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Article

Comparative Analysis of Two Pear Pests, Cacopsylla jukyungi and Cacopsylla burckhardti (Hemiptera: Psyllidae), Based on Complete Mitochondrial Genomes and Comparison to Confamilial Species

by
Ah Rang Kang
1,2,
Min Jee Kim
3,
Jeong Sun Park
2,
Ho-Jin Seo
1,
Jang-Hoon Song
1,
Kyung-Ho Won
1,
Eu Ddeum Choi
1 and
Iksoo Kim
2,*
1
Pear Research Institute, National Institute of Horticultural & Herbal Science, Rural Development Administration, Naju 58216, Korea
2
Department of Applied Biology, College of Agriculture & Life Sciences, Chonnam National University, Gwangju 61186, Korea
3
Experiment and Analysis Division, Honam Regional Office, Animal and Plant Quarantine Agency, Gunsan 54096, Korea
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(9), 2037; https://doi.org/10.3390/agronomy12092037
Submission received: 31 July 2022 / Revised: 24 August 2022 / Accepted: 25 August 2022 / Published: 27 August 2022
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
Mitochondrial genome sequences have been used in diverse fields of biology. In this study, we sequenced the complete mitochondrial genomes (mitogenome) of two pear pests: Cacopsylla jukyungi, the most damaging insect pest to commercial pears in South Korea, and Cacopsylla burckhardti (Hemiptera: Psyllidae). The two mitogenomes were compared to confamilial species to accumulate genetic information and understand evolutionary characteristics of the family Psyllidae. The 15,438 bp-and 14,799 bp-long complete mitogenomes of C. jukyungi and C. burckhardti, respectively, had many features typical of insect mitogenomes; however, at 1283 bp, the C. jukyungi mitogenome had an unusually long A+T-rich region, which was composed of two identical 540-bp repeat sequences. Among the intergenic spacer regions, the one located at the ND1 and trnS2 junction was relatively well conserved in length (mostly within 23–36 bp). This region had a high sequence identity in all Psyllidae, possessing a 5-bp consensus sequence (CGGTA), which is speculated to have a functional role. Though the A+T-rich region in available Psyllidae mitogenomes varied substantially in length (662–1430 bp) and sequence divergence, all species had a conserved sequence stretch at the 3′-end of srRNA, which is also speculated to have a functional role. Genetic divergence among genes indicated the lowest variability in srRNA, lrRNA, and COI, whereas ATP8 and ND6 showed the highest variability at both family and genus (Cacopsylla) levels. Our data provide evidence that the family Psyllidae, including current C. jukyungi and C. burckhardti, have evolutionary unique features that were previously undetected, along with the unique A+T-rich region structure in C. jukyungi.

1. Introduction

After a substantial revision of east Palaearctic pear psyllid taxonomy, the genus Cacopsylla Ossiannilsson, 1970 [1] (Hemiptera: Psyllidae) was determined to comprise four species in Korea: Cacopsylla burckhardti (Luo, Li, Ma & Cai, 2012) [2], Cacopsylla jukyungi (Kwon, 1983) [3], Cacopsylla maculatili (Li, 2011) [4], and Cacopsylla sandolbaea (Park & Lee, 1982) [5,6]. Among them, the polyvoltine C. jukyungi [3], which was previously misidentified as Cacopsylla pyricola (Foerster, 1848) [3,7], is the most damaging pest among commercial pears, particularly the southern type, Pyrus pyrifolia N., in Korea [8]. C. jukyungi overwinters under bark scale, after which it moves upward to tree branches, usually during February and March, and starts a new generation by ovipositing on branches. Ultimately, C. jukyungi will undergo 4–5 generations in a year [9,10]. The species displays seasonal dimorphism, converting from a light-colored form (summer form) to a dark overwintering form (winter form) in late summer and autumn in response to the shortening photoperiod [11]. The univoltine C. burckhardti [2], which was previously misidentified as Cacopsylla pyrisuga [7], is known to overwinter in the weeds around pear orchards. In South Korea, this psyllid enters commercial pear orchards in the spring, during which it damages pears, then disperses outside pear orchards [12]. A detailed, in-field lifecycle for C. burckhardti is not known. As is typical of psyllids, both nymphs and adults of C. burckhardti and C. jukyungi damage leaves and fruits by injecting a toxin while feeding that causes blackening and burning of the foliage (psylla shock) and by secreting honeydew that causes sooty mold and marks the fruit [13]. The other two pear psyllids occurring in Korea damage the northern-type pear (P. ussuriensis M.), which is a non-commercial pear, distributed infrequently in Korea [14].
Full and partial mitochondrial genome (mitogenome) sequences have been used for diverse purposes, such as evolutionary genomics, phylogenetic inference, and biogeographic inference [15,16,17,18,19]. Furthermore, mitogenomes have also been used to trace the origin of invasive species, evaluate donors for the genetic rescue of endangered species, and identify species, often using the mitochondrial COI gene segment [15,16,20,21,22,23,24].
Until now, the Psyllidae mitogenomes have been reported for 11 species. Four of them have been used for individual mitogenome reports, the main content of which includes genome annotation and nucleotide composition, along with phylogenetic inference, focusing on the phylogenetic position of the given species [25,26,27,28]. In this list, the C. burckhardti mitogenome, which has been reported this year, is also included [28]. On the other hand, the remaining seven species have been used to elucidate evolutionary relationships within the superfamily Psylloidea [29]. However, no study has been performed to illustrate the evolutionary characteristics of the family Psyllidae, particularly using a comparative approach, which will provide us with a further scrutinized understanding on mitogenome evolution.
To control pear-damaging psyllids, the prompt application of proper control strategies upon detection of psyllids is important. This requires rapid identification of Cacopsylla species, but the co-occurrence of different species, life stages, and seasonal forms in pear orchards often complicates species identification in the field, especially when identification is performed by non-specialists. Thus, molecular marker-based identification is often required. Moreover, pest control using pesticides is often limited by the number and type of commercial pesticides available to control pear pests, and eco-friendly cultivation requires minimal application of pesticides. To overcome such difficulties, integrated pest-control strategies are required, involving an ecological understanding of pests, such as population genetic structure and gene flow [30,31,32]. For this, molecular markers, which provide variable regions, are essential for acquiring a meaningful understanding of population dynamics.
In this study, the complete mitogenomes of C. jukyungi and C. burckhardti were sequenced and the two genomes were characterized in terms of gene arrangement, codon composition, genome structure, and conserved sequence stretch. Available complete mitogenome sequences of other Psyllidae were also considered by a comparative approach. Thus, the newly sequenced mitogenomes of C. jukyungi and C. burckhardti are expected to enrich our understanding of mitogenome evolution in the family Psyllidae. Moreover, we compared the sequence divergence of individual genes to obtain a better understanding of individual gene-level evolution, which will be useful for the selection of candidate genes for subsequent population genetic analyses.

