Lobaria pulmonaria (L.) Hoffm. and allied lichens have a rich selection of cohabiting, lichenicolous fungi (e.g. Etayo & Diederich Reference Etayo and Diederich1996; Martínez & Hafellner Reference Martínez and Hafellner1998; Wedin & Hafellner Reference Wedin and Hafellner1998; Bergmann & Werth Reference Bergmann and Werth2017; Suija et al. Reference Suija, Berger, Arsenault, Baines, Zimmermann and Etayo2024). Some of these species are known by their sexual stage, while others are identified by their asexual stage (e.g. Diederich et al. Reference Diederich, Lawrey and Ertz2018). Although they are described under different genera, they probably represent the same fungus, but the connection between their life stages has not yet been established. This study is inspired by the statement in Vicente et al. (Reference Vicente, Westberg, Isaksson, Anderberg, Frisch, Hammarström, Hansson, Jonsson and Svensson2024) that Chalara lobariae Etayo (Etayo & Diederich Reference Etayo and Diederich1996) is the asexual stage of the recently described Calycina alstrupii Suija & Motiej. (Suija & Motiejūnaitė Reference Suija and Motiejūnaitė2017), both growing on Lobaria pulmonaria. The Chalara-like anamorphic fungi have been associated with many ascomycetous classes, including Leotiomycetes (Wu & Diao Reference Wu and Diao2023), and are characterized by sporodochial or synnematal conidiomata, stalked, hyaline to pigmented conidiophores, terminal and phialidic conidiogenous cells with a long collarette and by forming hyaline, mainly cylindrical conidia (Wu & Diao Reference Wu and Diao2023; Diederich et al. Reference Diederich, Etayo, Brackel, Cannon, Zhurbenko, Diederich, Ertz and Braun2024; literature herein). They can be found in both terrestrial and submerged habitats where they act as, for example, saprotrophs, plant pathogens and endophytes, alongside and inside of various organisms (Wu & Diao Reference Wu and Diao2023), including lichens (Diederich et al. Reference Diederich, Etayo, Brackel, Cannon, Zhurbenko, Diederich, Ertz and Braun2024).

To test whether Chalara lobariae and Calycina alstrupii are indeed the same fungus, we amplified the DNA barcoding marker (nuITS) from three specimens of C. lobariae (UPS F-1143069, UPS F-1121135, UPS F-1121134) and compared these with those of C. alstrupii. DNA extraction and amplification were carried out at the molecular laboratory of the University of Tartu (TU). Using a scalpel, we took samples far enough from ascomata to minimize the risk of picking up DNA from fruiting bodies. We used the High Pure PCR Template Preparation Kit (Roche Applied Science®) and following the protocol provided by the manufacturer. We amplified the nuclear ribosomal internal transcribed spacer (nuITS) with the primer pair ITS0F and LA-W (Tedersoo et al. Reference Tedersoo, Jairus, Horton, Abarenkov, Suvi, Saar and Kõljalg2008). The PCR reaction mix (25 μl) consisted of 5 μl 5× HOT FIREPol Blend Master Mix (with 10 mM MgCl₂; Solis BioDyne, Tartu, Estonia), 0.5 μl of both primers (all 20 μM), 3 μl of target DNA and the rest made up of distilled water. PCR products were visualized on a 1% agarose gel stained with ethidium bromide, and for the purification of PCR products, 1 μl of FastAP and 0.5 μl of Exonuclease I (Thermo Scientific, Waltham, MA, USA) were added to each tube per 20 μl of the product. Both complementary strands were Sanger sequenced by Macrogen Inc. (Amsterdam, the Netherlands). CodonCode Aligner v. 8.0.2 (CodonCode Corporation®, Centerville, MA, USA) was used to check, assemble and manually adjust the resulting sequence fragments. The consensus sequences were compared with those publicly available in GenBank using the ‘blastn’ algorithm (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990). The extracted DNA samples are deposited in the DNA and Environmental Sample Collection of the Natural History Museum in Tartu University (TUE).

To visualize the results, we compiled an alignment with nine nuITS sequences: two each of Calycina alstrupii, Calycina lactea (Sacc.) Baral, R. Galán & Platas and Calycina languida (P. Karst.) Baral, R. Galán & Platas, and three of Chalara lobariae. We used the online version of IQ-TREE v. 1.6.12. (Nguyen et al. Reference Nguyen, Schmidt, von Haeseler and Minh2015), applying TN + I as the best-fit nucleotide substitution model selected according to AICc. Branch support was calculated according to ultrafast bootstrapping (Hoang et al. Reference Hoang, Chernomor, von Haeseler, Minh and Vinh2018) with 1000 iterations.

Results

We obtained nuITS sequences from all three specimens of Chalara lobariae, and all three were identical to those of Calycina alstrupii (Fig. 1). Several Chalara species have already been transferred to Calycina (Wu & Diao Reference Wu and Diao2023); with this study and following the ‘One Fungus = One Name’ principle (Taylor Reference Taylor2011), we combine Chalara lobariae in Calycina. Since Calycina alstrupii is the younger name (Suija & Motiejūnaitė Reference Suija and Motiejūnaitė2017), the new name for the fungus is Calycina lobariae.

Figure 1. The nuITS-based ML phylogeny showing sequences of anamorph and teleomorph strains of Calycina lobariae. The bootstrap values (BS) are indicated above the branches. GenBank Accession numbers precede the species names. The scale bar is proportional to the substitution rate.