Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi Beauveria bassiana and B. brongniartii

Abstract

The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus.
The complete mt genomes of B. bassiana (32,263 bp) and B. brongniartii (33,920 bp) were fully analysed. Apart from a typical gene content and organization, the Beauveria mt genomes contained several introns and had longer intergenic regions when compared with their close relatives. The phylogenetic diversity of a population of 84 Beauveria strains -mainly B. bassiana (n = 76) - isolated from temperate, sub-tropical and tropical habitats was examined by analyzing the nucleotide sequences of two mt intergenic regions (atp6-rns and nad3-atp9) and the nuclear ITS1-5.8S-ITS2 domain. Mt sequences allowed better differentiation of strains than the ITS region. Based on mt and the concatenated dataset of all genes, the B. bassiana strains were placed into two main clades: (a) the B. bassiana s. l. and (b) the "pseudobassiana". The combination of molecular phylogeny with criteria of geographic and climatic origin showed for the first time in entomopathogenic fungi, that the B. bassiana s. l. can be subdivided into seven clusters with common climate characteristics.
This study indicates that mt genomes and in particular intergenic regions provide molecular phylogeny tools that combined with criteria of geographic and climatic origin can subdivide the B. bassiana s.l. entomopathogenic fungi into seven clusters with common climate characteristics.

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RESEARCH ARTICLE
© 2010 Ghikas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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Research article
Phylogenetic and biogeographic implications
inferred by mitochondrial intergenic region
analyses and ITS1-5.8S-ITS2 of the
entomopathogenic fungi
Beauveria bassiana
and
B.
brongniartii
Dimitri V Ghikas
†
, Vassili N Kouvelis
†
and Milton A Typas*
Abstract
Background: The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect
hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control
agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt)
genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to
exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and
genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic
information to study the biogeography of the fungus.
Results: The complete mt genomes of B. bassiana (32,263 bp) and B. brongniartii (33,920 bp) were fully analysed. Apart
from a typical gene content and organization, the Beauveria mt genomes contained several introns and had longer
intergenic regions when compared with their close relatives. The phylogenetic diversity of a population of 84 Beauveria
strains -mainly B. bassiana (n = 76) - isolated from temperate, sub-tropical and tropical habitats was examined by
analyzing the nucleotide sequences of two mt intergenic regions (atp6-rns and nad3-atp9) and the nuclear ITS1-5.8S-
ITS2 domain. Mt sequences allowed better differentiation of strains than the ITS region. Based on mt and the
concatenated dataset of all genes, the B. bassiana strains were placed into two main clades: (a) the B. bassiana s. l. and
(b) the "pseudobassiana". The combination of molecular phylogeny with criteria of geographic and climatic origin
showed for the first time in entomopathogenic fungi, that the B. bassiana s. l. can be subdivided into seven clusters
with common climate characteristics.
Conclusions: This study indicates that mt genomes and in particular intergenic regions provide molecular phylogeny
tools that combined with criteria of geographic and climatic origin can subdivide the B. bassiana s.l. entomopathogenic
fungi into seven clusters with common climate characteristics.
Background
Beauveria Vuill. is a globally distributed genus of soil-
borne entomopathogenic hyphomycetes that is preferred
as a model system for the study of entomopathogenesis
and the biological control of pest insects [1]. The most
abundant species of the genus is Beauveria bassiana,
found in a wide host range of nearly 750 insect species,
with extended studies on host-pathogen interactions at
the molecular level and all the prerequisite knowledge for
its commercial production [2]. B. brongniartii, the second
most common species of the genus, has narrow host
specificity and is well-studied as the pathogen of the
European cockchafer (Melolontha melolontha), a pest in
permanent grasslands and orchards [3]. Strains of both
* Correspondence: matypas@biol.uoa.gr
1 Department of Genetics, Faculty of Biology, University of Athens,
Panepistimiopolis 15701, Athens, Greece
† Contributed equally
Full list of author information is available at the end of the article

