Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa.

The South African macadamia industry is severely affected by a complex of stink bugs, dominated by the two-spotted stink bug, Bathycoelia distincta Distant (Pentatomidae). This species was first discovered during the spring of 1984 in the Limpopo province. Although considerable effort has been spent trying to manage this pest, it continues to be a pest of concern for the macadamia industry. Information on the genetic diversity of this species is lacking, despite the potential relevance of such information for management strategies. The present study aimed to characterise the genetic diversity of B. distincta populations in South Africa. The Cytochrome c Oxidase Subunit 1 (COI) and cytochrome b (Cytb) gene regions were sequenced from individuals collected from the three main regions of macadamia production over three different seasons (2018-2020). An overall high haplotype diversity (COI = 0.744, Cytb = 0.549 and COI+Cytb = 0.875) was observed. Pairwise mean genetic distance between populations from each region varied from 0.2-0.4% in both datasets, which suggests the absence of cryptic species. The median joining network for both datasets consisted of one or two central haplotypes shared between the regions in addition to unique haplotypes observed in each region. Finally, low genetic differentiation (FST < 0.1), high gene flow (Nm > 1) and the absence of a correlation between genetic and geographic distance were estimated among populations. Overall, these results suggest that the B. distincta populations are not structured among the areas of macadamia production in South Africa. This might be due to its ability to feed and reproduce on various plants and its high dispersal (airborne) between the different growing regions of the country along with the rapid expansion of macadamia plantations in South Africa.

Introduction

The Pentatomidae is one of the largest families within the Heteroptera with more than 4,700 species distributed worldwide [1]. Phytophagous and polyphagous, Pentatomidae are a major concern to agricultural production around the world, including nut crops [2, 3]. In nut crops, stink bugs can cause direct feeding damage (e.g., nut abortion, discolouration, fruit drop) by insertion of their mouthparts into developing nuts inducing losses in yield and kernel nut quality [4-6]. Stink bugs can also cause indirect feeding damage by transmission of pathogens. This has been demonstrated especially in Nezara viridula (L.) with the transmission of Pantoea agglomerans into cotton bolls [7, 8].

In South Africa, several nuts are produced and exported worldwide, including macadamia nuts. Considered as the current world’s largest producer of macadamia nuts, the industry is continuously growing with about 5000 ha of trees planted annually [9]. However, this production is severely affected by various Heteroptera from the families Coreidae and Pentatomidae [10-12], causing approximately 15.23 million USD in losses due to nut damage annually [12, 13]. Among the different stink bug species occurring in macadamia orchards, Bathycoelia distincta Distant (Heteroptera: Pentatomidae) is the most abundant [10, 14, 15] and one of the most damaging pests for the industry due to its long proboscis which can cause kernel damage throughout the entire season [11, 16]. Bathycoelia distincta was discovered during the spring of 1984 in the Limpopo province and is now present in plantations in all macadamia production regions. Nymphs and adults have been recovered in the orchards even when no or few nuts are available on trees [10, 14], suggesting that this pest is breeding in the orchards and well established in all production areas in the country.

Control of B. distincta and other stink bug species has relied mainly on chemical insecticides. However, the prevalence of insecticide resistance and outbreaks of secondary pests coupled with increased environmental concerns have led to more integrated pest control approaches [17-19]. Some studies have been conducted to understand the seasonal occurrence [14] and the distribution patterns of B. distincta [20] to help with the implementation of IPM methods in South Africa [21]. Nevertheless, a comprehensive understanding of B. distincta population dynamics is lacking and required for the design of effective management strategies.

Population genetic studies can provide knowledge about the origin, migration patterns, genetic structure, and dynamics of pest populations. Exploring the genetic variability of a species among regional populations is also crucial to detect the existence of cryptic species complexes that could directly affect the efficiency of pest control efforts [22, 23]. Indeed, the utilisation of pheromones is often species-specific and may not work if subpopulations or cryptic species occur in the same geographical area, as has been demonstrated in various lepidopteran species [24-26]. Similarly, successful biological control depends on the use of species-specific parasitoid wasps. For example, a recent study discovered that cryptic species of the parasitoid Ganaspis brasiliensis have different affinities towards their hosts regardless of their food source, and as such impact the biological control of Drosophila suzukii (Matsumura) [27]. Another study on generalist parasitoids from the subfamily Aphidiinae revealed the presence of multiple cryptic species which are each in fact associated with a different host species [28]. Host preferences also exist in parasitoids of the Pentatomidae [29]. For example, Trissolocus utahensis (Ashmead) parasitizes more eggs of Chlorochroa uhleri (Stål) than Chlorochroa sayi (Stål) [30].

Population genetic studies have also highlighted genetic differentiation which may occur between insecticide resistant and susceptible populations. This may be a result of insecticide selection [31-33], management practices [34, 35], or other factors such as species isolation [36, 37]. Insecticide resistance has been reported for several Pentatomidae species [38]. For example, Euschistus heros (Fabricius) populations in Brazil have shown reduced susceptibility to various insecticides such as organophosphates and endosulfan [38, 39]. Concern regarding the reduced susceptibility of E. heros led to a large geographical survey and geostatistical analysis of bioassays with various insecticide formulations in order to map and identify areas with high risk of insecticide control failure [40].

The utilisation of mitochondrial DNA (mtDNA) has become an effective method to detect genetic variation among regional populations [41-44] due to its central role in metabolism and its high conservation rate across species in some sites, but high mutation rate in other sites to allow for species delimitation [41-43]. Although this method has been criticized [45-48], population genetic studies have been widely used among insect taxa in a variety of disciplines, including general ecology and evolution [49], to inform and improve management strategies. In addition, the mitochondrial Cytochrome c Oxidase Subunit 1 (CO1) gene region has been used to determine intraspecific divergence rates. To this end intraspecific divergence of 3 to 5% in Heteroptera has been found to delineate cryptic species within populations [50-53] in comparison to 2% intraspecific divergence rates for some other insect groups [42].

