https://orcid.org/0000-0003-2744-1239
Figures
Abstract
Pentatomidae, the most diverse family of Pentatomoidea, is found worldwide. Currently, the phylogenetic relationships among Pentatomidae tribes remain unstable, and subfamily divergence has not been estimated. Here, we sequenced and analyzed the complete mitochondrial genomes of two species of Lelia, and studied the phylogenetic relationships among Pentatominae tribes. We also selected three available fossil as the calibration points in the family, and preliminarily discussed the divergence time of Pentatomidae. Trees of Pentatomidae were reconstructed using the Bayesian inference method. Divergence times of Pentatominae were estimated based on the nucleotide sequences of protein-coding genes with a relaxed clock log-normal model in BEASTv.1.8.2. The results showed that the gene arrangements, nucleotide composition, and codon preferences were highly conserved in Lelia. Further, a phylogenetic analysis recovered Eysarcorini, Strachiini, Phyllocephalini, and Menidini as monophyletic with strong support, however, the monophyly of Antestiini, Nezarini, Carpocorini, Pentatomini and Cappaeini were rejected. Moreover, Pentatominae diverged from Pentatomidae soon after the origin of the Cretaceous Period, at approximately 110.38 Ma. This study enriches the mitochondrial genome database of Pentatomidae and provides a reference for further phylogenetic studies, and provides a more accurate estimate of divergence time.
Citation: Jia W, Chen J, Ge S, Zhang Z, Xiao Y, Qi L, et al. (2024) Phylogenetic and divergence analysis of Pentatomidae, with a comparison of the mitochondrial genomes of two related species (Hemiptera, Pentatomidae). PLoS ONE 19(10): e0309589. https://doi.org/10.1371/journal.pone.0309589
Editor: Pankaj Bhardwaj, Central University of Punjab, INDIA
Received: April 2, 2024; Accepted: August 15, 2024; Published: October 23, 2024
Copyright: © 2024 Jia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All files are available from the NCBI database (accession number: OR500703 and OR500704.)
Funding: This research was funded by the National Science Foundation of China (No.31872272); Natural Science Research General Project of Shanxi Province (Nos.202103021224331); the Research Project Supported by Shanxi Scholarship Council of China (2024-072).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pentatomidae, proposed by Leach (1815), is the largest family in the superfamily Pentatomoidea, and it contains 940 genera and 4,949 species in 10 subfamilies [1]. These insects are widely distributed and ubiquitous worldwide. Members of Pentatomidae also vary greatly in size; some are very small, only 2–3 millimeters, such as Spermatodes variolosus (Walker, 1867) and Sepontiella aenea (Distant, 1883), whereas others are very large, ranging from 20 to 30 millimeters such as Catacanthus incarnatus (Drury, 1773). Except for the subfamily Asopinae, which includes predatory species, nearly all species of the Pentatomidae are phytophagous, and many species are found in host plants, whereas some have beenfound to feed on dung or carrion. Owing to a lack of unique methods, the identification of subfamilies and tribes of Pentatomidae, and the construction of a stable taxonomic group have become major problems. In recent years, with the development of sequencing technology, molecular data have been widely used in phylogenetic research [2, 3]. Many researchers have studied the phylogenetic relationships between pentatomids at different classification levels. It is mainly through the sequencing of mitochondrial genome to supplement new molecular data information, build phylogenetic tree, and judge the phylogenetic location of sequenced species and the phylogenetic relationships within some taxa. For example, Yuan et al. (2015) [4] elucidated the phylogenetic relationships among 26 species of Pentatomomorpha based on mitochondrial genomes, demonstrating the monophyly of Pentatomoidea. Lian et al. (2022) [5] newly sequenced mitochondrial genome sequences of three species of the Phyllocephalini and analyzed their phylogenetic position within Pentatomidae. Ding et al. (2023) [6] recently sequenced the mitochondrial genome sequences of three Menida species, and clarified the phylogenetic position of Menida in Pentatominae. The phylogenetic research of Pentatomidae has always been a hot topic. Grazia et al. (2008) [7] supported the monophyly of Pentatomidae by a combination of morphological and molecular data. However, Roca-Cusaches et al. (2022) [8] used two mitochondrial genes, namely cox1 and 16S rRNA, and two molecular genes, 28S rRNA and 18S rRNA, to reconstruct a phylogenetic tree of Pentatomidae based on Bayesian Inference (BI) and Maximum Likelihood (ML) methods, and the results denied the monophyly of Pentatomidae. So far, further research is needed on the monophyly and internal relationships of Pentatomidae.
Fossil records provide evidence of the existence of ancient organisms, the most direct and important evidence of biological evolution, but there are also many ambiguous aspects associated with these [9]. Although Pentatomomorpha fossils have been studied for more than 100 years, the number of fossils for this large group remains limited. The earliest known Pentatomomorpha fossils were found in strata for the end of the Late Triassic in Mid west of China and the United Kingdom. Regarding Pentatomomorpha fossils, 14 families, 158 genera and 200 species have been reported [10]. The superfamily Pentatomoidea comprises 18 families worldwide, including two fossil families [1]. However, owing to the incomplete preservation of most fossils and the inability to observe certain key features, it is difficult to fully understand pentatomid origins and evolution [1]. Therefore, it is necessary to use molecular data for such estimations.
The genus Lelia was first established by Walker in 1867. Later, Reuter (1890) [11] solved the taxonomic problems associated with some genera, and Lelia was used as a valid generic name. Currently, it is a small genus containing only three species worldwide. Moreover, it is widely distributed, and can harm several crops. Members of this genus are typically broad and oval-shaped, and interspecific morphological differences can be identified based on the basal angle of the pronotum and scutellum and the number of spots on the dorsal surface of the body. Fan and Liu (2010) [12] performed a morphological study of this genus with a new species reported. Lelia decempunctata (cox1) was used to explore the phylogenetic relationships of Pentatomidae [8]. However, to date, a complete mitochondrial genomes of this genus has not been used to explore its phylogenetic relationships and to estimate its of divergence times.
