DNA/DNA hybridization studies of the carnivorous marsupials. I: The intergeneric relationships of bandicoots (Marsupialia: Perameloidea)

Summary A complete suite of comparisons among six bandicoot species and one outgroup marsupial was generated using the hydroxyapatite chromatography method of DNA/DNA hybridization; heterologous comparisons were also made with three other bandicoot taxa. Matrices of T_m's, modes, and T₅₀Hs were generated and corrected for nonreciprocity, homoplasy, and, in the case of T_m's, normalized percent hybridization; these matrices were analyzed using the FITCH algorithm in Felsenstein's PHYLIP (version 3.1). Uncorrected and nonreciprocity-corrected matrices were also jackknifed and analyzed with FITCH to test for consistency. Finally, sample scores for T_m, mode, and T₅₀H matrices were bootstrapped and then subjected to phylogenetic analysis. These manipulations were carried out, in part, to address criticisms of the statistics used to summarize DNA/DNA hybridization (especially T₅₀H) and the method itself. However, with the exception of an unresolved trichotomy among the twoEchymipera species andPeroryctes longicauda, all trees showed the same branchpoints. Except in the case of the tree generated from reciprocal-corrected T_m data, nodes were stable under jackknifing; and, again excepting the above-mentioned trichotomy, all nodes were supported by 95% or more of the bootstrapped trees. These results suggest that, despite arguments to the contrary, all three summary statistics can be valid for DNA/DNA hybridization data. Of taxonomic interest is the placement ofEchymipera spp. andPeroryctes longicauda together and separate from the more distantPeroryctes raffrayanus; the genusPeroryctes is thus at least paraphyletic. The trees further groupedEchymipera-plus-Peroryctes as the sister group ofIsoodon-plus-Perameles. Limited hybridizations withMacrotis lagotis suggest that its current position as representative of an entirely distinct family of perameloids is correct.This article was presented at the C.S.E.O.L. Conference on DNA-DNA Hybridization and Evolution, Lake Arrowhead, California, May 11–14, 1989     

DNA-DNA hybridization phylogeny of sand dollars and highly reproducible extent of hybridization values

Summary A DNA hybridization phylogeny of four sand dollars using a sea biscuit as an outgroup is presented. The study is unusual in that the normalized percent hybridization (NPH) values were all <50%, yet the same topology was obtained regardless of which distance metric was used, i.e., whether reciprocal distances were averaged or not, or whether or not a molecular clock was assumed. The tree also appears robust under jackknifing and bootstrapping. The extent of hybridization between homologous hybrids was measured with a five- to sevenfold higher precision than is typical, and by implication NPH was also measured with a higher than normal precision. The ability to measure highly reproducible NPH values offers the possibility of examining the phylogeny of more widely divergent species than typically studied using DNA hybridization techniques, using 1/NPH as a distance metric. The hypothesis of a molecular clock within the sand dollars was rejected, adding sand dollars to the growing list of groups where significant rate variation is known. A small fraction of the sand dollar genomes hybridized with the distantly related regular sea urchin Lytechinus. These slowly evolving sequences probably represent conserved exonic components of the genome. Offprint requests to: C.R. Marshall     

Estimation of hominoid phylogeny from a DNA hybridization data set

Summary Analysis of the expanded data set of Sibley and Ahlquist (1987) on primate phylogeny using a maximum likelihood mixed model analysis of variance method shows that there is significant evidence for resolving theHomo-Pan-Gorilla trifurcation in favor of aHomo-Pan clade. The resulting tree is close to that estimated by Sibley and Ahlquist (1984). The mixed model can be used to test a number of hypotheses about the existence of components of variance and the linearity of the relationship between branch length and expected distance. No evidence is found that there is a variance component for extract, or for the individual from which the extract was taken. A variance component for experiment does seem to exist, presumably arising as a result of error of measurement of the common standard from which all values in the same experiment were substracted. There is significant evidence that the relationship between total branch length between species and their expected distances is nonlinear, or else that the measurement error on larger distances is greater than on smaller ones. Allowing for the nonlinearity might cause one to infer the time of distant common ancestors as less remote than the measured hybridization values would imply if used directly.     

Molecular phylogeny of the Praomys complex (Rodentia: Murinae): a study based on DNA/DNA hybridization experiments.

