International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4701–4709
`
`DOI 10.1099/ijsem.0.000634
`
`Correspondence
`Pannida Khunnamwong
`pannida_minn@hotmail.com
`
`Description of Diutina gen. nov., Diutina
`siamensis, f.a. sp. nov., and reassignment of
`Candida catenulata, Candida mesorugosa,
`Candida neorugosa, Candida pseudorugosa,
`Candida ranongensis, Candida rugosa and
`Candida scorzettiae to the genus Diutina
`
`Pannida Khunnamwong,1 Noppon Lertwattanasakul,1
`Sasitorn Jindamorakot,2 Savitree Limtong1,3 and Marc-Andre´ Lachance4
`
`1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand
`2Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology
`(BIOTEC), Pathumthani, Thailand
`3Center for Advanced Studies in Tropical Natural Resources, National Research University
`Kasetsart University, Thailand
`4Department of Biology, University of Western Ontario, London, Ontario, Canada
`
`Three strains (DMKU-RE28, DMKU-RE43T and DMKU-RE123) of a novel anamorphic yeast
`species were isolated from rice leaf tissue collected in Thailand. DNA sequence analysis
`demonstrated that the species forms a sister pair with Candida ranongensis CBS 10861T but
`differs by 24–30 substitutions in the LSU rRNA gene D1/D2 domains and 30–35 substitutions
`in the ITS region. A phylogenetic analysis based on both the small and the large rRNA gene
`subunits confirmed this connection and demonstrated the presence of a clade that also
`includes Candida catenulata, Candida mesorugosa, Candida neorugosa, Candida
`pseudorugosa, Candida rugosa and Candida scorzettiae. The clade is not closely affiliated to
`any known teleomorphic genus, and forms a well-separated lineage from currently recognized
`genera of the Saccharomycetales. Hence, the genus Diutina gen. nov. is proposed to
`accommodate members of the clade, including Diutina siamensis f.a. sp. nov. and the
`preceding seven Candida species. The type strain is DMKU-RE43T (5CBS 13388T5BCC
`61183T5NBRC 109695T).
`
`Abbreviations:
`forma asexualis (asexual
`ITS,
`form);
`f.a.,
`transcribed spacer; LSU, large subunit; SSU, small subunit.
`
`internal
`
`The GenBank/EMBL/DDBJ accession numbers of the SSU and the
`LSU rRNA gene sequences of Diutina siamensis f.a. DMKU-RE43T,
`Diutina mesorugosa CBS 12656T, Diutina rugosa CBS 613T, Diutina
`pseudorugosa CBS 10433T, Diutina neorugosa CBS 12627T, Diutina
`ranongensis CBS 10861T, Diutina catenulata CBS 565T and Diutina
`scorzettiae CBS 10107T are KT336715–KT336722, respectively. The
`MycoBank numbers of Diutina rugosa comb. nov., Diutina catenulata
`comb. nov., Diutina mesorugosa comb. nov., Diutina neorugosa comb.
`nov., Diutina pseudorugosa comb. nov., Diutina ranongensis comb. nov.,
`Diutina scorzettiae comb. nov. and Diutina siamensis f.a., sp. nov. are
`MB 813768, MB 813778, MB 813786, MB 813787, MB 813788,
`MB 813874, MB 813789 and MB 813790, respectively.
`
`A supplementary figure is available with the online Supplementary
`Material.
`
`One mystery of yeast biodiversity is the role played by phylo-
`genetically isolated species that are recovered only infre-
`quently and from an eclectic array of substrates. One
`rightly wonders whether the remote position of rare species
`in the tree of life is an indication of impending extinction, or
`alternatively a sign that their true habitat remains elusive.
`In the last monograph on Candida (Lachance et al., 2011),
`Candida catenulata, Candida rugosa and Candida scorzettiae
`were reported to form an unaffiliated clade with an uncon-
`vincing connection to the genus Kodamaea. The species are
`known from a few isolates from clinical specimens, insects,
`rotting wood, dung, or spoiled food. Three more relatives
`not included in the monograph, Candida mesorugosa, Can-
`dida neorugosa and Candida pseudorugosa, have been iso-
`lated from clinical materials (Li et al., 2006; Paredes et al.,
`
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`P. Khunnamwong and others
`
`2012; Chaves et al., 2013) and another, Candida ranongensis,
`was found in a small number of estuarine water samples in
`Thailand (Am-In et al., 2011). Addition of new taxa did
`little to provide a more satisfactory placement of the clade
`from sequences of the D1/D2 domains of the large subunit
`rRNA gene. These are usually sufficient for the identification
`of existing yeast species and the discovery of new species, but
`a more reliable phylogenetic classification often requires a
`broader sequence sampling, as exemplified in some recent
`studies that used multilocus approaches for classification at
`the level of higher categories (Dea´k & Pe´ter, 2013; Kurtzman
`& Robnett, 2013; Sousa et al. 2014).
