Studies on Argentine Phylacia species (Hypoxylaceae) using a polythetic taxonomic approach

The current study is dedicated to the taxonomy of the genus Phylacia (Hypoxylaceae) in Argentina. Fieldwork in the north of the country provided several fresh collections that were studied, using a polyphasic approach. The secondary metabolite profiles of the specimens were generated by high-performance liquid chromatography hyphenated by diode array and mass spectrometry (HPLC–DAD/MS) of the stromata. This study confirmed the presence of secondary metabolites that are also found in the related genus Daldinia. The detection of binapththalene tetrol (BNT), daldinal B, and daldinol, which are also characteristic of certain species of Daldinia and Hypoxylon, further confirmed the chemotaxonomic affinities within the Hypoxylaceae. The phylogenetic affinities of several species were determined using a multi-gene genealogy based on ITS, LSU, TUB2, and RPB2 sequences, confirming that Phylacia is most closely related to Daldinia, Rhopalostroma, and Thamnomyces. The new species P. lobulata, which features a rather unique stromatal morphology and seems to exhibit apparent host specificity for the endemic tree Pseudobombax argentinum, is described.


Introduction
Phylacia Lev. constitutes a small genus of the Hypoxylaceae (Xylariales), which currently includes 12 species (Wijayawardene et al. 2022), which have almost exclusively been reported from the Neotropics. The most recent overview on the taxonomy and biogeography of the genus by Medel et al. (2006) emphasized on material from Mexico. However, this paper did not evaluate type specimens of previously described taxa. Neither did Rodrigues and Samuels (1989), who were the first to describe the anamorphs. These authors classified the conidial stages of three species to be "geniculosporium-like" (sensu Petrini and Petrini 1985), but their Figs. 14 and 15 clearly show that this was a misinterpretation. The conidiogeneous cells shown do not bear any geniculate scars that result from dehiscence of conidia (cf. figure 1C in Petrini and Petrini 1985), but they should rather be classified as nodulisporium-like (sensu Petrini and Petrini 1985-cf. their fig. 1E-as well as in the anamorph classification established by Ju and Rogers 1996). For the taxonomic history of the genus, however, the paper by Rodrigues and Samuels (1989) constitutes a very valuable and complete source. Therefore, we refer to this publication for details and will here only discuss the characteristics of Phylacia in view of modern, polythetic concepts.
Phylacia spp. are characterized by elongated ellipsoid translucent yellow-brownish ascospores arranged in globose, evanescent asci that break up early in development and are arranged in carbonaceous, cleistothecial ascomata, which have no ostiolar canal. The asci lack an apical apparatus, and spores are not actively discharged but released after rupture Section Editor: Marco Thines of the ascomatal apex (Rodrigues and Samuels 1989). These characteristics are highly atypical of the Xylariales, but it has meanwhile been established that the characteristics of the teleomorph are not necessarily in agreement with the phylogeny of this order (Jaklitsch et al. 2016;Wendt et al. 2018;Voglmayr et al. 2022). These modern, polyphasic taxonomic studies showed that in the Xylariales and other groups of Sordariomycetes, the teleomorphic characters have subordinate importance. Anamorphic states as well as chemotaxonomic features often agree better with the molecular phylogeny than the morphology of asci and ascospores. For example, chemotaxonomic studies have revealed great similarities of Phylacia to the genera Daldinia, Rhopalostroma, and Thamnomyces (Bitzer et al. 2008;Stadler et al. 2004Stadler et al. , 2010. The stromatal pigments of Phylacia closely resemble those of Daldinia, Thamnomyces, and Rhopalostroma (Stadler et al. 2004). A comparative study using cultures of numerous representatives of the stromatic Xylariales revealed a close chemotaxonomic relationship between the aforementioned genera, as well as Entonaema and Ruwenzoria, which all produced small polyketides like 1,8-naphthol, eutypinol and chromone derivatives, while Hypoxylon species lacked these compounds and produce mellein and isosclerone derivatives instead (Bitzer et al. 2008). This was later corroborated in a polyphasic study by Wendt et al. (2018), where Phylacia was placed in the family Hypoxylaceae, but this phylogeny did not include sequences of Phylacia. Other previous molecular phylogenetic studies that included a few sequences of this genus were only based on the ITS locus. In this study, we wish to fill this gap by creating additional data on Phylacia spp. from Argentina using morphological, chemotaxonomic, and molecular phylogenetic characters.

