2020 Volume 85 Issue 2 Pages 115-122
Chromosomal traits and their variation were investigated for a total of 435 plants from 23 local populations of the three subspecies of Chamaelirium hisauchianum. 334 out of 340 plants of subsp. kurohimense had 2n=44=12L+32S (where L and S denote ‘long’ and ‘short’ respectively) with the base number 22 (x1), and six others were all hyperploids with 2n=45 (2x1+1; five plants) or 46 (2x1+2; one plant). In subsp. hisauchianum, 46 out of 48 plants had 2n=42=14L+28S with the base number 21 (x2), and two others had 2n=42 (2x2) with an aberrant karyotype or 2n=43 (2x2+1). In subsp. minoense, 45 out of 47 plants, like subsp. hisauchianum, possessed 2n=42=14L+28S (x2=21), and two others showed 2n=43 (2x2+1) or 45 (2x2+3). All of the observed chromosomal variants exhibited structural heterozygosity and presumably arose by fission, fusion, and duplication probably derived from irregular chromosomal disjunction. The chromosomal variants found in all populations examined amounted to 2.3% on average. Pollen stainability with cotton blue in four chromosomal variants of subsp. kurohimense and minoense was lower than in chromosomally normal plants. Some topics relevant to the results were also discussed, comparing with the data previously reported for C. japonicum and C. koidzumianum that markedly differ in the mating system as well as some chromosomal traits.
Chamaelirium hisauchianum (Okuyama) N. Tanaka is one of the four species belonging to subsect. Chionographis in sect. Chionographis (Tanaka 2017). It is a perennial endemic to Japan, comprising three subspecies, hisauchianum, minoense and kurohimense. Subspecies kurohimense is distributed on the Japan Sea side of central and northern Honshu, while two other subspecies, hisauchianum and minoense, are confined respectively to small areas on the Pacific side of central Honshu. These subspecies are allopatric in distribution (Tanaka 1985, 2003, 2017). The populations of C. hisauchianum are reported as either hermaphroditic or gynodioecious, hence the individuals are hermaphroditic (including male or andromonoecious plants) or female (Tanaka 1985, 2003, 2016). The hermaphrodites are highly self-compatible (Tanaka 2003), whereas those of most populations of C. japonicum and all populations of C. koidzumianum of the same subsection investigated are highly self-incompatible (Tanaka 2003, 2020).
Chromosomal traits, such as chromosome number and karyotype, of the three subspecies of C. hisauchianum have been reported by Kurosawa and Hara (1960), Hara and Kurosawa (1962), Ajima (1976), and Tanaka and Tanaka (1977, 1979, 1980). According to these reports, subsp. kurohimense has 2n=44, while subsp. hisauchianum and minoense have 2n=42, thus markedly differing from C. japonicum and C. koidzumianum that possess 2n=24 (e.g. Hara and Kurosawa 1962, Tanaka and Tanaka 1977, 1979, Tanaka 2020). Except for subsp. minoense, all of the taxa so far investigated have been reported to have holocentric chromosomes.
Despite these investigations, we still have very limited knowledge of chromosomal variation at the population level of C. hisauchianum. In species having holocentric chromosomes, chromosomal variants originated by chromosomal fission are expected to occur, for fragmented chromosomes are likely to survive through mitoses and gametes with such fragments may participate in sexual reproduction. This deduction has recently been explored for C. japonicum and C. koidzumianum (Tanaka 2020), but not yet for C. hisauchianum that is contrastingly distinct from them in the mating system as well as some chromosomal traits such as karyotype and ploidy level. To fill this lack of knowledge, many individuals of C. hisauchianum were sampled from different local populations and their chromosomal traits and variation were investigated. In this paper, some topics relevant to the results are also discussed, comparing with the data previously reported for C. japonicum and C. koidzumianum.
