Scanning electron micrographs of preserved flowers. A, C, Gymnadenia frivaldii , Bulgaria: A, Details of gynostemium; C, excised labellum. B, D, Pseudorchis albida , Italy. B, details of gynostemium; D, excised labellum. Scale bars: 0.5 mm (A, B), 1 mm (C, D). Photos: P. Rudall. 

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... dried materi- al collected primarily for DNA extraction; they were sub- sequently stored in 70% ethanol. The spirit collection at RBG Kew yielded further alcohol-fixed inflorescences of G. frivaldii ( M. A. Clements 2784 , Greece; E. R. S. Winter 40140 , Bulgaria) and P. albida ( E. M. Payne 18500 , Scotland; J. J. Wood 346 , Italy). Selected flowers from each inflorescence were dehydrated through an alcohol series to 100% ethanol. They were then critical- point dried using a Balzer CPD 020 unit, mounted on stubs using double-sided tape, coated with gold and examined under a Hitachi cold-field emission SEM S- 4700-II at 2 kV. The resulting images were recorded dig- itally for subsequent manipulation in Adobe Photoshop. Comparison of the flowers of G. frivaldii originally preserved in spirit and silica gel demonstrated the absence of preservation-related artifacts. Molecular phylogenetic analysis. — Base-pair sequences were obtained from the widely used, rapidly mutating ITS region of rDNA (e.g., Baldwin & al., 1995; Hershkovitz & al., 1999) using the extraction protocols of Rogers & Bendich (1994) and the analytical procedures outlined in an earlier phylogenetic analysis of Orchidinae by Pridgeon & al. (1997). DNAs were ex- tracted from one Greek (RB 878) and one Bulgarian (RB 867) specimen of G. frivaldii (Appendix). Since the ITS sequences obtained from these two extremes of the Balkan distribution of the species were consistently identical in replicated analyses, no further specimens of this species were sequenced. The approximate phylogenetic position of G. frivaldii was determined by inserting the Bulgarian sequence into the pre-existing ITS matrix of Bateman & al. (2003), which contained sequences for 188 accessions of tribe Orchideae plus two outgroups, together aligned in MacClade 4.05 (Maddison & Maddison, 2002). Visual inspection of the G. frivaldii sequence was sufficient to show that its affinities were with Gymnadenia s.s. rather than with Pseudorchis s.s. Alignment by eye was straight –forward, as G. frivaldii lacked any insertion-deletion events (indels) other than those already observed in other closely related species of Gymnadenia . Subsequent analysis of the matrix using PAUP* 4.0b10 (Swofford, 2002) followed the heuristic procedures outlined by Bateman & al. (2003) and clearly demonstrated that frivaldii is indeed nested within Gymnadenia . Hence, a second, more focused phylogenetic analysis was then performed, abstracting all sequences of Gymnadenia s.l. from the matrix of Bateman & al. (2003) and adding several further sequences from a study of Gymnadenia s.l. originally conducted in 1996 (Bateman & al., unpubl.). In addition, ITS sequences were obtained from accessions of G. conopsea s.s., sampled from sites close to the G. frivaldii localities in Greece and Bulgaria, and also from a Bulgarian plant tentatively assigned to G. densiflora . In total, 14 ingroup accessions were included (Appendix), together repre- senting eight different ITS types. Since monophyly of Gymnadenia s.l. had already been confidently demonstrated by Bateman & al. (2003), a single outgroup was considered sufficient to polarise the characters; thus, a species representative of the basal group of the sister- genus, Dactylorhiza s.l., was selected as outgroup. The resulting matrix contained very few missing values; moreover, the relatively strong similarity of the 15 sequences facilitated alignment, generating only four indels, three of which were located near the 3' end of ITS 2: a 1 bp indel and a 2 bp indel separated the outgroup from the ingroup, and two 1 bp indels (one of which was autapomorphic) occurred within the ingroup. Thus, it was feasible to treat these few small indels as a fifth state, rather than necessitating use of one of the several gap-coding strategies available (cf. Simmons & Ochoterena, 2000). The alignment allowed comparison of a continuous run of bases from ITS1 through 5.8S to ITS2 that totalled 643 sites; of these, 63 (10%) were variable but only 19 were parsimony-informative. The modest number of entities analysed allowed rapid and reliable detection of all most-parsimonious trees using the branch-and-bound search strategy of PAUP. Branch support was estimated using both the decay index and fast bootstrap, via 1,000 replicates of a heuristic search employing stepwise addition. Morphology. — The morphology of Gymnadenia frivaldii is compared with that of Pseudorchis albida in Figs. 1–4. Those portions of the plant given least attention by previous authors who have provided technical descriptions of G. frivaldii (Györffy, 1904; Camus & Camus, 1929; Baumann, 1978; Moore, 1980b; Buttler, 1991; Delforge, 2001), specifically the tubers, spur and gynostemium, have been given greatest attention here. The fusiform tubers