# Karyomorphological Studies in Some Species ofParnassia L. (Saxifragaceae s.l.) in East Asia and Intraspecific Polyploidy of P. palustris L.

Authors: Tsuneo Funamoto; Katsuhiko Kondo
Journal: Journal of Botany
 (2012)

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## Abstract

Karyomorphological information is one of the most important characters for cytotaxonomy. We described karyomorphology of 14 species ofParnassia in East Asia. They had commonly the resting chromosomes of the simple chromocenter type and the mitotic prophase chromosomes of the proximal type. The somatic chromosome number of 2n=14 was shown in three species, that of 2n=18 was shown in six species, that of 2n=18 or 36 was shown in two species, that of 2n=32 was shown in one species, that of 2n=36 or 36+1~8 s was shown in one species, and that of 2n=18, 27, 36 or 45 was shown in one species. They were commonly monomodal (gradual) decrease in length from the largest to the smallest chromosomes. However, their absolute chromosome sizes were different from each other. Average chromosome sizes were recognized as three types small (1.21 μm), medium (1.7–2.36 μm), and large (3.1–3.75 μm). Parnassia palustris showed four cytotypes that could be likely cytogeographically correlated with habitats, polyploidy, and sizes of pollen grains.


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## Body

## 1. Introduction

The genusParnassia L., traditionally classified in the monotypic subfamily Parnassioideae, the family Saxifragaceae sensu lato [1], is currently placed in the family Parnassiaceae, closely related to Saxifragaceae in the order Celastrales [2, 3]. The genus Parnassia consists of approximately 70 species [4], that are perennial herbs and grow in wet grassy plains and forests in the temperate to arctic regions of the Northern Hemisphere. Among the species of this genus, 63 species are found in China [4], five species are distributed in Russia [5] of which four species are common to China and Russia, and three species grow in Japan [6].The chromosome numbers of 33 species ofParnassia have been documented as 2n=18, 27, 32, 33, 34, 35, 36, 37, 36 + 1~8s, 43, 44, 45, 48, and 54 including intraspecific polyploid and aneupoid (e.g., [24, 28–35]; Table 1).Table 1
Chromosome numbers ofParnassia previously reported.
SpeciesChromosome numberSelected bibliographyP. tenella Hook.f. et Thoms.2n=18Ding et al. 2005 [7]P. yunnanensis Franch. var. longistipitata Jine2n=32Funamoto et al. 1997 [8]P. esquirolii Lévl.2n=18Ding et al. 2005 [7]P. faberi Oliv.2n=18Ding et al. 2005 [7]P. chinensis Franch.2n=18Ding et al. 2005 [7]P. epunctulata J.T. Pan2n=18Ding et al. 2005 [7]P. venusta Jien2n=18Ding et al. 2005 [7]P. mysorensisHeyne ex Wigjt et Arn.2n=18Ding et al. 2005 [7]P. scaposa Mattf. var. yushuensis J.T. Pan2n=18Funamoto et al. 1996 [9]P. aff. cacuminum Hand.-Mazz. f. yushuensis Ku2n=18Funamoto et al. 2001 [10]P. pusilla Wall.n=9Malla et al. 1979 [11]P. oreophylla Hance2n=18,36Funamoto et al. 1994, 1996 [10, 12]P. nubicola Wall.n=9Malla et al. 1979 [11]P. subscaposa C.Y. Wu ex Ku2n=18Ding et al. 2005 [7]P. trinervis Drude2n=18Funamoto et al. 1996 [9]; Ding et al. 2005 [7]P. viridiflora Batalin2n=18,36Funamoto et al. 1998 [13], 2001 [10, 13]P. laxmannii Pall.2n=18,36Funamoto et al. 2002 [14]; Murín et al. 1984 [15]P. brevistyla (Brieg) Hand.-Mazz.2n=14Funamoto et al. 2001 [10]; Ding et al. 2005 [7]P. delavayi Franch.2n=14Funamoto et al. 1998, 2001 [10, 13]; Ding et al. 2005 [7]P. alpicola Makino2n=18Funamoto 1986 [16]P. bifolia Nekrass.2n=36,36+1~8sFunamoto et al. 2002 [14]; Ding et al. 2005 [7]P. wightiana Wall.2n=14,36Funamoto et al. 1998 [13]; Malla et al. 1981 [17]P. monochorifolia Franch.2n=48Ding et al. 2005 [7]P. foliosa Hook.f. et Thoms.2n=18Funamoto 1986 [16]P. grandifolia D.C.2n=32Gastony and Soltis 1977 [18]P. caroliniana Michaux2n=32Gastony and Soltis 1977 [18]P. asarifolia Vent.2n=32Gastony and Soltis 1977 [18]P. glauca Raf.2n=32,36Gastony and Soltis 1977 [18]; A. Löve and D. Löve 1980 [19]P. palustrisL.2n=18,27,32,33,34,35,36,37,43,44,45,54Hultgård 1987 [20]; Lövkvist and Hultgård 1999 [21]; Funamoto et al. 2006, 2008 [22, 23]P. obtusiflora Rupr.2n=36A. Löve and D. Löve 1982 [24]P. kotzebuei Cham.2n=18,36Packer 1964 [25]; Löve and Ritchie 1966 [26]P. fimbriata Koenig.2n=36Packer 1964 [25]P. townsendii Robinson2n=36Bye and Soltis 1979 [27]The present study contributes cytological data for the species ofParnassia, mainly in its East Asian distribution range, and based on karyomorphology with respect to resting nuclei, mitotic prophase chromosomes and the mitotic metaphase chromosomes, the distribution of intraspecific polyploidy and aneuploidy, and cytogeographic patterns.


