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Article

Whole-Genome Resequencing Reveals Phylogenetic Relationships and Sex Differentiation Mechanisms Among Fujian Cycas Species

by
Xinyu Xu
1,2,
Yousry A. El-Kassaby
2,
Sijia Liu
4,
Juan Zhang
3,
Lanqi Zhang
3,
Junnan Li
3,
Wenkai Li
3,
Kechang Zhang
5,
Minghai Zou
6,
Zhiru Lai
7,
Likuang Lin
7,
Yongdong Zhang
7,
Shasha Wu
1,* and
Bihua Chen
3,*
1
The Innovation and Application Engineering Technology Research Center of Ornamental Plant Germplasm Resources in Fujian Province, National Long-Term Scientific Research Base for Fujian Orchid Conservation, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Department of Forest and Conservation Sciences, Faculty of Forestry, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada
3
The Key Laboratory of Timber Forest, Breeding and Cultivation for Mountainous Areas in Southern China of China National Forestry and Grassland Bureau and the Key Laboratory of Forest Culture and Forest Product Processing Utilization of Fujian Province, Fujian Academy of Forestry Sciences, Fuzhou 350012, China
4
Fujian Satellite Data Development Co., Ltd., Fuzhou 350025, China
5
Zhao’an County Forestry Bureau, Zhao’an 363500, China
6
Sanming City Shaxian District Wildlife Center, Shaxian 365500, China
7
Yongtai County Forestry Bureau, Yongtai 350700, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(5), 488; https://doi.org/10.3390/horticulturae11050488
Submission received: 26 March 2025 / Revised: 26 April 2025 / Accepted: 29 April 2025 / Published: 30 April 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

:
Cycads, renowned as “living fossils”, are among the most ancient extant seed plants, playing a crucial role in understanding plant evolution and sex differentiation. Despite their importance, research on their genetics and sex differentiation remains scarce. This study investigates three species, represented by six samples, collected from various regions in Fujian Province, China, using whole-genome resequencing on the Illumina platform. The sequence data underwent rigorous quality control, alignment, and variant detection, focusing on SNP and InDel distribution and annotation. Among the studied species, Cycas revoluta exhibited the highest number of SNPs and the greatest heterozygosity values. Based on SNP data, phylogenetic trees and principal component analysis revealed distinct clusters, with the three C. revoluta samples forming one cluster, while the two C. szechuanensis samples and the C. taiwaniana sample were grouped separately. Gene function using COG and GO annotations, and KEGG enrichment analysis, all highlighted differences in genomic structure and functional gene distribution between male and female cycads. Notably, genes associated with sex differentiation, such as MADS-box and auxin-responsive protein genes, were shown, while other transcription factors showed distinct annotations and enrichment patterns based on sex. This study improves our understanding of genetic variation, evolutionary relationships, and gene enrichment in cycads, providing a foundation for conservation, cultivation, and insights into sex differentiation mechanisms in these ancient plants.

