BACKGROUND
the ascomycetes form the largest phylum in the fungal kingdom.
they exhibit a broad range of life styles, ranging from saprophytes to both plant and animal  pathogens.
mycosphaerella fijiensis, for instance, is the causal agent of 'black sigatoka', one of the most devastating diseases affecting banana and plantains worldwide  <cit> , while the opportunistic pathogen aspergillus fumigatus is the cause of allergy in human patients with atopic immune systems  <cit> .
with the increasing number of genomes of ascomycetes being available, the study of the evolutionary dynamics that give rise to pathogenicity becomes amenable.
the phylogeny of the ascomycetes and more specifically of the pezizomycotina is of particular interest in this respect as some of the pathogens are more closely related to their non-pathogenic counterparts than to other pathogens, emphasizing the importance of the environment in acquiring the pathogenic phenotype  <cit> .
recent studies showed that at least for some protein families the retention of duplicated genes is larger in pathogens than in non-pathogens.
the functions of such gene families with a larger size in pathogens than in non-pathogens are in line with the pathogenic phenotype.
authors agreed that the presence of pathogen-specific families or the specific increase in gene family size in pathogens created novel gene inventories that can be associated with phenotypic trait acquisitions  <cit> .
however, by only comparing gene inventories in the extant species, the evolutionary process that gave rise to the observed enlarged family sizes in pathogens remains unknown.
moreover, most studies which consider gene families at the level of the extant species only tend to explain observed differences in gene family sizes by gains rather than by losses.
as a result, the impact of gene loss during genome evolution is usually underestimated.
in this study we evaluated how both gains and losses affect the origin of phenotypic traits that resulted in pathogenicity in ascomycetes.
to this end, we focused on non-pathogenic and plant pathogenic pezizomycotina.
we reconstructed the ancestral states of extant gene families and studied their evolution in relation to their involvement in the phytopathogenic phenotype.
RESULTS
the phylogenetic relationship between the species used in our analysis is depicted in figure  <dig>  this tree, derived from a subset of ascomycete species, shows that not all pathogens cluster together in the species tree.
the topology in which some of the pathogens are more closely related to non-pathogenic counterparts is of particular interest to uncover the evolutionary mechanisms that resulted in differential phenotypic trait acquisition  and allows to find out which traits are common between evolutionary distant pathogens, whether such common traits have been acquired in each of the pathogens independently  or whether they have a common origin.
gene family size in extant species
for each gene family, a phylogenetic profile was constructed that indicates per species the number of genes belonging to the family.
transforming these profiles allowed extracting those families for which one or more species contained a considerably larger number of genes than observed on average for that family in the remainder of the species; i.e.
families that were selectively larger in a certain species or species set .
subsequent clustering of these families according to the mutual similarity of their normalized profiles identified clusters of families that all displayed a higher than average copy number in the same set of species.
results of this clustering are displayed in figure 2: some clusters represent families with a copy number that is larger than the family-average in one species only, while other clusters correspond to families that show a copy number that is higher than the average in several species.
the latter ones can be further subdivided in gene families with a larger size in species that form a monophyletic clade and those with a larger size in species that do not form a monophyletic clade.
of the  <dig>  analyzed gene families,  <dig> showed a copy number higher than the average exclusively in non-pathogenic species, while  <dig> families had a larger size only in the pathogens.
the function of these gene families that showed a copy number that is higher than the family-average in a subset of the species ) was related to the phenotype of the species in which the larger family size was observed.
only families for which the evolutionary history was consistently reconstructed by dollop and cafe are displayed.
description: go description of the families.
clustering: indicates whether an above the family-average gene copy number was observed in at least one species according to the cluster analysis.
evolutionary events in non-pathogens were not categorized.
family size contractions due to gene loss events that do not imply the entire loss of the gene family were not included in the table but covered in the 'dating gene duplication and loss events' section.
evolutionary events: 'gains' and 'losses' as inferred by dolop and cafe; 'significant expansion as inferred by cafe; 'enlarged gene family size' as inferred by clustering i.e.
size of the family in the indicated species is larger than the family average.
an 'x' indicates whether the event occurred in an ancestral node or in an extant species.
class: gene families were classified according to the evolutionary event that explains their origin.
the four classes  correspond to those shown in figure  <dig> and described in the main text.
remark 1: non-pathogen-specific gains were never observed.
remark 2: although theoretically cafe should be able to discover significant expansions or contractions of gene families in ancestral species, these were never observed.
all cases of significant expansions and contractions were observed in the extant species.
remark 3: note that the class distinction was made based on the gene content of the families and not based on their functional annotation.
this explains why families belonging to the same go category can belong to different classes  of the  <dig> families that were selectively larger in non-pathogens, and for  <dig> of  <dig>  the families that were selectively larger in pathogens.
for the non-pathogens it is mainly in the genome of a. oryzae that many families show a higher than family-average gene copy number .
those families are enriched in acid-ammonia ligase activity, required for the biosynthesis of essential amino acids and x-pro dipeptidyl-peptidase activity.
the latter activity is in line with the ability of a. oryzae to secrete proteolytic enzymes to the environment  <cit> .
for the pathogens, it is mainly f. graminearum, m. fijiensis and m. graminicola that show several families with lineage-specific larger sizes in remarkably similar functions ).
for gene families that exhibited a higher than average copy number in several species simultaneously, no families were found with a larger size in all four non-pathogens.
clusters xi, xii and xiii in figure 2a are examples of families  with a larger size in three of the four non-pathogens.
among the pathogenic species we found three protein families with a higher than the family-average gene copy number in all four pathogens .
these three protein families are involved in mannosidase activity, which is responsible for oligosaccharide catabolism and n-glycan processing.
where mannosidase activity is not essential for morphogenesis and cellular function in the non-pathogen a. nidulans, deleting the activity caused severe defect in conidial formation in the human pathogen aspergillus fumigatus  <cit> .
clusters x-xiv in figure 2b consist of  <dig> families with a larger size in three out of the four pathogens.
functional categories enriched in these protein families included peptidase, g-protein receptor, transferase and chitin binding activities, all of which have been related to pathogenicity in ascomycetes  <cit> .
