Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini.

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.

A molecular phylogeny for the frog genus Limnodynastes (Anura: myobatrachidae).

Members of the genus Limnodynastes are a prominent and widespread feature of the Australian frog fauna. Yet despite their potential to be informative about biogeographic history and mechanisms of speciation, the relationships among these taxa are not well known. We investigated phylogenetic relationships within the genus Limnodynastes via sequencing of mitochondrial (mt)DNA from current members of the genus Limnodynastes and the monotypic genus Megistolotis. a 450-bp fragment of the 16S rRNA gene and a 370-bp fragment of the protein-coding gene ND4 were used to infer a molecular phylogeny. We revise traditional species groupings and now recognize four species groups within Limnodynastes: the L. ornatus group (L. ornatus and L. spenceri), the L. peronii group (L. peronii, L. tasmaniensis, L. fletcheri, the L. depressus), the L. salmini group (L. salmini, L. convexiusculus, and L. lignarius), and the L. dorsalis group (L. dorsalis, L. terraereginae, L. dumerilii and L. interioris). The L. ornatus species group forms a highly distinctive clade that is a sister group to the other Limnodynastes groups. Pending broader phylogenetic studies it could be removed from the genus Limnodynastes. Our results concur with previous suggestions that Megistolotis lignarius is nested within Limnodynastes, and we therefore reclassify this species as Limnodynastes lignarius. Furthermore, specimens identified as L. depressus form a mtDNA lineage distinct from other species in the genus, confirming the validity of the species. Specimens of species from the L. dorsalis group (L. dorsalis, L. dumerilii, L. interioris, and L. terraereginae) are closely related such that L. dumerilii is paraphyletic with two other species. Finally, our study provides broad support for previous phylogenies based on microcomplement fixation.

Microsatellite variation and assessment of genetic structure in tea tree (Melaleuca alternifolia-Myrtaceae).

Analysis of five microsatellite loci in 500 Melaleuca alternifolia individuals produced 98 alleles that were useful for population genetic studies. Considerable levels of observed heterozygosity were recorded (HO = 0.724), with approximately 90% of the variability being detected within populations. A low level of selfing (14%) was suggested to be the principal cause of excess homozygosity in a number of populations (overall FIS = 0.073). This study showed low levels of inbreeding in certain populations as well as a significant isolation-by-distance model. Only two groups of populations (Queensland and New South Wales) constituted different genetic provenances as a result of geographical isolation. The M. alternifolia data suggest that microsatellite loci did not always arise by a stepwise mutation process but that larger jumps in allele size may be involved in their evolution.

A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis.

Using degenerate oligonucleotide primers based on conserved active site residues, we have isolated a cDNA encoding an aspartic protease from the nematode parasite Strongyloides stercoralis, an important, enteric pathogen of humans. cDNAs encoding the aspartic protease were isolated from the infective, third stage larvae of the parasite as well as from free-living, rhabditiform larvae. Based on comparisons of other aspartic proteases, the cDNA encoded a short signal peptide, an enzyme pro-segment of 35 amino acid residues, and mature enzyme of 337 residues. Homology alignments using the proenzyme sequence showed that the novel S. stercoralis zymogen was 36% identical to human pepsinogen A and 36% identical to pepsinogen C (progastricin) from humans and macaques. Phylogenetic analyses using the Phylip program and analysis of Glx/Asx and Leu/Ile ratios indicated that the proenzyme was closely related to pepsinogen A-like enzymes from the free-living nematode Caenorhabditis elegans and Haemonchous contortus, a nematode parasite of the gastro-intestinal tract of sheep. We have termed this novel enzyme strongyloidespepsin.

Molecular population genetics of the southern elephant seal Mirounga leonina.

