New Chromosome Counts Reveal Hidden Diversity in Italy’s Flora

Chromosome numbers may sound like a dry corner of botany, but they are among the most powerful tools scientists have for untangling the true diversity of plants. A newly published installment of an ongoing census of the Italian flora, appearing in the journal Italian Botanist, adds a fresh batch of chromosome counts for plants collected across the country, including the very first counts for six taxa and the first Italian counts for three more. The work, spanning Sardinian seasonal ponds, Apennine mountain meadows, and Calabrian rocky slopes, shows how cytogenetics continues to sharpen our picture of one of Europe’s richest floras.

The technical core of the study rests on a classic but still indispensable method: the chromosome squash preparation. For most of the species examined, researchers germinated seeds in the laboratory and harvested root tips, the regions of most active cell division. These tips were pre-treated with a colchicine solution, either 0.3% or 0.4% depending on the laboratory protocol, for about three hours. Colchicine disrupts the spindle fibers that normally pull chromosomes apart during cell division, effectively arresting cells in metaphase, the stage at which chromosomes are most condensed and easiest to count. The tissue was then fixed in Carnoy solution, an ethanol and acetic acid mixture that preserves cellular architecture, before hydrolysis in hydrochloric acid at 60 degrees Celsius and staining with Schiff reagent or leuco-basic fuchsine, dyes that bind specifically to DNA. The result is a single-layer preparation in which each cell’s chromosome complement can be observed and counted under the microscope.

The first of the new counts concerns Damasonium alisma, a striking aquatic plant of the water-plantain family, sampled from a temporary Mediterranean pond at Funtana Satoa on the island of Sardinia. The team counted 2n = 28 chromosomes, confirming that the Sardinian plants are tetraploid, with four copies of the basic chromosome set. This aligns with the established understanding that D. alisma is a tetraploid species distributed across England, France, Italy, Portugal, Russia, and Ukraine, and is considered the northern counterpart of the diploid D. bourgaei, which has 2n = 14. The finding matters because a previous count from Sicily, reported decades ago under a different name, showed a diploid number, and D. bourgaei itself has been recorded in Sicily. That raises the possibility of a misidentification in the earlier Sicilian record. The authors argue that clarifying the true distribution of these two taxa in Italy will require a more in-depth study combining morphological and karyological evidence.

Sardinian temporary ponds yielded another first: the chromosome number of Helosciadium crassipes, an umbellifer found in shallow seasonal wetlands that flood in winter or late spring. These habitats belong to a vegetation alliance associated with habitat 3120 of the European Habitats Directive, which protects oligotrophic waters with amphibious vegetation, a priority conservation type. Until now, the species’ chromosome number was entirely unknown. The count of 2n = 22, obtained from plants collected at Perdiana in the province of Oristano, fits neatly with numbers reported for the closely related H. inundatum species group and for most other members of the genera Helosciadium and Apium. For a plant whose wetland habitats are among Europe’s most threatened, even this basic piece of biological information adds to the foundation needed for conservation planning.

The mountain floras of southern and northern Italy contributed their own surprises, particularly among the hawkweeds of the genus Hieracium, a group notorious for its complexity. Hieracium terraccianoi, a hemicryptophytic herb endemic to the Calabrian side of the Pollino Massif within Pollino National Park, was counted for the first time from material gathered at the species’ type locality. The result, 2n = 36, corresponds to a tetraploid level and agrees with counts available for other members of Hieracium sect. Grovesiana, a cluster of similar morphotypes that has challenged botanists for decades.

Three more hawkweed counts, all yielding 2n = 27, or a triploid level, further illuminate this intricate genus. Hieracium hypochoeroides subsp. cilentanum, a rock-dwelling endemic of Monte Cervati in Campania counted at 1,860 meters elevation, belongs to an aggregate of apomictic taxa that likely evolved during the post-glacial period, many of them narrow endemics that survived as relicts in southern Europe. Apomixis, the asexual production of seeds without fertilization, allows triploid plants to reproduce despite their odd chromosome number, which would normally render them sterile. Hieracium lesimanum and Hieracium scopolioides, both endemic to Mount Lesima in the Ligurian Apennines and both known only from their type localities on the mountain’s northern slope, were also found to be triploid. For H. scopolioides, the count is consistent with the only known ploidy estimate in its section, while H. lesimanum’s number matches observations in related taxa of the H. prenanthoidea group in Europe. The repeated pattern of triploidy in these young, range-restricted endemics supports the view that apomictic reproduction has been a major engine of diversification in this genus, generating distinct lineages without the need for sexual recombination.

