Epipactis bucegensis—A Separate Autogamous Species within the E. helleborine Alliance

A new species of Epipactis from Bucegi Natural Park ROSCI0013, Southern Carpathians, Central Romania is described. Three medium-sized populations of Epipactis bucegensis (65–70 individuals in total) were discovered in the south-eastern, subalpine area of the park. To properly describe and distinguish the newly found taxon from other Romanian Epipactis, 37 morphological characters were measured directly from living plants and flowers. Moreover, a detailed taxonomic treatment and description with corresponding colour photos and line drawings illustrations of the holotype are also included. Epipactis bucegensis is an obligate autogamous species that partially resembles Epipactis muelleri, from which it differs in the basal distribution of leaves on the stem (vs. median distribution); near-erect leaf posture (vs. horizontally spread, arched downwards); lanceolate–acuminate, yellowish-green leaves (vs. oval–elongate, vivid-green leaves); bipartite labellum lacking the mesochile (vs. tripartite labellum); crimson-red, wide, ovoid–elongated, flattened hypochile (vs. dark-brown to black roundish hypochile); triangular, white epichile with a sharply tapering apex (vs. heart-shaped, greenish-yellow epichile with obtuse, roundish apex); and two wide-apart, purple, pyramidal calli (vs. two closely placed, attenuated, mildly wrinkled, greenish-yellow calli). Epipactis bucegensis is easily distinguished from all other European Epipactis taxa by the bipartite, wide labellum that lacks the mesochile. In addition, information regarding its distribution (maps), habitat, ecology, phenology and IUCN conservation assessments are provided.


Introduction
Genus Epipactis Zinn, 1757 belongs to Tribe Neottieae Lindl., 1826, Subfamily Epidendroideae Lindl., 1821, Family Orchidaceae Juss., 1789. The generic name, Epipactis, originates in the ancient Greek word epipaktís, a name given for the first time by Greek philosopher and botanist Theophrastus of Eresos (ca. 371-ca. 287 B.C.E.) to a herbaceous plant that curdled milk, possibly a member of the highly poisonous, unrelated genera of invasive plants Helleborus (Family Ranunculaceae, known as Hellebores) and Veratrum (Family Melanthiaceae, known as the False Hellebores). Since Epipactis orchids have been associated with the poisonous, invasive Hellebores from ancient times, the generic vernacular name of this genus remains, to this day, the Helleborines [1].
In this paper, we describe and illustrate a new autogamous species within the E. helleborine alliance named Epipactis bucegensis. The first encounter with Epipactis bucegensis took place in July 2009 during an orchid field study in the south-eastern part of Bucegi Natural Park, Southern Carpathians ( Figure 1C, red dots). At first sight, in the harsh light of the melting-hot summer days, the plants looked rather like a peculiar group of yellowish, withered Epipactis helleborine (L.) Crantz, perfectly camouflaged among the brownish, grassy, surrounding vegetation. However, the most striking features were the elongated inflorescences bearing several inconspicuous, creamy-white, closed flowers hanging on the pendant, yellowish ovaries, a clear indication of an autogamous species, different from the common, allogamous Epipactis helleborine (L.) Crantz, which is sporadically found in the area. After a closer examination, which involved manually opening several flowers, we also noticed the unusual, unique structure of the nectarless labellum, which completely lacked the middle narrowing junction, a feature that differentiated it from any other Romanian Epipactis species. Furthermore, the absence of the viscidium and the crumbling, disintegrating pollinia reinforced our initial supposition of a distinct, autogamous taxon. Furthermore, in the summer of 2022, several expeditions to Bucegi Natural Park were made, and during these field trips, two new populations of the same taxon were discovered. The most distinctive morphological features proved to be highly preserved and consistent, the new specimens showing little to no At first sight, in the harsh light of the melting-hot summer days, the plants looked rather like a peculiar group of yellowish, withered Epipactis helleborine (L.) Crantz, perfectly camouflaged among the brownish, grassy, surrounding vegetation. However, the most striking features were the elongated inflorescences bearing several inconspicuous, creamy-white, closed flowers hanging on the pendant, yellowish ovaries, a clear indication of an autogamous species, different from the common, allogamous Epipactis helleborine (L.) Crantz, which is sporadically found in the area. After a closer examination, which involved manually opening several flowers, we also noticed the unusual, unique structure of the nectarless labellum, which completely lacked the middle narrowing junction, a feature that differentiated it from any other Romanian Epipactis species. Furthermore, the absence of the viscidium and the crumbling, disintegrating pollinia reinforced our initial supposition of a distinct, autogamous taxon. Furthermore, in the summer of 2022, several expeditions to Bucegi Natural Park were made, and during these field trips, two new populations of the same taxon were discovered. The most distinctive morphological features proved to be highly preserved and consistent, the new specimens showing little to no variation regarding the yellowish aspect of the plants, the creamy-white closed flowers and the nectarless labellum completely lacking the middle junction. Undeniably, the newly discovered populations confirmed, once more, the occurrence of a persistent, new species, well-established within the south-eastern area of the park. Consequently, we chose to formally describe this new taxon as Epipactis bucegiana, with the confidence that, in the years to come, more Plants 2023, 12, 1761 4 of 31 undiscovered populations will be revealed within the park's greater area. Additionally, we provide information about its geographical distribution, habitat, ecology, phenology and IUCN conservation status, together with illustrations and photographs based on living specimens (the holotype).

