Assessment of quality and chemical composition of continental halophytic grasslands in south-east Europe

Continental halophytic grasslands are known for performing of range of ecosystem services especially remarkable in the regions where they are much distributed – in arid and semi-arid areas. Continental halophytic grasslands of the Central and South-East Europe are not considered as favourable for arable farming, however, traditional animal husbandry plays a crucial role in maintaining biodiversity and preserving these natural habitats. The particular interest of this study is the assessment of the chemical composition of biomass and the quality of insufficiently studied halophytic grassland communities of the central Balkans. In addition, the differences in pastoral value were monitored along the geographical gradient, i.e., between grasslands situated in the Pannonian plain (grasslands of Alliance Puccinellion limosae) and those distributed on the south Serbia (Alliance Festucion pseudovinae). The study was carried out at 18 representative sites, focusing on the saline grasslands of the two distinct regions – on the north and on the south of Serbia. The obtained results show that the halophytic grasslands have satisfying quality characteristics (average values: dry matter 93.67%, crude protein 8.66%, cellulose 30.36%, crude fat 2.27%) compared with other studied grasslands of saline habitats. There were no significant differences in quality and chemical composition of grasslands of the two regions, despite differences in floristic composition, indicating that salinity is the key determinant for pastoral value of the halophytic vegetation. The certain variations were attributed to the specific floristic composition related to ecological conditions and halophytic community characteristics.


Introduction
Halophytes represent a small group of plants which is widely dispersed within different plant families (Attia-Ismail, 2018) contributing for about 2% of the total terrestrial flora (Ben Hamed and Custodio, 2019). It has been estimated that up to one-third of halophytes, could be used as a forage (Aronson, 1985). Halophytic flora and vegetation are primarily associated to habitats containing concentration of the sodium salts in soil 2 solution higher than 200 mM (Flowers and Colmer, 2008). Such habitats have widespread geographic distribution, covering about 7-10% of the Earth's land surface (Dudal and Purnell, 1986) and are especially present but not limited to arid and semi-arid regions. Halophytes may occur in nature in the form of trees or shrubs, but commonly they are grasses and forbs of many could serve as livestock forage and are a major food sources in animal feeding in case they are dominant flora of a certain region (e.g. Middle East, countries of North Africa, India, parts of Australia, etc.). The Pannonian halophytic grasslands are among the bestpreserved grassland habitats in Europe, traditionally managed by moderate cattle and sheep grazing, as their poor soil quality and fluctuating water balance are unsuitable for intensive agriculture and forestry (Török et al., 2011;Valkó et al., 2014). Several studies also confirm active grazing and other types of use of European continental halophytic grasslands or in Mediterranean basin (Dajic-Stevanovic et al., 2008;Dítětová et al., 2016;Abd El-Hack et al., 2018).
The management, conservation and traditional grazing of Central European alkali grasslands by cattle has been the subject of several studies (Molnár and Borhidi, 2003;Török et al., 2014;Valkó et al., 2014;Tóth et al., 2018). However, there is insufficient information on the chemical composition and quality of inland halophytic vegetation, referring mainly to European region and its particular parts. The available studies on the quality of animal feed mostly targeted the chemical composition of selected plant species which are used as animal feed, such as those of genera Atriplex, Kochia, Juncus, Suaeda, Salsola, etc. In addition, individual halophytic species from coastal saline areas were screened for their nutritional quality (El-Shaer and Attia-Ismail, 2002), as well as some of terrestrial halophytes as a viable supplementary food source (Centofanti and Banuelos, 2019). It is known that species of the genus Atriplex are often used as an alternative animal feed, especially in conditions of deficiency of adder fodders, such as in early spring, during hot and dry summer months and late autumn (Osman et al., 2006). The average value of the several studied halophytes from the arid regions refers to about 10% of total proteins, 21.7% of the crude fiber and 27% for ash (Abd El-Hack et al., 2018). According to Temel et al. (2015), the majority of halophytes are underused due to insufficient knowledge about their chemical composition, as well as their potential nutritional value and yield. Moreover, it is known that salt affected soils are characterized by much lower fertility and other traits needed for production of traditional crops and therefore are mostly used for grazing (Kazemi and Eskandari, 2011). However, it is known that management of salt meadows involves grazing and mowing, where most of halophytic species have a satisfactory fodder value and are the basic resource for feeding domestic animals in drylands (Arzani et al., 2010;Rad et al., 2013;Oktay and Temel 2015).
In Europe, continental salt grasslands have a limited geographical distribution. Continental halophytic grasslands occur in the central Europe (Pannonian Plain) under strong influence of continental climate and in the southeast Europe (Balkan region, mainly southern parts of Serbia, Northern Macedonia and Bulgaria) under the influence of the sub-Mediterranean climate and are restricted to small patches. Besides climatic impacts, there are a number of ecological and geographical factors differentiating Pannonian and central Balkan halophytic grasslands, which primarily reflects on the floristic composition and vegetation types (Lukovic, 2019).
The focus of this study was the vegetation of the class Festuco-Puccinellietea and its two typical salt grassland alliances: Puccinellion limosae-Pannonian hypersaline open grasslands on solonetz and solonchack soils, composed of typical alkali grasses and Festucion pseudovinae-Pontic Pannonian salt-steppe pastures and saline steppic pastures on solonetz soils, composed of short grass communities dominated by short bunch and species tolerant to grazing and trampling (Borhidi et al., 2012;Mucina et al., 2016). These two alliances are common for Pannonian and central Balkan saline grasslands, while the occurrence of communities composed 3 of succulent euhalophytes such as Salicornia eurpaea, Suaeda maritima, Salsola soda and some other highly tolerant species.
The aim of this paper is to present the chemical composition and pastoral value of the most distributed continental saline grasslands along the geographical gradient from the north (the Pannonian Plain) to the south of Central Balkans, and to investigate possible differences in the quality of grasslands of the two main halophytic alliances Puccinellion limosae and Festucion pseudovinae.

