Taxonomic Approach for Species Diversity of Yeasts and Yeasts-like Fungi through D 1 / D 2 Region of Large Subunit Ribosomal DNA Sequences

The identification of yeasts or yeasts-like fungi and verify their diversity are principal aspect for bioindustry and ecosystem sustainability. Taxonomic approach provides identification tool to ensure the taxonomic position of yeasts and yeasts-like fungi which definitely set to utilization concerns. The aim of this study is to understanding the taxonomic position of yeasts and yeasts-like fungi from the distinctive of its sequences relationship. Yeasts and yeasts-like fungi strains were isolated through various culture dependent methods from natural resources samples of Karimun Besar Island, Province of Riau Islands, Indonesia. The identification process was performed through amplifying the accurate DNA-based in D1/D2 region of large subunit (26S) ribosomal DNA. As the result, a total of 85 isolates of yeasts and yeasts-like fungi were obtained with 16 closest related taxa through phylogenetic tree construction. Ascomycetous was the predominating group representing 91% of the total isolates sequences followed by Basidiomycetous (8%) and Zygomycetous (1%). The black yeasts (yeasts-like) known as Aureobasidium melanogenum was predominant species with represent to 54% of total isolates and present in particular habitat. Taxonomically, there are six isolates are represent to be novel taxa candidates which pretend to enhance genetic resources of yeasts and yeasts-like fungi especially from Indonesia. In addition, this information provides specific technique to reach specific yeasts or yeasts-like fungi species in nature by managing the sample collection and culture methods.

divergence fragment around ± 600 bp and sufficient for identify yeasts and yeasts-like species.Meanwhile, Scorzetti et al. (2002) emphasis the combination of some conserve genes like internal transcribed spacer (ITS) was required to analyze certain characteristics and perhaps able to describe a novel species.
Ecological and taxonomic yeasts in some area of Indonesia have been studied but not in outer islands yet (Sjamsuridzal et al., 2010).It considers to exploring the occurrence yeasts and yeasts-like in outer islands in Indonesia such Karimun Besar Island.This island is part of Kepulauan Riau Province as border of Indonesian country between Malaysia and Singapore.In this study, to understand the taxonomic position of the yeasts and yeasts-like fungi, we explore them by molecular approach to construct their diversity through the phylogenetic tree.The diversity of yeasts and yeasts-like fungi are necessary for industry, ecosystem management and genetic resources database from Indonesian natural resources.

Isolation method and preservation
Isolation was conducted in Biosystematic of Yeast Laboratory-Indonesian Culture Collection (InaCC), Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI).The isolation of samples were performed with various methods such as direct plating, serial dilution, balistospores-fall, millipore vacuum, and incubated into enrichment media.The isolation process was performed by following Sumerta and Kanti (2016) steps.The pure isolates were maintained in deep freezing -80 0 C with 10% glycerol and 5% trehalose.Some of this isolates were deposited in InaCC for public collection (http://inacc.biologi.lipi.go.id/).

Molecular identification
Amplification of the D1/D2 region of

INTRODUCTION
Yeasts have been exploited for many decades by scientist.They are important microbes for bioindustry and ecosystem services.They have essential roles on nutrient cycling (Botha, 2011), food (Aidoo et al., 2006), bioenergy (Buijs et al., 2013;Nielsen et al., 2013), health (Chi et al., 2009;Leathers et al., 2015), biological control (Pantelides et al., 2015), and environmental indicator (Vogel et al., 2007).However the number of yeasts and yeasts-like species that already studied, cultivated and preserved are about 1% from expected in nature (Fell et al., 2000).Therefore, many studies are needed to unravel the biodiversity and its ecological roles for bioindustry.Discovering biodiversity and elucidating the taxonomic position of yeasts and yeasts-like fungi are necessary for conservation, quality control, and ecological monitoring (Xu, 2016).Obviously, the biodiversity studies cover construction of taxonomical richness as an exhaustive mapping of community structure and predict their roles in ecosystem (Takashima et al., 2012).
Culture dependent technique may preserve the isolate for further analysis to discover many physiological characters and explore its bioindustry prospect.Thus, the taxonomic identity of the isolate is a compulsory to carry out.In polyphasic identification, taxa position of yeasts are commonly using some criteria based on septal morphology, cell wall composition, chemical characteristic, and conserve genes analysis (Fell et al., 2000;Kurtzman et al., 2011).Earlier study, identification of yeasts was only used conventional method based on morphological and chemical analyses.Those methods took many time and expensive (Fell et al., 2000;Xu, 2016).Recently, rapid identification method is carried out on DNA sequence amplification which supported by the emergence of scientific community such GenBank for database comparison.Based on conserve genes database, we are able to mapping the taxonomical position of our isolates.The conserve genes are more stable through mutation factor rather than other genes.

