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Article

Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan

by
Keita Watanabe
1,2,*,
Makoto Yaneshita
2,
Tetsuo Denda
3,
Masatsugu Yokota
3,
Shun K. Hirota
4,
Yoshihisa Suyama
5 and
Yoshihiko Tsumura
6
1
Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba 305-8572, Japan
2
Environment Research Section Urban Engineering Research Department, Taisei Corporation, Yokohama 245-0051, Japan
3
Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus, Nishihara 903-0213, Japan
4
Botanical Gardens, Osaka Metropolitan University, Katano 576-0004, Japan
5
Field Science Center, Graduate School of Agricultural Science, Tohoku University, Osaki 989-6711, Japan
6
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8786, Japan
*
Author to whom correspondence should be addressed.
Plants 2022, 11(22), 3015; https://doi.org/10.3390/plants11223015
Submission received: 10 October 2022 / Revised: 28 October 2022 / Accepted: 3 November 2022 / Published: 8 November 2022
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Abstract

:
Anthropogenic activities, such as the movement of plants through greening, can result in genetic disturbance that can interfere with local adaptation in wild populations. Although research is underway to prevent genetic disturbance associated with greening, genetic disturbance of intraspecific polyploidy, which is estimated to be present in 24% of vascular plants, has not been well studied. Liriope muscari is a polyploid complex with known diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) forms. The plants of this species tolerate dry and hot conditions and are therefore frequently used for greening and gardening. However, the distribution of this polyploid in Japan, its genetic structure, and genetic disturbance are not known. In this study, we investigated the polyploidy distribution and genetic structure in naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). Commercially produced individuals were also studied and compared with natural populations to assess any genetic disturbance of the ploidy complex in this species. Chromosome counts, cpDNA, and nDNA results showed three genetically and cytologically distinct groups in Japan: first, a tetraploid group in mainland Japan; second, a hexaploid group in the Ryukyu Islands; and third, a diploid and tetraploid group in the Ryukyu Islands. Significant isolation by distance was also detected within the three groups (p = 0.001). Genetic disturbance due to greening and gardening should be avoided among the three groups. Genetic disturbance can be reduced by using individuals derived from natural populations that are close to the sites used for greening and gardening. For commercially produced individuals, genetic disturbance is unlikely in the Kanto region, an area of high usage, while genetic disturbance is thought possible in the Ryukyu Islands.

1. Introduction

The genetic structure of a population is formed by various mechanisms including the interaction of gene flow, genetic drift, and natural selection [1]. The genetic structure of populations may include adaptations to the local environment [2], which may be the starting point for expansion into different environments and speciation. Such genetic structure is important for the maintenance of genetic diversity and biodiversity.
Anthropogenic activity, such as plant transfer through greening, can result in genetic disturbance, which can affect a natural population by disrupting its genetic structure and disturbing any adaptation to local conditions [3]. Several studies have been conducted to prevent genetic disturbance caused by revegetation [4,5,6,7].
Polyploidy is common among plants; approximately 24% of vascular plants are polyploid [8]. Polyploidization may be harmful in the short term by causing karyotype instability and reducing opportunities for propagation, etc., but in the long term it is thought to play an important role in the creation of diversity and species differentiation. It can do this by reducing the manifestation of deleterious genes that can result from the redundancy of multiple genomes, enabling sexual differentiation and adaptation to different environments [9,10,11]. In plants with intraspecific polyploidy, each polyploid with its distinct characteristics is sometimes geographically limited to a specific habitat due to changes in ecological niches and reproductive isolation [12,13,14]. Despite the importance of polyploid plants in evolutionary processes, few studies on genetic disturbances in polyploid plants exist in the literature [15,16].
Liriope muscari (Decne.) Bailey is an evergreen perennial plant found in Mainland China, Taiwan, South Korea, and Japan. It is a polyploid species comprising three forms: diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) [17,18,19,20,21]. L. muscari is a useful plant with many applications and has been used as a herbal medicine since ancient times [22]. It is often used for greening because of its tolerance to drought and high temperature, and its high phytoremediation effect on Zn, Cu, Pb, Ni, and Cd in planted soil has been reported [23]. As of 2021, approximately 420,900 plants had been produced in Japan [24]. It is propagated by means of seeds from its hermaphrodite flowers. Its pollinators are not well documented, but visits by Episyrphus and Parnara are known (Watanabe pers. obs). Seeds are thought to be spread by birds [25], and the raccoon dog Nyctereutes procyonoides viverrinus is known to feed on the seed [26]. As mentioned above, L. muscari, which is heavily used for greening and gardening, is likely to be subject to genetic disturbance. However, the distribution of the polyploids in Japan, its genetic structure, and genetic disturbance are not known.
Here, we report our investigations into the distribution of polyploidy and the genetic structure of naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). We also investigate commercially produced individuals to compare with the natural populations and discuss the genetic disturbance of the polyploidy complex in this species.

2. Results

2.1. Polyploidy Level

The polyploidy level of 116 individuals at 86 sites were identified as follows (Table 1, Figure 1): 18 were diploid (8 sites), 61 were tetraploid (49 sites), and 37 were hexaploid (29 sites) (Table 1, Figure 1). We estimated the chromosome number using multiple individuals at 21 sites. The diploid individuals were found sporadically in the Ryukyu Islands and Taiwan, the tetraploids were found in mainland Japan and the southern Ryukyu Islands, and the hexaploids were found only in the central Ryukyu Islands. All 18 commercial individuals of L. muscari obtained from six nurseries in the Kanto region were tetraploid. Of the three individuals obtained from the nursery on the main island of Okinawa, two were tetraploid and one was hexaploid (Figure 2).

