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Chloroplast DNA-based Genetic Variation of Rosa roxburghii in

Southwest China: Phylogeography and Conservation Implications

Min Lu , Huaishan Zhang , Huaming An

PII: S2468-0141(21)00002-9
DOI: https://doi.org/10.1016/j.hpj.2021.01.002
Reference: HPJ 241

To appear in: Horticultural Plant Journal

Please cite this article as: Min Lu , Huaishan Zhang , Huaming An , Chloroplast DNA-based Genetic
Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications, Hor-
ticultural Plant Journal (2021), doi: https://doi.org/10.1016/j.hpj.2021.01.002

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 Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China:
Phylogeography and Conservation Implications
Min Lua, Huaishan Zhanga,b, and Huaming Ana,b,*
a
Agricultural College, Guizhou University, Guiyang 550025, China
b
Guizhou Engineering Research Center for Fruit Crops, Guiyang 550025, China
Received 19 May 2020; Received in revised form 11 June 2020; Accepted 24 November 2020
Available online 2020

ABSTRACT
Rosa roxburghii Tratt. is a well-known commercial horticultural crop in China with nutritional and medicinal value. Wild
germplasms of this species are mainly distributed in Southwest China but the population is decreasing due to continuous
exploitation, habitat destruction, and fragmentation. Therefore, assessing the genetic diversity and phylogeography is essential for
efficient conservation. Herein, two chloroplast intergenic spacers (trnL-trnF and accD-psaI) were investigated in 255 individuals from
29 R. roxburghii populations and 18 haplotypes (H1–H18) were identified. High levels of haplotype diversity (Hd = 0.829) and
−3
nucleotide diversity (π = 1.3 × 10 ) were detected in these populations. Also, the genetic variation representing 86.4% of the total
variation was detected by an analysis of molecular variance. A significant correlation was established between genetic divergence
and geographic distance by the Mantel test (r = 0.204, P = 0.04, 9 999 permutations), suggesting the isolation-by-distance model. A
significantly higher Nst than Gst (Nst = 0.257, Gst = 0.136, P < 0.05) indicated the phylogeographic structure of R. roxburghii. Further
phylogeographic analysis revealed rapid range expansion in the population, probably between 647 073 and 217 848 years ago. The
primary processes shaping the genetic patterns of the R. roxburghii populations included restricted gene flow with isolation distance
within clades 1-8, 2-3, and overall, contiguous expansion within clades 1-3 and 3-2, past fragmentation, and/or long-distance
colonization within clades 1-9 and 2-2. Conservation priority should be given to the core populations GZ, FQ, DF, DS, xy, AL, LC, PB,
and XY in the Yunnan-Guizhou Plateau, NZ and MX in the Qingling-Bashan mountains, and MN in the Hengduan mountains, where
an in situ preservation and management strategy should be applied.
Keywords: chloroplast DNA; genetic diversity; core population; phylogeography; Rosa roxburghii

1. Introduction

Rosa roxburghii Tratt. (2n = 2x = 14), a well-known species in the Rosaceae family that is unique because of
the bristly spines covering the hypanthium and fruits, is also a major commercial fruit crop due to the nutritional
and medicinal components in the fruit, including ascorbic acid, superoxide dismutase, flavonoids,
polysaccharides, organic acids, amino acids, and dietary fiber (He et al., 1984; An et al., 2011; Liu et al., 2015; Lu

