MSPPKSSRGAPKFKWSHLWEPAIVNPVNLKSYTIPIFNLGDPYARAFHLSWLGFFVAFLSWFAFPPLIPDAIKSDLHLSAAQVANSNIIALCATFVVRVGVGPLVDQYGPRKVMAYLLILGAIPSGLAGTARSAEGLYVLRFFIGILGATFVPCQAWTSAFFDKNCVGTANALVGGWGNMGGGATFAIMTSLFQSLTQTYGLSTHVAWRAAFAIVPAPILLFVAVLTFIFGQDHPAGKWSERHTLPVAALAVQQ

1
Molecular evolution of nrt2, a nitrate
transporter gene, with an emphasis on Hebeloma
phylogeny
Jason C. Slot and David S. Hibbett Department of
Biology, Clark University, Worcester, MA
01610 Jslot_at_clarku.edu, dhibbett_at_clarku.edu
Presented at the Joint Meeting of the Mycological
Societies of America and Japan, August, 2005 in
Hilo, HI, USA
Clockwise from upper left Gymnopilus sp.,
Hebeloma velutipes, Alnicola escharoides,
Hebeloma sp. Courtesy of P.B. Matheny
Two copies of nrt2 Above is a schematic
representation of the NRT2 protein relative to
the cell membrane. The nitrate transporter bears
the signature 12-transmembrane helices and
certain highly conserved sequences characteristic
of members of the Major Facilitator Superfamily.
The arginine (R) residue in helix 2 (from the
left) is highly conserved across several kingdoms
of nitrate transporters. Following mutation
analysis in Aspergillus nidulans NRTA, it has
been suggested that this region most likely forms
part of the substrate binding site. The high
level of divergence around this residue between 2
copies of the protein within an individual is
worthy of note, suggesting that function might
differ between the copies.
Introduction The most ephemeral source of
nitrogen in the soil, nitrate, is transported
into cells by nitrate transporters of
differential affinities for nitrate. A nitrate
assimilation gene cluster containing a high
affinity nitrate transporter gene, nrt2 along
with nitrate and nitrite reductase genes was
recently characterized in Hebeloma
cylindrosporum. NO3- (environmental) --gt NO3-
(cellular) --gt NO2- --gt NH4 --gtOrganic
Nitrogen NRT2
NAR1 NIR1 Hebeloma cylindrosporum is an
ectomycorrhial basidiomycete belonging to a clade
that is characterized by a diversity of soil
nitrogen-status preferences. For example, H.
radicosum forms a tripartite symbiosis involving
mole latrines and H. syrjense is appropriately
called corpe-finder, while H. cylindrosporum is
found in sand and many other Hebeloma prefer
nitrogen-poor acidic soil. In addition to being
chemoecologically diverse, Hebeloma has been
problematic for traditional and molecular
systematists alike. Hebeloma, furthermore appears
to be an example of a recent acquisition of
mycorrhization, or a symbiotic stronghold within
a clade of many mycorrhiza-losers (such as
Agrocybe, Hypholoma and Gymnopilus). This study
explores the evolution of nrt2 sequences relative
to that of the Internal Transcribed Spacer, ITS,
sequences of nuclear rDNA. Patterns in
nitrate-assimilation gene evolution could improve
the understanding of the selective pressure
exerted by soil nitrogen availability, and the
relative significance of nitrate in the evolution
of Ectomycorrhizal relationships.
Above Tanglegram assembled in Gene Tree. On
the left is a 50 majority rule concensus of 2
most parsimonious its trees found by equally
weighted branch and bound. Support values in red
are posterior probabilities of Bayesian MCMC with
two hot and two cold chains for 2million
generations. Support values in black are derived
from 5000 neighbor joining bootstrap replicates
adjusted with kimura-2 parameter of the complete
its dataset shown below and left. On the right
is a neighbor joining boostrap phylogram of
inferred NRT2 amino acid sequences with 5000
replicates. Selected bootstrap values above 70
are indicated.