The mitochondrial genomes of insects are double-stranded circular DNA molecules (15–20kb) consisting of 37 genes, specifically 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a control region [13, 14]. In recent years, sequencing technology has developed rapidly, and increasing numbers of insect mitochondrial genomes have been sequenced. Although the functions and replication of the mitochondrial genome are controlled by the nucleus, due to its stable genetic composition, rapid evolution and relatively complete molecular information, it is still widely used in molecular evolution, phylogeny, population genetic structure and biogeographical research [2, 15].
In this study, we sequenced the whole mitochondrial genomes of two species of Lelia, analyzed the mitochondrial genome characteristics in detail, and determined the secondary structures of 12S rRNA and 16S rRNA. By comparing and analyzing the mitochondrial genome size, nucleotide composition, codon usage, and RNA structure, we further explored the phylogenetic position of among subfamilies within Pentatomidae. In addition, we used three available Pentatomidae fossil as a fossil calibration point and combined with previous research to estimate the divergence time for each tribe and subfamily. The results of this study will provide a reference for the phylogenetic analysis, identification, origin, and evolution of Pentatomidae.
Materials and methods
Sample collection and DNA extraction
Adult specimens of Lelia concavaemargo Fan & Liu, 2010 were collected from Baiyan Village, Mashan Town, Meitan County, Zunyi City, Guizhou Province (28°2′45″N, 107°35′2″E) on May 10, 2020. Adult specimens of L. decempunctata (Motschulsky, 1860) were collected from Yaoluoping Nature Reserve, Yuexi County, Anqing City, Anhui Province (31°0′56″N, 116°7′60″E) on July 29, 2019. All samples were immediately placed in anhydrous ethanol and stored in a refrigerator at −25°C until DNA extraction. Total DNA was extracted from thoracic tissue using a Genomic DNA Extraction Kit (Personalbio, Nanjing, China). The two complete mitogenome were submitted to GenBank (accession numbers: OR500703 and OR500704).
DNA sequencing, assembly, sequence annotation and analyses
A fluorescent dye (Quant it PicoGreen dsDNA Assay Kit) was used to determine the total amount of DNA. The total amount of DNA was 2.39 mg, and the concentration based on fluorescence was 47.80 ng/ml. The genomic library was constructed using the standard Illumina TruSeq Nano DNA LT library preparation process (Illumina TruSeq DNA Sample Preparation Guide). Whole mitochondrial genome sequencing was performed using an Illumina Novaseq 6000 platform with 400bp insert sizes and a read length of PE150. Fastp was used to evaluate the quality of the sequencing data [16]. Mitochondrial genomes were assembled and annotated using Geneious v. 11.0 software [17]. The reference sequence of Pentatoma semiannulata (NC_053653), used for annotation, was obtained from the Basic Local Alignment Search Tool (BLAST) in the NCBI database. The tRNA genes were identified using MITOS (http://mitos.bioinf.uni-leipzig.de/index.py/) with an invertebrate mitochondrial code [18]. The boundaries of the PCGs were determined using the Open Reading Frame Finder on the NCBI website (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The boundaries of the rRNA genes were identified based on the positions of adjacent genes and previously sequenced rRNA genes [19]. The exact location of the control region was determined based on the boundary of the neighboring genes.
The nucleotide composition and codon usage (RSCU) were analyzed using MEGA v. 11.0 [20]. DnaSP6 software [21] was used to enumerate the non-synonymous substitutions (Ka) and synonymous substitutions (Ks) of each PCG and to calculate the Ka/Ks values. Nucleotide skew was calculated as follows: AT skew = (A −T) / (A + T) and GC skew = (G − C) / (G + C) [18, 22, 23]. The Tandem Repeats Finder web server was used to predict the tandem repeat sequences in the control region [13]. To assess the neutral evolution of species of Pentatominae, we calculated the gene lengths and numbers of non-synonymous and synonymous mutations. Linear regression analyses were performed by comparing 13 PCGs, including the relationship between the number of non-synonymous mutations and the length of base alignments and the relationship between the number of synonymous mutations and the length of base alignments.
Phylogenetic analyses
Phylogenetic analyses were conducted using two newly sequenced species and 71 available Pentatomidae taxa from NCBI, with two Tessaratomidae species, Eusthenes cupreus (Westwood, 1837) and Mattiphus splendidus Distant, 1921, as outgroups (Table 1). The nucleotide sequences of the PCGs and two rRNAs were extracted using Geneious v. 11.0. We further imported the extracted genes into PhyloSuite v.1.2.3 [24], selected MAFFT for the alignment, and used MACSE to optimize the alignment results of the PCGs. We used Gblocks to prune the PCG sequences and TrimAL to prune the rRNA sequences. To determine whether the sequences contained phylogenetic information, we tested the nucleotide substitution saturation and plotted transition and transversion rates against the TN93 distances for the PCGs (all codon positions of the 13 PCGs) and PCGRNA (13 PCGs and two rRNAs) datasets, using DAMBE to further validate the feasibility of constructing a phylogenetic tree [25, 26]. The heterogeneity of sequence divergence in the two datasets was analyzed using AliGROOVE with a default sliding window size [27]. ModelFinder v.2.2.0 was used to provide the best-fit model (S1 Table) [28]. MrBayes v.3.2.7 was used to construct the BI tree [29]. Four independent Markov chains (three heated and one cold) were run for 20,000,000 generations, and samples were collected every 1000 generations. The initial 25% of trees were discarded as burn-in after an average standard deviation of less than 0.01. Phylogenetic trees were constructed using the PCGs and PCGRNA datasets, and the generated phylogenetic trees were visualized using the online editing tool Chipolt [30] (https://www.chiplot.online/).
