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Article

Diploid Ancestor Tracing of Allopolyploid Cultivars in Camellia reticulata Based on ITS and RPB2 Sequences

by
Zhifeng Fan
1,
Wei Zheng
2,*,
Chengmin Yan
2 and
Xiaodan Xu
2
1
Department of Architecture, City College, Kunming University of Science and Technology, Kunming 650500, China
2
Laboratory of Landscape Plants, Department of Landscape Architecture, Faculty of Architecture and City Planning, Kunming University of Science and Technology, Kunming 650500, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(1), 85; https://doi.org/10.3390/horticulturae11010085
Submission received: 11 December 2024 / Revised: 11 January 2025 / Accepted: 12 January 2025 / Published: 14 January 2025
(This article belongs to the Section Floriculture, Nursery and Landscape, and Turf)
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Abstract

:
Camellia reticulata is a well-known ornamental species with a type specimen that is an allohexaploid, possibly descended from diploid ancestors like C. saluenensis, C. pitardii, and diploid C. reticulata. With over 1500 years of cultivation, heritage C. reticulata cultivars show varied ploidy levels, including hexaploid, octoploid, and decaploid forms, though their diploid ancestors are still unidentified. This study aims to trace these diploid ancestors by cloning and sequencing ITS from 25 taxa and RPB2 from 21 taxa across various ploidy levels of C. reticulata and its traditional cultivars and related species, combined with their fruit morphology data. Analyses of the ITS and RPB2 sequences suggest that the diploid ancestors of C. reticulata and its traditional cultivars may include C. saluenensis, C. pitardii, and diploid C. reticulata, while excluding C. mairei and C. polyodonta. Morphological analysis showed that diploid C. reticulata has significantly larger fruit weight, diameter, and pericarp thickness compared to C. pitardii, C. saluenensis, and both tetraploid and hexaploid C. reticulata. Since diploid ancestors of allopolyploids are often classified as distinct taxa, we suggest that diploid C. reticulata be recognized as a new variety of C. pitardii, as its ITS sequences are closely aligned with C. pitardii. This study offers key insights into the origin, evolution, and breeding of C. reticulata.

1. Introduction

The tallest camellia, Camellia reticulata Lindl. (Theaceae), has been cultivated in China as an ornamental plant for over 1500 years [1,2,3] (Figure 1). It was initially identified in 1827 based on a cultivar with semi-double flowers, introduced to Western countries from Tengchong (Yunnan, China) by Captain Rawes in 1820 and John Damper Parks in 1824 [4]. It is renowned globally for its large flower size, vibrant and diverse colors, various cultivars, and extended blooming period [2,5,6]. In addition to its ornamental value, C. reticulata serves as a source of camellia oil [7]. The species, an evergreen tree naturally distributed in southwest China [4,8,9], is listed on the IUCN Red List of Threatened Species [10]. Its wild relatives, predominantly hexaploid, were discovered in the 1940s [11]. Subsequently, diploid and tetraploid members of C. reticulata were identified in the Jinshajiang Valley in 1994 and 1997, respectively [12,13]. In 2012, genomic in situ hybridization (GISH) provided compelling evidence for the allopolyploid origin of C. reticulata genomes, demonstrating that (1) diploid C. reticulata, C. pitardii, and C. saluenensis are progenitors of hexaploid C. reticulata; (2) hybridization between diploid C. reticulata and C. pitardii produced the allotetraploid C. reticulata; and (3) subsequent hybridization between allotetraploid C. reticulata and C. saluenensis formed the allohexaploid C. reticulata [14] (Figure 1). Consequently, hybridization and polyploidization are the primary mechanisms behind the origin of polyploid C. reticulata, and the diploid ancestors contributing to the polyploidy of C. reticulata include not only diploid C. reticulata itself.
In allohexaploid species, diploid ancestors are typically considered distinct taxa [15,16]. The pericarp thickness of diploid C. reticulata is substantially greater than that of C. saluenensis Stapf ex Bean (2x) and C. pitardii Cohen-Stuart (2x) [12]. Additionally, karyotypic analysis reveals that diploid C. reticulata possesses 24 metacentric (m) and 4 submetacentric (sm) chromosomes, while C. pitardii has 22 m and 6 sm, and C. saluenensis has 20 m and 8 sm chromosomes [13]. Although diploid C. reticulata shares morphological similarities with tetraploid and hexaploid C. reticulata, they differ in pericarp thickness [12] (though population data on fruit morphology is lacking). However, the phylogenetic position of diploid C. reticulata remains unresolved, which is a crucial question underlying its taxonomic treatment.
Globally, a total of 858 ornamental cultivars of C. reticulata are registered in the International Camellia Register database (https://camellia.iflora.cn/, accessed on 10 December 2024), with about 123 heritage cultivars currently under primary cultivation [2,5,6]. These heritage cultivars comprise hexaploid, octoploid, and decaploid [17], most of which lack detailed parental records [2], impeding genetic breeding and cultivation research. Furthermore, high-ploidy cultivars present challenges in propagation through cutting and exhibit more stamen petalization in reproductive organs, complicating genetic tracing using root tips or pollen-based GISH experiments.
Low-copy nuclear genes are highly applicable to studies on plant phylogenetics and parental tracing [18,19]. Furthermore, second-generation sequencing technologies have been successfully employed in studies of the family Theaceae [20,21,22,23,24,25]. However, species in the genus Camellia exhibit complex ploidy [26] and possess relatively large genomes, with polyploid genomes ranging from approximately 13.12 to 25.35 Gb [27]. As a result, genome sequencing for these species is cost-prohibitive. The chloroplast genome of Camellia is comparatively small, approximately 156,576–156,975 bp [28,29], and comprehensive chloroplast genome data are available for closely related species. This has facilitated its effective application in evolutionary analyses of cultivars in Camellia [30]. However, the chloroplast genome of Camellia exhibits typical maternal inheritance [31]. While it can infer maternal ancestry for C. reticulata cultivars [32], it cannot comprehensively reflect biparental genetic characteristics.
The internal transcribed spacer (ITS), comprising ITS1 and ITS2, serves as a vital marker for investigating phylogenetic relationships at lower taxonomic levels [33,34]. Over the past two decades, ITS has been extensively applied to research on the genetic structure [35], phylogeny [36,37,38,39,40,41,42], and taxonomy [43] of Camellia species. Nonetheless, pseudogene ITS copies and non-concerted evolution have been documented in some Camellia species [44,45]. These studies suggest that ITS polymorphism in Camellia is likely attributable to genome polyploidization and interspecific hybridization [39,44,45]. Consequently, randomly amplified ITS sequences may represent only one of the parental species. Fortunately, by employing heritage DNA cloning techniques to isolate ITS sequences from different parental species and excluding pseudogenes, researchers can utilize the non-concerted evolution of ITS to trace the diploid ancestors of allopolyploids [45]. Currently, the use of the ITS functional sequence to trace the ancestral materials of C. reticulata does not include the 2x C. reticulata.
The RPB2 gene, encoding the second largest subunit of RNA polymerase II, typically occurs in low-copy or single-copy states in most plants and offers superior phylogenetic resolution [46]. This gene plays a pivotal role in elucidating plant lineage divergence and hybridization events, demonstrating greater efficacy in capturing genetic differentiation compared to plastid markers [47]. Additionally, the duplication of the RPB2 gene in certain angiosperm lineages highlights polyploidy events influencing plant evolution [48]. In Camellia, RPB2 has effectively clarified the classification and evolutionary history of several monophyletic groups [49]. Consequently, RPB2 is regarded as an ideal marker for determining phylogenetic relationships and classifications. Applying RPB2 to investigate the phylogenetic relationships of polyploid C. reticulata and its closely related taxa can enhance understanding of species formation and varietal origins in polyploid C. reticulata.
This study utilized nrITS and RPB2 sequences to examine the evolutionary characteristics of C. reticulata and its polyploid cultivars. We reconstructed the phylogenetic relationships within section (sect.) Camellia, encompassing both wild and cultivated polyploid cultivars of C. reticulata. Furthermore, we performed morphological comparisons of fruits at the population level for C. reticulata with varying ploidy and related diploid species. The aim is to: (1) trace the diploid progenitors of heritage C. reticulata cultivars; (2) clarify the phylogenetic position of diploid C. reticulata.