General
All scientific names of fungi follow MycoBank (http:// www. mycob ank. org). No authorities or years of publication are given beside the taxonomic entries. Names of fungaria and culture collections are abbreviated as recommended in Index Herbariorum (http:// sweet gum. nybg. org/ scien ce/ ih). The chemotaxonomic studies of the stromata were carried out using the same methodology as reported recently by Cedeño-Sanchez et al. (2023).

Samples sources and morphological characterization
The fungal specimens surveyed in this study were collected in the subtropical montane forests of the Argentine Northwest. Microscopical and macroscopical morphology were examined and documented as described by Sir (2021). In addition, PVAlactophenol was used as a mounting medium to ascertain the presence or absence of the germ slit of the ascospores.
For examination of conidiophores, HPLC profiling and sequencing, cultures of the specimens were obtained from multispore isolates according to Kuhnert et al. (2017). The morphology of cultures was studied as described by Stadler et al. (2014), using phase contrastmicroscopy and differential interference contrast under × 400-1000 optical magnification. Colors of stromatal extracts and cultures were assigned according to Rayner (1970). Cultures designated STMA are stored at interim at the HZI Braunschweig under liquid nitrogen.

DNA extraction, PCR, and molecular phylogenetics
The EZ10 Spin Column Fungal Genomic DNA Mini Preps kit (Bio Basic INC.) was used to extract genomic DNA (gDNA) following the manufacturer's protocol. For extraction, either hyphal material was removed from a YMA plate with an inoculation loop and transferred to a reaction tube with a screw cap supplied with 5-10 precellys ceramic beads (Preqlab, Germany), or material was taken from a liquid culture containing 30 mL of YM medium, which was incubated at 140 rpm and 23 °C for 2-7 days in a 150-mL Erlenmeyer flask. The samples were homogenized, and the subsequent purification steps were carried out according to the manufacturer's protocol. Samples were stored at 4 °C until further use.
Sequences from a forward and a reverse read were processed using Geneious® 7.1.9 (Kearse et al. 2012). The electropherogram was checked for sequencing errors and trimmed manually. For TUB2 and RPB2 derived PCR sequences, sequencing was performed with four (T1, T2, T11, T22) and three different primers (fRPB25F, fRPB27cR, fRPB26F). The sequences were checked for authenticity using the NCBI (Sayers et al. 2022) BLAST® (Basic Local Alignment Search Tool; Altschul et al. 1990) program.
The MAFFT (Multiple Alignment with Fast Fourier Transform; v. 7.017) algorithm implemented in Geneious was used to align each locus separately (Katoh and Standley 2013). The L-INS-i algorithm with a 200PAM/k = 2 scoring matrix, a gap open penalty of 1.53 and an offset value of 0.123 were used. The alignments were automatically curated with gBlocks (Castresana 2000;Castresana 2002;Talavera and Castresana 2007) as implemented in the molecular sequence data management package PhyloSuite v1.2.2 (Zhang et al. 2020).
Molecular phylogenetic trees were inferred using IQTree2 (Minh et al. 2020) following a maximum likelihood and a Bayesian (MrBayes 3.2.7a; Ronquist et al. 2012) approach. The taxon selection followed Wendt et al. (2018) with additional sequences from Sir et al. (2016). Sequence data was retrieved from GenBank (https:// www. ncbi. nlm. nih. gov/ gene/). An appropriate substitution model was automatically selected by ModelFinder (Kalyaanamoorthy et al. 2017), following the Bayesian information criterion (BIC) for each alignment's partition (Chernomor et al. 2016) before tree reconstruction with 1000 non-parametric bootstrap replicates (BS, Felsenstein 1985). Additionally, PartitionFinder2 (Lanfear et al. 2016) was used to determine best-fit nucleotide substitution models restricted to the ones available in MrBayes 3.2.7a following the BIC criterion. Options for the Bayesian molecular phylogenetic inference were identical to the settings used by Matio Kemkuignou et al. (2022). The topologies were compared and support values ≥ 50% (BS) or ≥ 0.95 (posterior probability, pp) assigned to the respective bipartitions. Single-gene phylogenetic inferences were carried out following the maximum likelihood criterion with 1000 bootstrap replicates to check for congruency of the resolved Phylacia sequences. Support values were assigned following the previously stated strategy.

