Matthew R. Klooster

The Cleistogamous Breeding System: A Review of Its Frequency, Evolution, and Ecology in Angiosperms

Cleistogamy, a breeding system in which permanently closed, self-pollinated flowers are produced, has received increasing attention in recent years, but the last comprehensive review of this system was over 20 years ago. The goal of this paper is to clarify the different types of cleistogamy, quantify the number of families, genera, and species in which cleistogamy occurs, and estimate the number of times and potential reasons why cleistogamy has evolved within angiosperms. Cleistogamous species were identified through a literature survey using 13 online databases with references dating back to 1914; only those species well-supported by floral descriptions or empirical data were included in the data set. On the basis of this survey, we suggest the use of three different categories of cleistogamy in future studies: dimorphic, complete, and induced. Based on these categories, cleistogamy in general is present in 693 angiosperm species, distributed over 228 genera and 50 families. When analyzed on a family level across the angiosperms, the breeding system has evolved approximately 34 to 41 times. Theoretical investigations indicate that the evolution of cleistogamy in taxa may be influenced by the presence of heterogeneous environments, inbreeding depression and geitonogamy, and differential seed dispersal, as well as by various ecological factors and plant size. Cleistogamy will undoubtedly be discovered in additional species as the reproductive biology of more taxa is examined in the future. Such information will be invaluable for understanding the selective pressures and factors favoring the evolution of cleistogamy as well as the evolutionary loss of this breeding system, a subject that has received little attention to date.

Cryptic bracts facilitate herbivore avoidance in the mycoheterotrophic plant Monotropsis odorata (Ericaceae).

Plant coloration, shown to play a dynamic role in animal attraction, has been proposed as a means of defense, although these reports lack experimental evidence. This study empirically assesses defensive coloration in the mycoheterotrophic plant, Monotropsis odorata, which produces stems and flowers covered by dried vegetative bracts. Field studies were conducted using an experimental group of plants with bracts removed and a control group to evaluate the frequency of and fitness impacts associated with herbivory. Additionally, we quantitatively assessed the reflectance spectra of bracts, stems, and flowers of M. odorata relative to an ambient leaf litter substrate. Across the 2-yr study, the experimental group experienced a 20-27% higher mean herbivory rate and 7-20% lower mean fruit production relative to the control group. Bracts were shown to strongly resemble ambient leaf litter in spectral analyses, with stems and flowers having more conspicuous coloration. Results show that the presence of dried bracts effectively camouflages conspicuous stem and floral tissues, significantly reducing the frequency of floral and stem herbivory, and thereby increasing fruit set, a component of plant fitness. This study supports the principal hypothesis that coloration can play a fundamental role in plant defense.

Comparative analysis of the reproductive ecology of Monotropa and Monotropsis: Two mycoheterotrophic genera in the Monotropoideae (Ericaceae)

Studies of mycoheterotrophs, defined as plants that obtain carbon resources from associated mycorrhizal fungi, have fundamentally contributed to our understanding of the importance and complexity of symbiotic ecological interactions. However, to date, the reproductive ecology of these organisms remains empirically understudied, with existing literature presenting hypotheses about traits including a generalist pollination syndrome and autogamous self-pollination. To address this gap in our knowledge of the reproductive ecology of mycoheterotrophic plants, we comparatively analyzed three species of two monotropoid genera, Monotropa and Monotropsis. During three consecutive years of field observations and manipulations of four populations of Monotropa uniflora, seven of M. hypopitys (both red and yellow color forms), and two of Monotropsis odorata, we investigated flowering phenology, pollination ecology, breeding system, floral herbivory, and reproductive effort and output. Contrary to previous predictions, our results revealed that taxa are largely outcross-pollinated and specialized toward Bombus pollinators. Additionally, species differ in breeding system, timing and duration of reproductive development, fluctuations in reproductive effort and output, and fitness impacts of herbivory. This study is the first thorough investigation of the reproductive ecology of mycoheterotrophic species and provides insight into possible limitations in reproductive traits imposed by a mycoheterotrophic life history.

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.

