Wet Meadow Plant Communities of the Alliance Trifolion pallidi on the Southeastern Margin of the Pannonian Plain

: The article deals with wet meadow plant communities of the alliance Trifolion pallidi that appear on the periodically inundated or waterlogged sites on the riverside terraces or gentle slopes along watercourses. These plant communities are often endangered by inappropriate hydrological interventions or management practices. All available vegetation plots representing this vegetation type were collected, organized in a database, and numerically elaborated. This vegetation type appears in the southeastern part of the Pannonian Plain, which is still under the inﬂuence of the Mediterranean climate; its southern border is formed by southern outcrops of the Pannonian Plain and its northern border coincides with the inﬂuence of the Mediterranean climate (line Slavonsko Gorje-Fruška Gora-Vršaˇcke Planine). Numerical analysis established four plant associations— Trifolio pallidi–Alopecuretum pratensis , Ventenato dubii–Trifolietum pallidi , Ranunculo strigulosi–Alopecuretum pratensis , and Ornithogalo pyramidale–Trifolietum pallidi . Each association was elaborated in detail: diagnostic plant species, nomenclature, geographical distribution, climatic and ecological conditions, and possible division into subassociations. Results are presented in a distribution map, ﬁgures resulting from numerical analysis, and a synoptic table. The hydrological gradient was found as the most important factor shaping the studied plant communities. The article also brings new ﬁeld data on this vegetation type, which has not been sampled for decades and is in process of evaluation to be included as a special habitat type in the Habitat Directive.


Introduction
Wet meadows of Trifolion pallidi appear on clayey, mesotrophic to eutrophic soils on riverside terraces and gentle slopes along the rivers in the southeastern margin of the Pannonian Plain. Inundations of the sites during winter and spring are common, and the humidity of sites is often influenced by the high level of groundwater, which is frequently close to the surface. During the summer, the ground may dry up [1].
Hydrological factors such as seasonal floods and high water table in the soil significantly define the composition of plant communities exposed to such influences. The hydrological gradient has been generally accepted as the primary factor that determines the growth of different species and the structure of herbaceous wetland plant communities [2][3][4][5]. Water level fluctuation that is also characteristic of studied sites shapes the European Habitat Directive [26] but has been proposed for consideration in the accession process of Serbia [23].
The aim of the paper is to collect all available data about Trifolion pallidi communities and present the appearance of wet meadows of the alliance Trifolion pallidi on the southeastern margin of the Pannonian Plain. We tried to reveal the most important factors that enable the diversity of those meadows. We prepared an insight into its classification up to subassociation level taking into consideration also results of new synthetic works that have appeared recently. At the same time, we provided new field data on this habitat, which is under consideration within the European Habitat Directive [26].

Study Area
The study area extends along the southeastern margin of the Pannonian Basin, encompassing eastern parts of Croatia and the northern part of Serbia ( Figure 1). This area is under the influence of a moderate continental climate: a moderately warm and humid climate with warm summers [28,29]. The climatic data for three meteorological stations in the region are-Osijek mean annual temperature 11.0 • C and annual precipitation 655 mm; Novi Sad 10.9 • C and 647 mm and Valjevo 11.4 • C and 787 mm [28] (http://www.hidmet.gov.rs, accessed 31 October 2020). process of Serbia [23].
The aim of the paper is to collect all available data about Trifolion pallidi communities and present the appearance of wet meadows of the alliance Trifolion pallidi on the southeastern margin of the Pannonian Plain. We tried to reveal the most important factors that enable the diversity of those meadows. We prepared an insight into its classification up to subassociation level taking into consideration also results of new synthetic works that have appeared recently. At the same time, we provided new field data on this habitat, which is under consideration within the European Habitat Directive [26].