Divergence time estimate
Divergence times in Pentatomidae were estimated using the nucleotide sequences of PCGs with a relaxed clock log-normal model in BEAST version 1.8.2 [59]. The PCGs dataset was partitioned using ModelFinder v.2.2.0 [28], and the optimal nucleotide replacement model for each partition was estimated. Appropriate parameters in BEAUti, GTR model, and Yule prior were set for each subset to generate a runnable XML file in BEAST. To estimate the divergence time calibration, Asopus puncticollis Piton, 1940 (61.6–59.2 Ma), Eurydema Laporte de Castelnau, 1833 (102.24–72.14 Ma) and Pentatomidae (125.0–113.0 Ma), three reported fossils of Pentatomidae, were used to assign the age calibration [60–63]. Tracer v.1.7.2 [64] was used to confirm the convergence of the chain by running the final Markov chain twice every 2×108 generations and sampling every 10,000 generations, with the first 25% of the generations discarded as burn-in. The most effective sample sizes were >200. We used TreeAnnotator v.1.8.4 to obtain the largest branch tree credibility subsample tree. The 95% highest probability density (95%HPD) was displayed using the online editing tool Chipolt [30].
Results
Mitochondrial genomic structure
The total lengths of the Pentatomidae mitogenomes were 14,782–19,587 bp, and those of L. concavaemargo and L. decempunctata were 16,074 and 15,464 bp respectively (Fig 1). The mitogenomes of the two species were determined to be closed circular double-stranded DNA molecules containing 37 genes (13 PCGs, 22 tRNAs, and two rRNAs) and a control region. The arrangement of the mitochondrial genome was consistent with 23 genes located on the J-strand, and 14 genes on the N-strand. Moreover, the longest intergenic spacers (22 and 20 bp) of the two species were detected between nad1 and trnS2, and the longest overlapping region was located between trnW and trnC and had a length of 8 bp (AAGCTTTA). In addition, there were two conserved overlaps, with a 7 bp overlap between atp8/atp6 and nad4/nad4L (ATGATAA) (S2 Table). This has also been observed in other species of the Pentatomidae family. The nucleotide composition of the total mitogenomes showed a strong bias toward A and T bases; further, the AT skew was positive and the GC skew was negative, with A+T contents of 78.14% (L. concavaemargo) and 77.73% (L. decempunctata) (S3 Table).
Protein-coding genes
Similar to those of other Pentatomidae members, the nucleotide compositions of the 13 PCGs of these two species had high AT contents, specifically 77.34% (L. concavaemargo) and 77.38% (L. decempunctata), respectively (S3 Table). In these two species, nine genes were found to be encoded on the major strand (J-strand), whereas the other four were encoded on the minor strand (N-strand). Most PCGs used ATN (ATT/ATA/ATG/ATC) as the initiation codon, but some PCGs (cox1, atp8, nad6, nad1) used TTG as the initiation codon. Most PCGs ended with the complete termination codon TTA, except for cox1 and cox2, which ended with the incomplete stop codon T (S2 Table).
We also calculated the RSCU of PCGs for both species and a similar RSCU pattern was observed (Fig 2). Most of the codons with high frequency ended in A/T, and the most frequently used codon was UUA (Leu2). These results indicated that the codons of the PCGs of Lelia tended to end with A/T.
Codon families are shown on the x-axis and the frequency of RSCU on the y-axis.
In addition, we determined the Ka, Ks, and Ka/Ks ratios for the 13 PCGs of Pentatomidae to explore evolutionary patterns. The Ka/Ks ratio for all 13 PCGs was < 0.71, indicating that these genes were affected by purifying selection. Among the PCGs, atp8 evolved at the fastest rate (Ka/Ks = 0.71), whereas cox1 evolved at the slowest rate (Ka/Ks = 0.07) (Fig 3). Owing to its slow evolution rate, we determined that cox1 can be used for barcode analysis and classification. Linear regression analysis showed that non-synonymous and synonymous changes were significantly correlated with the gene length (R2 = 1.000, 0.996) (Fig 4).
Rate of non-synonymous substitutions (Ka), rate of synonymous substitutions (Ks), and ratio of rate of non-synonymous substitutions to rate of synonymous substitutions (Ka/Ks) are calculated for each PCG.
(a) Correlation between nonsynonymous mutations and length in bases of the genes. (b) Correlation between synonymous mutations and length in bases of the genes.
Transfer and ribosomal RNAs
The total tRNA lengths of L. concavaemargo and L. decempunctata were 1,479 bp and 1,481 bp, respectively (S3 Table). The 22 tRNAs showed high A + T contents of 78.30% (L. concavaemargo) and 78.12% (L. decempunctata), and the lengths of the tRNA genes ranged from 63 bp to 75 bp. Fourteen genes were located on the J-strand, and eight other genes were located on the N-strand (S2 Table). Only trnS1 and trnV lacked the dihydrouridine (DHU) arm, and the remaining 20 tRNA genes formed a typical cloverleaf structure in both species. However, in most Pentatomidae species, only trnS1 lacked the DHU arm. trnR showed the weakest conservation compared to the other genes in the two species of Lelia. Moreover, 17 wobble G-U pairs were found in 22 tRNAs from Lelia (Fig 5).
The conserved sites within Lelia are marked in red.
