Coniocybe Ach. Revisited

Calicioids form a research field that has encompassed ascomycetous fungi with stalked ascomata similar to those of the lichen genus Calicium. Early generic circumscriptions of calicioid lichens and fungi were mainly based on morphological and secondary chemistry information. After the introduction of molecular data, taxonomy in the group has been reconsidered. Here, based on a broad geographical sampling, Coniocybe Ach. was revised using molecular and morphological features. Three loci (ITS, LSU and rpb1) were compared to infer its phylogenetic position, and a total of 52 new sequences (14 ITS, 24 LSU and 14 rpb1) were produced. Apart from its type C. furfuracea, Coniocybe was revised and emended to also include C. brachypoda and C. confusa. In addition, a new species, Coniocybe eufuracea, was described, and a key to the species of Coniocybe was provided.


Introduction
Calicioid fungi has long been a research field of considerable interest.It includes the systematics of fungi having ascomata similar to those of Calicium Pers., i.e., stalked ascomata with a distinct head.
Coniocybe Ach.: Fr. was described by Acharius [2].The genus in Acharius' circumscription included Mucor furfuraceum (=C.furfuracea (L.) Ach.)-in fact the only crustose calicioid described by Linnaeus-and also C. stilbea Ach.(an illegitimate name [15]), C. brachypoda Ach.[2] and C. gracilenta (Ach.)Ach.Coniocybe was accepted by E. Fries [16] and also by Th.M. Fries [17] and Zahlbruckner [18], as in contrast to Chaenotheca having a poorly developed excipulum.For many decades the genus was maintained until a major revision of the taxonomy of calicioids were undertaken by Tibell [9].Taxonomy at that time relied mostly on morphologic and chemistry data, and, based on this, Coniocybe brachypoda and C. furfuracea were transferred to Chaenotheca.This wider concept of Chaenotheca will henceforth be called Chaenotheca s. lat.Somewhat later, a species very similar to C. furfuracea, was described under the name Chaenotheca confusa Tibell [19].
Molecular studies have proven increasingly important in elucidating taxonomic relationships of calicioid lichens by inference of their phylogeny [11,13].In a recent study based on Internal Transcribed Spacer (ITS) sequences, it was shown that, within Chaenotheca s. lat., there are well characterized clades [20] which were also given preliminary nicknames.Thus C. brachypoda and C. furfuracea were shown to belong to a group referred to as 'Coniocybe s. str.'.The species at that time included have slender, yellow-pruinose ascomata and are associated with Stichococcus sp.That study was limited to comparisons of species mainly from Europe.The inclusion of C. brachypoda, C. furfuracea and C. confusa in a resurrected Coniocybe as proposed below is a first step towards re-evaluating the taxonomy of Chaenotheca s. lat., in a context of a wider sampling of material.
The main focus of this study is the emendation of Coniocybe utilizing a combination of molecular and morphological features based on a wide geographical sampling.

Taxon Sampling
This study is partly based on African materials collected jointly by the authors in the Kilimanjaro and Arusha regions in 2018, while some other material was collected in Australia (Tasmania), in addition to a wide geographical representation of the species, mainly vouchered by herbarium material kept in UPS.

Morphological Features
Ascomata anatomy was observed on freezing microtome sections 10 to 15 µm thick and on squash preparations under the light microscope.The sections were mounted in water.The ascospores of some specimens were investigated under the scanning electron microscope (SEM).
When statistical estimates of sizes are given the following format is applied: mean (X) minus one standard deviation/(sd)/mean plus one standard deviation, number of observations (n), number of specimens observed (c).Unless otherwise stated, the number of observations is 40.

