Introduction
Megasporaceae was introduced by Lumbsch et al. (Reference Lumbsch, Feige and Schmitz1994) for the single genus Megaspora (Clauzade & Cl. Roux) Hafellner & V. Wirth. Previously, Hafellner (Reference Hafellner1984) had included Megaspora in Hymeneliaceae Körb. (Lecanorales Nannf.), which at that time included Aspicilia s. lat., but Lumbsch et al. (Reference Lumbsch, Feige and Schmitz1994) transferred the family to Pertusariales M. Choisy ex D. Hawksw. & O. E. Erikss. More recently, in a five-locus study of Lecanoromycetes, Miadlikowska et al. (Reference Miadlikowska, Kauff, Hofstetter, Fraker, Grube, Hafellner, Reeb, Hodkinson, Kukwa and Lücking2006) uncovered a strongly supported sister relationship between Aspicilia A. Massal. and Ochrolechia A. Massal. and included Aspicilia in Pertusariaceae Körb. Schmitt et al. (Reference Schmitt, Yamamoto and Lumbsch2006) further divided Pertusariaceae and grouped Aspicilia and Lobothallia (Clauzade & Cl. Roux) Hafellner with Megaspora in an expanded Megasporaceae.
Nordin et al. (Reference Nordin, Savić and Tibell2010) performed a two-locus (nuLSU, mtSSU) analysis focused on Megasporaceae and, although this had weak support for Aspicilia and was poorly resolved, they resurrected the genera Sagedia Ach. and Circinaria Link for two groups of species formerly included in Aspicilia while also excluding Aspilidea Hafellner from the family. In a revision of the vagrant ‘manna group’ members of Circinaria, Sohrabi et al. (Reference Sohrabi, Stenroos, Myllys, Søchting, Ahti and Hyvönen2013a) recovered a topology that supported the generic delimitations in Nordin et al. (Reference Nordin, Savić and Tibell2010). Sohrabi et al. (Reference Sohrabi, Leavitt, Rico, Halıcı, Shrestha and Stenroos2013b) later introduced the genus Teuvoa Sohrabi & S. D. Leav. for three species previously included in Lobothallia. Miadlikowska et al. (Reference Miadlikowska, Kauff, Högnabba, Oliver, Molnár, Fraker, Gaya, Hafellner, Hofstetter and Gueidan2014) suggested that Sagedia should be included within Aspicilia, and the expanded genus (Aspicilia + Sagedia) along with Circinaria, Lobothallia and Aspilidea (with low support), included in a highly supported Megasporaceae sister to Ochrolechiaceae R. C. Harris ex Lumbsch & I. Schmitt within Pertusariales. Subsequently, Oxneriaria S. Y. Kondr. & Lőkös was introduced for a large group of mostly Arctic species previously included in Aspicilia as sister to the Aspicilia s. str./Sagedia clade (Moniri et al. Reference Moniri, Gromakova, Lőkös and Kondratyuk2017). This permitted Sagedia to be recognized as a distinct genus. Zakeri et al. (Reference Zakeri, Divakar and Otte2017) resurrected the genus Aspiciliella M. Choisy for a group of species sibling to Circinaria/ Megaspora. A different approach was taken by McCune & Di Meglio (Reference McCune and Di2021) who investigated the Aspicilia reptans group in NW North America using a two-locus phylogeny (ITS, nuLSU) and revealed a topology in which most of these recently recognized genera, although monophyletic, were either unsupported or nested within other genera. Consequently, they recognized only Aspiciliella and Megaspora among the recently segregated genera, leaving all other species in Aspicilia s. lat. Finally, Paukov et al. (unpublished data) have proposed the new genus Atrostelia Paukov et al. (ined.) for a single species A. magnifica Davydov & Yakovchenko from the Republic of Tyva, Russia, which resolved as sister to Aspiciliella in their phylogenetic tree.
In recent years, Aspicilia s. lat. has been the subject of several regional studies. Thomson (Reference Thomson1997) broadly surveyed the Arctic North American species, Owe-Larsson et al. (Reference Owe-Larsson, Nordin, Tibell, Nash, Gries and Bungartz2007) described 19 new species as part of their treatment of the genus in SW North America, and Øvstedal et al. (Reference Øvstedal, Tønsberg and Elvebakk2009) included 38 Aspicilia species in their account of the lichens of Svalbard. However, no comprehensive study of the group has been undertaken since Magnusson (Reference Magnusson1939). Here we present a robust five-locus phylogeny of Megasporaceae, erect a new genus for an early diverging species in the family, describe two new species of Aspicilia and make new combinations at the species level in Aspilidea and Lobothallia. We also report the lichenicolous fungus Sagediopsis aspiciliae (Zopf ex Sacc. & D. Sacc.) Nik. Hoffm. & Hafellner as new to North America.
Materials and Methods
Taxon sampling
The majority of the specimens used in this study were collected by the first author between 2007–2017, but further specimens were obtained from herbarium loans and the collecting efforts of other researchers. Analysis focused on specimens from the Northern Hemisphere, with efforts concentrated on the group in western North America and the northern Rocky Mountains, but specimens from eastern Canada and Scandinavia are also included (Supplementary Material S1 & S2, available online). Additional sequences were downloaded from GenBank (Supplementary Material S1).
Morphological study
Hand-cut sections of apothecia and pycnidia were mounted in water and tested by the addition of 10% KOH (K) and 50% HNO3 (N). The presence of norstictic and stictic acids was inferred from positive reactions under the microscope with 10% KOH and para-phenylenediamine (Pd), respectively, and confirmed with thin-layer chromatography in solvent systems A and C (Orange et al. Reference Orange, James and White2001). Ascospore measurements and ratios are given as: (lowest recorded–)normal range(–highest recorded), except for Aspilidea spp. and Sagediopsis aspiciliae, which are given as (lowest recorded–) x̄ ± standard deviation (–highest recorded); n = number of measurements.
