A New Species of Scymnus (Coleoptera, Coccinellidae) from Pakistan with Mitochondrial Genome and Its Phylogenetic Implications

Simple Summary The study presents the new species Scymnus (Pullus) cardi sp. nov. and provides its mitochondrial genome. We describe the distinguishing features of S. (P.) cardi when compared with related species and discuss its habitat and feeding preferences. Additionally, based on the mitochondrial genomic dataset, the phylogeny of Coccinellidae is analyzed. The results confirm the position of the new species, which is nested within the genus Scymnus, and recovered subfamilies as monophyletic groups (such as Coccinellinae and Microweiseinae). These findings contribute to the understanding of Coccinellidae evolution and highlight the need for further taxonomic and genetic studies within the family. Abstract In this study, a new species of the subgenus Pullus belonging to the Scymnus genus from Pakistan, Scymnus (Pullus) cardi sp. nov., was described and illustrated, with information on its distribution, host plants, and prey. Additionally, the completed mitochondrial genome (mitogenome) of the new species using high-throughput sequencing technology was obtained. The genome contains the typical 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs) and a non-coding control region, and is arranged in the same order as that of the putative ancestor of beetles. The AT content of the mitogenome is approximately 85.1%, with AT skew and GC skew of 0.05 and −0.43, respectively. The calculated values of relative synonymous codon usage (RSCU) determine that the codon UUA (L) has the highest frequency. Furthermore, we explored the phylogenetic relationship among 59 representatives of the Coccinellidae using Bayesian inference and maximum likelihood methods, the results of which strongly support the monophyly of Coccinellinae. The phylogenetic results positioned Scymnus (Pullus) cardi in a well-supported clade with Scymnus (Pullus) loewii and Scymnus (Pullus) rubricaudus within the genus Scymnus and the tribe Scymnini. The mitochondrial sequence of S. (P.) cardi will contribute to the mitochondrial genome database and provide helpful information for the identification and phylogeny of Coccinellidae.

Pullus has been established by Mulsant [14] as a subgenus of Scynmus, with Coccinella subvillosa Goeze [15] as a type species, and this has been accepted by most authors [1,2,6,8,[16][17][18][19]. Taxonomic identification at the species level of subgenus Pullus is difficult with external appearance due to its small size and polymorphism, therefore identification has relied on male genital characteristics.Based on the characteristics of male genitalia, Chen et al. [2] divided the subgenus Pullus into five groups: hingstoni, subvillosus, impexus, perdere, and sodalis.Members of this genus are known to be general predators of aphids, adelgids, mealybugs, and scale insects [20].The nominative subgenus is characterized by the following combination of characteristics: antennae with 11 antennomeres (a short pedicle that is narrower than the scape); a prosternal process that is relatively narrow with convergent carinae, reaching to anterior margins; an abdomen with six ventrites; and the abdominal postcoxal line completed (a characteristic which separates it from the subgenus Scymnus).
In this paper, a new species, Scymnus (Pullus) cardi sp.nov., and a single new country record named Scymnus (Pullus) latifolius Poorani, 2018 of the group perdere are recorded from Pakistan.Here, we (i) describe this new species and compare its diagnostic characteristic with related species (Scymnus (Pullus) latifolius, (ii) analyze the mitochondrial genomic characteristics, and (iii) reconstruct the phylogenetic relationship of the family Coccinellidae.

