The cup fungus Pestalopezia brunneopruinosa is Pestalotiopsis gibbosa and belongs to Sordariomycetes.

Pestalopezia brunneopruinosa, the type species of Pestalopezia in Leotiomycetes, produces typical cup-shaped ascomata. Because its asexual morph has conidia comprised of five cells including apical and basal appendages and three pigmented median cells, it was first described as Pestalotia gibbosa, which belongs to Sordariomycetes. This contradiction has not been resolved due to the difficulty in isolating this fungus in culture. In this study, we isolated separate strains from the sexual morph and the asexual morph for molecular analysis. Phylogenetic trees of Sporocadaceae based on internal transcribed spacer, partial β-tubulin, and partial translation elongation factor 1-alpha sequence datasets revealed that both strains fall into the same taxon, in a clade in Pestalotiopsis sensu stricto alongside P. gaultheriae and P. spathulata. We provide the first evidence that fungi producing cup-shaped ascomata in Pestalotiopsis belong to Sordariomycetes, and we have proposed the transfer of Pestalopezia brunneopruinosa to Pestalotiopsis gibbosa.

Introduction

Pestalopezia brunneopruinosa (Zeller) Seaver is a leaf spot pathogen on salal (Gaultheria shallon Pursh) that produces asci on an apothecium as a sexual morph [1]. The asexual morph of Pestalopezia brunneopruinosa resembles that of Pestalotiopsis sensu lato (s. lat.) and was first described independently by Harkness [2] as Pestalotia gibbosa. Thus, it has been suspected that Pestalopezia brunneopruinosa and Pestalotia gibbosa are the same fungus, because the two fungi have been found in close proximity on the same leaves. Bonar [3] demonstrated that cultures from germinated ascospores of Pestalopezia brunneopruinosa produced conidia that were the same as that of Pestalotia gibbosa. Seaver [4] likewise concluded that Pestalopezia brunneopruinosa was the sexual morph of Pestalotia gibbosa. However, phylogenetic analyses of both fungi to clarify their relationship has not been previously conducted.

The genus Pestalotia was established by De Notaris [5]. Subsequently, Steyaert [6] split the genus Pestalotia into Pestalotia sensu stricto (s. str.) (conidia composed of 6 cells), Pestalotiopsis (5 cells) and Truncatella (4 cells), although many species were still retained in Pestalotia s. lat. without reconsideration. Recently, Pestalotiopsis s. lat. was further split into three genera, Pestalotiopsis s. st., Neopestalotiopsis, and Pseudopestalotiopsis, based on morphology and molecular phylogeny [7]. These fungi belong to Sporocadaceae within Sordariomycetes [8]. The Harkness description of Pestalotia gibbosa conidia (three pigmented median cells in five-celled versicoloured conidia, with septa darker than the rest of the cell), is similar to that of Neopestalotiopsis. However, the current taxonomic position of Pestalotia gibbosa is unclear, especially since the disposition of this fungus in Sordariomycetes was made without molecular data support. The sexual morph of Pestalotiopsis s. lat. was determined by Barr [9] to be Pestalosphaeria which produces three celled-ascospores and perithecial ascocarps. Réblová et al. [10] proposed using the name Pestalotiopsis rather than Pestalosphaeria as the currently accepted name, following recent botanical code changes, but there was no mention of the name Pestalopezia in this argument. Pestalopezia, Pestalotiopsis, and Pestalosphaeria, are, however, included in a “without-prejudice list of generic names of fungi for protection under the International Code”[11].

Pestalopezia brunneopruinosa, the sexual morph, was classified as a member of the Leotiomycetes [12] because it produces cup-shaped ascomata. Thus, the genus names of the sexual and asexual morphs are currently forced into different taxonomic classes. Beimforde et al. [13] conducted a phylogenetic analysis by combining fossil data and molecular data (18S rDNA, 28S rDNA, RPB1, and RPB2) and showed estimated lineages of both families diverged during the Permian or Carboniferous periods and Leotiomycetes and Sordariomycetes are sister clades. Their results indicate that these families, both of which produce inoperculate asci, are closely related in the molecular phylogenetic tree. However, there is no report that fungi belonging to Sordariomycetes can produce cup-shaped ascomata. The aim of this study was to clarify the taxonomic position of Pestalopezia brunneopruinosa with respect to Pestalotia gibbosa, and to determine the name for this fungus based on the concept of one fungus, one name [14, 15].

