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Mitogenome of Coprophanaeus ensifer and phylogenetic analysis of the Scarabaeidae family (Coleoptera)

Abstract

Several studies about the phylogenetic relationships of the Scarabaeinae subfamily (Coleoptera: Scarabaeidae) have been performed, but some phylogenetic uncertainties persist including the relationship and monophyly of different tribes and some genera. The aim of this study was to characterize the mitogenome of Coprophanaeus ensifer in order to establish its position within the Scarabaeidae family and to contribute to the resolution of some phylogenetic uncertainties. The mitogenome was sequenced on the Illumina HiSeq 4000, assembled using the Mitobim software and annotated in MITOS WebServer. The phylogenetic trees were reconstructed by Bayesian inference. The C. ensifer mitogenome is a molecule of 14,964 bp that contains the number and organization of the genes similar to those of most Coleoptera species. Phylogenetic reconstruction suggests monophyly of the tribe Phanaeini and supports the hypothesis that Coprini is a sister group of Phanaeini. The results also revealed the position of the tribe Oniticellini which is grouped with Onthophagini and Onitini. The geographic distribution of these species that form the most ancestral clade suggests with Scarabaeinae originated in Africa.

Keywords:
Dung beetle; mitochondrial genome; phylogenomics

Introduction

In the order Coleoptera, the family Scarabaeidae is particularly interesting due to its large adaptive radiation, as well as being formed by lineages with different feeding habits, including phytophagous, saprophagous and copro-necrophagous species (Gunter et al., 2016Gunter NL, Weir TA, Slipinksi A, Bocak L and Cameron SL (2016) If dung beetles (Scarabaeidae: Scarabaeinae) arose in association with dinosaurs, did they also suffer a mass co-extinction at the K-Pg boundary? PLoS One 11:e0153570. ). The species of this family were grouped into 19 subfamilies (Smith, 2009Smith ABT (2009) A review of the family-group names for the superfamily Scarabaeoidea (Coleoptera) with corrections to nomenclature and a current classification. Coleopt Bull 60:144-204. ); among these, Scarabaeinae are important because these beetles provide ecosystem services such as recycling of organic matter, biological control of agricultural pests, and secondary seed dispersal (Nichols et al., 2008Nichols E, Spector S, Louzada J, Larsen T, Amezquita S and Favila ME (2008) Ecological functions and ecosystem services provided by Scarabaeinae dung beetles. Biol Conserv 141:1461-1474. ). This subfamily is characterized by high species diversity, with about 6,200 species taxonomically grouped into 267 genera and 12 tribes (Tarasov and Génier, 2015Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671. ).

Most phylogenetic studies indicate that eight of the 12 Scarabaeinae tribes are monophyletic (Eucraniini, Eurysternini, Gymnopleurini, Oniticellini, Onitini, Phanaeini, Scarabaeini, and Sisyphini) and one is polyphyletic (Deltochilini) (Tarasov and Génier, 2015Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671. ). Contradictory results have been described for the remaining tribes (Ateuchini, Coprini, and Onthophagini), with morphological and/or molecular analyses suggesting monophyly of these tribes, although they are frequently reported as being paraphyletic or polyphyletic (Villalba et al., 2002Villalba S, Lobo JM, Martín-Piera F and Zardoya R (2002) Phylogenetic relationships of Iberian dung beetles (Coleoptera: Scarabaeinae): Insights on the evolution of nesting behavior. J Mol Evol 55:116-126.; Bai et al., 2011Bai M, McCullough E, Song K-Q, Liu W-G and Yang X-K (2011) Evolutionary constraints in hind wing shape in Chinese dung beetles (Coleoptera: Scarabaeinae). PloS One 6:e21600. ; Tarasov and Génier, 2015Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671. ; Tarasov and Dimitrov, 2016Tarasov S and Dimitrov D (2016) Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae). BMC Evol Biol 16:257. and references therein). Differences in the number of lineages have also been reported for Coprini, with descriptions of two or three lineages (Philips et al., 2004Philips TK, Pretorius E and Scholtz CH (2004) A phylogenetic analysis of dung beetles (Scarabaeinae: Scarabaeidae): Unrolling an evolutionary history. Invertebr Syst 18:53-88. ; Monaghan et al., 2007Monaghan MT, Inward DJ, Hunt T and Vogler AP (2007) A molecular phylogenetic analysis of the Scarabaeinae (dung beetles). Mol Phylogenet Evol 45:674-692. ).

The phylogenetic relationships of some Scarabaeinae tribes are also contradictory; for example, the relationships among the Deltochilini, Eurysternini, Onthophagini, Oniticellini, Onitini and Sisyphini tribes are frequently altered (Tarasov and Génier, 2015Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671. ). The position of the tribe Phanaeini is also uncertain since it formed a sister group with the tribe Eucraniini in molecular studies (Ocampo and Hawks, 2006Ocampo FC and Hawks DC (2006) Molecular phylogenetics and evolution of the food relocation behaviour of the dung beetle tribe Eucraniini (Coleoptera: Scarabaeidae: Scarabaeinae). Invertebr Syst 20:557-570. ; Monaghan et al., 2007Monaghan MT, Inward DJ, Hunt T and Vogler AP (2007) A molecular phylogenetic analysis of the Scarabaeinae (dung beetles). Mol Phylogenet Evol 45:674-692. ) and with the tribe Coprini in morphological studies (Philips et al., 2004Philips TK, Pretorius E and Scholtz CH (2004) A phylogenetic analysis of dung beetles (Scarabaeinae: Scarabaeidae): Unrolling an evolutionary history. Invertebr Syst 18:53-88. ). Furthermore, based on morphological analyses, Vaz-de-Mello (2007Vaz-de-Mello FZ (2007) Revision taxonomica e analysis phylogenetico de la tribu Ateuchini. D. Sc. Thesis, Instituto de Ecologia A.C., Xalapa, Veracruz, Mexico, 238 p.) showed that Phanaeini also formed a cluster with Eucraniini, Onitini, and Onthophagini.

