Elucidating Scarab Divergence in an Evolutionary-Ecological Context through the Comprehensive Analysis of the Complete Mitogenome of Anomala

Anomala Samouelle, 1819 is one of the specious genera of Coleoptera, with over 1000 known species, and includes some of the most destructive pests of crops or forests. Morphological convergence is a common phenomenon within this genus, making the identification of closely related species very difficult. To explore the phylogenetic placement of Anomalini and provide a basis for the classification and identification of Anomala, we comparatively analyzed the complete mitogenome of three Anomala species (A. exoleta, A. perplexa diana, and A. praecoxalis). Based on all accessible mitogenome data, we performed comparative mitochondrial genomics analysis of this genus and reconstructed the phylogenetic relationships of Scarabaeidae based on two datasets (protein-coding genes and amino acids) and two methods (Bayesian approach and maximum likelihood). The phylogenetic relationships found in this study highly support that the groups of Aphodiinae, Cetoniinae, Dynastinae, Rutelinae and Scarabaeinae are monophyletic. Interestingly, the phylogenetic clustering relationship was highly consistent with the Scarabaeidae diet, indicating that the herbivorous species and dung-eating species are clustered separately. The phylogenetic tree showed that the subfamily Melolonthinae and the genus Anomala are not monophyletic, suggesting that these two groups should be further revised with more data.


Introduction
Coleoptera is one of the most biodiversity-rich groups within insecta.Rutelinae, a subfamily of the family Scarabaeidae (Coleoptera, Polyphaga), is mainly distributed in the Indomalayan, Palaearctic, and Afrotropical regions.In addition, the biodiversity of Rutelinae is extremely rich, with over 4200 recorded species in 235 genera from seven tribes worldwide.In China alone, three tribes, 25 genera, and 526 species have been reported.Rutelinae beetles are voracious feeders, capable of consuming large amounts of food in a short period.They pose significant ecological and economic threats through their prolonged harmful activity and wide distribution.Adults of the family Rutelinae harm the buds, leaves, flowers, and fruits of plants [1].Their larvae are significant underground pests, causing harm to trees, lawns, crops, and other plant roots and adversely affecting the ecology and cash crop.Rutelinae were considered a distinct subfamily based on a few morphological features.The dominant distinguishing feature between scarab beetles and other subfamilies of the family scarab is moveable and asymmetrical claws, which are usually split [2].Based on current morphological and molecular evidence, it is believed that the Rutelinae and Dynastinae form a common lineage; however, their monophyletism needs to be verified.Howden (1982) reconstructed a phylogenetic relationship of Scarabaeoidea based on the morphological characteristics (the base of antennae had well-developed setae, the base of claws had no teeth, the size of claws varied, the abdominal valve [3][4][5][6][7] nodes were nearly parallel, etc.) and found that Rutelinae and Dynastinae gather in a cluster [3].Browne and Scholtz (1998) showed that Rutelinae and Dynastinae shared five common characteristics based on the morphology of the wing bases; however, they could not distinguish the two subfamilies by the wing bases [4].Multiple studies based on molecular evidence have yielded similar results [5,6].However, the phylogenetic relationship between Rutelinae and Dynastinae is still not well explained, and there is a lack of systematic research on the Rutelinae species.
With the development of molecular sequencing technology and the low cost of sequencing, DNA taxonomy has become one of the most promising methods to solve the taxonomic problems of this subfamily.For Rutelinae, molecular phylogenetics studies used to rely on mitochondrial single genes, nuclear gene fragments, or combinations of two or three genes.Mitochondrial genes such as cox1, cox2, 16S, 12S, cytb and nad1 are molecular markers frequently used in evolutionary analysis [7].However, a single gene not only contains less evolutionary information, which makes it difficult to completely analyze the phylogenetic relationships among species, but also exhibits differences in selection pressure and evolutionary rate among different genes, which may lead to "long branch attraction" and other issues.Mitochondria are semi-autonomous organelles that exist in eukaryotic cells.The mitochondrial genome serves as a molecular sequence that can autonomously perform genetic functions.It is widely used in species identification, population genetics, biogeography, and phylogeny [8,9].
Anomala is the largest genus of the Rutelinae, with over 1300 species worldwide, covering two-thirds of the species in the tribe Anomalini.Anomala also has the most taxonomic problems, especially in terms of identifying closely related species.To date, only 10 mitochondrial genomes from Rutelinae have been recorded in NCBI (of which Anomala has only three mitochondrial genome sequences).In this study, we sequenced, assembled, and annotated the mitochondrial genomes of three taxa of Anomala, namely A. exolete, A. perplexa diana, and A. praecoxalis.A comparative analysis of mitochondrial genomes was then conducted, and a phylogenetic tree of Scarabaeidae based on a Bayesian approach (BI) and method of maximum likelihood (ML) was constructed to analyze phylogenetic relationships.This approach primarily solves the following scientific problems: (1) determining the status of Rutelinae in the Scarabaeidae classification; (2) clarifying the basic characteristics of the mitochondrial genome of Anomala; and (3) assessing whether the monophyletic group of Anomala is formed.