Genetic diversity of Pentatomidae species has mostly been studied using the COI gene region [50–52, 54, 55], although additional genes encoded by the mitochondria such as the cytochrome b (Cytb) are also commonly used [49, 56]. The occurrence and genetic diversity of Halyomorpha halys (Stål) has been widely studied in several countries [57-62] and such information is also available for N. viridula [63-65]. For example, in Brazil, various studies examined population structure and variation among populations of N. viridula [66, 67], E. heros [68, 69] and Loxa spp. [70] from different geographic regions. The genetic variability of Chinavia hilaris (Say), C. uhleri, C. sayi, and Thyanta pallidovirens (Stål) has also been examined to determine the presence of cryptic species in order to assist with pest management in pistachio orchards [53].

The present study aimed to investigate the genetic diversity of Bathycoelia distincta in South Africa. We examined the COI and Cytb gene regions from individuals collected from different regions where macadamia is planted commercially. We determined the genetic variability of B. distincta populations within and between the three main macadamia production areas of South Africa. We also consider whether there is any evidence of cryptic species.

Materials and methods

Ethics statement

No endangered or protected species were involved in this study. No national permissions were required for this study. All work on this project was conducted with permission from landowners.

Sample collection

Stink bugs were collected from different macadamia farms in Limpopo, Mpumalanga, and Kwazulu-Natal from October 2017 to March 2020 (Tables 1 and 2). Insects were collected as adults, from macadamia trees after insecticide application using a beating cloth under the trees. Samples were preserved in ethanol (> 95%) at -20°C until molecular analysis.

Table 1

Sampling locations where B. distincta was collected between October 2017 to March 2020 in South Africa provinces.

Location	n1	Latitude (S)	Longitude (E)
Farm L1	38	23°04’41.1"	30°08’31.1"
Farm L2	28	23°05’34.6"	30°14’23.3"
Farm M1	45	25°04’50.4"	31°01’08.4"
Farm K1	46	31°01’41.2"	30°13’13.1"

1 Number of specimens collected and used in this study to determine genetic diversity.

Table 2

Distance in straight line (Km) among the different sampling sites of Bathycoelia distincta population.

Distance (Km) in straight line	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-	-	-	-
Farm L2	10	-	-	-
Farm M1	242	234	-	-
Farm K1	885	881	667	-

The two-spotted stink bug specimens were identified based on external morphological features described in literature [71] and also confirmed morphologically by an entomologist at the Agricultural Research Council—Plant Health and Protection (Pretoria, South Africa). Insects used for this study are conserved in our collection located at University of Pretoria. Vouchered specimens were pinned, accession number assigned (PENT00026-PENT00030) and deposited in the National Collection of Insects located at the Agricultural Research Council—Plant Health and Protection (Pretoria, South Africa).

DNA extraction, PCR amplification and sequencing

The genetic diversity of B. distincta was determined by analysing sequence data from sections of the mitochondrial COI and Cytb genes. DNA was extracted from leg tissue of 157 adult specimens (Table 1) using the NucleoSpin® DNA insect (Macherey-Nagel GmbH & Co. KG, Düren, Germany) kit, following the manufacturer’s protocol for tissue extraction. A negative control was also carried out with all the kit solutions but without insect tissue to check for contamination. The DNA quantity was measured using the Thermo Scientific NanoDrop® ND-1000 spectrophotometer (Wilmington, DE, USA). The quantity of DNA in samples ranged from 20–40 ng/μl.

The COI gene region was amplified using the universal forward LCO1490 (5′-GGTCAACAAA TCATAAAGATATTGG-3′) and reverse HCO2198 (5′-TAAACTTCAGGGTGACCA AAAAATCA-3′) primer [72], and the Cytb gene region was amplified using the forward (5′-GGATATGTTTTACCTTGAGGACA-3′) and the reverse (5′-GGAATTGATCGTAAGATTGCGTA-3′) primer [66, 73]. Polymerase Chain Reactions (PCR) were performed in a total volume of 25 μL. For amplification of the COI gene, each PCR contained 5 μL of 5X PCR Buffer (dNTPs and MgCl2 included), 0.5 μL Taq DNA Polymerase (Bioline, South Africa), 0.5 μL of each primer (10 mM), 16.5 μL of distilled water and 2 μL of 50–100 ng DNA. PCR cycling conditions consisted of denaturation at 94°C for 1 min, followed by five cycles with denaturation at 94°C for 1 min, annealing at 45°C for 90 sec, and extension at 72°C for 90 sec, followed by another 30 cycles with denaturation at 94°C for 1 min, annealing at 50°C for 90 sec, extension at 72°C for 1.30 min, and final extension at 72°C for 5 min. The amplification of the Cytb gene was performed with 50–100 ng of DNA, 1.5 μL of MgCl2, 1 μL of dNTPs (10 μM), 1 μL of each primer (10 mM), 2.5 μL of 10X PCR buffer, and 0.5 μL of FastStart Taq DNA Polymerase (Roche, Molecular Biochemicals, Manheim, Germany). The PCR cycles consisted of initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 30 sec, extension at 72°C for 2 min and final extension at 72°C for 10 min. With each run, negative controls without DNA in the PCR reaction were performed for PCR validation.

The PCR products were verified on agarose gel (1.5% w/v) with BioRad Gel Doc™ Ez Imager and then purified using the ExoSAP-IT™ (Applied Biosystems, Foster City, CA) PCR Product clean-up kit. Forward and reverse sequence reaction was prepared using the BigDye® Terminator Kit v3.1 (Applied Biosystems, Foster City, CA). Sequencing products were cleaned and precipitated using ethanol and NaAC and sequenced using an ABI Prism™ 3100 Automated Capillary DNA sequencer (Applied Biosystems) at the Bioinformatics Sequencing facility of the University of Pretoria (South Africa).

