New Chromosomal Data and Karyological Relationships in Geranium: Basic Number Alterations, Dysploidy, Polyploidy, and Karyotype Asymmetry

Abstract

Abstract Chromosomal data and karyological relationships provides valuable contributions to understanding speciation and karyotypic phylogeny. Because of the large number of species, wide distribution, morphological differences and chromosomal variations, Geranium is an important genus for determining the relationship between chromosomal alterations and karyotypic phylogeny. In the present study, the chromosomal data of 38 taxa are provided, nine of which are given for the first time (G. eginense, G. gracile, G. ibericum subsp. jubatum, G. lasiopus, G. libani, G. libanoticum, G. petri-davisii, G. ponticum, G. psilostemon), five present new chromosome numbers (G. asphodeloides, G. ibericum subsp. ibericum, G. molle subsp. molle, G. pretense, G. rotundifolium), and 24 agree with previous reports. Eleven different diploid numbers (2n = 18, 20, 22, 26, 28, 30, 32, 46, 48, 64, and 84) are detected. In basic numbers, infraspecific variations are encountered. The comprehensive variations of basic numbers and the relatively low rate of polyploid species showed in the present study promote the evolutionary significance of karyotype alterations by dysploidy mechanism. Regarding karyological relationships, G. sanguineum forms a monophyletic group by quite different karyological features, which are different basic number, diploid number, and karyotype sample and high ploidy level. Other clad consists of two subclades with a medium strong monophyletic group. In regression analyses, there are significant positive correlations between THL and 2n/ploidy levels. Asymmetry indices (CVCL and MCA) show weak positive correlations mainly caused by polyploidy. The most asymmetrical karyotypes are G. molle subsp. bruitium in intrachromosomal asymmetry and G. asphodeloides in interchromosomal asymmetry.

Full text

Vol.65: e22210354, 2022
https://doi.org/10.1590/1678-4324-2022210354
ISSN 1678-4324 Online Edition
Brazilian Archives of Biology and Technology. Vol.65: e22210354, 2022 www.scielo.br/babt
Article - Biological and Applied Sciences
New Chromosomal Data and Karyological
Relationships in Geranium: Basic Number Alterations,
Dysploidy, Polyploidy, and Karyotype Asymmetry
Esra Martin1
https://orcid.org/0000-0002-5484-0676
Ahmet Kahraman2
https://orcid.org/0000-0002-9344-1993
Tuncay Dirmenci3
https://orcid.org/0000-0003-3038-6904
Havva Bozkurt1
https://orcid.org/0000-0003-0223-2895
Halil Erhan Eroğlu4*
https://orcid.org/0000-0002-4509-4712
1Necmettin Erbakan University, Faculty of Science, Department of Biotechnology, Konya, Turkey; 2Uşak University,
Faculty of Science and Art, Department of Biology, Uşak, Turkey; 3Balıkesir University, Faculty of Necatibey Education,
Department of Biology Education, Balıkesir, Turkey; 4Yozgat Bozok University, Faculty of Science and Art, Department
of Biology, Yozgat, Turkey.
Editor-in-Chief: Alexandre Rasi Aoki
Associate Editor: Acácio Antonio Ferreira Zielinski
Received: 30-May-2021; Accepted: 24-Nov-2021.
*Correspondence: herhan.eroglu@bozok.edu.tr; Tel.: +90-354-2421021 (H.E.E.)
Abstract: Chromosomal data and karyological relationships provides valuable contributions to understanding
speciation and karyotypic phylogeny. Because of the large number of species, wide distribution,
morphological differences and chromosomal variations, Geranium is an important genus for determining the
relationship between chromosomal alterations and karyotypic phylogeny. In the present study, the
chromosomal data of 38 taxa are provided, nine of which are given for the first time (G. eginense, G. gracile,
G. ibericum subsp. jubatum, G. lasiopus, G. libani, G. libanoticum, G. petri-davisii, G. ponticum, G.
psilostemon), five present new chromosome numbers (G. asphodeloides, G. ibericum subsp. ibericum, G.
molle subsp. molle, G. pretense, G. rotundifolium), and 24 agree with previous reports. Eleven different
diploid numbers (2n = 18, 20, 22, 26, 28, 30, 32, 46, 48, 64, and 84) are detected. In basic numbers,
infraspecific variations are encountered. The comprehensive variations of basic numbers and the relatively
low rate of polyploid species showed in the present study promote the evolutionary significance of karyotype
alterations by dysploidy mechanism. Regarding karyological relationships, G. sanguineum forms a
monophyletic group by quite different karyological features, which are different basic number, diploid number,
and karyotype sample and high ploidy level. Other clad consists of two subclades with a medium strong
HIGHLIGHTS
• Karyological relationships are useful to infer processes of evolution and speciation.
• This paper reports chromosomal data of 38 taxa. First report (9) and new count (5).
• Dysploidy, polyploidy, and karyotype asymmetry are important parameters.
• Dysploidy and polyploidy variations are main factors in karyotype evolution of genus.

