Lasioloma antillarum (Ascomycota: Pilocarpaceae), a new lichenized fungus from the Antilles, and the importance of posterior annotations of sequence data in public repositories

Abstract: We describe the new lichenized fungus Lasioloma antillarum Lücking, Högnabba & Sipman from the Netherlands Antilles. The new species is characterized by a corticolous growth habit, apothecia with shortly tomentose margins, and rather small (35–50 × 12–16 µm), muriform ascospores in numbers of 2(–4) per ascus. The material had originally been identified as Calopadia phyllogena (Müll. Arg.) Vězda, with associated sequence data, but in phylogenetic analyses consistently fell outside the latter genus. Its revised identification as a species of Lasioloma is consistent with its phylogenetic position and underlines the necessity of posterior annotations in public sequence repositories, in order to correct previous identifications. Citation: Lücking R., Högnabba F. & Sipman H. J. M. 2021: Lasioloma antillarum (Ascomycota: Pilocarpaceae), a new lichenized fungus from the Antilles, and the importance of posterior annotations of sequence data in public repositories. – Willdenowia 51: 83–89. Version of record first published online on 23 March 2021 ahead of inclusion in April 2021 issue.

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(Lücking , 2008Lücking & Sérusiaux 2001;Sérusiaux & al. 2008;Neuwirth & Stocker-Wörgötter 2017). Calopa dia, 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-84 Lücking & al.: Lasioloma antillarum from the Antilles black apothecia with purple hypothecium and anastomosing, net-like paraphyses (Lücking 1999(Lücking , 2008. Thus far, few phylogenetic studies exist for Pilocar paceae, although the data available show an emerging picture of some genera being monophyletic and others paraor 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 Lasiolo ma 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).
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 Lecanoro mycetes 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 Calo padia have also been shown to produce a woolly prothallus (Lücking 1998(Lücking , 2008. Table 1. Voucher information and GenBank accession numbers for the specimens used in this study.

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 aff ect backbone topology and support, all sites were maintained to achieve maximum terminal resolution. No supported confl ict 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 identifi ed as Calopadia phyllogena (Sipman 54818) was supported as sister to a clade including La sioloma arachnoideum (Kremp.) R. Sant. from Costa Rica and two specimens originally identifi ed as L. arach noideum 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 identifi ed 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 refl ect 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 identifi ed as Lasioloma arach noideum by Wang & al. (2020) formed a clade separate from the neotropical specimen (Fig. 1). The photograph in Wang & al. (2020: 383, fi g. 4D) indicates that the sequenced material may represent L. phycophorum (Vain.) R. Sant., although the depicted specimen was not sequenced. Likewise, specimens identifi ed as Calopa dia foliicola (Fée) Vězda formed three separate clades (Fig. 1). Given that the species was described from the neotropics (Brazil; see Lücking 2008), the material  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. Diagnosis -Differing from Lasioloma spinosum in the broader ascospores and the corticolous instead of foliicolous growth habit.
Etymology -The epithet refers to the origin of the material in the Antilles.

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