DNA isolation and sequencing
Tissue samples for total DNA were extracted from 10–15 healthy apothecia and surrounding tissue. Two 3 mm steel beads were added to the sample tubes and frozen at −80 °C for 1 h. Samples were then mounted on the TissueLyser II (Qiagen, Germany) and ground in 30 s intervals for 1–2 min at 30/hz. DNA was extracted using the Qiagen DNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer's instructions, except for the following modifications: in the first step, samples were incubated in lysis buffer for 1 h and vortexed every 10 min; in the final step, the samples were eluted in 50 μl AE buffer twice. DNA quantity was tested on an Implen Nanodrop (Implen, München, Germany). Standard PCR amplifications were conducted in 25 μl reaction volumes using Ready-To-Go PCR Beads (GE Healthcare, UK) or Gotaq Green Master Mix (Promega) following manufacturers’ recommendations. All primers used in this study appear in Table 1. Amplifications were carried out in an Eppendorf Mastercycler Pro thermal cycler (Eppendorf North America, New York, USA) and performed using the protocols in Table 2. PCR products were cleaned using the Qiagen PCR Purification Kit (Qiagen, Germany) or Agencourt AMPure XP beads (Beckman Coulter, Inc., Brea, California, USA), following manufacturers’ instructions, and were visualized on 1% agarose gel stained with ethidium bromide. Sequencing reactions were performed by Eurofins Genomics (Louisville, Kentucky, USA).
Primers used in this study
PCR protocols used in this study
Sequence alignment
Sequences were quality checked and sequence ends were manually trimmed in Aliview (Larsson Reference Larsson2014). Each sequence was checked against the NCBI nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify that the desired organism had been sequenced. Alignments were visually checked in AliView and minor misalignments were manually adjusted.
Phylogenetic analyses
Maximum likelihood trees for each locus (not shown) were constructed in raxmlGUI 2.0 (Stamatakis Reference Stamatakis2014; Edler et al. Reference Edler, Klein, Antonelli and Silvestro2021), and the bootstrap support values compared for each clade. Using a 70% bootstrap value threshold, clades were compared and conflict was assumed to be significant where a monophyletic group was supported with bootstrap values ≥ 70% within one locus and the same group of taxa was supported ≥ 70% as non-monophyletic within another locus (Mason-Gamer & Kellogg Reference Mason-Gamer and Kellogg1996). Since no strongly supported conflicts were uncovered among the five loci, downstream relationships and analyses were performed on the concatenated dataset. Analyses were run using raxmlGUI 2.0 to reconstruct a maximum likelihood (ML) concatenated five-locus tree. We used Lepra albescens as the root and ran 1000 thorough maximum likelihood bootstraps with the substitution model set to GTRGAMMA.
Results
We obtained sequence data from 98 specimens, representing c. 52 taxa, for a total of 337 new sequences (Supplementary Material S1, available online). In total, 77 nuITS, 73 nuLSU, 75 mtSSU, 76 Mcm7 and 36 RPB2 sequences were recovered. The final concatenated alignment contained 172 sequences and 5676 positions.
Several conclusions can be drawn from the resulting phylogenetic tree (Fig. 1):
1) Aspilidea is basal to the Megasporaceae, with high support, and consists of two distinct taxa.
2) Aspicilia brucei Owe-Larss. & A. Nordin forms a well-supported clade between Aspilidea and the rest of the Megasporaceae.
3) The relationships among the genera (including Sagedia) are well supported except for Megaspora and Oxneriaria, the positions of which as siblings to Circinaria and Aspicilia respectively have poor support.
4) The infrageneric groups in Aspicilia and Oxneriaria have poor support.
5) Two undescribed species of Aspicilia are indicated.
6) As previously shown by Nordin et al. (Reference Nordin, Savić and Tibell2010), the presence of marginal lobes is confirmed not to be a genus-level character in Megasporaceae, and two species of Aspicilia without marginal lobes should be transferred to Lobothallia.
7) The sequenced collection of Sagedia mastrucata (Wahlenb.) A. Nordin et al. is not conspecific with previous sequenced collections of this species.
Five-locus (nuITS, nuLSU, mtSSU, Mcm7 and RPB2) concatenated maximum likelihood tree. Bootstrap support values ≥ 60% are indicated above branches. New species, new combinations and sequenced types are in bold. Coloured polygons correspond to the 11 currently accepted genera. Roman numerals correspond to the seven supported monophyletic clades. In colour online.
We deal with many of these below.
Taxonomy
A new genus for Aspicilia brucei
The species Aspicilia brucei, described from California (Owe-Larsson et al. Reference Owe-Larsson, Nordin, Tibell, Nash, Gries and Bungartz2007) and subsequently reported from France (Roux et al. Reference Roux, Nordin, Tibell and Sohrabi2011) and the Czech Republic (Vondrák et al. Reference Vondrák, Svoboda, Malíček, Palice, Kocourková, Knudsen, Mayrhofer, Thüs, Schultz and Košnar2022), was known to be morphologically and chemically similar to A. cinerea (L.) Körb. (Owe-Larsson et al. Reference Owe-Larsson, Nordin, Tibell, Nash, Gries and Bungartz2007). However, it differed from that species by having smaller ascospores and shorter, bacilliform conidia. Molecular data from two specimens of A. brucei, both from California and including an isotype (Owe-Larsson 9161), were sequenced and analyzed along with sequences from a Czech Republic specimen downloaded from GenBank. Aspicilia brucei was recovered as a highly supported group near the base of Megasporaceae, and the new genus Antidea is described to accommodate this monotypic clade.
Antidea T. B. Wheeler gen. nov.
MycoBank No.: MB 822149
Similar to Aspicilia in general morphology and in producing norstictic acid but differing in the smaller ascospores and shorter, bacilliform to filiform conidia. Also differing in its molecular sequence data.
