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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Evolution of <italic>CDC42</italic>, a putative virulence factor triggering
meristematic growth in black yeasts</title><author><name sortKey="Deng, S" sort="Deng, S" uniqKey="Deng S" first="S." last="Deng">S. Deng</name><affiliation><nlm:aff id="aff1"><italic>Department of Dermatology, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Xinjiang, China</italic></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Van Den Ende, A H G Gerrits" sort="Van Den Ende, A H G Gerrits" uniqKey="Van Den Ende A" first="A. H. G. Gerrits" last="Van Den Ende">A. H. G. Gerrits Van Den Ende</name><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Ram, A F J" sort="Ram, A F J" uniqKey="Ram A" first="A. F. J." last="Ram">A. F. J. Ram</name><affiliation><nlm:aff id="aff3"><italic>Institute of Biology, Department of Molecular Microbiology, Kluyver Center for Genomics of Industrial Fermentation, Leiden, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Arentshorst, M" sort="Arentshorst, M" uniqKey="Arentshorst M" first="M." last="Arentshorst">M. Arentshorst</name><affiliation><nlm:aff id="aff3"><italic>Institute of Biology, Department of Molecular Microbiology, Kluyver Center for Genomics of Industrial Fermentation, Leiden, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Gr Ser, Y" sort="Gr Ser, Y" uniqKey="Gr Ser Y" first="Y." last="Gr Ser">Y. Gr Ser</name><affiliation><nlm:aff id="aff5"><italic>Institut für Mikrobiologie und Hygiene, Department of Parasitology (Charité), Humboldt University, Berlin, Germany</italic></nlm:aff></affiliation></author><author><name sortKey="Hu, H" sort="Hu, H" uniqKey="Hu H" first="H." last="Hu">H. Hu</name><affiliation><nlm:aff id="aff6"><italic>Xinjiang Medical University, Urumqi, Xinjiang, China</italic></nlm:aff></affiliation></author><author><name sortKey="De Hoog, G S" sort="De Hoog, G S" uniqKey="De Hoog G" first="G. S." last="De Hoog">G. S. De Hoog</name><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><italic>Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands</italic></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19287534</idno><idno type="pmc">2610298</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610298</idno><idno type="RBID">PMC:2610298</idno><idno type="doi">10.3114/sim.2008.61.12</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000099</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000099</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evolution of <italic>CDC42</italic>, a putative virulence factor triggering
meristematic growth in black yeasts</title><author><name sortKey="Deng, S" sort="Deng, S" uniqKey="Deng S" first="S." last="Deng">S. Deng</name><affiliation><nlm:aff id="aff1"><italic>Department of Dermatology, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Xinjiang, China</italic></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Van Den Ende, A H G Gerrits" sort="Van Den Ende, A H G Gerrits" uniqKey="Van Den Ende A" first="A. H. G. Gerrits" last="Van Den Ende">A. H. G. Gerrits Van Den Ende</name><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Ram, A F J" sort="Ram, A F J" uniqKey="Ram A" first="A. F. J." last="Ram">A. F. J. Ram</name><affiliation><nlm:aff id="aff3"><italic>Institute of Biology, Department of Molecular Microbiology, Kluyver Center for Genomics of Industrial Fermentation, Leiden, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Arentshorst, M" sort="Arentshorst, M" uniqKey="Arentshorst M" first="M." last="Arentshorst">M. Arentshorst</name><affiliation><nlm:aff id="aff3"><italic>Institute of Biology, Department of Molecular Microbiology, Kluyver Center for Genomics of Industrial Fermentation, Leiden, The Netherlands</italic></nlm:aff></affiliation></author><author><name sortKey="Gr Ser, Y" sort="Gr Ser, Y" uniqKey="Gr Ser Y" first="Y." last="Gr Ser">Y. Gr Ser</name><affiliation><nlm:aff id="aff5"><italic>Institut für Mikrobiologie und Hygiene, Department of Parasitology (Charité), Humboldt University, Berlin, Germany</italic></nlm:aff></affiliation></author><author><name sortKey="Hu, H" sort="Hu, H" uniqKey="Hu H" first="H." last="Hu">H. Hu</name><affiliation><nlm:aff id="aff6"><italic>Xinjiang Medical University, Urumqi, Xinjiang, China</italic></nlm:aff></affiliation></author><author><name sortKey="De Hoog, G S" sort="De Hoog, G S" uniqKey="De Hoog G" first="G. S." last="De Hoog">G. S. De Hoog</name><affiliation><nlm:aff id="aff2"><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><italic>Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands</italic></nlm:aff></affiliation></author></analytic><series><title level="j">Studies in Mycology</title><idno type="ISSN">0166-0616</idno><idno type="eISSN">1872-9797</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The cell division cycle gene (<italic>CDC42</italic>) controlling cellular
polarization was studied in members of <italic>Chaetothyriales</italic>. Based on
ribosomal genes, ancestral members of the order exhibit meristematic growth in
view of their colonization of inert surfaces such as rock, whereas in derived
members of the order the gene is a putative virulence factor involved in
expression of the muriform cell, the invasive phase in human
chromoblastomycosis. Specific primers were developed to amplify a portion of
the gene of 32 members of the order with known position according to ribosomal
phylogeny. Phylogeny of <italic>CDC42</italic> proved to be very different. In all
members of <italic>Chaetohyriales</italic> the protein sequence is highly conserved.
In most species, distributed all over the phylogenetic tree, introns and
3<sup>rd</sup> codon positions are also invariant. However, a number of
species had paralogues with considerable deviation in non-coding exon
positions, and synchronous variation in introns, although non-synonomous
variation had remained very limited. In some strains both orthologues and
paralogues were present. It is concluded that <italic>CDC42</italic> does not show any
orthologous evolution, and that its paralogues haves the same function but are
structurally relaxed. The variation or absence thereof could not be linked to
ecological changes, from rock-inhabiting to pathogenic life style. It is
concluded that eventual pathogenicity in <italic>Chaetothyriales</italic> is not
expressed at the DNA level in <italic>CDC42</italic> evolution.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Stud Mycol</journal-id><journal-id journal-id-type="publisher-id">simycol</journal-id><journal-title>Studies in Mycology</journal-title><issn pub-type="ppub">0166-0616</issn><issn pub-type="epub">1872-9797</issn><publisher><publisher-name>CBS Fungal Biodiversity Centre</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19287534</article-id><article-id pub-id-type="pmc">2610298</article-id><article-id pub-id-type="publisher-id">0121</article-id><article-id pub-id-type="doi">10.3114/sim.2008.61.12</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Evolution of <italic>CDC42</italic>, a putative virulence factor triggering
meristematic growth in black yeasts</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Deng</surname><given-names>S.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>van den Ende</surname><given-names>A.H.G. Gerrits</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ram</surname><given-names>A.F.J.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Arentshorst</surname><given-names>M.</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Gräser</surname><given-names>Y.</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Hu</surname><given-names>H.</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>de Hoog</surname><given-names>G.S.</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1"><label>1</label><italic>Department of Dermatology, First Affiliated Hospital, Xinjiang Medical University, Urumqi, Xinjiang, China</italic></aff><aff id="aff2"><label>2</label><italic>CBS Fungal Biodiversity Centre, Utrecht, The Netherlands</italic></aff><aff id="aff3"><label>3</label><italic>Institute of Biology, Department of Molecular Microbiology, Kluyver Center for Genomics of Industrial Fermentation, Leiden, The Netherlands</italic></aff><aff id="aff4"><label>4</label><italic>Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands</italic></aff><aff id="aff5"><label>5</label><italic>Institut für Mikrobiologie und Hygiene, Department of Parasitology (Charité), Humboldt University, Berlin, Germany</italic></aff><aff id="aff6"><label>6</label><italic>Xinjiang Medical University, Urumqi, Xinjiang, China</italic></aff><author-notes><fn id="cor1"><label>*</label><p>Correspondence: G.S. de Hoog,
<email>de.hoog@cbs.knaw.nl</email></p></fn></author-notes><pub-date pub-type="ppub"><year>2008</year></pub-date><volume>61</volume><issue-title>Black fungal extremes</issue-title><fpage>121</fpage><lpage>129</lpage><permissions><copyright-statement>Copyright © Copyright 2008 CBS Fungal Biodiversity Centre</copyright-statement><license><p>You are free to share - to copy, distribute and transmit the work, under
the following conditions:</p><p><bold>Attribution:</bold> You must attribute
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<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode">http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode.</ext-link> Any of the above
conditions can be waived if you get permission from the copyright holder.
