Research Article 
Corresponding author: Pepy Bareka ( bareka@aua.gr ) Academic editor: Gennady Karlov
© 2016 Sofia Samaropoulou, Pepy Bareka, Georgia Kamari.
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.
Citation:
Samaropoulou S, Bareka P, Kamari G (2016) Karyomorphometric analysis of Fritillaria montana group in Greece. Comparative Cytogenetics 10(4): 679695. https://doi.org/10.3897/CompCytogen.v10i4.10156

Fritillaria Linnaeus, 1753 (Liliaceae) is a genus of geophytes, represented in Greece by 29 taxa. Most of the Greek species are endemic to the country and/or threatened. Although their classical cytotaxonomic studies have already been presented, no karyomorphometric analysis has ever been given. In the present study, the cytological results of Fritillaria montana Hoppe ex W.D.J. Koch, 1832 group, which includes F. epirotica Turrill ex Rix, 1975 and F. montana are statistically evaluated for the first time. Further indices about interchromosomal and intrachromosomal asymmetry are given. A new population of F. epirotica is also investigated, while for F. montana, a diploid individual was found in a known as triploid population. Paired ttests and PCoA analysis have been applied to compare the two species.
Fritillaria epirotica, Fritillaria montana, karyotype analysis, PCoA, endemics, Greek flora, karyograms
The genus Fritillaria Linnaeus, 1753 (Liliaceae) comprises approximately 150 taxa of geophytes, found in the temperate zones of the Northern Hemisphere (
According to its latest revision (
In Greece, the genus is also characterized by high diversity and is represented by a multitude of taxa (24 species and 5 subspecies), all belonging to the subgenus Fritillaria (
Out of the 29 taxa found or described in Greece so far, 18 taxa (14 species and 4 subspecies) are endemic to the country and no less than 17 species and 2 subspecies occur in the Aegean archipelago and the surrounding continental region (
Among the Fritillaria taxa occurring in continental Greece two species constitute the Fritillaria montana group (
Fritillaria epirotica is a very short plant (up to 15 cm) with dark purplish, obscurely tessellated flowers, which almost touch the ground and it grows on ophiolithic substrates, usually at high altitudes (up to 2600 m). On the contrary, F. montana is tall (up to 60 cm), characterized by alternate or subopposite linear, slightly canaliculated leaves, with dark purplish distinctly tessellated flowers, and it grows usually on limestone substrate at an altitude up to 1600 m.
Fritillaria epirotica is included in the Red Data Book of Rare and Threatened Plants of Greece (
Concerning the cytology of the genus, Fritillaria has been studied for many years due to the interest of its large chromosomes and vast genome size (
Recently many questions have arisen, regarding the classification and phylogeny of the genus, especially for the species appearing in Greece. Although several molecular phylogenetic studies have been published (
Living plants of the Fritillaria montana and F. epirotica populations were collected (Table
Origin, chromosome numbers (2n) and voucher number of Fritillaria material.
Taxon  Origin  2n  Voucher number, Herbarium 

