# Comparative genomics of emerging pathogens in the Candida glabrata clade.

Toni Gabaldón, Tiphaine Martin, Marina Marcet-Houben, Pascal Durrens, Monique Bolotin-Fukuhara, Olivier Lespinet, Sylvie Arnaise, Stéphanie Boisnard, Gabriela Aguileta, Ralitsa Atanasova, et al.

► To cite this version:

Toni Gabaldón, Tiphaine Martin, Marina Marcet-Houben, Pascal Durrens, Monique Bolotin-Fukuhara, et al.. Comparative genomics of emerging pathogens in the Candida glabrata clade.. BMC Genomics, 2013, 14 (1), pp.623. 10.1186/1471-2164-14-623. inserm-00871184

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# Comparative genomics of emerging pathogens in the *Candida glabrata* clade

Toni Gabaldón<sup>1,13*</sup>, Tiphaine Martin<sup>2</sup>, Marina Marcet-Houben<sup>1</sup>, Pascal Durrens<sup>2</sup>, Monique Bolotin-Fukuhara<sup>3</sup>, Olivier Lespinet<sup>3</sup>, Sylvie Arnaise<sup>3</sup>, Stéphanie Boisnard<sup>3</sup>, Gabriela Aguileta<sup>1</sup>, Ralitsa Atanasova<sup>4</sup>, Christiane Bouchier<sup>5</sup>, Arnaud Couloux<sup>6</sup>, Sophie Creno<sup>5</sup>, Jose Almeida Cruz<sup>7,12</sup>, Hugo Devillers<sup>3</sup>, Adela Enache-Angoulvant<sup>3,11</sup>, Juliette Guitard<sup>4</sup>, Laure Jaouen<sup>3</sup>, Laurence Ma<sup>5</sup>, Christian Marck<sup>8</sup>, Cécile Neuvéglise<sup>9</sup>, Eric Pelletier<sup>6</sup>, Amélie Pinard<sup>3</sup>, Julie Poulain<sup>6</sup>, Julien Recoquillay<sup>3</sup>, Eric Westhof<sup>7</sup>, Patrick Wincker<sup>6</sup>, Bernard Dujon<sup>10</sup>, Christophe Hennequin<sup>4</sup> and Cécile Fairhead<sup>3*</sup>

**Abstract**

**Background:** *Candida glabrata* follows *C. albicans* as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, *C. glabrata* being part of the *Nakaseomyces*, a group more closely related to *Saccharomyces cerevisiae*. Although *C. glabrata* was thought to be the only pathogenic *Nakaseomyces*, two new pathogens have recently been described within this group: *C. nivariensis* and *C. bracarensis*. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic *Nakaseomyces*, and compared them to other sequenced yeasts.

**Results:** Our results indicate that the two new pathogens are more closely related to the non-pathogenic *N. delphensis* than to *C. glabrata*. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing *N. delphensis* and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in *C. glabrata*.

**Conclusions:** Remarkably, some features thought to be the result of adaptation of *C. glabrata* to a pathogenic lifestyle, are present throughout the *Nakaseomyces*, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the *EPA* gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the *Nakaseomyces*.

**Sequence Accession Numbers:**

*Nakaseomyces delphensis*: CAPT01000001 to CAPT01000179

*Candida bracarensis*: CAPU01000001 to CAPU01000251

*Candida nivariensis*: CAPV01000001 to CAPV01000123

*Candida castellii*: CAPW01000001 to CAPW01000101

*Nakaseomyces bacillisporus*: CAPX01000001 to CAPX01000186

**Keywords:** *Candida glabrata*, Fungal pathogens, *Nakaseomyces*, Yeast genomes, Yeast evolution

<footnote>* Correspondence: toni.gabaldon@crg.eu; cecile.fairhead@u-psud.fr
¹Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003, Barcelona, Spain</footnote>

<footnote n="13">¹³Comparative Genomics Group, CRG-Centre for Genomic Regulation, Doctor Aiguader, 88, 08003, Barcelona, Spain</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
## Background

Opportunistic fungal pathogens have become a major source of life-threatening nosocomial infections. This situation is partly explained by modern medical progress, relying on large-spectrum antibiotics, immunosuppressive chemotherapy, and devices such as catheters, all of which have been shown to predispose to invasive candidiasis [1].

Among the emerging fungal pathogens, the incidence of *Candida glabrata* has progressively increased, and it is currently the second or third most prevalent cause of candidiasis. Despite its name, this yeast is phylogenetically closer to the model yeast *Saccharomyces cerevisiae* than to *C. albicans* [2], and is part of the *Nakaseomyces* genus. This genus originally included three other species of yeasts isolated only from the environment, namely *Nakaseomyces* (*Kluyveromyces* delphensis, *Candida castellii* and *Nakaseomyces* (*Kluyveromyces* bacillisporus [3]. Recently however, two pathogens have been added to the genus, *Candida nivariensis* and *Candida bracarensis* [4,5]. Because routine phenotypic tests, such as biochemical identification methods, are unable to identify these newly described species, leading most often to misidentification as *Zygosaccharomyces* (CH and AEA, unpublished) their true clinical relevance may be underestimated. Recently, collections of clinical isolates, phenotypically identified as *C. glabrata*, were screened with molecular methods, and *C. bracarensis* and *C. nivariensis* were found to represent less than 2.2% and less than 0.1% of the strains, respectively, with prevalence possibly varying across countries [6,7]. Interestingly, although *C. glabrata* is considered a commensal of the human gut [8], the ecological niches of *C. bracarensis* and *C. nivariensis* remain unknown. Of note, *C. nivariensis* has been isolated from flowering plants in Australia [9], pointing to the possibility that this species may colonize humans from an environmental source.

As it is often the case in fungi, loss, or scarcity of sexual reproduction is associated to species isolated in human patients. Nonetheless, the *Nakaseomyces* comprise at least one known “environmental” species in which no sex has been observed, *C. castellii* [10]. MAT-like loci and the HO gene, the key player of mating-type switching in *S. cerevisiae* [11], were known to be conserved in *C. glabrata* and *N. delphensis* [12].

The genomic sequence of *C. glabrata* has been available since 2004 [2], and its comparison to *S. cerevisiae* has served to discuss possible genomic and metabolic features related to the pathogenic nature of the former [13]. However, it was as yet unclear whether some of these features were also shared by other *Nakaseomyces* and how their presence actually related to the ability of the different species to become human pathogens. In the case of *Candida albicans*, this has been explored by

sequencing several of its close relatives [14]. To gain a similar insight into the specific features of *C. glabrata* and their relation to pathogenicity, we now report the complete sequencing of the five other known species in the *Nakaseomyces* group.

Our results show that all *Nakaseomyces* nuclear genomes are small, transposon-free and contain significantly less genes than *S. cerevisiae*. This is in contrast to their mitochondrial genomes which, with the exception of *C. glabrata*, are large and invaded by palindromic putative mobile elements, the GC inserts [15]. Loss of genes involved in several metabolic pathways as well as loss or amplification of some gene families, are shared by most, sometimes all, *Nakaseomyces* species, although some remain species-specific. Our molecular phylogenetic analysis supports the phylogeny of the *Nakaseomyces*, as published by Kurtzman, [3] ie all these species can be grouped together as a new genus with a single common ancestor. We also confirm that the genus can be clearly subdivided into two main lineages, where the lineage containing *C. castellii* and *N. bacillisporus* has followed a very divergent evolutionary path. The second group, which we will refer to as the ‘*glabrata* group’, contains the three pathogenic species and *N. delphensis*, which is more closely related to the two recently identified pathogens. This depicts a complex scenario suggesting multiple independent events of emergence of pathogenesis within this lineage, and the presence of a genomic repertoire that may facilitate the emergence of pathogenicity towards humans. Altogether our comparative analyses have enabled us to trace, at high levels of resolution, the genomic changes that occurred within this group and discuss how they relate to the pathogenic ability of the different species.

