Copies Of Mitochondrial Genes Research Articles

The “evolution” of the mitochondrial genome of the (<i>Phylloscopus borealis</i> sensu lato) occurs in its nuclear genome

Sequencing of a partial fragment of the ND5–cytb mithochondrial genes (1553 bp) and its nuclear copies was carried out to Phylloscopus borealis sensu lato, belonging to individual taxonomic groups from different parts of the range. It was shown that the majority of taxon-specific and unique mitochondrial substitutions in examinandus and xanthodryas forms were identical to those in nuclear copies of borealis mtDNA. Differences between mitochondrial haplotypes of examinandus and nuclear copies of mtDNA borealis were low (p = 0.02), at the same time the mtDNA genetic divergence in borealis–examinandus, borealis–xanthodryas and examinandus–xanthodryas significantly exceeded this value (p = 0.035, 0.044 and 0.046, respectively). A nuclear copy of the mitochondrial haplotype of the easternmost form of xanthodryas was first discovered in the nuclear genome of one borealis individual in the western part of the breeding range (Komi Republic). Alongside this, it was shown at the first time, the nuclear copies of xanthodryas mtDNA from Toyama Prefecture (Japan) were close to the mitochondrial haplotypes of borealis from Kytlym (Sverdlovsk region) (p = 0.018). Thus, the mutations emerging in the nuclear copies of mitochondrial genes are the source of most substitutions in the mitochondrial DNA of the studied forms. The origin of the mitochondrial haplotypes examinandus and xanthodryas from nuclear copies of mtDNA borealis and the close similarity of their nuclear genomes gives grounds to consider the mitogenomes of these forms as variants of the haplotype of the single species Ph. borealis sensu lato. With a high degree of probability, it can be argued that the divergence time of the haplotypes of the analyzed forms is significantly less than 2.5-3 million years, as previously assumed by a number of authors [Saitoh et al. 2010; Alström et al. 2011], and the “molecular clock” that do not take into account recombination events between the nuclear and mitochondrial genomes cannot be used in this case.

Quantitative assessment of reef foraminifera community from metabarcoding data.

Describing living community compositions is essential to monitor ecosystems in a rapidly changing world, but it is challenging to produce fast and accurate depiction of ecosystems due to methodological limitations. Morphological methods provide absolute abundances with limited throughput, whereas metabarcoding provides relative abundances of genes that may not correctly represent living communities from environmental DNA assessed with morphological methods. However, it has the potential to deliver fast descriptions of living communities provided that it is interpreted with validated species-specific calibrations and reference databases. Here, we developed a quantitative approach to retrieve from metabarcoding data the assemblages of living large benthic foraminifera (LBF), photosymbiotic calcifying protists, from Indonesian coral reefs that are under increasing anthropogenic pressure. To depict the diversity, we calculated taxon-specific correction factors to reduce biological biases by comparing surface area, biovolume and calcite volume, and the number of mitochondrial gene copies in seven common LBF species. To validate the approach, we compared calibrated datasets of morphological communities from mock samples with bulk reef sediment; both sample types were metabarcoded. The calibration of the data significantly improved the estimations of genus relative abundance, with a difference of ±5% on average, allowing for comparison of past morphological datasets with future molecular ones. Our results also highlight the application of our quantitative approach to support reef monitoring operations by capturing fine-scale processes, such as seasonal and pollution-driven dynamics, that require high-throughput sampling treatment.

Incomplete lineage sorting explains the low performance of DNA barcoding in a radiation of four species of Western European grasshoppers (Orthoptera: Acrididae: Chorthippus)

Abstract DNA barcoding often fails to identify species despite its undisputed advantages. Hybridization, sample contamination, incomplete lineage sorting and nuclear copies of mitochondrial genes (NUMTs) are often put forward as explanations but have seldomly been tested. Here I used available RNA-sequencing data to explore this issue in four Chorthippus grasshopper species. I was able to exclude NUMTs, contamination and recent hybridization as probable causes of the low barcoding performance. Using a phylogenetic method, I estimated the nuclear and mitochondrial mutation rates as 1.31 × 10−9–2.27 × 10−9 and 8.1 × 10−9–1.4 × 10−8 mutations/site/year, respectively. These grasshoppers therefore did not exhibit a particularly low mitochondrial mutation rate compared to other insect species. Using coalescence simulation, I was able to show that two simple demographic scenarios, with a divergence period of 1–3 Myr, provided a good fit to the mitochondrial genealogies in three of the four target species. Interestingly, the mitochondrial genealogy of Chorthippus mollis was inconsistent with a neutral evolution pattern, suggesting that it had undergone adaptive selection.

