Phenotypic diversity and provenance variation of Cupressus funebris: a case study in the Sichuan Basin, China

Introduction

Genetic diversity, the abundance of genetic information within a species, reflects its level of endangerment, environmental adaptability, and evolutionary potential (Frankham, 2012; Hoban et al., 2021; Li et al., 2022a), serving as a cornerstone guiding plant conservation and breeding efforts (Jones et al., 2006). As research techniques advance, scholars are increasingly utilizing contemporary molecular technologies to explore plant genetic diversity (Plomion et al., 2014; Motalebipour et al., 2016; Liu et al., 2022a; Qing et al., 2022), leading to substantial progress in unraveling the genetic basis and evolutionary history of diverse plant species (Lu et al., 1996; Wolf, Lindell & Backström, 2010). This achievement is deserving of recognition. However, during genetic variation and adaptive evolution in plants, the initial response manifested by plants towards their external environment is invariably observed at the phenotypic level. This is evident in the observable differences in plant traits, commonly referred to as phenotypic variation, which can be discerned without the use of specialized equipment.

The existence of phenotypic variation is essential for the emergence of phenotypic diversity, a key component of genetic diversity in morphological studies (Chowdhury, Jana & Schroeder, 2000). Intraspecific genetic variation and the phenotypic plasticity of plants contribute to phenotypic variation (Wu, 1998; Sugai et al., 2023). The adaptive evolution of plants in response to variable environments results in the divergence and formation of distinct morphological groups at the population level, which facilitates the inheritance of genetic variation to subsequent generations (González-Martínez, Krutovsky & Neale, 2006). This illustrates that phenotypic changes can, to some extent, reflect altered plant genetic information (Kuijper & Johnstone, 2021). Therefore, phenotypic variability was employed as a fundamental criterion for plant breeding selection. Meanwhile, the use of phenotypic evaluation represents a classical approach in genetic information research for assessing genetic diversity (Zhang et al., 2015). In comparison to alternative approaches for studying genetic diversity, while molecular techniques such as genomics could provide faster and more efficient information on the genetic variation of a species, our understanding of genetic information is always limited without the objective evaluation of phenotypic data (Beaton et al., 2022). Consequently, phenotyping remains a pivotal methodology for the advancement and utilization of plant resources.

The term “provenance” pertains to the geographic origin of seeds or other propagation materials. This concept holds significant importance in the genetic improvement of trees (Míguez-Soto & Fernández-López, 2015; Alizoti, Aravanopoulos & Ioannidis, 2019). Trees of the same species, when grown in diverse natural environmental conditions for prolonged periods, exhibit genetic traits and geographical variations that mirror local environmental conditions (Cortan, Nonic & Sijacic-Nikolic, 2019). As a result, these germplasm materials effectively represent the growth patterns of natural geographic populations and demonstrate greater genetic diversity compared to a single geographic population (Barzdajn, Kowalkowski & Chmura, 2016). Under uniform growth conditions, these genetic variations manifest themselves morphologically, resulting in differences in growth characteristics among species such as Alnus cremastogyne (Zheng et al., 2023) and variations in fruit size in Prunus avium (Popovic et al., 2020).

The Cupressus funebris (C. funebris), an indigenous species of Cupressus in China, exhibits remarkable adaptability to its environment. The species possesses well-developed lateral roots, enabling it to thrive even on infertile and shallow soils. This renders it a suitable candidate for pioneering afforestation and ecological restoration of barren mountainous regions (Wang et al., 2021; Li et al., 2022b). Furthermore, its graceful and upright posture has made C. funebris a popular choice for landscaping in China. Consequently, a significant wild seed collection project was initiated in Sichuan Province, China, during the 1970s and 1980s with the objective of afforesting barren mountains. The wild germplasm materials, which were investigated and collected during this project, exhibit significant phenotypic variations and abundant intraspecific genetic diversity. For instance, they display a range of cone sizes and diverse seed shapes, spanning from near-rhomboid to circular. Notably, some of the ancient trees investigated have trunk diameters exceeding 1 m, demonstrating extraordinary growth ability. Furthermore, these C. funebris exhibit distinct growth morphologies; some varieties boast slender, pendulous twigs reminiscent of weeping willow (Salix babylonica), while others are characterized by shorter, bushy twigs. In 2004, the government of Sichuan Province, China (Sichuan, 2011), designated C. funebris as a crucial tree species for the development of industrial raw material forests, emphasizing its significance for the cultivation of rare trees. Consequently, it is of utmost importance to conduct relevant assessments of genetic diversity in C. funebris. In the field of C. funebris research, the majority of studies have concentrated on the testing of the antibacterial properties of essential oil and the renovation of inefficient plantation forests (Romeo et al., 2008; Lohani et al., 2015; Xie et al., 2016; Wang et al., 2021; He et al., 2022; Wen et al., 2022; Yuan & Hao, 2023). However, there has been a paucity of research on genetic diversity in C. funebris. Only a few studies have employed SSR markers in parental breeding, assessed genetic information through microsatellite markers, and conducted straightforward comparisons of growth traits (Lu et al., 2014; Yang et al., 2016; Yang et al., 2023a). Moreover, there is a lack of pertinent morphological research, which may hinder the genetic improvement and utilization development of C. funebris.

