Gymnadenia winkeliana—A New Orchid Species to Romanian Flora

A novel species, Gymnadenia winkeliana, has been identified in the Bucegi Natural Park ROSCI0013, located in the Southern Carpathians of Central Romania. Two moderately sized populations of Gymnadenia winkeliana, totalling 120–140 individuals, were discovered inhabiting the alpine grasslands of the park, situated 2.000 m above sea level. To describe this newly found population as comprehensively as possible, 44 vegetative and floral organs/organ parts were directly studied and measured from living plants. Special attention was focused on the characteristics that proved to have taxonomic significance, particularly those involving distinctive details in the morphology of the leaves, perianth, labellum and gynostemium. A total of 223 characteristics were analysed encompassing the morphology of every organ of the plant, cytology and breeding system. Furthermore, comprehensive taxonomic treatment and description, accompanied by colour photographs illustrating the holotype, are provided. Voucher specimens were deposited at the Herbarium of the University of Agriculture and Veterinary Medicine, Bucharest (USAMVB Herbarium barcode: 40102, NEA); Gymnadenia winkeliana, a (micro)endemic species, is characterized as a putative allogamous, facultatively apomict that significantly differs from other Gymnadenia R.Br. species found in Romania. Notably, it distinguishes itself through its smaller habitus (reaching heights of up to 8–10 cm), its two-coloured, rounded/hemispherical inflorescence displaying a gradient of pink hues in an acropetal fashion (ranging from whitish-pink at the base to vivid-pink at the topmost flowers), and its limited distribution in high-altitude areas, encompassing approximately 8–10 km2 in the central area of the Bucegi Natural Park. This species has been under observation since 2005, with observed population numbers showing a significant increase over time, from ca. 50–55 (counted at the time of its discovery) to 120–140 individuals (counted in June 2023). Additionally, comprehensive information regarding the habitat, ecology, phenology and IUCN conservation assessments of Gymnadenia winkeliana are provided, including maps illustrating its distribution.

Etymology: the generic name Gymnadenia is a compound term that originates from the ancient Greek words gymnós-(meaning naked or bare) and -aden (referring to a gland), ad litteram meaning "with naked glands", a reference to the viscidia-the adhesive disks found at the base of the pollinia, also termed viscidial glands [3]-that, usually, are free, exposed Plants 2024, 13, 1363 2 of 47 and not contained within bursicles (pockets of protective tissue), hence the reference "with naked viscidia", used for the orchid species of this genus [3][4][5].Due to their noticeable sweet fragrance, Gymnadenia R.Br.orchids are commonly known as fragrant orchids [1,6,7].
Gymnadenia R.Br.aggregate, which encompasses the former genus Nigritella Rich., represents a circumpolar genus of terrestrial orchids that extends across temperate Eurasia to Central India.Its range spans from Portugal to Kamchatka, including regions, such as China, Japan, Mongolia, Siberia, the Himalayas and Iran [6,8,9].Estimates indicate that the total number of species within this aggregate ranges from 15-16 [6,10] to 25-26 [2,11] and 31 [12].In Romania, Gymnadenia R.Br.species occur mainly in alpine regions, of Eastern and Southern Carpathians, with a great diversity in Bucegi Natural Park (Figures 1 and 2).
Plants 2024, 13, x FOR PEER REVIEW 2 of 48 ad litteram meaning "with naked glands", a reference to the viscidia-the adhesive disks found at the base of the pollinia, also termed viscidial glands [3]-that, usually, are free, exposed and not contained within bursicles (pockets of protective tissue), hence the reference "with naked viscidia", used for the orchid species of this genus [3][4][5].Due to their noticeable sweet fragrance, Gymnadenia R.Br.orchids are commonly known as fragrant orchids [1,6,7].Gymnadenia R.Br.aggregate, which encompasses the former genus Nigritella Rich., represents a circumpolar genus of terrestrial orchids that extends across temperate Eurasia to Central India.Its range spans from Portugal to Kamchatka, including regions, such as China, Japan, Mongolia, Siberia, the Himalayas and Iran [6,8,9].Estimates indicate that the total number of species within this aggregate ranges from 15-16 [6,10] to 25-26 [2,11] and 31 [12].In Romania, Gymnadenia R.Br.species occur mainly in alpine regions, of Eastern and Southern Carpathians, with a great diversity in Bucegi Natural Park (Figures 1  and 2).Although classified as sister genera, the orchid taxa Gymnadenia R.Br. and Nigritella Rich.are easily distinguishable and are often recognized as distinct genera by many authors, primarily due to morphological evidence [13][14][15][16][17]. Members of Gymnadenia R.Br. are Although classified as sister genera, the orchid taxa Gymnadenia R.Br. and Nigritella Rich.are easily distinguishable and are often recognized as distinct genera by many authors, primarily due to morphological evidence [13][14][15][16][17]. Members of Gymnadenia R.Br. are characterized by a highly elongate inflorescence (which can reach up to 50 cm in length in certain individuals), with resupinate flowers carrying a slender, medium-sizedto-highly elongated spur, half-filled with abundant nectar [18,19].In contrast, Nigritella Rich.species have a short and compact/dense, head-like inflorescence (measuring 1-2.5 cm in length), very small flowers (Figures 3 and 4), with mild flower colour polymorphism (observed in some species) and non-resupinate flowers carrying a minute sac-like spur (Figure 5), in which much reduced amounts of nectar are secreted [6,14,15,20,21].However, despite their differences, the two genera share several common traits, including a deeply divided palmate-digitate tuber (Figure 5H), narrow unspotted leaves (Figure 5A) and similar morphologies of the gynostemium [3,6,20].
Gymnadenia R.Br.orchids are commonly found in alpine and sub-alpine regions, where they prefer habitats with abundant sunlight.They thrive in meadows, grasslands, rocky slopes and other exposed areas, often growing on calcareous/alkaline nutrient-poor (oligotrophic to mesotrophic) substrates (Figure 2A-C).Their habitat extends from altitudes of 1.600 to 2.300 m above sea level (m a.s.l.) [1,22,24,25].
In this paper, we describe and illustrate a new species within the Gymnadenia R.Br.aggregate (former genus Nigritella Rich.), named Gymnadenia winkeliana.The population was first documented in June 2005 by botanist Dan Anghelescu, as part of an orchid field study conducted on the alpine plateau in the central region of Bucegi Natural Park, located in the Southern Carpathian Mountains.The population was found at an altitude of approximately 2000 m above sea level (a.s.l.) (Figure 2B,C).
The main distinguishing features of Gymnadenia winkeliana, which immediately attracted his attention, were its small-to-medium habitus, distinct pale-pink colour of the flowers and hemispherical-to-subcylindrical shapes of the inflorescences (Figures 3A-F and 4A-K).These features were highly distinctive and very conserved within the 40-50 individuals discovered.
Since its discovery, the population has been under continuous observation and close monitoring.These key distinctive morphological characteristics of Gymnadenia winkeliana have remained consistent over time, with the new individuals displaying little-to-no variation.Over the last eighteen years, the initially rather reduced population more than doubled, indicating that the small, vulnerable plants were thriving and gradually expanding across a larger area.
Nevertheless, the species' limited area of occurrence and proximity to tourist resorts and cattle farms have been considered as potential significant threats.However, it was likely the confined area of its occurrence that kept it relatively hidden from the widespread human intervention, such as collectors, photographers, tourists and the constant presence of grazing animals often found in Bucegi Natural Park.
Consequently, we chose to formally describe this new taxon as Gymnadenia winkeliana, in confidence that further explorations may reveal additional undiscovered populations within the park's greater area.
Thus, the aims of the present study are as follows: (1) conduct detailed biometrical/morphometrical measurements of both vegetative and floral organs; (2) provide a comprehensive discussion on the key morphological, distinctive characteristics of Gymnadenia winkeliana; (3) analyse its potential facultative apomictic origin; (4) conduct a comprehensive study of the pollinator community present within its restricted habitat, documenting its main insect visitors and potential pollinators; (5) identify the most frequent pollinator(s) of this species and discuss the potential facultative allogamy employed by Gymnadenia winkeliana; (6) provide information on various aspects of its geographical range, habitat preferences, ecological interactions, phenology and IUCN conservation status, complemented by illustrations and photographs derived from living specimens (the holotype); (7) offer a complete taxonomical treatment of this newly identified species.
Given the significance and rarity of Gymnadenia winkeliana, we strongly advocate for its recognition as a new addition to the Romanian flora.

