﻿Taxonomic and ecological remarks on Solenopsisbivonae species complex (Campanulaceae)

﻿Abstract The populations usually attributed to Solenopsisbivonae (Tineo) M.B.Crespo, Serra & A.Juan are investigated from a taxonomical and morphological viewpoint. Within this species complex, four new subspecies occurring in Sicily and Calabria are recognized, such as subsp. bivonae, subsp. madoniarum, subsp. peloritana and subsp. brutia. In addition, a new species from Cyprus described as S.meikleana and S.bacchettae from Sardinia must be included in this group. The synonymy, typification, description, seed testa morphology, chorology, ecology, illustrations, conservation status, and examined specimens for each taxon are provided. Besides, the analytical keys, distribution maps, and phytosociological arrangement regarding these taxa are given too.

Taxonomic and ecological remarks on Solenopsis bivonae species complex (Campanulaceae) Introduction Solenopsis C.Presl is a very peculiar genus of Campanulaceae, belonging to subfam. Lobelioideae, distributed in the Mediterranean and Macaronesian territories. Within this genus, two well-distinct groups can be recognized, which differ in habit and in flower structure (Crespo et al. 1998;Brullo et al. 2023a, b). The first one is characterized by a caulescent or subcaulescent habit with leaves all inserted on the scape and flowers with corolla provided by lobes slightly divaricated at the top. Conversely, the second one shows a stemless habit with leaves arranged in basal rosette and flowers with corolla provided by lobes markedly patent at the top. The only exception is represented by a species showing intermediate characters between the two groups since it has an erect scapose habit and flower corolla with clearly divaricated lobes at the top. The first group includes only annual species, such as Solenopsis laurentia C.Presl, widespread in the Mediterranean area and the Canary Islands, represented by several subspecies examined by Brullo et al. (2023a), to which S. mothiana C. Brullo, Brullo & Giusso, showing a punctiform distribution in Sicily (Isola Grande dello Stagnone), must be added. According to Brullo et al. (2023b), the second group includes many more species, such as

Material and methods
The morphological investigations were conducted on wild plants collected in several Mediterranean territories (Sicily, South Italy, Sardinia, and Cyprus) and cultivated in the Botanical Garden of Catania (Italy). The morphological features were analyzed based on at least twenty individuals for each examined population, with well-developed vegetative and reproductive structures. The living material was observed under a Zeiss Stemi SV 11 Apo stereomicroscope at 6-66× magnification, provided with a drawing device. Electron micrographs (SEM) were obtained under a Zeiss EVOL LS10 scanning electron microscope at an accelerating voltage of 10 kV; ten seeds were directly mounted onto aluminum stubs with double adhesive tape and coated with gold prior to observation. The seed surface sculpturing terminology mainly followed Barthlott (1981Barthlott ( , 1984 and Gontcharova et al. (2009). The vouchers are deposited in the Herbarium of the University of Catania (CAT). The herbarium codes are according to Thiers (2020). Phytosociological investigations were carried out using the method of the Sigmatist school (Braun-Blanquet 1928), while for the syntaxonomical arrangement, Mucina et al. (2016) was followed. The conservation status of the species was calculated with GeoCAT (Geospatial Conservation Assessment Tool) software (Bachman et al. 2011) and according to IUCN guidelines (IUCN 2022 Crespo et al. 1998).
Iconography. Bivona-Bernardi (1806)  Phenology. Flowering late April to September, fruiting May to September. Distribution and ecology. According to herbarium investigations and our field survey, this taxon occurs in North-West Sicily (Fig. 10), in the surroundings of Palermo, especially along the banks of the Oreto River, where it is today very rare, while it is very widespread along the Sosio river near Chiusa Sclafani. Previously, it was reported from Alcamo, where unfortunately it is extinct. As concerns its ecological requirements, it is localized at an elevation between 10 and 250 m, growing on calcareous vertical wet rocky places affected by permanent dripping (Fig. 8A). The plant community characterized by this hygrophyte can be referred to the phytosociological class Adiantatea capilli-veneris Br.-Bl. in Br.-Bl., Roussine andNègre 1952 (cfr. Cambria 2020). This habitat is floristically differentiated by a moss carpet where grow also Adiantum capillus-veneris L. and Samolus valerandi L.
