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31
Studies in Mycology
available online at www.studiesinmycology.org
INTRODUCTION
DNA sequence-based phylogenetics has dramatically inuenced
both the taxonomy and systematics of the Botryosphaeriaceae
during the course of the past decade (Crous et al. 2006), as it has
done in most other groups of Fungi (James et al. 2006, Hibbett et
al. 2007). At a higher taxonomic level, DNA sequence data have
led to the recognition that the Botryosphaeriaceae represents a
distinct order within the Dothideomycetes, leading Schoch et al.
(2006) to introduce the Botryosphaeriales. The circumscription of
the Botryosphaeriales has suffered from insufcient sampling and
it was only recently that Minnis et al. (2012) provided molecular
evidence to show that the Planistromellaceae resides in this order.
In a subsequent study, Liu et al. (2012) provided a comprehensive
phylogenetic analysis of genera in the Botryosphaeriales and
they also concluded that, other than the Botryosphaeriaceae and
Planistromellaceae, a number of clearly dened evolutionary
lineages exist.
Apart from the Planistromellaceae, the genera traditionally
associated with Botryosphaeria and Phyllosticta have sexual
morphs that are clearly distinct phylogenetically, morphologically
and ecologically. However, both are still grouped within the
Botryosphaeriaceae. Members of the Botryosphaeria group are
common endophytes of leaf and woody tissue of many woody plant
species, have hyaline to dark ascospores, multilocular ascomata,
and a wide range of asexual morphs that typically lack a mucoid
sheath and apical appendage. Species in the Guignardia group (=
Phyllosticta) typically infect leaves and fruit, less commonly wood,
have unilocular ascomata with smaller ascospores that typically
have mucoid appendages, and Phyllosticta asexual morphs. The
Phyllostictaceae has been resurrected to accommodate this group
of taxa (see Wikee et al. 2013b, this volume).
Substantial changes to the denition of sexual and asexual
genera linked to the Botryosphaeriaceae have been made during
the past decade (e.g. Crous et al. 2006, Phillips et al. 2008, Liu et al.
2012). Only a selection of the most common examples is discussed
here. The rst DNA sequence data for the Botryosphaeriaceae
appeared to reveal a distinction between asexual morphs with
hyaline fusicoccum-like conidia and those with pigmented diplodia-
like conidia, termed sections Hyala and Brunnea (Jacobs & Rehner
1998, Denman et al. 2000, Zhou & Stanosz 2001). This distinction
became increasingly less obvious as sampling increased and it
was evident that conidial pigmentation is a feature that evolved
more than once. It was, for example, shown that dark, septate
and even muriformly septate dichomera-like conidia could be
synasexual morphs of well-known genera such as Fusicoccum
Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary
framework
B. Slippers1#*, E. Boissin1,2#, A.J.L. Phillips3, J.Z. Groenewald4, L. Lombard4, M.J. Wingeld1, A. Postma1, T. Burgess5, and P.W. Crous1,4
1Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa; 2USR3278-Criobe-CNRS-EPHE, Laboratoire
d’Excellence “CORAIL”, Université de Perpignan-CBETM, 58 rue Paul Alduy, 66860 Perpignan Cedex, France; 3Centro de Recursos Microbiológicos, Departamento de
Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal; 4CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8,
3584 CT Utrecht, The Netherlands; 5School of Biological Sciences and Biotechnology, Murdoch University, Perth, Australia
*Correspondence: B. Slippers, Bernard.Slippers@fabi.up.ac.za
# These authors contributed equally to this paper.
Abstract: The order Botryosphaeriales represents several ecologically diverse fungal families that are commonly isolated as endophytes or pathogens from various woody
hosts. The taxonomy of members of this order has been strongly inuenced by sequence-based phylogenetics, and the abandonment of dual nomenclature. In this study, the
phylogenetic relationships of the genera known from culture are evaluated based on DNA sequence data for six loci (SSU, LSU, ITS, EF1, BT, mtSSU). The results make
it possible to recognise a total of six families. Other than the Botryosphaeriaceae (17 genera), Phyllostictaceae (Phyllosticta) and Planistromellaceae (Kellermania), newly
introduced families include Aplosporellaceae (Aplosporella and Bagnisiella), Melanopsaceae (Melanops), and Saccharataceae (Saccharata). Furthermore, the evolution of
morphological characters in the Botryosphaeriaceae were investigated via analysis of phylogeny-trait association. None of the traits presented a signicant phylogenetic
signal, suggesting that conidial and ascospore pigmentation, septation and appendages evolved more than once in the family. Molecular clock dating on radiations within the
Botryosphaeriales based on estimated mutation rates of the rDNA SSU locus, suggests that the order originated in the Cretaceous period around 103 (45–188) mya, with
most of the diversication in the Tertiary period. This coincides with important periods of radiation and spread of the main group of plants that these fungi infect, namely woody
Angiosperms. The resulting host-associations and distribution could have inuenced the diversication of these fungi.
Key words: Aplosporellaceae, Melanopsaceae, molecular dating, Phyllostictaceae, Planistromellaceae, Saccharataceae, systematics.
Taxonomic novelties: New families – Aplosporellaceae Slippers, Boissin & Crous, Melanopsaceae Phillips, Slippers, Boissin & Crous, Saccharataceae Slippers, Boissin &
Crous.
Published online: 30 September 2013; doi:10.3114/sim0020. Hard copy: September 2013.
StudieS in Mycology 76: 31–49.
SlipperS et al.
32
and Neofusicoccum (Barber et al. 2005, Phillips et al. 2005).
Furthermore, dark, septate ascospores were shown to be a
polyphyletic character of several genera and more common than
previously believed (Phillips et al. 2008). As the true phylogenetic
diversity within the group emerged, a number of new genera were
described (e.g. Botryobambusa, Cophinforma, Neofusicoccum,
Neoscytalydium, Pseudofusicoccum, etc.) or older genera re-
dened (e.g. Auerswaldia, Barriopsis, Dothiorella, etc.) (e.g. Crous
et al. 2006, Phillips et al. 2008, Liu et al. 2012). The most recent
work by Liu et al. (2012) reviewed these genera, and this study
reects a growing consensus regarding the circumscription of the
majority of the genera (29 in total, of which sequence data are
available for 20).
DNA-based sequence analyses have also resulted in signicant
changes to the nomenclature, identication and circumscription of
species in the Botryosphaeriaceae. These changes have resulted
in the implementation of a single nomenclature for all morphs of
a species (Crous et al. 2006, Hawksworth et al. 2011, Wingeld
et al. 2012). For the Botryosphaeriaceae, this has included the
description of cryptic species based on DNA sequence data, where
morphological characters were not variable enough for this purpose
(Pavlic et al. 2009a, Sakalidis et al. 2011).
Insights gained from contemporary studies on the
Botryosphaeriaceae have led to uncertainty regarding the
application of names published in the older literature. The analyses
show for example that morphological characters typically used for
species identication (chiey conidia or ascospore dimensions,
shape, septation and pigmentation) are frequently unreliable.
Even ecological and geographical data are difcult to interpret,
with some species occurring on numerous hosts, and single
locations or hosts often yielding numerous co-occurring species
(Slippers & Wingeld 2007, Slippers et al. 2009). For this reason
(together with the signicant changes in generic descriptions
mentioned above) many, if not most, of the taxa dealt with before
the introduction of DNA sequence-based phylogenetic inference
will need to be redened (possibly neo- or epitypied), to allow
meaningful comparisons with currently applied names (also see
the discussion in Phillips et al. 2013, this volume). Where it is not
possible to follow this approach, older names may have to be
ignored and new species introduced that are supported by DNA
data (see Slippers et al. 2014).
The Botryosphaeriales is an important group of fungi due to
the ecological and economic signicance of many of its species.
All species are plant-associated, and many are classied
as pathogens, known to cause disease on a wide range of
ecologically and economically important plants (Mehl et al. 2012).
Some species are also known to cause opportunistic infections in
humans (de Hoog et al. 2000). Most species exist as endophytes
living in healthy plant tissues for extended periods of time (Slippers
& Wingeld 2007). Their roles as endophytes or pathogens often
overlap, as is for example found in the case of Diplodia sapinea.
This well-known pathogen of Pinus (Swart et al. 1991) is also a
common endophyte in branches, the trunks and seed cones of
these trees. In an extreme example, D. sapinea has been isolated
from the wood of Pinus in South Africa, where it must have existed
without causing disease subsequent to the tree being infected as
long as a decade previously (Bihon et al. 2011).
Unlike the case for D. sapinea, the ecological roles for the
majority of species of Botryosphaeriaceae are unknown. The
changes to the taxonomy of the group are already strongly
promoting an ability to characterise the diversity in this group. In
turn, this is providing an evolutionary framework making it possible
to study the ecological role that remains obscure for the majority of
these fungi.
In this paper, the phylogenetic relationships of all the
genera known from culture and considered to reside in the
Botryosphaeriales and Botryosphaeriaceae are determined based
on DNA sequence data for six loci. The Planistromellaceae is well
dened within the Botryosphaeriales. As expected, Phyllosticta (=
Guignardia) also forms a strongly supported monophyletic lineage,
recognised as the Phyllostictaceae (see Wikee et al. 2013b, this
volume). Saccharata, however, groups separately with respect to
all other genera in the Botryosphaeriales, as does Aplosporella,
Bagnisiella and Melanops. The nomenclatural changes necessary
to reect these distinctions are considered in this study. With the
well-supported phylogeny provided by these analyses, we also test
hypotheses regarding the evolution of major morphological features
typically used in taxonomy of the Botryosphaeriaceae. Finally, we
use the nuclear ribosomal subunit data to date the divergence in
the major groups of the Botryosphaeriales.