## Results

### Genome assemblies

Sequencing of the type strains of the five *Nakaseomyces* species; *Candida nivariensis* CBS9983, *Candida bracarensis* CBS10154, *Nakaseomyces* (*Kluyveromyces* delphensis CBS2170, *Candida castellii* CBS4332, and *Nakaseomyces* (*Kluyveromyces* bacillisporus CBS7720, was performed at Genoscope (Evry, France), using a combination of Illumina and 454 technologies (see Methods). Genome data has been deposited at the EMBL.

Final assemblies showed a close correspondence between scaffolds and chromosomes (Additional file 1), and were annotated for coding and non-coding genes (see Methods). Flow cytometry results show that species are haploid, except *N. bacillisporus* which is diploid (Additional file 2). All species have an haploid genome size of 10 to 12 Mb. Chromosome numbers, as estimated by Pulsed-Field Gel Electrophoresis (not shown), range from eight in *C. castellii*, the smallest yet recorded
number of chromosomes in post-WGD yeasts [16], to fifteen in *N. bacillisporus*; with the 'glabrata' group' exhibiting the least variation (10–13 chromosomes). Centromeres, similar in structure to *S. cerevisiae's*, i.e. composed of three short "centromere defining elements", CDEI, II and III [17]; were identified in all species but not in all scaffolds. Telomeric repeats identical to those in *C. glabrata* and the putative telomerase RNA-component were found in the 'glabrata' group'.

Several ncRNA genes are known to be surprisingly large in *C. glabrata*, such as the *RPR1* gene of RNAse P [18] and the above-mentioned *TLC1* gene [19]. This tendency to exceptionally large ncRNAs seems to be general in the *Nakaseomyces*, such as the 1368 nt-long *RPR1* gene in *N. delphensis* (only 369 nt-long in *S. cerevisiae*), and the 1937 nt-long U1 snRNA gene in *C. castellii* (568 nt-long in *S. cerevisiae*). Other structural genomic features are discussed in the supplementary

results (see Additional file 3). None of the species contain detectable active transposons in their nuclear genome.

## Phylogeny of Nakaseomyces

In order to clarify the phylogenetic relationships of the newly-sequenced *Nakaseomyces* and 17 other *Saccharomycotina*, we used two alternative phylogenomics approaches, namely i) a Maximum Likelihood (ML) analysis of a concatenated alignment of 603 protein families that have one-to-one orthologs in all the species considered, and ii) a super-tree approach based on the analysis of 4,965 individual gene trees, which finds the species topology that is most parsimonious in terms of implied duplication events in all the individual gene trees [20]. Both approaches yielded the same, highly resolved topology (Figure 1), which is largely

<figure><img src="image_2.png" /><figcaption>**Figure 1 Maximum likelihood species tree of 22 *Saccharomycotina* species.** The tree was reconstructed based on the analysis of a concatenated alignment of one-to-one orthologs of 603 widespread genes. Species names in red and with an asterisk indicate human fungal pathogens. Underlined species names correspond to the newly sequenced *Nakaseomyces* species. Important evolutionary events such as the Whole Genome Duplication (WGD) or the genetic code transition in the *Candida* clade (CTG) are marked on the tree. All aLRT-based supports were maximal and a single node with a bootstrap support below 100% is indicated. This topology is also the most parsimonious in terms of inferred duplications in 4,965 individual gene phylogenies, as assessed by a Gene Tree Parsimony approach implemented in duptree [20].</figcaption></figure>
congruent, for the shared species, with our current un-
derstanding of *Saccharomycotina* phylogeny [21].

This topology supports the the existence of the *Nakaseomyces* genus, and defines two clear sub-groups separated by an ancient split. The first group comprises the highly divergent *C. castellii* and *N. bacillisporus*, whereas the second group (i.e. the ‘*glabrata* group’) includes the three pathogenic species and *N. delphensis*. The average protein identity between orthologs of species in the different sub-groups ranges from 51 to 53% (Additional file 4), which is similar to that of orthologs in the *C. castellii/N. bacillisporus* (53%) and *C.glabrata/S.cerevisiae* (54%) pairs, pointing to large levels of divergence. The ‘*glabrata* group’, is more compact and shows higher identity levels (77-88%). Notably, the two newly identified pathogens, *C. nivariensis* and *C. bracarensis*, are both closer to *N. delphensis* than to the most frequently isolated pathogen, *C. glabrata* (Figure 1). Thus the emerging picture for the appearance of pathogenesis is complex, with plausible alternative scenarios involving either gain of the ability to infect humans at the base of the sub-clade followed by loss of the trait in *N. delphensis* or three independent events of acquisition of pathogenicity. These possibilities will be further discussed below.

The degree of synteny, i.e. gene order conservation be-
tween species, was found to correlate with phylogenetic
proximity, with the highest conservation occurring between
*C. bracarensis*, *C. nivariensis* and *N. delphensis* (Additional
file 5). Conservation of synteny of the *Nakaseomyces* in
general, relative to *S. cerevisiae* is low. Detailed analysis of
syntenic regions will be presented elsewhere (HD, TG, CF,
in preparation).

Mating types

As is the case for many fungal species described as asexual,
genes involved in sexual reproduction are known to be con-
served in *C. glabrata* [22-24]. In particular, although mating
has never been reported in *C. glabrata*, the two additional
*MAT* cassettes, *HMRa* and *HMLalpha*, and the *HO* gene
are present in its genome, and haploid isolates of both mat-
ing types are found. In *N. delphensis*, also a mainly haploid
species, these elements are also conserved [12,23] and
mating-type switch may occur in culture [12]. For the
remaining four species, three are considered asexual and
mainly haploid, but the last one, *N. bacillisporus*, is consid-
ered to be diploid and homothallic [25]. As mentioned
above, ploidy was confirmed by flow cytometry. All spe-
cies contain a well-conserved homolog of the *HO* gene
(Additional file 6), the most diverged encoded protein being
the one from *N. bacillisporus*, which exhibits a C-terminal
extension rich in proline and serine.

In all species, additional sequencing was needed in
order to obtain the cassettes. In *N. delphensis*, *N.
bacillisporus* and *C. castellii*, amplification of the *MAT*-

like cassette yielded both a and alpha fragments. This
raises the intriguing possibility that *C. castellii* switches
mating types in culture, whilst having no described sex-
ual cycle, or that it is sexual and goes through a diploid
phase. This will need further experimental analysis.
Figure 2 shows which cassettes are currently identified
in these genomes. Genes within cassettes, a1, alpha1 and
alpha2 are identified in all species, as is the Ho site,
which can be recognised at the YZ junction. In cases
where the three cassettes are identified, their configur-
ation is apparently similar to *C. glabrata's*: both *HML*-
and *MAT*-like cassettes are on the same scaffold, while
the *HMR*-like cassette is on a different one, except for
*N. delphensis*, where the three cassettes are on the same
scaffold. Thus, *Nakaseomyces* species follow the rule of
conservation of *HML* and *MAT* on the same chromo-
some, noticed by Gordon et al. [26].

