SEM differences in sporophyte micromorphology of Plagiothecium nemorale and P. longisetum (Plagiotheciaceae, Bryophyta)

Plagiothecium nemorale sensu lato is described as a variable Eurasian taxon. Recent studies indicate that this variability is reflected in both qualitative and quantitative features of its gametophyte and its genetic variation. This taxon comprises at least two separate species: P. nemorale (Mitt.) A. Jaeger and P. longisetum Lindb. The present paper examines whether the gametophyte variability of these species is reflected in the micromorphological variation of the sporophyte. The analysis revealed that the seta of P. nemorale is shorter than that of P. longisetum. In addition, regarding the exostome teeth, the lower portion of the outer surface is characterised by loosely-arranged, moniliform cristae in P. nemorale, with conical papillae on the upper side; however, in P. longisetum it possesses tightly arranged, epapillose cristae, with clearly flattened papillae on the upper side. In addition, the inner surface is more gently papillose in P. nemorale than in the latter. These findings not only describe hitherto unknown features of the sporophyte of these species, but also justify the resurrection of P. longisetus as separate from P. nemorale.


INTRODUCTION
As most species of the genus Plagiothecium Schimp. are sterile when collected (Greene, 1957), their taxonomic features tend to concern the morphological characteristics of their vegetative organs, particularly the qualitative features of the stem leaves (Wolski, 2018). Nevertheless, although previous works have focused mainly on the characteristics of the gametophyte, some also provide information on the sporophytes of these species (Lindberg, 1872;Jedlička, 1948Jedlička, , 1950Nyholm, 1965;Ireland, 1969;Iwatsuki, 1970;Buck & Ireland, 1989;Noguchi, 1994;Ochyra et al., 2008). However, these data refer only to features that are observable with the use of a light microscope. Only Ireland (1987Ireland ( , 2001 and Ignatov et al. (1996) have described the species of this genus using a Scanning Electron Microscope (SEM). Even so, their studies are fragmentary, referring only to a few selected species and sporophytic structures of these taxa.
One species belonging to the genus Plagiothecium is P. nemorale (Mitt.) A. Jaeger, which was described by Mitten (1859) as Stereodon nemoralis Mitt. Over the past 50 years, studies have found that P. nemorale was a very variable species, which has resulted in significant taxo-nomic problems (Greene, 1957;Nyholm, 1965;Ireland, 1969;Iwatsuki, 1970;Lewinsky, 1974); however, the causes of this variation were not identified. Eventually, a detailed qualitative and quantitative analysis of the intraspecific variability of the gametophyte of P. nemorale sensu lato distinguished at least two groups of specimens. The most important features supporting this division was found to be the length and width of the cells from the upper, middle, and lower parts of the stem leaves. This confirmed the hypothesis that P. nemorale had previously been too broadly described, and that it should be considered a complex (Wolski, 2017(Wolski, , 2018. These observations were later confirmed by further taxonomic and molecular research (Wolski & Nowicka-Krawczyk, 2020), which found that two groups of specimens distinguished by Wolski (2017Wolski ( , 2018 are in fact two separate species: P. nemorale sensu stricto and P. longisetum Lindb. Unfortunately, there remains a lack of detailed data on the sporophyte micromorphology of P. nemorale and P. longisetum. The present study was performed to determine whether the gametophyte variability demonstrated by these species is reflected in the micromorphology of SEM differences in sporophyte micromorphology of Plagiothecium nemorale and P. longisetum (Plagiotheciaceae, Bryophyta) the sporophyte.

MATERIALS AND METHODS
Sporophytes were separated into two groups depending on the qualitative and quantitative features of the gametophyte. The material was obtained from two separate species proposed by Wolski and Nowicka-Krawczyk (2020) -P. nemorale sensu stricto and P. longisetum. Specimens with symmetrical leaves, a denticulate leaf apex, as well as hexagonal and narrowly hexagonal leaf cells no longer than 100 µm in the central part of the leaf were considered P. nemorale sensu stricto. Material with asymmetrical leaves, an entire leaf apex, and elongated hexagonal leaf cells longer than 100 µm but not exceeding 150 µm in the central part of the leaf was classified as P. longisetum (Wolski, 2017(Wolski, , 2018Wolski & Nowicka-Krawczyk, 2020).
The examined sporophytes were collected from 50 specimens deposited in nine herbaria (BIL, BM, C, E, H, LOD, NY, POZG, S). In good condition and mature sporophytes were gently removed under a binocular microscope from homogeneous turf. The fragments of sporophytes were mounted onto aluminium stubs with double-sided adhesive tape and directly coated with a layer of gold in Quorum Sputter Coater SC7620. The coated sporophytes were observed, and relevant parts were photographed under a Carl Zeiss EVO LS 10 scanning electron microscope (15 kV accelerating voltage was used). Qualitative and quantitative analysis was performed of selected characteristics of the sporophyte samples: the setae (surface, color and length), capsules (orientation, shape, color, length, width), exothecial cells (shape, length, width), opercula (shape, length), exostome and endostome teeth (shape, color, length, width), cilia (length, surface).

