2018, Vol. 10
2. Key Laboratory of Stress Physiology and Ecology in Cold and Arid Regions, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, Gansu 730000, China;
3. Urat Desert-grassland Research Station, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou, Gansu 730000, China
Artemisia halodendron Turcz. ex Bess. (Asteraceae, Anthemideae, Subgen. Dracunculus) is one of the most common semishrub species in the Horqin Sandy Land in Northeast China. It is important for vegetation rehabilitation in Horqin Sandy Land because of its high ecological value, including that (1) it is the key species of the plant communities and landscapes studies in Horqin Sandy Land (Li, 1991); (2) it plays a key role in the vegetation-restoration process due to its high drought tolerance, anti-wind erosion properties, and sand-burial resistance (Dong et al., 2000 ; Li et al., 2002 ; Zhao et al., 2006 ). Previous studies on A. halodendron focused on aspects of population-distribution patterns (Chao et al., 1999 ; Cao et al., 2008 ), biomass allocation (Li et al., 2005 ), breeding distribution (Li et al., 2005 ), morphological characteristics and physiological adaptations (Zhou et al., 1999 ), root longevity (Huang et al., 2009 ), genetic diversity (Huang et al., 2011 , 2014), and establishment (Li et al., 2002 ) in Horqin Sandy Land. However, systematic comparison of genetic relationships of A. halodendron among populations from different hydrothermal regions has not yet been reported.
Horqin Sandy Land is located in the agropastoral transitional zone between the Inner Mongolian Plateau and the Northeast Plains (42°41′N–45°45′N, 118°35′E–123°30′E) and is one of the four largest sandy areas in northern China; it covers an area of approximately 139,300 km2, of which up to 71,884 km2 is desertified sandy land (Wang, 2003; Zhao et al., 2003 ). Landscape in this area is characterized by sand dunes that alternate with gently undulating lowland areas (Li et al., 2005 ). This area belongs to the continental semi-arid monsoon climate and is in the temperate zone, with a mean annual temperature (AMT) of 3–7 °C and mean annual rainfall (AP) of 350–500 mm (Zhao et al., 2003 ). Over recent decades, this region has undergone severe desertification (Li et al., 2000 , 2004) and has displayed the northern-moving phenomenon of the interlocked agropasturing area of North China in the most recent hundred years (Zhao et al., 2000 , 2002).
In the present study, we used chloroplast DNA (cpDNA) trnL–F to examine the genetic diversity ofA. halodendron. We specifically aimed to address the following questions: (1) What is the level of nucleotide diversity in A. halodendron from different hydrothermal regions? (2) How are the identified haplotypes distributed within and among populations? In particular, is there subdivision in the different hydrothermal-level populations (according to hydrothermal synthesis index)? We attempted to interpret the results to provide baseline genetic information pertinent to the restoration and management of degraded ecosystems in arid and semi-arid areas.
2 Materials and methods 2.1 SamplingA total of 243 individuals were sampled from 10 natural A. halodendron populations (Tables 1 and 2). Populations 1–6 were in the low-hydrothermal-synthesis index region (average 24.97), while populations 7–10 were in the high-hydrothermal-synthesis index region (average 31.98) (Tables 1 and 2). The equation of hydrothermal synthesis index was
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Table 1 The average monthly rainfall in 10 years of 10 populations of Artermisia halodendron in Horqin Sandy Land |
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Table 2 The monthly mean temperature in 10 years of 10 populations for Artermisia halodendron in Horqin Sandy Land |
Total genomic DNA was extracted using AxyPrep Genomic DNA Mini Kits (Axygen Inc., Beijing, China) following the manufacturer's instructions. DNA quality was checked on a 1.0% agarose gel. Several pairs of cpDNA primers designed by Hamilton (1999), Taberlet et al. (1991) , and Sang et al. (1997) were used in the initial screening. Two pairs of primers, trnL (5'-CGGAATTGGTAGACGCTACG-3') and trnF (5'-ATTTGAACTGGTGACACGAG-3') (Sang et al., 1997 ), identified sequence variations in the sampled individuals and therefore were used for all remaining individuals. Polymerase chain reaction (PCR) was performed in a 25-μL reaction volume, containing 40 ng of genomic DNA, 1.0 U of Taq polymerase (Axygen Inc., Beijing, China), 3 mmol/L MgCl2, 500 μmol/L each dNTP, 20 mmol/L Tris-HCl (pH 8.3), 100 mmol/L KCl, and 0.3 μmol/L primer. The amplification condition was an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, and a final 5-min extension step at 72 °C. The PCR products were determined by 1.0% agarose gel electrophoresis. The amplification products were purified using an AxyPrep PCR Purification Kit, following the manufacturer's protocol (Axygen Inc., Beijing, China). Purified DNA was sequenced by the MEIJI sequencing company in Shanghai, China, applying the PCR-primers as sequencing primers.