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fungal species have been exploited as biological control
agents (BCAs) [4,5].
As is usually the case for most mitosporic fungi, mor-
phological characters are inadequate for delimiting spe-
cies within a genus and this creates a continuing demand
of screening for additional taxonomic characters. Conse-
quently, through the years, several efforts have been
made to genetically characterize or differentiate Beau-
veria species and strains, using various tools, including
isozyme markers [6], karyotyping [7], vegetative compati-
bility groups [8], RAPD markers [9,10], rRNA gene
sequencing and intron analyses [11,12], RFLPs and
AFLPs [13-15], subtilisin protease genes [16], microsatel-
lites [17,18] and combinations of rRNA gene complex and
other nuclear genes [1,19,20]. These approaches provided
valuable information on polymorphisms in populations
of B. bassiana, with ITS sequences combined with other
nuclear gene sequences being more reliable in taxonomic
and phylogenetic studies [1,20,21]. Consequently, earlier
assumptions that Beauveria is strictly asexual have been
severely hampered by the recent discoveries of Cordyceps
teleomorphs associated with Beauveria [1,22,23]. Thus,
the extent to which the entire Beauveria genus is corre-
lated with sexual Cordyceps remains to be examined and
proved [1].
Mitochondrial DNA (mtDNA), due to its properties to
evolve faster than the nuclear DNA, to contain introns
and mobile elements and to exhibit extensive polymor-
phisms, has been increasingly used to examine genetic
diversity within fungal populations [24-26]. In other
mitosporic entomopathogenic fungi, such as Metarhiz-
ium [27], Lecanicillium [28] and Nomurea [29], mtDNA
data compared favourably to data based on ITS combined
with a single nuclear gene, for applications in phylogeny,
taxonomy and species or strain -identification. In Beau-
veria, the use of mtDNA RFLPs or partial mtDNA
sequences suggested that mtDNA can be equally useful
for such studies [2,30].
In recent years, molecular techniques have revolution-
ized taxonomical studies and have provided strong evi-
dence that some morphologically defined species consist
of a number of cryptic species that are independent lin-
eages with restricted distributions, for example, Metarhi-
zium anisopliae [31], Neurospora crassa [32], and
Pleurotus cystidiosus [33]. This has urged mycologists to
extend their studies on large samples of individuals
throughout the world, in order to establish robust phylog-
enies from the congruence of genealogies based on
appropriately polymorphic gene sequences and to test
hypotheses regarding the processes responsible for distri-
bution patterns. Thus, the notion of phylogenetic species
recognition and phylogeography was introduced as a
powerful method for answering questions about distribu-
tion in an evolutionary context [34-36]. Phylogeography
or phylogenetic biogeography emerge as the field that
aims to understand the processes shaping geographic dis-
tributions of lineages using genealogies of populations
and genes [37]. It is therefore, particularly important for
genera like Beauveria for which only a few studies exist
on strain variability and their geographic distribution and
phylogenetic origins [6,13,16,17,20].
This work was undertaken to serve a dual purpose.
Firstly, to further assess the usefulness of mtDNA
sequences as species diagnostic tool, alone or in combi-
nation with the more commonly studied rRNA gene
sequences (ITS), and secondly to infer relationships
among a large population of Beauveria species and
strains from different geographic origins, habitats and
insect hosts. To achieve these targets we have analyzed
the complete mt genomes of B. bassiana and B.
brongniartii, selected the two most variable intergenic
regions and constructed the phylogenetic relationships of
a number of isolates for determining their biogeographic
correlation.
Results
Gene content and genome organization
The mt genomes of the two Beauveria species had similar
sizes, i.e., B. brongniartii IMBST 95031 33,926 bp and B.
bassiana Bb147 32,263 bp, and both mapped circularly
(Fig. 1). They contained all the expected genes found in
typical mt genomes of ascomycetes (see Fig. 1; and Addi-
tional File 1, Table S1). Both genomes were compact and
preserved the four synteny units proposed for Sordario-
mycetes, i.e., rns-trn(1-5)-cox3-trn(1-5)-nad6-trn(2-9); nad1-
nad4-atp8-atp6; rnl-trn(11-12)-nad2-nad3 and nad4L-
nad5-cob-cox1 [38]. Important deduced differences in the
gene content of the two genomes were found only when
the intron number and insertion sites were included. This
was also the case for mtDNA genome sequence of
another B. bassiana isolate (Bb13) from China, recently
deposited in GenBank (EU371503; 29.96 kb). When com-
pared with our Bb147 mtDNA genome sequence, the two
genomes were identical in gene order and nucleotide
sequence (98-100%), for most of their sequence (approx.
28.1 kb). The difference in size -approx. 2.3 kb- was due
to the absence from Bb13 of two introns, located in cox1
and nad1 genes of Bb147. Minor sequence differences
were mostly in the intergenic regions with a preference to
AT-rich areas, and were to a large extent SNP transitions
(A/G and C/T) or single nucleotide insertions or dele-
tions. The remaining differences were due to small inser-
tions or deletions of 5-6 bp. The largest deletion (15bp)
and the lowest sequence homology (86%) were observed
in the intergenic region cox1- trnR2 (see Fig. 1).

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Introns
B. bassiana Bb147 contained five and B. brongniartii six
introns, contributing to their total mtDNA genome size
by 20.3% and 24.7%, respectively. All introns were group-I
members, located in rnl, cob, cox1, cox2 and nad1 (Fig. 1;
for details on exact positions of insertion and type of
intron sub-group see Additional File 1, Table S1). All
introns contained ORFs, i.e., the Rps3 homolog within
the rnl gene (BbrnlI and BbrrnlI2), putative GIY-YIG
homing endonucleases (BbcobI1, cox2I1 and nad1I1) and
the LAGLI-DADG endonuclease (Bbcox1I1 and
Bbrcox1I1). The insertion positions of these introns were
found to be conserved (identical sequences for at least 10
bp upstream and downstream of the insertion) for all
known fungal complete mt genomes examined (36 in
total). The only exception was the cox2 intron which was
rarely encountered in other fungi. Interestingly, the addi-
tional intron detected in rnl of the B. brongniartii IMBST
95031 mt genome (positions 806-2102 of NC_011194 and
Additional File 1, Table S1), was inserted at site not
encountered before among the other complete mt
genomes, i.e., the stem formed in domain II of rnl 's sec-
ondary structure. The target insertion sequence for the
intron was GATAAGGTTGTGTATGTCAA and its
intronic ORF encoded for a GIY-YIG endonuclease which
shared homology (57% identity at the amino acid level)
with I-PcI endonuclease of Podospora curvicolla (Acc.
No. CAB 72450.1).
Intergenic regions
Both mt genomes contained 39 intergenic regions
amounting for 5,985 bp in B. bassiana and 5,723 bp in B.
brongniartii, and corresponding to 18.6% and 16.9% of
their total mt genome, respectively. The A+T content was
very similar for these regions in both mt genomes
(~74.5%) and the largest intergenic region was located
between cox1-trnR2 with sizes 1,314 bp for B. bassiana
and 1,274 bp for B. brongniartii, respectively. Analysis of
these particular regions revealed large unique putative
ORFs (orf387 and orf368 for both genomes) with no sig-
nificant similarity to any other ORFs in Genbank. Addi-
tionally, many direct repeats were also located in the
same regions (maximum length 37 bp and 53 bp for B.
bassiana and B. brongniartii, respectively). All other
intergenic regions in the two mt genomes had approxi-
mately the same sizes but with reduced nucleotide iden-
tity (sometimes as low as 78%). Therefore, the potential
usefulness of mt intergenic sequence variation for intra-
and inter- species discrimination and phylogenetic stud-
ies of Beauveria was examined following an in silico anal-
ysis based on criteria of size, complexity and suitability
(for designing primers) of all Beauveria mt intergenic
regions. More specifically, smaller than 200 bp interenic
regions were excluded due to the few informative charac-
ters they contained, whereas ideal regions were consid-
ered those with sizes between 200-800 bp because they
can be easily cloned and/or obtained by PCR. Regions
containing trn genes -due to their cloverleaf structures-
and regions with dispersed repetitive elements were
avoided because their structures make them unsuitable
for designing primers for PCR amplification (for details of
all intergenic regions see Additional File 1, Table S1).
Thus, the most suitable intergenic regions following the
Figure 1 Genetic organization of (a) B. bassiana strain Bb147 and (b) B. brongniartii strain IMBST 95031 mtDNA. Protein-coding genes are
marked with black arrows, and all other genes with gray arrows. Introns are shown with white arrows. Arrows indicate transcription orientation.
Beauveria bassiana Bb147
32263 bp
rnl
rps3 trnT
trnE
trnM1
trnL1
trnA
trnF
trnK
trnL2
trnQ
trnH
trnM3
nad2
nad3
atp9
cox2
cox2i1
trnR
nad4L
nad5
cob
cob i1
trnC
cox1
cox1i1
tnR2
nad1
nad1i1
nad4
atp8
atp6
rns
trnY
trnD
trnS
trnN
cox3
trnG
nad6
trnV
trnI
trnS2
trnW
trnP
trnM2 rnl
rnl I1
trnT
trnE
trnM
trnM2
trnL
trnA
trnF
trnK
trnL2
trnQ
trnH
trnM3
nad2
nad3
atp9
cox2
trnR
nad4L
nad5
cob cob I1
cox1
cox1 I1
cox1 I2
trnR2
nad1
nad4
atp8
atp6
rns
trnY
trnD
trnS
trnN
cox3
trnG
trnV
trnI
trnS2
trnW
trnP
rps3
nad1 I1
Beauveria brongniartii IMBST 95031
3bp3926

