Conservation of genetic diversity hotspots of the high‐valued relic yellowhorn (Xanthoceras sorbifolium) considering climate change predictions

Abstract Genetic structure and major climate factors may contribute to the distribution of genetic diversity of a highly valued oil tree species Xanthoceras sorbifolium (yellowhorn). Long‐term over utilization along with climate change is affecting the viability of yellowhorn wild populations. To preserve the species known and unknown valuable gene pools, the identification of genetic diversity “hotspots” is a prerequisite for their consideration as in situ conservation high priority. Chloroplast DNA (cpDNA) diversity was high among 38 natural populations (H d = 0.717, K = 4.616, Tajmas’ D = −0.22) and characterized by high genetic divergence (F ST = 0.765) and relatively low gene flow (N m = 0.03), indicating populations isolation reflecting the species’ habitat fragmentation and inbreeding depression. Six out of the studied 38 populations are defined as genetic diversity “hotspots.” The number and geographic direction of cpDNA mutation steps supported the species southwest to northeast migration history. Climatic factors such as extreme minimum temperature over 30 years indicated that the identified genetic “hotspots” are expected to experience 5°C temperature increase in next following 50 years. The results identified vulnerable genetic diversity “hotspots” and provided fundamental information for the species’ future conservation and breeding activities under the anticipated climate change. More specifically, the role of breeding as a component of a gene resource management strategy aimed at fulfilling both utilization and conservation goals.


| INTRODUC TI ON
The International Union for Conservation of Nature and Natural Resources (IUCN) recognized the need for biodiversity conservation at its three levels: genetic, species, and ecosystem (McNeely, Miller, Reid, Mittermeier, & Werner, 1990). Conservation of genetic diversity is fundamental for species and ecosystem scales (Frankham, Ballou, & Briscoe, 2010). The importance of genetic diversity can be addressed as to reducing inbreeding depression (Frankham et al., 2010), providing fundamental resources for species evolution (Frankel & Bennett, 1970), enhancing population fitness, and decreasing extinction risk (Vellend & Geber, 2005). Although genetic diversity conservation was proposed as an explicit goal in 1993 (Burhenne-Guilmin & Casey-Lefkowitz, 1993), the conservation of genetic resources only focused on species of recognized value to humans. However, the value of vast genetic resources may still remain unknown and has been described as "sitting on the shelf" or "gene morgues" (Hoisington et al., 1999), such as those of yellowhorn (Xanthoceras sorbifolium).
Climate change may affect the genetic diversity within and among populations and thus impacting species' evolutionary potential (Vellend & Geber, 2005). The current observed rate of climate change is faster than that of postglacial warming (Willis & MacDonald, 2011) and is considered as a pervasive global problem (Hegerl et al., 2007). Thus, the reliance on long-term evolutionary factors for creating new genes is not an option. To preserve yellowhorn's known and unknown valuable gene pools, the identification of the species genetic diversity "hotspots" is a prerequisite for their consideration as in situ conservation high priority (Shafer, Cullingham, Cote, & Coltman, 2010;Weiss & Ferrand, 2007). Additionally, predicting the magnitude of climate change and its impact on the genetic diversity within these "hotspots" is essential for the species future utilization and conservation strategies.
Breeding practices often focus on a subset of the base population, thus effectively reducing the genetic diversity (Chaisurisri & El-Kassaby, 1994;El-Kassaby & Ritland, 1996;Stoehr & El-Kassaby, 1997); however, breeding activities should be viewed as a component of larger gene resource management strategy aimed at fulfilling both utilization and conservation goals. Wild genetic resources substantially contribute to conventional crop improvement efforts (e.g., wheats and maize (Hoisington et al., 1999)) and it should be noted that substantial numbers of the selected and deployed valuable varieties originated from landraces or nature individuals (Hoisington et al., 1999). Historically, tree selective breeding is a young practice started in 1950s, indicating that the state of forest tree genetic resources is still resembling their wild ancestors (White, Adams, & Neale, 2007). Most tree selective breeding efforts are mainly focused on economically valuable attributes with emphases on increasing yield and to a lesser extent quality. Exploring future tree genetic resources is anticipated to include additional attributes such as wood quality, drought, frost, and pest resistant or tolerance, and fruit yield and taste.
Chloroplast DNA (cpDNA) is haploid and nonrecombining genome (Comes & Kadereit, 1998), usually maternally inherited in most angiosperms such as Theaceae (Li, Awasthi, Yang, & Li, 2013) and some gymnosperms such as Ginkgo (Shen et al., 2004). cpDNA has been widely used in phylogeny, classification, and biogeography of many plant species (Olmstead & Palmer, 1997). In this study, we used cpDNA sequencing from five noncoding regions to assess haplotype diversity and relationship in 399 yellowhorn individuals representing 38 wild populations. We focused on three issues: (a) uncover the genetic diversity, genetic structure, and phylogenetic relationships; (b) identify the genetic diversity "hotspots" and conservation role under climate change; and (c) utility of breeding with valuable genotypes.
Most of the sampled areas are located on the Loess Plateau, an arid or semi-arid region, with 200-700 mm of annual precipitation, and occupy an elevational band between 800 and 2,200 m. From each individual tree, a fresh sample of 2-10 leaves was collected and stored in silica gel until further use. Due to the vegetative propagation nature of yellowhorn that often creates clonal clumps, sampled trees were intentionally separated by a minimum distance of 100 m (Song, Yin, Liu, Wang, & Jiao, 2011). All sampled locations were recorded using GPS HOLUX M-241.
Genetic abundance distribution and cluster for the 38 yellowhorn populations were conducted using the "heatmap" function in the "gplots" package (Warnes et al., ). Haplotype network and frequency were determined using "haploNet" function in the "pegas" package (Paradis, 2010). All analyses were conducted in R version 3.3.1 (R Development Core Team, 2010).

