The anatomical properties of leaves, stems, roots and root tubers of 27 taxa belonging to three orchid genera (Ophrys, Orchis and Dactylorhiza) were investigated to determine their diagnostic characteristics and assess interspecific and intergeneric relationships by means of cladistic analyses. Anatomical sections were done using different staining methods, such as Sartur, Phloroglucin-HCl, Delafield's Hematoxylin-Safranin, and Alcian Blue-Periodic Acid-Schiff. Additionally, lengths of raphides (in all the organs), cuticle thickness, cell size and stomata dimensions (in the leaf epidermes), as well as dimensions of starch granules (only in root tubers) were measured and statistically compared. During root tuber investigations, for every taxon the types of meristele were outlined and diagrammed. All morphometric data except starch grain dimensions, are generally not significantly different between the investigated taxa. The anatomical sections showed the following basic anatomical characteristics: Leaf surface was glabrous, stomata were abaxially located and mostly anomocytic. Cuticle was mostly striate. The chlorenchyma was homogeneous. The stem had a uniseriate epidermis with several rows of thick- or thin-walled, sclerenchymatous (or collenchymatous) cortical parenchyms, a pericycle, collateral vascular bundles within a central ground tissue. Orchis purpurea lacks a pericycle. The roots generally had a uniseriate epidermis, an exodermis, a parenchymatous cortex with occasional inclusions of fungal pelotons and hyphae, an endodermis bearing casparian strips, pericycle, and vascular cylinders. Root tubers, from the outside to the centre, had orderly velamen, exodermis, and ground tissue, which were scattered with meristeles, mucilage cells and assimilatory cells bearing cruciate starch grains. Based on the anatomical results, tables were created based on the important primary (species-specific) and secondary (common-specific, used only in some taxa) descriptive characteristics. Some of the primary descriptive characters include the following: Leaf-epidermal wall properties (thin/thick), chlorenchyma cell shape, chloroplast distribution, presence or absence of large lacunae; stem-presence/absence of pericycle, cortical thickness; root-cell shape of exodermis, presence/absence of tilosome passage cells, shapes of endodermal and/or pericycle cells, structures and distributions of archs; root tuber forms of starch grains and mucilage cells and meristele types. In general, leaf chlorenchyma and stem pericycle were found to be thicker and mucilage-water storage cells were more frequent in the Orchis and Dactylorhiza taxa. Cladistically analysed anatomical data support the hypothesis that Ophrys and Dactylorhiza are monophyletic, but contrast the triphyletic states of Orchis, which is very likely to be the reason for uncovering the other Orchis taxa. Finally, we strongly emphasize the importance of this kind of detailed anatomical study to solve identification problems of orchid taxonomy, which have wrongly been disregarded in recent works. (C) 2009 Elsevier GmbH. All rights reserved.