Introduction
Lichens of the genus Thamnolia Ach. ex Schaer. are widely distributed in all hemispheres and their wormlike thalli are easy to recognize by the chalk-white colour and gross morphology. They mainly occur in alpine and arctic environments where extensive colonies are often formed. Even if signs of past or infrequent recombination have been found (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017), Thamnolia has never been found fruiting and it is believed to disperse exclusively by thallus fragments (Andrei et al. Reference Andrei, Iacob and Pascale2006–2007) or mitospores formed in pycnidia (Lord et al. Reference Lord, Knight, Bannister, Ludwig, Malcolm and Orlovich2013).
Morphology, chemistry and geography have traditionally been used to recognize species in the genus. Thamnolia vermicularis (Sw.) Schaer., T. papelillo R. Sant. and T. juncea R. Sant. were thus recognized by thallus morphology. Variation in secondary chemistry within and between these species has resulted in the recognition of T. subuliformis (Ehrh.) W. L. Culb. (syn. T. subvermicularis Asahina), differing from T. vermicularis (in the above sense) by containing squamatic and baeomycesic acids (reacting UV+ yellow) rather than thamnolic acid (UV−). These two chemistries are hereafter referred to as UV+ and UV−, respectively. Sometimes T. subuliformis has been considered a variety and rarely a subspecies of T. vermicularis, an option supported by the fact that subsp. vermicularis and subsp. subuliformis on a large scale have different geographical distributions (Satô Reference Satô1965; Sheard Reference Sheard1977). It is noteworthy that the two chemistries found in T. vermicularis s. lat. also occur in both T. papelillo and T. juncea, and this has also been used as a justification for recognizing infraspecific taxa in these species (Santesson Reference Santesson2004).
Thamnolia has figured prominently in the debate about the recognition of chemotypes as species and previous phylogenetic studies based on molecular data have arrived at different conclusions regarding the use of chemistry in species recognition in this genus. First, Platt & Spatafora (Reference Platt and Spatafora2000) used two specimens of a different chemistry to T. vermicularis s. lat. in a phylogenetic study on non-sexual lichens in Icmadophilaceae. They concluded that the genetic distance between specimens of the two chemotypes was large enough to warrant the recognition of two different species. In two other studies (Nelsen & Gargas Reference Nelsen and Gargas2009; Lord et al. Reference Lord, Knight, Bannister, Ludwig, Malcolm and Orlovich2013) it was concluded that the chemotypes of T. vermicularis s. lat. are not reciprocally monophyletic, although a strongly supported clade composed only of individuals of a UV+ chemotype collected from the Aleutian Islands and Norway was demonstrated. In a recent paper (Leavitt et al. Reference Leavitt, Divakar, Crespo and Lumbsch2016), the authors performed a multispecies coalescent model analysis based on the dataset of Nelsen & Gargas (Reference Nelsen and Gargas2009) and argued for the recognition of the two chemotypes as different species. The authors of the latter study concluded that there is strong evidence for ‘T. vermicularis’ (UV−) and ‘T. subuliformis’ (UV+) being separate species and it was suggested that the lack of reciprocal monophyly of individuals with different chemistries was due to recent divergence and incomplete lineage sorting.
In our recent study, which was based on the analyses of six nuclear markers (genes or noncoding regions) of a worldwide sample of Thamnolia (including the Nelsen & Gargas (Reference Nelsen and Gargas2009) dataset), we showed the existence of three well-supported lineages with different chemistries and geographical distributions (Fig. 1) (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). One lineage (‘Lineage C’, ibid.) has a worldwide distribution and is partly sympatric with the other two lineages. It contains individuals of both chemotypes and also specimens of the morphologically characterized T. papelillo. The other two lineages (‘Lineages A’ and ‘B’) are allopatric and have different chemistries. ‘Lineage A’ was UV+ and geographically restricted to the tundra region of Eurasia and the Aleutian Islands. Interestingly it contains all the UV+ specimens that were grouping in the well-supported clade of the other two studies mentioned above (Nelsen & Gargas Reference Nelsen and Gargas2009; Lord et al. Reference Lord, Knight, Bannister, Ludwig, Malcolm and Orlovich2013). ‘Lineage B’ is UV− and has a rather limited distribution in the European Alps, Tatra Mountains and the Western Carpathians. The Nelsen & Gargas dataset of 2009 included both ‘Lineage A’ (UV+) and ‘Lineage C’ (both UV− and UV+ specimens), hence they based their conclusion on a heterogeneous sample (Leavitt et al. Reference Leavitt, Divakar, Crespo and Lumbsch2016).
