Dr. Jack Sites, Department of Biology

This award provided $20,000 to support of molecular phylogenetic work on a number of projects, including those related to my long-term focus on the “deep history” relationships of squamate reptiles (lizards and snakes), and the second more recent emphasis on phylogeographic (“shallow history”) studies of frogs and lizards. This second focus is in large measure a direct consequence of the NSF-PIRE project housed here at BYU (“Speciation in Patagonia: Establishing Sustainable International Collaborations in Evolution, Ecology, and Conservation Biology”; www.patagonia.byu.edu). In this project we focus on comparative phylogeography of selected co-distributed species in Patagonia. My lab is in charge of data collection/analyses of multiple species of frogs and lizards, conducting some field work (in Argentina & Chile), proper cataloging and curation of both specimen voucher and tissues, and coordinating student/faculty exchanges between collaborating institutions working on these groups.

I requested support last year for PhD student Arley Camargo ($3,500) to mentor undergraduate students, another $2,500 for post-doc Dr. Frank Fontanella to organize a field trip to the Appalachian region to collect snakes, with one BYU undergraduate student, $6,000 for support of several undergraduate students, and $8,000 for consumable supplies for molecular lab work for 3 projects described in some detail in the original proposal.

1 – Arley Camargo completed all lab work associated with his dissertation, and of the 3 required chapters, he has published the first (Camargo, A., B. Sinervo, & J.W. Sites, Jr. 2003. Lizards as model organisms for linking phylogeographic and speciation studies. Molecular Ecology 19:3250-3270). His second chapter has been edited by me and is now under review by his committee members (Camargo, A., M. Morando, L.J. Avila, & J.W. Sites, Jr. Empirical performance of multi-locus species tree methods under varying locus, base-pair, and individual sampling designs. This ms will be submitted shortly to the journal Systematic Biology). Arley is conducting analyses for his final manuscript, and I should see a draft in early December. He will defend his dissertation on 3 February, and plans an April graduation.

Beyond his own work, Arley mentored several undergraduates, including Greg Walker (now in medical school), and Amy Dawson (doing an independent research project currently in my lab). He also mentored several visiting Argentinian colleagues here at BYU this summer, and he team-taught with me and 3 other university faculty members a 5-day workshop in February of this year, at Univ. of Cordoba. We were NOT able to procure all of the specimens needed for the parthenogenetic study described in this MEG proposal, but these undergraduate students will be co-authors on other studies.

Arley initially supervised two students, Amy Dawson and Greg Walker, in the molecular lab, and taught both all DNA protocols for the successful extraction, amplification, sequencing, and editing of gene sequences for some of the projects in my lab. Both of these students worked with us long enough to attain independence, and they in turn became mentors for other incoming students. Greg is now in medical school, and Amy remains in my lab, mentoring a new undergraduate students. Both of these students will be co-authors on manuscripts currently in preparation, or soon to be written. Arley also mentored two other graduate students, one each from Argentina and Brazil, especially in providing guidance to the editing and alignment of nuclear gene sequences. Compared to genes from the mitochondrial genome, nuclear genes present unique and non-intuitive challenges to alignment and analyses, and Arley is currently the leader in my lab group for these efforts.

Lastly, Arley also worked directly with students, including three other undergraduates (Rustin Reed, Doug Brown, & Nathan Hanna), as well as visiting graduate students from Argentina (Cintia Medina & Melisa Olave), to curate thousands of tissue/DNA samples representing nearly a decade of field work in Patagonia. This requires duplicating all samples, preparing a computerized catalog to data base these collections, cross-linking the tissue catalog with the catalog of voucher specimens (housed in the Bean Life Science Museum and in collections in Argentina), and then when the molecular work is complete, transferring these samples from the WIDB to the ultra-low freezer storage facility in the BLSM. As a result of Arley’s efforts, we have been able to transfer perhaps 3,500 samples into the permanent archival collections in the BLSM.

In all of these cases, Arley mentored students on a daily basis, especially at the beginning of a project when they are most unfamiliar with protocols. He first taught students the techniques for DNA extraction from tissues, amplification of target DNA regions via PCR reactions, and DNA sequencing, and followed with details editing of DNA sequences and the preparation of DNA data for analyses such as phylogenetic reconstruction and species delimitation. In this context Arley explained clearly how information in DNA sequences can be used to test specific hypotheses about species limits, relationships among species, and population genetic histories. All of these points were reinforced in the reading/discussion of published papers in our weekly lab group meetings. For museum curation, Arley taught students the basics of cataloging new specimens (or tissues), and computerization of geo-referenced data associated with each entry.

