Mycologia, 104(6), 2012, pp. 1351–1368. DOI: 10.3852/12-056
# 2012 by The Mycological Society of America, Lawrence, KS 66044-8897
How well do ITS rDNA sequences differentiate
species of true morels (Morchella)?
Research, United States Department of Agriculture,
Agricultural Research Service, 1815 North University
Street, Peoria, Illinois 61604
Xi-Hui Du
Qi Zhao
Zhu L. Yang
Key Laboratory of Biodiversity and Biogeography,
Kunming Institute of Botany, Chinese Academy of
Sciences, Lanhei Road No. 132, Kunming 650201,
Yunnan Province, P.R. China
Graduate University of Chinese Academy of Sciences,
Beijing 100049, P.R. China
Abstract: Arguably more mycophiles hunt true
morels (Morchella) during their brief fruiting season
each spring in the northern hemisphere than any
other wild edible fungus. Concerns about overharvesting by individual collectors and commercial
enterprises make it essential that science-based
management practices and conservation policies are
developed to ensure the sustainability of commercial
harvests and to protect and preserve morel species
diversity. Therefore, the primary objectives of the
present study were to: (i) investigate the utility of the
ITS rDNA locus for identifying Morchella species,
using phylogenetic species previously inferred from
multilocus DNA sequence data as a reference; and (ii)
clarify insufficiently identified sequences and determine whether the named sequences in GenBank were
identified correctly. To this end, we generated 553
Morchella ITS rDNA sequences and downloaded 312
additional ones generated by other researchers from
GenBank using emerencia and analyzed them phylogenetically. Three major findings emerged: (i) ITS
rDNA sequences were useful in identifying 48/62
(77.4%) of the known phylospecies; however, they
failed to identify 12 of the 22 species within the
species-rich Elata Subclade and two closely related
species in the Esculenta Clade; (ii) at least 66% of the
named Morchella sequences in GenBank are misidentified; and (iii) ITS rDNA sequences of up to six
putatively novel Morchella species were represented in
GenBank. Recognizing the need for a dedicated Webaccessible reference database to facilitate the rapid
identification of known and novel species, we
constructed Morchella MLST (http://www.cbs.knaw.
nl/morchella/), which can be queried with ITS rDNA
sequences and those of the four other genes used in
our prior multilocus molecular systematic studies of
this charismatic genus.
Key words: Ascomycota, biodiversity, biogeography, emerencia, GCPSR, GenBank, phylogeny, species
limits
Karen Hansen
Cryptogamic Botany, Swedish Museum of Natural
History, P.O. Box 50007, SE-10405 Stockholm, Sweden
Hatıra Taşkın
Saadet Büyükalaca
Department of Horticulture, Faculty of Agriculture,
Çukurova University, Adana 01330, Turkey
Damon Dewsbury
Jean-Marc Moncalvo
Department of Ecology and Evolutionary Biology,
University of Toronto, Toronto, Ontario M5S2C6,
Canada
Department of Natural History, Royal Ontario
Museum, and Department of Ecology and Evolutionary
Biology, University of Toronto, Toronto,
Ontario M5S2C6, Canada
Greg W. Douhan
Department of Plant Pathology and Microbiology,
University of California Riverside, Riverside,
California 92521
Vincent A.R.G. Robert
Pedro W. Crous
CBS-KNAW Fungal Biodiversity Center, Utrecht,
the Netherlands
Stephen A. Rehner
Systematic Mycology and Microbiology Laboratory,
United States Department of Agriculture, Agricultural
Research Service, Beltsville, Maryland 20705
Alejandro P. Rooney
Crop Bioprotection Research Unit, National Center for
Agricultural Utilization Research, United States
Department of Agriculture, Agricultural Research
Service, 1815 North University Street, Peoria,
Illinois 61604
Stacy Sink
Kerry O’Donnell1
Bacterial Foodborne Pathogens and Mycology Research
Unit, National Center for Agricultural Utilization
INTRODUCTION
With the notable exception of true truffles in the
genus Tuber (Bonito et al. 2010), few fungal genera are
Submitted 17 Feb 2012; accepted for publication 16 May 2012.
1
Corresponding author. E-mail: [email protected]
1351
1352
MYCOLOGIA
as synonymous with epicurean cuisine as true morels
(Morchella, phylum Ascomycota). Literally thousands
of mycophiles scour mixed hardwood and coniferous
forests throughout the northern hemisphere in search
of these highly prized edible fungi during their short
and often sporadic fruiting season each spring. Adding
to their huge popularity is the dramatic growth of
commercial harvesting of morels into highly lucrative
cottage industries in several morel-rich countries,
including China, Turkey and the United States (Pilz
et al. 2007), together with significant advances toward
their cultivation on a commercial scale (Ower et al.
1986, Zhao et al. 2009).
Due to the increasing commercial demand, it is
imperative that comprehensive studies are conducted
to develop informed science-based conservation
policies and management practices that will ensure
that species diversity is maximally preserved and
commercial harvests are sustainable (see Pilz et al.
2007 and references therein). To this end, our
multilocus molecular phylogenetic analyses have
identified at least 62 phylogenetically distinct species
worldwide based on their genealogical exclusivity (Du
et al. 2012; O’Donnell et al. 2011; Taşkın et al. 2010,
2012). Robust hypotheses of their species limits,
employing genealogical concordance/discordance
phylogenetic species recognition (GCPSR; Dettman
et al. 2003, Taylor et al. 2000), provides the requisite
framework for assessing the utility of ITS rDNA for
identifying species so that their geographic distribution and area of endemism can be critically assessed
and so that unknown taxa can be detected and
identified for further taxonomic evaluation.
Even though the ITS rDNA region has been used as
the sole locus in more studies than any other for
assessing Morchella genetic diversity (Pagliaccia et al.
2011, Stefani et al. 2010, Taşkın et al. 2012 and
references therein), a search of GenBank using
emerencia (Ryberg et al. 2009) revealed that most
sequences were insufficiently identified (n 5 190),
and in the near absence of type studies, binomials
appear to have been arbitrarily applied to the vast
majority of those designated fully identified (n 5 127)
sensu Nilsson et al. (2005). Thus, the present study of
Morchella was conducted to: (i) determine how well
ITS rDNA sequence data resolve species we previously
delimited via GCPSR, (ii) assess whether the fully
identified sequences in GenBank were identified
correctly and identify ones classified as insufficiently
identified, (iii) mine metadata to better understand
species diversity and their geographic distributions
and (iv) develop a dedicated, Web-accessible informational database, including references to voucher
specimens and/or cultures (Agerer et al. 2000), that
aids DNA sequence-based identifications of true
morels via the Internet. We have accomplished the
latter objective by constructing Morchella MLST
(multilocus sequence typing, http://www.cbs.knaw.
nl/morchella/) and populated it with the data we
generated both in the current and our prior multilocus molecular systematic studies of this charismatic
genus.
MATERIALS AND METHODS
Source of sequence data.—We generated ITS sequence data
from 553 collections during extensive surveys of China (Du
et al. 2012), Turkey (Taşkın et al. 2010, 2012) and a global
survey of the genus (O’Donnell et al. 2011). In addition,
312 sequences were retrieved from GenBank with the genus
search tool in emerencia (Nilsson et al. 2005, Ryberg et al.
