MycoKeys 33: 69-84 (202 | ) r-reviewed open-access journa

doi: 10.3897/mycokeys.83.7 | 156 < MycoKkeys

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New species of Yamadazyma
from rotting wood in China

Wan-Li Gao!, Ying Li', Chun-Yue Chai', Zhen-Li Yan’, Feng-Li Hui’

I School of Life Science and Technology, Nanyang Normal University, Nanyang 473061, China 2 State
Key Laboratory of Motor Vehicle Biofuel Technology, Henan Tianguan Enterprise Group Co., Ltd., Nanyang
473000, China

Corresponding author: Feng-Li Hui (fenglihui@yeah.net)

Academic editor: Thorsten Lumbsch | Received 6 July 2021 | Accepted 6 August 2021 | Published 26 August 2021

Citation: Gao W-L, Li Y, Chai C-Y, Yan Z-L, Hui F-L (2021) New species of Yamadazyma from rotting wood in
China. MycoKeys 83: 69-84. https://doi.org/10.3897/mycokeys.83.7 1156

Abstract

Yamadazyma is one of the largest genera in the family Debaryomycetaceae (Saccharomycetales, Saccharo-
mycetes) with species mainly found in rotting wood, insects and their resulting frass, but also recovered
from flowers, leaves, fruits, tree bark, mushrooms, sea water, minerals, and the atmosphere. In the present
study, several strains obtained from rotting wood in Henan and Yunnan Provinces of China were iso-
lated. Based on morphology and a molecular phylogeny of the rDNA internal transcribed spacer region
(ITS) and the D1/D2 domain of the large subunit (LSU) rDNA, these strains were identified as three
new species: Yamadazyma luoyangensis, Y. ovata and Y. paraaseri; and three previously described species,
Y. insectorum, Y. akitaensis, and Y. olivae. The three new species are illustrated and their morphology
and phylogenetic relationships with other Yamadazyma species are discussed. Our results indicate a high
undiscovered diversity of Yamadazyma spp. inhabiting rotting wood in China.

Keywords
Debaryomycetaceae, phylogeny, rotting wood-inhabiting yeast, taxonomy, Yamadazyma

Introduction

The genus Yamadazyma Billon-Grand (1989) was erected to accommodate 16 spe-
cies previously assigned to the genus Pichia (Billon-Grand 1989). These species
have coenzyme Q-9 as their main ubiquinone, form hat-shaped ascospores, produce

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70 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

pseudohyphae, ferment sugars, and require an exogenous source of vitamins for growth
(Billon-Grand 1989; Kurtzman 2011). However, Yamadazyma was not initially accept-
ed as genus due to a phylogenetic analysis of D1/D2 LSU rDNA strongly suggesting its
polyphyletic nature (Kurtzman and Robnett 1998). Kurtzman and Suzuki (2010) ana-
lyzed phylogenetic relationships among species of Pichia and related genera based on
combined sequences of the D1/D2 LSU rDNA and SSU rDNA, and proposed a new
circumscription for Yamadazyma with six sexual species and 11 asexual species assigned
to the genus Candida (Kurtzman and Suzuki 2010). Yamadazyma was later resolved as a
well-supported monophyletic clade and a generally accepted genus in the family Debar-
yomycetaceae, order Saccharomycetales (Kurtzman and Suzuki 2010; Kurtzman 2011).
The monophyly of the Yamadazyma clade was also supported by combined analysis of
the ITS and D1/D2 LSU rDNA (Groenewald et al. 2011; Haase et al. 2017). In the
fifth edition of The Yeasts: A Taxonomic Study, Yamadazyma philogaea, the type species
of the genus, as well as Y. akitaensis, Y. mexicana, Y. nakazawae, Y. scolyti, Y. triangu-
laris, and 23 Candida species were placed in the Yamadazyma clade (Kurtzman 2011;
Lachance et al. 2011). Since then, a few novel Candida species have been described
from this clade, including C. kanchanaburiensis (Nakase et al. 2008), C. khao-thaluensis,
C. tallmaniae, C. oceani (Burgaud et al. 2011), and C. vaughaniae (Groenewald et al.
2011). In addition, many new species, e.g., Y phyllophila, Y. paraphyllophila, Y. siamen-
sis (Kaewwichian et al. 2013), Y terventina (Ciafardini et al. 2013), Y ubonensis (Jun-
yapate et al. 2014), Y. dushanensis (Wang et al. 2015), Y¥. epiphylla, Y. insecticola (Jinda-
morakot et al. 2015), Y¥. riverae (Lopes et al. 2015), Y¥. barbieri (Burgaud et al. 2016), ¥
endophytica (Khunnamwong and Limtong 2016), Y. kitorensis (Nagatsuka et al. 2016),
Y. laniorum (Haase et al. 2017), and ¥. cocois (Maksimova et al. 2020), have been pro-
posed as part of the genus, and three have already been transferred to Yamadazyma as
new combinations: Y olivae, Y. tumulicola, and Y. takamatsuzukensis (Nagatsuka et al.
2016). The Yamadazyma clade currently consists of 24 species of the genus Yamada-
zyma and 38 asexual species still assigned to the genus Candida, making it one of the
largest genera tentatively assigned to the family Debaryomycetaceae (Groenewald et
al. 2011; Kurtzman 2011; Maksimova et al. 2020). Among 24 species included in this
genus, 7 were sexual morphs, viz. Y¥. akitaensis, Y. mexicana, Y. nakazawae, Y. philogaea,
Y. riverae, Y. scolyti, and Y. triangularis (Kurtzman 2011; Lopes et al. 2015).
Yamadazyma species can be originally found in tropical, subtropical, and temper-
ate regions of different continents, but most known species appear to exist in Asia
and South America (Nakase et al. 2008; Groenewald et al. 2011; Kurtzman 2011;
Lachance et al. 2011; Kaewwichian et al. 2013; Junyapate et al. 2014; Jindamorakot
et al. 2015; Lopes et al. 2015; Wang et al. 2015; Burgaud et al. 2016; Khunnamwong
and Limtong 2016; Nagatsuka et al. 2016). The genus has been heavily studied in
Asia, and 17 species of Yamadazyma were previously reported in Japan and Thailand
(Nakase et al. 2008; Groenewald et al. 2011; Kurtzman 2011; Lachance et al. 2011;
Kaewwichian et al. 2013; Junyapate et al. 2014; Jindamorakot et al. 2015; Wang et al.
2015; Khunnamwong and Limtong 2016; Nagatsuka et al. 2016). By contrast, little
is known about Yamadazyma spp. in China. To date, only three Yamadazyma species

