Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Mutation rate and the cost of complexity

Two recent theoretical studies of adaptation suggest that more complex organisms tend to adapt more slowly. Specifically, in Fisher's "geometric" model of a finite population where multiple traits are under optimizing selection, the average progress ensuing from a single mutation decreases as the number of traits increases--the "cost of complexity." Here, I draw on molecular and histological data to assess the extent to which on a large phylogenetic scale, this predicted decrease in the rate of adaptation per mutation is mitigated by an increase in the number of mutations per generation as complexity increases. As an index of complexity for multicellular organisms, I use the number of visibly distinct types of cell in the body. Mutation rate is the product of mutational target size and population mutation rate per unit target. Despite much scatter, genome size appears to be positively correlated with complexity (as indexed by cell-type number), which along with other considerations suggests that mutational target size tends to increase with complexity. In contrast, effective population mutation rate per unit target appears to be negatively correlated with complexity. The net result is that mutation rate probably does tend to increase with complexity, although probably not fast enough to eliminate the cost of complexity.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Improved consensus network techniques for genome-scale phylogeny

Although recent studies indicate that estimating phylogenies from alignments of concatenated genes greatly reduces the stochastic error, the potential for systematic error still remains, heightening the need for reliable methods to analyze multigene data sets. Consensus methods provide an alternative, more inclusive, approach for analyzing collections of trees arising from multiple genes. We extend a previously described consensus network method for genome-scale phylogeny (Holland, B. R., K. T. Huber, V. Moulton, and P. J. Lockhart. 2004. Using consensus networks to visualize contradictory evidence for species phylogeny. Mol. Biol. Evol. 21:1459-1461) to incorporate additional information. This additional information could come from bootstrap analysis, Bayesian analysis, or various methods to find confidence sets of trees. The new methods can be extended to include edge weights representing genetic distance. We use three data sets to illustrate the approach: 61 genes from 14 angiosperm taxa and one gymnosperm, 106 genes from eight yeast taxa, and 46 members of a gene family from 15 vertebrate taxa.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Investigating the intron recognition mechanism in eukaryotes

Recent studies indicate that many introns, as well as the complex spliceosomal mechanism to remove them, were present early in eukaryotic evolution. This study examines intron and exon characteristics from annotations of whole genomes to investigate the intron recognition mechanism. Exon definition uses the exon as the unit of recognition, placing length constraints on the exon but not on the intron (allowing it a greater range of lengths). In contrast, intron definition uses the intron itself as the unit of recognition and thus removes constraints on internal exon length forced by the use of an exon definition mechanism. Thus, intron and exon lengths within a genome can reflect the constraints imposed by its splicing. This study shows that it is possible firstly to recover valid intron and exon information from genome annotation. We then compare internal intron and exon information from a range of eukaryotic genomes and investigate possible evolutionary length constraints on introns and exons and how they can impact on the intron recognition mechanism. Results indicate that exon definition-based mechanisms may predominate in vertebrates although the exact system in fish is expected to show some differences with the better characterized system from mammals. We also raise the possibility that the last common ancestor of plants and animals contained some type of exon definition and that this mechanism was replaced in some genes and lineages by intron definition, possibly as a result of intron loss and/or intron shortening.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Accurate inference and estimation in population genomics

Both intra- and interspecific genomic comparisons have revealed local similarities in the level and frequency of mutational variation, as well as in patterns of gene expression. This autocorrelation between measurements leads to violations of assumptions of independence in many statistical methods, resulting in misleading and incorrect inferences. Here I show that autocorrelation can be due to many factors and is present across the genome. Using a one-dimensional spatial stochastic model, I further show how previous results can be employed to correct for autocorrelation along chromosomes in population and comparative genomics research. When multiple hypothesis tests are autocorrelated, I demonstrate that a simple correction can lead to increased power in statistical inference. I present a preliminary analysis of population genomic data from Drosophila simulans to show the ubiquity of autocorrelation and applicability of the methods proposed here.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Molecular genetics of natural populations

