Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome

INTRODUCTIONNuclearpseudogenesofmitochondrial(mt)DNAwereinitiallydiscoveredintheearly80's[1--6].However,mechanismsforthegenerationofmtDNApseudogenesarestillnotclearandmayvaryindifferentcases.BothRNA--[7--8]andDNAmediated[9--11]processeshavebeensugges...     

Rooting phylogenetic trees with distant outgroups: a case study from the commelinoid monocots

Phylogenetic rooting experiments demonstrate that two chloroplast genes from commelinoid monocot taxa that represent the closest living relatives of the pickerelweed family, Pontederiaceae, retain measurable signals regarding the position of that family's root. The rooting preferences of the chloroplast sequences were compared with those for artificial sequences that correspond to outgroups so divergent that their signal has been lost completely. These random sequences prefer the three longest branches in the unrooted ingroup topology and do not preferentially root on the branches favored by real outgroup sequences. However, the rooting behavior of the artificial sequences is not a simple function of branch length. The random outgroups preferentially root on long terminal ingroup branches, but many ingroup branches comparable in length to those favored by random sequences attract no or few hits. Nonterminal ingroup branches are generally avoided, regardless of their length. Comparisons of the ease of forcing sequences onto suboptimal roots indicate that real outgroups require a substantially greater rooting penalty than random outgroups for around half of the least-parsimonious candidate roots. Although this supports the existence of nonrandomized signal in the real outgroups, it also indicates that there is little power to choose among the optimal and nearly optimal rooting possibilities. A likelihood-based test rejects the hypothesis that all rootings of the subtree using real outgroup sequences are equally good explanations of the data and also eliminates around half of the least optimal candidate roots. Adding genes or outgroups can improve the ability to discriminate among different root locations. Rooting discriminatory power is shown to be stronger, in general, for more closely related outgroups and is highly correlated among different real outgroups, genes, and optimality criteria.     

Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa

Although much progress has been made recently in teleostean phylogeny, relationships among the main lineages of the higher teleosts (Acanthomorpha), containing more than 60% of all fish species, remain poorly defined. This study represents the most extensive taxonomic sampling effort to date to collect new molecular characters for phylogenetic analysis of acanthomorph fishes. We compiled and analyzed three independent data sets, including: (i) mitochondrial ribosomal fragments from 12S and 16s (814bp for 97 taxa); (ii) nuclear ribosomal 28S sequences (847bp for 74 taxa); and (iii) a nuclear protein-coding gene, rhodopsin (759bp for 86 taxa). Detailed analyses were conducted on each data set separately and the principle of taxonomic congruence without consensus trees was used to assess confidence in the results as follows. Repeatability of clades from separate analyses was considered the primary criterion to establish reliability, rather than bootstrap proportions from a single combined (total evidence) data matrix. The new and reliable clades emerging from this study of the acanthomorph radiation were: Gadiformes (cods) with Zeioids (dories); Beloniformes (needlefishes) with Atheriniformes (silversides); blenioids (blennies) with Gobiesocoidei (clingfishes); Channoidei (snakeheads) with Anabantoidei (climbing gouramies); Mastacembeloidei (spiny eels) with Synbranchioidei (swamp-eels); the last two pairs of taxa grouping together, Syngnathoidei (aulostomids, macroramphosids) with Dactylopteridae (flying gurnards); Scombroidei (mackerels) plus Stromatoidei plus Chiasmodontidae; Ammodytidae (sand lances) with Cheimarrhichthyidae (torrentfish); Zoarcoidei (eelpouts) with Cottoidei; Percidae (perches) with Notothenioidei (Antarctic fishes); and a clade grouping Carangidae (jacks), Echeneidae (remoras), Sphyraenidae (barracudas), Menidae (moonfish), Polynemidae (threadfins), Centropomidae (snooks), and Pleuronectiformes (flatfishes).     