## 2. Materials and Methods

Total 400 samples of 14 species ofParnassia were collected in 75 localities in Russia, Mongolia, China, and Japan (Figure 1; Table 2) and were brought and cultivated in pots in shade place in the experimental garden of Showa Pharmaceutical University. Somatic chromosome observations were made in meristematic cells of fresh root tips. They were pretreated in 2 mM 8-hydroxyquinoline at ca 20°C for 4 hours and then fixed in 45% acetic acid at ca 2°C for 10 minutes. They were macerated in a mixture of 45% acetic acid and 1 N hydrochloric acid (1 : 1) at ca 60°C for 20–23 seconds and were then stained with 2% aceto-orcein at room temperature in moist chamber with 45% acetic acid for ca 30 minutes and were prepared by the conventional squash method. Measurements of chromosomes in 14 species were made in each average length in several good metaphase cells.Table 2
Chromosome numbers in 14 species ofParnassia studied here.
SectionSpeciesNumber of locality and samplesChromosome number (2n)SaxifragastrumP. yunnanensis var. Franch. longistipitata Jien2 localities 17 samples32P. scaposa Mattf. var. yushuensis J.T. Pan5 localities 17 samples18P. aff. cacuminum Hand.-Mazz. f. yushuensis Ku1 locality 5 samples18P. oreophila Hance3 localities 24 samples1 locality 2 samples1836P. trinervis Drude7 localities 28 samples18NectarotrilobosP. viridiflora Batalin1 locality 5 samples1 locality 3 samples1836P. laxmannii Pall.2 localities 11 samples18P. brevistyla (Brieg) Hand.-Mazz2 localities 7 samples14P. delavayi Franch.2 localities 5 samples14P. alpicolaMakino1 locality 1 sample18NectarabilobosP. bifolia Nekrass.5 localities 39 samples1 locality 5 samples3636 + 1s, 2s, 3s, 5s, 7s, 8s*AllolobosP. wightiana Wall.1 locality 4 samples14FimbripetalumP. foliosaHook.et Thoms.4 localities 25 samples18Parnassia(Nectarodroson)P. palustris L.28 localities 153 samples3 localities 12 samples 11 localities 35 samples 1 locality 2 samples1827 36 45*s: supernumerary chromosome.Figure 1
Collection sites of 14 species ofParnassia in China, South Siberia and Far East in Russia, Mongolian Altai, and Japan.Morphological types of the resting and the mitotic prophase chromosomes were classified for Tanaka [36, 37], and the classification of mitotic metaphase chromosomes followed Levan et al. [38]. Taxonomical treatment followed Czerepanov [5], Ku and Hultgård [4], and Akiyama [6]. The voucher specimens of the plants used in this study were deposited in Funamoto’s Herbarium in Showa Pharmaceutical University.