2009). Five additional sequences were retrieved by emerencia, including three of M. rufobrunnea, although one of
these (GQ304946) was misidentified as M. crassipes, possibly because the pileus of this species turns yellow during ascosporogenesis. The other two GenBank accessions
(EU480151.1, U61390.1) were unidentifiable sequences
unrelated to Morchella based on BLAST queries of
GenBank. Sequences of M. rufobrunnea were not analyzed
further because they represent a highly divergent basal
sister to the rest of Morchella. Thus, a total of 865 Morchella
ITS rDNA nucleotide sequences were analyzed.
Molecular methods.—Total genomic DNA was extracted
from herbarium specimens or freeze-dried mycelium
cultivated from pure cultures using a CTAB protocol
(Gardes and Bruns 1993, O’Donnell et al. 2011). PCR
amplification and sequencing of the nuclear ribosomal
transcribed spacer region (ITS rDNA) was conducted using
the ITS5 3 ITS4, ITS1 3 ITS4 (White et al. 1990) or ITS5 3
NL4 (O’Donnell et al. 2011) primer pairs. To completely
sequence the latter amplicon ITS4 and NL1 also were used
as internal sequencing primers (O’Donnell et al. 2011). All
PCR amplifications were performed with Platinum Taq
DNA Polymerase High Fidelity (Invitrogen Life Technologies, Carlsbad, California), after which amplicons were
purified with Montage96 filter plates (Millipore Corp.,
Bellerica, Massachusetts), and then sequenced with ABI
BigDye chemistry 3.1 (Applied Biosystems). ABI XTerminator was used to purify the sequencing reactions before
running them on an ABI 3730 genetic analyzer as described
in O’Donnell et al. (2011).
Sequence analyses.—The Morchella ITS rDNA sequences we
generated (n 5 553) and those downloaded from GenBank
(n 5 312) were too divergent to align across the breadth of
the genus. Therefore, the following three clade-specific
alignments were constructed: (i) Esculenta Clade (n 5 222:
164 new + 58 from GenBank, SUPPLEMENTARY TABLE I), (ii)
Elata Clade (n 5 200: 110 new + 90 from GenBank,
SUPPLEMENTARY TABLE I), and (iii) Elata Subclade (n 5 443:
348 + 164 from GenBank by others, SUPPLEMENTARY TABLE
III). A fourth clade of highly divergent M. rufobrunnea
sequences (n 516: 13 new + three from GenBank,
SUPPLEMENTARY TABLE IV) also was identified. However,
DU ET AL.: MORCHELLA ITS RDNA
because these sequences were highly divergent from the
aforementioned three clades, they were excluded from
further analysis. Sequence chromatograms generated in
this study were imported into Sequencher 4.9 (Gene Codes
Corp., Ann Arbor, Michigan), where they were edited,
aligned and exported as NEXUS files. Assignment of the
Morchella sequences downloaded from GenBank using
emerencia (Nilsson et al. 2005) to one of the four
aforementioned clades was based on an initial maximum
parsimony analysis conducted in PAUP* 4.0b10 (Swofford
2002). Sequences in each of the three clade-specific datasets
were aligned with MUSCLE 3.8 (Edgar 2004) in SeaView
4.3.0 (http://pbil.univ-lyon1.fr/software/seaview) and then
improvements in the alignments were made visually with
TextPad 5.1.0 for Windows (http://textpad.com/). To
improve phylogenetic resolution sequences of M. tomentosa
(Mel-1) and M. steppicola (Mes-1), the earliest diverging
species within the Elata and Esculenta clades respectively,
were used to root these phylogenies. Sequences of Mel-11
and Mel-25, the sister group of the Elata Subclade (Du et al.
2012; O’Donnell et al. 2011; Taşkın et al. 2010, 2012), were
used to root this phylogeny.
In addition to excluding two unidentifiable highly
divergent sequences from the emerencia search of GenBank
(EU480151.1, U61390.1), the 59 or 39 ends of three
sequences obtained from GenBank were trimmed to
remove poor sequence (93 bp from 59 end of FJ237237,
194 bp from 39 end of GQ223457, 101 bp from 59 end of
EU834821). To construct partitions that only contained
unique haplotypes for the final analyses, COLLAPSE 1.2
(http://darwin.uvigo.es/software/collapse) was used to
identify haplotypes within each dataset. Instead of using
percent identity to place unidentified sequences within
similarity clusters (Bonito et al. 2010, Horton and Bruns
2001), haplotypes were identified to species, where possible,
by including ITS rDNA sequences of the phylogenetic
species identified with multilocus DNA sequence data (Du
et al. 2012; O’Donnell et al. 2011; Taşkın et al. 2010, 2012).
Each partition was analyzed via maximum parsimony
(MP) in PAUP* (Swofford 2002) to obtain tree statistics,
including number of parsimony informative characters
(PIC); clade support was assessed based on 1000 MP
pseudoreplicates of the data (O’Donnell et al. 2011). Due
to the large number of MPTs, single phylograms were
chosen randomly to present the metadata and clade
support for the Esculenta (FIG. 1) and Elata Clades
(FIG. 2). However, due to the large number of ITS
haplotypes, a 70% majority rule bootstrap consensus was
chosen to present the Elata Subclade phylogeny (FIG. 3).
All DNA sequence data generated in this study was
deposited in GenBank (JQ723016–JQ723151), TreeBASE
(S12489, Tr51345-Tr51347) and Morchella MLST at CBSKNAW (http://www.cbs.knaw.nl/morchella/). In addition,
accession numbers identifying voucher specimens deposited in herbaria and cultures are listed (SUPPLEMENTARY
TABLES I–IV).
Construction of Morchella MLST.—This database was
created with the BioloMICS software (Robert et al. 2011).
All available molecular, specimen and/or culture data were
1353
PHYLOGENETICS
imported into the database. The database can be queried by
keywords or using one of the two available identification
tools. The first one consists of a modified version of BLAST
(Altschul et al. 1990). Single sequences can be aligned
against the reference database and ranked according to
a combination of parameters including similarity, BLAST
score, overlap percentage between query and reference
sequences and number of fragments. The second tool is
called polyphasic identification because it allows aligning
(pairwise alignments) one or several sequences from
different loci at the same time. A global similarity coefficient
is computed with this formula (Gower 1971):
n
1 X
Sjk ~ P
Si Wi
n
Wi i~1
i~1
Where Sjk is the global similarity coefficient for the
comparison of records j and k; Si is the local similarity
coefficient for the pairwise alignment of locus I; Wi is the
weight of locus I (no differential weighting is applied in the
Morchella database); n is the number of loci in the similarity
comparison. Results are ranked according to the combination of this global similarity coefficient, the number of loci
and the overlap percentage between query and reference
sequences. Phenetic trees based on one of the following
methods can be produced dynamically to help end users
locate the clade to which the unknown specimen belongs:
UPGMA, WPGMA, single linkage, complete linkage,
UPGMC, WPGMC, ward or neighbor joining using a fixed
model (Saitou and Nei 1987).
