The genus Stigmaeopsis (family Tetranychidae) has 11 species including the serious bamboo pest, S. nanjingensis. All Stigmaeopsis species are difficult to identify by their morphology, and the diagnostic character (the length of dorsal setae) can be used only to identify fresh specimens. To identify these species at the molecular level, we sequenced the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA and two nuclear ribosomal RNA genes (18S and 28S) of 20 strains of seven species of Stigmaeopsis [S. celarius, S. longus, S. miscanthi (both low- and high-aggression phenotypes), S. nanjingensis, S. tenuinidus, S. saharai and S. takahashii]. In maximum likelihood (ML) phylogenetic trees of both COI and combined 18S-28S genes, all but one Stigmaeopsis species could be identified as a monophyletic clade with high bootstrap values. The present results strongly suggested that the exceptional species, S. miscanthi, consists of three biologically different entities based on two phylogenetic trees. Though the phylogenetic trees did not comprehensively solve the phylogeny of Stigmaeopsis, a phylogenetic tree based on the combined nuclear genes showed a sibling relationship between two sub-social Stigmaeopsis species, S. miscanthi and S. longus. In addition, diagnostic PCR detected Wolbachia or Cardinium, which frequently affect mitochondrial haplotypes, in S. longus and S. nanjingensis. In the COI tree, S. longus was separated into two groups which were more consistent with their bacterial infection status than with their geographical distribution.
The genus Stigmaeopsis consists of 11 species in the family Tetranychidae (Saito et al. 2004, 2016; Flechtman 2012). All species appear on Poaceae species such as bamboo, dwarf bamboo, reed grass and silver grass. Identification of species in this genus has been based on both the length of the dorsal setae and distance between the bases of specific dorsal setae (Saito 2009). However, setae are frequently worn down by constant contact with threads in the ceiling of their nests. Thus, to accurately identify mites, they must be reared for more than one generation and many microscope slides must be prepared to make setae measurements in newly emerged females. Such morphological identification requires expertise and experience.
Another identification strategy is molecular analysis based on DNA sequences. The internal transcribed spacer 2 (ITS2) region of nuclear ribosomal RNA (rRNA) genes (Navajas & Boursot 2003; Noge et al. 2005; Ben-David et al. 2007) and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA) (Matsuda et al. 2012, 2013) have been used to identify spider mite species. Once molecular identification corresponds with morphological identification, it can be used to determine species rapidly and without a need for expertise in morphology. DNA-based molecular identification can also discover new invasive species (Wang & Qiao 2009) and cryptic species (Carew et al. 2011; Matsuda et al. 2013).
Except for the recently described S. temporalis Saito et Ito (Saito et al. 2016), five species of Stigmaeopsis have been identified and phylogenetically investigated in Japan. These five Japanese species were reported to belong to five distinct groups based on the COI gene of mtDNA (Ito & Fukuda 2009 Ito et al. 2011). Based on the 28S rRNA gene, these five species were clearly divided into two groups, which well corresponded to the host plants (bamboo species and Miscanthus grass species, respectively) (Sakagami et al. 2009). Some intraspecific groups were reported from two species, S. miscanthi and S. longus, based on phylogenetic analyses (Sakagami et al. 2009; Ito & Fukuda 2009; Ito et al. 2011). S. miscanthi has two behavioral phenotypes based on differences in male pugnacity against conspecific males. The two phenotypes have been called the low-aggression (LW) phenotype and the high-aggression (HG) phenotype (Saito & Sahara 1999; Sato et al. 2008, 2013), and these phenotypes formed distinct groups in phylogenetic trees of the 28S rRNA gene (Sakagami et al. 2009) and COI gene (Ito & Fukuda 2009; Ito et al. 2011). S. longus also forms two distinct groups in a phylogenetic tree based on the COI gene; one is distributed in the northern part of Japan (from Hokkaido Prefecture to Yamagata Prefecture) and the other is distributed in the southern part of Japan (from Yamagata Prefecture to the Kyushu district), suggesting that there were two routes of expansion of this species (Ito et al. 2011). Indeed, during the glacial age, many plants and animals in the Japanese Archipelago likely arrived from the Asian continent via land bridges between Sakhalin and Hokkaido Islands and between the Korean Peninsula and the Kyushu Islands (Masuda & Abe 2005).
To develop a DNA-based species identification method for the genus Stigmaeopsis, we determined the COI (618 bp), 18S (1863 bp) and 28S (671 bp) rRNA gene sequences of seven out of the 11 known species in the world (Flechtmann 2012; Saito et al. 2016). To better understand the phylogenetic relationship among these species, we also tried to detect the presence or absence of two intracellular endosymbiotic bacteria, Wolbachia and Cardinium, which frequently affect the genetic structure and diversity of the host species (Turelli et al. 1992; Gotoh et al. 2007; Yu et al. 2011; Zhang et al. 2013). Furthermore, these intracellular endosymbiotic bacteria frequently affect mitochondrial haplotypes of spider mite species (Zhang et al. 2013).
