Values are mean ± standard errors. EC: electrical conductivity, OM: organic matter. Superscript letters within each row showed significant variation at *p* < 0.05 (Duncan's test). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 at degree of freedom (*df*) for region (n − 1) = 4 and replications (n − 1) = 9.

The correlation between the vegetation composition and soil properties was assessed by canonical correspondence analysis (CCA). The CCA showed that inland sabkhas of the Qareenah region were separated on the upper left side of the CCA biplot and showed a close correlation to CaCO3, Mg, organic matter content, pH, and sulphate (Figure 4). In contrast, the inland sabkha of the Jouf region is segregated in the CCA biplot's lower left side, where they are affected by Ca and Clay. On the other hand, the coastal sabkha of the Jizan region was segregated on the lower right side of the CCA biplot, where it showed a correlation to silt contents. Finally, the inland sabkha of Qaseem and coastal sabkha of Salwa were separated on the central part of the CCA biplot, where they showed a positive correlation with salinity, moisture, K, bicarbonates, Na, and sand (Figure 4).

**Figure 4.** Canonical correspondence analysis (CCA) showing the correlation between the soil variables and dominant and important species representing the studied locations. Aca ger: *Acacia gerrardii*, Ael lag: *Aeluropus lagopoides*, Zyg coc: *Zygophyllum coccineum*, Sua aeg: *Suaeda aegyptiaca*, Zyg alb: *Zygophyllum album*, Tam nil: *T. nilotica*, Jun rig: *Juncus rigidus*, Rha str: *Rhazya stricta*, Phr aus: *Phragmites australis*, Cre cre: *Cressa cretica*, Lyc sha: *Lycium shawii*, Sal eur: *Salicornia europaea*, Pho dac: *Phoenix dactlifera*, Pan rep: *Panicum repens*, Cyp con: *Cyperus conglomeratus*, Aer jav: *Aerva javanica*, Zyg sim: *Zygophyllum simplex*. OM: organic matter, EC: electrical conductivity.

The Pearson's correlation analysis between soil variables and dominant, co-dominant and important species is shown in Figure 5. *A. lagopoides*, the most dominant species of the Qareenah, Qaseem, and Salwa regions, and second most dominant species of the Jouf and Jizan regions, showed a strong positive correlation to all tested soil parameters, except for CaCO3 (r = −0.07) and sand (r = −0.54). Similarly, *S. aegyptiaca* (the most dominant species of the Jizan region and the second most dominant species of the Qaseem region), *J. rigidus*, *L. shawii*, *P. dactylifera*, *P. australis,* and *S. europaea*, revealed a positive correlation for all tested characteristics, except for CaCO3 and sand contents.

**Figure 5.** Pearson's correlation heatmap between the soil variables and the dominant, co-dominant, and important associated plant species within the studied locations. EC: electrical conductivity, OM: organic matter, and MC: moisture content.

However, *T. nilotica*, the most dominant species of the Jouf Region, was negatively correlated with all the soil parameters, except for sand (r = 0.20). *Z. coccineum*, the second most dominant species of the Qareenah region, showed a negative correlation with most of the soil parameters, except for pH (r = 0.35), anions, and sand (r = 0.36). Among the important associated species, *J. rigidus*, *L. shawii*, *P. dactylifera*, *P. australis,* and *S. europaea* showed a strong positive correlation with almost all the soil parameters (Figure 5).

#### **3. Discussion**

Saudi Arabia is located within an arid and semi-arid zone. It is distinguished by its different ecosystems, including mountains, meadows, valleys, rocky and sandy deserts, and salt marshes [11,25]. Among salt marsh ecosystems, there are inland salt marshes and saline coastal habitats, called sabkhas. These sabkhas have scarce vegetation due to severe environmental conditions (wind exposure, high temperature, and high salinity). However, sabkha edges (transition towards sand) are characterized by well-defined zones, and each zone occupies a particular plant community. The salt-tolerant or halophytic plants, which constitute about 2% of the world's flora [20], grow in these habitats. The spatial distribution of plants in these vegetation zones is affected by soil salinity and soil composition. This explained the low plant diversity in the studied sabkha locations.

The floristic analysis of this study revealed the predominance of phanerophytes and chamaephytes, reflecting the domination of perennial halophytes over annuals and ephemerals in saline conditions. The perennial halophytes can tolerate high salt content in soil [33]. The predominance of Poaceae, Chenopodiaceae, and Mimosaceae in the studied habitats was in harmony with other previous studies in different saline habitats [25,34]. On the other hand, most of the recorded species belong to the Saharo-Arabian element. This finding is in harmony with other studies on salt marsh habitats [10].

Based on the data of diversity indexes, the Qareenah location showed the highest richness. This could be ascribed to the water factor, as this location is a wadi system and has a relative amount of water. In our previous work, a total of 111 plant species were recorded within this wadi, even some hydrophytes and ferns are flourished [25]. The diversification of the Qareenah location could be ascribed to the fact that it forms a woodland community

by colonizing various acacia species (*A. gerardii* Benth., *Acacia ehrenbergiana* Hayne, and *Acacia tortilis* (Forssk.) Hayne), along with *T. nilotica* and other xerophytes, such as *L. shawii* and *R. stricta*. Since this habitat is interconnected with wadis, these plants are the common plants in various wadis in desert habitats of Saudi Arabia [35–37].

On the other hand, the Qaseem location showed higher evenness and low richness. This location is a wetland, with a water content of 21.56%, that enables specific species (species that flourished in wetlands, i.e., with high water content) to colonize this habitat with a higher number of individuals. The soil analysis showed that the Qaseem location accomplished the highest moisture, clay, silt, salinity, Ca, Mg, Na, K, Cl, SO4, HCO3, and organic matter. The harsh soil conditions, such as high salinity, can be the factor of low species diversification due to the non-survival of annuals. The inland sabkha of Qaseem forms a brine on the soil, making it difficult for less salt-tolerant plants to grow. Usually, in saline environments, soil factors control plant species' growth and survival rates, thus, affecting vegetation patterns [38]. Soil factors also reduce plants' fecundity and germination ability, thus, shaping plant competition and population fitness [39,40]. Most of the dominant and associated species in this location are typical halophytes, such as *C. cretica*, *J. rigidus*, *L. shawii*, and *S. europaea,* which grow in high salt and wet habitats [39,41–43], thus, determining the vegetation zonation pattern of salt marshes.

The cluster analysis of the studied locations showed that the northern inland plain of Jouf is dissimilar to the other locations. This could be attributed to the environmental factors, where the soils of the Jouf location showed the lowest salinity. In this location, *T. nilotica* was determined as the dominant species. *T. nilotica* usually colonizes *A. lagopoides* patches, where the altitude is greater than 500 m ASL but grows in soil with low salinity [44].

Among all the regions, the southern coastal sabkha of Jizan has the least diverse vegetation. The soils in this location revealed the lowest moisture content, pH, organic matter, K, sulphate, bicarbonate, calcium carbonate. This could explain the low diversity of this location, as well as the presence of xerophytic plants, such as *C. conglomeratus*, *A. javanica*, and *Z. simplex*.

Overall, the vegetation analysis of the studied location revealed the dominance of *A. lagopoides* in all locations, where it was the most dominant species in the Qareenah, Qaseem, and Salwa locations, and second most dominant species in Jouf and Jizan locations. *A. lagopoides* is one of the most important halophytic grasses in Saudi Arabia. It is a salt excretive grass that grows in the form of patches or mats in highly saline and moistened soil, where it is characterized by structural adaptations and modifications [27,45]. It can tolerate the harsh and saline habitat by expelling the salts it gains, and the plant itself has a very low salt content, making it a palatable forage grass [46]. In addition, this grass has small and waxy leaves, as well as a network of roots and underground rhizomes that help the plant to survive in high salty conditions, even in the summer season, where the salinity becomes even higher [47]. According to our field observation, the *A. lagopoides* has phenotypic plasticity, where its morphology is changed from one location to another. This observation could be a way of adapting to the harsh conditions.

Within the studied locations, other halophytic plants (highly salt-tolerant species) were determined with high abundances, such as *S. aegyptiaca*, *Z. album*, *T. nilotica*, *S. aegyptiaca*, *C. cretica*, and *S. europaea*. These species flourish in saline habitats, where moisture and salinity shaped the community structure [25,48].

#### **4. Materials and Methods**

#### *4.1. Study Area*

The study was carried out from 2020 through 2021 around the entirety of Saudi Arabia to explore the vegetation zones of the halophyte *A. lagopoides* (Figure 6). *A. lagopoides* mosaic vegetation has been shaped in different eco-regions of Saudi Arabia based on soil properties of the habitat and the morphological adaptation of the indicator plant via phenotypic plasticity.

**Figure 6.** The populations of *Aeluropus lagopoides* (L.) Thwaites in different locations of Saudi Arabia (**a**–**c**), and different morphological growth forms (**d**–**i**). AP: *Aeluropus* patch, FMA: full mature *Aeluropus* patch, Cu: culm, GI: globose inflorescence, St: stolon.

We monitored this grass in five sabkha regions (Figure 7) of Saudi Arabia which represents both coastal and inland sabkhas as follows:


**Figure 7.** Map of Saudi Arabia showing the different locations of sampled *Aeluropus lagopoides* communities. Qareenah (Qar), Qaseem (Qas), Salwa (Sal), Jouf (Jou), and Jizan (Jiz).

The climate of Saudi Arabia is dry-hot and is classified as an arid region occupying about 5% of the world's arid zones [49]. It has low relative humidity except along the coastal zones, where it sometimes reaches 100%. The mean annual temperature is 33 ◦C during summer and 14 ◦C in winter, with a wide seasonal and diurnal variation [50]. The mean solar radiation was recorded highest for July and August and lowest for December and January except in Jizan. However, the rate of pan-evaporation was minimal along the coastal and high mountainous terrain and maximal in the interior due to the maximum presence of desert conditions. High rainfall variations and long drought periods have been recorded between the years without any rain. The climate data were collected between 1999 and 2019 from https://en.climate-data.org/asia/saudi-arabia-29/ (accessed on 15 May 2021) (Figure S2).

#### *4.2. Vegetative Sampling*

The vegetation sampling for each location was conducted from September to March when *A. lagopoides* was fully growing in each location and was in the full maturity stage. For each location and depending on species growth and form of vegetation composition of the studied area, 10 quadrats were selected randomly from each region (Table 3). The nested quadrat's area was 10 m × 10 m for shrubs and 5 m × 5 m for small shrubs and herbaceous species. These quadrats were chosen within the sabkha area dominated by patches of *A. lagopoides* population. The species in each quadrat were identified and named according to Chaudhary [35] and Miller et al. [51], as well as following the website http://www.powo.org (accessed on 1 February 2022). The plant density was determined according to Bonham [52], while the plant cover was estimated based on the scale of Braun-Blanquet [53]. To assess species dominance in each location, species importance value was calculated by the summation of relative density and relative cover of each species. The species life forms were identified according to Raunkiaer [54], while the chorotypes of all the species were made to assess the recorded species to World Geographical Groups.


**Table 3.** The coordinates and elevation of the different sample quadrats of different locations, Saudi Arabia.

#### *4.3. Soil Sampling and Analysis*

From each quadrat (n = 10) where vegetative sampling was conducted, three soil samples (0–30 cm depth) were collected from three random positions in plastic bags and pooled as a composite sample. All the soil samples were duly labeled and transferred to Range Science Lab, College of Food Science and Agriculture, King Saud University, Riyadh,

Saudi Arabia for further analyses. In addition, a portion of each sample was collected in moisture tins for the determination of soil moisture content by the weight-loss method. The soil samples were spread over separate plastic sheets, air-dried at room temperature, filtered through a 2 mm sieve to remove any debris, if present, and stored in a plastic bag until further analyses. Soil texture for sand, silt, and clay fractions were analyzed by the hydrometer method [55]. Soil organic matter (OM) was determined by wet combustion with dichromate at 450 ◦C [56]. Soil water extracts (1:5) were prepared for the estimation of soil electrical conductivity (EC) and pH [56]. Soluble inions (Cl and SO4) were determined by titration method, while the determination of soluble cations (Ca, Mg, Na, and K), using a flame photometer according to Rhoades [57].

#### *4.4. Data Analysis*

In this study, multivariate analysis was applied viz. classification and ordination. Based on the data of relative density and cover of all species inside the quadrats (n = 10) of each region, a matrix of species importance values (relative density + relative cover) was constructed and subjected to hierarchical cluster analysis for classification and detrended correspondence analysis (DCA) for ordination using PAST 4.03 software [58]. Species rarefaction for base species richness and abundance in all the studied regions was analyzed using PAST 3X. The soil variables for the studied regions were subjected to one-way ANOVA and the mean values were separated based on Duncan's test at 0.05 probability level to examine the significant difference among studied regions. In order to detect the relationship between plants (dominant and important plant species with high importance values) of the studied area on one hand and soil variable data, on the other hand, canonical correspondence analysis (CCA) was conducted using MultiVariate Statistical Package (MVSP Version 3.2, Kovach Computing Services, Pentraeth, Wales, UK) according to Ter Braak and Smilauer [59]. In the CCA analysis, two datasets were constructed; one regarding the importance values of the dominant and important species (performed like that for DCA) and the second of the soil parameters of the quadrats (n = 10) of each region. Also, Pearson's correlation heatmap between the soil variables and the dominant and important species was performed using the XLSTAT software program (version 2018, Addinsoft, NY, USA).

#### **5. Conclusions**

The present study revealed variance among the community structure of *A. lagopoides,* within different sabkhas in Saudi Arabia. The community of inland sabkhas (Qareenah and Qaseem locations) showed higher plant diversity compared to the coastal sabkhas. The plant diversity of the *A. lagopoides* communities is mainly shaped by the salinity and water content. Moreover, the survival and flourishment of the halophytic grass *A. lagopoides* within a wide soil range in sabkhas revealed the adaptability of this plant to the harsh environment, which could be ascribed to its structural adaptations and modifications, as well as the phenotypic plasticity. Since *A. lagopoides* has many economic potentialities, where it is utilized as fodder, stabilizes sand dunes, used for landscaping of urban areas, the conservation of these natural vegetation zone habitats is of vital importance. Also, this valuable plant could be integrated as a promising forage candidate in saline-affected areas, even in the summer dry season.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11050666/s1, Table S1: Floristic analysis of the recorded plant species in the studied sabkha locations of Saudi Arabia, Table S2: Vegetation composition of studied locations dominated with *A. lagopoides* in Saudi Arabia, Figure S1: Species richness and abundance based on the relative density of all the studies regions, Figure S2: Monthly climate data of the surveyed regions.

**Author Contributions:** Conceptualization, A.M.A. and A.A.A.-D.; software, A.M.A.-E.; formal analysis, A.M.A.-E., B.A.D. and A.M.A.; investigation, A.M.A., A.M.A.-E., S.L.A.-R., B.A.D. and A.A.A.-D.; writing—original draft preparation, A.M.A.-E., A.M.A. and B.A.D.; writing—review and editing, A.M.A., A.M.A.-E., S.L.A.-R., B.A.D. and A.A.A.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** Deanship of Scientific Research at King Saud University supported this work through research group no. RG-1441-302.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for supporting this work through research group no. RG-1441-302.

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

#### **References**


**Iva Apostolova 1,\*, Desislava Sopotlieva 1, Magdalena Valcheva 1, Anna Ganeva 1, Veselin Shivarov 1, Nikolay Velev 1, Kiril Vassilev 1, Tsvetelina Terziyska <sup>1</sup> and Georgi Nekhrizov <sup>2</sup>**


**Abstract:** This work represents the first study of the floristic diversity on Bulgaria's ancient mounds. The objective of this research was to assess the importance of the mounds for the preservation of the native vascular and cryptogam flora. Our sampling design included 111 ancient mounds distributed throughout the country. We recorded a total of 1059 vascular plants, 58 bryophytes and 61 lichen taxa. Despite their small area, the mounds were shown to preserve nearly a quarter of the Bulgarian flora. The vegetation cover on the mounds included 61% perennials indicating a long-term persistence and stability. The majority (98%) of the established vascular plants were native species. Although the conservation significance of the vascular plant species were not common, we recorded 2 critically endangered, 9 endangered and 14 Balkan endemics during the present study. The lichen *Arthopyrenia salicis* was recorded for the first time in Bulgaria and a new locality of the rare bryophyte *Ceratodon conicus* was discovered. The established compositional difference between plots from the northern and southern slopes of the mounds (88.95%) is a testament to the high local habitat diversity. The prevalence of species characteristic for *Festuco-Brometea* suggests that the mounds preserve fragments of native grasslands and steppes. The variation in cover of agricultural and other human modified areas in the mounds' immediate surroundings did not substantially affect their species richness. We argue that the ancient mounds should be taken into consideration in future green space planning.

**Keywords:** bryophytes; generalist plants; grassland specialists; historical monuments; invasive alien plants; kurgans; lichens; native plants

#### **1. Introduction**

Ancient mounds (also called tumuli or more commonly kurgans) were constructed in temperate Eurasia between 4th millennium BC and 4th century AD and used primarily for burial purposes. A remarkable number of these mounds have been preserved due to their spiritual and cultural importance [1–4]. Bulgaria is exceptionally rich in ancient mounds with a known number of approximately 50,000 [5]; 11,000 of these mounds have been registered in the Archaeological Map of Bulgaria (http://www.naim-bas.com/akb/ accessed on 3 November 2021). Some of Bulgaria's ancient mounds are remarkable historical monuments, including massive underground stone buildings often decorated with wall paintings (e.g., Kazanlak and Aleksandrovo tombs). The most attractive of these structures are important tourist destinations open to the public. At present, most of Bulgaria's ancient mounds are surrounded by vast agricultural lands [4]. Similar to ancient mounds in other European countries, these structures are often standing as sole "islands" of semi-natural vegetation in an otherwise human-modified landscape [3,6,7]. Along with field margins, road verges and buffer strips adjacent to arable land, the ancient mounds preserve small

**Citation:** Apostolova, I.; Sopotlieva, D.; Valcheva, M.; Ganeva, A.; Shivarov, V.; Velev, N.; Vassilev, K.; Terziyska, T.; Nekhrizov, G. First Survey of the Vascular and Cryptogam Flora on Bulgaria's Ancient Mounds. *Plants* **2022**, *11*, 705. https://doi.org/10.3390/ plants11050705

Academic Editor: Robert Philipp Wagensommer

Received: 11 February 2022 Accepted: 4 March 2022 Published: 6 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

semi-natural fragments and provide an opportunity for the long-term survival of indigenous flora. Moreover, ancient mounds consist of different microhabitats, which enrich the suitability for the development of an ecologically diverse flora [8,9]. The long-term persistence of the mounds within agricultural lands, primarily due to sacred and religious respect, naturally makes them a part of the Green and Blue Infrastructure defined at the European level as a "strategically planned network of natural and semi-natural areas with other environmental features designed and managed to deliver a wide range of ecosystem services" [10].

Recently, there has been an increased interest in burial mounds as biodiversity hotspots situated in an otherwise homogenous agricultural landscape [2,3,7,11–15]. Plants, and especially flowering plants (Angiosperms), are one of four groups of living organisms (along with Heteroptera, Symphyta and aculeate Hymenoptera) that have been shown to be best served for the biodiversity evaluation of cultivated areas [16]. Recent research has shown that ancient mounds preserve a remarkable plant diversity [3,11,13,15,17,18]. To date, no studies on the natural value of Bulgaria's ancient mounds have been conducted and no records of their floristic diversity are known to exist. At the beginning of the current research, we assumed that the cultural significance and principal sacrosanct nature of Bulgaria's ancient mounds, akin to other countries, provided long-term repository conditions for natural communities and that they served as refugia for indigenous flora in anthropogenically transformed areas. Considering cryptograms' signal for increased degree of community stability and naturalness [19], and in order to enrich the current biodiversity assessment, we included cryptograms (bryophytes and lichens) along with vascular plants in this survey.

The objectives of this study were (1) to collect completely novel information about the floristic diversity of Bulgarian ancient mounds and (2) to assess the potential of ancient mounds to preserve native vascular and cryptogam flora, despite being largely isolated.

#### **2. Materials and Methods**

#### *2.1. Study Objects and Study Area*

We used the Archaeological Map of Bulgaria (http://www.naim-bas.com/akb/ accessed on 3 December 2021) to select the study objects. Our selection criteria included mounds that were (1) undisturbed by archaeological investigation, (2) were clearly recognizable, and (3) were higher than 1 m and more than 9 m in diameter (Table 1). We visited a total of 111 ancient mounds spread out across the territory of Bulgaria (Figures 1 and 2). The mounds were located in the lowlands and hilly plains of the country between 60 and 900 m a.s.l. In our study, a larger mound base usually corresponded to a larger height. The correlation of height to diameter was *r* = 0.57518 (*p* < 0.05) and the correlation of height to 2D area (calculated as *πd*2/4, where "*d*" is diameter) was *r* = 0.61815 (*p* < 0.05). Therefore, we used the 2D area of the mounds as a representation of the relative mound size.

**Table 1.** Basic topographic parameters of the studied mounds (n = 111).


**Figure 1.** Map of Bulgaria with marked locations of the sampled mounds.

**Figure 2.** An example of a typical mound view (photo credit: I. Apostolova).

We visually estimated the percentage of grassland and woody vegetation cover of the mounds using Google Earth images. We chose a 10% threshold to facilitate the rough estimation in vegetation cover of the mounds. There were 86 mounds with more than 70% herbaceous cover, 16 with more than 70% forest vegetation cover and 9 with mixed vegetation cover.

In order to estimate the degree of mound isolation, we created a buffer area with a radius of 200 m around the base of each mound and calculated the land cover of natural vs. non-natural habitats within. The land cover types in the buffer area were obtained from the Land Parcel Identification System (LPIS) database maintained by the Ministry of Agriculture, Food and Forestry of Bulgaria and generalized as semi-natural vegetation and agricultural and other anthropogenic lands (for details see [4]). More than half of the studied mounds were highly isolated—80 mounds were surrounded by more than 70% of agricultural and other anthropogenic lands.

The investigated mounds fall within the temperate and the continental–Mediterranean climatic zones. The temperate zone, which incorporates the northern parts of the country, has an average mean annual temperature of 11.9 ◦C and annual precipitation of 573 mm (town of Pleven, 1971–2000), while the continental–Mediterranean zone, which is more typical for the southern part of the country, has an average mean annual temperature of 12 ◦C and annual precipitation of 637 mm (town of Haskovo, 1971–2000) [20]. A major part of the study area falls within the broadleaved deciduous forests zone (Map of Natural Vegetation of Europe, [21]), while the southern areas include Mediterranean vegetation fragments with typical plant species [22]. Some small areas in the north-east are influenced by steppe vegetation [23].

#### *2.2. Sampling Design, Data Collection and Data Analysis*

The field work was conducted during the maximum period for vegetation development (June and July) in 2019 and 2020. Our sampling design was focused on the major ecological differences exhibited at the northern and southern slopes of the mounds. This design considered findings reported by previous studies on burial mounds [9] regarding the difference in floristic composition at different exposures due to habitat heterogeneity. In order to obtain more detailed floristic data, we first sampled all species within 5 × 5 m plots situated on the northern and southern slopes (two plots per mound). We then carefully explored the remaining mound area and recorded any additional species until the floristic variety was exhausted. We used presence/absence species data, both at plot and at mound level, for our analyses.

Despite the fact that some taxa were identified to subspecies, we set the final plant list to species level. Some closely related species were joined in species aggregates (*Achillea millefolium* aggr.–including *A. millefolium*, *A. pannonica* and *A. setacea*) or determined at aggregate level (*Rubus hirtus* aggr.). Plants that were in a phenological stage unsuitable for correct species determination, or that were difficult to identify (e.g., *Taraxacum* spp.), were determined to genus level. The vascular plant species nomenclature follows The Euro+Med PlantBase [24], with the exception of *Brassica juncea*, which follows the Plant List [25]. Bryophyte nomenclature follows Hill et al. [26], and lichen nomenclature follows Nimis et al. [27].