Specimen Collection and DNA Extraction
A. exoleta specimens were collected from Xigu district, Lanzhou City, Gansu Province, China, on 28 June 2020; A. perplexa diana were collected from Mêdog County, Tibet, China, on 18 August 2020; and A. praecoxalis were collected from Dawei Mountain National Nature Reserve, Yunnan Province, China, on 2 May 2020, and instantly stored in anhydrous ethanol.The samples were transported to the laboratory for immediate storage at −20 • C until characterization or DNA extraction.These three species were accurately identified based on morphological characteristics.The extraction of DNA samples for sequencing was conducted using a DNeasy© Tissue Kit as per the specification manual of the manufacturer.

Mitogenome Sequencing, Assembly, and Annotation
The mitogenomes for three Anomala species were sequenced by Berry Genomics on a HiSeq 2500 platform (Illumina) with 150 bp paired-end reads.The average insert size was 350 bp, and 6 GB of clean data was obtained.The mitogenomes were assembled using Geneious Prime 2020.2.1 software [10] and were based on a mitochondrial reference sequence of A. rufiventris (OR208200).The assembled mitogenome sequence was compared with the homologous sequences of A. corpulenta NC069575, A. rufiventris OR208200, and A. russiventris NC065310 [11], which were obtained from GenBank, and the accuracy of the sequences was confirmed by performing a BLAST search on NCBI [12].The assembled mitogenome was annotated utilizing the MITOS network services and the invertebrate mitochondrial hereditary code [13].The placement of 22 tRNAs was controlled utilizing tRNAscan-SE version 1.21 and ARWEN version 1.2 [14,15].Two rRNA genes were confirmed based on the position of proximity tRNA genes and were defined as the other Anomala whose mitogenome sequences were previously published in GenBank.The circular mitogenomic map was created using OGDraw version 1.3.1 [16].

Sequence Analysis of Mitogenomes
Base composition of Anomala mitogenomes was assessed using DNASTAR Lasergene v7.1 (http://www.dnastar.com(accessed on 28 June Month 2024)).Strand asymmetries in Rutelinae mitogenomes were calculated using the following formulas: GC Codon usage patterns within the Anomala mitogenome were analyzed using Sequence Manipulation Suite based on genetic code 5.

Sequence Alignments and Phylogenetic Analyses
The Scarabaeidae of 30 representative species (all Rutelinae genomes available and one random species per other genera) were selected for systematic evolution as the ingroup, and two species, Sinodendron yunnanense (NC036157) and Pseudorhaetus sinicus (NC069553), were chosen as outgroups (Table 1) [11,[17][18][19][20][21][22][23][24][25].The two datasets (protein-coding genes [PCG]: 13; and amino acids [AA]: amino acid sequences of 13 PCGs) were used to analyze the phylogenetic relationships within Scarabaeidae.Initially, the alignment of each PCG was conducted utilizing the MAFFT arithmetic in the Translator X server [26].Ultimately, all sequences were evaluated and hand-corrected with MEGA7 [27].To test whether the two datasets were suited for high-level phylogenetic inferences, the heterogeneity in nucleotide divergence was evaluated via pairwise comparisons in a multiple sequence alignment using AliGROOVE v1.05 [28].Based on two different datasets containing 32 species, we used ML and BI methods to reconstruct phylogenetic relationships.An ML phylogenetic tree was reconstructed using IQ-TREE with 10,000 iterations via a superrapid bootstrap approximation [29].BI system phylogeny was executed by MrBayes 3.3 [30].The BI analysis used the default settings and simulated four standalone runs for an aggregate of 0.1 billion generations, sampling once per 1000 generations.When the convergence value was less than 0.01, the operation was stopped.The tree was stored every 1000 generations, and the first 25% was discarded based on the burn-in parameter.Finally, a 50% merge tree was formed.The phylogenetic trees were visualized using FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and beautified using Adobe Illustrator CC 22. 1. Software.