* Corresponding author. Tel.: +86 851 83855894 1

E-mail address: anhuaming@hotmail.com
Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of
Agricultural Sciences (CAAS)
Https://doi.org/10.1016/j.hpj.
2468-0141/2020 Chinese Society for Horticutural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural
Sciences (CAAS). Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under th CC BY-NC-ND
license. (Http://creativecommons.org/licenses/by-nc-nd/4.0/)
 Min Lu et al.
2
et al., 2015). To date, at least 150 000 hm of economic planting area of the cultivar ‘Guinong 5’ in China have
been reported, and a series of food and health products have been developed (Lu et al., 2016). R. roxburghii is
native to China, and its wild germplasm resources are widely distributed in the southwest, mainly Guizhou
Province and sporadically in adjacent provinces such as Sichuan, Chongqing, Hunan, Guangxi, Hubei, and Shaanxi
(Fan and Xia, 1997). Southwest China is regarded as one of the ancient relict areas throughout the Quaternary
(Qiu et al., 2011). It experiences both Pacific and Indian monsoons and comprises of mountains, plateaus, river
valleys, deep gorges, and basins. Also, the diverse climate and topography of Southwest China may be the main
reason why it has become one of the world’s biodiversity hotspots.
The breeding and sustainable industry development of R. roxburghii is based on the wild genetic resources.
Any loss of genetic diversity will affect its potential application value for humans. Various environmental stresses,
such as continuous exploitation, habitat destruction, and fragmentation, have caused excessive resource
destruction. For efficient conservation, it is imperative to assess the genetic diversity and phylogeography.
However, only limited studies have been carried out on R. roxburghii, using it as a reference to study the
phylogenetic relationships in the Rosa genus (Wen et al., 2003a, 2004; Tang et al., 2008; Deng et al., 2015;
Fougère-Danezan et al., 2015; Zhu et al., 2015; Wang et al., 2018; Jeon and Kim, 2019). Nonetheless, the genetic
diversity at the intra-specific level has only been investigated using random amplified polymorphic DNA (RAPD;
Wen et al., 2003b, 2003c) and simple sequence repeat (SSR; Yan et al., 2015a, 2015b; Zhang et al., 2017)
methods. A study on the population structure of R. roxburghii in Guizhou Province revealed that the majority of
genetic variation within populations was characterized by frequent gene exchange, high genetic consistency, and
small Nei’s genetic distance (Zhang et al., 2017), which provided information for the protection and exploitation
of R. roxburghii resources. Surprisingly, no information is available about the phylogeography of wild R.
roxburghii.
Phylogeographic studies within or among closely related species have increased exponentially in recent
years (Qiu et al., 2011). Quaternary population history has been inferred using molecular phylogeographic
evidence. Population history can be inferred from genetic information in the nuclear and cytoplasmic genomes.
Nuclear DNA markers, such as inter-simple-sequence repeats (ISSRs), amplified fragment length polymorphisms
(AFLPs), nuclear microsatellites (nSSRs), and DNA sequence variation at nuclear loci (nDNA), can be utilized; to
date, these have only rarely been explored, especially in Rosaceae plants (Qiu et al., 2011; Daneck et al., 2016).
Recently, chloroplast (cp) DNA sequences, especially the noncoding cpDNA regions accD-psaI and trnL-trnF, have
been frequently used in some Rosaceae species (Fér et al., 2007; Liu et al., 2013; Zong et al., 2014; Gao et al.,
2015). In this study, both trnL-trnF and accD-psaI cp intergenic spacers were cloned and sequenced in 29 R.
roxburghii populations in Southwest China to (1) judge the genetic diversity and genetic differentiation of R.
roxburghii resources, (2) infer the phylogeographic structure of R. roxburghii, and (3) assess the conservation
implications for R. roxburghii in Southwest China.

2. Materials and methods

2.1. Population sampling

In this study, 255 samples from 29 naturally occurring Rosa roxburghii populations were collected from
Southwest China in 2014–2016 (Table 1). A minimum distance of 20 m between individuals was maintained to
avoid sampling similar genotypes. Healthy young leaves were collected and dried in silica gel, stored in normal
2
Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications
temperature.