For each taxon, we attributed a set of characteristics regarding biological type, functional role, floristic element, conservation and native status (Supplementary ESM S1). Data regarding the biological type of vascular plants were extracted from national literature sources [28,29]. The biological types were grouped as follows: short lived (including annual and biannual plants), perennial (including biannual to perennial and perennial plants), dwarf-shrub, shrub and tree. The association of vascular plant species to higher rank syntaxa was defined following Mucina et al. [30]. In cases when more than a single phytosociological class was proposed for a certain diagnostic species, we selected the best representative for the country's vegetation based on our expertise. The diagnostic role of a species was used to assign each species to one of the following 3 functional groups: generalists, grassland specialists and forest specialists. We considered species to be generalists if they were diagnostic of synanthropic vegetation or if they had a broad distribution across different habitat types. The functional affiliation of species that were not assigned to a specific syntaxon was determined based on their most common habitat occurrence in the country. The determination of phytogeographical (floristic) elements for vascular plants follows Assyov and Petrova [31], for bryophytes—Ganeva and Düll [32] and for lichens—Wirth [33] and Nimis [34].

The native status of vascular plants was retrieved from Euro+Med PlantBase [24] because of the lack of such data for Bulgaria. Only species listed by Petrova et al. [35] were recognized as invasive alien plants. Vascular plants with conservation importance included Balkan and Bulgarian endemics ([31,36] complemented by The Euro+Med PlantBase [24]), Bulgarian red list species [37], species protected by the Bulgarian legislation (Appendix 3 of the Bulgarian Biological Diversity Act [38]) and other European and international documents (e.g., Council Directive 92/43/EEC [39], as well as the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [40]). The above mentioned attributes were not applicable for the taxa determined to genus level (N/A). The records of these species were not included in the analyses based on functional groups.

We calculated basic descriptive statistics for all biological characteristics of the registered vascular plants. We used similarity percentages analysis (SIMPER) [41] in PRIMER 7 [42] to determine the species that contributed the most to the floristic resemblances between mounds. The difference in species composition between the mounds was assessed by using the beta diversity index in PAST [43]. We used correlation analyses with Pearson correlation coefficient in STATISTICA 13 [44] to test for correlation between floristic richness (total vascular plant richness, richness of generalists and of grassland and forest specialists) and two other variables: 2D area and proportion of anthropogenic land in the buffer areas. We used plot level data to graphically express the differences in species richness of the different species groups between plots with northern and plots with southern exposure (by their mean values and standard deviation), also carried out in STATISTICA 13 [44].

#### **3. Results**

#### *3.1. Diversity and Species Characteristics*

#### 3.1.1. Vascular Plants

The list of registered vascular plants includes 1059 taxa (Supplementary ESM S1). The average number of species per mound was 69.9 ± 22.6 SD (min 27, max 152). We identified 971 plants to species level, accepted 3 taxa as aggregates or species groups, and identified 85 taxa to genus level. The floristic diversity was confined to 82 vascular plant families. Flowering plants (Angiosperms) made up the majority of the observed species diversity and only seven species belonged to other groups: one horsetail—*Equisetum hyemale*, two ferns—*Polystichum aculeatum*, *Pteridium aquilinum*, and four Gymnosperms—*Juniperus communis*, *J. oxycedrus*, *Pinus nigra*, *P. sylvestris*. There were 23 families represented by more than 10 taxa, the most species rich of these were Asteraceae—126 taxa (11.9% of the established taxa), Fabaceae—111 taxa (10.5%), Poaceae—106 taxa (10%), Lamiaceae—61 taxa (5.8%), Brassicaceae—58 taxa (5.5%), Caryophyllaceae—57 taxa (5.4%), Rosaceae—48 taxa (4.5%), Apiaceae—46 taxa (4.3%), Boraginaceae—31 taxa (2.9%) and Plantaginaceae—31 taxa (2.9%). Twenty-three other families (28.1%) were represented by a single species. The flora of the studied mounds was composed primarily of perennial herbaceous plants (61%), followed by short-lived plants, and a low number of shrubs and trees. Generalists and grassland specialists dominated the species composition of the mounds. A major part (98%) of the established vascular plants consisted of native species (Table 2). Only 21 plants belonged to other categories (alien (status unknown)—13, naturalized alien—5, in large-scale cultivation—2 and doubtfully native—1). Invasive alien plants included *Acer negundo*, *Ailanthus altissima*, *Amaranthus albus*, *Conyza canadensis*, *Cuscuta campestris*, *Datura stramonium*, *Erigeron annuus*, *Phytolacca americana*, *Robinia pseudoacacia*, *Sorghum halepense*, *Xanthium orientale* subsp. *italicum* and *X. strumarium*. These taxa represent 20% of all plants included in the list of invasive or potentially invasive alien plants in Bulgaria and three of them (*Acer negundo*, *Ailanthus altissima* and *Robinia pseudoacacia*) are among the "top 10" invasive alien plants in Bulgaria. The largest number of invasive plant species we registered on a single mound was four. The mound (ID 586) was situated in the central part of north Bulgaria (north of the town of Veliko Tarnovo). A substantial number of the studied mounds (40 or 35.4%) had at least one invasive alien plant.


**Table 2.** Biological characteristics of the registered vascular plants. Total number and descriptive statistics are given.

Critically endangered species present on the mounds included *Anchusa stylosa* and *Limonium asterotrichum*. Endangered plants present on the mounds included *Astragalus haarbachii*, *A*. *wilmottianus*, *Chamaecytisus frivaldszkyanus*, *C*. *kovacevii*, *Dianthus pallidiflorus*, *Erysimum cheiranthoides*, *Festuca thracica*, *Goniolimon besseranum* and *Jurinea ledebourii*. Fourteen of the registered taxa were Balkan endemics: *Achillea clypeolata*, *A*. *pseudopectinata*, *Armeria rumelica*, *Astragalus wilmottianus*, *Asyneuma anthericoides*, *Cytisus eriocarpus*, *Dianthus moesiacus*, *Festuca thracica*, *Heptaptera triquetra*, *Koeleria simonkaii*, *Dichoropetalum vittijugum*, *Polygala supina* subsp. *rhodopea* (syn. *P*. *rhodopaea* (Velen.) Janch.), *Scabiosa triniifolia* and *Thymus longidentatus*. Eleven plants were included in the CITES Convention. A mound near the town of Radomir (ID 185) contained the richest number of plants of conservation interest and maintained populations of 5 such species.

According to our SIMPER analyses, species composition similarity across plots with northern exposure was 13.25% and was 11.84% across plots with southern exposure. We found a remarkable dissimilarity between plots with northern and southern exposure— 88.95%. We registered 142 species unique to the plots with southern exposure. Species registered in more than three plots on southern slopes were *Camelina sativa*, *Heliotropium europaeum*, *Sedum hispanicum*, *Senecio leucanthemifolius*, *Valerianella dentata*, *Verbascum ovalifolium*, *V. densiflorum*, *Tribulus terrestris*, *Crepis sancta*, *Haplophyllum suaveolens*, *Herniaria incana*, *Thymelaea passerina* and *Filago arvensis*. Another 152 species were confined to plots with northern exposure. Taxa that exhibited higher frequency on the plots with northern exposure included *Astragalus glycyphylloides*, *Ficaria verna*, *Carlina vulgaris*, *Helleborus odorus*, *Leucanthemum vulgare*, *Phlomis tuberosa*, *Ranunculus polyanthemos*, *Anthoxanthum odoratum*,

*Helictochloa compressa*, *Quercus frainetto* and *Luzula campestris*. The mean species richness of grassland and forest specialists and generalists was similar between plots with northern and plots with southern exposure (Figure 3).

**Figure 3.** Differences in mean species richness (columns) and standard deviations (whiskers) of different species groups (A—all plant species, G—grassland specialists, F—forest specialists and N—generalists) between plots with northern (N) and plots with southern (S) exposure on the studied mounds.

Average dissimilarity between all pairs of mounds was 0.78 (Whittaker measure for beta diversity). Fifteen species were registered on more than 50% of the studied objects and included *Poa angustifolia* (77% of the studied mounds), *Achillea millefolium* aggr. (72.6%), *Eryngium campestre* (67.3%), *Galium verum* (64.6%), *Teucrium chamaedrys* (61.1%), *Tragopogon dubius* (60.2%), *Sanguisorba minor* (54%) and *Botriochloa ischaemum* (50.4%). According to our SIMPER analyses, these taxa along with *Prunus spinosa*, *Lactuca serriola*, *Dactylis glomerata*, *Falcaria vulgaris*, *Convolvulus arvensis*, *Crataegus monogyna*, *Elytrigia repens*, *Galium aparine* and *Potentilla recta* contributed the most to the floristic similarity between mounds. On the other hand, 480 species (45.3% of the total flora) were registered from one or two mounds. We found no relationship between the 2D area of the mounds and their floristic richness. With the enlargement of the 2D area of the mounds, the richness of forest specialists slightly increased (*r* = 0.28, *p* < 0.05). More than a half of the studied mounds (69) were highly isolated and surrounded by an agricultural matrix above the average in the buffer (Table 1). As the anthropogenically transformed lands around the mounds increased, the total number of registered species (*r* = −0.28, *p* < 0.05) as well as the total number of specialists (*r* = −0.34, *p* < 0.05), including grassland specialists (*r* = −0.33, *p* < 0.05), decreased.

The phytogeographical spectrum of the registered plants resembled that of the national flora (Figure 4). The number of species with Mediterranean distribution was the highest, followed by the plants with European and Eurasian distribution.

**Figure 4.** Phytogeographical spectrum of vascular plants on the mounds, compared to vascular plants in the Bulgarian flora. The national record was calculated by following [45].

Across all plants growing on the mounds, 853 were diagnostic of 40 different vegetation classes. Most numerous were the grassland specialists and generalists, and *Festuco-Brometea* diagnostic species constituted 44% of all identified plants (Figure 5). There were 193 species diagnostic of anthropogenic vegetation (classes *Artemisietea vulgaris*, *Papaveretea rhoeadis*, *Chenopodietea*, *Epilobietea angustifolii*, *Polygono-Poetea annuae* and *Sisymbrietea*) and their per mound abundance was relatively low (on average from 2.2 ± 1.8 SD for *Chenopodietea* to 7.87 ± 3.5 SD species for *Artemisietea vulgaris*). There were 100 species diagnostic for forest vegetation (classes *Alno glutinosae-Populetea albae*, *Carpino-Fagetea sylvaticae*, *Quercetea pubescentis*, *Quercetea robori-petraeae* and *Salicetea purpureae*). Plants diagnostic for *Quercetea pubescentis* prevailed with the highest average per mound presence (2.56 ± 2.5 SD). There were 206 species with wide ecological plasticity to which no diagnostic value toward a particular syntaxon was attributed.

#### 3.1.2. Bryophytes

We registered a total of 58 bryophyte taxa; 54 were identified to species level (Supplementary ESM S1). They belong to 39 genera and 16 families. Pottiaceae included the highest number of species, all of which were confined to dry, skeletal and sandy substrates. The second most diverse family was Bryaceae, a group which includes species with diverse ecological preferences. On the mounds, this family was represented by species typical of dry eroded terrains. No bryophytes were registered on 40 of the studied mounds (36.04%). In cases where bryophytes were found, their number ranged from 1 to 10 species (average 3.1 ± 2.1SD). The only typical epiphytic moss recorded during this study was *Orthotrichum pumilum*, which was found on trees of the genus *Quercus*. The rest of the listed species usually occupied the soil substrate and were occasionally also found on woody stems. *Abietinella abietina*, *Barbula unguiculata*, *Pterygoneurum ovatum*, *Rhynchostegium megapolitanum* and *Thuidium assimile* were confined to herbaceous habitats. *Bryum pallescens* has broad ecological affiliation, while *Atrichum undulatum*, *Fissidens taxifolius* and *Plagiomnium affine* were observed only within the forested northern slopes. Nearly all of the bryophytes registered during the present study are taxa commonly found in Bulgaria where they are known to occur in a variety of different plant communities, usually in lowland areas. Phytogeographically, most of the bryophytes (35 species) belong to the temperate region. A significant share of the registered bryophytes were cosmopolites (*Polytrichum*

*juniperinum*, *Polytrichum piliferum*, *Funaria hygrometrica*, *Schistidium apocarpum*, *Ceratodon purpureus*, *Weissia controversa*, *Syntrichia ruralis*, *Bryum argenteum*, *Pohlia nutans* and *Hypnum cupessiforme*). The *Ceratodon conicus* species, previously known from a single locality in Bulgaria (Vitosha Mt. [46]), deserves special attention. This taxon was registered on a mound in the Thracian lowland near the town of Plovdiv (ID249), a substantial distance from the previously reported location.

**Figure 5.** Vascular plants number associated with the phytosociological class level.

#### 3.1.3. Lichens

A total of 61 lichen taxa, 56 identified to species level, were registered on 52% of the studied mounds (Supplementary ESM S1). The highest number of recorded lichens were epiphytes (35 taxa), followed by epigean taxa (16 taxa), and by species with wide ecological breadth occurring on all substrate types (2 taxa). We recorded eight epilithic lichen taxa. The number of species registered on a mound ranged between 1 and 18 taxa (average 4.0 ± 2.1SD). Lichens were not registered on 57 of the studied mounds. The most common family found on mounds was Parmeliaceae, followed by Cladoniaceae and Physciaceae. Frequently registered lichen species were *Xanthoria parietina*, *Parmelia sulcata* and *Physcia adscendens*. *Arthopyrenia salicis* is recorded for the first time for the country. Until present, *Caloplaca cerinella* and *Catillaria nigroclavata* were known from single Bulgarian localities and are reported here for a second time. Phytogeographically, many of the lichens (31 taxa) are typical for the temperate region. In general, there is a clear preponderance of warmtemperate lichens. Arctic–alpine lichens, common for the Bulgarian alpine and subalpine zones, were not encountered during the present study. It is interesting to note that the new country record and the two other poorly recorded taxa were found together on a single mound (ID 562) situated in the eastern part of the country, near the Black Sea.

#### **4. Discussion**

#### *4.1. Species Diversity*

This article presents new information regarding the vascular and cryptogam (bryophytes and lichens) flora of ancient mounds in Bulgaria. The species diversity established during this study is a testament to the importance of ancient mounds for preserving the native

flora as previously emphasized for other parts of Eurasia [3,12,13,15]. According to the most recent report [47], the Bulgarian flora includes 4064 vascular plant species. The 1056 vascular plant taxa we recorded present nearly a quarter of the overall national plant diversity. These results emphasize the role of ancient mounds in the preservation of a high percentage of the national floristic diversity. Similar results have been previously reported in other European countries: a total of 346 plants were registered on 82 mounds in Hungary [18], which equals 15.5% of the national floristic diversity [48] and 721 species were registered on 106 mounds in Ukraine [12], which represents 11.6% of the national flora [49]. The flora of the studied ancient mounds mirrors the phytogeographical characteristics of the Bulgarian flora, i.e., the mounds contain a representative sample of the national plant species pool. The higher proportion of Mediterranean and European, as well as adventive, elements reflects the species pool of the lowlands. A similar finding, namely that ancient mounds reflect local biogeographical zones, was reported by Sudnik-Wójcikowska and Moysiyenko [50], in Ukraine.

We expected that the flora of the mounds in different countries will reflect local environmental conditions, species pools and human influence. Nevertheless, there was a surprising similarity regarding the number of species recorded per mound in different countries. Our results were close to the 72 species registered on the best documented mound, Csíp˝o-halom (Hortobágy, Great Hungarian Plain) [51]. The range of species established per mound in Poland has been found to be from 44 to 81 [52], and the reported number of species per mound in Ukraine has been found to be between 82 and 125 [12]. Similarities among the mounds in Europe did not solely concern their species diversity. Another similarity is the weak correlation between mound area and species richness, originally established by Deák et al. [53]. Our results corroborate these findings and strengthen the idea that mound protection should be of high priority irrespective of their size. Studies of ancient mounds often refer to these structures as islands in a sea of anthropogenically modified areas [12,14,15]. This is a fair comparison, given the fact that the increase in agricultural and other modified areas around the mounds does not significantly affect their biodiversity, a finding also confirmed by our study.

The outstanding floristic diversity of Bulgarian mounds, especially given the fact that most of them are located in areas characterized by intensive large-scale agriculture, supports our assumption that ancient mounds are valuable refugia for indigenous flora and that they play an important role in its conservation. The number of species we registered on one or two mounds was high (45.3% of the established flora) and it reflected the local species pool, a finding similar to that of Sudnik-Wójcikowska and Moysiyenko [8]. The high rate of dissimilarity between the sampled mounds is a reflection of the diversity of biogeographical zones in the country. The difference in species composition between plots with northern and those with southern exposure corresponds to the well-established effect of slope aspect on vegetation [54]. The abundance of generalists on the south facing slopes and the abundance of grassland specialists on the northern slopes support the findings of Deák et al. [9]. Forest specialists are more abundant on north facing slopes due to the ecological requirements of trees to milder climatic conditions and especially to higher air and soil moisture.

According to our results, 6.4% of the bryophytes native to the country can be found on the mounds. The new *Ceratodon conicus* locality, established nearly a century later and on a substantial distance away from the original locality, gives us a reason to believe in the potential of ancient mounds for the long-term preservation of biodiversity. The cover and diversity of lichen species depends primarily on suitable substrates and environmental factors. Although the saxicolous lichens represent one of the largest ecological groups of lichens, their preferred bare and stable rock substrates were not found on the studied mounds. Most of the saxicolous lichens found during this study appeared on artificial substrates of contemporary anthropogenic structures, such as geodetic points. Only a few species were found on pebbles that are not a suitable substrate for lichens because of their instability. The predominance of epiphytic lichens is not surprising as most of

the mounds had trees and shrubs. The terricolous taxa were rarely present due to their inability to compete with the well-established vegetation. The first record of the epiphytic lichen *Arthopyrenia salicis* from Bulgaria is noteworthy. To our knowledge, the closest *A. salicis* locality is in the Slovenian Julian Alps [55]. This species is especially frequent in northern and western Europe [56], where it occurs on the smooth bark of the trees, as experienced in our study. Lichens frequently established on the mounds are among the most common species characteristic of the lowland epiphytic communities on broadleaved trees in Bulgaria. However, the abundance of *Xanthoria parietina* and *Physcia adscendens* is indicative of a higher load of nitrogenous compounds [57]. Typical nitrophilous lichen community members, such as *Phaeophyscia orbicularis* and *Polycauliona polycarpa* [58], were also relatively frequently found on the studied mounds. We assume that these nitrophilous species might have been positively affected by the surrounding agricultural lands and treated with fertilizers.

#### *4.2. Species Characteristics*

The ecological characteristics of the established flowering plants strongly resemble the peculiarities of the national flora with a dominant presence of species typical for dry areas. These species use evolutionary advantages to survive via vegetative propagules (e.g., *Agropyron repens*, *Poa angustifolia*, *Botryochloa ischaemum*, *Achillea millefolium*, *Bryum dichotomum*, *B. klinggraeffii*, *B. moravicum*, *B. rubens* and *B. ruderale*), underground storage organs (e.g., *Arum maculatum*, *Crocus flavus* and *Ornithogalum* spp.) or via annual life cycles (e.g., *Trifolium striatum*, *Astragalus spruneri*, *Apera spica-venti*, *Arenaria serpyllifolia* and *Coronilla scorpioides*). Patches of bare ground and pits left after continuous illegal treasure hunting become suitable places where fruits or seeds of common trees and shrubs (*Pyrus pyraster*, *Prunus cerasifera*, *P. spinosa*) accidentally fall and develop free from competition. Open spaces also favor the establishment and continued persistence of certain bryophytes confined to eroded habitats (*Ceratodon purpureus*, *Polytrichim piliferum* and *Bryum argenteum*).

The role of ancient mounds in steppe preservation is continuously being reiterated in the published literature [3,6,7,17,59]. Our study confirms the prevalence of species characteristic of the *Festuco-Brometea* class associated with the mounds, similar to those reported for Ukraine [6]. This vegetation type includes dry grasslands and steppes from the sub-Mediterranean, nemoral and hemiboreal areas of Europe [30]. *Festuco-Brometea* is widespread in the lowlands and hilly plains of Bulgaria and unites mainly secondary herbaceous communities. Some exceptions occur in the north-eastern parts of the country where small fragments of true steppe communities are present. Given the anthropogenic origin of the mounds, a large number of anthropophytes are logically expected to be associated with these structures. We identified 19.9% of all species as related to anthropogenic vegetation of the classes *Artemisietea vulgaris*, *Papaveretea rhoeadis* and *Sisymbrietea*. Species characteristic of *Artemisietea vulgaris* provide a signal for ruderalization associated with dry habitats. Species diagnostic of *Papaveretea rhoeadis* and *Sisymbrietea* reveal the apparent influence of segetal vegetation in the mounds' immediate vicinity. The diagnostic features of the plant taxa registered on the mounds gives us a reason to characterize most of their vegetation as ruderalized steppes. Although the ancient mounds are considered to be important areas for the protection of steppe flora and vegetation [3], in Bulgaria they also preserve fragments of forest communities [4] characteristic of temperate lowlands. Most of the established trees and shrubs belong to *Quercetea pubescentis* and *Carpino-Fagetea sylvaticae*, two classes widely distributed on the territory of the country. The development of trees, which were visibly old in some places, is a result of the natural succession directed toward the potential vegetation of the Bulgarian lowlands under temperate climate [21]. This successional trend is also indicative of the apparent lack of management practices and activities as associated with the ancient mounds. The diversity of tree species and forest specialists was low compared to the overall floristic diversity. However, the increasing tree coverage leads to a decreased biodiversity under the canopy [60].

#### *4.3. Significance for Nature Conservation*

Preserved for 2–3 millennia, ancient mounds retain the natural flora as evidenced by the predominant presence of native species associated with these structures. The prevalence of perennials is a testament to the long-lasting stability of the local vegetation and creates conditions for the establishment of cryptogams. The lack of serious disturbances over prolonged periods, excluding archaeological investigations and damaging activities by treasure hunters, has favored the establishment of perennial species, respective of closed vegetation, which prevents the penetration of many neophytes and especially that of invasive alien plants. The common development of communities in vascular plants and cryptogams is considered an indication of sustainability [61].

Vascular plants of conservation importance were not common on the ancient mounds. The observed endemic species presented only 5.2% of the Balkan endemics reported for Bulgaria [36]. The sampled mounds preserve two critically endangered and nine endangered plants, which represent 1.8% of both categories evaluated at the national level [37].

Our results corroborate the notion that the undisturbed closed vegetation prevents establishment of alien invasive plants [62]. The presence of low neophytes and alien plant numbers has been previously reported for other archaeological sites [63]. Nevertheless, our results hint towards an impending threat given the proportion of mounds affected by the presence of even sole individuals of alien plant species. Although the presence of large agricultural fields isolates the mounds from immediate human influence, we found no evident relationship between the surrounding land use and the number of anthropophytes present on the mounds. Similar results were previously reported by Sudnik-Wójcikowska and Moysiyenko [64].

The existence of relatively well-preserved floristic diversity indicates that the mounds have the potential to provide not only cultural and spiritual but also other valuable ecosystem services (e.g., provisioning of biomass, maintenance of native plant populations and maintenance of physical, chemical and biological conditions of the locality) [65], which, in the face of accelerated fragmentation and land degradation, will become even more important in the future. Therefore, there is a clear need for further research on the topic. The high rate of isolation on the mounds along with their floristic richness, emphasize their remarkable role in the preservation of natural communities, and in providing connectivity when serving as stepping stones for species dispersal from and to other fragments of natural and semi-natural environments, as previously mentioned by others [7].