2. Materials and Methods

2.1. Insects

C. jukyungi and C. burckhardti were collected at Daap-myeon, Gwangyang city, Jeollanam-do Province, South Korea (35°04′00.5″ N, 127°44′02.5″ E) in April 2021. After live samples were transferred to the laboratory, samples were identified to species by examining their external morphology following Cho et al. [6], and also by sequencing the mitochondrial COI gene, corresponding to the DNA-barcoding region. Leftover specimen and DNA were deposited at Chonnam National University with accession numbers CNU15412 for C. jukyungi and CNU15404 for C. burckhardti.

2.2. Genomic DNA Extraction and DNA Barcode Sequencing

Genomic DNA was extracted from adult hind legs using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), isopropyl alcohol, and 70% ethanol, following the manufacturer’s instructions. For confident species identification, a 658-bp DNA barcoding region was amplified using the primer set LCO1490 and HCO2198 [33]. Polymerase chain reaction (PCR) was conducted under the following conditions: initial denaturation for 4 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 51–52 °C, and 1 min at 72 °C, with a subsequent final 7 min extension at 72 °C. AccuPower PCR PreMix (Bioneer, Daejeon, Korea) was used for PCR. To confirm successful DNA amplification, electrophoresis was conducted using 0.5× TAE buffer on a 0.5% agarose gel. PCR products were purified using a PCR Purification Kit (Qiagen, Hilden, Germany). DNA sequencing was performed using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA). All products were sequenced bi-directionally. A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 4 March 2022) showed 100% identity to DNA-barcoding sequences of both C. jukyungi (GenBank no. JF327670) and C. burckhardti (GenBank no. MK039639) that were also sampled from Korea.

2.3. Next-Generation Sequencing

For library construction, 50 ng of genomic DNA was isolated and randomly sheared using the Covaris System (Woburn, MA, USA) to generate inserts of ~300 bp fragments. Library construction was performed using the TruSeq Nano DNA Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The quality and size of DNA libraries were assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). Libraries were quantified by qPCR using the CFX96 Real-Time System (BioRad, Hercules, CA, USA). After normalization, sequencing of the prepared library was conducted on the MGISEQ-2000 sequencing platform (MGI Tech Co. Ltd., Shenzhen, China). A quality analysis of the raw sequence data was performed using FastQC software [34]. Adapter sequence reduction and trimming of 5′-and 3′-ends were performed using Skewer ver. 0.2.2. [35]. Base-calling errors or insertions/deletions (indels) were corrected from the filtered set of reads using the alignment-based error correction tool Karect [36]. Consequently, ~3.99 giga-bases (Gb) of nucleotides from 133 million C. jukyungi reads and ~4.38 Gb of nucleotides from 146 million C. burckhardti reads were obtained. The Phred quality score (Q) indicated that base call accuracy was 87.75% for C. jukyungi and 88.15% for C. burckhardti from the Q30 score.

2.4. Assembly and Gap Filling

The two mitogenomes were constructed using MITObim ver. 1.9 [37] by de novo assembly of the Illumina reads with Cacopsylla citrisuga [25]. The assembled mitogenomes were remapped with the whole genome sequence reads using Bowtie2 [38] before conducting manual curation. Mismatch calling and correction of the assembled sequences were conducted using GATK [39]. Finally, primary annotation of protein-coding genes (PCGs), tRNAs, rRNAs, and the A+T-rich region of each mitogenome was carried out using the MITOS Web Server [40] (http://mitos.bioinf.uni-leipzig.de/index.py, accessed on 10 April 2022).
Gap filling was usually not necessary, but the C. jukyungi A+T-rich region was unexpectedly long (1282 bp) and needed confirmation. Thus, a long fragment (LF) encompassing lrRNA and COI, in which the A+T-rich region is included, was amplified using a primer designed from the C. jukyungi mitogenome. Amplification of the LF was conducted using LA Taq (Takara Biomedical, Tokyo, Japan) under the following conditions: 94 °C for 4 min, 30 cycles at 98 °C for 10 s and 48 °C for 15 min, and a final extension step at 72 °C for 10 min. The PCR product was purified with a PCR purification kit (Bioneer, Daejeon, Korea) and sequenced using barcode-tagged sequencing technology (Celemics, Inc., Seoul, Korea) and the Illumina MiSeq platform (Illumina, San Diego, CA, USA).

2.5. Gene Annotation

The identification, boundary delimitation, and secondary structure folding of tRNAs were performed using the MITOS Web Server by following the protocols presented by Cameron [41]. Twenty-one tRNA genes were well-identified, but trnS1, which has a truncated dihydrouridine (DHU) arm, was detected using a hand-drawn secondary structure in conjunction with an alignment of the predicted trnS1 regions of C. citrisuga [25]. Start and stop codons of PCGs were further confirmed by alignment against mitochondrial PCGs of Psyllidae species. The nucleotide sequences of the PCGs were translated based on the invertebrate mitochondrial DNA (mtDNA) genetic code. Sequence data were deposited into the GenBank database under accession numbers ON553958 for C. jukyungi and ON411626 for C. burckhardti.