In the tribe Phanaeini, phylogenetic uncertainties have also been described within the genus Coprophanaeus d’Olsoufieff, 1924 (Maldaner et al., 2018Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2018) Molecular phylogeny of Coprophanaeus (Megaphanaeus) (Coleoptera: Scarabaeidae: Scarabaeinae) and the position of C. bellicosus. Insect Syst Evol 51:241-255. ). Phylogenetic analyses based on molecular markers (mitochondrial COI, COII and 16S genes) indicated Coprophanaeus to be paraphyletic, since the species of this genus form a clade with Diabroctis mirabilis and Sulcophanaeus faunus (Maldaner et al., 2018Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2018) Molecular phylogeny of Coprophanaeus (Megaphanaeus) (Coleoptera: Scarabaeidae: Scarabaeinae) and the position of C. bellicosus. Insect Syst Evol 51:241-255. ). Within the genus Coprophanaeus, taxonomic uncertainty also exists regarding C. (Megaphanaeus) ensifer (Maldaner et al., 2018Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2018) Molecular phylogeny of Coprophanaeus (Megaphanaeus) (Coleoptera: Scarabaeidae: Scarabaeinae) and the position of C. bellicosus. Insect Syst Evol 51:241-255. ). Individuals of this species exhibit a large variation in size, color and geographic distribution, suggesting the presence of cryptic species (Maldaner et al., 2019Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2019) Genetic and chromatic variation of Coprophanaeus (Megaphanaeus) ensifer (Germar, 1821) (Coleoptera: Scarabaeidae). Zool Anz 283:150-160. ).

Considering the phylogenetic and taxonomic uncertainties, more robust analyses (using larger datasets) of Scarabaeinae tribes, genera and species are necessary (Tarasov and Dimitrov, 2016Tarasov S and Dimitrov D (2016) Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae). BMC Evol Biol 16:257. ). In view of the absence of recombination and exclusively maternal inheritance in animals, mitochondrial DNA is used for phylogeographic inferences (Boore and Brown, 1998Boore JL and Brown WM (1998) Big trees from little genomes: Mitochondrial gene order as a phylogenetic tool. Curr Opin Genet Dev 8:668-674.), resolution of taxonomic uncertainties, and phylogenetic reconstructions (Vilstrup et al., 2011Vilstrup JT, Ho SY, Foote AD, Morin PA, Kreb D, Krützen M, Parra GJ, Robertson KM, Stephanis R, Vegorgh P et al. (2011) Mitogenomic phylogenetic analyses of the Delphinidae with an emphasis on the Globicephalinae. BMC Evol Biol 11:65. ). Specific mitochondrial genes (Baca et al., 2017Baca SM, Toussaint EFA, Miller KB and Short AEZ (2017) Molecular phylogeny of the aquatic beetle family Noteridae (Coleoptera: Adephaga) with an emphasis on data partitioning strategies. Mol Phylogenet Evol 107:282-292.; Nolasco-Soto et al., 2017Nolasco-Soto J, González-Astorga J, Monteros AE, Galante-Patiño E and Favila ME (2017) Phylogeographic structure of Canthon cyanellus (Coleoptera: Scarabaeidae), a neotropical dung beetle in the Mexican transition zone: Insights on its origin and the impacts of pleistocene climatic fluctuations on population dynamics. Mol Phylogenet Evol 109:180-190. ; Villastrigo et al., 2019Villastrigo A, Jäch MA, Cardoso A, Valladares LF and Ribera I (2019) A molecular phylogeny of the tribe Ochthebiini (Coleoptera, Hydraenidae, Ochthebiinae). Syst Entomol 44:273-288.), or the complete mitogenome can be used in such studies, conferring greater robustness to the analysis by providing a larger set of molecular data (Yuan et al., 2016Yuan M-L, Zhang Q-L, Zhang L, Guo Z-L, Liu Y-J, Shen Y-Y and Shao R (2016) High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences. Mol Phylogenet Evol 104:99-111. ; Nie et al., 2018Nie R-E, Breeschoten T, Timmermans MJTN, Nadein K, Xue H-J, Bai M, Huang Y, Yang X-K and Vogler AP (2018) The phylogeny of Galerucinae (Coleoptera: Chrysomelidae) and the performance of mitochondrial genomes in phylogenetic inference compared to nuclear rRNA genes. Cladistics 34:113-130.). Based on the mitogenome, phylogenetic uncertainties were resolved in different taxonomic groups, including the beetle family Chrysomelidae (Nie et al., 2018Nie R-E, Breeschoten T, Timmermans MJTN, Nadein K, Xue H-J, Bai M, Huang Y, Yang X-K and Vogler AP (2018) The phylogeny of Galerucinae (Coleoptera: Chrysomelidae) and the performance of mitochondrial genomes in phylogenetic inference compared to nuclear rRNA genes. Cladistics 34:113-130.).

The present study aimed to characterize the mitochondrial DNA of C. (M.) ensifer and to investigate the phylogenetic relationships within the family Scarabaeidae. In addition, this study provides molecular markers that can be used to test specific taxonomic hypothesis such as assessing the presence of cryptic species in C. (M.) ensifer.

Material and Methods

Collection of the biological sample

Coprophanaeus (M.) ensifer (Germar, 1821Germar EF (1821) Neue exotische Käfer beschrieben. Magazin Entomol 4:107-183. ) specimens were collected in Aldeia, Camaragibe (8º1’18” S; 34º58’52” W), Pernambuco, Brazil, with pitfall traps baited with rotten meat. This collection was authorized by IBAMA/SISBIO through the permanent license of zoological material of the class Insecta (16278 - 1). The specimens were identified at the Laboratory of Biodiversity and Insect Genetics (LGBI), University of Pernambuco (UPE), using the identification key of the genus Coprophanaeus (Edmonds and Zídek, 2010Edmonds WD and Zídek J (2010) A taxonomic review of the neotropical genus Coprophanaeus Olsoufieff, 1924 (Coleoptera: Scarabaeidae, Scarabaeinae). Insecta Mundi 129:1-111.) and by comparison with other specimens of the Entomological Collection of the University of Pernambuco (CEUPE), Laboratory of Biodiversity and Genetics of Insects (LBGI), Institute of Biological Sciences, University of Pernambuco, Pernambuco, Brazil (curator: Rita de Cássia de Moura). In addition, the identification was confirmed by the taxonomist Fernando Silva (UFPA). The specimens were sacrificed and stored in 100% alcohol until the time of DNA extraction. Only one specimen was used for this study.

