# Specific populations of the yeast Geotrichum candidum revealed by molecular typing

Noémie Jacques, Sandrine Mallet, Fatima Laaghouiti, Colin R. Tinsley, Serge Casaregola

► To cite this version:

Noémie Jacques, Sandrine Mallet, Fatima Laaghouiti, Colin R. Tinsley, Serge Casaregola. Specific populations of the yeast Geotrichum candidum revealed by molecular typing. Yeast, 2017, 34 (4), pp.165-178. 10.1002/yea.3223. hal-03665115

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Specific populations of the yeast *Geotrichum candidum* revealed by

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molecular typing

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Noémie Jacques#,
Sandrine Mallet#, Fatima Laaghouiti, Colin R. Tinsley
and Serge Casaregola*

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Micalis Institute, INRA, AgroParisTech, CIRM-Levures, Université Paris-Saclay, 78350

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Jouy-en-Josas, France.

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**Running title:** Specific populations of *Geotrichum candidum*

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**Abbreviations**: MLST, Multi Locus Sequence Typing, Sequence Type, ST; RAPD, Random
Amplification of Polymorphic DNA; LTR, Long Terminal Repeat; CLP, Chromosome
Length Polymorphism

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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study
are: HF558450 for *URA1* gene, HF558451 for *URA3* gene, HF558452 for *NUP116* gene,
HF558453 *SAPT4* (*YSP3*) gene, HF586414 for *PLB3* gene and HF558454 for the retro-
transposon LTR. The *G. candidum* MLST database can be accessed online at
http://pubmlst.org/geotrichum/

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# These authors participated equally to the work

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<footnote>*Corresponding author: serge.casaregola@grignon.inra.fr</footnote>
26 **Abstract**

27 *Geotrichum candidum* is an ubiquitous yeast, and an essential component in the production of
28 many soft cheeses. We developed a Multi-Locus Sequence Typing (MLST) scheme with five
29 retained loci (*NUP116*, *URA1*, *URA3*, *SAPT4*, *PLB3*) which were sufficiently divergent to
30 distinguish 40 Sequence Types (STs) among the 67 *G. candidum* strains tested. Phylogenetic
31 analyses defined five main clades; one clade was restricted to environmental isolates, three
32 other clades included distinct environmental isolates and dairy strains, while the fifth clade
33 comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or
34 dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group
35 of specialized European *G. candidum* strains. In addition, we developed a PCR inter-LTR
36 scheme, a fast and reproducible RAPD-like method for *G. candidum*, to type the closely
37 related dairy strains, which could not be distinguished by MLST. Overall, our findings
38 distinguished two types of dairy strains, one forming a homogeneous group with little genetic
39 diversity, and the other more closely related to environmental isolates. Neither regional nor
40 cheese specificity was observed in the dairy *G. candidum* strains analyzed. This present study
41 sheds light on the genetic diversity of both dairy and environmental strains of *G. candidum*
42 and thus extends previous characterizations that have focused on the cheese isolates of this
43 species.

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45 **Key words:** MLST, inter-LTR, *Geotrichium candidum*, dairy, population

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47
48 **Introduction**

The dimorphic yeast *Geotrichum candidum* (teleomorph *Galactomyces candidus*) is
commonly found in foodstuffs, either as part of their normal constitution or as a contaminant.
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It is present in raw milk (Desmasures *et al.*, 1997) and is marketed as a starter for cheese
making, because of its proteolytic activities and its organoleptic properties. For these reasons,
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53 *G. candidum* is desirable on the surface of semi-hard cheeses and mould-ripened or smeared
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55 soft cheeses (Boutrou and Gueguen, 2005), and is of considerable importance in the cheese-
making industry (Pottier *et al*., 2008).
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56 *G. candidum* displays substantial morphological diversity and a wide ecological distribution.
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The genetic basis of this variability has been investigated: work based on RAM-PCR and
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RAPD-PCR (Gente *et al.*, 2002; Gente *et al.*, 2006; Marcellino *et al.*, 2001) demonstrated a
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high degree of genetic diversity of *G. candidum*, to some extent correlated to its various
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ecological niches and morphological polymorphism. PFGE profiling of 13 strains revealed
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highly diverse chromosome numbers, and hence genome sizes. It was suggested that genome
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size correlated with strain morphology (Gente *et al.*, 2006). It was recently shown that *G.
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*candidum* displays an unusual intra-specific polymorphism in the ribosomal DNA sequences
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used for species identification (Alper *et al*., 2011).
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Among various typing methods, Multi-Locus Sequence Typing (MLST) is based on
comparisons of partial housekeeping gene sequences. It is accepted to be reliable and
reproducible, and has the advantage of portability, via the creation of a common database
which can be continuously enlarged and updated by independent laboratories (Bougnoux et
al., 2004; Taylor and Fisher, 2003). MLST is the most popular typing method for yeast and
has been applied to more than 10 species, including Candida albicans (Bougnoux et al.,
2002), Candida glabrata (Dodgson et al., 2003), Candida tropicalis (Tavanti et al., 2005),
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72 Candida dubliniensis (McManus et al., 2008), Candida krusei (Jacobsen et al., 2007),
73 *Saccharomyces cerevisiae* (Ayoub *et al.*, 2006; Fay and Benavides, 2005; Munoz *et al.*, 2009) and *Cryptococcus gattii* (Feng *et al.*, 2008). The first MLST scheme for *G. candidum* was developed using six gene fragments covering 3009 nucleotides, defining 17 ST among 40 strains (Alper *et al.*, 2013). This work confirmed the intra-specific variability observed in *G. candidum* (Gente *et al.*, 2002; Gente *et al.*, 2006; Marcellino *et al.*, 2001).

78 Long Terminal Repeats (LTRs) are short transposon-associated sequences of around 300-400 bp widely repeated in some genomes, like those of the genus *Saccharomyces* and other 79 Saccharomycotina yeasts (Bleykasten-Grosshans and Neuveglise, 2011; Neuveglise *et al.*, 80 2002), either as flanking parts of the cognate retrotransposon or as isolated elements resulting 81 from deletion of the retrotransposon through recombination between the two flanking LTRs. 82
83 The lattersolo elements are numerous in some genomes, and this feature has been used to 84 differentiate isolates within a single species by amplifying the regions between LTRs, using 85 primers in the conserved regions of LTRs (inter-LTR PCR). This approach has been applied 86 to various species including *S. cerevisiae* (Legras and Karst, 2003; Ness *et al.*, 1993), 87 *Debaryomyces hansenii* and *Kluyveromyces marxianus* (Sohier *et al.*, 2009).

