Diatrypella macrospora sp. nov. and new records of diatrypaceous fungi from Iran

Abstract

Descriptions and illustrations are provided for six diatrypaceous species from Iran. Diatrypella macrospora is introduced as new to science. It differs from all other Diatrypella species by its larger ascospores. New records for the Iranian mycota include Cryptovalsa rabenhorstii, Eutypella citricola, Peroneutypa scoparia and Quaternaria quaternata. Descriptions and illustrations of microscopic characters, ITS sequence data and taxonomic notes are provided for these newly reported taxa.

Full text

Phytotaxa 252 (1): 043–055
http://www.mapress.com/j/pt/
Copyright © 2016 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Kevin Hyde: 22 Feb. 2016; published: 8 Mar. 2016
http://dx.doi.org/10.11646/phytotaxa.252.1.4
43
Diatrypella macrospora sp. nov. and new records of diatrypaceous fungi from Iran
MEHDI MEHRABI1*, ROGHAYEH HEMMATI1, LARISSA N. VASILYEVA2 & FLORENT P. TROUILLAS3
1 Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Iran
2 Institute of Biology & Soil Science, Far East Branch of the Russian Academy of Sciences, Vladivostok 690022, Russia
3 Department of Plant Pathology, University of California, Davis, CA 95616, USA
*Corresponding author: mehrabimhd@yahoo.com
Abstract
Descriptions and illustrations are provided for six diatrypaceous species from Iran. Diatrypella macrospora is introduced
as new to science. It differs from all other Diatrypella species by its larger ascospores. New records for the Iranian mycota
include Cryptovalsa rabenhorstii, Eutypella citricola, Peroneutypa scoparia and Quaternaria quaternata. Descriptions and
illustrations of microscopic characters, ITS sequence data and taxonomic notes are provided for these newly reported taxa.
Key words: Diatrypaceae, ITS phylogeny, Iran, Taxonomy
Introduction
The family Diatrypaceae encompasses a wide range of morphologically diverse fungi, including pathogenic and
saprotrophic species that are commonly found on the dead wood or bark of their host plants (Glawe & Rogers
1984, Rappaz 1987, Trouillas & Gubler 2010, Trouillas et al. 2011, Pitt et al. 2013, Liu et al. 2015). The family
was characterized primarily on the basis of their perithecial ascomata, long stalked asci and allantoid ascospores
(Glawe & Rogers 1984). Kirk et al. (2008) lists 13 genera and 229 species in Diatrypaceae. Based on molecular and
morphological data, two saprotrophic genera, Diatrypasimilis J.J. Zhou & Kohlm. and Pedumispora K.D. Hyde &
E.B.G. Jones, which were reported from aquatic ecosystems, were added to the family by Klaysuban et al. (2014)
and Abdel-Wahab et al. (2014). More recently two additional genera were transferred to or established in the family,
namely Monosporascus Pollack & Uecker and Phaeoisaria Höhn. (Maharachchikumbura et al. 2015). According to
Maharachchikumbura et al. (2015), the family contains 15 genera.
Several asexual morph genera have been linked to the family Diatrypaceae, including Cytosporina Sacc., Libertella
Desm. and Naemospora Sacc. (Glawe & Rogers 1984). Unlike other groups of Ascomycetes, asexual morphs in this
family are not useful for identification, because the conidial states in the Diatrypaceae are relatively indistinguishable
(Glawe & Rogers 1984, Rappaz 1987, Senanayake et al. 2015).
Detailed descriptions of the family Diatrypaceae have been presented by several authors (Tiffany & Gilman 1965,
Glawe & Rogers 1984, Rappaz 1987, Vasilyeva & Stephenson 2004, 2005, Carmarán et al. 2006, Trouillas & Gubler
2010, Trouillas et al. 2011). Species delimitation in this family traditionally has been difficult due to plasticity of
morphological characteristics which are based mostly on stromata and perithecia characteristic as well as the number of
spores per ascus (Glawe & Rogers 1984, Rappaz 1987). The advent of DNA sequencing has provided new insights into
the phylogenetic relationships of these fungi. The phylogenetic relationships within Diatrypaceae are not well resolved
due to poor taxon sampling and lack of adequate sequence data. Most previous phylogenetic studies of this family were
based on the ribosomal internal transcribed spacer region (ITS) and partial sequence of the β-tubulin gene (Acero et al.
