Putative Control Region Research Articles

Evolutionary Rates, Divergence Rates, and Performance of Individual Mitochondrial Genes Based on Phylogenetic Analysis of Copepoda.

Copepoda is a large and diverse group of crustaceans, which is widely distributed worldwide. It encompasses roughly 9 orders, whose phylogeny remains unresolved. We sequenced the complete mitochondrial genome (mitogenome) of Sinergasilus major (Markevich, 1940) and used it to explore the phylogeny and mitogenomic evolution of Copepoda. The mitogenome of S. major (14,588 bp) encodes the standard 37 genes as well as a putative control region, and molecular features are highly conserved compared to other Copepoda mitogenomes. Comparative analyses indicated that the nad2 gene has relatively high nucleotide diversity and evolutionary rate, as well as the largest amount of phylogenetic information. These results indicate that nad2 may be a better marker to investigate phylogenetic relationships among closely related species in Copepoda than the commonly used cox1 gene. The sister-group relationship of Siphonostomatoida and Cyclopoida was recovered with strong support in our study. The only topological ambiguity was found within Cyclopoida, which might be caused by the rapid evolution and sparse taxon sampling of this lineage. More taxa and genes should be used to reconstruct the Copepoda phylogeny in the future.

Comparative mitochondrial genomics of American nectar-feeding long-nosed bats Leptonycteris spp. with insights into the phylogeny of the family Phyllostomidae

Among leaf-nosed bats (family Phyllostomidae), the genus Leptonycteris (subfamily Glossophaginae), contains three migratory and obligate nectar-dwelling species of great ecological and economic importance; the greater long-nosed bat L. nivalis, the lesser long-nosed bat L. yerbabuenae, and the southern long-nosed bat L. curasoae. According to the IUCN, the three species are categorized as ‘vulnerable’, ‘endangered’, and ‘near threatened’, respectively. In this study, we assembled and characterized in detail the mitochondrial genome of Leptonycteris spp. and examined the phylogenetic position of this genus in the family Phyllostomidae based on protein coding genes (PCGs). The mitogenomes of L. nivalis, L. curasoae, and L. yerbabuenae are 16,708, 16,758, and 16,729 bp in length and each encode 13 PCGs, 22 transfer RNA genes, 2 rRNA genes, and a putative control region (CR). Mitochondrial gene order is identical to that reported before for the family Phyllostomidae. All tRNAs exhibit a ‘cloverleaf’ secondary structure, except tRNA-Serine-1 that is missing the DHU arm in the three species. All PCGs are exposed to purifying selection with atp8 experiencing the most relaxed purifying selection as the ω ratio was higher for this gene compared to the other PCGs in each species. The CR of each species contains three functional domains: extended termination associated sequence (ETAS), Central, and conserved sequence block domain (CSB). A phylomitogenomic analysis revealed that Leptonycteris is monophyletic and most closely related to the genus Glossophaga. The analysis also supported the monophyly of the family Glossophaginae in the speciose family Phyllostomidae. The mitochondria characterization of these species provides relevant information to develop molecular markers for conservation purposes.

The mitochondrial genome of the endemic and endangered trumpet-nosed bat Musonycteris harrisoni (Chiroptera: Phyllostomidae)

ABSTRACT The trumpet-nosed bat Musonycteris harrisoni (family Phyllostomidae) is endemic to the western Pacific slope of Mexico, and is considered endangered given prolonged and continuous population decline. This study, for the first time, assembled and characterised in detail the mitochondrial genome of this species that is in need of genomic resources. The mitogenome of Musonycteris harrisoni is 16,668 bp in length and encodes 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a relatively long non-coding putative control region (CR) 1229 bp in length. Mitochondrial synteny is identical to that reported before in the family Phyllostomidae. As reported before in all phyllostomids, most mitochondrial genes are encoded on the heavy strand (H-strand), except for nad6 and eight tRNA genes that are encoded on the light strand (L-strand). All the tRNAs exhibit a ‘cloverleaf’ secondary structure, except tRNA Serine-1 that is missing the dihydrouridine (DHU) arm. All 13 mitochondrial PCGs are evolving under purifying selection, with atp8, nad4l and nad6 experiencing relaxed purifying selection in comparison to the other PCGs. Tandem repeats, microsatellites, and hairpin structures are observed in the CR of Musonycteris harrisoni, in agreement with what has been reported before in other cofamilial bats. The CR contains three functional domains: extended termination associated sequence (ETAS) domain, central domain, and conserved sequence block (CSB) domain. The mitochondrial genome sequence herein characterised completely and in detail represents a new genomic resource for M. harrisoni that will further the understanding of genetic diversity in this endangered species.