Phylogenetic tree analysis
As the taxonomic dependent analysis, sequences data were compared with known type strains.The MAFFT 7.304 (Katoh & Standley, 2013) was operated for multiple alignment then modified manually with MEGA 7.0 (Tamura et al., 2013).The phylogenetic position was constructed by Neighbor-Joining method and Kimura 2-parameter model with partial deletion.Bootstraps value generated by 1000 replication to determine evolutionary distance.Finnaly, the phylogenetic tree was enhanced in iTOL (https://itol.embl.de).

Occurrence of yeasts and yeasts-like fungi
Isolation process of pure cultures was very challenging due to luxurious growth of filamentous fungi.Particularly, isolation of yeasts from soil was very difficult even with adding fungi growth suppressing chemical.Various techniques were carried out to obtain yeasts that has special characters for biofuel and product development as well as to verify its physiological characters to predict its roles in natural habitat.Introduction of enriched media was intended to obtain yeasts and yeasts-like fungi that have ability to ferment glucose, xylan, and xylose.Hence, it also expected as prescreening process to obtain important isolates for bioproduct development and to study its ecological roles.
Occurrence of yeasts and yeasts-like fungi were observed in all types of samples.There are 85 isolates were collected mostly from leaf liters (Table 1).Fourteen isolates from leaf liters and decay woods were shown as candidate to hydrolyze organic material such xylan and xylose.Yeasts and yeasts-like fungi have cellulolytic enzymes that able to decomposing those organic material (Souza et al., 2013;Kanti, 2015).Those abilities would be important for second generation biofuel research (Morais et al., 2013).In other hands, isolation techniques have crucial role to gain particular isolates.Millipore vacuum filtration was the great method to collect yeasts in leaf liters sample.Each sample from natural sources has differences in physic-chemical and biological characteristic.Hence, special isolation techniques were required to culture under artificial condition.As yeasts and yeasts-like fungi have many industrial interests, we have to develop specific technique for success isolation of targeted yeasts and yeastslike fungi.