Consequently, this study evaluates the genetic diversity of 180 C. funebris trees across 19 traits within a seed orchard, utilizing five provenances and 60 half-sibling families, with the support of the national superior C. funebris germplasm base located in Santai County, Mianyang City, Sichuan Province. The objective of this study is to elucidate phenotypic variations and the degree of differentiation among C. funebris individuals, thereby strengthening the research framework on the genetic variation of this species. Furthermore, the study aims to provide valuable insights into the genetic management of C. funebris germplasm resources and contribute to the development and utilization of greening tree species.

Materials and Methods

Results

Discussion

The intricate natural environment, prolonged geographical isolation, and the process of natural selection are the primary factors facilitating phenotypic variation in woody plants (González-Martínez, Krutovsky & Neale, 2006). Cupressus funebris is a species widely distributed across various climatic zones in China, commonly inhabiting hilly areas, karst mountains, and river valleys (Lu et al., 2014). Phenotypic variation arises among different populations due to a combination of genetic factors and environmental influences in heterogeneous growth conditions.

The coefficient of variation and the Shannon-Wiener genetic diversity index are commonly employed to assess phenotypic variation, providing a direct indication of the variability within the subject of research (Zhang et al., 2022). In this study, we selected 19 relatively important and intuitive traits of C. funebris to analyze the variability and genetic diversity among 180 individuals in the C. funebris seed orchard. Our results uncovered substantial phenotypic diversity in the traits of C. funebris, aligning with the findings of phenotypic studies on Pinus koraiensis germplasm resources (Kaviriri et al., 2020).

Cupressus funebris, a gymnosperm with a long evolutionary history, can trace its origins back to the Mesozoic era (Spencer et al., 2015; Liu et al., 2022b). Having adapted to environmental changes such as mountain range formations, it has gradually evolved to thrive in arid, cold, and alpine climates, developing xeromorphic characteristics like low tree forms, compact crowns, and short-scaled leaves, which persist in some modern populations (Jiang & Wang, 1997). With climate changes and geographical expansion, C. funebris has demonstrated remarkable adaptability (Dakhil et al., 2019), particularly in watershed areas, where some populations have evolved superior growth traits such as larger tree forms, longer branches, and distinct scale leaves (Kvacek, Manchester & Schorn, 2000). This adaptive evolution has significantly contributed to the phenotypic diversity among C. funebris germplasm resources (Ma et al., 2019; Hu et al., 2023). The comprehensive genetic diversity index, measured at 1.97 in this study, underscores the robust adaptability of C. funebris. The 180 germplasm resources were sourced from mountainous regions surrounding basins and adjacent river basins, with notable differences in growth environments, including arid and high-altitude regions (e.g., BZ) and areas with mild and humid climates (e.g., NC). These differences in growth environments have led to notable variations in growth and seed-related traits (Mastretta-Yanes et al., 2012; Sahib et al., 2022; Zheng et al., 2023), as evidenced by the high coefficients of variation observed in wood volume (26.3%), cone volume (31.3%), and 100-grain weight (32.6%), respectively (Figs. S2A–S2E). In this study, the 180 germplasm resources possess diverse genetic backgrounds, stemming from various historical growth environments. Consequently, the high variability observed among these 180 germplasm resources is a concerted result of genetic background differences and local adaptive evolution. This means that some individuals exhibit typical xeromorphic characteristics, while others have undergone evolution in different traits in response to wetter growth environments (Marks, 2007; Blanco-Sánchez et al., 2024).

