A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species

Abstract

Abstract: The globally distributed genus Trichaptum is one of the most species-rich among polypores in terms of hosting other fungi. Among Trichaptum-associates, there is a group of mazaediate lichenized fungi (Coniocybomycetes, Ascomycota) that previously had an uncertain phylogenetic position. DNA sequences – mitochondrial small subunit (mtSSU), nuclear large subunit rDNA (nuLSU), and internal transcribed spacer (ITS) – were obtained from 29 specimens collected from Europe and North America. Maximum likelihood and Bayesian inference analyses of these three gene loci were used to infer phylogenetic position and relationships among lineages. Statistical tests were used to find which phenotypical characteristics distinguish species. The molecular sequence data provide evidence that the fungicolous specimens form a distinct lineage within Coniocybomycetes sister to the combined clade of Chaenotheca s. lat. and Sclerophora. Considering its phylogenetic placement and strict specialization, we describe a new genus – Chaenotricha. This fungicolous lineage contains three species based on molecular characteristics. Morphological characters mostly overlap except for spore size and stalk length of apothecia. We provide a new combination, Chaenotricha obscura, for the only previously described species for which we designate an epitype, and introduce a new species – Chaenotricha cilians. The third lineage remains undescribed because of a small sample size, which did not allow us to clearly delineate species boundaries.

Citation: Suija A, McMullin RT, Lõhmus P (2023). A phylogenetic assessment of a fungicolous lineage in Coniocybomycetes: Chaenotricha, a new genus of Trichaptum-inhabiting species. Fungal Systematics and Evolution 12: 255-269. doi: 10.3114/fuse.2023.12.13

INTRODUCTION

The surface of polypore sporocarps can be a substratum for many organisms such as epiphytic algae (Zavada & Simoes 2001, Stonyeva et al. 2015, Vondrák et al. 2023), non-lichenized (e.g., Hutchison 1987, Sun et al. 2019, Maurice et al. 2021) and lichenized fungi (Hawksworth et al. 2014), forming so-called epimycotic or fungicolous communities (Stonyeva et al. 2015, Maurice et al. 2021). For example, a globally distributed genus of poroid white-rotting fungi, Trichaptum (Hymenochaetales, Agaricomycetes, Basidiomycota; Larsson et al. 2006), may host more than 20 green algal (Chlorophyta) species (Mukhin et al. 2018) and is one of the richest among polypores in terms of associated fungicolous fungi (Maurice et al. 2021). Among these, there are at least three lichenized Chaenotheca species, C. gracillima (Spribille et al. 2010), C. trichialis (Selva 2014), and C. obscura (= C. balsamconensis; Merrill 1909, Allen & McMullin 2015, Selva & McMullin 2020), reported as growing on Trichaptum sporocarps.

The genus Chaenotheca, together with the genus Sclerophora, belongs to the early diverging lichenized lineage Coniocybomycetes, species of which are characterized by having stalked and mazaediate ascomata (Prieto et al. 2013), and their closest relatives belong to Lichinomycetes (Prieto et al. 2013, Díaz-Escandón et al. 2022). The oldest report of a Chaenotheca species growing on Trichaptum sporocarps was made by Fries (1865), who mentioned a Trichaptum-inhabiting variant called Chaenotheca brunneola ß cilians due to its similarities to eyelashes. Unfortunately, the original material of this taxon is lost (L. Tibell & A. Thell, pers. comm.). The second oldest evidence comes from North America, where Merrill (1909) described Calicium obscurum (= Chaenotheca obscura) growing on Trichaptum. In 2015, Allen & McMullin (2015) described C. balsamconensis from North America, but after examining Merrill's type material, Selva & McMullin (2020) concluded that this species is conspecific with C. obscura and synonymized accordingly. At the same time, a study by Suija et al. (2016) tested if morphologically similar, but ecologically distinct (wood inhabiting vs. fungal sporocarp-inhabiting) specimens belong to the same species – Chaenotheca brunneola. A single-gene (full-length ITS) analysis in that study showed that all Trichaptum- dwelling specimens, including the type of C. balsamconensis from North America, form a distinct lineage sister to the rest of Chaenotheca and Sclerophora species. Moreover, there was a clear distinction in nucleotide sequences between European and North American specimens, suggesting that these two may represent different species.

In the current study, we aimed to clarify the taxonomic position of these fungi within Coniocybomycetes. We analyzed slow- and fast-evolving ribosomal DNA markers as well as their morphological characteristics. We also sampled widely to better understand the distribution of these Trichaptum-inhabiting fungi.

MATERIAL AND METHODS

Taxon sampling and morphological examination

We examined chaenotheca-like lichenized fungi growing on Trichaptum sporocarps. The specimens are deposited in BILAS, CANL, DAU, M, NY, UPS, TRH and TUF (fungarium acronyms follow Index Herbariorum; https://sweetgum.nybg.org/science/ih/). In total, we studied the morphology and anatomy of 29 specimens collected from Europe and North America (Table 1), including an isotype of Chaenotheca obscura from the exsiccate series Merrill, Lich. Exs. Ser. II. 92. (M0205375 and CANL) and the holotype of C. balsamconensis (NY02359896).

Table 1.

Information about voucher specimens (taxon name, voucher ID, country of origin, lab code, and morpho ID), and NCBI accession codes of the new and downloaded DNA sequences (full-length ITS, nuLSU and mtSSU) used for reconstruction of phylogeny presented on Figs 1 and 2. "—" means sequence not generated or not available for this specimen. The type specimens are in bold.