Specimens and Taxonomy
The specimens were collected by sweeping net method in Pakistan and preserved in absolute ethanol at −20 • C. External morphology was observed with a Labomed microscope (CZM6).Male genitalia were dissected and cleared in 10% KOH solution by boiling several times and were then washed with 70% and 90% ethanol.Images were taken using a Nikon Digital camera (SMZ 1500) attached to the stereo microscope.Photographs were edited using Helicon Focus 8.1 (Helicon Soft Ltd., Kyiv, Ukraine.) and Adobe Photoshop 2020 (Adobe Inc., San Jose, CA, USA).
Measurements were made by using an ocular micrometer, which works at the millimeter (mm) scale.The following abbreviations were used: TL-total length from apical margin of clypeus to elytral apex; TW-total width across both elytra at widest part; TH-total height at highest part of elytra; HW-head width at widest part including eyes; PL-pronotal length from the middle of anterior margin to the base of pronotum; PWpronotal width at widest part; EL-elytral length along suture from base to apex including scutellum.
Insects 2024, 15, 371 3 of 15 Morphological terminology was determined following Ślipiński [10] and Chen et al. [1,2].Type specimens were deposited at the Department of Entomology, South China Agricultural University, Guangzhou, China.Some paratypes were deposited at the Pir Mehr Ali Shah -Arid Agriculture University, Rawalpindi, Pakistan.
The following acronyms are used in this paper for the specimens' depositories.
SCAU-Department of Entomology, South China Agricultural University, Guangzhou, China.

