Paramoeba aparasomata n. sp., a symbiont-free species,and its relative Paramoeba karteshi n. sp. (Amoebozoa,Dactylopodida)

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

Phylogeny of Rhigonematomorpha based on the complete mitochondrial genome of Rhigonema thysanophora (Nematoda: Chromadorea)

We determined the complete mitochondrial genome sequence of Rhigonema thysanophora, the first representative of Rhigonematomorpha, and used this sequence along with 57 other nematode species for phylogenetic analyses. The R. thysanophora mtDNA is 15 015 bp and identical to all other chromadorean nematode mtDNAs published to date in that it contains 36 genes (lacking atp8) encoded in the same direction. Phylogenetic analyses of nucleotide and amino acid sequence data for the 12 protein‐coding genes recovered Rhigonematomorpha as the sister group to the heterakoid species, Ascaridia columbae (Ascaridomorpha). The organization of R. thysanophora mtDNA resembles the most common pattern for the Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha clade in gene order, but with some substantial gene rearrangements. This similarity in gene order is in agreement with the sequence‐based analyses that indicate a close relationship between Rhigonematomorpha and Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha. These results are consistent with certain analyses of nuclear SSU rDNA for R. thysanophora and some earlier classification systems that asserted phylogenetic affinity between Rhigonematomorpha and Ascaridomorpha, but inconsistent with morphology‐based phylogenetic hypotheses that suggested a close (taxonomic) relationship between rhigonematomorphs and oxyuridomorphs (pinworms). These observations must be tempered by noting that few rhigonematomorph species have been sequenced and included in phylogenetic analyses, and preliminary studies based on SSU rDNA suggest the group is not monophyletic. Additional mitochondrial genome sequences of rhigonematids are needed to characterize their phylogenetic relationships within Chromadorea, and to increase understanding of mitochondrial genome evolution.     

Resequencing and comparison of whole mitochondrial genome to gain insight into the evolutionary status of the Shennongjia golden snub‐nosed monkey (SNJ R. roxellana)

Shennongjia Rhinopithecus roxellana (SNJ R. roxellana) is the smallest geographical population of R. roxellana. The phylogenetic relationships among its genera and species and the biogeographic processes leading to their current distribution are largely unclear. To address these issues, we resequenced and obtained a new, complete mitochondrial genome of SNJ R. roxellana by next‐generation sequencing and standard Sanger sequencing. We analyzed the gene composition, constructed a phylogenetic tree, inferred the divergence ages based on complete mitochondrial genome sequences, and analyzed the genetic divergence of 13 functional mtDNA genes. The phylogenetic tree and divergence ages showed that R. avunculus (the Tonkin snub‐nosed monkey) was the first to diverge from the Rhinopithecus genus ca. 2.47 million years ago (Ma). Rhinopithecus bieti and Rhinopithecus strykeri formed sister groups, and the second divergence from the Rhinopithecus genus occurred ca. 1.90 Ma. R. roxellana and R. brelichi diverged from the Rhinopithecus genus third, ca. 1.57 Ma. SNJ R. roxellana was the last to diverge within R. roxellana species in 0.08 Ma, and the most recent common ancestor of R. roxellana is 0.10 Ma. The analyses on gene composition showed SNJ R. roxellana was the newest geographic population of R. roxellana. The work will help to develop a more accurate protection policy for SNJ R. roxellana and facilitate further research on selection and adaptation of R. roxellana.     

Complete mitochondrial genome of the black‐tailed hornet,Vespa ducalis (Hymenoptera: Vespidae): Genomic comparisons in Vespoidea

