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Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera)
Journal Pre-proofs Research paper Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae) Igor Costa Amorim, Adriana de Souza Melo, Alexandre Freitas da Silva, Gabriel da Luz Wallau, Rita de Cássia de Moura PII: DOI: Reference:
S0378-1119(20)30031-7 https://doi.org/10.1016/j.gene.2020.144362 GENE 144362
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Gene Gene
Received Date: Revised Date: Accepted Date:
21 September 2019 8 January 2020 9 January 2020
Please cite this article as: I. Costa Amorim, A. de Souza Melo, A. Freitas da Silva, G. da Luz Wallau, R. de Cássia de Moura, Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae), Gene Gene (2020), doi: https://doi.org/10.1016/j.gene. 2020.144362
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Characterization of the mitogenome of Rhammatocerus brasiliensis and
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phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae)
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Igor Costa Amorim1; Adriana de Souza Melo1; Alexandre Freitas da Silva2; Gabriel da
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Luz Wallau*2; Rita de Cássia de Moura1*
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1 Laboratório
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Universidade de Pernambuco, Brasil, Rua Arnóbio Marques, 310, Santo Amaro- Recife,
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PE – Brazil. CEP: 50.100-130
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2
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Brasil.
de Biodiversidade e Genética de Insetos, Instituto de Ciências Biológicas,
Departamento de Entomologia, Instituto Aggeu Magalhães – FIOCRUZ, Recife, PE,
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E-mail
address:
[email protected];
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[email protected];
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[email protected] *.
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* Corresponding author
[email protected]; [email protected];
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ABSTRACT
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Acrididae family is characterized by diverse phylogenetic uncertainties, with
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different paraphyletic subfamilies. This study characterized the mitogenome of the
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grasshopper Rhammatocerus brasiliensis and determined its phylogenetic position in
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the family Acridadae. Sequencing was performed on an Illumina platform. The Short
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Oligonucleotide Analysis Package (SOAP) was used for genome assembly and the
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MITOS Web Server for annotation. Phylogenetic analysis was performed using mtDNA
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nucleic acid and protein sequences of R. brasiliensis and more 63 species belonging to
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12 subfamilies of Acridadae. Phylogenetic trees were reconstructed using Bayesian
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inference with a relaxed molecular clock to estimate the speciation divergence time
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between taxa. The mitochondrial genome of R. brasiliensis has 15,571 bp of length, is
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rich in AT (72%), and contains 37 genes, including 13 protein-encoding genes, 22 genes
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encoding transfer RNA and two genes encoding ribosomal RNA. In addition, we also
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have annotated intergenic spacers and gene overlaps. The phylogenetic trees based on
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nucleic acid and amino acid sequences showed similar topologies. Phylogenetic analysis
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revealed that R. brasiliensis is grouped as an early offset of the Acrididae family.
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Phylogenetic analyses also corroborated the presence of several paraphyletic
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subfamilies in the family Acrididae including Gomphocerinae. The positioning of R.
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brasiliensis in the mtDNA phylogenetic tree further supports paraphyly of this
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subfamily. Moreover, the basal position of R. brasiliensis suggests that Gomphocerinae
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probably originated in South America.
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Keywords: Mitochondrial genome; Grasshopper; Nucleic and amino acid phylogeny;
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Molecular clock.
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1. INTRODUCTION
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Gomphocerinae, a subfamily of Acrididae (Orthoptera) family, is composed of
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192 genus and 1.274 species (Song et al. 2018). In this subfamily, the species are
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distinguished due to presence of estridulatory mechanism that produces reproductive
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sounds, located in the internal femur region (Jago 1971). Despite several systematic
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studies, phylogenetic relationships remain largely uncertain into Gomphocerinae
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subfamily (Bugrov et al. 2006). Phylogenetic analysis inferred from mitochondrial
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genes revealed incongruent results, in which Gomphocerinae is a paraphyletic or
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monophyletic group (Bugrov et al. 2006; Chapco and Contreras 2011; Nattier et al.
51
2011). Thus, it suggests that the estridulatory mechanism may be a characteristic
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originated from convergent evolution which emerged in different moments during the
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Gomphocerinae subfamily diversification or that originated in the Gomphocerinae
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ancestral and was lost in some lineages (Chapco and Contreras 2011; Nattier et al.
55
2011).
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In recent years, the possibility of obtaining genomic sequences has enabled
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robust phylogenomics analyzes in insects, using mitochondrial (mitogenome) or nuclear
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genomes. In most eukaryotes, the mitogenome consists of a small circular molecule (15-
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20 kb) that encodes 37 genes being 13 of them related to oxidative phosphorylation, two
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mitochondrial ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs), necessary for
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the translation of proteins encoded by the mtDNA (Boore 1999). Additionally, it is
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composed of a non-coding region with initiation sites of the mtDNA replication and
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transcription (Boore 1999). In Gomphocerinae subfamily, mitogenomes were
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characterized and used in robust phylogenetic analyzes (Song et al 2018), but none of
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these covered the Rhammatocerus genus. A previous study suggested that
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Gomphocerinae is a paraphyletic group, grouping with species of Acridinae and
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Oedipodinae clades (Song et al. 2018).
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The genus Rhammatocerus Saussure, 1861 stands out in the subfamily
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Gomphocerinae due to wide distribution in the Neotropical region. This genus currently
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has 15 species (Santos and Assis-Pujol 2003) with large intraspecific variability which
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hinders their correct taxonomic identification (Clemente et al. 2012). Phylogenetic
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analysis inferred from molecular (partial genes of NADH dehydrogenase subunit V,
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16S ribosomal, Cytochrome b, Cytochrome oxidase subunit I and II genes) and
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morphological characters revealed their grouping with different Oedipodinae and
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Acridinae species. The results of the different studies revealed a phylogenetic
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incongruences, in which Rhammatocerus being one of the most basal or derived genus
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of Gomphocerinae (Chapco and Contreras 2011; Song et al. 2015). This phylogenetic
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uncertainty reveals that more robust analyzes are necessary in order to clarify its real
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positioning in the Acrididae tree, including phylogenies inferred from other molecular
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data such as mitogenomes.
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The present study aimed to characterize the mtDNA of Rhammatocerus
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brasiliensis (Bruner 1904) in order to compare the genomic organization and
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composition with other Acrididae species, and to establish its position in family
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Acridadae and subfamily Gomphocerinae using phylogenomic methods. Besides we
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also performed dating analysis to investigate the place and time of the Rhammatocerus
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lineage emergence.
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2. MATERIALS AND METHODS 2.1 Sample, DNA extraction and ethical aspects
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The R. brasiliensis specimen analyzed was collected manually in Itamaracá (07º
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45’ 00’’ S and 34º 05’ 10’’ W), Pernambuco, Brazil. The collection was authorized by
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IBAMA/SISBIO, permanent license Nº 16278-1 for the collection of zoological
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material. The specimen was kept in absolute alcohol and stored at -20°C until
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processing. DNA was extracted from femur region tissues from a single individual
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according to the protocol of Sambrook and Russel (2001).