DNA extraction and sequencing

DNA was extracted from pronotal tissue of one specimen using the phenol-chloroform protocol of Sambrook and Russel (2001Sambrook J and Russel WD (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York.). Genomic sequencing was performed by Macrogen Inc. on an Illumina HiSeq 4000 platform using the following parameters: preparation of the genomic library using the TruSeq DNA PCR-Free kit (350-bp insert size), an average read length of 150 bp, and a paired-end sequencing approach.

Characterization of the mitogenome of Coprophanaeus (M.) ensifer

The sequences obtained were first treated with the Trimmomatic program (Bolger et al., 2014Bolger AM, Lohse M and Usadel B (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120. ) to remove sequences with a quality less than Q20. Next, the mitochondrial genome was assembled using the Mitobim software (Hahn et al., 2013Hahn C, Bachmann L and Chevreux B (2013) Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads--a baiting and iterative mapping approach. Nucleic Acids Res 41:e129. ). The mitogenome of Sarophorus sp. (GenBank: JX412735.1) (Scarabaeinae) was used as reference to bait the reads in the first steps of Mitobim analysis. The consensus sequence was uploaded to the MITOS WebServer for gene annotation of the C. (M.) ensifer mitogenome using the genetic code 05 - invertebrate.

Phylogenetic analysis

The phylogenetic trees were reconstructed using two datasets: 1) complete nucleotide sequence of the mitogenome, except for the control region; 2) concatenated amino acid sequence of protein-coding genes of the mitogenome. In addition to the mtDNA of C. (M.) ensifer, we used the Scarabaeidae mitogenomes available at NCBINCBI - National center for biotechnology information , https://www.ncbi.nlm.nih.gov/
https://www.ncbi.nlm.nih.gov/...
until September, 2019. These mitogenomes included species of Scarabaeinae (32 spp.), Melolonthinae (6 spp.), Rutelinae (3 spp.), Cetoniinae (3 spp.), Aphodiinae (3 spp.), and Dinastinae (1 spp.). Additionally, three species of the family Lucanidae were included as outgroups (Prosopocoilus gracilis, Odontolabis cuvera fallaciosa, Dynodorcus curvidens hopei), since this family is considered sister group to Scarabaeidae, according to molecular and morphological evidence (Grebennikov and Scholtz, 2004Grebennikov VV and Scholtz CH (2004) The basal phylogeny of Scarabaeoidea (Insecta: Coleoptera) inferred from larval morphology. Invertebr Syst 18:321-348.; Timmermans et al., 2016Timmermans MJTN, Barton C, Haran J, Ahrens D, Culverwell CL, Ollikainen A, Dodsworth S, Foster PG, Bocak L and Vogler A (2016) Family-level sampling of mitochondrial genomes in Coleoptera: Compositional heterogeneity and phylogenetics. Genome Biol Evol 8:161-175. ; Yang et al., 2018Yang W, Zhang Y, Feng S, Liu L and Li Z (2018) The first complete mitochondrial genome of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) and its phylogenetic implications for the superfamily Scarabaeoidea. Int J Biol Macromol 118:1406-1413.). These sequences were retrieved from the National Center for Biotechnology Information database (NCBINCBI - National center for biotechnology information , https://www.ncbi.nlm.nih.gov/
https://www.ncbi.nlm.nih.gov/...
). The accession numbers are listed in Table S1 Table S1 - List of species used in the analyses and their respective NCBI accession number, tribe and subfamily. . The resulting sequences were aligned using MAFFTMAFFT version 7, https://mafft.cbrc.jp/alignment/server/
https://mafft.cbrc.jp/alignment/server/...
and then treated with Gblock 0.91b using default parameters (Castresana, 2000Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540-552.).

For phylogenetic analysis, the GTR G+I and MtREV G+I+F substitution models were used for nucleotide and protein sequences, respectively. These models were selected based on Akaike’s information criterion (AIC) using jModelTest v. 2.1.4 (Darriba et al., 2012Darriba D, Taboada GL, Doallo R and Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nat Methods 9:772. ) and ProtTest v. 3.4.2 (Abascal et al., 2005Abascal F, Zardoya R, and Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104-2105.). The trees were reconstructed by Bayesian inference on the CIPRES Science Gateway webserverCIPRES Science Gateway webserver v. 3.3, https://www.phylo.org
https://www.phylo.org...
using the following MCMC parameters: Number of generations of 10 million, sampling every 1,000 generations, four Markov chains, and burn-in of 20%. The tree was visualized and edited using Figtree v. 1.4.3 (Rambaut, 2016Rambaut A (2016) FigTree v1.4.3 software, 3 software, http://tree.bio.ed.ac.uk/software/figtree/ (accessed 3 March 2020).
http://tree.bio.ed.ac.uk/software/figtre...
).

Results and Discussion

Characterization of the mitogenome

The analysis of generated sequences allowed us to characterize the mitogenome of Coprophanaeus (M.) ensifer. A total of 1,190,241 reads were assembled in a mitogenome of 18,134 bp showing an average coverage depth of 9845x and average coverage breadth of 100%. Considering that the assembly and size of the molecule can be influenced by the repetitive nature of the control region (Gillett et al., 2014Gillett CPDT, Crampton-Platt A, Timmermans MJTN, Jordal BH, Emerson BC and Vogler AP (2014) Bulk de novo mitogenome assembly from pooled total DNA elucidates the phylogeny of weevils (Coleoptera: Curculionoidea). Mol Biol Evol 31:2223-2237. ), the size of the molecule without the control region would be 14.964 bp (Figure 1) (accession number: MW122514 and Supplementary File S1 File S1 - GenBank file of Coprophanaeus (M.) ensifer mitogenome, accession number MW122514. ). This is a common size when compared to the typical mitogenome of animals, with the complete mitochondrial genome of animals comprising 15 to 16 kb (Boore, 1999Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27: 1767-1780.) and the control region in representatives of Coleoptera generally comprising 1 kb (Sheffield et al., 2008Sheffield NC, Song H, Cameron SL and Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol 25:2499-2509. ).