RESULTS
Esculenta clade.—The 222-sequence Esculenta Clade
dataset included 58 sequences we deposited in
GenBank (O’Donnell et al. 2011; Taşkın et al. 2010,
2012) and 164 collected in the present study
(SUPPLEMENTARY TABLE I). COLLAPSE 1.2 analysis of
the 222-sequence dataset (1349 bp alignment) identified 125 unique haplotypes. The latter dataset was
used for all subsequent analyses, after 253 nucleotide
positions within the ITS1 were excluded as ambiguously aligned. Maximum parsimony (MP) analysis of
the 125-sequence Esculenta Clade dataset recovered
. 10 000 equally most parsimonious trees (MPTs)
1045 steps long (FIG. 1, consistency index 5 0.67,
retention index 5 0.96). The Esculenta Clade
phylogeny was rooted on sequences of M. steppicola
Mes-1 based on more inclusive analyses (O’Donnell
et al. 2011). Of the 27 species previously identified
with multilocus GCPSR, we were able to apply names
with confidence only to M. steppicola and five species
endemic to North America (FIG. 1, Kuo et al. 2012).
Parsimony analysis of the Esculenta Clade dataset
indicated that 212/222 (95.5%) of ITS sequences
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MYCOLOGIA
FIG. 1. One of . 10 000 equally most parsimonious trees (MPTs) inferred from Esculenta Clade (yellow morels) ITS sequences,
rooted on sequences of Morchella steppicola (Mes-1), the basal-most member of this clade (O’Donnell et al. 2011). The phylogram contains
the 125 unique haplotypes identified within the 222-sequence dataset (SUPPLEMENTARY TABLE I). Each sequence is identified by GenBank
or herbarium accession number or laboratory code (SUPPLEMENTARY TABLE I), geographic origin, name used as deposited in GenBank
and number of sequences in parentheses that comprise each haplotype represented by two or more sequences. ITS sequences identified
by an asterisk were used as a reference for each phylospecies based on prior multilocus GCPSR analysis (Du et al. 2012; O’Donnell et al.
2011; Taşkın et al. 2010, 2012). Because binomials can be applied with confidence to only six of the species, each phylogenetic species (i.e.
phylospecies) is identified by Mes followed by a unique Arabic number. Numbers to the right of the phylospecies identify the size and
number of sequences (in subscript) of the (ITS1) (5.8SrDNA) (ITS2) (complete ITS region) determined by DNA sequence analysis. Note
that ‘‘s’’ indicates the sequence was short and ‘‘?’’ indicates that the species and/or ITS length is unknown. The number above internodes
represents maximum parsimony bootstrap support $ 70% based on 1000 pseudoreplicates of the data. Phylospecies Mes-23 and Mes-24
cannot be distinguished with ITS data. Mes-27 from Sichuan Province, China, is featured.
DU ET AL.: MORCHELLA ITS RDNA
FIG. 1.
PHYLOGENETICS
1355
Continued.
could be identified to species. The exception involved
GenBank sequences of nine collections from China
and one from India, none of which were full length
(SUPPLEMENTARY TABLE I, FIG. 1). In analyses of the
Esculenta Clade dataset, five collections of Mes-23 or
Mes-24 from China could not be distinguished when
ambiguously aligned regions were included or excluded. In addition, three collections from Xingiang and
one from Jilin, which we listed as Mes-?, could be
Mes-21. As with the aforementioned unidentified
collections, additional data is needed to determine
the identity of one collection from India listed as Mes-?.
Six of the species (SUPPLEMENTARY TABLE I; i.e. M.
diminutiva Mes-2, M. esculentoides Mes-4, Mes-6, Mes-8,
Mes-9 and Mes-16) were represented by 15–42 sequences and comprised 132/222 (59.5%) of Esculenta Clade
sequences. Of the 27 Esculenta Clade species, M.
esculentoides, the most common yellow morel endemic
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MYCOLOGIA
FIG. 2. Phylogenetic relationships within Elata Clade (black morels) inferred by MP analysis of 60 unique ITS haplotypes
identified within the 200-sequence dataset (SUPPLEMENTARY TABLE II). The phylogram is one of . 10 000 MPTs 373 steps long,
rooted on sequences of Morchella tomentosa (Mel-1). Haplotype sequences are identified by a herbarium or GenBank accession
number or laboratory number (SUPPLEMENTARY TABLE II), geographic origin, the name used for the deposit in GenBank and
the number of sequences (in parentheses) that comprise each non-singleton haplotype. An asterisk identifies ITS sequences
that were used as a reference for each phylogenetic species (i.e. phylospecies) previously resolved by multilocus GCPSR data.
MP bootstrap support is identified above internodes when it was $ 70%. Nine of the species can be identified by binomials; the
10 known phylospecies (O’Donnell et al. 2011) are identified by Mel followed by a unique Arabic number. Two putatively
novel species represented by ITS sequences in GenBank are identified by NEW-1? and NEW-2? The size and number of
sequences of the (ITS1) (5.8S rDNA) (ITS2) (entire ITS region) analyzed is indicated to the right of each species. Note that
ITS sequences for M. populiphila (Mel-5) and NEW-1? from China are not full length. Mel-10 from Yunnan Province, China,
is featured.
DU ET AL.: MORCHELLA ITS RDNA
to North America, was represented by the most
sequences (n 5 42). By contrast, we were unable to
obtain ITS sequence data from collections of the
European endemic Mes-5, the only known phylogenetic species missing from the Morchella ITS dataset.
With ITS rDNA sequences of species identified
previously via multilocus GCPSR as a reference,
separate analyses using maximum parsimony and
COLLAPSE 1.2 revealed that 58 Esculenta Clade
sequences deposited in GenBank by other researchers
comprised at least 10 species (SUPPLEMENTARY TABLE
I). Three of the 58 accessions in GenBank were listed
as Morchella sp., with the remaining 55 reported as M.
crassipes (n 5 29), M. esculenta (n 5 16), M. spongiola
(n 5 9) and M. vulgaris (n 5 1). Our results,
however, indicate the former three names have been
misapplied broadly given that sequences of eight
species were deposited in GenBank as M. esculenta
and M. crassipes and four as M. spongiola (SUPPLEMENTARY TABLE I). All species represented by sequences deposited under the latter three names are nested
within the Esculenta Clade, whereas those listed as M.
esculenta included three species within the Esculenta
Clade, two within the Elata Clade and three within the
Elata Subclade (SUPPLEMENTARY TABLES I–III).
Furthermore, our analyses indicate that, at best,
only 18/55 (32.7%) of the Esculenta Clade sequences
in GenBank are correctly identified to species. We
found that 12 of the 16 accessions deposited as M.
esculenta are the putative North American endemic
M. esculentoides (Mes-4). Moreover, it is plausible
that some of the 18 sequences that we assumed to be
correct are actually misidentified. For these sequences
to be identified correctly the sequences of Mes-16
(n 5 10), Mes-17 (n 5 7) and M. esculentoides (Mes-4,
n 5 1) respectively would have to be authentic for M.
crassipes, M. spongiola and M. vulgaris. Studies of
original material, epi- or neo-typification of recent
material and additional sampling of Morchella in
Europe are needed to test whether this assumption is
correct. We were able to obtain complete sequences
of the ITS rDNA region for 24 of the 27 species within
the Esculenta Clade in the present study. In addition
to missing ITS rDNA data from the European
endemic Mes-5, the ITS1 and ITS2 were sequenced
only partially in GenBank accession GQ228465 from
India, the ITS1 was missing in Mes-18 from Turkey
and it was represented by a partial sequence in Mes-20
from Yunnan Province, China. Although six sequences downloaded from GenBank possessed insertions
(1–11 bp) and deletions (1–7 bp) within the 5.8S
rDNA (SUPPLEMENTARY TABLE V), these indels may
represent sequencing errors because the length of
this gene appears to be fixed at 156 bp in Morchella
based on the sequences we generated from 663
PHYLOGENETICS
1357
collections. However, without the ability to resequence the individuals from which these sequences
were generated, we cannot discount the possibility
that they represent rare variants within their populations of origin.
