Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 67-73, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-170 Citation: E. Mitrenina, M. Skaptsov, M. Kutsev, A. Kuznetsov, H. Ikeda, A. Erst (2020) A new diploid cytotype of Agri- monia pilosa (Rosaceae). Caryologia 73(1): 67-73. doi: 10.13128/caryolo- gia-170 Received: February 19, 2019 Accepted: February 23, 2020 Published: May 8, 2020 Copyright: © 2020 E. Mitrenina, M. Skaptsov, M. Kutsev, A. Kuznetsov, H. Ikeda, A. Erst. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. A new diploid cytotype of Agrimonia pilosa (Rosaceae) Elizaveta Mitrenina1, Mikhail Skaptsov2, Maksim Kutsev2, Alexander Kuznetsov1, Hiroshi Ikeda3, Andrey Erst1,4,* 1 Laboratory of Herbarium, National Research Tomsk State University, Tomsk, Russia 2 South-Siberian Botanical Garden, Altai State University, Barnaul, Russia 3 The University Museum, The University of Tokyo, Tokyo, Japan 4 Laboratory of Herbarium, Central Siberian Botanical Garden, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia *Corresponding author. E-mail: erst_andrew@yahoo.com Abstract. A new diploid cytotype of Agrimonia pilosa Ledebour (Rosaceae) collected in China has been revealed. Karyotype formula is 2n = 2x = 16 = 14m + 2sm. Previ- ously, chromosome numbers in A. pilosa established by other researchers were 2n = 28; 56; 70 with basic chromosome number x = 7. All the other members of genus Agrimo- nia Linnaeus have the same basic chromosome number. In the meanwhile, some mem- bers of fam. Rosaceae have different basic chromosome numbers: x = 8 (e.g., in genera Amygdalus L., Aphanes L., Cerasus Mill., etc.), x = 9 (e.g., in genera Adenostoma Hook. & Arn., Chamaebatia Benth., etc.), x = 17 (e.g., in genera Amelanchier Medik., Chae- nomeles Lindl., etc.). We suppose that the new basic chromosome number x = 8 was revealed in Agrimonia pilosa collected in China. Keywords. Agrimonia pilosa Ledebour, Rosaceae, chromosomes, karyotype, new cyto- type, flora of China. 1. INTRODUCTION Genus Agrimonia Linnaeus (fam. Rosaceae, subfam. Rosoideae) compris- es 15 to 25 species and some naturally occurring hybrids distributed main- ly in temperate regions throughout Europe, Asia, North America, Central America, the West Indies, southern South America, and the Southern Africa (Li et al. 2003; Chung 2008; Kline and Sørensen 2008; Angelo and Boufford 2012). The genus belongs to the tribe Sanguisorbeae DC divided into two subtribes – Agrimoniinae J. Presl and Sanguisorbeae Torr. & A. Gray. The first subtribe, along with genus Agrimonia, includes genera Aremonia Neck. ex Nestl., Hagenia J. F. Gmel., Leucosidea Eckl. & Zeyh., Spenceria Trimen (Potter et al. 2007). Last four genera are monotypic endemics (Chung 2008; Chung et al. 2012). Species of the subtribe display basic chromosome number x = 7 and different levels of ploidy (2x, 4x, 6x, 8x, 10x, 12x) correlating with geographic distribution patterns (Chung 2008; Rice et al. 2015). 68 Elizaveta Mitrenina et al. Several species in Agrimonia (e.g., A. pilosa, A. eupa- toria) are used for medicinal purposes. They have been reported to possess antibacterial (Muruzović et al. 2016), antiviral (Kwon et al. 2005), antitumor (Miyamoto et al. 1987; Tang et al. 2017), diuretic (Giachetti 1986) and antidiabetes properties (Swanston-Flatt et al. 1990; Kuc- zmannová et al. 2016), antioxidant (Chen and Kang 2014; Muruzović et al. 2016), immunomodulating (Bukovsky and Blanarik 1994), hepatoprotective (Park et al., 2004) and other effects. In flora of China, the genus comprises following species: Agrimonia coreana Nakai, Agrimonia eupato- ria Linnaeus subsp. asiatica (Juzepczuk) Skalický, Agri- monia nipponica Koidzumi var. occidentalis Skalický ex J. E., Agrimonia pilosa Ledebour (Agrimonia pilosa var. pilosa and Agrimonia pilosa var. nepalensis (D. Don) Nakai) (Li et al., 2003). In the current study, karyotype analysis of A. pilosa collected in China (Figure 1) has been conducted. A new diploid cytotype 2n = 16 and a new probable basic chro- mosome number x = 8 for the genus Agrimonia L. were revealed. Combination of chromosome investigation with morphological methods, molecular genetics methods and scanning electron microscopy gives possibility to obtain essential data to reach conclusions on plants sys- tematics and phylogeny. 