Preliminary studies on the diversity of the leaf-blade hair and stomata density in the Polypodium vulgare L. complex in Poland

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1 39 The-leaf-blade-hair-and-stomata-density-in-the-P.-vulgare-L.-complex-in-Poland 39 Preliminary studies on the diversity of the leaf-blade hair and stomata density in the Polypodium vulgare L. complex in Poland EDYTA M. GOLA 1, EWA SZCZÊŒNIAK 2 1 Department of Plant Morphology and Development, Institute of Experimental Biology, University of Wroc³aw, Kanonia 6/8, PL Wroc³aw; edytag@biol.uni.wroc.pl 2 Department of Biodiversity and Plant Cover Protection, University of Wroc³aw, Kanonia 6/8, PL Wroc³aw; ewaszcz@biol.uni.wroc.pl ABSTRACT: Polypodium vulgare complex in Central Europe consists of three taxa differing in the ploidy level, i.e. P. cambricum (2n), P. vulgare (4n) and P. interjectum (6n). In Poland, only two latter species have been noted, as well as the hybrid between them, P. mantoniae (5n). These ferns are morphologically similar and thus their unequivocal identification is based on the measurement of the nuclear DNA content or chromosome counting. An indirect tool for the taxon identification is the annulus structure, namely the number of indurated and basal cells. Here, we present an additional micromorphological feature, which is the presence of hairs at the leaf-blade surface. This feature has not been given in the manuals but it may possibly be useful in a taxon recognition. Further analyses and confirmation of the taxonomic value of this feature are however necessary due to limited material studied. ABSTRAKT: Kompleks Polypodium vulgare w Europie Œrodkowej sk³ada siê z trzech taksonów ró ni¹cych siê poziomem ploidalnoœci, tj. P. cambricum (2n), P. vulgare (4n) i P. interjectum (6n). W Polsce odnotowano tylko dwa ostatnie gatunki oraz ich mieszañca, P. mantoniae (5n). Paprocie te s¹ morfologicznie do siebie podobne i dlatego ich jednoznaczna identyfikacja opiera siê na pomiarze zawartoœci j¹drowego DNA lub okreœleniu liczby chromosomów. Poœrednio poszczególne taksony mo na identyfikowaæ w oparciu o strukturê zarodni (pierœcienia), poniewa liczba komórek zgrubia³ych i komórek podstawy pierœcienia jest sta³a i ró ni poszczególne taksony. W tej pracy prezentujemy wstêpne obserwacje nad obecnoœci¹ w³osków na powierzchni blaszki liœciowej. Cecha ta nie jest podawana w kluczach, ale potencjalnie mo e byæ przydatna przy okreœlaniu taksonu. Dalsze analizy i potwierdzenie wartoœci taksonomicznej tej cechy s¹ jednak konieczne ze wzglêdu na ograniczon¹ iloœæ zbadanego materia³u. Key words: Polypodium vulgare, P. interjectum, P. mantoniae, leaf micromorphology, hair, ploidy. Gola E.M., Szczêœniak E Preliminary studies on the diversity of the leaf-blade hair and stomata density in the Polypodium vulgare L. complex in Poland. In: E. Szczêœniak, E. Gola (eds), Genus Polypodium L. in Poland. Polish Botanical Society, Wroc³aw, p

2 40 Edyta-M.-Gola,-Ewa-Szczêœniak 40 Introduction In Central Europe, the Polypodium vulgare complex consists of mixedploidy species, namely diploid P. cambricum L. (syn. P. australe Fée), tetraploid P. vulgare L., hexaploid P. interjectum Shivas, and their interspecific hybrids (Shivas 1961; Valentine et al. 1993). These taxa are widely distributed throughout Europe (e.g. Shivas 1961; Dostal, Reichstein 1984; Valentine et al. 1993; Bureš et al. 2003; Helánová et al. 2004), although only two latter species, and their hybrid, P. mantoniae Rothm., has been reported in Poland (Szczêœniak et al. mscr.). All taxa within the complex are morphologically similar, thus the most conclusive method of their identification is flow cytometry or verification of the chromosome number, since they represent different cytotypes (ploidy levels). However, it has been shown in ferns that some micromorphological features, e.g., the size of the epidermal guard cells and