Typus generis: Antidea brucei (Owe-Larss. & A. Nordin) T. B. Wheeler.
Etymology
Antidea, an early Latin meaning of ‘to come before’ and ‘in the past’, is a reference to the early diverging position of the genus in the Megasporaceae.
Notes
This monotypic genus is morphologically very similar to some forms of A. cinerea. However, the single species, A. brucei, differs in having smaller ascospores (9–13 × 5–8 μm vs (10–)12–18(–22) × 7–11(–14) μm in A. cinerea), shorter conidia (6–10 μm vs (10–)12–19(–22) μm in A. cinerea), and non-moniliform to submoniliform paraphyses (moniliform in A. cinerea).
Antidea brucei (Owe-Larss. & A. Nordin) T. B. Wheeler comb. nov.
MycoBank No.: MB 822154
Aspicilia brucei Owe-Larss. & A. Nordin, Lichen Flora of the Greater Sonoran Desert Region (Tempe) 3, 73 (2007).
(Fig. 2)
New genus: Antidea brucei and Aspicilia cinerea. A & B, Antidea brucei (A, Knudsen 15069; B, Owe-Larsson 9161—isotype). C & D, Aspicilia cinerea (A, Wheeler 6277; B, Wheeler 7214). Scales = 1 mm. In colour online.
A full description of this species is given by Owe-Larsson et al. (Reference Owe-Larsson, Nordin, Tibell, Nash, Gries and Bungartz2007) and is not repeated here. See Fig. 2 for a visual comparison of Antidea brucei and Aspicilia cinerea.
Two new species of Aspicilia
Massalongo (Reference Massalongo1852) introduced the name Aspicilia for a disparate group of lichens with innate apothecia, including species currently placed in Bellemerea Hafellner & Cl. Roux, Ionaspis Th. Fr. and Rhizocarpon Ramond ex DC. Massalongo (Reference Massalongo1852) also introduced the new name Aspicilia polygonia (Vill.) A. Massal. based on Lichen polygonius Vill., which Choisy (Reference Choisy1929) implied to be the lectotype of the genus. Since Clauzade & Roux (Reference Clauzade and Roux1984) could not locate any material of this species in Villars’ herbarium, Laundon & Hawksworth (Reference Laundon and Hawksworth1988), in their proposal to conserve Aspicilia, selected the illustration of L. polygonius (Villars Reference Villars1789) as the lectotype. Unfortunately, it is impossible to determine to which species this illustration refers, and so the Committee for Fungi amended Laundon & Hawksworth's proposal to include as the conserved type for Aspicilia a Schaerer exsiccate specimen (Exc. Lich. Helv. #127) in Verona (VER) that had been cited by Massalongo (Reference Massalongo1852) in the protologue of his new genus (Gams Reference Gams1993). This specimen is referable to Aspicilia cinerea (L.) Körb. (basionym Lichen cinereus L.), which is the conserved type of the genus. Later, Jørgensen et al. (Reference Jørgensen, James and Jarvis1994) selected the same exsiccate number as the neotype for L. cinereus, but selected the specimen in Uppsala (UPS), rather than the one in Verona, as the type of the species.
Although Aspicilia was initially accepted by contemporary authors (Koerber Reference Koerber1855; Mudd Reference Mudd1861), later authors almost always treated it as a section in Lecanora Ach. (Zahlbruckner Reference Zahlbruckner1928; Magnusson Reference Magnusson1939, Reference Magnusson1951), and it was not until the 1970s that Aspicilia was again widely accepted as a separate genus (Poelt Reference Poelt1974; Roux Reference Roux1977; Clauzade & Roux Reference Clauzade and Roux1984; Hafellner Reference Hafellner1984).
Aspicilia s. str. was highly supported in our analysis, but the relationships within the genus were poorly resolved. We also identified two additional species in the genus that are described below.
Aspicilia indeterminata T. B. Wheeler sp. nov.
MycoBank No.: MB 853846
Similar to the A. cinerea group in its immersed apothecia, greyish thallus, olive green epihymenium, and production of norstictic acid, but differing in the larger ascospores, submoniliform paraphyses, pruinose apothecia, and lack of additional substances.
Type: USA, Montana, Beaverhead Co., Pioneer Mountains, Vipond Park, 45.730623°N, 112.863999°W, 2015 m, on quartzite talus, 23 June 2016, Wheeler 7241 (ASU).
(Fig. 3)
Newly described species. A & B, Aspicilia indeterminata (A, Wheeler 5460; B, Wheeler 7241—holotype). C & D, Aspicilia suavis (C, Wheeler 7226; D, Wheeler 7345—holotype). Scales = 1 mm. In colour online.
Hypothallus lacking. Thallus contiguous, areolate, dense, separated by deep cracks, non-determinate, 4–10 cm diam. Areoles greyish white to grey, epruinose to slightly white pruinose, angular, 0.2–1 mm diam., 0.1–0.5 mm thick. Upper cortex pseudoparenchymatous, 15–30 μm thick, cells to 4–6 μm diam. Algal layer continuous, undulating, 40–50 μm thick, algal cells chlorococcoid, mostly c. 10 μm diam. Medulla obscure, c. 200 μm tall.
Apothecia usually one per areole, lecanorine/aspicilioid, round, with a thin to thick margin the same colour as the thallus or slightly darker; disc sunken, concave, proper margin thin and slightly pruinose, mature disc black, weakly to densely white pruinose, 0.25–1.25 mm diam., eventually raised above the thallus. Exciple pseudoparenchymatous, cells 4–5 μm, dark grey to hyaline, up to 100 μm thick. Epihymenium 10–20 μm tall, olive green, K+ brown. Paraphyses submoniliform, top 2–3 cells rounded and expanded to 4 μm, occasionally branching and anastomosing, 2–3 μm thick. Hymenium 100–130 μm tall, I+ blue. Asci 8-spored, c. 90 × 25 μm, Aspicilia-type. Ascospores simple, hyaline, (15–)17–21(–22) × 10.5–13 μm, broadly ellipsoid. Hypothecium hyaline, 40–60 μm tall, with small oil droplets, algal layer not continuous below hypothecium.