Conservation status. Currently, this taxon's result is circumscribed in Sicily to two wet stands (Oreto and Sosio rivers), where it is very rare in the first locality and quite spread in the second one. Overall, this plant results in it being seriously threatened since it is linked to wetlands potentially subject to anthropic pressure, which tends to alter the water regime, prejudicing its survival. Therefore, in agreement with Conti et al. (1997), who quoted it as S. minuta subsp. nobilis, it can be treated as Endangered (EN), following IUCN criteria (IUCN 2022 Diagnosis. It differs from the type in having leaves arranged in a smaller rosette with shorter blade, shorter floral pedicel, provided with a single bracteole glabrous or with few apical hairs and 1-2 basal sessile glands, corolla smaller with upper lip lobes without glands in the ventral face and lower lip lobes shorter, obtuse, provided with dense and shorter papillae, with anther tube papillose at the basis and longer capsule. Conversely, the type is characterized by leaves arranged in a larger rosette with longer blade, longer floral pedicel, provided with (1)2 bracteoles with several hairs at the apex and 1-4 lateral stipulated glands, corolla larger with upper lip lobes with glands in the ventral face and lower lip lobes longer, acute, provided with lax and longer papillae, anther tube without papillae at the basis and shorter capsule.
Etymology. The epithet derives from Madonie, a massif of North Sicily, where this taxon is rather spread.
Phenology. Flowering late May to October, fruiting June to October. Distribution and ecology. Based on herbarium data and field investigations, this taxon is distributed in the Madonie massif, where it is localized in many places at 700-1600 m of altitude (Fig. 10), represented mainly by peat bogs, dominated by Sphagnum sp. pl., Aulacomnium palustre (Hedw.) Schwägr. and Polytrichum commune Hedw. Here, it characterized an orophilous plant community belonging to Scheuchzerio palustris-Caricetea fuscae R. Tx. 1937, as emphasized by Raimondo et al. (1980Raimondo et al. ( , 2021. Sometimes, as near Petralia Soprana or Piazza Armerina, it occurs also on calcareous vertical wet rocky places affected by permanent dripping, where it is a member of vegetation of the class Adiantetea capilli-veneris, dominated by Adiantum capillus-veneris and several bryophytes.
Conservation status. This taxon shows a scattered distribution, occurring mainly in some localities within the Madonie Regional Park. Besides, it is a species closely linked to small wet stands fed by water springs, whose collecting leads to the destruction of the habitat and the disappearance of the vegetation that characterizes it. It shows an EOO of 410 km 2 and an AOO of 20 Km 2 . Therefore, according to B criterion, we propose to consider this taxon as Endangered Diagnosis. It differs from the type in having longer bracteoles, glabrous, provided with apical gland, longer calyx with longer lobes, larger corolla with denser and spread glands in the ventral face, larger upper lip lobes and lower lip lobes, within the lower lip a yellow macula at the base, slightly bordered of redbrown, longer style and larger capsule. Conversely, the type is characterized by shorter bracteoles, hairy apex without gland, shorter calyx with shorter lobes, smaller corolla with more scattered glands in the ventral face, smaller upper lip lobes and lower lip lobes, within the lower lip a greenish-yellow macula at the base, slightly bordered of brown, shorter style and smaller capsule.
Etymology. The epithet derives from Peloritani, a chain of North-eastern Sicily, where this taxon is localized.
Phenology. Flowering June to August, fruiting July to August. Distribution and ecology. It grows on metamorphic vertical wet rocky stands affected by permanent dripping. It is a member of a plant community of the class Adiantetea capilli-veneris, dominated by Adiantum capillus-veneris, associated with Samolus valerandi L., Lysimachia nemorum L., Hypericum hircinum L. subsp. majus (Aiton) N. Robson and several bryophytes. In this stand, it is localized exclusively along a short watercourse of the Mela valley (Peloritani chain) at an elevation of 600-700 m (Fig. 10), where several individuals of this taxon were surveyed.