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above criteria for the population analyses were nad3-atp9
and atp6-rns.
Population and phylogenetic studies based on ITS1-5.8S-
ITS2 and intergenic mt region sequences
PCR amplicons for the ITS1-5.8S-ITS2 region showed lit-
tle variation in size, being almost identical for all B. bassi-
ana (480-482 bp) and B. brongniartii (478-481 bp)
isolates, but with sizeable differences for the other Beau-
veria species (471-512 bp). On the contrary, the inter-
genic nad3-atp9 and atp6-rns amplicons exhibited a
much greater variability in sizes even within B. bassiana
isolates, ranging from 259-332 bp for the former and 283-
483 bp for the latter (Additional File 2, Table S2 and Addi-
tional File 3, Table S3), thus providing excellent tools for
species or species-group identification. For example,
using high-resolution agarose electrophoresis (data not
shown), nad3-atp9 B. bassiana amplicons can be easily
differentiated from the other Beauveria species and at the
same time can be grouped into Clades A and C according
to their sizes and in congruence to the classification pro-
posed earlier [1] (Additional File 3, Table S3). Variability
for the other Beauveria species was even greater, ranging
from 84-302 bp and 249-441 bp for the nad3-atp9 and
atp6-rns, respectively. When analyzed, these differences
were found to be mainly due to deletions and/or addi-
tions of 3-5 nucleotides for nad3-atp9, scattered through-
out this region, and rarely due to single point mutations.
The atp6-rns sequence differences were primarily due to
a 4-bp repeat (GCTT) inserted in the corresponding
sequence up to 13 times (e.g., R184-483bp), thus provid-
ing in many cases excellent tools for isolate identification.
Amplicon sequences from all isolates listed in Addi-
tional File 2, Table S2 were used to draw phylogenetic
trees deduced from NJ analyses (Fig. 2, 3, 4 and 5), and
parsimony and Bayesian methods were applied to exam-
ine the sensitivity of the resulting trees and tree topolo-
gies. Trees remained largely invariant to these
manipulations and topologies were similar to a significant
extent for each gene region tested independently of the
phylogenetic method applied (symmetric difference val-
ues between the trees obtained with different methods
for the same dataset are shown in Additional File 4, Table
S4). Trees were rooted using as outgroups Aschershonia
sp. and/or Simplicillium lamelicolla (both members of
Hypocreales). Specifically, the phylogenetic tree pro-
duced from the ITS1-5.8S-ITS2 sequences obtained in
this work and known related sequences from the data-
banks, divided the majority of B. bassiana strains into
two major clades (Clade A and C), with marginal support
of each clade (Fig. 2). The only exception was three
strains (namely U259, O46 and IR582) that grouped
together, at the base of the remaining B. bassiana strains
with significant bootstrap (99 and 84% for the NJ and MP
analyses, respectively) and Posterior Probability support
(99% for the BI analysis). Similarly, the three B.
brongniartii strains, grouped with the respective
sequences obtained from GeneBank and produced a sis-
ter clade to B. bassiana, whereas the B. vermiconia and B.
amorpha strains were basal to B. bassiana and B.
brongniartii (Fig. 2). They all clearly clustered to a group
different from the other species of the order Hypocreales,
with significant NJ (97%) and MP (90%) bootstrap sup-
port. Based on 265 informative characters, 2,700 most
parsimonious trees were constructed with tree length of
1,106 steps [Consistency Index (CI) = 0.56, Homoplasy
Index (HI) = 0.44, Retention Index (RI) = 0.86, Rescaled
Consistency Index (RC) = 0.48]. The relatively small
number of informative characters may explain the mar-
ginal MP bootstrap and PP support. The remaining pre-
viously known Beauveria species (B. geodes, B. nubicola,
B. tundrense and B. parasiticum) grouped well with other
To ly p oc l a di um species as expected according to known
taxonomic criteria [39,40].
Both mt intergenic regions were more variable than the
nuclear ITS1-5.8S-ITS2 for the B. bassiana strains. MP
analyses were based on 232 and 343 informative charac-
ters and produced 7,700 most parsimonious trees with
tree lengths 750 (CI = 0.71, HI = 0.29, RI = 0.87, RC =
0.62) and 1,085 steps (CI = 0.68, HI = 0.37, RI = 0.87, RC
= 0.59) for the nad3-atp9 and atp6-rns regions, respec-
tively. B. bassiana strains clustered into the same two
groups (Clade A and C) and again the three isolates (SP
IR582, SP O46 and SP U259) were placed as a separate
group, as in the ITS1-5.8S-ITS2 trees (Fig. 3 and 4).
Strains of B. brongniartii were basal to those of B. bassi-
ana with a significant bootstrap and posterior probability
support (94%, 99% and 78% for NJ, MP bootstrap and BI,
respectively) in the nad3-atp9 analysis (Fig. 3), while in
the atp6-rns tree they presented an identical topology to
the ITS dataset, as a sister species to Clade A with a 100%
support for all methods applied (Fig. 4). Here again,
Beauveria species were clearly differentiated from other
Hypocreales species, with significant support (Fig. 3 and
4). In addition, mt datasets provided better support of
Clade C B. bassiana strains than their nuclear counter-
part, i.e., NJ (98%) and MP (90%) bootstrap support for
the nad3-atp9 dataset (Fig. 3), and 83% and 100%, respec-
tively, for atp6-rns (Fig. 4). For both mt intergenic regions
Clade C B. bassiana strains clustered as a sister group
with the two B. vermiconia strains (i.e., IMI 320027 and
IMI 342563), with the addition of the three independent
B. bassiana isolates in the case of nad3-atp9.
In relation to insect host order, a "loose host-associated
cluster" was observed only for Clade A strains, whereas
Clade C B. bassiana strains were more diverse and no
relation to host origin could be detected. Interestingly,
the association of B. bassiana strain clusters with their

