Stipa pennata subsp. ceynowae (Poaceae, Pooideae), a new taxon from Central Europe

Abstract Based on numerical analyses of macromorphological characters, scanning electron microscopy observation of leaves and lemma micromorphology, as well as field observations, Stipa pennata subsp. ceynowae was described here as a new taxon from Poland. It differs from the most similar S. pennata subsp. pennata and S. borysthenica mainly by its longer ligules of vegetative shoots. The affinities of this taxon are discussed and a morphological comparison with related species is provided. Illustrations and images of the micromorphological structures, as well as information about its distribution, habitat and conservation status are given.

During the taxonomic revision of the central European representatives of the Stipa pennata group, we came across herbarium specimens from Folusz near Szubin in Poland that greatly differ from the hitherto known species. On the basis of these sheets, Ceynowa-Giełdon (1976) distinguished Stipa joannis var. cujavica. Unfortunately, the name of this taxon was not validly published because the author provided only its brief description in Polish with no references to the type and place of its preservation. The aim of our study was to examine distinctiveness of individuals from Folusz in relation to other Central European taxa from S. pennata group by using multivariate morphometric analysis.

Materials and methods
Over 500 herbarium sheets with specimens from the Stipa pennata group  Table 1. Measurements were taken using a Nikon SMZ800 stereo microscope.
In accordance with the assumption of numerical taxonomy (Sokal and Sneath 1963), each specimen was treated as an Operational Taxonomic Unit (OTU). For testing the normal distribution of each characteristic, the Lilliefors and Shapiro-Wilk statistical tests were performed. Those that did not fulfill the criterion of normality were log-transformed. Next, the Pearson correlation coefficient was calculated; the characteristics in which a strong correlation was found (>0.9) were excluded from further analyses. To illustrate the relationship between the studied taxa and also to select the features that best describe the existing variability, a Principal Component Analysis (PCA) was conducted using all quantitative characteristics. According to the Kaiser criterion, factors with eigenvalues >1 were chosen (Kaiser 1960) and characteristics with the highest factor loadings of the first three principal components (r≥0.60) were determined. Subsequently, descriptive statistics of characters for all recognized taxa were calculated. Levene's test was using to assess the equality of variances. To reveal significant differences between means of particular characters across all examined taxa, one-way analysis of variance (ANOVA) and nonparametric Kruskal-Wallis test followed by Tukey's HSD test or multiple comparison test were calculated. All statistical analyses and calculations were performed using Statistica software, version 10 (Statsoft Inc. 2011). For observations in a scanning electron microscope, samples were coated with gold using a JFC-1100E Ion sputter manufactured by JEOL, then observed and photographed using a Hitachi S-4700 scanning electron microscope (SEM). The methods and terminology were adopted from Thomasson (1978Thomasson ( , 1981, Ellis (1979), Snow (1996) and Nobis (2013Nobis ( , 2014a.

Numerical analysis
The result of the Principal Component Analysis (PCA) revealed twelve characteristics with high factor loadings (r≥0.6) on the first three principal components. Together, the first three components accounted for 57.71% of the total variation. The first two components explained respectively 27.85% and 21.48% of the total variation ( Table 2). The scatterplot of the first two axes showed three group of points ( Figure 1). Seven characteristics, including AL, CL, CRL, LCL, LigC, LP, and NF, displayed the highest correlations with the first axis, grouping specimens of Stipa borysthenica on the left and S. pennata on the right side. The remaining characteristics (AwnL, Col 1 L, LC, LigIV, LV) highly influenced the second axis, separating specimens representing Stipa from Folusz. The results of the one-way ANOVA/Kruskal-Wallis test revealed significant differences in all examined characters ( Table 2). The results of the post hoc tests (Tukey's HSD test for variables with normal distribution and multiple comparison tests for characters with non-normal distribution) are presented in Table 3. The ranges of variability of the most important characteristics of the designated morphological groups corresponding to the two examined taxa and the population from Folusz are presented in Table 4.