The long-range interaction between two GNAS imprinting control regions delineates pseudohypoparathyroidism type 1B pathogenesis.

Genetic defects of GNAS, the imprinted gene encoding the stimulatory G protein α-subunit, are responsible for multiple diseases. Abnormal GNAS imprinting causes pseudohypoparathyroidism type 1B (PHP1B), a prototype of mammalian end-organ hormone resistance. Hypomethylation at the maternally methylated GNAS A/B region is the only shared defect in patients with PHP1B. In autosomal dominant (AD) PHP1B kindreds, A/B hypomethylation is associated with maternal microdeletions at either the GNAS NESP55 differentially methylated region or the STX16 gene located approximately 170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and the putative imprinting control regions (ICRs) NESP-ICR and STX16-ICR remain unknown. Here, we generated a human embryonic stem cell model of AD-PHP1B by introducing ICR deletions using CRISPR/Cas9. With this model, we showed that the NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that the SXT16-ICR is a long-range enhancer of NESP55 transcription, which originates from the maternal NESP-ICR. Furthermore, we demonstrated that the STX16-ICR is an embryonic stage-specific enhancer enabled by the direct binding of pluripotency factors. Our findings uncover an essential GNAS imprinting control mechanism and advance the molecular understanding of PHP1B pathogenesis.

The mitochondrial genomes of big-eared bats, Macrotus waterhousii and Macrotus californicus (Chiroptera: Phyllostomidae: Macrotinae)

In the species-rich family Phyllostomidae, the genus Macrotus (‘big eared’ bats) contains only two species; Macrotus waterhousii, distributed in western, central, and southern Mexico, Guatemala and some Caribbean Islands, and Macrotus californicus, distributed in the southwestern USA, and in the Baja California peninsula and the state of Sonora in Mexico. In this study, we sequenced and assembled the mitochondrial genome of Macrotus waterhousii and characterized in detail this genome and that of the congeneric M. californicus. Then, we examined the phylogenetic position of Macrotus in the family Phyllostomidae based on protein coding genes (PCGs). The AT-rich mitochondrial genomes of M. waterhousii and M. californicus are 16,792 and 16,691 bp long, respectively, and each encode 13 PCGs, 22 tRNA genes, 2 rRNA genes, and a putative non-coding control region 1,336 and 1,232 bp long, respectively. Mitochondrial synteny in Macrotus is identical to that reported before for all other cofamilial species. In the two studied species, all tRNAs exhibit a ‘typical’ cloverleaf secondary structure with the exception of trnS1, which lacks the D arm. A selective pressure analysis demonstrated that all PCGs are under purifying selection. The CR of the two species feature three domains previously reported in other mammals, including bats: extended terminal associated sequences (ETAS), central (CD), and conserved sequence block (CSB). A phylogenetic analysis based on the 13 mitochondrial PCGs demonstrated that Macrotus is monophyletic and the subfamily Macrotinae is a sister group of all remaining phyllostomids in our analysis, except Micronycterinae. The assembly and detailed analysis of these mitochondrial genomes represents a step further to continue improving the understanding of phylogenetic relationships within the species-rich family Phyllostomidae.

The first genomic resource for the ‘near threatened’ Neotropical otter Lontra longicaudis (Carnivora: Mustelidae): mitochondrial genome characterisation and insights into phylomitogenomic relationships in the family Mustelidae

ABSTRACT The Neotropical otter Lontra longicaudis is a top predator in aquatic ecosystems from North, Central, and South America and is subjected to substantial environmental distress due to historic and contemporary anthropogenic disturbance. Currently, L. longicaudis is considered ‘near threatened’ by the International Union for Conservation of Nature Red List of Threatened Species. This study renders the first genomic resource for L. longicaudis; its mitochondrial genome was assembled and characterised in detail. The AT-rich mitochondrial genome of L. longicaudis was 16,436 bp in length and encoded 13 protein coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a 994 bp long putative control region (CR). The heavy use of CTA (Leu), ATA (Met), and ATC (Ile) codons in PCGs contributes to the AT-rich nature of this genome. Out of 22 tRNAs, tRNA-Ser (AGY) (trnS1) lacked a typical ‘cloverleaf’ secondary structure. In the CR, numerous microsatellites and two tandem repeats were detected as well as multiple ‘hairpin’ structures. Ka/Ks values estimated for all but one PCG were < 1, indicating purifying selection in most PCGs. atp8 experienced neutral selection. A phylogenomic analysis based on PCGs confirms the monophyly of the family Mustelidae and also supports the monophyletic status of the subfamilies Guloinae, Helictidinae, Melinae, Mustelinae and Lutrinae; Ictonychinae was found to be polyphyletic. In the Lutrinae, L. longicaudis had an early branching position. The characterisation of a complete mitochondrial genome in the Neotropical otter L. longicaudis constitutes the first step towards supporting conservation efforts using genomic tools in this and other mustelids inhabiting aquatic neotropical environments under considerable contemporary anthropogenic disturbance.

