Forest Vegetation of Hardwood Tree Species along the Mirna River in Istria (Croatia)

background and purpose: The paper presents the forest vegetation of periodically flooded and wet forests of hardwood tree species along the Mirna River (Istria, Croatia). The main objective was to study the older and less influential stands, and to compare them among themselves and with related syntaxa of the Mediterranean and continental area. Materials and Methods: The research was conducted on the basis of 33 new phytocoenological relevés and 12 from previous studies, according to the principles of the standard Central European Phytocoenological School. Results and Conclusions: Based on 45 phytocoenological relevés two main vegetation types were found. In the lowest and periodically flooded habitats grow forests dominated by Fraxinus angustifolia and numerous hygrophilous species. In somewhat higher and drier localities, but with a high level of ground waters, grow mixed forests of Quercus robur, Fraxinus angustifolia, Ulmus minor and Carpinus betulus, with a greater presence of mesophilous species. The paper analyzes their mutual relationship, phytocoenological affiliation, as well as their position with regard to the related syntaxa of the Mediterranean and continental area. The results suggest isolation and a transitional character of the studied forests, which is a consequence of the biogeographical position in the north Mediterranean, of the ecological conditions, and to a lesser extent of anthropogenic influence.


InTRODUCTIOn
Mediterranean forests of hardwood trees are very rare nowadays, and one of the best-preserved ones is located on the Istrian Peninsula (Croatia) in the lower course of the Mirna River (better known as the Motovun Forest). The forests along the course of the Mirna River stretch over approximately 1100 ha, and the main tree species are Quercus robur, Fraxinus angustifolia and Ulmus minor, whereas the drier part also holds Acer campestre and Carpinus betulus. The basic ecological factor for the composition and growth of these forests is the periodically flooding and high groundwater, which reflects on the floristic composition and distribution of syntaxa. In addition, the biogeographical position is specific as it is in the north of the Mediterranean and close to the Dinaric massif, with a strong continental influence.
The first phytocoenological studies [1,2] presented the entire complex of the Motovun Forest as an autonomous association Querco robori-Carpinetum betuli "submediterraneum" within the southeast European alliance of oak-hornbeam forests Erythronio-Carpinion betuli. This was subsequently, following corrections to the name of the association, also accepted by other phytocoenologists [3,4]. Brullo and Spampinato [5] classify them in the association Querco roboris-Carpinetum betuli within the alliance Alno-Quercion roboris. Finally, Trinajstić [6] defined them descriptively as a new association Carici pendulae-Ouercetum roboris, also within the alliance Alno-Quercion roboris. Vukelić [7] advocates for more intensive pyhtocoenological research because the 2009 forest inventory suggests differences in the composition and structure of stands. According to Vukelić [7], it is not possible to cover the entire forest complex with a single syntaxa.
These different views on nomenclature and syntaxonomy have impelled us to conduct phytocoenological studies of these forest stands in 2016 and 2017. The objective was to survey older and less influential stands, and to compare them among themselves and with related syntaxa of the Mediterranean and continental area. The results of the research should help to define the forest vegetation of this important forest locality, but also of the northern part of the Mediterranean region. When it comes to these issues, the opinions of phytocoenologists are quite varied, which is also evidenced by the overviews of the forest vegetation of Europe or its specific regions [5,[8][9][10][11][12].

Research area
The Motovun Forest is situated in the western part of the Istrian Peninsula, in the valley of the Mirna River (43 km long) and its Butoniga tributary ( Figure 1). It is some fifteen kilometers away from the sea coast, and only a few more kilometers away from the Dinaric massif Ćićarija (peak Orljak, 1106 m). The terrain altitude ranges between 7 and 17 m, and the forest complexes stretch along 15 km, with the average width of approximately 500 m. Nowadays, the Motovun Forest is known Europe-wide as the habitat of the white and black truffle (Tuber magnatum and Tuber melanosporum), and the habitat of the Italian agile frog (Rana latastei). It is a part of the Natura 2000 ecological network, and 300 ha of the old forest were protected in 1963 as a "special reserve of forest vegetation". This area is characterized by a moderately warm and humid climate with hot summers. According to the data from the Botoniga weather station (29 m MASL, period 1986(29 m MASL, period -2015 the mean annual temperature is 13.0˚C, and the mean annual precipitation is 1004 mm. For comparison, the mean annual temperature in the area of Abruzzo and Molise in the central part of the Adriatic coast in Italy is higher by up to 4˚C, and precipitation is lower by up to 400 mm [13]