Study Area
The study area extends along the southeastern margin of the Pannonian Basin, encompassing eastern parts of Croatia and the northern part of Serbia ( Figure 1). This area is under the influence of a moderate continental climate: a moderately warm and humid climate with warm summers [28,29]. The climatic data for three meteorological stations in the region are-Osijek mean annual temperature 11.0 °C and annual precipitation 655 mm; Novi Sad 10.9 °C and 647 mm and Valjevo 11.4 °C and 787 mm [28] (http://www.hidmet.gov.rs, accessed 31 October 2020) Deciduous forests dominated by Hungarian oak (Quercus frainetto) appear in the area [30]. In the west, the area of research corresponds to the western localities of Hungarian oak in Slavonia (Kutjevo) [31]. In the north, the area includes a major part of Vojvodina. This area is still influenced by the Mediterranean climate, which arrives from the south ( Figure 1) along rivers (e.g., Morava) [32]. The southern boundary corresponds to the geomorphological border of the Pannonian Plain [33], which corresponds to the phytogeographic border of the Pannonian Plain toward the Illyrian region proposed [34] and the border of the central European region toward the Mediterranean region [35]. Deciduous forests dominated by Hungarian oak (Quercus frainetto) appear in the area [30]. In the west, the area of research corresponds to the western localities of Hungarian oak in Slavonia (Kutjevo) [31]. In the north, the area includes a major part of Vojvodina. This area is still influenced by the Mediterranean climate, which arrives from the south ( Figure 1) along rivers (e.g., Morava) [32]. The southern boundary corresponds to the geomorphological border of the Pannonian Plain [33], which corresponds to the phytogeographic border of the Pannonian Plain toward the Illyrian region proposed [34] and the border of the central European region toward the Mediterranean region [35].

Data
We collected all available relevés elaborated according to the standard central European method [36,37] dealing with wet meadows of Trifolion pallidi from the region under consideration. This study is based on a data set consisting of 181 relevés-146 relevés were used from the available literature sources [11,12,[38][39][40][41][42][43][44], and 35 were newly made in the field ( Figure A2). The relevés were stored in Turboveg [45] and elaborated in the Juice program [46].
Unweighted Ellenberg indicator values (EIV) [48] for nutrients and moisture were calculated for each relevé to facilitate ecological interpretation of clusters. Climatic conditions were estimated by climatic data extracted from the Worldclim database [49]. These variables and the number of species in relevés are presented as box-whisker diagrams. We also extracted longitude, latitude, and altitude from relevé material. We correlated all these variables with the floristic gradient presented by the first two axes of detrended correspondence analysis (DCA).