Analyses of the present dataset have highlighted
several noteworthy features of the ITS1 and ITS2
spacers within the Esculenta Clade. Mapping the
size of the ITS regions on the molecular phylogeny
(FIG. 1) revealed that the ITS1 more than doubled in
length during the evolutionary diversification of this
clade; the two basal-most lineages possessed the
shortest ITS1 spacer (285 bp in M. steppicola Mes-1
and 285–286 bp in M. virginiana Mes-3), whereas it
was largest in the two most derived species (610–
611 bp in Mes-8 and 617 bp in Mes-9). By contrast, the
ITS2 was remarkably conserved in length within the
Esculenta Clade in that the shortest (371 bp in Mes20) and longest (387 bp in Mes-25) spacers differed
by only 16 bp (FIG. 1). Intraspecific variation was
detected only within the ITS1 (1 bp) in five species
and ITS2 (1–2 bp) in four (FIG. 1). Analyses of the
ITS regions revealed that the ITS1 (313 PIC/501 bp
5 0.624 PIC/bp) was 2.77 times more informative
phylogenetically than the ITS2 (96 PIC/426 bp 5
0.225 PIC/bp). By comparison, the 5.8S rDNA,
excluding 13 putatively erroneous indel containing
nucleotide positions, was highly conserved in that it
possessed only 8 PIC/156 bp (5 0.051 PIC/bp).
To assess whether sequences of the ITS1 or ITS2, or
portions of these two spacers could be used to identify
Esculenta Clade species, we coded the 59, middle and
39 thirds of the ITS1 (59 5 251 bp, middle 5 251 bp,
39 5 252 bp) and 59 and 39 halves of the ITS2 (59 5
213 bp, 39 5 213 bp) as separate character sets and
analyzed them and the separate spacers individually
with maximum parsimony. These analyses revealed
that the ITS1 and 59 third of the ITS1 distinguished
the most species (22/26), whereas the 39 third of the
ITS1 (16/26) and 59 (16/26) and 39 (14/26) halves of
the ITS2 were the least discriminating (SUPPLEMENTARY TABLE VI).
Elata clade.—The 200 sequences in the Elata Clade
dataset included 90 that we downloaded from
GenBank and 110 generated in the present study
(SUPPLEMENTARY TABLE II). Validly published binomials can be applied with confidence to only three of
the species (i.e. 5 M. tomentosa Mel-1, M. semilibera
Mel-3 and M. punctipes Mel-4); however, names for six
species endemic to North America have been
proposed (FIG. 2, Kuo et al. 2012). Using COLLAPSE
1.2, we identified 60 unique haplotypes within the 200
sequence Elata Clade dataset (996 bp alignment)
for subsequent MP phylogenetic analysis. Before
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MYCOLOGIA
FIG. 3. Bootstrapped 70% majority rule cladogram inferred from maximum parsimony analysis of 106 unique ITS
haplotypes in the 443-sequence Elata Subclade (black morels) dataset (SUPPLEMENTARY TABLE III). Sequences of the sister taxa
Mel-11 and Mel-25 were used to root the phylogeny. Each haplotype is identified by GenBank or herbarium accession number
or laboratory code (SUPPLEMENTARY TABLE III), geographic origin and number of sequences (in parentheses) that comprise
each non-singleton haplotype. Haplotypes identified by an asterisk represent ITS sequences used as a reference for each
phylogenetically distinct species based on multilocus support for their genealogical exclusivity. Because binomials can be
DU ET AL.: MORCHELLA ITS RDNA
FIG. 3.
PHYLOGENETICS
1359
Continued.
r
applied with confidence to only four of the species, each phylogenetic species (i.e. phylospecies) is identified by Mel followed
by a unique Arabic number. Note that ITS sequences of four putatively novel Elata Subclade species (NEW-3?-NEW-6?) were
discovered in GenBank. The size and number of sequences of the (ITS1) (5.8SrDNA) (ITS2) (complete ITS region)
determined by DNA sequence analysis is indicated to the right of each phylospecies, except for five sequences where the ITS1
is short (s). Internodes that were supported by $ 70% maximum parsimony bootstrap intervals are indicated. Note that several
groups of phylospecies cannot be distinguished using ITS sequence data, with Mel-17/Mel-19/Mel-20/Mel-34, indicated by
gray highlight being the most notable. Mel-20 from Yunnan Province, China, is featured.
1360
MYCOLOGIA
conducting phylogenetic analyses, 102 ambiguously
aligned nucleotide positions within the ITS1 and 27
positions within the ITS2 were excluded from the
dataset. To minimize the number of ambiguously
aligned nucleotide characters, Elata Clade phylogeny
was rooted on sequences of Mel-1 based on more
inclusive analyses (O’Donnell et al. 2011). MP analysis
of the Elata Clade dataset revealed that all 200
sequences could be identified to species with the
ITS rDNA data (SUPPLEMENTARY TABLE II). However,
the genealogical exclusivity of two putatively novel
species designated NEW-1? and NEW-2? needs to be
accessed via GCPSR. Four species composed 79%
(158/200) of the sequences, with M. capitata (Mel-9,
n 5 53) and M. importuna (Mel-10, n 5 56) being the
best represented, followed by M. tomentosa (Mel-1, n
5 25) and M. septimelata (Mel-7, n 5 24). By contrast,
seven of the Elata Clade species each were represented by fewer than eight sequences (SUPPLEMENTARY
TABLE II), including two putatively novel species
designated NEW-1? from China and NEW-2? from
China and Germany (SUPPLEMENTARY TABLE II).
Moreover, only one-quarter (22/90) of the Elata
Clade sequences in GenBank were identified by
validly published binomials. Analysis of these accessions revealed that M. importuna (Mel-10) was
deposited under four names, one sequence of M.
sextelata (Mel-6) and one of M. importuna (Mel-10)
were deposited as M. esculenta and three sequences
of M. semilibera (Mel-3) were deposited as M. gigas.
Although the six sequences of M. tomentosa (Mel-1)
are authentic for this species, type studies are needed
to determine whether the eight sequences of Mel-10
that we obtained from GenBank as M. conica are
identified correctly. Even if we assume that they are,
at least 8/22 (36.4%) of the Elata Clade sequences
in GenBank can be inferred to be misidentified.
However, if M. conica is shown to represent a species
other than Mel-10, then 14–16 (63.6–72.7%) of the
sequences from this clade can be inferred to be
misidentified in GenBank.
