Kazachstania yasuniensis sp . nov . , an ascomycetous yeast species found in mainland Ecuador and on the Galápagos

Correspondence Stephen A. James steve.james@ifr.ac.uk National Collection of Yeast Cultures (NCYC), Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Colección de Levaduras Quito Católica (CLQCA), Centro Neotropical para Investigación de la Biomasa, Pontificia Universidad Católica del Ecuador, Escuela de Ciencias Biológicas, Quito, Ecuador Imaging and Microscopy Group (IMG), Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

The genus Kazachstania was first proposed by Zubkova in 1971 with the description of Kazachstania viticola, a yeast isolated from fermenting grapes in Kazachstan (Zubkova, 1971).Later in the same decade, the taxonomic status of Kazachstania viticola was reassessed by Von Arx et al. (1977), and it was considered to be a synonym of Saccharomyces dairenensis.However, the genus was reintroduced and redefined in 2003 (Kurtzman, 2003) to accommodate Kazachstania viticola (type species) and 19 other species from the genera Arxiozyma, Kluyveromyces, Pachytichospora and Saccharomyces (sensu lato).This resulted from a detailed multigene sequence analysis carried out by Kurtzman & Robnett (2003) to examine the phylogenetic relationships and genus boundaries of the approximately 80 species belonging to the 'Saccharomyces complex'.In the most recent (fifth) edition of The Yeasts, A Taxonomic Study, 32 species were listed as belonging to the genus (Vaughan-Martini et al., 2011).Since then, the genus has continued to increase in size as additional species have been discovered and described including Kazachstania bromeliacearum, Kazachstania ichnusensis, Kazachstania intestinalis, Kazachstania pyschrophila, Kazachstania rupicola, Kazachstania taianensis and Kazachstania wufongensis (Lee et al., 2009;Chen et al., 2010;Suh & Zhou, 2011;Arau ´jo et al., 2012;Cardinali et al., 2012;Kabisch et al., 2013;Safar et al., 2013).
The GenBank/EMBL/DDBJ accession numbers for the LSU D1/D2 sequences of strains CLQCA 20-132 T , CLQCA  are HG934855, LN650649, HG934856 and HG934857, respectively.Those for the ITS sequences are HG934852, LN650650, HG934853 and HG934854, respectively.Kurtzman (2003) concluded that the newly redefined genus was likely to be provisional, as the species assigned to it were clearly polyphyletic.A similar result was obtained more recently by Vaughan-Martini et al. (2011) using LSU D1/D2 sequences, where at least five separate subgroups/subclades, with varying statistical support, were identified (see Fig. 34.1 of Vaughan-Martini et al., 2011).The overall consensus is that as additional species are discovered and further multigene sequencing is carried out, stronger and more reliable species partitioning can be achieved (Kurtzman, 2003;Lu et al., 2004;Vaughan-Martini et al., 2011).If so, then this in turn will inevitably lead to a reclassification of the genus as presently defined, and result in the creation of a number of new sister genera (Kurtzman, 2003;Vaughan-Martini et al., 2011).
Since 2007, the Coleccio ´n de Levaduras Quito Cato ´lica (CLQCA) has been conducting a yeast bio-prospecting programme to catalogue and characterize the indigenous yeast species present in the many differing ecological habitats found in Ecuador, both on the mainland and on the Gala ´pagos Islands.To date, more than 3000 yeast strains have been collected and a number of novel species have been discovered and formally described, including Candida carvajalis (James et al., 2009), Saturnispora quitensis (James et al., 2011) and most recently Wickerhamomyces arborarius (James et al., 2014).In October 2013, the CLQCA carried out a preliminary study to catalogue the yeast diversity at several sites in the Yasunı ´National Park (Yasunı ´), a 9800 km 2 region of prime Amazonian rainforest habitat situated in eastern Ecuador, approximately 250 km from Quito.Yasunı ´is widely recognized as representing one of the most biologically diverse regions on Earth, and harbours the greatest variety of tree species found anywhere on the planet.Furthermore, many of the plants and animals found there are endemic to the region (e.g. the bat species Lophostoma yasuni).Yasunı ´is also incorporated within the territory of two indigenous tribes of people, the Tagaeri and Taromenan, who live in voluntary isolation from the outside world.One key objective of the Yasunı ´yeast-collecting project was to investigate whether or not it was possible to isolate ethanoltolerant species (e.g.Saccharomyces) in this arboreal habitat.In order to do this, a selective sampling and enrichment method, as developed by Sniegowski et al. (2002), was used.