MATERIALS AND METHODS
Isolates and DNA extractions
A total of 96 strains corresponding to 85 species were grown on
2 % potato dextrose agar (PDA) plates incubated at 25 ºC. Genomic
DNA was extracted from mycelium using the PrepManTM Ultra
protocol (Applied Biosystems). Sequences from additional species
were retrieved from GenBank. A total of 140 taxa were included in
the ingroup and six taxa in the outgroup (see Table 1 for details).
PCR and sequencing
A total of six partial gene portions were used in this study: the
nuclear ribosomal small subunit (SSU), the nuclear ribosomal
large subunit (LSU), the intergenic spacer (ITS), the translation
elongation factor 1-alpha (EF1), the β-tubulin gene (BT) and the
mitochondrial ribosomal small subunit (mtSSU).
The primers used were NS1 and NS4 (White et al. 1990)
for SSU, LROR and LR5 (Vilgalys Laboratory, Duke university,
www.biology.duke.edu/fungi/mycolab/primers.htm) for LSU, ITS-1
and ITS-4 (White et al. 1990) for ITS, EF-AF and EF-BR (Sakalidis et
al. 2011) for EF1, BT2A and BT2B (Glass & Donaldson 1995) for BT
and mrSSU1 and mrSSU3R (Zoller et al. 1999) for mtSSU. All PCR
reactions were conducted in 15 µL containing 1.5 mM of MgCl2, 0.5
mM of dNTP, 1 × nal concentration of buffer, 1 µM of each primer,
0.25 U of FastStart Taq Polymerase (Roche), 1.5 µL of DNA template
and Sabax sterilised water (Adcock Ingram) to complete up to 15 µL.
The cycling parameters were as follows: a rst step of denaturation at
95 ºC for 5 min followed by 35 cycles of (i) denaturation at 95 ºC for
60 s, (ii) annealing at optimal temperature (55 ºC for ITS, EF1, LSU
and 45 ºC for SSU, mtSSU, BT) for 80 s, (iii) elongation at 72 ºC for
90 s, and a nal elongation step of 5 min was applied.
Sephadex columns (Sigma-Aldrich) were used to clean the
samples both before and after the sequencing reactions. The
sequencing PCRs were performed in 10 µL containing 1 µL of PCR
product, 0.7 µL of Big Dye Terminator v. 3.1 (Applied Biosystems),
2.5 µL of sequencing buffer (provided with Big Dye), 1 µL of primer
(10 µM) and 4.8 µL of Sabax sterilised water. Cycling parameters
consisted of 25 cycles with three steps each: 15 s at 95 ºC, 15 s
at 55 ºC (for ITS, EF1, LSU) or 45 °C (for SSU, mtSSU, BT) and
4 min at 60 ºC. The sequencing PCR products were sent to a
www.studiesinmycology.org
Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
33
partner laboratory for sequencing of both strands (Sequencing
Facility, University of Pretoria).
Data analyses
Sequences were aligned using MAFFT v. 6 online (Katoh &
Toh 2008) and rened visually. In order to retrieve the genetic
information from indels, GapCoder (Young & Healy 2003) was used
to code the gaps contained in the EF1 and the mSSU alignments.
Analyses were run both with gaps coded and not coded.
The datasets were combined because this is believed to
increase phylogenetic accuracy (Bull et al. 1993, Cunningham
1997). The six phylogenies resulting from the six data sets were rst
inspected visually to check whether there were conicts between
the histories of the genes that would preclude the combination of
data. Additionally, the Incongruence Length Difference (ILD) test
(Farris et al. 1995a, b) also known as the partition homogeneity test
was run using PAUP v. 4.0b10 (Swofford 2003).
MrAIC (Nylander 2004) was used to determine the best t
model of nucleotide substitutions for each gene. Phylogenetic
relationships of the samples were investigated using both Maximum
Likelihood (ML) and Bayesian Inference (BI). The ML analysis was
conducted using PhyML online (Guindon et al. 2005). The reliability
of each node was assessed using the bootstrap (Felsenstein 1985)
resampling procedure (100 replicates).
The BI was conducted using the software BeaSt v. 1.7.4
(Drummond et al. 2012). The phylogenetic relationships were
estimated by running 10 000 000 generations and sampling
every 100th generation. Bayes Factors were computed to choose
between the different options available in BeaSt (four clock models:
strict clock, exponential or lognormal uncorrelated relaxed clocks,
and random local clock and two tree priors: Yule process or Birth-
Death speciation model). The Birth-Death speciation model and
a relaxed uncorrelated exponential clock were selected as best
tting our data. Six independent runs were performed and outputs
were combined using LogCombiner (in the BeaSt package). The
programme Tracer v. 1.5 (available on the BeaSt website) was used
to check that the effective sampling sizes (ESS) were above 200
(as advised by the programmers to ensure an accurate estimation
of phylogeny and parameters of interest). The programme Tree
Annotator (available in the BeaSt package) was used to summarize
the resulting trees using the maximum clade credibility option. The
nal tree was visualised in FigTree v. 1.4.
Molecular clock dating
Using a mean molecular rate of 1–1.25 % per lineage per 100 million
years, commonly accepted in fungi for the SSU gene (Berbee &
Taylor 2010), a rough estimation of the times to the most recent
common ancestors of groups of interest was assessed using BeaSt.
A normal distribution was used as prior and this was centred on a
95 % interval spanning 1.0–1.25 % (mean = 0.000113; standard
deviation = 0.000006). The dating of the distinct groups of interest
and their 95 % highest posterior density (HPD) were retrieved using
Tracer v. 1.5.
Analysis of phylogeny-trait association
In order to investigate the evolution of morphological characters
in the Botryosphaeriaceae, ancestral trait reconstructions and
tests for phylogenetic signal were conducted in Mesquite v. 2.74
(Maddison & Maddison 2010). The characters considered were
1) ascospore colour; 2) presence or absence of ascospore septa;
3) conidial colour; 4) presence or absence of conidial septa; and
5) presence or absence of a mucus sheath. Both parsimony and
ML reconstructions were used in Mesquite to test for phylogenetic
signal. The observed distribution of character states at the tips
of the phylogeny was compared to null distributions obtained
when reshufing the tip characters on the tree topology (10 000
times). Where the number of steps (or likelihood value) in the
observed trait reconstruction fell outside the 95 % range of the null
distribution, this was seen to indicate that character states are not
distributed randomly on the phylogeny (i.e. there is a phylogenetic
component).
RESULTS
Phylogenetic relationships
The alignment included a total of 4498 bp from six gene portions.
The results from the ILD test were not signicant and supported
a decision to combine the 6 gene datasets. The number of
polymorphic and parsimony informative sites ranged from 108 for
the SSU, 165 for the mtSSU, 170 for the LSU, 222 for BT, 335 for
the EF1 to 361 for the ITS.
The Maximum Likelihood (ML) and Bayesian Inference (BI)
phylogenetic reconstructions were similar, and the BI tree is
shown on Fig. 1. Species residing in the genera Aplosporella and
Bagnisiella (in one clade), Melanops, Saccharata and Kellermania
had a basal position on the tree with respect to other genera in the
Botryosphaeriales. The remaining genera in the Botryosphaeriales
clustered together with a bootstrap support value of 94 % and
Posterior Probability (PP) value of 1. The rst cluster to split from
the rest of the main group was formed by species of Phyllosticta,
hereafter treated as Phyllostictaceae (see Wikee et al. 2013b, this
volume). The main clade below Phyllostictaceae was dened as
Botryosphaeriaceae s. str. (0.99 PP and 89 % bootstrap).
Pseudofusiccocum was basal within the
Botryosphaeriaceae, followed by Endomelanconiopsis. The
remaining species formed a clade having strong bootstrap
support v (99 %) and could be further subdivided into four sub-
clades. Sub-clade 1 encompassed species of the genera Diplodia,
Neodeightonia, Lasiodiplodia, Macrovalsaria, Phaeobotryosphaeria,
Phaeobotryon, Barriopsis, Botryobambusa and Tiarosporella. The
recently described Auerswaldia lignicola clustered in sub-clade 1,
together with Lasiodiplodia. Sub-clade 2 encompassed species of
Neoscytalidium, Cophinforma, Botryosphaeria (= Fusicoccum) and
Macrophomina together with Dichomera saubinetti. Sub-clade 3
accommodated species in the genera Dothiorella, Spencermartinsia
and the recently described Auerswaldia dothiorella. Sub-clade 4
included species of Neofusicoccum and Dichomera.
Molecular dating
Based on the models used, families split between 57 (28–100) –
103 (45–188) mya. Divergence within some families was also very
ancient, such as the split between Pseudofusicoccum [65 (28–112)
mya] and Endomelanconiopsis [52 (27–78) mya] and the rest of the
Botryosphaeriaceae. The split between sub-clades 1 to 4 within the
Botryosphaeriaceae was estimated to be between 33 (15–55) and
44 (25–64) mya.
SlipperS et al.