Nothing in this license impairs or restricts the author's moral rights.</p></license></permissions><self-uri xlink:title="pdf" xlink:href="121.pdf"></self-uri><abstract><p>The cell division cycle gene (<italic>CDC42</italic>) controlling cellular
polarization was studied in members of <italic>Chaetothyriales</italic>. Based on
ribosomal genes, ancestral members of the order exhibit meristematic growth in
view of their colonization of inert surfaces such as rock, whereas in derived
members of the order the gene is a putative virulence factor involved in
expression of the muriform cell, the invasive phase in human
chromoblastomycosis. Specific primers were developed to amplify a portion of
the gene of 32 members of the order with known position according to ribosomal
phylogeny. Phylogeny of <italic>CDC42</italic> proved to be very different. In all
members of <italic>Chaetohyriales</italic> the protein sequence is highly conserved.
In most species, distributed all over the phylogenetic tree, introns and
3<sup>rd</sup> codon positions are also invariant. However, a number of
species had paralogues with considerable deviation in non-coding exon
positions, and synchronous variation in introns, although non-synonomous
variation had remained very limited. In some strains both orthologues and
paralogues were present. It is concluded that <italic>CDC42</italic> does not show any
orthologous evolution, and that its paralogues haves the same function but are
structurally relaxed. The variation or absence thereof could not be linked to
ecological changes, from rock-inhabiting to pathogenic life style. It is
concluded that eventual pathogenicity in <italic>Chaetothyriales</italic> is not
expressed at the DNA level in <italic>CDC42</italic> evolution.</p></abstract><kwd-group><kwd>Cell Division Cycle <italic>CDC42</italic></kwd><kwd><italic>Chaetothyriales</italic></kwd><kwd>chromoblastomycosis</kwd><kwd>muriform cell</kwd><kwd>paralogue evolution</kwd><kwd>phylogeny</kwd><kwd>virulence factors</kwd></kwd-group></article-meta></front><body><sec><title>INTRODUCTION</title><p>Human infection by agents of chromoblastomycosis is accompanied by dramatic
morphogenetic changes of fungal cells in tissue. The fungi concerned have the
ability of morphogenetic switching from polarized filamentous growth to
isodiametric expansion, leading to large spherical cells
(<xref ref-type="bibr" rid="ref26">Szaniszlo <italic>et al.</italic>1983</xref>). Subdivision of the cells gives rise to muriform cells
(<xref ref-type="bibr" rid="ref19">Matsumoto <italic>et al.</italic>1993</xref>), the invasive phase of the fungi concerned and triggering
hyperphasia characteristic for the disease. Chromoblastomycosis is exclusively
known to be caused by members of the ascomycete order <italic>Chaetothyriales</italic>(<xref ref-type="bibr" rid="ref10">Haase <italic>et al.</italic> 1999</xref>,
<xref ref-type="bibr" rid="ref1">Badali <italic>et al.</italic> 2008</xref>):
primarily by <italic>Cladophialophora</italic> and <italic>Fonsecaea</italic> species and
occasionally by species of <italic>Exophiala, Phialophora</italic> or
<italic>Rhinocladiella</italic>. The order comprises a large number of pathogens and
opportunists on warm- and cold-blooded vertebrates
(<xref ref-type="bibr" rid="ref13">de Hoog <italic>et al.</italic>2000</xref>), and hence is likely to have ancestral virulence
factors.</p><p>The order <italic>Chaetothyriales</italic> is remarkable in the fungal Kingdom, for
two reasons. First, a large number of the infections are observed in
individuals without known immune disorder. Of the about 77 species confirmed
to belong to the order by sequence data
(<xref ref-type="bibr" rid="ref2">Barr 1990</xref>,
<xref ref-type="bibr" rid="ref9">Gueidan <italic>et al.</italic> 2008</xref>),
about 33 have been encountered as etiologic agents of infections in
vertebrates (<xref ref-type="bibr" rid="ref13">de Hoog <italic>et al.</italic>2000</xref>, Zeng <italic>et al.</italic> 2006,
<xref ref-type="bibr" rid="ref1">Badali <italic>et al.</italic> 2008</xref>).
This high percentage of species with an infective potential is only matched by
the order <italic>Onygenales</italic> containing the dermatophytes and classical
systemic fungi. Second, the diversity in clinical pictures caused by members
of the <italic>Chaetothyriales</italic> is bewildering. Species of <italic>Onygenales</italic>are very consistent in their pathology, displaying a similar clinical course
by members causing cutaneous or systemic infections, whereas those of
<italic>Chaetothyriales</italic> encompass a wide diversity of diseases, which are
nevertheless more or less characteristic within a single species
(<xref ref-type="bibr" rid="ref13">de Hoog <italic>et al.</italic>2000</xref>). This pathogenic potential is particularly observed in the
more derived parts of the order, comprising the ascomycete family
<italic>Herpotrichiellaceae</italic> (<xref ref-type="bibr" rid="ref28">Untereiner
2000</xref>). Recently it was established that members of this same group
show `dual ecology', i.e., they also possess the uncommon ability to
assimilate monoaromatic pollutants
(<xref ref-type="bibr" rid="ref21">Prenafeta-Boldú <italic>et al.</italic>2006</xref>).</p><p>A second recurrent trend in the <italic>Chaetothyriales</italic> is
extremotolerance, i.e. growth on exposed surfaces, having a competitive
advantage at high temperature and dryness (Sterflinger <italic>et al.</italic> 1998,
<xref ref-type="bibr" rid="ref22">Ruibal 2004</xref>). Phylogenetic
trees published by Lutzoni <italic>et al.</italic>(<xref ref-type="bibr" rid="ref18">2001</xref>) and Gueidan <italic>et
al.</italic> (<xref ref-type="bibr" rid="ref9">2008</xref>) indicate that
some deep branches among the pathogenic black yeasts have a shared evolution
with rock-inhabiting fungi. A meristematic growth form, morphologically
similar to the muriform cells described above, may be expressed under adverse
environmental conditions of nutrient depletion, high temperature and dryness.
This suggests a functional change in the course of evolution, from an
ancestral rock-inhabiting lifestyle to a derived strategy in which ultimately
pathogenicity to vertebrate hosts enhances the fitness of species.</p><p>Polarized <italic>vs.</italic> isodiametric growth in fungi is regulated by the
Rho-related GTPase Cdc42p (Cdc = Cell Division Cycle), reviewed by Johnson
(<xref ref-type="bibr" rid="ref16">1999</xref>). Cdc42p is essential
for the reorganization of the actin cytoskeleton during the shift from
polarized to isodiametric growth. Upon activation, Cdc42p is recruited to the
plasma membrane to initiate actin nucleation. Localization and activity of
Cdc42p are mediated by guanine-nucleotide exchange factors (GEFs). In <italic>S.
cerevisiae</italic>, the only GEF for Cdc42p is Cdc24p, controlled by cell cycle
proteins (Cdc28p) and additional proteins (Cla4p and Bemp1;
<xref ref-type="bibr" rid="ref3">Bose <italic>et al.</italic> 2001</xref>). The
eleven current members of the CDC42 family display between 75 and 100 % amino
acid identity and are functionally as well as structurally homologous. In
filamentous fungi another member of this family is present, RacAp. This is a
well studied GTP-binding protein in mammalian cells and it has been shown that
RacAp is required for the formation of lamellipodia in fibroblast cells.