The Elata Clade dataset contained complete ITS
sequences for all of the species except M. populiphila
(Mel-5), where approximately one-half of the ITS1
spacer region was missing (FIG. 1). In contrast to what
was observed within the Esculenta Clade, the ITS
region was largest in the two basal-most taxa, M.
tomentosa (Mel-1, 834–835 bp) and M. frustrata (Mel2, 903–904 bp), and smallest in the five most derived
taxa, where it was 626–655 bp. By mapping the size of
the ITS1/5.8S rDNA/ITS2 on the ITS phylogeny
(FIG. 2) and the topologically concordant multilocus
phylogeny (O’Donnell et al. 2011 FIG. 3), our results
indicate that the ITS1 and ITS2 spacers both have
contracted during the evolutionary diversification of
the Elata Clade. Comparison of the two species with
the largest (M. tomentosa Mel-1, 834–835 bp and M.
frustrata Mel-2, 903–904 bp) and smallest (M.
septimelata Mel-7, 626–628 bp and Morchella sp. Mel8, 624 bp) spacers revealed that the ITS1 and ITS2
contracted respectively by a factor of 1.7–1.9 and 1.2–
1.3 during the evolution of the Elata Clade. Minor
length variation was observed within the ITS1 (2–5 bp)
in five species and within the ITS2 (2–3 bp) in three
(FIG. 2). Assessment of phylogenetic signal within the
ITS regions revealed that, in contrast to the Esculenta
Clade, the ITS2 (138 PIC/308 bp 5 0.448 PIC/bp)
was 1.08 times more phylogenetically informative
than the ITS1 (166 PIC/402 bp 5 0.413 PIC/bp)
within the Elata Clade. The 5.8S rDNA, by comparison, was highly conserved, possessing only 5 PIC/
156 bp (5 0.032 PIC/bp).
Finally, to determine how well the ITS1 and ITS2,
and equally divided portions of these two spacers,
performed in distinguishing species, we coded the 59
and 39 halves of the ITS1 (59 5 252 bp, 39 5 252 bp)
and ITS2 (59 5 168 bp, 39 5 167 bp) as separate
character sets and analyzed them and alignments of
each spacer separately via maximum parsimony. The
results of these analyses indicated that sequences from
each of these partitions can be used to identify all the
species or all but two closely related species pairs within
the Elata Clade (SUPPLEMENTARY TABLE VI).
Elata subclade.—The 443-sequence Elata Subclade
dataset included 164 sequences downloaded from
GenBank and 279 sequences generated in the present
study. We were able to apply binomials with confidence to only four of the 22 phylogenetically distinct
species previously identified within this subclade
using multilocus DNA sequence data (Du et al.
2012; O’Donnell et al. 2011; Taşkın et al. 2010,
2012). One of these was for M. angusticeps (Mel-15,
Kuo et al. 2012), a species restricted to eastern North
America, and three others were names recently
proposed for North American endemics (Kuo et al.
2012). Analysis using COLLAPSE 1.2 identifed 106
unique haplotypes (FIG. 3, SUPPLEMENTARY TABLE V).
The MP and COLLAPSE 1.2 analyses revealed that
only 10/22 species within this clade could be
identified with ITS rDNA sequence data (FIG. 3). No
sequences were excluded as ambiguously aligned
from the Elata Subclade dataset. Therefore, phylogenetic analyses and BLAST queries using ITS rDNA
sequences cannot be used to definitively identify 10
species because they share an identical allele with at
least one other species (FIG. 3).
The MP analysis also suggested that 21 of the
sequences obtained from GenBank might represent
as many as four putatively novel species that we
DU ET AL.: MORCHELLA ITS RDNA
designated NEW-3?–NEW-6? (F IG. 3); however,
GCPSR-based analyses are needed to determine
whether these lineages are genealogically exclusive.
One of these species (NEW-4?) appears to be a
previously unknown species on the basis of phylogenetic analyses of ITS + LSU rDNA sequence data
(Pagliaccia et al. 2011). Forty of the Elata Subclade
sequences downloaded from GenBank were deposited as Mel-12 (Pagliaccia et al. 2011); the remaining 55
sequences could not be identified to species (SUPPLEMENTARY TABLE III). Although 95 Elata Subclade
sequences were represented in GenBank, only 50 of
these were identified to species. However, our
analyses indicated that at least 35 of the 50 identifications are incorrect. Seven species were listed as M.
elata, four as M. angusticeps and three as M. conica
and M. esculenta (SUPPLEMENTARY TABLE III).
Complete sequences of the ITS rDNA region were
obtained for all species within the Elata Subclade,
except for an unassigned species (Morchella sp.,
GenBank accession number AF000970) and the
aforementioned putative species NEW-4? (GenBank
accession numbers JF319903–JF319906), which encompassed ITS sequences that were missing approximately 17 bp from the 59 end of the ITS1
(SUPPLEMENTARY TABLE III). Although the ITS1 spacer
within this subclade showed slight variation (213–
221 bp) in three species (FIG. 3), the ITS2 was fixed at
277 bp. The latter spacer is identical in length to the
ITS2 in Mel-11 and Mel-25, the sisters of the Elata
Subclade (O’Donnell et al. 2011), which was used to
root the Elata Subclade phylogeny. Intraspecific
variation was observed only within the ITS1 of Mel13 (213 or 217 bp), Mel-14 (217 or 221 bp) and a
putatively novel species (NEW-5?, 215–216 bp) represented by two collections incorrectly deposited in
GenBank as M. elata from India (SUPPLEMENTARY
TABLE III, Kanwal et al. 2011). A comparison of
phylogenetic signal within the ITS regions showed
that the ITS1 (65 PIC/231 bp 5 0.281 PIC/bp) was
2.3 times more informative than the ITS2 (34 PIC/
279 bp 5 0.122 PIC/bp). By contrast, only two
synapomorphic positions were found within the
highly conserved 5.8S rDNA (2 PIC/156 bp 5 0.013
PIC/bp).
Morchella MLST.—The Web-accessible Morchella
MLST database hosted at the CBS-KNAW was
constructed to aid molecular identification of morels
via the Internet with BLAST queries or phylogenetic
analysis. In contrast to GenBank, the Morchella
database will include only accession sequences for
which voucher specimens or cultures are available
from publically accessible herbaria or culture collections. This criterion is the same as that employed by
PHYLOGENETICS
1361
the FUSARIUM-ID (Geiser et al. 2004) and Fusarium
MLST (http://www.cbs.knaw.nl/fusarium) databases.
In addition, the Morchella MLST database houses
metadata that can be downloaded for further study.
The current version of Morchella MLST contains
broad taxonomic sampling from four loci (ITS and
LSU rDNA, RPB1, RPB2 and EF-1a). Two options are
available for identifying sequences via BLAST queries
of the database: (i) a single sequence from one of the
four loci can be compared with those housed in
Morchella MLST or Morchella MLST and GenBank
databases or (ii) multiple sequences can be used,
which is recommended especially to obtain a definitive identification for species within the Elata
Subclade. In addition to being able to conduct
BLAST queries, neighbor joining phylogenetic analysis can be conducted to infer evolutionary relationships of an unknown to species represented in the
database. The Website contains a portal by which
sequences can be added to the database under the
conditions that vouchers, including accession numbers, are made available from publically accessible
herbaria and/or culture collections.