34
Phyllostictaceae
0.98/100
26(1144)
1/94
87(40163)
1/100
0.98/
33(1555)
11(323)
1/100
1/100
0.99/89
0.77/
65(28112)
38(1659)
26(852)
1/99 1/100
44(2564) 35(1358)
P. podocarpi CBS 111647
G. philoprina CBS 901.69
P. beaumarisii CBS 535.87
G. bidwellii CBS 111645
P. yuccae CBS 117136
G. mangiferae CBS 115052
G. heveae CBS 101228
P. capitalensis CBS 226.77
P. capitalensis CBS 398.80
G. mangiferae CBS 100176
G. mangiferae CBS 115345
P. cornicola CBS 111639
P. minima CBS 111635
G. gaultheriae CBS 447.70
P. minima CBS 585.84
P. telopeae CBS 777.97
G. philoprina CBS 174.77
G. philoprina CBS 616.72
G. citricarpa CBS 828.97
P. citriasiana CBS 120486
P. hypoglossi CBS 101.72
P. stromaticum CMW 13434
P. andansoniae CMW 26147
P. ardesiacum CMW 26159
P. kimberleyense CMW 26156
D. versiformis CBS 118101
N. mangiferum CMW 7801
N. ribis CMW 7772
N. umdonicola CMW 14058
N. parvum CMW 9081
N. luteum CMW 10309
N. australe CMW 6837
N. vitifusiforme CMW 24571
N. eucalypticola CMW 6539
S. pretoriensis CMW 36480
S. pretoriensis CMW 36481
D. longicollis CMW 26166
D. brevicollis CMW 36464
D. brevicollis CMW 36463
Spencermartinsia sp. ICMP 16827
S. rosulata CMW 25389
Spencermartinsia sp. CBS 500.72
Spencermartinsia sp. CBS 302.75
S. viticola CBS 117009
D. thailandica MFLUCC 110438
D. dulcispinae CMW 36462
D. dulcispinae CMW 36460
D. oblonga CMW 25407
D. dulcispinae CMW 36461
D. iberica CBS 115041
Dothiorella sp. CBS 188.87
Dothiorella sp. CBS 252.51
Dothiorella sp. CBS 114124
Dothiorella sp. CBS 113091
Dothiorella sp. CBS 910.73
D. sarmentorum CBS 115038
D. saubinetti CBS 990.70
Botryosphaeria sp. CMW 25413
B. dothidea CMW 8000
B. corticis CBS 119047
B. fusispora MFLUCC 110507
B. fusispora MFLUCC 100098
B. agaves MFLUCC 100051
B. agaves MFLUCC 110125
F. ramosum CMW 26167
C. eucalypti MFLUCC 110425
C. eucalypti MFLUCC 110655
M. phaseolina CBS 227.33
N. novaehollandiae CMW 26170
N. dimidiatum IP 127881 Neoscytalidium
Macrophomina
Cophinforma
Botryosphaeria
Dothiorella/
Spencermar9nsia
Neofusicoccum
Pseudofusicoccum
Botryosphaeriaceae
3
4
2
Fig. 1. Phylogenetic relationships of the Botryosphaeriales using Bayesian reconstruction and six gene portions (LSU, SSU, ITS, EF1, BT and mtSSU). Numbers above
branches indicate bootstrap values/posterior probabilities. Numbers highlighted in red below branches indicate estimated dates in million years with the 95 % Highest Posterior
Density interval given in brackets. Clades 1–4 in the Botryosphaeriaceae are indicated by a circled number on the corresponding node.
www.studiesinmycology.org
Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
35
Melanopsaceae
Planistromellaceae
Aplosporellaceae
Saccharataceae
Endomelanconiopsis
Barriopsis
Phaeobotryon
Phaeobotryosphaeria
Tiarosporella
Botryobambusa
Neodeightonia
Diplodia
Lasiodiplodia
Botryosphaeriaceae
1/90
103(45188) 1/100
29(842)
0.98/97
52(2778)
1/100
1/96
1/
1/
1/99
1/63
1/100
0.66/
0.96/ 1/100
1/100
1/100
1/
75(34136)
57(28100)
38(1673)
B. fusicoccum MFLUCC 110143
B. fusicoccum MFLUCC 110657
T. urbis-rosarum CMW 36479
T. urbis-rosarum CMW 36478
L. crassispora CBS 118741
L. venezuelensis CMW 13512
L. rubropurpurea CBS 118740
L. pseudotheobromae CBS 116459
L. lignicola MFLUCC 110435
L. lignicola MFLUCC 110656
L. parva CBS 456.78
L. theobromae CBS 164.96
M. megalosporagi CMW 178149
M. megalosporagi CMW 178150
L. gonubiensis CMW 14077
D. corticola CBS 112549
D. sapinea CMW 190
D. scrobiculata CBS 118110
D. seriata CBS 112555
D. allocellula CMW 36469
D. allocellula CMW 36470
D. allocellula CMW 36468
D. cupressi CBS 168.87
D. tsugae CBS 418.64
D. rosulata CBS 116472
D. africana CBS 120835
D. mutila CMW 7060
N. palmicola MFLUCC 100822
N. phoenicum CBS 122528
Neodeightonia sp. MFLUCC 110026
N. subglobosa CBS 448.91
P. citrigena ICMP 16812
P. eucalypti MFLUCC 110579
P. porosa CBS 110496
P. visci CBS 186.97
P. mamane CBS 122980
P. cupressi IRAN 1445C
P. mamane CPC 12440
B. fusca CBS 174.26
B. iranianum IRAN 1448C
E. microspora CBS 353.97
E. endophytica CBS 120397
Kellermania sp.1 CPC 20390
K. confusa CBS 131723
K. macrospora CBS 131716
K. uniseptata CBS 131725
K. yuccifoliorum CBS 131726
K. yuccigena CPC 20627
K. yuccigena CPC 20623
K. yuccigena CBS 131727
Kellermania sp.2 CPC 20418
Kellermania sp.2 CPC 20388
K. yuccigena CPC 20386
K. anomala CBS 132218
K. nolinae CBS 131717
K. crassispora CBS 131714
K. plurilocularis CBS 131719
K. micranthae CBS 131724
K. dasylirionis CBS 131715
K. dasylirionicola CBS 131720
A. africana CMW 25424
A. papillata CMW 25427
A. prunicola CBS 121167
A. yalgorensis MUCC 512
B. examinans CBS 551.66
S. proteae CBS 115206
S. kirstenboschensis CBS 123537
S. capensis CBS 122693
Melanops sp. CBS 118.39
M. tulasnei CBS 116805
Fusicladium convolvularum CBS 112706
Fusicladium effusum CPC 4525
Fusicladium oleagineum CBS 113427
Amarenomyces ammophilae CBS 114595
Dothidotthia symphoricarpi CPC 12929
Dothidotthia aspera CPC 12933
1
0.0 0.1 0.2 0.3 0.4
Fig. 1. (Continued).
SlipperS et al.
36
Table 1. Isolates subjected to DNA analysis in this study.
Species Isolate No.1GenBank Accession No.
