Population structure, effective population size and adverse effects of stocking in the endangered Australian eastern freshwater cod Maccullochella ikei.

Microsatellite markers were used to examine spatio-temporal genetic variation in the endangered eastern freshwater cod Maccullochella ikei in the Clarence River system, eastern Australia. High levels of population structure were detected. A model-based clustering analysis of multilocus genotypes identified four populations that were highly differentiated by F-statistics (F(ST) = 0·09 - 0·49; P < 0·05), suggesting fragmentation and restricted dispersal particularly among upstream sites. Hatchery breeding programmes were used to re-establish locally extirpated populations and to supplement remnant populations. Bayesian and frequency-based analyses of hatchery fingerling samples provided evidence for population admixture in the hatchery, with the majority of parental stock sourced from distinct upstream sites. Comparison between historical and contemporary wild-caught samples showed a significant loss of heterozygosity (21%) and allelic richness (24%) in the Mann and Nymboida Rivers since the commencement of stocking. Fragmentation may have been a causative factor; however, temporal shifts in allele frequencies suggest swamping with hatchery-produced M. ikei has contributed to the genetic decline in the largest wild population. This study demonstrates the importance of using information on genetic variation and population structure in the management of breeding and stocking programmes, particularly for threatened species.

Genetic Differentiation among Karyotypic Forms of the Black Rat, RATTUS RATTUS.

The black rat, Rattus rattus, consists of five karyotypic forms-2n = 42 (high C-banding); 2n = 42 (low C-banding); 2n = 40; 2n = 38; 2n = 42 Mauritius. Here, we use isozyme electrophoresis and microcomplement fixation to elucidate the genetic distance and phylogenetic relationship among each of the various karyotypic forms of R. rattus and R. norvegicus . The results show that (1) the 2n = 42 Mauritius black rat (2n = 42Mau) is genetically very similar to the 2n = 38 form, suggesting that this island population has undergone very rapid chromosomal evolution; (2) the 2n = 40 form from the highlands of Sri Lanka is genetically distinct from the 2n = 38 form from the lowlands; the genetic difference is probably insufficient, however, to prevent future introgression; (3) the level of genetic differentiation occurring between the 2n = 42 forms on the one hand and the 2n = 38, 2n = 40 and 2n = 42 Mau forms on the other support the hybrid incompatability data in suggesting that the two groups are either full species or incipient species; (4) in contrast to data from amino acid composition of transferrin and from restriction endonuclease digests of mtDNA, the present data suggest that the various karyotypic forms of R. rattus are phylogenetically more closely related to each other than any is to R. norvegicus, and that they are related by a series 2n = 42 --> 2n = 40 --> 2n = 38; (5) the R. rattus/R. norvegicus divergence occurred 2-8 million years ago, whereas the various chromosomal forms of R. rattus diverged over the last 4 million years.

The structure and evolution of immunoglobulin kappa chain constant region genes in the genus Rattus.

We have cloned and sequenced the C-kappa (Ck) genes from seven species and subspecies of rats which have diverged over the past few million years in Australia. Comparisons of these sequences with each other and the Ck genes of the laboratory rat, Rattus norvegicus, indicate noncoding regions have accumulated fewer mutations than adjacent coding sequences, and amino acid replacing nucleotide substitutions in the coding regions have accumulated at a rate at least as great as silent changes. Exactly the opposite of both of these findings is observed when comparisons are made between Ck or other genes from more distantly related species, indicating that these features may be characteristic of Ck short-term evolutionary gene divergence. Changes in the coding regions of these genes result in a non-random distribution of amino acid substitutions on the three-dimensional alpha-carbon backbone of the Ck domain in the most serologically distinct forms of Ck. While phylogenetic relationships inferred from the Ck nucleotide sequences are in general agreement with those derived from other data, considerable differences are seen in rates of accumulation of Ck gene nucleotide substitutions vs rates of accumulation of enzyme polymorphisms.

Ribosomal DNA sequence comparison of Babesia and Theileria.

Previous studies on the taxonomy of Babesia spp. (phylum Apicomplexa) using morphological and life cycle characteristics have resulted in their classification into 3 subgenera, with the genus Theileria being most closely related to them. Using a strategy based on the direct sequence analysis of products derived by asymmetric PCR to determine the nucleotide sequences, we have tested the validity of this classification by sequencing the small subunit ribosomal RNA genes amplified from 2 Babesia species, namely Babesia bovis and Babesia rodhaini, and comparing these with previously published sequences of Theileria annulata and Babesia bigemina using Plasmodium falciparum as an outgroup. The results of this phylogenetic analysis support the recognition of at least 2 genera in Babesia--one to include B. bigemina and B. bovis, the other to include B. rodhaini.

The phylogeny of Neospora caninum.

