate (weak), ethanol and palatinose, but not L-sorbose, D- glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, cellobiose, salicin, arbutin, melibiose, lactose, inulin, starch, erythritol, ribitol, galactitol, myo-inositol, 5- keto-d-gluconate, D-glucuronate, D-galacturonate, DL-lactate, succinate, citrate, levulinate, L-malic acid and N- acetylglucosamine. Nitrate, nitrite, L-lysine, glucosamine and D-proline are not assimilated. Does not grow in vitamin-free media. Grows in 50 % glucose, but not in 60 % glucose or in 16 % NaCl. Growth on 10 % NaCl plus 5 % glucose is positive in most strains. All strains are sensitive to 0.01 % cycloheximide and do not grow in medium containing 1 % (v/v) acetic acid or methanol. he maximum temperature for growth is 32 uc. Urease hydrolysis and diazonium blue B reactions are negative. No starch-like substance is produced. Acid production is weak. Fig. 2. Lachancea lanzarotensis sp. nov. strain L2C-15. (a) Colonies on ME5 % agar after 7 days at 25 6C. (b) Budding cells in ME5 % broth for 2 days at 25 6C. (c d) Ascospores produced on V8 juice (c) and ME5 % agar (d) after 8 days at 25 6C. Microscopic characteristics were examined under differential interference contrast (Nomarski) optical microscope. Bars, 2 mm (a) and 10 mm (b d). 362 International Journal of Systematic and Evolutionary Microbiology 63 On: hu, 23 Nov :01:59

6 Lachancea lanzarotensis sp. nov. he type strain, L2C-15 (5CBS CEC ) was isolated from grape berries of Vitis vinifera L. cv. Listán Negro red grape variety in inajo, Lanzarote, Canary Islands, Spain, in Additional reference strains of L. lanzarotensis are 5BA-1 (5CBS CEC 13067), CMV1-9 (5CBS CEC 13072), 12BA-11 (5CBS CEC 13068), FLN2-4 (5CBS CEC 13069), GMV1-7 (5CBS CEC 13071) and GMV1-4 (5CBS CEC 13070). More details are given in able 1. Acknowledgements his work was supported by the Instituto Nacional de Investigación y ecnología Agraria y Alimentaria (INIA) and cofinanced with FEDER funds (Project RA ) at the Instituto Canario de Investigaciones Agrarias. S. S. González is supported by the Agencia Canaria de Investigación, Innovación y Sociedad de la Información, and F. Laich is supported by the Recursos y ecnologías Agrarias in coordination with the Comunidades Autónomas del Plan Nacional de Investigación Científica, Desarrollo e Innovación ecnológica program, financed with the involvement of the European Social Fund. References Altschul, S. F., Madden,. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAS and PSI-BLAS: a new generation of protein database search programs. Nucleic Acids Res 25, Barata, A., González, S. S., Malfeito-Ferreira, M., Querol, A. & Loureiro, V. (2008). Sour rot-damaged grapes are sources of wine spoilage yeasts. FEMS Yeast Res 8, Barata, A., Malfeito-Ferreira, M. & Loureiro, V. (2012). he microbial ecology of wine grape berries. Int J Food Microbiol 153, del-arco, M., Acebes, J. R., Pérez-de-Paz, P. L. & Marrero, M. C. (1999). Bioclimatology and climatophilous vegetation of Hierro (part 2) and La Palma (Canary Islands). Phytocoenologia 29, del-arco, M., Pérez-de-Paz, P. L., Acebes, J. R., González-Mancebo, J. M., Reyes Betancort, J. A., Bermejo, J. A., de-armas, S. & González González, R. (2006). Bioclimatology and climatophilous vegetation of enerife (Canary Islands). Ann Bot Fenn 43, Esteve-Zarzoso, B., Belloch, C., Uruburu, F. & Querol, A. (1999). Identification of yeasts by RFLP analysis of the 5.8S rrna gene and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 49, Fell, J. W., Statzell-allman, A. & Kurtzman, C. P. (2004). Lachancea meyersii sp. nov., an ascosporogenous yeast from mangrove regions in the Bahama Islands. Stud Mycol 50, Fleet, G. & Heard, G. M. (1993). Yeasts-growth during fermentation. In Wine Microbiology and Biotechnology, pp Edited by G. H. Fleet. Philadelphia: Harwood. Fleet, G., Prakitchaiwattana, C., Beh, A. & Heard, G. (2002). he yeast ecology of wine grapes. In Biodiversity and Biotechnology of Wine Yeasts, pp Edited by M. Ciani. India: Research Signpost. Kurtzman, C. P. & Robnett, C. J. (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73, Lachance, M. A. (2011). Lachancea Kurtzman (2003). In he Yeasts, a axonomic Study, 5th edn, vol. 2, pp Edited by C. P. Kurtzman, J. W. Fell &. Boekhout. New York, NY: Elsevier. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors (2007). CLUSAL W and CLUSAL_X version 2.0. Bioinformatics 23, Lee, C. F., Yao, C. H., Liu, Y. R., Hsieh, C. W. & Young, S. S. (2009). Lachancea dasiensis sp. nov., an ascosporogenous yeast isolated from soil and leaves in aiwan. Int J Syst Evol Microbiol 59, Mestre, M. C., Ulloa, J. R., Rosa, C. A., Lachance, M. A. & Fontenla, S. (2010). Lachancea nothofagi sp. nov., a yeast associated with Nothofagus species in Patagonia, Argentina. Int J Syst Evol Microbiol 60, Pereira, L. F., Costa, C. R., Jr, Brasileiro, B.. & de Morais, M. A., Jr (2011). Lachancea mirantina sp. nov., an ascomycetous yeast isolated from the cachaca fermentation process. Int J Syst Evol Microbiol 61, Reyes-Betancort, J. A., Wildpret, W. & León Arencibia, M. C. (2001). he vegetation of Lanzarote (Canary Islands). Phytocoenologia 31, Samson, R. A., Houbraken, J., hrane, U., Frisvad, J. C. & Andersen, B. (2010). Food and Indoor Fungi. CBS Laboratory Manual Series no. 2. Utrecht: CBS-KNAW Fungal Biodiversity Centre. amura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, Yarrow, D. (1998). Methods for the isolation, maintenance and identification of yeasts. In he Yeasts, a axonomic Study, 4th edn, pp Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Elsevier On: hu, 23 Nov :01:59