Unexpected Discovery of Thelypteris palustris (Thelypteridaceae) in Sicily (Italy): Morphological, Ecological Analysis and Habitat Characterization

Thelypteris palustris Schott (Thelypteridaceae), known as “marsh fern”, is infrequent in the Mediterranean area. The occurrence of this species is known for almost all the Italian regions (except for Sardinia and Sicily), but with rare and declining populations. During floristic fieldwork on the Sicilian wetlands, a new unknown population was found. The aim of this paper is to analyze the morphological traits of the species, as well as its ecological features and the floristic composition of the plant communities where it lives. According to IUCN guidelines, here we provide the regional assessment (Sicily) of T. palustris. To analyze its morphological features, many living plants were examined, with particular attention to the spore structure. A total of 179 plots (110 species) and 34 pools were sampled. Our results highlight the relic character of the species which is at the southernmost border of its distribution range. The micro-morphological investigations on the spores show that the Sicilian population belongs to the subsp. palustris. The floristic analysis confirms the clear dominance of perennial temperate-cold zones Eurasian taxa. Finally, a new association, Thelypterido palustris-Caricetum paniculatae, within the Caricion gracilis alliance (Phragmito-Magnocaricetea class) is described.


Introduction
Thelypteris palustris Schott (Thelypteridaceae), known as "marsh fern", is a deciduous species that represents one of the most complex species in the pteridophytes. Fernald [1] recognized four varieties of Thelypteris palustris: var. palustris of Eurasia (from Europe and NW Africa to eastern Himalayas and southern China); var. pubescens (G. Lawson) Fernald of northeastern United States, Canada, and eastern Asia; var. haleana Fernald of the southeastern United States and Bermuda; and var. squamigera (Schltdl.) Weath. of Africa, southern India, northern New Zealand [2]. Afterwards Tryon et al. [3], mainly analyzing the spore structure, recognized two species, one largely of the southern hemisphere (T. confluens (Thunb.) C.V.Morton = T. palustris var. squamigera) and the other, T. palustris, including two varieties (T. palustris var. palustris and T. p. var. pubescens), in the northern hemisphere.
This taxonomic view has been confirmed in recent times, so currently the genus Thelypteris includes two species, T. palustris in the northern hemisphere and T. confluens in the southern hemisphere, the only change regarding the rank of the two taxa within T. palustris that are now considered subspecies [4].
In Europe Thelypteris palustris (subsp. palustris) is known from several countries [5]. In Italy, this species is known for almost all regions, except for Sardinia and Sicily, although it is reported as an extinct or doubtful taxon for many territories [6][7][8].
The species has undergone a considerable decline throughout its distribution range, mainly due to habitat loss and reduction. Despite this significant decrease in area at the European level, it was recently classified as Least Concern (IUCN category) [9].
Our finding, during a survey in the Nebrodi Mountains (northern Sicily), is very interesting because the species is quite rare in the Italian territory since the habitats where it grows are in strong reduction, and even more because the Sicilian population represents the southernmost limit of its distribution range. The new finding is certainly unexpected because the flora of Sicily is one of the best studied in Italy and probably in Europe: consider that the start of a "modern" botanical exploration of the island date back to 1664 at least [22] Marginal habitats in the Mediterranean area represent sites of high ecological importance and a refuge for threatened plants (e.g., hygrophytes) like the case of Thelypteris palustris. In fact, these hydrophytic species are linked to peculiar ecological requirements and are highly susceptible to climate changes, and this could be led to their disappearance in the next years. The correct identification of T. palustris, as well as the floristic composition of the plant community where it grows and its ecology, are relevant issues for future conservation measures and monitoring actions of this species.