Methods DNA was extracted from either dried
fruit bodies or rapidly growing mycelium
according to a variation of the method
of____________. Sequences were PCR amplified
under a variety of conditions using 3-7 position
degenerate primers designed from a consensus of
H. cylindrosporum and P. chrysosporium nrt2
sequences. ITS products were generated with
ITS1F and ITS4 by Vilgalys et al. and include
ITS1, 5.8S and ITS2. Nrt2 sequences range from
1.2-2kb. PCR products were cloned with either
TA or TOPO TA"?PCR 2.1 vector, selected with Xgal
on LB Kanamycin agar. 10-20 clones were screened
with PCR and gel electrophoresis, then sequenced
with m13 primers and 2-6 internal primers using
BigDye ver1.1. Sequences were obtained on ABI
377 and ABI3700? Data was compiled using
Sequencher"e Inferred intron sequences were not
alignable and were removed prior to analysis.
Exon sequences were aligned manually in
MacCladeV. The large ITS alignment was first
done by D.K. Aanen, then modified by P.B. Matheny
before the sequences from this study were
included. Phylogenetic analyses were done in
PAUP IV and Mr.Bayes 3.3. Transmembrane Helical
domains were confirmed by the HMMTOP web-based
algorithm.
Discussions Phylogenetic Patterns Assuming the
tree topologies generated in these analyses are
an accurate estimate of the true gene
phylogenies, there are three main phenomena that
require explanation. 1.The most recent common
ancestor of the two nrt2 copies precedes the
divergence of the Hebeloma in this dataset, and
copy 2 is sister to Hebeloma. ?This suggests that
there was a duplication of the gene very early
in, or preceding the emergence of Hebeloma, but
after the divergence of Hebeloma and Gymnopilus.
? The lack of second copies of nrt2 in other
Hebeloma might suggest the loss or failure to
detect many sequences, but there are potentially
more plausible explanations based on the tree
topologies recovered here. 2. Copy 1 of nrt2 in
H. helodes is more closely allied with
H.velutipes and the sweet-smelling Hebelomas than
with copy 2 from the same dikaryon. ?This is in
direct conflict with the highly supported
monophyletic nature of the helodes/crustuliniforme
complex relative to the remaining Hebeloma in
the ITS phylogeny. If we were to accept that the
position of copy1 was strictly due to duplication
and loss, then we would be forced to reconstruct
a minimum of 24 losses based on these topologies
(as calculated by GeneTree). ? If we broaden the
possibilities of molecular evolution, however, we
might hypothesize hybridization or lateral gene
transfer. In this case, we could expect the
donor of copy1 to be closely related to H.
sacchariolens or H. velutipes. A possible case
of hybridization in the H.velutipes clade has, in
fact, been demonstrated (Aanen, 2001). ? Problems
with these hypotheses include the likelihood of
these events being of some debate, and also the
lack of additional corroborative molecular
evidence such as additional ITS sequences in H.
helodes. These are not necessarily fatal flaws,
however, as concerted evolution in ITS or uneven
crossing over during an ephemeral hybridization
could explain the failure to detect additional
ITS sequences. In the case of lateral transfer,
these additional factors need not be invoked,
although a mechanism is required. ? It should be
noted that H.helodes has been found associated
with a broad range of hosts, whereas potential
donors of second copies have been found on only
one or two hosts (Aanen, 2002) circumstantial
evidence of acquisition of new ecologies through
hybridization. ? A final explanation relies on
assuming that copy1 tracks the host phylogeny
with poor support, and copy2 represents a recent
duplication followed by a rapid divergence due to
the relaxing of constraints of selection.
