Ethnopharmacobotany and Diversity of Mediterranean Endemic Plants in Marmilla Subregion, Sardinia, Italy

Human populations in various regions across the world exploit the medicinal properties of plants to treat a wide variety of diseases. Areas with both high rates of endemic taxa and persisting traditional uses of the local botanical resources are key sites for the investigation of Traditional Botanical Knowledge (TBK). Commonly, in these areas, information regarding the medicinal properties of native plants has been transmitted orally from generation to generation, however, a rapid decline in this knowledge has been observed, which can be attributed to socio-economic changes in recent years. The Mediterranean basin is one such site, where human history is intimately entwined with nature. The unique geographical situation and unrivaled environmental heterogeneity of the area, have allowed both the development of diverse civilizations as well as providing the basis for the evolution of extraordinary biodiversity. The Mediterranean basin can therefore be considered a global hotspot of endemic vascular plants, and of traditional knowledge of medicinal and aromatic species. This study researches the historical subregion of Marmilla (central-southern Sardinia, Italy), which was chosen because of its specific cultural and demographic characteristics: i.e., prolonged isolation and extreme longevity of the inhabitants of the area. Semi-structured interviews were conducted with 145 people from the region, and 137 medicinal plants belonging to 62 families were identified, of which around 57,3% were taxa exclusive to the Mediterranean Basin. Findings showed that the most used parts of the plant were the leaves (49%), while as far as preparations are concerned, decoction (50%) was the most used to prepare medicinal formulations, making this the highest number of medico-botanical taxa reported in a study carried out in Sardinia using a similar methodology. In addition, this study contributes towards preventing the loss of TBK by documenting the medicinal traditions, passed down orally for centuries, in the words of the participants, shedding new light on the traditional knowledge of the inhabitants of the island. The findings lay the foundations for future applied studies in the fields of phytotherapy and phytochemical investigation.


Introduction
The variety of environments on Earth and the complex relationships among plants themselves and with the entire ecosystem led to the extreme biological diversity of these organisms [1,2]. As a result, highly diversified adaptation strategies have evolved, reflected either in phenotypic or chemical variability [3,4]. This is particularly true for medicinal plants, whose active ingredients have been used for centuries in folk tradition. Even today, they represent an essential source of study in the phytochemistry, biochemistry, and

Results
The results of the interviews were processed and presented graphically in Table 1. The species were firstly divided by family, then, for each species, several data were provided: vernacular name, part of the plant used and preparation, and finally the reported effect of the herbal drug. Emergent from the research was the use, in the Marmilla area, of 137 medicinal plants, belonging to 62 families. The family with the most significant number of taxa was Lamiaceae, followed by Asteraceae, Apiaceae, Rosaceae, and Ranunculaceae ( Figure 1). unique characteristic, it has never been thoroughly examined. The main objectives are i) to record the medicinal plants used, by monitoring the plant biodiversity of the territory; and ii) to codify the ethnobotanical use, preparation, and effects of the medicinal plants, preventing the loss of cultural heritage. Therefore, this work provides a valuable contribution to the ethnobotanical uses of Mediterranean endemic plants.

