Introduction
Gomphillaceae is the dominant element of foliicolous lichen communities in tropical rainforests, with well over 300 leaf-dwelling species known (Lücking et al. Reference Lücking, Sérusiaux and Vězda2005, Reference Lücking, Hodkinson and Leavitt2017; Lücking Reference Lücking2008; Xavier-Leite et al. Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022). The family is uniquely characterized by peculiar conidiomata, the so-called hyphophores, which are mostly setiform and produce hyphal drops that serve as asexual propagules (Vězda Reference Vězda1979; Sérusiaux & De Sloover Reference Sérusiaux and De Sloover1986; Vězda & Poelt Reference Vězda and Poelt1987; Lücking Reference Lücking1997, Reference Lücking2008; Lücking et al. Reference Lücking, Sérusiaux and Vězda2005). Several lineages have also evolved sterile thallus setae, probably derived from setiform hyphophores; these were previously included in the collective genus Tricharia Fée s. lat. (Santesson Reference Santesson1952; Vězda & Poelt Reference Vězda and Poelt1987; Lücking Reference Lücking1997).
The circumscription of the family, as well as the delimitation of genera contained within it, has undergone substantial changes since it was first described by Watson (Reference Watson1929) for the single genus, Gomphillus Nyl., and in its current sense by Vězda & Poelt (Reference Vězda and Poelt1987), who recognized ten genera. Given the sparse molecular data at the time (Lücking et al. Reference Lücking, Stuart and Lumbsch2004), Dennetière & Péroni (Reference Dennetière and Péroni1998) and Lücking et al. (Reference Lücking, Sérusiaux and Vězda2005) made an attempt to define genera based on a quantitative, cladistic analysis, and the latter authors increased the number of genera to 19. Until the year 2018, this number had increased to 27 (Lücking et al. Reference Lücking, Hodkinson and Leavitt2017; Diederich et al. Reference Diederich, Lawrey and Ertz2018). Recently, Xavier-Leite et al. (Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022) provided the first broad molecular phylogenetic approach for the Gomphillaceae, confirming most of the phenotypically defined genera but identifying an additional 19 genus-level clades which were subsequently formally instated (Xavier-Leite et al. Reference Xavier-Leite, Goto, Lücking and Cáceres2023, Reference Xavier-Leite, Goto, Cáceres and Lücking2024). The underlying phylogenetic study supported the notion that a combination of apothecial and hyphophore features characterizes genera in this family.
The genus Paratricharia Lücking (Lücking Reference Lücking1997) was established to accommodate a single species originally described in Tricharia Fée, T. paradoxa Lücking (Lücking Reference Lücking1991), which deviated from all other Gomphillaceae known at the time by forming apothecia with a carbonized zeorine margin, such as in the genus Aulaxina Fée, combined with robust, sterile black thallus setae, seen for example in the genus Tricharia as defined by Lücking et al. (Reference Lücking, Sérusiaux and Vězda2005). The new taxon further differed from other genera by producing a sterile columella in the centre of the apothecial disc, a feature otherwise known from the genus Ocellularia G. Mey. and its relatives in the sister family Graphidaceae (Kraichak et al. Reference Kraichak, Lücking and Lumbsch2015).
Thus far, the phylogenetic relationships of Paratricharia have been unresolved. Phenotype-based cladistic analysis predicted the placement in a clade containing the genera Aulaxina and Caleniopsis Vězda & Poelt, as well as Aplanocalenia Lücking et al. and Rolueckia Papong et al. (the latter as Caleniopsis; Lücking et al. Reference Lücking, Sérusiaux and Vězda2005). Phenotype-based phylogenetic binning predicted placement of the genus in the early diverging Calenia triseptata Zahlbr. clade, now placed in the new genus Caleniella Xavier-Leite et al. (Xavier-Leite et al. Reference Xavier-Leite, Goto, Cáceres and Lücking2024). These results ascribed a greater predictive power to apothecial features than to the presence of sterile, black setae, characteristic of the distantly placed genus Tricharia s. lat. in which the species was originally described (now divided into Batistomyces Xavier-Leite et al., Microxyphiomyces Bat. et al., Santricharia Xavier-Leite et al. and Tricharia; Xavier-Leite et al. Reference Xavier-Leite, Goto, Lücking and Cáceres2023), but not known in Aulaxina or Caleniopsis. Unfortunately, molecular data for Paratricharia had not been obtained for the study by Xavier-Leite et al. (Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022).
Here, we resolve the phylogenetic placement of Paratricharia employing newly generated sequences of the mtSSU and nuLSU markers, obtained from material collected in Costa Rica.
Material and Methods
The newly sequenced specimens of Paratricharia paradoxa were collected in 1992 and 1997 at La Selva Biological Station in Costa Rica. Since then, the material had been kept in the freezer at −4 to −20 °C after drying and pressing, which apparently helped to preserve the DNA since we had no problems in amplifying the mtSSU and nuLSU markers after DNA extraction. We also included three specimens of Gyalectidium, representing G. filicinum Müll. Arg. and G. imperfectum Vězda, collected by Claudia Hartmann in Costa Rica.