We sequenced the complete mitochondrial genome (mitogenome) of the black‐tailed hornet, Vespa ducalis (Hymenoptera: Vespidae). The genome was 15,779‐bp long and contained typical sets of genes [13 protein‐coding genes (PCGs), 22 tRNAs, and 2 rRNAs]. The V. ducalis A + T‐rich region was 166‐bp long and was the shortest of all sequenced Vespoidea genomes, including Vespa. The genome was highly biased toward A/T nucleotides—80.1 % in the whole genome, 77.8 % in PCGs, 83.4–85.6 % in RNAs, and 92.8 % in the A + T‐rich region. These values are well within the typical range for genes and regions of Vespoidea mitogenomes. Start and stop codons in several Vespa species—including V. ducalis—were diversified, despite these species belonging to the same genus. In comparison with the ancestral mitogenomes, Vespa mitogenomes—including that of V. ducalis—showed substantial gene rearrangement; however, we detected no gene rearrangement among Vespa species. We conducted phylogenetic reconstruction based on concatenated sequences of 13 PCGs and two rRNAs (12,755 bp ) in available species of Vespoidea—21 species in six subfamilies in two families (Vespidae and Formicidae). The Bayesian inference and maximum likelihood (ML) methods revealed that each family formed strong monophyletic groups [Bayesian posterior probability (BPP) = 1; ML, 100 %]. Moreover, V. ducalis and V. mandarinia formed a strong sister group (BPP = 1; ML, 94 %).     

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single‐celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra‐isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co‐evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.     

The complete mitochondrial genome of the grand jackknife clam, Solen grandis (Bivalvia: Solenidae): a novel gene order and unusual non-coding region

Molluscs in general, and bivalves in particular, exhibit an extraordinary degree of mitochondrial gene order variation when compared with other metazoans. The complete mitochondrial genome of Solen grandis (Bivalvia: Solenidae) was determined using long-PCR and genome walking techniques. The entire mitochondrial genome sequence of S. grandis is 16,784 bp in length, and contains 36 genes including 12 protein-coding genes (atp8 is absent), 2 ribosomal RNAs, and 22 tRNAs. All genes are encoded on the same strand. Compared with other species, it bears a novel gene order. Besides these, we find a peculiar non-coding region of 435 bp with a microsatellite-like (TA)₁₂ element, poly-structures and many hairpin structures. In contrast to the available heterodont mitochondrial genomes from GenBank, the complete mtDNA of S. grandis has the shortest cox3 gene, and the longest atp6, nad4, nad5 genes.     

Comparative mitochondrial genome analysis reveals the evolutionary rearrangement mechanism in Brassica

The genus Brassica has many species that are important for oil, vegetable and other food products. Three mitochondrial genome types (mitotype) originated from its common ancestor. In this paper, a B. nigra mitochondrial main circle genome with 232,407 bp was generated through de novo assembly. Synteny analysis showed that the mitochondrial genomes of B. rapa and B. oleracea had a better syntenic relationship than B. nigra. Principal components analysis and development of a phylogenetic tree indicated maternal ancestors of three allotetraploid species in Us triangle of Brassica. Diversified mitotypes were found in allotetraploid B. napus, in which napus‐type B. napus was derived from B. oleracea, while polima‐type B. napus was inherited from B. rapa. In addition, the mitochondrial genome of napus‐type B. napus was closer to botrytis‐type than capitata‐type B. oleracea. The sub‐stoichiometric shifting of several mitochondrial genes suggested that mitochondrial genome rearrangement underwent evolutionary selection during domestication and/or plant breeding. Our findings clarify the role of diploid species in the maternal origin of allotetraploid species in Brassica and suggest the possibility of breeding selection of the mitochondrial genome.     

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha‐proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early‐diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein‐coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.     

The mitochondrial genome of the water spider Argyroneta aquatica (Araneae: Cybaeidae)

We sequenced nearly the entire mitochondrial genome of Argyroneta aquatica, a wholly underwater‐living spider, thereby enhancing the available genomic information for Arachnida. The confirmed sequences contained the complete set of known genes present in other metazoan mitochondrial genomes. However, the mitochondrial gene order of A. aquatica was distinctly different from that of the most distant Chelicerata Limulus polyphemus (Xiphosura), probably because of a series of gene translocations and/or inversions. Comparison of arachnid mitochondrial gene orders for the purpose of phylogenetic inference is only minimally useful, but provides a strong signal in closely related lineages. To test the basal relationships and the evolutionary pattern of tRNA gene rearrangements among Arachnida, phylogenetic analyses using amino acid sequences of the 13 protein‐coding genes were performed. An interesting feature, the five 135‐bp tandem repeats and two 363‐bp tandem repeats, was identified in the putative control region. Although control region tandem repeats have been reported in many other arachnid and metazoan species, this is the first time it has been described in spiders.     

Characterization of the complete mitochondrial genome of Barilius malabaricus and its phylogenetic implications

Bariliine fishes are important components of the ornamental as well as subsistence fishery sectors in India. Many of the species in the genus Barilius are threatened by habitat loss and therefore need to be met with conservation initiatives. Effective conservation measures, however, require clarification of species identities and resolution of the validity of many species currently treated as synonymous or sub-species. The complete mitochondrial genome data provide better insight into phylogenetic information than the short fragment or single gene based analysis. Thus, we have sequenced the complete mitochondrial genome of Barilius malabaricus, one of the important fish species in the fresh water ornamental sector, for better understanding its phylogenetic status. The 16,519 bp mitochondrial genome consists of 37 genes which classifies as 22 tRNA, 13 protein coding and 2 ribosomal RNA genes and a control region. Overall, the mitochondrial genome bears the typical gene order and composition as in other fishes. Further, the COI, cytochrome b and 16S rRNA gene sequences revealed that, B. malabaricus is genetically closer to B. canarensis and B. bakeri compared to other Barilius species. Also, the Barilius species of west flowing rivers in Western Ghats were consistently recovered as a clade distinct from other species. We therefore suggest to retain the genus name Barilius for the species from the Western Ghats until a comprehensive analysis based on both morphological and molecular markers reveals the relationship between species now variously placed in the genera Barilius and Opsarius in greater detail.     

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein‐coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA‐Met gene was found located between the tRNA‐Gln and ND2 genes. On both the 5′ and 3′‐sides, the control region possessed distinct repeat regions; however, the CSB‐2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum‐likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).     

Complete mitochondrial genome of the wild silkmoth,Saturnia boisduvalii (Lepidoptera: Saturniidae)

We sequenced mitochondrial genome (mitogenome) of the wild silkmoth, Saturnia boisduvalii (Lepidoptera: Saturniidae), which occurs in mainland Korea, and compared it with other species in Bombycoidea to characterize the genomic evolution of the superfamily. We found that the composition and arrangement of genes in the 15,257‐bp S. boisduvalii genome are typical of the majority of Lepidoptera, and the genome is biased toward A/T nucleotides, as previously reported. Comparison of individual gene divergence among bombycoid species showed that ND6 was most variable (p‐distance = 0.21), whereas COI and COII were most conserved, indicating that of all the protein‐coding genes (PCGs) ND6 appear to have evolved most rapidly. Thus, other PCGs beside COI are potential alternative markers, where scrutinized discrimination among species is required.     

Sequencing of Camelina neglecta,a diploid progenitor of the hexaploid oilseed Camelina sativa

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.     

Phylogeography and lineage‐specific patterns of genetic diversity and molecular evolution in a group of North American skinks

Geography influences the evolutionary trajectory of species by mediating opportunities for hybridization, gene flow, demographic shifts and adaptation. We sought to understand how geography and introgression can generate species‐specific patterns of genetic diversity by examining phylogeographical relationships in the North American skink species Plestiodon multivirgatus and P. tetragrammus (Squamata: Scincidae). Using a multilocus dataset (three mitochondrial genes, four nuclear genes; a total of 3455 bp) we discovered mito‐nuclear discordance, consistent with mtDNA introgression. We further tested for evidence of species‐wide mtDNA introgression by using comparisons of genetic diversity, selection tests and extended Bayesian skyline analyses. Our findings suggest that P. multivirgatus acquired its mitochondrial genome from P. tetragrammus after their initial divergence. This putative species‐wide mitochondrial capture was further evidenced by statistically indistinguishable substitution rates between mtDNA and nDNA in P. multivirgatus. This rate discrepancy was observed in P. multivirgatus but not P. tetragrammus, which has important implications for studies that combine mtDNA and nDNA sequences when inferring time since divergence between taxa. Our findings suggest that by facilitating opportunities for interspecific introgression, geography can alter the course of molecular evolution between recently diverged lineages.     