`
`Our interest in the placement of the C. catenulata clade
`arose from an investigation of endophytic yeasts in rice
`leaf tissue in Thailand. Endophytic yeasts colonize healthy
`plant tissue without causing any apparent damage (Petrini,
`1991; Azevedo et al., 2000; Oliveira et al., 2012). Examples
`of endophytic species in the Ascomycota include Meyero-
`zyma guilliermondii, Candida oleophila, Candida railenensis,
`Cyberlindnera saturnus, Metschnikowia pulcherrima, Wick-
`erhamomyces anomalus, and Wickerhamiella siamensis
`(Nassar et al., 2005; Isaeva et al., 2010; Rodrı´guez et al.,
`2011; Oliveira et al., 2012; Khunnamwong et al., 2014).
`Endophytes with affinities in the Basidiomycota include
`Cryptococcus albidus, Cryptococcus flavescens, Cryptococcus
`laurentii, Cryptococcus magnus, Papiliotrema siamense,
`Occultifur tropicalis, Rhodotorula graminis, Rhodotorula
`mucilaginosa, Rhodotorula pinicola and Rhodotorula rubra
`(Gai et al., 2009; Abdel-Motaal et al., 2009; Isaeva et al.,
`2010; Khan et al., 2012; Akhtyamova & Sattarova, 2013;
`Khunnamwong et al., 2015; Surussawadee et al., 2014).
`
`Here, we report on three endophytic isolates that represent
`a sister species to C. ranongensis. The previously simple
`matter of providing a phylogeny and a standard description
`of the new species is now complicated by the recent abol-
`ition of the dual nomenclature hitherto used in mycology
`to designate asexual
`forms in a system where generic
`assignments were based on sexual cycle morphology
`(Miller et al., 2011; McNeill et al., 2012). Whereas existing
`members of the C. catenulata clade were formally assigned
`to the polyphyletic genus Candida, new rules dictate that a
`novel species should only be assigned to Candida if it
`belongs to a clade that includes Candida albicans and is
`currently typified by Candida tropicalis (Daniel et al.,
`2014). We therefore propose the genus Diutina gen. nov.
`to accommodate the novel species Diutina siamensis f.a.,
`sp. nov. and the transfer of other clade members to the
`new genus. Our attempts to place the genus Diutina into
`an existing family were fraught with difficulty and we
`choose to leave the family assignment open until the exist-
`ing family structure within the order Saccharomycetales is
`better clarified based on eventual phylogenomic data.
`
`Yeasts
`
`Yeast strains used in this study are listed in Table 1. Three
`(DMKU-RE28, DMKU-RE43T and
`endophytic strains
`
`DMKU-RE123) were isolated from tissue of surface-steri-
`lized rice leaves collected in Thailand by the method of
`Khunnamwong et al. (2014). Briefly, three grams of leaf
`tissue were cut into fragments (3|3 cm) and surface-ster-
`ilized by immersion in 70 % ethanol for 3 min and 5 %
`sodium hypochlorite for 3 min, followed by rinsing five
`times for 5 min in sterile deionized water. The sterilized
`into smaller fragments (0.5|0.5 cm),
`leaves were cut
`ground in a sterile mortar, and placed onto YM agar sup-
`plemented with 0.02 % chloramphenicol. Plates were incu-
`bated at 25 uC until yeast colonies appeared. Strain
`DMKU-RE28 came from a sample collected in Bang Len dis-
`trict, Nakhon Pathom province (14u 39 00 N 100u 109 600 E)
`on 12 January 2012. Strains DMKU-RE43T and DMKU-
`RE123 were obtained from samples collected 2 March 2012
`in U Thong district, Suphan Buri province (14u 249 390 N
`99u 539 220 E) and Kao Liao district, Nakhon Sawan pro-
`vince (15u 539 160 N 100u 59 470 E), respectively. The ex-
`type strains of C. catenulata CBS 565T, C. mesorugosa CBS
`12656T, C. neorugosa CBS 12627T, C. pseudorugosa CBS
`10433T, C. rugosa CBS 613T, and C. scorzettiae CBS 10107T
`were obtained from the Centraalbureau voor Schimmelcul-
`tures (CBS), Utrecht, the Netherlands. Strains UWOPS 91-
`643.2 and UWOPS 91-647.1 had been recovered from Droso-
`phila sp. feeding on soapberries (Sapindus saponaria) in
`Hawaii Volcanoes National Park, Hawaii, in 1991. Two
`strains (RS16 and DMKU-Y-24-3) are from estuarine
`water in Ranong, Thailand and peat swamp forest soil in
`Narathiwat, Thailand, respectively.
`
`DNA sequencing and phylogenetic analysis
`
`The sequences of the internal transcribed spacer (ITS)
`(ITS1-5.8S-ITS2) region and the D1/D2 region of the
`large subunit (LSU) rRNA gene of strains DMKU-RE28,
`DMKU-RE43T and DMKU-RE123 were determined from
`PCR products amplified from genomic DNA, using pri-
`mers ITS1 and ITS4 (White et al., 1990), and NL1 and
`NL4 (Kurtzman & Robnett, 1998), respectively. DNA
`extraction and amplification of the ITS and D1/D2 regions
`were performed as described previously (Limtong et al.,
`2007). The PCR product was checked by agarose gel elec-
`trophoresis and purified using a HiYield Gel/PCR DNA
`Fragments Extraction kit (RBC Bioscience), according to
`the manufacturer’s protocol. The purified product was
`submitted to Macrogen (Korea) for sequencing with the
`same primers. The sequence of the region spanning the
`small-subunit (SSU) rRNA gene through the LSU rRNA
`gene was determined by PCR amplification using the pri-
`mers NS7A, NL5A (Kurtzman & Robnett, 2003), SSU1f,
`SSU4r, SSU3f, SSU2r, LSU3f, LSU4r, LSU5f and LSU2r
`(Rosa et al., 2007). Sequencing templates were amplified
`directly from whole yeast cells and sequenced as described
`by Lachance et al. (1999). Amplified DNA was analysed in
`an ABI sequencer at the John P. Robarts Research Institute,
`London, ON, Canada. The sequences were assembled,
`edited and aligned with MEGA software version 6.06
`(Tamura
`et
`al.,
`2013).