| Phylogenetic relationships and analyses
Phylogenetic relationships based on cpDNA sequence data were conducted using the Bayesian phylogenetic analysis following

| Identifying climate variables associated with individual-based genetic diversity
We used canonical correspondence analysis (CCA) ordination to identify the climate variables that are correlated with the distribution of haplotype diversity. CCA was calculated by the "cca" function in package "vegan" in R 3.3.1 (Oksanen et al., 2007), and it is based on chi-squared distances and performs weighted linear mapping. In

| RE SULTS
3.1 | Genetic structure and genetic diversity "hotspots" identification

| Phylogenetic relationships and cluster among wild populations
Based on Bayes (posterior probabilities >0.9), maximum parsimony increasing from southwest to northeast (Figures 2 and 4).

| Genetic structure genetic diversity and phylogenetic relationships
In  (Gao et al., 2007)). This may be associated with the long evolutionary history for woody species. Genetic diversity is controlled by four processes, including mutation, drift, migration, and selection (Hoisington et al., 1999). Tajima's D is widely used to evaluate the pattern of demographic processes within species (genetic bottlenecks (or genetic drift) and founder effects), which may F I G U R E 5 Network for 24 cpDNA microsatellite haplotypes (solid bars indicate the number of mutational steps. See Figure 1 for haplotype colors) cause population structure and selection (Mahoney, 2004;Ptak & Przeworski, 2002). Negative and positive values of within populations Tajima's D indicate that populations have undergone demographic expansions and experienced bottlenecks, respectively (Tajima, 1989 Fruit of yellowhorn form capsules, those capsules disclose when ripe (July-August), the capsule shells become hard and woody.
These fruits contain on average 15-25 seeds (with hard seed coats) (Zhou & Liu, 2012), and mean single seed weight is around 0.87 g (Wang, Huang et al., 2017;Wang, Yang et al., 2017). As in most plant species, relatively heavy seed is dispersed within short distance (dozen meters) (Willson, 1993). The evolution of plants is affected by long-distance dispersal (LDD). LDD is influenced by several factors including the presence of open terrestrial landscapes and mediation by large animals, migratory animals, extreme meteorological events, ocean currents, and human transportation (Nathan et al., 2008) as well as extreme phenomenon such as glaciation and continental drift (Smith, 1993).