Population genetic analyses

Electropherograms for all sequences were visualised and a consensus sequence generated using the Biological Sequence Alignment Editor (BioEdit) software (version 7.0.5) [74] and aligned using the online software Multiple Alignment using Fast Fourier Transform (MAFFT) v.7 [75]. To account for different sequence lengths, sequences were trimmed at 642 bp for the COI gene and 443 bp for the Cytb gene. Sequences of both COI and Cytb were concatenated to yield a total length of 1085 bp. Sequences of COI (accession numbers: OM263477-OM263633), and Cytb (accession numbers: OM219650-OM219806) of B. distincta were deposited in NCBI GenBank (www.ncbi.nlm.nih.gov). All the COI sequences obtained in this study were also submitted in the BOLD database (http://boldsystems.org/) under the project “PBDSA-Bathycoelia distincta Pentatomidae South Africa” (ID numbers: PMSL01-66 sequences from Limpopo; PMSM01-45 sequences from Mpumalanga; PMSK01-46 sequences from Kwazulu-Natal).

The COI and Cytb haplotype networks were constructed using Population Analysis with Reticulate Trees (PopART) version 1.7 [76]. Uncorrected (p) pairwise mean genetic distances between populations were calculated using the Kimura 2-parameter substitution model with 1000 bootstraps replicated in MEGA version 7.0.21 [77]. Genetic diversity parameters were determined using DnaSP 5.10.01 [78] and included the number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (Pi), genetic differentiation (Fst) and gene flow (Nm) values. The levels of genetic differentiation can be categorized as FST > 0.25 (high differentiation), 0.15 to 0.25 (moderate differentiation), and FST < 0.05 (negligible differentiation) [79]. The levels of gene flow can be categorized as Nm >1 (high gene flow), 0.25 to 0.99 (intermediate gene flow), and Nm <0.25 (low gene flow). Tajima’s D [80] and Fu’s Fs [81] values were estimated to test for changes in population size of B. distincta using DnaSP (v5.10.01). Significant negative values generally suggest population expansion. One thousand simulations under a model of selective neutrality were used to generate Tajima’s D and Fu’s Fs values. To determine the occurrence of isolation by distance (IBD), Mantel tests between the genetic and geographic distances between each population and marker were conducted using GenAlEx 6.5 with 9999 permutations [82].

Results

Sequence variation

The final sequence aligned matrix of the COI gene from 157 individuals was 642 bp in length. Genetic diversity indices for the COI gene are shown in Table 3. A total of 40 polymorphic nucleotides were observed. Thirty-five haplotypes were identified amongst COI sequences. The farms L1 and L2 showed 14 and 13 haplotypes from a total of 38 and 28 samples, respectively. Only 11 and 8 haplotypes were obtained from 45 and 46 samples of the farms M1 and K1, respectively. The haplotype diversity was the highest in Limpopo (farm L2, Hd = 0.754) and the lowest in the Kwazulu-Natal (farm K1, Hd = 0.571). The estimated nucleotide diversity (Pi) was low overall, ranging from 0.00140 to 0.00261. When all samples were included, the total diversity was Hd = 0.739, but the nucleotide diversity was quite low Pi = 0.00206.

Table 3

Summary of molecular diversity indices and population expansion test statistics of COI, Cytb and COI+Cytb genes in Bathycoelia distincta populations.

Gene		n	h	S	k	Hd	Pi	D	FS
COI	Population
	Farm L1	38	14	15	0.989	0.606	0.00154	-2.32655**	-13.391**
	Farm L2	28	13	18	1.677	0.754	0.00261	-2.22585**	-8.615*
	Farm M1	45	11	13	1.285	0.725	0.00200	-1.73408	-5.511*
	Farm K1	46	8	11	0.899	0.571	0.00140	-1.89467*	-3.600**
All samples		157	35	40	1.316	0.739	0.00206	-2.46755 **	-43.196 **
Cytb	Population
	Farm L1	38	14	13	2.004	0.782	0.00452	-1.10959	-7.080
	Farm L2	28	11	8	1.587	0.746	0.00358	-0.99040	-5.860
	Farm M1	45	10	12	0.786	0.431	0.00177	-2.15056*	-7.174*
	Farm K1	46	7	9	0.391	0.246	0.00088	-2.30002**	-5.923**
All samples		157	34	30	1.174	0.549	0.00265	-2.31555 **	-33.271 **
COI+Cytb	Population
	Farm L1	38	23	28	2.993	0.936	0.00276	-1.90073*	-18.195*
	Farm L2	28	21	26	3.265	0.955	0.00301	-1.92193*	-17.779*
	Farm M1	45	19	25	2.071	0.831	0.00191	-2.11947*	-13.579*
	Farm K1	46	12	20	1.290	0.643	0.00119	-2.31566**	-6.770**
All samples		157	62	70	2.490	0.875	0.00229	-2.52468 ***	-86.981 **

For each population and gene marker, the number of specimens (n), number of haplotype (h), number of polymorphic (segregation) sites (S), average number of nucleotide differences (k), haplotype diversity (Hd), nucleotide diversity (pi) and Tajima’s D (D) and Fu’s Fs (FS) tests statistics are given. Values are significant at

* P ≤ 0.05;

** P ≤ 0.01;

*** P ≤ 0.001.

Genetic diversity indices for the Cytb gene are shown in Table 3. The final aligned sequence was composed of 443 nucleotides for the Cytb with a total of 30 polymorphic nucleotides observed. The total number of haplotypes was 34. The farm L1 revealed the highest number of haplotypes (14 haplotypes for 38 samples), while the farm K1 showed the lowest number of haplotypes (only 7 haplotypes for 46 samples). The total haplotype diversity was Hd = 0.549, ranging from 0.246 to 0.782. Low nucleotide diversity (Pi) was observed (Pi < 0.01). It was the highest in Limpopo (farm L1, Pi = 0.00452) and lowest in Kwazulu-Natal (farm K1, Pi = 0.00088).

The results of the combined analysis of both COI and Cytb genes are presented in Table 3. The 1085 paired bases analysed revealed 70 polymorphic nucleotides. In total, 62 haplotypes were obtained with haplotype diversity Hd = 0.875 and a nucleotide diversity Pi = 0.00229. The highest number of haplotypes was observed in Limpopo at the farm L1 with 23 haplotypes, while the lowest number was observed in Kwazulu-Natal with 12 haplotypes. However, the highest haplotype diversity and nucleotide diversity was observed in Limpopo at the farm L2 (Hd = 0.955, Pi = 0.00301). It was the lowest in Kwazulu-Natal (Hd = 0.643 and Pi = 0.00119).