2.6. Comparative Genome Analyses

For the comparative analysis, 11 Psyllidae mitogenomes were downloaded from the GenBank database. These sequences, along with the two mitogenome sequences obtained in the present study, were compared for several genomic characteristics. The A/T content of each gene and whole genome was calculated using DNASTAR (Madison, WI, USA). Codon usage was determined by MEGA 6 [42], and the gene overlap and intergenic spacer sequences were hand-counted. The genetic distance for each gene was calculated at within-Psyllidae and within-Cacopsylla levels using unrooted pairwise distances estimated with PAUP ver. 4.01b10 [43]. Genetic distances were then plotted with minimum, maximum, and median values presented with boxplots using JMP software ver. 15.0.0 (SAS Institute, Carry, NC, USA).

3. Results

3.1. General Mitochondrial Genome Features

Mitogenome sizes were 15,438 bp in C. jukyungi and 14,799 bp in C. burckhardti (Table 1). These sizes are within the range previously reported for complete Psyllidae mitogenomes, which range from 14,790 bp (Arytainilla spartiophila) to 16,047 bp (Russelliana solanicola) (Table 2). The two mitogenomes contained 37 genes (13 PCGs, 22 tRNA genes, and two rRNA genes), which is typical in animals, and one major non-coding A+T-rich region [44]. In both species, thirteen PCGs had the typical ATN start codon, whereas the stop codons were incomplete in COII, ND5, and ND4 in C. jukyungi and COII, COIII, ND5, and ND4 in C. burckhardti; in these cases, stop codons were present only as a single thymine (Table 1). The gene arrangement was identical to that typically observed in other insects [41].
The number of codons of the two species (3598 and 3597 in C. jukyungi and C. burckhardti, respectively) was well within the range found in other Psyllidae species (Table 2). Regions with the highest A/T content were, in descending order, the A+T-rich region, 82.20%; srRNA, 77.06%; lrRNA, 76.40%; tRNAs, 75.29%; whole genome, 73.69%; and PCGs, 72.10% in C. jukyungi. On the other hand, C. burckhardti A/T content was greater in tRNAs (76.38%) than in lrRNA (76.09%) compared to that of C. jukyungi. Similarly, other species of Psyllidae also showed variable order in A/T content among genes, such that srRNA, lrRNA, and tRNAs were variable, whereas those of the A+T-rich region, whole genome, and PCGs were consistent (Table 2).
A total of 22 tRNA genes (one for each amino acid and two for leucine and serine) were identified for each mitogenome sequenced in this study (Figure S1 for C. jukyungi and Figure S2 for C. burckhardti). All tRNAs except trnS1, which lacked the DHU loop, folded into the expected cloverleaf secondary structures in both species. This incomplete trnS1 structure has been detected frequently in the mitogenomes of other animals, including insects [45]. The postulated tRNA cloverleaf structure of C. jukyungi and C. burckhardti harbors an invariant 7 bp in the aminoacyl stem, 5 bp in the anticodon stem, and 7 bp in the anticodon loop, whereas the DHU and TΨC arms, particularly within the loops, were variable in length (3–5 bp in stems, but 0 bp in trnS1 DHU stems; 3–9 bp in loops; Figures S1 and S2). A total of 38 and 40 unmatched base pairs were detected in C. jukyungi and C. burckhardti tRNAs, respectively. Of these unmatched base pairs, 18 and 19 were G–U base-pairs, while 20 and 21 were non-Watson–Crick in C. jukyungi and C. burckhardti, respectively. The biased A/T content shown in the whole genome and genes reflects a biased usage of A/T-containing codons (Table 3). Among 64 codons, TTA (leucine), ATT (isoleucine), TTT (phenylalanine), and ATA (methionine), which are composed only of A and T nucleotides, were most frequently used, accounting for 31.43% of codons in C. jukyungi and 32.0% in C. burckhardti. In other species of Psyllidae, this bias is also obvious, with the frequency of these A/T-containing codons ranging from 28.91% in Cacopsylla coccinea to 36.59% in a Heteropsylla sp. (Table 3).

3.2. Intergenic Spacer Sequences

The genes of C. jukyungi and C. burckhardti mitogenomes were interleaved with 84 and 53 bp intergenic spacer sequences (ISSs), which were spread over 13 and 10 regions, respectively, ranging in size from 1 to 27 bp in both species (Table 4). In contrast, genes (including the A+T-rich region) overlapped by 39 and 43 bp in 9 and 10 regions in C. jukyungi and C. burckhardti, respectively, with the size of these overlapping sequences ranging from 1–12 bp in both species (Table 4). The majority of ISS are short (1–10 bp), with a few exceptions, and variable in their position and length. Other species of Psyllidae also present similar overlapping sequence and ISS patterns to those of the two species sequenced in this study, with some notable exceptions. Cyamophila willieti [26] has an exceptionally long 871 bp ISS at the trnI–trnQ junction, whereas other species have either no ISS, a 2–4-bp long ISS, or a 3-bp long overlap at this junction. Furthermore, Freysuila caesalpiniae and R. solanicola [30] also have longer ISS at the trnS2 and ND1 junction (273 and 469 bp, respectively; Table 4). An ISS at the trnS2 and ND1 junction is common in all species of Psyllidae except F. caesalpiniae and R. solanicola. The length of this ISS is highly conserved at 23–36 bp, with the length in the two species considered in this study both being 27 bp. Sequence alignment of this junction allowed us to detect a well-conserved stretch of penta-nucleotides, CGGTA, at the trnS2 and ND1 junction of all species, regardless of ISS length (Figure 1).

