Nomenclatural Synopsis, Revised Distribution and Conservation Status of Ranunculus gracilis (Ranunculaceae) in Italy

Ranuculus gracilis is endemic to the SE Euro-Mediterranean area and its presence in Italy is controversial. Based on analysis of the relevant literature, field surveys and examination of herbarium specimens, a revised distribution of this species in Italy is presented and its conservation status is assessed. Ranunculus agerii, described by Antonio Bertoloni from Bologna (Emilia-Romagna, Northern Italy), and R. schowii, described by Vincenzo Tineo from Vittoria and Terranova (Sicily), usually regarded as synonyms of R. gracilis, are here lectotypified and their taxonomic status discussed. Thanks to our study, the presence of R. gracilis in Italy is confirmed and, now, it is reported in a national conservation framework.


Introduction
Ranunculus L. is the largest genus in the family Ranunculaceae Juss., with a cosmopolitan distribution, consisting of about 1200 species (including also ca. 600 agamospecies) [1,2]. Based on morphological and molecular data, the genus Ranunculus was divided into two subgenera (subg. Auricomus and subg. Ranunculus) and 17 sections [2]. In Italy, the genus Ranunculus comprises 112 taxa (species, subspecies and agamospecies), of which 33 are endemic (four are extinct) and one is an alien species [3][4][5].
Ranuculus gracilis was described in 1814 by Edward Daniel Clarke from the East Aegean island of Kos (Greece) [6] and belongs to R. subg. Ranunculus sect. Ranunculastrum DC. [2]. This species is endemic to the SE Euro-Mediterranean area and it is distributed in Italy, the Balkan Peninsula, Turkey and Georgia [7,8]. Contrary to what is reported by Euro+Med Plantbase [7] and POWO [8], the presence of R. gracilis in Italy is controversial, i.e., [4,9]. According to the latest Italian Flora [9], it is present in a few localities in Sicily and Calabria, no longer recorded in Emilia-Romagna and cultivated in Umbria. On the contrary, following the Italian Checklist [4], it is recorded by mistake in Piemonte, Emilia-Romagna, Umbria, Sicily and doubtfully occurring in Puglia. Two species, described from Italy, are usually regarded as synonyms of R. gracilis (e.g., [9][10][11][12]): R. agerii Bertol. and R. schowii Tineo (with doubt). Ranunculus agerii was described in 1819 by Antonio Bertoloni from the areas around Bologna (Emilia-Romagna, northern Apennines, Italy) [13] and it is currently regarded as a synonym of R. gracilis (i.e., [7][8][9]12,[14][15][16][17]). Ranunculus schowii was described by Vincenzo Tineo in Gussone [18] from Vittoria and Terranova (Sicily) and regarded as a dubious synonym of R. gracilis or R. agerii by some authors (i.e., [10][11][12]) or as a synonym of R. monspeliacus L. subsp. monspeliacus (i.e., [8]). The purpose of this study is to critically review the presence of R. gracilis in Italy and to understand the taxonomic identity of R. agerii and R. schowii, two names that turned out to be, to the best of our knowledge, not yet typified.
The present contribution is part of an ongoing project promoted by the Italian Botanical Society, aimed at recognizing and typifying all the taxa described from Italy, in order to increase their systematic knowledge and promote further studies [19][20][21][22].

Materials and Methods
This study is based on an extensive analysis of relevant literature, field surveys and examination of herbarium specimens (including the original material) preserved in APP, BOLO, BR, CAT, CHE, CLF, DR, E, FI, G, GOET, JE, MW, NAP, P, PI, PAL, W and WU (the acronyms follow [23]). We performed a survey for original material for the name R. agerii at BOLO where Bertoloni's main collection is housed and at FI, G, NAP and PAL to trace the original material of the name R. schowii (see [24,25]). The original material for the name R. gracilis was searched at A, BM, CAN, CGE, ECON, GH, FI, K and SWN. The type designations herein follow the Shenzhen Code ( [26], hereafter ICN).
The revised Italian distribution of R. gracilis is based on examination of herbarium specimens. The distribution data and the occurrence status are given for the Italian administrative regions according to Bartolucci et al. [4].

The Long and Controversial History of R. gracilis in Italy
Over the years, the presence of R. gracilis in Italy has been controversial due to the confusion in its identification and to the unclear taxonomic relationships with some currently not accepted species [7,8], such as R. agerii Bertol., R. schowii Tin. and R. chaerophyllos L.
