BACKGROUND
change in gene inventory in pathogenic genomes is an important evolutionary signal.
previous studies have documented the relationship between virulence and differential gene gain and/or loss in bacteria and viruses  <cit> .
however, this phenomenon remains unexamined at a genomic scale in fungal pathogens.
our exploration of patterns of differential gene gain and loss in pathogenic fungal genomes was prompted by two possibly related observations.
first, gene counts in phytopathogenic euascomycete species and fungus-like plant parasites, such as species of phytophthora, are often higher than those for the most closely related non-pathogenic genomes  <cit> .
second, some of the additional genes identified in these pathogens are predicted to have roles in secondary metabolism and managing encounters with hosts  <cit> .
for instance, polyketide synthetases and non-ribosomal peptide synthetases are essential for toxin production, while g protein-coupled receptors and cytochrome p450s are critical for host perception and quenching infection-related oxidative stress  <cit> .
the differential expansion of a gene family by duplication in a particular species is termed lineage-specific gene family expansion   <cit> .
selection for virulence could induce lse among particular gene families  <cit> , as well as contraction among other families  <cit> .
size differences between the genomes of pathogenic and non-pathogenic species will depend on the relative rates of gene duplication, gene loss and horizontal transfer events.
two evolutionary trends that would result in larger genomes among pathogens are the consistent expansion of certain gene families  <cit> , as well as pathogens' apparent affinity for gene acquisition through horizontal transfer  <cit> .
however, it is also known that the number of genes in the genomes of opportunistic human fungal pathogens  <cit>  appears to be reduced, as compared to non-pathogenic relatives, suggesting that gene loss may also be increased among some pathogens  <cit> .
in the present study, we evaluated patterns of gene duplication in pathogens versus non-pathogens and in phylogenetically-informed paired species comparisons.
we subsequently explored potential functional differences among duplicate genes in pathogens as compared to non-pathogens.
in addition, we investigated trends of gene gain and loss in pathogenic fungal genomes.
RESULTS
altered patterns of gene duplication among diverse fungi
gene duplicates identified by genomehistory in the eleven sequenced fungal genomes were grouped into gene families via single linkage clustering procedures .
figure  <dig> and table  <dig> give the number of gene families of size two or greater that met all minimum threshold criteria, as well as the total number genes in gene families for each species.
u. maydis, a basally branching hemibiotrophic pathogenic basidiomycete lineage, had the fewest gene families and the least number of genes in gene families, while the opportunistic euascomycete pathogen a. flavus possessed the greatest number of gene families, and also the largest number of genes in gene families.
1the stringency and efficiency of rip-like processes varies among euascomycete genomes.
2asexual propagation is the most frequently observed reproductive mode in field settings.
however, asexual lineages often either have the potential for sexual reproduction, as indicated by the presence of different mating types in populations, and/or phylogenetic evidence for recombination and cryptic speciation.
we initially predicted greater proportions of duplicated genes would become fixed in pathogenic lineages as a result of increased preservation of duplicated genes by natural selection and/or higher rates of duplication.
in this coevolutionary arms race scenario, continually evolving host resistance would give rise to constant selective pressure for the preservation of duplications of genes relevant to virulence  <cit> .
we thus compared the distributions of gene family sizes in pathogens versus non-pathogens.
the distribution of gene family sizes in a genome is thought to follow a power law distribution  <cit> , and we therefore modeled the pooled set of pathogenic gene families as following this distribution.
similarly, we allowed the pooled set of non-pathogenic gene families to follow a power law distribution, with a potentially different value of the power law coefficient, a .
we then applied a likelihood ratio test to examine the null hypothesis that the value of a was the same in pathogens as in non-pathogens.
although we can reject the hypothesis of equal values of a , surprisingly, in view of our initial prediction, we find that non-pathogenic fungi in fact have slightly larger gene families than do the pathogens .
interestingly, when the two basidiomycete species  are excluded from the analysis, no overall significant differences in gene family size distribution are evident .
we also compared lineages that were primary pathogens  to their non-pathogenic relatives , finding again that the non-pathogens have larger gene families .
again, excluding the basidiomycete species results in no significant difference being found .
finally, we examined only the aspergillus species, comparing the opportunistic pathogens a. flavus and a. fumigatus to a. nidulans and a. oryzae.
no significant differences in gene family sizes are evident in the comparison between opportunistic pathogens and their non-pathogenic relatives .
the above approach is potentially flawed, because the species in question do not represent independent realizations of the same general stochastic process.
rather, the genomes are related by the phylogeny shown in figure  <dig> <cit> .
to control for this common ancestry, we performed phylogenetically independent contrasts for gene family size distributions for the six species pairs indicated in figure  <dig>  applying the same likelihood ratio test described above.
we find evidence for larger gene families in phytopathogenic species for two paired-species comparisons , while the other comparisons either showed either the opposite trend  or no significant differences .
in all cases, we used a significance threshold of α =  <dig> , which reflects application of a bonferroni correction for  <dig> hypothesis tests.
note that a maximum likelihood fit of another potential distribution describing the probability of observing a gene family of size x in a genome, the exponential distribution, visually provides a rather poorer explanation of these data .
