Bryorutstroemia (Rutstroemiaceae, Helotiales), a New Genus to Accommodate the Neglected Sclerotiniaceous Bryoparasitic Discomycete Helotium fulvum

The new genus Bryorutstroemia is established for the red-brown, stipitate, bryoparasitic discomycete Helotium fulvum Boud. Combined phylogenetic analysis of ITS and LSU rDNA and EF1α revealed that Bryorutstroemia fulva belongs to the sclerotiniaceous clade, which comprises the paraphyletic families Rutstroemiaceae and Sclerotiniaceae. Bryorutstroemia formed with Clarireedia a supported clade (Rutstroemiaceae s.l.), though with high distance. Bryorutstroemia closely resembles other Rutstroemiaceae in having uninucleate ascospores with high lipid content and an ectal excipulum of textura porrecta, but is unique because of its bryophilous lifestyle and is extraordinary with its thick-walled inamyloid ascus apex. Although B. fulva was described in 1897, very few records came to our notice. The present study summarizes the known distribution of the species, including 25 personal collections from the years 2001–2022. Bryorutstroemia fulva was most often encountered on Dicranella heteromalla, and rarely on other members of Dicranales or Grimmiales, while inducing necrobiosis of the leaves. A detailed description based on mainly fresh apothecia is provided together with a rich photographic documentation. Six new combinations are proposed based on our phylogenetic results and unpublished personal morphological studies: Clarireedia asphodeli, C. calopus, C. gladioli, C. henningsiana, C. maritima, and C. narcissi.


Introduction
Helotium fulvum was described by Boudier in 1897 based on his collection from Forêt de Carnelle north of Paris (France) [1]. The apothecia grew on sandy soil among Phascum, Dicranella, and other small mosses. They consistently arose from leaf axils (leaf bases) at the tip of stems of what was obviously a Dicranella. Reliable reports of the species in the literature are very sparse up to now and include collections from Great Britain [2] and Belgium [3]. During an ascomycete foray in Luxembourg in April 2001 [4], the first author collected and documented a single apothecium on Dicranella, which remained undetermined for many years. Because of its very short stipe, large, ellipsoid, multiguttulate ascospores, and large, inamyloid asci, an affinity with the genus Mniaecia Boud. was considered, despite its reddish-brown colour. Further fresh collections from Sweden (Småland), France (Bretagne), Germany (Sachsen), Czech Republic (regions of Ústí nad Labem, Liberec, Hradec Králové, Vysočina, Olomouc, Moravian-Silesian and Zlín), Poland (Silesia), and Hungary (near Budapest) all deviated from our first record in possessing comparatively long stipes. Apart from the brown, stipitate apothecia, a ± gelatinized ectal excipulum of textura (prismatica-)porrecta with ochre-brown, partially encrusted cortical

DNA Extraction, PCR Amplification and Sequencing
DNA was extracted from dried apothecia using the CTAB method described by Doyle et Doyle [7]. Apothecia were homogenised using a pestle and incubated in 300 µL of extraction buffer at 65 • C for one hour. The extract was subsequently purified in chloroform-isoamyl alcohol mixture (24:1), precipitated by isopropanol, washed in 70% ethanol, dried and finally dissolved in water and incubated with RNase for 30 min at 37 • C. DNA quality was checked using agarose gel electrophoresis. Three genomic regions including the internal transcribed spacers (ITS = ITS1-5.8S-ITS2 region) and the 28S subunit (LSU) of ribosomal DNA (rDNA) plus the translation elongation factor-1alpha (EF1α) were amplified and sequenced with the primers ITS1F [8] / ITS4 [9], LR0R/LR6 [10], and EF1-983F/EF1-1567R [11], respectively. PCR was performed with EliZyme FAST Taq MIX Red (Elisabeth Pharmacon, Brno, Czech Republic) following a standard protocol with 37 cycles and annealing temperature of 54 • C. The PCR products were purified by precipitation with polyethylene glycol (10% PEG 6000 and 1.25 M NaCl in the precipitation mixture) and sequenced from both directions using the same primer pairs by the Sanger method at Macrogen Europe, Amsterdam, the Netherlands.

