Peltigera serusiauxii (Lecanoromycetes, Ascomycota), a new species from Papua New Guinea and Malaysia

Peltigera serusiauxii is proposed here as a new species from Papua New Guinea and Sabah, northern Borneo (Malaysia). The species belongs to the polydactyloid clade of section Polydactylon. Because of its large thalli with a glabrous upper surface, this species was previously identified as P. dolichorhiza, but it differs by its polydactylon-type lower surface and the high amount of dolichorrhizin. It appears to be a strict specialist in its association with Nostoc phylogroup IX throughout its known distribution. This is one of many undescribed species remaining to be formally described within the genus Peltigera, especially in Asia and Australasia.


Introduction
Peltigera is a genus of ascomycetes that mainly form lichen thalli with cyanobacteria of the genus Nostoc. This group of cyanolichens is present on all continents and often develops large and conspicuous thalli. It is particularly diverse in boreal and tropical mountain forests (Vitikainen 1994;Martinez et al. 2003). Recent studies based on multi-locus phylogenies and species delimitation methods revealed a largely underestimated level of biodiversity throughout its distribution (Jüriado et al. 2017;Magain et al. 2017aMagain et al. , b, 2018Miadlikowska et al. 2018;, such that half of the currently accepted 150 species remain to be formally described. Undescribed species are distributed across all sections recognized by Miadlikowska & Lutzoni (2000).
In section Polydactylon, the name P. dolichorhiza (holotype collected in Colombia) has been widely applied to specimens forming large, brittle thalli with a glabrous upper surface in many parts of the world, including Africa (Swinscow & Krog 1988), Asia (e.g., Wei et al. 2009) and Australasia (e.g., Kantvilas & Mitchin 1989). Slightly over a decade ago, Sérusiaux et al. (2009) demonstrated that specimens morphologically identified as P. dolichorhiza in Papua New Guinea potentially represented at least four distinct evolutionary lineages. Species delimitations based on DNA sequences subsequently revealed that none of these four lineages include specimens from the Americas (Magain et al. 2017a, b), where P. dolichorhiza was originally described (Nylander 1860). Therefore, specimens previously identified as P. dolichorhiza in Asia and Australasia (including Papua New Guinea) represent distinct species (Magain et al. 2017a, b).
In fact, Peltigera in Papua New Guinea includes three putative species belonging to the dolichorhizoid clade of section Polydactylon (sensu Magain et al. 2017a), as well as a fourth putative species belonging to the polydactyloid clade. This last species, referred to as P. dolichorhiza s.lat. D by Sérusiaux et al. (2009), was subsequently recognized as Peltigera sp. 11 by Magain et al. (2017a, b) and shares a most recent common ancestor with P. polydactylon and P. nana. However, it has consistently been shown to be on a long branch, and is therefore considered to be phylogenetically distinct from all currently recognized Peltigera lineages (Magain et al. 2017a, b). Peltigera sp. 11 is one of the oldest lineages within section Polydactylon (Magain et al. 2017a). It also appears to be a strict specialist, having so far been found exclusively in association with Nostoc phylogroup IX (Magain et al. 2017a). It is fairly widespread in Papua New Guinea, where it is known from three provinces (Sérusiaux et al. 2009). Members of the polydactyloid clade occur mostly in Asia and Australasia, with the exceptions of the North American species Peltigera seneca and the primarily circumboreal species P. polydactylon (Magain et al. 2016(Magain et al. , 2017a. Here we describe a new species, Peltigera serusiauxii, to accommodate the taxon previously reported as P. dolichorhiza s.lat. D sensu Sérusiaux et al. (2009) and P. sp. 11 (Magain et al. 2017a, b). Additionally, we show that a recent collection from Mount Kinabalu, Sabah (northern Borneo, Malaysia), represents a distinct population of P. serusiauxii.