Recently, RacAp was found in basidiomycetes
(<xref ref-type="bibr" rid="ref8">Gorfer <italic>et al.</italic> 2001</xref>)
as well as in ascomycetes (<xref ref-type="bibr" rid="ref15">Hurtado <italic>et
al.</italic> 2000</xref>, <xref ref-type="bibr" rid="ref4">Boyce <italic>et
al.</italic> 2001</xref>, <xref ref-type="bibr" rid="ref30">Virag <italic>et
al.</italic> 2007</xref>), but RacAp orthologus are absent in <italic>S.
cerevisiae</italic> and have not been reported to be present in
<italic>Chaetothyriales</italic>. Analysis of transformants overexpressing a dominant
active allele of RacAp(<italic>racAG12V</italic>) displays unpolarized growth in
<italic>Aspergillus niger</italic>, resembling the muriform cells, the pathogenic
tissue-phase characteristic for chromoblastomycosis (A.F.J. Ram, unpublished
data). This form in the agents of chromoblastomycosis, and even the agent of
phaeohyphomycosis, <italic>Exophiala dermatitidis</italic>, is easily expressed in
culture by a low pH of the growth medium and conditions of calcium limitation
at more neutral pH (<xref ref-type="bibr" rid="ref20">Mendoza <italic>et
al.</italic> 1993</xref>, Karuppayil & Szaniszlo 1993,
<xref ref-type="bibr" rid="ref27">Szaniszlo <italic>et al.</italic>1993</xref>, <xref ref-type="bibr" rid="ref1">Badali <italic>et al.</italic>2008</xref>). The Cdc42 proteins act as molecular switches by responding
to exogenous and/or endogenous signals and relaying those signals to activate
downstream components of a biological pathway. Ye & Szaniszlo
(<xref ref-type="bibr" rid="ref32">2000</xref>) confirmed that Cdc42p
plays a unique regulatory role in the morphogenesis of the black yeast
<italic>E.dermatitidis</italic> during its phenotype transition from yeast to
isodiametric cells and muriform cells in vitro.. They also found that the
derived Cdc42 protein is highly conserved member of the Cdc42 subfamily. These
results suggest that the <italic>CDC42</italic> gene products seem to play an
important role in the regulation of stress-induced fungal cellular
morphogenesis. One of the aspects of this therefore is its implication in
human chromoblastomycosis.</p><p>When the functional change of Cdc42p, from extremotolerance to
pathogenicity, concerns an evolutionary adaptation, we might expect the
transition to be reflected at the DNA level. Comparing <italic>CDC42</italic> DNA gene
sequences with the phylogenetic scaffold based on ribosomal genes as the gold
standard, might thus provide additional insights about whether structural and
functional changes in the gene concern adaptive changes in ecological
strategies. In the present paper, tools are developed for the detection <italic>of
the CDC42 gene</italic> in <italic>Chaetothyriales</italic> in view of a phylogenetic
study of the <italic>CDC42</italic> gene, in parallel to genes with known phylogenetic
content.</p></sec><sec sec-type="materials|methods"><title>MATERIAL AND METHODS</title><sec><title>Strains and culture conditions</title><p>The isolates studied are listed in <xref ref-type="table" rid="tbl1">Table
1</xref>. A total of 32 members of <italic>Chaetothyriales</italic> including
basal lineages were obtained from the reference collection of the
Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre and grown on
Malt Extract Agar (MEA) at 25 °C.</p><p><table-wrap position="float" id="tbl1"><label>Table 1.</label><caption><p>Strains used in this study</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="top"><bold>Name</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>CBS no.</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Status</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Ortho-/para-</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Other reference</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Source</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Origin</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala dermatitidis</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 34100; NIH 8656
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human sputum
</td><td colspan="1" rowspan="1" align="left" valign="top"> Japan
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=292.49&link_type=CBS">CBS 292.49</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15696
</td><td colspan="1" rowspan="1" align="left" valign="top"> Faeces
</td><td colspan="1" rowspan="1" align="left" valign="top"> U.S.A.
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=207.35&link_type=CBS">CBS 207.35</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 28869; UAMH 3967
</td><td colspan="1" rowspan="1" align="left" valign="top"> Man, facial chromoblastomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> Japan; Osaka University
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala heteromorpha</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS 232.33</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> MUCL 9894; NCMH 17
</td><td colspan="1" rowspan="1" align="left" valign="top"> Wood pulp
</td><td colspan="1" rowspan="1" align="left" valign="top"> Sweden
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia mansonii</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=101.67&link_type=CBS">CBS 101.67</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 18659; IMI 134456
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Populus tremula</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> Sweden
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia munkii</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=615.96&link_type=CBS">CBS 615.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 16078
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Populus tremuloides</italic>, wood
</td><td colspan="1" rowspan="1" align="left" valign="top"> Canada, Alberta; south of Hinton
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia epimyces</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=606.96&link_type=CBS">CBS 606.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 16065
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Nectria</italic>, fruit bodies, on <italic>Pinus</italic> wood
</td><td colspan="1" rowspan="1" align="left" valign="top"> Canada; Ontario
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Rhinocladiella mackenziei</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=650.93&link_type=CBS">CBS 650.93</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> MUCL 40057
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, cerebral phaeohyphomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> Saudi Arabia
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia acutiseta</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=618.96&link_type=CBS">CBS 618.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 56428;ATCC 76482
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Dacrydium cupressinum</italic>, wood
</td><td colspan="1" rowspan="1" align="left" valign="top"> New Zealand; Saltwater State, Westland County
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia parasitica</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=123.88&link_type=CBS">CBS 123.88</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15347
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Hypoxylon cohaerens</italic> var. <italic>microsporum</italic>, on <italic>Quercus</italic>sp.
</td><td colspan="1" rowspan="1" align="left" valign="top"> France; Bois de Lourdes
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Rhinocladiella anceps</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=181.65&link_type=CBS">CBS 181.65</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> NT
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 18655; IMI 134453; MUCL 8233
</td><td colspan="1" rowspan="1" align="left" valign="top"> Soil under <italic>Thuja plicata</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> Canada, Ontario; Campbellville
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia villosa</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=616.96&link_type=CBS">CBS 616.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 56206
</td><td colspan="1" rowspan="1" align="left" valign="top"> Decorticated wood
</td><td colspan="1" rowspan="1" align="left" valign="top"> New Zealand
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Fonsecaea monophora</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=269.37&link_type=CBS">CBS 269.37</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 12659
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, chromoblastomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> South America
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phialophora verrucosa</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=286.47&link_type=CBS">CBS 286.47</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 9541; MUCL 9768
</td><td colspan="1" rowspan="1" align="left" valign="top"> Mycetoma hand, human
</td><td colspan="1" rowspan="1" align="left" valign="top"> Brazil
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phialophora americana</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=840.69&link_type=CBS">CBS 840.69</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> MUCL 15537
</td><td colspan="1" rowspan="1" align="left" valign="top"> Decaying timber
</td><td colspan="1" rowspan="1" align="left" valign="top"> Finland; Helsinki
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Cladophialophora carrionii</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=260.83&link_type=CBS">CBS 260.83</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 44535
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, chromoblastomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> Venezuela, Falcon State, Uganda
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Cladophialophora boppii</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=126.86&link_type=CBS">CBS 126.86</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15357
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, skin lesion, on limb
</td><td colspan="1" rowspan="1" align="left" valign="top"> Brazil
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala castellanii</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=158.58&link_type=CBS">CBS 158.58</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> NT
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 18657; IFM 4702; MUCL 10097
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human
</td><td colspan="1" rowspan="1" align="left" valign="top"> Sri Lanka
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala nigra</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=546.82&link_type=CBS">CBS 546.82</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15993
</td><td colspan="1" rowspan="1" align="left" valign="top"> Soil under ice
</td><td colspan="1" rowspan="1" align="left" valign="top"> Russia
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala bergeri</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=353.52&link_type=CBS">CBS 353.52</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15792
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, chromoblastomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> Canada
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala spinifera</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=899.68&link_type=CBS">CBS 899.68</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 18218; IHM 1767; NCMH 152
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, nasal granuloma
</td><td colspan="1" rowspan="1" align="left" valign="top"> U.S.A.