Morphological studies by electron microscopy on the protozoan Neospora caninum have shown that this organism possesses a subcellular structure typical of parasites classified in the family Sarcocystidae, subclass Coccidiasina of the phylum Apicomplexa. Using a strategy based on DNA sequence analysis of products derived by asymmetric PCR to determine the nucleotide sequences, we have tested the validity of this classification by comparing the small subunit ribosomal RNA (18S rRNA) gene sequences of N. caninum with those of other parasitic protozoa classified in the phylum Apicomplexa. The results of this analysis confirm the placing of N. caninum in the family Sarcocystidae and place it as a sister group to Toxoplasma gondii in the phylum Apicomplexa.

Phylogenetic relationships of the thylacine (Mammalia: Thylacinidae) among dasyuroid marsupials: evidence from cytochrome b DNA sequences.

DNA sequences from the mitochondrial cytochrome b gene were obtained from a museum specimen of the presumed extinct thylacine (Thylacinus cynocephalus) and were compared with homologous sequences from 13 representatives of the Australian marsupial family Dasyuridae. The relationship of the thylacine to dasyurids has been suggested by previous anatomical and molecular studies, but its position within the dasyuroid radiation has not been addressed with genetic data. Phylogenetic analysis of the sequences reported here suggests that the thylacine is a sister group to Dasyuridae and lends support to the hypothesis that Thylacinus represents an ancient Australian marsupial lineage. Relationships with Dasyuridae support the results of other recent molecular studies, particularly in showing the affinities of endemic New Guinean subfamilies with larger Australian clades.

Phylogenetic relationships of Sarcocystis species from sheep, goats, cattle and mice based on ribosomal RNA sequences.

Partial sequences of the small subunit ribosomal RNA of four species of Sarcocystis were obtained by reverse transcription of total cellular RNA. The semi-conserved regions of these four species were aligned with homologous sequences of two other Sarcocystis species and a range of other eukaryotes including Toxoplasma, Eimeria and Cryptosporidium. Parsimony analysis of the aligned sequences showed that Sarcocystis and Toxoplasma had a more recent common ancestor with Eimeria than with Cryptosporidium. The six Sarcocystis spp. did not cluster together in this analysis; two monophyletic groups were observed, one formed by the two Sarcocystis spp. with felids as definitive hosts, and another by the four Sarcocystis spp. with canids as definitive hosts. These two clades were segregated by Toxoplasma. An analysis of nucleotide divergence suggests that the difference between the two groups of Sarcocystis spp. is similar to that between invertebrates and vertebrates. The results obtained here question the validity of a separation of the genus Sarcocystis from Toxoplasma and refute classifications that place these two genera into two different subfamilies of the Sarcocystidae.

The phylogenetic relationships of Kronborgia (Platyhelminthes, Fecampiida) based on comparison of 18S ribosomal DNA sequences.

Approximately 580 bp at the 5' end of the small subunit RNA gene were amplified by PCR for 19 platyhelminth taxa, and Homo and Artemia were used as outgroups. These were analysed to test the hypothesis that fecampiids and Neodermata are sister groups. No evidence was found that the fecampiid Kronborgia isopodicola is closely related to the Neodermata or to the Rhabdocoela (in which the fecampiids are usually included). Morphological, including ultrastructural, characters and DNA data do not support a close relationship of fecampiids with any other platyhelminth taxon, although the DNA sequence analysis provides some evidence that the Acoela and Tricladida are closest. Fecampiids are sufficiently different from any other platyhelminth group to warrant establishment of a class, Fecampiida, for them. A diagnosis of the new class is given.

Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison.

Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa.

Conflicting phylogenetic hypotheses for the parasitic platyhelminths tested by partial sequencing of 18S ribosomal RNA.

Partial sequencing of the 18S ribosomal RNA in nine parasitic and one free-living species of platyhelminth was used to test hypotheses on the phylogenetic relationships among the major groups. The eucestodes, amphilinideans, gyrocotylideans and monopisthocotylideans appeared as a monophyletic assemblage in a cladistic analysis of the data, with a very close association between the gyrocotylideans and monopisthocotylideans. The polyopisthocotylidean monogeneans were paraphyletic to the monopisthocotylideans. The digeneans appeared to be a sister group to the monogeneans and eucestodes, while the temnocephalidean was closely related to the free-living polyclad.

Genetic characterization of three strains of Hymenolepis diminuta at 39 enzyme loci.

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.

Contributions to the phylogeny of Platyhelminthes based on partial sequencing of 18S ribosomal DNA.

