Saxiloba: a new genus of placodioid lichens from the Caribbean and Hawaii shakes up the Porinaceae tree (lichenized Ascomycota: Gyalectales)

The new genus Saxiloba is described with the two species S. firmula from the Caribbean and S. hawaiiensis from Hawaii. Saxiloba is characterized by a unique, placodioid thallus forming distinct lobes, growing on rock in shaded to exposed situations with a trentepohlioid photobiont and a fenestrate thallus anatomy with distinct surface lines. The material is often sterile, but Porina-like perithecia and ascospores had previously been described for the Caribbean taxon and were here confirmed for both species. Molecular sequence data also confirmed placement of this lineage in Porinaceae. Its position within that family supports the notion that Porinaceae should be subdivided into a larger number of genera than proposed in previous classification attempts. Compared to other Porinaceae, Saxiloba exhibits a unique morphology and anatomy that recalls taxa in the related family Graphidaceae and it substantially expands the known phenotypic variation within Porinaceae. The two recognized species are similar in overall morphology but, apart from their disjunct distribution and different substrate ecology, differ in lobe configuration, color and disposition of the crystal clusters and resulting surface patterns.

During a survey of three islands in Hawaii in 2013 to sample lichen material for molecular studies, RL and BM revised collections held at the National Tropical Botanical Garden herbarium (PTBG), including a strange lichen collected by TWF 24 years ago on shaded basalt on the island of Kauai. The taxonomic affinities of this lichen remained unresolved until similar material was found rather abundantly on shaded calcareous rock outcrops during an expedition to western Cuba (Pinar del Río) in April 2016, by RL, BM , CV and JG . While the overall morphology and anatomy of the two lichens suggested affinities with certain Graphidaceae, in particular the genera Leucodecton and Sanguinotrema (Frisch et al. 2006;Rivas Plata et al. 2010;Lücking et al. 2015), molecular sequence data unexpectedly placed the Cuban taxon within Porinaceae. Careful search for ascomata indeed revealed the presence of perithecia in a few specimens, producing 3-5-septate ascospores. Revision of taxa historically described from Cuba, as part of a checklist project together with CV and HJMS, turned up two names under which the lichen had been previously described, namely Verrucaria firmula Nyl. (Nylander 1892;nom. inval.) [≡ Porina firmula Nyl. ex Müll. Arg. (Müller 1885)] and Endopyrenium incrassatum Müll. Arg. (Müller 1885) [≡ Dermatocarpon incrassatum (Müll. Arg.) Zahlbr.].
Whereas Porinaceae is well-delimited at family level, both phenotypically and phylogenetically, its internal classification has been the subject of much debate with proposals ranging from recognizing few genera only, including a single large genus, Porina (Lücking 1998(Lücking , 2008Lücking et al. 2017), to distinguish several smaller genera characterized by perithecial morphology and thallus characters (Hafellner & Kalb 1995;Harris 1995Harris , 2005McCarthy & Malcolm 1997). Molecular sequence data support the separation into smaller genera, but the phenotypes hitherto distinguished at genus level do not necessarily form monophyletic groups, and many taxa have not yet been sampled (Baloch & Grube 2006, 2009Nelsen et al. 2014). Along with the recent recognition of a new genus, Flabelloporina, a possible future generic concept for Porinaceae was outlined (Sobreira et al. 2018). The placement of the newly discovered lineage further supports this solution. Also, the two lichens treated here add a new phenotype to the known morphological variation of this family. Since the phylogenetic analysis supports the uniqueness of this lineage, it is here described as a new genus, Saxiloba.

Material and methods
The sequenced material was collected in the province of Pinar del Río in westernmost Cuba, during a field trip by RL, BM, CV, and JG; voucher specimens are deposited in B and HAJB. The new sequences for this study were generated using the Sigma REDExtract-N-Amp Plant PCR Kit (St. Louis, Missouri, SA) for DNA isolation, following the manufacturer's instructions but with lower proportions for lower amounts of DNA. We targeted both the mitochondrial small subunit (mtSSU) and the nuclear large subunit rDNA (nuLSU), but only obtained sequences for the former; previous work suggests that nuLSU sequences are notoriously difficult to obtain for members of this family (Nelsen et al. 2014). The mtSSU was amplified using the primers SSU1R and SSU3R (Zoller et al. 1999). The 13-µL PCR reactions consisted of 6.0 µL of water, 0.1 µL of each PCR primer, 3.5 µL of REDExtract-n-AmpPCRReady Mix (Sigma-Aldrich) and 2.0 µL DNA. The PCR cycling conditions were as follows: 95°C for 5 min, followed of 35 cycles at 94°C for 45 s, after 50°C for 1 min, 72°C for 1.5 min, followed by a single 72°C final extension for 10 min.
