Introduction {#S1}
============

The yeast species *Geotrichum candidum* (teleomorph *Galactomyces candidus*) is commonly found in water, air, soil, cereals, ripened fruits, milk, and especially on the surface of mold- and smear-ripened cheeses like Camembert, Tilsit, and Pont-L'Évêque ([@B52]; [@B8]; [@B71]; [@B18]; [@B26]; [@B20]). *G. candidum* was long considered an undesirable microorganism because its presence in cheese was uncontrolled, but is now recognized for its contribution to cheese ripening, due to its capacity to consume lactic acid and generate alkaline products ([@B52]; [@B68]; [@B8]). This yeast also contributes to the development of flavor in the cheese due to its proteolytic and lipolytic activities ([@B9]; [@B31]). Additionally, *G. candidum* is a good competitor against unwanted microorganisms such as *Mucor* spp. and has an antagonistic action against pathogens such as *Listeria monocytogenes* ([@B22]; [@B8]). However, these various traits, especially those contributing to the development of cheese flavors and texture, are strain dependent. *G. candidum* species display a great diversity at the morphologic and metabolic levels ([@B35]; [@B44]; [@B40]; [@B54]). Their high level of polymorphism and widely variable phenotypic traits complicate the identification and characterization of *G. candidum* strains by traditional microbiological methods ([@B61]; [@B32]). Many indigenous strains of *G. candidum* have been isolated from the environment or from dairy products ([@B52]; [@B30]; [@B48]). However, the lack of genomic and functional information has limited their potential utility.

Different *G. candidum* strains can be discerned based on chromosome sizes or sequences of their housekeeping genes ([@B29]; [@B3]; [@B56]; [@B41]). The ability to identify distinct phylogenetic groups within the *G. candidum* species lead to the development of two independent multilocus sequence typing (MLST) schemes to genotype isolates of *G. candidum*, but those were never compared using the same set of strains \[MLST2013 ([@B3]) and MLST2017 ([@B41])\]. Moreover, only one genome of the *G. candidum* species has been partially sequenced and subjected to further gene prediction analysis ([@B56]).

The aim of this study was to combine phenotypic assays and whole genome sequencing to elucidate the phenotypic and genetic diversity of eight *G. candidum* strains that belong to distantly related groups from a previous study using MLST ([@B3]; [Table 1](#T1){ref-type="table"}). We also used the newly sequenced genomes to optimize a MLST scheme based on the two MLST schemes available in the literature ([@B3]; [@B41]). The phenotypic and genomic results obtained in this study increase the scientific knowledge on the ripening yeast *G. candidum* and, in the near future, could be used to improve strain selection for cheese ripening processes.

###### 

Origins and allelic profiles of the 41 *G. candidum* strains analyzed.

  Strain     Geo complex   Lineage   ST       Allele   Origin^a^   References                                                                  
  ---------- ------------- --------- -------- -------- ----------- ------------ ------- ------- ------- -------------------------------------- ------------------------
  15         A             3         1        1        1           1            1       1       1       Mold-ripened cheese, Canada            [@B2]
  20         A             2         2        1        2           1            1       2       2       Mold-ripened cheese, Canada            [@B2]
  21         C             1         3        2        3           2            2       2       3       Clotted carrot, Japan                  [@B34]
  28         A             5         4        1        1           1            1       1       4       Mold-ripened cheese, Canada            [@B2]
  34         A             2         2        1        2           1            1       2       2       Mold-ripened cheese, Spain             [@B2]
  37         A             2         2        1        2           1            1       2       2       Smear cheese, France                   [@B2]
  38         A             2         2        1        2           1            1       2       2       Mold-ripened cheese, France            [@B2]
  39         A             5         4        1        1           1            1       1       4       Smear cheese, France                   [@B2]
  **40**     **A**         **5**     **4**    **1**    **1**       **1**        **1**   **1**   **4**   **Smear cheese, France**               [@B21]
  48         B             4         5        1        1           2            3       2       4       Bioreactor contaminant                 [@B2]
  **70**     **C**         **1**     **3**    **2**    **3**       **2**        **2**   **2**   **3**   **Smear cheese, Canada**               [@B2]
  73         A             5         4        1        1           1            1       1       4       Smear cheese, France                   [@B29], [@B30], [@B32]
  74         B             4         6        1        4           2            1       3       5       Grass, France                          [@B29], [@B30], [@B32]
  75         B             4         7        1        5           2            1       4       4       Corn silage, France                    [@B29], [@B30], [@B32]
  76         A             5         8        1        1           1            1       2       4       Milk, France                           [@B30], [@B32]
  **77**     **A**         **3**     **1**    **1**    **1**       **1**        **1**   **1**   **1**   **Milk, France**                       [@B30], [@B32]
  **244**    **C**         **4**     **9**    **3**    **3**       **2**        **4**   **2**   **6**   **Milk, Canada**                       [@B48]
  **317**    **B**         **4**     **10**   **4**    **4**       **2**        **3**   **2**   **7**   **Organic milk, Canada**               [@B48]
  436        A             2         2        1        2           1            1       2       2       Industrial strain--company A           [@B2]
  562        A             5         4        1        1           1            1       1       4       Milk, MPP 1, Canada                    [@B48]
  **563**    **A**         **5**     **4**    **1**    **1**       **1**        **1**   **1**   **4**   **Milk, Canada**                       [@B48]
  645        A             5         4        1        1           1            1       1       4       Smear cheese, Canada                   [@B48]
  655        A             5         4        1        1           1            1       1       4       Mold-ripened cheese, Canada            [@B48]
  664        B             4         11       4        4           2            1       2       4       Milk, MPP 1, Canada                    [@B48]
  690        B             4         12       5        6           2            1       4       7       Milk, MPP 3, Canada                    [@B48]
  1024       A             3         13       5        1           1            1       1       1       Industrial strain--company B           [@B2]
  1025       A             3         13       5        1           1            1       1       1       Industrial strain--company B           [@B2]
  1026       A             4         14       1        1           1            1       2       8       Industrial strain--company B           [@B2]
  **1028**   **A**         **2**     **2**    **1**    **2**       **1**        **1**   **2**   **2**   **Industrial strain--company A**       [@B3]
  1031       A             3         13       5        1           1            1       1       1       Industrial strain--company A           [@B3]
  1032       A             3         13       5        1           1            1       1       1       Industrial strain--company A           [@B3]
  1033       A             3         13       5        1           1            1       1       1       Industrial strain--company A           [@B3]
  1034       A             2         2        1        2           1            1       2       2       Industrial strain--company A           [@B3]
  1035       C             1         15       3        4           2            2       2       3       Industrial strain--company A           [@B3]
  1036       A             2         2        1        2           1            1       2       2       Industrial strain--company A           [@B3]
  1037       A             3         13       5        1           1            1       1       1       Industrial strain--company A           [@B3]
  1038       A             5         4        1        1           1            1       1       4       Industrial strain--company A           [@B3]
  1039       A             5         4        1        1           1            1       1       4       Industrial strain--company A           [@B3]
  1040       A             5         4        1        1           1            1       1       4       Industrial strain--company A           [@B3]
  1041       A             3         1        1        1           1            1       1       1       Industrial strain--company A           [@B3]
  **1146**   **A**         **3**     **1**    **1**    **1**       **1**        **1**   **1**   **1**   **Artisanal sheep cheese, Slovakia**   **CBS 11176**

a

MPP: Milk Production Plant. Bold: Strains sequenced in this study.