Materials and Methods
Mites. Seven Stigmaeopsis species (15 strains and 5 strains previously reported by us) were used in this study (Table 1). The mite samples were maintained on leaf discs of the original host plants. The leaves were placed on a water-saturated polyurethane mat in a plastic dish (90 mm diameter, 20 mm depth). The mites were reared at 25°C and under a 16:8 h light:dark photoperiod until analysis. After the laboratory strains were established, newly emerged samples with complete dorsal setae were mounted in Hoyer's medium and identified under phase-contrast and differential interference-contrast microscopes. Sn_Nanping and Sn_Huanxi strains of S. nanjingensis (Ma & Yuan), Ste_Guiyan and Ste_Hongliao strains of S. tenuinidus (Zhang & Zhang), and Sm_(Cn) strain of S. miscanthi were established at China using the above-mentioned methods. More than ten individuals of each strain were preserved in 99 % ethanol and sent to Ibaraki University for molecular analysis. In addition, adult males and females of each strain with complete dorsal setae were mounted on slides in Hoyer's medium and sent to Ibaraki University for morphological identification. Some of the latter were used as voucher specimens.
Collection records of seven species of the genus Stigmaeopsis and two outgroup species used in this study.
Phylogenetic analysis. Mites were randomly selected from each strain or from 99 % ethanol-preserved strains. Total DNA was extracted from the whole body of each female with a Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Live female individuals for DNA samples and female individuals for voucher specimen were obtained from the same leaf discs. PCR primers are given in Table 2. PCR amplification was performed with the following profile: 3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 45°C (COI), 55°C (28S region) or 65°C (18S region) and 1.5 min at 72°C. An additional 10 min at 72°C was allowed for last strand elongation. The resultant DNA solutions were purified using MinElute PCR Purification Kit (Qiagen, Valencia, CA) and sequenced directly. Sequencing was carried out in both directions using the amplifying primers with a BigDye Terminator Cycle Sequencing Kit v. 3. 1 (Applied Biosystems, Foster City, CA) and on an ABI 3130×1 automated sequencer.
Primers used in polymerase chain reaction amplification and sequencing of the rDNA (18S and 28S regions) and mtDNA (COI gene).
All sequence data obtained were deposited in DDBJ/EMBL/GenBank International Nucleotide Sequence Databases (Table 1). Sequences obtained and cited from our previously published data (Matsuda et al. 2014) were aligned using CLUSTAL W (Thompson et al. 1994) and numbers or parsimony-informative sites were calculated using MEGA6 software (Tamura et al. 2013). Gaps included in the 28S and 18S sequences were treated using the complete deletion option in MEGA6. Sequences for Tetranychus urticae Koch (green form) and T. kanzawai Kishida (Matsuda et al.2014) were used as a single outgroup. The robustness of the branches was tested by bootstrap analysis (Felsenstein 1985) with 1,000 replications. Maximum likelihood (ML) trees of both mtDNA and nuclear genes were constructed with RAxML (Stamatakis 2006). For all analyses, we used the GTR Gamma model selected by the Akaike Information Criterion (AIC) using the program Kakusan4 (Tanabe 2011). The RAxML search was executed for the best-scoring ML tree in one single program run (the ‘-f a’ option) instead of the default maximum parsimony-starting tree. Statistical support was evaluated with 1,000 rapid bootstrap inferences. Then, genetic distances (Kimura 2 parameter distances) between the COI genes of Stigmaeopsis species were calculated using MEGA6.
Diagnostic PCR of Wolbachia/Cardinium infection. The DNA samples used for phylogenetic analysis were also used as diagnostic PCR templates. Primer pairs of diagnostic PCR of Wolbachia (wsp gene; Zhou et al. 1998) and Cardinium (16S rRNA gene; Morimoto et al. 2006) are given in Table 3. PCR amplification was performed with the following profile: 2 min at 95°C, followed by 35 cycles of 0.5 min at 95°C, 0.5 min at 52°C and 0.5 min at 72°C. An additional 5 min at 72°C was allowed for last strand elongation. PCR products were run on 2% agarose gels to observe DNA bands with the expected size (Wolbachia-wsp gene, 610 bp; Cardinium-16S rRNA gene, 470 bp).
Primers used in diagnostic PCR of Wolbachia and Cardinium infection.
COI gene of mtDNA. We obtained COI sequences of 15 strains of seven species determined in this study and five strains of five species from our previously published data (Table 1). None of the COI sequences contained insertions or deletions. After alignment, the COI fragment had 618 nucleotide sites, of which 282 were parsimony-informative sites.