Results
The results of the interviews were processed and presented graphically in Table 1. The species were firstly divided by family, then, for each species, several data were provided: vernacular name, part of the plant used and preparation, and finally the reported effect of the herbal drug. Emergent from the research was the use, in the Marmilla area, of 137 medicinal plants, belonging to 62 families. The family with the most significant number of taxa was Lamiaceae, followed by Asteraceae, Apiaceae, Rosaceae, and Ranunculaceae ( Figure 1). It is interesting to note that the majority (54.4%) of the local medicinal flora is Mediterranean endemic taxa (endemics sensu lato, [30] whereas four of the collected plants are endemic sensu stricto (Dipsacus ferox Loisel., Helichrysum microphyllum (Willd.) Cambess. Subsp. Tyrrhenicum (Bacch., Brullo & Giusso) Herrando, J.M. Blanco, L. Sáez Galbany, Scrophularia trifoliata L. and Stachys glutinosa L.), corresponding to 2.9% of the local medicinal flora. So overall, even if endemics sensu stricto represents 15% of the whole native flora of the island [31], it is striking that 57.3% of the local medicinal flora of Marmilla is represented by Mediterranean endemic taxa. It is interesting to note that the majority (54.4%) of the local medicinal flora is Mediterranean endemic taxa (endemics sensu lato, [30] whereas four of the collected plants are endemic sensu stricto (Dipsacus ferox Loisel., Helichrysum microphyllum (Willd.) Cambess. Subsp. Tyrrhenicum (Bacch., Brullo & Giusso) Herrando, J.M. Blanco, L. Sáez Galbany, Scrophularia trifoliata L. and Stachys glutinosa L.), corresponding to 2.9% of the local medicinal flora. So overall, even if endemics sensu stricto represents 15% of the whole native flora of the island [31], it is striking that 57.3% of the local medicinal flora of Marmilla is represented by Mediterranean endemic taxa.  Life Forms analysis of the medicinal plants (Table 2) revealed that the majority were hemicryptophytes with 32%, followed by phanerophytes (27%), therophytes (24%), geophytes (13%), while the least represented form was the chamaephytes, with 4%. Regarding the Chorotype, (    The data collected provided us with a complete overview of the part of the plant used, reported in Figure 2. In the vast majority of the medicinal plants analyzed, the herbal drugs were represented by the leaves (47.9%), while a minority is constituted by fruits (5%), seeds (5.7 %), and barks (2.9 %). cryptophytes, divided in bienn, biennal, caesp, caespitose, rept, reptant; ros, rosulate, and scap, scapose. T, therophytes, divided in scap, scapose, and rept, reptant.
The data collected provided us with a complete overview of the part of the plant used, reported in Figure 2. In the vast majority of the medicinal plants analyzed, the herbal drugs were represented by the leaves (47.9%), while a minority is constituted by fruits (5%), seeds (5.7 %), and barks (2.9 %). Subsequently, we investigated the preparations of the herbal drug ( Figure 3). According to the participants, the main preparations were decoction (50%) and infusion (30%), followed by internal use (8%), compress and cataplasm (4%), and lastly, external use and others (2%). Subsequently, we investigated the preparations of the herbal drug ( Figure 3). According to the participants, the main preparations were decoction (50%) and infusion (30%), followed by internal use (8%), compress and cataplasm (4%), and lastly, external use and others (2%).  Finally, we collected and organized the reported effects of medicinal plants (Table 1), deepened with the help of a local medical doctor during the interview. Overall, these preparations were used in Marmilla's folk medicine to treat pathologies of the digestive system, 33%, while the other principal uses regarded nervous, epidermal, and urogenital systems, 17%, 15%, and 14%, respectively (Figure 4). Finally, we collected and organized the reported effects of medicinal plants (Table 1), deepened with the help of a local medical doctor during the interview. Overall, these preparations were used in Marmilla's folk medicine to treat pathologies of the digestive system, 33%, while the other principal uses regarded nervous, epidermal, and urogenital systems, 17%, 15%, and 14%, respectively ( Figure 4).
As regards the digestive apparatus, the species used reported anti-diarrheal, laxative, and antispasmodic properties ( Figure 5). The effects at the uro-genital apparatus level span from diuretic, anti-inflammatory, and emmenagogue effects ( Figure 5). Finally, at the nervous system level, the properties concern analgesic, sedative, stimulating, and antineuralgic effects ( Figure 5).
Finally, we collected and organized the reported effects of medicinal plants (Table 1), deepened with the help of a local medical doctor during the interview. Overall, these preparations were used in Marmilla's folk medicine to treat pathologies of the digestive system, 33%, while the other principal uses regarded nervous, epidermal, and urogenital systems, 17%, 15%, and 14%, respectively ( Figure 4). As regards the digestive apparatus, the species used reported anti-diarrheal, laxative, and antispasmodic properties ( Figure 5). The effects at the uro-genital apparatus level span from diuretic, anti-inflammatory, and emmenagogue effects ( Figure 5). Finally, at the nervous system level, the properties concern analgesic, sedative, stimulating, and antineuralgic effects ( Figure 5). Compared to previous ethnobotanical research conducted on the island [20][21][22][23][24][25][26][27], an interesting finding relates to the number of plants provided compared to the people interviewed ( Figure 6). Compared to previous ethnobotanical research conducted on the island [20][21][22][23][24][25][26][27], an interesting finding relates to the number of plants provided compared to the people interviewed ( Figure 6).