Taxon name	Voucher ID	Country	Lab code	Morpho ID ITS	LSU	mtSSU
Chaenotheca biesboschii	A.v.d. Pluijm 3244 (UPS)	Netherlands		MK514539	—	—
	A.v.d. Pluijm 3244 (UPS)	Netherlands		MK376459	—	—
Chaenotheca brachypoda	Tibell 22193 (UPS)	Sweden		AF297963	—	—
	Tibell 17062 (UPS)	Sweden		AF297962	—	—
Chaenotheca brunneola	Tibell 22202 (UPS)	Sweden		AF297964	—	—
	TUF076414	Estonia	CB22	KX348121	—	—
	TUF076421	Estonia	CB14	KX348125	—	—
Chaenotheca chlorella	Tibell 16867 (UPS)	Sweden		—	AY804191	—
	Tibell 22372 (UPS)	Estonia		AF445356	—	—
	Tibell 22187 (UPS)	Sweden		AF297966	—	—
Chaenotheca chrysocephala	Tibell 21799 (UPS)	Sweden		AF298121	—	—
	PRA-Vondrak26008	Austria		OQ717362	—	—
Chaenotheca cinerea	TUF039194	Estonia	BF18	KX348119	—	—
	Tibell 22374 (UPS)	Estonia		AF421201	—	—
	Jonsson & Nordin (UPS)	Sweden		AF298122	—	—
Chaenotheca deludens	Tibell 16575	New Zealand		AF408678	—	—
Chaenotheca ferruginea	TUF089549	Estonia	CH463	OR661708	OR661698	—
	Tibell 22276 (UPS)	Sweden		AF298123	—	—
Chaenotheca furfuracea	Wedin 6366 (UPS)	Unspecified		—	JX000087	JX000121
	TUF091901	Estonia	AS899	OR661703	—	—
	Tibell 22364	Sweden		AF445357	—	—
Chaenotheca gracilenta	Wedin 7022 (S)	Unspecified		JX000100	JX000084	JX000119
	TUF030149	Estonia	BF30	KX348118	—	—
Chaenotheca gracillima	TUF091585	Estonia	CH289	OR661701	—	OR661671
	Tibell 16725 (UPS)	New Zealand		AF408682	—	—
	Tibell 17614 (UPS)	Argentina		AF408679	—	—
Chaenotheca hispidula	TUF051093	Latvia	CH361	OR661707	—	—
	Tibell 21900 (UPS)	India		AF298128	—	—
Chaenotheca hygrophila	TNS:YO9596	Japan		LC669601	—	—
	Thor 15612	Japan		AF298129	—	—
Chaenotheca laevigata	Tibell 21998b (UPS)	India		AF298131	—	—
	Tibell 22176 (UPS)	Sweden		AF298130	—	—
Chaenotheca nitidula	Tibell 21490 (UPS)	USA		AF492388	—	—
	Koffman 170 (UPS)	Canada		AF492387	—	—
Chaenotheca phaeocephala	Tibell 22291 (UPS)	Sweden		AF446045	—	—
	Tibell 21819 (UPS)	Sweden		AF445360	—	—
Chaenotheca sp.	Tibell 22113 (UPS)	India		AF298135	—	—
Chaenotheca sphaerocephala	Tibell 21939 (UPS)	India		AF298134	—	—
Chaenotheca stemonea	Tibell 22191 (UPS)	Sweden		AF408683	—	—
	WSL:BC-087-3	Switzerland		KX133006	—	—
Chaenotheca subroscida	Tibell 22150 (UPS)	Sweden		AF298136	—	—
	TUF049310	Estonia	CH760	OR661702	—	—
Chaenotheca trichialis	Prieto 3028 (S)	Unspecified		JX000102	JX000085	JX000120
	Tibell 22384 (UPS)	Sweden		AF421207	—	—
	KR-0051902	Unspecified		MW325680	—	—
Chaenotheca xyloxena	Selva 7753 (UMFK)	Canada			AF421213	—	—
	Tibell 22329 (UPS)	Sweden			AF421212	—	—
Chaenotricha cilians	TUF095043	Norway	AS972		OR661716	—	OR661678
	UPS-L-941561	Sweden	CH419		OR661713	OR661686	OR661659
	TUF091610	Estonia	CH99		—	OR661699	—
	TUF089479	Canada	CH484	1	—	—	OR661670
	TUF050023	Estonia	AS699	2	OR661715	OR661694	OR661676
	TUF076412	Estonia	CH168	3	KX348131	—	—
	TUF076423	Estonia	CH98	4	KX348120	OR661693	OR661672
	TUF091611	Estonia	CH290	5	—	—	OR661669
	TUF091612	Estonia	CH288	6	OR661721	OR661692	OR661665
	DAU0602050	Latvia	CH310	7	OR661717	—	OR661666
	DAU0602051	Latvia	CH311	8	—	—	OR661667
	DAU0602052	Latvia	CH312	9	—	—	OR661668
	TUF089401	Latvia	CH481	10	—	OR661689	OR661662
	TUF090000	Latvia	CH480	11	OR661712	OR661688	OR661661
	TRH-L-18707	Norway	CH435	12	OR661709	OR661687	OR661660
	TRH-L-18708	Norway	CH434	13	OR661711	OR661690	OR661663
	TUF050022	Norway	AS698	14	OR661714	OR661695	OR661675
	TUF095044	Norway	AS973	15	OR661719	—	OR661677
	BILAS	Russia	CH436	16	—	OR661691	OR661664
	UPS-L-867275	Sweden	CH418	17	OR661710	—	—
	UPS-L-872283	Sweden	CH420	18	OR661718	—	—
	TUF076413	Estonia	CB1		KX348130	—	—
	TUF076417	Estonia	CB3		KX348129	—	—
	TUF076416	Estonia	CB5		KX348128	—	—
	TUF076420	Estonia	CB15		KX348124	—	—
	TUF076419	Estonia	CB19		KX348123	—	—
	TUF076422	Estonia	CB21		KX348122	—	—
Chaenotricha obscura	TUF089391	Canada	CH488	24	OR661720	OR661684	OR661657
	NY02359896	USA	CH174	25	KX348132	OR661679	OR661652
	NY02439109 (epitype)	USA	CH175	26	KX348133	OR661680	OR661653
	CANL20337 (lectotype)	USA		27	—	—	—
Chaenotricha sp.	TUF089393	Canada	CH490	19	—	OR661681	OR661654
	TUF089480	Canada	CH485	20	—	OR661682	OR661655
	TUF089481	Canada	CH486	21	—	OR661696	OR661674
	TUF089547	Estonia	CH461	22	—	OR661683	OR661656
	TUF089548	Estonia	CH462	23	—	OR661685	OR661658
Lempholemma polyanthes	Zoladeski & Lutzoni 11294-L1(2/2) (CANL	Unspecified			—	AF356691	AY584709
Peltula auriculata	B. Büdel 24902	Venezuela			MF766344	MF766385	MF766303
	Herb. B. Büdel 24901	Venezuela			—	DQ832330	DQ922953
Peltula rodriguesii	B. Büdel 15901	Namibia			MF766373	—	—
Sclerophora amabilis	PRA-Vondrak24780	Czechia			OQ718083	—	—
	PRA-Vondrak24776	Czechia			OQ718082	—	—
Sclerophora farinacea	TUF086803	Estonia	SC406		OR661706	OR661700	—
	TUF055034	Estonia	BF32		OR661705	—	—
Sclerophora pallida	EDNA09-01585	United Kingdom		FR799288	—	—
	EDNA09-01513	United Kingdom		FR799287	—	—
Sclerophora peronella	TUF051090	Estonia	SC362	OR661704	OR661697	OR661673
	TUF038050	Estonia	BF16	KX348134	—	—

