The Lichenologist 44(6): 757–772 (2012) 6 British Society, 2012 doi:10.1017/S0024282912000448

Testing morphology-based delimitation of juniperinus and V. tubulosus () using three molecular markers

Kristiina MARK, Lauri SAAG, Andres SAAG, Arne THELL and Tiina RANDLANE

Abstract: The delimitation of two morphologically similar and not easily separable Vulpicida species, V. juniperinus and V. tubulosus, is analyzed using nuclear ITS and Mcm7, and mitochondrial SSU DNA sequences. Seventy-nine Vulpicida specimens, most from the two focal taxa, are included in the three-locus gene tree. The results from Bayesian and parsimony analyses are presented. There are strong conflicts between the single locus gene trees. Vulpicida juniperinus and V. tubulosus are divided into two clearly distinguished groups in the ITS and concatenated B/MCMC tree. However, these species are mixed in both , appearing polyphyletic. Currently accepted V. juniperinus and V. tubulosus are not distinct according to the loci studied. appears monophyletic based on the available sequences. Key words: gene tree, ITS, lichenized fungi, Mcm7, molecular , MrBayes, mtSSU, Accepted for publication 15 June 2012

Introduction J. Lai, V. juniperinus (L.) J.-E. Mattsson & M. J. Lai, V. pinastri (Scop.) J.-E. Mattsson, The morphological group of cetrarioid V. tilesii (Ach.) J.-E. Mattsson & M. J. Lai, V. (Parmeliaceae) with erect foliose/subfruticose tubulosus (Schaer.) J.-E. Mattsson & M. J. Lai thallus, marginal apothecia and pycnidia, and and V. viridis (Schwein.) J.-E. Mattsson & production of the -type lichenan, M. J. Lai. In 1993, the new Vulpicida contains nearly 150 species in over 20 genera was delimited based on morphology, anatomy, (Randlane et al. 1997, 2010), of which c. 100 chemistry, distribution and ecology (Mattsson species and 17 genera form a monophyletic & Lai 1993). The genus is distributed in the , the so-called ‘cetrarioid core’ (Thell temperate and arctic regions of the Northern et al. 2009; Nelsen et al. 2011). Many taxa in Hemisphere and its species are characterized the cetrarioid core group are narrowly cir- by a unique set of secondary metabolites, cumscribed relative to other genera in the pinastric and vulpinic acids, that are prod- Parmeliaceae and so the species and genus ucts of the shikimic acid pathway and cause delimitations of cetrarioid lichens are still in an intense yellow colour of the medulla focus. (Mattsson 1993). The morphological recogni- The genus Vulpicida J.-E. Mattsson & M. tion of the genus is easy due to this impressive J. Lai, belonging to the cetrarioid core, con- character, but its monophyly has not been sists of six species of lichenized fungi that shown with confidence and the evolutionary were formerly classified within Cetraria Ach: relationships between the species have re- V. canadensis (Ra¨sa¨nen) J.-E. Mattsson & M. mained unclear. Vulpicida species have been distinguished K. Mark, A. Saag and T. Randlane: University of Tartu, using morphological characters and, surpris- Institute of Ecology and Earth Sciences, Department of ingly, also distribution. In Northern Europe, Botany, Lai 38, 51005 Tartu, Estonia. three of the six species are known: V. juni- L. Saag (corresponding author): Estonian Biocentre, perinus, V. pinastri and V. tubulosus (Thell Riia 23b, 51010 Tartu, Estonia. Email: [email protected] A. Thell: The Biological Museums, Lund University, et al. 2011). Vulpicida pinastri is distributed Lund, Sweden. over the Northern Hemisphere whereas the 758 THE LICHENOLOGIST Vol. 44 two other species have narrower distribution been published. The previous studies with areas: V. juniperinus occurs in different parts cetrarioid lichens have shown that the Vulpi- of Eurasia, and V. tubulosus only on alvars of cida specimens sequenced were related to the the Baltic Sea islands and in the mountains taxa from and Cetraria (Mattsson of Central Europe (Randlane & Saag 2005). & Wedin 1998; Thell & Miao 1999; Wedin The distribution of the fourth taxon, V. tilesii, et al. 1999; Thell et al. 2002, 2009; Crespo is restricted to the arctic and alpine regions et al. 2007; Nelsen et al. 2011). However, of Asia and North America, according to in these studies, not all species and only Mattsson (1993); however, some herbarium few representatives from Vulpicida were in- samples from alpine areas in Europe are also cluded. labelled as V. tilesii. In this study we focus on two species: V. Vulpicida juniperinus and V. tubulosus are juniperinus and V. tubulosus. Distinguishing morphologically very similar, while V. pinastri between these is often very difficult using is easily recognized by being the only sore- morphology. Intermediate forms of V. juni- diate species in the genus. Historically, V. perinus and V. tubulosus in Estonia appear to juniperinus was considered to inhabit the be especially problematic. As a result, we branches of Juniperinus communis, and V. attempt to elucidate the evolutionary rela- tubulosus calciferous ground. Both of them tionships of these two taxa using molecular can actually grow on the ground as well as on methods. bark (Mattsson 1993). Similar overlapping is observed in the morphological characteris- Material and Methods tics that the authors of the genus used in the species descriptions to distinguish these taxa. Taxon sampling and morphology In the key for Vulpicida species Mattsson The DNA sequences from 79 specimens of eight (1993: 31–32) used the following characters species were used in this study, 76 of them from five to distinguish V. juniperinus and V. tubulosus: Vulpicida species (Appendix 1). As V. juniperinus and V. tubulosus are the focus of this study, most sequences shape of lobes and lobe margins, colour and come from these two taxa (63 specimens together with distribution. According to him the thallus of the morphological intermediates). Cetraria islandica was V. juniperinus is dorsiventral with thin lobe chosen as the outgroup, based on the recent phyloge- margins, yellow, and the species occurs in netic studies of the cetrarioid core group (Thell et al. 2009; Nelsen et al. 2011). arctic/alpine boreal Eurasia. The lobes of V. The morphology of V. juniperinus, V. pinastri, V. tilesii tubulosus are terete or dorsiventral with thick and V. tubulosus specimens (74) was observed using a margins. Vulpicida tubulosus is greenish yellow binocular microscope (Olympus SZ51). The main diag- and is distributed in the Central European nostic characters described by Mattsson (1993) were Alps and around the Baltic Sea. In the de- recorded: growth form of thallus, shape of lobes, thick- ness of lobe margins, presence and type of reproductive scription of V. juniperinus, Mattsson (1993: structures, and abundance and size of pycnidia. The 38) states that V. juniperinus has only mar- height and width of an average of five pycnidia per ginal pycnidia on projections, while V. tubu- thallus were measured in the case of V. juniperinus and losus can also have laminal and immersed V. tubulosus, but mostly less for V. pinastri and V. tilesii as pycnidia are scarce on these species. To analyze the pycnidia. Of anatomical characters, the width variance of pycnidial size between clades and morpho- of pycnidia was reported as the least overlap- logical types, a nested ANOVA was used in the program ping character between V. juniperinus (110 e Statistica 7.01 (Statsoft Inc.). For this, the height and 4 mm) and V. tubulosus (90e6 mm), and width of 343 pycnidia were measured on 61 specimens therefore could be used to separate the two from clades A1, A2 and B. For analysis within morpho- logical types, 29 specimens with typical V. juniperinus species. In the field, typical V. tubulosus or V. tubulosus morphology were selected (15 and 14 (growing on the ground) is distinguished specimens accordingly, as shown on the gene tree). from typical V. juniperinus (growing on com- Additionally, the colour of thallus and emergence and mon juniper) by more fruticose growth form location of pycnidia were observed on all specimens, but as there was no difference in these characters among with tubular/terete lobes. the focal taxa of our study, these data are not presented Until now, no molecular studies focusing here. From other diagnostic characters, substratum was on the phylogeny of Vulpicida species have also recorded. 2012 Vulpicida juniperinus and V. tubulosus—Mark et al. 759