`Phylogenetic
`trees were
`
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`Table 1. Properties of yeast strains examined in this study
`
`Diutina gen. nov.
`
`T, Type strain.
`
`Strain
`
`Diutina rugosa comb. nov.
`CBS 613T5ATCC 10571T
`Diutina catenulata comb. nov.
`CBS 565T
`Diutina pseudorugosa comb. nov.
`XH 1164T5AS 2.3107T5CBS
`10433T
`Diutina scorzettiae comb. nov.
`QuB-82T5CBS 10107T5NRRL
`Y-27665T
`RS16
`DMKU-Y24-3
`UWOPS 91-497.1
`UWOPS 91-493.2
`Diutina ranongensis comb. nov.
`RS28T5BCC 25964T5NBRC
`103861T5CBS
`10861T
`Diutina neorugosa comb. nov.
`UTHSC 10-2054T5CBS
`12627T
`Diutina mesorugosa comb. nov.
`L69DT5CBS 12656T
`Diutina siamensis f.a., sp. nov.
`DMKU-RE28
`
`DMKU-RE43T5BCC
`61183T5NBRC
`109695T5CBS 13388T
`DMKU-RE123
`
`Locality
`
`Source of sample
`
`Reference
`
`Unknown, USA
`
`Faeces of human
`
`Lachance et al. (2011)
`
`Puerto Rico, USA
`
`Faeces of a dysentery patient
`
`Lachance et al. (2011)
`
`Beijing, China
`
`Sputum of an ICU patient with acute
`pneumonia
`
`Li et al. (2006)
`
`Wageningen, Netherlands
`
`Rotten oak wood (Quercus robur L.)
`
`Ranong, Thailand
`Narathiwat, Thailand
`Hawaii, USA
`Hawaii, USA
`
`Estuarine water in a Thai mangrove forest
`Peat swamp forest soil
`Drosophila sp. on soapberry fruit
`Drosophila sp. on soapberry fruit
`
`Middelhoven &
`Kurtzman (2007)
`This study
`This study
`This study
`This study
`
`Ranong, Thailand
`
`Estuarine water in a Thai mangrove forest
`
`Am-In et al. (2011)
`
`San Antonio, Texas,
`USA
`
`Leg wound of female patient
`
`Paredes et al. (2012)
`
`Sao Paulo, Brazil
`
`Blood of comatose ICU patient
`
`Chaves et al. (2013)
`
`Nakhon Pathom,
`Thailand
`Suphan Buri, Thailand
`
`Rice leaf tissue (Oryza sativa)
`
`Rice leaf tissue (Oryza sativa)
`
`This study
`
`This study
`
`Nakhon Sawan,
`Thailand
`
`Rice leaf tissue (Oryza sativa)
`
`This study
`
`reconstructed using the maximum-likelihood (ML) algor-
`ithm included in MEGA software and the MrBayes plugin
`(Huelsenbeck & Ronquist, 2001) of the Geneious (Biomat-
`ters) program. Schizosaccharomyces pombe CBS 356T (Gen-
`Bank accession no. Q698936/ JQ689077) was used as
`outgroup in these analyses.
`
`Growth characteristics
`
`The growth characteristics (Table 1) were determined by
`replica plating following standard methods (Kurtzman
`et al., 2011). Casein hydrolysis was determined using a
`modification (Phaff et al., 1994) of the medium proposed
`by Ahearn et al. (1968). The medium contained 2.4 %
`skimmed milk powder, 0.3 % beef extract, 0.5 % tryptone,
`0.1 % glucose and 1.5 % agar. Lipid hydrolysis was deter-
`mined on a medium containing 0.4 % Tween 80, 1 % pep-
`tone, 0.1 % yeast extract, 0.5 % NaCl, 0.02 % CaCl2 and
`2 % agar. Growth at various temperatures was determined
`
`with replica plate cultures on YM agar incubated at 4 to
`12 uC at every even-numbered temperature and at one-
`from 28 to 41 uC, at
`degree intervals
`the microbial
`temperature facility of the University of Western Ontario
`Biotron. Ubiquinones were extracted from cells cultured
`in 500 ml Erlenmeyer flasks containing 250 ml yeast
`extract-peptone-dextrose (YPD) broth (1 % yeast extract,
`2 % peptone and 2 % dextrose) on a rotary shaker at
`28 uC for 48 h and purified according to the methods of
`Yamada & Kondo (1973) and Kuraishi et al. (1985). Isopre-
`nologues were identified by HPLC as described previously
`(Limtong et al., 2007). Formation of true hyphae or pseu-
`dohyphae of strains DMKU-RE28, DMKU-RE43T and
`DMKU-RE123 was investigated by cultivation for up to
`7 days on potato dextrose agar (PDA; 20 % potato infu-
`sion, 2 % dextrose and 1.5 % agar) in slide culture at
`25 uC. Ascospore formation was investigated by growing
`strains individually or in pairs on PDA, corn meal agar,
`5 % malt extract agar (5 % malt extract and 1.5 % agar),
`YM agar, and Yeast Carbon Base agar supplemented with
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`P. Khunnamwong and others
`
`0.01 % yeast extract (YCBY) at 25 uC for up to 6 weeks
`with periodic examination under the microscope.