For the same number of participants, a higher number of cited taxa may indicate good conservation of TBK, while a low number conversely indicates erosion. This was observed in Sardinia during the ethnobotanical survey of the Ligurian minority, the Tabarkin community in the Sulcis archipelago [22].
With the aim of representing these concepts numerically, we compared the R ratio between participants and taxa (I/T). Although this parameter is relative, it can be used to compare ethno-botanical studies confined to homogeneous territories (influenced by the same phenomena over time). For this, the R ratio was used for the comparison between the ethnobotanical studies of the island subregions and the single villages, such as Sarrabus, Campidano, Sulcis archipelago, and Marmilla, discussed in the present study. Compared to previous ethnobotanical research conducted on the island [20][21][22][23][24][25][26][27], an interesting finding relates to the number of plants provided compared to the people interviewed ( Figure 6). For the same number of participants, a higher number of cited taxa may indicate good conservation of TBK, while a low number conversely indicates erosion. This was observed in Sardinia during the ethnobotanical survey of the Ligurian minority, the Tabarkin community in the Sulcis archipelago [22].
With the aim of representing these concepts numerically, we compared the R ratio between participants and taxa (I/T). Although this parameter is relative, it can be used to compare ethno-botanical studies confined to homogeneous territories (influenced by the same phenomena over time). For this, the R ratio was used for the comparison between the ethnobotanical studies of the island subregions and the single villages, such as Sarrabus, Campidano, Sulcis archipelago, and Marmilla, discussed in the present study. This ratio, here called R, can provide a quick and useful parameter to verify TBK erosion in communities: for low R values, around zero and one, the number of species exceeds the number of participants, suggesting that traditions are well established in the area. Conversely, high R values, far above one, may suggest an onset of cultural erosion.
The present study has R values just above 1 (R = 1.05), a value that does not indicate erosion, also evidenced by the highest number of taxa among the studies under review, which may indicate a good conservation of TBK.
In general, the value of R can be used quickly and immediately for data comparison, however, it should be considered that it provides an indication that needs to be proven by knowledge of the phenomena occurring in the area.

Discussion
The present work provides extensive documentation of the medicinal plants used by the community of Marmilla, obtained through a blending arrangement of different disciplinary competencies. The participants here played the leading role, transmitting their local cultural knowledge. In addition, they visually recognized fresh plants or dried specimens, helping with plant identification and with translation from the vernacular to the scientific name. Subsequently, with the aid of the local doctor, the reported effects were identified in medical and diagnostic terms. The vernacular name was later documented and shown in the table since it is fundamental not only for identifying the plant by the community but also because it often represents the description of the plant itself, both from the morphological point of view and from the therapeutic action described. As reported in the results (Figure 3), leaves were the most used herbal drug. Interestingly, the bibliographic analysis revealed the use of plants containing toxic substances. In some instances, these substances are avoided by carefully choosing the herbal drug, such as the toxic alkaloids in the roots of Prospero autumnale (L.) Speta or the poisonous berries of Solanum nigrum L., rich in solasodine, a steroidal alkaloid [54], when un-ripe. In both these cases, the local population uses the leaves as a herbal drug, in which the toxic principle is absent. Again, the species Clematis vitalba L. contains saponins and alkaloids such as anemonine and protoanemonine, which are caustic and irritating. In this case [55], the local population refers to the use of roots and branches, softening the effect through topical use as an analgesic. A further example, Lysimachia arvensis (L.) U. Manns & Anderb. contains saponins, flavonoids, and tannins present in every part but mainly concentrated in the seeds, which can even cause severe phenomena of gastric irritations and contact dermatitis [56]. In this case, local people use the leaves as a poultice and as a decoction.