2.2. Chloroplast DNA Haplotypes

By combining the sequence data of four cpDNA regions that were analyzed, we identified nine haplotypes (m01, m02, m03, m04, m05, m06, m07, m08, and m09). The nucleotide substitutions and indels are shown in Table S1. The haplotype diversity (h) and nucleotide diversity (π) of L. muscari are shown in Table 2. TCS haplotype network based on a sequence with four regions combined showed that the central haplotype was m01. Haplotypes m02, m03, and m09 were confirmed as independent haplotypes, each with a single mutation step. Systematic connections were indicated between m04 and m05 and between m06, m07, and m08. In terms of the relationship between the haplotype network and the polyploids, of the nine haplotypes, a different polyploidy level was confirmed to be present within the same haplotype in m01, m03, and m07 (Figure 3b).

2.3. Nuclear DNA

The Mantel test for Mash distance to SNP-based genetic distance showed a significant correlation between data sets (p = 0.001). SNP analysis finally called 93 SNPs with a genotyping rate of over 90%, and sequence coverage averages were above 30 for all individuals. Stacks parameters adjusted during SNP call settings were -m = 8, R0.85, -n = 1. The statistics obtained from stacks-2.60 are shown in Table 3.
In the neighbor-joining tree output from Mashtree, three groups related to chloroplast DNA haplotypes and polyploidy were recognized: Group 1 includes mainland Japan and is tetraploid with major chloroplast DNA haplotypes m04 and m09; Group 2 is distributed in the Ryukyu Islands and is hexaploid with the major cpDNA haplotype m02; and Group 3, also distributed in the Ryukyu Islands, is diploid and tetraploid, with the major cpDNA haplotypes m01 and m03 (Figure 3a,b). Group 3 was identified as several closely related clusters composed of diploid and tetraploid (Figure 3a). PCoA plots also distinguished three groups (Figure 3c). Nuclear and chloroplast DNA results were generally consistent, but some discrepancies were observed. Haplotype m01 was found in all three groups, and haplotype m07 was commonly found in groups 2 and 3 (Figure 3a). Mash distance and geographic distance in the three groups were significantly correlated (p = 0.001). In contrast to the trend of genetic diversity for cpDNA, a trend of increasing genetic diversity with increasing polyploidy level was observed for nDNA (Table 2 and Table 3).

2.4. Genetic Characteristics of Commercially Produced L. muscari

The polyploidy, cpDNA haplotype, and nDNA characteristics of 18 L. muscari individuals obtained from nurseries 1–6 in the Kanto region were consistent with those naturally distributed around the nurseries. All were tetraploid, belonged to Group 1 identified by nDNA, and had cpDNA haplotypes m04, m05, and m09 (Figure 3b and Figure 4). Of the three individuals obtained from the nursery on Okinawa Island, one was hexaploid and had haplotype m02 and was included in Group 2. The remaining two individuals were tetraploid, had cpDNA haplotype m03, and belonged to Group 3. One individual in Group 2 matched the major type obtained from the Okinawa mainland, while two individuals in Group 3 had cpDNA haplotype m03, a type with a more southerly distribution (Figure 3b and Figure 4).
Genetic diversity of commercially produced L. muscari in nurseries in mainland Japan, with the main area of consumption near Tokyo, was comparable to the genetic diversity of Group 1 in mainland Japan in both cpDNA and nDNA (Table 2 and Table 3).

3. Discussion

3.1. Distribution of Polyploidy Complex

All L. muscari individuals sampled from mainland Japan were tetraploid in our study; but the Ryukyu Islands samples comprised a mix of three polyploids. However, the hexaploid L. muscari has been reported in Hiroshima, mainland Japan [17]; the hexaploid form is therefore probably distributed throughout mainland Japan but only at a low frequency. Previous studies have reported the diploid form in Zhejiang Province, China; tetraploid in Korea; and hexaploid in mainland Japan [17,19,20,27]. Combined with the present results, there may be considerable overlap in the distribution of polyploidy, with the diploid distributed from the Ryukyu Islands to Taiwan and Zhejiang Province, China; the tetraploid from mainland Japan to the Ryukyu Islands and Korea; and the hexaploid from mainland Japan to the Ryukyu Islands. In our study we were able to clarify the polyploidy distribution pattern roughly in Japan. However, due to the limited number of survey sites and individuals, the distribution of L. muscari polyploidy throughout its distribution range remains unknown in term of the frequency of polyploidy in each region.
Two closely related diploid–tetraploid pairs were identified in the neighbor-joining tree by Mash distance, suggesting that the tetraploids have multiple origins. The multiple origins of polyploidy has also been reported elsewhere [28,29]. The multiple origins of polyploidy may be one of the factors contributing to the geographic obscurity of the distribution of the L. muscari polyploid complex in the Ryukyu Islands.

