Spore morphology of Selaginella borealis , S. sanguinolenta and S. helvetica (Selaginellaceae, Lycopodiophyta)

the first time we have conducted a study of three species of the genus Selaginella P. Beauv. from Russia: S. sanguinolenta (L.) Spring, S. helvetica (L.) Link and S. borealis (Kaulf.) Rupr. using the method of scan ning electron microscopy (SEM). In a comparative analysis of the morphology of micro- and megaspores of S. sanguinolenta , S. helvetica and S. borealis , we first compiled an information on representatives of Selaginella from the territories of China and Russia. A distinctive feature of S. borealis and S. sanguinolenta based on the megaspore morphology is the different nature of the exosporium surface: S. borealis has the exosporium with rounded-polygonal tu -bercles, and S. sanguinolenta has the exosporium with roller-like folds rising above the sporoderma. S. sanguinolenta is characterized by following species-specific features of microspores: the presence of radially arranged convolutions on the proximal surface of the spore, outside the laesura, and convoluted folds on the distal surface of the microspores. S. helvetica is characterized by the following microspore morphology: the hemispherical distal side is in the equatorial position, and flat proximal side; the surface of the exosporium is granular, completely covered with rounded-polygo nal tubercles. In the research we confirmed the presence of S. borealis in China (Yunnan). It has been established that the morphology of S. sanguinolenta microspores from the Khabarovsk Territory, the Republics of Buryatia, Tuva and the Irkutsk Region is identical to the microspores of representatives of S. sanguinolenta from China (Yunnan); and the morphology of S. helvetica microspores from the territory of the Chita Region, Trans-Baikal and Primorye Territories is identical to the morphology of the species samples from Liaoning Province (China) .


Introduction
Genus Selaginella P. Beauv. (Selaginellaceae) is the largest family in the Lycopodiophyta (Zhou, Zhang, 2015). Representatives of Selaginellaceae are distributed almost worldwide, mainly in the tropics (Tryon, Lugardon, 1991, Zhou et al., 2015b. Representatives of the genus prefer not only the main habitats of the temperate, subtropical and tropical zones, including the desert, but also enter arctic areas and climb the mountains in the alpine belt (Jermy, 1990;Zhang et al., 2013;Zhou, Zhang, 2015).
In the territory of Russia, the genus is represented by 8 species (Tzvelev, 2004), of which the complex Selaginella borealis-S. sanguinolenta is most difficult. Many researchers recognize both these species as independent (Tzvelev, 2004), and some consider them as one polymorphic species (Zhang et al., 2013).
The spore morphology of Selaginella representatives is important in the systematics of the genus. Using the spore morphology researchers differentiate some taxonomic and complex species (Zhou et al., 2015a). Such conclusions were made by Zhou's research team based on 70 Selaginella species from China. Using the morphological characteristics of megaspores and/or microspores, the researchers divided the spores of the Chinese representatives of the genus into 15 types, and three more types were divided into different subtypes. Wherein some types reflect the species composition of the clades and subclades identified in further molecular genetic studies of Zhou et al. (2015b).
According to Japan samples the megaspore morphology of S. helvetica refers to seventh spore type -"The Selaginella pallidissima type" (species composition of the type: S. pallidissima, S. denticulata, S. helvetica, S. jugorum, S. laxistrobila, S. nipponica, S pallidissima, S. prostrata, S. pseudonipponica, S. rubella, S. tama-montana) (Zhou et al., 2015a). Megaspores of this type are characterized by a rounded or almost rounded shape, the presence of a wide laesura, occupying a length of 1/2-3/4 the size of the equatorial diameter on the proximal side. The proximal surface of the megaspore is warty, and the distal surface is covered with furrows with a curved ornament.
Similar characteristics on the S. helvetica megaspores are given by researchers on samples from Europe and North Asia (without specifying the herbarium samples from the Herbarium "S" of the Swedish Museum of Natural History). At the same time, there is an additional clarification in terms of the sizes of S. helvetica megaspores -350-400 μm in the morphological description (Korall, Taylor, 2006).
S. helvetica spores on samples from China (Liaoning province) are characterized by the presence of a hemispherical microspore. The microspore laesura occupies 3/4 the size of the equatorial diameter on the proximal side. The proximal and distal surfaces of microspores are warty, sometimes with ridges (Zhou et al., 2015a).

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Vaganov A. V. et al. Spore morphology of Selaginella Zhou et al. (2015a) used S. sanguinolenta specimens (including specimens of S. borealis, without a species separation of these two taxa) from Yunnan Province to study the morphology of spores. According to this study the megaspore morphology of S. sanguinolenta is related to the first spore type -"The Selaginella sanguinolenta type" (species composition of the type: S. nummularifolia, S. rossii, S. sanguinolenta). Megaspores of this type have a tetrahedral form; a laesura occupies a length of 3/4 the size of an equatorial diameter. The proximal and distal surfaces of megaspores are warty. The microspore form of S. sanguinolenta is also tetrahedral, as megaspore one. Laesuras are clearly defined and reach the equator in length. The proximal and distal microspore surfaces are wrinkled (Zhou et al., 2015a).
We should note, that Zhou et al. have proposed generalized morphological spore types features which characterize the group of species studied. This creates difficulties in identifying certain species using the characters of the spore morphology, that is especially important in species differentiation in such complex taxa as S. borealis and S. sanguinolenta. In this study, the task was to identify the speciesspecific features of the megaspore morphology of S. borealis and S. sanguinolenta representatives, as well as to clarify the information on the morphology of S. sanguinolenta and S. helvetica microspores.