2. MATERIALS AND METHODS Seeds of A. pilosa for cytological study and her- barium specimens were col lected in China, Bei- jing, Yun Xiu Gu Forest park, Rocky ledges (40°60’N; 117°41’E, 22 July 2016; collectors: Erst A.S., Erst T.V., Lian L., Bing L., Shi C) and were collected in Russia, West Sibiria, Tomsk, southern edge of the city, inunda- tion meadow (56°47’N; 85°03’E, 1 Sep. 2017; collector: Mitrenina E. Yu.). All herbarium materials are depos- ited in Novosibirsk (NS). 2.1. Karyotype analysis Mitotic metaphase chromosomes in root tips of seedlings were studied. Seeds were grown at Petri dishes with wet sand at room temperature after cold stratification at 3–4° C during 4 months. Newly formed roots about 1.0–1.5 cm long were pretreated in a 0.2% colchicine solution during 2 hours at room tempera- ture. Fixation was carried out in a mixture of absolute ethanol and glacial acetic acid (3 : 1). Root tips were stained in 1% aceto-haematoxylin, and the squashing method was employed for investigating of karyotype (Smirnov 1968). Chromosomes were counted in 127 mitotic cells of 5 A. pilosa seedlings collected in China and in 25 mitotic cells of 5 A. pilosa seedlings collected in Russia. Mitotic metaphase chromosome plates were observed by microscope Primo Star (Carl Zeiss, Germany) and pho- tographed by microscope AxioImager A.1 (Carl Zeiss, Germany) with software AxioVision 4.7 (Carl Zeiss, Germany) and CCD-camera AxioCam MRc5 (Carl Zeiss, Germany) at 1000× magnification at Laboratory for Ecology, Genetics and Environmental Protection (“Ecogene”) of National Research Tomsk State Universi- ty. For karyotyping, the software KaryoType (Altinordu et al. 2016) was used, and for figures, the software Ado- be Photoshop CS5 (Adobe Systems, USA) and Inkscape 0.92 (USA) was used. The measurements were performed on 10 metaphase plates. For analysis of karyotype, the nomenclature of Levan, Fredgam, and Sandberg (1964) has been used. Figure 1. Agrimonia pilosa Ledebour (Beijing, China). 69A new diploid cytotype of Agrimonia pilosa (Rosaceae) 2.2. Flow cytometry Flow cytometry with propidium iodide (PI) staining was implemented to determine relative DNA content. At least 10 seeds from each plant were taken for this study. Each seed was analysed separately. Seed buffer (Matzk et al. 2001) was used for nuclei extraction. Seeds was squashed by porcelain pestle and chopped with a sharp razor blade in the nuclei extraction buffer. The sam- ples were filtered through 50-μm nylon membrane into a sample tube. Flow cytometry with Partec CyFlow PA revealed the data on isolated nuclei fluorescence (Partec, GmbH) using the laser 532 nm wavelength, while loga- rithmic fluorescence data representation (logarithmic scale) was used to record the signals. To calculate the mean of peak, at least 1000 nuclei peaks with less than 2.5% CV indicator values were used. The final data did not exceed the DNA content of the mean sample by more than 3% (Kubešová et al. 2010). As an external standard was used Euryops chrysanthemoides (DC.) B. Nord, 2C = 2.70 pg, and internal standard Glycine max ‘Polanka’, 2C = 2.50 pg (Doležel et al. 1994; Skaptsov et al. 2016). We used the Statistica 8.0 software (StatSoft Inc.), Flowing Software 2.5.1 (Turku Centre for Biotech- nology) and CyView software for the flow cytometer data analysis (Partec, GmbH), and for the analysis of our results. The possible effect of secondary metabolites on the binding of the intercalating dye was evaluated by co- grinding in a nuclei extraction buffer of the samples and Allium fistulosum L. leaves. The resulting preparation was investigated three times within 10 minutes. In the absence of variations in the average values of the detec- tion channels of the A. fistulosum peak, it was believed that no effect was detected. Flow cytometry performed at the Laboratory of Bio- engineering of South-Siberian Botanical Garden, Altai State University. 