3.2. Genetic Structure of L. muscari in Japan

The three groups clearly differed in nDNA were also mostly consistent with the results for polyploidy and cpDNA, but partial discrepancies were observed in cpDNA. Incomplete lineage sorting and chloroplast capture are the main causes of mismatching between nuclear and chloroplast DNA [30,31,32]. In our present study, both processes are also possible; but given the wide distribution for haplotype m01, which was one of the two chloroplast DNA haplotypes that did not match the nuclear DNA results, and the fact that it was found in all three groups with different polyploidy, strongly suggests a high probability of incomplete lineage sorting.
The diverse results for polyploidy, chloroplast DNA, and nuclear DNA among the three groups suggest that a genetic barrier to gene flow exists between these groups. In general, geographic isolation and climatic conditions are known to be barriers to gene flow [33,34,35]. The adjacent Groups 1 and 2 are separated by the Strait of Tokara, which is known to have a different flora due to geographic isolation [35]. While Group 1 is tetraploid, Group 2 is hexaploid, and such differences in chromosome number are also a barrier to gene flow [36]. The boundary between Group 1 and Group 2 distributions is unclear, probably due to low sampling density, but multiple barriers may also prevent gene flow.
It is difficult to explain the geographic and climatic barriers to gene flow between Groups 2 and 3, whose distributions overlap in the Ryukyu Islands. Since Group 2 is hexaploid and Group 3 is diploid and tetraploid, a barrier to gene flow due to reproductive isolation at different polyploidy levels can be inferred. Group 3 includes diploid and tetraploid, but within the wild population the ploidy level is fixed to either diploid or tetraploid. Fixation of the polyploidy level in each local population suggests that diploid and tetraploid may be exclusive. It may be possible that gene flow is restricted between polyploids within Group 3.
The relationship between Mash distance and geographic distance showed a significant correlation for all groups. Restricted interbreeding between geographically distant individuals results in IBD [37]. L. muscari is a common species at low elevations within the range of our collection of samples. The possibility of restricted gene flow between diploids and tetraploids in Group 3 should be noted, but, in any case, it suggests that geographic proximity is important for interbreeding within groups.
We found lower cpDNA diversity of hexaploid individuals in the Ryukyu Islands compared to other polyploidy levels (h = 0.399, π = 0.00030). If both haplotype diversity and nucleotide diversity are low, the hexaploid may have experienced a more recent origin or a more restrictive bottleneck compared to other polyploidy levels. In either case, the hexaploid in the Ryukyu Islands is likely dependent on limited genetic sources. On the other hand, the higher genetic diversity of hexaploids compared to diploids and tetraploids in nDNA may reflect the increased diversity associated with genome duplication.

3.3. Taxonomic Confusion

The genus Liriope exhibits a certain amount of taxonomic confusion. L. tawadae is characterized by large plant size, broad and long leaf blades, and large flowers and long flower stalks, and has been reported in the Ryukyu Islands [38]. Due to the lack of morphological information, the relationship between L. tawadae and the result of this study is unclear. Some cpDNA haplotypes of L. muscari are shared with those of L. spicata (Watanabe, unpublished data), suggesting past hybridization. The situation is further complicated because L. spicata is known to be diploid, tetraploid, and hexaploid [19,21,39]. Further taxonomic reexamination, including related species, is therefore needed.

3.4. Potential of Anthropogenic Disturbance and Countermeasures

The commercially produced L. muscari was abundant near Tokyo; however, there was no obvious risk of genetic disturbance evident from our study. All L. muscari produced in nurseries near Tokyo were confirmed to be tetraploid and genetically close to naturally distributed individuals in the neighborhood. On the other hand, there is a possibility of genetic disturbance of L. muscari in the Ryukyu Islands due to greening. Despite our limited number of samples, we observed that genetically distinct L. muscari were being sold together. In addition, individuals from genetically distinct groups that were sold together also differed in their polyploidy levels, with one of the three individuals studied being Group 2 hexaploid and two being Group 3 tetraploids. The mixing of different polyploidy levels can cause additional problems. It has been noted that orthotopic growth of different polyploidy levels causes the eradication of minor polyploidy levels [40]. In fact, the polyploidy levels were fixed in populations where chromosome counts of multiple individuals were examined in this study. It should also be noted that pentaploids can easily be obtained by artificially crossing tetraploids and hexaploids from the Ryukyu Islands (Watanabe, unpublished data).
An effective means of preventing anthropogenic genetic disturbances of L. muscari is to avoid contact between genetically distinct groups. By selecting L. muscari that belong to the same genetic group as the natural population surrounding the proposed greening site, and by using seed collected from a single group during seedling production, contact between genetically distinct groups can be reduced. Considering within groups, genetic distance between geographically close individuals is close, so using individuals derived from natural populations that are geographically close to the proposed greening site is expected to further reduce genetic disturbance. Group 3 suggests multiple origins of the tetraploid, and although the situation may be complex, it is expected that the supply of individuals with the same polyploidy level from a natural population near the proposed site at the time of greening will reduce genetic disturbance.
Determining a level of genetic disturbance based on genetic information remains difficult for greening officials. With L. muscari in Japan, it is possible to recognize groups that should avoid contact with each other based on their geographic distribution and polyploidy levels. Three groups in Japan should avoid being genetically disturbed. The first group is the tetraploid distributed in mainland Japan. There are also two groups in the Ryukyu Islands that have overlapping geographic boundaries but can be distinguished by polyploidy level. The second group is the hexaploid of the Ryukyu Islands. The third group is the diploid and tetraploid of the Ryukyu Islands. At present it is difficult to estimate the polyploidy level of L. muscari by morphological features, but accumulating such morphological information will be an effective way to test for correspondence with the polyploidy level in order to aid their recognition in the field.

4. Materials and Methods

4.1. Collection of Materials

We collected between one and five individuals from 86 sites (totaling 116 individuals) in the natural distribution area, ranging from Niigata Prefecture in Japan to Taiwan. A further 21 commercially produced individuals were also collected: three each from two nurseries in Tokyo, four nurseries in Saitama Prefecture, and one nursery in Okinawa Prefecture (Table 1).