Nuclear mitochondrial pseudogenes in Austinograea alayseae hydrothermal vent crabs (Crustacea: Bythograeidae): effects on DNA barcoding

Members of the brachyuran crab family, Bythograeidae, are among the most abundant and common crabs in vent fields. However, their identification based on morphological characteristics often leads to incorrect species recognition due to a lack of taxonomic factors and the existence of sibling (or cryptic) species. For these reasons, we used DNA barcoding for vent crabs using mitochondrial cytochrome c oxidase subunit 1 (CO1). However, several nuclear mitochondrial pseudogenes (Numts) were amplified from Austinograea alayseae Guinot, 1990, using universal primers (Folmer primers). The Numts were characterized in six haplotypes, with 13.58–14.11% sequence divergence from A. alayseae, a higher nonsynonymous substitution ratio than true CO1, and the formation of an independent clade in bythograeids. In a neighbour‐joining tree, the origin of the Numts would be expected to incorporate into the nucleus at an ancestral node of Austinograea, and they mutated more slowly in the nucleus than CO1 in the mitochondria. This evolutionary process may have resulted in the higher binding affinity of Numts for the Folmer primers than CO1. In the present study, we performed long PCR for the amplification of CO1 in A. alayseae. We also present evidence that Numts can introduce serious ambiguity into DNA barcoding, including overestimating the number of species in bythograeids. These results may help in conducting taxonomic studies using mitochondrial genes from organisms living in hydrothermal vent fields.     

Nuclear mtDNA pseudogenes as a source of new variants of mitochondrial genes: A case study of Siberian rubythroat <Emphasis Type="Italic">Luscinia calliope</Emphasis> (muscicapidae,aves)

First evidence for the presence of copies of mitochondrial cytochrome b gene of the subspecies group Luscinia calliope anadyrensis–L. c. camtschatkensis in the nuclear genome of nominative L. c. calliope was obtained, which indirectly indicates the nuclear origin of the subspecies-specific mitochondrial haplotypes in Siberian rubythroat. This fact clarifies the appearance of mitochondrial haplotypes of eastern subspecies by exchange between the homologous regions of the nuclear and mitochondrial genomes followed by fixation by the founder effect. This is the first study to propose a mechanism of DNA fragment exchange between the nucleus and mitochondria (intergenomic recombination) and to show the role of nuclear copies of mtDNA as a source of new taxon-specific mitochondrial haplotypes, which implies their involvement in the microevolutionary processes and morphogenesis.     

Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes

The utility of mitochondrial DNA as a molecular marker for evolutionary studies is well recognized. However, several problems can arise when using mitochondrial DNA, one of which is the presence of nuclear mitochondrial pseudogenes, or Numts. Pseudogenes of cytochrome oxidase I were preferentially amplified from Acanthocephalus lucii (Acanthocephala) using a universal PCR approach. To verify the presence and abundance of pseudogenes, length heterogeneity analysis of the PCR fragments was performed. PCR products obtained with universal primers often contained fragments of different sizes. Cloned sequences from universal PCR products nearly always contained sequence abnormalities such as indels and/or stop codons. Based on these sequences, new primers were developed to specifically target mitochondrial DNA. Sequences obtained with these specific primers lacked abnormalities. Phylogenetic analysis produced a single most parsimonious tree in which pseudogenes obtained with universal primers grouped together as did putative mitochondrial DNA sequences obtained with specific primers. The pattern of codon bias observed in the pseudogenes suggests a single nuclear integration event from the mitochondria. This is the first reported occurrence of pseudogenes in an acanthocephalan, and it demonstrates the potential dangers associated with the use of universal primers.     

First evaluation of mitochondrial DNA as a marker for phylogeographic studies of Calcarea: a case study from Leucetta chagosensis

In most animals mitochondrial DNA (mtDNA) evolves much faster than nuclear DNA. Therefore, and because of its shorter coalescent time, mitochondrial (mt) markers provide better resolution to trace more recent evolutionary events compared to nuclear DNA. But in contrast to most other Metazoa, previous studies suggested that in sponges mitochondrial sequence evolution is much slower, making mtDNA less suitable for studies at the intraspecific level. However, these observations were made in the class Demospongiae and so far no data exist for calcareous sponges (Class Calcarea). We here provide the first study that evaluates intraspecific mt sequence variation in Calcarea. We focus on arguably the best-studied species Leucetta chagosensis, for which three nuclear DNA marker datasets existed previously. We here sequenced the partial mitochondrial cytochrome oxidase subunit III gene (cox3). Our analyses reveal an unexpected variability of up to 8.5% in this mitochondrial marker. In contrast to other sponges where this marker evolves considerable slower than the nuclear internal transcribed spacer region (ITS), we found that cox3 in L. chagosensis evolves about five times as fast as ITS. The variability is similar to that of nuclear intron data of the species. The phylogeny inferred with cox3 is congruent with other markers, but separates earlier reported genetic groups much more distinctively than nuclear DNA. This provides further evidence for cryptic speciation in L. chagosensis. All these features make calcarean mtDNA exceptional among sponges and show its suitability for phylogeographic studies and potential as a species-specific (DNA barcoding) marker to distinguish morphologically identical cryptic species.     

Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes

Nuclear inserted copies of mitochondrial origin (numts) vary widely among eukaryotes, with human and plant genomes harboring the largest repertoires. Numts were previously thought to be absent from fish species, but the recent release of three fish nuclear genome sequences provides the resource to obtain a more comprehensive insight into the extent of mtDNA transfer in fishes. From the sequence analyses of the genomes of Fugu rubripes, Tetraodon nigroviridis, and Danio rerio, we have identified 2, 5, and 10 recent numt integrations, respectively, which integrated into those genomes less than 0.6 million years (Myr) ago. Such results contradict the hypothesis of absence or rarity of numts in fishes, as (i) the ratio of numts to the total size of the nuclear genome in T. nigroviridis was superior to the ratio observed in several higher vertebrate species (e.g., chicken, mouse, and rat), and only surpassed by humans, and (ii) the mtDNA coverage transferred to the nuclear genome of D. rerio is exceeded only by human and mouse, within the whole range of eukaryotic genomes surveyed for numts. Additionally, 335, 336, and 471 old numts (>12.5 Myr) were detected in F. rubripes, T. nigroviridis, and D. rerio, respectively. Surprisingly, old numts are inserted preferentially into known or predicted genes, as inferred for recent numts in human. However, because in fish genomes such integrations are old, they are likely to represent evolutionary successes and they may be considered a potential important evolutionary mechanism for the enhancement of genomic coding regions.     

Using higher taxa as surrogates of species-level data in three Portuguese protected areas: a case study on Spheciformes (Hymenoptera)

Protected areas are the focus of most conservation efforts worldwide. Despite vast amount of investment in protected areas, biodiversity loss continues. This has led to increasing efforts to develop measures to assess the effectiveness of protected areas. The reliability of these measures depends on the quality of the information collected. However, because the resources available for the collection of information are limited, several strategies have been developed to reduce the resources necessary. In this study the combination of two resource reduction approaches—bioindicator and higher-taxa—is proposed. Spheciformes have been found to be useful as biodiversity, ecological and environmental indicators. Identification to the species level is usually very costly, but the use of genus-level information has been suggested. Tribe- and genus-level data for Spheciformes were assessed for their ability to predict the number of species independently of other variables—sampling area, geographic location, vegetation type, disturbance regime, and sampling effort—at three Portuguese protected areas. Tribe and genus-level data were found to be good indicators, with genus being the more reliable taxonomic level. Sampling effort was the only external variable that affected the relationship between species and higher-taxa richness. Genus-level data were also found to be useful for ranking sites according to richness or composition, and for determining richness-based and rarity-based complementary sets of sites for conservation. Using genus richness as a surrogate for species richness seems a promising approach for monitoring and contributing to the establishment of protected areas in Portugal and the entire Mediterranean region.     

Nuclear integrations: challenges for mitochondrial DNA markers

The combined use of mitochondrial DNA markers and polymerase chain reaction (PCR) techniques has greatly enhanced evolutionary studies. These techniques have also promoted the discovery of mitochondrial-like sequences in the nuclear genomes of many animals. While the nuclear sequences themselves are interesting, and capable of serving as valuable molecular tools, they can also confound phylogenetic and population genetic studies. Clearly, a better understanding of these phenomena and vigilance towards misleading data are needed.     

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.     