Significant progress in evolutionary genetics has been made by studying, on the one hand, patterns of DNA sequence polymorphism and, on the other, genetic architecture of complex adaptive traits. However, connections between nucleotide variants under selection and adaptively relevant phenotypes are missing. Such connections can be established using precise gene replacement. We review the recent successful introduction of this technique to the analysis of two evolutionarily interesting loci--Odysseus and desaturase2. Both genes have subtle phenotypes that nevertheless could be identified using gene replacement, demonstrating that effects of naturally occurring alleles can be measured in the laboratory. This is an important first step in connecting statistical signatures of selection with adaptation in nature. More candidate genes involved in adaptation, for example, through cloning of genes responsible for reproductive isolation, now need to be identified. Molecular genetic manipulation, DNA polymorphism analysis, and field studies then have to be integrated to provide fresh insights into the mechanisms of evolutionary change.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Unusual evolution of interspersed repeat sequences in the Drosophila ananassae subgroup

New repeat sequences were found in the Drosophila ananassae genome sequence. They accounted for approximately 1.2% of the D. ananassae genome and were estimated to be more abundant in genomes of its closely related species belonging to the Drosophila bipectinata complex, whereas it was entirely absent in the Drosophila melanogaster genome. They were interspersed throughout euchromatic regions of the genome, usually as short tandem arrays of unit sequences, which were mostly 175-200 bp long with two distinct peaks at 180 and 189 bp in the length distribution. The nucleotide differences among unit sequences within the same array (locus) were much smaller than those between separate loci, suggesting within-locus concerted evolution. The phylogenetic tree of the repeat sequences from different loci showed that divergences between sequences from different chromosome arms occurred only at earlier stages of evolution, while those within the same chromosome arm occurred thereafter, resulting in the increase in copy number. We found RNA polymerase III promoter sequences (A box and B box), which play a critical role in retroposition of short interspersed elements. We also found conserved stem-loop structures, which are possibly associated with certain DNA rearrangements responsible for the increase in copy number within a chromosome arm. Such an atypical combination of characteristics (i.e., wide dispersal and tandem repetition) may have been generated by these different transposition mechanisms during the course of evolution.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Insight into the diversity and evolution of the cryptomonad nucleomorph genome

The cryptomonads are an enigmatic group of marine and freshwater unicellular algae that acquired their plastids through the engulfment and retention of a eukaryotic ("secondary") endosymbiont. Together with the chlorarachniophyte algae, the cryptomonads are unusual in that they have retained the nucleus of their endosymbiont in a miniaturized form called a nucleomorph. The nucleomorph genome of the cryptomonad Guillardia theta has been completely sequenced and with only three chromosomes and a total size of 551 kb, is a model of nuclear genome compaction. Using this genome as a reference, we have investigated the structure and content of nucleomorph genomes in a wide range of cryptomonad algae. In this study, we have sequenced nine new cryptomonad nucleomorph 18S ribosomal DNA (rDNA) genes and four heat shock protein 90 (hsp90) gene fragments, and using pulsed-field gel electrophoresis and Southern hybridizations, have obtained nucleomorph genome size estimates for nine different species. We also used long-range polymerase chain reaction to obtain nucleomorph genomic fragments from Hanusia phi CCMP325 and Proteomonas sulcata CCMP704 that are syntenic with the subtelomeric region of nucleomorph chromosome I in G. theta. Our results indicate that (1) the presence of three chromosomes is a common feature of the nucleomorph genomes of these organisms, (2) nucleomorph genome size varies dramatically in the cryptomonads examined, (3) unidentified cryptomonad species CCMP1178 has the largest nucleomorph genome identified to date at approximately 845 kb, (4) nucleomorph genome size reductions appear to have occurred multiple times independently during cryptomonad evolution, (5) the relative positions of the 18S rDNA, ubc4, and hsp90 genes are conserved in three different cryptomonad genera, and (6) interchromosomal recombination appears to be rapidly changing the size and sequence of a repetitive subtelomeric region of the nucleomorph genome between the 18S rDNA and ubc4 loci. These results provide a glimpse into the genetic diversity of nucleomorph genomes in cryptomonads and set the stage for more comprehensive sequence-based studies in closely and distantly related taxa.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Genome-wide associations between hybrid sterility QTL and marker transmission ratio distortion

