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

Karyomorphological information is one of the most important characters for cytotaxonomy. We described karyomorphology of 14 species of Parnassia 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.


Introduction
The genus Parnassia 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 present study contributes cytological data for the species of Parnassia, 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.

Materials and Methods
Total 400 samples of 14 species of Parnassia 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  [27] 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.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.

Resting Nuclei and Mitotic Prophase
Chromosomes.Fourteen species of Parnassia 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.

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 of 2n = 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) Smallsized 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 medianand 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 medianand 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.
The plants with the chromosome number of 2n = 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 welldiversified 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].

Table 1 :
Chromosome numbers of Parnassia previously reported.

Table 2 :
Chromosome numbers in 14 species of Parnassia studied here.

Table 3 :
Karyotype characters in 14 species of Parnassia observed here.Collection sites of 14 species of Parnassia in China, South Siberia and Far East in Russia, Mongolian Altai, and Japan.