Phylogeny
ITS sequences were obtained from five collections of B. fulva, while LSU and EF1α were obtained from four ( Table 1). The S1506-intron is absent in all of them, according to the used ITS1F primer. The five sequences are fully identical in the overlapping parts. In BLASTn searches in GenBank, B. fulva had the highest ITS similarity to members of the Clarireedia clade: Clarireedia narcissi (90%), C. monteithiana and C. jacksonii (89.5%), C. asphodeli, C. calopus, C. henningsiana, C. homoeocarpa, C. maritima, and C. paspali (88-89%). Also in the LSU D1-D2 domain the highest similarity (95%) was to members of Clarireedia but also to Piceomphale, followed by other rutstroemiaceous taxa, including Rutstroemia firma (92.5-93.5%).
Helotium fulvum was only once recombined into another genus when Hengstmengel [19] suggested a relationship with the genus Hymenoscyphus. Our phylogenetic analysis of nuITS+LSU rDNA + EF1α (Figure 9 and Figure S4), in which we used Hymenoscyphus scutula (Pers.) W. Phillips (isolate G.M. 2014-12-25.2, ITS + LSU: MK674606) as outgroup, indicated a high distance between B. fulva and that species. Instead, B. fulva nested in the strongly supported sclerotiniaceous lineage as circumscribed by Baral [20] p. 173, a group which currently includes two families, Rutstroemiaceae and Sclerotiniaceae. Two further families in our dataset, Cenangiaceae and Chlorociboriaceae, clustered outside the sclerotiniaceous lineage.
In our Bayesian analysis, the paraphyletic family Rutstroemiaceae appears in three different clades (Figures 9 and 10). One clade (Rutstroemiaceae s.str.) comprises species growing on wood and bark but also on the leaves of trees; it includes two strongly supported subclades, one containing the type species of Rutstroemia, R. firma, and four other Rutstroemia spp., but also Torrendiella setulata, the other containing Lambertella subrenispora and Lanzia allantospora.
A different, strongly supported clade comprises species growing on monocots and also on dung. It represents the recently established genus Clarireedia L.A. Beirn et al. [21], with the type species C. homoeocarpa (F.T. Benn.) L.A. Beirn et al. (≡ Sclerotinia homoeocarpa F.T. Benn.), and includes species currently assigned to Rutstroemia but also Ciboria, Sclerotinia, and Stromatinia. Within Clarireedia, C. paspali clustered in our ITS+LSU analysis closest to B. fulva despite its comparatively high ITS distance (Figure 10), perhaps because the specimen lacks LSU, whereas in our ITS+LSU+EF1α analysis it clustered supported with other Clarireedia spp. (Figure 9).
The following new combinations are proposed to harmonize the nomenclature of species on monocots which cluster in the supported Clarireedia clade. The listed taxonomic synonyms are to be taken as tentative and require type studies for clarification. C. henningsiana (= R. paludosa) is here understood as a species on Cyperaceae and Juncaceae characterized by simple-septate asci, whereas C. calopus (= C. bennettii) and C. maritima represent species on Poaceae characterized by asci arising from croziers, C. maritima also by asci with inamyloid, moderately thick-walled apex (pers. obs.). We tentatively regarded R. cuniculi as a synonym of C. calopus because available ITS sequences in GenBank differed from those of C. calopus by only one nucleotide.
Clarireedia asphodeli (Duvernoy and Maire) Baral  A third clade is formed by the type species of Lambertella, L. corni-maris, and two more Lambertella spp., but also includes Bicornispora seditiosa, and with less support Rutstroemia longipes and Martininia panamaensis. A further, strongly supported clade represents the family Sclerotiniaceae, which includes in the present analysis members of Ciboria, Dumontinia, Monilinia, Pycnopeziza, Schroeteria, Sclerencoelia, and Sclerotinia, with partly high distances among the species.