4.2. Determination of Polyploid Level

For each of the 137 L. muscari individuals collected, we counted their chromosome number using the aceto-orcein squash method. The root tip meristems were placed in a 0.002 M 8-hydroxyquinoline solution and pretreated at room temperature for 4–5 h. Subsequently, they were left in acetic acid alcohol (3:1, anhydrous ethanol–glacial acetic acid) to harden for at least five hours at 4 °C. After hardening, they were disaggregated for approximately 40 s in a 60 °C disaggregation solution (2:1, 1N hydrochloric acid–45% acetic acid) and stained for 1 to 15 min in a 2% aceto-orcein solution, and then squashed on a glass slide. The number of chromosomes was counted in somatic cells at metaphase.

4.3. Chloroplast DNA Analysis

The total DNA was extracted following the CTAB method of Doyle and Doyle (1987) after removing polysaccharides using the method of Setoguchi and Ohba (1995) [41,42].
Using four pairs of primers developed by Taberlet, (1991), Denda and Yokota (2003), Nakamura et al. (2006), Liston and Kadereit (1995) [43,44,45,46], polymerase chain reaction (PCR) amplification was conducted for the following intron and intergenic spacers and regions: trnK 5′ intron: (5′-CTCAACGGTAGAGTACTCG-3′, 5′-CCAAAAACTTCCACAGGTTCG-3′), trnT-trnL: (5′-GCGATGCTCTAACCTCTGAG-3′, 5′-TAGCGTCTACCGATTTCGCC-3′), trnL-trnF: (5′-ATTTGAACTGGTGACACGAG-3′, 5′-ATTTGAACTGGTGACACGAG-3′), and atpB-rbcL: (5′-ACTTAGAGGAGCTCCCGTGTCAATC-3′, 5′-GAGTTACTCGGAATGCTGCC-3′) intergenic regions. PCR amplification was performed using a PCR Thermal cycler SP (Takara), and base sequence determination was performed using a CEQ 8800 capillary DNA sequencer (Beckman Coulter). The base sequence obtained in this manner was aligned using the default parameters of the ClustalW program implemented in the MEGA X software [47]. The chloroplast DNA haplotypes were detected based on the arrangement of 3021 bases for the four combined domains. In addition, using DnaSP version 6.12.03 [48], the haplotypic diversity (h) and nucleotide diversity (π) were calculated for each polyploid and each region in which differences in polyploid distribution had been determined [49]. A parsimony haplotype network diagram of chloroplast DNA was created using the PopART 1.7 TCS network based on the data set in which the base sequences for the trnK intron, the trnT-L, trnL-F and atpB-rbcL intergenic regions [50], and the matK gene region were combined. Insertion–deletion (INDEL) mutations were excluded from analysis on the TCS network, so a dataset was created with INDELs replaced with base substitutions. We excluded from the analysis all repeated insertion–deletion of sequences for which the homology of the mutations was unclear.

4.4. Nuclear DNA Analysis

Nuclear DNA was investigated by sequencing with MIG-seq analysis [51,52], which creates a reduced library of genomes for samples with known chromosome numbers and chloroplast DNA haplotypes. The region flanked by SSRs was PCR amplified using 8 primers in the first PCR, and the resulting amplicons were indexed in a second PCR. The indexed DNA library was sequenced using the MiSeq Reagent Kit v3 (150-cycle) (Illumina, San Diego, CA, USA). The obtained reads were filtered using Trimmomatic 0.39 [53]. Adapter sequences were removed using default settings and short reads were removed (MINLEN:79). Low quality reads were then removed and trimmed (SLIDINGWINDOW:4:15, CROP:79). To investigate genetic distances between individuals, we used Mashtree [54] to create a neighbor-joining tree based on Mash distance between individuals. Mashtree parameters were k-mer length 21 (--kmerlength 21), sketch k-mer count was set to 30,000 (--sketchsize 30,000), and k-mers with a count of less than 2 were excluded from the analysis (--mindepth 2).
The genetic analysis of polyploid data involves certain challenges. The main one is the difficulty in estimating allele frequencies of the polyploid. Many existing analysis methods require allele doses, for example, hundreds of coverages to recover tetraploid alleles with 90% confidence [55]. It is difficult to obtain this amount of information by normal greening. k-mer analysis using the MinHash method does not require allele dosage, thus alleviating the difficulties of polyploid analysis [56]. However, Mashtree-derived Mash distances provide information equivalent to genetic distances. They are obtained by evaluating the similarity of reads resolved into k-mers. To assess the similarity between individuals, Principal Coordinate Analysis (PCoA) using Mash distance was performed in GenAlex 6.502. Mantel tests were performed on GenAlex 6.502 for Mash distance and geographic distance to estimate isolation by distance (IBD) between the groups recognized by PCoA (Tables S2–S4). However, such data should be treated with caution when evaluating between different polyploidy levels using Mash distances, as they can be subject to bias [56]. To assess the plausibility of comparisons between different polyploidy levels using the Mash distance, a Mantel test was performed on the genetic distance obtained from single nucleotide polymorphisms (SNP) and the Mash distance (Table S5). The Mantel test was performed using GenAlex 6.502, and the SNP call was made using the denovo_map.pl pipeline from stacks-2.60 [57]. The parameters for estimating the genetic diversity of L. muscari were also obtained with stacks-2.60. Stacks is an analytical protocol for a diploid model, which is usually difficult to apply to a polyploid. Although several informative alleles are lost, stacks can be used to analyze polyploidy by treating them as diploid by linking copies derived from polyploidy [58].