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala jeanselmei</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=507.90&link_type=CBS">CBS 507.90</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 34123; <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=664.76&link_type=CBS">CBS
664.76</ext-link>; IHM 283; NCMH 123
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human
</td><td colspan="1" rowspan="1" align="left" valign="top"> Uruguay
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala oligosperma</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=725.88&link_type=CBS">CBS 725.88</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 16212
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, tumour of sphenoidal cavity
</td><td colspan="1" rowspan="1" align="left" valign="top"> Germany; Würzburg
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phialophora europaea</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=129.96&link_type=CBS">CBS 129.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> -/+
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 10389
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, chromoblastomycosis of toe
</td><td colspan="1" rowspan="1" align="left" valign="top"> Germany; Giessen
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Fonsecaea pedrosoi</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=271.37&link_type=CBS">CBS 271.37</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> NT
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> ATCC 18658;IMI 134458
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, chromoblastomycosis
</td><td colspan="1" rowspan="1" align="left" valign="top"> Argentina
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Coniosporium perforans</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=885.95&link_type=CBS">CBS 885.95</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 16308
</td><td colspan="1" rowspan="1" align="left" valign="top"> Marble
</td><td colspan="1" rowspan="1" align="left" valign="top"> Greece
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phaeoannellomyces elegans</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=122.95&link_type=CBS">CBS 122.95</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15343; NCMH 1286
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, skin infection of toe nail
</td><td colspan="1" rowspan="1" align="left" valign="top"> Canada; Toronto
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala pisciphila</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=661.76&link_type=CBS">CBS 661.76</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 16145
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Heterodera schachtii</italic> egg from cyst, recovered from soil
</td><td colspan="1" rowspan="1" align="left" valign="top"> Germany; Elsdorf
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phialophora reptans</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=113.85&link_type=CBS">CBS113.85</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 5543
</td><td colspan="1" rowspan="1" align="left" valign="top"> Food
</td><td colspan="1" rowspan="1" align="left" valign="top"> Sweden
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala mesophila</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=402.95&link_type=CBS">CBS 402.95</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> DH 15838
</td><td colspan="1" rowspan="1" align="left" valign="top"> Silicone, shower cabinet,
</td><td colspan="1" rowspan="1" align="left" valign="top"> Germany, Hamburg
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Cladophialophora modesta</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=985.96&link_type=CBS">CBS 985.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"> T
</td><td colspan="1" rowspan="1" align="center" valign="top"> +/-
</td><td colspan="1" rowspan="1" align="left" valign="top"> NCMH 108; UAMH 4004
</td><td colspan="1" rowspan="1" align="left" valign="top"> Human, brain
</td><td colspan="1" rowspan="1" align="left" valign="top"> U.S.A.; Chapel Hill
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala salmonis</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=157.67&link_type=CBS">CBS 157.67</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top">T
</td><td colspan="1" rowspan="1" align="center" valign="top">+/-
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Brain, Salmo clakii
</td><td colspan="1" rowspan="1" align="left" valign="top">Canada; Galgary
</td></tr></tbody></table></table-wrap></p></sec><sec><title>DNA extraction</title><p>Approximately 1 cm<sup>2</sup> mycelium of 30-d-old cultures was
transferred to a 2 mL Eppendorf tube containing 300 μL TES-buffer (Tris 1.2
% w/v, Na-EDTA 0.38% w/v, SDS 2 % w/v, pH 8.0) and about 80 mg of a silica
mixture (Silica gel H, Merck 7736, Darmstadt, Germany / Kieselguhr Celite 545,
Machery, Düren, Germany, 2 : 1, w/w). Cells were disrupted mechanically
in a tight-fitting sterile pestle for approximately 1 min. Subsequently 200
μL TES-buffer was added, the mixture was vortexed, 10 μL proteinase K
was added and incubated for 10 min at 65 °C. After addition of 140 μL
of 5 M NaCl and 1/10 vol CTAB 10 % (cetyltrimethylammoniumbromide) solution,
the material was incubated for 30 min at 65 °C. Subsequently 700 μL
SEVAG (24 : 1, chloroform: isoamylalcohol) was added to the solution and
shortly mixed by shaking, incubated for 30 min on ice water and centrifuged
for 10 min at 1125 × g. The supernatant was transferred to a new tube
with 225 μL 5 M NH<sub>4</sub>-acetate, incubated on ice water for 30 min.
and centrifuged again for 10 min at 1125 × g. The supernatant was
transferred to another Eppendorf tube with 0.55 vol isopropanol mixed
carefully by flipping and spin for 5 min at 1125 × g. Subsequently, the
pellet was washed with ice cold 70 % ethanol. After drying at room temperature
it was re-suspended in 48.5 μL TE buffer (Tris 0.12 % w/v, a-EDTA 0.04 %
w/v) plus 1.5 μL RNAse 20 U/mL and incubated for 15–30 min at 37
°C. DNAs were purified with GFX PCR DNA and Gel Band Purification Kit
(Amersham Biosciences) as recommended by the manufacturer.</p></sec><sec><title><italic>CDC42</italic> primer design</title><p>Primers specific for <italic>CDC42</italic> amplifications were selected using a
complete alignment of the amino acid sequences of species listed in
<xref ref-type="table" rid="tbl2">Table 2</xref>. Two highly conserved
areas were detected for <italic>CDC42</italic> that were absent from the Rac and Rho
gene families. Degenerated forward (<italic>CDC42</italic>-F1, <italic>CDC42</italic>-F2) and
reverse (<italic>CDC42</italic>-R1, <italic>CDC42</italic>-R2) primers were designed matching
the target regions. In addition, primers
(<xref ref-type="table" rid="tbl4">Table 4</xref>) were synthesized in
the same positions but that were specific for the published sequence of
<italic>Exophiala dermatitidis</italic>,
<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link> (NIH
8656 = AF162788) as control (<xref ref-type="bibr" rid="ref32">Ye &
Szaniszlo 2000</xref>). The resulting specific primers
<italic>CDC42</italic>-F1s, <italic>CDC42</italic>-F2s, <italic>CDC42</italic>-R1s and
<italic>CDC42</italic>-R2s (<xref ref-type="table" rid="tbl4">Table 4</xref>)
were subsequently tested with the aim to establish amplification conditions.