Three novel Yamadazyma species Fal

have been described in China, namely C. diospyri, Y. dushanensis, and Y. paraphyllophila
(Lachance et al. 2011; Kaewwichian et al. 2013; Wang et al. 2015). In this study, we
collected rotting wood samples from Yunnan and Henan Provinces in China. After
isolation and examination, three new species and three known species of Yamadazyma
were identified based on phenotypic characteristics and phylogenetic analysis, increas-
ing the species diversity of Yamadazyma in China.

Materials and methods

Sample collection and yeast isolation

Samples of rotting wood were collected in the Xishuangbanna Primeval Forest Park
(Yunnan Province, China) and the Tianchi Mountain National Forest Park (Henan
Province, China). The Xishuangbanna Primeval Forest Park (21°98'N, 100°88'E) is
500 m above sea level (MASL), with a hot and humid climate. The average annual tem-
perature is between 16 °C and 28 °C, and the average annual rainfall is above 1,100 mm.
The Tianchi Mountain National Forest Park (34°33'N, 112°28'E) is at 850 MASL, with
a continental monsoon climate, average annual temperature of 14-16 °C, and average
annual rainfall between 800 mm and 900 mm. Fifty decayed wood samples were col-
lected during July and August in 2018-2020. The samples were stored in sterile plastic
bags and transported under refrigeration to the laboratory over a period of no more than
24 h. ‘The yeast strains were isolated from rotting wood samples in accordance with the
methods described by Wang et al. (2015). Each sample (1 g) was added to 20 ml sterile
yeast extract-malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% pep-
tone, 1% glucose, pH 5.0 + 0.2) supplemented with 0.025% sodium propionate and
200 mg/L chloramphenicol in a 150 ml Erlenmeyer flask and then cultured for 3-10
days on a rotary shaker. Subsequently, 0.1 ml aliquots of the enrichment culture and ap-
propriate decimal dilutions were spread on YM agar plates and then incubated at 25 °C
for 3-4 days. Different yeast colony morphotypes were then isolated by repeated plating
on YM agar and then stored on YM agar slants at 4 °C or in 15% glycerol at — 80 °C.

Phenotypic study

Morphological and physiological properties were determined according to those used
by Kurtzman et al. (2011). The beginning of the sexual stage was determined by in-
cubating single or mixed cultures of each of the two strains on cornmeal (CM) agar,
5% malt extract (ME) agar, dilute (1:9) V8 agar, or yeast carbon base plus 0.01% am-
monium sulfate (YCBAS) agar at 15 and 25 °C for 6 weeks (Kurtzman 2011; Wang
et al. 2015). The assimilation of carbon and nitrogen compounds and related growth
requirements were tested at 25 °C. The effects of temperature from 25-40 °C were
examined in liquid and agar plate cultures. Photomicrographs were taken using a Leica
DM 2500 microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a Leica

2 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

DFC295 digital microscope color camera, with bright field, phase contrast, and DIC
optics. Novel taxonomic descriptions and proposed names were deposited in Myco-
Bank (http://www.mycobank.org; 8 June 2021) (Crous et al. 2004).

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from the yeast using an Ezup Column Yeast Genomic
DNA Purification Kit, according to the manufacturer’s instructions (Sangon Biotech,
Shanghai, China). The internal transcribed spacer (ITS) and the D1/D2 domain of the
large subunit (LSU) rDNA were respectively amplified using ITS5/ITS4 (White et al.
1990) and NL1/NL4 (Kurtzman and Robnett 1998) primers with cycling conditions
of 94 °C/30 s, 55 °C/50 s, 72 °C/60 s. All the PCR protocols had 35 cycles including
94 °C/5 min initial denaturation and 72 °C/10 min final extension.