2004, Carmarán et al. 2009, Trouillas & Gubler 2010, Trouillas et al. 2010a, 2010b, 2011, Luque et al. 2012, Chacón et
al. 2013, Liu et al. 2015). These studies suggest that the current taxonomic scheme for the Diatrypaceae may not reflect
the true phylogenetic relationship. Based on a four-gene phylogeny ( ITS, LSU, RPB2 and parts of a β-tubulin gene),
Trouillas et al. (2015) investigated taxonomy of the genus Cryptosphaeria in the western United States and described
C. multicontinentalis as new species.
In Iran, little work has been undertaken to investigate the diversity and taxonomy of the diatrypaceous fungi and

MEHRABI ET AL.
44 • Phytotaxa 252 (1) © 2016 Magnolia Press
only ten taxa have been recorded (Ershad 2009, Mehrabi et al. 2015, Jabbari Firoozjah et al. 2015, Hashemi et al.
2015, Pourmoghaddam et al. 2015). This paper is the second in a series reporting and describing diatrypaceous species
occurring in Iran.
Materials and methods
Fungal isolation and morphological studies
Branches harboring perithecia typical of Diatrypaceae were collected from various trees in different locations, mainly
from northern Iran. Isolates were grown from a single ascospore using the method of Trouillas et al. (2010). Dry
specimens are deposited in the herbarium of the Iranian Research Institute of Plant Protection (IRAN…F), and fresh
cultures are deposited at the Iranian Fungal Culture Collection (IRAN…C) of the same institute. Identification and
morphological observations were made using a compound light microscope (Nikon 80i) and stereo microscope
(Olympus SZH) and followed methods of Mehrabi et al. (2015). Measurements were obtained for 20 perithecia, 20
asci and 20 ascospores produced on naturally infected twigs. Sizes of 20 conidia were obtained from colonies grown
in Petri-dishes with PDA and incubated in the dark at 24°C. Colors of the surface and reverse sides of the pure fungal
colonies in Petri-dishes with Potato Dextrose Agar (PDA) were compared using a color chart (Rayner 1970).
FIGURE 1. One of the 48 most parsimonious trees obtained from the ITS sequence data. MP/NJ Bootstrap support values from 1000
replicates higher than 50% are reported at the nodes. The original strain numbers are noted after the species names. The ex-type strains are
in bold. The new sequences obtained in this study are indicated in highlight.

NEW RECORDS OF DIATRYPACEOUS FUNGI FROM IRAN Phytotaxa 252 (1) © 2016 Magnolia Press • 45
DNA extraction, amplification and sequence analysis
Fungal isolates were grown in the dark at 25 C for 7–14 d in a 50 mL liquid cultures of potato dextrose broth on a
rotary shaker (120 rpm). Mycelium was harvested by centrifugation, and the pellets stored at -20°C. DNA extraction
was conducted with an initial step of grinding the mycelia in liquid nitrogen as described by Liu et al. (2000). The
ITS1-5.8S-ITS2 region of the ribosomal DNA (rDNA) was amplified with primers ITS1 and ITS4 (White et al. 1990).
Polymerase chain reactions (PCR) were carried out in 25 µL reaction mixture containing 1 µL of each primer (10
pmol/µL, Takapouzist Inc.), 1.0 µL genomic DNA (30 ng/µL), 2.5 µL 10× high yield PCR buffer (Jena Bioscience,
Germany), 1.5 unit Taq polymerase(Jena Bioscience, Germany) , 1 mM MgCl2 and 0.5 mM dNTP.
