Population Genetic Diversity of Two Blue Oat Mite Species on Triticum Hosts in China

Simple Summary Blue oat mite species mainly include Penthaleus major, P. falcatus, P. tectus, and P. minor. Among them, P. major, P. falcatus and P. tectus are important pest mites on gramineous crops, which often occur in low-temperature and high-humidity environments. These three mites are difficult to distinguish in the field due to their small size and similar shape. Furthermore, they exhibit different resistance to chemical pesticides, making it challenging to control their outbreak. Worryingly, we only saw reports of frequent occurrence of P. major in winter wheat regions in China, and no other species of blue oat mites were found. Understanding the distribution pattern, genetic diversity, and genetic differentiation among populations of blue oat mite species in winter wheat regions of China is essential for developing effective control programs. In this study, we evaluated the distribution of blue oat mites in the major wheat regions in China and assessed the level of genetic diversity and genetic structure of blue oat mites from winter wheat regions of China using mitochondrial cytochrome c oxidase I (COI) sequences. Abstract Blue oat mite species, including Penthaleus major and P. tectus, are pests widely distributed across China that cause damage to winter wheat. This study evaluated the genetic diversity of P. major and P. tectus on Triticum hosts collected from 23 geographic locations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences. We identified nine haplotypes in 438 P. major individuals from 21 geographic locations and five haplotypes in 139 P. tectus individuals from 11 geographic locations. Meanwhile, P. major exhibits high values of haplotype diversity (Hd) and nucleotide diversity (Pi) (Hd = 0.534 > 0.5 and Pi = 0.012 > 0.005), representing a large stable population with a long evolutionary history. P. tectus shows low values of Hd and Pi (Hd = 0.112 < 0.5 and Pi = 0 < 0.005), which suggest recent founder events. Moreover, demographic analysis suggested that P. major and P. tectus have not undergone a recent population expansion. The lowest genetic variation was observed in Xiangzhou (XZ-HB), Zaoyang (ZY-HB), Siyang (SY-JS), and Rongxian (RX-SC), with only one species and one haplotype identified in over 30 individuals. Robust genetic differentiation was found in P. major compared to P. tectus, which provides a theoretical basis for the widespread distribution of P. major in China.


Data Analysis
The assembling and alignment of COI sequences were completed in DNASTAR LASERGENE 7.1.0 and MEGA 7.0, respectively [30][31][32]. Genetic diversity parameters were determined in DnaSP 5.10.01 [27], including the number of polymorphic sites (S), the total number of mutations (η), the number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (Pi), and the average number of nucleotide differences (K). A total of 12 haplotypes reported from Canada and Poland were used to analyze the phylogenetic relationships. Phylogenetic analyses were performed in MEGA 7.0 using the Neighbor-Joining method, while haplotype networks of P. major and P. tectus haplotypes were constructed in TCS 1.21 [33]. Pairwise F ST and gene flow (N m ) between each pair of the sampled locations and pairwise F ST and gene flow (N m ) among three geographic regions were calculated in ARLEQUIN 3.5 [34]. The mismatch distribution analysis was performed to detect historical population expansion events in P. major and P. tectus populations in DnaSP 5.10.01. A multimodal pattern implies that populations are at demographic equilibrium, whereas a unimodal pattern shows that populations are experiencing rapid demographic growth [35,36].

Distribution Pattern of Wheat Pest Mites
Two genera of mites were found in the main winter wheat regions of China during this survey, including Penthaleus spp. and Petrobia spp. (Figure 1). The study found that Penthaleus spp. were the main pest mites in winter wheat regions and were distributed in 11 provinces and a municipality, including Yunnan, Sichuan, Guizhou, Shaanxi, Hubei, Shanxi, Henan, Anhui, Shandong, Jiangsu, and Chongqing. The range of Petrobia spp. was small, primarily in Shanxi and Hebei. Moreover, the map revealed that both Penthaleus spp. and Petrobia spp. occurred in Shanxi Province. Penthaleus spp. was the most widely distributed; however, it was not found in Hebei. Among the 35 survey sites, Penthaleus spp. was found in 32 sites, accounting for up to 91.43%, while Petrobia spp. was only found in three sites. The population distribution range and number were significantly lower than Penthaleus spp.