we found a larger number of protein families with larger sizes in only two genomes .
for the largest subset of those families, gene families exhibited a larger size in two species that form a monophyletic clade: from the  <dig> families,  <dig> had a larger size in both related aspergillus species .
also for  <dig> of the  <dig> families in pathogenic species, the higher copy number was consistently shared by both closely related mycosphaerella species.
gene families with a larger size in two species that do not belong to a monophyletic clade in the species tree are cluster vi in figure 2a consisting of  <dig> protein families enriched in nuclease and fatty-acid synthase activities that show a higher copy number in the genomes of the two non-related non-pathogens t. reesei and a. oryzae and cluster vi of figure 2b consisting of  <dig> protein families enriched in n-acetyltransferase, oxidoreductase and tartrate dehydrogenase activities showing increased copy numbers in the non-related pathogenic genomes of f. graminearum and m. graminicola.
evolution of gene family size
phylogenetic profiling confirms what was also noted in previous studies, namely that the functionality of protein families with a larger gene copy number in extant pathogens is linked to pathogenicity.
the analysis of the phylogenetic profiles, however, does not indicate how these families ended up being extended in the extant pathogens.
for instance, the families with a larger copy number in multiple species that do not form a monophyletic clade can either originate through independent duplication events in non-related species  or by duplications in an ancestral species followed by gene loss in some of the descendants.
to understand the evolutionary dynamics of these common and specific traits that can be linked to pathogenicity, we applied a suite of complementary analyses.
first, we used dollop  <cit>  which maps for each gene family the evolutionary history of gene gains and losses.
by reconciling the binary profiles of gene families with the species tree, dollop infers the minimal gene set for the different ancestral nodes using a parsimony approach which assumes gene loss to be irreversible  <cit> .
dollop thus finds for every internal node in the tree whether the species derived from that node gained or lost a specific protein family .
however, as dollop uses binarized phylogenetic profiles that indicate whether a certain species contains a representative of a family, it does not contain information on the size of the protein family in each species.
cafe  <cit>  on the other hand makes also use of the number of genes in each family to model the evolution of gene families.
in doing so, cafe not only reports on gains and losses, but it also estimates the most likely number of genes for each family at each of the ancestral nodes of the species tree.
from these results the occurrence of expansions and contractions of gene families along the time points in the species tree can be derived.
cafe infers an expansion at a given node if the number of genes in a gene family augmented compared to its number in a previous ancestral time point.
a gain of a gene family can be considered as a particular case of an expansion for which the gene family was not present in the previous ancestral time point.
similarly, a contraction is a reduction in the number of genes in a gene family compared its number in the previous ancestral time point.
gene family loss is again a particular case of a contraction for which an ancestral gene family completely disappears in a descendant species.
the species tree, with for each node an indication on the global number of gained and lost gene families, as derived from dollop and cafe, is depicted in figure  <dig> .
to gain insights into the molecular functions and/or biological processes of the gene families that have been lost or gained or were expanded or contracted at each of the different time points, we analyzed the go functional categories for which the families were enriched.
the complete results of dollop and cafe, i.e.
a list of the evolutionary events for each of the time points  in the species tree can be found on the supporting information .
protein families for which both dollop and cafe were consistent in inferring the same evolutionary history are displayed in table  <dig>
general observations
in figure  <dig>  tp <dig> represents the last common ancestor of the eight species under consideration.
small discrepancies in absolute numbers between dollop and cafe result from the different underlying assumptions between both methods, but in general both methods predict similar tendencies.
since the origin of the ancestral species on tp <dig> and tp <dig>  gene families seem to have been continuously lost and this loss does not seem to have been compensated for by gene family gains .
when considering the species derived from tp <dig> , gene loss was significantly higher in the non-pathogens than in the pathogens .
for the second group of species , this tendency might be masked by the unusually large genome size that seems to have been maintained in the cultivated species a. oryzae  <cit> , resulting in gene loss being at least as prominent in the pathogenic mycosphaerella species than in the non-pathogenic aspergillus species .
in the following, we give a detailed analysis of the evolutionary events that resulted in the currently observed gene copy numbers in extant pathogens.
pathogen-specific gene family gains
with a pathogen-specific gain we refer to a gene family not present in a species that is ancestral to both pathogenic and non-pathogenic lineages , but that appears in one of the extant pathogens  or in an ancestral node that gives exclusively rise to pathogenic lineages  .
note that the term 'specific' used in the context of this paper should be interpreted as specific relative to the number of species analyzed in this study.
we found several examples of families that were selectively present in a pathogen or in set of pathogens that originated through this mechanism of family gain and of which the function can be related to pathogenicity.
examples of families that are gained in one specific extant pathogen are, for instance for m. grisea, families involved in subtilase and cutinase activity and families related to the polyketide biosynthesis pathway.
all of these gene families have been directly linked to the pathogenic phenotype in m. grisea  <cit> .
in the pathogen m. graminicola we observed a significant gain of families containing genes with peptidase and phospholipase a <dig> activities.
these functions are needed for the organization of infective structures in pathogenic fungi.
the expression of genes with phospholipase a <dig> activity is induced in pathogenic fungi during nutrient starvation, which is assumed to be an environmental trigger for host invasion  <cit> .
families specifically gained by m. fijiensis are enriched in metallo-exopeptidase activity which has been related to the morphological development of infective structures in the fungal pathogen ustilago maydis  <cit> .
in the pathogen f. graminearum we found some specific gained gene families related to lipid metabolism and peptidase activity that do not occur in any other species.
in f. graminearum we also observed the gain of specific gene families associated with transmembrane transporters.
the higher than average family size we observed in multiple extant pathogens that form a monophyletic clade in the species tree, are explained by gains in the common ancestor of these related species, maintained in the extant species .
an example is the gain of proteolysis related gene families in the common ancestor tp <dig> of the closely related m. fijiensis and m. graminicola species.
especially the gain of a family of zinc endopeptidases with astacin activity is interesting as it has been related to the activation of peptides that act as morphogenetic signal for foot activation in hydra  <cit> .