Amplifications were performed in a PC-320 PCR System (ASTEC Co., Japan), which was programmed for
denaturation at 94 C for 4 min, followed by 35 cycles at 94 C for 45 s, 58 C for 35 s and 72 C for 1 min 30 s, with a
final elongation step at 72 C for 10 min. PCR products were resolved on a 1.0 % agarose gel and visualized by staining
with ethidium bromide. Samples showing one single, obvious band of the anticipated length on the gel were selected
and sent out for purifying and sequencing in double directions (Macrogen company, South Korea). Sequences were
checked with FinchTV v. 1.4.0 (Geospiza Inc.). The sequences obtained in this study were compared to those available
in the GenBank database by using the BLASTn algorithm. Based on the BLASTn results, sequences were retrieved
from GenBank for the comparative phylogenetic analysis. GenBank accession numbers of the sequences used in this
study (Acero et al. 2004, Trouillas et al. 2010b, Trouillas et al. 2011, Chacón et al. 2013, Liu et al. 2015, Mehrabi et
al. 2015 ) are provided in Table 1.
The dataset was aligned in ClustalX v1.83 (Thompson et al. 1997) with default parameters. No segments
were excluded from the analyses. Phylogenetic analyses were performed using PAUP*v4.0b10 (Swofford 2003) for
neighbour-joining (NJ) and maximum-parsimony (MP) analyses. The NJ analysis was performed using Kimura-2-
parameter nucleotide substitution model (Kimura 1980). All characters were unordered and of equal weight. Bootstrap
values were obtained from 1000 NJ bootstrap replicates. MP analysis was performed with the heuristic search option
with 1000 random taxon additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All
characters were unordered and of equal weight and gaps were treated as missing data. Branches of zero length were
collapsed and all multiple, equally parsimonious trees were saved. The robustness of the most parsimonious trees was
evaluated by 1000 bootstrap replications (Hillis & Bull 1993). Trees were visualised with TreeView (Page 1996).
Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI), Homoplasy
index (HI) and rescaled consistence index (RC). Xylaria hypoxylon and Daldinia concentrica was selected as outgroup.
Sequences have been deposited in GenBank (Table 1). ITS sequence-alignment file is deposited in TreeBASE (www.
treebase.org) as accession S18861. The taxonomic novelty is deposited in MycoBank (Crous et al. 2004) and Face of
Fungi (www.facesoffungi.org).
TABLE 1. Species information used for the molecular phylogenetic analyses (new taxon and new records are bolded).
species host GenBank accession no. Origin
Cryptosphaeria ligniota Populus tremula AJ302418 Switzerland
Cryptosphaeria pullmanensis Vitis vinifera GQ293967 USA
Cryptosphaeria subcutanea Salix borealis AJ302420 Norway
Cryptovalsa ampelina Vitis vinifera GQ293901 Australia
Cryptovalsa rabenhorstii Vitis vinifera HQ692619 Australia
Cryptovalsa rabenhorstii Citrus sp. KR605649 Iran
Daldinia concentrica _ AF201708 _
Diatrype brunneospora Acacia longifolia HM581946 Australia
Diatrype bullata Salix sp.AJ302422 Switzerland
Diatrype disciformis Fagus sylvatica AJ302437 Switzerland
Diatrype disciformis Alnus sp. KR605644 Iran
Diatrype palmicola Caryota urens KP744439 Thailand
Diatrype spilomea Acer campestre AJ302433 Switzerland
...Continued on next page

MEHRABI ET AL.
