BACKGROUND
fungi  are an incredibly diverse and adaptable group of organisms which colonise all habitats on earth.
some are plant and animal pathogens of major economic importance and as stressed by kupferschmidt  <cit> , “fungi have now become a greater global threat to crops, forests, and wild animals than ever before.
they have killed countless amphibians, pushing some species to extinction, and they are threatening the food supply for billions of people.” the sequencing of fungal genomes  initially indicated that genomes of filamentous ascomycetes are relatively small , with few repetitive elements  and they contain  <dig> - <dig>  protein-encoding genes.
unlike symbiotic and parasitic bacteria, which usually have smaller genomes than their free-living relatives  <cit> , the first genome data from filamentous fungi did not indicate differences between genome size of fungal pathogens and that of saprophytes.
however, contrasting to what is observed in bacteria, recent sequence data indicate that some phytopathogens have drastically expanded genomes, mostly due to massive invasion by transposable elements .
the 45-mb genome of leptosphaeria maculans, the ascomycete that causes stem canker of crucifers contains 33% tes, the 120-mb genome of cereal downy mildew, blumeria graminis, contains 64% tes , and that of the 240-mb oomycete phytophthora infestans contains 74% tes  <cit> .
these data, suggesting convergent evolution towards bigger genomes in widely divergent fungal and oomycete species, are intriguing and the counterbalance between selective advantage conferred to the pathogen and the cost of maintaining this amount of “parasitic” dna is unknown.
analysis of l. maculans ‘brassicae’  genome  <cit>  revealed a genomic structure at this time only observed in mammals and other vertebrates: the base composition  varied widely along the chromosomes, but locally, was relatively homogeneous.
such structural features of chromosomes are termed “isochores”  <cit> .
in lmb, the high te proportion in conjunction with rip   <cit>  generates large at-rich regions , called at-isochores.
these at-isochores are scattered along the genome alternating with large gc-equilibrated regions , gc-isochores, containing 95% of the predicted genes and mostly devoid of tes.
at-isochores cover 36% of l. maculans genome, are mainly composed of mosaics of truncated and rip-degenerated tes and only contain 5% of the predicted genes, but 20% of those genes encode small secreted proteins  considered as putative effectors of pathogenicity  <cit> .
as only 4% of the genes located in gc-isochores encode ssps, we hypothesized that at-isochores were niches for putative effectors.
this was corroborated by the presence within these at-isochores of genes encoding known effectors, including four avirulence genes , avrlm1
<cit> , avrlm6
<cit> , avrlm4-7
<cit>  and avrlm11
<cit> .
moreover, genes within at-isochores are affected by rip, which can occasionally overrun the repeated region into adjacent single-copy genes, resulting in extensive mutation of the affected genes .
this genome environment favors a rapid response to selection pressure.
for example, effector genes acting as avirulence genes can be inactivated in a single sexual cycle due to rip or deletions due to large-scale genome rearrangements in at-isochores  <cit> .
in addition to genes encoding effectors and genes with no predicted function, at-isochores are also enriched in gene clusters responsible for the biosynthesis of secondary metabolites  <cit> .
both effector genes and secondary metabolite gene clusters are distributed discontinuously among ascomycetes and/or show species specificity  <cit> .
we have postulated that genome invasion by tes, followed by their degeneracy by rip shaping large at-isochores, is a recent evolutionary event that has contributed to the rise of a better adapted new species, and that maintaining large te-rich regions hosting pathogenicity determinants has favoured adaptation to new host plants along with generation of new virulence specificities  <cit> .
this hypothesis can be now tested by exploiting comparative genomics between lmb and other members of the leptosphaeria species complex, for which preliminary electrokaryotype and southern blot analyses suggested only limited te content .
l. maculans and l. biglobosa are dothideomycete phytopathogens specialized on crucifers and encompassing a series of ill-defined entities more or less adapted to oilseed rape/canola .
some of the species of the complex attack oilseed rape and are responsible for the main disease of the crop  .
the complex also encompasses related lineages only found on cruciferous weeds.
these include l. maculans ‘lepidii’  isolated from lepidium spp.
and l. biglobosa ‘thlaspii’  isolated from thlaspi arvense, but also found occasionally on brassica species in canada  <cit> .
the l. maculans and l. biglobosa lineages infecting b. napus  and l. biglobosa ‘canadensis’ ) share morphological traits, epidemiology, initial infection strategies, ecological niches and regardless of geographic distribution, are often found together in tissues of individual infected plants  <cit> .
in this paper we investigate when and how genome expansion took place in the l. maculans-l. biglobosa species complex, and the consequences it had on genome structure, adaptability and pathogenicity.
the genomes of five members of the species complex  were sequenced.
the genomic data were analysed and compared to those from other dothideomycete species to address the following questions:  do the different members of the species complex constitute separate biological species and did tes contribute to speciation?
did te invasion contribute to the rise of a better adapted and more adaptable species: i.e.
what was the incidence of te invasion on the generation, modification, loss or regulation of pathogenicity determinants?
RESULTS
phylogeny and estimates of divergence times
l. maculans and l. biglobosa belong to order pleosporales from the class dothideomycetes.
this class is the largest and the most phylogenetically diverse within pezizomycotina , and includes numerous plant pathogens such as species in the genera cochliobolus, phaeosphaeria, pyrenophora, mycosphaerella, zymoseptoria and venturia
<cit>  .figure  <dig>
chronogram of major classes in
ascomycota, with a focus on
dothideomycetes.
the chronogram was produced with beast from a data set of  <dig> truncated proteins.
branches with terminal grey triangles were collapsed and their number of leaves indicated in brackets after taxon labels.
numbers at nodes indicate mean node ages in millions of years and green bars indicate their 95% highest posterior density intervals.
a broken line indicates uncertain phylogenetic placement.
the chronogram presented in figure  <dig> , based on the alignment of  <dig> conserved proteins, has representatives from three of the four main classes in the pezizomycotina, but the main focus is on dothideomycetes.
this phylogeny is congruent with other more complete analyses  <cit>  and indicates that the leptosphaeria species diverged from the sampled plant pathogens of pleosporaceae  approximately  <dig> mya.
based on morphological features, l. maculans and l. biglobosa were considered as two pathotypes of the same species for more than 70 years  <cit>  until formal renaming in  <dig>  <cit> .
our data estimate that l. maculans and l. biglobosa diverged ca.
<dig> mya .
divergence times were also determined within the l. maculans and l. biglobosa lineages.
voigt et al.
<cit>  suggested that lbt was distinct from the other l. biglobosa sub-clades, which is supported by our current data since it diverged ca.
<dig> mya from the terminal lbb or lbc .
in terminal branches, divergence time between lmb and lml  and that of lbb and lbc  are comparable .
genome statistics
the reference genome of lmb , was sequenced in  <dig> using a whole genome shotgun strategy and sanger technology  <cit> .
here, next generation sequencing  technologies were used to sequence the genomes of five members of the species complex, including another isolate of lmb.
despite different sequencing and assembly strategies, the resequenced isolate of lmb  had a similar genome size and proportion of tes  compared to v <dig> .
<dig>  .table  <dig>
sequencing statistics and genome statistics for six members of the
leptosphaeria maculans-l. biglobosa
species complex
‘ancestral’ genome size a
a‘ancestral genome’ refers to the core genome without tes.
compared to the lmb isolates, the other members of the species complex had smaller genomes, ranging from  <dig>  to  <dig>  mb and only comprising  <dig> % to  <dig> % of tes.
these sequencing data are consistent with electrokaryotypes done using pulsed field gel electrophoresis  and results of dna hybridization with known tes .
the gc-content of the lmb reference genome was 45%, which is similar to that of lmb isolate wa <dig> .
in contrast, all other isolates of the species complex had genome gc-contents  that related more to those of other dothideomycetes , with a couple of exceptions discussed below  <cit> .
these data indicate that lmb is an exception in terms of te-content compared to other pleosporales including closely related members of the species complex .
only dothideomycete species of order capnodiales such as pseudocercospora fijiensis and cladosporium fulvum have genomes enriched in tes, resulting in low overall gc-content of their genome  <cit>  .
in the l. maculans-l. biglobosa species complex, the amount of non-repetitive dna was very similar ca.
28–29 mb .
this indicates that the size differences between genomes are essentially due to a difference in te amount and not due to expansion or loss of gene families, as found in other fungi  <cit> .
gene annotation predicted a comparable number  of protein-encoding genes in all genomes .
for each genome, the gene features were also very similar in terms of mean gene length, mean coding sequence length or proportion of genes with introns .
chromosomal organisation and synteny
lmb was estimated to have 17– <dig> chromosomes by combination of analysis of electrokaryotypes developed by pfge  and sequencing of the reference genome  <cit> .
the new genomic data described here, especially those of the well-assembled genome of lml  and optical maps of lmb, allowed this estimate to be refined by considering scaffolding errors on the reference assembly.
with the support of genetic mapping, the chromosome count was updated to  <dig> , including the dispensable chromosome  <cit>  only present in the two lmb isolates.
as a direct consequence to the overall gc-content of the genomes, the limited amount of tes in lml, lbb, lbc and lbt resulted in a major difference in chromosomal structure: the at-rich landscapes were mostly restricted to chromosome ends in lml, lbb or lbt .
thus, within the species complex, only the lmb genome had an isochore structure alternating large at-rich and gc-equilibrated regions.figure  <dig>
genome alignment and synteny analyses between
l. maculans
‘brassicae’
and
l. maculans
‘lepidii’
.
whole genome dotplot showing intrachromosomal inversions occurring in the genome of l. maculans ‘brassicae’  compared to that of l. maculans ‘lepidii’ .
inverted regions are circled in black and the number of inversions is mentioned close to the circle.
schematic zoomed representation of one supercontig_ <dig> region  and its syntenic region in lml.
the region contains three inversions and the location of bordering transposable elements in lmb are indicated as black boxes.
the arrows represent genes and their orientation.
genes with no orthologs are colored in grey.
comparative chromosome structure in
l. maculans
‘brassicae’
,
l. maculans
‘lepidii’
and
l. biglobosa
‘thlaspii’
:
the example of chromosome  <dig>   circos representation of synteny between the homologous chromosomes.
the black parts represent the te-rich, at-rich chromosomal regions;  gc content changes along the chromosomes showing the typical isochore structure of the l. maculans ‘brassicae’ chromosomes, absent form the other species.
repeat-masked nucleotide sequences of each genome were aligned with the others using mummer  <cit> .
the genome alignment between the two lmb isolates showed a perfect conservation of macrosynteny , as also observed by ohm et al.
<cit>  for isolates of cochliobolus heterostrophus.
comparisons between lmb and lbb genomes, or between lmb and lbt genomes showed a globally well-conserved synteny with many intrachromosomal inversions .
the alignment between lmb and the most closely related species, lml, is of particular interest since it showed a highly conserved macrosynteny pattern with only few major genomic rearrangements .
while large scale translocations were not seen,  <dig> intrachromosomal sequence inversions sized from  <dig>  to 355 kb were identified .
these inversions were scattered along all the chromosomes and encompassed one to more than a hundred genes .
the inversions within the chromosomes of the two isolates of lmb analysed were bordered by tes in 70% of the cases with tes located at 82 bp on average from the inversion breakpoints .
the alignments also allowed to infer the sequence polymorphism between the different members of the species complex.
the two lmb isolates had very similar genomes, only  <dig>  snps have been identified in the aligned non-repetitive regions .
in spite of highly syntenic genomes, the number of snps between lmb and lml was ca.
