Orchidaceae in Puglia (Italy): Consistency, Distribution, and Conservation

A study of the distribution of orchid species in Puglia, based on an analysis of 2084 bibliographic reports from 2000 to 2022, was carried out with the aim of revising and updating the information on the consistency of the Orchidaceae family in Puglia, with a special focus on assessing threatened species occurring inside and outside protected areas. The work presents a checklist of the Orchidaceae taxa (genera, species, and subspecies) found in the region, including observations on genera and species that present taxonomic challenges. A total of 113 taxa (i.e., species and subspecies), distributed across 16 genera, are listed in alphabetical order. The most representative genera were Ophrys (51 taxa), Serapias (15 taxa), and Epipactis (11 taxa). Additionally, 49 taxa (43.4%) were found to be endemic to Italy, with 21 of these, mostly belonging to the Ophrys genus, being exclusive to Puglia. Our study notes two different trends of distribution: a predominantly coastal distribution for orchid records located in southern Puglia (the Salento peninsula) and a more widespread distribution for the other provinces. Our study also shows that the greatest number of records locate orchids in protected areas with a positive correlation between their presence and habitats cited in Directive 92/43/EEC.


Introduction
The Orchidaceae family is a diverse group of plants and one of the most abundant among angiosperms, with over 28,000 accepted species and 763 genera. Many of these species are endemic to small areas and are considered rare and endangered [1,2]. Unfortunately, orchids are at a high risk of extinction, primarily due to the loss of their habitats and the effects of climate change [3]. However, due to their significant conservation value, Orchidaceae is a prominent plant family that receives attention from ecologists, biologists, and environmentalists worldwide [1]. In Europe, orchids are present in almost all habitats, and southern Europe, particularly the Mediterranean, has the highest species diversity. This region is considered the birthplace of certain orchid genera, such as Ophrys and Serapias, which boast remarkable diversity [3].
The Orchidaceae family is well represented in the native vascular flora of Italy, with 236 taxa (species and subspecies) within 27 genera. Among these, the Ophrys genus is particularly species-rich, with 103 taxa. When considering only Italian endemics, the Orchidaceae family is represented by 87 taxa, ranking below the Asteraceae, Plumbaginaceae, and Caryophyllaceae families. The Ophrys genus has 61 endemic taxa, with smaller numbers in the Hieracium, Limonium, and Centaurea genera [4]. The Puglia region has the highest orchid biodiversity, with 101 taxa out of 2552 native plants, followed by Tuscany (100 taxa) and Basilicata (97 taxa) [5]. In terms of Italian endemics, 31 species and subspecies of Orchidaceae occur in Puglia [4], many of which are related to eastern Mediterranean taxa, indicating its relative phytogeographical isolation with respect to other parts of southern peninsular Italy [6].
Puglia is an area of significant interest in terms of flora and vegetation due to its geological history and biogeographical location. The Orchidaceae family has been the focus of numerous botanical studies, including floristic surveys of specific areas and more in-depth investigations of the genetics and morphometry of certain taxa. These studies have often led to the discovery of new specific or subspecific taxa.
The earliest reports on the ancient local flora were conducted by researchers such as Baselice in 1812 for Gargano [7], Marinosci in 1870 [8], Groves in 1887 for Salento [9], and Palanza in 1900 for Bari area [10], all of which are considered pioneering. Later, more modern and systematic specialized studies of Mediterranean Orchidaceae were carried out, mainly by foreign authors. Nelson in 1968 [11]; the spouses Danesch (who described several new species and hybrids) in 1972 [12]; Baumann and Künkele in monographs on Ophrys and Serapias genera, respectively [13,14]; and Lorenz [15] for Serapias all made significant contributions. Additionally, cartographical studies were conducted by Gölz and Reinhard in 1982 for central-southern Puglia [16] and by Lorenz and Gembardt in 1987 for Gargano [17]. More recent research on orchids in Puglia has been published by Romolini and Souche [18], Delforge [19], and GIROS [20]. Souche in 2008 also provided a paper on the study of hybrid species [21].
The aim of this paper is to revise and update the information on the consistency of the Orchidaceae family in Puglia. This involves aligning the terminology with the latest taxonomy adopted by GIROS (Gruppo Italiano per la Ricerca sulle Orchidee Spontanee-Italian Group for Research on Spontaneous Orchids), examining the distribution in the region and recognizing the significance of conserving these species, which are frequently uncommon, endemic to restricted areas, and/or have their locus classicus in Puglia. These plants are employed as bioindicators in environmental management and the safeguarding of habitats.