5. Conclusions

In Japan, there are three groups of natural populations of L. muscari recognized by polyploidy, cpDNA, and nDNA: the first is the mainland tetraploid group; the second is the Ryukyu Islands hexaploid group; and the third is the Ryukyu Islands diploid and tetraploid group. For the reduction of potential risks regarding the destruction of the local adaptation of natural individuals around the greening area and for the establishment of the planted individuals, genetic disturbance associated with greening between these three groups must be avoided. In the Kanto region near large cities, the possibility of genetic disturbance due to greening is low because the cultivated products and the surrounding natural populations belong to the same group. On the other hand, in the Ryukyu Islands, individuals belonging to different groups were being sold in the same nursery, suggesting the possibility of genetic disturbance between groups due to greening. Within the three groups, distinct IBD could be identified in nDNA. Using individuals derived from natural populations that are geographically close to the proposed greening site is expected to further reduce genetic disturbance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11223015/s1, Table S1: Nucleotide substitutions and indels observed in the Liriope muscari; Table S2: Geographic distance (below) and Mash distance (above) for Group 1; Table S3: Geographic distance (below) and Mash distance (above) for Group 2; Table S4: Geographic distance (below) and Mash distance (above) for Group 3; Table S5: Marsh distance (below) and SNP-based genetic distance (above) for all individuals.

Author Contributions

Conceptualization, K.W., T.D., M.Y. (Masatsugu Yokota) and Y.T.; investigation, K.W., T.D., M.Y. (Masatsugu Yokota) and S.K.H.; methodology, K.W., M.Y. (Makoto Yaneshita), T.D., M.Y. (Masatsugu Yokota), S.K.H., Y.S. and Y.T.; software, S.K.H. and Y.S.; resources, Y.S. and Y.T.; data curation, K.W. and M.Y. (Makoto Yaneshita); writing—original draft preparation, K.W. and M.Y. (Makoto Yaneshita); writing—review and editing, T.D., M.Y. (Masatsugu Yokota) and Y.T.; visualization, K.W. and M.Y. (Makoto Yaneshita); supervision, Y.T.; project administration, K.W.; funding acquisition, K.W., M.Y. (Makoto Yaneshita), T.D., M.Y. (Masatsugu Yokota) and Y.T. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported in part by a Grant-in-Aid for Scientific Research No. 19570089 to T.D.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Sequence data have been submitted to the GenBank database (cpDNA: LC730908-LC730988, LC731009-LC731251, nDNA: DRR412610-DRR412690).