Prior to assembly, the obtained sequence reads were evaluated using BLASTn (Chen et al. 2015) and combined with selected sequences of Porinaceae from GenBank, using Coenogonium as outgroup (Table 1). All sequences were arranged in BIOEDIT 7 (Hall 1999) and aligned using MAFFT 7 (Katoh & Standley 2013). The phylogenetic tree was built by means of maximum likelihood with 1000 bootstrap pseudoreplicates using RAxML 8 (Stamatakis 2014). The best-scoring tree was visualized in FigTree 1.4 (Rambaut & Drummond 2012).

Results and discussion
Although based on a single marker, the mitochondrial small subunit rDNA (mtSSU), the molecular phylogeny of Porinaceae was well-resolved and supported including in the backbone (Fig. 1). Overall the topology was congruent with that obtained by Sobreira et al. (2018). The family formed two large clades with 77% and 70% support, respectively. One contained taxa with exposed, black or red perithecia, the other those with perithecia covered by thallus, except for the black-fruited Porina byssophila lineage, which clustered with support with Clathroporina and Myeloconis. Besides perithecial wall pigmentation, the lineages in the first large clade were largely distinguished by substrate (leaves vs. bark vs. rock) and biome ecology (tropical vs. temperate); most of these have generic names available. For instance, blackfruited tropical species on leaves clustered in the genus Trichothelium, whereas temperate-subtropical species on bark and rock, including aquatic taxa, formed separate lineages (Pseudosagedia, P. pacifica clade). Similarly, red-fruited, tropical, foliicolous species formed a lineage (Phragmopeltheca) separate from temperate, corticolous taxa (Segestria). In the clade characterized by thallus-dominated perithecial verrucae, lineages were chiefly separated by ascospore septation (muriform in Clathroporina and Myeloconis) and shape (narrow filiform in the P. dolichophora clade), as well as habitat ecology, thallus morphology and substrate (Porina s.str., Phyllophiale, P. guianensis clade, Phylloporina).
The unique, placodioid taxon from Cuba, Saxiloba firmula, was placed in the first large clade, which is congruent with its red perithecia (present in few specimens) resembling those of Phragmopeltheca and Segestria. However, it formed a strongly supported lineage on a long branch separate from the latter two, and monophyly of these three lineages was rejected with high significance (SH test employed in RAxML 8; p < 0.001). Together with the unique thallus morphology within the genus, we conclude that this lineage should be recognized at genus level, and introduce the genus Saxiloba for it. Description. Thallus saxicolous, placodioid, convex with distinct marginal lobes, but densely appressed to the substrate; surface smooth, but with a distinct network of reticulate to meandering lines (best seen when hydrated). Photobiont Trentepohlia. Thallus in section with large, triangular to rhomboid crystal clusters embedded into the photobiont layer (somewhat appearing like teeth within gums), the photobiont layer developed horizontally beneath and vertically between the crystal clusters; above with a prosoplectenchymatous cortex and below with a medulla and a thick, dark brown hypothallus layer. Perithecia immersed to erumpent or somewhat prominent, largely covered by thallus, ostiolar area orange-red to cherry-red or dark reddish brown, lacking a thallus cover and slightly translucent; paraphyses and asci as in other genera of the family. Ascospores transversely 3-5-septate, small.

Taxonomy
Etymology. Referring to the placodioid thallus with distinct marginal lobes, apparently growing exclusively on rock.
Distribution and ecology. Thus far only known from disjunct collections on islands in the Caribbean (Cuba, Isla de la Juventud, Puerto Rico) and the Hawaiian archipelago (Kauai); growing on calcareous or volcanic rock in shady to exposed situations.
Notes. Within Porinaceae, the new genus Saxiloba is readily distinguished by its placodioid thallus with columnar clusters of crystals embedded in a network of reticulate to meandering lines when seen from above. The unique thallus morphology and anatomy corresponds to its phylogenetic position on a separate, rather long branch. Notably, the largely thallus-covered perithecia of Saxiloba firmula resemble those of the larger second clade of the family, whereas those of S. hawaiiensis are more similar to those of red-fruited foliicolous species in the Phragmopeltheca clade.