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Figure 2 Phylogenetic trees constructed from unambiguously aligned ITS1-5.8S-ITS2 domain, as produced by NJ analysis. Clade credibility
using NJ calculated from 1K replicates (upper numbers in roman), parsimony BS support calculated from 100 replicates (first lower numbers in italics)
using PAUP and PPs produced by 2M generations (second lower numbers - in bold) using MrBayes, are shown. In the phylogenetics analysis of the
ITS1-5.8S-ITS2 region, fungal species names and sequences obtained from GenBank are shown with their accession numbers in the figure. Fungal
hosts are indicated as follows: in a circle, A, Araneida; C, Coleoptera; D, Diptera; H, Hemipetra; L, Lepidoptera; N, Nematoda; O, Orthoptera, T, Thys-
anoptera, R, Rotifera; ?, not known; in a square, H, Hymenoptera and no indication from soil or air. Geographic location is provided next to each isolate
together with blue, orange, green, purple and magenta colour for the isolates originated from Europe, Asia, America, Africa and Oceania, respectively.
B. brongniartii isolates are shown in yellow while isolates from all other Hypocreales species are provided in black.

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Figure 3 Phylogenetic trees constructed from unambiguously aligned nad3-atp9 inte rgeni c regi on, as produ ced by NJ ana lysis . Clade cred-
ibility using NJ calculated from 1K replicates (upper numbers in roman), parsimony BS support calculated from 100 replicates (first lower numbers in
italics) using PAUP and PPs produced by 1M generations (second lower numbers - in bold) using MrBayes, are shown. Fungal hosts, geographic loca-
tions and colour designations as in Fig. 2.

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Figure 4 Phylogenetic trees constructed from unambiguously aligned atp6-rns intergenic region, as produced by NJ analysis. Clade credi-
bility using NJ calculated from 1K replicates (upper numbers in roman), parsimony BS support calculated from 100 replicates (first lower numbers in
italics) using PAUP and PPs produced by 1M generations (second lower numbers - in bold) using MrBayes, are shown. Fungal hosts, geographic loca-
tions and colour designations as in Fig. 2.

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Figure 5 Phylogenetic trees constructed from unambiguously aligned combined DNA sequences of the mt interegenic regions and the ITS
domain as produced by NJ analysis. Clade credibility using NJ calculated from 1K replicates (numbers in roman), parsimonial BS support calculated
from 100 replicates (numbers in italics) using PAUP and PPs produced by 2M generations (numbers in bold) using MrBayes, are shown. Fungal hosts,
geographic locations and colour designations as in Fig. 2. The 3 symbol Köppen-Geiger climate classification is also provided as follows: Af, Tropical
Rain Forest; Am, Tropical Monsoon climate; Aw, Tropical wet and dry; BWh, Dry (arid and semiar id) desert low latitude climate; BWk, Dry (arid and semi-
arid) desert middle latitude climate; BSh, Dry (arid and semiarid) steppe low latitude climate; BSk, Dry (arid and semiarid) steppe middle latitude cli-
mate; Csa/Csb, Temperate Mediterranean climate; Cfa/Cwa, Temperate humid subtropical climate; Cfb/Cwb/Cfc, Temperate Maritime climate; Cwb,
Temperate with dry winters climate; Cfc, Temperate Maritime Subarctic climate; Dfa/Dwa/Dsa, Hot summer Continental climate; Dfb/Dwb/Dsb, Warm
summer Continental climate; Dfc/Dwc/Dsc, Continental Sub arctic climate; Dfd/Dwd, Continental Subarctic climate with extremely severe winters [41].