46 • Phytotaxa 252 (1) © 2016 Magnolia Press
TABLE 1. (Continued)
species host GenBank accession no. Origin
Diatrype stigma Fagus sylvatica AJ302435 Spain
Diatrype undulata Betula sp.AJ302436 Switzerland
Diatrypella favacea Betula sp.AJ302440 Netherlands
Diatrypella frostii Acer sp.AJ302441 _
Diatrypella iranensis Quercus brantii KM245033 Iran
Diatrypella macrospora Quercus brantii KR605648 Iran
Diatrypella prominens Plantanus sp.AJ302442 USA
Diatrypella pulvinata Quercus robur AJ302443 Netherlands
Diatrypella quercina Quercus faginea AJ302444 Spain
Diatrypella verruciformis Quercus sp.GQ293926 USA
Diatrypella vulgaris Citrus paradisi HQ692590 Australia
Eutypa armeniacae Vitis vinifera AJ302446 Italy
Eutypa laevata Salix sp.AJ302449 Switzerland
Eutypa lata Tilia sp.AJ302450 Switzerland
Eutypella australiensis Acacia longifolia HM581945 Australia
Eutypella caricae Ficus carica AJ302460 France
Eutypella cerviculata Alnus glutinosa AJ302468 Switzerland
Eutypella citricola Citrus paradisi HQ692589 Australia
Eutypella citricola Salix sp. KR605647 Iran
Eutypella cryptovalsoidea Ficus carica HQ692573 Australia
Eutypella leprosa Tilia sp.AJ30246 Switzerland
Eutypella microtheca Vitis vinifera HQ692569 Australia
Eutypella prunastri Prunus avium AJ302464 Switzerland
Eutypella semicircularis Alnus acuminata JQ517314 Panama
Eutypella vitis Vitis vinifera HQ288224 USA
Peroneutypa alsophila Arthrocnemum fruticosum AJ302467 France
Peroneutypa kochiana Atriplex halimus AJ302462 Spain
Peroneutypa scoparia Robinia pseudacacia AJ302465 France
Peroneutypa scoparia Gledischia sp.KR605646 Iran
Quaternaria quaternata Fagus sulvatica AJ302469 Switzerland
Quaternaria quaternata Fagus sp.KR605645 Iran
Xylaria hypoxylon _ AF194027 _
Results
Molecular phylogeny
The ITS sequences data for six taxa obtained in this study (GenBank accession numbers KR605644–KR605649)
were aligned with 39 sequences retrieved from GenBank wich included representative strains and types in the
Diatrypaceae.

NEW RECORDS OF DIATRYPACEOUS FUNGI FROM IRAN Phytotaxa 252 (1) © 2016 Magnolia Press • 47
The combined dataset after alignment resulted in 810 characters including alignment gaps, of which 479 were
constant, 91 were variable and parsimony-uninformative and 240 were parsimony-informative. A heuristic search of
the remaining 230 parsimony-informative characters resulted in 48 most parsimonious trees (TL = 1018, CI = 0.514,
RI = 0.722, RC = 0.371 and HI=0.486), each with the same topology. NJ analysis produced a tree with the same
overall topology as the MP trees. One of the MP trees with bootstrap support values is shown in Fig. 1. On the basis of
phylogenetic analysis Diatrypella macrospora formed a distinct lineage embedded in a larger clade with D. iranensis
and D. quercina. Identification of Cryptovalsa rabenhorstii, Diatrype disciformis, Eutypella citricola, Peroneutypa
scoparia and Quaternaria quaternata were supported by strong bootstrap values (Fig. 1).
Taxonomy
Diatrypella macrospora Mehrabi, R. Hemmati, L.N. Vasilyeva & F.P. Trouillas sp. nov. (Fig. 2)
MycoBank MB 813001, Facesoffungi number: FoF 01891
Differs from other Diatrypella species by the size of ascospores (12–20 × 1.7–3 μm).
Etymology:—Refers to the rather large ascospores.
Type:—IRAN, Kohkiluyeh & Buyer Ahmad Province, Sisakht City, on dead branches of Quercus brantii Lindl. (Fagaceae), 9 April 2014,
M. Mehrabi, KDQ15 (IRAN 16679F, Holotype), ex-type living cultures, IRAN 2344C.
Saprobic on oak branch. Sexual morph: Stromata immersed in the bark of dead branches (2 cm diameter), 1.5–2.5
mm diam, discrete, erumpent, circular, surface black, delimited by a black zone in the host tissues, with groups of 3–10
perithecia, white to yellow to light brown entostroma, Perithecia 400–700 μm diam, usually not compressed, hyaline to
black (with age), circinately arranged, monostichous, shining inside, globoid. Necks of the perithecia about 200–500
μm above the periderm, converge together, black. Ostioles black, opening separately. Asci elongate, sporiferous parts
110–150 (–160) × 10–15 µm, basal part filiform, up to 80 mm long, narrow when young and more or less cylindrical
when mature, with obtuse apex, poly spore. Ascospores (10–) 12–20 (–23) × 1.7–3 (–3.7) μm (av.=15.8 × 2.3 μm,
n=39), allantoid, subhyaline, yellowish in mass, thinwalled, smooth, aseptate, usually with two oil droplets. Paraphyses
elongate, filiform, septate.