Table 1 Description of the 29 sampling sites of wild Rosa roxburghii

Population code Locations Altitude/m Latitude (N) Longitude (E) Sample size
AL Anlong, Guizhou 1 258–1 334 25°11'29"–25°14'5" 105°19'23"–105°29'14" 13
BJ Bijie, Guizhou 1 529–1 886 27°07'2"–27°18'27" 105°04'5"–105°18'56" 5
DF Dafang, Guizhou 1 400–1 416 27°02'59"–27°04'16" 105°41'6"–105°41'10" 5
DS Dushan, Guizhou 885–917 25°20'5''–25°27'4'' 107°52'7''–107°54'11'' 5
FQ Fuquan, Guizhou 957–1 000 26°48'2"–26°48'48" 107°35'0"–107°36'10" 7
HS Huishui, Guizhou 946–1 032 25°44'7"–26°04'35" 106°34'26"–106°59'24" 10
HX Huaxi, Guizhou 1 000–1 474 26°20'9"–26°41'53" 106°43'11"–106°82'56" 18
LD Luodian, Guizhou 836–934 25°37'8"–25°43'58" 106°32'12"–106°40'20" 7
LZ Liuzhi, Guizhou 1 214–1 638 26°11'29"–26°29'40" 105°17'16"–105°32'5" 5
PB Pingba, Guizhou 1 243–1 400 26°21'40"–26°41'34" 106°12'37"–106°33'36" 20
PX Panxian, Guizhou 1 530–1 780 25°10'50"–25°54'53" 104°32'12"–104°50'14" 14
QL Qinglong, Guizhou 1 499–1 583 25°81'51"–25°83'29" 105°19'45"–105°21'54" 7
QX Qianxi, Guizhou 1 202–1 264 26°57'46"–27°01'27" 106°00'2"–106°06'27" 13
SC Shuicheng, Guizhou 1 698–1 796 26°47'32"–26°55'41" 104°52'43"–104°55'46" 4
SQ Shiqian, Guizhou 692–892 27°28'48"–27°35'30" 108°01'14"–108°12'24" 6
SY Suiyang, Guizhou 734–1 405 27°53'23"–29°54'21" 106°58'51"–107°19'59" 10
XY Xingyi, Guizhou 1 528–1 920 24°54'36"–25°57'6" 104°43'45"–104°50'17" 18
ZJ Zhijin, Guizhou 1 198–1 700 26°22'7"–26°35'39" 105°41'55"–105°44'58" 11
ZY Zunyi, Guizhou 778–981 27°26'53"–27°58'53" 106°54'39"–107°18'32" 12
ZT Zhaotong, Yunnan 1 380–1 391 27°39'5"–27°45'14" 104°48'8"–104°55'11" 5
JL Junlian, Sichuan 1 076–1 202 27°51'24"–27°53'15" 104°33'13"–104°35'4" 5
MN Mianning, Sichuan 1 769–1 828 28°28'50"–28°35'44" 102°04'5"–102°15'28" 11
xy Xuyong, Sichuan 1 050–1 155 27°50'54"–28°12'33" 105°16'39"–105°39'4" 8
CQ Chongqing 518–540 30°15'37"–30°15'46" 107°43'2"–107°45'13" 6
GZ Guzhang, Hunan 250–267 28°40'1"–28°41'3" 109°45'0"–109°46'31" 5
HY Huayuan, Hunan 522–570 28°26'3"–28°28'0" 109°25'21"–109°29'40" 6
LC Lichuan, Hubei 963–1 088 30°4'35"–30°16'54" 108°36'12"–108°56'13" 7
NZ Nanzheng, Shaanxi 528–586 32°56'43"–32°59'0" 106°45'41"–106°48'27" 6
MX Mianxian, Shaanxi 547–602 33°6'38"–33°7'27" 106°28'33"–106°31'32" 6

2.2. DNA extraction

A modified cetyltrimethyl ammonium bromide (CTAB) method (Porebski et al., 1997) was used to extract
total genomic DNA. The DNA was solubilized in TE buffer (EDTA and Tris-HCl, pH 8.0) and stored at –20 °C for later
analyses.

2.3. PCR amplification

The accD-psaI and trnL-trnF intergenic spacers were amplified according to Small et al. (1998) and Taberlet
et al. (1991), respectively. The PCR was carried out in a 20 mL volume containing 1× Taq PCR MasterMix (KT201,
Tiangen Biotech, Beijing, China), containing 50 ng of DNA template and 0.25 mmol · L-1 of each primer. The
amplification system was as follows: initial denaturation at 94 °C for 3 min; followed by 35 cycles of 94 °C for 40 s,
53.6 °C (trnL-trnF intergenic spacers) and 51.6 °C (accD-psaI intergenic spacers) for 40 s, 72 °C for 1 min, and a
final extension at 72 °C for 10 min. The PCR products were purified by 2% agarose gel electrophoresis using a
Dingguo Purification Kit (Dingguo, Beijing, China) and sequenced using the amplification primer pairs on an ABI
3730 automatic DNA sequencer (PerkinElmer) using the cycle sequencing protocols for DyeTM 3.1 (PerkinElmer).