The 25 pL total volume of PCR mixture contained 9.5 pL of ddH20, 12.5 uL of
2X PCR Master Mix (TIANGEN Co., China), 1 wL of DNA template, and 1 pL of
forward and reverse primers (10 uM each) in each reaction. PCR amplified products
were checked on 1% agarose electrophoresis gels stained with GoldView I nuclear
staining dye (1 pL/10 mL of agarose). Purification and sequencing of PCR products
were done by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. A consensus se-
quence for each gene region was assembled in SeqMan (DNAStar, Inc., Madison, WI,
USA). The newly-generated sequences were deposited in GenBank (https://www.ncbi.
nlm.nih.gov/genbank/ (accessed on 30 May 2021); Table 1).

Abbreviations:

CBS CBS-KNAW Collections, Westerdijk Fungal Biodiversity Institute, Utre-
cht, The Netherlands;

CECT the Spanish Type Culture Collection, Valencia, Spain;

VICC Vietnam Type Culture Collection, Hanoi, Vietnam;

NYNU_ Microbiology Lab, Nanyang Normal University, Henan, China;

T type strain.

Phylogenetic analysis

The sequences obtained in this study and the reference sequences downloaded from
GenBank (Table 1) were aligned using MAFFT v 7(https://maftt.cbre.jp/alignment/
server/large.html;) (Katoh et al. 2019) and manually edited using MEGA7 (Kumar et al.
2016). The best-fit nucleotide substitution models for individual and combined datasets
were selected using jModel Test v2.1.7 (Darriba et al. 2012) according to the Akaike in-
formation criterion. Phylogenetic analyses of combined gene regions (ITS and D1/D2
LSU) were performed using MEGA7 for maximum parsimony (MP) analysis (Kumar
et al. 2016) and PhyML v3.0 for maximum likelihood (ML) analysis (Guindon et al.
2010). Scheffersomyces coipomoensis (CBS 8178) and Babjeviella inositovora (CBS 8006)
were used as the outgroup taxa based on Haase et al. (2017) and Nagatsuka et al. (2016).

Three novel Yamadazyma species 73

Table |. Sequences used in molecular phylogenetic analysis. Entries in bold are newly generated in this study.

Species Strain no. Locality Sample GenBank accession numbers
ITS D1/D2

Candida aaseri CBS 1913" Norway Sputum AY821838 U45802
C. amphixiae CBS 9877" Panama Beetle EU491501 AY520327
C. andamanensis CBS 10859" Thailand Estuarine water AB525239 AB334210
C. atlantica CBS 5263" Portugal Shrimp egg AJ539368 U45799
C. atmosphaerica CBS 4547? Spain Atmosphere AJ539369 U45779
C. blattariae CBS 9876! Panama Cockroach FJ715435 AY640213
C. buinensis CBS 6796" Papua New Guinea Gelatinous exudate HQ283376 U45778
C. cerambycidarum CBS 98797 Panama Beetle AY964669 AY520299
C. conglobata CBS 20187 - Tubercular lung AJ539370 U45789
C. dendronema CBS 6270! South Africa Frass HQ283365 U45751
C. diddensiae CBS 2214" USA Shrimp AY580315 U45750
C. diospyri CBS 9769" China Kaki fruit AY450919 AY450918
C. endomychidarum CBS 98817 Panama Beetle AY964672 AY520330
C. friedrichii CBS 41147 Germany D-glucitol solution HQ283377 U45781
C. germanica CBS 4105° Germany Atmosphere HQ283366 AF245401
C. gorgasii CBS 9880" Panama Beetle AY964670 AY520300
C. insectorum CBS 6213! South Africa Frass HQ283372 U45791
C. insectorum NYNU 1672 China Rotten wood MZ314279 MZ314278
C. jaroonii CBS 107907 Thailand Frass AB360437 DQ404493
C. kanchanaburiensis CBS 11266! Thailand Mushroom NR_137581 KY106534
C. keroseneae CECT 13058! UK Aircraft fuel FJ235128 FJ357698
C. khao-thaluensis CBS 8535" Thailand Leaf HQ283374 HQ283383
C. koratica CBS 10789" Thailand Frass AB360443 AB354232
C. lessepsii CBS 99417 Panama Beetle AY964671 AY640214
C. membranifaciens CBS 19527 India Urine AJ606465 U45792
C. michaelii CBS 9878" Panama Beetle AY964673 AY520329
C. naeodendra CBS 60327 South Africa Frass AY580316 U45759
C. oceani CBS 11857! Atlantic Ocean Deep-sea coral NR_156008 GU002284
C. pseudoaaseri CBS 111707 Germany Blood culture JN241686 JN241689
C. sinolaborantium CBS 99407 Panama Beetle NR_111343 NG_055206
C. songkhlaensis CBS 107917 Thailand Frass AB360438 DQ404499
C. spencermartinsiae CBS 10894" Seawater Florida FJ008050 FJ008044
C. tallmaniae CBS 8575 French Guiana Flower HQ283378 HQ283385
C. tammaniensis CBS 8504" USA Frass HQ283375 AF017243
C. taylori CBS 8508! Belize Sea water FJ008051 FJ008045
C. temnochilae CBS 99387 Panama Beetle AY964678 AY242344
C. trypodendroni CBS 8505° Canada Beetle FJ153212 AF017240
C. vaughaniae CBS 8583" French Guiana Flower HQ283364 HQ283381
C. vrieseae CBS 10829! Brazil Bromeliad FJ755905 EU200785
Yamadazyma akitaensis CBS 67017 Japan Exudate DQ409164 U45766
Y. akitaensis NYNU 16719 China Rotten wood MZ314281 MZ314280
Y. barbieri CBS 143017 Brazil Sea water LT547714 LT547716
Y. cocois VTCC 920004" Vietnam Fruits of the coconut palm MN764369 MN764369
Y. dushanensis CBS 139147 China Rotten wood KM272249 KM272248
Y. endophytica CBS 141637 Thailand Corn leaf KT307981 KT307981
Y. epiphylla CBS 133847 Thailand Rice leaf LC006082 LC006026
Y. insecticola CBS 13382" Thailand Frass LC006081 DQ400379
Y. kitorensis CBS 14158! Japan Red viscous gel LC060995 LC060995
Y. laniorum CBS 14780' USA Bark KY588337 KY588136
Y. luoyangensis NYNU 201023° China Rotting wood MW365549 MW365545
Y. luoyangensis NYNU 201035 China Rotting wood MZ318445 MZ318422
Y. mexicana CBS 7066" Mexico Agria cactus AB054110 U45797
Y. nakazawae CBS 67007 Japan Exudate EU343867 U45748