The Natura 2000 and the network of protected areas are naturally considered as part of the EU's strategy for green and blue infrastructure, but new areas are expected to be added in the future [10]. Within the growing body of literature regarding the concept of green infrastructure, we perceive green infrastructure as green space planning [66]. Situated in anthropogenically transformed lands, the mounds present ideal features for greenspace preservation. Some of Bulgaria's ancient mounds are situated within urban areas, but we did not include them in our sampling. The presence of semi-natural (green) space within arable fields will certainly support an increase in environmental benefits. Ancient mounds are existing structures that only need maintenance and protection. Protection from further treasure hunting is required because such disturbances could facilitate the penetration of alien and woody plants. Reduction of shrubs (especially *Prunus spinosa*) and non-native trees (*Robinia pseudoacacia*, *Ailanthus altissima*) will help to maintain species-rich grasslands. Although species of conservation importance were rarely observed during our study, the mounds should not be neglected as important areas for plant protection, especially in the cases of highly fragmented semi-natural areas where different subpopulations are well separated from each other.

#### **5. Conclusions**

Millennia-old ancient mounds are a characteristic feature of the Bulgarian landscape and play an important role for the conservation of indigenous flora. Agricultural practices in the surrounding areas have little effect on the floristic richness of the mounds. In this study, we show that Bulgaria's ancient mounds preserve a considerable proportion of the national vascular and cryptogam flora. Further research will certainly offer new knowledge about the natural significance of the ancient mounds. Current legal preservation of the ancient mounds as archaeological monuments does not guarantee their proper management in terms of nature conservation. With the enlargement of the green infrastructure in the cultural landscape at European level, the significance of ancient mounds will most certainly increase.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11050705/s1. ESM S1: Original floristic data from 111 ancient mounds from Bulgaria.

**Author Contributions:** Conceptualization I.A.; methodology I.A. and D.S.; data collection I.A., D.S., M.V., N.V., K.V., A.G., T.T. and V.S.; formal analysis I.A., M.V. and D.S.; original draft preparation I.A., A.G. and V.S.; review and editing M.V., D.S. and G.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bulgarian National Science Fund (contract KΠ-06- H21/2, 2018).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data analyzed in this study are available in Supplementary ESM S1.

**Acknowledgments:** We wish to thank Balazs Deák for his inspiration to begin studying mounds in Bulgaria. We also thank Salza Palpurina for her help during the field work. We would like to acknowledge Kaloyan Ivanov, who kindly polished the English in this paper.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**Duilio Iamonico**

Ce.R.S.I.Te.S., University of Rome Sapienza, Viale XXIV Maggio 7, 04100 Latina, Italy; d.iamonico@yahoo.it

**Abstract:** The first inventory of the flora of Appia Antica Regional Park (Italy), one of the largest protected urban areas in Europe (4580 ha), its biological, ecological and biogeographical composition, and notes of the vegetation physiognomies and landscape are presented; physical characteristics of the territory (geomorphology, lithotypes, and phytoclimate) are also given. The landscape is defined by an agricultural matrix with natural and seminatural areas as patches, and riparian vegetation communities as corridors. The vegetation physiognomies are represented by types linked to the Mediterranean climate (mixed, Mediterranean, and riparian forests; scrubby, rocky, aquatic, and helophytic vegetation; anthropogenic communities). The floristic list includes 714 taxa (104 families and 403 genera). Therophytes prevail over hemicryptophytes; woody flora comprises about 30% of alien species. As regards chorotypes, together with a considerable number of Mediterranean species, there are many exotic species with wide distribution areas testifying to a long-lasting anthropic impact. Floristic novelties (european, national, and regional levels) for 21 taxa are reported. The extraordinary species diversity discovered (43% of flora of Rome and 20% of regional flora) is linked to the landscape heterogeneity, the characteristics of which are: (1) persistence of residual natural patches, (2) occurrence of quite well-preserved aquatic habitats and humid meadows, (3) a rich anthropogenic flora, (4) an interesting flora of archeological sites, (5) occurrence of species not common in Latium, (6) occurrence of populations of aliens in crops (which cause economic impact), (7) presence of aliens on archeological ruins (which cause economic-social impacts). The extensive set of data provided represents a general base framework for guiding future research efforts and landscape action plans consistent with environmental sustainability.

**Keywords:** alien status; biological records; Europe; Latium; Mediterranean flora; Rome; urban biodiversity

#### **1. Introduction**

Biodiversity loss is a phenomenon mainly related to the intensification of land use and management, as well as the conversion of natural lands to agricultural, forestry, and building areas [1]. This phenomenon is particularly intense in urban areas where several processes, e.g., loss of soil, biological invasions or pollution, are highly exacerbated due to human activities and have a significant impact on the native flora and residual fragments of natural vegetation [2–4]. In addition, as widely known, urbanization has been accelerating for several decades at an alarming rate around the world, and most of the world's population is now concentrated in urban areas [5]. Urbanization is one of the major causes of plant diversity loss at the local and regional scales [6].

Urban areas are "hotspots" of biological invasions, being not only key points of entry for many non-native species, but also playing an important role in the secondary spread of aliens towards surrounding territories, i.e., rural or natural landscapes [3,7]. However, at the same time, urban parks and natural areas in cities are important as detailed-scale biodiversity hotspots [8]. So, maintenance of the biodiversity in urban areas represents a very important conservation issue [9,10]. With this aim in mind, the first fundamental step for sustainable actions is to improve the floristic knowledge of these areas by preparing inventories of taxa [9,11]. Floristic catalogues provide useful data for subsequent studies,

**Citation:** Iamonico, D. Biodiversity in Urban Areas: The Extraordinary Case of Appia Antica Regional Park (Rome, Italy). *Plants* **2022**, *11*, 2122. https://doi.org/10.3390/ plants11162122

Academic Editors: Robert Philipp Wagensommer and Daniel Sánchez-Mata

Received: 11 July 2022 Accepted: 11 August 2022 Published: 15 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

e.g., distributional patterns [12], or for future actions aimed, for instance, at managing non-native taxa [13].

The flora of Mediterranean cities still remains poorly known [9]. Concerning Italy, most urban floras refer to cities of northern Italy, e.g., Milan in Lombardy region [14] or Trieste in Friuli-Venezia Giulia region [15]. Concerning central and southern Italy, few contributions have been published, e.g., for Naples in Campania region [16] or Rome in Lazio region [9].

Rome, and its administrative territory, is one of the largest and most populated urban areas in southern Europe, and it has been investigated since the 19th century from a botanical point of view. The first published floristic works of the Roman area were by Sebastiani in 1813 and 1815 [17,18] and Sebastiani and Mauri in 1818 [19], whereas the more recent list of spontaneous plants growing in Rome was published about 10 years ago [9]. The territory of Rome Municipality covers 1287 km2, and it is characterized by very high landscape heterogeneity dependent primarily on its location in the center of the Mediterranean basin. This location favors, in turn, the influence by various environmental factors, e.g., biogeographic effects (from western and eastern Europe), proximity of the Tyrrhenian Sea (on the west) and Apennine Mountains (on the east), different types of phytoclimates, 3000 years of human impact, etc. [9,20]. These environmental features contribute to a high floristic richness (1649 taxa according to [9]) as well as the occurrence of many types of natural vegetation communities and potential types, the dominant ones being the sub-Mediterranean deciduous oak woods with *Quercus cerris* L., *Q. frainetto* Ten., *Q. robur* L., and *Q. pubescens* Willd. Despite the quite extensive knowledge of flora of the Roman area, detailed studies on specific areas (e.g., those protected by regional or national laws) are still partially lacking. Some of these areas have been studied in recent years, e.g., the Monte Mario Natural Reserve in the northwestern part of the Rome Municipality [21], the Laurentino-Acqua Acetosa Natural Reserve in the southwestern part [22], or the Augazzano Urban Park [23] and Nomentum Natural Reserve [24] in the eastern part.

Appia Antica Regional Park, which is the topic of the present work, was not investigated in detail from a floristic point of view, despite being one of the larger protected areas of the Rome Municipality and, as an urban park, in the whole of Europe (with about 4580 ha). There is only a floristic catalogue on the Caffarella Valley that occupies only 190 ha (about 4% of the total area), but it was published 22 years ago [25].

As a part of ongoing studies on the flora of urban areas, with special attention to the Rome territory [21–23,26], I here present the flora of Appia Antica Regional Park, also giving notes on abiotic factors (climate, geology, geomorphology, etc.) as well as landscape structure and main vegetation physiognomies.

#### **2. Materials and Methods**

This work was based mainly on field surveys that were carried out during the period 2010–2022. Collected material was deposited mainly at the Herbarium RO (secondly at FI and HFLA). Further specimens, kept at A, AC, BM, BR, CAS, COL, F, FI, G, GH, GOET, HFLA, JE, K, MICH, MO, NA, P, RO, RSA, SI, UC, US, URT, and YU, were examined (acronyms of the herbaria follow *Index Herbariorum* [27]). Relevant botanical literature was also analyzed (citations occur throughout the text).

The taxa (species, subspecies, and variety ranks) were identified using the new edition of the *Flora of Italy* [28]; recent monographs were also considered for critical genera (e.g., *Amaranthus* [29] or *Vitis* [30]). The nomenclature follows the Italian Checklists of both native and non-native flora and the subsequent updates [31–33]. Concerning the genera *Amaranthus* and *Cyanus*, I considered, respectively, my recent monograph [29] and the new edition of the *Flora of Italy* [28]. Biological forms and chorotypes of each taxon follow the new edition of the *Flora of Italy* [28]; grouped chorotypes follow [26], except for the category "Aliens", which includes, in the present paper, all the non-native taxa. The alien status

and definitions of categories follow the recent Italian Checklist of non-native flora [31] and Pyšek and collaborators [34].

In the floristic list (Appendix A), the systematic order of the families follows Italian Checklists of both native and non-native flora [31,32]. Within each family, the taxa are ordered alphabetically. For each taxon, after the accepted scientific name, the following information is reported: endemic, cryptogenic, alien status.

Geological and geomorphological information and the pertinent nomenclature refer mainly to [35,36] and [37,38], respectively. Further, field observations were conducted.

Climatic and phytoclimatic data derive from the climate classification by Zepner and collaborators [39] and the map of the Roman area by Blasi and Michetti [40].

Landscape remarks are based on [41,42], but also on direct observations in the field.

Vegetation physiognomies were observed directly in the field, lacking a detailed phytosociological study of the park area (see also [41]). Nomenclature of the main syntaxonomic ranks follows the recent classification system for European vegetation [43].

Photographs are original and produced by the author of the present manuscript, except for the bottom picture of Figure 8 ("Cava di Fioranello"), which was produced using a drone and kindly provided by Lucio Virzì (Rome).

#### **3. Study Area**

#### *3.1. Geographical Context*

The Appia Antica Regional Park is located mostly in the southeastern part of the urban area of Rome Municipality (less than 5% of the territory is included in the Municipalities of Ciampino and Marino, toward the south) and was established in 1988 by the Regional Law No. 66. Later, in 2002, by the Regional Law 31 May 2002, an extension of the original area was proposed and approved. Currently, the park covers about 4580 ha; altitude ranges from about 15 m in the north sector of the park to 189 m a.s.l. in the south (Frattocchie locality, Marino Municipality).

The general shape of the park is a long wedge defined by one main axis, the Appia Antica street, which runs for about 16 km from northeast (Numa Pompilio square, in the vicinity of the archeological area of *Caracalla* thermal baths, in the historic center of Rome) to southwest (Ciampino and Marino Municipalities). Coordinates of the park are: 41◦50 00 latitude N, 12◦33 00 longitude E. The main boundaries of the park are (see Figure 1):


**Figure 1.** Map of Appia Antica Regional Park (boundary in red line) with main localities and streets; yellow line: Appia Antica street. Scale bar = 2 km.

#### *3.2. Geological Features*

The area of Appia Antica Regional Park had a geological history strictly linked with the activity of the Colli Albani volcano (currently known as *Castelli Romani*), which started about 600,000 years ago [26]. During the first phase (0.60–0.36 Ma), violent eruptions occurred, causing the formation of a large volcanic structure. This later collapsed, giving rise to the Tuscolano-Artemisian caldera. A second volcanic phase (0.27–0.10 Ma) was marked by the rise of a second volcanic structure, internal to the previous one. Finally, there was a third phase (0.10–0.01 Ma), which was characterized by intense hydromagmatic activity, creating the lakes Albano and Nemi plus several minor basins, drained in the past by man for agricultural purposes (Vallericcia, Laghetto, Valle Marciana, Prata Porci,

Pantano Secco). The territory of Appia Antica Regional Park, which is located from the north-western slope of *Castelli Romani* to the historic center of Rome (toward the north), is covered by lithotypes related to volcanic rocks with alkaline-potassic chemism [26,35]. Three main types occur: lavas, tuffs, and unconsolidated pyroclastic deposits (named also as *Pozzolane*). The former is related to the volcanic activity of the above-mentioned second phase, when just one erupted material flowed out of the Tuscolano-Artemisian caldera toward the current urban area of Rome. This lava was named *Colata di Capo di Bove* (literally "Lava flow of Bove Capo") and it appears in various parts of the park (Figure 2). The remaining area is covered by various types of tuffs and unconsolidated pyroclastic deposits (Figure 3). In addition to lavas, tuffs, and *Pozzolane*, lahar deposits occur in the *Acquedotti* area (east of the park) being part of the Ciampino Plain, the origin of which represents the most recent activity of Colli Albani volcano (25 Ka; [36]).

**Figure 2.** Lava *Colata di Capo di Bove* (south of Caffarella valley, north of the park).

**Figure 3.** Unconsolidated pyroclastic deposits (*Pozzolane*) in the northwestern sector of Caffarella valley (north of the park).

Holocene deposits are represented by alluvials and colluvials of sands, silty sand, or silty clayey sand related to the fluvial processes [35].

#### *3.3. Geomorphological Features*

As a whole, the natural landscape in which the city of Rome has developed was mainly molded by fluvial processes. In addition, polygenetic, structural, and gravitational forms also occurred and are widespread; finally, there are many landforms deriving from the millennian human activities [38].

The study area includes all four main types of landforms that can be found in the whole Roman area [38], i.e.,:

¾ Fluvial landforms: the study area is characterized by a more or less structured hydrographic network composed mainly of channels, mostly with steady water flows, natural ponds, and freshwater springs. The most complex networks can be found in the *Caffarella* (north of the park) and *Acquedotti* (east) areas.

The *Caffarella* area is a typical V-shaped valley molded by the river Almone and its tributaries (channels, locally named *Marrane*). Almone is a left-side tributary of the river Tevere originated in the Colli Albani volcano and joining the river Tevere in the *Ostiense* district (southwestern Rome); the total length of Almone is about 21 km, and its drainage basin is about 51 km2 [44]. In particular, the river Almone, which has a mean river flow of 1.7 m2/s, forms on some parts of its alluvial plain a series of meanders with deposition of material on the inside of each bend and erosion of the outside bank of the bend (Figure 4).

The *Acquedotti* area is flat with a main channel (named *Acqua Mariana*, that originates in Molara Valley in the Castelli Romani Regional Park [45] about 20 km toward the south) and secondary channels (Figure 4). *Acqua Mariana* is an artificial channel created during the 12th century by redirecting a tributary of the natural channel *Acqua Crabra* [36,44]. The hydrographic network was affected by erosion of lahar deposits distributed in the Ciampino plain (<23 Ma) [46].

**Figure 4.** Meander of river Almone (yellow line: flow direction): deposition of material on the inside bank of the bend (white arrow) and erosion of the outside bank (red arrow) (left side photo); secondary channel in Caffarella valley (right side photo).

¾ Structural landforms: these forms are the results of the fluvial erosion cut of the flat ignimbritic plateau generated after the eruption of the Colli Albani volcano (middle Pleistocene); surfaces are often bordered by cliffs affected by various weathering and denudation processes [37]. Some structural landforms can be observed in the Caffarella valley, where the volcanic plateau was cut by the Almone River (Figure 5).

**Figure 5.** Flat surface on east side of the Caffarella valley. Flood plain is at about 20 m a.s.l., top of the structural landform (red line) is at about 43 m a.s.l. Yellow line: base of the slope (45–80◦).

¾ Gravitational landforms: two main types were observed, i.e., falls, which occur especially in the Caffarella valley and Tor Marancia locality where there are some slopes with a high gradient (more than 80%, even vertical) (Figure 6) (no data occur in the map of the Italian landslide for the study area [47]) and sinkholes originating from the collapse of underground cavities (see below under "Man-made landforms") and the subsequent subsidence of the shallower layers of the soil [48]. The latter gravitational landforms have dimensions ranging from 1–6 m of depth and 1–12 m of diameter (pers. obs.) (Figure 6).

**Figure 6.** Gravitational landforms: fall on the east side of Caffarella valley (top photo); sinkhole, diameter about 12 m, depth about 5 m (bottom photo).

¾ Man-made landforms: the study area has been affected by several human activities mostly beginning thousand years ago. The marks of these activities are often juxtaposed with those related to the natural processes [37].

The most widespread human-made landforms are represented by a dense network of underground cavities, which are especially concentrated in the Caffarella area (north of the park). These cavities (Figure 7) were firstly used to extract material for construction of buildings and catacombs, i.e., lithic tuffs and unconsolidated pyroclastic deposits (*Pozzolane*), and to distribute and collect water [48]. The more recent use of these cavities (up to the 1990s) was as mushroom patches (genera *Pleurotus* (Fr.) P. Kumm. and *Agaricus* L. (*A. bisporus* (J.E.Lange) Imbach, named "champignon")).

**Figure 7.** Entrance of an underground cavity (Caffarella valley).

Further landforms occurring in Appia Antica Park are the mines (currently inactive). They started during the 6th–5th centuries BC [49] and are characterized by both straight scarps and step-like slopes. In some cases, the mines are no longer readily visible, being covered by the vegetation and/or subjected to naturalistic engineering operations ([50]; Figure 8). A famous mine (named *Cava di Fioranello*; Figure 8) occurs near the Ciampino airport (south of the park) and was used to extract basalt to prepare a type of cobblestone resembling truncated and square-based pyramids (locally named *sanpietrini*) that represents the traditional pavement of many streets and squares in the city of Rome [51]; Fioranello's mine is currently used for climbing sport activity.

**Figure 8.** Mines: abandoned mine of Pozzolana located near the Latina street (northeast of the park); its slope (yellow lines) is covered by natural vegetation (top photo); mine *Cava di Fioranello* of basalt, near Ciampino airport, south of the park (bottom photo).

In addition to underground cavities and mines, there are further man-made elements that deserve to be mentioned since they locally changed the landscape from both environmental and social points of view. These man-made landforms are the artificial lakes occurring in Caffarella valley and Acquedotti locality (Figure 9):


**Figure 9.** Artificial lakes occurring in Caffarella valley (top photo) and Acquedotti locality (bottom photo).

#### *3.4. Climate and Phytoclimate*

According to the online ClimateCharts.net (accessed on 11 July 2022) database, which is based on the Köppen-Geiger's climate classification [39], the study area would be included in the Temperate type ("C"), which is defined by a temperature of the hottest month of ≥10 ◦C and temperature of the coldest month ranging between 0 and 18 ◦C. In particular, the subtype is "Temperate without dry season" (code "Cfa"), defined by precipitation in the driest month in summer of >40 mm rain and temperature in the hottest month of ≥22 ◦C.

From the phytoclimatic point of view, the whole Roman area belongs to the Mediterranean region and the Meso Mediterranean type [40]. Appia Antica Regional Park is included in the following two subtypes:


**Figure 10.** Thermo-Pluviometric Bagnolous-Gaussen diagrams of the stations Ciampino (left side diagram) and Monte Mario (right side diagram). Blue lines refer to rainfalls; red lines refer to temperatures. Axis x refers to months (e.g., "J" = January).

#### *3.5. Landscape Remarks and Actual Vegetation Physiognomies*

The landscape of Appia Antica Regional Park is defined by an agricultural matrix (primarily wheat fields; Figure 11) that covers more than 50% of the total area. Natural patches are mostly *remnant*-type according to Forman and Godron [52], being caused by widespread disturbance from the matrix. These patches are represented in the study area by shrubs or forest residual areas (Figure 12) that resulted after both agricultural activities and grazing (sheep and cows) (the so-called *Anthropic determinism sensu* [53]). Other patches related to human activities, but not residual, are the mesophilous meadows

used as pastures (Figure 12) and/or for recreational activities. In further cases, patches exist since the natural environmental conditions did not allow easy and favorable land uses (the so-called *Natural determinism sensu* [53]). Examples are the humid meadow areas occurring in the Almone valley (north of the park) which remain due to the near-surface aquifer (Figure 12). Concerning the landscape corridors, they are represented mainly by channels (locally named *Marrane*) with banks covered by both herbaceous or shrubby vegetation only and riparian forests (Figure 13).

In addition to the natural and seminatural patches, the landscape of the park is characterized by many historical and archeological elements (Figures 14 and 15). The former are represented mainly by farmhouses (e.g., *Vaccareccia* (16th century), *Ex Mulino* and *Vigna Cardinali* (19th century)). The archeological elements consist of many types of monuments, e.g., aqueducts (e.g., *Aqua Claudia* (38–52 a.C.)), *Felix Aqueduct* (around half of the 1st century a.C.), *Antoniano Aqueduct* (around half of 2nd century a.C.)), catacombs (e.g., *San Callisto*, *San Domitilla*, *San Sebastiano* (half of 2nd century a.C.)], churchs [*Domine Quo Vadis* (medieval), *Sant'Urbano* (6th century a.C.)), nymphaeum (*Egeria nymphaeum*, 2nd century a.C.), tombs and sepulchres (e.g., *Latin tombs* (1st–2nd century a.C.), *Cecilia Metella sepulchre* (30–10 a.C.)), palaces (e.g., *Circus* and *Massenzio's imperial palace*, 2nd–3rd century a.C.), temples (e.g., *temple of God Redicolo*, second half of the 2nd century a.C.), walls (e.g., *Aureliane's wall* with *San Sebastiano door* (270–275 a.C.)), ways (e.g., *Appia Antica* (end of 4th century a.C.), *Latina* (end of 4th century a.C. to beginning of 3rd century a.C.)); medieval towers or fortifications (e.g., Tor Fiscale and Valca towers) also occur.

As a whole, this landscape composition reveals a high landscape fragmentation that occurred during the past, which caused a decrease in the environmental quality of the territory. In addition, the ecologic connectivity appears to be low, especially in the southern part of the park, where few corridors occur and the matrix occupies a higher percentage of the total area than in the northern part.

**Figure 11.** Landscape elements of Appia Antica Regional Park: matrix. Wheat field in locality Acquedotti.

**Figure 12.** Landscape elements of Appia Antica Regional Park: patches. Shrubs adjacent to crops (top photo); pasture (central photo); humid meadow (bottom photo).

**Figure 13.** Landscape elements of Appia Antica Regional Park: corridors. Riparian forest in Caffarella valley.

**Figure 14.** Historical farmhouses of Appia Antica Regional Park landscape: *Vaccareccia* (top photo); *Vigna Cardinali* (bottom photo).

**Figure 15.** Archeological elements of Appia Antica Regional Park landscape. First row (from left to right): *Aqua Claudia* aqueduct; *Druso*'s Arch (*Antoniano* aqueduct); catacomb *San Sebastiano* (entrance). Second row: church *Sant'Urbano*; *Egeria* nymphaeum; *Cecilia Metella* sepulcher. Third row: *Massenzio* ruins; temple of *God Redicolo*; *Aureliane*'s wall. Fourth row: *San Sebastiano*'s door; *Latina* way; medieval tower *Valca*.