The total lengths of the two rRNAs were 2,106 bp (L. concavaemargo) and 2,107 bp (L. decempunctata). Moreover, they were encoded on the N-strand and showed high AT contents of 79.63% (L. concavaemargo) and 79.83% (L. decempunctata) (S3 Table). The complete secondary structures are shown in Figs 6 and 7. In Lelia, 16S rRNA contained 92.12% conserved sites and 12S rRNA contained 93.97% conserved sites.
The conserved sites within Lelia are marked in red.
The conserved sites within Lelia are marked in red.
Control ergion
The control region was determined to be the largest non-coding region. In the mitochondrial genome of Pentatomidae, the longest control region was 4,651 bp, and there was a significant difference in length between these two species, with lengths of 1,378 bp (L. concavaemargo) and 762 bp (L. decempunctata). Moreover, the control regions were located between 12S rRNA and trnI. High AT contents of 81.28% (L. concavaemargo) and 73.75% (L. decempunctata) (S3 Table). In L. concavaemargo, five types of tandem repeat units were observed, with lengths of 3–68 bp, however, in L. decempunctata, only one type of tandem repeat unit was found, with a length of 56 bp (S4 Table).
Phylogenetic relationships
We next analyzed the substitution saturation and heterogeneity of the PCGs and PCGRNA dataset, before constructing a phylogenetic tree. The results showed that the Xia saturation index was below the critical values for a symmetric and asymmetric topologies (Iss < Iss.c, p < 0.05) (Fig 8), indicating that the nucleotide sequences of the two datasets were not saturated. The heterogeneity between both sequences is shown in blue, and the lightest part of the blue occurreds between the outgroup and the remaining sequences in the dataset (Fig 9), indicating that these datasets are suitable for further phylogenetic studies.
The substitution saturation analysis of two datasets (a) PCGs (b) PCGRNA.
Analysis of heterogeneity of sequence divergence for (a) PCGs and (b) PCGRNA dataset. The mean similarity score between sequences is represented by colored squares, based on AliGROOVE scores ranging from –1, which indicates a great difference in rates from the remainder of the data set (= heterogeneity, red color) to +1, which indicates rates that matched all other comparisons (blue color, as in this case).
We also constructed phylogenetic trees for Pentatomidae based on two datasets (PCGs and PCGRNA) using BI (Fig 10 and S1 Fig). The results showed that the topological structures of the two trees were reliable and that most clades had high posterior probabilities. A topology of PCG dataset is as follows: (Aeschrocorini + (Neojurtina + (Euschistus + ((Sciocoris + (Graphosoma + Dybowskyia)) + ((Caystrini + (Homalogonia + Halyini)) + (Halyomorpha + (Placosternum + Phyllocephalini))) + (Nezarini + (Anaxilaus + (Glaucias + Antestiini))) + ((Dolycoris + Aelini) + Eysarcorini) + ((Piezodorini + Brachymna) + ((Deroploa + Tholosanus) + Pentatomini)) + (Hoplistodera + Strachiini) + ((Catacanthus + Scotinophara) + (Menidini + Asopinae))))))). The topologies showed that Aeschrocorini was the earliest diverging lineage within Pentatomidae. Eysarcorini, Strachiini, and Menidini were recovered as monophyletic with strong support; however, the monophyly of Antestiini, Nezarini, Carpocorini, Pentatomini, Cappaeini, and Podopinae was rejected. Owing to the limited mitochondrial genomic data available in the NCBI database, the monophyly of the remaining tribes could not be accurately verified.
Numbers on nodes are the posterior probabilities (PP), lower than 0.6 is not displayed.
Divergence time estimate
Based on the topology of Pentatomidae recovered from the BEAST analysis, the age estimates, average, and 95% HPD for each subfamily and tribe are summarized in Fig 11. We used three type fossil information within the Pentatomidae to analyze and update the divergence time of family. Asopus puncticollis and Eurydema Laporte, as crown groups, and Pentatomidae as a stem groups showed that the divergence of Pentatomidae occurred from the Cretaceous to the Quaternary period.
Horizontal bars represent 95% credibility intervals of time estimates. Numbers on the nodes indicate the mean divergence times. The calibration point is the red dot in the figure.
Further, Pentatominae diverged from Pentatomidae soon after the origin of the Cretaceous period, at approximately 110.38 Ma (95% HPD: 138.38–83.77 Ma). After a period of evolution, Aeschrocorini and N. typica were the earliest to diverge, with a divergence time of 89.15 Ma (95% HPD: 109.75–68.95 Ma), which occurred in the Upper period of the Cretaceous. The divergence time of Lelia and Pentatoma was 46.81 Ma (95% HPD: 58.94–35.53 Ma), which occurred in the Eocene and Paleocene periods of the Paleogene. The divergence time of Phyllocephalinae was 67.60 Ma (95% HPD: 82.56–51.76 Ma), which occurred in the Upper Campanian period of the Cretaceous to the Eocene Ypresian period of the Paleogene. Scotinophara lurida was the first to differentiate in Podopinae, and this occurred 62.66 Ma (95% HPD: 77.29–47.61 Ma) in the Upper Campanian period of the Cretaceous to Eocene Lutetian period of the Paleogene. The divergence time of Asopinae and Menidini was 68.49 Ma (95% HPD: 77.29–47.61 Ma), which occurred in the Eocene Ypresian period of the Paleogene to the Upper Campanian period of the Cretaceous.
Discussion and conclusions
In this study, we compared two mitochondrial genomes, and the results showed that the gene arrangement was consistent with other published mitochondrial genomes of Pentatomidae [33, 42, 57].
The length of the control region was found to be closely related to the number of tandem repeat units. Other pentatomid species with different length of control regions and tandem repeats have been reported in previous studies [4, 44]. This finding strongly suggests that the length of the control region determines the length of the entire mitochondrial genes.
