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.

1. Introduction

Scymnus is one of the largest genera of ladybird beetles (Coleoptera, Coccinellidae, Scymnini), comprising more than 800 described species. This genus is composed of eight subgenera: Scymnus Kugelann, Pullus Mulsant, Didion Casey, Neopullus Sasaji, Parapullus Yang, Mimopullus Fürsch, Orthoscymnus Canepari, and Canalipullus Lafer [1,2]. Many taxonomists, such as Crotch [3], Sicard [4], Korschefsky [5], Kamiya [6], Sasaji [7], Gordon [8], Kovář [9], Ślipiński [10], Giorgi et al. [11] and Seago et al. [12], have established different classification systems for this genus. Recently, Che et al. [13] have classified it into the tribe Scymnini of the subfamily Coccinellinae.

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).

The members of subgenus Pullus are widely distributed all over the world, with 461 species described from the palaearctic (149) [16], nearctic (154) [8,21], neotropical (27) [22], Australian (5) [23], oriental (78) [24,25,26] and afrotropical regions (50) [27,28,29,30]. Currently, six species of Pullus have been recorded from Pakistan.

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.

2. Materials and Methods

2.1. 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; PW—pronotal width at widest part; EL—elytral length along suture from base to apex including scutellum.

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.

PMAS-AAU-	Pir Mehr Ali Shah-Arid Agriculture University, Rawalpindi, Pakistan.
SCAU-	Department of Entomology, South China Agricultural University, Guangzhou, China.

2.2. 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. K-mer 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.

Geneious Prime 2020.2.4 was used to perform gene annotations, check circularization, and extract the protein-coding gene and rRNAs from the mitochondrial genome [39]. A map of the mitogenomes was created using CGView Server (http://cgview.ca, visited on 25 January 2024) [40]. The base compositions of the mitochondrial genome were analyzed by MEGA v.11 [41]. Transfer RNAs (trnS1) composition and codon usage of the mitogenome of Scymnus (Pullus) cardi sp. nov. was compared with Scymnus (Pullus) loewii, (GenBank: MZ303019) and Scymnus (Pullus) rubricaudus (GenBank: MZ303020). The secondary structures and the anticodon of tRNAs of mitochondrial genomes were identified using MITOS Web Server (http://mitos2.bioinf.uni-leipzig.de/index.py (accessed on 15 May 2024)) [42] and tRNAscan-SE v1.3.1 [43]. The equations for computing AT-skew and GC-skew were as follows: GC-skew = [G% − C%]/[G% + C%] and AT-skew = [A% − T%]/[A% + T%] [44]. The codon and relative synonymous codon usage (RSCU) of 13 protein-coding genes (13 PCGs) were determined in Phylosuite [45,46]. The mitogenome of Scymnus (Pullus) cardi has been submitted to GenBank under Accession number PP639204, as shown in Table 1.

2.3. Phylogenetic Analyses

The phylogenetic position of Scymnus (Pullus) cardi and the high-level relationships of the family Coccinellidae were inferred from 59 Coccinellidae mitochondrial genome sequences, with Dastarcus helophoroides, Discolomatinae sp., and Tenebrio molitor used as outgroups (Table S1). The 13 protein-coding genes (PCGs) and 2 ribosomal RNAs (rRNAs) were extracted from each mitochondrial genome by Geneious Prime 2020.2.4 [39] and aligned separately using MUSCLE v.3.8 with default settings [47]. The phylogenetic analyses were carried out using the following two datasets: (1) 13 PCGs + 2 rRNAs, composed of the 13 protein-coding genes, including all codons and 2 ribosomal RNAs; (2) 13 PCGs_AA, wherein the 13 protein-coding genes were translated into amino acids. Gaps and ambiguous sites in the multiple alignment sequences were filtered using Gblocks 0.91 under default parameters [48]. The aligned gene datasets were then concatenated using SequenceMatrix v.1.9 [49]. The trees were inferred using PhyloBayes MPI v1.5a [50] and IQ-TREE v2.2.0 [51]. 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].