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monophyletic group. In regression analyses, there are significant positive correlations between THL and
2n/ploidy levels. Asymmetry indices (CVCL and MCA) show weak positive correlations mainly caused by
polyploidy. The most asymmetrical karyotypes are G. molle subsp. bruitium in intrachromosomal asymmetry
and G. asphodeloides in interchromosomal asymmetry.
Keywords: Geranium; chromosome alterations; ploidy levels; symmetrical karyotype.
INTRODUCTION
Geraniaceae is widely distributed around the world and is generally localized in subtropical and
temperate regions with five genera: Erodium L’Hér., California Aldasoro & C.Navarro & P.Vargas & L.Sáez
& Aedo, Pelargonium L’Hér., Monsonia L., and Geranium L. Geranium is represented by almost 400 species
and is localized in temperate areas and tropical elevations in many regions of the world except deserts, polar
regions and tropical plains [1]. In currently accepted classification, genus is divided into three subgenera:
subgen. Geranium, subgen. Robertium (Picard) Rouy, and subgen. Erodioidea (Picard) Yeo, [2]. In the
following years, while the subgenera included in this classification are accepted, the sectional classification
is reevaluated and new subsections were added as well [1,3-9]. As its systematic situation is always in
dispute, Geranium are excellent systems to use for determining diversification and speciation.
Chromosomal and karyological data contribution the properties determining interspecific relationships
and karyotype evolution. The primary chromosomal data are diploid number (2n), basic number (x), and
chromosome lengths. These properties could be replaced numerically through aneuploidy and polyploidy, as
well as through structural arrangements containing inversion, deletion, and translocation (which can change
chromosome number by dysploidy). All of these form interspecific and intraspecific variations, alter
centromere position and chromosome morphology, and affect karyotype asymmetries as intrachromosomal
asymmetry and interchromosomal asymmetry [10-17]. Geranium seems to be an important model for
understanding karyotype evolution due to global distribution, chromosome number variations (basic and
diploid), and various ploidy levels.
In genus Geranium, cytogenetic studies represent the variations of chromosome numbers from 2n = 14
in G. phaeum L. to 2n = 128 in G. robertianum L. and G. palmatum Cav., including intraspecific variations
[18-20]. According to the chromosome count database (CCDB, http://ccdb.tau.ac.il), the chromosome
numbers are reported from 91 species [18,20-38]. Fifty-two species are diploid; however, they exhibit five
different basic numbers: x = 11 (2n = 22), x = 12 (2n = 24), x = 13 (2n = 26), x = 14 (2n = 28), and x = 23 (2n
= 46). Twenty-three species are polyploid and reveal different polyploidy levels: triploidy (2n = 3x = 30, 39,
42), tetraploidy (2n = 4x = 32, 36, 48, 52, 56, 68), pentaploidy (2n = 5x = 50), and heptaploidy (2n = 7x = 84).
G. magellanicum Hook., G. palmatum, G. potentilloides L'Hér. ex DC., and G. robertianum indicate quite high
polyploidy (2n = 8x = 112, 128). Sixteen species are both diploid and polyploid [39]. Geranium seems to have
basic numbers ranging from as low as 7 up to 23 and more common x = 13 and 14. Probably some species
show dysploidy, which is an alteration of basic chromosome number generally [16,40]. Geranium taxa are
well characterized in point of all these chromosomal data. In some species, e.g. G. columbinum L. and G.
dissectum L., there is a large cytotaxonomic database [20-21,25,27,29,35]. However, there are still serious
shortcomings in the detailed karyotype data of genus Geranium and this prevents a more comprehensive
definition of major chromosome rearrangements in terms of karyotype evolution.
Seven sections, Batrachioidea W.D.J Koch, Divaricata Rouy, Dissecta Yeo, Lucida R. Knuth, Ruberta
Dumort., Unguiculata (Boiss.) Reiche, and Tuberosa (Boiss.) Reiche are distributed in Mediterranean region
and western Asia [1,3,7].
Sect. Batrachioidea and sect. Divaricata occurs six taxa, which are between Macaronesia and
Himalayas, Turkey, Caucasus, Iran, and north Africa [3]. In sect. Divaricata, diploid numbers are 2n = 20, 28
in G. albanum and 2n = 26, 28 in G. divaricatum Ehrh. and basic number is probably x = 14 [20-21,25,29,35].
In sect. Batrachioidea, the basic number is x = 13 by 2n = 26, 52 in diploid and polyploid species
[18,25,31,35]. Aedo and coauthors [3] reported that annual species, with various basic numbers, probably
evolved independently. In this context, the basic number (x = 13) could be seen as a derived character by
dysploidy.
Sect. Dissecta occurs four taxa, which are Lebanon, Turkey, between Sicily and Caucasus, and a
species distributed worldwide; it is probably indigenous to the Eurasian [7]. The only one chromosome
number is reported in G. dissectum (2n = 22) and G. sintenisii Freyn (2n = 26) [20,26,35]. By contrast, G.
asphodeloides Burm. has different chromosome numbers with 2n = 24, 28, 30, which are probably shaped
by dysploidy [20,31,37].