Distribution and Conservation Status in Italy
In Italy, the species is reported for almost all regions. Probably, it has never been found in some territories due to the reduction or disappearance of its natural habitat, or in some cases, also owing to incorrect reports. It is reported as extinct in Marche, as a doubtful record in Molise and Campania, and not found in recent times in Umbria, Valle d'Aosta and Abruzzo [7][8][9]. In southern Italy the species is highly localized, with an altitude range between 0 and 1000 m a.s.l., from the coast to the mountain, occurring in Puglia at Laghi Alimini, Otranto [23,24], in Calabria at Lago dell'Aquila, Reggio Calabria [13,25] and in Sicily at Serra della Testa (Nebrodi) (Figure 3). This population recently discovered in Sicily has extended its distribution range and represents the southernmost population of Italy.
According to the European Red List of Vascular Plants [26], the species is classified as Least Concern (LC). Currently, in Italy, Thelypteris palustris has been recently evaluated as vulnerable (VU) by Orsenigo et al. [27] based on the criterion B [28]. In Sicily, the total area occupied by Thelypteris palustris is about 0.62 ha. Despite its very small distribution area, it was not possible to carry out a detailed count of the individuals of the population due to the stoloniferous vegetative development of the species. Therefore, thanks to our data and according to the IUCN criterion B, we recommend considering Thelypteris palustris as Critically Endangered (CR B2abii, iii, iv) for Sicily, due to a very small AOO (4 km 2 ), the occurrence on one location, and possible decline of the population especially because of the grazing practices and water flow reduction due to climate change.
In Sicily, Thelypteris palustris falls within the sedges of the Caricion gracilis alliance (Magnocaricion elatae). This alliance, until now never reported in Sicily, groups plants communities growing on eutrophic clayey soils flooded for long time with a temperate Europe distribution.

Vegetation Ecology and Habitat
Overall, 15 different plant communities, each one with specific floristic compositions, were identified (Appendix A). Most of these plant communities were investigated by Brullo et al. [32] for Nebrodi Mounts. Therefore, we avoid a detailed description of the investigated communities. The wide sampling and cluster analysis allowed us to highlight the uniqueness and rarity of T. palustris in Sicily and define objectively the correct syntaxonomic framework. The cluster analysis of all relevés carried out on the Nebrodi Mounts showed 2 main groups ( Figure 4). The first group (cluster A) includes mainly the helophytic perennial vegetation of the Phragmito-Magnocaricetea class, while the second group (cluster B) includes the aquatic vegetation of the Lemnetea and Potametea classes [31]. Within the Phragmito-Magnocaricetea four alliances can be distinguished: the first one (A11) Phragmition communis includes the vegetation dominated by tall graminoid species subjected to regular, prolonged periods of flooding that grow on mineral meso-eutrophic, often muddy, soils; the second one, Magnocaricion elatae (A121) consist of plant communities of mesotrophic to dystrophic soils, often peaty and flooded for prolonged periods; the third alliance Caricion gracilis (A122) groups communities of eutrophic soils, flooded for prolonged periods; the fourth alliance Alopecuro-Glycerion spicatae (A2), that includes the vegetation of hygrophilous herblands of shallow montane pools characterized by large water-depth fluctuations at high altitudes of Sicily. This last alliance is grouped with a peculiar annual amphibious vegetation dominated by Lythrum portula which falls within the Nanocyperetalia order (Isoeto-Nanojuncetea). Within the second group (cluster B) two subclusters can be distinguished: the first one (B1) (Potametea pectinati) delimits the perennial macrophytic communities of fresh, mesotrophic to eutrophic, waters; while the second one subcluster (B2) includes (Lemnetea minoris) the floating pleustophyte communities eutrophic to hypertrophic waters.
Bray-Curtis ordination shows a marked correspondence with cluster analysis (

Floristic Composition and Phytosociological Insights of the Thelypteris palustris Population in Sicily
In the study area, Thelypteris palustris was found exclusively in a perennial wetland characterized by Carex paniculata L. and Juncus subnodulosus. This perennial vegetation grows on flat or slightly sloping surfaces, on clayey-silty acid soils, permanently wet and rich in organic matter. The structure is determined mainly by Carex paniculata, the dominant species in terms of biomass and number of individuals, joined to several hygrophilous species, as Galium palustre L. subsp. elongatum, Mentha aquatica L., Cirsium creticum (Lam. This later order includes hygrophilous communities domi-nated by helophytes (rushes and sedges) that grow in depressions in the supratemperate thermotype subjected to periodic submersions, on soils with low permeability and a rich silty-clayey component [33][34][35]. In addition, the association hosts floristic elements, very rare in Sicily, of high phytogeographic value (Figure 6), such as Epipactis palustris (L.) Crantz, Equisetum palustre L., Rhynchocorys elephas (L.) Griseb., Juncus conglomeratus L., Carex flacca Schreb. subsp. flacca, C. pallescens L., etc. The muscinal component also plays an important ecological role, particularly Calliergonella cuspidata (Hedw.) Loeske with a high degree of coverage and sociability. From the chorological and structural viewpoint, this vegetation highlights the relevance of the species with an Euroasiatic-Circumboreal distribution (34%), with geophytes (34%) and hemicryptophytes (61%) being the dominant life forms. This new association can be considered a southern vicariant of the Caricetum paniculatae, with a central and northern Italian distribution [36,37]. This last association shows structural affinities with Thelypterido palustris-Caricetum paniculatae owing to a high cover of Carex paniculata. However, the two plant communities can be clearly separated, based on many differential diagnostic species, such as Epipactis palustris, Equisetum palustre, Juncus subnodulosus, and Rhynchocorys elephas. From a bioclimatic point view the Thelypterido palustris-Caricetum paniculatae falls into the lower Supramediterranean belts with lower subhumid ombrotype [38], in contact with deciduous thermophilic Quercus cerris oak forests, referable to the Arrhenathero nebrodensis-Quercetum cerridis [39].