Similar to that observed in other pentatomid species, the mitochondrial genomes of the two species of Lelia exhibited a preference for an asymmetric nucleotide composition is thought to be caused by mutational pressure and natural selection [23]. Generally, in PCGs, cox1 is widely used in taxonomic studies of insects because it is a potential marker for species identification [65, 66]. The fact that the number of synonymous and non-synonymous mutations was highly correlated with the length of their respective genes was evidence of neutral evolution, which is consistent with that previously predicted for the mitochondrial genome [67].
In Lelia, the lack of DHU arm in trnS1 (AGN) is a typical feature of insect mitogenomes [68–70]. Apart from typical Watson–Crick pairings (G-C and A-U), some atypical G-U pairings can be transformed into fully functional proteins via post-transcriptional mechanisms [71, 72].
The phylogenetic results were consistent with those of traditional morphological research [1]. Lelia and Pentatoma have a close genetic relationship but very different morphologies. Moreover, the species of tribe Aeschrocorini was the first species to differentiate from Pentatomidae, and previous studies have reported similar result [39]. The phylogenetic trees constructed based on the two datasets formed the same topology on branch, as follow: (Nezarini + (Anaxilaus + (Glaucias + Antestiini))) and ((Dolycoris + Aelini) + Eysarcorini) (Fig 10 and S1 Fig). Antestiini and Nezarini are closely related but do not form a monophyletic group. However, because of the uncertainty of its location, Plautia Stål, 1864 has been temporarily placed in the Antestiini tribe; moreover the placement of the genus Plautia has been problematic and could bridge the gap between this tribe and Nezarini [1]. In Eysarcorini, Eysarcoris gibbosus (Jakovlev, 1904) was the first to diverge from other species; our results support the suggestion of Roca-Cusachs and Jung (2019) and Li et al. (2021) to transfer E. gibbosus to Stagonomus Gorski, 1852 [44, 73]. Morphologically, Eysarcorini and Carpocorini are extremely similar [1], yet the molecular data that we examined indicate that these two tribes are closely related. In previous studies, the sister group relationship between Menidini and Asopinae has been supported through nuclear and mitochondrial gene analyses [8]. In the phylogenetic tree that we constructed, owing to limited data and differences in the results between the two datasets, it was difficult to analyze the phylogenetic relationships and classification status of the remaining tribes.
This study was the first to estimate the divergence time of various tribes of the lower taxon Pentatomidae. We selected three fossils that are currently available as the calibration points for Pentatomidae. The earliest divergence of Pentatomidae occurred in the Lower Cretaceous, which is consistent with previous research [62]. The emergence of Asopinae indicates that the transition of their feeding habits from herbivorous to predatory is closely related to their divergence time. Except for Pentatominae, the divergence times of the other three subfamilies are relatively close (Fig 11). Moreover, previous studies have focused on higher taxa for divergence time estimations. In the absence of fossil evidence, we collected fossils within Pentatomidae as the calibration point, and these had a closer genetic relationship than that observed in previous studies on higher-order elements, making the research results more reliable; at the same time, the estimation of the divergence time of each branch node was more accurate, resulting in higher research significance.
In conclusion, our study not only examined the genus Lelia at the molecular level and identified its taxonomic position in phylogenetic relationships but also discussed the subfamily and tribe evolution in Pentatomidae. We also provide a theoretical basis for the evolutionary history of Pentatomidae. It is necessary to sequence more mitochondrial genomes and to discover more fossil data for further studies.
Supporting information
S1 Table. Partitions and models based on model finder of PCGs and PCGRNA.
https://doi.org/10.1371/journal.pone.0309589.s001
(XLSX)
S2 Table. Organization of the mitochondrial genomes of L. concavaemargo and L. decempunctata.
https://doi.org/10.1371/journal.pone.0309589.s002
(XLSX)
S3 Table. Nucleotide composition of the mitogenomes of L. concavaemargo and L. decempunctata.
https://doi.org/10.1371/journal.pone.0309589.s003
(XLSX)
S4 Table. Tandem repeats of the control region of the mitochondrial genomes of L. concavaemargo and L. decempunctata.
https://doi.org/10.1371/journal.pone.0309589.s004
(XLSX)
S1 Fig. The phylogenetic relationships of tribes within Pentatomidae reconstructed from DNA sequences of 13 protein coding and 2 rRNA mitochondrial genes using BI methods.
Numbers on nodes are the posterior probabilities (PP), lower than 0.6 is not displayed.
https://doi.org/10.1371/journal.pone.0309589.s005
(JPG)
Acknowledgments
Many thanks to Xiaofei Ding and Dan Lian for collecting the material specimens, and thanks Editage for linguistic assistance during the preparation and revision of this manuscript.
References
- 1. Rider DA, Schwertner CF, Vilímová J, Rédei D, Kment P, Thomas DB. Higher systematics of the Pentatomoidea. Invasive Stink Bugs and Related Species (Pentatomoidea): Biology, Higher Systematics, Semiochemistry, and Management CRC Press Boca Raton. 2018:25–204.