of particular interest are the different families, all involved in similar biological processes that were gained independently in multiple species, that are not each other close neighbors in the species tree, as these confer examples of convergent evolution.
for instance, f. graminearum and m. grisea both independently gained gene families that are different from each other, but all related to polyssacharide binding and chitinase activity.
chitinases are closely linked to growth and morphological development in fungi: cell expansion and division, spore germination, hyphal branching and septum formation all depend on enzymes with hydrolytic activities including chitinases  <cit> .
in previous work, a larger than expected size of the chitinase family was already observed for f. graminearum  <cit> .
similarly, the genomes of f. graminearum, m. fijiensis and m. grisea gained different sets of families all related to oxidative stress response, such as families involved in glutathione and thioredoxin peroxidase activities.
these gene families that were gained independently from each other in evolution could be involved in a generally conserved defence mechanism of fungi against plant mediated oxidative burst  <cit> .
we also observed different families related to transferase activity that were gained independently in f. graminearum, m. fijiensis and m. graminicola.
pathogen-specific gene family expansions
also gene families with an origin ancestral to both pathogens and non-pathogens that expanded in a pathogen-specific way can explain the larger size of gene families in extant pathogens .
based on cafe ,  <dig> such gene families showed a significant expansion in extant pathogenic species .
for instance, a gene family with methyltransferase activity that has previously been related to fungal growth and virulence  <cit>  showed a significant expansion in f. graminearum.
as methyltransferases methylate fungal specific glucosylceramides, i.e., molecules that serve as receptors for antifungal plant defensins, they can be directly linked to molecular interactions between the fungus and its host.
two more gene families with ribonuclease h and rna-directed dna polymerase activities showed a significant expansion in m. grisea.
not surprisingly, the function of these families is related to processing transposable elements which are, as indicated by the dollop analysis abundantly present in the genome of m. grisea.
insertional mutants of genes from both the ribonuclease h and rna-directed dna polymerase activities gene families are virulence defectives .
an ancestral family involved in the polyketide biosynthesis pathway was also found to have been expanded in m. grisea.
the relation of polyketide biosynthesis with pathogenicity in m. grisea is strongly supported by the work of jeon et al.
based on insertional mutagenesis .
experimental data was collected from two sources:  pathogen-host interaction database   <cit>  and  the work of jeon et al.
<cit> .
phenotype: based on mutant deviations in conidiation and appressorium formation compared to untransformed control.
effector: gene product or signaling molecule resulting from its enzymatic activity that triggers defensive mechanisms in the plant host.
hence, effectors are usually known as avirulence determinants.
although contractions of gene families in ancestral nodes can theoretically be inferred by cafe, we did not observe them.
non-pathogen-specific losses
both previous mechanisms of pathogen-specific gains and pathogen-specific expansions of ancestral gene families only account for  <dig> % of the families with enlarged size in pathogens .
for a considerable fraction of the families , the observed gene family sizes in the extant pathogens seems to originate from an ancestral gene family that got lost in extant non-pathogens, but that was selectively maintained in the pathogens .
with 'loss of a family in a particular species or ancestral node' we refer here to an ancestral gene family that lost all of its members in the species of interest descending from the ancestor.
the non-pathogen-specific loss of ancestral gene families is in line with the overall observed higher number of gene losses in non-pathogens than in pathogens  for the species we used in this study.
the most striking examples of non-pathogen-specific family losses are those observed for the families involved in transmembrane transport , lipid metabolism and chitin binding.
these families, shared by all pathogens, were present in a common ancestor on tp <dig>  but got lost in the non-pathogen lineages .
this indicates that transmembrane transport, lipid metabolism and chitin binding are essential and generic traits in determining pathogenicity in ascomycetes.
related to the evolutionary dynamics of gene families involved in lipid metabolism, it seems that not only these families were lost in the non-pathogenic branch , but also that the pathogen f. graminearum gained some extra families in addition to the ones it inherited from its ancestor on tp <dig> .
the fact that both non-pathogen-specific loss and species-specific gains are observed for lipid metabolism further strengthens the importance of this function in pathogenicity.
the relation between lipid metabolism and pathogenicity is further confirmed by the fact that disrupting the genes encoding for an extracellular lipase responsible for fatty-acid uptake, significantly reduced virulence in ascomycete pathogens  <cit> .
in addition, a gene expression study of f. graminearum isolated from infected plant tissue showed that genes involved in lipid biosynthesis and degradation were differentially expressed during host colonization  <cit> .
transmembrane transport mechanisms are known to play a key role during the early stages of host invasion by functioning as receptors for signals from the environment that trigger spore germination  <cit> .
a mutant of a gene coding belonging to this family of transmembrane transporters in m. graminicola showed a reduced virulence phenotype .
on the other hand, chitin binding proteins are known as avirulence receptors that interact with the plant host.
genes encoding chitin binding proteins were differentially expressed in a cdna library of m. fijiensis grown in the presence of banana leaves  <cit> .
other ancestral gene families present on tp <dig> that got lost in all studied non-pathogens but were retained in the pathogens are those involved in mannosidase activity, astacin activity and the g-coupled protein receptor.
this model of gene family loss in non-pathogens also explains why we observed members of the families related to n-acetyltransferase, oxidoreductase and tartrate dehydrogenase activities only in the genomes of the pathogens f. graminearum and m. graminicola that do not form a monophyletic clade in the species tree.
the utilization of tartrate has been linked to the induction of pathogenicity related genes in m. graminicola  <cit> .
these families originated in the ancestral tp <dig>  and got lost in all extant species other than f. graminearum and m. graminicola .
both f. graminearum and m. graminicola share wheat as common host  and those families only being retained in those two pathogens reinforces the importance of the pathogen-host interaction process in the selection of pathogenic traits.
in addition to the examples discussed so far where gene families have been collectively lost during evolution in all non-pathogens, some losses of gene families were more restricted to the non-pathogens of the evolutionary branch that started either from tp <dig> or the one that descended from tp <dig> .
some of the ancestral families that got lost in non-pathogens, but were specifically retained in pathogens underwent further expansions as predicted by cafe.