Materials and methods

Sample collection and isolation

Diseased leaves of salal (Fig 1) were collected from Sandcut Beach trail near Shirley, Vancouver Island BC, Canada in 2013. Several isolates that originated from single conidia in acervuli were cultured from diseased leaves. Isolates were also initiated from ascospores in an ascus, but ejected ascospores failed to individually germinate. Subsequent transfers from the ascus isolate were made from single conidia. Isolates obtained from the asexual morph: NOF 3175/TAP13K_P3, and from the sexual morph: NOF 3176/TAP13K_ca_as2 were maintained on PDA (potato dextrose agar, Eiken, Tokyo, Japan) at 15°C, examined to assess taxonomic position, and deposited in The Fungus Culture Collection of the Northern Forestry Centre, Edmonton, Alberta, Canada and Tamagawa University, Machida, Tokyo, Japan. A voucher specimen containing both apothecia and acervuli was deposited in the Pacific Forestry Centre Forest Pathology Herbarium (DAVFP 29689). Information of new combination in Nomenclature was deposited in the Mycobank (http://www.mycobank.org/defaultinfo.aspx?Page=Home: MB#824630).

Fig 1

A diseased leaf of salal (Gaultheria shallon) from Sandcut beach trail, Vancouver Island, BC, Canada.

DNA extraction and molecular analysis

DNA from each strain was extracted using the Qiagen DNA Mini Kit (Qiagen, Tokyo, Japan) following the manufacturer's protocol. Internal transcribed spacer (ITS), β-tubulin, and partial translation elongation factor 1-alpha (tef1) gene regions were amplified as described previously [16-19], using primers ITS1/ITS4, Bt2d/Bt2c, and pest_ef_f/EF1-1567R, respectively. These primers target regions that are approximately 550 bp, 560 bp, and 530 bp in size, respectively.

To confirm the culture was isolated from the sexual morph, DNA was extracted from a single apothecium from DAVFP 29689 by CTAB [20], and ITS was amplified using our designed primer PES3 (5’-GGCCTACCCTGTAGCGCCTT-3’) and ITS4.

Polymerase chain reaction (PCR) products were purified using ExoSAP-IT (GE Healthcare Japan, Tokyo, Japan) and sequenced using the ABI 310 DNA sequencer (ABI, Tokyo, Japan). These sequences have been deposited in the DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index-e.html: accession numbers are shown in Table 1).

Table 1

Source of species for molecular analyses and the DNA database accession number.