## 3. Results and Discussion

### 3.1. Resting Nuclei and Mitotic Prophase Chromosomes

Fourteen species ofParnassia in East Asian (Table 2) had common karyomorphological characters in the resting and the mitotic prophase chromosomes: the resting chromosomes were classified as the simple chromocenter type which had several small heteropycnotic bodies per nucleus according to Tanaka [36, 37], and the mitotic prophase chromosomes were classified as the proximal type in which the early condensed segments were confined to the proximal regions of both short and long arms or short arm, showing clear condensation transition from the proximal to the distal regions (Tanaka [36, 37]; Figure 2). There was no difference in the resting and the mitotic prophase chromosome types among in these 14 species, thus it was concluded that the genus Parnassia had the simple chromocenter types at the resting nucleus and the proximal types at the mitotic prophase chromosomes.Figure 2
Karyomorphological comparisons of resting and mitotic prophase chromosomes in 14 species ofParnassia. (a)–(n) The resting chromosomes; (a′)–(n′) the mitotic prophase chromosomes. (a) and (a′) P. yunnanensis var.longistipitata; (b) and (b′) P. scaposa var.yushuensis; (c) and (c′) P. aff.cacuminum f. yushuensis; (d) and (d′) P. oreophylla; (e) and (e′) P. trinervis; (f) and (f′) P. viridiflora; (g) and (g′)P. laxmannii; (h) and (h′) P.  brevistyla; (i) and (i′) P. delavayi; (j) and (j′) P. alpicola; (k) and (k′) P. bifolia; (l) and (l′) P. wrightiana; (m) and (m′) P.  foliosa; (n) and (n′) P. palustris. Scale bar = 5 μm.


### 3.2. The Mitotic Metaphase Chromosomes

Chromosome numbers in 33 species ofParnassia were from 2n=14 to 2n=54 (Table 1). Three species had the somatic chromosome number of 2n=14, five species had that of 2n=32, one species had that of 2n=48, and the other 24 species had that of 2n=18. Intraspecific polyploids were found in P. oreophylla, P. viridiflora, P. laxmannii, P. palustris, and P. obtusiflora, while intraspecific aneuploids were found in P. bifolia, P. wightiana, P. glauca, and P. palustris (Table 1). Particularly, Parnassia  palustris had various chromosome numbers from 2n=18 to 54 (e.g., [34, 35]; Table 1).Parnassia brevistyla (Figure 3(j)), P. delavayi (Figure 3(k)), and P. wightiana (Figure 3(n)) had 2n=14, P.  scaposa var. yushuensis (Figure 3(b)), P. aff. cacuminum f. yushuensis (Figure 3(c)), P. trinervis (Figure 3(f)), P. laxmannii (Figure 3(i)),  P. alpicola (Figure 3(l)), and P. foliosa (Figure 3(o)) had 2n=18, P. oreophylla (Figures 3(d) and 3(e)) and P. viridiflora (Figures 3(g) and 3(h)) had 2n=18 and 36, P. palustris had 2n=18, 27, 36, and 45 (Figures 3(p)–3(s)), P. yunnanensis var. longistipitata had 2n=32 (Figure 3(a)), and P. bifolia had 2n=36 and 36 + 1~8s (Figures 3(m) and 4).  Parnassia palustris in a plant collected in Scandinavia by Hultgård [20] and Lövkvist and Hultgård [21] was very rarely documented to have the chromosome numbers of 2n=27 and 45. The chromosome number of 2n=14 for P. wightiana [13] was different from the previous counts of 2n=36 [17] and 2n=32 [18], while that of P. glauca was also different from the previous count of 2n=36 [19]. Thus, these two species are necessary to have more cytological studies. A peculiar chromosome number of 2n=14 for Chinese species of Parnassia was reported by Funamoto et al. [10, 13] and Ding et al. [7]. The chromosome number of 2n=32 was previously reported in four North American species of Parnassia such as P. gandifolia, P. caroliniana, P. asarifolia, and P. glauca by Gastony and Soltis [18] and that was also found in Chinese species [8].Figure 3
Somatic metaphase chromosomes in 14 species ofParnassia. (a) P. yunnanensis var.longistipitata (2n=32); (b) P. scaposa var.yushuensis (2n=18); (c) P. aff.cacuminum f. yushuensis (2n=18); (d) and (e) P. oreophylla ((d) 2n=18; (e) 2n=36); (f) P. trinervis (2n=18); (g) and (h) P. viridiflora ((g) 2n=18; (h) 2n=36); (i) P. laxmannii (2n=18); (j) P.  brevistyla (2n=14); (k) P. delavayi (2n=14); (l) P. alpicola (2n=18); (m) P. bifolia (2n=36); (n) P. wrightiana (2n=14); (o) P.  foliosa (2n=18); (p) to (s) P. palustris ((p) 2n=18; (q) 2n=27; (r) 2n=36; (s) 2n=45). Arrow points satellite. Scale bar = 5 μm.Figure 4
Variations of supernumerary chromosomes ofParnassia bifolia. Arrow indicates satellites and arrows head indicates supernumerary chromosomes. (a) 2n=36. (b) 2n=36+1s; (c) 2n=36+2s; (d) 2n=36+3s; (e) 2n=36+5s; (f) 2n=36+7s; (g) 2n=36+8s. (s) supernumerary chromosomes. Scale bar = 5 μm.Aneuploid chromosome numbers of2n=36+1~8s for P. bifolia could include supernumerary chromosomes since these aneuploid plants had high pollen stainabilities (87.5–98.6%).