While often the toxic principle is cleverly avoided, in other cases, it is exploited just for this reason. Here, we found effects concerning the digestive system, as the instance of the latex of Euphorbia helioscopia L., used as an emetic and laxative [57], and the infusion obtained from the leaves of Daphne gnidium L., containing mezerein and daphnine, with vesicatory, rubefacient and purgative action [58]. Also interesting is the use of toxic principles which have an effect at the nervous system level, as reported for Hyoscyamus niger L., toxic in all its parts if ingested, causing convulsions, respiratory difficulties, and death [59]. Nevertheless, the local community uses it as a decoction in order to relieve symptoms of trigeminal neuralgia, Parkinson's disease, and senile tremor. Moreover, it has local anesthetic, antispasmodic, and analgesic activities. The effects are recognized and used at the pharmaceutical level to prepare antispasmodic and antineuralgic products acting on smooth muscles. Still, Hedera helix L. is used by the local community to treat neuritis and neuralgia, even though it is reported to be toxic if ingested as it contains triterpene saponins and alkaloids [60].
In summary, the common use of toxic plants in folk medicine may suggest local knowledge of the plant properties, developed through hundreds of years of trial and error, constituting a primitive clinical trial. However, it also highlights possible concerns regarding the safety and security of herbal drugs.
In respect of the reported effects, the targeted use against specific pathologies can also be linked to the ethnic characteristics of the population. As previously mentioned, the Sardinian human population has been isolated for a long time, modifying its genetic structure [61]. Furthermore, several studies have revealed the significant presence of inflammatory and autoimmune diseases, defining the island as an autoimmune hotspot [62] and the subject of numerous studies [63,64]. Notably, 19 plants are reported to have antiinflammatory properties, while eight plants are reported to have specific antirheumatic properties. As regards the latter, Ranunculaceae appear particularly noteworthy, as three plant species out of eight belong to this family and have been found to exhibit antirheumatic activity (Anemone hortensis L., Clematis flammula L., and Ranunculus macrophyllus Desf.). As a result, it can be assumed that the population's specific traits have therefore conditioned the relationship between the people and the usage of medicinal plants present in the territory.
Another aspect of this study is that nearly 60% of the plants locally used for medicinal aims are Mediterranean endemic taxa, of which four (2.9%) are narrow endemic plants, with a restricted range and extremely valuable as a source of new bioactive molecules [47]. Therefore, among the four narrow endemic plants that emerged, we conducted a literature research focusing on both similar ethnopharmacobotanical uses and pharmacological activity.
Dipsacus ferox Loisel has reported beneficial effects at the epidermic level, being used against desquamative dermatoses, eczema, folliculitis, urticaria, and psoriasis. A comparative literature review showed it to be used in different parts of the Island for food purposes [65,66], and by the Ogliastra community (Nuoro Province) as an antieczemic, confirming the benefits against epidermic inflammatory diseases [67].
From a chemical and pharmacological point of view, few studies have been found related to D. ferox, characterized by the presence of iridoids, distinctive compounds in Dipsacaceae [68]. More can be said about the pharmacological activity of Dipsaucus genus, which is reported to have anti-aging, anti-inflammatory, anti-bone fracture, hepatoprotective, and anti-myocardial infarction activity. Moreover, studies show its activity against inflammation-based diseases such as osteoarthritis [69][70][71].
Stachys glutinosa L. is employed by the Marmilla community as a cholagogue, diuretic, and hepatoprotective. Various and different ethnobotanical uses have also been found within the Island, from the simplest use against colds to antiseptic, antispasmodic, and sedative applications [20,67,72].
Essential oils and extracts of S. glutinosa have been chemically well characterized, while in vitro tests have shown mild antiproliferative activity against cellular tumor lines [73][74][75]. Further studies have then shown an affinity for opioid receptors, and good bacteriostatic activity against certain types of bacteria and fungi [76].