The content of secondary metabolites was studied in (Posada & Crandall 2001), Maxchi (Maynard Smith some specimens by means of TLC using solvent system 1992), Bootscan (Martin et al. 2005a), SiSscan (Gibbs A (Orange et al. 2001); TLC of all specimens was not et al. 2000), PhylPro (Weiller 1998) and 3Seq (Boni considered necessary as V. juniperinus and V. tubulosus et al. 2007). are chemically identical, according to previous knowledge For phylogenetic inference, we used methods from (Mattsson 1993; Thell et al. 2002). two paradigms. The single locus gene trees and the con- catenated trees were computed following the Bayesian DNA extraction and PCR amplification approach (B/MCMC) in MrBayes 3.1.2 (Huelsenbeck et al. 2000; Ronquist & Huelsenbeck 2003) and maxi- The thalli were carefully examined under a stereo- mum parsimony (MP) with nonparametric bootstrap- microscope for possible fungal infection. Pieces of the ping in PAUP* 4.0 (Swofford 2002). Bayesian support vegetative thallus were used for DNA extraction. The values can sometimes be overestimates, especially when samples were ground in 2 ml microtubes with 2–4 3 mm the tree branches are short. In contrast, bootstrap values stainless steel beads using a bead mill (Mixer Mill MM can be viewed as lower bounds of support values 400, Retsch). Subsequently, total genomic DNA was (Douady et al. 2003). We considered the clades with extracted using High Pure PCR Template Preparation bootstrap support b70% in MP and posterior prob- Kit (Roche), according to the manufacturer’s instructions abilities b95% as strongly supported. The phylogenetic with an extra phase separation step using chloroform. The trees were visualized using the program FigTree v1.3.1 following primers were used: ITS1F (Gardes & Bruns (Rambaut 2009). 1993) and ITS4 (White et al. 1990) for nuITS rDNA (ITS); Mcm7–709for and Mcm7–1348rev (Schmitt et al. Single locus gene trees and concatenated tees 2009) as well as LecMCM7f and LecMCM7r (Leavitt et al. 2011) for nuclear Mcm7 gene (Mcm7); mrSSU1 In B/MCMC, independent evolution models were and mrSSU3R (Zoller et al. 1999) for mitochondrial assumed for the matrix partitions. The models were SSU rDNA (mtSSU). The PCR mix consisted of 125 ml selected using standard AIC (Akaike Information Crite- PCR Master Mix 2x (Fermentas), containing 005 u/ml rion) in MrModeltest 2.3 (Nylander 2004) and corre- Taq DNA Polymerase, 4 mM MgCl2,04 mM of each sponding model form settings were applied in MrBayes. dNTP and reaction buffer, 125 pmol of both primers The partitions and selected models were: ITS1 – (in 2 ml of water), variable amounts of template and water GTR+G, 5.8S – K80+I, ITS2 – SYM+G, Mcm7 – up to the volume of 25 ml. PCR cycling parameters used K80+G, mtSSU – GTR+G. for amplifying ITS and mtSSU loci followed Wedin et al. The gaps were coded as standard characters via modi- (2009) and for Mcm7 Schmitt et al. (2009). The PCR fied complex indel coding using the software SeqState products were visualized on 15% agarose gels stained (Mu¨ller 2005, 2006). These numeric data were analyzed with ethidium bromide and purified via SAP/EXO treat- as separate partitions with the default models for stan- ment (Fermentas). The same primers were used for dard characters. sequencing and PCR amplification; both strands of DNA The ITS, Mcm7 and mtSSU regions were first ana- were sequenced using BigDye Terminator v3.1 Ready lyzed separately. The numbers of nucleotide positions Reaction Cycle Sequencing Kit (Applied Biosystems). and the numeric indel codes in the matrices were as The sequences were run on ABI 3730xl DNA Analyzer follows: 518 and 43 for ITS; 489 and 0 for Mcm7; 839 (Applied Biosystems). The sequencing procedures were and 11 for mtSSU. Then the data were concatenated carried out by the DNA Genotyping and Sequencing into the three-locus matrix, which consisted of 79 speci- Core Facility of the Estonian Biocentre and the Institute mens and 1835 nucleotide positions with 54 indel codes. of Molecular and Cell Biology at the University of Tartu Sequences of all 3 loci were present in the matrix for all (Estonia). The sequence traces were observed in 4Peaks 79 specimens. (mekentosj.com). In some cases, if the sequences of the In all analyses, the MrBayes default priors were used, two strands were not fully complementary, such bases except for the partition specific rates prior that was set to were inspected carefully and usually replaced with ambi- ‘variable’ (flat Dirichlet). Two simultaneous B/MCMC guity codes. All sequences were checked using GenBank analyses were run for 10 million (Mcm7, mtSSU) or BLAST. 12 million generations (ITS, three-locus dataset), both with four chains and starting from random trees. Trees Sequence alignments and phylogenetic analyses were sampled every 200 generations. The initial 4 million generations were discarded as burn-in from both runs All regions were aligned separately using the software and the majority-rule consensus tree with average branch MAFFT v6.850b (Katoh & Toh 2008) and MacClade lengths was calculated for the remaining trees using the 4.08 (Maddison & Maddison 2000). We removed the sumt command of MrBayes. The average standard devia- end of nuSSU, sometimes together with intron, from tion of split frequencies between simultaneous runs was the beginning of ITS alignment. 0005inthree-locusand0003 in other analyses; PSRF The recombination detection program RDP (Martin values all equalled 10. et al. 2005b) was used to scan for possible recombination The three-locus dataset was also analyzed by means events in all matrices. The following methods available of MP. Nonparametric bootstrap support (Felsenstein in this package were employed: RDP (Martin & Rybicki 1985) for each clade was estimated based on 1000 repli- 2000), GENECONV (Padidam et al. 1999), Chimaera cates, using the heuristic search option with 10 random 760 THE LICHENOLOGIST Vol. 44 sequence additions, TBR branch swapping and MulTrees were found between all loci. In the ITS tree, option in effect. The gaps were treated as fifth state. V. pinastri sequences form a strongly sup- ported monophyletic clade (Fig. 2) but, according to Mcm7, some V. pinastri spe- Results cimens group with a few V. tubulosus and V. DNA sequences and recombination tilesii samples (Fig. 3). While in the ITS tree V. juniperinus and V. tubulosus form two dis- Original sequences were obtained from 79 tinct intermixed clades, in the Mcm7 analysis specimens for ITS, Mcm7 and mtSSU (Ap- most sequences of these taxa are found in pendix 1). Before the ITS1 region, a nuSSU one clade with a smaller (also mixed) sub- intron was found in all Vulpicida samples ex- clade that has no match in the ITS tree. In cept two V. canadensis and two V. tilesii speci- the Mcm7 tree, Allocetraria appears closer to mens from North America (TIL 03, TIL the V. juniperinus/ pinastri/ tilesii/ tubulosus 04). No credible recombination events were group than V. canadensis, while in the ITS detected, either within or between loci. tree V. canadensis groups with one of the V. juniperinus/ tubulosus clades. According to Concatenated B/MCMC trees mtSSU, all Vulpicida specimens are united The B/MCMC consensus tree with poste- in one clade with low support but the inner rior probabilities (PP) and maximum parsi- structure of that is different from the ITS mony nonparametric bootstrap branch sup- and Mcm7 trees. port values (PBS), based on the three-locus matrix (ITS, Mcm7 and mtSSU), is shown Morphological characters in Fig. 1. Starting from the base, three well- supported clades can be distinguished: Allo- By assigning species epithets to the speci- cetraria (PP ¼ 100%; PBS ¼ 100%), Vulpi- mens, we tried to reflect the morphological cida canadensis (PP ¼ 100%; PBS ¼ 100%) traits of the thalli as much as possible and and a clade consisting of four other Vulpicida therefore intermediate specimens were marked species, V. juniperinus, V. pinastri, V. tilesii with more than one epithet in the order of their and V. tubulosus (PP ¼ 100%; PBS ¼ 96%). affinity (Appendix 1). The latter is divided into two major clades, The states of the recorded morphological both consisting of taxa focal to this paper. characters are given in Fig. 1. Both V. juni- One of the major clades (PP ¼ 100%; PBS ¼ perinus and V. tubulosus were found growing 72%) consists of V. pinastri (PP ¼ 100%; on bark as well as on the ground. The V. PBS ¼ 95%) and V. juniperinus/ tilesii/ tubu- juniperinus growth form is foliose to subfruti- losus groups (clade A; PP ¼ 100%; PBS ¼ cose, and V. tubulosus subfruticose to fruti- 83%) that in turn includes a few small clades cose. Few thalli had terete lobes only; most with strong Bayesian and bootstrap support. thalli of V. tubulosus had a mix of terete/ The second major clade (clade B; PP ¼ radial and flat/ dorsiventral lobes (Fig. 5.). 100%; PBS ¼ 99%) is more distant from V. Apothecia were found on only a few thalli. pinastri and unites the remaining V. juni- Vulpicida. pinastri is distinguished by having perinus and V. tubulosus specimens. soralia. Pycnidia were absent only on one studied thallus. No differences in thallus chem- Single locus B/MCMC trees istry between the tested specimens were found by means of TLC. The single locus trees are shown in Figures The height and width measurements of 2–4. The ITS tree (Fig. 2) was best resolved pycnidia were as follows [(min) averagee and supported, followed by Mcm7 (Fig. 3) standard deviation (max)]. Height in clades and the relatively less informative mtSSU A1+A2: (48) 118e26 (172) mm; clade B: (Fig. 4). Vulpicida juniperinus and V. tubulosus (72) 117e24 (168) mm; typical V. juniperinus: were always intermixed. Major topology con- (72) 125e24 (168) mm; typical V. tubulosus: flicts above 95% posterior probability level (80) 116e24 (160) mm. Width in clades 2012 Vulpicida juniperinus and V. tubulosus—Mark et al. 761