Fig. 1 Phylogenetic relationships in the genus Thamnolia based on a six-gene phylogeny (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). The three Thamnolia lineages are represented as collapsed clades. Distribution map of the three lineages with triangles corresponding to each lineage: orange=Lineage A, pink=Lineage B, green=Lineage C. Chemistry of lineages represented by rectangles adjacent to triangles: light blue=baeomycesic and squamatic acids (UV+), grey=thamnolic acid (UV−).
This paper aims to formally clarify the complex taxonomic and nomenclatural situation of Thamnolia, including the recognition and naming of three distinct clades as species. The aim could alternatively be expressed as it was by Minks (Reference Minks1874): “… eine eingehende und umfassenden, eine befriedigende Schilderung von Thamnolia vermicularis zu entwerfen und eine der empfindlichesten Lücken der Lichenologie zu ergänzen.” which adequately stressed the importance and complexity in understanding the taxonomy of Thamnolia.
Material and Methods
Sampling and phenotype descriptions
Twenty-two samples were newly investigated in this study and analyzed together with data from 65 samples included in the study by Onut-Brännström et al. (Reference Onuţ-Brännström, Tibell and Johannesson2017). Information about the samples is given in Table 1. Based on morphological characters, these were previously assigned to the ‘phenospecies/taxa’: T. vermicularis, T. subuliformis, T. papelillo var. papelillo, T. papelillo var. subsolida (M. Satô) R. Sant., T. juncea var. juncea, T. juncea var. subjuncea R. Sant and T. taurica (Wulfen) A. Massal. The newly investigated samples included the following types: holotypes of T. papelillo var. papelillo and T. juncea var. juncea; neotypes of T. vermicularis, T. subuliformis and T. taurica; an isotype of T. juncea var. subjuncea. Podetium chemistry was recorded by documenting the fluorescence under UV light (3500 Å): the podetium appears bright yellow when containing squamatic and baeomycesic acids (UV+), and dark red when containing only thamnolic acid (UV−).
Table 1 Thamnolia specimens used in this study together with their original identification and subsequent assignment to one of the three Thamnolia species. Sample ID includes the geographical origin and laboratory ID of the specimen. Accession numbers in bold indicate new sequences. Dashes=failed PCR or sequencing reactions; blanks=no attempt made to obtain marker
*UV− contains thamnolic acid; UV+ contains baeomycesic and squamatic acids.
DNA extraction, PCR and sequencing
Before DNA extraction, each podetium was investigated for fungal parasites and for each sample we used a 0·3–2·0 cm long part of a single podetium that seemed free of infection. We isolated total genomic DNA using the DNeasy Plant Mini Kit from Qiagen following the manufacturer’s recommendations but with the following important modifications. The samples were handled under sterile conditions while they were placed in individual tubes together with two 3 mm tungsten carbide beads after which the samples were dried at 47°C for 30–40 min. After drying, the tubes were placed in Qiagen TissueLyser II sample holders and kept for 30 min at −80°C, immediately followed by 1 min of shaking at 27 m s–1 in a Qiagen TissueLyser II machine. We incubated the samples for at least 1 h while inverting the tubes every 10 min. Between the last washing and the elution we placed the DNeasy Mini spin column in a new collection tube and centrifuged it for 2 min at 20000 g. Finally, we eluted our sample twice with 60 μl AE buffer. The two eluates were kept in separate tubes.
