Given its unique relationship with pollinators, the genus Ophrys L., a member of the Orchidaceae family, has caught the interest of numerous scientists, including Darwin himself [1], as a model organism for the study of floral evolution. The species of this genus are perennial herbaceous geophytes with a predominant distribution in the Mediterranean area, and one of the centers of diversity is the Balkan Peninsula [2]. The genus Ophrys is one of the genera that have had several taxonomic treatments and the most significant increase in the number of species in recent decades [3–6]. Depending on the methodology applied in the circumscription of the genus and the applied species concepts, the number of species varies significantly. Molecular methods, primarily sequencing different parts of the nuclear and plastid genomes, recognize up to ten species [6–9]. In contrast, there are 350 species [5], of which a considerable number have been recognized and described mostly based only on morphology. Some researchers have argued that the interactions between Ophrys species and their pollinators offer more valuable insights than neutral markers due to their crucial role in the process of speciation [10,11]. Ophrys species are known to attract a limited number of pollinator species [12] by emitting floral scents that mimic sex pheromones [13,14]. Variations in these floral scents can lead to shifts in pollinator species, potentially facilitating reproductive isolation among populations of the same species and promoting speciation [15]. Taxonomic confusion arising from conflicting views regarding which criteria should be used to delimit species in this genus could have significant consequences for conservation [16]. Ophrys sphegodes complex is one of the most taxonomically difficult groups within the genus, with the number of species varying from 3 to 48 [4,17]. Many of the newly proposed taxa seem to be artificial and merely extremes in the cline of variation [18]. On the other hand, several recently recognized taxa do appear to be isolated by flowering time and pollinator specificity [18]. Since the distribution and morphology of these species are not sufficiently documented, some authors have decided to treat this entire complex of species as several subspecies of O. sphegodes [19,20]. Ophrys sphegodes subsp. grammica (B. Willing & E. Willing) Kreutz was described as a subspecies of O. mammosa Desf. [21]. Delforge et al. [22] elevated it to the species status. Antonopoulos [23] and Delforge [4] also recognized this taxon as a species. In the Catalogue of European Orchids, Kreutz [24] introduced a new nomenclatural combination: O. sphegodes subsp. grammica. However, this name is more commonly used as a synonym for O. sphegodes subsp. mammosa (Desf.) Soo ex E. Nelson [19,20]. In Niketić et al. [25], the name O. sphegodes subsp. grammica is treated as a synonym of O. sphegodes subsp. taurica (Aggeenko) Soó ex Niketić & Djordjevic, which is a new nomenclatural combination for O. sphegodes subsp. mammosa. In Serbia seven taxa have been recorded from the genus Ophrys [26], including two from the O. sphegodes complex. Morphometric studies are traditionally used in species delimitation of orchids [27–30] as well as in genus Ophrys [31–34]. Phenotypic differentiation among taxa is often more significant than genetic data [35], but using morphometric characters for taxonomic purposes is relevant in the genus Ophrys [35]. This may be attributed to the fact that morphometric traits, which are likely subject to selection pressures to align with the morphology of pollinators [36], may undergo more rapid evolutionary changes and be less affected by hybridization compared to neutral genetic markers [15].

The aims of the study were: a) to clarify the taxonomic status of Ophrys sphegodes subsp. grammica based on comparative morphological and morphometric analyses, and to compare it with O. sphegodes subsp. taurica and the type subspecies, b) to provide a morphological description and habitat preferences for O. sphegodes subsp. grammica from the northernmost point of distribution (Serbia) and c) to estimate the IUCN threatened status of this taxon, new to Serbia.

Ophrys sphegodes subsp. grammica was discovered for the first time in Serbia in April 2023, on Svrljiške Mts., and data on its distribution, habitat preference, flowering period, population size, and morphology were obtained. One specimen from each site, without underground organs for vegetative propagation, was collected and deposited in the general collection of the Herbarium of the University of Novi Sad (BUNS) [37].