Plants of C. hisauchianum were collected in the 1980s from 23 localities (Table 1). These were individually potted and cultivated in the experimental nursery of Teikyo University at Hachioji, Tokyo. Herbarium specimens were prepared from many of them to serve as vouchers and for phenotypical studies and are currently preserved at the author’s herbarium at Hachioji, Tokyo. Taxonomic identification followed Tanaka (2017).
| Taxon/Locality code: Locality | Total no. of plants examined | Chromosome no. and karyotype (2n) | Ploidy1) |
|---|---|---|---|
| Subsp. kurohimense | |||
| T: Tohoku District | |||
| YK: Akita Pref., Yuri-honjyô-shi, Mt. Yakushi | 18 | 44=12L+32S | 2x1 |
| TO: Akita Pref., Yuri-honjyô-shi, Mt. Tôkô | 4 | 44=12L+32S | 2x1 |
| OS: Akita Pref., Yuri-honjyô-shi, Ôsawakyô | 3 | 44=12L+32S | 2x1 |
| C: Chûbu District | |||
| WK: Niigata Pref., Murakami-shi, Wakigawa | 10 | 44=12L+32S | 2x1 |
| IM: Niigata Pref., Murakami-shi, Imagawa | 22 | 44=12L+32S | 2x1 |
| KW: Niigata Pref., Murakami-shi, Kuwagawa | 40 | 44=12L+32S | 2x1 |
| 1 | 45=12L+32S+1f | 2x1+1 | |
| MA: Niigata Pref., Murakami-shi, Nogata, Majima | 8 | 44=12L+32S | 2x1 |
| TG: Niigata Pref., Shibata-shi, Tagai | 18 | 44=12L+32S | 2x1 |
| NI: Niigata Pref., Shibata-shi, Mt. Ninôji | 8 | 44=12L+32S | 2x1 |
| TK: Niigata Pref., Gosen-shi, Takôji | 12 | 44=12L+32S | 2x1 |
| GM: Niigata Pref., Minami-kanbara-gun, Tagami-machi, Gomadôyama | 31 | 44=12L+32S | 2x1 |
| OG: Niigata Pref., Santô-gun, Izumozaki-cho, Oginojyô | 8 | 44=12L+32S | 2x1 |
| HK: Niigata Pref., Nagaoka-shi, Hakama | 21 | 44=12L+32S | 2x1 |
| SU: Niigata Pref., Kashiwazaki-shi, Suganuma, Kanakura* | 1 | 44=12L+32S | 2x1 |
| 1 | 45=12L+33S | 2x1+1 | |
| HA: Niigata Pref., Nagaoka-shi, Mt. Hachikoku | 11 | 44=12L+32S | 2x1 |
| KO: Niigata Pref., Kashiwazaki-shi, Kôchi | 14 | 44=12L+32S | 2x1 |
| 1 | 45=12L+31S+2f | 2x1+1 | |
| YO-1: Niigata Pref., Kashiwazaki-shi, Tan’ne, Mt. Yoneyama | 35 | 44=12L+32S | 2x1 |
| 1 | 45=12L+32S+1f | 2x1+1 | |
| YO-2: Niigata Pref., Jyôetsu-shi, Kakizaki-cho, Mt. Yoneyama (Shimomaki-guchi) | 11 | 44=12L+32S | 2x1 |
| 1 | 45=12L+33S | 2x1+1 | |
| KH: Niigata Pref., Itoigawa-shi, Mt. Kurohime | 30 | 44=12L+32S | 2x1 |
| 1 | 46=11L+35S | 2x1+2 | |
| MJ: Niigata Pref., Itoigawa-shi, Mt. Myôjyô | 21 | 44=12L+32 | 2x1 |
| OA: Nagano Pref., Azumi-gun, Otari-mura, Kita-otari, Ôami | 8 | 44=12L+32 | 2x1 |
| Total 1 | 340 | ||
| Subsp. hisauchianum | |||
| K: Kantô District | |||
| KA : Saitama Pref., Han’nô-shi, Kawamata | 46 | 42=14L+28S | 2x2 |
| 1 | 42=13L+1L′+27S+1f | 2x2-ab2) | |
| 1 | 43=14L + 29S | 2x2+1 | |
| Total 2 | 48 | ||
| Subsp. minoense | |||
| C: Chûbu District | |||
| NE: Gifu Pref., Motosu-shi, Neo | 45 | 42=14L+28S | 2x2 |
| 1 | 43=14L+29S | 2x2+1 | |
| 1 | 45=14L+31S | 2x2+2 | |
| Total 3 | 47 |
*Material collected by Shun’itsu Iwano. 1) For chromosome base number, see the text. 2) 2x2-ab: 2x2 with aberrant karyotype.