FIGURE 2. Diatrypella macrospora from Quercus brantii, (1) Habit of ascostromata on bark. (inset: close-up of perithecia necks in
lateral view), (2) Longitudinal section through the stroma shows ostioles and perithecia (3,4) Anamorph (5) Ascus with ascocpores (6)
Ascospores (7) Conidia (8) Colony morphology on PDA.—Bars 1 = 1 mm. 2,3,4 = 500 μm. 5,6,7= 20 μm.

MEHRABI ET AL.
48 • Phytotaxa 252 (1) © 2016 Magnolia Press
Asexual morph: immersed in bark, acervuli interspersed amongst stromata, rare, 0.5–1 mm diam, flat to
subconical, hymenium labyrinthiform, yellow, with yellow stratum of spores. Conidia slender and strongly arcuate,
20–40(–44) × 0.6–0.8 μm.
Cultural characteristics:—Colonies circular to slightly irregular, cottony with moderately dense, fluffy aerial
mycelium, white on PDA, reverse primrose (23,,b), covering 75 mm Petri-dish after 2 weeks at 24°C.
Notes:—A literature review on Diatrypella (e.g., Saccardo 1882, Ellis & Everharts 1892, Berlese 1900, Chacon
2003, Vasilyeva & Stephenson 2005, Trouillas et al. 2011) highlighted the unique features of this taxon. Diatrypella
macrospora can easily be distinguished from other species of the genus by its large ascospores (12–20 μm). Diatrypella
macrospora is similar to D. persica Rick. in respect to ascospore size. However the latter has wider ascospores (14–17
× 4 μm vs. 12–20 × 1.7–3 μm, Saccardo 1913). The new species also differs in having prominent perithecial necks
emerging in groups. Phylogenetic analysis indicates that the new species belongs to Diatrypella and is closely related
to D. quercina and D. iranensis (bootstrap value = 82% in MP and 95% in NJ, Fig. 1). Morphologically, the new
species can easily be distinguished by having larger ascospores (6–7× 1–1.3 μm in D. iranensis and 8–12 × 2–3 μm in
D. quercina, Mehrabi et al. 2015, Croxall 1950). The new species also differs by its longer necks.
Cryptovalsa rabenhorstii (Nitschke) Sacc., Myc. Ven. 135, tab. XIV. Fig. 3
Basionym: Valsa rabenhorstii Nitschke Pyr. Germ.
Synonym: Sphaeria spiculosa var. robiniae Rabenh., in Exsicc. Klotzsch, Herb. myc.
FIGURE 3. Cryptovalsa rabenhorstii from Citrus sp., (1) Habit of ascostromata on bark (2) Longitudinal section through the stroma
shows ostioles and perithecia (3,4) Anamorph (5) Ascus with ascocpores (6) Ascospores (7) Conidia (8) Colony morphology on PDA.—
Bars 1 = 1 mm. 2,3,4 = 500 μm. 5,6,7= 20 μm.
Saprobic on decaying wood. Sexual morph: Stromata in the bark of dead branches (2 cm diam), effuse, covered by
the epidermis which is not discolored. Perithecia buried in the inner bark, black and shin inside, either scattered in
groups of 5–25 perithecia or rarely in larger groups, circinately or linear arranged, 400–600 µm diam, delimited by
a black zone in the host tissues, ostioles mostly sheltered around a white to yellowish ectostroma, ostioles poorly
emerging. Asci clavate, long and slender pedicellate, polysporous, 115–175 (–200) × 13–20 μm. Ascospores hyaline
when immature turning yellowish to light-brown at maturity, sub-allaintoid to cylindrical, smooth, aseptate (12−)13−15
(−16)×(2.5−)3−4(−5) μm.

NEW RECORDS OF DIATRYPACEOUS FUNGI FROM IRAN Phytotaxa 252 (1) © 2016 Magnolia Press • 49
Asexual morph: immersed in bark, pycnidiainterspersed amongst stromata, rare, 0.5–2 mm diam, subconical,
delimited by a black zone in the host tissues, hymenium labyrinthiform, light yellow, surface white to grey, powdery,
with yellow stratum of spores. Conidia filiform and arcuate, 17–22(–25) × 0.8–1 μm.