3. RESULTS Karyotype analysis of A. pilosa collected in China, Beijing has been conducted. All 127 investigated cells in 5 seedlings had diploid chromosome number 2n = 16 (Figure 2). By the software KaryoType (Altinordu et al. 2016) morphometric chromosome analysis (total chromosome length, short and long chromosome arms length, arm ratio) has been conducted (Table 1). Chro- mosome length ranged from 1.87 ± 0.17 µm to 2.14 ± 0.18 µm. Arm ratio varied from 1.03 to 1.81. Chromo- somes were classified into two groups: seven pairs with median centromeric position (metacentric chromo- Figure 2. Mitotic metaphase chromosomes of Agrimonia pilosa Ledebour (Beijing, China), 2n = 16. Scale bar = 10 µm. Table 1. Karyotype parameters of Agrimonia pilosa Ledebour, China (2n = 16). Chromosome pair Total length, µm, ± SD Long arm, µm, ± SD Short arm, µm, ± SD Arms ratio (long/ short) Chromosome type 1 2.14 ± 0.18 1.29 ± 0.12 0.85 ± 0.07 1.52 m 2 2.10 ± 0.24 1.10 ± 0.11 1.00 ± 0.13 1.10 m 3 2.04 ± 0.23 1.12 ± 0.13 0.92 ± 0.12 1.22 m 4 2.02 ± 0.24 1.15 ± 0.26 0.87 ± 0.11 1.32 m 5 1.95 ± 0.29 1.06 ± 0.17 0.89 ± 0.13 1.19 m 6 1.88 ± 0.17 1.05 ± 0.22 0.83 ± 0.08 1.27 m 7 1.87 ± 0.17 0.95 ± 0.08 0.92 ± 0.09 1.03 m 8 2.11 ± 0.27 1.36 ± 0.17 0.75 ± 0.10 1.81 sm Notes: m – metacentric chromosome; sm – submetacentric chromosome; ± SD – mean length ± standard deviation. 70 Elizaveta Mitrenina et al. somes, m; arm ratio 1–1.7), and one pair with sub-medi- an centromeric position (submetacentric chromosomes, sm; arm ratio 1.7–3.0). Some metacentric pairs were low- differentiated. They had almost equal length and arm ratio. Karyotype formula is 2n = 2x = 16 = 14m + 2sm (Figure 3). Karyotype asymmetry degree (Stebbins 1971): 1A. Secondary constrictions in 1–2 metaphase chromo- some pairs were revealed. Nucleolus number observed in mitotic interphase were 1–2 per cell. Chromosome counting in A. pilosa collected in Rus- sia, Tomsk has revealed typical octaploidic cytotype for the species: 2n = 8x = 56. By the flow cytometry method, we have found out relative DNA content in two agrimo- nies: 2C = 2.51 pg in A. pilosa (China) with 2n = 16, and 2C = 4.96 pg in A. pilosa (Russia) with 2n = 56 (Figure 4). 4. DISCUSSION According to the data of Chromosome Counts Data- base (Rice et al. 2015), Index to Plant Chromosome Numbers and other learned treatise (Iwatsubo et al. 1993; Chung 2008; Angelo and Boufford 2012; Kumar et al. 2014), diploid chromosome numbers in Agrimo- nia are known as 28; 42; 56; 70 и 84 (Table 2). The basic chromosome number in the genus x = 7. Currently, within Agrimonia only polyploids have been reported. As long as the lowest ploidy levels reported among spe- cies of Agrimonia are tetraploids, the lineage appears to have an ancient origin where diploids have gone extinct (Chung 2008). Such a basic chromosome number is common for many members of fam. Rosaceae (e.g., gen- era Geum L., Potentilla L., Rosa L., etc.). At the same time, there are other basic chromosome numbers in Rosaceae: х = 8 (e.g., in genera Amygdalus L., Aphanes L., Cerasus Mill., Exochorda Lindl, Padus Mill., Prunus L.), x = 9 (e.g., in genera Adenostoma Hook. & Arn., Chamaebatia Benth., Holodiscus Maxim.), x = 17 (e.g., in genera Amelanchier Medik., Chaenomeles Lindl., Kage- neckia Ruiz & Pav.) (Rice et al. 2015). Conventionally, subfamily classification was based on a combination of basic chromosome numbers and fruit types (Chung et al. 2012). Other genera belonging to subtribe Agrimoniinae, Figure 3. Idiogram of Agrimonia pilosa Ledebour (Beijing, China), 2n = 16. m – metacentric chromosome, sm – submetacentric chro- mosome. Figure 4. Flow cytometry histograms: a – Agrimonia pilosa (Tomsk, Russia); b – Agrimonia pilosa (Beijing, China); c – Agrimonia pilosa (Tomsk, Russia), Samp. 1 with internal standard (Glycine max (L.) Merr.), St.; d – Agrimonia pilosa (Tomsk, Russia), Samp. 1 with Agrimonia pilosa (Beijing, China), Samp. 2. Table 2. Chromosome numbers in the genus Agrimonia L. (Chung 2008; Angelo and Boufford 2012; Rice et al. 2015). Species Chromosome numbers Agrimonia coreana Nakai 24; 28 Agrimonia eupatoria L. 28; 42; 56; 70; 84 Agrimonia grandis Andrz. ex C. A. Mey. 42 Agrimonia gryposepala Wallroth 56 Agrimonia incisa Torr. & A. Gray 28 Agrimonia japonica (Miq.) Koidz. 56 Agrimonia nipponica Koidz. 28 Agrimonia parviflora Aiton 28 Agrimonia pilosa Ledeb. 28; 56; 70 Agrimonia x nipponica-pilosa Murata 42 Agrimonia procera Wallr. 56 Agrimonia pubescens Wallroth 28 Agrimonia repens L. 28 Agrimonia rostellata Wallroth 28 Agrimonia striata Michx. 