Root tip meristematic cells were used to observe chromosome traits. The method for making chromosomal preparations is the same as stated in Tanaka (2020). For observation of meiosis, very young spikes were excised and fixed in a 2 : 1 : 1 mixture of ethanol, acetic acid, and chloroform for over 1 h. A few immature anthers fixed were placed onto a glass slide, squeezed with a dissecting needle tip, stained with 1 or 2% aceto-orcein, and carefully pressed after a coverslip was mounted on the samples. For the method for investigating pollen stainability with cotton blue and the information about the microscope and camera used, see Tanaka (2020).
A total of 340 plants of subsp. kurohimense from 21 localities were chromosomally examined (Tables 1, 2). Of them, 334 plants had 44 chromosomes, of which 12 were relatively long (L) and 32 were short (S), although the distinction between the shortest L and the longest S was slight. The somatic chromosome composition or karyotype can be formulated as 2n=44=12L+32S (Fig. 1A), coinciding with previous reports (Ajima 1976, Tanaka and Tanaka 1979). At least five plants from five localities (C-GM, -HA, -HK, -KW, -NI; Table 1) observed regularly formed 22 bivalents at meiotic metaphase I (n=22, Fig. 2A), and the subsequent meiotic process and microsporogenesis proceeded normally, forming tetrad cells with n=22 (Fig. 2B) and sound pollen grains (Fig. 2C, Table 3). As two other species of the same subsection, C. japonicum and C. koidzumianum, are diploid, having 2n=24 with the base number (x) 12 (Tanaka and Tanaka 1979, 1980, Tanaka 2020), subsp. kurohimense with 2n=44 is interpreted as a hypotetraploid (4x−4) if assumed to share the same base number with the two diploids. On the other hand, the chromosomes in meiosis of subsp. kurohimense behave regularly like those of diploids, in this respect, this subspecies is viewed as a secondarily balanced diploid with the base number x=22 (x1).
| Taxon | 2n (rarely found) | Total no. of examined plants (populations) | Total no. of chromosomal variants (%) | Type of chromosomal variant (total no. of plants) |
|---|---|---|---|---|
| C. hisauchianum | ||||
| subsp. kurohimense | 44 (45, 46) | 340 (21) | 6 (1.8) | Hyperploid (6) |
| subsp. hisauchianum | 42 (42*, 43) | 48 (1) | 2 (4.2) | Hyperploid (1), structural heterozygote* (1) |
| subsp. minoense | 42 (43, 45) | 47 (1) | 2 (4.3) | Hyperploid (2) |
| Total 1 | 435 (23) | 10 (2.3) | ||
| C. japonicum** | 24 (25, 36) | 334 (34) | 3 (0.9) | Hyperploid (1), triploid (2) |
| C. koidzumianum** | 24 | 20 (3) | 0 (0) | — |
| Total 2 | 354 (37) | 3 (0.8) |
*Structural heterozygote without change in chromosome number. **Data from Tanaka (2020).