Study area
The studied area includes the continental Pannonian and the Central Balkan salt grasslands with focus on the territory of the Republic of Serbia. The vegetation of continental saline habitats is very well developed in the Pannonian Plain (Eliaš et al., 2013;Lukovic, 2019), while towards the south, the occurrence of saline soils and euhalophytic vegetation decreases (Zlatković et al., 2014). The sampling sites include 18 halophytic grasslands (13 on the territory of the Pannonian Plain and 5 in central Balkan) (Figure 1, Table 1). The plant material was collected for the chemical quality analysis. The sites were selected according to: 1. floristic and plant communities' diversity, 2. variety of salt soils (from wet to moderately wet salt marches and dry salt meadows), 3. subtle differences in soil chemical composition and 4. traditional management. Phytocoenological relevés were carried out at these selected sites in 2012, 2013 and 2014 according to the method of Braun-Blanquet (1964). The average size of the plots was 16m 2 . Plant material was collected by cutting the square plots of 1m 2 . The fresh plant material was measured in situ, while the dry biomass was measured after 7 days of drying at room temperature in the dark. The prepared material was grounded and stored in the dark, in well-sealed pots. Voucher specimens of plant species were deposited in the herbarium of the Department of Agrobotany, Faculty of Agriculture, University of Belgrade.
All phytocoenological relevés were stored in a vegetation database (Dajić Stevanović et al., 2012, update 2015 using the TURBOVEG software (Hennekens and Schamineé, 2001). The affiliation of relevés to a particular vegetation type (Table 1,

Chemical analysis
Chemical analysis of the main quality parameters of the plant material was carried out at the Institute for Fodder Plants in Kruševac (Serbia). Dried sampled plant material was analysed using the Weende analytical system: Hygroscopic moisture; Dry matter (DM); The content of raw ash was determined by dry incineration at 550 degrees; The content of crude protein (CP) was determined indirectly by the amount of total nitrogen by multiplying it by the factor 6.25, the Bremner modification (Bremner et al., 1965); crude cellulose (C) content was determined by successive analysis of the sample with a diluted solution of H2SO4 in NaOH 5 (Henneberg and Stohman, 1859); crude fat content (CF) was determined by Soxhlet extraction; BEM-free nitrogen extracts were obtained by subtracting 1000 g dry matter from the sum of: Crude ash, Crude fat, Crude protein and Crude cellulose; Determination of cellulose fractions of ash free neutral detergent fibre (NDF) and acid detergent fibre (ADF) was determined by the method of Van Soest (1984).