1. 2. 3. 4. 5. 6. 1 – SUBSTRATE 99 jun (Sweden; JUN 15) m s y y P2 A 105 l tub (Austria; TUB 34) l s y y n P1 107 terricolous 72 l r y y n m tub (Austria; TUB 51) P0 98 corticolous jun (Estonia; JUN 02a) m r y y P2 117 jun / til (Sweden; JUN 08B) l s y y P1 109 2 – FORM OF THALLUS jun (Norway; JUN 12B) l s y y P1 101 r foliose with adnate lobes jun / til (Austria; TUB 24) l s y y P1 102 jun / tub (Austria; TUB 28) l s yl y s foliose with raised lobes 75 P2 131 tub (Austria; TUB 29) l s y y n P2 119 (subfruticose) tub (Austria; TUB 30) l s yl y P1 124 n fruticose tub (Austria; TUB 32) l n yl y n P2 126 jun / tub (Austria; TUB 39) l r y y P2 110 3 – SHAPE OF LOBES jun (Austria; TUB 47) l s y y P2 115 y dorsiventral/flat jun / tub (Estonia; VSP 10) m s y y n P2 112 jun (Estonia; VSP 12) m s y y P2 119 l terete jun (Estonia; JUN 04) m s y y P2 116 A1 jun (Estonia; JUN 06) m r yl y P2 116 4 – LOBE MARGINS jun (Estonia; JUN 07) m s y y P2 A 124 clade A y thin tub (Estonia; JUN 14) l s y y n P1 109 n thick tub (Estonia; TUB 01) l n l - P1 106 jun (Estonia; TUB 02) m r y y 100 P2 A 111 tub (Estonia; TUB 03) l s yl y P2 115 5 – REPRODUCTIVE STRUCTURES tub (Estonia; TUB 19) l n l - P2 108 P0 few pycnidia tub (Estonia; TUB 21a) l n l - P2 107 P1 some pycnidia tub / jun / til (Austria; TUB 40) l s yl y n P0 110 P2 pycnidia abundant jun / tub (Austria; TUB 50) l s yl y P0 94 A apothecia jun / tub (Estonia; VSP 03) l r yl y P2 123 99 tub (Estonia; VSP 05) m n yl y n P2 101 S soralia jun (Estonia; VSP 14) m s y y P2 A 114 jun / tub (Estonia; VSP 15) l s y y n P2 114 juniperinus / tubulosus 6 – WIDTH OF PYCNIDIA jun (Austria; TUB 23) l s y y P2 114 (average, μ m) 99 jun / tub / til (Austria; TUB 25) l s yl y P2 98 jun / til (Austria; TUB 27) l r y y P2 106 A2 100 l s y y P2 118 - character missing jun (Austria; TUB 37) jun (Austria; TUB 45) l r y y P2 110 100 jun / til (Austria; TUB 52) l r y y P0 96 83 100 jun (Japan; JUN 20) m r y y P2 A 103 100 90 jun (Japan; JUN 21) m r y y P2 A 88 99 75 jun (Japan; JUN 22) m r y y n P2 A 80 57 99 til (USA; TIL 03B) l s y y P0 80 til (Canada; TIL 04) l s y y P0 65 100 80 100 til / jun (Russia, Buryatia Republic; TIL 05) l s y y P2 85 72 100 til / jun (Russia, Buryatia Republic; VSP 16)l s y y P0 108 100 pin (Estonia; PIN 08) m r y y P0 S 80 99 pin (Austria; PIN 09) l r y y P0 S 76 100