Sites Studied
The study sites were on wet to dry, calcareous substrates, next to deciduous to mixed woodland; altitude between 700-1100 m a.s.l. The populations occurred in sunny meadows and pasturelands, neighbouring margins of mixed forests covering subalpine slopes, close to urban sites ( Figure 1C, red dots).

Morphological Comparisons
Despite the modern molecular techniques, a quick and simple tool to recognize a taxon in field conditions is still required and thus, morphological comparisons prevail in plant identification [23]. Meanwhile, taking into consideration the great phenotypic plasticity of the genus, the macro-and micromorphological features that can be used in taxon delimitation should be carefully assessed. A detailed comparison emphasizing the most significant morphological characters that distinguish Epipactis bucegensis from the related species is shown in Table 1

Morphological Distinctness of Epipactis bucegensis
Epipactis bucegensis is morphologically comparable to the autogamous Epipactis muelleri Godfery and the allogamous Epipactis helleborine (L.) Crantz, but significantly differs from these species in several main characteristics of the vegetal and floral parts (Figures 5-8).
The lowermost leaf size and shape are species-specific features and thus pivotal in species delimitation/separation within the Epipactis genus [5,7,9]. As such, the lanceolateelongated, tapering Epipactis bucegensis basal leaf, different from the characteristic roundish-oval basal leaf of Epipactis helleborine (L.) Crantz, clearly differentiates/separates the two taxa as separate species. Epipactis bucegensis leaves' distribution on the stem (phyllotaxis) is mainly basal, very different from the middle-stem distribution characteristic to Epipactis muelleri Godfery and Epipactis helleborine (L.) Crantz. The leaf posture is spreading to erect, sheathing to a subtended angle of ca. 30° relative to the stem, differentiating it from Epipactis muelleri Godfery, in which the elongated, arched leaves spread horizontally, curving downwards. The leaf shape is elongate-lanceolate, acuminate, tapering at the tip, vs. the ovoid-elongate, acuminate leaves of Epipactis muelleri Godfery and the broadly ovoid to ovoid-elongated, horizontally spread leaves of Epipactis helleborine (L.) Crantz.