for those cases it is the combined action of non-pathogen-specific loss and pathogen-specific expansions that explains the observed large family size.
for instance the gene families involved in transmembrane transporters showed significant expansions in the pathogens m. graminicola and f. graminearum .
the same was true for the families involved in chitin binding and astacin activity that were both selectively expanded in m. fijiensis.
pathogen-specific gene family losses
in a few cases only, the acquisition of the pathogenic phenotype could be related to the loss of ancestrally present gene families in a pathogen-selective way.
a significant number of the families that disappeared in the ancestor of both mycospaerella species   are related to nitrogen catabolic processes, to the regulation of nitrogen utilization and sensing and to cellular responses towards nitrogen starvation.
nitrogen starvation has been related to virulence in ascomycete plant pathogens by acting as an environmental clue for the expression of disease symptoms in the pathosystem magnaporthe grisea - oryza sativa  <cit> .
loss of gene families involved in nitrogen metabolism, regulation and sensing could reduce the ability of the fungus to sense environmental nitrogen levels and mimic a continuous nitrogen starvation condition that might activate the pathogenicity transcriptional program in m. fijiensis and m. graminicola.
dating gene duplication and loss events
both dollop and cafe agree in predicting that the selective loss of families with an ancestral origin in non-pathogens  is the major cause for the observed differences in gene copy number between extant pathogenic and non-pathogenic species.
so far, predictions made by dollop and cafe only considered losses of entire gene families .
this is to be expected as dollop can only make predictions about 'presence' or 'absence'  states of entire protein families while for cafe the entire loss of a family provide statistically a stronger signal than the spurious loss of a single gene in a family.
to uncover whether also less drastic losses i.e., where part of the genes of a family got lost but not the entire family disappears, we relied on tree reconciliation.
we analyzed  <dig> families that have minimally two members in at least one of the species on tp <dig>  tp <dig>  tp <dig> and tp <dig> and in addition the family should have representatives in at least two other species .
we only considered the part of the species tree  where a non-pathogenic species always clusters with its pathogenic counterpart  as this topology allows directly assessing what proportion of the observed differences in gene family size between extant pathogens and non-pathogens are caused by the different evolutionary scenarios depicted in figure  <dig>  gene trees for all families were constructed.
by reconciling them with the species tree, we predicted the nodes at which duplication and loss events occurred during evolution of the studies species  <cit> .
to avoid biases introduced by large scale tree reconciliation  <cit> , we retained  <dig>  trees of which the topology was supported with a bootstrap value of at least 75%  and only considered the reconciliation results obtained from gene trees with a reliable topology .
the reconciliation analysis showed that for a relative small fraction of the gene families, larger gene family sizes in the extant pathogens f. graminearum and m. grisea resulted from expansions of ancestral or later gained gene families specifically occurring in these genomes  .
most expansions occurred selectively in the genome of m. grisea.
conversely, expansions of gene families in non-pathogens did barely occur.
the majority of the families with a larger gene copy number in extant pathogens  seem to have been the result of ancestral gene families of which the copies were selectively maintained in the pathogens, but got  lost in the non-pathogenic counterparts .
the observed gene family contractions in the non-pathogens were significantly higher than contractions observed in pathogenic gene families.
discussion
previous comparative approaches studied pathogenicity in ascomycetes mainly by comparing gene inventories in extant species.
in line with these studies we showed that gene families which exhibit a size larger than the family-average in pathogens are responsible for acquired phenotypic traits related to pathogenicity  <cit> .
however, by not only studying differences in gene family size between pathogens and non-pathogens, but by also analyzing the complete evolutionary history that gave rise to the observed differences in gene family size, we found that only a relatively small fraction of the gene families with a size that is selectively larger in pathogens could be explained by the combined effect of gene family gain with pathogen-specific expansions.
most of the families with such larger size in pathogens seemed to have been present already in the ancestor of both pathogens and non-pathogens.
the potential of genes and gene products needed to develop pathogenicity was already present in the ancestor of all current species and was selectively maintained in pathogens.
strikingly, it were not the pathogen-specific expansions of these ancestrally present gene families that explained the discrepancies in gene family size between pathogens and non-pathogens, but rather the selective loss of those ancestrally present gene families in non-pathogens.
species that did not develop pathogenicity seem to have reduced their genetic complexity compared to their ancestors.
gene families that evolved according to this gene loss theory are mainly involved in generic functions, conserved amongst all fungi such as transmembrane transport, lipase activity, mannosidase activity, g-coupled protein receptor and astacin activity.
only the number of families with those functions remained considerably higher in pathogens than in non-pathogens.
this observation is confirmed by previous molecular studies which showed indeed that a vast repertoire of genes, involved in virulence and pathogenicity, have general functions conserved among all fungal species   <cit> .
pathogenic fungi have to closely interact with their host.
it is, therefore, not surprising that the host-pathogen interaction further shaped the genomes of fungal pathogens in the ascomycetes.
we observed that the interaction with a specific environment resulted in the differential retention of gene families with similar functionalities in phylogenetically distant pathogenic species .
moreover, all families that are expanded in certain pathogens could be functionally linked to the specificities of the host-fungus interaction.
the latter observations emphasize the importance of the environment in explaining expansions in family size.
CONCLUSIONS
we found that most of the proteins needed to develop pathogenicity were already present in the ancestor of the ascomycetes lineages we used in our study, i.e.
non-pathogenic and plant pathogenic pezizomycotina.
species that did not develop pathogenicity reduced their genetic complexity compared to their ancestors or pathogenic counterparts.
we further showed that expansions of gained or already existing families in a species-specific way are important to fine-tune the specificities of the pathogenic host-fungus interaction.