The Phusion High-Fidelity DNA Polymerase Kit (Thermo Fisher Scientific Inc.) was used to amplify the following nuclear markers of the mycobiont: internal transcribed spacer (ITS), translation elongation factor 1-alpha (Efα), and a putative DEAD-box helicase (DEAD). The latter was introduced as a molecular marker for Thamnolia in Onut-Brännström et al. (Reference Onuţ-Brännström, Tibell and Johannesson2017). The primers used for each amplified region, their direction and relative positions are presented in Table 2. For herbarium specimens older than 20 years, we used undiluted DNA in the PCR reaction. For old herbarium specimens (>200 years), we amplified regions of a length of c. 500 bp using undiluted DNA and Thamnolia specific primers. All PCR products were cleaned with Exonuclease I (Thermo Fisher Scientific, SE) and Shrimp Alkaline Phosphatase (Affymetrix), prepared for sequencing with BigDye Teminator v3.1 Cycle Sequencing and BigDye XTerminator Purification Kits (Thermo Fisher Scientific Inc., Waltham, MA, USA) and sequenced on an ABI 3730xl machine. The reads were trimmed and corrected for errors using Geneious R9 (http://www.geneious.com).
Table 2 Primers for the nuclear markers used in this study together with the position of each forward and reverse primer relative to the alignment
*ITS=Internal Transcribed Spacer, Efα=Elongation Factor alpha, bp=base pairs. DEAD=DEAD-box helicase. **see Dryad for reference to alignment.
Preparing the two datasets
For the phylogenetic analyses, we divided our data into two sets. The first dataset comprised ITS sequences of all samples from which we were able to amplify and sequence the marker (Table 1). The second dataset contained a subsample of the first, for which we had complete information for at least two out of the three nuclear markers: ITS, Efα and DEAD (Table 1). In total, 56 new sequences were identified in this study (Table 1). For the analyses that required the use of outgroups, we used sequences previously obtained from three other species of Icmadophilaceae: Dibaeis baeomyces (L. f.) Rambold & Hertel, Icmadophila ericetorum (L.) Zahlbr., and Siphula ceratites (Wahlenb.) Fr. (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). The FASTA files of the nuclear markers obtained for the outgroups are deposited in the TreeBase repository (http://purl.org/phylo/treebase/phylows/study/TB2:S21678).
Phylogenetic analyses
The sequences were visualized with AliView (Larsson Reference Larsson2014) and aligned with MAFFT (Katoh Reference Katoh2013). When necessary, the sequences were trimmed manually and the missing nucleotides were replaced with N. Gaps were treated as phylogenetic information. All alignment files used for the subsequent analyses were deposited in TreeBase repository (http://purl.org/phylo/treebase/phylows/study/TB2:S21678). The nucleotide substitution models for ITS and Efα were selected using jModelTest (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012) while the amino acid substitution model for DEAD was chosen with MEGA v.06 (Tamura et al. Reference Tamura, Stecher, Peterson, Filipski and Kumar2013).
Three methods were used for phylogenetic analyses: Bayesian inference, maximum likelihood (ML) inference and haplotype networks. Simultaneous Bayesian inference of alignment and phylogeny implemented in Bali-Phy 2.3.8 (Redelings Reference Redelings2014) was used to infer a rooted phylogeny using D. baeomyces, S. ceratites and I. ericetorum as outgroup. For both datasets we chose R07 as the insertion/deletion model. Due to the high computational demand of Bali-Phy, the ITS dataset was collapsed into 33 haplotypes with the web tool FaBOX. For this phylogeny we used the TrNef + G as nucleotide substitution model and started 7 MCMC chains with a total of 70000 iterations. For the three-gene phylogeny we chose TrNef+G as the nucleotide substitution model for ITS and Efα, and the amino acid substitution model JTT for DEAD. We started 5 MCMC chains accumulating a total of 80000 iterations. The convergence of chains was detected and confirmed using the program Tracer (Rambaut et al. Reference Rambaut, Suchard, Xie and Drummond2014) and the informative parameters calculated by the ‘bp-analyze.pl’ script of Bali-Phy 2.3.8. After discarding 1500 trees for the ITS dataset and 2000 trees for the three-gene dataset as burn-in, the rest of the iterations were summarized in a 50% majority-rule consensus tree with posterior probabilities (PP) provided. The phylogenies were visualized using FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). ML phylogenies were obtained from the same datasets but for these analyses we aligned and trimmed both datasets in AliView by removing all the ambiguously aligned sites. The program Garli v.2 was used to construct the ML trees, each with 500 bootstrap iterations. For the ITS ML phylogeny we used K80+G as the nucleotide substitution model. In the case of the concatenated dataset we chose TrNef+I+G for ITS, TrNef+G for Efα and K80+G for DEAD region. The Neighbour-Net algorithm (Bryant & Moulton Reference Bryant and Moulton2004) implemented in SplitsTree v.4 (Huson & Bryant Reference Huson and Bryant2006) was used for calculating a non-rooted haplotype network of the ITS dataset.
