Genetic diversity of natural psammophilous populations of Hypogymnia physodes (L.) Nyl. on Polish seacoast dunes 1 of 6Published by Polish Botanical Society Acta Mycologica ORIGINAL RESEARCH PAPER Genetic diversity of natural psammophilous populations of Hypogymnia physodes (L.) Nyl. on Polish seacoast dunes Anetta Wieczorek1,2*, Magdalena Achrem2,3, Aleksandra Truszkowska1, Andrzej Łysko4, Agnieszka Popiela2,5 1 Department of Ecology and Environmental Protection, University of Szczecin, Wąska 13, 71-415 Szczecin, Poland 2 Center for Molecular Biology and Biotechnology, Faculty of Biology, University of Szczecin, Wąska 13, 71-415 Szczecin, Poland 3 Department of Cell Biology, University of Szczecin, Wąska 13, 71-415 Szczecin, Poland 4 Department of Environmental Protection and Management, Western Pomeranian University of Technology, Słowackiego 17, 71-434 Szczecin, Poland 5 Department of Botany and Nature Conservation, University of Szczecin, Felczaka 3c, 71-412 Szczecin, Poland * Corresponding author. Email: anetta.wieczorek@usz.edu.pl Abstract Hypogymnia physodes is a lichenized fungus of the family Parmeliaceae. The aim of this study was to compare the level of genetic diversity in eight psammophilous and three epiphytic populations of this species from the Baltic coast in Poland, based on randomly amplified polymorphic DNA (RAPD) markers. In the reactions with nine primers, 153 fragments were obtained, of which 133 were polymorphic. In one reaction, from 0 (for lich2 primer) to 55 (for C02 primer) amplicons were obtained. A Dice’s genetic similarity index matrix was constructed based on the results of RAPD marker polymorphism examination. The values of similarity indices ranged from 0.00 to 0.73. Results of this study confirm the separateness of all three epiphytic popula- tions from those found on sand dunes (100% support, UPGMA/1000 trees). Keywords genetic polymorphism; interpopulation variability; randomly amplified polymorphic DNA; RAPD; Hypogymnia physodes; sand dunes Introduction Hypogymnia physodes (L.) Nyl. is an epiphytic species of lichenized fungi of the family Parmeliaceae. This taxon also occurs, although less often, on bedrock, mosses, or soil. Hypogymnia physodes produces numerous soredia and reproduces mainly vegetatively. Recent molecular studies have shown that genetic diversity in the lichens reproducing vegetatively is high and not lower than in the lichens that reproduce sexually [1–3]. Hypothetically, the preference of lichens for one type of substrate can be reflected in their genetic constitution. Using molecular markers, Mattsson et al. [4] have distinguished among populations of H. physodes, growing on different types of trees, the haplotypes that more than others prefer a specific type of substrate. The primary objective of this study was to evaluate the interpopulation variability of epigeic populations of H. physodes growing on the sand dunes of the Polish seacoast and their preference for substrate. The genetic variability of H. physodes was studied using randomly amplified polymorphic DNA (RAPD) analyses, although RAPD analyses have been still rarely used in lichenized fungi [5–7], possibly due to their low reproducibility. DOI: 10.5586/am.1096 Publication history Received: 2016-04-27 Accepted: 2017-05-01 Published: 2017-07-17 This article was originally submitted to and processed by the Acta Agrobotanica (another journal of the Polish Botanical Society) editors. Upon consent of the authors and the editors of both journals, it was eventually published in Acta Mycologica. Handling editor Joanna Kaczmarek (Acta Agrobotanica), Institute of Plant Genetics, Polish Academy of Sciences, Poland Authors’ contributions AW: idea of the study, writing the manuscript, collection of material; MA: writing the manuscript, final version of the manuscript, genetic analyzes; AT: genetic analyzes, manuscript drafting; AŁ: collection of material, preparation of the distribution map; AP: collection of material, final version of the manuscript Funding The research has been conducted on the authors own expenses. Competing interests No competing interests have been declared. Copyright notice © The Author(s) 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits redistribution, commercial and non- commercial, provided that the article is properly cited. Citation Wieczorek A, Achrem M, Truszkowska A, Łysko A, Popiela A. Genetic diversity of natural psammophilous populations of Hypogymnia physodes (L.) Nyl. on Polish seacoast dunes. Acta Mycol. 