The pairwise distance comparison among B. distincta populations based on COI, Cytb and both COI+Cytb genes are shown in Table 4. Sequence divergence among the four populations by pairwise comparison ranged from 0.2–0.4%. The highest sequence divergence was found when farm L1-Cytb was compared with farm L2-Cytb. The lowest sequence divergence was observed between the farms M1-Cytb and K1-Cytb.

Table 4

Uncorrected “p” distance matrix between different locations based on COI, Cytb and COI+Cytb DNA sequences of Bathycoelia distincta from South Africa.

COI	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-
Farm L2	0.002	-
Farm M1	0.002	0.003	-
Farm K1	0.002	0.003	0.002	-
Cytb	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-
Farm L2	0.004	-
Farm M1	0.003	0.003	-
Farm K1	0.003	0.003	0.001	-
COI+Cytb	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-
Farm L2	0.003	-
Farm M1	0.003	0.003	-
Farm K1	0.003	0.003	0.002	-

Haplotype network analysis

To clarify the genetic relationship between B. distincta populations collected from various farms over different regions in South Africa, median-joining networks were generated (Figs 1–3). The obtained networks were generated with the genetic indices and neutrality tests calculated previously. The COI network (Fig 1) showed a star-like pattern with the two common haplotypes (Hap_C2 and Hap_C19) (S1 Table). The two common haplotypes included B. distincta collected from all the different farms in all three regions. They were separated by a single mutational change. For the Cytb, the generated haplotype network showed a dominant haplotype Hap_B1 where B. distincta collected from the four farms in all three regions are represented (Fig 2, S2 Table). Similar to the COI network, two common haplotypes Hap_CB3 and Hap_CB13 were found in the COI+Cytb median-joining network (Fig 3, S3 Table). The median-joining network of COI, Cytb, and COI+Cytb also showed a high number of unique haplotypes (35 for COI, 34 for Cytb, 62 for COI+Cytb), suggesting population expansions.

Fig 1

Median-joining haplotype network of Bathycoelia distincta for COI gene.

Each haplotype is represented by a circle. Relative sizes of the circle indicate haplotype frequency. Color patterns demonstrate samples collected from different farms and regions of South Africa (Limpopo: farm L1 (n = 38), farm L2 (n = 28); Mpumalanga farm M1 (n = 45); Kwazulu-Natal farm K1 (n = 46)). Crossbars indicate one mutational step.

Fig 3

Median-joining haplotype network of Bathycoelia distincta for COI+Cytb genes.

Each haplotype is represented by a circle. Relative sizes of the circle indicate haplotype frequency. Color patterns demonstrate samples collected from different farms and regions of South Africa (Limpopo: farm L1 (n = 38), farm L2 (n = 28); Mpumalanga farm M1 (n = 45); Kwazulu-Natal farm K1 (n = 46)). Crossbars indicate one mutational step.

Fig 2

Median-joining haplotype network of Bathycoelia distincta for Cytb gene.

Each haplotype is represented by a circle. Relative sizes of the circle indicate haplotype frequency. Color patterns demonstrate samples collected from different farms and regions of South Africa (Limpopo: farm L1 (n = 38), farm L2 (n = 28); Mpumalanga farm M1 (n = 45); Kwazulu-Natal farm K1 (n = 46)). Crossbars indicate one mutational step.

Neutrality test

The neutrality test was conducted using Tajima’s D and Fu’s Fs statistics (Table 3). The results of the tests indicated significant negative D and FS values for the total of the populations for each gene (COI: D = -2.467, P < 0.01, FS = -43.196, P < 0.01; Cytb: D = -2.315, P < 0.01, FS = -33.271, P < 0.01; COI+Cytb: D = -2.525, P < 0.001, FS = -86.981, P < 0.01), suggesting population expansion. Considering each population separately, all the farms presented significant negative values for the neutrality tests, indicating that there is an excess of rare mutations in B. distincta populations which can imply recent population growth. In concordance, the haplotype networks indicated that the different sequence types observed in South Africa would have derived from a common ancestral haplotype (Hap_2).

Genetic structure

Genetic distance (FST) and migration rates (Nm) of the B. distincta populations were calculated (Table 5). Based on COI sequence data, the pairwise FST among 4 pairs of B. distincta populations ranged from -0.005 to 0.291. Bathycoelia distincta samples from the Limpopo farms (L1 and L2) exhibited statistically significant genetic differentiation when compared to Mpumalanga (M1) and Kwazulu-Natal (K1) farms. Interestingly, similar results were obtained based on the COI+Cytb data set. Consistent results were also observed based on the Cytb dataset, which showed no significant differences (P < 0.05) of pairwise FST in most population pairs except for the farms L1 and L2 when compared to the farm K1. Regarding the migration rates (Nm) values, all population pairs were greater than one, except for the Limpopo farms (COI, Nm = -48.96), or between the Mpumalanga and Kwazulu-Natal farms (Cytb, Nm = -90.55). Finally, for each marker, the Mantel test showed no statistically significant IBD, indicating no positive correlation between the geographic and genetic distances among B. distincta populations (COI: r = 0.731; P > 0.05; CytB: r = 0.310, P > 0.05; COI+Cytb: r = 0.669, P > 0.05) (S1–S3 Figs). Our results suggested that more than one stink bug female per generation was estimated to migrate between all pairs of populations, except between Limpopo and Mpumalanga or Kwazulu-Natal.

Table 5

Genetic differentiation (FST) and gene flow (Nm) based on COI, Cytb and COI+Cytb DNA sequences of Bathycoelia distincta from three different regions of South Africa.

