Italian Botanist 9: 101-110 (2020) doi: 10.3897 /italianbotanist.9.54973 RESEARCH ARTICLE QQ g Italian Botanist http://italianbotanist.pensoft.net Chromosome numbers for the Italian flora: 9 Giovanni Astuti', Gianni Bedini!', Daniela Ciccarelli, Lijuan Liu', Manuel Tiburtini', Lorenzo Peruzzi' | Dipartimento di Biologia, Universita di Pisa, Via Derna 1, 56126 Pisa, Italy Corresponding author: Giovanni Astuti (gastuti@biologia.unipi.it) Academic editor: G. Domina | Received 2 June 2020 | Accepted 12 June 2020 | Published 8 July 2020 Citation: Astuti G, Bedini G, Ciccarelli D, Liu L, Tiburtini M, Peruzzi L (2020) Chromosome numbers for the Italian flora: 9. Italian Botanist 9: 101-110. https://doi-org/10.3897/italianbotanist.9.54973 Abstract In this contribution, new chromosome data obtained on material collected in Italy are presented. It in- cludes counts from six populations of three taxa within the genus Pulmonaria, two of which are endemic to Italy (P vallarsae subsp. apennina and P vallarsae subsp. vallarsae); the other is the widespread European P officinalis. In addition, two counts from Potentilla detommasii and Stachys thirkei, two eastern Mediter- ranean species, are also reported. Keywords cytogeography, cytotaxonomy, Emilia-Romagna, Friuli Venezia Giulia, Marche, Toscana, Trentino-Alto Adige How to contribute Texts concerning new chromosome data should be submitted electronically to Gio- vanni Astuti (gastuti@biologia.unipi.it), including indications on voucher specimens and methods used. Copyright Giovanni Astuti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 102 Giovanni Astuti et al. / Italian Botanist 9: 101-110 (2020) Chromosome counts Boraginaceae Pulmonaria vallarsae A.Kern. subsp. apennina (Cristof. & Puppi) Cecchi & Selvi Chromosome number. 27 = 22 (Figs 1a, 2) Voucher specimen. Italy. Toscana. Molino del Pallone (Sambuca Pistoiese, Pis- toia), sulla sponda toscana del flume Reno (WGS84: 44.100658N, 10.962573E), margine di bosco, ca. 500 m s.l.m., 5 April 2019, LZ. Lin & G. Astuti (PI n° 034194— 034202); Passo del Muraglione (San Godenzo, Firenze), sulla SS67 scendendo verso San Benedetto in Alpe (WG84: 43.935302N, 11.658088E), ca. 900 m s.l.m., 9 April 2019, L. Liu & G. Astuti (PI n° 034203-034214). Chromosome number. 27 = 28 (Fig. 1b) Voucher specimen. Italy. Toscana. Molino del Pallone (Sambuca Pistoiese, Pis- toia), sulla sponda toscana del flume Reno (WGS84: 44.100658N, 10.962573E), margine di bosco, ca. 500 m s.l.m., May 2015, P Pupillo (plant only temporarily cul- tivated at the Botanic Garden of Pisa University). Method. Squash preparations were made on root tips obtained from potted plants. Root tips were pre-treated with 0.4% colchicine for 3 hours and then fixed in Carnoy fixative solution for 1 hour. After hydrolysis in HCI 1N at 60 °C, the tips were stained in leuco-basic fuchsine. Observations. ‘The overall morphology of these plants is congruent with P vallar- sae subsp. apennina, a subspecies endemic to peninsular Italy (Cecchi and Selvi 2015). In the population from Molino del Pallone we found ten individuals showing 27 = 22 (Fig. la) and one individual with 27” = 28 chromosomes (Fig. 1b). Vosa and Pistolesi (2004) found a few individuals with an intermediate chromosome number (27 = 26) b vas Figure |. Pulmonaria vallarsae A.Kern. subsp. apennina (Cristof. & Puppi) L.Cecchi & Selvi from Moli- no del Pallone (Sambuca Pistoiese, Pistoia), 27 = 22 (a) and 27 = 28 (b). Scale bar: 10 pm. Chromosome numbers for the Italian flora 103 > Figure 2. Pulmonaria vallarsae A.Kern. subsp. apennina (Cristof. & Puppi) L.Cecchi & Selvi from Passo del Muraglione (San Godenzo, Firenze), 27 = 22. Scale bar: 10 pm. among plants showing 27 = 22 chromosomes at Passo del Muraglione. However, in the present study, we found there only plants with 27 = 22 chromosomes (12 individuals studied). In both cases, we highlight the rarity of chromosome numbers different from 2n = 22, which is typical of P vallarsae subsp. apennina, although a population showing 2n = 26 chromosomes has been recently recorded from Abruzzo (Astuti et al. 2019). Pulmonaria vallarsae A.Kern. subsp. vallarsae Chromosome number. 