Data Sampling
Studies of the forest vegetation were conducted following the principles of the standard phytocoenological school [14]. The field phytocoenological survey was conducted on 28 plots in the period from April to mid-July 2016, whereas five unpublished relevés of Fraxinus angustifolia stands originate from the ecological vegetation study of hydrological issues of the Motovun Forest [15]. In addition to these relevés, the statistical analysis also includes 12 relevés from previous studies [2]. Positions of new relevés in the WGS84 Coordinate Reference System are listed in the Appendix 1. The surface of the plots was 400 m², inclination 0˚, and the terrain altitude ranged between 10 and 18 m. The plant nomenclature is aligned with the Flora Croatica Database [16], and mosses with Atherton et al. [17]. Syntaxonomic nomenclature of higher units mostly follows the overviews by Biondi et al. [18] for Fraxinus angustifolia stands, and Košir et al. [19] for mixed hardwood stands. The local syntaxa from previous studies are presented in their original form, whereby a part of the syntaxa is described according to ICPN [20], and a part of them followed the multidimensional distribution of vegetation units [21]. The original names of the syntaxa with their authors and year of publication are listed in the supplementary materials (Appendix 2).

Data analysis
Vegetation relevés were entered into the TURBOVEG database [22]. Cover-abundance values of the species appearing in several layers were combined in the TURBOVEG program [22], with every plant species being considered with total cover-abundance, regardless of the number of structural layers of the individual relevé in which it appears. Hierarchical clustering was performed in the R package (www.r-project.org) [23]. Bray-Curtis dissimilarity index [24], and square-root transformation of species percentage coverabundance values were used. In the phytocoenological analysis, we separated our 33 analytical relevés into two types and compared them with the presence class of related phytocoenoses of the Mediterranean and continental part of the southwestern rim of the Pannonian Plain. The diagnostic species were determined using the JUICE 7.0 program [25], based on the analysis of fidelity measure.
The mosses were not recorded in the majority of studies, hence they were not taken into consideration in statistical analyses. Individual species and subspecies were consolidated under a species aggregate.

ReSUlTS anD DISCUSSIOn
The statistical analysis clearly classifies the 33 relevés of the Motovun Forest into two clusters or vegetation types. The first type with 23 phytocoenological relevés represents mixed stands of Quercus robur, Fraxinus angustifolia, Ulmus minor and Carpinus betulus, whereas the second type with 10 phytocoenological relevés is dominated by Fraxinus angustifolia. Both types were in further analyses separately compared to the floristically and chorologically related syntaxa of southern Europe and continental forests of the southwestern part of the Pannonian Plain -western part of the Pannonian sector of the Pannonian-Carpathian floristic province sensu [26]. All of the compared syntaxa are listed in Table 1 under their original names, authors, and numbers of relevés.

