Introduction
In a Festschrift volume of Bibliotheca Lichenologica dedicated to the late lichenologist Dr David Galloway, the enigmatic monospecific lichen genus Gallaicolichen Sérus. & Lücking was described from several locations in the Pacific Ocean, viz. the Hawaiian archipelago (USA), Queensland (Australia), Vanuatu and New Caledonia (Sérusiaux & Lücking Reference Sérusiaux and Lücking2007). It is also documented from the Philippines (Grant et al. Reference Grant, Webbink, von Konrat, Lumbsch and Gaswick2022), based on a gathering by the American botanist Adolph Daniel Edward Elmer, possibly the oldest known collection of the taxon. Recently, the species was detected in the Ryukyu Islands (Iriomote) in southern Japan (Miyazawa et al. Reference Miyazawa, Ohmura and Yamaoka2022).
The asexual reproductive structures produced by this species, somewhat resembling the goniocystangia described for opegraphoid lichens (Sérusiaux Reference Sérusiaux1985), were named peltidiangia, since they are morphologically unique: they produce distinctive, disc-shaped diaspores, known as peltidia, which are made of small arms of dichotomously branched cells of the photosynthetic partner (assumed to represent Phycopeltis). These arms are linked to a central foot and coiled up inwards to encapsulate a tiny, irregular layer of mycobiont hyphae. This pattern of co-dispersal of a fungus, wrapped by a photobiont diaspore, has no equivalent throughout the fungal kingdom; in other superficially similar structures, it is the fungal hyphae that enclose the photobiont cells.
In the present study, the systematic position of Gallaicolichen pacificus Sérus. & Lücking was resolved, due to the discovery of fertile specimens found on living leaves in New Caledonia, and to phylogenetic analyses of mitochondrial small subunit ribosomal RNA (mtSSU) sequences.
Material and Methods
Sampling material
This study is based on specimens of Gallaicolichen pacificus collected by EL and AS during a field trip dedicated to foliicolous lichen species in New Caledonia, in November and December 2022 (for specimen information see Supplementary Material File 1, available online). Specimens of Coenogonium luteum (Dicks.) Kalb & Lücking s. lat., Porina alba (R. Sant.) Lücking and P. fusca Lücking were collected by EL, ES and Nicolas Magain during a collecting trip focused on foliicolous lichens in Guadeloupe (Lesser Antilles), in January 2022. The leaves bearing the specimens were carefully dried for three weeks using a plant press with absorbent paper and placed in a box containing silica gel beads, then stored at −20 °C until DNA extraction. Collection of specimens was carried out under collection permit no. 395-2022/ARR/DDDT (EL) for the South Province of New Caledonia and nos 609011-18/2022/JJC (EL) and 609011-51/2022/JJC (AS) for North Province. For Guadeloupe, collection of specimens was carried out under the collection permit no. 2022-03 (EL) issued by the Guadeloupe National Park.
Morphological analysis
Specimens were examined with an Olympus SZX12 stereomicroscope. Macroscopic images were captured using a Keyence VHX-5000 digital microscope and a VH-Z20R/W/T lens. Hand-cut sections and squash preparations of thallus were mounted in water, a 5% aqueous potassium hydroxide solution (K), or in Lugol's iodine solution (1% I2) or with K pretreatment (KI) and studied using an Olympus BX51 compound microscope. The presence of crystals was investigated using polarized light. Measurements refer to dimensions in water and are given as follows: (min–)x̄(–max) (n = number of measurements). Microscopic images were captured using an Olympus BX51 compound microscope fitted with an Olympus SC50 digital camera.