Materials and methods
Taxon sampling, additional sequencing and phylogenetic analyses We extracted DNA from a single thallus of Peltigera (DNA number N8001) collected from Mount Kinabalu, Sabah (northern Borneo, Malaysia) in 2018, using a CTAB-based protocol (Cubero et al. 1999). We sequenced the complete internal transcribed spacer of the nuclear ribosomal RNA genes (ITS), β-tubulin, and the largest subunit of RNA polymerase II (RPB1) for the mycobiont, as well as the rbcLX region of the Nostoc photobiont from this thallus, following Magain et al. (2017a). Mycobiont sequences were added to a dataset including all available sequences of these three loci for Peltigera serusiauxii (P. dolichorhiza s.lat. D), as well as representatives of all known species from the polydactyloid clade (Magain et al. 2017a, b) retrieved from GenBank (Table 1). Peltigera dolichorhiza s.str. and three other putative species from Papua New Guinea from the dolichorhizoid clade were chosen for the outgroup (Table 1).
ITS, RPB1 and β-tubulin alignments were assembled using Mesquite v. 3.11 (Maddison & Maddison 2011) and aligned using MAFFT v7.305b (Katoh & Standley 2013) with default parameters. A 3-locus dataset was assembled using the combine.pl script of the PLexus package (Magain 2018). The dataset was divided into eleven subsets (ITS1, 5.8S, ITS2, β-tubulin 1 st , 2 nd and 3 rd codon positions and introns, RPB1 1 st , 2 nd and 3 rd codon positions and intron), and the best partition scheme for the phylogenetic analyses was determined using PartitionFinder 2.1.1 (Lanfear et al. 2016) by implementing the corrected Akaike information criterion (AICc) and the greedy algorithm. Maximum likelihood (ML) phylogenetic searches were implemented with RAxML version 8.2.12 (Stamatakis 2006; Stamatakis et al. 2008) using the CIPRES portal (Miller et al. 2010), with the GTRGAMMA model and the best partition scheme retrieved from the Partition-Finder analysis. Bootstrap support values were obtained based on 1000 pseudoreplicates.
Using ITS sequences, we provide DNA barcodes for the unambiguous recognition of P. serusiauxii from all other Peltigera species. The rbcLX sequence from the specimen collected on Mount Kinabalu was added to the rbcLX dataset from Magain et al. (2017a, Matrix 4). The dataset was partitioned by codon positions, and the ML phylogeny was generated using the same parameters as for the mycobiont.

Morphological and chemical studies
Ascospores were measured and photographed under a Nikon Eclipse 80i light microscope at 400×, mounting the material in 3% KOH. Thin-layer chromatography (TLC) was performed in phase C following Orange et al. (2010) for three specimens of P. serusiauxii: the type of the species, an additional representative from Papua New Guinea, and the specimen from Borneo.

Results and discussion
Phylogeny of the polydactyloid clade The PartitionFinder analysis suggested that the mycobiont dataset should be divided into two subsets: one composed of ITS1, ITS2, RPB1 3 rd codon position, β-tubulin 3 rd codon position and β-tubulin introns; the other composed of 5.8S, the 1 st and 2 nd codon positions of both protein-coding genes, and the intron in RPB1. The phylogeny of the polydactyloid clade ( Fig. 1A) is similar to the topology obtained by Magain et al. (2017a, b). Within this clade, Peltigera sp. 9, which likely corresponds to Peltigera dolichospora based on its unique chemistry (absence of tenuiorin and methyl gyrophorate; Magain et al. unpublished), is sister to the remaining taxa in the clade, whereas P. serusiauxii (P. sp. 11 in Magain et al. 2017a, b) represents the second split. Within the P. serusiauxii clade, the specimen from Borneo is sister to a strongly supported group composed of all specimens from Papua New Guinea. All interspecific relationships and clades representing species or putative species are highly supported (BS ≥ 90), with the exception of the unresolved relationships within the nana group (Fig. 1A). The specimen from Mount Kinabalu, Borneo (N8001), is assigned to P. serusiauxii until more material from this lineage becomes available. Additional phenotypic information will allow a re-evaluation of the taxonomic status of this population.
In the rbcLX Nostoc tree (Fig. S1), all P. serusiauxii symbionts, including the newly sequenced cyanobiont from the Malaysian specimen, belong to a strongly supported clade (Fig. 1B), which corresponds to phylogroup IX sensu Magain et al. (2017a). To date, all known representatives from Nostoc phylogroup IX have been found in association with the mycobiont of P. serusiauxii and vice versa, representing another rare case of one-to-one reciprocal specificity in the genus (Magain et al. 2017a. The strict specialist profile of P. serusiauxii is confirmed in its current expanded geographic range. Therefore, previous findings do not seem to be an artifact caused by the limited sampling of P. serusiauxii exclusively from Papua New Guinea (Magain et al. 2017a).

Chemistry, morphology and biogeography
Peltigera dolichorhiza s.lat. D was distinguished chemically by the abundance of dolichorrhizin, but was otherwise indistinguishable from P. dolichorhiza s.lat. A-C (Sérusiaux et al. 2009). In Papua New Guinea, P. dolichorhiza s.lat. D occurs as an epiphyte (including dead wood) between 1800 and 2400 meters. The specimen Table 1. Voucher information and GenBank accession numbers for specimens included in the phylogenetic analyses of the polydactyloid clade of the genus Peltigera, section Polydactylon (Fig. 1A). Newly acquired sequences are bolded.