Mitogenomic Characteristics of Anomala Species
This study reports the complete mitochondrial genome sequences of three Anomala species: A. exolete, A. perplexa diana, and A. praecoxalis, which were sequenced and found to be 17,066 bp, 16,857 bp, and 16,913 bp in length, respectively.Comparative genomic analysis of these mitochondrial genomes with those of A. corpulenta (NC069575), A. rufiventris (OR208200), and A. russiventris (NC065310) obtained from NCBI revealed high conservation in gene content and order.Mitochondrial genome length in the studied Rutelinae species varied from 15,601 bp (A.russiventris) to 17,240 bp (A.rufiventris).Comparative analysis with Drosophila yakuba revealed conserved gene order and orientation, with no evidence of gene rearrangements in any Anomala species.The mitochondrial genome of all six Anomala species encodes 37 genes, including 13 PCGs, 22 transfer ribonucleic acid (tRNA) genes, and two ribosomal ribonucleic acid (rRNA) genes.Nine PCGs and 14 tRNA genes are encoded on the J-strand, whereas the remaining four PCGs, eight tRNA genes, and two rRNA genes are encoded on the N-strand (Figure 1, Table 2).
In the sequenced mitochondrial genome of Rutelinae insects, ATT, TTA, TTT, and ATA were the main codons used, whereas GC-rich codons (such as GCG, CGG, and CTC) were used less frequently.Most PCGs start their transcription with ATN.Anomala insect gene transcription is often terminated using TAA, TAG, or an incomplete codon T-(Table 2).

Codon Usage Analysis
Energy expenditure and translation rate are directly affected by codon usage.Mitochondrial genes prefer specific codons that help optimize the efficiency of translation and conserve energy for cellular functions.We compared 10 mitogenomes to analyze the codon usage trends in different Rutelinae mitogenomes (Figure 2).This set comprised seven mitogenomes that had already been published in the database, along with three Anomala mitogenomes that we obtained in the current study.Our extensive analysis showed that the most common codon in the mitogenomes of 10 Rutelinae species was UUA (representing leucine; Leu).Rutelinae mitogenomes predominantly initiated translation of PCGs using the start codon ATG, whereas the most frequent stop codon was TAA, followed by TAG.In the sequenced mitochondrial genome of Rutelinae insects, ATT, TTA, TTT, and ATA were the main codons used, whereas GC-rich codons (such as GCG, CGG, and CTC) were used less frequently.Most PCGs start their transcription with ATN.Anomala insect gene transcription is often terminated using TAA, TAG, or an incomplete codon T-(Table 2).