Results and Discussion
DNA isolation and amplification from Thamnolia specimens
DNA extraction and amplification of nuclear markers were successful for the selected specimens of all chemical varieties and morphologies of Thamnolia that were collected from 1960 onwards (Table 1). We also succeeded in extracting DNA and amplifying the ITS region from the holotype of T. juncea ssp. juncea, and all three markers from the holotype of T. papelillo var. papelillo and from the neotype of T. vermicularis (of late 18th century origin). Unfortunately, we failed to amplify DNA from the neotypes of T. taurica and T. subuliformis, and the isotype of T. juncea var. subjuncea. Reasons for our failure might be deleterious storage and herbarium treatment practices and/or the small amount of tissue available to us for DNA isolation (fragments of podetia, 3–5 mm long).
Three phylogenetic species
It has previously been shown, based on a six-gene ML analysis (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017), that three mycobiont lineages occur in T. vermicularis s. lat. (Fig. 1) and furthermore that T. papellilo is part of one of these lineages, ‘Lineage C’. In this study, we included ITS sequences, and when possible Efα and DEAD markers, from Thamnolia type specimens, additional specimens belonging to ‘Lineage B’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017), T. papelillo and T. juncea (Table 1). All tree files resulting from these analyses are deposited in the TreeBase repository (http://purl.org/phylo/treebase/phylows/study/TB2:S21678). The Bayesian and ML analyses together with the haplotype network also showed the three lineages previously mentioned. Furthermore, all phylogenetic analyses revealed that both subspecies of T. juncea belonged to ‘Lineage C’ (Figs 3 & 4). The exclusively UV+ (‘Lineage A’) and exclusively UV− (‘Lineage B’) clades, which contained sequences from individuals formerly named T. vermicularis s. lat. or T. subuliformis s. lat., received strong posterior probabilities (PP) and bootstrap support (BP), both in the ITS (Fig. 3) and the three-gene phylogenies (Fig. 4). Only moderate support was obtained for the third clade (‘Lineage C’) in both Bayesian and ML analyses. This clade includes samples of both chemistries and different types of morphology, and the earlier analysis based on six nuclear markers (Fig. 1) showed a high BP support for it. Suspecting that the additional samples used in this study might have contributed to the diminished support for this clade, we performed an ML analysis of just three genes (ITS, Ef and DEAD) of the dataset from an earlier study (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). In this we obtained very similar supports to those presented in Fig. 4 (data not shown). Hence, we concluded that ‘Lineage C’ is monophyletic and the moderate support obtained in this study is due to a more limited selection of markers and not to a disturbance caused by a different sampling.
Given their genetic distinctiveness the three lineages (Fig. 1) shall, in our opinion, be considered separate species. The following names are proposed: ‘Lineage A’ as T. tundrae sp. nov.; ‘Lineage B’ as T. vermicularis and ‘Lineage C’ as T. subuliformis. Recognition of cryptic species in lichenized fungi has become rather uncontroversial in recent years (Crespo & Lumbsch Reference Crespo and Lumbsch2010; Lumbsch & Leavitt Reference Lumbsch and Leavitt2011; Molina et al. Reference Molina, Del-Prado and Kumar2011; Leavitt et al. Reference Leavitt, Moreau and Lumbsch2015), with Leavitt et al. also arguing for justifications for a strong emphasis on genetic data. In the present study the three species are distinct in phylogenies of a comprehensive sampling of 83 (ITS; Fig. 3) and 66 (ITS, Ef, DEAD; Fig. 4) collections of Thamnolia. The lineages were morphologically cryptic but they had different distributions (Fig. 2), photobiont specificity and, in part, secondary chemistry.