Cultural characteristics:—Colonies irregular, with abundant aerial mycelia reaching the lid of Petri-dishes,
white to primrose (23,,b) at the surface and primrose to amber (19,b) at the reverse after 2 weeks in the dark at 24 °C,
covering 75 mm Petri-dish after 5 days at 24°C.
Specimen examined:—IRAN, Mazandaran Province, Sari City, on dead branches of Citrus sp., 5 November
2014, M. Mehrabi, MNC72 (IRAN 16683F, IRAN 2346C).
Notes:—This species resembles closely C. rabenhorstii on Vitis vinifera in Australia (Trouillas et al. 2011).
However, this fungus has a black zone that surrounds the globose perithecia within the host tissue. The pathogenicity
of this species on grapevine has been confirmed (Pitt et al. 2013). The results of the phylogenetic analyses of the ITS
sequences supported the morphological identification (bootstrap value= 100% in MP and 99% in NJ, Fig. 1). To our
knowledge, no members of the genus Citrus have been reported as substrate for this species. The present record is the
first report of this species in Iran.
Eutypella citricola Speg., Anales del Museo Nacional de Buenos Aires 6: 245, 1898. Fig. 4
Saprobic on dead branch. Sexual morph: Stromata immersed in the bark of dead branches (1 cm diam), in pustules
scattered; 6–25 perithecial arranged in a stromata, rarely single, delineated with black line, 0.5–1.5 mm diam,
stromatal aggregations widely effused over branches; perithecia surrounded by white, powdery entostroma, sometimes
compressed with others, black, circinately arranged, 300–500 μm diam, ostioles raising and piercing the periderm,
rarly with elongated clusters of perithecial necks, about 100–300µm long, 3–6 sulcate. Asci octospori, clavate, 50–80
(–90) × 6–8 µm,. Ascospores allantoid, sometimes semicircular, with an oil droplet at each end, subhyaline, yellow
in mass, aseptate, 7–11(–12) × 1.7–2.3 µm. Paraphyses were elongate, filiform. Asexual morph: Conidia have been
observed on the surface of Petri-dish in light yellow mass, filiform, (11−)13–20(−23)×1–1.2 μm.
FIGURE 4. Eutypella citricola From possible Salix sp., (1) Habit of ascostromata on bark (inset: close-up of sulcate ostioles) (2)
Longitudinal section through the ascoma shows globose perithecia surrounded in white entostroma. (3) Ascus with ascocpores (4)
Ascospores (5) Conidia (6) Colony morphology on PDA.—Bars 1 = 1 mm. 2 = 500 μm. 3,4,5= 10 μm.

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50 • Phytotaxa 252 (1) © 2016 Magnolia Press
Cultural characteristics:—Colonies with abundant aerial mycelia reaching the lid of Petri-dishes, irregular,
cottony, forming numerous olivaceous black (27,,,,m) dots of melanized mycelium spread across the media, white on
PDA and whitish to primrose (23,,b) at the reverse after 2 weeks in the dark at 24 °C, after 4wk turning olivaceous black
(27,,,,m), beginning from the center of the colony, covering 75 mm Petri-dish after 5 days at 24°C.
Specimen examined:—IRAN, Guilan Province, Masal County, on dead branches of probably Salix sp., 5 August
2014, M. Mehrabi, GNS46 (IRAN 16682F, IRAN 2349C)
Notes:—This taxon was described by Trouillas et al. (2011) from a specimen collected in Australia. The specimen
studied here fits with the description of Trouillas et al. (2011) and its pathogenicity to grapevine has been confirmed (Pitt
et al. 2013). The phylogenetic analyses of the ITS sequences confirmed the morphological identification (bootstrape
value= 99% in MP and 99% in NJ, Fig. 1). This is the first report from Iran and raises concerns for plant health.
Peroneutypa scoparia (Schwein.) Carmarán & A.I. Romero, in Carmarán, Romero & Giussani, Fungal Diversity Res.
Ser. 23: 84 (2006). Fig. 5
Basionym: Sphaeria scoparia Schwein., Schr. naturf. Ges. Leipzig 1: 37 (1822).
Synonym: see Mycologica Helvetica 2: 285–648 (1987).
