Introduction

Pilocarpaceae is a moderately sized family of predominantly tropical lichenized fungi often found on living leaves, but with several lineages growing on other substrata (Kalb & Vězda 1987; Lücking 2008; Sérusiaux & al. 2008; Brand & al. 2014; Sanders 2014; Gumboski 2015; Lücking & al. 2017; Guzow-Krzemińska & al. 2019). It consists of two major groups, one formed by the former family Micareaceae and one corresponding to the former family Pilocarpaceae s.str. (Andersen & Ekman 2005; Miadlikowska & al. 2014; Kraichak & al. 2018; Aptroot & al. in Hyde & al. 2019). The latter includes a number of genera characterized by peculiar conidiomata, so-called campylidia, which are more or less hood-shaped and adapted to dispersal of the conidia by rain splash (Sérusiaux 1986, 1995; Lücking 2001, 2008; Sanders & al. 2016). These genera had formerly been assigned to the family Ectolechiaceae and the monogeneric family Lasiolomataceae (Hafellner 1984; Kalb & Vězda 1987; Lücking 1999).

Calopadia Vězda, Lasioloma R. Sant., Sporopodium Mont. and Tapellaria Müll. Arg. are the core genera in the campylidia-bearing lineages of Pilocarpaceae s.str. (Lücking 1999, 2008; Lücking & Sérusiaux 2001; Sérusiaux & al. 2008; Neuwirth & Stocker-Wörgötter 2017). Calopadia, Lasioloma and Tapellaria are similar to each other in thallus and ascoma morphology and share filiform conidia adapted to rain water dispersal. Lasioloma differs from the other two genera in the woolly prothallus, the pilose apothecial margins and the centrally branched conidia, whereas Tapellaria can be distinguished from Calopadia in the jet-black apothecia with purple hypothecium and anastomosing, net-like paraphyses (Lücking 1999, 2008).

Thus far, few phylogenetic studies exist for Pilocarpaceae, although the data available show an emerging picture of some genera being monophyletic and others para- or polyphyletic (Andersen & Ekman 2005; Miadlikowska & al. 2014; Kraichak & al. 2018; Aptroot & al. in Hyde & al. 2019; Wang & al. 2020). The genera Calopadia and Lasioloma have been resolved as closely related, whereas Tapellaria is phylogenetically more distant (Wang & al. 2020), in agreement with the different hamathecial anatomy of the latter. The most recent study resolved Lasioloma to be nested within a paraphyletic Calopadia (Wang & al. 2020), suggesting that the peculiar morphological features of Lasioloma evolved from a plesiomorphic residual corresponding to the morphology of Calopadia and the two genera should perhaps be merged. This topology had not been noticed before, as the complete set of taxa had not been analysed simultaneously in previous studies (Andersen & Ekman 2005; Miadlikowska & al. 2014; Aptroot & al. in Hyde & al. 2019).

Table 1.

Voucher information and GenBank accession numbers for the specimens used in this study.

Since this nested topology was caused by a single specimen identified as Calopadia phyllogena (Müll. Arg.) Vězda, collected in the Netherlands Antilles and first published in a broad-scale assessment of Lecanoromycetes as part of the AFTOL project (Miadlikowska & al. 2014), we set out to examine the taxonomic status of the underlying specimen, housed at B (Sipman 54818). We thereby envisioned three potential scenarios: (1) the specimen had been correctly identified, at least to genus level, making Calopadia indeed paraphyletic relative to Lasioloma; (2) the material consisted of a mixed collection, including genuine C. phyllogena but also thalli of Lasioloma that had accidentally been sequenced; (3) the material was misidentified and in reality represented a species of Lasioloma. The latter two options are not unlikely as mixed collections in these usually small lichens are common and some species and specimens of Lasioloma have reduced apothecial hairs, making them superficially similar to Calopadia. Some species of Calopadia have also been shown to produce a woolly prothallus (Lücking 1998, 2008).

Fig. 1.

