Jan E. Aagaard

RAPDs and allozymes exhibit similar levels of diversity and differentiation among populations and races of Douglas-fir

Thirty-six nuclear-encoded RAPD loci and 20 allozyme loci were studied to compare levels of diversity and differentiation among populations and races of the widespread North American conifer, Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RAPD assays used diploid seed embryo DNA from 22 to 36 trees in each of six populations that sampled the three major races (two populations per race). A comparable allozyme data set for nearby populations was constructed from a published study. RAPDs of organelle origin were excluded by hybridization of blotted RAPD gels with chloroplast and mitochondrial DNA-enriched probes. RAPD and allozyme markers had similar levels of diversity within populations (HS=0.22±0.03 and 0.16±0.03, respectively) and differentiation among populations (GST=0.34±0.07 and 0.29±0.07, respectively). When the allozyme data set was transformed into dominant, biallelic markers to study how RAPDs may bias diversity estimates, resampling studies showed that simulated HS and HT were reduced by half regardless of sample size. Because observed diversity for RAPDs was equivalent to, or higher than, that of allozymes, our simulations suggest that RAPD markers may contain substantially higher levels of inherent, but hidden, diversity. In contrast, the simulations showed that estimates of GST using RAPDs should not be significantly biased at the population sizes we employed.

Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins.

Mitochondrial DNA products among RAPD profiles are frequent and strongly differentiated between races of Douglas-fir

Racial differentiation and genetic variability were studied between and within the coastal, north interior, and south interior races of Douglas‐fir using RAPD and allozyme markers. Nearly half of all RAPD bands scored (13:45%) were found to be amplified from mitochondrial DNA. They exhibited maternal inheritance among hybrids and back‐crosses between the races, and were much more highly differentiated (GST= 0.62 for haplotype frequencies) than were allozymes (GST= 0.26). No evidence of hybridization or introgression was detected where the coastal and interior races come into proximity in central Oregon.

ZP Domain Proteins in the Abalone Egg Coat Include a Paralog of VERL under Positive Selection That Binds Lysin and 18-kDa Sperm Proteins

Identifying fertilization molecules is key to our understanding of reproductive biology, yet only a few examples of interacting sperm and egg proteins are known. One of the best characterized comes from the invertebrate archeogastropod abalone (Haliotis spp.), where sperm lysin mediates passage through the protective egg vitelline envelope (VE) by binding to the VE protein vitelline envelope receptor for lysin (VERL). Rapid adaptive divergence of abalone lysin and VERL are an example of positive selection on interacting fertilization proteins contributing to reproductive isolation. Previously, we characterized a subset of the abalone VE proteins that share a structural feature, the zona pellucida (ZP) domain, which is common to VERL and the egg envelopes of vertebrates. Here, we use additional expressed sequence tag sequencing and shotgun proteomics to characterize this family of proteins in the abalone egg VE. We expand 3-fold the number of known ZP domain proteins present within the VE (now 30 in total) and identify a paralog of VERL (vitelline envelope zona pellucida domain protein [VEZP] 14) that contains a putative lysin-binding motif. We find that, like VERL, the divergence of VEZP14 among abalone species is driven by positive selection on the lysin-binding motif alone and that these paralogous egg VE proteins bind a similar set of sperm proteins including a rapidly evolving 18-kDa paralog of lysin, which may mediate sperm-egg fusion. This work identifies an egg coat paralog of VERL under positive selection and the candidate sperm proteins with which it may interact during abalone fertilization.

Toward the Evolutionary Genomics of Gametophytic Divergence: Patterns of Transmission Ratio Distortion in Monkeyflower (Mimulus) Hybrids Reveal a Complex Genetic Basis for Conspecific Pollen Precedence

Abstract Conspecific pollen precedence (CPP) is a major component of reproductive isolation between many flowering plant taxa and may reveal mechanisms of gametophytic evolution within species, but little is known about the genetic basis and evolutionary history of CPP. We systematically investigated the genetic architecture of CPP using patterns of transmission ratio distortion (TRD) in F2 and backcross hybrids between closely related species of Mimulus (Phrymaceae) with divergent mating systems. We found that CPP in Mimulus hybrids was polygenic and was the majority source of interspecific TRD genome-wide, with at least eight genomic regions contributing to the transmission advantage of M. guttatus pollen grains on M. guttatus styles. In aggregate, these male-specific transmission ratio distorting loci (TRDLs) were more than sufficient to account for the 100% precedence of pure M. guttatus pollen over M. nasutus pollen in mixed pollinations of M. guttatus. All but one of these pollen TRDLs were style-dependent; that is, we observed pollen TRD in F1 and/or M. guttatus styles, but not in M. nasutus styles. These findings suggest that species-specific differences in pollen tube performance accumulate gradually and may have been driven by coevolution between pollen and style in the predominantly outcrossing M. guttatus.