Fig. 2 Distribution maps for the three Thamnolia species studied. A, Thamnolia subuliformis s. str. is found worldwide and sympatric with the two other species; B, Thamnolia vermicularis s. str. and Thamnolia tundrae were found only in the Northern Hemisphere and are allopatric with each other and sympatric with T. subuliformis s. str. T. vermicularis s. str. was recorded in alpine regions of the European Alps, Tatra Mts and Western Carpathians while T. tundrae was found only in Sweden, Norway, Siberian Russia and the Aleutian Islands.
Morphology and secondary chemistry
No morphological feature was found to be consistently characteristic of any of the three species. This, however, does not preclude the possibility that such features might be found in meticulous studies designed for that purpose. An interesting variation in morphology in Siberian Thamnolia material was pointed out by Kärnefelt & Thell (Reference Kärnefelt and Thell1995) and a similar range of variation was also encountered in T. subuliformis s. str. in the present study (e.g. Fig. 3). Further studies are thus needed to ascertain a possible connection between the morphological and genetic variation in their material. Secondary chemistry varies among the species in as much as two chemotypes (UV+ with squamatic and baeomycesic acids, and UV− with thamnolic acid) were found only in T. subuliformis s. str., whereas T. vermicularis s. str. (UV−) and T. tundrae (UV+) both had a uniform chemistry (Figs 3 & 4).
Fig. 3 Phylogenetic relationships in Thamnolia based on 84 ITS sequences. A, Bali-Phy phylogeny rooted with three outgroups (Dibaeis baeomyces, Siphula ceratites and Icmadophila ericetorum). Posterior probabilities are above branches, ML bootstrap support ≥65% below branches. Scale bar indicates branch lengths. B, unrooted SplitsTree haplotype network. Scale bar indicates branch length. Numbers signify the assigned haplotype (H) for each sample. Associated information including GenBank numbers can be found in Table 1. Red boxes indicate type specimens.
Fig. 4 Phylogenetic relationships in Thamnolia based on sequence data using ITS, Efα and DEAD from 52 samples. Type specimens are highlighted in red. Outgroups are Dibaeis baeomyces, Siphula ceratites and Icmadophila ericetorum. PP=posterior probabilities ≥70%; BP=bootstrap values ≥70% obtained by ML analyses. Scale bar indicates branch length.
The Species
Thamnolia subuliformis (Ehrh.) W. L. Culb.
Brittonia 15: 144 (1963). —Lichen subuliformis Ehrh. Beitr. Naturk. 3: 82 (1788); type: a neotype, not associated with any annotations allowing it to be considered for lectotypification, was designated by Culberson (Reference Culberson1963) on a specimen from Ehrhart’s exsiccata Plantae Cryptogamae Linneae (no. 30, M) from the Harz Mountains in Germany. According to Culberson it contains squamatic and baeomycesic acids (UV+). Unfortunately, we were not able to retrieve any sequence from a small fragment of the neotype. Because of its chemistry it is not likely to belong to ‘Lineage B’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017) (i.e. T. vermicularis s. str.), and given its origin and secondary chemistry we find it most probably belongs to ‘Lineage C’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017), which consequently has to be named T. subuliformis.
Chemistry. Thamnolia subuliformis has two chemotypes with specimens either containing baeomycesic and squamatic acids (UV+), or thamnolic acid (UV−).
Photobionts. Trebouxia impressa Ahmadjian, T. simplex Tschermak-Woess ‘clade 1’, T. simplex ‘clade 2’, T. vagua A. Voytsekhovich & A. Beck s. lat. (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017).
Notes. This species is morphologically cryptic versus T. tundrae and T. vermicularis s. str., but well characterized by molecular data obtained from six nuclear markers (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). Thamnolia subuliformis is a widespread and often locally abundant species, being circumpolar in the Arctic and occurring in many alpine areas of both the Northern and Southern Hemisphere. It is in part sympatric with T. vermicularis s. str. and T. tundrae (Fig. 2). It is unfortunate that T. subuliformis is here given a different interpretation than in most of the 20th century literature, where it has been used to designate UV+ specimens of T. vermicularis s. lat. However, Onut-Brännström et al. (Reference Onuţ-Brännström, Tibell and Johannesson2017) have shown that this assemblage of specimens is not monophyletic. The nomenclatural status of T. subuliformis as conceived by the neotypification could be challenged via an epitypification, but this would be a risky procedure since there is nothing in the protologue that would unequivocally tie the name to an epitype. To be certain about this name one would hope for the discovery and genetic characterization of type material connected to the protologue in such a way that the neotypification could be superseded, but only at the cost of further nomenclatural confusion.
