High genetic diversity and strong genetic structure of Strongyllodes variegatus populations in oilseed rape production areas of China

Background Strongyllodes variegatus (Fairmaire) is a major insect pest of oilseed rape in China. Despite its economic importance, the contribution of its population genetics in the development of any suitable protection control strategy for the management of oilseed rape crops is poorly studied. It is a much urgent need to prevent its spread to the rest of the world. Results Using the sequences of mitochondrial DNA cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) as genetic markers, we analyzed the population genetic diversity and structure of 437 individuals collected from 15 S. variegatus populations located in different oilseed rape production areas in China. In addition, we estimated the demographic history using neutrality test and mismatch distribution analysis. The high level of genetic diversity was detected among the COI and Cytb sequences of S. variegatus. The population structure analyses strongly suggested three distinct genetic and geographical regions in China with limited gene flow. The Mantel test showed that the genetic distance was greatly influenced by the geographical distance. The demographic analyses showed that S. variegatus had experienced population fluctuation during the Pleistocene Epoch, which was likely to be related to the climatic changes. Conclusion Overall, these results demonstrate that the strong genetic structure of S. variegatus populations in China, which is attributed by the isolation through the geographical distance among populations, their weak flight capacity and subsequent adaptation to the regional ecological conditions.


Background
The brown beetle, Strongyllodes variegates (Fairmaire) (Coleoptera: Nitidulidae), feeds on brassicaceous plant species [1,2], which often cooccurs with the pollen beetle, Meligethes aeneus [3]. The S. variegatus adults chew up owers, buds and leaves, and create crescent-shaped bites where the mature females lay eggs. After hatching, the larvae feed on mesophyll resulting in irregular bubble-shaped wounds before pupation in soil. The wounded leaves become necrotic and abscise prematurely [4,5]. Recently, the leaf damage of oilseed rape crops by this beetle has become more and more serious, so that it has become a major insect pest of oilseed rape crops. In spring 2013, S. variegatus population broke out in Hanshan, Anhui province, destructing 97% of oilseed rape leaves [6].
S. variegatus displays speci c ecological characteristics to temperature and photoperiod of geographical regions. In spring oilseed rape areas, it reproduces once or twice a year [2]. However, only two generations occur in winter oilseed rape areas [6]. In addition, S. variegates has a high reproductive ability [4] and can y 30~40 m in 2 min [2]. In Anhui, the overwintering adults begin to appear in March. When the temperature is more than 30℃, the adults stay in soil in summer, and some of them are mixed into the harvested rapeseed. They appear on cruciferous vegetables in September, then move to rape elds and cause damages in October. When the temperature is low in November, they move back to soil and overwinter in soil [4,6].
S. variegatus is generally distributed in the middle and lower reaches of the Yangtze River valley. It was rst found on the spring oilseed rape plants in Ningxia, Gansu province, China in 1993 [2], and then on the winter oilseed rape crops in Hanshan, Anhui province in 2008 [4]. For the past few years, we investigated oilseed rape production areas in China and found that this pest has spread to Chongqing municipality and Qinghai, Gansu, Sichuan, Shaanxi, Hubei, Anhui and Jiangsu provinces (unpublished). Currently, it is widely distributed around China but has not yet been found globally in the rest of the world except in China. The phylogeography and population genetics of S. variegates have not been studied. Consequently, it is in urgent demand to conduct the genetics studies and understand the genetic diversity and structure of S. variegatus populations in order to manage and control this pest.
Population genetic studies on crop pests can provide information on the spatial scales at which population structure is established and gene ow occurs. Such information can be used in de ning relevant strategies for pest control [7]. In addition, genetic diversity contains the information on past and present demography that could be useful to characterize the demographic history of crop pests [8]. In recent years, more and more molecular markers have been used to study insect population genetics, demonstrating the importance of phylogeographical approaches [9]. The insect molecular markers mainly include the sequences of nuclear DNA and mitochondrial DNA (mtDNA). Mitochondrial genes have a faster evolution rate than nuclear genes, and are more informative for studying phylogenetic evolution, especially the degree of inter-and intra-speci c population differentiation and the level of gene ow [9,10]. Thus, the fragments of the mtDNA cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) have been widely used among insect molecular markers to study population genetic variation and differentiation of insects, for example, Dendrolimus kikuchii, Chilo suppressalis and Agriosphodrus dohrni [11][12][13][14][15]. The COI and Cytb genes were also used to track the colonization routes of Halyomorpha halys and to identify the places where the insect has originated [16][17][18].
In this study, we use COI and Cytb genes to elucidate for the rst time the genetic diversity and structure of 15 S. variegates populations occurring on the oilseed rape production areas in China. We hypothesize that the populations would have a high level of genetic diversity and a clear genetic structure. At the same time, the e cient molecular data collected are used to assess if historical geographic events and associated ecological adaptations had played an important part in shaping the observed genetic and geographic patterns of this pest in China.