2. Materials and Methods

2.1. Experimental Materials

The diploid C. reticulata specimens were sourced from the Huaping population in Yunnan Province (collection numbers Zheng2406, 2420) and from potted plants cultivated at the Kunming Botanical Garden, Chinese Academy of Sciences. Mature and healthy leaves were harvested for DNA extraction. To ascertain the phylogenetic position of diploid C. reticulata in sect. Camellia, 86 ITS sequences from 11 additional species within sect. Camellia were obtained from NCBI. Following alignment and analysis, 61 functional ITS sequences were selected for further investigation. Ten RPB2 sequences, each corresponding to a distinct species within sect. Camellia, were also retrieved for analysis (Table 1). The study selected polyploid cultivars of C. reticulata, comprising hexaploid cultivars: ‘Xiaoguiye’, ‘Zaotaohong’, and ‘Dalicha’; octoploid cultivars: ‘Juban’, ‘Mudancha’, ‘Yunfengcha’, ‘Maye Yinhong’, ‘Honghua Youcha’, ‘Jing’ancha’, ‘Zipao’, and ‘Tongzimian’; and decaploid cultivars: ‘Chuxiong Dalicha’ and ‘Hentiangao’ (Figure 1). These materials encompass various ploidy levels of C. reticulata and its related species. The selected cultivars of C. reticulata are widely cultivated heritage cultivars, which better represent the genetic diversity of the species and serve as an ideal representation of its germplasm resources.

2.2. DNA Extraction, Amplification, and Sequencing

Total genomic DNA was extracted from leaf tissue samples using a modified CTAB method [50]. The PCR reaction mixture (20 µL total) comprised 0.2 mmol/L dNTPs, 2.0 mmol/L MgCl2, 0.5 µmol/L of each primer (forward and reverse), 1.2 U Taq DNA polymerase, and 50 ng template DNA. ITS regions were amplified using primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) [45]. Nuclear RPB2 introns were amplified utilizing primers RPB2-F2 (5′-CACCCCAGATATTATTGTGAACC-3′) and RPB2-R2 (5′-CTCGGGAATGGATCTTGTCAT-3′) [51]. Amplified products underwent sequencing using an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA) at Zanna Biological Company, Kunming, China. The newly obtained ITS and RPB2 sequences of Camellia were deposited in GenBank under accession numbers OQ600121 and PQ409355–PQ409404, respectively (Table 1).
For cloning and sequencing, recombinant plasmids were constructed utilizing purified PCR products, the PMD19-T vector, and ligase at 16 °C. These recombinant plasmids were subsequently transformed into DH5α-competent cells [45]. Following overnight incubation, positive clones were identified and sequenced. Simultaneously, direct sequencing of PCR products was conducted.