We selected 28 morpho-anatomical characters to describe the specimens (Supplementary Table S1). For each specimen, we recorded the thallus type (immersed or episubstratal), shape (granular or farinaceous) and presence of thallus cortex; if thallus was visible, we also tested thallus color reactions with standard spot tests with reagents following protocols described by Brodo et al. (2001): potassium hydroxide ca. 10 % solution (K), para-phenylenediamine ethanol solution (Pd) and commercial bleach containing sodium hypochlorite (C). The abbreviation KC refers to a color reaction after applying K and then C immediately afterwards to the same location. We examined up to five apothecia per specimen (the number examined depended on the availability/abundance of apothecia). We also recorded the location of the apothecia on the Trichaptum fruitbodies and stalk pigment reaction in K. For each apothecium, we described 13 characteristics: presence of pruina on the stalk, stalk color (dull or shining black), capitulum shape (spherical or obconical), development of excipulum (well or weakly), color of mazaedium (dark brown or black) and its structure (powdery or granular), height and width of the stalk and capitulum, shape of the asci (cylindrical or clavate) and its length (without stipe), shape of the ascospores (spherical, slightly elliptical or both), ascospore surface (smooth, with fissures or both) and diameter of ascospores. We calculated the average length-width ratio of the stalk based on up to five apothecia per specimen. Average size of ascospores and asci were calculated using 10 ascospores and four asci (if possible) for each apothecium to a maximum of 20 asci and 50 ascospores per specimen. Variable values were given as (min–)mean ±SD(–max) where SD is the standard deviation, and min and max are minimum and maximum values respectively.

We examined the anatomical characters of ascomata and ascospores in squash preparations under a Leica DM1000 LED compound light microscope. Our measurements were made in tap water with a precision of 1 μm using 10× objectives for apothecia and 100× (water) objectives for asci and ascospores. We produced scanning electron micrographs from air-dried material mounted on stubs coated with a thin layer of gold and observed using a FEI INSPECT Scanning Electron Microscope (The National Museum of Natural Sciences [MNCN, CSIC], Madrid). Our character selection and terminology of morphological and anatomical characteristics follows Schmidt (1970), Tibell (1980, 1999) and Allen & McMullin (2015).

Using a Mann-Whitney U Test, we assessed the difference in morphological and anatomical variables (except two variables with extremely low sample size) between two taxon groups with Statistica® v. 6.0 software (StatSoft 1984–2001).

DNA extraction, amplification, and sequencing

We extracted total DNA from 29 specimens growing on the sporocarps of Trichaptum abietinum, T. fuscoviolaceum and T. biforme and from several Chaenotheca and Sclerophora specimens (Table 1). For DNA extraction, we removed four to five ascomata per specimen from the substratum and placed them into a 1.5 mL test tube. We used a High Pure PCR Template Preparation Kit (Roche Applied Science®) and followed the protocol provided by the manufacturer.

We amplified two nuclear (full-length internal transcribed spacer [ITS] and partial large subunit [nuLSU]), and one mitochondrial (small subunit (mtSSU)) ribosomal DNA regions. To amplify these loci, we used the following primer pairs: ITS0F / ITS4, ITS0F / LA-W or ITS1 / LA-W (White et al. 1990, Tedersoo et al. 2008) for ITS, LR0R / LR7 or LR0R / LR5 (Vilgalys & Hester 1990) for nuLSU, and mrSSU1 / mrSSU3r (Zoller et al. 1999) for mtSSU. The PCR mix (25 μL) consisted of 5 μL 5× HOT FIREPol Blend Master Mix (Solis BioDyne, Tartu, Estonia), 0.5 μL of both primers, 3–8 μL of target-DNA and the rest of distilled water. We visualized the PCR products on a 1 % agarose gel stained with ethidium bromide. For the purification of PCR products, we added 1 μL of FastAP and 0.5 μL of Exonuclease I (Thermo Scientific, Waltham, MA, USA) to each tube and the tubes were incubated at 37 °C for 45 min, and the enzymes were deactivated by heating at 85 °C for 15 min. We sequenced both complementary strands of ITS using primer pairs ITS4 and ITS5 (White et al. 1990), and nuLSU with CTB6 (Garbelotto et al. 1997) and LR7, and mtSSU with the same primers as amplified. We performed DNA extraction, amplification, and purification in the molecular lab of mycology at the University of Tartu (TU, Estonia) and we Sanger sequenced the amplicons by Macrogen Inc. (Amsterdam, the Netherlands).

