In memoriam: Richard G. Harrison - his life and legacy.

Richard G. Harrison passed away unexpectedly on April 12th, 2016. In this memoriam we pay tribute to the life and legacy of an extraordinary scientist, mentor, friend, husband, and father.

Comparative proteomics uncovers the signature of natural selection acting on the ejaculate proteomes of two cricket species isolated by postmating, prezygotic phenotypes.

Two of the most well-supported patterns to have emerged over the past two decades of research in evolutionary biology are the occurrence of divergent natural selection acting on many male and female reproductive tract proteins and the importance of postmating, prezygotic phenotypes in reproductively isolating closely related species. Although these patterns appear to be common across a wide variety of taxa, the link between them remains poorly documented. Here, we utilize comparative proteomic techniques to determine whether or not there is evidence for natural selection acting on the ejaculate proteomes of two cricket species (Allonemobius fasciatus and A. socius) which are reproductively isolated primarily by postmating, prezygotic phenotypes. In addressing this question, we compare the degree of within-species polymorphism and between-species divergence between the ejaculate and thorax proteomes of these two species. We found that the ejaculate proteomes are both less polymorphic and more divergent than the thorax proteomes. Additionally, we assessed patterns of nucleotide variation for two species-specific ejaculate proteins and found evidence for both reduced levels of variation within species and positive selection driving divergence between species. In contrast, non-species-specific proteins exhibited higher levels of within-species nucleotide variation and no signatures of positive selection. Nucleotide and putative functional data for the two species-specific proteins, along with data for a third protein (ejaculate serine protease), suggest that all three of these genes are candidate speciation genes in need of further study. Overall, these patterns of proteome and nucleotide divergence provide support for the hypothesis that there is a causative link between selection-driven divergence of male ejaculate proteins and the evolution of postmating, prezygotic barriers to gene flow within Allonemobius.

Label-free DNA methylation analysis using opto-fluidic ring resonators.

The opto-fluidic ring resonator (OFRR) is a sensitive label-free optical biosensor that is uniquely well suited for photonic and fluidic integration. For the first time we have explored the utility of this novel instrument for the analysis of methylation in oligonucleotides using the MBD-2 (methyl binding) protein as the capture molecule. This application has strong relevance to cancer research and future clinical tools through the study of methylation patterns in important gene promoters. In this work we quantitatively characterized the OFRR's response to artificially methylated ssDNA and dsDNA as a function of the number of methylated cytosines and DNA concentration. The effect of hemi- versus fully methylated oligonucleotides was also investigated. Additionally, anti 5-methylcytidine antibody was also used as the capture molecule and compared with MBD-2. It is found that the antibody has stronger affinity for ssDNA, whereas MBD-2 is much better at binding dsDNA.

Demonstration of the coupling of optofluidic ring resonator lasers with liquid waveguides.

Optofluidic lasers are of particular interest for lab-on-a-chip-type devices, with broad spectral tunability, convenient microfluidic integration, and a small footprint. Optofluidic ring resonator (OFRR) lasers are advantageous in terms of size but typically generate nondirectional emission that is of minimal practical use. We introduce two unique geometries for soft-lithography-based OFRR lasers--side-coupled rings and spiral rings--both of which can be produced in polydimethyl siloxane substrates with contact molding. These rings utilize evanescent and direct butt-coupling, respectively, to effectively couple the OFRR laser emission into microfluidic channels. A laser threshold of a few to tens of microJ/mm(2) is achieved.

Fabry-Pérot cavity sensors for multipoint on-column micro gas chromatography detection.

We developed and characterized a Fabry-Pérot (FP) sensor module based micro gas chromatography (microGC) detector for multipoint on-column detection. The FP sensor was fabricated by depositing a thin layer of metal and a layer of gas-sensitive polymer consecutively on the endface of an optical fiber, which formed the FP cavity. Light partially reflected from the metal layer and the polymer-air interface generated an interference spectrum, which shifted as the polymer layer absorbed the gas analyte. The FP sensor module was then assembled by inserting the FP sensor into a hole drilled in the wall of a fused-silica capillary, which can be easily connected to the conventional gas chromatography (GC) column through a universal quick seal column connector, thus enabling on-column real-time detection. We characterized the FP sensor module based microGC detector. Sensitive detection of various gas analytes was achieved with subnanogram detection limits. The rapid separation capability of the FP sensor module assembled with both single- and tandem-column systems was demonstrated, in which gas analytes having a wide range of polarities and volatilities were well-resolved. The tandem-column system obtained increased sensitivity and selectivity by employing two FP sensor modules coated with different polymers, showing great system versatility.