Within the Murinae (Muridae: Rodentia), the African rats of the Praomys group, whose systematics has been studied through different approaches, have raised numerous taxonomic problems. Different taxa related to Praomys have successively been described, among which Mastomys, Myomys and Hylomyscus were considered either as separate genera or subgenera of Praomys. In order to clarify the relationships within the Praomys group, we conducted a series of DNA/DNA hybridization experiments involving different species of Praomys, Mastomys, Myomys and Hylomyscus plus other Murinae and a Cricetomyinae. This study indicates that the Praomys complex is a monophyletic entity clearly separated from the other African and Asian Murinae. If Mastomys and Hylomyscus appeared to be independent genera, the taxonomic situation of Praomys and Myomys is more difficult to ascertain. Indeed, Praomys tullbergi appears more closely related to Myomys daltoni than to another species of Praomys , namely P. jacksoni , suggesting paraphyly for Praomys. Furthermore, P. jacksoni is as distant from P. tullbergi as from any species of Mastomys. Additional species of Praomys and, especially, of Myomys , are needed for reaching a definitive conclusion on these latter taxa. The Praomys group is more related to Mus than to Rattus. To calibrate our molecular distances with geological time, we used a dating of 10 Myr for the Musi Rattus dichotomy. The inferred rate of molecular evolution suggests a dating of c. 8 Myr for the separation of the Praomys group from the Mus lineage.     

A quantitative assessment of congruence between molecular and nonmolecular estimates of phylogeny

Summary An important means of assessing the validity of phylogenetic hypotheses is to measure congruence between different studies of the same group. We applied statistical methods to assess patterns of congruence among phylogenies taken from the literature, using strict-consensus and quartet statistics to measure congruence for 48 pairs of phylogenies of various groups of birds and mammals. The strict-consensus measures were higher on average for distance-distance comparisons than for distance-parsimony or parsimony-parsimony comparisons, and for molecular-molecular comparisons than for molecular-morphological or morphological-morphological comparisons. However, the only factor that was consistently important statistically was the number of taxa involved in the comparison, with congruence decreasing as the number of taxa increased. Quartet indices, which measure network similarity regardless of root position, also showed an effect based on number of taxa but exhibited no trend toward higher congruence in molecular-molecular and distance-distance comparisons. The number of studies with jointly unresolved quartets was small, indicating that data sets varied in the degree to which they could resolve relationships at different phylogenetic levels.The levels of congruence between phylogenies, including those employing single-copy nuclear DNA hybridization data, appear to be higher than expected in random sets of trees, cannot be explained by nonindependence of data sets, and thus provide empirical support for the validity of both distance and parsimony methods of phylogenetic inference. In specific instances, low congruence pointed to mistakes in the application of certain methods, and to the existence of problem taxa in need of additional study.     

Phylogenetic relationships and natural hybridization in Triadica inferred from nuclear and chloroplast DNA analyses

Triadica (Euphorbiaceae) is a small genus endemic to East Asia and Southeast Asia, consisting of three species differentially adapted to heterogeneous habitats. To date, the phylogenetic relationships of this genus have not been resolved, and there has been no evidence for interspecific hybridization in Triadica. In this study, we sequenced the nrITS regions, two nuclear genes and a chloroplast gene to reconstruct the molecular phylogeny of Triadica and to test the hypothesis of natural hybridization between Triadica sebifera and Triadica cochinchinensis, and between T. sebifera and Tridica rotundifolia. Phylogenetic analysis showed that T. sebifera diverged first within this genus, and T. cochinchinensis and T. rotundifolia were sister species. Both of the two putative hybrids show chromatogram additivity at each of the two nuclear genes, providing convincing evidence for natural hybridization between T. sebifera and T. cochinchinensis, and between T. sebifera and T. rotundifolia. The chloroplast gene sequences of both hybrids were identical with that of T. sebifera, suggesting that T. sebifera was the maternal parent of the two hybrids. This is the first report of natural hybridization in Triadica, and the hybrids identified in this study should be a good starting point for further hybridization-based breeding in T. sebifera.     

DNA hybridization as a guide to phylogeny: Chemical and physical limits

Summary The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance.Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments.As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes.     

DNA/DNA hybridization studies of carnivorous marsupials. III. Relationships among species ofDidelphis (Didelphidae)