Different combinations of specific primers were then tested for the 32 strains
listed in <xref ref-type="table" rid="tbl1">Table 1</xref>.</p><p><table-wrap position="float" id="tbl2"><label>Table 2.</label><caption><p><italic>CDC42</italic> reference sequences taken from GenBank.</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="top"><bold>Species</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Gene</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>GenBank no. protein</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>GenBank no. DNA</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala dermatitidis</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> AAD46909
</td><td colspan="1" rowspan="1" align="center" valign="top"> AF162788
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Emericella nidulans</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> AAF24514
</td><td colspan="1" rowspan="1" align="center" valign="top"> AF217199
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Penicillium marneffei</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> AAK56917
</td><td colspan="1" rowspan="1" align="center" valign="top"> AF330694
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>RAC</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> AAN77094
</td><td colspan="1" rowspan="1" align="center" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Aspergillus niger</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>RAC</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> AAT09022
</td><td colspan="1" rowspan="1" align="center" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>Aspergillus nidulans</italic></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>RHO</italic></td><td colspan="1" rowspan="1" align="left" valign="top">XP-663344
</td><td colspan="1" rowspan="1" align="center" valign="top"></td></tr></tbody></table></table-wrap></p><p><table-wrap position="float" id="tbl4"><label>Table 4.</label><caption><p>Degenerate and specific primers designed in this study.</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="2" rowspan="1" align="left" valign="top"><bold>Degenerate primer:</bold></th><th colspan="1" rowspan="1" align="left" valign="top"></th><th colspan="1" rowspan="1" align="left" valign="top"></th></tr><tr><th colspan="2" rowspan="1" align="left" valign="top">Gene:
</th><th colspan="1" rowspan="1" align="left" valign="top">Amino acid sequence:
</th><th colspan="1" rowspan="1" align="left" valign="top">Nucleotide sequence:
</th></tr></thead><tbody><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-F1:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> M V V A T I
</td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-ATG GTI GTI GCI ACI ATH
</td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-F2:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> I G D E PYT
</td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-ATH GGI GAY GAR CCI TAY AC
</td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R1:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> R M A K EL G
</td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-CCI ARY ICY TTI GCC ATI CK
</td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R2:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Y K L K D VF
</td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-RAA IAC RTC YTT IAR YTT RTA
</td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><bold>Specific primers for <italic>CDC42</italic> orthologue:</bold></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr></tbody><tbody><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-F1s: 5′-ATG GTT GTC GCA ACG ATC
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>- F2s: 5′-GGA TTA CGA CCG GCT TCG
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R1s: 5′-CCA ACT CCT TGG CCA TTC
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R2s: 5′-AAA GAC GTC TTT GAG TTT GTA
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top">Primer combination
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic> F1s--R2s 698 bp
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic> F1s--R1s 639 bp
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic> F2s--R2s 372 bp
</td><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic> F2s--R1s 315 bp
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="2" rowspan="1" align="left" valign="top"><bold>Six specific backward primers for <italic>CDC42</italic> paralogue:</bold></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-F1s:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-ATG GTT GTC GCA ACG ATC
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>- R1d:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-GGA CTT GTG GGT CGT CA
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R2d:
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-TCA GCG ACG GAT GGG T
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R3d (<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS
232.33</ext-link>):
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-GTT CCA ACA ATC AGA CA
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R4d (<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=606.96&link_type=CBS">CBS
606.96</ext-link>):
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-TCC CAA CAA TCA GAC AT
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R5d (<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=616.96&link_type=CBS">CBS
616.96</ext-link>):
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> 5′-CTT GCG GGT AGT CAC GAA
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CDC42</italic>-R6d (<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=618.96&link_type=CBS">CBS
618.96</ext-link>):
</td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">5′-AAC AAT CAA ACA AGG CAC T
</td><td colspan="1" rowspan="1" align="left" valign="top"></td></tr></tbody></table></table-wrap></p></sec><sec><title>Amplification of <italic>CDC42</italic> orthologue</title><p>Approximately 100 ng of genomic DNA was used as a template in semi-nested
PCR with primer pair <italic>CDC42</italic>-F1s-R2s and followed by a second PCR using
the amplicon of the first PCR with primer <italic>CDC42</italic>-F1s-R1s. Both PCR
were performed in 25 μL PCR-mix consisting of GoTaq green master mix 7
μL (Promega, Leiden), MQ 13 μL, DMSO 1 μL, primers 1 μL each, DNA
2 μL, using a Biosystems 2720 thermal cycler with an initial cycle of 1 min
at 98 °C, subsequently 30 cycles of 30 s at 98 °C, 30 s at 54 °C,
and 1 min at 72 °C, and a final extension of 7 min at 72 °C.</p></sec><sec><title>Specific primers for <italic>CDC42</italic> paralogue</title><p>Backward primers were designed (<xref ref-type="table" rid="tbl4">Table
4</xref>) specific for each deviating motif using <italic>CDC42</italic>-F1s as
forward primer. With both PCR amplifications, approximately 100 ng of genomic
DNA was used as a template in first PCR with the forward primer combined with
the backward primer in six separate reactions, and nested with the same primer
pair. PCR was performed in a 25 μL PCR-mix consisting of GoTaq green master
mix 7 μL (Promega, Leiden), MQ 13 μL, DMSO 1 μL, primers 1 μL
each, DNA 2 μL using a Biosystems 2720 thermal cycler with an initial cycle
of 1 min at 98 °C. Program was as follows: 30 cycles of 30 s at 98 °C,
30 s at 54 °C and 1 min at 72 °C, and a final extension of 7 min at 72
°C. The sond PCRs used amplicons from the first PCR and a touch-down
program with an initial cycle of 1 min at 95 °C, subsequently 10 cycles of
30 s at 95 °C, 30 s at 62 °C/-1°C/cycle and 1 min at 72 °C,
followed by 20 cycles of 30 s at 95 °C, 30 s at 52 °C, and 1 min at 72
°C, and a final extension of 7 min at 72 °C in sond PCR.</p></sec><sec><title>Cloning of the <italic>CDC42</italic> paralogue</title><p><italic>CDC42</italic> paralogues were cloned using a cloning kit (pGEM-T vector;
Promega, Madison, WI, U.S.A.), according to the manufacturer's instructions.
We picked up one white colony to do direct PCR with primer M13 fw
[5′-GTA AAA CGA CGG CCA GT-3′], M13 rv [5′-GGA AAC AGC TAT
GAC CAT G-3]. PCR was performed in a 25 μL PCR-mix consisting of PCR buffer
10x 2.5 μL, MQ 15 μL, dNTP mix (1 mM) 2.5 μL, <italic>Taq</italic> polymerase
(1 U/μL) 1 μL, primers 1 μL each and one white colony. amplifications
were with a Biosystems 2720 thermal cycler using an initial cycle of 3 min at
94 °C, 28 subsequent cycles of 1 min at 93 °C, 1 min at 52 °C, and
2 min at 72 °C, and a final extension of 3 min at 72 °C. The resulting
<italic>CDC42</italic> paralogue sequences were compared to the previously obtained
orthologous <italic>CDC42</italic> gene sequences using
B<sc>io</sc>N<sc>umerics</sc> software v. 4.61 (Applied Maths, Kortrijk,
Belgium)</p></sec><sec><title>SSU and LSU amplification and sequencing</title><p>Primers
(<ext-link ext-link-type="uri" xlink:href="http://www.biology.duke.edu/fungi/mycolab/primers.htm">http://www.biology.duke.edu/fungi/mycolab/primers.htm</ext-link>)
shown in <xref ref-type="table" rid="tbl3">Table 3</xref> were used to
amplify part of the nuclear rDNA operon spanning the 3' end of the 18S r RNA
gene (SSU), then first internal transcribed spacer (ITS1), the 5.8S rRNA gene,
the second ITS region and 5' end of the 28S rRNA gene (LSU). The PCR
conditions followed the methods of Crous <italic>et al.</italic> (2006b). PCR
amplifications were performed as follows: 95 °C for 1 min, followed by 30
cycles consisting of 95 °C for 10 s, 50 °C for 5 s and 60 °C for 4
min. Reaction products were then purified with Sephadex G-50 fine (GE
Healthcare Bio-Sciences AB, Uppsala, Sweden) and sequencing was done on an ABI
3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.).