1. Introduction

Cycads, one of the oldest extant plant groups with an evolutionary history dating back approximately 300 million years, are often referred to as the “pandas of the plant world”. Throughout geological history, cycads were widely distributed and played a critical role in maintaining ecosystems and contributing to plant evolutionary processes [1]. Today, their distribution is mainly confined to tropical and subtropical regions. In China, around 20 cycad species naturally occur, with Fujian Province being the native region for Cycas revoluta, C. taiwaniana, and C. szechuanensis. The latter two species are critically endangered, making their conservation an urgent priority. In China, particularly in Fujian Province, cycads are widely cultivated as ornamental and landscape plants, underscoring the importance of conservation and research efforts [2]. Despite this, genomic research on cycads in China—and Fujian specifically—remains relatively sparse. While several studies have focused on phylogenetics or organelle genomes [3], few have addressed the genetic mechanisms underlying key biological traits such as sex differentiation, which is particularly important in dioecious species like cycads.
Sex differentiation is a natural phenomenon across the biological world, marking a key transition from vegetative growth to reproductive development in plants. Most plants are monoecious, meaning that male and female flowers are present on the same plant, while only about 6% of plants are dioecious, with male and female flowers occurring on separate individuals [4]. Plant sex differentiation occurs during flower bud formation and is regulated by a combination of sex-determining genes, sex chromosomes, and internal signals such as auxin, gibberellins, and carbohydrates, as well as environmental factors like temperature, photoperiod, and nutrition. Due to the complexity of sex determination, plants exhibit a wide variety of sex differentiation patterns [5,6,7]. Cycads are dioecious, with each individual producing either male or female cones, a characteristic distinct from other extant seed plants. Like ginkgo, cycads retain motile sperm, with pollination and fertilization being separate stages—a trait shared with ancestors of bryophytes, lycophytes, and ferns [8]. Additionally, cycad karyotypes have been studied, revealing the existence of an XY sex-determination system in a specific chromosomal region, as identified through whole-genome sequencing of the reference genome of C. panzhihuaensis [9]. In various dioecious plants, sex chromosomes have evolved independently multiple times, resulting in a diversity of sex-determination systems. To date, over 70 plant species have been found to possess sex chromosomes, including asparagus (Asparagus officinalis) [10], papaya (Carica papaya) [11], and spinach (Spinacia oleracea) [12]. The sex-determining genes located on sex chromosomes form the genetic basis for sex determination in dioecious plants. Recently, the successful cloning of sex-determining genes in the dioecious plant persimmon (Diospyros lotus) has provided insight into sex determination mechanisms, with both “two-gene” and “single-gene” models explaining these genetic processes [13,14].
This study aims to fill a critical knowledge gap by investigating the genomic variation and sex differentiation in native and cultivated cycad populations from Fujian Province using whole-genome resequencing (WGS). By examining sequence polymorphisms—including single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels)—and analyzing their distribution, phylogenetic relationships, and potential association with sex differentiation, this research provides one of the first genome-wide perspectives on sex-determination mechanisms in Fujian cycads. Furthermore, by integrating population-level genomic variation with functional annotation, our findings offer novel insights into the evolutionary history of cycads and provide a molecular basis for species conservation and breeding strategies.

2. Materials and Methods

2.1. Sample Collection and Sequencing

Cycas revoluta (CR-FZ-18, CR-FZ-19, CR-FZ-20), C. szechuanensis (CS-SX-28, CS-YT-09), and C. taiwaniana (CT-ZARG01) were collected from the Fujian Province, China (CR-FZ-18 (female), CR-FZ-19 (female), and CR-FZ-20 (male) Gushan, Fuzhou City; CS-SX-28 (female) Shaxian County, Sanming City; CS-YT-09 (female) Yongtai County, Fuzhou City; and CT-ZARG01 (female) Zhao’an County, Zhangzhou City) (Figure 1). CR-FZ-18, CR-FZ-19, and CR-FZ-20 are ancient trees, while CT-ZARG01 is cultivated. All samples were quickly frozen in liquid nitrogen and stored at −80 °C for future use, and sent to Biomarker Technology Co. Ltd. (Beijing, China) for sequencing. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method [15]. Concentration and quality of the total genomic DNA were measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). DNA libraries (350 bp) for Illumina/BGI sequencing were constructed according to the manufacturer’s instructions. Following library construction, sequencing was performed by a commercial service provider (B. for Beijing, China) on the Illumina HiSeq XTen/NovaSeq/BGI platform, with a read length of 150 bp.

2.2. Re-Sequencing Data Processing and Variation Detection

The raw reads (paired-end sequences) obtained from sequencing were quality-assessed and filtered. An index was established using the Cycas genome from C. panzhihuaensis. Clean data were then aligned to the reference genome using the BWA (v0.7.17) software [16]. SNPs (single nucleotide polymorphisms) and small InDels (small insertions and deletions) were detected primarily using GATK (v4.2.x) [17] software. The HaplotypeCaller module was used to generate gVCF (genome VCF) files for each sample, which are intermediate files used for variant detection. The CombineGVCFs module was employed to merge all individual gVCF files into a comprehensive gVCF file, which was then converted to a VCF file using the GenotypeGVCFs module. SNP and InDel sites were selected using the SelectVariants command. Variants were filtered using the VariantFiltration command and then compared with functional databases such as NR, SwissProt, GO, COG, and KEGG [18] via BLAST (2.13.0) to obtain gene annotations for functional analysis. Enrichment analysis was performed with filtering criteria of p-value ≤ 10−5 and FDR ≤ 0.01.