Molecular Methods
Total DNA was extracted from freshly collected material, or material temporarily kept at −20 • C by using the DNeasy Plant Mini Kit (Quiagen, Hilden, Germany), following the manufacturer's instructions.About 10 to 30 apothecia were carefully selected under a dissecting microscope, cleaned from foreign material and used for DNA extraction.Total DNA was used for PCR amplifications with the following primers ITS1F [21], ITS4 [22] for ITS; LROR and LR5 [23] for the partial 28S large subunit rDNA (LSU), and gRPB1-A and gRPB1-C for the partial RNA polymerase II largest subunit RPB1 (rpb1) [24].The AccuPower PCR PreMix (Bioneer, Daejeon, Republic of Korea) was used, adding 3 µL diluted DNA, 1.5 mL of each primer (10 mM), and water to a total volume of 20 µL.The PCR conditions for ITS and LSU were: initial denaturation for 4 min at 95 • C, followed by 35 cycles of 1 min at 94 • C, 1 min at 54 • C, 45 s. at 72 • C, and final elongation for 5 min at 72 • C. For rpb1, PCR amplifications were carried out using Illustra Hot Start PCR beads under the same PCR conditions.PCR products were visualized by electrophoresis on 1.5% agarose gels.Products were purified using Illustra™ (GE Healthcare UK Limited, Little Chalfont, UK) ExoStar buffer diluted 10×, following the manufacturer's protocol.Sequencing was conducted by Macrogen (www.macrogen.com[25].After assessment of their quality, the sequences were aligned by using MAFFT v7 (on-line server: https: //mafft.cbrc.jp/alignment/server/,accessed on 14 May 2024), with G-INS-1 Strategy (Slow; progressive method) and default parameters.
The study involved two datasets.The larger dataset consists of three marker region (ITS, LSU, rpb1) sequences representing 15 species of Chaenotheca s. lat.(with newly produced sequences in bold, Table 1).They represented the clades already demonstrated and nicknamed by Tibell et al. [20].Sclerophora farinacea was chosen as outgroup for the analyses.The second dataset, a smaller dataset, only included sequences for the species of Coniocybe s. str., and here Chaenotheca biesboschii was chosen as outgroup.
For both datasets, phylogenetic relationships and their posterior probabilities (PP) were inferred using a Bayesian approach, and additional support values were estimated using Maximum Likelihood Bootstrap Support (MLbs).For the Bayesian analyses, the most likely models of evolution were estimated using the Akaike Information Criterion (AIC) as implemented in Modeltest 3.7 [26].For the first dataset, the GTR + I + G model of evolution was employed for ITS and LSU, and HKY + I + G was used for rpb1.For the second dataset, the GTR + G model was implemented for ITS.A conflict among single-locus datasets was considered significant if a well-supported monophyletic group (posterior probability [PP] ≥0.95) was found to be well supported as non-monophyletic when different loci were used.Further analyses were performed after concatenation using SequenceMatrix v1.8.2 [27].
The Bayesian analysis was executed using MrBayes v3.2.6 [28], where two analyses of two parallel runs were carried out for 10 M generations.Each run included four chains, and trees were sampled every 1000 generations and 25% were discarded as burn-in.All runs converged on the same average likelihood score and topology.Maximum Likelihood (ML) estimates were carried out by RAxML v8.2.10 using the GTR + G + I model of site substitution [29].The branch support was acquired by maximum likelihood bootstrapping (MLbs) of 1000 replicates [30], and MLbs ≥ 70% were considered to be significant.The trees were visualized in FigTree v1.3.1 [31].

Phylogeny of Chaenotheca s. lat.
A phylogeny of Chaenotheca s. lat., based on concatenation of the three loci of species representing the different clades provisionally named in Tibell et al. [20], is presented below (Figure 1).The analyses included 12 species of Chaenotheca s. lat.There was no conflict among the trees obtained for the individual locus (see Supplementary Materials;

Phylogeny of Coniocybe
A phylogeny of Coniocybe is presented in Figure 2. The analysis includes sequences representing four species of Coniocybe, two of which (C. brachypoda, C. furfuracea) were shown to belong in 'Coniocybe s. str.' in Tibell et al. [20].In addition two further species belong to Coniocybe, viz.C. confusa, that is sequenced here for the first time, and C. eufuracea, newly described here.Coniocybe furfuracea is a species with a wide distribution occur- In this phylogeny, Coniocybe is distinct from the clades of Chaenotheca s. lat.and it has maximum support in Bayesian and ML analyses.It is close to the 'gracillima group' in agreement with the results of Tibell et al. [20].