Sequence data were adjusted using the SeqMan of Lasergene software (DNAStar
Inc., Madison, Wisconsin, U.S.A.).</p><p><table-wrap position="float" id="tbl3"><label>Table 3.</label><caption><p>Primer sequences for PCR amplification and sequencing of LSU and SSU.</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="top"><bold>Gene</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>PCR primers</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>Sequencing primers</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>References</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"> LSU rDNA
</td><td colspan="1" rowspan="1" align="left" valign="top"> LRORa, LR7b
</td><td colspan="1" rowspan="1" align="left" valign="top"> LRoR LR3R, LR5, LR7b
</td><td colspan="1" rowspan="1" align="left" valign="top"> a Rehner & Samuels (1994)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> b Vilgalys & Hester (<xref ref-type="bibr" rid="ref29">1990</xref>)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> SSU rDNA
</td><td colspan="1" rowspan="1" align="left" valign="top"> NS1a, NS24b
</td><td colspan="1" rowspan="1" align="left" valign="top"> BF83, Oli1, Oli9, BF951, BF963, BF1438, Oli3, BF1419 c
</td><td colspan="1" rowspan="1" align="left" valign="top"> c White <italic>et al.</italic> (<xref ref-type="bibr" rid="ref31">1990</xref>)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> b Gargas & Taylor (<xref ref-type="bibr" rid="ref7">1992</xref>)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">c de Hoog <italic>et al.</italic>(<xref ref-type="bibr" rid="ref12">2005</xref>)
</td></tr></tbody></table></table-wrap></p></sec><sec><title>Alignment and phylogenetic reconstruction</title><p>Phylogenetic analyses were carried out on data with a taxon sampling
representative of the order <italic>Chaetothyriales</italic> and including the three
genes nucLSU, nucSSU and <italic>CDC42</italic>. This dataset was used in order to
assess the phylogenetic evolution of <italic>CDC42</italic> gene on diverse species
within <italic>Chaetothyriales</italic>. SSU, LSU and <italic>CDC42</italic> data sets were
analyzed separately. Alignments were adjusted manually. Ambiguous regions were
excluded from the alignments. Concatenated sequences of LSU and SSU were
submitted to the Cipres Portal v.1.14
(<ext-link ext-link-type="uri" xlink:href="http://www.phylo.org/portal/Home.do">http://www.phylo.org/portal/Home.do</ext-link>)
using the RAxML web server. Maximum likelihood searches for the best-scoring
tree were made after the bootstrap estimate proportion of invariable sites
automatically determined the number of bootstrapping runs: if checked, RAxML
will automatically determine the point at which enough bootstrapping
replicates have been produced (<xref ref-type="bibr" rid="ref24">Stamatakis
<italic>et al.</italic> 2008</xref>). <italic>CDC42</italic> data were analyzed in the
same way. Bootstrap values equal to or greater than 80 % were considered
significant (<xref ref-type="bibr" rid="ref11">Hillis & Bull
1993</xref>).</p></sec></sec><sec><title>RESULTS</title><sec><title>Evaluation of designed primers for <italic>CDC42</italic></title><p>The degenerate primers designed proved to be insufficiently specific to
amplify the <italic>CDC42</italic> gene. Four oligonucleotide primer sets, i.e.
<italic>CDC42</italic>-F1s-R2s, <italic>CDC42</italic>-F1s-R1s, <italic>CDC42</italic>-F2s-R2s and
<italic>CDC42</italic>-F2s-R1s were synthesized and tested for amplification with
genomic DNA from 32 selected strains in the CBS reference collection
representing the order <italic>Chaetothyriales</italic>. Primer pairs
<italic>CDC42</italic>-F2s-R2s and <italic>CDC42</italic>-F2s-R1s provided poor results and
were excluded from the analysis. Primer sets <italic>CDC42</italic>-F1s-R2s yielded a
PCR product of 690 bp, whereas primer pair <italic>CDC42</italic>-F1s-R1s produced an
amplicon of 630 bp in length, spanning two introns. The primer sets were found
to be largely specific for their target groups. Semi Nested PCRs were
successfully employed, with the first PCR giving only very weak bands but
clear bands being visible with second PCR. With the second amplification the
ratio of concentration of inhibitors <italic>vs.</italic> template DNA allowed
amplification of the product. In general it was difficult to amplify the
desired <italic>CDC42</italic> gene fragments from strains of
<italic>Chaetothyriales,</italic> because of the following: (1) PCRs had frequently to
be repeated using the first amplicon, because the product from the first PCR
was mostly not visible on the gel; (2) Successful PCRs were only obtained with
GoTaq and DMSO, not with BioTaq; (3) Although the PCR programs were optimized
for amplification of <italic>Chaetothyriales</italic>, some strains had to be done
with touch-down programs in order to avoid generation of unspecific bands; (4)
Frequent heavy backgrounds in electropherograms suggested contaminated PCR
products. BLAST searches in GenBank showed that the sequences determined in
this study deviated maximally 7 % from the published <italic>CDC42</italic> sequence
of <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link>,
<italic>E. dermatitidis</italic> (AF162788) (<xref ref-type="bibr" rid="ref32">Ye
& Szaniszlo 2000</xref>).</p></sec><sec><title>Phylogenetic analysis</title><p>A phylogenetic tree was constructed for 32 members of
<italic>Chaetothyriales</italic> using concatenated SSU and LSU ribosomal genes, with
<italic>Conisporium perforans</italic> as an outgroup. This species was shown
previously to be basal to the <italic>Herpotrichiellaceae</italic> by Badali <italic>et
al.</italic> (<xref ref-type="bibr" rid="ref1">2008</xref>). Four of the
five clades recognized previously (e.g.,
<xref ref-type="bibr" rid="ref10">Haase <italic>et al.</italic> 1999</xref>)
were separated with high bootstrap support
(<xref rid="fig2" ref-type="fig">Fig. 2</xref>): <italic>Exophiala
dermatitidis</italic> clade (1), <italic>Fonsecaea pedrosoi</italic> clade (2) and
<italic>Exophiala spinifera</italic> clade (3), while Haase's clade 5 is now known to
represent the ancestral group (lineage 2) close to <italic>Ceramothyrium</italic>(<xref ref-type="bibr" rid="ref1">Badali <italic>et al.</italic> 2008</xref>)
and members of this clade 4 were not included in the present analysis.</p><p>DNA sequences of the partial of <italic>CDC42</italic> were strictly identical for
25 strains (<xref rid="fig1" ref-type="fig">Fig 1</xref>). In these
strains also the intron and the 3<sup>rd</sup> codon positions of the exons
were identical. The strains were distributed randomly over the ribosomal tree
(<xref rid="fig2" ref-type="fig">Fig. 2</xref>). No difference was
detected between <italic>Coniosporium perforans</italic><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=885.96&link_type=CBS">CBS 885.96</ext-link>,
selected as the ribosomal outgroup, and the most derived groups containing
e.g. <italic>Exophiala oligosperma</italic><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=725.88&link_type=CBS">CBS 725.88</ext-link>. In a
number of strains significant deviations were found
(<xref rid="fig1" ref-type="fig">Fig. 1</xref>), which were limited to
the third codon positions and to the introns
(<xref ref-type="table" rid="tbl5">Table 5</xref>). The strains
clustered in four ribosomal clades (1, 2, 3, 5;
<xref rid="fig2" ref-type="fig">Fig. 2</xref>). Nearest neighbours in
the <italic>CDC42</italic> trees were found to belong to the same ribosomal
clades.</p><p><table-wrap position="float" id="tbl5"><label>Table 5.</label><caption><p>Number of mutations in 89 codons of partial <italic>CDC42</italic> coding region of
ten strains which had paralogues. Amino acid changes from orthologue are