Numerical Analysis
To reduce noise in the data, outlier analysis was conducted using PC-ORD 5.0 (MjM Software Design, Gleneden Beach, OR, USA), and four relevés whose species composition deviated more than ±2 SD from the mean calculated Euclidean distance of all plots were excluded. Finally, 175 relevés were included in the analysis, which contained a total of 321 taxa of vascular plants.
Classification of relevés was performed using cluster analysis in PC-ORD 5.0. The OptimClass method [52] for identifying the optimal partition suggested square root transformation of cover values of species, Beta flexible (β = −0.25) for group linkage with the relative Sørensen index as the distance measure.
Diagnostic species were determined by calculating fidelity using the phi (Φ) coefficient. Only species with Φ > 0.4 and a probability under the random expectation of the observed pattern of species occurrence lower than 0.001 (Fisher's exact test) were considered diagnostic for associations and subassociations [53]. Diagnostic species of subassociations were calculated only within relevés classified to the relevant association. To calculate fidelity, the number of relevés for each (sub) association was virtually standardized to equal size [54].
Detrended correspondence analysis (DCA) was performed by the Vegan program [57] in the R program environment (https://cran.wu.ac.at/). The first two axes are presented on the diagram with passively projected climatic and ecological variables that show the highest correlation with the axes calculated by Spearman correlations. We prepared a synoptic table ( Figure A3) and a table of new unpublished relevés used in the analysis ( Figure A2); we also showed the distribution of individual syntaxa on the map and a map presenting literature and own field data ( Figure A1). Figure 2 showed that relevés can be divided into six clusters (groups). They are consistent with the existing classification scheme, with the exception of Ventenato-Trifolietum, which has not appeared in previous analyses. The diagram shows four associations, two of which are subdivided into subassociations ( Figure 2). These groups were the subject of further analysis.  Figure A3) as diagnostic species of associations and excluded some widely distributed species, such as Poa pratensis and Poa trivialis (in which the subspecies sylvicola is often not distinguished). Detrended correspondence analysis (DCA) was performed by the Vegan program [57] in the R program environment (https://cran.wu.ac.at/). The first two axes are presented on the diagram with passively projected climatic and ecological variables that show the highest correlation with the axes calculated by Spearman correlations. We prepared a synoptic table ( Figure A3) and a table of new unpublished relevés used in the analysis ( Figure A2); we also showed the distribution of individual syntaxa on the map and a map presenting literature and own field data ( Figure A1). Figure 2 showed that relevés can be divided into six clusters (groups). They are consistent with the existing classification scheme, with the exception of Ventenato-Trifolietum, which has not appeared in previous analyses. The diagram shows four associations, two of which are subdivided into subassociations ( Figure 2). These groups were the subject of further analysis. EIV values for moisture Figure 3 show that the wettest sites are those of Ranunulo-Alopecuretum typicum, Ventenato-Trifolietum, and Trifolio-Alopecuretum typicum. At the same time, it was found that the nutrient status is highest within Ranunuculo-Alopecuretum typicum and Trifolio-Alopecuretum typicum, while there are fewer nutrients within Ventenato-Trifolietum. It must be taken into account that the moisture of sites is a result of inundations and higher groundwater table in combination with soil characteristics (clay). Inundations also bring nutrients to the sites, but this is not always the case with high EIV values for moisture Figure 3 show that the wettest sites are those of Ranunulo-Alopecuretum typicum, Ventenato-Trifolietum, and Trifolio-Alopecuretum typicum. At the same time, it was found that the nutrient status is highest within Ranunuculo-Alopecuretum typicum and Trifolio-Alopecuretum typicum, while there are fewer nutrients within Ventenato-Trifolietum. It must be taken into account that the moisture of sites is a result of inundations and higher groundwater table in combination with soil characteristics (clay). Inundations also bring nutrients to the sites, but this is not always the case with high groundwater, so the two properties are not necessarily parallel. It can be seen that Ornithogalo-Trifolietum appears on the driest and nutrient poorest stands, while the remaining groups, such as Ranunuculo-Alopecuretum filipenduletosum and Trifolio-Alopecuretum rhinanthetosum, possess an intermediate position in respect to these ecological factors. It can be seen that altitude has a very narrow range of 100 m and does not play an important role in the differentiation of groups. This division is also shown by the ecological division of groups reflected on the first axis of DCA analysis (Table 1 and Figure 4).  . Diagram of Detrended Correspondence Analysis (DCA) of relevés with the centroid of groups and spider plots with passively projected EIV nutrient and EIV moisture and climatic variables-precipitation seasonality, mean temperature of the coldest quarter, mean annual temperature, precipitation in the driest quarter. Eigenvalues for the first two axes are 0.493 and 0.295, respectively. The legend is the same as in Figure 3.   The legend is the same as in Figure 3. Climatic conditions reflect the geographic position of the studied plant communities ( Figures 5 and 6). There is more precipitation (mean annual, of the driest quarters) in Trifolio-Alopecuretum (both subassociations) appearing in the northern part of central Serbia. At the same time, the temperature regime is more propitious in this region (annual mean temperature, mean temperature of the coldest quarter). The climatic conditions become more severe (precipitation seasonality) in the regions toward the central Pannonian Plain, where Ranunuculo-Alopecuretum (both subassociations), Ornithogalo-Trifolietum, and Ventenato-Trifolietum can be found. Macroclimatic conditions correspond to the second DCA axis (Table 1 and Figure 4) and separate communities according to their geographical position ( Figure 6). The number of plant species varies between the studied communities and reaches higher values in the subassociations ( Figure 5), which occur on slightly drier sites than typical forms ( Figure 3).

