A new Calicium on Ramboldia

Abstract

A lichenicolous species, Calicium ramboldiicola, growing on Ramboldia elabens is described. In phylogenetic analyses with 22 Calicium taxa, based on 121 sequences from five DNA regions (mtSSU, Mcm7, nuITS, nuLSU, β-tubulin), the new species formed a strongly supported clade with C. abietinum and C. verrucosum. Although the ascomata of the new species resemble miniatures of those of C. abietinum, no morphological synapomorphies for this clade were found. Calicium ramboldiicola is known from boreal and hemiboreal areas of northern Europe and north-eastern North America. It is the second known lichenicolous species of Calicium and the first found on a lichen in the Lecanorales.

Introduction

The genus Calicium Ach. in Caliciaceae Chevall. includes mazaediate fungi (Prieto et al. 2013) with stalked ascomata and mostly 1-septate, dark, thick-walled spores with distinctive surface ornamentation. A recent molecular study supports a wider circumscription of the genus to also include some species with sessile ascomata that were previously placed in Cyphelium Ach. (Prieto & Wedin 2017). However, Cyphelium sessile (Pers.) Trevis. and C. marcianum B. de Lesd., which are lichenicolous on species of Pertusaria and Lepra, were transferred to Acolium (Prieto & Wedin 2017).

Regional revisions of Calicium species in Australasia (Tibell 1987), Europe (Tibell 1999a), North America (Selva et al. 2023a, b), South America (Tibell 1996, 1998) and the Himalayas (Tibell 2006) have contributed descriptions of numerous species, and Calicium has thus been rather well investigated on a global scale. With the new circumscription and the recent description of additional species, it is known to include 36 species (Tibell & Knutsson 2016; Prieto & Wedin 2017; Selva et al. 2023a). Among these is the recently described Calicium episcalaris Tibell & Knutsson, parasitic on Hypocenomyce scalaris (Ach. ex Lilj.) M. Choisy (Tibell & Knutsson 2016; Selva et al. 2023b). All other described species of Calicium have a distinct, autonomous thallus that varies from thick and verrucose in some species to immersed in others.

Three of the authors were therefore surprised to encounter, independently of each other, what looked like a perfectly ‘classical’ small Calicium growing on the thallus of Ramboldia elabens (Fr.) Kantvilas & Elix in boreal and hemiboreal areas of northern Europe and north-eastern North America. The first collection was made by one of us (LT) in 1977 in Sweden. Since then, we have found the species at additional localities in Sweden, Norway and Canada, and we have also detected several other specimens in herbaria.

Material and Methods

Microscopy

Squash preparations and hand-cut sections of apothecia were mounted in water, 10% KOH (K), 1.5% Lugol's solution (I), and lactophenol cotton blue (LCB), and examined with light microscopy (LM). Measurements of ascospores and anatomical characters were made in water and are reported as (minimum–)a–b(–maximum) (n = number of measurements), where ‘a’ is the arithmetic mean minus 1 standard deviation (SD) and ‘b’ is the arithmetic mean plus 1 SD. Apothecial dimensions were measured in air-dried specimens examined with a stereomicroscope. For scanning electron microscopy (SEM), air-dried specimens were mounted on stubs, coated with gold in an Edwards S150A sputter coater, and examined at 15 kV in a JEOL2 JSM 6400 instrument at the University of New Brunswick.

Taxon sampling

Five specimens of the new species and a wide selection of species of the genus Calicium were included in the phylogenetic analysis (Table 1). Based on previous studies (Prieto & Wedin 2017), three members of the Caliciaceae (Diplotomma alboatrum (Hoffm.) Flot., Pyxine cocoes (Sw.) Nyl. and Pyxine sorediata (Ach.) Mont.) were used as outgroups.

Table 1. Details on taxa used in the phylogenetic analyses, including voucher information and GenBank Accession numbers. New species and sequences are in bold.

Molecular techniques

Sequences of Calicium were either downloaded from GenBank or produced in the laboratory for the new species. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen), according to the manufacturer's instructions. Five regions were amplified: nuITS, nuLSU, mtSSU, β-tubulin and Mcm7. The nuITS was amplified with the primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990), and the nuLSU with LR0R (Rehner & Samuels 1994), LR3, LR3R, LR6 and LR7 (Vilgalys & Hester 1990). We used the primers mtSSU1 and mtSSU3R for amplification of the mtSSU region (Zoller et al. 1999), Mcm7-709for and Mcm7-1348rev (Schmitt et al. 2009) for amplification of the Mcm7 region and Bt3-LM and Bt10-LM for the protein coding β-tubulin (Myllys et al. 2001).