*C. nivariensis* has two *HMR*-like cassettes, i.e. cassettes that contain type *a* information, and that are present in addition to the *MAT* locus, a situation already noted in other species [26]. Experimental testing of these cassettes will reveal what role, if any, these extra loci have, in organisms where sexual reproduction has not been characterized yet.

Variations in gene repertoires

The total numbers of protein-coding genes range from 5400 to 5900, which is similar to what is found in *C. glabrata* [2], and lower than in *S. cerevisiae*. Indeed, the number of true protein-coding genes in *S. cerevisiae* is estimated at around 5800, but this rises to around 6600 when dubious and other non-experimentally characterized ORFs are included, a figure more comparable to our predicted gene sets (SGD, 12 January 2012, http://www.yeastgenome.org, and Additional file 7). To assess the coverage of the predicted gene repertoires we tested for the presence of a set of 2,007 protein families previously found to be widespread in *Saccharomycotina* [21]. Our proteomes contained 99.4-100% of this core dataset, attesting for a high coverage in our procedures.

Intron-containing protein-coding genes are far fewer
in *C. glabrata* than in *S. cerevisiae* (129 vs 287, [27]).
This paucity of introns is shared by all *Nakaseomyces*,
which have intron counts lower than 230. Although ex-
perimental validation is needed, our data point to a re-
markable loss of introns in the *Nakaseomyces*.

To accurately trace the evolution of the genetic reper-
toires across the *Nakaseomyces*, and confidently estab-
lish orthology and paralogy relationships, we performed
an exhaustive phylogenomic analysis that included the
reconstruction of ML phylogenies for every gene
encoded in the *Nakaseomyces* genomes (i.e. the
phylome). These were used to detect orthology and par-
alogy relationships [28], and to map lineage-specific gene
<figure><img src="image_3.png" /><figcaption>**Figure 2 MAT-like cassettes in the sequenced genomes.** HMR-, HML- and MAT-like are represented as rectangles, with red ones containing a-type information and blue ones containing alpha-type information. MAT cassettes of each type are shown for *N. bacillisporus*, which is diploid, and bicolored MAT cassettes in haploid species indicate possible switching in culture. Approximate coordinates on corresponding scaffold are shown below each cassette.</figcaption></figure>

duplication [29] and gene loss events onto the species tree. Figures 3 and 4 and Additional file 8 summarize the main findings regarding the presence or absence of genes relevant to central processes. Lower gene numbers in the *Nakaseomyces* when compared to *S. cerevisiae* are reflected in the number of gene loss events indicated on the tree from Figure 3.

In particular, we have paid special attention to the retained copies of so-called ohnologs (i.e. gene duplicate pairs originated during the WGD event; [30]). In *S. cerevisiae*, only 551 ohnologous pairs are left in the contemporary genome, as most such pairs have lost one member [31]. This is also the case in the *Nakaseomyces* genomes, as in all post-WGD species. The exact complement of ohnologs retained in duplicates varies across species and is likely to reflect particular physiological differences.

A noteworthy example comes from central carbon metabolism: it has been suggested that the six ohnologous gene pairs encoding glycolytic enzymes, such as *ENO1/ENO2*, and *PYC1/PYC2*, conserved in *S. cerevisiae* and *C. glabrata*, play an important role in the Crabtree effect, ie fermentation even in the presence of high glucose and oxygen [32], a trait shared by all *Nakaseomyces* [33,34]. The genes involved are also in pairs in the ‘*glabrata* group’, but, in both *C. castellii* and *N. bacillisporus*, the situation is rather different, with single pyruvate kinase, enolase, and Glyceraldehyde-3-phosphate dehydrogenase genes (Figure 4,

and Additional file 9). Furthermore, *C. castellii* has two hexokinase genes (*HXK*), as compared to four (two pairs of ohnologs) in the other species. Other features of central carbon metabolism in the *Nakaseomyces* are mentioned below and in Additional file 3.

Comparison of the proteins encoded in *C. glabrata* and *S. cerevisiae*'s genomes had revealed several features specific to the former. In particular, since there are fewer genes in *C. glabrata* than in *S. cerevisiae*, there was a special interest in specific gene loss. Indeed, there are at least four entire multigenic families which are absent in all *Nakaseomyces* or represented by a sole member in *C. castellii* and/or *N. bacillisporus*: the *PHO*, *SNZ*, *SNO* and *PAU* families. It is noteworthy that the paralogous *PHO* family of acid phosphatases has been lost, while the rest of the *PHO* pathway is conserved. Functional analysis in *C. glabrata* has shown that the *PHO2* gene is present but not essential for regulation, and that the *PHO4* gene is poorly conserved at the sequence level, but is functional [35]. Phosphate-starvation induced phosphatase activity in *C. glabrata* has been identified and is encoded by *PMU1* homologs in tandem ([36], and see section on tandem arrays). The *SNZ* and *SNO* gene families are poorly characterized in *S. cerevisiae*, but their expression is known to be induced in stationary phase. Finally, the *PAU* family consists of at least 20 subtelomeric genes in *S. cerevisiae*, possibly involved in
<figure><img src="image_4.png" /><figcaption>**Figure 3** Summary of the main findings. The phylogenetic tree represents the evolution of the *Nakaseomyces* species, using *S. cerevisiae* as outgroup. Blue numbers indicate the number of genes that are predicted to have been gained at each lineage during the evolution of the *Nakaseomyces*. Red numbers indicate the yeast genes that have been lost. Species names coloured in red indicate the human pathogens, lighter colouring indicates recently-reported emerging pathogens. Coloured branches can be matched to the corresponding coloured boxes below, which list important events occurring at that lineage in the evolutionary history of *Nakaseomyces*. (Abbreviations: Ploidy: H: Haplobiontic, D: Diplobiontic; Sexual reproduction: H: homothallic, N.O.: not observed).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 4** Phylogenetic profiles of specific gene families and pathways. The phylogenetic tree represents the evolution of the *Nakaseomyces* species, with the pathogens colored. White boxes indicate absence of a particular family or pathway in a given species, while numbers in colored boxes indicate the number of paralogous copies of that gene family or the number of components of a given pathway. Intensity of the colors is proportional to the number of paralogs present.</figcaption></figure>
anaerobiosis [37] and similar to two other gene families, DAN and TIR, that encode cell-wall mannoproteins. There are no homologs to any of these genes in C. castellii and N. bacillisporus, while C. glabrata and C. nivariensis contain two copies of DAN-like genes, and the remaining species seem to contain only one homologous copy.

Also of particular interest was the loss of genes involved in de novo biosynthesis of nicotinic acid (BNA), which was hypothesized to result from the adaptation of C. glabrata to its human host [38]. Comparison to the newly sequenced Nakaseomyces has shown that the lack of BNA genes is common to them all (Additional file 8), regardless of their habitat, suggesting that the loss of this pathway is an ancient event. Notably, loss of BNA genes has also been observed in other, more distant species, such as Kluyveromyces lactis and other species, and seems to be a volatile trait [39]. In fact, all gene losses observed in C. glabrata relative to S. cerevisiae are shared by the 'glabrata group', whereas in the two other species gene absence is variable. A remarkable example concerns the genes necessary for allantoin catabolism. In S. cerevisiae, the subtelomeric DAL (degradation of allantoin) cluster consists of six genes, and was formed in the ancestor to S. cerevisiae and Naumovia castellii, but lost in C. glabrata [40]. The DAL cluster is absent from the 'glabrata group', but is present in both C. castellii and N. bacillisporus. Intriguingly, in these genomes, the cluster contains another gene involved in allantoin catabolism, DAL5, which, in S. cerevisiae, is not linked to the cluster (Additional file 10).

