The Lichenologist 44(6): 757–772 (2012) 6 British Lichen Society, 2012 doi:10.1017/S0024282912000448 Testing morphology-based delimitation of Vulpicida juniperinus and V. tubulosus (Parmeliaceae) 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 clades, appearing polyphyletic. Currently accepted V. juniperinus and V. tubulosus are not distinct according to the loci studied. Vulpicida pinastri appears monophyletic based on the available sequences. Key words: gene tree, ITS, lichenized fungi, Mcm7, molecular phylogenetics, MrBayes, mtSSU, taxonomy 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 lichens 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 Cetraria-type lichenan, M. J. Lai. In 1993, the new genus 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 clade, 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 Allocetraria 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.