are divided for approximately the distal half of their length into between two and four elongate lobes narrowing into filiform roots (Fig. 2A; see also Györffy, 1904, fig. 4). The flexuous above-ground portion of the stem varies in height from a few centime- tres up to 30 cm (Fig. 1A). Between two and four expanded leaves form an imperfect basal rosette only in plants occupying unusually exposed habitats; they are more typically distributed along the lower portion of the stem, as are the one or two bract-like leaves above. The expanded leaves are broadly lanceolate, robust and fleshy, 50–90 × 8–15 mm. The inflorescence is compact and conical, reaching 20–40 mm and containing 15–40 small flowers (Fig. 1B). The membraneous bracts are supplied by only a single vein and usually slightly exceed the compact ovaries. In some individuals the flowers are creamy white, but in most plants diffuse pink anthocyanins are present; they are most intense in the gynostemium, including the pollinia, and often extend to the bracts and the upper portion of the stem. The median sepal and lateral petals are curved forward to form a loose (in some individuals very loose) hood, whereas the lateral sepals are somewhat longer, opposite and spreading. The distinctive labellum ranges from shallowly three-lobed to entire, is at least shallowly concave near its attachment and recurved toward its apex (Figs. 1B, 4C). It is approximately equidimensional (3.5–4 mm × 3–3.5 mm) when artifi- cially flattened, and bears a small but distinct spur (Fig. 3A–C). Epidermal cells covering the thinner marginal zone appear considerably smaller than those in the fleshi- er main body of the labellum (Fig. 4C). Some previous descriptions of the spur are contra- dictory. Our macromorphological and SEM studies (Fig. 3A–C) reveal it to be short [1.5–2(–3) mm], slender (0.3–0.5 mm), cylindrical (Fig. 3A) or slightly tapering (Fig. 3B, C), slightly downcurved and considerably shorter than the ovary. It is clearly formed by upward invagination of the labellum (Fig. 4A). The gynostemium has rarely been described (e.g., Camus & Camus, 1929). In Fig. 4A it can clearly be seen to be unusually compact relative to most other members of subtribe Orchidinae. A pair of small pollinia (removed in Fig. 4A) are recessed in two adjacent “cowls” of thin- walled but deep thecae that are separated by a deeply invaginated, omegaform rostellum, which accommodates the two viscidia. The stigmatic surface extends laterally from immediately beneath the rostellum and above the spur entrance into two distinctive lappets. Distribution and ecology. — The distribution of G. frivaldii is centred on the most montane portion of the Balkans, thereby encompassing parts of several coun- tries: Greek Macedonia, Slavic Macedonia, eastern Albania and Transsylvanian Romania (e.g., Baumann, 1978; Moore, 1980b; Baumann & Künkele, 1982), with a smaller disjunct stronghold to the north in the Carpathiani Meridionalis of southwestern Bulgaria (Baumann, 1978). There is also an unconfirmed report of a single, incongruous outlying population in west-central Italy (Valentini & Montecchi, 1990). The species is strongly altitudinally constrained, spanning a range between 1000 m and 2500 m but char- acteristically occurring at 1700–2000 m, where it prefer- entially inhabits wet meadows and mountain seepages (Stapperfenne, 1966; Lakusic & Grgic, 1971; Baumann, 1978). These habitats and plant communities are analo- gous to those of the high meadows of the Alps; typical associated orchids include Pseudorchis albida , Gymnadenia conopsea s.s., species of Gymnadenia subgenus Nigritella , and Dactylorhiza cordigera . Gymnadenia frivaldii flowers in June and July, occasionally stretching into August at higher altitudes. The flowers are occasionally reported to be fragrant, and photographic evidence suggests that they contain nectar for approximately one half of their length (e.g., Baumann & Künkele, 1982, p. 373). Evidence of pollinators is anec- dotal; small moths and butterflies are implicated (Davies & al., 1983; Cingel, 1995). Molecular phylogenetics. — The molecular phylogenetic ITS studies of Pridgeon & al. (1997) and, more taxonomically comprehensively, Bateman & al. (2003) thoroughly analysed both Pseudorchis s.s., represented by P. albida and its segregate, P. straminea , and Gymnadenia s.l., represented by eight species. Together, these species spanned almost the entire morphological spectrum of this broadly delimited genus, which now encompasses both Gymnadenia s.s. and the morphologically distinct but molecularly very similar Nigritella . Generic circumscriptions of both Gymnadenia and Pseudorchis were unequivocally upheld, being mono- phyletic entities supported by both substantial molecular branch lengths (incorporating unique indels) and sufficient morphological synapomorphies. Gymnadenia s.l. is equally unequivocally sister to Dactylorhiza s.l., and this pair is in turn sister to the Platanthera clade. Pseudorchis is placed more ambiguously, being located between the Gymnadenia-Dactylorhiza and Platanthera clades, marginally closer to the latter. Thus, we ...