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Mycologia
ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: http://www.tandfonline.com/loi/umyc20
ITS phylogeny and taxonomy of Phyllachora
species on native Myrtaceae from the Brazilian
Cerrado
Maria Desterro Mendes dos Santos, Maria Esther de Noronha Fonseca,
Leonardo Silva Boiteux, Paulo Eduardo A.S. Câmara & Jose C. Dianese
To cite this article: Maria Desterro Mendes dos Santos, Maria Esther de Noronha Fonseca,
Leonardo Silva Boiteux, Paulo Eduardo A.S. Câmara & Jose C. Dianese (2016) ITS phylogeny
and taxonomy of Phyllachora species on native Myrtaceae from the Brazilian Cerrado, Mycologia,
108:6, 1141-1164
To link to this article: https://doi.org/10.3852/16-025
Published online: 30 Jan 2017.
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ITS phylogeny and taxonomy of Phyllachora species
on native Myrtaceae from the Brazilian Cerrado
Maria Desterro Mendes dos Santos
University of Brasília, Department of Phytopathology,
Campus Universitário Darcy Ribeiro, 70910-900 Brasília,
DF, Brazil
Maria Esther de Noronha Fonseca
Leonardo Silva Boiteux
EMBRAPA Hortaliças, Rodovia BR-060, Brasília/Anápolis,
Km 9, Fazenda Tamanduá, 70351-970 Brasília, DF, Brazil
Paulo Eduardo A.S. Câmara
University of Brasília, Department of Botany, Campus
Universitário Darcy Ribeiro, 70910-900 Brasília, DF, Brazil
Jose C. Dianese
1
University of Brasília, Department of Phytopathology,
Campus Universitário Darcy Ribeiro, 70910-900 Brasília,
DF, Brazil
Abstract: Nine Phyllachora species found on hosts
belonging to the family Myrtaceae native to the
Brazilian Cerrado were described and illustrated. We
sequenced nuc rDNA internal transcribed spacer
barcode regions for representatives of seven species
and conducted phylogenetic analyses, which provided
strong support for four new species that we describe
as Phyllachora cerradensis,P.ermidensis,P.furnasensis,
and P. myrciariae.Catacauma nigerrimum was recom-
bined into P. nigerrima, and a key to the common Phylla-
chora species on myrtaceous hosts from the Brazilian
Cerrado was also included.
Key words: barcode, Catacauma nigerimum, mycodi-
versity, Neotropical Ascomycetes, Phyllachorales
INTRODUCTION
In the Cerrado, a Brazilian savanna recognized as a bio-
diversity hotspot (Myers et al. 2000) where over 12 000
plant species are present (Mendonça et al. 2008), fungi
became the focus of mycological research in Brasília in
1993 (Dianese et al. 1993), with an early effort directed
to Phyllachorales (Medeiros 1994, Dianese et al. 1997).
This order accommodates biotrophic ascomycetes
that infect mostly plant leaves, mainly in tropical and
subtropical areas (Cannon 1991, Pearce et al. 1999).
Phyllachora (type species P. graminis [Pers.] Fuckel),
the type genus of the Phyllachoraceae, accommodates
species that grow immersed as a clypiate pseudostroma
in leaf tissues, varying from a subcuticular or
intraepidermal condition, to a generalized infection
of the entire section of the mesophyll, inducing charac-
teristic black shiny superficial symptoms commonly
designated as tar spots.
In Brazil the Phyllachorales were studied from
the early 1940s in well-illustrated papers by Viégas
(1943a, 1943b, 1944a, 1944b, 1944c, 1945a, 1945b,
1946, 1947) and Viégas and Teixeira (1945) describing
Phyllachora species on a range of host plants, in some
cases with the fungal species identified as members
of the genera Catacauma,Endodothella,orTrabutia
(SUPPLEMENTARY TABLE I).
Also important to the knowledge of the Brazilian
Phyllachoralles were the contributions by Batista and
collaborators, who described 11 new Phyllachora species
and expanded the study of 22 known ones. Among
other Phyllachorales they described new Phaeochorella,
Trabutia,Ophiodothella, and Sphaerodothis species
(Batista and Vital 1957; Batista 1958; Batista and Peres
1960; Batista et al. 1960, 1964a, 1964b, 1967; Batista
and Bezerra 1961) (SUPPLEMENTARY TABLE II).
Phyllachora mulleri Chardon was described on Eugenia
dodonaefolia, a host species from the Atlantic Forest,
absent in the Cerrado (Chardon et al. 1940). In addi-
tion, Costa et al. (2012) provided the neotypification
of the Phyllachora feijoae and a table with the character-
istics of Phyllachora species described on hosts belong-
ing in Myrtaceae.
Historically, Phyllachorales were considered an order
incertae sedis within Sordariomycetes (Zhang et al.
2006, Tang et al. 2007, Schoch et al. 2009, Lumbsch
and Huhndorf 2010, Zhang and Wang 2015), with
Wanderlei-Silva et al. (2003) providing the first set of
data on molecular characterization of the order.
Currently, Phyllachorales is inserted in the subclass
Sordariomycetidae comprising two families, Phaeochora-
ceae and Phyllachoraceae, on the basis of comparison of
sequences of the nuc rDNA 18S region (Maharachchi-
kumbura et al. 2015).
As the data on Phyllachora species from the Cerrado is
fragmentary and the taxonomy based on morphologi-
cal criteria, this paper is a first approach to the molecu-
lar phylogeny of some Phyllachora species associated
with myrtaceous hosts from the Cerrado for future
development of its poorly documented phylogenetic
interactions within the genus. Also, the possibility of
considering nuc rDNA internal transcribed spacer
(ITS) sequences as barcodes for Phyllachora species
was tested. The new taxa detected, and some known
Submitted 29 Jan 2016; accepted for publication 26 Sep 2016.
1
Corresponding author. E-mail: jcarmine@unb.br
Mycologia, 108(6), 2016, pp. 1141–1164. DOI: 10.3852/16-025
#2016 by The Mycological Society of America, Lawrence, KS 66044-8897
1141
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ones on Myrtaceae from the Cerrado, were molecularly
characterized, properly described, illustrated, and
organized in a dichotomous key.
MATERIALS AND METHODS
Collection.—The field collections of infected leaves showing
typical tar spots with the presence of healthy fungal ascomata
were dried in a standard plant press and designated as prop-
er vouchers deposited in the Mycological Collection of the
Herbarium UB (University of Brasília). Information on
voucher deposition or herbarium specimens of all new Phylla-
chora collections is provided (SUPPLEMENTARY TABLE III).
For each fresh sample, after careful selection of individual
pseudostroma containing well preserved ascomata, using a
stereomicroscope, materials were superficially disinfested
with 70% ethanol followed by about a 2 min rinse in running
tap water. Then, their centra containing asci, ascospores, and
paraphyses were mechanically exposed with an autoclaved
stylet, and were moistened to promote its mucous expansion,
and then collected for genomic DNA extraction.