COI	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-	-48.96	1.58	0.61
Farm L2	-0.00513	-	2.01	0.84
Farm M1	0.13647**	0.11052**	-	7.05
Farm K1	0.29150***	0.23025***	0.03427	-
Cytb	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-	5.07	4.44	2.75
Farm L2	0.04703	-	2.21	1.44
Farm M1	0.05335	0.10151	-	-90.55
Farm K1	0.08331*	0.14768*	-0.00277	-
COI+Cytb	Farm L1	Farm L2	Farm M1	Farm K1
Farm L1	-	9.56	2.45	1.08
Farm L2	0.02548	-	2.10	1.02
Farm M1	0.09256*	0.10654	-	11.32
Farm K1	0.18840*	0.19643**	0.02161	-

The data above and below the diagonal correspond to Nm and FST respectively.

*, ** and *** indicate significant difference at P<0.05, P< 0.01and P<0.001 respectively.

Discussion

Population genetic studies have played a significant role in the identification of cryptic species and the study of their genetic diversity [42, 51]. In this study, we investigated the population genetics of B. distincta for the first time. DNA sequences were analysed employing COI and Cytb markers from specimens collected in the three main macadamia production regions of South Africa. The highest genetic diversity was observed within the Limpopo province populations, the area from where the species was first discovered. Pairwise mean distance analysis between populations suggested the absence of cryptic species. The median joining network for both datasets consisted of a few central haplotypes shared among populations in South Africa, with several unique haplotypes (35 for COI, 34 for Cytb, 62 for COI+Cytb) among the 157 B. distincta individuals examined (Figs 1–3). Considering the direct relationship between haplotype frequency and the ages of haplotypes [83, 84], the existence of a star-like structure (central haplotype and several less frequently derived haplotypes) suggests that most of the haplotypes originated recently, and is indicative of a population expansion during the recent history of the species [85].

Bathycoelia distincta was originally discovered from the Limpopo region [14], and became a serious problem in South African macadamia areas in Limpopo and Mpumalanga in the early 2000s [10, 15, 86] and more recently in Kwazulu-Natal. The rapid and intensive increase in macadamia planted area in the last decade may be related to the genetic diversity observed in this study. Beck and Reese [87] proposed that insect survivorship, fecundity, growth rate and activity can be affected by the quantity and quality of the host. An abundance of hosts in a habitat will increase survival and fecundity and reduce mortality. As macadamia trees are the primary cultivated host for B. distincta, the increase in population size of this host would distinctly improve the fecundity and survival of the insect, and thus the extensive commercial cultivation of macadamia trees in recent years may have contributed to an expansion of B. distincta’s range in South Africa. Similar results have been obtained in E. heros populations in Brazil after the rapid expansion of the soybean-planted area, leading to an absence of genetic differentiation within populations [68], compared to the results obtained in the same area 14 years earlier [69]. This hypothesis is reinforced by the significant negative values found for the neutrality tests (Tajima’s D and Fu’s Fs), confirming a recent population expansion of B. distincta.

In our study, high gene flow (Nm > 1), lack of genetic differentiation (FST < 0.1) and no IBD (P > 0.05) were observed among the different populations (Table 5, S1–S3 Figs). Genetic differentiation between insect populations can be influenced by different factors such as the geographic distance between populations [53, 88, 89]. Although population differentiation by distance has been observed previously within Pentatomidae species [53, 57, 69], our results suggest that geographic distances did not affect the genetic structure of B. distincta. While limited genetic structure and high levels of population connectivity were expected for the farms located in Limpopo (distance between farms L1-L2 is 10 km), this was not expected for our regional scale investigation as the farms sampled are geographically distant, being up to 800 km apart (Table 2). Few studies have been conducted on the flight capacities of Pentatomidae using flight mills. Babu et al. [90] showed that Euschistus servus (Say) can fly a maximum distance of 15.9 km in 22 h, especially after overwintering emergence, whereas most individuals only flew between 0–1 km. For H. halys, adults can fly 5–7 km in 24 h and up to 117 km, with longer and faster flights achieved in summer [91-93], while nymphs can easily walk among host plants [94]. Considering that genetic isolation can also be higher in species with a limited capacity for dispersal [95], a high dispersal capacity in B. distincta could explain the genetic homogeneity among its populations in South Africa.

Factors other than flight ability might also facilitate movement of B. distincta. One such a factor is the distribution of suitable host plants (both wild and cultivated) in South Africa. Stink bugs can feed on several plant-hosts [2, 96] and higher levels of damage are often observed when forests and natural vegetation border crops [97, 98], a feature quite common in the South African landscape. In addition, the two-spotted stink bug population can reach high densities especially in the canopies [20], mainly from November to March when nuts are present but they can remain in the field even after the nuts have been harvested [10, 14]. After harvest B. distincta may disperse, looking for shelter to remain in diapause during winter. High levels of gene flow, as determined for B. distincta in this study, are determinants of the maintenance of high levels of genetic diversity and low population differentiation [99, 100]. This, in addition to its multivoltinism, may have contributed to high population densities, a wide distribution, and low levels of population differentiation throughout the country. Therefore, these long-distance dispersal events might occur in parallel with some anthropogenic driven dispersal (e.g., transport of seedlings or fruits among regions of South Africa).

Genetic divergence and nucleotide diversity in this study among the different locations was comparable to results for Hemiptera in previous studies. The intraspecific genetic divergence in this study was in the range of 0.2 to 0.4% (Table 4) and a low nucleotide diversity was observed (Pi < 3%) (Table 3). Previous studies observed intraspecific divergence of 4.7% between individuals of C. hilaris [53], and > 2% in N. viridula [54]. Nevertheless, an intraspecific genetic divergence value of 4.7% has been suggested to delineate cryptic species within populations of Heteroptera [51]. Considering the previous results obtained and the morphological and ecological similarity of B. distincta between the different locations analysed, we can confirm that our study did not show any evidence of cryptic species in its populations in South Africa. Knowledge of the existence of cryptic species is important for IPM because different species can respond differently to pest management strategies [23, 35]. For example, variation in the pheromone blends have been found among different populations of Diatraea saccharalis (Fabricius), suggesting that the trapping efficiency could vary among the regions [26]. Similarly, insect populations from different geographic regions can vary in their level of susceptibility to insecticides as it has been recently demonstrated in wireworm populations [101]. Thus, it would be useful to determine the effectiveness of future pheromone lures and other control methods across the wide geographical range of B. distincta.