DNA Extraction, Sequencing, Mitogenome Assembly, and Annotation
The genomic DNA was extracted from the selected adult specimens using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, eluted in 150 µL AE buffer, and kept at −20 • C until used.The mitochondrial genomes were sequenced using shotgun sequencing of total genomic DNA on the Illumina HiSeq.2500 platform (University of Liverpool) with libraries having a 350 bp insert size and 250 bp paired-end sequencing.The Illumina sequencing library was prepared for a pool of 60 ladybird beetle samples.Initially, low-quality and short reads were removed using Prinseq [31], and the remaining high-quality reads were preliminarily assembled using SPAdes genome assembler v3.15.5, MEGAHIT v1.2.9, and MaSuRCA v4.0.5, respectively.Kmer sizes for SPAdes and MEGAHIT were set as 21, 33, 55, 77, 99, 127 and kept at the default level for MaSuRCA [32][33][34][35].Based on the assembly results of the three assemblers above, the final contigs were assembled using Metassembler v1.5 [36].Contigs from the mixed library were associated with a specific species by matching them to a shotgun-sequenced cox1 gene fragment that was used as bait and was obtained with primers Ill_B_F [37] and Fol_degen_rev [38].Identification required a minimum of 99% identity in the Blast alignment.
In the PhyloBayes analyses (all three datasets above) the CAT-GTR model was used.Two parallel and independent tree searches were performed until the discrepancies were lower than 0.1 (maxdiff less than 0.1).A consensus tree was computed using the remaining trees from two runs after the initial 25% of trees were discarded as burn-in.Maximum likelihood phylogeny was inferred using IQ-TREE v2.2.0.The MEP model was used for the bootstrapping phase, and node support in all ML analyses was calculated using 1000 SH-aLRT repetitions and 1000 UFBoot2 bootstraps (-B 1000, -alrt 1000), respectively [52].
Diagnosis.Subgenus Pullus Mulsant species can be easily distinguished from other subgenera of Scymnus by the following combination of characteristics: the body is oval or elongated oval and the head capsule is rectangularly oval and finely punctate (Figure 1A); the prosternal process has well-developed carinae, that are convergent and reach to the anterior margin (Figure 1B); the antenna are composed of 11 antennomeres (Figure 1C); the mandible has double teeth, with the outer tooth slightly longer than the inner tooth (Figure 1D); the labrum with apical palpomere is narrow and shorter than the penultimate one (Figure 1E); a maxilla with terminal palpomere securiform and obliquely truncate apical (Figure 1F); a tibia without spur and a tarsal claw that is bifid (Figure 1G); an abdomen with six ventrites and an abdominal postcoxal line that is complete at the first ventrite, or in the male the fifth and sixth ventrites, and is usually moderately or strongly emarginate apically (Figure 2B).
Distribution.Worldwide.Scymnus (Pullus) cardi sp.nov. Figure 2A-H Type specimens.Holotype: male, Kot Sarang (33  Etymology.The name of this new species is derived from the Latin adjective cardio, meaning heart, referring to the penis guide as seen in the lateral view.The body (Figure 2A) is oval and convex, the dorsum surface is covered with dense pubescence.The head, mouthparts, and antennae are yellowish-brown.The pronotum, Etymology.The name of this new species is derived from the Latin adjective cardio, meaning heart, referring to the penis guide as seen in the lateral view.
The body (Figure 2A) is oval and convex, the dorsum surface is covered with dense pubescence.The head, mouthparts, and antennae are yellowish-brown.The pronotum, scutellum, elytra, and prothoracic hypomeron are brownish yellow.The elytral epipleuron is brown with dark brown outer and inner margins (Figure 2B,C).The under-side is dark brown.The leg (Figure 1G) is yellowish-brown, except for the apical part of the tibia, which is dark brown.
The head (Figure 2C) is small, 0.63 times the pronotal width, and has fine punctures that are 0.5-1.0diameters apart.The eye facets are dense, with an interocular distance that is 0.45× the head width.The pronotum is 0.77× the elytral width (PW/EW = 0.85/1.11).The eyes are densely faceted, with sparse hairs, and an interocular distance that is 0.44 times the head width.The pronotum is 0.74 times the elytral width, with pronotal punctures that are unevenly distributed and 0.5-2.5 diameter apart.The elytra are similar to the pronotum and are separated by 1.0-3.0diameters.The prosternal process (Figure 1B) is trapezoidal, with a length 2 time the basal width and with lateral margins that have distinct parallel carinae and which extend to the frontal region of the prosternum.The postcoxal lines (Figure 2D) at abdominal ventrite 1 are complete and reach 6/7ths of its length, while the male apical margins of the abdominal fifth ventrite are rounded.
Male genitalia (Figure 2E-H): the penis (Figure 2E) is stout.The penis capsule's inner arm is distinctly long and the outer ones are short and indistinct.The penis apex (Figure . 2F) is spoon shaped with a bifurcated large membranous appendage.The tegmen (Figure 2G,H) is stout, while the penis guide along the ventral view is robust and somewhat V-shaped, curving smoothly to join the apex and with two distinct keel at the outer and inner basal areas (Figure 2G).The penis guide along the lateral view is heart-like, broadly oval-shaped and wider at the middle, converging to a blunt apex (Figure 2H).The parameres are stout and oval, shorter than the penis guide and have long setae on the apical, outer and inner margins.
Female: external appearance is similar to the male, except for abdomen ventrite 5 (Figure 2I) posteriorly which is without a median tooth and ventrite 6 which is weakly arcuate.Female spermatheca (Figure 2J) are without a nodulus and ramus.
Diagnosis.This species can be easily separated from Scymnus (Pullus) latifolius by the brownish-yellow body (Figure 2A-C), and by the broad and strongly curved apex of the penis, which forms a hook-shaped structure (Figure 2D) in the cardi, while in latifolius the apex is also hook-shaped but is not strongly curved (Figure 3D).The penis guide along the lateral view is bulbous-like in latifolius (Figure 3E).In cardi the penis guide in the ventral view is somewhat V-shaped (Figure 2F) but is U-shaped and shovel-like in latifolius (Figure 3F).The penis guide along the lateral view is heart-like in cardi (Figure 2E).The parameres are slightly shorter than the penis guide in cardi (Figure 2E,F), while the opposite is true for latifolius (Figure 3E,F).

