Vol. 56, no. 3: 359-363, 2003 CARYOLOGIA Isoëtes (Isoetaceae), Ophioglossum and Botrychium (Ophioglossaceae) in Calabria (S Italy): more karyological and taxonomical data LORENZO PERUZZI*, GIULIANO CESCA, DOMENICO PUNTILLO Museo di Storia Naturale della Calabria ed Orto Botanico, Università della Calabria, 87030 Arcavacata di Rende (CS). Italy. Abstract - I. subinermis (Durieu) Cesca and Peruzzi, from a newly discovered Calabrian stand, was karyologically studied. Both chromosome number (2n = 22) and karyotype are presented. Systematic distinctiveness of I. subinermis from I. histrix Bory is confirmed. A new chromosome complement (2n = 44) is recorded for I. duriei Bory, confirming the karyological variability of this taxon. Ophioglossum lusitanicum L. (n = 120) and Botrychium lunaria L. (2n = 90) were karyologically studied too. Their chromosome numbers are reported as the first counts on Italian populations. Key words: Botrychium, karyology, Isoëtes, Ophioglossum, taxonomy. INTRODUCTION The genera Isoëtes (Isoetaceae), Ophioglossum and Botrychium (Ophioglossaceae) are all considered as taxonomic relics. The first is the only extant genus (incl. Stylites E. Amstutz) related with fossil mesozoic lycophytes, such as Pleuromeiales (TAYLOR and TAYLOR 1993); while the second and third ones are ferns with primitive organization, showing separate fertile and sterile segments of the frond. According to CESCA and PERUZZI (2001) there are three species of Isoëtes in Calabria (Southern Italy): I. duriei Bory, a polyploid species (2n = 55), I. histrix Bory (2n = 20), aneuploid, and I. subinermis (Durieu) Cesca and Peruzzi** (2n = 22), diploid. The three taxa have been often collected from the same areas, where they grew together. The recent discovery of a new population of I. subinermis in Calabria, near Crotone, * Corresponding author: fax +39 0984 838573; e-mail: peruzzi@unical.it gave the opportunity to verify the systematic position of this taxon with respect to I. histrix, which has a similar aspect. Two new populations of I. duriei Bory were also recently discovered (see Material and Methods), one of which is karyologically studied in this paper. According to KHANDELWAL (1990), the genus Ophioglossum includes species with the highest chromosome numbers known among plants (n = 720 in O. reticulatum L.). Ophioglossum lusitanicum L. is a rare little fern, sporadically occurring in acid grasslands of Middle-Southern Italy (PIGNATTI, 1982). This species was already known for Calabria (CESCA and PUNTILLO, 1982), but it was never karyologically studied in Italy up to now. Botrychium lunaria L. is a subcosmopolite species that rarely occurs in mountain grasslands in Italy (PIGNATTI, 1982); neither this species was karyologically studied of Italy. ** If the synonymy of this unit with I. sicula Tod., already proposed by Fiori (1926, 1943), will be shown to be correct, this latest name must be used for this plant. The problem deserves further close examinations. 360 PERUZZI, CESCA MATERIAL AND METHODS Living plants I. subinermis: Calabria, loc. Fratte, between Capo Colonna and Isola Capo Rizzuto, grassland together with Ferula, 20/II/2002, Puntillo (cult. Hort. Bot. Calabria University, acc. n. 51); I. duriei: Calabria, Sila, Rose (Cosenza), 1 Km Est in proximity to Statal Road n. 276, ca. 650 m a.s.l. loc. Giancorella, and PUNTILLO 16/III/2002, Puntillo; O. lusitanicum: Calabria, along the Gidora stream, 16/XI/1983, Puntillo; B. lunaria: Calabria, Mount Cozzo del Pellegrino, ca. 1600 m a.s.l., grassland, 10/V/2002, Peruzzi, Gargano et Donato. Karyological study Squash preparations were made from root tips (Isoëtes and Botrychium) or from mother cells of A B Fig. 1 – Metaphasic plate from root tips of Isoëtes subinermis, 2n = 22 (A); haploid idiogram of I. subinermis (B). Scale bars 5µm. Table 1 – Measurements made on 5 metaphasic plates, belonging to five specimens of Isoëtes subinermis (Durieu) Cesca and Peruzzi. Data were obtained from microphotographs, then reported in µm. Chromosomes Long arms (mean lenght of the homologue chromosomes, 2x) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Mean lenght (L) Short arms (mean lenght of the homologue chromosomes, 2x) Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Mean lenght (S) Total lenght (TL) Ratio Long arm / Short arm (L/S) Karyotype formula I II III IV V VI VII VIII IX X XI 1,70 1,23 1,06 1,27 1,14 1,28 1,89 1,42 1,43 1,46 1,58 1,56 1,69 1,39 1,38 1,31 1,36 1,43 1,42 1,38 1,13 1,48 1,21 1,32 1,35 1,53 1,26 1,34 1,34 1,36 1,35 1,28 0,91 1,14 1,00 1,14 1,15 1,07 0,99 1,03 0,90 1,03 1,46 1,40 1,11 1,25 1,03 1,25 1,46 1,12 0,85 1,24 1,13 1,16 1,34 1,02 0,70 0,93 0,88 0,97 1,08 0,90 0,60 1,00 0,75 0,87 1,58 1,18 0,97 1,19 1,09 1,20 2,48 0,74 0,99 0,54 0,71 0,47 0,69 2,25 0,79 0,98 0,67 0,82 0,71 0,79 2,22 0,91 0,79 0,79 0,64 0,70 0,77 2,09 0,73 0,63 0,39 0,76 0,55 0,61 1,98 1,05 0,96 0,61 0,95 0,83 0,88 1,91 0,67 0,66 0,57 0,68 0,61 0,64 1,89 0,60 0,69 0,55 0,56 0,51 0,58 1,74 0,71 0,88 0,60 0,83 0,71 0,75 1,72 0,64 0,56 0,39 0,56 0,66 0,56 1,43 1,06 2M 2,26 1,80 1,73 2,23 0,83 0,80 0,84 0,90 0,83 0,84 1,98 21,68 1,35 4m 1,17 1,96 1,99 1,31 8sm 4sm 1,54 4m KARYOLOGICAL DATA ON ISOËTES, OPHIOGLOSSUM AND BOTRICHIUM megaspores (Ophioglossum) of plants collected in situ, according to the following schedule: pretreatment in 0,5% colchicine solution for 4 hours; Carnoy fixing for 1 hour; hydrolysis in HCl 1 N for 7 minutes at 60°C; staining with leuco-basic fucsin for 3 hour. Karyotype formulas and terminology are according to LEVAN et al. (1964). At least five plates were measured in order to build the idiograms. 