How does mitochondria function contribute to aerobic performance enhancement in lizards?

Aims: Aerobic exercise typically enhances endurance across vertebrates so that chronically high energy demands can be met. Some known mechanisms of doing this include increases in red blood cell numbers, angiogenesis, muscle fiber adaptions, mitochondria biogenesis, and changes to cellular metabolism and oxidative phosphorylation. We used green anole lizards (Anolis carolinensis) to test for an effect of aerobic exercise on metabolism, mitochondria densities, and mitochondrial function. Methods: We first tested the response of green anoles to endurance training and pyrroloquinoline quinone (PQQ) supplementation, which has been shown to increase mitochondria biogenesis. We also conducted a mitochondrial stress test to determine how training affected mitochondrial function in skeletal muscle fibers. Results: Aerobic exercise led to increased endurance and decreased standard metabolic rate (SMR), while PQQ did not affect endurance and increased SMR. In a second experiment, aerobic exercise increased endurance and decreased resting metabolic rate (RMR) in both male and female green anoles. Higher counts of mitochondrial gene copies in trained lizards suggested additional mitochondria adaptations to achieve increased endurance and decreased metabolism. A mitochondrial stress test revealed no effect on baseline oxygen consumption rates of muscle fibers, but untrained lizards had higher maximal oxygen consumption rates with the addition of metabolic fuel. Conclusion: It is likely that trained lizards exhibited lower maximal oxygen consumption rates by developing higher mitochondria efficiency. This adaptation allows for high ATP demand to be met by making more ATP per oxygen molecule consumed. On the other hand, it is possible that untrained lizards prioritized limiting reactive oxygen species (ROS) production at rest, while sacrificing higher levels of proton leak and higher oxygen consumption rates when working to meet high ATP demand.

Metabarcoding for biodiversity inventory blind spots: A test case using the beetle fauna of an insular cloud forest.

Soils harbour a rich arthropod fauna, but many species are still not formally described (Linnaean shortfall) and the distribution of those already described is poorly understood (Wallacean shortfall). Metabarcoding holds much promise to fill this gap, however, nuclear copies of mitochondrial genes, and other artefacts lead to taxonomic inflation, which compromise the reliability of biodiversity inventories. Here, we explore the potential of a bioinformatic approach to jointly "denoise" and filter nonauthentic mitochondrial sequences from metabarcode reads to obtain reliable soil beetle inventories and address open questions in soil biodiversity research, such as the scale of dispersal constraints in different soil layers. We sampled cloud forest arthropod communities from 49 sites in the Anaga peninsula of Tenerife (Canary Islands). We performed whole organism community DNA (wocDNA) metabarcoding, and built a local reference database with COI barcode sequences of 310 species of Coleoptera for filtering reads and the identification of metabarcoded species. This resulted in reliable haplotype data after considerably reducing nuclear mitochondrial copies and other artefacts. Comparing our results with previous beetle inventories, we found: (i) new species records, potentially representing undescribed species; (ii) new distribution records, and (iii) validated phylogeographic structure when compared with traditional sequencing approaches. Analyses also revealed evidence for higher dispersal constraint within deeper soil beetle communities, compared to those closer to the surface. The combined power of barcoding and metabarcoding contribute to mitigate the important shortfalls associated with soil arthropod diversity data, and thus address unresolved questions for this vast biodiversity fraction.

Signatures of Selection on Mitonuclear Integrated Genes Uncover Hidden Mitogenomic Variation in Fur Seals.

Nuclear copies of mitochondrial genes (numts) are commonplace in vertebrate genomes and have been characterized in many species. However, relatively little attention has been paid to understanding their evolutionary origins and to disentangling alternative sources of insertions. Numts containing genes with intact mitochondrial reading frames represent good candidates for this purpose. The sequences of the genes they contain can be compared with their mitochondrial homologs to characterize synonymous to nonsynonymous substitution rates, which can shed light on the selection pressures these genes have been subjected to. Here, we characterize 25 numts in the Antarctic fur seal (Arctocephalus gazella) genome. Among those containing genes with intact mitochondrial reading frames, three carry multiple substitutions in comparison to their mitochondrial homologs. Our analyses reveal that one represents a historic insertion subjected to strong purifying selection since it colonized the Otarioidea in a genomic region enriched in retrotransposons. By contrast, the other two numts appear to be more recent and their large number of substitutions can be attributed to noncanonical insertions, either the integration of heteroplasmic mtDNA or hybridization. Our study sheds new light on the evolutionary history of pinniped numts and uncovers the presence of hidden sources of mitonuclear variation.

