Charlotte C. Germain-Aubrey

The Report of My Death was an Exaggeration: A Review for Researchers Using Microsatellites in the 21st Century

Microsatellites, or simple sequence repeats (SSRs), have long played a major role in genetic studies due to their typically high polymorphism. They have diverse applications, including genome mapping, forensics, ascertaining parentage, population and conservation genetics, identification of the parentage of polyploids, and phylogeography. We compare SSRs and newer methods, such as genotyping by sequencing (GBS) and restriction site associated DNA sequencing (RAD-Seq), and offer recommendations for researchers considering which genetic markers to use. We also review the variety of techniques currently used for identifying microsatellite loci and developing primers, with a particular focus on those that make use of next-generation sequencing (NGS). Additionally, we review software for microsatellite development and report on an experiment to assess the utility of currently available software for SSR development. Finally, we discuss the future of microsatellites and make recommendations for researchers preparing to use microsatellites. We argue that microsatellites still have an important place in the genomic age as they remain effective and cost-efficient markers.

Making next-generation sequencing work for you: approaches and practical considerations for marker development and phylogenetics

Recent reviews are setting the stage for the use of next-generation sequencing technologies in phylogenetic applications. However, the processes for developing new markers for phylogenetic analyses remain difficult to navigate for many researchers in plant systematics. We review several experimental approaches and practical considerations for developing new phylogenetic markers with next-generation sequencing technologies. We also outline a flexible framework for data acquisition that is readily adaptable to the needs of individual researchers and carefully consider cost-related issues that may be of concern to many laboratories in evolutionary biology. The next-generation and targeted sequencing approaches presented here offer considerable savings of time and money over the traditional PCR and Sanger sequencing approaches currently used in plant systematic research, particularly in cases involving large numbers of taxa and phylogenetic markers. Even with a limited research budget, next-generation sequence data can accommodate exploration of biological questions in ways that were not previously possible.

Digitization Workflows for Flat Sheets and Packets of Plants, Algae, and Fungi

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundations (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7–36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

A New Resource for the Development of SSR Markers: Millions of Loci from a Thousand Plant Transcriptomes

Premise of the study: The One Thousand Plant Transcriptomes Project (1KP, 1000+ assembled plant transcriptomes) provides an enormous resource for developing microsatellite loci across the plant tree of life. We developed loci from these transcriptomes and tested their utility. Methods and Results: Using software packages and custom scripts, we identified microsatellite loci in 1KP transcriptomes. We assessed the potential for cross-amplification and whether loci were biased toward exons, as compared to markers derived from genomic DNA. We characterized over 5.7 million simple sequence repeat (SSR) loci from 1334 plant transcriptomes. Eighteen percent of loci substantially overlapped with open reading frames (ORFs), and electronic PCR revealed that over half the loci would amplify successfully in conspecific taxa. Transcriptomic SSRs were approximately three times more likely to map to translated regions than genomic SSRs. Conclusions: We believe microsatellites still have a place in the genomic age—they remain effective and cost-efficient markers. The loci presented here are a valuable resource for researchers.

Niche divergence between diploid and autotetraploid Tolmiea

PREMISE OF STUDY Polyploidy is common in eukaryotes and is of major evolutionary importance over both short and long time-scales. Compared to allopolyploids, autopolyploids remain understudied; they are often morphologically cryptic and frequently remain taxonomically unrecognized, although there is increasing recognition of the high frequency of autopolyploidy in angiosperms. While autopolyploidy can serve as an instant speciation mechanism, little is known about the ecological consequences of this process. We describe the ecological divergence of a diploid-autotetraploid species pair in Tolmiea. METHODS We investigated whether abiotic niche divergence has shaped the current allopatric distribution of diploid T. diplomenziesii and its autotetraploid derivative, T. menziesii, in the Pacific Northwest of North America. We employed field measures of light availability, as well as niche modeling and a principal component analysis of environmental space. Within a common garden, we also investigated physiological responses to changes in soil moisture. KEY RESULTS Diploid and autotetraploid Tolmiea inhabit significantly different climatic niche spaces. The climatic niche divergence between these two species is best explained by a shift in precipitation availability, and we found evidence of differing physiological response to water availability between these species. CONCLUSIONS We found that spatial segregation of T. diplomenziesii and T. menziesii was accompanied by adaptation to changes in climatic regime. Tolmiea menziesii is not a nascent autotetraploid, having persisted long enough to be established throughout the Pacific Northwest, and therefore both polyploidization and subsequent evolution have contributed to the observed differences between T. menziesii and T. diplomenziesii.