F. montana  Mt. Vourinos (W Macedonia)  18  16765, UPA 
Mt. Kato Olympos (Thessalia)  18  SF1089, ACA cult. no. 253, UPA 

Mt. Boutsi (NW Macedonia)  27 and 18 (1 individual) 
SF1092, ACA 19865, UPA 

F. epirotica  Katara Pass (Epirus)  24  21348, UPA 7919, UPA 
Mt. Vasilitsa (N Pindos)  24  SF1076, ACA  
Mt. Smolikas (N Pindos)  24  SF1097, ACA  
Mt. Kratsovo (W Thessalia)  24  cult. no. 255, UPA 
The cytological study is based on the squash technique and the chromosome counts were made from root tip metaphases (
Before staining, the root tips were hydrolyzed in 1N HCl 60 °C for 15 min and stained in Feulgen for 3 hrs (
At least five metaphase plates of each species were analysed and indices were calculated with Microsoft Office Excel 2007, IBM SPSS Statistics version 22 and Past 3.03. Chromosome terminology follows
Fritillaria montana
Hoppe — 2n = 2x = 18 + 03B (Figs
Photomicrograph of mitotic metaphase plate of Fritillaria montana from Mt. Vourinos, 2n = 2x = 18. Bar = 10 µm.
Populations karyologically studied:
Greece: Macedonia: Nomos Kozanis: mons Vourinos, in declivibus orientalibus cacuminis, alt. 13001350 m, in apertis ad viam et in silva Abietis et Pinetis, solo ophiolithico, 9 Jul 1981, T.R. Dudley, D. Phitos, D. Tzanoudakis, Gr. Iatrou & D. Christodoulakis 16765 (UPA); Thessalia: Nomos Larissis: Mt. Kato Olympos, Livadaki, north of Kallipefki, alt. ca. 1407 m, 39°57'N; 22°29'E, 30 May 2015, S. Samaropoulou, I. Patrikios & K. Tamvakas SF1089 (ACA); Mt. Kato Olympos, Livadaki, alt. 1400 m, May 2006, K. Tamvakas 253 (UPA).
Fritillaria montana is the only Greek species with a basic chromosome number of x = 9, having 2n = 18 chromosomes (Fig.
The karyotype formula of the studied populations is given as 2n = 10m + 2st + 6t = 18 (Fig.
Studied species with karyomorphometric indices. Chromosome number (2n), total (TCL) and average (ACL) chromosome length, total haploid chromosome length (THL), maximum (max l + s) and minimum (min l + s) chromosome length, karyotype asymmetry indices (CV_{CL}, CV_{CI} and M_{CA}).
Species  F. montana  F. epirotica  