2.4. Data analysis

ClustalX 1.81 (Thompson et al., 1997) was used to combine, align the accD-psaI and trnL-trnF sequences,
and filter the alignments manually. Irrespective of size, insertions and deletions (InDels) were considered to be
 Min Lu et al.
single mutation events that were coded as substitutions (A or T). DnaSP 6 (Rozas et al., 2017) was used to
calculate the number of segregating sites (S), haplotype diversity (Hd), the number of haplotypes (N), and
nucleotide diversity (p). Neutrality tests were performed on all samples using DnaSP 6 for each population to
assess whether the combined sequences evolved neutrally; also, the mismatch distribution was examined.
Populations that have experienced demographic expansion are unimodal, while those of stable size exhibit a
multimodal mismatch distribution (Harpending, 1994). The expansion time (t) was calculated by the equation
Tau = 2ut = 2mTμt, in which Tau is the expansion variable of mismatch equilibrium calculated by DnaSP 6, mT is
the number of investigated nucleotides and m is the cpDNA mutation rate. Values of 1.01 × 10-9 and 3 × 10-9
substitutions per site/year were used as the lower and upper bounds of the cpDNA mutation rate in this study,
respectively (Graur and Li, 2000).
A molecular variance analysis (AMOVA) with 1 000 permutations and a Mantel test (Mantel, 1967) with
9 999 permutations were conducted using GenALEx 6.502 (Peakall and Smouse, 2012). The Mantel test was
implemented to identify significant association between Nei’s genetic distance (Nei, 1972) and geographic
distance. Using the program Permut 2.0 (Pons and Petit, 1996), the population differentiation was measured by
Gst, Nst with respect to the haplotypes, and compared by a test with 1 000 permutations. Nst and Gst are based on
ordered and unordered alleles, respectively, and a significantly higher level of Nst than Gst reveals the presence of
phylogeographic structure (Pons and Petit, 1996). The gene flow rate (Nem) among populations was calculated by
Gst = 1/(1 + 2Nem) (Wright, 1949).
A haplotype network was constructed in TCS 1.21 (Clement et al., 2000) under 95% statistical parsimony
criterion. GEODIS 2.6 (Posada et al., 2000) was used to carry out a nested clade analysis (NCA) based on the
haplotype network. The clade distance (Dc), nested clade distance (Dn), and interior-tip statistic (I-T) were
calculated to measure the geographic spread of each clade and the geographic distance to other clades in the
same higher-level nesting category. The permutation of the geographic sites of samples was used to compute the
probability of extreme values for these statistics. These measures were interpreted by consulting the latest
inference key pertaining to historical events from the GEODIS website (http://darwin.uvigo.es/
software/geodis.html).
Based on the pairwise genetic distances among populations, MEGA 7 (Kumar et al., 2016) was used to
construct a neighbor-joining phylogenetic tree. Moreover, based on the dendrogram of clusters, a
haplotype-preferred sampling strategy combined with stepwise clustering was selected to construct a core
population (Hu et al., 2000). The haplotype frequency was calculated by the formula: number of samples of
specific haplotype/number of total samples. Rare haplotypes, with frequency < 5%, were defined, and
populations with rarer haplotypes were preferred when selecting from each subgroup at the lowest level of
sorting. If the number of rare haplotypes was equal, the minimum value was selected.

3. Results

3.1. Genetic diversity of Rosa roxburghii by chloroplast DNA

The two cpDNAs surveyed across 255 R. roxburghii specimens (belonging to 29 populations) were aligned.
The total alignment length was 1 143 bp, and 9 nucleotide substitutions and 5 InDels revealed 18 haplotypes
(H1–H18). Ranging from 0.476 (population LC) to 0.933 (population MX) (Table 2), a high level of haplotype
diversity (Hd = 0.829) was detected, while both the LZ and SQ populations had only 1 haplotype (H8) each. Thus,
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Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications
the nucleotide diversity differed dramatically among these populations. The nucleotide diversity was maximal in
FQ (2.9 × 10−3) and minimal in LC (0.4 × 10−3) (excluding the monomorphic populations LZ and SQ) (Table 2).
Haplotype H8 was the most common, and found in 24 populations, while H16 was discovered in 15 populations
(Table 1).
Table 2 cpDNA haplotype distribution and related diversity statistics in the 29 populations