### 3.3. Chromosome Sizes and Basic Chromosome Numbers

Morphological characters of chromosomes in chromosome numbers, chromosome sizes, chromosome complements in centromeric positions, and chromosome alignment from the longest to the shortest one are important information for cytotaxonomy. The chromosome number of2n=14 was considered diploid x=7, that of 2n=32 was considered tetraploid x=8, and that of 2n=18 and 36 was considered diploid and tetraploid x=9, respectively, if these basic chromosome numbers of x=7, 8, and 9 were accepted. Plants of the basic chromosome number of x=7 are found in China, those of x=8 are found in China and North America, and those of x=9 are commonly found in the world. Their karyotypes had commonly monomodal gradation in chromosome length from the longest to the shortest chromosomes and had median and submedian centromeric chromosomes, rarely median, submedian, and subterminal centromeric chromosomes (Table 3; Figure 5). Thus, Parnassia could be divided by four groups at least from chromosome characters mentioned above. (1) Small-sized and median-centromeric chromosomes ranged from 1.6 to 1.0 μm long with average 1.21 μm long, and had the base number of x=8. (2) Medium-sized and median- and submedian-centromeric chromosomes ranged from 2.8 to 1.9 μm long with average 2.29 μm long and had the base number of x=9. (3) Large-sized and median- and submedian-centromeric chromosomes ranged from 3.6 to 2.7 μm long with average 3.10 μm long and had the base number of x=7. (4) Medium-sized and median-, submedian-, and subterminal-centromeric chromosomes ranged from 2.3 to 1.4 μm long with average 1.91 μm long, and had the base number of x=7. These cytotaxonomical groupings were not agreed with the taxonomical treatment by Ku [39]. More cytological information and another new technique are necessary to clarify and justify the cytosystematic relationships of Parnassia.Table 3
Karyotype characters in 14 species ofParnassia observed here.
SpeciesChromosome number (2n)Chromosome length (μm)FormLongest ± SDShortest ± SDTotal ± SDAverage ± SDP. yunnanensis var. longistipitata321.6±0.11.0±038.6±1.11.21±0.08mP. scaposa var. yushuensis182.8±0.31.9±0.241.2±3.82.29±0.21m + smP. aff. cacuminum f.yushuensis182.3±0.11.5±033.0±0.91.83±0.07mP. oreophyla182.8±0.21.8±0.240.6±3.22.26±0.27m + sm362.8±0.31.8±0.278.5±4.12.13±0.16m + smP. trinervis182.7±0.22.1±0.142.5±2.92.36±0.19m + smP. viridiflora182.0±0.11.6±0.130.7±1.21.71±0.11m + sm362.1±0.21.4±0.161.1±1.81.70±0.09m + smP. laxmannii182.3±0.31.6±0.134.9±1.71.94±0.1mP. brevistyla143.6±0.72.7±0.443.4±3.33.10±0.25m + smP. delavayi144.3±0.83.2±0.452.5±3.63.75±0.31m + smP. alpicola182.6±0.11.9±0.139.7±0.92.21±0.18m + smP. bifolia362.5±0.21.6±0.171.4±2.81.98±0.08m + smP. wightiana142.3±0.21.4±0.126.7±1.01.91±0.11m + sm + stP. foliosa182.8±0.31.9±0.239.5±2.82.19±0.13m + smP. palustris182.7±0.21.9±0.141.8±3.42.32±0.18m + sm272.8±0.21.9±0.161.2±3.92.27±0.15m + sm362.8±0.21.9±0.181.4±5.32.27±0.13m + sm452.7±0.11.8±099.4±2.52.21±0.07m + smSD: standard deviation; m: median-centromeric chromosome; sm: submedian-centromeric chromosome; st: subterminal-centromeric chromosome.Figure 5
Karyotype comparisons in 14 species ofParnassia. (a) P. yunnanensis var.longistipitata; (b) P. scaposa var.yushuensis; (c) P. aff.cacuminum f. yushuensis; (d) P. oreophylla; (e) P. trinervis; (f) P. viridiflora; (g) P. laxmannii; (h) P.  brevistyla; (i) P. delavayi; (j) P. alpicola; (k) P. bifolia; (l) P. wightiana; (m) P. foliosa; (n) P. palustris. Arrows indicate satellites. Scale bar = 5 μm.