Forests Dominated by Fraxinus angustifolia
The dendrogram on Figure 2 compares the presence classes of 16 Fraxinus angustifolia syntaxa in 23 columns. Of the syntaxa of the Mediterranean area, the most significant association is Carici remotae-Fraxinetum oxycarpae, while continental forests are represented by the association Leucojo-Fraxinetum angustifoliae. The dendrogram clearly separates Mediterranean forests of narrow-leaved ash from continental forests. In the first cluster which encompasses Mediterranean forests of narrow-leaved ash, two subclusters are clearly singled out. The studied forests along the Mirna River are classified in the sub-cluster 1a representing the association Carici remotae-Fraxinetum oxycarpae. Accordingly, we attached them to this association.
The association Carici remotae-Fraxinetum oxycarpae was established in the territory of Abruzzo in Italy [27,28], and later on also in other parts of the Apennine Peninsula [29][30][31][32][33][34][35] and southern Europe [36,37]. This community occupies lowland localities along river banks or depressions between deposits which are under the influence of the periodically flooding or high groundwater. According to the majority of cited papers, the diagnostically most important species for this association are: Fraxinus angustifolia, Carex remota, Ulmus minor, Rumex sanguineus, Ranunculus lanuginosus, Oenanthe pimpinelloides, Carex pendula and Carex divulsa.
In the studied forests along the Mirna River, the dominant ash stands stretch over several separate localities in the wettest and lowest parts of the studied area. These are shallower depressions with occasional surface waters. The tree layer is characterized by the complete domination of the narrow-leaved ash and co-domination of the European white elm, while the common oak is individually rarely present, and the common hornbeam is completely missing. In addition to the ash, elm and the mentioned diagnostic species, in the composition of the studied forests sociologically important are the species common in flooded and wet forests (such as Carex riparia, Lycopus europaeus, Cardamine pratensis, Leucojum aestivum, Galium palustre, Ranunculus repens, Lysimachia nummularia and others).
In its great area, the association Carici remotae-Fraxinetum oxycarpae demonstrates floristic variability, which was also demonstrated in the stands we studied in Croatia. They hold a number of species that have not been reported or are rare in other regions. These are primarily Crataegus laevigata, Cardamine pratensis, Deschampsia cespitosa, Lysimachia nummularia, Alisma plantago-aquatica and Lycopus europaeus. These species were singled out as differential for the new sub-association crataegetosum laevigatae, the holotype of which is relevé number 4 in Table 2 (according to Weber et al. [20]). Great diagnostic significance is also proper to Carex riparia, although it has already been set as a differential species of the sub-association Carici remotae-Fraxinetum oxycarpae caricetosum cuprinae [37].
Besides the association Carici remotae-Fraxinetum oxycarpae, in southern Europe several similar syntaxa have been described with the domination of Fraxinus angustifolia [5,[38][39][40][41][42][43]. Douda et al. [10] consider the majority of them to be synonyms for the association Lithospermo purpureocaerulei-Ulmetum minoris, which they included into the middle Mediterranean alliance Populion albae. The studied forests in Istria fit only partially into such a broadly understood association and its affiliation to the alliance Populion albae. They lack important diagnostic species for this association and alliance such as Bryonia dioica, Rubus ulmifolius, Iris foetidissima, Asparagus acutifolius, Celtis australis, Rubia peregrina, Smilax aspera, Oenanthe pimpinelloides, Rosa sempervirens, Tamus communis, Populus alba and others.
In the previous studies, the community Carici remotae-Fraxinetum oxycarpae was classified into different alliances: Alno-Ulmion, Alno-Quercion roboris, Alnion incanae, Carici remotae-Fraxinion oxycarpae, Populion albae [5,9,10,18,29,31,37]. Our research shows a high coincidence with the alliance Carici remotae-Fraxinion oxycarpae, as it is defined in the syntaxonomic overview of the vegetation of Italy [9,18]. In the cited papers, the species Fraxinus angustifolia subsp. oxycarpa, Ulmus minor subsp. minor, Ranunculus lanuginosus, Carex remota, Rumex sanguineus, Carex pendula and C. divulsa are listed as diagnostic species for it. Without going into the internal differentiation of the species Fraxinus angustifolia and Ulmus minor, the composition of species corresponds very well to the studied stands along the Mirna River. In the latest review of the vegetation of Europe [12], the Carici remotae-Fraxinion oxycarpae is listed as a synonym for the alliance Lauro nobilis-Fraxinion angustifoliae. However, the issue of syntaxonomy will continue to be the subject of many discussions.