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2.2 Sequencing, assembly, characterization and annotation of mtDNA
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The mitogenome of R. brasiliensis was sequenced on an Illumina HiSeq 2000
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platform using a pair-end approach, with an average read length of approximately 100
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bp. The sequences obtained were processed using the Trimmomatic program (Bolger et
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al. 2014) to exclude sequences with quality score < Q20. Genome assembly was
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performed using the SOAP denovo2 (Luo et al. 2012), with kmers of 65 bp and
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parameters un-mask, resolve repeats and fill gaps. A single mitochondrial contig was
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assembled and annotated on the MITOS Web Server (Bernt et al. 2013).
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2.3 Phylogenetic analysis
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Nucleic acid and protein sequences of the mtDNA of 63 species belonging to 12
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subfamilies of Acrididae, deposited in the National Center for Biotechnology
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Information (NCBI), were selected for phylogenetic analysis. The mtDNA sequences of
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three species of the Eumastacoidea superfamily, available in GenBank were used as
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outgroup. Multiple sequence alignment of the sequences was performed in the MAFFT
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Web Server (Katoh et al. 2017). The phylogenetic tree was reconstructed by Bayesian
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inference using the MrBayes 3.1.2 program (Huelsenbeck 2001). The GTR+I+G
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substitution model was used for nucleic acid sequences and the MtREV+I+G model for
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protein sequences. These models were selected based on Akaike’s information criterion
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(AIC) implemented in JModelTest (Posada 2008) and ProtTest (Abascal et al. 2005). In
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Bayesian analysis, MCMCs were run with 10,000,000 generations, four Markov chains,
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sampling at each 1,000 generations, and a burn-in period of 25% of each chain. Branch
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support was evaluated by the posterior probability of the remaining trees sampled. The
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tree was visualized using the Figtree v. 1.4.0 program.
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2.4 Divergence time estimation
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The time of divergence of Acrididae species was estimated through the software
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package BEAST 1.8.4 (Drummond and Rambaut 2007). The input file was generated in
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BEAUTI v.1.8.4, using the parameters of lognormal uncorrelated relaxed molecular
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clock and speciation model: Birth-Death Process. The original complete mitochondrial
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genomes were divided into three dataset: I - nucleic acid sequence of the entire
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mitogenome, exept for the highly repetitive controlling region; II - amino acid
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sequences of all predicted proteins were concatenated and analyzed together, using the
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substitution model MtREV+I+G, previously selected by ProtTest (Abascal et al. 2005);
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III - the predicted protein sequences were partitioned and their substitution model
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determined and set separately (Supplementary Table 1). Four calibration points obtained
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from the website (http://fossilworks.org/) were used. These dates were from the family
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Acrididae (59.3 Mya), from the subfamilies Acridinae (58.7 to 15.97 Mya),
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Melanoplinae (0.012 to 0.0 Mya) and Gomphocerus genus (0.126 to 0.012 Mya).
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Six independent runs were performed, with 100 million generations sampling at
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each 10.000 trees for the entire mitogenomes and concatenated proteins analysis. Two
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independent runs, with 60 million generations sampling were performed for partitioned
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protein analysis. The effective sample size (ESS) was checked in the Tracer v.1.7.1
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program to verify that the parameters were over 200, indicating sufficient sampling. The
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trees were combined using LogCombiner program and a burn-in 25% was applied. The
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maximum credible tree (MCC) was obtained in the TreeAnnotator program v.1.8.4 and
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the time scale tree was plotted using the R package, Phyloch.
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3. RESULTS AND DISCUSSION 3.1 Characterization of the mitochondrial genome
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Sequencing of the mtDNA produced 94,860 reads, 569.67 average depth and
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coverage breadth of 99.64% (>=100x). The mitogenome of R. brasiliensis comprises of
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15,571 bp (Accession Number NCBI - MK941832). The mitogenome size is similar to
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that of most insects, which ranges from 15-18 kb (Cameron 2014). For example,
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mitogenomes of the Acrididae species that varies from 15,443 bp in Oxya chinensis
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(Zhang and Huang 2008) to 16,293 bp in Choroedocus capensis (Li et al. 2018).
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Characterization of the mitogenome of R. brasiliensis revealed the presence of
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13 protein-encoding genes, seven of them related to the different subunits of NADH
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dehydrogenase (NADH1-6 and NADH4L), three to cytochrome c oxidase (Cox 1-3), two
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to ATP synthase subunits (ATP6, ATP8), and one to cytochrome b (CytB) (Figure 1;
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Table 1). The mitogenome also contains two rRNA genes (rRNAL, rRNAS) and 22
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tRNA genes. Regarding the orientation of these genes, eight tRNA genes, four protein-
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coding genes and two rRNA genes are located on the negative strand and the remaining
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14 tRNA genes and nine protein-coding genes on the positive strand (Figure 1; Table 1).
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The number, orientation and order of the genes of R. brasiliensis mitogenome
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are similar to those of different insect species, following the ancestral synteny (Boore
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1999). This mitogenome differ only in the order of the Asparagine and Lysine tRNA
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genes, considered a synapomorphy characteristic of Acrididae family (Song et al. 2015).
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In Acrididae, this configuration was observed in all species analyzed, including
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Gomphocerinae species, such as Phlaeoba tenebrosa (Song et al. 2014) and Gonista
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bicolor (Zhang et al. 2015).
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With respect to AT/GC content, a high percentage of A-T (72%) was observed
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in the mitogenome of R. brasiliensis. The high percentage is a common feature of
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animals mtDNA, including the different species of Acrididae, such as Oxya japonica
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japonica and O. agavisa robusta mitogenomes (Li et al. 2019). Intergenic spacers were
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found between 29 different gene regions, comprising a total of 383 bp. The largest
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spacer is located between the trnH and nad5 genes, with 61 bp. The presence of
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intergenic spacers is a common feature, as reported for mitogenomes of the grasshopper
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Traulia nigritibialis and Stenocatantops splendens (Li et al. 2018).