Figure 1.
Organization genetic of Coprophanaeus ensifer mitogenome. Protein-coding genes, tRNA genes and rRNA genes are shown in blue, green and purple, respectively. The arrows indicate the direction of the genes.

Mitogenome annotation of C. (M.) ensifer revealed two ribosomal RNAs (rRNA) and 22 transfer RNAs (tRNA), in addition to the following 13 protein-coding genes (CDS): ATP synthase membrane subunits 6 and 8 (ATP6 and 8), cytochrome b (CytB), cytochrome c oxidase 1 to 3 (COX 1-3), and NADH reductase subunits 1 to 6 (NAD1-6) and 4L (NAD4L) (Figure 1, Table 1). According to MITOS WebServerMITOS WebServer, http://mitos.bioinf.uni-leipzig.de/index.py
http://mitos.bioinf.uni-leipzig.de/index...
, 9 CDS and 14 tRNA are located on the positive strand (plus Strand), while 4 CDS, 2 rRNA and 8 tRNA are present on the negative strand (minus Strand) (Figure 1, Table 1). In addition, the positive strand has the lower content of guanine and thymine (G + T = 47%), indicating that this is the light strand (L), according to Lima and Prosdocimi (2017Lima NCB and Prosdocimi F (2017) The heavy strand dilemma of vertebrate mitochondria on genome sequencing age: number of encoded genes or G+ T content?. Mitochondrial DNA A DNA Mapp Seq Anal 28:300-302.). The number, orientation and order of the genes are similar to those of most insect species (Cameron, 2014Cameron SL (2014) Insect mitochondrial genomics: implications for evolution and phylogeny. Annu Rev Entomol 59:95-117. ; Sheffield et al., 2009Sheffield NC, Song H, Cameron SL and Whiting MF (2009) Nonstationary evolution and compositional heterogeneity in beetle mitochondrial phylogenomics. Syst Biol 58:381-394. ), as well as to those of the beetle species Dichotomius schiffleri (Amorim et al., 2017Amorim IC, Melo AS, Cruz GAS, Wallau GL and Moura RC (2017) Dichotomius (Luederwaldtinia) schiffleri (Coleoptera: Scarabaeidae) mitochondrial genome and phylogenetic relationships within the superfamily Scarabaeoidea. Mitochondrial DNA B 2:887-888.) and Priasilpha obscura (Sheffield et al., 2008Sheffield NC, Song H, Cameron SL and Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol 25:2499-2509. ). Within the family Scarabaeidae, they only differ from those observed in the genus Cyphonistes (Dynastinae), which possesses an inversion between the trnaA and trnaR genes, considered an apomorphy (Timmermans et al., 2016Timmermans MJTN, Barton C, Haran J, Ahrens D, Culverwell CL, Ollikainen A, Dodsworth S, Foster PG, Bocak L and Vogler A (2016) Family-level sampling of mitochondrial genomes in Coleoptera: Compositional heterogeneity and phylogenetics. Genome Biol Evol 8:161-175. ).

Table 1.
Gene annotation in the mitogenome of Coprophanaeus ensifer.

Twenty-four intergenic spacers were identified in the mitogenome of C. (M.) ensifer (Table 1). Among these, the spacer between NAD1 and RNAtS2 is a common feature of the superfamily Scarabaeoidea (Jeong et al., 2020Jeong JS, Kim MJ and Kim I (2020) The mitochondrial genome of the dung beetle, Copris tripartitus, with mitogenomic comparisons within Scarabaeidae (Coleoptera). Int J Biol Macromol 144:874-891. ). However, this spacer is larger in C. (M.) ensifer (83 bp) (Table 1) compared to other Scarabaeoidea species which harbor a maximum spacer of 31 bp (e.g., Eurysternus inflexus) (Jeong et al., 2020). In the mitogenome assembled, this region had coverage depth around 7800x which is similar to the average whole mitogenome coverage depth (9845x), strongly supporting the presence of this region.

The mtDNA of C. (M.) ensifer also contains four gene overlaps, totaling 46 bp (Table 1). Such overlaps have been reported in different Coleoptera species, including Cheirotonus mansoni (Shao et al., 2014Shao LL, Huang, DY, Sun XY, Hao JS, Cheng CH, Zhang W and Yang Q (2014) Complete mitochondrial genome sequence of Cheirotonus jansoni (Coleoptera: Scarabaeidae). Genet Mol Res 13:1047-1058.) and Amphimallon solstitiale (Yang et al., 2019Yang X-Z, Zhang L, Feng R-Q, Zhang L-J, Luo F-Z and Yuan M-L (2019) Mitochondrial genome of Amphimallon solstitale (Coleoptera: Scarabaeidae: Melolonthinae) and phylogenetic analysis. Mitochondrial DNA B 4:110-111. ). This type of overlapping is related to the evolution of mtDNA, which tends to reduce its size over time due to selective pressure, causing a reduction in intergenic spacers that can accumulate in gene overlap (Schneider and Ebert, 2004Schneider A and Ebert D (2004) Covariation of mitochondrial genome size with gene lengths: Evidence for gene length reduction during mitochondrial evolution. J Mol Evol 59:90-96. ; Sheffield et al., 2008Sheffield NC, Song H, Cameron SL and Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol 25:2499-2509. , 2010Sheffield NC, Hiatt KD, Valentine MC, Song H and Whiting MF (2010) Mitochondrial genomics in orthoptera using MOSAS. Mitochondrial DNA 21:87-104. ). C. (M.) ensifer, selective pressure apparently did not cause major reductions in the mitogenome, since numerous and large intergenic spacers and only four gene overlaps were observed (Figure 1, Table 1). This number is smaller than that of other species of the genus (Coprophanaeus sp.) whose mitogenome possesses 12 gene overlaps.