Materials and Methods {#S2}
=====================

Biological Material, Culture Conditions, and Genomic DNA Extraction {#S2.SS1}
-------------------------------------------------------------------

Eight *G. candidum* (identified in bold in [Table 1](#T1){ref-type="table"}) and three *Galactomyces* spp. strains were used in this study ([Table 2](#T2){ref-type="table"}). The eight *G. candidum* strains were selected as representatives of each branch in the phylogenetic tree obtained with MLST2013 ([@B3]), while the *Galactomyces* spp. strains were chosen to form an outgroup of other *Galactomyces* species. Considering that the genus *Galactomyces* is not well known, the choice of three strains of different species as outgroups allowed to draw a wider portrait of this genus. All strains were grown on YEG (Yeast Extract Glucose) agar plates \[10 g⋅L^--1^ of yeast extract (Thermo Fischer Scientific), 10 g⋅L^--1^ of [D]{.smallcaps}-glucose (EMD Chemicals) and 15 g⋅L^--1^ of Bacto agar (BD Diagnostics)\]. The strains were inoculated directly from 15% glycerol (*v/v*) stock cultures stored at −80°C. The plates were incubated in the dark for 4 days at 25°C. Isolated colonies were re-streaked on YEG agar plates or in YEG Broth and incubated for 7--9 days at 25°C in the dark. An additional 33 *G. candidum* strains isolated from dairy environment was used to validate the newly designed MLST scheme ([Table 1](#T1){ref-type="table"}).

###### 

Identification and origins of the *Galactomyces* spp. strains.

  Species                         Strain   Origin                                    References
  ------------------------------- -------- ----------------------------------------- ------------
  *Galactomyces geotrichum*       1147     Soil extract, Puerto Rico                 CBS 772.71
  *Galactomyces reessii*          1148     Cold water retting, Indonesia             CBS 179.60
  *Galactomyces citri-aurantii*   1150     Orange orchard soil extract, California   CBS 175.89

The mycelia on YEG Broth were harvested and grounded into fine powder in liquid nitrogen using a CryoMill apparatus (Retsch, Germany). Cryogenic grinding was performed at −196°C with an automatic pre-cooling step at a 5 Hz frequency followed by a 2 min grinding step at a 25 Hz frequency. Genomic DNA was extracted from 30 mg of grounded mycelium using Purelink RNA/DNA viral mini kit (Invitrogen) with the following modifications. Grounded mycelium was homogenized in 200 μl of a 0.9% NaCl solution, prior to the addition of the proteinase K and the lysis buffer. DNA concentration and quality were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, NC, United States). The purified DNA was stored at −80°C until used.

Isolation of Arthrospores, Phenotype Assessment, Growth at 35°C and Assimilation of Carbon Compounds {#S2.SS2}
----------------------------------------------------------------------------------------------------

The arthrospores of eight *G. candidum* and three *Galactomyces* spp. strains were isolated from the mycelium with a sterile cotton swab using 0.5% (*v/v*) Tween 80, suspended in YNB starvation broth \[6.7 g⋅L^--1^ of Bacto Yeast Nitrogen Base without amino acids (BD Diagnostics) and 100 g⋅L^--1^ of [D]{.smallcaps}-glucose (EMD Chemicals)\], and incubated for 48 h at 25°C in the dark ([@B47]). Microscopic images were taken, and spore concentration was determined using a hemocytometer and optical microscopy (BX61, Olympus). Thereafter, the solution was diluted with sterile carbon-free YNB solution to obtain a concentration of 1⋅10^6^ spores⋅ml^--1^. In order to compare the morphology between strains, isolated colonies were re-streaked in three points on YEG agar plates and photographed after 7 days of incubation at 25°C using a standardized photography setup ([@B59]).

Their ability to grow at 35°C was evaluated by inoculating *G. candidum/Galactomyces* spp. strains on GYP agar plates \[40 g⋅L^--1^ of [D]{.smallcaps}-glucose (EMD Chemicals), 5 g⋅L^--1^ of yeast extract (Thermo Fischer Scientific), 5 g⋅L^--1^ of Bacto peptone (BD Diagnostics) and 20 g⋅L^--1^ of Bacto agar (BD Diagnostics)\] for 4 days at 35°C in sealed plastic bags (containing O~2~) immersed in a water bath to ensure a constant temperature ([@B47]). Plates were inoculated in a single point with 1 μl from the starved and diluted yeast cell suspension (1⋅10^6^ spores⋅ml^--1^). Growth was assessed by the presence or the absence of a colony. Preliminary screening of carbon compound assimilation abilities was performed on the same 11 *G. candidum/Galactomyces* spp. strains using YT MicroPlates (BIOLOG) according to the manufacturer's protocol, with the following modifications. All wells of the microplate were filled with 100 μl of the starved and diluted yeast cell suspension (1⋅10^6^ spores.ml^--1^). Plates were incubated in the dark for 10 days at 25°C in unsealed plastic bags to avoid drying out of the wells. *Yarrowia lipolytica* LMA-23 was used as a positive control, while the negative control consisted of sterile distilled water. Yeast growth was determined after 3 and 10 days by optic density (OD) measurement with a BioTek microplate reader Synergy HI and Gen5^TM^ software v2.07. A scan area measurement of the OD was done at a wavelength of 590 nm. Carbon compounds, namely adonitol (Acros Organics), [D]{.smallcaps}-arabitol (Acros Organics), [D]{.smallcaps}-(-)-amygdalin (Acros Organics), bromosuccinic acid (Sigma-Aldrich), α-ketoglutaric acid (Sigma-Aldrich), [L]{.smallcaps}-(-)-malic acid (Acros Organics), [L]{.smallcaps}-glutamic acid (MP Biomedicals), for which variations in assimilation profiles were observed between strains (data not shown) were selected for further tests. Additional carbon sources, namely lactose (Laboratoire MAT), [D]{.smallcaps}-glucono-1,5-lactone (Sigma-Aldrich), sodium citrate (BDH), sodium DL-lactate (Sigma-Aldrich) were also selected because their assimilation allows the formal identification of *Geotrichum* and *Galactomyces* strains at the species level and because of their relevance in the dairy environment ([@B18]; [@B38]). Each carbon source was added at 0.5% concentration (w/v) in 4.9 ml of YNB culture media. For acidic or basic compounds, the pH of the solutions was adjusted between 5.2 and 5.6, with HCl (1N) or NaOH (1N). Spores of all *G. candidum* and *Galactomyces* spp. strains ([Tables 1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}) were then inoculated as described for the YT MicroPlates protocol, in triplicate, using the standard method of Wickerham and Burton and incubated for 21 days at 25°C in the dark ([@B74]; [@B47]). A 15 s vortex agitation was done on each test tube at days 7 and 21. Growth evaluation was determined by eyes using the descriptors established by [@B47].