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Gene overlap was observed between four pairs of genes in the R. brasiliensis
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mitogenome, comprising a total of 84 bp. Although the overlap between genes of the
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same mitochondrial DNA may cause transcriptional complications, it has been
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described in the mitogenome of different species of Acrididae, such as O. japonica
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japonica and T. nigritibialis (Li et al. 2019; Li et al. 2018). Surprisingly, in the
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mitogenome of R. brasiliensis was observed an overlap of 56 bp between the rRNAS
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and tRNAL1 genes, accounting for most of this tRNA, which has 64 bp. This overlap
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indicates that this genetic region produces only one of these RNAs for each transcribed
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molecule. However, this overlap may not represent transcriptional problems and lack
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tRNA in the cell, since the insect mitochondrial genomes have two copies this tRNA
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gene (Cameron 2014)
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3.2 Phylogenetic relationships and divergence time estimation
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Comparison of the trees reconstructed from nucleic and amino acid sequences
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showed similar topologies, suggesting that the amino acid sequences of the 13 protein-
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coding genes are also appropriate for phylogenetic analyses, in disagreement with
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studies reporting that amino acid sequences eliminate valuable phylogenetic signals in
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insects, including in Acrididae (Cameron 2014; Fenn et al. 2008). In the present study,
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these phylogenies differed mainly in the position of Acridinae species, being the most
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basal subfamily of Acrididae or more derived compared to Oedipodinae subfamily
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(Figure 2 – protein based; Supplementary Figure 1 – nucleotide based; Supplementary
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Figure 2; Supplementary Figure 3), as also observed in different studies (Chapco and
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Contreras 2011; Song et al. 2015). In addition, there were few differences in the
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positioning of some species within the clades, such as Tonkinacris sinensis and
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Yunnacris yunnaeus species in the Malanoplinae subfamily clade. Our result
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corroborates that additional phylogenetic analyzes including more markers are required
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to resolve this uncertainty. In both nucleotide and amino acid Bayesian analysis, the tree topology revealed
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that
Acridinae,
Cyrtacanthacridinae
and
Euprepocnemidinae
subfamilies
are
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monophyletic, while Catantopinae, Gomphocerinae, Melanoplinae, Oedipodinae and
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Oxyinae subfamilies are paraphyletic (Figure 2; Supplementary Figure 1). Regarding
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Calliptaminae, this subfamily has been shown to be paraphyletic and monophyletic in
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protein and nucleotide analysis, respectively (Figure 2; Supplementary Figure 1). Other
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studies also reported controversial results for Calliptaminae and the other paraphyletic
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subfamilies pointed above, in which these subfamilies may be monophyletic or
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paraphyletic (Li et al. 2019; Li et al. 2018; Song et al. 2018; Zhang et al. 2013). This
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controversy may be due to the different genes markers used in phylogenetic analysis or
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to the number of species of each subfamily analyzed, because the same phylogenetic
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methods and substitution model selection were used as the present study. According to
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Song et al. (2018) these subfamilies are expected to be paraphyletic because they are
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taxonomic dumping grounds that have not been correctly classified taxonomically.
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Although Gomphocerinae is paraphyletic in the present study (Figure 2), the
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species of this subfamily grouped into two main clades, suggesting that estridulatory
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mechanism emerged in different moments of Gomphocerinae diversification, as
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reported in literature (Chapco and Contreras 2011; Nattier et al. 2011; Song et al. 2018).
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However, these results also suggest that this mechanism was conserved in the different
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lineages after their emergence. Thus, this characteristic can be used as synapomorphy of
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the group that was classically classified as Gomphocerinae, but it does not allow
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distinguishing between clades of this subfamily.
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The Bayesian phylogenetic tree based on mitogenomes showed that R.
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brasiliensis is grouped in the basis of the species of Acridadae, except by Acridinae and
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Oedipodinae species (Figure 2; Figure 3). No consensus exists in the different studies
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regarding the position Rhammatocerus species. However, phylogenetic analysis inferred
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from morphological (Wingless) and molecular characters (NADH dehydrogenase
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subunit V, 16S ribosomal, Cytochrome b, Cytochrome oxidase subunit I and II genes)
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revealed that species of the genus Rhammatocerus (R. peragrans, R. pictus and R.
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schistocercoides) are basal in Gomphocerinae (Chapco and Contreras 2011). The
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present study brought more robust phylogenetic analysis for Rhammatocerus species,
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because it is the first to use a large set of molecular data giving further support to the
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basal status of the Rhammatocerus to several other Acrididae family and the ancient
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diversification of this genus.
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Regarding to estimate the divergence time, the different analyzes obtained the
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same result showing that Acrididae family originated between the Upper Jurassic and
11 239
the Upper Cretaceous, but most likely in the Lower Cretaceous period (Figure 3;
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Supplementary Figure 2; Supplementary Figure 3). These results disagree with the
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previous studies which reported the origin of this family in the Paleocene epoch of the
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Paleogene period (Guzmán et al. 2017; Song et al. 2018). This difference may be related
243
to the number and species analyzed, since most clades with available mitogenomes do
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originate in the Paleocene epoch of the Paleogene (Figure 3). Moreover, associating this
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recent time estimates with the absence mitogenomes of an ancient genus such as
246
Rhammatocerus may have biased the analysis of the previous studies.
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In the present study, phylogenetic results suggest that the most basal species
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(Acridinae and Oedopodinae species) belong to the old world (Figure 2; Figure 3). In
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this analysis, the oldest species of the New World is R. brasiliensis, which probably
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originated 87 million years ago (Figure 3). This finding supports a hypothesis proposed
251
by Amedegnato (1993), which suggest the Acrididae family originated in the Old
252
World. However, this hypothesis is disagreement with the results recently reported by
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Song et al. (2018), which suggests the Acrididae family originated in South America
254
and later spreaded worldwide. This difference must be due to the few species of the new
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world with characterized mitogenome and consequently not covered in our analysis,
256
mainly the absence of the Leptysminae, Marelliinae, Ommatolampidinae, Pauliniinae
257
and Rhytidochrotinae subfamilies, which indicated the origin of Acridadae in South
258
America (Song et al. (2018)).
259
In the species taxonomically classified as Gomphocerinae, Orinhippus tibetanus
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is most basal, grouped within the Oedopodinae clade (Figure 2; Figure 3), as reported
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by different molecular phylogenetic analyzes (Gao et al. 2017; Song et al. 2018). Based
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on this phylogenetic positioning Gao et al. (2017) proposed that O. tibetanus be
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reclassified as a member of the Oedopinae subfamily. Considering this reclassification,
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the results suggest that R. brasiliensis is the oldest species classified as Gomphocerinae
265
(Figure 3). This result suggests Gomphocerinae subfamily originated in South America,
266
disagreeing with a new hypothesis about its origin in the Palearctic region, later
267
colonizing the New World (Song et al. 2018). This hypothesis was suggested based in
268
pattern of diversity and biogeographical analysis, even though the basal species of
269
Gomphocerinae were from New World. Thus, more robust analyzes including a large
270
diversity of Gomphocerinae species from New World (such as R. brasiliensis), may
271
increasingly support the hypothesis of the origin this subfamily in South America.
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Origin in the South American region has been proposed for other Acrididae subfamilies,
273
such as the Melanoplinae subfamily (Amédégnato et al. 2003).
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The sequencing and characterization of the mitochondrial genome of R.
275
brasiliensis, as well as its use for establishing the position of this species within
276
Acrididae, provides new and important information to the correct classification its
277
paraphyletic subfamilies. In addition, phylogenetic analysis using the R. brasiliensis
278
mitogenome suggests that species classified as Gomphocerinae probably originated in
279
South America but that it may have recolonized the new world multiple times.
280 281
4. Funding
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This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal
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de Nível Superior – Brasil – (CAPES) [Coordination for the Improvement of Higher
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Education Personnel] – Cod. 001 and by supported by post-doctoral fellowship to
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Amorim, IC (PNPD-20130558); Fundação de Amparo a Ciência e Tecnologia do
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Estado de Pernambuco [Science and Technology Foundation of the State of
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Pernambuco] through a doctoral fellowship to Melo, AS (FACEPE IBPG: 0769-
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2.02/14) and da Silva, AF (FACEPE IBPG: IBPG-1127-2.13/18); Conselho Nacional
13 289
de Desenvolvimento Científico e Tecnológico [National Council for Scientific and
290
Technological Development]
291
305298/2014-3).
through a PQ-2 grant to Moura, RC (CNPq:
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Huelsenbeck JP (2001) MrBayes : A program for the Bayesian inference of phylogeny. Dna Seq. 17, 1–12. Jago ND (1971) A review of the Gomphocerinae of the world with a key to the genera (Orthoptera, Acrididae).