Area of Occupancy (AOO)-Extent of Occurrence (EOO)
Gymnadenia winkeliana is micro-endemic with a restricted distribution, forming a unique population of ca.120-140 individuals found exclusively in a single location, in the northern-central area of Bucegi Natural Park.Therefore, its Area of Occupancy (AOOthe total area within which a species is known to occur) coincides with its Extent of Occurrence (EOO-the total area encompassing all known locations of a species) (IUCN, 2024).The population was found to be spread over an alpine plateau (grassland) with an AOO/EOO of ca.2.5-5 km 2 ; locus classicus GPS of 45  1C, red dots).

Location Studied
Bucegi Natural Park is characterized by an unpredictable microclimate, with low temperatures and strong winds.In the northern-central area, situated at altitudes of 2.500-2.000m a.s.l., the climate is predominantly cold, wet and frequently overcast (Figure 2B,C).Snow and frost persist for 7-8 months, with an average annual temperature of −3 • C (at Omu Peak, 2.505 m a.s.l.).Rainfall is common, often accompanied by cold temperatures and hail [26].Conversely, the southern area (2.300-1.800m a.s.l.) experiences a warmer and drier climate, with more sunshine and less wind and an average annual temperature of 0 • C [23].

Sites Studied
The study sites were located on moderately damp-to-dry, calcareous substrates, at an altitude ranging between 1.990 and 2.010 m above sea level (m a.s.l.).The Gymnadenia winkeliana population occurred in full-sun alpine meadows and pasturelands, rich in alpine herbaceous species and visited by a diverse community of pollinators (Figure 1C, indicated by red dots, Figure 2A-C).

Population Studied
A population of approximately 120-130(140) individuals of Gymnadenia winkeliana species is included in this study (Figure 2A).The population was found to be spread over an alpine plateau (grassland), on an area measuring approximately 2.5-5 km 2 currently in the Bucegi Mountains Natural Park (Figure 1C, red dots).The population, initially discovered at the locus classicus in 06.2005, counted ca.50-55 individuals, a number that increased to 80-100 individuals (2012-2020) and then to ca. 120-130(140), although the number typically remained below 200 individuals (n < 200).The population numbers vary due to the dormancy periods of this species, which typically last one vegetative season from our observations.Furthermore, plants tend to be more numerous and robust in years characterized by abundant snowfalls and rainy springs, rather than in drought years.Moreover, it is possible that the initial population numbers were higher, as the areas frequently used as sheep and cattle fields and a portion of the vegetation may have already been damaged by grazing animals.

Species Studied
Gymnadenia winkeliana individuals were studied according to the morphology, habitat, flowering time, geographic location and accessibility.Measurements of the vegetative and floral parts were made from living plants and fresh flowers.To describe this newly found population as comprehensively as possible, 44 vegetative and floral organs/organ parts were directly studied and measured from living plants.Special attention was focused on the characteristics that proved to have taxonomic significance, particularly those involving distinctive details in the morphology of the leaves, perianth, labellum and gynostemium.A total of 223 characteristics scored encompassed the morphology of every organ of the plant, cytology and breeding system.The characteristics (listed in Table 1) described in detail are the roots (8), stem (9), leaves and bracts (36), inflorescence and flower (16), sepals and petals (17), labellum and spur (30), gynostemium (13), anther (13), pollinia and pollinarium (27), stigma (19), ovary (12), fruit (6), seed and embryo (15), chromosomes (2), flowering time and reproductive strategies (7).For laboratory stereomicroscope biometrical measurements, several flower-bract units were extracted from a position located approximately one-third to halfway from the base of the inflorescence.Only flowering parts (individual flowers and 1-2 inflorescences) of the plants were sampled, leaving the vegetative parts for persistence and continued growth.

Morphometric/Biometric Data
In the light of so much contradictory DNA-based evidence [29], detailed morphological measurements continue to be the primary method for plant identification.Considering the phenotypic plasticity and variations within the genus, it is essential to thoroughly evaluate the macro-and micromorphological characteristics that can be utilized for taxon delimitation.A comprehensive study of the key morphological characteristics, pollinator, flowering phenology, chromosomes and reproductive strategy of Gymnadenia winkeliana is listed in Table 1 and illustrated in detail in Figures 3-7.

Pollination Monitoring
Since Gymnadenia winkeliana is restricted to its locus classicus, all insect monitoring was conducted exclusively within the above AOO (Area of Occupancy).During our pollination surveys, conducted over several years, 2017-2023, and spanning the months of June and July, when the flowers are in full antheses; we documented a diverse range of insect pollinators/visitors, members of the Orders Coleoptera (beetles), Diptera (flies, mosquitoes), Hymenoptera (ants, bees, wasps) and Lepidoptera (moths, butterflies), all part of the Class Insecta (Figures 8-10).

Pollination Monitoring
Since Gymnadenia winkeliana is restricted to its locus classicus, all insect monitoring was conducted exclusively within the above AOO (Area of Occupancy).During our pollination surveys, conducted over several years, 2017-2023, and spanning the months of June and July, when the flowers are in full antheses; we documented a diverse range of insect pollinators/visitors, members of the Orders Coleoptera (beetles), Diptera (flies, mosquitoes), Hymenoptera (ants, bees, wasps) and Lepidoptera (moths, butterflies), all part of the Class Insecta (Figures 8-10).In total, we documented 23 families comprising 47 species of insects that were recorded pollinating and visiting the inflorescences of Gymnadenia winkeliana.The total number of pollinator/visitor insect species of each Order is summarised below: (I) Coleoptera-Accidental pollinators: 5 families, 7 species; (II) Diptera-Pollinators (efficient) and potential pollinators: 7 families 21 species; (III) Hymenoptera-Pollinators and potential pollinators: 2 families, 6 species; (IV) Lepidoptera-Pollinators (highly efficient): 9 families, 13 species.Of the species included in this study, approximately 15% were Coleoptera, 44.6% were Diptera, 12.76% were Hymenoptera and 27.65% were Lepidoptera (Figures 8-10).

