Acta Societatis Botanicorum Poloniae DOI: 10.5586/asbp.3564 ORIGINAL RESEARCH PAPER Publication history Received: 2017-04-26 Accepted: 2017-11-15 It is worth checking old data – validation of Published: 2017-12-18 Asplenium onopteris L. presence in the most Handling editor Krzysztof Spalik, Faculty of Biology, Biological and Chemical northeastern sites in Europe (Sudetes, SW Research Centre, University of Warsaw, Poland Poland) Authors’ contributions ES designed the project, 1 2 3 performed feld research, Ewa Szczęśniak *, Edyta M. Gola , Iwona Jędrzejczyk literature survey; EMG 1 Department of Botany, Institute of Environmental Biology, University of Wrocław, H. performed morphological Sienkiewicza 21, 50-335 Wrocław, Poland analyses; IJ performed fow 2 Department of Developmental Plant Biology, Institute of Experimental Biology, University of cytometry analyses; ES, EMG, Wrocław, Kanonia 6/8, 50-328 Wrocław, Poland and IJ wrote the manuscript 3 Department of Plant Genetics, Physiology and Biotechnology, UTP University of Science and Technology in Bydgoszcz, Prof. S. Kaliskiego 7, 85-789 Bydgoszcz, Poland Funding * Corresponding author. Email: [email protected] The project was supported by the University of Wrocław grants Nos. 1068/S/IBŚ/17 and 1068/S/ IBE/17. Abstract Competing interests In Poland, isolated serpentine rocks are exclusive habitats of some Asplenium species, EMG is a deputy editor of the reaching here their north or northeastern border range. One of them was Asplenium Acta Societatis Botanicorum onopteris, a diploid European species native to Mediterranean and Atlantic areas. Poloniae; other authors: no Since the nineteenth century, Polish out-of-range sites of A. onopteris have been competing interests have been declared quoted in literature without critical verifcation. Tus, to verify occurrence of this species in Poland, we analyzed the nuclear DNA content and micromorphological Copyright notice features as well as critically reviewed the literature data. We proved that all individu- © The Author(s) 2017. This is an als from Polish populations resembling A. onopteris were tetraploids and should Open Access article distributed be classifed as A. adiantum-nigrum. In addition, we validated a taxon silesiacum under the terms of the Creative Commons Attribution License, reported as co-occurring with A. onopteris. Te proposed diagnostic features are which permits redistribution, insufcient to indisputably delimit this taxon, and distinguishing it as a separate commercial and non- unit is not justifed. Analyses of the DNA content revealed also the presence of a commercial, provided that the triploid A. ×centovallense, a new hybrid for Polish fora. article is properly cited. Citation Keywords Szczęśniak E, Gola EM, Asplenium onopteris; Asplenium silesiacum; Asplenium ×centovallense; Asplenium Jędrzejczyk I. It is worth adiantum-nigrum complex; fow cytometry; micromorphological features checking old data – validation of Asplenium onopteris L. presence in the most northeastern sites in Europe (Sudetes, SW Poland). Acta Soc Bot Pol. 2017;86(4):3564. https://doi. org/10.5586/asbp.3564 Introduction Digital signature Te European Asplenium adiantum-nigrum complex includes three related and morpho- This PDF has been certifed using digital signature with a trusted timestamp to logically variable taxa: diploid A. cuneifolium Viv. and A. onopteris L. and their stable assure its origin and integrity. A verifcation tetraploid hybrid A. adiantum-nigrum L. [1]. Asplenium cuneifolium is associated with trust dialog appears on the PDF document when it is opened in a compatible PDF serpentine rocks in Southern Europe, up to southern Germany and southwestern Poland reader. Certifcate properties provide further details such as certifcation time [2]. Te most frost-resistant, circumpolar, and cosmopolitan A. adiantum-nigrum can and a signing reason in case any alterations colonize also other types of rock [3]; its European range covers the Atlantic and sub- made to the fnal content. If the certifcate is missing or invalid it is recommended to Mediterranean area up to western Norway, southwestern Poland, Slovakia, Ukraine, verify the article on the journal website. and Romania [4]. Asplenium onopteris is the most thermophilous species, tolerant to the substratum type, with a continuous range in Macaronesia, northwestern Africa, and the Mediterranean area [4,5]. Te species has also been confrmed in a discontinuous location in Ireland [5,6] and reported from Poland [6,7]. In Poland, all species of the complex with the only Central European population of A. onopteris and the locus clas- sicus of A. silesiacum Milde are associated exclusively with serpentine rock outcrops in the Sudetes. Te latter taxon is currently included in A. adiantum-nigrum though Published by Polish Botanical Society 1 of 10 Szczęśniak et al. / Asplenium onopteris L. presence in Poland (Central Europe) its origin, taxonomic rank, and diagnostic features vary and are sometimes contradic- tory [8–10]. Both last mentioned taxa were of particular interest for botanists due to suggested endemism and the