1 Introduction

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, systemat-ic 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 of A. 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 Sampling

A 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). Themean temperature) (Bailey, 1979). For each population, 17 to 30 individuals (spaced 30 m apart) were sampled (Table 3). Young and healthy leaves were randomly sampled from individuals and immediately stored with silica gel in ziplock plastic bags until DNA extractions were carried out. We chose A.lavandulifolia as the out-group.

Table 1 The average monthly rainfall in 10 years of 10 populations of Artermisia halodendron in Horqin Sandy Land

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2.2 Molecular methods

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.

Table 2 The monthly mean temperature in 10 years of 10 populations for Artermisia halodendron in Horqin Sandy Land

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2.3 Data analyses

这时，一队民工小跑着赶了上来，有扛子弹的、扛手榴弹的、扛炮弹的，也有扛担架的。民工中间夹有炊事班老王，正一头开水一头馒头挑了过来。

最后，从理论模型到理论。苏佩斯主张理论是理论模型的集合，理论中所探讨的对象实质上就是在模型中所处的位置。一个理论之下拥有多种理论模型，模型与模型之间因为同构而共同组成这一理论，从而在理论内部形成一种共享结构的关系，这种共享结构的关系就是对于理论的解释，从而形成了理论中的定理。

Notes: Sequences are numbered from the 5' to the 3' end in each region; – indicates lack.

由此推论，对任何一个违法乱纪者而言，查处真的是一种爱护。从这个角度讲，被查处者不论受到的是处分或者处罚，都要有这种清醒认识，做到口无怨言、心存感激。这样，才有助于痛改前非，重新做人。

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世界石油史的事例雄辩地说明，某个油田、油区，某种类型的油气生命力的衰退并不意味着整个石油工业的衰竭。社会需求推动着找油新思路的探索，推动着科技水平的创新、提高，于是石油工业呈现着波浪式上升的发展方式，一次次出现“山穷水尽疑无路”的困惑，一次次闯出“花明柳暗又一村”的美景。立足于实践形成于科学研究的石油地质新理论、找油新思路，勘探和开采油气的新技术就是实现其发展的关键。而这些关键问题的解决和它们产生实效却又以适合的体制为前提。显然，深化改革带来的合适体制、政策是发展的动力，反之则产生强大的阻力。

3 Results

3.1 Haplotype distribution and genetic diversity

Sequence 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).

DNA 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.

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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

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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).

Figure 1 Geographic distribution of the seven haplotypes found on 243 individuals for trnL–F observed in Artemisia 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

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 variationare 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.

3.2 Phylogenetic analyses and genetic structure

Maximum parsimony analysis resulted in a single tree (length=14 and consistency index =0.5834). Two clades with low bootstrap support were identified: one consisting of the Hap C, G, F, E, and D, with four haplotypes (Hap C, G, F, and Hap D) distributed in the low-hydrothermal-level region and one haplotype(Hap E) in the high-hydrothermal-level region; and the other consisting of the rest of Hap A and Hap B,both occurring in the high-hydrothermal-level region(Figure 2).

Figure 2 Phylogenetic relationships of the identified haplotypes of Artemisia halodendron in Horqin Sandy Land

Note: d.f., degrees of freedom.

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.

Table 5 Nucleotide variation and haplotype diversity at trnL-F in 10 populations of Artemisia halodendron from the low-hydrothermal-level region (populations 1–6), high-hydrothermal-level region (populations 7–10),and entire species' range (all populations) in the Horqin Sandy Land

Notes: n, sample size; S number of segregating sites; π, nucleotide diversity (Nei and Li, 1979; Nei, 1987); Nh, number of haplotypes; Hd, Nei's haplotype diversity; k, mean pairwise nucleotide differences.

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Table 6 Results of analyses of molecular variance (AMOVAs) of haplotype frequencies for populations and regional populations of Artemisia halodendron in Horqin Sandy Land

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The analysis of molecular variance (AMOVA)showed that around 17.54% of the variation was attributed to the low-/high-hydrothermal-level regions differentiation, and between- populations variation accounted for just over one-half (55%) of the total variation, indicating very strong differentiation and little gene flow between regions and populations (Table 6).Within the low- and high-hydrothermal-level regions,between-populations differentiation was respectively 81.52% and 93.77% (Table 6). Overall, these results strongly indicate that haplotypes are geographically structured across the species' distribution range. FST values were 0.728, 0.815, and 0.938 at the species level, low-hydrothermal-level region, and high-hydrothermal-level region, respectively.