| Taxon/Locality code* | 2n/karyotype (ploidy) | Total no. of plants examined | Total no. of pollen grains examined/plant: range (mean) | Pollen grains well stained/plant: range (mean) (%) | Plants examined: Collection number, date |
|---|---|---|---|---|---|
| Subsp. kurohimense | |||||
| C-KW | 44=12L+32S (2x1) | 1 | 823 | 98.1 | No. 4, 20 May 1980 |
| 45=12L+32S+1f (2x1+1) | 1 | 1103 | 57.8 | No. J200-13, 13 June 1980 | |
| C-HK | 44=12L+32S (2x1) | 1 | 825 | 99.4 | No. 7, June 1981 |
| C-KO | 44=12L+32S (2x1) | 4 | 564–1236 (795.8) | 98.9–99.3 (99.1) | No. J100-2, -4, -9, -10;20 June 1980 |
| 45=12L+31S+2f (2x1+1) | 1 | 919 | 44.4 | No. 4, 19 June 1981 | |
| C-YO-1 | 44=12L+32S (2x1) | 1 | 726 | 98.5 | Yoneyama-saru 3, 1 Apr. 1987 |
| 45=12L+32S+1f (2x1+1) | 1 | 858 | 86.4 | Yoneyama-ko A-3, June 1981 | |
| C-KH | 44=12L+32S (2x) | 1 | 783 | 98.9 | No.1040-5, 11 July 1980 |
| Subsp. hisauchianum | |||||
| C-KA | 42=14L+28S (2x2) | 3 | 505–781 (666.3) | 96.2–99.8 (98.4) | Nos. 235-1, -6, 260-11, 22 June 1980 |
| Subsp. minoense | |||||
| C-NE | 42=14L+28S (2x2) | 5 | 565–661 (608.4) | 99.5–100.0 (99.8) | Nos. 210-4, -6, 220-2, -3, -4, 4 June 1980 |
| 43=14L+29S (2x2+1) | 1 | 1828 | 93.9 | No. 350-6, 4 June 1980 |
Six others deviated in chromosome number and karyotype from the plants with 2n=44=12L+32S. All of the six variants were hyperploids, of which five were 2n=45 (2x1+1; Fig. 1B–F), and one was 2n=46 (2x1+2; Fig. 1G). Except for one plant whose sex is unknown, four plants were hermaphroditic (☿) and one was female (♀) (Fig. 1). These plants had the following karyotypes: — Two plants from the localities C-YO-1 (Yoneyama-ko A-3, ☿, 1 Apr 1987; Fig. 1B) and C-KW (no. J200-13, ☿, 13 June 1981; Fig. 1F) had 2n=45=12L+32S+1f (f: fragmented chromosome). Two plants from C-YO-2 (no. 400-6, ☿, 20 June 1980; Fig. 1C) and C-SU (no. 2, sex unknown, June 1980; Fig. 1D) possessed 2n=45=12L+33S. As having one excessive S chromosome that probably originated by duplication resulted from irregular chromosomal disjunction, they may be trisomic as to one of S chromosomes. One plant from C-KO (no. 4, ☿, June 1981; Fig. 1E) appears to have 2n=45=12L+31S+2f, the two fragments of which might have arisen by fission in one original S. One plant from C-KH (no. 1040-1, ♀, 11 June 1980; Fig. 1G) appears to have 2n=46=11L+35S, of which the three excessive S chromosomes might have originated by fission in one original L chromosome and addition in S chromosomes.
Subsp. hisauchianumA total of 48 plants of subsp. hisauchianum from one locality (K-KA) were chromosomally studied (Tables 1, 2). Of them, 46 plants had 2n=42=14L+28S (Fig. 3A), according with previous reports (Kurosawa and Hara 1960, Hara and Kurosawa 1962, Tanaka and Tanaka 1979). The chromosomes regularly form 21 bivalents at meiotic metaphase I, and the subsequent meiotic process and microsporogenesis proceed normally (Table 3), as reported by Tanaka and Tanaka (1980). Subsp. hisauchianum is, accordingly, regarded as a diploid with the base number of x=21 (x2). On the other hand, it is also interpreted as a hypotetraploid (2n=4x−6) if assumed to share the same base number (x=12) with the two diploids, C. japonicum and C. koidzumianum.