- 2. Simon C, Buckley TR, Frati F, Stewart JB, Beckenbach AT. Incorporating molecular evolution into phylogenetic analysis, and a new compilation of conserved polymerase chain reaction primers for animal mitochondrial DNA. Annu Rev Ecol Evol Syst. 2006;37:545–79. https://doi.org/10.1146/annurev.ecolsys.37.091305.110018
- 3. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the entomological Society of America. 1994;87(6):651–701. https://doi.org/10.1093/aesa/87.6.651
- 4. Yuan M-L, Zhang Q-L, Guo Z-L, Wang J, Shen Y-Y. Comparative mitogenomic analysis of the superfamily Pentatomoidea (Insecta: Hemiptera: Heteroptera) and phylogenetic implications. BMC genomics. 2015;16(1):1–16. https://doi.org/10.1186/s12864-015-1679-x
- 5. Lian D, Wei J, Chen C, Niu M, Zhang H, Zhao Q. Comparative analysis and phylogeny of mitochondrial genomes of Pentatomidae (Hemiptera: Pentatomoidea). Frontiers in Genetics. 2022;13:1045193. pmid:36437937
- 6. Ding X, Chen C, Wei J, Gao X, Zhang H, Zhao Q. Comparative mitogenomics and phylogenetic analyses of the genus Menida (Hemiptera, Heteroptera, Pentatomidae). ZooKeys. 2023;1138:29. https://doi.org/10.3897/zookeys.1138.95626
- 7. Grazia J, Schuh RT, Wheeler WC. Phylogenetic relationships of family groups in Pentatomoidea based on morphology and DNA sequences (Insecta: Heteroptera). Cladistics. 2008;24(6):932–76. pmid:34892882
- 8. Roca‐Cusachs M, Schwertner CF, Kim J, Eger J, Grazia J, Jung S. Opening Pandora’s box: molecular phylogeny of the stink bugs (Hemiptera: Heteroptera: Pentatomidae) reveals great incongruences in the current classification. Systematic Entomology. 2022;47(1):36–51. https://doi.org/10.1111/syen.12514
- 9. Xihai Y, Yanfeng W, Yanqing L. On indistinct nsect origin, insect evolulion and reason. Journal of Yanan University (Natural Science Edition). 2003;22(1):81–4.
- 10. Yao Y, Ren D. Phylogeny and Origins and Evolution of Pentatomomorpha. Palaeontological Society of China. 2009.
- 11. Reuter O. Adnotationes hemipterologicae. Revue d’Entomologie. 1890;9(8):248–54.
- 12. Fan Z-H, Liu G-Q. The genus Lelia Walker, 1876, with the description of one new species (Hemiptera: Heteroptera: Pentatomidae: Pentatominae). Zootaxa. 2010;2512(1):56–62–56–62. https://doi.org/10.11646/ZOOTAXA.2512.1.4
- 13. Boore JL. Animal mitochondrial genomes. Nucleic acids research. 1999;27(8):1767–80. pmid:10101183
- 14. Cameron SL. Insect mitochondrial genomics: implications for evolution and phylogeny. Annual review of entomology. 2014;59:95–117. http://doi.org/10.1146/annurev-ento-011613-162007 pmid:24160435
- 15. Zhu X-Y, Xin Z-Z, Wang Y, Zhang H-B, Zhang D-Z, Wang Z-F, et al. The complete mitochondrial genome of Clostera anachoreta (Lepidoptera: Notodontidae) and phylogenetic implications for Noctuoidea species. Genomics. 2017;109(3–4):221–6. pmid:28435087
- 16. Chen Y, Chen Y, Shi C, Huang Z, Zhang Y, Li S, et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience. 2018;7(1):gix120. pmid:29220494
- 17. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9. pmid:22543367
- 18. Bernt M, Donath A, Jühling F, Externbrink F, Florentz C, Fritzsch G, et al. MITOS: improved de novo metazoan mitochondrial genome annotation. Molecular phylogenetics and evolution. 2013;69(2):313–9. pmid:22982435
- 19. Boore JL. The use of genome-level characters for phylogenetic reconstruction. Trends in Ecology & Evolution. 2006;21(8):439–46. pmid:16762445
- 20. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution. 2021;38(7):3022–7. pmid:33892491
- 21. Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular biology and evolution. 2017;34(12):3299–302. pmid:29029172
- 22. Perna NT, Kocher TD. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. Journal of molecular evolution. 1995;41:353–8. pmid:7563121
- 23. Hassanin A, Leger N, Deutsch J. Evidence for multiple reversals of asymmetric mutational constraints during the evolution of the mitochondrial genome of Metazoa, and consequences for phylogenetic inferences. Systematic biology. 2005;54(2):277–98. pmid:16021696
- 24. Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, et al. PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular ecology resources. 2020;20(1):348–55. http://doi.org/10.1111/1755-0998.13096
- 25. Xia X, Lemey P. Assessing substitution saturation with DAMBE. The phylogenetic handbook: a practical approach to DNA and protein phylogeny. 2009;2:615–30. http://doi.org/10.1017/CBO9780511819049.022