88 In this study, we exploit the completed genome sequence of the *G. candidum* strain CLIB 918 89 (=ATCC 204307) (Morel *et al.*, 2015) to develop protocols for typing this species and to gain 90 insights on the phylogenetic relationship between isolates. 91
## 92 Materials and methods

## 94 Yeast strains and growth conditions.

95 Isolates used in this study are listed in Table 1. "FM" strains were provided during the "Food

96 Microbiomes" project (http://www.agence-nationale-recherche.fr/?Project=ANR-08-ALIA-

97 0007) by producers of starter cultures and cheeses, and were given FM numbers to keep their
98 origin confidential; UCMA strains were provided by the University of Caen (France) and

99 CLIB strains by CIRM-Levures (Thiverval-Grignon, France;

100 http://www6.inra.fr/cirm/Levures). Most of the *G. candidum* isolates were from dairy
101 products and are involved in the ripening of various cheeses, some are from other habitats
102 such as soil; these strains were isolated in various places in France and the rest of the world.

103 Other *G. candidum* strains were from Centralbureau voor Schimmelcultures (Utrecht, the
104 Netherlands), Museum National d'Histoire Naturelle, Laboratoire de Cryptogamie (Paris,
105 France), VTT Technical Research Center (Finland), Kasetsart University (Bangkok,
106 Thailand), DSMZ (Braunschweig, Germany), BCCM/MUCL (Louvain la Neuve, Belgium),
107 NCYC (Norwich, United Kingdom) and NBRC (Chiba, Japan). Routinely, yeast strains were
grown in YPD (Yeast Peptone Dextrose: yeast extract 10 g l⁻¹, bacto peptone 10 g l⁻¹, glucose
108 10 g l⁻¹) at 28°C with shaking. For solid medium, 14 g l⁻¹ of agar was added to YPD.
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## 111 DNA extraction.

112 Cultures grown in 3 ml YPD medium overnight at 28°C were centrifuged at 2,500 g for 3
113 min, and the cell pellets were washed in 750 µl 50 mM EDTA. Each cell pellet was
114 resuspended in 200 µl lysis buffer (1 % SDS, 2 % triton, 100 mM NaCl, 50 mM EDTA,
115 50mM Tris, pH=8), 200 µl chloroform/phenol (pH=8) and 300 mg glass beads and
116 mechanically shaken by vortexing for 4 min. Then, 200 µl TE buffer was added and the
samples centrifuged for 5 min at 12,000 g. The aqueous phase was transferred to a new tube, and two chloroform extractions were carried out. DNA was precipitated with an equal volume of 100% ethanol and centrifuged for 4 min at 12,000g. The pellet was rinsed with 400 µl of 70 % ethanol, dried at room temperature for 15 min, resuspended in 50 µl TE buffer, and incubated with 2 µl RNase (10 mg ml⁻¹) for 30 min at 37°C. The DNA concentration was quantified on a 0.8% agarose gel (wt/vol) with 1X TAE electrophoresis buffer containing 0.2 mg ml⁻¹ ethidium bromide, run at 100 V in a SUB-CELL GT electrophoresis system (Bio-Rad) for 60 min.

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126 PCR amplification.

The primers used in this study, based on the complete genome sequence of G. candidum CLIB 918 (Morel et al., 2015) were designed using Primer3 (http://fokker.wi.mit.edu/primer3). For inter-LTR PCR fingerprinting, sequences with similarities to a G. candidum retrotransposon LTR identified by Morel et al., (2015) were aligned using MAFFT7 (Katoh and Standley, 2013). Primers were designed in the conserved regions of the aligned sequences. The primers used were GC_LTR3 (5'-TCTTTCCTTTAATTAATCAA) and GC_LTR5 (5'-TGAAGTGAACCAACATAA). For MLST, the primers used were NUP116_for (5'-ACCGCTACAACTGGATTTGG), NUP116_rev (5'-GAGACCTGTTTGAGGGCTTG), PLB3_for (5'-AAGAATATCTGGGATCTTTC), PLB3_rev (5'-TGAAGAAGAAGTACCAAGAA), SAPT4_for (5'-ATCATTAACACCCCGGCATA), SAPT4_rev (5'-GTGTCACCAAGCAGAGCAAA), URA1_for (5'-CAAGCCAATTGTGCTGAGAA), URA1_rev (5'-GGTGTCGTAGGGCAGTTGAT), URA3_for (5'-GCCAAAAAGACCAACCTGTG), URA3_rev (5'-CCTCATCCATACGGTTCTGC).

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For MLST, between 25 and 50 ng of genomic DNA was amplified in a 50 µl PCR reaction mix containing 0.8 mM dNTP mixture, 0.1 µM forward and reverse primers in the manufacturer's recommended buffer and 1 U TaKaRa Ex Taq. Reactions were run on a 2720 thermal cycler (Applied Biosystems) as follows: 5 min at 94°C followed by 30 cycles of 30 sec at 94°C, 40 s at temperatures between 35 and 50°C and 30 sec at 72°C, with a final extension step of 7 min at 72 °C.

For inter-LTR PCR fingerprinting, 2 µl of DNA preparation (containing 20 ng) was added to a 23 µl PCR reaction mix containing 0.8 mM dNTP mixture, 0.8 µM GC1_LTR and GC2_LTR primers in the manufacturer's recommended buffer and 0.625 U TaKaRa Ex Taq.