Molecular phylogenetic inference
Concurrently, a molecular phylogeny was inferred from a MAFFT aligned and gblocks cured multilocus dataset, which in the end consisted of 421, 1279, 957, and 1143 sites for ITS, LSU, RPB2, and TUB2, respectively (totalling a data matrix of 3800 positions). The topologies resulting from maximum likelihood and Bayesian inference strategies were compared, and support values generated from the Bayesian approach were mapped onto the maximum likelihood tree (lLn = − 38156.2560, Fig. 1). The topologies were rooted against an outgroup consisting of one representative each from the Xylariaceae and the Graphostromataceae (Xylaria, Hypoxylon, and Graphostroma platystomum), compared and found to be identical, with the exception of a polytomy found in the tree inferred from the Bayesian approach in the node featuring the analyzed Phylacia sequences (data not shown) forming a moderately bootstrap supported clade (81% BS).

Chemotaxonomy
In total, seven methanolic stromatal extracts were analyzed by HPLC-UV-Vis-ESI-MS and evaluated for the occurrence of secondary metabolites. Major constituents, characterized by clearly discernible peaks in the 210 nm trace, were compared with our in-house database of stromatal secondary metabolites described for different representatives of the Hypoxylaceae (data not shown). From a total of eleven discernible peaks relating to compounds, only compounds 2, 6, and 10 could be identified as daldinal B, BNT, and daldinol, respectively, with the help of standards, while 1 resembles entonalactam A. The remainder could not be safely assigned to any of the known metabolites that were previously obtained from stromata of the Hypoxylaceae (cf. Helaly et al. 2018). The compound detection patterns were further assigned to three chemotypes (as summarized in Fig. 3 and Table 2). It would be necessary to collect more material and do destructive preparative work and subsequent NMR spectroscopy to unambigiously identify these yet unknown compounds.

Taxonomic part
Phylacia lobulata Sir & C. Lamb., sp. nov Diagnosis -Differs from all other Phylacia spp. by having lobed stromata and ascospores almost cylindrical with the wall slightly wider on the center of the spores.
Culture -Colonies on OA covering Petri dish in 2 weeks, at first whitish becoming Olivaceous Grey (121) velvety to felty, inconspicuously zonate with entire margins, reverse greenish black (124). Sporulation regions at the center, scattered. Conidiogeneous structure identical to that described above from the stroma.
Distribution and known host -Phylacia lobulata is restricted to the northernmost area in the Argentine Yungas (Jujuy y Salta province). Frequently, the materials were encountered on dead branches of Pseudobombax argentinum (R.E. Fr.) A. Robyns, "soroche" (Malvaceae), (LIL 159605). Possibly, this fungus is host-specific to this plant.
Additional Notes -This fungus is clearly a member of Phylacia for its cleistocarpic stomata and deliquescent asci without apical apparatus, originating from geniculate ascogeneous hyphae (Medel et al. 2006). The lobed stromata and the ascospores with thickening walls towards the spore center are the most salient discriminatory features for distinguishing it from all other known species of Phylacia. The latter show cylindrical, hemispherical, pulvinate, clavate, conical, pyriform, subglobose, or turbinate stroma and have ascospores with homogeneously thickened walls (Dennis 1957;Fournier and Lechat 2015;Medel et al. 2006).
Culture -Colonies on OA covering Petri dish in 2 weeks, at first whitish becoming Olivaceous Grey (121) at the center and with entire and Pure Yellow (14) margin; velvety to felty, reverse greenish black (124). Sporulation not observed.
Known distribution and host -Phylacia surinamensis is common in the urban areas of Tucuman province and is also encountered in natural reserves from Jujuy province (Argentina). The fresh stromata of this species have been found on recently dead branches and trunks of Ceiba sp. (Malvaceae). This fungus was previously recorded in Brazil (Amazonas), Guatemala, Mexico, and Surinam (Medel et al. 2006). Notes -This taxon is characterized by having densely caespitose stromata and by its elliptical ascospores. Its stromata are usually cylindrical with flattened or slightly concave apex and grouped on a broad stromatic base (Dennis 1957). The Argentine materials show stromata cylindrical with slight constrictions at the center; in some cases, their shape can be almost clavate.
Phylacia cylindrica has a similar stromatal shape as P. surinamensis, but these species differ by the colors of KOHextractable pigments (purple vinaceous vs green) and by their ascospore size (Lacerda et al. 2018). The ascospores in Phylacia taxa are apparently devoid of germ slits; Dennis (1957) however illustrated an ascospore with a notable germ slit for P. surinamensis. Lacerda et al. (2018) studied the type specimen and recognized a conspicuous germ slit in old ascospores of this species. Medel et al. (2006) also mentioned the presence of this feature for a collection of P. sagrana (Mont.) Mont. from Costa Rica. Fournier and Lechat (2015) also reported germ slits in ascospores of three   species: P. bomba, P. cf. sagrana, and P. korfii, when the spores were mounted in PVA-lactophenol. The salient discriminatory features of P. surinamensis, P. globosa, and P. lobulata are summarized and compared in Table 4 and illustrated in Fig. 6.

