New mitochondrial genomes of 39 soil dwelling Coleoptera from metagenome sequencing.

High-throughput DNA methods hold great promise for the study of the hyperdiverse arthropod fauna of the soil. We used the mitochondrial metagenomic approach to generate 39 mitochondrial genomes from adult and larval specimens of Coleoptera collected from soil samples. The mitogenomes correspond to species from the families Carabidae (6), Chrysomelidae (1), Curculionidae (9), Dermestidae (1), Elateridae (1), Latridiidae (1), Scarabaeidae (3), Silvanidae (1), Staphylinidae (12), and Tenebrionidae (4). All the mitogenomes followed the putative ancestral gene order for Coleoptera. We provide the first available mitogenome for 30 genera of Coleoptera, including endogean representatives of the genera Torneuma, Coiffaitiella, Otiorhynchus, Oligotyphlopsis, and Typhlocharis.

The mitochondrial metagenomics approach (MMG) provides a cost-effective method for sequencing mitochondrial genomes from numerous species (Andújar et al. 2015; Crampton-Platt et al. 2015). Total genomic DNA from multiple specimens, either extracted individually or in bulk, is shotgun sequenced in a metagenomics mixture, followed by assembly with standard genomic assemblers, from which whole mitochondrial genomes emerge preferentially due to their high copy number relative to most of the nuclear genome. This ‘genome skimming’ approach was used to sequence the mitogenomes of beetle specimens collected from soil samples of the southern Iberian Peninsula at Sierra de Grazalema (36.7N, −5.4W), Sierra de Cabra (37.4N, −4.3W) and Sierra Madrona (38.4N, −4.3W) (see Andújar et al. 2015) by following the ‘Floatation-Berlese-Floatation’ (FBF) protocol (Arribas et al. 2016). Briefly, aliquots of the DNA extracts from 1494 specimens (vouchered at the Natural History Museum London) were pooled to generate 3 pools with roughly equimolar DNA concentration per specimen, after the dsDNA concentration of extracts was measured (Qubit 2.0 Fluorometer, Life Technologies Corp., Carlsbad, CA). Further, TruSeq DNA libraries were constructed and sequenced in the Illumina MiSeq platform (Illumina Inc., San Diego, CA) (2 × 300 bp; 800–950 bp insert size).

The output was processed and assembled in three assemblers as indicated in Andújar et al. (2017). The resulting contigs were subjected to super-assembly in Geneious 7.1.9 (http://www.geneious.com) using the de novo assembly function and showed wide overlap. The procedure resulted in more than 200 mitogenomes longer than 5000 bp, of which 39 were selected for further annotation and identification to species or genus level. Thirty-four of these include the full set of protein-coding, rRNA and tRNA genes (>15,000 bp), of which 17 were complete circular mitogenomes. The remaining 17 were not circularized due to difficulties with the assembly of the control region. Five additional mitogenomes were incomplete by the lack of one or two genes (sequence length between 12,221 and 14,453 bp).

The mitogenomes were annotated using gene predictions with MITOS (Bernt et al. 2013) and manually refined in Geneious. All mitogenomes were structured following the putatively ancestral gene order for the Coleoptera. Mitogenomes assembled from the shotgun mixture were linked to particular specimens using the cox1 barcode sequences obtained from the same specimens with PCR-Sanger sequencing. For those cases where Sanger sequencing failed (5/39), validation was performed by unambiguous match to the species level on BOLD Public Data Portal (Ratnasingham and Hebert 2007, accessed on 20th March 2019). The mitogenomes correspond to 39 different species from the families Carabidae (6), Chrysomelidae (1), Curculionidae (9), Dermestidae (1), Elateridae (1), Latridiidae (1), Scarabaeidae (3), Silvanidae (1), Staphylinidae (12) and Tenebrionidae (4), and include representatives from 37 genera. For 30 of these genera, we provide the first available mitogenome and only two species (Oryzaephilus surinamensis and Hypera postica) have an available mitogenome. The new mitogenomes include endogean representatives of the genera Torneuma, Coiffaitiella, Otiorhynchus, Oligotyphlopsis, and Typhlocharis. For further details on specimens and mitogenomes see Figure 1, Tables 1 and 2, and GenBank Accession Numbers.