SSU LSU ITS EF1 BT mtSSU
Amarenomyces ammophilae CBS 114595 N/A KF766314 KF766146 KF766394 N/A N/A
Aplosporella africana CMW 25424, CBS 121777 KF766283 KF766366 KF766196 N/A N/A KF766475
Aplosporella papilata CMW 25427, CBS 121780 KF766284 N/A KF766197 N/A N/A KF766476
Aplosporella prunicola CBS 121167 KF766229 KF766315 KF766147 N/A N/A KF766440
Aplosporella yalgorensis MUCC512 N/A EF591944 EF591927 EF591978 EF591961
N/A
Bagnisiella examinans CBS 551.66 EU167562 KF766316 KF766148 GU349056 KF766126 KF766441
Barriopsis fusca CBS 174.26 KF766230 KF766317 KF766149 KF766395 EU673109 N/A
Barriopsis iranianum IRAN 1448C KF766231 KF766318 KF766150 FJ919652 KF766127 N/A
Botryobambusa fusicoccum MFLUCC110143 JX646826 JX646809 JX646792 JX646857 N/A N/A
MFLUCC110657 JX646827 JX646810 JX646793 JX646858 N/A N/A
Botryosphaeria agaves MFLUCC100051 JX646824 JX646807 JX646790 JX646855 JX646840 N/A
MFLUCC110125 JX646825 JX646808 JX646791 JX646856 JX646841 N/A
Botryosphaeria corticis CBS 119047 KF766232 EU673244 DQ299245 EU017539 EU673107 N/A
Botryosphaeria dothidea CMW 8000, CBS 115476 KF766233 KF766319 KF766151 AY236898 AY236927 FJ190612
Botryosphaeria fusispora MFLUCC100098 JX646823 JX646806 JX646789 JX646854 JX646839 N/A
MFLUCC110507 JX646822 JX646805 JX646788 JX646853 JX646838 N/A
Botryosphaeria ramosa CMW 26167 KF766253 KF766333 KF766168 EU144070 N/A N/A
Botryosphaeria sp.CMW 25413 KF766252 KF766332 KF766167 N/A N/A N/A
Cophinforma eucalypti MFLUCC110425 JX646833 JX646817 JX646800 JX646865 JX646848 N/A
Dichomera saubinetii CBS 990.70 KF766236 DQ377888 KF766153 KF766396 N/A N/A
MFLUCC110655 JX646834 JX646818 JX646801 JX646866 JX646849 N/A
Dichomera versiformis CMW 15210, CBS 118101 KF766237 KF766321 KF766154 N/A KF766128 N/A
Diplodia africana CBS 120835 KF766238 KF766322 KF766155 KF766397 KF766129 KF766442
Diplodia allocellula CBS 36468 N/A JQ239410 JQ239397 JQ239384 JQ239378 N/A
CBS 36469 N/A JQ239411 JQ239398 JQ239385 JQ239379 N/A
CBS 36470 N/A JQ239412 JQ239399 JQ239386 JQ239380 N/A
Diplodia corticola CBS 112549 KF766239 KF766323 KF766156 AY573227 DQ458853 KF766443
KF766398
Diplodia cupressi CBS 168.87 KF766240 EU673263 KF766157 DQ458878 DQ458861 KF766444
Diplodia mutila CMW 7060 KF766241 KF766324 KF766158 AY236904 AY236933 N/A
Diplodia rosulata CBS 116472 EU673212 DQ377897 EU430266 EU430268 EU673131 N/A
Diplodia sapinea CMW 109 KF766242 KF766325 KF766159 AY624251 AY624256 N/A
Diplodia scrobiculata CMW 189, CBS 118110 KF766243 KF766326 KF766160 N/A N/A KF766445
Diplodia seriata CBS 112555 KF766244 KF766327 KF766161 AY573220 DQ458856 N/A
Diplodia tsugae CMW 100325, CBS 418.64 KF766234 DQ377867 DQ458888 DQ458873 DQ458855 N/A
Dothidotthia aspera CPC 12933 EU673228 EU673276 N/A N/A N/A N/A
Dothidotthia symphoricarpi CPC 12929 EU673224 EU673273 N/A N/A N/A N/A
Dothiorella brevicollis CMW 36463 N/A JQ239416 JQ239403 JQ239390 JQ239371 N/A
CMW 36464 N/A JQ239417 JQ239404 JQ239391 JQ239372 N/A
Dothiorella dulcispinae CMW 36460 N/A JQ239413 JQ239400 JQ239387 JQ239373 N/A
CMW 36461 N/A JQ239414 JQ239401 JQ239388 JQ239374 N/A
CMW 36462 N/A JQ239415 JQ239402 JQ239389 JQ239375 N/A
Dothiorella iberica CBS 115041 KF766245 AY928053 AY573202 AY573222 EU673096 N/A
Dothiorella longicollis CMW 26166, CBS 122068 KF766246 KF766328 KF766162 EU144069 KF766130 KF766447
Dothiorella oblonga CMW 25407, CBS 121765 KF766247 KF766329 KF766163 N/A N/A KF766448
Dothiorella sarmentorum CBS 115038 KF766248 DQ377860 AY573206 AY573223 EU673101 KF766446
Dothiorella sp.CBS 114124 EF204515’ EF204498’ N/A N/A N/A N/A
CBS 113091 EF204516’ EF204499’ N/A N/A N/A N/A
Dothiorella sp. (=Diplodia acerina) CBS 910.73 EU673160 EU673234 EU673315 EU673282 N/A N/A
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Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
37
Table 1. (Continued).
Species Isolate No.1GenBank Accession No.
SSU LSU ITS EF1 BT mtSSU
Dothiorella sp. (=Diplodia coryli) CBS 252.51 EU673162 EU673235 EU673317 EU673284 EU673105 N/A
Dothiorella sp. (=Diplodia juglandis) CBS 188.87 EU673161 DQ377891 EU673316 EU673283 EU673119 N/A
Dothiorella thailandica MFLUCC110438 JX646829 JX646813 JX646796 JX646861 JX646844 N/A
Endomelanconiopsis endophytica CBS 120397 KF766249 EU683629 KF766164 EU683637 KF766131 KF766449
Endomelanconiopsis microspora CBS 353.97 KF766250 KF766330 KF766165 EU683636 N/A KF766450
Fusicladium convolvularum CBS 112706 AY251124 N/A AY251082 N/A N/A N/A
Fusicladium effusum CPC 4525 N/A EU035430’ AY251085 KF766428 N/A N/A
Fusicladium oleagineum CBS 113427 KF766251 KF766331 KF766166 N/A N/A N/A
Guignardia bidwellii (= Phyllosticta
parthenocissi)
CBS 111645 EU673223 DQ377876 EU683672 EU683653 FJ824777 N/A
Guignardia citricarpa (= Phyllosticta
citricarpa)
CBS 828.97 KF766254 KF766334 FJ538318 FJ538376 N/A N/A
Guignardia gaultheriae CBS 447.70 N/A KF766335 KF766169 KF766400 N/A FJ190646
Guignardia heveae (= Phyllosticta
capitalensis)
CBS 101228 KF766255 KF766336 FJ538319 FJ538377 N/A KF766452
Guignardia mangiferae (= Phyllosticta
capitalensis)
CBS 100176 N/A KF766337 FJ538321 FJ538379 N/A N/A
CBS 115052 N/A KF766338 FJ538321 FJ538379 N/A N/A
CBS 115345 N/A KF766339 FJ538331 FJ538389 N/A N/A
Guignardia philoprina (= Guignardia
rhodorae)
CBS 901.69 KF766258 KF766342 KF766172 KF766403 N/A N/A
Guignardia philoprina (= Phyllosticta
foliorum)
CBS 174.77 KF766256 KF766340 KF766170 KF766401 N/A KF766453
Guignardia philoprina (= Phyllosticta
philoprina)
CBS 616.72 KF766257 KF766341 KF766171 KF766402 N/A N/A
Kellermania anomala CBS 132218 KF766259 KF766343 KF766173 KF766404 KF766133 KF766454
Kellermania confusa CBS 131723 KF766260 KF766344 KF766174 KF766405 KF766134 KF766455
Kellermania crassispora CBS 131714 KF766261 KF766345 KF766175 KF766406 KF766135 KF766456
Kellermania dasylirionicola CBS 131720 KF766262 KF766346 KF766176 KF766407 KF766136 KF766457
Kellermania dasylirionis CBS 131715 KF766263 KF766347 KF766177 KF766408 KF766137 KF766458
Kellermania macrospora CBS 131716 KF766264 KF766348 KF766178 KF766409 KF766138 KF766459
Kellermania micranthae CBS 131724 KF766265 KF766349 KF766179 KF766410 KF766139 KF766460
Kellermania nolinae CBS 131717 KF766266 KF766350 KF766180 KF766411 KF766140 KF766461
Kellermania plurilocularis CBS 131719 KF766267 KF766351 KF766181 KF766412 KF766141 KF766462
Kellermania sp. 1 CPC 20390 KF766268 KF766352 KF766182 KF766413 KF766142 KF766463
Kellermania sp. 2 CPC 20418 KF766269 KF766353 KF766183 KF766414 N/A KF766464
CPC 20386 KF766273 KF766357 KF766187 KF766418 N/A KF766467
CPC 20388 KF766274 KF766358 KF766188 KF766419 N/A KF766468
Kellermania uniseptata CBS 131725 KF766270 KF766354 KF766184 KF766415 KF766143 KF766465
Kellermania yuccifoliorum CBS 131726 KF766271 KF766355 KF766185 KF766416 KF766144 N/A
Kellermania yuccigena CBS 131727 KF766272 KF766356 KF766186 KF766417 KF766144 KF766466
CPC 20623 KF766275 KF766359 KF766189 KF766420 N/A KF766469
CPC 20627 KF766276 KF766360 KF766190 KF766421 N/A KF766470
Lasiodiplodia crassispora CBS 118741 EU673190 DQ377901 DQ103550 EU673303 EU673133 N/A
Lasiodiplodia gonubiensis CMW 14077, CBS 115812 KF766277 KF766361 KF766191 DQ458877 DQ458860 N/A
Lasiodiplodia lignicola MFLUCC110435 JX646830 JX646814 JX646797 JX646862 JX646845 N/A
MFLUCC110656 JX646831 JX646815 JX646798 JX646863 JX646846 N/A
Lasiodiplodia parva CBS 456.78 KF766278 KF766362 KF766192 EF622063 N/A KF766471
Lasiodiplodia pseudotheobromae CBS 116459 KF766279 EU673256 KF766193 EF622057 EU673111 KF766481
Lasiodiplodia rubropurpurea CBS 118740 EU673191 DQ377903 DQ103553 EU673304 EU673136 N/A
Lasiodiplodia theobromae
(Botryosphaeria rhodina in CBS)
CBS 164.96 EU673196 EU673253 AY640255 AY640258 EU673110 N/A
SlipperS et al.
38
Table 1. (Continued).
Species Isolate No.1GenBank Accession No.