Population Distribution of Blue Oat Mites
In this study, the dominant population of blue oat mite species on winter wheat in China was identified ( Figure 2; Table 1). Two species of blue oat mites were found in 577 samples, including P. major and P. tectus. Among them, P. major accounted for 75.91%, more than P. tectus. P. major and P. tectus were found throughout three natural geographic groups, including Southwestern China (SW), the Middle and Lower Yangtze Valleys (YV), and the Yellow and Huai Valleys (YH). Of the 23 sites surveyed, P. major was found in 21 survey sites, with the exceptions being Rongxian (RX-SC) and Ruicheng (RC-SX). The distribution of P. tectus is similar to that of P. major, but it is not always found in the same sites. At nine sites, P. major and P. tectus co-occurred, including Santai (ST-SC), Nanchong (NC-SC), Dangyang (DY-HB), Xuzhou (XZ-JS), Donghai (DH-JS), Bozhou (BZ-AH), Changfeng (CF-AH), Shanzhou (SZ-HN), and Tanghe (TH-HN). Additionally, only P. tectus was found at two survey sites, including Rongxian (RX-SC) and Ruicheng (RC-SX).

Population Distribution of Blue Oat Mites
In this study, the dominant population of blue oat mite species on winter wheat in China was identified ( Figure 2; Table 1). Two species of blue oat mites were found in 577 samples, including P. major and P. tectus. Among them, P. major accounted for 75.91%, more than P. tectus. P. major and P. tectus were found throughout three natural geographic groups, including Southwestern China (SW), the Middle and Lower Yangtze Valleys (YV), and the Yellow and Huai Valleys (YH). Of the 23 sites surveyed, P. major was found in 21 survey sites, with the exceptions being Rongxian (RX-SC) and Ruicheng (RC-SX). The distribution of P. tectus is similar to that of P. major, but it is not always found in the same sites. At nine sites, P. major and P. tectus co-occurred, including Santai (ST-SC), Nanchong (NC-SC), Dangyang (DY-HB), Xuzhou (XZ-JS), Donghai (DH-JS), Bozhou (BZ-AH), Changfeng (CF-AH), Shanzhou (SZ-HN), and Tanghe (TH-HN). Additionally, only P. tectus was found at two survey sites, including Rongxian (RX-SC) and Ruicheng (RC-SX).

Haplotype Diversity, Nucleotide Diversity, and Haplotype distribution
The COI gene fragment, which was amplified from 577 individuals in 23 different locations across three central winter wheat regions, ranged from 657 to 658 bp ( Table 2). Of the 438 individuals of P. major, nine haplotypes (MH1-MH9) were identified with 22 polymorphic nucleotide sites. Nineteen of these sites were parsimony-informative, and three were singleton sites. The A/T content was significantly higher (70.30%) than the C/G content (29.70%). Meanwhile, 139 P. tectus individuals generated five haplotypes (TH1-TH5) with four polymorphic nucleotide sites. Four of these sites were parsimony-informative sites and none were singleton sites. The A/T content was significantly higher (69.90%) than the C/G content (30.10%). The Hd of P. major ranged from 0.000 in the seven populations (NC-SC, XZ-JS, SY-JS, BZ-AH, XZ-HB, ZY-HB, TH-HN) to 0.708 CF-AH population (Table 3). Haplotype diversity of P. major was identified in 12 of the 21 locations. For the other nine locations, only one haplotype was found. The Hd of P. tectus ranged from 0.000 in the six populations (RX-SC, ST-SC, NC-SC, XZ-JS, SZ-HN, TH-HN) to 0.467 CF-AH population. Additionally, haplotype diversity of P. tectus was only observed in three of the 11 locations. For the other eight locations, only one haplotype was found. Furthermore, for all individuals, P. major had large Hd and Pi values (Hd > 0.5 and Pi > 0.005), indicating a large stable population with an extended evolutionary history. In contrast, P. tectus had smaller Hd and Pi values (Hd < 0.5 and Pi < 0.005), implying recent founder events [37][38][39][40].

Phylogenetic Relationship and Haplotype Network
The Neighbor-Joining method was used to construct the phylogenetic tree with COI sequences of P. major, P. tectus, and 12 published COI sequences of Penthaleus sp. (Figure 3 into two branches distinct from those in Canada and Poland, while the 14 haplotypes in China were split into two branches, representing P. major (PMH1-PMH9) and P. tectus (PTH1-PTH5), respectively.