Pycnidia c. 150 × 100 μm, rare, inconspicuous. Conidia straight, 14–15 × 1–1.5 μm.
Spot tests and chemistry
Spot tests: K+ red crystals in the type specimen, but K− in the additional specimens; however, norstictic acid was present in all specimens analyzed with TLC.
Etymology
Named for the indeterminate thalli, and confusion it created when initially discovered.
Ecology and distribution
Currently known only from Montana, where it has been collected from granitic boulders and quartzite talus in dry, forested montane sites, from 1500–2015 m altitude.
Notes
Aspicilia indeterminata is an indistinctive, thin to thick, greyish Aspicilia with sunken discs. In the type specimen the discs are weakly pruinose, in contrast to the densely pruinose discs of other specimens. It is close to A. verrucigera Hue in general morphology, conidia and spore size, but differs in lacking stictic acid and having an epihymenium that is distinctively green to olive green, as opposed to olive-brown. Aspicilia cinerea differs in having smaller ascospores, moniliform paraphyses, an olive-brown epihymenium and non-pruinose apothecial discs.
Additional specimens examined
USA: Montana: Lewis and Clark Co., Orofino Gulch Road, 46.542127°N, 112.097662°W, 1525 m, on Tertiary granite boulders, 2012, Wheeler 5460, 5464, 5470, 5471, 5472 (hb. Wheeler).
Aspicilia suavis T. B. Wheeler sp. nov.
MycoBank No.: MB 853847
Similar to Aspicilia laevata (Ach.) Arnold in its thin, greenish grey, smooth, weakly rimose to uncracked thallus, immersed apothecia, habitat near mountain streams in shaded locales, olive green epihymenium and production of stictic and norstictic acids, but differing in the slightly larger ascospores, submoniliform paraphyses, and multilocus sequence data.
Type: USA, Montana, Ravalli Co., Sapphire Mountains, Skalkaho-Rye Road near the summit, 48.985375°N, 113.859990°W, 2145 m, on granodiorite boulders in small stream, 16 September 2016, Wheeler 7345 (ASU).
(Fig. 3)
Hypothallus lacking, to thin and greyish. Thallus continuous, thin, smooth and uncracked, or becoming sub-rimose when older and thicker, non-determinate, 4–18 cm diam. Surface greenish grey to dark olive green, epruinose. Areoles continuous, 0.5–3 mm diam., 0.1–0.25 mm thick. Upper cortex pseudoparenchymatous, 20–35 μm thick. Algal layer continuous, 20–40 μm thick, algal cells chlorococcoid, mostly c. 10 μm diam. Medulla obscure, whitish.
Apothecia 1–3 per areole, aspicilioid, round, sunken but with a 100–130 μm thick raised thalline margin the same colour as the thallus or slightly darker, proper margin thick and darker than the thallus tissue, disc dark green to black, epruinose, 0.25–1.0 mm diam., eventually raised above the thallus. Exciple pseudoparenchymatous, cells 4–5 μm, dark grey to hyaline, up to 130 μm thick. Epihymenium 15–20 μm tall, olive blue-green. Paraphyses non-moniliform to submoniliform, 2–3 μm thick, top 1–2 cells rounded and expanded up to 3 μm, coherent even in KOH. Hymenium 100–130 μm tall, I+ blue. Asci 8-spored, c. 100 × 30 μm, Aspicilia-type. Ascospores simple, hyaline, (19–)21–24(–26) × 12–15 μm, broadly ellipsoid. Hypothecium hyaline, olive green in upper part, 80–125 μm tall, algal layer not continuous below hypothecium.
Pycnidia c. 175 × 100 μm, common, inconspicuous black ostiole. Conidia long, straight, (19–)21–23(–25) × 1–1.25 μm.
Spot tests and chemistry
Spot tests: K+ yellow diffusion, Pd+ red; stictic acid (major) and norstictic acid (trace) present in both specimens when analyzed by TLC.
Etymology
Named for the beautiful smooth greenish thalli and calm, idyllic streamside mountain settings where it is often found.
Ecology and distribution
Currently known only from Montana, where it has been collected from granodiorite and argillite boulders in the splash zone of mountain lakes and streams from 1700–2200 m altitude.
Notes
Aspicilia suavis is an indistinctive, thin, greenish grey Aspicilia with sunken discs that is found on splash zone siliceous boulders in montane and alpine habitats. In the type specimen the thallus is distinctly green, while in other specimens it is paler greenish grey. The discs are epruinose, and raised in smooth, steep-sided mounds. It is very close to A. laevata in general morphology, conidia shape and size, chemistry, and preference for shady streamside sites, but it has larger ascospores (19–26 μm vs 13–20 μm). However, molecular data place A. laevata in a poorly supported clade with A. epiglypta (Norrl. ex Nyl.) Hue, A. subradians (Nyl.) Hue, A. indissimilis (H. Magn.) Räsänen and others, and A. suavis in an unsupported ‘pacifica group’ clade. Aspicilia aquatica (Fr.) Körb. s. lat. can be similar and occurs in similar habitats, but it has moniliform paraphyses, smaller conidia and a paler yellowish thallus that lacks a thalline chemistry. Much of what has been identified in western North America as A. aquatica and A. laevata will most likely be referable to A. suavis.
Additional specimens examined
USA: Montana: Lake Co., Mud Lake, Mission Mountains, 47.615719°N, 113.994752°W, 1768 m, on argillite boulders along southern shore, 2016, Wheeler 7225 and 7226 (hb. Wheeler).