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Zhang HL, Zhao L, Zheng ZM, Huang Y (2013) Complete Mitochondrial Genome of
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388
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17 389
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18 390 391
Table Table 1. Genetic annotation of Rhammatocerus brasiliensis mitogenome. Gene D-Loop trnI (atc) trnQ (caa) trnM (atg) NADH2 trnW (tga) trnC (tgc) trnY (tac) Cox1 trnL2 (tta) Cox2 trnD (gac) trnK (aag) ATP8 ATP6 Cox3 trnG (gga) NADH3 trnA (gca) trnR (cga) trnN (aac) trnS1 (agc) trnE (gaa) trnF (ttc) NADH5 trnH (cac) NADH4 NADH4L trnT (aca) trnP (cca) NADH6 CytB trnS2 (tca) NADH1 trnL1 (cta) rRNAL trnV (gta) rRNAS
Start 1 751 822 893 977 1981 2041 2113 2183 3711 3785 4467 4536 4622 4777 5459 6252 6324 6672 6741 6810 6876 6943 7012 7089 8811 8893 10221 10490 10555 10624 11151 12292 12402 13332 13339 14705 14780
Stop Strand Length 750 + 750 819 + 69 890 69 961 + 69 1954 + 978 2048 + 68 2105 65 2178 66 3691 + 1509 3776 + 66 4465 + 681 4532 + 66 4606 + 71 4780 + 159 5445 + 669 6247 + 789 6317 + 66 6668 + 345 6737 + 66 6807 + 67 6875 + 66 6942 + 67 7008 + 66 7075 64 8750 1662 8877 67 10215 1323 10481 261 10554 + 65 10618 64 11133 + 510 12260 + 1110 12362 + 71 13310 909 13395 64 14723 1385 14775 71 15571 792
19 392
Figures legends
393 394
Figure 1. Organization genetic of Rhammatocerus brasiliensis mitogenome. D-loop,
395
Protein-coding genes, rRNA genes and tRNA genes are shown in blue, purple, green
396
and red, respectively. The arrows indicate the direction of the genes.
397 398
Figure 2. Bayesian inference of the superfamily Acrididae based on amino acid
399
sequences of the concatenated 13 protein-coding genes of the mitochondrial genomes.
400
Species of Eumastacoidea superfamily available in GenBank, were used as outgroup.
401
The colors indicate the different Acrididae families and asteristic (*) the paraphyletic
402
subfamilies.
403 404
Figure 3. Divergence time estimation of the different lineages of Acrididae inferred by
405
BEAST and based on partitioned 13 predicted protein sequences of the mitochondrial
406
genomes.
407 408 409 410 411 412 413 414 415 416 417
Abbreviations list mtDNA – Mitochondrial DNA; rRNAs - Ribosomal RNAs; tRNAs - Transfer RNAs; NADH1-6/ NADH4L – NADH dehydrogenase subunit 1 - 6 and NADH dehydrogenase subunit 4L; Cox 1-3 - Cytochrome c oxidase subunit 1 – 3; ATP6/ATP8 - ATP synthase subunits 6 and 8; CytB - Cytochrome b ;
20 418
ABSTRACT
419
Acrididae family is characterized by diverse phylogenetic uncertainties, with
420
different paraphyletic subfamilies. This study characterized the mitogenome of the
421
grasshopper Rhammatocerus brasiliensis and determined its phylogenetic position in
422
the family Acridadae. Sequencing was performed on an Illumina platform. The Short
423
Oligonucleotide Analysis Package (SOAP) was used for genome assembly and the
424
MITOS Web Server for annotation. Phylogenetic analysis was performed using mtDNA
425
nucleic acid and protein sequences of R. brasiliensis and more 63 species belonging to
426
12 subfamilies of Acridadae. Phylogenetic trees were reconstructed using Bayesian
427
inference with a relaxed molecular clock to estimate the speciation divergence time
428
between taxa. The mitochondrial genome of R. brasiliensis has 15,571 bp of length, is
429
rich in AT (72%), and contains 37 genes, including 13 protein-encoding genes, 22 genes
430
encoding transfer RNA and two genes encoding ribosomal RNA. In addition, we also
431
have annotated intergenic spacers and gene overlaps. The phylogenetic trees based on
432
nucleic acid and amino acid sequences showed similar topologies. Phylogenetic analysis
433
revealed that R. brasiliensis is grouped as an early offset of the Acrididae family.
434
Phylogenetic analyses also corroborated the presence of several paraphyletic
435
subfamilies in the family Acrididae including Gomphocerinae. The positioning of R.
436
brasiliensis in the mtDNA phylogenetic tree further supports paraphyly of this
437
subfamily. Moreover, the basal position of R. brasiliensis suggests that Gomphocerinae
438
probably originated in South America.
439 440
Keywords: Mitochondrial genome; Grasshopper; Nucleic and amino acid phylogeny;
441
Molecular clock.
442 443
21 444 445 446 447 448 449 450 451 452 453 454
Author contributions Igor Costa Amorim: Conceptualization; mitogenome characterization and gene annotation, phylogenetic analysis and original draft Writing Adriana de Souza Melo: Mitogenome characterization, gene annotation and review of original draft Alexandre Freitas da Silva: Analysis of divergence time and review of original draft Gabriel da Luz Wallau: Conceptualization, project administration, software supervision and review of original draft Rita de Cássia de Moura: Project administration and review of original draft
455 456 457 458 459 460 461 462 463
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
464 465 466 467 468 469 470 471 472 473 474 475 476 477
Highlights (máximo de 85 caracteres com espaçamento) 1 - The Rhammatocerus brasiliensis mitogenome is similar to that of most insects 2 - The R. brasiliensis phylogenetic positioning supports the paraphily of Gomphocerinae 3 - R. brasiliensis is the oldest species of New World, with mitogenome characterized 4 - Gomphocerinae probably originated in South America.
Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae)
22 478 479
Igor Costa Amorim1; Adriana de Souza Melo1; Alexandre Freitas da Silva2; Gabriel da
480
Luz Wallau*2; Rita de Cássia de Moura1*
481 482
1 Laboratório
483
Universidade de Pernambuco, Brasil, Rua Arnóbio Marques, 310, Santo Amaro- Recife,
484
PE – Brazil. CEP: 50.100-130
485
2
486
Brasil.
de Biodiversidade e Genética de Insetos, Instituto de Ciências Biológicas,
Departamento de Entomologia, Instituto Aggeu Magalhães – FIOCRUZ, Recife, PE,
487 488
E-mail
address:
[email protected];
489
[email protected];
490
[email protected] *.
