Selection on plant male function genes identifies candidates for reproductive isolation of yellow monkeyflowers.

Understanding the genetic basis of reproductive isolation promises insight into speciation and the origins of biological diversity. While progress has been made in identifying genes underlying barriers to reproduction that function after fertilization (post-zygotic isolation), we know much less about earlier acting pre-zygotic barriers. Of particular interest are barriers involved in mating and fertilization that can evolve extremely rapidly under sexual selection, suggesting they may play a prominent role in the initial stages of reproductive isolation. A significant challenge to the field of speciation genetics is developing new approaches for identification of candidate genes underlying these barriers, particularly among non-traditional model systems. We employ powerful proteomic and genomic strategies to study the genetic basis of conspecific pollen precedence, an important component of pre-zygotic reproductive isolation among yellow monkeyflowers (Mimulus spp.) resulting from male pollen competition. We use isotopic labeling in combination with shotgun proteomics to identify more than 2,000 male function (pollen tube) proteins within maternal reproductive structures (styles) of M. guttatus flowers where pollen competition occurs. We then sequence array-captured pollen tube exomes from a large outcrossing population of M. guttatus, and identify those genes with evidence of selective sweeps or balancing selection consistent with their role in pollen competition. We also test for evidence of positive selection on these genes more broadly across yellow monkeyflowers, because a signal of adaptive divergence is a common feature of genes causing reproductive isolation. Together the molecular evolution studies identify 159 pollen tube proteins that are candidate genes for conspecific pollen precedence. Our work demonstrates how powerful proteomic and genomic tools can be readily adapted to non-traditional model systems, allowing for genome-wide screens towards the goal of identifying the molecular basis of genetically complex traits.

Duplicate abalone egg coat proteins bind sperm lysin similarly, but evolve oppositely, consistent with molecular mimicry at fertilization.

Sperm and egg proteins constitute a remarkable paradigm in evolutionary biology: despite their fundamental role in mediating fertilization (suggesting stasis), some of these molecules are among the most rapidly evolving ones known, and their divergence can lead to reproductive isolation. Because of strong selection to maintain function among interbreeding individuals, interacting fertilization proteins should also exhibit a strong signal of correlated divergence among closely related species. We use evidence of such molecular co-evolution to target biochemical studies of fertilization in North Pacific abalone (Haliotis spp.), a model system of reproductive protein evolution. We test the evolutionary rates (d(N)/d(S)) of abalone sperm lysin and two duplicated egg coat proteins (VERL and VEZP14), and find a signal of co-evolution specific to ZP-N, a putative sperm binding motif previously identified by homology modeling. Positively selected residues in VERL and VEZP14 occur on the same face of the structural model, suggesting a common mode of interaction with sperm lysin. We test this computational prediction biochemically, confirming that the ZP-N motif is sufficient to bind lysin and that the affinities of VERL and VEZP14 are comparable. However, we also find that on phylogenetic lineages where lysin and VERL evolve rapidly, VEZP14 evolves slowly, and vice versa. We describe a model of sexual conflict that can recreate this pattern of anti-correlated evolution by assuming that VEZP14 acts as a VERL mimic, reducing the intensity of sexual conflict and slowing the co-evolution of lysin and VERL.

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.

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.

Functional diversification and evolution of antifreeze proteins in the antarctic fish Lycodichthys dearborni.

Antifreeze proteins (AFPs) have independently evolved in many organisms. AFPs act by binding to ice crystals, effectively lowering the freezing point. AFPs are often at high copy number in a genome and diversity exists between copies. Type III antifreeze proteins are found in Arctic and Antarctic eel pouts, and have previously been shown to evolve under positive selection. Here we combine molecular and proteomic techniques to understand the molecular evolution and diversity of Type III antifreeze proteins in a single individual Antarctic fish Lycodichthys dearborni. Our expressed sequence tag (EST) screen reveals that at least seven different AFP variants are transcribed, which are ultimately translated into five different protein isoforms. The isoforms have identical 66 base pair signal sequences and different numbers of subsequent ice-binding domains followed by a stop codon. Isoforms with one ice-binding unit (monomer), two units (dimer), and multiple units (multimer) were present in the EST library. We identify a previously uncharacterized protein dimer, providing further evidence that there is diversity between Type III AFP isoforms, perhaps driven by positive selection for greater thermal hysteresis. Proteomic analysis confirms that several of these isoforms are translated and present in the liver. Our molecular evolution study shows that paralogs have diverged under positive selection. We hypothesize that antifreeze protein diversity is an important contributor to depressing the serum freezing point.