Conservation status. This taxon is known for one stand of the Peloritani chain, along a short wet wall, where about one hundred well-developed individuals were observed. This population is very isolated and inaccessible and it does not seem subject to immediate threats. It shows an EOO of 4 km 2 and an AOO of 4 Km 2 . Therefore, according to the B criterion (IUCN 2022), we propose to consider this taxon as Critically Endangered category [(CR -B1ab(iii)+2ab(iii))]. Diagnosis. It differs from the type in having leaves arranged in a smaller rosette, shorter leaves, with oblanceolate to oblanceolate-spathulate blade, shorter petiole, shorter floral pedicel, glabrous bracteoles, located in the upper half, provided with sessile apical gland, two basal glands and 0-2 lateral glands, corolla in the lower lip with a green macula at the base and provided with three dark blue spots above, lobes papillose up to the apex, longer staminal filaments, glabrous anther tube, longer style, slightly tuberculate capsule, reddish-brown and larger seeds. Conversely, the type is characterized by leaves arranged in a larger rosette, longer leaves, with spathulate blade, longer petiole, longer floral pedicel, bracteoles hairy at the apex, located in the middle, provided with 1-4 stipulated lateral glands, corolla in the lower lip with a greenish-yellow macula at the base, without spots, lobes papillose for more than the lower half, shorter staminal filaments, anther tube hairy at the apex, shorter style, smooth capsule, brownish and smaller seeds.
Phenology. Flowering June to September, fruiting June to September. Etymology. The specific epithet refers to "Brutia," the Latin name of Calabria, territory where this taxon grows.
Distribution and ecology. This taxon was surveyed in the lower reaches of Lao river (North Calabria), at elevations of 130-350 m, where it grows on rocky metamorphic outcrops (Fig. 10). It likes humid and shady stands covered by a dense moss carpet, associated mainly to Adiantum capillus-veneris. As for the other subspecies previously examined, it is linked to hygrophilous communities of the Adiantetea capilli-veneris too. From a phytogeographical point of view, it should be noted that this taxon is the only Solenopsis with a perennial habit, localized in a continental territory since all the others occur exclusively in big Mediterranean islands (Crespo et al. 1998). Conservation status. The populations of this subspecies are rare and all circumscribed to the banks of Lao river in North-West Calabria. Based on recent field surveys, its presence in the three hitherto known locations has been confirmed in only one of them (near Papasidero), while in the other two, it seems to have disappeared (Laino-Castello and Laino-Borgo). It shows an EOO of 9.51 km 2 and an AOO of 12 Km 2 . Therefore, in addition to its rarity and the considerable reduction of its current range, according to B criterion (IUCN 2022), we propose to consider this taxon as Endangered [EN -B1ab(iii)+2ab(iii)].
Additional specimens examined (  Diagnosis. It differs from Solenopsis bivonae in having glabrous and longer bracteoles, provided with apical sessile glands and 1--2 glands per side, pale blue or pale violet corolla, with upper lip lobes without papillae, lower lip lobes oblong, smaller, provided with shorter glands, anther tube shorter and papillose at the base, shorter style and longer capsule. Conversely, S. bivonae is characterized by shorter bracteoles, hairy at the apex and with 1--4 glands per side, bluish-lilac corolla, with upper lip lobes with papillae in the ventral face, lower lip lobes linear-lanceolate, larger, provided with longer glands, anther tube longer, without basal papillae, longer style and shorter capsule.
Iconography. Meikle (1985), plate 65. Phenology. Flowering March to October, fruiting April to October. Etymology. It is dedicated to Robert Desmond Meikle (1923Meikle ( -2021, author of the "Flora of Cyprus," who dealt with the taxonomy of the genus Solenopsis. Distribution and ecology. This species occurs in western Cyprus, where it is localized in very moist environments such as river banks, springs, waterfalls, and dripping walls (Fig. 10). Usually, it grows from hills to mountain belts up to an elevation of 1600 m, on ophiolitic rocky outcrops covered by bryophyte carpets and ferns, particularly Adiantum capillus-veneris. This vegetation can be referred to the Adiantetea capilli-veneris class for its floristic and ecological peculiarity.
Conservation status. This species, endemic to Cyprus, shows a scattered distribution in the western part of the island. It is a perennial hygrophyte, usually occurring in the wet rocky stands, which are always subject to dripping waters. Regarding conservation, the habitat characterized by this species is subject to synanthropic threats, represented mainly by the uptake of springs or the waters of streams, which allow its survival. The species shows an EOO of 1298 km 2 and an AOO of 288 Km 2 . Therefore, according to B criterion (IUCN 2022), we propose to consider this taxon as Endangered [EN -B1ab(iii)+2ab(iii)].
Iconography. Brullo et al. (2023b), Fig. 1. Phenology. Flowering May to August, fruiting June to September. Etymology. This species is dedicated to Gianluigi Bacchetta, an active botanist from Cagliari University and an expert on the Sardinian flora. Brullo et al. (2023b), Solenopsis bacchettae is distributed in central-east Sardinia, where it is localized on carbonatic substrates (Fig. 10). It grows exclusively on damp soils along or near small streams with fresh water at 700-1000 m a.s.l., where it is a member of a plant community rich in endemic hygrophilous species.