2.3. Genetic Evolution Analysis

The MEGA X (v10.2.6) [19] software was used to construct phylogenetic trees for C. revoluta (CR-FZ-18, CR-FZ-19, CR-FZ-20), C. szechuanensis (CS-SX-28, CS-YT-09), and C. taiwaniana (CT-ZARG01) based on the neighbor-joining method and the Kimura 2-parameter model, with 1000 bootstrap replicates. Population structure was analyzed using Admixture (v1.3.0) [20] software based on the SNP data. The number of subpopulations (K values) was pre-set from 1 to 10 for clustering, and cross-validation was performed to determine the optimal number of clusters based on the lowest cross-validation error rate. Principal component analysis (PCA) was conducted using EIGENSOFT (v7.2.1) [21] software based on the SNP data to visualize the species clustering. PCA provided insights into which Cycas species were relatively close or distant, aiding in the evolutionary analysis.

3. Results

3.1. Whole-Genome Resequencing of Fujian’s Cycas

Here, we conducted whole-genome resequencing of six samples representing C. revoluta (CR-FZ-18, CR-FZ-19, CR-FZ-20), C. szechuanensis (CS-SX-28, CS-YT-09), and C. taiwaniana (CT-ZARG01) collected from four locations in Fujian Province, China (Figure 1). The total data generated amounted to 367.85 Gbp of clean data, with significant variation in clean read count and clean base count across samples (Table 1). The Q30 scores ranged from 92.50 to 98.10%, and GC content showed relatively low variation, ranging from 34.66 to 37.42%, indicating genetic similarity. The mapping rate was high, ranging from 95.83 to 99.39%, with C. szechuanensis (CS-SX-28) having the highest mapping rate at 99.36%. The correct mapping rate varied from 77.15 to 79.27%. The average sequencing depth was 5× or 6×, with C. revoluta (CR-FZ-20) having a slightly higher average depth of 6×. The 1× coverage ranged from 79.52 to 82.63%, indicating relatively uniform genome coverage (Figure S1). These data provide robust support for downstream genomic analyses.

3.2. SNP and InDel Detection and Annotation

The high-quality sequences obtained were used to detect and annotate SNPs and InDels across the six samples. Significant differences were observed in the total number of SNPs across the species, ranging from 73,584,380 to 96,229,565, with C. taiwaniana (CT-ZARG01) exhibiting the highest SNP count with 96,229,565 SNPs. The SNP variants were classified into six categories, with C > T and T > C transitions occurring more frequently than other types. Among these, C > T transition was the most common across all species/samples (Figure S2). The transition/transversion (Ti/Tv) ratios were consistently between 3.31 and 3.35, indicating a relatively stable mutation pattern. Heterozygosity levels varied significantly and ranged from 4,216,254 to 21,541,040 (Table S1a). CR-FZ-19 had the lowest heterozygosity (4,713,333), while C. taiwaniana (CT-ZARG01) had the highest (21,541,040). In contrast, homozygosity levels also varied widely, ranging from 69,368,126 to 74,688,525. CR-FZ-18 had the highest homozygosity (70,849,639). C. szechuanensis (CS-SX-28, CS-YT-09) and C. taiwaniana CT-ZARG01 exhibited significantly higher heterozygosity ratios of 21.15, 21.00, and 22.38%, respectively. Meanwhile, C. revoluta (CR-FZ-18 and CR-FZ-19) displayed lower heterozygosity ratios, at 6.23 and 5.72%, respectively.
The analysis of the genomic circos plot provided a comprehensive view from the outer to the inner layers, including chromosome coordinates, gene density distribution, SNP density distribution, and InDel density distribution (Figure 2a). Gene density distribution indicated that genes are highly clustered in specific chromosome regions, such as chr8_part2, which may represent gene-rich areas or functionally significant gene clusters. SNP density distribution revealed that certain chromosomal regions exhibited significantly higher polymorphism compared to others. InDel density distribution further highlighted the structural variation within the genome, showing a concentration of InDels in regions with high gene density. These integrated data demonstrate the relationship between gene density and genetic variation (SNPs and InDels), suggesting that higher mutation frequencies or selection pressures may be present in gene-rich regions. Figure 2b,c show the results of COG functional classification and GO classification analysis, respectively. The category with the highest number of genes is “G: Carbohydrate transport and metabolism” (approximately 650 genes), followed by “E: Amino acid transport and metabolism” and “J: Translation, ribosomal structure, and biogenesis”. Conversely, categories such as “A: RNA processing and modification” and “Z: Cytoskeleton” had the fewest gene annotations. Figure 2c presents the GO classification analysis, where genes are categorized into biological process, cellular component, and molecular function. The most prevalent biological processes are “metabolic processes” and “cellular processes”, indicating their central role in the genome. In terms of cellular components, “cellular components” and “organelles” constitute the major proportions, reflecting the critical role of these genes in cellular structure and function. For molecular functions, “catalytic activity” and “binding activity” are the predominant categories, underscoring the importance of these genes in various biochemical reactions within the organism. Overall, COG and GO classification analyses collectively reveal the functional diversity and complexity of biological processes in the genome, providing foundation for further research into gene functions.

