3.2.1. Anatomy

Leaf anatomy of each of the three taxa was characterized by the number of vascular bundles of 7–9 (Figure 6). The adaxial leaf surface was covered by trichomes. The position of the sclerenchyma band was annular at the three species. The only difference we found was that one to three indenting epidermal cells interrupted the sclerenchyma ring in the leaf of *F. tomanii* at both sides near the middle vascular bundle.

**Figure 6.** SEM pictures of the typical leaf cross sections. (**A**) *F. vaginata* (**B**) *F. pseudovaginata* (**C**) *F. tomanii*. White arrows show the interruptions of the continuous sclerenchyma ring at *F. tomanii*. The line represents 100 μm.

#### 3.2.2. Phytoliths of the Leaves

Approximately 500–1000 phytolith microphotos were taken of every species with a total of 9000 (3000 per species) classified silica bodies in them (Figure 7). Several small pieces of silicified tissue were found because the cells adhered to each other sufficiently during the extraction process. Five phytolith morphotypes were counted including (following the ICPN 2.0) grass silica short-cell phytoliths (GSSCP, RONDELS), epidermal long cells with different ornaments (ELONGATE ENTIRE, ELONGATE SINUATE, ELONGATE DENTATE) and silicified trichomes (ACUTE BULBOSUS) (Table 1). There were no significant differences found in the frequency of the GSSCPs and trichomes between the species. The frequency of the ELONGATE cells was bigger at *F. pseudovaginata* and *F. tomanii* than at *F. vaginata* but the differences are not considerable. However, the distribution of the ELONGATE morphotypes represented some differences. Most of the ELONGATE phytoliths of *F. vaginata* were ELONGATE ENTIRE morphotype (81.2%) but most of the ELONGATE phytoliths of *F. pseudovaginata* and *F. tomanii* belonged to the ELONGATE SINUATE morphotype (Table 1). As a result of the hierarchical cluster analysis (Figure 8), *F. pseudovaginata* and *F. tomanii* were close to each other based on their phytolith assemblages.

**Figure 7.** Light microscopic pictures of phytoliths. (**A**) ELONGATE DENTATE (**B**) ELONGATE ENTIRE (**A**,**B**) Silicified epidermal long cells) (**C**) RONDEL (silicified epidermal short cell), (**D**) epidermal tissue fragments with short cells and ELONGATE SINUATE phytoliths in them. (v) *F. vaginata* (p) *F. pseudovaginata* (h) *F. tomanii*. The line represents 20 μm.

**Table 1.** Frequency (%) of phytolith morphotypes in the leaves of *Festuca* species (sum of all the phytoliths is 100%). Frequency of the ELONGATE morphotypes means the percentage of the amount of ELONGATE cells (sum of the ELONGATE cells is 100%). GSSCP = grass silica short-cell phytolith.


**Figure 8.** Hierarchical cluster dendrogram showing *Festuca* species grouping based on their phytolith assemblages (single linkage, Euclidean distance). V: *F. vaginata*, P: *F. pseudovaginata*, T: *F. tomanii.*

#### 3.2.3. Micromorphological Characters of the Epidermis

Because *F. tomanii* leaves have silver coloration that can help to identify this taxon, differences in the micromorphological features of the abaxial leaf epidermis were expected to be found. The abaxial, dorsal epidermis of the leaves of *F. vaginata* was smooth, with short cells and the stomata submerged (Figure 9). Only a few short trichomes (20–30 μm) were found in the abaxial epidermis of the *F. pseudovaginata* leaves, sparsely at the leaf margins. However, the abaxial epidermis of the *F. tomanii* leaves had longer trichomes (with 30–100 μm) occurring more frequently (Figure 9).

**Figure 9.** SEM pictures of the abaxial leaf surfaces: (**A**) *F. vaginata* (**B**) *F. pseudovaginata* (**C**) *F. tomanii*. Embedded figures are light microscopic pictures of phytoliths: EE ELONGATE ENTIRE phytoliths typical for *F. vaginata*, ES ELONGATE SINUATE phytoliths typical for *F. pseudovaginata* and *F. tomanii*, t trichomes typical for the abaxial surfaces of leaves at two latest species. The line represents 100 μm.

Moreover, silicified long cells were found in the abaxial surfaces of *F. tomanii* leaves under a stereomicroscope but there were no similar silicified cells in the epidermis of *F. vaginata* or *F. pseudovaginata* (Figure 10). EDX measurements supported the higher silicon content of *F. tomanii* leaves. The mean Si atom % values of this leaf surface were the following: at *F. vaginata* 3.65 atom %, *F. pseudovaginata* 3.50 atom % and at *F. tomanii* 14.2 atom %.

