DNA hybridization,cladistics, and the phylogeny of phalangerid marsupials

Summary Single-copy DNA/DNA hybridization experiments and numerical cladistic analyses of anatomical characters were used to investigate relationships among nine phalangerid (Marsupialia) species from four different genera. Both rate-dependent and rate-independent analyses of molecular data indicate that species ofTrichosurus form one clade and thatStrigocuscus, Phalanger, andSpilocuscus form a second. Within the latter group,Spilocuscus is excluded from aStrigocuscus-Phalanger calde, which, in turn, is not fully resolved on a jackknife strict consensus tree. Minimum-length Dollo, Wagner, and Camin-Sokal parsimony trees based on 35 anatomical characters, in contrast, suggest placement ofStrigocuscus withTrichosurus rather than withSpilocuscus andPhalanger. However, there are two derived characters that support the alternative arrange ofStrigocuscus withSpilocuscus andPhalanger and one character that further unitesStrigocuscus andPhalanger. Thus, DNA hybridization results are not inconsistent with the distribution of derived character states among anatomical characters, only with minimum-length trees based on character data.     

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.     

Post-weaning cranial ontogeny in two bandicoots (Mammalia,Peramelomorphia, Peramelidae) and comparison with carnivorous marsupials

The ontogeny of the skull has been studied in several marsupial groups such as didelphids, microbiotheriids, and dasyurids. Here, we describe and compare the post-weaning ontogeny of the skull in two species of bandicoots, Echymipera kalubu (Echymiperinae) and Isoodon macrourus (Peramelinae), analyzing specific allometric trends in both groups, describing common (and specific) patterns, and discussing them on functional and phylogenetic grounds. Growth patterns were analyzed both qualitatively and quantitatively, including bivariate and multivariate analyses of allometry. We also evaluated character transformation and phylogenetic signals of the allometric patterns in several groups of marsupials and some placentals. We identified morphological changes between juvenile and adult stages in both species of peramelids, many related to the development of the trophic apparatus. Notable differences were detected in the patterns of growth, suggesting divergences in ontogenetic trajectories between both species. Both bivariate and multivariate methods indicate that positive allometries in E. kalubu apply to longitudinal dimensions, whereas in I. macrourus, positive allometries are restricted to vertical dimensions of the skull. The comparison of the allometric trends of two bandicoots with previously studied taxa reveals that although peramelids exhibit a particularly short gestation period and divergent morphology compared to other marsupials, their pattern does not show any particular trend. Some allometric trends seem to be highly conserved among the species studied, showing weak phylogenetic signal. Marsupials in general do not show particular patterns of post-weaning skull growth compared with placentals.     

Phylogenetic relationships of the endemic malagasy carnivoreCryptoprocta ferox (Aeluroidea): DNA/DNA hybridization experiments

A molecular and morphological study of several living aeluroid Carnivora was completed to evaluate the evolutionary relationships of the endemicCryptoprocta ferox, a carnivore living on the island of Madagascar. The molecular analysis, based on DNA/DNA hybridization experiments, suggests thatCryptoprocta is more closely related to the Herpestidae (as represented byMungos andIchneumia) than it is to the Viverrinae (Genetta), Paradoxurinae (Paguma, Paradoxurus), Felidae (Felis, Panthera), or Hyaenidae (Crocuta). Based on bootstrapping procedures applied to the individual DNA/DNA results, three branching patterns were observed which differ only by the relative position of the Felidae within the Aeluroidea. The amounts of genetic divergence measured between pairs of compared taxa have been transformed into millions years datings by the molecular clock concept, and this was done by establishing a molecular time scale based on the fossil record of the aeluroid Carnivora.     

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.     

Molecular Relationships of the Extinct Pig-Footed Bandicoot Chaeropus ecaudatus (Marsupialia: Perameloidea) Using 12S rRNA Sequences

The pig-footed bandicoot, Chaeropus ecaudatus, is presumed to be extinct as no specimens have been collected or seen since early this century. Usually classified as a specialized member of the family Peramelidae, there is nevertheless still some doubt as to its taxonomic affinities, because this animal is highly specialized and shows several uniquely derived characters. We report here the first attempt to determine the molecular relationships of this animal using mitochondrial 12S rRNA sequences derived from spirit-preserved museum specimens. Phylogenetic analysis shows that the sequence derived from the Chaeropus sample is clearly that of a bandicoot. Within the bandicoot clade, the pig-footed bandicoot is quite distinct from all other taxa. Divergence-time estimates from the 12S rRNA sequences suggest that Chaeropus diverged from the other bandicoot genera in the late Oligocene or early Miocene and that bandicoots diverged from other Australian families in the late Paleocene–early Eocene.     