491 492 493 494
* Corresponding author
[email protected]; [email protected];
23
495
496
24
497
S0378-1119(20)30031-7 https://doi.org/10.1016/j.gene.2020.144362 GENE 144362
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
21 September 2019 8 January 2020 9 January 2020
Please cite this article as: I. Costa Amorim, A. de Souza Melo, A. Freitas da Silva, G. da Luz Wallau, R. de Cássia de Moura, Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae), Gene Gene (2020), doi: https://doi.org/10.1016/j.gene. 2020.144362
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© 2020 Published by Elsevier B.V.
1
Characterization of the mitogenome of Rhammatocerus brasiliensis and
2
phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae)
3 4
Igor Costa Amorim1; Adriana de Souza Melo1; Alexandre Freitas da Silva2; Gabriel da
5
Luz Wallau*2; Rita de Cássia de Moura1*
6 7
1 Laboratório
8
Universidade de Pernambuco, Brasil, Rua Arnóbio Marques, 310, Santo Amaro- Recife,
9
PE – Brazil. CEP: 50.100-130
10
2
11
Brasil.
de Biodiversidade e Genética de Insetos, Instituto de Ciências Biológicas,
Departamento de Entomologia, Instituto Aggeu Magalhães – FIOCRUZ, Recife, PE,
12 13
address:
[email protected];
14
[email protected];
15
[email protected] *.
16 17
* Corresponding author
[email protected]; [email protected];
2 18
ABSTRACT
19
Acrididae family is characterized by diverse phylogenetic uncertainties, with
20
different paraphyletic subfamilies. This study characterized the mitogenome of the
21
grasshopper Rhammatocerus brasiliensis and determined its phylogenetic position in
22
the family Acridadae. Sequencing was performed on an Illumina platform. The Short
23
Oligonucleotide Analysis Package (SOAP) was used for genome assembly and the
24
MITOS Web Server for annotation. Phylogenetic analysis was performed using mtDNA
25
nucleic acid and protein sequences of R. brasiliensis and more 63 species belonging to
26
12 subfamilies of Acridadae. Phylogenetic trees were reconstructed using Bayesian
27
inference with a relaxed molecular clock to estimate the speciation divergence time
28
between taxa. The mitochondrial genome of R. brasiliensis has 15,571 bp of length, is
29
rich in AT (72%), and contains 37 genes, including 13 protein-encoding genes, 22 genes
30
encoding transfer RNA and two genes encoding ribosomal RNA. In addition, we also
31
have annotated intergenic spacers and gene overlaps. The phylogenetic trees based on
32
nucleic acid and amino acid sequences showed similar topologies. Phylogenetic analysis
33
revealed that R. brasiliensis is grouped as an early offset of the Acrididae family.
34
Phylogenetic analyses also corroborated the presence of several paraphyletic
35
subfamilies in the family Acrididae including Gomphocerinae. The positioning of R.
36
brasiliensis in the mtDNA phylogenetic tree further supports paraphyly of this
37
subfamily. Moreover, the basal position of R. brasiliensis suggests that Gomphocerinae
38
probably originated in South America.
39 40
Keywords: Mitochondrial genome; Grasshopper; Nucleic and amino acid phylogeny;
41
Molecular clock.
3 42
1. INTRODUCTION
43
Gomphocerinae, a subfamily of Acrididae (Orthoptera) family, is composed of
44
192 genus and 1.274 species (Song et al. 2018). In this subfamily, the species are
45
distinguished due to presence of estridulatory mechanism that produces reproductive
46
sounds, located in the internal femur region (Jago 1971). Despite several systematic
47
studies, phylogenetic relationships remain largely uncertain into Gomphocerinae
48
subfamily (Bugrov et al. 2006). Phylogenetic analysis inferred from mitochondrial
49
genes revealed incongruent results, in which Gomphocerinae is a paraphyletic or
50
monophyletic group (Bugrov et al. 2006; Chapco and Contreras 2011; Nattier et al.
51
2011). Thus, it suggests that the estridulatory mechanism may be a characteristic
52
originated from convergent evolution which emerged in different moments during the
53
Gomphocerinae subfamily diversification or that originated in the Gomphocerinae
54
ancestral and was lost in some lineages (Chapco and Contreras 2011; Nattier et al.
55
2011).
56
In recent years, the possibility of obtaining genomic sequences has enabled
57
robust phylogenomics analyzes in insects, using mitochondrial (mitogenome) or nuclear
58
genomes. In most eukaryotes, the mitogenome consists of a small circular molecule (15-
59
20 kb) that encodes 37 genes being 13 of them related to oxidative phosphorylation, two
60
mitochondrial ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs), necessary for
61
the translation of proteins encoded by the mtDNA (Boore 1999). Additionally, it is
62
composed of a non-coding region with initiation sites of the mtDNA replication and
63
transcription (Boore 1999). In Gomphocerinae subfamily, mitogenomes were
64
characterized and used in robust phylogenetic analyzes (Song et al 2018), but none of
65
these covered the Rhammatocerus genus. A previous study suggested that
4 66
Gomphocerinae is a paraphyletic group, grouping with species of Acridinae and
67
Oedipodinae clades (Song et al. 2018).
68
The genus Rhammatocerus Saussure, 1861 stands out in the subfamily
69
Gomphocerinae due to wide distribution in the Neotropical region. This genus currently
70
has 15 species (Santos and Assis-Pujol 2003) with large intraspecific variability which
71
hinders their correct taxonomic identification (Clemente et al. 2012). Phylogenetic
72
analysis inferred from molecular (partial genes of NADH dehydrogenase subunit V,
73
16S ribosomal, Cytochrome b, Cytochrome oxidase subunit I and II genes) and
74
morphological characters revealed their grouping with different Oedipodinae and
75
Acridinae species. The results of the different studies revealed a phylogenetic
76
incongruences, in which Rhammatocerus being one of the most basal or derived genus
77
of Gomphocerinae (Chapco and Contreras 2011; Song et al. 2015). This phylogenetic
78
uncertainty reveals that more robust analyzes are necessary in order to clarify its real
79
positioning in the Acrididae tree, including phylogenies inferred from other molecular
80
data such as mitogenomes.
81
The present study aimed to characterize the mtDNA of Rhammatocerus
82
brasiliensis (Bruner 1904) in order to compare the genomic organization and
83
composition with other Acrididae species, and to establish its position in family
84
Acridadae and subfamily Gomphocerinae using phylogenomic methods. Besides we
85
also performed dating analysis to investigate the place and time of the Rhammatocerus
86
lineage emergence.
87 88 89
2. MATERIALS AND METHODS 2.1 Sample, DNA extraction and ethical aspects
5 90
The R. brasiliensis specimen analyzed was collected manually in Itamaracá (07º
91
45’ 00’’ S and 34º 05’ 10’’ W), Pernambuco, Brazil. The collection was authorized by
92
IBAMA/SISBIO, permanent license Nº 16278-1 for the collection of zoological
93
material. The specimen was kept in absolute alcohol and stored at -20°C until
94
processing. DNA was extracted from femur region tissues from a single individual
95
according to the protocol of Sambrook and Russel (2001).
