The evolution of insect male genitalia has received much attention, but there is still a lack of data on the macroevolutionary origin of its extraordinary variation. We used a calibrated molecular phylogeny of 71 of the 150 known species of the beetle genus Limnebius to study the evolution of the size and complexity of the male genitalia in its two subgenera, Bilimneus, with small species with simple genitalia, and Limnebius s.str., with a much larger variation in size and complexity. We reconstructed ancestral values of complexity (perimeter and fractal dimension of the aedeagus) and genital and body size with Bayesian methods. Complexity evolved more in agreement with a Brownian model, although with evidence of weak directional selection to a decrease or increase in complexity in the two subgenera respectively, as measured with an excess of branches with negative or positive change. On the contrary, aedeagus size, the variable with the highest rates of evolution, had a lower phylogenetic signal, without significant differences between the two subgenera in the average change of the individual branches of the tree. Aedeagus size also had a lower correlation with time and no evidence of directional selection. Rather than to directional selection, it thus seems that the higher diversity of the male genitalia in Limnebius s.str. is mostly due to the larger variance of the phenotypic change in the individual branches of the tree for all measured variables.

There are increasing concerns regarding bat mortality at wind energy facilities, especially as installed capacity continues to grow. In North America, wind energy development has recently expanded into the Lower Rio Grande Valley in south Texas where bat species had not previously been exposed to wind turbines. Our study sought to characterize genetic diversity, population structure, and effective population size in Dasypterus ega and D. intermedius, two tree-roosting yellow bats native to this region and for which little is known about their population biology and seasonal movements. There was no evidence of population substructure in either species. Genetic diversity at mitochondrial and microsatellite loci was lower in these yellow bat taxa than in previously studied migratory tree bat species in North America, which may be due to the non-migratory nature of these species at our study site, the fact that our study site is located at a geographic range end for both taxa, and possibly weak ascertainment bias at microsatellite loci. Historical effective population size (NEF) was large for both species, while current estimates of Ne had upper 95% confidence limits that encompassed infinity. We found evidence of strong mitochondrial differentiation between the two putative subspecies of D. intermedius (D. i. floridanus and D. i. intermedius) which are sympatric in this region of Texas, yet little differentiation using microsatellite loci. We suggest this pattern is due to secondary contact and hybridization and possibly incomplete lineage sorting at microsatellite loci. We also found evidence of some hybridization between D. ega and D. intermedius in this region of Texas. We recommend that our data serve as a starting point for the long-term genetic monitoring of these species in order to better understand the impacts of wind-related mortality on these populations over time.

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms.

The mitochondrial genomes (mitogenomes) of scale insects are less known in comparison to other insects, which hinders the phylogenetic and evolutionary studies of Coccoidea and higher taxa. Herein, the complete mitogenomes of Unaspis yanonensis, Planococcus citri and Ceroplastes rubens were sequenced for Coccoidea. The 15,220-bp long mitogenome of U. yanonensis contained the typical set of 37 genes including 13 PCGs, 22 tRNA genes and two rRNA genes; the 15,549-bp long mitogenome of P. citri lacked the tRNA gene trnV; the 15,387-bp long mitogenome of C. rubens exhibited several shortened PCGs and lacked five tRNA genes. The mitochondrial gene arrangement of the three mitogenomes was different from other scale insects and Drosophila yakuba. Most PCGs used standard ATN (ATA, ATT, ATC and ATG) start codons and complete TAN (TAA or TAG) termination codons. The ND4L had the highest evolutionary rate but COX1 and CYTB were the lowest. Most tRNA genes had cloverleaf secondary structures, whereas the reduction of dihydrouridine (DHU) arms and TψC arms were detected. Tandem repeats, stem-loop (SL) structures and poly-[TA]n stretch were found in the control regions (CRs) of the three mitogenomes. The phylogenetic analyses using Bayesian inference (BI) and maximum likelihood methods (ML) showed identical results, both supporting the inner relationship of Coccoidea as Coccidae + (Pseudococcidae + Diaspididae).