Acknowledgments

We thank Koh Nakamura of Hokkaido University, and Goro Kokubugata of the National Museum of Nature and Science, Miki Takai, Masanori Kondo, Taku Mizuno, and Sayaka Watanabe, for their assistance in the investigation and analysis for this study. We thank Nanako Suzuki for her advice and Naoshi Shimizu of Gunze Green Co. Ltd. for collecting individuals from horticultural stores and providing other materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 11 03015 g001 550
Figure 1. Chromosomes at mitotic metaphase of three polyploidy levels of L. muscari. (a): diploid (2n = 36, sample name: wk179), (b): tetraploid (2n = 72, sample name: tm179), (c): hexaploid (2n = 108, sample name: wk006). Scale bars are 5 µm.
Figure 1. Chromosomes at mitotic metaphase of three polyploidy levels of L. muscari. (a): diploid (2n = 36, sample name: wk179), (b): tetraploid (2n = 72, sample name: tm179), (c): hexaploid (2n = 108, sample name: wk006). Scale bars are 5 µm.
Plants 11 03015 g001
Plants 11 03015 g002 550
Figure 2. Geographic distributions of polyploidy levels observed in L. muscari.
Figure 2. Geographic distributions of polyploidy levels observed in L. muscari.
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Plants 11 03015 g003 550
Figure 3. Geographical genetic structure of L. muscari using cpDNA and nDNA: (a) Neighbor-joining tree of L. muscari. Colored circles indicate differences in chloroplast DNA haplotypes (b). The numbers indicate the polyploidy level (2x: diploid 4x: tetraploid 6x: hexaploid). The color-coded areas indicate the three groups observed in the PCoA of the Mash distance (c). (b) Map of geographic distributions of haplotypes and polyploidy levels observed in L. muscari. Numbers indicate polyploidy level (2: diploid, 4: tetraploid, 6: hexaploid). TCS network of 9 cpDNA haplotypes (m01, m02, m03, m04, m05, m06, m07, m08, m09) observed in cytotypes (2x, 4x, 6x). Each line connecting two haplotypes represents a single mutation step. Circles indicate sample size. The color-coded areas indicate the distribution areas where individuals belonging to the three groups observed in the PCoA of the Mash distance (c). (c) PCoA analysis of Mash distance. The different colors of the plots indicate the chloroplast DNA haplotypes. The numbers on each axis indicate the percentage of variance. The color-coded areas represent plots of individuals belonging to the three visually recognized groups. (d) Correlation between Mash distance and geographic distance for the three groups observed in the PCoA of the Mash distance.
Figure 3. Geographical genetic structure of L. muscari using cpDNA and nDNA: (a) Neighbor-joining tree of L. muscari. Colored circles indicate differences in chloroplast DNA haplotypes (b). The numbers indicate the polyploidy level (2x: diploid 4x: tetraploid 6x: hexaploid). The color-coded areas indicate the three groups observed in the PCoA of the Mash distance (c). (b) Map of geographic distributions of haplotypes and polyploidy levels observed in L. muscari. Numbers indicate polyploidy level (2: diploid, 4: tetraploid, 6: hexaploid). TCS network of 9 cpDNA haplotypes (m01, m02, m03, m04, m05, m06, m07, m08, m09) observed in cytotypes (2x, 4x, 6x). Each line connecting two haplotypes represents a single mutation step. Circles indicate sample size. The color-coded areas indicate the distribution areas where individuals belonging to the three groups observed in the PCoA of the Mash distance (c). (c) PCoA analysis of Mash distance. The different colors of the plots indicate the chloroplast DNA haplotypes. The numbers on each axis indicate the percentage of variance. The color-coded areas represent plots of individuals belonging to the three visually recognized groups. (d) Correlation between Mash distance and geographic distance for the three groups observed in the PCoA of the Mash distance.
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Plants 11 03015 g004 550
Figure 4. PCoA of wild individuals and cultivars by Mash distance. The different shapes of the gray symbols represent the three groups identified in wild individuals. The colored symbols indicate differences in nurseries. Nurseries 01–06 are in two adjacent prefectures in the Kanto region of Japan (Saitama and Tokyo). Nursery 07 is in Okinawa Prefecture, Japan.
Figure 4. PCoA of wild individuals and cultivars by Mash distance. The different shapes of the gray symbols represent the three groups identified in wild individuals. The colored symbols indicate differences in nurseries. Nurseries 01–06 are in two adjacent prefectures in the Kanto region of Japan (Saitama and Tokyo). Nursery 07 is in Okinawa Prefecture, Japan.
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Table
Table 1. Plant materials and localities of samples used in this study. Na: sample names, 2n: chromosome numbers, Hap: cpDNA haplotype names.
Table 1. Plant materials and localities of samples used in this study. Na: sample names, 2n: chromosome numbers, Hap: cpDNA haplotype names.
LocalityNa2nHapAccession Numbers
cpDNAnDNA
trnK 5′ IntrontrnT-trnLtrnL-trnFatpB-rbcL
Natural distribution area
Mainland Japan and Ryukyu Islands
Kakudayama, Niigata City, Niigata Pref.wk39072m09LC730908LC731009LC731090LC731171DRR412610