Species	Culture No.	Location	Host	GenBank accession
				ITS	β-tubulin	tef1
Pestalotiopsis gibbosa (syn. Pestalotia gibbosa, this study)	NOF 3175/TAP13K_P3	Canada	Gaultheria shallon	LC311589	LC311590	LC311591
P. gibbosa (syn. Pestalopezia brunneopruinosa, this study)	NOF 3176/TAP13K_ca_as2*	Canada	Gaultheria shallon	LC311586	LC311587	LC311588
Pestalotiopsis adusta	ICPM 6088	Fiji	On refrigerator door PVC gasket	JX399006	JX399037	JX399070
P. anacardiacearum	IFRDCC 2397	China	Mangifera indica	KC247154	KC247155	KC247156
P. arceuthobii	CBS 434.65	USA	Arceuthobium campylopodum	KM199341	KM199427	KM199516
P. arengae	CBS 331.92	Singapore	Arenga undulatifolia	KM199340	KM199426	KM199515
P. australasiae	CBS 114126	New Zealand	Knightia sp.	KM199297	KM199409	KM199499
P. chamaeropis	CBS 186.71	Italy	Chamaerops humilis	KM199326	KM199391	KM199473
P. clavata	MFLUCC 12–0268	China	Buxus sp.	JX398990	JX399025	JX399056
P. colombiensis	CBS 118553	Colombia	Eucalyptus eurograndis	KM199307	KM199421	KM199488
P. diploclisiae	CBS 115587	Hong Kong	Diploclisia glaucescens	KM199320	KM199419	KM199486
P. ericacearum	IFRDCC 2439	China	Rhododendron delavayi	KC537807	KC537821	KC537814
P. furcata	MFLUCC 12–0054	Thailand	Camellia sinensis	JQ683724	JQ683708	JQ683740
P. gaultheriae	IFRD 411–014	China	Gaultheria forrestii	KC537805	KC537819	KC537812
P. grevilleae	CBS 114127	Australia	Grevillea sp.	KM199300	KM199407	KM199504
P. hollandica	CBS 265.33	The Netherlands	Sciadopitys verticillata	KM199328	KM199388	KM199481
P. humus	CBS 336.97	Papua New Guinea	Soil	KM199317	KM199420	KM199484
P. kenyana	CBS 442.67	Kenya	Coffea sp.	KM199302	KM199395	KM199502
P. monochaeta	CBS 144.97	The Netherlands	Quercus robur	KM199327	KM199386	KM199479
P. neglecta (this study)	TAP1100*/MAFF239735	Japan	Quercus myrsinaefolia	AB482220	LC311599	LC311600
P. novae-hollandiae	CBS 130973	Australia	Banksia grandis	KM199337	KM199425	KM199511
P. oryzae	CBS 353.69	Denmark	Oryza sativa	KM199299	KM199398	KM199496
P. papuana	CBS 331.96	Papua New Guinea	Coastal soil	KM199321	KM199413	KM199491
P. parva	CBS 265.37	-	Delonix regia	KM199312	KM199404	KM199508
P. pallidotheae	MAFF 240993*	Japan	Pieris japonica	NR111022	LC311584	LC311585
P. portugalica	CBS 393.48	Portugal	-	KM199335	KM199422	KM199510
P. rhododendri	IFRDCC 2399	China	Rhododendron sinogrande	KC537804	KC537818	KC537811
P. scoparia	CBS 176.25	-	Chamaecyparis sp.	KM199330	KM199393	KM199478
P. spathulata	CBS 356.86	Chile	Gevuina avellana	KM199338	KM199423	KM199513
P. telopeae	CBS 114161	Australia	Telopea sp.	KM199296	KM199403	KM199500
Pestalotiopsis sp.1 (this study)	TAP0K00Kin	Japan	Osmanthus fragrans var. aurantiacus	LC311595	LC311596	LC311597
Pestalotiopsis sp.2 (this study)	TAP0E0SA*	Japan	Camellia sasanqua	LC311592	LC311593	LC311594
Pseudopestalotiopsis cocos	CBS 272.29	Java, Indonesia	Cocos nucifera	KM199378	KM199467	KM199553
Ps. theae	MFLUCC 12-0055/CPC 20281	Thailand	Camellia sinensis	JQ683727	JQ683711	JQ683743
Ps. myanmarina	NBRC 112264*	Myanmar	Averrhora carambola	LC114025	LC114045	LC114065
Ps. vietnamensis	NBRC 112252	Vietnam	Fragaria sp.	LC114034	LC114054	LC114074
Neopestalotiopsis australis	CBS 114159	Australia	Telopea sp.	KM199348	KM199432	KM199537
N. cubana	CBS 600.96	Cuba	Leaf litter	KM199347	KM199438	KM199521
N. foedans	CGMCC 3.9123	China	Mangrove plant	JX398987	JX399022	JX399053
N. honoluluana	CBS 114495	USA: Hawaii	Telopea sp.	KM199364	KM199457	KM199548
N. javaensis	CBS 257.31	Indonesia: Java	Cocos nucifera	KM199357	KM199437	KM199543
N. natalensis	CBS 138.41	South Africa	Acacia mollissima	KM199377	KM199466	KM199552
N. piceana	CBS 394.48	UK	Picea sp.	KM199368	KM199453	KM199527
N. protearum	CBS 114178/STE-U 1765	UK	Picea sp.	KM199368	KM199453	KM199527
N. saprophytica	MFLUCC 12–0282	China	Magnolia sp.	JX398982	JX399017	JX399048
N. surinamensis	CBS 450.74	Zimbabwe	Protea eximia	KM199351	KM199465	KM199518
N. zimbabwana	CBS 111495	Zimbabwe	Leucospermum cunciforme	JX556231	KM199456	KM199545
Seiridium camelliae	SD096/MFLUCC 12–0647	China	Camellia reticulata	JQ683725	JQ683709	JQ683741
Seiridium sp. 1 (this study)	TAP121	Japan	Hamamelis japonica	LC311607	LC311608	LC311609
Seiridium sp. 2 (this study)	TAP1041	Japan	Chamaecyparis obtusa	LC311610	LC311611	LC311612
Seiridium sp. 3 (this study)	TAP3355	Japan	Tilia cordata	LC311601	LC311602	LC311603
Seiridium sp. 4 (this study)	TAP881	Japan	Rhododendron keiskei	LC311604	LC311605	LC311606