`
`Species delineation and
`phylogenetic placement
`
`Analysis of the sequences of the ITS/5.8S rRNA gene region
`and the D1/D2 region of the LSU rRNA gene indicated that
`the three endophytic yeast strains represent a single species.
`Strains DMKU-RE43T and DMKU-RE28 were identical
`and differed from strain DMKU-RE123 by three nucleotide
`substitutions in the D1/D2 region. The ITS regions of
`strains DMKU-RE43T and DMKU-RE123 were identical
`and differed by a single substitution and an indel from
`that of strain DMKU-RE28. A phylogenetic analysis of con-
`catenated small and large subunit rRNA genes (Fig. 1)
`placed the species next to C. ranongensis CBS 10861T
`but the two differ by 24–30 substitutions (4.7–5.9 %) and
`9–10 indels in the D1/D2 region (511 nt) and by 30–35
`substitutions (7.1–8.3 %) and 21–38 gaps in the ITS
`region (414 nt). We therefore conclude that the endophytes
`represent a distinct sister species to C. ranongensis. The
`analysis further shows that these species have no close affi-
`nity to the genus Candida as it is to be redefined (Daniel
`et al., 2014) and represented in Fig. 1 by Lodderomyces elon-
`gisporus. Instead, the two species form a well-defined clade
`with Candida catenulata, C. mesorugosa, C. neorugosa, C.
`pseudorugosa, C. rugosa, and C. scorzettiae. Consistent
`with this, we propose the genus Diutina gen. nov. to
`accommodate members of the clade.
`
`As the reality and placement of the genus Diutina gen. nov.
`is of some importance, we also subjected the data to a
`Bayesian analysis (Fig. S1, available in the online Sup-
`plementary Material), which confirmed the conclusions
`drawn from the maximum-likelihood tree presented in
`Fig. 1. The cohesiveness of the clade is supported by a pos-
`terior probability of 1 in one case and a bootstrap value of
`92 % in the other. Next arises the question of family assign-
`ment. Our analyses include representatives of all currently
`proposed families
`of
`the
`order
`Saccharomycetales
`(Kurtzman & Robnett, 2013), but fails to link the genus
`Diutina gen. nov. reliably to an existing family. As it hap-
`pens, our analysis puts in question the integrity of the
`family Metschnikowiaceae
`sensu Kurtzman (2011a),
`which is poorly supported by either approach. The maxi-
`mum-likelihood tree presented in Fig. 1 has bootstrap
`values less than 50 % for clades that contain the four
`genera currently assigned to the family Metschnikowiaceae
`(Metschnikowia, Clavispora, Kodamaea and Aciculoconi-
`dium) or these together with Diutina gen. nov. Moreover,
`the Bayesian analysis (Fig. S1) suggests a poorly supported
`(0.71) family Metschnikowiaceae with a paraphyletic struc-
`ture with respect to Diutina gen. nov. In the interest of
`nomenclatural stability, which is a fundamental but too
`often neglected objective of good systematics, we choose
`to leave the family unassigned until a reliable genome-
`based phylogeny is in hand. The weakness of current
`
`family assignments based on phylogeny is substantiated
`by the uncertainty of placement of certain genera. For
`example,
`the genus Hyphopichia, represented here by
`Hyphopichia heimii, pairs up with Wickerhamia fluorescens
`in our maximum-likelihood analysis (Fig. 1), well within
`the family Debaryomycetaceae. At variance with this, our
`Bayesian analysis (Fig. S1) positions the genus Hyphopichia
`in an early emerging position with respect to a clade that
`combines
`the family Metschnikowiaceae with Diutina
`gen. nov., but with low support. In reply to the eventual
`objection that ribosomal genes are not sufficiently reliable,
`it should be noted that two published multilocus analyses
`position Hyphopichia as a sister taxon to the Metschniko-
`wiaceae in one instance (Kurtzman, 2011b) and well
`inside the Debaryomycetaceae in the other (Kurtzman
`and Robnett, 2013). The old cliche´,
`‘more work is
`needed’, appears entirely appropriate.
`
`As part of this study, we examined four strains, from our
`collections, that exhibited an affinity to Diutina gen. nov.
`(DMKU-Y24-3, RS16, UWOPS 91-643.2 and UWOPS
`91-647.1). The four strains were identical
`in their ITS
`and D1/D2 sequences and differed from C. scorzettiae by
`six substitutions in the ITS region and four in the D1/D2
`region. This degree of divergence sits at the very limit of
`what might be considered enough to warrant the descrip-
`tion of a novel species. A neighbour-joining analysis
`based on the sequences of the ITS region and the D1/D2
`region of the five strains (data not shown) showed that
`there is no phylogenetic justification for separation, and
`so the isolates are assigned to C. scorzettiae. The growth
`profiles of the strains supports the species assignment.