In the present study, Scrophularia trifoliata L. is reported to have purgative and emetic effects, and to be used against Grave's disease and related heart disorders. Interestingly, it was found that this endemic plant is used by different local populations in Sardinia to treat various diseases, often related to inflammatory or autoimmune conditions, such as Grave's disease in Marmilla, antirheumatic activity in Ussassai, Urzulei and Villagrande Strisaili (Nuoro Province), Escolca (South-Sardinia Province), and anti-inflammatory activity in Aggius (Sassari Province) [20,21,25,26]. Its extracts have shown anti-HIV activity in vitro [77][78][79], while the genus Scrophularia has antioxidant and anti-inflammatory activity [78][79][80]. Notably, several studies reported how S. striata Boiss. inhibits the production of NO [81] and pro-inflammatory cytokines [82]. When produced in excess, these molecules are associated with several diseases such as chronic inflammatory, septic shock, and autoimmune diseases. Due to its immunomodulatory activity, its potential effects against COVID-19 inflammation have been highlighted [83].
In general, ethnobotanical uses appeared to be confirmed by tested pharmacological properties. Notwithstanding, there are still few studies for some plants, especially endemic ones, which due to their potential in terms of chemical biodiversity, deserve to be further characterized.
Another element to consider in this regard is the preservation of biodiversity. The uncontrolled collection of wild plants for medicinal purposes might be harmful to the conservation of local populations. The medicinal plants here obtained have been compared both to the European and Italian red lists [92,93] and to the up-to-date IUCN online source (https://www.iucnredlist.org), and from this comparison, 71 Least Concern (LC), 4 Data Deficient (DD), 4 Near Threatened (NT), and 1 Vulnerable (VU) species were found.
The Red List presents critical indicators of biodiversity status, highlighting the threatened species. Nevertheless, classification is still an ongoing process that requires the collaborative efforts of researchers, in fact, only a fraction of known species is currently categorized.
In the present research, 80 plants out of 137 were already assessed with the IUCN criteria. Particularly, here emerged the need for the protection of the Near Threatened plants, Helosciadium crassipes W.D.J.Koch ex Rchb., Marrubium vulgare L., Oenanthe fistulosa L., and Scrophularia trifoliata L. Moreover, further precautions should be used when the medicinal effect is found at the level of bulbs and roots. In this case, harvesting the plant may pose a risk to the plant's survival. The orchid Ophrys apifera Huds. is a perfect case in point.
In conclusion, on the one hand, the preservation of TBK should be monitored for the conservation of biodiversity, particularly in hotspots of global biodiversity like the Mediterranean basin and its large islands, through raising public awareness about endangered species and avoiding their uncontrolled collection. On the other hand, it is possible that strong conservation stems from a combination of demographic and geomorphological characteristics of the territory. From the demographic point of view, Sardinia has been recognized as one of the four Blue Zones globally, characterized by extreme longevity. Specifically, Marmilla is adjacent to the focus territory of the Sardinian Restricted Blue zone, therefore, it is conceivable that the population's high seniority, combined with these historical, geomorphological, and cultural characteristics of sub-regions, has led to good conservation of TBK over time. From this perspective, it is even more interesting to discover the natural remedies, which, together with diet and life habits, contribute to maintaining such a long-lived community.

Historical and Ethnographic Background
The territory of Marmilla plays a principal role in the present research, and the ethnobotanical interviews revealed that medicinal plants employed in the local tradition are commonly collected still today.