Heteroplasmy and nuclear copies of mitochondrial genes (NUMTs) identified in the zone of introgressive hybridization of Norway spruce and Siberian spruce

Heteroplasmy and nuclear copies of mitochondrial genes (NUMTs) identified in the zone of introgressive hybridization of Norway spruce and Siberian spruce

Characterization of mitogenomes from four Mucorales species and insights into pathogenicity.

Mucorales, as one major order of Zygomycetes fungi, can infect human beings and cause serious consequence. We have noticed the pathogenicity of Mucorales is closely related to energy metabolism, while mitochondria play the role of energy factories in almost all biological activities. Virulence of M irregularis, M hiemalis, L corymbifera and R arrhizus were verified in Galleria mellonella larvae, as well as mitochondrial gene copies analysed with RT-qPCR. Mitogenomes of the four Mucorales species were sequenced based on illumina NovaSeq technology to study their characteristic features and functional regions. Variant virulence of M irregularis, M hiemalis, L corymbifera and R arrhizu were verified by clinical retrospective data and our G mellonella infection models, also copies of mitochondrial genes indicated the significant associations with pathogenicity. A total of 274.18 clean reads were generated to be assembled; the complete mitogenomes of the four Mucorales species were obtained with totally different length. After the genomes annotated and compared, M irregularis was found more similar with M hiemalis than those of L corymbifera and R arrhizus, especially the small (rrns) and large (rrnl) subunits of mitochondrial ribosomal RNA (rRNA) genes. The GC content, ncRNAs and the distribution of the SNPs and InDels were also compared, and the GC content rate of fungi seems to be related to the fungal thermal adaptability. In addition, linear mitogenomes of these four Mucorales showed diverse arrangements of orf genes and directionality of some conserved gene elements. This study uncovered the pathogenicity variances among the four Mucorales species and the relationship between their mitogenomic features and clinical pathogenicity. Further studies like spatial structure of mitochondrial genomes and the comprehensive analysis of transcription regulation are needed.

Quantitative determination of mutton adulteration with single-copy nuclear genes by real-time PCR

Mitochondrial genes were generally adopted for PCR-based meat adulteration authentication due to their excellent specificity to species and numerous copies in one cell. However, the number of mitochondrial gene copies varies according to cells and tissues, which leads to quantification errors for meat adulteration. To address this problem, single-copy nuclear genes were selected to develop a quantitative method for identifying mutton adulteration in this study. Both single-copy genes specific to sheep species and single-copy reference genes show good linearity between Ct values and series diluted DNA concentrations, with the correlation coefficients of 0.9999 and 0.9993, respectively. Meanwhile, a constant (correction factor) was introduced to transform DNA concentrations into mutton proportions in adulterated meat. With this method, simulated mutton-pork, mutton-chicken and mutton-duck adulteration samples could be accurately quantified with the recovery rates of 89.56%, 107.13% and 95.20%, respectively.

Copy numbers of mitochondrial genes change during melon leaf development and are lower than the numbers of mitochondria

Melon is a useful plant species for studying mitochondrial genetics because it contains one of the largest and structurally diverse mitochondrial genomes among all plant species and undergoes paternal transmission of mitochondria. We used droplet digital (dd) PCR in combination with flow cytometric determination of nuclear DNA quantities to determine the absolute per-cell copy numbers of four mitochondrial genes (nad9, rps1, matR, and atp6) across four stages of melon leaf development. The copy numbers of these mitochondrial genes not only varied during leaf development but also differed among each other, and there was no correlation between the copy numbers of the mitochondrial genes and their transcript levels. The gene copy numbers varied from approximately 36.8 ± 4.5 (atp6 copies in the 15th leaf) to approximately 82.9 ± 5.7 (nad9 copies in the 9th leaf), while the mean number of mitochondria was approximately 416.6 ± 182.7 in the 15th leaf and 459.1 ± 228.2 in the 9th leaf. These observations indicate that the leaf cells of melon do not contain sufficient copies of mitochondrial genes to ensure that every mitochondrion possesses the entire mitochondrial genome. Given this cytological evidence, our results indicate that mtDNA in melon exists as a sub-genomic molecule rather than as a single-master circle and that the copy numbers of individual mitochondrial genes may vary greatly. An improved understanding of the molecular mechanism(s) controlling the relative prevalence and transmission of sub-genomic mtDNA molecules should provide insights into the continuity of the mitochondrial genome across generations.