The natural vegetation of Appia Antica Regional Park is represented by several types, more or less linked to the Mediterranean climate that characterizes the study area. Although a detailed vegetation study of the park is lacking, ongoing surveys (Iamonico in prep.) allow presenting a general view of the main vegetation physiognomies occurring in the park. ¾ Zonal vegetation:

	- > Zonal temperate broad-leaved forests:
		- 1. Quercetea pubescentis Doing-Kraft ex Scamoni et Passarge 1959 (Figure 16): mixed forest communities of deciduous species mainly occurring in northern areas of the park; common species are: *Quercus ilex* L. subsp. *ilex*, *Q. pubescens*, *Q. petraea* (Matt.) Liebl., *Fraxinus ornus* L., *Acer campestre* L. among trees, and *Crataegus monogyna* Jacq., *Euonymus europaeus* L., *Cornus sanguinea* L. subsp. *sanguinea*, and *Viburnum tinus* L. among shrubs;

**Figure 16.** Broad-leaved forests (*Quercetalia pubescenti-petraeae*) in Caffarella valley.

	- 3. Robinietea Jurko ex Hadac et Sofron 1980: it comprises anthropogenic woody vegetation (Figure 17) characterized by an high presence of *Robinia pseudoacacia* L. and or *Ailanthus altissima* (Mill.) Swingle. Other common species are: *Sambucus nigra* L., *Ulmus minor* Mill. subsp. *minor* (among trees), *Rubus ulmifolius* Schott (among shrubs); herb layer is dominated by nitrophilous taxa, e.g., *Galium aparine* L. and *Urtica dioica* L. These types of vegetation occur sparsely throughout the study area;
	- 4. Crataego-Prunetea Tx. 1962: scrub vegetation occurring as patches among the cultivated fields or along the margins of the forests. The main communities occurring in the study area are those with *Cornus sanguinea* subsp. *sanguinea*, *Crataegus monogyna*, *Euonymus europaeus*, *Prunus spinosa* L. subsp. *spinosa*, *Rhamnus alaternus* L. subsp. *alaternus*, *Rosa canina* L., *Rubus ulmifolius*, etc. (Figure 18), whereas in the areas more affected by human pressures, monophytic communities with *Rubus ulmifolius* occur (Figure 18). Further shrub communities are those dominated by *Paliurus spina-christi* Mill. (which can be found in Caffarella valley in areas that are not or only marginally affected by pasture; Figure 19), *Spartium junceum* L. (often on the top of cliffs; Figure 19), and groups of species (*Cornus sanguinea* subsp. *sanguinea*, *Ligustrum vulgare* L., *Euonymus europaeus*, *Crataegus monogyna*, *Ulmus minor* subsp. *minor*) that occupy the anthropic sinkholes originating from the collapse of underground cavities;

**Figure 17.** Anthropogenic woody vegetation (Robinieta): *Robinia pseudoacacia* dominated community (Caffarella valley).

**Figure 18.** Scrub vegetation of the nemoral zone (Crataego-Prunetea): mixed scrub, locality Caffarella (top photo); monophytic communities with *Rubus ulmifolius*, locality Acquedotti (bottom photo).

**Figure 19.** Scrub vegetation in the nemoral zone (Crataego-Prunetea): *Paliurus spina-christi* community, locality Caffarella (top photo); *Spartium junceum* community, locality Tor Marancia (bottom photo).

	- 5. Molinio-Arrhenatheretea Tx. 1937: anthropogenic managed pastures, meadows and tall-herb meadow fringes on fertile deep soils at low and mid-altitudes.
		- Cool Temperate Group of Alliances:
			- ¾ Cynosurion cristati Tx. 1947 (Arrhenatheretalia elatioris Tx. 1931): mesophilous grasslands, grazed and mown once, growing on well-drained mineral/nutrient-rich soils. These communities are common throughout the park, particularly in the areas affected by human recreational use (Figure 20).

**Figure 20.** *Lolium perenne* dominated community, Cynosurion cristati (Divino Amore locality).

	- > Zonal mediterranean forests and scrub:
		- 6. Quercetea ilicis Br.-Bl. ex A. Bolós et O. de Bolós in A. Bolós y Vayreda 1950: thermo-mesomediterranean oak forests and associated Mediterranean macchia. Small forest patches with *Quercus ilex* subsp. *ilex* (Figure 21) or *Q. suber* L. and Mediterranean macchia occur. Holm oak forests are present in Caffarella and Tor Marancia localities. Only one patch of corn oak forest remains (the so-called *Boschetto Farnese* = Farnese's wood, included in the private farmstead "Farnesiana"); this forest is represented by a mixed evergreen/deciduous species where *Q. suber* grows together with *Q. pubescens* and *Q. ilex* subsp. *ilex*, whereas the shrub layer is composed by *Ulmus minor* subsp. *minor*, *Rubus ulmifolius*, *Crataegus monogyna*, *Prunus spinosa* subsp. *spinosa*, *Euonymus europaeus*, *Rhamnus alaternus* subsp. *alaternus*, *Cornus sanguinea* subsp. *sanguinea*, and *C. mas* L. Finally, the residual patches of Mediterranean macchia, which are dominated by *Arbutus unedo* L., *Phillyrea latifolia* L., and *Quercus ilex* subsp. *ilex* (Figure 21).

**Figure 21.** Quercetea ilicis: Olm Hoak forest in Caffarella valley (top photo); Mediterranean macchia in Acquedotti locality (bottom photo).

	- > Alluvial forests and scrub:
		- 7. Alno glutinosae-Populetea albae P. Fukarek et Fabijanic 1968: Mediterranean riparian communities on soils with high water table; this type is well represented in the park along rivers and channels, especially in the central and north zones of Caffarella valley and Tor Marancia locality, where forests are dominated by *Populus nigra* L., *Salix alba* L. subsp. *alba*, and *Fraxinus angustifolia* Vahl. subsp. *oxycarpa* (M.Bieb ex Willd.) Franco & Rocha Afonso (Figure 22).

**Figure 22.** Riparian forest along river Almone (Caffarella locality).

	- > Adiantetea Br.-Bl. et al. 1952: communities dominated by *Adiantum capillusveneris* L. and bryophytes that grow on siliceous dripping cliffs; they are common along deeper channels and springs (Figure 23).
		- 8. Cymbalario-Parietarietea diffusae Oberd. 1969: thermo-nitrophilous Mediterranean chasmophytic vegetation of walls and cliffs; quite distributed on both natural (volcanic cliffs) and anthropic (aqueducts, walls) surfaces. Frequent species are: *Antirrhinum majus* L., *Capparis orientalis* Veill., *Cymbalaria muralis* G.Gaertn., B.Mey. & Schreb. subsp. *muralis*, *Ficus carica* L., *Fumaria capreolata* L., *Parietaria judaica* L., *Reichardia picroides* Roth, *Sonchus tenerrimus* L., *Umbilicus rupestris* (Salisb.) Dandy (Figure 24).

**Figure 23.** Adiantetea on the volcanic cliffs of channel Acqua Mariana (Acquedotti locality).

**Figure 24.** Cymbalario-Parietarietea diffusae on the ancient Roman Claudio aqueduct (Acquedotti locality).

	- 9. Lemnetea O. de Bolos et Masclans 1955: pleustophytic vegetation that colonizes fresh waters; in the territory of the park, these communities mainly occur on weakly flowing waters (channels or lakes) where the alien *Lemna minuta* Kunth dominates the autochthonous *L. minor* L. (Figure 25).

**Figure 25.** *Lemna minuta* community, Lemnetea (Acquedotty locality).

	- 10. Phragmito-Magnocaricetea Klika in Klika et Novak 1941: perennial helophytic communities colonizing lacustrine and fluvial areas on eu- to mesotrophic soils of freshwater bodies; the communities mostly occur in the northern sector of the park along channels or around ponds and artificial lakes (Figure 26). Common species are: *Alisma plantago-aquatica* L., *Apium nodiflorum* (L.) Lag., *Arundo donax* L. (which often forms monophytic communities; Figure 27), *Equisetum telmateja* Ehrh., *Lymniris pseudacorus* (L.) Fuss., *Nasturtium officinalis* R.Br., *Phragmites australis* (Cav.) Tin. ex Steud., *Symphytum officinale* L., *Typha latifolia* L. (which sometimes forms monophytic communities; Figure 28), *Veronica anagallis-aquatica* L.

**Figure 26.** Phragmito-Magnocaricetea on channel Acqua Mariana (Acquedotti locality) with *Alisma planta-go-aquatica* (top photo) and *Veronica anagallis-aquatica* dominated (bottom photo).

**Figure 27.** *Arundo donax* community (Divino Amore locality).

**Figure 28.** *Typha latifolia* community (Caffarella valley).

• Anthropogenic vegetation: secondary vegetation communities that derive from direct or indirect results of human action; they are represented by several types occurring throughout the study area; most of these types are meadow for-mations. The main types observed are listed as follows:

11. Papaveretea rhoeadis S. Brullo et al. 2001 (= Secalinetea Br.-Bl. In Br.-Bl. et al. 1952 = Stellarietea mediae Tx. et al. in Tx. 1950): annual weed segetal vegetation of arable crops on base-rich soils; it is widely distributed in Appia Antica Regional Park since crops represent the landscape matrix (Figure 29).

**Figure 29.** Segetal vegetation dominated by *Glebionis segetum* (L.) Fourr. and *Papaver rhoeas* L., Papaveretea rhoeadis (Acquedotty locality).

12. Sisymbrietea Gutte et Hilbig 1975: anthropogenic vegetation of animal shelters and disturbed ruderal sites (Figure 30).

**Figure 30.** *Trisetaria panicea* dominated community, Sisymbrietea (Caffarella locality).

13. Polygono-Poetea annuae Rivas-Mart. 1975: nitrophilous pioneer vegetation of trampled habitats. This type of vegetation is common, especially on roadsides and crevices of paved roads (Figure 31).

**Figure 31.** Polygono-Poetea annuae: therophyte communities on crevices of paved Appia Antica street; occurred taxa: *Amaranthus retroflexus* L., *Cynodon dactylon* (L.) Pers., *Euphorbia prostrata* Aiton, *Herniaria glabra* L. subsp. *glabra*, *Polycarpon tetraphyllum* subsp. *tetraphyllum*, *Portulaca oleracea* L. subsp. *oleracea*, *Solanum nigrum* L.

14. Artemisietea vulgaris Lohmeyer et al. in Tx. ex von Rochow 1951: perennial meso-xerophilous ruderal vegetation. The most common communities found in the park are dominated by *Silybum marianum* (L.) Gaertn. (margins of fields and uncultivated lands; Figure 32), *Conium maculatum* L. (banks of water courses; Figure 32), and *Sambucus ebulus* L. (humid and disturbed soils near rivers and channels; Figure 32).

**Figure 32.** Artemisietea vulgaris: *Silybum marianum* community, Caffarella valley (top photo); *Conium maculatum* community, Acquedotti locality (central photo); *Sambucus ebulus* community, Acquedotti locality (bottom photo).

15. Chenopodietea Br.-Bl. in Br.-Bl. et al., 1952: weed segetal and ruderal vegetation of man-made habitats. This group includes many types in the park, the most common are the Mediterranean annual grasslands of Hordeion murini Br.-Bl. in Br.-Bl. et al. 1936 and Securigero securidacae-Dasypyrion villosi Cano-Ortiz, Biondi et Cano in Cano-Ortiz et al. ex Di Pietro in Di Pietro et al., 2015. The former is represented by ruderal grasslands occurring in nutrient-rich soils characterized by a high presence of humans (Figure 33). The second group (Securigero securidacae-Dasypyrion villosi) includes the therophytic anthropogenic grasslands in fallow-land habitats of the central regions of the Apennine Peninsula; this type of vegetation is dominated by *Dasypyrum villosum* (L.) P.Candargy (Figure 33), representing one of the most widespread anthropogenic steppe formations in the Roman countryside [54].

**Figure 33.** Chenopodietea: Hordeion-murini (Acquedotty locality; top photo); Securigero securidacae-Dasypyrion villosi (Acquedotti locality; bottom photo).

16. Epilobietea angustifolii Tx. et Preising ex von Rochow 1951 (=Galio-Urticetea Passarge 1967): tall-herb semi-natural perennial vegetation on disturbed forest edges and nutrient-rich riparian fringes (Figure 34).

**Figure 34.** *Galega officinalis* L. dominated community, Epilobietea angustifolii (Acquedotti locality).

#### **4. Results**

#### *4.1. General Statistics of the Flora*

The flora of Appia Antica Regional Park is composed by 714 taxa (including species and subspecies; see Appendix A), belonging to 104 families and 403 genera. These include 11 Lycopodiophyta and ferns and allies (six families and seven genera), three gymnosperms (*Pinus pinea* L., *P. halepensis* Mill. and *Cupressus sempervirens* L., as aliens), one Magnoliidae (*Laurus nobilis* L.), 557 angiosperm eudicots (81 families, 308 genera), and 142 angiosperm monocots (13 families, 85 genera).

The richest families are (Figure 35): Asteraceae Bercht. & J.Presl (81 taxa; 54 genera), Fabaceae Lindl. (74 taxa; 26 genera), and Poaceae Barnhart (69 taxa; 43 genera), followed by Caryophyllaceae Juss. (29 taxa; 12 genera), Lamiaceae Martinov (28 taxa; 14 genera), Brassicaceae Burnett (24 taxa; 19 genera), Rosaceae Juss. (23 taxa; 14 genera), and Apiaceae Lindl. (21 taxa; 16 genera). Eleven families include 10 (Chenopodiaceae Vent. and Euphorbiaceae Juss.) to 17 (Plantaginaceae Juss.) taxa. Fifty families comprise two to nine taxa. Finally, 34 families are monospecific for the flora.

The richest genera are (Figure 36): *Trifolium* (20 taxa); *Silene* and *Vicia* (10); *Amaranthus* and *Medicago* (8); *Carex*, *Euphorbia*, and *Quercus* (7); *Allium*, *Geranium*, *Ranunculus*, and *Veronica* (6); *Crepis*, *Erodium*, *Lathyrus*, and *Rumex* (5).

According to definition given by Fanfarillo and collaborators [54], the segetal flora of Appia Antica Regional Park includes 348 taxa (corresponding to 49.01% of the total flora), of which 14 (1.97% of the total flora) are strictly segetal (species that only occur in segetal habitats), whereas 25 (3.52% of the total flora) are characteristic segetal (species characteristic of segetal habitats that also commonly colonize other habitats) (Table 1). Three species (*Chenopodium vulvaria* L., *Tribulus terrestris* L., and *Xanthium spinosum* L.) are new additions to the Italian segetal flora, being not listed by [55]. The richness (percentage) of the segetal flora of Appia Antica Park is much higher than that of the entire segetal flora

of Italy, i.e., 10.10% (percentage of Italian segetal flora over the Italian vascular flora), 1.61% (percentage of characteristic segetal), and 0.59% (percentage of strictly segetal). These data reveal a high rate of agricultural areas in the park, which, in fact, occupy more than 50% of the territory (see above under the Section "3.5. Landscape Remarks and Actual Vegetation Physiognomies").

**Figure 35.** Number of genera (orange columns) and taxa (dark-green columns) per family; only the richest families (more than 20 taxa (species and subspecies)) are displayed. Abbreviations: Aste. = Asteraceae; Faba. = Fabaceae; Poac. = Poaceaeae; Cary. = Caryophyllaceae; Lami. = Lamiaceae; Bras. = Brassicaceae; Rosa. = Rosaceae; Apia. = Apiaceae.

**Figure 36.** Number of taxa per richest genera [more than four taxa (species and subspecies) per genus).

**Table 1.** Strictly (marked with an asterisk) and characteristic segetal species occurring in the territory of Appia Antica Regional Park (names in alphabetical order).

\* *Alopecurus myosuroides* Huds, subsp. *myosuroides Anisantha diandra* (Roth) Tutin ex Tzvelev *Anthemis arvensis* L. subsp. *arvensis Anthemis cotula* L. \* *Aphanes arvensis* L. *Avena sterilis* L. subsp. *sterilis* \* *Ballota nigra* L. subsp. *meridionalis* (Bég.) Bég. *Buglossoides arvensis* (L.) I.M.Johnst. \* *Cyanus segetum* L. \* *Delphinium consolida* L. subsp. *consolida* \* *Ervilia hirsuta* (L.) Opiz \* *Ervum tetraspermum* L. \* *Euphorbia exigua* L. subsp. *exigua* \* *Euphorbia falcata* L. subsp. *falcata Filago germanica* (L.) Huds. *Gladiolus italicus* Mill. *Herniaria glabra* L. *Herniaria hirsuta* L. subsp. *hirsuta Lamium purpureum* L. *Lathyrus annuus* L. *Lathyrus aphaca* L. subsp. *aphaca* \* *Legousia speculum-veneris* (L.) Chaix subsp. *speculum-veneris Lysimachia arvensis* (L.) U.Manns & Anderb. subsp. *arvensis Matricaria chamomilla* L. *Muscari comosum* (L.) Mill. *Myosotis arvensis* (L.) Hill subsp. *arvensis Neslia paniculata* (L.) Desv. subsp. *thracica* (Velen.) Bornm. *Orobanche crenata* Forssk. \* *Papaver dubium* L. \* *Papaver hybridum* L. \* *Papaver rhoeas* L. *Phalaris paradoxa* L. *Rapistrum rugosum* (L.) All. *Sinapis alba* L. subsp. *alba Sinapis arvensis* L. subsp. *arvensis* \* *Spergula arvensis* L. *Valerianella eriocarpa* Desv. *Veronica arvensis* L. *Veronica polita* Fr. *Vicia bithynica* (L.) L.

#### *4.2. Life Form Analysis*

The vascular flora of Appia Antica Regional Park is characterized by a prevalence of therophytes (287 taxa, corresponding to 40.20% of the total flora), followed by the hemicryptophytes (201 taxa, 28.15%) (Figure 37). The ratio T/H is 1.41, being quite high in comparison with other areas of Rome municipality (Figure 38). These data highlight the xeric conditions of Appia Antica Regional Park in pastures, uncultivated lands, and synanthropic environments that represent the most common habitats. A similar environmental situation occurs in Laurentino and Aguzzano Parks [high therophyte/hemicryptophyte (T/H) ratios], whereas in Monte Mario, Pineto, and Veio Parks (which occur on the north of the Rome area), mesophilous habitats are well represented and the T/H ratio is, therefore, lower due to the higher percentage of hemicryptophytes in comparison with that of the floras of Appia Antica, Laurentino, and Aguzzano Parks. To note, the T/H ratio for Appia Antica Park is also higher than that of the Caffarella valley (1.32), which is included in the studied area, representing about 4% (ca. 190 ha) of the total area.

**Figure 37.** Percentage (axis x) of plant life form spectra of the vascular flora of Appia Antica Regional Park. T: therophytes; H: hemicryptophytes; P: phanerophytes; G: geophytes; Ch: chamaephytes; I: idrophytes; He: helophytes.

**Figure 38.** T/H ratio (y axis) of some parks (x axis) in Rome Province.

The phanerophytes in Appia Antica Regional Park, reaching 14.85% (106 taxa) of the total flora, together with a fair percentage of rhizomatous geophytes (42 taxa, 5.88%), typical of forest habitats, highlight the rather rich flora of woody habitats. However, about 1/3 of the phanerophytes (5.04%) are aliens (Table 2), and most of them do not occur in wooded areas [e.g., *Agave americana* L., *Campsis radicans* (L.) Bureau, *Eucalyptus camaldulensis* Dehnh. subsp. *camaldulensis*, *Lantana camara* L, *Maclura pomif era* (Raf.) C.K. Schneid., *Malus domestica* L., *Passiflora caerulea* L., *Ziziphus jujuba* Mill.]. In fact, as a whole, forests represent only residual areas in the territory of the park, and several patches are actually shrubs (see Section "3.5. Landscape Remarks and Actual Vegetation Physiognomies").


**Table 2.** Phanerophytes (P) occurring in the territory of Appia Antica Regional Park.

Helophytes and hydrophytes represent 1.96% of the total flora (4 and 10 taxa, respectively) highlighting the presence of aquatic habitats (especially in the northern sector of the park). Of note, among rhizomatous geophytes, several grow in these habitat [e.g., *Adiantum capillus-veneris* L., *Eleocharis palustris* (L.) Roem. et Schult., *Limniris pseudacorus* (L.) Fuss, *Symphytum tuberosum* L. subsp. *angustifolium* (A.Kerner) Nyman, etc.], thus revealing the relevant occurrence of aquatic and strictly related habitats.

Finally, the low percentage of chamaephytes (3.08%, 22 taxa) can be explained by the scarcity of rocky habitats (more widespread in the mountain belt and on windy peaks in Latium region) and the absence of garrigues (occurring especially along coasts).

#### *4.3. Geographical Analysis*

The chorological spectrum (Figure 39) shows a prevalence of Eurimediterranean species (162 taxa, corresponding to 22.69% of the total flora). By considering the Mediterranean component *sensu lato* (Euri- plus Stenomediterranean), the percentage reaches 38.94% [288 taxa (162 euri-, 22.69%; 116 steno-, 16.25%)], which is congruent with the phytoclimatic background of the studied area (Meso-Thermomediterranean climate type and oak series as potential vegetation). This fact is also confirmed by both the high value of the Eurimediterranean/Eurasian species ratio (1.91, which is high in comparison with the ratios of other Parks included in Rome Province; Figure 40), and the low percentage of Nordic species (27 taxa, 3.78%); of note, the Eurimediterranean/Eurasian ratio for Appia Antica Park is also higher than that of the Caffarella valley (1.35), which is included in the studied area, representing about 4% (ca. 190 ha) of the total area. The percentage of the wide distribution species (Cosmopolitan, Subcosmopolitan, and Tropical; 110 taxa) plus the aliens (102 taxa) is high, namely 29.70% (15.41% and 14.29%, respectively); this value is mainly due to human impact, which causes the occurrence and spreading of r-selected species. Finally, the eastern component (SE-European, Pontic, and Turanian s.lat.), typical of the flora of Central Italy and particularly of Lazio, totals 5.32% (38 taxa); similar floristic backgrounds were observed in the whole region (see [26]). Finally, the endemic taxa are very few (only 3 taxa, for 0.42%), probably due to the lack of habitats characterized by biogeographical insularity.

**Figure 39.** Chorological spectrum of the vascular flora of Appia Antica Regional Park.

**Figure 40.** Eurimediterranean/Eurasian ratio (y axis) of some parks (x axis) in Rome Province.

Concerning the alien taxa, they total 102, corresponding to 14.29% of the total flora (native taxa account for 85.71%). Of 52 families, each one contains at least one non-native taxon; 13 of these 46 families are aliens, including one species only (Basellaceae Raf., Bignoniaceae Juss., Cactaceae Juss., Cannaceae Juss., Cupressaceae Gray, Cleomaceae Bercht. & J. Presl, Ebenaceae Gürke, Meliaceae Juss., Nyctaginaceae Juss., Passifloraceae Juss. ex Roussel, Phytolaccaceae R.Br., Pittosporaceae R.Br., Platanaceae T.Lestib.). The families rich in alien species include Asteraceae (10 taxa), followed by Poaceae and Amaranthaceae Juss. (7), Fabaceae (5), Rosaceae and Solanaceae Juss. (4), Araceae Juss., Asparagaceae Juss., Convolvulaceae Juss., Moraceae Gausich., and Oxalidaceae (3), and Apocynaceae Juss., Arecaceae Bercht. & J.Presl, Brassicaceae, Chenopodiaceae Vent., Euphorbiaceae Juss., Pinaceae Spreng. ex F.Rudolphi, and Vitaceae (2); the other families include one alien taxon.