PCR conditions were as follows: 94°C for 5 min, 30 cycles of 94°C for 30 s, 37°C for 40 s and 72°C for 2 min, followed by a final extension at 72°C for 7 min. PCR products were visualized on a 1.2% agarose gel (wt/vol) 15 cm in length with 1X TAE electrophoresis buffer containing 0.2 mg ml⁻¹ ethidium bromide, run at 100 V in a SUB-CELL GT electrophoresis system (Bio-Rad) for 90 min. The molecular marker was Gene ruler DNA Ladder Mix (Thermo Scientific). Acquisition of data by the Bionumerics software 6.5 (Applied Maths, Belgium) was as described in (Sohier et al., 2009). Briefly, electrophoresis patterns were normalized to compensate for migration bias, non-linear background was subtracted from the patterns and comparison was based on the rolling disk principle. Inter-LTR variability not being appropriate for phylogenetic analysis, and the object being only to discriminate strains within an MLST sequence type, interpretation of the Bionumerics output was by visual observation ; no clustering algorithm was performed.

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163 **Synonymous and non-synonymous base ratio**

Codon by codon analysis was performed using MEGA7 (Kumar *et al.*, 2016). Pairwise peptide comparisons were conducted with MEGA7 (codon based Z-test for selection) and
166 with the package *Dnasp* (Librado and Rozas, 2009). The likelihood analysis was performed
167 using the REL algorithm of the HyPhy package implemented in *DataMonkey* (Pond and
168 Frost, 2005).

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170 **DNA sequence determination and phylogenetic analysis**

171 PCR fragments were sequenced on both strands by Eurofins MWG Operon (Ebersberg,
172 Germany). Sequences were assembled with the phred/phrap/consed package (Ewing and
173 Green, 1998; Ewing *et al.*, 1998; Gordon *et al.*, 1998) and analyzed with BLAST
174 implemented at NCBI (http://www.ncbi.nlm.nih.gov). Sequence alignments were generated
175 using MAFFT7 (Katoh and Standley, 2013). Phylogenetic trees were constructed with the
176 Neighbor-Joining program implemented in MEGA6 (Tamura *et al.*, 2013). Phylogenetic trees
177 were visualized with NJplot (Perriere and Gouy, 1996). Bionumerics software 6.5 (Applied
178 Maths, Belgium) was used for the acquisition of inter-LTR profiles and the integration of
179 MLST, inter-LTR and phenotypic data.

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181 **Phylogenetic analysis with Structure**

182 The MLST data were used to predict the population structure on the basis of the clustering
183 program *Structure* (Pritchard *et al.*, 2000). Analyses were performed with burn-in of 80,000
184 cycles and 80,000 rounds of calculation. In accordance with the authors' recommendations,
185 the best estimation of the number of populations, K, was taken as the value of K for which the
186 difference in log(probability of data) between K and K+1 diminished abruptly (*ie.*, the log
187 probability ceased to increase rapidly). The population structure was thus calculated with K =
188 5 populations. For all other parameters, the recommended default values were used.

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190 Phylogenetic analysis using *Splits tree*
191 The MLST data were used to predict genetic exchanges with the program *Splits tree* (Huson and Bryant, 2006). Recommended default values were used.
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194 **Results**

195 **Design of a MLST scheme for *G. candidum***.

196 A literature survey was undertaken to identify genes which have been used for MLST
197 schemes in previous studies on yeasts (Bougnoux et al., 2002; Dodgson et al., 2003; Jacobsen
198 et al., 2007; McManus et al., 2008; Munoz et al., 2009; Tavanti et al., 2005). The sequences
199 of 28 *G. candidum* orthologs of such genes were extracted from the genome of CLIB 918.

200 PCR primers were designed to amplify fragments of around 500 bp and were tested on 67
201 strains, which were considered to be haploid on the basis of their mating type characteristics
202 (Morel et al., 2015). The study population included 34 *G. candidum* strains isolated from
203 cheese, 4 from milk, 1 from the dairy environment, 1 from cow's udder, 3 dairy product
204 contaminants and 17 isolates from diverse origins (fruit, human faeces, industrial plant,
205 industrial waste...). The latter group contained three *G. silvicola* strains, now reassigned to
206 the *G. candidum* species. A preliminary analysis was performed on closely related dairy
207 strains from France to select the most divergent markers. Five markers were selected as
208 showing the greatest sequence divergence between strains: *URA1*, *URA3*, *SAPT4* (*YSP3* in *S.
209 cerevisiae*), *NUP116* and *PLB3*. Data for the five MLST markers are presented in Table 2.
210 The PCR fragments analyzed were between 393 bp for *PLB3* and 501 bp for *SAPT4*, yielding
211 a concatenated sequence length of 2254 bp which contained 90 polymorphic sites.

212 Table 2 summarizes information concerning the diversity at each locus. The number of
213 polymorphic sites, only consisting in substitutions, varied between 11 for *URA1* and 28 for
214 *PLB3*. The number of allelic profiles between loci was five for *SAPT4* and nine for *PLB3*,
215 *URA1*, *URA3* and *NUP116*. The average divergence for the alleles ranges from 0.2 % for
216 *SAPT4* to 4.7 % for *PLB3* (Supplementary Table 1).

217 Analysis using MEGA7 showed evidence of positive selection in 4 of the 14 variable codons
218 of the *NUP116* sequence, in 2 of the 28 codons of *PLB3*, in 2 of 21 of *SAPT4*, in 1 of 11 of
URA1 and in 1 of 16 of URA3, though without good statistical support (P ≈ 0.7). Pairwise peptide comparisons showed a positive overall dN - dS for the sequences of NUP116 and of PLB3 in strain MUCL8652 when compared with about half of the other strains (P=0.16).

Analysis with the package DnaSP gave values of dN/dS always smaller than unity, excepting cases where dS was zero. In the case of NUP116 over 90% of the values of dN/dS were smaller than 0.25; for the other four genes, over 90% were less than 0.15. A likelihood approach showed 2 positively selected sites (Bayes factors > 100) in the sequence of SAPT4 and none in the other proteins. Considering the feeble evidence from the codon by codon analysis and the Z-test, the absence of evidence from the analysis in Dnasp and the prediction of 2/21 codons subject to selection in SAPT4 by the likelihood analysis, we may conclude that the large majority of sites used in the analysis are not subject to positive selection.

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## Geographical and industrial influence on strain grouping.