Antagonism of macrophage migration inhibitory factor decreases cyclophosphamide cystitis in mice.

AIMS: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine found pre-formed in the urothelium. During inflammation, MIF is released into the bladder lumen and bladder MIF mRNA is upregulated. Since MIF also has tautomerase activity and blocking tautomerase activity also blocks MIF's biological activity, we hypothesized that blocking MIF's tautomerase activity would prevent bladder inflammation. Therefore, we examined the effects of a MIF tautomerase inhibitor (ISO-1; also blocks biological activity) on cyclophosphamide (CYP)-induced cystitis in mice. METHODS: Mice receiving CYP (300 mg/kg; i.p.) to induce cystitis or saline (control) were treated either with ISO-1 (20 mg/kg; i.p.; daily) or vehicle (20% DMSO; i.p.; daily) for 2 days. After 2 days, micturition volume and frequency in awake mice were recorded and also mechanical sensitivity to abdominal stimulation using von Frey monofilaments. Bladders were collected under anesthesia and examined histologically, nerve growth factor levels were assayed in bladder homogenates, and production of inflammatory cytokines in the bladder was determined using a targeted array. RESULTS: CYP treatment resulted in decreased micturition volume, increased frequency, decreased threshold, increased histological signs of cystitis, increased bladder NGF levels and production of inflammatory cytokines when compared to the control group. Treatment with ISO-1 prevented or greatly decreased all these changes. CONCLUSION: Antagonizing MIF's activity with a systemic MIF tautomerase inhibitor was able to prevent or greatly reduced chemical cystitis in mice, thus indicating the MIF mediates bladder inflammation in this model. MIF represents a novel and important modulator of cystitis.

Rapid tandem-column micro-gas chromatography based on optofluidic ring resonators with multi-point on-column detection.

We demonstrated a novel tandem-column micro-gas chromatography (microGC) based on optofluidic ring resonator (OFRR). The OFRR is a thin-walled fused silica capillary whose interior surface is coated with a polymeric stationary phase. The circular cross section of the OFRR forms the micro-ring resonator and supports whispering gallery modes (WGMs). Via tapered optical fibers in contact with the OFRR, the WGM can be excited externally at any positions along the OFRR capillary, thus enabling multi-point, on-column, real-time detection of vapor molecules flowing through the OFRR. In the present OFRR-based tandem-column-based microGC implementation, a 180 cm long conventional GC column coated with a nonpolar stationary phase was followed by a relatively short OFRR column coated with a polar phase. Two detection positions, one at the inlet of the OFRR and the other a few centimeters downstream, were used to monitor the separation achieved by the first and the second column, respectively. Owing to the multi-point on-column detection that provides complementary retention time information on each chemical compound, co-eluted analytes can be well separated and identified on at least one detection channel and no modulation is needed at the interface of tandem columns. Separation and detection of twelve analytes with various volatilities and polarities within four minutes were demonstrated. In addition, the chromatograms obtained from three different locations along the OFRR column demonstrated the system's capability of on-column monitoring of the separation process for the target analyte in a vapor mixture. Our results will lead to the development of a rapid, simple, and portable microGC system with significantly improved selectivity and chemical identification capabilities.

Label-free analysis of DNA methylation using optofluidic ring resonators.

We demonstrate the utility of the opto-fluidic ring resonator (OFRR) sensor for analyzing methylated oligonucleotides. Cytosine methylation, a regular epigenetic function in cellular growth and metabolism, may have ties to abnormal suppression of key genes involved with cellular proliferation. Such behavior is suspected to be strongly related to the occurrence of several types of cancers. The OFRR is demonstrated as a tool both for detecting DNA hybridization and methylated cytosines residues.

PDMS embedded opto-fluidic microring resonator lasers.

