Notes on the distribution, ecology, associated vegetation and conservation status of Gymnadenia (Orchidaceae) in Kosovo

Abstract Four species of Gymnadenia are native to Kosovo: G. conopsea, G. frivaldii, G. nigra, and G. odoratissima. In this study, field expedition data, phytosociological relevés, herbarium specimens along with extensive literature sources were used to analyse vegetation and ecological characteristics, habitat types, distributional patterns as well as provide notes on conservation. Gymnadenia conopsea is distributed throughout the country, from lowlands to the alpine belt. It was recorded in various plant communities. Gymnadenia frivaldii grows in the alpine zone of mountains, close to streams and in wet meadows. Its relevés belong to the class Scheuchzerio-Caricetea fuscae. This species is classified as “Near Threatened” (NT) in Kosovo. Gymnadenia nigra grows in subalpine and alpine grassland on preferably calcareous substrate. It has been found in almost all mountains reaching >2000 m a.s.l., and occurs in different plant communities belonging to the class: Elyno-Seslerietea. Gymnadenia odoratissima was recorded from one locality only in Kosovo, on the massif of Maja e Zezë, Sharri Mts. It was growing in degraded beech forest and meadows on silicate bedrock. Its floristically diverse relevés associate with the class: Mulgedio-Aconitetea. Of the four studied species, G. frivaldii deserves more conservation attention because of its fragile populations.


Introduction
As a result of various human activities in recent years, many plant species have become vulnerable, threatened or even extinct. In Kosovo, as in many other parts of Europe, increased agricultural and forestry activities, combined with a cessation of the traditional management of meadows, centralization of farming and expansion of urban areas has deprived many plant species from their natural habitats. The consequence of this being the confinement of once large plant populations to smaller areas, mainly in remote and fragile habitats.
Several studies have shown that habitat fragmentation and small, scattered plant populations adversely affect the genetic structure of a species (Young et al. 1996;Bateman et al. 2003). Fragmented populations are exposed to a higher risk of restricted gene flow and loss of genetic polymorphism (Franklin 1980;Frankel and Soulé 1981;Berisha et al. 2015). In the long term, probably the main adverse effect will be the inability of the species to adapt or respond to ever-changing environmental conditions (Ellstrand 1992).
The aim of different conservation programs nowadays is to preserve natural habitats and with it the existing level of genetic diversity, especially in the case of rare and endangered species (Ellstrand et al. 1993;Gray 1996). Knowing that in developing countries like Kosovo, available resources for the conservation of nature are limited, the identification of conservation priorities is of crucial importance.
Orchidaceae are a very diverse and species-rich family of plants, that represent nearly 10% of all angiosperm species. They are characterized by considerable floral diversity and a unique and often intricate pollination biology. The research focus on the biology of these plants has been mainly directed towards their pollination, adaptations to pollinators, evolution of pollination traits and the evolutionary outcomes of their unique biology (Schiestl et al. 1999;Huber et al. 2004;Cozzolino and Widmer 2005). Additionally, as the family contains numerous rare, threatened and endangered species, studies on members of the group are of fundamental importance in plant conservation efforts (Case et al. 1998;Ávila-Díaz and Oyama 2007;Tsiftsis et al. 2008Tsiftsis et al. , 2019. In the present study, the genus Gymnadenia R.Br. (Orchidaceae) in Kosovo is studied with emphasis on its distribution, species composition, diversity, ecological and associated vegetation characteristics. Gymnadenia is represented in the country by four naturally occurring species: G. conopsea (L.) R.Br., G. frivaldii Hampe ex Griseb., G. nigra (L.) Rchb.f., and G. odoratissima (L.) Rich. Of these, only G. frivaldii is of conservation concern, being categorized as "Near Threatened" (NT) in the Kosovarian Red List of plant species. The main aims of the study were to: a) study the distribution of the four Gymnadenia species in Kosovo and understand their distributional patterns; b) conduct phytosociological relevés in order to provide information about the most common plant communities where these species grow and generally analyze their syntaxonomy; c) provide ecological characteristics for each species concerning EUNIS habitat type preferences; and d) assess conservation implications for G. frivaldii.

