Peter M. McGuire

Phylogeography of the West Indian manatee (Trichechus manatus): how many populations and how many taxa?

To resolve the population genetic structure and phylogeography of the West Indian manatee (Trichechus manatus), mitochondrial (mt) DNA control region sequences were compared among eight locations across the western Atlantic region. Fifteen haplotypes were identified among 86 individuals from Florida, Puerto Rico, the Dominican Republic, Mexico, Colombia, Venezuela, Guyana and Brazil. Despite the manatee’s ability to move thousands of kilometres along continental margins, strong population separations between most locations were demonstrated with significant haplotype frequency shifts. These findings are consistent with tagging studies which indicate that stretches of open water and unsuitable coastal habitats constitute substantial barriers to gene flow and colonization. Low levels of genetic diversity within Florida and Brazilian samples might be explained by recent colonization into high latitudes or bottleneck effects. Three distinctive mtDNA lineages were observed in an intraspecific phylogeny of T. manatus, corresponding approximately to: (i) Florida and the West Indies; (ii) the Gulf of Mexico to the Caribbean rivers of South America; and (iii) the northeast Atlantic coast of South America. These lineages, which are not concordant with previous subspecies designations, are separated by sequence divergence estimates of d = 0.04–0.07, approximately the same level of divergence observed between T. manatus and the Amazonian manatee (T. inunguis, n = 16). Three individuals from Guyana, identified as T. manatus, had mtDNA haplotypes which are affiliated with the endemic Amazon form T. inunguis. The three primary T. manatus lineages and the T. inunguis lineage may represent relatively deep phylogeographic partitions which have been bridged recently due to changes in habitat availability (after the Wisconsin glacial period, 10 000 BP), natural colonization, and human‐mediated transplantation.

Chromosome painting in the manatee supports Afrotheria and Paenungulata

BackgroundSirenia (manatees, dugongs and Stellars sea cow) have no evolutionary relationship with other marine mammals, despite similarities in adaptations and body shape. Recent phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida manatee.ResultsThe human autosomal and X chromosome paints delimited a total of 44 homologous segments in the manatee genome. The synteny of nine of the 22 human autosomal chromosomes (4, 5, 6, 9, 11, 14, 17, 18 and 20) and the X chromosome were found intact in the manatee. The syntenies of other human chromosomes were disrupted in the manatee genome into two to five segments. The hybridization pattern revealed that 20 (15 unique) associations of human chromosome segments are found in the manatee genome: 1/15, 1/19, 2/3 (twice), 3/7 (twice), 3/13, 3/21, 5/21, 7/16, 8/22, 10/12 (twice), 11/20, 12/22 (three times), 14/15, 16/19 and 18/19.ConclusionThere are five derived chromosome traits that strongly link elephants with manatees in Tethytheria and give implicit support to Paenungulata: the associations 2/3, 3/13, 8/22, 18/19 and the loss of the ancestral eutherian 4/8 association. It would be useful to test these conclusions with chromosome painting in hyraxes. The manatee chromosome painting data confirm that the associations 1/19 and 5/21 phylogenetically link afrotherian species and show that Afrotheria is a natural clade. The association 10/12/22 is also ubiquitous in Afrotheria (clade I), present in Laurasiatheria (clade IV), only partially present in Xenarthra (10/12, clade II) and absent in Euarchontoglires (clade III). If Afrotheria is basal to eutherians, this association could be part of the ancestral eutherian karyotype. If afrotherians are not at the root of the eutherian tree, then the 10/12/22 association could be one of a suite of derived associations linking afrotherian taxa.

Microsomal activation of estrogens and binding to nucleic acids and proteins

Natural and synthetic estrogens can be activated by rat liver microsomes to bind covalently to polyguanylic acid, single-stranded DNA, nucleotides and proteins. Incubation of polyG, estrone and liver microsomes (0.5 nmole cytochrome P-448 or P-450) from 3-methylcholanthrene-induced, phenobarbital-induced or control rats showed that the former microsomes gave better net binding of estrogens to polyG than the other two. Estradiol incubated with 3MC-induced microsomes did bind to DNA but marginally to polyG. Mestranol and estrone sulfate, both constituents of oral contraceptive formulations, bound to polyG whereas progesterone and cholesterol did not bind. We present also preliminary data on the characterization of estrogen-nucleic acid interactions using nucleases, proteinase K and high-pressure liquid chromatography.

