Rates of single-copy DNA evolution in herons

DNA-DNA hybridization was used to discover the extent of single-copy DNA similarity among 13 species of herons and one ibis. Genetic distances among taxa were summarized as Tm values in a folded matrix. From this matrix, trees with the same branching pattern were constructed by least squares under one of two assumptions: (1) that sister branches are equal in length and (2) that sister branches are not necessarily equal in length. The residual sums of squares of these trees were compared by F-test to see whether the branches of the tree built under assumption (2) fit the matrix data significantly better than those of the tree built under assumption (1). By this method the existence of different rates of DNA evolution in different heron lineages was established. Bittern single-copy DNA has evolved at a rate approximately 25% faster, and boat-billed heron (Cochearius) and rufescent tiger heron (Tigrisoma lineatum) DNA has evolved approximately 19% slower, than that of day and night herons. It appears that the differences in rates of DNA evolution may increase proportionally with genealogical distance.      

Rates of single-copy DNA evolution in phalangeriform marsupials

DNA/DNA hybridization was used to investigate the relationships of taxa representing the phalangeriform marsupial families Acrobatidae, Burramyidae, Macropodidae, Petauridae, Phalangeridae, and Pseudocheiridae and (as an outgroup) the bandicoot family Peramelidae. In the course of this, a marked rate slowdown was noted in the burramyid lineage represented by Cercartetus caudatus; ANOVA (with Tukey's test) and F-ratio tests of both corrected and uncorrected data matrices confirmed this rate disparity. As burramyids are small, short-generation-time phalangeriforms, these data present a striking counterexample to the common view that rates of change in DNA sequences are inversely correlated with generation time.     

Rapid assessment of single-copy nuclear DNA variation in diverse species

We investigated the use of PCR primers designed to conserved exons within nuclear DNA to amplify potentially variable regions such as introns or hypervariable exons from a wide range of species. We then explored various approaches to assay population-level variation in these PCR products. Primers designed to amplify regions within the histone H2AF, myoglobin , MHC DQA , and aldolase (ALD) genes gave clean amplifications in diverse mammals (DQA) , and in birds, reptiles and mammals ( aldolase, H2AF, myoglobin ). The sequenced PCR products generally, but not always, confirmed that the correct locus had been amplified. Several primer sets produced smaller size fragments consistent with preferential amplification of intronless pseudogenes; this was confirmed by sequencing seal and reptile H2AF PCR products. Digestion with randomly selected four-base recognizing enzymes detected variation in some cases but not in others. In species/gene combinations with either low (e.g. seal H2AF, ALD-A ) or high (e.g. skink ALD-1 ) nucleotide diversity it was more efficient to sequence a small number of distantly related individuals (e.g. one per geographic population) and from these data to identify informative or potentially informative restriction enzymes for 'targeted' digestion. We conclude that for studies of population-level variation, the optimal approach is to use a battery of primers for initial PCR of both mtDNA and scnDNA loci, select those that give clean amplifications, and sequence one sample from each population to (i) confirm gene identity, (ii) estimate the amount of variation and, (iii) search for diagnostic restriction sites. This will allow determination of the most efficient approach for a large-scale study.     

Reconstructing recent human evolution.

The two most distinct models of recent human evolution, the multiregional and the recent African origin models, have different retrodictions concerning specific archaic-recent population relationships. The former model infers multiple regional archaic-modern connections and the ancient establishment of regional characteristics, whereas the latter model implies only an African archaic-all modern relationship, with recent (late Pleistocene) development of regionality. In this paper, four late archaic groups from Europe, southwest Asia, Africa and East Asia are compared with various fossil and recent Homo sapiens crania or cranial samples. The results of Penrose shape comparisons narrowly favour a late archaic African-modern special relationship over an East Asian-modern one, with European and southwest Asian Neanderthal groups much more distant. No specific archaic-recent regional relationships are indicated in the shape analyses, nor in separate examinations of patterns of regionality, which indicate a recent origin for present day regionality. The Skhul-Qafzeh sample provides an excellent shape intermediate between the archaic and recent samples.     

Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure

Traditional classifications of agaric fungi involve gross morphology of their fruit bodies and meiospore print-colour. However, the phylogeny of these fungi and the evolution of their morphological and ecological traits are poorly understood. Phylogenetic analyses have already demonstrated that characters used in traditional classifications of basidiomycetes may be heavily affected by homoplasy, and that non-gilled taxa evolved within the agarics several times. By integrating molecular phylogenetic analyses including domains D1–D3 and D7–D8 of nucLSU rDNA and domains A–C of the RPB1 gene with morphological and chemical data from representative species of 88 genera, we were able to resolve higher groups of agarics. We found that the species with thick-walled and pigmented basidiospores constitute a derived group, and hypothesize that this specific combination of characters represents an evolutionary advantage by increasing the tolerance of the basidiospores to dehydration and solar radiation and so opened up new ecological niches, e.g. the colonization of dung substrates by enabling basidiospores to survive gut passages through herbivores. Our results confirm the validity of basidiospore morphology as a phylogenetic marker in the agarics.     

Reconstructing reticulate evolution in species-theory and practice.

We present new methods for reconstructing reticulate evolution of species due to events such as horizontal transfer or hybrid speciation; both methods are based upon extensions of Wayne Maddison's approach in his seminal 1997 paper. Our first method is a polynomial time algorithm for constructing phylogenetic networks from two gene trees contained inside the network.We allow the network to have an arbitrary number of reticulations, but we limit the reticulation in the network so that the cycles in the network are node-disjoint ("galled"). Our second method is a polynomial time algorithm for constructing networks with one reticulation, where we allow for errors in the estimated gene trees. Using simulations, we demonstrate improved performance of this method over both NeighborNet and Maddison's method.     

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics

This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.      

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Generating single-copy nuclear gene data for a recent adaptive radiation

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.     

Allopolyploid origin of Cardamine asarifolia (Brassicaceae): incongruence between plastid and nuclear ribosomal DNA sequences solved by a single-copy nuclear gene

Interspecific hybridization and polyploidization have played central roles in plant diversification. However, technical difficulties in the analyses of low-copy genes have limited the study of the origins of hybrid and polyploid plants. Here, we present a phylogenetic analysis of the hexaploid Cardamine asarifolia, distributed in the southern European Alps and northern Apennines. Our study included all relevant taxa of the genus found in Europe. A marked discrepancy was revealed between the trnL-trnF region of cpDNA and internal transcribed spacer (nrDNA ITS) sequences. To solve the incongruence, we sequenced a single-copy nuclear CHS gene (chalcone synthase) using a novel method to design homoeologue-specific PCR primers to bypass artefacts caused by artificial recombination of homoeologues during PCR and/or cloning. Three homoeologues were isolated from C. asarifolia, providing evidence for its allopolyploid origin. One homoeologue, showing the same phylogenetic position as the ITS sequences, most likely originated from an extinct parent. Furthermore, we documented recurrent polytopic hybridizations between C. asarifolia and diploid C. amara. The allohexaploidization and the following hybridization with a diploid species exemplify the ongoing dynamic processes of speciation in the genus Cardamine.     

Quantitative nuclear DNA differences associated with genome evolution in Guizotia (Compositae)

The present communication deals with 2C nuclear genome size variation in a fairly small genus Guizotia. Twenty-four accessions belonging to six species, out of seven known, were analysed in order to elucidate the extent of DNA variation both at an intra—as well as interspecific level. At the intraspecific level none of the species exhibited significant differences in their genome size. Between the species, the 2C DNA amounts ranged from 3.61 pg in G. reptans to 11.37 pg in G. zavattarii; over three-fold DNA variation is evident. Apparently these interspecific DNA differences have been achieved independent of the numerical chromosomal change(s), as all the Guizotias share a common chromosome number 2n=2x=30. The cultivated oilseed crop, G. abyssinica (7.57 pg), has accommodated nearly 78% extra DNA in its chromosome complement during the evolutionary time scale of its origin and domestication from the wild progenitor G. schimperi (4.25 pg). The extent of genomic DNA difference(s) between the species has been discussed in the light of their interrelationships and diversity.     

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.     

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.     

Using mitochondrial and nuclear DNA markers to reconstruct human evolution

Molecular genetic data have greatly improved our ability to test hypotheses about human evolution. During the past decade, a large amount of nuclear and mitochondrial data have been collected from diverse human populations. Taken together, these data indicate that modern humans are a relatively young species. African populations show the largest amount of genetic diversity, and they are the most genetically divergent population. Modern human populations expanded in size first on the African continent. These findings support a recent African origin of modern humans, but this conclusion should be tempered by the possible effects of factors such as gene flow, population size differences, and natural selection. BioEssays 20:126–136, 1998. © 1998 John Wiley & Sons, Inc.     