3.3. The A+T-Rich Region

The A+T-rich region of C. jukyungi was 1282 bp in size, which is nearly two-fold larger than that of C. burckhardti (662 bp; Table 2). Likewise, the size of this region is highly variable among species in Psyllidae, ranging from 355 bp (Heteropsylla sp.) to 1430 bp (R. solanicola) (Table 2). The C. jukyungi sequenced in this study showed exceptional structure, having the second-longest A+T-rich region in the family. It had two identical 540-bp repeat sequences surrounded by 46, 52, and 104 bp of non-repeat sequences, which do not have any homology (Figure 2A).
The longest R. solanicola A+T-rich region is more notable, having both a tandem repeat region and a peculiar poly-running microsatellite DNA-like region (Figure 2B). The tandem repeat region consisted of nine complete copies (49 bp) and one partial copy (28 bp), which lacks the end part. Eight of the nine copies are identical, but the fifth copy has one substitution (C ⇨ T) compared to the common copy. This repeat region is surrounded by 184 and 59 bp of non-repeat sequences. This 460-bp long repeat region is one source of longer sequences in this species, but the species additionally has a microsatellite DNA-like region (Figure 2B). This microsatellite-like region is unusual in Psyllidae, in that each copy is blended with different types of nucleotide repeats, such as CA, TA, GT, and ATA, infrequently interrupted by non-repeat sequences. This region also spans ~323 bp, making it another source of longer A+T-rich regions in this species. In other species of Psyllidae, such a microsatellite-like region also is present, but the length is much shorter than that of R. solanicola and is not blended with different nucleotide repeats as much as those of R. solanicola (data not shown).
In a comparison of the A+T-rich region, one highly conserved sequence stretch (conserved element [CE]), which spans ~33 bp, was detectable in Psyllidae, including in the two species analyzed in this study (Figure 2C). Located closer to the 5′-end of srRNA (beginning of the A+T-rich region), the CE was composed of poly-T and poly-A sequences, each of which abuts non-poly-running sequences. This CE was present in all species of Psyllidae, and was highly conserved in sequence identity and position in the family. In the case of C. jukyungi, which has two identical 540-bp repeat sequences, this CE also was duplicated within the repeats (Figure 2A).

3.4. Individual Gene Divergence

The genes srRNA, lrRNA, and COI, listed in order of increasing variation, provided the lowest variation of the 15 mitochondrial genes (13 PCGs and 2 rRNA genes) in the family Psyllidae (average median: 15.13–17.26%; Figure 3A). On the other hand, ATP8 provided the most variation, followed by ND6 and ND2 (average median: 36.89–27.55%). In the genus Cacopsylla, srRNA, lrRNA, and COI once again provided the lowest variation (10.86–14.86%), whereas ATP8, ND6, ND3 (=ND5), listed in order of decreasing variation, provided the highest variation (31.19–21.82%). Between two geographic samples of C. burckhardti, only seven genes provided variation, ranging from 1–4 bp, with the first- and the second-highest variation existing in ND5 (4 bp) and COI (3 bp), respectively (Figure 3B).

4. Discussion

4.1. Potential Motif Sequences in Intergenic Spacer Sequences

In contrast to the variability in length and position of the ISSs, one located at the trnS2 and ND1 junction is conserved in majority of Psyllidae and, more importantly, all species of Psyllidae consistently have penta-nucleotides, CGGTA at the junction (Figure 1). Considering that the evolutionary pressure on nucleotide substitution is higher in the ISS than in genic regions, an identical sequence stretch in an ISS may not be maintained in all members of Psyllidae if no functional role is granted. Previously, the ISS at the trnS2 and ND1 junction in insects was reported to contain conserved motif sequences, specifically TTAGTAT in the order Lepidoptera, TAGTA in the order Coleoptera, and TAGTA in the hemipteran superfamily Fulgoroidea, with a slight sequence alteration in some species [17,46,47,48]. This motif sequence has been noted for its role as a binding site for the mitochondrial transcription termination peptides (mtTERM), which terminate the transcription of PCGs [49,50]. Consistently located after CytB, which is the last PCG encoded in the major strand in the circular mitogenome, the motif sequence was identified as the site responsible for the binding of the mtTERM that signals the termination of transcription after CytB is transcribed. Currently, we strongly speculate that these penta-nucleotide stretches found in Psyllidae may have such a functional role; however, further sampling of this region, along with studies of the region’s functional role, will be required to determine the precise location and length of the motif.

4.2. The A+T-Rich Region Structure and Conserved Element

The size variation of the A+T-rich region among Psyllidae was substantial (355–1430 bp; Table 2), and such large variation resulted from the presence of large repeat sequences in C. burckhardti and R. solanicola (Figure 2A,B). One of the common interpretations of the occurrence of such identical repeats in animal mitogenomes includes slipped-strand mispairing, in concert with unequal crossing over during DNA replication, resulting in an expanded repeat [51,52,53].
Previous studies have analyzed the A+T-rich region of some hemipteran groups, although no species in Psyllidae were considered [17,54,55]. These studies also found that hemipteran species often have a long A+T-rich region (>1500 bp in most and >2000 bp in a few species) and have tandem repeat sequences of variable copy number and length. For example, three species of Flatidae in the hemipteran infraorder Fulgoromorpha had 1702–1836 bp-long A+T-rich regions. These A+T-rich regions are commonly structured into four smaller regions, composed of one repeat region, a large non-repeat region, another repeat region, and a short non-repeat region [17]. However, copy number and length in each repeat region differed among all species, although each species commonly had four dividable regions. Therefore, the structural composition of the A+T-rich region in the Fulgoromorpha, particularly Flatidae, may require further analysis before generalizations can be made. Similarly, generalizing about the A+T-rich region in Psyllidae also is difficult in that, for the two species that have the longest A+T-rich regions (R. solanicola and C. jukyungi), these regions’ structure differs. In addition, other species in Psyllidae do not have any notable repeat elements. Thus, an extended exploration of the A+T-rich region of Psyllidae will be necessary to generalize the structural composition of this region across the family.
Due to the high variability in length and composition of the A+T-rich region in Hemiptera, detection of CE, except for simple poly-running sequences, has not been feasible [17,54,55]. However, a comparison of this region allowed us to detect a CE within the region (Figure 2C). Such a CE in the insect A+T-rich region has been searched for because it contains a stretch of sequences that are responsible for functional roles such as signaling for replication and transcription initiation [56,57,58]. In the insect order Lepidoptera, an ATAGA motif that is located immediately upstream of the poly-T sequence, close to the 5′-end of srRNA in the A+T-rich region, has been suggested to be the precise position of the origin of replication. The poly-T sequence has been suggested to function as a structural signal for protein recognition in the initiation of replication for minor-strand mtDNA in Bombyx mori Linnaeus (Bombycidae: Lepidoptera) [58]. Indeed, both the motif and poly-T sequences have been found to be well-conserved in diverse taxonomic groups within Lepidoptera, although the length of poly-T sequence varies [24,47,59]. Currently, no functional study of the hemipteran mitogenome that includes the CE of the A+T-rich region is available. However, the findings that the CE was located at a relatively conserved position with high sequence homology in all species of Psyllidae may suggest that the CE has a functional signal, although current understanding is too limited to make such a conclusion.

