Horizontal Transfer of a Nitrate Assimilation Gene
Cluster and Ecological Transitions in Fungi: A
Phylogenetic Study
Jason C. Slot*, David S. Hibbett
Department of Biology, Clark University, Worcester, Massachusetts, United States of America
High affinity nitrate assimilation genes in fungi occur in a cluster (fHANT-AC) that can be coordinately regulated. The clustered
genes include nrt2, which codes for a high affinity nitrate transporter; euknr, which codes for nitrate reductase; and NAD(P)Hnir, which codes for nitrite reductase. Homologs of genes in the fHANT-AC occur in other eukaryotes and prokaryotes, but they
have only been found clustered in the oomycete Phytophthora (heterokonts). We performed independent and concatenated
phylogenetic analyses of homologs of all three genes in the fHANT-AC. Phylogenetic analyses limited to fungal sequences
suggest that the fHANT-AC has been transferred horizontally from a basidiomycete (mushrooms and smuts) to an ancestor of
the ascomycetous mold Trichoderma reesei. Phylogenetic analyses of sequences from diverse eukaryotes and eubacteria, and
cluster structure, are consistent with a hypothesis that the fHANT-AC was assembled in a lineage leading to the oomycetes and
was subsequently transferred to the Dikarya (Ascomycota+Basidiomycota), which is a derived fungal clade that includes the
vast majority of terrestrial fungi. We propose that the acquisition of high affinity nitrate assimilation contributed to the success
of Dikarya on land by allowing exploitation of nitrate in aerobic soils, and the subsequent transfer of a complete assimilation
cluster improved the fitness of T. reesei in a new niche. Horizontal transmission of this cluster of functionally integrated genes
supports the ‘‘selfish operon’’ hypothesis for maintenance of gene clusters.
Citation: Slot JC, Hibbett DS (2007) Horizontal Transfer of a Nitrate Assimilation Gene Cluster and Ecological Transitions in Fungi: A Phylogenetic
Study. PLoS ONE 2(10): e1097. doi:10.1371/journal.pone.0001097
reconstruct the history of the fHANT-AC and its constituent
genes. We first searched available eukaryotic genome sequences
for homologs of genes in fHANT-AC. We then estimated the
organismal phylogeny of ascomycetes, basidiomycetes, and
oomycetes using rRNA and RPB2 gene sequences. We contrasted
the organismal phylogeny with results of independent and
concatenated phylogenetic analyses of genes in the fHANT-AC,
which we performed at two phylogenetic scales: within the fungi
(rooted with the heterokont Phytophthora), and across diverse
eukaryotes and eubacteria (rooted with selected prokaryotes).
Phylogenetic analyses within the fungi suggest that the nitrate
assimilation cluster in the ascomycete T. reesei was obtained by
HGT from a basidiomycete and corresponds to a change in
nutritional mode. This is the best evidence to date that horizontal
transfer of a metabolic pathway can facilitate niche shift in fungi.
Less conclusive phylogenetic and structural evidence from analyses
across diverse eukaryotes and eubacteria cannot reject a heterokont
origin of the nitrate assimilation cluster in Dikarya.
INTRODUCTION
A cluster of nitrate assimilation genes occurs in the ascomycetes
Aspergillus nidulans [1] and Pichia angusta [2] and in the
basidiomycetes Hebeloma cylindrosporum, which is mycorrhizal, and
Phanerochaete chrysosporium [3], which is a wood-decayer. This
cluster, abbreviated fHANT-AC, encodes a high affinity nitrate
transporter (NRT2, TCDB 2.A.1.8.5) along with a nitrate
reductase (EUKNR, EC1.7.1.3) and a ferredoxin-independent
assimilatory nitrite reductase (NAD[P]H-NIR, EC1.7.1.4). The
latter two proteins are required for the reduction of nitrate to
ammonium [4]. Ascomycetes and basidiomycetes are sister taxa
that together form a clade called Dikarya, which contains the vast
majority of described species of filamentous, terrestrial fungi.
Without regard to clustering, nrt2 is widely distributed in bacteria,
plants, heterokonts and other groups, but not in opisthokonts
outside Dikarya [5]. Euknr is restricted to eukaryotes, and absent
from opisthokonts outside Dikarya, and NAD(P)H-nir is known to
occur in Dikarya, heterokonts and bacteria.
The clustering of functionally related genes may be selectively
favorable because it facilitates coordinated expression of the
constituent genes [6]. The ‘‘selfish operon’’ hypothesis puts forth
that clusters can result from the selective maintenance of fitnessimproving genes following horizontal gene transfer (HGT) events
[7]. The clustering of genes in selfish operons greatly increases
success of HGT events when a phenotypic advantage or ability to
exploit a new niche is conferred to the recipient [8]. Gene clusters
may facilitate the acquisition of entire metabolic pathways in the
fungi [9,10]. Examples mainly involve secondary metabolite
clusters, which do not necessarily require HGT between fungal
species to explain their distribution [11], and pathogenicity gene
clusters [9,12]. Transfers of supernumerary chromosomes and
large fragments of chromatin remain the most compelling
mechanisms [12,13,14] for the transfer of metabolic pathways.
After discovering a potential horizontal transfer of nrt2
previously [5], we conducted a series of phylogenetic analyses to
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Academic Editor: James Fraser, University of Queensland, Australia
Received July 20, 2007; Accepted October 8, 2007; Published October 31, 2007
Copyright: ß 2007 Slot, Hibbett. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work was made possible by a National Science Foundation
Doctoral Dissertation Improvement grant (DEB0608017, to DSH and JCS). The
National Science Foundation played no role in the production of data or of the
manuscript and this manuscript does not imply its approval of the research or
publication of the results.
Competing Interests: The authors have declared that no competing interests
exist.
* To whom correspondence should be addressed. E-mail: [email protected]
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HGT of Nitrate Gene Cluster
RESULTS
Phylogenetic Analyses Suggest Horizontal Gene
Transfer of the fHANT-AC
Distribution of Genes of the fHANT-AC in Eukaryotes
is Patchy
A comparison of the organismal phylogeny and the phylogeny of
fHANT-AC within fungi provides support for the hypothesis that
fHANT-AC has been transferred horizontally from basidiomycetes to an ascomycete (Fig. 1a). In separate and combined
analyses of nuclear ribosomal RNA genes (rDNA) and translated
rpb2 (RPB2) sequences using Bayesian, maximum likelihood (ML)
and maximum parsimony (MP) methods (BPP = Bayesian Posterior Probabilities, MLB = Maximum Likelihood Bootstrap,
MPB = Maximum Parsimony Bootstrap), we recovered species
relationships that are congruent with previous molecular phylogenies [17]. We received strong support (1.0 BPP, 100 MPB
combined analysis and 100MLB rDNA and RPB2 analyses unless
otherwise noted) for the monophyly of Basidiomycota (RPB2 = 97
MLB, rDNA = 63 MLB) including Ustilago maydis, the ascomycetes
(rDNA = 81 MLB), Pezizomycotina (non-yeast ascomycete
lineages), and Sordariomycetes including Trichoderma reesei.
The phylogeny of the fHANT-AC genes in fungi is similar to
the organismal phylogeny inferred with rRNA and RPB2 genes,
with one striking exception: in independent and combined
analyses, the three genes of the fHANT-AC of the ascomycete
T. reesei were strongly supported (1.0 BPP, 100 MPB, 100 MLB) as
Homologs of each of the three genes of the fHANT-AC (SI Table
S1) are limited to Viridiplantae, Rhodophyta, heterokonts
(oomycetes+diatoms in this dataset) and Dikarya, with one
exception in Nematostella vectensis (Metazoa), which possesses
bacteria-like nrt2 and nitrite reductase homologs (http://genome.jgipsf.org/Nemve1/Nemve1.home.html). Viridiplantae and Rhodophyta possess genes homologous to the dikarya/heterokont nrt2
and euknr, but not the fungal/heterokont NAD(P)H-nir. Instead,
these groups maintain a ferredoxin-dependent nitrite reductase
gene (cpnir, EC1.7.7.1) of probable plastid/cyanobacterial origin
[15]. Diatoms possess the three Dikarya/oomycete homologs in
addition to cpnir (http://genome.jgi-psf.org/Thaps3/Thaps3.
home.html). Diverse eubacteria possess nrt2 and NAD(P)H-nir
homologs, but not euknr which has an exclusively eukaryotic
distribution [16]. From the Populus trichocarpa genome, we
recovered a b proteobacteria-like NAD(P)H-nir. Homologs of the
dikarya/oomycete genes are absent from most Dikarya yeasts
(with the exception of Pichia angusta), filamentous fungi outside
Dikarya (Rhizopus oryzae and Phycomyces blakesleeanus), and the
chytrid Batrachochytrium dendrobatis.
Figure 1. Phylogeny and clustering of the fHANT-AC. (a) Organismal phylogeny based on rRNA and RPB2 genes (right), and gene phylogeny of the
fHANT-AC cluster (left), along with data on internal gene order and extent of clustering (center). Phylograms are from 50% majority rule Bayesian
consensus. Shading of genes indicates confirmed expression of sequences in GenBank (dbEST:CF878787.1, CB906451.1, CF872359, CF865713,
CB895628). Thick black branches denote strong support for the complete combined sequence (Bayesian posterior probabilities [BPP].0.95,
maximum parsimony bootstrap percentages [MPB].85), and thick gray branches denote strong support from two of three individual genes (BPP 0.99
and/or MPB 70%). Strong support from maximum likelihood bootstraps ([MLB].80%) is indicated by an *. Actual support values for critical nodes are
in Table 1. Clades labeled A (Ascomycota), B (Basidiomycota) and S (Sordariomycetes) follow James et al. (2006). Contributing phylogenetic analyses
are in SI Fig. S1a,b. (b) Description of open reading frames flanking the fHANT-AC in T. reesei and U. maydis. Complete descriptions are available at
genome project websites (see SI Table S1).
doi:10.1371/journal.pone.0001097.g001
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HGT of Nitrate Gene Cluster
Table 1. Alignments including fungal and Phythophthora sequences.