Complete mitochondrial genome of the Chinese spiny lobster <Emphasis Type="Italic">Panulirus stimpsoni</Emphasis> (Crustacea: Decapoda): genome characterization and phylogenetic considerations

The genetics and molecular biology of the commercially important Chinese spiny lobster, Panulirus stimpsoni are little known. Here, we present the complete mitochondrial genome sequence of P. stimpsoni, determined by the long polymerase chain reaction and primer walking sequencing method. The entire genome is 15,677 bp in length, encoding the standard set of 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The overall A + T content of the genome is 65.6%, lower than most malacostracan species. The gene order is consistent with the pancrustacean ground pattern. Several conserved elements were identified from P. stimpsoni control region, viz. one [TA(A)]n-block, two GA-blocks and three hairpin structures. However, the position of [TA(A)]n-block and number of hairpin structure are different from those in the congeneric P. japonicus and other decapods. Phylogenetic analyses using the concatenated nucleotide and amino acid sequences of 13 protein-coding genes do not support the monophyly of suborder Pleocyemata, which is in contrast to most morphological and molecular results. However, the position of Palinura and Astacidea is unstable, as represented by the basal or sister branches to other Reptantia species. P. stimpsoni, as the second species of Palinura with complete mitochondrial genome available, will provide important information on both genomics and conservation biology of the group.     

Mitogenomics reveals high synteny and long evolutionary histories of sympatric cryptic nematode species

Species with seemingly identical morphology but with distinct genetic differences are abundant in the marine environment and frequently co‐occur in the same habitat. Such cryptic species are typically delineated using a limited number of mitochondrial and/or nuclear marker genes, which do not yield information on gene order and gene content of the genomes under consideration. We used next‐generation sequencing to study the composition of the mitochondrial genomes of four sympatrically distributed cryptic species of the Litoditis marina species complex (PmI, PmII, PmIII, and PmIV). The ecology, biology, and natural occurrence of these four species are well known, but the evolutionary processes behind this cryptic speciation remain largely unknown. The gene order of the mitochondrial genomes of the four species was conserved, but differences in genome length, gene length, and codon usage were observed. The atp8 gene was lacking in all four species. Phylogenetic analyses confirm that PmI and PmIV are sister species and that PmIII diverged earliest. The most recent common ancestor of the four cryptic species was estimated to have diverged 16 MYA. Synonymous mutations outnumbered nonsynonymous changes in all protein‐encoding genes, with the Complex IV genes (coxI‐III) experiencing the strongest purifying selection. Our mitogenomic results show that morphologically similar species can have long evolutionary histories and that PmIII has several differences in genetic makeup compared to the three other species, which may explain why it is better adapted to higher temperatures than the other species.     

Species‐specific mitochondrial gene rearrangements in biting midges and vector species identification

Abstract Partial mitochondrial gene sequences of 16 Culicoides species were determined to elucidate phylogenetic relations among species and to develop a molecular identification method for important virus vector species. In addition, the analysis found mitochondrial gene rearrangement in several species. Sequences of the mitochondrial genome region, cox1–trnL2–cox2 (1940–3785 bp) of 16 Culicoides and additional sequences were determined in some species, including whole mitochondrial genome sequences of Culicoides arakawae. Nine species showed common organization in this region, with three genes cox1–trnL2–cox2 and a small or no intergenic region (0–30 bp) between them. The other seven species showed translocation of tRNA and protein‐coding genes and/or insertion of AT‐rich non‐coding sequences (65–1846 bp) between the genes. The varied gene rearrangements among species within a genus is very rare for mitochondrial genome organization. Phylogenetic analyses based on the sequences of cox1+cox2 suggest a few clades among Japanese Culicoides species. No relationships between phylogenetic closeness and mitochondrial gene rearrangements were observed. Sequence data were used to establish a polymerase chain reaction tool to distinguish three important vector species from other Culicoides species, for which classification during larval stages is not advanced and identification is difficult.