pin (Estonia; PIN 07) m r y y P0 S 64 clade 100 96 pin (Japan; PIN 04) m r y y P1 S 76 95 100 pin (Japan; PIN 03) m r y y P0 S 80 pin (Japan; PIN 05) m r y y S - 100 pin (Japan; PIN 06) m r y y P0 S 73 pinastri 99 tub (Sweden; TUB 10) l s yl n P2 100 l s yl n 59 tub (Sweden; TUB 16) P2 113 jun (Estonia; VSP 08) m s y y P2 113 jun/tub (Sweden; JUN 16)* m s y y P2 A 113 92 tub (Sweden; TUB 08b) m r y n P2 106 58 tub / jun (Sweden; TUB 14) m s y y n P2 90 100 100 jun / tub (Sweden; TUB 15) m r y y P2 96 96 79 jun/tub/til (Austria; TUB 41)l s yl y P2 118 clade B jun (Estonia; VSP 06) l r y y P2 110 jun (Estonia; JUN 13) m s y y P2 113 tub (Estonia; TUB 04) l n yl y n P2 126 tub (Estonia; TUB 05) l n l - P2 97 tub (Sweden; TUB 11) m s l - P2 A 91 72 tub (Sweden; TUB 12) l s yl y n P2 116 tub (Sweden; TUB 13) l n l - P2 97 53 jun (Sweden; TUB 17) m r y y P2 A 112 tub (Estonia; TUB 20B) l n l - P2 95 l n l - 99 tub (Estonia; VSP 01) P1 109 jun (Estonia; VSP 07) m r y y P2 109 67 jun / tub (Estonia; VSP 09) l s y y P2 105 70 juniperinus / tubulosus jun (Austria; TUB 31) l r y y P1 125 100 tub (Austria; TUB 33) l s y y n P1 114 99 tub (Austria; TUB 35) l s yl y P1 96 jun / til (Austria; TUB 38) l s y y P0 92 100 can (USA; CAN 01a) 100 can (USA; CAN 01b) 100 A. flavonigrescens (China; AFL 01) 100 A. stracheyi (China; AST 02) C. islandica (Sweden; ISL 01) 0.01

Fig. 1. The majority-rule (50%) consensus tree from Bayesian MCMC analysis based on nuclear ITS, Mcm7 and mitochondrial SSU combined dataset together with observed morphological characters. Abbreviations of species names: can – Vulpicida canadensis; jun – V. juniperinus; pin – V. pinastri; til – V. tilesii; tub – V. tubulosus. Location and laboratory code given in brackets. Bayesian posterior probabilities are indicated above branches and the maxi- mum parsimony nonparametric bootstrap values below branches. Branches with posterior probabilities above 095 and bootstrap proportions above 70% are marked in bold. Scale bar shows the number of changes per site. Morpho- logically characteristic V. juniperinus and V. tubulosus specimens marked with shaded background. 762 THE LICHENOLOGIST Vol. 44

V. juniperinus (Austria; TUB 23) 99 V. juniperinus/tubulosus/tilesii (Austria; TUB 25) V. juniperinus/tilesii (Austria; TUB 27) 100 V. juniperinus (Austria; TUB 37) V. juniperinus (Austria; TUB 45) V. juniperinus/tilesii (Austria; TUB 52) 96 V. tilesii (USA; TIL 03B) V. tilesii (Canada; TIL 04) 100 V. tilesii/juniperinus (Russia; TIL 05) V. tilesii/juniperinus (Russia; VSP 16) V. juniperinus (Estonia; JUN 02a) V. juniperinus (Estonia; JUN 04) V. juniperinus (Estonia; JUN 06) V. juniperinus (Estonia; JUN 07) V. juniperinus/tilesii (Sweden; JUN 08B) V. juniperinus (Norway; JUN 12B) V. tubulosus (Estonia; JUN 14) V. juniperinus (Sweden; JUN 15) V. juniperinus (Japan; JUN 22) V. tubulosus (Estonia; TUB 01) V. juniperinus (Estonia; TUB 02) V. tubulosus (Estonia; TUB 03) V. tubulosus (Estonia; TUB 19) 100 V. tubulosus (Estonia; TUB 21a) V. juniperinus/tilesii (Austria; TUB 24) V. juniperinus/tubulosus (Austria; TUB 28) V. tubulosus (Austria; TUB 29) V. tubulosus (Austria; TUB 30) V. tubulosus (Austria; TUB 32) V. tubulosus (Austria; TUB 34) V. juniperinus/tubulosus (Austria; TUB 39) V. tubulosus/juniperinus/tilesii (Austria; TUB 40) 98 V. juniperinus (Austria; TUB 47) V. juniperinus/tubulosus (Austria; TUB 50) V. tubulosus (Austria; TUB 51) V. juniperinus/tubulosus (Estonia; VSP 03) V. tubulosus (Estonia; VSP 05) V. juniperinus/tubulosus (Estonia; VSP 10) V. juniperinus (Estonia; VSP 12) 100 V. juniperinus (Estonia; VSP 14) V. juniperinus/tubulosus (Estonia; VSP 15) 82 V. juniperinus (Japan; JUN 20) V. juniperinus (Japan; JUN 21) V. pinastri (Japan; PIN 03) 100 V. pinastri (Japan; PIN 04) V. pinastri (Japan; PIN 05) 100 V. pinastri (Japan; PIN 06) 90 V. pinastri (Estonia; PIN 07) V. pinastri (Estonia; PIN 08) V. pinastri (Austria; PIN 09) V. tubulosus (Estonia; TUB 05) 98 V. tubulosus (Sweden; TUB 10) V. tubulosus (Sweden; TUB 12) V. tubulosus (Sweden; TUB 16) 59 V. juniperinus (Estonia; VSP 08) 100 V. tubulosus (Austria; TUB 33) V. juniperinus/tilesii (Austria; TUB 38) V. juniperinus (Estonia; JUN 13) V. juniperinus (Sweden; JUN 16) V. tubulosus (Estonia; TUB 04) V. tubulosus (Sweden; TUB 08b) V. tubulosus (Sweden; TUB 11) V. tubulosus (Sweden; TUB 13) 100 V. tubulosus/juniperinus (Sweden; TUB 14) V. juniperinus/tubulosus (Sweden; TUB 15) V. juniperinus (Sweden; TUB 17) V. tubulosus (Estonia; TUB 20B) V. juniperinus (Austria; TUB 31) 69 V. tubulosus (Austria; TUB 35) V. juniperinus/tubulosus/tilesii (Austria; TUB 41) V. tubulosus (Estonia; VSP 01) V. juniperinus (Estonia; VSP 06) V. juniperinus (Estonia; VSP 07) V. juniperinus/tubulosus (Estonia; VSP 09) 100 V. canadensis (USA; CAN 01a) V. canadensis (USA; CAN 01b) 100 Allocetraria flavonigrescens (China; AFL 01) Allocetraria stracheyi (China; AST 02) Cetraria islandica (Sweden; ISL 01) 0.2 Fig. 2. The B/MCMC majority rule (50%) consensus tree based on nuclear ITS dataset. Scale bar shows the number of changes per site. 2012 Vulpicida juniperinus and V. tubulosus—Mark et al. 763