Phylogenetic Relationship and Haplotype Network
The Neighbor-Joining method was used to construct the phylogenetic tree with COI sequences of P. major, P. tectus, and 12 published COI sequences of Penthaleus sp. (Figure 3). Among the 26 haplotypes of Penthaleus sp., Clade P1 (PMH1-PMH9 and PTH1-PTH5 from China) and Clade P2 (other samples from Canada and Poland) were identified. The COI sequence of Eupodes minutus with GenBank accession number DQ675139.1 was chosen as the outgroup. The phylogenetic tree showed that the Penthaleus sp. in China were divided into two branches distinct from those in Canada and Poland, while the 14 haplotypes in China were split into two branches, representing P. major (PMH1-PMH9) and P. tectus (PTH1-PTH5), respectively. The haplotypes of P. major and P. tectus populations are shown by the TCS network, with varying colors and sizes of circles representing different sampling sites and the number of individuals detected. MH3 is the most crucial haplotype for P. major, and TH1 is the most abundant haplotype for P. tectus (Figure 4).  The haplotypes of P. major and P. tectus populations are shown by the TCS network, with varying colors and sizes of circles representing different sampling sites and the number of individuals detected. MH3 is the most crucial haplotype for P. major, and TH1 is the most abundant haplotype for P. tectus (Figure 4).

Population Differentiation and Genetic Structure
Population pairwise fixation index (FST) and gene flow (Nm) investigated the diversity in 21 P. major populations and 11 P. tectus populations (Tables 4 and 5). The pairwise FST tests of P. major populations indicated significant differentiation in 120 of 210 location

Population Differentiation and Genetic Structure
Population pairwise fixation index (F ST ) and gene flow (N m ) investigated the diversity in 21 P. major populations and 11 P. tectus populations (Tables 4 and 5). The pairwise F ST tests of P. major populations indicated significant differentiation in 120 of 210 location pairs based on mitochondrial COI gene. Table 4. Pairwise F ST (below diagonal) and gene flow N m (above diagonal) among 21 populations of P. major in winter wheat of China.  Gene flow between SW and YV of P. major and between SW and YH of P. tectus show a relatively low degree due to the low N m value (N m < 1), suggesting that genetic drift might result in genetic differentiation (Tables 6 and 7). For three P. major natural geographic groups, higher gene flow (N m > 1) was found between SW and YH, while for P. major and P. tectus, high gene flow (N m > 3) was found between YV and YH. In addition, for P. tectus, high gene flow (N m = 4.23) was also found between SW and YV.  AMOVA results of P. major indicated significant molecular genetic variations among the locations within geographic regions (55.74%) ( Table 8). The variation within sites was 42.20%. Only 2.05% variation was found among geographic regions. However, for P. tectus, the AMOVA results indicated that a significant portion of the molecular genetic variation was found within locations (88.43%). The variation among the sites within geographic regions was 9.21%. Only 2.35% variation was found among geographic areas.

Demographic Analysis
Demographic analysis showed that the overall mismatch distribution for all sampled localities showed the multimodal profile ( Figure 5), suggesting that P. major populations in China might not have undergone a sudden demographic expansion. The actual observation results are compatible with the predicted constant model when assuming a constant population size for P. tectus, demonstrating that the population status is consistent with the expected model assumption. Altogether, results suggested that the population size of P. major and P. tectus have not changed over time.
Insects 2023, 14, x FOR PEER REVIEW 10 of 14 42.20%. Only 2.05% variation was found among geographic regions. However, for P. tectus, the AMOVA results indicated that a significant portion of the molecular genetic variation was found within locations (88.43%). The variation among the sites within geographic regions was 9.21%. Only 2.35% variation was found among geographic areas.

Demographic Analysis
Demographic analysis showed that the overall mismatch distribution for all sampled localities showed the multimodal profile ( Figure 5), suggesting that P. major populations in China might not have undergone a sudden demographic expansion. The actual observation results are compatible with the predicted constant model when assuming a constant population size for P. tectus, demonstrating that the population status is consistent with the expected model assumption. Altogether, results suggested that the population size of P. major and P. tectus have not changed over time.

Discussion
Blue oat mites, which are widely distributed in southern Australia, are an important agricultural pest species in Australia [4,5]. Electrophoresis results suggest that there are differences between species in terms of clonal diversity [11,13,14]. Previous research has also showed that Penthaleus spp. respond differently to pesticides [26,38]. Therefore, clarifying the distribution range and population density of different species is essential for adequate control.
In this study, Penthaleus spp. and Petrobia spp. were found on winter wheat, consistent with previous research reports [23]. A large number of blue oat mites (Penthaleus spp.) were found in three major winter wheat regions of China, including P. major and P.