DISCUSSION
Identification of Morchella phylogenetic species using
ITS rDNA sequence data.—The present study was
modeled after one conducted on a grass-associated
clade of Colletotrichum (Crouch et al. 2009) in which
species limits were inferred from multilocus GCPSR
to provide a framework for determining the utility of
ITS data for differentiating species from each other
via molecular phylogenetics and by BLAST queries of
a dedicated database for morels, Morchella MLST.
The primary objective of the present study was to
generate an inclusive set of taxonomically validated
ITS sequences for morels and to evaluate the accuracy
of this single, widely used locus for the dual purpose
of phylogenetic reconstruction and species identification (Du et al. 2012; O’Donnell et al. 2011; Taşkın
et al. 2010, 2012). To assess their utility within
Morchella we generated 553 ITS rDNA sequences
representing 61 of the 62 known phylogenetically
distinct species and downloaded another 312 sequences from GenBank with emerencia. Although
efforts to obtain ITS rDNA sequence data from the
European endemic Mes-5 were unsuccessful, we
predict that the ITS length will match that of the
closely related species Mes-6/Mes-7/Mes-21, where it
is conserved at 1089 bp.
Our analyses elucidated the strengths and limitations of ITS as the sole locus for distinguishing species
within Morchella. The present study revealed that the
ITS data could be used to distinguish 77.4% (48/62)
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MYCOLOGIA
of the known phylogenetic species within Morchella.
However, only 68.4% (592/865) of the sequences
analyzed could be assigned to one of those species.
ITS sequences resolved most species within the Elata
and Esculenta clades, but fewer than half of the species
within the Elata Subclade. Moreover, as a result of
mining ITS data in GenBank, the existence of six
putatively novel species was revealed, although their
genealogical exclusivity needs to be critically accessed
via GCPSR. Two of the potentially novel species were
nested within the Elata Clade, where ITS sequences
differentiated the 10 known phylogenetic species (Mel1 through Mel-10) and all 200 sequences analyzed from
this clade. Another noteworthy finding was that
sequences of the ITS1 (504 bp) or ITS2 (335 bp)
and even the 59 or 39 halves of either spacer resolved all
of the species, or all but two closely related sister
species within the Elata Clade (SUPPLEMENTARY TABLE
VI). We also discovered that ITS data performed well
within the Esculenta Clade in that 92.3% (24/26) of
the phylogenetic species were diagnosable and 95.5%
(212/222) of the sequences analyzed could be
assigned to one of these species. In addition, our
analyses revealed that sequences of the ITS1 (754 bp),
or only a 251 bp fragment comprising the 39 third of
the ITS1, could be used to identify 22/26 of the species
within this clade (SUPPLEMENTARY TABLE VI). While it
was possible to differentiate close to three-quarters of
the Esculenta Clade species (19/26) with complete
ITS2 sequences, the 59 and 39 halves of the ITS2
differentiated only 50–60% of the species.
One important finding is ITS sequence data has
limited utility in differentiating species within the
species-rich Elata Subclade. Counting sequences of
two sister species used to root the phylogeny (i.e. Mel11 and Mel-25), our results show that only 50% (12/
24) of species in the Elata Subclade + Mel-11/Mel-25
could be identified with the ITS data and only 44.7%
(198/443) of the sequences analyzed could be
assigned to one of these species. We attribute the
failure of the ITS to resolve species within the Elata
Subclade to its relatively recent origin in the middle
Miocene, about 12.15 Mya (95% HPD: 9.46–15.48)
(Du et al. 2012) and to its subsequent rapid radiation
into a species-rich lineage comprising one-third of the
true morels (O’Donnell et al. 2011). Rapid radiations
result in short internal branch lengths in many gene
sequence phylogenies; these short branches have little
phylogenetic information (Kraus and Miyamoto
1991) and may cluster randomly due to short-branch
attraction (Nei 1996). Thus, understanding species
diversity within the Elata Subclade will require
identification of additional marker loci with higher
phylogenetic signal (Lopez-Giraldez and Townsend
2011) to resolve closely related species and the
backbone of the phylogeny; it is likely that only
rapidly evolving loci will be informative due to the
short-branch phenomena described previously.
Our results also should serve as a cautionary note to
not assume a priori 3% ITS interspecific variability
within Morchella and other fungi (Nilsson et al. 2006).
While it is convenient to define species or phylotypes
phenetically in fungal environmental surveys (Peay
et al. 2008), or in systematic studies focused on
mining metadata from GenBank accessions (Bonito
et al. 2010, Ryberg et al. 2008), our results and those
of others (Crouch et al. 2009, Roe et al. 2010, Will
et al. 2005) strongly indicate that this approach is
likely to significantly underestimate species diversity.
Fortunately, in instances where researchers querying
Morchella MLST are unable to identify unknowns with
ITS data, they can conduct additional searches of this
reference database using sequences of one or more of
the three single-copy protein-coding genes (i.e. RPB1,
RPB2, EF-1a), which were developed initially within
the framework of the AFTOL project (James et al.
2006, Matheny 2005, Reeb et al. 2004).
Evolution of the ITS regions.—The current study adds
to our growing knowledge of ITS evolution and
variability within the Fungi. In contrast to the
significant ITS divergence reported for diverse species
inferred from GenBank data (Nilsson et al. 2008), we
detected surprisingly little intraspecific variability
within the ITS region in Morchella; the highest we
observed was only 1.3% in M. esculentoides (Mes-4).
However, had we grouped sequences by the names
used to identify them in GenBank, this estimate
would have been considerably higher because multiple species are accessioned under the same name in
this database. Our analyses of GenBank accessions,
for example, revealed that those listed as M. esculenta,
M. crassipes and M. elata each comprised eight
different phylogenetic species (SUPPLEMENTARY TABLES
I–III). To further illustrate problems caused by
misidentified sequences in GenBank (Bridge et al.
2003, Vilgalys 2003), Nilsson et al. (2008) reported
3.1% ITS divergence in Fusarium solani; however, this
estimate is flawed because our GCPSR studies
revealed that this morphospecies comprises more
than 50 distinct phylogenetic species (O’Donnell
2000, O’Donnell et al. 2008, Zhang et al. 2006). In
contrast to the aforementioned survey of ITS variation within diverse fungal species, our analyses
showed that the highest ITS divergence for any
species within the F. solani species complex was only
0.1% in F. falciforme. Estimates of ITS variation can be
affected significantly by putative paralogs and intragenomic variation, as reported in diverse fungi
(Lindner and Banik 2011, O’Donnell et al. 1997,
DU ET AL.: MORCHELLA ITS RDNA
Simon and Weiß 2008), but these were not detected
within the ITS of Morchella. In addition, we did not
detect any gene tree-species tree discordance between
the morel ITS rDNA and the RPB1, RPB2 and EF-1a
phylogenies.
Results of the present study fit experimental data in
budding and fission yeast that indicate the ITS1 is
generally less constrained evolutionarily than the
ITS2 (Abeyrathne and Nazar 2005, van Nues et al.
1995). Early in our studies we discovered that ITS
length variation was primarily due to length mutations within the ITS1 (O’Donnell et al. 1993), so we
used variation at this locus to screen cultures and
herbarium specimens via PCR to sample for the
greatest global genetic diversity (O’Donnell et al.