In the last decades, Italy as well as other developed countries have registered a decrease in the population size of many local horse breeds. The continuous crossbreeding has determined the dilution of genetic heritage of several native breeds. The Italian Heavy Draught Horse (IHD) is the only autochthonous Italian coldblooded horse among these breeds; therefore, it represents a resource to be preserved. In 1927, the first generation of this breed was officially created by crossing different Heavy Draught horses with local mares and recorded in a Studbook.

Chloroplast genomes provide insufficient phylogenetic information to distinguish between closely related sugarcane cultivars, due to the recent origin of many cultivars and the conserved sequence of the chloroplast. In comparison, the mitochondrial genome of plants is much larger and more plastic and could contain increased phylogenetic signals. We assembled a consensus reference mitochondrion with Illumina TruSeq synthetic long reads and Oxford Nanopore Technologies MinION long reads. Based on this assembly we also analyzed the mitochondrial transcriptomes of sugarcane and sorghum and improved the annotation of the sugarcane mitochondrion as compared with other species.

Mitochondrial genomes of twelve species of Trigonopterus weevils are presented, ten of them complete. We describe their gene order and molecular features and test their potential for reconstructing the phylogeny of this hyperdiverse genus comprising > 1,000 species. The complete mitochondrial genomes examined herein ranged from 16,501 bp to 21,007 bp in length, with an average AT content of 64.2% to 69.7%. Composition frequencies and skews were generally lower across species for atp6, cox1-3, and cob genes, while atp8 and genes coded on the minus strand showed much higher divergence at both nucleotide and amino acid levels. Most variation within genes was found at the codon level with high variation at third codon sites across species, and with lesser degree at the coding strand level. Two large non-coding regions were found, CR1 (between rrnS and trnI genes) and CR2 (between trnI and trnQ), but both with large variability in length; this peculiar structure of the non-coding region may be a derived character of Curculionoidea. The nad1 and cob genes exhibited an unusually high interspecific length variation of up to 24 bp near the 3' end. This pattern was probably caused by a single evolutionary event since both genes are only separated by trnS2 and length variation is extremely rare in mitochondrial protein coding genes. We inferred phylogenetic trees using protein coding gene sequences implementing both maximum likelihood and Bayesian approaches, each for both nucleotide and amino acid sequences. While some clades could be retrieved from all reconstructions with high confidence, there were also a number of differences and relatively low support for some basal nodes. The best partition scheme of the 13 protein coding sequences obtained by IQTREE suggested that phylogenetic signal is more accurate by splitting sequence variation at the codon site level as well as coding strand, rather than at the gene level. This result corroborated the different patterns found in Trigonopterus regarding to A+T frequencies and AT and GC skews that also greatly diverge at the codon site and coding strand levels.

Evolutionary timescales can be estimated from genetic data using the molecular clock, often calibrated by fossil or geological evidence. However, estimates of molecular rates in mitochondrial DNA appear to scale negatively with the age of the clock calibration. Although such a pattern has been observed in a limited range of data sets, it has not been studied on a large scale in metazoans. In addition, there is uncertainty over the temporal extent of the time-dependent pattern in rate estimates. Here we present a meta-analysis of 239 rate estimates from metazoans, representing a range of timescales and taxonomic groups. We found evidence of time-dependent rates in both coding and non-coding mitochondrial markers, in every group of animals that we studied. The negative relationship between the estimated rate and time persisted across a much wider range of calibration times than previously suggested. This indicates that, over long time frames, purifying selection gives way to mutational saturation as the main driver of time-dependent biases in rate estimates. The results of our study stress the importance of accounting for time-dependent biases in estimating mitochondrial rates regardless of the timescale over which they are inferred.

The dog has been the first domesticated animal to have a central role in human society from ancient times to present day. Although there have been numerous investigations of dog phylogeny and origin, genetic data of dogs in the region of the Balkan Peninsula (South-Eastern Europe) are still scarce. Therefore, the aim of the present study was to perform phylogenetic analysis of three native Bulgarian dog breeds. A total of 130 samples were analyzed at HVR1 (hypervariable region, D-loop region). The samples were taken from two hunting dog breeds (Bulgarian Hound Dog: Barak, n = 34; Bulgarian Scenthound Dog: Gonche, n = 45) as well as from a Bulgarian Shepherd Dog (n = 51). The first two breeds are reared in a flat region of the country (the Northern part of Bulgaria, the Danubian Plain), while the last breed is a typical representative of the mountainous part of the country. The results have shown the presence of almost all main clades-A, B, C and D-in the three dog breeds taken together, except clades E and F, as expected. With regard to haplogroups distribution, there are clear differences among investigated breeds. While hunting breeds exhibit a prevalence of clade C, the mountainous Shepherd dog shows presence of the D2 haplogroup but absence of the C clade. In conclusion, the present study has been the first to investigate the mitochondrial DNA diversity of native dog breeds in Bulgaria. The results have revealed a clear difference of haplogroups dissemination in native hunting and shepherd dogs, which suggests a dual independent phylogenetic origin, without hybridization events between these dogs.