A second species of Aspilidea
The genus Aspilidea was introduced for the single species A. myrinii (Fr.) Hafellner (Hafellner & Türk Reference Hafellner and Türk2001). However, the anomalous position of the species in Aspicilia was previously noted by Roux (Reference Roux1977), who included it with Bellemerea but did not make a formal new combination. Later, Clauzade & Roux (Reference Clauzade and Roux1984) acknowledged that A. myrinii was not congeneric with Bellemerea but did not suggest an alternative placement. Aspilidea myrinii has a superficial resemblance to the type species of Bellemerea, B. alpina (Sommerf.) Clauzade & Cl. Roux, in that both species have immersed apothecia with a pseudothalline margin and a thallus containing norstictic acid with an amyloid (I+ violet) medulla. However, they differ in important anatomical characteristics, most notably that the ascus of Bellemerea has a distinctive amyloid tube-like structure (Porpidia-type) and belongs in Lecideaceae, whereas the ascus of Aspilidea lacks the tube-like structure and belongs in Megasporaceae.
Hafellner & Türk (Reference Hafellner and Türk2001) separated Aspilidea from Aspicilia by the I+ pale purple medulla of the thallus, the euamyloid reaction of the hymenial jelly (hemiamyloid in Aspicilia), fine features of the ascus structure, conspicuous pycnidia with conidiophores of type II–III according to Vobis (Reference Vobis1980), and different lichenicolous fungi.
In our analysis, Aspilidea is early diverging in the Megasporaceae and, as in Miadlikowska et al. (Reference Miadlikowska, Kauff, Högnabba, Oliver, Molnár, Fraker, Gaya, Hafellner, Hofstetter and Gueidan2014) but here with high support, is included in the family. The clade included previously sequenced collections from Europe as well as new specimens from Newfoundland, Quebec and Alaska that represent an additional species, for which the epithet ‘subadunans’ is available.
Aspilidea subadunans (Vain.) T. B. Wheeler, J. W. McCarthy & Fryday comb. nov.
MycoBank No.: MB 853844
Lecanora myrinii var. subadunans Vain. [as ‘myrini’], Meddeland. Soc. Fauna Fl. Fenn. 6, 169 (1881).—Aspicilia subadunans (Vain.) Räsänen, Ann. Bot. Soc. Zool.-Bot. Fenn. ‘Vanamo’ 12(1), 78 (1939) nomen non funga.—Aspicilia myrinii var. subadunans (Vain.) Oxner [as ‘myrini’], in Kopaczevskaja et al., Opredelitel’ Lishaĭnikov SSSR Vypusk (Handbook of the Lichens of the USSR) (Leningrad) 1, 191 (1971); nom. inval. (ICNafp Art. 41.5); type: ‘In rupe syenitica loco aprico ventoso in regione subalpina montis Päänuorunen in Lapponia Rossica.’ (TUR!—holotype).
Comparison of the thalli of Aspilidea species. A, C & E, Aspilidea subadunans (A, TUR–V-05894; C, McCarthy 4153; E, McCarthy 4352). B, D & F, Aspilidea myrinii (B, O–L-20820; D, Tønsberg 42336; F, O–L-175683). Scales = 1 mm. In colour online.
Comparison of the ascospores of Aspilidea species. A, C & E, Aspilidea subadunans (A, McCarthy 3195; C, McCarthy 4161; E, TUR–V-05894). B, D & F, Aspilidea myrinii (B, O–L-175683; D, O–L-190124; F, O–L-207353) Scales = 10 μm. In colour online.
Hypothallus thin to thick, usually lacking, but in some specimens grey-black, occasionally visible between areoles and as a thin zone at the thallus margin. Thallus areolate, contiguous, separated by thin cracks, nondeterminate, up to 20 cm diam.; sometimes bimorphic with a thin primary thallus and a secondary thallus of ±dispersed convex areoles. Areoles cream-white to bluish grey, epruinose to faintly white pruinose, often shiny with a thin epinecral layer, irregular, even to irregularly ridged, 0.5–1.0 mm diam., 0.1–0.75 mm thick, 1–2 apothecia per areole. Upper cortex pseudoparenchymatous, 30–40 μm thick, epinecral layer thin, 3–7 μm thick, indistinct. Algal layer thin, discontinuous, irregular, 40–60 μm thick, algal cells chlorococcoid, 8–10 μm diam.
Apothecia 1–2 per areole, lecanorine, round, with thick outer thallus-coloured margin and a thinner inner darker ring coloured like the disc; immature disc initially sunken, soon becoming elevated and prominent with a narrowing base, margin smooth; mature disc smooth to slightly roughened, black, epruinose to blue-grey pruinose, 0.25–1.25 mm diam. Exciple pseudoparenchymatous, cells 4–6 μm, outer layer brownish, inner layer hyaline, 80–130 μm thick. Hymenium 80–120 μm, I+ blue; epihymenium 20–25 μm tall, dark brown to black, K+ brownish, N+ greenish. Paraphyses not branching, non-moniliform, thin, 1 μm thick, apices unexpanded, with pale brown tips. Asci 8-spored, 50–60 × 15–25 μm, Aspicilia type. Ascospores simple, hyaline, narrowly ellipsoid, (11.3–)16.77 ± 2.50(–22.0) × (4.9–)7.14 ± 1.19(–11.0) μm, l/w ratio (1.65–)2.39 ± 0.42(–3.33); n = 103. Hypothecium hyaline, 80–100 μm, hyaline above, grading into greyish medulla below, algal layer not continuous below hypothecium.
Pycnidia c. 150 × 100 μm, rare to common, with a black, slightly elevated ostiole; conidia short bacilliform, (5–)5.5–6.5(–7) × 1–1.5 μm.