functional distribution of gene duplicates in pathogens versus non-pathogens
to elucidate potential functional differences in duplicated genes in pathogenic versus non-pathogenic genomes, we compared the distribution of go terms between the two groups.
we initially selected twenty-two different go terms  representing functional categories that are relevant to fungal pathogenesis, as well as others related to basal metabolic processes.
we compared the proportions of gene duplicates associated with a go term for pathogens and non-pathogens.
when all eleven taxa were considered, two go terms differed significantly in the number of gene duplicates observed for pathogens versus non-pathogens, after allowing for a 20% false discovery rate .
the "receptor activity" and "hydrolase activity" terms showed significantly greater numbers of gene duplicates in pathogenic species than in non-pathogenic lineages.
when the same analysis was repeated for the nine euascomycete genomes, we identified three additional functional categories where pathogenic species had a greater than expected number of gene duplicates: "nucleic acid binding," "carbohydrate binding" and "regulation of transcription."
we also compared the number of gene duplicates associated with terms in the generic go slim ontology for pathogens and non-pathogens.
this survey revealed no significant distinctions among pathogens versus non-pathogens for all taxa.
when only euascomycete species were considered, we found that gene duplicates associated with following six terms were over represented among pathogens, again controlling for an fdr of 20%: "hydrolase activity," "extracellular region," "carbohydrate metabolism," "nucleobase, nucleoside, nucleotide and nucleic acid metabolism," "carbohydrate binding" and "catalytic activity" .
‡ significance values in this column are p-values obtained in chi-square tests
§significance values presented in this column are corrected for type  <dig> error
functional distribution of gene duplicates in species pairs
for the four species pairs that showed global differences in the magnitude of gene duplication , we surveyed the functional distribution of gene duplicates using the generic go slim ontology.
the rice blast fungus, m. grisea, showed higher average gene family size than its non-pathogenic relative n. crassa .
correspondingly, we found four go terms that are overrepresented in pathogenic m. grisea, as compared to exclusively saprophytic n. crassa .
further, while pathogenic f. graminearum had larger gene families than did non-pathogenic t. reesei , we found five overrepresented go terms in f. graminearum and an equal number of overrepresented terms in t. reesei .
when we compared the species pairs where the non-pathogenic taxon possessed more gene duplicates globally, , we found significantly more gene duplicates associated with a total of eighteen particular go terms in the a. nidulans-s. nodorum test pair, and fourteen terms in p. chrysosporium , respectively .
significant differences in paired species comparisons were determined after applying corrections for multiple tests, as above.
‡ enrichment in pathogens and non-pathogens across four different pairwise comparisons
† denotes duplicate enrichment for the term in non-pathogenic t. reesei
‡ significance values in this column are p-values obtained in chi-square tests
§significance values presented in this column are corrected for type  <dig> error
expansion or contraction of gene families in pathogens
to determine whether a given gene family showed significant expansion or contraction, we employed a binomial test .
sixteen gene families show significant expansion in pathogens, while five gene families appear to be contracted.
functional identities, as well as gene family sizes are presented for all significantly expanded or contracted gene families in additional files  <dig> and  <dig>
some significantly expanded gene families of particular interest are those with predicted hydrolytic, transporter or oxidioreductase activities, as well as those involved with carbohydrate metabolism .
examples of expanded gene families in the hydrolytic functional class included those with predicted chitin deactylase, cutinase, amino peptidase and feruloyl esterase activities, among others.
specific examples of expanded gene families that belong to the oxidoreductase functional class included those with predicted galactose oxidase and tyrosinase activities.
the transporter functional class of expanded gene families included instances of sugar porters, malic acid transporters, neutral amino acid permeases and l-fucose permeases, among others.
for gene families that were deemed significantly contracted, the main functional categories, as indicated by go and genbank annotations, also include hydrolases, oxidases and transporters.
however, the specific biological roles of particular gene families that showed contraction in pathogens differ from those that showed significant expansion .
for instance, in the oxidoreductase functional class, homologues of orda  appear to be depleted in pathogens.
in aflatoxin-producing species of aspergillus, this gene catalyzes the last reaction in the biosynthesis of this secondary metabolite  <cit> .
other interesting examples include gene families that were predicted to be components of the vaculolar atpase complex, in addition to those encoding glucose oxidase precursors .
expanded gene families in species that have more duplicated genes
for the four cases where phylogenetic contrast analyses indicated significant differences in gene family sizes in paired species comparisons , gene family expansion was also examined using an approach similar to that above .
five gene families are significantly expanded in m. grisea, relative to non-pathogenic n. crassa.
these gene familiesinclude those predicted to have oxidoreductase, transporter, peroxidase and melanin biosynthesis functions .
four gene families are significantly expanded in f. graminearum versus t. reesei, including those predicted to have transporter, endoglucanase and methyl transferase activities .
interestingly, the significantly expanded gene family with methyl transferase functional attributes has substantial homology to laea, a gene that may play a global regulatory role in secondary metabolism in aspergillus species  <cit> .
there are two significantly expanded gene families in a. nidulans, relative to s. nodorum .
one family is predicted to have phosphorylase activity, while the other encodes genes with atp-binding cassette  transporter function .
p. chrysosporium has eight significantly expanded gene families, relative to pathogenic u. maydis.
these eight families include genes with predicted oxidoreductase and peptidase functions, as well as genes with roles in carbohydrate metabolism .
