Radiation of the Australian flora: What can comparisons of molecular phylogenies across multiple taxa tell us about the evolution of diversity in present-day communities?

The Australian fossil record shows that from ca. 25 Myr ago, the aseasonal-wet biome (rainforest and wet heath) gave way to the unique Australian sclerophyll biomes dominated by eucalypts, acacias and casuarinas. This transition coincided with tectonic isolation of Australia, leading to cooler, drier, more seasonal climates. From 3 Myr ago, aridification caused rapid opening of the central Australian and zone. Molecular phylogenies with dated nodes have provided new perspectives on how these events could have affected the evolution of the Australian flora. During the Mid-Cenozoic (25-10 Myr ago) period of climatic change, there were rapid radiations in sclerophyll taxa, such as Banksia, eucalypts, pea-flowered legumes and Allocasuarina. At the same time, taxa restricted to the aseasonal-wet biome (Nothofagus, Podocarpaceae and Araucariaceae) did not radiate or were depleted by extinction. During the Pliocene aridification, two Eremean biome taxa (Lepidium and Chenopodiaceae) radiated rapidly after dispersing into Australia from overseas. It is clear that the biomes have different histories. Lineages in the aseasonal-wet biome are species poor, with sister taxa that are species rich, either outside Australia or in the sclerophyll biomes. In conjunction with the fossil record, this indicates depletion of the Australian aseasonal-wet biome from the Mid-Cenozoic. In the sclerophyll biomes, there have been multiple exchanges between the southwest and southeast, rather than single large endemic radiations after a vicariance event. There is need for rigorous molecular phylogenetic studies so that additional questions can be addressed, such as how interactions between biomes may have driven the speciation process during radiations. New studies should include the hither-to neglected monsoonal tropics.

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

Phylogeny of the Limnophilinae (Limoniidae) and early evolution of the Tipulomorpha (Diptera)

Tipulomorpha (craneflies) comprise one of the largest subgroups of Diptera, but its phylogeny at different levels has been poorly explored. This study presents the most comprehensive cladistic analysis of the group ever made, with emphasis on the genera and subgenera of the subfamily Limnophilinae (Limoniidae), assumed to include some of the earliest lineages of Tipulomorpha sensu stricto and therefore important for the understanding of the early patterns in the evolution of the craneflies. Eighty-eight characters of the male imago were scored for 104 exemplar species. The most parsimonious trees were searched using implied weighting, in the framework of a sensitivity analysis with different values of k (2 to 6). The dataset based on the characters of adult male morphology showed high levels of homoplasy and yielded very incongruent and unstable phylogenetic results, which are very sensitive to changes in analytical parameters. In the preferred and most parsimonious phylogenetic hypothesis, the Pediciidae is the sister-group of all other Tipulomorpha sensu stricto. The results indicate the paraphyly of the Limoniidae with respect to the Cylindrotomidae and Tipulidae, which are considered sister-groups. The Limoniidae subfamilies Limnophilinae, Limoniinae and Chioneinae are considered non-monophyletic. The study allowed a reconstruction of the possible ground plan condition of selected features of the adult male morphology of craneflies. The genera/subgenera Epiphragma (Epiphragma), Acantholimnophila, Shannonomyia, Limnophila (Arctolimnophila), Eloeophila, Conosia, Polymera, Polymera (Polymerodes), Prionolabis, Eutonia, Phylidorea (Phylidorea), Metalimnophila, Gynoplistia (Cerozodia), Gynoplistia (Dirhipis), Nothophila, Pseudolimnophila (Pseudolimnophila), Pilaria and Ulomorpha are considered monophyletic, but in general are defined by combinations of very homoplastic character states. Two Temperate Gondwanan clades, (Tonnoirella + (Edwardsomyia + (Tinemyia + (Rhamphophila + (Nothophila))))) and ((Notholimnophila + Bergrothomyia) + (Mesolimnophila + (Chilelimnophila + Ctenolimnophila))) are recovered. The genera Limnophila, Neolimnomyia, Gynoplistia (sensu lato) and Hexatoma (sensu lato) are considered non-monophyletic. The systematic position and some morphological characters of `problematic` taxa, such as Dactylolabis, Elephantomyia, Helius and Atarba are discussed on the light of the proposed phylogeny and the analysis of the characters. Character states are richly illustrated. A detailed study of the morphology of the male genitalia is made, and several genera and species have the morphology of the male genitalia illustrated for the first time.