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

Family	Subfamily	Tribe	Species	Accession Numbers	Size bp	A + T%	AT% of All PCGs	References
Coccinellidae	Coccinellinae	Coccinellini	Aiolocaria hexaspilota	MK583344	17,549	80.2	79.7	[53]
	Coccinellinae	Coccinellini	Anatis ocellata	KX035143	17,092	79.3	78.2	Unpublished
	Coccinellinae	Coccinellini	Anisosticta novemdecimpunctata	KT876880	15,289	78.9	77.9	[54]
	Coccinellinae	Coccinellini	Calvia championorum	KX132085	17,575	78.2	77.3	Unpublished
	Coccinellinae	Coccinellini	Calvia decemguttata	KX087252	16,425	78.4	77.2	Unpublished
	Coccinellinae	Coccinellini	Calvia muiri	MF992928	17,126	78.3	77.2	[55]
	Coccinellinae	Coccinellini	Cheilomenes sexmaculata	MW845811	17,297	78	77.2	[56]
	Coccinellinae	Coccinellini	Coccinella lama	MW029464	18,932	77.1	77.2	[57]
	Coccinellinae	Coccinellini	Coccinella septempunctata	OU015583	19,413	76.3	77.1	Unpublished
	Coccinellinae	Coccinellini	Coccinella transversoguttata	OK624419	17,575	77.9	77.2	Unpublished
	Coccinellinae	Coccinellini	Coleomegilla maculata	KJ778881	17,516	77.5	75.8	[58]
	Coccinellinae	Coccinellini	Cycloneda sanguinea	KJ778883	15,118	79	77.6	[58]
	Coccinellinae	Coccinellini	Cycloneda munda	KJ778882	14,292	77.4	76.3	[58]
	Coccinellinae	Coccinellini	Eriopis connexa	MG253268	17,652	79.5	78.7	Unpublished
	Coccinellinae	Coccinellini	Eriopis patagonia	MN509443	15,720	80.1	79.2	[59]
	Coccinellinae	Coccinellini	Harmonia axyridis	KR108208	16,387	78.7	77.4	[60]
	Coccinellinae	Coccinellini	Harmonia eucharis	MW029462	17,441	76	74.9	[57]
	Coccinellinae	Coccinellini	Harmonia quadripunctata	KX087296	18,051	76.5	75.5	Unpublished
	Coccinellinae	Coccinellini	Hippodamia convergens	KX755331	18,419	78.4	77.2	Unpublished
	Coccinellinae	Coccinellini	Hippodamia tredecimpunctata	KJ778889	17,275	77.6	76.1	[58]
	Coccinellinae	Coccinellini	Hippodamia undecimnotata	KX087298	15,587	77.5	76.1	Unpublished
	Coccinellinae	Coccinellini	Hippodamia variegata	MK334129	17,823	77.5	76.6	[61]
	Coccinellinae	Coccinellini	Lemnia saucia	MK574678	14,106	78.9	78.1	[62]
	Coccinellinae	Coccinellini	Megalocaria dilatate	MZ983384	18,608	79.2	78.5	Unpublished
	Coccinellinae	Coccinellini	Oenopia dracoguttata	MW029467	19,220	78.1	77.3	[57]
	Coccinellinae	Coccinellini	Oenopia sauzeti	MW530420	17,630	80.1	78.2	Unpublished
	Coccinellinae	Coccinellini	Oenopia formosana	OR804096	17,885	79.1	77.9	Unpublished
	Coccinellinae	Coccinellini	Olla v-nigrum	MZ303015	14,448	76.9	75.6	Unpublished
	Coccinellinae	Coccinellini	Propylea japonica	KM244660	15,027	79.1	78.6	[63]
	Coccinellinae	Coccinellini	Propylea quattuordecimpunctata	MF992931	17,471	79.4	78.6	Unpublished
	Coccinellinae	Coccinellini	Propylea sp.	KX132084	15,915	79.6	78.7	Unpublished
	Coccinellinae	Coccidulini	Cryptolaemus montrouzieri	KT874575	17,010	79.5	78.4	Unpublished
	Coccinellinae	Coccidulini	Coccidula rufa	JX412767	10,589	76.9	76.6	Unpublished
	Coccinellinae	Chilocorini	Chilocorus bipustulatus	MN053054	12,229	79.1	78.9	[55]
	Coccinellinae	Chilocorini	Chilocorus rubidus	OQ130027	16,801	78.3	77.1	Unpublished
	Coccinellinae	Epilanchnini	Afissula kambaitana	MF992930	14,082	77.3	76.7	Unpublished
	Coccinellinae	Epilanchnini	Epilachna admirabilis	MN053053	17,445	79.9	78	[55]
	Coccinellinae	Epilanchnini	Henosepilachna pusillanima	KJ131489	16,216	78.2	76.8	[64]
	Coccinellinae	Epilanchnini	Henosepilachna vigintioctopunctata	MG584727	17,057	79.2	78.2	[55]
	Coccinellinae	Epivertini	Epiverta chelonia	ON209194	17,347	75.8	74.2	[65]
	Coccinellinae	Hyperaspini	Brachiacantha groendali	MZ303003	15,499	79.6	77.9	Unpublished
	Coccinellinae	Hyperaspini	Hyperaspis festiva	MZ303012	15,999	78.2	77.3	Unpublished
	Coccinellinae	Noviini	Rodolia quadrimaculata	MN053055	12,660	75.7	75.3	[55]
	Coccinellinae	Psylloborini	Illeis bistigmosa	MZ325765	17,840	77.5	76.6	[66]
	Coccinellinae	Psylloborini	Illeis cincta	MF992929	15,856	76.4	75.3	[55]
	Coccinellinae	Psylloborini	Illeis koebelei	OK012004	17,054	77.2	75.2	Unpublished
	Coccinellinae	Psylloborini	Psyllobora lenta	MZ303017	14,462	77.4	76.1	Unpublished
	Coccinellinae	Psylloborini	Vibidia duodecimguttata	MT114193	19,627	75.4	74.6	[67]
	Coccinellinae	Scymnini	Nephus (Bipunctatus) includens	MN164642	16,638	80.1	78.8	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) oblongosignatus	MT445723	16,647	79.6	77.7	[68]
	Coccinellinae	Scymnini	Nephus (Geminosipho) reunioni	MN164643	17,031	79	77.1	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) apolonia	MN164644	16,430	77.5	76.4	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) voeltzkowi	MN164646	17,060	79.9	78.6	[68]
	Coccinellinae	Scymnini	Scymnus (Pullus) cardi sp. nov.	PP639204	15,416	78.5	77.2	THIS STUDY
	Coccinellinae	Scymnini	Scymnus (Pullus) loewii	MZ303019	15,629	79.4	78.4	Unpublished
	Coccinellinae	Scymnini	Scymnus (Pullus) rubricaudus	MZ303020	14,520	78.8	78.3	Unpublished
	Coccinellinae	Subcoccinellini	Subcoccinella vigintiquattuorpunctata	KT780695	14,645	76.1	75.2	Unpublished
	Microweiseinae	Microweiseini	Coccidophilus cariba	MN447521	15,343	78.3	77.4	[69]
Bothrideridae	Bothriderinae	Dastarcini	Dastarcus helophoroides	NC_024271	15,878	79.1	77.5	[70]
Discolomatidae	Discolomatinae	-	Discolomatinae sp.	JX412748	14,141	77.7	77	Unpublished
Tenebrionidae	Tenebrioninae	Tenebrionini	Tenebrio molitor	KF418153	15,785	72.4	69.3	[71]