In a ML tree based on the COI sequences, six of the seven Stigmaeopsis species (all except S. miscanthi) formed a monophyletic clade with high bootstrap values (Fig. 1). The interspecific genetic distances of the genus Stigmaeopsis (8.4–14.7%) were clearly higher than intraspecific genetic distances (0.0–2.5%) with one exception (Table 4). The exception was S. miscanthi, which showed low intra-group genetic distances (0.0–0.8%) but abnormally high inter-group genetic distances. The latter were 9.4–9.6% between Sm_(HG) and Sm_(LW), 8.1–8.5% between Sm_(HG) and Sm_(Cn) and 8.7% between Sm_(Cn) and Sm_(LW). The clade of S. miscanthi was divided into three groups as Sm_(HG), Sm_(LW) and Sm_(Cn) with deeply separated branches.
S. longus consisted of two groups within a monophyletic clade, and the split did not appear to be due to geographical distance because the Sl_Sapporo strain (Hokkaido Prefecture, Japan) and the Sl_Ami train (Ibaraki Prefecture, Japan) belonged to the same group, whereas the Sl_Date strain (Hokkaido Prefecture, Japan) made a different group. The highest intraspecific genetic distance was 2.5% (Table 4).
Nuclear rRNA genes. We obtained 18S and 28S rRNA gene sequences of 15 strains of seven species determined in this study and five strains of five species from our previously published data (Table 1). Both nuclear genes contained a number of insertions and deletions. After alignment, the 18S fragment had 1,863 nucleotide sites, of which 495 were parsimony-informative sites. Similarly, after alignment, the 28S fragment had 671 nucleotide sites, of which 201 were parsimony-informative.
In a ML tree based on the combined 18S and 28S rRNA sequences (Fig. 2), all but one Stigmaeopsis species formed a monophyletic clade with high bootstrap values. The exception was S. miscanthi, which was divided into three groups as it was in the COI tree. In contrast, all three strains of S. longus, which consisted of two groups in the COI tree, formed a single clade with a high bootstrap value. Furthermore, a sibling relationship between two sub-social Stigmaeopsis species, S. miscanthi and S. longus, was supported in the combined ML tree (74% bootstrap values, Fig. 2).
Bacterial infection. Two strains of S. longus (Sl_Sapporo and Sl_Ami) and one strain of S. nanjingensis (Sn_Nanping) were positive for Wolbachia and one strain of S. longus (Sl_Date) was positive for Cardinium (Table 1). None of the remaining strains were positive for either bacterium.
Per cent of intra- and inter specific genetic distances (Kimura 2 parameter distances) in COI gene of mtDNA among seven Stigmaeopsis species.
In this study, we examined the efficiency of DNA-based identification of seven morphologically similar species of Stigmaeopsis (five Japanese species and two Chinese species). Identifications were based on the sequences of the COI gene of mtDNA and two nuclear rRNA genes (28S and 18S regions). The 28S and 18S regions were combined to construct the phylogenetic tree by the ML method. In both trees, all but one Stigmaeopsis species formed a monophyletic clade with high bootstrap values (Figs. 1 and 2). The exception was S. miscanthi, which was divided into three groups in both trees. Our results contribute to the molecular-based rapid identification method of this genus, which includes the severe bamboo pest species, S. nanjingensis (Zhang et al. 2000). Recently, S. nanjingensis invaded Europe (Pellizari & Duso 2009) and severely damaged bamboo species, especially Mo so bamboo, Phyllostachys pubescens Mazel ex Houz, which is used in construction, furniture and food. Our findings should enable rapid identification of this species in the early stage of their invasions.
Hypotheses about the evolutional relationship of Stigmaeopsis species have been based on molecular analysis, morphology and host plants (Sakagami et al. 2009; Ito & Fukuda 2009; Ito et al. 2011). In this study, we were unable to determine a comprehensive phytogeny of Stigmaeopsis. The deep-level relationships were especially unresolved, as shown by the insufficient bootstrap values of our phylogenetic trees of mtDNA and combined nuclear genes (Figs. 1 and 2). Interestingly, the combined 18S and 28S tree showed a sibling relationship between two sub-social Stigmaeopsis species, S. miscanthi and S. longus (74% bootstrap values, Fig. 2). Sakagami et al. (2009) made a similar conclusion based on just the 28S rRNA gene. However, this sibling relationship was not strongly supported by the results of mtDNA (26% bootstrap values, Fig. 1). Finding the appropriate outgroup of Stigmaeopsis may help to solve the phylogenetic relationship of Stigmaeopsis mites. In recent years, the range of the genus Stigmaeopsis was extended. Flechtmann (2012) transferred S. malkovskii (Wainstein) and S. meghalensis (Gupta & Gupta) from Schizotetranychus to Stigmaeopsis. Further, Saito et al. (2016) described two novel Stigmaeopsis species (S. temporalis and S. tegmentalis Saito et Lin). Further investigations of the phylogenetic relationship of Stigmaeopsis species including these latter added species are needed.
