CARYOLOGIA Vol. 56, no. 3
Vol. 56, no. 3: 359-363, 2003
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.
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:
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.
MATERIAL AND METHODS
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,
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
Squash preparations were made from root tips
(Isoëtes and Botrychium) or from mother cells of
Fig. 1 – Metaphasic plate from root tips of Isoëtes subinermis, 2n = 22 (A); haploid idiogram of I. subinermis (B). Scale bars
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.
Long arms (mean lenght of the
homologue chromosomes, 2x)
Mean lenght (L)
Short arms (mean lenght of the
homologue chromosomes, 2x)
Mean lenght (S)
Total lenght (TL)
Ratio Long arm / Short arm (L/S)
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.
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).
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
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.
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.
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
Botrychium lunaria – This plant shows a 2n
= 90 chromosome complement (Fig. 4A, B).
Our count is the first for Italian populations, and agrees with numbers reported by
several authors from elsewhere (M ANTON
1950; WAGNER 1955; WAGNER and LORD 1956;
LÖVE and LÖVE 1961, 1976; LÖVE et al. 1971;
ENGELSKJON 1979; SAHASHI 1979; DALGAARD
1989). Our data confirm the extreme karyological homogeneity of this species. Different sporadically reported countings, as 2n = 96
(HAGERUP 1941), 2n = ca. 80 (ZHUKOVA and
PETROVSKY 1975) and 2n = ca. 60 (GAGNIDZE
et al., 1997), are probably mistaken.
KARYOLOGICAL DATA ON ISOËTES, OPHIOGLOSSUM AND BOTRICHIUM
BROWNLIE G., 1955 – Chromosome numbers in New
Zealand fern. Transactions of the Royal Society,
New Zealand, 85: 213-216.
CESCA G., PERUZZI L., 2001 – Isoëtes (Lycophytina,
Isoetaceae) with terrestrial habitat in Calabria
(Italy). New karyological and taxonomical data.
Flora Medit., 11: 303-309.
CESCA G., PUNTILLO D., 1982 – Notizie sulla flora
pteridologica della Calabria. In: Cesca G., “Contributi alla conoscenza delle piante di Calabria
10-13”. Giorn. Bot. Ital., 116 (Suppl. 1): 182186.
DALGAARD V., 1989 – Additional chromosome numbers in vascular plants from the Disko Bugt area
(west Greenland). Willdenowia, 19: 199-213.
ENGELSKJON T., 1979 – Chromosome numbers in
vascular plants from Norway, including Svalbard.
Opera Bot., 52: 1-38.
FIORI A., 1926 – “Nuova Flora Analitica d’Italia”, 1:
FIORI A., 1943 – “Flora Italica Cryptogama”, 5:
GAGNIDZE R., MTSKHVETADZE D., GVINIASHVILI
T., AVAZNELI T., 1997 – IOPB chromosome data
11. Newslett. Int. Organ. Pl. Biosyst. (Oslo),
GIOVANNINI A., GIORDANI A., GARBARI F., 2001 –
The chromosome number of Isöetes duriei Bory
(Lycopodiophyta, Isoetaceae). Webbia, 56: 219221.
HAGERUP O., 1941 – Nordiske Kromosom Tal. I.
Bot. Tidsskr., 45: 385-395.
KHANDELWAL S., 1978 – Cytological and Physiological studies on Ophioglossum L. Ph.D thesis of
Jiwaji University, Gwalior, India.
–, 1987 – Further studies on morphology, biochemistry, cytology and fine structure of spores of
Ophioglossum Linn. D.Sc thesis of Jiwaji University, Gwalior, India.
–, 1990 – Chromosome evolution in the genus
Ophioglossum L. Bot. Jour. Linn. Soc., 102: 205217.
JALAS J., SUOMINEM J. (Eds.), 1972 – “Atlas Florae
Europaeae”, 1: 31. Cambridge.
LEVAN A., FREDGA K., SANDBERG A. A., 1964 –
Nomenclature for centromeric position on chromosomes. Hereditas, 52: 201-220.
LÖVE A., LÖVE D., 1961 – Some chromosome numbers of Icelandic ferns and fern allies. Amer. Fern
Jour., 51: 127-128.
–, 1976 – In IOPB chromosome number reports LIII.
Taxon, 25: 483- 500.
LÖVE A., LÖVE D., KAPOOR B. M., 1971 – Cytotaxonomy of a century of rocky moutain orophytes.
Arctic Alp. Res., 3: 139-165.
MANTON I., 1950 – “Problems of cytology and evolution in the pteridophyta”. Cambridge.
–, 1953 – Cytological evolution of the fern flora of
Ceylon. Symposium Society Experimental Biology, Evolution, 7: 174-178.
MONTGOMERY L., KHALAF M., BAILEY J.P., GORNAL
K.J., 1997 – Contributions to a cytological catalogue of the British and Irish flora, 5. Watsonia,
NINAN C.A., 1956 – Cytology of the Ophioglossaceae. Current Science, 25: 161-162.
–, 1958 – Studies on the cytology and phylogeny of
the pteridophytes. VI. Observations on the
Ophioglossaceae. Cytologia, 23: 291-315.
PIGNATTI S., 1982 – “Flora d’Italia”, 1: 45. Bologna.
S AHASHI N., 1979 – Chromosome number in
Botrychium lunaria from Japan. J. Jap. Bot. 54: 60.
TAYLOR T. N., TAYLOR E. L., 1993 – “The Biology
and Evolution of Fossil Plants”. Englewood
Cliffs, New Jersey.
TROÌA A., 2001 – The genus Isoëtes L. (Lycophyta,
Isoëtaceae): synthesis of karyological data. Webbia, 56: 201-218.
TROÌA A., BELLINI E., 2001 – Karyological observations on Isoëtes duriei Bory (Lycophyta,
Isoetaceae) in Sicily. Bocconea, 13: 397-400.
VERMA S.C., 1956 – Cytology of Ophioglossum. Current Science, 25: 398-399.
–, 1957 – Cytology of Ophioglossum coriaceum A.
Cunn. Cytologia, 22: 393-403.
WAGNER W.H., 1955 – Cytotaxonomic observations
on North American ferns. Rhodora, 57: 219-240.
WAGNER W.H., LORD L.P., 1956 – The morphological and cytological distinctness of Botrychium
minganense and B. lunaria in Michigan. Bull.
Torrey Club, 83: 261-280.
ZHUKOVA P.G., PETROVSKY V.V., 1975 – Chromosome numbers of some Western Chukotka plant
species. Bot. Zhurn., 61: 963-969.
Received November 11, 2002; accepted June 14, 2003