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Sect. Lucida, Ruberta and Unguiculata occur 12 taxa, which are Macaronesia, Turkey, southern Spain,
Morocco, southern France, and western Asia [9]. The only one chromosome number is reported in G.
glaberrimum Boiss. & Heldr. (2n = 30), G. cataractarum Coss. (2n = 36), G. dalmaticum (Beck) Rech. (2n =
46), and G. maderense Yeo (2n = 68) [26,29,41]. By contrast, G. lucidum L. has different chromosome
numbers with 2n = 20 and 2n = 40 to 42 (probably dysploidy) [20-21,24,29,35]. In addition, other species
show the high ploidy levels by 2n = 64, 68, 92, 112, and 128 [20-21,24,31-32,35].
In sect. Tuberosa, subsect. Mediterranea occurs ten taxa, which are spread in the Caucasus, Turkey,
Iran, northwestern Africa, and western Europe [1]. The only one chromosome number is reported in G.
bohemicum L., G. gymnocaulon DC. (2n = 28), and G. ibericum Cav. (2n = 56) [18,20,22,35]. By contrast, G.
lanuginosum Lam. has different chromosome numbers with 2n = 42 and 2n = 48 [21,29]. van Loon [29]
reported that chromosome number is probably shaped by dysploidy. In addition, G. platypetalum Fisch. & C.
A. Mey. has tetraploids and pentaploids [18]. It was considered that the basic number is x = 14 in subsect.
Mediterranea [29]. Subsect. Tuberosa occurs seven taxa, two of them are recorded as 2n = 28 [27-28]. The
basic number looks the same as for subsect. Mediterranea.
In the present study, it is aimed to provide a detailed survey of chromosomal variations and to contribute
to the cytotaxonomy, karyotype evolution and karyotypic phylogeny of Geranium. In summary: (i) we
determined chromosome numbers and made karyotype analyses of almost all Turkish Geranium; (ii) we
examined to ploidy levels and possible aneuploid forms to explain the variations in monoploid diploid sets;
(iii) we showed the karyotype asymmetries through the latest parameters for the first time; (iv) we established
a dendrogram with combined data to determine the interspecific relationships; and (v) we made the
regression analysis of chromosomal data versus karyomorphological data.
MATERIAL AND METHODS
Plant material
Distribution map is generated by Google Maps (Figure 1). Thirty-eight Geranium taxa were collected
from their natural habitats across Turkey. Exsicates were deposited at the herbarium of the Department of
Biological Sciences, at the Middle East Technical University (METU) in Ankara. The distribution regions,
collection information and chromosomal status are given in Table 1.
Figure 1. Distribution map of the studied species in Turkey. (1) G. asphodeloides; (2) G. bohemicum; (3) G. collinum;
(4) G. columbinum, G. lucidum; (5) G. dissectum; (6) G. divaricatum; (7) G. eginense; (8) G. glaberrimum; (9) G. gracile,
G. sanguineum, G. sintenisii; (10) G. gymnocaulon; (11) G. ibericum; (12) G. lanuginosum, G. macrorrhizum; (13) G.
lasiopus, G. purpureum, G. robertianum; (14) G. libani; (15) G. libanoticum; (16) G. macrostylum, G. tuberosum; (17)
G. molle; (18) G. palustre, G. pretense; (19) G. petri-davisii; (20) G. platypetalum; (21) G. ponticum; (22) G. psilostemon;
(23) G. pusillum; (24) G. pyrenaicum; (25) G. rotundifolium; (26) G. sibiricum; (27) G. subcaulescens; (28) G. sylvaticum.

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Table 2. The karyological parameters and formulae used for chromosome characterization.
Species
Distribution regions and collection information
Chromosomal
status
G. asphodeloides
Trabzon, Hayrat, Küçük Mesoraş, 2055 m, 06.08.2015, A. Kahraman
2185 (B)
Studied
G. bohemicum
Bolu, between Bolu and Yedigöller, 1433 m, 14.07.2015, A. Kahraman
2157
Studied
G. collinum
Stephan ex Willd.
Ardahan, Haçuvan village (Hasköy), 1500 m, 04.08.2015, A. Kahraman
2170
Studied
G. columbinum
Karabük, between Karabük and Bartın, 667 m, 12.07.2015, A.
Kahraman 2141
Studied
G. dissectum
Antalya, between Akseki and İbradı, 955 m, 11.05.2014, A. Kahraman
1774
Studied
G. divaricatum
Sivas, between Akdağmadeni and Şarkışla, Çamlıca village, 1386 m,
24.06.2015, A. Kahraman 2109
Studied
G. eginense Hausskn.
& Sint. ex R.Knuth
Erzincan, Kemaliye, Karanlık Canyon road, 890 m, 15.06.2016, A.
Kahraman 2387(B)
Unstudied
G. glaberrimum
Antalya, Gündoğmuş, Gelesandra plateau, 1473 m, 13.05.2014, A.
Kahraman 1789
Studied
G. gracile
Ledeb. ex Nordm.
Gümüşhane, towards to Zigana gateway, 1696 m, 17.07.2014, A.
Kahraman 1879
Unstudied
G. gymnocaulon
Artvin, Şavşat, 2210 m, 18.07.2016, A. Kahraman 2412
G. ibericum
Trabzon, between Hayrat and Sarmaşık village (Büyük Mesoraş), 1815
m, 19.07.2014, A. Kahraman 1900
Unstudied
G. lanuginosum
Balıkesir, Erdek, between Ormanlı and Ballı, 70-100 m, 19.05.2016, A.
Kahraman 2361
Studied
G. lasiopus
Boiss. & Heldr.
Antalya, Akseki, Güzelsu, 1190 m, 14.05.2014, A. Kahraman 1791
Unstudied
G. libani P.H.Davis
Hatay, Belen, Güzelyayla, 980-1050 m, 30.05.2015, A. Kahraman 2076
Unstudied
G. libanoticum Schenk
Antalya: between Derebucak-İbradı, 1343 m, 23.04.2016, A. Kahraman
2311
Unstudied
G. lucidum
Karabük, between Karabük and Bartın, 667 m, 12.07.2015, A.
Kahraman 2144
Studied
G. macrorrhizum L.
Balıkesir, Erdek, between Ormanlı and Ballı, 70-100 m, 19.05.2016, A.
Kahraman 2362
Studied
G. macrostylum Boiss.
Antalya, Elmalı, Uzungeriş hill, 2116 m, 20.06.2014, A. Kahraman 1837
Studied
G. molle L.
Manisa, between Kula and Salihli, 129 m, 06.04.2014, A. Kahraman
1701
Studied
G. palustre L.
Kars, between Sarıkamış and Karaurgan, 2139 m, 04.08.2015, A.
Kahraman 2174
Studied
G. petri-davisii Aedo
Kahramanmaraş, Höbek mountain, 2107 m, 01.07.2015, A. Kahraman
2130
Unstudied
G. platypetalum
Artvin, between Şavşat and Ardahan, 2275 m, 03.08.2015, A.
Kahraman 2167
Studied
G. ponticum (P.H.Davis
& J.Roberts) Aedo
Gümüşhane, Zigana mountain, from Kepenek hill to Yayla village (Alas),
2322 m, 17.07.2014, A. Kahraman 1886
Unstudied
G. pratense L.
Kars, between Sarıkamış and Karaurgan, 2139 m, 04.08.2015, A.
Kahraman 2175
Studied
G. psilostemon Ledeb.
Gümüşhane, between Kayaiçi village (Toroslu) and Yağmurdere, 1613
m, 18.07.2014, A. Kahraman 1891
Unstudied
G. purpureum Vill.
Antalya, between Manavgat and Akseki, 303 m, 11.05.2014, A.
Kahraman 1770
Studied
G. pusillum Burm.f.
Burdur, road of Fethiye-Denizli, between Çavdır and Acıpayam, 1030
m, 04.05.2015, A. Kahraman 2060
Studied
G. pyrenaicum Burm.f.
Karabük, Gölören village, 1058 m, 11.07.2015, A. Kahraman 2137
Studied
G. robertianum
Antalya, between Manavgat and Akseki, 534 m, 11.05.2014, A.
Kahraman 1772
Studied
G. rotundifolium L.
Muğla, Ortaca, 0 m, 03.05.2015, A. Kahraman 2058
Studied