We report three sets of DNA hybridization experiments conducted to determine relationships among species ofDidelphis (D. albiventris, D. marsupialis, D. virginiana). The 1989 and 1991 sets had fewer replicates per cell than the 1990 series (3.4 and 5.4 vs 9), but in 1991 we distinguished two populations ofD. marsupialis and utilized several individuals for each heterologous comparison. BothPhilander opossum andLutreolina crassicaudata were used as outgroups in 1989, but onlyLutreolina was included in subsequent sets. For each set, we calculated all four standard indices of thermal stability (T _mode,T _m,T ₅₀ H, and NPH) and constructed trees by least-squares (FITCH) and neighbor-joining methods, both before and after correction for asymmetric reciprocal cell values. Subsets of the 1989 data lacking eitherPhilander orLutreolina were analyzed similarly. To explore measurement imprecision, the corrected and uncorrected matrices for each of the four indices were bootstrapped 100 times for the 1989 set and subsets and 1000 times for the 1990 and 1991 sets. Again, for the 1990 and 1991 data, an additional 100 bootstrapped distances were fitted to user trees representing the three possible pairings ofDidelphis spp. to determine the significance of the FITCH branch lengths. The successive experimental sets generated increasingly consistent evidence for pairingD. marsupialis withD. albiventris. The 1989 experiments involved just 85 comparisons, and only T _mode's pairedD. marsupialis withD. albiventris (at bootstrap percentages of 70% or above), but did so whetherPhilander orLutreolina or both were included as the outgroup(s). FITCH and neighbor-joining trees had identical topologies for T _mode's but sometimes differed for the other measures. In contrast, all but one of the corresponding FITCH and neighborjoining trees matched for the 1990 and 1991 data, and three of the four distance measures (T _mode, T _m, and T ₅₀ H) unitedD. marsupialis withD. albiventris at bootstrap percentages averaging 81%; NPH gave a different result for 1990, associatingD. marsupialis withD. virginiana. Further, all but 2 of the 16 matrices for 1990 and 1991 gave mean bootstrapped branch lengths for a consensus pairing that were positive and at least one standard deviation from zero, despite the very short internodes recovered. These results illustrate that the potential of DNA hybridization for resolving very close relationships depends on both the index and the experimental design employed. We conclude that of the three species,D. albiventris andD. marsupialis shared a more recent common ancestor and estimate thatDidelphis spp. have diverged at about 0.39% in nucleotide sequence per myr.Deceased.     

On the phylogeny of Euryteinae (Crustacea,Copepoda, Cyclopoida), with description of one new species from Korea

Euryte koreana sp. nov. is described from the shallow littoral on the East Coast of South Korea, and represents the first record of the subfamily Euryteinae Monchenko, 1974 in the Pacific Ocean north of the tropics. It belongs to a group of species that have the caudal rami length/width ratio of around four, but differs from all congeners by a number of morphological features. Detailed drawings and extensive scanning electron micrographs of many characters of ornamentation provided for this species should serve as a benchmark for distinguishing closely related species in this subfamily with conservative macro-morphology. A morphologically distinct population from anchialine caves in Mallorca, identified previously as Euryte longicauda Philippi, 1843, is described as another new species: Euryte jaumei sp. nov. To test the phylogenetic relationships of its members and previous hypotheses about generic placement of two species associated with scleractinian corals, several cladistic analyses are performed on all 16 currently recognized species of Euryteinae and two outgroups, Neocyclops australiensis Karanovic, 2008 and Troglocyclops janstocki Rocha and Iliffe, 1994, using 25 morphological characters. All resulting trees suggest a close relationship between the two commensal species and them as a sister clade to all other Euryteinae. They are transferred into a newly erected genus Coraleuryte gen. nov., as C. bellatula (Humes, 1991) comb. nov. and C. verecunda (Humes, 1992) comb. nov., and a revised diagnoses is provided for the genus Euryte Philippi, 1843. Obtained cladograms also show that Ancheuryte Herbst, 1989 is nested deeply within the Euryte clade, so the genus is synonymized and its only species is transferred, as E. notabilis (Herbst, 1989) comb. nov. A key to species of Euryteinae is also provided.     

DNA meter: Energy tunable,quantitative hybridization assay

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop‐containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre‐binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex‐bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof‐of‐principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 408–417, 2013.     

Testing the generation time hypothesis using DNA/DNA hybridization between artiodactyls

Heterogeneous rates of molecular change between some mammalian lineages are commonly explained by contrasts in generation time length. Here the generation time hypothesis is tested by comparing the relative rates of molecular change in related artiodactyl taxa differing by their generation time. A demographic model based on allometric relations with the adult body weight is used to estimate the cohort generation time in Bovidae and Cervidae families (Artiodactyla, Mammalia). Two pairs of closely related taxa (two cervids, two bovids) were selected, each showing clear ratios (1.5 to 3.5 times) in their generation time. Rates of genetic change in non-repeated nuclear DNA were estimated by DNA/DNA hybridization experiments performed among these ruminants and a camelid outgroup. Relative rate tests were applied to the two pairs of ingroup taxa differing by their generation time, in order to test if shorter generation time would correspond to higher rate of molecular change. Contradictory statistical results did not show a greater accumulation of nucleotide changes in the lineage leading to the short generation time species. The recorded differences in branch lengths of sister taxa were either conflicting or too small (relative to the contrasted generation times) to reveal a generation time effect. Alternative hypotheses are suggested to explain these preliminary results.     