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

Family	Subfamily	Tribe	Species	Accession Numbers	Size bp	A + T%	AT% of All PCGs	References
Coccinellidae	Coccinellinae	Coccinellini	Aiolocaria hexaspilota	MK583344	17,549	80.2	79.7	[53]
	Coccinellinae	Coccinellini	Anatis ocellata	KX035143	17,092	79.3	78.2	Unpublished
	Coccinellinae	Coccinellini	Anisosticta novemdecimpunctata	KT876880	15,289	78.9	77.9	[54]
	Coccinellinae	Coccinellini	Calvia championorum	KX132085	17,575	78.2	77.3	Unpublished
	Coccinellinae	Coccinellini	Calvia decemguttata	KX087252	16,425	78.4	77.2	Unpublished
	Coccinellinae	Coccinellini	Calvia muiri	MF992928	17,126	78.3	77.2	[55]
	Coccinellinae	Coccinellini	Cheilomenes sexmaculata	MW845811	17,297	78	77.2	[56]
	Coccinellinae	Coccinellini	Coccinella lama	MW029464	18,932	77.1	77.2	[57]
	Coccinellinae	Coccinellini	Coccinella septempunctata	OU015583	19,413	76.3	77.1	Unpublished
	Coccinellinae	Coccinellini	Coccinella transversoguttata	OK624419	17,575	77.9	77.2	Unpublished
	Coccinellinae	Coccinellini	Coleomegilla maculata	KJ778881	17,516	77.5	75.8	[58]
	Coccinellinae	Coccinellini	Cycloneda sanguinea	KJ778883	15,118	79	77.6	[58]
	Coccinellinae	Coccinellini	Cycloneda munda	KJ778882	14,292	77.4	76.3	[58]
	Coccinellinae	Coccinellini	Eriopis connexa	MG253268	17,652	79.5	78.7	Unpublished
	Coccinellinae	Coccinellini	Eriopis patagonia	MN509443	15,720	80.1	79.2	[59]
	Coccinellinae	Coccinellini	Harmonia axyridis	KR108208	16,387	78.7	77.4	[60]
	Coccinellinae	Coccinellini	Harmonia eucharis	MW029462	17,441	76	74.9	[57]
	Coccinellinae	Coccinellini	Harmonia quadripunctata	KX087296	18,051	76.5	75.5	Unpublished
	Coccinellinae	Coccinellini	Hippodamia convergens	KX755331	18,419	78.4	77.2	Unpublished
	Coccinellinae	Coccinellini	Hippodamia tredecimpunctata	KJ778889	17,275	77.6	76.1	[58]
	Coccinellinae	Coccinellini	Hippodamia undecimnotata	KX087298	15,587	77.5	76.1	Unpublished
	Coccinellinae	Coccinellini	Hippodamia variegata	MK334129	17,823	77.5	76.6	[61]
	Coccinellinae	Coccinellini	Lemnia saucia	MK574678	14,106	78.9	78.1	[62]
	Coccinellinae	Coccinellini	Megalocaria dilatate	MZ983384	18,608	79.2	78.5	Unpublished
	Coccinellinae	Coccinellini	Oenopia dracoguttata	MW029467	19,220	78.1	77.3	[57]
	Coccinellinae	Coccinellini	Oenopia sauzeti	MW530420	17,630	80.1	78.2	Unpublished
	Coccinellinae	Coccinellini	Oenopia formosana	OR804096	17,885	79.1	77.9	Unpublished
	Coccinellinae	Coccinellini	Olla v-nigrum	MZ303015	14,448	76.9	75.6	Unpublished
	Coccinellinae	Coccinellini	Propylea japonica	KM244660	15,027	79.1	78.6	[63]
	Coccinellinae	Coccinellini	Propylea quattuordecimpunctata	MF992931	17,471	79.4	78.6	Unpublished