The first herbarium samples collected in Italy that can be referred with certainty to R. gracilis date back to the 16th century. There are two specimens without collection locality preserved in the "En Tibi" herbarium, made by Francesco Petrollini in Bologna around 1558, kept in Leiden (an image of the specimen is available at https://data.biodiversitydata.nl/ naturalis/specimen/L.2110949 (accessed on 10 September 2022)) and in the controversial "Cibo" herbarium kept in Rome in Biblioteca Angelica, recently also attributed to Petrollini [27,28]. According to Stefanaki et al. [28], it is evident that many specimens in the "En Tibi" herbarium were collected in the area of Bologna, where Petrollini had his place of residence.
Towards the end of 1500, this plant was also collected by Nicolas Ager (or Agerius) in the Bologna hills. He sent samples to Jean Bauhin, who gave a brief description of this plant in the Phytopinax published by Caspar Bauhin as "Ranunculus racemosa radice Io. Bauhini . . . Reperitur in montibus Bononiensibus" [29]. Later, it was reported by the Bauhin brothers as "Ranunculus grumosa radice folio ranunculi bulbosi . . . Hic Ranunculus agris Bononiensibus familiaribus est, & à D. Agerio collectus" in the Prodromos theatri botanici [30], "Ranunculus grumosa radice folio ranunculi bulbosi" in the Pinax theatri botanici [31], "Ranunculus racemosa radice . . . Agerio siccam dedit pro Ranunculo Chelidoniae radice" in the Historia plantarum universalis [32] and by Parkinson [33] as "Ranunculus grumosa radice Bononiensis". Later, Linnaeus [34], mistakenly included the polynomial published by C. Bauhin in the Prodromus [30] and in the Pinax [31] in R. chaerophyllos L. Antonio Bertoloni was the first to accurately describe [13] the plant from the areas around Bologna (Emilia-Romagna) as "Ranoncolo Bolognese", dedicating it to Nicolas Ager, with the name R. agerii. After the description, R. agerii was treated as synonym of R. chaerophyllos L. by Arcangeli [35] and Cesati et al. [36], a name of uncertain application [37]. Later, Fiori et al. [10] and Fiori [11] re-evaluated R. agerii as a good species, recording it not only for its locus classicus, but also for Sicily and quoting R. gracilis and, with doubt, R. schowii Tineo, only in [11] as synonyms. Pons [38] recorded R. agerii (syn. R. gracilis) for several localities around Bologna in Emilia-Romagna and for Catania in Sicily. Tutin [14] and Tutin and Akeroyd [17] in Flora Europaea quoted R. gracilis (syn. R. agerii) for Italy and Sicily. Zangheri [39] reported R. gracilis (syn. R. agerii) for Sicily and as naturalized in Northern and Central Italy. Pignatti [15] reported R. gracilis (syn. R. agerii) in Sicily and no longer recorded for Emilia-Romagna. Greuter et al. [40] in the Med-Checklist quoted R. gracilis (syn. R. agerii) for Sicily and as doubtfully native in Italy. Jalas and Suominen [16] quoted R. gracilis (syn. R. agerii) for Calabria and Sicily and with doubt in Emilia-Romagna. Peruzzi and Passalacqua [41] reported R. gracilis for the Balkan Peninsula, Turkey, and Crete, while, the Italian records of Calabria and Sicily, should be referred to R. monspeliacus L. subsp. aspromontanus (Huter, Porta & Rigo) Peruzzi & N.G.Passal. Conti et al. [42] reported R. gracilis without synonyms as doubtfully occurring in Italy in Piemonte, Emilia-Romagna and Sicily. In the same year, Scoppola and Spampinato [43] recorded R. gracilis (syn. R. agerii) for Sicily and as indicated by mistake in Emilia-Romagna. Later, Conti et al. [44] reported R. gracilis as indicated by mistake in Italy, updating the occurrence status in Conti et al. [42]. Recently, Pignatti et al. [9] quoted R. gracilis (syn. R. agerii) as present in a few localities in Sicily, Calabria, no longer recorded in Emilia-Romagna and cultivated in Umbria. On the contrary, according to Bartolucci et al. [4], in the updated checklist of Italian vascular Flora, the species was recorded by mistake in Piemonte, Emilia-Romagna, Umbria, Sicily and as doubtfully occurring in Puglia. Recently, Guarino and La Rosa [45] in the Digital Flora of Italy included in the 4 th volume of Flora of Italy [46], recorded R. gracilis for Calabria, alien in Umbria and as doubtfully occurring in Sicily and Emilia-Romagna.  Figure 1B).