### 3.4. Intraspecific Polyploid and Aneuploid

Intraspecific polyploid of2n=18 and 36 for P. oreophylla, P. viridiflora, and P. laxmannii, and that of 2n=18, 27, 36, 45, and 54 for P. palustris were reported, especially this polyploidy for P. palustris was found in Scandinavia [20, 21] and Great Britain [34, 35], and 2n=27 and 45 chromosomes were also found in Russia [22, 23]. For example, 156 samples of this species were collected in 23 localities in Altai Mountains, Tuva and Krasunoyarsk region in Russia, of which 113 samples (72.4%) in 18 sites had 2n=18 chromosomes, 12 samples (7.7%) in three sites had 2n=27 chromosomes, 29 samples (18.6%) in five sites had 2n=36 chromosomes, and two samples (1.3%) in one site had 2n=45 chromosomes (Figure 6; [22, 23]; Funamoto and Smirnov unpublished).Figure 6
Map of collection sites and polyploidy ofParnassia palustris in Altai Mountains and the adjacent regions. Chromosome numbers of 2n=18 (⬤), 2n=27 (▲), 2n=36 (★), and 2n=45 (■).According to pollen stainability and sizes of pollen grains ofParnassia  palustris, plants of 2n=18 and 36 had high pollen stainabilities (81.4–98.2%), while those of 2n=27 and 45 had very low pollen stainabilities (0.74–0.85%). Sizes of pollen grains of the species were 27.3–30.3 μm × 25.8–28.1 μm in plants of 2n=18, 30.1–32.5 μm × 27.6–30.2 μm in plants of 2n=27, and 31.8–33.6 μm × 30.1–31.3 μm in plants of 2n=36, respectively. Significant t-test in sizes of pollen grains showed significant difference between the plants of 2n=18 and 2n=36 with P<0.05 and P<0.01 levels, respectively. Sizes of pollen grains could be correlated with polyploid levels [22, 23]. Similar results were reported with Japanese Deutzia crenata [40–42] and Japanese Hydrangea paniculata [43].The plants with the chromosome number of2n=18 were commonly found along the vegetation edges such as in the disturbed riverine, forest meadows and tundra in Russian and Mongolian Altai, and Tuva in Russia, those with the chromosome number of 2n=27 were found in placid lake side and together with the plants of the chromosome number 2n=18 in Russian Altai, while those with the chromosome number of 2n=36 were commonly found in narrow stream in mountain slope and mountain meadows in Russian Altai and Krasnoyarsk Region in Russia, and those with the chromosome number of 2n=45 were rarely found together with the plants with the chromosome number of 2n=36 in Russian Altai. Thus, Parnassia  palustris in Altai Mountains and adjacent regions in Russia seemed one of the well-diversified species with respect to chromosome characters. These cytological phenomena speculated that the two plant strains with the chromosome numbers of 2n=27 and 2n=45 might be isolated by pollination between nondisjunction diploid gametes producing diploid and tetraploid ovules (2x and 4x) and normal male gamete for diploid pollen (x), since any plant with the chromosome number of 2n=27 has never been found within the populations of the plants with the chromosome number of 2n=36. This factor might be caused by various geographical conditions and extremes of climate temperature [22, 23].