Thamnolia tundrae Brännström & Tibell sp. nov.
MycoBank No.: MB 821537
Thallus white, hollow, cylindrical, sometimes branched and/or tufted, erect or prostrate. Thallus PD+ yellow, K+ yellow, UV+ white, containing baeomycesic and squamatic acids. Differs from morphologically similar species in molecular sequences.
Type: Sweden, Jämtland, Åre par., Täljstensvalen, 63°14'36''N, 12°27'06''E, 2012, A. Larsson 95 (UPS L-812491—holotype). GenBank numbers: MF149104; MF143819; MF143810.
Fig. 5 Thamnolia tundrae (holotype, UPS-voucher). The holotype is UV+ (baeomycesic and squamatic acids) and has a cylindrical morphology.
Chemistry. Containing baeomycesic and squamatic acids.
Photobionts. Trebouxia simplex ‘clade 1’ and T. simplex ‘clade 2’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017).
Notes. This species is morphologically cryptic versus T. subuliformis s. str. and T. vermicularis s. str., but well characterized by molecular data obtained in this study (Figs 3 & 4) and from 6 nuclear markers; it corresponds to ‘Lineage A’ (Fig. 1) as described in Onut-Brännström et al. (Reference Onuţ-Brännström, Tibell and Johannesson2017). It is known mainly from the arctic tundra region of Eurasia reaching the Aleutian Islands. It is partly sympatric with T. subuliformis s. str. but allopatric with T. vermicularis s. str. Thamnolia tundrae (as ‘Lineage A’ in Onut-Brännström et al. 2017) was suggested to have survived the latest glaciation in coastal refugia adjacent to its present distribution. A map of its distribution is given in Fig. 2.
Thamnolia vermicularis (Sw.) Schaer.
Enum. Crit. Lich. Eur.: 243 (1850).
Lichen vermicularis Sw. Meth. Musc.: 37 (1781): ‘Habitat in alpibus Lapponicis inter gramina et muscos, fruticulis prostratis et diffusis, ascarides primo intuitu referentibus’. Neotype: a neotype was designated by Culberson (Reference Culberson1963, Fig. 2) on a specimen from the Swartz herbarium (S-L1596). It has few annotations and no close link to the protologue. Parts of the nuclear markers ITS and DEAD were obtained from the neotype and it belongs to ‘Lineage B’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). GenBank: MF149097; MF143817; MF143815
(Figs 2B & 3, sample 569.1_Tv_NEOTYPE)
Chemistry. Contains thamnolic acid.
Photobiont. Trebouxia simplex ‘clade 2’ (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017).
Notes. This species is morphologically cryptic versus T. subuliformis and T. tundrae, but well characterized by molecular data obtained from ITS (Fig. 3), the 3 nuclear markers (Fig. 4) and also in the six-gene phylogeny (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017). It is UV− and corresponds to ‘Lineage B’ (Fig. 1), and is known from the high alpine region of the European Alps, Tatra Mts and the Western Carpathian Mts of Romania. It is partly sympatric with T. subuliformis s. str. but allopatric with T. tundrae, this is a species suggested to have survived the latest glaciation (Onut-Brännström et al. Reference Onuţ-Brännström, Tibell and Johannesson2017) adjacent to its present distribution (Fig. 2). There is an obvious discrepancy between the protologue of T. vermicularis and the neotypification that Culberson was, however, not aware of at the time of neotypification. It was described from ‘Lapponia’, where T. vermicularis as typified by the neotype is so far not known to occur. For nomenclatural purposes, however, Culberson’s neotypification has to be followed until type material more closely connected to the protologue has been found and subjected to genetic analysis.
Further Nomenclatural Notes
Thamnolia juncea R. Sant.
Symb. Bot. Upsal. 34(1): 396 (2004); type: Papua New Guinea, Central Prov., Tapini subdistrict, Mount Strong, summit area, 08°00'S, 147°00'E, M. Coode & P. Stevens (UPS L-136871—holotype). GenBank: MF149084.