Two others deviated from the plants with 2n=42=14L+28S: —One (no. 25, ♀, 22 May 1986; Fig. 3B) was a diploid (2x2) with 2n=42=13L+1L′+27S+1f, including one particularly long L (designated as L′) and one f. There is the possibility that one original S fragmented into two, of which one became f, and the other fused with one original L, forming one L′. The other (no. 260-7, ☿, 22 June 1980; Fig. 3C) was a hyperploid (2x2+1) with 2n=43=14L+29S. Possessing one excessive S, it may be trisomic as to one of S chromosomes.
Subsp. minoenseA total of 47 plants from one locality C-NE of ssp. minoense were chromosomally investigated (Tables 1, 2). Forty-five of them consistently had 2n=42=14L+28S (Fig. 4C, D). Chromosomes measured for one metaphase plate in Fig. 4D showed that they range in length from 3.0 to 1.3 µm (L: 3.0–2.1 µm, S: 1.9–1.3 µm) with the mean 1.9 µm, and their total length was 80.0 µm. These measurements are well comparable to those for subsp. hisauchianum having the same chromosome number and a similar karyotype (Tanaka and Tanaka 1979). The 42 chromosomes regularly form 21 bivalents at metaphase I in meiosis, and the microsporogenesis proceeds normally (Table 3), as reported by Hara and Kurosawa (1962). Accordingly, subsp. minoense is regarded as a diploid with the base number x2=21. It is also viewed as a hypotetraploid (2n=4x−6) if assumed to share x=12 with diploid species, C. japonicum and C. koidzumianm.
Two others deviated from the plants with 2n=42=14L+28S: One (no. 350-6, ☿, 4 June 1980; Fig. 4F) was a hyperploid (2x2+1) with 2n=43=14L+29S. Having one excessive S, this plant may be trisomic regarding one of S chromosomes. The other (no. 350-7, sex unknown, 4 June 1980; Fig. 4G) was also a hyperploid (2x2+3) with 2n=45=14L+31S, including three excessive S. In the habitat these two plants occurred nearby (the distance between them was approximately less than 0.5 m). It is hence probable that the plant no. 350-7 arose as a consequence of fertilization involving gametes with excessive S chromosomes originally derived from the plant no. 350-6. Plant no. 350-7 might therefore be pentasomic as to one of S chromosomes.
Chromatin in the interphase nucleus (Fig. 4A) formed numerous small darkly stained heteropycnotic bodies as well as highly dispersed lightly stained domains. The heteropycnotic bodies looked somewhat more distinct and often slightly larger than those found in C. japonicum and C. koidzumianum (Tanaka and Tanaka 1979, Tanaka 2020), and the number per nucleus varied among nuclei but consistently far exceeded the somatic chromosome number. Chromosomes at prophase (Fig. 4B) were unevenly contracted along the length, forming a beaded configuration. Chromosomes at metaphase (Fig. 4C, D, F, G) and anaphase (Fig. 4E) had no portion corresponding to a localized centromere in cells treated with 8-hydroxyquinoline (Fig. 4C) before fixation or fixed directly without pretreatment (Fig. 4D–G). At anaphase, all pairs of sister chromatids separated and migrated parallel toward opposite spindle poles, suggesting the presence of diffuse centromeres along their length (Fig. 4E). There was no chromosome bearing a secondary constriction or satellite (Figs. 4C–G). These chromosomal traits in the mitotic cell cycle, including the holocentric nature of chromosomes, are reported here for the first time for this subspecies, and were quite similar to those of subsp. hisauchianum and kurohimense (Tanaka and Tanaka 1979).
C. hisauchianum as a species including three subspeciesPlants of C. hisauchianum are known to be sexually differentiated into two types, hermaphrodites and females, as stated earlier. However, no chromosomal difference was found between the two types in any of the three subspecies (Fig. 4C, D).