Saprobic on decaying wood. Sexual morph: Stromata in the bark or wood of dead branches (1.5 cm diam), numerous,
densely aggregated, erumpent from bark, 0.5–2 mm diam., surrounded by a thin, black stratum, with a black line
on horizontal section. Perithecia 5–25 in a stroma, globose, about 300–600 µm diam., black inside, with elongated
clusters of perithecial beaks, about 0.2–1 mm long. Asci clavate, crowded, 20–33(–40) × 3.5–4(–4.7) µm. Ascospores
allantoid, minute, strongly curved, hyaline, 3–4.5 × 1 µm, with a oil droplet in each end. Paraphyses were elongate,
filiform. Asexual morph: Conidia have been observed on the surface of Petri-dish, infrequent, filiform, (9–) 10–14
(–16) × 1–1.5 µm.
FIGURE 5. Peroneutypa scoparia from Gledischia sp. (1) Habit of ascostromata on bark. (2) Longitudinal section through the stroma
shows ostiolar necks and perithecia (3) Ascus with ascocpores (4) Ascospores (5) Conidia (6) Colony morphology on PDA.—Bars 1 = 1
mm. 2 = 500 μm. 3,4,5= 10 μm.

NEW RECORDS OF DIATRYPACEOUS FUNGI FROM IRAN Phytotaxa 252 (1) © 2016 Magnolia Press • 51
Cultural characteristics:—Colonies circular with regular margin, white on PDA with a smoke gray (21,,,,f)
central part, cottony, reverse primrose (23,,b) and olivaceous black(27,,,,m) in the middle after 2 wk in the dark at 24 °C,
covering 75 mm Petri-dish after 6 days at 24°C
Specimen examined:—IRAN, Guilan Province, Rezvanshahr County, on dead branches of Gledischia sp., 5
August 2014, M. Mehrabi, GNG35 (IRAN 16684F, IRAN 2345C).
Notes:—Berlese (1968) erected the genus Peroneutypa for fungi with small, clavate asci and perithecia with long
necks. This genus was resurrected by Carmarán et al. (2006) based on ascus morphology and other morphological
characters. The Iranian material examined is in agreement with the description of Peroneutypa scoparia by Ellis &
Everharts (1892) and Vasilyeva & Stephenson (2005). To our knowledge, the host genus Gledischia is reported as
a new substrate for this species. Our isolate appears to be closely related to P. scoparia based on DNA phylogenies
(bootstrap value = 99% in MP and 99% in NJ, Fig. 1). This species is reported from Iran for the first time.
Quaternaria quaternata (Pers.) J. Schröt., in Cohn, Krypt.-Fl. Schlesien (Breslau) 3.2(4): 451 (1897). Fig. 6
Basionym: Sphaeria quaternata Pers. in Observ. mycol. (Lipsiae) 1: 64 (1796).
Synonym: Eutypella quaternata (Pers.) Rappaz in Mycol. helv. 2(3): 502 (1987).
FIGURE 6. Quaternaria quaternata from Fagus sp., (1) Habit of ascostromata on bark. (2) Longitudinal section through the stroma
shows ostioles and perithecia (3) Ascus with ascocpores (4) Ascospores (5) Colony morphology on PDA.—Bars 1 = 1 mm. 2 = 500 μm.
3,4= 10 μm.
Saprobic on decaying wood. Sexual morph: Stromata densely immersed in the bark of dead branches (1.5 cm

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52 • Phytotaxa 252 (1) © 2016 Magnolia Press
diameter), dotted at the surface with tiny black ectostroma; 2–6 perithecial arranged in a stromata in the bark, circular,
1–2 mm diam, stromatal aggregations widely effused over branches; perithecia black, circinately arranged, 300–600
μm diam. Asci octospori, almost cylindrical or slightly clavate, (70–)90–130(–155) × 6–7.5 µm. Ascospores uni or
biseriate, allantoid, dilute brownish, : (8–)9–13(–14) × 2–3 µm. Paraphyses were elongate, filiform. Asexual morph:
Undetermined.