Midterm evaluation of hemodynamics of the Top Hat supraannular aortic valve.

The CarboMedics Top Hat supraannular aortic valve provides an orifice-to-annulus ratio of 1:1 in most patients, and enhances patient outcomes. This study compared the midterm echocardiographic parameters of 52 patients undergoing aortic valve replacement with a Top Hat valve with those in other studies of Top Hat or CarboMedics intraannular valves. Echocardiography was performed 6-48 months after surgery with Top Hat sizes 21-27. Parameters evaluated included mean gradient, peak gradient, effective orifice area, and effective orifice area index. Preoperative and postoperative echocardiographic measurements were compared in 38 patients with aortic valve stenosis. Hemodynamic data were comparable to those of other studies, but the Top Hat prosthesis implanted was significantly larger (by a mean of 3.29 mm) than the valve size indicated using an intraannular valve sizer in 48 patients. Mean effective orifice area improved significantly from 0.73 cm(2) preoperatively to 2.04 cm(2) postoperatively. In patients with preoperative aortic valve stenosis, postoperative mean peak gradient was significantly reduced. The larger orifice-to-annulus ratio of the Top Hat valve improved hemodynamic parameters assessed by echocardiography up to 4 years postoperatively, with a lower transvalvular gradient, hence decreased left ventricular workload.

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.

Bindin from a sea star.

The genetic basis for the evolution of development includes genes that encode proteins expressed on the surfaces of sperm and eggs. Previous studies of the sperm acrosomal protein bindin have helped to characterize the adaptive evolution of gamete compatibility and speciation in sea urchins. The absence of evidence for bindin expression in taxa other than the Echinoidea has limited such studies to sea urchins, and led to the suggestion that bindin might be a sea urchin-specific molecule. Here we characterize the gene that encodes bindin in a broadcast-spawning asterinid sea star (Patiria miniata). We describe the sequence and domain structure of a full-length bindin cDNA and its single intron. In comparison with sea urchins, P. miniata bindin is larger but the two molecules share several general features of their domain structure and some sequence features of two domains. Our results extend the known evolutionary history of bindin from the Mesozoic (among the crown group sea urchins) into the early Paleozoic (and the common ancestor of eleutherozoans), and present new opportunities for understanding the role of bindin molecular evolution in sexual selection, life history evolution, and speciation among sea stars.

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.

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 F(1) 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.

Aortic valve replacement: is there an implant size variation across Europe?

BACKGROUND AND AIMS OF THE STUDY: Prompted by anecdotal evidence and observations by surgeons, an investigation was undertaken into the potential differences in implanted aortic valve prosthesis sizes, during aortic valve replacement (AVR) procedures, between northern and southern European countries. METHODS: A multi-institutional, non-randomized, retrospective analysis was conducted among 2,932 patients who underwent AVR surgery at seven tertiary cardiac surgery centers throughout Europe. Demographic and perioperative variables including valve size and type, body surface area (BSA) and early mortality were collected. Group analysis by patient geographic distribution and by annular diameter of the prosthesis utilized was conducted. Patients with a manufacturer's labeled prosthesis size > or = 21 mm were assigned to the 'large' aortic size subset, while those with a prosthesis size < 21 mm were assigned to the 'small' aortic size subset. Effective orifice area indices were calculated for all patients to assess the geographic distribution of patient-prosthesis mismatch. Univariable and multivariable logistic regression analyses adjusting for possible confounding variables were performed. RESULTS: Prostheses with diameter < 21 mm were implanted at almost twice the rate in southern Europe compared to the north (56.4% versus 26.7%, p < 0.01). The mean valve size was also smaller in southern compared to northern European patients (21.6 +/- 2.1 mm versus 23.4 +/- 2.2 mm, p < 0.01). There were no regional differences in the distribution of either gender or BSA. In the multivariable model, south European patients were seven times more likely to receive a smaller-sized aortic valve (OR = 6.5, 95% CI = 4.82-8.83, p < 0.01), and thus the odds of developing patient-prosthesis mismatch were increased two-fold in southern European patients (OR = 1.9, 95% CI = 1.25-2.80, p = 0.02). However, neither geographic distribution nor valve size were significantly associated with operative mortality. CONCLUSION: The study results demonstrated differences in implanted aortic valve size, between the participating northern and southern European countries. Imbalances in the prevalence of rheumatic heart disease, health resource availability and variations in surgical practice throughout Europe might be possible etiological causes.