Forty sequence types (ST) were defined amongst the 67 strains, based on the possession of identical concatenated sequences. Overall, 31 strains were the sole member of their ST, while the other 36 strains were distributed among the remaining 9 STs: ST 21 and ST 23 each contained two strains, ST 14 and ST 19 contained three strains, ST 7, ST 18, ST 29 and ST 30 contained five strains and ST 22 contained six strains (Table 3). The pairwise divergence between STs (averaged over the concatenated sequences) was from 0.04 % to 2.57 % and average divergence for all STs was 0.96 %.

Phylogenetic trees were constructed by neighbor joining following alignment of the sequences using MAFFT7 (Figures 1 and 2). The tree constructed with ST type sequences, shown in Figure 1, differentiated five main clades. Two well-separated clades 1 and 2 included mainly environmental isolates and dairy strains. Clade 1 contained three STs represented by a single isolate and one (ST 7) which contained five strains. Clade 2 contained
five single-isolate STs. A third clade also heterogeneous with regard to the origin of the strains contained 14 single-strain STs and one (ST 14) composed of three strains. Clade 4 contained three single-strain STs and consisted of two strains isolated from soil and one from fruit. Finally, in contrast to the previous clades, clade 5 contained only strains involved in cheese making (either from cheese made with raw milk or from starters used in the cheese industry), with the exception of two isolates (FM 268 and FM 269, in ST 18 and ST 27 respectively) which were isolated from human stools. It may be reasonable to propose that these latter isolates have a cheese origin, considering their position in the tree and their geographical origin. Isolates assigned to clade 5 were less divergent; seven of the thirteen STs contained from two to six strains.

The slovakian strain CBS 11176 was classified among cheese isolates of clade 5. This strain was first described as *Geotrichum bryndzea* (Sulo *et al.*, 2009), but it was recently shown to belong to *Gal. candidus*, the teleomorph of *G. candidum* (Gроенewald *et al.*, 2012); the position of this strain in the phylogenetic tree shown in Figure 2 confirmed the recent taxonomic change.

## 260 Population analysis

Since *G. candidum* is a sexually reproducing species, we performed analyses using the programs *Structure* and *Splits tree*. *Structure* clusters the isolates based on their respective genetic profiles which are considered in terms of the relative contributions from a predefined number of hypothetical ancestral genomes. *Splits tree* calculates a phylogenetic tree which permits the visualization of genetic exchanges in addition to mutations.

Preliminary analysis of the data with *Structure* suggested that the data were best explained under the hypothesis of five ancestral genotypes. The results of clustering on this basis (Figure 3a) distinguish the previously defined clades 1 and 5, and suggest that the clade 2
269 contains hybrid organisms with genetic contributions originating in clades 1 and 3. Individual 270 gene trees for each of the markers also indicated genetic exchanges between isolates 271 (Supplementary Figure 1). As suggested by the above Neighbor-Joining analysis (Figures 1 272 and 2), the clade 3 is composed of two subgroups, while clade 4 branches from this group. 273 Clades 1 and 5 contained almost exclusively dairy strains, or strains isolated from human 274 faeces, presumably as a consequence of having eaten dairy products, while clades 2, 3 and 4 275 contained a mixture of dairy and environmental strains. *Splits tree* gave similar results (Figure 276 3b), separating the strains into groups which, with few exceptions, corresponded to the clades 277 previously defined. The analysis separated clades 1 and 5 and illustrated the derivation of 278 clades 2 and 4 from the clade 3. Again, the distinction of clades 1 and 5, of dairy origin, from 279 the other groups, of which environmental strains constituted about half the isolates, suggests 280 that dairy strains have developed separately from non-dairy strains. 281

282 Inter-LTR PCR distinguishes most of the *G. candidum* dairy strains.

283 Although MLST was very informative about the divergence of environmental strains and 284 dairy strains, it was not clear whether the large ST groups were due to clonality or limits to 285 the discrimination of the MLST markers. We therefore developed an additional typing 286 method more sensitive to genetic changes occurring at short time-scales. 287

287 Sequence analysis of the complete genome of *G. candidum* CLIB 918 revealed the presence 288 of a Long Terminal Repeat (LTR)-retrotransposon likely related to the yeast Ty families 289 (Morel *et al.*, 2015). The corresponding 385 bp long LTR sequences found in the genome of 290 CLIB 918 were aligned with MAFFT7 and oligonucleotide primers were designed on the 291 basis of conserved regions (Supplementary Figure 2). These were used to amplify genomic 292 DNA from the 36 strains which were in multi-strain STs (listed in Table 3). Various primer 293 pairs and various PCR conditions were tested; those leading to the most discriminating results
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(GC_LTR3 and GC_LTR5) were retained. In the subsequent analysis all the inter-LTR PCRs were repeated at least twice as previously described (Sohier *et al.*, 2009).

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Results of electrophoresis are shown in Figure 3 and Supplementary figure 3. The inter-LTR profiles were compared to the MLST tree obtained previously to highlight the variability of strains within the STs, now observable with inter-LTR analysis. The amplified bands ranged from 200 to 4000 bp and the profiles of the various strains differed in number, size and relative intensity of fragments. In the majority of cases, strains within the multi-strain ST were distinguished by inter-LTR analysis. Thus, 28 strains were uniquely discriminated by a combination of MLST and inter-LTR, while two strains of ST 29 (CLIB 1239 and CLIB 1241) shared the same inter-LTR profiles, as did FM 76 and FM 77 in ST 18, and CLIB 1244, CLIB 1245, CLIB 1246, CLIB1247 and CLIB 1253 in ST 30. The two strains of ST 29 originated from the Franche-Comté region and the five strains in ST 30 were isolated from the Haute-Savoie; their classification into the same clusters is therefore coherent. In conclusion, the combination of MLST and inter-LTR allowed precise discrimination between cheese strains.

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As described previously (Marcellino *et al.*, 2001), we observed a limited, but reproducible, phenotypic diversity in terms of carbon source assimilation (galactose, lactose and ribose; Figure 3). The capacity to utilize ribose is rarely found in *G. candidum*. We found that six of the 37 isolates could utilize ribose. They were mainly associated with ST 18 (4 of 5 isolates tested). Only two, unrelated strains were lactose-negative. Galactose assimilation was absent from ST 30 and from two thirds of the isolates belonging to the sister clade ST 22, these being all cheese strains from the region Haute-Savoie region.