discussion
change in gene inventory and fungal genome evolution
differential gene gain and loss clearly play definitive roles in both degree of virulence, and in determining host range  <cit> .
although there are numerous examples of gene family expansion in pathogenic lineages of fungi  <cit> , to date, gene duplication trends across whole genomes have not been analyzed.
our analysis sought to understand whether the sum of gene family expansions have given rise to an overall increase in numbers of gene duplicates in the genomes of pathogenic fungi.
we observed no such trend when all pathogenic species were compared to the non-pathogens, nor when we performed phylogenetically-informed paired species comparisons.
instead, the paired-species comparisons showed no clear association between gene family size distributions and pathogenicity.
an example of this lack of association is apparent in the aspergillus species comparisons, where we found no significant differences in gene family size distributions, a result that derives from both the opportunistic nature of the pathogens, as well as the close phylogenetic relationships of the species in question  <cit> .
another potential explanation for similarity among the complement of gene duplicates in genomes of the aspergilli is the recent suggestion that host animals, and humans in particular, do not generate the sort of co-evolutionary arms-race characteristic of the plant hosts of the other pathogenic species considered  <cit> .
a confounding factor for our analyses is that gene duplication's ability to drive biological innovation is diminished in some fungi by the process of repeat-induced point mutation  <cit> .
rip is a pre-meiotic homology-based mechanism that introduces characteristic point mutations into sequences present in multiple copies in a genome, and may have evolved to limit the deleterious effects of mobile genetic elements  <cit> .
all euascomycete genomes surveyed here likely possess some degree of rip  <cit> .
however, the severity and efficiency of the process varies, with n. crassa possibly possessing the most stringent form  <cit> .
we note that n. crassa shows the smallest average gene family size among the species studied, as would be expected, given rip's severe pruning of duplicates in this organism.
there is no evidence for rip in either of the basidiomycete genomes examined in this study, although it has recently been documented in anther smut  <cit> .
some of the differences illustrated in figure  <dig>  are possibly due to variation in rip stringency rather than pathogenicity.
although quite variable, the approximate divergence times among the six pathogenic and five non-pathogenic species examined are reasonable for large-scale comparative studies of gene duplication in euascomycete and basidiomycete lineages  <cit> .
functional patterns of duplicate gene enrichment in fungal genomes
enrichment of gene duplicates over particular functional categories in the set of twenty-two go terms, as well as those in the generic go slim ontology appear consistent with requirements of pathogenic lifestyles  <cit> .
these results parallel those of previous studies, which have demonstrated that fungal pathogenesis is associated with increased catalytic potential among enzymes such as hydrolases, relatively larger repertoires of receptors, and the expansion of secreted proteins, as well as carbohydrate recognition and binding gene families  <cit> .
for example, we found an increased number of duplicates associated with transport functions in f. graminearum, the causal agent of head blight in cereals, as compared to non-pathogenic t. reesei , a result consistent with the known relative deficit of carbohydrate catalysis genes in t. reesei  <cit> .
some of the differences observed between non-pathogenic p. chrysosporium and the hemibiotrophic corn smut fungus u. maydis appear relevant to the markedly divergent ecology and development these basidiomycete species  <cit> .
the over-represented terms "carbohydrate binding," "extracellular region," "response to abiotic stimulus," "multicellular organismal development," "peptidase activity," as well as "amino acid and derivative metabolic process" are associated with the suite of traits that make the white rot fungus, p. chrysosporium, amenable to industrial applications, such as lignocellulose and organopollutant degradation .
moreover, the observed enrichment of gene duplicates dedicated to particular developmental programs, such as muticellularity, is also consistent with the differences in morphological complexity between these two organisms in their fruiting body structures; u. maydis produces no fruiting body, per se, only teliospore-filled tumors on a host, whereas p. chrysosporium possesses a resupinate fleshy fruiting body.
that u. maydis showed no enrichment for gene duplicates for any go term was consistent with recent analyses of this genome, which indicated relatively few duplicates  <cit> .
when phylogenetic relationships are not considered, a cohort of go terms that is common to pathogens and non-pathogens in all four paired-species comparisons becomes evident .
these three terms are: electron transport, generation of precursor metabolites and energy, as well as catalytic activity.
biological explanations for this observation include two possibilities that are not mutually exclusive: first, that genes associated with these three terms are not peculiar to pathogenesis, and/or secondly, that genes associated with these terms possess inherently greater plasticity in copy number in fungal genomes.
given numerous accounts of duplication in pathogenic and non-pathogenic fungi, copy number plasticity in certain functional categories would seem the more plausible explanation.
our results, both from comparisons across all taxa, as well as paired species comparisons, suggest an association between organisms' lytic potential  and receptor  repertoires and pathogenesis .
moreover, even when a pathogen had fewer gene duplicates overall , enrichment of terms associated with lytic activities , management of oxygen toxicity , as well as terms possibly relevant to protein secretion  were still evident in the pathogenic lineage .
gene family expansion or contraction in pathogens
gene families that were expanded in pathogens largely fall into two functional categories: those encoding lytic enzymes and those encoding putative transporters .