..................................................................................................................................................
Dataset
Clade
Fungi
Ascomycetes
Pezizomycetes
T. reesei+Basidiomycetes
Sordariomycetes
Ustilago maydis+T. reesei
NRT2
1.0B 100L 100P1
-
-
NS2B NSL NSP
-
1.0B 100L 99P
EUKNR
1.0B 100L 100P
-
-
1.0B 99L NSP
-
1.0B 100L 100P
NAD(P)H NIR
1.0B 100L 100P
-
-
NSB 58L 97 P
-
1.0B 100L 100P
HANT-AC
1.0B 100L 100P
Reject3 p,0.0001
-
1.0B 96L 91P
-
1.0B 100L 100P
nlsu
1.0B 100L 100P
1.0B 96L 95P
1.0B 92L 88P
-
1.0B 100L 100P
-
nssu
1.0B 100L 100P
1.0B NSL NSP
1.0B 92L 92P
-
1.0B 100L 99P
-
RPB2
1.0B 100L 100P
1.0B 100L 99P
1.0B 100L 99P
-
1.0B 100L 98P
Reject p,0.0001
RDNA4+RPB2
1.0B 100P
1.0B 100P
1.0B 100P
-
1.0B 100P
-
rDNA
100L
81L
81L
-
100L
Reject p,0.05
1
Support values (B-Bayesian posterior probabilities, L-maximum likelihood bootstraps, P-maximum parsimony bootstraps.
No support from this method.
3
Shimodaira-Hasegawa test significance level p,0.05.
4
nssu+nlsu concatenated.
doi:10.1371/journal.pone.0001097.t001
2
heterokonts and 25-29 (depending on gene distribution) eukaryotic
lineages are not inconsistent with a heterokont origin of the fungal
HANT-AC, although patchy distribution prevents a strong
conclusion from phylogenetic analyses. NAD(P)H-NIR sequences
from three species of Phytophthora (oomycetes) and Phaeodactylum
and Thalassiosira (diatoms) do not form a heterokont clade.
Phytophthora NAD(P)H-NIR sequences are sister to fungal
sequences (1.0 BPP, 81 MPB, 57 MLB), and heterokonts plus
fungi are supported as monophyletic by MP analyses (77 MPB),
but not by ML or Bayesian analyses. Analyses of NRT2 suggest
a red alga+heterokont+fungi clade (1.0 BPP, ,50 MPB, 74 MLB)
that excludes green algae and plants in a strongly supported (1.0
BPP, 92 MPB, 99 MLB) eukaryote clade. These results are
sister to those of the basidiomycete Ustilago maydis. Additionally,
a Shimodaira-Hasegawa test (Table 1) rejected the monophyly of
ascomycete nitrate assimilation clusters when T. reesei sequences
were included (p,0.0001). Together, these analyses indicate
strong conflict between the organismal and nitrate assimilation
gene phylogenies that is best explained by HGT of the fHANTAC from the Ustilaginales to Trichoderma. A search of the EST
database at GenBank confirms that at least two of the horizontally
transferred genes (nrt2 and euknr) are expressed in T. reesei. We
recovered no evidence of vertically derived homologs of these
genes in T. reesei
Higher-level analyses (Fig. 2) of HANT-AC proteins including
diverse eubacteria sequences along with sequences from fungi,
Figure 2. Distribution and phylogeny of HANT-AC gene homologs. (a) Unrooted phylograms of maximum likelihood analyses performed in RaxMLVI-HPC ver. 2.2.3. Support values for critical nodes can be found in Table 2. Contributing phylogenetic analyses are in SI Fig. 2a,b,c. (b) Distribution
and clustering of HANT-AC genes in eukaryotes. Clustering indicated if most or all genomes in clade show clustering. Phylogeny of eukaryotes is
adapted from Baldauf [57]. 1A homolog similar to bacterial sequences is present in Nematostella vectensis. 2N. vectensis may also possesses bacterialike nitrite reductase homologs.
doi:10.1371/journal.pone.0001097.g002
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HGT of Nitrate Gene Cluster
consistent with previous analyses extensively focused on fungi,
which suggested that fungal NRT2 sequences form a monophyletic
group that is nested within a paraphyletic assemblage of sequences
from heterokonts [5]. Analyses of EUKNR sequences , which were
restricted to the eukaryotic distribution of the gene, revealed little
phylogenetic resolution outside the fungi. We found no synapomorphic insertion or deletion events in any of these sequences that
exclusively support a sister relationship between fungi and
oomycetes. Future analyses that include eukaryotic and fungal
lineages not represented in our survey could falsify the hypothesis
of an ancient transfer from heterokonts to dikarya.
rearrangement is common, these rearrangements do not necessarily disassemble the cluster. We propose three scenarios to
explain the disassembly of the fHANT-AC in ascomycetes (SI Fig.
S3B, C, D). If cluster formation is assumed to be an unlikely event,
then that would favor a scenario (SI Fig. S3D) that only requires
the assembly of the fHANT-AC to have occurred once. Metabolic
gene clusters have been reported in several fungal lineages
[22,23,24,25,26,27], and one occurrence of rapid assembly of
a cluster has been suggested in yeast [28]. However, there is
currently no consensus regarding methods of measuring the
relative rates of cluster assembly, disassembly and horizontal
transfer in fungi.
Gene Clustering of fHANT-AC genes is Conserved,
but Order is Not
The Anomalous fHANT-AC in Trichoderma reesei
coincides with Niche Shift
While fHANT-AC genes are usually clustered, gene orders within
and flanking the cluster are not well conserved between lineages
(Fig. 1a). A comparison of genes flanking the fHANT-AC (Fig. 1b)
in Ustilago maydis and T. reesei genomes did not reveal additional
genes that appear to have been obtained by HGT. Most genes on
the T. reesei scaffold 28 are highly similar to ascomycete genes (data
not shown). In addition, the Cip2 gene that occurs three genes
downstream from the nrt2 gene is a confirmed T. reesei sequence
[18]. These results suggest that the incongruous fHANT-AC was
incorporated by chromosomal recombination involving a small
number of genes rather than by acquisition of an additional
chromosome. Genes flanking the fHANT-AC in T. reesei appear to
be related to carbohydrate metabolism. We speculate that the
occurrence of this cluster could influence carbohydrate metabolism by either upregulating or inhibiting transcription of these
genes, and thereby contribute downstream to the superlative
propensity of T. reesei to produce cellulases, e.g.in [19].
T. reesei, an ascomycete adapted to wood decay occurs in a genus
consisting largely of endophytes and parasites [29]. T. reesei strain
QM9414, the subject of the T. reesei genome project, was derived
from QM6a, a strain originally defined by its inability to grow on
nitrate as a sole nitrogen source, likely due to defects in nitrite
reductase [30]. Several sexually competent strains of T. reesei,
however, are known to utilize nitrate [30]. We know of no direct
evidence of nitrate assimilation by other Trichoderma species,
although the presence of nitrate has been linked to physiological
responses [31,32,33] and suppression of growth [34] in some
species. Attempts to amplify nrt2 from various Trichoderma species
using PCR were not successful. A shift away from a tightly
regulated nitrate assimilation pathway may have preceded the
acquisition of a basidiomycete fHANT-AC by T. reesei. Analyses of
gene synteny (Fig. 1a) suggest that loss of the ascomycete nitrate
assimilation genes in T. reesei was preceded by the disassembly of
the cluster in Sordariomycetes. In one study, Trichoderma species
(not including T. reesei) and Sclerotinia sclerotiorum (which also lacks
an intact cluster) were shown to utilize ammonium preferentially,
and possibly exclusively (in Trichoderma), over nitrate in ammonium
nitrate feeding experiments [35]. The effect was also observed, but
less pronounced, for other sordariomycetes (Fusarium spp.). These
observations suggest that there was a change in the mode of nitrate
assimilation in the evolution of sordariomycetes. This could have
favored the acquisition of the basidiomycete fHANT-AC and
subsequent loss of vertically inherited genes when T. reesei switched
to a nitrogen-deprived, woody substrate. The horizontal transmission leading to the T. reesei cluster is a plausible scenario
because some Trichoderma species are intracellular parasites of
basidiomycetes [36,37].
DISCUSSION
The fHANT-AC persists with genomic rearrangement
The only groups found to maintain the three genes of the fHANTAC in a cluster (as of June 2007) are the dikarya fungi and
oomycete heterokonts (SI Table S1). Euknr and nrt2 are widely
distributed in dikarya fungi, chromalveolates (alveolates+heterokonts), and plants, whereas NAD(P)H-nir does not occur outside
heterokonts and Dikarya (Fig. 2). We cannot yet falsify the
hypothesis that the fHANT-AC cluster originated in the lineage
leading to heterokonts (SI Fig. S3A) and was subsequently
transferred to Dikarya. Because of the limited sample of nondikarya fungi (SI Table S1) we cannot rule out the possibility of an
earlier fungal origin. Nitrate assimilation clusters also occur in at
least two green algae [20,21], but each of these clusters contains
a unique complement of genes that are not all homologous to
those in the fHANT-AC. The convergent evolution of a nitrate
assimilation cluster in green algae could suggest a general selective
advantage to clustering of nitrate assimilation genes.