Spot tests and chemistry
Medulla K+ yellow to red crystals in section, I± pale brownish violet. TLC: norstictic acid.
Ecology and distribution
A northern species known from 100–1250 m altitude. In North America, it is known from Alaska in the west to Newfoundland in the east (Fig. 6). It has been collected from argillite, schist, phyllite, and quartzite.
Known distribution of Aspilidea subadunans in North America. In colour online.
To assess the distribution of this species and A. myrinii in North America, we examined all known collections of the latter species in North American herbaria available to us (c. 50). All were misidentified. They were revised as A. subadunans, Aspicilia cinerea s. lat. and Aspicilia spp., as well as Bellemerea alpina, Lecidea lactea Flöke ex. Schaer., L. swartzioidea Nyl. and, in one case, a heavily grazed Rhizocarpon species (Supplementary Material S2, available online). Thus, A. myrinii has not been correctly reported from North America. We also examined A. myrinii collections from Norway, Sweden and Finland, only one of which proved to be A. subadunans (UPS L-520804; Supplementary Material S2).
Notes
Aspilidea subadunans is a distinctive species owing to its thick, white to cream-coloured thallus and prominent, thick-margined apothecia often with pruinose discs. It is similar to A. myrinii in morphology and chemistry, having a yellowish to greyish white thallus, small bacilliform conidia and producing norstictic acid, but it differs in the elevated apothecia with prominent, often pruinose discs with double margins and narrower ascospores (Figs 4 & 5, Table 3).
Comparison of ascospore dimensions of Aspilidea species. Measurements are given as (lowest recorded–)x̄ ± standard deviation(–highest recorded); n = number of spores measured
North American specimens have mostly been labelled as Aspicilia arctica Lynge ex Oxner, A. cinerea or Aspilidea myrinii. Aspicilia arctica and A. cinerea have long filiform conidia (15–23 μm) in contrast to the short bacilliform conidia (5–7 μm) of A. subadunans, as well as a hemiamyloid hymenium; Aspilidea myrinii has non-pruinose, black, angular or irregular, immersed to slightly sessile apothecia in contrast to the raised, thick-margined apothecia of A. subadunans. All three species differ from A. subadunans in having broadly ellipsoid ascospores.
When Räsänen (Reference Räsänen1939) raised Vainio's name to the rank of species, the exsiccatae specimen he cited was Aspilidea myrinii not A. subadunans but this does not invalidate the combination (ICNafp Art. 7.3). Räsänen (Reference Räsänen1939) also considered Aspicilia cinerea var. sallensis Räsänen to be a synonym of Lecanora myrinii var. subadunans Vain. However, the syntypes in H (H9503235, H9503236, H9503237) have been annotated as A. myrinii and our examination of these collections confirmed that they are referable to A. myrinii not A. subadunans. We designate here one of these syntypes (H5903235) as the lectotype, with the other two collections being isolectotypes (MycoBank No. MBT 10020079).
Selected additional specimens examined
See Supplementary Material S2 and map showing the distribution of A. subadunans in North America (Fig. 6).
New combinations in Lobothallia
Lobothallia was originally described as a subgenus of Aspicilia by Clauzade & Roux (Reference Clauzade and Roux1984) but was elevated to genus by Hafellner (Reference Hafellner1991). Although initially proposed for a small group of species with elongate marginal lobes, as has been shown with other recent additions to Lobothallia (Nordin et al. Reference Nordin, Savić and Tibell2010; Kou et al. Reference Kou, Li and Ren2013; Roux et al. Reference Roux, Nordin and Bertrand2016), the extent of lobation is not an informative phylogenetic character for the genus. Based on our phylogeny, we here transfer two more species without extended marginal lobes to the genus.
Lobothallia determinata (H. Magn.) T. B. Wheeler comb. nov.
MycoBank No.: MB 822174
Lecanora determinata H. Magn., Lichens from Central Asia 1, 96 (1940).—Aspicilia determinata (H. Magn.) N. S. Golubk., Nov. Syst. Niz. Rast. 9, 236 (1972); type: China, Kansu [Gansu], Chia-yii-kuan, ad campum, Huang-tsao-ying, alt. 1580 m, ‘on coarse-grained sandstone, HCl+ bubbling’, 27.9.1930, Birger Bohlin Lich. No. 7 (S—holotype).
(Fig. 7A)
New combinations in Lobothallia. A, L. determinata (Wheeler 6017). B, L. peltastictoides (Knudsen 14420). Scales = 1 mm. In colour online.
Lobothallia peltastictoides (Hasse) T. B. Wheeler comb. nov.
MycoBank No.: MB 822175
Lecanora peltastictoides Hasse., Bryologist 17, 63 (1914).—Aspicilia peltastictoides (Hasse) K. Knudsen & Kocourk., Mycotaxon 124, 354 (2013); type: USA, California, Palm Springs, on granite, 1901, Hasse 861 (FH—holotype).
(Fig. 7B)
Sagedia mastrucata (Wahlenb.) A. Nordin, Savić & Tibell
The collection of Sagedia mastrucata that we sequenced did not cluster with sequences from other collections of this species that we downloaded from GenBank. Clearly either our collections (Wheeler 6430, 6434; hb. Wheeler) or those sequenced previously are incorrectly identified. The lectotype of Lichen mastrucatus Wahlenb. (Nordin et al. Reference Nordin, Tibell and Owe-Larsson2007) and the description given by Wahlenberg (Reference Wahlenberg1812) match our specimen collected from Varangerfjord in northern Norway, near the type locality of Lichen mastrucatus. It is probable, therefore, that previously sequenced specimens from Scandinavia and elsewhere identified as S. mastrucata are a different, presumably undescribed species. These are included in our phylogenetic tree (Fig. 1) as S. aff. mastrucata.