Distribution and ecology. According to
Conservation status. This species shows a scattered distribution, currently represented by few locations, where an estimated population of around 1000 individuals occurs. Based on IUCN (2022) criteria, Brullo et al. (2023b) proposed to treat it as an endangered species (EN).

Seed micromorphology
According to literature (Murata 1992(Murata , 1995Haridasan and Mukherjee 1993;Serra and Crespo 1997;Crespo et al. 1998;Brullo et al. 2013Brullo et al. , 2023b, the ornamentations of the seed coat in the Lobelioideae, subfamily of Campanulaceae, show a relevant taxonomical value and phylogenetic importance. Overall, the testa structure of mature seeds within this subfamily shows well-defined and constant ornamentations in every taxon. The seed coat sculptures are characterized by long, narrow cells (50-150 μm long) separated by longitudinal furrows. From the SEM observations, the seeds of Solenopsis bivonae subsp. bivonae (Fig. 11A1) have an ellipsoid-fusiform shape, narrowing towards the basal and apical ends, having a size of 0.40-0.45 × 0.20-0.25 mm. As concerns its seed testa, the cells have periclinal walls distinctly convex, 4-5 μm wide, crossed by a marked convex central ridge 1.4-1.8 μm wide, with anticlinal walls linear and deeply grooved (Fig. 11B1-C1). The seeds of S. bivonae subsp. madoniarum (Fig. 11A2, A3) show an obovoid-ellipsoid shape, rounded at the apical end, with a size of 0.40-0.46 × 0.24-0.26 mm. As concerns its seed testa, the cells have periclinal walls distinctly convex, 5.5-8.0 μm wide, crossed by a marked convex central ridge 0.8-1.6 μm wide, with anticlinal walls linear and deeply grooved ( Fig. 11B2-C2, B3-C3). The seeds of S. bivonae subsp. peloritana (Fig. 11A4) have an ellipsoid shape, rounded at the apical end, with a size of 0.45-0.50 × 0.24-0.26 mm. As concerns its seed testa, the cells have periclinal walls distinctly convex and smooth, 6.4-10.0 μm wide, without a central ridge, with anticlinal walls linear and deeply grooved (Fig. 11B4-C4). The seeds of S. bivonae subsp. brutia (Fig. 11A5) have an ellipsoid shape, rounded at the apical end, with a size of 0.46-0.50 × 0.26-0.30 mm. As concerns its seed testa, the cells have periclinal walls distinctly convex, 4.4-6.0 μm wide, crossed by a marked convex central ridge 1.4-2.0 μm wide with a row of distinct tubercles and with anticlinal walls linear and deeply grooved (Fig. 11B5-C5). The seeds of S. meikleana (Fig. 11A6) have a broadly ellipsoid shape, rounded at the apical end, with a size of 0.40-0.46 × 0.24-0.29 mm. Regarding its seed testa, the cells have periclinal walls slightly convex, 5.0-8.3 μm wide, crossed by an evanescent convex central ridge 0.8-1.2 μm wide, and with anticlinal walls linear and slightly grooved (Fig. 11B6-C6). The seeds of S. bacchettae (Fig. 11A7) have an ellipsoid shape, rounded at the apical end, with a size of 0.50-0.52 × 0.30-0.32 mm. As concerns its seed testa, the cells have periclinal walls usually quite flat, 4.0-4.5 μm wide, crossed by a slightly convex central ridge 1.0-1.6 μm wide and with anticlinal walls linear and slightly grooved (Fig. 11B7-C7). Figure 11. SEM images of seed shape (A ×250) and detail of seed testa (B ×1000 and C ×2000) regarding:

Phytosociological remarks
Based on our field observations during the surveys on the populations belonging to the Solenopsis bivonae group, it was possible to verify that they were always localized in very specialized humid habitats, limited to very circumscribed surfaces. As previously highlighted, three main habitats can be recognized, where usually the examined populations of Solenopsis occur. In particular, they are represented by dripping rocky walls, peat bogs, and edges of streams or springs. As concerns the wet rocky environments, the surface is usually covered by a bryophytic layer, where individuals of Adiantum capillus-veneris more or less densely grow. According to Deil (1995Deil ( , 1996Deil ( , 1998, these habitats represent conservative environments that remain very stable in time and space, unaffected by climate change in neither geological nor current climatic variation in the Mediterranean area. Besides, these wet stands host several vicarious taxa having a Tertiary origin (Deil 1995(Deil , 1996(Deil , 1998 belonging, in particular, to Primula, Hypericum sect. Adenosepalum, Pinguicola, and relictual tropical ferns, such as Woodwardia radicans (L.) Sm., Pteris vittata