Clustering of nitrate assimilation genes is observed in varying
degrees within Dikarya (Fig. 1a). In Basidiomycota where the
genes are present, the clustered orientation of the three genes
predominates with one known exception. A retroviral sequence
interrupts the cluster between nrt2 and NAD(P)H-nir in Laccaria
bicolor, but the genes remain in the same locus. The cluster has
disassembled in certain ascomycetes. At least two lineages have lost
the clustering of the three genes while maintaining the genes
themselves, most notably the Sordariomycetes, of which T. reesei is
a member. Botryotinia fuckeliana maintains a cluster of two of the
genes, while the closely related Sclerotinia sclerotiorum lacks clustering
entirely. The orientation of the genes within the cluster is not
phylogenetically conserved within Dikarya, suggesting that while
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Analysis of the HANT-AC Suggests Widespread Loss,
Convergence or a ‘‘Selfish Operon’’
The distribution of HANT-AC genes could suggest either
widespread loss outside Dikarya, heterokonts and plants or an
HGT origin of the fungal homologs. We can reject the null
hypotheses (Table 2) that chromalveolate and plant NRT2 form
a clade that excludes fungi (p = .038), that heterokont NAD(P)HNIR are monophyletic exclusive of fungi (p,.0001), and that an
organismal phylogeny based on five nuclear protein coding genes
(SI Fig. S2d), restricted to taxa bearing HANT-AC genes, is
consistent with the fungi+Phytophthora clade (p,.0001) suggested by
NAD(P)H-NIR analyses. Each of these results is consistent with an
HGT origin of these genes in fungi. The eukaryotic NAD(P)H-nir
could have been derived from a mitochondrial source, which
would require multiple losses in different lineages of eukaryotes, or
there could have been a more recent transfer from bacteria to
a lineage leading to the heterokonts. The absence of euknr from
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HGT of Nitrate Gene Cluster
Table 2. Alignments including diverse eukaryotic sequences.
..................................................................................................................................................
Dataset
Clade
Fungi
Heterokonts
Ascomycetes
T. reesei+
Basidiomycetes
Fungi+
Phytophthora
Fungi+
Heterokonts
Ustilago+
T. reesei
Chromalveolates+
Plantae
Reject p = .038
NRT2
1.0B, 100L, 100P1 1.0B, 100L, 80 P
NT2
NT
-
-
NT
EUKNR
1.0B, 100L, 99P
-
-
1.0B, 100L, 98P
-
-
1.0B, 100L, 100P
1.0B, 100L, 100P
NAD(P)H NIR
1.0B, 100L, 100P
Reject3 P,.0001
-
1.0B
1.0B, 97 L, 91P
84P
1.0B, 100L, 100P
NT
5nuc. prot. 4
1.0B, 100L, 100P
1.0B, 100L, 100P
NT
NT
Reject p,.0001
Reject p = .008
NT
1.0B, 100L, 100P
1
Support values (B-Bayesian posterior probabilities, L-maximum likelihood bootstraps, P-maximum parsimony bootstraps.
Clade not tested by this alignment.
Shimodaira-Hasegawa test significance level p,.05.
4
Five nuclear proteins (a-tubulin, b-tubulin, RPB1, RPB2 and EF1-a) were derived from genome projects as described in methods.
doi:10.1371/journal.pone.0001097.t002
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3
increase iron uptake [43]. In contrast, oomycete plant pathogens
could out-compete their host for nitrate in iron-limited conditions.
Alternatively, the absence of photosystems to efficiently replenish
reduced ferredoxin in fungi and oomycetes could have necessitated
convergent acquisition of a respiration-linked source of electrons
for nitrite reduction.
Co-regulation of the three genes in the fHANT-AC in H.
cylindrosporum was recently demonstrated [3]. This was earlier
demonstrated in the ascomycetous yeast, Pichia angusta [2]. The
intergenic region between NAD(P)H-nir and euknr in A. nidulans
serves as a bidirectional promoter for both genes [44,45]. These
findings collectively suggest that the fHANT-AC resembles
a eukaryotic operon. The ‘‘selfish operon’’ is a coordinately
regulated cluster of genes, which receive improved fitness because
they are more readily maintained when transferred between
organisms [8]. While not truly a ‘‘selfish’’ element due to its likely
positive influence on host fitness, evidence of HGT of the fHANTAC makes it a candidate for a ‘‘selfish’’ eukaryotic operon. The
current results suggest that acquisition of nutritional operons may
promote ecological shifts in fungi. Differential utilization of the
fHANT-AC in various strains of T. reesei could suggest this species
is currently undergoing such an evolutionary event.
other opisthokonts also suggests that this gene was secondarily
acquired in Dikarya, although phylogenetic analyses do not clearly
identify a source. The existence in Phytophthora of a cluster
(oHANT-AC) homologous to the fHANT-AC, some of whose
components appear to be vertically derived, is the strongest
evidence of a heterokont origin of the fungal genes, implying that
a cluster could have facilitated HGT. We present an hypothesis of
the origin of the fHANT-AC in SI Fig. S3, which parsimoniously
accounts for the observed distribution of HANT-AC genes in
eukaryotes. There we show a single origin of the cluster in
heterokonts from genes present in common ancestors of heterokonts and plants, followed by a transfer to the ancestor of Dikarya.
Recent studies have suggested closer genetic links between
oomycetes and fungi than would be predicted by organismal
phylogeny. One suggested that a number of shared EST sequences
relate to a mutual tendency toward pathogenicity [38]. It was not
clear if this is evidence of genomic convergence or genetic
exchange. Another study suggested that at least four genes have
been transferred from ascomycete fungi to oomycetes [39]. A
failure to detect homologous sequences in other heterokonts and
non-dikarya fungi in that study leaves the origin of these sequences
ambiguous, requiring the invocation of additional prokaryotic
donors. In our study, the presence of homologous genes in other
heterokonts and related lineages, but not in other fungi suggests
that the direction of the hypothetical transfer of the fHANT-AC
would be from a heterokont to a common ancestor of dikarya
fungi. A limited sample of non-dikarya fungi leaves an earlier
fungal origin also possible. In this case, the restriction of clustered
HANT-AC genes to oomycetes suggests that the heterokonts could
have produced the species that donated the fHANT-AC to dikarya
fungi. While the current study alone is not conclusive as to genetic
exchange between these groups, it contributes to a developing
hypothesis of an ancient, intimate association involving Dikarya
and heterokonts.
The alternate hypothesis is that the HANT-AC arose
convergently in fungi and oomycetes under similar environmental
pressures. For example, in aerobic soils, reduced iron for
denitrification is limited, leading to elevated nitrate [40]. Ironcontaining ferredoxin is required to donate electrons in plant
nitrite reduction, but this function can be performed by NAD(P)H
(derived from cellular respiration) in fungi and heterokonts; they
could be partly freed from iron limitations through this
mechanism. That certain diatoms are dominant in high-nitrate,
low-iron environments [41] seems to support this. This nutritional
mode could benefit photosynthetic partners in ectomycorrhizal
and lichen symbioses under aerobic conditions. In fact, ectomycorrhizal fungi both down-regulate plant nitrite reductase [42] and
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A key acquisition in colonization of dry land?
The Dikarya experienced a neoproterozoic diversification [46]
that was not paralleled in other fungal lineages, including some
that also associate with plants [17]. We speculate that the
horizontally acquired ability to derive nitrogen from oxidized
substrates, and to thus benefit photosynthetic hosts, may have been
a key innovation that allowed the Dikarya to diversify in terrestrial
habitats as free-living saprotrophs or mycorrhizal symbionts.
MATERIALS AND METHODS
Phylogenetic Analysis of Organisms Containing the
High Affinity Nitrate Assimilation Gene Cluster
(fHANT-AC) using rRNA and RPB2 gene sequences
We used a combination of tblastn [47] and searches for annotated
genes to obtain phylogenetically relevant sequences for species
noted with an * in SI Table S1. Substitutions were made for Pichia
angusta RNA polymerase II second largest subunit (RPB2,
AAS67502, Pichia guilliermondii) and Hebeloma cylindrosporum (partial
nuclear small ribosomal RNA subunit gene [nssu] AY745703 and
rpb2 DQ472718 from H. velutipes specimen PBM 2277) where
genome projects were not available. We generated a partial
nuclear large ribosomal RNA subunit gene (nlsu) sequence from
H.cylindrosporum strain CBS558.96 (EF219136) using the primers
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HGT of Nitrate Gene Cluster
LROR and LR5 (sequencing was performed on an ABI377
Automated DNA Sequencer using BigDye ver1.1; Applied
Biosystems, Foster City, CA, USA). We concatenated nlsu and
nssu nucleotide sequences and the inferred amino acid sequence of
RPB2 for all available fungal genomes. Sequences were aligned
with ClustalX [48], and adjusted manually. We created independent alignments (SI Table S2) for each gene by excluding
two of the three genes, and a combined analysis that included all
three genes. Ambiguously aligned characters were excluded from
further analysis. Each alignment was analyzed by (A) 1000
maximum parsimony bootstrap replicates with ten random
addition sequence replicates per bootstrap replicate, saving
multiple trees at each replicate in PAUP* 4.0b [49], (B) two
Bayesian analyses using mixed protein models for amino acids and
a GTR plus gamma model of evolution for nucleotides in MrBayes
ver. 3.2 [50,51,52]; we ran the Bayesian analyses for one million
generations, sampled trees every 100 generations and removed
trees sampled before likelihoods for two runs converged and
stabilized as the burnin, and (C) 100 Maximum Likelihood
Bootstrap replicates of RPB2 with mixed protein models and of
combined rDNA with mixed GTR models as implemented in
RaxML-VI-HPC ver. 2.2.3. We also performed (D) maximum
likelihood searches of a combined rDNA dataset and an RPB2
dataset in RaxML-VI-HPC ver. 2.2.3 [53] (mixed GTR and
protein models) for the optimal topologies and also for the best
topology in which T. reesei sequences were constrained to form
a clade with U. maydis. We then used a Shimodaira-Hasegawa test
[54], as implemented in TreePuzzle ver. 5.2 [55], to compare the
likelihoods of resulting topologies.