Morphological Distinctness of Epipactis bucegensis
Epipactis bucegensis is morphologically comparable to the autogamous Epipactis muelleri Godfery and the allogamous Epipactis helleborine (L.) Crantz, but significantly differs from these species in several main characteristics of the vegetal and floral parts (Figures 5-8).
The lowermost leaf size and shape are species-specific features and thus pivotal in species delimitation/separation within the Epipactis genus [5,7,9]. As such, the lanceolateelongated, tapering Epipactis bucegensis basal leaf, different from the characteristic roundishoval basal leaf of Epipactis helleborine (L.) Crantz, clearly differentiates/separates the two taxa as separate species. Epipactis bucegensis leaves' distribution on the stem (phyllotaxis) is mainly basal, very different from the middle-stem distribution characteristic to Epipactis muelleri Godfery and Epipactis helleborine (L.) Crantz. The leaf posture is spreading to erect, sheathing to a subtended angle of ca. 30 • relative to the stem, differentiating it from Epipactis muelleri Godfery, in which the elongated, arched leaves spread horizontally, curving downwards. The leaf shape is elongate-lanceolate, acuminate, tapering at the tip, vs. the ovoid-elongate, acuminate leaves of Epipactis muelleri Godfery and the broadly ovoid to ovoid-elongated, horizontally spread leaves of Epipactis helleborine (L.) Crantz.  The leaf colour, especially in young individuals, is yellowish to yellowish-green, different from the light-to deep-green leaves of the compared species (Figures 2B and  4A,C,E). The colour of the sepals and petals is white to whitish-yellow, vs. the whitishgreen to greenish-yellow tepals of Epipactis muelleri Godfery (Figures 2A-E, 4B, 5A and  7A,B).
Epipactis bucegensis's unique labellum structure represents its main distinctive feature, making it easily distinguishable, not only from Epipactis muelleri Godfery, but from all other European Epipactis species (Figures 2D,E, 3A,F,I,M, 5A and 7A). Specifically, the labellum is bipartite, formed of only two parts, the hypochile and epichile, with a completely absent mesochile. By comparison, Epipactis muelleri Godfery and Epipactis helleborine (L.) Crantz have tripartite labella, with a well-defined mesochile, the narrow junction between the hypochile and epichile. The complete absence of the mesochile is the most distinctive feature of the species (Figures 2D,E, 3I,M, 4A,C,E and 7A,B,D,E). The hypochile shape is characteristically wide, ovoid and flattened, vs. the orbicular, cup-like, deep la- The leaf colour, especially in young individuals, is yellowish to yellowish-green, different from the light-to deep-green leaves of the compared species ( Figures 2B and 4A,C,E). The colour of the sepals and petals is white to whitish-yellow, vs. the whitish-green to greenish-yellow tepals of Epipactis muelleri Godfery (Figures 2A-E, 4B, 5A and 7A,B).
Epipactis bucegensis's unique labellum structure represents its main distinctive feature, making it easily distinguishable, not only from Epipactis muelleri Godfery, but from all other European Epipactis species (Figures 2D,E, 3A,F,I,M, 5A and 7A). Specifically, the labellum is bipartite, formed of only two parts, the hypochile and epichile, with a completely absent mesochile. By comparison, Epipactis muelleri Godfery and Epipactis helleborine (L.) Crantz have tripartite labella, with a well-defined mesochile, the narrow junction between the hypochile and epichile. The complete absence of the mesochile is the most distinctive feature of the species (Figures 2D,E, 3I,M, 4A,C,E and 7A,B,D,E). The hypochile shape is characteristically wide, ovoid and flattened, vs. the orbicular, cup-like, deep labellum of Epipactis muelleri Godfery. The hypochile inner wall is shiny, dry, crimson-purple-coloured and unusually wrinkled, completely different from that of Epipactis muelleri Godfery, which is deep, cup-shaped, roundish, shiny, smooth, blackish-brown and mildly nectar-secreting. The epichile is reduced, triangular, flat, smooth and tapering, vs. the wide-obtuse, deeply wrinkled epichile of Epipactis muelleri Godfery and Epipactis helleborine (L.) Crantz. Its colour is invariably bright white vs. the greenish-yellow epichile of Epipactis muelleri Godfery. The two basal calli found at the base of the epichile are also highly specific, pyramid-shaped, tooth-like, prominent, wide apart, crimson-purple-coloured and smooth/non-wrinkled, vs. the significantly wrinkled and attenuated, greenish-yellow (very rarely pale-pinkishwashed) calli of Epipactis muelleri Godfery (Figures 3I, 4B, 5A and 7A,B).
The gynostemium (column) is specific, with the anther significantly angled relative to the stigma (typical of an obligate autogamous species), differentiating it from the erect gynostemium of the allogamous Epipactis helleborine (L.) Crantz. The stigma shape is rectangular, wider than long, bilobed, roof-like and entirely flat, vs. the quadrangular, bilobed, deeply V-shaped and concave one in Epipactis muelleri Godfery.
Epipactis bucegensis can be distinguished from Epipactis helleborine (L.) Crantz by its modified anther morphology associated with its pollination strategy, obligate autogamy ( Figures 6-8). The rostellum and viscidium are completely absent, which distinguishes Epipactis bucegensis from the allogamous Epipactis helleborine (L.) Crantz, in which the rostellum and viscidium structures are well-developed and functional ( Figures 5A,B,E,F and 6A,B,E,F). The clinandrium is absent, the highly friable pollinia lying free in the anther, crumbling onto the upper part of the stigmatic surface, vs. the more compact pollinia of Epipactis helleborine (L.) Crantz, enclosed in the clinandrium and well-separated from the stigmatic cavity by the roof-like rostellum ( Figures 5A,B,E,F and 6A,B,D,E).
The purple-pigmented flower pedicel base of Epipactis bucegensis clearly distinguishes it as a separate species from Epipactis muelleri Godfery, in which the pedicels' bases are yellowish to light-green ( Figure 8A-D,F). The pedicel-base pigmentation is an essential morphological feature (key) in Epipactis species delimitation/separation [5,7]. The fruit of Epipactis bucegensis is also specific, highly distinct from Epipactis muelleri Godfery. In mature stages, it is pear-shaped, dark-green, purple-washed and strongly ridged on the surface, vs. the elongated, light-green, smoother-surfaced fruit of Epipactis muelleri Godfery ( Figure 8A,B). Epipactis bucegensis was also closely compared to the European autogamous species described in detail in the comprehensive, abundantly illustrated database of the Arbeitskreis Heimische Orchideen Bayern e.V. [10], but no similar taxon was observed. The most important feature that distinguishes Epipactis bucegensis from all other European Epipactis taxa is the bipartite, wide labellum that totally lacks the mesochile ( Figures 3I, 4B, 5A and 7A). Therefore, given the significant morphological distinction, its reproductive isolation and its consistent establishment in Bucegi Natural Park, we consider Epipactis bucegensis to be a separate (obligate) autogamous species within the Epipactis helleborine alliance.