DNA extraction, target genomic regions, and PCR profiles.—Total
genomic DNA was extracted by the mini-CTAB protocol
(Doyle and Doyle 1987). The nuc rDNA ITS1-5.8S-ITS2
region (ITS) and 18S regions (White et al. 1990) were ampli-
fied by nested PCR after a 50-fold dilution of the first PCR
reaction. The PCR amplification mix had a total volume of
20 mL and contained 16thermophilic buffer, 2 mM MgCl
2
,
1 U PlatinumHTaq DNA Polymerase (Invitrogen, Carlsbad,
California), 0.75 mg/mL BSA, 0.16 mM of each dNTP,
0.2 mM of each primer and 4 ng of DNA. The PCR profile
was: 94 C (1 min), primer-specific annealing temperature
(SUPPLEMENTARY TABLE IV) for 45 s, 72 C (1 min) for 35 cycles,
always preceded by an initial melting step of 5 min at 94 C and
a final extension cycle of 72 C for 7 min. Both ITS and 18 S
sequences were deposited in GenBank.
Sequencing and phylogenetic analyses.—The PCR products
(amplicons) were purified with a PureLink PCR Purification
KitH(Invitrogen, Carlsbad, California). Clean PCR products
were used in cycle sequencing reactions with the Big-Dye
Terminator Kit (Applied Biosystems, Foster City, California).
The resulting products were purified by ethanol precipitation
and analyzed in an ABI 3100 (Applied Biosystems, Foster
City, California). Forward and reverse strands were
sequenced. Sequences were assembled by SEQMAN II 5.05
(DNAStar, Madison, Wisconsin). All sequences were submit-
ted to GenBank (http://www.ncbi.nlm.nih.gov/). Accession
numbers of all sequences are listed (SUPPLEMENTARY
TABLE III), including additional DNA sequences available at
the GenBank that were used in the phylogenetic analyses.
All datasets were initially aligned using ClustalX (Higgins
and Sharp 1988), then manually adjusted with Phy-De
(Müller et al. 2006), exported as Nexus files, and deposited
in TreeBASE under No. S19754.
Phylogenetic analyses were carried out by maximum parsi-
mony (MP), maximum likelihood (ML), and Bayesian infer-
ence (BI). For MP analyses PAUP* 4.0b10 for Macintosh
was used (Swofford 2002). Heuristic searches were done with
100 random additional replicates and tree-bisection-reconnec-
tion (TBR) branch swapping, saving a maximum of 10 000
trees; all characters were unordered and equally weighted,
and gaps were treated as missing data. For ML analyses, GARLI
0951 for Macintosh (Zwickl 2006) was used. The best-fit model
of evolution for each locus was obtained with JModeltest 2.1.1
(Guindon and Gascuel 2003, Darriba et al. 2012), using the
Akaike information criteria. Clade support was evaluated by
the nonparametric bootstrap (Felsenstein 1985) with 1000
replicates. Bayesian inferences were carried out by Mr Bayes
3.2.1 (Ronquist et al. 2012), four Markov Chain Monte Carlo
were run for 5 000 000 generations and sampled every 1000
generations in two parallel runs. Convergence was established
by ensuring that the average standard deviation of split fre-
quency was ,0.01. Also, the software Tracer 1.5 (Rambaut
and Drummond 2013) was used to determine when the tree
sampling stabilized. The first 25% of the trees were discarded
as burn in. A majority rule consensus tree was constructed
from the resulting trees, as shown (FIG. 1).
RESULTS
Phylogenetic analysis.—The new species (P. cerradensis,
P. ermidensis,P. furnasensis,P. myrciariae)andthree
known species (P. myrciae,P.subcircinans,P. truncatis-
pora) were keyed, described, and illustrated and had
their ITS rDNA segments sequenced and subjected
to phylogenetic analyses. Phyllachora nigerrima n.
comb. and P. myrciae-rostratae were characterized only
by morphology.
A total of 22 new ITS sequences were generated
for this study, and the results from MP, ML and BI
were congruent, differing only slightly in resolution
(FIG. 1). The class Sordariomycetes was here clearly
recognized as monophyletic with good support, with
bootstrap for parsimony (BP) 598, bootstrap for like-
lihood (BL) 593, and posterior probability (PP) 5
1.0. Within this class, nine clades were also recognized
with good support (FIG. 1). Thus, results of the ITS
phylogeny showed several orders as monophyletic,
but the Phyllachora species studied followed a polyphy-
letic distribution showing a lack of resolution or statis-
tical support that would allow a better understanding
of sister-group relationships for most orders, but three
clades containing members of the Phyllachorales were
retrieved as clearly monophyletic, as follows:
1. Clade I. With good support (BP 5100, BL 5100,
PP 51.0), contains several specimens belonging
in Phyllachora (viz. P. subcircinans,P. truncatisporum,
and two new species: P. cerradensis sp. nov. and
P. myrciariae sp. nov.). Within this clade it is possi-
ble to recognize three subclades with good sup-
port; the first one (BP 5100, BL 5100, I 51.0)
is composed of P. subcircinans specimens,parasitic
exclusively on Psidum species. Both of the remain-
ing subclades infect Myrcia species. The second
1142 MYCOLOGIA
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FIG. 1. Maximum likelihood based on ITS sequences obtained from Phyllachora species found on different myrtaceous hosts from
the Cerrado. Numbers above branches are Bootstrap for maximum parsimony, bootstrap for maximum likelihood, and posterior
probabilities from Bayesian inference, respectively. Numbers ,50 for bootstrap and 0.95 for Bayesian inference are not shown.
SANTOS ET AL.: ITS PHYLOGENY OF PHYLLACHORA SPECIES 1143
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subclade (BP 5100, BL 599, BI 51.0) contains
P. cerradensis sp. nov. with a basal P. myrciariae sp.
nov., and the third one (BP 5100, BL 596,
and BI 50.95) comprises only P. myrciae, with
P. truncatisporum appearing as a basal taxon within
this clade (FIG. 2).
2. Clade II. Has good support values (BP 599, BL 5
99, PP 51.0) for two Phyllachora representatives (P.
ermidensis sp. nov. and P. furnasensis sp. nov.), both
infecting Eugenia species that are described as new
taxa with strong morphological support (FIG.3).
3. Clade III. With good support (BP 597, BL 590,
BI 51.0) contains Cerrado collections from non
myrtaceous hosts: P. graminis, type species of the
genus Phyllachora obtained from an unidentified
Poaceae, and P. qualeae, a parasite of Qualea multi-
flora (Vochysiaceae) (FIG. 4).
Still, the Sordariales appeared as a clade with good
support (BP 5100, BL 5100, PP 50.97) (FIG. 1) con-
taining the genera Gelasinospora,Neurospora, and Sor-
daria. This clade is sister to Clade II but again without
statistical support. The Order Meliolales represented
by two Asteridiella species shows good support for maxi-
mum parsimony and Bayesian inference (BP 5100,
BI 51.0).The representatives of the Hypocreales
were distributed in two separate clades, one with
good support (BP 597, BL 599, BI 51.0) with two
genera (Claviceps,Balansia), morphologically and
physiologically distinct from Pseudonectria and Nectria,
these two grouped in another clade showing moderate
to good support (BP
5
75, BL 579, BI 50.98). The
Ceratocystis species were shown to be well-established
representatives of the monophyletic Microascales,
with good support (BP 5100, BL 5100, BI 51.0).