Molecular analysis
Well-preserved specimens of Coenogonium luteum s. lat. (Lebreton 1584a), Gallaicolichen pacificus (Lebreton 1811a, Lebreton 1812b), Porina alba (Lebreton 879a, Lebreton 880a1) and P. fusca (Lebreton 879b, Lebreton 880a2), lacking any visible symptoms of fungal infection, were selected for DNA extraction. Specimens of G. pacificus were possessing peltidiangia (sensu Sérusiaux & Lücking Reference Sérusiaux and Lücking2007). Due to the small size, the material was extracted using the Phire Plant Direct PCR Kit from Thermo Fisher Scientific® (Massachusetts, USA), following the manufacturer's instructions. DNA was extracted at the laboratories of the Botanical Institute, University of Liège, Belgium. The primer pairs mrSSU1 and mrSSU3R (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999) were used to amplify the mtSSU, with the following PCR conditions: initial denaturation for 5 min at 98 °C, followed by 40 cycles of denaturation for 5 s at 98 °C, annealing for 5 s at 58 °C, elongation for 20 s at 72 °C and a final elongation for 1 min at 72 °C. PCR products were visualized by electrophoresis on a 2% agarose gel, purified with ExoSAP-IT™ (IT PCR clean-up protocol) and sequenced by Macrogen® (Maastricht, the Netherlands). Sequence fragments were assembled with Geneious Prime v. 2022.2.2 (Biomatters, Auckland, New Zealand). Sequences were then subjected to a BLAST search (Altschul et al. Reference Altschul, Madden, Schaffer, Zhang, Zhang, Miller and Lipaman1997) in GenBank, using megaBLAST, to detect potential contamination.
Sequence alignment and phylogenetic analysis
After confirmation of the newly generated sequences as members of the Porinaceae, a dataset of mtSSU sequences was assembled for resolving the exact placement of the newly sequenced taxa in a broader phylogeny of the family Porinaceae. Sequences retrieved from GenBank were selected mainly from previous studies of the family (Sobreira et al. Reference Sobreira, Cáceres, Maia and Lücking2018; Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020; Ertz & Diederich Reference Ertz and Diederich2022; Sanders et al. Reference Sanders, De Carolis, Ertz, de los Ríos and Muggia2023), for a total of 122 sequences in the matrix. Four accessions of Coenogonium were selected as outgroup. Sequences were aligned with MAFFT v. 7 online (Katoh et al. Reference Katoh, Rozewicki and Yamada2019) and the alignment was checked manually using Mesquite v. 2023.3.81 (Maddison & Maddison Reference Maddison and Maddison2023). Ambiguous regions were delimited using the online version of Gblocks v. 0.91b (Castresana Reference Castresana2000) at http://phylogeny.lirmm.fr/, allowing for gap positions within the final blocks, and carefully checked manually (Supplementary Material Files 2 & 3, available online). Consequently, a total of 568 positions were conserved for the phylogenetic analysis. We inferred the best-scoring maximum likelihood tree and bootstrap support values based on 1000 pseudoreplicates in the same run using RAxML-HPC2 v. 8.2.12 (Stamatakis Reference Stamatakis2006; Stamatakis et al. Reference Stamatakis, Hoover and Rougemont2008), with the GTRGAMMA model and default settings as implemented on the CIPRES portal (Miller et al. Reference Miller, Pfeiffer and Schwartz2010) (Supplementary Material File 4, available online). Phylogenetic trees were visualized using FigTree v. 1.4.4 (Rambaut Reference Rambaut2018). Maximum likelihood bootstrap values (BS) > 70% were considered significant.
Results
Phylogenetic analyses
The backbone topology of the molecular phylogeny of Porinaceae is poorly supported (Fig. 1), which is consistent with recent phylogenetic reconstructions (Sobreira et al. Reference Sobreira, Cáceres, Maia and Lücking2018; Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020; Orange et al. Reference Orange, Palice and Klepsland2020; Ertz & Diederich Reference Ertz and Diederich2022; Sanders et al. Reference Sanders, De Carolis, Ertz, de los Ríos and Muggia2023). The 14 lineages highlighted in Sobreira et al. (Reference Sobreira, Cáceres, Maia and Lücking2018) are supported, along with five new clades added by Lücking et al. (Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020), Ertz & Diederich (Reference Ertz and Diederich2022) and Sanders et al. (Reference Sanders, De Carolis, Ertz, de los Ríos and Muggia2023).
Best-scoring maximum-likelihood (ML) tree of Porinaceae based on the mtSSU marker, showing the position of Gallaicolichen pacificus highlighted in green and the new sequences produced in the present study in bold. Potential genus-level lineages sensu Sobreira et al. (Reference Sobreira, Cáceres, Maia and Lücking2018) are indicated. Scores for branches with ML bootstrap values < 50 are not shown. In colour online.