Marmilla is a historical subregion of central-southern Sardinia, located between 39 • 47 and 39 • 30 North latitude and between 8 • 47 and 9 • 12 East longitude (Figure 7), presenting a territorial extension of 415 sqKm [94]. The Flumini Mannu river morphologically defines its borders to the south-east, Giara of Gesturi to the north, and Monte Arci to the west. It consists of two sub-areas, called "Alta Marmilla" and "Bassa Marmilla". The first one belongs to the province of Oristano and extends between two natural monuments: the Giara plateau and the Monte Arci, a volcanic massif, rich in obsidian, volcanic glass used by prehistoric populations for the production of tools. It borders to the south with the second sub-area, "Bassa Marmilla", which belongs to the province of Medio Campidano. west. It consists of two sub-areas, called "Alta Marmilla" and "Bassa Marmilla". The first one belongs to the province of Oristano and extends between two natural monuments: the Giara plateau and the Monte Arci, a volcanic massif, rich in obsidian, volcanic glass used by prehistoric populations for the production of tools. It borders to the south with the second sub-area, "Bassa Marmilla", which belongs to the province of Medio Campidano. From an historical point of view, there is evidence that dates back to the Middle Bronze Age (ca. 1600-1500 BCE), demonstrating that Marmilla has been inhabited since ancient times. Indeed, the territory is characterized by several 'Nuraghes', typical buildings of the so-called Nuragic civilization, including 139 different Nuragic sites. Among others, the Nuragic site of Barumini deserves a particular mention, representing the leading Nuragic site in Sardinia and being included in the United Nations Educational, Scientific and Cultural Organization's [UNESCO] list of World Heritage Sites in 1997. Further historical evidence is linked to the colonization in the first century CE by the Romans and in the Middle Age by the Kingdom of Arborea, with the construction of Marmilla's Castle, an area of strategic and military importance. The territory is characterized by a predominantly agricultural economy and a permanent resident population. The present work focused on five municipalities, Furtei, Gesturi, Segariu, Tuili, and Villamar, small villages with a population between 1000 and 2500 inhabitants, with an overall average of 1500 inhabitants. Similarly, to the whole Marmilla territory, these villages are affected by high emigration rates and older demographic profiles. In addition, the weakness of the economic structure is resulting in progressive depopulation [94]. From an historical point of view, there is evidence that dates back to the Middle Bronze Age (ca. 1600-1500 BCE), demonstrating that Marmilla has been inhabited since ancient times. Indeed, the territory is characterized by several 'Nuraghes', typical buildings of the so-called Nuragic civilization, including 139 different Nuragic sites. Among others, the Nuragic site of Barumini deserves a particular mention, representing the leading Nuragic site in Sardinia and being included in the United Nations Educational, Scientific and Cultural Organization's [UNESCO] list of World Heritage Sites in 1997. Further historical evidence is linked to the colonization in the first century CE by the Romans and in the Middle Age by the Kingdom of Arborea, with the construction of Marmilla's Castle, an area of strategic and military importance. The territory is characterized by a predominantly agricultural economy and a permanent resident population. The present work focused on five municipalities, Furtei, Gesturi, Segariu, Tuili, and Villamar, small villages with a population between 1000 and 2500 inhabitants, with an overall average of 1500 inhabitants. Similarly, to the whole Marmilla territory, these villages are affected by high emigration rates and older demographic profiles. In addition, the weakness of the economic structure is resulting in progressive depopulation [94].

Environmental Background
Sardinia is located in the center of the western Mediterranean basin. The territory shows remarkable differences from geological and morphological points of view, dividing the island into sectors. The result of the ancient geological history of the island is clearly visible in the central-eastern part of the island, in which there are the most ancient rocks of the Paleozoic age, from the Ercinic orogeny. This orogenic phase has led to metamorphism and magmatism phenomena, forming the Sardinian basement. Subsequently, Sardinia drifted away from southern France with the Alpine orogeny, separating permanently from the European block, and settling in the center of the Mediterranean basin. The centralwestern part extends the Sardinian-Campidanese graben, a sedimentation basin consisting primarily of marine sedimentary rocks interspersed with volcanites, resulting from the erosion of the neighboring areas. These ancient geological processes have shaped the island, which, from a morphological point of view, has a reduced mountainous component due to the consequent erosion. In contrast, the smoothened hills confer more than 50% hilly components, with altimetry between 200 and 700 m [95]. This heterogeneity, especially in the inner territories, limited the communities' exchanges. The climate is the Mediterranean Pluviseasonal Oceanic type, with evident seasonality, consisting of hot and dry summer and mild winter. However, the union of different factors (among others, temperature, continentality, and precipitation) results