De-Xing Zhang

Mitochondrial pseudogenes: evolution's misplaced witnesses

Nuclear copies of mitochondrial DNA (mtDNA) have contaminated PCR-based mitochondrial studies of over 64 different animal species. Since the last review of these nuclear mitochondrial pseudogenes (Numts) in animals, Numts have been found in 53 of the species studied. The recent evidence suggests that Numts are not equally abundant in all species, for example they are more common in plants than in animals, and also more numerous in humans than in Drosophila. Methods for avoiding Numts have now been tested, and several recent studies demonstrate the potential utility of Numt DNA sequences in evolutionary studies. As relics of ancient mtDNA, these pseudogenes can be used to infer ancestral states or root mitochondrial phylogenies. Where they are numerous and selectively unconstrained, Numts are ideal for the study of spontaneous mutation in nuclear genomes.

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.

Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects.

Population‐genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population‐genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above‐mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele‐discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.

Insect mitochondrial control region: A review of its structure, evolution and usefulness in evolutionary studies

Abstract The control region is the only major non-coding region in the mitochondrial genome of insects. It is heavily biased to A+T nucleotides and seems to evolve under a strong directional mutation pressure. Among insects, this region is variable in both size and nucleotide sequence and may contain tandem repetition which is often associated with heteroplasmy. Tandem repetition appears to undergo concerted evolution and copy number variation indicates a high mutation rate. In contrast, the nucleotide substitution rate in this region is likely to be much reduced due to high A+T content and directional mutation pressure. Insect mitochondrial control regions are not necessarily the most variable region in the genome in terms of nucleotide substitution, and may not evolve faster than single-copy nuclear non-coding sequences. These observations have implications for the use of this region as a genetic marker in evolutionary studies. Contrary to earlier expectation, this region may have limited usefulness for both inter- and intra-specific analyses, depending on the structure and evolutionary patterns of a particular sequence. As some structural elements have been observed to be highly conserved between phylogenetically very distant insect taxa, it is of great interest to study the molecular evolution of this region in the entire class, Insecta.

Evolution and structural conservation of the control region of insect mitochondrial DNA

The control regions of mitochondrial DNA of two insects, Schistocerca gregaria and Chorthippus parallelus, have been isolated and sequenced. Their sizes are 752 by and 1,512 bp, respectively, with the presence of a tandem repeat in C. parallelus. (The sequences of the two repeats are highly conserved, having a homology of 97.5%.) Comparison of their nucleotide sequences revealed the presence of several conserved sequence blocks dispersed through the whole control region, showing a different evolutionary pattern of this region in these insects as compared to that in Drosophila. A highly conserved secondary structure, located in the 3′ region near the small rRNA gene, has been identified. Sequences immediately flanking this hairpin structure rather than the sequences of this structure themselves are conserved between S. gregaria/C. parallelus and Drosophila, having a sequence consensus of “TATA” at 5′ and “GAA(A)T” at 3′. The motif “G(A)nT” is also present in the 3′ flanking sequences of mammalian, amphibian, and fish mitochondrial L-strand replication origins and a potential plant mitochondrial second-strand-replication origin, indicating its universal conservation and functional importance related to replication origins. The stem-and-loop structure in S. gregaria/C. parallelus appears to be closely related to that found in Drosophila despite occupying a different position, and may be potentially associated with a second-strand-replication origin. This in turn suggests that such a secondary structure might be widely conserved across invertebrates while their location in the control region may be variable. We have looked for such a conserved structure in the control regions of two other insects, G. firmus and A. mellifera, whose DNA sequences have been published, and their possible presence is discussed.Mitochondrial control regions characterized to date in five different insect taxa (Drosophila, G. firmus, A. mellifera, S. gregaria, and C. parallelus) may be classed into two distinct groups having different evolutionary patterns. It is observed that tandem repetition of regions containing a probable replication origin occurred in some species from disjunct lineages in both groups, which would be the result of convergent evolution. We also discuss the possibility of a mechanism of “parahomologous recombination by unequal crossing-over” in mitochondria, which can explain the generation of such tandemly repeated sequences (especially the first critical repetition) in the control region of mtDNA, and also their convergent evolution in disjunct biological lineages during evolution.

Assessment of the universality and utility of a set of conserved mitochondrial COI primers in insects

A set of mitochondrial COI primers has been studied by genomic PCR and many primer combinations shown to work universally well across Insects. They are able to amplify various amplicons with different variability which enables the selection of a particular amplicon as a suitable DNA marker for a project. The potential usefulness of different amplicons is examined, with analysis on published study cases employing these regions. With respect to their variability, amplicons UEA5/UEA6, UEA7/UEA8 and UEA5/UEA8 could be useful for low‐ to mid‐level phylogenetic analysis, i. e. from species, genus to‐perhaps family level depending on taxa involved. UEA5/UEA6 will be too conserved for intraspecific studies. Amplicons UEA3/UEA4 and UEA9/UEA10 would be better suited to low‐level phylogenetic investigations, such as analysis of relationships among closely related species and population genetic studies. However, these guidelines should not be over‐generalized for the reasons given. Amplification conditions of various primer combinations, and general problems in the use of conserved PCR primers are discussed.