The Mitochondrial Genome
Genome organization.The mitogenome of Scymnus (Pullus) cardi (GenBank: PP639204) is 15416 bp in length, with an A + T content of 78.5%.As with other beetle mitogenomes, the nucleotide composition of the S. (P.) cardi has an obvious A + T bias (Table 1).The newly sequenced mitogenome contains 37 genes (13 PCGs, 2 rRNAs, and 22 tRNAs) and 1 control region (876 bp, A + T ratio 85.1%).Nine of the PCGs and 14 tRNAs are encoded on the positive strand (forward strand) and the remaining 4 PCGs, 8 tRNAs, and 2 rRNAs are located on the negative strand (reverse strand) (Table S1 and Figure 4).
The gene arrangement of the new mitogenome is similar to those of the other Coccinellidae mitochondrial genomes, studied by Kim et al.
Insects 2024, 15, x FOR PEER REVIEW 1 in the anticodon loop might be considered to be indicative of those of more ancient species [77].
The control region (A + T-rich region) of Scymnus (Pullus) cardi is located betwe 12S rRNA and trnI-trnG-trnM cluster (Tables S1 and S2 and Figure 5) with a high content of 86% (Tables S1 and S2, Figure 5).Codon Usage.The relatively synonymous codon usage (RSCU) values for the in the mitogenome of the Scymnus (Pullus) cardi were analyzed and compared here Scymnus (Pullus) loewii, and Scymnus (Pullus) rubricaudus and showed very high simi A and U were more frequently used than G and C. The four most frequently used c in three species of genus Scymnus are UUA (Leu2), UCU (Ser2), CGA (Agr), and (Gly) (Figure S3).

Phylogenetic Analyses
The phylogenetic analyses of 15 mitochondrial genes from 59 ladybird species outgroups confirm that the sequenced species Scymnus (Pullus) cardi sp.nov. is nes the subgenus Pullus, with 100% support value.Furthermore, our results indicate th genus Scymnus is the sister group of Nephus, with a high support value (BS = 100% 1) (Figure 6).These results were recovered in all concatenated datasets using max likelihood (ML) and Bayesian inference (BI) analyses.
Based on the 13 PCGs and 2 rRNAs datasets, the phylogenetic trees were r structed by 2 inference methods (ML and BI).The BI trees and ML analyses present same topology (Figure 6).The results exhibit a strongly resolved relationship be tribes, with high posterior probability support values (PP = 1), except for Cheilom Oenopia (PP = 0.63), and Megalocaria + Anatis (Calvia + Lemnia + Coelophora + Propylea) 0.50) in tribe Coccinellini.PCGs, rRNAs, tRNAs, and control region.The total length of the 13 PCGs of Scymnus (Pullus) cardi is 11,060 bp and begins with the typical mitogenome codon ATN (N represents C, G, and T), except for the ND2 gene with AAT, and the ND1, ND4 and ND6 genes with TAT.All stop codons of the 13 PCGs were TAA/TAG or incomplete stop codons by T, except ND1 with CTG (Table S1), which are observed in the mitogenomes of beetles [59,62,75].
The large ribosomal RNA (16S rRNA) of Scymnus (Pullus) cardi is 1276 bp in size, with an A + T content of 82%, and its small ribosomal RNA (12S rRNA) is 793 bp, with an A + T content of 82.6% (Tables S1 and S2 and Figure 5).The two rRNA genes are located between tRNA-Leu1 and tRNA-Val and tRNA-Val and the control region, meaning that i resembles the mitogenome of previously studied ladybird beetles [59,62,65].
The 22 tRNAs have a size range of 57 bp (trnS1) to 70 bp (trnK) and are of 1413 bp in total length.All of the tRNA present a canonical cloverleaf secondary structure with the conventional four arms, except the trnS1 (tRNA-Serine1), which lacks the D-stem or loop in the dihydrouridine (DHU) arm, as in many beetle species (Figure S1).The tRNAs that include trnW, trnE, trnH, and trnP have smaller T-loop motifs.The tRNAs that include trnQ, trnC, trnA, trnR, trnV, and trnS2 have smaller D-loop motifs (Figure S1).The length of the trnS1 genes in Scymnus (Pullus) cardi, Scymnus (Pullus) loewii, and S. (P.) rubricaudus range from 57 bp to 55 bp and all these species have a UCUs codon in the anticodon loop (AC loop) (Figure S4), as in many ladybird beetle species [76].The structure of the UCUs in the anticodon loop might be considered to be indicative of those of more ancient insect species [77].
The control region (A + T-rich region) of Scymnus (Pullus) cardi is located between the 12S rRNA and trnI-trnG-trnM cluster (Tables S1 and S2 and Figure 5) with a high A + T content of 86% (Tables S1 and S2, Figure 5).
Codon Usage.The relatively synonymous codon usage (RSCU) values for the PCGs in the mitogenome of the Scymnus (Pullus) cardi were analyzed and compared here with Scymnus (Pullus) loewii, and Scymnus (Pullus) rubricaudus and showed very high similarity.A and U were more frequently used than G and C. The four most frequently used codons in three species of genus Scymnus are UUA (Leu2), UCU (Ser2), CGA (Agr), and GGA (Gly) (Figure S3).