Classification analysis
The DCA diagram shows the separation of groups along two gradients-axis 1 represents the ecological gradient, and axis 2 represents the macroclimatic (in our case also geographic) gradient ( Figure 4 and Table 1).
Wet meadows of Trifolion pallidi appear along the southeastern margin of the Pannonian Plain because there is a certain influence of the Mediterranean climate from the south (e.g., along river Morava), which gradually diminishes toward the north (Table 1, Figure 6). Along this gradient, we can distinguish two associations of wet meadows that are widely distributed in the area, namely, Trifolio-Alopecuretum thriving in areas with a milder climate in the northern part of central Serbia and Ranunculo-Alopecuretum appearing northwards in the area in which the climate becomes harsher, i.e., in the area approximately till the Climatic conditions reflect the geographic position of the studied plant communities (Figures 5 and 6). There is more precipitation (mean annual, of the driest quarters) in Trifolio-Alopecuretum (both subassociations) appearing in the northern part of central Serbia. At the same time, the temperature regime is more propitious in this region (annual mean temperature, mean temperature of the coldest quarter). The climatic conditions become more severe (precipitation seasonality) in the regions toward the central Pannonian Plain, where Ranunuculo-Alopecuretum (both subassociations), Ornithogalo-Trifolietum, and Ventenato-Trifolietum can be found. Macroclimatic conditions correspond to the second DCA axis (Table 1 and Figure 4) and separate communities according to their geographical position ( Figure 6). The number of plant species varies between the studied communities and reaches higher values in the subassociations ( Figure 5), which occur on slightly drier sites than typical forms (Figure 3). The DCA diagram shows the separation of groups along two gradients-axis 1 represents the ecological gradient, and axis 2 represents the macroclimatic (in our case also geographic) gradient ( Figure 4 and Table 1).
Axis 1 shows the separation along EIV moisture and nutrient. Ventenato-Trifolietum, Trifolio-Alopecuretum typicum and Ranunculo-Alopecuretum typicum can be found in the milder climate in the northern part of central Serbia and Ranunculo-Alopecuretum appear-ing northwards in the area in which the climate becomes harsher, i.e., in the area approximately till the line from Slavonsko Gorje over Fruška Gora to Vršačke Planine. Two associations have been described on small areas and represent local site conditions-Ventenato-Trifolietum in extremely wet conditions along the Danube and Drava River and Ornithogalo-Trifolietum in the driest conditions on Vršačke Planine. This distinction between the associations is predominately on a (macro)climatic scale.

Discussion
We confirmed the findings of Ilijanić [10,13], who reported that wet meadows of Trifolion pallidi appear in the eastern part of Croatia and northern Serbia. Ilijanić [13] also anticipated that such vegetation could also be found in southern Romania and northern Bulgaria, but it has not yet been reported from those regions [58,59]. However, some plant communities of a transitional character have been found there (e.g., [60]).
The present analysis is based on 20% of new field data. The new data did not produce their own clusters but were merged among existing syntaxa ( Figures A1 and A2). This indicates that no major change has appeared in species composition during different pe-