<dig>  million.
a similar number of snps between aligned regions is found when comparing other members of the species complex one to another, suggesting that what have been previously considered as sub-species might actually be distinct species.table  <dig>
alignment length and snp counts between the genomes of members of the
leptosphaeria maculans-leptosphaeria biglobosa
species complex
l. maculans
‘brassicae’
l. maculans
‘lepidii’
l. biglobosa
‘brassicae’
l. biglobosa
‘canadensis’
l. biglobosa
‘thlaspii’
repertoire of transposable elements
even though tes were highly degenerated by rip, the use of the repet pipeline followed by extensive manual annotation allowed identification and classification of a large proportion of the repeats present in the genomes of lmb, lml, lbb and lbt .
one hundred and twenty-one consensus sequences have been identified of which  <dig>  were classified as class i or class ii tes .
of these,  <dig> represented copy variants of te families identified in several genomes and grouped into  <dig> larger families while the  <dig> remaining families were invariant between species .
tes represented ca.
30% of the lmb genome but only 4% and less in the other genomes .
nevertheless, despite the major differences in genome coverage between lmb and related species, a similar number of te families  were identified in each of the annotated genomes .
class i elements were the most abundant in all genomes  and up to 83% of the repetitive fraction in the genome of lmb .
uncertainties remain due to the high proportion of non-categorized repeats in the genomes of lml, lbb, lbc and lbt  and higher proportion of unresolved nucleotides in the assemblies compared to the reference lmb v <dig> .
<dig> .
regardless of these uncertainties, our data support the fact that lmb is an exception in terms of tes and further indicate that the expansion of class i tes  is remarkable in this species compared to related ones .table  <dig>
transposable element content in genomes of isolates of the
leptosphaeria maculans-leptosphaeria biglobosa
species complex
evolutionary dynamics of transposable elements
previously, using an alignment-based phylogenetic approach with te sequences deprived of the rip-affected sites, we postulated that te expansion in lmb genome took place between  <dig> and  <dig> mya for tes such as dtm_sahana, rlg_rolly, rlg_olly, rlg_rolly and rlc_pholy while others such as rlg_dolly had been resident and maintained in the genomes for very long times   <cit> .
mining for te sequences in the entire ascomycota phylum showed that tes homologous to those present in the species complex were only found in class dothideomycetes and were restricted to the order pleosporales, with two exceptions, rlg_dolly and dtt_krilin, found in p. fijiensis a member of order capnodiales .
as mentioned previously, this species, like lmb, is te-rich and has a low gc-content .figure  <dig>
distribution of selected transposable elements
within the dothideomycete phylogeny.
the phylogeny is a simplified version of that in figure  <dig>  rxx and dxx correspond to retrotransposons and dna transposons families respectively.
the species in which the family has been identified are highlighted along with the corresponding branches.
colors indicate different times of invasion and are similar to those in additional file 14: figure s <dig>
according to the chronogram in figure  <dig>  these two te families have been present in the dothideomycete lineage for at least  <dig> mya.
this assumption is consistent with previous data that suggested rlg_dolly as the most ancient te present in the lmb genome  <cit> .
however it remains uncertain whether these two te families invaded the fungal genomes before the capnodiales-pleosporales separation and then were widely lost in the capnodiales or if their presence only in p. fijiensis is incongruent with vertical inheritance.
the remaining te families are likely to represent surges of genome invasion at different times before and after the divergence of the pleosporales .
among all the te families identified in the l. maculans-l. biglobosa species complex, 66% are specific to the species complex .
class i elements often showed an erratic phylogenetic distribution and presence/absence patterns in terminal nodes or terminal branches of the phylogeny.
in contrast, class ii elements generally were better conserved amongst the pleosporales.
in addition, complete copies of dna transposons were often maintained in all species in which they were present while only truncated copies or small-sized remnants were present for many class i elements .
for example, dtt_molly  was present in all species of pleosporales except the l. maculans lineage .
in many cases, two closely related species were missing one well-conserved te family, further exemplifying the highly dynamic gain and loss patterns of tes ).
lastly, some te families  were only found in distantly related species of the leptosphaeriaceae and pleosporaceae.
this indicates multiple losses, independent invasion of the genomes or horizontal transfer events .
transposable element dynamics in l. maculans‘brassicae’
fifty one families, mainly corresponding to non-categorized repeats, were specific to terminal branches of the species complex.
the number of these families varied from ca.
<dig> for the te-poor genomes to  <dig> for the te-rich lmb genome, thus supporting the hypothesis of recent invasion of the lmb genome by families of tes absent from other members of the species complex.
the presence of lmb-specific te families such as rlx_ayoly  and rlg_rolly   <cit>   indicate a recent invasion of lmb genome followed by a massive expansion of these families.
comparisons of the te mosaics between the two isolates of lmb showed that  <dig> % of them are conserved indicating that this massive te expansion occurred before, or at the onset of intraspecies divergence.
in contrast, some families like dtm_sahana showed highly diverse insertion patterns with only 12% of identical locations in the two lmb genomes .
this strongly suggests, as previously postulated  <cit>  that dtm_sahana is one of the most recent genome invaders and that waves of transposition activity took place after separation between the two lmb isolates.
this simple picture indicating coincidence between genome invasion by new te families and speciation becomes less straightforward when considering the three retrotransposons that account for most of the v <dig> .
<dig> genome expansion along with rlg_rolly: rlc_pholy , rlg_polly  and rlg_olly   <cit> .
these three retrotransposons have been present in the pleosporales phylogeny for at least  <dig> mya, but they only represent a little fraction of the genomes of other pleosporales species or other members of the species complex to-date .
classification of the tes in families and phylogenetic analyses further indicated that the tes associated with intrachromosomal inversions  between lmb and lml were mainly l. maculans- and even lmb-specific tes, i.e.
recent te-invaders such as dtm_sahana or rlx_ayoly .
gene conservation
of the  <dig>  proteins predicted in the leptosphaeria species complex, 75% were grouped into  <dig>  families using orthomcl  <cit> .
then, the core proteome of the sequenced species, encompassing  <dig> proteins, was established .figure  <dig>
conservation of protein-encoding genes in the
leptosphaeria maculans-
l. biglobosa
species complex.
lmb, l. maculans ‘brassicae’; lml, l. maculans ‘lepidii’; lbb, l. biglobosa ‘brassicae’; lbt, l. biglobosa ‘thlaspii’; lbc, l. biglobosa ‘canadensis’.
to identify species-specific proteins, all the proteins that were not grouped into the  <dig>  families were compared by blast at the nucleotide  and protein level  against the other genomes and at the protein level  against the set of ungrouped proteins of the other species.
the analysis allowed us to discriminate three classes of protein sequences:  species-specific sequences,  sequences present in at least one other species and  sequences for which presence or absence remained unresolved with our criteria .
in addition, the occurrence of pseudogenes was investigated when lack of matching predicted protein sequence was associated with blastn or tblastn hits .
each genome shared on average  <dig>  proteins with  the others, which represents  <dig> - <dig> % of the predicted proteins depending on the species.
usually, these sequences were conserved in organisms outside of the species complex since  <dig> - <dig> % of them have homologies in the nr database or harbour known protein domains.
the remaining species-specific proteins, which represented  <dig> - <dig> % of the genomes  had very few predicted functions since  <dig> - <dig> % of them had no automated functional annotations.
as a consequence, explaining species-specific biological or pathogenicity traits was not straightforward.
however, two categories of species-specific genes could be identified and are analysed in more details below: genes encoding putative effectors and cluster of genes encoding secondary metabolites.
interestingly, the lmb genome was more enriched in specific sequences than all other members of the species complex and these genes were located in at-isochores:  <dig> % of the  <dig> genes of lmb located in at-isochores were species-specific, compared to only  <dig> % of the genes located in gc-isochores.
pathogenicity gene analysis: effectors and secondary metabolites
candidate effectors
the number of small secreted proteins  amongst the whole predicted proteome of each genome was similar in all the sequenced leptosphaeria genomes, ranging from  <dig> in lbc to  <dig> in lml.
they represented ca.
6% of the predicted proteins and 60% of the predicted secretome of each genome.
their features  were very similar in all leptosphaeria isolates .
their encoding genes were scattered along the chromosomes and had a similar gc-content  not different from the other predicted genes of the genomes .
however, as previously found, gc content varied as a function of genome location, and ssp genes in at isochores had an average  <dig> % gc content  <cit> .table  <dig>
characteristics of genes encoding small secreted proteins  and other predicted proteins in isolates of the
leptosphaeria maculans-leptosphaeria biglobosa
species complex
arepeat induced point mutation  index as measured by the tpa/apt ratio.
the repertoire of ssp-encoding genes appeared to be in part extremely plastic and its conservation was consistent with phylogenetic distance with  <dig> % of the ssp-encoding genes of v <dig> .
<dig> conserved in the other lmb isolate, wa <dig>   <dig> % conserved in lml, and  <dig>  to  <dig> % conserved in l. biglobosa isolates .
in the core proteome, 20% of ssps have a predicted function, and may relate to factors linked with pathogenicity such as carbohydrate-degrading enzymes .
however, the greater was the species-specificity, the less obvious was the potential function of ssp-encoding genes.
ssp-encoding genes did not generally occur as multigene families in the l. maculans-l. biglobosa complex, but a few of them  had paralogs, usually conserved in all genomes.
they corresponded to proteins usually belonging to multigenic families such as cazys and were mainly located in gc-isochores in the lmb genome.
only one effector gene with avirulence activity had an easily recognisable paralog in the lmb genome: avrlm4- <dig> located within an at-isochore had a paralog located 30 kb upstream on the same chromosome.
avirulence effectors
seven ssp genes conferring an avirulence phenotype on a series of genotypes of brassica spp., i.e.
interacting in gene-for-gene interactions with the plant surveillance machinery, have been cloned in lmb.
all are located in at-isochores, and they generally showed a high level of diversification compared to their closest homologs , while usually maintaining a specific pattern of cysteine spacing .
they mostly showed a patchy distribution along the phylogenies or were specific to a few members of the species complex.
six of these had recognisable homologs in other fungal genomes:
avrlmmex  and avrlm11
<cit>  are specific to the leptosphaeriacae, with avrlmmex having homologs in every member of the species complex except lml .
the avrlm11-encoding gene is located on a conditionally dispensable chromosome  present in some isolates of lmb and absent from other isolates of the species complex  <cit> .
avrlm <dig>  however, showed homologies with a ssp predicted in the lbt genome  while three additional genes of the cdc also had orthologs in the genome of lbt.
these three genes were grouped in the lbt genome at a different location to that of the avrlm <dig> homolog.
sequences related to avrlm <dig>  <cit>  and avrlm4- <dig>  <cit>  were in a few species of class dothideomycetes only.
an avrlm <dig> homolog was in the set of predicted proteins of lbt but not in other members of the species complex .
interestingly, the orthologous gene was also located in a large but poorly assembled at-rich region, devoid of other predicted genes.
both avrlm <dig> and its lbt ortholog showed homology with two ssps with unknown function in the pleosporales species pyrenophora teres f. teres .
as described above, avrlm4- <dig> had a paralog in the lmb genomes .
this duplication did not occur in the other members of the species complex since either avrlm4- <dig> or lmcds <dig> had one-copy homologs in these genomes .
outside of the species complex, avrlm4- <dig> or lmcds <dig> had homology with two ssps with no predicted function of the botryosphaeriales species macrophomina phaseolina .
in contrast to the previous examples, avrlm6
<cit>  and lema_p <dig>   had homologs outside of the dothideomycetes, but only in a few species of class sordariomycetes .
within the species complex, these two proteins had ssp orthologs in lml and lbt .
secondary metabolite gene clusters
similar to genes encoding effectors, secondary metabolite biosynthetic genes such as non-ribosomal peptide synthase  and polyketide synthase  genes showed examples of extreme specificity of occurrence and complete conservation within the species complex.
npss and pkss are multimodular genes that are keys in the biosynthesis of secondary metabolites.