genome sequences, aligned with mafft ver. 5.861 [56] using the
default settings. Long ends of the alignment were trimmed and
a neighbor joining analysis was performed in PAUP* 4.0b. Thirtynine clades of NRT2 and eighty-three clades of NAD(P)H-NIR
were then analyzed as described above for the fHANT-AC genes,
except 500 maximum likelihood bootstraps were performed on
each gene separately. We confirmed that major clades from
previous NRT2 analyses [5] were represented. Twenty-nine
eukaryotic EUKNR sequences were also analyzed as described
for the other protein sequences. Constraint analyses as described
above forced the monophyly of heterokont NAD(P)H-NIR
sequences. We performed analyses as described for a combined
dataset of a-tubulin, b-tubulin, RNA polymerase II largest and
second largest subunits, and elongation factor 1-alpha limited to
taxa bearing homologs of genes in the fHANT-AC. We then
performed constraint analyses as described above, which forced
either fungi and Phytophthora or fungi and heterokonts to be
monophyletic.
Analysis of gene order and location in fungi
We used the browser at the T. reesei and Ustilago maydis genome
websites to catalog genes flanking the fHANT-AC in each species.
We cataloged genes of up to four inferred or hypothetical proteins
for each species. We used tBlastn [47] with a concatenated amino
acid sequence of the three proteins encoded by the fHANT-AC of
Coprinopsis cinerea in order of nitrate metabolism (NRT2-EUKNRNAD(P)H-NIR) to obtain the location and relative direction of
transcription of each gene in the eukaryotic genomes and bacterial
genomes shown in supporting materials, Table S1. We also
performed tBlastn using the individual genes, and used 45%
amino acid similarity to determine the absence of homologs for
each gene in eukaryotes and 40% amino acid similarity in
bacteria. This was repeated using the Arabidopsis thaliana ferredoxin-dependent nitrite reductase (NP_179164.1) as the query.
Analysis of the nitrate assimilation gene cluster in
fungi
We concatenated amino acid sequences inferred from three genes
in the fHANT-AC (nrt2, euknr and NAD(P)H-nir) of Hebeloma
cylindrosporum. We used tBlastn [47] with this sequence to search
genome projects shown in supporting materials (SI Table S1) for
homologous sequences. We obtained H. cylindrosporum and P.
angusta fHANT-AC sequences, and T. reesei EST sequences from
GenBank. The identity of each sequence was verified by a blastp
search of GenBank to determine the most similar sequences and
conservation of functional domain architecture. We assembled an
alignment consisting of concatenated amino acid sequences from
the fHANT-AC for all available fungal genomes (as of May, 2006,
shown with an * in SI Table S1) and also Phytophthora sojae and
Phytophthora ramorum genomes, which were included as the
outgroup. Alignments and analyses for independent genes and
combined sequences were conducted as described for amino acid
sequences in the phylogenetic analysis. We performed maximum
likelihood searches of the combined fHANT-AC dataset using
mixed protein models in RaxML-VI-HPC (see above) for the
optimal topology and also for the best topology in which
ascomycete sequences were constrained to be monophyletic. We
then compared the likelihoods of resulting topologies using
a Shimodaira-Hasegawa test as described above for RPB2.
SUPPORTING INFORMATION
Figure S1 Figure S1 depicts phylogenetic trees of fungal
sequences, rooted with an oomycete (heterokont) outgroup. Figure
S1a shows phylogenetic trees based on independent and combined
analyses of ribosomal RNA and RPB2 gene loci, which reflect the
organismal phylogeny. Figure S1b presents phylogenetic trees
based on independent and combined analyses of genes in the
fHANT-AC cluster. All analyses of the organismal phylogeny
group Trichoderma reesei with Gibberella zeae (ascomycetes), and
all analyses of genes in the fHANT-AC cluster group Trichoderma
reesei with Ustilago maydis (basidiomycetes).
Found at: doi:10.1371/journal.pone.0001097.s001 (0.49 MB
PDF)
Figure S2 Maximum likelihood trees showing relationships
among (a) NRT2, (b) EUKNR and (c) NAD(P)H NIR amino
acid sequences from eukaryotic and eubacteria genomes. (d)
Maximum likelihood trees showing relationships among concatenated nuclear gene products (a- and b-tubulin, rpb1, rpb2 and
ef1-a), from eukaryotic genomes. Branch lengths reflect number of
changes per site.
Found at: doi:10.1371/journal.pone.0001097.s002 (0.88 MB
PDF)
Analyses of components of the fHANT-AC in diverse
eukaryotes and prokaryotes
We conducted higher level analyses of NRT2, EUKNR and
NAD(P)H-NIR amino acid sequences retrieved from eukaryotic
genome projects along with bacterial sequences from GenBank.
For NRT2, approximately 200 bacterial sequences and for
NAD(P)H-NIR approximately 500 bacterial sequences were
initially obtained. These were combined with the eukaryotic
PLoS ONE | www.plosone.org
Figure S3 Evolution of the nitrate assimilation cluster in Fungi
and other eukaryotes. A. Evolution of the nitrate assimilation
cluster across the eukaryote phylogeny. B. Cluster evolution within
the fungi. C, D. Alternative reconstructions of cluster dissociation
and formation events in fungi. 1. Nrt2 and NAD(P)H nir were
6
October 2007 | Issue 10 | e1097
HGT of Nitrate Gene Cluster
1
Highest blastp sequence in GenBank, 88% similar to
AAQ21342, uncultured bacterium nitrite reductase. 2 Highest
tblastn hit in GenBank feature in acc. #CP000082 Psychrobacter
arcticus 273-4, nitrate transporter. 3 Tblastn 62%similar to
Arabidopsis thaliana GenBank acc. # NM_127123, nitrite
reductase, 42% similar to feature in NT_165926, Aspergillus
terreus sulfite reductase, beta subunit. 4 Tblastn 69% similar to
489AA of C.cinerea NAD(P)Hnir. 5 Locus ZP_00980374, nitrite/
sulfite reductase, Tblastn 44% similar to Arabidopsis thaliana
GenBank acc. #NP_179164.
Found at: doi:10.1371/journal.pone.0001097.s004 (0.11 MB
DOC)
acquired from bacteria, and euknr was derived from the sulfite
oxidase gene family in the nuclear genome[1]. 2. The common
ancestor of the Chromalveolates and Plantae retained nrt2,
NAD(P)H-nir, and euknr. 3. NAD(P)H-nir was lost in the lineage
leading to Plantae, after the divergence of Chromalveolates, and
Cp-nir was acquired during the primary endosymbiotic origin of
the chloroplast (not necessarily in that order). 4. Cp-nir was
horizontally transferred from the Rhodophytes to the Heterokonts
(Stramenopiles) during a secondary endosymbiotic origin of
chloroplasts [2]. Cp-nir was retained in the lineage leading to
the diatoms, but was lost in the lineage leading to the Oomycetes.
5. Nrt2, NAD(P)H-nir, and euknr were each lost in the lineage
leading to the Alveolates. 6. The nitrate assimilation cluster,
including nrt2, NAD(P)H-nir, and euknr, was formed in the
lineage leading to the Oomycetes. 7. The nitrate assimilation
cluster was horizontally transferred from the lineage leading to the
Oomycetes to the lineage leading to Dikarya. 8. The nitrate
assimilation cluster was horizontally transferred from the lineage
leading to Ustilago maydis (Basidiomycota) to the lineage leading
to Trichoderma reesei (Ascomycota). The Ascomycota-derived
components of the nitrate assimilation cluster were lost in the
lineage leading to Trichoderma reesei (this could have occurred
before or after the horizontal transfer event). 9. The nitrate
assimilation cluster became dissociated twice during the evolution
of Ascomycota. Aspergillus retains an ancestral clustered condition. Two component genes of the nitrate assimilation cluster (euknr and NAD(P)H-nir) were rejoined in the lineage leading to
Botryotinia fuckeliana. 10. Alternatively (B), the nitrate assimilation cluster became dissociated only once in the evolution of
Ascomycota, and was reconstituted in the lineage leading to
Aspergillus; euk-nr and NAD(P)H-nir were rejoined in the lineage
leading to Botryotinia fuckeliana. 11. Another possible scenario
(D) suggests that the nitrate assimilation cluster became dissociated
in four separate events in the Ascomycota, and that the clustered
condition in Aspergillus spp. and Botryotinia fuckeliana are both
plesiomorphic. Although this scenario involves the same number
of breakpoint/association events as those described in (B) and (C),
it does not require that clusters of genes be reconstituted after
having been disrupted. 12. All three component genes of the
nitrate assimilation cluster were lost in the lineages leading to
Cryptococcus neoformans (Basidiomycota) and Saccharomyces
cerevisiae (Ascomycota). Vertical order of branching points does
not reflect absolute timing of such events. 1. Stolz JF, Basu P
(2002) Evolution of nitrate reductase: molecular and structural
variations on a common function. Chembiochem 3: 198–206. 2.