Materials and methods
The studied species According to available literature sources, herbarium specimens as well as based on our own field data, in Kosovo there are four native Gymnadenia species. This study deals with: G. conopsea (L.) R. Br., G. frivaldii Hampe ex Griseb., G. nigra (L.) Rchb.f., and G. odoratissima (L.) Rich.
In this context, it is important to briefly discuss the taxonomic position of the species Gymnadenia nigra (L.) Rchb.f. Based on a study of this species by Teppner and Klein (1990), it was found that the natural distribution of G. nigra [homotypic synonym: Nigritella nigra (L.) Rchb. p.] is only in Scandinavia. Whereas in the Balkans G. nigra has been repeatedly erroneously reported and these specimens are in fact: , on which we relied for the current study, such a conclusion is not yet supported. Consequently, we referred to the species as: G. nigra.

Revision of herbarium material and distribution data
In total, 179 herbarium specimens (from the Herbarium of the Faculty of Natural Sciences, University of Prishtina) as well as private herbarium collection of F. Rexhepi (41 specimens) -(see Appendix 2 for details) were studied; the majority of these samples were collected by our team during the compilation of the Red Book of the vascular flora of the Republic of Kosovo (Millaku et al. 2013(Millaku et al. ) between 2009(Millaku et al. and 2013 Due to the fact that during the work for the Red Book, the main focus were certain natural habitats that were rich in endemic plants, some regions of the country remained poorly sampled. To compensate for this, we have conducted twenty-five additional expeditions (between 2014 and 2020) to those poorly explored areas, in order to be more confident that the presented data will allow for general conclusions about the investigated genus in Kosovo.
Plant samples were finally identified by F. Millaku, using identification keys and other relevant literature sources (Diklić 1976;Gölz and Reinhard 1986;Tutin 2010).

Vegetation data
To gain an overview of where the studied Gymnadenia species grow, in terms of phytosociological plant communities and certain related ecological preferences, a total of 15 phytosociological relevés were made (Appendix 1). Based on our field experience as well as from general knowledge from plant ecology, efforts have been made to conduct appropriate and representative phytosociological relevés.
Standard principles and methods of the Zürich-Montpellier school (Braun-Blanquet 1964;Mueller-Dombois and Ellenberg 1974) were applied. Depending on the habitat type, plot sizes of 10 × 10 m and 5 × 10 m respectively were used. For each plot, a complete list of vascular plants was recorded, alongside with cover-abundance values on a five-degree scale (Braun-Blanquet 1932). For the nomenclature of plant taxa, the Euro+Med Plant Base (Euro+Med 2006+) was followed. Relevés were made at elevations ranging from 140 to 2501 m a.s.l.; four relevés were made on communities with G. conopsea, four relevés on communities with G. frivaldii, four relevés on communities with G. nigra and three relevés on communities with G. odoratissima.
Based on diagnostic species, efforts have been made to offer a syntaxonomical classification of these relevés up to the level of Alliance. For this purpose, Mucina et al. (2016), Rexhepi (1994 and Sarić and Kojić (1984) were followed.

Data analysis
Using data obtained from herbarium specimens, phytosociological relevés as well as from literature, we were able to analyze the following parameters: altitude preferences, a set of ecological values (nutrients, pH reaction, moisture, continentality, temperature and light) from accompanying species of plants, using Pignatti indicator values (Pignatti et al. 2005). EUNIS corresponding natural habitat types (Davies et al. 2004) were defined for each species. Additionally, data on the overall species richness (from relevés) and richness of endemic taxa were compared. All of the geographical distributional data were mapped to show the distribution of Gymnadenia species in Kosovo (Fig. 1). R software (R Core Team 2013) for statistical computing was used to do the comparative analysis and generate the graphs.

Distribution of Gymnadenia species in Kosovo based on herbarium and field data
Based on the studied herbarium specimens, data collected from field surveys as well as literature sources, the presence of four Gymnadenia species is confirmed in a total of 88 different localities across Kosovo.
Within these localities, G. conopsea is clearly the most abundant species. Its presence has been confirmed in 60 localities and considering its ecological preferences, the species may have an even broader distribution across the country. Gymnadenia nigra is confirmed in 28 localities while G. frivaldii in 26 localities in Kosovo. Gymnadenia odoratissima is known so far to be present in only one locality in Kosovo (Appendix 2, Fig. 1).