Nineteen main clades can be recognized within the Porinaceae. The largest one, with 87% BS, includes the lineages named Phyllophiale, Phylloporina, the Porina dolichophora clade, the Porina guianensis clade, and Porina s. str. Additionally, there is another well-supported clade with 97% BS, comprising Clathroporina and Myeloconis. A separate clade with 98% support includes Porina hibernica P. James & Swinscow and P. pseudohibernica Tretiach, closely related to the P. collina and P. byssophila clades (sensu Sobreira et al. Reference Sobreira, Cáceres, Maia and Lücking2018). Another clade containing P. leptalea (Durieu & Mont.) A. L. Sm. forms a well-supported clade with the P. fusca clade, and finally, a clade with P. rupicola Ertz & Diederich, P. internigrans (Nyl.) Müll. Arg. s. lat. and P. nuculoides Ertz & Diederich is closely related to the recently described genus Saxiloba (Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020).
However, several lineages lack robust support in the deeper nodes. The sister relationship between the clade Porina covidii Ertz & Diederich and ‘Porina cf. malmei’, the clade P. nanoarbuscula Ertz et al. and P. microcoralloides Ertz et al. is weakly supported (BS = 48). The placement of some species remains unresolved, for example Porina mamillosa (Th. Fr.) Zahlbr., P. rivalis Orange and P. pacifica Brodo.
Based on the named species in the tree, the genus Porina appears to be resolved as paraphyletic because the lineages corresponding to recently accepted or suggested genera (e.g. Clathroporina, Flabelloporina, Myeloconis, Phragmopeltheca, Phyllophiale, Phylloporina, Pseudosagedia, Segestria, Saxiloba, Trichothelium, Zamenhofia, etc.) are nested within a backbone of species still classified within Porina. However, this depends on the definition of what constitutes the genus Porina. In a narrower definition, Porina could be circumscribed to include all species with a crystallostratum, a clade supported in the analyses by Sobreira et al. (Reference Sobreira, Cáceres, Maia and Lücking2018) and Lücking et al. (Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020) but not for instance in Ertz & Diederich (Reference Ertz and Diederich2022). The inconsistencies include two specimens of P. internigrans and one specimen of Porina nuculoides, both species that have a crystallostratum, placed outside this clade. Porina could alternatively be defined as the subclade including the P. nucula, P. dolichophora, Phyllophiale and Phylloporina clades (i.e. excluding Clathroporina, Myeloconis and the rogue samples of P. internigrans and P. nuculoides). In its most narrow definition, it would correspond to the P. nucula clade, containing the type species. One of the main challenges regarding genus-level classification is the notion that gross morphology generally correlates with phylogeny, but some oddly placed taxa challenge this assumption.
The placement of the Coenogonium luteum, Porina fusca and P. alba accessions recently collected in Guadeloupe falls with sequences of the same species; however, the long branches suggest that some of these are species complexes. Both accessions of Gallaicolichen pacificus are resolved as sister to Porina guianensis with strong support (BS = 92) (Fig. 1). The clade formed by Gallaicolichen and P. guianensis is closely related to the larger clades of Phylloporina and Phyllophiale (BS = 71), all representing largely foliicolous taxa or species growing on smooth bark. Furthermore, all these taxa share the production of a crystallostratum, a thick layer of crystals in the ascomatal wall, as also found in the related P. dolichophora and P. nucula clades, in addition to Clathroporina and Myeloconis.
Taxonomy
A detailed description of Gallaicolichen pacificus with only peltidiangia (i.e. without ascomata) was provided by Sérusiaux & Lücking (Reference Sérusiaux and Lücking2007; Fig. 2); it can now be completed with the description of its perithecia and ascospores (Figs 3 & 4).
Gallaicolichen pacificus; historical collections studied in Sérusiaux & Lücking (Reference Sérusiaux and Lücking2007). A, holotype collected in Hawaii by C. W. Smith in 1995 (LG12004-RTR00A). B, peltidia mounted in water. C, lateral view of a young peltidiangia with its inner margin made of raised, not agglutinated hyphae. D, mature peltidia with tips of mycobiont hyphae developed on the other side and emerging through the dichotomously branched Phycopeltis cells. Scales: A = 500 μm; B & C = 20 μm; D = 5 μm. Images by Damien Ertz & Elise Lebreton (A) and Emmanuël Sérusiaux (B–D) (Bibliotheca Lichenologica 95, 510 (Reference Sérusiaux and Lücking2007)). In colour online.