Bhattacharya D, Yoon HS, Hackett JD (2004) Photosynthetic
eukaryotes unite: endosymbiosis connects the dots. Bioessays 26:
50–60.
Found at: doi:10.1371/journal.pone.0001097.s003 (0.93 MB
PDF)
Table S1
Table S2 For each dataset, two simultaneous Bayesian analyses
were run for one million generations, using MrBayes ver. 3.1.2
[1,2]. Amino acid data was analyzed under mixed protein models,
and nucleotide data under a GTR+Gamma model. Trees were
sampled every 100 generations. Trees sampled before likelihood
convergence and stabilization of independent chains (usually 500–
2000) were removed as the burnin. Each alignment in SI Table 2
was also analyzed by maximum parsimony bootstrapping and
maximum likelihood bootstrapping. In parsimony analyses,
performed in PAUP*4.0b [3], characters were equally weighted.
1000 bootstrap replicates were performed. Each bootstrap
replicate performed a heuristic search with 10 random addition
sequence replicates, with the multitrees option turned on, using the
TBR branch-swapping algorithm. Likelihood analyses were
performed under mixed protein models or mixed GTR (for
nucleotides). 100 bootstraps were performed on fungal alignments
(Fig. S1), and 500 on eukaryotic alignments (Fig. S2) in RaxMLVI-HPC ver. 2.2.3[4]. Literature Cited: 1. Huelsenbeck JP,
Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic
trees. Bioinformatics 17: 754–755. 2. Ronquist F, Huelsenbeck JP
(2003) MrBayes 3: Bayesian phylogenetic inference under mixed
models. Bioinformatics 19: 1572–1574. 3. Swofford DL (2003)
PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. 4. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and
mixed models. Bioinformatics 22: 2688–2690.
Found at: doi:10.1371/journal.pone.0001097.s005 (0.03 MB
DOC)
ACKNOWLEDGMENTS
We would like to thank Z. Wang and several anonymous reviewers for
helpful suggestions, and P. Matheny, M. Binder and C. Khatchikian for
editorial help.
Author Contributions
Conceived and designed the experiments: DH JS. Performed the
experiments: JS. Analyzed the data: JS. Wrote the paper: DH JS.
REFERENCES
5. Slot JC, Hallstrom K, Matheny PB, Hibbett DS (2007) Diversification of NRT2
and the Origin of Its Fungal Homolog. Mol Biol Evol.
6. Price MN, Huang KH, Arkin AP, Alm EJ (2005) Operon formation is driven by
co-regulation and not by horizontal gene transfer. Genome Res 15: 809–819.
7. Lawrence JG (1997) Selfish operons and speciation by gene transfer. Trends
Microbiol 5: 355–359.
8. Lawrence J (1999) Selfish operons: the evolutionary impact of gene clustering in
prokaryotes and eukaryotes. Curr Opin Genet Dev 9: 642–648.
9. Rosewich UL, Kistler HC (2000) Role of Horizontal Gene Transfer in the
Evolution of Fungi. Annu Rev Phytopathol 38: 325–363.
10. Walton JD (2000) Horizontal gene transfer and the evolution of secondary
metabolite gene clusters in fungi: an hypothesis. Fungal Genet Biol 30:
167–171.
1. Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Cole GE, et al. (1990) Isolation
and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation
in Aspergillus nidulans. Gene 90: 181–192.
2. Brito N, Avila J, Perez MD, Gonzalez C, Siverio JM (1996) The genes YNI1 and
YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast
Hansenula polymorpha, are clustered and co-ordinately regulated. Biochem J
317(Pt 1): 89–95.
3. Jargeat P, Rekangalt D, Verner MC, Gay G, Debaud JC, et al. (2003)
Characterisation and expression analysis of a nitrate transporter and nitrite
reductase genes, two members of a gene cluster for nitrate assimilation from the
symbiotic basidiomycete Hebeloma cylindrosporum. Curr Genet 43: 199–205.
4. Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi.
Microbiol Mol Biol Rev 61: 17–32.
PLoS ONE | www.plosone.org
7
October 2007 | Issue 10 | e1097
HGT of Nitrate Gene Cluster
11. Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG (2003) Phylogenomic
analysis of type I polyketide synthase genes in pathogenic and saprobic
ascomycetes. Proc Natl Acad Sci U S A 100: 15670–15675.
12. Han Y, Liu X, Benny U, Kistler HC, VanEtten HD (2001) Genes determining
pathogenicity to pea are clustered on a supernumerary chromosome in the
fungal plant pathogen Nectria haematococca. Plant J 25: 305–314.
13. Kavanaugh LA, Fraser JA, Dietrich FS (2006) Recent evolution of the human
pathogen Cryptococcus neoformans by intervarietal transfer of a 14-gene
fragment. Mol Biol Evol 23: 1879–1890.
14. He C, Rusu AG, Poplawski AM, Irwin JA, Manners JM (1998) Transfer of
a supernumerary chromosome between vegetatively incompatible biotypes of the
fungus Colletotrichum gloeosporioides. Genetics 150: 1459–1466.
15. Luque I, Flores E, Herrero A (1993) Nitrite reductase gene from Synechococcus
sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite
reductases. Plant Mol Biol 21: 1201–1205.
16. Stolz JF, Basu P (2002) Evolution of nitrate reductase: molecular and structural
variations on a common function. Chembiochem 3: 198–206.
17. James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, et al. (2006)
Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature
443: 818–822.
18. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, et al. (2003)
Transcriptional regulation of biomass-degrading enzymes in the filamentous
fungus Trichoderma reesei. J Biol Chem 278: 31988–31997.
19. Seiboth B, Hartl L, Salovuori N, Lanthaler K, Robson GD, et al. (2005) Role of
the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in
cellulase induction by lactose. Appl Environ Microbiol 71: 851–857.
20. Derelle E, Ferraz C, Rombauts S, Rouze P, Worden AZ, et al. (2006) Genome
analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many
unique features. Proc Natl Acad Sci U S A 103: 11647–11652.
21. Quesada A, Galvan A, Schnell RA, Lefebvre PA, Fernandez E (1993) Five
nitrate assimilation-related loci are clustered in Chlamydomonas reinhardtii.
Mol Gen Genet 240: 387–394.
22. Howlett BJ, Idnurm A, Heitman J (2007) Fungal pathogenesis: gene clusters
unveiled as secrets within the Ustilago maydis code. Curr Biol 17: R87–90.
23. Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, et al. (2002) Molecular
cloning and characterization of an ML-236B (compactin) biosynthetic gene
cluster in Penicillium citrinum. Mol Genet Genomics 267: 636–646.
24. Fleetwood DJ, Scott B, Lane GA, Tanaka A, Johnson RD (2007) A complex
ergovaline gene cluster in epichloe endophytes of grasses. Appl Environ
Microbiol 73: 2571–2579.
25. Zhang S, Monahan BJ, Tkacz JS, Scott B (2004) Indole-diterpene gene cluster
from Aspergillus flavus. Appl Environ Microbiol 70: 6875–6883.
26. Laich F, Fierro F, Martin JF (2002) Production of penicillin by fungi growing on
food products: identification of a complete penicillin gene cluster in Penicillium
griseofulvum and a truncated cluster in Penicillium verrucosum. Appl Environ
Microbiol 68: 1211–1219.
27. Carbone I, Jakobek JL, Ramirez-Prado JH, Horn BW (2007) Recombination,
balancing selection and adaptive evolution in the aflatoxin gene cluster of
Aspergillus parasiticus. Mol Ecol.
28. Wong S, Wolfe KH (2005) Birth of a metabolic gene cluster in yeast by adaptive
gene relocation. Nat Genet 37: 777–782.
29. Samuels GJ (2006) Trichoderma: Systematics, the Sexual State, and Ecology.
Phytopathology 96: 195–206.
30. Lieckfeldt E, Kullnig C, Samuels GJ, Kubicek CP (2000) Sexually competent,
sucrose- and nitrate-assimilating strains of Hypocrea jecorina (Trichoderma
reesei) from South American soils. . Mycologia 92: 374–380.
31. Ostrikova NA, Konovalov SA (1983) Influence of nitrogen sources on cellulase
biosynthesis by a mutant strain Trichoderma viride 44. Applied Biochemistry
and Microbiology 19: 392–395.
32. Saikia R, Azad P (2001) Effect of Certain Carbon and Nitrogen Sources on the
Antagonistic Activities of some Biocontrol Agents against Colletotrichum
falcatum Went. Environment and Ecology 19: 849–852.
33. Jayaraj J, Ramabadran R (1998) Effect of certain nitrogenous sources on the in
vitro growth, sporulation and production of antifungal substances by
Trichoderma harzianum. Journal of Mycology and Plant Pathology 28: 23–25.
34. Wakelin SA, Sivasithamparam K, Cole ALJ, Skipp RA (1999) Saprophytic
growth in soil of a strain of Trichoderma koningii. New Zealand Journal of
Agricultural Research 42: 337–345.
PLoS ONE | www.plosone.org
35. Celar F (2003) Competition for ammonium and nitrate forms of nitrogen
between some phytopathogenic and antagonistic soil fungi. Biological Control
28: 19–24.