74 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

Species Strain no. Locality Sample GenBank accession numbers

ITS D1/D2

Y. olivae CBS 111717 Greece Fermenting olive FJ715432 FJ715430
Y. olivae NYNU 167106 China Rotting wood MZ314288 MZ314282
Y. olivae NYNU 209116 China Rotting wood MZ318443 MZ318444
Y. ovata NYUN 191125" China Rotting wood MT990560 MT990559
Y. ovata NYUN 19130 China Rotting wood MZ318424 MZ318425
Y. ovata NYUN 19116 China Rotting wood MZ318442 MZ318423
Y. paraaseri NYNU 1811114" China Rotting wood MK682794 MK682805
Y. paraaseri NYNU 181033 China Rotting wood MZ318421 MZ318460
Y. paraphyllophila CBS 99287 China, Taiwan Pencil wood leaf AY559447 AY562397
Y. philogaea CBS 6696" South Africa Soil AB054107 U45765
Y. phyllophila GBS12572" Thailand Corn leaf AB734050 AB734047
Y. riverae CBS 141217 Brazil Rotting wood KP900044 KP900043
Y. scolyti CBS 4802" USA Frass EU343807 U45788
Y. siamensis CBS 12573" Thailand Sugarcane leaf AB734049 AB734046
Y. takamatsuzukensis CBS 10916! Japan Air AB365470 AB365470
Y. tenuis CBS 6157 Russia Beetle HQ283371 U45774
Y. terventina CBS 12510° Italy Olive oil JQ247717 JQ247717
Y. triangularis CBS 4094" Japan Tamari soya EU343869 U45796
Y. tumulicola CBS 10917! Japan Stone chamber AB365463 AB365463
Y. ubonensis CBS 128597 Thailand Tree bark NR_155998 AB759913
Scheffersomyces coipomoensis CBS 81787 - - NR_111424 U45747
Babjeviella inositovora CBS 8006" - - NR_111018 U45848

MP analysis was run using a heuristic search option of 1,000 search replicates
with random-addition of sequences and tree bisection and reconnection (TBR) as the
branch-swapping algorithm. Gaps were treated as missing data. Bootstrapping with
1,000 replicates was performed to determine branch support (Felsenstein 1985). Par-
simony scores of tree length (TL), consistency index (CI), retention index (RI), and
rescaled consistency (RC) were calculated for each generated tree. ML analysis was per-
formed using a GTR site substitution model, including a gamma-distributed rate het-
erogeneity and a proportion of invariant sites (Guindon et al. 2010). Branch support
was evaluated using a bootstrapping method of 1,000 bootstrap replicates (Hillis and
Bull 1993). The phylogenies from MP and ML analyses were displayed using Mega7
and FigTree v1.4.3 (Rambaut 2016), respectively. ML and MP bootstrap support val-

ues above 50% are shown as first and second positionS above nodes, respectively.