Library Preparation, DNA Sequencing and Genome Assembly {#S2.SS3}
-------------------------------------------------------

Sequencing library construction was performed from 50 ng of total genomic DNA using the Illumina Nextera DNA library preparation kit (Illumina, San Diego, CA, United States) and Nextera index kit (Illumina), according to the manufacturer's instructions. Purified libraries were quantified using PicoGreen (Promega, Madison, WI, United States), diluted at 2 μM, multiplexed and sequenced on the Illumina HiSeq 2000 platform (101 bp paired-end reads). For strain LMA-244, the Illumina MiSeq platform (300 bp paired-end reads) was also used. FASTQ file generation and demultiplexing were performed using bcl2fastq v1.8.4 (Illumina, San Diego, CA, United States). Both single- and paired-end reads generated were quality filtered using FastQc v0.11.4 and trimmed with Trimmomatic v0.35 in order to cut adapters or indexes, to remove low quality bases (quality score \<10) from the end of the reads, and to discard reads below 21 nt ([@B4]; [@B7]). Reads that remained unpaired were retained as single-end reads. Genome assembly was performed using SPAdes v3.11.1 ([@B6]). The genome of strain CLIB 918 as reference was used for the assembly of the eight *G. candidum* genomes ([@B56]) and *de novo* sequencing and assembly was performed for the *Galactomyces* strains. Based on the optimization of the total number of contigs, the number of contigs ≥1,000 bp and the N50 value, the k-mer option -k 21, 33, 55, 77 were used for all strains.

Scaffolds were filtered using the khmer software with a length cut-off of 1,000 bp ([@B17]). Genome assembly statistics and completeness were assessed using QUAST web interface and BUSCO v3 ([@B36]; [@B73]). BUSCO measures the completeness of a genome assembly by comparing predicted genes in the assembly to a dataset of near-universal single-copy orthologs selected from OrthoDB v9 ([@B73]).

Genomic Content Comparison {#S2.SS4}
--------------------------

A measure of the similarity between all the strains was generated by comparing the k-mer (31 nt) content (presence or absence) of the whole assembled genomes (scaffolds) using Ray Surveyor software, which is a functionality of Ray v3.0.0 ([@B19]). Considering the set of all 31 bp k-mers of an assembled genome *i*, *A~*i*~* = (*k*~1~, *k*~2~, ..., *k~*l**A*~*), and the set of all 31 bp k-mers of an assembled genome *j, A*~*j*~ = (*k*~1~, *k*~2~,..., *k~*l**B*~*), the set of all 31 bp k-mers shared between the assembled genomes *i* and *j* is defined as *K*~*i,j*~ = (*A*~*i*~ ∩ *A*~*j*~). In order to have comparable values because of the varying genome sizes of the *G. candidum* and *Galactomyces* spp. strains, the normalized count of all 31 bp k-mers shared between the assembled genomes *i* and *j*, defined as $K_{i,j}^{\prime} = \frac{|k_{i,j}|}{\sqrt{{|k_{ii}|} \times {|k_{jj}|}}}$, was rather used. Hence, a similarity heatmap was generated with gplots packages in the R environment using the values of the normalized set *K′~*i,j*~* of shared 31 bp k-mers for all pairwise strain combinations ([@B72]). These normalized set of values were also transformed in Euclidean distance metrics with the distance matrix computation function in the R environment and used to generate a UPGMA dendrogram with MEGA v6 ([@B70]; [@B63]).

MLST Loci Selection {#S2.SS5}
-------------------

Two MLST schemes were developed and published independently for *G. candidum* isolates typing. MLST2013 targets six housekeeping genes coding for an alanyl-tRNA synthetase (*ALA1*), a pyruvate kinase (*CDC19*), an acetyl-CoA acetyltransferase (*ERG10*), a glutaminyl-tRNA synthase (*GLN4*), a phosphoglucoisomerase (*PGI1*), and a phosphoglucomutase *(PGM2*) ([@B3]). MLST2017 targets five loci coding for a nucleoporin (*NUP116*), a dihydroorotate dehydrogenase (*URA1*), an oritidine-5′-phosphate decarboxylase (*URA3*), a yeast subtilisin-like protease III (*SAPT4*), and a phospholipase B (*PLB3*) ([@B41]).

All 11 loci targeted in MLST2013 and MLST2017 were amplified for the eight isolates of *G. candidum* (identified in bold in [Table 1](#T1){ref-type="table"}), using the primers available in [Table 3](#T3){ref-type="table"} and the following parameters: initial denaturation step of 2 min at 94°C, followed by 35 cycles (denaturation 30 s at 94°C, annealing for 30 s at 56°C and extension of 1 min at 72°C) and a final extension of 3 min at 72°C. PCR products were visualized by electrophoresis on 0.8% agarose gel, stained with Gel Red, and illuminated under UV light. For all primer sets, a single band was obtained. PCR fragments were sequenced using an ABI 3730×l Data Analyzer (Thermo Fisher Scientific, Waltham, MA, United States) at the Plate-forme de séquençage et de génotypage des génomes (Centre de recherche du CHU de Québec, Université Laval, Québec, Canada).

###### 

Primer sequences of 11 MLST target loci.

  Locus      Primer sequence 5′→ 3′      Locus size (bp)   References
  ---------- --------------------------- ----------------- ------------
  *ALA1*     Fwd-GCTCTTCGTGAGGTTCTTGG    424               [@B3]
             Rev-ACCTCGTAGGCATCAGTGCT                      
  *CDC19*    Fwd-CGCCAGTCAGAGAAGGAATA    355               [@B3]
             Rev-GTCGACCTGGTTCTTGACAC                      
  *ERG10*    Fwd-AACACAACATTTCCCGTGAG    652               [@B3]
             Rev-AGAGCTTAGCGTCAGCACTGA                     
  *GLN4*     Fwd-TGTTCTCAGAGGGTTTCCTG    558               [@B3]
             Rev-CCACATCTGAGGATTGTCGT                      
  *PGI1*     Fwd-ACCGCTGAGACTCTTCGCAA    472               [@B3]
             Rev-CTCCATGGAAAGCTGCTGGA                      
  *PGM2*     Fwd-GAACGGTGTCTACGGTCTTG    548               [@B3]
             Rev-TCAATGTACAGACGGATGGTC                     
  *NUP116*   For-ACCGCTACAACTGGATTTGG    425               [@B41]
             Rev-GAGACCTGTTTGAGGGCTTG                      
  *URA1*     For-CAAGCCAATTGTGCTGAGAA    465               [@B41]
             Rev-GGTGTCGTAGGGCAGTTGAT                      
  *URA3*     For-GCCAAAAAGACCAACCTGTG    470               [@B41]
             Rev-CCTCATCCATACGGTTCTGC                      
  *SAPT4*    For-ATCATTAACACCCCGGCATA    501               [@B41]
             Rev-GTGTCACCAAGCAGAGCAAA                      
  *PLB3*     For-AAGAATATCTGGGATCTTTC    393               [@B41]
             Rev-TGAAGAAGAAGTACCAAGAA                      

The dendrogram obtained with Ray Surveyor software was used as a reference to compare the phylogenetics trees constructed with the MLST2013 and MLST2017 schemes. Once the optimum consensus MLST scheme was designed, all six target loci (*ALA1, CDC19, SAPT4, GLN4, PGI1*, and *PGM2*) ([Table 3](#T3){ref-type="table"}) were amplified, using the same PCR conditions, on an additional set of 33 *G. candidum* strains ([Table 1](#T1){ref-type="table"}).

MLST Data Treatment and Bioinformatics Analysis {#S2.SS6}
-----------------------------------------------

The Staden Package was used for alignment, edition and construction of consensus sequences based on the ABI sequence chromatograms ([@B69]). Consensus sequences were entered in Unipro UGENE v1.31.1 software and multiple sequence alignments were performed using MUSCLE ([@B25]; [@B57]). Sequences were trimmed to be in frame and encode the same number of amino acids. Evolutionary histories were inferred using the PhyML Maximum-likelihood method with bootstrap test of 1,000 replicates ([@B27]).