Molecular phylogeny of gibbons inferred from mitochondrial DNA sequences: Preliminary report

We analyzed the 896 base-pair (bp) mitochondrial DNA (mtDNA) sequences for seven gibbons, representative of three out of four subgenera. The result from our molecular analysis is consistent with previous studies as to the monophyly of subgenus Hylobates species, yet the relationship among subgenera remains slightly ambiguous. A striking result of the analysis is the phylogenetic location of Kloss's gibbon (H. klossii). Kloss's gibbon has been considered to be an initial off-shoot of the subgenus Hylobates because of its morphological primitiveness. However, our molecular data strongly suggest that Kloss's gibbon speciated most recently within the subgenus Hylobates. Correspondence to: S. Horai     

Rearrangements of mitochondrial transfer RNA genes in marsupials

Summary The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNA^Tyr gene at the same position as in placental mammals andXenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNA^Tyr gene has been evolving under more constraints than the other tRNA genes.Deceased July 21, 1991     

Molecular homology and DNA hybridization

Summary We reviewed the concept of homology, which can broadly be defined as a correspondence between characteristics that is caused by continuity of information (Van Valen 1982). The concept applies widely in molecular biology when correspondence is taken to mean a genetic relationship resulting from a unique heritable modification of a feature at some previous point in time. Such correspodence can be established for features within a single organism as well as between organisms, making paralogy a valid form of molecular homology under this definition. Molecular homology can be recognized at a variety of organizational levels, which are intedependent. For example, the recognition of homology at the site level involves a statement of homology at the sequence level, and vice versa. This hierarchy, the potential for nonhomologous identity at the site level, and such processes as sequence transposition combine to yield a molecular equivalent to complex structural homology at the anatomical level. As a result, statements of homology between heritable units can involve a valid sense of percent homology.We analyzed DNA hybridization with respect to the problems of recognizing homology and using it in phylogenetic inference. Under a model requiring continuous divergence among compared sequences, DNA hybridization distances embed evolutionary hierarchy, and groups inferred using pairwise methods of tree reconstruction are based on underlying patterns of apomorphic homology. Thus, symplesiomorphic homology will not confound DNA hybridization phylogenies. However, nonhomologous identities that act like apomorphic homologies can lead to inaccurate reconstructions. The main difference between methods of phylogenetic analysis of DNA sequences is that parsimony methods permit hypotheses of nonhomology, whereas distance methods do not.This article was presented at the C.S.E.O.L. Conference on DNA-DNA Hybridization and Evolution, Lake Arrowhead, California, May 11–14, 1989     

Immobilization and hybridization of DNA in a sugar polyacrylate hydrogel

Using a non-contact microarrayer, amine-terminated probe oligonucleotides representing 20-, 50-, and 70-mer fragments of the fliC gene were covalently coupled into three-dimensional regions in a "sugar polyacrylate" hydrogel based on poly(6-acryloyl-beta-O-methyl galactopyranoside-co-aminopropyl methacrylamide). The arrayer deposited the solution containing ssDNA probes in discrete regions on the surface of the gel (i.e. as a droplet with a ca. 450 microm diameter), allowing penetration and attachment of the ss DNA within the three dimensional region of the gel. The attachment was mediated by the homobifunctional crosslinker bis-succinimidyl suberate. Confocal microscopy showed the density of attached probe DNA was greatest in the interior-most regions of the gel volume. Target ssDNA (20- and 70-mer) was able to diffuse through the gel and undergo successful hybridization with the probes. For target ssDNA in the concentration range 0.19 microM to 6.0 microM, there was a linear correlation between DNA concentration and the fluorescence of the gel region where hybridization occurred.     

DNA hybridization assays using metal-enhanced fluorescence

We describe a new approach to DNA hybridization assays using metal-enhanced fluorescence. Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic time-dependent 12-fold increase in fluorescence intensity during hybridization. Proximity to silver particles resulted in a decreased fluorescence lifetime. This effect is thought to be the result of enhanced fluorescence from fluorescein near metallic silver particles. Hybridization could thus be measured from the decay kinetics of the emission, which can be measured independently from the emission intensity. These results suggest the use of silver particles as a general approach to measure DNA hybridization as a method to increase the sensitivity of DNA detection.