2011), using the universal PCR primers ITS5 3 ITS4
(White et al. 1990). In the present study we
discovered that the ITS1 more than doubled in
length during the evolutionary diversification of the
Esculenta Clade whereas its length contracted by 50%
within the Elata Clade. Significant length variation
has been documented within the ITS region in other
fungi (reviewed in Hausner and Wang 2005), with the
greater than threefold increase reported within the
ITS1 of Cantharellus being one of the most dramatic
(Feibelman et al. 1994). One of the more noteworthy
findings of the present study was that ITS2 length was
highly conserved during the estimated 68.7 million
year (95% HPD interval: 49.19–93.03] evolutionary
history of the Esculenta Clade (Du et al. 2012), with
the shortest and longest spacers differing in length by
only 16 bp. Although not as highly conserved, the
ITS2 only varied by 53 bp across the phylogenetic
breadth of the Elata Clade. Although MUSCLE
(Edgar 2004) produced fewer gaps in the final
alignment when compared to Clustal W (Thompson
et al. 1994) and MAAFT 6 (Katoh and Toh 2008)
(data not shown), considerable manual adjustments
were required and even then one-third and one-fifth
of the ITS1 nucleotide positions were excluded
respectively from the Esculenta and Elata Clade
datasets due to ambiguity in the alignment. Consistent with uniform reports of the 5.8S rDNA being
highly conserved in the Fungi (Nilsson et al. 2008),
our analyses indicate the 5.8S rDNA was fixed at
156 bp within Morchella and in the hypogeous
(Trappe et al. 2010) and other epigeous members
of the Morchellaceae (Hansen and Pfiser 2006,
O’Donnell et al. 1997). Conservation of the 5.8S
rDNA also was reported in an extensive survey of the
core group of Peziza, where it was fixed at 157 bp
(Hansen et al. 2002). Based on our findings that six of
the Morchella sequences we downloaded from GenBank had length mutations within the 5.8S rDNA that
necessitated inserting indels within the alignments
PHYLOGENETICS
1363
(SUPPLEMENTARY TABLE V), and three other ITS
sequences could be used only once, bad sequence
data at the 59 or 39 end were removed, it would be
highly desirable if automated processes at GenBank
were in place to identify sequences with probable
sequencing errors before they end up in the database.
Of course, this necessitates the adoption of a
sequence standard for each species or strain to which
a deposited sequence could be compared, in which
case rare variant alleles that encompass insertions or
deletions also must be taken into account. While such
a method could be based on models of nucleotide
substitution, they do not incorporate the probability
that an insertion or deletion occurs due to the
intractability of that problem (i.e. While the likelihood of an ARG transition can be predicted based
on observations from the sequence data, how can the
probability be calculated that a nucleotide will be
inserted between any two nucleotides?). This problem
requires that an alternative solution be found.
Can GenBank and emerencia be used to identify
Morchella species?— With 66% or more of the
sequences misidentified, Morchella may have the
dubious distinction of having the highest percentage
of poorly annotated named fungal ITS sequences in
GenBank. This estimate significantly eclipses the
often cited ‘‘upwards of 20%’’ reported as the worst
case scenario for certain groups of the Fungi (Bridge
et al. 2003, Nilsson et al. 2006, Vilgalys 2003). Our
startling discovery prompts three obvious questions:
(i) Why are Morchella sequences so poorly annotated
in GenBank?; (ii) Can these databases be used for
species identifications, and if so, how? And (iii) is the
poor state of Morchella annotation in GenBank closer
to the rule rather than the exception?
Lack of knowledge of species boundaries and
the absence of type studies both have contributed to
the large number of misidentifications. Contrary to
the general assumption, our studies indicate that
Morchella species are not cosmopolitan in distribution
(Du et al. 2012; O’Donnell et al. 2011; Taşkın et al.
2010, 2012). However, because Kanwal et al. (2011)
incorrectly assumed M. crassipes, M. elata and M.
angusticeps were present in India, and did not identify
their accession, GQ228474, as M. tomentosa (Mel-2),
75% (12/16) of the ITS sequences they deposited in
GenBank were misidentified. Once misidentified
sequences such as these are accessioned, they then
can serve as the reference for subsequent BLAST
queries of GenBank and identifications using emerencia (Nilsson et al. 2006). Another potential source
of error is the inclination of individual researchers to
deposit sequences under a validly published binomial
regardless of whether the sequence is actually
1364
MYCOLOGIA
assignable to that species. However, because almost
every GCPSR-based study, including our own, has
discovered widespread cryptic speciation, consistent
with the prediction that only , 7% of fungal diversity
has been discovered (Hawksworth 2004), it is essential
that sequences of phylogenetically distinct species be
identified by some standardized informal nomenclature when they are deposited in GenBank and
emerencia, especially when applying a binomial is
problematic. To this end, we distinguished sequences
of phylogenetic species within the Esculenta and Elata
Clades respectively by Mes and Mel followed by a
unique Arabic number, thereby facilitating DNA
sequence-based identifications of Morchella in GenBank. A BLAST query of GenBank matching one of
these sequences identifies the organism (e.g. ‘‘Morchella sp. Mel-14’’ represented by GU551435) and
provides a clarifying note (e.g. note 5 ‘‘phylogenetic
species Mel-14’’). However, because at least 66% of
the named sequences in GenBank are misidentified,
interpreting BLAST results can be complicated.
We found emerencia (Nilsson et al. 2005) to be
extraordinarily useful in retrieving Morchella ITS
sequences represented in GenBank because it also
found several from environmental studies that were
listed ‘‘uncultured fungus’’ (Taylor et al. 2008) and
one as ‘‘Pezizomycetes sp.’’ (SUPPLEMENTARY TABLE
III). Results of the present study, however, show that
emerencia cannot be used to obtain reliable species
identifications for Morchella because two-thirds of the
‘‘fully identified sequences’’ (i.e. those with binomials) it retrieved from GenBank as a reference were
misidentified. To provide an example of the problems
we encountered when we queried emerencia with
separate sequences of phylogenetic species Morchella
sp. Mel-20 (GU551406, O’Donnell et al. 2011;
HM056383, Taşkın et al. 2010), the best BLAST
matches were identified respectively as M. esculenta
(EU600241) and M. elata (EF081000). This heuristic
should serve to alert users of emerencia, as cautioned by its authors (Nilsson et al. 2005), that
‘‘fully identified’’ and correctly identified are not
synonymous.
Because the factors that contribute to poorly
annotated sequences in GenBank, such as widespread
cryptic speciation and failure to include type specimens as a reference, are hardly unique to Morchella,
it seems likely that the percentage of misidentified
sequences for certain groups of fungi in GenBank
could easily exceed the current estimate of 20%
(Bridge et al. 2003). Looking to the future, it is clear
that multilocus GCPSR-based studies will play an
ever-increasing role in assessing the genetic diversity
of fungi, increasing our ability to optimally mine
sequence data in GenBank. By constructing an ITS
dataset that comprised all but one of the Morchella
species inferred via GCPSR, we were able to identify
endophytes of Bromus (Baynes et al. 2012) and
Juniperus scopulorum (http://www.ncbi.nlm.nih.gov/
nuccore/GQ153010) in western North America, a
putative ectomycorrhizal associate of the terrestrial
orchid Gymnadenia conopsea in Germany (Stark et al.