In comparison to the research conducted by Yang et al. (2023a), the coefficients of variation for tree height, diameter at breast height, under branch height, and crown width of 9-year-old C. funebris ranged from 20.92% to 52.73%. These values were notably higher than the variability observed in the growth traits examined in this study, which ranged from 10.3% to 26.3%. Such discrepancies are likely attributable to differences in the age of the C. funebris samples. As trees during the juvenile stage exhibit more vigorous growth (Ununger, Ekberg & Kang, 1988), it is expected that growth traits would exhibit greater variability. Furthermore, in this stage, traits such as tree height, diameter at breast height, and crown width are particularly susceptible to environmental influences (Santos-del-Blanco et al., 2013), resulting in even greater variability. Additionally, phenotypic plasticity of plants is most pronounced during the early stages of growth (Wu et al., 2021a). As growth progresses and the tree matures, these traits tend to stabilize. Consequently, the observed variability in C. funebris growth traits in this study was less pronounced than that reported by Yang et al. (2023a) as was predictable.

After conducting ANOVA analyses, it was evident that there existed significant (p < 0.05) or highly significant (p < 0.01) differences in the majority of traits, not only among but also within the various provenances. Furthermore, a pivotal discovery was made: the intra-provenance variation was found to contribute more substantially to the overall variability compared to the variation between provenances, as showed in Fig. S4D. This observation is consistent with prior findings in phenotypic studies of Pinus yunnanensis (Xu et al., 2016). The observed phenotypic variations among distinct populations of C. funebris can be attributed to environmental factors, which result in morphological adaptations. This phenomenon, known as phenotypic plasticity (Wu, 1998), highlights the remarkable adaptability of C. funebris across diverse regions to their respective environments. Conversely, genetic differentiation is the primary factor responsible for the observed phenotypic differences within comparable regions (Hao et al., 2019).

In this study, traits in C. funebris exhibited variations influenced by both natural environmental factors and genetic components. With regard to growth-related traits, the observed phenotypic variation among different provenances exceeded that within a single provenance. This indicates that both differences in the growth environment and genetic factors contribute to the variability in growth traits (Fig. S4C). In contrast, for branch, leaf, and seed traits, the phenotypic variation within a single provenance was found to significantly exceed that among different provenances. This suggests that these traits were primarily influenced by genetic variations (Fig. S4D).

Previous studies have revealed significant disparities in the underlying mechanisms through which environmental and genetic variations influence growth and reproductive traits. Environmental factors exert a greater influence on growth traits, resulting in morphological differentiation (Puy et al., 2021), whereas genetic factors play a more pivotal role in shaping reproductive traits (Drosse et al., 2014). These findings suggest that selecting superior varieties based on the morphological characteristics of their cones and seeds is an effective and reliable approach when undertaking genetic breeding improvements for forest trees.

This study expands the fundamental phenotypic dataset within the genetic variation research framework of C. funebris. The coefficient of variation and genetic diversity index for phenotypic traits in C. funebris were 16.9% and 1.97, respectively, indicating substantial diversity. These findings suggest a strong genetic basis for selective breeding in short-term processes (Laakili et al., 2016; Tchokponhoué et al., 2020). However, cultivating superior seeds in a seed orchard is a lengthy process that involves a continuous depletion of genetic variability. The ongoing selection process will inevitably lead to a reduction in the genetic foundation of C. funebris, resulting in a significant loss of genetic resources (Siepielski & Benkman, 2010). Therefore, the continuous evaluation and supplementation of genetic resources based on phenotypic characteristics, physiological dynamics, and genetic differences will be a long-term process, aimed at improving superior varieties through breeding. This represents our forthcoming work.

Correlation analysis is a fundamental method for investigating the interrelationships among diverse phenotypic traits. This study identified substantial correlations among growth traits, cone traits, and seed traits, which is consistent with earlier research (Zheng et al., 2023). The analysis of scale factors, including the ratio of tree height to crown height (H/CH) and the ratio of crown height to crown width (CH/CW), uncovered distinct growth patterns within C. funebris. Notably, the branch height exhibited a significant negative correlation with crown height and CH/CW (p < 0.01), indicating that trees with broader crowns undergo more robust natural pruning.

Furthermore, the significant negative correlation between H/CH and CH/CW (p < 0.01) suggests that trees with a lower ratio of tree height to crown height tend to exhibit less lateral growth. This finding indicates a preference for vertical growth in C. funebris within the seed orchard setting. This trend may be linked to the high density of seedling planting during the initial C. funebris establishment, which has led to intense intra-specific competition for sunlight and other resources. Consequently, vertical growth is favored over lateral growth in C. funebris populations, as previously reported by Saito, Kawamura & Takeda (2012) and Hitsuma et al. (2021).