Morphological Changes to Autogamy
Orchids of the genus Epipactis that transition from allogamy to autogamy have to go through various overall morphological changes. To enable autogamy, the pollen should be able to reach the stigma. This is achieved by various adaptations of the flower morphology [30]. The transition from chasmogamous to cleistogamous flowers and some modifications in the architecture of the gynostemium and pollinia structure enable the flowers to switch the pollination strategy from allogamy to (near-) obligate autogamy [31,32]. Allogamous Epipactis species attract their specific pollinators with several floral signals, such as flower shape, coloration and complex floral scents (floral volatiles), and reward them with copious amounts of nectar [33]. Nectar is mainly secreted in the concave basal part of the labellum, known as the hypochile (Figures 4D, 5E and 7D). The transition from allogamy to autogamy/cleistogamy is regarded as a more efficient way for the plant to use its energetic/nutritional resources [34].
Epipactis bucegensis is an obligate autogamous species that does not require the presence of pollinators, showing all the particular morphological transformations of a typical selfing species. Its flowers are cleistogamous, pendant, scentless and inconspicuously coloured. By blocking the anthocyanin pigment synthesis, the flowers become whitishcreamy to yellowish-green, perfectly camouflaging the plant against the brownish-greenish background of the hot summer, sun-burnt, grassy vegetation characteristic of its preferred habitat (Figures 2 and 4A,B; note: in Figure 2D,E, for the purpose of this study, some of the flowers were hand/manually opened to clearly show the morphology of the floral parts).
However, despite being an obligate autogamous species, Epipactis bucegensis has not lost the ability to produce faint traces of floral nectar (Figures 2A-D, 3B,F,I,J,M, 4B, 5A and 7A,B). The finding was rather surprising since orchids commonly use nectar to attract their pollinators. We found only minute droplets of nectar that accumulated inside the hypochile of the one to two topmost, young flowers ( Figure 2E). Minute nectar production was reported several times in other autogams, such as Epipactis albensis Nováková and Rydlo [17,33,35], Epipactis muelleri Godfery [11] and Epipactis leptochila (Godfery) Godfery [30]. These obligate autogams are relatively young species that recently diverged from within the evolutionarily active Epipactis helleborine alliance [36]. Moreover, recent studies showed that the chemical composition of Epipactis albensis Nováková and Rydlo nectar and scent is partially similar to those of the closely related allogamous species Epipactis helleborine (L.) Crantz, further proving its evolutionary origin [33]. The above examples constitute indicative examples of species that transition from ancestral allogamous, insect-pollinating species to obligate autogamy. While still retaining some early features, such as nectar and scent production, these orchids became obligate autogamous/cleistogamous, making insects' visits nearly impossible [30]. The synthesis of floral attractants or stimuli, i.e., olfactive (scent, odours), food (nectar, food bodies, exudates) and visual stimuli (pigments, colours, shapes, sizes), is highly energy-costly for the plants [37,38]. Once their production is terminated/ceased, the spared nutrients are used by the plants to produce higher numbers of mature, fertile seeds, crucial for their survival and proliferation, a stage regarded as particularly difficult for newly emerged taxa (such as Epipactis bucegensis) in the full process of colonising new, nutrient-poor niches [39].
Further, the gynostemium also suffered several morphological transformations. Similar to other autogams, the gynostemium of Epipactis bucegensis completely lost the apical structure of the column, termed the clinandrium or anther-bed ( Figures 6A,B and 7C,D) an indicative characteristic of autogamous species [40]. In allogamous species, this spacious, hollow structure, situated above the stigma, houses the pair of pollinia, preventing the pollen tetrads from falling off the anther ( Figures 5E,F, 6E,F and 7A,B). At dehiscence, due to the lack of the clinandrium, the pollinia, which lay freely in the anther, are projected forwards, falling onto the underlying stigmatic cavity (Figures 3B,C,E-G, 5B, 6A,B and 7A,B). The sessile anther angles even more relative to the stigma, further inclining the pollinia, which can thus easily contact the stigmatic surface ( Figures 3C,G, 5B, 6B and 7A,B). The same pollination strategy is employed by other autogams, e.g., Epipactis muelleri Godfery ( Figures 4C,D,  5C,D and 6C,D).
Additionally, the pollinia of Epipactis bucegensis gradually lost coherence and became more friable (Figures 3H, 5B, 6A,B and 7C), disintegrating into individual tetrads or groups of tetrads [28,39]. More compact pollinia, e.g., the pollinia of Epipactis helleborine (L.) Crantz ( Figures 5E,F, 6E,F and 7D,F), prevent the pollen from falling on the stigmatic cavity [41]. When the pollinia are less coherent, the pollen grains crumble on the stigmatic surface, enabling rapid self-pollination [42]. In the case of Epipactis bucegensis, the friability of pollinia is also environmentally dependent. Quite often, external factors, such as high temperatures, humidity and air currents, were reported to influence their friability [43]. In Romania, in July, the outside temperatures may reach 38-40 • C, which causes the pollinia to expand and become even more friable. Apart from the external factors, the flowers hang on fairly long and flexible pedicels, very sensitive to any externally generated movements, such as wind or water drops, which may swing the flowers in all directions. Such movements further increase the disintegration of the mealy pollinia, which crumble onto the viscous stigmatic cavity, situated just below the anther.
Selfing in Epipactis bucegensis is also efficiently promoted by the complete lack of a rostellum, the swollen apical part of the median stigmatic lobe [44], which is well-developed in allogamous Epipactis species (Figures 5F, 6E,F and 7E). According to Uphof (1968), 'a characteristic of the cleistogamic orchid flower is a very rudimentary rostellum or its absence' [45]. In allogamous Epipactis taxa, a well-developed rostellum creates the most important physical barrier between the male/pollinia and female/stigma parts of the flower, preventing selffertilization [2,46,47]. In most self-pollinated orchids, however, this structure either does not develop, as in Epipactis bucegensis ( Figure 6A,B), or, as in Epipactis muelleri Godfery ( Figures 5C,D and 6C,D) it develops incompletely or sometimes disintegrates during flowering [48]. An important feature in autogams is that, in the absence of the rostellum, the stigmatic cavity usually becomes more active and hypersecreting, being covered in abundant, viscous stigmatic exudate. This is easily observed in Epipactis bucegensis, in which the stigma and, in particular, the lateral prominent stigmatic lobes are heavily loaded with viscous, translucid stigmatic exudate ( Figure 2E). Just after the impregnation of the pollen grains with the stigmatic secretions, the pollen tetrads start to germinate, producing elongated tubes that grow, fertilizing the ovules ( Figures 5B, 6A-D and 7A,B). The pollinia are thus fixed in the anther, immobile, continuously shedding tetrads, a feature that can be observed in many autogamous species [49]. Robatsch (1983) estimated that 60% of Epipactis orchids are autogamous, characterized by having powdery pollen that falls onto the stigma [50,51] due to degeneration of the rostellum and relatively low nectar and odour production. In cross-pollinated species, the tip of the rostellum produces adhesive substances, forming a viscidium [52]. In allogamous, insectdependent Epipactis orchids, the viscidium is a protruding sphere-like extension composed of a milky, adhesive liquid, surrounded by a viscidial membrane ( Figures 5E,F, 6E,F and 7D-F(a-c)), which connects the viscidium to the pollinarium [36,44]. The main role of the viscidium is to adhere to the pollinators' bodies and dislodge the pollinia from the anther during pollination ( Figure 7F(a,b)). The presence of a large, viscous viscidium ensures that the pollinia are removed by pollinators and hence, the level of autogamy is decreased.