2009), hypothesize the existence of six putatively
novel species from ITS sequences in GenBank
(SUPPLEMENTARY TABLES II–III) and identify threequarters of the Morchella accessions in GenBank to
phylospecies. The latter revealed: (i) several species
with intercontinental distributions, including four
fire-adapted species native to western North America
(Du et al. 2012, O’Donnell et al. 2011, Taşkın et al.
2012); (ii) the putative North American endemic M.
esculentoides (Mes-4) in central Europe (Kellner et al.
2005) and (iii) that Morchella sp. (Mes-16) appears to
be the most widely distributed species, based on
collections from China (Du et al. 2012), Israel
(Masaphy et al. 2010), Turkey (Taşkın et al. 2012),
New Zealand (GenBank JF423317), India (Kanwal
et al. 2011), three African countries (Degreef et al.
2009), and Hawaii and Java (O’Donnell et al. 2011).
As proposed for true truffles in the genus Tuber
(Bonito et al. 2010) and diverse ectomycorrhizal fungi
(Vellinga et al. 2009), our working hypothesis is that
most if not all disjunct distributions in Morchella are
due to human introductions of species into nonindigenous areas. Examples include the North American endemics M. esculentoides (Mes-4) and M.
importuna (Mel-10), which were discovered respectively in Europe and Turkey (Taşkin et al. 2012) and
Europe, Turkey and China (Du et al. 2012, Taşkin et
al. 2012). We theorize that their introduction into
Europe and China might have been mediated by
relatively recent anthropogenic activities, which
should minimize the likelihood that they represent
later synonyms of taxa described from European and
Chinese collections (Kuo et al. 2012).
The relatively recent discoveries that morels
(Baynes et al. 2012) and Verpa (Stark et al. 2009) can
grow endophytically in plants and form mycorrhizallike associations with angiosperms (Stark et al. 2009)
and gymnosperms (Dahlstrom et al. 2000) suggest that
economically important plants likely serve as vectors
for the vertical transmission of morels across oceans.
With the whole genome sequencing of one species
within the Elata Clade nearing completion (J. Spatafora pers comm) and others likely to follow in the near
future, rich genetic resources such as these should
provide a wealth of molecular markers for phylogeographic and phylogenetic studies needed to distinguish between human-mediated introductions and
long distance dispersal. Phylogenomic analyses should
DU ET AL.: MORCHELLA ITS RDNA
help elucidate the underlying genetic basis of niche
adaption of the Esculenta and Elata Clades respectively
to temperate deciduous and coniferous forests, help
guide genetic studies focused on strain improvement
for commercial cultivation and advance our understanding of species diversity and distribution so that
this invaluable genetic resource can be managed to
prevent overharvesting, thereby promoting long term
species conservation.
Morchella MLST.—We were motivated to construct
Morchella MLST based on our discovery that at least
two-thirds of the named ITS sequences of Morchella in
GenBank were misidentified and because this problem is exacerbated when emerencia is applied (Nilsson
et al. 2006), given that the latter database uses poorly
annotated named sequences in GenBank as the
reference to re-identify sequences, including those
of distinct phylogenetic species inferred to be
genealogically exclusive from multilocus DNA sequence data (O’Donnell et al. 2011; Taşkın et al.
2010, 2012). Because many people who deposit
sequences use the names in GenBank as a reference,
the system unfortunately is designed to perpetuate
itself (Bidartondo et al. 2008, Vilgalys 2003). To
obviate potential problems created by poorly annotated sequences in GenBank we constructed the
Internet-accessible Morchella MLST to provide a
means by which the scientific community can
contribute to and access high quality electropherograms of voucher specimens and/or cultures that are
accessible to interested researchers (Kang et al. 2010).
An added advantage of a dedicated database is that
the informal nomenclatural system that we developed
for distinguishing the phylogenetically distinct species
by clade (i.e. Mes or Esculenta and Mel for Elata/Elata
Subclade) followed by a unique Arabic number can
be implemented. This informal system was adopted
out of necessity because our studies surprisingly
revealed that most of the species in North America,
Turkey and Asia are novel, negating the assumption
that European taxa are cosmopolitan, and because of
the broad uncertainty on how to apply validly
published names. The utility of Morchella MLST, as
for any reference library dedicated to species identification and discovery, is the dense taxon sampling in
which species were resolved a priori by multilocus
GCPSR. One of the overarching goals of Morchella
MLST is to stimulate integrative taxonomic studies so
that all of the phylospecies receive specific epithets,
even in instances where they cannot be reliably
diagnosed phenotypically (Kuo et al. 2012).
A comprehensive nomenclatural and comparative
morphotaxonomic study of these species is needed to
be able to apply European names of Morchella to
PHYLOGENETICS
1365
species delimited via GCPSR. Roughly 82 valid
European species names of Morchella are available,
excluding many subspecific epithets (see Index
Fungorum http://www.indexfungorum.org/), the
majority of which are based on material from France,
Czechoslovakia, Italy, the Netherlands and Sweden.
Because many of these names date back to 1800 and
early 1900, it is likely that original material either does
not exist or that it will be much too old to yield useful
DNA sequence data. These names should be lectotypified as needed, or if neither material nor an illustration
exists a neotype should be selected (the latter with
newly collected material, including cultures and multilocus DNA sequence data). If an original type is too old
to yield useful DNA or is an illustration, it will be
necessary to designate a recent collection (with cultures
and DNA sequence data) as an epitype to be able to
stabilize the name, considering the high morphological
plasticity of macroscopic characters within species of
Morchella and stasis in several microscopic characters
across the genus. The neotype or epitype preferably
should be from the type locality or country. Europe is
the poorest sampled area in our surveys, and to fully
explore the diversity and boundaries of these species a
more thorough molecular and morphological study of
fresh collections from for example France, Czech
Republic, Slovakia, Italy, the Netherlands, Sweden,
Germany and Spain will be essential. Circumscriptions
and synonymies of iconic European species, such as M.
esculenta, M. crassipes, M. deliciosa, M. conica, M. elata,
M. distans and M. hiemalis, need to be clarified. Types
of a number of recently published taxa from Europe
(e.g. Jacquetant 1984, Jacquetant and Bon 1984) need
to be located and subjected to similar morphological
and molecular phylogenetic analyses.
ACKNOWLEDGMENTS
Special thanks are due Nathane Orwig for processing DNA
sequences in the NCAUR DNA core facility. The research visit
of X.-H.D. to NCAUR in 2011 was supported by the National
Basic Research Program of China (No. 2009CB522300), the
Hundred Talents Program of the Chinese Academy of
Sciences, Joint Funds of the National Natural Science
Foundation of China and the Yunnan provincial government
(No. U0836604). The mention of trade names or commercial
products in this publication is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U.S. Department of
Agriculture. The USDA is an equal opportunity provider
and employer.
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species from North America were described in Clowez (2012), at least six species described in Kuo et al. (2012)
and whose names were used herein (i.e. M. virginiana [Mes-3], M. esculentoides [Mes-4], M. populiphila [Mel-5],
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Clowez (2012). Molecular phylogenetic studies are in progress to determine which phylospecies is represented by
each type included in the latter publication (Franck Richard and Mathieu Sauve pers comm).