4.3. Individual Gene Divergence

The species in the genus Cacopsylla have relatively higher sequence variation even in the genes with lower variation (10.86–14.86% in srRNA, lrRNA, and COI). Thus, the result of current mitogenome comparison emphasizes that pest species identification in the genus can casually be possible with any mitochondrial gene, although two other Cacopsylla species occurring in Korea (C. maculatili and C. sandolbaea) are additionally required for their mitogenome sequences for further accurate estimation of sequence divergence. Given the limited availability in mitogenome sequences for conspecifics, further data are required, but available data indicate that COI and ND5, which were not highly variable relative to other genes at both the family and genus levels, were the most variable genes at the intra-species level. These results suggest that evolutionary pressure for nucleotide substitution among mitochondrial genes differs among taxonomic levels. Thus, for population-level studies, particularly those involving C. burckhardti, COI and ND5 could be suitable markers to uncover population structures if mtDNA is considered. On the other hand, ATP8 and ND6 can be considered for phylogenetic inference up to the family level if gene segments of mtDNA are considered.
In the case of Fulgoroidea, in which 2–8 mitogenomes are available per species, a higher genetic divergence was detected in ATP6, ND5, and COI in Laodelphax striatellus; ND5, lrRNA, and COIII in Lycorma delicatula; ND2, ND4L, and ATP6 in Nilaparvata lugens; and ND4, ND2, and srRNA in Sogatella furcifera [17]. This result differs from that of the C. burckhardti mitogenome, for which the three most variable genes were ND5, COI, and COIII, although these genes appeared at least once as highly variable genes in L. striatellus and L. delicatula. These results further reinforce the importance of understanding variability among mitochondrial genes of target species to draw meaningful results in the study of intraspecific variation. One of the utilities of mitogenome sequences is their role as molecular markers for intraspecific variation. These markers are involved in diagnostics (e.g., origins of inset-driven foods), history tracing (e.g., origins of invasive populations, genetically compatible populations for restoration), and population dynamics (e.g., genetic diversity and gene flow), in which one or more portions of the mitochondrial genic regions are used [17,18,22,23,24,60,61]. In such studies, COI is often used to trace population history and dynamics as well as conduct species identification using DNA barcoding [21,62]. Nevertheless, this gene alone infrequently provided low variability and low numbers of haplotypes within species, particularly in introduced species [24,63,64,65,66,67]. In such cases, understanding the variability among mitochondrial genes may provide the knowledge needed to select the proper genes for the study of population structure and diversity.

5. Conclusions

The addition of two mitogenome sequences to the Psyllidae family that contains notorious pear pests will enhance our understanding of the mitogenome characteristics of the family, particularly by comparative approach, which we employed firstly for the family in this study. The Psyllidae including the two mitogenomes sequenced in this study have several features shared with other members (e.g., codon usage, arrangement, presence of motif sequences at the trnS2 and ND1 junction, and presence of a conserved element in the A+T-rich region). However, the family Psyllidae, in detail, has divergent characteristics compared to non-Psyllidae members: conserved CGGTA at the trnS2 and ND1 junction, a conserved element in the A+T-rich region, and different groups of genes with higher or lower intra-specific variation. Furthermore, the C. jukyungi sequenced in this study is unique in that the A+T-rich region has two identical 540-bp repeat sequences surrounded by non-repeat sequences, whereas the majority of other species in Psyllidae do not have repeat sequences. Our data suggest that sequencing of C. jukyungi and C. burckhardti mitogenomes and comparing their genomic characteristics to the family Psyllidae offers a promising avenue for pursuing further mitogenome sequences of the family members and extended taxonomic groups for a better understanding of the evolution of mitogenome characteristics.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/agronomy12092037/s1, Figure S1: Predicted secondary cloverleaf structures for the tRNA genes of Cacopsylla jukyungi; Figure S2: Predicted secondary cloverleaf structures for the tRNA genes of Cacopsylla burckhardti.