Results

Molecular phylogeny

The alignment based on the combined nuclear dataset (ITS and D1/D2 LSU) includ-
ed 65 taxa and two outgroup taxa (Scheffersomyces coipomoensis and Babjeviella inosito-
vora), and was comprised of 1,103 characters including gaps (576 for ITS and 527 for
D1/D2 LSU) in the aligned matrix. Of these characters, 351 were constant, 455 vari-
able characters were parsimony-uninformative, and 297 characters were parsimony-

Three novel Yamadazyma species 7,

9000) Fomodagrad Inavangersrs WY M1023
Fanadogu inonangensis SYD FOLOSS
Tomadagma mecca CBS TG
Tamadequina terventing CBS 12510
Formadazy mo duskanensis CBS 13914
S|) Yamadazrma ovara NYNU 19130
PS) Famuadlagemas oats WETS LOL
Fomor aver NYC 191135
Candide my podendrom CBS 8505
00/00 | Fomadagimd paraasert SYN 181033
S508 (— 1Famadagma parassert WEN 1$11114
EO Camda anrert CBS 1913
Candida preudenaserd CHS 11170
Foe | | 6 Candide andamanensts CBS 10859
Candida comgiobara CBS 2018
oeyog Candide insecnortion NY LGT?
Candida muectonim CHS 6213
9000 Famedaqjma nveroe CES 14131
Famuidazima seat CBS 4502
SO07 -—— Yimadasma nes CBS 615
Fomedacirea iene CBS 14750
Camtide kanchurburiensts CBS 11256
soy — Candida denironema CBS 6270
3B 660. |-— Candicia veughaniag CBS 8583
ob Candide diddensiag CBS 2214
Candida maeodeuira CBS 6092
Caniida genmonicr CBS 4105
COR | Fomadayme akties SYN LGTY
4/08 || Tarmadasyma abineensis CBS 670
: Famadasjmea matacowoe CBS 6700
Ov] Tormadam plilogees CBS 6698
Famadasime womens CBS 12859
Ted Famadazyma piylephile CBS 12572
pay. L— Famadiopme poraphy Tephila CBS 9903
Tomas Somencis CBS 19575
—__— Fimeadayma iorensis CBS 14158
ogoo Tamadagina aint ia CHS 133534
Fomadagma endapigtics CBS 14163
él. Candida michaeli CBS D378
Candida gorges CES 9250
rtm 30 Candia cerambrendarum CBS 9879
. Candida lesepat CHS 994]
Candida vriesere CBS 10829
Candida amphtciae CBS 877
=] (eS3) L Candida endanychidarum CBS 9831
Tf Candida borarica CBS 10789
Candida Mioo-rhalvensts CHS $535
ogy | amnndacymma alee WYNT 209116
Famadagma oliver WYN 16T1OM
Formedacimea alte CBS 11171
O05 - Candida membrongfaciens CBS 1952
at Conia friedriciat CHS 4114
eaves 7 TL 998, — Candids songhilaencis CBS 10791
fats Candida jaroormi CBS 10790
Adj Candida @eggai CHS 9769
Candida Alamarive CBS 9876
AT! | | o7s Candia bainensis CBS 6796
. Candida relimaniae CBS 8575
Camda karosengae CECT 13058
5ME1 te Famadaqy ma tumnlicola CBS 10817
Canute tommmaniensis CBS 8504
Oi —Cimaida spencermanincias CBS 10854

Qi L$ Caeticta avionics CBS $263

58) L_concticia amorphnerien CBS 4547

Famundaqane barbiert CBS 14301
TaT4 Candida ocean CBS 11857
og/0g - Candida taylor CBS 8308
Famadacirad tahemacicuten: CBS 1016
Timaacuma cocets WIOC 820004
Tamadaqma invecnicala CHS 15382
Fimundacime mumguians CES 4004
— Candida tamneckilae CHS 9995
5. Candide smolaboraminen CES 904)
Scheferrenices copomaainis CBS 8178
Saijetela inasieverd CHS BONG
10
a |

Figure |. Maximum likelihood phylogenetic tree of Yamadazyma inferred from the combined ITS and
D1/D2 LSU dataset and rooted with Scheffersomyces coipomoensis (CBS 8178) and Babjeviella inositovora
(CBS 8006). ML and MP bootstrap support values above 50% are respectively shown at the first and

second positions. Newly sequenced collections are in blue boldface.

76 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

informative. The heuristic search using MP analysis generated the most parsimonious
tree (TL = 979, CI = 0.297, RI = 0.653, RC = 0.248). The best model applied in the
ML analysis was GIR+1+G. The ML analysis yielded a best scoring tree with a final
optimization likelihood value of —11,006.61. Both methods for phylogenetic tree in-
ference resulted in a similar topology. Therefore, only the best scoring PhyML tree is
shown with BS and BT values simultaneously in Figure 1.

According to the phylogenetic tree (Figure 1), three known species, Yo insecto-
rum, Y. akitaensis, and Y. olivae, were part of Yamadazyma. Yamadazyma luoyangensis,
Y. ovata, and Y. paraaseri are new to science based on the distinct and well-supported
molecular phylogenetic placement and morphological differences with their closest
described relatives (Table 2). Phylogenetically, Y /uoyangensis clustered together with
Y¥. ovata and other species, including Y. mexicana, Y. terventina, Y¥. dushanensis, and C.
trypodendroni, while Y. paraaseri was closely related to C. aaseri with high bootstrap
support (98% ML/98% MP).