We used Sequencher v. 4.10.1. (GeneCodes Corp.®, Ann Arbor, MI, USA) or CodonCode Aligner v. 8.0.2 (CodonCode Corporation®, Centerville, MA, USA) to check, assemble, and manually edit the sequence fragments. To avoid misidentifications, we compared the consensus sequences with those available in the nucleotide database of the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) using the 'megablast' algorithm (Altschul et al. 1990). We deposited the newly generated DNA sequences in NCBI and UNITE (Abarenkov et al. 2010) data repositories. The sequenced voucher specimens are in DAU, TUF, NY, TRH, UPS, and the extracted DNA samples in the DNA and Environmental Sample Collection of the Natural History Museum in Tartu University (TUE). We also provide UNITE Species Hypotheses (SH; Kõljalg et al. 2013) at a distance value of 1.5 %, and a reference sequence for each recognized taxon.

Phylogenetic analysis

We successfully generated 72 new sequences (18 ITS, 24 nuLSU and 30 mtSSU; Table 1). Sequence blasting in NCBI indicated that our specimens belong to Coniocybomycetes. We therefore downloaded sequences of Chaenotheca and Sclerophora from NCBI and UNITE data repositories (Table 1) to assess the phylogenetic position of these Trichaptum-habiting specimens. We included the closest relatives to Coniocybomycetes, species of Lichinomycetes – Peltula auriculata and P. rodriguesii to root the ITS-based phylogeny, and P. auriculata and Lempholemma polyanthes to root the three-locus phylogeny. Approximately 50 Chaenotheca species and seven Sclerophora species are described (Index Fungorum; https://www.indexfungorum.org; accessed 6 Sep. 2023; and salient literature), of them DNA sequences are available for 21 and four species respectively in publicly accessible repositories.

We aligned sequences with the online version of MAFFT v. 7 (Katoh et al. 2019; https://mafft.cbrc.jp/alignment/server/) using default options and then manually adjusted them in Seaview v. 3.2 (Gouy et al. 2010) or AliView v. 1.27 (Larsson 2014). We refined the nuLSU and mtSSU alignments, i.e., eliminated poorly aligned positions and divergent regions by using Gblocks v. 0.91b (Talavera & Castresana 2007; http://www.phylogeny.fr/). In Gblocks, we used relaxed settings by allowing gap positions within the final blocks and less strict flanking positions. From the ITS alignment, we used ITSx (Bengtsson-Palme et al. 2013) in the PlutoF workbench (https://plutof.ut.ee) for extraction of neighboring conservative rDNA regions.

We reconstructed single-gene phylogenies with Maximum Likelihood (ML) using IQ-TREE v. 2 (Trifinopoulos et al. 2016; http://iqtree.cibiv.univie.ac.at) to detect possible conflicts among individual genes. We selected GTR+I+G4+F as the nucleotide substitution model and tested branch support with ultrafast bootstrapping (Minh et al. 2013) by applying 1 000 iterations. No incongruences were found, so we concatenated the mtSSU, nuLSU and ITS alignments. In the concatenated alignment, we considered gaps as missing characters. Next, we analyzed these two datasets, i.e., the concatenated mtSSU+nuLSU+ITS (37 specimens; 2 229 characters, of which 714 bp are mtSSU, 816 bp nuLSU, and 669 bp ITS) and ITS (69 sequences: 737 characters, of which 117 were parsimony informative) datasets. We used both datasets to 1) assess the phylogenetic position of the fungicolous lineage in relation to Sclerophora and Chaenotheca s. lat.; and 2) evaluate the species limits within the fungicolous group. We deposited the alignments in the TreeBASE repository (study ID S30840).

Next, based on the concatenated alignment, we inferred the phylogenetic relationships and the tree confidence by using two different methodologies: the Metropolis coupled Markov Chain Monte Carlo (MCMC) approach implemented in MrBayes v. 3.2.1. (Ronquist et al. 2012) and Maximum Likelihood (ML) in RAxML (Stamatakis 2006), using RAxML-NG v. 1.0.0 software (Kozlov et al. 2019; https://raxml-ng.vital-it.ch/). We calculated the best-fit nucleotide substitution model using PartitionFinder v. 2.1.1 (Lanfear et al. 2012). The best-fit models according to the lowest value of the Akaike Information Criterion (AICc) were TrN+I+G for nuLSU and GTR+I+G for mtSSU and ITS. The settings for MCMC were as follows: two parallel, simultaneous runs with four incrementally heated chains starting with a random tree; ngen = 1 M generations, samplefreq and diagnfreq = 500, printfreq = 2 000. We ran the analysis until the standard deviation of split frequencies (SDSF) was below 0.01 and the potential scale reduction factor (PSRF) was close to 1 indicating convergence of the chains. We discarded the first 25 % as 'burn-in' and a consensus tree and posterior probabilities (PP) were calculated from the remaining tree distribution. We calculated Maximum Likelihood (ML) using a GTR+F0+G nucleotide substitution model, bootstrap support of the ML topology was obtained using bootstrapping with 1 000 pseudo-replicates (bootstrap cut-off was 0.01). In the phylogenetic trees, we considered clades supported when posterior probabilities (PP) were ≥ 0.95 and bootstrap values (BS) ≥ 70 %. Our consensus trees were visualized using FigTree v. 1.4.4 (Rambaut 2014) and annotated with Adobe Illustrator v. 13.0.0 CS3®.

RESULTS AND DISCUSSION

The analysis of two datasets (concatenated mtSSU+nuLSU+ITS and ITS only) indicate that the Trichaptum-dwelling specimens form a distinct, highly supported lineage sister to the Chaenotheca-Sclerophora clade (mtSSU+nuLSU+ITS: BS = 100 %, PP = 1; ITS: BS = 99 %) and in all combinations the Sclerophora clade is nested within Chaenotheca s. lat. (Figs 1, 2). In both analyses, the closest relatives are species in the Chaenotheca brunneola group (i.e., C. brunneola, C. deludens, C. ferruginea, C. hygrophila, C. sphaerocephala and C. stemonea), but this relationship is supported only in the ITS-based phylogeny (Figs 1, 2). Following the smaller genus concepts of Tibell et al. (2019) based on an ITS-phylogeny and morphology, and instead of incorporating all species of Coniocybomycetes under the single name Chaenotheca, we describe a new genus – Chaenotricha – to accommodate all Trichaptum-specialists (see the Taxonomy section). This allows us to retain Sclerophora as a separate taxonomic unit without combining it with Chaenotheca.