Here we describe the discovery of seven novel strains representing the genus Kazachstania isolated at separate sites on mainland Ecuador and in the Gala ´pagos archipelago, and provide a formal taxonomic description of a novel species of the genus Kazachstania, Kazachstania yasuniensis sp.nov., to accommodate them.Six ethanoltolerant Kazachstania strains were isolated from substrates collected in the Yasunı ´National Park.All were isolated by enrichment culturing using a medium containing 7.6 % (v/ v) ethanol (Sniegowski et al., 2002).One strain (CLQCA 20-132 T ) was isolated from rotten wood, two strains  were isolated from soil samples, and a further three strains  were isolated from decaying fruits.A seventh strain, CLQCA 24SC-045, was subsequently identified following a re-examination of Kazachstania strains previously collected during a 2009 trip to four of the human-inhabited islands of the Gala ´pagos (i.e.Floreana, Isabela, San Cristobal and Santa Cruz).Strain CLQCA 24SC-045 was found at Los Gemelos (approx.600 m above sea level) on Santa Cruz Island, and was isolated from a sample of rotten wood collected from a daisy tree (Scalesia pedunculata), a tree species endemic to the Gala ´pagos.
The seven yeast strains were characterized biochemically, morphologically and physiologically according to the standard methods described by Kurtzman et al. (2011).Growth temperature testing was determined by cultivation on YM (yeast extract-malt extract) agar.Sporulation tests were performed on corn meal agar, Gorodkowa agar, potassium acetate agar and YM agar, and plates were incubated at 25 u C for 1 month in individual and mixed cultures.
The variable D1/D2 domains of the LSU rRNA gene and ribosomal internal transcribed spacer (ITS) region were amplified by PCR directly from whole yeast cell suspensions as described previously by James et al. (1996).The LSU D1/D2 domain was amplified and sequenced using primers NL1 and NL4 (O'Donnell, 1993).The ITS region was amplified using primers ITS5 and ITS4, and sequenced using these primers as well as internal primers ITS2 and ITS3 (White et al., 1990).The amplified DNA was checked by 1.0 % agarose gel electrophoresis, purified and concentrated using QIAquick PCR purification spin columns (Qiagen).A NanoDrop 1000 spectrophotometer (Thermo Scientific) was used for measuring DNA concentration and samples were sequenced by a commercial sequencing facility (Eurofins MWG Operon, Germany).Sequence traces were edited manually and consensus sequences generated using the program SEQMAN, version 7 (DNASTAR).The LSU D1/D2 sequences were compared pairwise using a FASTA similarity search (Pearson & Lipman, 1988), and were aligned with the sequences of closely related taxa, retrieved from the EMBL sequence database, using the multiple alignment program CLUSTAL W (Thompson et al., 1994) included in the DNAMAN software package, version 5.1.5(Lynnon BioSoft).A phylogenetic tree was reconstructed from the combined sequences of the LSU D1/D2 and ITS regions (including 5.8S rDNA) using the neighbour-joining method (Saitou & Nei, 1987), with the Jukes-Cantor distance measure, and Kazachstania aquatica used as the outgroup species.Confidence limit values were estimated from bootstrap analyses of 1000 replicates (Felsenstein, 1985).