Bold accession numbers were obtained in this study.

* indicates strain producing sexual morph.

The results of the preliminary sequence homology search using BLAST were that the two Vancouver Island salal isolates, NOF 3175/TAP13K_P3 and NOF 3176/TAP13K_ca_as2, fell into Pestalotiopsis s. str. Additional sequence data for phylogenetic analysis were obtained from 7 other previously unpublished strains (listed in bold in Table 1), and 43 other strains published in previous studies [7, 21]. To generate phylogenies based on ITS, β-tubulin, and tef1 sequences, Seiridium spp., members of Amphisphaeriaceae (outgroup) and Phlogicylindriaceae, were chosen because they are phylogenetically close to Sporocadaceae. The dataset of each genomic region (ITS, β-tubulin, and tef1) was aligned using MAFFT [22]. All positions containing gaps and missing data were deleted from the analysis. The strength of internal branches from the resulting tree was tested using the bootstrap analysis [23] with 1,000 replications.

Sequence data comprising the aligned dataset were subjected to maximum-likelihood (ML), neighbor-joining (NJ) and maximum-parsimony (MP) phylogenetic analyses using MEGA software Version 7 [24]. Molecular analyses using the ML method were performed using HKY+G+I nucleotide substitution model for ITS, β-tubulin, and tef1. Initial trees for the heuristic searches were automatically generated by applying the NJ and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with a higher log-likelihood value. Evolutionary history was inferred using the NJ method [25]. The tree was drawn to scale with branch-length units equivalent to those of the evolutionary distances used to infer phylogeny. Evolutionary distances were computed using the Kimura 2-parameter method [26] as the number of base substitutions per site. MP trees were generated using the tree-bisection-regrafting (TBR) algorithm and search level 3, which generates initial trees by randomly adding sequences (10 replicates). Consistency, retention, homoplasy, and composition indices were calculated for parsimony-informative sites. The resulting trees were printed using TreeView v. 1.6.6 [27] and, together with the alignments, deposited as S21431 in TreeBASE (https://www.treebase.org/treebase-web/home.html).

Morphological observations

Morphological observations were made from symptomatic salal leaves collected in 2013 (DAVFP 29689) and from a single dried herbarium specimen DAVFP 11308. The latter was collected in 1959, also from Vancouver Island, and determined as Pestalopezia brunneopruinosa by W. Ziller (S1 Fig). The asexual and sexual morphs were observed and measured in water using light microscopy (BX 51, Olympus Tokyo, Japan).

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number [urn:lsid:mycobank.org:Mycobank:824630] contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories:PubMed Central, LOCKSS.

Results

Phylogenetic analysis

In addition to the Vancouver Island collections preliminarily identified as Pestalotia gibbosa (NOF 3175/TAP13K_P3, Culture from conidia) and Pestalopezia brunneopruinosa (NOF 3176/TAP13K_ca_as2, Culture from ascospores), a total of 52 strains, including Pestalotiopsis (30 strains with two obtained from sexual morphs), Neopestalotiopsis (11 strains), and Pseudopestalotiopsis (4 strains including one obtained from the sexual morph), were examined (accession numbers shown in Table 1). The sequence matrix used for phylogenetic analyses contained at least 1258 nucleotide positions for final data set from sequences 550 bp of ITS, 560 bp of β-tubulin, and 530 bp of tef1. In ML method, the highest log-likelihood was -6657.97. The optimal tree generated using the NJ method had a branch-length of 0.665. An MP tree had a length of 909, consistency index of 0.547, retention of 0.87 and composite index of 0.509. Only the ML tree (Fig 2) is shown here, because the ML, NJ, and MP methods generated similar topologies.