Characterization of microsatellite loci in the myco-heterotrophic plant Monotropa hypopitys (Ericaceae) and amplification in related taxa

The myco‐heterotroph Monotropa hypopitys is a perennial, circumboreally distributed herb of significant importance in studies of nonphotosynthetic plant biology. To address a deficiency in our knowledge of myco‐heterotroph population genetics, 11 microsatellite markers were developed using a cost‐effective, nonradioactive protocol. Multiplex reactions revealed polymorphism in the red and yellow colour forms of M. hypopitys with an average of 2.69 alleles per primer. Many primers additionally amplified in the congener Monotropa uniflora and five other closely related genera. This is the first report of microsatellite primer development and amplification in the Monotropoideae (Ericaceae).

Characterization of Microsatellite Markers for Pinedrops, Pterospora andromedea (Ericaceae), from Illumina MiSeq sequencing

Premise of the study: Pterospora andromedea (Ericaceae) is a mycoheterotrophic plant endemic to North America with a disjunct distribution. Eastern populations are in decline compared to western populations. Microsatellite loci will allow comparison of genetic diversity in endangered to nonthreatened populations. Methods and Results: Illumina MiSeq sequencing resulted in development of 12 polymorphic microsatellite loci from 63 perfect microsatellite loci tested. One polymorphic locus was obtained from a traditional enrichment method. These 13 loci were screened across two western and two eastern populations. For western and eastern populations, respectively, number of alleles ranged from one to 10 and one to four, and observed heterozygosity ranged from 0.000 to 0.389 and 0.000 to 0.143. Conclusions: These are the first microsatellite loci developed for Pterospora. They will be useful in conservation efforts of the eastern populations and for examination of population genetic parameters at different geographic scales and comparison with mycorrhizal fungal hosts.

Development of microsatellite markers for the medicinal plant Isodon rubescens (Lamiaceae) and related species

PREMISE OF THE STUDY Microsatellite markers were developed for the medicinal plant Isodon rubescens to investigate genetic variability of the species and, in future studies, to assess its relation to the content of pharmacologically active chemicals produced by the plant. METHODS AND RESULTS Eleven primer pairs were identified and tested in multiple populations of I. rubescens and related species (I. henryi, I. enanderianus, I. lophanthoides) from the Peoples Republic of China. The primers amplified dinucleotide repeats and had between two and 12 alleles per locus in a given population. CONCLUSIONS These microsatellites will be useful for understanding patterns of phytochemical variation in I. rubescens and have the potential to be applied to research on evolutionary processes in other species of the genus Isodon.

Microsatellite primers for the fungi Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) from 454 shotgun pyrosequencing.

Premise of the study: Rhizopogon kretzerae and R. salebrosus (Rhizopogonaceae) are ectomycorrhizal fungi symbiotic with pines and the mycoheterotrophic plant Pterospora andromedea (Ericaceae). Microsatellite loci will allow population genetic study of fungal hosts to P. andromedea. Methods and Results: Shotgun pyrosequencing of R. kretzerae DNA resulted in primer development of 23 perfect microsatellite loci and screened across two populations each for R. kretzerae and R. salebrosus. Twelve loci were polymorphic in R. kretzerae populations, and 11 loci cross-amplified in R. salebrosus populations. For R. kretzerae and R. salebrosus, number of alleles was one to eight and one to nine, respectively, and observed heterozygosity ranged from 0.00–0.57 and 0.00–0.70, respectively. Conclusions: These are the first microsatellite loci developed for any species within Rhizopogon subgenus Amylopogon. These microsatellite loci will be used in conservation genetic studies of rare to endangered eastern populations and to compare plant and fungal population genetic structure at different hierarchical levels.

Cleistogamy Review Dataset - Species Level

This is the raw data detailing the type of cleistogamy reported within 628 individual species in the scientific literature, as of October 2005. This data underlies the following study: Culley, Theresa M and Matthew R Klooster (2007) The cleistogamous breeding system: A review of its frequency, evolution, and ecology in angiosperms. The Botanical Review 73(1): 1-30.