Partial sequencing of the 18S ribosomal DNA gene of one nemertean and 13 free-living and parasitic Platyhelminthes (556 nucleotides), and of one nemertean and 20 Platyhelminthes (556 nucleotides) was used to test several hypotheses concerning the phylogenetic relationships of Platyhelminthes. The following conclusions were reached: the Neodermata is monophyletic; Trematoda (Aspidogastrea and Digenea) is monophyletic, although a sister group relationship of the Aspidogastrea and all other Neodermata cannot be definitely ruled out; the Cestoda comprising the Eucestoda, Amphilinidea and Gyrocotylidea is monophyletic; it is unresolved whether the Monogenea is paraphyletic; neither Gyrocotylidea and Monopisthocotylea nor Gyrocotylidea and Monogenea as a whole are sister groups; Anoplodiscus is a monopisthocotylean monogenean; none of Proseriata, Pterastericolidae/Umagillidae, Kalyptorhynchia, Rhabdocoela as a whole, Dalyelliida or the Temnocephalidae is the sister group of the Neodermata; there is some evidence that a larger taxon consisting of Proseriata, Tricladida and Rhabdocoela may be the sister group of the Neodermata but definitive evidence for a sister group relationship between the Neodermata and any turbellarian taxon is lacking; Rhabdocoela and Lecithoepitheliata are not closely related; it is unresolved whether the Rhabdocoela is monophyletic; Umagillidae, Pterastericolidae and Temnocephalidae belong to one monophylum; the Temnocephalidae are very close to the dalyelliids; Tricladida and Rhabdocoela are sister groups, the exact position of the Catenulida and Nemertini in relation to the Platyhelminthes has not been resolved, although Catenulida and Lecithoepitheliata may belong to one clade.

A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis.

The present study employs allozyme electrophoresis to characterize and inter-relate 61 isolates of Naegleria. Diploidy was confirmed, with heterozygotes observed at 29 of the 33 loci established and in all but two isolates. With a single exception, isolates clustered at two levels of similarity, either below 21% or above 52%. It is argued that such a major discontinuity provides a sound biological basis for a species concept in Naegleria. On this basis the present species-level taxonomy does not reflect the genetic diversity of the genus. The study recognized 18 genetic groups of species rank. The subspecies N. australiensis italica deserves specific rank; additional thermophilic species not closely related to N. fowleri and N. lovaniensis are recognized; and N. gruberi as currently conceived is a complex of 10 species, at least five of which are represented in the formal culture collections. Most species are genetically too different for relationships to be elucidated by allozyme electrophoresis, supporting the view that some of the times of divergence within the genus are extremely ancient.

The probable basis of the relationship between growth rate and winter mortality in the lizard Amphibolurus ornatus (Agamidae).

At Bakers Hill, differential winter mortality of juvenile slowgrowing Amphibolurus ornatus is correlated with the incidence of frosts (Bradshaw, 1971). The present study shows that the probability an individual will spend the night in a refuge that is safe from the lethal effect of a frost is directly related to the individual's size. Thus should frosts occur during any particular winter, juvenile slow-growers, will suffer a higher mortality than juvenile fast-growers. At Tuttanning, animals do not segregate according to size, and consequently juvenile slow-growers do not suffer increased mortality during frosty winters.

Phylogenetic relationships of the apicomplexan protist Sarcocystis as determined by small subunit ribosomal RNA comparison.

Reverse transcription of total cellular RNA was used to obtain the partial nucleotide sequence of the small subunit ribosomal RNA (srRNA) of Sarcocystis gigantea. The sequence was compared with the homologous sequences of 24 other eukaryotes. Phylogenetic analysis of the semiconserved regions by 4 different tree-building methods using bacteria as an outgroup all concur in showing monophyly of Sarcocystis gigantea and Toxoplasma gondii to the exclusion of all other taxa for which homologous sequences are available.

Evolutionary lineages of RT1.Ba in the Australian Rattus.

In this study, the evolutionary history of the variable second exon of RT1.Ba and its adjoining intron b are compared across a number of species and subspecies of the Australian RATTUS: Three lineages are identified in the second intron across a range of Rattus species. Two of these lineages, separated by the insertion of a probable rodent short interspersed nucleotide element and by point mutations outside the indel region, are both found in each of the major clades of the endemic Australian RATTUS: This pattern of ancestral polymorphism is reflected in the adjoining exon 2 sequences, although phylogenetic constraints confirm that the clustering is not identical to that of the associated intron sequences. In addition, the coding sequences show evidence of the retention of ancestral polymorphism, with identical exon sequences found in two divergent species, and some indication of gene conversion detected for the exon sequences.

Rat kappa-chain allotypes. III. Serological and genetic analysis of RI-1b cross-reactivity in Australian Rattus.

The presence of rat kappa-chain allotype specificities (RI-1a and b) has been studied in 13 subspecies of the seven native Australian species of Rattus. RI-1a reactivity was not detected among these rats. On the other hand, extensive cross-reactivity was seen with RI-1b, some sera cross-reacting totally (R. leucopus cooktownensis), some not at all (R. colletti), and the remainder showing at least two distinct levels of partial cross-reactivity, confirming the existence of multiple specificities for RI-1b. Three subspecies show polymorphism with respect to RI-1b cross-reactivity (R. sordidus, R. colletti, and R. l. leucopus) and in one case (leucopus) breeding studies have indicated that there is allelic inheritance of this trait. The segregation of RI-1b reactivity has been studied in crosses and backcrosses made between species differing in their RI-1b reactivity, and the results are consistent with the existence of codominant alleles at a single locus. The fact that these species differ extensively in their karyotype opens the door to possible chromosomal localization of this and other genetic traits.