## 3.1. Resting Nuclei and Mitotic Prophase Chromosomes

Fourteen species ofParnassia in East Asian (Table 2) had common karyomorphological characters in the resting and the mitotic prophase chromosomes: the resting chromosomes were classified as the simple chromocenter type which had several small heteropycnotic bodies per nucleus according to Tanaka [36, 37], and the mitotic prophase chromosomes were classified as the proximal type in which the early condensed segments were confined to the proximal regions of both short and long arms or short arm, showing clear condensation transition from the proximal to the distal regions (Tanaka [36, 37]; Figure 2). There was no difference in the resting and the mitotic prophase chromosome types among in these 14 species, thus it was concluded that the genus Parnassia had the simple chromocenter types at the resting nucleus and the proximal types at the mitotic prophase chromosomes.Figure 2
Karyomorphological comparisons of resting and mitotic prophase chromosomes in 14 species ofParnassia. (a)–(n) The resting chromosomes; (a′)–(n′) the mitotic prophase chromosomes. (a) and (a′) P. yunnanensis var.longistipitata; (b) and (b′) P. scaposa var.yushuensis; (c) and (c′) P. aff.cacuminum f. yushuensis; (d) and (d′) P. oreophylla; (e) and (e′) P. trinervis; (f) and (f′) P. viridiflora; (g) and (g′)P. laxmannii; (h) and (h′) P.  brevistyla; (i) and (i′) P. delavayi; (j) and (j′) P. alpicola; (k) and (k′) P. bifolia; (l) and (l′) P. wrightiana; (m) and (m′) P.  foliosa; (n) and (n′) P. palustris. Scale bar = 5 μm.


## 3.2. The Mitotic Metaphase Chromosomes

Chromosome numbers in 33 species ofParnassia were from 2n=14 to 2n=54 (Table 1). Three species had the somatic chromosome number of 2n=14, five species had that of 2n=32, one species had that of 2n=48, and the other 24 species had that of 2n=18. Intraspecific polyploids were found in P. oreophylla, P. viridiflora, P. laxmannii, P. palustris, and P. obtusiflora, while intraspecific aneuploids were found in P. bifolia, P. wightiana, P. glauca, and P. palustris (Table 1). Particularly, Parnassia  palustris had various chromosome numbers from 2n=18 to 54 (e.g., [34, 35]; Table 1).Parnassia brevistyla (Figure 3(j)), P. delavayi (Figure 3(k)), and P. wightiana (Figure 3(n)) had 2n=14, P.  scaposa var. yushuensis (Figure 3(b)), P. aff. cacuminum f. yushuensis (Figure 3(c)), P. trinervis (Figure 3(f)), P. laxmannii (Figure 3(i)),  P. alpicola (Figure 3(l)), and P. foliosa (Figure 3(o)) had 2n=18, P. oreophylla (Figures 3(d) and 3(e)) and P. viridiflora (Figures 3(g) and 3(h)) had 2n=18 and 36, P. palustris had 2n=18, 27, 36, and 45 (Figures 3(p)–3(s)), P. yunnanensis var. longistipitata had 2n=32 (Figure 3(a)), and P. bifolia had 2n=36 and 36 + 1~8s (Figures 3(m) and 4).  Parnassia palustris in a plant collected in Scandinavia by Hultgård [20] and Lövkvist and Hultgård [21] was very rarely documented to have the chromosome numbers of 2n=27 and 45. The chromosome number of 2n=14 for P. wightiana [13] was different from the previous counts of 2n=36 [17] and 2n=32 [18], while that of P. glauca was also different from the previous count of 2n=36 [19]. Thus, these two species are necessary to have more cytological studies. A peculiar chromosome number of 2n=14 for Chinese species of Parnassia was reported by Funamoto et al. [10, 13] and Ding et al. [7]. The chromosome number of 2n=32 was previously reported in four North American species of Parnassia such as P. gandifolia, P. caroliniana, P. asarifolia, and P. glauca by Gastony and Soltis [18] and that was also found in Chinese species [8].Figure 3
Somatic metaphase chromosomes in 14 species ofParnassia. (a) P. yunnanensis var.longistipitata (2n=32); (b) P. scaposa var.yushuensis (2n=18); (c) P. aff.cacuminum f. yushuensis (2n=18); (d) and (e) P. oreophylla ((d) 2n=18; (e) 2n=36); (f) P. trinervis (2n=18); (g) and (h) P. viridiflora ((g) 2n=18; (h) 2n=36); (i) P. laxmannii (2n=18); (j) P.  brevistyla (2n=14); (k) P. delavayi (2n=14); (l) P. alpicola (2n=18); (m) P. bifolia (2n=36); (n) P. wrightiana (2n=14); (o) P.  foliosa (2n=18); (p) to (s) P. palustris ((p) 2n=18; (q) 2n=27; (r) 2n=36; (s) 2n=45). Arrow points satellite. Scale bar = 5 μm.Figure 4
Variations of supernumerary chromosomes ofParnassia bifolia. Arrow indicates satellites and arrows head indicates supernumerary chromosomes. (a) 2n=36. (b) 2n=36+1s; (c) 2n=36+2s; (d) 2n=36+3s; (e) 2n=36+5s; (f) 2n=36+7s; (g) 2n=36+8s. (s) supernumerary chromosomes. Scale bar = 5 μm.Aneuploid chromosome numbers of2n=36+1~8s for P. bifolia could include supernumerary chromosomes since these aneuploid plants had high pollen stainabilities (87.5–98.6%).