Taxon
Voucher ITS ß-tubulin The lower surface of specimens from Papua New Guinea is variable, but in most cases it resembles the lower surface of P. polydactylon, with dark brown veins reaching the end of lobes, but well spaced and not delimiting small white oval interstices (Figs 2B & 3B). The lower surface of the specimen from Borneo has very dark and well-delimited veins, sometimes forming a dense network defining small white oval interstices, reminiscent of the lower surface of P. occidentalis. In some specimens with more widely spaced interstices, the lower surface resembles P. polydactylon (Fig. 3D).
Specimens of P. dolichorhiza s.lat. A and C (the latter shown as P. sp. 4 in Figure 1A), from the dolichorhizoid clade, generally belong to the dolichorhiza morphotype with thinner and more brittle thalli and paler veins. However, a relatively high degree of plasticity is present, with overlaps between species, preventing these character states from being diagnostic at species level. The upper surface of specimens of P. dolichorhiza s.lat. B (P. sp. 5 in Fig. 1A) is greenish brown to dark brown when dry, dark brown when wet, smooth and typically shiny, and the lobe margins are brittle and usually fragmented in herbarium specimens. The lower surface is pale orange brown near the margins, becoming black towards the thallus center where the black veins become fused and tend to cover the entire surface. The network of orange veins with rather small interstices is clearly visible towards the thallus margins but becomes more diffused, with large white interstices in the intermediary zone between the margins and the thallus center, similar to P. serusiauxii.
Strong affinities between lichens of Mount Kinabalu and Papua New Guinea are common, as many species such as Pertusaria epitheciifera, Thelotrema subweberi and Placopsis auriculata occur in both areas (Sipman 1993). The genus Sporopodiopsis (Sérusiaux 1997) includes two species, one from Papua New Guinea (S. mortimeriana) and one from Mount Kinabalu (S. sipmanii).
The chemistry of the P. serusiauxii specimen from Mount Kinabalu is similar to that of the Papua New Guinea specimens, with the presence of tenuiorin, methyl gyrophorate, dolichorrhizin and zeorin (Fig. S2).
Diagnosis: P. serusiauxii differs from other glabrous species in Asia and Papua New Guinea by its P. polydactylon-like lower surface (in which the veins reach the lobe tips, and the interstices exceed the width of the veins toward the thallus center but become narrower than the veins towards the lobe tips), its abundant production of dolichorrhizin, and its ITS barcodes GTAGCTGTG in ITS1 and AAATAG in ITS2.

Reference sequence: KX897392
Description. Thallus large, 6-8(-10) cm across, with lobes 1-2 cm across, upper surface grey, light brown D or dark brown when dry, dark grey to dark brown when wet, smooth but undulating, rather thin and fragile. Margins regular, flat or slightly raised. Vegetative propagules not seen. Lower surface with whitish interstices strongly contrasting with light brown to dark brown veins almost always extending to margins, becoming darker towards thallus center; veins forming a well-delimited network with large white interstices, veins rather narrow (0.5-1 mm), slightly raised, usually much narrower than the white interstices (2-)3-4 mm wide, especially towards center of thallus, whereas veins become broad and often wider than the interstices towards the margins (~1 mm wide each). Rhizines sparse, usually located near margins, quite long (0.5-)0.8-1(-1.2) cm, dark brown to black, simple to fasciculate. Apothecia abundant when present; brown, small and somewhat circular, usually 1-2 mm long and 1.5-2.5 mm wide, but up to 6 mm long and 4 mm wide, usually on top of a somewhat triangular raised lobe, usually folding on itself on upper surface when dry. Ascospores 8 per ascus, fusiform to acicular, (60-)80-110 × 3.5-5 μm, 4-11 septate, hyaline. Pycnidia not seen.

Notes.
No validly published epithet could be found for this species delimited as distinct in the species delimitation analyses by Magain et al. (2017b). Peltigera serusiauxii represents an independent lineage in the polydactyloid clade (Fig. 1A). Usually, thalli of P. serusiauxii are more robust than thalli of P. sp. 4 and P. dolichorhiza s.lat. A. As compared to P. sp. 5, thalli of P. serusiauxii have darker veins that extend to the margins and form a well-delimited brown network with large white interstices. However, ITS barcodes are needed to reliably distinguish P. serusiauxii from other species in Papua New Guinea. On Mount Kinabalu (Malaysia), P. serusiauxii differs in the vein pattern from other co-occurring species, including P. neopolydactyla 3 and an unnamed species related to P. sp. 4 and P. sp. 5 (Magain unpublished). Peltigera serusiauxii is endemic to Southeast Asia where it has so far been reported exclusively from Malaysia and Papua New Guinea.
documents. We also thank the UMS graduate students Foo She Fui and Mohd Hafiz Syukri for field assistance. We acknowledge the Sabah Biodiversity Center (SABC) and Sabah Parks for issuing collecting and export permits. The specimen from Borneo was collected by Nicolas Magain under SABC Access License JKM/MBS.1000-2/2 JLD.6 (101) and exported under Transfer License JKM/MB.1000-2/3 JLD.3 (70). IDM acknowledges support from the United States National Science Foundation Graduate Research Fellowship Program under grant number DGE 1644868. This work was financially supported by a grant from the National Science Foundation's Geneology of Life program (NSF GoLife DEB-1541548) to FL and JM. . TLC in phase C of three specimens of P. serusiauxii: two specimens from Papua New Guinea (N1532 and type N1533) and the specimen from Malaysia (N8001). Download file