Statistical data analysis
Since the sample size was too small to calculate statistical differences between the analysed groups, a nonparametric Mann-Whitney U test was performed. Data were analysed using the SPSS 26.0 software (SPSS, IBM Corp., 2019). Floristic richness (number of species per relevé) calculated using the Shannon-Wiener (H) diversity index in Juice software version 7.1 (Tichý, 2002). For species selection within biological groups, as well as within alliances, coverage coefficient expressed on a scale (r, +, 1-5) was used. Only the species with estimated coverage higher than 1 were considered (Figures 3, 4). The results of comparison between groups were visually displayed using the Population Pyramid histogram and level of similarity between groups calculated using the Jaccard index. The results of chemical analysis of halophytic grassland samples were graphically presented using the ordinal analysis method (DCA) in CANOCO (Braak and Smilauer, 2002).

Phytocoenological and floristic characteristics of the studied areas
The halophytic vegetation of the Central Balkans comprises 35 plant communities within the two classes Therosalicornietea and Festuco-Puccinellietea occurring in the Pannonian Plain and central Balkan (Dajić Stevanović et al., 2016;Lukovic, 2019). These communities develop within different types of saline habitats, from saline salt marshes to wet/moderately wet salines following a moisture gradient. The ecologically important and very vulnerable are the annual succulent communities on extremely salt-affected soils from the class Therosalicornietea of very limited distribution. Of practical importance is the more spread vegetation of salt meadows and meadow-steppes of the class Festuco-Puccinellietea.
Comparing two alliances, Puccinelion limosae had higher average values for Ash (7.18%), CP (8.  20.15 DM = dry matter (in %); CP = crude protein; CF = crude fat; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; Fresh mass (gm -2 ); dry mass (gm -2 ); H=Shannon-Wiener diversity index (%); code (L1-L18) corresponding to sites in table 1; minimum values expressed in yellow; maximum value expressed in turquoise blue Differences in chemical composition along the geographic gradient The relationships between halophytic grasslands and the values of the main nutritional parameters were visualized using the DCA ordination diagram (Figure 3). The graph contains passively projected values of the results obtained in the laboratory for each relevé. The results of the chemical analyses were plotted on the DCA ordination diagram.
DCA diagram shows the distribution of each phytocoenological relevé of halophytic grasslands along the 1 st and 2 nd ordination axes. The relevés distributed along the 1 st axis and its relation with quality parameters could be explained by correlation with Ash, BEM and CP for central Balkan vegetation, while pannonian halophytic grasslands related to C, DM and ADF. The relevés along the 2 nd axis related to quality parameters correlated to CF and NDF.
The differences between the Pannonian halophytic grassland grouped on the left side and the Central Balkan halophytic grasslands on the right side are evident. Additionally, the Pannonian halophytic grassland could be perceived as two separate groups: 1. Vegetation that mainly develop to moderate wet to dry solonchaks of all. Puccinellion limosae and 2. Vegetation of all. Festucion pseudovinae on slightly salinized soils, mostly solonetz which is correlated to ash and cellulose content.
The distinction between the Pannonian and the Central Balkan halophytic grasslands is the result of differential species that are typical for north (n.) and south (s.) regions (e.g., n. Camphorosma annua -s. Camphorosma monspeliaca, n. Festuca pseudovina -s. Festuca valesiaca, etc.). Although statistical tests did not confirm any significant differences, yet subtle differences do exist. The Central Balkan salt grasslands had higher mean values for CP (7.35%), Ash (9.82%), BEM (52.17%), NDF (60.05%), fresh mass (440.2g), while the Pannonian halophytic grasslands had higher mean values for DM (93.7%), C (30.11%), CF (2.3%), ADF (38.2%), dry mass (170.85g). These results refer to species composition and their abundance within the sampling points. The Shannon-Wiener diversity index showed small differences concerning the Pannonian halophytic grasslands (2.20%) and the Central Balkan (2.04%) ones. The main botanical groups (grasses, legumes, other forbs) with selected species based on their abundance in phytocoenological relevés were analysed using Jaccard's similarity index and visually represented using Pyramidal graph to express the differences between the Pannonian and the Central Balkan halophytic grasslands (Figure 4). The results showed that the similarity index is 0.38 for grasses, 0.14 for legumes and 0.15 for other forbs. The absence of some species such as Festuca pseudovina, Agrostis stolonifera, Bromus hordaceus, Lotus tenuifolius, Camphorosma annua, Aster tripolium ssp. pannonicus, Salicornia europaea, Suaeda maritima and etc., in the central Balkan halophytic grasslands is evident, while the Pannonian halophytic grasslands lack in presence of Bromus commutatus, Festuca valesiaca, Trifolium patens, and Camphorosma monspeliaca.