As reported previously by Vaughan-Martini et al. (2011), the four member species of the Kazachstania unispora subclade (viz.Kazachstania aerobia, Kazachstania servazzii, Kazachstania solicola and Kazachstania unispora) are closely related to one another, and form a distinct species group within the genus.Reliable taxonomic resolution of these species of the genus Kazachstania based on LSU D1/D2 sequences is at best limited and in the case of Kazachstania aerobia and Kazachstania solicola impossible.Levels of sequence divergence in this rDNA region range from zero (between Kazachstania aerobia and Kazachstania solicola) to six nt substitutions (between Kazachstania servazzii and Kazachstania unispora).However, despite their close phylogenetic relationships based on LSU D1/D2 sequences, the four current members of the Kazachstania unispora subclade as well as the Ecuadorian novel species can be readily distinguished from one another by ITS sequencing.Levels of ITS sequence divergence are significantly greater, ranging from 22 nt substitutions and 3 indels (in 659 nt) between Kazachstania aerobia and Kazachstania servazzii to 36 nt substitutions and 15 indels (in 671 nt) between Kazachstania solicola and Kazachstania unispora.In the case of the novel species, this taxon differs from its two closest relatives Kazachstania servazzii and Kazachstania unispora by 26 nt substitutions and 7 indels (in 659 nt), and by 28 nt substitutions and 7 indels (655 nt), respectively.Furthermore, the levels of ITS sequence divergence are such that the seven Ecuadorian strains can be differentiated into three separate subgroups based upon the type of substrate from which each was isolated.The two strains isolated from rotten wood (CLQCA 20-132 T and CLQCA 24SC-045) can be distinguished from the two soil strains  based on two nt substitutions in the ITS1 region, and from the three decaying/rotten fruit strains based on three nt substitutions in the ITS2 region.Interestingly, and perhaps rather unexpectedly, CLQCA 20-132 T and CLQCA 24SC-045 were found to have identical ITS sequences, despite the fact that one was isolated on the mainland in eastern Ecuador (CLQCA 20-132 T ) while the other was isolated approximately 1700 km to the west in the Gala ´pagos archipelago.A comparative alignment of the ITS sequences for all seven novel Kazachstania strains is shown in Fig. S1  At present, there are relatively few ITS sequences available for strains belongings to the Kazachstania unispora subclade.In their study of yeast biota involved in silage deterioration, Lu et al. (2004) characterized both Kazachstania aerobia strains (NS14 T and NS26) upon which the species description is based and found them to have identical ITS sequences.Likewise, in their phylogenetic study of the 'Saccharomyces complex', Kurtzman & Robnett (2003) examined two strains of Kazachstania unispora, including the type strain (NRRL Y-1556 T ), and found them to have identical ITS sequences.The ITS sequences for two additional Kazachstania unispora strains, one from fermented orange juice and the other from nasal mucus (GenBank accession nos AF321542 and AF455430, respectively) differ from that of the type strain by a single indel in the ITS2 region.Collectively, these data would suggest that the ITS sequences of these species of the genus Kazachstania are well conserved and exhibit very limited intra-specific variation.This supports our proposal that the seven Ecuadorian strains clearly belong to a distinct species, rather than simply representing South American variants of Kazachstania unispora.
The levels of LSU D1/D2 sequence divergence exhibited by members of the Kazachstania unispora subclade are extremely low (ranging from 0 to 6 nt substitutions), which makes accurate species delineation difficult.However, as Fig. 1 demonstrates, far better and more statistically significant resolution can be achieved by combining LSU D1/D2 and ITS sequences.Using this approach, the four known species along with the novel Ecuadorian species can be readily distinguished from one another.This includes Kazachstania aerobia and Kazachstania solicola which, as reported previously by Wu and Bai (2005), have identical D1/D2 sequences.Indeed, as this and previous studies have shown, ITS sequencing represents a far more reliable method of species discrimination for Kazachstania unispora and its close relatives (Lu et al., 2004;Wu & Bai, 2005).
Based upon the origins of the seven strains reported here, it would seem plausible to speculate that the ecological niche of Kazachstania yasuniensis sp.nov. is possibly an arboreal habitat.Although the strains were isolated from three different substrates, namely decaying fruits, rotten wood and soil, all were found in densely wooded environments.The six Yasunı ´strains were collected at separate sites within the Amazonian rainforest region of eastern Ecuador, while the Gala ´pagos strain was found in a Scalesia forest in the highlands of Santa Cruz Island.
An ecological analysis of the Ecuadorian yeast strains registered in the CLQCA database was performed recently in order to develop a simple mathematical model for calculating how well individual yeast species have adapted to the differing habitats found in Ecuador (Carvajal Barriga et al., 2014).In this study, a set of 881 yeast strains, representing 104 species, was analysed using a mathematical approach which focused on the number of different natural regions of Ecuador each species was found to colonize as well as the number of different types of substrate from which they had been isolated.From these analyses it was possible to calculate the relative specialization index (Si) for each species.The Si value measures the degree of specialization related to the habitats and substrates studied.Thus, the higher the Si value, the more specialized the yeast species.In Ecuador it was possible to find yeast species exhibiting Si values ranging from 0.02 (generalist) to 0.92 (specialist) (Carvajal Barriga et al., 2014).