Study Area
The study area is situated in the Nebrodi Mounts, Sicily's largest mountain complex (Figure 7). They are located in the N-E part of the island, between the west side of the Peloritani Mountains and the east side of the Madonie Mounts, constituting the extension of the Apennine ridge on the island. They are a mountain range without major roughness that reaches its maximum altitude at Monte Soro (1847 m a.s.l.). From a geological point of view, this territory is mainly made up of sedimentary successions belonging to different periods. The dominance of Flysch is mostly noted, the oldest sediments belonging to the Alpine Tethys Units [40], they are Cretaceous in age and are represented by deep-water flyschs and scaly clays. Most of the outcropping rocks are part of the so-called Flysch of Monte Soro (upper Tithonian, lower Cretaceous) and Numidian (lower Oligocene Miocene) [41].  Table S1), with zonation of the regional park (zone A: red; zone B: orange; zone C: blue; zone A is the most protected zone).
The outcrop of clayey layers favors the formation of humid environments and ponds, and lakes originate where the orographic conditions allow it. The existence of humid environments on the Nebrodi is possible because of favourable climatic conditions that characterize this mountain area that is the most mesic and rainy in Sicily, being affected by average annual rainfall between 1000 and 1400 mm. According to Rivas Martínez et al. [42], the bioclimate of this area is supra-Mediterranean lower middle-humid bioclimatic conditions [38]. Although these small wetlands can have a relatively short lifespan due to landfills, climate changes, etc., for some of these areas in the Nebrodi Mounts an existence has been documented since the end of the last glaciation (about 10,000 years ago) when it seems that the climate had become wetter in Sicily [43]. The climatic and geomorphological conditions of the Nebrodi Mounts make it the area with the greatest wooded coverage and with the highest values of biodiversity in Sicily [44]. In particular, this territory is characterized by very extensive oak forests (Quercus cerris L.) at medium altitudes, and beech woods (Fagus sylvatica L.) at higher altitudes. However, grazing meadows and small wetlands (mostly natural) are the main discontinuities in the forest cover of this territory.

Data Sets and Data Processing
The morphological study regarding Thelypteris palustris was carried out on living material (15 specimens), all coming from Nebrodi Mounts territory. The collected samples were kept at the Catania Herbarium (CAT). For scanning electron microscopy (SEM) images, samples of spores were transferred from herbarium specimens to aluminum SEM stubs coated with double sided carbon tape. The stubs were then sputter-coated with gold and imaged digitally using a Zeiss EVO LS10, with an accelerating voltage of 30 kV, in the Center for Microscopy at the University of Catania. The morphological terminology used in the description follows Lellinger [45], while to spore nomenclature follows Tryon & Lugardon [46].
To analyze the structure and floristic composition of the marsh vegetation in the Nebrodi Mounts, 34 pools were examined. A total of 179 unpublished phytosociological relevés (110 species) were collected, personally sampled in the period April 2018-June 2021. The floristic composition and cover of species in each plot were determined by using the standard method of relevés [47]. All the relevés were classified using classification and ordination methods. Numerical analysis was performed using the software package "PC-ORD", 6.08 software. A multivariate analysis (Linkage method: Ward's, Distance measure: Sorensen (Bray-Curtis) was applied. Bray-Curtis ordination (Distance measure: Jaccard) takes into account different quantitative data, such as vegetation coverage (%), altitude, number of species (N. sp.), Altitude (m a.s.l.), Slope ( • ), Aspect, and Simpson/Shannon index. Quantum GIS software version 3.6 and GPS Garmin Montana was used to geolocate the surveyed wetlands.
For the risk assessment at the regional scale (Sicily), we followed the IUCN protocol and the most recent guidelines for its application [28]. In particular, we applied the IUCN criterion B by estimating trends in the Area of Occupancy (AOO), that is, the area covered by a taxon. AOO was assessed by using a 2 × 2 km grid [48]. Syntaxa classification follows Biondi et al. [33], and Mucina et al. [49]. Taxonomic nomenclature follows Bartolucci et al. [9] and Pignatti [50][51][52][53].