Other notable examples of gene gain/loss involve the
translation machinery: both the number of ohnologous
ribosomal protein (RP) gene pairs and the RP gene regu-
lators, CRF1 and SFP1, vary between species, with no
correlation between absence/presence of regulator genes
and number of ohnologous RP gene pairs (Figure 4,
Additional files 3 and 11).

Central carbon metabolism again provides examples of
gene gain/loss events; all *Nakaseomyces* genomes con-
tain only two copies of *ADH* genes, which, both by simi-
larity and by conservation of synteny, correspond to
orthologs of *ADH1* and *ADH3*, the *S. cerevisiae* genes
that encode, respectively, the cytoplasmic and the mito-
chondrial activities converting acetaldehyde into ethanol.
*ADH2*, which in *S. cerevisiae* is specialized in the conver-
sion of ethanol to acetaldehyde, has no ortholog in the
*Nakaseomyces*. It is possible that bi-directional activities
exist, or that alternative enzymes take over this conver-
sion (i.e. co-option). For example, *S. cerevisiae* has add-
itional alcohol dehydrogenase genes, in particular the
family of aryl *alcohol* dehydrogenases encoded by seven
subtelomeric *AAD* genes and the non-subtelomeric
*YPL088W* gene [41]. *C. glabrata* possesses an array of

three such genes in tandem, while other *Nakaseomyces*
have several dispersed copies, except *C. castellii* which
harbors a single such gene. Experimental analysis is
needed to tell which enzymes catalyze which reactions
in *Nakaseomyces*, and even in *S. cerevisiae*, in which en-
zymatic activities are still in the process of being charac-
terized [42]. As for anaerobic growth, all *Nakaseomyces*
have the ability to grow in micro-aerobiosis, as tested by
standard laboratory methods (not shown). Two pairs of
regulators are described as essential to anaerobiosis in *S.
cerevisiae*; the *ECM22/UPC2* pair and the *SUT1/SUT2*
pair. These genes are conserved in all *Nakaseomyces*
except *C. castellii*. There are also pairs of genes that dif-
fer by their expression under aerobic and anaerobic
growth, such as *COX5A/COX5B* and of *CYC1/CYC7* in
*S. cerevisiae*. In contrast, all *Nakaseomyces* retain a sin-
gle member of each of these ohnologous pairs, raising
the question of their regulation.

Genes involved in virulence

In *C. glabrata*, the *EPA* genes, a family of glycosyl-
phosphatydylinositol (GPI)-anchored cell-wall protein
(CWP) genes [43], are the best characterized genes in-
volved in adhesion to human epithelia [44,45], an ability
associated to virulence in diverse pathogens [46]. Not-
ably, our search for homologs of *C. glabrata* *EPA* genes
in the newly sequenced *Nakaseomyces* (see methods), re-
vealed higher numbers of such genes in the three patho-
genic species. More specifically, in the most prevalent
pathogen, *C. glabrata*, the type strain harbors 18 mem-
bers of this family. Seven new variants of *EPA* genes are
found in a different *C. glabrata* strain, BG2 (data kindly
provided by Brendan Cormack). The other two patho-
genic *Nakaseomyces*, *C. bracarensis* and *C. nivariensis*
have respectively, 12 and 9 members of the *EPA* family.
In contrast, the non-pathogenic *N. delphensis* harbors a
single copy. These differences cannot be attributed to
differences in the quality of assemblies, which were simi-
lar in all newly sequenced species. Of the two remaining
*Nakaseomyces* species, only *C. castellii* contains three
homologs of the *EPA* genes, while *N. bacillisporus*
presented only one distant homolog that clustered with
PWP (PA-14 containing Wall Protein, adhesin gene) and
adhesin-like protein genes in *C. glabrata*. These proteins,
more similar to the floculin homologs in *S. cerevisiae*, are
only distantly related to Epa proteins [47,48]. A closer in-
spection of the corresponding trees in the phylome and of
a composite tree constructed with all members of the
identified *EPA*-like members (Figure 5a), revealed that
a significant fraction of the adhesin gene copies in
*C. glabrata* emerged from lineage-specific duplications in
this species, whereas many duplications in *C. bracarensis*
and *C. nivariensis* are shared. This independent expansion
of *EPA*-like genes in *C. glabrata* and the emerging
<figure><img src="image_6.png" /><figcaption>**Figure 5** Trees of the three groups of adhesins containing more than one member. Phylogenetic trees were reconstructed using the same approach as for the trees present in the phylome, and are visualized using ETE [50], A) proteins grouped with the known *C. glabrata* EPA genes, B) proteins similar to the yeast Flo protein, C) group of unknown adhesins, and D) sequences related to AWP adhesins. Labels indicate protein names and are colored depending on the species they belong to: *C. glabrata* in dark blue, a second strain of *C. glabrata* (BG2) in light blue, *C. bracarensis* in red, *C. nivariensis* in green, *N. delphensis* in purple, *N. bacillisporus* in yellow and *C. castellii* in orange. Logos at the lower right part of the image represent the conservation in each of the adhesin groups of sites that are considered important in the structure of *EPA1* [51].</figcaption></figure>

pathogens supports the idea of an independent emergence of pathogenesis and may explain the important differences in prevalence across these species. Strikingly, the non-pathogenic member of the 'glabrata group', *N. delphensis* possesses a sole representative of this family, although the phylogenetic scenario implies that the common ancestor of this species and the two emergent pathogens would have had a higher number of copies. This scenario implies specific losses of *EPA*-like genes in *N. delphensis*, and a possible secondary loss of adhesion capabilities in this species. Interestingly, our search for adhesin-like proteins uncovered the presence of a group of four proteins in *N. delphensis* with poor similarity with the *EPA*-like family (Figure 5c). Whether this group represents highly divergent members -or even pseudogenes- of the *EPA*-like family in *N. delphensis*, remains to be further investigated. In any case, their high levels of sequence divergence, particularly at the N-terminal region known to harbor the ligand-binding domain in Epa [49], suggest they cannot be functionally equivalent.

Other genes shown to be involved in virulence in *C. albicans* and/or *C.glabrata*, such as the phospholipase B

gene, or the Super Oxide Dismutase genes (*SOD* genes), exhibit variable presence/absence in the *Nakaseomyces*, with no obvious correlation to pathogenicity of the species.

## Genes in tandem arrays

Tandem arrays are a special genomic arrangement of certain gene families that imply specific amplification mechanisms and positive selection. They can either be shared by all members of a species, such as the globin genes in mammals [52], or display polymorphism in terms of number of copies, such as the *CUP1* gene in *S. cerevisiae*, whose amplification is positively selected in copper-containing medium [53]. A few dozen tandems were identified in the *Nakaseomyces* genomes (HD, TG and CF, ms in preparation). The species with the largest tandem arrays is *C. glabrata*, with two arrays of eight genes each (Figure 6), the *MNT3* array (shown to be variable in different strains, [54]) and the *YPS* array [55], and an array of five genes, coding for a protein of unknown function predicted to be involved in carbohydrate metabolism. None correspond to tandem arrays of more than two genes in other *Nakaseomyces*. Two other arrays
A MNT3

<figure><img src="image_7.png" /></figure>

B YPS

<figure><img src="image_8.png" /></figure>

**Figure 6** Tandem repeats of eight genes identified in *C. glabrata* and their homologs in the 'glabrata group'. A: *MNT3* homologs in *C. glabrata* are present in a tandem repeat of eight genes and in a tandem of two genes, with additional isolated copies. Closest species exhibit a tandem repeat of two genes with mixed synteny at the borders. B: *YPS* homologs in *C. glabrata* are present in a tandem repeat of eight genes, with additional isolated copies. Closest species exhibit a tandem repeat of two genes with conserved synteny at the borders.

of three genes are specific to *C. glabrata*, the aryl alco-
hol dehydrogenase gene array, and the *PMU1* array.
These findings indicate that most tandem arrays found
in *C. glabrata* originated specifically in this lineage. This,
together with the finding that some of these arrays are
variable across strains, suggests that the amplification of
these families may be driven by (directional) selection.
Functions encoded by these families, are thus good can-
didates for finding possible physiological advantages
underlying the success of *C. glabrata* as an opportunistic
pathogen. In the case of the *YPS* cluster, the genes have
been shown to be involved in virulence [55]. *PMU1* en-
codes the phosphate-starvation inducible phosphatase
activity, which has been hypothesized to be a specific
adaptation of *C. glabrata* to its mammalian host [36].