Low genetic diversity and minimal population substructure in the endangered Florida manatee: implications for conservation

Abstract Species of management concern that have been affected by human activities typically are characterized by low genetic diversity, which can adversely affect their ability to adapt to environmental changes. We used 18 microsatellite markers to genotype 362 Florida manatees (Trichechus manatus latirostris), and investigated genetic diversity, population structure, and estimated genetically effective population size (Ne). The observed and expected heterozygosity and average number of alleles were 0.455 ± 0.04, 0.479 ± 0.04, and 4.77 ± 0.51, respectively. All measures of Florida manatee genetic diversity were less than averages reported for placental mammals, including fragmented or nonideal populations. Overall estimates of differentiation were low, though significantly greater than zero, and analysis of molecular variance revealed that over 95% of the total variance was among individuals within predefined management units or among individuals along the coastal subpopulations, with only minor portions of variance explained by between group variance. Although genetic issues, as inferred by neutral genetic markers, appear not to be critical at present, the Florida manatee continues to face demographic challenges due to anthropogenic activities and stochastic factors such as red tides, oil spills, and disease outbreaks; these can further reduce genetic diversity of the manatee population.

Cytotoxic activity of an octadecenoic acid extract from Euphorbia kansui (Euphorbiaceae) on human tumour cell strains

We have investigated the cytotoxic and antitumour activity of an octadecenoic acid extract, mainly containing oleic and linoleic acids, from Euphorbia kansui on human gastric (SGC‐7901), hepatocellular carcinoma (BEL‐7402), and leukaemia (HL‐60) tumour cell strains. Significant and dose‐dependent antiproliferation effects were observed on tumour cells from the dose of 3.2 μg mL−1, which were comparable with or better than those of the common antitumour agent 5‐fluorouracil. Results from the clone formation assay and flow cytometry indicated that the mixture of octadecenoic acids resulted in a dose‐dependent reduction in the number of tumour cells and significantly inhibited cell proliferation, with induced apoptosis and G0/G1 phase cell cycle arrest. Also, the octadecenoic acids could not only cause cell apoptosis/necrosis but also functionally and structurally damage the tumour cell membrane and cell ultra‐structures. These observations encourage further clinical evaluation of the inhibitory effects of octadecenoic acids on various forms of cancer.

Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells

SummarytsAF8 cells area temperature-sensitive (ts) mutant of BHK cells that arrest in the G1 phase of the cell cycle at the non-permissive temperature of 40.6 °C. Previous reports had suggesed that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II. We now show that the direct microinjection of purified RNA polymerase 11 into nuclei of tsAF8 cells corrects the ts defect and allows these cells to enter the S phase of the cell cycle.

Molecular phylogeny of the genus Paramesotriton (Caudata : Salamandridae)

To elucidate the phylogeny of the genus Paramesotriton (Caudata: Salamandridae), we investigated three mitochondrial DNA gene fragments (1207 bp in total) of cytochrome b, ND2, and ND4 for its six recognized species. The phylogenetic relationships within Paramesotriton were reconstructed by maximum parsimony (MP) and maximum likelihood (ML) methods. Phylogenetic trees (MP and ML trees) that were constructed from the combined data set of the three gene fragments indicated that all six species of Paramesotriton formed a monophyletic group, with P. caudopunctatus as basal to the other five species. This result suggests that P. fuzhongensis is a valid species in Paramesotriton.

Molecular phylogeny of five species of Dremomys (Rodentia : Sciuridae), inferred from cytochrome b gene sequences

Analyses of the mitochondrial cytochrome b gene (1140 bp) showed that Dremomys lokriah, D. pernyi, D. pyrrhomerus, D. rufigenis and D. gularis all are separate species. Dremomys pyrrhomerus showed 8.5% sequence variation from D. rufigenis, and the level of estimated sequence divergence observed among D. gularis, D. lokriah and D. pernyi was > 11%. With Tamiops and Callosciurus as the outgroup taxa, in both maximum likelihood and Bayesian analyses, the five Dremomys species formed one strongly supported monophyletic group and D. pyrrhomerus is closely related to D. rufigenis. The derived divergence times and fossil record suggested that the present geographical distributions of Dremomys owe much to the uplifting of the Himalayas and the successive glacial and interglacial in the Pliocene–Pleistocene.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species‐specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross‐species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker‐panels for each species. These cross‐species microsatellite marker‐panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.

Depurination of yeast 26S ribosomal RNA by recombinant ricin A chain

The nature of the modification of yeast ribosomes by the recombinant form of the ricin A chain has been examined. Evidence is presented that the 26S rRNA molecule is depurinated at a specific site and that the activity is inhibited by antibody raised to ricin A chain. It thus appears that the recombinant form of this toxin retains the depurination activity of the native molecule. These results are consistent with the model that the site of depurination is in a highly conserved sequence forming a loop on the surface of the ribosome, a domain involved in elongation factor-dependent binding of aminoacyl-tRNA.