Figure 1.

Phylogenetic tree from maximum-likelihood analysis showing the relationships of the 39 newly generated mitogenomes. Circles in branch tips indicate the locality where each specimen was collected (Sierra de Grazalema: black; Sierra de Cabra: grey and Sierra Madrona: white). Shaded frames according with beetle families. GenBank accession numbers are in brackets.

Table 1.

Additional data for the 39 mitogenomes of Coleoptera in this study.

GB accession	Voucher code*	Family	Species	FG**	FSP***	Life stage	Identification
MK692552	BMNH 1041149	Carabidae	Microlestes mauritanicus	x	x	Adult	J.L. Lencina det.
MK692553	BMNH 1042258	Tenebrionidae	Oochrotus unicolor	x	x	Adult	J.L. Lencina det.
MK692554	BMNH 1041892	Curculionidae	Torneuma sp.	x	x	Adult	C. Hernando det.
MK692556	BMNH 1044019	Staphylinidae	Achenium seditiosum	x	x	Adult	V. Assing det.
MK692557	BMNH 1041971	Carabidae	Typhlocharis sp.	x	x	Adult	C. Andújar det.
MK692559	BMNH 1041157	Carabidae	Microlestes reitteri	x	x	Adult	J.L. Lencina det.
MK692560	BMNH 1042672	Staphylinidae	Othius myrmecophilus		x	Larva	BOLD match > 99%
MK692567	BMNH 1042021	Staphylinidae	Oligotyphlopsis sp.	x	x	Adult	C. Hernando det.
MK692568	BMNH 1042062	Curculionidae	Tychius pusillus	x	x	Adult	BOLD match > 99%
MK692574	BMNH 1042482	Carabidae	Trechus Obtusus	x	x	Larva	BOLD match > 99%
MK692579	BMNH 1041943	Staphylinidae	Tachyporus nitidulus	x	x	Adult	V. Assing det.
MK692585	BMNH 1041967	Elateridae	Cardiophorus signatus	x	x	Adult	J.L. Lencina det.
MK692586	BMNH 1041911	Curculionidae	Coiffaitiella sp.	x	x	Adult	C. Hernando det.
MK692587	BMNH 1043732	Curculionidae	Elliptacalles longus	x	x	Adult	BOLD match > 99%
MK692591	BMNH 1041150	Scarabaeidae	Ammoecius elevatus	x	x	Adult	J.L. Lencina det.
MK692592	BMNH 1041990	Curculionidae	Torneuma sp.	x	x	Adult	C. Hernando det.
MK692593	BMNH 1042238	Tenebrionidae	Scaurus uncinus	x	x	Adult	J.L. Lencina det.
MK692597	BMNH 1043977	Staphylinidae	Atheta sp.			Adult	V. Assing det.
MK692599	NA	Staphylinidae	Ocypus aethiops		x	NA	BOLD match > 99%
MK692601	BMNH 1042249	Staphylinidae	Medon sp.			Adult	V. Assing det.
MK692603	BMNH 1042190	Staphylinidae	Micrillus testaceus	x	x	Adult	V. Assing det.
MK692605	BMNH 1042074	Curculionidae	Hypera postica			Adult	BOLD match > 99%
MK692606	BMNH 1042031	Tenebrionidae	Cnemeplatia atropos	x	x	Adult	J.L. Lencina det.
MK692607	BMNH 1042209	Scarabaeidae	Pleurophorus caesus	x	x	Adult	J.L. Lencina det.
MK692609	BMNH 1041982	Curculionidae	Protapion trifolii	x	x	Adult	BOLD match > 99%
MK692616	BMNH 1041162	Staphylinidae	Geostiba sp.	x	x	Adult	V. Assing det.
MK692625	NA	Chrysomelidae	Cryptophagus pilosus	x	x	NA	BOLD match > 99%
MK692626	BMNH 1042569	Carabidae	Calathus granatensis		x	Larva	BOLD match > 99%
MK692638	NA	Staphylinidae	Lomechusa pubicollis	x	x	NA	BOLD match > 99%
MK692642	BMNH 1041893	Silvanidae	Oryzaephilus surinamensis			Adult	J.L. Lencina det.
MK692645	NA	Curculionidae	Echinodera andalusiensis	x	x	NA	BOLD match > 99%
MK692646	BMNH 1042067	Curculionidae	Otiorhynchus sp.	x	x	Adult	C. Hernando det.
MK692648	BMNH 1042068	Scarabaeidae	Esymus pusillus	x	x	Adult	J.L. Lencina det.
MK692661	BMNH 1042524	Staphylinidae	Anotylus inustus	x	x	Larva	BOLD match > 99%
MK692677	BMNH 1041924	Latridiidae	Corticaria sp.	x	x	Adult	J.L. Lencina det.
MK692678	BMNH 1042175	Dermestidae	Thorictus sp.	x	x	Adult	J.L. Lencina det.
MK692681	NA	Carabidae	Syntomus foveatus	x	x	NA	BOLD match > 99%
MK692702	BMNH 1042255	Staphylinidae	Mocyta fungi	x	x	Adult	BOLD match > 99%
MK692707	BMNH 1042182	Tenebrionidae	Centorus elongatus	x	x	Adult	J.L. Lencina det.

