2023 Volume 88 Issue 1 Pages 55-59
Paeonia (Paeoniaceae) has an ancient origin, unique genetic evolution, and an isolated taxonomic status. China is the genetic origin of the genus Paeonia and an area rich in wild Paeonia resources. However, since herbaceous peony is not as well studied as woody peony, especially some species that are currently endemic to China. One such species that is endemic to China is Paeonia mairei, which is valuable germplasm for evolution research and cultivar improvement. Despite this, the karyotype of P. mairei remains inconclusive. In this study, root tips of plants from eight populations of P. mairei were used to examine their ploidy and to assess karyotype diversity. The results indicate that all eight P. mairei populations were tetraploid (2n=4x=20=12m+4sm+4st) with a 2A karyotype. Karyotype asymmetry ranged from 60.61% for P6 (Liangfengya, Foping County) to 63.12% for P4 (Ningqiang County). The chromosome length ratio ranged from 1.7398 (Dongchuan District) to 1.9660 (Dayi County) while the AI ranged from 4.4535 (Ningqiang County) to 5.6085 (Foping County). These findings indicate that the population in Foping County is relatively evolved whereas that in Ningqiang County is relatively primitive. Cluster analysis of karyotypic parameters showed that P3 (Dayi County) and P7 (Miaogou, Foping County) populations differed greatly from the other six populations, which could be clustered into two geographic branches. Our findings provide a scientific basis for exploring origin and evolutionary characteristics of P. mairei.
Paeonia, the only genus in the Paeoniaceae, has an isolated taxonomy and ancient origin (Pan 1995, Zhou et al. 2021). Paeonia is comprised of 33 species that are mainly distributed in the northern temperate zone, although wild species are mainly distributed in temperate zones of the northern hemisphere while individual species may extend to cold temperate zones (Pan 1995, Hong 2011). China is the genetic origin or center of Paeonia and one of the distribution centers of subgenus Paeonia wild resources. However, there is limited research and analysis of these resources while their use and application are underdeveloped, undermining attempts to protect and utilize wild Chinese peony resources. Paeonia lactiflora is the only wild species that is used for medicinal and ornamental purposes, while similar in-depth studies on other Paeonia species have not been performed. Insufficient attention has been paid to P. mairei, an endemic species from China. Given that P. mairei displays early flowering and has a thick stem, single branch, and single flower, these horticultural characteristics have tremendous potential for developing early flowering and cut-flower cultivars. It is thus important to study the evolution of the genus Paeonia for breeding purposes and for developing related medicinal and horticultural industries.
P. mairei is a perennial herb with carrot-shaped roots, single and glabrous stems in which lower leaves are biternate, leaflets are oblong-ovate or oblong-lanceolate, glabrous, usually acuminate or even caudate at the apex, flowers are terminal, there are 7–9 obovate petals, and pink to red, the disk is yellow while the stigmas are red, the involucrate bracts are leaf-like or linear, seeds are black and oblong-spherical, flowering occurs in April and May (Hong 2011). P. mairei is endangered and is suffering from a shrinking distribution range (Yang et al. 2017a, b). The first karyotype analysis of a P. mairei cultivar revealed that it was diploid (La Cour 1952). A karyotype analysis of two wild populations of P. mairei in Sichuan and one wild population in Shanxi, in China, indicated the species as being tetraploid (Hong et al. 1988). It noted differences in ploidy among three P. mairei populations, but since these populations were located in the northwest and north of the distribution limits of P. mairei, they postulated that diploid populations might exist in the south and southeast of the distribution range of P. mairei, recommending extensive sampling to determine whether ploidy was in any way associated with geographic distribution.