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Cont. Table 2
G. sanguineum L.
Gümüşhane, Zigana road, Zigana village, 1412 m, 17.07.2014, A.
Kahraman 1877
Studied
G. sibiricum L.
Trabzon, Uzungöl area, 1100 m, 19.07.2016, A. Güngör & A. Kahraman
2414A
Studied
G. sintenisii
Gümüşhane, towards to Zigana gateway, 1704 m, 17.07.2014, A.
Kahraman 1880
Studied
G. subcaulescens
L Hér. ex DC.
Isparta, Aksu, Dedegöl mountain, Melikler plateau, 1815 m, 17.06.2014,
A. Kahraman 1831
Studied
G. sylvaticum L.
Düzce, between Düzce and Sakarya, Kaynaşlı, 413 m, 08.04.2015, A.
Karaman 1978
Studied
G. tuberosum L.
Antalya, Elmalı, Uzungeriş hill, 2138 m, 20.06.2014, A. Karaman 1838
Studied
Chromosome preparation
The seeds were germinated between moist Whatman papers in Petri dishes. The root tips were
pretreated in α-mono-bromonaphthalene at 4°C for 16 h. Then, the roots were fixed with Carnoy’s fixative
(absolute alcohol:glacial acetic acid - 3:1, v:v) at 4°C for 24 h and stored in 70% ethanol at 4°C until use. The
fixed roots were hydrolyzed in 1 N HCl at 60°C for 12 min, stained in 2% aceto-orcein, and squashed for
observations [16,42].
Karyotype analysis
At least ten metaphase cells were investigated to determine chromosome numbers. The chromosomal
measurements were made using the Software Image Analyses (Bs200ProP) loaded on a personal computer.
The following parameters were used to characterize the chromosomes numerically (Table 2). Karyotype
formulae were by chromosome morphology based on centromere position according to Levan and coauthors
[43]. The ideograms were drawn based on chromosome arm length (arranged large to small). Some data
obtained from Havva Bozkurt's master thesis were used in the article [44]. In Table 2, karyotype asymmetry
was estimated by two parameters: interchromosomal asymmetry (CVCL) and intrachromosomal asymmetry
(MCA) [45-46].
Table 2. The karyological parameters and formulae used for chromosome characterization.
Karyotypic parameters
Abbreviations and formulae
Long arm
L
Short arm
S
Total chromosome length
TL = LA + SA
Arm ratio
AR = LA / SA
Centromeric index
CI = [(SA) / (LA + SA)] × 100
Mean haploid length
MHL
Total haploid length
THL
Relative length
RL = [(LA + SA) / THL] × 100
Median chromosome
Submedian chromosome
Subtelocentric chromosome
Terminal chromosome
m, LA / SA = 1.0 – 1.7
sm, LA / SA = 1.7 – 3.0
st, LA / SA = 3.0 – 7.0
t, LA / SA = 7.0 – ∞
Mean centromeric asymmetry
(Intrachromosomal asymmetry)
MCA = [mean (LT – ST) / (LT + ST)] × 100
LT, total length of long arms
ST, total length of short arms
Coefficient variation of chromosome length
(Interchromosomal asymmetry)
CVCL = (SCL / XCL) × 100
SCL, standard deviation
XCL, mean chromosome length
Karyological relationships and regression analysis
Karyological relationships were evaluated by following seven parameters: basic number (x), diploid
number (2n), ploidy level, karyotype formula, total haploid length (THL), mean centromeric asymmetry (MCA),
and coefficient of variation of chromosome length (CVCL). A dendrogram showing karyological relationships
was drawn by bootstrap values (BV) with UPGMA software, chord coefficient. The dendrogram contains 12
Geranium species with detailed chromosomal data. In dendrogram, parameters are classified in the following
order: THL [10 ˂ THL ≤ 20 (1), 20 ˂ THL ≤ 30 (2), 30 ˂ THL ≤ 40 (3), 40 ˂ THL (4)]; MCA [10 ˂ MCA ≤ 15 (1),
15 ˂ MCA ≤ 20 (2), 20 ˂ MCA ≤ 25 (3)], and CVCL [10 ˂ CVCL ≤ 20 (1), 20 ˂ CVCL ≤ 30 (2), 30 ˂ CVCL ≤ 40 (3)].