Discussion
We confirmed the findings of Ilijanić [10,13], who reported that wet meadows of Trifolion pallidi appear in the eastern part of Croatia and northern Serbia. Ilijanić [13] also anticipated that such vegetation could also be found in southern Romania and northern Bulgaria, but it has not yet been reported from those regions [58,59]. However, some plant communities of a transitional character have been found there (e.g., [60]).
The present analysis is based on 20% of new field data. The new data did not produce their own clusters but were merged among existing syntaxa ( Figures A1 and A2). This indicates that no major change has appeared in species composition during different periods of sampling. It supports the estimation of [1], who found out that only a small percentage of the area of these communities is degraded (up to 20%), whereas the degree of degradation is slight (to moderate). We cannot judge about the extension of these communities from our data.
The Trifolion pallidi communities are distributed in the southeastern margin of the Pannonian Plain. The northern border coincides with the influence of the Mediterranean climate that comes from the south [32] and it extends from Slavonsko Gorje over Fruška Gora to Vršačke Planine. The transitional character of Ventenato-Trifolietum will be discussed below. The southern border coincides with the border of the Pannonian Plain [33].
Thermophilous deciduous oak forests of the alliances Quercion frainetto and Quercion pubescenti-petraeae and mesic oak-hornbeam forests of Erythronio-Carpinion appear in the area of distribution of Trifolion pallidi, but steppe woodlands of Aceri-Quercion, which appear on loess plateaus toward the north, cannot be found [30,61,62].
The hydrological gradient obtained on the base of EIV moisture values significantly correlated with axis 1 of DCA (Table 1), so we can confirm that the moisture gradient turned out as the most important factor in shaping the composition of the studied plant communities. Soil moisture and nutrient content have been shown to be the most important factors that determine the species composition of wet grasslands [63].
Any measures that would significantly change the extent or frequency of inundations of these sites or the level of groundwater would negatively influence the studied plant communities [64] and could eventually lead to changes of floristic composition and to substantial loss of regional biodiversity. The changes in hydrology would in the worst case cause the vanishing of this habitat type.
A distinction within associations on a local scale also exists, reflecting ecological site conditions. Two subassociations can be distinguished within each of the two broadly distributed associations; each reflects wetter and more nutrient-rich site conditions, on the one hand, and less wet and nutrient poorer conditions on the other. Ranunuculo-Alopecuretum typicum and Trifolio-Alopecuretum typicum can thus be found on wetter and more nutrientrich sites and Ranunuculo-Alopecuretum filipenduletosum and Trifolio-Alopecuretum rhinanthetosum on less wet and nutrient poorer sites. The other two associations appear on a limited area and in fairly unique ecological site conditions, so they were not subdivided into subassociations. This distinction on a subassociation level is recognized on a local scale.  [12], this relevé belongs to the subassociation rhinanthetosum rumelici. Article 19a [51] stated that a lectotype must be chosen from relevés assigned to the subassociation typicum, if such a subassociation exists. In Cincović [12], such a subassociation does exist and this makes the lectotypification invalid. We select here as lectotype of the association Trifolio pallidi-Alopecuretum pratensis: Cincović 1959 [12]: Table 3, relevé 14, lectotypus hoc loco. This relevé is also the autonym of the subassociation typicum (Art. 13b). At the same time, we typify the subassociation Trifolio pallidi-Alopecuretum pratensis rhinanthetosum rumelici Cincović 1959, as lectotype: Cincović 1959 [12]: Table 3, rel. 23, lectotypus hoc loco. This association appears in the northern part of central Serbia. It covers about half of the grassland area in the region and forms a large part of wet meadows along rivers in northwestern Serbia. These plant communities appear over fairly broad ecological conditions, from regularly inundated riverbanks to extreme sites that are barely reached by inundation water. The level of groundwater also varies significantly in these sites [12].