Morphological Characterization of Gymnadenia winkeliana
The main criteria used in the identification of Gymnadenia winkeliana included (1) the shape of the inflorescence and flowers, (2) the shape and form of the labellum, (3) the shape and form of the gynostemium, (4) the type of lower bract margins (entire, papillate, serrate, etc.) and ( 5) the presence or absence of denticles on bracts.These characteristics are important for distinguishing Gymnadenia winkeliana from other orchid species and are commonly used by botanists and taxonomists for the accurate identification of species in the field [3,31].The presence or absence of denticles on bracts was often used as a diagnostic characteristic [4,[32][33][34][35].Nevertheless, while denticulation of bracts has been traditionally used as a diagnostic characteristic for identification, its reliability can vary, leading some authors to question its usefulness as a qualitative characteristic [3,5].Klein [24] also emphasized the importance of flower colouration as a taxonomically relevant characteristic for some of the species of the subgenus Nigritella Rich., stressing its consistency across species.With few exceptions ([e.g., Gymnadenia rhellicani (Teppner & E. Klein) Teppner & E. Klein, which shows a wide variety of nuances [5,7]), flower colour is often a more reliable and consistent characteristic than denticulation, making it a valuable tool for the identification of these alpine orchids [10,11,18].
The main morphological characteristics studied are listed below.Their potential taxonomical value is discussed.
Habitus and Stem.Similar to other species of the subgenus Nigritella Rich., Gymnadenia winkeliana is a perennial that remains dormant as an underground tuber in the winter and produces a single short stem, at the beginning of the vegetative season.The conserved short stature of Gymnadenia winkeliana, typically reaching heights of about 8.5-10.5 cm, with occasional specimens reaching up to 14 cm, serves as a distinctive trait.This characteristic helps differentiate it from other orchid species with taller or more variable growth habits.The flowering stems are characteristically erect, slender, flexuous (may aid in adaptation to windy or harsh environmental conditions in the alpine habitat), ridged, glabrous (lacking hairs, trichomes or glandular, epidermal structures) and entirely vivid-green, with no purple pigmentation at the upper parts (absence of anthocyanin pigments).The vividgreen coloration of the stems, without any purple pigmentation at the upper parts due to the absence of anthocyanin pigments, is a distinctive characteristic, aiding in its accurate identification and differentiation from other orchid species (Figure 3A-E).
Basal leaves.Gymnadenia winkeliana is a summer-green orchid, i.e., its leaves emerge at the beginning of the vegetative season in spring (typically in May or later if snow persists at higher altitudes) and remain green throughout the summer.Their numbers vary, ranging from 6 to 10(12), and are arranged in a basal rosette.The variation in the number of leaves provides some degree of variability within the population but likely follows a consistent pattern within individuals.The leaves are grass-like, narrowly lanceolate with acuminate tips.The deeply keeled structure and spreading angle relative to the stem (approximately 40 • -45 • ) are likely adaptations for minimizing water loss in alpine habitats.In larger leaves, a central vein and faint parallel venation (multiple ribs) may be observed.The leaves are entirely green with a vivid green upper side and a yellowish-green underside and lack any purple markings (maculae).The leaf surface is smooth, glossy and deep green.The margins are yellowish-green, uniformly smooth, entire and straight, with no undulations present.These characteristics may help differentiate Gymnadenia winkeliana from other orchid species with different leaf textures and margin shapes (Figure 5A).
Cauline leaves.The 4-7(8) cauline leaves are arranged alternately or in two vertical rows (distichous) evenly spaced along the stem.Unlike the basal leaves, they are triangularlanceolate sheaths, erect to slightly arched and relatively rigid.They are vivid green with dark-red-to-purple-brown edges and tips, indicating the presence of anthocyanin pigments in the marginal epidermal cells.Their surface is textured, displaying a central vein (midrib).The leaf margins are serrated (presenting an irregular margin) or edged with fine, hyaline, translucent, conical-elongate, tooth-like papillae.The papillae are closely spaced and irregularly distributed, with some leaves having denser coverage than others.The irregular distribution of papillae along the leaf margins, with some leaves having denser coverage than others, suggests variability within the population (Figures 3A-C,F and 5A).
Bracts.The flower bracts are shorter than the cauline leaves but longer than the flowers.They are narrowly triangular and have acuminate tips.The basal bracts spread horizontally at an angle of approximately 90 • relative to the stem (perpendicular to the stem), while the median and top bracts are angled at approximately 40 • -45 • relative to the inflorescence rachis.The bracts exhibit a consistent greenish-brown coloration, with pronounced purple pigmentation at the tips and margins.The margins are finely serrated by numerous, elongate, translucent papillae (denticles), which are typically evenly spaced.While they measure between 0.04 and 0.14(0.28)mm in size, they show moderate variability and create an irregular or serrated margin (irregularly denticulate) (Figures 3E, 4A, 5D and 6D,E).
Stomata.Like all species in the subgenus Nigritella Rich., Gymnadenia winkeliana bears hypoamphistomatic leaves, with stomata present on both sides of the leaf, predominantly on the lower, abaxial surface [36].The adaxial surface of the bracts presents numerous anomocytic stomata (a stomatal type in which the subsidiary cells surrounding a stomate are not differentiated from the other epidermal cells [37]), restricted to the apical area [36].On the adaxial surface (upper surface) of the bract, stomata are restricted to the apical area, towards the tapering, strongly purple-pigmented tip (Figures 5P and 6D,E).
Inflorescences.The inflorescences are densely packed racemes (the flowers are arranged along a central axis, with each flower attached by a short stalk or pedicel), typically carrying an average of 60 to 80 flowers (floriferous).The inflorescences undergo mild changes in shape and size throughout their developmental stages.They start as pyramidal during bud stages, then become near-spherical to hemispherical (subcylindrical) at full anthesis (when flowers are fully open), and finally become ovate to subovate during the fruiting stages.Nevertheless, the size and shape slightly vary among individuals and populations.While there is slight variation in the inflorescence size and shape among individuals, these features are consistent across the species.The inflorescences exhibit a distinctive two-coloured appearance, with a gradient of pink hues.The basal flowers are white or pale pink, while the topmost flowers and buds display a range of shades from pale pink to dark pink.The light pink coloration of the inflorescences is highlighted as a key/main characteristic of the species, serving as a distinguishing feature from other Gymnadenia R.Br.species in Romania (Figures 3A-F, 4A-L and 5A-E).
Flowering pattern.The flowers open sequentially from the base upwards in an acropetal manner, meaning that the lower flowers open first, followed by those above them, until reaching the topmost flowers and buds.The inflorescences are very floriferous, bearing 40-60(80) small, pinkish flowers (Figures 4L and 5A).
Individual Flower.Similar to the inflorescences, individual flowers exhibit a colour gradient ranging from white to whitish-pink at the base, transitioning to pink and deeppink towards the tip of the inflorescence.This gradient creates a visually striking display of colour variation within the inflorescences, which helps in species identification in the field.While the general colour pattern follows a consistent gradient, there is slight variation among individuals, with some displaying a more pronounced pinkish hue at the base.The flowers are wide open (chasmogamous), star-shaped, with spreading petals, which enhances their attractiveness to pollinators.Similar to all other species in the subgenus Nigritella Rich., the flowers of Gymnadenia winkeliana are non-resupinate (untwisted, i.e., flowers maintain their original orientation throughout development, without undergoing a twisting or rotational movement) (Figures 4L, 5C-E and 6A,B).