The greatest gene overlap in C. (M) ensifer mtDNA is found between the tRNAL1 and rRNAL genes, corresponding to 37 bp of the 69 bp of this tRNA. Gene overlapping can lead to problems in the polycistronic transcription of mitogenomes (Sheffield et al., 2008Sheffield NC, Song H, Cameron SL and Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol 25:2499-2509. , 2010), as it prevents the release of full-length RNAs of each overlapping gene from the same transcript (Boore, 1999Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27: 1767-1780.). Considering this, each transcript of C. (M) ensifer mitogenome will produce only one functional tRNAL1 or rRNAL molecule. However, this effect may be minimized due to the presence of a second gene for leucine tRNA (tRNAL2), as proposed for the grasshopper Rhammatocerus brasiliensis (Amorim et al., 2020Amorim IC, Melo AS, Silva AF, Wallau GL and Moura RC (2020) Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera). Gene 731:144362.). In addition, an overlap between the atp6 and atp8 genes was observed, which is common in metazoans (Campbell and Barker, 1999Campbell NJ and Barker SC (1999) The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region. Mol Biol Evol 16:732-740. ) and is probably a plesiomorphic feature of this group.

Phylogenetic analysis

Among the different subfamilies analyzed in the present study, only the subfamily Melolonthinae was found to be paraphyletic (Figure 2 and Figure S1 Figure S1 - Phylogenetic reconstructions of the family Scarabaeidae based on nucleotide sequences of the mitochondrial genome. ). This result agrees with other phylogenies obtained based on the mitogenome (Song and Zhang, 2018Song N and Zhang H (2018) The mitochondrial genomes of phytophagous scarab beetles and systematic implications. J Insect Sci 18:11. ). Specifically in the subfamily Dynastinae, only the mitogenome of Ciphonistes vallatus has been sequenced and it was therefore not possible to confirm whether this group is monophyletic, as suggested in molecular studies using mitochondrial (16S rRNA, 12S rRNA, and COI) and nuclear markers (28S, LSU, and rRNA) (Gunter et al., 2016Gunter NL, Weir TA, Slipinksi A, Bocak L and Cameron SL (2016) If dung beetles (Scarabaeidae: Scarabaeinae) arose in association with dinosaurs, did they also suffer a mass co-extinction at the K-Pg boundary? PLoS One 11:e0153570. ).

Figure 2.
Phylogenetic reconstructions of the family Scarabaeidae based on mitochondrial protein sequences. These reconstructions were performed by Bayesian inference using the MtREV G+I+F substitution model. The colors of each clade distinguish the Scarabaeidae subfamilies as well as Scarabaeinae tribes. Coprophanaeus ensifer is highlighted in blue in the phylogeny.

The analysis of protein and nucleotide sequences resulted in different phylogenetic relationships between the Scarabaeidae subfamilies (Figure 2, Figure S1 Figure S1 - Phylogenetic reconstructions of the family Scarabaeidae based on nucleotide sequences of the mitochondrial genome. and S2 Figure S2 - Comparison between phylogenies generated based on nucleotide sequences and concatenated protein sequences of mitochondrial genes ). Such differences have been reported in several studies, including those on the family Scarabaeidae (Jeong et al., 2020Jeong JS, Kim MJ and Kim I (2020) The mitochondrial genome of the dung beetle, Copris tripartitus, with mitogenomic comparisons within Scarabaeidae (Coleoptera). Int J Biol Macromol 144:874-891. ). Nucleotide analysis revealed the topology (Dynastinae (Cetoniinae (Melolonthinae (Rutelinae (Aphodiinae + Scarabaeinae))))) (Figure 2). On the other hand, the results of protein analysis showed the topology (Melolonthinae (Cetoniinae (Rutelinae + Dynastinae))) (Scarabaeinae + Aphodiinae). The tree topology based on amino acid sequences was similar to that described in other studies (Song and Zhang, 2018Song N and Zhang H (2018) The mitochondrial genomes of phytophagous scarab beetles and systematic implications. J Insect Sci 18:11. ). Considering this topology and the higher resolution at the nodes, the use of amino acid sequences is suggested for the phylogenetic placement of Scarabaeidae. This is contrary to the hypothesis of Cameron (2014Cameron SL (2014) Insect mitochondrial genomics: implications for evolution and phylogeny. Annu Rev Entomol 59:95-117. ) that states the loss of phylogenetic signal for amino acid sequences in taxonomic groups within the class Insecta.

Among the seven tribes analyzed of the subfamily Scarabaeinae, only Eurysternini, Onitini, Oniticellini and Phanaeini were monophyletic by the two approaches (Figure 2, Figure S1 Figure S1 - Phylogenetic reconstructions of the family Scarabaeidae based on nucleotide sequences of the mitochondrial genome. ). The same result was reported for molecular analyses based on nuclear (28S) and mitochondrial genes (COI and rrnL) (Monahgan et al., 2007). In particular, the tribe Coprini was found to be paraphyletic and monophyletic in the nucleotide and protein trees, respectively (Figure 2, Figure S1 Figure S1 - Phylogenetic reconstructions of the family Scarabaeidae based on nucleotide sequences of the mitochondrial genome. ). Contradictory results are frequently reported for this tribe, which is monophyletic in analyses using COI and COII (Villalba et al., 2002Villalba S, Lobo JM, Martín-Piera F and Zardoya R (2002) Phylogenetic relationships of Iberian dung beetles (Coleoptera: Scarabaeinae): Insights on the evolution of nesting behavior. J Mol Evol 55:116-126.), but commonly paraphyletic in studies using sufficient taxon sampling, for example, in a phylogeny of nuclear (18S rDNA, 28SrDNA, CAD, and topoisomerase I) and mitochondrial genes (16S and COI) (Tarasov and Dimitrov, 2016Tarasov S and Dimitrov D (2016) Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae). BMC Evol Biol 16:257. ).