For the new consensus MLST scheme (MLST2019), the number of polymorphic sites, the dN/dS ratio (dN is the number of non-synonymous substitutions per non-synonymous site and dS is the number of synonymous substitutions per synonymous site), and the average number of nucleotide differences between populations were determined using DnaSP software v6 ([@B64]). The allelic profile of each locus was determined based on nucleotide polymorphisms. The allelic profile of each locus was then assigned a unique number in order of discovery (e.g., *Ala1-1, Ala1-2*...). Afterward, each unique combination of six loci numbers (1-1-1-1-1-1) was used to determine the sequence type (ST; e.g., ST1) of each strain ([@B43]).

Phylogenetic Analysis {#S2.SS7}
---------------------

The MLST2019 data were used to predict the population structure of *G. candidum* by using the STRUCTURE v2.3.4 software ([@B62]). Analyses were performed with a length of Burnin period of 80,000 and 80,000 rounds of calculation after the Burnin period. The number of assumed populations was determined by running simulations with *K* = 2 up to *K* = 10 with five iterations for each assumed population ([@B60]), and was best estimated when the log (probability of data) ceased to increase rapidly. The population structure was then calculated with the optimal ancestral subpopulation (*K* = 5) with default parameters, including 30 iterations of the analysis for *K* = 5. Recombination analysis was performed for the whole data set, within and between the Geo complexes using the NeighborNet method and the *phi*-test for recombination implemented in the software SplitsTree v4 ([@B12]; [@B39]). Additionally, the linkage disequilibrium between and within the loci was verified with the Index of Association (*I*~*A*~) and the Standardized Index of Association (*I*~*A*~^*S*^) using the LInkage ANalysis (LIAN) software v3.7 ([@B66]; [@B37]).

Data Availability {#S2.SS8}
-----------------

Raw sequence data for the Whole Genome Shotgun projects were deposited at GenBank under the Bioprojects accession numbers [PRJNA482576](PRJNA482576) for LMA-40, [PRJNA482605](PRJNA482605) for LMA-70, [PRJNA482610](PRJNA482610) for LMA-77, [PRJNA482613](PRJNA482613) for LMA-244, [PRJNA482616](PRJNA482616) for LMA-317, [PRJNA490507](PRJNA490507) for LMA-563, [PRJNA482619](PRJNA482619) for LMA-1028, [PRJNA490528](PRJNA490528) for LMA-1146, [PRJNA486748](PRJNA486748) for LMA-1147, [PRJNA486749](PRJNA486749) for LMA-1148, and [PRJNA486756](PRJNA486756) for LMA-1150.

MLST partial gene sequences were deposited at GenBank under the accession numbers from [MH745581](MH745581) to [MH745588](MH745588) for *ALA1*, from [MH745589](MH745589) to [MH745596](MH745596) for *CDC19*, from [MH745597](MH745597) to [MH745604](MH745604) for *ERG10*, from [MH745605](MH745605) to [MH745612](MH745612) for *GLN4*, from [MH745613](MH745613) to [MH745620](MH745620) for *NUP116*, from [MH745621](MH745621) to [MH745628](MH745628) for *PGI1*, from [MH745629](MH745629) to [MH745636](MH745636) for *PGM2*, from [MH745637](MH745637) to [MH745644](MH745644) for *PLB3*, from [MH745645](MH745645) to [MH745685](MH745685) for *SAPT4*, from [MH745686](MH745686) to [MH745693](MH745693) for *URA1*, and from [MH745694](MH745694) to [MH745701](MH745701) for *URA3*.

Results {#S3}
=======

Colony Morphology {#S3.SS1}
-----------------

Macroscopic and microscopic observations on YEG culture media revealed three different morphotypes among the 11 *G. candidum* and *Galactomyces* spp. strains (i.e., yeast-like, intermediate or mold-like morphotypes) ([Figure 1](#F1){ref-type="fig"}), as previously described ([@B8]). Strains LMA-77, 563, 1028, and 1148 were characterized by mold-like, white, filamentous, resistant-to-the-touch and not-greasy colonies, with a predominance of vegetative hyphae, whereas LMA-1146 and 1150 were characterized by yeast-like, cream-colored, not resistant-to-the-touch and greasy colonies, with abundant production of arthrospores. Strains LMA-40, 70, 244, 317, and 1147 showed an intermediate morphology, characterized by white-felted, slightly resistant-to-the-touch and not-greasy colonies, with abundant production of arthrospores.

![Morphology of *Geotrichum candidum* and *Galactomyces* spp. strains on YEG culture media. (Left) Macroscopic observation of colonies. (Right) Microscopic observation of arthrospores. **(A)** Mold-like (example: LMA-1028). **(B)** Intermediate (example: LMA-244). **(C)** Yeast-like (example: LMA-1146).](fmicb-11-00737-g001){#F1}

Carbon Assimilation Profile {#S3.SS2}
---------------------------

These 11 *G. candidum* and *Galactomyces* spp. strains were previously identified by rDNA sequencing ([@B2]). To confirm their identification at the species level, we performed carbon assimilation tests in YT MicroPlates and growth tests at 35°C on GYP agar ([Table 4](#T4){ref-type="table"}). Most of the carbon compounds tested showed similar assimilation profiles for all *G. candidum* and *Galactomyces* spp. strains. A positive assimilation was observed for glucose, galactose, sorbose, xylose, glycerol, and succinate. A negative assimilation was observed for inuline, sucrose, raffinose, melibiose, lactose, trehalose, maltose, methyl-glucoside, cellobiose, salicin, rhamnose, ribose, gluconate, glucosamine, and xylitol. A variable assimilation profile was observed for [D]{.smallcaps}-arabitol, α-ketoglutarate, malate, [L]{.smallcaps}-arabinose, adonitol, citrate, gluconolactone, lactate, and bromosuccinate. Among these variable assimilation profiles, α-ketoglutarate and malate could be used to distinguish *G. candidum* strains (*G. candidus*) from other *Galactomyces* species. Additionally, all *G. candidum* strains showed variable assimilation profiles for five carbon compounds (adonitol, [D]{.smallcaps}-arabitol, α-ketoglutaric acid, malic acid, and sodium citrate) ([Figure 2](#F2){ref-type="fig"}, right panel). Apart from strain LMA-1028, all *G.* candidum strains grew positively at 35°C. On the contrary, none of the three *Galactomyces* spp. strains grew at this temperature (Data not shown).

###### 

Carbon assimilation profile for the *G. candidum/Galactomyces* spp. strains.

