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

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 (CV_CL and M_CA) 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

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.

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.

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 Mediterra nean 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].

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.

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
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 (CV_CL) and intrachromosomal asymmetry (M_CA) [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 (M_CA), and coefficient of variation of chromosome length (CV_CL). 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)]; M_CA [10 ˂ M_CA ≤ 15 (1), 15 ˂ M_CA ≤ 20 (2), 20 ˂ M_CA ≤ 25 (3)], and CV_CL [10 ˂ CV_CL ≤ 20 (1), 20 ˂ CV_CL ≤ 30 (2), 30 ˂ CV_CL ≤ 40 (3)].

For regression analysis, linear models were calculated between three predictor variables (x, 2n, and ploidy levels) and four dependent variables (MHL, THL, CV_CL, and M_CA) 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 (M_CA) varies from 14.18 (G. petri-davisii) to 20.76 (G. molle subsp. bruitium), which refer to symmetric karyotypes. Interchromosomal asymmetry (CV_CL), 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, CV_CL, M_CA). 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.

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.

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 (CV_CL and M_CA). * 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
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 (CV_CL), mean centromeric asymmetry (M_CA), 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 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. 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)]; M_CA [10 ˂ M_CA ≤ 15 (1), 15 ˂ M_CA ≤ 20 (2), 20 ˂ M_CA ≤ 25 (3)], and CV_CL [10 ˂ CV_CL ≤ 20 (1), 20 ˂ CV_CL ≤ 30 (2), 30 ˂ CV_CL ≤ 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.

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].

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 (CV_CL and M_CA) show weak positive correlations mainly caused by polyploidy with 2n/ploidy levels, that the correlation of M_CA is twice as high as CV_CL. 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, CV_CL values show continuous variation. The most symmetric and asymmetric karyotypes are different between CV_CL and M_CA by very weak positive correlation (r = 0.014) (Figure 6). The most symmetrical karyotypes are G. petri-davisii (M_CA = 14.18) and G. collinum (CV_CL = 13.93), which have basic number of x = 14. The most asymmetrical karyotypes are G. molle subsp. bruitium (M_CA = 20.76) and G. asphodeloides (CV_CL = 30.38), which have basic number of x = 13.

Figure 6
Scatter diagram between M_CA and CV_CL. (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 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.