PCR amplifications were carried out using Illustra™ Hot Start Mix RTG PCR beads (GE Healthcare, UK) in 25 μl, adding 3–6 μl of diluted genomic DNA, 10 μM of each primer and distilled water. The following amplification programme was used: initial denaturation at 95 °C for 15 min, 35 cycles of 95 °C for 45 s, 54–56 °C for 50 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products were purified with ExoSAP-IT (USB Corporation, Santa Clara, California, USA) and sequenced using the same amplification primers.

Sequences were assembled and edited using Sequencher v. 4.10.1. (Genes Codes Corporation, Ann Arbor, Michigan, USA) and subsequently aligned using MacClade v. 4.01 (Maddison & Maddison 2001). Ambiguous regions (sensu Lutzoni et al. 2000) and introns were delimited manually and excluded from phylogenetic analyses.

Phylogenetic analyses

Each individual gene region was analyzed using maximum likelihood (ML) implemented in RAxML v. 8.2.12 (Stamatakis 2014) with a GTRGAMMA model for tree inference and bootstrapping with a GTRCAT model and 1000 replicates. Gene-tree incongruence was checked by comparing maximum likelihood bootstrap values (ML-BS) between the individual gene trees. After checking there was no conflict among clades, data were combined into a single concatenated data matrix. The combined maximum likelihood (ML) analysis was run with seven distinct partitions (nuITS, nuLSU, mtSSU, first and second codon positions of the Mcm7 and β-tubulin and the third codon position of the Mcm7 and β-tubulin) as individual analyses. All analyses were run on the CIPRES Science Gateway v. 3.3 (Miller et al. 2010). The resulting trees were visualized and edited in Figtree v. 1.4.4. (Rambaut 2010).

Results

The concatenated data matrix consisted of 25 taxa, 22 being Calicium species, and three outgroups. Calicium was represented by 109 sequences, of which 15 are new. In the analysis (Fig. 1), Calicium was strongly supported, and within the genus two major clades are apparent. Calicium ramboldiicola, together with C. abietinum and C. verrucosum Tibell, belongs to a strongly supported subclade. The other lichenicolous Calicium species, C. episcalaris, belongs to the same major clade as C. ramboldiicola.

Figure 1. Phylogeny of Calicium using a combination of five loci (nuITS, nuLSU, mtSSU, Mcm7 and β-tubulin) resulting from maximum likelihood (ML) in RAxML, with bootstrap values shown above nodes. Strongly supported branches (ML ≥ 70) and C. ramboldiicola are shown in bold.

Taxonomy

Calicium ramboldiicola Tibell, S. R. Clayden & Wedin

MycoBank No.: MB 856766

Resembling Calicium abietinum but occurring on Ramboldia elabens, lacking a distinct thallus, and with smaller apothecia, shorter asci, and ascospores with more coarsely and irregularly cracked ornamentation.

Type: Sweden, Lule Lappmark, Jokkmokk parish, River Pärlälven, W of Lake Nårvejaure, valley of the brook Laddonbäcken, on decorticated fallen Pinus, 66°36ʹ22ʺN, 19°16ʹ54ʺE, alt. c. 360 m, 2021, Wedin 9898 (S—holotype; CANL—isotype). The type has a full representation of all five DNA regions used in this study, and was chosen for this reason.

(Fig. 2)

Figure 2. Calicium ramboldiicola. A, apothecia arising from thallus and apothecia of Ramboldia elabens (Clayden 21343). B, longitudinal section of mature apothecium, LM (Clayden 21343). C, immature and mature apothecia, arising from thin to endoxylic area of thallus of R. elabens, SEM (Clayden 24619). D, mature ascospores, liberated from asci, LM (Clayden 28675). E, capitulum in oblique view, with wall rupturing apically and exposing the mazaedium, SEM (Clayden 24619). F, mature ascospore with surface ornamentation formed by irregular cracking, SEM (Clayden 21343). G, mature ascospore constricted at septum and with coarsely ornamented surface, SEM (Clayden 21343). LM = light microscopy; SEM = scanning electron microscopy. Scales: A = 1 mm; B & C = 100 μm; D = 10 μm; E = 50 μm; F & G = 5 μm. In colour online.

Thallus not evident, immersed in the thallus of Ramboldia elabens, not causing obvious necrosis or discoloration of the host lichen.