Southern elephant seals breed on sub-Antarctic islands and have a circumpolar distribution. We assayed mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) variation in the three main populations in the south Atlantic, south Indian, and south Pacific oceans, and a smaller continental population in South America. Population structure of mtDNA was strong and not consistent with isolation by distance. The nDNA loci, although less informative, were consistent with the mtDNA results. Geographic structure appears to be dominated by historical processes, not contemporary gene flow. Uncorrected levels of nucleotide diversity for mtDNA control region I (2.86%) and nDNA (0.09%) were similar to those in humans and mice. Mutation rates for control region I (75 x 10(-9) substitutions per site per year) and nDNA (1.23 x 10(-9)) were similar to those in other mammals. Female effective population size and total effective population size were roughly equal at approximately 4 x 10(4), indicating a twofold greater rate of drift for mtDNA. Effective breeding sex ratio of four to five females per male was estimated from nucleotide diversity and mutation rates for mtDNA and nDNA, and was much less than behavioral observations would suggest. There was no evidence for selection at any of the assayed loci.

Phylogeography of Bufo marinus from its natural and introduced ranges.

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.

Evolutionary dynamics of genetic variation in Epstein-Barr virus isolates of diverse geographical origins: evidence for immune pressure-independent genetic drift.

The question whether immune pressure exerted by cytotoxic T lymphocytes (CTLs) can influence the long-term evolution of genetically stable viruses such as Epstein-Barr virus (EBV) has generated considerable scientific interest, primarily due to its important implications for the overall biology of the virus. While arguing for a role of CTLs in the evolution of viruses, it is important to differentiate between genetic variation in virus and immune recognition of these variant virus by CTLs. To assess the role of genetic selection in the long-term evolution of EBV, we have analyzed a large panel of type 1 EBV isolates from African, Southeast Asian, Papua-New Guinean (PNG), and Australian Caucasian individuals. Seven different regions of the EBV genome, which include nine CTL epitopes restricted through a range of HLA class I alleles, were sequenced and compared. Although numerous nucleotide changes were identified within these isolates, comparison of synonymous and nonsynonymous substitutions in the CTL epitope indicated that the genetic variation was generated mostly independently of immune selection pressure. Surprisingly, an inverse correlation between genetic variation within certain CTL epitopes and the frequency distribution of HLA alleles that present the CTL epitopes was seen, suggesting that the evolutionary pressures on the CTL epitopes of the virus may be toward their conservation rather than their inactivation. Furthermore, molecular evolutionary genetic analysis of nucleotide sequences revealed that viral isolates from PNG are evolving as a lineage distinct from isolates from African, Southeast Asian, and Australian Caucasian individuals.

The expressed class II alpha-chain genes of the marsupial major histocompatibility complex belong to eutherian mammal gene families.

The major histocompatibility complex (Mhc) is a multigene family found in vertebrates. Mhc genes code for heterodimeric cell-surface molecules involved in presentation of peptides to T-lymphocytes. There are two classes of Mhc, and in eutherian mammals four main families of class II genes have been recognized; DR, DQ, DP, and DN/DO. Each class II family contains genes that code for one or more alpha and beta chains. Do the class II genes of marsupial mammals belong to any of these eutherian mammal class II families? The results to date are conflicting. The expressed class II beta-chain genes could not be satisfactorily assigned to any eutherian class II gene family and were designated as new gene families, while, conversely, a partial sequence of an expressed alpha-chain gene was clearly very similar to the DNA gene of eutherian mammals. The aim of this study was to conduct a more thorough analysis of the alpha-chain genes in a marsupial by obtaining full-length sequences of all the expressed alpha-chain genes in the red-necked wallaby, Macropus rufogriseus. Two class II alpha-chain genes were isolated from a spleen-derived cDNA library, and both have the potential to code for fully functional MHC molecules. Phylogenetic analysis indicated they belonged to previously identified eutherian class II families and are designated as Maru-DRA and Maru-DNA. Northern blot data indicated processed transcript sizes of approximately 1.6 kb for Maru-DRA and approximately 2.5 kb for Maru-DNA and that the latter was expressed at a lower level than the former. The phylogeny shows that the DR, DQ, DP, and DN/DO gene families diverged prior to the divergence of the marsupial and eutherian mammal lineages.

Hierarchical structure of mitochondrial DNA gene flow among humpback whales Megaptera novaeangliae, world-wide.