Mt. Tsukuba, Tsukuba City, Ibaraki Pref.tm09672m09LC730909LC731010LC731091LC731172DRR412611
tm00172
tm17972
Mt. Mayumi, Hitachiota City, Ibaraki Pref.wk27072
Arakawa river, Nagatoro Town, Saitama Pref.tm00572m05LC730910LC731011LC731092LC731173DRR412612
Kamitanadare, Kisai Town, Saitama Pref.wk01872
Sendabori, Matsudo City, Chiba Pref.wk24672m04LC730911LC731012LC731093LC731174DRR412613
Horiuchinai, Ichikawa City, Chiba Pref.wk50372m04LC730912LC731013LC731094LC731175DRR412614
Mogusa, Tama City, Tokyo Pref.tm00372
Hane, Hamura City, Tokyo Pref.wk54672m09LC730913LC731014LC731095LC731176DRR412615
Motohachioji, Hachioji City, Tokyo Pref.wk47572m04LC730914LC731015LC731096LC731177DRR412616
Horiuchi, Hayama Town, Kanagawa Pref.wk39172m04LC730915LC731016LC731097LC731178DRR412617
Hakone, Hakone Town, Kanagawa Pref.wk24572m04LC730916LC731017LC731098LC731179DRR412618
Siokawa, Kani City, Gifu Pref.tm19872m09LC730917LC731018LC731099LC731180DRR412619
Imodani, Hashimoto City, Wakayama Pref.tm00672m09LC730918LC731019LC731100LC731181DRR412620
Hasemiya, Kimino Town, Wakayama Pref.wk49572m09LC730919LC731020LC731101LC731182DRR412621
wk49672m09LC730920LC731021LC731102LC731183DRR412622
Mt. Kurama, Sakyou Ward, Kyoto Pref.wk27872
Mt. Takao, Kashiwara City, Osaka Pref.tm00872
Higashiune, Akou City, Hyogo Pref.wk06272m09LC730921LC731022LC731103LC731184DRR412623
Asagoe, Okayama City, Okayama Pref.wk06772
wk06872m01LC730922LC731023LC731104LC731185DRR412624
Mt. Ogonzan, Hiroshima City, Hiroshima Pref.wk07372m01LC730923LC731024LC731105LC731186DRR412625
Chuocho, Hikari City, Yamaguchi Pref.wk08072m09LC730924LC731025LC731106LC731187DRR412626
Tyuzankei, Shimonoseki City, Yamaguchi Pref.tm01272
Nagaonohana, Hagi City, Yamaguchi Pref.wk02272m09LC730925LC731026LC731107LC731188DRR412627
Onoyama, Sanyo Onoda City, Yamaguchi Pref.wk02472
Kishinoue, Mannou Town, Kagawa Pref.wk05072m01LC730926LC731027LC731108LC731189DRR412628
Sugeta, Ohzu City, Ehime Pref.wk16572m04LC730927LC731028LC731109LC731190DRR412629
Nagahama seashore, Hukuoka City, Fukuoka Pref.wk00972m09LC730928LC731029LC731110LC731191DRR412630
wk01072
wk01172
Mt. Ihara, Maebaru City, Fukuoka Pref.wk01372
Senbutsudo, Kokura City, Fukuoka Pref.tm01472
Mt. Kagamiyama, Karatsu City, Saga Pref.wk01572
Hae, Tano Town, Miyazaki Pref.wk25572
Okutsu, Kobayashi City, Miyazaki Pref.wk25672
Tatara, Ozu City, Kumamoto Pref.wk13472
Ino, Kikuchi City, Kumamoto Pref.wk13772m09LC730929LC731030LC731111LC731192DRR412631
Mt. Tokozan, Izumi City, Kagoshima Pref.wk12672m09LC730930LC731031LC731112LC731193DRR412632
Hatimanjinja, Kanoya City, Kagoshima Pref.wk11872m09LC730931LC731032LC731113LC731194DRR412633
Tomori, Amami City, Kagoshima Pref.tm169108
tm173108m08LC730932LC731033LC731114LC731195DRR412634
Oazasetsuta, Amami City, Kagoshima Pref.wk182108m07LC730933LC731034LC731115LC731196DRR412635
Mt. Amagi, Amagi Town, Kagoshima Pref.tm022108m02LC730934LC731035LC731116LC731197DRR412636
Syoda, Tokunoshima Town, Kagoshima Pref.tm019108m02LC730935LC731036LC731117LC731198DRR412637
San, Tokunoshima Town, Kagoshima Pref.wk548108
Kibiru, Wadomari Town, Kagoshima Pref.tm021108
Taminazaki, China Town, Kagoshima Pref.tm020108m02LC730936LC731037LC731118LC731199DRR412638
Rikugidara, Izena Vil., Okinawa Pref.tm168108
Mt. Chizin, Izena Vil., Okinawa Pref.tm086108m02LC730937LC731038LC731119LC731200DRR412639
Mt. Gusuku, Ie Vil., Okinawa Pref.wk028108m02LC730938LC731039LC731120LC731201DRR412640
wk029108
Cape Hedo, Kunigami Vil., Okinawa Pref.tm038108
tm144108
Uka river, Kunigami Vil., Okinawa Pref.tm03736
tm14536m06LC730939LC731040LC731121LC731202DRR412641
tm14636
Haramata river, Higashi Vil., Okinawa Pref.wk23636
wk23736
wk23836m06LC730940LC731041LC731122LC731203DRR412642
Bise, Motobu Town, Okinawa Pref.tm149108
Mt. Awa, Motobu Town, Okinawa Pref.wk17236m07LC730941LC731042LC731123LC731204DRR412643
Kushi, Nago City, Okinawa Pref.tm029108
Henoko, Nago City, Okinawa Pref.tm148108
Mt. Nago, Nago City, Okinawa Pref.wk17936
wk18036m07LC730942LC731043LC731124LC731205DRR412644
Cape Maeda, Onna Vil, Okinawa Pref.tm031108
tm032108m02LC730943LC731044LC731125LC731206DRR412645
wk505108m01LC730944LC731045LC731126LC731207DRR412646
Ojana, Ginowan City, Okinawa Pref.wk006108m02LC730945LC731046LC731127LC731208DRR412647
Sashikisinzato, Nanjo City, Okinawa Pref.wk24272m01LC730946LC731047LC731128LC731209DRR412648
Cape Kyan, Itoman City, Okinawa Pref.wk020108m02LC730947LC731048LC731129LC731210DRR412649
Ugu seashore, Aguni Vil., Okinawa Pref.tm152108
tm153108m02LC730948LC731049LC731130LC731211DRR412650
West side of Gityuyama, Tonaki Vil., Okinawa Pref.tm092108
Iri, Tonaki Vil., Okinawa Pref.tm095108
Womozaki, Tonaki Vil., Okinawa Pref.tm161108m02LC730949LC731050LC731131LC731212DRR412651
Mt. Ara, Kumejima Town, Okinawa Pref.tm15036m01LC730950LC731051LC731132LC731213DRR412652
wk36836
Hiyajo, Kumejima Town, Okinawa Pref.tm151108
Ishiki seashore, Nanjo City, Okinawa Pref.tm082108m02LC730951LC731052LC731133LC731214DRR412653
Hiraraogami, Miyakojima City, Okinawa Pref.wk295108m02LC730952LC731053LC731134LC731215DRR412654
Maesato, Miyakojima City, Okinawa Pref.tm140108m02LC730953LC731054LC731135LC731216DRR412655
Nishihennazaki, Miyakojima City, Okinawa Pref.tm142108
wk297108
Setozaki, Miyakojima City, Okinawa Pref.wk292108m02LC730954LC731055LC731136LC731217DRR412656