FIG. 2. Synapomorphies shown in Phyllachora clade I, in which all species show subepidermal, intradermal stromata without a
deeper invasion of the mesophyl, and clavate asci; three species appear specifically infecting Myrcia species, one on Myrciaria
species, and one on different Psidium species. Phyllachora myrciae showing characteristically lunate ascospores, and two types of
conidia (ellipsoidal and falcate) formed in separate conidiomata, but both showing phialidic conidiogenesis; P. truncatispora
on M. camapuanensis has sublunate ascospores with both ends showing characteristic wall thickenings; P. cerradensis on M. torta
shows elliptical ascospores with a predominantly longer dimension when compared to P. subcircinans, which is found only on
Psidium host species; finally P. myrciariae shows ascospores close in form to those of P. cerradensis, but additionally forms fusoid-
clavate phialidic conidia.
FIG. 3. Synapomorphies shown in Phyllachora clade II containing P. furnasensis and P. ermidensis, both on Eugenia host species,
with pseudostromata occupying the entire mesophyll of the infected leaves, both species showing cylindrical asci; however with
clear differences in ascospore shape as P. furnasensis shows fusoid ascospores and thickened cell wall at both ends, easily
segregated from oblong-fusoid ascospores of P. ermidensis.
1144 MYCOLOGIA
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The monophyletic genus Glomerella (Glomerellaceae,
Glomerellales) had moderate to good support (BP 5
76, BL 577 and BI 51.0). Similarly, the monophyletic
Ophiostoma and a basal Fragosphaeria, showing BP 587
and BL 571, were confirmed here as belonging to
Order Ophiostomatales.
TAXONOMY
Nine Phyllachora species found infecting myrtaceous
hosts in the Cerrado (P. nigerrima [Viégas] Santos;
R.B. Medeiros & Dianese comb. nov.; P. ermidensis
Santos; R.B. Medeiros & Dianese sp. nov.; P. furna-
sensis Santos; R.B. Medeiros & Dianese sp. nov.;
P.subcircinans Speg.; P. myrciariae Santos & Dianese
sp. nov.; P. myrciae [Lév.] Sacc; P. cerradensis Santos;
R.B. Medeiros & Dianese sp. nov; P. myrciae-rostratae
Viégas; and P. truncatispora [Viégas] Bat & Per.)
were morphologically identified by the key shown
below, which is followed by the description and illus-
tration of each species.
KEY TO COMMON PHYLLACHORA SPECIES ON
MYRTACEAE FROM THE CERRADO
1. Pseudostromata trans-mesophyllic, occupying the
entire section of the host leaf, on Campomanesia,or
on Eugenia species .............................2
19. Pseudostromata subcuticular, intradermal or
subepidermal .................................4
2. Ascospores, elliptical, biguttulate on Campomanesia
species, pseudostromata showing a typical textura
prismatico-intricata . ...................P. nigerrima
29. Ascospores elliptico-fusoid, fusoid or obclavate, not
biguttulate on Eugenia species ....................3
3. Asci without an apical thickening, ascospores short
elliptico-fusoid, uniseriate, verrugulose, centrally unigut-
tulate, 10–15 64–7mmonE. complicata......P. ermidensis
39. Asci without an apical thickening, ascopores fusoid
with acute wall thickening at both ends, uniseriate,
smooth, guttulate, 15–19 65–8mm ......P. furnasensis
4. Host species belonging to the genus Psidium,
ascospores short-ellipsoidal, oblong to subglobose,
centrally guttulate ...................P.subcircinans
49. Host species belonging in genera Myrcia or Myrciaria ...5
5. Parasitic on Myrcia species .......................6
59. Parasitic on Myrciaria delicatula; ascospores 14–19 6
5–8mm, elliptical, obliquely biseriate, irregularly gut-
tulate, with a mucoid shield; asci 63–90 611–15 mm,
cylindrical-clavate, short pedicellate, apex obtuse with-
out a conspicuous apical apparatus ....... P. myrciariae
6. Ascospores typically lunate, on several Myrcia
species..............................P. myrciae
69. Ascospores showing other shapes..................7
7. Ascospores 15–22 66–9mm, elliptic-oblong,
bisseriate; asci 69–92 617–28 mm, fusoid, short pedi-
cellate, thin-walled without a conspicuous apical appa-
ratus; asexual morph conidiomatal, conidiogenous
cells phialidic; conidia mostly clavate, small (3–46
1.5–2mm),on .......................P. cerradensis
79. Parasitic on other Myrcia species ..................8
8. Ascospores 7–14 64–8mm, elliptic-oblong,
transversally to obliquely unisseriate; centrally guttu-
late; asci 82–112 67–10 mm, cylindrical to subclavate,
apex obtuse to truncate, without a conspicuous
apical apparatus; asexual morph not seen, on Myrcia
rostrata .........................P. myrciae-rostratae
89. Ascospores 18–24 66–9mm, lunate-reniform to
half-moon shape, biseriate, with accute wall thicken-
ings at both ends; asci 89–117 614–20 mm, clavate,
long-pedicellate, with rounded apex without an api-
cal apparatus; assexual morph 350–400 660–70
mm, condiomatal, ostiolate, apparently spermatial,
conidiogenous cells phialidic, conidia filiform, 12–
20 61–1.5 mm, straight or curved, on Myrcia cama-
puanensis.........................P. truncatispora
TAXONOMY
Phyllachora nigerrima (Viégas) Santos, R.B. Medeiros
& Dianese comb. nov. FIG.5
MycoBank MB813641
Basionym: Catacauma nigerrimum Viégas, Bragantia
4:139. 1944.
Typification: BRAZIL. MINAS GERAIS: Belo Hori-
zonte, Fazenda Baleia, on leaves of Campomanesia ada-
mantium (Myrtaceae), 19 Jan 1943, A.P. Viégas
(holotype IACM 4175).
Sexual morph: Pseudostromata 1–3mmdiam,sub-
globose, sparse, sometimes coalescent, amphigenous,
occupying the entire section of the mesophyll, bright
black, with some carbonaceous superficial areas,
multilocular. Ascomata perithecial, subglobose to
FIG. 4. Synapomorphies in Phyllachora clade III, in which a Brazilian collection identified as P. graminis, the type species of
the genus, appears with P. qualeae. Both species show amphigenous pseudostromata; however, in P. qualeae the asci are typically
cylindrical with uniseriate oblong ascospores, whereas P. graminis shows clavate asci containing biseriate ascopores.