2.3. Phylogenetic Analysis

The ITS and RPB2 sequences were aligned using the MAFFT method in Geneious R10 [52]. Phylogenetic trees were constructed using the Maximum Likelihood (ML) method [53], utilizing the HKY model for genetic distance calculation. Polyspora axillaris (Roxburgh ex Ker Gawler) Sweet and Polyspora dalglieshiana (Craib) Orel, Peter G. Wilson, Curry & Luu served as outgroups (Table 1). Bootstrap resampling was performed with 1000 replicates, and a support threshold of 50% was established.
Separate phylogenetic analyses were conducted for functional ITS copies and RPB2 sequences of C. reticulata cultivars and their closely related species. Furthermore, comparative analyses were performed to examine base differences and base consistency rates between C. reticulata cultivars and diploid relatives within sect. Camellia [8], utilizing functional ITS and RPB2 sequences. The comparative analyses encompassed the following taxa: C. japonica L., C. chekiangoleosa Hu, C. pitardii, C. saluenensis, C. polyodonta F.C. How ex Hu, C. semiserrata C.W. Chi, C. azalea C.F. Wei, C. mairei (H. Léveillé) Melchior, C. subintegra T.C. Huang ex Hung T. Chang, C. edithae Hance, C. yunnanensis (Pitard ex Diels) Cohen-Stuart, C. drupifera Loureiro, and C. oleifera C. Abel.

2.4. Morphological Analysis of Diploid Camellia reticulata

Considering that C. reticulata displays highly similar morphological characteristics across various ploidy levels, with the main difference being pericarp thickness [12], this investigation examined fruit morphology at the population level for three ploidy types of C. reticulata, as well as two diploid progenitors (C. pitardii and C. saluenensis) (Table 2).
Ten individuals were randomly selected from each population, and for data collection, three ripe fruits were randomly chosen from the uppermost branches of each selected individual. Measurements were taken of fruit weight, transverse and longitudinal diameters, and pericarp thickness at the midpoint of each fruit. The number of seeds per fruit was enumerated. Owing to the irregular shape of the seeds, only the weight of individual seeds was recorded. The different ploidy levels of C. reticulata populations were referenced from previous research results [12,13,14].

3. Results

3.1. Analysis of ITS Sequences in Camellia reticulata Cultivars and Related Species

Phylogenetic analysis based on ITS sequences indicates that the polyploid cultivars of C. reticulata, together with C. pitardii and its varieties, C. saluenensis, 2x C. reticulata, and C. mairei and its varieties, cluster into a major clade.
The ITS sequence length of 2x C. reticulata was 617 bp, with an average GC content of 68.40% and a base pair identity of 99.65%. Within clade I, formed by functional ITS sequences of C. reticulata cultivars, the functional sequences of different cultivars did not cluster into a single clade but were grouped into two subclades, designated as A and B, with high support values. As illustrated in Figure 2, clade A comprises 18 clones from cultivars: two clones from ‘Xiaoguiye’, four from ‘Honghua Youcha’, three from ’Juban’, two from ‘Mudancha’, two from ‘Yunfengcha’, four from ‘Zaotaohong’, and one from ‘Maye Yinhong’. These clustered with C. pitardii var. cryptoneura, C. pitardii var. yunnanica, four clones of C. pitardii, and three clones of 2x C. reticulata. Clade B contains 11 clones from cultivars, including one clone each from ‘Juban’, ‘Mudancha’, ‘Yunfengcha’, and ‘Zaotaohong’, two clones from ‘Maye Yinhong’, and five from ‘Jing’ancha’. These clustered with six clones of C. saluenensis, one of C. mairei var. lapidea, and one of C. mairei. Notably, all ITS copies of the octoploid cultivar ‘Honghua Youcha’ and the hexaploid cultivar ‘Xiaoguiye’ were located in clade A, whereas all ITS copies of the octoploid cultivar ‘Jing’ancha’ were located in clade B. Functional ITS sequences from the remaining five cultivars (6x ‘Zaotaohong’ and 8x ‘Juban’, ‘Mudancha’, ‘Yunfengcha’, and ‘Maye Yinhong’) were distributed in both clades A and B. Another subclade C of clade I consists of two clones of C. polyodonta, C. mairei, and its variety C. mairei var. lapidea. Clade II, consisting of C. chekiangoleosa, C. japonica, C. edithae, C. azalea, C. semiserrata, and C. yunnanensis, lacked any wild or cultivated C. reticulata specimens. This absence suggests that the diploid progenitors of C. reticulata polyploid cultivars did not include these species.
Within sect. Camellia, the analysis of base differences between diploid species (Table 3) revealed notable variations. The highest number of differences (75) was observed between C. edithae and C. saluenensis. Conversely, the smallest difference (9) was found between C. japonica and C. chekiangoleosa, followed by C. polyodonta and C. saluenensis (19). Significantly, the base differences between 2x C. reticulata and other species in sect. Camellia ranged from 9 to 70, with C. pitardii exhibiting the smallest difference (9) and highest sequence identity (98.64%).