Material and methods
A material for the study was the spore samples from the herbarium material of the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences (NS), G. G. Popov Herbarium of the Central Siberian Botanical Garden of the SB RAS (NSK), Irkutsk State University (IRKU), Siberian Institute of Plant Physiology and Biochemistry SB RAS (IRK). In total, we selected 10 samples for the study on 3 species of the genus Selaginella, from the territory of Republics of Tuva and Buryatia, Irkutsk and Chita Regions, Khabarovsk and Primorye Territories. The spore studies were conducted in the laboratory of the Institute for Water and Environmental Problems (IHEP SB RAS, Barnaul), using a Hitachi S 3400 N electronic scanning microscope from Hitachi High-Technologies Corp. Spores were applied on double sided tape, fastened on metal object tables with a diameter of 10 mm. The spore surface was treated with a gold-palladium mixture in an Emitech SC 7620/QT S vacuum evaporation unit for about 6 minutes.
All spore samples were examined under high vacuum. The studied samples were scanned with an increase from ×400 (general view of the spores) to ×14 000 (study of the surface of exosporium).
The analysis of spores was carried out according to the following morphological features, μm: 1equatorial diameter; 2 -the polar axis; 3 -laesura length; 4 -laesura width; 5 -diameter of the tubercles on the proximal side of the spore; 6 -diameter of the tubercles on the dorsal side of the spore; 7diameter of the tubercles on the proximal side of the spore near the laesuras; 8 -the width of the curved folds on the distal side; 9 -the width of the convolutions on the proximal side. The measurements were carried out in a 20-fold repetition.

Morphological descriptions of micro-and megaspores of the Selaginella species.
Selaginella sanguinolenta (L.) Spring SEM description (Fig. 1.). The spores in the proximal-polar and distal-polar positions are rounded-triangular, not lobed. The equatorial diameter is 46.76 (from 32.8 to 64.3) μm. The polar axis is 26.3 (from 25.4 to 27.3) μm. In the equatorial position, the distal side is hemispherical, proximal -conical. Laesura arms are straight, 15.72 (from 13.4 to 17.0) μm long, 2.27 (from 1.8 to 2.9) μm wide. The surface on the proximal side of the spores out of the laesuras is flat, or with the presence of convolutions radially located from the edges of the laesuras, 1.3 (0.8 to 2.2) μm wide. There are convoluted folds, 3.74 (from 1.8 to 7.1) μm wide, across the distal surface of the spore. The surface of the exosporium is rough, without outgrowths.   steep eastern slope. 22 VII 1974, No. 333. I. Krasnoborov, V. Khanminchun. NS0003983" (NS).
Selaginella sanguinolenta (L.) Spring SEM description (Fig. 3.). The spores in proximal-polar and distal-polar positions are rounded. The equatorial diameter is 303.0 (from 297 to 312) µm. The exosporium is covered with roller-like folds, rising above the sporoderm, reaching a width of 8.3 (from 5.8 to 12.2) µm. The distance between adjacent roller-like folds reaches the width of the folds themselves. The surface of the exosporium is rough.
The main distinctive feature of S. borealis and S. sanguinolenta based on the characteristics of the megaspore morphology is the nature of the exosporium surface. Representatives of S. borealis have the exosporium with rounded-polygonal tubercles of 3.8-4.9 µm in diameter, unlike the S. sanguinolenta exosporium covered with roller-like folds rising above sporoderm and reaching a width of 8.3 µm. The S. borealis exosporium surface is granular and S. sanguinolenta one is rough. The form of the megaspore of S. borealis is rounded to tetrahedral, whereas S. sanguinolenta has the megaspore form only rounded. The size of the equatorial diameter differs in S. borealis and S. sanguinolenta slightly -296.2 µm and 303 µm, respectively.
In a comparative analysis of the megaspore samples of our study with the samples presented in the work of Zhou et al. (2015a), images B, C and D of fig. 1 of S. sanguinolenta megaspores do belong to S. sanguinolenta, and picture A of fig. 1 -to S. borealis with characteristic tubercles on the exosporium surface and the tetrahedral form of megaspore peculiar only to it. Thus, our study has confirmed the presence of S. borealis in the territory of Yunnan (China).
We have also established that the SEM data and the description of the microspore morphology of representatives of S. sanguinolenta from the Khabarovsk Territory, the Republics of Buryatia, Tuva and the Irkutsk Region are identical to the microspore morphology of S. sanguinolenta from China (Yunnan). Species-specific features of microspores characterizing S. sanguinolenta are the presence of radially arranged convolutions on the proximal surface of the spore, outside the laesura, and convoluted folds on the distal surface of the microspore.
As a result of our study, the microspore morphology and images of S. helvetica representatives from the Chita Region, Trans-Baikal and Primorye Territories turned out to be identical to the morphology of the studied specimens from Liaoning province (China) in the work of Zhou and co-authors. Species-specific features of microspores characterizing S. helvetica are hemispherical distal side in the equatorial position and flat proximal side; the surface of the exosporium granular, completely covered with rounded-polygonal tubercles.