Description of Individual Plant Communities
These meadows can easily be recognized in the landscape at the end of spring by the grey-green color of flowering Alopecurus pratensis. Communities are two-layered. The upper layer is formed by Alopecurus pratensis, Festuca pratensis, Rumex crispus, Cichorium intybus, and Thalictrum flavum, while in the lower layer, Trifolium pallidum, Trifolium patens, and Poa pratensis appear. The lower layer is denser than the upper one and forms a close canopy, so other plant species have difficulty appearing there [11].
The subassociation rhinanthetosum rumelici is differentiated by Anthoxanthum odoratum, Carex spicata, Cynosurus cristatus, Danthonia alpina, Rumex acetosa, and Trifolium pratense. It appears at the limit of the inundation area on alluvial terraces and is rarely inundated. In addition, the level of groundwater is deep, between 3 m and 5 m below the surface during the vegetation season. This subassociation encompasses drier forms of communities [12].
The subassociation typicum is differentiated by Lysimachia nummularia, Mentha pulegium, Trifolium patens, and Verbena officinalis. It includes the majority of the other three subassociations described by Cincović [11,12] and Škvorc et al. [23], as caricetosum Cincović 1956 nom. inval. (Art. 4), trifolietosum hybridi Cincović 1959 and lysimachietosum nummulariaeĆuk in Škvorc et al. 2020. It appears in the inundation area along rivers and is periodically under water every year at the end of spring and beginning of summer although the duration depends on the amount of water in the particular year. The groundwater table is high-from only 10 cm to 180 cm below the surface [12].
Nomenclatural remark. We selected the lectotype of the association Ventenato dubii-Trifolietum pallidi as Ilijanić 1968 [38]: Table 3, rel. 15, lectotypus hoc loco. The association has been described from the area along the Drava River north of Osijek. These plant communities appear on the riverbanks of the small Krašica River, on the boundary of the inundation area, but the soil is heavy and clayey and well maintains humidity. It mediates among Molinion, Deschampsion (syn. Cnidion), and Trifolion pallidi [23,38,40]. The floristic composition is a mixture of the diagnostic species of all three alliances: Molinion (Iris sibirica, Gentiana pneumonanthe), Deschampsion (Serratula tinctoria, Gratiola officinalis), and Trifolion pallidi (Trifolium pallidum, Oenanthe silaifolia). Three therophytic species (Trifolium pallidum, Ventenata dubia, and Trifolium striatum) can be observed, which were considered to be characteristic species of this association [38]. Communities are (co)dominated by Alopecurus pratensis. This association has been divided into two subassociations-poetosum palustris and lotetosum corniculati Ilijanić [38].
In a broader analysis [23], the relevés of this association were partly classified among outliers and partly classified within Trifolio-Alopecuretum. The results of this large-scale analysis and the appearance of many Trifolion pallidi species persuaded us to include these transitional relevés in the analysis, where this association occurred. Additional analysis should be done in the future to clarify its synsystematic position. The present analysis does not support its further division into subassociations. Nomenclatural remark. This association was invalidly described by Vučković [43] as Ranunculus stevenii-Alopecuretum but was later validated by Aćić et al. [65] as Ranunculo acris-Alopecuretum pratensis with Ranunculus acris L. 1753 as the name-giving taxon. We think that the species Ranunculus strigulosus (formerly misinterpreted as Ranunculus steveni Andrz. 1814) from the Ranunculus acris complex better describes the association because Ranunculus strigulosus Schur 1866 is a typical species of wet meadows in southeastern Europe. This concept is accepted in various regional floras [66] [51].
The association appears over a large area between Slavonsko Gorje and Vršačke Planine. It appears on deep soils that are wet, fertilized, and periodically inundated. According to an analysis made by Vučković [43], a comparison of this association with the Trifolio-Alopecuretum, thriving in the northern part of central Serbia, shows more hemicryptophyes and fewer therophytes, as well as more Eurasian and fewer sub-Mediterranean species; the differences are still more pronounced in comparison with associations appearing further toward the south (classified within Trifolion resupinati).
The subassociation filipenduletosum vulgaris is differentiated by Briza media, Filipendula vulgaris, Fragaria viridis, Galium verum, Leontodon hispidus, Rhinanthus rumelicus, and Tragopogon pratensis. It appears on drier sites that are nutrient poorer. Within this subassociation are also classified Ranunculo-Alopecuretum rumicetosum acetosae Vučković 1991 nom. inval. The association possesses a very limited distribution area, being found only on the southern slopes of Vršačke Planine. It is named after two sub-Mediterranean plant species, Trifolium pallidum and Ornithogalum pyramidale. The sites are drier and nutrient poorer than those of the previous associations and it is transitional to dry grasslands of Festuco-Brometea [43]. Two subassociations have been described within this association-Ornithogalo-Trifolietum trifolietosum pallidi Vučković 1991 nom. inval. (Art. 5) and Ornithogalo-Trifolietum rhinanthetosum rumelici Vučković 1991 nom. inval. (Art. 5), but our analysis did not confirm the division into lower syntaxa.

Conclusions
The article presents species-rich plant communities of the wet meadows that harbor a major part of regional biodiversity. The continuation of mowing and safeguarding against inappropriate hydrological interventions are key factors for maintaining these communities [71,72]. The main threats to these habitats are changes of traditional management practice, which could be abandonment leading to reforestation, or intensification of agriculture through fertilization and drainage [1]. Changes in hydrological conditions such as changes in the extent or frequency of inundations or changes in the level of groundwater on these sites would negatively influence these plant communities. The changes in hydrology would in the worst-case cause the vanishing of this habitat type [64].
We have provided new field data about this habitat type that has not been sampled for decades. They prove that it is still of good quality, worth protecting, and it deserves integration in the Habitat Directive. Data Availability Statement: Study is based on published material [11,12,[38][39][40][41][42][43][44] and new field data Figure A2.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. Data Availability Statement: Study is based on published material [11,12,[38][39][40][41][42][43][44] and new field data Figure A2.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.