Opto-fluidic ring resonator (OFRR) dye lasers are embedded in low index polydimethylsiloxane (PDMS) to achieve enhanced portability, mechanical stability, and potential integration with conventional soft lithography based microfluidics for development of micro total analysis systems. The OFRR retains high Q-factors (> 10(6)) and exhibits low lasing threshold (<1 microJ/mm(2)). Fiber prisms and tapered optical fibers are used to directionally couple out the laser emission. At 2.2 microJ/mm(2) pump intensity, the laser output from the fiber prism is 80 nW, corresponding to 50% power extraction efficiency. A microarray structure of parallel OFRRs is also demonstrated, allowing simultaneous multi-color emissions.

Genetic architecture of conspecific sperm precedence in Allonemobius fasciatus and A. socius.

The evolution of barriers to gene exchange is centrally important to speciation. We used the crickets Allonemobius fasciatus and A. socius to investigate the genetic architecture of conspecific sperm precedence (CSP), a postinsemination prezygotic reproductive barrier. With amplified fragment-length polymorphism (AFLP) markers and controlled crosses we constructed linkage maps and estimated positions of QTL associated with CSP. The majority of QTL have low to moderate effects, although a few QTL exist in A. socius with large effects, and the numbers of QTL are comparable to numbers of genes accounting for species differences in other studies. The QTL are spread across many unlinked markers, yet QTL placed with linked markers are on a small number of linkage groups that could reflect the role of the large Allonemobius sex chromosome in prezygotic isolation. Although many QTL had positive effects on conspecific sperm utilization several QTL also exerted negative effects, which could be explained by intraspecific sexual conflict, sperm competition, or epistasis of introgressed genes on novel backgrounds. One unexpected outcome was that A. socius CSP alleles have a stronger effect than those from A. fasciatus in hybrid females, causing hybrids to behave like A. socius with regard to sperm utilization. Implications of this asymmetry in the Allonemobius hybrid zone are discussed.

Identification and comparative analysis of accessory gland proteins in Orthoptera.

Accessory reproductive gland proteins (Acps) in Drosophila evolve quickly and appear to play an important role in ensuring the fertilization success of males. Moreover, Acps are thought to be involved in establishing barriers to fertilization between closely related species. While accessory glands are known to occur in the males of many insect groups, the proteins that are passed on to females by males during mating have not been well characterized outside of Drosophila. To gain a better understanding of these proteins, we characterized ESTs from the accessory glands of two cricket species, Allonemobius fasciatus and Gryllus firmus. Using an expressed sequence tag (EST) approach, followed by bioinformatic and evolutionary analyses, we found that many proteins are secreted and, therefore, available for transfer to the female during mating. Further, we found that most ESTs are novel, showing little sequence similarity between taxa. Evolutionary analyses suggest that cricket proteins are subject to diversifying selection and indicate that Allonemobius is much less polymorphic than Gryllus. Despite rapid nucleotide sequence divergence, there appears to be functional conservation of protein classes among Drosophila and cricket taxa.

Clustering of contact zones, hybrid zones, and phylogeographic breaks in North America.

A recent test for the existence of suture zones in North America, based on hybrid zones studied since 1970, found support for only two of the 13 suture zones identified by Remington in 1968 (Swenson and Howard 2004). One limitation of that recent study was the relatively small number of hybrid zones available for mapping. In this study, we search for evidence of clustering of contact zones between closely related taxa using data not only from hybrid zones but from species range maps of trees, birds, and mammals and from the position of phylogeographic breaks within species. Digital geographic range maps and a geographic information system approach allowed for accurate and rapid mapping of distributional data. Areas of contact between closely related species and phylogeographic breaks within species clustered into areas characterized by common physiographic features or predicted by previously hypothesized glacial refugia. The results underscore the general importance of geographic barriers to dispersal (mountain chains) and climate change (periods of cooling alternating with periods of warming, which lead to the contraction and expansion of species ranges) in species evolution.

Do suture zones exist?

