Polystichum setiferum at the Northeastern Limit of Its Distribution Range

During field studies in 2019 in the Beskid Śląski mountain range in southern Poland, one individual morphologically resembling Polystichum setiferum was found. Nuclear DNA content analysis confirmed the species identity. e new stand of P. setiferum corresponds to historical literature reports and herbarium specimens from the Czech Republic and Poland, and extends the northeastern limit of the distribution of this species. As the historical stands in both countries have not been confirmed, the newly discovered stand is at present strongly isolated, as the nearest extant locations of P. setiferum in Austria, southern Hungary, and western Romania are nearly 500 km away. erefore, this unique stand deserves special attention and protection.


Introduction
Polystichum setiferum (Forssk.) Moore ex Woynar. is a fern species with an Atlantic and sub-Mediterranean distribution, from Canary Islands, northern Africa to Ireland in the west, to the Balkans and the Caucasus Mountains in the east (Dostál & Reichstein, 1984). Its northeastern limit of distribution in central Europe has been supposed until recently to lie in Hungary and Romania (Dostál & Reichstein, 1984). Although this species was reported from regions close to the Polish-Czech border as early as in the nineteenth century (Milde, 1855(Milde, , 1865 under different names (e.g., Aspidium aculeatum Sw.), it was regarded not to occur in the Czech Republic and Poland by contemporary floras (Dostál & Reichstein, 1984;Šourková, 1997;Szafer et al., 1988). is was due to the lack of herbarium documentation and similarity of some forms of Polystichum aculeatum (L.) Roth to P. setiferum, leading to frequent misidentification (Dostál & Reichstein, 1984). Nevertheless, a recent detailed morphological study of herbarium materials allowed the identification of P. setiferum in herbarium collections dated 1881-1935 from the territory of the Czech Republic (Ekrt, 2016). ese individuals came from Moravskoslezské Beskydy Mountains (Mt Kněhyně and Mt Smrk), Moravský kras (Olomučany), Jeseníky Mountains (Rýmařov, Mt Praděd, Velkákotlina), and Rychlebské hory (Lázně Jeseník) ( Figure 1). Currently, P. setiferum has not been confirmed at these locations, and it is regarded as an extinct species in the Czech flora (Kaplan et al., 2017). e species occurs on moderately acidic, humid soil in beech and mixed forests in moderately high mountains with mild winters (Dostál & Reichstein, 1984). Polystichum setiferum is a diploid species (2n = 82), which is one of the parental species of the allotetraploid P. aculeatum (2n = 164) (Dostál & Reichstein, 1984). erefore, these morphologically similar species can be unequivocally distinguished by chromosome count or nuclear DNA content analysis.  Based on past and recent data, we undertook field surveys for P. setiferum in the Opawskie Mountains and in the western part of the Beskid Śląski mountain range (Silesian Beskid), areas close to the historical locations of the species in the Eastern Sudetes and the Western Carpathians, respectively. e aim of our study was to verify the presence of P. setiferum in Poland in light of the historical data.

Material and Methods
Field studies aimed at finding P. setiferum in the Opawskie Mountains and Beskid Śląski were conducted between 2018 and 2020. According to our observations, the best season for the search of P. setiferum is winter when all other species besides those of Polystichum are gone.
Analysis of the morphological characteristics, listed in Table 1  Herbarium studies were performed at the herbaria of the Jagiellonian University (KRA) and the Institute of Botany, Polish Academy of Sciences (KRAM).
Stomatal cell length (n = 100) was determined using leaf fragments placed between glass slides in a water drop, and was measured using a Delta Optical microscope, model Genetic Pro, with an ocular micrometer and a calibration glass slide (Opta-Tech, Poland).  chopped leaves in a Petri dish with 1 mL of Galbraith's buffer (Galbraith et al., 1983) supplemented with propidium iodide (PI; 50 μg cm −3 ), ribonuclease A (50 μg cm −3 ), and 1% (v/v) polyvinylpyrrolidone (PVP). e suspension was passed through a 50-μm mesh nylon filter and analyzed using a CyFlow SL Green (Partec GmbH, Germany) flow cytometer equipped with a high-grade solid-state laser with green light emission at 532 nm, long-pass filter RG 590 E, DM 560 A, as well as side (SSC) and forward (FSC) scatters. Analyses were performed on three-four samples of each taxon. For each sample, the DNA content was established in 3,000-5,000 nuclei. Histograms were analyzed using the soware FloMax (Partec GmbH). e coefficient of variation (CV) of the G 0 /G 1 peak of Polystichum sp. ranged between 3.8% and 5.2%. Nuclear DNA content was calculated using the linear relationship between the ratio of the 2C peak positions Polystichum/Allium on a histogram of fluorescence intensities.

Results and Discussion
Field studies performed during 2018-2020 in Poland, in regions of historical occurrence of P. setiferum, led to the identification of one individual in the western part of the Beskid Śląski mountain range (Figure 1), morphologically corresponding well to this species (Figure 2 and Figure 3). It was found by the first author on  e identification was based on the investigation of the morphological characteristics distinguishing P. setiferum and P. aculeatum (Table 1). e most pronounced morphological characteristics of P. setiferum differentiating it from P. aculeatum are the relatively long petiole, leaf blade truncated at the base, and stalked, hair-tipped pinnules.
Stomata length or stomatal cell (guard cell) length could also be useful in differentiating the two species (Ekrt, 2016). e results of the stomatal cell measurements of P. setiferum and P. aculeatum from the stand ( Figure 5) show that the length of stomatal cells of the putative P. setiferum individual was evidently lower than that of P. aculeatum plants. e length ratio of P. setiferum to P. aculeatum stomatal cells was 0.8, which corresponds well with the stomata length ratio of specimens from the Czech Republic (0.8) (Ekrt, 2016) and those originating from southern Europe (0.76) (Ekrt, 2016).
In addition to morphological characteristics, the most unequivocal evidence discriminating the two species is the chromosome number count or the corresponding nuclear DNA content measurements. Nuclear DNA content (2C values) of the putative P. setiferum and P. aculeatum growing in the neighborhood, collected from the new stand, were 15.46 and 28.77 pg, respectively (Table 2). To the best of our knowledge, this is the first study to estimate the genome size of these species (Leitch et al., 2019). e results revealed that 2C values of the investigated species differed nearly twofold, corresponding well with their ploidy level (Dostál & Reichstein, 1984   studies is the same as that of the diploid American species, P. acrostichoides (Michx.) Schott (15.5 pg/2C) (Bainard et al., 2011). ese data indicate that the analyzed P. setiferum individual indeed represents this diploid species and not tetraploid P. aculeatum or its triploid hybrid with P. setiferum, that is, P. ×bicknellii (Christ) Hahne, which should have a 2C value intermediate between those of the parental species.
An additional search for P. setiferum specimens among those of P. aculeatum collected in Poland in the KRA and KRAM herbaria did not result in finding P. setiferum even though hundreds of P. aculeatum sheets were deposited in these herbaria. is indicates that P. setiferum must have already been a very rare species in the past.
e present finding of P. setiferum determines its northeastern limit of distribution in Europe, which is a remnant of the more widespread occurrence of this species in the past. However, it cannot be excluded that the discussed stand is a result of a relatively new colonization by spores originating from other presently unidentified or extinct sites located in Poland or the Czech Republic.
As it is the only extant stand of P. setiferum in the area of the Sudetes and the Carpathian Mountains, it deserves special attention and protection.