The genetic structure of humpback whale populations and subpopulation divisions is described by restriction fragment length analysis of the mitochondrial (mt) DNA from samples of 230 whales collected by biopsy darting in 11 seasonal habitats representing six subpopulations, or 'stocks', world-wide. The hierarchical structure of mtDNA haplotype diversity among population subdivisions is described using the analysis of molecular variance (AMOVA) procedure, the analysis of gene identity, and the genealogical relationship of haplotypes as constructed by parsimony analysis and distance clustering. These analyses revealed: (i) significant partitioning of world-wide genetic variation among oceanic populations, among subpopulations or 'stocks' within oceanic populations and among seasonal habitats within stocks; (ii) fixed categorical segregation of haplotypes on the south-eastern Alaska and central California feeding grounds of the North Pacific; (iii) support for the division of the North Pacific population into a central stock which feeds in Alaska and winters in Hawaii, and an eastern or 'American' stock which feeds along the coast of California and winters near Mexico; (iv) evidence of genetic heterogeneity within the Gulf of Maine feeding grounds and among the sampled feeding and breeding grounds of the western North Atlantic; and (v) support for the historical division between the Group IV (Western Australia) and Group V (eastern Australia, New Zealand and Tonga) stocks in the Southern Oceans. Overall, our results demonstrate a striking degree of genetic structure both within and between oceanic populations of humpback whales, despite the nearly unlimited migratory potential of this species. We suggest that the humpback whale is a suitable demographic and genetic model for the management of less tractable species of baleen whales and for the general study of gene flow among long-lived, mobile vertebrates in the marine ecosystem.

The marsupial MHC: the tammar wallaby, Macropus eugenii, contains an expressed DNA-like gene on chromosome 1.

In the placental mammal major histocompatibility complex (MHC) three main families of class II genes, DR, DQ, and DP, have been recognized. Each family contains genes that code for one or more A- and B-chains. Recent evidence has indicated that a fourth family can be described, the DN/DO family. These four families arose sometime early in mammalian evolution. Our purpose was to deduce the MHC of an early mammalian ancestor of marsupials and eutherians. Using primers designed to conserved regions in exon 2 and exon 3 of the DQA gene we amplified an 830-bp band from the total genomic DNA of the marsupial, Macropus eugenii (tammar wallaby). However, sequence analysis of cloned genomic products showed that the primers had amplified three genes, two of which appeared to be alleles at one locus, while the other gene belonged to a closely related locus. Phylogenetic analysis showed that both these loci were most closely related to the human (HLA-DNA) and mouse (H-20a) DNA genes, with a bootstrap support of 78%. Expression of only one locus could be detected by RT-PCR from spleen RNA. In situ hybridization to tammar wallaby chromosomes mapped these genes to one region on the long arm of chromosome 1, indicating the position of the MHC in marsupials. Related A-chain genes were detected in monotremes, and human by southern blotting, and very faint bands were observed in the chicken. Hybridization with a tammar DNA-like gene on several marsupial species showed evidence of at least three DNA-like loci in the tammar wallaby, at least one in the koala, but none in the kowari. This indicates that the organization of the class II MHC may be more dynamic in marsupial than in placental mammals, but, in contrast to a previous study on the MHC of a marsupial, we cannot conclude that the class II gene families of placental and marsupial mammals evolved from different ancestral genes.

Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny.

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.

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.

Limited MHC polymorphism in the southern elephant seal: implications for MHC evolution and marine mammal population biology.

Genes of the major histocompatibility complex (MHC) are highly polymorphic in most terrestrial mammal populations so far studied. Exceptions to this are typically populations that lack genome-wide diversity. Here I show that two populations of the southern elephant seal (Mirounga leonina) have low DNA restriction fragment length polymorphism at MHC loci when compared with terrestrial mammals. Limited studies on MHC polymorphism in two cetacean species suggest this is a feature of marine mammal populations in general. MHC polymorphism is thought to be maintained by balancing selection, and several types of disease-based and reproductive-based mechanisms have been proposed. For the three marine mammal species examined, the low MHC polymorphism cannot be explained by low genome-wide diversity, or by any reproductive-based selection pressure. It can, however, be explained by diminished exposure to pathogenic selection pressure compared with terrestrial mammals. Reduced exposure to pathogens would also mean that marine mammal populations may be susceptible to occasional pathogen-induced mass mortalities.