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-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     

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.     

Experimental analysis of variance for DNA hybridization: I. Accuracy

We used tissues of the Virginia opossum (Didelphis virginiana) to examine the experimental accuracy of DNA hybridization statistics of thermal stability (T_mode, T_m, T₅₀H, and NPH) with respect to systematic biases in counting radioactivity in elution fractions, and column position and loading order of hybrids in the thermal elution device. We failed to detect any change in the mean melting temperatures among five replicate ¹²⁵I-labeled hybrids counted over 72 h. Furthermore, column position in the automated thermal elution device (TED) did not bias the statistics of aliquots loaded over a few minutes from a single large mother hybrid. On the other hand, the normalized percentage hybridization (NPH) increased as much as 3–5% for aliquots loaded during 1 h from a similar mother hybrid. A parallel but less consistent increase was noted for T₅₀H, which incorporates a measure of NPH. This NPH effect disappeared when hybrids were prepared individually and diluted and loaded in turn—the usual procedure in our laboratory. Replicate distances measured as NPH appear to be sensitive to departures from the normal-distribution assumption of least-squares regression. We recommend that replicate cell values of NPH be transformed to improve their fit to a normal distribution prior to analysis by least-squares phylogenetic algorithms such as those available in Felsenstein's PHYLIP package. Thus, potential sources of inaccuracy in DNA hybridization data can be avoided with simple precautions.     

The constitution of the order centrospermae: rRNA-DNA hybridization studies among betalain- and anthocyanin-producing families

Ribosomal RNA homologies and the thermal stabilities of rRNA/DNA hybrids among ten species of the Centrospermae (including three from the family Caryophyllaceae and seven from five betalain-producing families), three other angiosperms, and one fern, suggest that the betalain-producing families are phylogenetically closer to each other than to the anthocyanin-producing families which are examined.     

The basicranial region of marsupicarnivores (Marsupialia), interrelationships of carnivorous marsupials, and affinities of the insectivorous marsupial peramelids

The basicranial region is examined in detail in didelphids, dasyurids, thylacinids, borhyaenids, myrmecobiids, and comparisons are made with notoryctids and peramelids. Three dasyurid genera ( Sminthopsis, Antechinus and Planigale ) were doubly injected with latex and basicranial foramina identified partly on the basis of the vessels which they transmit. The resulting terminology differs in several significant ways from terminologies currently in use. A basic terminology for sinuses, recesses, foramina and other structures in the basicranial region is developed from this new information combined with existing systems used for eutherian mammals and mammal-like reptiles.
It is concluded that on the basis of basicranial structure didelphids are structurally ancestral to all other marsupicarnivores, directly or indirectly. They are directly ancestral to thylacinids, borhyaenids, dasyurids and peramelids. Myrmecobiids are structural descendents of dasyurids. Notoryctids are not marsupicarnivores and may be diprotodonts. No special relationships exist between thylacinids and dasyurids or between dasyurids and peramelids. All groups except didelphids show unique specializations in some members and several show evidence of convergence in sinus development and structure of the foramen ovale.     

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.     

Resolution of portions of the kangaroo phylogeny (Marsupialia: Macropodidae) using DNA hybridization