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For regression analysis, linear models were calculated between three predictor variables (x, 2n, and
ploidy levels) and four dependent variables (MHL, THL, CVCL, and MCA) using the software Past 4.04. Then,
a scatter diagram was drawn between interchromosomal asymmetry and the intrachromosomal asymmetry.
RESULTS
Chromosomal data
Figure 2 shows the metaphase chromosomes of Geranium taxa. Chromosome counts in 38 taxa are
listed in Table 3; nine are reported here for the first time, five present new chromosome numbers, and 24
have similar number with previous reports. Eleven different diploid numbers (2n = 18, 20, 22, 26, 28, 30, 32,
46, 48, 64, and 84) are detected. Chromosomes of Geranium are small. Mean haploid length varies from
0.83 μm in G. ibericum to 2.11 μm in G. columbinum. The smallest total haploid length is 18.43 μm in G.
lucidum, and the highest value is 71.56 μm in G. sanguineum. The smallest chromosome size among the
taxa detected detailed chromosomal measurements is 0.87 μm, in G. rotundifolium. The largest chromosome
size is 3.77 μm in G. asphodeloides (Table 4).
Basic numbers, ploidy levels and polyploidy
In genus Geranium, there are generally 2 common basic numbers, which are x = 13 and most commonly
x = 14. In the present study, the basic numbers are x = 13 in 11 taxa and x = 14 in 18 taxa. In addition, the
other basic numbers and ploidy levels are x = 8 in G. purpureum and G. robertianum with ploidy levels of 4x
and 8x; x = 9 in G. columbinum; x = 10 in G. lucidum and G. glaberrimum with ploidy level of 3x; x = 11 in G.
dissectum; x = 12 in G. lanuginosum and G. sanguineum with ploidy levels of 4x and 7x; and x = 23 in G.
macrorrhizum (Table 3 and Figure 3). In basic numbers, infraspecific variations are encountered in Geranium.
Karyotypes and karyotype asymmetry
In Table 4, twelve taxa have metacentric and submetacentric chromosomes and only x = 7 heptaploid
G. sanguineum has subtelocentric chromosomes; there are no telocentric chromosomes. Four different
karyotype samples are detected, which are M-m-sm (in two taxa), m (in only one taxon), m-sm (in nine taxa),
and m-sm-st (in only one taxon).
Intrachromosomal asymmetry (MCA) varies from 14.18 (G. petri-davisii) to 20.76 (G. molle subsp.
bruitium), which refer to symmetric karyotypes. Interchromosomal asymmetry (CVCL), indicating the karyotype
heterogeneity, varies from 13.93 (G. collinum) to 30.38 (G. asphodeloides).
Interspecific relationships
Figure 4 presents a dendrogram including chromosomal data of 12 Geranium species. The dendrogram
consists of two main clades. Firstly, G. sanguineum is separated as a monophyletic group by quite different
karyological features, which are different basic number (x = 12), diploid number (2n = 84), and karyotype
sample (m-sm-st) and high ploidy level (Clade I). Clade II consists of two subclades with a medium strong
monophyletic group (BV = 64). Subclade 1 contains different karyotype sample (M-m-sm) and relatively more
intrachromosomal asymmetry. Subclade 2 is branched by low bootstrap values and contains nine taxa by
strong karyological variations.
Regression analysis
In Figure 5, linear regression models are presented by scatter plots, which refer to the independent (x,
2n, ploidy levels) and dependent variables (MHL, THL, CVCL, MCA). The following parameters are listed under
each plot: regression slope, standard error, r value, and test statistic. Dotted lines in E and F represent
significant linear regression, and solid lines in the other plots show not significant correlations. The significant
regression models (E and F) have p values < 0.0001 that are significant after adjusting the p value for multiple
testing. In other regression models, p values are quite high.

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Figure 2. Metaphase chromosomes of Geranium species. (1) G. asphodeloides; (2) G. bohemicum; (3) G. collinum; (4)
G. columbinum; (5) G. dissectum; (6) G. divaricatum; (7) G. eginense; (8) G. glaberrimum; (9) G. gracile; (10) G.
gymnocaulon; (11) G. ibericum; (12) G. lasiopus; (13) G. libani; (14) G. libanoticum; (15) G. lucidum; (16) G.
macrorrhizum; (17) G. macrostylum; (18) G. molle subsp. bruitium; (19) G. molle subsp. molle; (20) G. palustre; (21) G.
petri-davisii; (22) G. platypetalum; (23) G. ponticum; (24) G. pretense; (25) G. psilostemon; (26) G. purpureum; (27) G.
pusillum; (28) G. pyrenaicum; (29) G. robertianum; (30) G. rotundifolium; (31) G. sanguineum; (32) G. sibiricum; (33) G.
sintenisii; (34) G. subcaulescens; (35) G. sylvaticum; (36) G. tuberosum. Scale bar 10 µm.