In conclusion, this study of B. distincta collected within the three main macadamia production regions in South Africa revealed a high genotypic diversity and a lack of genetic differentiation among localities. The high genotypic diversity suggests favourable environmental conditions for reproduction and growth of the species in its native range. The high gene flow observed, even across a wide geographic area, appears to be the major ecological force shaping the overall genetic pattern observed. Regional population dynamics of B. distincta are likely linked to the rapid expansion of macadamia planted areas. To our knowledge this is the first study on the population genetics of B. distincta. This information provides a critical starting point for understanding this species in South Africa and might assist future development of pest management strategies that incorporate pheromones and biological control. Furthermore, future studies using other genetic and genomic tools and including a larger sample size and geographic range, could help understand the movement of on B. distincta populations between orchards and native plants, and within regions and over shorter time scales. Such genetic and genomic tools are currently under-utilized as a resource for stink bug management and our study provides a foundation for such further work.

Isolation by distance of Bathycoelia distincta populations for COI marker (Mantel test, r = 0.731, P > 0.05).

(TIF)

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Isolation by distance of Bathycoelia distincta populations for Cytb marker (Mantel test, r = 0.310, P > 0.05).

(TIF)

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Isolation by distance of Bathycoelia distincta populations for COI+Cytb combined marker (Mantel test, r = 0.669, P > 0.05).

(TIF)

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List of the individual for each haplotype generated in the study for the COI marker.

(DOCX)

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List of the individual for each haplotype generated in the study for the Cytb marker.

(DOCX)

Click here for additional data file.

List of the individual for each haplotype generated in the study for the COI+Cytb combined markers.

(DOCX)

Click here for additional data file.

7 Apr 2022

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Reviewer #1: The manuscript entitled "Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa" by Fourie et al described the genetic diversity of B. distincta populations in South Africa using COI and Ctyb gene markers. The population analysis using different methods have been conducted using the two markers separately and combined. It is a nice study and the manuscript provided novel information on the two-spotted stink bug. I have a couple of suggestions as below:

1) The information on how to identify the sting bug is lacking in the manuscript. The method used for the morphological identification should be added, such as which morphological keys were used and identified by whom…

2) Suggest the authors to submit the COI sequence to BOLD databases as there are no COI sequence available in the databases until now.

3) For the name of the haplotype, similar names were used for COI, cytb and COI-cytb combined. I think it will be clearer if the authors can use slightly different names to distinct them, for example, Hap_C1 for COI, Hap_B1 for cytB, Hap_CB1 for combined (Note: just my suggested name systems, do not need to follow my name conventions)? In this way, it will be easy for readers to follow when Hap_B1 mentioned, they will know it is talking about cytB haplotype.

Minor comments:

1. Table 1, Table 2 and Figure1 are presenting the same information, it seems that figure 1 is not necessary.

2. Lines 159-160: suggest providing the primer names for the two cytb primers.

3. Lines 157-172: the authors used two different PCR mastermixes to amplify the COI and cytb genes. Just wondering the reasons for the authors not to use the same matermix for amplify the two genes. According to my experience, generally it will be able to amplify the two genes under the same PCR compositions.

4. Lines 177-181: the detail pressure on how to sequence is not necessary as nowadays, it is standard protocol for the sequencing provider to run the sequence.

5. Line 371: D. saccharalis mentioned in the first time, the genus name should be used.

.Reviewer #2: This paper looks at population genetics of Bathycoelia distincta. It is well-written and concise. However, I have a few concerns regarding the work described. These need clarification before the paper can be fully accepted.

Regarding the M&M, the authors appear to not include any controls. This include several insect outgroups (other Heteroptera) as well as several Pentatomids other than those in the same Family. I would also include several within the same Family. Secondly, the sequencing for each individual must be replicated a minimum of three times (forward and reverse) for each individual and each primer set. Failure to do so can result in misalignment and mis-sequencing. Finally, no indication is given as to where the insects tested are stored or the voucher specimens are located. Thus, it appears from first reading that the experiments were not conducted as rigorously as they could have and the appropriate controls are missing.

Reviewer #3: "Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa"

Overall, this paper is very well written, and makes an important contribution to a newly emerging insect pest of macadamia in South Africa.

There are a few very minor grammar suggestions.

line 64 edit 'damaging pest' to 'damaging pests'

line 76 remove the , after dynamics

Methods:

I have one major comment. The Fst values between K and L population are highest. These populations are geographically most distant. I suggest running a Mantel test, to examine the association between geographic distance and genetic divergence. It looks to be significant. This would be expected. Genalex can be used to run this test

Discussion: Line 379. Why do the authors believe there are so many new haplotypes? The star pattern in the haplotype tree shows they all branch off one or two main haplotype. What might cause many new mutations and haplotypes? Insecticides?

Some discussion of why there are so many new haplotypes would add to the manuscript.

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Reviewer #1: Yes: Dongmei Li

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Two-spotted stink bug-review comment.docx

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25 Apr 2022

Dr Patrizia Falabella

Academic Editor

PLOS ONE

Submission of Revised Manuscript

Thank you for your positive feedback regarding our manuscript, entitled “Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa”. We believe that the suggestions made by the reviewers have improved the scientific quality and clarity of our manuscript.

In this revision, we incorporated all the suggestions made by the reviewers, which has resulted in a number of minor changes to our manuscript.

Please find attached our revised manuscript. Our response to the comments and suggestions made by the reviewers can be found in the document below titled “Response to Reviewers”, a marked copy of the manuscript can be found in the document titled “Revised Manuscript with Track Changes” and the unmarked version of the manuscript can be found in the document titled “Manuscript”.

We thank you in advance for your assistance in handling this manuscript further.

Kindest regards,

Journal requirements

Please note that the page and line numbers referred to in our responses represent those found in the newly submitted manuscript. Furthermore, specific changes are indicated in italics, where appropriate.

1) Please ensure that your manuscript meets PLOS ONE’s style requirements, including those for file naming.