Ecological characteristics and species preferences
Based on the obtained data from 15 phytosociological relevés, general data on each species preferences were evident. In terms of species distribution at different elevations, it was ascertained that the studied species show a narrow distributional preference (Fig. 2). It was established that G. conopsea had a preference for lower altitudes (<1000 m a.s.l.), though it had a wider distributional range. Gymnadenia frivaldii showed a greater preference for higher altitudes (>2100 m a.s.l.) and had a wider distribution range compared to G. nigra. As for G. odoratissima, this parameter shows a narrow median weight due to the fact that is known from a single locality at ~1700 m a.s.l.
Concerning soil fertility and nutrients availability, it was established that all four Gymnadenia species communities grow on relatively poor soils, with an average value of 3. In this context, G. nigra communities can be distinguished with the lowest median weight compared to G. frivaldii and G. odoratissima that prefer slightly more nitrogen rich soils (Fig. 3, boxplot 1). As for soil pH reaction (Fig. 3, boxplot 2), it was observed that four studied species communities have a general preference for alkaline soils. Though six relevés were made on silicate soils, the occasionally deep, accumulated soil has a significant effect on reducing acidic influence of soil pH. In terms of soil moisture (Fig. 3, boxplot 3), it was established that plant communities of the four Gymnadenia species have a general preference for medium-wet soils, with G. frivaldii in particular, preferring wetter soils. With regard to climate-continentallity (C) values, it was clear that there are no oceanic species and the average (Fig. 3, boxplot 4) value of 5 is general for all studied species communities. The temperature (T) preferences (Fig. 3, boxplot 5) associate also with the community occurrences at respective elevations above sea level. But, additionally it relates to cold or warm habitats of Europe. In this context, apparent distinction in preferences has been observed namely between communities of G. conopsea (6) and G. nigra (3). As for light preferences (Fig. 3, boxplot 6), it was obvious that all studied communities have a clear preference for growing under full light, a preference particularly pronounced in the case of G. nigra.
Regarding general species richness of all plant communities of the four respective species (Fig. 4, boxplot 1), communities of G. nigra and G. odoratissima were particularly distinguished by a large number of plant species. The other analyzed parameter, the number of endemic plant taxa per plant communities of a species, showed that plant communities of G. nigra (Fig. 4, boxplot 2) were particularly rich in endemics, while those of G. conopsea are notably poor. In this regard, G. frivaldii communities were rich in endemics too.

Vegetation characteristics
From 15 phytosociological relevés, after syntaxonomical analysis (relying on differential/characteristic species), it was noted that they belong to six different Classes (Appendix 1), with Elyno-Seslerietea Br.-Bl. 1948 (Class of vegetation that entails swards of Alpine and subalpine ranges of Europe) being the most abundant, in terms of relevé numbers.
Four relevés were made with G. conopsea communities. Three relevés fall into the alliance Chrysopogono-Danthonion calycinae Kojić 1959 (Dry grasslands on deep soils) and the other one into the alliance Trifolion resupinati Micevski 1957 (Vegetation of wet meadows).  Four relevés were made with G. frivaldii communities. As related with its typical natural habitats, the recorded plant communities (3) belonged to the alliance Narthecion scardici Horvat ex Lakušić 1968 (Relict moderately-rich fens of the Balkans) and the remaining one belongs to the alliance Cirsion appendiculati Horvat et al. 1937 (Tall-herb vegetation on acidic soils along mountain streams).
Additional four relevés were made with plant communities of G. nigra. They all fell into the alliance Seslerio juncifoliae-Caricion firmae Trinajstić 2005 (Alpine calcicolous sedge swards in wind-exposed habitats in the alpine belt of the Illyrian region and the Northern Dinarides). These four relevés were characterized by an exceptionally high number of associated endemic plant taxa (Fig. 4, boxplot 2).
Finally, a set of three relevés were made with plant communities where also G. odoratissima was growing. Due to a confined habitat (in Maja e Zezë, Sharri Mts.), it was quite difficult to record the relevé data. Two of these relevés, after analysis have shown affiliation to the alliance Cirsion appendiculati Horvat et al. 1937 (tall-herb vegetation on acidic soils along mountain streams and water springs at high altitudes), while the third one belonged to the alliance Epilobion angustifolii Oberd. 1957 (tallherb perennial semi-natural vegetation on acidic soils of forest margins).