The mitochondrial genomes (mitogenomes) of Formosatettix qinlingensis, Coptotettix longjiangensis and Thoradonta obtusilobata (Orthoptera: Caelifera: Tetrigoidea) were sequenced in this study, and almost the entire mitogenomes of these species were determined. The mitogenome sequences obtained for the three species were 15,180, 14,495 and 14,538 bp in length, respectively, and each sequence included 13 protein-coding genes (PCGs), partial sequences of rRNA genes (rRNAs), tRNA genes (tRNAs) and a A + T-rich region. The order and orientation of the gene arrangement pattern were identical to that of most Tetrigoidea species. Some conserved spacer sequences between trnS(UCN) and nad1 were useful to identify Tetrigoidea and Acridoidea. The Ka/Ks value of atp8 between Trachytettix bufo and other four Tetrigoidea species indicated that some varied sites in this gene might be related with the evolution of T. bufo. The three Tetrigoidea species were compared with other Caelifera. At the superfamily level, conserved sequences were observed in intergenic spacers, which can be used for superfamily level identification between Tetrigoidea and Acridoidea. Furthermore, a phylogenomic analysis was conducted based on the concatenated data sets from mitogenome sequences of 24 species of Orthoptera in the superorders Caelifera and Ensifera. Both maximum likelihood and bayesian inference analyses strongly supported Acridoidea and Tetrigoidea as forming monophyletic groups. The relationships among six Tetrigoidea species were (((((Tetrix japonica, Alulatettix yunnanensis), Formosatettix qinlingensis), Coptotettix longjiangensis), Trachytettix bufo), Thoradonta obtusilobata).

Butterflies and moths (Lepidoptera) are becoming model organisms for genetics and evolutionary biology. Decoding the Lepidoptera genomes, both nuclear and mitochondrial, is an essential step in these studies. Here we describe a protocol to assemble mitogenomes from Next Generation Sequencing reads obtained through whole-genome sequencing and report the 15,338 bp mitogenome of Lerema accius. The mitogenome is AT-rich and encodes 13 proteins, 22 transfer-RNAs, and two ribosomal-RNAs, with a gene order typical for Lepidoptera mitogenomes. A phylogenetic study based on the protein sequences using both Bayesian Inference and Maximum Likelihood methods consistently place Lerema accius with other grass skippers (Hesperiinae).

Despite the significant progress that has been made in the genome sequencing of Beauveria species, mitochondrial genome (mitogenome) used to examine genetic diversity within fungal populations. Complete mitogenomes of Beauveria species can be easily sequenced and assembled using various sequencing techniques. However, since mitogenome annotations are mainly derived from similar species comparison and software prediction, and are not supported by RNA-seq transcripts data, it leads to problems with the accuracy of mitochondrial annotations and the inability to understand RNA processing. In this study, we assembled and annotated the mitogenome of eight Beauveria strains using Illumina DNA and RNA sequencing data. The circular mitogenome of eight Beauveria strains ranged from 26,850 bp (B. caledonica strain ATCC 64970) to 35,999 bp (B. brongniartii strain GYU-BMZ03), with the intronic insertions accounting for most of the size variation, thus contributing to a total mitochondrial genome (mitogenome) size of 7.01% and 28.95%, respectively. Intron number variations were not directly related to the evolutionary relationship distance. Besides ribosomal protein S3 (rps3), most introns are lost too quickly and lack the stability of protein-coding genes. The short RNA-seq reads from next-generation sequencing can improve the mitochondrial annotation accuracy and help study polycistronic transcripts and RNA processing. The transcription initiation sites may be located in the control region. Most introns do not serve as taxonomic markers and also lack open reading frames (ORFs). We assumed that the poly A tail was added to the polycistronic transcript before splicing and one polycistronic transcript (trnM (1)-trnL (1)-trnA-trnF-trnK-trnL (2)-trnQ-trnH-trnM (2)-nad2-nad3-atp9-cox2-trnR (1)-nad4L-nad5-cob-trnC-cox1-trnR (2)-nad1-nad4-atp8-atp6-rns-trnY-trnD-trnS-trnN-cox3-trnG-nad6-trnV-trnI-trnS-trnW-trnP-rnl(rps3)-trnT-trnE-trnM (3)) was first processed from the mitogenome and was subsequently processed into smaller mono-, di-, or tricistronic RNAs.