27 = 22 (Figs 3, 4) Voucher specimen. Italy. Trentino-Alto Adige. Pian delle Fugazze (Vallarsa, Trento) (WGS84: 45.760208N, 11.171882E), ca. 1160 ms.l.m., 17 April 2019, ZL. Liu & G. Astuti (PI n° 034215—-034219); Alla sorgente del Cop, Bellaria di Cei (Villa Lagarina, Trento) (WGS84: 45.96022N, 11.04163E), ca. 900 m s.l.m., 17 July 2019, L. Liu & G. Astuti (PI n° 034220034222). Method. Squash preparations were made on root tips obtained from potted plants. Root tips were pre-treated with 0.4% colchicine for 3 hours and then fixed in Carnoy fixative solution for 1 hour. After hydrolysis in HCI 1N at 60 °C, the tips were stained in leuco-basic fuchsine. Observations. Pulmonaria vallarsae subsp. vallarsae is endemic to Trentino-Alto Adige and Veneto (Cecchi and Selvi 2015), and it was described by Kerner (1878) on plants occurring in Vallarsa, the valley of the river Leno situated southeast of Rovereto, which is the /ocus classicus for this species (Puppi and Cristofolini 1996). Pian delle Fu- gazze, the northern summit of the valley, is among the localities cited in the protologue. 104 Giovanni Astuti et al. / Italian Botanist 9: 101-110 (2020) Figure 3. Pulmonaria vallarsae A.Kern. subsp. vallarsae from Pian delle Fugazze (Vallarsa, Trento), 2n = 22. Scale bar: 10 um. v- . Figure 4. Pulmonaria vallarsae A.Kern. subsp. vallarsae from Bellaria di Cei (Villa Lagarina, Trento), 2n = 22. Scale bar: 10 pm. All the five individuals sampled here displayed the typical chromosome number of P vallarsae (including P vallarsae subsp. apennina), 2n = 22, without any variation. This chromosome number was also found in the three individuals sampled in the other pop- ulation at Bellaria di Cei, which is ca. 40 km far from the locus classicus of this species. Chromosome numbers for the Italian flora 105 Pulmonaria hirta L. Chromosome number. 27 = 28 (Fig. 5) Voucher specimen. Italy. Emilia-Romagna. Parco di Monte Paderno (Bologna) (WGS84: 44.452272N, 11.320769E), ca. 280 ms.l.m., 3 April 2019, L. Lin & G. Astuti (PI n° 034223—034226). Chromosome number. 27 = 22 (Fig. 6) Voucher specimen. Italy. Emilia-Romagna. Parco di Monte Paderno (Bologna) (WGS84: 44.452272N, 11.320769E), ca. 280 ms.l.m., 3 April 2019, L. Lin & G. Astuti (PI n° 034227—034228). Method. Squash preparations were made on root tips obtained from potted plants. Root tips were pre-treated with 0.4% colchicine for 3 hours and then fixed in Carnoy fixative solution for 1 hour. After hydrolysis in HCI 1N at 60 °C, the tips were stained in leuco-basic fuchsine. Observations. These plants display intermediate morphological features between the typical P hirta and the typical P vallarsae subsp. apennina (Puppi and Cristofolini 1996; Cecchi and Selvi 2015), although showing a closer resemblance to the former species, whose range spreads from SE France to C Italy (Cecchi and Selvi 2015). Four out of six samples in this population were found to have 27 = 28 chromosomes, which is typical for P Airta (Astuti et al. 2019; Pupillo et al. 2019), whereas the remaining two individuals were found to have 27 = 22 chromosomes. Plants with 27 = 22 and 28 chromosomes grow together and do not show any pattern relating chromosome num- ber to a specific morphological syndrome, as already observed by Vosa and Pistolesi (2004) for other populations. Figure 5. Pulmonaria hirta L. from Parco di Monte Paderno (Bologna), 27 = 28. Scale bar: 10 um. 106 Giovanni Astuti et al. / Italian Botanist 9: 101-110 (2020) ws @ Figure 6. Pulmonaria hirta L. from Parco di Monte Paderno (Bologna), 27 = 22. Scale bar: 10 um. Ae ~~ * = rr a © " > - * . ; Figure 7. Pulmonaria officinalis L. subsp. officinalis from Castelmonte (Prepotto, Udine), 2” = 16. Scale bar: 10 pm. Pulmonaria officinalis L. subsp. officinalis Chromosome number. 