Taxonomic References and Data Analysis
To evaluate the consistency and distribution of Orchidaceae in Puglia, an updated list of reports (Table S1) was created. Due to the high degree of nomenclature variation and the considerable number of new species, and in order to minimize attribution errors, records were sought only among new data published in the period 2000-2022, mainly, but not only, belonging to specialist papers (i.e., mainly from GIROS and GIROS Orch. Spont. Eur.). For each analysed paper, floristic list, occurring site, and geographic coordinates of the study site were noted. If the geographical coordinates were not given, they were subsequently obtained on the basis of other information on the occurring site cited. For all records, the geographic coordinates were converted into WGS 84 system coordinates. Table S1 contains all the previously mentioned information except for the coordinates of records that were used for cartographic processing, which were not included for conservation purposes and to avoid misuse.
For each taxon, the corotype was also added, taken either from GIROS [20] or from recent publications regarding new species.
Overly generic records (e.g., Serapias without mentioning species attributes) were not considered in the dataset.
The reports of Ophrys tardans for Bosco Cuturi [26] and Bosco Difesa Grande [27] were considered to be Ophrys expansa (Lumare & Medagli) Biagioli, Kreutz, Lumare, Medagli & De Simoni, as stated by Lumare and Medagli [28].  Considering the single reports obtained by bibliographical research, Table 4 lists the number of records for each genus by province. For the province of Bari, Ophrys, Anacamptis, and Serapias were the most frequently reported genera, as they were in other provinces excluding Barletta-Andria-Trani and Foggia. The latter, with the highest number of genera in the region, showed a similar trend but with the addition of the genera Epipactis and Orchis. In contrast, the province of Barletta-Andria-Trani had the lowest number of reports. Considering the corotypes of the analysed orchids, there are 49 taxa (species and subspecies) endemic to Italy, with 620 records, and 3 subendemic taxa (i.e., with a restricted distribution that includes Italy and a small area beyond its border), with 23 records. With 36 taxa, Ophrys is the richest genus in terms of endemism, followed by Serapias (8 taxa), Epipactis (4 taxa), and Anacamptis (1 taxon). Ophrys also accounts for the only three subendemics. Table 5 shows the number of records for each genus by province for taxa (species and subspecies) endemic to Italy. In this case, the province of Lecce has the highest number of endemic taxa, followed by the provinces of Foggia and Bari. As previously mentioned, all endemics belong to the genera Ophrys and Serapias, except in the province of Foggia, which also has endemics belonging to Anacamptis and Epipactis.  Table 6 shows the correlation between genera and habitats cited in Directive 92/43 EEC. Taxa within the genus Ophrys are seen to occupy the highest number of habitats (10), ranging from humid environments (i.e., habitat 1410) to xeric Mediterranean grasslands and forests. This is obviously also related to the high species diversity of this genus. In contrast, some genera are associated with a specific habitat group, as in the case of Himantoglossum and Spiranthes, which grow in xeric Mediterranean grasslands, and Epipactis, Limodorum, Platanthera, Neottia, Cephalanthera, Coeloglossum, and Epipogium, which prefer woody habitats.