Pieces of thalli from three different specimens were removed and placed in 1.5 ml Eppendorf tubes. Total genomic DNA was extracted at the laboratories of the Botanischer Garten und Botanisches Museum, Freie Universität Berlin, employing the Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (St Louis, Missouri, USA), following the manufacturer's instructions but using lower amounts of reagents to obtain a lower amount of DNA extract.
Electrophoresis on a 1.5% agarose gel was used to assess the quality of the extracted DNA. In order to amplify the mitochondrial small subunit rRNA (mtSSU), the primer pair mrSSU1 and MSU7 (Zhou & Stanosz Reference Zhou and Stanosz2001) was used, with the following settings for the PCR: initial denaturation for 10 min at 94 °C, 45 s at 94 °C, 45 s at 50 °C, followed by 35 cycles of 45 s at 94 °C, then 10 min at 72 °C. Using the primer pair LR3 and LR0R (Vilgalys & Hester Reference Vilgalys and Hester1990), the nuclear large subunit rRNA (nuLSU) was amplified through PCR as follows: initial denaturation for 3 min at 95 °C, 45 s at 95 °C, 45 s at 54 °C, 1 min at 72 °C, then 35 cycles of 45 s at 95 °C, and a final elongation for 10 min at 72 °C. PCR products were purified with ExoSAP-IT™ (IT PCR Cleanup protocol) and sent for sequencing to Macrogen Europe (Amsterdam, the Netherlands; https://dna.macrogen.com/en).
The new sequences obtained were added to a subset of the concatenated mtSSU-nuLSU alignment provided by Xavier-Leite et al. (Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022), for a total of 85 terminals representing the main clades of the Gomphillaceae (Table 1). We removed one mtSSU sequence previously identified as Calenia triseptata (MZ827298), since a recheck revealed it to represent Aulaxina quadrangula. Instead of using Fissurina (Graphidaceae) as an outgroup, we performed ingroup routing with the genus Gyalidea, an early diverging clade within the family (Xavier-Leite et al. Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022), to reduce alignment ambiguity. The resulting alignment, with a length of 949 bp for the mtSSU region and 660 bp for the nuLSU region, was manually inspected in BioEdit v. 7.2.0 (Hall Reference Hall1999, Reference Hall2011). Since only a small number of narrow, ambiguously aligned regions were identified using the HoT scores approach on the Guidance web server (http://guidance.tau.ac.il) (Penn et al. Reference Penn, Privman, Ashkenazy, Landan, Graur and Pupko2010a, Reference Penn, Privman, Landan, Graur and Pupkob), we did not remove them prior to analysis.
Table 1. GenBank Accession numbers of Gomphillaceae material used in this study, with voucher information, including collectors for the newly generated sequences (highlighted in bold)
The final concatenated alignment was analyzed with a maximum likelihood (ML) approach using RAxML v. 8.2.0 (Stamatakis Reference Stamatakis2014) locally, employing the reversible GTR model with 1000 non-parametric bootstrap replicates. To test for potential monophyly of Paratricharia with Tricharia s. lat., we performed a Shimodaira-Hasegawa (SH) test as implemented in RAxML v. 8.2.0.
Results
The topology in our best-scoring ML tree matched the topology obtained in our previous study (Xavier-Leite et al. Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022), with the exception of the Caleniella triseptata clade, now positioned as an early diverging clade and no longer part of the Aulaxina clade; the previous result was caused by the erroneous mtSSU sequence now removed (MZ827298). Further early diverging clades included the genera Rolueckia, Taitaia Suija et al., and Corticifraga D. Hawksw. & R. Sant., the remaining genera forming a large, yet unsupported clade (Fig. 1). Within that clade, the topology largely matched that found in our previous analysis, with the difference that the Calenia-Echinoplaca complex formed a late-diverging clade; however, the backbone topology was overall not supported.
Figure 1. Best-scoring maximum likelihood tree of Gomphillaceae using the mtSSU and nuLSU markers, based on selected terminals representing most genus-level clades, showing placement of the genus Paratricharia. Bootstrap support values are shown below branches (to genus-level clades only). In colour online.
The three specimens of Gyalectidium clustered within their respective species, G. filicinum and G. imperfectum (Fig. 1). Paratricharia paradoxa was recovered with absolute support as sister to the genus Caleniopsis, this clade being strongly supported as sister to a clade containing the genera Aulaxina s. str. and Aulaxinella (Fig. 1). Paratricharia was not found to be closely related to either Tricharia s. str. or the T. vainioi R. Sant. and T. santessonii R. Sant. clades (Microxyphiomyces, Santricharia), the three groups that produce sterile, black setae. The SH test also rejected the monophyly of Paratricharia with Microxyphiomyces, Santricharia or Tricharia s. str. Since Paratricharia agrees with the latter three genera in the presence of robust, sterile, black setae, while its apothecial features are more in agreement with those of Aulaxina (Fig. 2), our results support the view that apothecial characters are more reliable in predicting the phylogenetic placement of this taxon. Paratricharia is now the fifth clade known to produce such large, sterile, black setae.