Popula- Haplotype
Sample π/
tion S N Hd
size H-1 H-2 H-3 H-4 H-5 H-6 H-7 H-8 H-9 H-10 H-11 H-12 H-13 H-14 H-15 H-16 H-17 H-18 (× 10-3)
code
AL 13 3 2 5 3 3 4 0.782 1.0
BJ 5 1 3 1 2 3 0.700 0.7
DF 5 1 1 2 1 4 4 0.900 1.4
DS 5 2 2 1 2 3 0.800 0.9
FQ 7 2 1 2 1 1 7 5 0.905 2.9
HS 10 1 2 1 1 5 3 5 0.756 0.9
HX 18 2 3 1 1 4 1 6 5 7 0.837 1.2
LD 7 2 1 2 2 3 4 0.857 1.2
LZ 5 5 0 1 0 0
PB 20 2 1 7 1 9 4 5 0.695 0.8
PX 14 2 1 1 8 2 3 5 0.671 0.9
QL 7 2 1 1 2 1 3 5 0.905 1.4
QX 13 1 1 3 8 3 4 0.603 0.7
SC 4 2 2 1 2 0.667 0.6
SQ 6 6 0 1 0 0
SY 10 1 1 3 1 4 4 5 0.800 1.1
XY 18 2 1 6 7 2 4 5 0.752 0.9
ZJ 11 3 2 6 2 3 0.655 0.9
ZY 12 1 2 2 3 1 3 4 6 0.879 1.2
ZT 5 3 2 1 2 0.600 0.5
JL 5 1 1 2 1 3 4 0.900 1.4
MN 11 3 4 4 3 4 0.727 1.8
xy 8 4 3 1 2 3 0.679 0.7
CQ 6 3 3 1 2 0.600 0.5
GZ 5 1 1 3 2 3 0.700 0.7
HY 6 1 1 3 1 3 4 0.800 1.2
LC 7 5 2 1 2 0.476 0.4
NZ 6 1 3 2 2 2 0.600 1.1
MX 6 1 1 1 2 1 5 5 0.933 1.8
Total 255 8 13 5 5 5 15 21 82 17 4 4 3 4 6 4 57 1 1 9 18 0.829 1.3

3.2. Genetic differentiation of Rosa roxburghii

The genetic variation existed within populations according to AMOVA, representing 86.4% of the total
variation (Table 3). This indicated high genetic consistency across populations (Table S1). The genetic identity
between MX and LZ, MX and SY was 0.97. Owing to the abundant 0 values for genetic identity between MN and
other populations and between GZ and some other populations (Table S1), Nei’s genetic distances could not be
calculated, and hence, MN and GZ were excluded from the Nei’s genetic distance matrix (Table S2). A significant
correlation between genetic and geographic distances (Fig. 1) was detected by the Mantel test (r = 0.204, P =
0.04, 9 999 permutations). Additionally, a significantly higher Nst than Gst (Nst = 0.257, Gst = 0.136, P < 0.05) was
observed, revealing a phylogeographic structure of R. roxburghii.
 Min Lu et al.
Table 3 Analyses of Molecular Variation (AMOVA) and gene flow in R. roxburghii populations based on cpDNA sequences
Source of variation d.f. Variance components Percentage of variation/% P
Among populations 28 0.039 13.60 ≤ 0.005
Within populations 226 0.248 86.40 ≤ 0.005
The gene flow rate Nem = 3.176

Fig. 1 The correlation between Nei’s genetic distance and geographic distance for 27 populations (excluding the MN and GZ populations)

3.3. Phylogeographic structure of Rosa roxburghii

The mismatch distributions were unimodal (Fig. 2), supporting the theory of population expansion events in
R. roxburghii. The expansion time was calculated using the equation Tau = 2ut = 2mTμt using the lower and
upper bounds of the cpDNA mutation rate. The overall population expansion of R. roxburghii might have
occurred between 647 073 and 217 848 years ago.

Fig. 2 Mismatch distribution of the 29 Rosa roxburghii populations

The solid line represents the expected distribution under a model of sudden demographic expansion, while the dashed line
represents the observed distribution.