SANTOS ET AL.: ITS PHYLOGENY OF PHYLLACHORA SPECIES 1145
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FIG.5. A–J. Phyllachora nigerrima on Campomanesia adamantium (Cambess.) O. Berg. (Syn. C. coerulea Cambess.) (Myrtaceae), UB
Mycol. Col. 1392. A. Leaf symptoms with arrow indicating a pseudostroma (bar 52cm).B,C.Surfaceofapseudostroma(bar5
5 mm). D. Section through a hypophyllous pseudostroma showing two ascomata (bar 5100 mm).E.Ahypophyllousascoma
immersed in the pseudostroma (bar 5200 mm).F.Detailofpartofanascoma(bar550 mm). G. Textura prismatica of the
pseudostroma (bar 520 mm). H. Cylindrical asci (bar 550 mm). I. Uniseriate asci (bar 520 mm).J.Ascospores(bar510 mm).
1146 MYCOLOGIA
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globose, with an almost flat base, seated on a layer of
lacunose parenchyma, peridium 40 mmthick;locules
300–400 6400–450 mm, clypeate; clypei opaque,
black, epidermal 15–25 mm thick. Asci 100–130 67–
9mm, cylindrical, straight, short-pedicellate, with
clearly thickened apex, paraphysate; paraphyses
filiform, septate, hyaline, ,2mm wide. Ascospores
10–16 65–6mm, ellipsoidal, uniseriate, hyaline,
biguttulate.
Asexual morph: Seen only on the holotype. Conidio-
mata epiphyllous, black, depressed, opening by irregu-
lar fissures; conidiophores branched, hyaline bearing
conidiogenous cells that produce abundant masses
of curved or sigmoid, apparently spermatial, cells,
,1mm wide.
Original illustration: Viégas (1944). Bragantia 4:354,
FIGS.a–d; 355, FIGS.e–i.
Other specimens examined: BRAZIL. DISTRITO FEDERAL:
Brasília, Península Norte, 31 May 1992, José C. Dianese 347,
UB Mycol. Col. 1392.
Phyllachora ermidensis Santos, R.B. Medeiros &
Dianese sp. nov. FIG.6
MycoBank MB813644
Etymology: Epithet derived from type locality, the Dom
Bosco Ermida (ermida means chapel in Portuguese) in
Brasília.
Sexual morph: Pseudostromata 1–3 mm diam, black,
shiny, glabrous, mostly circular, slightly domed above
the leaf surface, escutiform, immerse occupying the
entire section of the leaf, amphigenous, sparse, multilo-
cular (4–8 locules), surrounded by a brown edge. Asco-
mata perithecial, solitary or gregarious, ampulliform to
pyriform, immersed in pseudostroma occupying both
palisade and spongy parenchyma, ostiolate, clypeate;
ostiole conspicuous, periphysate; clypei 47–63 mm
thick, covering the entire pseudostroma; peridium
13–26 mm thick showing 3–5 layers of dark prismatic
cells; locules 157–245 6200–287 mm; hymenium occu-
pying the basal wall of the ascomata. Asci 77–103 68–
13 mm, cylindrical, 8-spored, pedunculate, thin walled
in all developmental stages, devoid of apical structures,
apex obtuse truncated, paraphysate; paraphyses 2–3
mm wide, abundant, hyaline, filiform, septate, with
thin-walled. Ascospores 10–15 64–7mm, unicellular,
hyaline, elliptical, obliquely uniseriate, commonly with
a conspicuous guttule in the middle region.
Asexual morph: Not seen.
Typification: BRAZIL. DISTRITO FEDERAL: Brasília,
Dom Bosco Chapel, on leaves of Eugenia complicata
O. Berg (Myrtaceae), 15u47951″S647u48938″W, 29
Jun 2012, Maria D. M. Santos 185 (holotype UB Mycol.
Col. 22191).
Other specimen examined: BRAZIL. DISTRITO FEDERAL:
Planaltina, Águas Emendadas Ecological Reserve, 22
Jan 1998, C.A. Inácio 40, UB Mycol. Col. 5944.
Brazlândia, Fazenda Palestina, 22 Aug 1992, Ricardo B. de
Medeiros 130, UB Mycol. Col. 2239.
Phyllachora furnasensis Santos, R.B. Medeiros &
Dianese sp. nov. FIG.7
MycoBank MB813645
Etymology: Epithet derived from type locality: at the
margin of the Furnas Dam.
Sexual morph: Pseudostromata 1–3mmdiam,black,
amphigenous, circular, semi-pulvinate, initially subepider-
mal epiphyllous, then growing deep through the meso-
phyll, sparse, flat, multilocular, glabrous, shiny.
Ascomata perithecial, ovoid to ampulliform, immersed
in black pseudostromata inside the host mesophyll, ostio-
late, clypeate; ostioles periphysate; clypei between 50–70
mmthick;locules125–250 6175–262 mm; peridium 12–
18 mm thick, consisting of three or more layers of compact
melanized cells clearer than the pseudostroma; hymeni-
um formed at the bottom wall of the ascomata. Asci
89–120 69–17 mm, typically cylindrical, paraphysate;
paraphyses 2–3mm wide, abundant, hyaline, filiform, sep-
tate, thin walled. Ascospores 15–19 65–8mmobliquely
uniseriate, unicellular, hyaline, elliptical, fusiform, irregu-
lar surface, with acute wall thickeningsatbothtips.
Asexual morph: Not seen.
Typification: BRAZIL. MINAS GERAIS: São José da
Barra, near cabins at Furnas Dam, 20u34924″S6
46u3925.7″W, on leaves of Eugenia punicifolia DC (Kunth),
(Myrtaceae), 27 Dec 2011, Rita C. Pereira-Carvalho 106
(holotype UB Mycol. Col. 22130).
Other specimens examined: BRAZIL. MATO GROSSO DO
SUL: Campo Grande, 22 Jul 1993, José C. Dianese 1035,UB
Mycol. Col. 4396. MATO GROSSO: Chapada dos Guimarães,
13 Apr 1996, Mariza Sanchez 1723, UB Mycol. Col. 11624.
Comment: Phyllachora ermidensis and P. furnasensis
were the only new species identified in this work with
pseudostromata occupying the entire leaf section. How-
ever, the latter shows semi-pulvinate pseudostromata
instead of flatly scutiform. Furthermore, major differ-
ences in shape and size of asci and ascospores help to
separate the two species. The most significant differ-
ence is that the format of the fusoid ascospores of P.
furnasensis shows terminal appendices or wall thicken-
ings, thus definitively separating the two species.
Phyllachora subcircinans Speg. Revista Argent. Hist.
Nat. 1:413. 1891. FIG.8
Syn. Catacauma subcircinans (Speg.) Theiss. & Syd.
Annals Mycol. 13(3/4):394. 1915.
MycoBank MB211961
Sexual morph: Pseudostromata 1–4 mm diam, charac-
teristically subcuticular partially intraepidermal, cir‐
cular to irregular, gregarious, coalescent, glabrous,
shiny, multilocular (2–4 locules), epiphyllous, occasion-
ally amphigenous, sparse, often covering most of the
leaf surface. Ascomata perithecial, ampulliform,
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FIG.6. A–F. Phyllachora ermidensis on Eugenia complicata from the holotype. A. Leaf symptoms (bar 52 cm). B. Pseudostromata
showing ascomata with extruding ascospore cirri (bar 52 mm). C. Amphigenous distribution of the ascomata inside the
pseudostromata (bar 5100 mm). D. Paraphysate asci (bar 520 mm). E. Uniseriate unitunicate asci (bar 510 mm). F. Ascospores
(bar 510 mm).