 the ascomycetes form the largest phylum in the fungal kingdom. they exhibit a broad range of life styles, ranging from saprophytes to both plant and animal pathogens. mycosphaerella fijiensis, for instance, is the causal agent of'black sigatoka', one of the most devastating diseases affecting banana and plantains worldwide, while the opportunistic pathogen aspergillus fumigatus is the cause of allergy in human patients with atopic immune systems. with the increasing number of genomes of ascomycetes being available, the study of the evolutionary dynamics that give rise to pathogenicity becomes amenable. the phylogeny of the ascomycetes and more specifically of the pezizomycotina is of particular interest in this respect as some of the pathogens are more closely related to their non-pathogenic counterparts than to other pathogens, emphasizing the importance of the environment in acquiring the pathogenic phenotype. recent studies showed that at least for some protein families the retention of duplicated genes is larger in pathogens than in non-pathogens. the functions of such gene families with a larger size in pathogens than in non-pathogens are in line with the pathogenic phenotype. authors agreed that the presence of pathogen-specific families or the specific increase in gene family size in pathogens created novel gene inventories that can be associated with phenotypic trait acquisitions. however, by only comparing gene inventories in the extant species, the evolutionary process that gave rise to the observed enlarged family sizes in pathogens remains unknown. moreover, most studies which consider gene families at the level of the extant species only tend to explain observed differences in gene family sizes by gains rather than by losses. as a result, the impact of gene loss during genome evolution is usually underestimated. in this study we evaluated how both gains and losses affect the origin of phenotypic traits that resulted in pathogenicity in ascomycetes. to this end, we focused on non-pathogenic and plant pathogenic pezizomycotina. we reconstructed the ancestral states of extant gene families and studied their evolution in relation to their involvement in the phytopathogenic phenotype.  the phylogenetic relationship between the species used in our analysis is depicted in figure this tree, derived from a subset of ascomycete species, shows that not all pathogens cluster together in the species tree. the topology in which some of the pathogens are more closely related to non-pathogenic counterparts is of particular interest to uncover the evolutionary mechanisms that resulted in differential phenotypic trait acquisition and allows to find out which traits are common between evolutionary distant pathogens, whether such common traits have been acquired in each of the pathogens independently or whether they have a common origin. gene family size in extant species for each gene family, a phylogenetic profile was constructed that indicates per species the number of genes belonging to the family. transforming these profiles allowed extracting those families for which one or more species contained a considerably larger number of genes than observed on average for that family in the remainder of the species; i.e. families that were selectively larger in a certain species or species set. subsequent clustering of these families according to the mutual similarity of their normalized profiles identified clusters of families that all displayed a higher than average copy number in the same set of species. results of this clustering are displayed in figure 2: some clusters represent families with a copy number that is larger than the family-average in one species only, while other clusters correspond to families that show a copy number that is higher than the average in several species. the latter ones can be further subdivided in gene families with a larger size in species that form a monophyletic clade and those with a larger size in species that do not form a monophyletic clade. of the analyzed gene families, showed a copy number higher than the average exclusively in non-pathogenic species, while families had a larger size only in the pathogens. the function of these gene families that showed a copy number that is higher than the family-average in a subset of the species ) was related to the phenotype of the species in which the larger family size was observed. only families for which the evolutionary history was consistently reconstructed by dollop and cafe are displayed. description: go description of the families. clustering: indicates whether an above the family-average gene copy number was observed in at least one species according to the cluster analysis. evolutionary events in non-pathogens were not categorized. family size contractions due to gene loss events that do not imply the entire loss of the gene family were not included in the table but covered in the'dating gene duplication and loss events' section. evolutionary events:'gains' and'losses' as inferred by dolop and cafe;'significant expansion as inferred by cafe;'enlarged gene family size' as inferred by clustering i.e. size of the family in the indicated species is larger than the family average. an'x' indicates whether the event occurred in an ancestral node or in an extant species. class: gene families were classified according to the evolutionary event that explains their origin. the four classes correspond to those shown in figure and described in the main text. remark 1: non-pathogen-specific gains were never observed. remark 2: although theoretically cafe should be able to discover significant expansions or contractions of gene families in ancestral species, these were never observed. all cases of significant expansions and contractions were observed in the extant species. remark 3: note that the class distinction was made based on the gene content of the families and not based on their functional annotation. this explains why families belonging to the same go category can belong to different classes of the families that were selectively larger in non-pathogens, and for of the families that were selectively larger in pathogens. for the non-pathogens it is mainly in the genome of a. oryzae that many families show a higher than family-average gene copy number. those families are enriched in acid-ammonia ligase activity, required for the biosynthesis of essential amino acids and x-pro dipeptidyl-peptidase activity. the latter activity is in line with the ability of a. oryzae to secrete proteolytic enzymes to the environment. for the pathogens, it is mainly f. graminearum, m. fijiensis and m. graminicola that show several families with lineage-specific larger sizes in remarkably similar functions ). for gene families that exhibited a higher than average copy number in several species simultaneously, no families were found with a larger size in all four non-pathogens. clusters xi, xii and xiii in figure 2a are examples of families with a larger size in three of the four non-pathogens. among the pathogenic species we found three protein families with a higher than the family-average gene copy number in all four pathogens. these three protein families are involved in mannosidase activity, which is responsible for oligosaccharide catabolism and n-glycan processing. where mannosidase activity is not essential for morphogenesis and cellular function in the non-pathogen a. nidulans, deleting the activity caused severe defect in conidial formation in the human pathogen aspergillus fumigatus. clusters x-xiv in figure 2b consist of families with a larger size in three out of the four pathogens. functional categories enriched in these protein families included peptidase, g-protein receptor, transferase and chitin binding activities, all of which have been related to pathogenicity in ascomycetes. we found a larger number of protein families with larger sizes in only two genomes. for the largest subset of those families, gene families exhibited a larger size in two species that form a monophyletic clade: from the families, had a larger size in both related aspergillus species. also for of the families in pathogenic species, the higher copy number was consistently shared by both closely related mycosphaerella species. gene families with a larger size in two species that do not belong to a monophyletic clade in the species tree