96 97
2.2 Sequencing, assembly, characterization and annotation of mtDNA
98
The mitogenome of R. brasiliensis was sequenced on an Illumina HiSeq 2000
99
platform using a pair-end approach, with an average read length of approximately 100
100
bp. The sequences obtained were processed using the Trimmomatic program (Bolger et
101
al. 2014) to exclude sequences with quality score < Q20. Genome assembly was
102
performed using the SOAP denovo2 (Luo et al. 2012), with kmers of 65 bp and
103
parameters un-mask, resolve repeats and fill gaps. A single mitochondrial contig was
104
assembled and annotated on the MITOS Web Server (Bernt et al. 2013).
105 106
2.3 Phylogenetic analysis
107
Nucleic acid and protein sequences of the mtDNA of 63 species belonging to 12
108
subfamilies of Acrididae, deposited in the National Center for Biotechnology
109
Information (NCBI), were selected for phylogenetic analysis. The mtDNA sequences of
110
three species of the Eumastacoidea superfamily, available in GenBank were used as
111
outgroup. Multiple sequence alignment of the sequences was performed in the MAFFT
112
Web Server (Katoh et al. 2017). The phylogenetic tree was reconstructed by Bayesian
113
inference using the MrBayes 3.1.2 program (Huelsenbeck 2001). The GTR+I+G
114
substitution model was used for nucleic acid sequences and the MtREV+I+G model for
6 115
protein sequences. These models were selected based on Akaike’s information criterion
116
(AIC) implemented in JModelTest (Posada 2008) and ProtTest (Abascal et al. 2005). In
117
Bayesian analysis, MCMCs were run with 10,000,000 generations, four Markov chains,
118
sampling at each 1,000 generations, and a burn-in period of 25% of each chain. Branch
119
support was evaluated by the posterior probability of the remaining trees sampled. The
120
tree was visualized using the Figtree v. 1.4.0 program.
121 122
2.4 Divergence time estimation
123
The time of divergence of Acrididae species was estimated through the software
124
package BEAST 1.8.4 (Drummond and Rambaut 2007). The input file was generated in
125
BEAUTI v.1.8.4, using the parameters of lognormal uncorrelated relaxed molecular
126
clock and speciation model: Birth-Death Process. The original complete mitochondrial
127
genomes were divided into three dataset: I - nucleic acid sequence of the entire
128
mitogenome, exept for the highly repetitive controlling region; II - amino acid
129
sequences of all predicted proteins were concatenated and analyzed together, using the
130
substitution model MtREV+I+G, previously selected by ProtTest (Abascal et al. 2005);
131
III - the predicted protein sequences were partitioned and their substitution model
132
determined and set separately (Supplementary Table 1). Four calibration points obtained
133
from the website (http://fossilworks.org/) were used. These dates were from the family
134
Acrididae (59.3 Mya), from the subfamilies Acridinae (58.7 to 15.97 Mya),
135
Melanoplinae (0.012 to 0.0 Mya) and Gomphocerus genus (0.126 to 0.012 Mya).
136
Six independent runs were performed, with 100 million generations sampling at
137
each 10.000 trees for the entire mitogenomes and concatenated proteins analysis. Two
138
independent runs, with 60 million generations sampling were performed for partitioned
139
protein analysis. The effective sample size (ESS) was checked in the Tracer v.1.7.1
7 140
program to verify that the parameters were over 200, indicating sufficient sampling. The
141
trees were combined using LogCombiner program and a burn-in 25% was applied. The
142
maximum credible tree (MCC) was obtained in the TreeAnnotator program v.1.8.4 and
143
the time scale tree was plotted using the R package, Phyloch.
144 145 146
3. RESULTS AND DISCUSSION 3.1 Characterization of the mitochondrial genome
147
Sequencing of the mtDNA produced 94,860 reads, 569.67 average depth and
148
coverage breadth of 99.64% (>=100x). The mitogenome of R. brasiliensis comprises of
149
15,571 bp (Accession Number NCBI - MK941832). The mitogenome size is similar to
150
that of most insects, which ranges from 15-18 kb (Cameron 2014). For example,
151
mitogenomes of the Acrididae species that varies from 15,443 bp in Oxya chinensis
152
(Zhang and Huang 2008) to 16,293 bp in Choroedocus capensis (Li et al. 2018).
153
Characterization of the mitogenome of R. brasiliensis revealed the presence of
154
13 protein-encoding genes, seven of them related to the different subunits of NADH
155
dehydrogenase (NADH1-6 and NADH4L), three to cytochrome c oxidase (Cox 1-3), two
156
to ATP synthase subunits (ATP6, ATP8), and one to cytochrome b (CytB) (Figure 1;
157
Table 1). The mitogenome also contains two rRNA genes (rRNAL, rRNAS) and 22
158
tRNA genes. Regarding the orientation of these genes, eight tRNA genes, four protein-
159
coding genes and two rRNA genes are located on the negative strand and the remaining
160
14 tRNA genes and nine protein-coding genes on the positive strand (Figure 1; Table 1).
161
The number, orientation and order of the genes of R. brasiliensis mitogenome
162
are similar to those of different insect species, following the ancestral synteny (Boore
163
1999). This mitogenome differ only in the order of the Asparagine and Lysine tRNA
164
genes, considered a synapomorphy characteristic of Acrididae family (Song et al. 2015).
8 165
In Acrididae, this configuration was observed in all species analyzed, including
166
Gomphocerinae species, such as Phlaeoba tenebrosa (Song et al. 2014) and Gonista
167
bicolor (Zhang et al. 2015).
168
With respect to AT/GC content, a high percentage of A-T (72%) was observed
169
in the mitogenome of R. brasiliensis. The high percentage is a common feature of
170
animals mtDNA, including the different species of Acrididae, such as Oxya japonica
171
japonica and O. agavisa robusta mitogenomes (Li et al. 2019). Intergenic spacers were
172
found between 29 different gene regions, comprising a total of 383 bp. The largest
173
spacer is located between the trnH and nad5 genes, with 61 bp. The presence of
174
intergenic spacers is a common feature, as reported for mitogenomes of the grasshopper
175
Traulia nigritibialis and Stenocatantops splendens (Li et al. 2018).