2.3 Data analysesDNA sequences were aligned using the CLUSTAL X program (Thompson et al., 1997 ), with subsequent manual adjustments in MEGA4 (Tamura et al., 2007 ). A matrix of combined sequences was constructed for the 243 individuals that we examined, and different cpDNA sequences were identified as haplotypes.
Basic population genetic parameters were estimated for three groups of populations: the low-hydrothermal-level region (populations 1–6); the high-hydrothermal-level region (populations 7–10); and finally, all populations. All parameters were calculated with DNASP 5.10.01 (Librado and Rozas, 2009), including the number of segregating sites (S), the number of haplotypes (Nh), the haplotype diversity (Hd), the average number of nucleotide differences per site between two sequences in a sample, π (Nei and Li, 1979; Nei, 1987), and the average number of pairwise nucleotide differences (k).
Phylogenetic analyses of cpDNA haplotypes were performed with maximum parsimony (MP), using PAUP version 4.0 (Swofford, 2002). Heuristic search was implemented with 100 random additional sequence replicates, tree-bisection-reconnection (TBR) branch swapping, MULPARS option, and ACCTRAN optimization. To evaluate the relative robustness of the clades found in the most parsimonious tree, bootstrap analysis was conducted using 1,000 replicates with a simple taxon addition. Genetic differentiation among populations at the three different sampling levels was estimated by pairwise FST values (Wright, 1951). AMOVA was performed to analyze the source of variation among populations, using Arlequin 3.0 (Excoffier et al., 2005 ) with 1,000 replicates of bootstrap.
3 Results 3.1 Haplotype distribution and genetic diversitySequence data were obtained for one loci from on average 101, 142, and 243 individuals from the low-hydrothermal-level region, the high-hydrothermal-level region, and the entire species' range, respectively. The length of the aligned trnL–trnF DNA sequences (including trnL and the trnF spacer region) ranged between 849 and 863 bp with two insertions. The analysis of cpDNA variation identified seven haplotypes (HapA-HapG) (Table 4). Haplotype C was the most abundant, occurring in three populations, followed by haplotypes A, B, D, and G, which occurred in two populations; and the remaining haplotypes were found in only a single population (Table 3 and Figure 1). We confirmed the division of the native range into two areas using hydrothermal synthesis index data (Table 3). Six populations in the low-hydrothermal-level region of the species were dominated by four different haplotypes. The other four populations in the high-hydrothermal-level region of the species were dominated by three different haplotypes (Table 3 and Figure 1).
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Table 3 Origin of materials and number of samples for 10 populations of Artemisia halodendron from the Horqin Sandy Land |
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Figure 1 Geographic distribution of the seven haplotypes found on 243 individuals for trnL–F observed inArtemisia halodendron of the Horqin Sandy Land. Pie charts indicate the frequency of haplotypes within each population, and unique alleles are indicated by different colours. NDRS indicates Naiman Desertification Research Station, China Academy of Sciences. Base map data produced in 2000 |
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Table 4 Variable sites of the aligned sequences of trnL-F in seven haplotypes of Artemisia halodendron in the Horqin Sandy Land |
We identified a total of two, three, and three segregating sites for the low-hydrothermal-level region, the high-hydrothermal-level region, and all populations, respectively. Summary statistics of sequence variation are given in Table 5. Overall haplotype diversity (Hd) and nucleotide sequence (π) diversity for A. halodendron were 0.706±0.001 and 0.0013±0.0001, respectively. At the regional level, haplotype diversity and nucleotide diversity between two regions varied between 0.318 (low-hydrothermal-level region) and 0.671 (high-hydrothermal-level region), and between 0.0006 (low-hydrothermal-level region) and 0.0015 (high-hydrothermal-level region), respectively. The population from the high-hydrothermal-level region had higher haplotype diversity and nucleotide diversity than the population from the low-hydrothermal-level region.
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