these genes are usually organised in clusters that include genes encoding enzymes such as methyl transferases, cytochrome p <dig> monooxygenases, hydroxylases that modify pathway intermediates.
a total of  <dig> nps genes were identified across the five members of the species complex  and most  were shared by all five.
sirp, the nps involved in the production of the epipolythiodioxopiperazine toxin, sirodesmin pl  <cit> , is only found in lmb and lml, which is relevant with biological data since l. biglobosa spp.
do not produce this toxin.
globally, the distribution of nps was well conserved between species in final branches , whereas lbt had an intermediate pattern of nps.
the species-specific nps-genes in the complex may be the results of horizontal gene transfer or newborn pathogenicity determinants .figure  <dig>
conservation of key secondary metabolite biosynthesis genes in the
leptosphaeria maculans
-l. biglobosa
evolutionary series.
non-ribosomal peptide synthases ;  polyketide synthase  genes.
the colour of shading indicates those genes present  or absent  in the corresponding species.
the phylogenetic tree on the left is a simplified version of that in figure  <dig>  and the calculated divergence times  are indicated.
unlike the situation with nps genes, there was a high degree of diversity in number and types of pks genes identified across the members of the species complex .
in addition, and except for the examples mentioned below, homologs of pkss were rare or absent in other dothideomycetes and often had closest relatives in sordariomycetes such as fusarium spp.
or colletotrichum spp., or in aspergilli .
a total of  <dig> pks genes were identified of which only six were found in all species.
lmb and lml had a very similar complement of pks genes with  <dig> pks in lmb and only two missing in lml, one of which was truncated.
twenty-three different pkss were in l. biglobosa species and several of them were species-specific: six were only found in lbt which reflects the distinctiveness of lbt from other members of the species complex .
the pks and nps biosynthetic gene clusters conserved between lmb and lml and between lbb and lbc displayed a high level of synteny .
of the  <dig> clusters conserved in all the sequenced members of the species complex, eight were embedded in highly syntenic regions.
this included genes widely conserved in the dothideomycetes , and also nps <dig> and nps <dig> that are only conserved within the species complex.
in the eight other cases, secondary metabolite clusters showed microsynteny in lmb and lml, while they were reorganized in lbb and lbc.
once again, lbt showed an intermediate genomic pattern with some clusters being syntenic with those present in lmb and lml  and some clusters showing microsynteny with lbb and lbc .
horizontal gene transfer has been proposed for the discontinuous distribution of several fungal secondary metabolite gene clusters  <cit>  and a clear example of this is seen in the situation of pks <dig>  amongst the five species, this pks was only present in lbb, and reciprocal best matches showed that it had > 90% identity at both the nucleotide and protein level to a pks from a very distantly related fungus, the eurotiomycete, arthroderma otae, which is a dermatophyte .
the five upstream genes showed high sequence similarity  to genes upstream of the a. otae pks, although this microsynteny was interrupted by the presence of three genes in a. otae.
several of the homologous genes had predicted functions consistent with biosynthesis of a secondary metabolite .
genes downstream of pks <dig> in lbb were also present in genomes of some of the other leptosphaeria species .figure  <dig>
organisation of the genes surrounding the polyketide synthase,
pks <dig>
of
l. biglobosa
‘brassicae’.
pks <dig> and upstream genes show > 90% identity with genes from arthroderma otae.
each square represents a single gene .
genes that are present in different species are highlighted in the same colour.
ends of contigs are represented with a circle whilst arrows indicate that the contig continues.
lmb, l. maculans ‘brassicae’; lml, l. maculans ‘lepidii’; lbb, l. biglobosa ‘brassicae’; lbt, l. biglobosa ‘thlaspii’; lbc, l. biglobosa ‘canadensis’.
genes common to lbb and a.otae have the following predicted functions: gene  <dig> has a retinol dehydrogenase  motif; genes  <dig> and  <dig> have a methyltransferase motif, gene  <dig> has no conserved domains and gene  <dig> is a dimethyl allyl transferase.
transposable elements and pathogenicity genes
of the  <dig> genes located in at-isochores in the lmb genome,  <dig> genes  were surrounded by tes , whereas the others were located at the transitional regions between at- and gc-isochores.
among these genes,  <dig> were conserved in other members of the species complex.
in order to know if these genes were already present at the same location before the te expansion, we investigated whether the region containing these genes and their closest flanking genes in lmb were syntenic in the other genomes.
for six genes, this analysis was impossible due to scaffolding differences of the assembly of the other genomes.
for the remaining  <dig> genes,  <dig> did not encode ssps and 81% of them showed a conserved microsynteny in the other genomes of the species complex, suggesting that the te expansion did not influence the organisation of these small regions.
among the  <dig> genes encoding ssps, six genes  were not colocalized with their flanking genes while these latters were, suggesting that those six genes were translocated within the genome.
strikingly, five of these six translocated genes were characterized avirulence genes: avrlm <dig>  avrlm4- <dig>  avrlm <dig>  avrlmmex, and lema_p <dig> .
furthermore, a sixth avirulence gene, avrlm <dig>  was present in the  <dig> genes analyzed but could not be analyzed further due to poor assembly of its region in lbt where it is likely to be hosted in an at-rich genome environment.
microarray and rna-seq data were generated to identify the top  <dig> genes produced in planta, during the primary infection stage, by two species able to infect oilseed rape plants: lmb and lbb.
among the top  <dig> genes expressed at seven days post inoculation, 45% encoded ssps in the lmb genome, whereas this proportion decreases to 21% for lbb .
of these ssp-encoding genes, 30% represent species-specific genes in lmb genome, with very few  are species-specific genes in lbb.
rna-seq gene expression values for lmb and lbc orthologs were coupled with proximity of tes in the gene environment.
orthologous genes of lmb and lbc are more highly up-regulated in planta  when a te is located close to their promoter, than when it is not nearby .figure  <dig>
effect of presence of repetitive element adjacents to promoter of orthologs on gene expression
in planta
compared to in axenic culture.
in planta upregulation ratio ) calculated from rna-seq data for orthologs in l. maculans ‘brassicae’  and l. biglobosa ‘canadensis’  during growth in planta and in vitro.
ortholog pairs were categorised according to the presence or absence of repeat elements adjacent to the promoter region of both genes.
positive up-regulation ratios indicate higher expression during growth in planta.
the categories were: no lmb no lbc , no lmb yes lbc , yes lmb no lbc , yes lmb yes lbc .
discussion
in this study, we used comparative genomic approaches to reassess speciation issues in the l. maculans-l. biglobosa species complex and to evaluate the role of tes in genome reshaping and the rise of species with improved pathogenic abilities.
our data demonstrate that  each entity of the species complex actually is a distinct species;  tes likely contributed to speciation by generating chromosomal inversions;  te invasions/burst are recent at the evolution scale;  tes contributed to build two-speed genomes and most likely to translocate effector genes within plastic regions of the genome, with consequences on gene diversification, better adaptation to selection pressure and gene expression.
all in all, te bursts and their consequence on genome structure favored adaptation and pathogenicity towards oilseed rape by allowing generation of novel effector genes and their concerted expression during the first steps of plant infection.
reassessing speciation within the l. maculans-l. biglobosaspecies complex
fungi are estimated to include over  <dig>  million species  <cit> .
their description and classification have been long based on macro- or microscopic criteria, such as morphology or reproduction mode.
the availability of genomic data for a large number of fungal species has allowed the inference of new phylogenetic relationships between close or even unrelated taxa previously aggregated in the same genera.
this is exemplified by the mycosphaerella genera and its new classification  <cit> .
here, we firstly re-investigated speciation issues in the l. maculans-l. biglobosa species complex based on sequence data analyses.
the estimated divergence times between the terminal clades  are consistent with divergence time between species in closely related genera   and strongly indicate all leptosphaeria isolates analysed here belong to different species.
this hypothesis is supported by high numbers of snps  detected between each lineage of the species complex, which are in a comparable order of magnitude than those observed between different species of cochliobolus
<cit>  further support for this hypothesis is provided by the degree of mesosynteny, i.e.
the conservation within chromosomes of gene content but not order or orientation, between the members of the species complex  <cit> .
mesosynteny, firstly identified in dothideomycetes, was recently postulated to be a mode of chromosomal evolution specific to fungi  <cit> .
comparative analyses of  <dig> dothideomycete species, and a simulation-based approach indicated that serial random inversions within the chromosome lead with time to extensive reshuffling of gene order within homologous chromosomes from one species to a distantly related one  <cit> .
focusing on relatively short divergence times, our analyses are consistent with this modelling-based hypothesis.
the number of genome rearrangements  between lmb and lml is many fewer than what has been reported between cochliobolus sativus and c. heterostrophus, where there were as many as  <dig> inversions in a single scaffold  <cit> .
this represents an earlier step towards mesosynteny than what was described for the cochliobolus species.
te regulation in ascomycete genomes
sequencing confirmed that the genome of lmb contains a larger proportion of tes than those of the other members of the species complex, but also those of other pleosporales.
heavily te-invaded genomes can be found in ascomycetes such as blumeria spp., in basidiomycetes or in oomycetes  <cit> .
often related species all show heavily te-invaded genomes as exemplified by downy mildew fungi or rust fungi  <cit> .
an intermediate situation is found in fungi in order capnodiales of the dothideomycetes that have a series of distantly related species with either te-poor or te-invaded genomes  <cit> .
compared to these examples, pleosporales are an exception since all species sequenced to-date, including those of the species complex sequenced here show te-poor genomes and te expansion is only observed in lmb.
in phytophthora spp.
and downy mildew fungi, the absence of irreversible defense systems able to regulate the te activity results in an evolutionary trade-off in which the fitness cost linked with genome “obesity” eventually counter-selects individuals for which the fitness deficit is higher than the selective advantage conferred by enhanced genome plasticity.
compared to the blumeria or phytophthora examples, the expansion of tes in the genome of lmb  remains limited .
these data question about how tes were controlled through time by lmb and, in general, by most ascomycete species.
rip is likely the most efficient genome defense mechanism against tes and has been experimentally demonstrated to be currently active in l. maculans
<cit> .
evidences for past or current rip activity has been reported in most ascomycete species, suggesting that rip is an ancestral mechanism common to  ascomycete fungi  <cit> , with occasional patterns of secondary losses like observed in blumeria spp.