listed</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="top"><bold>Accession No.</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Species name</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Total codon</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>1<sup>st</sup> base</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>2<sup>nd</sup> base</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>3<sup>rd</sup> base</bold></th><th colspan="1" rowspan="1" align="left" valign="top"><bold>Amino acid change</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=618.96&link_type=CBS">CBS 618.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia acutiseta</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 38
</td><td colspan="1" rowspan="1" align="left" valign="top"> CAA → TCA/Q → S
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS 232.33</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala heteromorpha</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="center" valign="top"> -
</td><td colspan="1" rowspan="1" align="left" valign="top"> 33
</td><td colspan="1" rowspan="1" align="left" valign="top"> -
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=260.83&link_type=CBS">CBS 260.83</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Cladophialophora carrionii</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 39
</td><td colspan="1" rowspan="1" align="left" valign="top"> CAA → TCG/Q → S
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=126.86&link_type=CBS">CBS 126.86</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Cladophialophora boppii</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 39
</td><td colspan="1" rowspan="1" align="left" valign="top"> CAA → TCG/Q → S
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=286.47&link_type=CBS">CBS 286.47</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Phialophora verrucosa</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 3
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 46
</td><td colspan="1" rowspan="1" align="left" valign="top"> CAA → TCC/Q → S
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=606.96&link_type=CBS">CBS 606.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia epimyces</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> -
</td><td colspan="1" rowspan="1" align="center" valign="top"> -
</td><td colspan="1" rowspan="1" align="left" valign="top"> 35
</td><td colspan="1" rowspan="1" align="left" valign="top"> -
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=207.35&link_type=CBS">CBS 207.35</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala dermatitidis</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2
</td><td colspan="1" rowspan="1" align="center" valign="top"> -
</td><td colspan="1" rowspan="1" align="left" valign="top"> 33
</td><td colspan="1" rowspan="1" align="left" valign="top"> -
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Exophiala dermatitidis</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="center" valign="top"> -
</td><td colspan="1" rowspan="1" align="left" valign="top"> 23
</td><td colspan="1" rowspan="1" align="left" valign="top"> -
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=616.96&link_type=CBS">CBS 616.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia villosa</italic></td><td colspan="1" rowspan="1" align="center" valign="top"> 89
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1
</td><td colspan="1" rowspan="1" align="left" valign="top"> 5
</td><td colspan="1" rowspan="1" align="left" valign="top"> GAC → TAC/D → Y
</td></tr><tr><td colspan="1" rowspan="1" align="center" valign="top"><ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=615.96&link_type=CBS">CBS 615.96</ext-link></td><td colspan="1" rowspan="1" align="left" valign="top"><italic>Capronia munkii</italic></td><td colspan="1" rowspan="1" align="center" valign="top">89
</td><td colspan="1" rowspan="1" align="center" valign="top">-
</td><td colspan="1" rowspan="1" align="center" valign="top">-
</td><td colspan="1" rowspan="1" align="left" valign="top">5
</td><td colspan="1" rowspan="1" align="left" valign="top">-
</td></tr></tbody></table><table-wrap-foot><fn><p>Q glutamin; S serin; D asparaginic acid; Y tyrosin</p></fn></table-wrap-foot></table-wrap></p><p>In order to verify the different motifs obtained, specific primers were
developed for each sequence type of 32 strains. Amplification of the invariant
type was relatively straightforward: the same sequence was obtained
consistently. However, specific amplifications of deviating types were mostly
unsuccessful. Strains <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS
232.33</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=618.96&link_type=CBS">CBS
618.96</ext-link> consistently provided a deviating sequence. Two strains of
<italic>Exophiala dermatitidis</italic>(<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link> and
<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=207.37&link_type=CBS">CBS 207.37</ext-link>),
<italic>Capronia epimyces</italic> (<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=606.96&link_type=CBS">CBS
606.96</ext-link>) and <italic>Cladophialophora boppii</italic>(<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=126.86&link_type=CBS">CBS 126.86</ext-link>)
revealed two deviating sequences, one identical to the core sequence of 25
strains, another clearly deviating in intron and 3<sup>rd</sup> codon
sequences. Cloning results proved that the deviating genotypes were common in
many strains. In some strains the invariant type could not be amplified
(<xref ref-type="table" rid="tbl1">Table 1</xref>)</p><p><fig position="float" id="fig1"><label>Fig. 1.</label><caption><p>Initial tree constructed for 32 members of <italic>Chaetothyriales</italic> based
on partial <italic>CDC42</italic> sequences.</p></caption><graphic xlink:href="121fig1"></graphic></fig></p><p><fig position="float" id="fig2"><label>Fig. 2.</label><caption><p>Tree constructed for 32 members of <italic>Chaetothyriales</italic> obtained from a
ML analysis of two combined loci (SSU and LSU) using RAxML. Bootstrap support
values were estimated based on 500 replicates and are shown above the branches
(thick branch for values ≥ 90 %). The tree was rooted using
<italic>Coniosporium perforans</italic>,
<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=885.95&link_type=CBS">CBS 885.95</ext-link>.</p></caption><graphic xlink:href="121fig2"></graphic></fig></p></sec></sec><sec><title>DISCUSSION</title><p>CDC42 is an essential GTPase that is ubiquitously expressed in eukaryotes,
where it participates in the regulation of the cytoskeleton and a wide range
of cellular processes, including cytokinesis, gene expression, cell cycle
progression, apoptosis, and tumorogenesis. It transduces signals to the actin
cytoskeleton to initiate and maintain polarized growth and to promote
mitogen-activated protein morphogenesis. In filamentous fungi the
<italic>CDC42</italic> gene product is involved in the transition of hyphae to
isodiametrically growing cells. This transition becomes apparent in the
formation of meristematic cells under conditions of environmental stress, such
as with growth on rock, but also concerns muriform cells, the invasive form in
human chromoblastomycosis.</p><p>In the evolution of <italic>Chaetothyriales</italic>, we witness a functional
change from a rock-inhabiting life style prevalent in <italic>Coniosporium</italic>and relatives (<xref rid="fig2" ref-type="fig">Fig. 2</xref>;
Sterflinger <italic>et al.</italic> 1998) to an increased ability to infect humans and
other vertebrates, e.g. in cases of chromoblastomycosis. Both life styles are
characterized by isodiametric growth at least during part of the life cycle.
Thus a major ecological and functional transition has taken place, the same
characteristics of isodiametric expansion becoming applied in an entirely
different setting. Exposure on rock is an ancestral condition in
<italic>Coniosporium</italic> (<xref ref-type="bibr" rid="ref18">Lutzoni <italic>et
al.</italic> 2001</xref>, <xref ref-type="bibr" rid="ref9">Gueidan <italic>et
al.</italic> 2008</xref>), while the pathogenic role in agents of
chromoblastomycosis in <italic>Cladophialophora, Fonsecaea</italic> and
<italic>Phialophora</italic> is a derived condition
(<xref ref-type="bibr" rid="ref1">Badali <italic>et al.</italic> 2008</xref>).