The taxon Ophrys mammosa subsp. grammica was discovered in the area of Kastoria Prefecture in northwestern Greece by Willing and Willing [21], and since then it has been considered endemic to northwestern Macedonia, with Mount Grammos as the center of its range [23]. More recently, populations of this taxon have also been recorded in other regions of Greece, on mountain ranges in eastern Macedonia [43] and in southern Pindos, the Ionian Islands, and Crete [2,23]. Although Delforge [4] suggests that this species may also occur in Albania, to our knowledge, its occurrence has not been documented in Albania, North Macedonia, or Bulgaria. Its range outside Greece has been found only in the territory of Montenegro [44,45]. The finding of the subspecies O. sphegodes subsp. grammica in the Svrljiške Mts. is its first record for Serbia. Moreover, this record represents the northernmost point of range for this taxon. According to Antonopoulos [23], O. sphegodes subsp. grammica flowers throughout May and the first half of June, where Delforge [4] gives the flowering period from late March to June. All three populations in Serbia were found in late April (23rd and 24th). The populations at Dolac and Ljubatovica, located at lower altitudes(294–350 m a.s.l.), were in full bloom, while the population at Donji Rinj (884 m a.s.l.) was just beginning to flower. The habitats where this subspecies has been discovered in Serbia are usually warm and dry, with substrates that have a slightly acidic to slightly basic reaction and are low in nutrients. The occurrence of O. sphegodes subsp. grammica in such habitats is consistent with the statements in the literature about this taxon and the genus in general. In Greece, O. sphegodes subsp. grammica grows mainly on skeletal soils formed by erosion of limestone or serpentine, It can occur on short grasslands, in shrublands, oak forests, pine forests, pine forest clearings, and alpine meadows at elevations up to 1200 m above sea level [4,23,43]. Despite having somewhat differing perspectives on the O. mammosa complex, Devillers and Devillers-Terschuren [3] and Delforge [4] both identify O. sphegodes subsp. grammica as a species within it. Although some authors split O. mammosa into several species, Pedersen and Faurholdt [19] threat the whole complex as one subspecies of O. sphegodes. Various forms of data, such as molecular, morphometric, and chemical, often do not align [35], resulting in differing interpretations of species boundaries. Methods based on genetic data are then judged as more reliable; however, speciation sometimes leaves no signature at the level of neutral markers, and in that case, neutral marker-based methods may fail to detect species boundaries by excluding the data that are the most informative [35]. Although some floral features may resemble the body forms of female wasps, other floral features most likely serve other purposes, such as effective pollinium transfer [46]. Rakosy et al. [47] proposed that there is strong pollinator-mediated selection for the labellum’s morphological characteristics, which pollinators use for functional interaction. On the other hand, relaxed selection and other stochastic factors are more likely to have an impact on features that are not utilized in effective interaction. Collecting data on the range of morphological variability, as well as their pattern of variability, is crucial to understand which traits are involved in population divergence and to establish the evolutionary scenario underlying the speciation process [48]. According to Delforge [4] pattern of coloration of O. grammica (basal field green-orange, lighter than centre of lip) could define a group consisting of O. grammica, O. herae M. Hirth & H. Spaeth, O. cretensis (H. Baumann & Künkele) Paulus, and O. caucasica Woronow ex Grossh. There is confusion in identification between O. grammica and O. herae, especially in the costal parts of Greece [23]. The main difference according to Paulus [49] between these two species is their pollinators, O. grammica is pollinated by Andrena nigroaenea, while O. herae is pollinated by Andrena thoracica. Identification of O. grammica is more certain in higher altitudes of continental Greece [2]. Apart from the different pollinators, O. herae has a larger and darker labellum and flowers earlier than O. grammica [23]. Ophrys cretensis is considered endemic to the Greek islands, occurring mainly in Crete [19]. According to Antonopoulos [23], this species flowers later than O. herae and has a different pollinator (Andreana vachali subsp. creticola). On the other hand, the presence of O. grammica in Crete was also confirmed. This calls into question the separation of these taxa, and suggests the possibility of a single widespread taxon. From the results of this study, it is evident that the populations of O. sphegodes subsp. taurica are morphologically significantly different from those of O. sphegodes subsp. grammica, and it is clear that the names of these two taxa should not be used as synonyms. Despite their geographical proximity, no O. sphegodes subsp. grammica populations from our sample, including one from Greece, were grouped with O. sphegodes subsp. taurica in the morphospace of CD analysis. The fact that a greater number of studied characters fall into the zone of intermediate variability, with values closer to the lower limits of the zone, suggests that certain morphological characteristics are rather stable. The height of O. sphegodes subsp. grammica specimens from Serbia varies considerably, as does the number of flowers, and differ from the values given in the literature for this taxon. The lateral sepals are never distinctly bicoloured, as in O. sphegodes subsp. taurica, although pink pigment occasionally occurs along the margins and central nerve, which also occurs in the type subspecies. The flower of O. sphegodes subsp. grammica are smaller than those of the other two taxa. It has a pale, predominantly olive-green lip and long, dense grey to white hairs on the shoulders and basal swellings (Fig. 1). The lip has a broad yellow-green margin and is shorter than the dorsal sepal, both characteristics that distinguish it from the other two taxa. In addition, this subspecies differs from the type subspecies by the front edge, which is acuminate. The general coloration of the flower differs markedly from O. sphegodes subsp. taurica, both in the colour of the sepal and lip, and in the colour of the basal field and pseudoeyes. The basal field is pale green or orange-green and always lighter than the rest of the lip, while the colour of the pseudoeyes is often the same as the basal field.

The results of this study show that the O. sphegodes subsp. grammica is a morphologically well-defined taxon, which is best seen from the size and shape of the labellum, as well as the colour of the basal field and labellum. This taxon should not be equatd with O. sphegodes subsp. taurica, since there is a greater number of morphological characters that clearly separate them. The near overlap in the values of analysed morphological characters with O. sphegodes subsp. sphegodes indicates that this taxon should not be treated as a species. This study provides a foundation for future research on the distribution, ecology, and morphology of the Ophrys sphegodes complex in the Balkan Peninsula. A more thorough analysis of the morphology of these taxa should reveal additional features that distinguish similar taxa in this region and help to clarify the taxonomy of this complex.