Distributional patterns, habitat type and general environmental characteristics
As expected, all four species of the genus Gymnadenia follow different distribution patterns in Kosovo. Gymnadenia conopsea is a widely distributed species in Europe. In south-eastern Europe, it is common in the Carpathians, Hungary, Romania, the Balkan Peninsula and up to southern Russian and Ukrainian peninsula of Crimea. In all of these areas, it tends to occur at higher elevations, usually >800 m a.s.l. (Meekers et al. 2012). In Kosovo it represents the most common species of the genus. It was recorded in different habitats ranging from 750 m up to 1550 m a.s.l. As we had records on silicate as well as calcareous substrates, it indicates that this species can successfully grow in either one of these substrate types. Due to the wide distribution of the species, we are convinced that especially G. conopsea must have an even wider distribution than we depict on the map (Fig. 1). From the available data, it was confirmed in the following EUNIS Habitat types in Kosovo: E2.1 Permanent mesotrophic pastures and aftermath-grazed meadows; E3.3 Sub-mediterranean humid meadows; E1.2 Perennial calcareous grassland and basic steppes; E1.7 Non-Mediterranean dry acid and neutral closed grassland; E1.73 Deschampsia flexuosa grasslands; E4.31 Alpic Nardus stricta swards and related communities; and E4.4 Calcareous alpine and subalpine grassland.
From this genus in Kosovo, undoubtedly one of the rarest and most fragile species is the Balkan endemic G. frivaldii. It is worth noting that this species previously has been assigned to the genus Pseudorchis Ség. (syn.: Leucorchis E.May) and just recently, molecular based analysis has confirmed that it belongs to the genus Gymnadenia (Bateman et al. 2003). Gymnadenia frivaldii is a species with a relatively small range in Europe limited to high-mountain belts on the Carpathians and the central and eastern Balkans (Delforge 2006). It has been reported for Albania, Kosovo, North Macedonia, Greece, Romania, Bulgaria, Montenegro, and Serbia (Diklić 1976;Bateman et al. 2006;Millaku ed. 2013, Djordjević et al. 2016, 2017Berisha et al. 2020). This species prefers to grow on silicate substrates in fen communities and wet meadows of subalpine and alpine areas; occasionally it has also been recorded in pastures (of calcareous substrates too) as well as shrubs of the same altitudes in the mountains (Millaku ed. 2013). It has been reported (Djordjević et al. 2016) that this species is an indicator of an entire class (Scheuchzerio palustris-Caricetea fuscae Tx. 1937) of sedge-moss vegetation of fens in our region. The elevation distributional range was 1500-2600 m a.s.l. From the available data, mainly from the herbarium specimens, it was confirmed in the following EUNIS Habitat types in Kosovo: D2.2 Poor fens and soft-water spring mires; D2.22 Carex nigra, Carex canescens, Carex echinata fens; D2.26 Eriophorum angustifolium fens; D2.282 Balkan Willemetia fens; D2.3 Transition mires and quaking bogs and D2.38 Sphagnum and Eriophorum rafts.
Gymnadenia nigra was recorded on calcareous substrates of sub-alpine and alpine grasslands, or almost on all mountains exceeding 2000 m a.s.l. Based on the available data, it was confirmed in the following EUNIS Habitat types in Kosovo: E4.4 Calcareous alpine and subalpine grasslands; E1.7 Non-Mediterranean dry acid and neutral closed grassland and E1.72 Agrostis-Festuca grassland. All four relevés (Appendix 1) with this species, syntaxonomically fall into the Alliance: Seslerio juncifoliae-Caricion firmae Trinajstić 2005 (Alpine calcicolous sedge swards in wind-exposed habitats), though it has been reported also in different plant communities growing on limestone substrates (Rexhepi 1994). Also G. nigra previously has been assigned to Nigritella Rich. and Orchis Tourn. ex L., but phylogenetic studies (Bateman et al. 2003) have proved that it belongs to the genus Gymnadenia.
Gymnadenia odoratissima, a species very similar to G. conopsea, has an extensive distribution range in Europe. It is recorded from Spain in the west up to Ukraine in the east. Common in the mountain ranges of central Europe, up to the Sweden in the north and has been recorded also in Greece to the south. In Kosovo, Maja e Zezë massif represents the only known habitat of this species. It was recorded on silicate substrate, scattered in an area of ~1700 m 2 , at an elevation ranging from 1680 m up to 1800 m a.s.l. Its corresponding habitat type was that of (EUNIS -E5.5721) Moesian Balkan thistle tall herb communities, and at the forest (rather degraded) margins of (EUNIS -G1.6933), namely the Balkan range subalpine beech forests.