3.2. Analysis of RPB2 Sequences in Camellia reticulata Cultivars and Related Species

The alignment of the RPB2 sequences indicates that the number of base differences between the 2x C. reticulata and C. pitardii is the least among the examined species. Furthermore, polyploid cultivars of C. reticulata, along with the three natural ploidy levels of C. reticulata, C. pitardii, C. mairei, C. saluenensis, and C. polyodonta, form a major clade.
A total of 38 RPB2 sequences from C. reticulata cultivars were deposited in GenBank (Table 1). These sequences ranged in length from 1024 to 1041 base pairs, with a GC content of 32.03% and a base pair identity of 98.70%. A major clade comprised clones from 27 C. reticulata polyploid cultivars (6x, 8x, and 10x), three natural polyploids, C. reticulata (2x, 4x, and 6x), C. pitardii, C. mairei, C. saluenensis, and C. polyodonta, exhibiting intermixing between cultivars and species (Figure 3). Within this clade, pairwise identity reached 99.12%, suggesting substantial evolutionary consistency in RPB2 sequences.
Camellia chekiangoleosa, C. japonica, C. edithae, C. oleifera, C. drupifera, and C. yunnanensis formed another major clade, which lacked any C. reticulata cultivars (Figure 3). This observation suggests that the diploid progenitors of C. reticulata polyploid cultivars did not include these species, despite their shared sectional classification.
Within the existing diploid RPB2 sequences in sect. Camellia, the maximum base difference was observed between C. japonica and C. saluenensis (66), while the minimum difference was found between C. japonica and C. edithae (13) (Table 4). Significantly, base differences between diploid C. reticulata and other species within sect. Camellia ranged from 10 to 63, with the smallest difference (10) and highest sequence identity (99.00%) recorded in comparison to C. pitardii.

3.3. Fruit Morphology Comparison of Diploid Camellia reticulata and Congeneric Species

Morphological data show that the fruits of 2x C. reticulata are significantly larger than those of 4x and 6x C. reticulata, C. pitardii, and C. saluenensis.
In terms of fruit morphological traits (Figure 4), 2x C. reticulata exhibited a significantly higher average fruit weight of 101.45 ± 17.36 g (here and further, data were presented as average ± standard deviation) compared to C. pitardii, C. saluenensis, and 4x and 6x C. reticulata (Figure 5, Table S1). Regarding seed characteristics, C. pitardii produced the heaviest seeds (average 1.50 g), with no significant difference observed between 2x and 4x C. reticulata but significantly heavier than those of C. saluenensis and 6x C. reticulata (Figure 5, Table S1). Notably, seed weight did not differ significantly among the three ploidy levels of C. reticulata. 2x C. reticulata demonstrated the largest measurements in transverse diameter, vertical diameter, and pericarp thickness (5.98 ± 0.61 cm, 5.47 ± 0.46 cm, and 1.66 ± 0.25 cm, respectively), significantly exceeding those of C. pitardii and 4x and 6x C. reticulata. In contrast, C. saluenensis displayed the smallest measurements, significantly lower than those of C. pitardii and all three ploidy levels of C. reticulata. The average seed number per fruit in all three ploidy levels of C. reticulata was considerably higher than in C. pitardii and C. saluenensis. Among the ploidy levels, 2x C. reticulata had the highest seed number per fruit (9.38 ± 0.62), significantly higher than 6x C. reticulata, although neither showed a significant difference from 4x C. reticulata.
The analysis of fruit morphological characteristics revealed that during the allopolyploidization process of C. reticulata, the transverse and vertical diameters of the fruit, pericarp thickness, and seed number exhibited a progressive decrease with increasing ploidy levels (Figure 4). The diploid C. reticulata produces larger fruits, demonstrating significantly greater single fruit weight, transverse diameter, vertical diameter, and pericarp thickness compared to its 4x and 6x counterparts, as well as C. pitardii and C. saluenensis.

