The results of investigations in these laboratories of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines as human CCR5 antagonists have recently been disclosed. To facilitate further development of these antagonists, we have developed a pharmacophore model based on the structure-activity relationships (SAR) and a human CCR5 receptor docking model using the crystal structure of rhodopsin as a template [Palczewski, K., et al. (2000) Science 289, 739-745]. Guided by the receptor docking model, we have mapped the compounds' site of interaction with CCR5 using site-directed mutagenesis experiments. Our results are consistent with a binding site for the two series that is located within a cavity near the extracellular surface formed by transmembrane helices 2, 3, 6, and 7. This site is overlapping yet distinct from that reported for another antiviral agent which binds to CCR5 [Dragic, T., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 5639-5644].

A suite of comparisons among ten radiolabelled dasyurid species and one outgroup bandicoot was generated using the hydroxyapatite chromatography method of DNA-DNA hybridisation; comparisons were also made with four other dasyurid taxa. Square matrices of DELTA-T(m)s, DELTA-Modes, and DELTA-T50H's were complied and corrected for reciprocity, additivity, and, in the case of DELTA-T(m)'s, normalised percentages of hybridisation. These matrices were analysed using the FITCH algorithm in Felsenstein's PHYLIP (Version 3.1), and all distinct topologies were jackknifed to test for internal consistency. Additionally, uncorrected DELTA-T(m), DELTA-Mode, and DELTA-T50H datasets were bootstrapped and subjected to phylogenetic analysis to assess measurement imprecision. FITCH trees from folded matrices including unlabelled species or those for which heteroduplex comparisons were incomplete were also calculated and jack-knifed, both before and after correction. With the exception of limited measurements to Dasyuroides byrnei and Dasykaluta rosamondae, which showed affinities with Dasyurus spp., the final tree was fully resolved: Sminthopsis crassicaudata and S. murina, together with the more distant Planigale maculata, are the sister-group to all other dasyurids examined, which in turn comprise two clades. One of these includes Dasyurus, Dasyuroides, and Dasykaluta; the other, 'true' Antechinus (A. flavipes, A. stuartii, A. swainsonii) as a sister-group to Antechinus melanurus plus Murexia longicaudata, with Phascogale tapoatafa representing a probable sister-group to all Antechinus with Murexia. DNA-DNA hybridisation provides no support for the genus Satanellus: most of the trees linked Dasyurus albopunctatus with D. maculatus instead of D. hallucatus. Similarly, Antechinus flavipes and A. stuartii appear to be closer to each other than either is to A. swainsonii. The historical biogeographic significance of the adopted phylogeny is considered, and it is concluded that the putative early Miocene separation of Australia and New Guinea was probably too early to account for the independent evolution of the New Guinean clade.

coalescence methods were developed to address the negative impacts of incomplete lineage sorting on species tree estimation with concatenation. Coalescence methods are statistically consistent if certain requirements are met including no intralocus recombination, neutral evolution, and no gene tree reconstruction error. However, the assumption of no intralocus recombination may not hold for many DNA sequence data sets, and neutral evolution is not the rule for genetic markers that are commonly employed in phylogenomic coalescence analyses. Most importantly, the assumption of no gene tree reconstruction error is routinely violated, especially for rapid radiations that are deep in the Tree of Life. With the sequencing of complete genomes and novel pipelines, phylogenetic analysis of retroposon insertions has emerged as a valuable alternative to sequence-based phylogenetic analysis.Retroposon insertions avoid or reduce several problems that beset analysis of sequence data with summary coalescence methods: 1) intralocus recombination is avoided because retroposon insertions are singular evolutionary events, 2) neutral evolution is approximated in many cases, and 3) gene tree reconstruction errors are rare because retroposons have low rates of homoplasy. However, the analysis of retroposons within a multispecies coalescent framework has not been realized. Here, we propose a simple workaround in which a retroposon insertion matrix is first transformed into a series of incompletely resolved gene trees. Next, the program ASTRAL is used to estimate a species tree in the statistically consistent framework of the multispecies coalescent. The inferred species tree includes support scores at all nodes and internal branch lengths in coalescent units. As a test case, we analyzed a retroposon dataset for palaeognath birds (ratites and tinamous) with ASTRAL and compared the resulting species tree to an MP-EST species tree for the same clade derived from thousands of sequence-based gene trees. The MP-EST species tree suggests an empirical case of the 'anomaly zone' with three very short internal branches at the base of Palaeognathae, and as predicted for anomaly zone conditions, the MP-EST species tree differs from the most common gene tree. Although identical in topology to the MP-EST tree, the ASTRAL species tree based on retroposons shows branch lengths that are much longer and incompatible with anomaly zone conditions. Simulation of gene trees from the retroposon-based species tree reveals that the most common gene tree matches the species tree. We contend that the wide discrepancies in branch lengths between sequence-based and retroposon-based species trees are explained by the greater accuracy of retroposon gene trees (bipartitions) relative to sequence-based gene trees. Coalescence analysis of retroposon data provides a promising alternative to the status quo by reducing gene tree reconstruction error that can have large impacts on both branch length estimates and evolutionary interpretations.

Comparison among eight pseudocheirid species and two outgroup petaurids were made by means of the hydroxyapatite chromatography method of DNA hybridisation. Matrices of DELTAT(m) and DELTAT(m)H-C values were analysed with the FITCH algorithm in Felsenstein's PHYLIP (Version 3.3). Jackknifing and bootstrapping were applied to determine the stability of resulting topologies. All the phylogenetic analyses produced trees that support (1) the monophyly of the Pseudocheirus herbertensis complex, (2) the monophyly of Pseudocheirus, (3) a close relationship between Hemibelideus and Petauroides, and (4) a close relationship between Pseudochirops archeri and Pseudochirops cupreus. Rates of single-copy DNA evolution are slightly faster in Pseudocheirus, Hemibelideus, and Petauroides than in Pseudochirops. Hybridisation evidence also provides a framework for understanding the timing of the pseudocheirid radiation and suggests that the divergence between extant genera dates back to about 36 million years ago.

DNA sequence alignments provide the majority of data for inferring phylogenetic relationships with both concatenation and coalescence methods. However, DNA sequences are susceptible to extensive homoplasy, especially for deep divergences in the Tree of Life. Retroposon insertions have emerged as a powerful alternative to sequences for deciphering evolutionary relationships because these data are nearly homoplasy-free.In addition, retroposon insertions satisfy the 'no intralocus recombination' assumption of summary coalescence methods because they are singular events and better approximate neutrality relative to DNA sequences commonly applied in phylogenomic work.Retroposons have traditionally been analyzed with phylogenetic methods that ignore incomplete lineage sorting (ILS). Here, we analyze three retroposon data sets for mammals (Placentalia, Laurasiatheria, Balaenopteroidea) with two different ILS-aware methods. The first approach constructs a species tree from retroposon bipartitions with ASTRAL, and the second is a modification of SVD-Quartets. We also develop a χ 2 Quartet-Asymmetry Test to detect hybridization using retroposon data. Both coalescence methods recovered the same topology for each of the three data sets. The ASTRAL species tree for Laurasiatheria has consecutive short branch lengths that are consistent with an anomaly zone situation. For the Balaenopteroidea data set, which includes rorquals (Balaenopteridae) and gray whale (Eschrichtiidae), both coalescence methods recovered a topology that supports the paraphyly of Balaenopteridae. Application of the χ 2 Quartet-Asymmetry Test to this data set detected 16 different quartets of species for which historical hybridization may be inferred, but significant asymmetry was not detected in the placental root and Laurasiatheria analyses.