The mitochondrial genomes of Statilia maculata and S. nemoralis (Mantidae: Mantinae) with different duplications of trnR genes

Different mitochondrial gene copies or gene rearrangements are observed in some interspecific mitochondrial genomes (mitogenomes), but few in intraspecific mitogenomes. In this study, we sequenced the complete mitogenomes of Statilia maculata and Statilia nemoralis (Mantinae: Mantini). The genetic distance of the whole genomes between these two species was just 0.5%, suggesting that S. maculata and S. nemoralis might be the same species. The mitogenome of S. maculata had five copies of trnR genes (three copies were identical and the other two had some base substitutions), whereas the mitogenome of S. nemoralis had six trnR genes. Five, six or seven tandem copies of trnR genes in S. maculata were found in different localities, respectively. We further sequenced the ND3-ND5 region of fifty individuals hatched from one ootheca (Tianjin) and we found that all individuals had five identical tandem copies of trnR genes and same sequences. Hence, we concluded that different copies of trnR genes can occur in the same species. The mitogenomes of S. maculata and S. nemoralis with other mitogenomes of Mantinae species published were used to construct BI and ML phylogenetic trees. In this study, both BI and ML phylogenetic trees showed that Mantini and Statilia were monophyletic whereas Paramantini was paraphyletic.

Inter-Specimen Imbalance of Mitochondrial Gene Copy Numbers Predicts Clustering of Pneumocystis jirovecii Isolates in Distinct Subgroups.

The molecular detection of Pneumocystis jirovecii is an important therapy-relevant tool in microbiological diagnostics. However, the quantification of this pathogen in the past has revealed discordant results depending on the target gene. As the clinical variety of P. jirovecii infections ranges between life-threatening infections and symptom-free colonization, the question arises if qPCRs are reliable tools for quantitative diagnostics of P. jirovecii. P. jirovecii positive BALs were quantitatively tested for the copy numbers of one mitochondrial (COX-1) and two nuclear single-copy genes (KEX1 and DHPS) compared to the mitochondrial large subunit (mtLSU) by qPCR. Independent of the overall mtLSU copy number P. jirovecii clustered into distinct groups based on the ratio patterns of the respective qPCRs. This study, which compared different mitochondrial to nuclear gene ratio patterns of independent patients, shows that the mtLSU gene represents a highly sensitive qPCR tool for the detection of P. jirovecii, but does not display a reliable target for absolute quantification.

Complete mitochondrial genome sequences of the northern spotted owl (Strix occidentalis caurina) and the barred owl (Strix varia; Aves: Strigiformes: Strigidae) confirm the presence of a duplicated control region.

We report here the successful assembly of the complete mitochondrial genomes of the northern spotted owl (Strix occidentalis caurina) and the barred owl (S. varia). We utilized sequence data from two sequencing methodologies, Illumina paired-end sequence data with insert lengths ranging from approximately 250 nucleotides (nt) to 9,600 nt and read lengths from 100–375 nt and Sanger-derived sequences. We employed multiple assemblers and alignment methods to generate the final assemblies. The circular genomes of S. o. caurina and S. varia are comprised of 19,948 nt and 18,975 nt, respectively. Both code for two rRNAs, twenty-two tRNAs, and thirteen polypeptides. They both have duplicated control region sequences with complex repeat structures. We were not able to assemble the control regions solely using Illumina paired-end sequence data. By fully spanning the control regions, Sanger-derived sequences enabled accurate and complete assembly of these mitochondrial genomes. These are the first complete mitochondrial genome sequences of owls (Aves: Strigiformes) possessing duplicated control regions. We searched the nuclear genome of S. o. caurina for copies of mitochondrial genes and found at least nine separate stretches of nuclear copies of gene sequences originating in the mitochondrial genome (Numts). The Numts ranged from 226–19,522 nt in length and included copies of all mitochondrial genes except tRNAPro, ND6, and tRNAGlu. Strix occidentalis caurina and S. varia exhibited an average of 10.74% (8.68% uncorrected p-distance) divergence across the non-tRNA mitochondrial genes.