## 3.3. Chromosome Sizes and Basic Chromosome Numbers

Morphological characters of chromosomes in chromosome numbers, chromosome sizes, chromosome complements in centromeric positions, and chromosome alignment from the longest to the shortest one are important information for cytotaxonomy. The chromosome number of2n=14 was considered diploid x=7, that of 2n=32 was considered tetraploid x=8, and that of 2n=18 and 36 was considered diploid and tetraploid x=9, respectively, if these basic chromosome numbers of x=7, 8, and 9 were accepted. Plants of the basic chromosome number of x=7 are found in China, those of x=8 are found in China and North America, and those of x=9 are commonly found in the world. Their karyotypes had commonly monomodal gradation in chromosome length from the longest to the shortest chromosomes and had median and submedian centromeric chromosomes, rarely median, submedian, and subterminal centromeric chromosomes (Table 3; Figure 5). Thus, Parnassia could be divided by four groups at least from chromosome characters mentioned above. (1) Small-sized and median-centromeric chromosomes ranged from 1.6 to 1.0 μm long with average 1.21 μm long, and had the base number of x=8. (2) Medium-sized and median- and submedian-centromeric chromosomes ranged from 2.8 to 1.9 μm long with average 2.29 μm long and had the base number of x=9. (3) Large-sized and median- and submedian-centromeric chromosomes ranged from 3.6 to 2.7 μm long with average 3.10 μm long and had the base number of x=7. (4) Medium-sized and median-, submedian-, and subterminal-centromeric chromosomes ranged from 2.3 to 1.4 μm long with average 1.91 μm long, and had the base number of x=7. These cytotaxonomical groupings were not agreed with the taxonomical treatment by Ku [39]. More cytological information and another new technique are necessary to clarify and justify the cytosystematic relationships of Parnassia.Table 3
Karyotype characters in 14 species ofParnassia observed here.
SpeciesChromosome number (2n)Chromosome length (μm)FormLongest ± SDShortest ± SDTotal ± SDAverage ± SDP. yunnanensis var. longistipitata321.6±0.11.0±038.6±1.11.21±0.08mP. scaposa var. yushuensis182.8±0.31.9±0.241.2±3.82.29±0.21m + smP. aff. cacuminum f.yushuensis182.3±0.11.5±033.0±0.91.83±0.07mP. oreophyla182.8±0.21.8±0.240.6±3.22.26±0.27m + sm362.8±0.31.8±0.278.5±4.12.13±0.16m + smP. trinervis182.7±0.22.1±0.142.5±2.92.36±0.19m + smP. viridiflora182.0±0.11.6±0.130.7±1.21.71±0.11m + sm362.1±0.21.4±0.161.1±1.81.70±0.09m + smP. laxmannii182.3±0.31.6±0.134.9±1.71.94±0.1mP. brevistyla143.6±0.72.7±0.443.4±3.33.10±0.25m + smP. delavayi144.3±0.83.2±0.452.5±3.63.75±0.31m + smP. alpicola182.6±0.11.9±0.139.7±0.92.21±0.18m + smP. bifolia362.5±0.21.6±0.171.4±2.81.98±0.08m + smP. wightiana142.3±0.21.4±0.126.7±1.01.91±0.11m + sm + stP. foliosa182.8±0.31.9±0.239.5±2.82.19±0.13m + smP. palustris182.7±0.21.9±0.141.8±3.42.32±0.18m + sm272.8±0.21.9±0.161.2±3.92.27±0.15m + sm362.8±0.21.9±0.181.4±5.32.27±0.13m + sm452.7±0.11.8±099.4±2.52.21±0.07m + smSD: standard deviation; m: median-centromeric chromosome; sm: submedian-centromeric chromosome; st: subterminal-centromeric chromosome.Figure 5
Karyotype comparisons in 14 species ofParnassia. (a) P. yunnanensis var.longistipitata; (b) P. scaposa var.yushuensis; (c) P. aff.cacuminum f. yushuensis; (d) P. oreophylla; (e) P. trinervis; (f) P. viridiflora; (g) P. laxmannii; (h) P.  brevistyla; (i) P. delavayi; (j) P. alpicola; (k) P. bifolia; (l) P. wightiana; (m) P. foliosa; (n) P. palustris. Arrows indicate satellites. Scale bar = 5 μm.