In the present study, the phylogenetic relationships between Scarabaeinae tribes obtained with the two approaches (amino acid and nucleotide sequences) were: (Onthophagini + Oniticellini) Onitini) Eurysternini) (Coprini+ Phanaeini) (Figure 2). This result is similar to the phylogeny obtained based on one nuclear gene (28S) and two mitochondrial genes (COI and rrnL) (Monaghan et al., 2007Monaghan MT, Inward DJ, Hunt T and Vogler AP (2007) A molecular phylogenetic analysis of the Scarabaeinae (dung beetles). Mol Phylogenet Evol 45:674-692. ). The presence of one monophyletic clade composed of Onthophagini and Oniticellini has also been reported in morphological and molecular studies (COI and 28S genes) (Philips, 2016Philips TK (2016) Phylogeny of the Oniticellini and Onthophagini dung beetles (Scarabaeidae, Scarabaeinae) from morphological evidence. ZooKeys 579:9-57.; Mlambo et al., 2015Mlambo S, Sole CL and Scholtz CH (2015) A molecular phylogeny of the African scarabaeinae (Coleoptera: Scarabaeidae). Arthropod Syst Phylogeny 73:303-321. ). On the other hand, the sister group of this clade differs between morphological and molecular studies, including Sisyphini + Epirinus and Onitini (as observed in the present study), respectively (Monaghan et al., 2007; Tarasov and Genier, 2015Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671. ). This incongruity may be related to the lack of morphological synapomorphies characterizing these tribes, as suggested by Tarasov and Dimitrov (2016).

Coprophanaeus ensifer was clustered with another representative of this genus (Coprophanaeus sp.), which belongs to the tribe Phanaeini, and with species of the tribe Coprini (Canthidium sp. and Dichotomius schiffleri) (Figure 2). The phylogenetic proximity between these tribes has also been reported in other studies using morphological and molecular markers (Philips et al., 2004Philips TK, Pretorius E and Scholtz CH (2004) A phylogenetic analysis of dung beetles (Scarabaeinae: Scarabaeidae): Unrolling an evolutionary history. Invertebr Syst 18:53-88. ; Tarasov and Dimitrov, 2016Tarasov S and Dimitrov D (2016) Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae). BMC Evol Biol 16:257. ). However, this result diverges from the hypothesis of Zunino (1983Zunino M (1983) Essai préliminaire sur l’évolution des armures génitales des Scarabaeinae, par rapport à la taxonomie du groupe et à l’évolution du comportement de nidification [Col. Scarabaeidae]. Bull Soc Entomol France 88:531-542.) based on genitalia traits that suggests Phanaeini to be a sister group of Onitini. Morphological analysis also revealed a closer proximity of Phanaeini to Eucraniini (Philips and Scholtz, 2004Philips TK, Edmonds WD and Scholtz CH (2004) A phylogenetic analysis of the new world tribe Phanaeini (Coleoptera: Scarabaeidae: Scarabaeinae): Hypotheses on relationships and origins. Insect Syst Evol 35:43-63. ). Such positioning could not be verified in our study since there is no Eucraniinimi mitogenome sequenced so far.

In the present study, C. ensifer is part of the most basal clade of Scarabaeinae, which is formed by species of Ateuchini, Coprini and Phanaeini tribes (Figure 2). These species show a neotropical distribution, especially in South America (Edmonds and Zídek, 2010Edmonds WD and Zídek J (2010) A taxonomic review of the neotropical genus Coprophanaeus Olsoufieff, 1924 (Coleoptera: Scarabaeidae, Scarabaeinae). Insecta Mundi 129:1-111.; Valois et al., 2017Valois MC, Vaz-De-Mello FZ and Silva FAB (2017) Taxonomic revision of the Dichotomius sericeus (Harold, 1867) species group (Coleoptera: Scarabaeidae: Scarabaeinae). Zootaxa 4277:503-530. ; Cupello, 2018Cupello M (2018) On the types species of the new world dung beetle genus Canthidium erichson, 1847 (Coleoptera: Scarabaeidae: Scarabaeinae), with an annotated checklist of species. Zootaxa 4388:451-486.), except for Sarophorus sp. (Ateuchini) which is most basal and is found mainly in South Africa (Frolov and Scholtz, 2003Frolov AV and Scholtz CH (2003) Revision of the Afrotropical dung beetle genus Sarophorus Erichson (Coleoptera: Scarabaeidae). Afr Entomol 11:183-198.).

The geographic distribution of these species that form the most ancestral clade suggests with Scarabaeinae originated in Africa, as suggested by Sole and Sholtz (2010Sole CL and Scholtz CH (2010) Did dung beetles arise in Africa? A phylogenetic hypothesis based on five gene regions. Mol Phylogenet Evol 56:631-641. ). After its origin, colonization in South America must have occurred by the common ancestor of Sarophorus sp. and species of Ateuchini, Coprini and Phanaeini tribes. Furthermore, these results do not suggest a fauna exchange between South America and Africa during the early evolution of Scarabaeinae, as suggested by Gunter et al. (2016Gunter NL, Weir TA, Slipinksi A, Bocak L and Cameron SL (2016) If dung beetles (Scarabaeidae: Scarabaeinae) arose in association with dinosaurs, did they also suffer a mass co-extinction at the K-Pg boundary? PLoS One 11:e0153570. ). However, this may be related to a limited number of mitogenomes characterized in Scarabaeinae.

The characterization and analysis of the C.ensifer mitogenome showed a genomic organization and the number of genes similar to those from the majority of Coleoptera species analyzed. Spacers and gene overlaps were also observed in this mitogenome. Regarding phylogenetic uncertainties in Scarabaeinae, the phylogenetic results suggest proximity between the tribes Phanaeini and Coprini, and place the tribe Oniticellini proximity to Onthophagini and Onitini. Despite this, inconsistencies were observed between the protein and nucleotide phylogenetic trees. ​Therefore, additional data from nuclear markers is necessary to elucidate the phylogeny of the Scarabaeidae family. The analysis suggests the origin of the Scarabaeinae subfamily in Africa. The results of this study provide a basis for future phylogenetic analyses of Coprophanaeus in order to gain a better understanding of its evolutionary history.