Marker transmission ratio distortion (TRD) in genetic mapping populations is frequently ascribed to selection against allelic combinations that cause hybrid incompatibility. Accordingly, genomic regions of TRD should be nonrandomly associated (colocated) with loci that underlie hybrid incompatibility. To directly test this hypothesis, we evaluated the genome-wide qualitative and quantitative agreement between chromosomal regions exhibiting marker TRD and those known to contain hybrid incompatibility quantitative trait locus (QTL). Incompatibility data came from a near-isogenic line (NIL) analysis of pollen and seed sterility in a cross between two Solanum (formerly Lycopersicon) species. We assessed (1) whether these incompatibility loci are colocated with markers that show significant TRD in two earlier generations preceding these introgression lines and (2) whether the magnitude of marker distortion quantitatively matches the estimated strength of selection against each incompatibility locus. We found evidence that TRD regions are chromosomally colocated with hybrid incompatibility loci more frequently than is expected by chance: pollen sterility QTLs were most closely associated with distorted heterozygote frequencies in later-generation backcrosses. Nonetheless, there was no evidence for an association between TRD and seed sterility and little evidence of a quantitative association between the magnitude of marker TRD and the fitness effects of heterospecific alleles at each chromosomal location. We propose and test a model (the "dance partner" model) to explain several cases where regions of TRD are not associated with hybrid incompatibility loci. Under this model, some NILs containing greater than one heterospecific introgression may not express hybrid incompatibility phenotypes because they carry both appropriate genetic dance partners required for a fully functional interaction. Accordingly, negative interactions expressed in earlier backcross generations are masked in these double-introgression NILs. Based on this model, we identify the location of several new putative pairwise interactors underlying hybrid incompatibility in this species cross.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Lineage-specific expansions and contractions of the bitter taste receptor gene repertoire in vertebrates

The sense of bitter taste plays a critical role in how organisms avoid generally bitter toxic and harmful substances. Previous studies revealed that there were 25 intact bitter taste receptor (T2R) genes in humans and 34 in mice. However, because the recent chicken genome project reported only three T2R genes, it appears that extensive gene expansions occurred in the lineage leading to mammals or extensive gene contractions occurred in the lineage leading to birds. Here, I examined the T2R gene repertoire in placental mammals (dogs, Canis familiaris; and cows, Bos taurus), marsupials (opossums, Monodelphis domestica), amphibians (frogs, Xenopus tropicalis), and fishes (zebrafishes, Danio rerio; and pufferfishes, Takifugu rubripes) to investigate the birth-and-death process of T2R genes throughout vertebrate evolution. I show that (1) the first extensive gene expansions occurred before the divergence of mammals from reptiles/birds but after the divergence of amniotes (reptiles/birds/mammals) from amphibians, (2) subsequent gene expansions continuously took place in the ancestral mammalian lineage and the lineage leading to amphibians, as evidenced by the presence of 15, 18, 26, and 49 intact T2R genes in the dog, cow, opossum, and frog genome, respectively, and (3) contractions of the gene repertoire happened in the lineage leading to chickens. Thus, continuous gene expansions have shaped the T2R repertoire in mammals, but the contractions subsequent to the first round of expansions have made the chicken T2R repertoire narrow. These dramatic changes in the repertoire size might reflect the daily intake of foods from an external environment as a driving force of evolution.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Coalescent-based estimation of population parameters when the number of demes changes over time

We expand a coalescent-based method that uses serially sampled genetic data from a subdivided population to incorporate changes to the number of demes and patterns of colonization. Often, when estimating population parameters or other parameters of interest from genetic data, the demographic structure and parameters are not constant over evolutionary time. In this paper, we develop a Bayesian Markov chain Monte Carlo method that allows for step changes in mutation, migration, and population sizes, as well as changing numbers of demes, where the times of these changes are also estimated. We show that in parameter ranges of interest, reliable estimates can often be obtained, including the historical times of parameter changes. However, posterior densities of migration rates can be quite diffuse and estimators somewhat biased, as reported by other authors.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Reconstructing the origins and dispersal of the Polynesian bottle gourd (Lagenaria siceraria)