Karyotype analyses are an important method for revising species classification and studying phylogenetic relationships (Hong 2021). By analyzing the karyotypic parameters, cytological evidence for plant taxonomy can be obtained, and this is of great significance to understanding genome size, species origin, and evolution, and is also important evidence for classification and identification. Herbaceous peonies have rich germplasm diversity in terms of external morphology and ploidy (Ji et al. 2014, Hao et al. 2016, Yang et al. 2017a). Considerable cytological work has been carried out on Paeonia. Dark (1936) found that the basic chromosome number was x=5. Ding and Liu (1991) studied P. obovata, finding that tetraploid (2n=4x=20) species existed in China. Hong et al. (1988) and Sang et al. (2004) found that P. veitchii and P. anomala were both diploid (2n=2x=10). When the proportion of chromosome with arm ratio more than 2 : 1 in the chromosome set is between 0.1 and 0.5, using the ratio of the longest chromosome to the shortest one, the karyotype of a species can be judged, and when the ratio is less than 2 : 1, it is referred to as 2A, but when the ratio is between 2 : 1 and 4 : 1, it is referred to as 2B (Stebbins 1971). Most peonies are 2A (Sang et al. 2004), whereas only Hong et al. (1988) and Liu et al. (2016) found 2B among individuals of a P. obovata population. Research on the karyotype of P. mairei is lacking, so its ploidy is uncertain. In this study, a karyotype analysis, ploidy, and cluster analysis of karyotype characteristics morphology of eight P. mairei populations with a wide geographic distribution range were determined. The resulting information may provide a cytological basis for exploring the ploidy and genetic characteristics, as well as phylogeny and origin, of P. mairei.
In 2021, a field survey was conducted in southwestern China, covering most of the geographic distribution of P. mairei, and eight populations from seven counties were identified (Table 1). About five plants per population were carefully removed and transported to Beijing, then planted in the Beijing Forestry University greenhouse.
| P code | Locality | Latitude, longitude |
|---|---|---|
| P1 | Yinmin, Dongchuan District, Yunnan Province | E102°56′30″, N26°16′30″ |
| P2 | Yaoshan Nature Reserve, Qiaojia County, Yunnan Province | E103°07′29″, N27°11′58″ |
| P3 | Xiling Snow Mountain, Dayi County, Sichuan Province | E103°12′36″, N30°41′50″ |
| P4 | Hanshuiyuan Scenic Area, Ningqiang County, Shanxi Province | E106°07′29″, N32°46′25″ |
| P5 | Manping, Maiji District, Gansu Province | E106°28′20″, N34°18′25″ |
| P6 | Liangfengya, Foping County, Shanxi Province | E107°51′11″, N33°40′54″ |
| P7 | Miaogou, Foping County, Shanxi Province | E107°53′33″, N33°30′36″ |
| P8 | Wangshun mountain, Lantian County, Shanxi Province | E109°30′22″, N34°06′42″ |
Actively growing root tips (1–3 cm) were pretreated with saturated p-dichlorobenzene for 24 h in the dark at 4°C, fixed in Carnoy’s solution (ethanol : acetic acid=3 : 1, v/v) for 24 h, and then stored in 70% ethanol at 4°C until use. Root tips were hydrolyzed in 1 M HCl at 60°C for 10 min, washed with distilled water three times, and stained with improved carbon fuchsin for 5–10 min. Karyokinetic observations were made and documented by a Leica Application Suite version 3 (Leica Microsystems, Wetzlar, Germany). Chromosome measurements were made using five well-spread metaphase plates per population. Karyotype analyses were performed using the criteria described by Chen et al. (2003). Karyotype symmetry was classified according to Stebbins (1971). Karyotype asymmetry (long arm length/total genome length×100) was determined according to Arano (1963). The index of relative length (IRL) (chromosome length/mean genome length) and composition of the relative length of the genome (CRL) (chromosome length/genome length) were calculated according to Kuo et al. (1972). Chromosome morphology was determined using an arm ratio (long arm/short arm) and classified as either m (1.01–1.70), sm (1.71–3.00), or st (3.01–7.00) (Levan et al. 1964). The ratio of the longest to the shortest chromosome (Lt/St) and centromere index percentage (short arm length/chromosome length×100) was also calculated. The asymmetric index (AI) was calculated as CVCL×CVCI/100, where CVCL is the relative variation in chromosome length, and CVCI is the relative variation in the centromeric index (Paszko 2006).
Based on eight parameters (average arm ratio, relative length, karyotype asymmetry coefficient, chromosome length ratio, average centromeric index, AI, CVCL, and CVCI), a system clustering method was adopted. Using R software, the data was the first standardized by the scale function, the Euclidean distance among samples was calculated using the dist function, and the final distance matrix was calculated by the hclust function to perform an unweighted pair-group method with arithmetic means (UPGMA) analysis (Li et al. 2019).