Discussion
Our study presents the findings of bibliographical research on orchids in Puglia covering the years from 2000 to 2022. We opted to focus on data from the last two decades, mainly from specialized journals, because they provided more precise determinations, particularly for taxonomically critical genera such as Ophrys, Serapias, and Epipactis. Additionally, there have been ongoing nomenclatural changes, and other taxa have been added in recent years. In this study, we considered not only the presence of orchids but also their location, distribution, and preferred habitats.
Thanks to this research project, the orchidological biodiversity of Puglia has been updated to include 113 taxa (species and subspecies), of which 49 (43.4%) are endemic to Italy. These numbers exceed those reported in previous studies. For instance, the authors of [4] reported only 31 endemic taxa, and those of [209] reported 42 taxa, while our research found 52 endemic/subendemic taxa. The same trend was observed in the overall number of species. Among the endemics, 21 taxa (species and subspecies), mostly belonging to the genus Ophrys, are exclusive to Puglia. This growth is also consistent with what was recently asserted in [210].
The distribution of records was used to draw up a map of orchids across Puglia using geolocation data extracted from publications and other information that helped us to geolocate the study areas ( Figure 1, Table S1). As can be seen, most of the records for the Salento peninsula are situated near the coastline, indicating that a significant portion of the biodiversity of this area in terms of environments and habitats is concentrated there. Indeed, the Salento peninsula is characterized mainly by urban areas and agroecosystems, which have eroded almost all the biodiversity located inland. In contrast, the records are more dispersed in the rest of Puglia, where woody environments are more extensive and abundant in addition to coastal and transitional environments. The distribution of records was used to draw up a map of orchids across Puglia using geolocation data extracted from publications and other information that helped us to geolocate the study areas ( Figure 1, Table S1). As can be seen, most of the records for the Salento peninsula are situated near the coastline, indicating that a significant portion of the biodiversity of this area in terms of environments and habitats is concentrated there. Indeed, the Salento peninsula is characterized mainly by urban areas and agroecosystems, which have eroded almost all the biodiversity located inland. In contrast, the records are more dispersed in the rest of Puglia, where woody environments are more extensive and abundant in addition to coastal and transitional environments. It is worth noting that a significant majority of orchid records are found within protected areas such as parks, SCIs, and SACs ( Figure 2). This reinforces the significance of these areas not only for conservation purposes but also as areas that can be used for research activities by naturalists and scientists.
Based on our data, the province with the highest number of records is Lecce, followed by Bari, Foggia, Taranto, Brindisi, and Barletta-Andria-Trani (Table 4). The limited availability of data for the BT province can not only be attributed to its recent establishment in 2009 but also to its small size and because it has clearly been poorly investigated by botanists and naturalists.
Referring to Table 2 and considering Lorenz and Gembardt's [17] published report on Spiranthes spiralis in the province of Foggia, the distribution of genera by province is presented in Figure 3. It is worth noting that a significant majority of orchid records are found within protected areas such as parks, SCIs, and SACs ( Figure 2). This reinforces the significance of these areas not only for conservation purposes but also as areas that can be used for research activities by naturalists and scientists.   Based on our data, the province with the highest number of records is Lecce, followed by Bari, Foggia, Taranto, Brindisi, and Barletta-Andria-Trani (Table 4). The limited availability of data for the BT province can not only be attributed to its recent establishment in 2009 but also to its small size and because it has clearly been poorly investigated by botanists and naturalists.
Referring to Table 2 and considering Lorenz and Gembardt's [17] published report on Spiranthes spiralis in the province of Foggia, the distribution of genera by province is presented in Figure 3.
The trend observed in the distribution of species and reports by province seems to differ from the distribution of genera depicted in Figure 3. Despite having a lower number of genera, the provinces of Bari and Lecce exhibit a higher richness of taxa (species and subspecies) than the province of Taranto. Meanwhile, Foggia maintains its status as the province with the highest species richness (Figure 4).
In terms of species reports, the province of Bari has the highest number of reports for the genera Ophrys and Himantoglossum, while the province of Lecce has the majority of reports for the genera Serapias and Anacamptis, and the province of Foggia has the highest number of reports for the genera Orchis and Epipactis.
Puglia serves as the southernmost boundary for certain Italian species such as Anacamptis palustris, Ophrys funerea, O. lucifera, O. parvimaculata, and O. minipassionis. However, it also marks the northernmost boundary for other taxa such as Epipactis aspromontana, E. schubertiorum, Ophrys celiensis, O. sipontensis, and O. tarentina, as stated by Pezzetta [209].   The trend observed in the distribution of species and reports by province seems to differ from the distribution of genera depicted in Figure 3. Despite having a lower number of genera, the provinces of Bari and Lecce exhibit a higher richness of taxa (species and subspecies) than the province of Taranto. Meanwhile, Foggia maintains its status as the province with the highest species richness (Figure 4). In terms of species reports, the province of Bari has the highest number of reports for the genera Ophrys and Himantoglossum, while the province of Lecce has the majority of reports for the genera Serapias and Anacamptis, and the province of Foggia has the highest number of reports for the genera Orchis and Epipactis.
Puglia serves as the southernmost boundary for certain Italian species such as Ana-

Conclusions
In conclusion, our study provides an updated and comprehensive checklist of orchid species and subspecies found in Puglia, including taxonomic observations and assessments of threatened species within and outside of protected areas. Our study, based on a thorough analysis of bibliographic reports spanning over two decades, contributes significantly to the understanding of Orchidaceae diversity in the region.
The checklist of 113 taxa (species and subspecies) across 16 genera also provides a comprehensive overview, highlighting taxonomic challenges and the prevalence of certain genera such as Ophrys, Serapias, and Epipactis compared to other genera. The presence of 49 taxa endemic to Italy, with a substantial number exclusive to Puglia, underscores the region's significance for orchid biodiversity and conservation. In this context, it must be highlighted that all new species novelties were added in the checklist, including that of Ophrys panormitana [180], for which further investigations should be made to exclude the hybridogenic origin of the plants, as only one plant was found with several tufts of vegetative origin, which would suggest a morphological convergence rather than the presence of a new species, in this case, endemic to Sicily.
The distribution patterns reveal distinct trends, with a strong coastal preference in the south of Puglia and a more widespread distribution in other provinces. Importantly, this study underscores the importance of protected areas as critical habitats for orchid populations. Overall, this research enhances our knowledge of Puglia's orchid flora and emphasizes the need for conservation efforts to safeguard these valuable plant species.