A total of 84 genera include at least one alien taxon. *Amaranthus* is the richest one, with 6 alien species, followed by *Erigeron* L., *Oxalis* L., and *Solanum* L. (3), and *Cuscuta* L., *Dysphania* R.Br., *Euphorbia* L., *Galinsoga* Riuz. & Pav., and *Pinus* L. (2); the other 75 genera each include one alien taxon.

Most of the exotic taxa are native to America (51 taxa, representing 51.00% of the total aliens), followed by the Asia (19 taxa, 19.38%); Europe and Africa, including, respectively, 11 (11.22%) and 8 (8.16%) taxa, and two (2.04%) taxa native to Australia (Figure 41). Concerning the alien status, most of the non-native taxa are casual (50, corresponding to 49.02% of the total aliens) and naturalized (40, 39.22%), whereas only 12 species (11.76%) can be considered as invasive (Figure 42).

**Figure 41.** Share of aliens plant species and subspecies (percentages in axis y) by thier per origin.

**Figure 42.** Share of casual (CAS), naturalized (NAT), and invasive (INV) alien plant species and subspecies (percentages in axis y) occurring in Appia Antica Regional Park.

#### *4.4. Floristic Notes*

More than 10 years of field surveys allowed the discovery of several floristic novelties or confirmations at the European (1 species), national (3 species), regional (10 species), and local (7 species) levels; all of these novelties refer to alien species (Table 3; see Appendix B).

**Table 3.** Floristic novelties discovered during field surveys. Names are ordered alphabetically per type of novelty.


#### 4.4.1. New Record for Europe and First One Out of the Native Range

*Denisophytum bessac* **(Choiv.) E.Gagnon & G.P.Lewis** (≡ *Caesalpinia bessac* Chiov.): a population of this species (monophytic shrub community; Figure 43) was first observed during the spring of 2016 on the west side of Caffarella valley. I initially identified this population as belonging to the genus *Caesalpinia* L., mainly based on the flower and leaf morphologies [56]. However, on the basis of a recent taxonomic work [57], a new classification of the *Caesalpinia* group was proposed, recognizing 26 genera (some newly described, other ones resurrected or redelimitated). By using the diagnostic key (genus rank) provided by Gagnon and collaborators [57], the identification of the Roman population (erect shrubs armed, with leaves bipinnate terminating with a pair of pinnae, flowers yellow, and fruits dehiscent and unarmed) was restricted to *Caesalpinia* or *Denisophytum* R. Vig. Note that these two genera are clearly separated from the molecular point of view (see Figure 3A in [57]) and the resurrection of *Denisophytum* is well supported. On the other hand, these two genera are morphologically similar, as highlighted by the authors ("no reliable diagnostic characters have been found to differentiate these two genera" [57] pag. 45). The only character considered to distinguish *Denisophytum* and *Caesalpinia* is the color of the flowers, which are yellow (sometimes with red markings on the standard, i.e., the median petal) in *Denisophytum* and orange, red, green, or white (rarely yellow or pink) in *Caesalpinia* ([57] pag. 27). Furthermore, on the basis of the detailed emended descriptions given, the two genera differ by the length of the fruits (pods), which are 18–49 mm long in *Denisophytum* ([57] pag. 45) and 34–120 mm long in *Cesalpinia* ([57] pag. 43). Flowers in the Roman population are all yellow, with minute red spots on the standard (Figure 43); pods

(pers. obs.) are up to 50 mm long (never less than 40 mm). All things considered, I here identify the Roman population as a *Denisophytum* species.

The identification at species rank was quite difficult. Currently, *Denisophytum* comprises eight species, but unfortunately, no diagnostic key at species rank was provided by Gagnon and collaborators [57]. Furthermore, no further comprehensive key of *Denisophytum* taxa was found in the literature, and the descriptions of the taxa, when existing, are not very detailed. Hence, I decided to check the protologues of all eight of these species and examine all of the original material that I was able to trace. On the basis of this research (the complete work is still ongoing), I assembled the following data:

<sup>¾</sup> *D. bessac* (Choiv.) E. Gagnon & G.P. Lewis (≡ *Caesalpinia bessac* Chiov.): Chiovenda described this species (sub *Caesalpinia bessac*) in his *Flora Somala* [58], providing a detailed description that matches the plants found in Caffarella valley. The same author described also *C. eriantherum* Chiov. (see below) as morphologically similar to *C. bessac*. These two species differ from each other by the size of their leaflets, which are longer in *C. bessac* (basal leaflets 9–12 mm long vs. 4–5 mm long in *C. eriantherum*; distal leaflets 10–20 mm long vs. 7–8 mm long in *C. eriantherum*).

#### **Syntype found**: FI001388.


**Syntypes found**: FI001390 (var. *eriantherum*), FI001392, and K000232357 (var. *pubescens*).

¾ *D. madagascariense* R. Vig.: it is the only unarmed species in *Denisophytum* [57] pag. 45, while Caffarella plants have curved thorns along the shoot.

#### **Syntypes found**: P00131739.

<sup>¾</sup> *D. pauciflorum* (Griseb.) E. Gagnon & G.P. Lewis (≡ *Libidibia pauciflora* Griseb.): this species is different from all other species in having few flowers per raceme ("recemis simplicibus laxis paucifloris (v. pedunculis 1floris)" in the protologue [61]) and corolla slightly longer than the calyx ("calycis... corolla paullo superantibus"), whereas my specimens have many flowers per raceme (up to 30, never less than 20), and corollas are 5–7 times longer than the calyx.

**Syntypes found**: GH00065814, P02142660, P02142661, UC936921, US00382832, and YU001398;

<sup>¾</sup> *D. rosei* (Urb.) E. Gagnon & G.P. Lewis (≡ *Caesalpinia rosei* Urb.): it has leaflets glaucous in the abaxial surface ("foliole... in siccu supra obscure viridia, subtus valde pallida, glaucescentia" in the protologue [62]). Plants found in Rome show leaves green on both surfaces.

#### **Syntypes found**: NY00022764 and US00479309;

<sup>¾</sup> *D. sessilifolium* (S.Watson) E. Gagnon & G.P. Lewis (≡ *Caesalpinia sessilifolia* S.Watson): it is characterized in having sessile leaves, each one with two or three pairs of pinnae

according to the protologue [63]. Caffarella plants have leaves that are petiolate with mostly five pairs of pinnae (rarely four).

**Syntypes found**: A00061947, AC00319854, BR0000005110933, CAS0001542, COL000092321, F0057403F, GH00059873, GOET004917, JE00004880, MICH1107159, MO125071, NA0026234, P02940720, PH00010119, RSA0003187, SI001822, US00344744, and US00345006.

<sup>¾</sup> *D. stuckertii* (Hassl.) E. Gagnon & G.P. Lewis (≡ *Caesalpinia stuckertii* Hassl.): the diagnosis and description given in the protologue [64] are congruent with the morphology of the plants found in Caffarella valley, except for the fruit, which was described as "oblongum basi et apice acutum", whereas fruits in Caffarella's plants have apexes that are obtuse-rounded (Figure 41); also, the illustration given by Gagnon and collaborators [57] pag. 44, Figure 12 displays a typical fruit of *D. stuckertii*, confirming that Roman plants cannot be assigned to this *Denisophytum* species. Further, Gagnon and collaborators' illustration shows stipules that are clearly different from those of the Roman plants (foliaceous with two lobes vs. narrow, not lobed).

#### **Syntype found**: G00364837.

All things considered, the population found in Appia Antica Regional Park is identifiable as *Denisophytum bessac*, a species native to central Somalia and currently unknown elsewhere [65]. This record represents, therefore, not only the first one for Europe, but also the first discovery outside the native range of this Somalian species.

**Figure 43.** *Denisophytum bessac* on the western side of Caffarella valley; flower and fruit (bottom-left insets), pinna (bottom-right inset).

#### 4.4.2. New Records or Changes in Alien Status for Italy

*Aloe maculata* **All. subsp.** *maculata*: this species, currently considered as casual in Italy [33], was recorded as casual in Latium for the first time in 2012 on the basis of a population found in Caffarella valley [66]. It is currently still considered as casual alien for the region [31,67]. After continuous monitoring over the years, it was observed that this population blooms regularly, is able to maintain itself by both vegetative and sexual reproduction (Figure 44), and has spread. I here consider *Aloe maculata* as naturalized in the Latium region, and this status is reported here for the first time at the national level.

*Euphorbia pulcherrima* **Willd. ex Klotzsch**: this species was recorded for Italy only in the Campania region based on a single population found in Naples city [16,68]. Recently, *Euphoribia pulcherrima* was excluded from the region (and Italy) since the site in Naples was destroyed (a new garage was built) [69]. My discovery on a riverbed of channel Acqua Mariana (Acquedotti locality) represents the only Italian site in which the species (casual) certainty occurs (Figure 45).

*Rosa chinensis* **Jacq. var.** *semperflorens* **(Curtis) Koehne**: I first observed, in spring 2015 in Caffarella valley, a small population of a rose cultivar growing in a shrub community dominated by *Rubus ulmifolius*. During the subsequent years, I again observed this population and found another one not far from the former (Figure 46). As a whole, these two populations seem to flourish occasionally in the park, and, therefore, their presence can be considered as casual.

Concerning the identification of this rose, I note first that it is a so-called modern rose having flowers with 17–20 petals [70]. On the basis of the treatment of the genus in *Flora of China* [71], as well as the *European Garden Flora* [72], plants found in Caffarella are identifiable as *Rosa chinensis* s.l. showing the following morphology: shrubs, leaves evergreen, each one with 3–5 leaflets, stipules adnate to petiole, hypanthium globose, flowers double, red-scarlet, usually solitary, up to 10 cm in diameter, not fragrant. Three varieties were recognized by Ku and Robertson [71], i.e., var. *chinensis*, var. *spontanea* (Rehder & E. H. Wilson) T. T. Yu & T. C. Ku, and var. *semprerflorens* (Curtis) Koehne. Var. *spontanea* differs from the other two by the flowers, which are single, whereas var. *chinensis* and var. *semperflorens* have flowers double or semi-double. Morphological differences between these two latter varieties refer to branches (robust in var. *chinensis* vs. slender in var. *semperflorens*), flowers (several and rarely solitary vs. solitary, rarely in fascicles of two or three), and petals (red, pink, or white vs. deep red or deep purple). The plants I found display slender branches and flowers solitary with petals deep red. Therefore, they are identifiable as var. *semperflorens* according to *Flora of China*.

According to Ku and Robertson [71], *Rosa chinensis* var. *semperflorens* has a cultivated origin and it is widely used in China. POWO [73] does not recognize infraspecific taxa of *Rosa chinensis*, recording it as native in South-Central China (it corresponds to var. *spontanea* in *Flora of China*, which is the only native variety from Guizhou, Hubei, and Sichuan) and as alien in other parts of Asia (India, Korea, Kazakhstan, Laos, Nepal, Pakistan, Uzbekistan, and Vietnam), North America (Alabama), Central America (Guatemala), Australia (Queensland and Western Australia), Europe (Belgium, Bulgaria, and Greece) and Oceania (Cook Islands and Guinea Islands); further (not reported in POWO), it was recorded as casual in Slovakia [74]. The occurrences in Europe are based on [75] for Belgium, [76] for Greece, and [77] for Bulgaria. Concerning Italy [33], *R. chinensis* is doubtfully recorded in Elba island (Tuscany, Central Italy) where the species "was possibly cultivated" [78]. So, my discovery in Rome represents the first certain record of *R. chinensis* s.l. in Italy.

**Figure 44.** *Aloe maculata* All. subsp. *maculata* (Caffarella valley).

**Figure 45.** *Euphorbia pulcherrima* on riverbed of channel Acqua Mariana (Acquedotti locality).

**Figure 46.** *Rosa chinensis* var. *semperflorens* in Caffarella valley.

#### 4.4.3. New Records or Confirmations for Latium

*Heliotropium amplexicaule* **Vahl.**: it is recorded in northern and peninsular Italy and Sicily [33]. Four sites are on the Italian peninsula, one in Tuscany (Pisa Province [79]) and three in Campania (Naples and Salerno Provinces [80]). For Latium, it was indicated as no longer recorded in Rome based on an old collection (April 1928, herbarium RO; [67,81]). I found in 2017 a population in the central reservation of Appia Nuova street (Figure 47), and observed it again in 2018 and 2022. The species is casual for Latium, representing the fifth record for the Italian peninsula.

**Figure 47.** *Heliotropium amplexicaule* in the central reservation of Appia Nuova street.

*Hydrangea macrophylla* **(Thunb.) Ser.**: this species was recorded in Italy [33] in the north (Lombardy, Trentino-Alto Adige, and Veneto regions, as casual), center (Tuscany region, as naturalized), and South (Campania region, as casual). The population found (first observation in 2015) grows along the channel Acqua Mariana (Acquedotti locality), blooms regularly, and sustains itself especially by vegetative reproduction; of note, the plants are regularly pruned (one or two times per year), but they rapidly re-grow and flowers appear after about 2 months (Figure 48). It can be considered a naturalized species and represents the first record for the Latium region.

*Hydrangea macrophylla* is a species native to Japan, and it is characterized in having high morphological variability [82,83], which led to the publication of several infraspecific names [84]; in fact, various authors accepted the recognition of infraspecific taxa (subspecies, varieties, and forms; see e.g., [82,85,86]), but the infraspecific variability is still incompletely known. Furthermore, a species related to *H. macrophylla*, i.e., *H. serrata* (Thunb.) Ser. ex DC., shows, in turn, an high phenotypic variability [87] and the relationship between these two species and their infraspecific taxa would need further study [82–87]. Lacking final conclusions about this group, I here prefer to avoid the use of infraspecific taxa and consider *H. macrophylla* as separate from *H. serrata*, according to POWO [84].

**Figure 48.** *Hydrangea macrophylla* along Acqua Mariana channel (Acquedotti locality).

*Ruellia simplex* **C. Wright**: this species is currently known in Italy as casual and recorded only in two regions, i.e., Apulia (southern italian peninsula) and on the island of Sardinia [33]. These findings refer to single localities, one per region, i.e., Otranto (Lecce Province) for Apulia [88] and Serramanna (Medio Campidano Province) for Sardinia [89]. The population found in Appia Antica Regional Park (Acquedotti locality, along the channel Acqua Mariana; Figure 49) represents the first record for the Latium region and the third one at the national level. The few individuals found were first observed in 2020, and they do not seem to be able to spread. So, *Ruellia simplex* is here considered as casual for Latium.

**Figure 49.** *Ruellia simplex* along channel Acqua Mariana (Acquedotti locality).

*Trachelospermum jasminoides* **(Lindl.) Lem.** (≡ *Rhynchospermum jasminoides* Lindl.): this species is recorded in Italy as casual and only in two regions [33], i.e., Lombardy (just one site in Mantova Province; [90]) and Sardinia [just one site in Cagliari Province [91]). A small population was found in the Acquedotty locality on the cliff of channel *Acqua Mariana* (Figure 50), and it represents the first record for the Latium region and peninsular Italy (casual species) and the third one for Italy.

**Figure 50.** *Trachelospermum jasminoides* on cliff along Acqua Mariana channel (Acquedotti locality).

#### 4.4.4. Changes or Confirmation of Alien Status for Latium

*Campsis radicans* **(L.) Bureau**: this species was reported as casual alien for Latium in *Flora of Italy* [33], whereas in volume no. 1 of *Atlante della flora vascolare del Lazio* [67], it was considered as naturalized. Actually, the first indication of naturalization of *Campsis radicans* in Latium was in 2014 [66] on the basis of a population found in Caffarella valley. Of note, the *Flora of Rome* [9] does not list this species. I here confirm the occurrence and naturalization of *Campsis radicans* in Appia Antica Regional Park in Caffarella locality. Furthermore, a new population was recently found in the Acquedotti locality of the park (Figure 51).

**Figure 51.** *Campsis radicans* in Acquedotti locality.

*Canna indica* **L.**: this species is currently considered as casual alien in the Latium region [33,67]. I found many populations of *Canna indica*, especially in the northern sector of the park (Caffarella valley and Acquedotti locality; Figure 52) where, during the years, the number of individuals increased, thus showing that the populations are able to spread. All of the plants found bloom regularly. *C. indica* is here considered as naturalized in Latium.

*Cyperus alternifolius* **L. subsp.** *flabelliforme* **Kük.**: this taxon was considered as a casual alien in Latium [33,67], and also indicated for Rome (sub *Cyperus involucratus* Rottb.). In Appia Antica Regional Park there are many individuals occurring especially in the northern sector (Tor Marancia, Caffarella, and Acquedotti localities), which have highly increased in number over the years. All of the plants found bloom regularly and the populations have spread (Figure 53). *C. alternifolius* subsp. *flabelliforme* is a naturalized species throughout the whole study area.

**Figure 52.** *Canna indica* along channel Acqua Mariana, near Claudio's aqueduct (Acquedotti locality).

**Figure 53.** *Cyperus alternifolius* subsp. *flabelliforme* along channel Acqua Mariana as part of holophyte community of Phragmito-Magnocaricetea (Acquedotti locality).

*Kalanchoe daigremontiana* **Raym.**: species reported as casual for Latium [33,67], but not cited in *Flora of Rome* [9]. The populations found in Appia Antica Regional Park are not only able to sustain themselves, but they have spread (Figure 54). *Kalanchoe daigremontiana* is a naturalized non-native species in Latium.

**Figure 54.** *Kalanchoe daigremontiana* in *Arundo donax* community (Caffarella valley).

*Melia azedarach* **L.**: this species was indicated as doubtfully spontaneous in *Atlante della flora vascolare del Lazio* [67], whereas it is casual for the region in Italian and Roman floras [9,33]. I here confirm the occurrence of *Melia azedarach* as casual in Latium, having found individuals in shrubs and forest communities and along paths (Figure 55).

**Figure 55.** *Melia azedarach* (red arrow) in Caffarella valley (left and top-right photos); young individual in Acquedotti locality (bottom-right photo).

*Punica granatum* **L.**: it is currently considered as casual in Latium [33,67] and Rome [9], whereas in *Flora vascolare del Lazio* by Anzalone and collaborators [92], *Punica granatum* is reported as naturalized, with various localities listed (including Rome). In Caffarella valley, I traced a population along a channel consisting of well-developed individuals (2–5 m tall, with truck of 15–20 cm in diameter) that have bloomed and fruited regularly for more than 10 years (Figure 56). Further scattered individuals were observed in Caffarella and Acquedotti localities. The species is naturalized in the park.

**Figure 56.** *Punica granatum* in Caffarella valley.

4.4.5. New Records or Confirmations for Rome

*Anreedera cordifolia* **(Ten.) Steenis**: this species was not listed in the *Flora of Rome* [9], whereas in *Atlante della flora vascolare del Lazio* [67] it was indicated generically for "Roma città" (= Rome city). Two populations growing in a *Rubus ulmifolium* dominated community (Caffarella valley and Acquedotti locality; Figure 57) were found some years ago, and they appear to be well-established; the species is naturalized. This discovery confirms the occurrence of *Anreedera cordifolia* in Rome.

*Bidens subalternans* **DC.**: this species was not listed in either the *Flora of Lazio* by Anzalone and collaborators [92] or the *Flora of Rome* [9], whereas the more recent *Atlante della flora vascolare del Lazio* [67] indicates *Bidens subalternans* DC. as casual in Tivoli and Genzano cities and the Aurunci Mountains. Recently, the species was considered as naturalized in Latium in Frosinone and Rome Provinces [93]. In any case, the species was not reported for Rome city. My discovery (a dense population growing on a little bridge of channel *Acqua Mariana*) represents, therefore, the first one for the city, and it can be considered as casual for the moment (Figure 58).

**Figure 57.** *Anreedera cordifolia* on *Rubus ulmifolium* dominated community (Acquedotti locality).

**Figure 58.** *Bidens subalternans* in Acquedotti locality.

*Chlorophytum comosum* **(Thumbs.) Jacques**: this species was recently recorded for the first time in Latium (Viterbo Province, Orte Scalo locality) as casual [91]. My discovery on the banks of channel *Acqua Mariana* (Acquedotti locality; Figure 59) represents the first one for Rome Province and the second at the regional level. I first observed the population in 2017, and it persisted during the years (last observation in June 2022), but the population does not spread and is, therefore, considered as casual.

**Figure 59.** *Chlorophytum comosum* along channel Acqua Mariana (Acquedotti locality).

*Diospyrus kaki* **L.**: this species was reported for Latium in both *Flora of Italy* [33] and, along the river Tevere, in *Flora of Lazio* [92]; contrarily, it was not listed either in *Flora of Rome* [9] or in *Atlante della flora vascolare del Lazio* [67]. Some individuals, which bloom and fruit regularly, were traced in Caffarella valley (Figure 60), thus confirming the occurrence of *Diospyrus kaki* in the city.

*Papaver somniferum* **L.**: this species was listed in the *Flora of Rome* as "doubtfully alien" [9], but not later reported for the Italian capital in *Atlante della flora vascolare del Lazio* [67]. Scattered individulas were found in Caffarella valley, thus confirming the occurrence of *P. somnifermum* in Rome (Figure 61).

*Passiflora caerulea* **L.**: this species was reported in the *Flora of Rome* as casual [9], but not later indicated for the city in *Atlante della flora vascolare del Lazio* [67]. I found various individuals growing on the Claudio's aqueduct (Figure 62), thus confirming the occurrence (as casual) of *Passiflora caerulea* in Rome.

*Zantedeschia aetiopica* **(L.) Spreng.**: this African species is not currently reported for Rome [9,67]. In Appia Antica Regional Park, individuals were found along channels in Caffarella valley and, especially, in the Acquedotty locality, where the species is able to spread along the channel Acqua Mariana (Figure 63). I hereby consider this alien as naturalized.

**Figure 60.** *Diospyrus kaki* along a channel in Caffarella valley.

**Figure 61.** *Papaver somniferum* in Caffarella valley.

**Figure 62.** *Passiflora caerulea* (blue arrow) associated with *Capparis orientalis* (red arrow) on the ancient Roman Claudio's aqueduct.

**Figure 63.** *Zantedeschia aetiopica* along channel Acqua Mariana (Acquedotti locality).

#### 4.4.6. Species No Longer Recorded

Literature analysis and herbaria investigations allow the verification of nine species collected in the past in Appia Antica Regional Park, but no longer recorded (see Appendix B). I never found any population of these species during the field surveys. Seven species are native, and two (*Ehrhata erecta* Lam. and *Tarenaya spinosa* (Jacq.) Raf.) are allochthonous (native, respectively, to South America and Africa).

*Astragalus glycyphyllos* **L.**: this species was recorded in Caffarella valley in the *Centuriae XII* of *Florae Romanae Prodromus* [19] pag. 240 and not listed among the flora of Caffarella valley [25]. No specimen collected in the territory of Appia Antica Regional Park was traced. *A. glycyphyllos* is not a common species in the territory of Rome Municipality, although it is very common in the Latium region as a whole [92]. Although the study area was investigated in depth for more than 10 years through field surveys, further field investigations are needed to verify its occurrence in the park.