Scanning microscope observation
The results of SEM observations showed that the general patterns of the lemma micromorphology of Stipa from Folusz are typical for the genus Stipa (cf. Barkworth and Everett 1987, Nobis 2013, 2016b (Figure 2). Fundamental (long) cells are elongated, rectangular to a more or less square in shape. The side walls of long cells are raised and undulate, but often hidden under a thick layer of wax that hin- ders observation. Silica bodies are quite common, reniform to oblong or ovate, while cork cells are sparse or absent. Hooks are frequent, oriented towards the lemma apex, whereas prickles are completely absent. Lemma apex is glabrous (Figure 2A-D).
The adaxial surface of leaves of the vegetative shoots is ribbed and densely covered by short prickles, long cells and silica bodies ( Figure 2E). Whereas the abaxial surface is dominated by long cells with admixtures of silica bodies and sparsely distributed prickles ( Figure 2F).
Etymology. The name of taxon honors the collector-Prof. Mirosława Ceynowa-Giełdon, who first noted the distinctiveness of Stipa individuals from Folusz.
Distribution and habitat. Stipa pennata subsp. ceynowae is an endemic taxon, known only from Folusz settlement near Szubin in Kuyavia region (northern Poland). It grows on a dune hill surrounded by wet meadows occurring in the Gąsawka River Valley. The subspecies occurs on small fragment of dry, sandy grassland adjoining oak and pine stands. At the locality, the following species grow together with Stipa: Achillea pannonica Scheele, Asperula tinctoria L., Avenula pratensis (L.) Dumort., Betula pendula  Nienartowicz et al. 2014). Currently, S. pennata subsp. ceynowae should be considered as a critically endangered (CR) species-to date, only several flowering individuals have survived (tufts with 8, 10, 11 and 14 culms), occupying a very small area of dry grassland. Lack of grazing has resulted in increased ground cover by layer of "steppe felt", which hamper seeds germination and seedlings growth. Also, tree seedlings pose a threat by shading the grasslands. Similarly, as in the case of other dry grassland species-survival depends on the preservation of suitable habitat conditions, which can be achieved through active protection. Due to the extremely small size of the population, it seems reasonable to apply the methods of ex situ conservation, including in vitro propagation.
Additional specimens studied ( -Giełdon (1976) distinguished Stipa joannis var. cujavica (nom. inval.) based on the longer hairless part of the awn, longer vegetative leaves and longer upper cauline leaves than in the case of the typical variety. Although the Principal Component Analysis supports the usefulness of these characteristics (Table 2), their larger size can also be found in individuals of S. pennata subsp. pennata and, after examining a great number of individuals, they seem to be insufficient to distinguish this taxon based on its description. According to our results, the internal vegetative leaves ( Figure 4D) in specimens of S. pennata subsp. ceynowae have distinctly longer ligules (usually 4.1-5.2 mm in length) than the other closely related species from Poland, namely S. borysthenica and S. pennata subsp. pennata (in both cases, usually reaching of 1.3-2.2 mm in length; Table 4). Our research carried out on a large number of herbarium specimens (from the geographical range of these taxa), as well as on the findings of other authors (Bor 1970, Tzvelev 1976, Martinovský 1977, 1980, Conert 1998, Gonzalo et al. 2013, Nobis et al. 2016b, confirm that all known taxa closely related with S. pennata do not have such long ligules of their vegetative shoots. Ligules of a similar length or even longer are observed in other species of the section Stipa occurring in Central Asia, namely: S. turkestanica subsp. turkestanica, S. macroglossa subsp. macroglossa and S. macroglossa subsp. kazachstanica (Gonzalo et al. 2013, Nobis et al. 2016b). However, S. pennata subsp. ceynowae cannot be confused with any of them due to its definitely longer anthecium, callus, awn, culm and vegetative leaves, as well as to its distribution, limited only to Central Europe.

Ceynowa
Stipa kirghisorum, is another species that is morphologically similar to both S. pennata subsp. pennata and S. pennata subsp. ceynowae. However, S. kirghisorum differs from the two above-mentioned taxa by the strongly scabrous abaxial surface of leaves of the vegetative shoots, shorter anthecium (13.1-)14.5-16.0(-17.8) mm and ventral line of hairs terminating (0.5-)1.4-3.1(-4.6) mm below the top of the lemma, as well as its general range that is limited to the Central Asia (Nobis et al. 2016b).
Stipa pennata subsp. ceynowae is somewhat similar to two other European species from ser. Penicillifera. First is S. styriaca that is also characterized by having long awn up to 445 mm and anthecium 17.5-21.5 mm, but in contrast to S. pennata subsp. ceynowae it has densely pubescent leaf sheaths (with 0.2-0.8 mm hairs) (Martinovský 1977). The second species is S. danubialis that differs from Stipa pennata subsp. ceynowae by having pilose column (lower part of awn) and anthecium 23-25 mm long (Martinovský 1977).
Due to its long awn, Stipa pennata subsp. ceynowae could be also confused with S. pulcherrima that occurs in Central Europe too. However, it can easily be distinguished by its ventral lines of hairs terminating at 1/2-2/3 of lemma length, shorter anthecium (17.4-20.0 mm) and longer ligules on vegetative shoots, while S. pulcherrima is characterized by ventral lines reaching the base of the awn, anthecium 18-25 mm long and ligules of the vegetative shoots not exceeding 2 mm long (Martinovský 1980).
The results of ANOVA and post-hoc tests confirm separateness of the taxon from Folusz (Tables 2, 3). Stipa pennata subsp. ceynowae differs from S. pennata subsp. pennata and from S. borysthenica in a statistically significant way by 13 and 10 characters respectively (Table 3).
A key to identification of feather grasses (Stipa) in Poland