- 26. Xia X, Xie Z. DAMBE: software package for data analysis in molecular biology and evolution. Journal of heredity. 2001;92(4):371–3. pmid:11535656
- 27. Kück P, Meid SA, Groß C, Wägele JW, Misof B. AliGROOVE–visualization of heterogeneous sequence divergence within multiple sequence alignments and detection of inflated branch support. BMC bioinformatics. 2014;15(1):1–15. pmid:25176556
- 28. Kalyaanamoorthy S, Minh BQ, Wong TK, Von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nature methods. 2017;14(6):587–9. pmid:28481363
- 29. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic biology. 2012;61(3):539–42. pmid:22357727
- 30. Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree Visualization By One Table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Research. 2023:gkad359. pmid:37144476
- 31. Wu Y, Yang H, Zhou W, Song F, Cai W, Li H. Characterization of the complete mitochondrial genome of Arma custos (Hemiptera: Pentatomidae). Mitochondrial DNA Part B. 2020;5(3):2624–6. https://doi.org/10.1080/23802359.2020.1780985
- 32. Liu Y, Li H, Song F, Zhao Y, Wilson JJ, Cai W. Higher‐level phylogeny and evolutionary history of Pentatomomorpha (Hemiptera: Heteroptera) inferred from mitochondrial genome sequences. Systematic Entomology. 2019;44(4):810–9. http://doi.org/10.1111/syen.12357
- 33. Zhao Q, Wang J, Wang M-Q, Cai B, Zhang H-F, Wei J-F. Complete mitochondrial genome of Dinorhynchus dybowskyi (Hemiptera: Pentatomidae: Asopinae) and phylogenetic analysis of Pentatomomorpha species. Journal of Insect Science. 2018;18(2):44. pmid:29718506
- 34. Guo Y, Xiao J, Li D, Wang J. The complete mitochondrial genome of the stink bug Eocanthecona furcellata (Hemiptera: Pentatomidae). Mitochondrial DNA Part B. 2021;6(10):3085–6. pmid:34595345
- 35. Mu Y-L, Zhang C-H, Zhang Y-J, Yang L, Chen X-S. Characterizing the complete mitochondrial genome of Arma custos and Picromerus lewisi (Hemiptera: Pentatomidae: Asopinae) and conducting phylogenetic analysis. Journal of Insect Science. 2022;22(1):6. pmid:35039857
- 36. Zhao Q, Wei J, Zhao W, Cai B, Du X, Zhang H. The first mitochondrial genome for the subfamily Asopinae (Heteroptera: Pentatomidae) and its phylogenetic implications. Mitochondrial DNA Part B. 2017;2(2):804–5. pmid:33473988
- 37. Goncalves LT, Pezzi PH, Bianchi FM. Four new stink bug mitogenomes corroborate the internal inconsistencies in the classification of Pentatomidae (Hemiptera). Zootaxa. 2022;5120(1):128–42. pmid:35391177
- 38. Zhao Q, Cassis G, Zhao L, He Y, Zhang H, Wei J. The complete mitochondrial genome of Zicrona caerulea (Linnaeus)(Hemiptera: Pentatomidae: Asopinae) and its phylogenetic implications. Zootaxa. 2020;4747(3):zootaxa. 4747.3. 8-zootaxa. .3. 8. pmid:32230102
- 39. Jia W, Wei J, Niu M, Zhang H, Zhao Q. The complete mitochondrial genome of Aeschrocoristuberculatus and A. ceylonicus (Hemiptera, Pentatomidae) and its phylogenetic implications. ZooKeys. 2023;1160:145. http://doi.org/10.3897/zookeys.1160.100818
- 40. Xu S, Wu Y, Liu Y, Zhao P, Chen Z, Song F, et al. Comparative mitogenomics and phylogenetic analyses of Pentatomoidea (Hemiptera: Heteroptera). Genes. 2021;12(9):1306. pmid:34573288
- 41. Wang Y, Duan Y, Yang X. The complete mitochondrial genome of Plautia crossota (Hemiptera: Pentatomidae). Mitochondrial DNA Part B. 2019;4(2):2281–2. pmid:33365505
- 42. Lee W, Kang J, Jung C, Hoelmer K, Lee SH, Lee S. Complete mitochondrial genome of brown marmorated stink bug Halyomorpha halys (Hemiptera: Pentatomidae), and phylogenetic relationships of hemipteran suborders. Molecules and Cells. 2009;28:155–65. pmid:19756390
- 43. Zhang Q-L, Yuan M-L, Shen Y-Y. The complete mitochondrial genome of Dolycoris baccarum (Insecta: Hemiptera: Pentatomidae). Mitochondrial DNA. 2013;24(5):469–71. pmid:23391217
- 44. Li R, Li M, Yan J, Bai M, Zhang H. Five mitochondrial genomes of the genus Eysarcoris Hahn, 1834 with phylogenetic implications for the Pentatominae (Hemiptera: Pentatomidae). Insects. 2021;12(7):597. pmid:34209052
- 45. Chen Q, Niu X, Fang Z, Weng Q. The complete mitochondrial genome of Eysarcoris guttigerus (Hemiptera: Pentatomidae). Mitochondrial DNA Part B. 2020;5(1):687–8. pmid:33366704
- 46. Zhao Q, Chen C, Liu J, Wei J. Characterization of the complete mitochondrial genome of Eysarcoris aeneus (Heteroptera: Pentatomidae), with its phylogenetic analysis. Mitochondrial DNA Part B. 2019a;4(2):2096–7. pmid:33365424
- 47. Jiang P. Studies on the comparative mitochondrial genomics and phylogeny of Heteroptera (insecta: Hemiptera). PhD, China Agricultural University, Beijing. 2017.