All of the ten chromosomal variants of the three subspecies showed no remarkable phenotypical difference from chromosomally normal plants of the respective subspecies.
Pollen stainabilityPollen stainability with cotton blue was examined for 11 plants of C. hisauchianum subsp. kurohimense, three plants of subsp. hisauchianum, and six plants of subsp. minoense (Table 3). In general, pollen grains stained well in blue were globose in shape and looked normal (fertile) (Fig. 2C, D), whereas those unstained or faintly stained were more or less distorted and apparently unviable (Fig. 2D). In eight plants of subsp. kurohimense with the normal karyotype 2n=44=12L+32S from C-KW, -HK, -KO, -YO-1, -KH (Fig. 2C), pollen stainability was high, ranging from 98.1 to 99.4% (98.9% on average). In contrast, in three chromosomal variants from C-KW (Fig. 2D), -KO, -YO-1, it was lower, ranging from 44.4 to 86.4% (62.9% on average). The percentage of well-stained pollen grains in chromosomally normal plants (2n=42=14L+28S) of subsp. hisauchianum from C-KA (three plants) and subsp. minoense from C-NE (five plants) were respectively high, ranging from 96.2 to 99.8% (the mean 98.4%) in the former, and from 99.5 to 100.0% (the mean 99.8%) in the latter. In one hyperploid with 2n=43=14L+29S of subsp. minoense, the percentage was 93.9 and only slightly lower than normal plants. The percentage of well-stained pollen grains in four chromosomal variants of both subsp. kurohimense and minoense varied widely from 44.4 to 93.9% (the mean 70.6%), probably reflecting varying degrees of chromosomal aberration.
In subsp. kurohimense, one hyperploid with 2n=45 from C-KW (no. J200-1, 13 June 1980) had one large (dyad-sized), well-stained pollen grain (0.1%). One chromosomally normal plant with 2n=44 from C-HK (no. 7, June 1981) also had one dyad-sized, well-stained pollen grain (0.1%). Another chromosomally normal plant with 2n=44 from C-KH (no. 1040-5, 11 July 1980) had two dyad-sized, well-stained pollen grains (0.3%) and one monad-sized, faintly stained pollen grains (0.1%).
The number of chromosomal variants found in the three subspecies of C. hisauchianum is summarized in Table 2. The percentage of the variants in populations varied from 1.8 to 4.3% (2.3% on average) between the three subspecies, and was slightly higher than that (0.8% on average) in two diploid species with 2n=24, C. koidzumianum (no variants) and C. japonicum (0.9%) (Table 2; Tanaka 2020). The hermaphrodites and andromonoecious plants of C. hisauchianum are highly self-compatible and hence deemed as autogamous, in contrast, those of C. japonicum and C. koidzumianum are highly self-incompatible and viewed as allogamous (Tanaka 2003). Accordingly, the rate of chromosomally anomalous seeds set by chromosomal variants of C. hisauchianum is expected to be higher than in the two self-incompatible plants. The higher occurrence of chromosomal variants in C. hisauchianum may partly be ascribed to the higher self-compatibility of the same species.
As stated in a previous paper (Tanaka 2020), plants of Chamaelirium propagate themselves mostly by sexual means, and it takes a comparatively long period (at least three or four years) for their seedlings to reach sexual maturity, and hence it is highly probable that the offspring produced by sexual reproduction are subject to natural selection and many of them eventually die. That is, the survival rate of the seeds or seedlings is presumably very low. This low survival rate is supposedly one of the main causes of the comparatively low occurrence of chromosomal variants in populations not only of C. japonicum and C. koidzumianum but also of C. hisauchianum (Table 2).