in highly diverse isobioclimates, with a total of 43 [95]. This variability is reflected in a considerable diversity of environments that have contributed to the development of high endemism over the centuries. At present, Sardinia's native flora is constituted of 2300-2500 species and subspecies [96,97] of which 15% are considered endemic to the island [31]. Considering vegetational, physiographic, bioclimatic, and biogeographical factors, the latest studies divided Sardinia into 23 main vegetation series. The main ones are the woodland formations, distinguished in oaks (Quercus coccifera L., Quercus ilex L., Quercus suber L., Quercus gr. pubescens Willd.), junipers (Juniperus communis L., Juniperus macrocarpa Sm., Juniperus oxycedrus L., Juniperus turbinata Guss.), wild olive (Olea europea L.,), pines (Pinus halapensis Mill., Pinus pinaster Aiton, Pinus pinea L.) and other tree species covering smaller areas (Acer monspessulanum L., Castanea sativa Mill., Ilex aquifolium L., Laurus nobilis L., Taxus baccata L.) [98]. Another typical vegetation type is the Mediterranean maquis, with evergreen formations exceeding 4 m [99]. The main exponents are Arbutus unedo L., Chamaerops humilis L., Myrtus communis L., Pistacia lentiscus L., Rhamnus alaternus L., Salvia rosmarinus Schleid., Smilax aspera L. and many species of the genera Cistus L. and Genista L. Finally, the garrigue, shrubby vegetation formed by low plants that grow in an isolated way and can be found up to 1400 m of altitude. This vegetation is represented mainly by Helichrysum italicum (Roth) G.Don subsp. tyrrhenicum

Data Collection
Ethnobotanical research was carried out in the municipalities of Furtei, Gesturi, Segariu, Tuili, and Villamar. The survey occurred from February 2017 to March 2018, and the participants' sample was selected purposively over 65 years of age. It involved a final sample of 145 people, 94 men and 51 women (64.8% men and 35.2% women). Semistructured interviews with pre-formulated questions were conducted, according to the survey method used by [100,101] through guide interviews aimed at compiling a preformulated form. The surveys were performed both in Italian and vernacular languages. The subsequent systematic classification of the species was carried out following the guidelines of Flora d'Italia [102]. For the updated classification of the plant families, we followed the Bartolucci et al. classifications [96]. Regarding the survey field, the acquired data are processed and compared with the plant samples used (whole plant or parts of it).
The plant matrices were stored with the most appropriate methodology according to the plant, if complete, portioned, chopped, fresh or dried, packaged or otherwise. The difficulty at this stage lies in the degree of botanical knowledge of the people interviewed in identifying plant species and the need for translation from the vernacular name to the scientific name corresponding to the plant matrix indicated. As for the dried plant drugs used in traditional medicine, conservation techniques relevant to classical pharmacognosy were used. In the laboratory, the samples were identified following standard phytognosy techniques, when possible. Where it was not possible, for example, because of mixtures of different plant drugs, we proceeded to the recognition of drugs and their subsequent identification by analysis of macroscopic (shape, size, etc.) and microscopic (absence, presence of starch, etc.) characteristics, sensory (bitter, sweet, aromatic, etc.) and tests with chemical solvents and reagents. For the identification, we also used the consultation of relevant literature and the Cagliari Herbarium (CAG) of the University of Cagliari. Finally, for a more precise diagnosis of the diseases reported by the participants, we used the cooperation of local medical doctors and their valuable knowledge about the health of their patients.

Data Analysis
The data collected during the interviews were processed on Microsoft Excel and organized in Table 1, divided into plant species, vernacular name, traditional use, herbal drug, and preparation.
The Excel spreadsheet was further processed, grouped by homogenous categories (family, preparation, endemism, etc.), expressed in percentage, and reported in the figures.
The ratio between the No. of participants and No. of emerged medico-botanical taxa in the present article was calculated. Then, it was compared with the other ethnobotanical studies in Sardinia and reported in Figure 6.

Conclusions
This paper provides extensive documentation of the ethnobotanical culture of the Marmilla subregion. The geographic and cultural isolation characterizes Sardinia and its subregions, providing its communities with unique cultural and social peculiarities. In addition, numerous studies report how isolation also shaped the population genetically. Therefore, it is even more interesting to understand the complex relationships between the population and Sardinia's rich floristic biodiversity resulting from centuries of trial and error.
The study adds a piece to Sardinian and Mediterranean ethnobotany that is of increasing relevance given the rapid decline of folk traditions. TBK preserved in the older generations is at risk of disappearing at an accelerated pace due to the recent global coronavirus outbreak.
In addition to its fundamental role in documenting and codifying the cultural heritage of the Marmilla Subregion, the present work may also provide new targets for phytochemical and phytotherapeutic research.