The cpDNA haplotype network constructed in TCS comprised of clades 1-1 to 1-9 (nine one-step clades),
clades 2-1 to 2-4 (four two-step clades), and clades 3-1 and 3-2 (two three-step clades) according to NCA (Fig. 3).
Clade 3-1 had 8 haplotypes and a relatively restricted geographic distribution, while clade 3-2 contained 10

6
Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications
haplotypes, thereby encompassing most of the populations. Clades 1-1 and 1-7 were not included in the analyses
of geographic correlations because NCA could be performed when different polymorphism (haplotypes) was
detected in each clade. Then, using significant Dc, Dn, or I–T values, the latest inference key was applied to
deduce the demographic events in the R. roxburghii populations. Intriguingly, no statistically significant distances
within clades were noted in 1-2, 1-4, 1-5, 1-6, 2-1, 2-4, and 3-1 (Table S3); hence, the null hypothesis of no
geographical association of haplotypes could not be rejected, and the analysis moved on to another clade at the
same or higher level. The final results showed that the primary processes underlying these analyses included
restricted gene flow with isolation by distance (within clades 1-8, 2-3 and overall), contiguous expansion (within
clades 1-3 and 3-2), and past fragmentation and/or long-distance colonization (within clades 1-9 and 2-2) (Table
4).

Fig. 3 Haplotype network of chloroplast DNA of Rosa roxburghii and nested clade analysis of haplotypes H1–H18
The 18 haplotypes Hl–H18 are represented by different colors; small, open circles represent missing haplotypes; lines joining these two haplotypes represent
mutant events.

Table 4 Nested clade analysis and phylogeographic inferences made from the haplotype network of Rosa roxburghii
Clade χ2 statistic P Clade Key Results
1-3 26.00 0.003 1-19-20-2-11-12-No Contiguous range expansion
1-8 9.88 0.220 1-2-3-4-No Restricted gene flow with isolation by distance
1-9 64.17 0.006 1-2-3-5-15-No Past fragmentation and/or long distance colonization
2-2 90.00 0.000 1-19-20-2-3-5-15-No Past fragmentation and/or long distance colonization
2-3 21.09 0.088 1-2-3-4-No Restricted gene flow with isolation by distance
3-2 67.75 0.000 1-2-11-12-No Contiguous range expansion
Total 57.13 0.002 1-2-3-4-No Restricted gene flow with isolation by distance
Note: Numbers in the column Clade Key indicated the choices made in the latest inference key.
 Min Lu et al.
3.4. Construction of a core population of Rosa roxburghii
As described above, high genetic consistency was observed among populations, while the genetic variation
in R. roxburghii existed mainly within the populations. Thus, it was considered reasonable that an independent
population could be considered as a minimum element for conservation priority. Subsequently, a core population
could be constructed logically. As there were many 0 values for the genetic identity between MN and the other
populations and between GZ and some other populations (Table S1), Nei’s genetic distances could not be
calculated. This phenomenon indicated that the genetic distance between MN and GZ was maximal, and hence,
these two populations should be prioritized while selecting the core population. Additionally, a neighbor-joining
phylogenetic tree was constructed using MEGA 7 based on the pairwise genetic distance among populations,
except MN and GZ (Fig. S1), and subsequently, the haplotype-preferred sampling strategy was adopted. Among
the 18 haplotypes, 12 were defined as rare haplotypes with < 5% frequency: H1, H3, H4, H5, H10, H11, H12, H13,
H14, H15, H17, and H18 (Table S4). Populations with rare haplotypes were preferred when selecting from each
subgroup at the lowest level of sorting. If the number of rare haplotypes was equal, the minimum value was
selected (Table S4). When the number of core populations was decreased to 12, all 18 haplotypes were included;
thus, 12 was used as the core population number (Fig. S2). The core populations were MN, GZ, FQ, NZ, DF, DS, xy,
AL, MX, LC, PB, and XY (Fig. 4), successively, among which GZ, FQ, DF, DS, xy, AL, LC, PB, and XY were located in
the Yunnan-Guizhou Plateau region, NZ and MX were in the Qingling-Bashan mountains region, and MN was in
the Hengduan mountains region.