SSU LSU ITS EF1 BT mtSSU
Lasiodiplodia venezuelensis CMW 13512 KF766280 KF766363 KF766194 EU673305 N/A N/A
Macrophomina phaseolina CBS 227.33 KF766281 KF766364 KF766195 KF766422 N/A KF766473
Macrovalsaria megalosporagi CMW 178150 FJ215707 FJ215701 N/A KF766399 N/A N/A
CMW 178149 FJ215706 FJ215700 N/A N/A N/A N/A
Melanops sp. (Botryosphaeria quercuum
in CBS)
CBS 118.39 FJ824763 DQ377856 FJ824771 FJ824776 FJ824782 N/A
Melanops tulasnei CBS 116805 KF766282 KF766365 FJ824769 KF766423 FJ824780 KF766474
Neodeightonia palmicola MFLUCC100822 HQ199223 HQ199222 HQ199221 N/A N/A N/A
Neodeightonia phoenicum CBS 122528 KF766285 EU673261 KF766198 EU673309 EU673116 N/A
Neodeightonia sp. MFLUCC110026 JX646837 JX646821 JX646804 JX646869 JX646852 N/A
Neodeightonia subglobosa CBS 448.91 KF766286 DQ377866 KF766199 EU673306 EU673137 N/A
Neofusicoccum australe CMW 6837 KF766287 KF766367 KF766200 AY339270 AY339254 KF766477
Neofusicoccum eucalypticola CMW 6539, CBS 115679 KF766288 KF766368 KF766201 AY615133 AY615125 N/A
Neofusicoccum lutea CMW 10309 KF766289 KF766369 KF766202 KF766424 DQ458848 N/A
Neofusicoccum mangiferum CMW 7801 KF766290 KF766370 KF766203 KF766425 AY615174 KF766479
Neofusicoccum parvum CMW 9081 KF766291 KF766371 KF766204 KF766426 AY236917 KF766480
Neofusicoccum ribis CMW 7772, CBS 115475 KF766292 KF766372 KF766205 DQ677893 AY236906 KF766481
Neofusicoccum umdonicola CMW 14058, CBS 123645 KF766293 KF766373 KF766206 KF766427 KF766145 KF766482
Neofusicoccum vitifusiforme CMW 24571 KF766235 KF766320 KF766152 FJ752707 N/A N/A
Neoscytalidium dimidiatum IP127881 AF258603 DQ377925’ AY819727 EU144063 FM211167 N/A
Neoscytalidium novaehollandiae CMW 26170, CBS 122071 KF766294 KF766374 KF766207 EF585580 N/A N/A
Phaeobotryon cupressi IRAN 1445C KF766295 N/A KF766208 N/A N/A N/A
Phaeobotryon mamane CPC 12440 EU673184 EU673248 EU673332 EU673298 EU673121 KF766483
KF766209
CBS 398.80 KF766301 KF766378 KF766213 KF766430 N/A KF766486
Phaeobotryosphaeria citrigena
(Botryosphaeria fusca in CBS)
ICMP 16812 EU673180 EU673246 EU673328 EU673294 EU673140 N/A
Phaeobotryosphaeria eucalypti MFLUCC110579 JX646835 JX646819 JX646802 JX646867 JX646850 N/A
Phaeobotryosphaeria porosa CBS 110496 KF766297 KF766375 KF766210 N/A EU673130 N/A
Phaeobotryosphaeria visci CBS 186.97 KF766298 KF766393 KF766211 EU673293 EU673128 N/A
Phyllosticta beaumarisii CBS 535.87 KF766299 KF766376 KF766212 KF766429 N/A KF766484
Phyllosticta capitalensis CBS 226.77 KF766300 KF766377 FJ538336 FJ538394 N/A KF766485
CBS 398.80 N/A N/A N/A N/A N/A N/A
Phyllosticta citriasiana CBS 120486 KF766302 KF766379 FJ538360 FJ538418 N/A N/A
Phyllosticta cornicola CBS 111639 N/A KF766380 KF766214 KF766431 N/A KF766487
Phyllosticta hypoglossi CBS 101.72 KF766303 KF766381 FJ538365 FJ538423 N/A N/A
Phyllosticta minima CBS 585.84 N/A KF766382 KF766216 KF766433 N/A N/A
Phyllosticta minima (= Phyllosticta
rubrum)
CBS 111635 N/A EU754194 KF766215 KF766432 N/A N/A
Phyllosticta podocarpi CBS 111647 KF766304 KF766383 KF766217 KF766434 N/A N/A
Phyllosticta telopeae CBS 777.97 N/A KF766384 KF766218 KF766435 N/A N/A
Phyllosticta yuccae CBS 117136 KF766305 KF766385 KF766219 KF766436 N/A N/A
Pseudofusicoccum adansoniae CMW 26147, CBS 122055 KF766306 KF766386 KF766220 EF585571 N/A KF766488
Pseudofusicoccum ardesiacum CMW 26159, CBS 122062 KF766307 KF766387 KF766221 EU144075 N/A KF766489
Pseudofusicoccum kimberleyense CMW 26156, CBS 122058 KF766308 KF766388 KF766222 EU144072 N/A KF766490
Pseudofusicoccum stromaticum CMW 13434, CBS117448 KF766309 KF766389 KF766223 KF766437 EU673094 N/A
Saccharata capensis CMW 22200, CBS 122693 N/A KF766390 KF766224 EU552095 N/A KF766491
Saccharata kirstenboschensis CBS 123537 KF766310 FJ372409 KF766225 N/A N/A KF766492
Saccharata proteae CBS 115206 KF766311 DQ377882 KF766226 KF766438 N/A KF766493
Spencermartinsia pretoriensis CMW 36480 N/A JQ239418 JQ239405 JQ239392 JQ239376 N/A
CMW 36481 N/A JQ239419 JQ239406 JQ239393 JQ239377 N/A
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Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
39
Table 1. (Continued).
Species Isolate No.1GenBank Accession No.
SSU LSU ITS EF1 BT mtSSU
Spencermartinsia rosulata CMW 25389, CBS 121760 KF766312 KF766391 KF766227 KF766439 N/A KF766494
Spencermartinsia sp. (Botryosphaeria sp.
in ICMP)
ICMP 16827 EU673171 EU673241 EU673322 EU673289 EU673144 N/A
Spencermartinsia sp. (Diplodia
medicaginis in CBS)
CBS 500.72 EU673167 EU673237 EU673318 EU673285 EU673118 N/A
Spencermartinsia sp. (Diplodia
spegazziniana in CBS)
CBS 302.75 N/A EU673238 EU673319 EU673286 EU673135 N/A
Spencermartinsia vitícola CBS 117009 KF766313 KF766392 KF766228 AY905559 EU673104 N/A
Tiarosporella urbis-rosarum CMW 36478 N/A JQ239421 JQ239408 JQ239395 JQ239382 N/A
CMW 36479 N/A JQ239422 JQ239409 JQ239396 JQ239383 N/A
1CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CMW: Tree Pathology Co-operative Program, Forestry and Agricultural Biotechnology Institute,
University of Pretoria, South Africa; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Iran; MFLUCC: Mae Fah Luang University Culture
Collection, Chiang Mai, Thailand; MUCC: Culture Collection, Laboratory of Plant Pathology, Mie University, Tsu, Mie prefecture, Japan.
Table 2. Test of phylogenetic signal for each of the 5 traits investigated using Parsimony and Maximum Likelihood reconstructions.
Phylogenetic Signal Parsimony (number of steps) Maximum Likelihood (-log likelihood values)
Observed Expected Mean (Range) P-value Observed Expected Mean (Range) P-value
Ascospore colour 3 5,89 (3-7) ns 11,35 12,87 (10,6-13,7) ns
Ascospore septation 4 5,29 (3-6) ns 11,7 12,79 (8,9-13,7) ns
Conidial colour 6 8,99 (5-13) ns 15,51 17,76 (15,1-18,4) ns
Conidial septation 8 8,90 (5-12) ns 18,02 17,77 (15,8-18,6) ns
Mucus 4 3,98 (2-4) ns 13,59 14,09 (8,0-18,1) ns
Analysis of phylogeny-trait association
None of the ve morphological traits that were investigated had a
signicant phylogenetic signal (Table 2; Fig. 2A–E).
Taxonomy
Based on the phylogenetic distinctions found in this study, as well
as the morphological and in some cases ecological distinction
between the major groups in the Botryosphaeriales, six families
are recognised. Of these, the Planistromellaceae (accommodating
Kellermania) and Phyllostictaceae (accommodating Phyllosticta)
are accepted as previously described (Minnis et al. 2012, Wikee
et al. 2013b, this volume). The Botryosphaeriaceae is redened,
while the Aplosporellaceae, Saccharataceae and Melanopsaceae
are newly described.
Botryosphaeriaceae Theis. & P. Syd., Ann. Mycol. 16: 16.
1918.
Type genus: Botryosphaeria Ces. & De Not., Comment. Soc.
Crittog. Ital. 1: 211. 1863.
Type species: B. dothidea (Moug. : Fr.) Ces. & De Not., Comment.
Soc. Crittog. Ital. 1: 212. 1863.
Genera included based on support by DNA sequence data:
Barriopsis, Botryobambusa, Botryosphaeria (= Fusicocccum,
incl. Dichomera pro parte), Cophinforma, Dothiorella, Diplodia,
Endomelanconiopsis, Lasiodiplodia (incl. Auerswaldia,
Macrovalsaria), Macrophomina, Neodeightonia, Neofusicoccum
(incl. Dichomera pro parte), Neoscytalidium, Phaeobotryon,
Phaeobotryosphaeria, Pseudofusicoccum, Spencermartinsia,
Tiarosporella.
Genera lacking DNA sequence data: Auerswaldiella,
Leptoguignardia, Microdiplodia, Phyllachorella, Pyrenostigma,
Septorioides, Sivanesania, Thyrostroma, Vestergrenia (Liu et al.
2012).
Ascostromata uni- to multilocular, solitary or in clusters, fully
or partially erumpent at maturity, with multi-layered, dark
brown walls, infrequently embedded in stromatic tissue. Asci
bitunicate, ssitunicate, chiey 8-spored, with a thick endotunica
and well-developed apical chamber, short stipitate, clavate.