Chromosome number  2n = 2x = 18  2n = 3x = 27  2n = 2x = 24 
Karyotype formula  10m + 2st + 6t  15m + 3st + 9t (10m + 4st + 4t, 1 individual) 
2m + 2sm + 14st + 6t 
TCL (µm) (SD) 
316.34 (30.22) 
363.23 (53.47) 
324.39 (51.12) 
THL (µm) (SD) 
158.17 (15.11) 
121.08 (17.82) 
162.2 (25.56) 
ACL (µm) (SD) 
17.57 (1.68) 
13.45 (1.98) 
13.52 (2.13) 
max l + s (µm)  24.41  22.86  18.44 
min l + s (µm)  11.26  8.00  10.00 
CV_{CL} (SD) 
25.26 (1.12) 
31.07 (3.61) 
16.85 (2.43) 
CV_{CI} (SD) 
56.21 (0.99) 
54.79 (3.57) 
51.66 (2.99) 
M_{CA} (SD) 
41.42 (0.35) 
40.41 (1.18) 
63.33 (1.25) 
Fritillaria montana
Hoppe — 2n = 3x = 27 and 2n = 2x = 18 (1 individual) (Figs
Photomicrograph of mitotic metaphase plate of Fritillaria montana from Mt. Boutsi, 2n = 3x = 27. Bar = 10 µm.
Populations karyologically studied:
Greece: Macedonia: Nomos Florinas: Montes Triklarion, in declivibus boreooccidentalibus cacuminis Boutsi, in apertis saxosis calc., alt. 14501550 m, 19 May 1987, D. Phitos & G. Kamari 19865 (UPA); Mt. Boutsi, alpine meadow, calcareous substrate, alt. ca. 1549 m, 40°38'33"N; 21°09'25"E, 2 Jun 2015, S. Samaropoulou, I. Patrikios & A. Ioannou, sub Samaropoulou SF1092 (ACA).
The triploid population previously reported for the first time by
It is noteworthy that a diploid individual was found for the first time at the studied triploid population. The karyotype of this individual comprises 2n = 10m + 4st + 4t = 18 chromosomes, with an additional pair of acrocentric (st) chromosomes compared with the other diploid populations studied and without Bchromosomes in contrast with the population of Mt. Vourinos. The secondary constrictions were also unclear as in the triploid individuals.
Fritillaria epirotica
Turrill ex Rix — 2n = 2x = 24 (Figs
Photomicrograph of mitotic metaphase plate of Fritillaria epirotica from Mt. Smolikas, 2n = 2x = 24. Bar = 10 µm.
Populations karyologically studied:
Greece: Epirus: Nomos Ioanninon: Katara Pass, prope ekchionistikos stathmos, alt. 1750 m, in apertis (Pinus Linnaeus, 1753; Buxus Linnaeus, 1753 etc), solo serpentinico, 4 May 1990, D. Phitos & G. Kamari 21348 (UPA); Eparchia Metsovou, Katara Pass, close to the second snowplow station, c. 13.5 km of Metsovon along the road to Trikala, slopes with Pinus nigra Arnold, 1785 and Buxus sempervirens Linnaeus,1753; ophiolithic substrate, alt. c. 1640 m, 39°47'N; 21°13'E, 24 Jun 1998, Th. Constantinidis 7919 (UPA); Macedonia: Nomos Grevenon: Mt. Vasilitsa, alt. 1764 m, 17 May 2015, G. Kofinas s.n. (cult. no. SF76, ACA); Mt. Smolikas, alt. 2200 m, Aug 2015, G. Kofinas s.n. (cult. no. SF97, ACA). Thessalia: Nomos Trikalon: Ep. Kalampakas, Mt. Chasia (Kratsovo), stony slopes close to a forest road, c. 3.0–3.5 km from Kakoplevri village, serpentine, alt. c. 1100–1180 m, 39°48'N; 21°24'E, 15 Jun 2000, D. Phitos, G. Kamari & Th. Constantinidis s.n. (cult. no. 235, UPA); Ep. Kalampakas, Mt. Chasia (Mt. Kratsovon), c. 3.1 km WNW of Kakoplevri village on the foothills of the mountain, hills with low Buxus sempervirens and Juniperus oxycendrus Linnaeus,1753; serpentine substrate, alt. 1120–1160 m, 39°49'N; 21°24'E, 24 Jul 2006, Th. Constantinidis s.n. (cult. no. 235, UPA).
Unlike Fritillaria montana, F. epirotica has the same basic somatic number as the rest of the Greek Fritillaria taxa, x = 12. The karyotype consists of 2n = 2m + 2sm + 14st + 6t = 24 chromosomes (Figs
Karyomorphometric indices of marker chromosomes for each species, marker chromosome pairs (numbered according to their chromosome length), long arm’s length (l), short arm’s length (s), chromosome length (l + s) with minimum and maximum prices, r index, Centromeric index, Arm difference ratio, Rlength.
Species  Fritillaria montana  Fritillaria epirotica  

Chromosome number  2n = 18  2n = 24  
marker chromosomes  Pair no. 1  Pair no. 5  Pair no. 1  Pair no. 2  Pair no. 3  Pair no. 5 
l (µm) (SD) 
12.84 (1.00) 
10.71 (1.09) 
9.68 (1.05) 
10.51 (1.04) 
12.19 (1.07) 
10.87 (1.23) 
s (µm) (SD) 
11.15 (0.79) 
6.93 (0.67) 
6.86 (0.82) 
5,27 (0.63) 
1.60 (0.44) 
1,82 (0.36) 
l + s (µm) (SD) 
23.99 (1.71) 
17.65 (1.66) 
16.53 (1.73) 
15.77 (1.49) 
13.79 (1.18) 
12.42 (1.51) 
min l + s (µm)  20.88  15  13.53  12.94  11.47  8.40 
max l + s (µm)  26.47  20  19.12  18.24  15.59  15 
rindex l/s 
1.15  1.54  1.40  2.03  8.06  6.24 
Centromeric index l/l + s 
0.54  0.61  0.58  0.67  0.88  0.85 
Arm difference ratio l  s/l + s 
0.70  0.21  0.17  0.34  0.77  0.71 
Rlength l + s/Sn(l + s) 
0.08  0.06  0.05  0.05  0.04  0.04 
According to paired ttests made (Table
Paired ttests between the three species regarding the TCL and ACL, along with degrees of freedom (df) and Significance (Sig) for every parameter. Bold characters are used for P values (Sig 2tailed) under 0.01, which reveal significant statistical difference.
Species in comparison  TCL  ACL  