Pollination Monitoring
True Epipactis pollinators are usually large, strong insects capable of carrying the heavy load of pollinia. Our observations included various hymenopterans-wasps (family Vespidae), bees (family Apidae), bumblebees (mainly genus Bombus) and ants (family Formicidae); coleopterans-beetles (Cerambycidae and Oedemeridae families); and large dipterans-forest flies (family Anthomyiidae). They usually feed on copious amounts of nectar secreted by allogamous Epipactis species such as Epipactis helleborine (L.) Crantz, Epipactis purpurata Sm., Epipactis distans Arv.-Touv. and Epipactis atrorubens (Hoffm.) Besser [19]. In Epipactis bucegensis, the viscidium is not formed as a consequence of the absence of the rostellum (Figures 6A,B and 8B). Similarly, the rostellum is absent in Epipactis muelleri Godfery ( Figures 5D and 6C,D). Thus, the complete lack of the rostellum-viscidium structure(s), accompanied by the friable pollinia and hypersecreting stigma, resulted in very efficient self-pollination, consequently reducing the chances of pollen being transported by insects. As a result, the cleistogamous flowers of Epipactis bucegensis self-pollinate during the early stages or even before anthesis (in the bud stages). This was confirmed by the fact that, during the 10-12 days of field research, we did not observe any true pollinating insects visiting the flowers of Epipactis bucegensis. Nevertheless, the flowers were accidentally visited only by sporadic small forest flies of the family Drosophilidae ( Figure 4B, white arrow) and red ants, Myrmica rubra (family Formicidae, Figure 8A, red arrow). These random visitors are only food foragers, searching for nectar or floral exudates during their visits. They are not true orchid pollinators, since they are too small to carry or displace the heavy pollinia from the anther. In one instance, a small female spider ( Figure 8A, white arrow) was observed to reside in one of the inflorescences, using it as a hunting site for its small dipteran prey. Spiders (order Araneae) are the most common predators in orchids, found to inhabit the inflorescences of many orchid species, successfully preying on their pollinators [53].
Thus, the inconspicuously coloured, nectarless, scentless, cleistogamous flowers of Epipactis bucegensis show all the characteristic features of a typical obligate autogam capable of forming healthy, new populations, completely independent of the presence of pollinating insects. Nevertheless, autogamy is rarely absolute. There is always a chance that an insect of a suitable size, usually a food forager, either a true pollinator or a visitor, occasionally visits the nearly closed (cleistogamous) flowers of Epipactis bucegensis. Because the species does not produce a viscidium, even when the flowers are penetrated by insects, the pollinia do not attach to their bodies. Instead, due to the insects' disturbance and movements, the pollinia disintegrate even more, spreading onto the stigmatic surface, and thus, selfpollinating the flowers.
The early swelling of the ovaries is also a clear indication of early autogamy [54][55][56][57]. Even before the topmost flowers reach maturity, the basal ovaries are already swollen, while still keeping the withered flowers hanging on the capsules. Within 2-5 days, almost all ovaries develop into dark-green, purple-tinted, pear-shaped, swollen fruits (Figures 2A,B and 8A,B). The fruit set is very high, up to 90-98% (in 65 counts), a characteristic of autogamous species. In a few individuals, the upper 1-2 flowers remain non-self-pollinated, being eventually aborted by the plant. Once the fruits start to swell, the initial yellowish-green colour of the ovaries and leaves gradually changes to dark green ( Figure 8A,B). This indicates a significant increase in the photosynthetic activity of the plants, which start to produce higher amounts of carbohydrates to accomplish the maturation of the fruits and seeds, thus assuring their successful reproduction and proliferation. Similar quick and efficient self-pollination strategies were observed in other autogamous Epipactis species, such as Epipactis muelleri Godfery, Epipactis albensis Nováková and Rydlo, Epipactis leptochilla (Godfery) Godfery and Epipactis phyllanthes G.E.Sm. [28,29,36,58].