Fig 2

Maximum-likelihood (ML) tree with length 743 determined by analysis of the combined ITS, β-tubulin, and tef1 sequence matrix.

Numbers (ML/NJ/MP) and hyphens on the branches represent the bootstrap values (%) for each node, calculated from 1,000 replicates; only values > 80% are shown. NJ: neighbor-joining, MP: Maximum-parsimony. *: ex-holotype cultures. Blue texts indicate strains producing sexual morphs.

Pestalotia gibbosa and Pestalopezia brunneopruinosa were placed in the same clade with Pestalotiopsis gaultheriae (ML/NJ/MP: 100/100/100). Pestalotiopsis spathulata was also closely placed to Pestalotia gibbosa and Pestalopezia brunneopruinosa with highly supported bootstrap values (ML/NJ/MP: 100/100/99). Furthermore, the ITS sequence obtained from Pestalotia gibbosa (NOF 3175/TAP13K_P3) and Pestalopezia brunneopruinosa (NOF 3176/TAP13K_ca_as2) were the same as the ITS sequence obtained from DNA extracted directly from an apothecium of DAVFP 29689 (epitype specimen) (S2 Fig).

Morphological comparisons

Our observations of the apothecia from DAVFP 11308 and 29689 are similar to those of Seaver’s description [4] of Pestalopezia brunneopruinosa, with few exceptions. Seaver’s ascospore measurements were slightly larger than the Vancouver Island DAVFP (VI) specimens at 7–10 x 14–20 um, plus we observed in the VI collections that mature ascospores eventually darken to brown rather than remaining hyaline (Fig 3, S3 Fig). We also observed a ring-shaped ascus apparatus in DAVFP 29689 which stained blue in Melzer’s reagent, but only in scattered mature asci. These morphological variations are relatively minor and likely reflective of the state of maturity of Seaver’s material (S1 Table). We also compared our observations and measurements of the conidial states of DAVFP 11308 and 29689 from leaves to published descriptions and specimens of conidia of Pestalopezia brunneopruinosa, Pestalotiopsis gaultheriae, and P. spathulata (Table 2). With the exception that P. spathulata has fewer and longer appendages [7], all are morphologically very similar.

Fig 3

Morphological characteristics of Pestalotiopsis gibbosa (Epitype).

(A) Apothecia (arrowheads). (B) Acervuli (arrows). (C) A vertical section of an apothecium. (D) Asci containing ascospores. (E) Apical ring of ascus tip staining blue in Melzer’s reagent. (F) Mature ascospores (G) A Section of an acervulus. (H) Conidia. Bars (A), (B) = 2 mm, (C)–(E) = 20 μm.

Table 2

Morphological comparison of asexual morphs of Pestalotiopsis gibbosa and related species.

Species	Three median cells			Apical appendages
	Size (length × width, μm)	Length (μm)	Colour	Number	Size (length, μm)	Tip
Pestalopezia brunneopruinosa [2]	25–30 × 8–10.5	16–20	concolorous, olivaceous	2–4 (3)	30–60	knobbed
Pestalopezia brunneopruinosa (DAVFP 11308)	22.5–32 × 8–13.5	13–20	versicolorous, dark brown	1–4 (3)	20–48	knobbed
Pestalotiopsis gibbosa (DAVFP 29689)	24–31 × 7.5–10	15.5–22.5	versicolorous, dark brown	2–4 (3)	22–61	knobbed
Pestalotiopsis gaultheriae [28]	23–31 × 7–9.5	–	versicolorous, dark brown	3	15–50	knobbed
Pestalotiopsis spathulata [7]	24–32 × 7.5–9.5	13–20	versicolour	2–5	17–25	knobbed

Taxonomy

Pestalotiopsis gibbosa (Harkn.) Kyoko Watan., Nozawa & B. Callan, comb. nov. [urn:lsid:mycobank.org:Mycobank:824630]

