Requienella populi sp. nov. (Requienellaceae, Xylariales) from the bark of living aspen trees in Western Norway

Abstract

The new species Requienella populi in the Requienellaceae is described from Western Norway. Multigene analysis of the four molecular markers ITS, LSU, RPB2 and TUB revealed it as a strongly supported sister clade within the genus. The new species appears to be restricted to old aspen Populus tremula trees and can be morphologically distinguished by submuriform and somewhat smaller ascospores compared to the other species of the genus. A table comparing species of Requienella is provided. The Requienellaceae received a moderate statistical support as a sister group to the Cainiaceae in our analysis and the circumscriptions of the two families need to be studied further using additional genetic markers.

Citation: Andreasen M, Jordal JB, Nordén B (2025). Requienella populi sp. nov. (Requienellaceae, Xylariales) from the bark of living aspen trees in Western Norway. Fungal Systematics and Evolution 15: 225–234. doi: 10.3114/fuse.2025.15.10

INTRODUCTION

As part of a biodiversity mapping project in Norway, we focused our interest on bark-living species on old aspen trees, Populus tremula. During fieldwork in the oceanic parts of Western Norway we encountered a species macroscopically reminiscent of Requienella fraxini, which we knew well from previous studies. Requienella is a small genus of prominent bark-living Ascomycota with only three species hitherto known, R. fraxini, R. seminuda and the newly published R. shangrilana, the two former being specific to trees in the Oleaceae (on Fraxinus and Olea, respectively) while the latter was encountered from wood of an unknown host species. Macroscopically R. fraxini and R. seminuda are easily identified in the field by their prominently protruding cone-shaped black papillae surrounded by white rings of amorphous matter, an aspect shared with our new species but seemingly not with R. shangrilana. Under the microscope, however, the species on aspen differed clearly from the three previous species of the genus by having submuriform ascospores.

Requienella has been considered a member of the Dothideomycetes due to its bitunicate asci, or of the Pyrenulales due to the distoseptate ascospores with lenticular lumina. However, Jaklitsch et al. (2016) showed that it belongs to the Xylariales based on molecular evidence. For an exhaustive historical account of the systematic treatment and a detailed morphological account of Requienella, please refer to Jaklitsch et al. (2016).

We here describe the new species as R. populi based on morphological data and multigene analysis of four molecular markers. To aid identification, we provide a table comprising all species of Requienella.

MATERIALS AND METHODS

Sampling and morphological investigation

Ascomata were rehydrated with autoclaved water and studied using a Nikon SMZ 745T stereomicroscope and a Nikon Eclipse Ci-L or a Zeiss Axio Imager A2 compound microscope. Images of ascomata were captured with a Nikon DS-Fi2 or Tucsen DigiRetina 16 camera, using stacking software Lite Helicon Focus 8 v. 8.2.2. Microslides were created with contents of the ascomata mounted in sterile water or 5 % KOH. Photomicrographs were produced using a Zeiss Axiocam 503 camera and measurements were made with Zeiss AxioVision v. 4.9.1 software (Carl Zeiss AG), and images were processed in GIMP v. 2.10.34 (Kimball & Mattis 1996).

DNA extraction and sequencing

Genomic DNA was extracted from hymenial material, placed in inhibiting buffer solution, and sent to Eurofins, Germany for DNA isolation, amplification, and Sanger sequencing of the nuclear ribosomal DNA (nrDNA) regions of internal transcribed spacer (ITS) containing ITS1, 5.8S NRDNA and ITS2 and the 28S large subunit nrDNA (LSU), RNA polymerase II second largest subunit (RPB2) and the beta-tubulin gene (TUB) using the primer pairs ITS4/ITS5 (White et al. 1990), LR5/V9G (Vilgalys & Hester 1990/De Hoog & Gerrits van den Ende 1998), fRPB2-5/rRPB2-7C (Novakova et al. 2012), and Bt2a/Bt2b (Hsieh et al. 2005), respectively.