Mixed Forests Dominated by Quercus robur, Fraxinus angustifolia, Ulmus minor and Carpinus betulus
Mixed hardwood forests of the studied area are presented with 23 new relevés in Table 3. They grow in drier terrains which are up to 2 m higher than the previous community. They are flooded only in some areas and very rarely, only in extremely wet years in early spring or late fall. The type of soil is pseudogley on alluvial deposits, and such conditions have also reflected on the floristic composition with fewer hygrophytes and more mesophilous species.
The first phytocoenological relevés of such stands were published by Lausi [44] from northeastern Italy along the Po River, and somewhat later by Bertović [2] from the valley of the Mirna River in the Croatian part of Istria. They described them as autonomous associations under different but invalid names, hence Marinček [45] suggested the name Asparago tenuifolii-Quercetum roboris. The majority of papers classify the studied forests into the alliance Erythronio-Carpinion [1][2][3][4]19]. However, Brullo & Spampinato [5] and Trinajstić [6] classify them into the alliance Alno-Quercion roboris. Due to the different nomenclature and syntaxonomic characterization and their status, we surveyed them phytocoenologically in more detail and in further analyses  Figure 3). The compared syntaxa originate from the north-Adriatic Mediterranean area and southwestern rim of the Pannonian Plain. The statistical analysis demonstrated the separation of the compared syntaxa into two main clusters: the first one represents drier communities with a significant presence of mesophilous species from alliances within the order Fagetalia, and the other one contains communities in periodically flooded and moist habitats with the main subassociation Genisto elatae-Quercetum roboris caricetosum remotae (Table 1 numbers 15 a-b, 16 a-f, and  19) within the alliance Alnion incanae. In the first cluster, one can distinguish between three sub-clusters: the first one represents Carpinetum communities of central and eastern Croatia and the studied area (1a); the second one more humid syntaxa of Slovenia classified in the alliances Alnion incanae and Fraxino angustifoliae-Carpinion betuli (1b); and the third one the association Fraxino pannonicae-Quercetum roboris from northern Italy, which is classified in the alliance Alno-Quercion roboris (1c).
The studied forests in the valley of the Mirna River demonstrate affiliation with the sub-cluster 1a, where the dominant forests are oak-hornbeam forests of drier type of central and eastern Croatia. They grow along the course of the Sava River and are connected to the studied forests in Istria, primarily by the great presence of Carpinus betulus, Acer campestre and other mesophilous species. The floristic and sociological relationships between individual cluster groups were analyzed based on the fidelity of species using the JUICE 7.0 program [25]. We took a total of 216 phytocoenological relevés in the analysis from the main syntaxa representing an individual group (   Pres. deg. Figure 1  24  25  27  28  26  29  32  30  33  31   Altitude in m  14  14  14  14  14  10  14  11  12  12 Releve area (00 m²
On the basis of previous vegetation research and this analysis, we have classified mixed forests of hardwood broadleaved trees along the Mirna River in the association Asparago-tenuifolii-Quercetum roboris. Its area is in the Mediterranean region of the north-western part of Slovenia and Croatia, and in the eastern part of Venezia Giulia. The diagnostic species of the association are Ruscus aculeatus, Lonicera caprifolium, Carex pendula, Primula vulgaris and Arum italicum. Although it is of extrazonal character, the cover of the species of the Illyrian floral geoelement is high. The sub-Mediterranean climate benefits their relatively thermophilic character.
The species Asparagus tenuifolius is absent from our phytocenological relevés, but is abundantly present in the surrounding zonal forests of the downy oak and the European hop-hornbeam. It does not respond well to wet and occasionally flooded habitats.
Future research of forests in the river valleys of the North Adriatic littoral will show the justifiability of classifying the studied forest stands of hardwood broadleaved trees into the association Asparago tenuifoli-Quercetum roboris and the alliance Erythronio-Carpinion betuli. Based on our analyses and recent phytocenological trends, their separation into new syntaxa is for now unfounded. The association Asparago tenuifolii-Quercetum roboris was studied in two larger localities, hence its analysis in the hierarchical phytocoenological system is not yet warranted. The study of the stand along the Mirna River can be divided into two subtypes (Table 3): the first subtype of 12 relevés (subtype Carex remota, relevés 1-12 in Table 3) is distributed on wetter habitat, shallow depressions and very mild slopes, and according to the drier subtype, the prominent differential species are Carex remota, Deschampsia cespitosa, Prunus spinosa, Lysimachia nummularia, Ranunculus lanuginosus. In addition to them, there is also an increased presence of other hygrophilous species, especially Ulmus minor.
Relevés 13-23 in Table 3 represents a drier subtype (subtype Vinca minor) of mixed hardwood stands especially distributed on highest terrains along the Mirna River. The differential species according to the first subtype are Vinca minor, Listera ovata, Symphytum tuberosum, Galeobdolon luteum, Euphorbia amygdaloides, Polygonatum multiflorum and Pulmonaria officinalis. To a lesser extent, other species of less humid habitats also have diagnostic significance, and in relation to the previous subtype there is a higher presence of the common oak and common hornbeam.
It should be emphasized that the studied hardwood forests along the Mirna River represent a permanent vegetation stage conditioned by the hydrological regime, primarily by groundwater. For that reason, the presence of the species Ulmus minor and Fraxinus angustifolia is higher than in other Carpinetum communities of southeast Europe.

COnClUSIOnS
The phytocoenological study of periodically flooded and wet forests along the Mirna River in Istria and their comparisons with related syntaxa resulted in defining two associations. Fraxinus angustifolia forests with numerous hygrophilous species are classified in the association Carici remotae-Fraxinetum oxycarpae with the new sub-association crataegetosum laevigatae. The mixed hardwood forests belong to the association Asparago tenuifolii-Quercetum roboris distributed in the planar zone in broader river lowlands of the central part of the northern Mediterranean area. Both associations in the studied area lack numerous termophilous species from related syntaxa of southern Europe, as well as many hygrophilous species from related continental flooded and wet forests. The reasons primarily lie in the biogeographical position, ecological conditions and anthropogenic influences in the studied area. It is located at the northern most part of the Mediterranean, deeply retracted into the European continent. It is also marked by Mediterranean and continental climate impacts, especially modified by the 15 km distant Dinaric mountains with numerous species of the Illyrian floristic geoelement. These reasons have caused a certain isolation and transitional character of the area, which, in turn, has reflected on the floristic composition of the studied forests.
The study results add to the knowledge of the composition and character of flooded and wet forests in this part of Europe, and will also serve to resolve the issues with their syntaxonomy. In addition, they are important in the preservation of these rare and endangered habitat types.