We generated a DNA hybridization matrix comparing eleven 'true' kangaroos (Macropodinae) and two outgroup marsupials, the rufous rat-kangaroo Aepyprymnus rufescens (Potoroinae) and the brush-tailed phalanger Trichosurus vulpecula (Phalangeridae). A small matrix included additional species of the genus Macropus (large kangaroos and wallabies). The results indicate that the New Guinean forest wallaby Dorcopsulus vanheurni, and the quokka Setonix brachyurus, represent successively closer sister-groups of other macropodines. The remaining taxa examined form two clades: the tree kangaroo Dendrolagus matschiei with die pademelons Thylogale and rock wallabies Petrogale, and Macropus including the swamp wallaby Wallabia bicolor. The smaller matrix of five Macropus species and Wallabia (with Dorcopsulus as an outgroup) pairs the red-necked wallaby M. rufogriseus and Parry's wallaby M. parryi, with the eastern grey kangaroo M. giganteus as their nearest relative; and associates the red kangaroo M. rufus and wallaroo M. robustus, with Wallabia as their sister-taxon. In the larger study, we found mat inclusion of both outgroups provided little resolution among the macropodines, judging by jackknife and bootstrap tests. When Aepyprymnus was deleted, the Dendrolagus-Thylogale-Petrogale association obtained; with Trichosurus eliminated instead, the Wallabia-Macropus group was recovered. Only analysis of the eleven ingroup taxa by themselves gave a topology which supported both major clades. Our findings suggest that, at least for DNA hybridization studies, when ingroup taxa are separated by very short internodes experimental error in outgroup-to-ingroup distances may seriously compromise determination of ingroup affinities as well as the position of the root. We recommend that in such cases separate analyses with the outgroups sequentially eliminated and rigorous validation of die topology at each step should be conducted.     

Molecular phylogenetic relationships of two extinct potoroid marsupials, Potorous platyops and Caloprymnus campestris (Potoroinae: Marsupialia)

Complete 12S rRNA and partial cytochrome b (cytb) gene sequences have been obtained from museum samples of two recently extinct potoroids-Potorous platyops and Caloprymnus campestris. Phylogenetic analyses based on these mitochondrial DNA sequences suggest that the broad-faced potoroo (P. platyops) was a close relative of the recently discovered Potorous longipes and the recently re-discovered Potorous gilberti. Although the extinct desert rat-kangaroo (C. campestris) was clearly resolved as a member of the subfamily Potoroinae, its precise relationships vis a vis other living potoroines are unclear. We confirmed that the rufous rat-kangaroo (Aepyprymnus rufescens) is sister to all living Bettongia species, but the molecular data provide no support for a sister relationship between A. rufescens and C. campestris as suggested by on the basis of four shared morphological characters. Molecular dating analyses suggest that the initial radiation of potoroinae seems to have occurred soon after its origin in the early Miocene. Within Potoroinae, C. campestris diverged from other taxa approximately 16 million years ago. P. platyops diverged from P. longipes+P. gilberti approximately 14-15 million years ago.     

Use of DNA/DNA hybridization techniques to authenticate the production of new Azolla-Anabaena symbiotic associations

Abstract The establishment of Azolla-Anabaena 'recombination' symbioses by grafting the Anabaena -containing indusium (cap) from the donor megasporocarp onto a decapitated megasporocarp from an Anabaena -free recipient has been recently reported. We have generated strain- and species-specific DNA probes for unequivocally establishing the identity of Anabaena azollae strains isolated from Azolla-Anabaena symbioses (whether new, i.e. heterosymbioses or natural i.e. homosymbioses). Eight out of nine heterosymbioses tested proved to contain the expected heterosymbiont while one recombined association was shown to contain both its own homosymbiont and a heterosymbiotic Anabaena strain. In addition, a species-specific plant probe was isolated from the total DNA of Azolla microphylla roots and used to establish the identity of a host plant used for recombination experiments.     

Body temperature variation of free-ranging and captive southern brown bandicoots Isoodon obesulus (Marsupialia: Peramelidae)

To investigate patterns of thermoregulation in free-ranging and captive southern brown bandicoots Isoodon obesulus, we measured abdominal body temperature (T_b) of five free-ranging bandicoots over 42 days using implanted data loggers and T_b of three captive bandicoots over 3 months using implanted temperature-sensitive radio transmitters. Bandicoots in the wild had a mean T_b of 36.5±1.0 °C (range 33.4–39.8 °C) and showed a pronounced nychthemeral pattern with two distinct temperature phases. T_b increased at 13:30±2.6 h each day and remained high for 10.65±2.07 h, suggesting a crepuscular and early evening activity pattern. Daily T_b variation of I. obesulus would save considerable energy by reducing daytime thermoregulatory costs in the wild. Captive bandicoots had a similar mean body temperature (36.9±0.2°C) and range (33.0–39.9°C) as free-ranging bandicoots. However, the nychthemeral T_b pattern of captive bandicoots was different from free-ranging bandicoots, with a less pronounced daily cycle and the nocturnal rise in T_b occurring mainly at sunset and the daily decline occurring mainly at dawn.