= Pestalotia gibbosa Harkn. Bull. Calif. Acad. Sci. 2: 439, 1887 MB#191515

= Dermatea brunneopruinosa Zeller, Mycologia 26: 291, 1934 MB#259032

= Pestalopezia brunneopruinosa (Zeller) Seaver, Mycologia 34: 300, 1942 MB#289174

≡Pestalotiopsis gaultheriae Y.M. Zhang, Maharachch. & K.D. Hyde, Sydowia 65: 121, 2013 MB#803236

Epitype (Fig 3)

DAVFP 29689, Sandcut Beach trail, Shirley, Vancouver Island BC, Canada, 48.4173°N, 124.0185°W March 5, 2013, on leaves of G. shallon Pursh collected by B. Callan and M. Brannigan. Ex-epitype NOF 3176/TAP13K_ca_as2 was isolated from a conidium transferred from a colony originating from a single ascus.

Ascocarp: Apothecium developing on the upper surface of pale tan to light brown necrotic areas of attached living leaves, sessile or with short stalk approximately 0.5–2 mm in diameter, cup-shaped, with a wood brown to yellowish brown furfuraceous exterior. Hymenium fuscous when immature, becoming black at maturity because of the dark tips of paraphyses forming the epithelium; asci: 115–150 μm in length (including a short stalk) × 11–15 μm in diameter (n = 20), eight-spored, unitunicate, cylindrical, with slightly pointed apex, apical apparatus ring-shaped and staining blue in Melzer’s reagent, but only when fully mature; ascospores: 5–8 × 11–16 μm (n = 20), ellipsoidal to ovate, at first hyaline, becoming dark brown when mature, one-seriate; paraphyses: slender and clavate, light brown at their tips in Melzer’s reagent.

Conidiomata: Acervuli erumpent through the upper surface of the leaf epidermis, frequently in a zonate pattern in necrotic lesions. Lesions frequently coalescing, turning the leaf almost entirely brown while still attached to the stem. Conidiomata from leaves, subglobose to oval, immersed, then erumpent, black, up to 150–219 μm wide (n = 10); Conidiogenous cells directly lining the acervular wall, hyaline, cylindrical, annellidic; Conidia: 24–31 × 7.5–10 μm (n = 30), pyriform, curved, four-septate and slightly constricted at the septa, which are darker than the body of the cells; median three cells 15.5–22.5 μm long (n = 30) in total, pigmented; two upper pigmented cells fuscous, darker than lower pigmented cell, 15.5–22.5 μm long (n = 30); apical cell: hyaline, conical with two to four (mostly three) apical appendages arising from the apical crest. Apical appendages typically swollen at the tip, unbranched, filiform, 22–61 μm long (n = 30). Basal cell hyaline, conical, with a single, tubular, unbranched, centric appendage.

Additional specimen examined: DAVFP 11308 (S3 Fig), Cowichan Lake, Vancouver Island, BC, Canada, April 23, 1959, on leaves of G. shallon Pursh collected and determined as Pestalopezia brunneopruinosa by W. Ziller.

Note: The Holotype was O. S. C. Herb., 8096 in the original description of Dermatea by Zeller in 1934 [1].This description did not mention the color of mature ascospores. DAVFP 11308 collected by Ziller (as Pestalopezia) in 1959 contains mature ascomata and is in sufficiently intact state to observe brownish ascospores. However neither sample was suitable for DNA extraction, and hence we established an epitype. Since obtaining cultures that originate from single ascospores is difficult, we initiated our culture (NOF 3176/TAP13K_ca_as2) from a monoconidial isolate that was obtained from hyphae grown from ascospores of a single ascus. We were able to germinate single ascospores ejected from mature ascocarps onto agar, but the resulting germinants failed to grow beyond an initial germ tube. We designated the epitype of Pestalotiopsis gibbosa as DAVFP 29689. We consider P. gaultheriae Y.M. Zhang, Maharchch. & K.D. Hyde [28] to be a synonym of P. gibbosa, but the authors [28] were unable to obtain living cultures from the specimen of P. gaultheriae.

Discussion

Our morphological observations and sequence results confirm that Pestalopezia brunneopruinosa and Pestalotia gibbosa are the same fungus. Conidia of Pestalotia gibbosa are strikingly similar to those of Neopestalotiopsis species because the three median cells of the conidia are versicoloured, and they could be classified into the genus Neopestalotiopsis based on morphology. However, in this study, we demonstrate by genomic analysis that P. gibbosa should be transferred to Pestalotiopsis s. str., even though its sexual morph is an apothecium.