Fig. 4 Consensus neighbor-joining tree of Rosa roxburghii based on the genetic distances of the 10 core populations
(excluding the MN and GZ population)

4. Discussion

4.1. Genetic diversity of Rosa roxburghii populations

In this study, the two cpDNA intergenic fragments in wild R. roxburghii populations displayed a high degree
of diversity (Hd = 0.829), which was higher than that in other Rosaceae species, such as R. omeiensis (Gao et al.,
2015) in the Qinghai-Xizang Plateau region (Hd = 0.799) and Qilian-Qin mountains region (Hd = 0.598), Pyrus
betulaefolia (Hd = 0.807), as reported by Zong et al. (2014), and P. pashia (Hd = 0.718) and P. calleryana (Hd =
0.719) as reported by Liu et al. (2012, 2013). Moreover, the nucleotide diversity of R. roxburghii (π = 1.3 × 10−3)
was nearly identical to that of P. betulaefolia (π = 1.31 × 10−3) and was higher than that of P. pashia (π = 0.85 ×
10-3), P. calleryana (π = 1.05 × 10−3) (Liu et al., 2012, 2013), and R. omeiensis (Gao et al., 2015) in the

8
Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications
Qinghai-Xizang plateau region (π = 0.78 × 10−3) and Qilian-Qin mountains region (π = 0.47 × 10−3). This might be
due to the difference in the species differences or extensive sampling coverage.
Among populations, the genetic diversity varied significantly. Populations MX and FQ displayed high levels of
haplotype and nucleotide diversity, while LZ and SQ showed no variation. R. roxburghii harbored significant
genetic variation within populations (86.4%) as compared to P. calleryana (77.0%; Liu et al., 2012), P. pashia
(59.2%; Liu et al., 2013), P. betulaefolia (27.7%; Zong et al., 2014), and R. omeiensis (Gao et al., 2015) in the
Qilian-Qin mountains region (26.3%) and Qinghai-Xizang plateau region (17.0%). Additionally, most of the cp
haplotypes were shared by more than two populations, which also indicated weak population genetic
differentiation (Gst = 0.136). Petit et al. (2003) demonstrated that seed dispersal mechanisms played a critical role
in shaping the genetic structure of maternally inherited cpDNA. An efficient seed dispersal mechanism among
populations may account for the low population differentiation. The materials in this experiment were mainly
collected from the side of the road where there was frequent human activity, followed by mountain slopes and
brook sides, where few people tread. People, birds, animals, and flowing water may be contributors to the high
gene flow rate (Nem = 3.176) among these populations. Additionally, the wide distribution of the main haplotypes
H8 and H16 in the Yunnan-Guizhou plateau may have been caused by the high flow rate of the genes that
modulates the genetic structure of R. roxburghii and weakens the geographical restrictions, leading to weak
correlations between the geographical distribution and neighbor-joining clustering results.

4.2. Geographic distribution of cpDNA polymorphisms in Rosa roxburghii populations

The most common haplotype H8 was the center of the haplotype network and dispersed over a large area,
which implied its ancient origin (Posada and Crandall, 2001). Interestingly, in the LZ and SQ populations, H8 was
the only haplotype, and the absence of cpDNA variation in these two populations indicated that they were
derived from adjacent populations after a genetic drift or a founder event through colonization rather than being
relicts (Zong et al., 2014). A comprehensive analysis of genetic and geographic distances suggested that the LZ
population was probably colonized from PB, and SQ from ZY.
Populations in refugia usually display exclusive haplotypes and more genetic diversity than the migratory
populations (Heuertz et al., 2004). Thus, the populations FQ and MX played major roles in the genetic
composition of adjacent populations and seemed to be relicts of Quaternary glaciation. MX, which was located
north of Qinling and south of the Bashan mountains, was the northernmost population. The FQ population was
located on the Yunnan-Guizhou plateau, a crucial biodiversity hotspot, the uplifting of which has generated
extremely complex topography and climates and likely created opportunities for divergence and speciation in
isolated populations through geographical barriers and habitat heterogeneity (Chen et al., 2017). Both these
populations were suggested to have had refugia in Southwest China (Qiu et al., 2011). The versatile biomes in
these areas may have provided protective environments that preserved the genetic diversity of R. roxburghii. The
MN population was localized in the eastern margin of the Qinghai Tibet plateau, which belongs to the Hengduan
mountains, and was also suggested to have had refugia in Southwest China (Qiu et al., 2011). However, in the
current study, the MN population contained H9, H11, and H13, among which H11 and H13 were rare haplotypes.
Additionally, H8 was suggested to be the ancient original haplotype but was not found in MN, implying that the
MN population in the Hengduan mountains could not be a refugia, rather colonized from the Yunnan-Guizhou
plateau or Qinling and Bashan mountains. In the MN population, the genetic drift was caused by the founder
effect. The haplotype H9 was suggested to be fixed; thus, H11 and H13 may have been generated later owing to
 Min Lu et al.
the specific environment requirement, which consists of a series of grand north-south trending ridges alternating
with deep valleys.