Lichenicolous fungus on Aspilidea subadunans
A lichenicolous fungus with large perithecia (c. 0.2–0.4 mm diam.) and simple ellipsoid ascospores ((8.7–)12.0 ± 1.40(–15.1) × (4.9–)6.6 ± 0.73(–9.1) μm, l/w ratio (1.24–)1.84 ± 0.31(–2.94), n = 68) growing on several collections of A. subadunans was identified as Sagediopsis aspiciliae Nik-Hoffm. & Hafellner (Hoffmann & Hafellner Reference Hoffmann and Hafellner2000) (Fig. 8). This has been reported previously on A. myrinii in Europe but is reported here for the first time from North America.
Sagediopsis aspiciliae (McCarthy 4423). A, thallus of Aspilidea subadunans with perithecia of S. aspiciliae. B, ascospores of S. aspiciliae. Scales: A = 1 mm; B = 10 μm. In colour online.
Two species of Sagediopsis have been reported from Aspilidea myrinii: S. aspiciliae and S. fissurisedens Hafellner (Triebel Reference Triebel1993). Sagediopsis fissurisedens has larger perithecia (0.4–0.7 mm diam.) than S. aspiciliae and fusiform, 3-septate ascospores, 12–17 × 5–8 μm. We have not observed S. fissurisedens on any specimen of A. subadunans, or S. aspiciliae on any specimen of A. myrinii s. str. Given these observations and Hafellner's statement in Hafellner & Türk (Reference Hafellner and Türk2001) that the type specimen of Lecanora myrinii var. subadunans Vain. supported a different species of Sagediopsis to that reported from A. myrinii (Hafellner & Türk Reference Hafellner and Türk2001), we suspect that previous reports of the occurrence of S. aspiciliae on A. myrinii are misidentifications of the host species and that the two species of Sagediopsis occur only on the two different species of Aspilidea. However, we note that the Sagediopsis sp. that occurs on the type species of L. myrinii var. subadunans has larger ascospores (14–18 × 8–11 μm, x̄ 14.8 × 9.7 μm, l/w = 1.5, n = 23) than are reported in the literature for S. aspiciliae and observed by us on other collections of this species.
Selected specimens examined
See Supplementary Material S2 (available online).
Discussion
This study is the largest comprehensive phylogenetic analysis of the family Megasporaceae so far performed. The family formed a well-supported monophyletic group composed of seven highly supported clades, corresponding to the genera Aspilidea, Antidea, Lobothallia/Teuvoa, Circinaria/Megaspora/Aspiciliella/Atrostelia, Sagedia, Oxneriaria and Aspicilia (Fig. 1: Roman numerals I–VII). All clades were recovered as monophyletic and highly supported, with their relationships to each other also strongly supported, with the exception of the position of Megaspora and Oxneriaria as siblings to Circinaria and Aspicilia s. str., respectively. This analysis confirms the monophyly of the family, placing it with high support as sister to Ochrolechiaceae.
Systematic position of Aspilidea and Antidea
Our phylogeny shows that Megasporaceae is closely related to Ochrolechiaceae and that two genera, Aspilidea and Antidea, occupy a basal position in Megasporaceae close to Ochrolechiaceae. Although the two families have some similarities, they also differ in several respects.
1) Morphology
The thalli of Ochrolechiaceae are pale creamy white in colour whereas those of Megasporaceae are predominantly pale to dark grey. Antidea has a grey thallus and resembles a species of Megasporaceae whereas Aspilidea has a pale cream-coloured thallus and more closely resembles Ochrolechiaceae.
Apothecia in Ochrolechiaceae are sessile to substipitate, discoid, lecanorine with a distinct, well-developed proper exciple, whereas those in Megasporaceae are immersed, sunken to sessile with a poorly developed exciple. The apothecia of Antidea and Aspilidea resemble those of Megasporaceae.
Ascospores in Ochrolechiaceae have thick walls and are large to very large (35–65(–300) × (18–)19–32(–100) μm in Ochrolechia) whereas those of Megasporaceae are thin-walled and smaller ((8–)15–25(–35) × 5–15(–26) um). The ascospores of Aspilidea and Antidea are thin-walled and within the range of Megasporaceae, and are much smaller than those of Ochrolechiaceae.
The epihymenium in Ochrolechiaceae is either hyaline or pale brown, whereas in Megasporaceae it is usually olivaceous (K+ brown, N+ green; Caesiocinerea-green). Consequently, the apothecial discs of Ochrolechiaceae are pale-coloured (yellow to pink to orange) whereas those of Megasporaceae are darkly pigmented (dark grey to black). Both Antidea and Aspilidea have an olivaceous epihymenium and dark-coloured apothecia.
Paraphyses in Ochrolechiaceae are thin, densely branching and anastomosing whereas in Megasporaceae they are thick, moniliform to submoniliform, unbranched to weakly branched, and not densely anastomosing. Both Antidea and Aspilidea have relatively narrow paraphyses but in Antidea they are submoniliform, suggesting a closer relationship to Megasporaceae. Aspilidea has non-moniliform paraphyses but in Aspilidea myrinii they are branched and anastomosing whereas those of A. subadunans are unbranched and not anastomosing.
Asci of Ochrolechiaceae have thick, multilayered, strongly amyloid walls, whereas Megasporaceae has thin-walled asci with a thin outer coat K/I+ blue, and K/I− wall and apical dome. The asci of Aspilidea and Antidea resemble those of Megasporaceae.
Aspilidea resembles Ochrolechiaceae in having a euamyloid (I+ blue) hymenium, whereas it is hemiamyloid (I+ orange-brown) in Megasporaceae and Antidea.
Many Ochrolechia species can have sterile vertical tissues dividing the hymenium. These are unknown in Megasporaceae, Aspilidea and Antidea.