4. Discussion

4.1. Diploid Progenitors of Heritage Camellia reticulata Cultivars

In this study, the ITS sequences of C. reticulata did not constitute a monophyletic group but were distributed across two distinct clades. Clade A comprised numerous ITS clones of C. reticulata cultivars, along with diploid C. reticulata and C. pitardii and its varieties C. pitardii var. cryptoneura and C. pitardii var. yunnanica. Clade B, also containing many C. reticulata cultivar ITS clones, included C. saluenensis and C. mairei and its variety C. mairei var. lapidea. Clade B aligned with findings from studies on C. polyodonta [45]. These results suggest that the ancestors of C. reticulata and its polyploid cultivars may involve C. pitardii and its varieties (C. pitardii var. cryptoneura and C. pitardii var. yunnanica), diploid C. reticulata, C. saluenensis, and C. mairei and its variety C. mairei var. lapidea.
In clade B, the ITS sequences most closely related to those of C. reticulata cultivars were C. saluenensis, C. mairei, and C. mairei var. lapidea (Figure 2). Notably, all six ITS clones of C. saluenensis cluster within clade B, and this species is diploid [13,27,54,55,56]. Among the four ITS clones of C. mairei and its variety, two clustered with clade B (MF171087, EU579733), while the other two grouped with C. polyodonta in clade C (EU579727, FJ432108). Morphologically, C. mairei and C. polyodonta share similar characteristics, including filaments covered with soft hairs and comparable pericarp thickness [8], which aligns with our ITS results. Min (2000) proposed that C. mairei and C. polyodonta might represent polyploid complexes derived from C. pitardii. However, C. polyodonta is a diploid species, with 2n = 30 [7,27,57,58,59]. In contrast, C. mairei and its variety are polyploids, with 4x = 60 [27], 6x = 90 [11,59], or 8x = 120 [60]. These findings suggest that C. mairei and its variety might be polyploids derived from C. polyodonta, with a genetic background involving C. saluenensis. Regarding distribution, C. mairei and C. mairei var. lapidea occur in northwestern Guangdong, Guangxi, Guizhou, southern Hunan, southern Sichuan, and southeastern Yunnan, at altitudes ranging from 400 to 2300 m [9]. Camellia polyodonta is found in western Guangdong, eastern and northeastern Guangxi, and southwestern Hunan, at altitudes from 100 to 1000 m [9]. There is some overlap in the distribution areas of these two species. Additionally, both species overlap with the distribution of C. saluenensis (western Guizhou, southwestern Sichuan, and central and western Yunnan, at altitudes of 1200–3200 m). This geographical overlap supports the hypothesis that C. mairei and C. mairei var. lapidea may represent a polyploid complex that evolved through hybridization and polyploidization involving C. polyodonta and C. saluenensis. Future research could further validate this hypothesis through GISH or genomic hybridization analysis. In conclusion, given that C. mairei and C. mairei var. lapidea are polyploids, the diploid ancestors of C. reticulata and its cultivars likely do not involve C. mairei and C. mairei var. lapidea.
RPB2 exhibits relatively few variable sites, potentially due to consistent evolution [61]. In this study, the RPB2 gene in polyploid cultivars of C. reticulata demonstrated a high degree of evolutionary consistency, suggesting these polyploid cultivars have undergone limited mutations in the RPB2 sequence, resulting in a relatively stable evolutionary pattern. This consistency may originate from the common diploid ancestor of these polyploid cultivars. Additionally, phylogenetic analysis based on the RPB2 gene revealed the differentiation between C. reticulata and other species in sect. Camellia. The RPB2 sequences of polyploid C. reticulata cultivars formed distinct evolutionary branches from those of C. chekiangoleosa, C. japonica, C. edithae, C. oleifera, C. vietnamensis, and C. yunnanensis (Figure 3), indicating significant genetic divergence between these species and C. reticulata during phylogenetic evolution. This result suggests that the polyploid cultivars of C. reticulata do not involve these species as their diploid ancestors, further supporting the unique genetic background and phylogenetic structure of C. reticulata. No apparent differentiation was observed between the RPB2 sequences of polyploid cultivars and natural diploid C. reticulata, suggesting these cultivars may have maintained a high degree of genetic consistency during polyploidization through selfing or gene flow. Within the C. reticulata cultivar cluster, the relationships involved diploid C. reticulata, C. pitardii, C. saluenensis, C. mairei, and C. polyodonta, reflecting a complex phylogenetic relationship. Among these, C. polyodonta is a diploid species with 2x = 30 [7,57,58,59]. However, C. polyodonta occurs in western Guangdong, eastern and northeastern Guangxi, and southwestern Hunan at altitudes of 100–1000 m and does not overlap with the distribution of C. reticulata (1000–3200 m, W Guizhou, SW Sichuan, and Yunnan) [9], suggesting that the diploid ancestor of C. reticulata and its cultivars may not involve C. polyodonta.
Previous research utilizing GISH on meiotic pollen mother cells and young root apex indicated that the genome of the polyploid complex of C. reticulata had been infiltrated by genetic components from C. japonica [62,63]. However, the phylogenetic analyses based on ITS and RPB2 in this study collectively suggest that the diploid ancestor of C. reticulata and its polyploid cultivars did not involve C. japonica, contradicting earlier findings. Geographically, C. japonica is predominantly distributed in eastern China [9] and lacks overlapping distribution with C. reticulata, reducing the likelihood of natural gene exchange between the two species. Furthermore, GISH studies on meiotic pollen mother cells hypothesized that C. yunnanensis might also be a parental species of the polyploid complex of C. reticulata [62]. However, this study refutes that hypothesis, concluding that the diploid ancestor of C. reticulata and its polyploid cultivars did not involve C. yunnanensis.
In this study, the fruit size and pericarp thickness of tetraploid C. reticulata exhibited intermediate characteristics between those of diploid C. reticulata and C. pitardii. Similarly, the fruit size and pericarp thickness of hexaploid C. reticulata were intermediate between those of tetraploid C. reticulata and C. saluenensis, demonstrating a typical genetic integration pattern. In the Jinshajiang Valley of southwestern Sichuan and northern Yunnan, C. reticulata displays significant morphological diversity, including variations in leaf size and the size and number of bracts, sepals, and petals, as well as the length of stamens and pistils. These variations are evident not only among different populations but also within the same population and even on different parts of individual plants [8], suggesting that the Jinshajiang Valley may be the center of origin and differentiation for C. reticulata. Research has indicated the presence of numerous tetraploid C. reticulata populations in the Jinshajiang Valley [13]. Furthermore, several wild germplasm previously classified as new species of C. paucipetala, C. jinshajiangica, C. brevicolumna, C. brachygyna, and C. borealiyunnancia [64] were subsequently incorporated into C. reticulata [8,9]. These species were presumed to be octoploid based on flow cytometry analysis [27]. These wild germplasm resources may be associated with the origin of the octoploid cultivars of C. reticulata.
Tracing the ancestry of polyploid cultivars is a complex endeavor. Utilizing two significant molecular markers and morphological evidence, we have confirmed that the primary mechanism behind the early formation of polyploid cultivated varieties of C. reticulata is allopolyploidization, and we have identified several potential diploid progenitors. However, this study did not explicitly identify the ancestors of specific ploidy levels or individual cultivars of C. reticulata. In future research, the ancestral parents of the polyploid cultivated varieties can be further elucidated by identifying the subgenomes of the allopolyploids, comparing the karyotypes of the target polyploid species with those of their potential diploid parents, and employing strategies such as whole-genome sequencing and the analysis of unique repetitive elements.