2017;52(1):1096. https:// doi.org/10.5586/am.1096 Digital signature This PDF has been certified using digital signature with a trusted timestamp to assure its origin and integrity. A verification trust dialog appears on the PDF document when it is opened in a compatible PDF reader. Certificate properties provide further details such as certification time and a signing reason in case any alterations made to the final content. If the certificate is missing or invalid it is recommended to verify the article on the journal website. mailto:anetta.wieczorek%40usz.edu.pl?subject=Genetic%20diversity%20of%20natural%20psammophilous%20populations%20of%20Hypogymnia%20physodes%20%28L.%29%20Nyl.%20on%20Polish%20seacoast%20dunes https://doi.org/10.5586/am.1096 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.5586/am.1096 https://doi.org/10.5586/am.1096 2 of 6© The Author(s) 2017 Published by Polish Botanical Society Acta Mycol 52(1):1096 Wieczorek et al. / Genetic diversity of Hypogymnia physodes based on RAPD Material and methods Lichen material Samples of Hypogymnia physodes were collected on the sand dunes of the Polish seacoast (Fig. 1) from 11 sites in July 2014. The analysis was performed on dried and cleaned material. DNA extraction and RAPD analysis Genomic DNA from each population was isolated using the DNeasy Plant Mini Kit (Qiagen, USA). RAPD analysis was then carried out according to the modified method of Willliams et al. [8]. Amplification reactions were carried out in 25 µL final volume of reaction mixture containing: 50 ng DNA, 1× Green GoTaq Reaction Buffer (Promega, USA), 0.2 mM dNTPs (Fermentas, Lithuania), 0.28 mM MgCl2, 5 mM primers, 0.04 U GoTaq DNA Polymerase (Promega). The thermal profile of the reaction was deter- mined experimentally and included initial DNA denaturation at 95°C for 11 min, 40 cycles of DNA denaturation at 93°C for 30 s, primer annealing at 37°C for 40 s, DNA chain elongation at 72°C for 80 s, and final elongation at 72°C for 5 min. Amplification was carried out twice in an MJ Mini Gradient Thermal Cycler (Bio-Rad, USA) on two samples of DNA from each genotype. The resulting amplification products were separated on 2.0% agarose gel with ethidium bromide (5 µg/mL; Sigma-Aldrich, USA). Data analysis To visualize, document, and analyze the results obtained, a set of Gel Doc XR and Quantity One 4.6.5 software (Bio-Rad) were used. In the populations, we assumed the existence of Hardy–Weinberg equilibrium. The genetic similarity of the studied popula- tions of Hypogymnia physodes was also determined using similarity index, according to Dice’s formula (1945), as described by Nei and Li [9]. The genetic similarity matrix was used to construct the UPGMA dendrogram using the FreeTree and TreeView 1.6.6 software [10,11]. Fig. 1 Study area and location of sampled Populations 1–11 of Hypogymnia physodes. 3 of 6© The Author(s) 2017 Published by Polish Botanical Society Acta Mycol 52(1):1096 Wieczorek et al. / Genetic diversity of Hypogymnia physodes based on RAPD Results This study examined the variation of 11 populations represented by 45 specimens on the basis of 133 loci, i.e., RAPD bands, in which a model of total dominance was assumed. No band was defined as a recessive allele and, on this ground, the probable percentage of recessive alleles in the population was calculated on the basis of Bayesian method that estimates the allele frequency based on the diversity of RAPD fragments, separately in all the analyzed loci in each population. The total number of analyzed PCR products was 153 and included polymorphic products whose size ranged from 155 to 1500 base pairs (Tab. 1, Fig. 2). The number of the polymorphic products amplified by one (individual) primer ranged from 0 in lich2 to 55 in C02. The assay effectiveness index (effective multiplex ratio; EMR) was also determined, as a ratio of the number of polymorphic products to the number of applied primers, which amounted to 14.78. The highest similarity values were recorded for population from Gąski: 73% similar- ity to the population from Łeba and 70% similarity to the population from Pobierowo (Tab. 2). The analyses showed the lowest genetic similarity for populations from Wisełka and Międzywodzie versus the populations from Pobierowo and Darłówko (Tab. 2). Overall, the lowest genetic similarity (0–45%) was characteristic for the Hypogymnia physodes population from Międzywodzie. Dendrogram (Fig. 3) revealed two separate clusters: the first group is formed by the lichen populations from Międzyzdroje, Międzywodzie, and Wisełka, found on the bark of pine trees, whereas the second one includes the other analyzed populations of H. physodes, occur- ring on sand. This is confirmed by clade stability tests, with the use of bootstrap resampling methods. Discussion The study of Molnár