Sequence alignment and phylogenetic analyses

Sequence editing, assembly and concatenations were done using Geneious Prime v. 2025.0.2 (Kearse et al. 2012) and deposited in GenBank (Table 1), and the alignments were uploaded to Figshare (www.figshare.com; doi: 10.6084/m9.figshare.28266224). Sequence data from Han et al. 2024 and Jaklitsch et al. 2016 were downloaded from GenBank. Preliminary alignments were made using MAFFT v. 7.490 (Katoh & Standley 2013) with standard settings as incorporated in Geneious Prime. All alignments were inspected and manually adjusted. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI). Substitution models for each locus were determined based on the AICc model selection criterion (small-sample-size corrected version of Akaike information criterion) as implemented in PartitionFinder v. 2.1.1 (Lanfear et al. 2016). The search was set to ‘all’ and branch lengths set to ‘linked’. The ML analyses were performed on aligned sequences using RAxML v. 8.2.11 (Stamatakis 2014) as implemented in Geneious. Rapid Bootstrapping and search for best-scoring ML tree algorithms were used with GTR-GAMMA-I substitution model and Bootstrap analyses obtained by 1 000 bootstrap replications.

Table 1.

Fungal taxa, strains and GenBank accessions used of Apiosporaceae, Barrmaeliaceae, Cainiaceae, Graphostromataceae, Hypoxylaceae, Lopadostomaceae, Requienellaceae and Xylariaceae, along with representatives from Hypocreales as outgroup. The sequences generated in this current study are indicated in bold. Type strains or type specimens are labelled with HT (holotype), ET (epitype), LT