Gallaicolichen pacificus (Lebreton 1886c; LG12002-PTR00A). A, specimen with only peltidiangia. B, two agglutinated thalli, one with only perithecia and the other with only peltidiangia. C & D, specimens with both peltidiangia and perithecia. Scales = 0.5 mm. In colour online.
Gallaicolichen pacificus (Lebreton 1886c; LG12002-PTR00A), mounted in water. A, cross-section of perithecia showing crystallostratum and algiferous layer covering the yellowish involucrellum. B, crystallostratum visible with polarized light. C, ascus with ascospores. D, ascospores (with gelatinous sheath). E, thallus with trentepohlioid algal cells arranged in rows in plates. Scales: A & B = 20 μm; C–E = 10 μm. In colour online.
Gallaicolichen pacificus Sérus. & Lücking
In Sérusiaux & Lücking, Bibliotheca Lichenologica 95, 510 (2007); type: USA, Hawaii, Hamakua, Kolekola Park, on leaves of Syzygium cuminii, 11 vii 1995, C. W. Smith s. n. (LG12004-RTR00A).
Description provided by Sérusiaux & Lücking (Reference Sérusiaux and Lücking2007): ‘Thallus foliicolous, epiphyllous, composed of rounded patches, 0.5–1.2(–1.7) mm in diam., pale greenish yellow to pale yellowish gray, or very pale yellowish, with a slightly lobulate margin, surface somewhat shiny, smooth or very slightly uneven (under high magnification), coalescing when contiguous, up to 20 μm thick, formed of a loose network of interwoven hyphae and large, regular plates of photobiont cells, containing large (up to c. 25 × 10 μm) oxalate crystals, mostly present in mature parts and absent near the margins; cortex sometimes developed, sometimes completely absent, formed of a single layer of polygonal cells, sometimes with a slightly brownish wall, 5–7 × 2–3 μm; prothallus usually present, membranaceous and colourless, rarely bluish or brownish. Photobiont: a species of Phycopeltis (Trentepohliaceae) with greenish brown, c. 8–10(–11) × 4–5 μm cells, regularly and radiately arranged rows in plates. Goniocystangia-like structures always present (here named peltidiangia), 1–5(–8) per thallus patch, the first one developing at the center, almost perfectly circular, 0.1–0.15(–0.2) mm in diam. and c. 0.13–0.15 mm high, formed by a rather thick erect margin with its inner part typically whitish and made of raised, not agglutinated hyphae and its outer part usually covered by the thallus. Diaspores (here named peltidia) numerous, usually filling up the peltidiangia cavity, disc-like, c. 25–55 μm in diam. and c. 10–15 μm thick, with a regular pattern of organization: small arms of dichotomously branched (1–2 branching points) cells of Phycopeltis, all linked to a central foot-like structure and coiled up inwards on the other side where they encompass a tiny, regularly arranged layer of mycobiont cells; 1–2 extremities of hyphal filaments usually present between algal arms and easily seen on the diaspores outer surface; extremities of algal arms slightly but distinctly inflated, and refringent.’
Perithecia solitary, dispersed, hemispherical to ±subglobose, 0.2–0.3 mm diam., 0.1–0.2 mm high, glabrous, surface smooth, with applanate top. Ostiole apical, rather inconspicuous, periostiolar area pale orange. Crystallostratum of calcium oxalate crystals, yellowish, K+ orange-red, c. 30–45 μm thick, covered by a 15–20 μm layer of algiferous tissue. Involucrellum fused with excipulum, densely covered with the same crystals 10–15 μm thick. Hamathecium hyaline, not inspersed, paraphyses simple, non-branched and non-anastomosing, 1–1.5 μm. Asci narrowly obclavate to cylindrical clavate, I−, c. 92.5 × 12 μm (n = 2); ascus apex slightly truncate with a ring structure. Ascospores 8 per ascus, (4–)6–7-septate, fusiform, (24.5–)26(–28) × (4–)5(–5.5) μm (n = 30), 4–7 times as long as wide, colourless with c. 3.5 μm thick gelatinous sheath.
Pycnidia not observed.
Chemistry
TLC not performed.