99 V. juniperinus (Sweden; JUN 16) V. tubulosus (Austria; TUB 51) V. juniperinus (Sweden; JUN 15) V. tubulosus (Sweden; TUB 08b) V. tubulosus (Sweden; TUB 10) 81 V. tubulosus/juniperinus (Sweden; TUB 14) V. juniperinus/tubulosus (Sweden; TUB 15) V. tubulosus (Sweden; TUB 16) 100 V. tubulosus (Austria; TUB 34) V. juniperinus/tubulosus/tilesii (Austria; TUB 41) V. juniperinus (Estonia; VSP 08) V. juniperinus (Estonia; VSP 06) V. juniperinus (Estonia; JUN 02a) V. juniperinus (Estonia; JUN 04) V. juniperinus (Estonia; JUN 06) V. juniperinus (Estonia; JUN 07) V. juniperinus/tilesii (Sweden; JUN 08B) V. juniperinus (Norway; JUN 12B) V. juniperinus (Estonia; JUN 13) V. tubulosus (Estonia; JUN 14) V. tubulosus (Estonia; TUB 01) V. juniperinus (Estonia; TUB 02) V. tubulosus (Estonia; TUB 03) V. tubulosus (Estonia; TUB 04) V. tubulosus (Estonia; TUB 05) V. tubulosus (Sweden; TUB 11) V. tubulosus (Sweden; TUB 12) V. tubulosus (Sweden; TUB 13) V. juniperinus (Sweden; TUB 17) V. tubulosus (Estonia; TUB 19) 96 V. tubulosus (Estonia; TUB 20B) V. tubulosus (Estonia; TUB 21a) V. juniperinus/tilesii (Austria; TUB 24) V. juniperinus/tubulosus (Austria; TUB 28) V. tubulosus (Austria; TUB 29) V. tubulosus (Austria; TUB 30) V. juniperinus (Austria; TUB 31) V. tubulosus (Austria; TUB 32) V. tubulosus (Austria; TUB 33) V. juniperinus/tubulosus (Austria; TUB 39) V. tubulosus/juniperinus/tilesii (Austria; TUB 40) V. juniperinus (Austria; TUB 47) V. juniperinus/tubulosus (Austria; TUB 50) V. tubulosus (Estonia; VSP 01) V. juniperinus/tubulosus (Estonia; VSP 03) V. tubulosus (Estonia; VSP 05) V. juniperinus (Estonia; VSP 07) V. juniperinus/tubulosus (Estonia; VSP 09) V. juniperinus/tubulosus (Estonia; VSP 10) V. juniperinus (Estonia; VSP 12) 92 V. juniperinus (Estonia; VSP 14) V. juniperinus/tubulosus (Estonia; VSP 15) V. juniperinus (Austria; TUB 23) V. juniperinus/tubulosus/tilesii (Austria; TUB 25) 92 V. juniperinus/tilesii (Austria; TUB 27) V. juniperinus (Austria; TUB 37) V. juniperinus (Austria; TUB 45) V. juniperinus/tilesii (Austria; TUB 52) 100 V. pinastri (Japan; PIN 03) V. pinastri (Japan; PIN 05) V. pinastri (Japan; PIN 06) V. pinastri (Japan; PIN 04) 100 V. pinastri (Estonia; PIN 07) 100 V. pinastri (Estonia; PIN 08) V. pinastri (Austria; PIN 09) 99 V. tilesii/juniperinus (Russia; TIL 05) V. tubulosus (Austria; TUB 35) V. juniperinus/tilesii (Austria; TUB 38) V. tilesii/juniperinus (Russia; VSP 16) 97 96 V. juniperinus (Japan; JUN 21) V. juniperinus (Japan; JUN 22) 99 V. juniperinus (Japan; JUN 20) V. tilesii (USA; TIL 03B) V. tilesii (Canada; TIL 04) 100 Allocetraria flavonigrescens (China; AFL 01) Allocetraria stracheyi (China; AST 02) 100 V. canadensis (USA; CAN 01a) V. canadensis (USA; CAN 01b) Cetraria islandica (Sweden; ISL 01) 0.2 Fig. 3. The B/MCMC majority rule (50%) consensus tree based on nuclear Mcm7 dataset. Scale bar shows the number of changes per site. 764 THE LICHENOLOGIST Vol. 44

V. juniperinus (Estonia; JUN 02a) V. juniperinus/tilesii (Sweden; JUN 08B) V. juniperinus (Norway; JUN 12B) V. juniperinus (Sweden; JUN 15) V. tubulosus (Estonia; TUB 05) V. tubulosus (Sweden; TUB 13) V. juniperinus (Sweden; TUB 17) V. juniperinus/tilesii (Austria; TUB 24) V. juniperinus/tubulosus (Austria; TUB 28) 98 V. tubulosus (Austria; TUB 29) V. tubulosus (Austria; TUB 30) V. juniperinus (Austria; TUB 31) V. tubulosus (Austria; TUB 32) V. tubulosus (Austria; TUB 33) V. tubulosus (Austria; TUB 35) V. juniperinus (Austria; TUB 37) V. juniperinus/tubulosus (Austria; TUB 39) V. juniperinus (Austria; TUB 47) V. tubulosus (Austria; TUB 51) V. juniperinus/tubulosus (Estonia; VSP 10) V. juniperinus (Estonia; VSP 12) V. juniperinus (Estonia; JUN 04) V. juniperinus (Estonia; JUN 06) V. juniperinus (Estonia; JUN 07) V. juniperinus (Estonia; JUN 13) V. tubulosus (Estonia; JUN 14) V. juniperinus (Sweden; JUN 16) V. tubulosus (Estonia; TUB 01) V. juniperinus (Estonia; TUB 02) V. tubulosus (Estonia; TUB 03) V. tubulosus (Estonia; TUB 04) V. tubulosus (Sweden; TUB 08b) V. tubulosus (Sweden; TUB 10) V. tubulosus (Sweden; TUB 11) V. tubulosus (Sweden; TUB 12) 95 V. tubulosus/juniperinus (Sweden; TUB 14) V. juniperinus/tubulosus (Sweden; TUB 15) V. tubulosus (Sweden; TUB 16) V. tubulosus (Estonia; TUB 19) V. tubulosus (Estonia; TUB 20B) V. tubulosus (Estonia; TUB 21a) V. tubulosus/juniperinus/tilesii (Austria; TUB 40) V. juniperinus/tubulosus/tilesii (Austria; TUB 41) V. juniperinus/tubulosus (Austria; TUB 50) V. tubulosus (Estonia; VSP 01) V. juniperinus/tubulosus (Estonia; VSP 03) V. tubulosus (Estonia; VSP 05) V. juniperinus (Estonia; VSP 06) V. juniperinus (Estonia; VSP 07) V. juniperinus (Estonia; VSP 08) V. juniperinus/tubulosus (Estonia; VSP 09) V. juniperinus (Estonia; VSP 14) V. juniperinus/tubulosus (Estonia; VSP 15) 96 V. juniperinus (Japan; JUN 20) V. juniperinus (Japan; JUN 21) V. juniperinus (Japan; JUN 22) 100 V. canadensis (USA; CAN 01a) V. canadensis (USA; CAN 01b) 97 V. tilesii (USA; TIL 03B) V. tilesii (Canada; TIL 04) 96 V. tilesii/juniperinus (Russia; TIL 05) V. tilesii/juniperinus (Russia; VSP 16) V. pinastri (Japan; PIN 03) V. pinastri (Japan; PIN 04) 70 V. pinastri (Japan; PIN 05) V. pinastri (Japan; PIN 06) V. pinastri (Estonia; PIN 07) V. pinastri (Estonia; PIN 08) V. pinastri (Austria; PIN 09) V. juniperinus (Austria; TUB 23) V. juniperinus/tubulosus/tilesii (Austria; TUB 25) V. juniperinus/tilesii (Austria; TUB 27) V. tubulosus (Austria; TUB 34) V. juniperinus/tilesii (Austria; TUB 38) V. juniperinus (Austria; TUB 45) V. juniperinus/tilesii (Austria; TUB 52) 100 Allocetraria flavonigrescens (China; AFL 01) Allocetraria stracheyi (China; AST 02) Cetraria islandica (Sweden; ISL 01) 0.2 Fig. 4. The B/MCMC majority rule (50%) consensus tree based on mitochondrial SSU dataset. Scale bar shows the number of changes per site. 2012 Vulpicida juniperinus and V. tubulosus—Mark et al. 765