distance to the continuous range. Our research primarily focused on biology and ecology of peripheral populations of the complex due to their biological importance (e.g., [11–13]), in order to estimate the habitat conditions limiting the A. onopteris occurrence to only four out of several serpentine rock outcrops in Poland. The long-term continuous monitoring of A. onopteris populations revealed enormous morphological variability related to the fern age and habitat conditions. Terefore, the main goal of the presented research was to validate the identity of the ferns quoted in the literature as the only Central European populations of A. onopteris. Also, we aimed to elucidate the current status of the co- occurring taxon “silesiacum”. We applied three approaches: (i) a critical review of the published data; (ii) estimation of the nuclear DNA content by fow cytometry, and (iii) morphological analyses. Material and methods To check the changes in the nomenclature of the revised taxa, we have reviewed all available publications since the frst paper by Milde [14]. Te feld research was conducted between 1996 and 2016 on all populations of A. onopteris and A. silesiacum reported in the Sudetes (SW Poland). As comparative material, we used A. adiantum-nigrum and A. cuneifolium sampled in the neighbor- ing serpentine rock outcrops and A. onopteris collected in its continuous distribution range, in Pitigliano (Tuscany, Italy), Bari Sardo (Sardinia, Italy), and Tenerife (Canary Islands, Spain). To determine the species identity, we estimated the nuclear DNA content and mea- sured the size of the spores and stomata. Due to the rarity of the A. adiantum-nigrum complex in Poland, only one leaf was taken from each tuf. Leaves were collected from all ferns resembling A. onopteris and A. silesiacum sensu Milde (41 samples). As a reference, 31 leaves of A. onopteris, 31 of A. cuneifolium, and 34 of A. adiantum-nigrum were analyzed. Te nuclear DNA content was analyzed for all taxa studied from the Sudetes popula- tions (18 leaves of potential A. onopteris plants, eight of A. cuneifolium, and 10 of A. adiantum-nigrum), and additionally for A. onopteris from Pitigliano (three leaves). Samples were prepared according to Galbraith et al. [15], with some modifcations. Prior to the genome size analysis, the presence of propidium iodide (PI) staining inhibitors in plant tissues was tested [16]. Leaves of Vicia faba ‘Inovec’ (2C = 26.90 pg) [17] were used as an internal standard. Plant tissues of the target taxa and internal standard were chopped in 1 mL of Tris-MgCl2 nucleus isolation bufer supplemented with 2% (w/v) polyvinylpyrrolidone (PVP-10), PI (50 μg/mL), and ribonuclease A (50 μg/mL). For each sample, 7000–10 000 nuclei were measured using a Partec CyFlow SL Green fow cytometer (Partec GmbH, Germany). At least fve replicates were performed for each taxon. Te obtained histograms were analyzed using FloMax sofware (Partec GmbH, Germany). Te genome size was estimated using the linear relationship between the ratio of the target taxon and internal standard 2C peak positions on the histograms. Te mean coefcients of variation of the 2C DNA content were estimated for all samples. Te 1C genome size was calculated afer the conversion of values into base pair numbers using the formula 1 pg = 978 Mbp [18]. Te size of stomata and spores was analyzed in voucher materials. Prior to the mea- surements, basal fragments of dried pinnae were soaked with warm water for ca. 1 h and the strips of abaxial epidermis were peeled. Specimens were analyzed and documented by an Olympus Microscope BX50 – DP71 camera – Cell^B sofware system (Olympus Optical, Poland). Te length of the spores (with perispore) and stomata was measured with AxioVision LE 4.2 sofware (Carl Zeiss MicroImaging GmbH, Germany). For each taxon, six to eight specimens were analyzed. For each specimen, 100 randomly selected stomata and 30 spores, when available, were measured. Te results were analyzed in Statistica 12. © The Author(s) 2017 Published by Polish Botanical Society Acta Soc Bot Pol 86(4):3564 2 of 10 Szczęśniak et al. / Asplenium onopteris L. presence in Poland (Central Europe) Results Review of published information Te presence of Asplenium onopteris in the Sudetes refers mainly to one population from a serpentine rock outcrop in the Gozdnik Hill (previously Weinberg, Winna Góra; Ślęża Massif, Sudetes Foreland) discovered and recorded by Milde as A. sile- siacum [14,19]. Tis name was maintained during the subsequent division of the A. adiantum-nigrum group into three species [19]: (i) A. adiantum-nigrum with fve varieties, (ii) A. serpentini Tausch (currently A. cuneifolium Viv.) with four forms, and (iii) A. silesiacum Milde, for which A. adiantum-nigrum subsp. silesiacum was given as a synonym. Next, the last taxon was categorized following Heufer’s [20] classifcation as a form of A. adiantum-nigrum subsp. onopteris (L.) Heuf. [21–24]. Luerrsen [25], based on herbarium materials from Central Europe, also divided A. adiantum-nigrum into three subspecies: adiantum-nigrum, serpentini, and onopteris, the latter with var.