Author Contributions

Conceptualization, A.R.K., H.-J.S., J.-H.S., K.-H.W., and I.K.; Data curation, M.J.K., and I.K.; Formal analysis, M.J.K., J.S.P., and E.D.C.; Funding acquisition, A.R.K., H.-J.S., J.-H.S., and K.-H.W.; Investigation, I.K.; Methodology, I.K.; Project administration, A.R.K., and K.-H.W.; Resources, A.R.K., H.-J.S., J.-H.S., E.D.C., and I.K.; Supervision, H.-J.S., J.-H.S., and K.-H.W.; Validation, M.J.K. and I.K.; Visualization, M.J.K., J.S.P., and I.K.; Writing—original draft, A.R.K., M.J.K., H.-J.S., J.-H.S., and I.K.; Writing—review & editing, A.R.K., M.J.K., J.S.P., H.-J.S., J.-H.S., and I.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from the National Institute of Horticultural and Herbal Science (NIHHS) funded by the Rural Development Administration (RDA) of the Republic of Korea [PJ01509505].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome sequence data supporting this study’s findings are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov under the accession nos. ON553958 for C. jukyungi and ON411626 for C. burckhardti. The associated BioProject, Sequence Read Archive, and Bio-Sample numbers are PRJNA843483 for both species, SRX16308894 and SAMN29783646 for C. jukyungi, and SRX15496259 and SAMN28744766 for C. burckhardti, respectively.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Alignment of the internal spacer located between trnS2 and ND1 of 13 Psyllidae species, including Cacopsylla jukyungi and C. burckhardti, which were considered in this study. The shaded nucleotides indicate a conserved stretch of penta-nucleotides (CGGTA). The underlined nucleotides indicate the adjacent partial sequences of trnS2 and ND1. The arrows indicate the transcriptional direction. *, consensus sequences.
Figure 1. Alignment of the internal spacer located between trnS2 and ND1 of 13 Psyllidae species, including Cacopsylla jukyungi and C. burckhardti, which were considered in this study. The shaded nucleotides indicate a conserved stretch of penta-nucleotides (CGGTA). The underlined nucleotides indicate the adjacent partial sequences of trnS2 and ND1. The arrows indicate the transcriptional direction. *, consensus sequences.
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Figure 2. Schematic map of the two longest A+T-rich regions detected in Psyllidae species. (A) Cacopsylla jukyungi (this study). (B) Russelliana solanicola (Percy et al., 2018). Yellow boxes, copies with substitutions. (C) An alignment of a conserved element (CE) detected in the A+T-rich region of Psyllidae. The arrows indicate the transcriptional direction of the neighboring two genes. *, consensus sequences.
Figure 2. Schematic map of the two longest A+T-rich regions detected in Psyllidae species. (A) Cacopsylla jukyungi (this study). (B) Russelliana solanicola (Percy et al., 2018). Yellow boxes, copies with substitutions. (C) An alignment of a conserved element (CE) detected in the A+T-rich region of Psyllidae. The arrows indicate the transcriptional direction of the neighboring two genes. *, consensus sequences.
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Figure 3. Sequence divergence of each protein-coding gene (PCG) and rRNA in Psyllidae. (A) Boxplot distribution of within-genus (Cacopsylla) and within-family (Psyllidae) genetic divergence for 13 PCGs and two rRNAs in Psyllidae. Top, middle, and bottom of bars indicate maximum, average, and minimum divergence (%), respectively; the red horizontal line represents the median of sequence divergence (%) in Psyllidae. (B) Divergence between two Cacopsylla burckhardti.
Figure 3. Sequence divergence of each protein-coding gene (PCG) and rRNA in Psyllidae. (A) Boxplot distribution of within-genus (Cacopsylla) and within-family (Psyllidae) genetic divergence for 13 PCGs and two rRNAs in Psyllidae. Top, middle, and bottom of bars indicate maximum, average, and minimum divergence (%), respectively; the red horizontal line represents the median of sequence divergence (%) in Psyllidae. (B) Divergence between two Cacopsylla burckhardti.
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Table
Table 1. Summary of Cacopsylla jukyungi and C. burckhardti mitochondrial genomes.
Table 1. Summary of Cacopsylla jukyungi and C. burckhardti mitochondrial genomes.
Gene* D** ACCacopsylla jukyungiCacopsylla burckhardti
Nucleotide
Position (Size)		Anticodon PositionStart/Stop
Codon		Nucleotide
Position (Size)		Anticodon PositionStart/Stop
Codon
trnIFGAT1–66 (66)33–35 1–66 (66)33–35
trnQRTTG71–136 (66)106–108 71–136 (66)106–108
trnMFCAT142–207 (66)174–176 142–206 (65)174–176