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insect host origin was more consistent with the nad3-
atp9 data, than with data derived from atp6-rns analysis.
Concatenated sequence analysis and evidence for host and
climate associations of the clades
To fully integrate and exploit all the above information, a
tree was constructed based on the concatenated ITS1-
5.8S-ITS2, atp6-rns and nad3-atp9 sequences. Parsimony
analysis provided more than 10,000 trees after exploiting
575 informative characters and the tree length was based
on 1,895 steps (CI = 0.612, HI = 0.388, RI = 0.858, RC =
0.576). Analysis of the same dataset with NJ and BI meth-
ods produced similar trees with identical topologies
wherever there was a strong support (Fig. 5). As in every
tree produced by the analysis of a single gene region, B.
bassiana strains grouped again into the same two major
groups. The three isolates that were placed basally to the
remaining B. bassiana remained independent, with sig-
nificant bootstrap support (NJ: 99%, Fig. 5; see also DNA
sequence percentage identity in comparisons of members
of Clade A2 with members of Clades A and C in Addi-
tional File 5, Table S5). The most interesting feature of the
concatenated data tree was that B. bassiana strains of
Clade A could be divided further into seven distinct sub-
groups that showed a "loose" association with their host
(Fig. 5). This association was strengthened if the fungi
were clustered according to their geographic and climatic
origin (Fig. 6). More precisely, sub-groups 1, 3, 4 and 6
contained strains from Europe with five, nine, three and
twelve members, respectively (Additional File 3, Table
S3). Sub-group 1 strains were derived from France, Hun-
gary and Spain (with a single strain from China). They
were all isolated from a Lepidopteran host and originated
from temperate maritime and continental microthermal
climates (Cfb/Dwb) according to the Köppen-Geiger
classification [41]. The three strains of sub-group 2 were
isolated from Oceania (one from Australia and two from
Papua New Guinea). To these, an Indian (Cfa), a Chinese
(Cfa) and a Spanish (Csa) strain were also added, i.e., fun-
gal strains from regions with temperate humid subtropic
and Mediterranean climates, resembling the climate of
the Oceanic Cfa [41]. Sub-groups 3 and 4 consisted
almost exclusively of European strains (9 and 3, respec-
tively) from regions with Mediterranean climate, such as
Spain, Portugal and Italy. On the other hand, 12 strains
from regions of Europe with maritime temperate climates
(Cfb) formed a well-supported group (87 and 92% NJ and
MP bootstrap and 94% PP support) presented as sub-
group 6. All nine strains of sub-group 5 were from
regions with dry arid, semiarid (BSh, BSk and BWk) and
temperate (Csa and Csb) climates in Asia and Europe,
while the South American (6) from tropic (Af, Am and
Aw) and dry arid/semiarid (BSh) climates may be named
as sub-group 7.
Discussion
Fungal mt genome size shows high divergence among the
Pezizomycotina, ranging from 100.3 Kb for Podospora
anserina (NC_001329) to 24.5 Kb for the entomopatho-
gen Lecanicillium muscarium (AF487277). Beauveria mt
genomes sizes were similar to those of other fungi of the
order Hypocreales, e.g., Fusarium oxysporum (34.5 Kb;
AY945289) and Hypocrea jecorina (42.1 Kb; NC_003388),
but they were significantly larger (~40%) than the mt
genomes of the other two known entomopathogenic
fungi of the order, i.e., M. anisopliae (24.7 kb) [27] and L.
muscarium (24.5 kb) [42]. Since the Beauveria mtDNAs
contained the same protein and rRNA coding genes -also
identical in sizes- with all above mt genomes, their larger
sizes can be attributed to more introns and to longer
intergenic regions.
Compared to mt genomes of plants and animals, fungal
mt genomes are significantly richer in group I and II
introns [43]. Divergence in intron content is a common
feature among mt genomes of Pezizomycotina. At one
extreme is the mt genome of P. anserina which contains
41 introns [44] and at the other are several fungi that con-
tain a single intron in the rnl genes of their mt genomes
(i.e., L. muscarium and M. anisopliae). The recently
released mt genome of another B. bassiana isolate
(EU371503) also presented fewer introns than the
genomes that we analyzed. These data support and
extend previous evidence for intronic variability among
strains of the same Beauveria species [14,16]. However,
introns usually share common futures like insertion sites,
encoded ORFs and total length [43,45]. Exceptions are
noteworthy, not only because they suggest tools for the
discrimination of the fungus but also because they pro-
vide information valuable to our understanding of fungal
evolution [46-48]. In that respect, intron Bbrrnl1 inserted
within domain II of rnl's secondary structure was located
in a novel (unique) site amongst the 36 Ascomycota com-
plete mt genomes examined (Additional File 6, Table S6).
Even though introns have been found in the same domain
in Basidiomycota, for example Agrocybe aegerita [49], the
uniqueness of this insertion site is of great importance to
ascomycetes, as it may be a result of horizontal intron
transfer. The fact that this intron encodes for a GIY-YIG
homing endonuclease which shares homology with ORFs
in introns located in different genes in other fungal
genomes further strengthens the hypothesis of horizontal
transfer. Yet, such a hypothesis remains to be experimen-
tally tested.
Recently, a thorough attempt was made to determine
associations of morphological characteristics with molec-
ular data in Beauveria species [1]. Based on ITS1-5.8S-
ITS2 and EF-1a sequences 86 exemplar isolates were
examined and assigned to six major clades (A-F), where
all known Beauveria species were included. B. bassiana

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isolates were grouped into two unrelated and morpholog-
ically indistinguishable clades (Clades A and C), while B.
brongniartii formed a third sister clade to the other two
(designated as Clade B). A new species, B. malawiensis,
was later introduced and placed as sister clade to clade E
[50], and several other B. bassiana isolates pathogenic to
the coffee berry borer from Africa and the Neotropics
were added to Clades A and C [22]. Our results from the
ITS1-5.8S-ITS2 dataset are in full agreement with the
grouping into Clades A-C and this division of B. bassiana
isolates into two distinct clades is further supported by
the mt intergenic region and the concatenated datasets
with the best so far known bootstrap values. Mt genomes
present different evolutionary rates compared to the
nuclear [51] and topologies provided by one evolutionary
pathway may not always indicate the correct relation-
ships. As indicated by our findings, combining informa-
tion from two independent heritages (nuclear and mt)
may offer the possibility to resolve phylogenetic ambigui-
ties. Thus, the two unrelated and morphologically indis-
tinguishable B. bassiana clades proposed by Rehner and
Buckley [1], i.e., the "B. bassiana s.l.", which contains the
authentic B. bassiana (Clade A), and the "pseudobassi-
ana" clade, which remains to be described (Clade C), are
fully supported by our combined mt and nuclear data.
Equally well supported by bootstrap is the placement of
B. brongniartii strains as a sister clade to B. bassiana. The
consistent clustering of the three B. bassiana isolates (our
Clade A2 in Fig. 5 and Additional File 5, Table S5), which
grouped basally to other B. bassiana with any datasets,
indicates that this group is possibly a cryptic complex of
B. bassiana. Experimental work with these and other sim-
ilar isolates will be needed to substantiate this hypothesis.
A generally accepted notion that insect hosts are
related to certain genotypes of entomopathogenic fungi
has been tested in several occasions in the past for B.
bassiana and B. brongniartii. However, only a few cases
supported a host - fungal genotype specificity. For
instance, associations have been reported between B.
brongniartii and Melolontha melolontha, M. hippocastani
or Hoplochelus marginalis [17,52]. A common B. bassi-
ana genotype was detected in isolates from Ostrinia
nubilalis [10] and from Alphitobius diaperinus [53]. More
often, B. bassiana isolates collected from the same insect
species were found to be genetically dissimilar [54,55] or
showed cross-infectivity [56]. Similarly, fungal isolates
derived from different insect species, families or orders
clustered together [57]. Our results from the concate-
nated mt and nuclear gene datasets come to an agree-
ment with the latter view, since molecular variability
showed no general correlation between strains and host
and/or geographic origin. This indicates that B. bassiana
is a generalized insect pathogen, and is in agreement
which its world-wide distribution, the vast variety of
hosts from which it has been isolated and its entomo-
pathogenic and/or endophytic characteristics [1,58]. It is
only in rare occasions that a particular genotype, like
Clade A sub-group 1 isolates (Fig. 6; Table 1), may be
Figure 6 Grouping of B. bassiana sensu lato strains (Clade Α) as well as Clade C and A2, according to their geographic distribution, climate
conditions and molecular data (concatenated datasets from ITS1-5.8S-ITS2, nad3-atp9 and atp6-rns). The 3 symbol Köppen-Geiger climate
classification is as shown in Fig. 5.
Subgroup -2
(Af/Cfa)
Clade A
: Subgroup -1 (5)
: Subgroup -2 (6)
: Subgroup -3 (9)
: Subgroup -4 (3)
: Subgroup -5 (9)
: Subgroup -6 (12)
: Subgroup -7 (5)
:Clade C (15)
:Clade A2(3)
Subgroup -7
(Af/Am/Aw/Bsh) Subgroup -5
(Bsh/Bsk/Bwk/Csa/Csb)
Subgroup -6
(Cfb)
Subgroup -1
(Cfb/Dwb)
Subgroup -4
(Csa) Subgroup -3
(Csa/Bsh/Bsk)