Remington (1968) argued that 13 suture zones exist in North America. Remington defined a suture zone as, "a band of geographic overlap between major biotic assemblages, including some pairs of species or semispecies which hybridize in the zone" (p. 322). Although initially controversial, the idea that suture zones exist has picked up momentum over the past decade, due largely to the phylogeographic work of Hewitt, Avise, and their colleagues. Nevertheless, the reality of suture zones has not yet been subjected to rigorous analysis using statistical and geographic information system (GIS) approaches. To test for the existence of Remington's suture zones, we first identified 117 terrestrial hybrid zones in Canada and the United States through a literature search for the key words "cline," "contact zone," "hybrid zone," and "hybridization" in articles published between 1970 and 2002. The 117 hybrid zones were mapped using a GIS approach and compared with a digitized version of Remington's original suture zone map. Overall, there does appear to be an association between hybrid zones and suture zones, but this association is largely attributable to clustering of hybrid zones in only two of the 13 suture zones recognized by Remington. The results suggest that evolutionary biologists should retain some skepticism toward Remington's suture zones.

The genetics of reproductive isolation: a retrospective and prospective look with comments on ground crickets.

An intriguing aspect of the current renaissance in investigations of the genetics of reproductive isolation is that it has been dominated by studies that resemble work done in the 1930s, 1940s, and 1950s. The dominant model organism (Drosophila), research approaches, and traits of interest (sterility and inviability of hybrids) all harken back to this earlier era. Herein, we explore the factors that led to a rebirth of interest in the genetics of reproductive isolation and to the adoption of the approaches of an earlier generation of biologists. At the same time, we appeal for more intensive investigations of traits that reproductively isolate closely related species, inclusion of a greater range of organisms in studies of reproductive isolation, and focus on a broader range of questions surrounding speciation. We end with a description of ongoing quantitative trait loci (QTL) studies of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and Allonemobius socius. We have found several QTL with large effects on variance in patterns of sperm utilization in backcross females. Moreover, some QTL have an antagonistic effect on conspecific sperm, a finding that lends support to the hypothesis that rapid evolution of conspecific sperm precedence is a by-product of sexual conflict.

VARIATION IN GENETIC ARCHITECTURE OF CALLING SONG AMONG POPULATIONS OF ALLONEMOBIUS SOCIUS, A. FASCIATUS, AND A HYBRID POPULATION: DRIFT OR SELECTION?

Predictions using quantitative genetic models generally assume that the variance-covariance matrices remain constant over time. This assumption is based on the supposition that selection is generally weak and hence variation lost through selection can be replaced by new mutations. Whether this is generally true can only be ascertained from empirical studies. Ideally for such a study we should be able to make a prediction concerning the relative strength of selection versus genetic drift. If the latter force is prevalent then the variance-covariances matrices should be proportional to each other. Previous studies have indicated that females in the two sibling cricket species Allonemobius socius and A. fasciatus do not discriminate between males of the two species by their calling song. Therefore, differences between the calling song of the two males most likely result from drift rather than sexual selection. We test this hypothesis by comparing the genetic architecture of calling song of three populations of A. fasciatus with two populations of A. socius. We found no differences among populations within species, but significant differences in the G (genetic) and P (phenotypic) matrices between species, with the matrices being proportional as predicted under the hypothesis of genetic drift. Because of the proportional change in the (co)variances no differences between species are evident in the heritabilities or genetic correlations. Comparison of the two species with a hybrid population from a zone of overlap showed highly significant nonproportional variation in genetic architecture. This variation is consistent with a general mixture of two separate genomes or selection. Qualitative conclusions reached using the phenotypic matrices are the same as those reached using the genetic matrices supporting the hypothesis that the former may be used as surrogate measures of the latter.

REINFORCING SELECTION IS EFFECTIVE UNDER A RELATIVELY BROAD SET OF CONDITIONS IN A MOSAIC HYBRID ZONE.