Onosanrin, Miyakojima City, Okinawa Pref.tm077108m01LC730955LC731056LC731137LC731218DRR412657
wk291108
Otakikoen, Miyakojima City, Okinawa Pref.tm07636
tm07836m07LC730956LC731057LC731138LC731219DRR412658
tm07936
tm14336
wk29836
Nobarudake, Miyakojima City, Okinawa Pref.wk30136m01LC730957LC731058LC731139LC731220DRR412659
Umarezatonoutaki, Miyakojima City, Okinawa Pref.wk334108
Mt. Makiyama, Miyakojima City, Okinawa Pref.tm11172
wk28972
Kuninakautaki, Miyakojima City, Okinawa Pref.wk25272m01LC730958LC731059LC731140LC731221DRR412660
Toriike, Miyakojima City, Okinawa Pref.tm04172m01LC730959LC731060LC731141LC731222DRR412661
tm04472
tm13472
Misakiutaki, Ishigaki City, Okinawa Pref.wk17172m03LC730960LC731061LC731142LC731223DRR412662
Yutsun river, Taketomi Town, Okinawa Pref.tm11772
wk30972m01LC730961LC731062LC731143LC731224DRR412663
Komi, Taketomi Town, Okinawa Pref.wk31172m03LC730962LC731063LC731144LC731225DRR412664
wk31272
Aira river, Taketomi Town, Okinawa Pref.wk03072m03LC730963LC731064LC731145LC731226DRR412665
Thindahanata, Yonaguni Town, Okinawa Pref.tm11572
Agarizaki, Yonaguni Town, Okinawa Pref.wk21572m03LC730964LC731065LC731146LC731227DRR412666
Mt. Kubura, Yonaguni Town, Okinawa Pref.tm11372
tm11472
Nama seashore, Yonaguni Town, Okinawa Pref.wk21172m03LC730965LC731066LC731147LC731228DRR412667
Yonaguni, Yonaguni Town, Okinawa Pref.wk21272m03LC730966LC731067LC731148LC731229DRR412668
Taiwan
Chingching-tsaoyan, Lanyu, Taitungtm09836m03LC730967LC731068LC731149LC731230DRR412669
Nurseries
Nursery 01, Kawaguchi City, Saitama Pref.wk46072m05LC730968LC731069LC731150LC731231DRR412670
wk46172m04LC730969LC731070LC731151LC731232DRR412671
wk46272m04LC730970LC731071LC731152LC731233DRR412672
Nursery 02, Kawaguchi City, Saitama Pref.wk46372m09LC730971LC731072LC731153LC731234DRR412673
wk46472m04LC730972LC731073LC731154LC731235DRR412674
wk46572m05LC730973LC731074LC731155LC731236DRR412675
Nursery 03, Kawaguchi City, Saitama Pref.wk46672m05LC730974LC731075LC731156LC731237DRR412676
wk46772m04LC730975LC731076LC731157LC731238DRR412677
wk46872m04LC730976LC731077LC731158LC731239DRR412678
Nursery 04, Yorii Town, Saitama Pref.wk46972m04LC730977LC731078LC731159LC731240DRR412679
wk47072m04LC730978LC731079LC731160LC731241DRR412680
wk47172m05LC730979LC731080LC731161LC731242DRR412681
Nursery 05, Musashimurayama City, Tokyo Pref.wk50072m09LC730980LC731081LC731162LC731243DRR412682
wk50172m04LC730981LC731082LC731163LC731244DRR412683
wk50272m09LC730982LC731083LC731164LC731245DRR412684
Nursery 06, Chohu City, Tokyo Pref.wk47272m09LC730983LC731084LC731165LC731246DRR412685
wk47372m09LC730984LC731085LC731166LC731247DRR412686
wk47472m09LC730985LC731086LC731167LC731248DRR412687
Nursery 07, Nishihara City, Okinawa Pref.wk49772m03LC730986LC731087LC731168LC731249DRR412688
wk49872m03LC730987LC731088LC731169LC731250DRR412689
wk499108m02LC730988LC731089LC731170LC731251DRR412690
Table
Table 2. cpDNA haplotype diversity and nucleotide diversity observed in L. muscari. N: number of samples, NH: number of haplotypes, h: haplotype diversity, π: nucleotide diversity.
Table 2. cpDNA haplotype diversity and nucleotide diversity observed in L. muscari. N: number of samples, NH: number of haplotypes, h: haplotype diversity, π: nucleotide diversity.
GroupsPolyploidyNNHhπ
Natural distribution area
 Group 1: Mainland Japan4x2430.5830.00062
 Group 2: Ryukyu Islands6x1840.3990.00030
 Group 3: Ryukyu Islands2x, 4x1840.7390.00041
   Group 3–1 2x840.8210.00044
   Group 3–24x1020.5330.00018
Nurseries
 Nursery 01–06: Mainland Japan4x1820.4710.00063
 Nursery 07: Ryukyu Islands4x210.0000.00000
 Nursery 07: Ryukyu Islands6x11--
Table
Table 3. nDNA Genetic diversity of L. muscari. N: number of samples, He: genetic diversity, Ho: observed heterozygosity, FIS: inbreeding coefficient, π: nucleotide diversity.
Table 3. nDNA Genetic diversity of L. muscari. N: number of samples, He: genetic diversity, Ho: observed heterozygosity, FIS: inbreeding coefficient, π: nucleotide diversity.
GroupsPolyploidyNHeHoFISπ
Natural distribution area
 Group 1: Mainland Japan4x240.002620.00283−0.000520.00268
 Group 2: Ryukyu Islands6x180.003770.003820.000610.00390
 Group 3: Ryukyu Islands2x, 4x180.003480.002560.003740.00360
   Group 3-1 2x80.001780.001040.002940.00196
   Group 3-24x100.002540.00299−0.000930.00262
Nurseries
 Nursery 01-06: Mainland Japan4x180.002540.00299−0.000930.00262
 Nursery 07: Ryukyu Islands4x20.002340.00371−0.000180.00359
 Nursery 07: Ryukyu Islands 6x10.003010.0060200.00602
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Watanabe, K.; Yaneshita, M.; Denda, T.; Yokota, M.; Hirota, S.K.; Suyama, Y.; Tsumura, Y. Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan. Plants 2022, 11, 3015. https://doi.org/10.3390/plants11223015

AMA Style

Watanabe K, Yaneshita M, Denda T, Yokota M, Hirota SK, Suyama Y, Tsumura Y. Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan. Plants. 2022; 11(22):3015. https://doi.org/10.3390/plants11223015

Chicago/Turabian Style

Watanabe, Keita, Makoto Yaneshita, Tetsuo Denda, Masatsugu Yokota, Shun K. Hirota, Yoshihisa Suyama, and Yoshihiko Tsumura. 2022. "Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan" Plants 11, no. 22: 3015. https://doi.org/10.3390/plants11223015

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