Fig. 5. Morphology of the focal taxa, Vulpicida juniperinus and V. tubulosus.A,V. tubulosus growing on the ground in its typical locality, on the alvar of Atla, Island Saaremaa, Estonia; B, V. tubulosus with fruticose thallus and raised terete lobes (TU-43527); C, V. juniperinus/tubulosus with foliose thallus, and both terete and flat lobes (TU, Leppik 38); D, V. juniperinus/tubulosus/tilesii with foliose thallus and raised flat lobes (GZU-67830); E, V. juniperinus with fo- liose thallus and adnate flat lobes (LD-1452912). Scales: A ¼ 5 mm; B–E ¼ 1 mm. 766 THE LICHENOLOGIST Vol. 44

A1+A2: (80) 112e19 (160) mm; clade B: accepted V. juniperinus and V. tubulosus cannot (80) 105e20 (164) mm; typical V. juniperinus: be separated using the DNA characters of (80) 115e18 (164) mm; typical V. tubulosus: our studied loci. (72) 102e19 (144) mm. We found no distinctive morphological Nested ANOVA analysis was carried out characters for clades A and B except the to compare the sizes of pycnidia between: 1) width of pycnidia. However, this difference clades A1+A2 and clade B, and 2) morpho- is small and the large variability and overlap logically typical specimens of V. juniperinus of the measurements between the groups make and V. tubulosus that were first determined this character impractical. Also, in many cases without measurements of pycnidia. There was the width of pycnidia is clearly incompatible significant variation of the height (F59 282 ¼ with the rest of the characters, in respect of 1999, P < 0001) and width (F59 282 ¼ the morphological species concepts currently 1440, P ¼ 0027) of pycnidia among spe- applied. As the width and height of pycnidia cimens, but only the variance of pycnidial are significantly different between morpho- width was significant among clades A and B species, the significant difference in the width (F1282 ¼ 10810, P < 0001). There was also of pycnidia between the two clades could be significant variation in the height (F1144 ¼ partly due to uneven dispersal of morpholog- 4893, P < 0029) and width (F1144 ¼ ical types between clades. 18460, P < 0001) of pycnidia among mor- There seems to be no significant correla- phological groups. tion between the clades of the concatenated trees and geography concerning V. juniperinus Discussion and V. tubulosus specimens from Europe. Vulpicida juniperinus specimens from Japan Despite the conflicts between loci, our con- form a strongly supported monophyletic sub- catenated three-locus tree is surprisingly well group. Between the subclades of V. pinastri, resolved and supported, clearly better than no geographic distinction was detected. any single locus tree. However, we cannot Sequences of V. pinastri form a strongly be sure if this method handles the conflicts supported clade. This group is easily separa- correctly. The results of the analysis are ble from others also by morphology (soralia dominated by the strong signal from ITS. It on thalli). The few (four) V. tilesii specimens shows that V. juniperinus and V. tubulosus sampled do not form a monophyletic clade in specimens are divided into two clearly dis- the concatenated or single locus trees but tinguished groups by the DNA characters. cluster together with V. juniperinus and V. One of these groups (clade A) is more closely tubulosus specimens in clade A. The position related to the monophyletic V. pinastri than of V. tilesii needs to be clarified in the future to the remaining V. juniperinus and V. tubulosus using a greater number of specimens. sequences (clade B). The two morphospecies Morphologically similar but genetically are mixed between the clades and V. juni- distinct species are sometimes hidden under perinus and V. tubulosus appear polyphyletic a single name. Such phylogenetic species are in the concatenated, as well as in single locus, frequently referred to as ‘cryptic’ (Funk & analyses. A tendency can be seen that more Omland 2003; Crespo & Pe´rez-Ortega 2009). specimens with the typical V. juniperinus The discordance between traditional morpho- morphology are located in clade A than in logical species concepts and DNA based phy- clade B, which includes more morphologi- logeny is not uncommon (Lumbsch & Leavitt cally characteristic V. tubulosus specimens. 2011). One could conclude, based on our ITS However, representatives of both morpho- and concatenated trees, that V. pinastri splits species are clearly present in both clades. two morphologically indistinguishable cryptic Moreover, a few morphological V. junipe- species that correspond to clades V. junipe- rinus and V. tubulosus specimens have identi- rinus/ tubulosus A and V. juniperinus/ tubulosus cal sequences in all three loci. Thus, the B. However, this might not necessarily be the main outcome of our study is that currently case. Relatively short branches in the phylo- 2012 Vulpicida juniperinus and V. tubulosus—Mark et al. 767 genetic trees and little differences in other those show traces of the alternate version of characters indicate that we might be dealing the locus. with a young species complex consisting of Besides paralogy or recombination, it is V. juniperinus and V. tubulosus together with also possible that the clades A and B really V. tilesii and V. pinastri. In young diverging are in an early stage of speciation that is taxa, a common cause of the apparent poly- reflected only in ITS and not yet in the other phyly in gene trees is the incomplete lineage two markers because of different molecular sorting (Grube & Kroken 2000; Taylor et al. evolution rates between loci. But in this case 2000; Funk & Omland 2003; Knowles & we still would not call them species before Carstens 2007). Also, when there is insuffi- other loci support it. We share the view of cient time for the fixation of gene lineages, Grube & Kroken (2000) that the species the random division of allele copies during should be separated based on single locus speciation can lead to different gene histories only if the clades clearly correlate with between loci (Hudson & Coyne 2002), and phenotypic characters or biogeographic dis- thus to the incongruence between the single tribution. locus gene trees that we found in this study. The results of our analyses do not support One of the major biological reasons for the two traditional species separated by mor- polyphyly of a species is considered to be phology. As the samples of these species are recombination, which can be detected using intermixed in all gene trees, distinguishing numerous algorithms. We found no credible V. juniperinus and V. tubulosus according to recombination events within or between loci; the current descriptions is not justified. We however, we cannot completely rule out their do not propose nomenclatural changes in presence in our matrix, as different recom- this paper because the phylogenetic situation bination detection methods show distinct in the genus Vulpicida is complex, and to performances depending on the amount of overcome the conflicts between the one- recombination, genetic diversity and other locus trees, more loci and more phylogenetic properties of the sequence data (Posada & methods should be employed. Before we Crandall 2001). can delimit any taxa with confidence, other As the division of V. juniperinus and V. Vulpicida species, especially V. tilesii, need to tubulosus sequences into the clades A and B be sufficiently sampled. is only present in the ITS tree, not Mcm7 We thank the collectors of the specimens, especially and mtSSU one-locus trees, one could con- Emma Sandler Berlin and Ede Leppik, and the curators sider this to be the result of paralogy: two of GZU, LD, MAF and UPS for the loans. The study slightly different copies of the same locus in was financially supported by the Estonian Science Foun- one genome. Even though we cannot exclude dation (grants JD173, 7470 and 9109), and the Euro- pean Union through the European Regional Devel- this option entirely without applying cloning, opment Fund (Center of Excellence FIBIR). Some of we are not seeing clear signs of paralogy in the phylogenetic analyses were carried out in the High our study. From some V. juniperinus and V. Performance Computing Center of the University of tubulosus specimens, the ITS region was in- Tartu. dependently amplified and sequenced twice References with different template dilutions (some also Boni, M. F., Posada, D. & Feldman, M. W. (2007) An extracted twice) resulting in identical se- exact nonparametric method for inferring mosaic quences (4 from both clades A and B). From structure in sequence triplets. Genetics 176: 1035– few other repeated PCRs the sequences of 1047. parasitic fungi in addition to the ones of Crespo, A. & Pe´rez-Ortega, S. (2009) Cryptic species and species pairs in lichens: a discussion on the rela- Vulpicida were obtained, as identified by tionship between molecular phylogenies and mor- BLAST, but never any possible paralogs. phological characters. Anales del Jardı´n Bota´nico de The variable sites in the ITS matrix distin- Madrid 66: 71–81. guishing clades A and B are all based on Crespo, A., Lumbsch, H. T., Mattsson, J.-E., Blanco, O., Divakar, P. K., Articus, K., Wiklund, E., Bawin- high quality sequence traces and none of gan, P. A. & Wedin, M. (2007) Testing morphology- 768 THE LICHENOLOGIST Vol. 44