1148 MYCOLOGIA
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FIG.7. A–H. Phyllachora furnasensis on Eugenia punicifolia DC (Kunth) (Myrtaceae), from the holotype (UB Mycol. Col.
22130). A. Leaf symptoms (bar 51 cm). B. Subpulvinate pseudostromata (bar 52 mm). C. Amphigenous distribution of the
ascomata inside the pseudostromata (bar 550 mm). D. Paraphysate asci (bar 520 mm). E. Uniseriate unitunicate asci (bar 5
20 mm). F–H. Ascospores showing characteristically tickened walls at both ends (arrows) (bars 510 mm).
SANTOS ET AL.: ITS PHYLOGENY OF PHYLLACHORA SPECIES 1149
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immersed in pseudostromatic tissue, ostiolate, clipeate;
ostioles conspicuous, períphysate; peridium 15–21 mm
thick showing layers of compressed melanized cells;
clypei 53–82 mm thick, well developed, black; locules
329–417 6193–275 mm; hymenium coating the base
and the side wall of the ascomata. Asci 73–100 612–
19 mm, unitunicate, cylindrical, short-pedunculate,
8-spored, obtuse apex, without a conspicuous apical
FIG.8. Phyllachora subcircinans on Psidium australe var. argenteum (Berg) Ladrum (UB Mycol. Col. 21238). A. Leaf symptoms
(bar 52 cm). B. Pseudostromata (bar 51 mm). C. Cross section through amphiginous pseudostroma (bar 5100 mm).
D. Paraphysate asci (bar 530 mm). E. Biseriate unitunicate ascus (bar 510 mm). F. Asci at different stages of maturation (bar 5
10 mm). G. Ascospores (bar 510 mm).
1150 MYCOLOGIA
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apparatus, paraphysate; paraphyses 2–3.5 mm wide,
numerous, filiform, hyaline, septate, unbranched, thin
wall. Ascospores 11–16 67–9mm, unicellular, hyaline
when immature to light olivaceous at maturity, mostly
uniseriate, sometimes with biseriate areas, oblong to
ellipsoid, guttulate in the middle portion, with irregu-
larly shaped guttules, thin wall surrounded by a gelati-
nous sheath.
Asexual morph: Not seen.
The four accesses of P. subcircinans were grouped in
a well-supported subclade based on ITS sequences with
all specimens studied infecting five Psidium species, as
seen below.
Specimens examined: (SUPPLEMENTARY TABLE V).
Phyllachora myrciariae Santos & Dianese sp. nov.
FIG.9
MycoBank MB813646
Etymology: Epithet derived from host genus Myrciaria.
Sexual morph: Pseudostromata 0.5–1.5 mm diam.,
black, circular, intraepidermal, sparse, occasionally
coalescent, prominent, unilocular, epiphyllous, gla-
brous, shiny. Ascomata perithecial, ampulliform,
immersed in the pseudostromata, paraphysate, ostio-
late, clypiate; ostioles periphysate; clypei 56–63 mm
thick, black; peridium 19–24 mm thick formed by
slightly compressed and melanized cells; locules 161–
432 6126–267 mm. Asci 63–90 611–15 mm, unituni-
cate, clavate-fusoid, short-pedunculate, 8-spored,
obtuse apex, without apical structures, paraphysate;
paraphyses 2–3mm wide, numerous, dispersed among
the asci, filiform, hyaline, septate, unbranched, thin-
walled. Ascospores 14–19 65–8mm, unicellular, hya-
line, obliquely biseriate, elliptical, irregularly guttulate,
covered by mucoid sheath.
Asexual morph: Not observed.
Typification: BRAZIL. DISTRITO FEDERAL: Brasília,
Brasília Botanical Garden, 15u53909.1″S647u49926.4″
W, on leaves of Myrciaria delicatula (DC) O. Berg (Myr-
taceae), 24 May 2011, Maria D.M. Santos 181 (holotype
UB Mycol. Col. 21781).
Other specimen examined: BRAZIL. MATO GROSSO DO
SUL: Campo Grande, Parque dos Poderes, on leaves of Myr-
ciaria delicatula, 18 Aug 1996, José C. Dianese 2760, UB Mycol.
Col. 12255.
This is the first report of a Phyllachora species associ-
ated with plants belonging to genus Myrciaria. As there
is no record of a Phyllachora species infecting members
of different myrtaceous host genera, and considering
the phylogenetic characteristics shown (FIGS. 1, 2),
the data strongly indicate that the specimen studied
belongs to a new Phyllachora species.
Phyllachora myrciae (Lév.) Sacc, Syll. Fung. (Abellini)
2:597. 1883. FIGS. 10, 11
Syns.: Catacauma myrciae (Lév.)Theiss.&Syd.,Ann.Mycol.
13(3/4):393. 1915. Dothideamyrciae Lév., Ann. Sci. Nat.
Bot. 5:264. 1846.
MycoBank MB170028
Other illustrations in: Viégas, Bragantia III:49–
72. 1948.
Sexual morph: Pseudostromata 3–6 mm diam, circu-
lar, scutiform, intraepidermal, black, glabrous, shiny,
occasionally coalescent, multilocular (3–12 locules),
amphigenous. Ascomata perithecial, globose to ampul-
liform, ostiolate, multiloculate; locules 205–485 6148–
207 mm; hymenium mainly basal, sometimes lining
portions of the side wall; ostioles periphysate; clypei
56–70 mm thick, black. Asci 89–117 613–19 mm, unitu-
nicate, fusoid, pedicellate, 8-spored, apex tapered with
a slight thickening, paraphysate; paraphyses 2.5–4.5 mm
wide, scattered among the asci, hyaline, abundant,
septate, branched. Ascospores 14–18 65–7mm asep-
tate, hyaline, typically lunate, thin-walled, smooth,
biseriate to multisseriate.
Asexual morph: Conidiomata spermacial or conidial,
black, immersed in same pseudostromata containing
the ascomata. Spermatial locules 380–456 699–172
mm, ampulliform, ostiolate; spermatogenous cells
14–18 62.5–3mm, hyaline, phialidic, covering internal
wall of the conidiomata; spermatia 17–19 61.5–2mm,
falcate, curved, aseptate, hyaline, with rounded subtrun-
cate ends. Conidial locules 250–275 668–80 mm; coni-
diogenous cells 6–763–4mm, holoblastic, covering
the base of the conidiomata; conidia 15–16 65–6mm,
oblong.
Specimens examined: (SUPPLEMENTARY TABLE VI).
Phyllachora myrciae was originally described as Dothi-
dea myrciae Lév. on Myrcia sp. in undefined locality in
Brazil (Saccardo 1883). In the Cerrado the same spe-
cies was well-illustrated and described by Viégas
(1948) on the basis of material collected in 1936 with
host then identified as E. bimarginata, that is now con-
firmed as a Myrcia species; and in 1943 on M. vestita.