Active Speciation within the Epipactis Genus
Epipactis is regarded as an evolutionarily young genus that, recently, has undergone a rapid process of diversification and speciation [23,59], with numerous new (mostly) autogamous species being described. According to Delforge (2006), during the last glaciation, these species had their distribution restricted to the south, to the Iberian, Italian and Balkan peninsulas, as well as the Caucasus. With the amelioration of the climate, which began at around 10,000 B.C.E, the beechwoods moved slowly northwest, reaching Scandinavia at around 500 C.E. This recent arrival in mid-Europe may explain why Epipactis seems to be in the process of evolutionary radiation and why the taxonomic treatment of the genus is rather challenging [5] Based on extensive phylogenetic analyses, it was suggested that the newly emerged, near-obligate autogams had repeatedly radiated across Europe from within the more widespread, putative universal ancestral species, the predominantly allogamous Epipactis helleborine sensu stricto (s.s.). According to Sramkó et al. (2019), Epipactis helleborine (L.) Crantz is, most probably, the direct ancestor of at least ten recently derived species, the majority of them near-obligate autogams, such as Epipactis leptochila Godfery) Godfery, Epipactis greuteri H.Baumann and Künkele, Epipactis muelleri Godfery, Epipactis albensis Nováková and Rydlo and Epipactis dunensis (T.Stephenson and T.A.Stephenson) Godfery. In evolutionary terms, these facultative/near-obligate autogams were supposed to have undergone a fairly recent, rapid separation from their ancestral genetic background [36]. Authentic speciation events can lead to the formation of autogams from allogams, although autogams are believed to constitute evolutionary dead-ends, no autogam ever being able to generate further autogamous species, as reported previously [23,29,[60][61][62][63]. Consequently, this excludes the possibility of an eventual radiation/emergence of Epipactis bucegensis from obligate autogams, such as Epipactis muelleri Godfery. Nevertheless, further detailed phylogenetic analyses are needed to elucidate the potential direct ancestral species of Epipactis bucegensis, the time of radiation and its phylogenetic relationships within the aggregate. As such, the Epipactis helleborine alliance represents an example of an active evolutionary clade, within which speciation events have occurred comparatively recently, mainly through transitions from allogamy to autogamy [24,26,64].
It is well-known that self-compatible Epipactis orchids are well adapted to switch from allogamy to autogamy, depending on the degree of the environmental factors' adversity, which may accelerate the process [2,36,47]. Thus, the natural pressure imposed by the external factors may accelerate this transition process, causing autogamy to occur with increasingly high frequency in successive flowering seasons, ultimately leading to genetic drift, i.e., the change in the frequency of an existing gene variant (allele) in a population due to random chance [65], also known as allelic drift or the Wright effect [66]. There are many examples of species that can act as both cross-pollinating (pollinator-dependant) and autopollinating, depending on various external factors of their natural habitats. Thus, even in the obligately allogamous species, autogamy was shown to incidentally take place [31,33,67]. Both autogamous and allogamous flowers within the same Epipactis helleborine (L.) Crantz plant were reported several times [3,42,50,68,69]. Additionally, it was reported that, as an adaptation to extreme conditions, obligate allogams, such as Epipactis helleborine ssp. neerlandica (Verm.) Buttler and Epipactis helleborine subsp. orbicularis (K.Richt.) E.Klein (now Epipactis distans Arv.-Touv.), can change their mode of pollination from allogamy towards autogamy [54]. In temperate regions, they are allogamous and well-visited by insects [35,70]. However, in xerophilous regions, they may become facultative autogams even before anthesis [54][55][56][57]. Therefore, the actual pollination syndrome or the reproductive strategy can be significantly influenced by floral ontogeny (age of the flowers), environment (temperature, high or low humidity, drying winds, etc.) or both [30,61,62]. Nevertheless, the evolutionary (morphological) transition from obligate allogams to obligate autogams is the result of a combination of developmental genetic, epigenetic and ecophenotypic factors, as a consequence of both prolonged natural selection pressure and genetic drift [36].
It must be mentioned that the evolutionary shift from cross-fertilisation to self-fertilization is one of the most frequent evolutionary transitions in plants. It is believed that autogamy is employed by approximately 10-15% of flowering plants [71] as an adaptation to growing in harsh, unfamiliar habitats where, usually, the specific pollinating insects are lacking [31]. There have also been numerous reports of autogamy in the orchid family [72]. Among the temperate orchids, apart from the Epipactis genus, self-pollination (facultative and/or obligate) has been found in several other genera such as Ophrys L., Pseudorchis Ség., Neottia Guett., Cephalanthera Rich., Chamorchis Rich. and Corralorhiza Gagnebin [19,39,73]. The more extreme the conditions in which an orchid grows (biotope, habitat and/or climate changes, presence/absence of pollinators, etc.), the higher the chances that it will turn towards autogamy as a survival strategy. Anthropogenic factors, mainly the destruction and loss of the original habitats (agriculture, urban expansions, deforestation, etc.), leaving only small suitable patches for the orchids, probably also contributed to the switch of pollination mode and reproductive strategy [30]. Regardless of the presence or absence of pollinators, independence from insects offers orchids an opportunity to conquer new habitats, assuring unconditional, certain reproductive success [71,[74][75][76]. Shady woodlands with comparatively impoverished ground floras, where pollinator visits are likely to be less frequent, are the preferred habitats of most of the autogams. Hence, the increased ability of self-pollinating orchids to colonise new ecological niches may explain the large geographic area that the newly formed autogamous Epipactis species can occupy [36].

