Dispersed cuticles and conducting tissue of Sphenophyllum BRONGNIART from the Westphalian D of Kalinovo , Donets Basin , Ukraine

Upper Westphalian coals are usually rich in organic-matter of different plant taxa, including gymnosperms. The assemblage from Kalinovo (Eastern Ukraine) is exceptional in being dominated by Sphenophyllum, both cuticles and conducting tissues. Sphenophyllum cuticles are easily distinguished by their parallel oriented cells with sinuous anticlinal walls, and paracytic stomata on the abaxial cuticle. Tracheids with multiseriate bordered pits that occur in Sphenophyllum can be also found in the Calamitaceae and some pteridosperms. However, rectangular shaped remains of parenchyma cell strips along the radial wall of tracheids are only known in Sphenophyllum.


introduction
The genus Sphenophyllum BROnGnIART belongs to the sphenopsids and is characterized by linear, spatulate or fanshaped leaves that extend up to several cm in length.The leaves are arranged along the axis in nodal whorls of six or nine.The name Sphenophyllum is used for both impressioncompression and petrified Permo-Carboniferous samples.
Although more than 100 compression-impression and petrified Sphenophyllum species have been described in the world, cuticles are known only from 15 of those species.The cuticles known so far, have been prepared by maceration of coalified Sphenophyllum leaves; studied directly on fossils by means of optical techniques (BARTHEl, 1997); or studied in sections of coal balls (GOOD, 1973).This paper brings unique information on sphenophyllalean cuticles and conducting tissue obtained directly from the coal.Unfortunately, taxonomic affiliation was not possible because the leaf outline is not preserved.Similar cuticles have only been previously observed in Sphenophyllum priveticense lIBERTÍn et al., 2014.We presume that both species lived under simi-sandstones (AIzEnvERG et al. 1975).Mudstones with roots and coal seam no.n 3 (0.3 m thick) are located above the sandstone.A sample from this coal seam from the Kalinovo locality, which is situated about 55 km W. from Luhansk in the Donets Basin (Fig. 2 et al. 1975).This flora is of Asturian (Westphalian D) age and is the youngest Westphalian flora found in the Donets Basin.

MAteriAl And Methods
In order to obtain cuticles authors applied a new method.A 2.5 g sample of coal was macerated in Schulze's reagent: 35 ml concentrated (65%) nitric acid (HNO 3 ) and 1.5 g of potassium chlorate (KClO 3 ) for 2 days and 18 hours.The black residue was fully washed under running water in a sieve and then treated with 10% potassium hydroxide (KOH) for up to one hour.During this process, the "coal matter" was completely dissolved and only the cuticles and vascular tissue remained.The cuticles were stained in Safranin, Bismarck brown, Malachite green or neutral red, and mounted in Glycerine Jelly, or were attached to an SEM stub for observation under a scanning electron microscope (SEM).
About 30 foliar fragments of cuticles and a similar number of conducting tissue fragments were mounted on six slides.About ten cuticular fragments are longer than 1 mm (up to 1.3 mm), and represent both the adaxial and abaxial leaf surfaces.Some fragments of conducting tissue were up to 3 (4) mm long.
The dispersed cuticles are stored in slides no.595/1-6.limestone n 3 in the Isayevskaya Suite (Formation) is overlain by a 7 m thick sequence of unfossiliferous bluishgrey claystones and siltstones and about 30 m of fine-grained

