David A. Morrison

EVOLUTION OF THE GENUS LEISHMANIA REVEALED BY COMPARISON OF DNA AND RNA POLYMERASE GENE SEQUENCES

Previous hypotheses of Leishmania evolution are undermined by limitations in the phylogenetic reconstruction method employed or due to the omission of key parasites. In this experiment, sequences of the gene encoding the DNA polymerase alpha catalytic polypeptide (POLA) were analysed phylogenetically in combination with those encoding the RNA polymerase II largest subunit gene (RPOIILS) to infer a comprehensive phylogeny of Leishmania. Nineteen species of parasites were studied, comprising representatives of each Leishmania species-complex (Leishmania Leishmania tropica, Leishmania Leishmania donovani, Leishmania Leishmania mexicana, Leishmania Leishmania hertigi and Leishmania Viannia braziliensis), as well as parasites of questionable taxonomy (Leishmania herreri, Sauroleishmania adleri, Sauroleishmania deanei, Sauroleishmania gymnodactyli and Sauroleishmania tarentolae). The analyses presented here provide strong support for the hypothesis that the Leishmania that infect reptiles (also known as Sauroleishmania) evolved from mammalian Leishmania. One implication of this finding is that the taxonomic definition of Leishmania should be broadened to encompass characteristics of the reptilian parasites. However, this taxonomic revision is complicated in that Leishmania (L.) hertigi, Leishmania (L.) deanei and Leishmania herreri, which exhibit some biological properties of Leishmania, are more closely related to Endotrypanum on the basis of these sequence comparisons. Consequently, the taxonomic discrimination between Leishmania that infect mammals, Leishmania that infect reptiles and Endotrypanum may be more problematic than has been previously thought. Since our resulting phylogenetic hypothesis is supported by the analyses of two different genes, we speculate on the origin and evolutionary expansion of this lineage of kinetoplastid protozoa.

The conceptual basis for a new classification of the coccidia

At the joint meeting of the 8th International Coccidiosis Conference and the Annual Scientific Meeting of the Australian Society for Parasitology in Palm Cove, Australia, in July 2001, a Controversial Roundtable was held on New classification of coccidia. The aim of this Roundtable was to stimulate and encourage discussion and debate on current classification schemes for the group of parasitic protozoa known as the eimeriid coccidia. In the past, such classifications have been based only on phenotypic characters such as morphology, ultrastructure, life cycles, and host specificity. However, over the past 10-15 years, molecular phylogenetic studies on taxa of the eimeriid coccidia have revealed that several of the families, subfamilies, and genera that have been erected based on non-molecular characters are paraphyletic. Therefore, this Roundtable was an important forum for initial discussions on how a new and more comprehensive classification of the eimeriid coccidia, which takes into consideration both phenotypic and molecular characters, can be devised. The stimulus came from invited speakers who gave introductions into selected areas of taxonomy and classification. Following these introductions, a more general discussion with the audience addressed potential steps that may be taken in future work. This review is the immediate outcome of the Roundtable. It describes advantages and disadvantages of the use of phenotypic or molecular characters as the base for taxonomic schemes for eimeriid coccidia. It gives specific examples for drawbacks of current classifications based only on phenotypic characters as well as potential pitfalls associated with the use of only molecular phylogenies. It addresses current controversies as well as rules of taxonomy and nomenclature relevant for the eimeriid coccidia. Finally, it recommends the establishment of an international group of scientists to meet on a regular basis, stimulate further discussions, and give direction on how the final goal, i.e. a proposal for a revised, and widely accepted, classification of the eimeriid coccidia, may be achieved.

Conservation conflicts over burning bush in south-eastern Australia

Current fire management practices in the fire-prone vegetation of south-eastern Australia are based mainly on the concept of hazard reduction via the use of periodic low-intensity fires to maintain the amount of flammable fuel within specified (low) limits. We examined the possible conflict between the requirements of fire management for hazard reduction and requirements for species conservation in the dry-sclerophyll shrublands and woodlands of the Sydney region. Our data indicate that potentially severe fire hazards (fine fuel loads of ≥10 t.ha−1) can reappear in the woodland and shrubland vegetation within 2–4 years after low-intensity fires, such as are typical of the fuel-reduction burns usually prescribed. Our data also show that low-intensity fires will have significant effects on the species composition of the communities if they occur with an inter-fire interval of less than 7–8 years, causing a significantly reduced abundance of long-lived woody shrub species. n nThere is thus a clear conflict in south-eastern Australia between fire management practices based solely on prescribed burning for hazard reduction and the fire management practices necessary to maintain ecosystem biodiversity, and this conflict is greatest for fire-sensitive shrub species. The conflict between these two vegetation management objectives cannot be resolved by a simple compromise, as prescribed fires with inter-fire intervals any greater than 4 years will allow a potentially severe fire hazard to exist, while a burning regime with any inter-fire intervals less than 8 years will result in loss of biodiversity. This conflict means that it will probably not be possible to achieve simultaneously both hazard reduction and species conservation for any specified managed area.

Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites.

Molecular phylogeny of Besnoitia and the genetic relationships among Besnoitia of cattle, wildebeest and goats.

Knowledge on parasites of the genus Besnoitia is sparse, which are classified in the subfamily Toxoplasmatinae of the phylum Apicomplexa. This arrangement hypotheses that Besnoitia represents the sister group to species such as Toxoplasma gondii and Hammondia hammondi. In order to test this hypothesis, phylogenetic analyses of 18S ribosomal DNA (rDNA) from Besnoitia, Hammondia, Isospora, Frenkelia, Eimeria, Neospora, Sarcocystis and Toxoplasma were performed. The 18S rDNA of Besnoitia besnoiti, Besnoitia jellisoni and Eimeria alabamensis were amplified by PCR and sequenced. Phylogenetic analyses by parsimony and maximum-likelihood methods showed Besnoitia to be reproducibly the sister group to a clade containing Hammondia, Neospora and Toxoplasma. Furthermore, Besnoitia of cattle, wildebeest and goats had identical ITS1 rDNA sequences, which questions the use of the taxon Besnoitia caprae to describe the Besnoitia found in goats.

AN ASSESSMENT OF SOME IMPROVED TECHNIQUES FOR ESTIMATING THE ABUNDANCE (FREQUENCY) OF SEDENTARY ORGANISMS

The traditional sampling method for estimating frequency (the number of sub-quadrats containing a basal part of the organisms) is compared, using both computer simulations and direct comparison in the field, to two new methods that use a compound series of variable-sized concentric sub-quadrats. Both the new frequency-score and the new importance-score methods are closer approximations of density than is the standard frequency method, and the estimates produced by both of the new methods are less affected by the choice of sub-quadrat size and the spatial distribution (dispersion) of the organisms (i.e. clumping and regularity). Thus, the two nested-quadrat methods appear to ameliorate the usual frequency limitations associated with sub-quadrat size and organism dispersion, by the use of a range of different sub-quadrat sizes. This is important in community studies, where the component species may show a wide range of densities and dispersions. Both of the new methods are easily employed in the field. The importance-score method involves no more sampling effort than does standard qualitative (presence-absence) sampling, and it can therefore be used to sample a larger quadrat area than would normally be used for frequency sampling. This makes the method much more cost-effective as a means of estimating abundance, and it allows a greater number of the rarer species to be included in the sampling. The frequency-score method is more time-consuming, but it is capable of detecting more subtle community patterns. This means that it is particularly useful for the study of species-poor communities or where small variations in composition need to be detected.

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.

The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

Analysis of dinucleotide frequency and codon usage in the phylum Apicomplexa

Dinucleotide frequency (DiF) and codon usage (cu) were analysed in gene sequences from four parasitic protozoa, Babesia bovis, Theileria parva, Toxoplasma gondii and Eimeria tenella, of the phylum Apicomplexa. In keeping with the genome hypothesis, cu was found to be non-random and species specific in these organisms, although cu among members of the same subclass was found to be very similar. Several low-usage (lu) codons were identified, and the usage of lu codons appears to be related to the taxonomic position of the organisms under study. A comparison of the observed/expected DiF ratios obtained from gene coding regions revealed a low frequency of the TA and CG dinucleotides in all organisms studied. A comparison of these DiF ratios with those found in rRNA-encoding genes and in introns, showed that in the parasites, B. bovis and Th. parva (representing the piroplasms), the low frequency of dinucleotides appeared to be the result of coding pressure alone. In T. gondii and E. tenella (representing the coccidia), however, coding pressure could not completely explain differences in DiF.

Phylogenetic tree-building

Cladistic analysis is an approach to phylogeny reconstruction that groups taxa in such a way that those with historically more-recent ancestors form groups nested within groups of taxa with more-distant ancestors. This nested set of taxa can be represented as a branching diagram or tree (a cladogram), which is an hypothesis of the evolutionary history of the taxa. The analysis is performed by searching for nested groups of shared derived character states. These shared derived character states define monophyletic groups of taxa (clades), which include all of the descendants of the most recent common ancestor. If all of the characters for a set of taxa are congruent, then reconstructing the phylogenetic tree is unproblematic. However, most real data sets contain incongruent characters, and consequently a wide range of tree-building methods has been developed. These methods differ in a variety of characteristics, and they may produce topologically distinct trees for a single data set. None of the currently-available methods are simultaneously efficient, powerful, consistent and robust, and thus there is no single ideal method. However, many of them appear to perform well under a wide range of conditions, with the exception of the UPGMA method and the Invariants method.