Species	Family	Strain no.	Status	GenBank accession no.				Reference
ITS	LSU	RPB2	TUB
Acrocordiella occulta	Requienellaceae	RS9	LT	KT949893	KT949893	n/a	n/a	Jaklitsch et al. (2016)
	Requienellaceae	RS10	—	KT949894	KT949894	n/a	n/a	Jaklitsch et al. (2016)
Acrocordiella omanensis	Requienellaceae	SQUCC 13852	PT	MG584569	MG584571	n/a	n/a	Maharachchikumbura et al. (2018)
	Requienellaceae	SQUCC 15091	HT	MG584568	MG584570	n/a	n/a	Maharachchikumbura et al. (2018)
Acrocordiella photiniicola	Requienellaceae	MFLU 17-1552	HT	MW240627	MW240556	MW658617	MW775583	Samarakoon et al. (2022)
	Requienellaceae	HKAS 102287	—	MW240628	MW240557	n/a	MW775584	Samarakoon et al. (2022)
Acrocordiella yunnanensis	Requienellaceae	HKAS 111922	HT	MW424507	MW424505	n/a	n/a	Dissanayake et al. 2021
	Requienellaceae	HKAS 111923	—	MW424497	MW424506	n/a	n/a	Dissanayake et al. 2021
Arecophila bambusae	Cainiaceae	HKUCC 4794	—	NA	AF452038	NA	NA	Kang et al. (1999)
Arecophila clypeata	Cainiaceae	GZUCC0110	HT	MT742129	MT742136	MT741732	n/a	Li et al. (2022)
Arecophila zhaotongensis	Cainiaceae	GMBCC1145	HT	OR995740	OR995747	OR995579	n/a	Han et al. (2024)
Arecophila xishuangbannaensis	Cainiaceae	GMB-W1283	HT	OR995736	OR995743	n/a	n/a	Han et al. (2024)
Arthrinium caricicola	Apiosporaceae	ALV16691	—	MK014871	MK014838	n/a	MK017977	Crous et al. 2020
	Apiosporaceae	CBS 145903	ET	MN313782	MN317266	n/a	MN313861	Crous et al. 2020
Barrmaelia rappazii	Barrmaeliaceae	Cr2 = CBS 142771	HT	MF488989	MF488989	MF488998	MF489017	Voglmayr et al. (2018)
Barrmaelia rhamnicola	Barrmaeliaceae	BR = CBS 142772	ET	MF488990	MF488990	MF488999	MF489018	Voglmayr et al. (2018)
Cainia desmazieri	Cainiaceae	CAI	—	KT949896	KT949896	n/a	n/a	Jaklitsch et al. (2016)
Cainia graminis	Cainiaceae	CBS 136.62	—	MH858123	AF431949	n/a	n/a	Vu et al. (2019)
Camillea obularia	Graphostromataceae	ATCC 28093	—	KY610384	KY610429	KY624238	KX271243	Wendt et al. (2018)
Camillea tinctor	Graphostromataceae	YMJ 363	—	JX507806	n/a	n/a	JX507795	Mirabolfathy et al. (2013)
Endocalyx cinctus	Cainiaceae	NBRC 31306	—	MZ313191	MZ313152	n/a	n/a	Delgado et al. (2022)
Graphostroma platystomum	Graphostromataceae	CBS 270.87	HT	JX658535	AY083827	KY624296	HG934108	Stadler et al. (2014)
Hypocrea gelatinosa	Hypocreales	NBRC 104900	ET	JN943358	JN941453	n/a	n/a	Schoch et al. (2012)
Hypoxylon fragiforme	Hypoxylaceae	MUCL51264	ET	KM186294	KM186295	KM186296	KX271282	Daranagama et al. (2015)
Hypoxylon fuscum	Hypoxylaceae	α22-093	—	PV029874	n/a	n/a	n/a	This study
	Hypoxylaceae	CBS 113049	HT	NR172215	n/a	n/a	n/a	Jaklitsch et al. (2016)
Hypoxylon investiens	Hypoxylaceae	CBS 118185	—	KC968924	KY610451	KY624260	KC977269	Wendt et al. (2018)
Hypoxylon rubiginosum	Hypoxylaceae	α23-050	—	PV029870	n/a	n/a	n/a	This study
	Hypoxylaceae	MUCL 52887	HT	NR155152	NG059785	n/a	n/a	Wendt et al. (2018)
Jackrogersella multiformis	Hypoxylaceae	CBS 119016	ET	KC477234	KT281893	KY624290	KX271262	Wendt et al. (2018)
Kretzschmaria deusta	Xylariaceae	CBS 163.93	—	KC477237	KY610458	KY624227	KX271251	Stadler et al. (2014)
Longiappendispora chromolaenae	Cainiaceae	MFLUCC 17-1485	HT	NR169723	NG068714	n/a	n/a	Mapook et al. (2020)
Lopadostoma turgidum	Lopadostomataceae	LT2	ET	KC774618	KC774618	KC774563	MF489024	Jaklitsch et al. 2014
Lopadostoma dryophilum	Lopadostomataceae	LG21	ET	KC774570	KC774570	KC774526	MF489023	Jaklitsch et al. 2014
Nectria cinnabarina	Hypocreales	CBS 125165	—	n/a	HM484562	n/a	n/a	Hirooka et al. (2011)
Obolarina dryophila	Graphostromataceae	MUCL 49882	—	GQ428316	GQ428316	KY624284	GQ428322	Pažoutová et al. (2010)
Paramphibambusa bambusicola	Cainiaceae	GMBCC1142	HT	OR995739	OR995746	OR995578	n/a	Han et al. (2024)
Poronia pileiformis	Xylariaceae	WSP 88113001	ET	GU324760	n/a	GQ853037	GQ502720	Hsieh et al. (2010)
Poronia punctata	Xylariaceae	CBS 656.78	HT	KT281904	KY610496	KY624278	KX271281	Wendt et al. (2018)
Requienella fraxini	Requienellaceae	RS2	—	KT949909	KT949909	n/a	n/a	Jaklitsch et al. (2016)
	Requienellaceae	RS3	HT	KT949910	KT949910	n/a	n/a	Jaklitsch et al. (2016)
	Requienellaceae	RS7	—	KT949911	KT949911	n/a	n/a	Jaklitsch et al. (2016)
Requienella populi	Requienellaceae	α23-061	PT	PV029871	n/a	n/a	n/a	This study
	Requienellaceae	α23-076	HT	PV029872	PV029875	n/a	n/a	This study
	Requienellaceae	α23-076a	—	PV029873	PV029876	n/a	n/a	This study
Requienella seminuda	Requienellaceae	RS12	—	KT949912	KT949912	n/a	n/a	Jaklitsch et al. (2016)
	Requienellaceae	RS13	—	KT949913	KT949913	n/a	n/a	Jaklitsch et al. (2016)
Requienella shangrilana	Requienellaceae	HKAS 130302	HT	PP584755	PP584828	n/a	n/a	Dissanayake et al. (2024)
	Requienellaceae	HKAS 130303	—	PP584756	PP584829	n/a	n/a	Dissanayake et al. (2024)
Seynesia erumpens	Cainiaceae	SMH 1291	—	n/a	AF279410	AY641073	n/a	Bhattacharya et al. (2000)
Stromatonectria caraganae	Hypocreales	CBS 125579	—	n/a	HQ112288	n/a	n/a	Jaklitsch & Voglmayr (2010)
Xylaria hypoxylon	Xylariaceae	CBS 122620	ET	KY610407	KY610495	KY624231	KX271279	Wendt et al. (2018)