4.2. Phylogenetic Position of Diploid Camellia reticulata

Within clade A, the ITS sequences from four C. pitardii clones and three diploid C. reticulata clones were intertwined, suggesting a close phylogenetic relationship between diploid C. reticulata and C. pitardii. C. reticulata is predominantly found in Yunnan Province and southern Sichuan Province [4,8], with overlapping distributions of diploid, tetraploid, and hexaploid populations in the Jinshajiang Valley. The tetraploid and hexaploid C. reticulata, which are widely distributed in the Jinshajiang Basin and Yunnan Province, were initially collectively referred to as C. pitardii var. yunnanica Sealy [65]; these species actually have the same morphology as the hexaploid C. reticulata form. simplex Sealy found in Tengchong, Yunnan Province [8]. Camellia reticulata Lindl., named for its double flowers, may have evolved from single-flowered types through prolonged selection. Taxonomically, C. reticulata and C. reticulata form. simplex were integrated into the varieties of C. pitardii [8]. However, as a renowned ornamental plant, the name C. reticulata Lindl. has been widely accepted for nearly two centuries, leading to its continued use in classifications [8,9]. The discovery of diploid C. reticulata revealed notable differences from diploid relatives such as C. saluenensis and C. pitardii in morphology, although diploid C. reticulata closely resembles tetraploid and hexaploid populations, differing primarily in its thicker fruit pericarp [12]. This study further found that the fruit diameter and pericarp thickness of diploid C. reticulata were significantly greater than those of tetraploid and hexaploid populations. GISH analysis confirmed that diploid C. reticulata, C. pitardii, and C. saluenensis are progenitors of hexaploid C. reticulata [14]. The diploid ancestors of allopolyploid hexaploids are generally treated as separate species [15,16]. The ITS sequence analysis revealed a close phylogenetic relationship between diploid C. reticulata and C. pitardii, with base pair differences ranging from 9 to 14 bp, comparable to differences observed between C. japonica and C. chekiangoleosa (9 bp). Consequently, there is potential to consider diploid C. reticulata as an independent taxon.
Camellia pitardii encompasses two additional varieties beyond the original variety: C. pitardii var. compressa (2n = 120) [27,59] and C. pitardii var. cryptoneura (2n = 90) [56]. C. pitardii var. compressa is distributed in northern Guizhou, western Hubei, and northwestern Hunan at elevations between 700 and 1100 m. C. pitardii var. cryptoneura occurs in northeastern Guangxi and southern Hunan at elevations ranging from 500 to 1400 m. In contrast, diploid C. reticulata is found in southern Sichuan and northern Yunnan, at altitudes between 1650 and 2800 m, with no distributional overlap with the two C. pitardii varieties. Morphologically, diploid C. reticulata can attain heights exceeding 10 m, surpassing C. pitardii and its varieties (3–9 m) [9]. The leaves of diploid C. reticulata can reach a maximum length of 16.3 cm, exceeding those of C. pitardii (10.0 cm), C. pitardii var. compressa (14.0 cm), and C. pitardii var. cryptoneura (12.0 cm) [9]. Similarly, the maximum leaf width of diploid C. reticulata (8.6 cm) surpasses that of C. pitardii (3.5 cm), C. pitardii var. compressa (6.4 cm), and C. pitardii var. cryptoneura (4.0 cm) [9]. In summary, diploid C. reticulata exceeds C. pitardii and its varieties in plant height, leaf length, and leaf width. Comparative analysis of ITS sequences reveals that nucleotide differences between C. pitardii varieties and the original species range from 8 to 14 bp, comparable to the differences between diploid C. reticulata and C. pitardii (9–14 bp). These findings suggest that classifying diploid C. reticulata as a varieties of C. pitardii might be more appropriate.
Among the three diploid progenitors of hexaploid C. reticulata, C. saluenensis predominantly exists as a shrub, C. pitardii manifests as a small tree [9], while diploid C. reticulata can reach heights exceeding 10 m, characterizing it as a typical tree. Thus, diploid C. reticulata likely serves as the primary contributor to the height trait observed in hexaploid C. reticulata. However, only two populations of diploid C. reticulata were identified, each consisting of fewer than 100 individuals [12,66]. If diploid C. reticulata can be classified independently, it would be considered a critically small population, which may increase public awareness and potentially benefit conservation efforts for the species.