ND2F 208–1179 (972) ATA/TAA207–1178 (972) ATA/TAA
trnWFTCA1202–1264 (63)1232–1234 1177–1239 (63)1208–1210
trnCRGCA1266–1326 (61)1295–1297 1242–1302 (61)1271–1273
trnYRGTA1328–1389 (62)1358–1360 1303–1364 (62)1333–1335
COIF 1390–2922 (1533) ATG/TAA1366–2898 (1533) ATG/TAA
trnL2FTAA2922–2989 (68)2953–2955 2898–2961 (64)2928–2930
COIIF 2990–3653 (664) ATA/T2962–3625 (664) ATA/T
trnKFCTT3654–3723 (70)3685–3687 3626–3695 (70)3657–3659
trnDFGTC3729–3794 (66)3763–3765 3694–3754 (61)3724–3726
ATP8F 3795–3947 (153) ATC/TAA3755–3907 (153) ATT/TAA
ATP6F 3941–4615 (675) ATG/TAA3901–4575 (675) ATG/TAA
COIIIF 4615–5394 (780) ATG/TAG4575–5352 (778) ATG/T
trnGFTCC5400–5457 (58)5430–5432 5353–5414 (62)5387–5389
ND3F 5458–5808 (351) ATA/TAA5415–5765 (351) ATT/TAA
trnAFTGC5811–5870 (60)5839–5841 5767–5827 (61)5795–5797
trnRFTCG5872–5932 (61)5903–5905 5831–5891 (61)5862–5864
trnNFGTT5932–5998 (67)5962–5964 5891–5955 (65)5921–5923
trnS1FGCT5999–6052 (54)6017–6019 5956–6009 (54)5974–5976
trnEFTTC6053–6114 (62)6085–6087 6010–6069 (60)6040–6042
trnFRGAA6103–6165 (63)6133–6135 6058–6120 (63)6088–6090
ND5R 6166–7786 (1621) ATT/T6121–7741 (1621) ATT/T
trnHRGTG7786–7842 (57)7812–7814 7739–7800 (62)7767–7769
ND4R 7845–9087 (1243) ATG/T7801–9049 (1249) ATG/T
ND4LR 9081–9362 (282) ATT/TAG9043–9324 (282) ATT/TAG
trnTFTGT9370–9428 (59)9400–9402 9332–9392 (61)9364–9366
trnPRTGG9429–9490 (62)9458–9460 9393–9454 (62)9422–9424
ND6F 9493–9978 (486) ATT/TAA9457–9942 (486) ATA/TAA
CytBF 9972–11,117 (1146) ATA/TAG9936–11,078 (1143) ATA/TAA
trnS2FTGA11116–11,177 (62)11,146–11,148 11,079–11,142 (64)11,109–11,111
ND1R 11,205–12,119 (915) ATA/TAG11,170–12,084 (915) ATA/TAA
trnL1RTAG12,120–12,182 (63)12,151–12,153 12,085–12,147 (63)12,116–12,118
lrRNAR 12,183–13,339 (1157) 12,148–13,297 (1150)
trnVRTAC13340–13402 (63)13,371–13,373 13,298–13,359 (62)13,328–13,330
srRNAR 13,403–14,156 (754) 13,361–14,137 (777)
A+T-rich region14,157–15,438 (1282) 14,138–14,799 (662)
tRNAs are denoted as one-letter symbols in accordance with the IUPAC-IUB single-letter amino acid codes, except those encoding leucine and serine, which are labeled L1 for the CTN codon family, L2 for the TTR codon family, S1 for the AGN codon family, and S2 for the TCN codon family. * D, direction (F and R, forward and reverse transcriptional directions, respectively) and ** AC, anticodon.
Table
Table 2. Characteristics of 13 mitochondrial genomes of Psyllidae species.
Table 2. Characteristics of 13 mitochondrial genomes of Psyllidae species.
TaxonSize (bp)A/T Content (%)PCGsrRNAlrRNAtRNAA+T-Rich RegionGenBank
Accession No.		References
No. Codons aAT (%)Size (bp)AT (%)Size (bp)AT (%)Size (bp)AT (%)Size (bp)AT (%)
Psyllidae
Acizzinae
Acizzia uncatoides14,95772.75359670.6874777.24114575.9140475.7878985.17MG989217[29]
Aphalaroidinae
Freysuila caesalpiniae15,32775.62359874.7774677.21114677.31138776.3594783.95MG989225[29]
Russelliana solanicola16,04772.23359770.9374775.64114776.20137275.73143079.16MG989236[29]
Ciriacreminae
Heteropsylla sp.15,28477.06359775.3274879.68115179.15140177.6635591.83MG989226[29]
Psyllinae
Arytainilla spartiophila14,79075.73359574.5874477.55114677.84137376.3369686.78MG989220[29]
Cacopsylla jukyungi15,43873.69359872.1075477.06115776.40138075.29128282.20ON553958This study
Cacopsylla burckhardti14,79973.69359772.2077777.48115076.09137676.3866282.93ON411626This study
Cacopsylla burckhardti14,79873.69361272.1877677.45115076.17137776.3366283.08OK574466[27]
Cacopsylla citrisuga14,90672.65360871.0378077.56115675.61137975.3473983.76MT990978[25]
Cacopsylla coccinea14,83272.04360170.4477378.01115476.43138374.6969579.28KP245955[28]
Cacopsylla pyri14,88673.85359772.3475577.35115577.75138576.7566082.42MG989222[29]
Cyamophila willieti15,80973.84359772.0974477.28117776.13138774.8484480.69MN364946[28]
Psylla alni14,97173.66359872.0471176.93114577.73136775.4989182.72MG989235[29]
Average15,14273.88359972.3675477.42115276.82138275.9281983.38
a Termination codons were excluded from the total codon count.
Table
Table 3. Frequency of the four most common codons in Psyllidae.
Table 3. Frequency of the four most common codons in Psyllidae.
SpeciesCodon (No/%)
TTA (L)ATT (I)TTT (F)ATA (M)Total
Acizzinae
   Acizzia uncatoides258/7.17293/8.11276/7.64224/6.201051/29.10
Aphalaroidinae
   Freysuila caesalpiniae328/9.12384/10.67280/7.78256/7.121248/34.69
   Russelliana solanicola287/7.98312/8.67244/6.78223/6.201066/29.64
Ciriacreminae
  Heteropsylla sp.392/10.90368/10.23316/8.79240/6.671316/36.59
Psyllinae
   Arytainilla spartiophila316/8.79354/9.85337/9.37204/5.671211/33.69