<cit> .
since rip induces irreversible changes in tes, thus disabling their transposition mechanisms, it is therefore confusing how massive transposition activity could have taken place recently in lmb genome for ltr retrotransposon families potentially present in the pleosporales lineage since  <dig> millions years.
our data suggest that these te families were not inactivated by rip or that they were reactivated.
three main and complementary hypotheses, discussed below, could explain these data:  loss and regain of rip activity with involvement of another epigenetic mechanism to control te transposition events,  biological traits preventing rip activity, i.e.
absence of sexual reproduction and  recent and massive events of horizontal transfer .
other inactivation mechanisms, also linked to the sexual cycle, have been described in fungi such as methylation induced premeiotically  in the ascomycete ascobolus immersus  <cit>  and in the basidiomycete coprinus cinereus  <cit> .
mip is an epigenetic mechanism which methylates but does not irreversibly mutate duplicated sequences.
the fact that it is conserved between ascomycetes and basidiomycetes, raises the possibility that rip evolved from mip or from a similar silencing process  <cit> .
the breakdown of such genome defense mechanism seemed to be linked with bursts of transposition in many species, resulting in genome rearrangements and eventually speciation  <cit> .
thus, we can hypothesize that a reversible epigenetic mechanism was previously active in lmb genome and relieved under a stress-inducing event at the time of speciation, leading to the unleashing of transposition activity.
the fact that all te families are affected by rip  suggests that epigenetic mechanisms were replaced by rip as an irreversible control mechanism.
rip is only active during meiosis, therefore prolonged growth in absence of sex would prevent rip inactivation of tes.
lmb undergoes prolific sexual reproduction  <cit> , but asexual populations exist that are either adapted to other crucifers such as cabbage or present in environments where short growth season of the host plant prevent the fungus from completing its sexual cycle  <cit> .
asexual growth could also be imposed within a founder population with only one mating type.
we can thus hypothesize that the rise of the lmb species was followed by a long phase of asexual behaviour in order to propagate rapidly the newly-born species, without preventing expansion of tes until sexual reproduction became prevalent with the need to diversify genotypes to adapt changing environmental conditions.
under this hypothesis, the other members of the species complex known to also use profusely sexual reproduction  never had such a long phase of asexual behaviour and efficiently limited te expansion using rip.
tes can invade a genome through ht  <cit>  and, in animals, arthropods vectors are allowing ht between species  <cit> .
microbes sharing the same diverse ecological niche with lmb , but whose biodiversity is currently unexplored, or the host plants themselves are candidates vectors for such ht of tes to lmb genome.
our phylogenetic analyses did not allow us to establish whether such ht events occurred in the dothideomycete phylogeny, but multiple cases of patchy distribution of te families do not contradict this hypothesis.
in addition, even with rip being active and occurrence of sexual reproduction, constant donation of tes could occur from a donor that was present over an extended period of many million years, such as a virus or an intracellular symbiotic bacteria  <cit> .
the donor tes would not be affected by rip, as it is protected in the vector, but each new introduction would be riped and inactivated as it arrived.
long term addition could result in the establishment and the expansion of te-rich regions.
we acknowledge that this is extremely speculative, as currently massive and recent introduction and expansion of tes cannot be distinguished from multiple introductions, due to extensive sequence degeneracy following rip.
speciation and te expansion
in many eukaryotic lineages, links between bursts of transposition and speciation are observed, but it is difficult to determine whether these bursts were responsible for speciation or if they were a consequence of it  <cit> .
in this study, two events were predicted to have taken place during the same period of time:  the speciation event separating lmb from lml and  the introduction in the lmb genome of new te families followed by the expansion of ancient and recent te famillies.
the effects of invasion by species-specific tes on speciation events are difficult to sort out since introduction of new te families were observed several times during evolution within the dothideomycete lineage while bursts of transposition did not occur in any other pleosporales species.
however, the analysis of te distribution was based only on tes identified in the members of the l. maculans-l. biglobosa species complex, which precluded inference of relationships between tes and speciation in the other pleosporales genera.
moreover, as mentioned previously, the patchy distribution of some te families, especially ltr retrotransposons, could be evidence of ht of tes between species  <cit>  and thus disrupt the evolutionary history of these elements.
in lmb 70% of the intrachromosomal inversions were bordered by tes specific to the l. maculans clade.
ohm et al.
<cit>  suggested that inversion breakpoints in dothideomycetes genomes were associated with simple sequence repeats , a feature that was not apparent when comparing lmb and lml genomes.
these data may indicate a major role for tes rather than ssrs in genome reshaping and reshuffling at the chromosomal level, similar to the documented incidence of tes on chromosomal inversions in more complex eukaryotes such as drosophila
<cit>  or mammals  <cit> .
this first step towards mesosynteny clearly ascribes a role to species-specific tes in this evolutionary mechanism that will eventually generate non-homologous sections of chromosomes and isolate part of the genome from meiotic recombination.
can genome data explain biological and phytopathological specificities?
tes are involved in adaptation of phytopathogens to new hosts by promoting hgt, as exemplified by the transfer of a host selective toxin, toxa between phaeosphaeria nodorum and pyrenophora tritici-repentis, thus generating a new disease because of better adaptation of strains of p. tritici-repentis to their host plant, wheat  <cit> .
tes also promote the birth of new pathogenicity traits via gene duplication and translocation eventually generating large multigene families of effectors as exemplified by the downy mildew fungi and phytophthora species  <cit> .
our data were indicative of hgt between phylogenetically divergent species mostly for secondary metabolite gene clusters, of which some species-specific are located within at isochores in lmb, but information on the involvement of tes in these hgt phenomena were sparse.
such hgt is seen in another member of the species complex, lbb, in the situation of pks <dig> and surrounding genes of the cluster only found in the eurotiomycete a. otae.
the consequences of this on lbb pathogenicity  are not known.
one other intriguing example is that of nps <dig>  the five-module nps thought to be involved in the production of the transiently produced depsipeptide, phomalide.
phomalide is postulated to be an important pathogenicity determinant as it has been proposed to act as an host-selective toxin   <cit> .
the gene cluster is located in an at-rich subtelomeric region of the genome of lmb and has no known homologs.
for ssp-encoding genes, indication of hgt were sparse due to high level of sequence divergence with possible homologs within or outside the species complex.
we confirmed here that effector genes did not belong to recognisable multigene families, and that tes are unlikely to promote multiple duplications.
this is consistent with the known impact of rip on restriction of gene innovation mediated by duplications  <cit> .
however, in cases where homologs could be identified, another impact of tes on ssp genes could be evidenced here for an important part of the effector genes known to be also behaving as avirulence determinants during plant pathogen interaction.
microsynteny analyses between the members of the species complex highlighted the fact that, in the lmb genome,  <dig> of  <dig> characterized avirulence genes present in at-rich isochores have been translocated from another location in the genome to these te-rich environments in the course of evolution.
this is reminiscent at a different scale of what is observed in m. oryzae, where a paralog of avr-pita  is found at an invariant genome location, while avr-pita and other variants may be absent or present at different chromosomal locations, or on different chromosomes, depending on the isolate  <cit> .
in m. oryzae, both patterns of complete loss of the gene and patterns of multiple translocations were ascribed to the close association of avr-pita with a retrotransposon.
the authors postulated that other avirulence genes of m. oryzae may have the same behaviour and also be subjected to multiple translocations in the genomes and that this maintains a pool of isolates with the avirulence gene when selection is exerted so that it can be easily disseminated in populations when the selection is no more present.
in our study, these translocations were not identified between lmb isolates but between members of a species complex displaying contrasted te-content in their genome.
this suggests that during the te expansion, genes  were subjected to te-mediated translocations, resulting in many cases in their isolation within highly dynamic te-rich compartments.
we thus highlight here another mechanism from what is reported in species for which te are drivers of gene innovation via generation of multigene families: in lmb, probably because of the activity of rip, duplication only rarely occurs, but tes contribute to displace the genes of importance in novel dynamic regions of the genome.
on this basis the question is to understand what can be the selective advantage of displacing effector genes in such plastic genomic regions and how this contributes to enhanced pathogenicity towards oilseed rape.
while being morphologically very similar  <cit>  and adapted to crucifer hosts, the more divergent species of the species complex, lmb, lbb and lbc have very similar host range and pathogenic behaviour.
they are pathogenic towards brassica species and share most of their infection strategies and specificity of life cycle  <cit> .
also, the divergence time which predate speciation  between l. biglobosa and l. maculans correlates well with current estimates of the whole-genome triplication events that facilitated the origin of brassica crops centered at  <dig>  mya  <cit> .
this could indicate the l. maculans-l. biglobosa lineage co-evolved with its host to become adapted to these newly-born triplicated brassica plants and that adaptation to brassica predates enhanced pathogenicity towards oilseed rape observed in lmb.
under this hypothesis, the very restricted host range of lml  would be a secondary adaptation to a crucifer weed, possibly linked with its expanded repertoire of ssp-encoding genes compared to the other members of the species complex.
in m. oryzae, effector repertoire is associated with host range expansion; loss of a single effector avr-co <dig> enables this fungus to colonize rice, as well as foxtail millet  <cit> .
perhaps the expanded repertoire of effectors in lml contributes to its narrower host range and its specialisation to lepidium sp.
compared to other members of the species complex.
while pathogenic strategies and life cycle characteristics have been conserved between lmb and lbb/lbc for  <dig> million years, lmb shows some pathogenicity traits that may have been acquired more recently.
two main differences exist between the species: aspects of leaf lesions and ability to develop the stem canker.
according to the leaf symptoms they cause in the field or in controlled environments, lbb and lbc often have been considered as “weakly pathogenic”  <cit> .
however lbb and lbc often cause large lesions on cotyledons: lmb causes grey tissue collapse with no visual evidence of plant responses, suggesting the fungus has deployed mechanisms to suppress plant defenses, while the l. biglobosa isolates cause limited tissue collapse containing numerous large-size black spots suggesting the presence of some plant defense responses  <cit>  .
lmb is the most damaging pathogen of b. napus as it causes cankers at the base of the stem that cause the plant to lodge, resulting in significant losses at harvesting, while lbc and lbb cause limited upper stem lesions, of much lesser incidence on yield.
these differences in pathogenic abilities were difficult to relate to gene content since most of genes, which are species-specific and thus potentially involved in species-specific life traits mostly did not have any attributed function.
in contrast, genes with possible function in disease  were conserved between the different members of the species complex.
in addition, the amount of ssp-encoding genes was comparable between the three species.
however, most of the species-specific effector genes of lmb expressed early during the plant infection are closely associated with tes in the lmb genome and located in large at-rich isochores.
it is possible that the displacement in at-isochores resulted in accelerated sequence diversification of effector genes due to action of rip on non-duplicated sequences hosted in the at-rich environment  <cit> .
the lower at content of these genes compared to that of other genes encoding ssps in the gc isochores of lmb  <cit>  or of genes encoding ssp in other members of the species complex  would be in favor of such an environment-mediated accelerated rate of diversification of the genes and thus explain the enrichment in species-specific effector genes in at-isochores of lmb.
our data also indicate a link between proximity of tes and in planta gene expression.
eventually, moving effector genes in the at isochores would favor in planta expression of effector genes and since ca.
<dig> candidate effector genes are located in  of at-isochores results in concerted expression of these at the onset of plant infection  <cit> .
we previously showed that at-isochores create heterochromatic landscapes responsible for an epigenetic control of the effector gene expression  <cit> .
here, we showed that patterns of expression of effector genes differ between lmb and lbb.
amongst the top  <dig> genes expressed following cotyledon at seven dpi, 45% encode ssps in the lmb genome, whereas this proportion decreases to 21% for lbb .
the finding that 30% of the top  <dig> lmb genes up regulated in planta represent species-specific genes located in at-isochores, compared to very few in lbb, suggests that the concerted expression of these species-specific ssps in lmb play a major role at the onset of the plant colonisation and may contribute to the development of the typical grayish-green tissue collapse on leaves, possibly by suppressing the plant pti , a function commonly attributed to effector genes expressed in the earliest stages of plant infection  <cit> .
the lmb-brassica co-evolution and the early expression of effector genes in planta to suppress pti eventually allowed the plant to build a second line of defense response, effector triggered immunity  that seems to occur in the lmb-b.
napus interaction, but not in the interaction with lbb or lbc  <cit> .
as a consequence, gene-for-gene interactions are observed in the lmb-brassica interactions, but not in interactions of brassica species with l. biglobosa species.
in contrast to the lmb case, the dark lesions caused by lbb or lbc  suggest that the plant initiates pti and that the fungus cannot efficiently suppress it.
in this case, speed of growth of the fungus and multiple re-infection points nevertheless allow more or less efficient subsequent tissue colonisation.
apart from an extensively described role of at-isochores in rapid adaptation to plant resistance selection  <cit> , and probable role of hgt, tes thus are likely to have played a major role in the course of evolution by moving effector genes to novel plastic genomes environments promoting sequence diversification and concerted expression of effector genes at the onset of plant infection, eventually resulting in the success of lmb as a destructive pathogen of oilseed rape and other brassicas.