176
Gene overlap was observed between four pairs of genes in the R. brasiliensis
177
mitogenome, comprising a total of 84 bp. Although the overlap between genes of the
178
same mitochondrial DNA may cause transcriptional complications, it has been
179
described in the mitogenome of different species of Acrididae, such as O. japonica
180
japonica and T. nigritibialis (Li et al. 2019; Li et al. 2018). Surprisingly, in the
181
mitogenome of R. brasiliensis was observed an overlap of 56 bp between the rRNAS
182
and tRNAL1 genes, accounting for most of this tRNA, which has 64 bp. This overlap
183
indicates that this genetic region produces only one of these RNAs for each transcribed
184
molecule. However, this overlap may not represent transcriptional problems and lack
185
tRNA in the cell, since the insect mitochondrial genomes have two copies this tRNA
186
gene (Cameron 2014)
187 188
3.2 Phylogenetic relationships and divergence time estimation
9 189
Comparison of the trees reconstructed from nucleic and amino acid sequences
190
showed similar topologies, suggesting that the amino acid sequences of the 13 protein-
191
coding genes are also appropriate for phylogenetic analyses, in disagreement with
192
studies reporting that amino acid sequences eliminate valuable phylogenetic signals in
193
insects, including in Acrididae (Cameron 2014; Fenn et al. 2008). In the present study,
194
these phylogenies differed mainly in the position of Acridinae species, being the most
195
basal subfamily of Acrididae or more derived compared to Oedipodinae subfamily
196
(Figure 2 – protein based; Supplementary Figure 1 – nucleotide based; Supplementary
197
Figure 2; Supplementary Figure 3), as also observed in different studies (Chapco and
198
Contreras 2011; Song et al. 2015). In addition, there were few differences in the
199
positioning of some species within the clades, such as Tonkinacris sinensis and
200
Yunnacris yunnaeus species in the Malanoplinae subfamily clade. Our result
201
corroborates that additional phylogenetic analyzes including more markers are required
202
to resolve this uncertainty. In both nucleotide and amino acid Bayesian analysis, the tree topology revealed
203 204
that
Acridinae,
Cyrtacanthacridinae
and
Euprepocnemidinae
subfamilies
are
205
monophyletic, while Catantopinae, Gomphocerinae, Melanoplinae, Oedipodinae and
206
Oxyinae subfamilies are paraphyletic (Figure 2; Supplementary Figure 1). Regarding
207
Calliptaminae, this subfamily has been shown to be paraphyletic and monophyletic in
208
protein and nucleotide analysis, respectively (Figure 2; Supplementary Figure 1). Other
209
studies also reported controversial results for Calliptaminae and the other paraphyletic
210
subfamilies pointed above, in which these subfamilies may be monophyletic or
211
paraphyletic (Li et al. 2019; Li et al. 2018; Song et al. 2018; Zhang et al. 2013). This
212
controversy may be due to the different genes markers used in phylogenetic analysis or
213
to the number of species of each subfamily analyzed, because the same phylogenetic
10 214
methods and substitution model selection were used as the present study. According to
215
Song et al. (2018) these subfamilies are expected to be paraphyletic because they are
216
taxonomic dumping grounds that have not been correctly classified taxonomically.
217
Although Gomphocerinae is paraphyletic in the present study (Figure 2), the
218
species of this subfamily grouped into two main clades, suggesting that estridulatory
219
mechanism emerged in different moments of Gomphocerinae diversification, as
220
reported in literature (Chapco and Contreras 2011; Nattier et al. 2011; Song et al. 2018).
221
However, these results also suggest that this mechanism was conserved in the different
222
lineages after their emergence. Thus, this characteristic can be used as synapomorphy of
223
the group that was classically classified as Gomphocerinae, but it does not allow
224
distinguishing between clades of this subfamily.
225
The Bayesian phylogenetic tree based on mitogenomes showed that R.
226
brasiliensis is grouped in the basis of the species of Acridadae, except by Acridinae and
227
Oedipodinae species (Figure 2; Figure 3). No consensus exists in the different studies
228
regarding the position Rhammatocerus species. However, phylogenetic analysis inferred
229
from morphological (Wingless) and molecular characters (NADH dehydrogenase
230
subunit V, 16S ribosomal, Cytochrome b, Cytochrome oxidase subunit I and II genes)
231
revealed that species of the genus Rhammatocerus (R. peragrans, R. pictus and R.
232
schistocercoides) are basal in Gomphocerinae (Chapco and Contreras 2011). The
233
present study brought more robust phylogenetic analysis for Rhammatocerus species,
234
because it is the first to use a large set of molecular data giving further support to the
235
basal status of the Rhammatocerus to several other Acrididae family and the ancient
236
diversification of this genus.
237
Regarding to estimate the divergence time, the different analyzes obtained the
238
same result showing that Acrididae family originated between the Upper Jurassic and
11 239
the Upper Cretaceous, but most likely in the Lower Cretaceous period (Figure 3;
240
Supplementary Figure 2; Supplementary Figure 3). These results disagree with the
241
previous studies which reported the origin of this family in the Paleocene epoch of the
242
Paleogene period (Guzmán et al. 2017; Song et al. 2018). This difference may be related
243
to the number and species analyzed, since most clades with available mitogenomes do
244
originate in the Paleocene epoch of the Paleogene (Figure 3). Moreover, associating this
245
recent time estimates with the absence mitogenomes of an ancient genus such as
246
Rhammatocerus may have biased the analysis of the previous studies.
247
In the present study, phylogenetic results suggest that the most basal species
248
(Acridinae and Oedopodinae species) belong to the old world (Figure 2; Figure 3). In
249
this analysis, the oldest species of the New World is R. brasiliensis, which probably
250
originated 87 million years ago (Figure 3). This finding supports a hypothesis proposed
251
by Amedegnato (1993), which suggest the Acrididae family originated in the Old
252
World. However, this hypothesis is disagreement with the results recently reported by
253
Song et al. (2018), which suggests the Acrididae family originated in South America
254
and later spreaded worldwide. This difference must be due to the few species of the new
255
world with characterized mitogenome and consequently not covered in our analysis,
256
mainly the absence of the Leptysminae, Marelliinae, Ommatolampidinae, Pauliniinae
257
and Rhytidochrotinae subfamilies, which indicated the origin of Acridadae in South
258
America (Song et al. (2018)).
259
In the species taxonomically classified as Gomphocerinae, Orinhippus tibetanus
260
is most basal, grouped within the Oedopodinae clade (Figure 2; Figure 3), as reported
261
by different molecular phylogenetic analyzes (Gao et al. 2017; Song et al. 2018). Based
262
on this phylogenetic positioning Gao et al. (2017) proposed that O. tibetanus be
263
reclassified as a member of the Oedopinae subfamily. Considering this reclassification,
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the results suggest that R. brasiliensis is the oldest species classified as Gomphocerinae
265
(Figure 3). This result suggests Gomphocerinae subfamily originated in South America,
266
disagreeing with a new hypothesis about its origin in the Palearctic region, later
267
colonizing the New World (Song et al. 2018). This hypothesis was suggested based in
268
pattern of diversity and biogeographical analysis, even though the basal species of
269
Gomphocerinae were from New World. Thus, more robust analyzes including a large
270
diversity of Gomphocerinae species from New World (such as R. brasiliensis), may
271
increasingly support the hypothesis of the origin this subfamily in South America.
272
Origin in the South American region has been proposed for other Acrididae subfamilies,
273
such as the Melanoplinae subfamily (Amédégnato et al. 2003).
274
The sequencing and characterization of the mitochondrial genome of R.
275
brasiliensis, as well as its use for establishing the position of this species within
276
Acrididae, provides new and important information to the correct classification its
277
paraphyletic subfamilies. In addition, phylogenetic analysis using the R. brasiliensis
278
mitogenome suggests that species classified as Gomphocerinae probably originated in
279
South America but that it may have recolonized the new world multiple times.