36. Rocha-Ramirez V, Omero C, Chet I, Horwitz BA, Herrera-Estrella A (2002)
Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in
mycoparasitic coiling and conidiation. Eukaryot Cell 1: 594–605.
37. Sarrocco S, Mikkelsen L, Vergara M, Jensen DF, Lubeck M, et al. (2006)
Histopathological studies of sclerotia of phytopathogenic fungi parasitized by
a GFP transformed Trichoderma virens antagonistic strain. Mycol Res 110:
179–187.
38. Randall TA, Dwyer RA, Huitema E, Beyer K, Cvitanich C, et al. (2005) Largescale gene discovery in the oomycete Phytophthora infestans reveals likely
components of phytopathogenicity shared with true fungi. Mol Plant Microbe
Interact 18: 229–243.
39. Richards TA, Dacks JB, Jenkinson JM, Thornton CR, Talbot NJ (2006)
Evolution of filamentous plant pathogens: gene exchange across eukaryotic
kingdoms. Curr Biol 16: 1857–1864.
40. Stemmler SJ, Berthelin J (2003) Microbial activity as a major factor in the
mobilization of iron in the humid tropics. European Journal of Soil Science 54:
725–733.
41. Tsuda A, Takeda S, Saito H, Nishioka J, Nojiri Y, et al. (2003) A mesoscale iron
enrichment in the western subarctic Pacific induces a large centric diatom
bloom. Science 300: 958–961.
42. Bailly J, Debaud JC, Verner MC, Plassard C, Chalot M, et al. (2007) How does
a symbiotic fungus modulate expression of its host-plant nitrite reductase? New
Phytol 175: 155–165.
43. Leyval C, Reid CPP (1991) Utilization of microbial siderophores by mycorrhizal
and non-mycorrhizal pine roots. New Phytologist 119: 93–98.
44. Muro-Pastor MI, Gonzalez R, Strauss J, Narendja F, Scazzocchio C (1999) The
GATA factor AreA is essential for chromatin remodelling in a eukaryotic
bidirectional promoter. Embo J 18: 1584–1597.
45. Punt PJ, Strauss J, Smit R, Kinghorn JR, van den Hondel CA, et al. (1995) The
intergenic region between the divergently transcribed niiA and niaD genes of
Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally. Mol Cell Biol 15: 5688–5699.
46. Heckman DS, Geiser DM, Eidell BR, Stauffer RL, Kardos NL, et al. (2001)
Molecular evidence for the early colonization of land by fungi and plants.
Science 293: 1129–1133.
47. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25: 3389–3402.
48. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The
CLUSTAL_X windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
49. Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and
Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
50. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics 17: 754–755.
51. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19: 1572–1574.
52. Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F (2004) Parallel Metropolis
coupled Markov chain Monte Carlo for Bayesian phylogenetic inference.
Bioinformatics 20: 407–415.
53. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic
analyses with thousands of taxa and mixed models. Bioinformatics 22:
2688–2690.
54. Shimodaira H, Hasegawa M (1999) Multiple Comparisons of Log-Likelihoods
with Applications to Phylogenetic Inference. Mol Biol Evol 16: 1114–1116.
55. Schmidt HA, Strimmer K, Vingron M, von Haeseler A (2002) TREE-PUZZLE:
maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502–504.
56. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for
rapid multiple sequence alignment based on fast Fourier transform. Nucleic
Acids Res 30: 3059–3066.
57. Baldauf SL (2003) The deep roots of eukaryotes. Science 300: 1703–1706.
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Figure S1a
Phylogenetic analyses of organisms (BPP,MLB,MPB)
Combined
nlsu, nssu, RPB2
MLB for rDNA only
Trichoderma reesei
1.0,100,100
Gibberella zeae
1.0,100,100
1.0,100,100
1.0,96,89
nssu
Neurospora crassa
1.0,100,100
1.0,100,100
AY752972 Hebeloma velutipes
1.0,92,89
Phanerochaete chrysosporium
Magnaporthe grisea
Ustilago maydis
Botryotinia fuckeliana
Phaeosphaera nodorum
Sclerotinia sclerotiorum
1.0,92,72
1.0,100,100
Phaeosphaera nodorum
-,62,-
1.0,81,100-
.95,61,-
Aspergillus nidulans
Magnaporthe grisea
1.0/100
1.0,100,92
Pichia sp.
.84,73,-
.95,91,83
1.0,99,98
Phanerochaete
chrusosporium
Laccaria bicolor
1.0,100,100
1.0,100,66
1.0,-,68
1.0,100,100
nlsu
1.0,99,100
1.0, 98,100
1.0,96,77
Pichia stipitis
Phytophthora sojae
1.0,100.100
Phytophthora ramorum
Neurospora crassa
Chaetomium globosum
1.0,100,100
RPB2
Neurospora crassa
1.0,100,97
1.0,92,88
1.0,76,55
Phaeosphaera nodorum
1.0,100,99
Aspergillus fumigatus
Aspergillus nidulans
1.0,96,95
Aspergillus fumigatus
1.0,100,100
Phaeosphaera nodorum
1.0,94,98
1.0,100,100
Botryotinia fuckeliana
Sclerotinia sclerotiorum
.93,51,-
1.0,100,99
Chaetomium globosum
Magnaporthe grisea
1.0,100,100
1.0,100,87
Aspergillus nidulans
PAU75524 Pichia angusta
AAS67502 Pichia guilliermondii
Laccaria bicolor
Ustilago maydis
AF041494 Coprinopsis cinerea
1.0,97,73
EF219136 Hebeloma
cylindrosporum
1.0,99,99
Phytophthora sojae
Phanerochaete chrysosporium
DQ472718Hebeloma velutipes
.81,73,-
Ustilago maydis
Phytophthora ramorum
1.0,100,100
Laccaria bicolor
Phanerochaete chrysosporium
1.0,100,100
1.0,97,100
Botryotinia fuckeliana
Sclerotinia sclerotiorum
Trichoderma reesei
Gibberella zeae
1.0,77,59
Trichoderma reesei
Gibberella zeae
1.0, 100,100
Aspergillus nidulans
Hebeloma sp.
AF362554 Magnaporthe grisea
1.0,100,100
Neurospora crassa
Aspergillus fumigatus
Phytophthora sojae
1.0,100,100
Chaetomium globosum
Coprinopsis cinerea
Phytophthora ramorum
1.0,100,100
Trichoderma reesei
Gibberella zeae
Ustilago maydis
1.0,63,77
Botryotinia fuckeliana
Sclerotinia sclerotiorum
Aspergillus fumigatus
1.0,100,100
Coprinopsis cinerea
Laccaria bicolor
1.0,99,100
Chaetomium globosum
1.0,96,97
1.0/100
.91,52,64
1.0,100,100
Coprinopsis cinerea
Phytophthora ramorum
Phytophthora sojae
Figure S1b.