Phylogeny of Coniocybe
A phylogeny of Coniocybe is presented in Figure 2. The analysis includes sequences representing four species of Coniocybe, two of which (C. brachypoda, C. furfuracea) were shown to belong in 'Coniocybe s. str.' in Tibell et al. [20].In addition two further species belong to Coniocybe, viz.C. confusa, that is sequenced here for the first time, and C. eufuracea, newly described here.Coniocybe furfuracea is a species with a wide distribution occurring on several continents.It has a characteristic spore ornamentation of reticulate ridges as investigated by scanning electron microscopy.The Tanzanian collections SGT 426 and SGT 431 of C. eufuracea are genetically very similar (Figure 2), but differ slightly from other collections of the species.However, a much wider sampling from all parts of the distribution area is required for resolving relationships within this species (or possibly species complex) and both molecular data, secondary chemistry and morphology need to be chartered in detail, which is beyond the scope of this investigation.Thallus crustaceous; ascomata with long stalks and rounded capitula (Figure 3) with inconspicuous or missing excipulum; asci catenulate, with croziers; spores spherical, nonseptate, small, pale brown, with an ornamentation of minute irregularly arranged ridges (Figure 4); mazaedium well developed; secondary metabolites vulpinic acid derivatives; photobiont Stichococcus sp.Thallus crustaceous; ascomata with long stalks and rounded capitula (Figure 3) with inconspicuous or missing excipulum; asci catenulate, with croziers; spores spherical, nonseptate, small, pale brown, with an ornamentation of minute irregularly arranged ridges (Figure 4); mazaedium well developed; secondary metabolites vulpinic acid derivatives; photobiont Stichococcus sp.Thallus immersed; apothecia short, 0.4-1.4mm high, covered by a dense greenish pruina; mazaedium dark to medium brown, ± pruinose; capitulum spherical, 0.1-0.2mm diam., with poorly developed excipulum; stalk 0.04-0.08mm wide, covered with pruina; spores medium brown, spherical to somewhat cuboid, 3.0-4.5 µm diam., with a very minute ornamentation of tiny ridges and conspicuous, larger irregular cracks (Figure 4A,B); photobiont: Stichococcus sp.
Figures 3B and 4A,B.Note: Characterised by having rather short apothecia, an unusually immersed thallus and a rather dark brown mazaedium with, at least in young stages, a yellowish green pruina covering the mazaedium.Capitulum 0.1-0.2mm diam.The spores are spherical to cuboid, 3-4.5 µm diam.and have an ornamentation of minute, irregularly arranged ridges not visible under the light microscope and larger, irregular cracks with SEM (Figure 4B) that are well within the resolution of the light microscope.
The images of C. brachypoda in Tibell [32] with Figure 44, agree well with this insofar that in the transmission electron microscopy image, while Figure 44A shows gaps in the outermost spore wall corresponding to cracks visible in our SEM view (Figure 4B), while the ridge ornamentation in Figure 44B is minute and only barely discernible.These then most likely represent C. brachypoda.However, for the New Zealand material, the thallus was described as episubstratic and green [32], which might indicate that at least some of the material used for the description in fact refers to misidentified C. confusa.Coniocybe brachypoda grows on bark and wood in shaded and humid situations.A very widely distributed species in the Northern Hemisphere and also known from New Zealand, whereas Australian [32] and South American [19]  Thallus superficial and well developed, farinose to minutely granular, yellowish green; apothecia long and slender, 2.3-3.0 mm high, covered by a dense greenish pruina; mazaedium pale brown, ± pruinose; capitulum spherical, 0.3-0.4mm diam, with poorly developed excipulum forming a small collar at the base when young, covered by numerous hair-like crystals; stalk 0.10-0.15mm wide, pruinose; Spores spherical, 2.5-3.5 µm diam.with an ornamentation of minute ridges and provided with distinct cracks visible under the light microscope ( [19] with Figure 10E) and under SEM (Figure 4C); photobiont: Stichococcus sp.
Figures 3C and 4C (see also Tibell [19] with Figure 10E).Habitat.On tree trunks and decorticated stumps in dark and humid situations.Distribution.Widely distributed in the Southern Hemisphere.Vouchered by molecular data from specimen from Australia.
Note.Characterized by having a farinose to minutely granular, greenish yellow thallus; long-stalked apothecia covered by a greenish-yellow pruina; a hemispherical to almost spherical capitulum with poorly developed excipulum; catenulate asci; and spherical to cuboid spores 2.5-3.5 µm diam.having a minutely fissured surface.Very similar to C. furfuracea, but differing in having higher ascomata, larger capitula and larger spores provided with distinct cracks visible under the light microscope.Known from temperate South America and Australasia.
Specimen them in Europe, viz.C. furfuracea and the newly described C. eufuracea, its sister species.To resolve the nomenclatural situation of C. furfuracea an epitypification is suggested.This is not without complication, since this species in Acharius'sense might just as well have been C. eufuracea, but, until our suggestion has been proven wrong, we find the suggested epitypification a reasonable tentative solution.In the protologue, Solander was given as the collector and we find it suitable to epitypify based on a recent Swedish collection for which some molecular information is available.
Habitat.In dark and humid situations, particularly on rootlets and soil of uprooted trees and decorticated stumps in coniferous forests, more rarely on deciduous trees.
Distribution.Wide distribution in cool temperate to temperate areas of the Northern Hemisphere (Eurasia, North America).Vouchered by molecular data from specimens from India, Sweden and Switzerland.