CONCLUSIONS
in fungi, genome invasion by tes is often postulated to have contributed to the success of phytopathogens, mutualists and endophytes and, as stated by raffaele & kamoun  <cit> , phytopathogens often have expanded genomes compared to their free-living relatives.
the l. maculans-l. biglobosa system also shows that tes have contributed to intrachromosomal inversions, likely resulting in speciation as already described for more complex eukaryotes.
in lmb, as in other phytopathogens, the hosting of effector genes in te-rich genome environment contributed to sequence diversification and probably adaptation to new hosts or better adaptation to the existing host.
it currently contributes to a highly dynamic plasticity and diversification of these genes.
in lmb, rip mutation increases the speed with which the detrimental genes can be mutated to extinction, rendering migration of low importance compared to the ability to generate variants locally in a single sexual cycle.
to-date, this process has not been documented in other fungal species.
the hosting of effector genes in such dynamic genome environments has a dual advantage for phytopathogens: immediate adaptation to a new source of resistance  consistent with the speed of birth and dissemination of new virulent populations, and long-term enhanced ability to duplicate and diversify effector genes to facilitate adaptation to the host or adaptation to new hosts.
recent advances suggested that the association of effector genes with tes also has an impact on epigenetic, heterochromatin-based regulation of their expression.
this, along with evolutionary consequences of this finding, provide a new and promising field for research.

 fungi are an incredibly diverse and adaptable group of organisms which colonise all habitats on earth. some are plant and animal pathogens of major economic importance and as stressed by kupferschmidt, “fungi have now become a greater global threat to crops, forests, and wild animals than ever before. they have killed countless amphibians, pushing some species to extinction, and they are threatening the food supply for billions of people.” the sequencing of fungal genomes initially indicated that genomes of filamentous ascomycetes are relatively small, with few repetitive elements and they contain - protein-encoding genes. unlike symbiotic and parasitic bacteria, which usually have smaller genomes than their free-living relatives, the first genome data from filamentous fungi did not indicate differences between genome size of fungal pathogens and that of saprophytes. however, contrasting to what is observed in bacteria, recent sequence data indicate that some phytopathogens have drastically expanded genomes, mostly due to massive invasion by transposable elements. the 45-mb genome of leptosphaeria maculans, the ascomycete that causes stem canker of crucifers contains 33% tes, the 120-mb genome of cereal downy mildew, blumeria graminis, contains 64% tes, and that of the 240-mb oomycete phytophthora infestans contains 74% tes. these data, suggesting convergent evolution towards bigger genomes in widely divergent fungal and oomycete species, are intriguing and the counterbalance between selective advantage conferred to the pathogen and the cost of maintaining this amount of “parasitic” dna is unknown. analysis of l. maculans ‘brassicae’ genome revealed a genomic structure at this time only observed in mammals and other vertebrates: the base composition varied widely along the chromosomes, but locally, was relatively homogeneous. such structural features of chromosomes are termed “isochores”. in lmb, the high te proportion in conjunction with rip generates large at-rich regions, called at-isochores. these at-isochores are scattered along the genome alternating with large gc-equilibrated regions, gc-isochores, containing 95% of the predicted genes and mostly devoid of tes. at-isochores cover 36% of l. maculans genome, are mainly composed of mosaics of truncated and rip-degenerated tes and only contain 5% of the predicted genes, but 20% of those genes encode small secreted proteins considered as putative effectors of pathogenicity. as only 4% of the genes located in gc-isochores encode ssps, we hypothesized that at-isochores were niches for putative effectors. this was corroborated by the presence within these at-isochores of genes encoding known effectors, including four avirulence genes, avrlm1, avrlm6, avrlm4-7 and avrlm11. moreover, genes within at-isochores are affected by rip, which can occasionally overrun the repeated region into adjacent single-copy genes, resulting in extensive mutation of the affected genes. this genome environment favors a rapid response to selection pressure. for example, effector genes acting as avirulence genes can be inactivated in a single sexual cycle due to rip or deletions due to large-scale genome rearrangements in at-isochores. in addition to genes encoding effectors and genes with no predicted function, at-isochores are also enriched in gene clusters responsible for the biosynthesis of secondary metabolites. both effector genes and secondary metabolite gene clusters are distributed discontinuously among ascomycetes and/or show species specificity. we have postulated that genome invasion by tes, followed by their degeneracy by rip shaping large at-isochores, is a recent evolutionary event that has contributed to the rise of a better adapted new species, and that maintaining large te-rich regions hosting pathogenicity determinants has favoured adaptation to new host plants along with generation of new virulence specificities. this hypothesis can be now tested by exploiting comparative genomics between lmb and other members of the leptosphaeria species complex, for which preliminary electrokaryotype and southern blot analyses suggested only limited te content. l. maculans and l. biglobosa are dothideomycete phytopathogens specialized on crucifers and encompassing a series of ill-defined entities more or less adapted to oilseed rape/canola. some of the species of the complex attack oilseed rape and are responsible for the main disease of the crop. the complex also encompasses related lineages only found on cruciferous weeds. these include l. maculans ‘lepidii’ isolated from lepidium spp. and l. biglobosa ‘thlaspii’ isolated from thlaspi arvense, but also found occasionally on brassica species in canada. the l. maculans and l. biglobosa lineages infecting b. napus and l. biglobosa ‘canadensis’ ) share morphological traits, epidemiology, initial infection strategies, ecological niches and regardless of geographic distribution, are often found together in tissues of individual infected plants. in this paper we investigate when and how genome expansion took place in the l. maculans-l. biglobosa species complex, and the consequences it had on genome structure, adaptability and pathogenicity. the genomes of five members of the species complex were sequenced. the genomic data were analysed and compared to those from other dothideomycete species to address the following questions: do the different members of the species complex constitute separate biological species and did tes contribute to speciation? did te invasion contribute to the rise of a better adapted and more adaptable species: i.e. what was the incidence of te invasion on the generation, modification, loss or regulation of pathogenicity determinants?  phylogeny and estimates of divergence times l. maculans and l. biglobosa belong to order pleosporales from the class dothideomycetes. this class is the largest and the most phylogenetically diverse within pezizomycotina, and includes numerous plant pathogens such as species in the genera cochliobolus, phaeosphaeria, pyrenophora, mycosphaerella, zymoseptoria and venturia.figure chronogram of major classes in ascomycota, with a focus on dothideomycetes. the chronogram was produced with beast from a data set of truncated proteins. branches with terminal grey triangles were collapsed and their number of leaves indicated in brackets after taxon labels. numbers at nodes indicate mean node ages in millions of years and green bars indicate their 95% highest posterior density intervals. a broken line indicates uncertain phylogenetic placement. the chronogram presented in figure , based on the alignment of conserved proteins, has representatives from three of the four main classes in the pezizomycotina, but the main focus is on dothideomycetes. this phylogeny is congruent with other more complete analyses and indicates that the leptosphaeria species diverged from the sampled plant pathogens of pleosporaceae approximately mya. based on morphological features, l. maculans and l. biglobosa were considered as two pathotypes of the same species for more than 70 years until formal renaming in. our data estimate that l. maculans and l. biglobosa diverged ca. mya. divergence times were also determined within the l. maculans and l. biglobosa lineages. voigt et al. suggested that lbt was distinct from the other l. biglobosa sub-clades, which is supported by our current data since it diverged ca. mya from the terminal lbb or lbc. in terminal branches, divergence time between lmb and lml and that of lbb and lbc are comparable. genome statistics the reference genome of lmb, was sequenced in using a whole genome shotgun strategy and sanger technology. here, next generation sequencing technologies were used to sequence the genomes of five members of the species complex, including another isolate of lmb. despite different sequencing and assembly strategies, the resequenced isolate of lmb had a similar genome size and proportion of tes compared to v..table sequencing statistics and genome statistics for six members of the leptosphaeria maculans-l. biglobosa species complex ‘ancestral’ genome size a a‘ancestral genome’ refers to the core genome without tes. compared to the lmb isolates, the other members of the species complex had smaller genomes, ranging from to  mb and only comprising % to % of tes. these sequencing data are consistent with electrokaryotypes done using pulsed field gel electrophoresis and results of dna hybridization with known tes. the gc-content of the lmb reference genome was 45%, which is similar to that of lmb isolate wa. in contrast, all other isolates of the species complex had genome gc-contents that related more to those of other dothideomycetes, with a couple of exceptions discussed below. these data indicate that lmb is an exception in terms of te-content compared to other pleosporales including closely related members of the species complex. only dothideomycete species of order capnodiales such as pseudocercospora fijiensis and cladosporium fulvum have genomes enriched in tes, resulting in low overall gc-content of their genome. in the l. maculans-l. biglobosa species complex, the amount of non-repetitive dna was very similar ca. 28–29 mb. this indicates that the size differences between genomes are essentially due to a difference in te amount and not due to expansion or loss of gene families, as found in other fungi. gene annotation predicted a comparable number of protein-encoding genes in all genomes. for each genome, the gene features were also very similar in terms of mean gene length, mean coding sequence length or proportion of genes with introns. chromosomal organisation and synteny lmb was estimated to have 17– chromosomes by combination of analysis of electrokaryotypes developed by pfge and sequencing of the reference genome. the new genomic data described here, especially those of the well-assembled genome of lml and optical maps of lmb, allowed this estimate to be refined by considering scaffolding errors on the reference assembly. with the support of genetic mapping, the chromosome count was updated to, including the dispensable chromosome only present in the two lmb isolates. as a direct consequence to the overall gc-content of the genomes, the limited amount of tes in lml, lbb, lbc and lbt resulted in a major difference in chromosomal structure: the at-rich landscapes were mostly restricted to chromosome ends in lml, lbb or lbt. thus, within the species complex, only the lmb genome had an isochore structure alternating large at-rich and gc-equilibrated regions.figure genome alignment and synteny analyses between l. maculans ‘brassicae’ and l. maculans ‘lepidii’. whole genome dotplot showing intrachromosomal inversions occurring in the genome of l. maculans ‘brassicae’ compared to that of l. maculans ‘lepidii’. inverted regions are circled in black and the number of inversions is mentioned close to the circle. schematic zoomed representation of one supercontig_ region and its syntenic region in lml. the region contains three inversions and the location of bordering transposable elements in lmb are indicated as black boxes. the arrows represent genes and their orientation. genes with no orthologs are colored in grey. comparative chromosome structure in l. maculans ‘brassicae’, l. maculans ‘lepidii’ and l. biglobosa ‘thlaspii’: the example of chromosome circos representation of synteny between the homologous chromosomes. the black parts represent the te-rich, at-rich chromosomal regions; gc content changes along the chromosomes showing the typical isochore structure of the l. maculans ‘brassicae’ chromosomes, absent form the other species. repeat-masked nucleotide sequences of each genome were aligned with the others using mummer. the genome alignment between the two lmb isolates showed a perfect conservation of macrosynteny, as also observed by ohm et al. for isolates of cochliobolus heterostrophus. comparisons between lmb and lbb genomes, or between lmb and lbt genomes showed a globally well-conserved synteny with many intrachromosomal inversions. the alignment between lmb and the most closely related species, lml, is of particular interest since it showed a highly conserved macrosynteny pattern with only few major genomic rearrangements. while large scale translocations were not seen, intrachromosomal sequence inversions sized from to 355 kb were identified. these inversions were scattered along all the chromosomes and encompassed one to more than a hundred genes. the inversions within the chromosomes of the two isolates of lmb analysed were bordered by tes in 70% of the cases with tes located at 82 bp on average from the inversion breakpoints. the alignments also allowed to infer the sequence polymorphism between the different members of the species complex. the two lmb isolates had very similar genomes, only snps have been identified in the aligned non-repetitive regions. in spite of highly syntenic genomes, the number of snps between lmb and lml was ca. million. a similar number of snps between aligned regions is found when comparing other members of the species complex one to another, suggesting that what have been previously considered as sub-species might actually be distinct species.table alignment length and snp counts between the genomes of members of the leptosphaeria maculans-leptosphaeria biglobosa species complex l. maculans ‘brassicae’ l. maculans ‘lepidii’ l. biglobosa ‘brassicae’ l. biglobosa ‘canadensis’ l. biglobosa ‘thlaspii’ repertoire of transposable elements even though tes were highly degenerated by rip, the use of the repet pipeline followed by extensive manual annotation allowed identification and classification of a large proportion of the repeats present in the genomes of lmb, lml, lbb and lbt. one hundred and twenty-one consensus sequences have been identified of which were classified as class i or class ii tes. of these, represented copy variants of te families identified in several genomes and grouped into larger families while the remaining families were invariant between species. tes represented ca. 30% of the lmb genome but only 4% and less in the other genomes. nevertheless, despite the major differences in genome coverage between lmb and related species, a similar number of te families were identified in each of the annotated genomes. class i elements were the most abundant in all genomes and up to 83% of the repetitive fraction in the genome of lmb. uncertainties remain due to the high proportion of non-categorized repeats in the genomes of lml, lbb, lbc and lbt and higher proportion of unresolved nucleotides in the assemblies compared to the reference lmb v.. regardless of these uncertainties, our data support the fact that lmb is an exception in terms of tes and further indicate that the expansion of class i tes is remarkable in this species compared to related ones.table transposable element content in genomes of isolates of the leptosphaeria maculans-leptosphaeria biglobosa species complex evolutionary dynamics of transposable elements previously, using an alignment-based phylogenetic approach with te sequences deprived of the rip-affected sites, we postulated that te expansion in lmb genome took place between and mya for tes such as dtm_sahana, rlg_rolly, rlg_olly, rlg_rolly and rlc_pholy while others such as rlg_dolly had been resident and maintained in the genomes for very long times. mining for te sequences in the entire ascomycota phylum showed that tes homologous to those present in the species complex were only found in class dothideomycetes and were restricted to the order pleosporales, with two exceptions, rlg_dolly and dtt_krilin, found in p. fijiensis a member of order capnodiales. as mentioned previously, this species, like lmb, is te-rich and has a low gc-content.figure distribution of selected transposable elements within the dothideomycete phylogeny. the phylogeny is a simplified version of that in figure  rxx and dxx correspond to retrotransposons and dna transposons families respectively. the species in which the family has been identified are highlighted along with the corresponding branches. colors indicate different times of invasion and are similar to those in additional file 14: figure s according to the chronogram in figure  these two te families have been present in the dothideomycete lineage for at least mya. this assumption is consistent with previous data that suggested rlg_dolly as the most ancient te present in the lmb genome. however it remains uncertain whether these two te families invaded the fungal genomes before the capnodiales-pleosporales separation and then were widely lost in the capnodiales or if their presence only in p. fijiensis is incongruent with vertical inheritance. the remaining te families are likely to represent surges of genome invasion at different times before and after the divergence of the pleosporales. among all the te families identified in the l. maculans-l. biglobosa species complex, 66% are specific to the species complex. class i elements often showed an erratic phylogenetic distribution and presence/absence patterns in terminal nodes or terminal branches of the phylogeny. in contrast, class ii elements generally were better conserved amongst the pleosporales. in addition, complete copies of dna transposons were often maintained in all species in which they were present while only truncated copies or small-sized remnants were present for many class i elements. for example, dtt_molly was present in all species of pleosporales except the l. maculans lineage. in many cases, two closely related species were missing one well-conserved te family, further exemplifying the highly dynamic gain and loss patterns of tes ). lastly, some te families were only found in distantly related species of the leptosphaeriaceae and pleosporaceae. this indicates multiple losses, independent invasion of the genomes or horizontal transfer events. transposable element dynamics in l. maculans‘brassicae’ fifty one families, mainly corresponding to non-categorized repeats, were specific to terminal branches of the species complex. the number of these families varied from ca. for the te-poor genomes to for the te-rich lmb genome, thus supporting the hypothesis of recent invasion of the lmb genome by families of tes absent from other members of the species complex. the presence of lmb-specific te families such as rlx_ayoly and rlg_rolly indicate a recent invasion of lmb genome followed by a massive expansion of these families. comparisons of the te mosaics between the two isolates of lmb showed that % of them are conserved indicating that this massive te expansion occurred before, or at the onset of intraspecies divergence. in contrast, some families like dtm_sahana showed highly diverse insertion patterns with only 12% of identical locations in the two lmb genomes. this strongly suggests, as previously postulated that dtm_sahana is one of the most recent genome invaders and that waves of transposition activity took place after separation between the two lmb isolates. this simple picture indicating coincidence between genome invasion by new te families and speciation becomes less straightforward when considering the three retrotransposons that account for most of the v. genome expansion along with rlg_rolly: rlc_pholy, rlg_polly and rlg_olly. these three retrotransposons have been present in the pleosporales phylogeny for at least mya, but they only represent a little fraction of the genomes of other pleosporales species or other members of the species complex to-date. classification of the tes in families and phylogenetic analyses further indicated that the tes associated with intrachromosomal inversions between lmb and lml were mainly l. maculans- and even lmb-specific tes, i.e. recent te-invaders such as dtm_sahana or rlx_ayoly. gene conservation of the proteins predicted in the leptosphaeria species complex, 75% were grouped into families using orthomcl. then, the core proteome of the sequenced species, encompassing proteins, was established.figure conservation of protein-encoding genes in the leptosphaeria maculans- l. biglobosa species complex. lmb, l. maculans ‘brassicae’; lml, l. maculans ‘lepidii’; lbb, l. biglobosa ‘brassicae’; lbt, l. biglobosa ‘thlaspii’; lbc, l. biglobosa ‘canadensis’. to identify species-specific proteins, all the proteins that were not grouped into the families were compared by blast at the nucleotide and protein level against the other genomes and at the protein level against the set of ungrouped proteins of the other species. the analysis allowed us to discriminate three classes of protein sequences: species-specific sequences, sequences present in at least one other species and sequences for which presence or absence remained unresolved with our criteria. in addition, the occurrence of pseudogenes was investigated when lack of matching predicted protein sequence was associated with blastn or tblastn hits. each genome shared on average proteins with the others, which represents - % of the predicted proteins depending on the species. usually, these sequences were conserved in organisms outside of the species complex since - % of them have homologies in the nr database or harbour known protein domains. the remaining species-specific proteins, which represented - % of the genomes had very few predicted functions since - % of them had no automated functional annotations. as a consequence, explaining species-specific biological or pathogenicity traits was not straightforward. however, two categories of species-specific genes could be identified and are analysed in more details below: genes encoding putative effectors and cluster of genes encoding secondary metabolites. interestingly, the lmb genome was more enriched in specific sequences than all other members of the species complex and these genes were located in at-isochores: % of the genes of lmb located in at-isochores were species-specific, compared to only % of the genes located in gc-isochores. pathogenicity gene analysis: effectors and secondary metabolites candidate effectors the number of small secreted proteins amongst the whole predicted proteome of each genome was similar in all the sequenced leptosphaeria genomes, ranging from in lbc to in lml. they represented ca. 6% of the predicted proteins and 60% of the predicted secretome of each genome. their features were very similar in all leptosphaeria isolates. their encoding genes were scattered along the chromosomes and had a similar gc-content not different from the other predicted genes of the genomes. however, as previously found, gc content varied as a function of genome location, and ssp genes in at isochores had an average % gc content.table characteristics of genes encoding small secreted proteins and other predicted proteins in isolates of the leptosphaeria maculans-leptosphaeria biglobosa species complex arepeat induced point mutation index as measured by the tpa/apt ratio. the repertoire of ssp-encoding genes appeared to be in part extremely plastic and its conservation was consistent with phylogenetic distance with % of the ssp-encoding genes of v. conserved in the other lmb isolate, wa % conserved in lml, and to % conserved in l. biglobosa isolates. in the core proteome, 20% of ssps have a predicted function, and may relate to factors linked with pathogenicity such as carbohydrate-degrading enzymes. however, the greater was the species-specificity, the less obvious was the potential function of ssp-encoding genes. ssp-encoding genes did not generally occur as multigene families in the l. maculans-l. biglobosa complex, but a few of them had paralogs, usually conserved in all genomes. they corresponded to proteins usually belonging to multigenic families such as cazys and were mainly located in gc-isochores in the lmb genome. only one effector gene with avirulence activity had an easily recognisable paralog in the lmb genome: avrlm4- located within an at-isochore had a paralog located 30 kb upstream on the same chromosome. avirulence effectors seven ssp genes conferring an avirulence phenotype on a series of genotypes of brassica spp., i.e. interacting in gene-for-gene interactions with the plant surveillance machinery, have been cloned in lmb. all are located in at-isochores, and they generally showed a high level of diversification compared to their closest homologs, while usually maintaining a specific pattern of cysteine spacing. they mostly showed a patchy distribution along the phylogenies or were specific to a few members of the species complex. six of these had recognisable homologs in other fungal genomes: avrlmmex and avrlm11 are specific to the leptosphaeriacae, with avrlmmex having homologs in every member of the species complex except lml. the avrlm11-encoding gene is located on a conditionally dispensable chromosome present in some isolates of lmb and absent from other isolates of the species complex. avrlm however, showed homologies with a ssp predicted in the lbt genome while three additional genes of the cdc also had orthologs in the genome of lbt. these three genes were grouped in the lbt genome at a different location to that of the avrlm homolog. sequences related to avrlm and avrlm4- were in a few species of class dothideomycetes only. an avrlm homolog was in the set of predicted proteins of lbt but not in other members of the species complex. interestingly, the orthologous gene was also located in a large but poorly assembled at-rich region, devoid of other predicted genes. both avrlm and its lbt ortholog showed homology with two ssps with unknown function in the pleosporales species pyrenophora teres f. teres. as described above, avrlm4- had a paralog in the lmb genomes. this duplication did not occur in the other members of the species complex since either avrlm4- or lmcds had one-copy homologs in these genomes. outside of the species complex, avrlm4- or lmcds had homology with two ssps with no predicted function of the botryosphaeriales species macrophomina phaseolina. in contrast to the previous examples, avrlm6 and lema_p had homologs outside of the dothideomycetes, but only in a few species of class sordariomycetes. within the species complex, these two proteins had ssp orthologs in lml and lbt. secondary metabolite gene clusters similar to genes encoding effectors, secondary metabolite biosynthetic genes such as non-ribosomal peptide synthase and polyketide synthase genes showed examples of extreme specificity of occurrence and complete conservation within the species complex. npss and pkss are multimodular genes that are keys in the biosynthesis of secondary metabolites. these genes are usually organised in clusters that include genes encoding enzymes such as methyl transferases, cytochrome p monooxygenases, hydroxylases that modify pathway intermediates. a total of nps genes were identified across the five members of the species complex and most were shared by all five. sirp, the nps involved in the production of the epipolythiodioxopiperazine toxin, sirodesmin pl, is only found in lmb and lml, which is relevant with biological data since l. biglobosa spp. do not produce this toxin. globally, the distribution of nps was well conserved between species in final branches, whereas lbt had an intermediate pattern of nps. the species-specific nps-genes in the complex may be the results of horizontal gene transfer or newborn pathogenicity determinants.figure conservation of key secondary metabolite biosynthesis genes in the leptosphaeria maculans -l. biglobosa evolutionary series. non-ribosomal peptide synthases; polyketide synthase genes. the colour of shading indicates those genes present or absent in the corresponding species. the phylogenetic tree on the left is a simplified version of that in figure  and the calculated divergence times are indicated. unlike the situation with nps genes, there was a high degree of diversity in number and types of pks genes identified across the members of the species complex. in addition, and except for the examples mentioned below, homologs of pkss were rare or absent in other dothideomycetes and often had closest relatives in sordariomycetes such as fusarium spp. or colletotrichum spp., or in aspergilli. a total of pks genes were identified of which only six were found in all species. lmb and lml had a very similar complement of pks genes with pks in lmb and only two missing in lml, one of which was truncated. twenty-three different pkss were in l. biglobosa species and several of them were species-specific: six were only found in lbt which reflects the distinctiveness of lbt from other members