  Genus                       *Geotrichum candidum*   *Galactomyces* sp.                                                       
  --------------------------- ----------------------- -------------------- ------ ----- ----- ------ ----- ----- ------ ------ ------
  Strain (LMA)                77                      1146                 1028   40    563   317    70    244   1147   1148   1150
  Geo complex                 A                       A                    A      A     A     B      C     C                   
  ST                          ST1                     ST1                  ST2    ST4   ST4   ST10   ST3   ST9                 
  **Substrate**                                                                                                                
                                                                                                                               
  [D]{.smallcaps}-arabitol    W                       \+                   −      \+    −     \+     −     \+    −      −      −
  α-ketoglutarate             W                       \+                   W      \+    W     \+     −     −     −      −      −
  Malate                      \+                      W                    W      W     W     \+     W     \+    −      −      −
  Glucose                     \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  Inuline                     −                       −                    −      −     −     −      −     −     −      −      −
  Sucrose                     −                       −                    −      −     −     −      −     −     −      −      −
  Raffinose                   −                       −                    −      −     −     −      −     −     −      −      −
  Melibiose                   −                       −                    −      −     −     −      −     −     −      −      −
  Galactose                   \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  Lactose                     −                       −                    −      −     −     −      −     −     −      −      −
  Trehalose                   −                       −                    −      −     −     −      −     −     −      −      −
  Maltose                     −                       −                    −      −     −     −      −     −     −      −      −
  Methyl-glucoside            −                       −                    −      −     −     −      −     −     −      −      −
  Cellobiose                  −                       −                    −      −     −     −      −     −     −      −      −
  Salicin                     −                       −                    −      −     −     −      −     −     −      −      −
  Sorbose                     \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  Rhamnose                    −                       −                    −      −     −     −      −     −     −      −      −
  Xylose                      \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  [L]{.smallcaps}-arabinose   −                       −                    −      −     −     −      −     −     −      −      W
  Ribose                      −                       −                    −      −     −     −      −     −     −      −      −
  Glycerol                    \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  Adonitol                    −                       −                    \+     \+    −     W      −     −     −      −      \+
  Mannitol                    \+                      \+                   \+     \+    \+    \+     \+    \+    \+     −      \+
  Succinate                   \+                      \+                   \+     \+    \+    \+     \+    \+    \+     \+     \+
  Citrate                     −                       −                    −      −     W     W      W     W     W      −      \+
  Gluconate                   −                       −                    −      −     −     −      −     −     −      −      −
  Glucosamine                 −                       −                    −      −     −     −      −     −     −      −      −
  Xylitol                     −                       −                    −      −     −     −      −     −     −      −      −
  Gluconolactone              \+                      \+                   \+     \+    \+    \+     \+    \+    \+     −      W
  Lactate                     \+                      \+                   \+     \+    \+    \+     \+    \+    W      \+     \+
  Bromosuccinate              \+                      \+                   −      \+    \+    \+     \+    \+    N/A    N/A    N/A
  Amygdalin                   W                       W                    W      W     W     W      W     W     N/A    N/A    N/A
  Glutamate                   \+                      \+                   \+     \+    \+    \+     \+    \+    N/A    N/A    N/A

+: Positive assimilation; −: Negative assimilation; W: Weak assimilation; N/A: Not available.

![Genomic similarity of three *Galactomyces* spp. and eight *G. candidum* strains represented by a heatmap obtained from the Euclidean distance between the normalized set of k-mers (31 bp fragments) shared between the partial genomes of *Galactomyces* spp. and *G. candidum* strains. Phenetic tree analyses were conducted in MEGA6. (Right panel) Ability of *G. candidum/Galactomyces* spp. strains to grow on carbon compounds (+ for positive assimilation, − for negative assimilation, and W for weak assimilation).](fmicb-11-00737-g002){#F2}

Sequencing, Genome Assembly and Genomic Content Comparison {#S3.SS3}
----------------------------------------------------------

The statistics for the eight *G. candidum* genome assemblies ([Table 1](#T1){ref-type="table"} in bold) were highly variable, except for the assembly size, the GC content and the BUSCO completeness ([Table 5](#T5){ref-type="table"}). Genome sizes varied from 23.1 Mb (LMA-40) to 24.2 Mb (LMA-70), with an average GC content of 42%. Similarly, the statistics for the three *Galactomyces* spp. *de novo* assemblies were highly variable depending on the species, except for the BUSCO completeness. The genome length varied between 22 Mb (*Galactomyces geotrichum* LMA-1147) and 26 Mb (*Galactomyces citri-aurantii* LMA-1150), and the GC content between 37% (*Galactomyces reessii* LMA-1148) and 40% (*G. geotrichum* LMA-1147). Although different strains and/or species were assembled, for all partial genomes, the BUSCO completeness percentage was close to 90% which suggests a good quality draft genome for all isolates sequenced.

###### 

Assembly statistics for the genome of *Geotrichum candidum* and *Galactomyces* spp. strains.

  LMA strain   Mean coverage   Assembly size (Mb)   Total no. of scaffolds   Scaffold N50 (bp)   Longest scaffold (bp)   GC content (%)   BUSCO Completeness (%)
  ------------ --------------- -------------------- ------------------------ ------------------- ----------------------- ---------------- ------------------------
  40           63×             23.10                1,268                    32,639              167,475                 41.8             90.3
  70           70×             24.17                1,450                    44,909              195,322                 41.4             90.2
  77           58×             23.32                1,488                    29,977              158,856                 41.7             90.5
  244          114×            23.20                836                      137,000             210,021                 41.5             90.3
  317          19×             23.36                1,370                    35,529              209,353                 41.8             90.3
  563          49×             23.30                1,427                    31,326              124,845                 41.7             89.9
  1028         34×             23.42                1,332                    36,124              191,607                 41.7             90.5
  1146         54×             23.35                1,363                    32,892              152,162                 41.7             90.3
  1147         64×             21.96                3,121                    13,090              102,582                 40.4             89.8
  1148         105×            22.56                2,775                    13,420              64,496                  36.6             89.5
  1150         55×             25.98                2,668                    16,289              69,389                  39               90.9

The similarity among the partially assembled genomes of the *G. candidum* and *Galactomyces* spp. strains was assessed by comparing their k-mer content, using the software implementation Ray Surveyor ([@B19]). The dendrogram generated from the *K*′*~*i,j*~* Euclidean distances and the heatmap representation of the normalized similarity values (*K*′*~*i,j*)~* uncovered separate groups within the strains ([Figure 2](#F2){ref-type="fig"}, left and center). First, the *G. candidum* strains (LMA-40, LMA-70, LMA-244, LMA-317, LMA-563, LMA-1028, and LMA-1146) were clearly separated from the other *Galactomyces* (strains LMA-1147, LMA-1148, and LMA-1150). These two groups shared 3% or less of their k-mer content. Even though *G. candidum* strains were closely related, they could be separated into three major groups based on their k-mer content ([Figure 2](#F2){ref-type="fig"}).

Multilocus Sequence Typing Schemes for *G. candidum* {#S3.SS4}
----------------------------------------------------

We observed similarities between both previous MLST schemes, according to the clustering of the strains ([Supplementary Figures S1](#DS1){ref-type="supplementary-material"}--[S3](#DS1){ref-type="supplementary-material"}). LMA-40, -77, -563, -1028, and -1146 were grouped together whereas LMA-70 and -244 were grouped together at the opposite side of the tree. On the other hand, LMA-317 clustered differently in the MLST2017 scheme compared to MLST2013. In MLST2017, LMA-317 clustered in a third group of isolates located between the other complexes. Also, for LMA-40, -77, -563, -1028, and -1146, clustering between strains of the same clade was different in the two schemes. This observation led us to evaluate the resolution level of both schemes ([Supplementary Figures S1](#DS1){ref-type="supplementary-material"}, [S2](#DS1){ref-type="supplementary-material"}) by constructing a maximum-likelihood phylogenetic tree for each scheme. We compared both trees to the dendrogram that we constructed using the distance matrix based on whole genome content ([Figure 2](#F2){ref-type="fig"} left panel). Both MLSTs showed similar clustering to the one using whole genomic content, but none had the same precision as the whole genome comparison. Following these observations, we selected six (6) target loci from both MLST schemes to develop an improved genotyping method for *G. candidum* isolates ([Supplementary Figure S3](#DS1){ref-type="supplementary-material"}), named MLST2019. We selected the loci *ALA1, CDC19, SAPT4, GLN4, PGI1*, and *PGM2* because they clustered the isolates the same way as the shared k-mer comparison using the whole genomic content. We then genotyped 33 additional *G. candidum* strains using MLST2019.