Codon Usage Analysis
Energy expenditure and translation rate are directly affected by codon usage.Mitochondrial genes prefer specific codons that help optimize the efficiency of translation and conserve energy for cellular functions.We compared 10 mitogenomes to analyze the codon usage trends in different Rutelinae mitogenomes (Figure 2).This set comprised seven mitogenomes that had already been published in the database, along with three Anomala mitogenomes that we obtained in the current study.Our extensive analysis showed that the most common codon in the mitogenomes of 10 Rutelinae species was UUA (representing leucine; Leu).Rutelinae mitogenomes predominantly initiated translation of PCGs using the start codon ATG, whereas the most frequent stop codon was TAA, followed by TAG.Anomala's 13 PCGs are extremely conserved in length, except for cox1, nad5, and nad6, with the others being approximately the same length (Figure 3a).The GC content of these 13 PCGs in the mitochondrial genomes of the six Anomala species was greatest for cox1, while atp8 and nad4l had the lowest GC content relative to other PCGs (Figure 3b).Except for nad4, nad4L, nad5, and nad6, all other PCGs had negative values of AT skew.Except for atp6 and atp8, which showed differences of AT skew between different species, other PCGs had similar AT skew values (Figure 3c).The GC skew of atp8 was found to be positive in five species (A.praecoxalis, A. russiventris, A. corpulenta, A. exoleta, and A. perplexa diana), whereas the GC skew of all genes in other species was negative (Figure 3d).
13 PCGs in the mitochondrial genomes of the six Anomala species was greatest for cox1, while atp8 and nad4l had the lowest GC content relative to other PCGs (Figure 3b).Except for nad4, nad4L, nad5, and nad6, all other PCGs had negative values of AT skew.Except for atp6 and atp8, which showed differences of AT skew between different species, other PCGs had similar AT skew values (Figure 3c).The GC skew of atp8 was found to be positive in five species (A.praecoxalis, A. russiventris, A. corpulenta, A. exoleta, and A. perplexa diana), whereas the GC skew of all genes in other species was negative (Figure 3d).

Phylogenetic Relationships
Based on the calculation results obtained from the AliGROOVE 1.06 software, the heterogeneity of PCG and AA datasets in the mitogenomic data of Scarabaeidae was found to be weak (Figure 4).Hence, the two datasets could be used to reconstruct a phylogenetic tree.

Phylogenetic Relationships
Based on the calculation results obtained from the AliGROOVE 1.06 software, the heterogeneity of PCG and AA datasets in the mitogenomic data of Scarabaeidae was found to be weak (Figure 4).Hence, the two datasets could be used to reconstruct a phylogenetic tree.In this study, we used two methods (BI and ML) to construct phylogenetic trees from two datasets (PCG and AA).The support rate of the BI-based method was higher than that of the ML-based method.Phylogenetic trees reconstructed using these methods were highly consistent across different datasets.In all phylogenetic relationships, the monophyletic nature of all Scarabaeidae subfamilies, except for Melolonthinae, was well supported (BP, bootstrap percentage = 100, PP, Posterior Probability = 1).Specifically, Scarabaeinae and Aphodiinae, as well as Rutelinae and Cetoniinae, formed sister groups.Within Rutelinae, the monophyly of Popillia was well verified (BP = 100, PP = 1).However, the monophyly of Anomala was questioned, as it formed different branches from M. splen- In this study, we used two methods (BI and ML) to construct phylogenetic trees from two datasets (PCG and AA).The support rate of the BI-based method was higher than that of the ML-based method.Phylogenetic trees reconstructed using these methods were highly consistent across different datasets.In all phylogenetic relationships, the monophyletic nature of all Scarabaeidae subfamilies, except for Melolonthinae, was well supported (BP, bootstrap percentage = 100, PP, Posterior Probability = 1).Specifically, Scarabaeinae and Aphodiinae, as well as Rutelinae and Cetoniinae, formed sister groups.Within Rutelinae, the monophyly of Popillia was well verified (BP = 100, PP = 1).However, the monophyly of Anomala was questioned, as it formed different branches from M. splendens and C. plagiicollis (Figure 5 and Figures S1-S3).