are cluster vi in figure 2a consisting of protein families enriched in nuclease and fatty-acid synthase activities that show a higher copy number in the genomes of the two non-related non-pathogens t. reesei and a. oryzae and cluster vi of figure 2b consisting of protein families enriched in n-acetyltransferase, oxidoreductase and tartrate dehydrogenase activities showing increased copy numbers in the non-related pathogenic genomes of f. graminearum and m. graminicola. evolution of gene family size phylogenetic profiling confirms what was also noted in previous studies, namely that the functionality of protein families with a larger gene copy number in extant pathogens is linked to pathogenicity. the analysis of the phylogenetic profiles, however, does not indicate how these families ended up being extended in the extant pathogens. for instance, the families with a larger copy number in multiple species that do not form a monophyletic clade can either originate through independent duplication events in non-related species or by duplications in an ancestral species followed by gene loss in some of the descendants. to understand the evolutionary dynamics of these common and specific traits that can be linked to pathogenicity, we applied a suite of complementary analyses. first, we used dollop which maps for each gene family the evolutionary history of gene gains and losses. by reconciling the binary profiles of gene families with the species tree, dollop infers the minimal gene set for the different ancestral nodes using a parsimony approach which assumes gene loss to be irreversible. dollop thus finds for every internal node in the tree whether the species derived from that node gained or lost a specific protein family. however, as dollop uses binarized phylogenetic profiles that indicate whether a certain species contains a representative of a family, it does not contain information on the size of the protein family in each species. cafe on the other hand makes also use of the number of genes in each family to model the evolution of gene families. in doing so, cafe not only reports on gains and losses, but it also estimates the most likely number of genes for each family at each of the ancestral nodes of the species tree. from these results the occurrence of expansions and contractions of gene families along the time points in the species tree can be derived. cafe infers an expansion at a given node if the number of genes in a gene family augmented compared to its number in a previous ancestral time point. a gain of a gene family can be considered as a particular case of an expansion for which the gene family was not present in the previous ancestral time point. similarly, a contraction is a reduction in the number of genes in a gene family compared its number in the previous ancestral time point. gene family loss is again a particular case of a contraction for which an ancestral gene family completely disappears in a descendant species. the species tree, with for each node an indication on the global number of gained and lost gene families, as derived from dollop and cafe, is depicted in figure. to gain insights into the molecular functions and/or biological processes of the gene families that have been lost or gained or were expanded or contracted at each of the different time points, we analyzed the go functional categories for which the families were enriched. the complete results of dollop and cafe, i.e. a list of the evolutionary events for each of the time points in the species tree can be found on the supporting information. protein families for which both dollop and cafe were consistent in inferring the same evolutionary history are displayed in table general observations in figure tp represents the last common ancestor of the eight species under consideration. small discrepancies in absolute numbers between dollop and cafe result from the different underlying assumptions between both methods, but in general both methods predict similar tendencies. since the origin of the ancestral species on tp and tp gene families seem to have been continuously lost and this loss does not seem to have been compensated for by gene family gains. when considering the species derived from tp, gene loss was significantly higher in the non-pathogens than in the pathogens. for the second group of species, this tendency might be masked by the unusually large genome size that seems to have been maintained in the cultivated species a. oryzae, resulting in gene loss being at least as prominent in the pathogenic mycosphaerella species than in the non-pathogenic aspergillus species. in the following, we give a detailed analysis of the evolutionary events that resulted in the currently observed gene copy numbers in extant pathogens. pathogen-specific gene family gains with a pathogen-specific gain we refer to a gene family not present in a species that is ancestral to both pathogenic and non-pathogenic lineages, but that appears in one of the extant pathogens or in an ancestral node that gives exclusively rise to pathogenic lineages. note that the term'specific' used in the context of this paper should be interpreted as specific relative to the number of species analyzed in this study. we found several examples of families that were selectively present in a pathogen or in set of pathogens that originated through this mechanism of family gain and of which the function can be related to pathogenicity. examples of families that are gained in one specific extant pathogen are, for instance for m. grisea, families involved in subtilase and cutinase activity and families related to the polyketide biosynthesis pathway. all of these gene families have been directly linked to the pathogenic phenotype in m. grisea. in the pathogen m. graminicola we observed a significant gain of families containing genes with peptidase and phospholipase a activities. these functions are needed for the organization of infective structures in pathogenic fungi. the expression of genes with phospholipase a activity is induced in pathogenic fungi during nutrient starvation, which is assumed to be an environmental trigger for host invasion. families specifically gained by m. fijiensis are enriched in metallo-exopeptidase activity which has been related to the morphological development of infective structures in the fungal pathogen ustilago maydis. in the pathogen f. graminearum we found some specific gained gene families related to lipid metabolism and peptidase activity that do not occur in any other species. in f. graminearum we also observed the gain of specific gene families associated with transmembrane transporters. the higher than average family size we observed in multiple extant pathogens that form a monophyletic clade in the species tree, are explained by gains in the common ancestor of these related species, maintained in the extant species. an example is the gain of proteolysis related gene families in the common ancestor tp of the closely related m. fijiensis and m. graminicola species. especially the gain of a family of zinc endopeptidases with astacin activity is interesting as it has been related to the activation of peptides that act as morphogenetic signal for foot activation in hydra. of particular interest are the different families, all involved in similar biological processes that were gained independently in multiple species, that are not each other close neighbors in the species tree, as these confer examples of convergent evolution. for instance, f. graminearum and m. grisea both independently gained gene families that are different from each other, but all related to polyssacharide binding and chitinase activity. chitinases are closely linked to growth and morphological development in fungi: cell expansion and division, spore germination, hyphal branching and septum formation all depend on enzymes with hydrolytic activities including chitinases. in previous work, a larger than expected size of the chitinase family was already observed for f. graminearum. similarly, the genomes of f. graminearum, m. fijiensis and m. grisea gained different sets of families all related to oxidative stress response, such as families involved in glutathione and thioredoxin peroxidase activities. these gene families that were gained independently from each other in evolution could be involved in a generally conserved defence