Phylogenetic Analyses
The phylogenetic analyses of 15 mitochondrial genes from 59 ladybird species and 3 outgroups confirm that the sequenced species Scymnus (Pullus) cardi sp.nov. is nested in the subgenus Pullus, with 100% support value.Furthermore, our results indicate that the genus Scymnus is the sister group of Nephus, with a high support value (BS = 100%, PP = 1) (Figure 6).These results were recovered in all concatenated datasets using maximum likelihood (ML) and Bayesian inference (BI) analyses.
Insects 2024, 15, x FOR PEER REVIEW 11 of 16 The 13 PCGs_AA partition scheme produced a slightly different topology in the BI tree, with Anatis found to be sister to Megalocaria (Halyzia + Vibidia + Psyllobora + Illeis) with a moderate support value (PP = 0.89), while in ML, Megalocaria was not recovered as sister of the Anatis (Figure S2).Similarly, In the 13 PCGs + 2rRNAs dataset, the clade Coccinella was found to be a sister to Aiolocaria + Cheilomenes with a high support value (BS = 89%, BL = 1), which is not observed in the other datasets (13 PCGs_AA).In the 13 PCGs + 2rRNAs dataset, both ML and BI analyses place the tribe Novini as the basal group in the subfamily Coccinellinae (Figure 6), whereas, in the 13 PCGs_AA dataset, both ML and BI analyses supported the tribe Coccidulini together with Epilachnini, which diverges as an early sister group in Coccinellinae (Figure S2).

Discussion
In the present study, we describe the new species Scymnus (Pullus) cardi of Scymnus and provide its mitochondrial genome, which gives comprehensive information for the identification of this species.Based on the 13 PCGs and 2 rRNAs datasets, the phylogenetic trees were reconstructed by 2 inference methods (ML and BI).The BI trees and ML analyses presented the same topology (Figure 6).The results exhibit a strongly resolved relationship between tribes, with high posterior probability support values (PP = 1), except for Cheilomenes + Oenopia (PP = 0.63), and Megalocaria + Anatis (Calvia + Lemnia + Coelophora + Propylea) (PP = 0.50) in tribe Coccinellini.
The 13 PCGs_AA partition scheme produced a slightly different topology in the BI tree, with Anatis found to be sister to Megalocaria (Halyzia + Vibidia + Psyllobora + Illeis) with a moderate support value (PP = 0.89), while in ML, Megalocaria was not recovered as sister of the Anatis (Figure S2).Similarly, In the 13 PCGs + 2rRNAs dataset, the clade Coccinella was found to be a sister to Aiolocaria + Cheilomenes with a high support value (BS = 89%, BL = 1), which is not observed in the other datasets (13 PCGs_AA).In the 13 PCGs + 2rRNAs dataset, both ML and BI analyses place the tribe Novini as the basal group in the subfamily Coccinellinae (Figure 6), whereas, in the 13 PCGs_AA dataset, both ML and BI analyses supported the tribe Coccidulini together with Epilachnini, which diverges as an early sister group in Coccinellinae (Figure S2).