Typification of the Names
Nomenclatural notes: Antonio Bertoloni [13] described R. agerii, providing a detailed description, quoting a precise collection locality and citing an illustration "Tab VI". In BOLO, where Bertoloni's main collection is housed, we traced only one herbarium sample with two mounted individuals collected in 1818 on Monte Donato ( Figure 1) that can be considered as part of the original material, as well as the illustration "Tab VI" cited in the protologue (Figure 1) (Art. 9.4 of the ICN). The herbarium sample kept in BOLO is complete, well conserved and agrees with the protologue and is selected here as a lectotype for the name R. agerii.
Taxonomic notes: based on the original material studied, R. agerii should be regarded as a heterotypic synonym of R. gracilis.   Figure 2).
Nomenclatural notes: Vincenzo Tineo in Gussone [18] described R. schowii, providing a detailed description, quoting a precise collection locality and citing an unpublished illustration "Tin. ined.". In order to trace the original material, we checked the PAL herbarium, where Tineo's main collection is housed. We also searched in FI, G, NAP and P, where duplicates by Tineo's collections are kept. Lojacono Pojero [48] wrote that he saw the only authentic specimen of R. schowii in H. Pan. (i.e., Herbarium Panormitanum), today PAL. In the Herbarium Mediterraneum Panormitanum (PAL), this sample is no longer present (G. Domina, pers. comm.). In NAP (ex-Herbarium Gussone Sicilia), where duplicates by Tineo's specimens are usually hosted, we did not trace any samples but only the unpublished illustration ("Ranunculus schowii Tin./V. Cartoccio dis./1845"; NAP barcode NAP0000512) cited in the protologue. The illustration is also labelled with a representation label [49] "12a. Ranunculus schowii Tin./Aprile, Majo", where "12a" is a reference to the position of the species within the genus in Gussone's Synopsis. We were not able to trace original material in FI, G and P. The unpublished illustration in NAP (NAP0000512) is the only element belonging to the original material (Art. 9.4 of the ICN), agrees with the protologue and is here designated as the lectotype for the name R. schowii (Figure 2).
Taxonomic notes: based on the protologue and the lectotype, R. schowii seems to have unique characteristics, only marginally close to particular forms of R. isthmicus Boiss. The individual depicted in the illustration (lectotype) shows fusiform root tubers, basal leaves tripartite with entire or lobed (only at the apex) segments and deflexed sepals at flowering. Further studies to assess the morphological variability in this species and to clarify its taxonomic status will be needed. In the case of synonymy of R. isthmicus Boiss. (published in 1846, [50]) and R. schowii (published in 1845, [18]), the latter would have priority and it should be advisable to proceed with a formal conservation proposal for the name R. isthmicus.
Distribution: endemic to the SE Euro-Mediterranean area, distributed in Italy, Balkan Peninsula, Turkey and Georgia. The presence of R. gracilis in the latter country was reported by Grossheim [54], under the name R. agerii Bertol., for a single locality (Akhaltsikhe district; see Map No. 82 included in [54]), but, in our opinion, this report requires further checks. In Italy, in the current state of knowledge, it is present only in Lazio based on our finding reported here, no longer recorded in Emilia-Romagna and Sicily, recorded by mistake in Calabria and Puglia and formerly cultivated in botanical gardens in Toscana and Umbria (Figure 3). The presence of the species in each Italian administrative region is discussed below: • Piemonte: the species was cited as doubtfully occurring in Piemonte by Conti et al. [42] and as recorded by mistake by Bartolucci et al. [4]; it was never recorded for the region (D. Bouvet and A. Selvaggi, pers. comm.). • Emilia-Romagna: the presence of the species is confirmed by several old herbarium specimens kept in BOLO, CHE, FI, G, P, PI and RO (see specimens examined). Furthermore, the 16th century samples preserved in the "En Tibi" and "Cibo" herbaria were also collected in Emilia-Romagna near Bologna (Stefanaki et al. 2018(Stefanaki et al. , 2019. No recent herbarium samples or bibliographic records have been found (see, [55]); therefore, the species should be considered as no longer recorded. Targeted field research will be needed before considering the species as locally extinct. • Toscana: the species was never recorded for the region. We traced an old herbarium specimen in FI, collected in the Botanical Garden of the University of Firenze, where the species was probably cultivated. • Umbria: the species was recorded for the region in the past [9,11,15] as naturalized