Four species of the sclerotiniaceous lineage clustered outside the four above-mentioned clades (Figures 9 and 10): Bryorutstroemia fulva formed with Clarireedia a strongly supported clade, though with high distance. Scleromitrula shiraiana is morphologically similar to Ciboria but it clustered unsupported in Figure 9 but formed a moderately supported sister clade to Rutstroemia s.str. in Figure 10. As in other published analyses [22], Piceomphale bulgarioiodes clustered with "Cenangium" acuum distant from all other sclerotiniaceous taxa, despite its morphological similarity with Ciboria and an ascus structure of the Sclerotinia-type. The two species form the "Piceomphale-clade", which is difficult to assign to a family, but may be better recognized in the sclerotiniaceous lineage than in Cenangiaceae to which Encoelia furfuracea belongs [22].
A phylogenetic tree generated with MEGA6 (ML, GTR+G+I, 1000 replicates, Figure S4), based on the very same dataset as in Figure 9, gave a similar tree topology though with only weak support for Rutstroemia s.str. and moderate support for Lambertella s.str. Again, B. fulva clustered sister to Clarireedia, though with only moderate support and by forming with C. paspali an unsupported clade. Contrary to the Bayesian analysis, Martininia panamaensis clustered strongly supported with Lambertella in the ML ITS+LSU analysis of Baral et al. [23] but unresolved in Figure S4, and Scleromitrula shiraiana clustered unresolved in both Baral et al. [23] and in Figure S4.

Morphological Remarks
Bryorutstroemia fulva is characterized by deep reddish-brown, stipitate or rarely subsessile apothecia, a gelatinized ectal excipulum of textura porrecta covered by ochre-brown cortical hyphae with short outgrowths, inamyloid asci arising from simple septa, and large, multiguttulate, ellipsoid ascospores. Especially the latter varied among the collections, particularly in width, some being predominantly narrowly ellipsoid, the others more broadly ellipsoid. The living paraphyses usually looked empty and colourless by lacking vacuolar bodies (VBs), but sometimes they contained groups of lipid bodies (LBs). The pale to bright ochre-brown cortical hyphae of the receptacle and stipe often had an encrusted surface but were occasionally smooth.
In order to summarize the most important differences between Bryorutstroemia and related genera, the following key is provided. It needs to be taken as provisional, as the taxonomy of Rutstroemiaceae is still insufficiently solved and nomenclatural changes in the circumscription of the family and its members can be expected.
Provisional key to the recognized genera of Rutstroemiaceae s.l.
The type clade of Clarireedia homoeocarpa is closely related to R. maritima (Roberge ex Desm.) Dennis and R. asphodeli (Duvernoy and Maire) R. Galán and Matočec, here classified as Clarireedia maritima and C. asphodeli, whereas the remaining three species (Clarireedia jacksonii C. Salgado et al., C. monteithiana C. Salgado et al., C. paspali) represent a distinct group of genotypes which includes strains that are misnamed as Sclerotinia homoeocarpa in GenBank (based on our ML analysis of ITS rDNA, not shown).
Delimitation of the families Sclerotiniaceae and Rutstroemiaceae within the sclerotiniaceous lineage is still not clear in all respects. In the morphology-based classification defined by ascospores with a low vs. high lipid content coupled with globose vs. prismatic excipular cells, respectively, both families are paraphyletic (Figures 9 and 10). Hereafter, Scleromitrula, Martininia, and Piceomphale share characters with the core clade of Sclerotiniaceae, while Lambertella and Clarireedia share characters with the core clade of Rutstroemiaceae. Additionally, Bryorutstroemia shares characters with Rutstroemiaceae, for which it could represent an ancestor on a phylogenetically old host, although the tree topology of Figure 9 suggests an evolution from mainly woody plants to monocots and mosses. The current concept that characterizes Rutstroemiaceae by a stroma and Sclerotiniaceae by sclerotia [25] largely coincides with the morphology-based concept, but both concepts include some problematic genera.