线粒体假基因用作分子化石推算两个榕小蜂姐妹种的分化时间（英文）

线粒体假基因(nuclear mitochondrial pseudogenes, NUMTs)是指由生物体的线粒体基因组转移至核基因组内的DNA片段。由于其独立进化的特点, NUMTs在用于系统发育分析时是一把双刃剑。我们用基于PCR扩增的方法研究了对叶榕Ficus hispida上两姐妹种榕小蜂Philotrypesis pilosa和Philotrypesis sp.中起源于线粒体Nad1 12S片段的NUMTs。该两姐妹种榕小蜂由同域物种形成过程产生, 它们生活在同一生态环境里（即同一榕果内）, 因此可以用作很好的模型来研究在相同生态环境里物种的行为学及遗传学细微差异的进化。这些深入研究都依赖于对两个物种分化时间的正确估算。通过对所获取的NUMTs进行进化分析, 我们发现： 1）这些NUMTs都是最近引入核基因组事件; 2）NUMTs引入事件发生在物种分化之前。由于这些NUMTs引入核内时间尚短, 其碱基替换速率与线粒体基因相似, 而节肢动物线粒体基因的平均碱基替换速率约为2.3×10^-8 替换/位点·年。根据这些进化历史特征可帮助我们将这两个姐妹种榕小蜂的分化时间追溯至0.40-0.48百万年以前。结果提示, 一些线粒体假基因可以很好的用作分子化石来推断一些重要进化事件如物种形成。     

Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: a case study from Naucleeae (Rubiaceae)

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of previous studies therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

Tetraphenylphosphonium-selective electrode as a tool for evaluating mitochondrial permeability transition pore function in isolated rat hepatocytes

The changes in mitochondrial membrane potential (Deltapsi(m)) were used as an indicator for evaluating the mitochondrial permeability transition pore (MPTP) function. We found that in situ mitochondria in digitonin-permeabilized hepatocytes were coupled and responded to the addition of substrates, inhibitors and uncouplers. Ca(2+)-induced Deltapsi(m) dissipation was caused by MPTP opening because this process was inhibited by cyclosporin A. MPTP opening was enhanced by the pro-oxidant tert-butyl hydroperoxide.     

Conservation strategies for poorly surveyed taxa: a dung beetle (Coleoptera, Scarabaeidae) case study from southern Africa

Despite being the focus of an international research effort spanning decades, the spatial distribution of southern African scarab beetles remains poorly documented. As well as reinforcing the magnitude of the challenge facing biodiversity scientists, this raises real concerns about best practice conservation strategies in the absence of detailed distribution information. However, dung beetles appear to be well represented in established conservation areas. This apparent contradiction could be ascribed to anthropogenic transformation, successful conservation efforts, the presence of dung generalists and reserve-biased or mesic-biased dung beetle collection efforts. It is suggested that all of the above contribute to the observed pattern to varying degrees. The implications of selecting areas that are either rich in species, contain rare species or contain taxonomically distinct species from a group whose taxonomy is well known but for which inadequate distribution data exist are explored. Best practice, in the face of inadequate data, appears to revolve around a subtle interplay between advantages and disadvantages associated with data interpolation techniques, reserve selection algorithms that use criteria more robust than database rarity (such as taxonomic distinctiveness) and the long-term economic costs of proceeding with the data at hand versus investing in biological surveys.     

Isolated populations or isolated taxa? A case study in narrowly-distributed snapdragons (Antirrhinum sect. Sempervirentia) using RAPD markers

RAPD fingerprinting was used to study species boundaries in narrowly distributed endemic species in Antirrhinum section Sempervirentia. Based on RAPD data, similarity values within species were relatively high but pair-wise similarity values among species were low. Partitioning of the overall RAPD variation using AMOVA showed that most of the variation was found among species (58.06%), whereas the remaining phenotypic diversity was distributed among populations (25.18%) and among individuals within populations (16.76%). Comparison of the matrices of geographical distances and phenetic distances (1-Dice index) among populations using the Mantel test showed a moderate, but statistically significant correlation (r=0.588, P < 0.01), suggesting that isolation by distance is responsible for the distribution of genetic variation among Antirrhinum populations. Phenetic relationships among Antirrhinum samples based on a Dice similarity matrix showed a clear taxonomic pattern, confirming the grouping of individuals within their own populations and the clustering of populations within species. Individuals of A. charidemi, A. valentinum and A. subbaeticum, from subsection Valentina, made up a discrete group, whereas the samples belonging to subsection Sempervirentia (A. petegasii, A. sempervirens, A. microphyllum, A. pulverulentum) clustered together. RAPD data are entirely congruent with the subsection classification scheme proposed by Fernández Casas (1997) in section Sempervirentia. However, A. subbaeticum, treated as a synonym of A. valentinum by Fernández-Casas (1997), showed an unique RAPD profile characterized by the highest number of fixed species-specific markers found in section Sempervirentia. Thus, although A. valentinum appeared the most closely related species to A. subbaeticum, molecular data suggested that this species merits taxonomic distinction.