To date, five ctenophore species' mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria.

The species of Lasiopodomys Lataste 1887 with their related genera remains undetermined owing to inconsistent morphological characteristics and molecular phylogeny. To investigate the phylogenetic relationship and speciation among species of the genus Lasiopodomys, we sequenced and annotated the whole mitochondrial genomes of three individual species, namely Lasiopodomys brandtii Radde 1861, L. mandarinus Milne-Edwards 1871, and Neodon (Lasiopodomys) fuscus Büchner 1889. The nucleotide sequences of the circular mitogenomes were identical for each individual species of L. brandtii, L. mandarinus, and N. fuscus. Each species contained 13 protein-coding genes (PCGs), 22 transfer RNAs, and 2 ribosomal RNAs, with mitochondrial genome lengths of 16,557 bp, 16,562 bp, and 16,324 bp, respectively. The mitogenomes and PCGs showed positive AT skew and negative GC skew. Mitogenomic phylogenetic analyses suggested that L. brandtii, L. mandarinus, and L. gregalis Pallas 1779 belong to the genus Lasiopodomys, whereas N. fuscus belongs to the genus Neodon grouped with N. irene. Lasiopodomys showed the closest relationship with Microtus fortis Büchner 1889 and M. kikuchii Kuroda 1920, which are considered as the paraphyletic species of genera Microtus. TMRCA and niche model analysis revealed that Lasiopodomys may have first appeared during the early Pleistocene epoch. Further, L. gregalis separated from others over 1.53 million years ago (Ma) and then diverged into L. brandtii and L. mandarinus 0.76 Ma. The relative contribution of climatic fluctuations to speciation and selection in this group requires further research.

In this article, we present the nearly complete mitochondrial genome (mitogenome) of the weevil beetle Apion squamigerum (Curculionoidea, Brentidae), assembled using data from Illumina next generation sequencing (NGS). This mitogenome was found to be very large, with the total length of 18,562 bp. Two trnM genes were identified. A large non-coding intergenic spacer spanning 1,949 bp occurred between trnI and trnM2. Combined with 111 existing weevil mitogenomes, we conducted phylogenetic reconstructions based on various datasets under maximum likelihood and Bayesian inference. Firstly, phylogenetic analyses robustly supported a sister group of A. squamigerum and Rhopalapion longirostre, namely, that two species of Apioninae (Brentidae) formed a clade. Within the entire Curculionoidea, the Nemonychidae diverged firstly, following the families Anthribidae and Attelabidae. In addition, a large clade comprising the sister families Brentidae and Curculionidae was strongly supported in all trees.

Scelimeninae is a key member of the pygmy grasshopper community, and an important ecological indicator. No mitochondrial genomes of Scelimeninae have been reported to date, and the monophyly of Scelimeninae and its phylogenetic relationship within Tetrigidae is still unclear. We sequenced and analyzed eight nearly complete mitochondrial genomes representing eight genera of Scelimeninae. These mitogenomes ranged in size from 13,112 to 16,380 bp and the order of tRNA genes between COII and ATP8 was reversed compared with the ancestral order of insects. The protein-coding genes (PCGs) of tetrigid species mainly with the typical ATN codons and most terminated with complete (TAA or TAG) stop codons. Analyses of pairwise genetic distances showed that ATP8 was the least conserved gene within Tetrigidae, while COI was the most conserved. The longest intergenic spacer (IGS) region in the mitogenomes was always found between tRNASer(UCN) and ND1. Additionally, tandem repeat units were identified in the longest IGS of three mitogenomes. Maximum likelihood (ML) and Bayesian Inference (BI) analyses based on the two datasets supported the monophyly of Tetriginae. Scelimeninae was classified as a non-monophyletic subfamily.