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associated with a particular host (Ostrinia nubilalis). In
the case of B. brongniartii and under the light of previous
analyses of larger fungal populations [17,52], the associa-
tion between fungal genotypes and a particular host seem
to be stricter.
An increasing number of studies point towards a broad
correlation of fungal isolates with their place of origin
and/or habitats [e.g., [18,21,30,59,60]]. Obviously, the fac-
tors that can influence B. bassiana population structures
are many and can include: climate conditions, the range
of temperatures in which the various isolates can grow in
nature, humidity levels, UV exposure, habitat, cropping
system and soil properties [18,27,59,61]. In addition, col-
lection strategies, numbers of isolates tested, gene loci
used and molecular methods applied for population anal-
yses can greatly contribute to the variability recorded.
Thus, although the description of the effects of a single
variable on the population of entomopathogenic fungi in
a habitat can give significant and useful ecological and
agronomical information, there may be relationships
among the different variables that must be studied in
detail to adequately understand the source of genetic
variability in these fungi [59,61]. Therefore, to increase
our potential to detect correlations between molecular
markers and environmental variables, we incorporated
climate conditions in our analyses, based on the most
widely accepted classification system, the Köppen-Geiger
climate classification [41]. This approach allowed fungal
isolates that were otherwise outside of a particular cluster
to be embodied in this cluster. Also, with few exceptions,
strains isolated from distant geographic regions, which
however shared similar climatic conditions, clustered
together. If an explanation had to be proposed, the isola-
tion by distance (allopatry) cannot be ruled out [22]. Dur-
ing the last decade molecular phylogenetic studies
concerning fungal taxa which are considered to be wide-
spread have resulted in the recognition of allopatric cryp-
tic sibling species [33,62]. The suggestion that some
morphologically defined species consist of a number of
cryptic species that are independent lineages with
restricted distributions [36], may explain the phylogeo-
graphic distribution of the three B. bassiana isolates des-
ignated in group A2 in this work. In other words, even
though they are morphologically indistinguishable from
the rest B. bassiana isolates, all three have the same host
and are originated from Asia (i.e., Iran, Turkey and
Uzbekistan) with similar climate (Bsk/Csa/Dsa).
It may be argued, and indeed it is the case, that the fun-
gal isolates studied in this work are geographically
"biased", since they are predominantly isolated from
insects found in Europe (40) and Asia (19), and to a lesser
extend from other places in North and South America,
Africa and Oceania (16 isolates). However, even with this
worldwide distribution of the isolates studied, continental
drifts, geological barriers, host restrictions and human
activities may contribute to long-distance dispersal and
result to mixed sub-grouping classification. For instance,
sub-group 2 (Fig. 6) contains the Oceanic isolates, one
from India and one from Britain. While the "Indian" iso-
late may be considered as an evolutionary result of the
opening of the Weddell Sea when eastern (including Aus-
tralia, New Zealand and India) and western Gondwana
(including Africa and Northern South America) sepa-
rated [63], the "British" isolate may only be explained by
accepting long-distance dispersal due to the human inter-
vention as the most probable way. In similar studies
where fungal distribution was caused by the breakup of
Pangaea to New and Old World, like the Pleurotus cystid-
iosus group [33], Schizophyllum [64] and Lentinula [65],
Table 1: Data from the phylogenetic analyses
ITS1-5.8S-ITS2 atp6-rns nad3-atp9 Concatenated
Total characters 640 687 496 1823
Constant characters 258 222 155 642
Variable characters 117 122 109 382
Informative characters 265 343 232 799
Tree length 1106 1085 750 2918
Consistency Index (CI) 0.56 0.68 0.71 0.64
Homoplasy Index (HI) 0.44 0.37 0.29 0.36
Retention Index (RI) 0.86 0.87 0.87 0.83
Rescaled Consistency Index (RC) 0.48 0.59 0.62 0.53
Parsimonious trees 2700 7700 7700 4100
Data obtained from the phylogenetic analyses of the nuclear ITS1-5.8S-ITS2 and the two mitochondrial intergenic regions atp6-rns and nad3-
atp9 for all isolates examined in this study.