Mitochondrial Genome of “Spotted Numbfish” Narcine timlei (Bloch &amp; Schneider, 1801) and Phylogenetic Relationships among Order Torpediniformes

In this study, we report, for the first time, mitochondrial genome of Narcine timlei (Bloch &amp; Schneider, 1801) and its phylogenetic relationships within the order Torpediniformes. Narcine timlei is a medium-sized ray that occurs in nearshore waters of the Indo-Pacific and is classified as “‘vulnerable” category on the IUCN Red List. The mitogenome is assembled from short Illumina reads (150 bp paired end reads). It is 17,964 bp long and includes 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes. The gene order, size, and nucleotide composition are largely consistent with mitogenomic characteristics of previously reported other Narcine spp. The slightly larger mitogenome length of N. timlei than other Narcine spp. may be due to the presence of a putative control region of 1,916 bp with three tandem repeats. Phylogenetic reconstruction using concatenated PCGs (n = 13) of 9 Torpediniformes based on maximum likelihood and Bayesian inference analysis revealed identical topologies. The tree showed two main clades: one clade containing members of the family Narcinidae and the second sister clade consisting of the families Narkidae and Torpedinidae. Our result supports the monophyletic nature of Narcinidae based on mtDNA. The information obtained in this study will contribute to a better understanding of the population genetics, phylogenetic analysis, conservation, and evolutionary biology research of N. timlei.

High-throughput sequencing yields a complete mitochondrial genome of the rice thrips, Stenchaetothrips biformis (Thysanoptera: Thripidae)

Rice thrips, Stenchaetothrips biformis (Bagnall, 1913), are one of the destructive pests of rice. Here, the complete mitochondrial genome of S. biformis was sequenced using high-throughput sequencing. The mitogenome is 15,359 bp long with an A + T content of 76.94%, which contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), 2 ribosomal RNA genes (rRNAs) and 2 putative control regions (CRs). The phylogenetic analysis showed that S. biformis is closely related to Thrips imaginis and Thrips palmi. This new mitochondrial genome data can be better used to provide a basis for studies of the mitochondrial evolution of Thysanoptera.

Mitochondrial genome of Leocrates chinensis Kinberg, 1866 (Annelida: Hesionidae)

We report the complete mitochondrial genome of Leocrates chinensis Kinberg, 1866 – the type species of the genus. It is 15061 bp long, and contains 13 protein-coding genes (PCGs), 22 tRNA genes (tRNAs), and 2 rRNA genes (rRNAs), and 1 putative control region. Phylogenetic analysis indicated that L. chinensis was placed as sister to Sirsoe methanicola (BS = 100) of the same family Hesionidae.

Mitochondrial genomes assembled from non-invasive eDNA metagenomic scat samples in the endangered Amur tiger Panthera tigris altaica.

The Amur or Siberian tiger Panthera tigris altaica (Temminck, 1844) is currently restricted to a small region of its original geographical range in northwestern Asia and is considered 'endangered' by the IUCN Red List of Threatened Species. This solitary, territorial, and large top predator is in major need of genomic resources to inform conservation management strategies. This study formally tested if complete mitochondrial genomes of P. tigris altaica can be assembled from non-enriched metagenomic libraries generated from scat eDNA samples using the Illumina sequencing platform and open-access bioinformatics pipelines. The mitogenome of P. tigris altaica was assembled and circularized using the pipeline GetOrganelle with a coverage ranging from 322.7x to 17.6x in four different scat eDNA samples. A nearly complete mitochondrial genome (101x) was retrieved from a fifth scat eDNA sample. The complete or nearly complete mitochondrial genomes of P. tigris altaica were AT-rich and composed of 13 protein coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a putative control region. Synteny observed in all assembled mitogenomes was identical to that reported before for P. tigris altaica and other felids. A phylogenomic analysis based on all PCGs demonstrated that the mitochondrial genomes assembled from scat eDNA reliably identify the sequenced samples as belonging to P. tigris and distinguished the same samples from closely and distantly related congeneric species. This study demonstrates that it is viable to retrieve accurate whole and nearly complete mitochondrial genomes of P. tigris altaica (and probably other felids) from scat eDNA samples without library enrichment protocols and using open-access bioinformatics workflows. This new genomic resource represents a new tool to support conservation strategies (bio-prospecting and bio-monitoring) in this iconic cat.