lytic enzymes, such as feruloyl esterases, cutinases, aminopeptidases and endoglucanases are known to be significant in successful plant parasitism interactions, as such genes have demonstrated roles in plant cell wall decomposition  <cit> .
serine proteinases and the regulatory p domain of the subtilisin-like proprotein convertases have been implicated in mutualistic, as well as pathogenic, interactions with grasses  <cit> .
moreover, the importance of fungal chitin deactylases in entomopathology has been empirically demonstrated  <cit> .
expanded families that were predicted to have diverse transporter functions included genes that encode l-fucose and neutral amino acid permeases, as well as mfs, malic acid and sugar transporters, respectively .
l-fucose permeases have not only been implicated in galactose transport and sphigolipid metabolism, but also in development of resistant sclerotia in a. flavus, while neutral amino acid permeases and mfs transporters are possibly involved in efflux of non-ribosomal peptides and other secondary metabolites required for virulence  <cit> .
permeases may also be critical for the initial assault upon a host, as well as assimilation of host-derived carbohydrates  <cit> .
the remaining expanded families included those encoding tyrosinases, galatose oxidase precursors and transmembrane receptors, all of which have been implicated in pathogenic life styles  <cit> .
tyrosinases catalyze initial steps in melanin biosynthesis  <cit> .
melanization in fungi is frequently, if not always, essential for virulence  <cit> .
it is notable that another gene in the melanin synthesis pathway was also found to be over duplicated in m. grisea, relative to non-pathogenic n. crassa  <cit> .
galactose oxidation yields peroxide  <cit> , a reactive oxygen species known to be produced by fungal pathogens during infection  <cit> .
recent work in m. grisea described a phalanx of receptors that are linked to pathogenesis and host perception  <cit> .
the five families that showed gene losses in pathogens included those with predicted roles in secondary metabolism .
one intriguing case is that of o-methylsterigmatocystin oxidoreductase, which catalyzes the last step in aflatoxin biosynthesis.
while only a few species of aspergillus are known to produce aflatoxin, numerous euascomycete and basidiomycete lineages possess homologues of this gene, as well as others in the aflatoxin biosynthesis pathway  <cit> .
the wide phylogenetic distribution of these genes suggests not only that that these genes are ancient, but also that they are possibly important in secondary metabolism.
we speculate that contraction of this family in pathogenic lineages may be the result of random gene deletions after relaxation of selective pressure that maintains production of this toxin  <cit> .
recent genomic and population studies that focused on the aflatoxin biosynthesis cluster in species of aspergillus would seem to support this conjecture, as orda, an o-methylsterigmatocystin oxidoreductase, appears to have undergone more extensive loss following duplication than any other gene in the cluster  <cit> .
gene family expansion in species pairs
gene families that were expanded in two paired-species comparisons also had either expected or previously demonstrated roles in virulence, host-pathogen interaction or chemical defense  <cit> .
for example, homologues of laea, a methyltransferase, are over duplicated in f. graminearum.
this gene appears to be a global regulator of secondary metabolism in species of aspergillus  <cit> .
it is interesting to speculate whether an expanded repertoire of upstream regulators of secondary metabolism in f. graminearum could reflect either greater numbers of target genes or refined regulatory control of secondary metabolic networks in this primary pathogen .
in the second instance where a phytopathogen had overall greater levels of gene duplication, five gene families were expanded.
gene families encoding diverse cytochrome p450s, mfs transporters, as well as those predicted to have trihydroxynaphthalene reductase activity were expanded in m. grisea, as compared to exclusively saprophytic n. crassa .
the white rot fungus p. chrysosporium had eight gene families that were significantly expanded, relative to basal basidiomycete u. maydis .
these eight expanded families encode genes that have diverse oxidoreductase activities, protease and endo- <dig>   <dig> -beta-glucanase functions, consistent with genes previously reported to be present in high copy numbers in this genome  <cit> .
a large complement of such genes in this organism's genome is likely a reflection of its saprotropic wood-decay ecology.
CONCLUSIONS
here, we present results on patterns of gene duplication and differential gene gain and loss in pathogenic fungal genomes.
the scope of this study captures at least of one billion years of fungal evolution  <cit> .
no general relationship between pathogenicity and the magnitude of gene duplication was evident, but differences in duplicate gene retention among certain functional classes were consistent with known and predicted requirements of virulent lifestyles  <cit> .
gene family expansion in species with overall higher levels of duplication also showed functional trends that could be reconciled with organismal life histories  <cit> .
the observed differences in overall levels of duplication between phytopathogenic lineages and their non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi.