Discussion
Here we have emended Acharius' description of Coniocybe to also include the occurrence of catenulate asci and a very unusual type of spore micro-ornamentation consisting of short irregularly arranged ridges.Along with molecular data, this has led to the exclusion of some species originally included in the genus and we have also been able to add two species unknown to Acharius, one of them, C. eufuracea, a new species.
It is interesting that Acharius' recognition of Coniocybe stands up quite well to scrutinizing by genetic investigations some 200 years later, insofar that both C. brachypoda and 'C.furfuracea' (although this species in Acharius' sense might just as well have been C. eufuracea) were included, and the features of these species very much put their mark on the generic description.This emphasized the farinose thallus, the long, thin and flexuous stalks, the small, spherical capitulum with its knob-like central part, a rather pale mazaedium, and the occurrence of a pruina.He also commented that the species occur in dark and humid sites.In Acharius' own words (in Swedish): 'Ehuru vid första påseende en visss formal likhet visar sig imellan detta och nyss förut beskrifna Slägtes (i.e., Calicium) arter, så upptäckas dock snart vid en nogare uppmärksamhet den väsendtliga skillnad, som är dem imellan. ..'-in short, careful attention convinced Acharius about the considerable difference between Coniocybe as compared to Calicium.Yes, we have to acknowledge Acharius' careful attention!However, apart from C. brachypoda and 'C.furfuracea', Acharius also included 'C.stilbea' = Sclerophora pallida (Pers.)Y.J. Yao & Spooner and Calicium aciculare Ach.= Chaenotheca hispidula (Ach.)Zahlbr in Coniocybe.Both these latter species, although different from Calicium in having a pale brown mazaedium, rather poorly agree with Acharius' description of Coniocybe.The inclusion of C. gracilenta Ach.is, in contrast, more excusable since, in its ascoma morphology, it is quite similar to that of Coniocybe, but DNA information is at odds with this classification and has shown that, although it belongs in Chaenotheca s. lat., it is outside Coniocybe (Figure 1).

Figure 1 .
Figure 1.Consensus tree based on a Bayesian and Maximum Likelihood (ML) analysis of concatenated ITS, nuLSU and rpb1 of Chaenotheca s. lat.showing the phylogenetic position of Coniocybe.The tree was rooted using Sclerophora farinacea and S. coniophaea.The two support values associated with each internal branch correspond to posterior probability (PP) and bootstrap support (bs), respectively.Branches in bold indicate a support of PP ≥ 95% and an MLbs ≥ 70%.An asterisk on a bold branch indicates that this node has a support of 100% for both support estimates.A dash instead of an MLbs value indicates that the node of the Bayesian tree was not recovered by ML bootstrapping.Coniocybe is highlighted by a shaded box.

Figure 1 .
Figure 1.Consensus tree based on a Bayesian and Maximum Likelihood (ML) analysis of concatenated ITS, nuLSU and rpb1 of Chaenotheca s. lat.showing the phylogenetic position of Coniocybe.The tree was rooted using Sclerophora farinacea and S. coniophaea.The two support values associated with each internal branch correspond to posterior probability (PP) and bootstrap support (bs), respectively.Branches in bold indicate a support of PP ≥ 95% and an MLbs ≥ 70%.An asterisk on a bold branch indicates that this node has a support of 100% for both support estimates.A dash instead of an MLbs value indicates that the node of the Bayesian tree was not recovered by ML bootstrapping.Coniocybe is highlighted by a shaded box.

J 13 Figure 2 .
Figure 2. Phylogenetic relationships of 4 species of Coniocybe based on a Bayesian and Maximum Likelihood (ML) analysis of an ITS dataset.The tree was rooted using Chaenotheca biesboschii and Chaenotheca gracillima.The two support values associated with each internal branch correspond to posterior probabilities (PP) and maximum likelihood bootstrap support (MLbs) proportions, respectively.Branches in bold indicate a support of PP ≥ 95% and MLbs ≥ 70%.An asterisk on a bold branch indicates that this node has a support of 100% for both support estimates.

Figure 2 .
Figure 2. Phylogenetic relationships of 4 species of Coniocybe based on a Bayesian and Maximum Likelihood (ML) analysis of an ITS dataset.The tree was rooted using Chaenotheca biesboschii and Chaenotheca gracillima.The two support values associated with each internal branch correspond to posterior probabilities (PP) and maximum likelihood bootstrap support (MLbs) proportions, respectively.Branches in bold indicate a support of PP ≥ 95% and MLbs ≥ 70%.An asterisk on a bold branch indicates that this node has a support of 100% for both support estimates.

Table 1 .
Species and GenBank accession numbers of sequences used in the DNA analyses.Newly produced sequences in bold.