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317
318 **Discussion**

319 We report the development of tools for investigating the evolution of *G. candidum* and for
320 studying industrial strains used in cheese production. The MLST analysis we describe here
321 involving five single copy genes confirms the high genetic diversity reported for *G.
322 candidum*. Previous typing studies based on RAM-PCR, RAPD and Chromosome Length
323 Polymorphism (CLP) techniques suggest substantial diversity within the species (Marcellino
324 et al., 2001; Gente et al., 2002; Gente et al., 2006), which was again evidenced by the
325 variability of the ribosomal DNA sequences observed by (Alper et al., 2011). Our results
326 demonstrate a large degree of genetic variation in the five genes considered (between 2.4 and
327 7.1 % of polymorphic sites, this being higher than the 0.9 to 3.2 % found in the study of
328 (Alper et al., 2013).

329 In this work, *G. candidum* strains could be readily differentiated on the basis of the partial
330 sequences of five genes: 40 different STs were obtained for 67 isolates based on 90
331 polymorphic sites within the 2254 bp. This compares favorably with the 17 STs distinguished
332 in the MSLT scheme of Alper *et al.* (2013) using 58 polymorphic sites in 6 loci (3009 bp), our
333 choice of candidate genes being facilitated by the availability of the whole genome sequence
334 of *G. candidum*. In addition, the analysis was performed on a larger number of isolates from a
335 wide range of geographical or ecological origins, including the environment, industrial plant
336 or food contaminant, which permitted a more general view of the phylogenetic diversity of
337 the species.

338 Twenty-eight of the strains studied here have previously been studied by RAPD (Marcellino
339 et al., 2001). The previous RAPD results and our findings are generally coherent, although
340 there were some differences. The three strains, CLIB 1267 (GC129 in Marcellino et al.,
341 2001), CLIB 1258 (=GC101) and CLIB 1237 (=GC37), isolated from milk used respectively
342 for manufacturing Camembert (Normandy), St Nectaire (Auvergne) and Chaource cheeses
343 (Champagne-Ardenne), were previously found by (Marcellino *et al.*, 2001) to be closely related to other cheese strains. However, we found that they are well separated from other 344 cheese strains and closely related to environmental strains (Figure 2). Another difference 345 concerns the yeasts that we assign to ST30 (composed of isolates from ripening cheese from 346 Annecy). The isolate CLIB 1253 (=GC90) was suggested to be different from the other strains 347 that we classified as ST 30 (Marcellino *et al.*, 2001). We clearly show here that the five 348 strains belonging to ST 30 are indistinguishable on the basis of MLST, and inter-LTR profile. 349 In addition they have identical phenotypic characteristics (this work), and we have previously 350 shown that they carry the same mating type (Morel *et al.*, 2015). These strains were isolated 351 from two different cheeses, Reblochon and Tomme de Savoie, made in different dairy 352 facilities. Our results therefore indicate that these are clones of the same strain present at more 353 than one particular site and in more than one cheese, suggesting that *G. candidum* isolates are 354 not region or cheese specific. 355

356 The 67 isolates studied here were classified in 40 STs. A total of 31 isolates were typed by 357 MLST alone as they all display a unique ST. Though all of the environmental isolates could 358 be distinguished, there were a number of dairy isolates (eight isolates in clades 1, 2 or 3 and 359 28 isolates belonging to the dairy clade 5) that were not typed by this approach. We were able 360 to improve strain differentiation by developing inter-LTR PCR for *G. candidum*, which 361 successfully differentiated all but nine of the strains that were not separated by MLST. Inter- 362 LTR PCR is simple, reliable and fast, as demonstrated previously for other species (Ness *et 363 al.*, 1993; Legras and Karst, 2003; Sohier *et al.*, 2009) and may therefore be useful for strain 364 differentiation, for example, in such applications as analysis of yeast population dynamics 365 during cheese ripening. 366

366 It has been reported that *G. candidum* is highly variable as assessed by chromosome 367 separation using PFGE (Gente *et al.*, 2006). However, CLP very often results from ectopic
recombination between similar sequences, such as transposons (Zolan, 1995; Casaregola et al., 1998; Rachidi et al., 1999). Accordingly, we observed a very large number of various kinds of transposons in the genome of *G. candidum* (Morel *et al.*, 2015). The discrepancy between MLST and RAPD may be inherent to the techniques used because methods based on the amplification of repeats do not measure genetic diversity directly, but rather, like PFGE, indicate chromosomal differences. It has been demonstrated, for example, that the species *Schizosaccharomyces pombe* displays substantial CLP which is not associated with either sequence or phenotypic variability (Brown *et al.*, 2011). In this study, strains which could not be separated on the basis of sequence divergence, *i.e.* MLST, displayed little phenotypic diversity; they are therefore presumably closely related. The use of inter-LTR PCR permitted a fine-scale discrimination of these related isolates within a single ST.

Our MLST analysis differentiated a clade containing dairy isolates from those containing environmental isolates mixed with dairy isolates, in agreement with the suggestion that *G. candidum* species could be separated into two groups, on the basis of rRNA variability and genomic sequence diversity (Alper *et al.*, 2013). The observation of these authors concerns dairy yeasts, and may correspond to the two types of dairy yeasts that we evidenced (clade 5 vs. clades 2, 3 and 4). In summary, our work has brought to light a population of dairy yeasts which is clearly distinct from the clades containing a mixture of dairy and environmental strains. This is reminiscent of population genomics findings for *S. cerevisiae* (Liti et al., 2009; Schacherer et al., 2009), in which specialized isolates formed separated clades. *G. candidum* clade 5 is reminiscent of the *S. cerevisiae* European wine group in which strains are closely related and specialized in wine making.