in the Botanical Garden of the University of Perugia. We traced the herbarium specimen linked to the old report by Fiori [11] in FI (see Specimens examined). • Lazio: during field investigations carried out in the territory of Anagni (Frosinone, Central Italy) in March and April 2022, we discovered the species on Mt. Campitelli ( Figure 3). Our finding corroborates the old report for this area by Sibilia ([56], under the name R. agerii) and confirms the presence of this species in Italy. The Sibilia record has never been incorporated in the regional floras [4,[57][58][59]. In the current state of knowledge, this is the only population present in Italy. • Puglia: the species was recorded by Di Pietro and Misano [60]. This record was later regarded as doubtful by Bartolucci et al. [4]. We were not able to trace herbarium specimens linked to this record and the species should be regarded as probably indicated by mistake (R. Di Pietro, pers. comm.). • Calabria: according to Peruzzi and Passalacqua [41], the Calabrian records should be referred to R. monspeliacus subsp. aspromontanus. • Sicily: the species was reported in Sicily from different localities by Giardina et al. [12]: between Vittoria and Terranova based on the description of R. schowii [18], between Catania and Misterbianco based on Strobl [61] and from Polizzi Generosa [62]. We have shown that R. schowii is not related to R. gracilis; therefore, the report of the latter between Vittoria and Terranova is erroneous. We traced, in PAL, the sample collected in Polizzi Generosa ("sotto il paese di Polizzi Generosa vicino all'acquedotto, 30 April 1990, Raimondo and Certa"), which should be referred to R. paludosus. A specimen cited in Wikiplantbase Sicilia [63] as R. gracilis and stored in PAL (No. 43515) collected at Busambra belongs to R. paludosus as well as a specimen in CAT No. 048272 (Monte Lauro, 9/V/1991, Brullo et al.). The only datum that we were able to confirm is the indication by Strobl [61], for the Amenano between Catania and Misterbianco at the foot of Etna, thanks to the tracing of an old herbarium sample stored in FI, collected in 1874 by Heidenreich at Misterbianco (quoted also by Fiori et al. [10]). Based on our data, R. gracilis should be considered as no longer recorded in Sicily. Targeted field research will be needed before considering the species as locally extinct.  Chromosome number: 2n = 16 [64]. Conservation status: Ranunculus gracilis currently occurs outside the NATURA 2000 network on Mt. Campitelli (Anagni, Frosinone) in Lazio (Central Italy). The populations in Emilia-Romagna (Northern Italy) and Sicily, confirmed by old herbarium specimens, have not been observed for over 120 years. The area of occupancy (AOO) is 4 km 2 , calculated with GeoCAT (Geospatial Conservation Assessment Tool) software [65]. The species actually occurs in one location and a decline in the AOO was observed, considering the possible extinction of some populations. According to IUCN [66] criterion B2ab(i,ii,iv), the species is assessed as Critically Endangered (CR) at the regional level (Italy).

Conclusions
Nomenclature plays a central role in the description of the diversity of life on our planet and the typification process is essential for any taxonomic study. At the same time, floristic research and the study of herbarium collections are of crucial importance in biodiversity conservation of vascular plants and are necessary to collect data for planning the correct conservation strategies. Our study on R. gracilis in Italy allowed us, primarily, to evaluate the taxonomic identity of R. agerii and R. schowii, both described from Italian territory. After typification, R. agerii should be regarded as a heterotypic synonym of R. gracilis, while R. schowii showed a combination of unique characters, close in some ways to atypical forms of R. isthmicus, and needs further studies to assess the morphological variability in the species and to clarify its taxonomic status. Thanks to our contribution, the presence of R. gracilis in Italy is confirmed, expanding the distribution range of this endemic species to the SE Euro-Mediterranean towards the west. In Italy, R. gracilis is present, in the current state of knowledge, with a single population at risk of extinction found in Lazio (Central Italy). Furthermore, we were able to confirm the historical presence of the species, based on the study of herbarium collections, in the Emilia-Romagna (Northern Italy) and Sicily, where it has not been observed for over 120 years. It will now be possible to plan specific field surveys to verify whether R. gracilis is still present in these areas or is to be considered extinct. Furthermore, the species is now reported in the national conservation framework.