Taxonomy

Yamadazyma luoyangensis C.Y. Chai & F.L. Hui, sp. nov.
MycoBank No: 840099
Figure 2

Type. Cuina, Henan Province, Luoyang City, Song County, in rotting wood from a
forest park, September 2020, J.Z. Li & Z.T Zhang (holotype NYNU 201023', culture
ex-type CBS 16666, CICC 33509).

Etymology. The species name /uoyangensis refers to the geographical origin of the
type strain of this species.

Description. The cells are ovoid to ellipsoid (2-4 x 3.5—7 um) and occur singly
or in pairs after being placed in YM broth for three days at 25 °C (Figure 2A). Bud-
ding is multilateral. After three days of growth on YM agar at 25 °C, the colonies
are white to cream-colored, buttery, and smooth, with entire margins. After seven
days at 25 °C ona Dalmau plate culture with CM agar, pseudohyphae are formed,
but true hyphae are not (Figure 2B). Asci or signs of conjugation are not observed
on sporulation media. Glucose, galactose, trehalose, and cellobiose are ferment-
ed, but maltose, sucrose, melibiose, lactose, melezitose, raffinose, D-xylose, and
inulin are not. Glucose, galactose, p-glucosamine, D-ribose, D-xylose, L-arabinose,
p-arabinose, L-rhamnose, sucrose, maltose, trehalose, methyl «-p-glucoside, cel-
lobiose, salicin, arbutin, melezitose, inulin, glycerol, erythritol, ribitol, p-glucitol,
p-mannitol, galactitol, p-glucono-1, 5-lactone, 5-keto-p-gluconate, p-gluconate,
succinate, citrate, and ethanol are assimilated. No growth is observed in L-sorbose,
melibiose, lactose, rafiinose, myo-inositol, 2-keto-p-gluconate, p-glucuronate, DL-
lactate, or methanol. In nitrogen-assimilation tests, growth is present on ethyl-
amine, L-lysine, glucosamine, and p-tryptophan, while growth is absent on nitrate,
nitrite, cadaverine, creatine, creatinine, and imidazole. Growth is observed at 35 °C

Three novel Yamadazyma species fies

10 vim ~ . » 10m
" : 4
=

Figure 2. Yamadazyma luoyangensis (NYNU 201023, holotype) A budding cells after three days in YM
broth at 25 °C B pseudohyphae on cornmeal agar after seven days at 25 °C. Scale bars: 10 um.

Table 2. Physiological characteristics of the new Yamadazyma species and their closely related taxa.

Characteristics Y¥. luoyangensis Y. mexicana Y.ovata C.trypodendroni _ Y. paraaseri C. aaseri
Fermentation of
p-Glucose + + + + - v

Assimilation of

t-Sorbose = a rs a % 2
p-Glucosamine + + + = op -
t-Rhamnose + + a + 7 =
Melibiose = Vv + 4, = =
Lactose = + = * 4+ Vv
Raffinose = + © a J z
Inulin + = = 2 4 *
Xylitol + + #5 = 4
Galactitol + v + es xt
2-Keto-b-Gluconate . S + + st us
Cadaverine 4 n - + ni i

Growth tests
10%Nacl/5%glucose + + + v wy 7 +
Growth at 37 °C - + + = Bye +

+, positive reaction; —, negative reaction; s, slow positive reaction; v, variable reaction; n, data not available.

but not at 37 °C. Growth in the presence of 10% NaCl with 5% glucose is present,
but growth in the presence of 0.01% cycloheximide and 1% acetic acid is absent.
Starch-like compounds are not produced. Urease activity and diazonium blue B
reactions are negative.

Additional isolate examined. Cuina, Henan Province, Luoyang City, Song
County, in rotting wood from a forest park, September 2020, J.Z. Li & Z.T Zhang,
NYNU 201035.

GenBank accession numbers. Holotype NYNU 201023! (ITS: MW365549;
D1/D2 LSU: MW365545); additional isolate NYNU 201035 (ITS: MZ318445; D1/
D2 LSU: MZ318422).

78 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

Notes. Two isolates representing Y /uoyangensis were resolved in a well-sup-
ported clade and are most closely related to Y. mexicana (Figure 1). Yamadazyma
luoyangensis can be distinguished from Y. mexicana based on ITS and D1/D2 LSU
loci (4/592 in ITS and 10/531 in D1/D2 LSU). Physiologically, Y. luoyangensis dif-
fers from Y. mexicana by its ability to assimilate inulin and 5-keto-p-gluconate and
its inability to assimilate lactose, raffnose, and 2-keto-p-gluconate. Additionally,
Y. mexicana grows at 37 °C, while Y luoyangensis does not (Table 2) (Kurtzman
20 11,

Yamadazyma ovata C.Y. Chai & F.L. Hui, sp. nov.
MycoBank No: 840100
Figure 3

Type. Cutna, Henan Province, Luoyang City, Song County, in rotting wood from a
forest park, September 2019, J.Z. Li & Z.T Zhang (holotype NYNU 1911251, culture
ex-type CBS 16655, CICC 33500).

Etymology. The species name ovata refers to the ovoid cell morphology of the
type strain.