3.3. Population Structure and Genetic Analysis

In general, the error rate decreases with increasing values of K, with the optimal K value determined by the lowest cross-validation (CV) error (Figure 3a). At K = 8, the four Cycas varieties, including the reference genome of C. panzhihuaensis, were grouped into six clusters: CR-FZ-18 and CR-FZ-20 (C. revoluta) formed one group (Figure 3b), while CR-FZ-19 (C. revoluta), CS-SX-28 and CS-YT-09 (C. szechuanensis), CT-ZARG01 (C. taiwaniana), and the C. panzhihuaensis reference genome (CP-PRJNA734434) were each assigned to separate groups. Notably, CS-SX-28 and CS-YT-09 exhibited some overlap. The relationship between samples and species indicated that CR-FZ-18 and CR-FZ-20 had very high Q1 probabilities, suggesting they belong to the same species. Conversely, CR-FZ-19, CS-SX-28, CS-YT-09, CT-ZARG01, and CP-PRJNA734434 were identified as distinct groups. Sample CT-ZARG01 showed a Q2 probability of 0.584212 and a relatively high Q6 probability (0.379774), indicating significant mixing between Q2 and Q6. CS-SX-28 had a higher Q5 probability but also exhibited substantial components in Q8 (Table 2).
Phylogenetic analysis revealed that CR-FZ-18, CR-FZ-19, and CR-FZ-20 belong to the same lineage (C. revoluta), with CR-FZ-18 and CR-FZ-20 being more closely related. Meanwhile, CS-SX-28 and CS-YT-09 (C. szechuanensis), CT-ZARG01 (C. taiwaniana), and CP-PRJNA734434 (C. panzhihuaensis) formed a separate lineage, with CS-SX-28 and CS-YT-09 (C. szechuanensis) showing higher relatedness (Figure 3c). This result is consistent with the phylogenetic tree. PCA results (Figure 3d) divided the seven populations into four clusters: CR-FZ-18, CR-FZ-19, and CR-FZ-20 (C. revoluta) clustered together, while CS-SX-28 formed a distinct subcluster and CS-YT-09 (C. szechuanensis) formed another cluster; CT-ZARG01 (C. taiwaniana) was a third cluster; and CP-PRJNA734434 (C. panzhihuaensis) was a fourth cluster (Figure 3e). This clustering result is consistent with the NJ phylogenetic tree constructed from whole-genome SNP data.

