**4. Discussion**

#### *4.1. Distribution and Habitat of V. dingleri*

The results of this study indicate that the geographical distribution of the species *V. dingleri* lies in the Mediterranean area, at a low altitude of 100–200 m asl, on, generally, south-faced, very rocky slopes of highly degraded, overgrazed areas, in low hill sites. The climate of its habitat belongs to the Csa type, with a mean annual precipitation of 602 mm and a mean annual temperature of 14.6 ◦C. The species habitat is characterized by very shallow, alkaline soils, rich in organic matter, nitrogen, phosphorus, and potassium. The area consists of a grazing area, close to villages and century-old agricultural land. This means that the species habitat is not an isolated area, but the species co-exists with traditional anthropogenic activities.

From a floristic point of view, the species appears in a specific degraded shrub community where the dominant floristic elements, such as the species *P. spina-christi*, *O. europea ssp. europaea*, and *Q. coccifera*, demonstrate the presence of grazing for a long time. A quite rich herb stratum also appears, consisting of the species *Eu. dendroides*, *V. dingleri*, *P. bellardii*, and many species common in open grazing areas, such as *A. barbata*, *A. sphaerocephalon*, *C. bursa-pastoris*, and *B. tectorum*, which indicates species preference for open grazing, degraded slope areas. According to the inventory data, the species appears locally in a very limited area. Few individuals were recorded in the two years of this study, less than the limit of 500 that is currently considered the minimum number (effective population size) of individuals necessary to secure a species population genetic variability [28,29]. This creates strong uncertainty for the species future perspectives. Such low-sized populations are extremely vulnerable to extinction; however, the available data show that the species has persisted in the area for almost a century (since 1926) and grows between the cities of Kavala and Xanthi, northern Greece, just above the plate agricultural land at the foothills of the mountains, on low-altitude, rocky slopes and very shallow soils. Any specific explanation for this very restricted distribution of the species is still unknown. Probably, this may depend on species regeneration ecology and specific requirements for its propagation in the field. This species' restricted occurrence in a single well-defined area within a small part of the Mediterranean region is a characteristic element of the Mediterranean endemism [30]. Compared with *Verbascum pseudonobile*, another range-restricted *Verbascum* species, which, according to the GBIF, has been recorded in 14 localities in northern Greece, around 41.1◦ N, 23.624.8◦ E, it seems that *V. dingleri* is specialized to grow in drier habitats with mild winters, close to the sea (Aegean Sea), while *V. pseudonobile* thrives in colder habitats with a more continental climate. Thus, the species distributions are not overlapping.

#### *4.2. Species Regeneration Ecology*

The species fruit contained a quite high number (58.2 ± 3.37) of minute normal seeds. According to the germination experiments, no indication of seed dormancy was observed. Seeds quite highly germinated (up to 80.0%), depending on the fruit, at 20 ◦C, like many other species of the genus *Verbascum* [21,22]. In addition, the seed germination rate of *V. dingleri* in the open-air nursery, with climatic conditions similar to the natural ones, depended on the fruit from which the seeds were extracted. The seed of some fruits showed a germination of 30%, while the seeds of other fruits did not germinate. The seeds germinated and produced seedlings after approximately 30 days from planting in the middle of spring (April 2017), while their growth was slow and very similar for all produced plants.

## *4.3. Species Conservation*

Both laboratory and nursery results analysis revealed that the seeds of *V. dingleri* do not present dormancy and germinate under favorable environmental conditions (for the species). The seeds of the species showed an average percentage of 30–40% germination at 20 ◦C, a temperature common in the species habitat area, especially during spring or autumn, when many species regenerate in the fields in temperate zones and with climate type Csa. Thus, we conclude that the species regeneration in the field

is theoretically feasible at any time after dispersal. However, in natural conditions, a combination of ecological factors such as temperature, light, and water availability play a key role in regulating plant species germination and seedling emergence. Seeds behavior in response to these crucial environmental factors greatly di ffers among species, depending on each species eco-physiological attributes [7,8]. Many studies have proven that the temperature greatly influences seed germination, and almost in all species, high temperatures slow down the germination [3,4,31]. Direct sunlight also plays an important role in seed germination behavior [32] as well as in early-stage survival of many non-drought-resistant plant species. Probably, during summer, a combination of high temperatures and high intensity of direct sunlight may be a crucial factor a ffecting a species regeneration success in the field.

On the other hand, the low amount of precipitation, not only during summer (30–40 mm in the area of the species occurrence, according to the data from the nearest meteorological station of Kavala city), but also during the period of seed dispersion in nature, may be a crucial determining factor for species regeneration. Soil water stress commonly reduces seed germination, since this physiological process is sensitive to water availability, especially during the first stage of germination (swelling of seeds through water adsorption). However, seeds of *V. dingleri* show a tolerance to desiccation, being able to germinate in the presence of a moisture content of approximately 10% (data not shown). Considering that the seeds can survive desiccation, we conclude that *V. dingleri* seeds are orthodox, like the seeds of other species of the genus *Verbascum* [21,22,33]. Thus, in field conditions, it is expected that the tolerance of *V. dingleri* seeds to desiccation could contribute to keep them viable until the time of autumn rains. The findings of the current studies on the seed germination behavior of this species seem not to be able to explain the restricted species occurrence and its narrow endemism. Perhaps, it could be assumed that *V. dingleri* effective regeneration and habitat expansion is determined mainly by seedling propagation ability rather than seed germination in the field. It is worth pointing out that, for the soils of the general area of Kavala city [34,35], some extreme values are recorded for As, Pb, and Zn (which are enriched 7.6, 3.3, and 2.7 times, respectively, in comparison to the values of normal USA soils), even though the majority of the elements in the soils have concentrations within normal ranges. Furthermore, these elements are found at their highest concentrations in the vicinity of the industrial zone of Kavala.

The knowledge of the favorable conditions for early plant growth (germination and seedling emergence) of a rare plant species is definitely necessary for taking the appropriate measures for species conservation and the establishment of population restoration programs [10,36,37]. In the case of endangered species whose habitats are subjected to intensive anthropogenic disturbances, in situ and ex situ species conservation strategies can be suggested, and information about the reintroduction of the species by seedlings produced from seeds collected and e ffectively treated for germination can be provided [38]. Our study demonstrates that in situ and ex situ conservation and reintroduction of *V. dingleri* using seedlings produced from the seeds collected from a natural population is theoretically feasible.

Specifically, our findings demonstrate that ex situ propagation of *V. dingleri* is feasible from seeds, resulting in the conservation of the species diversity. Thus, these findings can contribute to the advancement of artificial seedlings' production that can be used either for ex situ species conservation or for species reintroduction when the natural population is seriously endangered. However, further research is needed to determine the key factors leading to a satisfactory in situ seedling establishment of this species. More knowledge for e ffective seed germination and production of high-quality seedlings is crucial for the conservation of this extremely threatened species [9].

**Author Contributions:** Conceptualization, P.G.; methodology, P.G., M.T., C.D. and A.S.; formal analysis, M.T.; investigation, T.K. (Theodoros Kalapothareas), T.K. (Theodoros Karydopoulos), C.D., A.S. and K.P.; writing—original draft preparation, P.G.; and writing—review and editing, M.T.

**Conflicts of Interest:** The authors declare no conflict of interest.