We applied the MLST2019 scheme to a total of 41 strains of *G. candidum* (for which 40/41 were the same as MLST2013) and observed 15 STs distributed into three complexes, namely GeoA, GeoB, and GeoC ([Figure 3](#F3){ref-type="fig"}). GeoA complex, supported by a bootstrap value of 1, contained 31 isolates, whereas GeoB complex, supported by a bootstrap value of 0.688, contained 6 isolates and GeoC complex, supported by a bootstrap value of 1, contained 4 isolates. Most *G. candidum* strains (31/41) clustered within GeoA, which includes all the commercial starters except LMA-1035 and all the cheese isolates except LMA-70. Furthermore, the GeoA isolates had either the filamentous, intermediate or yeast-like morphotype. The GeoB complex was composed of *G. candidum* isolates with an intermediate or yeast-like morphotype and these were divided into two sub-complexes, according to their isolation source. Dairy strains LMA-317, 664, and 690 isolated from milk produced in Canada clustered separately from the non-dairy strains LMA-48, -74, and -75, isolated respectively from a bioreactor, grass and corn silage ([Table 1](#T1){ref-type="table"}). The GeoC complex was composed of strains isolated from various environments and were not clustered according to their isolation source. These strains had either a filamentous (LMA-21) or an intermediate (LMA-70, -244, and -1035) morphotype on YEG agar. LMA-1035 was a commercial ripening starter, and LMA-21, -70, and -244 were respectively isolated from rotting carrots, smear cheese and milk ([Table 1](#T1){ref-type="table"}).

![Phylogram of the MLST2019 scheme (*ALA1, CDC19, SAPT4, GLN4, PGI1*, and *PGM2*) constructed with the PhyML Maximum Likelihood method with 1,000 bootstraps (bootstrap values are shown next to the main nodes) on 41 isolates of *Geotrichum candidum*. Phylogenetic analyses were performed with Unipro UGENE v1.31.1.](fmicb-11-00737-g003){#F3}

The combined sequences of the six loci used in the MLST2019 scheme generated a concatenated contig of 2,859 bp with 64 polymorphic sites. The number of polymorphic sites varied from 5 for *CDC19* to 18 for *GLN4* and consisted mostly of synonymous substitutions. The number of non-synonymous substitution sites varied between 1 and 2 for most loci and up to 4 sites for the *PGI1* locus. The allelic profile for each locus was 2 for *SAPT4*, 4 for *GLN4* and *PGI1*, 5 for *ALA1*, 6 for *CDC19*, and 8 for *PGM2*. The proportion of variable sites varied between 1.27 and 3.23%. For each locus, the number of alleles was always lower than the number of polymorphic sites, which resulted in a number of variable alleles per variable sites ranging from 0.13 to 1.20. The dN/dS ratio was always close to 0 and mostly lower than 0.2, except for *PGI1* ([Table 6](#T6){ref-type="table"}).

###### 

Loci characteristics targeted in the consensus MLST scheme used to genotype 41 isolates of *G. candidum*.

  Locus     Locus size (bp)   Number of polymorphic sites\*   Number of alleles   Proportion of variable sites (%)   Number of alleles per variable site   dN/dS
  --------- ----------------- ------------------------------- ------------------- ---------------------------------- ------------------------------------- -------
  *ALA1*    424               8 (1)                           5                   1.89                               0.63                                  0.000
  *CDC19*   355               5 (1)                           6                   1.41                               1.20                                  0.171
  *SAPT4*   501               16 (2)                          2                   3.19                               0.13                                  0.079
  *GLN4*    558               18 (2)                          4                   3.23                               0.23                                  0.028
  *PGI1*    472               6 (4)                           4                   1.27                               0.67                                  0.616
  *PGM2*    548               11 (1)                          8                   2.01                               0.73                                  0.048

\*Numbers in parentheses are the number of non-synonymous substitution sites detected in the locus.

Evaluation of the Population Structure of *G. candidum* {#S3.SS5}
-------------------------------------------------------

Using MLST2019 ST profiles in STRUCTURE software, we statistically estimated the number of ancestral genotypes (*K*) of the yeast *G. candidum*. The STs were clustered in 5 subpopulations (*K* = 5), called lineages or clades (1, 2, 3, 4, and 5) ([Figure 4](#F4){ref-type="fig"} and [Table 1](#T1){ref-type="table"}). When compared to MLST2019 clusters (GeoA, B, and C), lineages 2, 3, and 5 form the GeoA complex while lineage 1 corresponds to GeoC. The lineage 4 corresponds mostly to GeoB except for strains LMA-1026 and LMA-244, which come from GeoA and GeoC, respectively ([Figure 3](#F3){ref-type="fig"} and [Table 1](#T1){ref-type="table"}). The subpopulations observed did not seem to cluster the strains either by their ability to assimilate carbon sources, their colonial morphology or their isolation source, although lineages 2, 3, and 5 do not contain environmental isolates ([Table 1](#T1){ref-type="table"} and [Figure 4](#F4){ref-type="fig"}).

![Estimation of the population structure of 41 *G. candidum* strains based on their sequence type profile. For each of the strains, on the abscissa, the estimated contribution, on the ordinate, to its genotype from each of five hypothetical ancestral genotypes is represented by bar plot of different colors. The source of isolation of each strain is shown above the histogram: cheese strain (C), environmental strain (E), industrial strain (I), milk (M). Each color represents a lineage in the *G. candidum* population. Lineage 1 (60% red and 40% yellow), lineage 2 (green), lineage 3 (blue), lineage 4 (close to 60% yellow and 40% red), and lineage 5 (purple).](fmicb-11-00737-g004){#F4}

All MLST2019 loci and Geo complexes were investigated for genetic exchanges by a NeighborNet analysis, based on nucleotide sequences. The six targeted loci (*ALA1, CDC19, SAPT4, GLN4, PGI1*, and *PGM2*) showed a tree-like subpopulation structure, which suggests that the allele diversity is due to clonal descent within a given locus rather than recombination ([Supplementary Figure S4](#DS1){ref-type="supplementary-material"}). The NeighborNet analysis was then performed for GeoA, GeoB, and GeoC complexes separately, for all pairs of complexes GeoA-B, GeoB--C, and GeoA--C, and for all complexes together GeoA--B--C ([Figure 5](#F5){ref-type="fig"}). Tree-like structures were observed within GeoA, and GeoC complexes, and between GeoA and GeoC. The GeoB individual complex and all other comparisons (GeoA--B, GeoB--C, and GeoA--B--C) showed parallelogram-shaped (network-like) structures, suggesting intergenic recombination (data not shown). The recombination hypothesis was verified doing a *phi*-test in SplitsTree ([Table 7](#T7){ref-type="table"}; [@B11]). The recombination events were supported statistically within the GeoB complex (*p* = 0.03), between the GeoA and the GeoB complexes (*p* ≤ 0.001) and on the whole data set (*p* ≤ 0.001) while the absence of recombination was statistically supported for the GeoA (*p* = 1.0), between GeoA and GeoC (*p* = 0.2) and between the GeoB and GeoC (*p* = 0.3). Because only four strains formed the GeoC complex, not enough data was available to perform the *phi*-test.

###### 

Recombination and linkage disequilibrium statistics.