*Catabrosa aquatica* **(L.) P.Beauv.**: this species was recorded in Caffarella valley in *Egeria nymphaeum* ("nella Grotta della Ninfa Egeria" = in the cave of Nymph Egeria) in the *Centuriae XII* of *Florae Romanae Prodromus* sub *Aira aquatica* L., [19] pag. 38. *C. aquatica* was not listed in the flora of either Caffarella valley [25] or Rome city, in the *Flora of Lazio* [92]. No specimen collected in Caffarella valley was traced. However, I traced one specimen (included in Montelucci's Herbarium at RO; collection number 5621) bearing a plant collected by G. Montelucci in April, 20 (year 1944) at "Via Appia Nuova, lungo fossetto tra Ciampino e S. Maria delle Mole. Luoghi aquitrinosi" (= Appia Nuova street, along channel between Ciampino and S. Maria delle Mole. swampy sites). The area around these two localities (Ciampino and S. Maria delle Mole) would represent the southernmost part of Appia Antica Regional Park, at least partially (the part on the west side of Appia Nuova street). However, the landscape configuration has deeply changed in this sector of the park over time, especially with new buildings. Therefore, the "swampy sites" cited by G. Montelucci in the label, which could be at that time, were probably destroyed; I will verify the absence of this type of habitat during field trips. The species is potentially extinct in the territory of the park.

*Ehrharta erecta* **Lam.**: this species is listed in the flora of Caffarella valley [25] as no longer recorded. This indication was based on a specimen (deposited at RO; Figure 64) collected by G. G. B. Cuboni on 17 March 1876 at "Valle della Ninfa Egeria" (= Valley of Ninfa Egeria). *E. erecta* is an alien species for Italy [31], and its ability to reproduce is probably very low in Latium; this could be the reason why this species was no longer recorded.

*Linaria pelisseriana* **(L.) Mill.**: this species was recorded in Caffarella valley in the *Centuriae XII* of *Florae Romanae Prodromus* [19] pag. 203; it was not cited in the flora of Caffarella valley [25]. Only one specimen (deposited at RO) collected in the territory of Appia Antica Regional Park (Ardeatina street on 15 May 1892) was traced (Figure 65). *L. pelisseriana* is a therophyte with a very short life cycle (flowering time March–April [28]), being also very rare in the territory of Rome Municipality. However, it is common in Latium [92]. Although the study area was investigated in depth for more than 10 years through field surveys, I cannot consider this species as extinct (or potentially so), and I think that further field trips are necessary to verify its presence in the park.

*Parapholis cylindrica* **(Willd.) Romero Zarco**: this species was recorded in Caffarella valley (sub *Rottboellia subulata* Savi) in the *Centuriae XII* of *Florae Romanae Prodromus* ([19], pag. 62]), but not reported in the flora of Caffarella valley [25] and Rome [9], or Rome city in the *Flora of Lazio* [92]. Only one specimen (deposited at RO) collected in the territory of Appia Antica Regional Park (Caffarella on XIX century) was traced (Figure 66). *P. cylindrica* is a species growing mainly along Mediterranean coasts (rarely on inlands) on subsaline

and clay soils [28]. Subsaline soils are not present in the territory of the park to date, and it is possible that the habitat was lost or destroyed. The species, which is considered rare in Italy as a whole [28], is potentially extinct in Appia Antica Regional Park.

*Polycnemum heuffelii* **Láng**: the occurrence in Italy of this European species (distributed from Poland to Greece) is based on just one specimen (deposited at RO; Figure 67), collected by A. Cacciato along Appia Pignatelli street on 6 August 1966 [94]. The Flora of Italy [33] report this species as cryptogenic. The habitat in which this species was discovered (roadsides) was exposed to various types of human activities during the time; this factor could have caused the extinction of the Roman population.

*Silene gallinyi* **Rchb.**: this species was recorded in Caffarella valley (sub *Silene trinervia* Sebast. & Mauri) in *Florae Romanae Prodromus* [19]. *S. trinervia* was there firstly described with *Locus classicus* including the locality Caffarella ("Alla Caffarella presso il Fonte di Egeria..." = At Caffarella near Egeria's spring). This *Silene* species was not listed in the flora of Caffarella valley [25]. After Herbaria checking (including RO, where original collections of A. Sebastiani and E. Mauri are currently deposited [95,96]), I verified that the last collections of this species were dated June–July 1980 (three specimens in Anzolone's Herbarium at RO; Figure 68). *S. gallinyi* is a species that can be easily distinguished from the other *Silene* taxa occurring in the territory of the park [*S. bellidifolia* Jacq., *S. conica* L., *S. gallica* L., *S. italica* (L.) Pers. subsp. *italica*, *S. latifolia* Poir., *S. nocturna* L., *S. pendula* L., *S. vulgaris* (Moench) Garcke subsp. *tenoreana* (Colla) Soldano & F.Conti, *S. vulgaris* (Moench.) Garcke subsp. *vulgaris*]. Despite this, anf the more than 10 years of field surveys, some carried out specifically to search for *S. gallinyi*, I did not yield positive results. My opinion is that this species is extinct throughout the park.

*Stachys germanica* **L. subsp.** *germanica*: this species was recorded in Caffarella valley in *Egeria nymphaeum* ("presso la Grotta di Egeria" = near the cave of Egeria) in the *Centuriae XII* of *Florae Romanae Prodromus* ([19] pag. 194). *S. germanica* s.str. was not listed in the flora of Caffarella valley [25]. Five specimens (at RO) collected in the territory of Appia Antica Regional Park [Caffarella valley (May 1829) and in uncultivated lands near Cecilia Metella sepulcher (3 June 1922)] were traced (Figure 69). *S. germanica* subsp. *germanica* is not a common species in the territory of Rome Municipality, although it is very common in the Latium region as a whole [92]. Plants of this species are easy to see in field due both to their size (30 to 60 cm on average) and their hairiness (densely white-woolly). Despite these facts, I never found it during >10 years of field surveys. The species is potentially extinct in the territory of the park.

*Tarenaya spinosa* **(Jacq.) Raf.**: this species is recorded in the Latium region based on a specimen (deposited at RO; Figure 70) collected by A. Cacciato along Appia Pignatelli street in 1966 [78,92]. It was not reported in the *Flora fo Rome* [9]. I never found any individual of *Tarenaya spinosa* during field surveys. *T. spinosa* is no longer found for two possible reasons: a low ability to reproduce and modification of its habitat (roadsides) by humans.

**Figure 64.** Specimen of *Ehrharta erecta* collected in Caffarella valley on 17 March 1876 (RO-Herbarium Romano no. 51234).

**Figure 65.** Specimen of *Linaria pelisseriana* collected along Ardeatina street on 15 May 1892 (RO-Herbarium Romano no. 34429).

**Figure 66.** Specimen of *Parapholis cylindrica* collected in Caffarella valley in the 19th century (RO-Herbarium Romano no. 55564).

**Figure 67.** Specimen of *Polycnemum heuffelii* collected along Appia Pignatelli street in august 1966 (RO-Herbarium Romano no. 5993).

**Figure 68.** Specimen of *Silene gallinyi* (sub *S. trinervia*) collected in Caffarella valley in July 1980 (RO-Herbarium Anzalone no. 13276).

**Figure 69.** Specimen of *Stachys germanica* L. subsp. *germanica* collected in Caffarella valley in May 1829 (RO-Herbarium Romano no. 38523).

**Figure 70.** Specimen of *Tarenaya spinosa* collected along Appia Pignatelli street in august 1965 (RO-Herbarium Romano s.n.).

4.4.7. Species Having Loci Classici and/or Nomenclatural Types Collected in Appia Antica Regional Park

*Biarum tenuifolium* **(L.) Schott subsp.** *tenuifolium*: the basionym *Arum tenuifolium* L. was described by Linnaeus in the first edition of his *Species Plantarum* [97], where the provenance "*Habitat in* Romae" was reported. The Linnaean name was recently lectotypified by Iamonico [98] on a specimen preserved at BM (barcode BM000647349); further, an epitype collected in the Caffarella valley (20 August 2015) was designated (the specimen kept at HFLA; Figure 71) to serve as an interpretative type according to the current concept in *Arum* (see e.g., [99]).

**Figure 71.** Epitype of *Biarum tenuifolium* subsp. *tenuifolium* (HFLA no. 4861).

*Epilobium lanceolatum* **Sebast. & Mauri**: this species was described in 1818 in *Florae Romanae Prodromus* [19] pag. 138. *Locus classicus* includes the locality Caffarella ("In umbrosis, ad oras nemorum, sepes circa Romam frequens. Copiosamente intorno ai boschetti della Caffarella presso la Grotta di Egeria..." = In the shades, at the edge of the woods, hedges about Rome. Copiously around small forest of Caffarella near Egeria's cave). Iamonico and collaborators [100] designated as lectotype a specimen (deposited at RO; Figure 72) collected by E. Mauri and A. Sebastiani on 3 June 1812.

**Figure 72.** Lectotype of *Epilobium lanceolatum* (RO no. 19436).

*Silene trinervia* **Sebast. & Mauri**: this species was described in 1818 in *Florae Romanae Prodromus* ([19] pag. 152), and *locus classicus* includes the locality Caffarella ("Alla Caffarella presso il Fonte di Egeria..." = At Caffarella near Egeria's spring). Lacking specimens of original material, Iamonico [101] designated an iconography (Table II in [19]) as lectotype; further, a specimen (deposited at RO; Figure 73) collected by E. Mauri in Caffarella locality in July 1832 was designated as epitype. *Silene trinervia* is currently a heterotypic synonym of *S. gallinyi* Rchb.

**Figure 73.** Epitype of *Silene trinervia* (RO-Herbarium Romano no. 6762).

*Typha latifolia* **L.**: on the basis of a recent study on some Linnaean names of aquatic plants by Iamonico and Iberite [102], an epitype was designated for this Linnaean name. The exsiccatum, deposited at RO (Figure 74), was collected in Caffarella valley by D. Iamonico on 14 October 2020. The population from which the epitype was collected is that shown in Figure 28 of the present paper.

**Figure 74.** Epitype of *Typha latifolia* (RO s.n.).

#### 4.4.8. Other Notable Species

*Amaranthus hypochondriacus* **L.**: this species, indicated as casual alien for Italy [33], was recently reported as naturalized in the Latium region [103] based on two populations occurring in Roma and Frosinone Provinces. The population in Rome refers to 10–15 individuals growing in the territory of Appia Antica Regional Park, i.e., in the Acquedotti locality along the channel Acqua Mariana (Figure 75). The presence of *A. hypochondriacus* has been documented for 6 years.

**Figure 75.** *Amaranthus hypochondricus* in Acquedotti locality.

*Colocasia esculenta* **(L.) Schott**: this tropical/subtropical Asian species was recently recorded in Latium for the first time as naturalized (first alien status for Italy). The population was found in the Acquedotti locality along channel *Acqua Mariana* (first observation in 2015, last one in 2019 according to Iamonico [104]). The author, by comparing the climate features of Rome and the native distribution range of *C. esculenta*, showed that the occurrence of this species in Rome is probably linked to micro-climatic factors, i.e., (1) soil (sandy sediments submerged during autumn and winter seasons, well-drained, and partially soaked during spring and summer); (2) brightness (low light intensity, which characterizes the site over almost the entire day); (3) air (high humidity related to both the morphology of the site (a gorge) and the close occurrence of a small waterfall (height: 2.5–3.0 m).

I continue to control the population and found it also during the next years (2021 and 2022; Figure 76).

**Figure 76.** *Colocasia esculenta* along channel Acqua Mariana (Acquedotti locality).

*Lemna minuta* **Kunth**: this South American species was first discovered in the Latium region based on collections made in 2007, of which one refers to the Caffarella valley [105]. I am continuing to control the various populations, founding many others both in Caffarella valley and in other parts (mostly in Acquedotti and Tor Marancia localities) where the abundance of the autochthonous *L. minor* decreased over time. Note that the populations of *L. minuta* even survived well through snowfall (Figure 77).

**Figure 77.** *Lemna minuta* in aquatic habitat of *Egeria nymphaeum* during snowfall in February 2018 (Caffarella valley).

*Lupinus albus* **L. subsp.** *graecus* **(Boiss. et Spruner) Franco & Pinto da Silva**: 11 localities of this taxon currently occur in the Latium region, one referring to Caffarella valley [25,106]. I monitored this population over the years, but in 2018 the volcanic slope on which it occurred was destroyed to build paths (Figure 78). During the next 2 years, a part of the area previously occupied by *L. albus* subsp. *graecus* was recolonized by the South American alien *Nassella neesiana* (Trin. et Rupr.) Barkworth (Figure 78); the population of *L. albus* subsp. *graecus* recorded by [25,106] is extinct. Fortunately, I found another population not far from the lost one (never seen before 2018) composed of tens of individuals blooming and fruiting regularly (Figure 79).

**Figure 78.** *Lupinus albus* subsp. *graecus* in Caffarella valley: population lost (**top** photo, dated 2017); *Nassella neesiana* dominated community (**bottom** photo, dated 2022).

**Figure 79.** *Lupinus albus* subsp. *graecus* in Caffarella valley: new population found.

*Plumbago auriculata* **Lam.**: this species was recently recorded in Latium (year 2016) as casual [107]. My discovery in the northeastern sector of Caffarella valley represents the second record for Latium. The population found occurs in shrubby vegetation dominated by *Rubus ulmifolium* and *Sambucus nigra* (Figure 80).

**Figure 80.** *Plumbago auriculata* in shrubby vegetation (Caffarella locality).

#### **5. Discussion and Conclusions**

The present study provides the first comprehensive inventory of the flora of Appia Antica Regional Park, one of the largest protected urban areas in Europe.

The data presented revealed an extraordinary species diversity of the flora, which comprises 714 taxa representing about 43% of the flora of Rome (1649 taxa according to [9]) and about 20% of the flora of the Latium region (3593 taxa according to [108]). This notable datum is mainly linked to the high landscape heterogeneity of the park, which comprises several types of habitats, from those strictly natural (e.g., broad-leaved forests, Mediterranean macchia, Mediterranean riparian woods, humid meadows, helophytic vegetation, etc.) to those strictly anthropogenic (i.e., the segetal and ruderal communities and floras of, respectively, arable crops and disturbed/human-made sites). The main landscape environmental characteristics of the studied area can be, therefore, summarized as:

(1) the persistence of residual patches of natural vegetation (especially in the northern sector of the park), notably concerning those forests (Figure 15) that reveal a rich woody flora (106 taxa of Phanerophytes, corresponding to 14.85% of the total flora). Some patches also have high cultural-historical value, e.g., the so-called "Bosco Sacro" (= Sacread Wood) with various centenarian individuals of *Quercus ilex* subsp. *ilex* (see also [25]); other trees, found throughout the park, are very large and have, therefore, high nature conservation value (Figure 81);

**Figure 81.** Large individual of *Quercus ilex* subsp. *ilex* in Caffarella valley.

(2) the occurrence of quite well preserved aquatic habitats and humid meadows (mostly in Caffarella valley and Tor Marancia locality; Figure 82) that include taxa and vegetation communities not so common in urban areas where their general decline is a widely acknowledged trend across not only Rome [9], but also worldwide [109,110]. Of note, some of these habitats are included in the Annex I of the Habitat Directive of 21 May 1992 (formally known as Council Directive 92/43/EEC on the Conservation of natural habitats and of wild fauna and flora), e.g., that named "*Salix alba* and *Populus alba* galleries" (code 92A0; see Figure 22).

**Figure 82.** Humid meadow dominated by *Ranunculus repens* L. (Caffarella valley).

(3) a rich anthropogenic flora occuring in cultivated lands (segetal taxa; Figure 29) and human-made habitats (ruderal taxa), e.g., those trampled (Figure 31) and/or grasslands and pastures occurring on nutrient-rich soils (e.g., Figures 33 and 34). Notably, this type of flora plays an important role as a source of floristic richness in metropolitan areas [9,111–113]. It is also noteworthy that some species of conservation interest grow in anthropogenic environments, as crop wild relatives [114,115].

The anthropogenic flora includes, in addition to native species well-adapted to human disturbance, exotic taxa that decrease the quality of the biodiversity and can negatively impact the natural vegetation. Some naturalized and invasive species are particularly dangerous in the territory of the park and threaten native ones; examples are:


**Figure 83.** *Lemna minuta* monophytic community on a pond in Caffarella valley.

**Figure 84.** *Fallopia baldschuanica* on shrubby vegetation along a path running near Appia Nuova street.

**Figure 85.** Anthropogenic woody vegetation dominated by *Ailanthus altissima* (Caffarella valley).

**Figure 86.** *Lonicera japonica* on *Crataegus monogyna* (**left** photo) and *Viburnum tinus* (**right** photo) (Acquedotti locality).

Concerning the segetal flora, some allochthonous species impact the wheat crops from an economic point of view, since populations densely grow on fields, imposing significantly increased costs for their management and reducing wheat yields [29,117]. *Amaranthus retroflexus* L. and *A. hybridus* L. are the main dangerous species observed in the park (Figure 87).

**Figure 87.** Dense population of young individuals of *Amaranthus sp. pl.* on a crop (Acquedotti locality).

(4) the flora of archeological sites: these areas have a significant role in enhancing plant diversity in cities, being refuges of natural flora and vegetation in the urban ecosystems [9,118–120].

Appia Antica Regional Park is rich in archeological elements (e.g., aqueducts, catacombs, churches, tombs, sepulchers, temples, etc.) that also significatively contribute to the landscape structure (see Section "3.5. Landscape remarks and actual vegetation physiognomies"). Several taxa occur on both the top and vertical walls of the ruins (see also [120]), and they consist of herbs (therophytes, hemicryptophytes, and geophytes), subshrubs (chamaephytes), shrubs (caespitose phanerophytes), or even trees (scapose phanerophytes). Various species have cliffs or rocky places as their natural habitat and found the same dry environmental conditions by growing on archeological remains, which, therefore, can be considered as secondary habitats for them; examples of species with different biological forms that occur in the park are (Figure 88): *Fumaria officinalis* L. subsp. *officinalis* (therophyte), *Umbilicus rupestris* (Salisb.) Dandy (bulbose geophyte), *Micromeria graeca* (L.) Benth. ex Rchb. (suffruticose chamaephyte), *Capparis orientalis* (nano-phanerophyte), *Hedera helix* L. subsp. *helix* (liana), and *Olea europea* L (scapose phanerophyte).

In some cases, the archeological sites allow the presence of species not common in the whole region of Latium. An example is *Parietaria lusitanica* L. subsp. *lusitanica*, which occurs in Rome territory mostly in the northern sector, whereas only three scattered populations are currently recorded toward the south, and one of them is located in Appia Antica Regional Park on walls along Appia Antica street near Cecilia Metella sepulcher [121].

Finally, alien species also occur on these sites and they can inflict damage on their structures, mainly due to their roots, which induce both chemical and mechanical forms of deterioration [122]. Examples for Appia Antica Park are *Ailanthus altissima*, *Phoenix canariensis* H.Wildpret, and *Robinia pseudoacacia*, which can be observed at the base or on the vertical walls of ancient Roman aqueducts (Figure 89).

**Figure 88.** *Fumaria officinalis* subsp. *officinalis* on Felix's Aqueduct (top-left photo); *Umbilicus rupestris* on the vertical wall of the aqueduct located in front of the *Egeria* nymphaeum, in Caffarella valley (top-right photo); *Micromeria graeca* on Claudio's Aqueduct (central-left photo); *Capparis orientalis* on Felix's Aqueduct (central-right photo); *Hedera helix* subsp. *helix* on Felix's Aqueduct (bottom-left photo); *Olea europaea* on Claudio's Aqueduct (bottom-right photo).

**Figure 89.** *Ailanthus altissima* (**top** photo) and *Phoenix canariensis* (**bottom** photo) at the base of Felix's Aqueduct (Tor Fiscale and Acquedotti localities, respectively).

In conclusion this study, by providing (1) an extensive set of floristic data on the species diversity of Appia Antica Regional Park, including its structure in biological, ecological and biogeographical terms and floristic novelties at the regional, national and European levels, (2) an overview of the landscape structure and vegetation physiognomies, and (3) an emphasis on the non-native flora and its ecological, social and economic impacts on autochthonous flora, archeological ruins and crops, gives a general base framework for guiding future scientific and applied researches and landscape action plans. Concerning basic scientific studies, the first one should investigate the vegetation communities in detail (based on the physiognomies listed in the present paper and the Land Use map published by Iamonico [40]) carried out on phytosociological plots. With regard to applied research and landscape action plans, there are many possibilities, e.g., eradication of dangerous alien species (see e.g., [123]), planning of education paths for natural flora and vegetation (see e.g., [124]), urban forestry (see e.g., [125]), etc. All of these research efforts and actions have, as their final aim, the conservation of biodiversity in terms of environmental sustainability [126], providing, in turn and by green infrastructure and ecosystem services implementation, benefits for urban residents in the form of improved human health and well-being (see e.g., [127,128]).

**Funding:** No external funding was obtained for this research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** I acknowledge the Directors and Curators of the herbaria mentioned for their support during my visits or for loans of specimens/photographs, with special thanks to D. Magri, M. Iberite, A. Tilia, and M. Pierfranceschi (Herbarium RO). Thanks are due M. Del Monte (Univerisity of Rome Sapienza, Italy) for the information on geomorphological literature; to E. Fanfarillo (University of Siena, Italy) for the discussion about the segetal flora; to M. Arianoutsou (University of Athens, Greece) and F. Verloove (Meise Botanic Garden, Belgium) for the information about the occurrence of *Rosa chinensis* in Greece and Belgium; to F. Luebert (University of Bonn) for the help in identification of *Heliotropium amplexicaule*. I also extend my thanks to L. Virzì (Rome) for permission to reproduce his picture of the mine "Cava di Fioranello" (Figure 8).

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


#### **Appendix A**

Inventory of the taxa occurring in Appia Antica Regional Park. Names of families in nonbold and uppercase; groups higher than families in bold and uppercase. Abbreviations: A: alien (with CAS: casual taxon; NAT: naturalized taxon; INV: invasive taxon); E: endemic; C: cryptogenic; NC: no longer recorded (not confirmed, but documented by the literature and/or herbaria specimens) S: segetal taxon (with, if occurring, sS: stritcly segetal taxon; cS: characteristic segetal taxon).

**FERNS And ALLIES** EQUISETACEAE Michx. ex DC. *Equisetum arvense* L., S *Equisetum ramosissimum* Desf., S *Equisetum telmateia* Ehrh., S

DENNSTAEDTIACEAE Lotsy *Pteridium aquilinum* (L.) Kuhn subsp. *aquilinum*

NEPHROLEPIDACEAE Pic. Serm. *Nerpholepis cordifolia* (L.) C.Persl, A NAT

ASPLENIACEAE Newman *Asplenium onopteris* L. *Asplenium scolopendrium* L. subsp. *scolopendrium Asplenium trichomanes* L. subsp. *quadrivalens* D.E. Meyer

#### **GYMNOSPERMS**

PINACEAE Spreng. ex F.Rudolphi *Pinus halepensis* Mill., A CAS *Pinus pinea* L., A CAS

CUPRESSACEAE Gray *Cupressus sempervirens* L., A CAS

#### **ANGIOSPERMS**

LAURACEAE Juss. *Laurus nobilis* L.

ARACEAE Juss. *Arum italicum* Mill. subsp. *italicum*, S *Biarum tenuifolium* (L.) Schott subsp. *tenuifolium Colocasia esculenta* (L.) Schott, A NAT *Lemna gibba* L. *Lemna minor* L. *Lemna minuta* Kunth, A INV *Zantedeschia aethiopica* (L.) Spreng., A NAT

ALISMATACEAE Vent. *Alisma lanceolatum* With. *Alisma plantago-aquatica* L.

DIOSCOREACEAE R.Br. *Dioscorea communis* (L.) Caddick & Wilkin

SMILACACEAE Vent. *Smilax aspera* L.

ORCHIDACEAE Juss.