`
`Ecology
`
`Members of the proposed genus originate from a diversity
`of sources (Table 1), but an ecological generalization does
`not seem possible in view of the small number of known
`specimens.
`Isolation substrates
`include a leg wound
`(C. neorugosa),
`faeces (C. rugosa and C. catenulata),
`sputum (C. pseudorugosa), blood (C. mesorugosa), rotten
`oak wood
`(C.
`scorzettiae),
`and
`estuarine water
`(C. ranongensis), as well as soil and spoiled food (Li
`et al., 2006; Middelhoven & Kurtzman, 2007; Am-In
`et al., 2011; Lachance et al., 2011; Paredes et al., 2012;
`Chaves et al., 2013). The present study adds three strains
`of a novel species isolated from rice leaf tissue and four
`strains of C. scorzettiae recovered from estuarine water,
`soil, and fruit flies. None of the isolates are thought to be
`the causative agent of disease, although some have been
`recognized as opportunistic pathogens (Li et al., 2006;
`Lachance et al., 2011; Chaves et al., 2013; Kobayashi
`et al., 2013). Limtong & Kaewwichian (2015) have reported
`the isolation of C. rugosa and C. catenulata from the surface
`of rice leaves and the latter was once recovered from a
`stingless bee in Brazil (Lachance et al. 2011). The question
`of the significance of rare, phylogenetically isolated species
`in the want of a defined habitat remains open.
`
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`Diutina gen. nov.
`
`0.02
`
`99
`
`100
`
`63
`
`58
`
`81
`
`64
`
`57
`97
`
`Saccharomyces cerevisiae CBS 1171T (Z75578/JQ689017)
`Saccharomycodes ludwigii CBS 821T (JQ698892/JQ689025)
`Wickerhamomyces canadensis CBS 1992T (EF550438/JQ689007)
`Kuraishia capsulata CBS 1993T (JQ698883/EF550270)
`Pichia membranifaciens CBS 197T (JQ698896/EF550227)
`100
`Martiniozyma abiesophila CBS 5366T (JQ698894/EF550212)
`Candida boidinii CBS 2428T (JQ698882/JQ689009)
`53
`Ambrosiozyma monospora CBS 2554T (JQ698881/EU011590)
`66
`Ogataea minuta CBS 1708T (JQ698880/EU011618)
`76
`Citeromyces matritensis CBS 2764T (JQ698918/JQ689054)
`Nakazawaea holstii CBS 4140T (JQ698919/JQ689055)
`Peterozyma toletana CBS 2504T (JQ698921/JQ689057)
`Pachysolen tannophilus CBS 4044T (JQ698920/JQ689056)
`Cephaloascus fragrans CBS 121.29T (JQ698916/JQ689052)
`Babjeviella inositovora CBS 8006T (JQ698917/JQ689053)
`Debaryomyces hansenii CBS 767T (JQ698910/JQ689041)
`Candida multigemmis CBS 6524T (AB013535/JQ689037)
`Meyerozyma guilliermondii CBS 2030T (JQ698913/JQ689047)
`Millerozyma farinosa CBS 185T (AB054281/JQ689046)
`Scheffersomyces stipitis CBS 5773T (JQ698912/JQ689044)
`Kurtzmaniella cleridarum CBS 8793T (JQ698907/JQ689038)
`Priceomyces haplophilus CBS 2028T (JQ698908/JQ689039)
`Schwanniomyces occidentalis CBS 819T (JQ698911/JQ689042)
`Yamadazyma philogaea CBS 6696T (JQ698914/JQ689048)
`89
`Lodderomyces elongisporus CBS 2605T (JQ698906/JQ689035)
`Spathaspora passalidarum CBS 10155T (DQ232894/JQ689036)
`Wickerhamia fluorescens CBS 4565T (JQ698905/JQ689034)
`Hyphopichia heimii CBS 6139T (JQ698904/JQ689033)
`Saccharomycopsis capsularis CBS 2519T (JQ698884/JQ689010)
`Lipomyces starkeyi CBS 1807T (JQ698932/JQ689072)
`Sporopachydermia lactativora CBS 6192T (JQ698922/JQ689058)
`Nadsonia fulvescens CBS 2596T (JQ698923/JQ689059)
`Trichomonascus petasosporus CBS 9602T (GU597332/JQ689064)
`Diddensiella santjacobensis CBS 8183T (AB018150/JQ689062)
`Middelhovenomyces petrohuensis CBS 8173T (AB018148/JQ689061)
`Middelhovenomyces tepae CBS 5115T (AB018153/JQ689063)
`Ascoidea rubescens CBS 116.35T (JQ698885/JQ689011)
`Kodamaea ohmeri CBS 5367T (GU597327/GU597323)
`Aciculoconidium aculeatum CBS 5578T (JQ698899/JQ689029)
`Clavispora lusitaniae CBS 6936T (JQ698900/JQ689030)
`Metschnikowia bicuspidata CBS 5575T (JQ698902/JQ689032)
`Diutina catenulata CBS 565T (KT336721)
`Diutina ranongensis CBS 10861T (KT336720)
`Diutina siamensis f.a. DMKU-RE43T (KT336715)
`Diutina scorzettiae CBS 10107T (KT336722)
`Diutina mesorugosa CBS 12656T (KT336716)
`Diutina rugosa CBS 613T (KT336717)
`Diutina pseudorugosa CBS 10433T (KT336718)