Maximizing prosthetic valve size with the Top Hat supra-annular aortic valve.

BACKGROUND AND AIM OF THE STUDY: The CarboMedics Top Hat supra-annular aortic valve allows a one-size (and often two-size) increase over the standard intra-annular valve. This advantage should minimize the risk of patient-prosthesis mismatch, where the effective prosthetic valve orifice area is less than that of a normal valve. It is suggested that the ability to implant Top Hat valves having greater size, relative to standard intra-annular valves, may currently be under-utilized. Further, there has been some concern that Top Hat implantation can cause obstruction of the coronary ostia. This study evaluates the authors' clinical experience with Top Hat supra-annular aortic valve size selection, and the technical aspects of implantation. METHODS: Between January 1999 and October 2005, a total of 251 consecutive patients underwent 252 aortic valve replacements with Top Hat supra-annular mechanical prostheses (CarboMedics, Inc., Arvada, CO, USA) at two institutions. Size frequency distribution was compared to published series, and to the manufacturer's US registry. The ventriculoaortic junction (VAJ) size was available in 234 patients, and compared to the size of the Top Hat valve implanted. RESULTS: Valves implanted were 2 mm larger than the VAJ in 56% of patients (130/234), 3 mm larger in 1.7% (4/234), and 4 mm larger in 42% (98/234). Numbers of 19-mm and 21-mm valves were less (p <0.001) in the present series (22%) than in published series (52%) and the US registry (46%). No patient required unplanned coronary bypass, and 30-day mortality was 2.0% (5/251), indicating a good safety profile for the valves implanted in this series. CONCLUSION: The general distribution of implant sizes in the US indicates that cardiac surgeons may be under-sizing the Top Hat supra-annular aortic valve during aortic valve replacement, thereby missing the full advantage of its unique design for lowering the transvalvular gradient and, consequently, left ventricular workload, leading to a reversal of left ventricular hypertrophy.

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.

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.

Evolution of reproductive proteins from animals and plants.

Sexual reproduction is a fundamental biological process common among eukaryotes. Because of the significance of reproductive proteins to fitness, the diversity and rapid divergence of proteins acting at many stages of reproduction is surprising and suggests a role of adaptive diversification in reproductive protein evolution. Here we review the evolution of reproductive proteins acting at different stages of reproduction among animals and plants, emphasizing common patterns. Although we are just beginning to understand these patterns, by making comparisons among stages of reproduction for diverse organisms we can begin to understand the selective forces driving reproductive protein diversity and the functional consequences of reproductive protein evolution.

Accuracy and power of the likelihood ratio test for comparing evolutionary rates among genes.

Sequences for multiple protein-coding genes are now commonly available from several, often closely related species. These data sets offer intriguing opportunities to test hypotheses regarding whether different types of genes evolve under different selective pressures. Although maximum likelihood (ML) models of codon substitution that are suitable for such analyses have been developed, little is known about the statistical properties of these tests. We use a previously developed fixed-sites model and computer simulations to examine the accuracy and power of the likelihood ratio test (LRT) in comparing the nonsynonymous-to-synonymous substitution rate ratio (omega = dN/dS) between two genes. Our results show that the LRT applied to fixed-sites models may be inaccurate in some cases when setting significance thresholds using a chi(2) approximation. Instead, we use a parametric bootstrap to describe the distribution of the LRT statistic for fixed-sites models and examine the power of the test as a function of sampling variables and properties of the genes under study. We find that the power of the test is high (>80%) even when sampling few taxa (e.g., six species) if sequences are sufficiently diverged and the test is largely unaffected by the tree topology used to simulate data. Our simulations show fixed-sites models are suitable for comparing substitution parameters among genes evolving under even strong evolutionary constraint (omega approximately 0.05), although relative rate differences of 25% or less may be difficult to detect.