The mitotic metaphase of the well-dispersed chromosomes of eight populations of P. mairei is shown in Fig. 1. Base numbers were x=5. No aneuploidy was observed in any populations and the ploidy was identical, i.e., all populations were tetraploid (2n=4x=20=12m+4sm+4st), with 20 chromosomes. The karyotypes of all populations were 2A. According to the relative length coefficient and Kuo classification standard, the chromosomes of P2, P3, P4, P5, and P6 were divided into four types: long chromosome L (≥1.26), medium and long chromosome M2 (1.01–1.25), medium and short chromosome M1 (0.76–1.00) and short chromosome S (＜0.76). P1, P7, and P8 did not have short chromosome S (＜0.76) (Table 2).
| P-code | Chromosome number and ploidy | Relative lengthz | IRL | Mean arm ratioy | Lt/Stx | AI (%)w | Karyotypev | Average centromere index (%)u | CVCLt | CVCIs | AIr |
|---|---|---|---|---|---|---|---|---|---|---|---|
| P1 | 2n=4x=20 | 15.54–27.01 | 2L+6M2+12M1 | 1.9550 | 1.7398 | 61.78 | 2A | 0.3770 | 17.0179 | 26.2007 | 4.4589 |
| P2 | 2n=4x=20 | 14.63–27.39 | 2L+6M2+10M1+2S | 1.9223 | 1.8458 | 62.32 | 2A | 0.3750 | 19.5145 | 24.7776 | 4.8352 |
| P3 | 2n=4x=20 | 13.24–27.02 | 3L+7M2+6M1+4S | 1.8352 | 1.9660 | 62.15 | 2A | 0.3792 | 22.0194 | 23.0478 | 5.0750 |
| P4 | 2n=4x=20 | 14.24–26.36 | 2L+7M2+9M1+2S | 1.9282 | 1.8821 | 63.12 | 2A | 0.3752 | 17.3239 | 25.7070 | 4.4535 |
| P5 | 2n=4x=20 | 14.29–25.85 | 2L+8M2+8M1+2S | 1.9227 | 1.8238 | 60.61 | 2A | 0.3810 | 16.6291 | 26.9840 | 4.4872 |
| P6 | 2n=4x=20 | 13.93–25.93 | 2L+8M2+8M1+2S | 1.8474 | 1.8384 | 60.57 | 2A | 0.3803 | 19.4967 | 24.5868 | 4.7936 |
| P7 | 2n=4x=20 | 15.78–27.71 | 3L+6M2+11M1 | 1.9818 | 1.7598 | 62.27 | 2A | 0.3753 | 19.8111 | 28.3100 | 5.6085 |
| P8 | 2n=4x=20 | 15.26–26.40 | 2L+8M2+10M1 | 1.8543 | 1.7829 | 61.04 | 2A | 0.3784 | 18.7740 | 23.4974 | 4.4114 |
ZChromosome length/total chromosome length. yLong arm/short arm. xRatio of longest chromosome to shortest chromosome. wClassification of karyotypes concerning their degree of asymmetry according to Stebbins (1971). vLong arm length/total genome length×100. uShort arm length/chromosome length×100. tRelative variation in chromosome length. sRelative variation in the centromeric index. rIndex to measure karyotype asymmetry proposed by Paszko (2006), where AI=(CVCL×CVCI)/100
These results indicate that there were differences in relative length, IRL, chromosome length ratio (Lt/St), average arm ratio, average centromere index, karyotype asymmetry coefficient, and the AI among eight P. mairei populations (Table 2). The relative length of chromosomes was between 13.24% (P3) and 27.71% (P7). The length ratio of chromosomes ranged from 1.7398 (P1) to 1.9660 (P3). The mean arm ratio ranged from 1.8352 (P3) to 1.9818 (P7). Karyotype asymmetry ranged from 60.61% (P5) to 63.12% (P6). The average centromeric index ranged from 37.5% (P2) to 38.10% (P5). The AI ranged from 4.4114 (P8) to 5.6085 (P7).