In addition to shedding light on the population structure and probable evolution of the species *G. candidum*, our work provides information and methods with potential to facilitate genetic improvement and studies of genetic diversity within this species. For example, it provides a
393 framework which will make it easier to correlate genetic divergence with industrially
394 important characteristics of *G. candidum*, such as aroma production, morphology and growth
395 characteristics.
396
397 Acknowledgements

398
399 This work received funding from the Agence National pour la Recherche grant “Food
400 Microbiomes” (ANR-08-ALIA-007-02). This publication made use of the Geotrichum MLST
401 website (http://pubmlst.org/geotrichum/) developed by Keith Jolley and sited at the University
402 of Oxford (Jolley and Maiden, 2010). The development of this site has been funded by the
403 Wellcome Trust. We thank Christelle Louis-Mondésir for expert technical assistance. We are
404 grateful to Prof. Savitree Limtong (Kasetsart University, Thailand) for providing us with the
405 isolate NT12. We are grateful to Guillaume Morel (supported by a CIFRE fellowship with
406 CNIEL) for the extraction from the genome sequence of CLIB 918 of the genes of interest.
407 We also thank the Centre National Interprofessionnel de l'Economie Laitière (CNIEL) and
408 the Syndicat Professionnel des Producteurs d'Auxiliaires pour l'Industrie Laitière (SPPAIL)
409 for providing us with dairy strains.

410
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503
504
<table><caption>**Table 1** List of strains studied</caption><thead><tr><th>Species</th><th>Isolate</th><th>Other names</th><th>Origin</th><th>Source</th></tr></thead><tbody><tr><td>Geotrichum bryndzae<sup>a</sup></td><td>CLIB 1366<sup>T</sup></td><td>CBS 11176<sup>T</sup></td><td>Slovakia</td><td>Bryndza cheese</td></tr><tr><td>Geotrichum candidum</td><td>CLIB 1368<sup>T</sup></td><td>CBS 615.84<sup>T</sup></td><td>Ile de France</td><td>Brie cheese</td></tr><tr><td>//</td><td>CLIB 918</td><td>ATCC 204307</td><td>Normandy</td><td>Pont l'Evêque cheese</td></tr><tr><td>//</td><td>CLIB 1235</td><td>GC12</td><td>Normandy</td><td>Camembert cheese</td></tr><tr><td>//</td><td>CLIB 1236</td><td>GC 21</td><td>Normandy</td><td>Goat's cheese</td></tr><tr><td>//</td><td>CLIB 1237</td><td>GC37</td><td>Normandy</td><td>Camembert cheese</td></tr><tr><td>//</td><td>CLIB 1239</td><td>GC43</td><td>Franche-Comté</td><td>Mont d'Or cheese</td></tr><tr><td>//</td><td>CLIB 1240</td><td>GC44</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1241</td><td>GC45</td><td>Franche-Comté</td><td>Mont d'Or cheese</td></tr><tr><td>//</td><td>CLIB 1242</td><td>GC42</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1243</td><td>GC51</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1244</td><td>GC59</td><td>Haute-Savoie</td><td>Tomme de Savoie cheese</td></tr><tr><td>//</td><td>CLIB 1245</td><td>GC60</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1246</td><td>GC63</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1247</td><td>GC64</td><td>Haute-Savoie</td><td>Tomme de Savoie cheese</td></tr><tr><td>//</td><td>CLIB 1248</td><td>GC74</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1251</td><td>GC79</td><td>Burgundy</td><td>Epoisses cheese</td></tr><tr><td>//</td><td>CLIB 1252</td><td>GC84</td><td>Burgundy</td><td>Epoisses cheese</td></tr><tr><td>//</td><td>CLIB 1253</td><td>GC90</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1255</td><td>GC96</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1256</td><td>GC97</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1257</td><td>GC100</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1258</td><td>GC101</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1260</td><td>GC105</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1262</td><td>GC110</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1263</td><td>GC120</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1267</td><td>GC129</td><td>Champagne-Ardenne</td><td>Chaource cheese</td></tr><tr><td>//</td><td>CLIB 1270</td><td>GC146</td><td>Auvergne</td><td>Saint Nectaire cheese</td></tr><tr><td>//</td><td>CLIB 1274</td><td>GC164</td><td>Haute-Savoie</td><td>Reblochon cheese</td></tr><tr><td>//</td><td>CLIB 1283</td><td>UCMA 103</td><td>Normandy</td><td>Pont l'Evêque cheese</td></tr><tr><td>//</td><td>CLIB 1285</td><td>UCMA 3936</td><td>Normandy</td><td>Dairy</td></tr><tr><td>//</td><td>CLIB 1361</td><td>CBS 182.33</td><td>Italy</td><td>Yoghurt</td></tr><tr><td>//</td><td>CLIB 1415</td><td>LCP 51.590</td><td>Spain</td><td>Sand</td></tr><tr><td>//</td><td>FM 03</td><td></td><td>unknown</td><td>Cheese contaminant</td></tr><tr><td>//</td><td>FM 115</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 122</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 125</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 127</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 128</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 136</td><td></td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>FM 212</td><td></td><td>France</td><td>Corn silage</td></tr></tbody></table>
<table><tr><td>//</td><td>FM 214</td><td>Normandy</td><td>Cow's milk</td></tr><tr><td>//</td><td>FM 260</td><td>Normandy</td><td>Cow's udder</td></tr><tr><td>//</td><td>FM 268</td><td>Normandy</td><td>Stools</td></tr><tr><td>//</td><td>FM 269</td><td>Normandy</td><td>Stools</td></tr><tr><td>//</td><td>FM 270</td><td>Normandy</td><td>Stools</td></tr><tr><td>//</td><td>FM 29</td><td>Auvergne</td><td>Goat's cheese</td></tr><tr><td>//</td><td>FM 30</td><td>Auvergne</td><td>Goat's cheese</td></tr><tr><td>//</td><td>FM 31</td><td>Auvergne</td><td>Goat's cheese</td></tr><tr><td>//</td><td>FM 34</td><td>Auvergne</td><td>Goat's cheese</td></tr><tr><td>//</td><td>FM 76</td><td>Normandy</td><td>Cow's milk</td></tr><tr><td>//</td><td>FM 77</td><td>unknown</td><td>unknown</td></tr><tr><td>//</td><td>CBS 117138</td><td>Italy</td><td>Municipal compost</td></tr><tr><td>//</td><td>CBS 11628</td><td>South Africa</td><td>Soil</td></tr><tr><td>//</td><td>CBS 476.83</td><td>Senegal</td><td>Soil</td></tr><tr><td>//</td><td>CBS 557.83</td><td>Egypt</td><td>Fruit</td></tr><tr><td>//</td><td>DSM 10452</td><td>Germany</td><td>Sauerkraut</td></tr><tr><td>//</td><td>DSM 13629</td><td>United Kingdom</td><td>Polyurethane</td></tr><tr><td>//</td><td>MUCL 11539</td><td>United Kingdom</td><td>Polluted water</td></tr><tr><td>//</td><td>MUCL 14462</td><td>United States</td><td>Squash, Curcubita</td></tr><tr><td>//</td><td>MUCL 881</td><td>Belgium</td><td>Milk</td></tr><tr><td>//</td><td>MUCL 8652</td><td>Belgium</td><td>Soaked hay</td></tr><tr><td>//</td><td>NBRC 5368</td><td>unknown</td><td>Butter</td></tr><tr><td>//</td><td>NCYC 49</td><td>unknown</td><td>Milk</td></tr><tr><td>Geotrichum silvicola<sup>a</sup></td><td>CLIB 1378<sup>T</sup></td><td>CBS 9194<sup>T</sup></td><td>Brazil</td></tr><tr><td>//</td><td></td><td>NT 12</td><td>Thailand</td></tr><tr><td>//</td><td></td><td>VTTC 4559</td><td>Sweden</td></tr></table>