Highly conserved nuclear copies of the mitochondrial control region in the desert locust Schistocerca gregaria: some implications for population studies

Animal mitochondrial DNA has proved a valuable marker in intraspecific systematic studies. However, if nucleotide sequence heterogeneity exists at the individual level, its usefulness will be much reduced. This study demonstrates that the presence of highly conserved non‐coding mitochondrial sequences in the nuclear genome of Schistocerca gregaria greatly impairs the use of mtDNA in population genetic studies. Caution is called for in other organisms; and it seems necessary to check for conserved nuclear copies of mitochondrial sequences before launching into a large scale analysis of populations using mtDNA as a genetic marker. Experimental procedures are suggested for this purpose.

Measuring population differentiation using GST or D? A simulation study with microsatellite DNA markers under a finite island model and nonequilibrium conditions

The genetic differentiation of populations is a key parameter in population genetic investigations. Wright’s FST (and its relatives such as GST) has been a standard measure of differentiation. However, the deficiencies of these indexes have been increasingly realized in recent years, leading to some new measures being proposed, such as Jost’s D (Molecular Ecology, 2008; 17, 4015). The existence of these new metrics has stimulated considerable debate and induced some confusion on which statistics should be used for estimating population differentiation. Here, we report a simulation study with neutral microsatellite DNA loci under a finite island model to compare the performance of GST and D, particularly under nonequilibrium conditions. Our results suggest that there exist fundamental differences between the two statistics, and neither GST nor D operates satisfactorily in all situations for quantifying differentiation. D is very sensitive to mutation models but GST noticeably less so, which limits D’s utility in population parameter estimation and comparisons across genetic markers. Also, the initial heterozygosity of the starting populations has some important effects on both the individual behaviours of GST and D and their relative behaviours in early differentiation, and this effect is much greater for D than GST. In the early stages of differentiation, when initial heterozygosity is relatively low (<0.5, if the number of subpopulations is large), GST increases faster than D; the opposite is true when initial heterozygosity is high. Therefore, the state of the ancestral population appears to have some lasting impacts on population differentiation. In general, GST can measure differentiation fairly well when heterozygosity is low whatever the causes; however, when heterozygosity is high (e.g. as a result of either high mutation rate or high initial heterozygosity) and gene flow is moderate to strong, GST fails to measure differentiation. Interestingly, when population size is not very small (e.g. N ≥ 1000), GST measures differentiation quite linearly with time over a long duration when gene flow is absent or very weak even if mutation rate is not low (e.g. μ = 0.001). In contrast, D, as a differentiation measure, performs rather robustly in all these situations. In practice, both indexes should be calculated and the relative levels of heterozygosities (especially HS) and gene flow taken into account. We suggest that a comparison of the two indexes can generate useful insights into the evolutionary processes that influence population differentiation.

Unexpected relationships of substructured populations in Chinese Locusta migratoria.

BackgroundHighly migratory species are usually expected to have minimal population substructure because strong gene flow has the effect of homogenizing genetic variation over geographical populations, counteracting random drift, selection and mutation. The migratory locust Locusta migratoria belongs to a monotypic genus, and is an infamous pest insect with exceptional migratory ability – with dispersal documented over a thousand kilometers. Its distributional area is greater than that of any other locust or grasshopper, occurring in practically all the temperate and tropical regions of the eastern hemisphere. Consequently, minimal population substructuring is expected. However, in marked contrast to its high dispersal ability, three geographical subspecies have been distinguished in China, with more than nine being biologically and morphologically identified in the world. Such subspecies status has been under considerable debate.ResultsBy multilocus microsatellite genotyping analysis, we provide ample genetic evidence for strong population substructure in this highly migratory insect that conforms to geography. More importantly, our genetic data identified an unexpected cryptic subdivision and demonstrated a strong affiliation of the East China locusts to those in Northwest/Northern China. The migratory locusts in China formed three distinct groups, viz. (1) the Tibetan group, comprising locusts from Tibet and nearby West China high mountain regions; this is congruent with the previously recognized Tibetan subspecies, L. m. tibetensis; (2) the South China group, containing locusts from the Hainan islands; this corresponds to the Southeast Asia oriental tropical subspecies L. m. manilensis; (3) the North China group, including locusts from the Northwest and Northern China (the Asiatic subspecies L. m. migratoria), Central China and Eastern China regions. Therefore, the traditional concept on Locusta subspecies status established from Uvarov in 1930s needs to be revised. The three groups of locusts probably have separate evolutionary histories that were most likely linked to Quaternary glaciations events, and derived from different ancestral refugial populations following postglacial expansions.ConclusionThe migratory locust populations in China have differentiated into three genetically distinct groups despite high dispersal capability. While this clarified long-standing suspicions on the subspecific diversification of this species in China, it also revealed that the locusts in the vast area of East China are not the oriental subspecies but the Asiatic subspecies, an unexpected substructuring pattern. The distribution pattern of the three locust groups in China may be primarily defined by adaptive differentiation coupled to Quaternary glaciations events. Our results are of general significance both for locust research and for phylogeographical study of flora and fauna in China, illustrating the potential importance of phylogeographical history in shaping the divergence and distribution patterns of widespread species with strong dispersal ability.

Isolation of Animal Cellular Total DNA

A general method for the isolation of total DNA from animal tissue is described below. It is modified from the original procedure described by Gross-Bellard et al. (1) using proteinase K, SDS and phenol/chloroform. First, tissue or the whole body of a small organism is rapidly frozen in liquid nitrogen and ground to a fine powder. The processed tissue is then suspended in an extraction buffer containing proteinase K, SDS and EDTA which are used to digest cellular proteins or inactivate nucleases. By successive phenol/chloroform extractions, the mixture containing DNA is deproteinized and the DNA finally recovered by ethanol precipitation.