4.3. Demographic history of Rosa roxburghii

The overall population expansion of R. roxburghii was estimated to have occurred between 647 073 and
217 848 years ago in the middle Pleistocene. As Liu et al. (2013) reported, a rapid range expansion could be
expected by a star-like network of cp haplotypes. This phenomenon was supported by the mismatch distribution
analysis. Unimodal distributions represented that populations have experienced demographic expansion. The
overall expansion time of R. roxburghii was earlier than that of the R. sericea complex (Gao et al., 2015) and
Pyrus (Liu et al., 2013; Zong et al., 2014). However, the estimated times in this study should be treated with
caution because no fossils have been used to calibrate the estimates. A lower-level clade would experience
demographic events as new as or newer than those of the higher-level clades nested within it in NCA (Watanabe
et al., 2006). Thus, within the R. roxburghii populations in Southwest China, gene flow restriction by distance and
contiguous expansion has occurred repeatedly over a prolonged period.

4.4. Conservation priority of Rosa roxburghii populations

In recent decades, in some areas of China, populations of wild R. roxburghii have been destroyed by climate
change and continuous urbanization, and the genetic diversity of wild R. roxburghii relatives has been eroded
gradually (Zhang et al., 2017). Thus, it is urgent to conserve the germplasms of this species. The genetic diversity
of R. roxburghii was partitioned predominantly within populations, consistent with the previous results from
EST-SSR markers (Zhang et al., 2017). Compared to other species with variation among populations, such as Pyrus
betulaefolia (Zong et al., 2014) and R. omeiensis (Gao et al., 2015), the conservation strategy for R. roxburghii
could be mostly concentrated within populations. The strategy of constructing a core collection was used as a
reference (Hu et al., 2000), leading to the concept of a core population. The results showed that conservation
priority should be given to the core populations, such as FQ, PX, JL, LC, LZ, and DS in the Yunnan-Guizhou plateau,
NZ and MX in the Qingling-Bashan mountains, and MN in the Hengduan mountains. The high genetic diversity
within these populations is generated by specific environments, and hence, in situ conservation and
management strategy, such as building natural preservation zones, should be employed.

5. Conclusions

Wild-type germplasms of Rosa roxburghii Tratt. are decreasing. Two cp intergenic spacers (trnL-trnF and
accD-psaI) were investigated in 255 specimens from 29 R. roxburghii populations to assess the genetic diversity
and phylogeography for efficient conservation. High levels of haplotype and nucleotide diversities were detected
in these populations. The primary processes shaping the genetic patterns of R. roxburghii populations included
restricted gene flow with isolation by distance, contiguous expansion, past fragmentation, and/or long-distance
colonization. Conservation priority should be given to the core populations GZ, FQ, DF, DS, xy, AL, LC, PB, and XY
in the Yunnan-Guizhou Plateau, NZ and MX in the Qingling-Bashan Mountains, and MN in the Hengduan
Mountains.

Data archiving statement

The accD-psaI and trnL-trnF sequences have been submitted to GenBank and the accession numbers were
10
Chloroplast DNA-based Genetic Variation of Rosa roxburghii in Southwest China: Phylogeography and Conservation Implications
from MG201858–MG201866.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Grant No.
31660558), the Department of Science and Technology of Guizhou Province (Grant Nos. 2020Y113, 20164016
and 20175788) and and the Construction Program of Biology First-class Discipline in Guizhou (Grant No.
GNYL2017009).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:
10.1016/j.hpj.2020.

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