<footnote>a previous species denomination. GC, strains described in Marcelino et al. (2001).</footnote>
<table><caption>**Table 2** Characteristics of the five loci studied</caption><thead><tr><th>Locus</th><th>Size of examined<br>amplicon (bp)</th><th>Sequence<br>divergence (%)</th><th>Number of<br>polymorphic sites</th><th>Number of allelic<br>profile per locus</th></tr></thead><tbody><tr><td>NUP116</td><td>425</td><td>3.29</td><td>14</td><td>9</td></tr><tr><td>URA1</td><td>465</td><td>2.36</td><td>11</td><td>9</td></tr><tr><td>URA3</td><td>470</td><td>3.40</td><td>16</td><td>9</td></tr><tr><td>SAPT4</td><td>501</td><td>4.19</td><td>21</td><td>5</td></tr><tr><td>PLB3</td><td>393</td><td>7.12</td><td>28</td><td>9</td></tr></tbody></table>
<caption>1 Table 3 MLST profiles for each strains of this study and distribution of isolates in STs</caption>

2

<table><thead><tr><th rowspan="2">Strains</th><th colspan="5">Markers</th><th rowspan="2">STs</th></tr><tr><th>NUP116</th><th>PLB3</th><th>SAPT4</th><th>URA1</th><th>URA3</th></tr></thead><tbody><tr><td>NT 12</td><td>6</td><td>2</td><td>2</td><td>5</td><td>4</td><td>1</td></tr><tr><td>CLIB 1267</td><td>2</td><td>2</td><td>4</td><td>1</td><td>1</td><td>2</td></tr><tr><td>CLIB 1237</td><td>2</td><td>2</td><td>2</td><td>2</td><td>1</td><td>3</td></tr><tr><td>LCP 51.590</td><td>2</td><td>2</td><td>5</td><td>2</td><td>3</td><td>4</td></tr><tr><td>CLIB 1235</td><td>1</td><td>2</td><td>1</td><td>2</td><td>1</td><td>5</td></tr><tr><td>CBS 182.33</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>6</td></tr><tr><td>CLIB 1236</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>7</td></tr><tr><td>CLIB 1251</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>7</td></tr><tr><td>CLIB 1252</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>7</td></tr><tr><td>CLIB 1263</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>7</td></tr><tr><td>FM 125</td><td>2</td><td>2</td><td>1</td><td>2</td><td>1</td><td>7</td></tr><tr><td>CBS 9194</td><td>4</td><td>3</td><td>3</td><td>3</td><td>1</td><td>8</td></tr><tr><td>FM 122</td><td>3</td><td>7</td><td>2</td><td>2</td><td>1</td><td>9</td></tr><tr><td>CLIB 1283</td><td>2</td><td>1</td><td>2</td><td>1</td><td>1</td><td>10</td></tr><tr><td>FM 270</td><td>2</td><td>1</td><td>2</td><td>4</td><td>3</td><td>11</td></tr><tr><td>FM 03</td><td>5</td><td>4</td><td>2</td><td>2</td><td>1</td><td>12</td></tr><tr><td>FM 212</td><td>2</td><td>4</td><td>2</td><td>2</td><td>1</td><td>13</td></tr><tr><td>FM 136</td><td>1</td><td>4</td><td>2</td><td>2</td><td>1</td><td>14</td></tr><tr><td>CLIB 1258</td><td>1</td><td>4</td><td>2</td><td>2</td><td>1</td><td>14</td></tr><tr><td>CBS 117138</td><td>1</td><td>4</td><td>2</td><td>2</td><td>1</td><td>14</td></tr><tr><td>VTTC 4559</td><td>1</td><td>4</td><td>2</td><td>2</td><td>5</td><td>15</td></tr><tr><td>CLIB 1274</td><td>2</td><td>5</td><td>1</td><td>2</td><td>1</td><td>16</td></tr><tr><td>FM 260</td><td>3</td><td>4</td><td>1</td><td>2</td><td>3</td><td>17</td></tr><tr><td>FM 214</td><td>2</td><td>1</td><td>1</td><td>2</td><td>3</td><td>18</td></tr><tr><td>FM 268</td><td>2</td><td>1</td><td>1</td><td>2</td><td>3</td><td>18</td></tr><tr><td>FM 34</td><td>2</td><td>1</td><td>1</td><td>2</td><td>3</td><td>18</td></tr><tr><td>FM 76</td><td>2</td><td>1</td><td>1</td><td>2</td><td>3</td><td>18</td></tr><tr><td>FM 77</td><td>2</td><td>1</td><td>1</td><td>2</td><td>3</td><td>18</td></tr><tr><td>CBS 11176</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>19</td></tr><tr><td>CLIB 1248</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>19</td></tr><tr><td>CLIB 1260</td><td>2</td><td>1</td><td>1</td><td>2</td><td>1</td><td>19</td></tr><tr><td>CBS 615.84</td><td>3</td><td>1</td><td>3</td><td>2</td><td>1</td><td>20</td></tr><tr><td>CLIB 1257</td><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>21</td></tr><tr><td>FM 128</td><td>1</td><td>1</td><td>1</td><td>2</td><td>1</td><td>21</td></tr><tr><td>CLIB 1240</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>CLIB 1242</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>CLIB 1243</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>CLIB 1255</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>FM 127</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>MUCL 881</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>22</td></tr><tr><td>FM 29</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>23</td></tr><tr><td>FM 31</td><td>1</td><td>1</td><td>1</td><td>1</td><td>3</td><td>23</td></tr></tbody></table>
<table><tr><td>FM 30</td><td>1</td><td>6</td><td>1</td><td>1</td><td>3</td><td>24</td></tr><tr><td>CLIB 1285</td><td>2</td><td>1</td><td>1</td><td>4</td><td>1</td><td>25</td></tr><tr><td>FM 115</td><td>3</td><td>6</td><td>5</td><td>1</td><td>1</td><td>26</td></tr><tr><td>FM 269</td><td>2</td><td>1</td><td>1</td><td>1</td><td>3</td><td>27</td></tr><tr><td>CLIB 918</td><td>2</td><td>1</td><td>1</td><td>1</td><td>2</td><td>28</td></tr><tr><td>CLIB 1239</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>29</td></tr><tr><td>CLIB 1241</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>29</td></tr><tr><td>CLIB 1256</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>29</td></tr><tr><td>CLIB 1262</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>29</td></tr><tr><td>CLIB 1270</td><td>2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>29</td></tr><tr><td>CLIB 1244</td><td>5</td><td>1</td><td>1</td><td>1</td><td>1</td><td>30</td></tr><tr><td>CLIB 1245</td><td>5</td><td>1</td><td>1</td><td>1</td><td>1</td><td>30</td></tr><tr><td>CLIB 1246</td><td>5</td><td>1</td><td>1</td><td>1</td><td>1</td><td>30</td></tr><tr><td>CLIB 1247</td><td>5</td><td>1</td><td>1</td><td>1</td><td>1</td><td>30</td></tr><tr><td>CLIB 1253</td><td>5</td><td>1</td><td>1</td><td>1</td><td>1</td><td>30</td></tr><tr><td>DSM 10452</td><td>2</td><td>2</td><td>1</td><td>2</td><td>6</td><td>31</td></tr><tr><td>MUCL 11539</td><td>2</td><td>4</td><td>2</td><td>2</td><td>6</td><td>32</td></tr><tr><td>CBS 11628</td><td>7</td><td>7</td><td>2</td><td>6</td><td>7</td><td>33</td></tr><tr><td>CBS 476.83</td><td>8</td><td>7</td><td>2</td><td>6</td><td>8</td><td>34</td></tr><tr><td>CBS 557.83</td><td>7</td><td>8</td><td>2</td><td>9</td><td>8</td><td>35</td></tr><tr><td>NBRC 5368</td><td>9</td><td>4</td><td>2</td><td>7</td><td>1</td><td>36</td></tr><tr><td>MUCL 8652</td><td>3</td><td>9</td><td>2</td><td>1</td><td>1</td><td>37</td></tr><tr><td>DSM 13629</td><td>2</td><td>6</td><td>2</td><td>7</td><td>1</td><td>38</td></tr><tr><td>MUCL 14462</td><td>2</td><td>3</td><td>5</td><td>8</td><td>9</td><td>39</td></tr><tr><td>NCYC 49</td><td>2</td><td>2</td><td>1</td><td>2</td><td>2</td><td>40</td></tr></table>