Description:
Adaxial cuticle: The cells are elongate, more or less fusiform or elongate tetragonal in shape with coarsely sinuous anticlinal walls that affect the original cell shape.The cells are oriented parallel to the veins, and are 150-300 μm long and 25-50 μm wide.The costal (vein) areas are usually prominent with original black vascular tissue (Pl. 1, fig. 2), which is about 100 μm wide.When this tissue is not preserved (Pl. 1, fig.1), the costal area is about 50 μm wide and shows narrow cells only 15-20 μm wide.
Abaxial cuticle: The cells have essentially the same shape and dimensions as the cells of the adaxial cuticle.The difference is in the presence of stomata (Pl. 1,.The stomata tend to be concentrated in "bands" along the veins and they are oriented parallel to the veins.The guard cell pairs are fusiform or elliptical, 26-32 μm long and 10-14 μm wide (Pl. 1,figs. 4,6,Pl. 2,fig. 4).Each stoma has two lateral subsidiary cells (paracytic stomatal type), one of which is shorter -78-90 μm long; and the longer is 176-192 μm long.Both cells have approximately the same width -20-35 μm.
The cuticles in scanning electron microscope have very fine striated periclinal walls in outer view (Pl.2, fig. 1) and the anticlinal walls are prominent in the inner view (Pl.2, figs.2-4B).
Using the same approach as described by POOlE & KüRSCHnER (1999) stomatal density and stomatal index were counted from several cuticle fragments; the area of each fragment was 0.1-0.15mm 2 .The stomatal density of Sphe-nophyllum sp. is 83-90 stomata per mm 2 and the stomatal index is 20.5-23.
Conducting tissue: Fourteen tracheids from slides 595/1, 2, 3 and 5 were studied.Fragments of tracheids have walls perforated mainly by multiseriate bordered pits (Pl. 3,figs. 1,3), which locally become in reticulate cell wall thickening (Pl.3, fig.4).Bordered pits are free, and according to the number of their rows and tight configuration they belong to the alternate pitting surface type .The number of bordered pit rows on the tracheid wall is (2)-4-6.Bordered pits have a circular to elliptical shape and approximately the same dimension.The tracheids are 60-100-( 125) mm wide.The thickening on some tracheids is interrupted by the walls of parenchyma cells that form a rectangular outline on the tracheid (Pl.3, fig.2).Due to the fragmentary size of the preserved tracheids, it was not possible to determine their original total length.

reMArKs
Fragments of the xylem -tracheids were found in association with the Sphenophyllum cuticles.According to TAYlOR et al. (2009, p. 206), annular or helical thickenings of tracheids are most often found in the earliest matured primary xylem.Secondary xylem is made up predominantly of pitted tracheids, although some plant groups also have secondary xylem tracheids.Bordered pits in tracheal elements are according to NěMEjC (1963, p.76) very rare in many pteridophylls (Pteridophyta).It is common in equisetopsids (Equisetopsida) and in many pteridosperms and gymnosperms.

Sphenophyllum cuticles And their coMPArison
Most species of Sphenophyllum BROnGnIART have irregu larly isodiametric to elongate epidermal cells that usually posses sinuous anticlinal walls.Stomatal complexes occur on the abaxial surface of the leaf and consist of two guard cells with polar and circumpolar thickenings, and two paracytic lateral subsidiary cells.Subsidiary cells may have the same shape and size as normal epidermal cells, or they can differ.One subsidiary cell is usually distinctly larger than the other (TAYlOR et al. 2009).These characteristics fit very well with the cuticles described above.Individual species differ in the distribution of the stomata, the orientation and size of the guard cells, and the shape, arrangement and size of the intercostal cells (Table 1).BARTHEl (1997) distinguished two groups among the Sphenophyllum species.Group 1 consists of Sphenophyllum cuneifolium, S. emarginatum, S. majus ABBOT (=S.geinitzii STORCH), S. thonii, S. speciosum.S. longifolium and S. saxonicum.This group is hypostomatic, with sinuous anticlinal walls and larger cells.Group 2 consists of Sphenophyllum oblongifolium and the anatomically preserved species S. quadrifidum REnAUlT and S. reedae GOOD.In this group, the epidermis has rectangular cells with straight anticlinal walls.The Sphenophyllum sp.described in the present paper belongs to Group 1.The elongate cells with coarsely sinuous anticlinal walls occur in many species: e.g.Sphenophyllum emarginatum, S. cuneifolium, S. thonii, S. majus ABBOT (non BROnn) (=S.geinitzii), S. kobatake and S. zwickawense.However, the cell size in the present species (150-300 μm) is rather large compared to all the species mentioned above, where it is usually only up to 200 μm.The exception is the newly described Sphenophyllum priveticense lIB-ERTÍn, BEK et DRáBKOvá, 2014, in which the cells are 125-350 μm long and so more comparable to our samples of Sphenophyllum sp.However, they differ in other characteristics (see Tab. 1) and it is unlikely that they belong to the same species, especially as Sphenophyllum priveticense is of early Bolsovian age whereas the present Sphenophyllum sp. is of latest Asturian age.
Sphenophyllum guard cells are usually reniform and the outline of the pair is elliptical.Their size differs according to species, however some overlap exists.Our Sphenophyllum sp. has medium sized guard cells among the sphenophylls, 28-32 μm in length.The smallest ones are known from permineralised S. multirame (only 15 μm long) and the largest ones are known in four species S. emarginatum (31-57 μm), S. apiciseratum (34-54 μm), S. koboense (27-54 μm) and S. priveticense (45 μm).An important feature is also the shape of the subsidiary cells.Many species have two lateral subsidiary cells per stoma (usually one of them is larger than the other); they are of the same shape as normal epidermal cells in Sphenophyllum sp., S. emarginatum, S. thonii, S. speciosum, S. longifolium, S. apiciseratum, S. koboense and S. priveticense.According to lIBERTIn et al. ( 2014), the stomatal complex of S. priveticense is anomocytic, but it seems that all those mentioned above have paracytic stomata.In other species, the subsidiary cells differ significantly from the ordinary epidermal cells: Sphenophyllum cuneifolium, S. thonii, S. geinitzii (S. majus ABBOT), S. saxonicum, S. multirame and S. reedae.Some species have trichomes and papillae on the adaxial surface of the leaves: Sphenophyllum majus, S. speciosum, S. saarensis, S. trichomatosum STUR and probably also S. sewardii BATEnBURG (lIBERTÍn et al. 2014).No trichomes or emergences have been observed on cuticles from Sphenophyllum sp. from Kalinovo.