单拷贝核基因在昆虫分子系统学中的应用

单拷贝核基因(single-copy nuclear gene,scnDNA)是指基因组中拷贝数目少,只有1个或几个拷贝的核基因,大多数是属于生物体内组成型表达的持家基因。单拷贝核基因是分子系统学中的一种极有价值的分子标记,在构建生命树的主干及主干和末梢之间的中间分枝中将起极其重要的作用。文章主要介绍了单拷贝核基因在昆虫分子系统学中的应用情况、一些单拷贝核基因的特点以及应用单拷贝核基因研究昆虫分子系统学时存在的问题及注意事项。     

Untangling the hybrid origin of the Chinese tea roses: evidence from DNA sequences of single-copy nuclear and chloroplast genes

The tea-scented China roses largely correspond to the three recognized double-petaled Rosa odorata (Andrews) Sweet (Rosoideae, Rosaceae) varieties, which are the ancestors of modern hybrid tea roses and had a definite and permanent influence on the evolution of modern garden roses. Here the hypothesis of a hybrid origin of the tea-scented China roses between R. odorata var. gigantea and R. chinensis was tested. Two single-copy nuclear genes of the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the chloroplast-expressed glutamine synthetase (ncpGS) together with two plastid loci (trnL-F and psbA-trnH) were sequenced for representative accessions of four R. odorata varieties, R. chinensis, and 28 other Rosa species. Phylogenetic relationships were estimated from two nuclear loci using maximum parsimony and Bayesian analyses, and a haplotype network was constructed on the combined plastid data using NETWORK. For GAPDH and ncpGS loci, the clonal sequences of the three double-petaled varieties were clustered into two clades, one clade with R. odorata var. gigantea, and the other with partial sequences of R. chinensis, which suggested that the tea-scented China roses were hybrids between R. odorata var. gigantea and R. chinensis. Two plastid loci suggested that R. odorata var. gigantea could be the maternal parent and R. chinensis the paternal parent.     

Reconstructing cranial evolution in an extinct hominin

Homo erectus is the first hominin species with a truly cosmopolitan distribution and resembles recent humans in its broad spatial distribution. The microevolutionary events associated with dispersal and local adaptation may have produced similar population structure in both species. Understanding the evolutionary population dynamics of H. erectus has larger implications for the emergence of later Homo lineages in the Middle Pleistocene. Quantitative genetics models provide a means of interrogating aspects of long-standing H. erectus population history narratives. For the current study, cranial fossils were sorted into six major palaeodemes from sites across Africa and Asia spanning 1.8–0.1 Ma. Three-dimensional shape data from the occipital and frontal bones were used to compare intraspecific variation and test evolutionary hypotheses. Results indicate that H. erectus had higher individual and group variation than Homo sapiens, probably reflecting different levels of genetic diversity and population history in these spatially disperse species. This study also revealed distinct evolutionary histories for frontal and occipital bone shape in H. erectus, with a larger role for natural selection in the former. One scenario consistent with these findings is climate-driven facial adaptation in H. erectus, which is reflected in the frontal bone through integration with the orbits.     

Reconstructing patterns of reticulate evolution in plants

Until recently, rigorously reconstructing the many hybrid speciation events in plants has not been practical because of the limited number of molecular markers available for plant phylogenetic reconstruction and the lack of good, biologically based methods for inferring reticulation (network) events. This situation should change rapidly with the development of multiple nuclear markers for phylogenetic reconstruction and new methods for reconstructing reticulate evolution. These developments will necessitate a much greater incorporation of population genetics into phylogenetic reconstruction than has been common. Population genetic events such as gene duplication coupled with lineage sorting and meiotic and sexual recombination have always had the potential to affect phylogenetic inference. For tree reconstruction, these problems are usually minimized by using uniparental markers and nuclear markers that undergo rapid concerted evolution. Because reconstruction of reticulate speciation events will require nuclear markers that lack these characteristics, effects of population genetics on phylogenetic inference will need to be addressed directly. Current models and methods that allow hybrid speciation to be detected and reconstructed are discussed, with a focus on how lineage sorting and meiotic and sexual recombination affect network reconstruction. Approaches that would allow inference of phylogenetic networks in their presence are suggested.