 change in gene inventory in pathogenic genomes is an important evolutionary signal. previous studies have documented the relationship between virulence and differential gene gain and/or loss in bacteria and viruses. however, this phenomenon remains unexamined at a genomic scale in fungal pathogens. our exploration of patterns of differential gene gain and loss in pathogenic fungal genomes was prompted by two possibly related observations. first, gene counts in phytopathogenic euascomycete species and fungus-like plant parasites, such as species of phytophthora, are often higher than those for the most closely related non-pathogenic genomes. second, some of the additional genes identified in these pathogens are predicted to have roles in secondary metabolism and managing encounters with hosts. for instance, polyketide synthetases and non-ribosomal peptide synthetases are essential for toxin production, while g protein-coupled receptors and cytochrome p450s are critical for host perception and quenching infection-related oxidative stress. the differential expansion of a gene family by duplication in a particular species is termed lineage-specific gene family expansion. selection for virulence could induce lse among particular gene families, as well as contraction among other families. size differences between the genomes of pathogenic and non-pathogenic species will depend on the relative rates of gene duplication, gene loss and horizontal transfer events. two evolutionary trends that would result in larger genomes among pathogens are the consistent expansion of certain gene families, as well as pathogens' apparent affinity for gene acquisition through horizontal transfer. however, it is also known that the number of genes in the genomes of opportunistic human fungal pathogens appears to be reduced, as compared to non-pathogenic relatives, suggesting that gene loss may also be increased among some pathogens. in the present study, we evaluated patterns of gene duplication in pathogens versus non-pathogens and in phylogenetically-informed paired species comparisons. we subsequently explored potential functional differences among duplicate genes in pathogens as compared to non-pathogens. in addition, we investigated trends of gene gain and loss in pathogenic fungal genomes.  altered patterns of gene duplication among diverse fungi gene duplicates identified by genomehistory in the eleven sequenced fungal genomes were grouped into gene families via single linkage clustering procedures. figure and table give the number of gene families of size two or greater that met all minimum threshold criteria, as well as the total number genes in gene families for each species. u. maydis, a basally branching hemibiotrophic pathogenic basidiomycete lineage, had the fewest gene families and the least number of genes in gene families, while the opportunistic euascomycete pathogen a. flavus possessed the greatest number of gene families, and also the largest number of genes in gene families. 1the stringency and efficiency of rip-like processes varies among euascomycete genomes. 2asexual propagation is the most frequently observed reproductive mode in field settings. however, asexual lineages often either have the potential for sexual reproduction, as indicated by the presence of different mating types in populations, and/or phylogenetic evidence for recombination and cryptic speciation. we initially predicted greater proportions of duplicated genes would become fixed in pathogenic lineages as a result of increased preservation of duplicated genes by natural selection and/or higher rates of duplication. in this coevolutionary arms race scenario, continually evolving host resistance would give rise to constant selective pressure for the preservation of duplications of genes relevant to virulence. we thus compared the distributions of gene family sizes in pathogens versus non-pathogens. the distribution of gene family sizes in a genome is thought to follow a power law distribution, and we therefore modeled the pooled set of pathogenic gene families as following this distribution. similarly, we allowed the pooled set of non-pathogenic gene families to follow a power law distribution, with a potentially different value of the power law coefficient, a. we then applied a likelihood ratio test to examine the null hypothesis that the value of a was the same in pathogens as in non-pathogens. although we can reject the hypothesis of equal values of a, surprisingly, in view of our initial prediction, we find that non-pathogenic fungi in fact have slightly larger gene families than do the pathogens. interestingly, when the two basidiomycete species are excluded from the analysis, no overall significant differences in gene family size distribution are evident. we also compared lineages that were primary pathogens to their non-pathogenic relatives, finding again that the non-pathogens have larger gene families. again, excluding the basidiomycete species results in no significant difference being found. finally, we examined only the aspergillus species, comparing the opportunistic pathogens a. flavus and a. fumigatus to a. nidulans and a. oryzae. no significant differences in gene family sizes are evident in the comparison between opportunistic pathogens and their non-pathogenic relatives. the above approach is potentially flawed, because the species in question do not represent independent realizations of the same general stochastic process. rather, the genomes are related by the phylogeny shown in figure. to control for this common ancestry, we performed phylogenetically independent contrasts for gene family size distributions for the six species pairs indicated in figure applying the same likelihood ratio test described above. we find evidence for larger gene families in phytopathogenic species for two paired-species comparisons, while the other comparisons either showed either the opposite trend or no significant differences. in all cases, we used a significance threshold of α =, which reflects application of a bonferroni correction for hypothesis tests. note that a maximum likelihood fit of another potential distribution describing the probability of observing a gene family of size x in a genome, the exponential distribution, visually provides a rather poorer explanation of these data. functional distribution of gene duplicates in pathogens versus non-pathogens to elucidate potential functional differences in duplicated genes in pathogenic versus non-pathogenic genomes, we compared the distribution of go terms between the two groups. we initially selected twenty-two different go terms representing functional categories that are relevant to fungal pathogenesis, as well as others related to basal metabolic processes. we compared the proportions of gene duplicates associated with a go term for pathogens and non-pathogens. when all eleven taxa were considered, two go terms differed significantly in the number of gene duplicates observed for pathogens versus non-pathogens, after allowing for a 20% false discovery rate. the "receptor activity" and "hydrolase activity" terms showed significantly greater numbers of gene duplicates in pathogenic species than in non-pathogenic lineages. when the same analysis was repeated for the nine euascomycete genomes, we identified three additional functional categories where pathogenic species had a greater than expected number of gene duplicates: "nucleic acid binding," "carbohydrate binding" and "regulation of transcription." we also compared the number of gene duplicates associated with terms in the generic go slim ontology for pathogens and non-pathogens. this survey revealed no significant distinctions among pathogens versus non-pathogens for all taxa. when only euascomycete species were considered, we found that gene duplicates associated with following six terms were over represented among pathogens, again controlling for an fdr of 20%: "hydrolase activity," "extracellular region," "carbohydrate metabolism," "nucleobase, nucleoside, nucleotide and nucleic acid metabolism," "carbohydrate binding" and "catalytic activity". ‡ significance values in this column are p-values obtained in chi-square tests §significance values presented in this column are corrected for type error functional distribution of gene duplicates in species pairs for the four species pairs that showed global differences in the magnitude of gene duplication, we surveyed the functional distribution of gene duplicates using the generic go slim ontology. the rice blast fungus, m. grisea, showed higher average gene family size than its non-pathogenic relative n. crassa. correspondingly, we found four go terms that are overrepresented in pathogenic m. grisea, as compared to exclusively saprophytic n. crassa. further, while pathogenic f. graminearum had larger gene families than did non-pathogenic t. reesei, we found five overrepresented go terms in f. graminearum and an equal number of overrepresented terms in t. reesei. when we compared the species pairs where the non-pathogenic taxon possessed more gene duplicates globally,, we found significantly more gene duplicates associated with a total of eighteen particular go terms in the a. nidulans-s. nodorum test pair, and fourteen terms in p. chrysosporium, respectively. significant differences in paired species comparisons were determined after applying corrections for multiple tests, as above. ‡ enrichment in pathogens and non-pathogens across four different pairwise comparisons † denotes duplicate enrichment for the term in non-pathogenic t. reesei ‡ significance values in this column are p-values obtained in chi-square tests §significance values presented in this column are corrected for type error expansion or contraction of gene families in pathogens to determine whether a given gene family showed significant expansion or contraction, we employed a binomial test. sixteen gene families show significant expansion in pathogens, while five gene families appear to be contracted. functional identities, as well as gene family sizes are presented for all significantly expanded or contracted gene families in additional files and some significantly expanded gene families of particular interest are those with predicted hydrolytic, transporter or oxidioreductase activities, as well as those involved with carbohydrate metabolism. examples of expanded gene families in the hydrolytic functional class included those with predicted chitin deactylase, cutinase, amino peptidase and feruloyl esterase activities, among others. specific examples of expanded gene families that belong to the oxidoreductase functional class included those with predicted galactose oxidase and tyrosinase activities. the transporter functional class of expanded gene families included instances of sugar porters, malic acid transporters, neutral amino acid permeases and l-fucose permeases, among others. for gene families that were deemed significantly contracted, the main functional categories, as indicated by go and genbank annotations, also include hydrolases, oxidases and transporters. however, the specific biological roles of particular gene families that showed contraction in pathogens differ from those that showed significant expansion. for instance, in the oxidoreductase functional class, homologues of orda appear to be depleted in pathogens. in aflatoxin-producing species of aspergillus, this gene catalyzes the last reaction in the biosynthesis of this secondary metabolite. other interesting examples include gene families that were predicted to be components of the vaculolar atpase complex, in addition to those encoding glucose oxidase precursors. expanded gene families in species that have more duplicated genes for the four cases where phylogenetic contrast analyses indicated significant differences in gene family sizes in paired species comparisons, gene family expansion was also examined using an approach similar to that above. five gene families are significantly expanded in m. grisea, relative to non-pathogenic n. crassa. these gene familiesinclude those predicted to have oxidoreductase, transporter, peroxidase and melanin biosynthesis functions. four gene families are significantly expanded in f. graminearum versus t. reesei, including those predicted to have transporter, endoglucanase and methyl transferase activities. interestingly, the significantly expanded gene family with methyl transferase functional attributes has substantial homology to laea, a gene that may play a global regulatory role in secondary metabolism in aspergillus species. there are two significantly expanded gene families in a. nidulans, relative to s. nodorum. one family is predicted to have phosphorylase activity, while the other encodes genes with atp-binding cassette transporter function. p. chrysosporium has eight significantly expanded gene families, relative to pathogenic u. maydis. these eight families include genes with predicted oxidoreductase and peptidase functions, as well as genes with roles in carbohydrate metabolism. discussion change in gene inventory and fungal genome evolution differential gene gain and loss clearly play definitive roles in both degree of virulence, and in determining host range. although there are numerous examples of gene family expansion in pathogenic lineages of fungi, to date, gene duplication trends across whole genomes have not been analyzed. our analysis sought to understand whether the sum of gene family expansions have given rise to an overall increase in numbers of gene duplicates in the genomes of pathogenic fungi. we observed no such trend when all pathogenic species were compared to the non-pathogens, nor when we performed phylogenetically-informed paired species comparisons. instead, the paired-species comparisons showed no clear association between gene family size distributions and pathogenicity. an example of this lack of association is apparent in the aspergillus species comparisons, where we found no significant differences in gene family size distributions, a result that derives from both the opportunistic nature of the pathogens, as well as the close phylogenetic relationships of the species in question. another