The Molecular Basis of Sex: Linking Yeast to Human

Species-specific recognition between egg and sperm, a crucial event that marks the beginning of fertilization in multicellular organisms, mirrors the binding between haploid cells of opposite mating type in unicellular eukaryotes such as yeast. However, as implied by the lack of sequence similarity between sperm-binding regions of invertebrate and vertebrate egg coat proteins, these interactions are thought to rely on completely different molecular entities. Here, we argue that these recognition systems are, in fact, related: despite being separated by 0.6-1 billion years of evolution, functionally essential domains of a mollusc sperm receptor and a yeast mating protein adopt the same 3D fold as egg zona pellucida proteins mediating the binding between gametes in humans.

RAPD markers of mitochondrial origin exhibit lower population diversity and higher differentiation than RAPDs of nuclear origin in Douglas fir

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity (HS = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population (GST = 0.18 and 0.05, respectively) and racial levels (GST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.

Duplication of floral regulatory genes in the Lamiales

Duplication of some floral regulatory genes has occurred repeatedly in angiosperms, whereas others are thought to be single-copy in most lineages. We selected three genes that interact in a pathway regulating floral development conserved among higher tricolpates (LFY/FLO, UFO/FIM, and AP3/DEF) and screened for copy number among families of Lamiales that are closely related to the model species Antirrhinum majus. We show that two of three genes have duplicated at least twice in the Lamiales. Phylogenetic analyses of paralogs suggest that an ancient whole genome duplication shared among many families of Lamiales occurred after the ancestor of these families diverged from the lineage leading to Veronicaceae (including the single-copy species A. majus). Duplication is consistent with previous patterns among angiosperm lineages for AP3/DEF, but this is the first report of functional duplicate copies of LFY/FLO outside of tetraploid species. We propose Lamiales taxa will be good models for understanding mechanisms of duplicate gene preservation and how floral regulatory genes may contribute to morphological diversity.

Relaxed selection among duplicate floral regulatory genes in Lamiales.

Polyploidization is a prevalent mode of genome diversification within plants. Most gene duplicates arising from polyploidization (paralogs) are typically lost, although a subset may be maintained under selection due to dosage, partitioning of gene function, or acquisition of novel functions. Because they experience selection in the presence of other duplicate loci across the genome, interactions among genes may also play a significant role in the maintenance of paralogs resulting from polyploidization. Previously, we identified duplicates of the genes LFY/FLO and AP3/DEF that directly interact in a floral regulatory pathway and are thought to be the result of ancient polyploidization in the Lamiales (> 50 mya). Although duplicates of MADS box genes including AP3/DEF are common throughout the angiosperm lineage, LFY/FLO duplicates in Lamiales are the first reported outside of tetraploid taxa. In order to explore hypotheses for the joint preservation of these interacting floral regulatory genes including novel LFY/FLO paralogs, here we clone FLO and DEF duplicates from additional Lamiales taxa and apply codon substitution models to test how selection acts on both genes following duplication. We find acceleration in the ratio of nonsynonymous-to-synonymous nucleotide substitutions for one (FLO) or both (DEF) paralogs that appears to be due to relaxed purifying selection as opposed to positive selection and shows a different pattern among functional domains of these genes. Several mechanisms are discussed that might be responsible for preservation of co-orthologs of FLO and DEF in Lamiales, including interactions among the genes of this regulatory pathway.

THE TEGULA TANGO: A COEVOLUTIONARY DANCE OF INTERACTING, POSITIVELY SELECTED SPERM AND EGG PROTEINS

Reproductive proteins commonly show signs of rapid divergence driven by positive selection. The mechanisms driving these changes have remained ambiguous in part because interacting male and female proteins have rarely been examined. We isolate an egg protein the vitelline envelope receptor for lysin (VERL) from Tegula, a genus of free‐spawning marine snails. Like VERL from abalone, Tegula VERL is a major component of the VE surrounding the egg, includes a conserved zona pellucida (ZP) domain at its C‐terminus, and possesses a unique, negatively charged domain of about 150 amino acids implicated in interactions with the positively charged lysin. Unlike for abalone VERL, where this unique VERL domain occurs in a tandem array of 22 repeats, Tegula VERL has just one such domain. Interspecific comparisons show that both lysin and the VERL domain diverge via positive selection, whereas the ZP domain evolves neutrally. Rates of nonsynonymous substitution are correlated between lysin and the VERL domain, consistent with sexual antagonism, although lineage‐specific effects, perhaps owing to different ecologies, may alter the relative evolutionary rates of sperm‐ and egg‐borne proteins.