Analysis of evolutionary rates in the lineages leading to
the 'glabrata group'

As mentioned above, the ‘glabrata group’ contains sev-
eral species with the ability to infect humans. We have
previously discussed changes that occurred within this
lineage in terms of acquisition and loss of genes. We
next set out to investigate whether we could identify sig-
natures of possible positive selection in the form of

genes with accelerated rates in the branch preceding the
diversification of the ‘glabrata group’. For this we fo-
cused on 2,153 genes predicted as one-to-one orthologs
shared by all *Nakaseomyces* species and *S. cerevisiae*.
Using the species’ phylogeny, we used a likelihood ratio
test (LRT) to compare two nested rate models (Add-
itional file 12). For 991 of the 2153 genes (43%), a model
including a different rate in the branch leading to the
‘glabrata group’ was favored, and in 35 of them there
was evidence for accelerated rates, suggestive of positive
selection (i.e. *d*<sub>N</sub>/*d*<sub>S</sub> rate >1, with an average of 3). In
contrast, we did not find a significant rate acceleration
in either the *S. cerevisiae*, *N. bacillisporus* or *C. castelli*
lineages, nor within the ‘glabrata group’ itself, where the
average *d*<sub>N</sub>/*d*<sub>S</sub> rate was 0.04 indicating high levels of
purifying selection. These findings suggest that prior to
the diversification of the ‘glabrata group’, there was an
increase in the non-synonymous substitution rate in at
least 35 genes (1.6% of the tested genes). Among these
we did not find families that have been related to patho-
genesis in *C. albicans*. Finally, we focused on the
‘glabrata group’ to identify genes accelerated in parallel
in the three lineages leading to the three pathogenic
species or exclusively in the *C. glabrata* lineage. We
identified 94 proteins with evidence for different select-
ive constrains in pathogenic species and *N. delphensis*,
although this difference was mostly due to stronger purify-
ing selection, rather than positive selection, in the patho-
gens. With respect to the *C. glabrata* specific branch, only
four genes presented *dN/dS* >1. Overall, these results
would suggest a burst of nonsynonymous substitution
rates in a significant number of genes preceding the diver-
gence of the '*glabrata* group' and a subsequent stasis
within the group itself. Nevertheless, genes that are posi-
tively selected in these lineages constitute good candidates
for testing potential roles in virulence.

Discussion

Even though experimental data will be needed to clarify
the relationship between genome content and adaptation
to the environment, we show that many gene variations
observed occur in families of genes encoding cell wall
proteins (PAU, EPA, and other ministallite-containing
genes), as well as proteins involved in carbohydrate me-
tabolism. These classes of genes have already been
shown to be involved in adaptation of yeast species to
particular biotechnological niches such as the MEL and
MAL genes in Saccharomyces species, [56,57], the SUL
genes in lager yeasts [58] or in adaptation to the human
host such as the EPA genes in C. glabrata [55] and the
ALS genes in C. albicans [59]. Some of these genes fit
the definition of contingency genes, i.e. genes that en-
code products that mediate the cell's response to its en-
vironment and that evolve faster than allows the average
mutation rate of other genes [60]. Rapid evolution is
supposed to be facilitated by internal repeats, such as
within the EPA genes, and by subtelomeric location, sit-
uations which enhance recombination frequency and
generate new alleles and possibly new genes.

Our data firmly support the existence of the
*Nakaseomyces* genus, but nonetheless, two of the spe-
cies, *C. castellii* and *N. bacillisporus*, have diverged in
many ways, first by the low conservation of their
orthologous genes, when compared to the four other
*Nakaseomyces*, and also by their paucity of tandems, and
by their frequent variation in gene numbers compared
to the ‘*glabrata* group’ (such as in “ohnologous” riboso-
mal protein gene pairs). Furthermore, our analyses
shows that the two emerging pathogens, *C. nivariensis*
and *C. bracarensis*, are most closely related to the non-
pathogenic *N. delphensis*, and all descend from an ances-
tor that has also given rise to *C. glabrata*. In accordance
to their phylogenetic relationship, most genomic par-
ticularities observed in *C. glabrata* as compared to
*S. cerevisiae*, are also shared by the two emerging patho-
gens and *N. delphensis*. Some others, such as the ab-
sence of the nicotinic acid synthesis pathway are even
common to all *Nakaseomyces*, and thus represent

ancient *Nakaseomyces* traits, whose origin must have
pre-dated the adaptation to the human host of some of
the members of the group. Hence, the scenario for the
emergence of pathogenicity within the *Nakaseomyces*,
and the underlying genomic and metabolic changes
must be reinterpreted in the light of these new genomic
data. These findings also highlight the utility of in-
creased taxonomic samplings when correlating genomic
and phenotypic differences. Considering that most gen-
omic and metabolic features previously thought to be
particular of *C. glabrata* are shared by the three most
closely related *Nakaseomyces*, of which two are not nat-
ural inhabitants of the human gut, the most plausible ex-
planation is that they result from adaptations to
environments other than the human gut. The alternative
scenario in which human commensalism is an ancestral
trait that was lost in *C. nivariensis* and *N. delphensis*
seems unlikely given the large evolutionary distances in-
volved and the relatively recent origin of our species.
Furthermore, since these four related species include
one that has never been identified as a human pathogen,
and two that have only been recently reported as oppor-
tunistic pathogens, the link to the emergence of patho-
genesis for their specific genomic features should be
indirect. Nevertheless, the presence of three related spe-
cies able to infect humans within this sub-group of
*Nakaseomyces* is in stark contrast to the almost
complete absence of this ability in the rest of the genus
as well as in the related *Saccharomyces* and
*Kluyveromyces* groups. Thus, it seems that some of the
particularities shared by *C. glabrata* and the three re-
lated species may represent pre-adaptations (exaptations
in evolutionary terms) that may have facilitated, but not
directly triggered, the emergence of pathogenecity to-
wards humans. Although further research is needed to
identify which traits may have been particularly import-
ant for the emergence of pathogenesis within the
*Nakaseomyces*, several of the traits shared by *C. glabrata*
and the three closest relatives are firm candidates. These
include genes that likely underwent positive selection
specifically in this lineage, of which some have homologs
implicated in pathogenesis in *C. albicans*. In addition,
the multiple parallel expansions of the *EPA* genes,
known to be important for the ability of *C. glabrata* to
adhere to human cells, represent a clear example of a
genomic change that correlates with the pathogenic trait.
Our data supports the fact that the emergence of patho-
genesis relies on a combination of genomic alterations,
rather than changes in a single gene family.