spores (Shivas 1961; Beck et al. 2010; Gabriel y Galán et al. 2010) and a vigor of spores (with abortive ones prevailing in some hybrid taxa of Polypodium, e.g., Kott, Peterson 1974; Kott, Britton 1982; Ivanova 2006) are positively correlated with the ploidy level. Therefore such traits are often used apart from the laboratory analyses as a practical tool for an indirect indication of the ploidy level. The most reliable and consistent trait for species delimitation in the P. vulgare complex is the structure of annulus, i.e. the number of basal and indurated cells (Bureš et al. 2003; Helánová et al. 2004; Ivanova 2006). Interestingly, it is a well-established feature for European polypodies, but not for the North American P. virginianum group, for which no significant relationship was found between the annulus structure and the cytotype (Kott, Britton 1982). Our introductory studies on the P. vulgare complex distribution revealed the occurrence of three taxa from this group in the Sudety Mts., i.e. P. vulgare, P. intejectum and P. mantoniae, whose identity has been confirmed by flow cytometry (Szczêœniak et al. mscr.). During detailed micromorphological analyses of these taxa, we noticed the presence of various leaf hairs, a feature which according to our knowledge has not been given in the manuals of the P. vulgare complex. In addition, in the Hegi s Flora (Dostal, Reichstein 1984) the leafblades of P. vulgare are said to be bald and glabrous. Moreover, we observed differences in the density of stomata distribution in all taxa tested. Although it is generally accepted that the size of stomata is correlated with the ploidy level in ferns (Kott, Britton 1982; Helánová et al. 2004; Beck et al. 2010; Gabriel y Galán et al. 2010), but the stomata density is hardly ever taken into consideration in Polypodium (Kott, Britton 1982). Here, we present preliminary results on the hair occurrence with the discussion on the possible diagnostic value of this feature for identification of the taxa within the P. vulgare complex,

3 41 The-leaf-blade-hair-and-stomata-density-in-the-P.-vulgare-L.-complex-in-Poland 41 and the initial analyses of differences in the density of hair and stomata distribution. Material and methods The plant material was collected in the Sudety Mts and their foreland during the initial research on the P. vulgare complex distribution. Specimens of the taxa P. interjectum, P. vulgare, and P. mantoniae, whose identification was confirmed by flow cytometry (Szczêœniak et al. mscr.), were used in further analyses. Dried pinnae, coming from the middle part of fully developed, sori-bearing leaves, were soaked with warm water for at least 15 min, and then the strips of adaxial and abaxial epidermis were peeled with a sharp razor and forceps. Fresh leaves of P. vulgare were used as a comparison. In general, five specimens of P. interjectum, ten of P. mantoniae and six of P. vulgare were analyzed. Epidermal peels were observed in a microscope Olympus BX50 and the images were taken by a DP71 camera with a Cell ^B software (Olympus S.A., Poland). The number of stomata and hairs was counted in the field of vision and then their frequencies were calculated per a 1mm 2 of the surface. Results and discussion In the course of the studies, the leaf epidermis was compared in P. interjectum, P. vulgare, and their hybrid, P. mantoniae. The adaxial side of the leaves was relatively uniformly developed in all taxa examined, covered by puzzle-shaped pavement cells (Fig. 1 A-C). These cells were slightly elongated parallel to the pinna long axis with exception of P. mantoniae, whose cells were a bit more rectangular comparing to the parental species (Fig. 1B). Also, the cell wall undulation in the hybrid was slightly less pronounced and sinuses less pointed than in two other species. The abaxial epidermis was more diversified than the other leaf surface. It contained pavement cells, guard cells forming stomata, subsidiary cells surrounding the guard cells, elongated marginal cells, and hairs. Except for the guard cells and trichomes, cell outlines were undulated, similar in shape to those at the adaxial leaf side. Stomata and hairs were scattered on the entire abaxial leaf surface; they were absent only directly above the veins and at the leaf margins. The stomata size was related to the ploidy level, with the biggest guard cells in the hexaploid P. interjectum (stomata measurements in specimens studied