2 – Dr. Frank Fontanella – with additional support from the Dept. of Biology and the Bean Life Science Museum, Dr. Fontanella was able to take 2 BYU undergraduates with him to western N. Carolina for 2+ weeks of intensive field work. The students were Doug Brown & Dan Pearce. Their goal was the focused collection of ringneck snakes near a contact zone between two strongly-differentiated populations, and they were successful in this endeavor. Upon return to BYU, both Frank & I mentored these students in curation procedures for specimens and tissues. Dan has graduated, but Doug has continued with curatorial work and is now in the molecular lab. He will be part of a phylogenetic study of North American “heterodontine” snakes (this group includes ringnecks), and this should lead to a high quality manuscript on which Doug will be a co-author.

3 – Other students – My second PhD student, Fernenda Werneck, indirectly benefitted from this project by working as a mentor to visiting students from Argentina, and just this fall term, by working closely with incoming PhD student Cesar Aguilar. Cesar is from Peru and has not had any prior molecular lab bench training, so Fernanda is teaching him all of the basics, with a focus now on extraction and testing DNAs from lizard samples that Cesar brought with him from Peru. To date about 85% of these samples contain high quality DNA, and Cesar has just begun learning PCR protocols for amplifying target gene regions.

General Student Outcomes

After initial training, students are able to perform lab tasks independently, but occasionally need further supervision for planning, guidance, and troubleshooting. The computer lab portion of these projects has trained students on software that is widely used in alignment of DNA sequences, and most of the students are now competent in proofreading DNA sequences, preparing and formatting data, and at a very basic level, some of the analyses for reconstructing phylogenetic trees. Students mentored in curation and data-basing methods are now fully versed in these methods, but also in the larger context of the value of professionally curated collections of voucher specimens, for research and teaching. The participation of several students in these projects will, with sufficient commitment, assure them of co-authorship in presentations and on scientific papers. My lab has a strong record in this area: 10 of 35 papers published or submitted in the last 5 yrs (2006-2010) included BYU undergraduate co-authors.

Evaluation of how well the academic objectives of the proposal were met

I described several research projects in the original proposal, and for some of these the data collection is complete or in progress (ringneck snakes), while for one other (parthenogenetic species of Liolaemus), we failed to obtain adequate samples to undertake the genetic studies. However, at any given time my lab has perhaps 10 – 12 projects in play, including those from the previous MEG (2009) that continue beyond the life of any single MEG. Several of the manuscripts currently in review were actually started 2 yrs ago, and the undergraduate students involved are gone, but they are now seeing their names on author lines. As in other years, more BYU undergraduate students participated in other projects that were not described in the original proposal, and details of their academic deliverables are summarized below. My two graduate students, Arley Camargo and Fernanda Werneck, submitted major manuscripts that are now published or in press, which form core parts of their dissertation projects, and both secured substantial funding from on and off- campus sources. Collectively, I think my lab met or exceeded the academic objectives of the MEG proposal, but this needs to be understood in the context of the kinds of studies we carry out, and the turn-around time for most of our projects. The time needed to complete these studies, analyze and write up the results, and then carry manuscripts through submission, revision, and eventual publication, is normally about two years. This means that almost all BYU undergraduates leave my lab and are well into the next phase of their education before they see their own papers published.

Evaluation of the mentoring environment

Undergraduate students joining my group normally have three options for mentored learning experiences, one of these being museum curatorial work in the Herpetological Research Collections at the Bean Museum (cataloging new specimens, geo-referencing new localities, responding to requests for data, etc.). A second option is to participate in the PIRE – Patagonia Project exchange experience for a semester abroad; most of the cost for such an experience is supported by the NSF award for this project, but not for lab work at BYU upon return of the students. This is the final year for this project. The third and preferred option for most students is a research experience in the molecular lab, and for all students making a commitment for serious work under this option, I contribute to their mentored learning in all aspects of molecular lab training (DNA extraction, PCR amplification of targeted gene products, cleaning and sequencing of these products, editing of sequences), and some of the statistical analyses and participation in writing the paper. During the fall and winter terms we hold weekly lab group meetings in which students participate in reading and discussion of papers, or critique of manuscripts or presentations prepared by members of my lab group. Students working on spin-off projects from the PIRE-Patagonia program are exposed to the more general issues addressed by the larger scope of that project, and the same is true for spin-off projects from the Squamate Tree-of-Life Project (now completed). Beyond the normal topics of individual student projects and lab issues, we also discuss things like career options, how to prepare a resume, and similar topics that students frequently suggest to the group.