  Geo group   *Phi*    I~*A*~   I~*A*~^*S*^   *p*-value
  ----------- -------- -------- ------------- -----------
  A           1.0      0.21     0.4           0.5
  B           0.03     --0.33   --0.07        0.9
  C           NA       --0.63   --0.13        1
  A--B        ≤0.001   0.32     0.06          0.05
  B--C        0.3      --0.2    --0.04        0.8
  A--C        0.2      1.9      0.37          0.0001
  A--B--C     ≤0.001   0.36     0.07          0.03

![NeighborNet analysis of the combined six loci (*ALA1, CDC19, SAPT4, GLN4, PGI1*, and *PGM2*) obtained from 41 *G. candidum* isolates. Numbering in the figure corresponds to sequence type. Parallelogram formation indicate recombination events. NeighborNet analysis was performed with SplitsTree version 4.](fmicb-11-00737-g005){#F5}

Finally, we verified the linkage disequilibrium of all loci within and between the Geo complexes by using the value of the Index of Association (*I*~*A*~) and the Standardized Index of Association (*I*~*A*~^*S*^) calculated using LIAN software. Both indexes measure the extent of linkage equilibrium within a population by quantifying the amount of intergenic recombination and by detecting association among alleles at different loci ([@B1]). If the value of the indexes differs significantly from zero, it is a sign of linkage disequilibrium and of a clonal population, while a value of 0 is a sign of linkage equilibrium due to frequent recombination ([@B75]). The *I*~*A*~*^*S*^* values were close to 0 for GeoB, GeoA--B, GeoB--C, and GeoA--B--C complexes, and were significantly different from 0 for GeoA (0.4, *p* = 0.5), GeoC (−0.13, *p* = 1), and GeoA--C (0.37, *p* ≤ 0.0001) complexes, confirming what was observed in the NeighborNet analysis ([Table 7](#T7){ref-type="table"}).

Discussion {#S4}
==========

Phenotypic Identification and Characterization {#S4.SS1}
----------------------------------------------

The strains used in this study were successfully associated to *G. candidum* or a *Galactomyces* species following their phenotypic characterization ([@B18]). The strains LMA-1147 (*G. geotrichum*), LMA-1148 (*G. reessii*), and LMA-1150 (*G. citri-aurantii*) had the typical carbon assimilation profiles and the ability to grow at 35°C. Based on the same criteria, all other isolates except LMA-1028 were positively identified as *G. candidum*. It was also possible to distinguish *G. candidum* strains from the *Galactomyces* species, based on their weak or positive utilization of α-ketoglutaric acid and malic acid ([Table 4](#T4){ref-type="table"}), as already observed ([@B46]). In addition, the pairwise comparison of the shared k-mer content (31-mers) confirmed the identification and phylogeny of the four species studied ([Figure 2](#F2){ref-type="fig"}).

The *G. candidum* strains displayed variability in the assimilation of a few carbon compounds ([Figure 2](#F2){ref-type="fig"}, right panel) which could reflect subgroups with different functional traits. In contrast with previous reports, we found that LMA-40 can use adonitol and did not detect variable assimilation of DL-lactate and D-mannitol among the *G. candidum* strains ([@B52]; [@B3]). In addition, using traditional microbiological tests, we observed for the first time the variable assimilation of malic acid and α-ketoglutaric acid, the weak assimilation of amygdalin, and the positive assimilation of bromosuccinic acid in *G. candidum*. Some of the carbon compounds tested in this study can be metabolized during cheese ripening by different microorganisms, including *G. candidum* strains. Lactate assimilation by *G. candidum* during the early phase of cheese ripening has been described several times and is positively associated with deacidification of the cheese surface, which is mandatory for the growth of acid-sensitive bacteria ([@B28]; [@B52]; [@B33]; [@B49]; [@B53]; [@B51]; [@B5]; [@B8]; [@B14]; [@B23]). Citrate metabolism in lactic acid bacteria leads to the production of CO~2~ and diacetyl, which contributes to the typical aspect and flavor of Gouda, Cottage, Quark, and Cheddar cheeses ([@B28]; [@B55]; [@B53]). However, citrate utilization as the sole carbon source by *G. candidum* strains was found to be weak or negative ([Figure 2](#F2){ref-type="fig"}, right panel), suggesting that metabolites produced by *G. candidum* through citrate catabolism would not be major contributors to the typical flavor of surface-ripened cheese varieties ([@B52]; [@B49]; [@B51]). Glutamic acid, which can be consumed by all *G. candidum* strains tested, is produced through the transamination of [L]{.smallcaps}-methionine. This enzymatic reaction, in the presence of an amino-acid acceptor, usually the α-ketoglutaric acid, contributes to ammonia and aroma production by *G. candidum* strains during cheese ripening ([@B10]; [@B8]; [@B16]; [@B51]; [@B53]). Finally, no relationship was observed between the genetic population structure and the phenotypic characteristics.

Genome Comparisons and MLST Analysis {#S4.SS2}
------------------------------------

Before this study, the only genome sequence of *G. candidum* available in public databases was for the strain CLIB 918, which was isolated from Pont-L'Evêque cheese ([@B56]). The draft genome assemblies for eight additional *G. candidum* strains presented in this study contained from 836 to 1,488 scaffolds (≥1,000 bp) with N50 values between 29,977 and 137,000 bp ([Table 4](#T4){ref-type="table"}). The high number of short scaffolds observed might arise from the short reads produced through the Illumina HiSeq technology and the presence of repeated regions, which are abundant in eukaryotic genomes ([@B58]; [@B76]; [@B45]). Using the *G. candidum* CLIB 918 draft genome sequence as a reference allowed major improvements in the scaffolding step for the *G. candidum* strains. The assembly size, N50 value and length of the longest scaffold increased with CLIB 918 as a reference, in comparison to assemblies without CLIB 918 ([Supplementary Table S1](#DS1){ref-type="supplementary-material"}). Overall, the three *Galactomyces* spp. draft genome assemblies provided similar estimations of the genome sizes and GC contents to those previously proposed using the thermal denaturation method ([@B67]). These assemblies contain close to 3,000 scaffolds (≥1,000 bp) with N50 value around 13,000 bp. The absence of an available reference genome for the scaffolding of *Galactomyces* spp. strains could explain the more fragmented assemblies, compared to those of *G. candidum* strains.

Sequencing the whole genome of eight *G. candidum* and three *Galactomyces* spp. strains allowed us to use the pairwise comparison of their shared k-mer content as the reference clustering method for optimizing current MLST schemes. Two previous MLST schemes were compared for the genotyping of a common set of eight *G. candidum* isolates ([@B3]; [@B41]). Genotyping with MLST2013 gave rise to two groups of isolates, as previously obtained, whereas a third group was obtained with MLST2017 ([@B3]; [@B41]). Although a few subgroups differ between MLST2013 and MLST2017, both schemes clustered LMA-70 and LMA-244 together and apart from the other *G. candidum* strains.

Considering that neither MLST2013 nor MLST2017 allowed the same sub-grouping as the shared k-mer content, a selection of six loci was used to create an optimized MLST scheme (MLST2019) that would increase the discrimination power between the isolates. The MLST2019 scheme uses the loci *ALA1*, *CDC19, SAPT4, GLN4, PGI1*, and *PGM2*, which, once concatenated, generate a sequence of 2,859 bp. The evolution rate was assessed using the dN/dS ratio for each locus (between 0.000 and 0.616) and suggests that they are not subjected to positive selection, making them suitable gene targets for MLST ([@B65]; [@B77]).

