A study of the distribution of orchid species in Puglia, based on an analysis of 2084 bibliographic reports from 2000 to 2022, was carried out with the aim of revising and updating the information on the consistency of the Orchidaceae family in Puglia, with a special focus on assessing threatened species occurring inside and outside protected areas. The work presents a checklist of the Orchidaceae taxa (genera, species, and subspecies) found in the region, including observations on genera and species that present taxonomic challenges. A total of 113 taxa (i.e., species and subspecies), distributed across 16 genera, are listed in alphabetical order. The most representative genera were Ophrys (51 taxa), Serapias (15 taxa), and Epipactis (11 taxa). Additionally, 49 taxa (43.4%) were found to be endemic to Italy, with 21 of these, mostly belonging to the Ophrys genus, being exclusive to Puglia. Our study notes two different trends of distribution: a predominantly coastal distribution for orchid records located in southern Puglia (the Salento peninsula) and a more widespread distribution for the other provinces. Our study also shows that the greatest number of records locate orchids in protected areas with a positive correlation between their presence and habitats cited in Directive 92/43/EEC.

This study presents an updated analysis of cytogenetic data for several species within the 40,42-chromosome genera of the subtribe Orchidinae. The research includes insights into the distribution of heterochromatin obtained using C-banding and fluorochrome techniques. Our investigation confirmed variation in the distribution of heterochromatin and repetitive DNA sequences among species pertaining to Neotinea s.l. and Orchis s.str. These variations also potentially contribute to the diversification of these species. Cytogenetic analyses of the Neotinea group demonstrated that both H33258 and DAPI staining result in blocks of fluorescent regions on numerous chromosomes. Particular attention was paid to the cytological composition of the polyploid Neotinea commutata, focusing on its potential origin. Based on the karyological results acquired, a hypothesis concerning the origin of N. commutata is proposed. The most noteworthy revelations regard the O. mascula complex. In these species, the telomeric areas of all chromosome sets display extensive heterochromatin. Fluorochrome staining revealed telomeric blocks on many chromosomes that were not seen with Giemsa staining. This highlighted a distinct feature of O. mascula, where particularly large C-bands surrounding the centromeric regions of multiple chromosomes were found. However, in O. mascula, O. provincialis, O. pauciflora, and O. patens, C+ chromatin may not show a significant response to fluorochrome Hoechst or DAPI+ staining. The unique cytomorphological arrangement observed in the O. mascula species, unlike other members of the O. mascula complex, suggest epigenetic phenomena. Additional data are presented for the genera Dactylorhiza and Gymnadenia. A deeper understanding of the diversity of chromosomal structures among these orchids promises to shed light on the mechanisms underlying speciation, adaptation, and the remarkable diversity characteristic of the Orchidaceae family.

This article provides a summary of the current knowledge on the cytogenetics of four genera, which are all composed of 36 chromosomes, within the Orchidinae subtribe (Orchidaceae). Previous classical studies have revealed differences in karyomorphology among these genera, indicating genomic diversity. The current study includes an analysis of the current knowledge with an update of the karyotype of 47 species with 36 chromosomes from the genera Anacamptis, Serapias, Himantoglossum, and Ophrys. The study discusses comparisons of karyotypes among these genera that used traditional techniques as well as karyotype asymmetry relationships with various asymmetry indices. Additionally, the study reports new findings on polyploidy in Anacamptis pyramidalis and Serapias lingua, which were observed through karyotype and meiotic metaphase analyses in EMC. Moreover, the study detected B chromosomes for the first time in A. papilionacea and A. palustris. The article also describes the use of fluorescent in situ hybridization in some specimens of A. papilionacea and A. collina to locate different sites of the 18S-5.8S-25S rDNA and 5S rDNA ribosomal complexes on chromosomes. The information derived from these cytogenetic analyses was used to refine the classification of these orchids and identify evolutionary relationships among different species and genera.