Discussion
Blue oat mites, which are widely distributed in southern Australia, are an important agricultural pest species in Australia [4,5]. Electrophoresis results suggest that there are differences between species in terms of clonal diversity [11,13,14]. Previous research has also showed that Penthaleus spp. respond differently to pesticides [26,38]. Therefore, clarifying the distribution range and population density of different species is essential for adequate control.
In this study, Penthaleus spp. and Petrobia spp. were found on winter wheat, consistent with previous research reports [23]. A large number of blue oat mites (Penthaleus spp.) were found in three major winter wheat regions of China, including P. major and P. tectus. The distribution range and population density of P. major were broader and higher than P. tectus, suggesting that controlling pest mites on wheat is very difficult because different species have different resistance levels. The risk is that the sympatric occurrence of different species will make control more difficult. We also found high levels of genetic diversity in the P. major population by analyzing mitochondrial cytochrome oxidase subunit I sequences. The distribution proportion of P. major was significantly higher than P. tectus, and the total number of P. major was as high as 75.91% in 577 samples, consistent with the study of Australian scholars [41]. P. tectus only occurs in limited areas and is recorded only from Australia and South Africa [12]. In China, P. tectus was first recorded in this study and was widely distributed only in a few samples, such as Rongxian (RX-SC) and Nanchong (NC-SC).
The number of haplotypes, haplotype diversity (Hd), and nucleotide diversity (Pi) are used for assessing diversity in population-level COI sequencing surveys [42][43][44][45]. These metrics are useful for biodiversity assessment because they can be influenced by a variety of factors [46]. In the current study, the Hd and Pi values suggested that P. tectus may not be an ancient and stable population compared to P. major. The Hd and Pi among all P. tectus samples were lower than those of P. major. The Hd of P. major in Changfeng (CF-AH) was higher than in this study of other locations. In addition, the Pi of P. major in Changfeng (CF-AH) was up to 0.014. Among the P. tectus populations, the Hd and Pi of Changfeng (CF-AH) were the highest (Hd = 0.467, π = 0.001). Compared to P. tectus, the genetic variation of P. major was at a relatively high level, which was confirmed by allozyme loci [13,14].
Clonal diversity is often high in obligate asexual organisms and most research on asexual organisms has focused on the role of environmental heterogeneity in promoting clonal diversity [13,46,47]. The blue oat mite P. major is asexual and lacks a sexual relative. Populations consist of numerous clonal genotypes and clone frequencies change over time and space [13]. Previous studies have also provided direct evidence for negative frequency-dependent selection maintaining clonal variation in P. major [15]. In our research, haplotype 3 (MH3) was found in 17 P. major populations and haplotype 1 (TH1) was the most abundant in P. tectus populations. The different haplotypes represent different genetic backgrounds, and clone competition, differential resource utilization, and pesticide resistance likely cause the haplotypes' diversity [48][49][50]. A larger haplotype variation was found in P. major than P. tectus populations, indicating that it has a stronger evolutionary potential for adaptation to future environmental changes and chemical pesticides.
We observed significant differentiation of P. major among regions, locations within regions, and within locations. The percentage of variation was only 2.05% between regions, but as much as 42.20% within locations. For P. tectus, the percentage of variation was up to 88.43% within locations. AMOVA analysis indicated a low level of genetic differentiation among regions of P. major (F CT = 0.02, p = 0.3323) and P. tectus (F CT = 0.02, p = 0.2063), which agrees with previous studies [14]. This may be due to the long-range movement of diapause eggs through winds or human assistance in summer [5,51].

Conclusions
In this study, the occurrence pattern of blue oat mites was clarified in three major winter wheat regions. The results indicated that the occurrence of blue oat mites was particularly serious in the Yellow and Huai River winter wheat zone of China, especially during the early spring season. Among the pest mites, P. major was found to be the most common in winter wheat, followed by P. tectus. Although P. tectus had not been previously reported in China, this study found that it occurred in all three wheat regions, and there was an evident co-occurrence between the P. major and P. tectus. Furthermore, the COI sequence analysis revealed that P. major boasts more genotypes and higher genetic diversity than P. tectus. It suggested that P. major may have stronger adaptability. Further studies should focus on exploring the tolerance of P. major and P. tectus to temperature, the fitness on different host plants, and the resistance to various chemicals. This way, we can develop a more reasonable scheme for regional precise control of these mites.