Inbreeding-Friend or Foe?
In nature, most plant and animal species have evolved various mechanisms to avoid inbreeding. Inbreeding produces increased homozygosity of recessive partially deleterious mutants and by chance in small populations, such as isolated populations of autogamous plants, these alleles can become fixed [77]. Repetitive autogamy leads to population inbreeding depression, generally expressed by an increased frequency and accumulation of recessive lethal or mildly deleterious mutations. Consequently, the individuals experience significantly reduced viability and fecundity, which ultimately leads to a sudden decline in population numbers [75]. In the early 19th century, Darwin argued that outcrossed offspring of plants are usually fitter and better adapted to survive than those produced by self-fertilization [78,79]. He considered that flowering plants evolved well-adapted features to enable outcrossing, thus avoiding inbreeding depression caused by selfing, as the predominant mode of reproduction [80][81][82].
Despite the commonly believed disadvantages of inbreeding, studies/observations of dominantly allogamous species vs. the dominantly autogamous species within the Epipactis section revealed that there is no noticeable deleterious effect of selfing in the recently formed autogams [39]. According to Sramkó et al. (2019), inbreeding depression in Epipactis lineages may be either counterbalanced by outbreeding or cleared out from the autogams by natural selection that acts on the unmasked deleterious recessives. At the same time, the average distributional areas or population sizes/counts proved not to be significantly different between the already established allogams and recently radiated autogams. Thus, it was suggested that the great genetic diversity of Epipactis helleborine (L.) Crantz, together with its greater phylogenetic range, enabled it to function rather successfully as a source of the future novel (autogamous, cleistogamous) species [36].

The Role of Cleistogamy in Active Speciation
In the case of geographically localized populations that suffer genetic isolation from their progenitors, active speciation may take place, generating new lineages, mostly with a tendency towards producing cleistogamous flowers, i.e., flowers that do not open and are self-fertilized in the bud [2,42], a tendency strongly expressed by Epipactis bucegensis. Cleistogamy prevents the access of insects, invariably leading to obligate autogamy. Nevertheless, some authors further suggested that this transition in the breeding system was unidirectional, the allogams never arising from autogams, which makes the autogamous Epipactis species potentially evolutionary dead-ends [29,[60][61][62]. Varying degrees of autogamy were reported in several other groups, e.g., the Spiranthes sinensis (Pers.) Ames species complex, in which autogamy has contributed to intraspecific morphological variability and, in some instances, speciation [63]. A typical feature of obligately self-pollinating taxa is that the newly emerged group(s) are highly homogenous, while there are considerable differences between different populations [27][28][29]47]. Squirrell et al. (2002) noted that: 'With each generation of complete selfing, homozygosity increases by 50%. In this fashion, a large genetic distance arises rapidly between progenitor and derivative species' [28]. This has led to an increase in speciation, mostly represented by local (micro)endemic forms, demonstrating the plasticity of the genus and the dynamics of its evolution [11].
The cleistogamous, micro-endemic Epipactis bucegensis may represent an example of a recently genetically separated autogam that eventually colonized new habitats and successfully reproduced and proliferated, independent of the pollinators' presence. Discovered 14 years ago, Epipactis bucegensis proved to form stable, large, healthy populations in the south-eastern part of Bucegi Natural Park, at the same time presenting highly preserved specific characters that showed little to no variability. The essential morphological features (keys) in Epipactis species separation, such as the creamy-white, pendant, cleistogamous flowers; the unique structure of the labellum lacking the mesochile; the distinctive pyramidal/triangular purple-coloured labellar calli; and the purple-pigmented base of the pedicel and fruit represent species-specific characters, which significantly distinguish it from the related Epipactis taxa.
Therefore, our thorough approach strongly supports the recognition of Epipactis bucegensis as a morphologically, phenologically and ecologically distinct species within the Epipactis helleborine aggregate.

Sites Studied
The studies were conducted in three subalpine areas within the Bucegi Natural Park, a protected area included within Natura 2000 site ROSCI0013, IUCN category V (Protected Landscape, Law No. 5, 6.03.2000), covering Prahova, Dâmbovit , a and Bras , ov Counties, Southern Carpathians, Central Romania, with an area of ca. 32.663 ha/326.63 km 2 and the highest elevation (elev.) at Omu Peak of 2505-2514 m a.s.l (above sea level).

Populations Counts
The first population of Epipactis bucegensis, counting a total of 5-6 individuals, was discovered by NEA on 26 July 2009 in Prahova County, Bucegi Natural Park, elev. 810-960 m a.s.l. Its occurrence was subsequently monitored in 2010 and 2011, counting 3-4 and 6-7 individual plants, respectively. Several digital photographs were taken, but neither detailed measurements nor formal descriptions were performed at the time. Unfortunately, further monitoring of the first Epipactis bucegensis population was not possible as the area was destroyed and most of the present flora was lost due to real estate development. Nevertheless, on 17 July 2022, during a botanical field study, two new populations were discovered by LB and MB in the south-eastern part of the park, in Dămbovit , a County, elev. 820-980 m a.s.l. Together, the two newly discovered populations contained a total of ca. 60-75 individuals (ca. 45-55 and 10-15 individuals/population). The plants were found occurring individually or in groups of 2-6 siblings. The initial population numbers might have been higher since the areas were used as cattle fields and part of the vegetation was already destroyed by the grazing animals.