1

2
1 **Figure legends**

2 **Figure 1:** Neighbor-Joining tree of the 40 STs generated from the analysis of the
3 concatenation of the five selected sequences in 67 *G. candidum* isolates. Bootstrap values
4 (1000 repetitions) are indicated at the main nodes. The evolutionary distances were computed
5 using the Kimura 2-parameter method and are expressed as number of base substitutions per
6 site. The rate variation among sites was modeled with a gamma distribution (shape parameter
7 = 5). The analysis involved 40 nucleotide sequences with a total of 2254 positions in the final
8 dataset. Evolutionary analyses were conducted using MEGA6 (Tamura *et al.*, 2011). Numbers
9 in brackets indicate the number of isolates with the same ST. Bar, 0.001 substitutions per site.

10 **Figure 2:** Neighbor-Joining tree of 67 *G. candidum* isolates based on the concatenated
11 sequences. Details of the calculations are as for figure 1.. The analysis involved 67 nucleotide
12 sequences with a total of 2254 positions in the final dataset. Evolutionary analyses were
13 conducted in MEGA6 (Tamura *et al.*, 2011). Strain numbers in bold are from dairy origin.
14 Strains with boxed numbers were isolated from stools. Strains with numbers in italic are of
15 unknown origin. Bar, 0.001 substitutions per site.

16 **Figure 3:** Population structure in connection with the phylogenetic analysis of 67 strains of
17 the 67 *G. candidum* strains. a) Clustering of the strain genotypes by *Structure*. For each of the
18 strains, presented on the abscissa, the estimated contribution (ordinate) to its genotype from
19 each of five hypothetical ancestral genotypes is represented as rectangles of different colours.
20 The origins of each strain are shown above the histogram: “D” milk or dairy, “E”
21 environment, “F” non-dairy food processing, “H” human stools, “n” not known. b)
22 Phylogenetic analysis of the yeasts using *Splits tree*. Strain names are colour-coded as for the
23 histogram labels in a) (D, E, F, H and n): blue, milk or dairy; orange, environmental strains;
24 green, non-dairy food processing; pink, human stools; grey, origin unknown. Groups 1 to 5
1

are highlighted for comparison with the results of the Neighbor-Joining phylogenetic tree of
Figure 2, and of the analysis with Structure.

2

3

4 **Figure 4:** Composite-analysis of the 36 isolates which were in non-unique STs. The MLST
dendrogram was generated as described in the Materials and Methods and is shown on the
left. The inter-LTR profile of each strain was super-imposed on the MLST tree. Variability of
the inter-MLST profile must therefore be observed within each represented ST. The ability to
assimilate galactose, lactose and ribose is indicated by a black rectangle. The geographical
origin and the substrate of isolation of the strains and the ST to which the strains belong are
shown at the right.

5

6

7

8

9

10

11
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4</figcaption></figure>