Apothecia on the areoles and rarely the apothecia of R. elabens, stalked to nearly sessile, with a black capitulum and dark olive-brown stalk, shiny except for the black mazaedium, lacking pruina, (0.25–)0.30–0.44(–0.55) mm tall (n = 30), all parts K−, I−. Stalk 0.08–0.20 mm diam., consisting of densely interwoven thick-walled hyphae with lumina 0.7‒2 μm wide, central part dark olive-brown, grading to pale brown to hyaline in outer part, with an outermost clear gelatinous layer 5–10 μm wide. Capitulum initially globose to obovoid, becoming flat-topped-cupulate, (0.15–)0.17–0.27(–0.35) mm diam. (n = 30). Excipulum to c. 50 μm thick at base, narrowing to c. 20 μm laterally, structure continuous with that of the stalk, of ±isodiametric to somewhat elongated cells with strongly thickened walls, inner part dark olive-brown, outermost hyaline gelatinous layer thinner than on the stalk. Hypothecium c. 30–60 μm tall, slightly concave at top, upper part concolorous with inner part of exciple and stalk, lower part hyaline to very pale brown. Asci cylindrical to narrowly clavate, (34–)35.8–41.8(–43) × (4–)4.2–5.4(–6) μm (n = 10), with uniseriate but somewhat irregularly orientated spores. Ascospores initially greenish grey, soon becoming dark brown, ellipsoid, 1-septate, constricted or not at septum, surface initially smooth to finely roughened, developing coarse irregular cracks, (9.5–)10.8–13.9(–16.5) × (5–)5.7–7.2(–8) μm (n = 73).

Etymology

Named after the genus of the host lichen, Ramboldia elabens.

Distribution

Calicium ramboldiicola is currently known from Norway and Sweden in northern Europe, and from the eastern Canadian provinces of New Brunswick and Québec in north-eastern North America. The occurrences in Canada are in intermediate oceanic–continental and continental (OC and C₁) sectors of the hemiboreal bioclimatic zone (sensu Tuhkanen 1984; Clayden 2010). In Scandinavia, C. ramboldiicola has a more distinctly boreal distribution. To date, it has been found at eight localities, spanning c. 10° of latitude, from the southern to northern sub-zones of the boreal zone and their elevational counterparts as defined by Ahti et al. (1968). These occurrences are in areas with intermediate oceanic–continental (OC, O₁) climates, not the more highly oceanic sectors (O₂, O₃) of westernmost Scandinavia.

Further study is needed to determine whether the distribution of C. ramboldiicola conforms more fully to that of its host lichen. Ramboldia elabens occurs widely in boreal and temperate-montane areas of the Northern Hemisphere (Kantvilas & Elix 2007). In North America, it has a mainly Appalachian–Great Lakes distribution, with outliers as far west as the Black Hills of South Dakota (Wetmore 1967). Reports of R. elabens from areas of North America west of the Black Hills are based on misidentifications of other lichens (Pérez-Ortega et al. 2010). In Eurasia, it occurs discontinuously from Scandinavia and subalpine western and central Europe (Hafellner 1993) to Japan (Inoue 1982) and the Russian Far East (e.g. Konoreva et al. 2018).

Ecology

Calicium ramboldiicola has been found exclusively on the crustose lichen Ramboldia elabens growing on hard exposed wood (‘lignum’) of conifers, including standing snags and fallen trees. Ramboldia elabens also occurs less frequently on hard, weathered wood of built structures such as old barns or fences, but no occurrences of C. ramboldiicola are known on host thalli occupying such substrata. In Scandinavia, all samples are from very old Pinus sylvestris L. snags in old-growth forest localities. In New Brunswick, the habitat at one of the localities is an open, south-west-facing, granitic boulder talus slope with patches of Picea rubens Sarg. and Pinus strobus L. At the other, the habitat is an open, wet, ombrotrophic bog, with scattered low thickets of Larix laricina (Du Roi) K. Koch and Picea mariana (Mill.) Britton et al. Associated lignicolous lichens at one or both localities in New Brunswick include Imshaugia aleurites (Ach.) S. L. F. Mey., Mycoblastus alpinus (Fr.) Kernst., M. sanguinarius (L.) Norman, Ochrolechia mahluensis Räsänen, O. pseudopallescens Brodo, Pertusaria sulcata Dibben, Xylographa disseminata Willey, and X. vitiligo (Ach.) J. R. Laundon.

Although the thallus of R. elabens is often moderately thick, with contiguous, somewhat lumpy areoles contrasting with the black, shiny apothecia, it can also be thin and even to mostly endoxylic towards the thallus margin or more extensively. Apothecia of Calicium ramboldiicola are present mainly on well-developed areoles of R. elabens (Fig. 2A), but also on more or less endoxylic portions of some thalli (Fig. 2C), and less commonly on the host apothecia. Because of the small amount or age of the material available for study, we have not examined areoles histologically to assess whether the hyphae of C. ramboldiicola are distinguishable from those of R. elabens, or whether there are differences in the abundance or appearance of photobiont cells in areoles of R. elabens with or without apothecia of C. ramboldiicola. Older apothecia of R. elabens sometimes develop uneven or wrinkled discs, but these changes occur in thalli with or without C. ramboldiicola. Apothecia of C. ramboldiicola are sporadically present on eroded or senescent areoles of the host lichen, but these occurrences may be accidental. That is, the deterioration of such areoles could have been caused by agents other than the Calicium.