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Table 3. The chromosome counts of the taxa in present and previous studies. In addition, the karyological data with
basic number (x), ploidy levels, mean haploid length (MHL), total haploid length (THL), and asymmetry indices (CVCL
and MCA). * probably dysploidy.
Taxa
(alphabetically)
Previous results
Present results
Note
2n
References
2n
x
Ploidy
MHL
THL
CVCL
MCA
G. asphodeloides
24, 28*
[20,31,37]
26
13
2x
1.25
32.49
30.38
16.25
New
count
G. bohemicum
28
[20,35]
28
14
2x
Equal
number
G. collinum
28
[38]
28
14
2x
0.87
24.28
13.93
18.38
Equal
number
G. columbinum
18
[20-21,25,
27,29,35]
18
9
2x
2.11
18.99
20.98
17.92
Equal
number
G. dissectum
22
[20,35]
22
11
2x
1.72
18.88
25.42
18.85
Equal
number
G. divaricatum
26, 28*
[20,25,35]
28
14
2x
0.99
27.74
Equal
number
G. eginense
26
13
2x
First
report
G. glaberrimum
30
[26]
30
10
3x
Equal
number
G. gracile
26
13
2x
0.94
24.54
First
report
G. gymnocaulon
28
[22]
28
14
2x
Equal
number
G. ibericum
subsp. ibericum
56
[18]
28
14
2x
0.83
23.19
New
count
G. ibericum
subsp. jubatum
28
14
2x
0.92
26.68
First
report
G. lanuginosum
42, 48*
[18,21]
48
12
4x
Equal
number
G. lasiopus
26
13
2x
First
report
G. libani
28
14
2x
1.00
27.94
First
report
G. libanoticum
28
14
2x
First
report
G. lucidum
20, 40,
41, 42,
43, 44*
[20,24,
26,35]
20
10
2x
1.84
18.43
15.57
15.62
Equal
number
G. macrorrhizum
46, 92
[20,31]
46
23
2x
Equal
number
G. macrostylum
28
[27]
28
14
2x
Equal
number
G. molle
subsp. bruitium
26, 52
[20]
26
13
2x
1.72
22.21
19.01
20.76
Equal
number
G. molle
subsp. molle
26
[24-25]
28
14
2x
1.71
23.92
24.65
15.26
New
count
G. palustre
28, 56
[20,25,35]
28
14
2x
Equal
number
G. petri-davisii
28
14
2x
2.32
32.41
21.97
14.18
First
report
G. platypetalum
28, 42
[18]
28
14
2x
1.91
26.67
27.79
18.46
Equal
number
G. ponticum
26
13
2x
First
report
G. pratense
24, 28,
56
[36]
26
13
2x
New
count

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Cont. Table 3
G. psilostemon
26
13
2x
1.03
26.72
First
report
G. purpureum
32, 64
[20,24,31-
32,35]
32
8
4x
0.90
28.95
Equal
number
G. pusillum
26
[20,25,35]
26
13
2x
1.99
25.82
25.81
19.39
Equal
number
G. pyrenaicum
26, 28,
52*
[18,25,31,
35]
26
13
2x
1.06
27.49
Equal
number
G. robertianum
32, 52,
54, 56,
64,128*
[21,24,35]
64
8
8x
Equal
number
G. rotundifolium
26, 46
[21,23-
24,27,35]
28
14
2x
1.78
24.88
25.28
14.43
New
count
G. sanguineum
52,56,
82,84*
[20-21,29]
84
12
7x
1.70
71.56
25.32
19.31
Equal
number
G. sibiricum
28
[35]
28
14
2x
Equal
number
G. sintenisii
26
[26]
26
13
2x
Equal
number
G. subcaulescens
28, 56
[26,31,33]
28
14
2x
0.96
26.91
Equal
number
G. sylvaticum
24, 28,
56*
[30,34]
28
14
2x
1.58
22.10
20.49
15.20
Equal
number
G. tuberosum
28
[28]
28
14
2x
Equal
number
Table 4. The karyological features of the studied Geranium taxa.
Taxa
KF
SC (μm)
LC (μm)
RL (%) SC–LC
CI (min–max)
G. asphodeloides
24m + 2sm
1.21
3.77
3.72–11.60
35.74–47.32
G. collinum
22m + 6sm
1.40
2.19
5.77–9.02
27.32–47.95
G. columbinum
14m + 4sm
1.33
2.97
7.00–15.64
35.43–46.20
G. dissectum
22m
1.13
2.53
5.99–13.40
37.43–47.16
G. lucidum
18m + 2sm
1.36
2.31
7.38–12.53
32.03–49.17
G. molle subsp. brutium
20m + 6sm
1.04
2.17
4.68–9.77
33.55–47.12
G. molle subsp. molle
26m + 2sm
0.95
2.36
3.97–9.87
35.91–48.59
G. petri-davisii
26m + 2sm
1.51
3.45
4.66–10.64
36.00–46.42
G. platypetalum
22m + 6sm
1.01
2.76
3.79–10.35
26.73–48.67
G. pusillum
22m + 4sm
1.31
3.12
5.07–12.08
29.11–47.60
G. rotundifolium
2M + 22m + 4sm
0.87
2.47
3.50–9.93
26.32–50.00
G. sanguineum
66m + 16sm + 2st
0.90
2.73
1.26–3.81
24.84–48.21
G. sylvaticum
2M + 22m + 4sm
1.09
2.25
4.93–10.18
34.22–50.00
Abbreviations: karyotype formula (KF), shortest chromosome (SC), longest chromosome (LC), relative length (RL), total
haploid length (THL), mean chromosome length (MCL), centromeric index (CI), coefficient of variation of chromosome
length (CVCL), mean centromeric asymmetry (MCA), median point (M), median (m), submedian (sm).
DISCUSSION
Variations of chromosome number
Various chromosome numbers such as 2n = 18, 20, 22, 26, 28, 30, 32, 46, 48, 64, and 84 are detected
with dominant numbers of 2n = 26 and 28. The chromosome numbers of nine taxa are reported here for the
first time: G. eginense, G. gracile, G. lasiopus, G. ponticum, and G. psilostemon (2n = 26), G. ibericum subsp.
jubatum, G. libani, G. libanoticum, and G. petri-davisii (2n = 28). These are the most common diploid
chromosome numbers of genus.
The chromosome numbers of five taxa represent new cytotypes: G. asphodeloides and G. pratense (2n
= 26), G. ibericum subsp. ibericum, G. molle subsp. molle, and G. rotundifolium (2n = 28). The chromosome
numbers are 2n = 24, 28 in G. asphodeloides, 2n = 56 in G. ibericum subsp. ibericum, 2n = 26 in G. molle
subsp. molle, and 2n = 26, 46 in G. rotundifolium [18, 20-21, 23-25, 27, 31, 35, 37]. The chromosome numbers
of 24 taxa are the same as in previous reports. Our study did not confirm far-reaching discrepancies of
chromosome numbers in Geranium taxa that were reported by previous studies (Table 3: G. lucidum, G.
robertianum etc). Winterfeld and coauthors [17] reported that such findings might result from the preparation