Confirming this mechanism requires the uncovering
of additional copy2 sequences to determine the
level of similarity in the orthologs and whether
there are differential rates of evolution. In
this scenario, either ITS or nrt2 (or both) is a
poor indicator of host phylogeny, as they would
have very different topologies. Resolution of
this issue at a minimum requires additional
methods of determining nrt2 copy number in the
species in this dataset. 3. The topology of the
NRT2 tree appears to conflict with that of ITS
along a moderately to poorly supported node,
which places H.radicosum as monophyletic with H.
cylindrosporum and H. edurum/sinuosum. ?This is
a result that warrants further investigation as
it could suggest accelerated evolution in nrt2
along the branch to the H.radicosum terminal. H.
radicosum is one of the nitrophilic species in
this clade, implying that it relies on a more
reduced form of nitrogen, thus relaxing selective
pressure on nrt2, in turn leading to poor
bootstrap support for any clades that contain it.
Future studies should include sampling the
remaining nitrophilic Hebeloma to eventually test
relative rates of evolution. ? Different methods
have suggested different topologies in NRT2
phylogeny, however both parsimony and distance
analyses of amino acids suggest the topology
presented above. It is possible that third base
saturation with homoplasy is responsible for
alternative topologies in branch and bound of the
exon nucleotide sequences. Furthermore, ITS
appears to be a poor choice for resolving the
backbone of the Hebeloma clade, implying that it
is a poor indicator of species phylogeny.
Structural Comparison of Copies In Aspergillus,
the duplication of nrt appears to predate the
divergence of A. nidulans and A. fumigatus. It
has been demonstrated that nrtA and nrtB in
Aspergillus nidulans are expressed under
different concentrations of environmental
nitrate, thereby giving the species environmental
plasticity. Similarly, in Hebeloma, the
sequence similarity between two paralogous copies
is less than that between orthologous copies in
any other two species. This could certainly be
an effect of sampling, however the level of
divergence between the two copies is still
noteworthy, and does not appear to be due to the
existence of a pseudogene or merely random
mutation, as key functional residues are well
conserved, most substitutions are conservative,
the gene appears to be fully functional, and
substitutions are clustered in specific
locations. The most notable substitutions occur
in putative transmembrane helix 2, surrounding
the arginine residue named R87 (Unkles, 2004).
This residue has been found to be essential to
nitrate transport and is most likely the site of
nitrate binding (Unkles, 2004). To have a high
concentration of substitutions located around
R87, while R87 and its position remain intact
could suggest a retained, yet differential
capacity for binding nitrate. Aspergillus
nidulans NRTB, which is suggested to be more
active in high ambient nitrate, with 1/10th the
Km for binding nitrate(Unkles, 2004), shares a
common FV signature 2 residues upstream of R87
with copy1 in H. helodes (but not copy2 or NRTA),
which is more allied with the high affinity
transporter of H. cylindrosporum, a weak
suggestion that copy 1 is the higher affinity
locus. There is also a high degree of divergence
in the putative intracellular loop which could be
involved in regulation. It is therefore plausible
that H. helodes has acquired by hybridization or
duplication, a system of nitrate transport with
variable affinity which enhances survival under
fluctuating soil nitrogen status. Under what
conditions is each expressed? Do both sequences
occur in a cluster, and if so, is it the same
cluster or a distant locus? Are there additional
copies of nitrate and nitrite reductase? Is there
a correlation between gene expression and
mycorrhization?
References Jargeat, Patricia et al.
Characterixation and expression analysis of a
nitrate transporter and nitrate reductase genes,
two members of a gene cluster for nitrate
assimilation from the symbiotic basidiomycete
Hebeloma cylindrosporum. Curr Genet (2003) 43
199-205. Marmeisse, R., et al. Hebeloma
cylindrosporum-a model species to study
ectomycorrhizal symbiosis from gene to
ecosystem. New Phytologist (2004) 163
481-498. Aanen, Duur K., et al. Phylogenetic
relationships in the genus Hebeloma based on
ITS1 and 2 sequences, with special emphasis on
the Hebeloma crustuliniforme complex.