Vegetation analysis
Of the four relevés with G. conopsea (Relevés 1-4, Appendix 1), relevés 1, 3 and 4 belonged to the Alliance: Chrysopogono-Danthonion calycinae Kojić 1959, with the following species being dominant: Polygala major Jacq., Hypochaeris maculata L., Festuca nigrescens Lam., Danthonia alpina Vest. and Sanguisorba minor Scop. The richest relevé in terms of number of plant taxa was relevé no. 4., with 36 recorded plant taxa. Relevé no. 2 belonged to the Alliance: Trifolion resupinati Micevski 1957. It was recorded at a lower elevation (140 m) compared with the other ones of this group. The most dominant species in this relevé were: Hordeum secalinum Schreb. and Trifolium fragiferum L. As it is known, G. conopsea has a wider, more complex distributional range, though we aimed at offering its most common plant communities for comparative reasons. It was not our aim to define its syntaxonomical status, though this study can assist in that matter.
Of the three relevés with G. odoratissima (Relevés 13-15, Appendix 1), relevés 13 and 14 most likely should belong to the Alliance: Cirsion appendiculati Horvat et al. (1937), with the following taxa recorded as dominant ones: Barbarea balcana Pančić, Eriophorum latifolium Hoppe, Caltha palustris L., Cirsium appendiculatum Griseb., Cardamine pratensis L., and Helianthemum nummularium (L.) Mill. While the other remaining relevé on silicates (relevé no. 15) most likely should belong to the Alliance: Epilobion angustifolii Oberd. 1957. Epilobium angustifolium L. is the most dominant plant taxon in this relevé, as a characteristic species for the forest edges on silicate as well as a potential indicator of previously burned habitats or man-induced deforestation. High cover-abundance values on this relevé had also: Salix caprea L., Pimpinella saxifraga L. and Avenella flexuosa (L.) Drejer.
In the context of the vegetation diversity assessment, from only 15 relevés it was noticeable a very diverse vegetation affiliation by these four species of Gymnadenia. Four relevés belonged to Elyno-Seslerietea, three relevés to Festuco-Brometea, Scheuchzerio palustris-Caricetea fuscae and Mulgedio-Aconitetea respectively, and finally one releve belonged to Epilobietea angustifolii and Molinio-Arrhenatheretea respectively.
Commonly, all strategies for measuring biodiversity involve protecting a single species or several species of a given genus. Vegetation ecology contributes towards a better understanding of species indices of threats and a variety of interrelated paradigms in ecology (Cornell and Karlson 1996;Austin 1999). Furthermore, it also gives an incomparable insight into understanding the complex relationships between plant diversity, vegetation cover and site conditions (Wiesmair et al. 2017).

Conservation aspects
Although all of the four investigated Gymnadenia species are included in the European Red List of Vascular Plants (Bilz et al. 2011), all categorized as "LC" [Least Concern], more attention should be paid to those species that are more vulnerable and have fragmented habitats.
From this point of view, G. frivaldii is characterized by limited populations and a small number of mature individuals, with the exception of those in the Mts. of Gjeravica and Dobrosh. Nonetheless, since species populations are observed to be stable, its categorization as Near Threatened [NT] (Millaku ed. 2013) in Kosovo is completely reasonable.
Due to the large number of associated endemic species and their importance, we suggest that G. nigra populations in Kosovo also be assessed against conservation criteria and that monitoring measures be taken. In addition, the single population of G. odoratissima should be carefully monitored, as it represents the only habitat of the species in Kosovo.

Conclusions
Like all members of the Orchidaceae, Gymnadenia species are under pressure in the wild, primarily for their ornamental merits. These species also face many other threats in the wild, but are mainly affected by the loss, degradation or increasing fragmentation of their natural habitats. This habitat fragmentation is caused by human impacts on the natural environment.
Due to the fact that populations of G. frivaldii grow in habitats near watercourses and wet meadows, the conservation and management of these resources is directly related to the sustainability of the habitats that host them and many accompanying species.
The data presented in this study can help in the decision-making processes of the relevant agencies to implement appropriate conservation programs as well as further research.

References
Austin MP (1999) The potential contribution of vegetation ecology to biodiversity research.