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Molecular Identification of Unknown Yellowstone Shrews
Nathan Stack & Dr. Hayley Lanier
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Acknowledgements Wyoming INBRE Field collection Lab assistance
Dr. Scott Seville Dr. Zac Roehrs Meredith Roehrs Kevin Crouch-Jacobs Ashkia Campbell Andy Kulikowski, II Lab assistance Dr. John Chase Dr. Motriuk-Smith UW Core facility UW Macromolecular Analysis Core
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Introduction Ecological & biodiversity studies
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Introduction Ecological & biodiversity studies Challenges
Difficult to see Hard to collect
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Introduction Ecological & biodiversity studies Challenges Collection
Difficult to see Hard to collect Collection Different methods Types of traps
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Introduction Ecological & biodiversity studies Challenges Collection
Difficult to see Hard to collect Collection Different methods Types of traps Identification of cryptic species External characters
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Introduction Ecological & biodiversity studies Challenges Collection
Difficult to see Hard to collect Collection Different methods Types of traps Identification of cryptic species External characters
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Introduction Ecological & biodiversity studies Challenges Shrews
Difficult to see Hard to collect Collection Different methods Types of traps Identification of cryptic species External characters Shrews Hard to determine species in the field Look very similar
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Introduction
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Introduction
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Sorex Unicuspids 1st upper incisor – large hooked, posterior cusp appears to be distinct tooth.
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Sorex unicuspids 1st upper incisor – large hooked, posterior cusp appears to be distinct tooth. Multiple species share the same unicuspid formula.
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Yellowstone Project 1988 fire in Greater Yellowstone Area (GYA)
~570,000 ha Shrews – features to specialize on eating insects. Short dense hair, no auditory bulla, zygomatic arch. Soricidae family w/ true shrews. 3 subfamilies.
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Yellowstone Project 1988 fire in Greater Yellowstone Area (GYA) Shrews
~570,000 ha Shrews Mammals Must eat large quantities of invertebrates. Invertebrate abundance Shrews – features to specialize on eating insects. Short dense hair, no auditory bulla, zygomatic arch. Soricidae family w/ true shrews. 3 subfamilies.
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Yellowstone Project 1988 fire in Greater Yellowstone Area (GYA) Shrews
~570,000 ha Shrews Mammals Must eat large quantities of invertebrates Invertebrate abundance Species identification Field Lab Molecular identification methods DNA barcoding © L. Olson Shrews – features to specialize on eating insects. Short dense hair, no auditory bulla, zygomatic arch. Soricidae family w/ true shrews. 3 subfamilies.
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DNA Barcoding
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DNA Barcoding
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DNA Barcoding
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DNA Barcoding
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Yellowstone Project Shrews – features to specialize on eating insects. Short dense hair, no auditory bulla, zygomatic arch. Soricidae family w/ true shrews. 3 subfamilies.
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Methods & Materials Collection
222 shrews trapped, 173 specimens (& tissues) kept Sherman traps & pitfall traps
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Methods & Materials Extraction Genomic DNA DNeasy Blood & Tissue Kit
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Methods & Materials PCR optimization
Custom cycles designed based on thermal gradients DNA amplification Polymerase chain reaction (PCR) using thermocycler Primers Cytochrome b Breast Cancer Susceptibility 1 Gene Apoplipoprotein B gene
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Methods & Materials Electrophoresis
After amplification, gels visualized DNA visualization
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Methods & Materials Sequencing DNA cleanup & sequencing preparation
Exosap Sequencing
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp.
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 3 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 3 78 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 3 78 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 3 78 15 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Originally designated species
Results Total of 50 samples sequenced Actual Species Originally designated species Number Captured Sorex cinereus Sorex monticolus Sorex spp. 24 2 4 - 71 3 78 15 1 Sorex sp. that stayed sp. contamination issue. 22 species had more than one band, which complicated resulting chromatograms
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Discussion East facing burn East facing control
More shrews in both types of plots. They now make up 25-35% of species diversity.
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Discussion
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Discussion
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Discussion 28 sequences were clear enough to have nucleotide BLAST results on NCBI with a similar species 5 identified correctly in the field 4 misidentified in the field 19 newly identified to species More species to be verified or identified More species from 2014 and past years waiting for molecular verification of identifications.
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Beyond shrews… Habitat change Molecular techniques have many uses
Impacts biodiversity Molecular techniques have many uses Universal tool Any species Habitats are changing quickly, and this has impacts on biodiversity These types of techniques can be used to very quickly assess diversity in an area.
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