Combined
NRT2, EUKNR,
NAD(P)H NIR
HANT-AC analyses (BPP,MLB,MPB)
1.0,100,100
Hebeloma cylindrosporum
NRT2
1.0,100,100
Laccaria bicolor
1.0,100,100
1.0,93,91
-,-,58
Coprinopsis cinerea
Ustilago maydis
1.0,100,100
Phanerochaete chrysosporium
CAA11229 Pichia angusta
.82,-,55
Trichoderma reesii
Phaeosphaera nodorum
Pichia angusta
1.0,63,78
1.0,100,100
Aspergillus fumigatus
1.0,100,100
1.0/90
Botryotinia fuckeliana
1.0,-,80
Sclerotinia sclerotiorum
1.0,100,100
1.0/62
Magnaporthe grisea
1.0/61
Ustilago maydis
1.0,100,99
Trichoderma reesii
phytophthora ramorum
1.0,100,100
phytophthora sojae
.99,84,80
EUKNR
CAB60010 Hebeloma
cylindrosporum
Laccaria bicolor
1.0,100,100
1.0,73,-
phytophthora sojae
1.0,100,99
NAD(P)H NIR
1.0,100,100
1.0,98,98
Coprinopsis cinerea
1.0,100,100
Ustilago maydis
1.0,100,100
Trichoderma reesii
1.0,100,99
1.0,90,81
1.0,100,100
.99,60,-
Aspergillus nidulans
1.0,100,100
Botryotinia fuckeliana
1.0,100,100
1.0,100,100
1.0,-,-
Magnaporthe grisea
1.0,59,-
1.0,95,74
Neurospora crassa
phytophthora sojae
Botryotinia fuckeliana
Magnaporthe grisea
1.0,79,-
Chaetomium globosum
phytophthora ramorum
Aspergillus fumigatus
Sclerotinia sclerotiorum
Gibberella zeae
1.0,100,100
Phaeosphaera nodorum
.89,51,-
.98,-,-
Sclerotinia sclerotiorum
1.0,100,72
1.0,100,100
CAA11230 Pichia angusta
.74,-,-
Aspergillus nidulans
1.0,80,74
1.0,84,73
Trichoderma reesii
Phaeosphaera nodorum
Aspergillus fumigatus
Laccaria bicolor
Phanerochaete chrysosporium
Ustilago maydis
CAA11232 Pichia angusta
CAB60008 Hebeloma
cylindrosporum
Coprinopsis cinerea
Phanerochaete chrysosporium
1.0,99,98
Magnaporthe grisea
Neurospora crassa
Neurospora crassa
phytophthora ramorum
Sclerotinia sclerotiorum
Gibberella zeae
1.0,97,78
Gibberella zeae
1.0,100,100
Botryotinia fuckeliana
Chaetomium globosum
.99,-,77
Chaetomium globosum
1.0/69
Aspergillus nidulans
Aspergillus fumigatus
1.0,69,63
Aspergillus nidulans
1.0,100,100
1.0,83,96
1.0,85,59
1.0,100,100
1.0,70,76
Phaeosphaera nodorum
1.0,100,99
Laccaria bicolor
Coprinopsis cinerea
Phanerochaete chrysosporium
1.0,96,91
CAB60009 Hebeloma
cylindrosporum
1.0,64,57
Gibberella zeae
Chaetomium globosum
Neurospora crassa
1.0,100,100
phytophthora ramorum
phytophthora sojae
10
HT
C
HT
HT
7
Basidiomycota
8
9
Cluster
formation
(partial)
12
En
2
HT
Cluster
dissociation
9
Ascomycota
HT
11
HT
horizontal transfer
En
endosymbiosis
Cp
chloroplast
12
Cluster
dissociation
9
Ascomycota
D
Pichia angusta
6
3
Saccharomyces cerevisiae
1
4
Aspergillus fumigatus
En
Aspergillus nidulans
7
Cluster
formation
Phaeospora nodorum
B
Viridiplantae
Rhodophytes
Alveolates
Diatoms
Oomycetes
Heterokonts
Sclerotinia sclerotiorum
HT
Botryotinia fuckeliana
9
Neurospora crassa
HT
Ascomycota
Dikarya
Chaetomium gibbosum
Basidiomycota
Glomeromycota
Zygomycota
Chytridiomycota
Microsporidia
Mycetozoa
Animalia
Fungi
Magnaporthe grisea
Gibberella zeae
Trichoderma reesei
Ustilago maydis
Phanerochaete chrysosporium
Laccaria bicolor
Coprinopsis cinerea
Hebeloma cylindrosporum
Cryptococcus neoformans
A
Figure S3
Opisthokonts
Chromalveolates
Plantae
5
Cp
nrt2
euk-nr
NAD(P)H nir
Cp nir
nuclear phylogeny
nitrate assimilation cluster
cluster formation/dissociation
gene loss
cluster loss
Ascomycota
Figure S2a
NRT2 (BPP,MLB,MPB)
Thalassiosira pseudonana2
1.0,100,100
Thalassiosira pseudonana
Phaeodactylum tricornutum
1.0,100,100
Phaeodactylum tricornutum2
1.0,-,63
Thalassiosira pseudonana3
1.0, -,74
Phaeodactylum tricornutum5
1.0,100,80
Phaeodactylum tricornutum4
Phaeodactylum tricornutum3
Phaeodactylum tricornutum6
Sporobolomyces roseus
1.0,82,Hebeloma cylindrosporum
1.0,70,64
Ustilago maydis
1.0,100,100
Fusarium graminearum
1.0,100,100
Aspergillus nidulans
-,60,Pichia angusta
Phytophthora sojae
1.0,75,Phytophthora ramorum
1.0,100,100
Phytophthora sojae3
1.0,93,89
Phytophthora sojae2
1.0,74,-
Cyanidioschyzon merolae
1.0,99,87
Galdieria sulphuraria
1.0,99,92
Brassica napus
1.0,100,100
Oryza sativa
Ostreococcus lucimarinus
1.0,100,100
Chlamydomonas reinhardtii
1.0,100,100
ABO39202 Ralstonia solanacearum
1.0,79,64
YP_111252 Burkholderia pseudomallei
1.0,100,100
YP_046561 Acinetobacter sp.
ZP_01056324 Roseobacter sp.
ZP_01718566 Algoriphagus sp.
ZP_01736732 Marinobacter sp.
1.0,100,100
YP_001171434 Pseudomonas stutzeri
-,63,Nematostella vectensis
1.0,85,94
NP_241478 Bacillus halodurans
1.0,98,90
-,57,60
YP_435142 Hahella chejuensis
1.0,92,91
ZP_01220155 Photobacterium profundum
ABD83940 Arthrospira maxima
ZP_00107423 Nostoc punctiforme
YP_001226203 Synechococcus sp.
0.1
Figure S2b
EUKNR (BPP,MLB,MPB)
Stagonospora nodorum
1.0,100,100
Aspergillus fumigatus
.93,89,74
.93,81,53
Aspergillus nidulans
Botryotinia fuckeliana
1.0,100,100
Sclerotinia sclerotiorum
1.0,100,100
Neurospora crassa
Chaetomium globosum
1.0,100,99
Magnaporthe grisea
-,-,66
Fusarium graminearum
Pichia angusta
Hebeloma cylindrosporum
1.0,98,56
Laccaria bicolor
1.0,100,100
1.0,100,100
Coprinopsis cinerea
-,-,63
Sporobolomyces roseus
1.0,82,-
Phanerochaete chrysosporium
1.0,100,98
Ustilago maydis
1.0,100,100
Trichoderma reesei
1.0,74,-
.93,55,-
Phytophthora sojae
1.0,100,100
Phytophthora ramorum
Chlamydomonas reinhardtii
.98,59,-
Populus trichocarpa2
1.0,100,100
Populus trichocarpa1
1.0,100,100
Brassica napus
1.0,100,100
Oryza sativa
Thalassiosira pseudonana
1.0,100,100
Phaeodactylum tricornutum
Ostreococcus tauri
1.0,100,100
Ostreococcus lucimarinus
Cyanidioschyzon merolae
0.1
Figure S2c
NAD(P)H NIR (BPP<MLB<MPB)
1.0,99,99
0.1
1.0,100,100 NP_800565 Vibrio parahaemolyticus
1.0,100,100 ZP_01259818 Vibrio alginolyticus
ZP_01233250 Vibrio angustum
.99,59,YP_957466 Marinobacter aquaeolei
1.0,70,1.0,93,87
NP_743862 Pseudomonas putida
ZP_01612971 Alteromonadales bacterium
1.0,95,73
ZP_01110083 Alteromonas macleodii
YP_130707 Photobacterium profundum
1.0,100,100 NP_800497 Vibrio parahaemolyticus
1.0,93,79 ZP_01259899 Vibrio alginolyticus
.98,64,YP_129641 Photobacterium profundum
1.0,100,100
YP_001140138 Aeromonas salmonicida
1.0,100,100YP_072221
Yersinia pseudotuberculosis
.97,56,ZP_00830568 Yersinia frederiksenii
1.0,100,100
NP_756005 Escherichia coli
YP_391426 Thiomicrospira crunogena
1.0,99,97
1.0,100,100 ZP_01561020 Burkholderia cenocepacia
Burkholderia cenocepacia
1.0,100,100 ZP_00979466
EAY70617 Burkholderia dolosa
1.0,97,90
YP_775824 Burkholderia cepacia
1.0,62,YP_111251 Burkholderia pseudomallei
1.0,-,ZP_01509563 Burkholderia phytofirmans
1.0,81,78
YP_728936 Ralstonia eutropha
1.0,100,100
ZP_00945794 Ralstonia solanacearum
1.0,99,91
1.0,59,ZP_01581018 Delftia acidovorans
YP_692806 Alcanivorax borkumensis
1.0,100,100
YP_884025 Mycobacterium avium
NP_334670 Mycobacterium tuberculosis
1.0,100,-,61,YP_706301 Rhodococcus sp.
1.0,90,YP_001105998 Saccharopolyspora erythraea
1.0,99,92
ZP_00993512 Janibacter sp.
YP_873502 Acidothermus cellulolyticus
NP_826838 Streptomyces avermitilis
100,94,YP_593900 Deinococcusgeothermalis
1.0,100,100
Fusarium verticilliodes
1.0,100,100
Fusarium graminearum
1.0,58,Nectria haemolytica
1.0,53,Chaetomium globosum
1.0,95,93
Magnaporthe grisea
1.0,92,87
Neurospora crassa
1.0,62,Botryotinia fuckeliana
1.0,100,100
1.0,100,100
Sclerotinia sclerotiorum
Stagonospora nodorum
1.0,100,100
Mycosphaerella graminicola
Aspergillus fumigatus
1.0,97,76
Aspergillus terreus
Aspergillus niger
1.0,100,100
Aspergillus nidulans
1.0,100,95
Laccaria bicolor
1.0,100,100
Hebeloma cylindrosporum
1.0,64,Coprinopsis cinerea
1.0,100,96
1.0,100,100
Phanerochaete chrysosporium
Sporobolomyces roseus
1.0,100,100
Ustilago maydis
1.0,97,91
Trichoderma reesei
Pichia angusta
-,60,Phytophthora ramorum
1.0,100,100
Phytophthora sojae
-,-,84
Phytophthora infestans
1.0,100,100
Phaeodactylum tricornutum
Thalassiosira pseudonana
1.0,98,99
ZP_00631188 Paracoccus denitrificans
1.0,100,100
AAQ18184
Rhodobacter capsulatus
.78,60,73
ZP_01586147 Dinoroseobacter shibae
1.0,100,100
ZP_01227782 Aurantimonas sp.
1.0,100,100
ZP_01415115 Sinorhizobium medicae
1.0,98,95
YP_767592 Rhizobium leguminosarum
1.0,87,74
1.0,87,85
YP_317337 Nitrobacter winogradskyi
1.0,73,NP_419432 Caulobacter crescentus
1.0,100,92
YP_616313 Sphingopyxis alaskensis
AAM73544 Azospirillum brasilense
YP_578189 Nitrobacter hamburgensis
1.0,100,76
1.0,100,98
ZP_00976113 Pseudomonas aeruginosa
1.0,92,YP_363824 Xanthomonas campestris
1.0,100,100
YP_001155780 Polynucleobacter sp.