The infraorder Cephalobomorpha is a diverse and ecologically important nematode group found in almost all terrestrial environments. In a recent nematode classification system based on SSU rDNA, Cephalobomorpha was classified within the suborder Tylenchina with Panagrolaimomorpha, Tylenchomorpha and Drilonematomorpha. However, phylogenetic relationships among species within Tylenchina are not always consistent, and the phylogenetic position of Cephalobomorpha is still uncertain. In this study, in order to examine phylogenetic relationships of Cephalobomorpha with other nematode groups, we determined the complete mitochondrial genome sequence of Acrobeloides varius, the first sequenced representative of Cephalobomorpha, and used this sequence for phylogenetic analyses along with 101 other nematode species. Phylogenetic analyses using amino acid and nucleotide sequence data of 12 protein-coding genes strongly support a sister relationship between the two cephalobomorpha species A. varius and Acrobeles complexus (represented by a partial mt genome sequence). In this mitochondrial genome phylogeny, Cephalobomorpha was sister to all chromadorean species (excluding Plectus acuminatus of Plectida) and separated from Panagrolaimomorpha and Tylenchomorpha, rendering Tylenchina non-monophyletic. Mitochondrial gene order among Tylenchina species is not conserved, and gene clusters shared between A. varius and A. complexus are very limited. Results from phylogenetic analysis and gene order comparison confirms Tylenchina is not monophyletic. To better understand phylogenetic relationships among Tylenchina members, additional mitochondrial genome information is needed from underrepresented taxa representing Panagrolaimomorpha and Cephalobomorpha.

We describe Arge bella Wei & Du sp. nov., a large and beautiful species of Argidae from south China, and report its mitochondrial genome based on high-throughput sequencing data. We present the gene order, nucleotide composition of protein-coding genes (PCGs), and the secondary structures of RNA genes. The nearly complete mitochondrial genome of A. bella has a length of 15,576 bp and a typical set of 37 genes (22 tRNAs, 13 PCGs, and 2 rRNAs). Three tRNAs are rearranged in the A. bella mitochondrial genome as compared to the ancestral type in insects: trnM and trnQ are shuffled, while trnW is translocated from the trnW-trnC-trnY cluster to a location downstream of trnI. All PCGs are initiated by ATN codons, and terminated with TAA, TA or T as stop codons. All tRNAs have a typical cloverleaf secondary structure, except for trnS1. H821 of rrnS and H976 of rrnL are redundant. A phylogenetic analysis based on mitochondrial genome sequences of A. bella, 21 other symphytan species, two apocritan representatives, and four outgroup taxa supports the placement of Argidae as sister to the Pergidae within the symphytan superfamily Tenthredinoidea.

To determine the Dysgonia stuposa mitochondrial genome (mitogenome) structure and to clarify its phylogenetic position, the entire mitogenome of D. stuposa was sequenced and annotated. The D. stuposa mitogenome is 15,721 bp in size and contains 37 genes (protein-coding genes, transfer RNA genes, ribosomal RNA genes) usually found in lepidopteran mitogenomes. The newly sequenced mitogenome contained some common features reported in other Erebidae species, e.g., an A+T biased nucleotide composition and a non-canonical start codon for cox1 (CGA). Like other insect mitogenomes, the D. stuposa mitogenome had a conserved sequence 'ATACTAA' in an intergenic spacer between trnS2 and nad1, and a motif 'ATAGA' followed by a 20 bp poly-T stretch in the A+T rich region. Phylogenetic analyses supported D. stuposa as part of the Erebidae family and reconfirmed the monophyly of the subfamilies Arctiinae, Catocalinae and Lymantriinae within Erebidae.

The stag beetle Lucanus cervus (Coleoptera: Lucanidae) is widely distributed in Europe. Habitat loss and fragmentation has led to significant reductions in numbers of this species. In this study, we sequenced the complete mitochondrial genome of L. cervus and reconstructed phylogenetic relationships among Lucanidae using complete mitochondrial genome sequences.