Acknowledgements

This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance code 001) and by a postdoctoral fellowship granted to I.C. Amorim (PNPD-88882.317331/2019-01). We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scientific initiation fellowship granted to C.A.A. Mello and the productivity fellowship (PQ-2 303902/2019-1) granted to G.L. Wallau. A.F. Silva was the recipient of a doctoral fellowship from Fundação de Amparo em Ciência e Tecnologia do Estado de Pernambuco (FACEPE IBPG: IBPG-PBPG-1127-2.13/18).

References

  • Amorim IC, Melo AS, Cruz GAS, Wallau GL and Moura RC (2017) Dichotomius (Luederwaldtinia) schiffleri (Coleoptera: Scarabaeidae) mitochondrial genome and phylogenetic relationships within the superfamily Scarabaeoidea. Mitochondrial DNA B 2:887-888.
  • Amorim IC, Melo AS, Silva AF, Wallau GL and Moura RC (2020) Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera). Gene 731:144362.
  • Abascal F, Zardoya R, and Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104-2105.
  • Baca SM, Toussaint EFA, Miller KB and Short AEZ (2017) Molecular phylogeny of the aquatic beetle family Noteridae (Coleoptera: Adephaga) with an emphasis on data partitioning strategies. Mol Phylogenet Evol 107:282-292.
  • Bai M, McCullough E, Song K-Q, Liu W-G and Yang X-K (2011) Evolutionary constraints in hind wing shape in Chinese dung beetles (Coleoptera: Scarabaeinae). PloS One 6:e21600.
  • Bolger AM, Lohse M and Usadel B (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120.
  • Boore JL and Brown WM (1998) Big trees from little genomes: Mitochondrial gene order as a phylogenetic tool. Curr Opin Genet Dev 8:668-674.
  • Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Res 27: 1767-1780.
  • Cameron SL (2014) Insect mitochondrial genomics: implications for evolution and phylogeny. Annu Rev Entomol 59:95-117.
  • Campbell NJ and Barker SC (1999) The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: Fivefold tandem repetition of a coding region. Mol Biol Evol 16:732-740.
  • Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540-552.
  • Cupello M (2018) On the types species of the new world dung beetle genus Canthidium erichson, 1847 (Coleoptera: Scarabaeidae: Scarabaeinae), with an annotated checklist of species. Zootaxa 4388:451-486.
  • Darriba D, Taboada GL, Doallo R and Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nat Methods 9:772.
  • Edmonds WD and Zídek J (2010) A taxonomic review of the neotropical genus Coprophanaeus Olsoufieff, 1924 (Coleoptera: Scarabaeidae, Scarabaeinae). Insecta Mundi 129:1-111.
  • Frolov AV and Scholtz CH (2003) Revision of the Afrotropical dung beetle genus Sarophorus Erichson (Coleoptera: Scarabaeidae). Afr Entomol 11:183-198.
  • Germar EF (1821) Neue exotische Käfer beschrieben. Magazin Entomol 4:107-183.
  • Gillett CPDT, Crampton-Platt A, Timmermans MJTN, Jordal BH, Emerson BC and Vogler AP (2014) Bulk de novo mitogenome assembly from pooled total DNA elucidates the phylogeny of weevils (Coleoptera: Curculionoidea). Mol Biol Evol 31:2223-2237.
  • Grebennikov VV and Scholtz CH (2004) The basal phylogeny of Scarabaeoidea (Insecta: Coleoptera) inferred from larval morphology. Invertebr Syst 18:321-348.
  • Gunter NL, Weir TA, Slipinksi A, Bocak L and Cameron SL (2016) If dung beetles (Scarabaeidae: Scarabaeinae) arose in association with dinosaurs, did they also suffer a mass co-extinction at the K-Pg boundary? PLoS One 11:e0153570.
  • Hahn C, Bachmann L and Chevreux B (2013) Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads--a baiting and iterative mapping approach. Nucleic Acids Res 41:e129.
  • Jeong JS, Kim MJ and Kim I (2020) The mitochondrial genome of the dung beetle, Copris tripartitus, with mitogenomic comparisons within Scarabaeidae (Coleoptera). Int J Biol Macromol 144:874-891.
  • Lima NCB and Prosdocimi F (2017) The heavy strand dilemma of vertebrate mitochondria on genome sequencing age: number of encoded genes or G+ T content?. Mitochondrial DNA A DNA Mapp Seq Anal 28:300-302.
  • Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2018) Molecular phylogeny of Coprophanaeus (Megaphanaeus) (Coleoptera: Scarabaeidae: Scarabaeinae) and the position of C. bellicosus. Insect Syst Evol 51:241-255.
  • Maldaner ME, Vaz-de-Mello FZ, Takiya DM and Ferreira DC (2019) Genetic and chromatic variation of Coprophanaeus (Megaphanaeus) ensifer (Germar, 1821) (Coleoptera: Scarabaeidae). Zool Anz 283:150-160.
  • Mlambo S, Sole CL and Scholtz CH (2015) A molecular phylogeny of the African scarabaeinae (Coleoptera: Scarabaeidae). Arthropod Syst Phylogeny 73:303-321.
  • Monaghan MT, Inward DJ, Hunt T and Vogler AP (2007) A molecular phylogenetic analysis of the Scarabaeinae (dung beetles). Mol Phylogenet Evol 45:674-692.
  • Nichols E, Spector S, Louzada J, Larsen T, Amezquita S and Favila ME (2008) Ecological functions and ecosystem services provided by Scarabaeinae dung beetles. Biol Conserv 141:1461-1474.
  • Nie R-E, Breeschoten T, Timmermans MJTN, Nadein K, Xue H-J, Bai M, Huang Y, Yang X-K and Vogler AP (2018) The phylogeny of Galerucinae (Coleoptera: Chrysomelidae) and the performance of mitochondrial genomes in phylogenetic inference compared to nuclear rRNA genes. Cladistics 34:113-130.