4 Discussion and conclusion

In this study, we examined nucleotide variation at two nuclear loci of A. halodendron sampled from the Horqin Sandy Land. We confirmed the division of the native range into two areas, using hydrothermal synthesis index data (Table 3). The analysis of cpDNA variation identified seven haplotypes (Table 4 and Figure 2). 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 2).

Many studies have demonstrated that endemic species tend to possess high levels of genetic diversity (Wang et al., 2010; Ge et al., 2011). Compared with similar studies (Wang et al., 2011), the level of haplotype diversity (Hd=0.706) and the nucleotide diversity (π=0.0013) within the whole populations of A. halodendron were somewhat lower than that of the endemic plants in northern of China. Such a low Hd value and π value indicated that this species was very adapted to the sandy land environment, and this character might have made it become the dominant and constructive species in Horqin Sandy Land.At the same time, the populations from the low-hydrothermal-level region exhibited lower haplotype diversity and more limited nucleotide diversity, and were a subset of that observed in the populations from the the high-hydrothermal-level region. These results lend support to the scenario described by Huang et al.(2011) using ISSR markers and Huang et al. (2013)using psbA-trnH. This fact might be due to environmental differences (Li et al., 2009; Bao et al., 2010;Liu et al., 2010; Li and Wu, 2011). The low-hydrothermal-level region is a region of severe desertification (Zhao et al., 2000; Bao et al., 2010). In the process of ecological restoration, species increasing adapt to the local environment, which, in turn, reduces the genetic diversity (Wang et al., 2009; Huang et al.,2011).

Phylogenetic relationships analysis of the cpDNA sequences collected from A. halodendron in the different hydrothermal-level regions in Horqin Sandy Land did not entirely cluster according to populations or hydrothermal-level regions. This result is similar to that of some other studies (Chen et al., 2009; Hu et al., 2010; Zhou et al., 2010; Zhang et al., 2015). The MP tree indicated that the seven haplotypes formed two clades: the haplotypes in clade I all came from the high-hydrothermal-level region (63% bootstrap support); the haplotypes (except Hap E) in clade II came from the low-hydrothermal-level region (58%bootstrap support). This cluster process was closely related to the hydrothermal gradients in Horqin Sandy Land. The results of phylogenetic relationships analysis show that hydrothermal conditions change determines the increase of the haplotypes distribution difference.

The spatial analyses of genetic variation in A.halodendron in the different hydrothermal-level regions in Horqin Sandy Land indicate that the between-populations genetic differentiations are high within the low-, the high-hydrothermal-level region,and the total distribution range (Table 6). Our data revealed significant genetic differences among populations in different hydrothermal level regions. The increase in population genetic differentiation might be related to strong human disturbance, high rates of habitat fragmentation, and decreasing population size in Horqin Sandy Land (Zhao et al., 2000; Wang et al.,2010). It is generally accepted that genetic differentiation would have increased among the native populations due to the changed environment (Wang et al.,2009).

（1）四年正月初一日，鑽玉華院撰制玉冊訖，宣詔三官四聖，於上元節日，並赴七寶瓊臺。（《太上說玄天大聖真武本傳神呪妙經註》卷一，《中华道藏》30/530）

In conclusion, there are significant genetic differences between A. halodendron populations from different hydrothermal-level regions. This information about genetic variation has important implications for restoring and managing the degraded ecosystems in arid and semi-arid areas. It is particularly important to understand the genetic variation of extant populations over the entire range of the species' distribution. Additional research is needed to determine levels of genetic variation in A. halodendron throughout its entire distributional range.

The authors thank all the members of Naiman Desertification Research Station, China Academy of Sciences (CAS), for their help in the field work. We acknowledge the China Meteorological Administration (Beijing, China) for help on the meteorological data information support. This study was financially supported by research projects 2016YFC0500907, 2017FY100205, 41201561,Y551821001, and 145RJYA269.

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