MOLECULAR SYSTEMATICS AND BIOGEOGRAPHY OF MESOAMERICAN FLYING SQUIRRELS HUMBOLDT STATE UNIVERSITY By Nicholas John Kerhoulas A Thesis Presented to The Faculty of the Department of Biological Sciences In Partial Fulfillment Of the Requirements for the Degree Masters of Arts In Biology May 2008 MOLECULAR SYSTEMATICS AND BIOGEOGRAPHY OF MESOAMERICAN FLYING SQUIRRELS By Nicholas John Kerhoulas We certify that we have read this study and that it conforms to acceptable standards of scholarly presentation and is fully acceptable, in scope and quality, as a thesis for the degree of Master of Arts. Brian S. Arbogast, Major Professor Date Joseph M. Szewczak, Committee Member Date W. Bryan Jennings, Committee Member Date Jeffrey W. White, Committee Member Date Michael R. Mesler, Graduate Coordinator Date Chris A. Hopper, Interim Dean Date Research, Graduate Studies ABSTRACT MOLECULAR SYSTEMATICS AND BIOGEOGRAPHY OF MESOAMERICAN FLYING SQUIRRELS Nicholas John Kerhoulas Populations of flying squirrels occupying oak and oak-pine forests of the Mesoamerican highlands represent the least studied and most poorly known members of the genus Glaucomys. Traditionally, these Mesoamerican populations have been considered to be southern disjuncts of the southern flying squirrel, G. volans, a species that is also widespread across the deciduous and mixed-deciduous forests of eastern North America. The small number (less than 65) of Mesoamerican flying squirrel museum specimens in existence has made discerning the systematic and biogeographic relationships of these populations a challenge. In an effort to clarify the systematic position and biogeographic history of the Mesoamerican flying squirrels, ancient DNA techniques were used to extract, amplify, and sequence a 571-bp segment of the mtDNA cytochrome b gene from 34 of museum specimens. These represented five of six recognized subspecies of Mesoamerican flying squirrels. Mesoamerican flying squirrel data was combined with homologous sequences from representative populations of Glaucomys from across the rest of North America. This combine data set was phylogenetically analyzed using likelihood and Bayesians approaches. Results of phylogenetic analyses indicate that iii Mesoamerican flying squirrels form a sister monophyletic group to populations of G. volans from eastern North America. Within Mesoamerica, there are two distinct mtDNA lineages of flying squirrels: one comprising of populations from the Sierra Madre Oriental, Oaxacan highlands and Sierra Madre de Chiapas, and one comprising of populations from the Sierra Madre del Sur. These phylogenetic relationships suggest that divergence of Mesoamerican populations from those in eastern North America preceded a series of subsequent divergence events within Mesoamerica. iv ACKNOWLEDGEMENTS I would like to thank the many people who provided expertise and support, making this project possible. My major advisor Dr. Brian Arbogast helped me immensely throughout all aspects of this project from acquisition of tissue samples to analyzing and interpreting data. Anthony Baker provided crucial help optimizing DNA extractions and PCR methods, as well as providing and maintaining the low template areas necessary for extraction and amplification of the DNA used in this project. I would also like to thank my committee members Dr. Bryan Jennings, Dr. Joe Szewczak, and Dr. Jeffrey White, for their valuable input and support. I am grateful to the following museums for providing Mesoamerican flying squirrel tissue samples: the Field Museum of Natural History, Kansas State University, Louisiana State University, and the United States National museum. I would also like to thank the following organizations for funding: Humboldt State University and the American Society of Mammalogists. v TABLE OF CONTENTS ABSTRACT.................................................................................................................. iii ACKNOWLEDGEMENTS.............................................................................................v TABLE OF CONTENTS ...............................................................................................vi LIST OF TABLES ........................................................................................................vii LIST OF FIGURES......................................................................................................viii INTRODUCTION...........................................................................................................1 METHODS ...................................................................................................................11 Samples.....................................................................................................................11 Extractions ................................................................................................................12 PCR and Sequencing .................................................................................................13 Data Analysis ............................................................................................................14 RESULTS .....................................................................................................................21 DISCUSSION ...............................................................................................................26 Phylogeographic relationships within Glaucomys ......................................................26 Genetic support of Mesoamerican subspecies ............................................................28 Biogeographic history of G. volans in Mesoamerica ..................................................30 Conclusions...............................................................................................................34 LITERATURE CITED..................................................................................................37 vi LIST OF TABLES Table Page 1 Locations and accession numbers of Glaucomys samples used in this study. Museum abbreviations are as follows: Louisiana State University (LSU), Field Museum of Natural History (FMNH), United States National Museum (USNM), Kansas University (KU), Burke Museum of Natural History and Culture (BM)......................................................................................17 2 Primers used to amplify four overlapping segments of the cytochrome-b gene for members of the genus Glaucomys. The primer L14724 is from Irwin et al. (1991) all other primers were designed for this study. ..........................20 vii LIST OF FIGURES Table Page 1 Geographic distribution of Glaucomys volans in North America and Mesoamerica. Adapted from The Smithsonian Book of North American Mammals.................................................................................................................7 2 General distribution of Mesoamerican oak and pine-oak forests shown in shades of green. Labels refer to the seven mountain and highland regions referred to in this study. ...........................................................................................8 3 Aerial view of Mesoamerica, with shaded areas representing the six subspecies of Mesoamerican G. volans proposed by Goodwin (1961). Subspecies represented by the shaded areas are: G.v. chontali (yellow), G.v. goldmani (orange), G.v. underwoodi (aqua), G.v herreranus (brown), G.v. madrensis (blue), and G.v oaxacensis (red).......................................................9 4 Aerial view of Mesoamerica, with shaded areas representing the four subspecies of Mesoamerican G. volans proposed by Diersing (1980). Subspecies represented by the shaded areas are: G.v. goldmani (orange), G.v. madrensis (blue), G.v oaxacensis (green), G.v. guerreroensis (red). ...............10 5 Visual representation of the overlapping segments amplified by the 8 primers used to amplify a 571-bp segment in Glaucomys. Distance between vertical lines is 100-bp, created in Amplify 3X. .......................................19 6 Phylogram constructed using Bayesian analysis, rooted with the three closest relatives to Glaucomys. Branch lengths represented by numbers above branches. The first number below the line represents the Bayesian posterior probability support for that branch, the second represents the maximum likelihood bootstrap support for that branch. Topologies identical to outgroup-rooted ML tree with the exception of the Pacific Coastal clade of G. sabrinus, which was not supported in the ML analysis. Mesoamerican abbreviations: SMOr = Sierra Madre Oriental, ChiHi = Chiapas highlands, SMdC = Sierra Madre de Chiapas, OaxHi = Oaxaca highlands, and SMdS = Sierra Madre del Sur.........................................................24 viii 7 Midpoint rooted maximum likelihood tree. Numbers above and below represent branch lengths and maximum likelihood bootstrap support for that branch. Mesoamerican abbreviations: SMOr = Sierra Madre Oriental, ChiHi = Chiapas highlands, SMdC = Sierra Madre de Chiapas, OaxHi = Oaxaca highlands, and SMdS = Sierra Madre del Sur. ...........................................25 8 Ultrametric tree, time scale estimated from four calibration points based on Kimura two-parameter corrected distances for third position nucleotide data and rates of sequence divergence of 10 – 15 % / 106 years. Mesoamerican abbreviations: SMOr = Sierra Madre Oriental, ChiHi = Chiapas highlands, SMdC = Sierra Madre de Chiapas, OaxHi = Oaxaca highlands, and SMdS = Sierra Madre del Sur.........................................................36