Identification and phylogenetic analysis
Verification of species richness and abundance of yeasts and yeasts-like fungi were conducted by molecular approach.The 85 isolates were delineated by phylogenetic tree of sequences of D1/D2 region LSU ribosomal DNA (Figure 1).That region was quite variable and difficult to align (Kurtzman & Robnett, 1998).The bootstraps value with 1000 replication was performed to solve it.As the result, the monophyletic tree was constructed with high bootstrap support (>50%) among each clades.Otherwise, the bootstrap value under 50% was healed to showed significant level among each branch.It convinced significant distinct of each other clades.
The phylogenetic tree delineates the relationship among yeasts and yeasts-like fungi with their type strains to propose certain position.Most of isolates represent to Ascomycetous 91%, Basidiomycetous (8%) and only 1% member of Zygomycetous (Figure 1).
In total, they were classified to 16 closest related species with strong bootstraps.The strong bootstraps were provided by high homology among 99-100% in similarity analysis result.Thirteen isolates were recognized to true yeasts which closely related to Saccharomyces cerevisiae, Candida glabrata, Schwanniomyces polymorphus, Cyberlindera fabianii, Kodamaea ohmeri, Candida orthopsilopsis, Sporobolomyces poonsokiae, Kazachtania africana, Wickerhamomyces rabaulensis, Pichia kudriavsevii, Pseudozyma hubeiensis, Bullera formosensis, and Kluveromyces aestuarii.Three species were belonged to yeasts-like fungi i.e.Aureobasidium melanogenum, Aureobasidium thailandense, and Umbelopsis ovata.In other hands, there are 6 isolates were showed poor similarity analysis result.It was displayed on Figure 1 which were labeled to A and B. The tree on those labels was pruned to Figure 2 with some additional closely type strains.Isolates number Y15Kr022, Y15Kr023, Y15Kr024, Y15Kr026, and Y15Kr027 were 92% closely related to Candida ghanaensis CBS: 8798 (acc.number KY106464).They were clustered to strong sister clade (Figure 2A) The high bootstraps emphasized they were quite different than closest related species and supposed to be novel taxa.Meanwhile isolate Y15Kr055 was delineated a distinct branch with Rodotorula clade (Figure 2B).Based on its similarity analysis result, it was 96% closely related to Rhodotorula paludigena CBS 6566 (acc.number NG_042383).That's mean Y15Kr055 could be recognized to novel species in Rhodotorula group.On these cases, further study should be conducted to ensure those claims such amplify another conserve regions, and chemotaxonomy test.Internal transcribed spacer (ITS) is one of common region that can be used to recognize them into species level (Scorzetti et al., 2002).Molecular approaches analyses are effective and efficient method for identification than phenotypic approaches.Earlier studies showed that phenotypic characters of yeasts are variable and difficult to define genera level than filamentous fungi (Takashima et al., 2012).Combine many approaches able to representing dimorphism among yeasts and filamentous fungi life histories (Fell et al., 2000).In some cases, phenotypic identification does not linier with genotype approach (Kurtzman & Suzuki, 2010).As well found in genera of Aureobasidium (Zalar et al., 2008;Peterson et al., 2013).For example, Rich et al. (2016) study reported that A. pullulans strains which delineated by phylogenetic tree showed they have distinct clade with each color formation and organic material production such as xylanase, feruloyl esterase, and pullulan.As the result, every sample showed different species abundance and species richness (Table 1).Leaf liters sample has most abundance isolates and covered into 10 closely related taxa.The A. melanogenum was dominant representing to 54% of total isolates particularly in leaf liters.They were common in every type of samples except soil and decay woods.Aureobasidium also called yeasts-like fungi or black yeasts due to they able to change color become blackish which formerly it was white, cream, light pink, or light brown colony (Chi et al., 2009).Actually, they occur in usual or extreme condition such presence in absolute anaerobes substrate (Biedunkiewicz et al., 2013).Also Babic et al. (2016) reported that yeast and yeast-like can be found in house hold appliances.Therefore, they can be persisting in many substrates with specific roles (Bhadra et al., 2008).
Certain taxonomic position serves the economic potency information that can be used to initializing research or industrial purposes.Earlier study showed that several species in this study have been used for many bioindustry interests.For example, A. melanogenum isolated from the mangrove ecosystem able to produce biopolymer pullulan (Ma et al., 2015).The basidiomycetous yeast, P. hubeiensis has ability to produce mannosylerythritol lipids (MEL), the biosurfactant for cosmetic (Konishi et al., 2011).The phytase producing yeast such K. ohmeri is important for food and livestock industry (Li et al., 2008).In energy sector, A. melanogenum also able to produces a large amount of long chain alkane oil that can be used for biodiesel production (Liu et al., 2014).Fuel from bioethanol can be conducted to the wild-type strains of S. cerevisiae that ferment glucose, mannose, fructose, and galactose from plant hydrolysate by different pathway (Maris et al., 2006).Meanwhile, P. kudriavsevii has significance ethanol production during high temperature fermentation (Yuangsaard et al., 2013).Other important Ascomycetous yeast, Schwanniomyces polymorphus was able to xylose-fermenting and/or produce xylanase (Morais et al., 2013).
The yeasts and yeasts-like fungi from natural resources of Karimun Island are diverse with many economic and industrial values.The good diversity data facilitates the availability of important yeasts and yeast-like fungi in Indonesia especially in Indonesian public culture collection (InaCC) that we able to access freely for any purposes.In other hands, some strains were taxonomically visible to enhance the genetic database for novel taxa from Indonesian resources.In further, they should be reanalysis for describing their new taxonomic position by using other conserve genes, chemotaxonomy and physiology test.

CONCLUSION
Several taxonomically and economically important yeasts and yeasts-like fungi were isolated from Karimun Besar Island, Indonesia with various culture dependent methods.They were mostly belonging to Ascomycetous yeast which Aureobasidium melanogenum found abundance particularly in leaf liters.Several isolates with taxonomic analysis on D1/D1 large subunit ribosomal DNA sequences supposed to candidate for novel taxa that should to reanalysis in further.

ACKNOWLEDGEMENT
This study was supported by LIPI Project grant on Exploration of Bioresources in Outer Islands of Indonesia fiscal year 2015.The authors express gratitude to Indonesian Culture Collection (InaCC) for its facilities to support this study.We also would like to thanks Mrs. Yeni Yuliani and Mrs. Mia Kusmiati for the great help in field and laboratory work.

Figure 1 .
Figure 1.The Neighbor-Joining phylogenetic tree of yeast and yeast-like fungi diversity in Karimun Besar island.It showed closely related species among Karimun Island's yeasts isolates and type strains.The big grey dots mean strong bootstraps.

BFigure 2 .
Figure 2. Pruned phylogenetic trees of Karimun Besar Island isolates were supposed to novel taxa.A) Strong sister clade of Karimun Besar isolates from Candida ghanaensis CBS: 8798; B) Separately branch of Y15Kr055 with Rhodotorula clade.

Table 1 .
Distribution of yeast and yeast-like species based on isolation method and samples * refer to number of isolates