Undoubtedly, *C. glabrata* is the most prevalent patho-
gen among the *Nakaseomyces*. This increased ability to in-
fect immunocompromised humans is probably related to
some of the specific changes observed in the *C. glabrata*
lineage, as compared to the other *Nakaseomyces*. These
include specific losses, expansions of some gene fam-
ilies, particularly as tandem arrays, and even the acquisi-
tion of horizontally transferred genes [61]. Remarkably,
some of these traits specific to *C. glabrata* have been re-
lated to virulence, such as the largest expansion of gene
families involved in cell adhesion (EPA) and in phos-
phate starvation (PMUI). That the *EPA* genes have been
expanded in *C. glabrata* independently of the emergent
pathogens is consistent with the important differences
of prevalence and suggest an independent emergence of
increased adherence to the human epithelium, an im-
portant virulent trait.

Considering all these data, one can speculate on the
possible scenarios depicting the origin of pathogenesis
within the *Nakaseomyces*. To start with, our phylogeny
does not support the monophyly of *C. glabrata* and the
two emerging pathogens. Two competing scenarios may
explain this pattern: a single origin of pathogenesis to-
wards humans followed by loss of the trait in *N.
delphensis* or, alternatively, at least two independent
events of emergence of pathogenesis. Although the first
scenario is more parsimonious in terms of the number
of phenotypic shifts, the second hypothesis seems more
likely in the light of the parallel expansions of the *EPA*
genes, and the fact that the pathogens other than *C.
glabrata* have only been recently identified, suggesting
these are recently emerged rather than derived patho-
gens. A plausible scenario for the emergence of patho-
genesis within the *Nakaseomyces*, compatible with our
data, comprises the following steps: an ancestral envir-
onmental yeast with specific genomic features, gives rise
to species adapted to being commensals of humans, of
which some can evolve into opportunistic pathogens. A
certain level of adaptation to the mammalian gut may
have represented a selective advantage to yeast species
that are naturally present in edible parts of plants (i.e.
fruits), since this may have facilitated dispersion by the
animals consuming the plants. Increased levels of adap-
tation to the mammalian gut environment may have
resulted in species that persist in the gut and gradually
adapt to a particular host (e.g. human). Once reached
this point, particular features of some species may pro-
vide them with the ability to colonize an immuno-
compromised host. Such features could be related to the
ability to adhere to the host, persist in tissues other than
the gut and to overcome the (debilitated) host immune
system. Intriguingly, *C. nivariensis* seems not to be a in-
habitant of the human gut, and may colonize human pa-
tients from an environmental source. Although further
research is needed to clarify this, emergence of patho-
genesis from environmental species would suggest that
prior commensalism with humans is not a pre-requisite
for developing infection capacity in *Candida* spp. Never-
theless, such species found in the environment may also

be associated to other mammals. Clearly, additional data
on ecological distribution of these *Nakaseomyces* species
is needed to sort out these alternative hypotheses.

Conclusions

Figure 3 shows a summary of the main findings de-
scribed in this work. Comparative genomics analyses
support the hypothesis that pathogenicity arose several
times in the *Nakaseomyces*, and that this group repre-
sents a true genus, with common ancestral traits that
may be favorable to adaptation to the human host.

Methods

Strains and DNA preparation

All strains are the type strains of the corresponding spe-
cies [4,5,8,10,62,63], obtained from the CBS collection.
All media used were prepared as for *S. cerevisiae*: glu-
cose and glycerol-based complete media, broth and
solid. Cultures were performed at 30°C or 37°C, with
agitation for broth cultures.

Anaerobic growth was tested by inoculating Sabouraud
plates and using the Oxoid™ Anaerogen system [64]. Plates
were examined after 120 h of incubation.

Petite mutants were obtained by exposing cells to Eth-
idium Bromide [65]. Petite mutants completely lacking
mitochondrial (mt) DNA (ρ⁰ mutants) were used for the
flow cytometry experiments so that mt DNA did not
interfere with measures, since mt DNA content can be
quite high in these species.

For the same reason, DNA for sequencing was pre-
pared by standard zymolyase extraction followed by sep-
aration on a CsCl gradient with bis-benzimide [66]. The
upper mt DNA band was discarded and the lower band
used for sequencing. This procedure allowed the mt
DNA sequence to be acquired nonetheless at an accept-
able fold-coverage.

Ploidy determination by flow cytometry

An aliquot of 4 mL from a fresh yeast culture at 1–2 10⁶
cells/mL is mixed with 9.2 mL of pure ethanol and incu-
bated at 4C overnight. After centrifugation, cells are
washed in 50 mM, pH7 Sodium Citrate and resuspended
at 10⁸ cells/mL. An aliquot of 200 µL is treated with
RNAse by adding 2 µL of a 100 mg/mL solution and in-
cubating 2 hrs at 37°C. Half is then labeled by adding
400 µL of Propidium Iodide at 50 µg/mL in 50 mM So-
dium Citrate, and incubating 20mn in the dark. The
sample is then ready for the flow cytometer.

Sequence and annotation of protein-coding genes

Sequencing was done by the Genoscope (Evry, France).
Briefly, a whole genome shotgun (WGS) strategy associ-
ating different types of sequencing technologies was
performed for each strain. An mean 24.8 genome
equivalent (from 15.4 up to 41) was achieved using 454 GSFlx approach using a mixture of 8 kb mate pairs sequencing and single reads. Genome assembly was performed using 454 Newbler software. Subsequently, a mean 70 genome equivalent coverage for each strain was obtained using Illumina GAIIx technology 36 or 76 bp single reads, and these data were used to correct assembly errors [67].

Probably because of the complete sequence identity between segments of the three *MAT*-like cassettes, most of these loci were absent from the automated assembly generated from the Illumina and 454 reads. Only three loci were present in the assemblies: one *HMRa* from *C. bracarensis* and two *HMRa* from *C. nivariensis*. We therefore searched for sequence gaps in regions of synteny with the cassettes from *C. glabrata*. In all cases except one, there was indeed a gap in the region where the cassette was expected to be, by synteny conservation. The *HMR* cassette from *N. bacillisporus* is still missing from the assembly. Fragments were amplified by PCR and sequenced by Sanger sequencing.

Annotation of protein-coding genes was performed with an in-house procedure, which consists of two phases: syntactical annotation (prediction and location of protein coding genes), followed by functional annotation of each element based on comparison with known sequences. The first phase calls upon 6 gene prediction algorithms: CONRAD [68], AUGUSTUS [69], GETORF [70], SNAP [71], GENEMARK [72], GENEID [73], using the same training set of gene sequences, which contains genes with and without introns, for those which needed a training step; the intron-containing genes being defined by comparison to intron-bearing genes of *C. glabrata* and *S. cerevisiae*. All predictions as well as tBLASTn alignments to proteomes of reference species and Uniprot, and PSI-tBLASTn alignments to PSSM representative of Génolevures protein families are integrated using JIGSAW [74]. Then, all gene models from the 6 prediction algorithms plus JIGSAW are put together and filtered to eliminate gene models having unrealistic introns. The overlap conflicts between elements and validation of gene models are solved by taking into account predicted gene models, other chromosomal elements already validated, and similarity regions, strands and frames. The resulting gene models are then submitted to functional annotation, based on a decision tree inspired by previous semi-automated annotation projects held by the Génolevures Consortium (which used BLASTp alignments to proteomes of reference species and Uniprot).