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GenBank Accession Numbers Laboratory Taxon code Specimen locality; collector and voucher information ITS Mcm7 mtSSU

Allocetraria flavonigrescens AFL 01 China, Tibet, prov. Sichuan; Obermayer 08146, 4 viii 2000 (GZU) JX144030 JX143872 JX143951 A. stracheyi AST 02 China, Tibet, prov. Sichuan; Obermayer 143, 4 viii 2000 (GZU) JX144031 JX143873 JX143952 Cetraria islandica ISL 01 Sweden, O¨ land; Pihu & Reier, 8 vi 2006 (TU) JX144032 JX143874 JX143953 Vulpicida canadensis CAN 01b USA, California; Suija, 10 vii 2008 (TU45115) JX144033 JX143875 JX143954 V. canadensis CAN 01a USA, California; Suija, 10 vii 2008 (TU45115) JX144034 JX143876 JX143955 V. juniperinus JUN 02a Estonia, Saaremaa; Leppik & To˜rra, 18 ix 2008 (LD1261755) JX144035 JX143877 JX143956

V. juniperinus JUN 04 Estonia, Saaremaa; Thell, 18 ix 2008 (LD1277668) JX144036 JX143878 JX143957 LICHENOLOGIST THE V. juniperinus JUN 06 Estonia, Saaremaa; Randlane, 14 ix 2007 (LD1277728) JX144037 JX143879 JX143958 V. juniperinus JUN 07 Estonia, Hiiu mk.; Suija 892, 2 vii 2008 (TU) JX144038 JX143880 JX143959 V. juniperinus JUN 12B Norway, Storfjord; Nordin, 09 viii 2003 (UPS257546) JX144039 JX143881 JX143960 V. juniperinus JUN 13 Estonia, Saaremaa; Leppik 33, 24 viii 2009 (TU) JX144040 JX143882 JX143961 V. juniperinus JUN 15 Sweden, Ha¨rjedalen; Fro¨de´n, 25 viii 2009 (LD1452912) JX144041 JX143883 JX143962 V. juniperinus JUN 16 Sweden, Ha¨rjedalen; Fro¨de´n, 23 viii 2009 (LD1458792) JX144042 JX143884 JX143963 V. juniperinus JUN 20 Japan, Hokkaido; Thor 24261, 13 vi 2010 (UPS) JX144043 JX143885 JX143964 V. juniperinus JUN 21 Japan, Hokkaido; Thor 26077, 23 vi 2010 (UPS) JX144044 JX143886 JX143965 V. juniperinus JUN 22 Japan, Hokkaido; Thor 24395, 13 vi 2010 (UPS) JX144045 JX143887 JX143966 V. juniperinus TUB 17 Sweden, Gotland; Sandler Berlin, 11 iii 2009 (LD1331035) JX144046 JX143888 JX143967 V. juniperinus TUB 23 Austria, Steiermark; Hafellner & Muggia 67160, 26 x 2006 (GZU) JX144047 JX143889 JX143968 V. juniperinus TUB 31 Austria, Steiermark; Bilovitz 3650, 18 vii 2007 (GZU) JX144048 JX143890 JX143969 V. juniperinus TUB 37 Austria, Steiermark; Hafellner 68689, 24 vi 2007 (GZU) JX144049 JX143891 JX143970 V. juniperinus TUB 45 Austria, Steiermark; Hafellner 62921, 06 ix 2003 (GZU) JX144050 JX143892 JX143971 V. juniperinus TUB 47 Austria, Steiermark; Hafellner 62536, 28 ix 2003 (GZU) JX144051 JX143893 JX143972 V. juniperinus VSP 06 Estonia, Saare mk.; Ju¨riado 471, 10 ix 2009 (TU) JX144052 JX143894 JX143973 V. juniperinus VSP 07 Estonia, Saare mk.; Ju¨riado 471, 10 ix 2009 (TU) JX144053 JX143895 JX143974 V. juniperinus VSP 08 Estonia, Saaremaa; Leppik 39, 22 ix 2009 (TU) JX144054 JX143896 JX143975 V. juniperinus VSP 12 Estonia, Hiiumaa; Ju¨riado & Leppik, 13 vi 2010 (TU) JX144055 JX143897 JX143976 V. juniperinus VSP 14 Estonia, Hiiumaa; Ju¨riado & Leppik 48, 17 vi 2010 (TU) JX144056 JX143898 JX143977 V. juniperinus TUB 02 Estonia, Saaremaa; Randlane, 14 ix 2007 (TU43526) JX144057 JX143899 JX143978 V. juniperinus/tilesii JUN 08B Sweden, Lule Lappmark; Westberg, 29 vii 2004 (LD1277466) JX144058 JX143900 JX143979 V. juniperinus/tilesii TUB 24 Austria, Steiermark; Hafellner 67904, 13 x 2006 (GZU) JX144059 JX143901 JX143980 V. juniperinus/tilesii TUB 27 Austria, Steiermark; Hafellner 67754, 11 ix 2006 (GZU) JX144060 JX143902 JX143981

V. juniperinus/tilesii TUB 38 Austria, Steiermark; Hafellner & Muggia 68028, 30 ix 2006 (GZU) JX144061 JX143903 JX143982 44 Vol. V. juniperinus/tilesii TUB 52 Austria, Steiermark; Hafellner 64383, 24 ix 2005 (GZU) JX144062 JX143904 JX143983 V. juniperinus/tubulosus TUB 15 Sweden, Gotland; Sandler Berlin, 11 iii 2009 (LD1325643) JX144063 JX143905 JX143984 V. juniperinus/tubulosus TUB 28 Austria, Steiermark; Hafellner 67217, 09 ix 2006 (GZU) JX144064 JX143906 JX143985 Appendix 1. Continued 2012