Discussion
In the present study, we describe the new species Scymnus (Pullus) cardi of Scymnus and provide its mitochondrial genome, which gives comprehensive information for the identification of this species.
Scymnus (Pullus) cardi is allied with the clade of Scymnus (Pullus) latifolius and Scymnus (Pullus) loewii nested in subgenus Pullus.Based on the brief description of S. (P.) latifolius by Poorani and Lalitha [78], the most relevant characteristics that distinguish these species are the body appearance and male genitalia.S. (P.) cardi has a yellowish-brown body, while S. (P.) latifolius has a dark brown body with an elytral discal area of yellow to light brown.The male genitalia of S. (P.) cardi has a spoon-shaped structure at the penis apex, and a V-shaped structure of the penis guide in the ventral view, whereas, in S. (P.) latifolius the penis apex has a weak and slight curve, and the penis guide in the ventral view is U shaped.These distinct characteristics of the male genitalia of S. (P.) cardi differentiate this new species from the six recorded species of the subgenus Pullus in Pakistan [20,26,79,80].The type locality of S. (P.) cardi is Kot Sarang, located in district Chakwal of Punjab province.The type specimen of S. (P.) cardi was collected from the Ziziphus nummularia plants of the family Rhamnaceae and was found feeding on Myzus persicae (Aphididae), while the paratypes collected from Kallar Kahar (Chakwal) were found feeding on both Aphis gossypii and Myzus persicae (Aphididae) of Ziziphus nummularia.Specimens of S. (P.) latifolius were collected from district Rawalakot (33 • 40.668N, 73 • 34.456 E, 478 m) of Azad Jammu and Kashmir.Previous studies have indicated that most of the reported Pakistani species of the subgenus Pullus feed on members of the families Apididae and Pseudococcidae [79,80].According to the paper of Poorani and Lalitha [78], S. (P.) latifolius predates on three species of the family Pseudococcidae (Ferrisia virgata, M. hirsutus, and Paracoccus marginatus) of mulberry and eggplant in India.
To provide more genetic information about the new species, its mitochondrial genome was obtained using next-generation sequencing.Based on 59 taxa and 3 outgroups, we explored the phylogenetic relationships of Coccinellidae using two different methods (IQ-TREE and Phylobayes) based on two datasets (13 PCGs+2r RNAs, and 13 PCGs_AA), which recovered this family into two main clades (Coccinellinae and Microweiseinae).These results strongly support the placement of the new species S. (P.) cardi in the genus Scymnus Kugelann, 1794, with a high support value (BS = 100%, PP = 1) (Figure 6).
Che et al. [13] conducted a comprehensive study on the phylogenetic relationships within the family Coccinellidae, providing strong support for the notion of the monophyly of several tribes within the subfamily Coccinellinae.Their research consistently found Coccinellini to be monophyletic, which contrasts with previous studies [11,55,57,76,81,82] that have suggested it to be the sister group of Chilocorini.Our results also recovered a similar branching pattern showing Coccinellini as a sister to Chilocorini based on the mitogenome data (Figure 6).
The monophyly of Coccinellidae and several genera within it were supported here.Our study suggests that increased taxa sampling is necessary to comprehensively evaluate the phylogenetic relationships of Coccinellidae in the future.

Conclusions
The combination of morphological and mitogenomic data allows us to describe a new species of family Coccinellidae, Scymnus (Pullus) cardi, and identify its phylogenetic relationship.In this study, we utilized high-throughput sequencing technology to reconstruct the mitochondrial genome S. (P.) cardi, which is 15416 bp in length and expresses high AT bias.Our phylogenetic analysis revealed the phylogenetic position of S. (P.) cardi and indicated that it belongs to the genus Scymnus in the tribe Scymnini.The S. (P.) cardi mitogenome reported here adds to the number of Coccinellidae mitochondrial genomes available for future work.Additionally, our sequenced mitogenome with morphology was helpful in the identification of S. (P.) cardi.This study will contribute to future work on population genetics and the evolution of the genus Scymnus Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/insects15050371/s1, Figure S1 S1: Summary of the mitogenome of Scymnus (Pullus) cardi sp.nov.; Table S2: Nucleotide composition of the Scymnus (Pullus) cardi sp.nov.mitogenome.Protein-coding genes (PCGs), ribosomal RNAs (rRNAs), and transfer RNAs (tRNAs).