t  df  Sig (2tailed)  t  df  Sig (2tailed)  
F. epirotica
2n = 2x = 24 
F. montana
2n = 2x =18 
0.379  18  0.709  4.347  18  0.000 
F. epirotica
2n = 2x = 24 
F. montana
2n = 3x = 27 
1.427  16  0.173  0.057  16  0.955 
F. montana 2n = 2x = 18 
F. montana
2n = 3x = 27 
1.947  10  0.080  3.898  10  0.003 
Paired ttests between the three species regarding the CV_{CL} and M_{CA}, along with degrees of freedom (df) and Significance (Sig) for every parameter. Bold characters are used for P (Sig 2tailed) under 0.01, which reveal significant statistical difference.
Species in comparison  CV_{CL}  M_{CA}  

t  df  Sig (2tailed)  t  df  Sig (2tailed)  
F. epirotica
2n = 2x = 24 
F. montana
2n = 2x = 18 
8.598  18  0.000  44.847  18  0.000 
F. epirotica
2n = 2x = 24 
F. montana
2n = 3x = 27 
9.754  16  0.000  34.473  16  0.000 
F. montana 2n = 2x = 18 
F. montana
2n = 3x = 27 
4.066  10  0.002  1.995  10  0.074 
In the present study a detailed karyomorphological analysis of Fritillaria montana and Fritillaria epirotica, in material from Greece, was implemented focusing specifically to the study of the inter and intrachromosomal asymmetry, as well as the detailed analysis of the marker chromosomes.
The study of marker chromosomes (Table
Marker chromosomes were observed in both F. epirotica with 2n = 2x = 24 and F. montana with 2n = 2x = 18 chromosomes. However, when it comes to triploid karyotypes of the same species, the secondary constrictions are not stable in number and position.
Fritillaria epirotica (2n = 24) has four marker chromosome pairs (Fig.
Fritillaria montana (2n = 18) has two marker chromosome pairs with secondary constrictions. The karyotype formula given here (2n = 10m + 2st + 6t = 18) differs from the previously reported karyotype of 2n = 10m + 8t = 18 chromosomes given by
The triploid chromosome number of F. montana (2n = 3x = 27) is known in Greece from only one population, but it has also been reported from Italy by
Paired ttests have revealed similarities among the three karyotypes. Especially the similarity between TCL of the diploid F. epirotica 2n = 24 and F. montana 2n = 18 reinforces the hypothesis, apart from the secondary constrictions, that the second species has derived after successive chromosomal reconstructions and Robersonianfusions. Less similar indices of TCL between F. montana 2n = 2x = 18 and F. montana 2n = 3x = 27 can also be explained since it is known that polyploidy usually comes with gene loss and genome amount reduction (
The results concerning the heterogeneity of centromere position CV_{CI} and the intrachromosomal asymmetry M_{CA} are nothing but expected. Following the explanation of this index by
In total, the multivariate analysis PCoA confirms all above findings. More precisely, it presents all the accessions belonging to the same species close to each other. The two cytotypes of Fritillaria montana overlap, while the two species are clearly separated (Fig.
PCoA analysis based on six quantitative karyologial parameters. Triangle depicts F. epirotica, 2n = 2x = 24; dots F. montana, 2n = 2x = 18 and x F. montana, 2n = 3x = 27.
Generally, karyological characteristics, as chromosome number, ploidy level, centromere position, and the number and location of satellites and secondary constrictions, can be used in elucidating taxonomical relationships of several plant taxa (