REFERENCES

• ¹
  Aedo C, Garcia MA, Alarcon ML, Aldasoro JJ, Navarro C. Taxonomic revision of Geranium subsect. Mediterranea (Geraniaceae). Syst Bot. 2007;32(1):93-128.
• ²
  Yeo PF. Fruit-discharge-type in Geranium (Geraniaceae): its use in classification and its evolutionary implications. Bot J Linn Soc. 1984;89(1):1-36.
• ³
  Aedo C, Aldasoro JJ, Navarro C. Taxonomic revision of Geranium sections Batrachioidea and Divaricata(Geraniaceae). Ann Mo Bot Gard. 1998;85(4):594-630.
• ⁴
  Aedo C. The genus Geranium L. (Geraniaceae) in North America. I. annual species. An Jardin Bot Madrid. 2000;58(1):39-82.
• ⁵
  Aedo C. Taxonomic revision of Geranium sect. Brasiliensia (Geraniaceae). Syst Bot. 2001;26(2):205-15.
• ⁶
  Aedo C, Aldasoro JJ, Navarro C. Revision of Geranium sections Azorelloida, Neoandina and Paramensia (Geraniaceae). Blumea. 2002;47(2):205-97.
• ⁷
  Aedo C, Fiz O, Alarcon ML, Navarro C, Aldasoro J. Taxonomic revision of Geranium sect. Dissecta (Geraniaceae). Syst Bot. 2005;30(3):533-58.
• ⁸
  Aedo C, de la Estrella M. Taxonomic revision of Geranium subsect. Tuberosa (Boiss.) Yeo (Geraniaceae). Israel J Plant Sci. 2006;54(1):19-54.
• ⁹
  Aedo C. Taxonomic Revision of Geranium Sect. Ruberta and Unguiculata (Geraniaceae). Ann Mo Bot Gard. 2017;102(3):409-65.
• ¹⁰
  Schubert I. Chromosome evolution. Curr Opin Plant Biol. 2007;10(2):109-15.
• ¹¹
  Guerra M. Chromosome numbers in plant cytotaxonomy: concepts and implications. Cytogenet Genome Res. 2008;120(3-4):339-50.
• ¹²
  Schubert I, Lysak MA. Interpretation of karyotype evolution should consider chromosome structural constraints. Trends Genet. 2011;27(6):207-16.
• ¹³
  Guerra M. Cytotaxonomy: The end of childhood. Plant Biosyst. 2012;146(3):703-10.
• ¹⁴
  Sirin E, Bozkurt M, Uysal T, Ertugrul K. Karyomorphological features of Turkish Centaurea (subgenus Cyanus, Asteraceae) species and its taxonomic importance. Turk J Bot. 2019;43(4):538-50.
• ¹⁵
  Çelik M, Bagci Y, Martin E, Eroglu HE. Karyotype analysis and karyological relationships of Turkish Bunium species (Apiaceae). Arch Biol Sci. 2020;72(2):203-9.
• ¹⁶
  Martin E, Kahraman A, Dirmenci T, Bozkurt H, Eroglu HE. Karyotype evolution and new chromosomal data in Erodium: chromosome alteration, polyploidy, dysploidy, and symmetrical karyotypes. Turk J Bot. 2020;44(3):255-68.
• ¹⁷
  Winterfeld G, Ley A, Hoffmann MH, Paule J, Röser M. Dysploidy and polyploidy trigger strong variation of chromosome numbers in the prayer-plant family (Marantaceae). Plant Syst Evol. 2020;306(36):1-17.
• ¹⁸
  Darlington CD, Wylie AP. Chromosome atlas of flowering plants. London: George Allen and Unwin; 1956.
• ¹⁹
  Nagl W. Über Endopolyploidie, restitutionskernbildung und kernstrukturen im suspensor von angiospermen und einer gymnosperme. Österr Bot Zeitschrift. 1962;109(4-5):431-94. German.
• ²⁰
  Petrova A, Stanimirova P. Karyological study of some Geranium (Geraniaceae) species growing in Bulgaria. Bocconea. 2003;16(2):675-82.
• ²¹
  Warburg EF. Taxonomy and relationship in the Geraniales in the light of their cytology. New Phytol. 1938;37(2):130-59.
• ²²
  Tumajanov II, Beridze RK. A karyological investigation of some representatives of the upper alpine adnival floras of the Great Caucasus. Bot Zurn. 1968;53:58-68.
• ²³
  Dahlgren R, Karlsson TH, Lassen P. Studies on the Flora of the Balearic Islands, I. Chromosome numbers in Balearic angiosperms. Bot Not. 1971;124:249-69.
• ²⁴
  Löve A, Kjellqvist E. Cytotaxonomy of Spanish plants. IV. Dicotyledons: Caesalpiniaceae- Asteraceae. Lagascalia. 1974;4(2):153-211.
• ²⁵
  Murin A. In Index of chromosome numbers of Slovakian flora. Part 4. Acta Fac Rerum Nat Univo Comen Bot. 1974;23:1-23.
• ²⁶
  Guittoneau GG. Contributions à l'étude caryosystématique et phylogénétique des Géraniacées dans le Bassin Méditerranéen, Colloques Internatl. 1975;235:195-205. French.
• ²⁷
  Strid A, Franzen R. In chromosome number reports LXXIII. Taxon. 1981;30:829-42.
• ²⁸