5. Conclusions

The heritage cultivars of C. reticulata have diploid ancestors, including C. pitardii, C. saluenensis, and 2x C. reticulata, but not C. mairei, C. polyodonta, C. japonica, or C. yunnanensis. The 2x C. reticulata demonstrates potential as an independent taxon, and it may be most appropriate to classify it as a new variety of C. pitardii. This study demonstrates that the origin of C. reticulata and its cultivars is rooted in allopolyploidization, accompanied by hybridization and gene introgression from other Camellia species throughout the evolutionary process. These findings provide essential foundational insights for the breeding of novel cultivars of C. reticulata.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11010085/s1, Table S1: Comparison of the characters between Camellia reticulata and their ancestors.

Author Contributions

Conceptualization, W.Z. and X.X.; methodology, W.Z.; software, Z.F.; validation, W.Z. and X.X.; formal analysis, Z.F.; investigation, W.Z. and C.Y.; resources, W.Z. and C.Y.; data curation, W.Z. and Z.F.; writing—original draft preparation, Z.F.; writing—review and editing, W.Z. and X.X.; visualization, Z.F.; supervision, W.Z.; project administration, W.Z.; funding acquisition, W.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32060691 and 31660228) and Natural Science Research Foundation of Kunming University of Science and Technology (KKZ3202456200).

Data Availability Statement

The newly obtained ITS and RPB2 sequences of Camellia are available in GenBank (https://www.ncbi.nlm.nih.gov/, accessed on 10 December 2024) under accession numbers OQ600121 and PQ409355–PQ409404, respectively.