Best-scoring maximum-likelihood tree of Calopadia and Lasioloma based on a 4-marker concatenated alignment (mtSSU, nuLSU, ITS, RPB1). The target specimen is supported sister to Lasioloma arachnoideum.

Material and methods

To re-assess the phylogenetic placement of Calopadia phyllogena (Sipman 54818), we downloaded all available sequences from GenBank of the genera Calopadia and Lasioloma, with Sporopodium antoninianum Elix, Lumbsch & Lücking as outgroup, representing four markers (mtSSU, nuLSU, ITS, RPB1; Table 1). Separate alignments were prepared using MAFFT 7 (Katoh & Standley 2013) and potentially ambiguously aligned regions were assessed using the Guidance Web Server (Penn & al. 2010). Given that only few ambiguously aligned sites were detected and these did not affect backbone topology and support, all sites were maintained to achieve maximum terminal resolution. No supported conflict was detected between topologies from the individual markers and so the concatenated alignment was subjected to maximum likelihood tree search in RAxML 8 (Stamatakis 2014), under the universal GTR-Gamma model, with 1000 bootstrap pseudoreplicates.

The underlying specimen of Calopadia phyllogena (Sipman 54818) was re-examined morphologically and anatomically using a LEICA Zoom 2000 dissecting microscope and a ZEISS Axioskope compound microscope. Secondary chemistry was assessed following Orange & al. (2010).

Results and Discussion

In our 4-marker phylogeny, the sequenced specimen originally identified as Calopadia phyllogena (Sipman 54818) was supported as sister to a clade including Lasioloma arachnoideum (Kremp.) R. Sant. from Costa Rica and two specimens originally identified as L. arachnoideum from Thailand (Fig. 1; see below). The above clade was separate from a strongly supported clade on a long stem branch including all other sequenced species of Calopadia (Fig. 1). Revision of the underlying material identified as C. phyllogena revealed that it does not represent a species of Calopadia, as evident from the apothecial anatomy and the branched conidia, but indeed corresponds to the genus Lasioloma. We can therefore conclude that with current available data, Calopadia and Lasioloma are reciprocally monophyletic. Closer inspection of the material further revealed that the specimen in question represented an undescribed species in the genus Lasioloma, which is formally introduced below.

This case highlights the necessity of critically revising voucher material of sequences that exhibit unexpected phylogenetic relationships, and the need to properly identify underlying voucher material in sequence data. In the present case, with the information provided, we were able to readily trace the voucher specimen and assess its taxonomic status. In order to reflect the updated taxonomy, it is also necessary to update the corresponding sequence records, which can currently be done only by the original submitter.

The phylogeny also indicates further need for taxonomic revision of sequenced material. Thus, the Thai specimens originally identified as Lasioloma arachnoideum by Wang & al. (2020) formed a clade separate from the neotropical specimen (Fig. 1). The photograph in Wang & al. (2020: 383, fig. 4D) indicates that the sequenced material may represent L. phycophorum (Vain.) R. Sant., although the depicted specimen was not sequenced. Likewise, specimens identified as Calopadia foliicola (Fée) Vězda formed three separate clades (Fig. 1). Given that the species was described from the neotropics (Brazil; see Lücking 2008), the material from Thailand (Wang & al. 2020) needs to be revised. The photograph provided by Wang & al. (2020: 383, fig. 4B), corresponding to one of the three specimens (KYW0035) forming the terminal clade, fits C. foliicola except for the plane apothecial disc (distinctly convex in C. foliicola), so there appears to be some indication of more or less cryptic speciation in this genus, combined with geographic signal.

Fig. 2.

Morphology and anatomy of Lasioloma antillarum (holotype). – A: thallus with apothecia and 1 campylidium (middle left); B: apothecium enlarged; C: section through apothecium showing hypothecium and excipulum; D: lateral excipulum enlarged showing irregular surface and protruding hairs; E, F: part of hymenium with asci and immature ascospores; G: mature ascospore. – Scale bars: A, B = 1 mm; C = 50 µm; D = 20 µm; E, F, G = 10 µm.