Conclusions
The species we found in Sicily seems to be very rare in the island, unless new discoveries that might be made in the future. It is localized in a microrefuge area that means, according to Rull [54], a small area with local favorable environmental features, in which small populations can survive outside their main distribution area, protected from the unfavorable regional environmental conditions. T. palustris grows in contexts which, even if somewhat subject to disturbing factors such as grazing, maintain good natural characteristics. However, these places are vulnerable to further disturbances, such as drainage, and above all to the decrease in rainfall triggered by climate change as detected for Sicily [55,56] that could jeopardize its precarious survival. Therefore, even the microrefuge area may not be enough to guarantee the existence in Sicily of this species; in any case, it will need to be monitored over time. This is a general trend that can undermine a species that, although with a large distribution range, is linked to peculiar environmental conditions. These circumstances could fail especially in semi-arid areas such as around the Mediterranean basin where climate change overlaps the usual intense anthropogenic disturbance that particularly affects wetlands [57]. In the Mediterranean area, the populations of T. palustris are likely to be declining following the general trend of destruction and degradation of shallow wetlands. It is not considered common anywhere in its Mediterranean range. It is very rare in Morocco and in Algeria. In Morocco it is known from two localities only: Bou Charen and near to Açilah in the western Rif. In Algeria, T. palustris is known from three localities, including Senhadja in Numidie. It is widespread in Turkey, but its habitats are under threat and because of this in the future this taxon may be threatened [58]. In Italy, as we have shown, many reports are old and no longer reconfirmed. The species, going south, is highly localized, occurring only in Puglia, Calabria, and Sicily ( Figure 3), with only one sub-population for each region. Therefore, this recently discovered in Sicily extend its distribution area and represents the southernmost population of Italy.
Plants characterizing wet environments represent one of the most threatened groups of the Mediterranean flora [59][60][61][62][63][64][65]. For the reasons quoted above, these areas require urgent and effective conservation policies not only to safeguard the biodiversity but also for the important ecosystem services they perform [66]. An example can be our study that illustrates the environmental context of the T. palustris populations and the morphological features of the species. Moreover, it clarifies some ecological requirements which are relevant issues for future conservation measures for this species, especially in the Mediterranean areas where the extremely scattered distribution with isolated populations make it vulnerable to disappearance. Unlike the northern European populations, quite widespread, the risk of local-scale extinction is really high in all the Mediterranean populations.
The floristic composition of the Sicilian Thelypteris palustris plant community shows a clear affinity with the common sedge communities of the northern Europe. Perhaps, this plant community is a relict vegetation type of the last glacial stage, which currently is localized exclusively in the humid stands of the Nebrodi Mounts. These microrefuges were originated by peculiar geological characteristics of the territory, with a humid supramediterranean bioclimate that facilitate the growth of these hygrophilous species [43].
In conclusion, our study has made possible to highlight the unexpected occurrence in Sicily of the marsh fern Thelypteris palustris, growing together with some floristic elements of the highest nature value, such as Equisetum palustre, Epipactis palustris, Utricularia australis R. Br., Rhynchocorys elephas, Juncus conglomeratus, J. subnodulosus, Carex paniculata, C. flacca subsp. flacca, C. pallescens, and Hypericum tetrapterum. These vascular species, linked to wetlands, show in Sicily a narrow distribution range due to a strong reduction of their habitat in recent decades. Although they are included in the "A" zone of the Nebrodi Park ( Figure 7) and within the Natura2000 site SAC ITA030014, targeted conservation and monitoring actions would be desirable, aimed at the long-term conservation of the floristic component and especially their humid habitats.