Description. ‘The cells are ovoid to ellipsoid (2-3 x 3—6.5 um) and occur singly
or in pairs after growth in a YM broth for three days at 25 °C (Figure 3A). Bud-
ding is multilateral. After three days of growth on YM agar at 25 °C, the colonies
are white to cream-colored, buttery, and smooth with entire margins. After nine
days at 25 °C, on a Dalmau plate culture with CM agar, pseudohyphae consisting
of elongated cells with lateral buds are formed (Figure 3B). True hyphae are not ob-
served. Asci or signs of conjugation are not observed on sporulation media. Glucose,
galactose, and trehalose are fermented, but maltose, sucrose, melibiose, lactose, cel-
lobiose, melezitose, raffinose, D-xylose, and inulin are not. Glucose, galactose, L-sor-
bose, p-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, sucrose, maltose,
trehalose, methyl «-p-glucoside, cellobiose, salicin, melibiose, melezitose, glycerol,
erythritol, ribitol, xylitol, p-glucitol, p-mannitol, p- galactitol, p-glucono-1, 5-lac-
tone, 2-keto-p-gluconate, p-gluconate, succinate, citrate, and ethanol are assimi-
lated. No growth is observed in t-rhamnose, lactose, rafinose, inulin, myo-inositol,
p-glucuronate, pi-lactate, or methanol. In nitrogen-assimilation tests, growth is
present on L-lysine, creatine, glucosamine, and D-tryptophan, while growth is ab-
sent on nitrate, nitrite, ethylamine, cadaverine, creatinine, or imidazole. Growth
is observed at 37 °C, but not at 40 °C. Growth in the presence of 16% NaCl with
5% glucose is present, but growth in the presence of 0.01% cycloheximide and 1%
acetic acid is absent. Starch-like compounds are not produced. Urease activity and
diazonium blue B reactions are negative.

Additional isolates examined. Cuina, Henan Province, Luoyang City, Song
County, in rotting wood from a forest park, September 2019, J.Z. Li & Z.T Zhang,
NYNU 19116, NYNU 19130.

Three novel Yamadazyma species 79

10 pm 10 om

Figure 3. Yamadazyma ovata (NYNU 191125, holotype) A budding cells after three days in YM broth
at 25 °C B pseudohyphae on cornmeal agar after nine days at 25 °C. Scale bars: 10 pm.

GenBank accession numbers. Holotype NYNU 1911257 (ITS: MT990560; D1/
D2 LSU: MT990559); additional isolates NYNU 19116 (ITS: MZ318442; D1/D2
LSU: MZ318423), and NYNU 19130 (ITS: MZ318424; D1/D2 LSU: MZ318425).

Notes. We generated sequences for three isolates of Y. ovata, NYNU 191125,
NYNU 19116, and NYNU 19130. This new species is phylogenetically most closely
related to C. trypodendroni (Figure 1). Yamadazyma ovata can be distinguished from
C. trypodendroni based on ITS and D1/D2 LSU loci (15/565 in ITS and 8/532 in
D1/D2 LSU). Physiologically, Y ovata can be differentiated from C. trypodendroni
based on growth in L-sorbose, p-glucosamine, melibiose, and p-glucono-1, 5-lac-
tone, all of which are positive for Y. ovata and negative for C. trypodendroni (Table 2)
(Lachance et al. 2011).

Yamadazyma paraaseri C.Y. Chai & EL. Hui, sp. nov.
MycoBank No: 840101
Figure 4

Type. Cuina, Yunnan Province, Jinghong City, Mengyang Town, in rotting wood
from a tropical rainforest, July 2018, K.E Liu & Z.W. Xi (holotype NYNU 1811114",
culture ex-type CBS 16010, CICC 33365).

Etymology. The species name paraaseri refers to its phylogenetic similarity to C. aaseri.

Description. ‘The cells are ovoid to elongate (2—2.5 x 3—8.5 um) and occur singly
or in pairs after being placed in YM broth for three days at 25 °C (Figure 4A). Budding
is multilateral. After three days of growth on YM agar at 25 °C, the colonies are white
to cream-colored, buttery, and smooth, with entire margins. After two weeks at 25 °C
on a Dalmau plate culture with CM agar, pseudohyphae consisting of elongated cells
with lateral buds are formed (Figure 4B). True hyphae are not observed. Asci or signs of

80 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

A

B 10 pm

10 pm

Figure 4. Yamadazyma paraaseri (NYNU 1811114, holotype) A budding cells after three days in YM
broth at 25 °C B pseudohyphae on cornmeal agar after two weeks at 25 °C. Scale bars: 10 pm.

conjugation are not observed on sporulation media. Fermentation of sugars is absent.
Glucose, galactose, L-sorbose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arab-
inose, sucrose, maltose, trehalose, methyl «-p-glucoside, cellobiose, salicin, arbutin, lac-
tose, melezitose, inulin, glycerol, erythritol, ribitol, p-glucitol, p-mannitol, p-gluconate,
pL-lactate, succinate, citrate, and ethanol are assimilated. No growth is observed in L-
rhamnose, melibiose, rafhinose, xylitol, galactitol, myo-inositol, p-glucono-1, 5-lactone,
2-keto-p-gluconate, D-glucuronate, or methanol. In nitrogen-assimilation tests, growth
is present on ethylamine, L-lysine, glucosamine, and p-tryptophan, while growth is
absent on nitrate, nitrite, cadaverine, creatine, creatinine, and imidazole. Growth is
observed at 37 °C but not at 40 °C. Growth in the presence of 0.01% cycloheximide,
10% NaCl with 5% glucose and 1% acetic acid is absent. Starch-like compounds are
not produced. Urease activity and diazonium blue B reactions are negative.