Figure 2. Morphology of selected genera and species of Gomphillaceae. A, Paratricharia paradoxa. B, Aulaxina submuralis. C, Caleniopsis laevigata. D, Tricharia carnea. E, Microxyphiomyces vainioi. F, Batistomyces pallidus. In colour online.
Discussion
Our study confirmed our previous assessment suggesting that apothecial features are more informative regarding the phylogenetic placement of Paratricharia paradoxa than the presence of sterile, black setae, based on which the species had originally been described in the genus Tricharia s. lat. (Lücking Reference Lücking1991). Instead, molecular data resolved this taxon close to Caleniopsis and Aulaxina, notably in the same area of the tree as a previous cladistic analysis based on phenotype data alone, as well as a phenotype-based binning approach, had suggested (Lücking et al. Reference Lücking, Sérusiaux and Vězda2005; Xavier-Leite et al. Reference Xavier-Leite, Goto, Cáceres and Lücking2024).
Given that phenotypic characters in Paratricharia are conflicting, with the apothecial features most similar to Aulaxina, the thallus features most similar to Tricharia, plus a columella as unique autapomorphy (Lücking Reference Lücking1997, Reference Lücking2008), the previous placement close to Aulaxina based on phenotype characters alone, using two different approaches, and its confirmation with molecular data, seems surprising. However, studies in other lichenized fungi have shown the predictive power of phenotype features when analyzed in the proper context, such as in the Graphidaceae or Arthoniales (Berger et al. Reference Berger, Stamatakis and Lücking2011; Lücking & Kalb Reference Lücking and Kalb2018; Perlmutter et al. Reference Perlmutter, Rivas Plata, LaGreca, Aptroot, Lücking, Tehler and Ertz2020).
Our results support the findings by Xavier-Leite et al. (Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022, Reference Xavier-Leite, Goto, Cáceres and Lücking2024) that apothecial features are more conserved in the Gomphillaceae, thus having greater predictive power in the phylogenetic placement of taxa and showing a greater correlation with larger clades within the family. Hyphophore and thallus features are highly informative for genus-level clades, but similar phenotypes also evolved independently in different areas of the tree. Thus, in addition to Paratricharia, sterile, black setae are apomorphies for four genus-level clades in the family: Tricharia s. str., the T. vainioi clade (Microxyphiomyces), the T. santessonii clade (Santricharia) and the T. pallida Vězda clade (Batistomyces; see Xavier-Leite et al. Reference Xavier-Leite, Goto, Lücking and Cáceres2023). Similarly, genera with red-brown (Rolueckia, Rubrotricha Lücking et al.) or white setae (e.g. Aderkomyces Bat., Arthotheliopsis Vain., Echinoplaca Fée, Psathyromyces Bat. & Peres, Roselviria Xavier-Leite et al.) are not necessarily closely related (Xavier-Leite et al. Reference Xavier-Leite, Cáceres, Aptroot, Moncada, Lücking and Goto2022, Reference Xavier-Leite, Goto, Lücking and Cáceres2023).
The function of these sterile setae is not known but for them to have evolved independently in so many clades, one would assume strong environmental pressure. Setiform hyphophores probably evolved to facilitate dispersal of the diahyphae in the semi-aquatic environment that leaf surfaces display during rainfall (Lücking Reference Lücking2001, Reference Lücking2008), but sterile setae do not bear diahyphae. Notably, in almost all cases, the sterile setae are longer and more robust than the hyphophores in the same species (Vězda Reference Vězda1979; Sérusiaux & De Sloover Reference Sérusiaux and De Sloover1986; Vězda & Poelt Reference Vězda and Poelt1987; Lücking Reference Lücking1997, Reference Lücking2008).
Acknowledgements
The material of Paratricharia paradoxa was collected as part of a research project funded by the DAAD (Kennziffer 320/605/500/1; Jahresstipendium; 1991–1992) and the Deutsche Forschungsgemeinschaft (Kennzeichen LU 597/1-1; ‘Neotropical Foliicolous Lichens: Taxonomy, Systematics, Biogeography, Diversity, Ecology, and Applications’; 1995–1997). Claudia Hartmann kindly provided the material of Gyalectidium. Colleagues and staff at La Selva Biological Station in Costa Rica are warmly thanked for their hospitality and support. The sequencing work for this study was supported by the Verein der Freunde des Botanischen Gartens Berlin. We thank Elise Lebreton for assisting in rechecking the underlying sequence data used for this study and discovering the problematic mtSSU sequence of Caleniella triseptata (MZ827298).
Author ORCIDs
Robert Lücking, 0000-0002-3431-4636; Bibiana Moncada, 0000-0001-9984-2918.
Open access