To examine topological incongruence among data sets, ML bootstrapping analyses were carried out on each of the single-gene data sets. Topological incongruence was assumed if conflicting tree topologies were supported by ≥ 70 % ML support. Since topological incongruence could not be observed, maximum likelihood (ML) bootstrapping analyses were carried out on the concatenated four-locus dataset using the same settings as for the single-gene analyses. The BI analyses were performed with MrBayes v. 3.2.6 (Huelsenbeck & Ronquist 2001) with substitution models for different regions selected with the AICc parameter. Metropolis-coupled Markov chain Monte Carlo (MCMC) runs were performed for 1 M generations with trees sampled every 200 generations. Convergence of the MCMC procedure was assessed and effective sample (EES) size scores > 200 checked by using the MrBayes build in Tracer v. 1.6 (Rambaut et al. 2018). The first 10 % of trees were discarded as burn-in, and the remaining trees were used to calculate 50 % majority rule trees and to determine Bayesian posterior probabilities (BPP) for individual branches. Output trees were edited with Inkscape v. 1.4 (Harrington et al. 2003).

RESULTS

Phylogenetic analyses

We obtained consensus sequences from three strains for the ITS and two strains for the LSU markers (Table 1). In addition, we added two strains of the ITS markers from identified species of the Hypoxylaceae to the dataset. The concatenated alignment for Apiosporaceae, Barrmaeliaceae, Cainiaceae, Graphostromataceae, Hypoxylaceae, Lopadostomaceae, Requienellaceae and Xylariaceae comprised 4 372 nucleotide characters, including gaps (ITS1, 5.8S and ITS2: 1–740; LSU: 741–1 595; RPB2: 1 596–2 684; TUB: 2 658–4 372). In total, the alignment was composed of 48 strains of the ITS, 46 strains of LSU, 19 of RPB2, 19 TUB and the following outgroup taxa: Hypocrea gelatinosum (NBRC 104900), Nectria cinnabarina (CBS 125165), and Stromatonectria caraganae (CBS 125579).

The maximum likelihood (ML) analysis of the combined datasets yielding the best scoring trees for Xylariales had an MLn of –37393.72 (Fig. 1). The Bayesian inference (BI) analysis showed congruence with the topology of the ML analyses, and for simplicity, only the ML trees are shown. Values for both ML bootstrap (MLB) above 50 % and Bayesian posterior probabilities (BPP) higher than 0.90 are given at the nodes. The alignments had 60.93 % undetermined nucleotide gaps.

Fig. 1.

Phylogeny of representative of Xylariales based on ITS, LSU, RPB2 and TUB combined sequence data revealed by RAxML (MLn = - 37393.72). Numbers above branches indicate Maximum likelihood RAxML bootstrap values above 50 % and Bayesian posterior probabilities higher than 0.90 at first and second position, respectively. Newly obtained strains are shown in bold. Shortened branches are marked with crossing lines and indications (×2) of how many times the branch has been shortened. Branch length equals substitutions per site.

Representatives from the order Hypocreales formed a fully supported clade (ML 100 % and BPP 1) and outgroup to all included taxa from Xylariales, but the reconstruction of Xylariales is not entirely settled as the analysed dataset inferred less significant support for some families and genera. Requienellaceae and Cainiaceae received high support (ML 92 % and BPP 1) as sister group to Graphostromataceae, Barrmaeliaceae and Xylariaceae, but their sister group relationships were not significantly supported (ML 43 % and BPP 0.7, data not shown). Our analyses showed support for our new species represented by three strains, namely α23-061, α23-076 and α23-076a respectively, within Requienellaceae (see Fig. 1). The topology within Requienella is highly supported and monophyletic.