**Figure 10.** Stereomicroscopic pictures of the abaxial leaf surfaces: (**A**) *F. vaginata* (**B**) *F. pseudovaginata* (**C**) *F. tomanii*, with the EDX element spectrum diagrams near them. SLC: silicified long cells in the epidermis of *F. tomanii*. The line represents 50 μm.

#### *3.3. Determination of Ploidy Level*

The ploidy levels of *Festuca* spp. were determined using flow cytometric analyses. The measurements revealed the differences of DNA content among the tested samples (Figure 11). The relative DNA content was two times higher in samples of *Festuca pseudovaginata* (Figure 11B) and *Festuca tomanii* (Figure 11C) than in the sample of the diploid *Festuca vaginata* species (Figure 11A).

**Figure 11.** Flow cytometric analyses of *Festuca* spp.: histograms demonstrate the relative DNA content of (**A**) *Festuca vaginata*, (**B**) *Festuca pseudovaginata* and (**C**) *Festuca tomanii*.

## **4. Discussion**

One of the most important identification keys to these *Festuca* taxa was the length of the awn of the lemma. Awns of *F. vaginata* were missing or very short (0.2–0.4 mm), confirming the findings of [25,26] and [34]. Awn of the lemma of *F. pseudovaginata* is longer (1.2–1.8 mm) according to [76]. Awn of the lemma of *F. tomanii* is significantly longer than 2 mm [77], which was confirmed by the present study results.

Among the inflorescence parameters there were morphological markers that were not suitable for distinguishing the three species examined, such as the length of the generative stem, floral number of spikelet, length of upper glume and hairiness. Beyond these marks, *F. vaginata* was distinguished by the other parameters studied, including the longer inflorescence branch and the significantly longer lower inflorescence branch. The spikelet was the shortest of the three taxa examined. According to the spikelet, *F. pseudovaginata* and *F. tomanii* can be distinquished. The studies in [78,79] highlighted this fact. According to [78,79], samples must be taken from one particular point of the panicle. The present data confirmed [78,79] finding that a spikelet at a given position should be examined. In the case of F. goals and no hypotheses, the fourth spikelet at the lower inflorescence branch was longer than the fourth spikelet at the apex of the inflorescence, which is also a good distinguishing morphological parameter of identification.

The distribution of the different phytolith morphotypes did not answer the question of what caused the silver coloration of the *F. tomanii* leaves. There were no more ELONGATE phytoliths or trichomes in the *F. tomanii* leaves than in the two other species but the phytolith analysis highlighted the differences between the micromorphological features of the abaxial epidermis surfaces of the studied *Festuca* leaves, namely that most of the ELONGATE phytoliths of *F. vaginata* were the ELONGATE ENTIRE morphotype but most of the ELONGATE phytoliths of *F. pseudovaginata* and *F. tomanii* belonged to the ELONGATE SINUATE morphotype. This finding supports the results of the inflorescence data analysis of these species and confirm the usefulness of the quantitative phytoliths analysis for revealing a new taxonomic character to distinguish different species of a grass genus [77]. As all the three taxa had trichomes in the adaxial surfaces of their leaves, we could not find considerable differences among the species concerning the number of the trichomes in the phytolith assemblages.

However, it was a reliable parameter in their identification key that the abaxial surfaces of the *F. tomanii* leaves bore trichomes, which may be the reason for the silvery epidermis. It is not clear why the silicified long epidermal cells observed under a stereo microscope were not represented in the phytolith assemblages of *F. tomanii* in larger numbers. On the other hand, the EDX measurements proved the high silica content of the abaxial epidermis of *F. tomanii* leaves, with more than triple Si atom % value.

Based on the length of the spikelet, the size of *F. tomanii,* the individuals in the middle of the Kiskunság were smaller, probably due to the adaptation to the drier and warmer habitat [80,81] Another strategy to adapt to it is the more intensive silica accumulation [82–85], which is also a characteristic feature of *F. tomanii*. As this taxon has special morphological and anatomical characters according to its distinct area and habitat.

Based on our results, we confirmed the appearance of *F. vaginata* in natural grasslands and discovered new occurrences of *F. pseudovaginata* and *F. tomanii*. *F. pseudovaginata* inhabits only the Pannon region; we found endemic and natural stands of it, but in its secondary habitats it was confirmed as a completely new species. Furthermore, taxa of disturbed vegetations are currently being examined. These habitats are potential hotspots of speciation.

On bare soil surfaces of areas exposed to anthropogenous effects, two species of the genus *Festuca* became dominant.