mechanism of fungi against plant mediated oxidative burst. we also observed different families related to transferase activity that were gained independently in f. graminearum, m. fijiensis and m. graminicola. pathogen-specific gene family expansions also gene families with an origin ancestral to both pathogens and non-pathogens that expanded in a pathogen-specific way can explain the larger size of gene families in extant pathogens. based on cafe, such gene families showed a significant expansion in extant pathogenic species. for instance, a gene family with methyltransferase activity that has previously been related to fungal growth and virulence showed a significant expansion in f. graminearum. as methyltransferases methylate fungal specific glucosylceramides, i.e., molecules that serve as receptors for antifungal plant defensins, they can be directly linked to molecular interactions between the fungus and its host. two more gene families with ribonuclease h and rna-directed dna polymerase activities showed a significant expansion in m. grisea. not surprisingly, the function of these families is related to processing transposable elements which are, as indicated by the dollop analysis abundantly present in the genome of m. grisea. insertional mutants of genes from both the ribonuclease h and rna-directed dna polymerase activities gene families are virulence defectives. an ancestral family involved in the polyketide biosynthesis pathway was also found to have been expanded in m. grisea. the relation of polyketide biosynthesis with pathogenicity in m. grisea is strongly supported by the work of jeon et al. based on insertional mutagenesis. experimental data was collected from two sources: pathogen-host interaction database and the work of jeon et al.. phenotype: based on mutant deviations in conidiation and appressorium formation compared to untransformed control. effector: gene product or signaling molecule resulting from its enzymatic activity that triggers defensive mechanisms in the plant host. hence, effectors are usually known as avirulence determinants. although contractions of gene families in ancestral nodes can theoretically be inferred by cafe, we did not observe them. non-pathogen-specific losses both previous mechanisms of pathogen-specific gains and pathogen-specific expansions of ancestral gene families only account for % of the families with enlarged size in pathogens. for a considerable fraction of the families, the observed gene family sizes in the extant pathogens seems to originate from an ancestral gene family that got lost in extant non-pathogens, but that was selectively maintained in the pathogens. with'loss of a family in a particular species or ancestral node' we refer here to an ancestral gene family that lost all of its members in the species of interest descending from the ancestor. the non-pathogen-specific loss of ancestral gene families is in line with the overall observed higher number of gene losses in non-pathogens than in pathogens for the species we used in this study. the most striking examples of non-pathogen-specific family losses are those observed for the families involved in transmembrane transport, lipid metabolism and chitin binding. these families, shared by all pathogens, were present in a common ancestor on tp but got lost in the non-pathogen lineages. this indicates that transmembrane transport, lipid metabolism and chitin binding are essential and generic traits in determining pathogenicity in ascomycetes. related to the evolutionary dynamics of gene families involved in lipid metabolism, it seems that not only these families were lost in the non-pathogenic branch, but also that the pathogen f. graminearum gained some extra families in addition to the ones it inherited from its ancestor on tp. the fact that both non-pathogen-specific loss and species-specific gains are observed for lipid metabolism further strengthens the importance of this function in pathogenicity. the relation between lipid metabolism and pathogenicity is further confirmed by the fact that disrupting the genes encoding for an extracellular lipase responsible for fatty-acid uptake, significantly reduced virulence in ascomycete pathogens. in addition, a gene expression study of f. graminearum isolated from infected plant tissue showed that genes involved in lipid biosynthesis and degradation were differentially expressed during host colonization. transmembrane transport mechanisms are known to play a key role during the early stages of host invasion by functioning as receptors for signals from the environment that trigger spore germination. a mutant of a gene coding belonging to this family of transmembrane transporters in m. graminicola showed a reduced virulence phenotype. on the other hand, chitin binding proteins are known as avirulence receptors that interact with the plant host. genes encoding chitin binding proteins were differentially expressed in a cdna library of m. fijiensis grown in the presence of banana leaves. other ancestral gene families present on tp that got lost in all studied non-pathogens but were retained in the pathogens are those involved in mannosidase activity, astacin activity and the g-coupled protein receptor. this model of gene family loss in non-pathogens also explains why we observed members of the families related to n-acetyltransferase, oxidoreductase and tartrate dehydrogenase activities only in the genomes of the pathogens f. graminearum and m. graminicola that do not form a monophyletic clade in the species tree. the utilization of tartrate has been linked to the induction of pathogenicity related genes in m. graminicola. these families originated in the ancestral tp and got lost in all extant species other than f. graminearum and m. graminicola. both f. graminearum and m. graminicola share wheat as common host and those families only being retained in those two pathogens reinforces the importance of the pathogen-host interaction process in the selection of pathogenic traits. in addition to the examples discussed so far where gene families have been collectively lost during evolution in all non-pathogens, some losses of gene families were more restricted to the non-pathogens of the evolutionary branch that started either from tp or the one that descended from tp. some of the ancestral families that got lost in non-pathogens, but were specifically retained in pathogens underwent further expansions as predicted by cafe. for those cases it is the combined action of non-pathogen-specific loss and pathogen-specific expansions that explains the observed large family size. for instance the gene families involved in transmembrane transporters showed significant expansions in the pathogens m. graminicola and f. graminearum. the same was true for the families involved in chitin binding and astacin activity that were both selectively expanded in m. fijiensis. pathogen-specific gene family losses in a few cases only, the acquisition of the pathogenic phenotype could be related to the loss of ancestrally present gene families in a pathogen-selective way. a significant number of the families that disappeared in the ancestor of both mycospaerella species are related to nitrogen catabolic processes, to the regulation of nitrogen utilization and sensing and to cellular responses towards nitrogen starvation. nitrogen starvation has been related to virulence in ascomycete plant pathogens by acting as an environmental clue for the expression of disease symptoms in the pathosystem magnaporthe grisea - oryza sativa. loss of gene families involved in nitrogen metabolism, regulation and sensing could reduce the ability of the fungus to sense environmental nitrogen levels and mimic a continuous nitrogen starvation condition that might activate the pathogenicity transcriptional program in m. fijiensis and m. graminicola. dating gene duplication and loss events both dollop and cafe agree in predicting that the selective loss of families with an ancestral origin in non-pathogens is the major cause for the observed differences in gene copy number between extant pathogenic and non-pathogenic species. so far, predictions made by dollop and cafe only considered losses of entire gene families. this is to be expected as dollop can only make predictions about'presence' or'absence' states of entire protein families while for cafe the entire loss of a family