Notes. The holotype is UV− and has long, solid and unbranched podetia; molecular information based on the ITS region indicates that it belongs to T. subuliformis (Fig. 3, haplotype 1). This is supported by the three-gene phylogeny where Efα and DEAD were amplified from another specimen (NG_WilhelmMt_585.1_Tjj) with a similar morphology and collected from the same area as the holotype (Fig. 4, Table 1).
Thamnolia juncea R. Sant. var. subjuncea R. Sant.
Symb. Bot. Upsal. 34(1): 397 (2004); type: Papua New Guinea, Morobe Prov., Sarawaged Range, C. Keysser 67 (W—holotype; UPS L-136869—isotype).
Notes. Thamnolia juncea var. subjuncea differs from T. juncea var. juncea in being UV+. Unfortunately, we were not able to retrieve sequences from the isotype but molecular information based on the ITS region (Fig. 3, haplotypes 6, 7, 8) and three molecular markers (Fig. 4, NG_WilhelmMt_574.1_Tjs) obtained from a morphologically and chemically similar specimen from Papua New Guinea indicates that it belongs to T. subuliformis (Table 1).
Thamnolia papelillo R. Sant.
Symb. Bot. Upsal. 34(1): 394 (2004); type: Peru, Dept. Junín, Prov. Jauja, 30 km NNW of Jauja, B. & R. Santesson & R. Moberg P20:1 (UPS L-136872—holotype). GenBank: MF149093; MF143828; MF143806.
Notes. The holotype is UV− and has flattened, solid and dichotomously branched podetia; molecular information from this study (Figs 3 & 4, sample PE_Jauja_579_Tpp_HOLOTYPE) indicates that it is a taxonomic synonym of T. subuliformis.
Thamnolia papellilo R. Sant. var. subsolida (Satô) R. Sant.
Symb. Bot. Upsal. 34(1): 395 (2004).—Thamnolia subuliformis var. subsolida Satô, Bryologist 71: 50 (1968); type: Peru, Junín, between Tarma and Jauja, 1965, F. Maekawa (TNS—holotype).
Notes. According to Santesson (Reference Santesson2004) T. papellilo var. subsolida contains squamatic and baeomycesic acids. The holotype was not studied, but molecular data obtained from Peruvian samples identified by Santesson as T. papelillo var. subsolida in this study (Table 1, Figs 3 & 4) indicate it to be a synonym of T. subuliformis.
Thamnolia subvermicularis Asahina
J. Jap. Botany 13: 317 (1937). Typification was suggested by Kashiwadani & Kurokawa (Reference Kashiwandani and Kurokawa2003) but there is no reference to this material in the protologue and thus it cannot be accepted. The alleged type not seen by us.
Notes. Characterized by having squamatic and baeomycesic acids. There are no sequence data available for the type but, given its origin and secondary chemistry, we consider it most probably to be a taxonomic synonym of T. subuliformis.
Lichen tauricus Wulfen in Jacquin
Collectanea Bot. 2: 177 (1789); type: Austria, Carinthia; a neotype (‘Lichen Tauricus. alijo subuliformis, alijo etiam vermicularis’) was designated by Culberson (Reference Culberson1963) on a specimen from the Dawson Turner herbarium in London (BM). The specimen has few annotations, all however seemingly in the same hand (Wulfen’s?), but it is not otherwise closely connected to the protologue as both collecting date and locality are missing. The neotype, according to Culberson, contains thamnolic acid. We were not able to retrieve sequences from a fragment of the neotype but, given its origin and secondary chemistry, we find it most likely to be a taxonomic synonym of T. vermicularis.
We thank Nahid Heidari and Markus Hiltunen for their help with DNA isolation, PCR amplification and sequencing of the old and recent herbarium material. We are grateful to the curators of the following herbaria for providing specimens for study: BM, M, and UPS (both types and recent collections). We would also like to thank all the collectors who helped to obtain Thamnolia specimens from different parts of the world, and in particular Anders Larsson, Mats Thulin and Alexander Paukov, who collected specimens of T. tundrae from Sweden and Russia, and Anca Dragu who collected specimens of T. vermicularis from Romania. Our great appreciation goes to two anonymous reviewers for their useful comments and corrections.
