Anatomical and Genetic Variation of Western Oxyloma (Pulmonata: Succineidae) Concerning the Endangered Kanab Ambersnail (Oxyloma haydeni kanabense) in Arizona and Utah Scientific Investigations Report 2013–5164 U.S. Department of the Interior U.S. Geological Survey Cover: Foreground photograph: Kanab ambersnail (Oxyloma haydeni kanabense). Background photograph: Vasey’s Paradise waterfalls, Colorado River, Arizona. Photograph taken by Jeff Sorensen, Arizona Game and Fish Department. Anatomical and Genetic Variation of Western Oxyloma (Pulmonata: Succineidae) Concerning the Endangered Kanab Ambersnail (Oxyloma haydeni kanabense) in Arizona and Utah By Melanie Culver, Hans-Werner Herrmann, Mark Miller, Barry Roth, and Jeff Sorenson Scientific Investigations Report 2013–5164 U.S. Department of the Interior U.S. Geological Survey U.S. Department of the Interior SALLY JEWELL, Secretary U.S. Geological Survey Suzette M. Kimball, Acting Director U.S. Geological Survey, Reston, Virginia: 2013 For more information on the USGS—the Federal source for science about the Earth, its natural and living resources, natural hazards, and the environment, visit http://www.usgs.gov or call 1–888–ASK–USGS. For an overview of USGS information products, including maps, imagery, and publications, visit http://www.usgs.gov/pubprod To order this and other USGS information products, visit http://store.usgs.gov Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner. Suggested citation: Culver, Melanie, Herrmann, Hans-Werner, Miller, Mark, Roth, Barry, and Sorenson, Jeff, 2013, Anatomical and genetic variation of western Oxyloma (Pulmonata: Succineidae) concerning the endangered Kanab ambersnail (Oxyloma haydeni kanabense) in Arizona and Utah: U.S. Geological Survey Scientific Investigations Report, 2013–5164, 66 p., http://pubs.usgs.gov/sir/2013/5164/. iii Contents Abstract ...........................................................................................................................................................1 Introduction.....................................................................................................................................................2 Background Information on Subspecies Trinomial .........................................................................4 Molecular Genetic Markers Used .....................................................................................................4 Study Objectives ...................................................................................................................................5 Purpose and Scope ..............................................................................................................................5 Methods ..........................................................................................................................................................5 Specimen Collection ............................................................................................................................5 Shell Morphology .................................................................................................................................7 Soft Tissue Anatomy .............................................................................................................................8 DNA Isolation.........................................................................................................................................8 Genetic Analyses ..................................................................................................................................8 Mitochondrial DNA ......................................................................................................................8 Data Generation ..................................................................................................................8 Nuclear DNA.................................................................................................................................9 Single Copy ..........................................................................................................................9 Markers from Other Studies ....................................................................................9 Genomic Library .........................................................................................................9 Data Analyses—Mitochondrial DNA and Nuclear Single Copy DNA ............10 Microsatellite (Single Locus) ..........................................................................................10 Genomic Library .......................................................................................................11 PCR Conditions .........................................................................................................11 AFLP (Multilocus) ..............................................................................................................11 Data Generation .......................................................................................................11 Data Analyses ..........................................................................................................11 Results ...........................................................................................................................................................12 Specimen Collection ..........................................................................................................................12 Shell Morphology ................................................................................................................................12 Principal Components Analysis ...............................................................................................12 Discriminant Analysis................................................................................................................18 Soft Tissue Anatomy ...........................................................................................................................18 DNA Isolation.......................................................................................................................................18 Genetic Analyses ...............................................................................................................................18 Mitochondrial DNA ....................................................................................................................26 Nuclear DNA...............................................................................................................................30 Single Copy ........................................................................................................................38 Microsatellite (Single Locus) ..........................................................................................42 AFLP (Multilocus) ..............................................................................................................50 iv Contents—Continued Discussion ....................................................................................................................................................52 Shell Morphology ................................................................................................................................52 Soft Tissue Anatomy ...........................................................................................................................52 Morphology and Anatomy—Taxonomic-Nomenclatural Considerations .................................53 Genetics................................................................................................................................................53 Each Marker Separate ..............................................................................................................54 Markers Combined ....................................................................................................................56 Conservation Implications .................................................................................................................56 Future Research ..........................................................................................................................................57 Summary........................................................................................................................................................57 Acknowledgments ......................................................................................................................................57 References Cited..........................................................................................................................................58 Glossary of Molecular Genetic Terms ......................................................................................................63 Appendix A. Genbank Accession Numbers ...........................................................................................65 Appendix B. DNA Sequences for Random Ambersnail Nuclear Clone 356 ......................................65 Appendix C. DNA Sequences for Ribosomal First and Second