Yellowhorn is an ancient woody perennial species belonging to
Xanthoceroideae family that evolved during the Late Cretaceous  (Zheng et al., 2007). Population's genetic diversity showed substantial reduction from northeast to southwest regions (Figures 2 and 4) and may be due to the isolation by mountains and rivers forcing yellowhorn to evolve to adapt to the new environments during its northeastward migration. According to the tropical origin hypothesis, H1 is the most ancient haplotype, the pattern of mutation steps highly support the northeastward migration route ( Figure 5).

| Genetic diversity conservation under climate change
In the present time, the rate and magnitude of warming may be comparable to that of postglacial warming (Solomon, 2007;Willis & MacDonald, 2011). Climate warming can reduce genetic richness by indirectly reducing landscape and species diversity (Chapin III et al., 2000). Understanding among and within genetic diversity indices will help identifying the genetic diversity "hotspots" and unique haplotypes. Moreover, identifying climate factors which contribute to the distribution of genetic diversity may provide ideas for genetic conservation under climate change. In light of the present study, genetic "hotspot" conservation is the most efficient way to protect the yellowhorn's genetic diversity (i.e., the identified six wild yellowhorn populations harboring higher haplotype diversity). Climate variables TD (temperature difference between MWMT (mean warmest month temperature) and MCMT (mean coldest month temperature)) and EMT (extreme minimum temperature over 30 years) in the identified haplotype diversity "hotspots" are expected to increase by 3-5°C in the following 50 years (Figure 7). TD showed the difference between mean warmest month temperature (MWMT) and mean coldest month temperature (MCMT) is increasing, while the extreme minimum temperature over 30 years (EMT) is increasing, which indicate that extreme weather conditions may occur more than contemporary observed. While yellowhorn is adapted to warm climate as its original distribution is in tropical region; however, the observed and predicted climate change are expected to cause greater challenges.
Surprisingly, all moisture-related climatic variables were nonsignificant indicating that temperature alone plays a major role in the species contemporary and future distributions and confirming the species drought resistance .
The cpDNA genome is characterized by its slow evolution caused by low mutation rate (Palmer & Herbon, 1988). Thus, the observed high haplotype diversity (F ST > 0.2) that is coupled with very low gene flow indicates that yellowhorn populations have been isolated for a very long time and may undergo further habitat fragmentation.
Furthermore, while the mode of inheritance of yellowhorn's chloroplast is unknown, it is expect to be material as most angiosperms (Reboud & Zeyl, 1994), thus limiting gene flow due to the species large seed (Wang, Huang et al., 2017;Wang, Yang et al., 2017), further contributing to fragmentation.
Wild yellowhorn often found in Versant Soleil occupying forest edges presumably for sunlight accessibility (personal field observation). This habitat distribution type is more sensitive to edge effects and habitat fragmentation which may be exasperated by global warming (Reinmann & Hutyra, 2017), and consequently contributing to woody plant ecosystem's properties and thus changing the habitat of animals associated with long-distance dispersal (LDD). Thus, the migration and evolution of yellowhorn are expected to face natural and anthropogenic biogeographic barriers (Lumibao, Hoban, & McLachlan, 2017).

To achieve Aichi Biodiversity Targets (Goal A) in Convention
on Biological Diversity (https://www.cbd.int/sp/targets/), the Chinese National Natural Conservation Areas program is providing the most restrictive laws to protect wild plant and animal species (Wang, Huang et al., 2017;Wang, Yang et al., 2017), and this in its wild populations (Wang, Huang et al., 2017;Wang, Yang et al., 2017). Thus, we suggest the establishment of yellowhorn protected areas in the identified six genetic diversity "hotspots," and this initiative could help conserving the existing genetic diversity within these unique gene pools (Jenkins & Joppa, 2009;Maxted, Dulloo, Ford-Lloyd, Iriondo, & Jarvis, 2008). Additionally, maintaining and conserving the yellowhorn ecosystem will aid in the conservation and protection of those animals associated with the species seeds dispersal. Furthermore, while efforts are dedicated to the in situ conservation of yellowhorn in its wild estate within protected ecosystems, a complementary ex situ conservation efforts such as gene banks are also advocated (Cohen, Williams, Plucknett, & Shands, 1991;Vavilov, 1927).
F I G U R E 7 Significant climate variables changing in recognized genetic diversity "hotspots" for next 50 years