Scent.The flowers emit a distinctly sweet, vanilla-like fragrance.This fragrance is likely produced by volatile compounds released by the flowers (which include vanillin and vanillyl ethyl ether), which are perceived by pollinators [6].We noticed that the fragrance is particularly intense during the morning hours/first part of the day, indicating that scent production may be influenced by environmental factors, such as temperature and humidity.Morning hours are often a peak activity time for many pollinators, so the timing of scent emission aligns with pollinator foraging behaviour.The sweet and vanilla-like fragrance is likely an adaptation to attract potential pollinators.Due to their remarkable chocolate and/or vanilla fragrance, the species in the subgenus Nigritella Rich.are known as the European Vanilla Orchids or simply the Vanilla Orchids [1,16,23].Flower longevity.The anthesis typically lasts for a period of 4 to 7 days, with some flowers remaining open for up to 10 days.The longevity of the flowers is greatly influenced by environmental factors, such as temperature, rainfall, wind and others.
Perianth segments.The perianth segments, sepals and lateral petals spread laterally, have acute/acuminated, arched tips and exhibit similar colouration that may vary depending on their position within the inflorescence.The sepals are slightly longer and approximately twice as wide at the base as the lateral petals.The median sepal is broader and more pronounced, oriented downward due to the non-resupination of the flower.This positioning may serve as a landing platform for potential insect pollinators or visitors, facilitating pollination interactions (Figures 5C-G,I,J and 6A,B).
Labellum.It is non-resupinate, hence its upward orientation, narrower rhomboidal in shape, with an acuminate tip.It is shallowly three-lobed, consisting of a central lobe and two lateral lobes, with a bulbous base containing the gynostemium.The central lobe (the epichile) is elongate and triangular, with slightly undulating, smooth margins and no markings (spots or dots) present on its surface.The lateral lobes are scalloped and rounded, but less pronounced than the central lobe.The middle segment of the labellum (the mesochile) is saddle-shaped and forms a narrow tube-like junction, approximately 0.6-0.7 mm wide, created by the incurved, narrowing edges of the lateral lobes.In some cases, these edges almost touch each other.The narrowing tunnel leads to the centre of the flower where the reproductive structure, the gynostemium, is situated.The basal part of the labellum (the hypochile) is broad, bulbous and rounded, terminating in a saccate, short spur (Figure 5F,G).
Spur.The spur is nectariferous, translucent-white, straight (not curved or arched) and filled up to ¼-½ of its length with nectar.The presence of nectar in the spur serves as an attractant to pollinators, potentially contributing to the reproductive success of the orchid.Additionally, the shape and size of the spur, along with other floral characteristics, such as the labellum, have been frequently used as discriminatory features in the identification of various species within the genus Nigritella Rich.[33,34,[38][39][40] (Figures 5D,E,I,L and 7A).
Gynostemium.In Orchidaceae, the reproductive structures-the unique, fertile anther and the stigma-are fused into a singular, columnar central structure known as the gynostemium.In Gymnadenia winkeliana, the gynostemium is thick, cylindrical and translucent in appearance.The gynostemium is positioned horizontally or slightly facing downwards, with the stigma located above the anther due to the non-resupination characteristic of the flower, above or adjacent to the entrance of the spur.The downwards (non-resupinated) orientation of the gynostemium, which places the stigmatic cavity above the anther, makes self-pollination impossible, a main characteristic of all species of the subgenus Nigritella Rich.Laterally, the gynostemium features two verrucose gynostemial auricles (Figures 5I,J,L,M, 6B,C and 7A).
Pollinia.Each theca contains an ovoid-elongate, yellow, massulate (granular) pollinium, formed by individual densely packed massulae.The massulae are ovoid blocks of pollen grains (tetrads) held together by elastoviscin [41,42].At the terminal end, the pollinia form a thin, long, translucent-yellow structure called the caudicle that typically measures more than 30% of the length of the pollinium.This caudicle serves as a connecting structure between the pollinium and the viscidial disk.The viscidial disk is a circular, adhesive structure located at the end of the caudicle.It allows the pollinarium to adhere to the proboscides of potential pollinators, aiding in pollen transfer during pollination [41] (Figures 5M,N,R, 6C and 7A,C,E).
Pollinarium.The pollen dispersal unit (PDU), also known as a pollinarium, is composed of a massulate pollinium, a caudicle, and a free viscidial disk [41][42][43].Unlike other species of orchids, the viscidial disk in Gymnadenia winkeliana is enclosed or contained within a rudimentary membranous bursicle.In older flowers, it becomes more exposed and freer to adhere to potential pollinators.There are two pollinaria per anther, one in each theca, containing all the pollen of the flower.All species of the genus Gymnadenia R.Br.possess massulate pollinia/pollinaria as part of their reproductive structures [44].Additionally, in some species, the viscidia may be contained in rudimentary bursicles [5,11] (Figures 5M,N, 6C and 7C).
Stigma.Due to the non-resupinated feature of the flower, the stigma is located above the anther (the gynostemum points downwards).The placement of the stigma above the anther makes spontaneous self-pollination impossible [5,41].The stigmatic cavity is concave elliptical in shape, translucent white and covered with stigmatic exudate, indicating a wet stigma.In massulate orchids, like Gymnadenia R.Br.species, the stigma typically consists of three lobes: one central (rostellum) and two lateral lobes.The lateral lobes are broad, flap-like structures that spread laterally [9,45,46] (Figures 5L,M, 6C and 7A).
Rostellum.The rostellum functions as a barrier between the anther, which contains the pollinia pair, and the stigma, preventing even more the occurrence of (accidental) selfpollination in the non-resupinate Nigritella Rich.species.The median lobe, also referred to as the rostellar flap, is prominent and protrudes between the two thecae containing the pollinaria, making self-pollination impossible [45].The well-defined separation created by the highly developed rostellar fold indicates that Gymnadenia winkeliana relies on insect pollinators (entomophilous) for cross-pollination, making it a potentially facultative allogamous species (Figures 5L,M, 6C and 7A).
Ovary.The ovary is ovoidal, unilocular/monocarpellar (consisting of a single chamber, lacking the septum) and syncarpous (composed of fused carpels).The ovary is epigynous, meaning it is enclosed within the receptacle with the floral parts arising above it [47].The ovary is sessile, lacking a flower pedicel, and does not twist since Gymnadenia winkeliana has non-resupinate flowers.Placentation is parietal, meaning that the placenta develops along the fused margins of adjacent carpels on the parietal wall of the ovary [48].In the majority of the plants observed, the basal ovaries were significantly swollen, still having the unwithered/still fresh, anthetic flowers hanging, a putative indication of a species characterized by an early onset of apomixis [47] (Figures 5E,J,K,O and 7B).
Ovules.The ovules are translucent-white to whitish-green, anatropous (inverted during development by the bending of the funicle (stalk) attaching it to the carpel wall) and tenuinucellate (the nucellus is reduced to one layer of cells, which surrounds the megaspore mother cell), with one or two integuments [47] (Figures 5O and 7B,D).
Fruit.The indehiscent fruit is an elongate-ovoidal green pod, characterized by three highly pronounced longitudinal ridges (three lines of dehiscence).Fruiting occurs in June-July, with a relatively high fruit set rate, estimated at approximately 40-68(80)%, based on 45 counts (Figures 3E and 5J).
Seed capsule.Seed maturation typically occurs between July and August, with the last capsules dehiscing in September to October.The seed capsule is brownish, elongate-ovoidal and slightly larger than the fruit pod.
Seeds.The seeds are very small (microseeds) and numerous (microspermy), elongatedovoidal to fusiform in shape, tapering from the middle to the tips, up to twice as long as wide [17] (Figure 5R).
Testa.The seeds are covered in a brownish reticulated testa.The testal cells are rectangular-elongated, with non-sinuous anticlinal walls.The testa is open at the micropyllar end and closed at the chalazal end (micropyle-the opening at the apex of the ovule where the integument does not completely cover the nucellus and allows the pollen tube to enter the ovule; chalaza-the basal part of the ovule where the integuments and nucellus are attached [47] (Figure 5R).
Embryo.Centrally, the seeds contain a spherical-ovoidal rudimentary, whitish-yellow, endospermless embryo.The endosperm in orchid seeds is nuclear (reduced) and nonfunctional, as it either fails to develop/form or degenerates after the fertilization of the polar nuclei [49] (Figure 5R).