  • Nolasco-Soto J, González-Astorga J, Monteros AE, Galante-Patiño E and Favila ME (2017) Phylogeographic structure of Canthon cyanellus (Coleoptera: Scarabaeidae), a neotropical dung beetle in the Mexican transition zone: Insights on its origin and the impacts of pleistocene climatic fluctuations on population dynamics. Mol Phylogenet Evol 109:180-190.
  • Ocampo FC and Hawks DC (2006) Molecular phylogenetics and evolution of the food relocation behaviour of the dung beetle tribe Eucraniini (Coleoptera: Scarabaeidae: Scarabaeinae). Invertebr Syst 20:557-570.
  • Philips TK, Edmonds WD and Scholtz CH (2004) A phylogenetic analysis of the new world tribe Phanaeini (Coleoptera: Scarabaeidae: Scarabaeinae): Hypotheses on relationships and origins. Insect Syst Evol 35:43-63.
  • Philips TK, Pretorius E and Scholtz CH (2004) A phylogenetic analysis of dung beetles (Scarabaeinae: Scarabaeidae): Unrolling an evolutionary history. Invertebr Syst 18:53-88.
  • Philips TK (2016) Phylogeny of the Oniticellini and Onthophagini dung beetles (Scarabaeidae, Scarabaeinae) from morphological evidence. ZooKeys 579:9-57.
  • Sambrook J and Russel WD (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York.
  • Schneider A and Ebert D (2004) Covariation of mitochondrial genome size with gene lengths: Evidence for gene length reduction during mitochondrial evolution. J Mol Evol 59:90-96.
  • Shao LL, Huang, DY, Sun XY, Hao JS, Cheng CH, Zhang W and Yang Q (2014) Complete mitochondrial genome sequence of Cheirotonus jansoni (Coleoptera: Scarabaeidae). Genet Mol Res 13:1047-1058.
  • Sheffield NC, Song H, Cameron SL and Whiting MF (2008) A comparative analysis of mitochondrial genomes in Coleoptera (Arthropoda: Insecta) and genome descriptions of six new beetles. Mol Biol Evol 25:2499-2509.
  • Sheffield NC, Song H, Cameron SL and Whiting MF (2009) Nonstationary evolution and compositional heterogeneity in beetle mitochondrial phylogenomics. Syst Biol 58:381-394.
  • Sheffield NC, Hiatt KD, Valentine MC, Song H and Whiting MF (2010) Mitochondrial genomics in orthoptera using MOSAS. Mitochondrial DNA 21:87-104.
  • Smith ABT (2009) A review of the family-group names for the superfamily Scarabaeoidea (Coleoptera) with corrections to nomenclature and a current classification. Coleopt Bull 60:144-204.
  • Sole CL and Scholtz CH (2010) Did dung beetles arise in Africa? A phylogenetic hypothesis based on five gene regions. Mol Phylogenet Evol 56:631-641.
  • Song N and Zhang H (2018) The mitochondrial genomes of phytophagous scarab beetles and systematic implications. J Insect Sci 18:11.
  • Tarasov S and Dimitrov D (2016) Multigene phylogenetic analysis redefines dung beetles relationships and classification (Coleoptera: Scarabaeidae: Scarabaeinae). BMC Evol Biol 16:257.
  • Tarasov S and Génier F (2015) Innovative bayesian and parsimony phylogeny of dung beetles (Coleoptera, Scarabaeidae, Scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10:e0116671.
  • Timmermans MJTN, Barton C, Haran J, Ahrens D, Culverwell CL, Ollikainen A, Dodsworth S, Foster PG, Bocak L and Vogler A (2016) Family-level sampling of mitochondrial genomes in Coleoptera: Compositional heterogeneity and phylogenetics. Genome Biol Evol 8:161-175.
  • Valois MC, Vaz-De-Mello FZ and Silva FAB (2017) Taxonomic revision of the Dichotomius sericeus (Harold, 1867) species group (Coleoptera: Scarabaeidae: Scarabaeinae). Zootaxa 4277:503-530.
  • Vaz-de-Mello FZ (2007) Revision taxonomica e analysis phylogenetico de la tribu Ateuchini. D. Sc. Thesis, Instituto de Ecologia A.C., Xalapa, Veracruz, Mexico, 238 p.
  • Villalba S, Lobo JM, Martín-Piera F and Zardoya R (2002) Phylogenetic relationships of Iberian dung beetles (Coleoptera: Scarabaeinae): Insights on the evolution of nesting behavior. J Mol Evol 55:116-126.
  • Villastrigo A, Jäch MA, Cardoso A, Valladares LF and Ribera I (2019) A molecular phylogeny of the tribe Ochthebiini (Coleoptera, Hydraenidae, Ochthebiinae). Syst Entomol 44:273-288.
  • Vilstrup JT, Ho SY, Foote AD, Morin PA, Kreb D, Krützen M, Parra GJ, Robertson KM, Stephanis R, Vegorgh P et al (2011) Mitogenomic phylogenetic analyses of the Delphinidae with an emphasis on the Globicephalinae. BMC Evol Biol 11:65.
  • Yang W, Zhang Y, Feng S, Liu L and Li Z (2018) The first complete mitochondrial genome of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) and its phylogenetic implications for the superfamily Scarabaeoidea. Int J Biol Macromol 118:1406-1413.
  • Yang X-Z, Zhang L, Feng R-Q, Zhang L-J, Luo F-Z and Yuan M-L (2019) Mitochondrial genome of Amphimallon solstitale (Coleoptera: Scarabaeidae: Melolonthinae) and phylogenetic analysis. Mitochondrial DNA B 4:110-111.
  • Yuan M-L, Zhang Q-L, Zhang L, Guo Z-L, Liu Y-J, Shen Y-Y and Shao R (2016) High-level phylogeny of the Coleoptera inferred with mitochondrial genome sequences. Mol Phylogenet Evol 104:99-111.
  • Zunino M (1983) Essai préliminaire sur l’évolution des armures génitales des Scarabaeinae, par rapport à la taxonomie du groupe et à l’évolution du comportement de nidification [Col. Scarabaeidae]. Bull Soc Entomol France 88:531-542.

Internet Resources Section

Publication Dates

  • Publication in this collection
    09 Aug 2021
  • Date of issue
    2021

History

  • Received
    12 Nov 2020
  • Accepted
    02 May 2021
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