The expansive vertical growth of C. funebris, which creates ample growing space and increased light availability, is reflected in annual branches and leaves (St. Clair, 1994). As depicted in Fig. S5, this characteristic displays a significant positive correlation between crown height and annual branch length (p < 0.05), as well as between tree height and leaf angle (p < 0.05). These findings form a basis for selecting and advancing superior varieties of C. funebris. Moreover, the findings provide valuable insights for the management of growth in seed orchards and plantation forests.

Principal component analysis (PCA) is an effective method for reducing the number of indicators while maximizing the explanatory power of the original indicator information (Jolliffe & Cadima, 2016). This method is commonly employed in the comprehensive evaluation process of seed selection and breeding (Khadivi-Khub, Sarooghi & Abbasi, 2016; Yang et al., 2023b). During our preliminary investigation, we observed notable differences in the morphological characteristics of C. funebris branches within the seed orchard. However, these specific branch morphological variations have not been significantly investigated in previous research studies (Papageorgiou et al., 2005; Yang et al., 2023a). In this study, we observed significant inter-individual differences in C. funebris, particularly in leaf angle, annual branch angle, and annual branch length after dimension reduction (Table S4). The results of the principal component analysis corroborated our observations. Furthermore, the principal component analysis results demonstrated that there was no significant correlation or consistent pattern between each principal component and the provenance of C. funebris (Figs. S6D and S6E). Similar results were observed in the clustering analysis, where a total of 180 individual C. funebris were categorized into four groups, each comprising individuals from diverse provenances (Fig. S7). This indicated that the clustering results did not significantly correlate with the provenances.

The patterns of trait variation in individual forest trees can be summarized into three types: geographic, continuous, and random (McKown et al., 2014; Suvanto et al., 2016; Queiroz et al., 2021; Wu et al., 2021b). The final type, random variation, is less prevalent in related studies. In this study, the phenotypic variation of C. funebris exhibited a random pattern that was not geographically related. This pattern was consistent with that observed in Pinus yunnanensis (Liu et al., 2022c). Apart from the adaptive morphological variations stemming from differences between the original habitat and the current environment (Singh et al., 2013), the growth characteristics and reproductive biology of C. funebris also contributed significantly to the random variation patterns observed in this species. Due to its extensive distribution and high adaptability, it was often difficult to discern geographical isolation between populations of C. funebris (Jiang & Wang, 1997). Moreover, C. funebris, as a wind-pollinated coniferous species, exhibits a characteristic that creates favorable conditions for gene flow and even facilitates interspecific gene introgression through hybridization (Petrova et al., 2018). Consequently, the frequent exchange of genetic material and the presence of adaptive genetic variation serve as primary drivers for the observed random patterns of variation in diverse C. funebris populations. Moreover, according to Wójkiewicz, Litkowiec & Wachowiak (2016), despite the considerable distances between populations of wind-pollinated coniferous trees, as long as they are not isolated, there are no notable genetic differences among them. Based on this, it can be hypothesized that similar patterns of variation are likely to occur in natural populations of coniferous or congeneric trees that exhibit growth characteristics similar to C. funebris. This finding has the potential to further our understanding of the geogenetics of these tree species and facilitate conservation and management efforts.

The results of the clustering analysis revealed that the four groups of C. funebris exhibited distinct phenotypic differences. Group III exhibited a graceful tree morphology, making it ideally suited for integration into urban greening projects. In contrast, group IV exhibited remarkable growth and seed traits, rendering it an excellent candidate for forestry breeding and genetic enhancement programs. These findings will provide a solid foundation for the classification and utilization of C. funebris.

Conclusions

The abundant genetic diversity of C. funebris serves as a prerequisite for generating variant populations and facilitating continuous selective breeding, thereby enabling the screening of populations with distinct characteristics that hold immense potential for applications in ecological restoration, afforestation, timber production, and urban greening. This study investigated the genetic diversity of C. funebris through a comprehensive analysis of phenotypic traits. The results revealed a high degree of genetic variation among 180 germplasm materials sourced from five populations. Cluster analysis identified four distinct groups, each characterized by unique combinations of traits including tree height, wood volume, branch and leaf morphology, cone and seed traits. These findings underscore the richness of genetic resources available for the improvement of C. funebris. The identified groups represent valuable genetic pools that can be harnessed for the development of superior cultivars. Future research should focus on elucidating the genetic architecture of key traits and evaluating the performance of these groups across diverse environmental conditions.

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