Except for the occasionally present perithecia, which immediately reveal its systematic affinities, the genus would not be considered a member of Porinaceae, but bears resemblance with lichens in some other related and unrelated families. Most similar are certain species in the genus Leucodecton in the related family Graphidaceae, in particular L. phaeosporum (Rivas Plata et al. 2010;Rivas Plata & Lumbsch 2011), which are readily distinguished by the apothecioid, pore-like ascomata with apically thick-walled asci forming brown, muriform ascospores, and in the sterile state by the absence of distinct marginal lobes.
The particular thallus anatomy found in Saxiloba can be compared to what has been coined 'window lichens' ('Fensterflechten';Vogel 1955;Follmann 1965), in analogy to the 'stone plants' ('Fensterpflanzen') in the former family Mesembryanthemaceae (now Aizoaceae). This anatomy was redescribed in much detail from a presumably monospecific genus, Labyrintha implexa (Malcolm et al. 1995), recently recombined as Poeltidea implexa (Fryday & Hertel 2014), a rock-dwelling taxon in the unrelated family Lecideaceae. Malcolm et al. (1995) made no reference of the earlier treatments of 'window lichens' by Vogel (1995) and Follmann (1965), but Vondrák & Kubásek (2013) and Fryday & Hertel (2014) showed that this anatomy appears to have evolved in several unrelated lineages, although it is not frequent. Vertical stacks of algal cells separated by columnar crystal clusters or vertical stacks of fungal hyphae have been interpreted as adaptation to xeric habitats and/or high solar insolation (Vondrák & Kubásek 2013;Fryday & Hertel 2014). This hypothesis seems to fit the Hawaiian species, Saxiloba hawaiiensis, whereas S. firmula grows more frequently in shady conditions. Under such conditions, one could also imagine the vertical crystals to aid in light distribution across a thicker photobiont layer, thus enabling the accumulation of higher biomass under low light. Description. Thallus saxicolous on calcareous rocks, up to 5-10(-20) mm diam. with regularly radiating lobes with closely contiguous tips; lobes 0.5-0.1 mm wide; surface silvery grey-green with a network of reticulate lines around isodiametric to elongate chambers (best seen when hydrated). Thallus in section 200-300 μm thick with large, up to 100 μm high and broad, triangular to rhomboid crystal clusters embedded into the photobiont layer, the latter developed horizontally beneath and vertically between the crystal clusters; above with a 20-30 μm thick, prosoplectenchymatous cortex and below with a 30-50 μm thick medulla and a 40-70 μm thick, dark brown hypothallus layer. Perithecia erumpent from the thallus and almost up to the ostiolar area covered with a thick thallus layer, up to 0.5 mm diam.; excipulum 25-35 μm thick, outer parts paraplectenchymatous, pale yellowish, K+ deep orange-red, inner parts prosoplectenchymatous, colourless; involucrellum only developed around the ostiolar area, 30-60 μm thick, paraplectenchymatous, reddish brown with a distinct reddish tinge when seen from the outside. Ascospores 8 per ascus, oblong-fusiform, transversely 3-5(-7)-septate, 15-20(-25) × 3-4(-6) μm, hyaline. Pycnidia rare, up to 0.1 mm diam., appearing as brownish red warts. Conidia oblong-bacillar, non-septate, 3-4 × 1 μm, hyaline.

Key to the known species of Saxiloba
Distribution and ecology. Thus far known from Cuba, including Isla de la Juventud (also known as Isle of Pines or Isla de Pinos) and further reported from the Bahamas and Puerto Rico (Müller 1885;Riddle 1923;Gannutz 1970;Mercado-Díaz 2009). Possibly a Caribbean endemic, in Cuba quite common on calcareous rocks in mostly shaded microhabitats in the dry forests of western and central Cuba. In Puerto Rico, it would be expected in similar habitats, but has not been found in recent surveys. According to Gannutz (1970), the Puerto Rican material was collected at El Yunque National Forest, a rather wet forest with a geology dominated by volcanic rock. This would broaden the ecological range for Saxiloba firmula, but since we have not been able to obtain the material corresponding to this record, we are considering these data with care.