Identification of centromeres was done by searching with fuzznuc from the Emboss suite [70], for sequences homologous to *S. cerevisiae* consensus centromere sequence, ie "[AG]TCA[TC][AG]TG[AC][TC]N(73,167)G

[GT]N(7,15)TTCCGAA" [75], and by manually checking for conservation of synteny in case of multiple hits within a single scaffold. Telomeric repeats were searched for by using the repeat motif from *C. glabrata*, *CAGCACCCAGACCCCA*, as blastn queries against the genomic sequences. This also revealed the putative template inside the *TLC1* gene.

### ncRNA discovery and annotation

tRNA genes were identified by both cloverleaf structure detection [76] and tRNAscan-SE [77]. BLAST [78] and Infernal [79] searches were performed on each of the five *Nakaseomyces* genomes for other ncRNA genes; using annotated ncRNAs from the Genolevures database [80] as queries for BLAST, and the covariance models from RFam database [81] for the Infernal search. The hits of both searches were combined according to the respective ncRNA family and extended in order to detect contiguous hits corresponding to a same candidate. All hits from the same families were aligned and manually checked. Hits were accepted as candidates if: i) the sequence agrees with known structural features, guiding sequences (for snoRNAs) and conserved sequence motifs for homologous molecules and ii) known synteny was verified.

### Phylogenomics

A phylome, the complete collection of phylogenetic trees for each gene in a genome, was reconstructed for each one of the six *Nakaseomyces* species (five newly sequenced and the reference *C. glabrata* genome). The phylomes include 16 other species: *Saccharomyces cerevisiae*, *S. mikatae*, *S. (Naumovia) castellii*, *S. kluyveri*, *S. bayanus*, *Vanderwaltozyma polyspora*, *Lachancea thermotolerans*, *Ashbya gossypii*, *Candida dubliniensis*, *Kluyveromyces lactis*, *Candida albicans*, *Debaryomyces hansenii*, *Zygosaccharomyces rouxii*, *Yarrowia lipolytica*, *Pichia stipitis*, *Lachancea waltii*. Phylomes were reconstructed using the pipeline described in [82]. In brief, for all genes in each *Nakaseomyces* genome, a Smith-Waterman search [83] was used to retrieve homologs using an e-value cut-off of <10⁻⁵, and considering only sequences that aligned with a continuous region representing more than 50% of the query sequence.

Once the sets of homologous sequences were defined, phylogenetic trees were reconstructed as follows. Selected sequences were aligned using three different programs: MUSCLE v3.7 [84], MAFFT v6.712b [85], and DIALIGN-TX [86]. Alignments were performed in forward and reverse direction (i.e. using the Head or Tail approach [87]), and the six resulting alignments were combined using M-COFFEE [88]. The resulting combined alignment was subsequently trimmed with trimAl
v1.3 [89] using a consistency score cutoff of 0.1667 and
a gap score cutoff of 0.9.

The selection of the evolutionary model best fitting
each protein alignment was performed as follows: A
phylogenetic tree was reconstructed using a Neighbour
Joining (NJ) approach as implemented in BioNJ [90];
The likelihood of this topology was computed, allowing
branch-length optimisation, using seven different models
(JTT, LG, WAG, Blosum62, MtREV, VT and Dayhoff),
as implemented in PhyML v3.0 [91]. The two evolution-
ary models best fitting the data were determined by
comparing the likelihood of the used models according
to the AIC criterion [92]; Then, ML trees were derived
using these two models. All trees and alignments have
been deposited in PhylomeDB [82] and can be browsed
on-line (www.phylomedb.org, phylome codes 78 to 83).
Trees were scanned to i) define orthology and paralogy
relationships using a phylogeny-based, species overlap
approach [28]; ii) detect and date duplication events
[29], including large expansions of gene families; and iii)
transfer functional annotations from one-to-one
orthologs in S. cerevisiae. Unless indicated otherwise, all
operations with phylogenetic trees were performed using
scripts implemented within the ETE package [50].

To reconstruct a species phylogeny, alignments of 603
proteins that had a single ortholog in all species consid-
ered in the phylome were concatenated into a single
trimmed alignment of 288,995 positions. A Maximum
Likelihood tree was reconstructed using phyML using
the same parameters indicated for the trees in the
phylome and the LG model. Branch support values were
computed using the aLRT approach (see above) and based
on an analysis of 100 bootstrap repetitions. In addition, a
super-tree was reconstructed from the 4,965 gene phylog-
enies contained in the *N. delphensis* phylome, using a tree
parsimony approach as implemented in DupTree [20].
Both approaches yielded identical topologies.

Average levels of sequence identity between orthologs
were computed as follows: Each orthologous pair was
aligned with MUSCLE v3.7 and the level of sequence
identity was measured with trimAl V1.3 [89] as the
number of identical residues over the length of the
shortest protein.

Substitution rate acceleration along specific lineages

In order to investigate how selective pressure varied
along specific lineages in the phylogeny and whether
positive selection was involved in the evolution and di-
versification of the *Nakaseomyces*, we used a subset of
the original data. The subset analyzed included 7 species:
*the 6 Nakaseomyces* and *S. cerevisiae* as outgroup. Follow-
lowing the same automated pipeline previously de-
scribed to construct the *Nakaseomyces* phylome, we
retrieved all the shared orthologous genes present in a

single copy in all 7 genomes (i.e., one-to-one orthologs),
we concatenated their respective alignments and esti-
mated a phylogenetic tree using maximum likelihood.
The resulting topology is consistent with that of the spe-
cies tree in Figure 1. Using this phylogeny, we tested
whether the rate of evolution of the one-to-one
orthologs had accelerated along specific branches of the
tree (i.e., affecting different species in the group), which
would be consistent with either the relaxation of select-
ive constraints or with the action of positive selection.
We used the program codeml in the paml 4 package
[93] to estimate the *d*<sub>N</sub>*d*<sub>S</sub> rate ratio variation in each in-
dividual one-to-one ortholog, along particular branches
in the tree. Subsequently, we compared pairs of nested
models by means of a likelihood ratio test (LRT) where
the degrees of freedom correspond to the difference in
the number of parameters estimated in each model, and
the distribution of the *d*<sub>N</sub>*d*<sub>S</sub> rate is assumed to follow a
chi square distribution. Values of *d*<sub>N</sub>*d*<sub>S</sub> larger than 5
were filtered out.

We hypothesized an acceleration in gene evolution
rate, and possible cases of positive selection, preceding
the diversification of the 'glabrata group', that were per-
haps involved in the capability of these species to be-
come pathogenic. In LRT A (Additional file 12) we
compare: i) A model that assumes an overall *d*<sub>N</sub>*d*<sub>S</sub> rate
ratio for all branches in the tree (omega 1) and a differ-
ent rate for the branch that is ancestral to the 'glabrata
group' (omega 2), where we hypothesize a rate acceler-
ation; and ii) an alternative model that assumes one *d*<sub>N</sub>
*d*<sub>S</sub> ratio for the basal branches including *S. cerevisiae*, *C.
castellii* and *N. bacillisporus* (omega 1), a different *d*<sub>N</sub>*d*<sub>S</sub>
rate in the branch ancestral to the 'glabrata group'
(omega 2), and another *d*<sub>N</sub>*d*<sub>S</sub> rate for the genus itself
(omega 3). In this test, we were interested in verifying
whether there was a significant increase in the *d*<sub>N</sub>*d*<sub>S</sub>
rate in the branch ancestral to the 'glabrata group' rela-
tive to the overall rate and whether this increase also oc-
curred in the different species within the group.