Ecology and distribution
Gallaicolichen pacificus is reported from various regions of the Pacific Ocean: Hawaiian archipelago, Queensland in Australia, Vanuatu and New Caledonia (Sérusiaux & Lücking Reference Sérusiaux and Lücking2007). Additionally, it has been observed in the Philippines and the Ryukyu Islands, Japan (Miyazawa et al. Reference Miyazawa, Ohmura and Yamaoka2022). In New Caledonia, G. pacificus has been observed in several locations in the South and North Provinces of Grande Terre, in high numbers on phorophytes in forests at mid-altitude (150–600 m elev.), as well as along forest edges and in riparian habitats. The majority of specimens exhibit only peltidiangia without ascomata. The occurrence of both perithecial ascomata and peltidiangia on the same thallus is uncommon. When perithecia are present, peltidiangia are reduced (Fig. 3B). A similar phenomenon has also been observed in Porina alba, where the thallus, when producing perithecia, exhibits few disc-shaped isidia, and vice versa (Lücking Reference Lücking2008).
Notes
Porina atriceps (Vain.) Vain., P. epiphylla Fée, P. lucida R. Sant, P. karnatakensis Makhija et al. and P. subepiphylla Lücking & Vězda share similar perithecia morphology and 7-septate ascospores. However, they differ in spore size and the presence of a reddish or blackish brown spot around the ostiole (Lücking & Vězda Reference Lücking and Vězda1998). In the absence of peltidiangia, Gallaicolichen pacificus could be considered an intermediate species between P. epiphylla and P. lucida. Gallaicolichen pacificus has broader spores than P. epiphylla and shorter spores than P. lucida. Porina subepiphylla also develops larger spores.
Porina epiphylla belongs to a taxonomically difficult group of species, with intermediate forms being common, and should be treated as P. epiphylla s. lat. when identification is uncertain (Lücking Reference Lücking2008).
Specimens examined with ascomata
New Caledonia: Prov. Sud: Mts Koghis, Pic Malaoui tropical rainforest in a creek, collected on Cyathea sp. leaves, 540 m elev., 22°10ʹ57.468ʺS, 166°30ʹ34.667ʺE, 2022, E. Lebreton 1886c (LG12002-PTR00A); Mts Koghis départ du sentier de randonnée juste après la guérite, 500 m elev., 22°10.774ʹS, 166°30.576ʹE, 2022, E. Lebreton 1901e (LG11997-KTR00A).
Specimens examined without ascomata
New Caledonia: Prov. Sud: Mts Koghis-Dumbéa, tropical rainforest with Cryptocarya macrocarpa, Bureavella wakere, Hernandia cordigera, Cyathea intermedia, 15 km NNE of Nouméa, 22°14ʹS, 166°30ʹE, 550 m elev., 23 viii 1994, K. & A. Kalb s. n. (LG12003-QTR00A); Mts Koghis vallée des fougères, 530 m elev., 22°10.631ʹS, 166°30.519ʹE, 2022, E. Lebreton 1811a (LG14602-PPV00A; GenBank Accession no. |PP080177), E. Lebreton 1812b (LG14601-OPV00A; GenBank Accession no. PP080178); Mt Dore, sentier de randonnée du Pic Malaoui par Yahoué, près de la rivière, 138 m elev., 22°11.742ʹS, 166°30.072ʹE, 2 xi 2022, E. Lebreton s. n. (LG12001-OTR00A); Sarraméa, sentier de randonnée du plateau du Dogny, 662 m elev., 21°37.273ʹS, 165°52.189ʹE, 2022, E. Lebreton 1925b (LG11993-GTR00A). Prov. Nord: Pouébo, Mt Amos, patch de forêt humide, 573 m elev., 20°18.135ʹS, 164°26.278ʹE, 13 xi 2022, E. Lebreton s. n. (LG11992-FTR00A); Koné, Massif du Koniambo, Vallée de la Confiance, foliicolous in lowland rainforest, along the river, 21.0298°S, 164.8218°E, 2022, A. Simon 1204 (O, O-L-400621) with A. Evankow, R. Haugan, E. Möller & E. Timdal.—Philippines: Luzon: Sorsogon Province, Irosin, Mt Bulusan, A. D. E. Elmer 15932 (F).