The molecular lab-based research projects require that the students become proficient in laboratory techniques, literature reviews, data analysis, PowerPoint presentations, and at least some aspects of manuscript writing. In so doing, students develop the confidence necessary to pursue additional training (e.g. post-graduate work), and they gain the capacity to think critically, problem solve, and develop more effective oral and written communication skills. Critical thinking and problem solving skills develop as the students troubleshoot PCR/sequencing protocols, participate in study design (such as species and population sampling efforts), and generate and analyze their data. Student communication skills are sharpened as they read the literature, contribute in weekly group meetings, apply for scholarships (ORCA), and help write up results of their work for publication.

My 2010 lab group included post-doc Dr. Frank Fontanella, 4th yr PhD student Arley Camargo (Uruguay), 3rd yr PhD student Fernanda Werneck (Fulbright fellow from Brazil), Argentinian PhD student Florencia Breitman (here for 7 months) – all well into analyses/write up of dissertation chapters (3 are required), and as of August 2010, beginning PhD student Cesar Aguilar. We carry over from last year to this year undergraduates Amy Dawson in the molecular lab and Doug Brown in the museum, along with 3 new students: Rachel Arendt and Nathan Hannah (molecular), Eric Baker (museum); Doug Brown has just moved into the molecular lab, and Eric will transition in as well in January. Two more students, Tabitha Brown and Lesley Hardman, are working with me at the moment to develop research projects for a semester abroad experience in Puerto Madryn, Argentina (winter 2011; with Sites collaborators Drs. M. Morando & L.J. Avila. The intellectual environment of my research group was enhanced by several short- term visitors, including: (1) Drs. Luciano J. Avila & Mariana Morando (CENPAT), 3-month fellowships to complete some molecular work on phylogeography of Argentinian lizards; (2) Dr. Pedro Victoriano (Chile; U. Concepcion) for 2 weeks, to complete molecular work on phylogeography of Chilean lizards; and (3) graduate students from both of these groups: PhD students Natalia Feltrin, Cintia Medina, & Melisa Olave here for 3 months from Argentina (supervised by Drs. Morando & Avila), and MS student Tania Coronado for 1 week of molecular work (supervised by Dr. Victoriano). The BYU undergraduate students were exposed to the work of these scholars and their students, both in the lab every day, in weekly lab meetings, and in some social events, and learned things that they would otherwise never have been exposed to. My evaluation of the mentoring environment is that in 2010 it was VERY good, due in large part to the large number of BYU students participating in multiple activities, and the number of visitors here to pursue their own research projects. Some additional details will be given below.

Students participating, their activities, and academic deliverables completed or in progress

As an example of the point I made above in section 2, the research projects completed by 2009 MEG (Tom Beckstead, Tyler Williams) and PIRE students (Nicole Koontz), are now either accepted for publication (Mendelson, Mulcahy, Williams, & Sites. “A phylogeny and evolutionary natural history of Mesoamerican toads [Anura: Bufonidae]” ; Cladistics (accepted with revision); Nunez, Koontz, Rabanal, Fontanella, & Sites. “Amphibian phylogeography in the Antipodes: refugia and postglacial colonization explain mitochondrial haplotype distributions in the Patagonian frog Eupsophus calcaratus [Cycloramphidae]”. Molecular Phylogenetics & Evolution (accepted with revision), or they have been revised and re-submitted (Mulcahy, Beckstead, Sites. “Molecular systematics of the Leptodeirini (Colubroidea, Dipsadidae) revisited: nuclear loci with concatenation and coalescent methods.” resubmitted to Copeia after revision). Of the 2010 MEG supported personnel, PhD student A. Camargo has submitted his second dissertation chapter to his committee (“Empirical performance of multi-locus species tree methods under varying locus, base-pair, and individual sampling designs.”), and we have received pre-submission “friendly reviews” from the labs of two colleagues. I anticipate that this paper will be submitted (to Systematic Biology) within the next 2-3 weeks, and Arley is completing the analyses for his final chapter. Fernanda Werneck is collecting molecular data for her second dissertation chapter, and should be analyzing/writing this manuscript early next year. She will be mentoring at least one undergraduate student for assistance with her final chapter, and continuing to mentor C. Aguilar. Post-doc Frank Fontanella has initiated two molecular projects that will involve 3 undergraduates (E. Baker, D. Brown, & N. Hannah mentioned above), and some of these have been submitted as ORCA proposals. I have interviewed one new student, and depending on our success with this proposal, ORCA proposals, and two external proposals now under review, will be looking for additional students in 2011.