Extent of Occurrence (EOO)
The populations were found growing nearby, at a distance of 3-5 km, with an EOO of ca. 10-15 km 2 each ( Figure 1C, red dots).

Morphological Comparisons
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, a total of 117 morphological characters were compared, out of which 37 morphological characters were measured directly from living plants and flowers. The morphological characters used for the study included most of the characters used previously [83]. Special attention was given to the characters that proved to be taxonomically informative and those that involve the differentiating details in the morphology of the leaves, gynostemium, labellum, pollinia, ovary and fruit. The measurements are examples of the new taxon, Epipactis bucegensis, and its related species, the autogamous Epipactis muelleri Godfery and the allogamous Epipactis helleborine (L.) Crantz.

Pollination Monitoring
Monitoring was conducted for a total of 4-6 h per day, between 17 and 21 July 2022 when most of the flowers were in full anthesis. Nevertheless, the cleistogamous flowers were never fully opened; hence, pollinator presence/attraction was rather scarce. The observer (NA) was initially located approximately 2-3 m from the subjects (groups or individual plants). Once various insects were observed to patrol and/or approach the flowers, they were (intended) to be recorded in digital photographs (note: no insects were collected or harmed in any way during the study).

Digital Photographic Equipment
Digital 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 24.0-70.0 mm 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 2023, Zerene Stacker Software, Vers.2021-11-16 [84].

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.

Conclusions
Autogamy is a common reproductive mechanism used by many species of flowering plants, including the complex orchid genus Epipactis, as an adaptation to colonise new habitats. This monophyletic clade, with numerous, mostly newly evolved autogamous species is presently undergoing evolutionary radiation driven by a large spectrum of genotypic (genetic and/or epigenetic factors, genetic drift), phenotypic (ecophenotypic) and environmental factors (habitat changes, climate changes, presence/absence of true pollinators and specific mycorrhizae). Ancestor species, such as Epipactis helleborine s.l., have been shown to have, rather frequently and recently, generated many isolated, local autogamous (often cleistogamous) forms. These, generally viewed as examples of incipient speciation from within the parental genetic background, are often as widespread and ecologically successful as allogams, a result of a high level of initial/incipient genetic variation [29], which gives them the potential to evolve into new taxa [36].
Thus, due to the great phenotypic plasticity of the genus in response to environmental requirements, the formation of micro-endemic populations with different reproductive mechanisms led, in recent years, to noticeable, fast changes within the taxonomy of the Epipactis genus [85]. Novel morphological adaptations to new, isolated habitats are constantly described, often making the recently emerged taxa the subject of much discussion [60,86] and Epipactis one of the most taxonomically complex and dynamic orchid genera in Europe.
Cytogenetics: Chromosome numbers are very variable within the genus, with a basic chromosome number x = 10 [84]. The species might be similar in chromosome number to its relative Epipactis muelleri Godfery 2n = 4 [5]; nevertheless, this still needs to be determined.
Flowering period: The species has been observed exclusively in its natural habitat flowering from the beginning to mid-July. The flowers' longevity is very short to absent, self-pollination/autogamy occurring before the anther dehiscence, while still in the bud stages (cleistogamy). Nevertheless, we noticed closed flowers still hanging on the developing/swollen fruit capsules for several days before showing clear signs of flower senescence (flower wilting or shedding of the floral parts).
Habitat: Epipactis bucegensis prefers a cool subalpine climate, with moderate humidity, in full sun to partial shade, on dry to moist, neutral to calcareous/alkaline substrates. It also grows in open woodland, next to forest edges, in mixed (deciduous and coniferous) forests, grasslands, shrublands and anthropogenic habitats, such as rural and urban roadsides, lawns or private estates.
Ecology: Individuals of the species have been found occurring either as isolated adult plants, separated by a distance of ca. 10-30 m, or aggregated, forming small-to medium-sized groups (usually n < 10) composed of several siblings and one to three adult plants. Our field observations suggest that plants usually synchronize their blooming, most of them flowering during the hottest summer season, which usually corresponds to the subalpine areas of the park may lead to discovering new populations of Epipactis bucegensis. In recent years, Bucegi Natural Park proved to harbour undiscovered taxa, such as the newly discovered Nigritella nigra subsp. bucegiana Hedrén, Anghel. and R.Lorenz, subsp. nov. [87]. At the same time, our future research includes several similar habitats outside the Bucegi Mountains Natural Park protected area that may be suitable to Epipactis bucegensis occurrence, since they are important biological reserves for threatened species [88]. It must, however, be emphasized that this micro-endemism is restricted to an area subject to rapid deforestation due to abrupt urban expansion and increased anthropogenic activities, such as cattle farming, agriculture, tourism and real estate development. According to the EU Biodiversity Strategy (2020-2050), which works towards restoring natural environments by stopping the destruction of ecosystems and loss of biodiversity [89], effective measures should be implemented in order to protect and preserve these fragile habitats that harbour rare endemic species. Consequently, we are proposing this taxon, which is restricted exclusively to one mountain range, to be treated as 'Endangered' (EN) following the Red List criteria of the IUCN Standards and Petitions Committee of the IUCN [90].