28; 56 71A new diploid cytotype of Agrimonia pilosa (Rosaceae) disregarding Agrimonia, have following chromosome numbers and ploidy: Aremonia – 2n = 5x = 35 and 2n = 6x = 42, Hagenia – 2n = 6x = 42, Leucosidea and Spen- ceria – 2n = 2x =14 (Ikeda et al. 2006; Chung et al. 2012; Rice et al. 2015). Previously, a karyotype of A. pilosa var. japonica was examined by Iwatsubo et al. (1993). All studied plants had 2n = 56. Chromosomes at metaphase ranged 1.2–2.5 µm in length and 1.0–2.5 in arm ratio. These were clas- sified into two groups: 21 metacentric pairs, and seven submetacentric pairs. One submetacentric pair had a satellite on the short arm. According to other scientific data, somatic chromosome number of A. pilosa are 2n = 28 and 2n = 70 (Table 2). We had revealed a new diploid cytotype in A. pilo- sa collected in China with 2n = 16. Apparently, these plants exhibit diploid karyotype and new basic chro- mosome number x = 8 for the genus and subtribe. That chromosome number 2n = 16 was determined in all 127 investigated root meristematic cells. We suppose there were no B chromosomes for diploid cytotype 2n = 14. Dysploidy arising from chromosomes fusions (Escudero et al. 2014) is also unlikely, having our data on chromo- somes length and morphology are relevant to the results previously obtained on A. pilosa with 2n = 56 by Iwat- subo et al. (1993). We suppose that a haploidization of genome took place in A. pilosa specimen collected in China. According to classification developed by Kimber and Riley (1963), this event relates to aneupolyhaploidia, that is haploidization of polyploid form associated with aneuploidy. In addition to karyotypè s divergence from typical polyploid A. pilosa with 2n = 56, the investigated herbar- ium specimen exhibits reduced dimensions, fruits, and seeds. This corresponds with the revealed less ploidy lev- el of the plant because polyploidy is followed by «gigas»- effect (Ramsey and Ramsey 2014). We do not exclude the possibility that this specimen could be related to a new taxon. Our flow cytometry studies revealed that C-values in two investigated A. pilosa differ at a factor of two, approximately. This result was unexpected to us due to the fact that the number of chromosomes didǹ t corre- late with relative DNA content in two examined agrimo- nies. Unfortunately, we had no A. pilosa specimen with chromosome number 2n = 28 to determine relative DNA content and to compare it with data obtained. The sizes of the monoploid genome were found to be equal 1Сх = 1.25 pg for samples with 2n = 16, and 1Сх = 0.62 pg for samples with 2n = 56 which indicates a significantly more ancient origin of diploid populations, according to the genome downsizing theory (Leitch, Bennett 2004). Due to other studies reports, DNA loss in polyploid series is usually at the level of 15.4% (Zenil-Fergusson et al. 2016), whereas in our case, such a significant decrease may indicate complex molecular- genetic processes and DNA loss during the evolution of the Agrimonia pilosa genome. Studies of many eukary- otic genomes show that noncoding regions of DNA can be lost in the polyploidization process (Shaked et al. 2001). Some cytological studies show a loss of hetero- chromatin, whole chromosomes or their segments after polyploidization (Gustafson and Bennett 1982; Song et al. 1995; Chen and Ni 2006; Xiong et al. 2011). 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Niamat Ali1, Bashir A Ganai2 Population genetic studies in wild olive (Olea cuspidata) by molecular barcodes and SRAP molecular markers Rayan Partovi1, Alireza Iaranbakhsh1,*, Masoud Sheidai2, Mostafa Ebadi3 In Vitro Polyploidy Induction in Persian Poppy (Papaver bracteatum Lindl.) Saeed Tarkesh Esfahani1, Ghasem Karimzadeh1,*, Mohammad Reza Naghavi2 Long-term Effect Different Concentrations of Zn (NO3)2 on the Development of Male and Female Gametophytes of Capsicum annuum L. var California Wonder Helal Nemat Farahzadi, Sedigheh Arbabian*, Ahamd Majd, Golnaz Tajadod A karyological study of some endemic Trigonella species (Fabaceae) in Iran Hamidreza Sharghi1,2, Majid Azizi1,*, Hamid Moazzeni2 Karyological studies in thirteen species of Zingiberacaeae from Tripura, North East India Kishan Saha*, Rabindra Kumar Sinha, Sangram Sinha