	Coccinellinae	Coccinellini	Propylea sp.	KX132084	15,915	79.6	78.7	Unpublished
	Coccinellinae	Coccidulini	Cryptolaemus montrouzieri	KT874575	17,010	79.5	78.4	Unpublished
	Coccinellinae	Coccidulini	Coccidula rufa	JX412767	10,589	76.9	76.6	Unpublished
	Coccinellinae	Chilocorini	Chilocorus bipustulatus	MN053054	12,229	79.1	78.9	[55]
	Coccinellinae	Chilocorini	Chilocorus rubidus	OQ130027	16,801	78.3	77.1	Unpublished
	Coccinellinae	Epilanchnini	Afissula kambaitana	MF992930	14,082	77.3	76.7	Unpublished
	Coccinellinae	Epilanchnini	Epilachna admirabilis	MN053053	17,445	79.9	78	[55]
	Coccinellinae	Epilanchnini	Henosepilachna pusillanima	KJ131489	16,216	78.2	76.8	[64]
	Coccinellinae	Epilanchnini	Henosepilachna vigintioctopunctata	MG584727	17,057	79.2	78.2	[55]
	Coccinellinae	Epivertini	Epiverta chelonia	ON209194	17,347	75.8	74.2	[65]
	Coccinellinae	Hyperaspini	Brachiacantha groendali	MZ303003	15,499	79.6	77.9	Unpublished
	Coccinellinae	Hyperaspini	Hyperaspis festiva	MZ303012	15,999	78.2	77.3	Unpublished
	Coccinellinae	Noviini	Rodolia quadrimaculata	MN053055	12,660	75.7	75.3	[55]
	Coccinellinae	Psylloborini	Illeis bistigmosa	MZ325765	17,840	77.5	76.6	[66]
	Coccinellinae	Psylloborini	Illeis cincta	MF992929	15,856	76.4	75.3	[55]
	Coccinellinae	Psylloborini	Illeis koebelei	OK012004	17,054	77.2	75.2	Unpublished
	Coccinellinae	Psylloborini	Psyllobora lenta	MZ303017	14,462	77.4	76.1	Unpublished
	Coccinellinae	Psylloborini	Vibidia duodecimguttata	MT114193	19,627	75.4	74.6	[67]
	Coccinellinae	Scymnini	Nephus (Bipunctatus) includens	MN164642	16,638	80.1	78.8	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) oblongosignatus	MT445723	16,647	79.6	77.7	[68]
	Coccinellinae	Scymnini	Nephus (Geminosipho) reunioni	MN164643	17,031	79	77.1	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) apolonia	MN164644	16,430	77.5	76.4	[68]
	Coccinellinae	Scymnini	Nephus (Nephus) voeltzkowi	MN164646	17,060	79.9	78.6	[68]
	Coccinellinae	Scymnini	Scymnus (Pullus) cardi sp. nov.	PP639204	15,416	78.5	77.2	THIS STUDY
	Coccinellinae	Scymnini	Scymnus (Pullus) loewii	MZ303019	15,629	79.4	78.4	Unpublished
	Coccinellinae	Scymnini	Scymnus (Pullus) rubricaudus	MZ303020	14,520	78.8	78.3	Unpublished
	Coccinellinae	Subcoccinellini	Subcoccinella vigintiquattuorpunctata	KT780695	14,645	76.1	75.2	Unpublished
	Microweiseinae	Microweiseini	Coccidophilus cariba	MN447521	15,343	78.3	77.4	[69]
Bothrideridae	Bothriderinae	Dastarcini	Dastarcus helophoroides	NC_024271	15,878	79.1	77.5	[70]
Discolomatidae	Discolomatinae	-	Discolomatinae sp.	JX412748	14,141	77.7	77	Unpublished
Tenebrionidae	Tenebrioninae	Tenebrionini	Tenebrio molitor	KF418153	15,785	72.4	69.3	[71]

