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.

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.

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).

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.