Additional isolate examined. Cuina, Yunnan Province, Jinghong City, Meng-
yang Town, in rotting wood from a tropical rainforest, July 2018, K.E Liu & Z.W. Xi,
NYNU 181033.

GenBank accession numbers. Holotype NYNU 1811114! (ITS: MK682794;
D1/D2 LSU: MK682805); additional isolate NYNU 181033 (ITS: MZ318421; D1/
D2 LSU: MZ318460).

Notes. Two strains representing Y paraaseri were clustered in a well-supported
clade and were phylogenetically related to C. aaseri [7]. Yamadazyma paraaseri can
be distinguished from C. aaseri based on ITS and D1/D2 LSU loci (8/573 in ITS
and 8/531 in D1/D2 LSU). Physiologically, the ability to assimilate p-glucosamine
and inulin and the inability to assimilate xylitol and p-glucono-1, 5-lactone are the
primary differences between Y. paraaseri and its closest relative, C. aaseri. Additionally,
C. aaseri can grow in 10% NaCl with 5% glucose, while Y paraaseri cannot (Table 2)
(Lachance et al. 2011).

Three novel Yamadazyma species 81

Discussion

In this work, six Yamadazyma species were identified based on morphology and mo-
lecular phylogeny. All species were isolated from rotting wood collected in Henan
and Yunnan Provinces, China. Yamadazyma luoyangensis, Y. ovata, and Y. paraaseri are
proposed as new species in Yamadazyma due to their well-supported phylogenic posi-
tions and distinctive physiological traits. Also, three known species of Yamadazyma, Y.
insectorum, Y. akitaensis, and Y. olivae, were clearly identified by both morphological
and molecular approaches.

In the past, methods of species identification of Yamadazyma were based only
on morphology and physiological characters such as the shape of ascospores and
reactions in standard growth and fermentation tests (Billon-Grand 1989). Recent
molecular phylogenetic analyses demonstrate that determining species boundaries
using only morphology and physiological characters is not possible due to their
variability under changing environmental conditions (Kurtzman 2011; Lachance
et al. 2011). D1/D2 LSU sequence is an appropriate marker to identify species of
Yamadazyma species through phylogenetic analysis, as revealed by Kurtzman and
Robnett (1998). Many Yamadazyma species are described based on a polyphasic
approach together with morphological and physiological characterization (Suh et
al. 2005; Kurtzman 2007; Imanishi et al. 2008; Nagatsuka et al. 2009; Am-In et
al. 2011). However, none to only two substitutions are present in D1/D2 LSU
sequences of the ex-type strains of the closest related species within Yamadazyma,
such as C. diddensiae and C. naeodendra, Y. akitaensis and Y. nakazawae as well as C.
jaroonii and C. songkhlaensis (Groenewald et al. 2011; Wang et al. 2015). The ITS
sequences show more variation between these closely related well-defined species in
contrast to the low nucleotide differences in D1/D2 LSU sequences (Groenewald
et al. 2011). Although D1/D2 LSU sequence is still an appropriate region to use
for higher level taxon delimitations, it is clear that this sequence alone is not suf-
ficient for species delimitation in the Yamadazyma clade. The ITS sequence is thus
a good additional marker to obtain a better understanding of relatedness among
Yamadazyma species.

Yamadazyma species have a worldwide distribution and are isolated from diverse
substrates. They can be found in flowers, leaves, fruits, tree bark, mushrooms, sea
water, mineral and atmosphere, but most known species appear to exist in rotting
wood, insects and their resulting frass (Groenewald et al. 2011; Kurtzman 2011).
This clade also includes the clinically significant species C. aaseri, C. conglobata,
C. pseudoaaseri, and Y. triangularis (Kurtzman 2011; Lachance et al. 2011). These
studies expanded our knowledge on the substrates where Yamadazyma species can
occur, but on the other hand demonstrated the complicated ecological function of
this genus. In this study, three known species and three new species were identified
from rotting wood in China. Further research will focus on Yamadazyma diversity
from a wide range of substrates.

82 Wan-Li Gao et al. / MycoKeys 83: 69-84 (2021)

Acknowledgements

We sincerely thank Dr. Jing-Zhao Li, Dr. Zheng-Tian Zhang, Dr. Kai-Fang Liu, and
Dr. Zhi-Wen Xi for their help with collecting specimens. This project was supported by
Grant No. 31570021 from the National Natural Science Foundation of China, China,
No. 2018001 from the State Key Laboratory of Motor Vehicle Biofuel Technology,
Henan Tianguan Enterprise Group Co., Ltd., China.

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