With regard to Kazachstania yasuniensis sp.nov., the Si value was calculated to be 0.62.This meant it grouped with the majority of yeast species (67 %) analysed which were found to be highly specialized and restricted to a small number of habitats and substrates, both on mainland Ecuador and in the Gala ´pagos archipelago (Carvajal Barriga et al., 2014).Other species sharing the same Si value as Kazachstania yasuniensis sp.nov.included Candida ecuadorensis, Candida natalensis, Candida oleophila, Geotrichum silvicola, Hanseniaspora meyeri, Rhodotorula glutinis and Wickerhamiella occidentalis.Although the actual distribution of each of these species differed from that of Kazachstania yasuniensis sp.nov.
From the same study it was also possible to establish a correlation between the percentage of plant species that were originally from the mainland and which had subsequently migrated, via different means of dispersal (e.g.birds), to the oceanic archipelago.In 1976, Porter (1976) determined that ~30 % of all vascular plant species found in the Gala ´pagos have a Neotropical origin.Remarkably in their more recent study, Carvajal Barriga et al. (2014) identified that 31 % of the yeast species isolated on the Gala ´pagos Islands were also found on mainland Ecuador.This would indicate that plant dispersal may have played an important role in the dispersal of yeast species from the mainland to the archipelago.However, it is as yet unclear as to how a species such as Kazachstania yasuniensis sp.nov., which to date has only been found in the Ecuadorian Amazon, could have been introduced into the Gala ´pagos Islands.Further sampling will be need to be carried out in order to gain a better insight into the origins and distribution of this novel species of the genus Kazachstania, and to establish how it may have been dispersed from the mainland to the Gala ´pagos archipelago.
Physiologically, Kazachstania aerobia, Kazachstania servazzii, Kazachstania solicola, Kazachstania unispora and Kazachstania yasuniensis sp.nov.are very similar to one another.Table S1 lists the key characteristics that can be used to differentiate between these five Kazachstania species.With regard to the novel species, the assimilation of trehalose and ethanol as well as growth on ethylamine hydrogen chloride and sodium chloride (10 %) appear to be variable growth characteristics.Kazachstania yasuniensis sp.nov.differs from its closest genealogical relatives: from Kazachstania unispora (Fig. 1) by its ability to assimilate sucrose (positive or delayed) and inability to grow at 37 u C, and from Kazachstania servazzii (Fig. 1) by its ability to IP: 54.70.40.11 On: Thu, 13 Dec 2018 05:02:37 grow in the presence of 0.01 % cycloheximide and inability to assimilate glycerol.In view of the fact that these five species have such similar overall phenotypic profiles, making accurate discrimination difficult, we strongly recommend that ITS sequencing should be adopted as a more reliable and robust method for determining species identity.
The Kazachstania unispora subclade, which with the description of Kazachstania yasuniensis sp.nov.now comprises five closely related species, represents a distinct and statistically well-supported species group within the genus Kazachstania (Fig. 1).The five species have similar overall phenotypes, and whilst not a distinct characteristic of the subclade each typically forms persistent asci which are transformed directly from vegetative cells and contain one spheroidal ascospore each (Lu et al., 2004;Wu & Bai, 2005;Vaughan-Martini et al., 2011;this study).Results from the present study as well as from previous studies would strongly suggest that these five species represent a separate genus (Lu et al., 2004;Wu & Bai, 2005).However, it is also evident that while the genus as currently defined appears to be polyphyletic (Kurtzman, 2003;Kurtzman & Robnett, 2003;Wu & Bai, 2005;Vaughan-Martini et al., 2011), further multigene sequencing is still required to establish clear and well-defined genus boundaries prior to any future reclassification of these yeasts.
The type strain, CLQCA 20-132 T (5CBS 13946 T 5NCYC 4008 T ), was isolated in October 2013 from a rotten wood sample collected in the Yasunı ´National Park, Ecuador.The Mycobank deposit number is MB810753.
Fig.1.Neighbour-joining dendrogram based on the combined sequences of the LSU D1/D2 and ITS regions (including 5.8S rDNA) of Kazachstania yasuniensis sp.nov.and its closest relatives.Species names are followed by CBS, CLQCA or NRRL strain accession numbers and, respectively, the EMBL/GenBank accession numbers for the LSU D1/D2 and ITS regions.Kazachstania aquatica was used as the outgroup species for the analysis.Bootstrap values of ¢50 %, determined from 1000 replicates, are shown at branch nodes.Bar, 1 base substitution per 100 nt.