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and identification of the studied samples or use of obsolete species description and would allow observation
of chromosome number variations in the future.
Figure 3. Monoploid ideograms of Geranium species.
Basic number alterations, dysploidy and ploidy levels, polyploidy
Basic chromosome numbers of x = 13 and 14 dominate in Geranium taxa, but basic numbers of x = 8,
9, 10, 11, 12, and 23 characterize several taxa. Many taxa contain basic number variations possibly caused
by the dysploidy mechanism. The dysploidy is likely to have happened depending the fusion of metacentric
chromosomes or reciprocal translocations in ancestral karyotypes including dominant basic numbers. Basic
number alterations are x = 12, 14 in G. asphodeloides, G. lanuginosum, and G. sylvaticum; x = 13, 14 in G.

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divaricatum and G. pyrenaicum; x = 10, 11, 14 in G. lucidum; x = 8, 13, 14 in G. robertianum; and x = 12 ,13,
14 in G. pratense and G. sanguineum [18,20-21,24-26,29-31,34-37].
Figure 4. The dendrogram showing karyological relationships of 12 Geranium species. Numbers at branches indicate
bootstrap values. Main clades and subclades are shown in circle. Seven changes are x, 2n, ploidy level, karyotype
formula, THL [10 ˂ THL ≤ 20 (1), 20 ˂ THL ≤ 30 (2), 30 ˂ THL ≤ 40 (3), 40 ˂ THL (4)]; MCA [10 ˂ MCA ≤ 15 (1), 15 ˂ MCA
≤ 20 (2), 20 ˂ MCA ≤ 25 (3)], and CVCL [10 ˂ CVCL ≤ 20 (1), 20 ˂ CVCL ≤ 30 (2), 30 ˂ CVCL ≤ 40 (3)]. a, sect Geranium;
b, sect Dissecta; c, sect Ruperta; d, sect Tuberosa; e, sect Batrachioidea; f, sect Subacaulia.
According to the previous reports, there are many diploid and polyploid reports that include various basic
numbers (x = 8, 9, 10, 11, 12, 13, 14, and 23) [18,20-37]. In the present study, G. glaberrimum, G. purpureum,
G. lanuginosum, G. sanguineum, and G. robertianum are polyploid species by 2n = 3x = 30, 2n = 4x = 32,
2n = 4x = 48, 2n = 7x = 84, and 2n = 8x = 64, respectively. Polyploidy, is an important mechanism regarding
speciation and evolution of plants, occurs in two ways, which are autopolyploidy with genome duplication in
only one species and allopolyploidy with genome duplication between species. It is reported that polyploidy
rates are increased by glaciation, altitudes, and high latitudes although not always [47-48]. For example,
polyploid taxa have a wide distribution ranging from 70 to 1470 m.

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Figure 5. Regression analysis of chromosomal data (basic number, diploid number, ploidy levels) versus
karyomorphological data (mean haploid length, total haploid length, interchromosomal asymmetry, and
intrachromosomal asymmetry) in Geranium taxa. Parameters: square regression slope, triangle standard error, circle r
value, star test statistic. Dotted lines (significant linear regression) and regular lines (not significant linear regression).
The comprehensive variations of basic numbers and the relatively low rate of polyploid species showed
in the present study promote the evolutionary significance of karyotype alterations by dysploidy mechanism.
Despite widespread opposite models, dysploidy may cause relatively long-term persistence in the
evolutionary process compared to polyploid changes. The results are inconsistent with previous reports of
dysploidy and polyploidy, which are highlighting the evolutionary role of the polyploidy. There are limited
studies reporting that the common mechanism in species diversification is dysploidy [16-17,49-50].