3. Results and Discussion

3.1. Taxonomy

Genus Scymnus Kugelann, 1794

Scymnus Kugelann, 1794: 545 [72]. Type species: Scymnus nigrinus Kugelann, 1794 [72], subsequently designated by Westwood [73].

Distribution. Worldwide.

Subgenus Pullus Mulsant, 1846

Figure 1A–G

Scymnus (Pullus) Mulsant, 1846: 241 [14]. Type species: Coccinella subvillosa Goeze, 1777 [15], subsequently designated by Korschefsky [5].

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°1.540′ N, 72°24.212′ E, 477 m), Chakwal, Punjab, 06. viii.2016, Rashid A., leg (SCAU). Paratypes: Khyber Pakhtunkhwa: 1 male, Mardan, Mardan Rd. (34°4.077′ N, 72°15.233′ E, 322 M), 30.ix.2016, Rashid A., leg. (SCAU): Punjab: 2 males, 3 females, Chakwal, Kallar Kahar (Izhar Farm) (32°54.370′ N, 72°30.151′ E, 416 m), 20.viii.2017, Z. Iqbal leg. (PMAS-AAU).

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.

Description. TL: 1.40–1.60 mm; TW: 1.01–1.21 mm; TH: 0.64–0.77 mm; EL/EW: 1.02–1.03; TL/TW: 1.32–1.41; HW/PW: 0.62–0.67; PL/PW: 0.49–0.51.

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 0.5–1.0 diameters 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 1.0–3.0 diameters. 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).

Prey. This species predates Myzus persicae (Sulzer) and Aphis gossypii Glover (Hemiptera: Aphididae) (current study).

Host Plant. This species was collected from Ziziphus nummularia (Burman) (Rosales: Rhamnaceae) (current Study).

Distribution. Pakistan: Chakwal (Kallar Kahar).

3.2. 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. [74], Behere et al. [64], Niu et al. [60], Seo et al. [53], Hao et al. [61], Zhou et al. [62], Salazar and Nattier [59] and Zhang et al. [65], and contain the typical set of mitochondrial genes present in insects.

The general composition of the GC/AT ratio of the Scymnus (Pullus) cardi mitogenome is 21.5/78.5%, PCGs 22.8/77.2%, rRNAs 17.8/82.1%, and tRNAs 21.1/78.9%, with negative GC skew and positive AT skew (Table S2 and Figure 5). The AT skew and GC skew of 13 PCGs in S. (P.) cardi range from 0.26 (ND1) to −0.13 (ND3) and −0.05 (COX1) to −0.38 (ND6).

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).

3.3. 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.

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).

4. 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°40.668′ N, 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 tribes Chilocorini, Epilachnini, Hyperaspini, Scymnini, and Coccidulini are often considered paraphyletic or polyphyletic [57,82]. Our mitochondrial genome data supported a sister-group relationship between Coccidulini and the clade Epilachnini (Epivertini + Subcoccinellini), consistent with the mitochondrial sequence findings of Zhang et al. [65]. However, Scymnini and Hyperaspini were found to be paraphyletic in our analyses, which was also observed in previous studies [11,13,55,82].

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.

5. 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