Cytogenetics
The chromosome numbers within the Gymnadenia R.Br.genus are known to vary significantly, between diploidy and polyploidy (tri-, tetra-and pentaploidy), with a basic chromosome number of x = 20 [19,31].Due to the early swelling of the ovaries (a potential sign of apomixis), Gymnadenia winkeliana has been suggested to be a polyploid species, more specifically putatively tetraploid, with a chromosome count of 2n = 4x = 80 (polyploidy is usually associated with apomixis).Further research is required to confirm the exact chromosome number of Gymnadenia winkeliana.

Asexual Reproduction Vegetative Propagation
Gymnadenia winkeliana may have the potential for asexual reproduction through vegetative propagation via root tubers.This is evidenced by numerous compact clusters of 2-4(6) adjacent plants emerging from the same rhizome or multiple root tubers, a phenomenon previously reported by [13,16,44,45].Although vegetative reproduction is seemingly uncommon, the production of extra tubers can result in clumps of (genetically identical) flowering stems adjacent to each other in some individuals, as shown (Figure 3A,B,F).Gymnadenia winkeliana seems to employ, rather often, vegetative propagation since clusters of 2-4 plants were rather often encountered within the population.

Apomixis
All members of the subgenus Nigritella Rich.have non-resupinate flowers.Consequently, the gynostemium is slightly downward-oriented, positioning the stigmatic cavity directly above the anther, thereby preventing self-pollination.Furthermore, these species possess a well-developed stigmatic median lobe, known as the rostellum, which acts as an additional barrier against accidental self-pollination.As a result, due to the absence of self-pollination, the polyploid Nigritella Rich.species employed apomixis.
Our observations showed that, in the majority of flowering individuals, the young basal blooms (at the very beginning of anthesis) presented already significantly swollen ovaries.A transversal section through the basal, young flowers' ovaries revealed ovules in advanced stages of development, already developing into immature seeds with a distinctly reticulate testa (Figure 7B,D).Nevertheless, we cannot conclude with certainty whether the observed swollen ovaries in Gymnadenia winkeliana are the result of apomixis.Still, considering the limited presence of pollinators, we hypothesize that the high occurrence of fruiting ovaries in young plants might suggest the early onset of apomixis.This assumption is based on the fact that, similar to all other species within the subgenus Nigritella Rich., self-pollination is not possible in Gymnadenia winkeliana.This is due to two main factors: (1) non-resupination, which positions the anther above the stigmatic cavity, and (2) the significant development of the rostellum, which acts as a barrier preventing any contact between the pollinia and the stigmatic cavity.
Apomixis is a form of asexual reproduction that results in the production of seeds without the occurrence of meiosis or fertilization [50][51][52].As a consequence, through this alternative reproductive mode, clonal seeds (genetically identical to the mother plants) are produced [53].
Apomixis can be categorized into two types based on the origin of the embryo: gametophytic apomixis and sporophytic apomixis (adventitious embryo) [51,54].
In the former genus Nigritella Rich., apomixis was shown to be sporophytic and exclusively observed in polyploid species [13,16,34,47,55].While diploid Nigritella Rich.species reproduce sexually (allogamy), their polyploid counterparts reproduce asexually through apomixis [56,57].Although the association between apomixis and polyploidy was observed several times, [24,34,40], apomixis has only been confirmed in the former genus Nigritella Rich.within the subtribe Orchidinae (subfamily Orchidoideae) [13].Consequently, while the presence of a diploid Gymnadenia winkeliana cannot be ruled out, given the connection between apomixis and polyploidy, it is plausible to consider that Gymnadenia winkeliana might be a polyploid member of the genus.
In sporophytic apomixis, also known as adventitious embryony, embryos develop directly from the somatic tissues of the ovules, located outside the embryo sac, i.e., from either the nucellar (nucellar embryony) or the integumental cells (integumentary embryony) of the ovule [53,55,58,59].Additionally, in sporophytic apomixis, the formation of the embryo sac was reported to proceed normally, with the megaspore mother cell (MMC) undergoing multiple meiotic and mitotic cycles, resulting in the development of a sevencelled, eight-nucleate/octanucleate embryo sac [47,60,61].As a result, embryo sac formation is not directly associated with apomixis, leading to the production of a functional haploid egg cell [58,62].One of the first types of adventitious (nucellar) embryony has been described by Afzelius (1932) [63] in the tetraploid Gymnadenia nigra (L.) Rchb.f., in which simultaneously with the development of the female gametophyte (arrested at the nuclear stage), one or two nucellar embryos were also formed [47].

Do apomicts still need pollinators?
Once formed within the ovule, the adventitious embryos engage in competition with the sexual embryo for nutrients [55,59].In order for their development to progress further, it has been demonstrated that their survival significantly depends on the double fertilization of the sexual embryo sac [52,64,73].This process not only generates the zygote and the triploid nucleus (resulting in reduced endosperm, in Orchidaceae) but also triggers the developmental start of the ovule [73][74][75].This initiation is facilitated by complex nutrient and growth signals originating from both the egg cell and sperm cells [58,65,76].Therefore, in most facultative apomicts, the formation of the embryo sac may still require the presence of pollen on the stigma to initiate the process of embryo sac development [77].In orchids, in general, microsporogenesis proceeds normally, and the male sexual function is maintained not only for double and triple fertilisation, but also because pollination is essential for ovule development [75,78].The reliance on pollination for ovule and embryo development might serve as an additional, yet underexplored, constraint influencing the scarcity of apomixis in orchids, which is a relatively rare phenomenon in the family [65,79].
The requirement for pollination in facultative apomixis may explain why facultative apomicts, i.e., Gymnadenia winkeliana, a putative apomict, still exhibit all the characteristics needed for allogamy (pollination by insects), such as showy flowers, strong fragrances (vanilla, chocolate), abundant nectar and well-developed pollinaria [19,31].Therefore, despite being regarded as a potential facultative apomict, Gymnadenia winkeliana may still rely, to some extent, on pollinators for the development and maturation of its fruits and seeds, whether they are formed through sexual or asexual reproduction pathways [80,81].