Discussion
In the absence of ascomata or molecular data, Gallaicolichen pacificus would probably not be assigned to the Porinaceae, a similar case being the recently described genus Saxiloba (Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020). As for Gallaicolichen pacificus, several hypotheses have been proposed based on morphological similarities, such as the resemblance of the peltidiangia to the goniocystangia found in the Opegrapha lambinonii aggr. (Sérusiaux Reference Sérusiaux1985), and the resemblance of the thallus to Porina alba (Lücking et al. Reference Lücking, Streimann and Elix2001) or species of Opegraphaceae (Opegrapha) and Roccellaceae (Mazosia) (Lücking & Kalb Reference Lücking and Kalb2001; Sérusiaux & Lücking Reference Sérusiaux and Lücking2007). The discovery of ‘fertile’ thalli, producing both peltidiangia and perithecia, together with studies of the perithecial structure and molecular data with the mtSSU marker, strongly support its assignment in the Porinaceae.
This family is characterized by perithecioid ascomata with unbranched paraphyses, thin-walled unitunicate asci, and hyaline ascospores with thin walls and septa, all features displayed by G. pacificus. Furthermore, the ascomata features support the close relationship of this species with the Porina epiphylla aggr., characterized by perithecia with a yellowish involucrellum turning orange-red when treated with K, the presence of a crystallostratum covering algiferous thallus tissue, and septate ascospores (Hafellner & Kalb Reference Hafellner and Kalb1995; Lücking & Vězda Reference Lücking and Vězda1998; Lücking & Cáceres Reference Lücking and Cáceres1999; Lücking Reference Lücking2008; Ertz & Diederich Reference Ertz and Diederich2022).
Gallaicolichen pacificus can be recognized easily based on the unique thallus structure and the co-dispersal structures named peltidiangia. These propagules share morphological characteristics with those produced by Porina alba, P. fusca, P. mirabilis and the Opegrapha lambinonii aggr. Their morphology is characterized by a disc-like structure, supposedly developed by the plate-forming alga Phycopeltis within the thallus. In the case of P. alba and its relatives, the disc-shaped propagules extend into a sucker that requires inversion for attachment, and they are produced individually. Within the propagule, a singular young discoid thallus of the algal symbiont is present, featuring radial, dichotomously branched filaments (Sanders Reference Sanders2002). The branching pattern of the algae is consistent with morphological descriptions provided by Karsten (Reference Karsten1891) and Thompson & Wujek (Reference Thompson and Wujek1997) for non-lichenized Phycopeltis. In the Opegrapha lambinonii aggr., multiple propagules, referred to as goniocysts, are produced together within a single, dedicated structure similar to that produced by G. pacificus (Sérusiaux & Lücking Reference Sérusiaux and Lücking2007). The propagules in both cases remain small and coin-like. However, in G. pacificus, the mycobiont cells are predominantly embedded or nearly so by algal cells, whereas it is the reverse for species in the Opegrapha lambinonii aggr. where the alga is trapped within the hyphal envelope of the propagules (Sérusiaux Reference Sérusiaux1985). The distinctive feature observed in G. pacificus, where the photobiont partner organizes the propagules and envelops its mycobiont partner, is unique among structures containing both the photobiont and mycobiont of lichenized fungi.
Its closest relative in our phylogeny (Fig. 1), Porina guianensis, has a different thallus morphology. While both species have a non-carbonaceous perithecial wall, a crystallostratum and transverse-septate ascospores, this is also the case for most other taxa within Porina s. str., Phyllophiale and Phylloporina. A unique feature of P. guianensis is the finely verrucose thallus. This, combined with a radiating photobiont, is otherwise also known in P. mazosioides Lücking & Vězda, P. epiphylloides Vězda and P. longispora Vězda, species belonging to another clade. Thus, although these two taxa form a separate clade within the P. nucula complex, the genus name Gallaicolichen is available for this clade. However, no morphological or anatomical characters can be used as autapomorphies to support the genus Gallaicolichen with these two species. Similar problems are found with other morphologically unique taxa, such as Flabelloporina, Myeloconis and Saxiloba, which form parts of phylogenetically separate clades that, however, also include species with other morphologies (Nelsen et al. Reference Nelsen, Lücking, Andrew, Aptroot, Cáceres, Mercado-Díaz, Rivas Plata and Lumbsch2014; Sobreira et al. Reference Sobreira, Cáceres, Maia and Lücking2018; Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020; Ertz & Diederich Reference Ertz and Diederich2022).