• The manuscript and file names of the manuscript was reviewed in order to ensure that all the PLOS ONE’s style requirements are met.

2) In your methods section, please provide additional information regarding the permits you obtained for the work.

• No permit is needed in South Africa. An additional section has been added Lines 130-132: “Ethics statement. No endangered or protected species were involved in this study. No national permissions were required for this study. All work on this project was conducted with permission from landowners”.

3) Funding statement modifications.

• The funding statement was updated online and in the cover letter.

4) We note that the grant information you provide in the “funding information” and “Financial Disclosure” sections do not match.

• As mentioned above, funding information was updated and corrected.

5) We note that Figure 1 in your submission contain map images which may be copyrighted.

• As suggested by the reviewer 1, Figure 1 was removed from the manuscript.

Response to Reviewers

Please note that the page and line numbers referred to in our responses represent those found in the newly submitted manuscript. Furthermore, specific changes are indicated in italics, where appropriate.

Reviewer 1:

1) The reviewer suggested that information on how to identify the stink bug species was lacking and that the method for morphological identification and morphological keys should be added.

• Lines 140-146: We included more detail about the method for morphological identification by adding, “The two-spotted stink bug specimens were identified based on external morphological features described in literature [72] and also confirmed morphologically by an entomologist at the Agricultural Research council – Plant Health and Protection (Pretoria, South Africa). Insects used for this study are conserved in our collection located at University of Pretoria. Vouchered specimens were pinned, accession number assigned (PENT00026-PENT00030) and deposited in the National Collection of Insects located at the Agricultural Research Council – Plant Health and Protection (Pretoria, South Africa).”

2) The reviewer suggested to submit our COI sequences to BOLD database.

• Our COI sequences were deposited to BOLD database under accession numbers PMSL01-66 sequences from Limpopo, PMSM01-45 sequences from Mpumalanga, PMSK01-46 sequences from Kwazulu-Natal. This was also stated in the revised manuscript Lines 196-199.

3) The reviewer suggested to use different names to distinguish between haplotypes.

• This is a valid suggestion. We have revised the names used for each haplotype into “Hap_C1” for COI, “Hap_B1” for Cytb and “Hap_CB1” for combined COI-Cytb as suggested.

4) The reviewer suggested a number of minor comments.

1. Table 1, Table 2 and Figure 1 are presenting the same information, it seems that figure 1 is not necessary: Figure 1 has been removed.

2. Lines 159-160, the reviewer suggests to provide the primer names for the two cytb primers: The two Cytb primers used in this study were developed by Muraji et al. 2000 and used in the study of Li et al. 2014. In both studies, no primer names were listed.

3. Lines 157-172 the authors used two different PCR mastermixes: Our initial tests of CytB amplification using a standard Taq DNA polymerase resulted in non-specific amplification. We therefore switched to using a FastStart Taq to increase the specificity. This was also added in the revised manuscript Line 176.

4. Lines 177-181 the reviewer suggested that the details for the sequencing was not needed: We removed the following from the manuscript “using 5.8 µL distilled water, 1 µL Big Dye, 1 µL sequencing buffer, 0.2 µL primer and 2 µL purified PCR product. Cycle sequencing conditions included initial denaturation of 2 min at 96°C followed by 25 cycles of 10 s at 96°C, 5 s at 50°C and 4 min at 60°C”.

5. Line 371 D. saccharalis mentioned for the first time: We are grateful to the reviewer for picking up this error. We have changed the species name to “Diatraea saccharalis” (line 394).

Reviewer 2:

1) The reviewer asked to include controls in the materials and methods such as several insect outgroups (other Heteroptera) as well as several Pentatomids other than those in the same family.

• We included more details in line 180: “With each run, negative and positive PCR controls were performed for PCR validation.”

• This is a valid point, however this study was focused specifically on the genetic diversity of

Bathycoelia distincta in South Africa and. It is our opinion that it was is not necessary to include an outgroup. For example, similar studies, published in PLoS ONE, such as Karsten et al. 2013 and Low et al. 2014 (references cited below) which were also focused on one species, did not include outgroups.

- Karsten M, van Vuuren BJ, Barnaud A, Terblanche JS (2013) Population Genetics of Ceratitis capitata in South Africa: Implications for Dispersal and Pest Management. PLoS ONE 8(1): e54281. doi:10.1371/journal.pone.0054281

- Low VL, Adler PH, Takaoka H, Ya’cob Z, Lim PE, et al. (2014) Mitochondrial DNA Markers Reveal High Genetic Diversity but Low Genetic Differentiation in the Black Fly Simulium tani Takaoka & Davies along an Elevational Gradient in Malaysia. PLoS ONE 9(6): e100512. doi:10.1371/journal.pone.0100512

2) The reviewer specified that the sequencing must be replicated a minimum of three times (forward and reverse) for each individual and each primer set.

• Each individual was sequenced in both directions (i.e. forward and reverse primer) using an ABI Prism™ 3100 Automated Capillary DNA sequencer (Applied Biosystems). The electropherograms were visualised, base calling accuracies were checked and a consensus sequence was generated. This type of sequencing provides highly accurate results (99.99%) compared to other next generation sequencing technologies such as Illumina and/or PacBio. Sequencing each sample with each individual primer set is not compulsory in this type of study. In addition, recent studies published in PLOS ONE journal this year (references below) did not repeat the sequencing of the gene markers COI and Cytb three times. For these reasons, we did not repeat our sequencing.

• The following sentence was added in the revised manuscript line 190: “Electropherograms for all sequences were visualised and a consensus sequence generated”.

Tsoupas A, Papavasileiou S, Minoudi S, Gkagkavouzis K, Petriki O, Bobori D, et al. (2022) DNA barcoding identification of Greek freshwater fishes. PLoS ONE 17(1): e0263118. https://doi.org/10.1371/journal.pone.0263118

Arnaout Y, Djelouadji Z, Robardet E, Cappelle J, Cliquet F, Touzalin F, et al. (2022) Genetic identification of bat species for pathogen surveillance across France. PLoS ONE 17(1): e0261344. https://doi.org/10.1371/journal.pone.0261344

3) The reviewer noted that no indication is given as to where insects tested are stored or the voucher specimens are located.