and Farkas [12], in which the five loci sequences were analyzed, found no significant genetic diversity of the H. physoides population. This is also confirmed by studies by Mattsson et al. [4]. Tab. 1 Primers employed with their sequence, the number of RAPD markers obtained, as well as the number and percentage (P) of polymorphic markers for each primer. Primer name Sequence 5'–3' Total No. of products No. of polymorphic products P (%) C02 GTGAGGCGTC 55 55 100 D03 GTCGCCGTCA 23 23 100 D08 GTGTGCCCCA 24 23 96 E07 AGATGCAGCC 12 9 75 lich2 AACGCGCAAC 6 0 0 lich3 GTAGACCCGT 8 3 37 lich4 AAGAGCCCGT 13 12 92 lich5 CCCGTCAGCA 10 6 60 OPA 18 AGGTGACCGT 2 2 100 Total 153 133 Fig. 2 Agarose gel electrophoresis of PCR product from DNA od Hypogymnia physodes obtained with primer C02. 4 of 6© The Author(s) 2017 Published by Polish Botanical Society Acta Mycol 52(1):1096 Wieczorek et al. / Genetic diversity of Hypogymnia physodes based on RAPD However, our analyzes showed a high population differentiation depending on the presence on the bark or on the dunes. As a result of the reactions being performed, 87% of the products were polymorphic, which indicates a high genetic diversity of the analyzed populations of Hypogymnia physodes. Among the lichen populations grow- ing on tree bark, forming the first group, the highest genetic similarity was observed between individuals from Międzywodzie and Międzyzdroje (64%); a lower genetic similarity was found between other populations, not exceeding 50% (Tab. 2). Such a high genetic diversity between local populations of lichens has been already described [2,13]. Moreover, another studies have shown even higher intraspecific genetic diversity than noted in our research, e.g., in the lichen species Lobothallia alphoplacaand, L. radiosa [14] or in the fungus Pisolithus tinctorius [15]. The reason of such high genetic may be the habitat whose isolation reduces the flow of genes between populations [16–18]. The high variation is an important trait because it plays a role under environmental changes, determining the survival of organisms [14,19,20]. The evaluation of two analyzed groups of H. physodes, the first growing on the bark of pine trees and the second on the sand, confirms the hypothesis that lichens are sensi- tive to substrate features [21]. It is believed that some genotypes are closely related to a preferential substrate, which can be associated with the process of colony formation [22]. The fact that the type of substrate can affect lichen variation is confirmed by other studies of H. physodes, in which an intraspecific diversity resulting from different amounts of nutrients in the substrate has been shown [4]. A high diversity, as in H. physoides, between populations preferring different substrate was also observed in Xanthoria parietina [17]. Sequence analysis of the intergenic spacer (IGS) and internal transcribed spacer (ITS) sequences revealed significant differentiation between seven populations Tab. 2 Dice’s similarity matrix of Hypogymnia physodes populations. Population 1Miz 2Wis 3Miw 4Pob 5Pus 6Sia 7Gas 8Dar 9Ust 10Leb 2Wis 0.48 3Miw 0.64 0.43 4Pob 0.28 0.00 0.00 5Pus 0.58 0.32 0.39 0.28 6Sia 0.50 0.20 0.36 0.62 0.35 7Gas 0.63 0.46 0.45 0.48 0.70 0.55 8Dar 0.41 0.06 0.08 0.66 0.53 0.54 0.51 9Ust 0.44 0.23 0.30 0.40 0.60 0.58 0.54 0.46 10Leb 0.58 0.35 0.32 0.48 0.62 0.62 0.73 0.64 0.66 11Lub 0.56 0.33 0.36 0.62 0.41 0.40 0.68 0.45 0.25 0.48 10Leb 7Gos 5Pus 9Ust 4Pob 8Dar 6Sia 11Lub 1Miz 3Miw 2Wis 34 18 24 9 18 41 24 56 23 100 0,1 G R O U P I G R O U P I I Fig. 3 UPGMA dendrogram of Hypogymnia physodes populations based on RAPD markers. 5 of 6© The Author(s) 2017 Published by Polish Botanical Society Acta Mycol 52(1):1096 Wieczorek et al. / Genetic diversity of Hypogymnia physodes based on RAPD Xanthoria parietina in different habitat (on the bark and rock) but, in contrast to our results, it was not high between the studied populations within the same habitat type [17]. Results reported by Gaya et al. 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Proc Natl Acad Sci USA. 2015;112(37):11600–11605. https://doi.org/10.1073/pnas.1507072112 https://doi.org/10.2307/2656585 https://doi.org/10.1111/j.1365-294X.2006.02880.x https://doi.org/10.3161/104.062.0206 https://doi.org/10.1038/sj.hdy.6800133 https://doi.org/10.1098/rstb.2005.1680 https://doi.org/10.1016/B978-0-12-044950-7.50017-9 https://doi.org/10.1890/0012-9658(2006)87[2037:QDAELI]2.0.CO;2 https://doi.org/10.1073/pnas.1507072112 Abstract Introduction Material and methods Lichen material DNA extraction and RAPD analysis Data analysis Results Discussion References 2017-07-17T17:50:13+0100 Piotr Otręba