Complete mitochondrial genome of Acrossocheilus yunnanensis (Cypriniformes: Cyprinidae: Acrossocheilus) and its phylogenetic analysis

Acrossocheilus yunnanensis is an endemic species in China. In this study, the complete mitochondrial genome of A. yunnanensis was determined. It was 16,587 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a putative control region. Phylogenetic analysis showed that A. yunnanensis was clustered with A. monticola.

Comparative mitogenomic analyses provide evolutionary insights into the retrolateral tibial apophysis clade (Araneae: Entelegynae).

The retrolateral tibial apophysis (RTA) clade is the largest spider lineage within Araneae. To better understand the diversity and evolution, we newly determined mitogenomes of ten RTA species from six families and performed a comparative mitogenomics analysis by combining them with 40 sequenced RTA mitogenomes available on GenBank. The ten mitogenomes encoded 37 typical mitochondrial genes and included a large non-coding region (putative control region). Nucleotide composition and codon usage were well conserved within the RTA clade, whereas diversity in sequence length and structural features was observed in control region. A reversal of strand asymmetry in nucleotide composition, i.e., negative AT-skews and positive GC-skews, was observed in each RTA species, likely resulting from mitochondrial gene rearrangements. All protein-coding genes were evolving under purifying selection, except for atp8 whose Ka/Ks was larger than 1, possibly due to positive selection or selection relaxation. Both mutation pressure and natural selection might contribute to codon usage bias of 13 protein-coding genes in the RTA lineage. Phylogenetic analyses based on mitogenomic data recovered a family-level phylogeny within the RTA; {[(Oval calamistrum clade, Dionycha), Marronoid clade], Sparassidae}. This study characterized RTA mitogenomes and provided some new insights into the phylogeny and evolution of the RTA clade.

The complete mitochondrial genome of Montiporavietnamensis (Scleractinia, Acroporidae).

Montiporavietnamensis Veron, 2000 (Cnidaria, Anthozoa, Scleractinia, Acroporidae) is an uncommon, but distinctive species of stony coral. The complete mitochondrial genome of M.vietnamensis was sequenced in this study for the first time, based on 32 pairs of primers newly designed according to seven species in the family Acroporidae. The mitogenome of M.vietnamensis has a circular form and is 17,885 bp long, including 13 protein-coding genes (PCGs), 2 tRNA (tRNAMet, tRNATrp), 2 rRNA genes and a putative control-region. The base composition of the complete mitogenome was 24.8% A, 14.2% C, 24.2% G and 36.8% T, with a higher AT content (61.6%) than GC content (38.4%). Based on 13 protein-coding genes, a Maximum Likelihood phylogenetic analysis showed that M.vietnamensis is clustered in the genus Montipora which belongs to the family Acroporidae. More stony coral species should be sequenced for basic molecular information and to help confirm the taxonomic status and evolutionary relationships of Scleractinia in the future.

The complete mitochondrial genome of Eristalinus viridis (Coquillett, 1898) (Diptera: Syrphidae) and its phylogenetic implications

The complete mitochondrial genome of Eristalinus viridis (Coquillett, 1898) was obtained for the first time using Next Generation Sequencing (NGS). The mitogenome assembly of E. viridis is 15,640 bp in length and its annotation confirms the presence of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and one putative control region. The results of the phylogenetic analyses using Maximum Likelihood and Bayesian inference recover a highly supported sister relationship between E. viridis and Mallota bellus.

Characterization and phylogenetic analysis of the complete mitochondrial genome of Notostomum cyclostomum

Notostomum cyclostomum is a parasite that lays eggs on the snow crab shells and causes various diseases by parasitizing fish. Although there have been some studies on the life history of this parasite and the associated fish diseases, little is known about the molecular biology of this parasite. Thus, here we report the mitochondrial genome of N. cyclostomum, which is 16,972 bp long and contains 13 mitochondrial protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative control region with a 92% AT-rich sequences between tRNA-R and tRNA-H. Phylogenetic analysis using 13 PCGs confirmed that N. cyclostoum belongs to the family Pisciodlidae. This is the first study revealing the complete mitochondrial genome sequence of N. cyclostomum.

