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The recent increase in accessibility and scale of genetic data available through next-generation sequencing (NGS) technology has transformed biological inquiry. As a direct result, the application and analysis of NGS data has quickly become an important skill for future scientists. However, the steep learning curve for applying NGS technology to biological questions, including the complexity of sample preparation for sequencing and the analysis of large data sets, are deterrents to the integration of NGS into undergraduate education. Here, we present a course-based undergraduate research experience (CURE) designed to aid in overcoming these limitations through NGS investigations of prokaryotic diversity. Specifically, we use 16S rRNA sequencing to explore patterns of diversity stemming from student-directed hypothesis development. This CURE addresses three learning objectives: (1) it provides a forum for experimental design hypothesis generation, (2) it introduces modern genomic tools through a hands-on experience generating an NGS data-set, and (3) it provides students with an introductory experience in bioinformatics.

Disentangling the contemporary and historical factors underlying the spatial distributions of species is a central goal of biogeography. For species with broad distributions but little capacity to actively disperse, disconnected geographical distributions highlight the potential influence of passive, long-distance dispersal (LDD) on their evolutionary histories. However, dispersal alone cannot completely account for the biogeography of any species, and other factors-e.g. habitat suitability, life history-must also be considered. North American ice worms ( Mesenchytraeus solifugus) are ice-obligate annelids that inhabit coastal glaciers from Oregon to Alaska. Previous studies identified a complex biogeographic history for ice worms, with evidence for genetic isolation, unexpectedly close relationships among geographically disjunct lineages, and contemporary migration across large (e.g. greater than 1500 km) areas of unsuitable habitat. In this study, we analysed genome-scale sequence data for individuals from most of the known ice worm range. We found clear support for divergence between populations along the Pacific Coast and the inland flanks of the Coast Mountains (mean FST = 0.60), likely precipitated by episodic ice sheet expansion and contraction during the Pleistocene. We also found support for LDD of ice worms from Alaska to Vancouver Island, perhaps mediated by migrating birds. Our results highlight the power of genomic data for disentangling complex biogeographic patterns, including the presence of LDD.

Abstract Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at http://www.ambystoma.org.

Abstract Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories. Results Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Conclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

Comparative population genetic studies provide a powerful means for assessing the degree to which evolutionary histories may be congruent among taxa while also highlighting the potential for cryptic diversity within existing species.In the Rocky Mountains, three confamilial stoneflies (Zapada glacier, Lednia tumana, and Lednia tetonica; Plecoptera, Nemouridae) occupy cold alpine streams that are primarily fed by melting ice. Lednia tumana and L. tetonica are sister species diagnosed from systematic morphological differences, and they are endemic to areas surrounding Glacier National Park and Grand Teton National Park, respectively, in the U.S. Rocky Mountains. Zapada glacier is also present in alpine streams from Glacier National Park to the Teton Range, sometimes co‐occurring with either Lednia species.We used mitochondrial sequence data to clarify species boundaries, compare population genetic patterns, and test demographic models in a coalescent framework for the three stoneflies. We addressed four questions: (1) Is there genetic support for the morphology‐based species boundaries in Lednia? (2) Is there genetic support for cryptic, or as‐yet undescribed, diversity within Z. glacier? (3) Do similar geographic distributions and ecological requirements yield spatial congruence of genetic structure between high‐elevation Lednia and Z. glacier populations? (4) Is there evidence for contemporary gene flow among isolated populations in either group?Our results supported the existing taxonomy with Z. glacier and the two Lednia species differing in their depths of divergence among study regions (e.g. maximum sequence divergence within Z. glacier = 1.2% versus 5% between L. tumana and L. tetonica). However, spatial population genetic patterns were broadly congruent, indicating stonefly populations isolated on mountaintop islands. Coalescent modelling supported the possibility of rare, extremely limited contemporary gene flow among Z. glacier populations, with no support for gene flow between L. tumana and L. tetonica.The focal stoneflies and associated assemblages occupy the highest elevation, coldest permanent alpine streams in the study region. This lotic habitat type faces an uncertain future under a diminishing alpine cryosphere. Given spatial congruence of genetic structure demonstrating unique biodiversity associated with individual alpine islands, we encourage conservation management strategies be developed and applied at corresponding spatial scales. [ABSTRACT FROM AUTHOR]

Perhaps the most important recent advance in species delimitation has been the development of model-based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next-generation sequence data sets provides powerful insights into genome-wide patterns of divergence during speciation. However, applying complex models to large data sets is time-consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight-locus, 137-individual data set and an 89-locus, seven-individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow-scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range. [ABSTRACT FROM AUTHOR]

Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing ( PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander ( Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research. [ABSTRACT FROM AUTHOR]