Results
Genetic variation of S. variegatus populations Seventy haplotypes of the COI gene and 67 haplotypes of the Cytb gene were identi ed from the 15 populations. The S. variegates COI fragment (652bp) and Cytb fragment (421bp) have 45 (6.9%) and 40 (9.5%) variable sites with 28 and 23 parsimony informative sites, respectively ( Table 1). The base composition of the two genes is adenine (A) and thymine (T) (67.5% and 73.3%, respectively) biased, which is common for insect mitochondrial genes. The haplotype diversity (Hd) ranges from 0.424 to 0.913 (mean = 0.865) and the nucleotide diversity (π) ranges from 0.00072 to 0.00462 (mean = 0.00427) for the COI gene (Table 1). Similarly, the Hd ranges from 0.464 to 0.833 (mean = 0.834) and π ranges from 0.00119 to 0.00539 (mean = 0.00479) for the Cytb gene (Table 1).
The haplotype distribution and haplotype network analyses (see below) of both COI and Cytb genes revealed that S. variegates populations could be divided into three major geographical distribution regions or haplogroups: the northwestern China For the haplotype network of the COI gene, there was only one common haplotype (H1) in three haplogroups. The haplotype 2 (H2) was only detected and abundant in the CC haplogroup. The haplotype 3 (H3) was only discovered in the CE haplogroup. There were six common haplotypes (H4-H9) between the NW haplogroup and the CC haplogroup. A total of ve missing haplotypes was observed in all populations ( Fig. 2a). Similarly, for the haplotype network of the Cytb gene, there were two common haplotypes (H1, H4) in three haplogroups. The haplotype 2 (H2) was most abundant and only detected in the CC haplogroup. The haplotype 3 (H3) was only discovered in the CE haplogroup. The haplotypes 5-6, 7, 8-9 (H5-H6, H7, H8-H9) were common in the NW and the CC haplogroups, the NW and the CE haplogroup, the CC and the CE haplogroup, respectively. A total of four missing haplotypes was observed in the CC haplogroup (Fig. 2b).

Population genetic differentiation
A strong genetic divergence was observed across populations (F ST = 0.425, P < 0.0001, Table 2). The F CT value among three regions (NW, CC and CE) was highly signi cant (F CT = 0.470, P< 0.0001, Table 2), further demonstrating that S. variegates populations in China is divided into three regions. A signi cant genetic differentiation was observed among populations within the regions (F SC = 0.072, P< 0.0001, Table 2), and within the populations (F ST = 0.508, P< 0.0001, Table 2) based on the combined data of the COI and Cytb genes. The percentages of genetic variation within the populations (60.16% in the populations between NW and CC regions, and 56.00% in the populations between NW and CE regions) were signi cantly higher than those of the comparisons between the regions (33.89% between NW and CC regions, 33.88% between NW and CE regions) ( Table 2). However, the percentage of genetic variations between CC and CE regions (54.95%) was higher than 42.82% within the populations (  Table 4), and the gene ow among the regions was estimated extremely low (Nm < 1, Table 4), suggesting a limited gene ow among the regions. The results are greatly consistent with those obtained by the analysis of molecular variance (AMOVA) described in above sections.
The Mantel test based on the combined data of the COI and Cytb genes revealed a signi cant correlation between the genetic distance (F ST /(1-F ST )) and the geographical distances among all populations (r = 0.500, P < 0.0001, Fig. 3).