Notes. Saxiloba firmula was first introduced as nomen nudum by Nylander (1876) in the genus Verrucaria and shortly after validated by Müller (1885) in the genus Porina. Müller (1885: 401) did not properly describe the unique thallus morphology of the species, likely because the type material had originally been scraped off the substrate and is fragmented into numerous, small pieces in the lectotype and isolectotypes. Müller (1885: 377) described the same species also as Endopyrenium incrassatum, possibly because of the few, young ascomata ('apothecia pauca valde juvenilia offerunt') with small (18-20 × 4-6 μm), 1-septate ascospores, not realizing the identity of the material. McCarthy (1993) did not mention the name in his treatment of saxicolous Porina species; he annotated the type material in 1992 as 'probably not Porina'. Riddle (1923) reported the species from Isla de la Juventud (Isle of Pines). One of the two cited collections was reported as '... Cerro San Juan del Mar, Columbia ...' (Riddle 1923: 71), which may cause confusion, as there is indeed such a location in Colombia. However, the material refers to Cerros de San Juán in the Columbia community on Isla de la Juventud (Kallunki 1980;Boom 1996;Sastre de Jesús & Santiago-Valentín 1996). In the same paper, Riddle (1923) described P. subfirmula Riddle, also from Isla de la Juventud. That species has, however, a very different, crustose thallus morphology (isotype in NY, NY01219352, Britton & Wilson 15741 checked). Müller (1885) described (and illustrated the ascospores on the type material as predominantly 5-septate and 20-30 μm long. In our recently collected material, we found only few thalli with perithecia with the ascospores mostly 3-septate and 15-20 μm long. Annotations on NY specimens by R.C. Harris also indicate 3-septate ascospores and a discrepancy with Müller's description (J. Lendemer, pers. comm. 2020). Revision of the lectotype showed the ascospores to be mostly 3-septate, more rarely 4-5-septate, and 15-20(-25) μm long. It is therefore unclear how Müller (1885) derived his observations, although based on annotations by R. C. Harris, the material from the Bahamas apparently also features ascospores with more numerous septa (J. Lendemer, pers. comm. 2020 Description. Thallus saxicolous on basalt, up to 10 mm diam. with irregularly branched lobes usually leaving interspaces; lobes 1.0-1.5 mm wide; surface yellowish olive with meandering, labyrinthine lines forming a network of partially open, elongate to jigsaw-puzzle-shaped chambers. Thallus in section 400-800 μm thick with large, up to 300 μm high and up to 150 μm high broad, narrowly rhomboid crystal clusters embedded into the photobiont layer, the latter developed horizontally beneath and vertically between the crystal clusters; above with a 20-30 μm thick, prosoplectenchymatous cortex and below with an up to 300 μm thick medulla and a 30-50 μm thick, brownish black hypothallus layer. Perithecia immersed in the thallus and almost up to the ostiolar area covered with a thick thallus layer, up to 0.6 mm diam.; excipulum 25-35 μm thick, outer parts paraplectenchymatous, yellowish, K+ deep orange-red, inner parts prosoplectenchymatous, colourless; involucrellum only developed around the ostiolar area, 30-50 μm thick, paraplectenchymatous, reddish brown with a distinct reddish tinge when seen from the outside. Ascospores 8 per ascus, oblong-fusiform, transversely 3-septate, 15-20 × 3.5-4.5 μm, hyaline. Pycnidia not observed.
Distribution and ecology. Thus far known only from the type locality on the island of Kauai in Hawaii, like the type species growing on shaded rocks, but on volcanic basalt.
Notes. The material here described as Saxiloba hawaiiensis was collected by TWF and colleagues more than 25 years ago, but left unidentified. During a visit by RL and BM to the National Tropical Botanic Garden on Kauai, the material drew our attention. With the subsequent discovery of a similar lichen in Cuba, we were able to finally put a name on the Hawaiian lichen as well, especially as upon close inspection, we discovered the presence of perithecia producing asci and ascospores typical of Porinaceae. Saxiloba hawaiiensis shares with S. firmula the placodioid thallus with the striking surface pattern formed by the columnar thallus crystals. Although we do not have sequence data for this taxon, the irregular lobe configuration, the more robust lobes, the differences in the patterns of the surface lines surrounding the crystalline clusters, and the different substrate and disjunct distribution leave no doubt that this species is distinct from the Caribbean taxon.