SPORE MORPHOLOGY OF POLYPODIUM ALEUTICUM A . E . BOBROV ( POLYPODIACEAE ) AND RELATED SPECIES

Spores of type specimens of Polypodium aleuticum A.E. Bobrov are studied by SEM-method. The morphology of the spores has been studied and described for comparative purposes. The results showed characteristic features of P. aleuticum exospores different from P. glycyrrhiza D.C. Eaton, which gives the basis for acknowledgment the independence of the investigated species.


INTRODUCTION
Polypodium aleuticum A.E. Bobrov was described by A. E. Bobrov in 1964 from USA "Unalaschka, Langsdorf".The species is distributed in the Aleutian Islands (Attu, Kodiak, Unalaska) and Alaska (island Sitka).Since its publication, the species was not being recognized by many researchers because of its similar morphological characters with the other north-american species P. glycyrrhiza.In the Flora of North America (Haufler et al., 1993) P. aleuticum is provided as synonym of P. glycyrrhiza.In our opinion, these are two completely different and independent species in spite of close morphological characters (Shalimov et al., 2015).The first studies of the morphology of the spores of P. aleuticum were performed by A.E. Bobrov (1964) and based on the data of light microscopy (LM).
The purpose of the research is to clarify the taxonomic positon of P. aleuticum.To achieve this objective, we studied differences in the spore structure of the taxa, since the spore morphology and exospores structure are the characters stable enough in the fern systematics and have unique structural features at the genus and species level.

MATERIALS AND METHODS
The study material was the specimens of spores from the herbarium material of Herbarium collection LE in V. L. Komarov Botanical Institute (BIN RAS, Saint-Petersburg, Russia) and Herbarium ALTB of Altai State University (Barnaul, Russia).For the study we chose 2 specimens: P. aleuticum and P. glycyrrhiza.As well as issued species we added some representatives of Polypodium L. from the Russian Far East.We used morphometric parameters of P. sibiricum Sipl., P. kamelinii Shmakov and P. fauriei Christ studied by authors in 2011 (Shalimov et al., 2011).Studies of the spores of P. aleuticum were carried out in the laboratory of the Center for collective use of BIN RAS using Analytical Scanning Electron Microscope JEOL JSM-6390LA.The spores of P. glycyrrhiza were studied in the laboratory of the Institute for Water and Environmental Problems (IWEP SB RAS, Barnaul), on the scanning electron microscope -S 3400 Spore morphology of Polypodium aleuticum and related species N, Hitachi High Technologies Corp. Spores were applied to the double sided tape, then attached to the metal stage with a diameter of 10 mm.In both studies, the surface of spores was treated with a mixture of gold-palladium in a vacuum sputtering installation JEOL SPUTTER JFC 1100 (BIN RAS) and Emitech SC 7620 / QT S (IWEP SB RAS), deposition time was 7-10 min.All spore samples were studied in high vacuum mode.Scanning of the samples was carried out with the increase from ×400 (a general view of spores) to ×14 000 (exospores surface).The parameters of spores were measured in 20-fold repetition.Measurements were carried out on the following values: 1 -major equatorial diameter, μm; 2 -polar axis, μm; 3 -aperture length, μm; 4 -minor equatorial diameter (spore width), μm; 5 -height of tubercles on the contour of spores, μm.The morphometric data were processed using discriminant analysis in the program Statistica 7.0 for Windows.The descriptive terms we used in this study follow the definitions used by Tryon & Lugardon (1991).Eaton, 1856, Amer. J. Sci., ser. 4, 22: 138, p. p.;Tzvelev, 2004, Bot. Zhurn. 89, 10: 1648, p. p. (Fig. 1: 1a, 1b).

DISCUSSION
The studied sample of P. aleuticum differs by exospores surface from P. glycyrrhiza.P. aleuticum has strongly verrucate exospores surface (Fig. 1: 1a, 1b), whereas P. glycyrrhiza (Fig. 1: 1c, 1d) exospores surface is low verrucate, with tubercles oblong or roundish form (Tryon & Lugardon 1991, fig 131.20.).In our opinion, it is quite distinguishable feature in the morphology of spore of these two species.At the base of tubercles of P. glycyrrhiza and P. aleuticum exospores surface is smooth, sometimes slightly rugate in P. aleuticum.Similar slightly rugate exospores surface at the base of the tubercles could be seen in other North-American species P. virginianum L. studied in Tryon & Lygardon (1991, fig. 131.19).
Spores morphology of P. aleuticum is clearly different from representatives of Polypodium from the Russian Far East.The exospores of P. aleuticum has verrucate form, P. fauriei and P. kamelinii have the tubercles from rounded to oblong in shape, and exospores of P. sibiricum is low echinate tubercles (Fig. 2).Two species: P. kamelinii and P. sibiricum have perispore; the first species has exfoliative perispore, the second one -echinate.The results of a more detailed morphological description of spores are presented in the research paper of Shalimov et al. (2011).
Discriminant analysis of spore morphometric characters of Polypodium representatives from the Pacific coast of the Russian Far East and the west of United States has revealed clear differences P. glycyrrhiza and P. aleuticum (Fig. 3).The dispersal cloud of P. aleuticum occupies a middle position between P. glycyrrhiza Shalimov A.P., Shmakov A.I. and P. fauriei; it may indicate the involvement of genes of both species in the formation of the genome of P. aleuticum in the Aleutian Islands.
The dispersal cloud of P. sibiricum on the sum of morphometric characters occupies a separate position from the rest of studied species.The median position of P. kamelinii in the species dispersal cloud between P. fauriei and P. sibiricum can talk about its possible origin from these two species, but not from P. glycyrrhiza.
The spores of the studied Polypodium species have the clear morphological differences at the species level, reflecting the graph (Fig. 3).Each species on the sum of morphometric characters has a separate cloud,   with a slight overlay P. aleuticum on P. glycyrrhiza and P. fauriei.The main characters of the differences in the spore structure of Polypodium species are the form and surface of exospores, the spore parameters: major and minor equatorial diameter, polar axis, aperture length.
The results of this research confirm the independence of both species, but further study of P. aleuticum from the Aleutian Islands and Alaska is required to establish its area of distribution.

Fig. 3 .
Fig.3.The objects projection of groups P. fauriei, P. sibiricum, P. kamelinii, P. aleuticum, P. glycyrrhiza in the axes of the canonical variables obtained from discriminant analysis of total morphometric parameters of spores.