3.4. Sexual Differentiation and Genomic Variation in Cycas

The Cycas samples CR-FZ-18, CR-FZ-19, and CR-FZ-20 (C. revoluta), collected from the same location, show notable differences in terms of sex differentiation. CR-FZ-20 is a male Cycas, while CR-FZ-18 and CR-FZ-19 are female Cycas. Female Cycas produce large megasporophylls that are typically located at the top or center parts of the plant (Figure 4a). In contrast, male plants produce smaller, cylindrical or oval microsporophylls, which are usually found at the top of the plant (Figure 4b). As shown in Table 1 and Table 2, the Clean_Reads and Clean_Base values for female Cycas are significantly higher than those for male Cycas. Additionally, non-synonymous SNP and InDel mutation genes are more prevalent in female Cycas. Furthermore, during the detection of small InDels between the samples and the reference genome, male Cycas (CR-FZ-20) exhibited a higher number of insertions and deletions in both the whole genome and coding regions compared to female Cycas (CR-FZ-18, CR-FZ-19) when compared to the C. panzhihuaensis reference genome (Table S1b).
Combining KEGG analysis with previous research, several genes related to sex differentiation were identified. Female Cycas CR-FZ-18 annotated one MADS-box gene (CYCAS_010388, K09264), nine auxin-responsive protein SAUR71 genes in pathway K14488, three DELLA protein RGL1 genes in pathway K14494, three auxin-responsive protein SAUR36-like genes in pathway K14488, three transcription factor MYB77 genes in pathway K09422, seven GH3 auxin-responsive promoter genes in pathway K14487, and twenty transcription factor MYC2 genes in pathway K13422 (Table S2). Female Cycas CR-FZ-19 annotated nine auxin-responsive protein SAUR71 genes in pathway K14488, three DELLA protein RGL1 genes in pathway K14494, four auxin-responsive protein SAUR36-like genes in pathway K14488, three transcription factor MYB77 genes in pathway K09422, eight GH3 auxin-responsive promoter genes in pathway K14487, one additional GH3 auxin-responsive promoter gene in pathway K14506, and thirty-one transcription factor MYC2 genes in pathway K13422 (Table S2). Male Cycas CR-FZ-20 annotated one MADS-box gene (CYCAS_010388, K09264), ten auxin-responsive protein SAUR71 genes in pathway K14488, three DELLA protein RGL1 genes in pathway K14494, three auxin-responsive protein SAUR36-like genes in pathway K14488, three transcription factor MYB77 genes in pathway K09422, one GH3 auxin-responsive promoter gene in pathway K14487, one additional GH3 auxin-responsive promoter gene in pathway K14506, and thirty-one transcription factor MYC2 genes in pathway K13422 (Table S2). A notable difference is that the female Cycas CR-FZ-19 annotated an auxin-responsive protein SAUR36-like gene (CYCAS_000607, K14488) which was absent in the male Cycas CR-FZ-20.