  Van Loon JC, Oudemans JJMH.. In IOPB chromosome number reports LXXV. Taxon. 1982;31:343-44.
• ²⁹
  Van Loon JC. Chromosome numbers in Geranium from Europe, II. The annual species. Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen C. 1984;87:279-96.
• ³⁰
  Dmitrieva SA. Chisla khromosom nekotorych vidov rastenij Berezinskogo Biosfernogo Zapovednika. Zapov Belorussii Issl. 1986;10:24-8. Russian.
• ³¹
  Baltisberger M. Cytological investigations of some Greek plants. Fl Medit. 1991;1:157-73.
• ³²
  Luque T, Lifante ZD. Chromosome numbers of plants collected during Iter Mediterraneum I in the SE of Spain. Bocconea. 1991;1:303-64.
• ³³
  Baltisberger M. Two interesting chromosome numbers from the Balkans. IOPB Newsletter. 1993;20:12-5.
• ³⁴
  Montgomery L, Khalaf M, Bailey JP, Gornal KJ. Contributions to a cytological catalogue of the British and Irish flora, 5. Watsonia, 1997;21:365-68.
• ³⁵
  Albers F, Pröbsting W. Chromosomenatlas. In: Wisskirchen R, Haeupler H, editors. Standardliste der Farn- und Blütenpflanzen Deutschlands. Stuttgart: Bundesamt für Naturschutz & Verlag Eugen Ulmer; 1998. p. 562-616.
• ³⁶
  Kumar P, Singhal VK. Chromosome number and secondary chromosomal associations in wild populations of Geranium pratense L. from the cold deserts of Lahaul-Spiti (India). Tsitol Genet. 2013;47(2):56-65.
• ³⁷
  Probatova NS. Chromosome numbers of some plant species of the Primkorsky Territory and the Amur River basin. Bot Zurn. 2006;91:785-804.
• ³⁸
  Baltisberger M, Voelger M. Sternbergia sicula. IAPT/IOPB chromosome data 1. Taxon. 2006;55:443-45.
• ³⁹
  Rice A, Glick L, Abadi S, Einhorn M, Kopelman NM, Salman-Minkov A, et al. The Chromosome Counts Database (CCDB) - a community resource of plant chromosome numbers. New Phytologist. 2015;206(1):19-26.
• ⁴⁰
  Fiz O, Vargas P, Alarcón ML, Aldasoro JJ. Phylogenetic relationships and evolution in Erodium (Geraniaceae) based on trnL-trnF sequences. Syst Bot. 2006;31(4):739-63.
• ⁴¹
  Yeo PF. Two new Geranium species endemic to Madeira. Boletim do Museu Municipal do Funchal. 1969;23:25-35.
• ⁴²
  Eroglu HE, Altay D, Budak Ü, Martin E. Karyotypic phylogeny and polyploidy variations of Paronychia (Caryophyllaceae) taxa in Turkey. Turk J Bot. 2020;44(3):245-54.
• ⁴³
  Levan AK, Fredga K, Sandberg AA. Nomenclature for centromeric position on chromosomes. Hereditas. 1964;52(2):201-20.
• ⁴⁴
  Bozkurt H. Türkiye'den Geraniaceae taksonlarinin kromozom analizleri. [dissertation]. Konya: Necmettin Erbakan University; 2018.
• ⁴⁵
  Paszko B. A critical review and a new proposal of karyotype asymmetry indices. Plant Syst Evol. 2006;258:39-48.
• ⁴⁶
  Peruzzi L, Eroglu HE. Karyotype asymmetry: again, how to measure and what to measure? Comp Cytogenet. 2013;7(1):1-9.
• ⁴⁷
  Metzgar J, Stamey M, Ickert-Bond S. Genetic differentiation and polyploid formation within the Cryptogramma crispa complex (Polypodiales: Pteridaceae). Turk J Bot. 2016;40(3):231-40.
• ⁴⁸
  Demirci Kayiran S, Özhatay FN. A karyomorphological study on the genus Muscari Mill. growing in Kahramanmaras (Turkey). Turk J Bot. 2017;41(3):289-98.
• ⁴⁹
  Mandakova T, Lysak MA. Post-polyploid diploidization and diversification through dysploid changes. Curr Opin Plant Biol. 2018;42:55-65.
• ⁵⁰
  Winterfeld G, Becher H, Voshell S, Hilu K, Röser M. Karyotype evolution in Phalaris (Poaceae): the role of reductional dysploidy, polyploidy and chromosome alteration in a wide-spread and diverse genus. PLoS ONE. 2018;13(4):e0192869.
• ⁵¹
  Baltisberger M, Hörandl E. Karyotype evolution supports the molecular phylogeny in the genus Ranunculus (Ranunculaceae). Perspect Plant Ecol Evol Syst. 2016;18:1-14.

• Funding:

  This research was funded by TUBITAK, grant number 113 Z 099.

Edited by

Editor-in-Chief:

Alexandre Rasi Aoki

Associate Editor:

Acácio Antonio Ferreira Zielinski

Publication Dates

• Publication in this collection
  16 May 2022
• Date of issue
  2022

History

• Received
  30 May 2021
• Accepted
  24 Nov 2021