of the species complex. the pks and nps biosynthetic gene clusters conserved between lmb and lml and between lbb and lbc displayed a high level of synteny. of the clusters conserved in all the sequenced members of the species complex, eight were embedded in highly syntenic regions. this included genes widely conserved in the dothideomycetes, and also nps and nps that are only conserved within the species complex. in the eight other cases, secondary metabolite clusters showed microsynteny in lmb and lml, while they were reorganized in lbb and lbc. once again, lbt showed an intermediate genomic pattern with some clusters being syntenic with those present in lmb and lml and some clusters showing microsynteny with lbb and lbc. horizontal gene transfer has been proposed for the discontinuous distribution of several fungal secondary metabolite gene clusters and a clear example of this is seen in the situation of pks amongst the five species, this pks was only present in lbb, and reciprocal best matches showed that it had > 90% identity at both the nucleotide and protein level to a pks from a very distantly related fungus, the eurotiomycete, arthroderma otae, which is a dermatophyte. the five upstream genes showed high sequence similarity to genes upstream of the a. otae pks, although this microsynteny was interrupted by the presence of three genes in a. otae. several of the homologous genes had predicted functions consistent with biosynthesis of a secondary metabolite. genes downstream of pks in lbb were also present in genomes of some of the other leptosphaeria species.figure organisation of the genes surrounding the polyketide synthase, pks of l. biglobosa ‘brassicae’. pks and upstream genes show > 90% identity with genes from arthroderma otae. each square represents a single gene. genes that are present in different species are highlighted in the same colour. ends of contigs are represented with a circle whilst arrows indicate that the contig continues. lmb, l. maculans ‘brassicae’; lml, l. maculans ‘lepidii’; lbb, l. biglobosa ‘brassicae’; lbt, l. biglobosa ‘thlaspii’; lbc, l. biglobosa ‘canadensis’. genes common to lbb and a.otae have the following predicted functions: gene has a retinol dehydrogenase motif; genes and have a methyltransferase motif, gene has no conserved domains and gene is a dimethyl allyl transferase. transposable elements and pathogenicity genes of the genes located in at-isochores in the lmb genome, genes were surrounded by tes, whereas the others were located at the transitional regions between at- and gc-isochores. among these genes, were conserved in other members of the species complex. in order to know if these genes were already present at the same location before the te expansion, we investigated whether the region containing these genes and their closest flanking genes in lmb were syntenic in the other genomes. for six genes, this analysis was impossible due to scaffolding differences of the assembly of the other genomes. for the remaining genes, did not encode ssps and 81% of them showed a conserved microsynteny in the other genomes of the species complex, suggesting that the te expansion did not influence the organisation of these small regions. among the genes encoding ssps, six genes were not colocalized with their flanking genes while these latters were, suggesting that those six genes were translocated within the genome. strikingly, five of these six translocated genes were characterized avirulence genes: avrlm avrlm4- avrlm avrlmmex, and lema_p. furthermore, a sixth avirulence gene, avrlm was present in the genes analyzed but could not be analyzed further due to poor assembly of its region in lbt where it is likely to be hosted in an at-rich genome environment. microarray and rna-seq data were generated to identify the top genes produced in planta, during the primary infection stage, by two species able to infect oilseed rape plants: lmb and lbb. among the top genes expressed at seven days post inoculation, 45% encoded ssps in the lmb genome, whereas this proportion decreases to 21% for lbb. of these ssp-encoding genes, 30% represent species-specific genes in lmb genome, with very few are species-specific genes in lbb. rna-seq gene expression values for lmb and lbc orthologs were coupled with proximity of tes in the gene environment. orthologous genes of lmb and lbc are more highly up-regulated in planta when a te is located close to their promoter, than when it is not nearby.figure effect of presence of repetitive element adjacents to promoter of orthologs on gene expression in planta compared to in axenic culture. in planta upregulation ratio ) calculated from rna-seq data for orthologs in l. maculans ‘brassicae’ and l. biglobosa ‘canadensis’ during growth in planta and in vitro. ortholog pairs were categorised according to the presence or absence of repeat elements adjacent to the promoter region of both genes. positive up-regulation ratios indicate higher expression during growth in planta. the categories were: no lmb no lbc, no lmb yes lbc, yes lmb no lbc, yes lmb yes lbc. discussion in this study, we used comparative genomic approaches to reassess speciation issues in the l. maculans-l. biglobosa species complex and to evaluate the role of tes in genome reshaping and the rise of species with improved pathogenic abilities. our data demonstrate that each entity of the species complex actually is a distinct species; tes likely contributed to speciation by generating chromosomal inversions; te invasions/burst are recent at the evolution scale; tes contributed to build two-speed genomes and most likely to translocate effector genes within plastic regions of the genome, with consequences on gene diversification, better adaptation to selection pressure and gene expression. all in all, te bursts and their consequence on genome structure favored adaptation and pathogenicity towards oilseed rape by allowing generation of novel effector genes and their concerted expression during the first steps of plant infection. reassessing speciation within the l. maculans-l. biglobosaspecies complex fungi are estimated to include over million species. their description and classification have been long based on macro- or microscopic criteria, such as morphology or reproduction mode. the availability of genomic data for a large number of fungal species has allowed the inference of new phylogenetic relationships between close or even unrelated taxa previously aggregated in the same genera. this is exemplified by the mycosphaerella genera and its new classification. here, we firstly re-investigated speciation issues in the l. maculans-l. biglobosa species complex based on sequence data analyses. the estimated divergence times between the terminal clades are consistent with divergence time between species in closely related genera and strongly indicate all leptosphaeria isolates analysed here belong to different species. this hypothesis is supported by high numbers of snps detected between each lineage of the species complex, which are in a comparable order of magnitude than those observed between different species of cochliobolus further support for this hypothesis is provided by the degree of mesosynteny, i.e. the conservation within chromosomes of gene content but not order or orientation, between the members of the species complex. mesosynteny, firstly identified in dothideomycetes, was recently postulated to be a mode of chromosomal evolution specific to fungi. comparative analyses of dothideomycete species, and a simulation-based approach indicated that serial random inversions within the chromosome lead with time to extensive reshuffling of gene order within homologous chromosomes from one species to a distantly related one. focusing on relatively short divergence times, our analyses are consistent with this modelling-based hypothesis. the number of genome rearrangements between lmb and lml is many fewer than what has been reported between cochliobolus sativus and c. heterostrophus, where there were as many as inversions in a single scaffold. this represents an earlier step towards mesosynteny than what was described for the cochliobolus species. te regulation in ascomycete genomes sequencing confirmed that the genome of lmb contains a larger proportion of tes than those of the other members of the species complex, but also those of other pleosporales. heavily te-invaded genomes can be found in ascomycetes such as blumeria spp., in basidiomycetes or in oomycetes. often related species all show heavily te-invaded genomes as exemplified by downy mildew fungi or rust fungi. an intermediate situation is found in fungi in order capnodiales of the dothideomycetes that have a series of distantly related species with either te-poor or te-invaded genomes. compared to these examples, pleosporales are an exception since all species sequenced to-date, including those of the species complex sequenced here show te-poor genomes and te expansion is only observed in lmb. in phytophthora spp. and downy mildew fungi, the absence of irreversible defense systems able to regulate the te activity results in an evolutionary trade-off in which the fitness cost linked with genome “obesity” eventually counter-selects individuals for which the fitness deficit is higher than the selective advantage conferred by enhanced genome plasticity. compared to the blumeria or phytophthora examples, the expansion of tes in the genome of lmb remains limited. these data question about how tes were controlled through time by lmb and, in general, by most ascomycete species. rip is likely the most efficient genome defense mechanism against tes and has been experimentally demonstrated to be currently active in l. maculans. evidences for past or current rip activity has been reported in most ascomycete species, suggesting that rip is an ancestral mechanism common to ascomycete fungi, with occasional patterns of secondary losses like observed in blumeria spp.. since rip induces irreversible changes in tes, thus disabling their transposition mechanisms, it is therefore confusing how massive transposition activity could have taken place recently in lmb genome for ltr retrotransposon families potentially present in the pleosporales lineage since millions years. our data suggest that these te families were not inactivated by rip or that they were reactivated. three main and complementary hypotheses, discussed below, could explain these data: loss and regain of rip activity with involvement of another epigenetic mechanism to control te transposition events, biological traits preventing rip activity, i.e. absence of sexual reproduction and recent and massive events of horizontal transfer. other inactivation mechanisms, also linked to the sexual cycle, have been described in fungi such as methylation induced premeiotically in the ascomycete ascobolus immersus and in the basidiomycete coprinus cinereus. mip is an epigenetic mechanism which methylates but does not irreversibly mutate duplicated sequences. the fact that it is conserved between ascomycetes and basidiomycetes, raises the possibility that rip evolved from mip or from a similar silencing process. the breakdown of such genome defense mechanism seemed to be linked with bursts of transposition in many species, resulting in genome rearrangements and eventually speciation. thus, we can hypothesize that a reversible epigenetic mechanism was previously active in lmb genome and relieved under a stress-inducing event at the time of speciation, leading to the unleashing of transposition activity. the fact that all te families are affected by rip suggests that epigenetic mechanisms were replaced by rip as an irreversible control mechanism. rip is only active during meiosis, therefore prolonged growth in absence of sex would prevent rip inactivation of tes. lmb undergoes prolific sexual reproduction, but asexual populations exist that are either adapted to other crucifers such as cabbage or present in environments where short growth season of the host plant prevent the fungus from completing its sexual cycle. asexual growth could also be imposed within a founder population with only one mating type. we can thus hypothesize that the rise of the lmb species was followed by a long phase of asexual behaviour in order to propagate rapidly the newly-born species, without preventing expansion of tes until sexual reproduction became prevalent with the need to diversify genotypes to adapt changing environmental conditions. under this hypothesis, the other members of the species complex known to also use profusely sexual reproduction never had such a long phase of asexual behaviour and efficiently limited te expansion using rip. tes can invade a genome through ht and, in animals, arthropods vectors are allowing ht between species. microbes sharing the same diverse ecological niche with lmb, but whose biodiversity is currently unexplored, or the host plants themselves are candidates vectors for such ht of tes to lmb genome. our phylogenetic analyses did not allow us to establish whether such ht events occurred in the dothideomycete phylogeny, but multiple cases of patchy distribution of te families do not contradict this hypothesis. in addition, even with rip being active and occurrence of sexual reproduction, constant donation of tes could occur from a donor that was present over an extended period of many million years, such as a virus or an intracellular symbiotic bacteria. the donor tes would not be affected by rip, as it is protected in the vector, but each new introduction would be riped and inactivated as it arrived. long term addition could result in the establishment and the expansion of te-rich regions. we acknowledge that this is extremely speculative, as currently massive and recent introduction and expansion of tes cannot be distinguished from multiple introductions, due to extensive sequence degeneracy following rip. speciation and te expansion in many eukaryotic lineages, links between bursts of transposition and speciation are observed, but it is difficult to determine whether these bursts were responsible for speciation or if they were a consequence of it. in this study, two events were predicted to have taken place during the same period of time: the speciation event separating lmb from lml and the introduction in the lmb genome of new te families followed by the expansion of ancient and recent te famillies. the effects of invasion by species-specific tes on speciation events are difficult to sort out since introduction of new te families were observed several times during evolution within the dothideomycete lineage while bursts of transposition did not occur in any other pleosporales species. however, the analysis of te distribution was based only on tes identified in the members of the l. maculans-l. biglobosa species complex, which precluded inference of relationships between tes and speciation in the other pleosporales genera. moreover, as mentioned previously, the patchy distribution of some te families, especially ltr retrotransposons, could be evidence of ht of tes between species and thus disrupt the evolutionary history of