Fifteen years' clinical experience with the CarboMedics prosthetic heart valve.

BACKGROUND AND AIM OF THE STUDY: The CarboMedics bileaflet prosthetic heart valve was first implanted as part of a prospective clinical study at the authors' institution in November 1987. The patient cohort included was part of a multicenter trial set up by the manufacturer for an FDA application. The present report details findings over a 15-year period, with a continuous follow up on this patient cohort. METHODS: Between November 1987 and August 1990, 132 patients (68 males, 64 females; median age 56 years; range: 12-74 years) received a CarboMedics heart valve prosthesis. All patients were included in the study, whether surgery was elective or emergency, first time or reoperation. There were 69 aortic, 49 mitral and 12 double (aortic and mitral) valve replacements. Two patients had isolated tricuspid valve replacement. Concomitant surgery was performed in 15 patients. Anticoagulation with warfarin was started on postoperative day 1. After discharge, patients were examined regularly as outpatients for up to five years. Subsequent follow up was obtained prospectively by questionnaires to the patients' general practitioners and with telephone calls to the patients. Actuarial estimates of survival and freedom from morbid events were calculated using the Kaplan-Meier method; 95% confidence limits for the distribution function were calculated according to the Greenwood formula. RESULTS: Complete follow up information was available on 94% of the patients; total follow up was 1,270.3 patient-years (pt-yr). Actuarial survival at 15 years was 51+/-4.9% overall; 56+/-6.2% for single aortic, 51+/-8.0% for single mitral, and 30+/-15.9% for double valve replacements. Actuarial rates of freedom from complications were: valve thrombosis 100%, embolism 89+/-3.3%, and all anticoagulant-related bleeding 76+/-4.5%. The linearized rates per 100 pt-yr were: embolism 0.94 (aortic 0.74, mitral 1.25); anticoagulant-related bleeding 2.28; paravalvular leakage overall 0.24 (aortic 0.29); prosthetic valve endocarditis overall 0.24 (aortic 0.29, mitral 0.21). There was no hemolysis, prosthetic valve dysfunction, or structural deterioration. CONCLUSION: Over a 15-year time frame, the CarboMedics prosthetic heart valve has proven to be a highly reliable device with no structural failures and a low incidence of valve-related complications.

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.

Fourteen years' experience with the CarboMedics valve in young adults with aortic valve disease.

BACKGROUND AND AIM OF THE STUDY: Homografts and the Ross procedure are recommended by many surgeons for aortic valve replacement (AVR) in young adults. The study aim was to monitor patient outcome over a 14-year period after implanting mechanical CarboMedics prosthetic heart valves in young adults with aortic valve disease. METHODS: Between November 1987 and December 2000, 55 patients (42 males, 13 females; median age 33 years; range 15-40 years) each received a CarboMedics valve in the aortic position. All patients were included on a consecutive and non-selected basis. Concomitant surgery was performed in five patients. Anticoagulation was commenced on postoperative day 1. After discharge, patients were followed up by their cardiologist or general practitioner using questionnaires and telephone calls. Actuarial estimates of survival and freedom from morbid events were calculated using the Kaplan-Meier method. RESULTS: No patient died during surgery. Complete follow up data were available for all patients; total follow up was 404 patient-years (pt-yr). The 30-day mortality rate was zero. Actuarial survival at 14 years was 90 +/- 4.9%, and actuarial freedom from all valve-related events 92 +/- 3.7%. No valve thrombosis or major bleeding events were identified. Linearized rates per 100 pt-yr were: embolism 0.25; paravalvular leakage 0.25; prosthetic endocarditis 0.25; and reoperation 0.50. There was no hemolysis, prosthetic valve dysfunction, or structural deterioration. CONCLUSION: Over a 14-year period, implantation of the CarboMedics prosthetic heart valve has proven to be an excellent solution for AVR in young adults with aortic valve disease.