Many hybrid zones have a mosaic structure, yet we know of no theoretical work that examines the impact of mosaicism on the outcome of evolution. We developed a computer simulation model designed to test whether the outcome of reinforcing selection differs in a mosaic and a clinal hybrid zone. Our model was a one-dimensional stepping-stone model. The mosaic and clinal hybrid zones that we modeled were, respectively, a mosaic maintained by differential fitness of the interacting taxa in patchy habitats and a tension zone. We modeled changes in gene frequency at two biallelic loci, A and B. Hybrids at the A locus were selected against. An allele at the B locus caused assortative mating at the A locus, which promoted reinforcement; there was a selective cost to this allele. In a mosaic hybrid zone, spatial variation in the fitness of A-locus homozygotes in different patches caused gene and genotype frequencies at the A and B loci to differ greatly from those in a tension zone. Compared to a tension zone, a mosaic hybrid zone had a broader region in which hybrids could be formed and, thus, a broader region in which the assortative-mating allele provided a net selective advantage (via decreased production of the less fit A-locus hybrids). This caused the assortative-mating allele to be favored under a broader set of conditions in a mosaic hybrid zone than in a tension zone. In mosaic and tension hybrid zones, both low and high levels of migration could prevent the establishment of the allele that promoted reinforcement, but the allele could establish under a wider range of migration rates in a mosaic than in a tension zone. In a tension zone, both low and high levels of selection against A-locus hybrids could prevent the establishment of the assortative-mating allele. In a mosaic hybrid zone, the assortative-mating allele established under lower levels of selection against hybrids than in a tension zone, and high levels of selection did not impede the establishment of this allele. Overall, our work illustrates how the structure of a hybrid zone can alter the outcome of an important evolutionary process, in this case, reinforcement.

GENETIC VARIATION IN CRICKET CALLING SONG ACROSS A HYBRID ZONE BETWEEN TWO SIBLING SPECIES.

The sibling ground crickets Allonemobius fasciatus and A. socius meet along a mosaic hybrid zone at ≈ 40°N latitude in eastern North America. In this paper we report the findings of a genetic analysis of calling-song variation within and among six cricket populations sampled along a transect through the hybrid zone in southern New Jersey. We compared aspects of the calling song of both wild-caught and laboratory-reared crickets to test the hypothesis that population differences in song observed in the wild were genetically based. We found significant, species-level differences in all aspects of the calling song, and these differences persisted even after a generation of common-garden rearing in the laboratory, supporting the hypothesis that interspecific variation observed in the wild largely reflects genetic differentiation between the two taxa. A discriminant function analysis indicated that individual crickets could be assigned to the proper taxon with less than 10% error, supporting the premise that calling song could be used by female crickets as a mechanism for species recognition. One population, collected from within the hybrid zone and containing significant numbers of hybrid individuals, was intermediate in its calling song, presumably reflecting this population's mixed genetic makeup. In this hybrid zone population, song phenotype was highly correlated to a hybrid index score generated using species-specific alleles at four diagnostic allozyme markers, suggesting a multigenic basis to calling-song variation in these crickets as well as linkage disequilibrium between markers and song. Based on an analysis of laboratory-reared full-sib families, broad-sense heritabilities for calling-song characteristics were generally significant in the two A. socius populations, whereas many components of song showed no significant family effects in the three A. fasciatus populations. The genotypically mixed, hybrid zone population showed very high heritabilities for most calling-song components, which likely reflect the influence of interspecific gene flow on genetic variation for quantitative traits.

CONSPECIFIC SPERM PRECEDENCE IS AN EFFECTIVE BARRIER TO HYBRIDIZATION BETWEEN CLOSELY RELATED SPECIES.

Conspecific sperm precedence is widespread in animals, appears to evolve rapidly, and is thought to have the potential to prevent hybridization between closely related species. However, to date no study has tested the isolating potential of such a barrier in mixed populations of two taxa under conditions in which other potential barriers to gene flow are controlled for or are prevented from operating. We tested the isolating potential of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and A. socius in population cage experiments in which the frequency of the two species was varied. Despite the observation of abundant interspecific matings, the proportions of hybrid progeny were low and differed statistically from the proportions expected in the absence of conspecific sperm precedence. The results demonstrate that conspecific sperm precedence can severely limit gene flow between closely related species, even when one species is less abundant than the other.

HOW DISCRETE ARE OAK SPECIES? INSIGHTS FROM A HYBRID ZONE BETWEEN QUERCUS GRISEA AND QUERCUS GAMBELII.

The white oaks Quercus gambelii and Q. grisea overlap in distribution in New Mexico and Arizona. Within the region of overlap, there are numerous instances of contact between the two taxa. In some areas of contact morphologically, intermediate trees are common, whereas in others, morphologically intermediate trees are rare or absent. We describe a set of RAPD markers that distinguish between the two species and use these markers to examine patterns of gene exchange in an area of contact in the San Mateo Mountains of New Mexico. The markers are highly coincident with morphology and confirm that hybridization between the two species takes place. Despite the occurrence of hybrids, both species remain distinct, even in areas of sympatry, and marker exchange appears to be limited.