Morphological character matrix

Detailed morphological characters of the species of Requienellaceae are shown in Table 2.

Table 2.

Character matrix for species of Requienella.

Species	Ascomata			Papilla	Peridium	Asci	Ascospores			Host	Distribution
Ascoma position	Neck above host surface	Size	Form and size	Size	Size	Disto-septation	Form
Requienella fraxini	Erumpent	0.2–0.9 mm	0.45–1 mm diam, 0.5–1.1 mm high	Conical, more or less acute, shiny black, apically 26–160 μm wide papilla	15–30 μm wide at the base, thickened to 150 μm and hard in upper regions	153–206 × 20–30(–33) μm, oblong to narrowly clavate	(23.3–)26.7–31.5(–36) × (8.0–)9.5–12(–14.5) μm	3(–5)	Ellipsoid, oblong to fusiform, yellow, finally brown withpaler ends	Fraxinus excelsior	Europe
Requienella populi	Erumpent	0.1–0.3 mm	0.55–1 mm diam, 0.6–1.1 mm high	Conical with rounded base or more or less globose with a prominent, conical, more or less acute, shiny black, apically 25–150 μm wide papilla	(22.5–)21.6–32.5(–36.9) μm diam wide at the base, thickened to (49.2–)56.6–94.3(–99) μm and hard in upper regions	(139–)143.8–160(–175.2) × (17–)20.4–25.9(–27.4) μm, oblong to narrowly clavate, with thick-walled apex	(21–)25.1–31.4(–33.5) × ((9–)10.1–12.4(–13.6) μm (n = 50)	Submuriform, 5 transverse and 1–4 longitudinal	Ellipsoid, oblong to fusiform, brown with paler ends	Populus tremula	Norway (Oceanic?)
Requienella seminuda	Erumpent	0.2–0.4 mm	(0.35–)0.45–0.9(–1.1) mm diam, 0.6–1.2 mm high	Papillate to conical, shiny black, apex typically 25–160 µm wide, round, blunt to pointed, sometimes flattened and 90–300 µm diam	15–40 µm wide at the base, thickened to 160 µm in upper regions	(148–)158–178(–182) × (21.7–)23.5–29(–32.5) μm, oblong to subfusiform	(25.3–)28.3–32.7(–37) × (9.8–)10.8–12.7(–13.3) μm	(3–)5(–7)	Ellipsoid, inequilateral, brown	Olea europae	Europe (mediterranean)
Requienella shangrilana	Immersed	0 mm	0.48–0.62 mm diam, 0.4–0.52 mm high	N/A	25–40 µm wide, thickened in upper regions	100–160 × 10–18 μm, cyllindrical, unitunicate	20–30 × 9–12 μm	3(–4)	Ellipsoid, inequilaternal, narrowly rounded to nearly acute at the ends, at first hyaline, greyish when young, olivaceous to medium brown when mature	Unknown	China

Taxonomy

Requienellaceae Boise, Mycologia 78: 37. 1986. MycoBank MB 81336.

Type genus: Requienella Fabre, Ann. Sci. Nat., Bot. ser. 6, 15: 55. 1883. MycoBank MB 4676.

Ascomata perithecioid, immersed or erumpent, subglobose; ostiolar neck inconspicuous or massive and strongly erumpent, black; extra ascomatal tissue present. Hamathecium comprising two types of apically free paraphyses differing in length and width. Asci bitunicate, fissitunicate, cylindrical, subfusiform to narrowly clavate, with thick-walled apex and wide ocular chamber comprising a slightly refractive, inversely funnel-shaped dome turning slightly reddish in Congo Red, containing 8 uni- to biseriately arranged ascospores. Ascospores ellipsoid to oblong, olivaceous or brown, sometimes with paler ends, with several transverse distosepta and large lumina, sometimes with additional thin longitudinal septa.