Pseudoparaphyses intermixed with asci, hyaline, septate,
frequently constricted at septa, hyphae-like, branched or not,
frequently deliquescing at maturity. Ascospores 2–3 seriate,
hyaline to pigmented, smooth to verruculose, septate or not, fusoid
to ellipsoid or ovoid, with or without a mucoid sheath or rarely with
appendages. Asexual morphs mostly have uni-, rarely multilocular
pycnidial conidiomata, infrequently embedded in stromatic
tissue. Conidiophores mostly reduced to conidiogenous cells.
Conidiogenous cells hyaline, phialidic, proliferating percurrently or
via periclinal thickening, with or without collarettes. Conidia hyaline
to pigmented, aseptate, one or multi-septate, sometimes muriform,
smooth or striate, thin to thick-walled, and sometimes with mucoid
sheaths or appendages. Synasexual morphs coelomycetous or
hyphomycetous (see Crous et al. 2006). Spermatogonia similar
to conidiomata in anatomy. Spermatogenous cells ampulliform to
lageniform or subcylindrical, hyaline smooth, phialidic. Spermatia
developing in conidiomata or spermatogonia, hyaline, smooth,
granular, subcylindrical or dumbbell-shaped, with rounded ends.
SlipperS et al.
40
Ascospore colour Ascospore septation Conidial colour
Conidial septation Mucus
Fig. 2. Ancestral state reconstruction using parsimony and mapping the ve traits onto the phylogenetic tree. Traits are coded as presence (in black) or absence (in white).
www.studiesinmycology.org
Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
41
Saccharataceae Slippers, Boissin & Crous, fam. nov.
MycoBank MB805794. Fig. 3.
Type genus: Saccharata Denman & Crous, In: Crous et al., CBS
Biodiversity Ser. (Utrecht) 2: 104. 2004.
Type species: S. proteae (Wakef.) Denman & Crous, In: Crous et
al., CBS Biodiversity Ser. (Utrecht) 2: 104. 2004.
Genus supported by DNA sequence data: Saccharata.
Ascomata pseudothecial, unilocular, solitary or in clusters, with
multilayered dark brown walls, infrequently embedded in stromatic
tissue, with upper ascomatal layer darkened and thickened.
Asci bitunicate, ssitunicate, 8-spored, with a thick endotunica,
stalked or sessile, clavate, with a well-developed apical chamber.
Pseudoparaphyses intermixed with asci, hyaline, septate, hyphae-
like, branched or not. Ascospores hyaline to pigmented, granular,
septate or not, ellipsoid to ovoid, without mucoid appendages or
sheath. Asexual morph has unilocular pycnidial conidiomata,
infrequently embedded in stromatic tissue with thickened,
darkened upper layer. Conidiophores sparingly branched, hyaline,
subcylindrical, or reduced to conidiogenous cells. Conidiogenous
cells hyaline, smooth, phialidic, proliferating via periclinal thickening
or percurrent proliferation, with or without collarettes. Conidia
hyaline, thin-walled, granular, fusoid, aseptate. Synasexual morph
formed in separate conidiomata, or in same conidiomata with
asexual morph. Synasexual conidia pigmented, thick-walled, nely
verruculose, ellipsoid or oval, aseptate. Spermatogonia similar to
conidiomata in anatomy. Spermatogenous cells ampulliform to
lageniform or subcylindrical, hyaline smooth, phialidic. Spermatia
developing in conidiomata or spermatogonia, hyaline, smooth,
granular, subcylindrical or dumbbell-shaped, with rounded ends.
Aplosporellaceae Slippers, Boissin & Crous, fam. nov.
MycoBank MB805795. Fig. 4.
Type genus: Aplosporella Speg., Anal. Soc. cient. argent. 10(5–6):
158. 1880.
Type species: A. chlorostroma Speg., Anal. Soc. cient. argent.
10(5–6): 158. 1880.
Fig. 3. Saccharataceae (Saccharata proteae, CBS 121406). A. Symptomatic leaves with tip die-back. B. Supercial view of immersed ascomata, showing clypeus-like structure.
C, D. Asci and ascospores. E–G. Conidiogenous cells and paraphyses. H, I. Conidia and spermatia. Scale bars = 10 µm.
SlipperS et al.
42
Genera supported by DNA sequence data: Aplosporella,
Bagnisiella.
Ascomata pseudothecial, mostly multilocular with multilayered
dark brown walls, embedded in stromatic tissue. Asci bitunicate,
with a thick endotunica, stalked or sessile, clavate, with a well-
developed apical chamber, intermixed with hyaline, septate,
hyphal-like pseudoparaphyses, branched or not. Ascospores
hyaline to pigmented, septate or not, ellipsoid to ovoid, without
mucoid appendages or sheath. Asexual morphs with uni- to
multilocular pycnidial conidiomata, embedded in stromatic tissue.
Conidiophores reduced to conidiogenous cells. Conidiogenous
cells hyaline, phialidic, proliferating percurrently or with periclinal
thickening at apex. Paraphyses present or absent, hyaline, smooth-
walled, septate, branched or not, hyphae-like. Conidia ellipsoid to
subcylindrical, initially hyaline becoming pigmented, aseptate, thin-
walled and smooth, becoming thick-walled and spinulose.
Fig. 4. Aplosporellaceae (Aplosporella prunicola, CBS 121167). A, B. Oozing spore masses from submerged conidiomata. C. Transverse section through multilocular conidioma.
D, E. Conidiogenous cells. F. Paraphyses. H. Conidia and branched paraphyses. G–J. Conidia. Scale bars: A–C = 250 μm, F = 20 μm, D, E, G–J = 10 μm (adapted from Damm
et al. 2007).
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Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
43
Melanopsaceae Phillips, Slippers, Boissin & Crous, fam.
nov. MycoBank MB805796. Figs 5, 6.
Type genus: Melanops Nitschke ex Fuckel, In: Fuckel, Jahrb.
Nassau. Ver. Naturk. 23–24: 225. 1870.
Type species: Melanops tulasnei Nitschke, In: Fuckel, Jahrb.
Nassau. Ver. Naturk. 23–24: 225. 1870.
Genus supported by DNA sequence data: Melanops (see Phillips
& Alves 2009).
Ascomata pseudothecial, multiloculate, immersed, partially
erumpent at maturity, black, subglobose, thick-walled; wall
composed of thick-walled textura angularis. Asci 8-spored,
bitunicate, ssitunicate, stipitate, clavate. Pseudoparaphyses
hyaline, thin-walled, hyphal-like, septate. Ascospores hyaline,
aseptate, thin-walled, ellipsoid to rhomboid, with a persistent
mucus sheath. Conidiomata indistinguishable from ascomata and
often formed in the same stroma. Paraphyses hyaline, septate,
branched or not, liform, arising from between the conidiogenous
cells. Conidiophores hyaline, smooth, 1–2-septate, branched
or not, or reduced to conidiogenous cells. Conidiogenous cells
subcylindrical, hyaline, branched or unbranched, discrete, formed
from the inner wall of the conidioma, proliferating percurrently at
apex, or with periclinal thickening. Conidia hyaline, aseptate, fusoid,
with a persistent mucus sheath, rarely with minute marginal frill.
Phyllostictaceae Fr. (as “Phyllostictei”), Summa veg.
Scand., Section Post. (Stockholm): 420. 1849.
Type genus: Phyllosticta Pers., Traité sur les Champignons
Comestibles (Paris): 55. 147. 1818.
Type species: P. convallariae Pers., Traité sur les Champignons
Comestibles (Paris): 148. 1818.
Genus supported by DNA sequence data: Phyllosticta (see Wikee
et al. 2013b, this volume).
Planistromellaceae M.E. Barr, Mycotaxon 60: 433. 1996.
Fig. 7.
Type genus: Planistromella A.W. Ramaley, Mycotaxon 47: 260.
1993.
Type species: P. yuccifoliorum A.W. Ramaley, Mycotaxon 47: 261.
1993 (= Kellermania yuccifoliorum)
Genus supported by DNA sequence data: Kellermania (= Alpakesa,
Piptarthron, Planistroma, Planistromella, Septoplaca (possibly),
see Minnis et al. 2012).
Ascomata pseudothecial, multi- or uniloculate, immersed to
erumpent, solitary to gregarious, with papillate, periphysate
ostiole; walls of several layers of dark brown textura angularis.
Hamathecium mostly lacking pseudoparaphyses at maturity. Asci
8-spored, bitunicate, ssitunicate, thick-walled, oblong to clavate
or subcylindrical, stipitate, with well-developed ocular chamber.
Ascospores 1–3-seriate, hyaline or pale brown, guttulate, ellipsoid
to broadly obovoid, aseptate or with 1–2 transverse septa, thin-
walled, with or without a gelatinous sheath. Conidiomata pycnidial
to acervular, subepidermal, dark brown, immersed to semi-
erumpent, solitary to gregarious; wall comprising several layers with
cells of dark brown textura angularis, becoming hyaline towards
the inner region. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells ampulliform to sub-cylindrical, hyaline, smooth,
phialidic, proliferating via percurrent proliferation or periclinal
thickening. Conidia obclavate to ellipsoid-cylindrical, aseptate or
transversely multiseptate, hyaline to brown, smooth to verruculose,
with or without one or more apical appendages, a persistent
mucoid sheath, and a basal marginal frill. Spermatogonia similar
to conidiomata in anatomy. Spermatogenous cells ampulliform to
lageniform or subcylindrical, hyaline smooth, phialidic. Spermatia
developing in conidiomata or spermatogonia, hyaline, smooth,
granular, sub-cylindrical or dumbbell-shaped, with rounded ends.