Colonising Unfamiliar Habitats.
Although apomixis is rare in Orchidaceae, it has been recognized as a strategy that could potentially provide reproductive assurance independently from pollinators or pollen vectors [68,77,82].Therefore, apomixis has been thought to be a major advantage for species colonizing areas that were previously glaciated or located at high altitudes [74,83,84].In such environments, a limited number of highly fit, recently formed/evolved genotypes may rapidly spread over larger areas, particularly at elevated altitudes [83,85,86].
The micro-endemic Gymnadenia winkeliana represents a remarkable example of a potential facultative apomict that has successfully colonized a high-altitude harsh habitat characterized by a cold, rainy microclimate, with short, stormy summers, and with a limited presence of pollinators (Figure 2B,C).The significant increase in population numbers reinforces its potential facultative apomictic nature, which, combined with its remarkable adaptability, allowed it to colonize new, unfamiliar ecological niches.
Gymnadenia winkeliana shares similarities with several other allogamous European species of its genus, at the same time, retaining all the morphological features of a sexual reproductive species.Its brightly coloured chasmogamous flowers, the pleasant vanilla sweet scent (longdistance attractant for pollinators) and the nectar secreted in its saccate spur (reward for potential pollinators) are the main characteristics of an entomophilous species, making this species a good candidate for allogamy.Additionally, its well-developed, functional pollinia and putative sporophytic type of apomixis may enable cross-pollination.Consequently, Gymnadenia winkeliana may also be considered a putative facultative allogamous (crosspollinated) species, relying on entomophily (insect pollination) to produce fruit and seeds sexually, thus reassuring its genetic diversity within its constantly/gradually increasing population.Autogamy is impossible in this species due to non-resupination, which places the stigma above the anther (gynostemium faces downward) [3,5].Since Gymnadenia winkeliana produces nectar and rewards its insect pollinators/visitors, the pollination method employed is a generalized food-foraging strategy (insects are attracted by food, i.e., nectar) [80,81].This strategy is largely employed by most of the rewarding orchids, accounting for approximately two-thirds of all species in the family [87][88][89][90][91].
In this strategy, generalist insects are drawn to the sweet vanilla scent emitted by orchids and exploit available food or nutrient sources, such as nectar or floral exudates.Simultaneously, rewarding orchids increase their chances of pollination and seed production due to the insects' constant food-foraging behaviour, always in search of food sources [92][93][94].This pollination strategy contributes to the ecological success of both generalist insects and rewarding orchids [95][96][97][98].
Gymnadenia winkeliana is found at very high altitudes, ca.1.900-2.010m a.s.l., in Bucegi Natural Park, thriving in an alpine habitat characterized by a harsh microclimate.Summers in this region are typically cool, marked by frequent rainfall and storms, with sporadic overnight snowfall being common, particularly during May and June when Nigritella Rich.orchids are in full bloom (Figure 2B,C).Consequently, the pollinator communities in this area are rather modest, consisting of fewer species with even fewer insect representatives serving as potential orchid pollinators or visitors.During our pollinator monitoring studies, we encountered several species, as documented in Table 2, including various Lepidopteran, Coleopteran, Hymenoptera and Dipteran insect species.Similar observations were previously reported by Faegri and Van Der Pijl (1979) [99], Vöth (2000) [100] and Claessens andKleynen (2011, 2016) [3,5].These data strongly support our initial hypothesis that Gymnadenia winkeliana may be a facultative allogamous species.Order Coleoptera Linnaeus, 1758 (beetles): Several species of Coleopterans were observed visiting the flowers of Gymnadenia winkeliana.These were mostly members of Cantharidae Imhoff, 1856 (soldier beetles) (Figure 8D,E), Chrysomelidae Latreille, 1802 (leaf beetles) and Elateridae Leach, 1815 (click beetles) (Figure 8G) families, primarily phytophagous or pollen-foraging species.Phytophagous beetles are herbivorous insects that feed on plant material, including leaves, stems, flowers and roots.In some instances, they may directly harm orchids by consuming their tissues, leading to reduced plant fitness or even death.While not all phytophagous beetles feed destructively, some species may inadvertently serve as pollinators for orchids.These beetles may visit orchid flowers in search of food or shelter and, in the process, transfer pollen from one flower to another, facilitating pollination.During our studies from 2017 to 2023, there were no instances where Coleopterans were observed carrying pollinia, although they may serve as accidental pollinators for high-altitude orchid species.However, we did observe several pollination events in which Dactylorhiza viridis (L.) R.M.Bateman, Pridgeon & M.W.Chase, occurring alongside Gymnadenia winkeliana, was efficiently pollinated by Rhagonycha lignosa (Müller, O.F., 1764), a member of the Cantharidae Imhoff, 1856 family of beetles frequently found in the same location [1,22,30] (Figure 8B-E,G).
Order Diptera Linnaeus, 1758 (flies, gnats, mosquitoes): Several Dipterans were observed visiting Gymnadenia winkeliana.Small Dipterans, such as Empididae Latreille, 1804, (Figures 8A and 9B) are frequent visitors to orchids and efficient pollinators, especially in environments where other pollinators are scarce, such as alpine grasslands and wetlands [28,101] or mountain habitats with cold climates [102,103].Some species are also predators or carnivores, preying on larvae, but they may also forage on nectar and floral exudates [104,105].On several occasions, species, such as Empis ciliata Fabricius, 1787 (black dance fly) or Empis trigramma Wiedemann in Meigen, 1822 (yellow dance fly) from the family Empididae Latreille, 1804 (dagger flies) (Figures 8A and 9B) were observed carrying multiple pollinaria attached to their long proboscides.While dagger flies may not be as specialized as other pollinators, like moths or butterflies, their visits to orchid flowers can still lead to pollination, including both cross-pollination and geitonogamy [27,28,106].
Common flies belonging to the families Anthomyiidae Robineau-Desvoidy 1830 (houseflies), Muscidae Latreille, 1802 (house flies or stable flies) and Syrphidae Latreille, 1802 (hoverflies or syrphids) were frequently observed visiting Gymnadenia winkeliana inflorescences, being highly abundant in its specific alpine habitat (Figure 9A-H,J,K).These insects are generalized food foragers and are attracted to any floral attractants in their constant search for food.In several instances, they may inadvertently pollinate orchids as accidental pollinators.However, no species of flies were observed carrying any Gymnadenia winkeliana pollinia.Members of the Muscidae Latreille, 1802 family may be considered accidental/potential pollinators of Gymnadenia winkeliana, as they were observed previously [3,5,106].We documented syrphids carrying pollinia of Dactylorhiza maculata (L.) Soó, 1962, Dactylorhiza fuchsii (Druce) Soó and Anacamptis coriophora (L.) R.M.Bateman, Pridgeon & M.W.Chase on multiple occasions (unpublished results) (Figures 8A, 9A-H,J,K and 10H), Order Hymenoptera Linnaeus, 1758 (wasps, bees, ants): Generally, bees and bumblebees from the family Apidae Latreille, 1802 are considered the most efficient orchid pollinators [1].However, our observations have shown that they are relatively scarce in the high-altitude habitats specific to Gymnadenia winkeliana.This scarcity may contribute to the low frequency of orchid pollination in these areas.During our study, only two instances were recorded in which a single pollinarium was observed attached to the proboscides of Bombus pratorum (Linnaeus, 1761) (early bumblebee) and Apis mellifera Linnaeus, 1758 (European honey bee) (Figure 9I).Therefore, bees and bumblebees appear to play a minor role in the pollination of Gymnadenia winkeliana.While ants from the family Formicidae Latreille, 1809 are not typically regarded as primary pollinators, like moths and butterflies, they may have a minor role in the pollination of certain orchid species, particularly in temperate regions (Figure 8A,F).This is because the pollinaria of temperate orchids are small enough to be removed and transported to another flower by such small insects.We observed several Gymnadenia winkeliana inflorescences inhabited by black aphids, members of the superfamily Aphidoidea Geoffroy, 1762.These small sap-sucking insects infest young orchid inflorescences and typically feed on the plant's sap by piercing the tissues with their needle-like mouthparts (Figure 8F).Ants and aphids often engage in a mutualistic relationship known as trophobiosis [107,108].Aphids secrete a sugary substance called honeydew, which ants harvest as a food source [109].In this relationship, ants protect aphids from predators and parasites [108].As a result, Gymnadenia winkeliana inflorescences were frequently visited by ants from the family Formicidae Latreille, 1809, such as Formica fusca Linnaeus, 1758 (silky ant), Myrmica rubra (Linnaeus, 1758) (common red ant, European fire ant) and Myrmica schencki Viereck, 1903 (flower ant).On the orchid inflorescences, ants were observed to gently tap the black aphids with their antennae to stimulate honeydew secretion, while also patrolling the opened flowers foraging for nectar or floral exudates.No ants were observed to directly pollinate Gymnadenia winkeliana, but accidental pollination may occur.Myrmecophily (ant pollination), was observed in the case of Epipactis palustris (L.) Crantz and Neottia ovata (L.) Bluff & Fingerh., which were recorded as being pollinated by ants attending aphid "farms" on their inflorescences [1,106].Gymnadenia winkeliana inflorescences bearing black aphids were also observed to be briefly visited by beetles of the family Coccinellidae Latreille, 1807 (Ladybugs, Ladybirds), such as Coccinella septempunctata Linnaeus, 1758 (seven-spot ladybird), and Hippodamia variegata Goeze, 1777 (variegated ladybug).Ladybirds are carnivorous insects and typically visit orchids in search of aphid colonies, on which they prey.Although they are frequently encountered on orchid inflorescences, they are not true orchid pollinators but may be considered potential accidental pollinators (Figures 8A,F and 9I).
In conclusion, Lepidopterans were the dominant taxa of pollinators in regards to the overall number of species recorded, proving to have stronger interactions with Gymnadenia winkeliana, potentially suggesting a more specific relationship between the two parties.The newly identified species, Gymnadenia winkeliana, demonstrates a preference for several species of moths and butterflies as pollinators, with Boloria pales (Nymphalidae Rafinesque, 1815) being the most frequent and efficient visitor of the inflorescences (Figure 10A-C,F,K).Despite possessing atypical lepidopteran flowers characterized by a short, rounded spur, butterflies and moths proved to be the primary (potential) pollinators of the alpine Gymnadenia winkeliana.Our observations of the pollinator community within the restricted study area support our initial hypothesis that Gymnadenia winkeliana may be regarded as a putative generalist pollinator, capable of employing facultative allogamy when pollinators are available.Generalized pollination strategies have been associated with the extensive diversification of orchid species (Ray and Gillett-Kaufman, 2022).These observations are in line with previous studies that show that species of the former genus Nigritella Rich.may potentially switch from asexual (apomixis) to sexual reproduction (allogamy).Hence, their potential for employing both reproduction strategies provides them with greater reproductive flexibility, enabling them to adapt to and colonise diverse new habitats and overcome various reproductive challenges.