The difficulty of conducting phylogenetic analysis on sclerotiniaceous fungi based on rDNA data alone became obvious when trying to resolve the position of Schroeteria [23]. Multigene analyses probably better resolve phylogenetic affinities in this group. However, in a preliminary analysis of the EF1α gene with MEGA6 (TN+G, not shown), which comprised members of Helotiales (mainly sclerotiniaceous taxa), Pezizales, Phacidiales, Rhytismatales, Dothideomycetes, Eurotiomycetes, and Sordariomycetes, B. fulva clustered with Sordariomycetes, though with a high distance. B. fulva formed a clade with Clarireedia only when non-helotialean sequences were excluded from the analysis ( Figure S3). Despite this curious result, BLAST search (megablast) for EF1α (strain Z.S. 19/2021) yielded Rutstroemia firma as the second most similar species (85.3%, query cover 61%), with the highest similarity of 88% (query cover 51%) to Spathularia (Rhytismatales) and 83.5-83.6% (query cover 68%) to Sordaria (Sordariomycetes) and Lasiobolidium (Pezizales). BLASTn search, however, yielded R. firma on top with 84.4% similarity (80% query cover). The EF1α sequences obtained from B. fulva strongly deviate at various positions from any other group of Ascomycota, which impedes a reasonable conclusion about its phylogenetic relationships. EF1α sequences obtained from four collections of B. fulva in this study were about the same length of 500 nucleotides and fully identical (except for nine ambiguities in C.N. 103), thus confirming the reliability of the result.
We have encountered B. fulva mainly during the colder season, i.e., from November to May, but three of our records were from July. Collections from August by J. Poelt [29], September by A. Yelland [17], and October by D.M. Henderson [29] also exist. Although only a few records have been published, B. fulva seems to be common in colline to montane regions with acidic bedrock, which was exemplified in the present study for Czechia (24 collections during 2021-2022). The presently known distribution ( Figure 11) is certainly incomplete. However, as the most frequent host D. heteromalla prefers acidic pH and grows most often on acidic forest soil or less often on sandy soil or directly on silicate boulders [30], the fungus might be rarer in areas with neutral to basic soil.
In France (Bretagne), the Netherlands, Luxembourg, Germany, Poland (Silesia), and Czechia the host was always Dicranella, which mainly grew on acidic sandstone (Figure 2: 1a), but also on sandy or loamy soil over sandstone, slate (Ordovician shale), silt, orthogneiss, migmatite, or granulite, etc. Sometimes the moss grew on soil on an uprooted fallen tree. The vegetation was preferably an acidic pure coniferous forest (predominantly Picea but also Pinus), also mixed with Betula or Fagus, etc. In Divadlo, the main vegetation was a Vaccinio myrtilli-Pinetum sylvestris, in Stohánek (Figure 3: 1) a Vaccinio vitis-idaeae-Quercetum with Pinus sylvestris and Quercus petraea, less often Q. robur, with admixture of Betula pendula, Sorbus aucuparia, and Frangula alnus, but also Dicrano-Pinion with the dominant P. sylvestris admixed with Quercus petraea, Betula pendula, Frangula alnus or Sorbus aria. Collections were often from the margins of forest pathways and also in ditches at the edges of roads. At the French sites the host moss occurred in close association with Diplophyllum albicans, Calypogeia, and Cephalozia, etc. Especially when growing on rock, the plant community in which Bryorutstroemia fulva parasitizes Dicranella may be classified as Dicranellion heteromallae [31]. In Sweden B. fulva grew on Bucklandiella (Figure 8: 2a) or Dicranum covering silicate stonewalls, and at the Hungarian site it grew in cushions of D. scoparium occurring scattered on open soil in an acidophilous Quercus petraea forest (Figure 8: 1a). admixture of Betula pendula, Sorbus aucuparia, and Frangula alnus, but also Dicrano-Pinion with the dominant P. sylvestris admixed with Quercus petraea, Betula pendula, Frangula alnus or Sorbus aria. Collections were often from the margins of forest pathways and also in ditches at the edges of roads. At the French sites the host moss occurred in close association with Diplophyllum albicans, Calypogeia, and Cephalozia, etc. Especially when growing on rock, the plant community in which Bryorutstroemia fulva parasitizes Dicranella may be classified as Dicranellion heteromallae [31]. In Sweden B. fulva grew on Bucklandiella (Figure 8: 2a) or Dicranum covering silicate stonewalls, and at the Hungarian site it grew in cushions of D. scoparium occurring scattered on open soil in an acidophilous Quercus petraea forest (Figure 8: 1a).