potential explanation for similarity among the complement of gene duplicates in genomes of the aspergilli is the recent suggestion that host animals, and humans in particular, do not generate the sort of co-evolutionary arms-race characteristic of the plant hosts of the other pathogenic species considered. a confounding factor for our analyses is that gene duplication's ability to drive biological innovation is diminished in some fungi by the process of repeat-induced point mutation. rip is a pre-meiotic homology-based mechanism that introduces characteristic point mutations into sequences present in multiple copies in a genome, and may have evolved to limit the deleterious effects of mobile genetic elements. all euascomycete genomes surveyed here likely possess some degree of rip. however, the severity and efficiency of the process varies, with n. crassa possibly possessing the most stringent form. we note that n. crassa shows the smallest average gene family size among the species studied, as would be expected, given rip's severe pruning of duplicates in this organism. there is no evidence for rip in either of the basidiomycete genomes examined in this study, although it has recently been documented in anther smut. some of the differences illustrated in figure are possibly due to variation in rip stringency rather than pathogenicity. although quite variable, the approximate divergence times among the six pathogenic and five non-pathogenic species examined are reasonable for large-scale comparative studies of gene duplication in euascomycete and basidiomycete lineages. functional patterns of duplicate gene enrichment in fungal genomes enrichment of gene duplicates over particular functional categories in the set of twenty-two go terms, as well as those in the generic go slim ontology appear consistent with requirements of pathogenic lifestyles. these results parallel those of previous studies, which have demonstrated that fungal pathogenesis is associated with increased catalytic potential among enzymes such as hydrolases, relatively larger repertoires of receptors, and the expansion of secreted proteins, as well as carbohydrate recognition and binding gene families. for example, we found an increased number of duplicates associated with transport functions in f. graminearum, the causal agent of head blight in cereals, as compared to non-pathogenic t. reesei, a result consistent with the known relative deficit of carbohydrate catalysis genes in t. reesei. some of the differences observed between non-pathogenic p. chrysosporium and the hemibiotrophic corn smut fungus u. maydis appear relevant to the markedly divergent ecology and development these basidiomycete species. the over-represented terms "carbohydrate binding," "extracellular region," "response to abiotic stimulus," "multicellular organismal development," "peptidase activity," as well as "amino acid and derivative metabolic process" are associated with the suite of traits that make the white rot fungus, p. chrysosporium, amenable to industrial applications, such as lignocellulose and organopollutant degradation. moreover, the observed enrichment of gene duplicates dedicated to particular developmental programs, such as muticellularity, is also consistent with the differences in morphological complexity between these two organisms in their fruiting body structures; u. maydis produces no fruiting body, per se, only teliospore-filled tumors on a host, whereas p. chrysosporium possesses a resupinate fleshy fruiting body. that u. maydis showed no enrichment for gene duplicates for any go term was consistent with recent analyses of this genome, which indicated relatively few duplicates. when phylogenetic relationships are not considered, a cohort of go terms that is common to pathogens and non-pathogens in all four paired-species comparisons becomes evident. these three terms are: electron transport, generation of precursor metabolites and energy, as well as catalytic activity. biological explanations for this observation include two possibilities that are not mutually exclusive: first, that genes associated with these three terms are not peculiar to pathogenesis, and/or secondly, that genes associated with these terms possess inherently greater plasticity in copy number in fungal genomes. given numerous accounts of duplication in pathogenic and non-pathogenic fungi, copy number plasticity in certain functional categories would seem the more plausible explanation. our results, both from comparisons across all taxa, as well as paired species comparisons, suggest an association between organisms' lytic potential and receptor repertoires and pathogenesis. moreover, even when a pathogen had fewer gene duplicates overall, enrichment of terms associated with lytic activities, management of oxygen toxicity, as well as terms possibly relevant to protein secretion were still evident in the pathogenic lineage. gene family expansion or contraction in pathogens gene families that were expanded in pathogens largely fall into two functional categories: those encoding lytic enzymes and those encoding putative transporters. lytic enzymes, such as feruloyl esterases, cutinases, aminopeptidases and endoglucanases are known to be significant in successful plant parasitism interactions, as such genes have demonstrated roles in plant cell wall decomposition. serine proteinases and the regulatory p domain of the subtilisin-like proprotein convertases have been implicated in mutualistic, as well as pathogenic, interactions with grasses. moreover, the importance of fungal chitin deactylases in entomopathology has been empirically demonstrated. expanded families that were predicted to have diverse transporter functions included genes that encode l-fucose and neutral amino acid permeases, as well as mfs, malic acid and sugar transporters, respectively. l-fucose permeases have not only been implicated in galactose transport and sphigolipid metabolism, but also in development of resistant sclerotia in a. flavus, while neutral amino acid permeases and mfs transporters are possibly involved in efflux of non-ribosomal peptides and other secondary metabolites required for virulence. permeases may also be critical for the initial assault upon a host, as well as assimilation of host-derived carbohydrates. the remaining expanded families included those encoding tyrosinases, galatose oxidase precursors and transmembrane receptors, all of which have been implicated in pathogenic life styles. tyrosinases catalyze initial steps in melanin biosynthesis. melanization in fungi is frequently, if not always, essential for virulence. it is notable that another gene in the melanin synthesis pathway was also found to be over duplicated in m. grisea, relative to non-pathogenic n. crassa. galactose oxidation yields peroxide, a reactive oxygen species known to be produced by fungal pathogens during infection. recent work in m. grisea described a phalanx of receptors that are linked to pathogenesis and host perception. the five families that showed gene losses in pathogens included those with predicted