| Breeding program based on utilization of genetic resources
Mountains and plateaus account to 70% of land area of China (Baiping, Shenguo, Ya, Fei, & Hongzhi, 2004). Considering the habitat of yellowhorn (tolerance to high pH, clay, sandy, loam, average, medium or well-drained soil (Li et al., 2010)), there is large potential for planting yellowhorn. The main advantage of planting yellowhorn is the species ability to grow on marginal sites, thus does not compete on fertile land distant for other crops creating an economic and environmental "win-win" scenario (Wang, Huang et al., 2017;Wang, Yang et al., 2017). To enhance to development of yellowhorn planting, the Chinese government sponsored the "11th Five-Year Plan" to ensure planting of more than 105 ha per year until 2020 (Yu et al., 2017). The present challenge is the identification of the best yellowhorn "varieties," thus embarking on a breeding and selection program is expected to address this issue.
In reality, the selection and breeding of yellowhorn have started in 1970s, with traditional breeding methods following the recurrent selection scheme with its selection, breeding, and testing cycles.
Presently, with the availability of affordable genomic markers and advanced computational methods, a more efficient breeding methods such as genomic selection (GS) could be implemented (Meuwissen, Hayes, & Goddard, 2013 Ratcliffe et al., 2015). It should be highlighted that the peculiar reproductive nature of yellowhorn (known as the species with "thousand flowers but one fruit" (Ding & Ao, 2008)) and alternative deployment methods through vegetative propagation are required to overcome the reproductive approach drawbacks (El-Kassaby & Klapště, 2015). We feel that the adoption of genomic selection for identifying superior individuals and the implementation of vegetative propagation scheme (e.g., root cutting (Yao, Qi, & Yin, 2013)) will lead to a faster yellowhorn population development. and these could be dealt with tree aging and statistical methods that account for spatial heterogeneity (Cappa et al., 2017). Previous research has shown that yellowhorn oil contains unsaturated very long chain fatty acids (VLCFAs: oleic, linoleic, gondoic, erucic, and nervoic acid (Zhang et al., 2010)) which is been widely used as edible oil, and in cosmetics, medicine, and biofuel (Venegas-Calerón, Ruíz-Méndez, Martínez-Force, Garcés, & Salas, 2017). Thus, these attributes will form the foundation for phenotyping of economically valuable attributes during the implementation of GS. To maximize the chances for success, we propose implementing GS on the six identified "hotspots" wild yellowhorn populations.
Finally, it should be emphasized that breeding efforts are complementary to conservation, so it is expected that the majority of the unknown valuable attributes of yellowhorn will be safeguarded with this initiative.

| CON CLUS ION
The genetic structure and diversity of 38 yellowhorn wild populations were assessed using 399 individuals representing 7 provinces covering the species' natural range. We use cpDNA microsatellite haplotype variation to determine the species contemporary variation and its population differentiation as affected by postglacial migration and the anticipated global warming. Six genetic diversity "hotspots" were identified and deemed important for high conservation priority. A utilization (breeding) and conservation initiative is proposed.

ACK N OWLED G M ENTS
The authors are grateful to Tongli Wang for his advice on ClimateAP. Thanks

CO N FLI C T O F I NTE R E S T
None declared. contributed to data analysis. R.Z., Q.W., W.G, and Y.A.E. wrote, edited, and reviewed the MS.

DATA ACCE SS I B I LIT Y
Not applicable.