4 42 Edyta-M.-Gola,-Ewa-Szczê niak 42 Fig. 1. Micromorphology of the leaf epidermis in the taxa studied of the Polypodium vulgare complex. A, D, G, H Polypodium vulgare L., B, E, I, J P. mantoniae Rothm., C, F, K, L P. interjectum Shivas. A-C the adaxial leaf surface showing puzzle-shaped pavement cells. D-F the abaxial surface of the leaf. Different types of the cells are present: pavement cells, stomata of different size and frequency, and hairs. G-L variability of hairs: mace shaped two-celled hairs (G, I, K), three-celled hairs with one branch (L), and multiple-branched hairs (H, J). The dark content of the hair cells can result from the production and/or accumulation of phenolic compounds (G-L). Ryc. 1. Mikromorfologia epidermy blaszki li ciowej u badanych taksonów z kompleksu Polypodium vulgare. A, D, G, H Polypodium vulgare L., B, E, I, J P. mantoniae Rothm., C, F, K, L P. interjectum Shivas. A-C doosiowa powierzchnia li cia pokazuj¹ca puzzlowaty kszta³t komórek w³a ciwych epidermy. D-F powierzchnia odosiowa li cia. Widoczne komórki w³a ciwe epidermy, aparaty szparkowe, ró ni¹ce siê wielko ci¹, i w³oski. G-L ró norodno æ w³osków: maczugowate dwukomórkowe w³oski (G, I, K), trzykomórkowe w³oski o pojedynczym rozga³êzieniu (L) i w³oski wieloramienne (H, J). Ciemna zawarto æ komórek w³osków mo e byæ wynikiem gromadzenia i/lub produkcji zwi¹zków fenolowych (G-L).

5 43 The-leaf-blade-hair-and-stomata-density-in-the-P.-vulgare-L.-complex-in-Poland 43 are presented in Szczêœniak et al. mscr), resulting in the lowest frequency of stomata in this taxon, opposite to their dense packing in P. vulgare, characterized by the smallest guard cells (Fig. 1D-F; Table 1). The hybrid presented intermediate values to its parental species. Since the frequency of stomata remains inversely proportional to their sizes (Kott, Britton 1982; Kessler et al. 2007; Gabriel y Galán et al. 2010), density of stomata in the field of vision can indirectly suggest, which ploidy level occurs in a given specimen. However, exhaustive studies on different ferns have shown that the stomata density can be much more variable within the species than between different taxa (Kessler et al. 2007; Gabriel y Galán et al. 2010), and also our preliminary results seem to confirm a variation of stomata packing in Polypodium taxa (Table 1), thus this feature requires careful evaluation. Table 1. Average density of stomata and glandular hairs per 1mm 2 of the surface in the Polypodium taxa studied. Tabela 1. Œrednia gêstoœæ aparatów szparkowych i w³osków gruczo³owych na 1mm 2 powierzchni w badanych taksonach Polypodium. Taxon (Takson) Stomata density (Gêstoœæ aparatów szparkowych) [min-max] Hair density (Gêstoœæ w³osków) % of branched hair (% w³osków rozga³êzionych) P. vulgare 58 [54-62] 3.9 >?1% P. mantoniae 53 [48-60] 3.5 ~22% P. interjectum 31 [24-48] 3.3 ~5% The interesting feature observed in taxa studied was the presence of the hairs. All the hairs in taxa studied contained characteristic brown content suggesting the glandular nature of trichomes (Fig.1G-K). Possibly, they can be the sites, other than rhizomes, for accumulation of phenolic compounds, typical of P. vulgare (Bagniewska-Zadworna et al. 2008), P. mantoniae (Zenkteler, Jêdrzejczyk 2012), and other Polypodium taxa (Peterson, Kott 1974), and which can play a role during the desiccation process. The