Potential Occurrence of Intra-and Intergeneric Hybrids
Despite the diverse pollinator community in the study area and frequent interactions between various insect species and sympatric orchids, no hybrids were observed between Gymnadenia winkeliana and other diploid (2n = 2x = 40) related species, like Pseudorchis albida (L.) Á.Löve & D.Löve, Pseudorchis albida subsp.tricuspis (Beck) E.Klein and Gymnadenia conopsea (L.) R.Br., the only representative of the genus Gymnadenia R.Br. in the habitat.While hybrids could potentially be produced, as pollinators were observed carrying Gymnadenia winkeliana pollinaria between sympatric individuals, there appears to be a post-pollination mechanism (either pre-or postzygotic) acting as a barrier, thus preventing cross-pollination and hybrid formation.One possible explanation could be the notable disparity in population sizes between the parental species.While Gymnadenia winkeliana was found in relatively large numbers, the population of Gymnadenia conopsea (L.) R.Br.consisted of only 2-4 mature or flowering individuals at the specific location.Additionally, although Pseudorchis albida subsp.tricuspis (Beck) E.Klein was more abundant in the habitat, there were considerable distances between its populations and those of Gymnadenia winkeliana, spanning approximately 30-50(60) meters.This spatial separation could act as a barrier to cross-pollination, especially in a habitat characterized by a specific microclimate, characterized by frequent rains, strong winds and low temperatures, which may limit the movement of pollinators over longer distances.
Consequently, it is important to note that the processes involved in the formation of such hybrids are still under investigation, and the possibility of their occurrence remains open.
Other highly morphologically distinctive members of the subgenus Nigritella Rich.present in Romanian Carpathians are the diploid (2n = 2x = 40) Gymnadenia carpatica (Zapał.)Teppner, E.Klein & Zag., a Romanian sub-endemic present in Eastern Carpathians, exclusively confined to Northern Romania and Ukraine (Figure 11A), and the tetraploids (2n = 4x = 80) Gymnadenia miniata (Crantz) Janch, a uniformly bright-red species (Figure 11B) and the dark-red to dark-brown Gymnadenia austriaca (Teppner & E.Klein) P.Delforge (Figure 11C) and Nigritella nigra subsp.bucegiana Hedrén, Anghel.& R.Lorenz (Figure 11D), all present in Southern and Eastern Carpathians.The study of the former genus Nigritella Rich.continues as ongoing work, with the list remaining open to potential new taxonomical additions, still waiting to be discovered within the vastness of the Romanian Carpathians, a region that remains relatively unexplored to date.nia winkeliana, spanning approximately 30-50 (60) meters.This spatial separation could act as a barrier to cross-pollination, especially in a habitat characterized by a specific microclimate, characterized by frequent rains, strong winds and low temperatures, which may limit the movement of pollinators over longer distances.
Consequently, it is important to note that the processes involved in the formation of such hybrids are still under investigation, and the possibility of their occurrence remains open.
Other highly morphologically distinctive members of the subgenus Nigritella Rich.present in Romanian Carpathians are the diploid (2n = 2x = 40) Gymnadenia carpatica (Zapał.)Teppner, E.Klein & Zag., a Romanian sub-endemic present in Eastern Carpathians, exclusively confined to Northern Romania and Ukraine (Figure 11A), and the tetraploids (2n = 4x = 80) Gymnadenia miniata (Crantz) Janch, a uniformly bright-red species (Figure 11B) and the dark-red to dark-brown Gymnadenia austriaca (Teppner & E.Klein) P.Delforge (Figure 11C) and Nigritella nigra subsp.bucegiana Hedrén, Anghel.& R.Lorenz (Figure 11D), all present in Southern and Eastern Carpathians.The study of the former genus Nigritella Rich.continues as ongoing work, with the list remaining open to potential new taxonomical additions, still waiting to be discovered within the vastness of the Romanian Carpathians, a region that remains relatively unexplored to date.

Sites Studied
The studies were conducted within the alpine areas of the Bucegi Natural Park ROSCI0013, a protected area IUCN category V (Protected Landscape, Law No. 5, 6.03.2000), part of the Natura 2000 site, covering Prahova, Dâmbovit , a and Brasov Counties, Southern Carpathians, Central Romania.The Park has an area of ca.32.663 ha/326.63km 2 , with the highest elevation (elev.) at Omu Peak of 2.505-2.514m a.s.l (above sea level) (see previous orchidological studies within the same protected area by [8,101] (Figures 1A-C and 2B,C).

Populations Studied
The Gymnadenia winkeliana population was first discovered at locus classicus, in 2005, during an orchidological expedition, containing approximately 50-55 individuals (information obtained from Dan Anghelescu, personal communication, 2005Anghelescu, personal communication, -2016)).Over time, the population size increased consistently, reaching approximately 80-100 individuals between 2012 and 2020, and approximately 120-130(140) individuals from 2020 to 2023.The initial population numbers may have been higher, considering that the areas were constantly utilized as cattle fields and a portion of the vegetation had already been destroyed by the grazing animals (Figure 1C, red dots and Figure 2A).

Extent of Occurrence (EOO)
The population was found to be spread over an alpine plateau (grassland) with an EOO of ca.2.5-5 km 2 , locus classicus GPS of 45

Morphological Measurements
Measurements of the vegetative and floral parts were made from living plants and fresh flowers.To describe this newly found population as comprehensively as possible, 175 morphological features were directly studied and measured from living plants and flowers.The morphological characteristics used for the study included most of the characteristics used previously [8,28].Special attention was focused on the characteristics that proved to have taxonomic significance, particularly those involving distinctive details in the morphology of the leaves, perianth, labellum and gynostemium.Only flowering parts of the plants were sampled, leaving the vegetative parts for persistence and continued growth.The 223 characteristics scored encompassed the morphology of every organ of the plant, cytology and breeding system.No organ was represented by fewer than three characteristics.The characteristics (listed in Table 1) described in detail are the roots (8), stem (9), leaves and bracts (36), inflorescence and flower (16), sepals and petals (17), labellum and spur (30), gynostemium (13), anther (13), pollinia and pollinarium (27), stigma (19), ovary (12), fruit (6), seed and embryo (15), chromosomes (2), flowering time and reproductive strategies (7).