- 48. Ji H, Xu X, Jin X, Yin H, Luo J, Liu G, et al. Using high-resolution annotation of insect mitochondrial DNA to decipher tandem repeats in the control region. RNA biology. 2019;16(6):830–7. pmid:30870076
- 49. Chen W, Zhang L, Cao Y, Yuan M. Palomena viridissima The complete mitochondrial genome of (Hemiptera: Pentatomidae) and phylogenetic analysis. Mitochondrial DNA Part B, Resources. 2021;6(4):1326–7. pmid:33889740
- 50. Hua J, Li M, Dong P, Cui Y, Xie Q, Bu W. Comparative and phylogenomic studies on the mitochondrial genomes of Pentatomomorpha (Insecta: Hemiptera: Heteroptera). BMC genomics. 2008;9:1–15. http://doi.org/10.1186/1471-2164-9-610
- 51. Wang Y-C, Li G-L, Liu X-Y, He Q-J, Yi C-H, Yang C, et al. The complete mitochondrial genome of Pycanum ochraceum Distant 1893 (Hemiptera: Tessaratomidae). Mitochondrial DNA Part B. 2021;6(12):3383–5. pmid:34778560
- 52. Zhao L, Wei J, Zhao W, Chen C, Gao X, Zhao Q. The complete mitochondrial genome of Pentatoma rufipes (Hemiptera, Pentatomidae) and its phylogenetic implications. ZooKeys. 2021;1042:51. pmid:34163290
- 53. Zhao W, Zhao Q, Li M, Wei J, Zhang X, Zhang H. Comparative mitogenomic analysis of the Eurydema genus in the context of representative Pentatomidae (Hemiptera: Heteroptera) taxa. Journal of insect science. 2019b;19(6):20. pmid:31841604
- 54. Zhao W, Zhao Q, Li M, Wei J, Zhang X, Zhang H. Characterization of the complete mitochondrial genome and phylogenetic implications for Eurydema maracandica (Hemiptera: Pentatomidae). Mitochondrial DNA Part B. 2017b;2(2):550–1. pmid:33490465
- 55. Chen C, Wei J, Ji W, Zhao Q. The first complete mitochondrial genome from the subfamily Phyllocephalinae (Heteroptera: Pentatomidae) and its phylogenetic analysis. Mitochondrial DNA Part B. 2017;2(2):938–9. http://doi.org/10.1080/23802359.2017.1413313
- 56. Wang J, Zhang L, Yang X-Z, Zhou M-Q, Yuan M-L. The first mitochondrial genome for the subfamily Podopinae (Hemiptera: Pentatomidae) and its phylogenetic implications. Mitochondrial DNA Part B. 2017;2(1):219–20. pmid:33473775
- 57. Song W, Li H, Song F, Liu L, Wang P, Xun H, et al. The complete mitochondrial genome of a tessaratomid bug, Eusthenes cupreus (Hemiptera: Heteroptera: Pentatomomorpha: Tessaratomidae). Zootaxa. 2013;3620(2):260–72–72. http://doi.org/10.11646/zootaxa.3620.2.4 pmid:26120708
- 58. Xu S, Wu Y, Cai W, Song F. The complete mitochondrial genome of the lychee stinkbug Mattiphus splendidus (Hemiptera: Tessaratomidae). Mitochondrial DNA Part B. 2020;5(1):321–2. http://doi.org/10.1080/23802359.2019.1703609
- 59. Drummond AJ, Rambaut A. Bayesian evolutionary analysis by sampling trees. The phylogenetic handbook: a practical approach to phylogenetic analysis and hypothesis testing. 2009:564–74. http://doi.org/10.1186/1471-2148-7-214
- 60. Piton LE. Paléontologie du gisement éocène de Menat (Puy-de-Dôme): flore et faune: Imprimeries P. Vallier Clermont-Ferrand, France; 1940.
- 61. Zhao W. DNA barcoding and mitochondrial genomes of Chinese species of the genus Eurydema (Hemiptera: Pentatomidae).: PhD, Shanxi Agricultural University, Shanxi.; 2018.
- 62. Li H, Leavengood JM Jr, Chapman EG, Burkhardt D, Song F, Jiang P, et al. Mitochondrial phylogenomics of Hemiptera reveals adaptive innovations driving the diversification of true bugs. Proceedings of the Royal Society B: Biological Sciences. 2017;284(1862):20171223. http://doi.org/10.1098/rspb.2017.1223
- 63. Song F, Li H, Shao R, Shi A, Bai X, Zheng X, et al. Rearrangement of mitochondrial tRNA genes in flat bugs (Hemiptera: Aradidae). Scientific Reports. 2016;6(1):25725. https://doi.org/10.1038/srep25725
- 64. Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic biology. 2018;67(5):901–4. pmid:29718447
- 65. Françoso E, Zuntini AR, Ricardo PC, Silva JPN, Brito R, Oldroyd BP, et al. Conserved numts mask a highly divergent mitochondrial-COI gene in a species complex of Australian stingless bees Tetragonula (Hymenoptera: Apidae). Mitochondrial DNA Part A. 2019;30(7):806–17. http://doi.org/10.1080/24701394.2019.1665036
- 66. Noh P, Kim WJ, Song J-H, Park I, Choi G, Moon BC. Rapid and simple species identification of cicada exuviae using COI-Based SCAR assay. Insects. 2020;11(3):168. pmid:32155837
- 67. Ballard JWO, Whitlock MC. The incomplete natural history of mitochondria. Molecular ecology. 2004;13(4):729–44. pmid:15012752
- 68. Navajas M, Conte YL, Solignac M, Cros-Arteil S, Cornuet J-M. The complete sequence of the mitochondrial genome of the honeybee ectoparasite mite Varroa destructor (Acari: Mesostigmata). Molecular biology and evolution. 2002;19(12):2313–7. pmid:12446822
- 69. Wolstenholme DR. Animal mitochondrial DNA: structure and evolution. International review of cytology. 1992;141:173–216. pmid:1452431
- 70. Shi A, Li H, Bai X, Dai X, Chang J, Guilbert E, et al. The complete mitochondrial genome of the flat bug Aradacanthia heissi (Hemiptera: Aradidae). Zootaxa. 2012;3238(1):23–38. http://doi.org/10.11646/zootaxa.3238.1.2
- 71. Chao JA, Patskovsky Y, Almo SC, Singer RH. Structural basis for the coevolution of a viral RNA–protein complex. Nature structural & molecular biology. 2008;15(1):103–5. http://doi.org/10.1038/nsmb1327
- 72. Pons J, Bauzà-Ribot MM, Jaume D, Juan C. Next-generation sequencing, phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae (Amphipoda: Crustacea). BMC genomics. 2014;15:1–16.
- 73. Roca-Cusachs M, Jung S. Redefining Stagonomus Gorski based on morphological and molecular data (Pentatomidae: Eysarcorini). Zootaxa. 2019;4658(2):zootaxa.4658.2.10. pmid:31716750