coMPArison oF conductinG eleMents
The characteristic pitting of the tracheid walls in the studied samples corresponds to the description of secondary xylem anatomy of the Sphenophyllum plant as mentioned in  BATEN BUrG, 1981).BOUREAU (1964, p. 98, 99), who figured Sphenophyllum with multiseriate bordered pits.He also figured the distribution of parenchyma cell strips along the radial walls of a tracheid.It has been shown that strips of parenchyma cells created a rectangular contour on the radial wall of the tracheids.These contours are the only remnants of parenchyma cells.

conclusion
Cuticles and conducting tissue of Sphenophyllum BROnG-nIART have for the first time been identified in the dispersed cuticle spectrum.Many Sphenophyllum species have the same cuticular pattern, so it is difficult to identify Sphenophyllum species only using cuticles, as noted by BARTHEl (1997) Sphenophyllum sp. was discovered in Kalinovo coal, and S. priveticense was found in a tuff that buried the peat-swamp "in situ" and also contained many peat-forming floral elements.It is likely, therefore, that both Sphenophyllum sp. and S. priveticense lived in similar habitats.lIBERTÍn et al.
(2014) supposed that Sphenophyllum priveticense lived in peat-swamps with a high water table and also in slightly drier habitats with a transition to an Omphalophloios-phase. Maybe the larger cell dimension is caused by living in such an environment.
It is not possible to systematically classify the studied tracheid fragments of the genus Sphenophyllum sp.based on tracheid thickening.A similar structure of tracheids is also common in the other plant groups from the Pennsylvanian (Calamitaceae, pteridosperms).Any palaeoecological interpretation based on the tracheids is not possible, as their width depends on vegetation conditions, and we are not sure if all the studied tracheids in fact belong to this Sphenophyllum.

AcKnoWledGeMent
This study was part of the International Geoscience Programme (IGCP Project 575) and the GACR project P210/10/0232.The authors are obliged to V. I. POlETAEv, n.BOYARInA and A. SCHEGOlEv (Kiev, Ukraine) for guiding in the field and to C.j. ClEAl (Cardiff, UK) for checking of the text.

Figure 1 :
Figure 1: Stratigraphy of the Pennsylvanian of the Donets Basin.The coal sample was taken from the upper part of the section.Adapted from PrivAlov et al. (2005).

Figure 2 :
Figure 2: Map of Ukraine with Kalinovo locality highlighted.
Although the Kalinovo species has medium-sized guard cells that fit with several species, together with Sphenophyllum priveticense lIBERTÍn, BEK et DRáBKOvá, 2014, it has the longest cells that have been reported so far in a Sphenophyllum species, 300 -350 μm long, in contrast to usually no more than 200 μm long seen in other species.Despite the similarity of their cuticles, the Sphenophyllum sp. from Kalinovo and Sphenophyllum priveticense cannot be regarded as conspecific as the former is stratigraphically much younger.