Pollination Monitoring
Monitoring was conducted for a total of 5-6 h per day, mostly between June-July (2017-2023), when most of the flowers were in full anthesis.The observer (NA) initially positioned themselves approximately 2-3 meters away from the subjects, whether they were in groups or individual plants.Digital photographs and insect pollinators/visitors records were taken upon observing different insects patrolling or approaching the flowers.It is important to note that no insects were collected or harmed in any way during the study.A comprehensive list of insect pollinators and visitors is given in Table 2.

Explant Collection
Wherever possible, several bract-ovary-flower units were removed from a position approximately one-third to halfway from the base of the inflorescence (Figure 5C-E,I,J).This was performed to minimize the impact of the pink colour gradient and the decrease in flower size observed from the base to the apex of the inflorescence, a characteristic of this species.The removal of fewer flowers/inflorescences does not affect the viability of the plants, since the leaves and roots remain intact.The plants collected in the field were kept in vials with fresh water, at 4 • C, for approximately 14 h, before being submitted to stereomicroscopy for biometrical measurements.Immature seeds (from indehiscent fruit/seed capsules) were collected 5-6 weeks after the peak of antheses.Seed capsules were opened and fresh seeds were submitted to stereomicroscopy for biometrical measurements.

Stereomicroscopy
The flower-bract units were analysed and biometrically measured under a stereomicroscope, before and after dissection (for floral parts and reproductive organs biometry).

Maps
The map was created using ArcGIS Pro 3.1 software; the maps and elevation services were provided by the entities mentioned in the copyright [8]

Conclusions
This monophyletic group [9,46,114], which encompasses numerous recently evolved species, is currently undergoing an evolutionary radiation.This radiation is driven by a wide range of factors, including genotypic (such as genetic and epigenetic factors, genetic drift), phenotypic (ecophenotypic) and environmental influences (such as habitat alterations, climate fluctuations and the presence or absence of true pollinators and specific mycorrhizal associations).Hence, the remarkable ability of European vanilla orchids to exhibit diverse phenotypic responses to environmental demands has led to significant and rapid changes in the taxonomy of the genus Gymnadenia R.Br. in recent years, driven by the emergence of micro-endemic populations with varying reproductive strategies.Constantly evolving morphological adaptations to novel and isolated habitats are frequently documented, often sparking extensive discussion and making the newly emerged taxa the focus of attention.Consequently, in the last decade, the aggregate Gymnadenia R.Br. has become one of the most taxonomically complex and debated orchid genera in Europe.

Figure 4 .
Figure 4. Gymnadenia winkeliana N.Anghelescu, L.Balogh, M.Balogh & N.Kigyossy inflorescence details. (A-L) Details of various inflorescences.The inflorescences are densely packed, near-spherical to subcylindrical racemes (at full anthesis), exhibiting a distinctive gradient of pink hues, with white or pale pink basal flowers and pink to dark pink topmost flowers and buds.The specific colouration is highlighted as a main distinguishing characteristic of the species.(A,B,F-I) Inflorescences showing swollen basal ovaries, a typical sign of early apomixis onset.(L) Detail of the white-to-pale pink basal flowers of the inflorescences; the flowers are non-resupinate, chasmogamous, with purple-red pigmented bracts, longer than the flowers; on one of the flowers, a pollinarium may be seen, a sign of entomophily/allogamy (insect pollination).Photographs by Nora E. Anghelescu (A,B) 29 June 2020, (C) Nicoleta Kigyossy 29 June 2021, (D-J) 26-29 June 2021, (L) 3 July 2023, BNP, Romania.

Figure 4 .
Figure 4. Gymnadenia winkeliana N.Anghelescu, L.Balogh, M.Balogh & N.Kigyossy inflorescence details. (A-L) Details of various inflorescences.The inflorescences are densely packed, near-spherical to subcylindrical racemes (at full anthesis), exhibiting a distinctive gradient of pink hues, with white or pale pink basal flowers and pink to dark pink topmost flowers and buds.The specific colouration is highlighted as a main distinguishing characteristic of the species.(A,B,F-I) Inflorescences showing swollen basal ovaries, a typical sign of early apomixis onset.(L) Detail of the white-to-pale pink basal flowers of the inflorescences; the flowers are non-resupinate, chasmogamous, with purple-red pigmented bracts, longer than the flowers; on one of the flowers, a pollinarium may be seen, a sign of entomophily/allogamy (insect pollination).Photographs by Nora E. Anghelescu (A,B) 29 June 2020, (C) Nicoleta Kigyossy 29 June 2021, (D-J) 26-29 June 2021, (L) 3 July 2023, BNP, Romania.

Figure 10 .
Figure 10.Gymnadenia winkeliana N.Anghelescu, L.Balogh, M.Balogh & N.Kigyossy-Lepidopteran and Dipteran pollinators and visitors.(A-C,F,K) Boloria pales (Nymphalidae Rafinesque, 1815), Figure 10.Gymnadenia winkeliana N.Anghelescu, L.Balogh, M.Balogh & N.Kigyossy-Lepidopteran and Dipteran pollinators and visitors.(A-C,F,K) Boloria pales (Nymphalidae Rafinesque, 1815), Flowering time.The flowering time typically spans from late June to mid-July, based on field observations conducted from 2004 to 2023.Our field observations indicate that the plants often tend to synchronize their blooming, flowering simultaneously.The majority of flowering occurs during the hottest part of the summer season, typically in June and July, with the longest daylight hours in temperate Romania (Figures 2A, 3A-F and 4A-L).

4. 10 .
Digital Photographic EquipmentDigital images of individual plants and floral parts were taken using Nikon D3 and Nikon D850 camera bodies equipped with Nikon Micro NIKKOR 60 mm and NIKKOR 24.0-70.0mm lenses.Additional equipment included a Manfrotto Tripod and Litra Torches 2.0s.An adapted Helion FB tube was used for automated focus bracketing.The images were analysed using Adobe Photoshop ® CC 2024, Zerene Stacker Software, Version 2021-11-16[78].

Funding:
This work was supported by the Faculty of Horticulture, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Blvd, District 1, 011464 Bucharest, Romania.Data Availability Statement: Data are contained within the article.Acknowledgments: The authors cordially thank Coltoiu Alexandru (Director, Bucegi Natural Park Administration-APN, National Directorate of Forests-Romsilva) and Daniel Ungureanu (IT specialist of APN Bucegi) for granting the Research Permit No. 1887/CAN/22.07.2021-2023 to investigate the orchid flora present in the Bucegi Natural Park and for providing the detailed maps and geographical coordinates (Global Positioning System-GPS) of the greater Bucegi Natural Park area.We also thank Adrian Ruicănescu Ph.D., C.S.II, Institute of Biological Research, Cluj-Napoca (for Coleopterans identification), Bogdan Tomozei Ph.D., Professor at Ion Borcea Museum of Nature and Science, Bacău and Ioan Tăus , an, Ph.D., Lucian Blaga University, Sibiu (for Hymenopterans identification), Corneliu Pârvu, Ph.D., Grigore Antipa National Museum of Natural History, Bucharest and Melania Stan, PhD, Deputy Director, Grigore Antipa National Museum of Natural History, Bucharest (for Dipterans identification), Szekely Levente, PhD, entomologist, Bras , ov (for Lepidopterans identification), Bodgan Palade and Florin Palade for constructing the distribution map of Gymnadenia winkeliana.
Interest:The authors declare no conflicts of interest.