This paper presents a comprehensive study of the size and conservation status of the only Italian population of Centaurea pumilio (Asteraceae) and the threats to its survival. The population is located on a short stretch of sandy shoreline along the Ionian coast of Puglia, near Torre S. Giovanni (Ugento, Lecce). It was estimated in the 1990s to number about 500 plants, but in recent years a significant reduction, bringing the population to fewer than 100 individuals, has been observed. This study involved a census of the individuals (differentiating young plants from adult and reproductive ones) conducted with a precision GPS tool, phytosociological analysis and high-definition orthophoto image acquisition using a drone. Concerning the latter, to evaluate anthropic pressure from tourism, data were acquired in spring 2023 and autumn 2024 and compared using GIS geoprocessing, showing a significant increase in the area occupied by footpaths. GIS analysis also revealed that the survival of C. pumilio is strongly linked to the intensity of the walking routes, which have fragmented the population into small and isolated clusters. On the basis of all the collected data, the current conservation status of the species in Italy was assessed as critically endangered. Finally, our study provides a series of suggestions to improve the conservation status of the species and strategies to reduce the risk of extinction in Italy.

Based on our karyological findings in the Anacamptis Rich., Ophrys L., and Serapias L. genera, we have identified chromosomal markers within some hybrids and elucidated their interrelationships. Mitotic chromosomes of fifteen taxa were analyzed using the conventional Feulgen staining method. Only for Anacamptis ×gennarii (Rchb. f.) H.Kretzschmar, Eccarius & Dietr. [A. morio (L.) R.M.Bateman, Pridgeon & M.W.Chase × A. papilionacea (L.) R.M.Bateman, Pridgeon & M.W.Chase] and its parental species were some data obtained and reported with the banding method with Giemsa, Hoechst 33258 fluorochrome, and the FISH techniques. Our research involved new chromosomal measurements of fifteen taxa, including six hybrids, along with schematic representations. Morphometric parameters, i.e., M_CA and CV_CL, were used to evaluate karyotype asymmetry. Of meaning were the analyses performed on chromosomal complements of selected hybrids, which distinctly revealed marker chromosomes present in one or both putative parental species. Among the parents identified in some hybrids, Ophrys tenthredinifera Willd. has shown some interest due to the presence in its karyotype of a pair of chromosomes (n.1) showing a notable secondary constriction on the long arm. Indeed, one of the homologs is clearly distinguishable in the analyzed hybrids, where it clearly emerges as one of the putative parents. Given the challenges in detecting certain karyomorphological features within the Orchidinae subtribe using alternative methods, such as Giemsa C-banding or fluorescence banding, the Feulgen method remains valuable for cytogenetic characterization. It helps us to understand the genomes of hybrids and parental species, thus contributing to a deeper understanding of their genetic composition.

This work presents a summary of cytogenetic data, including new information, on several species within the tribe Neottieae, with an update of the karyotype for 23 species belonging to the genera Cephalanthera, Limodorum, Epipactis, and Neottia (including Listera). Each of these four genera also presents distinctive chromosomal features, such as bimodal karyotypes. Our research includes insights into the distribution of constitutive heterochromatin, measured using C-banding and, in some cases, specific fluorochromes for the detection of A-T- and G-C-rich DNA. In the Epipactis group, it is noteworthy that when using the Giemsa banding technique, certain species (e.g., E. placentina, E. meridionalis) with a chromosome number of 2n = 38 were observed to exhibit a conspicuous wide band of constitutive heterochromatin on the long arm of the third pair in a subcentromeric position, resembling what has been observed in E. helleborine. These differences also have the potential to contribute to the diversification of these species. Based on the karyological results obtained, a hypothesis regarding the origin of certain species within the E. helleborine group is proposed. Additionally, karyological analyses conducted on a specimen of E. microphylla revealed chromosome counts ranging from 36 to 40. Somatic metaphases exhibited evident structural alterations in certain chromosomes, showing rearrangements probably caused by translocation phenomena. Based on the data obtained from the species within the studied genera, it is conceivable that variations in chromosomes, both structurally and in the distribution of constitutive heterochromatin, exert a significant influence on the evolution of the karyotype. Moreover, in many entities belonging to the Neottieae tribe, these processes may also contribute to the diversification of the phenotype in some instances.