YP_981642 Polaromonas naphthalenivorans
1.0,99,92
YP_001098778 Herminiimonas arsenicoxydans
1.0,64,82
ZP_01125768 Nitrococcus mobilis
1.0,100,100
ZP_01179445 Bacillus cereus
1.0,100,97
YP_036299 Bacillus thuringiensis
1.0,99,96
YP_189546 Staphylococcus epidermidis
YP_077721 Bacillus licheniformis
YP_821483 Solibacter usitatus
Figure S2d
Alpha-tubulin, beta-tubulin, RPB1, RPB2, EF1alpha AA (BPP,MLB,MPB)
Phytophthora sojae
1.0, 100, 100
Phytophthora ramorum
1.0, 100,100
Thalassiosira pseudonana
1.0, 100,100
Phaeodactylum tricornutum
1.0, 100,100
Chlamydomonas reinhardtii
1.0, 100,100
Ostreococcus lucimarinus
1.0, 100,100
Oryza sativa
1.0, 98,99
Brassica napus
Aspergillus sp.
1.0, 100,100
Fusarium graminarum
1.0,85,60
Pichia sp
1.0, 100,100
Hebeloma sp.
1.0,94,80
Sporobolomyces roseus
Ustilago maydis
Cyanidioschyzonmerolae
500 changes
Table S1: Distribution of high affinity nitrate assimilation genes.
Genome project
URL
ecology
genes
Galdieria sulphuraria
http://genomics.msu.edu/galdieria/
autotroph
nrt2 euknr cpnir
Cyanidioschyzon merolae
Viridiplantae
http://merolae.biol.s.u-tokyo.ac.jp/
autotroph
nrt2 euknr
Arabidopsis thaliana
http://www.tigr.org/tdb/e2k1/ath1/
autotroph
nrt2 euknr cpnir
Zea mays
http://compbio.dfci.harvard.edu/tgi/cgi- autotroph
bin/tgi/Blast/index.cgi
nrt2 euknr cpnir
Medicago trunculata
http://compbio.dfci.harvard.edu/tgi/cgi- autotroph
bin/tgi/Blast/index.cgi
nrt2 euknr cpnir
Triticum aestivium
http://www.tigr.org/tdb/e2k1/tae1/
autotroph
nrt2 euknr cpnir
Oryza sativa
http://www.tigr.org/tdb/e2k1/tae1/
autotroph
nrt2 euknr cpnir
http://genome.jgipsf.org/Poptr1/Poptr1.home.html
Chlamydomonas reinhadtii
http://genome.jgipsf.org/Chlre3/Chlre3.home.html
Ostreococcus lucimarinus v2.0 http://genome.jgipsf.org/Ost9901_3/Ost9901_3.home.ht
ml
Ostreococcus tauri v2.0
http://genome.jgipsf.org/Ostta4/Ostta4.home.html
Heterokonts
autotroph
ectomycorrhizal
autotroph
nrt2 euknr NADPHnir cpnir
autotroph
nrt2 euknr cpnir
autotroph
nrt2 euknr cpnir
*Phytophthora sojae v1.1
http://genome.jgipsf.org/sojae1/sojae1.home.html
*Phytophthora ramorum v1.1 http://genome.jgipsf.org/ramorum1/ramorum1.home.htm
l
Thalassiosira pseudonana v3.0 http://genome.jgipsf.org/thaps1/thaps1.home.html
Phaeodactylum tricornutum
http://genome.jgi-psf.org/cgiv2.0
bin/runAlignment?db=Phatr2&advance
d=1
Metazoa
plant pathogen
4nrt2 euknr NADPHnir
plant pathogen
3nrt2 euknr NADPHnir
autotroph
nrt2 euknr NADPHnir cpnir
autotroph
nrt2 euknr NADPHnir cpnir
Homo sapiens
heterotroph
Rhodophyta
Populus trichocarpa v1.1
Nematostella vectensis
Xenopus tropicalis v4.1
Ciona intestinalis v2.0
Fugu rubripes v4.0
http://www.ensembl.org/Homo_sapiens
/index.html
http://genome.jgipsf.org/Nemve1/Nemve1.home.html
http://genome.jgipsf.org/Xentr4/Xentr4.home.html
http://genome.jgipsf.org/Cioin2/Cioin2.home.html
http://genome.jgipsf.org/Takru4/Takru4.home.html
heterotroph
heterotroph
animal parasite
heterotroph
1
nrt2 euknr cpnir
nrt2
2
Microsporidia
Encephalitozoon cuniculi
Fungi
(sample of projects searched)
Batrachochytrium_dendrobati
dis
Cryptococcus neoformans
Phycomyces blakesleeanus
Rhizopus oryzae
Glomus (EST)
Candida lusitanian
Coccidioides immitis
Pichia stipitis v2.0
Aspergillus niger v1.0
*Laccaria bicolor v1.0
Nectria haematococca v1.0
*Phanerochaete
chrysosporium v2.0
*Trichoderma reesei v1.0
Coprinopsis cinerea
Ustilago maydis
Sporobolomyces roseus
Gibberella zeae
Magnaporthe grisea
Chaetomium globosum
Neurospora crassa
Botryotinia fuckeliana
Sclerotinia sclerotiorum
Phaeosphaera nodorum
Aspergillus nidulans
Aspergillus fumigatus
http://www.cns.fr/externe/English/Proje intracellular
ts/Projet_AD/AD.html
parasite
http://www.broad.mit.edu/annotation/ge
nome/batrachochytrium_dendrobatidis
http://www.broad.mit.edu/annotation/ge
nome/cryptococcus_neoformans/Home.
html
http://genome.jgipsf.org/Phybl1/Phybl1.home.html
http://www.broad.mit.edu/annotation/ge
nome/rhizopus_oryzae/Home.html
http://darwin.nmsu.edu/~fungi/
animal pathogen
http://www.broad.mit.edu/annotation/ge
nome/candida_lusitaniae/Home.html
http://www.broad.mit.edu/annotation/ge
nome/coccidioides_immitis/Home.html
http://genome.jgipsf.org/Picst3/Picst3.home.html
http://genome.jgipsf.org/Aspni1/Aspni1.home.html
http://genome.jgipsf.org/Lacbi1/Lacbi1.home.html
http://genome.jgipsf.org/Necha1/Necha1.home.html
http://genome.jgipsf.org/Phchr1/Phchr1.home.html
http://gsphere.lanl.gov/trire1/trire1.hom
e.html
animal symbiont
animal symbiont
heterotroph
saprotroph
heterotroph
saprotroph
endomycorrhizal
animal symbiont
methylotrophic
heterotroph
heterotroph
saprotroph
ectomycorrhizal
nrt2 euknr NADPHnir
plant pathogen
nrt2 euknr NADPHnir
heterotroph
saprotroph
heterotroph
saprotroph
(fungal/ plant
parasite?)
http://www.broad.mit.edu/annotation/ge heterotroph
nome/coprinus_cinereus/Home.html
saprotroph
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/ustilago_maydis/Home.html
http://genome.jgi-psf.org/cgiplant pathogen
bin/runAlignment?db=Sporo1&advance
d=1
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/fusarium_graminearum/Home.ht
ml
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/magnaporthe_grisea/Home.html
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/chaetomium_globosum/Home.ht
ml
http://www.broad.mit.edu/annotation/ge heterotroph
nome/neurospora/Home.html
saprotroph
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/botrytis_cinerea/Home.html
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/sclerotinia_sclerotiorum/Home.ht
ml
http://www.broad.mit.edu/annotation/ge plant pathogen
nome/stagonospora_nodorum/Home.ht
ml
http://www.broad.mit.edu/annotation/ge heterotroph
nome/aspergillus_nidulans/Home.html saprotroph
nrt2 euknr NADPHnir
http://www.sanger.ac.uk/Projects/A_fu
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
nrt2 euknr NADPHnir
animal pathogen nrt2 euknr NADPHnir
migatus/
Alveolata
Tetrahymena thermophila
http://www.tigr.org/tdb/e2k1/ttg/
heterotroph
Plasmodium falciparum
http://www.tigr.org/tdb/e2k1/pfa1/
animal parasite
http://genome.jgipsf.org/Monbr1/Monbr1.home.html
heterotroph
Choanoflagellate
Monosiga brevicollis
Amoeboflagellate
Naegleria gruberi v1.0
http://genome.jgipsf.org/Naegr1/Naegr1.home.html
Bacteria
Synechococcus WH8102
Burkholderia cenocepacia
pc184
http://genome.jgiphototroph
psf.org/finished_microbes/synw8/synw
8.home.html
http://www.broad.mit.edu/annotation/ge endobacterium
nome/burkholderia_cenocepacia/Home.
html
3
Nrt2 cpnir
4
5
Nrt2 NAD(P)Hnir cpnir
Table S2: Description of alignments analyzed in this study.
Figure
Alignment
Included
characters (#)
S1a
Combined nssu, nlsu,
4301, 3637 (rDNA
RPB2
likelihood)
S1a
nssu
1824
S1a
nlsu
1813
S1a
RPB2
1085
S1b
fHANT-AC combined
2381
S1b
NRT2 (fungal)
468
S1b
EUKNR(fungal)
851
S1b
NAD(P)H NIR (fungal)
1062
S2a
NRT2 (eukaryote)
424
S2b
EUKNR (eukaryote)
785
S2c
NAD(P)H NIR (eukaryote) 1082
Parsimony informative
characters (#)
1283
374
403
534
1651
386
597
668
325
547
855