A Computational- and Storage-Cloud for Integration of Biodiversity Collections

A core mission of the Integrated Digitized Biocollections (iDigBio) project is the building and deployment of a cloud computing environment customized to support the digitization workflow and integration of data from all U.S. non-federal biocollections. iDigBio chose to use cloud computing technologies to deliver a cyber infrastructure that is flexible, agile, resilient, and scalable to meet the needs of the biodiversity community. In this context, this paper describes the integration of open source cloud middleware, applications, and third party services using standard formats, protocols, and services. In addition, this paper demonstrates the value of the digitized information from collections in a broader scenario involving multiple disciplines.

Are microsatellite fragment lengths useful for population-level studies? The case of Polygala lewtonii (Polygalaceae).

Premise of the study: Microsatellites, despite being commonly used population-level markers, contain biases because scoring relies solely on fragment length. Their complexity can lead to homoplasy, the effects of which are poorly understood. We investigated the impact of using fragment lengths, repeats, or flanking region sequences on common population-level analyses. Methods: Five polymorphic microsatellite markers amplified across the central Florida scrub endemic Polygala lewtonii (Polygalaceae) and its close, widespread congener P. polygama. We genotyped 147 individuals of P. lewtonii and 156 of P. polygama, and sequenced the amplicons of four markers across all individuals. We ran basic statistics, spatial clustering analysis, historical demographics, and migration tests. Results: One population of intermediate morphology was genetically clearly identified as P. polygama, making it the southernmost population of this species. Statistics were comparable between the fragment length and repeat numbers, with some notable differences. Flanking regions exhibited surprisingly high polymorphism between species, and between geographically distant conspecific populations. Discussion: The increasing use of markers developed in one species and amplified in another is only a good practice if precautions are taken, notably the sequencing of the fragments between species and populations. Flanking region sequences are a useful marker at the interspecific level.

Using Comparative Biogeography to Retrace the Origins of an Ecosystem: The Case of Four Plants Endemic to the Central Florida Scrub

Premise of research. Plant communities assemble through historical, adaptive, and stochastic processes, with their relative contributions varying among communities. This study traces the origin of the highly threatened Florida scrub ecosystem, a series of relic sand dunes with exceptionally high endemism. We used dated phylogenies with four endemic angiosperms to test two hypotheses on the origins of the Florida scrub: the western hypothesis, which emphasizes the role of mid-Pliocene fragmentation of the xeric belt between southwestern North America and Florida, and the eastern hypothesis, which emphasizes the glacial cycles of the Pleistocene. Methodology. Augmenting existing phylogenies, we sequenced the nuclear internal transcribed spacer region and several plastid loci of species of Prunus (Rosaceae), Polygala (Polygalaceae), Persea (Lauraceae), and Ilex (Aquifoliaceae) to phylogenetically place the Florida scrub endemics and identify their putative sister taxa. We used topology tests, ancestral area reconstruction analyses, and time-calibrated trees to evaluate the western and eastern hypotheses both geographically and temporally. Pivotal results. Results for Polygala and Ilex support the eastern hypothesis for the origin of the central Florida endemics, while data for Persea and Prunus are ambiguous. However, all species show an eastern ancestral distribution to some degree. Molecular dating analyses suggest that extant Ilex opaca populations diverged in the Pleistocene, with older Pliocene origins for the North American Persea clade, Polygala lewtonii, and Prunus geniculata. Conclusions. Taken together, our results support eastern North America as the dominant origin of the plant species of the central Florida scrub. However, contrary to the current eastern hypothesis, molecular dating suggests that the origins of the four endemics predate the last glacial cycles. These results have implications for the age of the Florida scrub itself as well as its component species, suggesting that the Florida scrub or its precursor existed before Pleistocene glaciation. Furthermore, the more ancient age of this assemblage of species than envisioned by most accounts suggests that interspecific interactions in this community are not recently formed (i.e., since the end of the Pleistocene) and may have been established over much longer periods of geological history. These fundamental associations argue for enhanced conservation efforts of both the individual species and the persistent remnants of the scrub community.

Microsatellite marker development for the federally listed Prunus geniculata (Rosaceae)1

PREMISE OF THE STUDY Prunus geniculata is a federally listed species endemic to the central Florida scrub. To investigate levels and geographic partitioning of genetic variation in this species, we developed microsatellite markers for P. geniculata and its sister species, P. maritima. METHODS AND RESULTS Following a protocol optimized in our laboratory, we designed 36 primer pairs, of which 8 amplified across both species and showed informative polymorphism. Product size varied from 163 to 370 bp, and the number of alleles ranged from 5 to 27. He across all populations ranged from 0.604 to 0.9155. CONCLUSIONS Eight loci were useful at the population level and will be used for conservation genetic and phylogeographic studies of Prunus geniculata and P. maritima. Furthermore, from five to all eight of these loci can also be amplified in several other North American Prunus species.