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several exceptions to this pattern were observed in each
study and they were usually explained by rare, but recent
long - distance dispersal. Thus, gene flow among geo-
graphically distant populations of B. bassiana may be
attributed to the long-distance dispersal of fungal spores
through a variety of different direct or indirect means
including wind, migratory insect vectors, rainfall, flood-
ing and human traffic. On the other hand, the fact that
several B. bassiana isolates belonging to different phylo-
genetic clades have been found in the same geographic
location (e.g., Fig. 5, clades 3 and 4) may indicate a sym-
patric diversification. There appears to be no single mor-
phological, physiological, host range, or genetic marker
characteristic that can alone resolve molecular phyloge-
nies in B. bassiana. Therefore, a strictly vicariant scenario
may be not supported with these datasets and the occur-
rence of long - distance dispersal may be an alternate fea-
sible scenario which renders the genus Beauveria
cosmopolitan with several cryptic species, as already have
been shown in other fungal taxa [66-68]. Nevertheless, in
view of the ecological complexities of this entomopatho-
genic fungus, it is evident that terminal lineages can only
be found if experiments are performed using more hier-
archical parameters (climate, habitat, ecology and bioge-
ography) in combination with multiple gene analyses that
include data both from nuclear and mitochondrial genes.
Conclusions
The complete mt genomes of B. bassiana and B.
brongniartii analysed in this work had the typical gene
content and organization found in other Ascomycetes of
the order Hypocreales, but contained more introns and
longer intergenic regions. The latter features can serve as
tools for inter- and intra- species specific analysis within
the genus Beauveria. Two mt intergenic regions (nad3-
atp9 and atp6-rns) provided valuable sequence informa-
tion and good support for the discrimination of Beau-
veria species and the division of 76 B. bassiana isolates
into two groups, namely the B. bassiana sensu lato and
the B. bassiana "pseudo-bassiana". These findings were in
agreement with phylogenetic inferences based on ITS1-
5.8S-ITS2 and demonstrated that mt sequences can be
equally useful with the universally approved ITS1-5.8S-
ITS2 for phylogenetic analysis. Further, mt sequence phy-
logenies constantly supported the formation of a third B.
bassiana group, clearly differentiated from the rest, thus
hinting for the presence of cryptic species within B. bassi-
ana. Concatenated data sets of sequences from the three
regions studied (i.e., the two mt and the nuclear ITS
sequences) supported the above conclusions and often
combined with criteria of isolate and geographic and cli-
matic origins offered a better resolution of the B. bassi-
ana s.l. strains and showed for the first time in
entomopathogenic fungi, that B. bassiana s.l. strains can
be subdivided into seven distinct sub-groups with com-
mon climate characteristics.
Methods
Strains, growth conditions, and DNA extraction
Seventy six strains of Beauveria bassiana, 3 of B.
brongniartii and 14 strains of 9 other Beauveria species,
together with one representative from each of 11 species
belonging to the order Hypocreales were examined and
are listed in Additional File 2, Table S2 (a fungal collec-
tion kept in the Department of Genetics and Biotechnol-
ogy, Athens University, Greece). All fungal isolates were
derived from single conidial spores grown on Potato Dex-
trose Agar (PDA) plates and all cultures were started
from single spore isolations. Liquid cultures were in 250
ml flasks containing 50 ml of medium, inoculated with a
spore suspension to reach 105/ml final spore concentra-
tion, on an orbital shaker at 150 rev min-1, 25°C, for 3-4
days. Mycelia were removed by vacuum filtration, lyo-
philized for 2-4 days, and ground in liquid nitrogen using
a mortar and pestle. Small quantities of ground mycelia
(50-100 mg) were used for the extraction of DNA as
described [69].
Construction of libraries, PCR amplification and sequencing
of the complete mt genomes
Isolation and digestion of nuclear and mtDNA from B.
bassiana strain Bb147 and B. brongiartii strain IMBST
95031 were performed as previously described [69].
EcoRI and HindIII restricted fragments of CsCl-purified
mtDNA were ligated into vector pBluescript KS+ (Strata-
gene, Cedar Creek, TX), analysed, subcloned and
sequenced, thus covering over 78-80% of their complete
mtDNA. The rest of the mtDNA and overlapping junc-
tions were determined through sequence analysis of long-
expand PCR amplicons. For this purpose, previously
designed primers were used as follows: nad1B, cox3B,
atp6A [42], cox2R, LSUER [27], LSUSF [38], and NMS1,
NMS2 [70]. The primer pairs and respective amplicon
sizes are shown in Additional File 7 (Additional File 7,
Table S7). No sequence differences were observed
between cloned fragments and PCR amplicons for the
overlapping regions. PCR amplifications were performed
with the proof-reading polymerase Herculase (Strata-
gene), in a PTC-200 Gradient Peltier Thermal Cycler (MJ
Research, Waltham, MA), according to the manufac-
turer's instructions. PCR products were cloned in vector
pDrive (QIAGEN, Hilden, Germany), subcloned as
smaller fragments to pBluescript SKII and sequenced.
Sequencing was performed with the Thermo Sequenase
Primer Cycle Sequencing kit (Amersham Biosciences,
Amersham, UK), and the reactions were analyzed at a
LICOR 4200 IR2 automated sequencer. All fragments
were sequenced in both directions. DNA similarity