Fig. 1.

Maximum likelihood (ML) phylogeny based on rDNA ITS sequences generated for this study and derived from NCBI and UNITE database (Table 1) and showing the position of Chaenotricha in relation to other "taxa" within Coniocybomycetes. The names of the clades follow Tibell et al. (2019); bootstrap support values (BS) ≥ 70 % are above branches. Letters "H" and "E" in brackets indicate the holotype of Chaenotheca balsamconensis (= Chaenotricha obscura) and epitype of Chaeotricha obscura, respectively.

Fig. 2.

Three-locus phylogeny (mtSSU+nuLSU+ITS) based on the Maximum Likelihood consensus tree showing the position of Chaenotricha within Coniocybomycetes and three clades within it corresponding to Chaenotricha species. The branches with posterior probabilities (PPs) ≥ 0.95 and bootstrap values (BS) ≥ 70 % are considered as supported and indicated with a thicker line. Support values are above branches. Species of Lichinomycetes form an outgroup. Type specimens are in bold. CH174 – holotype of Chaenotheca balsamconensis; CH175 – epitype of Chaenotricha obscura.

The Bayesian and ML trees were topologically concordant and revealed three lineages within Chaenotricha (Fig. 2), instead of the previously recognized two (Suija et al. 2016). One of the groups includes the holotype of C. balsamconensis (= C. obscura, combined with Chaenotricha here), and two other specimens from USA and Canada. The second group includes 18 specimens from Europe (Estonia, Latvia, Norway, Sweden, European Russia), and one from Canada, and the third group is from Canada and Estonia (five specimens). The specimens in the second group have smaller ascospores and shorter stalk lengths compared to C. obscura (Fig. 3, Table 2). Also, the pairwise comparison of ITS sequences revealed that there are 23 parsimony informative characters (4.6 % of 504 bp, n = 25) separating these two groups as distinct species (Table 3). The third group is also well-supported molecularly, but we failed to obtain ITS sequences from any of these specimens. However, the sequences of slow-evolving genes were divergent enough not to incorporate these specimens under the names C. cilians or C. obscura (Fig. 2, Table 3). We found that the morphological characteristics of these specimens are somewhat intermediate between C. obscura and C. cilians (Fig. 3, Table 2), but because of the small sample size, we are only describing the two species that are morphologically distinct for now.

Fig. 3.

Average values of anatomical (A) and morphological (B, C) characters and the proportion of apothecia with stalks that have a K+ red reaction (D) of the studied specimens of Chaenotricha cilians (•), Chaenotricha sp. (■) and C. obscura (▲). Specimen ID numbers correspond to vouchers in Table 1. Five apothecia were surveyed per specimen, except for ID #4 (four apothecia), #3, #26–27 (three), and #20, #24, #25 (one). Type specimen of C. cilians is ID #6. Spore data are missing for #20.

Table 2.

Morphological and anatomical apothecial characters for three Chaenotricha species. Sample size (N) represents the number of specimens studied (for each specimen, up to five apothecia were studied; also see corresponding illustrations in Fig. 3).

Character	Chaenotricha cilians					Chaenotricha sp.					Chaenotricha obscura
	N	min	Mean	max	SD	N	min	Mean	max	SD	N	min	Mean	max	SD
Stalk with K+ red (%)	18	0	31	80	32	5	0	44	100	46	4	0	58	100	50
Stalk+capitulum length (mm)	18	0.4	0.7	1.0	0.2	5	0.6	0.8	1.0	0.2	4	0.8	1.3	2.3	0.7
Stalk length (mm)	18	0.3	0.6	0.9	0.2	5	0.5	0.7	0.8	0.1	4	0.6	1.1	1.9	0.6
Stalk width (mm)	18	0.04	0.06	0.08	0.01	5	0.05	0.07	0.10	0.02	4	0.05	0.08	0.14	0.04
Stalk length to width ratio	18	6	10	14	2	5	6	10	13	3	4	10	14	18	3
Capitulum length (mm)	18	0.03	0.08	0.12	0.03	4	0.10	0.12	0.13	0.01	4	0.05	0.19	0.40	0.15
Capitulum width (mm)	18	0.1	0.2	0.3	0.0	4	0.2	0.3	0.3	0.0	4	0.1	0.3	0.6	0.2
Mazaedium length (mm)	18	0.03	0.08	0.16	0.04	4	0.08	0.14	0.22	0.06	2	0.05	0.07	0.08	0.02
Ascus length (µm)	17	16	18	20	1	4	18	20	21	1	2	16	23	30	10
Ascospore diameter (µm)	18	3.8	4.4	5.0	0.3	4	4.6	5.0	5.5	0.4	4	5.2	5.4	5.6	0.2

Table 3.

An overview of the single-gene alignments (full-length ITS, nuLSU, and mtSSU) for the Trichaptum-specialized specimens characterized by the number of sequences in the alignment, number of base pairs in the alignment (length), number and percentage of variable nucleotide positions and parsimony informative nucleotide positions.

Locus	No. of sequences	Length (bp)	Variable (%)	Informative (%)
ITS	25	504	25 (5.0)	23 (4.6)
nuLSU	18	1 242	38 (3.1)	18 (1.5)
mtSSU	25	818	7(1.3)	5 (0.9)

Taxonomy

Chaenotricha Suija, McMullin & P. Lõhmus, gen. nov. MycoBank MB 850355. Figs 4–7; fig. 1 in Allen & McMullin (2015).

Fig. 7.