Additional specimens examined

Canada: New Brunswick: Kings Co., Canadian Forces Base Gagetown, Nerepis Hills, 1 km NE of confluence of Askwith Brook with Nerepis River, 45.442°N, 66.298°W, alt. 140 m, on decorticated snag of Picea rubens, 2010, Clayden 21343 (NBM); ibid., 2022, Clayden 28675 (NBM); Sunbury Co., Bull Pasture Bog Protected Natural Area, 46.0524°N, 66.3113°W, alt. 100 m, lignicolous on snag of Larix laricina, 2014, Clayden 24619 (NBM). Québec: Parc de la Vérendrye, Réservoir Cabonga, Îles Bruyères, 47°14ʹ30ʺN, 76°34ʹ30ʺW, sur un vieux tronc de conifère en bordure du réservoir, 1991, Bastien 789 (QFA).—Norway: Troms: Storfjord, 13.5 km SSE of Skibotn, along Skibotnelva, above Gustavsvingen, 69°16ʹN, 20°28ʹE, alt. 120–200 m, 2003, Wedin 7234 (UPS). Buskerud: Nore of Uvdal, Viken, Godfarfoss S, 60.4312°N, 8.605°E, 2021, Reiso (O L-2289081).—Sweden: Dalarna: Los parish, Gönhammaren, on dry, still standing Pinus, Forsslund (UPS); Ore parish, Jässelåsen, Rärmyren, 61°14ʹN, 15°16ʹE, alt. 370 m, on dry, still standing Pinus snags, 2003, Hermansson 13315 (UPS); Svärdsjö parish, E of Orrtjärnen, on dry snag in a bog, 2024, Skog (S). Jämtland: Undersåker parish c. 15.5 km S of Undersåker church, south side of Lake Håckrenmagasinet, c. 570 m E of Furubergskojan, 63.19080°N, 13.42355°E, 2020, Johannesson & Wadstein (UPS). Lule Lappmark: Gällivare parish, Linaåive Nature Reserve, E of Parajaure, 67°16ʹN, 20°26ʹE, alt. 490 m, on decorticated branches of fallen, dead Pinus, 1977, Tibell 6916 (UPS); ibid., Jokkmokk par., Karats area, SW of Mt Luspevarasj, 66.62713°N, 18.81745°E, elev. 440 m, 2021, Westberg SCNB150 (UPS).

Discussion

Fungi with stalked ascomata resembling those of Calicium are a heterogeneous group from a phylogenetic standpoint, but they are often studied together and thus form a defined research area (Temu et al. 2024). Although lichenicolous species occur in many calicioid genera, especially Chaenothecopsis and Sphinctrina (both in the Mycocaliciales; Tibell & Wedin 2000), Calicium ramboldiicola is one of rather few lichenicolous species discovered in Caliciaceae. In this family, Acolium marcianum (B. de Lesd.) M. Prieto & Wedin and A. sessile (Pers.) Arnold are associated with Pertusaria and Lepra species (Pertusariaceae and Variolariaceae, respectively; both Pertusariales), whereas the species described here is found with Ramboldia (Ramboldiaceae, Lecanorales; Miadlikowska et al. 2014). This is only the second Calicium species known to occur on Lecanoromycetes, the other being Calicium episcalaris, on Hypocenomyce (Ophioparmaceae, Umbilicariales). Other genera of Caliciaceae including small numbers of lichenicolous species are Diplotomma and Tetramelas (Cannon et al. 2021).

The ascomata of C. ramboldiicola are small but otherwise similar to those of other species in Calicium, such as C. glaucellum. In our analysis (Fig. 1), C. ramboldiicola is sister to C. abietinum, and indeed resembles a miniature of that species, with much smaller ascomata but different spore ornamentation: C. ramboldiicola has an irregularly cracked spore surface, while that of C. abietinum presents with minute, distinct warts. The clade including C. abietinum, C. ramboldiicola and C. verrucosum has high support (Fig. 1), but apart from a general similarity in ascoma morphology, we have not been able to find any specific morphological synapomorphies.

Calicium ramboldiicola also resembles C. pinastri in its small size, lack of pruina, short cylindrical asci, and spore ornamentation. However, the apothecial stalks in C. pinastri are black, lacking the brown hue that is usually present in C. ramboldiicola. Also, C. pinastri is a mainly corticolous species, in Europe occurring especially on trunks of Pinus sylvestris (Tibell 1999a, b). Our phylogenetic analysis (Fig. 1) indicates that these two species are not closely related to one another within Calicium.

Ramboldia elabens is currently red-listed in Sweden and categorized as near-threatened (SLU Artdatabanken 2020). As C. ramboldiicola is known only from this host, it makes the new lichenicolous species extremely rare per se. Similar to R. elabens, it probably occurs only in localities of very high conservation value. Thus, it should be given consideration as a threatened species and probably placed in a higher category than its host.