Phyllachora myrciae is shown on a broad host spectrum
of species but within the genus Myrcia causing tar spots
in six different species (M. decrescens,M. dictyophylla,
M. guianensis,M. nivea,M. variabilis,M. vestita), consis-
tently forming in all of them the same type of pseudos-
troma, precisely drawn by Viégas (1944), with fusoid
asci containing the typically lunate ascospores, and fal-
cate spermatial conidia on a pseudostromatic conidio-
mata as shown (FIG. 10).
Phyllachora cerradensis Santos, R.B. Medeiros &
Dianese sp. nov. FIGS. 12, 13
MycoBank MB813647
Sexual morph: Pseudostromata 2-4 mm diam, scuti-
form to hemispheric, intraepidermal to subepidermal,
also occupying the palisade mesophyll layer, sparse or
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FIG.9. A–F. Phyllachora myrciariae on Myrciaria delicatula (DC) O. Berg (Myrtaceae), from the holotype. A. Leaf symptoms
(bar 55 mm). B. Pseudostromata (bar 51 mm). C. Cross section through an epiphyllous pseudostroma (bar 550 mm).
D. Paraphysate asci (bar 520 mm). E. Biseriate unitunicate asci (bar 510 mm). F. Ascospores (bar 510 mm).
1152 MYCOLOGIA
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gregarious, occasionally coalescing, prominent, uniloc-
ular to multilocular (2–5). Exclusively epiphyllous,
glabrous, shiny. Ascomata perithecial, ampulliform to
globose, clypeate; clypei 68–95 mm thick, black; locules
260–362 6168–264 mm, ostiolate; ostioles periphysate;
hymenium coating the basal and portion of the side
walls; peridium 17–21 mm thick, consisting of three or
more layers of compacted melanized cells, clearly
distinguished from the pseudostromatic texture.
Asci 69–92 617–28 mm, unitunicate, fusoid, short-
pedunculate, 8-spored, obtuse apex, without apical
thickening, thin-walled in all stages of development,
paraphysate; paraphyses 1.6–3mm wide, numerous,
hyaline, septate, branched, thin-walled. Ascospores
15–22 66–9mm, unicellular, hyaline, elliptic-oblong,
biseriate, with microguttulate cytoplasm, covered by a
thin gelatinous sheath.
Asexual morph: Conidiomata often inserted into a dis-
tinct area of the pseudostromatic tissue; locules 89–
175 646–100 mm, paraphysate, globose to ampulliform.
Conidiogenous cells hyaline, phialidic, lageniform, with
tapered ends (10–12 61.5–2.5 mm). Conidia 3–56
1.5–2mm, hyaline, smooth, mostly clavate, elliptical to
fusiform.
Typfication: On leaves of Myrcia torta DC (Myrtaceae).
BRAZIL. DISTRITO FEDERAL: Brasília, Botanic
FIG. 10. A–F. Phyllachora myrciae sexual morph on Myrcia pallens (DC) O. Berg (Myrtaceae), (UB Mycol. Col. 21292). A. Leaf
symptoms (bar 53 cm). B. Pseudostromata (bar 53 mm). C. Cross section through an epiphyllous multilocular pseudostroma
(bar 5100 mm). D. Paraphysate asci (bar 520 mm). E. Asci with mature ascospores (bar 510 mm). F. Ascospores (bar 510 mm).
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Garden, 15u5399.1″S647u49926.4″W, 24 May 2011,
W.R.O. Soares 24 (holotype UB Mycol. Col 21908).
Other specimens examined: BRAZIL. DISTRITO FEDERAL:
15u44910.5″S647u5390.3″W, 24 May 2011, Maria D.M. San-
tos 183, UB Mycol. Col. 21780; Fazenda Água Limpa, 14 Jan
93, R.B. Medeiros 218, UB Mycol. Col. 3115; 16 Oct 1992, Cle-
ber Furlanetto 73, UB Mycol. Col. 2703; IBGE Natural Reserve
15u56943.3″S647u56926.9″W, 12 Sep 2007, Mariza Sanchez
4380, UB Mycol. Col. 20735; Ecological Reserve of IBGE,
5 Apr 2001, Mariza Sanchez 3992, UB Mycol. Col. 18342. Bra-
zlândia: APA da Cafuringa, 27 Aug 1992, José C. Dianese 553,
UB Mycol. Col. 2237. 22 Sep 1992, R.B. Medeiros 129,UB
Mycol. Col. 2238. Planatina: Estação Ecológica de Águas
Emendadas, 22 Jan 1998, Carlos A.P. de Souza 342, UB Mycol.
Col. 16014. GOIÁS: Cristalina, Fazenda Nova India, 10 Apr
1993, R.B. Medeiros 353, UB Mycol. Col. 5304. Mineiros: Par-
que Nacional das Emas, 11 Apr 1997, Mariza Sanchez 2596,
UB Mycol. Col. 14219. MATO GROSSO: Rodonópolis,
4 Nov 1993, R.B. Medeiros 388, UB Mycol. Col. 5478; MATO
GROSSO DO SUL: Campo Grande, Parque dos Poderes,
18 Aug 1996, C.A. Inácio 539, UB Mycol. Col. 12315. Parque
dos Poderes, near EMPAER headquarters, 18 Aug 1996, Mar-
iza Sanchez 1964, UB Mycol. Col. 12364. MINAS GERAIS:
Miranda, Miranda Hydroelectric Plant, 11 Apr 1993, José C.
Dianese 1480, UB Mycol. Col. 5569.
Phyllachora cerradensis was found on M. torta in the
DISTRITO FEDERAL and four different states (Goiás,
Mato Grosso, Mato Grosso do Sul, Minas Gerais).
The main characteristic of this species is the shape of
the conidia (mostly clavate), and clearly elliptical
ascospores formed within fusoid asci. Phyllachora cerra-
densis was preliminarily studied, but not properly
described, by Medeiros (1994), but then the asexual
morph, now reported as an important characteristic,
was not observed.
Phyllachora myrciae-rostratae Viégas, Bragantia IV:75.
1944. FIG.14
MycoBank MB289541
Sexual morph: Pseudostromata 2–7 mm diam, elongat-
ed, irregular, black, initially subcuticular becoming
intraepidermal, sparse, slightly domed and raised, multi-
locular (3–8 locules), epiphyllous, shiny, glabrous but
showing widespread bright pinpoints revealing the posi-
tion of the ostioles of the ascomata, sometimes showing
a central larger pseudostroma encircled by a group of
smaller pseudostromata. Ascomata perithecial, ampulli-
form, ovoidal or pyriform, isolated, immersed in pseudo-
parenchymatous tissue, dark brown; locules 337–571 6
FIG. 11. A–E. Phyllachora myrciae assexual morph on Myrcia pallens (DC) O. Berg (Myrtaceae), (UB Mycol. Col. 21292). A.
Cross section through a spermatial locule (bar 550 mm). B. Spermatogenous cells (arrows) (bar 550 mm). C. Spermatia (bar 5
10 mm) D. Cross section through a conidioma (bar 550 mm). E. Conidia (bar 510 mm).