Micromorphological characters of Chaenotricha cilians (TUF050023). A, B. Base of the stalk. C. K+ bleeding red reaction of the apothecium. D. Hypothecium and hamathecium consisting of asci at different developmental stages and paraphyses, and single liberated ascospores. E. Exciple. F. Stalk structure (note periclinally arranged hyphae and uneven surface of the stalk). G. Developing asci, paraphyses and ascospores H. Ascus (immature) developing singly from the ascogenous cells. I. Immature ascus with developing ascospores. J. Mature ascospores. Scale bars: A, C = 50 µm; B, F = 20 µm; D, E, G = 10 µm; H–J = 5 µm.

Etymology: The name combines two genus names – Chaenotheca, a genus in which the type species was originally settled, and Trichaptum, a genus on which the lichenized species grow.

Type species: Chaenotricha obscura (G. Merr.) Suija, McMullin & P. Lõhmus

Typus: Calicium (Allodium) obscurum Merrill. Merrill, Lichenes Exsiccati prepared by G.K. Merrill no. 92. USA, Rockland, Maine, on dead fungus, 5 Sep. 1909, G.K. Merrill (lectotype designated here CANL 20337!, vidi, MBT 10016450; isotype M0205375!, vidi).

Diagnosis: Species in this genus grow exclusively on sporocarps of Trichaptum, which distinguishes it from the rest of Coniocybomycetes. It differs from the species in the Chaenotheca brunneola group by having ascospores without fissures and cracks.

Description: Thallus immersed or inconspicuous, forming loose associations with unicellular green algae on the surface of Trichaptum sporocarps or infrequently episubstratal forming ecorticate, granular aggregations of hyphae and algae. Ascomata stalked, stalk dark brown to black, mostly shiny, straight to somewhat curved, consisting of periclinally arranged brown hyphae, surface uneven, stalk K– or K+ red (color bleeds from the stalk). Capitulum spherical to obconical, epruinose. Mazaedium powdery. True excipulum and hypothecium well-developed, brown to dark brown, formed as a continuation of the stalk, with similar hyphal structure. Hamathecium consists of asci dissolving at the early stage of development, and paraphyses. Asci cylindrical, raising singly and directly from the ascogenous hyphae, no croziers, consisting of eight uniseriately arranged ascospores, stalked. Paraphyses hyaline, straight, not swollen at tips, without septa. Ascospores aseptate, at the early stage of development hyaline, brown when mature, smooth, spherical to irregularly spherical. Asexual morph not observed.

Chaenotricha obscura (G. Merr.) Suija, McMullin & P. Lõhmus, comb. nov. MycoBank MB 850356.

Basionym: Calicium obscurum G. Merr., Bryologist 12: 107. 1909. Synonyms: Chaenotheca obscura (G. Merr.) Nádv., Stud. Bot. Čechoslov. 5: 124. 1942.

Chaenotheca balsamconensis J.L. Allen & McMullin, Bryologist 118: 55. 2015.

Epitype: USA, Michigan, Chippewa county, Hiawatha National Forest, FS3343 1.5 mi E of jct w/ MI-123, 1.9 mi NE of Trout Lake, 4.3 mi NW of Old Dick (45.21444°N, 84.889722°W), bog dominated by Pinus banksiana with additional hardwoods (Acer, Betula, Populus, Salix) and conifers (Abies, Larix, Picea), on T. abietinum on dead Pinus banksiana, leg. J.C. Lendemer, #45283-A (epitype designated here NY02439109, vidi, MBT 10016475).

Species hypothesis: SH1265129.09FU.

Reference sequence from the epitype: ITS (GenBank KX348133), other available gene sequences nuLSU (GenBank OR661680), mtSSU (GenBank OR661653).

Materials examined: Canada, Lunenburg County, ca. 1 km N of Crouse's Settlement, ca. 1 km NE of Crouse's Settlement Road, 8 m E of Old Wood's Road (44.3561°N, 64.4050°W), coastal mature mixed-wood forest, on T. abietinum, on a dead Abies balsamea, leg. F. Anderson, det. T. R. McMullin (TUF089391). USA, North Carolina, Yancey County: Mount Mitchell State Park, Balsam Cone summit and vicinity, ca. 2 mi N of Mount Mitchell, ca. 3 mi W of US80 (35.7894°N, 82.2559°W), spruce (Picea)-fir (Abies) forest with Betula, Rhododendron, and Sorbus on top of narrow ridge with scattered, large rock outcrops, on T. abietinum, on dead Abies, leg. J.L. Allen & J.C. Lendemer, J. Allen #4108 (holotype of C. balsamconensis NY02359896).

Chaenotricha cilians Suija, McMullin & P. Lõhmus, sp. nov. MycoBank MB 850357. Figs 5–7, and fig. 1 in Suija et al. (2016).

Fig. 5.

Chaenotricha cilians sp. nov. A. Ascomata of C. cilians on the upper side of the Trichaptum fruitbodies (TUF091612, holotype). B. Ascomata on the hymenophore layer of the Trichaptum fruitbodies (TUF050023). Scale bars = 20 mm.

Etymology: The epithet cilians was used by Theodor Magnus Fries (1832–1913) for a variant of Chaenotheca brunneola inhabiting Trichaptum sporocarps. The name was adopted by him due to the resemblance to eyelashes.

Diagnosis: This species differs from Chaenotricha obscura by having shorter stalks on average 0.6 mm vs. 1.1 mm, and slightly smaller ascospores, on average 4.5 µm vs. 5.5 µm. The species is similar to Chaenotheca brunneola (lignicolous) except inhabiting fruitbodies of Trichaptum spp., having exclusively cylindrical asci with uniseriately arranged smooth ascospores and stalks K– or with a K+ red reaction (bleeds from the stalk).

Typus: Estonia, Tartu Co., Kardla village (58.4204°N 26.5604°E), Aegopodium boreo-nemoral forest site type, 66-yr-old Norway spruce dominated forest, on Trichaptum on a Picea abies snag, 2 Jun. 2017, leg. P. Lõhmus, Kardla ID27 (holotype TUF091612).

Species hypothesis: SH1265130.09FU.

Reference sequence: ITS (UNITE: UDB0801842; GenBank OR661721), other gene sequences nuLSU (GenBank OR661692), mtSSU (GenBank OR661665).

