Jessica R. Brzyski

An Empirical Review: Characteristics of Plant Microsatellite Markers that Confer Higher Levels of Genetic Variation

During microsatellite marker development, researchers must choose from a pool of possible primer pairs to further test in their species of interest. In many cases, the goal is maximizing detectable levels of genetic variation. To guide researchers and determine which markers are associated with higher levels of genetic variation, we conducted a literature review based on 6782 genomic microsatellite markers published from 1997–2012. We examined relationships between heterozygosity (He or Ho) or allele number (A) with the following marker characteristics: repeat type, motif length, motif region, repeat frequency, and microsatellite size. Variation across taxonomic groups was also analyzed. There were significant differences between imperfect and perfect repeat types in A and He. Dinucleotide motifs exhibited significantly higher A, He, and Ho than most other motifs. Repeat frequency and motif region were positively correlated with A, He, and Ho, but correlations with microsatellite size were minimal. Higher taxonomic groups were disproportionately represented in the literature and showed little consistency. In conclusion, researchers should carefully consider marker characteristics so they can be tailored to the desired application. If researchers aim to target high genetic variation, dinucleotide motif lengths with large repeat frequencies may be best.

Reproductive allocation between the sexes, across natural and novel habitats, and its impact on genetic diversity

Allocation to reproductive mode (sexual and asexual) can vary depending on environmental conditions but is often examined at the population level, whereas selection acts upon the individual. We examined individual variation in reproductive mode to identify how the interaction of sex and the environment affect population genetic diversity. Using the plant Marchantia inflexa, we tested whether reproductive allocation pattern varies consistently between males and females and among plants collected from different environments, and determined if morphological responses were the result of individual plasticity or genetic differences. We then quantified genetic variability between the different environments and between the sexes. Male and female plants were collected from two strikingly different habitats within the same region: along natural sites (rivers) and along novel human-modified sites (roadsides). Using a common garden approach, we found a strong sex by habitat interaction: male and female responses differed significantly by their source habitat. For females, relative to river-collected, road-collected plants had higher growth and asexual reproduction, while the pattern was reversed, although not significant, for males. Genetic differentiation was significant between the two habitats with no evidence of individual differences in plasticity for growth, but there was a genotype effect for asexual propagule production. Males and females did not differ genetically; but river-collected plants with lower sexual potential were more diverse than roadside-collected plants, possibly the result of founder events. These results show that individual variation in reproduction is controlled by the interaction of both the environment and genetics. Due to different selection pressures between natural and novel habitats, there are observable differences in life history traits with an associated evolutionary response to the novel habitat.

An Efficient Technique for Primer Development and Application that Integrates Fluorescent Labeling and Multiplex PCR

Premise of the study: Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction. Methods and Results: This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol. Conclusions: This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material.

Isolation and characterization of microsatellite markers in the rare clonal plant, Spiraea virginiana (Rosaceae).

UNLABELLED PREMISE OF THE STUDY Microsatellite markers were developed in Spiraea virginiana, a federally threatened native shrub found along stream banks, to identify clonal genotypes and measure population genetic variability. • METHODS AND RESULTS Eleven primer sets were developed using a non-radioactive protocol. These revealed a moderate level of genetic variation, as indicated by the number of alleles per locus (range = 1-4) and an average observed heterozygosity of 0.595. Select loci also amplified successfully in the related species Spiraea japonica. • CONCLUSION Development of the markers described here is critical for the genetic identification of clonal plants as a first step in demographic analyses, and is necessary for the future conservation of this rare species. Amplification of the markers in S. japonica suggests their potential utility in research regarding this species.

Beaver (Castor canadensis) Impacts on Herbaceous and Woody Vegetation in Southeastern Georgia

Abstract North American beavers alter habitat properties such as stream flow, faunal composition and plant community structure. In North America, the majority of studies on impacts by beavers have occurred in the northeast or western regions. This study examined the effect of wood-cutting and foraging by beaver on the surrounding plant community in Southeastern Georgia. To determine their impact, beaver modified habitat and nearby, but non-impacted riparian habitat, were sampled in a matched pair design. Vegetation surveys were performed, and species richness, density, relative abundance and canopy cover were calculated for every site. Richness of herbaceous vegetation was higher at distances closer to shore while richness of large woody vegetation increased with distance from shore with no difference found between beaver and control sites. The composition of herbaceous or woody vegetation did not differ significantly between beaver and control sites; species had an equal chance of occurring in either site. Canopy cover was similar at the two site types. Non-native species were rare and did not differ by site type. Beavers were in low density and had a relatively benign impact on the plant community. Density of woody plants was higher at beaver sites, suggesting that beavers may favor areas of abundant woody vegetation. Beaver impact may be less in southern regions than in northern ones. This may reflect the lower density of beavers, but impact also could be due to the year round availability of and access to vegetation.

Seed Germination in the Riparian Zone: The Case of the Rare Shrub, Spiraea virginiana (Rosaceae)

ABSTRACT   Conservation of rare species often involves restoration efforts to enhance or expand existing populations, a critical step of which is seed germination. Seed germination can be especially problematic in riparian zones where habitat disturbance through flooding can be a regular and frequent event. Spiraea virginiana Britton, a riparian clonal shrub, is a rare species that is particularly susceptible to problems at the seed stage; it is generally assumed to lack effective seed recruitment, although seeds are sometimes observed in the field. Therefore, we examined the effects of growth substrate and cold stratification on the number of days required for seeds to germinate and overall seed germination success. Overall, total seed germination for S. virginiana was low at only 10% and cold stratification had a significant effect on the initiation of germination; as the number of days of cold stratification increased, the time necessary for seeds to germinate decreased. Although seedlings of S. virginiana have not been observed in nature, this study indicates that seeds can germinate and thus recruitment by seed is at least theoretically possible. We recommend that a seed germination protocol using sterile soil with cold (5°C), stratification treatment of at least 30 d should be used in future propagation and restoration efforts.