4. Discussion

Cycads, among the oldest extant seed plants, harbor extensive genetic information. China is home to approximately 20 species of the genus Cycas, with three primary species found in Fujian: C. revoluta, C. szechuanensis, and C. taiwaniana. Due to the long maturation period and small population size of Cycas, particularly the rarity of male plants, studying their population evolution and genetic distribution is challenging [22]. The molecular mechanisms underlying sex differentiation in these plants are not well understood. This study presents a novel approach by using the C. panzhihuaensis genome as a reference for resequencing the Cycas species from Fujian, thereby obtaining high-quality genomic data and conducting evolutionary analyses. Additionally, we have explored genes related to sex differentiation, providing new insights for the conservation of Cycas.
In this study, resequencing of six samples representing three species revealed numerous SNPs and InDels, with the highest number of non-synonymous SNPs and InDel genes observed in sample CS-YT-09 (C. szechuanensis), suggesting that this sample’s genome may have undergone higher mutation rates or selective pressure. Conversely, the CR-FZ series samples (C. revoluta) exhibited lower levels of variation, potentially reflecting the environmental complexity and genetic diversity of their populations. Previous studies have noted higher variability in the rDNA regions of cycads compared to other plants, with SNPs primarily showing C  >  T substitutions in symmetric CG and CHG contexts, and these substitutions are highly methylated, leading to the genetic diversity observed in cycads [23]. The predominant SNP types in these samples are consistent with previous findings. Moreover, geographical differences were noted, with CS-SX-28 and CS-YT-09 (C. szechuanensis) exhibiting relatively higher SNP and InDel counts. This might be due to their more complex growing environments or limited genetic exchange, leading to higher mutation accumulation. For instance, in a study of 43 beech (Fagus sylvatica) populations across different environments, SNPs were significantly associated with environmental variables [24]. The higher precipitation in Shaxian (where CS-SX-28 is located) compared to Yongtai (where CS-YT-09 is found) suggests that regional environmental adaptation may have led to unique genomic variations in these cycads. These variations could be related to stress resistance and reproductive capacity, and further functional studies on these variant genes could elucidate their roles in cycad evolution and adaptation. Furthermore, CR-FZ-18, CR-FZ-19, and CR-FZ-20 (C. revoluta) are ancient trees, whereas CT-ZARG01 (C. taiwaniana) is cultivated. Interestingly, despite being cultivated, CT-ZARG01 exhibited substantial genetic diversity, potentially due to diverse propagation sources or historical hybridization. The gene variations in ancient trees may be related to their long growth periods, accumulating more mutations, similar to the leaf trait variations in ancient ginkgo trees which are associated with population origin and human activities [25]. Thus, the differences in cycad genomic variations reflect the impact of geographic location and population history (ancient vs. cultivated).
On the other hand, as dioecious plants, Cycas show significant differences in sex distribution in Fujian, with higher number of female plants and fewer males, impacting reproduction and population recovery and complicating conservation efforts. This study explored sex differentiation-related SNPs in ancient male (CR-FZ-20) and female (CR-FZ-18, CR-FZ-19) (C. revoluta) cycads. Notably, the MADS-box gene CYCAS_010388 (K09264), which might be a key gene for sexual differentiation. Previous genome sequencing of C. panzhihuaensis identified a sex differentiation gene MADS-Y, and the CYCAS_010388 gene is likely homologous to MADS-Y [9]. Similar sex-determining factors have been identified in asparagus (TDF1 and SOFF) [26], and similar regulatory patterns are reported in Phoenix dactylifera [27], Vitis amurensis [28], and Actinidia chinensis [29]. Additionally, both female and male cycads annotated genes in pathways related to auxin-responsive proteins (SAUR71, SAUR36-like), DELLA proteins (RGL1), transcription factors (MYB77, MYC2), and GH3 auxin-responsive promoters. These gene families or response factors have been studied in Arabidopsis thaliana [30,31], Spinacia oleracea [32], and Carica papaya [33], potentially influencing plant growth, development, and sex differentiation. However, due to limitations in this study, some variant genes could not be well localized, and future research could focus on locating sex-determining genes on chromosomes and exploring the evolution of sex chromosomes.
To enhance conservation and understanding of Cycas in Fujian, genetic and evolutionary analyses of these samples were conducted. Notably, genetic ancestry analysis and phylogenetic tree analysis indicated that CR-FZ-18 and CR-FZ-20 cluster in the same ancestral group (Q1), and they are expected to be in the same or adjacent branches in the phylogenetic tree, reflecting a shared evolutionary history. In contrast, CR-FZ-19, though geographically or taxonomically similar to other CR samples, belongs to a completely different group (Q4), potentially reflecting genetic divergence or adaptation to different ecological environments. A study on twelve Taiwanese cycad populations on Hainan Island revealed three distinct groups, possibly due to climatic differences on the island, similar to CR-FZ-19’s situation [34]. The mixed ancestral components exhibited by CS-SX-28 and CT-ZARG01 may correspond to the differentiation and crossover of branch positions in the phylogenetic tree, suggesting historical gene flow events between different populations. Both CS-SX-28 and CS-YT-09 are C. szechuanensis, but the two samples come from different geographical locations. In other cycads, individuals of Cycas dolichophylla have been reported to show significant genetic admixture with Cycas bifida, Cycas changjiangensis, and Cycas balansae [35]. Strong hybridization signals were detected between the ancestors of C. dolichophylla and C. szechuanensis, as well as between C. taiwaniana and C. changjiangensis. C. szechuanensis is distributed in both Sichuan and Fujian, China, with a large geographical span [36], indicating evolutionary diversity and gene flow within C. szechuanensis. As C. panzhihuaensis also originates from Sichuan, this explains the homologous relationship between CS-SX-28, CT-ZARG01, and C. panzhihuaensis (CP-PRJNA734434). This homology likely reflects shared ancestry or gene flow events between these species, particularly given their overlapping geographical ranges. This finding aligns with previous studies showing some homology between C. panzhihuaensis and C. szechuanensis, as well as with other cycads. It is worth noting that previous research suggested a closer phylogenetic relationship between C. panzhihuaensis and other cycads compared to C. szechuanensis [37].
Overall, this study reveals the preliminary characteristics of the genetic structure and sex differentiation of Cycas populations in Fujian, providing foundational data to support the conservation and breeding of regional Cycas resources. However, several limitations remain. First, the number of male samples is insufficient, which restricts the depth of analysis regarding sex-associated genetic variations. Second, the number of samples from Fujian is relatively small and lacks comprehensive geographic coverage, making it difficult to fully represent the region’s genetic diversity. Future studies should expand both the sample size and geographic range, with particular emphasis on elucidating the mechanisms underlying non-flowering male individuals and exploring the adaptive and evolutionary strategies of Cycas in different ecological niches.