To investigate whether pathogenic and non-
pathogenic species were subjected to different selective
pressure, we built two more LRTs to analyse another
subset of species focusing on the 'glabrata group', we
therefore excluded *S. cerevisiae*, and *C. castellii*, and
used *N. bacillisporus* as the outgroup (Additional file
12). In LRT B we compared i) a model with two rates,
one for the 'glabrata group', and one for the outgroup
(*N. bacillisporus*); with ii) a model with four rates, one
for the pathogenic species, one for the single non-
pathogenic species in the subset (*N. delphensis*), one for
the branch ancestral to the 'glabrata group', and one for
the outgroup. In LRT C we compared i) a model with
two rates, one for the 'glabrata group', and one for the
outgroup (*N. bacillisporus*); with ii) a model with four
rates, one for the 'glabrata group', one for the branch
ancestral to the 'glabrata group', one for the C. glabrata
species itself and one for the outgroup.

Detection of adhesin genes

Putative adhesins were detected in the newly sequenced *Nakaseomyces* using a similarity search based on the known *EPA* genes in *C. glabrata* and the *FLO* genes in *S. cerevisiae*. Hits were filtered using the same thresholds applied during phylome reconstruction. Additionally, all proteomes were scanned for the presence of the Pfam domain PF10528, which is related to adhesins in fungi. The search was performed using HMMER v3, and proteins containing this domain with an e-value below 1e-05 were added to the selection of adhesins. Proteins were then scanned for undetermined regions as their sub-telomeric location can cause problems in the assembly. Proteins containing more than 33% of undetermined regions were excluded from further analysis (this affected 8 proteins, of which 1 in *C. castelli*, 2 in *N. delphensis*, 4 in *C. bracarensis*, and 1 in *C. nivariensis*). Putative adhesins were then clustered using the TribeMCL [94] algorithm as implemented in scps (inflation = 1.5) [95]. Clusters were then used to infer phylogenetic trees. First the repetitive regions of each sequence were masked using SEG [96]. Alignments were then reconstructed using MUSCLE v3.7 [84] followed by a maximum likelihood tree reconstruction as implemented in PhyML. The LG model was used along with four rate categories and invariant positions inferred from the data.

**Additional files**

Additional file 1: Assembly data.

**Additional file 2: Flow cytometry of the *Nakaseomyces*.** Species names are indicated above each panel. 1C, 2C, 4C indicate peaks corresponding to the DNA content of, respectively, one, two and four haploid genomes.

**Additional file 3: Supplementary text containing additional results and references. Table S1.** tRNA genes in the *Nakaseomyces* genomes. Table S2. GC inserts in the mitochondrial genomes of *C. bracarensis* and *C. nivariensis*.

**Additional file 4: Pair-wise species identity between orthologous protein-coding genes.** Each histogram represents the numbers of orthologous gene pairs according to their percentage of identity.

**Additional file 5: Number of synteny blocks according to the mean length of synteny blocks.** All pairwise comparisons of the genomes of the *Nakaseomyces* and *S. cerevisiae* (out-group) are shown. Genomes were divided into three groups according to their location in the phylogenetic tree (Figure 1): the 'glabrata group' (red); *C. castelli* and *N. bacillisporus* (green); *S. cerevisiae* (blue). Each dot corresponds to one pairwise comparison and is colored according to the groups of the two compared genomes.

**Additional file 6: Alignment of putative Ho proteins.** "LAGLIDADG" motifs and Nuclear Localization Signals are boxed.

**Additional file 7: Histograms of Nakaseomyces genes according to the length of encoded proteins.**

**Additional file 8: Genes from S. cerevisiae that are absent from C. glabrata and their absence/presence in the Nakaseomyces.**

**Additional file 9: Glycolytic enzymes in the Nakaseomyces.**

**Additional file 10: The DAL cluster.** The cluster from *S. cerevisiae* is shown at top. The cluster containing the additional *DAL5* gene in *C. castelli* and *N. bacillisporus* is shown below, using the gene nomenclature from *S. cerevisiae*. In these two genomes, the cluster differs only by the synteny on the left. Genes are represented by arrows, genes in black are *DAL* genes.

**Additional file 11: Duplicated ribosomal protein genes in the Nakaseomyces.**

**Additional file 12: Scenarios for positive selection tests.**

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TG and CF analysed genome data and wrote the paper. AC, EP, JP, PW produced genome data. TM, PD produced tools for automatic genome annotation and analysis. OL produced tools for genome analysis and analysed genome data. SA, SB, LJ, AP, JR produced and analysed MAT cassettes data. RA, SA, LJ, CF produced PFGE and ploidy data. MBF, MMH, GA, RA, CB, SC, JAC, HD, AEA, JG, LM, CM, CN, EW, BD, CH analysed genome data. All authors read and approved the final manuscript.

Acknowledgements

We thank Brendan Cormack for sharing unpublished results about *EPA* genes and access to sequences. We thank our colleagues from the Genolevures network for their continued enthusiasm and support. TG's research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007- 2013)/ERC Grant Agreement n.310325, a Grant from the Qatar National Research Fund grant (NPRP 5-298-3-086), and by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161). CF's research is funded in part by an "Attractivité" grant from the University Paris Sud. GA is a recipient of a Marie Curie grant (FP7-PEOPLE-2010-IEF-No.274223). SB, HD and RA are recipients of, respectively, a shared post-doctoral grant and a Ph. D. grant, from the Région Ile-de-France's DIM Malinf program. JAC was supported by the Ph.D. Program in Computational Biology of the Instituto Gulbenkian de Ciência, Portugal (sponsored by Fundação Calouste Gulbenkian, Siemens SA, and Fundação para a Ciência e Tecnologia; SFRH/ BD/33528/2008).

Author details

¹Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003, Barcelona, Spain. ²Université de Bordeaux 1, LaBRI, INRIA Bordeaux Sud-Ouest (MAGNOME), F-33405, Talence, France. ³Institut de Génétique et Microbiologie, UMR8621 CNRS-Université Paris Sud, Bât 400, UFR des Sciences, F 91405, Orsay Cedex, France. ⁴APHP, Hôpital St Antoine, Service de Parasitologie-Mycologie, and UMR S945, Inserm, Université P. M. Curie, Paris, France. ⁵Département Génomes et Génétique, Institut Pasteur, Plate-forme Génomique, rue du Dr. Roux, F-75015, Paris, France. ⁶CEA, IG, DSV, Genoscope, 2 rue Gaston Crémieux, 91057, Evry Cedex, France. ⁷Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, F-67084, Strasbourg Cedex, France. ⁸Institut de biologie et technologies de Saclay (iBiTec-S), 91191, Gif-sur-Yvette cedex, France. ⁹INRA, UMR 1319 Micalis, F-78850, Thiverval-Grignon, France. ¹⁰Institut Pasteur, Unité de Génétique moléculaires des levures, UMR3525 CNRS, UFR927, Université P. M. Curie, 25 rue du Docteur Roux, F75724, Paris Cedex15, France. ¹¹APHP, Hôpital Bicêtre, Service de Microbiologie, Paris, France. ¹²Present adress: Champalmaud Foundation, Av. Brasília, 1400-038, Lisboa, Portugal. ¹³Comparative Genomics Group, CRG-Centre for Genomic Regulation, Doctor Aiguader, 88, 08003, Barcelona, Spain.

Received: 8 April 2013 Accepted: 31 July 2013

Published: 14 September 2013
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doi:10.1186/1471-2164-14-623

Cite this article as: Gabaldón et al.: Comparative genomics of emerging pathogens in the *Candida glabrata* clade. BMC Genomics 2013 **14**:623.