Discussion
This study reports several new records of Phylacia collected in Argentina that were analyzed by a polyphasic approach using a multigene molecular phylogenetic inference and a chemotaxonomic study of their stromatal constituents by HPLC-DAD-MS. The affinities of the genus to Daldinia and Thamnomyces were already described by Ju et al. (1997), which was later corroborated by a chemotaxonomic study on the type and authentic specimens by Stadler et al. (2004). In that paper, binaphthalene derivatives such as BNT, azaphilones of the daldinin type, and daldinal derivatives were reported from stromatal extracts of P. bomba, P. globosa, P. poculiformis, P. sagraena, P. surinamensis, and P. turbinata. These secondary metabolites are commonly encountered in certain species of Daldinia (D. childiae group; cf. Stadler et al. 2014), but also occur in the Hypoxylon fuscum complex (Stadler et al. 2008b;Lambert et al. 2021). This paper is thus the first to use an integrative approach to the taxonomy of the genus that relies on a significant number of freshly collected specimens. Another chemotaxonomic study of numerous strains that now belong in the Hypoxylaceae included a culture of Phylacia sagrana (CBS 119992), which produced several small polyketides, present also in the concurrently studied cultures of Daldinia spp., but apparently absent in cultures of Annulohypoxylon, Hypoxylon, and other genera now classified as Jackrogersella or Pyrenopolyporus (Bitzer et al. 2008; see also Wendt et al. 2018 for the current taxonomy of these  Bars: a, b, c, g, h = 10 µm; d, e, i, j = 5 µm genera and species). The secondary metabolite profile of this Phylacia culture was most similar to that of D. caldariorum, to which it also showed the closest phylogenetic relationship in an ITS-based phylogenetic tree. A similar pattern was described for a subsequent study focusing on Thamnomyces (Stadler et al. 2010), where an additional species of Phylacia (P. poculiformis) was included. The sequences of the two Thamnomyces spp. and both Phylacia species were shown to resolve within the same phylogenetic clade. The current study confirmed and settled the phylogenetic affinities of the genus using, for the first time for Phylacia, a multi-gene genealogy and resolving its placement inside the Hypoxylaceae. These results confirm previous studies as rDNA-derived sequence information was repeatedly shown to be of questionable utility. For the Hypoxylaceae, it was shown that polymorphisms of the rDNA cistron on the one hand, and high redundancies of ITS sequences across several species complexes occur on the other hand (see Stadler et al. 2020 andMaharachchikumbura et al. 2021 for an extensive discussion of this matter).
Our investigation on chemotaxonomically informative secondary metabolites in the stromatal extracts yielded compounds with five different UV-Vis spectra types. In P. surinamensis Sir 1056, a metabolite (1) occurred whose UV-Vis and mass spectra are reminiscent of the reported spectra for entonalactam A, isolated and reported from the stromata of an Australian fungus assigned to Entonaema sp. by Choomuenwai et al. (2015). However, since the authors did not specify how the fungus was identified and did not deposit a voucher, the identity of the material remains unclear. In the same specimen, three other compounds were found that were not detected in the other Phylacia extracts, of which one compound (2) resembled daldinal B (also isolated by Choomuenwai et al. 2015), while the others (8 and 10) showed a characteristic triple-UV maxima in the shape of a crown, indicative of naphthalene derivatives. The mass (616 Da) of 8 suggests a dimer of 6 (318 Da), but could not be assigned to a known compound, while 10 resembled