The carnation family contributed two notable results. Dianthus carthusianorum subsp. tenorei, a subspecies endemic to Italy and distributed through the southern-central Apennines from Marche to Calabria, was counted for the first time from material collected at Monte Pollino at 2,000 meters. The standard count of 2n = 30 confirms the numbers obtained for other subspecies in Italy and central-eastern Europe. Intriguingly, the researchers also spotted cells with 2n = 60 chromosomes within the same root tip, a phenomenon known as endopolyploidy, in which cells duplicate their DNA without dividing. Endopolyploidy is common in many Dianthus species and, while it does not change the germ line’s genetic makeup, it reflects an additional layer of genomic flexibility in this charismatic genus of ornamental and wild flowers.

The second caryophyllid finding concerns Illecebrum verticillatum, a sub-Atlantic species of temporary ponds across Europe and North Africa. In Italy, the plant occurs in Piedmont, Sardinia, and Tuscany, and the Tuscan populations are particularly rare, with recent confirmations restricted to a single area at Montalbano in the municipality of Carmignano. Material from that area showed 2n = 10, a low chromosome number already known from elsewhere in Europe but recorded here for the first time in Italian populations. Meanwhile, in Piedmont, the team obtained the first Italian count for Crupina vulgaris, a widespread southern European composite sampled near Acqui Terme. The result, 2n = 30, matches counts from other European populations and provides a useful reference point for distinguishing the species from its relatives: the more Mediterranean C. crupinastrum has 2n = 28, while C. intermedia, an allopolyploid of hybrid origin, has 2n = 58. Because these species overlap in Italy, chromosome data offer a critical line of evidence for identifying plants and tracing how hybridization has shaped the group’s evolution.

What emerges from this cumulative exercise is a portrait of the Italian flora as a laboratory of evolutionary processes. Polyploidy, apomixis, endopolyploidy, and hybridization each leave fingerprints in chromosome numbers, and every new count helps place a species on the map of these processes. Tetraploid aquatic plants in Sardinian ponds, triploid hawkweeds clinging to Apennine and Apuan summits, and diploid rarities in disappearing wetlands each tell a story about how lineages originate, persist, and sometimes retreat to the edges of their former ranges. Chromosome counts also have immediate practical value: they help resolve taxonomic ambiguities, flag likely misidentifications in old records, and inform conservation assessments for narrow endemics whose entire known range may span a single mountain slope or a single pond.

The study is the thirteenth contribution to a long-running series dedicated to chromosome numbers for the Italian flora, and the series continues to accept new data. Researchers are invited to submit contributions including voucher specimens and methods, ensuring that each count remains traceable to a physical herbarium or germplasm record. That rigor, with seeds deposited in germplasm banks and herbarium specimens preserved for verification, is what transforms a simple count of chromosomes into durable scientific evidence. As Mediterranean temporary ponds shrink and mountain endemics face shifting climates, documenting the genomic architecture of Italy’s plants is not merely bookkeeping. It is a race to understand, and ultimately to protect, a living heritage written in chromosomes.

Subject of Research: Chromosome numbers of plant taxa in the Italian flora

Article Title: Chromosome numbers for the Italian flora: 13

Article References: Astuti, G., Bagella, S., Bajona, E., Barone, G., Becca, G., Caria, M. C., Di Gristina, E., Fainelli, F., Franzoni, J., Giacò, A., Orsenigo, S., Paliy, M., Rivieccio, G., Urbani, M., & Peruzzi, L. (2023). Chromosome numbers for the Italian flora: 13. Italian Botanist, 16, 135-148. https://doi.org/10.3897/italianbotanist.16.116700

Image Credits: AI Generated

DOI: 10.3897/italianbotanist.16.116700

Keywords: chromosome counts, Italian flora, cytogenetics, polyploidy, apomixis, Hieracium, endemic plants, Sardinia, temporary ponds, Dianthus, plant taxonomy, karyology