`Diutina neorugosa CBS 12627T (KT336719)
`Yarrowia lipolytica CBS 6124T (JQ698926/JQ689067)
`Magnusiomyces magnusii CBS 108.12T (JQ698929/JQ689070)
`Phaffomyces opuntiae CBS 7010T (JQ698927/JQ689068)
`Starmerella bombicola CBS 6009T (JQ698924/JQ689065)
`Candida blankii CBS 1898T (GU597329/DQ438223)
`Dipodascus albidus CBS 766.85T (GU597336/GU597326)
`Tortispora caseinolytica CBS 7881T (GU597337/JQ689075)
`Schizosaccharomyces pombe CBS 356T (JQ698936/JQ689077)
`
`78
`100
`
`92
`
`100
`
`92
`
`99
`
`99
`
`100
`
`100
`100
`
`86
`
`59
`
`60
`
`98
`
`Fig. 1. Phylogenetic tree based on concatenated SSU and LSU rRNA genes showing the placement of Diutina siamensis
`f.a. sp. nov. with respect to related species and genera. The tree was reconstructed using the maximum-likelihood method in
`MEGA 6.0 software package. Distances were fitted to Kimura’s two-parameter model. Numbers at the nodes are percentage
`bootstrap values from 1000 pseudoreplicates. GenBank accession numbers are given in parentheses. Schizosaccharomyces
`pombe CBS 356T (GenBank accession nos Q698936/JQ689077) was used as the outgroup. Bar, patristic distance
`of 0.02.
`
`http://ijs.microbiologyresearch.org
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`

`P. Khunnamwong and others
`
`Taxonomy
`
`Description of Diutina Khunnamwong,
`Jindamorakot, Limtong & Lachance gen. nov.
`Diutina (Di.u9ti.na. L. fem. n. Diutina long-lasting, expressing
`the hope that the genus is being thus named in perpetuity and
`will not fall victim to frivolous nomenclatural changes).
`
`Growth is by multilateral budding. Cells are ovoid to ellipsoi-
`dal. Pseudohyphae are produced. Ascospore formation has
`not been observed. Sugars may or may not be fermented.
`Glucose, galactose, ethanol, glucitol, succinic acid, N-acetyl-
`glucosamine and hexadecane are assimilated. Ethylamine,
`lysine and cadaverine are utilized as sole nitrogen sources,
`but nitrate and nitrite are not. DBB and urease reactions
`are negative. Starch-like compounds are not produced.
`
`Phylogenetic placement: Saccharomycetales, Saccharomy-
`cotina, Ascomycota.
`
`The type species of the genus is Diutina rugosa (Anderson)
`Khunnamwong, Jindamorakot, Limtong & Lachance.
`
`The MycoBank number is MB 813756.
`
`Description of Diutina siamensis
`Khunnamwong, Jindamorakot, Limtong &
`Lachance sp. nov.
`Diutina siamensis (si.am.en9sis. N.L. fem. adj. siamensis of
`Siam, the old name of Thailand, where the type strain
`was isolated).
`Growth occurs in YM broth. After 3 days at 25 uC, cells are
`subglobose to elongate (2–3|3–18.5 mm) and occur singly
`or in pairs. Budding is multilateral (Fig. 2a). After 3 days
`on YM agar at 25 uC, the streak culture is white-cream,
`soft with a smooth surface and has an entire margin. Pseu-
`dohyphae and true hyphae are formed abundantly in slide
`culture on PDA after 7 days at 25 uC (Fig. 2b). Ascospore
`formation has not been observed in any of the strains
`singly or in pairs on PDA agar, corn meal agar, 5 % malt
`extract agar or YM agar at 15 uC and 25 uC for up to
`6 weeks. Fermentation is absent. The major ubiquinone is
`Q-9. The growth characteristics are given in Table 2.
`The type strain is DMKU-RE43T, which was isolated from
`rice leaf tissue (Oryza sativa) collected from U Thong dis-
`trict, Suphan Buri province, Thailand. The type strain has
`been deposited at the Centraalbureau voor Schimmelcul-
`tures (CBS), Netherlands, as strain CBS 13388T (5BCC
`61183T5NBRC 109695T).
`
`The MycoBank number is MB 813790.
`
`New species combinations
`
`Diutina rugosa (H. W. Anderson) Khunnamwong,
`Jindamorakot, Limtong & Lachance comb. nov.
`
`Basionym: Mycoderma rugosum, H. W. Anderson. J Infect
`Dis 21, 341 (1917).
`
`Fig. 2. Micrographs of Diutina siamensis f.a., sp. nov. DMKU-
`RE43T. (a) Budding cells on YM agar after 3 days at 25 8C. (b)
`Pseudohyphae formed on PDA after 7 days at 25 8C. Bar, 10 mm.
`
`Type strain: CBS 613T, from human faeces (Table 1).