361 A Herbarium specimens All the vouchers of the studied plants are conserved in the Herbarium of the University of Calabria (CLU). In addition, we checked the followings specimen: I. duriei: Italy, Calabria: Cotronei (Crotone) alla centrale ENEL vicino le Terme Vecchie, UTM XD 58.38, alt. 130 m s.l.m., 11/I/1998, Puntillo (CLU). B RESULTS AND DISCUSSION Isoetes subinermis – This species shows a diploid chromosome complement 2n = 22 (Fig. 1A). The measurements carried out (Table 1) have enabled us to build the haploid idiogram (Fig. 1B). Karyotype formula can be expressed as follows: 2n = 2x = 22 = 2M + 8sm + 4m + 4sm + 4m. According to the synthesis of karyological knowledge of Isoëtes made by TROÌA (2001), detailed karyotype analysis in this genus appear to be very rare or absent. In fact, it is very difficult to obtain with these plants good metaphase plates where centromeres are evident, and the occurrence in this work is quite unusual. Population from the new stand of I. subinermis have confirmed the taxonomic status of this species. In fact, all the studied plants show a diploid complement, and the related morphologic features (CESCA and PERUZZI 2001), such as phyllopodia with short prickles trunked at the base and tubercolate-coniculate megaspores, which permit to definitely distinguish this species from I. histrix, aneuploid, showing phyllopodia with long lateral prickles and tuberculate megaspores. In support to this view, a 2n = 20 count for the latter was recently reported for British and Irish Islands too (M ONTGOMERY et al. 1997). I. subinermis, considering the dry stand where we observed the studied population (which, in Calabria, is the only not admixed with I. histrix or I. duriei), appear to be a species more adapted to the Mediterranean climate than I. histrix, which has a Steno-MediterraneanAtlantic distribution (J ALAS and S UOMINEM 1972). Fig. 2 – Metaphasic plates from root tips of Isoëtes duriei, 2n = 44 (A)and relative drawing (B). Scale bars 5µm. Isoetes duriei – This species shows a tetraploid chromosome complement 2n = 44 (Fig. 2A, B). Only the chromosome numbers 2n = 55, 110, ca. 120 were reported up to now for this species (CESCA and PERUZZI 2001; GIOVANNINI et al. 2001; TROÌA and BELLINI 2001) in plants from Calabria, Tuscany and Sicily, respectively. Our count, 2n = 44, is new for this taxon. This species seems to represent a polyploid series, based on x = 11. I. duriei has a W-Mediterranean-Atlantic distribution with oriental limit in Italy and few isolated stands in Greece (JALAS and SUOMINEM 1972). Lower known ploidy levels occur in Calabria, and it could lead us to think this species originating in S Italy, and then spread westwards. However, the cytogeographic knowledge of this taxon is still too partial to allow us drawing any type of conclusion: karyological data outside Italy in fact totally lack. 362 PERUZZI, CESCA and PUNTILLO Fig. 3 – Chromosomes of Ophioglossum lusitanicum L. showing n = 120 bivalents (A); and relative drawing (B). Scale bars 5µm. Fig. 4 – Metaphasic plate from root tips of Botrychium lunaria, 2n = 90 (A); and relative drawing (B). Scale bars 5µm. Ophioglossum lusitanicum – This species shows an haploid chromosome complement n = 120 (Fig. 3A, B). According to KHANDELWAL (1990), n = 120 would be a neobasic number in Ophioglossum, and every extant species has at least some representatives with this complement. This neobasic number should be derived by palaeobasic number n = 30, produced by three distinct ultimate base numbers (4, 5, 6). In Ophioglossum the ploidy level does not appear to be at present related with morphological and ecological plasticity, thus this genus would represent an evolutionary dead end. Our count is the first for Italian populations, and agrees with data by KHANDELWAL (1987) himself (with Indian provenance) and by MANTON (1950) who counted n = 123-130 in material from England. The same species showed chromosome complements n = 240 (M ANTON 1953; NINAN 1956, 1958; KHANDELWAL 1978, 1987) in Indian material, n = 360 (B ROWNLIE 1955; KHANDELWAL , 1978) in plants from New Zealand and India, n = 510 (V E R M A 1956, 1957) in plants of Australian provenance. Indeed, n = 120 cytotype appears to be restricted to Eurasia. Botrychium lunaria – This plant shows a 2n = 90 chromosome complement (Fig. 4A, B). 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