Description of the results/findings of the projects

General results of some projects are described above, but we do not yet have manuscripts from the projects initiated in 2010.

How the budget was spent

By far the bulk of the budget was spent either on consumable chemicals/supplies for the molecular work, and student salaries. This included the $3,500 stipend for A. Camargo, which was matched by the department, and the $2,500 for F.Fontanella’s field work in the east (partially matched by the Bean Museum). Another $4,000 or so went to student salaries (some of which was also matched by the department and the museum), and all of the rest covered consumable supplies for the molecular work. Some of the current students are new, and as I require of all new students, they are spending their first semester participating as volunteers. This will change in January, with most of these students either shifting to salaried positions or working for 494R credit, and the two PIRE students will be supported by that award for the semester spent in Argentina. With the number of projects and those brought by all of the visitors (Drs. Avila, Morando, Victoriano, and their students), the Brazilian couple, and side projects conducted by post-doc Dr. Dan Mulcahy, I spent close to $28,000 for lab supplies and consumables in 2010 (from multiple sources).

Summary of papers, manuscripts, and presentations supported by MEG funds for 2010

Bold font = BYU undergraduate, bold* = BYU graduate student, bold italics = Argentinian or Chilean student

Papers published, in press, or accepted

• Sinclair, E.A., J.B. Pramuk, R.L. Bezy, K.A. Crandall, and J.W. Sites, Jr. 2010. DNA evidence for non-hybrid origins of parthenogenesis in natural populations of vertebrates. Evolution 64:1346-1357.
• Noonan, B.P., and J.W. Sites, Jr. 2010. Tracing the origins of Iguanid lizards and Boine snakes of the Pacific. American Naturalist 175:61-72.
• Avila, L.J., C.H.F. Perez, M. Morando, and J.W. Sites, Jr. 2010. A new species of Liolaemus (Reptilia: Squamata) from southwestern Rio Negro province, northern Patagonia, Argentina. Zootaxa 2434:47-59.
• Sinervo, B., F.M. de la Cruz, D.B. Miles, B. Heulin, E. Bastiaans, M. Villagran-Santa Cruz, R. Lara-Resendiz, N. Martínez-Méndez, M.L. Calderon-Espinosa, R.N. Mesa-Lázaro, H. Gadsden, L.J. Avila, M. Morando, I.J. de la Riva, P. Victoriano- Sepulveda, C.F.D. Rocha, N. Ibargüengoytía, C.A. Puntriano, M. Masson, V. Lepetz, T.A. Oksanen, D.G. Chapple, A.M. Bauer, W.R. Branch, J. Clobert, and J.W. Sites, Jr. 2010. Erosion of lizard diversity by climate change and altered thermal niches. Science 328:894-899.
• Camargo, A.*, B. Sinervo, and J.W. Sites, Jr. 2010. Lizards as model organisms for linking phylogeographic and speciation studies. Molecular Ecology 19:3250- 3270.
• Wiens, J.J., C.A. Kuczynski, T. Townsend, T.W. Reeder, D. Mulcahy, and J.W. Sites, Jr. 2010. Combining phylogenomics and fossils in higher-level squamate phylogeny: molecular data change the placement of fossil taxa. Systematic Biology 59:xxx- xxx (DOI:10.1093/sysbio/syq048).
• Avila, L.J., M. Morando, D.R. Perez, and J.W. Sites, Jr. 2010. A new species of the Liolaemus elongatus group (Squamata: Iguania: Liolaemini) from Cordillera del Veinto, northwestern Patagonia, Neuquén, Argentina. Zootaxa (in press).
• Werneck, F.P.*, G.C. Costa, G.R. Colli, D.E. Prado, and J.W. Sites, Jr. 2011. Revisiting the Seasonally Dry Tropical Forest historical distribution: new insights based on paleo-distribution modeling and palynological evidence. Global Ecology and Biogeography 19:xxx-xxx.
• Nuñez, J.J., N. Koontz, F. Rabanal, F. Fontanella, and J.W. Sites, Jr. Amphibian phylogeography in the Antipodes: refugia and postglacial colonization explain mitochondrial haplotype distributions in the Patagonian frog Eupsophus calcaratus (Cycloramphidae). (accepted, Molecular Phylogenetics & Evolution).
• Mendelson, J.R., III, D.G. Mulcahy, T.S. Williams, and J.W. Sites, Jr. A phylogeny and evolutionary natural history of Mesoamerican toads (Anura: Bufonidae). (accepted with revision, Cladistics).