Description: Thallus inconspicuous, mycobiont hyphae loosely connected with cells of trebouxioid algae on the sporocarp surface. Ascomata developed on the upper surface and at the edge of the fungal sporocarp. Stalk epruinose, dark brown, K–, (0.35–)0.4 ± 0.07(–0.5) mm in length × (0.045–)0.05 ± 0.01(–0.06) mm in width, length to width ratio (6–)7 ± 1(–8). Capitulum spherical to obconical, (0.10–)0.13 ± 0.01(–0.14) mm in diameter (n = 5). Excipulum well developed, mazaedium dark brown, powdery. Asci cylindrical, born singly on a stalk, (15–)19.3 ± 4.4(–30) µm in length (n = 20); some measured asci (n = 9) had a stipe, (5–)5.6 ± 1.7(–10) µm long. Ascospores arranged within the ascus uniseriately, hyaline when young, brown, smooth, spherical to irregularly spherical, (3–)4.5 ± 0.6(–5) µm diam (n = 50). Asexual morph not observed.

Ecology and Distribution: Chaenotricha cilians grows on the sporocarps of three Trichaptum species, T. abietinum, T. fuscoviolaceum and T. biforme. So far, the distribution includes European countries (Denmark, Estonia, Latvia, Lithuania, Norway, Russia, Sweden) but there is also one record from Canada. Chaeotricha cilians is reported from hemiboreal and boreal forests and bog areas, on Trichaptum sporocarps inhabiting standing dead trunks, mainly of Norway spruce and Scots pine and rarely on birch.

Notes: Eighteen specimens were examined (see Table 1), and on a few occasions ascomata were produced on the hymenophore surface (Fig 4B), and in those cases, we did not find associations with algae using a compound light microscope. Moreover, we did not locate living or dead algal cells in the area around the ascomata. Life-style switching is common among fungi, and optional and weak lichenization has been demonstrated for several groups of ascomycetes (e.g., Wedin et al. 2006, Pérez-Ortega et al. 2016). Our results suggest that C. cilians may be an example of optional lichenization. Genome screening, metatranscriptomics, and other techniques may provide further information about the relationships of this tri-partite association.

Fig. 4.

Isotype of Chaenotricha obscura from Merrill, Lich. Exs. Ser. I 92 (CANL). Scale bar = 0.5 mm.

The intensity of the K+ red pigment reaction of the stalks and the degree of shininess can vary within and among specimens (Fig. 3D, Table 2). The length of apothecia (and other anatomical and morphological characters) may vary slightly among specimens (Fig. 3, Table 2); for example, the type specimen has 0.2 mm shorter stalks than the average length measured for 18 specimens (Table 2). The species differs significantly from Chaenotricha obscura by its 1 µm smaller spores, respectively (Table 2; Mann-Whitney U test, U = 7.00, p = 0.013) and in the length of the stalk, which is significantly shorter than that of C. obscura (on average 0.5 mm, Table 2; U = 0, p = 0.002), However, because of very unequal (and small) sample sizes of species groups, the results of statistical tests should be interpreted with caution.

Materials examined (selected): Denmark, North Denmark Region, Thisted Municipality, Nationalpark Thy (56.9769°N, 8.4274°E), on Trichaptum sp. on Pinus contorta, 14 Oct. 2023, leg. P. Lõhmus & A. Suija (TUF095157); ibid., Thagaard plantation (56.9817°N, 8.4283°N), on T. abietinum on standing corticated Pinus cf. mugo, 14 Oct. 2023, leg. N. Johansson (TUF095158). Estonia, Pärnu county, Saarde community (58.1224°N, 25.0914°E), on T. abietinum on a Picea abies snag, 18 Aug. 2014, leg. P. Lõhmus (TUF076419; TUF076420); Surju comm. (58.3099°N, 24.9826°E), on T. abietinum on a P. sylvestris snag, 26 May 2014, leg. P. Lõhmus (TUF076423); Tartu co., Puhja comm. (58.3337°N, 26.2631°E), on T. abietinum on snag of P. sylvestris, 10 Jun. 2014, leg. P. Lõhmus (TUF076422); Tartu comm., Valmaotsa village, Se∏i-Sillaotsa hiking trail, Alam-Pedja Nature Reserve, (58.4411°N, 26.2662°E), on Trichaptum sp. on a snag of Betula, 7 Aug. 2022, leg. A. Suija & M. Suija (TUF050023). Latvia, Alūksne Municipality, Liepna Parish, young boggy birch forest (57.4110°N 27.4843°E), on T. abietinum on P. sylvestris, unknown collection date, leg. R. Moisejevs (DAU0602050); ibid. (DAU0602051); Jēkabpils Municipality, Sauka Parish (56.3187°N 25.3954°E), on T. abietinum on P. sylvestris, unknown collection date, leg. R. Moisejevs (DAU0602052); Drabeši parish (57.2638°N, 25.1108°E), spruce forest, on Trichaptum sp. on natural spruce stump, 2020, leg. P. Degtjarenko & R. Moisejevs (TUF090000). Lithuania, Trakai district, Plomenai bog. close to Sibirka village (54.6425°N, 24.9004°E), on sporocarps of Trichaptum sp. growing on Pinus, 6 Feb. 2022, leg. M. Ryla (TUF095099, ex BILAS 11108). Norway, Steinkjer Municipality, W of Strukstadmyra (63.9873°N, 11.5801°E), boreal rainforest, on Trichaptum on dead spruce (P. abies), 9 Aug. 2018, leg. A. Frisch (TRH-L-18707); Vefsn Municipality, Langmoen, NW of Fustvatnet (65.912°N, 13.28372°E), on Trichaptum sp. on a spruce snag, 26 Jun. 2018, leg. A. Frisch (TRH-L-18706); Grong Municipality, Solemsmoen naturreservat, Kvernbekken (64.5760°N, 12.5557°E), boreal rainforest, on T. abietinum on P. abies, 16 Aug. 2019, leg. H. Holien (TRH-L-18708); Nordre Follo, Ås (59.6715°N,10.8846°E), on T. fuscoviolaceum on dead P. sylvestris in Sphagnum bog, 28 Jan. 2023, leg. A. K. Ruud (TUF050022); Fredrikstad Municipality, Askedalstangen (59.1367°N,11.0780°E), on T. fuscoviolaceum on pine, 5 Nov. 2022, leg. A.G. Helle (TUF095043); Moss, Vardasen nature reserve (59.3529°N,10.6764°E), on T. fuscoviolaceum on pine, 29 Jan. 2023, leg. A.G. Helle & M. Angard (TUF095044). Russia, Krasnoznamensky District, SE to Krasnoznamensk, forest "Michurinsky", near Kaban'e bog" (54.8892°N, 22.5622°E), old-growth pine forest with Sphagnum spp. and Carex sp., with mosses and Vaccinium myrtillis on hummocks, with young birches and spruce undergrowth, with upturned trees and big log, on T. biforme on trunk of Betula sp., 27 Sep. 2019, leg. I. Stepanchikova & D. Himelbrant (BILAS). Sweden, Uppsala, Kvarnbo (59.8410°N, 17.5668°E), on T. fuscoviolaceum, 21 Apr. 2019, leg. R. Elleby (UPS-L-941561); ÅmÅl Municipality, Edelskog par., Baljasen Nature Reserve, ca. 750 m NW of the folk museum Petersborg (59.0812°N, 12.4657°E), on T. abietinum on the trunk of P. abies, 14 Apr. 2017, leg. M. Westberg & C. Kannesten (UPS-L-867275); Uppsala, Hammarparken, (59.8424°N, 17.5981°E), on T. fuscoviolaceum, 16 Mar. 2019, leg. H. Lernefalk & B. Kühn (UPS-L-941560); Uppland, Vänge par., Fiby urskog (59.8899°N, 17.3525°E), on T. abietinum on P. abies, 7 Apr. 2016, leg. J. C. Zamora, M. Svensson, S. Ekman, M. Westberg & G. von Hirschheydt (UPS-L-872283).