DISCUSSION
Using the DNA sequence data for the six loci analysed in this study,
together with unique morphological and ecological characteristics
(as discussed below), we have distinguished six families in the
Botryosphaeriales. The Planistromellaceae that has recently been
dened based on DNA sequence data (Minnis et al. 2012) has been
retained. Furthermore, the Aplosporellaceae, Melanopsaceae, and
Saccharataceae are distinguished from the Botryosphaeriaceae
and introduced as novel families. These families are also
phylogenetically distinct from the Phyllostictaceae, which has been
dened in a separate study (Wikee et al. 2013b, this volume).
Botryosphaeriaceae
The Botryosphaeriaceae as it has been dened in this study
includes the type genus Botryosphaeria (asexual morph
Fusicoccum), as well as 16 other genera. This group corresponds
to a group traditionally referred to as botryosphaeria-like. This
term is, however, now understood to be much more restricted,
including B. dothidea and a few closely related species (as dened
in Slippers et al. 2004, Crous et al. 2006, Phillips et al. 2013,
this volume). Using Botryosphaeria to refer to the assemblage
of genera including Diplodia, Lasiodiplodia, Neofusicoccum and
others, of which the sexual morphs were formerly described in
Botryosphaeria, is thus taxonomically incorrect. These groups are
now referred to using a single genus name, which is typically the
asexual morph, irrespective of whether a sexual morph is known or
not. This convention has been applied subsequent to the taxonomic
changes introduced by Crous et al. (2006), and is also consistent
with the recent decisions to abolish the dual nomenclatural system
for fungal taxonomy (Hawksworth et al. 2011, Wingeld et al. 2012).
The data presented in this study, together with those emerging
from more focused earlier studies, as well as the recent changes
resulting in the abandonment of a dual nomenclature, necessitates
reducing a number of genera in the Botryosphaeriaceae to
synonymy with others. Most importantly, there is no longer just
cause to maintain Botryosphaeria and Fusicoccum as distinct
genera. In the interests of maintaining taxonomic stability, and the
fact that B. dothidea is the type species of the order and family, it is
recommended that Botryosphaeria be retained (Slippers et al. 2004,
Schoch et al. 2006, Phillips et al. 2013, this volume). Botryosphaeria
must thus be redened to include species where only the asexual
(Fusicoccum and dichomera-like) morphs are known. Species such
as F. ramosum and Dichomera saubinetti must then be redened in
Botryosphaeria. For F. ramosum, an ex-type isolate was available
and it has thus been redescribed in Botryosphaeria in a companion
paper (Phillips et al. 2013, this volume).
SlipperS et al.
44
Fig. 5. Melanopsaceae (Melanops tulasnei, LISE 95179). A. Stroma erumpent through bark. B, C. Sections through stromata revealing ascomata and conidiomata. D. Ascus.
E. Asci and pseudoparaphyses. F. Pseudoparaphyses. G, H. Ascus tips viewed under differential interference contrast (G) and phase contrast (H). I–K. Ascospores. Scale bars:
A–C = 250 µm, D, E = 20 µm, F–K = 10 µm (adapted from Phillips & Alves 2009).
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Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
45
Dichomera is polyphyletic and most likely also includes
synasexual morphs of other genera. Two of the species included
in this analysis, D. eucalypti and D. versiformis, clearly group in
Neofusicoccum and we consider them as synonyms of species
in this genus. Dichomera saubinetti grouped with Botryosphaeria
and should be redescribed in this genus. Unfortunately no ex-type
isolates of these species are presently available.
A number of genera grouped in Lasiodiplodia s. lat. in our
analyses and are possibly synonyms of this genus. Macrovalsaria
(see Sivanesan 1975) clearly grouped amongst species of
Lasiodiplodia. This was also pointed out by Liu et al. (2012), but
they did not nd the available LSU and SSU data sufciently
convincing to make taxonomic changes. Isolates of Macrovalsaria
however, grouped extremely closely with L. theobromae and we
view them as representing a synonym of Lasiodiplodia, rather than
Lasiodiplodia being polyphyletic. Lasiodiplodia and Macrovalsaria
are both tropical fungi. Our analyses differ from those of Liu et al.
(2012), indicating that Auerswaldia is a synonym of Lasiodiplodia.
This was also conrmed in Phillips et al. (2013, this volume), who
redescribed A. lignicola as L. lignicola. These ndings suggest that
Fig. 6. Melanopsaceae (Melanops tulasnei, LISE 95179). A. Section through conidiomata. B. Conidiogenous layers with developing conidia among paraphyses. C. Immature
conidiogenous cells. D. Paraphyses. E. Conidiogenous cell with percurrent proliferations (arrowed). F. Conidia. G. Conidium in indian ink, revealing sheath. H. Conidium
attached to conidiogenous cell with mucus sheath (arrowed). Scale bars: A = 200 µm, B–D, F, G = 10 µm, E, H = 5 µm (adapted from Phillips & Alves 2009).
SlipperS et al.
46
the taxonomy of Lasiodiplodia needs to be re-evaluated, but for
the present we do not recognize Auerswaldia as a genus in the
Botryosphaeriaceae.
Diplodia juglandis and D. corylii both grouped in Dothiorella,
which is consistent with the results of a previous study (Phillips
et al. 2008). Type specimens were not available for these species
and they were, therefore, not re-described. These species require
epitypication. It is likely that a number of other Diplodia species will
similarly reside in Dothiorella or Spencermartinsia, or vice versa,
given the confusion of these names in the past (Phillips et al. 2005,
2008). The conidia of these genera remain difcult to distinguish,
which can create problems when interpreting older descriptions or
poorly preserved herbarium specimens.
There was no statistically signicant pattern that could be
discerned in the Botryosphaeriaceae with respect to the evolution
of hyaline or pigmented conidia or ascospores. These characters
appear to be more or less randomly spread amongst the clades
of this section of the phylogenetic tree for the family. This would
suggest that these characters predate the divergence of the
genera in this family, and that they have been independently lost
or suppressed (character not expressed under all conditions) in
different groups. This would also explain the “appearance” of darker
or even dark muriform conidia in genera such as Botryosphaeria
and Neofusicoccum that were traditionally considered not to
have such synasexual morphs (Barber et al. 2005, Phillips et al.
2005b, Crous et al. 2006). Clearly these characters, which have
traditionally been commonly used for phylogenetic and taxonomic
purposes, have very little phylogenetic and taxonomic value above
the genus level.
The distinction and more narrow denition of the
Botryosphaeriaceae is important when considering the
economic and ecological importance of this group. Many of the
Botryosphaeriaceae species share a common ecology in being
endophytic and latent pathogens in virtually all parts of woody
plants (Slippers & Wingeld 2007). While not all species have been
isolated as endophytes, or from all plant parts, most of those that
have been carefully studied have conformed to this pattern, and it is
thus expected for the group as a whole. Many of the genera in the
family are also very widespread, with wide host ranges and broad
levels of environmental tolerance (e.g. N. parvum, N. australe,
B. dothidea, L. theobromae, L. pseudotheobromae). Sakalidis et
al. (2013) for example reported N. parvum from 90 hosts in 29
countries on six continents. This broad ecological range, together
with their cryptic nature as endophytes, makes these fungi important
to consider as a group prone to being spread with living plant
material. Ample evidence exists that these fungi can infect both
native and non-native trees, once they have been introduced into
a region. The observed (Dakin et al. 2010, Piškur et al. 2011) and
expected (Desprez-Loustau et al. 2006) increase of the importance
of this group due to pressure on plant communities as a result of
climate change provides another reason to focus future efforts on
characterising the diversity, distribution and pathogenicity of this
group of fungi.
Aplosporellaceae
An unexpected outcome of this study was the consistent connection
between Aplosporella and Bagnisiella and their distinction from
other members of the Botryosphaeriales. While it has been
suggested that some Aplosporella spp. might be asexual morphs
of Bagnisiella, this connection has never been proven. Neither of
these genera were treated in molecular phylogenetic re-evaluations
of the Botryosphaeriaceae until very recently. The rst analyses to
include DNA sequence data for Aplosporella (Damm et al. 2007,
Liu et al. 2012) and Bagnisiella (Schoch et al. 2009) hinted at a
distant relationship with other Botryosphaeriales. However, none of
these studies included both genera. The phylogenetic relationship
between these genera revealed in this study is further supported
by their remarkably similar multiloculate sporocarps, expressed
in both the asexual morphs and sexual morphs, which is thus
Fig. 7. Planistromellaceae (Kellermania yuccigena, CPC 20627). A. Conidiomata sporulating on OA. B, C. Conidiogenous cells showing percurrent proliferation. D. Spermatia.
E–G. One-septate macroconidia with apical appendages. Scale bars = 10 µm.
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Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework
47
not the product of parallel evolution in two distinct groups. There
are, however, undoubtedly many species of Aplosporella and
Bagnisiella that would not be connected to the phylogenetic clade
identied here, because both genera are heterogeneous and likely
contain unrelated species.
The data presented in this study suggest that Aplosporella
and Bagnisiella are not only related, but that they should be
synonymized. Both genera were described in 1880, and historical
precedence can thus not be used to choose an appropriate genus.