Taxonomic treatment

Lasioloma antillarum Lücking, Högnabba & Sipman, sp. nov. – MycoBank MB 838956. – Fig. 2.

Holotype: Netherlands Antilles, Saba, Summit of Mt Scenery, along trail to western lookout, 17°38′05″N, 63°14′22″W, 825 m, disturbed mossy forest (hurricane damage), on bark, 8 Mar 2007, H. J. M. Sipman 54818 (B 60 0143940).

Diagnosis — Differing from Lasioloma spinosum in the broader ascospores and the corticolous instead of foliicolous growth habit.

Description — Thallus corticolous, 1–3 cm in diam., 30–50 µm thick, centrally continuous but toward margin with dispersed, irregular patches; surface smooth to uneven, white to pale grey. Photobiont chlorococcoid. Apothecia sessile, rounded, 0.5–0.8 mm in diam., 300–400 µm high; disc plane to slightly concave, dark brownish grey, non-pruinose; margin thick, slightly prominent, creamcoloured, very shortly tomentose. Excipulum distinctly paraplectenchymatous, laterally 50–80 µm broad, below hypothecium 200–300 µm high, colourless, laterally with irregular surface and protruding, short hairs formed by single, unbranched hyphae. Hypothecium 30–60 µm high, dark brown to brown-black. Apothecial base hyaline. Epithecium thin, 5–10 µm high, yellowish brown. Hymenium 80–90 µm high, colourless. Paraphyses branched and slightly anastomosing. Asci 80–90 × 20–25 µm. Ascospores 2(–4) per ascus, ellipsoid to fusiform, muriform, with 7–9 transverse and 1–3 longitudinal septa per segment, 35–50 × 12–16 µm, 2.7–3.5 × as long as broad, slightly constricted at middle septum, colourless. Campylidia sessile, 0.7–1 mm broad, 1–1.3 mm long (high); lobe well-developed, hood-shaped, dark aeruginous grey, non-pruinose; socle not apparent. Wall composed of 2 layers, outer layer para- to prosoplectenchymatous with thick-walled cells, hyaline, 20–30 µm thick, inner layer paraplectenchymatous, dark aeruginous, 20–30 µm thick, layers bent around each other and separated by an additional inner, prosoplectenchymatous, yellowish, 15–25 µm thick layer to form a 5-layered wall 100–130 µm thick. Conidiogeneous cells lining inner wall surface. Conidia filiform, branched from centre and with 4 or 5 branches, each branch 3–5-septate, 30–40 × 1.5–2.5 µm, main branch slightly longer and thicker than others. Secondary chemistry: no substances detected by TLC.

Etymology — The epithet refers to the origin of the material in the Antilles.

Remarks — The new species is characterized by apothecia with rather short hairs and mostly 2-spored asci with small, muriform ascospores. Most species of Lasioloma have single-spored asci with large, muriform ascospores (Santesson 1952; Lücking & Sérusiaux 2001; Lücking 2008). Only three species have been described with smaller spores and 2–8-spored asci, namely L. inexspectatum R. Sant. & Lücking (Santesson & Lücking 1999), L. pauciseptatum Van den Boom (van den Boom & al. 2018) and L. spinosum Hafellner & Vězda (Vězda 1994). The first, described from Africa, differs in its smaller, 7-septate ascospores, whereas L. pauciseptatum from Suriname produces 4–8-spored asci and also distinctly smaller ascospores (see key below). Most similar in morphology and anatomy is L. spinosum, a foliicolous taxon from Indonesia. Besides the deviating distribution and substrate ecology, the latter has much narrower ascospores, 7–9 × as long as broad (see key below).

Key to the known species of Lasioloma

In the following key, all validly described species in the genus are included. Lasioloma heliotropicum Bat. & M. P. Herrera is not a validly published name (no description, no reference to original material; Batista & Cavalcanti 1964) and its status could not be established (Lücking & al. 1998).