The complete mitochondrial genome of the sand dollar Astriclypeus mannii (Verrill, 1867) (Echinoidea: Astriclypeidae) in the subtidal sand flat in Jeju Island off the south coast of Korea

We sequenced the complete mitochondrial genome of sand dollar Astriclypeus mannii (Verrill 1867) (Echinoidea: Astriclypeidae) occurring in the subtidal sand flat in Jeju Island off the south coast of Korea. The mitochondrial genome was 15,744 bp in length and contained 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, and 140 nucleotides representing the putative control region. We reconstructed the concatenated phylogenetic tree based on 13 PCGs of 18 echinoderms, including A. mannii. From the maximum likelihood clustering, A. mannii was grouped in the order Echinolampadacea. The complete mitochondrial sequence of A. mannii for the first time in this study provide valuable insight in understanding the evolution and phylogenetic analysis of echinoids (sea urchins).

Characterization of 15 Earthworm Mitogenomes from Northeast China and Its Phylogenetic Implication (Oligochaeta: Lumbricidae, Moniligastridae)

Earthworms are an important ecological group, especially in agricultural regions in Northeast China. However, fewer studies focus on this group of organisms compared with other faunal groups. Here, we sequenced 15 new mitogenomes of Aporrectodea tuberculata Eisen, 1874, A. trapezoides Duges, 1828, Eisenia nordenskioldi Eisen, 1878 and Drawida ghilarovi Gates, 1969 in Northeast China using a high-throughput sequencing platform. These incomplete linear and double-stranded mitogenomes vary from 14,998 bp to 16,123 bp in size and include 37 genes and a putative control region. Intraspecific genetic divergence was quantified in the lumbricid species, and a control region in D. ghilarovi was reported for the first time by comparison to the mitogenomes of the congeners. Phylogenetic analysis based on coding genes and ribosomal DNA datasets using BI and ML inferences showed the non-monophyly of Aporrectodea and polyphyly of E. nordenskioldi. Future works should examine the taxonomy, phylogeny and population genetics not only of Lumbricidae but also the other earthworm families on the global scale using mitogenomic and nuclear data.

First record of the complete mitochondrial genome of Tubifex tubifex (Müller) 1774 (Annelida; Clitellata; Oligochaeta) and phylogenetic analysis

The complete mitochondrial genome of Tubifex tubifex was analyzed using the MGISEQ-2000 platform. The size of the complete mitochondrial genome was 15,972 bp. Data pertaining to the genome, such as the presence of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a putative control region were submitted to NCBI (MW690579). A phylogenetic tree was constructed with the sequences of the 13 PCGs using the maximum-likelihood method. Despite only a few references available on the complete mitochondrial genome of other aquatic oligochaetes, our phylogenetic analysis revealed that the freshwater oligochaetes T. tubifex and Limnodrilus hoffmeisteri are in a cluster different from that of the earthworm group.

The complete mitochondrial genome of Pseudopleuronectes herzensteini

Pseudopleuronectes herzensteini belonging to Pleuronectiformes (family Pleuronectidae) is important in the fishery industry. However, the molecular biology of this valuable fish has hardly been reported. Thus, here we report the complete mitochondrial genome of P. herzensteini. The mitochondrial DNA (mtDNA) of P. herzensteini is 16,719 bp long and contains 13 mitochondrial protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a putative control region between tRNA-P and tRNA-F distinguished by a single short noncoding region. Phylogenetic analysis using PCGs confirmed that this mtDNA sequence belongs to the family Pleuronectidae. This is the first study reporting the complete mitochondrial genome sequence of P. herzensteini.

Characterization and phylogenetic analysis of the complete mitochondrial genome of the firemouth cichlid, Thorichthys meeki (Perciformes: Cichlidae)

Here, we report information on the complete mitochondrial genome of the firemouth cichlid, Thorichthys meeki (Brind 1918). Illumina HiSeq genome sequencing produced the assembly of a circular mitogenome of 16,527 base pairs (bp) from T. meeki consisting of 46.8% GC nucleotides, 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a putative control region as shown in the typical teleost gene composition. The gene order of the T. meeki mitogenome was identical to that of other cichlid species. A maximum likelihood phylogenetic tree based on mitochondrial PCGs showed a close relationship of T. meeki with Thorichthys aureus (Gunther 1862) within Heroini tribe.