Discussion
In this study, we obtained complete mitochondrial genomes of three species (A.exolete, A. perplexa diana, and A. praecoxalis) from subfamily Rutelinae of the Scarabaeidae.We then performed comparative mitochondrial genomics analysis of this genus based on two datasets (PCG and AA) and two methods (Bayesian approach and method of ML), and the phylogenetic relationships of Scarabaeidae were reconstructed.The high support of this study indicated that the groups of Aphodiinae, Cetoniinae, Dynastinae, Rutelinae, and Scarabaeinae were monophyletic.Most genera of these subfamilies are monophyletic groups, which is consistent with previous studies [17].The systematic relationships constructed in this study show that scarab insects with the same diet congregate, that is, the dung-eating Scarabaeinae and Aphodiinae cluster into a branch, whereas the herbivorous subfamilies Cetoniinae, Dynastinae, Melolonthinae, and Rutelinae form another cluster, which is consistent with previous studies [16,19].This study supports the idea that fecaleating behavior predates herbivorous eating, which aligns with previous findings by Ayivi et al. and Song and Zhang [16,19].The two subfamilies of Dynastinae and Rutelinae are sister groups of each other, which is consistent with the prevailing view based on morphological and molecular evidence [30].Simultaneously, our phylogenetic relationship analysis indicates that the subfamily Melolonthinae is not monophyletic, with Apogonia being isolated from other Melolonthinae, which is consistent with previous reports [5,17,19,20,31,32].
In addition, the phylogenetic tree showed that the Anomala species clustered into three branches.In the first branch, A. corpulenta and M. splendens formed sister species, whereas A. perplexa diana and A. exoleta formed another sister group.The second branch was formed by A. praecoxalis and A. russiventris, whereas the third branch was formed by A. rufiventris and C. plagiicollis.The second and third branches are sister groups to each

Discussion
In this study, we obtained complete mitochondrial genomes of three species (A.exolete, A. perplexa diana, and A. praecoxalis) from subfamily Rutelinae of the Scarabaeidae.We then performed comparative mitochondrial genomics analysis of this genus based on two datasets (PCG and AA) and two methods (Bayesian approach and method of ML), and the phylogenetic relationships of Scarabaeidae were reconstructed.The high support of this study indicated that the groups of Aphodiinae, Cetoniinae, Dynastinae, Rutelinae, and Scarabaeinae were monophyletic.Most genera of these subfamilies are monophyletic groups, which is consistent with previous studies [17].The systematic relationships constructed in this study show that scarab insects with the same diet congregate, that is, the dung-eating Scarabaeinae and Aphodiinae cluster into a branch, whereas the herbivorous subfamilies Cetoniinae, Dynastinae, Melolonthinae, and Rutelinae form another cluster, which is consistent with previous studies [16,19].This study supports the idea that fecaleating behavior predates herbivorous eating, which aligns with previous findings by Ayivi et al. and Song and Zhang [16,19].The two subfamilies of Dynastinae and Rutelinae are sister groups of each other, which is consistent with the prevailing view based on morphological and molecular evidence [30].Simultaneously, our phylogenetic relationship analysis indicates that the subfamily Melolonthinae is not monophyletic, with Apogonia being isolated from other Melolonthinae, which is consistent with previous reports [5,17,19,20,31,32].

Figure 1 .
Figure 1.Circular maps of the mitogenomes of three newly sequenced Anomala species.

Figure 3 .
Figure 3. Variation in length and base composition of the 13 protein-coding genes (PCGs) among five Anomala mitochondrial genomes.(a) PCG length variation; (b) GC content of PCGs; (c) AT skew; (d) GC skew.

Figure 3 .
Figure 3. Variation in length and base composition of the 13 protein-coding genes (PCGs) among five Anomala mitochondrial genomes.(a) PCG length variation; (b) GC content of PCGs; (c) AT skew; (d) GC skew.

Figure 4 .
Figure 4. Heterogeneity of AA (left) and PCG (right) datasets in Scarabaeidae mitogenomes.Color gradients from dark red (−1) to dark blue (+1) represent levels of heterogeneity between sequences, with darker shades indicating greater heterogeneity.

Figure 4 .
Figure 4. Heterogeneity of AA (left) and PCG (right) datasets in Scarabaeidae mitogenomes.Color gradients from dark red (−1) to dark blue (+1) represent levels of heterogeneity between sequences, with darker shades indicating greater heterogeneity.

Figure 5 .
Figure 5. Phylogenetic trees of Scarabaeidae inferred by the MrBayes 3.2.6 method-based amino acid sequences of PCGs.

Table 1 .
Information of samples for phylogenetic analyses.

bp) Anticodon Start Codon Stop Codon Intergenic Nucleotides *
Figure 1.Circular maps of the mitogenomes of three newly sequenced Anomala species.