Two rare Peltigera species new to the Canadian Arctic, P. islandica and P. lyngei

. Peltigera islandica and P. lyngei are rarely reported lichens. Previously, P. islandica was known from British Columbia, Estonia, and Iceland, and P. lyngei from Amchitka Island (Alaska), Gough Island (South Atlantic), Iceland, Siberia and Svalbard. Both species are reported here for the first time from the Canadian Arctic and from the second localities in North America. Peltigera lyngei is also reported for the first time from Canada. The iden tities of these species are confirmed morphologically, chemically, and with molecular data. Phylogenetic relationships are inferred using the ITS region. The widespread, but scattered, distribution of both species suggests that they may be underreported throughout their range.


Introduction
Peltigera (Peltigerales, Ascomycota) is a cosmopolitan genus of relatively large foliose macrolichens (Martínez et al. 2003). It can be a taxonomically difficult genus with subtle morphological differences among some species, and many molecularly defined taxa appear to lack clear corresponding morphological characters (Magain et al. 2016;Miadlikowska et al. 2018). Consequently, some species may be overlooked in the field and have distributions that are poorly understood.
Peltigera islandica is an example of a species with a widespread, but scattered, distribution. It is known only from western Canada, Estonia, and Iceland (Jüriado et al. 2017;Manoharan-Basil et al. 2016). Since this species was recently described, new information about its range would not be surprising as more survey work is conducted. Peltigera lyngei is another species with a scattered distribution. It is known from Amchitka Island (Alaska), Gough Island (South Atlantic), Iceland, Siberia and Svalbard (Øvstedal et al. 2009;Dillman et al. 2012). However, it was described 90 years ago (Gyelnik 1932), so it is either a rare species or it has been overlooked, possibly both. Nevertheless, what we currently know about these two species is that they are rarely reported and only from widely dispersed localities.
During on-going lichen surveys in Nunavut, Canada, new localities for P. islandica and P. lyngei were discovered that are reported here for the first time from the Canadian Arctic. These records fill gaps in our understanding of their distribution and they illustrate the need for continued survey work in the Canadian Arctic to gather fundamental baseline biodiversity data in a quickly changing environment. The Arctic is warming faster than any other region on Earth (IPCC 2007;Kaufman et al. 2009), and knowing what species are present is essential for understanding the impacts of climate change.

Metabolites, morphology, and deposition
Specimen morphology was examined microscopically using a stereoscope. Secondary metabolites were determined using thin layer chromatography following Culberson and Kristinsson (1970) and Orange et al. (2001) in solvents A, B', and C. Thallus fragments were extracted in hexane for ~5 min. All specimens examined have been deposited at the Canadian Museum of Nature (CANL) and duplicates at Duke University (DUKE).

DNA extraction
DNA extraction was conducted at the Canadian Centre for DNA barcoding (CCDB) following CCDB protocols outlined by Ivanova et al. (2008Ivanova et al. ( , 2011. In summary, a small amount (~5 mm 2 ) of dry lichen tissue was removed from the sample using a stereoscope and fine tipped forceps while ensuring that there were no vegetative propagules (i.e., soredia or isidia) from other lichens and no lichenicolous fungi. A fragment of the lichen was placed into racked sterile mini tube strips with a 3.17 mm stainless steel bead in each tube and then sealed with a sterile cap strip. The fragment was then ground into fine powder using a Tissue Lyser (Qiagen, USA) with rack adapters at 28 Hz for 30 seconds, then rotated and ground for an additional 30 seconds. The ground material was then incubated with 2× CTAB buffer at 65°C for 1 hour and DNA was then extracted using semi-automated method employing glass fiber filtration (Fazekas et al. 2012;Ivanova et al. 2008). The final concentration of the eluted DNA was 20-40 ng/μL.

PCR and sequencing
Fungal primers ITS-1F (Gardes & Bruns 1993) and ITS 4 (White et al. 1990) were used for amplification of the ITS1, 5.8S and ITS2 regions, commonly named ITS (Internal Transcribed Spacers 1 and 2). The PCR conditions for rbcL described by  were followed for the ITS region. The thermocycle profile for the ITS region consisted of 94°C for 2 min, 40 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min. PCR products were visualized on a 2% agarose gel using an E-Gel96® Pre-cast Agarose Electrophoresis System (Invitrogen). Bidirectional sequencing using the same PCR primers was done with the BigDye® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Life Technologies) on an ABI 3730xl Genetic Analyzer (Applied Biosystems, Life Technologies) following Ivanova and Grainger (2007). Bidirectional sequences were assembled in CodonCode 4.2.2 and manually edited.