The origin of the Polynesian bottle gourd (Lagenaria siceraria), an important crop species in prehistoric Polynesia, has remained elusive. Most recently, a South American origin has been favored as the bottle gourd could have been introduced from this continent with the sweet potato by Polynesian voyagers around A.D. 1,000. To test the hypothesis of an American origin for the Polynesian bottle gourd, we developed seven markers specific to bottle gourd (two chloroplast and five nuclear). The nuclear markers were developed using a new technique where polymorphic inter simple sequence repeat (ISSR) markers are converted into single-locus polymerase chain reaction and sequencing markers--an approach that will be useful for developing markers in other taxa. All seven markers were sequenced in 36 cultivars of bottle gourd from Asia, the Americas, and Polynesia. The results support a dual origin for the Polynesian bottle gourd: the chloroplast markers are exclusively of Asian origin, but the nuclear markers show alleles originating in both the Americas and Asia. Because hybridization of Polynesian bottle gourds with post-European introductions cannot be excluded, ancient DNA from archaeological material will be useful for further elucidating the prehistoric movements of this species in Polynesia. This work has implications not only for the dispersal of the Polynesian bottle gourd but also for the domestication and dispersal of the species as a whole.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Baleen whale phylogeny and a past extensive radiation event revealed by SINE insertion analysis

Baleen whales (suborder Mysticeti) comprise 11 extant species that are classified into four families. Although several phylogenetic hypotheses about these taxa have been proposed, their phylogenetic relationships remain confused. We addressed this problem using short interspersed repetitive element (SINE) insertion data, which now are regarded as almost ideal shared, derived characters at the molecular level. We reconstructed the phylogenetic relationships of baleen whales by characterizing 36 informative SINE loci. One of the intriguing conclusions is that balaenopterids and eschrichtiids radiated very rapidly during a very short evolutionary period. During this period, speciation occurred in balaenopterids and eschrichtiids while newly inserted SINE loci remains polymorphic. Later on, these SINEs were sorted incompletely into each lineage. Thus, there are now inconsistencies among species regarding the presence or absence of a given SINE. This is in sharp contrast to the phylogeny of toothed whales, for which no SINE inconsistencies have been found. Furthermore, we found monophyletic groupings between humpback and fin whales as well as between (sei+Bryde's) whales and blue whales, both of which have not previously been recognized. The comprehensive SINE insertion data, together with the mitochondrial DNA phylogeny that was recently completed (Sasaki, T., M. Nikaido, H. Healy et al. 2005. Mitochondrial phylogenetics and evolution of mysticete whales. Syst. Biol. 56:77-90; Rychel, A. L., T. W. Reeder, and A. Berta. 2004. Phylogeny of mysticete whales based on mitochondrial and nuclear data. Mol. Phylogenet. Evol. 32:892-901), provide a nearly complete picture of the evolutionary history of baleen whales.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.     

Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites

Cysteine proteinase inhibitors of the cystatin superfamily have several important functions in plants, including the inhibition of exogenous cysteine proteinases during herbivory or infection. Here we used a maximum-likelihood approach to assess whether plant cystatins, like other proteins implicated in host-pest interactions, have been subject to positive selection during the course of their evolution. Several amino acid sites were identified as being positively selected in cystatins from either Poaceae (monocots) and Solanaceae (dicots). These hypervariable sites were located at strategic positions on the protein: on each side of the conserved glycine residues in the N-terminal trunk, within the first and second inhibitory loops entering the active site of target enzymes, and surrounding the larfav motif, a sequence of unknown function conserved among plant cystatins. Supporting the assumption that positively selected, hypervariable sites are indicative of amino acid sites implicated in functional diversity, mutants of the 8th cystatin unit of tomato multicystatin including alternative residues at positively selected sites in the N-terminal trunk exhibited highly variable affinities for the cysteine proteases papain, cathepsin B and cathepsin H. Overall, these observations support the hypothesis that plant cystatins have been under selective pressure to evolve in response to predatory challenges by herbivorous enemies. They also indicate the potential of site-directed mutagenesis at positively selected sites for the generation of cystatins with improved binding properties.