  Cacopsylla jukyungi (this study)316/8.78329/9.14292/8.12194/5.391131/31.43
  Cacopsylla burckhardti (this study)315/8.76325/9.04299/8.31212/5.891151/32.00
   Cacopsylla burckhardti315/8.72324/8.97300/8.31 213/5.90 1152/31.89
   Cacopsylla citrisuga321/8.90297/8.23296/8.20188/5.211120/30.54
   Cacopsylla coccinea271/7.53287/7.97281/7.80202/5.611041/28.91
   Cacopsylla pyri311/8.65326/9.06295/8.20208/5.781140/31.69
   Cyamophila willieti284/7.90337/9.37281/7.81202/5.621104/30.69
   Psylla alni307/8.53339/9.42287/7.98192/5.341125/31.27
Average309/8.59329/9.13291/8.08212/5.891143/31.70
Table
Table 4. Overlapping and intergenic spacer sequences in the mitochondrial genomes of Psyllidae species.
Table 4. Overlapping and intergenic spacer sequences in the mitochondrial genomes of Psyllidae species.
RegionABCDEFGHIJKLM
trnI-trnQ(I)4(I)4 (I)4(I)4(I)4(I)2(I)4(I)871(I)4(O)3(O)3(O)3
trnQ-trnM(I)5(I)5(I)12(I)5(I)5(I)5(I)3(I)4(I)1(I)4(O)1(I)2(O)1
trnM-ND2 (I)5 (I)1(I)1(I)1 (O)1(I)1(I)1(I)1
ND2-trnW(I)22(O)2 (I)36(I)22(I)46(O)3 (O)1(I)2(O)3(I)7
trnW-trnC(I)1(I)2(O)2(I)2(I)3(I)1(I)4(I)2(I)1(I)6 (I)1(I)2
trnC-trnY(I)1 (I)2 (I)1 (I)5(I)1
trnY-COI (I)1 (I)4(I)1(I)1(I)4(I)2 (I)4(I)9(I)2(I)15
COI-trnL2(O)1(O)1(O)1 (O)1(I)3(O)2(O)6 (I)3(O)6(I)5(O)2
trnL2-COII (O)1 (I)1(I)1(I)1 (O)1(I)1(I)1(I)1
COII-trnK (I)1 (O)1(O)1(O)1 (I)1(O)1(O)1(O)1
trnK-trnD(I)5(O)2(O)2(I)17(I)3(I)5(O)2(I)2(O)2(O)2 (O)2
trnD-ATP8 (I)9(I)9(I)1(I)1(I)1 (O)1(I)1(O)7(I)1
ATP8-ATP6(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7
ATP6-COIII(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1(O)1
COIII-trnG(I)5 (O)1(I)2(O)1(O)1(I)1(I)1(O)1(I)2(O)1
trnG-ND3 (O)3(I)1(I)1(I)1 (I)2(I)1(I)1(I)1
ND3-trnA(I)2(I)1(I)1(O)1 (I)3(O)3(I)2 (O)1(O)3(I)1(O)3
trnA-trnR(I)1(I)3(I)3(I)1(I)1(I)1 (I)1(I)1(I)1 (I)6
trnR-trnN(O)1(O)1(O)1(O)1(O)1 (O)1 (O)1(O)1 (I)4(O)1
trnN-trnS1 (O)2 (O)2 (O)2
trnS1-trnE (O)5(I)1 (I)2(I)2 (O)5(I)1(I)6(I)3(I)3
trnE-trnF(O)12(O)12(O)12(O)12(O)12(O)12(O)12(I)1(O)12(O)12(O)13 (I)1
trnF-ND5 (I)1(I)2(O)1 (O)1(O)1(O)1(O)1
ND5-trnH(O)1(O)3 (O)3(O)1(O)1(O)1 (I)1(O)1(O)1(I)8
trnH-ND4(I)2 (O)5(O)4(O)4(O)4 (I)1 (O)4
ND4-ND4L(O)7(O)7(O)49(O)49(O)49(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7
ND4L-trnT(I)7(I)7(I)1(I)1(I)28 (I)1(I)2 (I)1
trnT-trnP (O)1(O)1
trnP-ND6(I)2(I)2(I)2(I)2(I)2(I)3(I)3(I)3(I)2(I)1(I)3(I)2(I)4
ND6-CytB(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7(O)7
CytB-trnS2(O)2 (O)2(O)2(I)3(O)3(O)3 (O)1(O)2(O)2(O)3
trnS2-ND1(I)27(I)27(I)27(I)26(I)28(I)28(I)23(I)27(I)26(I)36(I)273(I)33(I)469
ND1-trnL1 (O)1(O)1(O)1 (I)1(O)1(O)1(O)1
trnL1-lrRNA
lrRNA-trnV (O)3 (I)5(I)6(O)21(I)3(I)2(I)1(I)6
trnV-srRNA (I)1(I)1(I)1(I)1 (O)1 (O)1(O)1(O)2(I)2(O)2
srRNA-A+T-rich region (O)1(O)1(O)24 (I)12 (I)20(I)11(I)14(I)12
A+T-rich region-trnI (I)11(I)6(I)11 (I)2(I)23(I)16
Total nucleotides(O)39(O)43(O)90(O)85(O)116(O)41(O)57(O)40(O)66(O)45(O)60(O)48(O)41
(I)84(I)53(I)54(I)110(I)107(I)112(I)70(I)69(I)904(I)94(I)334(I)96(I)539
O, overlapping sequences; and I, intergenic spacer sequences. Empty columns indicate that neighboring genes are abutting to each other. A, Cacopsylla jukyungi (this study); B, Cacopsylla burckhardti (this study); C, Cacopsylla burckhardti; D, Cacopsylla citrisuga; E, Cacopsylla coccinea; F, Cacopsylla pyrisuga; G, Arytainilla spartiophila; H, Psylla alni; I, Cyamophila willieti; J, Acizzia uncatoides; K, Freysuila caesalpiniae; L, Heteropsylla sp.; M, Russelliana solanicola.
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Kang, A.R.; Kim, M.J.; Park, J.S.; Seo, H.-J.; Song, J.-H.; Won, K.-H.; Choi, E.D.; Kim, I. Comparative Analysis of Two Pear Pests, Cacopsylla jukyungi and Cacopsylla burckhardti (Hemiptera: Psyllidae), Based on Complete Mitochondrial Genomes and Comparison to Confamilial Species. Agronomy 2022, 12, 2037. https://doi.org/10.3390/agronomy12092037

AMA Style

Kang AR, Kim MJ, Park JS, Seo H-J, Song J-H, Won K-H, Choi ED, Kim I. Comparative Analysis of Two Pear Pests, Cacopsylla jukyungi and Cacopsylla burckhardti (Hemiptera: Psyllidae), Based on Complete Mitochondrial Genomes and Comparison to Confamilial Species. Agronomy. 2022; 12(9):2037. https://doi.org/10.3390/agronomy12092037

Chicago/Turabian Style

Kang, Ah Rang, Min Jee Kim, Jeong Sun Park, Ho-Jin Seo, Jang-Hoon Song, Kyung-Ho Won, Eu Ddeum Choi, and Iksoo Kim. 2022. "Comparative Analysis of Two Pear Pests, Cacopsylla jukyungi and Cacopsylla burckhardti (Hemiptera: Psyllidae), Based on Complete Mitochondrial Genomes and Comparison to Confamilial Species" Agronomy 12, no. 9: 2037. https://doi.org/10.3390/agronomy12092037

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