## 3.4. Intraspecific Polyploid and Aneuploid

Intraspecific polyploid of2n=18 and 36 for P. oreophylla, P. viridiflora, and P. laxmannii, and that of 2n=18, 27, 36, 45, and 54 for P. palustris were reported, especially this polyploidy for P. palustris was found in Scandinavia [20, 21] and Great Britain [34, 35], and 2n=27 and 45 chromosomes were also found in Russia [22, 23]. For example, 156 samples of this species were collected in 23 localities in Altai Mountains, Tuva and Krasunoyarsk region in Russia, of which 113 samples (72.4%) in 18 sites had 2n=18 chromosomes, 12 samples (7.7%) in three sites had 2n=27 chromosomes, 29 samples (18.6%) in five sites had 2n=36 chromosomes, and two samples (1.3%) in one site had 2n=45 chromosomes (Figure 6; [22, 23]; Funamoto and Smirnov unpublished).Figure 6
Map of collection sites and polyploidy ofParnassia palustris in Altai Mountains and the adjacent regions. Chromosome numbers of 2n=18 (⬤), 2n=27 (▲), 2n=36 (★), and 2n=45 (■).According to pollen stainability and sizes of pollen grains ofParnassia  palustris, plants of 2n=18 and 36 had high pollen stainabilities (81.4–98.2%), while those of 2n=27 and 45 had very low pollen stainabilities (0.74–0.85%). Sizes of pollen grains of the species were 27.3–30.3 μm × 25.8–28.1 μm in plants of 2n=18, 30.1–32.5 μm × 27.6–30.2 μm in plants of 2n=27, and 31.8–33.6 μm × 30.1–31.3 μm in plants of 2n=36, respectively. Significant t-test in sizes of pollen grains showed significant difference between the plants of 2n=18 and 2n=36 with P<0.05 and P<0.01 levels, respectively. Sizes of pollen grains could be correlated with polyploid levels [22, 23]. Similar results were reported with Japanese Deutzia crenata [40–42] and Japanese Hydrangea paniculata [43].The plants with the chromosome number of2n=18 were commonly found along the vegetation edges such as in the disturbed riverine, forest meadows and tundra in Russian and Mongolian Altai, and Tuva in Russia, those with the chromosome number of 2n=27 were found in placid lake side and together with the plants of the chromosome number 2n=18 in Russian Altai, while those with the chromosome number of 2n=36 were commonly found in narrow stream in mountain slope and mountain meadows in Russian Altai and Krasnoyarsk Region in Russia, and those with the chromosome number of 2n=45 were rarely found together with the plants with the chromosome number of 2n=36 in Russian Altai. Thus, Parnassia  palustris in Altai Mountains and adjacent regions in Russia seemed one of the well-diversified species with respect to chromosome characters. These cytological phenomena speculated that the two plant strains with the chromosome numbers of 2n=27 and 2n=45 might be isolated by pollination between nondisjunction diploid gametes producing diploid and tetraploid ovules (2x and 4x) and normal male gamete for diploid pollen (x), since any plant with the chromosome number of 2n=27 has never been found within the populations of the plants with the chromosome number of 2n=36. This factor might be caused by various geographical conditions and extremes of climate temperature [22, 23].



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*Source: 874256-2012-05-15.xml*