1154 MYCOLOGIA
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FIG. 12. A–E. Phyllachora cerradensis sexual morph on Myrcia torta DC (Myrtaceae), from the holotype. A. Leaf symptoms
(bar 50.5 cm). B. Pseudostromata (bar 52 mm). C. Cross section through an epiphyllous multilocular pseudostroma (bar 5
50 mm). D. Asci (bar 510 mm). E. Mature ascospores (bar 510 mm).
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200–308 mm, ostiolate; ostioles conspicuous, periphysate,
clypeate; clypei 65–113 mm thick, black; peridium 30–
40 mm thick formed by compactly arranged strongly
melanized cells; hymenium coating the bottom surface
and the lower portion of the sidewall of the ascomata.
Asci 82–112 67–10 mm, unitunicate, cylindrical, subcla-
vate, 8-spored, short-pedunculate, thicker-walled when
immature, apex obtuse to truncate, lacking apical struc-
ture, paraphysate; paraphyses 2.5–3.5 mm wide, abun-
dant, hyaline, septate, threadlike. Ascospores 7–14 6
4–8mm, unicellular, elliptic-oblong, hyaline, guttulate,
with granular protoplasm and showing a median guttule,
obliquely uniseriate.
Asexual morph: Conidiomata hemispheric to subglo-
bose, inserted into the same pseudostroma as the sexu-
al morph; locules 298–400 6112–126 mm, elliptical.
Conidiogenous cells phialidic, distributed on the bot-
tom surface of the conidiomata. Conidia 5–761.5–
2.5 mm, hyaline, smooth, ellipsoid, sometimes oblong.
Specimens examined: on leaves of Myrcia splendens (SW) DC
(Myrtaceae). BRAZIL. DISTRITO FEDERAL: Brasília: IBGE
Ecological Reserve, 15u55982.8″S647u53902.9″W, 13 Sep
1999, Zuleide Martins Chaves 835, UB Mycol. Col. 18018;
15u5597,6″S647u53903.5″W, 22 Oct 1999. C.A.P. Souza 647,
UB Mycol. Col. 18078. Brasília National Park, 15u43944.5″S
647u5695.37″W, 1 Feb 2006, Mariza Sanchez 4215,UB
Mycol. Col. 20060; 29 Nov 1990, Ana Lucia Nunes Fialho 4,
UB Mycol. Col. 447.
After the description of P.myrciae-rostratae by Viégas
(1944) this species was studied by Medeiros (1994),
and found in Brazil also on Myrtus communis (Mendes
et al. 1998). However, neither Viégas (1944) nor
Medeiros (1994) observed the presence of amphigen-
ous pseudostromata or the conidial morph of the spe-
cies, here found in all M. splendens samples studied,
collected in two important Cerrado natural reserves
in Brasília.
Phyllachora truncatispora (Viégas) Bat & H. Maia, in
Batista, Peres & Maia, Atas Inst. Micol. Univ. Recife
4:70. 1967.
Syn. Catacauma truncatisporum Viégas, Bragantia 4(1–6):
145. 1944. FIGS.14,15
MycoBank MB336697
Sexual morph: Pseudostromata 2–5 mm diam, black,
circular, discrete, sparse, sometimes coalescent, gla-
brous, shiny, intraepidermal to subepidermal, epiphyl-
lous, multilocular. Ascomata perithecial globose to
ovoid, immersed in pseudostroma; locules 160–200 6
100–350 mm. Asci 69–117 614–25 mm, short-peduncu-
late, tapered toward the apex, paraphysate; paraphyses
2.5–5mm wide, numerous, filiform, hyaline, septate,
FIG. 13. A–D. Phyllachora cerradensis assexual morph on Myrcia torta, from the holotype (UB- Mycol. Coll. 21908). A, B.
Sections through a pseudostroma (bars 550 mm). C. Conidigenous cells (bar 55mm). D. Conidia (bar 55mm).
1156 MYCOLOGIA
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FIG. 14. A–I. Phyllachora myrciae-rostratae on Myrcia splendens (SW) DC (Myrtaceae) (UB Mycol. Col. 20060). A. Leaf symptoms
(bar 51 cm). B. Pseudostroma showing protruding ostioles (bar 52 mm). C. Cross section through an epiphyllous
pseudostroma (bar 5100 mm). D. Paraphysate asci (bar 550 mm). E, F. Asci (bars: E 510 mm, F 55mm). G. Ascospores (bar 5
5mm). H. Section through a conidioma (bar 550 mm). I. Conidia (bar 55mm).
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FIG. 15. A–E. Phyllachora truncatispora on Myrcia camapuanensis N. Silveira (Myrtaceae) (UB Mycol. Col. 14083). A. Leaf
symptoms (bar 51 cm). B. Pseudostromata (bar 53 mm). C. Cross section through an epiphyllous multilocular pseudostroma
(bar 5100 mm). D. Paraphysate asci (bar 520 mm). E, F. Asci with mature ascospores (bars 510 mm). G. Ascospore (bar 55mm).
1158 MYCOLOGIA
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thin-walled. Ascospores 18–26 67–8mm, sublunate to
fusoid, hyaline, smooth-walled, showing characteristic
wall thickenings at both acute ends.
Asexual morph: Pseudostromata 1–1.5 mm diam, spher-
ical, scattered to confluent, multilocular. Conidiomata
immersed, oblong, ostiolate; locules 350–400 660–70
mm diam. Conidiophores 30–35 mm. Conidia 12–20 6
1–1.5 mm, filiform, hyaline, sometimes curved.
Specimens examined: BRAZIL. SÃO PAULO: Pirassununga,
on leaves of indeterminate Myrtaceae, 27 May 1943, W.A.
Rodrigues, Viégas Fungarium, Herbarium IACM 4246 (holo-
type). GOIÁS, Emas National Park, Água Ruim, on leaves of
Myrcia camapuanensis N. Silveira (Myrtaceae), 7 Apr 1997,
Ludwig Pfenning 27, UB Mycol. Col. 13932. 9 Apr 1997, José
C. Dianese 3007, UB Mycol. Col. 13857. 11 Apr 1997, Ludwig
Pfenning 73, UB Mycol. Col. 14083. Mariza Sanchez 2618,UB
Mycol. Col. 14252.
The shape of the ascospores of P. truncatispora
are unique among all Phyllachora species known on
Myrtaceae, as reported by Viégas (1944). The asexual
morph, a stromatic coelomycete forming filiform,
curved, hyaline conidia, tapered on both ends, was
observed on the type material only.
FIGURES 2–4 clearly show the connections of the
data on ITS phylogeny with host genera, and the
main synapomorphies (shape of the stromata, asci,
and ascospores) detected for each member of the dif-
ferent Phyllachora clades.
DISCUSSION
The class Sordariomycetes is here shown to be monophy-
letic with good support, corroborating the previous
findings of Zhang et al. (2006) and Untereiner et al.