Comparative analysis of the quality of halophytic alliances
Since the sample size was small, a non-parametric Mann-Whitney U test was performed. The purpose of this test was to determine if there were statistically significant differences in chemical composition between the Puccinelion limosae and Festucion pseudovinae alliances. The results of the test showed that there were no statistically significant differences in the observed parameters between the compared alliances (p < 0.05). However, there were subtle differences in species composition that lead to differences in quality within the studied alliances. The results of the comparative analysis of the main functional groups (grasses, legumes and other forbs) within the Puccinellion limosae and Festucion pseudovinae alliances showed a similarity index of 0.62 for grasses, 0.43 for legumes and 0.36 for other forbs. The floristic differences were plotted in a Pyramidal graph ( Figure 5).

Discussion
Analysis of the chemical composition of halophytic grasslands compared to quality standards for conventional grasses, legumes and other forbs, show that a mixture of halophytes has an intermediate to low quality ranging from 3 to 5 according to the proposed scale (Marsalis et al., 2009). The results also show that there are no statistically significant differences between the studied halophytic grasslands in terms of geographical distribution from the Pannonian Plain to the Central Balkans, and between the Puccinellion limosae and Festucion pseudovinae alliances. The minor differences in the chemical composition of halophytic grasslands are the result of floristic diversity and small variations in ecological factors that condition plant communities' patterns.