most common in all taxa examined were simple mace-shaped hairs, which were built of two, occasionally three, cells (Fig. 1G, J). Sporadically in P. interjectum (about 5% of all hairs) and P. vulgare (less than 1%), threecelled branched hairs occurred (Fig. 1K; Table 1). Hairs of P. vulgare rarely had an additional branch, resulting in threefold-branched trichomes (Fig. 1H). The most interesting, few-times branched trichomes were characteristic of P. mantoniae (Fig. 1I). They were the biggest ones among all observed hairs, with more branches, and were relatively more frequent than branched hairs

6 44 Edyta-M.-Gola,-Ewa-Szczêœniak 44 in the parental species (Table 1). The frequencies of different hair occurrence, given here, are however only preliminary and require further detailed studies, including more specimens from various habitats, to enable the evaluation of this feature value for species delimitation. Furthermore, the habitat impact on the hair and stomata densities cannot be excluded since the environmental factors strongly affect both features (Kessler et al. 2007). For European polypodies, the specific information on the epidermis diversity is lacking. Nevertheless, similar to presented here, differences in the adaxial epidermal cell shape, size and waviness were reported for the American Polypodium ploidal series which included three cytotypes of P. virginianum (di-, tri- and tetraploids). Also, the hair presence was observed in this group, although only simple and three-celled-branched trichomes resembling those of P. interjectum were found (Kott, Britton 1982). It is known that in the model plant, Arabidopsis, one of the pathways regulating trichome branching is related to the repetitive endoreduplication events and the number of hair branches strongly depends on the cell ploidy (e.g. reviews by Hülskamp 2004; Ishida et al. 2008). Thus it cannot be ruled out that the number of branches and frequency of multi-branched trichomes can be related to different cytotypes of polypodies. In conclusion we can say, that although the slight differences in the micromorphology of epidermal cells and diversity of hairs are not the most significant and sufficient to delimit the taxon, nevertheless they indicate the requirement for further detailed comparative studies within the entire P. vulgare complex. References BAGNIEWSKA-ZADWORNA A., ZENKTELER E., KAROLEWSKI P., ZADWORNY M Phenolic compound localisation in Polypodium vulgare L. rhizomes after mannitol-induced dehydration and controlled desiccation. Plant Cell Rep. 27: BECK J.B., WINDHAM M.D., YATSKIEVYCH G., PRYER K.M A diploid-first approach to species delimination and interpreting polyploid evolution in the fern genus Astrolepis (Pteridaceae). Systematic Botany 35: BUREŠ P., TICHY L., WANG F., BARTOŠ J Occurrence of Polypodium mantoniae and new localities for P. interjectum in the Czech Republic confirmed using flow cytometry. Preslia 75(4): DOSTÁL J., REICHSTEIN T Polypodiaceae. In: CONERT H., HAMANN U., SCHULTZE- MOTEL W., WAGENITZ G. (eds.), Illustrierte Flora von Mitteleuropa. Band 1. Pteridophyta. Teil 1. Verlag Paul Parey, Berlin, Hamburg, pp GABRIEL Y GALÁN J.M., PRADA C., ROLLERI C.H., LAHOZ-BELTRÁ R., MARTÍNEZ-CALVO C Biometry of stomata in Blechnum species (Blechnaceae) with some taxonomic and ecological implications for the ferns. Rev. Biol. Trop. 59:

7 45 The-leaf-blade-hair-and-stomata-density-in-the-P.-vulgare-L.-complex-in-Poland 45 HELÁNOVÁ K., BUREŠ P., ŠMARDA P., HOROVÁ L., Polypodium mantoniae (P. interjectum P. vulgare) new hybrid in Romania, confirmed using flow cytometry. Contribuþii Botanice 39: HÜLSKAMP M Plant trichomes: a model for cell differentiation. Nature Reviews 5: ISHIDA T., KURATA T., OKADA K., WADA T A genetic regulatory network in the development of trichomes and root hairs. Annual Reviews of Plant Biology 59: IVANOVA D Polypodium interjectum and P. mantoniae (Polypodiaceae: Pteridophyta), new to the Bulgarian flora. Phytologia Balcanica 12: KESSLER M., SIORAK Y., WUNDERLICH M., WEGNER C Patterns of morphological leaf traits among pteridophytes along humidity and temperature gradients in the Bolivian Andes. Plant Function & Evolutionary Biology 34: KOTT L.S., PETERSON R.L A comparative study of gametophyte development of the diploid and tetraploid races of Polypodium virginianum. Canadian Journal of Botany 52: KOTT L.S., BRITTON D.M A comparative study of sporophyte morphology of the three cytotypes of Polypodium virginianum in Ontario. Canadian Journal of Botany 60: PETERSON R.L., KOTT L.S The sorus of Polypodium virginianum: some aspects of the development and structure of paraphyses and sporangia. Canadian Journal of Botany 52: SHIVAS M. G Contributions to the cytology and taxonomy of species of Polypodium in Europe and America. II. Taxonomy. J. Linn. Soc. Bot. 58: SZCZÊŒNIAK E., PIELECH R., JÊDRZEJCZYK I., RECZYÑSKA K., ŒWIERKOSZ K. Occurence of Polypodium interjectum Shivas and P. mantoniae Rothm. in Polish Sudety Mts. (2012), mscr. VALENTINE D. H., AKEROYD J. R., JERMY A. C Polypodium L. In: TUTIN T. G., BURGES N. A., CHATER A. O., EDMONDSON J. R., HEYWOOD V. H., MOORE C. M., VALENTINE D. H., WALTERS S. M., WEBB D. A. (eds.), Flora Europaea 1. Lycopodiaceae to Platanaceae. Second edition. Cambridge University Press, pp ZENKTELER E., JÊDRZEJCZYK I Morphology and anatomy of the rhizome of Polypodium xmantoniae Shivas. In: E. SZCZÊŒNIAK, E. GOLA (eds.), Genus Polypodium L. in Poland. Polish Botanical Society, Wroc³aw, pp

8 46 Edyta-M.-Gola,-Ewa-Szczêœniak 46 Wstêpne obserwacje zró nicowania w³osków blaszki liœciowej oraz gêstoœci aparatów szparkowych w kompleksie Polypodium vulgare w Polsce Badania mikromorfologii liœcia prowadzono u trzech przedstawicieli kompleksu Polypodium vulgare, wystêpuj¹cych w Polsce, tj. P. vulgare i P. interjectum, oraz u ich mieszañca - P. mantoniae. Paprocie nale ¹ce do tej grupy charakteryzuj¹ siê znacznym podobieñstwem morfologicznym, dlatego te jednoznaczne okreœlenie ich to samoœci wymaga zastosowania metod analizy kariotypu lub zawartoœci j¹drowego DNA, poniewa taksony te reprezentuj¹ ró ne cytotypy (poziomy ploidalnoœci). Niektóre z cech mikromorfologicznych, np. wielkoœæ aparatów szparkowych, zarodników, ich ywotnoœæ, s¹ pozytywnie skorelowane z poziomem ploidalnoœci i dlatego s¹ wykorzystywane do poœredniej identyfikacji taksonu. Najbardziej wiarygodnymi i sta³ymi cechami morfologicznymi dla taksonów kompleksu P. vulgare jest struktura pierœcienia, tj. liczba komórek zgrubia³ych i bazalnych (podstawy) pierœcienia. Wstêpne badania morfologii liœcia wykaza³y obecnoœæ w³osków na odosiowej (spodniej) stronie blaszki liœciowej u wszystkich badanych paproci. Charakteryzowa³o je ciemnobrunatne zabarwienie, sugeruj¹c zawartoœæ polifenoli i charakter wydzielniczy w³osków. U wszystkich badanych taksonów dominowa³y proste, maczugowate, zwykle dwukomórkowe w³oski. U P. interjectum i rzadziej u P. vulgare wystêpowa³y tak e trzykomórkowe w³oski o pojedynczym rozga³êzieniu. Forma mieszañcowa, P. mantoniae, charakteryzowa³a siê znacznie wiêkszym udzia³em w³osków (ponad 20%) o kilku rozga³êzieniach. Równoczeœnie w³oski te by³y najwiêksze spoœród wszystkich wystêpuj¹cych u badanych taksonów. Wiadomo, e u modelowej roœliny, Arabidopsis, jeden ze szlaków regulacji procesu rozga³êzienia trichomów zwi¹zany jest z powtarzaj¹cymi siê endoreduplikacjami, a liczba ramion w³osków zale y od ploidalnoœci komórek. Dlatego te nie mo na wykluczyæ, e liczba rozga³êzieñ i czêstotliwoœci wystêpowania w³osków wieloramiennych mog¹ byæ skorelowane z ró nymi cytotypami Polypodium. Przedstawione wyniki maj¹ jednak tylko wstêpny charakter i wymagaj¹ dalszych szczegó³owych badañ, obejmuj¹cych wiêksz¹ liczbê osobników z ró nych siedlisk, aby umo liwiæ ocenê przydatnoœci tej cechy do identyfikacji gatunku.