British records of H. fulvum from leaf axils of Dicranella heteromalla were figured by Dennis [2] (as 'D. heteromera') and Ellis et Ellis [32], but no collection data were given (see Figure 1b,c). The database of the British Mycological Society [17] indicates two collections, one from Gloucestershire made in 1991 and one without data. Dennis mentioned the negative ascus iodine reaction and considered an affinity with Rutstroemia, but also referred to a "small group of similar species parasitic on bryophytes, for which a separate genus may perhaps be needed" (Dennis probably meant the later erected genus Bryoscyphus Spooner). The almost identical measurements by Dennis and Ellis et Ellis (l.c., asci 150-180 × 13-16 µm, ascospores 16-21 × 6-9 µm) concur well with the present data. The ascospores were illustrated with two large and some smaller LBs, probably because the material was studied in a rehydrated state.   De Meulder [3] described and illustrated a personal collection of H. fulvum on Dicranella cerviculata collected in 1992 in Belgium (Figure 1d). Ten days after this collection was made, the first author received a part of the dried specimen from the collector. Despite the short time span, no living elements could be found. The obtained measurements differed from De Meulder's data by much narrower paraphyses ( †2-2.2 vs. †3-4 µm), slightly shorter asci ( †130-158 × 14-18 vs. †137.5-175 × 12.5-18 µm), and distinctly narrower ascospores ( †15-20 × 6-7.5 vs. *15.5-22.75 × 7.5-8.7 µm). De Meulder might have studied a rehydrated apothecium with still living spores, judging from the larger size, and also from the included partly large LBs which were likely formed by the fusion of smaller ones during rehydration. Paraphysis width is hardly over 1.5 µm when evaluated from De Meulder's drawing, hence his given width of 3-4 µm should be an error or a mere copy of Boudier's data, whereas values around †1.5-2.5 µm would have been closer to what was here observed in the other collections.
Bryorutstroemia fulva may be confused with Bryoscyphus dicrani because of a similar ascospore size and shape and inamyloid asci. However, confusion is only possible when comparing herbarium specimens in which B. dicrani may attain a reddish-brownish colour due to secondary pigmentation of the multiguttulate contents of paraphyses and excipular cells. In the living state, B. dicrani has white apothecia, binucleate ascospores with a lower lipid content (OCI 2-3), and multiguttulate paraphyses and cortical excipular cells due to strongly refractive vacuolar bodies (VBs). A further difference lies in the asci which are also inamyloid but arise from croziers.
Hengstmengel [19] studied a collection on Brachythecium rutabulum from the Netherlands (Drenthe, Rolde, Deurzerbroek). We have seen no documentation of this collection, but we consider the possibility that it might be a misidentification, judging from the deviating host.

Other Bryicolous Species of the Sclerotiniaceous Lineage
Bryorutstroemia fulva is exceptional within the sclerotiniaceous lineage by its ecological restriction to acrocarpous mosses. Only a very small number of other bryicolous discomycetes with a clear affinity to the sclerotiniaceous lineage are known up to now. One of them is Sclerotinia atrostipitata Svrček from Czechia, which was described as emerging from a 2 mm large subglobose sclerotium among rhizoids of Ceratodon purpureus, with globose excipular cells, amyloid asci, and comparatively small, ellipsoid-ovoid, eguttulate ascospores [36]. Svrček's remark of an attachment of the sclerotium to the moss rhizoids might be an argument for a real connection to the moss, but interactions at the cellular level have not been assessed. The North American Sclerotinia incondita (Ellis) Sacc. mentioned by Svrček likewise grew among mosses, but its description which includes four-spored asci is too brief to permit any conclusion.