`
`The MycoBank number is MB 813768.
`
`Diutina catenulata (H. A. Diddens & J. Lodder)
`Khunnamwong, Jindamorakot, Limtong &
`Lachance comb. nov.
`
`Basionym: Candida catenulata H. A. Diddens & J. Lodder.
`Die anaskosporogenen Hefen, Zweite Halfte, 486 (1942).
`Type strain: CBS 565T, from faeces of a dysentery patient
`(Table 1).
`
`The MycoBank number is MB 813778.
`
`Diutina mesorugosa (G. M. Chaves,
`G. R. Tercarioli, A. C. B. Padovan, R. C. Rosas,
`R. C. Ferreira, A. S. Melo & A. L. Colombo)
`Khunnamwong, Jindamorakot, Limtong &
`Lachance comb. nov.
`
`Basionym: Candida mesorugosa G. M. Chaves, G. R.
`Terc¸arioli, A. C. B. Padovan, R. C. Rosas, R. C. Ferreira,
`A. S. Melo & A. L. Colombo. Med Mycol 51, 231 (2013).
`
`4706
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`

`

`Diutina gen. nov.
`
`Table 2. Growth characteristics of species of Diutina gen. nov.
`
`Species: 1, Diutina rugosa comb. nov.; 2, Diutina catenulata comb. nov.; 3, Diutina mesorugosa comb. nov.; 4, Diutina neorugosa comb. nov.; 5,
`Diutina pseudorugosa comb. nov.; 6, Diutina ranongensis comb. nov.; 7, Diutina scorzettiae comb. nov.; 8, Diutina siamensis sp. nov. All species
`assimilate glucose, galactose, ethanol, glucitol, succinate, N-acetylglucosamine and hexadecane, but not inulin, sucrose, raffinose, melibiose, lactose,
`melezitose, methyl a-D-glucoside, sorbose, L-arabinose, D-arabinose, methanol, erythritol, xylitol, galactitol, inositol, citrate, malate, tannin, acetone
`or ethyl acetate. All species utilize ethylamine, lysine and cadaverine as sole nitrogen sources, but not nitrate or nitrite. DBB and urease reactions are
`negative. Starch-like compounds are not produced. Acid formation is absent. All species grow at 28 and 33 8C. All species grow in the absence of
`amino acids, but not in the absence of vitamins. All species grow on YM agar with phosphoric acid, pH 2.5, but not pH 2.0. None of the species
`grow on YM agar with 0.5 % acetic acid or in the presence of 8 % ethanol, or on Yeast Nitrogen Base with 1 % phenylalanine as sole carbon and
`nitrogen source. None of the species hydrolyse gelatin. +, Positive; 2, negative; S, slow; W, weak; V, variable.
`
`Characteristic
`
`Fermentation of glucose
`Assimilation of carbon compounds
`Trehalose
`Maltose
`Starch
`Cellobiose
`Salicin
`Rhamnose
`Xylose
`Ribose
`1-Propanol
`2-Propanol
`1-Butanol
`Glycerol
`Ribitol
`Mannitol
`Lactate
`Gluconate
`Glucono-g-lactone
`D-glucuronate
`2-Ketogluconate
`Glucosamine
`Hexadecane
`Growth on/at:
`NaCl (5%)
`NaCl (10%)
`21)
`Cycloheximide (10 mg l
`21)
`Cycloheximide (100 mg l
`21)
`Cycloheximide (1000 mg l
`21)
`Digitonin (8 mg l
`Cetyl trimethylammonium bromide (CTAB) (50 mg l
`Acetic acid (0.5%)
`Ethanol (6%)
`Growth temperature
`Minimum (8C)
`Maximum (8C)
`Growth at:
`34 8C
`35 8C
`36 8C
`37 8C
`38 8C
`39 8C
`40 8C
`41 8C
`Hydrolysis of:
`Casein
`Tween 80
`
`21)
`
`1
`
`2
`
`2
`2
`w
`2
`
`W
`
`W
`+
`w
`2
`w
`w
`+
`2
`+
`2
`+
`
`W
`2
`2
`2
`s
`
`+
`2
`2
`2
`2
`+
`2
`2
`+
`
`12
`$41
`
`+
`+
`+
`+
`+
`+
`+
`+
`
`2
`2
`
`2
`
`2
`
`2
`2
`2
`2
`2
`2
`2
`w
`w
`2
`2
`w
`2
`w
`2
`
`W
`2
`2
`2
`
`S
`w
`
`+
`2
`+
`
`+
`
`S
`
`S
`2
`2
`2
`
`8
`36
`
`+
`+
`+
`2
`2
`2
`2
`2
`
`+
`2
`
`3
`
`2
`
`2
`2
`2
`2
`2
`2
`+
`w
`+
`w
`w
`s
`2
`+
`2
`2
`2
`
`W
`2
`2
`s
`
`+
`2
`
`W
`2
`2
`
`S
`2
`2
`+
`
`$12
`$41
`
`+
`+
`+
`+
`+
`+
`+
`+
`
`2
`2
`
`4