280 281
4. Funding
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This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal
283
de Nível Superior – Brasil – (CAPES) [Coordination for the Improvement of Higher
284
Education Personnel] – Cod. 001 and by supported by post-doctoral fellowship to
285
Amorim, IC (PNPD-20130558); Fundação de Amparo a Ciência e Tecnologia do
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Estado de Pernambuco [Science and Technology Foundation of the State of
287
Pernambuco] through a doctoral fellowship to Melo, AS (FACEPE IBPG: 0769-
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2.02/14) and da Silva, AF (FACEPE IBPG: IBPG-1127-2.13/18); Conselho Nacional
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de Desenvolvimento Científico e Tecnológico [National Council for Scientific and
290
Technological Development]
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305298/2014-3).
through a PQ-2 grant to Moura, RC (CNPq:
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Table Table 1. Genetic annotation of Rhammatocerus brasiliensis mitogenome. Gene D-Loop trnI (atc) trnQ (caa) trnM (atg) NADH2 trnW (tga) trnC (tgc) trnY (tac) Cox1 trnL2 (tta) Cox2 trnD (gac) trnK (aag) ATP8 ATP6 Cox3 trnG (gga) NADH3 trnA (gca) trnR (cga) trnN (aac) trnS1 (agc) trnE (gaa) trnF (ttc) NADH5 trnH (cac) NADH4 NADH4L trnT (aca) trnP (cca) NADH6 CytB trnS2 (tca) NADH1 trnL1 (cta) rRNAL trnV (gta) rRNAS
Start 1 751 822 893 977 1981 2041 2113 2183 3711 3785 4467 4536 4622 4777 5459 6252 6324 6672 6741 6810 6876 6943 7012 7089 8811 8893 10221 10490 10555 10624 11151 12292 12402 13332 13339 14705 14780
Stop Strand Length 750 + 750 819 + 69 890 69 961 + 69 1954 + 978 2048 + 68 2105 65 2178 66 3691 + 1509 3776 + 66 4465 + 681 4532 + 66 4606 + 71 4780 + 159 5445 + 669 6247 + 789 6317 + 66 6668 + 345 6737 + 66 6807 + 67 6875 + 66 6942 + 67 7008 + 66 7075 64 8750 1662 8877 67 10215 1323 10481 261 10554 + 65 10618 64 11133 + 510 12260 + 1110 12362 + 71 13310 909 13395 64 14723 1385 14775 71 15571 792
19 392
Figures legends
393 394
Figure 1. Organization genetic of Rhammatocerus brasiliensis mitogenome. D-loop,
395
Protein-coding genes, rRNA genes and tRNA genes are shown in blue, purple, green
396
and red, respectively. The arrows indicate the direction of the genes.
397 398
Figure 2. Bayesian inference of the superfamily Acrididae based on amino acid
399
sequences of the concatenated 13 protein-coding genes of the mitochondrial genomes.
400
Species of Eumastacoidea superfamily available in GenBank, were used as outgroup.
401
The colors indicate the different Acrididae families and asteristic (*) the paraphyletic
402
subfamilies.
403 404
Figure 3. Divergence time estimation of the different lineages of Acrididae inferred by
405
BEAST and based on partitioned 13 predicted protein sequences of the mitochondrial
406
genomes.
407 408 409 410 411 412 413 414 415 416 417
Abbreviations list mtDNA – Mitochondrial DNA; rRNAs - Ribosomal RNAs; tRNAs - Transfer RNAs; NADH1-6/ NADH4L – NADH dehydrogenase subunit 1 - 6 and NADH dehydrogenase subunit 4L; Cox 1-3 - Cytochrome c oxidase subunit 1 – 3; ATP6/ATP8 - ATP synthase subunits 6 and 8; CytB - Cytochrome b ;
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ABSTRACT
419
Acrididae family is characterized by diverse phylogenetic uncertainties, with
420
different paraphyletic subfamilies. This study characterized the mitogenome of the
421
grasshopper Rhammatocerus brasiliensis and determined its phylogenetic position in
422
the family Acridadae. Sequencing was performed on an Illumina platform. The Short
423
Oligonucleotide Analysis Package (SOAP) was used for genome assembly and the
424
MITOS Web Server for annotation. Phylogenetic analysis was performed using mtDNA
425
nucleic acid and protein sequences of R. brasiliensis and more 63 species belonging to
426
12 subfamilies of Acridadae. Phylogenetic trees were reconstructed using Bayesian
427
inference with a relaxed molecular clock to estimate the speciation divergence time
428
between taxa. The mitochondrial genome of R. brasiliensis has 15,571 bp of length, is
429
rich in AT (72%), and contains 37 genes, including 13 protein-encoding genes, 22 genes
430
encoding transfer RNA and two genes encoding ribosomal RNA. In addition, we also
431
have annotated intergenic spacers and gene overlaps. The phylogenetic trees based on
432
nucleic acid and amino acid sequences showed similar topologies. Phylogenetic analysis
433
revealed that R. brasiliensis is grouped as an early offset of the Acrididae family.
434
Phylogenetic analyses also corroborated the presence of several paraphyletic
435
subfamilies in the family Acrididae including Gomphocerinae. The positioning of R.
436
brasiliensis in the mtDNA phylogenetic tree further supports paraphyly of this
437
subfamily. Moreover, the basal position of R. brasiliensis suggests that Gomphocerinae
438
probably originated in South America.
439 440
Keywords: Mitochondrial genome; Grasshopper; Nucleic and amino acid phylogeny;
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Molecular clock.
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Author contributions Igor Costa Amorim: Conceptualization; mitogenome characterization and gene annotation, phylogenetic analysis and original draft Writing Adriana de Souza Melo: Mitogenome characterization, gene annotation and review of original draft Alexandre Freitas da Silva: Analysis of divergence time and review of original draft Gabriel da Luz Wallau: Conceptualization, project administration, software supervision and review of original draft Rita de Cássia de Moura: Project administration and review of original draft
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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights (máximo de 85 caracteres com espaçamento) 1 - The Rhammatocerus brasiliensis mitogenome is similar to that of most insects 2 - The R. brasiliensis phylogenetic positioning supports the paraphily of Gomphocerinae 3 - R. brasiliensis is the oldest species of New World, with mitogenome characterized 4 - Gomphocerinae probably originated in South America.
Characterization of the mitogenome of Rhammatocerus brasiliensis and phylogenetic analysis of the family Acrididae (Orthoptera: Gomphocerinae)
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Igor Costa Amorim1; Adriana de Souza Melo1; Alexandre Freitas da Silva2; Gabriel da
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Luz Wallau*2; Rita de Cássia de Moura1*
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1 Laboratório
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Universidade de Pernambuco, Brasil, Rua Arnóbio Marques, 310, Santo Amaro- Recife,
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PE – Brazil. CEP: 50.100-130
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2
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Brasil.
de Biodiversidade e Genética de Insetos, Instituto de Ciências Biológicas,
Departamento de Entomologia, Instituto Aggeu Magalhães – FIOCRUZ, Recife, PE,
487 488
address:
[email protected];
489
[email protected];
490
[email protected] *.
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* Corresponding author
[email protected]; [email protected];
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