Requienella Fabre, Ann. Sci. Nat., Bot., sér. 6, 15: 55. 1883.

Synonym: Trematomyces Schrantz, Bull. Soc. Mycol. France 76: 324. 1961.

Type species: Requienella seminuda (Pers.) Boise.

Requienella populi Andreasen & Nordén, sp. nov. MycoBank MB 857429. Fig. 2.

Fig. 2.

Requienella populi. A–H, J, M–T, V, Y, X. TRH-F-25081 - α23-076 - holotype. I, K, L, U, V. TRH-F-14022 - α23-061 - paratype. A–C. Ascomata in face and lateral views. D, E. Ascomata in vertical section. F. Hymenium and ascomata in vertical section. G. Peridium of basal type (upper) and lateral type (lower) overlapping. H, I. Asci with paraphyses. J, K. Asci. L, M. Ascus apices (in Cotton Blue). N. Young asci with paraphyses and free apical ends of paraphyses. O–R. Immature ascospores (O–Q in water; R in 5 % KOH). S–V, X. Ascospores (S–V in water; X in 5 % KOH). Scale bars: A = 0.6 mm; B = 0.3 mm; C, F = 0.2 mm; D, E = 0.5 mm; G = 20 µm; H, I = 30 µm; J, K, N = 50 µm; L, M, O–V, X = 10 µm.

Etymology: With reference to the host species Populus tremula.

Ascomata immersed, with upper part erumpent 0.1–0.3 mm (n = 15) above the bark surface, solitary or aggregated in small numbers, 0.6–1.1 mm high, 0.55–1.0 mm diam (n = 15), conical with rounded base or more or less globose with a prominent, conical, more or less acute, shiny black, apically 25–150 μm (n = 15) wide papilla, circular in transverse section, black; often surrounded by white amorphous tissue. Peridium (21.5–)22.6–32.5(–36.9) μm diam (n = 8) wide at the base, thickened to (49.2–)56.6–94.3(–99.0) μm (n = 8) and hard in upper regions, dark brown, consisting of 1 × 2–7 μm rectangular pseudoparenchymatous cells. Hamathecium consisting of three parts i) 2–4 μm wide, apically free paraphyses containing oil drops when vital, and similarly long as the asci, ii) sparsely branched, 1.5–3 μm wide, apically free ‘pseudotrabeculae’ nearly reaching the ostiolum, iii) variously curved periphyses within the ostiolar canal, all immersed in a gel matrix. Asci (139.0–)143.8–160.0(–175.2) × (17.0–)20.4–25.9(–27.4) μm (n = 15) bitunicate, fissitunicate, oblong to narrowly clavate, with thick-walled apex, wide ocular chamber comprising a slightly refractive, inversely funnel-shaped dome, 8–10 μm long, 5–9 μm wide at the base, turning blue in Cotton Blue and reddish in Congo Red, demarcated by a basal plate, with short simple stipe, containing 8 uni- to biseriately arranged ascospores. Ascospores (21.0–)25.1–31.4(–33.5) × (9.0–)10.1–12.4(–13.6) μm (n = 50), ellipsoid, oblong to fusiform, submuriform, first hyaline, 1-celled, with narrow sheath, becoming septate and yellow, finally brown with paler ends, with 5 thick distosepta, lenticular lumina and faintly punctate perispore, often with 1–4 thin longitudinal distosepta, sometimes two in the same compartment/segment, turning darker olivaceous in KOH and lumina becoming smaller, more angular and longitudinal distosepta more evident.

Typus: Norway, Møre og Romsdal, Molde, Prestaksla nature reserve, on coarse bark of living Populus tremula in mixed forest (Pinus sylvestris, Betula pubescens, Corylus avellana, Salix caprea) with low herb vegetation, 21 Sep. 2022, J.B. Jordal, α23-076 (holotype TRH-F-25081; culture lost, JB22-66); 23 Jun. 2024, J.B. Jordal, P.G. Larsen & S. Vatne, topotype TRH-F-14021 (JB24-P2); Møre og Romsdal, Jordalsgrenda, on coarse bark of aspen in mixed forest, 21 Sep. 2022, J.B. Jordal, paratype TRH-F-14022 (α23-061 - culture lost).