The majority of the more than 200 species associated with the well-known genus Pestalotiopsis s. lat. are typified by the asexual morph, while only a few (14) have known sexual states producing perithecial ascocarps typified by the genus Pestalosphaeria [7, 21]. Réblová et al. [10] have recommended use of Pestalotiopsis rather than Pestalosphaeria, but this recommendation did not take into consideration the potential of either Neopestalotiosis or Pseudopestalotiopsis also having teleomorphs genetically related to Pestalosphaeria; and the small (three known species), obscure genus Pestalopezia was not mentioned at all in this recommendation. All species of Pestalosphaeria were considered to be linked to Pestalotiopsis s. str. after the three genera Neopestalotiopsis, Pseudopestalotiopsis, and Pestalotiopsis were separated from Pestalotiopsis s. lat. [7]. Silvério et al [29] in 2016 and Nozawa et al. [17] in 2017, found the sexual morphs of Neopestalotiopsis and Pseudopestalotiopsis, both in agreement with the description of Pestalosphaeria. Hence, they reported that Pestalotiopsis s. str., Neopestalotiopsis, and Pseudopestalotiopsis produce the same sexual morph. However, the relationship of these fungi to Pestalopezia, characterized by the production of apothecia, was not considered in these works. In this study, we obtained strains from conidia of Pestalotia gibbosa and from ascospores of Pestalopezia. In phylogenetic analyses based on ITS, β-tubulin, and tef1, both strains were placed with Pestalotiopsis s. str. (Fig 2) although the morphological characteristics of conidia were strikingly similar to those of conidia of Neopestalotiopsis (Fig 3). Hence, the name of Pestalotia gibbosa should be changed to Pestalotiopsis gibbosa. Although Pestalopezia Seaver 1942 precedes Pestalotiopsis Steyaert 1949, we recommend using Pestalotiopsis s. str. as this name is more widely known and therefore likely to be better accepted. The species name gibbosa (1887) is older than brunneopruinosa (1942).With our strains, P. gaultheriae belongs to same clade with high bootstrap values (MP/ML/NJ: 100/100/100, Fig 2). Pestalotiopsis gaultheriae was established as a new species based on morphology and molecular data of ITS, β-tubulin and tef1 sequences, which were directly obtained from the fungi on a leaf of salal. However, our sequence data demonstrated that P. gaultheriae was a synonym of Pestalotiopsis gibbosa. In sordariomycetes, there is no fungi producing cup–shaped ascomata. According to results of Zhuang et al [30] based on a phylogenetic tree of RNA secondary structures and on the estimated morphologies from their phylogenetic tree, ascomata having exposed hymenia are estimated as ancestral morphs. Even Pestalotiopsis s. lat. produces closed ascomata, and only the clade of Pestalotiopsis gibbosa produces open ascomata, nested among other taxa with closed ascomata. In this study, we were unable to determine whether this is the ancestral morph or a reversion morph. Our results provide the first evidence that Sordariomycetes include species that produce cup-shaped ascomata.

Specimen of DAVFP 11308.

This specimen is preserved in the Forest Pathology Herbarium at the Pacific Forestry Center, Victoria, BC, Canada.

(TIF)

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Multiple alignment of ITS sequences among Pestalopezia brunneopruinosa (NOF 3176/TAP13K_ca_as2), Pestalotiopsis gibbosa (NOF 3175/TAP13K_P3), and extract DNA directly from an apothecium on DAVFP 29689.

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Morphological characteristics of Pestalotia gibbosa (DAVFP 11308).

(A) Apothecia; (B) Acervuli; (C) Asci containing mature ascospores (arrow) on the layer of an apothecium; (D) Asci and ascospores (stained with iodine); (E) Conidial formation on the upper layer of an acervulus; and (F) Conidia. Bars (A), (B): 2 mm, (C): 100 μm, (D)–(F): 20 μm.

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Morphological comparison of sexual morph of Pestalopezia brunneopruinosa and related species.

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