Cultural characteristics:—Colonies white and fulvous (13 , i) in the center on PDA, with dense, chiefly immersed,
felty mycelium, circular to slightly irregular, reverse sienna (13i) and black particularly in the colony center after 2
weeks in the dark at 24 °C, covering 75 mm Petri-dish after 16 days at 24°C.
Specimen examined:—IRAN, Guilan Province, Rasht City, on dead branches of Fagus sp., 11 July 2013, M.
Mehrabi, GNF13 (IRAN 16681F, IRAN 2348C).
Notes:—Quaternaria was proposed by Tulasne & Tulasne (1863). Quaternaria quaternata was described as the
type species of the genus Quaternaria and later proposed to be included in genus Eutypella (Rappaz 1989). However,
Gams (1994) has not confirmed the conservation of the name Eutypella against Quaternaria. Molecular analysis by
Acero et al. (2004) supported maintaining Quaternaria as an independent genus. Vasilyeva (2011) concluded that this
genus can be distinguished as a separate genus not only because its name precedes that of Eutypella, but because the
stromata of its members are cryptosphaeroid in their appearance and develop within the bark parenchyma. Based on both
morphology and molecular sequence data, the occurrence of Quaternaria quaternata in Iran was confirmed with 100%
bootstrap values in MP and NJ analysis (Fig.1). In our analysis, Quaternaria genus formed a clade with Cryptovalsa
genus and this topology has been obtained previously (Trouillas et al. 2011). Vasilyeva (2011) has suggested that
Quaternaria is the eutypelloid counterpart of Cryptosphaeria, but our analysis did not support this hypothesis.
FIGURE 7. Diatrype disciformis from Alnus sp. (1) Habit of ascostromata on bark. (2) Transverse section through the ascoma shows
globose perithecia surrounded in white entostroma (3) Ascus with ascocpores (4) Ascospores (5) Colony morphology on PDA.—Bars 1
= 2 mm. 2 = 1 mm. 3,4= 10 μm.

NEW RECORDS OF DIATRYPACEOUS FUNGI FROM IRAN Phytotaxa 252 (1) © 2016 Magnolia Press • 53
Diatrype disciformis (Hoffm.) Fr., Summa veg. Scand., Section Post. (Stockholm): 385 (1849). Fig. 7
Basionym: Sphaeria disciformis Hoffm. , Veg. Crypt. 1: 15 (1787).
Saprobic on decaying wood with bark. Sexual morph: Stromata erumpent from bark of dead branches (8 mm
diameter), disc-shaped, 1.5–2 mm diam., flat or slightly convex, dark at the surface, whitish inside, base sunk to the
wood and circumscribed by a black line. Ostioles dark, at the same level as the discs surface, sulcate. Perithecia 10–23
(or more) in a stroma, dark, globoid to subgloboid, 300–600 µm diam. asci elongate, clavate, 45–70 (–90) × 3.5–5(–6)
µm, octospori, ascospore allantoids, hyaline, aseptate, 5–7 × 1–1.7 µm. Paraphyses were elongate, filiform. Asexual
morph: Undetermined.
Cultural characteristics:—Colonies circular to slightly irregular, white on PDA, cottony, reverse of colonies
primrose (23 ,, b) after 2 weeks in the dark at 24°C, covering 75 mm Petri-dish after 8 days at 24°C.
Specimen examined:—Iran, Guilan Province, Rasht City, on dead branches of Alnus sp., 11 July 2013, M.
Mehrabi, GNA14 (IRAN 16680F, IRAN 2347C).
Notes:—This species seems to be extremely rare outside Europe and restricted to Fagus sp. ( Vasilyeva 2011,
Rappaz 1987). The Iranian material examined was consistent with D. disciformis as described by Ellis & Everhart
(1892). This species has also described by Senanayake et al. (2015) on Ostrya carpinifolia from Italy. The material
studied here fits also with the description of Senanayake et al. (2015). The phylogenetic analyses of the ITS sequences
supported the morphological identification (bootstrap value= 93% in MP and 99% in NJ, Fig. 1). This species was
recently reported from Fagus orientalis in Iran (Pourmoghaddam et al. 2015). The present description is the first report
of this species from Alnus sp. in Iran.
Acknowledgments
We are grateful to the Research Institute of Modern Biological Techniques (University of Zanjan) for the use of their
equipment.
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