Cluster analysis of chromosomes and karyotypes of P. mairei populationsUPGMA-based cluster analysis of the eight P. mairei populations (Fig. 2) indicated that when the genetic distance is 2.5, P2 and P4 are clustered together, P6 and P8 are clustered together, and P1 and P5 are clustered separately. When the distance is 3.2, the result indicated four main clusters: cluster 1 (P3), cluster 2 (P7), cluster 3 (P1, P2, P4), and cluster 4 (P5, P6, P8). Clusters 3 and 4 belong to the eastern and western populations, respectively, while P3 and P7 may have evolved separately and more rapidly in eastern and western branches. When the genetic distance is 5.5, the result indicated three main clusters: cluster 1 (P1, P2, P4, P5, P6, P8), cluster 2 (P7), and cluster 3 (P3). When the genetic distance is 6, P1, P2, P4, P5, P6, P7, P8 clustered into a single clade, and P3 clustered into a single clade.
The basic chromosome number of Paeonia is x=5, all of them being euploid (Wang 2010). Polyploidy is an important aspect of evolutionary diversification and is one of the most important cytogenetic mechanisms in plant evolution and rapid speciation (Stebbins 1971, Levin 2002, Jian et al. 2013). La Cour (1952) carried out karyotype analyses of P. mairei for the first time, showing that it was diploid, although the experimental material was a cultivar. Hong et al. (1988) analyzed the karyotypes of three populations in Sichuan and Shanxi, and found that P. mairei was tetraploid. Similarly, our study shows that only tetraploid chromosomes were found in eight geographically diverse P. mairei populations. This suggests that diploids do not exist in wild P. mairei populations, all having a 2n=4x=20=12m+4sm+4st karyotype.
Chromosome morphology is the main karyotypic feature used to study relationships and evolutionary trends among subspecies or populations. Most herbaceous and tree peonies belong to 2A (Sang et al. 2004), although Hong et al. (1988) and Liu et al. (2016) found 2B cases in a P. obovata population and a hybrid of ‘Zhu ShaPan’ and ‘Cream Delight,’ respectively. The eight P. mairei populations in this study had the same chromosome number and karyotype, i.e., 2A. UPGMA analysis suggests that P7 evolved faster than other populations, based on the mean arm ratio, whereas P3 evolved more rapidly than other populations, based on chromosome length ratio. According to Stebbins (1971), the evolutionary trend of karyotypes in the plant kingdom is from symmetry to asymmetry. Thus, relatively primitive plants in systematic evolution have relatively symmetric karyotypes, but evolutionarily more advanced plants may have an asymmetric karyotype (Stebbins 1971, Deng et al. 2009, 2011). In our study, the karyotype asymmetry of eight P. mairei populations ranged from 60.57 to 63.12%: P2, P4, and P7 have poor karyotype symmetry and a high degree of evolution, while P5, P6, and P8 have better karyotype symmetry so that is relatively primitive.
In addition, the AI, which has a high degree of precision and sensitivity to assess karyotype asymmetry, with higher values considered to indicate higher levels of karyotypic heterogeneity (Paszko 2006, Zhang et al. 2013), was used to assess karyotype asymmetry. The AI can also indicate an evolutionary trend, so primitive species usually have a lower AI, whereas a plant with a higher AI value would indicate that it is more evolutionarily advanced (Deng et al. 2011, Gao et al. 2012, Zhang et al. 2013). The AI of P. mairei ranged from 4.4114 to 5.6085, ranked as P7＞P1＞P4＞P5＞P2＞P8＞P6＞P3, with P3 and P7 (5.0750 and 5.6085, respectively) having a relatively higher degree of evolution, in contrast to P8, P1, and P4 (4.4114, 4.4588, and 4.4535, respectively), with a relatively lower degree of evolution.
UPGMA-based genetic distances of karyotypic data (Fig. 2) suggest that P. mairei is ecologically divided into two branches: one branch in Sichuan and Yunnan (eastern populations) and one branch in Shaanxi (western populations). Average temperature, annual temperature differences, and precipitation are higher in the eastern region (Chen et al. 2020). Our findings indicate karyotype-based differences among eight populations, although additional research is needed, such as molecular phylogeny and genomic in situ hybridization, to identify the role of the ecological environment in genetic diversification.
Populations in this paper were investigated and transplanted after receiving the approval of the forestry bureau of the nature reserve. This work was financially supported by the National Natural Science Foundation (grant #: 32071817).