Invading from the garden? A comparison of leaf herbivory for exotic and native plants in natural and ornamental settings

Abstract  The enemies release hypothesis proposes that exotic species can become invasive by escaping from predators and parasites in their novel environment. Agrawal et al. (Enemy release? An experiment with congeneric plant pairs and diverse above‐ and below‐ground enemies. Ecology, 86, 2979–2989) proposed that areas or times in which damage to introduced species is low provide opportunities for the invasion of native habitat. We tested whether ornamental settings may provide areas with low levels of herbivory for trees and shrubs, potentially facilitating invasion success. First, we compared levels of leaf herbivory among native and exotic species in ornamental and natural settings in Cincinnati, Ohio, United States. In the second study, we compared levels of herbivory for invasive and noninvasive exotic species between natural and ornamental settings. We found lower levels of leaf damage for exotic species than for native species; however, we found no differences in the amount of leaf damage suffered in ornamental or natural settings. Our results do not provide any evidence that ornamental settings afford additional release from herbivory for exotic plant species.

Does sexual reproduction matter for a rare clonal species in frequently disturbed habitats?1

Abstract Sexual reproduction is often considered essential for evolutionary change, with many plants possessing mechanisms to promote outcrossing and produce genetically variable offspring. Many plant species, however, are also capable of asexual reproduction, but its relative role in population dynamics is often overlooked. Given that sexual reproduction depends in part on mate availability and therefore plant density, would asexual reproduction predominate in rare species, especially those found in highly disturbed habitats? To explore this question, we used field and genetic techniques to investigate the reproductive system of a rare, clonal shrub, Spiraea virginiana Britton, which inhabits the frequently disturbed riparian zone. We performed controlled hand pollinations within and among individuals both within and across populations. We also quantified allelic richness and heterozygosity at a locus purported to control self-incompatibility (SI). Hand pollinations resulted in low total fruit production but more infructescences were produced with outcross pollen (20%) than with self-pollen (2.3%). Low allelic richness (2–7 alleles/population) was found at the putative SI-locus; variation at this locus did not correspond directly with the pollination results, suggesting either the SI system is leaky or that the examined SI-locus is not primarily responsible for incompatibility in this species. This study shows that rarity combined with clonality within a highly disturbed habitat may promote a combination of asexual and sexual reproduction. Although sexual reproduction can occur, it may be limited by a scarcity of available mates for S. virginiana, thereby resulting in primarily asexual reproduction in the species.

Characterization of 12 polymorphic microsatellite markers in the liverwort Marchantia inflexa (Marchantiaceae)

PREMISE OF THE STUDY Microsatellite markers were developed in Marchantia inflexa, a haploid liverwort with unisexual individuals, to identify clonal genotypes and measure population genetic variability. METHODS AND RESULTS Twelve polymorphic primer sets were developed from three enriched genomic libraries. Primers were fluorescently labeled, and alleles were identified by fragment analysis. These primers were tested in four natural populations and revealed a moderate level of genetic variation within four populations, as indicated by the number of alleles per locus (range = 1-5). CONCLUSIONS Development of polymorphic markers is crucial to the identification of individuals and will allow additional research into this species, particularly on its population genetics and metapopulation dynamics.

The impact of asexual and sexual reproduction in spatial genetic structure within and between populations of the dioecious plant Marchantia inflexa (Marchantiaceae)

Background and Aims In dioecious plants, sexual reproduction requires close proximity to potential mates, but clonal growth can increase this distance and, therefore, reduce the probability of mating. Reduction in sexual propagules can lead to decreased dispersal and gene flow between populations. Gene flow and clonal growth may be further influenced by the size of the habitat patch. The effects of habitat size and reproductive mode (sexual or asexual reproduction) on spatial genetic structure and segregation of the sexes were tested by quantifying the distributions of genotypes and the sexes using the dioecious liverwort Marchantia inflexa. Methods Plants were sampled from five pairs of small-large habitat patches to identify within- and among-population spatial genetic structure using 12 microsatellite markers. Spatial distributions were calculated as the likelihood that pairs of individuals were the same sex or genotype, and it was determined how that likelihood was affected by habitat patch size (small/large). Key Results Asexual reproduction dominates within populations, and asexual dispersal also occurred across populations. Spatial segregation of the sexes was observed within populations; males were more likely to be near individuals of the same sex than were females. Although the likelihood of both sexes being near members of the same sex was similarly greater on small habitat patches, on large habitat patches male genotypes were almost 15 % more likely to be near clonemates than were female genotypes. Conclusions The results show a sex difference in clonal clumping that was dependent upon habitat size, suggesting differential colonization and/or survival between males and females. The sexes and genotypes being structured differently within and among populations have implications for the persistence of populations and the interactions between them. This study demonstrates that studying only the sexes and not their genotypes (or vice versa) can limit our understanding of the extent to which reproductive modes (sexual or asexual) influence genetic structure both within and between populations.