Demographic analyses
The Tajima's D values obtained with either single or combined data of the two genes in the NW region were negative, but not signi cant (P > 0.05, Table 1). The Tajima's D and Fu's Fs values in the CC and CE regions were negative and highly signi cant (P < 0.05, Table 1), whereas the CE region showed signi cant sum of squares deviation (SSD) values (P < 0.05, Fig. 4, S2). Thus, for the NW and CE regions, the sudden expansion hypothesis was rejected. However, the distributions of the pairwise differences obtained with single and combined gene data in the CC region were unimodal with non-signi cant SSD and Harpending's raggedness index (Rag) values (Fig. 4, S2), suggesting an expansion event in the CC region. The tau values (τ), a rough estimate of the population expansion, were approximately 3.842 (COI data), 2.016 (Cytb data), and 1.595 (COI + Cytb data) mutation units for the CC region. For the NW and CE regions, τ was 1.344 and 0.766 in the data of the COI gene, 3.693 and 0.875 in the data of the Cytb gene, and 2.628 and 1.875 in the combined data of the COI and Cytb genes (Fig. 4, S2).

Discussion
Using two mitochondrial genes, we investigated the genetic diversity and structure of 437 individuals collected from 15 S. variegates populations from different oilseed rape production areas in China. The results exhibited a high genetic diversity and clear genetic structure of S. variegates populations in China.
Based on the analyses of the mtDNA sequences, haplotype distribution, haplotype networks and AMOVA, three genetically diverse and geographically distinct regions of S. variegates distribution in China are classi ed, namely the northwestern China (NW) region, the central China (CC) region, and the central and eastern China (CE) region. A high proportion of total genetic variance is attributed to the variations within the populations (49.18%) and among the regions (47.01%). This indicates that the largest source of variation might not be due to the geographical barriers among the regions but to the variations among individuals within the populations. It was reported previously that the variations among individuals within the populations had a signi cant effect on the genetic structure of Chilo suppressalis [19]. This contrasts with the studies of Myotis myotis and Plecotus austriacus [20,21], which showed that the geographical barrier was the most important effect. Other factors could also play a signi cant role on the genetic structure. Chen and Dorn analyzed the genetic variation of Cydia pomonella populations in Switzerland and found that host speci city, geographic isolation, intrinsic ight capacity and anthropogenic measures could all shape the population structure [22].
A limited gene ow (Nm < 1) was revealed among the regions by the current study. It is known that once populations have become genetically differentiated, their genetic divergence status can be maintained if they have differentially adapted to regional ecological conditions, since geographic variation in selection can act as a strong barrier to gene ow [23]. Our analysis also suggested a large gene ow among the populations within the CC and CE regions. This may be due to the geographical isolation as the Mantel test results showed that the gene ow between the populations was greatly in uenced by geographical distance. This strong isolation-by-distance relationship in our study may be also due to the limited ight capacity of S. variegates. It was reported that S. variegates can y 30~40 m in 2 min [2]. However, the ight ability of S. variegates is less than tens of kilometres and would not be enough to weaken the isolation-by-distance relationships and to increase the potential for allopatric or parapatric speciation [24,25]. On the other hand, the three regions shared common haplotypes, suggesting small amounts of gene ow among the regions. This may be because some of adults are mixed into the harvested rapeseed over summer [4,6]. Human intervention in the method of alternating seed breeding in a different location of oilseed rape crops could also play an important role in the mixing of populations from distant geographic regions and provide the conditions for the gene ow among the regions [6].
Gene ow in insects has been reported to increase with mobility, which is more pronounced on herbaceous plants, and this feature is strong especially in agricultural pests [26]. The large genetic variation within populations was also found for the pollen beetle, Meligethes aeneus, another oilseed rape pest [9,[27][28][29]. However, no population structure of the pollen beetle could be found in ve provinces of Sweden [28]. M. aeneus is found to have high altitude ights (up to ca 200 m) at speci c points during the year and low-altitude ights at multiple periods [29], which could help to disperse over large distances with the assistance of prevailing wind currents [30], resulting in the high gene ow similar to the diamondback moths, Plutella xylostella [31].
Both the neutrality test and the mismatch distribution analysis indicated a population expansion in the CC region. Furthermore, the phylogeographic patterns of the COI and Cytb haplotype networks are roughly composed of three "star-like" clusters. In China, the management practices against S. variegates have primarily focused on using chemicals. The investigation of the genetic diversity of S. variegates populations can provide a useful guide for controlling this pest. Furthermore, localized populations with similar genetic structure should be considered as a same management unit for most effective control [38]. For isolated populations, various management methods should be used, especially, a variety of chemical pesticides with different properties and modes of action. Additional research will be carried out using other molecular markers, such as nuclear genes, or even faster evolutionary markers, such as microsatellites to obtain better understanding of the population genetic structure and evolutionary history of S. variegates in China, and in the rest of the world if the pest would occur in future.