these elements. in lmb 70% of the intrachromosomal inversions were bordered by tes specific to the l. maculans clade. ohm et al. suggested that inversion breakpoints in dothideomycetes genomes were associated with simple sequence repeats, a feature that was not apparent when comparing lmb and lml genomes. these data may indicate a major role for tes rather than ssrs in genome reshaping and reshuffling at the chromosomal level, similar to the documented incidence of tes on chromosomal inversions in more complex eukaryotes such as drosophila or mammals. this first step towards mesosynteny clearly ascribes a role to species-specific tes in this evolutionary mechanism that will eventually generate non-homologous sections of chromosomes and isolate part of the genome from meiotic recombination. can genome data explain biological and phytopathological specificities? tes are involved in adaptation of phytopathogens to new hosts by promoting hgt, as exemplified by the transfer of a host selective toxin, toxa between phaeosphaeria nodorum and pyrenophora tritici-repentis, thus generating a new disease because of better adaptation of strains of p. tritici-repentis to their host plant, wheat. tes also promote the birth of new pathogenicity traits via gene duplication and translocation eventually generating large multigene families of effectors as exemplified by the downy mildew fungi and phytophthora species. our data were indicative of hgt between phylogenetically divergent species mostly for secondary metabolite gene clusters, of which some species-specific are located within at isochores in lmb, but information on the involvement of tes in these hgt phenomena were sparse. such hgt is seen in another member of the species complex, lbb, in the situation of pks and surrounding genes of the cluster only found in the eurotiomycete a. otae. the consequences of this on lbb pathogenicity are not known. one other intriguing example is that of nps the five-module nps thought to be involved in the production of the transiently produced depsipeptide, phomalide. phomalide is postulated to be an important pathogenicity determinant as it has been proposed to act as an host-selective toxin. the gene cluster is located in an at-rich subtelomeric region of the genome of lmb and has no known homologs. for ssp-encoding genes, indication of hgt were sparse due to high level of sequence divergence with possible homologs within or outside the species complex. we confirmed here that effector genes did not belong to recognisable multigene families, and that tes are unlikely to promote multiple duplications. this is consistent with the known impact of rip on restriction of gene innovation mediated by duplications. however, in cases where homologs could be identified, another impact of tes on ssp genes could be evidenced here for an important part of the effector genes known to be also behaving as avirulence determinants during plant pathogen interaction. microsynteny analyses between the members of the species complex highlighted the fact that, in the lmb genome, of characterized avirulence genes present in at-rich isochores have been translocated from another location in the genome to these te-rich environments in the course of evolution. this is reminiscent at a different scale of what is observed in m. oryzae, where a paralog of avr-pita is found at an invariant genome location, while avr-pita and other variants may be absent or present at different chromosomal locations, or on different chromosomes, depending on the isolate. in m. oryzae, both patterns of complete loss of the gene and patterns of multiple translocations were ascribed to the close association of avr-pita with a retrotransposon. the authors postulated that other avirulence genes of m. oryzae may have the same behaviour and also be subjected to multiple translocations in the genomes and that this maintains a pool of isolates with the avirulence gene when selection is exerted so that it can be easily disseminated in populations when the selection is no more present. in our study, these translocations were not identified between lmb isolates but between members of a species complex displaying contrasted te-content in their genome. this suggests that during the te expansion, genes were subjected to te-mediated translocations, resulting in many cases in their isolation within highly dynamic te-rich compartments. we thus highlight here another mechanism from what is reported in species for which te are drivers of gene innovation via generation of multigene families: in lmb, probably because of the activity of rip, duplication only rarely occurs, but tes contribute to displace the genes of importance in novel dynamic regions of the genome. on this basis the question is to understand what can be the selective advantage of displacing effector genes in such plastic genomic regions and how this contributes to enhanced pathogenicity towards oilseed rape. while being morphologically very similar and adapted to crucifer hosts, the more divergent species of the species complex, lmb, lbb and lbc have very similar host range and pathogenic behaviour. they are pathogenic towards brassica species and share most of their infection strategies and specificity of life cycle. also, the divergence time which predate speciation between l. biglobosa and l. maculans correlates well with current estimates of the whole-genome triplication events that facilitated the origin of brassica crops centered at mya. this could indicate the l. maculans-l. biglobosa lineage co-evolved with its host to become adapted to these newly-born triplicated brassica plants and that adaptation to brassica predates enhanced pathogenicity towards oilseed rape observed in lmb. under this hypothesis, the very restricted host range of lml would be a secondary adaptation to a crucifer weed, possibly linked with its expanded repertoire of ssp-encoding genes compared to the other members of the species complex. in m. oryzae, effector repertoire is associated with host range expansion; loss of a single effector avr-co enables this fungus to colonize rice, as well as foxtail millet. perhaps the expanded repertoire of effectors in lml contributes to its narrower host range and its specialisation to lepidium sp. compared to other members of the species complex. while pathogenic strategies and life cycle characteristics have been conserved between lmb and lbb/lbc for million years, lmb shows some pathogenicity traits that may have been acquired more recently. two main differences exist between the species: aspects of leaf lesions and ability to develop the stem canker. according to the leaf symptoms they cause in the field or in controlled environments, lbb and lbc often have been considered as “weakly pathogenic”. however lbb and lbc often cause large lesions on cotyledons: lmb causes grey tissue collapse with no visual evidence of plant responses, suggesting the fungus has deployed mechanisms to suppress plant defenses, while the l. biglobosa isolates cause limited tissue collapse containing numerous large-size black spots suggesting the presence of some plant defense responses. lmb is the most damaging pathogen of b. napus as it causes cankers at the base of the stem that cause the plant to lodge, resulting in significant losses at harvesting, while lbc and lbb cause limited upper stem lesions, of much lesser incidence on yield. these differences in pathogenic abilities were difficult to relate to gene content since most of genes, which are species-specific and thus potentially involved in species-specific life traits mostly did not have any attributed function. in contrast, genes with possible function in disease were conserved between the different members of the species complex. in addition, the amount of ssp-encoding genes was comparable between the three species. however, most of the species-specific effector genes of lmb expressed early during the plant infection are closely associated with tes in the lmb genome and located in large at-rich isochores. it is possible that the displacement in at-isochores resulted in accelerated sequence diversification of effector genes due to action of rip on non-duplicated sequences hosted in the at-rich environment. the lower at content of these genes compared to that of other genes encoding ssps in the gc isochores of lmb or of genes encoding ssp in other members of the species complex would be in favor of such an environment-mediated accelerated rate of diversification of the genes and thus explain the enrichment in species-specific effector genes in at-isochores of lmb. our data also indicate a link between proximity of tes and in planta gene expression. eventually, moving effector genes in the at isochores would favor in planta expression of effector genes and since ca. candidate effector genes are located in of at-isochores results in concerted expression of these at the onset of plant infection. we previously showed that at-isochores create heterochromatic landscapes responsible for an epigenetic control of the effector gene expression. here, we showed that patterns of expression of effector genes differ between lmb and lbb. amongst the top genes expressed following cotyledon at seven dpi, 45% encode ssps in the lmb genome, whereas this proportion decreases to 21% for lbb. the finding that 30% of the top lmb genes up regulated in planta represent species-specific genes located in at-isochores, compared to very few in lbb, suggests that the concerted expression of these species-specific ssps in lmb play a major role at the onset of the plant colonisation and may contribute to the development of the typical grayish-green tissue collapse on leaves, possibly by suppressing the plant pti, a function commonly attributed to effector genes expressed in the earliest stages of plant infection. the lmb-brassica co-evolution and the early expression of effector genes in planta to suppress pti eventually allowed the plant to build a second line of defense response, effector triggered immunity that seems to occur in the lmb-b. napus interaction, but not in the interaction with lbb or lbc. as a consequence, gene-for-gene interactions are observed in the lmb-brassica interactions, but not in interactions of brassica species with l. biglobosa species. in contrast to the lmb case, the dark lesions caused by lbb or lbc suggest that the plant initiates pti and that the fungus cannot efficiently suppress it. in this case, speed of growth of the fungus and multiple re-infection points nevertheless allow more or less efficient subsequent tissue colonisation. apart from an extensively described role of at-isochores in rapid adaptation to plant resistance selection, and probable role of hgt, tes thus are likely to have played a major role in the course of evolution by moving effector genes to novel plastic genomes environments promoting sequence diversification and concerted expression of effector genes at the onset of plant infection, eventually resulting in the success of lmb as a destructive pathogen of oilseed rape and other brassicas.  in fungi, genome invasion by tes is often postulated to have contributed to the success of phytopathogens, mutualists and endophytes and, as stated by raffaele & kamoun, phytopathogens often have expanded genomes compared to their free-living relatives. the l. maculans-l. biglobosa system also shows that tes have contributed to intrachromosomal inversions, likely resulting in speciation as already described for more complex eukaryotes. in lmb, as in other phytopathogens, the hosting of effector genes in te-rich genome environment contributed to sequence diversification and probably adaptation to new hosts or better adaptation to the existing host. it currently contributes to a highly dynamic plasticity and diversification of these genes. in lmb, rip mutation increases the speed with which the detrimental genes can be mutated to extinction, rendering migration of low importance compared to the ability to generate variants locally in a single sexual cycle. to-date, this process has not been documented in other fungal species. the hosting of effector genes in such dynamic genome environments has a dual advantage for phytopathogens: immediate adaptation to a new source of resistance consistent with the speed of birth and dissemination of new virulent populations, and long-term enhanced ability to duplicate and diversify effector genes to facilitate adaptation to the host or adaptation to new hosts. recent advances suggested that the association of effector genes with tes also has an impact on epigenetic, heterochromatin-based regulation of their expression. this, along with evolutionary consequences of this finding, provide a new and promising field for research. 
through comparative and evolutionary genomics, five members of the leptosphaeria maculans-leptosphaeria biglobosa species complex, having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of tes on genome shaping, speciation, and on the rise of better adapted pathogens. many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements. comparative genomicsfungal plant pathogentransposable elementseffector genesspeciationadaptation to hostissue-copyright-statement© the author 2014 tes, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta. invasion of l. maculans ‘brassicae’ genome by tes followed by bursts of te activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the pleosporales lineage. these tes had an impact at the structural level by creating large te-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation.
 many plant-pathogenic fungi have a tendency towards genome size expansion, mostly driven by increasing content of transposable elements. through comparative and evolutionary genomics, five members of the leptosphaeria maculans-leptosphaeria biglobosa species complex, having different host ranges and pathogenic abilities towards cruciferous plants, were studied to infer the role of tes on genome shaping, speciation, and on the rise of better adapted pathogens.  l. maculans ‘brassicae’, the most damaging species on oilseed rape, is the only member of the species complex to have a te-invaded genome compared to the other members genomes. these tes had an impact at the structural level by creating large te-rich regions and are suspected to have been instrumental in chromosomal rearrangements possibly leading to speciation. tes, associated with species-specific genes involved in disease process, also possibly had an incidence on evolution of pathogenicity by promoting translocations of effector genes to highly dynamic regions and thus tuning the regulation of effector gene expression in planta.  invasion of l. maculans ‘brassicae’ genome by tes followed by bursts of te activity allowed this species to evolve and to better adapt to its host, making this genome species a peculiarity within its own species complex as well as in the pleosporales lineage. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords comparative genomicsfungal plant pathogentransposable elementseffector genesspeciationadaptation to hostissue-copyright-statement© the author 2014 