Two behavioral phenotypes of S. miscanthi, the low-aggression (L W) and high-aggression (HG) phenotypes based on male aggressiveness, were previously reported (Saito & Sahara 1999; Sato et al. 2008, 2013). The two phenotypes were basically shown to be two separate clades based on their nuclear 28S gene (Sakagami et al. 2009) and COI gene of mtDNA (Ito & Fukuda 2009). In our study, these two phenotypes also formed different clades and the Sm_(Cn) strain of S. miscanthi formed a third clade (Figs. 1 and 2), which is predicted to have an intermediate-aggression phenotype (between the LW and HG phenotypes) from the morphology of the male weapon (Sato et al. unpublished). Especially in the COI tree, the Sm_(Cn) strain and the other two strains formed deeply separated branches. Furthermore, the abnormally high inter-group genetic distances between Sm_(HG) and Sm_(LW), between Sm_(HG) and Sm_(Cn) and between Sm_(Cn) and Sm_(LW) (Table 4) support the existence of the species-level separation among these three strains. To see whether the Sm_(Cn) strain belongs to S. miscanthi will require an accurate re-examination of morphological and behavioral characters as well as crossing experiments.
Both Wolbachia and Cardinium were detected in different strains of S. longus. Wolbachia was also found in the Sn_Nanping strain of S. nanjingensis. This is the first record of Wolbachia infection in Stigmaeopsis mites. On the other hand, Cardinium infection was previously recorded in S. celarius Banks and S. longus (Nakamura et al. 2009). In general, these intracellular bacteria and the mitochondria are maternally inherited, so that the mitochondrial genome will be in strong linkage with the endosymbionts. Furthermore, in mites, Wolbachia is mainly known for inducing reproductive alterations such as cytoplasmic incompatibility (CI) and parthenogenesis induction (Weeks & Breeuwer 2001; Gotoh et al. 2003). Likewise, Cardinium causes CI (Gotoh et al. 2007; Ros & Breeuwer 2009) and feminization of mites that are genetically male (Weeks et al. 2001). Interestingly, in the mtDNA tree, the clade of S. longus was consistent with the bacterial infection status: the Wolbachia-infected Sl_Sapporo strain (Hokkaido Prefecture) and the Sl_Ami strain (Ibaraki Prefecture) formed the same group, and the Cardinium-infected Sl_Date-strain (Hokkaido Prefecture) belonged to a different group. However, all three strains belonged to the same clade in the nuclear gene tree. Likewise, in Tetranychus truncatus Ehara, Zhang et al. (2013) showed that the distribution of mtDNA haplotypes was associated with Wolbachia-infection status rather than with geographical distribution. In the genus Stigmaeopsis, the phenotypes induced by these bacteria are unknown, although in S. longus, the bacteria might be responsible for a divergence of the COI gene. From the viewpoint of invasive biology, the finding that the invasive pest mite, S. nanjingensis, is infected with Wolbachia in their naturally distributed area might help to identify their source.
Based on an analysis of the COI gene, Ito et al. (2011) divided S. longus into two regional subgroups, the northern group distributed from Hokkaido Prefecture, which was their northernmost area in Japan, to Yamagata Prefecture and the southern group distributed from Yamagata Prefecture to the Kyushu district. If this division is correct, the Sl_Sapporo strain collected in Hokkaido Prefecture should belong to the northern group. However, analysis of our COI dataset combined with the COI dataset of Ito et al. (2011) showed that the Sl_Sapporo strain belonged to the southern group (data not shown). This division is consistent with the observation that the genetic distance between the Sl_Sapporo strain and the Sl_Date strain (2.5%) was the same as that between the Sl_Ami strain and the Sl_Date strain (2.5%; Table 4). These data strongly suggest that S. longus was divided by Wolbachia/Cardinium infection rather than by a north-south regional difference. We cannot exclude the possibility that S. longus has a widely distributed cryptic species that cannot be identified by 18S and 28S gene sequences or differentiated by morphological characters. Further phylogenetic studies of a large number of infected and uninfected samples from different regions are needed to settle this question.
We are deeply grateful to Drs. Y. Kitashima and Jie Ji for collecting spider mites. This work was supported in part by JSPS KAKENHI Grant Number JP25292033, and supported from China Recruitment Program of Global Experts (Foreign Experts) (no. 2012-323) and the Sate Administration of Foreign Experts Affairs Key Project for Introduction of Foreign Expert (no. SZ2013003).
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