`
`+
`
`2
`2
`2
`2
`2
`2
`+
`w
`s
`w
`w
`w
`W
`+
`2
`2
`2
`2
`2
`2
`w
`
`+
`
`S
`+
`2
`2
`2
`2
`2
`+
`
`6
`40
`
`+
`+
`+
`+
`+
`+
`+
`2
`
`W
`2
`
`5
`
`+
`
`2
`2
`2
`2
`2
`2
`+
`w
`w
`w
`w
`w
`2
`2
`2
`2
`
`W
`2
`2
`2
`w
`
`+
`
`W
`+
`2
`2
`
`S
`2
`2
`2
`
`6
`$41
`
`+
`+
`+
`+
`+
`+
`+
`+
`
`2
`2
`
`6
`
`2
`
`+
`
`+
`2
`2
`2
`
`W
`w
`+
`+
`s
`+
`w
`2
`+
`+
`2
`
`S
`
`S
`
`S
`2
`+
`
`+
`
`W
`+
`
`+
`2
`2
`2
`+
`
`S
`
`12
`36
`
`+
`+
`w
`2
`2
`2
`2
`2
`
`2
`2
`
`7
`
`2
`
`+/W
`+
`w/s
`W/+/S
`W/2
`2
`w/+/s
`+/w
`+/s
`w/2
`+/w
`w/s
`W/S
`+
`2
`
`V
`
`W/S
`2
`
`W
`2
`+/s
`
`v
`W/2
`+
`
`+
`
`V
`2
`+
`
`V
`2
`
`8
`
`2
`
`S
`+
`w/s
`2
`2
`2
`w/s
`w
`+
`w
`+/w
`s
`W
`+
`2
`W/+
`+
`W/2
`S
`2
`s
`
`w/+
`2
`+
`
`+
`2
`2
`+
`2
`2
`
`8
`33234
`
`12
`38240
`
`v
`2
`2
`2
`2
`2
`2
`2
`
`+
`+
`+
`+
`+
`v
`v
`2
`
`2
`+
`
`2
`S/2
`
`http://ijs.microbiologyresearch.org
`
`4707
`
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`Ex. 1038
`Page 7 of 9
`
`

`

`P. Khunnamwong and others
`
`Type strain: L69DT (5CBS 12656T), from blood of coma-
`tose ICU patient (Table 1).
`
`The MycoBank number is MB 813786.
`
`Diutina neorugosa (K. Paredes, D. A. Sutton,
`J. Cano & J. Guarro) Khunnamwong, Jindamorakot,
`Limtong & Lachance comb. nov.
`
`Education Research Promotion and National Research University
`Project of Thailand, the Graduate School, Kasetsart University, Thai-
`land, and the Natural Sciences and Engineering Research Council of
`Canada. Thanks to Ms. Somjit Am-In (Bioresources Technology
`Unit, National Center for Genetic Engineering and Biotechnology
`(BIOTEC), Pathumthani, Thailand) for the isolation of strain RS16.
`M. A. L. acknowledges the granting of collection permits from the
`Hawaii Volcanoes National Park and the collection assistance of
`J. M. Bowles and W. T. Starmer.
`
`Basionym: Candida neorugosa K. Paredes, D. A. Sutton,
`J. Cano, A. W. Fothergill.
`
`References
`
`S. D. Lawhon, S. Zhang, J. P. Watkins & J. Guarro. J Clin
`Microbiol 50, 2397 (2012).
`Type strain: UTHSC 10-2054T (5CBS 12627T), from leg
`wound of a patient (Table 1).
`
`The MycoBank number is MB 813787.
`
`Diutina pseudorugosa (J. Li, Y. Xu & F. Y. Bai)
`Khunnamwong, Jindamorakot, Limtong &
`Lachance comb. nov.
`
`Basionym: Candida pseudorugosa J. Li, Y. Xu & F. Y. Bai.
`J Clin Microbiol 44, 4486 (2006).
`Type strain: XH 1164T (AS 2.3107T5CBS 10433T), from
`sputum of an ICU patient (Table 1).
`
`The MycoBank number is MB 813788.
`
`Diutina ranongensis (S. Am-In, S. Limtong,
`W. Yongmanitchai & S. Jindamorakot)
`Khunnamwong, Jindamorakot, Limtong &
`Lachance comb. nov.
`
`Basionym: Candida ranongensis S. Am-In, S. Limtong,
`W. Yongmanitchai & S. Jindamorakot. Int J Syst Evol
`Microbiol 61, 454 (2011).
`(5BCC 25964T5NBRC 103861T5CBS
`Type: RS28T
`10861T), from estuarine.
`
`Water, Thailand (Table 1).
`
`The MycoBank number is MB 813874.
`
`Diutina scorzettiae (W. J. Middelhoven & C. P.
`Kurtzman) Khunnamwong, Jindamorakot,
`Limtong & Lachance comb. nov.
`
`Basionym: Candida scorzettiae W. J. Middelhoven & C. P.
`Kurtzman. Antonie van Leeuwenhoek 92, 233 (2007).
`Type strain: CBS 10107T (5NRRL Y-27665T), from rotten
`oak wood (Table 1).
`
`The MycoBank number is MB 813789.
`
`Acknowledgements
`
`This work was supported by the Thailand Research Fund through the
`TRF Research-Team Promotion Grant (RTA5480009), the Royal
`Golden Jubilee PhD Progr