*Anacamptis coriophora* (L.) R.M.Bateman, Pridgeon & M.W.Chase *Anacamptis morio* (L.) R.M.Bateman, Pridgeon & M.W.Chase *Anacamptis papilionacea* (L.) R.M.Bateman, Pridgeon & M.W.Chase *Ophrys apifera* Huds. *Ophrys incubacea* Bianca *Ophrys sphegodes* Mill. subsp. *sphegodes Ophrys tenthredinifera* Willd. *Orchis laxiflora* Lam. *Serapias lingua* L. *Serapias parviflora* Parl. *Serapias vomeracea* (Burm. fil.) Briq. *Spiranthes spiralis* (L.) Chevall.

IRIDACEAE Juss. *Crocus biflorus* Mill. *Gladiolus italicus* Mill., cS *Iris germanica* L., A NAT *Limniris pseudacorus* (L.) Fuss *Romulea bulbocodium* (L.) Sebast. et Mauri *Romulea columnae* Sebast. et Mauri

ASPHODELACEAE Dumort. *Aloe maculata* All., A NAT *Asphodelus ramosus* L. subsp. *ramosus*

AMARYLLIDACEAE J.St.-Hil. *Allium ampeloprasum* L. *Allium chamaemoly* L. subsp. *chamaemoly Allium neapolitanum* Cirillo *Allium roseum* L., S *Allium triquetrum* L. *Allium vineale* L., S *Ipheion uniflorum* (Lindl.) Raf., A CAS *Leucojum aestivum* L., A CAS *Narcissus pseudonarcissus* L., A CAS *Narcissus tazetta* L. subsp. *tazetta*, S *Sternbergia lutea* (L.) Ker-Gawler

ASPARAGACEAE Juss. *Agave americana* L., A CAS *Asparagus acutifolius* L. *Asparagus offininalis* L.subsp. *officinalis Bellevalia romana* (L.) Reichenb., S *Chlorophytum comosum* (Thunb.) Jacques, A CAS *Muscari comosum* (L.) Mill., cS *Muscari neglectum* Guss. ex Ten.

*Loncomelos narbonense* (L.) Raf., S *Ornithogalum divergens* Boreau, S *Ruscus aculeatus* L. *Yucca gloriosa* L., A CAS

ARECACEAE Bercht. & J.Presl *Chamaerops humilis* L., A CAS *Phoenix canariensis* H. Wildpret, A CAS

TYPHACEAE Juss. *Sparganium erectum* L. *Typha latifolia* L.

CANNACEAE Juss. *Canna indica* L., A NAT

JUNCACEAE Juss. *Juncus articulatus* L. *Juncus inflexus* L. subsp. *inflexus Luzula campestris* (L.) DC. subsp. *campestris Luzula forsteri* (Sm.) DC.

CYPERACEAE Juss. *Carex distachya* Desf. *Carex distans* L. *Carex divisa* Hudson *Carex flacca* Schreb. subsp. *erythrostachys* (Hoppe) Holub *Carex hirta* L. *Carex otrubae* Podp. *Carex pendula* Hudson *Cyperus alternifolius* L. subsp. *flabelliformis* Kük., A NAT *Cyperus badius* Desf. *Cyperus longus* L. *Cyperus rotundus* L., S *Eleocharis palustris* (L.) Roem. et Schult., S *Scirpoides holoschoenus* (L.) Soják

POACEAE Barnhart *Agrostis stolonifera* L. subsp. *stolonifera*, S *Aira cupaniana* Guss., S *Aira elegans* Willd. subsp. *elegans Alopecurus myosuroides* Hudson subsp. *myosuroides*, sS *Anisantha diandra* (Roth) Tutin ex Tzvelev, cS *Anisantha madritensis* (L.) Nevski subsp. *madritensis*, S *Anisantha rigida* (Roth) Hyl., S *Anisantha sterilis* (L.) Nevski, S *Arundo donax* L., A INV *Arundo plinii* Turra, S *Avena barbata* Pott. ex Link subsp. *barbata Avena sativa* L. subsp. *sativa*, A NAT *Avena sterilis* L. subsp. *sterilis*, cS *Brachypodium distachyon* (L.) P. Beauv., S *Brachypodium rupestre* (Host) Roemer et Schultes, S *Brachypodium sylvaticum* (Huds.) Beauv. subsp. *sylvaticum* *Bromopsis erecta* (Huds.) Fourr., S *Bromus commutatus* Schrad., S *Bromus hordeaceus* L. subsp. *hordeaceus*, S *Catabrosa aquatica* (L.) P. Beauv., NC *Catapodium rigidum* (L.) C.E. Hubb., S *Cynodon dactylon* (L.) Pers., S *Dactylis glomerata* L. subsp. *glomerata*, S *Dactylis glomerata* L. subsp. *hispanica* (Roth) Nyman *Dasypyrum villosum* (L.) P. Candargy, S *Digitaria sanguinalis* (L.) Scop., S *Echinochloa crus-galli* (L.) Beauv., S *Ehrharta erecta* Lam., A CAS *Eleusine indica* (L.) Gaertn. *Elymus repens* (L.) Gould subsp. *repens*, S *Festuca danthonii* Asch. & Graebn. subsp. *danthonii*, S *Festuca ligustica* (All.) Bertol., S *Festuca myuros* L. subsp. **myuros**, S *Gaudinia fragilis* (L.) Beauv., S *Holcus lanatus* L., S *Hordeum bulbosum* L. *Hordeum murinum* L. subsp. *leporinum* (Link) Arcang., S *Hordeum murinum* L. subsp. *murinum*, S *Hordeum secalinum* Schreb. *Hyparrhenia hirta* (L.) Stapf subsp. *hirta Imperata cylindrica* (L.) Raeusch. *Lagurus ovatus* L. *Lolium arundinaceum* (Schreb.) Darbysh. subsp. *arundinaceum Lolium multiflorum* Lam. subsp. *multiflorum*, S *Lolium perenne* L., S *Lolium rigidum* Gaudin subsp. *rigidum*, S *Macrobriza maxima* (L.) Tzvelev, S *Nassella neesiana* (Trin. et Rupr.) Barkworth, A NAT *Oloptum miliaceum* (L.) Röser & H.R.Hamasha *Parapholis cylindrica* (Willd.) Romero Zarco, NC *Paspalum distichum* L. *Phalaris aquatica* L., S *Phalaris paradoxa* L., cS *Phleum pratense* L. subsp. *pratense*, S *Phragmites australis* (Cav.)Trin. ex Steud. subsp. *australis*, S *Phyllostachys reticulata* (Rupr.) K.Koch, A NAT *Poa annua* L., S *Poa bulbosa* L., S *Poa pratensis* L., S *Poa trivialis* L., S *Polypogon monspeliensis* (L.) Desf., S *Rostraria cristata* (L.) Tzvelev *Setaria italica* (L.) P.Beauv. subsp. *viridis* (L.) Thell., A NAT, S *Setaria verticillata* (L.) P.Beauv. *Sorghum halepense* (L.) Pers., S *Trisetaria panicea* (Lam.) Paunero, S *Triticum aestivum* L., A CAS *Triticum negletum* (Req. ex Bertol.) Greuter, S *Triticum vagans* (Jord. & Fourr.) Greuter, S

RANUNCULACEAE Juss. *Anemone hortensis* L., S *Clematis vitalba* L. *Delphinium consolida* L. subsp. *consolida*, sS *Delphinium halteratum* Sm. subsp. *halteratum Ficaria verna* Huds. subsp. *ficariiformis* (F.W.Schultz) B.Walln., S *Nigella damascena* L., S *Ranunculus bulbosus* L., S *Ranunculus lanuginosus* L., S *Ranunculus parviflorus* L., S *Ranunculus peltatus* Schrank *Ranunculus repens* L., S *Ranunculus sardous* Crantz, S *Thalictrum aquilegiifolium* L.

PLATANACEAE T.Lestib. *Platanus hispanica* Mill. ex Münchh.

PAPAVERACEAE Juss. *Chelidonium majus* L. *Fumaria capreolata* L. subsp. *capreolata*, S *Fumaria officinalis* L. subsp. *officinalis*, S *Papaver dubium* L., C, sS *Papaver hybridum* L., sS *Papaver rhoeas* L., C, sS *Papaver somniferum* L.

CRASSULACEAE J.St.-Hil. *Kalanchoe daigremontiana* Raym., A NAT *Petrosedum sediforme* (Jacq.) Grulich subsp. *sediforme Phedimus stellatus* (L.) Raf. *Sedum caespitosum* (Cav.) DC. *Sedum cepaea* L. *Umbilicus horizontalis* (Guss.) DC. *Umbilicus rupestris* (Salisb.) Dandy

SAXIFRAGACEAE Juss. *Saxifraga trydactilites* L., S

VITACEAE Juss. *Parthenocissus quinquefolia* (L.) Planch., A NAT *Vitis vinifera* L., A NAT

ZYGOPHYLLACEAE R.Br. *Tribulus terrestris* L.

FABACEAE Lindl. *Acacia dealbata* Link, A NAT *Astragalus glycyphyllos* L., S, NC *Astragalus hamosus* L., S *Astragalus pelecinus* (L.) Barneby *Bituminaria bituminosa* (L.) C.H.Stirt. *Cercis siliquastrum* L. subsp. *siliquastrum Cytisus villosus* Pourr.

*Denisophytum bessac* (Choiv.) E.Gagnon & G.P.Lewis, A NAT *Emerus major* Mill. subsp. *major Ervilia hirsuta* (L.) Opiz, sS *Ervum tetraspermum* L., sS *Galega officinalis* L., S *Gleditsia triacanthos* L., A CAS *Hymenocarpos circinnatos* (L.) Savi *Lathyrus annuus* L., cS *Lathyrus aphaca* L. subsp. *aphaca*, cS *Lathyrus cicera* L., S *Lathyrus clymenum* L., S *Lathyrus latifolius* L., S *Lathyrus oleraceus* Lam. subsp. *biflorus* (Raf.) H.Schaef., Coulot & Rabaute *Lotus corniculatus* L., S *Lotus ornithopodioides* L. *Lotus tenuis* Waldst. & Kit. ex Willd., S *Lupinus angustifolius* L. *Lupinus albus* L. subsp. *graecus* (Boiss. & Spruner) Franco & P.Silva *Medicago arabica* (L.) Huds., S *Medicago falcata* L. subsp. *falcata*, S *Medicago lupulina* L., S *Medicago minima* (L.) L., S *Medicago orbicularis* (L.) Bartal., S *Medicago polymorpha* L. *Medicago rigidula* (L.) All., S *Medicago sativa* L. *Onobrychis viciifolia* Scop. *Ononis spinosa* L. subsp. *antiquorum* (L.) Arcangeli *Ornithopus compressus* L., S *Robinia pseudoacacia* L., A INV *Scorpiurus muricatus* L., S *Securigera cretica* (L.) Lassen, S *Securigera securidaca* (L.) Degen et Dörfler, S *Spartium junceum* L. *Trifolium angustifolium* L. subsp. *angustifolium*, S *Trifolium arvense* L., S *Trifolium campestre* Schreb., S *Trifolium cherleri* L., S *Trifolium echinatum* M. Bieb., S *Trifolium incarnatum* L. subsp. *incarnatum Trifolium incarnatum* L. subsp. *molinerii* (Balb. ex Hornem.) Ces. *Trifolium ligusticum* Balb. ex Loisel. *Trifolium micranthum* Viv. *Trifolium nigrescens* Viv. subsp. *nigrescens*, S *Trifolium pratense* L. subsp. *pratense*, S *Trifolium repens* L., S *Trifolium resupinatum* L., S *Trifolium scabrum* L., S *Trifolium squarrosum* L. *Trifolium stellatum* L., S *Trifolium subterraneum* L., S *Trifolium suffocatum* L. *Trifolium tomentosum* L., S *Trifolium vesiculosum* Savi

*Trigonella alba* (Medik.) Coulot & Rabaute *Trigonella altissima* (Thuill.) Coulot & Rabaute *Trigonella smalii* Coulot & Rabaute *Vicia angustifolia* L., S *Vicia bithynica* (L.) L., cS *Vicia dasycarpa* Ten., S *Vicia faba* L., A CAS *Vicia hybrida* L., S *Vicia incana* Gouan *Vicia melanops* Sm. *Vicia narbonensis* L., S *Vicia sativa* L. *Vicia serratifolia* Jacq. ROSACEAE Juss. *Aphanes arvensis* L., sS *Agrimonia eupatoria* L. subsp. *eupatoria*, S *Crataegus monogyna* Jacq. *Geum urbanum* L. *Malus domestica* (Suckow) Borkh., A CAS *Malus sylvestris* Mill. *Potentilla recta* L., S *Potentilla reptans* L., S *Poterium sanguisorba* L. subsp. *balearicum* (Bourg. ex Nyman) Stace, S *Prunus cerasifera* Ehrh. var. pissardii (Carrière) C.K.Schneid *Prunus domestica* L., A CAS *Prunus spinosa* L. *Pyracantha coccinea* M.Y.Roemer *Pyrus communis* L. subsp. *pyraster* (L.) Ehrh. *Pyrus spinosa* Forssk. *Rhaphiolepis bibas* (Lour.) Galasso & Banfi, A CAS *Rosa canina* L. *Rosa chinensis* Jacq. var. *semperflorens* (Curtis) Koehne *Rosa gallica* L. *Rosa sempervirens* L. *Rubus caesius* L. *Rubus ulmifolius* Schott *Sorbus domestica* L.

RHAMNACEAE Juss. *Paliurus spina-christi* Mill. *Rhamnus alaternus* L. *Ziziphus jujuba* Mill., A CAS

ULMACEAE Mirb. *Ulmus minor* Mill.

CANNABACEAE Martinov *Celtis australis* L. subsp. *australis Humulus lupulus* L.

MORACEAE Gaudich. *Broussonetia papyrifera* (L.) Vent., A NAT *Ficus carica* L.

*Maclura pomifera* (Raf.) C.K. Schneid., A CAS *Morus alba* L., A CAS

URTICACEAE Juss. *Parietaria judaica* L. *Parietaria lusitanica* L. subsp. *susitanica Urtica dioica* L., S *Urtica membranacea* Poir. *Urtica pilulifera* L. *Urtica urens* L., S

FAGACEAE Dumort. *Castanea sativa* Mill. *Quercus cerris* L. *Quercus dalechampii* Ten. *Quercus frainetto* Ten. *Quercus ilex* L. subsp. *ilex Quercus pubescens* Willd. subsp. *pubescens Quercus robur* L. subsp. *robur Quercus suber* L.

JUGLANDACEAE DC. ex Perleb *Juglans regia* L., A CAS

CUCURBITACEAE Juss. *Bryonia dioica* Jacq. *Cucurbita maxima* Duchense subsp. *maxima*, A CAS *Ecballium elaterium* (L.) A. Rich., S

CELASTRACEAE R.Br. *Euonymus europaeus* L.

OXALIDACEAE R.Br. *Oxalis articulata* Savigny, A NAT, S *Oxalis corniculata* L., S *Oxalis dillenii* Jacq., A NAT, S *Oxalis pes-caprae* L., A NAT, S

VIOLACEAE Batsch *Viola alba* Besser subsp. *denhardtii* (Ten.) W.Becker, S *Viola arvensis* Murray, S *Viola reichenbachiana* Jordan ex Boreau

SALICACEAE Mirb. *Populus alba* L. subsp. *alba Populus nigra* L. *Salix alba* L. subsp. *alba*

PASSIFLORACEAE Juss. ex Roussel *Passiflora caerulea* L., A CAS

LINACEAE DC. ex Perleb *Linum usitatissimum* L. subsp. *angustifolium* (Huds.) Thell., S *Linum strictum* L.

HYPERICACEAE Juss. *Hypericum perforatum* L., S

EUPHORBIACEAE Juss. *Euphorbia characias* L. *Euphorbia exigua* L. subsp. *exigua*, sS *Euphorbia falcata* L. subsp. *falcata*, sS *Euphorbia helioscopia* L. subsp. *helioscopia*, S *Euphorbia peplus* L., S *Euphorbia platyphyllos* L. *Euphorbia prostrata* Aiton, A NAT, S *Euphorbia pulcherrima* Willd. ex Klotzsch, A CAS *Euphorbia terracina* L., S *Mercurialis annua* L., S

GERANIACEAE Juss. *Erodium acaule* (L.) Bech. & Thell. *Erodium ciconium* (L.) L'Hér., S *Erodium cicutarium* (L.) L'Hér., S *Erodium malacoides* (L.) L'Hér. subsp. *malacoides*, S *Erodium moschatum* (L.) L'Hér. *Geranium columbinum* L., S *Geranium dissectum* L., S *Geranium molle* L., S *Geranium robertianum* L., S *Geranium rotundifolium* L., S *Geranium sanguineum* L., S

ONAGRACEAE Juss. *Epilobium hirsutum* L. *Epilobium lanceolatum* Sebast. & Mauri *Epilobium parviflorum* Schreber, S *Epilobium tetragonum* L. subsp. *tournefortii* (Michalet) H.Lév., S

LYTHRACEAE J.St.-Hil. *Lythrum hyssopifolia* L., S *Lythrum salicaria* L., S *Punica granatum* L., A CAS

MYRTACEAE Juss. *Eucalyptus camaldulensis* Dehnh. subsp. *camaldulensis*, A CAS

ANACARDIACEAE R.Br. *Pistacia lentiscus* L. *Pistacia terebinthus* L. subsp. *terebinthus*

SAPINDACEAE Juss. *Acer campestre* L. *Acer monspessulanum* L. subsp. *monspessulanum Acer negundo* L., A NAT

RUTACEAE Juss. *Ruta chalepensis* L.

SIMAROUBACEAE DC. *Ailanthus altissima* (Mill.) Swingle, A INV

MELIACEAE Juss. *Melia azedarach* L., A CAS

MALVACEAE Juss. *Althaea cannabina* L. *Malva multiflora* (Cav.) Soldano, Banfi & Galasso, S *Malva nicaeensis* All., S *Malva punctata* (All.) Alef. *Malva sylvestris* L., S

RESEDACEAE Martinov *Reseda alba* L., S *Reseda lutea* L. subsp. *lutea*, S *Reseda phyteuma* L. subsp. *phyteuma*, S *Tarenaya spinosa* (Jacq.) Raf., A CAS

CAPPARACEAE Juss. *Capparis orientalis* Veill.

BRASSICACEAE Burnett *Alliaria petiolata* (Bieb.) Cavara et Grande, S *Arabis hirsuta* (L.) Scop. *Berteroa obliqua* (Sm.) DC. *Brassica oleracea* L., A CAS *Bunias erucago* L., S *Calepina irregularis* (Asso) Thell., S *Capsella bursa-pastoris* (L.) Medik., S *Capsella rubella* Reuter, S *Cardamine hirsuta* L., S *Diplotaxis erucoides* (L.) DC subsp. **erucoides**, S *Diplotaxis tenuifolia* (L.) DC., S *Draba verna* L. *Lepidium graminifolium* L. *Lepidium virginicum* L., A CAS *Lunaria annua* L., A CAS *Lunaria rediviva* L. *Microthlaspi perfoliatum* (L.) F.K.Mey., S *Nasturtium officinale* W.T. Aiton *Neslia paniculata* (L.) Desv. subsp. *thracica* (Velen.) Bornm., cS *Raphanus raphanistrum* L. subsp. *landra* (Moretti ex DC.) Bonnier & Layens, S *Rapistrum rugosum* (L.) All., cS *Sinapis alba* subsp. *alba*, cS *Sinapis arvensis* L. subsp. *arvensis*, cS *Sisymbrium officinale* (L.) Scop.

SANTALACEAE R.Br. *Osyris alba* L.

PLUMBAGINACEAE Juss. *Plumbago auriculata* Lam., A CAS *Plumbago europaea* L.

POLYGONACEAE Juss. *Fallopia baldschuanica* (Regel) Holub, A NAT *Fallopia dumetorum* (L.) J. Holub, S *Persicaria amphibia* (L.) Delabre *Persicaria lapathifolia* (L.) Delarbre *Persicaria maculosa* Gray, S *Polygonum aviculare* L., S *Polygonum rurivagum* Jord. ex Boreau, S *Rumex bucephalophorus* L. subsp. *bucephalophorus*, S *Rumex conglomeratus* Murray, S *Rumex crispus* L., S *Rumex obtusifolius* L. subsp. *obtusifolius*, S *Rumex pulcher* L. subsp. *pulcher*, S CARYOPHYLLACEAE Juss. *Arenaria leptoclados* (Reichenb.) Guss., S *Arenaria serpyllifolia* L., S *Cerastium brachypetalum* Desp. ex Pers., S *Cerastium glomeratum* Thuill., S *Cerastium ligusticum* Viv., S *Dianthus armeria* L. subsp. *armeria Herniaria glabra* L., cS *Herniaria hirsuta* L. subsp. *hirsuta*, S *Petrorhagia saxifraga* (L.) Link subsp. *saxifraga*, S *Petrorhagia dubia* (Raf.) G.López & Romo *Polycarpon tetraphyllum* (L.) subsp. *diphyllum* (Cav.) O. Bolòs et Font Quer *Polycarpon tetraphyllum* L. subsp. *tetraphyllum*, S *Sagina apetala* Ard. subsp. *apetala Saponaria officinalis* L. *Silene bellidifolia* Jacq. *Silene conica* L., S *Silene gallica* L., S *Silene gallinyi* Rchb., NC *Silene italica* (L.) Pers. subsp. *italica*, S *Silene latifolia* Poir., S *Silene nocturna* L., S *Silene pendula* L. *Silene vulgaris* (Moench) Garcke subsp. *tenoreana* (Colla) Soldano & F.Conti, S *Silene vulgaris* (Moench.) Garcke subsp. *vulgaris*, S *Spergularia rubra* (L.) J.Presl & C.Presl, S *Stellaria media* (L.) Vill. subsp. *media*, S *Stellaria neglecta* Weihe, S *Stellaria pallida* (Dumort.) Crép. *Spergula arvensis* L., sS CHENOPODIACEAE Vent.

*Atriplex patula* L., S *Beta vulgaris* L. subsp. *vulgaris Chenopodiastrum murale* (L.) S.Fuentes, Uotila & Borsch, S *Chenopodium opulifolium* Schrader ex Koch et Ziz, S *Chenopodium album* L., S *Chenopodium vulvaria* L.

*Dysphania ambrosioides* (L.) Mosyakin & Clemants, A NAT *Dysphania multifida* (L.) Mosyakin & Clemants, A CAS *Lipandra polysperma* (L.) S.Fuentes, Uotila & Borsch, S *Oxybasis urbica* (L.) S.Fuentes, Uotila & Borsch

AMARANTHACEAE Juss. *Amranthus albus* L., A NAT *Amaranthus blitoides* S. Watson, A NAT *Amaranthus blitum* L. subsp. *blitum Amaranthus deflexus* L., A INV, S *Amaranthus graecizans* subsp. *silvestris* (Vill.) Brenan, S *Amaranthus hybridus* L., A INV, S *Amaranthus retroflexus* L., A INV, S *Amaranthus viridis* L., A INV *Polycnemum heuffelii* Láng" C, NC

PHYTOLACCACEAE R.Br. *Phytolacca americana* L., A INV

NYCTAGINACEAE Juss. *Mirabilis jalapa* L., A NAT

BASELLACEAE Raf. *Anredera cordifolia* (Ten.) Steenis, A NAT

PORTULACACEAE Juss. *Portulaca oleracea* L., S

CACTACEAE Juss. *Opuntia ficus-indica* (L.) Mill., A NAT

CORNACEAE Bercht. & J.Presl *Cornus mas* L. *Cornus sanguinea* L. subsp. *sanguinea*

EBENACEAE Gürke *Diospyros kaki* Thunb., A CAS

HYDRANGEACEAE Dumort. *Hydrangea macrophylla* (Thunb.) Ser., A NAT

PRIMULACEAE Batsch ex Borkh. *Cyclamen hederifolium* Aiton *Cyclamen repandum* Sm. *Lysimachia arvensis* (L.) U.Manns & Anderb. subsp. *arvensis*, cS *Lysimachia foemina* (Mill.) U.Manns & Anderb., S

ERICACEAE Juss. *Arbutus unedo* L.