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Chromosome structure, karyotype asymmetry and regression analysis
In genus Geranium, the chromosomal data are generally based on reports of basic and diploid number.
Chromosomes are comparatively small (LC < 4 μm and generally MHL < 2 μm). Winterfeld and coauthors
[17] reported that the decreasing chromosome length accompanies increasing dysploidy. In addition, total
haploid lengths are comparatively small except G. sanguineum, which has high chromosome number (2n =
84). Centromeric index, intrachromosomal asymmetry and interchromosomal asymmetry in whole
complements are variable. In summary, the examined karyotypes show continuous variation.
Regression analyses are established by basic number, diploid number, and ploidy levels versus
karyotype data, such as mean haploid length, total haploid length, interchromosomal asymmetry, and
intrachromosomal asymmetry. There are significant positive correlations between THL and 2n/ploidy levels,
meaning that an increase in THL is linked with an increase in 2n/ploidy levels. Contrary to the THL, MHL
shows very weak positive/negative correlations with all predictor variables. Very weak correlations raise the
necessity of comparing chromosome data by a molecular phylogenetic analyses. Asymmetry indices (CVCL
and MCA) show weak positive correlations mainly caused by polyploidy with 2n/ploidy levels, that the
correlation of MCA is twice as high as CVCL. Asymmetry incidences are relatively increasing with genome
duplication.
In intrachromosomal asymmetry, the symmetrical karyotypes are dominant. The most asymmetrical
karyotypes are G. molle subsp. bruitium, G. pusillum, and G. sanguineum, which is a polyploid taxon. In
addition, G. sanguineum is the only taxon including subtelocentric chromosomes which may be due to the
reciprocal translocations of the metacentric/submetacentric chromosomes. In interchromosomal asymmetry,
CVCL values show continuous variation. The most symmetric and asymmetric karyotypes are different
between CVCL and MCA by very weak positive correlation (r = 0.014) (Figure 6). The most symmetrical
karyotypes are G. petri-davisii (MCA = 14.18) and G. collinum (CVCL = 13.93), which have basic number of x
= 14. The most asymmetrical karyotypes are G. molle subsp. bruitium (MCA = 20.76) and G. asphodeloides
(CVCL = 30.38), which have basic number of x = 13.
Figure 6. Scatter diagram between MCA and CVCL. (A) G. asphodeloides; (B) G. collinum; (C) G. columbinum; (D) G.
dissectum; (E) G. lucidum; (F) G. molle subsp. molle; (G) G. molle subsp. bruitium; (H) G. petri-davisii; (I) G.
platypetalum; (J) G. pusillum; (K) G. rotundifolium; (L) G. sanguineum; (M) G. sylvaticum.
Geranium taxa have different patterns in terms of asymmetry degrees: G. lucidum, G. petri-davisii, and
G. sylvaticum possess relatively low intrachromosomal and interchromosomal asymmetry; seven taxa have
a higher interchromosomal asymmetry; and seven taxa have a higher intrachromosomal asymmetry. G.
dissectum, G. platypetalum, G. pusillum, and G. sanguineum show distribution both higher interchromosomal
and higher intrachromosomal asymmetry. All taxa except G. sanguineum have symmetric karyotypes

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including metacentric or submetacentric chromosomes. It was recorded that the chromosome asymmetry
increases in karyotype evolution [51]. The fact that Turkish Geranium taxa have symmetrical karyotypes may
indicate that these taxa are at the first levels of karyotype evolution and Turkey is an important distribution
center for genus Geranium.
Interspecific relationships
There is no detailed research recording karyotype evolution and karyological relationships among
Turkish Geranium taxa. The studies consist of some karyotype analyses including basic and diploid
chromosome numbers. G. sanguineum forms a monophyletic group by quite different karyotypic features
such as different basic and diploid numbers, subtelocentric chromosomes, and high polyploidization (clade
I). The other species form a medium monophyletic group (clade II). G. rotundifolium and G. sylvaticum shape
the subclade 1 by some variations, which are different karyotype sample and relatively more
intrachromosomal asymmetry. Subclade 2 is branched by low bootstrap values and contains nine taxa by
strong karyological variations. First branched species are G. lucidum (x = 10, 2n = 20) and G. collinum (lowest
karyotype heterogeneity). The five species that have been separated so far are species of the sect. Geranium
and sect. Ruperta. Then other sections are separated, which are sect. Tuberosa, sect. Batrachioidea, sect.
Dissecta, and sect. Subcaulia. The only exception to the sect. Geranium is G. columbinum (x = 9, 2n = 18).
CONCLUSION
The following is an overview of the data included in the present study: (i) first record of diploid
chromosome numbers in nine taxa; (ii) new diploid counts different from previous records in five taxa; (iii)
detailed chromosomal data in 13 taxa; (iv) first record of karyotype asymmetry by symmetric karyotypes; and
(v) karyological variations as a result of polyploidy and especially dysploidy. Dysploidy and polyploidy
variations may be the main factors in karyotype evolution of the genus as our results indicate to some degree.
Funding: This research was funded by TUBITAK, grant number 113 Z 099.
Conflicts of Interest: The authors declare no conflict of interest.
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