Conidia in Ochrolechiaceae are elongate cylindrical to bacilliform (4–6 μm), whereas those in Megasporaceae are bacilliform to filiform long (5–40 μm). The conidia in both Aspilidea and Antidea are bacilliform (5–8 μm). Although there is some overlap in conidia size between Ochrolechia and Antidea and Aspilidea, other genera placed in Megasporaceae also have shorter conidia (e.g. Lobothallia and Circinaria).
2) Secondary metabolite chemistry
Ochrolechiaceae usually produces orcinol depsides, depsidones, tridepsides or xanthones (gyrophoric, lecanoric, olivetoric, 5-O-methylhiascic, 4,5-di-O-methylhiascic, 4-O-demethylmicrophyllinic and hiascic acids, and lichexanthone), whereas Megasporaceae produce aspicilin and norstictic, connorstictic, hyposalazinic, hypostictic, stictic and substictic acids, or no substances. Aspilidea and Antidea both produce norstictic acid indicating a closer affinity with Megasporaceae.
3) Ecology
Megasporaceae is predominantly saxicolous whereas Ochrolechiaceae is also corticolous, lignicolous and terricolous. Both Antidea and Aspilidea are only saxicolous.
Given its grey thallus, euamyloid hymenium and submoniliform paraphyses, we consider the systematic position of Antidea to be firmly within Megasporaceae, but the position of Aspilidea is more ambivalent. The genus, apparently, occupies an intermediate position between the two families but we are reluctant to erect a new family for a single genus containing only two species without further evidence. Although some characters of Aspilidea suggest an affinity with Ochrolechiaceae, most point towards it being closer to Megasporaceae and so we include it in that family. However, we acknowledge that this may change when the systematics of Pertusariales are more fully explored.
Status of the genera
Two of the clades mentioned above are composed of more than one genus and it is worth considering the status of these genera. The two genera in the Lobothallia clade, Lobothallia and Teuvoa, share several morphological characters including short conidia and small ascospores. However, the recently described genus Teuvoa differs from Lobothallia by growing on lignin, and lacking thalline chemistry and a subhymenial algal layer. If Teuvoa is accepted at the genus level, then a case could be made for erecting additional genera to accommodate L. determinata and, perhaps, the L. peltastictoides group, resulting in the Lobothallia clade being split up into four genera. Alternatively, Lobothallia could be expanded to include Teuvoa, but here we take the conservative approach and retain Lobothallia and Teuvoa as distinct genera.
Similarly, the Circinaria clade also includes the genera Megaspora, Aspiciliella and Atrostelia, which could either be subdivided into separate genera or subsumed into Circinaria, but we again take the conservative approach and retain them as separate genera.
Given the arbitrary level at which genera are recognized, another approach would be to recognize the smaller clades inside Circinaria and Lobothallia as subgenera, or even to return them all to Aspicilia and recognize the individual clades I–VII as subgenera.
Conclusions
The phylogeny presented here represents a solid foundation for future research on Megasporaceae. The inter-family groups are well defined and supported, and the relationships among them, with the exception of Oxneriaria and Megaspora, are also well supported. However, worldwide, Megasporaceae comprises c. 300 species (Outline of Fungi 2024; Wijayawardene et al. Reference Wijayawardene, Hyde, Dai, Sánchez-García, Goto, Saxena, Erdoğdu, Selçuk, Rajeshkumar and Aptroot2022) but our phylogeny includes only 88 (i.e. c. 30%). The Aspicilia/Oxneriaria clade is particularly poorly represented, with only 50 out of an estimated global total of c. 200 being included and it is probable that the generic divisions proposed here will need adjustment as sampling improves. In addition, the intergeneric groups within Aspicilia and Oxneriaria are still poorly supported and unsettled, and further work involving different species and additional loci are required to elucidate these relationships.
Acknowledgements
Funding for this project was provided from various sources. TBW was funded by a University of Montana Institute on Ecosystems Graduate Enhancement Fellowship. Funding for JMc was generously provided by the Jesuits of Canada, Jesuit Conference of Canada and the United States, Bishop Douglas Crosby, OMI (Diocese of Hamilton, Ontario), Sisters of St Joseph of Toronto, Kenneth Yeung, Frank Morneau, Jerry McCullough and Robert Wong, SJ. We are grateful to the custodians of the many herbaria who have loaned us collections in their care for this study: ALA, ASU, BG, CANL, FH, H, MIN, O, QFA, SBBG, TUR, UC, UCR, UPS, WIS, WTU, and the private herbaria of Curtis Björk, Trevor Goward, Kerry Knudsen and Peter Nelsen. TBW would like to thank Dan Vanderpool, John McCutcheon and Toby Spribille, who were instrumental in the early stages of this study. JMc thanks Tor Tønsberg for the gift of a specimen of Aspilidea myrinii from Norway.
It gives us great pleasure to dedicate this paper to Teuvo Ahti, who has been a constant source of advice and information throughout our lichenological journeys. In particular, JMc expresses his sincere gratitude to Teuvo (Ted) for his generous mentorship, support and friendship over the years, and for his unwavering dedication to lichen research in Newfoundland and Labrador for more than 68 years. At the invitation of the Government of Newfoundland and Labrador, Teuvo first arrived in Newfoundland in the summer of 1956 as an undergraduate student studying the quality of Newfoundland caribou habitat, particularly the availability of reindeer lichens. His nine visits to Newfoundland reflect Teuvo's deep attachment to the people and land of the province. He has named two Cladonia species after the province, Cladonia terrae-novae Ahti (1960) and Cladonia labradorica Ahti & Brodo (1981).
Author ORCIDs
Tim Wheeler, 0000-0003-0668-8662; John McCarthy, 0009-0003-1531-300X; Alan Fryday, 0000-0002-5310-9232.
Competing Interests
The authors declare none.
Supplementary Material
The Supplementary Material for this article can be found at https://doi.org/10.1017/S0024282924000239.
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