Phylogenetic analyses
All newly generated sequences were subjected to BLAST searches using NCBI database (https://blast.ncbi.nlm. nih.gov/Blast.cgi) to confirm their putative identity. To validate the morphological and BLAST identifications of the specimens, their preliminary placement within the genus Peltigera was determined by the Evolutionary Placement Algorithm (EPA; Berger & Stamatakis 2011) based on the ITS sequences as implemented in the Tree-Based Alignment Selector toolkit (T-BAS version 2.1, available at http://tbas.hpc.ncsu.edu; Carbone et al. 2017Carbone et al. , 2019 using first the global phylogeny of the genus Peltigera (Chagnon et al. 2019;Carbone et al. 2019) and subsequently the phylogenies for the Polydactylon and Peltigera + Retifoveatae sections (Magain et al. 2017Carbone et al. 2019) as the reference trees. For each EPA analysis we implemented the GTR substitution model (Rodríguez et al. 1990) with gamma distribution parameter (GTRGAMMA) and calculated likelihood weights with a placement cut-off distance of 10. We also performed a follow up search of the best tree and bootstrap analyses (1000 replicates (Katoh & Standley 2010), and the GTRGAMMA nucleotide substitution model and backbone constraint on the multifurcating reference trees (where internodes with bootstrap support <70% were collapsed) were implemented. Based on the preliminary EPA placement of P. lyngei within the Polydactylon clade, we selected and downloaded from T-BAS the 8-locus alignments for the Dolichorhizoid clade, excluding the scabrosella group (Magain et al. 2017, Fig. 1). We adjusted the ambiguous regions manually and added the two ITS sequences for P. lyngei. We completed maximum likelihood search as implemented in IQ-TREE v. 2.1.3 (Nguyen et al. 2015) by estimating the best fit partitioning scheme and the substitution models (ModelFinder; Kalyaanamororthy et al. 2017) followed by inferring the best tree and bipartitions support (UFBoot; Minh et al. 2013) using the following command line: iqtree2 -s combined.phy -m MFP+MERGE -p codons.txt -bb 1000 -bnni -pre combined. The following three partitions and corresponding models were used in IQ-TREE ML search: HKY+F+R2 for ITS + beta-tubulin introns + beta-tubulin 3rd codon position + EFT2.1 introns + EFT 2nd codon position + EFT2.1 3rd codon position + RPB1 introns + RPB1 2nd codon position + RPB1 3rd codon position + COR1b + COR3 + COR16; HKY+F for beta-tubulin 1st codon position + beta-tubulin 2nd codon position + EFT2.1 1st codon position + RPB1 1st codon position; F81+F: for LSU. We also completed RAxML analyses (as implemented on the CIPRES portal) using the best partitioning scheme estimated by ModelFinder in IQ-TREE, GTRGAMMA substitution model across all partitions and performing 1000 bootstrap replicates.

Phylogenetic analyses
The two ITS sequences for the putative P. islandica (LICHN485-19, GenBank ON943472, and LICHN175-19, Genbank ON943469) blasted with 100% Query Cover and 100% Identity to the sequences of P. islandica from Estonia (LT852849) and Iceland (KJ413238). In the absence of P. lyngei sequence data in GenBank, BLAST results for our ITS sequences (LICHN026-19, Genbank ON943470, and LICHN088-19, GenBank ON943471) were inconclusive with 100% Query Cover and 96-94% Identity with the individuals of multiple species from the Polydactylon section, e.g., Peltigera hymenina, P. neopolydacyla, and P. pacifica. In addition to a few nucleotide differences, the ITS of the putative P. lyngei contains a 22 base pairs long insertion in the ITS1 compared to the sequences of the most similar species. The EPA analyses using the genus Peltigera reference phylogeny placed P. islandica in the section Peltigera, P. canina clade (Clade 9 in Magain et al. 2018) and P. lyngei in the section Polydactylon, Dolichorhizoid clade (Magain et al. 2017) (Fig. 1). Although, their respective placements did not receive high support based on the ITS sequences alone using the EPA and maximum likelihood (RAxML) tools as implemented in T-BAS, we feel confident about the identity of P. islandica based on the morphology, BLAST results, and the presence of the species specific unique nucleotide motif (16 nucleotides) in the hypervariable region of the ITS1 (Miadlikowska et al. 2003;Magain et al. 2018). Maximum likelihood analyses grouped our two collections of P. lyngei together and placed them sister to P. hymenina within the hymenina group in the Dolichorhizoid clade; however, this phylogenetic relationship does not have strong support (Fig. 1). Morphologically, P. lyngei resembles P. malacea because of its almost veinless lower surface and P. scabrosa because of its scabrous upper surface (Vitikainen 1994). Therefore, P. lyngei was assumed to have a close affinity to P. malacea in section Peltidea or P. scabrosa in section Polydactylon, Scabrosoid clade (see discussion under P. lyngei in Vitikainen 1994). The revealed phylogenetic placement of this species in Section Polydactylon, Dolichorhizoid clade, hymenina group, is somewhat surprising. While other scabrid species do occur in the Dolichorhizoid clade (i.e., Peltigera pulverulenta, P. scabrosella, and some morphotypes of P. truculenta), there are no other scabrid species in the hymenina group. Moreover, species in the hymenia group mostly occur around the Pacific Ocean in temperate and tropical regions -Peltigera hymenina (Ach.) Delise is the only exception (Magain et al. 2017;Martínez et al. 2003). Additional loci should be sequenced and the type material for P. lyngei should be included to confirm the identity and phylogenetic placement of this morphospecies.

New reports
Peltigera islandica T. Goward & S.S. Manoharan-Basil (Fig. 2) Notes. Peltigera islandica was recently described from Canada and Iceland (Manoharan-Basil et al. 2016  DUKE). Here, we report it for the first time from Nunavut. We found no chemical substances with TLC, which corresponds with the negative spot test results reported by Manoharan-Basil et al. (2016). Peltigera islandica is distinguished from other species of Peltigera by its Nostoc primary photobiont, laminal tomentum, lobes 5-10 mm wide with downturned tips, and an emerald green upper surface when wet. Specimens from Nunavut have the hypervariable ITS1 region (Miadlikowska et al. 2003;Magain et al. 2018) identical to the motif found in other specimens of P. islandica from Iceland and British Colombia (as P. sp. 20 in Magain et al. 2018), which is unique to this species (GGGTTCGTATGTGCCC; Magain et al. 2018;Manoharan-Basil et al. 2016

Peltigera lyngei
Gyeln. (Fig. 3) Notes. Although the sequences we generated do not match any known species, the secondary metabolites and morphology are consistent with P. lyngei, for which no previous reference sequences exist. Peltigera lyngei was described from Svalbard (Gyelnik 1932) and reported from North America for the first time from Labrador (Kallio & Kärenlampi 1966), but Dillman et al. (2012) revised that specimen to P. malacea. Dillman et al. (2012)