Sporophyte description of Plagiothecium nemorale and P. longisetum
The reddish-brown seta is smooth and straight in both species. The capsule is inclined to horizontal and is usually cylindrical (Fig. 1). Even when dry, the capsule is smooth and usually dark brown, 2.6-3.5 × 1-1.2 mm. The exothecial cells are thin-walled, hexagonal to longhexagonal, 34-68 × 21-51 µm. The operculum is usually straight, 0.8-1.8 mm in length, longconic to rostrate, and the annulus is composed of 2-3 rows of cells, 50-64 × 21-35 µm. The double peristome is well developed (Fig. 1). The exostome teeth are lanceolate, narrowly triangular, and bright orange to yellowish, 400-600 × 80-119 µm. The outer surface of the exostome teeth in the lower part is striolate, and clearly papillose in the upper part. The exostome teeth themselves are trabeculate at the back, 10-26 µm (Figs 2-3). The endostome teeth are yellowish clearly segmented and form a triangular prism shape, 360-600 µm. At the base, the segments are sparsely papillose, much more densely so in the upper part, and clearly and intensively papillose inside (Fig. 4). The cilia, 300 µm in length, are clearly nodose with large swellings at cell boundaries, and clearly papillose along their entire length. The spores are spherical, 9-12 µm in diameter, and quite strongly papillose.
No explicit variation was observed for the shape, arrangement, length, or width of the capsule; the shape or dimensions of the exothecial cells, the dimensions or micromorphology of the endostome teeth, or the spores. However, in the group of studied specimens, some variability was noted in the length of the seta, and in the micromorphology of the surface of the exostome teeth. In addition, even the same trabecular areas can be differentially papillose, both quantitatively and qualitatively, with their ornamentation varying with regard to density (sparse or dense), diameter (thinner, thicker) and shape (straight, curved); sometimes even bifid papillae are present. The surface is heterogeneously papillose, varies according to the specimen being tested, which can be densely or rarely spiculose. It also depends on the position of the trabecula,  with the lower positions much more weakly and loosely papillose than the higher locations (Figs 2-3).
The seta of Plagiothecium nemorale is short (3-3.5 cm), the lower portion of the outer surface of the exostome teeth is charcterised by looselyarranged, moniliform cristae, while the upper side has conical papillae, and the inner surface is much more gently papillose (Figs 2, 5, 6). In contrast, the seta of P. longisetum is markedly longer (i.e. up to 5.5 cm). The lower portion of the outside of the exostome teeth of P. longisetum has tightly arranged, epapillose cristae, while the upper side has clearly flattened papillae, and their inner surface is much more strongly papillose (Figs. 3, 7, 8).

DISCUSSION
Many species of Plagiothecium have never been the object of SEM research. Although three studies have examined the micromorphology of species of Plagiothecium (Ireland, 1987;Ignatov et al., 1996;Ireland, 2001), the cited articles did not address the sporophytes of P. nemorale.
In sporophyte micromorphology P. nemorale and P. longisetum differ significantly from other species. The outer and inner surfaces of the exostome teeth of P. cavifolium, P. laetum and P. denticulatum are quite similar, with both surfaces having small, sparse papillae . The structure of the sporophyte has been associated with the taxonomic status of Plagiotheciaceae species Ignatov et al., 1998). According to these authors, the deviating sporophyte features of P. latebricola and P. piliferum justify their exclusion from the genus Plagiothecium .
Our findings indicate that the structures of the exostome teeth of the tested samples are variable. The lower part of the outer surface of the exostome is epapillose or moniliform. In the examined specimens, noticeable quantitative and qualitative differences were observed regarding the upper part of the outer surface of the exostome, as well as the papillose trabeculae of the inner surface of the exostome teeth.
A combination of our present findings with those of previous studies of the gametophyte of P. nemorale and P. longisetum (Wolski, 2017(Wolski, , 2018Wolski & Nowicka-Krawczyk, 2020) suggest that the following characteristics are indicative of P. nemorale sensu stricto: symmetrical leaves, a denticulate leaf apex, hexagonal or narrowly hexagonal leaf cells not longer than 100 µm in the central part of the leaf, a short seta, a sporophyte with loosely-arranged, moniliform cristae on the lower portion of the outer surface of the exostome teeth, the presence of conical papillae in the upper portion, as well as a gently papillose inner surface. In contrast, for P. longisetum, the gametophyte specimens are characterised by asymmetrical leaves, an entire leaf apex, the cells in the central part of the leaf elongated, hexagonal longer than 100 µm but not exceeding 150 µm, a long seta, tightly arranged epapillose cristae on the bottom side of the outside of the exostome teeth with clearly flattened papillae on the upper side, and a much more strongly papillose inner surface. These associations between the respective states remain constant, despite the variation in other features observed for the studied species.
Our findings provide new information on the micromorphology of the exostome and endostome teeth in the studied species. These data not only confirm the hypothesis of Wolski (2017Wolski ( , 2018) that currently P. nemorale is too widely described, but also -as indicated by Wolski & Nowicka-Krawczyk (2020) -that two distinct species can be distinguished within P. nemorale sensu lato. Furthermore, our study confirms that gametophyte variability is clearly reflected in the micromorphology of the sporophyte.