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searches were performed with Basic Local Alignment
Search Tool (BLAST 2.2.14) [71]. The tRNAs were pre-
dicted by tRNAscan-SE 1.21 [72]. Intron identification
and characterization utilized the intron prediction tool
RNAweasel [73].
Phylogenetic analysis
The ITS1-5.8S-ITS2 region of the nuclear rRNA gene-
complex and two mtDNA intergenic regions, namely
nad3-atp9 and atp6-rns, were examined in all isolates.
DNA extracts from each isolate were used as templates
for amplification with primers VLITS1 with VLITS2 for
the ITS region [74], atp6F and rnsR for the atp6-rns, and
nad3F with atp9R for the nad3-atp9 mt intergenic
regions [27]. All reactions were performed with Hercu-
lase polymerase (Stratagene) in a PTC-200 (MJ Research)
thermocycler according to the manufacturer's protocol,
with a minor modification involving lower annealing
temperature (45°C) for all three pairs. Sequencing was
performed as above. DNA sequence alignments were
made using CLUSTALW [75] with the multiple alignment
parameters set to default and then edited by visual
inspection (the matrix of the concatenated dataset and its
partitions is provided in Additional File 8). Maximum
parsimony (MP), Neighbor-Joining (NJ) and Bayesian
inference (BI) analyses were employed in order to create
the phylogenetic trees. The PAUP* program ver. 4.0b10
[76] was used for NJ (using the Kimura-2 parameter
model) and MP analyses with 1,000 and 10,000 replicates,
respectively, and with random addition of taxa and tree-
bisection reconnection branch swapping [76]. Reliability
of nodes was assessed using 1,000 and 100 bootstrap iter-
ations for NJ and MP analyses, respectively. The BI was
performed with MrBayes ver. 3.1 [77]. A gamma distribu-
tion model of site variation was used, calculated with
PAML [78]. For ITS1-5.8S-ITS2, nad3-atp9, atp6-rns and
concatenated data sets, alpha (a) was 0.529, 0.966, 1.311
and 0.693, respectively. Two independent MCMCMC
searches were run for each data set using different ran-
dom starting points (number of generations: 1,000,000
for all datasets except for the concatenated set, where 2
million generations were needed) with sampling every
100 generations. Convergence was checked visually by
plotting likelihood scores vs. generation for the two runs
[the first 25% samples from the cold chain (relburnin =
yes and burninfrac = 0.25) were discarded]. Based on this
analysis, the burn-in was set to 10,000, as this was found
to be clearly sufficient for the likelihood and the model
parameters to reach equilibrium. Distances between trees
produced by the same dataset but different method were
computed with the Symmetric Difference of Robinson
and Foulds [79] as implemented in program Treedist of
the PHYLIP v.3.69 package [80].
Nucleotide sequence accession numbers
The complete sequence of B. bassiana strain Bb147 and
B. brongniartii strain IMBST 95031 have been submitted
to GenBank [GenBank: EU100742 and GenBank:
NC_011194, respectively]. Also, nucleotide sequences for
ITS and mtDNA intergenic regions were submitted to
GenBank database [GenBank: FJ972917-FJ972972, Gen-
Bank: FJ973054-FJ973076 and GenBank: EU086417-
EU086434 for the ITS region, GenBank: FJ972973-
FJ973028, GenBank: FJ973077-FJ973101 and GenBank:
EU086435-EU086455 for intergenic region nad3-atp9
and GenBank: FJ972862-FJ972916, GenBank: FJ973029-
FJ973053 and GenBank: EU086396-EU086416 for the
intergenic region atp6-rns].
Additional material
Authors' contributions
DVG contributed to design and performed the experiments and analysis of the
complete mt genomes and helped in the population study. VNK contributed
to design, performed experiments on the population study and the phyloge-
netic analyses. MAT designed research and supervised all the work. All authors
contributed to the manuscript and approved the final version.
Acknowledgements
The authors wish to thank Dr. S. Kathariou (North Carolina State University) for
critically reading this manuscript. They also wish to thank Dr. Humber (USDA,
Ithaca, NY, USA), Dr. E. Quesada-Moraga (University of Cordoba, Spain), Dr. D.
Moore (CABI, UK), Drs. Y. Couteaudieur and Dr. A. Vey (INRA, France), Dr. C.
Tkaszuk (Research Centre for Agricultural and Forest Environment Poznae,
Poland), Dr. E. Kapsanaki-Gotsi (University of Athens, Greece), and Dr. E. Beerling
(Applied Plant Research, Division Glasshouse Horticulture, Wageningen, The
Netherlands), for kindly providing the ARSEF, EABb, SP, Bb and Bsp, PL, ATHUM
and (Fo-Ht1) isolates, respectively. The authors acknowledge the support of
the European Commission, Quality of Life and Management of Living
Resources Programme (QoL), Key action 1 on Food, Nutrition and Health QLK1-
Additional File 1 Genetic content of the (a) B. bassian a Bb147 mt
genome (EU100742) and (b) B. brongniartii IMBST 95031 mt genome
(NC_011194).
Additional File 2 The strains used in this study, their hosts, geograph-
ical/climate origin.
Additional File 3 PCR amplicon sizes (in nucleotides) of all B. bassiana
isolates studied for the mt intergenic regions nad3-atp9 and atp6-rns.
ITS1-5.8S-ITS2 amplicons are not shown because they were more or less
identical (ranging from 480-482 nt for all strains).
Additional File 4 Values of symmetric difference between the phylo-
genetic trees produced from ITS1-5.8S-ITS2, nad3-atp9, atp6-rns and
the concatenated dataset with NJ, BI and MP methods.
Additional File 5 DNA sequence comparisons (% identity) of ITS1-
5.8S-ITS2, nad3-atp9 and atp6-rns intergenic regions for representa-
tive isolates of B. bassi ana Clades A, A2, C. Isolates from Clade A and its
subgroups, in green cells (and number in parentheses); isolates from Clade
C and Clade A2 in yellow and blue cells, respectively.
Additional File 6 The complete mt genomes of fungi used in compari-
son with Beauveria mt genomes. The complete mt genomes of fungi
used in this study (all in red), their taxonomy, accession numbers, genome
length, number of proteins and structural RNAs. All other presently known
fungal complete mt genomes are shown in black.
Additional File 7 PCR primer pairs used for the amplification of the
complete mt genomes of B. bassiana Bb 147 and B. brongniartii IMBST
95031 and approximate amplicon sizes in bp.
Additional File 8 Matrix of concatenated dataset and genes/regions
partitions used for the construction of the phylogenetic trees.

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CT-2001-01391 (RAFBCA) and the Greek Secretariat of Research (project
'National Biotechnology Networks').
Author Details
Department of Genetics, Faculty of Biology, University of Athens,
Panepistimiopolis 15701, Athens, Greece
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Received: 5 February 2010 Accepted: 16 June 2010
Published: 16 June 2010
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doi: 10.1186/1471-2180-10-174
Cite this article as: Ghikas et al., Phylogenetic and biogeographic implica-
tions inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2
of the entomopathogenic fungi Beauveria bassiana and B. brongniartii BMC
Microbiology 2010, 10:174