In recent publications the ribosomal tree based on SSU and LSU exhibits a
basal lineage that has become individualized more clearly and referred to as
the separate family <italic>Chaetothyriaceae</italic> within the
<italic>Chaetothyriales</italic> (<xref ref-type="bibr" rid="ref1">Badali <italic>et
al.</italic> 2008</xref>). It contains primarily species from rock, but also
those occasionally colonizing human skin or causing mild cutaneous infections
(Li <italic>et al.</italic> 2008). The majority of species reported from human
chromoblastomycosis (<italic>Phialophora verrucosa</italic> and <italic>Cladophialophora
carrionii</italic>) are united in a separate clade which can be attributed to the
family <italic>Herpotrichiellaceae</italic>(<xref ref-type="bibr" rid="ref28">Untereiner 2000</xref>). An eventual
directional evolution of CDC should reflect both ecologies including
rock-inhabiting and pathogenic life styles.</p><p><fig position="float" id="fig3"><label>Fig. 3.</label><caption><p>Tree constructed for 10 strains based on the partial exon of the
<italic>CDC42</italic> paralogue.</p></caption><graphic xlink:href="121fig3"></graphic></fig></p><p>Our research was initiated with the development of primer sets to detect
<italic>CDC42</italic> in <italic>Chaetothyriales.</italic> We constructed four primer sets
from protein coding regions in which the PCR product would span at least one
intron. Four degenerate primer sets were tested for their ability to amplify
<italic>CDC42</italic> in members of <italic>Chaetothyriales.</italic> Specific primer sets
were subsequently synthesized. Only two sets worked well such that they
amplified PCR products matching the published <italic>CDC42</italic> sequence of
<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=525.76&link_type=CBS">CBS 525.76</ext-link>,
<italic>Exophiala dermatitidis</italic> (AF162788) and that were clearly different
from genes that encode functionally related proteins such as Rac and Rho.
Further amplification in related species was done with these primer sets
(<italic>CDC42</italic>-F1s-R2s and <italic>CDC42</italic>-F1s-R1s).</p><p>The majority of the strains, irrespective of their phylogenetic position
based on rDNA, were invariable in <italic>CDC42</italic>. Part of the strains,
however, showed considerable deviation in non-coding positions and introns
(<xref ref-type="table" rid="tbl5">Table 5</xref>). Since the
relationship with the ribosomal tree of the combined dataset was not directly
obvious, we were uncertain whether the deviating sequences were homologous
with <italic>CDC42</italic>, despite consistent highest scores in GenBank. For most
<italic>CDC42</italic> amplifications, PCRs were difficult and had to be repeated
several times due to frequent heavy background in the electropherogram
suggesting contaminated PCR products. In order to verify the different motifs
obtained, we developed specific primers for each sequence type. PCR efficiency
and sequencing was particularly difficult in the deviating sequences, even
when specific backward primers were used. Amplification of the invariant type
was relatively straightforward: the same sequence was obtained consistently,
with <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=618.96&link_type=CBS">CBS 618.96</ext-link>,
<ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS 232.33</ext-link> as the
only exceptions (<xref ref-type="table" rid="tbl1">Table 1</xref>).
However, specific amplifications of deviating haplotypes were mostly
unsuccessful. In two strains the deviating sequence was obtained in addition
to the invariant type. This strongly suggests that the deviating type was a
paralogue resulting from gene duplication. When the fragments were cloned,
ortho- and paralogues were obtained repeatedly, the orthologue remaining
absent from <ext-link ext-link-type="uri" xlink:href="http://www.studiesinmycology.org/cgi/external_ref?access_num=232.33&link_type=CBS">CBS
232.33</ext-link>.</p><p><fig position="float" id="fig4"><label>Fig. 4.</label><caption><p>Tree constructed for 9 strains based on the sequenced intron of the
<italic>CDC42</italic> paralogue.</p></caption><graphic xlink:href="121fig4"></graphic></fig></p><p>The <italic>CDC42</italic> orthologue evolution shows an unexpected topology. The
analyzed exons showed very limited non-synonymous change throughout the entire
phylogenetic history of <italic>Chaetothyriales</italic>, even between such remote
species as <italic>Coniosporium perforans</italic> and <italic>Exophiala
dermatitidis</italic>. A very strong constraint is observed on the gene, the exon
having remained identical and the protein structure having remained unchanged.
Remarkably, also intron and 3<sup>rd</sup> codon sequences had remained
identical (<xref rid="fig1" ref-type="fig">Fig. 1</xref>). The gene
was subject of strong functional and structural constraints.</p><p>In main traits the evolution of the <italic>CDC42</italic> paralogue was comparable
to that of the scaffold of the multilocus phylogeny of the
<italic>Chaetothyriales</italic> (<xref ref-type="bibr" rid="ref1">Badali <italic>et
al.</italic> 2008</xref>). SSU, LSU and <italic>CDC42</italic> were analysed using
the same set of strains (Figs
<xref rid="fig1" ref-type="fig">1</xref>,
<xref rid="fig2" ref-type="fig">2</xref>,
<xref rid="fig3" ref-type="fig">3</xref>,
<xref rid="fig4" ref-type="fig">4</xref>). SSU and LSU gene evolution
is likely to reflect the phylogenetic history of <italic>Chaetothyriales</italic>. The
paralogue showed considerable evolution in intron and 3<sup>rd</sup> codon
positions, the tertiary structure being relaxed, allowing considerable
mutation. The translated protein sequence, however, showed only limited
non-synonymous change (<xref ref-type="table" rid="tbl5">Table 5</xref>Figs <xref rid="fig3" ref-type="fig">3</xref>,
<xref rid="fig4" ref-type="fig">4</xref>). Non-synonymous change is
observed in <italic>Phialophora</italic> and <italic>Cladophialophora</italic> agents of
chromoblastomycosis, but not in <italic>Fonsecaea</italic> even though all three cause
the same disease. Hence these mutations cannot be linked to selection of an
adapted genotype.</p><p>We hypothesise that both types are expressed with identical proteins, with
gene duplication as an ancient event, and that subsequent structural evolution
has taken place in the paralogue without loss of function. In the majority of
the species investigated only one of the types was detected, suggesting that
ortho- or paralogues may be lost. When non-synonymous <italic>vs.</italic> synonymous
nucleotide divergence is high between species, functional divergence is
assumed to be high, due to positive selection and/or relaxed selective
constraint (e.g. <xref ref-type="bibr" rid="ref6">Friedman & Hughes
2004</xref>). When this ratio is low, the functional properties of the
gene products involved are thought to be conserved, because selective
constraint is high and there is little or no positive selection (e.g.
<xref ref-type="bibr" rid="ref23">Sehgal & Lovette 2003</xref>). In
<italic>CDC42</italic>, both ortho- and paralogues show a very low ratio, indicating
high functional and structural selective constraint.</p><p>In summary, we hypothesise that members of <italic>Chaetothyriales</italic> may
have two duplicate <italic>CDC42</italic> genes, producing exactly the same protein,
but having a conserved <italic>versus</italic> relaxed tertiary structure, which
involves 3<sup>rd</sup> codon positions as well as introns. The evolution that
takes place in the paralogue follows the main traits of evolution seen in
ribosomal genes, but the genes are considerably more variable and difficult to
align. The possibility is not excluded that in the course of evolution one of
the duplicate genes – either ortho- or paralogue – was lost in
individual species. In that case <italic>CDC42</italic> would be a poor phylogenetic
marker, as trees will consist of non-homologous genes. From the fact that both
orthogue and paralogue have a considerable constraint at the protein level, it
can be concluded that any eventual change of function of <italic>CDC42</italic> in the
course of evolution of <italic>Chaetothyriales</italic> (using <italic>CDC42</italic> for
rock-inhabiting or pathogenic life styles, respectively) is not reflected at
the DNA level. The limited number of non-synonymous changes in the paralogue
do not coincide with evolution in species causing chromoblastomycosis, and
thus do not suggest any positive selection of a functional change from
rock-inhabiting life styles to pathogenicity. Any role of <italic>CDC42</italic> as a
virulence factor has thus not been proven.</p></sec></body><back><ack><p>We are indebted to K. Voigt, P.J. Szaniszlo and G. Walther for useful
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