5. Conclusions

This study conducted whole-genome resequencing and genetic evolution analysis of three Cycas species from various geographic locations in Fujian Province, China. Phylogenetic tree analysis and genetic ancestry analysis revealed the impact of different geographic locations and population histories on the genetic structure of Cycas plants. The study found significant genomic variation between the Cycas from Fujian and Sichuan, with extensive SNP and InDel variations detected across samples, exhibiting varying levels of genetic diversity. KEGG enrichment analysis identified key genes potentially associated with sex differentiation, notably the CYCAS_010388 gene, which may play a crucial role in the sexual differentiation of Cycas. Additionally, genes related to hormone responses and transcription factors were also found to potentially influence the growth, development, and sex differentiation of Cycas. This research provides new insights into the evolution, sex differentiation mechanisms, and conservation strategies of Cycas species. The findings are significant for the protection and improvement of Cycas, highlighting the need for targeted conservation efforts and further studies to understand the underlying genetic mechanisms.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11050488/s1, Figure S1: Sample chromosome coverage depth distribution map; Figure S2: SNP mutation distribution map; Table S1a: Detected SNP statistics; Table S1b: The number of insertions and deletions (InDels) in differentially expressed genes between male and female Cycads; Table S2: KEGG annotation of differentially expressed genes between male and female Cycads.

Author Contributions

Methodology, X.X., Y.A.E.-K., S.L., J.Z., L.Z., J.L. and W.L.; software, X.X. and Y.A.E.-K.; resources, K.Z., M.Z., Z.L., L.L. and Y.Z.; conceptualization, Y.A.E.-K., B.C. and S.W.; writing—original draft, X.X. supervision, Y.A.E.-K., B.C. and S.W.; writing—review and editing, Y.A.E.-K., B.C. and S.W; project leader: B.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China Government Central Finance, project name: Conservation of Biological and Species Resources “Collection, Conservation, Propagation and Uutilization of Cycas taiwaniana and C. szechuanensis Resources” Project No.: Cai Zi Huan (2023) No. 115 and Min Cai Zi Huan Zhi (2023) No. 60. The fund recipient: Fujian Academy of Forestry Sciences.

Data Availability Statement

The resequencing data have been deposited in the Sequence Read Archive (SRA) database in NCBI (BioProject accession numbers: PRJNA1030051).

