Diversity and Resource Project of Yeast Flora in Japan Based on the Comparison between the Data of Yeast Species Isolated from Iriomote and Rishiri Islands

The islands of Japan stretch from the subarctic to the subtropics, and their geographical features extend from the south to north. Different types of vegetation exist in different areas, because of which there is abundant diversity among the microorganisms living in Japan. Therefore, Japan is considered rich in microorganism resources, such as yeast. However, microorganism resources have not yet been comprehensively investigated.

To understand the diversity and distribution characteristics of yeast flora in Japan, 1,021 yeast strains were isolated from soil and plant samples collected in Iriomote Island in Okinawa Prefecture and Rishiri Island in Hokkaido Prefecture. These isolates were analyzed using the partial sequence of a gene that codes for ribosomal RNA (the D1/D2 domain of the large subunit (LSU) rRNA gene, which is widely used for the identification of yeast species and hereinafter referred to as the D1/D2 domain). Based on this sequence, these isolates were classified into 183 species. This number is equivalent to 14% of the number of yeast species (1,312 species) registered in the fifth edition of The Yeasts: A Taxonomic Study (2011), a standard reference book on yeast taxonomy at present, indicating that a wide variety of yeast species exist in Japan. Approximately half of the 183 species were estimated to be new species. Moreover, only 15 species were isolated in both Iriomote and Rishiri Islands. A significance test^∗1 based on the sequence data showed that yeast species in Japan differ considerably according to the area inhabited.

For the conservation of biological diversity and the continuous use of biological resources, organisms should be analyzed at the genus level, which is one rank higher than species in taxonomy. However, in the current classification of yeast, even when a yeast strain is found to be a new species, the genus to which the yeast strain belongs cannot be identified based on its base sequence owing to the problems existing in the classification system. Therefore, the diversity of yeast is difficult to estimate at the genus level or higher. In the ecological analysis of bacteria, a method has been introduced in which bacteria with sequence similarities below 95% in the 16S rRNA gene are considered different species (Ludwig et al., 1998). However, this method has not yet been applied to the ecological analysis of fungi.

Recently, we proposed a method, in which the D1/D2 domain was analyzed using yeast species that belonged to the genus Saccharomyces, which had been reclassified based on multigene analysis, and allied yeast species (the family Saccharomycetaceae), and the results were compared with each other to clarify the diversity of yeasts at the genus level. In this method, the D1/D2 domain was used as an index of the similarity between base sequences. A rarefaction analysis^∗2 using the index values as thresholds showed that yeast isolates exhibited unique patterns according to the sampling location and the isolation source. Thus, the diversity of yeast differed greatly according to the area where the yeast was isolated at the genus level or higher. Using the index, approximately one-sixth of the species isolated in this study were revealed as candidates for new species.

The "general microorganisms" resource project, which handles various resources of bacteria, archaea, yeasts, and filamentous fungi, must provide resources with the correct scientific names. As is already known, the International Code of Botanical Nomenclature was revised in 2011 (Melbourne Code^∗3). Consequently, dual nomenclature, which has been used for higher fungi including yeasts—according to which genera and species of ascomycota and basidiomycota, whose polytypic life cycles and morphologies coexist, are pragmatically classified and differentiated using different scientific names in the sexual and asexual stages—has been shifted to unified nomenclature (one scientific name for one species). Consequently, some scientific names currently used for yeasts and filamentous fungi will be changed according to the list that will be created by the International Nomenclature Committee. Therefore, we will revise our online catalogue of strains, in which yeasts and filamentous fungi can be searched for using both their current and new names. When a new species that belongs to a new genus is discovered, similar to our study’s methods, a scientific name must be given to the new genus. For this, we are attempting to construct a system to classify genera based on genome sequence data.

We will successively publish strains used in the present study from JCM at http://www.jcm.riken.jp/.

This study was performed based on "Comparing the microbial diversity in subtropical and cold latitudes in Japan with the goal of establishing a database for microbial inventory," the first Research Grant awarded by the Institute for Fermentation, Osaka.

References
Takashima, M, Sugita, T, Van, BH, Nakamura, M, Endoh, R, Ohkuma, M. (2012) Taxonomic richness of yeasts in Japan within subtropical and cool temperate areas. PLoS ONE 7 (11): e50784. doi:10.1371/journal.pone.0050784