• The insects used in our study are conserved in our facility and some of the specimens were sent to a taxonomist at the Agricultural Research Council (ARC) in Pretoria for identification. Four of our specimens was deposited in the National Collection of Insects under the voucher numbers PENT00026-PENT00030.

• We added additional text Lines 140-146: “The two-spotted stink bug specimens were identified based on external morphological features described in literature [72] and also confirmed morphologically by an entomologist at the Agricultural Research Council – Plant Health and Protection (Pretoria, South Africa). Insects used for this study are conserved in our collection located at University of Pretoria. Vouchered specimens were pinned, accession number assigned (PENT00026-PENT00030) and deposited in the National Collection of Insects located at the Agricultural Research Council – Plant Health and Protection (Pretoria, South Africa).”

Reviewer 3:

1) The reviewer noted a few grammatical errors.

• Line 62: we corrected “damaging pest” to “damaging pests”

• Line 74: we removed “,” after dynamics

2) The reviewer suggested to conduct a Mandel test to examine the association between geographic distance and genetic divergence.

• Mantel tests were conducted and revealed no significant differences for each marker.

• The regression figures between the genetic and geographical distance among the populations for each genetic marker were added as supporting information: S1_Fig for COI, S2_Fig for Cytb and S3_Fig for COI+Cytb.

• Additional information related to these results were added in the manuscript:

o Lines 39-40: ….”absence of correlation between genetic and geographic distance”….

o Lines 212-214: “To determine the occurrence of isolation by distance (IBD), Mantel tests between the genetic and geographic distances between each population and marker were conducted using GenAlEx 6.5 with 9999 permutations [83]”

o Line 300: the title of the paragraph was renamed “Genetic structure”

o Lines 310-313: “Finally, for each marker, the Mantel test showed no statistically significant IBD, indicating no positive correlation between the geographic and genetic distances among B. distincta populations (COI: r = 0.731; P > 0.05; CytB: r = 0.310, P > 0.05; COI+Cytb: r = 0.669, P > 0.05) (S1-S3 Figs). (COI: r = 0.731; P > 0.05; CytB: r = 0.310, P > 0.05; COI+Cytb: r = 0.669, P > 0.05) (S1-S3 Figs)”.

o Line 353: …”and no IBD (P > 0.05)”….

o Line 354: …”S1-S3 Figs.”

3) The reviewer addressed some questions regarding line 379 - “what might cause many new mutations and haplotypes? Insecticides?” - and suggested adding some discussion to the manuscript.

• We believe that the presence of many haplotypes is linked to the species being in its native range. The species is also currently the most dominant and damaging pest of macadamia. Over time the species adapted to feed on macadamia and since nymphs are often present in orchards it suggests that macadamia orchards are a favourably habitat for reproduction and growth. Although insecticides could also likely explain some of the many haplotypes present, no information regarding insecticide resistance is currently available and we therefore did not comment on this in the manuscript

• The following sentence was added in the revised manuscript Lines 403-404: “The high genotypic diversity suggests favourable environmental conditions for reproduction and growth of the species in its native range.”

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

12 May 2022

17 May 2022

Submission of Revised Manuscript

Thank you for your positive feedback regarding our manuscript, entitled “Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa”. We believe that the suggestions made by the reviewers (both rounds) have improved the scientific quality and clarity of our manuscript. In this revision, we incorporated the suggestions made by reviewer 1, which has resulted in minor changes to our manuscript.

Please find attached our revised manuscript. Our response to the comments and suggestions made by the reviewer can be found in the document below titled “Response to Reviewers”, a marked copy of the manuscript can be found in the document titled “Revised Manuscript with Track Changes” and the unmarked version of the manuscript can be found in the document titled “Manuscript”.

We thank you in advance for your assistance in handling this manuscript further.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references

We have carefully reviewed our references and noted that reference [9] “DALRRD. Ressource Centre of the Department of Agriculture, Land Reform and Rural Development 2020. Available from: https://www.dalrrd.gov.za/.” are no longer available on-line. This reference was subsequently removed from our manuscript. The reference was not replaced since the information are also captured in reference [10].

- L56: "[9]" has been removed and as such the numbers from [9] onwards changed.

Finally, we noted a small type error in the SAMAC reference (previously numbered as [10] but [9] in the revised manuscript) and as such was updated to:

- L458-459: " SAMAC (South African Macadamia Association). Industry statistics on the South African Macadamia Industry 2021. 2021. Available online at: https://www.samac.org.za/industry-statistics/"

Response to Reviewers

Reviewer 1:

The reviewer had two minor points:

1) The sequence submitted in BOLD need to be publicly available before publication:

- We made the sequences publicly available as requested. You can retrieve the sequences under the project “PBDSA-Bathycoelia distincta Pentatomidae South Africa”.

2) Line 180 "With each PCR run, negative and positive PCR controls were performed for PCR validation"- what is the negative control? which species used, how about positive control? could the authors specify what DNA used for the controls? Was non-template control used for the PCR reactions?

- The negative control used consisted of sample without DNA. This was to ensure that the master mix was not contaminated. This was also stated in the revised manuscript Lines 180-181. “negative controls without DNA in the PCR reaction”. We removed the mention of a positive control from the manuscript. The positive control was added to each run as an internal control. The positive control comprised of B. distincta DNA that previously amplified and was used as a control to ensure that all the required PCR reagents were added to the master mix correctly.

Submitted filename: Response to Reviewers.docx

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20 May 2022

Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa

PONE-D-22-02292R2

Dear Dr.  Gerda Fourie,

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Additional Editor Comments (optional):

I suggest to insert in the manuscript a brief sentence in which it has reported the availability of the sequence in the project “PBDSA-Bathycoelia distincta Pentatomidae South Africa", with a specific website link.

Reviewers' comments:

2 Jun 2022

PONE-D-22-02292R2

Genetic diversity of the two-spotted stink bug Bathycoelia distincta (Pentatomidae) associated with macadamia orchards in South Africa

Dear Dr. Fourie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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