Conflicts of Interest

Author Sijia Liu was employed by the company Fujian Satellite Data Development Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Horticulturae 11 00488 g001
Figure 1. Map of sample collection locations for Cycas species in Fujian, China (CR-FZ-18, CR-FZ-19, and CR-FZ-20 (C. revoluta) from Gushan, Fuzhou, CS-SX-28 (C. szechuanensis) from Shaxian district, CS-YT-09 (C. szechuanensis) from Yongtai County, and CT-ZARG01 (C. taiwaniana) from Zhao’an County).
Figure 1. Map of sample collection locations for Cycas species in Fujian, China (CR-FZ-18, CR-FZ-19, and CR-FZ-20 (C. revoluta) from Gushan, Fuzhou, CS-SX-28 (C. szechuanensis) from Shaxian district, CS-YT-09 (C. szechuanensis) from Yongtai County, and CT-ZARG01 (C. taiwaniana) from Zhao’an County).
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Figure 2. Distribution of various types of mutations on chromosomes and COG annotation of mutated genes. (a) Distribution of various types of variations for each sample on the chromosomes. From outer to inner: chromosome coordinates, gene density distribution, SNP density distribution, InDel density distribution; (b) COG annotation classification for variation genes; (c) GO annotation clustering of variation genes.
Figure 2. Distribution of various types of mutations on chromosomes and COG annotation of mutated genes. (a) Distribution of various types of variations for each sample on the chromosomes. From outer to inner: chromosome coordinates, gene density distribution, SNP density distribution, InDel density distribution; (b) COG annotation classification for variation genes; (c) GO annotation clustering of variation genes.
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Figure 3. Phylogenetic relationship of Cycas species from Fujian. (a) Cross-validation error rate of K value, the red dot represents the minimum value. (b) Clustering results of samples corresponding to each K value. (c) NJ phylogenetic tree of four Cycas species. (d) Principal component analysis of the four Cycas 4 species. (e) Phylogenetic heatmap.
Figure 3. Phylogenetic relationship of Cycas species from Fujian. (a) Cross-validation error rate of K value, the red dot represents the minimum value. (b) Clustering results of samples corresponding to each K value. (c) NJ phylogenetic tree of four Cycas species. (d) Principal component analysis of the four Cycas 4 species. (e) Phylogenetic heatmap.
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Figure 4. Photographs of microsporophyll and megasporophyll of C. revoluta. (a) Represents male microsporophyll, (b) represents female megasporophyll with seeds.
Figure 4. Photographs of microsporophyll and megasporophyll of C. revoluta. (a) Represents male microsporophyll, (b) represents female megasporophyll with seeds.
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Table 1. Whole-genome resequencing data quality statistics for three Cycas species.
Table 1. Whole-genome resequencing data quality statistics for three Cycas species.
Sample IDCR-FZ-18CR-FZ-19CR-FZ-20CS-SX-28CS-YT-09CT-ZARG01
SpeciesC. revolutaC. revolutaC. revolutaC. szechuanensisC. SzechuanesesC. taiwaniana
Clean_Reads195,298,221189,740,456241,606,263199,116,827201,245,198203,092,913
Clean_Base58,284,172,56056,681,698,61471,996,807,31859,685,488,47660,324,130,51160,877,892,614
Q30 (%)93.3192.5094.0797.9698.1097.95
GC (%)36.9537.3637.4234.7835.1534.66
Mapped (%)96.4395.8395.9499.3698.9899.39
Properly_mapped (%)78.9177.2879.2778.7177.1578.62
Average depth556555
Coverage_1X (%)81.5581.1482.6379.5279.9179.97
Table 2. Relationship between cycas species.
Table 2. Relationship between cycas species.
Sample IDSpeciesQ1Q2Q3Q4Q5Q6Q7Q8Group
CR-FZ-18C. revoluta0.999930.000010.000010.000010.000010.000010.000010.00001Q1
CR-FZ-19C. revoluta0.000010.000010.000010.999930.000010.000010.000010.00001Q4
CR-FZ-20C. revoluta0.999930.000010.000010.000010.000010.000010.000010.00001Q1
CS-SX-28C. szechuanensis0.000010.000010.000010.000010.8751590.000010.000010.124781Q5
CS-YT-09C. szechuanensis0.000010.000010.000010.000010.000010.000010.000010.99993Q8
CT-ZARG01C. taiwaniana0.000010.5842120.000010.000010.000010.3797740.0359640.00001Q2
CP-PRJNA734434C. panzhihuaensis0.000010.000010.999930.000010.000010.000010.000010.00001Q3
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Xu, X.; A. El-Kassaby, Y.; Liu, S.; Zhang, J.; Zhang, L.; Li, J.; Li, W.; Zhang, K.; Zou, M.; Lai, Z.; et al. Whole-Genome Resequencing Reveals Phylogenetic Relationships and Sex Differentiation Mechanisms Among Fujian Cycas Species. Horticulturae 2025, 11, 488. https://doi.org/10.3390/horticulturae11050488

AMA Style

Xu X, A. El-Kassaby Y, Liu S, Zhang J, Zhang L, Li J, Li W, Zhang K, Zou M, Lai Z, et al. Whole-Genome Resequencing Reveals Phylogenetic Relationships and Sex Differentiation Mechanisms Among Fujian Cycas Species. Horticulturae. 2025; 11(5):488. https://doi.org/10.3390/horticulturae11050488

Chicago/Turabian Style

Xu, Xinyu, Yousry A. El-Kassaby, Sijia Liu, Juan Zhang, Lanqi Zhang, Junnan Li, Wenkai Li, Kechang Zhang, Minghai Zou, Zhiru Lai, and et al. 2025. "Whole-Genome Resequencing Reveals Phylogenetic Relationships and Sex Differentiation Mechanisms Among Fujian Cycas Species" Horticulturae 11, no. 5: 488. https://doi.org/10.3390/horticulturae11050488

APA Style

Xu, X., A. El-Kassaby, Y., Liu, S., Zhang, J., Zhang, L., Li, J., Li, W., Zhang, K., Zou, M., Lai, Z., Lin, L., Zhang, Y., Wu, S., & Chen, B. (2025). Whole-Genome Resequencing Reveals Phylogenetic Relationships and Sex Differentiation Mechanisms Among Fujian Cycas Species. Horticulturae, 11(5), 488. https://doi.org/10.3390/horticulturae11050488

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