Additional materials examined: Norway, Møre og Romsdal, Molde, Rislia, On bark of living Populus tremula in weak low herb forest, 27 Jun. 2022, M. Norby Lorentzen & J.B. Jordal, TRH-F-25063; Vestland (Sogn og Fjordane), Lærdal, Hausen, on bark of living Populus tremula, 22 Aug. 2022, M. Norby Lorentzen, TRH-F-25067 (MNL202201); Møre og Romsdal, Høystakklia, on coarse bark of living Populus tremula, 12 Oct. 2020, J.B. Jordal, TRH-F-14023 (JB20-P73); Møre og Romsdal, Molde, Prestaksla nature reserve, on bark of living Populus tremula in mixed forest (Pinus sylvestris, Betula pubescens, Corylus avellana, Salix caprea), 23 Jun. 2024, J.B. Jordal & P.G. Larsen & S. Vatne, TRH-F-14024 (JB24-P1); Møre og Romsdal, Molde, Prestaksla nature reserve, on bark of living Populus tremula in mixed forest (Pinus sylvestris, Betula pubescens, Corylus avellana, Salix caprea), 23 Jun. 2024, J.B. Jordal & P.G. Larsen & S. Vatne, TRH-F-14025 (JB24-P3); Møre og Romsdal, Molde, Tjellefonna west, on coarse bark of old living Populus tremula in deciduous forest, 1 Jul. 2024, J.B. Jordal, TRH-F-14026 (JB24-P4).

Culture characteristics: Ascospores germinated on MEA within 72 h. Growth of cultures reaching 0.5–0.7 cm diam after 4 wk at 20 °C, subcircular, with irregular margins, white, turning slightly yellow, reverse brown. No asexual morph observed.

Ecology: On coarse bark of living trunks of old trees of Populus tremula.

Distribution: Requienella populi was so far found only in the oceanic parts of Western Norway.

Notes: The presence of 1–4 longitudinal distosepta alongside molecular data and host relations clearly separate R. populi from R. seminuda and R. fraxini. The conical papillae are less markedly protruding than in R. fraxini. see also Table 2.

DISCUSSION

The ecology of the three known European species of Requienella seem to be defined by host relations. However, the host relations of R. shangrilana remains to be studied. Our new species on Populus tremula (Salicaceae) formed a highly supported sister clade relative to the other two European species of Requienella, occurring on Olea europaea and Fraxinus excelsior (Oleaceae), respectively. The association of these species to living hosts may indicate that they have co-evolved with their hosts. They belong to a little-known but species-rich community of Ascomycota with unknown nutritional modes, apparently not causing harm to the living tree and possibly being commensals or endophytic symbionts (Bowd et al. in press). Other species in this community on rough bark of old aspen trees include Amphisphaerella dispersella, Caesiodiscus populicola, Lasiobelonium corticale, Melaspilea bagliettoana and Caliciopsis calicioides. Pictures of the Populus habitat can be seen in Jordal et al. (2014).

The finding of the new species R. populi and several others during recent years illustrates that much is still unknown about the funga of Northern Europe. One area that appears to be particularly promising for future exploration is Western Norway with its oceanic forests. For instance, R. populi sometimes occurs with Crassistoma norvegicum, another newly described species on aspen in western Norway (Voglmayr et al. 2024). In Norway, the geographical distribution of R. populi overlaps with that of R. fraxini, which has a broad distribution in northern and southern Europe, but not with R. seminuda, which occurs in the mediterranean region. Requienella populi was only collected in the Western, oceanic parts of Norway. It was not systematically searched for in other areas. However, as we have not previously encountered it during various field surveys, we suspect that it may in fact have an oceanic distribution.

Our phylogenetic analyses showed relatively low support for the Requienellaceae as sister group to the Cainiaceae with ML 43 % and BPP 0.7 support. The addition of several strains to the dataset from newly published species of Acrocordiella and Arecophila resulted in reduced support. Most of these strains are represented only by ITS and LSU genetic markers and we would expect that the addition of further genetic markers for more taxa within the families of Requienellaceae and Cainiaceae would provide a more stable topology.