provide statistically a stronger signal than the spurious loss of a single gene in a family. to uncover whether also less drastic losses i.e., where part of the genes of a family got lost but not the entire family disappears, we relied on tree reconciliation. we analyzed families that have minimally two members in at least one of the species on tp tp tp and tp and in addition the family should have representatives in at least two other species. we only considered the part of the species tree where a non-pathogenic species always clusters with its pathogenic counterpart as this topology allows directly assessing what proportion of the observed differences in gene family size between extant pathogens and non-pathogens are caused by the different evolutionary scenarios depicted in figure gene trees for all families were constructed. by reconciling them with the species tree, we predicted the nodes at which duplication and loss events occurred during evolution of the studies species. to avoid biases introduced by large scale tree reconciliation, we retained trees of which the topology was supported with a bootstrap value of at least 75% and only considered the reconciliation results obtained from gene trees with a reliable topology. the reconciliation analysis showed that for a relative small fraction of the gene families, larger gene family sizes in the extant pathogens f. graminearum and m. grisea resulted from expansions of ancestral or later gained gene families specifically occurring in these genomes. most expansions occurred selectively in the genome of m. grisea. conversely, expansions of gene families in non-pathogens did barely occur. the majority of the families with a larger gene copy number in extant pathogens seem to have been the result of ancestral gene families of which the copies were selectively maintained in the pathogens, but got lost in the non-pathogenic counterparts. the observed gene family contractions in the non-pathogens were significantly higher than contractions observed in pathogenic gene families. discussion previous comparative approaches studied pathogenicity in ascomycetes mainly by comparing gene inventories in extant species. in line with these studies we showed that gene families which exhibit a size larger than the family-average in pathogens are responsible for acquired phenotypic traits related to pathogenicity. however, by not only studying differences in gene family size between pathogens and non-pathogens, but by also analyzing the complete evolutionary history that gave rise to the observed differences in gene family size, we found that only a relatively small fraction of the gene families with a size that is selectively larger in pathogens could be explained by the combined effect of gene family gain with pathogen-specific expansions. most of the families with such larger size in pathogens seemed to have been present already in the ancestor of both pathogens and non-pathogens. the potential of genes and gene products needed to develop pathogenicity was already present in the ancestor of all current species and was selectively maintained in pathogens. strikingly, it were not the pathogen-specific expansions of these ancestrally present gene families that explained the discrepancies in gene family size between pathogens and non-pathogens, but rather the selective loss of those ancestrally present gene families in non-pathogens. species that did not develop pathogenicity seem to have reduced their genetic complexity compared to their ancestors. gene families that evolved according to this gene loss theory are mainly involved in generic functions, conserved amongst all fungi such as transmembrane transport, lipase activity, mannosidase activity, g-coupled protein receptor and astacin activity. only the number of families with those functions remained considerably higher in pathogens than in non-pathogens. this observation is confirmed by previous molecular studies which showed indeed that a vast repertoire of genes, involved in virulence and pathogenicity, have general functions conserved among all fungal species. pathogenic fungi have to closely interact with their host. it is, therefore, not surprising that the host-pathogen interaction further shaped the genomes of fungal pathogens in the ascomycetes. we observed that the interaction with a specific environment resulted in the differential retention of gene families with similar functionalities in phylogenetically distant pathogenic species. moreover, all families that are expanded in certain pathogens could be functionally linked to the specificities of the host-fungus interaction. the latter observations emphasize the importance of the environment in explaining expansions in family size.  we found that most of the proteins needed to develop pathogenicity were already present in the ancestor of the ascomycetes lineages we used in our study, i.e. non-pathogenic and plant pathogenic pezizomycotina. species that did not develop pathogenicity reduced their genetic complexity compared to their ancestors or pathogenic counterparts. we further showed that expansions of gained or already existing families in a species-specific way are important to fine-tune the specificities of the pathogenic host-fungus interaction. 
previous studies in ascomycetes have shown that the function of gene families of which the size is considerably larger in extant pathogens than in non-pathogens could be related to pathogenicity traits. moreover, most studies which consider gene families in extant species only tend to explain observed differences in gene family sizes by gains rather than by losses, hereby largely underestimating the impact of gene loss during genome evolution. by analyzing the evolutionary history of gene families we found that most gene families with an enlarged size in pathogens were present in an ancestor common to both pathogens and non-pathogens. in our study we used a selection of recently published genomes of ascomycetes to analyze how gene family gains, duplications and losses have affected the origin of pathogenic traits. however, by only comparing gene inventories in extant species, no insights can be gained into the evolutionary process that gave rise to these larger family sizes in pathogens. we further show that expansion of gained or already existing families in a species-specific way is important to fine-tune the specificities of the pathogenic host-fungus interaction. non-pathogen-specific losses largely outnumbered pathogen-specific losses. species that did not develop pathogenicity seemed to have reduced their genetic complexity compared to their ancestors. the majority of these families were selectively maintained in pathogenic lineages, but disappeared in non-pathogens.
 previous studies in ascomycetes have shown that the function of gene families of which the size is considerably larger in extant pathogens than in non-pathogens could be related to pathogenicity traits. however, by only comparing gene inventories in extant species, no insights can be gained into the evolutionary process that gave rise to these larger family sizes in pathogens. moreover, most studies which consider gene families in extant species only tend to explain observed differences in gene family sizes by gains rather than by losses, hereby largely underestimating the impact of gene loss during genome evolution.  in our study we used a selection of recently published genomes of ascomycetes to analyze how gene family gains, duplications and losses have affected the origin of pathogenic traits. by analyzing the evolutionary history of gene families we found that most gene families with an enlarged size in pathogens were present in an ancestor common to both pathogens and non-pathogens. the majority of these families were selectively maintained in pathogenic lineages, but disappeared in non-pathogens. non-pathogen-specific losses largely outnumbered pathogen-specific losses.  we conclude that most of the proteins for pathogenicity were already present in the ancestor of the ascomycete lineages we used in our study. species that did not develop pathogenicity seemed to have reduced their genetic complexity compared to their ancestors. we further show that expansion of gained or already existing families in a species-specific way is important to fine-tune the specificities of the pathogenic host-fungus interaction. 