GenBank Accession Numbers Laboratory Taxon code Specimen locality; collector and voucher information ITS Mcm7 mtSSU

V. juniperinus/tubulosus TUB 39 Austria, Steiermark; Hafellner, Muggia & Hafellner 68563, JX144065 JX143907 JX143986 22 ix 2007 (GZU)

V. juniperinus/tubulosus TUB 50 Austria, Steiermark; Hafellner 62960, 16 viii 2003 (GZU) JX144066 JX143908 JX143987 juniperinus Vulpicida V. juniperinus/tubulosus VSP 03 Estonia, Saaremaa; Leppik 38, 13 vii 2009 (TU) JX144067 JX143909 JX143988 V. juniperinus/tubulosus VSP 09 Estonia, Saaremaa; Ju¨riado & Leppik 51, 12 vii 2010 (TU) JX144068 JX143910 JX143989 V. juniperinus/tubulosus VSP 10 Estonia, Hiiumaa; Ju¨riado & Leppik 112, 13 vi 2010 (TU) JX144069 JX143911 JX143990 V. juniperinus/tubulosus VSP 15 Estonia, Hiiumaa; Ju¨riado & Leppik, 13 vi 2010 (TU) JX144070 JX143912 JX143991 V. juniperinus/tubulosus/tilesii TUB 25 Austria, Steiermark; Hafellner 67830, 11 ix 2006 (GZU) JX144071 JX143913 JX143992 V. juniperinus/tubulosus/tilesii TUB 41 Austria, Steiermark; Hafellner 62681, 20 ix 2003 (GZU) JX144072 JX143914 JX143993 V. pinastri PIN 03 Japan, Hokkaido; Thor 26072, 23 vi 2010 (UPS) JX144073 JX143915 JX143994 V. pinastri PIN 04 Japan, Hokkaido; Thor 24387, 13 vi 2010 (UPS) JX144074 JX143916 JX143995 V. pinastri PIN 05 Japan, Hokkaido; Thor 24305, 13 vi 2010 (UPS) JX144075 JX143917 JX143996

V. pinastri PIN 06 Japan, Hokkaido; Thor 24396, 13 vi 2010 (UPS) JX144076 JX143918 JX143997 and V. pinastri PIN 07 Estonia, Vo˜rumaa; Mark, 15 i 2011 (TU) JX144077 JX143919 JX143998

V. pinastri PIN 08 Estonia, Vo˜rumaa; Mark, 15 i 2011 (TU) JX144078 JX143920 JX143999 tubulosus—Mark V. V. pinastri PIN 09 Austria, Steiermark; Hafellner 62921, 06 ix 2003 (GZU) JX144079 JX143921 JX144000 V. tilesii TIL 03B USA, Colorado; Westberg, 7 vii 1998 (LD1330563) JX144080 JX143922 JX144001 V. tilesii TIL 04 Canada, Nunavut; Wong 4686, 14 vi 1999 (TU, CANL) JX144081 JX143923 JX144002 V. tilesii/juniperinus TIL 05 Russia, Southern Siberia, Republic of Buryatia; JX144082 JX143924 JX144003 Urbanavichus, 11 viii 2007 (MAF28040) V. tilesii/juniperinus VSP 16 Russia, Republic of Buryatia; Urbanavichus, 11 viii 2007 (TU) JX144083 JX143925 JX144004 V. tubulosus JUN 14 Estonia, Saaremaa; Leppik 38, 27 viii 2009 (TU) JX144084 JX143926 JX144005 V. tubulosus TUB 01 Estonia, Saaremaa; Randlane, 14 ix 2007 (TU43527) JX144085 JX143927 JX144006 V. tubulosus TUB 03 Estonia, Saaremaa; Marmor, 18 ix 2008 (LD1261815) JX144086 JX143928 JX144007 V. tubulosus TUB 04 Estonia, Saaremaa; Thell, 18 ix 2008 (LD1278868) JX144087 JX143929 771 JX144008 al. et V. tubulosus TUB 05 Estonia, Saaremaa; Leppik & To˜rra, 18 ix 2008 (LD1261875) JX144088 JX143930 JX144009 V. tubulosus TUB 08b Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1325523) JX144089 JX143931 JX144010 V. tubulosus TUB 10 Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1332348) JX144090 JX143932 JX144011 V. tubulosus TUB 11 Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1325583) JX144091 JX143933 JX144012 V. tubulosus TUB 12 Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1337388) JX144092 JX143934 JX144013 V. tubulosus TUB 13 Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1332288) JX144093 JX143935 JX144014 V. tubulosus TUB 16 Sweden, Gotland; Sandler Berlin, 12 iii 2009 (LD1332408) JX144094 JX143936 JX144015 V. tubulosus TUB 19 Estonia, Saaremaa; Leppik 27, 19 viii 2009 (TU) JX144095 JX143937 JX144016 V. tubulosus TUB 20B Estonia, Saaremaa; Thell, 18 ix 2008 (LD1265415) JX144096 JX143938 JX144017 V. tubulosus TUB 21a Estonia, Saaremaa; Leppik 38, 27 viii 2009 (TU) JX144097 JX143939 JX144018 Appendix 1. Continued 772

GenBank Accession Numbers Laboratory Taxon code Specimen locality; collector and voucher information ITS Mcm7 mtSSU

V. tubulosus TUB 29 Austria, Steiermark; Hafellner, Muggia & Hafellner 68653, JX144098 JX143940 JX144019 09 vi 2007 (GZU) V. tubulosus TUB 30 Austria, Steiermark; Hafellner, Muggia & Hafellner 68595, JX144099 JX143941 JX144020 20 v 2007 (GZU) V. tubulosus TUB 32 Austria, Steiermark; Hafellner 67278, 03 vii 2005 (GZU) JX144100 JX143942 JX144021 V. tubulosus TUB 33 Austria, Steiermark; Hafellner 67228, 23 vii 2005 (GZU) JX144101 JX143943 JX144022 V. tubulosus TUB 34 Austria, Steiermark; Hafellner 67803, 13 ix 2006 (GZU) JX144102 JX143944 JX144023 V. tubulosus TUB 35 Austria, Steiermark; Bilovitz 3630, 18 vii 2007 (GZU) JX144103 JX143945 JX144024 V. tubulosus TUB 51 Austria, Steiermark; Hafellner 64407, 19 vi 2005 (GZU) JX144104 JX143946 JX144025 LICHENOLOGIST THE V. tubulosus VSP 01 Estonia, Saaremaa; Leppik & Ju¨riado 21, 7 viii 2009 (TU) JX144105 JX143947 JX144026 V. tubulosus VSP 05 Estonia, Saaremaa; Leppik & Ju¨riado 13, 14 vii 2009 (TU) JX144106 JX143948 JX144027 V. tubulosus/juniperinus TUB 14 Sweden, Gotland; Sandler Berlin, 11 iii 2009 (LD1330975) JX144107 JX143949 JX144028 V. tubulosus/juniperinus/tilesii TUB 40 Austria, Steiermark; Hafellner 63560, 11 ix 2004 (GZU) JX144108 JX143950 JX144029 o.44 Vol.