L., P. cretica L. and Osmunda regalis L. Indeed, the current floristic composition of these peculiar hygrophilous communities results from evolutionary processes within the single taxa rather than recent changes in the environmental and ecological conditions. Therefore, the plant communities within which these species now grow must be considered the impoverished remains of those dating back to the Tertiary. Due to the climatic changes during the Quaternary and the recent Holocene, these phytocoenoses generally occupy much smaller areas than in the past, remaining almost constant in their floristic composition. At the same time, the taxa that characterize them have undergone significant speciation processes, always remaining linked to the same ecological context and maintaining their phytosociological role. These communities, due to their floristic set, structure, and ecological requirements, must be referred to the phytosociological class Adiantetea capilliveneris Br.-Bl. in Br.-Bl., Roussine and Nègre 1952, syntaxon distributed mainly in the Mediterranean area and Western Asia (Braun-Blanquet et al. 1952;Brullo et al. 1989;Deil 1989Deil , 1998de Foucault 2015 Structure and ecology: This association occurs at an elevation of 10-250 m a.s.l. in the calcareous rocky walls subject to dripping by groundwater, partially covered by a bryophytic carpet mainly represented by Eucladium verticillatum, Pellia endiviifolia, Rhynchostegiella tenella, and Scorpiurum circinatum. It is differentiated physiognomically by the dominance of Solenopsis bivonae subsp. bivonae, which with its leaf rosettes covers most of these small surfaces, usually mixing with Adiantum capillus-veneris and Samolus valerandi. The stands colonized by this vegetation are localized especially along water-courses in the cooler and shadier places.
Distribution: The association was surveyed along the Sosio river near Chiusa Sclafani, where it is quite frequent, and the Oreto River near Palermo, where, however, it is currently very rare.
Characteristic species: Solenopsis bivonae subsp. madoniarum. Structure and ecology: This association is localized in a habitat very similar to those colonized by the previous one but linked to stands with higher elevation (700-1200 m a.s.l.). This vegetation shows a lower coverage of Adiantum capillus-veneris and a more developed bryophytic layer, characterized by Eucladium verticillatum and Pellia endiviifolia. This habitat is represented by vertical rocky walls with dripping waters coming from small springs.
Distribution: This association is quite rare, and was observed in a few mountain localities, like near Piazza Armerian and Petralia Soprana. C-Adianto capilli-veneris-Solenopsietum peloritanae ass. nov. hoc loco (Table 1,
Characteristic species: Solenopsis bivonae subsp. peloritana. Structure and ecology: It is a sub-mountain association closely linked to metamorphic vertical rocky walls with dripping groundwaters at an elevation of 600-700 m a.s.l. The bryophytic layer is represented by Pellia epiphylla and Conocephalum conicum, where Adiantum capillus-veneris, Solenopsis bivonae subsp. peloritana and Samolus valerandi grow, often with high values of coverage.
Distribution: This association is exclusive of a small stand in the Tyrrhenian slope of the Peloritani range.
Characteristic species: Solenopsis meikleana and Carex troodi Turril. Structure and ecology: This association usually is linked to wetlands represented mainly by waterfalls and dripping walls, often near the spring, where it grows on ophiolitic substrata at an elevation of 1000-1600 m a.s.l. The vegetation is localized prevalently in the stands not directly affected by the water flow, liking less damp surfaces. In the bryophytic layer, the more frequent species are Eucladium verticillatum, Pellia epiphylla, Eurhynchium praelongum, and Scorpiurum circinatum, while among the vascular plants, the endemic Solenopsis meikleana and Carex troodi are dominant, growing together with Adiantum capillus-veneris.
Distribution: This association is endemic to the western part of the island of Cyprus, which is localized in very specialized damp habitats.

Key to the taxa belonging to the Solenopsis bivonae group
Basing on the morphological diacritical characters listed in Table 3, the following analytical key has been performed. Bracteoles 3-5.5 mm long; corolla 12-14.5 mm long, with upper lip 5-6 mm long and lower lip 8-9 mm long; style 6.5-7 mm long ..