Genbank Accession Numbers, voucher codes, taxonomic identification, life stage, and information on whether the provided mitogenomes are the first available for the genus (FG column) and for the species (FSP column).

All mitogenomes were obtained by bulk sequencing of a mix of specimens. Voucher code refers to the specimen with PCR-Sanger sequencing that matches (100% similarity) the obtained mitogenomes. Vouchers are not available for mitogenomes if PCR-Sanger sequencing failed for a particular specimen.

FG: Marked with ‘x’ if the mitogenome is the first available for the genus.

FSP: Marked with ‘x’ if the mitogenome is the first available for the species.

Table 2.

Sampling localities for the 39 mitogenomes of Coleoptera in this study.

GB accession	Locality*	Latitude (N)	Longitude (W)	Altitude (Meters)	Habitat
MK692552	La Dehesilla, Benaocaz, Cádiz, Spain	36.7074	−5.4570	480	Olea europaea field
MK692553	N-420 km 105, Fuencaliente, Ciudad Real, Spain	38.4445	−4.3247	730	Grassland-riverside
MK692554	Arroyo del Espino, El Bosque, Cádiz, Spain	36.7613	−5.5069	275	Riverside
MK692556	Nava de Cabra, Cabra, Córdoba, Spain	37.4856	−4.3634	995	Grassland
MK692557	La Dehesilla, Benaocaz, Cádiz, Spain	36.7074	−5.4567	470	Grassland
MK692559	La Dehesilla, Benaocaz, Cádiz, Spain	36.7074	−5.4570	480	Olea europaea field
MK692560	Robledo de las Hoyas, Fuencaliente, Ciudad Real, Spain	38.4371	−4.3413	950	Quercus faginea forest
MK692567	Arroyo del Bosque, El Bosque, Cádiz, Spain	36.7667	−5.5011	290	Riverside
MK692568	Llanos del Republicano, Villaluenga del Rosario, Cádiz, Spain	36.6817	−5.3574	810	Grassland
MK692574	Llanos del Republicano, Villaluenga del Rosario, Cádiz, Spain	36.6907	−5.3639	925	Quercus suber forest
MK692579	Huerta Hedionda, Tavizna, Benaocaz, Cádiz, Spain	36.7192	−5.4850	360	Olea europaea field
MK692585	Colada de la Breña, Benaocaz, Cádiz, Spain	36.7070	−5.4704	430	Quercus suber forest
MK692586	El Pinsapar, Puerto del Pinar, Grazalema, Cádiz, Spain	36.7726	−5.4240	1115	Abies pinsapo forest
MK692587	Puerto del Boyar, Grazalema, Cádiz, Spain	36.7536	−5.3939	1120	Grassland
MK692591	La Dehesilla, Benaocaz, Cádiz, Spain	36.7074	−5.4570	480	Olea europaea field
MK692592	Arroyo del Bosque, El Bosque, Cádiz, Spain	36.7667	−5.5011	290	Riverside
MK692593	Cortijo del Navazuelo, Carcabuey, Córdoba, Spain	37.4852	−4.3412	1035	Grassland
MK692597	Ermita Nta. Sra. de la Sierra, Cabra, Córdoba, Spain	37.4905	−4.3813	1145	Pinus halepensis
MK692599	Sierra de Cabra, Córdoba, Spain	NA	NA	NA	NA
MK692601	Ladera de la Casa de Cipriano, Fuencaliente, Ciudad Real, Spain	38.4190	−4.3138	765	Quercus suber forest
MK692603	Nava de Cabra, Cortijo de los Benítez, Cabra, Córdoba, Spain	37.4856	−4.3634	995	Grassland