Chaenotricha sp.

Five specimens that we examined form a well-supported clade in the three-marker phylogenetic tree (Fig. 2). These specimens have an intermediate set of morphological characteristics between the other two Chaenotricha species. They are similar to C. cilians in stalk length, and other morpho-anatomical characters (Fig. 3, Table 2), but they differ by having larger (average 5 µm) ascospores similar to those to C. obscura (for additional results see the Notes of C. cilians).

Distribution: Five localities in North America (Canada, USA) and Europe (Estonia) are known.

Specimens examined: Canada, Lunenburg County, ca. 1 km N of Crouse's Settlement, ca. 1 km NE of Crouse's Settlement Road, 3 m E of Old Wood's Road (44.3555°N, 64.4045°W), coastal mature mixed-wood forest, on T. abietinum, on an Abies balsamea snag, 30 Aug. 2020, leg. R.T. McMullin (TUF089393); Ontario, Thunder Bay District, Sibley Peninsula, Sleeping Giant Provincial Park, between park cabin 5 and the Marie Louise Lake Campground, (48.4584°N, 88.7368°W), 16 Oct. 2018, leg. R.T. McMullin (TUF089481). Estonia, Saaremaa, Lussu village (58.4575°N, 22.4370°E), on T. fuscoviolaceum on P. sylvestris, 12 Nov. 2019, leg. M. Nõmm (TUF089547); Järise village (58.4920°N, 22.3916°E), on T. fuscoviolaceum, on log of P. sylvestris, 31 Oct. 2019, leg. M. Nõmm (TUF089548). USA, Haywood County, Great Smoky Mountains National Park, McKee Branch Trail, 0.48 km (linear) SE of junction with Caldwell Fork Trail (35.5952°N, 83.0991°W), mature mixed-wood forest, deciduous dominated in protected river valley, on T. abietinum, 27 Oct. 2017, leg. R.R. McMullin (TUF089480).

Fig. 6.

Scanning electron microscopy of Chaenotricha cilians (specimen TRH-L-18708). A. Ascomata (note the obovate shape of the mazaedium). B. Exciple surrounding the hamathecium. C. Periclinally arranged hyphae of the stalk. D. Base of ascomatal stalk. Scale bars: A = 300 µm; B, D = 50 µm; C = 20 µm.

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

Table S1.

List of evaluated morphological characters.

Character	Character state
Thallus	1–immersed, 2–episubstratal
Thallus shape	1–granular, 2–farinose
Thallus ecorticate	1–ecorticate, 2–corticate
Thallus reaction with K	0–negative, 1–positive
Thallus reaction with C	0–negative, 1–positive
Thallus reaction with KC	0–negative, 1–positive
Thallus reaction with P	0–negative, 1–positive
Stalk reaction with K	0–no reaction, 1–red (reaction bleeds from the stalk)
Pruina on ascomata and/or stalk	0–no pruina, 1–with pruina
Stalk position on polypore fruitbody	1–above, 2–at border, 3–under, on basiciocarp gills
Color of stalk	1–dark brown to black, 2–shining black
Shape of capitulum	1–sphaerical, 2–obconical
Excipulum development	1–well developed (excipulum edge thick and well visible), 2–less-developed (edge vague)
Color of mazaedium	1–dark brown, 2–black
Surface of mazaedium	1–powdery, 2–granular
Length of stalk (μm)
Length of capitulum (μm)
Length of mazaedium (μm)
Diameter of stalk (μm)
Diameter of capitulum (μm)
Shape of asci	1–cylindrical, 2–clavate
Length of stipe (μm)
Length of asci (μm)
Shape of ascospores	1–round, 2–slightly ellipsoid, 3–both round and ellipsoid
Ascospore arrangement in ascus	1–uniseriate, 2–biseriate
Color of ascospores	1–hyaline, 2–brown
Surface of ascospores	1–only with fissures, 2–only smooth, 3–both with fissures and smooth surface occur
Size of ascospores (μm)

obconical