Data Availability Statement:
The following information was supplied regarding the availability of DNA sequences: the new mitogenomes are deposited in GenBank of NCBI and the accession numbers are PP639204.

Figure 4 .
Figure 4. Circle maps of the mitochondrial genomes of Scymnus (Pullus) cardi sp.nov.Different genes are distinguished with different colors.PCGs, rRNAs, tRNAs, and control region.The total length of the 13 PCGs of Scymnus (Pullus) cardi is 11,060 bp and begins with the typical mitogenome codon ATN (N represents C, G, and T), except for the ND2 gene with AAT, and the ND1, ND4 and ND6 genes with TAT.All stop codons of the 13 PCGs were TAA/TAG or incomplete stop codons by T, except ND1 with CTG (TableS1), which are observed in the mitogenomes of beetles[59,62,75].The large ribosomal RNA (16S rRNA) of Scymnus (Pullus) cardi is 1276 bp in size, with an A + T content of 82%, and its small ribosomal RNA (12S rRNA) is 793 bp, with an A +

Figure 4 .
Figure 4. Circle maps of the mitochondrial genomes of Scymnus (Pullus) cardi sp.nov.Different genes are distinguished with different colors.

Figure 6 .
Figure 6.Phylogenetic relationships between S. (P.) cardi sp.nov.(highlighted in red and the tip is bold) and 61 other beetles.The tree was inferred by the maximum likelihood (ML) method based on 13 PCGs and 2 ribosomal RNAs (13 PCGs + 2rRNAs).Scale bar refers to a phylogenetic distance of 0.045 nucleotide substitutions per site.Node numbers show bootstrap support values (left) and Bayesian posterior probability support values (right).Different background colors indicate different tribes.

Figure 6 .
Figure 6.Phylogenetic relationships between S. (P.) cardi sp.nov.(highlighted in red and the tip is bold) and 61 other beetles.The tree was inferred by the maximum likelihood (ML) method based on 13 PCGs and 2 ribosomal RNAs (13 PCGs + 2rRNAs).Scale bar refers to a phylogenetic distance of 0.045 nucleotide substitutions per site.Node numbers show bootstrap support values (left) and Bayesian posterior probability support values (right).Different background colors indicate different tribes.

:
The predicted secondary structures of 22 tRNA genes inferred for Scymnus (Pullus) cardi sp.nov.The red circle dot indicates a mismatched base, and the blue line represent a matched base; Figure S2.Phylogenetic relationships between S. (P.) cardi sp.nov.(highlighted in red and the tip is bold) and 61 other beetles.Tree inferred by maximum likelihood (ML) method based on 13 PCGs being translated into amino acids (13 PCG_AA).Scale bar refers to a phylogenetic distance of 0.045 nucleotide substitutions per site.Node numbers show bootstrap support values (left) and Bayesian posterior probability support values (right).Different background colors indicate different tribes.(--) indicates that the node is not recovered by BI analysis; Figure S3: Relative synonymous codon usage (RSCU) of Scymnus (Pullus) cardi sp.nov., Scymnus (Pullus) loewii, and Scymnus (Pullus) rubricaudus mitochondrial genomes; Figure S4: The predicted secondary cloverleaf structure for the trn-Ser1 genes of Scymnus (Pullus) cardi sp.nov., Scymnus (Pullus) loewii, and Scymnus (Pullus) rubricaudus.Table

Table 1 .
List of reference mitochondrial genomes chosen for phylogenetic analysis.