A total of ten chromosomal variants were found in populations of the three subspecies (Tables 1, 2). Of them, eight had odd-numbered, heterozygous karyotypes (Figs. 1B–F, 3C, 4F, G), while two others had even-numbered, heterozygous karyotypes; 2n=46=11L+35S in subsp. kurohimense (Fig. 1G), and 2n=42=13L+1L′+27S+1f in subsp. hisauchianum (Fig. 3B). As evident from these data, all of the variants observed were chromosomally unstable, structural heterozygotes. Seldom propagating themselves by asexual means, such heterozygotes, like other normal individuals, is to be lost in due course from populations. Judging from the total absence of chromosomally balanced stable variants in populations examined, there may be only a very small chance that new structural homozygotes are generated by heterozygotes and established in populations (Tanaka 2020). Since the occurrence of chromosomal variants was a little higher in self-compatible C. hisauchianum (Table 2), the possibility of their generating new homozygotes is expected to be also stronger than in self-incompatible C. japonicum and C. koidzumianum. Heterozygotes found were quite a few even in C. hisauchianum (Table 2), yet their presence is still significant in that they are potential generators of new chromosomally balanced variants.
The low pollen viability in chromosomal variants (Table 3) must also be a key factor for reducing the chance of chromosomal variants being newly produced. In three chromosomally normal plants of subsp. kurohimense (2n=44) from C-KW, -HK, and C-KH, large, well stained, seemingly viable pollen grains were very rarely observed (0.1, 0.1, 0.3%, respectively). The production of unreduced gametes in the form of dyads or monads may potentially lead to the formation of polyploids and serve as a factor for enhancing chromosome variation, but such large pollen grains appear to be too few to participate in sexual reproduction.
Types of chromosomal aberration observedOf a total of ten chromosomal variants found in C. hisauchianum including three subspecies, nine were hyperploids (Figs. 1B–G, 3C, 4F, G) and one was a structural heterozygote without a change in chromosome number (Fig. 3B, Tables 1, 2). These variants appear to have arisen by chromosomal mutations such as fission, fusion including translocation, and duplication probably resulted from an irregular chromosomal disjunction in meiosis. When compared with the chromosome number and size in species with 2n=24 (e.g. C. japonicum), there is no doubt that a single polyploidization event also occurred in the origination of C. hisauchianum. In plants with holocentric chromosomes, hyperploids originated by chromosome fragmentation are expected to occur, for fragmented chromosomes are likely to be retained through mitoses owing to their diffuse centromeres. Actually, in populations of C. hisauchianum several hyperploids with fragmented chromosomes were observed: —three plants of subsp. kurohimense with 2n=45 from C-YO-1 (Fig. 1B), C-KO (Fig. 1E) and C-KW (Fig. 1F), and one plant of subsp. hisauchianum with 2n=42 (including one fragment) from C-KA (Fig. 3B). Various types of chromosomal mutation observed in C. hisauchianum have also been reported to occur in species of Cyperaceae or Juncaceae known to have holocentric chromosomes: —e.g. polyploidization in Carex siderosticta (Tanaka 1939, Hoshino and Tanaka 1977) and Luzula spicata (Juncaceae) (Nordenskiörd 1951), chromosomal fission in C. oxyandra (Hoshino 1992), fusion in C. conica (Hoshino and Waterway 1994), duplication in Eleocharis kamtschatica (Yano and Hoshino 2006), and duplication, fission, and fusion in C. blepharicarpa (Hoshino and Okamura 1994).
On the other hand, the phenomenon known as agmatoploidy (Malheiros-Gardé and Garde 1950) or endonuclear polyploidy (Nordenskiörd 1951) was suggested to occur in Luzula as a special type of polyploidy or chromosome rearrangement that involves the fragmentation of an entire chromosome complement, generating a diploid with double its original chromosome number (Guerra 2016). However, this type of polyploidy in the strict sense (i.e., complete agmatoploidy excluding partial agmatoploidy, or endonuclear polyploidy excluding a half completed endonuclear polyploidy) has never been observed in Chamaelirium.
I cordially thank the late Mr. Shun’itsu Iwano, a botanist in Niigata Prefecture, Japan, who kindly provided me with living materials from a locality in the same prefecture. I also sincerely thank reviewers for valuable comments and corrections.