Manuscripts in review

• Hamilton, B.T., R. Hart, and J.W. Sites, Jr. Feeding ecology of the milksnake (Lampropeltis triangulum, Colubridae) in the western United States (re-submitted after revision, Journal of Herpetology).
• Fontanella, F., N. Feltrin, L.J. Avila, J.W. Sites, Jr., and M. Morando. Early stages of divergence: phylogeography, climate modeling, and niche differentiation in the South American lizard Liolaemus petrophilus (Squamata: Tropiduridae). (re- submitted after revision, Journal of Biogeography).
• Sinervo, B.R., D.B. Miles, F.R. Méndez-De la Cruz, B. Heulin, Ignacio De La Riva, Carlos Frederico Duarte Rocha, Norberto Martînez, Rafael Lara-Resendiz, Nora R. Ibargüengoytía. and Jack W. Sites, Jr. Response to Comment by Henle, K., O. Schweiger, A. Harpke, and J. Settele. (in review, Science).
• Mulcahy, D.G., T.H. Beckstead, and J.W. Sites, Jr. Molecular systematics of the Leptodeirini (Colubroidea, Dipsadidae) revisited: nuclear loci with concatenation and coalescent methods. (re-submitted after revision, Copeia).
• Mulcahy, D.G., B.P. Noonan, T. Moss, T.M. Townsend, T.W. Reeder, J.W. Sites, Jr., and J. W. Wiens. Comparing the performance of dating methods using phylogenomic data in squamate reptiles. (in review, Systematic Biology).

Manuscripts in revision or in preparation (analyses complete, ms substantially complete)

• Breitman, M.F., L.J. Avila, J.W. Sites, Jr., and M. Morando. Lizards from the end of the world: phylogenetic relationships of the Liolaemus lineomaculatus section (Squamata: Iguania: Liolaemini). (in revision, Molecular Phylogenetics & Evolution).
• Morando, M., L.J. Avila, M. Hawkins, and J.W. Sites, Jr. Molecular phylogeny of Phymaturus (Squamata: Liolaemini). (in prep. for Molecular Phylogenetics & Evolution).
• Camargo, A., M. Morando, L.J. Avila, and J.W. Sites, Jr. (in prep.). Empirical performance of multi-locus species tree methods under varying locus, base-pair, and individual sampling designs. (intended for Systematic Biology).
• Heideman, N.J.L., J.W. Sites, Jr., D.G. Mulcahy, M.G.J. Hendricks, and S.R. Daniels. Cryptic diversity uncovered in the threatened fossorial skinks Scelotes gronovii and S. kasneri: conservation implications. (in prep. for Molecular Ecology).
• Pramuk, J.B., R.L. Bezy, B.P. Noonan, E.A. Sinclair, K. de Queiroz, and J.W. Sites, Jr. Phylogenetic relationships within the Xantusiidae (Squamata): using trees to address evolutionary questions at multiple levels. (in prep. for Molecular Phylogenetics & Evolution).

Oral/poster presentations at professional meetings

• Mendelson, J.R., D.G. Mulcahy, T. Williams, and J.W. Sites, Jr. Phylogenetic relationships among Central American bufonids. (July ’10; oral; Herpetolgists’ League meeting, Providence, RI).
• Mulcahy, D.G., B. Noonan, T. Moss, T. Townsend, T. Reeder, S. Smith, C. Kuczynski, J. Wiens, and J.W. Sites, Jr. Basal relationships among squamate reptiles based on 25 protein encoding nuclear loci. (July ’10; oral; Herpetolgists’ League meeting, Providence, RI).