Conclusions
The current study provides the rst population genetic analysis of S. variegates, a serious pest of oilseed rape crops. The high variability observed using the COI and Cytb molecular markers indicates that the markers are useful for measuring the genetic patterns in S. variegates populations. The distinct distribution of S. variegates populations in China could be divided into three genetic haplogroups and geographical regions with the limited gene ow among them. The distribution of this species in oilseed rape production areas in China is mainly structured by the isolation through geographical distance among the populations and their weak ight capacity. The population expansion signature in the CC region might be related to the climatic changes during the Pleistocene. The phylogenetic information obtained from this study could be used to guide the development of suitable protection control strategies against the insect pests of oilseed rape crops.

Methods
Sampling A total of 437 S. variegates individuals was collected from 15 populations in China (Fig. 1). The sample sizes ranged from 24 to 37 individuals per population except eight individuals for the ESHB population (Table S2). All S. variegates individuals were freshly collected from the elds and immediately stored in absolute ethyl ethanol at -20℃ before molecular analysis. The PCR products were subjected to electrophoresis on a 1.5 % agarose gel (UltraPure Agarose, Invitrogen) containing 10,000× stock GelRed (Biotium) diluted at 1:10,000, visualized on a BioDoc-it imaging system (UVP), puri ed from the gel using ExoSAP-IT (USB, USA), and bidirectionally sequenced (using the above primers) on an ABI 3730XL Automated Sequencer using the BigDye Terminator Cycle Sequencing The population genetic structure was assessed with AMOVA in Arlequin3.5 according to the degree of differentiation between the regions (F CT ), between the populations within the regions (F SC ), and between all populations (F ST ). The pairwise F ST analyses among the populations and the regions were carried out with signi cance tests based on 1,000 permutations using Arlequin3. 5 [46]. In order to test isolation by distance, the matrices of the genetic distance F ST /(1-F ST ) and the geographic distance (ln) between all 15   Ethics approval and consent to participate Not applicable.

Consent for publication
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Competing interests
The authors declare that they have no competing interests.   a Regions as defined in Fig. 1.

Additional Files
Additional file 1: Table S1 Geographical  Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
Haplotype networks estimated from the sequences of (a) the COI gene and (b) the Cytb gene. The circles represent haplotype, the numbers in the circle represent name of haplotype, the small black circles represent missing haplotypes that were not observed, the circle size denotes the total haplotype frequency, while each slice represents the haplotype frequency in different populations, and the lines between linked haplotypes correspond to one mutation. Three haplotype regions are indicated by three different colors: the NW region (red), the CC region (yellow) and the CE region (green).

Figure 3
Scatter plots of genetic divergence against geographical distance. The genetic divergence FST/(1-FST) and the geographic distance (ln) were compared using the Mantel test with 10,000 permutations. There is a strong correlation between the genetic divergence and the geographical distance in the pairwise comparisons of all populations (r = 0.500, P < 0.0001).

Figure 4
Pairwise mismatch distributions based on the combined data of the COI and Cytb genes for three derived regions. The x coordinate represents the number of pairwise differences among sequences, and the y coordinate represents the frequencies of pairwise differences in each region. The signi cance values (p) of the parameters were evaluated with 1,000 simulations; PSSD: P value for SSD (sum of squared deviations) PR: P value for Rag (Harpending's raggedness index); τ: the index of population expansion.

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