RUBIACEAE Juss. *Cruciata laevipes* Opiz, S *Galium album* Mill. (*G. erectum* Syme) subsp. *album*, S *Galium aparine* L., S

*Galium murale* (L.) All. *Galium parisiense* L. *Rubia peregrina* L. *Sherardia arvensis* L., S *Theligonum cynocrambe* L. *Valantia muralis* L.

GENTIANACEAE Juss. *Blackstonia perfoliata* (L.) Huds. subsp. *perfoliata Centaurium erythraea* Rafn subsp. *erythraea*, S *Centaurium maritimum* (L.) Fritsch *Centaurium tenuiflorum* (Hoffmanns. et Link) Fritsch subsp. *acutiflorum* (Schott) Zeltner, S

APOCYNACEAE Juss. *Nerium oleander* L., A CAS *Trachelospermum jasminoides* (Lindl.) Lem., A CAS *Vinca major* L. subsp. *major Vinca minor* L.

CONVOLVULACEAE Juss. *Convolvulus arvensis* L., S *Convolvulus cantabrica* L. *Convolvulus sepium* L. *Convolvulus silvaticus* Kit. *Cuscuta campestris* Yunck, A NAT *Cuscuta cesattiana* Bertol., A CAS *Ipomoea indica* (Burm.) Merr., A CAS

SOLANACEAE Juss. *Datura stramonium* L., S *Lycium chinense* Mill., A NAT *Solanum chenopodioides* Lam., A NAT *Solanum dulcamara* L. *Solanum lycopersicum* L., A NAT *Solanum nigrum* L., S *Solanum pseudocapsicum* L., A CAS *Solanum villosum* Mill., S

BORAGINACEAE Juss. *Anchusa azurea* Mill., S *Anchusa undulata* L. subsp. *hybrida* (Ten.) Bég., S *Borago officinalis* L., S *Buglossoides arvensis* (L.) I.M.Johnst., cS *Cynoglossum creticum* Mill., S *Echium italicum* L. subsp. *italicum Echium parviflorum* M\ch, S *Echium plantagineum* L., S *Echium vulgare* L., S *Myosotis arvensis* (L.) Hill subsp. *arvensis*, cS *Myosotis ramosissima* Rochel subsp. *ramosissima*, S *Symphytum officinale* L., S *Symphytum tuberosum* L. subsp. *angustifolium* (A.Kerner) Nyman, S

HELIOTROPIACEAE Schrader *Heliotropium amplexicaule* Vahl., A CAS *Heliotropium europaeum* L., S

OLEACEAE Hoffmanns. & Link *Fraxinus angustifolia* Vahl subsp. *oxycarpa* (Willd.) Franco et Rocha Afonso *Fraxinus ornus* L. subsp. *ornus Ligustrum lucidum* W.T. Aiton, A CAS *Ligustrum vulgare* L. *Phillyrea latifolia* L.

PLANTAGINACEAE Juss. *Antirrhinum majus* L. *Antirrhinum tortosuom* Bosc ex Lam, S *Callitriche stagnalis* Scop. *Cymbalaria muralis* G. Gaertn., B. Mey. et Scherb. subsp. *muralis Linaria pelisseriana* (L.) Mill., NC *Linaria purpurea* (L.) Mill., S *Linaria vulgaris* Mill. subsp. *vulgaris*, S *Misopates calycinum* Rothm. *Plantago afra* L. subsp. *afra*, S *Plantago lagopus* L. *Plantago lanceolata* L., S *Plantago major* L., S *Veronica anagallis-aquatica* L., S *Veronica arvensis* L., cS *Veronica cymbalaria* Bodard, S *Veronica hederifolia* L., S *Veronica persica* Poir., S, A INV *Veronica polita* Fr., cS

SCROPHULARIACEAE Juss. *Scrophularia auriculata* L. *Scrophularia peregrina* L. *Verbascum blattaria* L. *Verbascum sinuatum* L., S *Verbascum thapsus* L. subsp. *thapsus*

LAMIACEAE Martinov *Ballota nigra* L. subsp. *meridionalis* (Bég.) Bég., sS *Clinopodium menthifolium* (Host) Merino subsp. *ascendens* (Jord.) Govaerts, S *Clinopodium vulgare* L., S *Lamium amplexicaule* L., S *Lamium bifidum* Cirillo subsp. *bifidum*, S *Lamium maculatum* L. *Lamium purpureum* L., cS *Lycopus europaeus* L. *Marrubium vulgare* L. *Melissa officinalis* L. subsp. *altissima* (Sm) Arcang., S *Mentha aquatica* L. subsp. *aquatica*, S *Mentha pulegium* L. subsp. *pulegium*, S *Mentha suaveolens* Ehrh. subsp. *suaveolens*, S *Micromeria graeca* (L.) Benth. ex Rchb. *Origanum vulgare* L. subsp. *vulgare*

*Prunella laciniata* (L.) L. *Salvia clandestina* L. *Salvia verbenaca* L., S *Thymbra capitata* (L.) Cav. *Micromeria graeca* (L.) Benth. ex Rchb. *Micromeria juliana* (L.) Benth. ex Rchb. *Stachys arvensis* (L.) L., S *Stachys germanica* L. subsp. *germanica*, S, NC *Stachys ocymastrum* (L.) Briq., S *Stachys romana* (L.) E.H.L.Krause, S *Stachys sylvatica* L. *Teucrium chamaedrys* L. subsp. *chamaedrys Teucrium flavum* L. subsp. *flavum*

OROBANCHACEAE Vent. *Bellardia trixago* (L.) All., S *Bellardia viscosa* (L.) Fisch. & C.A.Mey., S *Odontites vernus* (Bellardi) Dumort. subsp. *serotinus* Corb. *Orobanche crenata* Forssk., cS *Orobanche hederae* Vaucher ex Duby *Parentucellia latifolia* (L.) Caruel, S *Phelipanche nana* (Reut.) Soják

VERBENACEAE J.St.-Hil. *Lantana camara* L., A CAS *Verbena officinalis* L., S

ACANTHACEAE Juss. *Acanthus mollis* L. subsp. *mollis Ruellia simplex* C. Wright, A CAS

BIGNONIACEAE Juss. *Campsis radicans* (L.) Bureau, A NAT

CAMPANULACEAE Juss. *Campanula erinus* L., S *Campanula rapunculus* L., S *Jasione montana* L. subsp. *montana Legousia speculum-veneris* (L.) Chaix subsp. *speculum-veneris*, sS

ASTERACEAE Bercht. & J.Presl *Anacyclus radiatus* Loisel. subsp. *radiatus*, S *Andryala integrifolia* L., S *Anthemis arvensis* L. subsp. *arvensis*, cS *Anthemis cotula* L., cS *Arctium minus* (Hill) Bernh. *Artemisia arborescens* (Vaill.) L. *Artemisia verlotiorum* Lamotte, S *Artemisia vulgaris* L., S *Bellis annua* L. subsp. *annua Bellis perennis* L., S *Bellis sylvestris* Cirillo *Bidens subalternans* DC., A CAS *Calendula arvensis* (Vill.) L., S

*Carduus micropterus* (Borbàs) Teyber subsp. *perspinosus* (Fiori) Arènes *Carduus nutans* L. subsp. *nutans*, S *Carduus pycnocephalus* L. subsp. *pycnocephalus*, S *Carlina corymbosa* L., S *Carthamus lanatus* L. *Centaurea calcitrapa* L., S *Centaurea jacea* L. subsp. *angustifolia* (DC.) Gremli, S *Centaurea napifolia* L., S *Centaurea solstitialis* L. subsp. *solstitialis*, S *Chondrilla juncea* L. *Cladanthus mixtus* (L.) Chevall. *Cota tinctoria* (L.) J.Gay subsp. *tinctoria*, S *Cichorium intybus* L., S *Cirsium arvense* (L.) Scop., S *Cirsium creticum* (Lam.) d'Urv. subsp. *triumfetti* (Lacaita) Werner *Coleostephus myconis* (L.) Cass. ex Rchb.f. *Cyanus segetum* L., sS *Crepis bursifolia* L. *Crepis neglecta* L. subsp. *neglecta*, S *Crepis sancta* (L.) Bornm. subsp. *nemausensis* (P.Fourn.) Babc., S, A NAT *Crepis setosa* Haller f., S *Crepis vesicaria* L., S *Dittrichia graveolens* (L.) Greuter *Dittrichia viscosa* (L.) Greuter subsp. *viscosa Erigeron bonariensis* L., A NAT, S *Erigeron canadensis* L., A CAS, S *Erigeron sumatrensis* Retz., A INV, S *Eupatorium cannabinum* L. subsp. *cannabinum*, S *Filago germanica* (L.) Huds., cS *Galactites tomentosus* Moench, S *Galinsoga parviflora* Cav., A CAS, S *Galinsoga quadriradiata* Ruiz et Pav., A NAT, S *Glebionis segetum* (L.) Fourr., S *Helianthus tuberosus* L., A CAS, S *Hypochaeris achyrophorus* L., S *Hypochoeris radicata* L. *Jacobaea aquatica* (Hill) G. Gaertn., B. Mey. & Scherb. *Jacobaea erratica* (Bertol.) Fourr. *Lactuca sativa* L. subsp. *serriola* (L.) Galasso, Banfi, Bartolucci & Ardenghi, S *Leontodon tuberosus* L. *Matricaria chamomilla* L., cS *Onopordum acanthium* L. subsp. *acanthium Onopordum illyricum* L., S *Pallenis spinosa* (L.) Cass. subsp. *spinosa*, S *Pentanema squarrosum* (L.) D.Gut.Larr., Santos- Vicente, Anderb., E.Rico & M.M.Mart.Ort., S *Petasites hybridus* (L.) G.Gaertn., B.Mey. & Scherb. subsp. *hybridus Helminthotheca echioides* (L.) Holub *Picris hieracioides* L. subsp. *hieracioides*, S *Picris hieracioides* L. subsp. *spinulosa* (Guss.) Arcang. *Pulicaria dysenterica* (L.) Bernh., S *Reichardia picroides* (L.) Roth, S *Rhagadiolus stellatus* (L.) Gaertn., S *Scolymus hispanicus* L., S *Senecio leucanthemifolius* Poir., S

*Senecio vulgaris* L., S *Silybum marianum* (L.) Gaertn., S *Sonchus asper* (L.) Hill subsp. *asper*, S *Sonchus oleraceus* L., S *Sonchus tenerrimus* L. *Symphyotrichum squamatum* (Spreng.) G.L.Nesom, A NAT *Taraxacum* F.H.Wigg. sect. **Taraxacum**, S *Tragopogon porrifolius* L., S *Tussilago farfara* L., S *Tyrimnus leucographus* (L.) Cass. *Urospermum dalechampii* (L.) F.W. Schmidt, S *Urospermum picroides* (L.) Scop. ex F.W. Schmidt, S *Xanthium spinosum* L., A NAT **Xanthium italicum** Moretti, S

VIBURNACEAE Rafinesque *Sambucus ebulus* L. *Sambucus nigra* L. *Viburnum tinus* L. subsp. *tinus*

CAPRIFOLIACEAE Juss. *Lonicera etrusca* Santi *Lonicera japonica* Thunb., A NAT

DIPSACACEAE Juss. *Dipsacus fullonum* L. *Knautia arvensis* (L.) Coult., S *Knautia collina* Jord. *Knautia integrifolia* (L.) Bertol. subsp. *integrifolia*, S *Sixalix atropurpurea* (L.) Greuter et Burdet

PITTOSPORACEAE R.Br. *Pittosporum tobira* (Thunb.) W.T. Aiton, A CAS

VALERIANACEAE Batsch *Centranthus ruber* (L.) DC. subsp. *ruber Valerianella eriocarpa* Desv., cS

ARALIACEAE Juss. *Hedera helix* L. subsp. *helix*

APIACEAE Lindl. *Ammi majus* L., S *Ammoides pusilla* (Brot.) Breistr. *Anethum piperitum* Ucria, S *Angelica sylvestris* L. *Anthriscus sylvestris* (L.) Hoffm. *Chaerophyllum temulum* L., S *Conium maculatum* L., S *Daucus carota* L. subsp. *carota*, S *Daucus carota* L. subsp. *maximus* (Desf.) Ball, S *Eryngium campestre* L., S *Ferula communis* L. subsp. *communis*, S *Ferula glauca* L.

*Helosciadium nodiflorum* (L.) W.D.J.Koch subsp. *nodiflorum Oenanthe pimpinelloides* L., S *Oenanthe silaifolia* M.Bieb. *Opopanax chironium* (L.) W.D.J.Koch *Smyrnium olusatrum* L. *Tordylium apulum* L., S *Tordylium maximum* L., S *Torilis arvensis* (Hudson) Link subsp. *arvensis*, S *Torilis japonica* (Houtt.) DC., S

#### **Appendix B**

Selected specimens collected during the field surveys. The list (alphabetical order of the scientific names) refers to floristic novelties, notable species, no longer recorded taxa, and taxa having *loci classici* and/or nomenclatural types collected in Appia Antica Regional Park (see Section "4.4. Floristic Notes").

#### *Aloe maculata* **All. subsp.** *maculata*

Italy, Latium, Roma, Parco Regionale dell'Appia Antica, Valle della Caffarella, incolto, 27 m a.s.l., 22 April 2012, *G. Nicolella s.n.* (RO); *ibidem*, 23 April 2017, *D. Iamonico* (RO).

#### *Amaranthus hypochondriacus* **L.**

Lazio, Rome Province, Rome city, Appia Antica Regional Park, locality Acquedotti, channels, 56 m a.s.l., 6 October 2016, *D. Iamonico s.n.* (RO!); ibidem, 18 October 2020 (RO!).

#### *Anredera cordifolia* **(Ten.) Steenis**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, on *Rubus ulmifolium* dominated community, 41◦51 04 N, 12◦33 12 E, 6 October 2019, *D. Iamonico s.n.* (RO).

#### *Biarum tenuifolium* **(L.) Schott. subsp.** *tenuifolium***.**

Italy. Lazio region, Rome Province, Rome city, Appia Antica Regional Park, shrubs vegetation dominated by *Paliurus spina-christi* Mill., 40–42 m a.s.l., 20 August 2015, *D. Iamonico s.n.* (HFLA No. 4861!).

#### *Bidens subalternans* **DC.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, on a little bridge of channel Acqua Mariana, 41◦51 02 N, 12◦33 21 E, 23 November 2020, *D. Iamonico s.n.* (RO).

#### *Campsis radicans* **(L.) Bureau**

Roma, P.R. dell'Appia Antica, Valle della caffarella, marrana, 23 April 2011, *D. Iamonico s.n.* (RO!); ibidem, 1 September 2013 (RO!); Acquedotti locality, 10 August 2021, *D. Iamonico s.n.* (RO!).

#### *Canna indica* **L.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, banks of channel Acqua Mariana, 15 June 2018, *D. Iamonico s.n.* (RO).

#### *Catabrosa aquatica* **(L.) P.Beauv.**

Italy, Latium, Roma, Via Appia Nuova, lungo fossetto tra Ciampino e S. Maria delle Mole. Luoghi aquitrinosi, 20 April 1944, *G. Montelucci s.n.* (RO-Herbarium Montelucci, sub *Aira aquatica* L.).

#### *Chlorophytum comosum* **(Thumbs.) Jacques**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, banks of channel Acqua Mariana, 15 May 2019, *D. Iamonico s.n.* (RO).

#### *Colocasia esculenta* **(L.) Schott**

Lazio region, Rome, Appia Antica Regional Park, locality Acquedotti, channels, 58 m a.s.l., 8 August 2015, *D. Iamonico s.n.* (HFLA!); ibidem, 19 m a.s.l., 9 May 2020 (RO!).

#### *Cyperus alternifolius* **L. subsp.** *flabelliforme* **Kük.**

Lazio region, Rome, Appia Antica Regional Park, Caffarella valley, channels, 25 May 2017 *D. Iamonico s.n.* (RO!); locality Acquedotti, channels, 56 m a.s.l., 15 May 2022, *D. Iamonico s.n.* (RO!).

#### *Denisophytum bessac* **(Choiv.) E.Gagnon & G.P.Lewis**

Italy, Latium, Rome, Appia Antica Regional Park, via Appia Pignatelli, shrubs, 24 April 2017, *D. Iamonico s.n.* (RO); *ibidem*, 25 May 2022, *D. Iamonico s.n.* (RO).

#### *Diospyrus kaki* **L.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, banks of channel Acqua Mariana, 5 October 2020, *D. Iamonico s.n.* (RO).

#### *Ehrharta erecta* **Lam.**

Italy, Latium, Roma, Valle della Ninfa Egeria, 17 March 1876, *G. G.. Cuboni s.n.* (RO-Herbarium Romano no. 51234).

#### *Epilobium lanceolatum* **Sebast. & Mauri**

Italy, Lazio, Roma nei prati della Caffarella, 3 June 1812, *F. A. Sebastiani* (RO-Herbarium Romano, n. 19436); Lazio, Via delle Vigne presso Albano, June 1853, *E. Rolli* (RO-Herbarium Romano, n. 19443).

#### *Euphorbia pulcherrima* **Willd. ex Klotzsch**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, on riverbed of channel Acqua Mariana, 3 June 2022, *D. Iamonico s.n.* (RO).

#### *Heliotropium amplexicaule* **Vahl.**

Italy, Latium, Rome, April 1928, *s.c.* (RO-HG, no. 32109); Rome, Appia Antica Regional Park, along on central reservation of the Appia Nuova street, 3 June 2022, *D. Iamonico s.n.* (RO).

#### *Hydrangea macrophylla* **(Thunb.) Ser.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, on riverbed of channel Acqua Mariana, 7 August 2016, *D. Iamonico s.n.* (RO); *ibidem*, 29 December 2020, *D. Iamonico s.n.* (RO).

#### *Kalanchoe daigremontiana* **Raym.**

Lazio region, Rome, Appia Antica Regional Park, Caffarella valley, *Arando donax* L. dominated community, 20 June 2020, *D. Iamonico s.n.* (RO!).

#### *Lemna minuta* **Kunth**

Roma, Parco Regionale dell'Appia Antica, loc. Vaccareccia Caffarella, channel and artificial reservoir, 23 m a.s.l., 7 September 2007, *Iamonico s.n.* (RO!, HFLA!)

#### *Linaria pelisseriana* **(L.) Mill.**

Italy, Latium, Roma, Via Ardeatina, 15 May 1892, *L. Salomonsohn s.n.* (RO-Herbarium Romano no. 34429).

#### *Lupinus albus* **L. subsp.** *graecus* **(Boiss. et Spruner) Franco & Pinto da Silva**

Italy, Latium, Rome, Appia Antica Regional Park, Caffarella valley, meadows, 25 April 2021, *D. Iamonico s.n.* (RO).

#### *Melia azedarach* **L.**

Lazio region, Rome, Appia Antica Regional Park, Caffarella valley, shrubs, 25 May 2018, 22 m a.s.l., *D. Iamonico s.n.* (RO!); locality Acquedotti, anthropogenic meadows, 57 m a.s.l., 2 July 2022, *D. Iamonico s.n.* (RO!).

#### *Papaver somniferum* **L.**

Lazio region, Rome, Appia Antica Regional Park, Caffarella valley, meadows, 7 May 2016, *D. Iamonico s.n.* (RO!).

#### *Parapholis cylindrica* **(Willd.) Romero Zarco**

Italy, Latium, Roma, Alla Caffarella, s.d. (XIX century), *s.c. s.n.* (RO-Herbarium Romano no. 55564, sub *Rottboellia subulata* Savi).

#### *Passiflora caerulea* **L.**

Italy, Latium, Rome, Appia Antica Regional Park, Claudio's aqueduct (Porta Furba), 7 June 2022, *D. Iamonico s.n.* (RO).

#### *Plumbago auriculata* **Lam.**

Italy, Latium, Rome, Appia Antica Regional Park, Caffarella valley, *Rubus ulmifolius* dominated community, 20 June 2020, *D. Iamonico s.n.* (RO).

#### *Polycnemum heuffelii* **Láng**

Italy, Latium, Roma, lungo la via Appia Pignatelli, July 1980, *B. Anzalone s.n.* (RO-Herbarium Romano no. 5993).

#### *Punica granatum* **L.**

Italy, Latium, Rome, Appia Antica Regional Park, Caffarella valley, channels, 23 April 2011, *D. Iamonico s.n.* (RO); *ibidem*, 27 June 2012, *D. Iamonico s.n.* (RO); *ibidem*, 01 September 2013, *D. Iamonico s.n.* (RO), *ibidem*, 29 June 2022, *D. Iamonico s.n.* (RO).

#### *Rosa chinensis* **Jacq. var.** *semperflorens* **(Curtis) Koehne**

Italy, Latium, Rome, Appia Antica Regional Park, Caffarella valley, shrub community with *Rubus ulmifolius* Schott and *Parthenocissus quinquefolia* Planch, 23 April 2017, *D. Iamonico s.n.* (RO).

#### *Ruellia simplex* **C.Wright**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, banks of channel Acqua Mariana, 3 June 2022, *D. Iamonico s.n.* (RO).

#### *Sagina apetala*

Italy, Lazio region, Rome city, Appia Antica Region Park, Acquedotti sector, among cobblestones ("basolato") of the ancient Roman road "Latina", 19 May 2020, *Iamonico s.n.* (RO).

#### *Silene gallinyi* **Rchb.**

Italy, Latium, Roma, alla Caffarella (presso catacombe di Pretestato), 6 August 1966, *A. Cacciato s.n.* (RO-Herbarium Anzalone no. 13276), sub *Silene trinervia* Sebast. & Mauri).

#### *Stachys germanica* **L. subsp.** *germanica*

Italy, Latium, Roma, Caffarella, May 1829, *P. Sanguinetti s.n.* (RO-Herbarium Romano no. 38523); incolti aridi della Via Appia Antica pr. La Torre di Cecilia Metella, 3 June 1922, *G. Lusina s.n.* (RO-Herbarium Romano no. 38548); *ibidem* (RO-Herbarium Romano no. 38549); *ibidem* (RO-Herbarium Romano no. 38550); *ibidem* (RO-Herbarium Romano no. 38551).

#### *Tarenaya spinosa* **(Jacq.) Raf.**

Italy, Latium, Roma, macerie a via Appia Pignatelli, 6 September 1966, *B. Anzalone s.n.* (RO-Herbarium Romano s.n., sub *Cleome pungens* Willd); Roma, in via Appia Pignatelli, 12 August 1966, *De Persio s.n.* (RO-Herbarium Romano s.n., sub *Cleome pungens* Willd); Roma, sull'Appia Pignatelli, 14 Semptember 1967, *A. Cacciato s.n.* (RO-Herbarium Romano s.n., sub *Cleome pungens* Willd).

#### *Trachelospermum jasminoides* **(Lindl.) Lem.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, on cliff of channel Acqua Mariana, 15 May 2019, *D. Iamonico s.n.* (RO).

#### *Typha latifolia* **L.**

Italy, Lazio region, Rome administrative province, Appia Antica Regional Park, Caffarella valley, wetlands, 20 m a.s.l., 14 October 2020, *D. Iamonico s.n.* (RO).

#### *Zantedeschia aetiopica* **(L.) Spreng.**

Italy, Latium, Rome, Appia Antica Regional Park, Acquedotti locality, banks of channel Acqua Mariana, 16 May 2019, *D. Iamonico s.n.* (RO).