27 = 16 (Fig. 7) Voucher specimen. Italy. Friuli Venezia Giulia. Lungo la SP31 sotto il santuario di Castelmonte (Prepotto, Udine), (WGS84: 46.092828N, 13.516041E), ca. 580 m s.l.m., 21 March 2019, L. Liu e G. Astuti (PI n° 034229034237). Observations. Plants in this population show some morphological features that are intermediate between P officinalis and P vallarsae, especially in terms of leaf shape and maculation. However, the cordate leaf base and the presence of aculeoli (very short Chromosome numbers for the Italian flora 107 conical hairs) clearly place these plants within P officinalis subsp. officinalis (Bolliger 1982), a taxon that is widespread in Europe. This is also confirmed by the chromo- some number 2” = 16, found in all nine individuals studied, which is typical of P officinalis (Sauer 1975; Bolliger 1982). Because of the faint maculation of most of the plants, these plants resemble P officinalis subsp. marzolae G.Astuti, Peruzzi, Cristof. & P.Pupillo, known so far for Trentino-Alto Adige only. However, the presence of ac- uleoli in the population studied here does not match with their absence in P officinalis subsp. marzolae (Astuti et al. 2014). Therefore, these plants could be safely ascribed to a faintly spotted form of P officinalis. L. Liu, G. Astuti, G. Bedini, D. Ciccarelli, L. Peruzzi Rosaceae Potentilla detommasii Ten. Chromosome number. 27 = 14 (Fig. 8) Voucher specimen. Italy. Basilicata. Belvedere del Malvento (Viggianello, Po- tenza), (WGS84: 39.902987N, 16.136537E), ca. 1620 ms.l.m., 13 August 2018, ZL. Peruzzi (seeds collected in the field and stored at the Seed Bank of the Department of Biology, University of Pisa). Method. Squash preparations were made on root tips obtained from germinating seeds. Root tips were pre-treated with 0.4% colchicine for 3 hours and then fixed in Carnoy fixative solution for 1 hour. After hydrolysis in HCl 1N at 60 °C, the tips were stained in leuco-basic fuchsine. Observations. This species spreads from peninsular Italy to Turkey, but it is not recorded for Croatia and Montenegro (Kurtto 2009). Here we report the first chromo- some count for P detommasii from Italy, which agrees with the only other count avail- able for this species (Markowa 1971), confirming its diploid status. G. Astuti, L. Peruzzi Figure 8. Potentilla detommasii Ten. from Belvedere del Malvento (Viggianello, Potenza), 2” = 14. Scale bar: 10 um. 108 Giovanni Astuti et al. / Italian Botanist 9: 101-110 (2020) Figure 9. Stachys thirkei K.Koch, 2n = 30 from Monte Rosato (Fermo). Scale bar: 10 um. Lamiaceae Stachys thirkei K.Koch Chromosome number. 27 = 30 (Fig. 9) Voucher specimen. Italy. Marche. Monte Rosato (Fermo) (WGS84: 43.129556N, 13.702611E), prato arido a sud del bosco su substrato argilloso, 88 m, 6 June 2015, M. Tiburtini (FI). Method. Squash preparations were made on root tips obtained from cultivated plants. Root tips were pre-treated with 0.4% colchicine for 4 h and then fixed in Car- noy solution for 1 h. After hydrolysis in IN HCl at 60 °C, the tips were stained with leuco-basic fuchsine. Observations. This species grows in the eastern Mediterranean, showing a main distribution range in the Balkans, but extending eastwards to Turkey and westwards to Italy (Ball 1972). This species has been recorded for Marche (Bartolucci et al. 2019), where it has been found in only two localities. The specimen investigated here comes from one of these localities, and our count agrees with a previous report for Italy (Falciani and Fiorini 1996). This chromosome number is also typical of all the species belonging to Salvia sect. Eriostomum (Hoffmans. & Link) Dumort. (Falciani 1997). G. Astuti, M. Tiburtini Acknowledgements Many thanks are due to the following people for their help in locating populations of Pulmonaria: Paolo Pupillo and Giancarlo Marconi (Bologna) for Molino del Pallone, Parco di Monte Paderno and Pian delle Fugazze; Filippo Prosser (Rovereto) for Bellaria di Cei; Giuseppe Pistolesi (Pisa) for Passo del Muraglione. We are also indebted to Sil- via Zublena and Francesco Roma-Marzio at the Botanic Garden and Museum of Pisa Chromosome numbers for the Italian flora 109 for their help in collecting root tips of Stachys thirkei. The authors gratefully acknowl- edge the financial support of “Progetto di Ricerca di Ateneo” (PRA) of the University of Pisa, grant number PRA_2018_15, for the chromosome counts of Pulmonaria. References Astuti G, Liu L, Peruzzi L (2019) Chromosome numbers for the Italian flora: 7. 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