MK692605	Casa de la Viñuela, Cabra, Córdoba, Spain	37.4852	−4.3861	1020	Quercus faginea forest
MK692606	Llanos del Republicano, Villaluenga del Rosario, Cádiz, Spain	36.6857	−5.3648	910	Quercus ilex forest
MK692607	Arroyo del Palancar, Carcabuey, Córdoba, Spain	37.4628	−4.2676	525	Riverside
MK692609	Arroyo del Bosque, El Bosque, Cádiz, Spain	36.7667	−5.5011	290	Riverside
MK692616	Llanos del Campo, Benamahoma, Cádiz, Spain	36.7556	−5.4556	642	Quercus ilex forest
MK692625	Sierra de Grazalema, Cádiz, Spain	NA	NA	NA	NA
MK692626	Llanos del Republicano, Villaluenga del Rosario, Cádiz, Spain	36.6907	−5.3639	925	Quercus suber forest
MK692638	Sierra Madrona, Ciudad Real, Spain	N.A.	N.A.	N.A.	N.A.
MK692642	El Boyar, Cortijo del Santo, Grazalema, Cádiz, Spain	36.7549	−5.4194	920	Quercus ilex forest
MK692645	Sierra de Grazalema, Cádiz, Spain	NA	NA	NA	NA
MK692646	Ermita Nta. Sra. de la Sierra, Cabra, Córdoba, Spain	37.4905	−4.3813	1145	Pinus halepensis
MK692648	Nava de Cabra, Cabra, Córdoba, Spain	37.5067	−4.3671	968	Quercus ilex forest
MK692661	Camino Viejo a la Ermita, Cabra, Córdoba, Spain	37.4811	−4.3885	970	Grassland
MK692677	Arroyo del Espino, El Bosque, Cádiz, Spain	36.7613	−5.5069	275	Riverside
MK692678	Cortijo del Navazuelo, Carcabuey, Córdoba, Spain	37.4852	−4.3412	1035	Grassland
MK692681	Sierra de Grazalema, Cádiz, Spain	NA	NA	NA	NA
MK692702	Collado del Navazuelo, Carcabuey, Córdoba, Spain	37.4801	−4.3347	995	Olea europaea field
MK692707	Nava de Cabra, Cortijo de los Benítez, Cabra, Córdoba, Spain	37.4856	−4.3634	995	Grassland

All mitogenomes were obtained by bulk sequencing of a mix of specimens, and voucher codes to particular specimens assigned by the PCR-Sanger sequencing that matches (100% similarity) the obtained mitogenomes (see Table 1). Metagenomes not linked to a particular vouchered specimen are assigned to a locality but lack detailed information (precise coordinates, altitude, and habitat).

For the 39 newly generated mitogenomes, the 13 protein-coding genes (PCGs) were extracted using Geneious and individually aligned using the FFT-NS-i-x2 algorithm of MAFFT (Katoh et al. 2002). Individual gene alignments were trimmed and concatenated to get a final dataset of 39 taxa and 12,940 bp. This alignment was used for phylogenetic inference using Maximum-likelihood in IQ-TREE (Nguyen et al. 2015), performed through the IQ-TREE web server (Trifinopoulos et al. 2016) without data partitioning, allowing the software to determine the best-fit substitution model and estimating an ultrafast bootstrap approximation with 10,000 replicates. The obtained tree showed the expected relationships among the families within Coleoptera, including the monophyly of the suborders Adephaga and Polyphaga and the monophyly of all families (with more than one mitogenome) (Figure 1).