Acknowledgments

We are grateful to Jun Wen from the Department of Botany, Smithsonian Institution, US, for her guidance on taxonomy. We also thank Zhonglang Wang from Kunming Institute of Botany, Chinese Academy of Sciences for assistance with sample collections.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Floral characteristics of representative species and cultivars. (A): diploid Camellia reticulata, (B): C. pitardii, (C): C. saluenensis, (D): C. reticulata (6x), and (E): C. reticulata ‘Tongzimian’ (8x), (F): C. reticulata ‘Juban’ (8x).
Figure 1. Floral characteristics of representative species and cultivars. (A): diploid Camellia reticulata, (B): C. pitardii, (C): C. saluenensis, (D): C. reticulata (6x), and (E): C. reticulata ‘Tongzimian’ (8x), (F): C. reticulata ‘Juban’ (8x).
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Figure 2. The ML tree of C. reticulata cultivars and congeneric species based on ITS sequences. The scale represents the differentiation measure reflected by the branch length. The same colorful font denotes the same species and its varieties.
Figure 2. The ML tree of C. reticulata cultivars and congeneric species based on ITS sequences. The scale represents the differentiation measure reflected by the branch length. The same colorful font denotes the same species and its varieties.
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Figure 3. The ML tree of C. reticulata (cultivars and wild type) and congeneric species based on RPB2 sequences.
Figure 3. The ML tree of C. reticulata (cultivars and wild type) and congeneric species based on RPB2 sequences.
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Figure 4. Fruit morphology of C. reticulata during hybridization polyploidy.
Figure 4. Fruit morphology of C. reticulata during hybridization polyploidy.
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Figure 5. Comparison of fruit morphological characteristics between C. reticulata and its ancestors. (A): Fruit weight, (B): Number of seeds per fruit, (C): Seed weight, (D): Fruit transverse and vertical diameter, and (E): Pericarp thickness. The whiskers on each bar represent the standard deviation; the same letter indicates that the trait difference among species was not significant. In subfigure (D), the blue bars denote fruit transverse diameter, and the yellow bars denote vertical diameter. Analysis of variance was used to examine differences in traits between species.
Figure 5. Comparison of fruit morphological characteristics between C. reticulata and its ancestors. (A): Fruit weight, (B): Number of seeds per fruit, (C): Seed weight, (D): Fruit transverse and vertical diameter, and (E): Pericarp thickness. The whiskers on each bar represent the standard deviation; the same letter indicates that the trait difference among species was not significant. In subfigure (D), the blue bars denote fruit transverse diameter, and the yellow bars denote vertical diameter. Analysis of variance was used to examine differences in traits between species.
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Table 1. ITS and RPB2 GenBank accessions of Camellia reticulata cultivars and outgroup species.
Table 1. ITS and RPB2 GenBank accessions of Camellia reticulata cultivars and outgroup species.
CodeCultivars and SpeciesCultivation SiteITS GenBank AccessionsRPB2 GenBank Accessions
1‘Xiaoguiye’ (6x)Kunming, ChinaJX500461-2-
2‘Zaotaohong’ (6x)Kunming, ChinaJX457339, JX500464, JX500466-8-
3‘Dalicha’ (6x)Kunming, China-PQ409391-5, PQ409399
4‘Juban’ (8x)Kunming, ChinaJX500457-60PQ409381, PQ409383
5‘Mudancha’ (8x)Kunming, ChinaJX500474-6-
6‘Yunfengcha’ (8x)Kunming, ChinaJX500477-9-
7‘Maye Yinhong’ (8x)Kunming, ChinaJX500480-2-
8‘Honghua Youcha’ (8x)Kunming, ChinaJX500483-6PQ409386-8
9‘Jing’ancha’ (8x)Kunming, ChinaJX500469-73-
10‘Zipao’ (8x)Kunming, China-PQ409371-4
11‘Tongzimian’ (8x)Kunming, China-PQ409375-6, PQ409378-80
12‘Chuxiong Dalicha’ (10x)Kunming, China-PQ409400-4
13‘Hentiangao’ (10x)Kunming, China-PQ409389-90
14C. reticulata (2x)Huaping and Yanbian, ChinaOQ600121, OR958721, OR984214PQ409365
15C. reticulata (4x)Yanbian, China-PQ409367-9
16C. reticulata (6x)Kunming, ChinaHM061380-401PQ409362-4, MZ488707
17C. pitardiiSW ChinaEF646288, EU579758-9, FJ432115MZ488701
18C. pitardii var. cryptoneuraSW ChinaEF646291-
19C. pitardii var. yunnanicaSW ChinaEF646289-
20C. saluenensisSW ChinaEU579767-8, HM061329-31, HM061353PQ409359-60
21C. maireiSW and S ChinaMF171087, FJ432108MZ488720
22C. mairei var. lapideaSW and S ChinaEU579733, MF171087-
23C. polyodontaC and S ChinaEF646285, EU579760MZ488711
24C. subintegraC and S ChinaEU579776-
25C. chekiangoleosaSE ChinaFJ432101MZ488676
26C. japonicaS and E China, S JapanEF649690MZ488695
27C. edithaeSE ChinaEU579695-6MZ488714
28C. azaleaS ChinaEU579681-
29C. semiserrataS ChinaEF649688, EU579770, EF649691-
30C. oleiferaS China, SE Asia-MZ488672
31C. drupiferaS China-MZ488656
32C. yunnanensisSW ChinaAY096015, AF456256PQ409355-6
33Polyspora dalglieshianaLaos, Thailand-MZ488732
34Polyspora axillarisSW ChinaAY214930-
Table 2. Geographic distribution of various ploidy levels in C. reticulata and its ancestral species.
Table 2. Geographic distribution of various ploidy levels in C. reticulata and its ancestral species.
TaxonLocationLongitudeLatitudeAltitude
C. reticulata 2xHuaping, YunnanE 101°09′37″N 26°38′01″1830
C. reticulata 2xYanbian, SichuanE 101°18′40″N 27°06′63″2110
C. reticulata 4xXichang, SichuanE 102°21′11″N 27°43′30″2423
C. reticulata 6xPanlong, YunnanE 102°43′13″N 25°23′30″2426
C. pitardiiFuyuan, YunnanE 104°25′17″N 25°52′05″2144
C. saluenensisSongming, YunnanE 102°44′13″N 25°13′27″2420
Table 3. Comparison of ITS sequence distances between diploid C. reticulata and related diploid species.
Table 3. Comparison of ITS sequence distances between diploid C. reticulata and related diploid species.
SpeciesC. pitardiiC. reticulata 2xC. subintegraC. saluenensisC. polyodontaC. semiserrataC. azaleaC. edithaeC. chekiangoleosaC. japonica
C. pitardii-98.6492.6692.8392.9791.0890.9089.7189.7689.64
C. reticulata 2x9-92.7892.9492.8091.3391.1489.9689.7289.60
C. subintegra4948-95.7395.4392.1392.3889.9991.6491.52
C. saluenensis484728-97.1193.3191.9588.8689.9189.79
C. polyodonta47483019-91.8690.6589.3988.8890.33
C. semiserrata6058524454-94.5891.4592.0192.03
C. azalea626051546336-93.0092.8492.86
C. edithae70706775715747-94.1294.57
C. chekiangoleosa6969556767534839-98.62
C. japonica70705668645348369-
Note: A two-decimal value denote the base consistency rate (%), while the integer value represent the number of base differences.
Table 4. Comparison of RPB2 sequence distance between diploid C. reticulata and diploid-related species.
Table 4. Comparison of RPB2 sequence distance between diploid C. reticulata and diploid-related species.
SpeciesC. pitardiiC. reticulata 2xC. saluenensisC. polyodontaC. edithaeC. japonica
C. pitardii-99.0098.4998.6594.4694.16
C. reticulata 2x10-98.7098.6594.0793.77
C. saluenensis1513-97.9493.5693.47
C. polyodonta141421-94.0293.72
C. edithae56606561-98.70
C. japonica5963666413-
Note: A two-decimal value denote the base consistency rate (%), while the integer value represent the number of base differences.
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Fan, Z.; Zheng, W.; Yan, C.; Xu, X. Diploid Ancestor Tracing of Allopolyploid Cultivars in Camellia reticulata Based on ITS and RPB2 Sequences. Horticulturae 2025, 11, 85. https://doi.org/10.3390/horticulturae11010085

AMA Style

Fan Z, Zheng W, Yan C, Xu X. Diploid Ancestor Tracing of Allopolyploid Cultivars in Camellia reticulata Based on ITS and RPB2 Sequences. Horticulturae. 2025; 11(1):85. https://doi.org/10.3390/horticulturae11010085

Chicago/Turabian Style

Fan, Zhifeng, Wei Zheng, Chengmin Yan, and Xiaodan Xu. 2025. "Diploid Ancestor Tracing of Allopolyploid Cultivars in Camellia reticulata Based on ITS and RPB2 Sequences" Horticulturae 11, no. 1: 85. https://doi.org/10.3390/horticulturae11010085

APA Style

Fan, Z., Zheng, W., Yan, C., & Xu, X. (2025). Diploid Ancestor Tracing of Allopolyploid Cultivars in Camellia reticulata Based on ITS and RPB2 Sequences. Horticulturae, 11(1), 85. https://doi.org/10.3390/horticulturae11010085

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