Quality of halophytic grasslands
Nutritional value: research shows that the quality of halophytic vegetation can vary due to several factors -the chemical composition of the plants themselves, the tissue morphology or their genetic structure; however, the maturity stage of the plant is also very important (Mountousis et al., 2008;Asaadi and Dadkhah 2010;Atasoglu et al., 2010). In addition, external factors are very important, especially climate (rainfall, wind, sunlight, temperature) and soil composition, which can greatly affect the amount of protein and cellulose in plants (Nasrullah et al., 2003). The chemical composition of any animal feed (including halophytes) is the first indicator of its nutritional value. The nutritional value is determined by the concentration of these nutrients (Lalman, 2017). The main quality indicators are the total amount of protein, fat and cellulose. Our results show that there is no statistically significant difference in the chemical composition of the quality parameters among the studied sites, but there are subtle variations in chemical composition caused by species composition in conjunction with ecological conditions (soil properties, moisture content and climate). Other studies reported that there are differences in nutrient levels depending precisely on the maturity stage of the particular studied plant (Ahmadi et al., 2013). Accordingly, this study was conducted at the same vegetation stage and the results obtained are not dependent on the maturity stage but rather on other external factors. Moreover, the crude protein content of legumes is higher than that of grasses (Minson, 1990), while the crude protein content of forbs is intermediate, between the values of legumes and grasses (Cook, 1972;Krysl et al., 1984;Meyer and Brown, 1985;Ruyle, 1993).
The crude proteins: The crude protein content varied from 4.42% to 12.23%, which is comparable with the previous results of other studies on halophytic flora e.g., Tawfik et al., 2015;Attia-Ismail, 2018, etc.).
The biomass samples from the locality L8-Trešnjevac, which had the highest value in crude protein content, consist of species that had very high values in protein content in individual analyses in other regions of the world: Atriplex nummularia was appointed by 13.68% of the total protein content (Tawfik et al., 2015) to 20.69% in Atriplex repanda (Attia-Ismail, 2015). Atriplex species have good protein content  and sites with dominance of this genus could be suitable for grazing. Regarding the species Bothrichloa ischaemum the protein content ranged between 14% in the leaves of this plant to 10.8% in the stems, while the species Bothrichloa bladhii has a crude protein value of 14.77% in the vegetative stage, which decreased with the age of the plant to 8.31% (Villanueva-Avalos, 2008). Species of the genus Lotus, which also confine to this community, have very high values of crude protein -14.86. The species Artemisia monosperm was characterized by 12.98% of the crude proteins (Tawfik et al., 2015), whereas Camphorosma monspeliaca varied in crude protein content from 5.39% in the flowering phase to 11.43% in the early vegetative phase (Ahmadi et al., 2013). The values of crude proteins in the genus Scorzonera varied from 6.63% (S. suberosa) to 10.05% in S. temmentos depending on the species (Tuncturk et al., 2015). The high values of crude proteins (11.62%) at site L5-Suva česma were also attributed to species composition, since it was showed that some have high crude protein values, as estimated for (Lotus ssp.: 14.86%, and Carex ssp.:15% (Catling et al., 1994), Cynodon dactylon: 8.91% (Munir et al., 2020)). According to studies of Abou El Nasr et al. (1996) and Badri and Ludidi (2020), where nutritional value of succulent plant parts in fresh or air-dried form was analysed, the silage is the most nutritious feed that can meet the energy and protein requirements of animals. Cellulose: high levels of cellulose were present in the biomass from localities L7-Žabalj 2 (37.59) and , which included the prevalence of the following species -Cynodon dactylon, Puccinellia distans, Camphorosma annua, Scorzonera cana, and Plantago tenuiflora for the area of Žabalj, and Cynodon dactylon, Luzula campestris, Puccinellia distans, Polygonum aviculare, Festuca ovina, and Trifolium fragiferum for L13-Ruski Krstur. The obtained values for cellulose are significantly higher than the data found in the literature for individual species that are part of the biomass of these sites (e.g., for Cynodon dactylon: 25.87%) (Munir et al., 2020). If we compare our results with the results obtained for non-saline productive grasslands of the central Balkan, it could be seen that pastures of the community Festucetum vallesiaceae had an uniform protein content ranging from 7.60 to 9.53% and cellulose values ranging from 27.49 to 29.55% (Tomić et al., 2009). Even though, in the central Balkan study region there was no species such as Agrostis stolonifera, for example, as a carrier of good quality, the studied halophytic grasslands could be considered to provide the biomass of good forage value. The crude fat: Total fat comprises a group of nutritious fats that play a very important role in the animal body (Liamadis, 2000) and are a very important energy component in animal feed (Bauman et al., 2003). The amount of fat in biomass is usually low (less than 3% of dry matter) (Coleman and Henry, 2002;Bruinenberg, 2003;Koutsoukis et al., 2016). The lowest values for fat in biomass samples were obtained for the plant material from the area of L12-Bački Brestovac (1.04), which contains the species Agrostis stolonifera, Puccinellia distans, Luzula campestris, Aster tripolium ssp. pannonicus, Festuca pseudovina, Lotus tenuis, Cynodon dactylon, and Camphorosma annua, while the highest value for fat was found in plant material from L8-Trešnjevac (3.82). However, the obtained average values were about 3%, which agrees very well with the results of other authors (Kandrelis, 2016). The factors that affect feed quality include environmental factors, palatability, nutrient content (chemical composition), presence of different secondary metabolites, feeding value (voluntary intake by animals, digestibility of nutrients) and finally animal performance (Attia-Ismail, 2018). In saline habitats the halophytes have the advantage because of salinity tolerance (Ahmadi et al., 2013) and are highly competitive with non-halophytes. The analysis of the main quality parameters was used to determine biomass quality (crude protein content ranged from 12.23% to 4.42%, cellulose content from 37.59% to 23.34% and fat content from 3.82% to 1.04%) indicating that South-east European halophytic vegetation is acceptable for animal feeding. The studied sites with generally higher cellulose values belong to the slightly salinized solonetz with dominance of grasses, while the heavily salinized solochaks are typical for dominance of euhalophytes exhibiting higher values of protein content. As showed in previous studies, the pastoral values vary along the soil salinity gradient (Dajic-Sevanovic et al., 2008). According to Masters (2015), halophytic vegetation has some limitations in animal feeding, mainly due to palatability, and less due to to the chemical composition. Field observations indicated that vegetation of salt-affected soils, ranging from slightly saline to severely saline, is under grazing or mowing management. Some species of saline grasslands have moderate to high nutrition quality (e.g. genus Lotus, Atriplex, Meliolotus, etc. (Marinoni et al., 2019), which points out the possible use of saline grasslands as fodder source.
Halophytic plants occur in different saline habitats upon specific environmental conditions, season, and geographic region, and vary significantly in their chemical composition and nutritional value (Attia-Ismail, 2018). They are characterised by complexity and polymorphism of saline habitats (Grigore et al., 2014).
Ecological, biogeographical and climate conditions, contributed to the certain differences in biomass quality between the studied Pannonian and central Balkan saline grasslands based, due to differences in species composition. Central Balkan saline grasslands are distributed in areas of drier climate and sub-Mediterranean climatic influence, where several low-protein grass species dominated (e.g., Puccinellia festuciformis ssp. convoluta, Puccinellia limosa, Bromus commutatus etc.). On the contrary, wet/moderately wet saline grasslands in Pannonia tend to be dominated by cellulose-and fat-rich grasses (Agrostis stolonifera, Puccinellia 12 limosa, Fesuca pseudovina). According to Dajic-Stevanovic et al., (2008), halophytic communities with salttolerant plants such as Achillea millefolium subsp. collina, Trifolium filiforme and Agropyrum repens are classified as very good according to pastoral value, while biomass with dominance of Lotus tenuis or Trifolium fragiferum is excellent. The lowest nutritional value has a group of species belonging to the salt tolerant species (e.g., Suaeda maritima, Salicornia europaea and some other Amranthaceae. The main reason for the low variation in pastoral quality between the studied regions is the fact that saline grasslands are usually composed of small number of species, where only one or few of them highly dominate, (Landucci et al., 2015) while most of other plants of floristic spectra are designated with abundance less than 5%. Species that are carriers of high fodder quality could not be detected. The second characteristic of halophytic grasslands is the rapid spatial change/shift of plant communities in a small area due to variations in ecological conditions, especially in the soil salt content as the key determinant (Luković et al., 2020). Small differences in salt content and the soil moisture frequently result in occurrence of specific vegetation units distributed on very small patches (average 1 m 2 to 16 m 2 ), with few or even only one dominant species (Stevanović et al., 2019). Furthermore, the high abundance of some grasses of respectable forage quality, such as Puccinellia limosa, Festuca pseudovina or Festuca valesiaca, Agrostis stolonifera points out the significance of halophytic grasslands for animal feeding. Moreover, some other herbs of salt affected soils, such as Artemisia santonicum, Statice gmelini, Camphorosma annua, Camphorosma monspeliaca, which account for the main amount of biomass, followed by legume species from the genus Trifolium or Lotus with small abundance contributed to the overall quality value of halophytic grasslands which is therefore not exceptional.
Finally, continental halophytic grasslands showed satisfied quality, even though did not express differences in quality along the geographic gradient, as well as between studied alliances. With intention to explain lack of differences and factors that contributing to, we highlighted floristic patterns and complex nature of halophytic vegetation as important factor.