daldinol. Compound 6 could be assigned to BNT and occurred in all studied specimen. Overall, the spectra of the metabolites of P. surinamensis shared similarities with the ones reported for the holotype by Stadler et al. (2004). The UV-Vis spectrum of compound 3 in P. globosa stromata was reminiscent of daldinin derivatives, like daldinin F, but was much smaller in comparison (346 versus 460 Da for daldinin F). All other compounds (4, 5, 7, 9, and 11) shared a similar UV-Vis pattern, which resembled that of daldinone B, but eluted either at a more hydrophilic (4 and 5) or more lipophilic (7, 9, and 11) mobile phase gradient, respectively. Furthermore, compounds 4, 7, and 11 could only be detected in P. globosa, while 5 and 9 only occurred in P. lobulata. All in all, comparison with the data of the previous study by Stadler et al. (2004) was difficult because comparative data relied mostly on ancient type specimens. In some of them, artifacts, like obvious degradation products and even compounds that may represent insecticides that were eventually added to the specimens for preservation, were detected. However, results like the detection of daldinals in P. surinamiensis are quite significant as that compound seems to have remained stable not only in the holotype specimen of this species for 150 years (Stadler et al. 2004), but also in several ancient specimens of Daldinia childiae (Stadler et al. 2014).
We did not dispose of sufficient material of the valuable specimens that would have allowed for the isolation of the unknown metabolites by preparative HPLC and confirm their chemical structures by means of nuclear magnetic resonance (NMR) spectroscopy and high-resolution MS. However, we have included retention times, mass ionization patterns, and UV/Vis spectra for all the unknown metabolites in the SI (Supplementary Figs. S1-S11) in a hope that these data can aid in future attempts to accomplish such tasks.
Our study also revealed new phylogenetically relevant evidence because we have included some taxa that were not formerly characterized using the current multi-locus approach. For example, Entonaema liquescens formed a well-supported cluster with D. korfii, a taxon that had not been included in recent phylogenies. This was rather unexpected because of the strongly diverging morphology of the respective taxa. The sequenced Entonaema liquescens culture presumably originated from a specimen collected in Kansas by R. Lichtwardt in 1979 (Rogers 1982;Stadler et al. 2008a). To date, it is the only one available of the genus. It was deposited by Jack D. Rogers in ATCC, following the first report on the anamorph of this genus (Rogers 1982). Concerns regarding the authenticity of this Entonaema strain have already been raised (Wibberg et al. 2021;Kuhnert et al. 2021). It was reported that the genome of the strain showed rather high affinities to that of D. concentrica. However, the biosynthesis gene clusters encoding for mitorubrin type azaphilones, which are omnipresent in the stromata of E. liquescens, were not detected in the genome of the ATCC strain. This phenomenon needs further study and requires authentic cultures of E. liquescens that can be studied for comparison.
Funding Open Access funding enabled and organized by Projekt DEAL. This work was funded by the DFG (Deutsche Forschungsgemeinschaft) priority program "Taxon-Omics: New Approaches for Discovering and Naming Biodiversity" (SPP 1991). We are also grateful to the LifeScience Foundation (Munich) for a PhD stipend for C.L.
Data availability All additional data (except for the DNA sequence data, which are deposited in GenBank (https:// www. ncbi. nlm. nih. gov/ genba nk/)) are available in the manuscript or the supplementary information.