roles in secondary metabolism. one intriguing case is that of o-methylsterigmatocystin oxidoreductase, which catalyzes the last step in aflatoxin biosynthesis. while only a few species of aspergillus are known to produce aflatoxin, numerous euascomycete and basidiomycete lineages possess homologues of this gene, as well as others in the aflatoxin biosynthesis pathway. the wide phylogenetic distribution of these genes suggests not only that that these genes are ancient, but also that they are possibly important in secondary metabolism. we speculate that contraction of this family in pathogenic lineages may be the result of random gene deletions after relaxation of selective pressure that maintains production of this toxin. recent genomic and population studies that focused on the aflatoxin biosynthesis cluster in species of aspergillus would seem to support this conjecture, as orda, an o-methylsterigmatocystin oxidoreductase, appears to have undergone more extensive loss following duplication than any other gene in the cluster. gene family expansion in species pairs gene families that were expanded in two paired-species comparisons also had either expected or previously demonstrated roles in virulence, host-pathogen interaction or chemical defense. for example, homologues of laea, a methyltransferase, are over duplicated in f. graminearum. this gene appears to be a global regulator of secondary metabolism in species of aspergillus. it is interesting to speculate whether an expanded repertoire of upstream regulators of secondary metabolism in f. graminearum could reflect either greater numbers of target genes or refined regulatory control of secondary metabolic networks in this primary pathogen. in the second instance where a phytopathogen had overall greater levels of gene duplication, five gene families were expanded. gene families encoding diverse cytochrome p450s, mfs transporters, as well as those predicted to have trihydroxynaphthalene reductase activity were expanded in m. grisea, as compared to exclusively saprophytic n. crassa. the white rot fungus p. chrysosporium had eight gene families that were significantly expanded, relative to basal basidiomycete u. maydis. these eight expanded families encode genes that have diverse oxidoreductase activities, protease and endo- -beta-glucanase functions, consistent with genes previously reported to be present in high copy numbers in this genome. a large complement of such genes in this organism's genome is likely a reflection of its saprotropic wood-decay ecology.  here, we present results on patterns of gene duplication and differential gene gain and loss in pathogenic fungal genomes. the scope of this study captures at least of one billion years of fungal evolution. no general relationship between pathogenicity and the magnitude of gene duplication was evident, but differences in duplicate gene retention among certain functional classes were consistent with known and predicted requirements of virulent lifestyles. gene family expansion in species with overall higher levels of duplication also showed functional trends that could be reconciled with organismal life histories. the observed differences in overall levels of duplication between phytopathogenic lineages and their non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. 
to determine if patterns of gene duplication differed between pathogens and non-pathogens, we identified gene families across nine euascomycete and two basidiomycete species. expanded gene families included those predicted to be important in melanin biosynthesis, host cell wall degradation and transport functions. gene family size distributions were fit to power laws to compare gene duplication trends in pathogens versus non-pathogens. fungal phytopathogens showed globally altered patterns of gene duplication, as indicated by differences in gene family size distribution. surveys of the functional distribution of gene duplicates indicated that pathogens show enrichment for gene duplicates associated with receptor and hydrolase activities, while euascomycete pathogens appeared to have not only these differences, but also significantly more duplicates associated with regulatory and carbohydrate binding functions. we also identified sixteen examples of gene family expansion and five instances of gene family contraction in pathogenic lineages. we hypothesize that the observed patterns of gene duplicate enrichment, gene family expansion and contraction reflect adaptation within pathogenic life histories. to date, differential gene gain and loss have not been studied at genomic scales in fungal pathogens, despite this phenomenon's known importance in virulence in bacteria and viruses. differences in the overall levels of gene duplication in phytopathogenic species versus non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. contracted families included those encoding genes involved in toxin production, genes with oxidoreductase activity, as well as subunits of the vacuolar atpase complex.
 duplication, followed by fixation or random loss of novel genes, contributes to genome evolution. particular outcomes of duplication events are possibly associated with pathogenic life histories in fungi. to date, differential gene gain and loss have not been studied at genomic scales in fungal pathogens, despite this phenomenon's known importance in virulence in bacteria and viruses.  to determine if patterns of gene duplication differed between pathogens and non-pathogens, we identified gene families across nine euascomycete and two basidiomycete species. gene family size distributions were fit to power laws to compare gene duplication trends in pathogens versus non-pathogens. fungal phytopathogens showed globally altered patterns of gene duplication, as indicated by differences in gene family size distribution. we also identified sixteen examples of gene family expansion and five instances of gene family contraction in pathogenic lineages. expanded gene families included those predicted to be important in melanin biosynthesis, host cell wall degradation and transport functions. contracted families included those encoding genes involved in toxin production, genes with oxidoreductase activity, as well as subunits of the vacuolar atpase complex. surveys of the functional distribution of gene duplicates indicated that pathogens show enrichment for gene duplicates associated with receptor and hydrolase activities, while euascomycete pathogens appeared to have not only these differences, but also significantly more duplicates associated with regulatory and carbohydrate binding functions.  differences in the overall levels of gene duplication in phytopathogenic species versus non-pathogenic relatives implicate gene inventory flux as an important virulence-associated process in fungi. we hypothesize that the observed patterns of gene duplicate enrichment, gene family expansion and contraction reflect adaptation within pathogenic life histories. these adaptations were likely shaped by ancient, as well as contemporary, intimate associations with monocot hosts. 