The continuous expansion of morphological diversity within the Porinaceae
Hafellner & Kalb (Reference Hafellner and Kalb1995) were the first to separate Porinaceae from the order Pyrenulales, a large and heterogeneous order at the time, and to assign it to its own order, the Trichotheliales (since the name Trichotheliaceae was used for the family). Autapomorphic characters for this order included hamathecial elements made of unbranched paraphyses and periphyses; an ascus wall with a single functional layer; apical structures with an external chitinoid ring structure; poroid ascus dehiscence; hyaline ascospores with thin septa. Based on pigment chemistry and ascus tip structure, as well as the presence of perithecial setae, these authors distinguished five genera at the time: Clathroporina, Porina, Pseudosagedia, Trichothelium and Zamenhofia. Later, McCarthy (Reference McCarthy2003) published an extensive catalogue of all species of Porinaceae, recognizing only three genera (Polycornum, Porina and Trichothelium) and 394 species, especially diverse in the tropics and subtropics.
More recently, Nelsen et al. (Reference Nelsen, Lücking, Andrew, Aptroot, Cáceres, Mercado-Díaz, Rivas Plata and Lumbsch2014) demonstrated the placement of Myeloconis within Porinaceae. Sobreira et al. (Reference Sobreira, Cáceres, Maia and Lücking2018), providing a first larger phylogeny of the family, suggested distinguishing at least 15 genus-level lineages, including the new genus Flabelloporina, with F. squamulifera (Breuss et al.) Sobreira et al., presenting a squamule-bearing thallus (see Lücking et al. (Reference Lücking, Breuss, Nelsen, Navarro, Aptroot, Chaves, Trest and Will-Wolf2013) for original description as Porina squamulifera). Another new genus, Saxiloba, was described based on a unique, rock-dwelling thallus with photobiont chambers separated by crystalline walls, with two species, S. firmula (Müll. Arg.) Lücking et al. from the Caribbean and S. hawaiiensis Lücking et al. from Hawaii (Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020).
The Porinaceae are widely distributed worldwide, colonizing various substrata, such as living leaves, rocks and tree trunks or tiny branches. Most members exhibit a crustose thallus with a smooth or verrucose surface, along with the presence or absence of a dark, shiny, or white prothallus (Figs 5 & 6). An impressively diverse range of forms, colours and structures have been identified within this family and their taxonomic relevance has been debated (McCarthy & Malcolm Reference McCarthy and Malcolm1997; Baloch & Grube Reference Baloch and Grube2006; Nelsen et al. Reference Nelsen, Lücking, Andrew, Aptroot, Cáceres, Mercado-Díaz, Rivas Plata and Lumbsch2014; Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020; Ertz & Diederich Reference Ertz and Diederich2022). Gallaicolichen pacificus now adds a further morphological dimension to the Porinaceae.
Morphological diversity of Porinaceae. A, Saxiloba firmula (Lücking et al. 43034), thallus placodioid. B, Flabelloporina squamulifera (Lücking et al. 39729), thallus bearing flabelliform squamules. C & D, Pseudosagedia crocynioides (R. C. Harris) R. C. Harris (Ertz 24301), byssoid thallus and black perithecia. E, Myeloconis guyanensis P. M. McCarthy & Elix (Cáceres 324), thallus with yellow medulla. F, Porina florensii Diederich & Ertz (Ertz 24283), thallus bearing cylindrical and branched isidia. G, Porina byssophila (Körb. ex Hepp) Zahlbr. (Ertz 26674), black perithecia. H, Porina leptosperma Müll. Arg. (Lebreton 184a), orange perithecia. I, Porina rubentior (Stirt.) Müll. Arg. (Lebreton 188a), cherry red perithecia. Note: specimens are not presented at the same scale. In colour online.
Morphological diversity of Porinaceae. A, Porina alba (left) (Lebreton 880a1) and Porina fusca (right) (Lebreton 880a2) growing side by side, both thalli bearing disc-shaped isidia. B, Trichothelium minus Vain. (Lebreton 196b), black perithecia with 4–6 pale perithecial setae. C, Porina rubescens (Lücking) Hafellner & Kalb (Lebreton 193c), brownish red perithecia with 5–8 pale perithecial setae. D, Trichothelium argenteum Lücking & L. I. Ferraro (Lücking 91-1374), black perithecia with numerous perithecial setae. E, Porina nitidula Müll. Arg. (Lebreton 72a), black perithecia, shortly pilose to tomentose. F, Porina mazosioides (Ertz 24342), perithecia yellow with a crystallostratum. G, Porina subinterstes (Nyl.) Müll. Arg. (Lebreton 108a), thallus with glossy dark prothallus. Note: specimens are not presented at the same scale. In colour online.