(2013). Zhang et al. (2006) has shown that the orders
Diaporthales, Sordariales, Chaetosphaeriales, Conio-
chaetales, Boliniales, Hypocreales, Halosphaeriales,
Microascales, Melanosporales, Xylariales, and Lul-
worthiales all belong within the subclass Sordariomyceti-
dae, most of them being monophyletic taxa, but
according to them, Microascales is probably paraphy-
letic. Untereiner et al. (2013) corroborates most of the
previous findings, but members of the Cerrado Phylla-
chorales are being phylogenetically investigated for the
first time in this work, and the ITS data is not sufficient
to provide the proper allocation of the order within the
Sordariomycetidae.
All seven clades within Sordariomycetes represented
here have good support and correspond to previously
recognized orders within the class. It was not possible,
however, to establish sister-group relationships among
those clades (orders), as most of the nodes lack statisti-
cally significant support, especially on the backbones.
This lack of support shows up as very short branches
and may indicate a rapid period of diversification early
in their history, which is not an uncommon event and
has been reported in animals (Martin et al. 2000) as
well as plants (Shaw et al. 2003). However, this needs
better investigation and is beyond the scope of this
paper, centered on the ITS phylogeny of Phyllachora
species.
Most of the Orders considered nested within the
Sordariomycetes are monophyletic. Some of them
like Sordariales, Boliniales, and Hypocreales have
been shown here to lack support when included with
all of their representatives; however, there is clearly a
monophyletic core for those Orders, and their mono-
phyly cannot be rejected here.
ITS sequences were not obtained for two species
(P. myrciae-rostratae,P. nigerrima) among the nine Phylla-
chora species studied. The order Phyllachorales, as
currently circumscribed by our ITS data, is not mono-
phyletic. Three clades (FIG. 1) contain representatives
of this order (clades I–III). The representative of the
type species (P. graminis) is nested within clade III
that therefore will always carry the name Phyllachora;
the clades I and III, containing most of the Phyllachora
species sampled here, need names. However, because
of the absence of statistical support on the backbone,
we decided to keep the name Phyllachora for all three
clades, even understanding that the group is not mono-
phyletic, until more evidence is gathered. In a recent
study on Polystigma, then considered a Phyllachora-relat-
ed genus, the species Polystigma amygdalinumis, a taxon
previously placed in Phyllachorales, was shown not to
group with Phyllachora species in Sordariomycetidae
but fitted within Xylariomycetidae (Habibi et al.
2015). This indicates the taxonomical instability still
present.
In clade I of the three Phyllachlora clades, pseudostro-
mata are always subcuticular to intra-epidermal without
a deeper penetration into the palisade parenchyma. In
this clade, three subclades are shown as well supported,
with two of them as sister subclades, both with hosts
belonging in genus Myrcia (P. myrciae,P. cerradensis sp.
nov.). In the third subclade is allocated P. subcircinans,
a parasite on Psidium species. Each of the three sub-
clades must be treated as representing individual
species, supported both molecularly and by solid synap-
omorphies. Thus, P. subcircinans shows obclavate asci
with oblong, short-ellipsoidal to subglobose ascospores,
whereas in P. cerradensis, ascospores are ellipsoidal and
guttulate. Conversely, the typically lunate ascospores
of P. myrciae are formed inside clavate asci. Besides
these three groups, a fourth species is inserted into
clade I, P. trucantispora, the most basal species, sister
to all other three, which however is morphologically
unique by presenting sub-lunate to fusoid ascospores
with thickened walls at its acute apices.
The two members of clade II (P. furnasensis sp. nov.,
P. ermidensis sp. nov.) are recognized by the presence
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FIG. 16. A–E. Phyllachora graminis Nitschke ex Fuckel on a non-identified Poaceae from Brasília National Park (UB Mycol.
Col. 21307). A. Leaf symptoms (bar 52 mm). B. Cross section through a pseudostroma (bar 530 mm). C, D. Paraphysate asci
(bars 520 mm). E. Ascospore (bar 55mm). F. Conidioma (bar 520 mm). G. Conidiogenous cells and spermatia (bar 510 mm).
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FIG. 17. A–E. Phyllachora qualeae (Viégas) Bat. & Peres on Qualeae multiflora Mart (UB Mycol. Coll. 21145). A. Leaf symptoms
(bar 52 cm). B. Pseudostroma (bar 52 mm). C. Section across a pseudostroma (bar 5100 mm). D. Asci associated with few
paraphyses (bar 520 mm). E. Asci (bar 510 mm). F. Ascospores within asci (bar 55mm).
SANTOS ET AL.: ITS PHYLOGENY OF PHYLLACHORA SPECIES 1161
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of a pseudostroma that crosses all the mesophyll
(FIGS. 5, 6). This is considered as one of several putative
synapomorphies for this clade, with the fact that
their host species belong exclusively in genus Eugenia
(Myrtaceae). In addition, the species P. furnasensis
have fusoid-clavate asci with fusiform ascospores
tapering toward the thick walled apices, a characteristic
that differentiates it from the second species within
this clade (P. ermidensis), which have cylindrical asci
without thickened apices. Consequently, they can be
recognized as distinct species, in agreement with the
ITS data.
Clade III, containing a collection from Brazil
belonging to P. graminis (FIG. 16), easily recognized
by the presence of amphigenous pseudostromata that
grows across the entire section of the leaf, occupying
the epidermal tissue on both sides of the leaf, with
broadly clavate lightly pitted ascospores formed within
fusoid asci. Phyllachora qualeae fell in the same clade III,
but its asci are typically long, straight, and cylindrical,
containing short-ellipsoidal uniseriate ascospores
(FIG. 17).
This paper constitutes a first approach to the molec-
ular phylogeny of Phyllachora species associated with
hosts of the family Myrtaceae, focusing on plants most-
ly endemic to the Brazilian Cerrado. The molecular
data is additionally supported by morpho-taxonomical
analyses that allowed for the splitting of the genus in
well consolidated species when considering the syna-
pomorfies present in the different groups. In addition,
it provides new information on the host preferences of
these species and the importance of ascospore, asci,
and pseudostroma morphology in the segregation of
the different species within the genus. Furthermore,
those sequences constitute an important tool to be
used in comparing Phyllachora species from Myrtaceae
with those found infecting other host species, as the
morphological and phylogenetic species concepts
herein do not overlap, and in most cases they are
potentially meaningful for species identification. The
present morphological concept accepted for the group
of species described correlates with the ITS phyloge-
netic segregation indicated in the phylogenetic tree
(FIG. 1). However, future studies should include
sequences of other rDNA regions, as well as those of
other genes to provide for an ample phylogenetic
account of the entire Phyllachoralles, which by now
remains as an order within Sordariomycetidae based
only on comparison of sequences of the 18S rDNA
region, although Polystigma, traditionally set in Phylla-
chorales, was placed by Maharachchikumbura et al.
(2015) in Xylariomycetidae. Finally, the ITS data pro-
vided helps on the segregation of the species described
on Myrtaceae from the Cerrado and is shown as a
strong barcode candidate for Phyllachora species.
ACKNOWLEDGMENTS
The authors thank Dr Zuleide Martins Chaves for editing
and improving the illustrations, Prof Danilo Batista Pinho
for manuscript review, and CNPq for a PhD fellowship to
the senior author and research grant to the last co-author
through PPBIO-Cerrado.
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