Conclusions
The Pannonian Plain is the largest area of saline soils and continental halophytic vegetation in the Europe. In addition, there are sporadic patches of continental saline habitats in the south Balkans, and due to geographic and climatic impacts, obvious differences in floristic composition and vegetation types exist. In the studied region, different environmental conditions provide a wide range of halophytic flora and vegetation occurring on different types of salt affected soils, including highly salinized solonchaks to solonetz soil types, Halophytes may play a role in "saline agriculture" as animal feed or human food, energy crops for fuel production, oils, pharmaceuticals, etc., but are an important source of animal feed in this and similar regions. The chemical values of the main nutrients of the halophytic biomass vary depending on the floristic composition. The results show that there are no statistically significant differences in the quality of chemical composition between two distinct study sites, but only a small range of variation due to differences in floristic composition. The possible relations between salt-affected soil type, floristic patterns, chemical analysis and suitability for grazing/mowing or management of halophytic grasslands could be a challenge for further studies.

Authors' Contributions
Conceptualization: ML, UŠ, JV, JR, GT, MK, ZDS. Field work and collection of samples: ML, JV, MK; chemical analysis: JR; statistical analysis: UŠ; GIS data preparing and editing text: GT; conceived the idea and designed the work and supervision: ZDS.

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Ethical approval (for researches involving animals or humans) Not applicable.