The systematics and biology of the south African gall-inducing scale insect, Calycicoccus merwei brain (Hemiptera : Coccoidea : Eriococcidae)

The scale insect genus Calycicoccus Brain has a single described species, C. merwei Brain, which is endemic to southeastern South Africa. Females of C. merwei induce small, mostly conical galls on the foliage of their host tree, Apodytes dimidiata E. Meyer ex Arn. (Icacinaceae), which has a wider, mostly coastal distribution, than that currently known for the scale insect. Calycicoccus has been placed in the family Eriococcidae and may be related to the South American genus Aculeococcus Lepage. No other native eriococcid species have been described so far in South Africa, although the family is diverse in other Gondwanan regions. This paper summarizes the biology of C. merwei, redescribes the adult female, describes the adult male, the second-instar female and the first-instar nymphs for the first time, and reconsiders the phylogenetic relationships of the genus. The adult female is shown to have unusual abdominal segmentation, in that segment I is present both dorsally and ventrally, but a segment is absent ventrally on the middle abdomen. First-instar nymphs are sexually dimorphic; males have a larger and relatively narrower body, larger mouthparts, longer antennae and legs, and more thoracic dorsal setae compared with females. Molecular data from nuclear small-subunit ribosomal DNA (18S) and elongation factor 1 alpha (EF-1a) show C. merwei to have no close relatives among the Eriococcidae sampled to date. Instead, the Calycicoccus lineage is part of a polytomy near the base of the Eriococcidae. Molecular dating of the node suggests that the Calycicoccus lineage diverged from other eriococcids more than 100 Mya. These data support the placement of Calycicoccus as the only genus in the subfamily Calycicoccinae Brain.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

THE TEMPO AND MODE OF EVOLUTION OF TRANSPOSABLE ELEMENTS AS REVEALED BY MOLECULAR PHYLOGENIES RECONSTRUCTED FROM MOSQUITO GENOMES

Although many mathematical models exist predicting the dynamics of transposable elements (TEs), there is a lack of available empirical data to validate these models and inherent assumptions. Genomes can provide a snapshot of several TE families in a single organism, and these could have their demographics inferred by coalescent analysis, allowing for the testing of theories on TE amplification dynamics. Using the available genomes of the mosquitoes Aedes aegypti and Anopheles gambiae, we indicate that such an approach is feasible. Our analysis follows four steps: (1) mining the two mosquito genomes currently available in search of TE families; (2) fitting, to selected families found in (1), a phylogeny tree under the general time-reversible (GTR) nucleotide substitution model with an uncorrelated lognormal (UCLN) relaxed clock and a nonparametric demographic model; (3) fitting a nonparametric coalescent model to the tree generated in (2); and (4) fitting parametric models motivated by ecological theories to the curve generated in (3).

High rates of molecular evolution in hantaviruses

Hantaviruses are rodent-borne Bunyaviruses that infect the Arvicolinae, Murinae, and Sigmodontinae subfamilies of Muridae. The rate of molecular evolution in the hantaviruses has been previously estimated at approximately 10(-7) nucleotide substitutions per site, per year (substitutions/site/year), based on the assumption of codivergence and hence shared divergence times with their rodent hosts. If substantiated, this would make the hantaviruses among the slowest evolving of all RNA viruses. However, as hantaviruses replicate with an RNA-dependent RNA polymerase, with error rates in the region of one mutation per genome replication, this low rate of nucleotide substitution is anomalous. Here, we use a Bayesian coalescent approach to estimate the rate of nucleotide substitution from serially sampled gene sequence data for hantaviruses known to infect each of the 3 rodent subfamilies: Araraquara virus ( Sigmodontinae), Dobrava virus ( Murinae), Puumala virus ( Arvicolinae), and Tula virus ( Arvicolinae). Our results reveal that hantaviruses exhibit shortterm substitution rates of 10(-2) to 10(-4) substitutions/site/year and so are within the range exhibited by other RNA viruses. The disparity between this substitution rate and that estimated assuming rodent-hantavirus codivergence suggests that the codivergence hypothesis may need to be reevaluated.

Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes

Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties Of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (Delta C(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in Delta C(p) on should include effects arising from the prot inlsolvent interface.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

The evolution of controlled multitasked gene networks: The role of introns and other noncoding RNAs in the development of complex organisms

Eukaryotic phenotypic diversity arises from multitasking of a core proteome of limited size. Multitasking is routine in computers, as well as in other sophisticated information systems, and requires multiple inputs and outputs to control and integrate network activity. Higher eukaryotes have a mosaic gene structure with a dual output, mRNA (protein-coding) sequences and introns, which are released from the pre-mRNA by posttranscriptional processing. Introns have been enormously successful as a class of sequences and comprise up to 95% of the primary transcripts of protein-coding genes in mammals. In addition, many other transcripts (perhaps more than half) do not encode proteins at all, but appear both to be developmentally regulated and to have genetic function. We suggest that these RNAs (eRNAs) have evolved to function as endogenous network control molecules which enable direct gene-gene communication and multitasking of eukaryotic genomes. Analysis of a range of complex genetic phenomena in which RNA is involved or implicated, including co-suppression, transgene silencing, RNA interference, imprinting, methylation, and transvection, suggests that a higher-order regulatory system based on RNA signals operates in the higher eukaryotes and involves chromatin remodeling as well as other RNA-DNA, RNA-RNA, and RNA-protein interactions. The evolution of densely connected gene networks would be expected to result in a relatively stable core proteome due to the multiple reuse of components, implying,that cellular differentiation and phenotypic variation in the higher eukaryotes results primarily from variation in the control architecture. Thus, network integration and multitasking using trans-acting RNA molecules produced in parallel with protein-coding sequences may underpin both the evolution of developmentally sophisticated multicellular organisms and the rapid expansion of phenotypic complexity into uncontested environments such as those initiated in the Cambrian radiation and those seen after major extinction events.

A total-evidence phylogeny of ticks provides insights into the evolution of life cycles and biogeography

We inferred the phylogeny of 33 species of ticks from the subfamilies Rhipicephalinae and Hyalomminae from analyses of nuclear and mitochondrial DNA and morphology. We used nucleotide sequences from 12S rRNA, cytochrome c oxidase I, internal transcribed spacer 2 of the nuclear rRNA, and 18S rRNA. Nucleotide sequences and morphology were analyzed separately and together in a total-evidence analysis. Analyses of the five partitions together (3303 characters) gave the best-resolved and the best-supported hypothesis so far for the phylogeny of ticks in the Rhipicephalinae and Hyalomminae, despite the fact that some partitions did not have data for some taxa. However, most of the hidden conflict (lower support in the total-evidence analyses compared to that in the individual analyses) was found in those partitions that had taxa without data. The partitions with complete taxonomic sampling had more hidden support (higher support in the total-evidence analyses compared to that in the separate-partition analyses) than hidden conflict. Mapping of geographic origins of ticks onto our phylogeny indicates an African origin for the Rhipicephalinae sensu lato (i.e., including Hyalomma spp.), the Rhipicephalus-Boophilus lineage, the Dermacentor-Anocentor lineage, and the Rhipicephalus-Booophilus-Nosomma-Hyalomma-Rhipicentor lineage. The Nosomma-Hyalomma lineage appears to have evolved in Asia. Our total-evidence phylogeny indicates that (i) the genus Rhipicephalus is paraphyletic with respect to the genus Boophilus, (ii) the genus Dermacentor is paraphyletic with respect to the genus Anocentor, and (iii) some subgenera of the genera Hyalomma and Rhipicephalus are paraphyletic with respect to other subgenera in these genera. Study of the Rhipicephalinae and Hyalomminae over the last 7 years has shown that analyses of individual datasets (e.g., one gene or morphology) seldom resolve many phylogenetic relationships, but analyses of more than one dataset can generate well-resolved phylogenies for these ticks. (C) 2001 Academic Press.

Interaction between natural and sexual selection during the evolution of mate recognition

The interaction between natural and sexual selection is central to many theories of how mate choice and reproductive isolation evolve, but their joint effect on the evolution of mate recognition has not, to my knowledge, been investigated in an evolutionary experiment. Natural and sexual selection were manipulated in interspecific hybrid populations of Drosophila to determine their effects on the evolution of a mate recognition system comprised of cuticular hydrocarbons (CHCs). The effect of natural selection in isolation indicated that CHCs were costly for males and females to produce. The effect of sexual selection in isolation indicated that females preferred males with a particular CHC composition. However, the interaction between natural and sexual selection had a greater effect on the evolution of the mate recognition system than either process in isolation. When natural and sexual selection were permitted to operate in combination, male CHCs became exaggerated to a greater extent than in the presence of sexual selection alone, and female CHCs evolved against the direction of natural selection. This experiment demonstrated that the interaction between natural and sexual selection is critical in determining the direction and magnitude of the evolutionary response of the mate recognition system.