The evidence for genetic diversity could indicate an adaptive divergence, a population differentiation under selection, between the wild strains and the cheese ripening strains of *G. candidum*, as observed for other fungi used in the agri-food industry such as *Penicillium roqueforti* ([@B24]), *Penicillium camemberti* ([@B15]), and *Saccharomyces cerevisiae* ([@B50]). This hypothesis is supported by the observation that most of the non-dairy strains are clustered in GeoB and GeoC complexes, whereas GeoA complex contains all industrial cheesemaking strains studied here, except for LMA-1035, and other isolates from milk or cheese. When analyzing the population structure of the *G. candidum* isolates, we observed that the Geo complexes ([Figures 3](#F3){ref-type="fig"}, [5](#F5){ref-type="fig"}) were similar, but there was a discordance in comparison to the estimation of the ancestral subpopulations (lineages) ([Figure 4](#F4){ref-type="fig"}). The five *G. candidum* lineages were clustered accordingly to MLST2019, except for linage 4 which contains all GeoB strains and includes LMA-244 and LMA-1026. According to the concatenated MLST loci and the whole genome sequences, these strains are part of GeoC and GeoA complexes, respectively ([Figures 3](#F3){ref-type="fig"}, [5](#F5){ref-type="fig"}). The fact that LMA-244 and -1026 share alleles with strains of the complex GeoB could hint at a speciation of environmental strains (GeoB) to form new complexes GeoA and GeoC ([@B24]). Additionally, the calculation of *I*~*A*~ and *I~*A*~^*S*^* indices, both close to 0, supports the hypothesis that the strains studied are in linkage equilibrium and that recombination did play a role in increasing the STs diversity, which was statistically demonstrated, except for GeoB--C ([Table 7](#T7){ref-type="table"}; [@B42]; [@B13]). In this particular case, the low number of strains (LMA-21, -70, -244, and -1035) in the GeoC complex might explain the discordance between both analysis. The isolation and genome sequencing of more strains clustering in GeoC would be needed to further characterize the species *G. candidum*. Moreover, a lower diversity was observed in the GeoA complex than in GeoB and GeoC, as represented by the fewer number of branches and their smaller size ([Figure 3](#F3){ref-type="fig"}). A similar phenomenon has also been observed for domesticated *P. roqueforti* strains, for which cheese complexes had fewer and smaller branches compared to the other clusters ([@B24]). Furthermore, GeoC isolates were distant from the GeoA/B complexes on a phylogenetic level, and isolates from the environment (LMA-21) and the dairy industries (LMA-70, -244, and -1235) were obtained for the GeoC complex. These findings suggest that the GeoC complex could represent a new pool of *G. candidum* strains with an interesting biotechnological potential for the development of cheese products with typical properties.

Conclusion {#S5}
==========

In conclusion, the aim of this study was to combine traditional microbiological techniques with new whole genome shotgun sequencing to illuminate the phylogenetic relationships among *G. candidum* and *Galactomyces* spp. strains of environmental and dairy origin. Thereby, we report the draft assembly of 11 *Geotrichum*/*Galactomyces* spp. strains, increasing the amount of genomic data available for *G. candidum* and other *Galactomyces* species, for which no genome sequence assemblies were available. These strains were identified based on their ability to grow under different conditions and we propose a new MLST scheme to optimally genotype *G. candidum* isolates, because it better represents the whole genomic content of this species and allows a clear visualization of its population structure. Further studies are needed to test the adaptive divergence hypothesis and future comparative genomics studies will shed light on the differences between GeoC and GeoA/B. Also, the sequencing of additional genome, especially from non-dairy niche, are likely to improve the new MLST scheme and give a better understanding of the diversity within each complex. Phenotypic and genomic tools such as whole genome sequencing and the MLST scheme substantially improve our knowledge on this ripening yeast and could ultimately allow a better selection and control of *G. candidum* strains throughout the ripening of cheeses.

Data Availability Statement {#S6}
===========================

The datasets generated for this study can be found in the raw sequence data for the Whole Genome Shotgun projects were deposited at GenBank under the Bioprojects accession numbers [PRJNA482576](PRJNA482576) for LMA-40, [PRJNA482605](PRJNA482605) for LMA-70, [PRJNA482610](PRJNA482610) for LMA-77, [PRJNA482613](PRJNA482613) for LMA-244, [PRJNA482616](PRJNA482616) for LMA-317, [PRJNA490507](PRJNA490507) for LMA-563, [PRJNA482619](PRJNA482619) for LMA-1028, [PRJNA490528](PRJNA490528) for LMA-1146, [PRJNA486748](PRJNA486748) for LMA-1147, [PRJNA486749](PRJNA486749) for LMA-1148, and [PRJNA486756](PRJNA486756) for LMA-1150. MLST partial gene sequences were deposited at GenBank under the accession numbers from [MH745581](MH745581) to [MH745588](MH745588) for ALA1, from [MH745589](MH745589) to [MH745596](MH745596) for CDC19, from [MH745597](MH745597) to [MH745604](MH745604) for ERG10, from [MH745605](MH745605) to [MH745612](MH745612) for GLN4, from [MH745613](MH745613) to [MH745620](MH745620) for NUP116, from [MH745621](MH745621) to [MH745628](MH745628) for PGI1, from [MH745629](MH745629) to [MH745636](MH745636) for PGM2, from [MH745637](MH745637) to [MH745644](MH745644) for PLB3, from [MH745645](MH745645) to [MH745685](MH745685) for SAPT4, from [MH745686](MH745686) to [MH745693](MH745693) for URA1, and from [MH745694](MH745694) to [MH745701](MH745701) for URA3.

Author Contributions {#S7}
====================

VP, SV, M-HL, ED-B, MF, and SL were involved in planning the study and writing the manuscript. SV performed phenotypic characterization, DNA extraction, and genome assembly and comparative genomics analysis. M-HL performed DNA libraries for HiSeq sequencing. VP performed DNA extraction for MLST schemes comparison, MLST analysis, and genome assembly and comparative genomics analysis. P-LP and JC helped with the development of the bioinformatic scripts used for genome analysis.

Conflict of Interest {#conf1}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). Vincent Perkins was the recipient of the of a Levier Doctoral scholarship awarded from the INITIA Foundation. SV was the recipient of the Québec Graduate Scholarship (FRQ-NT) and the Canada Graduate scholarship (NSERC). Eric Dugat-Bony was supported by the AgreenSkills+ fellowship program which has received funding from the EU's Seventh Framework Programme under grant agreement N° FP7-609398 (AgreenSkills+ contract).

The realization of this work could not have been possible without the great help of Dr. Frédéric Raymond and the technical support of the Calcul Québec team, Félix-Antoine Fortin and Maxime Boissonneault. Computations were made on the supercomputer Cedar from the Simon Fraser University, managed by Calcul Québec and Compute Canada. The operation of this supercomputer is funded by the Canada Foundation for Innovation (CFI), Ministère de l'Économie, des Sciences et de l'Innovation du Québec (MESI) and le Fonds de recherche du Québec -- Nature et technologies (FRQ-NT).

Supplementary Material {#S10}
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The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2020.00737/full#supplementary-material>

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[^1]: Edited by: Baltasar Mayo, Consejo Superior de Investigaciones Científicas (CSIC), Spain

[^2]: Reviewed by: Pallavi Singh, Northern Illinois University, United States; Manuela Lopez Villavicencio, Muséum National d'Histoire Naturelle, France

[^3]: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