Together with the assignment of the genera Myeloconis and Trichothelium, and the recent recognition of Flabelloporina (Sobreira et al. Reference Sobreira, Cáceres, Maia and Lücking2018), Saxiloba (Lücking et al. Reference Lücking, Moncada, Sipman, Sobreira, Viñas, Gutíerrez and Flynn2020) and Gallaicolichen (this paper) within the Porinaceae, a pattern of generic delimitation seems to be emerging. However, as mentioned above, the otherwise seemingly clear correlations of phylogeny with morphology are being challenged by some oddly placed taxa and so no strongly supported consensus throughout the whole family can be obtained with the data currently available. Therefore, several authors opted to retain Porina s. lat. in their recent studies (Orange et al. Reference Orange, Palice and Klepsland2020; Diederich & Ertz Reference Ertz and Diederich2022; Sanders et al. Reference Sanders, De Carolis, Ertz, de los Ríos and Muggia2023) waiting for wider sampling and, in particular, sequences from more loci.
Acknowledgements
EL and ES thank Dr Nicolas Magain for his scientific support and help during fieldwork in Guadeloupe. EL and AS would like to thank Philippe Bourdeau (President, Société Mycologique de Nouvelle-Calédonie) for his guidance during fieldwork in Mts Koghis. Emilie Ducouret, Damien Brouste, Pierre-Louis Stenger, Antoine Lespagnol and Dr Fabian Carriconde (Mycologist, Institut Agronomique néo-Calédonien) are thanked for their help in organizing the field trip in New Caledonia. Credit goes to Dr David Bruy (NOU herbarium curator, Institut de Recherche pour le Développement) for providing the plant presses used to dry foliicolous lichens. Dominique Fleurot from ASPMHNC (Association pour la Sauvegarde du Patrimoine Minier et Historique du Nord Calédonien) is thanked by AS for his insightful recommendations concerning the sampling locality in Koné.
Author Contribution
EL, ES and RL conceived and designed the study. EL and AS carried out fieldwork. EL conducted laboratory work and performed phylogenetic analyses. CS provided supplementary specimens. EL and DE conducted morphological analysis and captured the images. EL wrote the draft of the manuscript and designed the figures. All authors read and reviewed the manuscript.
Author ORCIDs
Elise Lebreton, 0000-0002-4628-6521; Damien Ertz, 0000-0001-8746-3187; Robert Lücking, 0000-0002-3431-4636; Antoine Simon, 0000-0002-7766-5751; Emmanuël Sérusiaux, 0000-0002-0456-0131.
Competing Interests
The authors declare there are no conflicts of interest.
Financial Support
This scientific research was carried out as part of a doctoral thesis (EL) funded by FRIA F.R.S.-FNRS (www.frs-fnrs.be; credit no. 1.E.087.21F), the French Association of Lichenology (www.afl-lichenologie.fr; donation) and the National Parc of Guadeloupe (www.guadeloupe-parcnational.fr; convention de subvention no. 2021-34, appel à projets scientifiques). The field trip in New Caledonia was financed by the National Inventory of the Natural Heritage, Paris (www.inpn.mnhn.fr; appel à projets INPN 2022 pour la contribution à la connaissance naturaliste), with support from PatriNat (OFB, CNRS, MNHN). AS acknowledges funding from the European Union HORIZON-MSCA-2021-PF-01 under grant agreement no. 101068774 (FRAME).
Supplementary Material
The Supplementary Material for this article can be found at https://doi.org/10.1017/S0024282924000124.
Supplementary Material File 1. Species names, voucher specimens and GenBank Accession numbers of mtSSU sequences used in this study.
Supplementary Material File 2. Matrix with Porinaceae mtSSU sequences before exclusion of ambiguous regions.
Supplementary Material File 3. Matrix with Porinaceae mtSSU sequences after exclusion of ambiguous regions.
Supplementary Material File 4. Phylogenetic tree of the Porinaceae in newick format.