Aplosporella includes many more species (352) than Bagnisiella
(65) (www.MycoBank.org, accessed August 2013). More species
have also recently been described in the former genus, possibly
because the asexual structures are more common than the sexual
structures (as is found in other Botryosphaeriales). An argument
based on taxonomic stability, relevance and frequency of occurrence
is thus favoured and has led us to decide that Bagnisiella should be
reduced to synonymy with Aplosporella.
Examination of the distribution of species of Aplosporella and
Bagnisiella is complicated by the fact that the literature is old (which
means the taxonomic accuracy is difcult to judge) and commonly
lacking relevant information. However, most of the well-known and
recently characterised species, and all species included here, are
from the Southern Hemisphere (Damn et al. 2007, Taylor et al.
2009). This result suggests the possibility of a Southern Hemisphere
and Gondwanan origin and divergence pattern, which should be
considered in future studies based on more robust sampling.
Melanopsaceae
Melanops tulasnei and an undescribed Melanops sp. grouped most
basal in the Botryosphaeriales, together with the Aplosporellaceae,
Planistromellaceae and Saccharataceae. This group is unique
amongst these families in having persistent mucous sheath
around its ascospores and conidia (Phillips & Alves 2009). Conidia
in M. tulasnei are typically hyaline and fusoid and it resembles
the Aplosporellaceae in having multiloculate ascomata and
conidiomata, often with locules at different levels. Little is known
regarding the ecology and distribution of this group, given the
paucity of recent reports that could be veried using DNA sequence
data. It appears similar, however, to other Botryosphaeriales that
infect woody tissue of plants, and sporulates on the dead tissue.
Whether it is pathogenic or endophytic is not known.
Saccharataceae
Saccharata (the only genus in the Saccharataceae) grouped
separately from all other families that were basal in the phylogenetic
tree, suggesting a long, separate evolutionary history. The genus
was rst described by Crous et al. (2004) from Proteaceae in
the South Western Cape region of South Africa. Subsequently,
three additional species were added to the genus, two also from
Proteaceae and one from Encephalartos (Marincowitz et al. 2008,
Crous et al. 2008, 2009). All known species are thus from the same
region on indigenous ora. These species are typically associated
with leaf spots and stem cankers and they appear to be pathogens.
Separate studies have also shown that they are endophytes
(Swart et al. 2000, Taylor et al. 2001), similar to members of the
Botryosphaeriaceae.
Apart from its restricted distribution and host range, Saccharata
is also unique in its asexual morphology, which includes a hyaline,
fusicoccum-like and a pigmented diplodia-like asexual morph.
These characters are shared with its related families; fusicoccum-
like conidia in Melanopsaceae and pigmented diplodia-like conidia
in Aplosporellaceae. It is clear that these variations in conidial
morphology are very old (tens of millions of years) ancestral
characters that must have existed prior to the divergence of this
group from other Botryosphaeriales. It is thus remarkable how
similar, especially in the fusicoccum-like conidia and ascospore
morphology, the spores of these fungi have remained over
time. The diplodia-like state is somewhat different from other
Botryosphaeriales in that the conidia are typically almost half the
size of other Diplodia conidia. We do not currently have enough
data to address the selective pressure that could have played a
role in the development these interesting morphological changes.
Phyllostictaceae and Planistromellaceae
Phyllosticta clearly warrants a separate family to accommodate
this morphologically and ecologically unique, widespread and
economically important genus in the Phyllostictaceae. These fungi
typically infect leaves and fruit, rather than woody tissue, and they
can cause serious damage (Glienke et al. 2011, Wong et al. 2012).
Species of Phyllosticta are also known to have an endophytic phase
(Wikee et al. 2013a), as is true for most other Botryosphaeriales.
The Phyllostictaceae is also morphologically unique in terms of the
ascospores and conidia (Van der Aa & Vanev 2002). The species
included in this study are only representative of a small extent of
the diversity in this group and a more complete analysis of the
Phyllostictaceae is presented in Wikee et al. (2013b, this volume).
The Planistromellaceae has previously been recognised as
distinct within the Botryosphaeriales (Minnis et al. 2012) and this
is supported by the analyses in the present study. The family is
currently considered to include only species of Kellermania.
Species in the Planistromellaceae have unique conidia with fairly
long appendages that are quite distinct from other genera in the
Botryosphaeriales. Kellermania spp. are mostly leaf infecting,
and one species has been associated with Yucca Leaf Blight
in California and Florida in the USA (Horst 2008). Species are
commonly collected sporulating on dead leaves of Agavaceae,
and appear to be endophytic, as they have been isolated from
healthy leaves in many countries where Yucca spp. are grown as
ornamentals (P.W. Crous, unpubl data). Minnis et al. (2012) have
also shown apparent patterns of host specicity and co-evolution
with major plant lineages. As is true for many other families in
the Botryosphaeriales, additional sampling is needed for a better
understanding of species diversity, host range and geographic
distribution.
Molecular dating
The molecular dating on the radiations within the Botryosphaeriales
in this study, based on general estimated mutation rates of the
rDNA SSU locus by Taylor & Berbee (2010), must be viewed
as preliminary. From the dating that was conducted, the group
appears to have originated in the Cretaceous period around 103
(45–188) mya, with most of the subsequent diversication within
the families occurring in the Tertiary period. This date is well within
the estimated date of the emergence of the Dothideomycetes
(Berbee & Taylor 2010, Gueidan et al. 2011). This coincides with
important periods of Angiosperm radiation and spread, which is the
main group of plants on which these fungi are found and that might
be expected to have inuenced the diversication of these fungi. Of
SlipperS et al.
48
particular relevance is the rapid diversication of the Eurosid and
other dominant woody Angiosperm groups and their prominence
in Angiosperm dominated forests from around 110 mya onwards
(Soltis et al. 2008, Fawcett et al. 2009, Wang et al. 2009, Bell et
al. 2010). De Wet et al. (2008) pointed out that the members of
the Botryosphaeriaceae are most diverse on Angiosperms, and
showed that ancestral state reconstruction suggests that this is the
main group of plants on which the Botryosphaeriaceae co-evolved.
A much smaller number of species, especially those in Diplodia,
occur commonly on coniferous hosts and appear to have emerged
and diversied more recently. They also tend to be more host-
specic than some of the other genera discussed above that are
more common on Angiosperms (De Wet et al. 2008, Sakalidis et
al. 2013), suggesting a specic acquired trait that allowed them to
infect coniferous hosts.
The major changes in dominant plant hosts in forests
globally during the Cretaceous period would be expected to have
inuenced fungal evolution beyond the Botryosphaeriales, and this
indeed appears to be the case. For example, studies on another
Dothideomycetes order, the sooty molds in the Capnodiales, also
appear to have been inuenced by the rise of Angiosperm forests
in the Cretaceous period (Schmidt et al. 2013). Furthermore,
the divergence of a prominent fungal complex, Fusarium, was
estimated to be later at around 93 mya, and is also thought to have
been inuence by this Angiosperm divergence (O’Donnell et al.
2013).
Molecular dating, together with the geographic distribution
(and restriction) of different groups in the Botryosphaeriales should
provide rich information to explore in future to understand the
patterns that have shaped the diversity of this important group
of plant-associated fungi. For example, the Saccharataceae
has previously been known only from southern Africa, and is
most diverse on the Proteaceae. Recent research has shown,
however, that it has also been introduced as endophyte into other
countries where South African Proteaceae are now being cultivated
(Marincowitz et al. 2008). It is known that this plant family, which
has a high endemic richness in southern Africa, has evolved in the
region for more than 100 million years (Barker et al. 2007). This
date allows for the estimated 57 (28–100) mya (based on rDNA
SSU) of separation of the Saccharataceae to have evolved with
these endemic plant hosts in the region.
Diversication within the most diverse family, the
Botryosphaeriaceae, occurred between 52–65 (27–112) mya for the
two earliest diverging (and least diverse) genera, Pseudofusicoccum
and Endomelanconiopsis. These dates correspond to the
diversication between some other families in the order [e.g.
the Aplosporellaceae, Melanopsaceae, Planistromellaceae and
Saccharataceae that split between 57–75 (28–136) mya]. At present,
however, there does not appear sufciently robust morphological or
ecological validation for a further split of the Botryosphaeriaceae
to accommodate Pseudofusicoccum and Endomelanconiopsis in
distinct families. If these genera were to be considered as residing
in distinct families, the question would arise as to whether further
family level distinction in the Botryosphaeriaceae is necessary. The
other major lineages within the Botryosphaeriaceae (clades 1–4)
appeared around 11–35 (3–58) mya. The most recent diversication
for which there was support was within Neofusicoccum clade, dated
at around 11 (3–23) mya.
This study has provided a systematic framework for future
taxonomic and ecological studies of the Botryosphaeriales. It
has also highlighted a number of interesting host association and
geographic patterns amongst the genera that are worthy of further
investigation. It is hoped that this framework, in conjunction with
the growing body of DNA–based sequence data reecting the
species diversity and their distribution will ultimately lead to a model
supporting an improved understanding of the co-evolution of woody
plants and their fungal endophytes/latent pathogens.
ACKNOWLEDGEMENTS
We thank members of the Tree Protection Co-operative Programme (TPCP), the
DST/NRF Centre of Excellence in Tree Health Biotechnology (CTHB), THRIP
(project no. TP2009061100011) and the University of Pretoria, South Africa for
nancial support. We also thank Ms Katrin Fitza and Ms Fahimeh Jami for assistance
with submissions of sequences.
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