Mycologia (2002) 92(2)269-281. Unkles, Sheila
E., et al. Two perfectly conserved arginine
residues are required for substrate binding in a
high-affinity Yang, et al., (in press) New Asian
species of the genus Anamika (euagarics,
hebelomatoid clade) based on morphology and
ribosomal DNA sequences. nitrate transporter.
PNAS (2004) 101(50) 17549-17554. Unkles, Shelia
E., et al., Apparent genetic redundancy
facilitates ecological plasticity for nitrate
transport. The EMBO Journal (2001) 20(22)
6246-6255. Aanen, Duur K., et al., A widely
distributed ITS polymorphism within a
biological species of the ectomycorrhizal fungus
Hebeloma velutipes. (2001). Mycol. Res. 105 (3)
284-290
Acknowledgements I express the sincerest
appreciation to Dr. Brandon Matheny for his
expertise and insight regarding basidiomycete
taxonomy and molecular methods. Thanks also to
Zhang Wang for help with PCR troubleshooting and
Dr. Manfred Binder for help with phylogenetic
analysis among other pitfalls. Also thanks to
Lisa Bukovnik at the sequencing facility at
Duke University for enabling me to build my
data set during times of adverse sequencing
conditions. THIS WORK WAS SUPPORTED BY NSF GRANT
DEB-0228657
An hypothesis of the phylogeny of NRT2 in Fungi
The nrt2 gene phylogeny presented here agrees
with the broad, underlying phylogeny of fungal
evolution, with more than one origin of second
loci suggested. Inferred amino acid sequences
were used to generate similar trees with distance
and parsimony methods. This is a neighbor
joining tree adjusted with Kimura-2 parameter.
Branch lengths are mean character differences.
Bootstrap values were obtained by 5000
neigbor-joining replicates, adjusted with
Kimura-2 parameter.
Hypothesis of Hebeloma phylogeny based on ITS
sequences The above tree is a 50 majority rule
consensus of 5000 neighbor joining bootstrap
replicates, adjusted with kimura-2 parameter.
The monophyly of Hebeloma is not well supported
here. (Yang, in press) Taxa used in this study
are indicated.
Below The complete amino acid sequence of NRT2
(Jargeat et al, 2003). Inferred transmembrane
helical motifs are shown as published and
confirmed by HMMTOP. The putative protein kinase
C phosphorylation site is indicated. Primers --gt
used in this study are indicated by direction of
replication. Conserved intron positions are
noted by a . Most sequences represented
here cover the ranges, F1-R3 and F1-R2. The
inferred intracellular loop is also shown.
F1
F3
R1.5
R1
F2
R2
R2.5
R3
MSPPKSSRGAPKFKWSHLWEPAIVNPVNLKSYTIPIFNLGDPYARAFHLS
WLGFFVAFLSWFAFPPLIPDAIKSDLHLSAAQVANSNIIALCATFVVRVG
VGPLVDQYGPRKVMAYLLILGAIPSGLAGTARSAEGLYVLRFFIGILGAT
FVPCQAWTSAFFDKNCVGTANALVGGWGNMGGGATFAIMTSLFQSLTQTY
GLSTHVAWRAAFAIVPAPILLFVAVLTFIFGQDHPAGKWSERHTLPVAAL
AVQQG
HQIPNIQPEKSLKSSSESSKDEKDPEGNAAVTVRPAIADEDLALVKSTVD
VAINEPLTLKTTVKILTNPLTWLPALAYLTTFGVELAIDSKFADVLFVLF
SKRRPGFDQTTAGYYTSILGLLNLVTRPAGGYFGDLVYRHYGTNGKKAWT
LLCGLIMGAALVAGGFYMQNNRTSGDEQLSVLMGVFSVAAIFSEFGNGAN
FALVPHCNAYNNGVMSGLVGSFGNLGGIIFALVFRFQTEVGKAFWIMGVI
SIGIN
ALLIPVSVPAL(521)