Phylogeography and demographic history of Siberian rubythroat Luscinia calliope

Phylogeographic analysis on the basis of individual marker variability provides insight into the history and mechanisms of the range formation of widely distributed species. A preliminary study of the mtDNA cytochrome b gene in Siberian rubythroat Luscinia calliope revealed the existence of three well-differentiated haplogroups, including one western and two eastern haplogroups. Continuing the study of the genetic markers of the species, we found that, in western part of the range, represented by the nominative geographic race, there were almost exclusively haplotypes of western group. In eastern populations of Khabarovsk krai, Chukotka, Kamchatka, and Sakhalin, haplotypes of all groups are mixed in different proportions. At the same time, the populations of Hokkaido and Iturup islands are exclusively represented by individuals with eastern haplotypes. Comparison of the identified nuclear copies of mitochondrial genes and construction of the phylogenetic network of haplotypes on the basis of cloned and initial sequences showed that two groups of eastern haplotypes (one of which geographically corresponded to L. c. anadyrensis and L. c. camtschatkensis and the second corresponded to L. c. sachalinensis) originated from nuclear pseudogenes of L. c. calliope through intergenomic recombination. In this regard, we propose a new hypothesis for the establishment of the modern range of this species, according to which the Siberian rubythroat dispersal from South Siberia occurred in two stages. At first, the species expanded its range to the northeast in the direction of the Kolyma and Koryak uplands. During the settling of these areas of northeastern Asia, a recombination between the mitochondrial and nuclear DNA took place, which led to the forming of a new haplotype, which was widespread in the emerging breeding populations. Birds with recombinant haplotypes populated the territories of Chukotka and Kamchatka, and then gradually occupied the Kuril Islands and, eventually, reached Hokkaido. At the next stage, Siberian rubythroat, probably, appeared in Sakhalin Island during spring migration, where some individuals stopped for breeding. Settling of the island was accompanied by similar intergenomic recombination and rapid fixation of a new recombinant haplotype with its subsequent spread across Sakhalin. The insular way of dispersal is completely repeated by modern migrants.

Striking pseudogenization in avian phylogenetics: Numts are large and common in falcons

Nuclear copies of mitochondrial genes (numts) are a well-known feature of eukaryotic genomes and a concern in systematics, as they can mislead phylogenetic inferences when inadvertently used. Studies on avian numts initially based on the chicken genome suggest that numts may be uncommon and relatively short among birds. Here we ask how common numts are in falcons, based on recently sequenced genomes of the Saker falcon (Falco cherrug) and Peregrine falcon (F. peregrinus). We identified numts by BLASTN searches and then extracted CYTB, ND2 and COI sequences from them, which were then used for phylogeny inference along with several sequences from other species in Falconiformes. Our results indicate that avian numts may be much more frequent and longer than previously thought. Phylogenetic inferences revealed multiple independent nuclear insertions throughout the history of the Falconiformes, including cases of sequences available in public databases and wrongly identified as authentic mtDNA. New sequencing technologies and ongoing efforts for whole genome sequencing will provide exciting opportunities for avian numt research in the near future.

Mitochondrial Retroprocessing Promoted Functional Transfers of rpl5 to the Nucleus in Grasses.

Functional gene transfers from the mitochondrion to the nucleus are ongoing in angiosperms and have occurred repeatedly for all 15 ribosomal protein genes, but it is not clear why some of these genes are transferred more often than others nor what the balance is between DNA- and RNA-mediated transfers. Although direct insertion of mitochondrial DNA into the nucleus occurs frequently in angiosperms, case studies of functional mitochondrial gene transfer have implicated an RNA-mediated mechanism that eliminates introns and RNA editing sites, which would otherwise impede proper expression of mitochondrial genes in the nucleus. To elucidate the mechanisms that facilitate functional gene transfers and the evolutionary dynamics of the coexisting nuclear and mitochondrial gene copies that are established during these transfers, we have analyzed rpl5 genes from 90 grasses (Poaceae) and related monocots. Multiple lines of evidence indicate that rpl5 has been functionally transferred to the nucleus at least three separate times in the grass family and that at least seven species have intact and transcribed (but not necessarily functional) copies in both the mitochondrion and nucleus. In two grasses, likely functional nuclear copies of rpl5 have been subject to recent gene conversion events via secondarily transferred mitochondrial copies in what we believe are the first described cases of mitochondrial-to-nuclear gene conversion. We show that rpl5 underwent a retroprocessing event within the mitochondrial genome early in the evolution of the grass family, which we argue predisposed the gene towards successful, DNA-mediated functional transfer by generating a "pre-edited" sequence.

Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens.

Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii’s physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene.

A Nightmare for Males? A Maternally Transmitted Male-Killing Bacterium and Strong Female Bias in a Green Lacewing Population

For maternally transmitted microbes, a female-biased host sex ratio is of reproductive advantage. Here we found a strong female bias in a field population of the green lacewing, Mallada desjardinsi (Insecta; Neuroptera). This bias was attributed to the predominance of individuals harboring a maternally inherited male-killing bacterium that was phylogenetically closely related to the plant-pathogenic Spiroplasma phoeniceum and Spiroplasma kunkelii. Among 35 laboratory-reared broods produced by wild-caught females, 21 broods (60%)—all infected with Spiroplasma—consisted of only females (940 individuals). Among 14 broods consisting of both males and females (516 and 635 individuals, respectively), 4 broods were doubly infected with Spiroplasma and Rickettsia, 6 broods were singly infected with Rickettsia, and 3 broods were uninfected (remaining one brood was unknown). Mortality during embryonic and larval development was prominent in all-female broods but not in normal sex ratio broods. Following antibiotic treatment on all-female broods, mortality was significantly reduced and the sex ratio was restored to 1:1. Strong expression and high prevalence of this male-killer is remarkable considering its low density (~10−5–10−4 cells per host mitochondrial gene copy based on quantitative PCR). In addition, a bacterium closely related to Rickettsia bellii was present in 25 of 34 broods (73.5%), irrespective of the sex ratio, with the infection density comparable to other cases of endosymbiosis (~10−2–10−1 cells per mitochondrial gene copy). Higher density of Rickettsia than Spiroplasma was also demonstrated by electron microscopy which visualized both Spiroplasma-like cells and Rickettsia-like cells inside and outside the ovarian cells.

Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithioidea, Pleuroceridae)

Pronounced mitochondrial heterogeneity within putative species of Pleuroceridae has prevented meaningful systematic revisions of this critically imperilled freshwater family. Previous studies have demonstrated that this mitochondrial diversity often produces polyphyletic species on mitochondrial gene trees, but its significance is unclear. Hypotheses advanced to explain this pattern have included cryptic species, retained ancestral polymorphisms and introgression; other possible explanations such as doubly uniparental inheritance or the presence of pseudogenes have not been given due consideration. Previous analyses have not included adequate sampling, neither in terms of number of individuals nor in geographic coverage, to adequately test any of these hypotheses. To fully characterize mitochondrial heterogeneity in pleurocerids and robustly assess possible causal explanations, we collected 239 individuals representing four putative species from seven sites and sequenced the COI and 16S rRNA mitochondrial genes and the H3 nuclear gene for all individuals. We also used whole‐genome shotgun sequencing to construct and annotate a mitochondrial genome for one individual. Characters with demonstrated utility in morphospecies delineation of gastropods (head–foot coloration, shell and radular morphology, pallial oviduct anatomy) were examined for a subset of individuals to determine whether morphology co‐varied by haplotype clade. We found pronounced mitochondrial heterogeneity at both the population level and species level in three of the species examined, but our data reject paralogous nuclear copies of mitochondrial genes (NUMTs) and doubly uniparental inheritance as causal mechanisms, and there was no evidence of cryptic diversity. Mutation rates were found to differ significantly among mitochondrial lineages, and population genetic statistics revealed a signature of balancing selection that could be acting to maintain this diversity. The observed pattern is similar to that seen in lineages with inherited endosymbionts like Wolbachia infections, which merits further investigation. Although questions remain concerning the precise cause(s) of intraspecific mitochondrial diversity in pleurocerids, nuclear and/or genomic data, combined with anatomical and life history investigations in an integrative phylogenetic context, is the most promising avenue for resolving pleurocerid systematics.

Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription.