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 . 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 , there are two distinct mtDNA lineages of flying squirrels: one comprising of populations from the Sierra

Madre Oriental, Oaxacan highlands and Sierra Madre de , 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 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

ix INTRODUCTION

Over the last decade, molecular phylogenetic studies have greatly clarified the systematics and biogeography of the flying squirrels (Genus Glaucomys;

Arbogast 1999, Bidlack and Cook 2001, 2002, Demboski et al. 1998, Peterson and

Stewart 2006). In particular, these studies indicate: (1) that there are at least three distinct evolutionary lineages of Glaucomys in North America (two within the northern flying squirrel, G. sabrinus, and one corresponding to populations of the southern flying squirrel, G. volans, from the eastern United Sates and southeastern Canada); and (2) that the evolutionary history of Glaucomys appears to have been shaped importantly by

Quaternary changes in the geographic distributions of the coniferous and deciduous forest biomes of North America. However, there remains a critical gap in our knowledge of the evolution and biogeography of New World flying squirrels because none of the previous molecular phylogenetic studies have included populations from the highlands of

Mesoamerica ( to ). Only six populations and approximately 65 specimens of Mesoamerican flying squirrels are known, all from high-elevation oak and pine-oak forests of Mexico, , and Honduras (Figure 1; Dolan and Carter 1977).

Analysis of morphological traits suggest that these populations represent four to six disjunct subspecies of the southern flying squirrel, G. volans (Braun 1988, Diersing 1980,

Goodwin 1961). However, the evolutionary relationships among Mesoamerican flying squirrels, as well as their relationships to populations of Glaucomys from North America, remain enigmatic due to the small number of specimens, sparse distributional data, and a

1 2 lack of genetic data. Furthermore, there are no published data on genetic diversity, population dynamics, or conservation status of Mesoamerican flying squirrels. This lack of information is especially troubling given that much of the high-elevation pine-oak forest these flying squirrels are thought to inhabit has suffered extensive damage over the last century. The major impact has been from human land use, in which large areas of these highlands have been deforested for commercial logging, cultivation, and grazing.

As a result, the World Wildlife Fund considers this one of the most threatened biomes in the world (WWF Global 200).

The oak and pine-oak forests of Mesoamerica occur as a series of disjunct, high elevation populations ranging from Mexico to Honduras. In Mexico, this includes the

Sierra Madre Occidental, Sierra Madre Oriental, Trans-Mexican Volcanic belt, Sierra

Madre del Sur, Oaxaca highlands, and Chiapas highlands, as well as the Sierra Madre de

Chiapas, extending from Chiapas Mexico to Honduras (Figure 2). These disjunct regions of high elevation oak and pine-oak habitats are considered to be remnants of more contiguous forests that also extended throughout lower elevation areas during cooler periods of the Pleistocene (Dolan and Carter 1977, Martian and Harrell 1957). Studies of several groups of small mammals (e.g., Reithrodontomys, Peromyscus, Habromys,

Cratogeomys, Pappogeomys, and Neotoma) from these highland areas of Mesoamerica have found high levels of genetic diversity and the presence of several cryptic species

(Carleton et al. 2002, Demastes et al. 2002, Edwards and Bradley 2002, Harris et al.

2000, Sullivan et al. 2000). 3 From a biogeographic perspective, the , a lowland separating the Sierra Madre de Chiapas and the Chiapas highlands from Mexican mountain ranges and highland areas, appears to have played an important role in the evolutionary diversification of several groups of small mammals (Carleton et al. 2002,

Sullivan et al. 2000). For example, Sullivan et al. (2000) found that populations of

Reithrodontomys sumichrasti, Peromyscus aztecus, and P. hylocetes divided by the

Isthmus of Tehuantepec formed distinct mtDNA clades. Populations of Reithrodontomys microdon were also found to form distinct clades on both sides of the isthmus (Arellano et al. 2005).

Due to the discontinuous nature of the high-elevation oak and pine-oak habitats in

Mesoamerica, as well as the presence of flying squirrels both north and south of the

Isthmus de Tehuantepec, the number and geographical boundaries of distinct evolutionary lineages of Mesoamerican flying squirrels is unclear. Using morphological differences, Goodwin (1961) proposed that Mesoamerican populations of G. volans could be divided into six subspecies: G.v. chontali from the southeastern portion of the Sierra

Madre del Sur, G.v. goldmani from the northern Sierra Madre de Chiapas, G.v. underwoodi from the southern Sierra Madre de Chiapas, G.v. herreranus from the southern Sierra Madre Oriental, G.v. madrensis from the middle Sierra Madre

Occidental, and G.v. oaxacensis from the eastern and northwestern Sierra Madre del Sur

(Figure 3). Subsequent investigation lead Diersing (1980) to propose: (1) that G.v. herreranus and G.v. underwoodi be synonymized with G.v. goldmani; (2) that eastern populations of G.v. oaxacensis be combined with G.v. chontali under the G.v. oaxacensis 4 subspecies epithet; and (3) that the western-most populations of G.v. oaxacensis be recognized as a new subspecies, G.v. guerreroensis (Figure 4).

Biogeographic implications of previous studies suggest that populations of both species of Glaucomys were forced into southern refugia in response to glacial advances during the Pleistocene (Arbogast 1999, Arbogast et al. 2005, Dolan and Carter 1977).

Using genetic data to examine the systematics of flying squirrels north of Mexico,

Arbogast (1999) found three distinct evolutionary lineages. Glaucomys sabrinus comprises two lineages (a widespread Continental lineage and a more geographically restricted Pacific Coast lineage), whereas G. volans from eastern North America represented a third lineage. The Continental lineage of G. sabrinus likely sought refuge in the southern U. S. during glacial maxima; in concurrence with this hypothesis, the most basal members of the Continental lineage of G. sabrinus are those presently restricted to high-elevation mountaintops in the southern Appalachian mountains, at the extreme southeastern edge of the species' range (Arbogast 1999). Glaucomys volans appears to have followed a similar trend, migrating to one or more southern refugia in the southern U.S. and/or Mesoamerica. Arbogast (1999) noted surprisingly low levels of within-species mtDNA variation in populations of G. volans from across the eastern U.S., a pattern that is consistent with a recent population expansion following a severe historical population bottleneck. However, because of a lack of genetic data on flying squirrels from Mesoamerica, it has not been possible to evaluate an equally plausible hypothesis: that the lack of genetic variation in populations of G. volans in eastern North

America is due to a population expansion following a recent founder event from 5 Mesoamerica. Thus, determining the evolutionary position of Mesoamerican flying squirrels within Glaucomys could have important implications for understanding the biogeographic history of this genus.

The use of molecular markers provides an opportunity to examine geographic patterns of genetic variation in Mesoamerican flying squirrels and help clarify the systematics and biogeography of the group. The cytochrome-b (cyt-b) gene of mitochondrial DNA (mtDNA) is widely used in molecular phylogenetic studies of mammals. Mitochondrial markers are well suited for examining intrageneric and intraspecific phylogenetic relationships because they undergo rapid geographic sorting.

This is due to several factors, foremost the relatively high mutation rate and small effective population size of the mtDNA genome relative to the nuclear genome (Avise

2000). In particular, the widespread use of the cyt-b gene has lead to a wealth of data on levels of genetic diversity in mammals, as well as estimates rates of molecular evolution for this region of mtDNA (Bradley and Baker 2001, Irwin et al. 1991). When used in conjunction with branch lengths on a phylogenetic tree and/or corrected estimates of genetic divergence between two evolutionary lineages, these rates of mutation can be used to estimate the time of divergence between two lineages (Arbogast et al. 2002,

Avise 2000). The extensive use of the cyt-b region of mtDNA in previous phylogenetic analyses of flying squirrels (Arbogast 1999, 2005, Bidlack and Cook 2001, Demboski et al. 1998, Petersen and Stewart 2006, Yu et al. 2006) also makes it an attractive marker for the current study because it allows for new data to be combined with existing data to 6 obtain a broad evolutionary and biogeographic framework for analyzing the systematics and biogeography of Mesoamerican flying squirrels.

Using mtDNA cyt-b sequences generated from museum specimens, this study aimed to address five major goals: (1) to determine the phylogenetic relationship of

Mesoamerican flying squirrels with other members of Glaucomys found in North

America; (2) to determine the number of distinct genetic lineages of flying squirrels in

Mesoamerica; (3) to evaluate whether genetic data support subspecies boundaries based on morphological analyses of Mesoamerican flying squirrels; (4) to examine the role of

Mesoamerican flying squirrels in the biogeographic pattern of New World flying squirrels in general; and (5) to compare the biogeographic patterns observed in

Mesoamerican flying squirrels with other Mesoamerican mammals. 7

Figure 1. Geographic distribution of Glaucomys volans in North America and Mesoamerica. Adapted from The Smithsonian Book of North American Mammals. 8

Figure 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. 9

Figure 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). 10

Figure 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). METHODS

Samples

Phylogenetic analyses were based on a 571-basepair (bp) segment of the cyt-b gene. A total of 74 individuals from the genus Glaucomys were included in the phylogenetic analyses (Table 1). Samples came from museum specimens, previously extracted template, and published sequences. Mesoamerican flying squirrel tissue was obtained from museum specimens. Two G. volans individuals from Texas were extracted from frozen tissue. Previously extracted template was used for 29 samples of both G. volans and G. sabrinus from north of Mexico (see Arbogast 1999, 2005 for methods).

Twenty-one sequences of G. sabrinus were obtained from GenBank (Bidlack and Cook

2001, Cook et al. 2001, Demboski et al. 1998).

The risk of contamination is elevated when working with low-yield DNA from museum specimens. To minimize this risk all extractions and polymerase chain reactions

(PCR) were conducted in a facility dedicated to low template procedures. PCR thermocycling, gel visualization, and PCR product storage was conducted in a separate facility. Before performing an extraction or PCR reaction, I showered and donned clean clothing. Negative controls were included throughout all aspects of the extraction and

PCR process.

11 12

Extractions

Mesoamerican flying squirrel tissue extractions were performed in a Labconco

Purifier/filtered hood with ultraviolet (UV) irradiation located in the aforementioned lab.

The hood and all tools were wiped down with RNAce Away® and irradiated for 30 minutes with UV light for both before and after each use. A small portion (1-2 mm3) of each museum specimen skin sample was used for DNA extraction. Sterilized scissors and forceps were used to clip the subsample and place it into a 1.5 ml microcentrifuge tube. Between each sample, scissors and forceps were flamed, the laboratory bench was wiped down, and gloves were changed. Before beginning the lysing process, a 24-hour ethanol wash was performed on tissue samples. The washing regime consisted of 1 ml of

100% ethanol changed every three hours for 24 hours. These ethanol washes were used to leach out contaminates held over from preparation and preservation as well as to remove surface contaminates. To facilitate the release and subsequent removal of any surface contaminants, samples were vortexed vigorously for 15 seconds before and after each ethanol exchange. Upon completion of the final wash cycle, the ethanol was removed and samples were air-dried in the hood. Once dry, the samples were extracted following the standard DNeasy tissue extraction protocol (Qiagen Inc., Valencia,

California) with the following exceptions: Buffer AE was diluted 1:10 and preheated to

70° C, the first elution was 50 !l, and the second elution 100 !l with a 10-minute period between adding the elution buffer and centrifuging the sample. A negative control was included through all wash cycles and extractions. Washes and extractions were performed on eight or fewer samples at a time. Frozen tissue samples from fresh tissues 13 (i.e., Texas samples) were extracted using the standard DNeasy Kit tissue extraction protocol in a separate location.

PCR and Sequencing

PCR was used to amplify a 571-base pair (bp) segment of the mitochondrial cyt-b gene. In order to amplify the entire 571-bp segment from the museum skin samples of

Mesoamerican flying squirrels, four small (<250-bp), overlapping internal segments were amplified (Figure 5). To achieve this I used one universal primer (Irwin et al. 1991) and seven Glaucomys-specific primers that I designed (Table 2). The conservative nature of cyt-b gene allowed the design of perfect or near perfect primers. Primer design was aided by the following programs: Primer3 (Rozen and Skaletsku 2000), Amplify 3X (Engels

1993), and the Sequence Manipulation Suite (Stothard 2000). The entire 571-bp segment was amplified in one reaction for samples from high-quality DNA extractions from previous studies (e.g. Arbogast 1999, Arbogast et al. 2005) and frozen tissue samples.

PCR reactions consisted of 12.5 !l AmpliTaq Gold PCR master mix, 7.5-9.5 !l ddH2O, 2 !l each primer, 0.5-1 !l template for a total of 25 !l. Thermal-cycling parameters were: 94° for 5 min, 40° or 45° for 1 min 30 sec (Table 3), 72° for 1 min, 50 cycles, 72° for 5 min. A two percent agarose gel was used to visualize PCR products.

All PCR cleanup and sequencing was outsourced to the High-Throughput Genomics

Unit, Seattle, WA, with the exception of four samples which were purified using the

QIAquick PCR purification kit (Qiagen Inc., Valencia, California) and sequenced at the

CSUPERB Microchemical Core Facility, San Diego, CA. 14 Data Analysis

Sequence data quality was visually inspected using the program FinchTV

(Geospiza Inc.). Special care was taken to check for the presence of multiple chromatogram peaks that could indicate contamination. Consolidation of overlapping sequences and alignment was performed by hand using the program Se-Al (version 2.0,

Rambaut 1995). Only unique taxa were used in phylogenetic analyses. Duplicate haplotypes were detected using the program Collapse (version 1.2, http://darwin.uvigo.es). Molecular phylogenies were inferred using maximum likelihood

(ML) and Bayesian methods. The computer programs PAUP* (version 4.0b Altivec,

Swofford 1998) and MrBayes (version 3.2-cvs, Huelsenbeck and Ronquist 2001,

Huelsenbeck et al. 2001) were used to infer ML and Bayesian phylogenies. The programs Modeltest (version 3.7, Posada and Crandall 1998) and MrModeltest (version

2.2, Nylander 2004) were used to select the best-fit model of nucleotide substitution for

ML and Bayesian models, respectively. The model selected by the Akaike information criterion (AIC) was used in all cases.

Both outgroup and midpoint rooting were used to construct ML phylogenetic trees. For the former, three taxa of Eurasian flying squirrels were used as outgroups

(Eoglaucomys, Hylopetes, and Petinomys). Because these Eurasian species are relatively distantly related to the New World flying squirrels (possibly confounding the rooting of the phylogenetic tree; Arbogast et al., 2001), I also used midpoint rooting to examine relationships within Glaucomys. Starting trees were constructed using random addition of taxa. A heuristic search with tree-bisection-reconstruction (TBR) branch swapping 15 was used to find the best phylogenetic tree under the ML criterion. To evaluate nodal support, 1,000 full-heuristic ML bootstrap replicates were performed (Felsenstein 1985).

I tested whether the data were consistent with a molecular clock using a likelihood ratio test (Huelsenbeck and Rannala 1997).

The Bayesian analysis was rooted with the same three Eurasian flying squirrel species as the ML analyses. Bayesian analysis consisted of two concurrent, independent analyses with different starting trees. Four chains, three heated and one cold, were used to improve Markov chain Monte Carlo sampling of the target distribution. Sampling consisted of 2x106 Markov chain Monte Carlo generations sampled every 100 generations. This sampling regime resulted in an average standard deviation of split frequencies below 0.01, indicating that the two runs had converged. The first 5,000 trees constructed were omitted as burn-in. Nodal support of Bayesian phylogenetic trees was assessed using posterior probabilities.

Pair-wise sequence divergence values were calculated using two methods, the

Kimura two-parameter model (Kimura 1980) and by taking the sum of the shortest branch lengths between two populations (Arbogast et al. 2006). The Kimura two- parameter distances were calculated using the program PAUP* and data from the third nucleotide position only. Branch lengths from the outgroup-rooted ML tree were used to calculate the sum of the shortest branch lengths. Rates of sequence divergence for the third position data (Kimura two-parameter model) were estimated at 10 - 15% / 106 years

(Arbogast 1999). The measure of shortest ML branch length included data from all codon positions. To accommodate the various mutation rates found between codon 16 positions a rate of 15% / 106 years of was assumed for third position data and 0.6% / 106 years for first and second position data (Irwin 1991). Combining these mutation rates relative to their abundance in the data yielded an all position mutation rate of 5.5% / 106 years. 17 Table 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).

Museum / GenBank Species Country State/Provence Locality Haplotype Accession Number G. volans Mexico San Luis Potosi Santa Barbarita LSU-4189 1 G. volans Mexico San Luis Potosi Santa Barbarita LSU-4190 1 G. volans Guatemala Chimaltenango Tecpan FMNH-41763 2 G. volans Mexico Chiapas Ococingo FMNH-64179 3 G. volans Mexico Chiapas Ococingo FMNH-64181 3 G. volans Mexico Chiapas Ococingo FMNH-64185 3 G. volans Mexico Chiapas Comitian KU-61317 3 G. volans Mexico Chiapas San Christobal de las Casas KU-61318 3 G. volans Mexico Chiapas San Christobal de las Casas KU-66574 3 G. volans Mexico Chiapas San Christobal de las Casas KU-66575 3 G. volans Mexico Chiapas San Christobal de las Casas KU-66576 3 G. volans Mexico Chiapas Ococingo FMNH-64183 4 G. volans Mexico Chiapas San Christobal de las Casas KU-66571 5 G. volans Mexico Chiapas Teopisca KU-68833 6 G. volans Mexico Chiapas Teopisca KU-68834 7 G. volans Mexico Guerrero Agua del Obisbo KU-99758 8 G. volans Mexico Guerrero Omilteme USNM-329701 9 G. volans Mexico Guerrero Omilteme USNM-329702 9 G. volans Mexico Guerrero Omilteme USNM-329703 9 G. volans Mexico Guerrero Omilteme USNM-329705 9 G. volans Mexico Guerrero Omilteme USNM-329706 9 G. volans Mexico Guerrero Omilteme USNM-329704 10 G. volans U.S. Florida Archbold Biological Station FL-6* 11 G. volans U.S. Florida Archbold Biological Station FL-102* 12 G. volans U.S. Florida Archbold Biological Station FL-8* 12 G. volans U.S. Florida Archbold Biological Station FL-100* 13 G. volans U.S. Florida Archbold Biological Station FL-104* 14 G. volans U.S. Missouri Cape Girardeau AJS-059* 15 G. volans U.S. Missouri Cape Girardeau AJS-060* 15 G. volans U.S. Missouri Cape Girardeau AJS-061* 15 G. volans U.S. Missouri Cape Girardeau AJS-062* 15 G. volans U.S. Pennsylvania Powdermill Biological Station PA-5* 15 G. volans U.S. Pennsylvania Powdermill Biological Station PA-7* 15 G. volans U.S. Pennsylvania Powdermill Biological Station PA-2* 16 G. volans U.S. Pennsylvania Powdermill Biological Station PA-9* 17 G. volans U.S. Texas Harrison Co. USNM-569025 18 G. volans U.S. Texas Harrison Co. USNM-569026 19 18 Table 1 continued.

Museum / GenBank Species Country State/Provence Locality Haplotype Accession Number G. sabrinus U.S. California Plumas Co. VM-2547** 20 G. sabrinus U.S. California Plumas Co. VM-2562** 21 G. sabrinus U.S. Utah Summit Co. LSU-M3013 22 G. sabrinus U.S. Utah Summit Co. LSU-M3014 22 G. sabrinus U.S. Alaska Helm Bay AF271669*** 23 G. sabrinus U.S. Alaska Revillagigedo Island AF271673*** 23 G. sabrinus U.S. Alaska Anita Bay AF359213*** 23 G. sabrinus U.S. Alaska Revillagigedo Island AF359216*** 23 G. sabrinus U.S. Alaska Helm Bay AF359217*** 23 G. sabrinus U.S. Alaska Helm Bay AF359218*** 23 G. sabrinus Canada Alberta Near Edmonton LSU-M3442 23 G. sabrinus Canada Alberta Near Edmonton LSU-M3444 24 G. sabrinus U.S. Washington Jefferson Co. BM-200068 25 G. sabrinus U.S. California San Bernardino Co. LSU-M5742 26 G. sabrinus U.S. Idaho Latah Co. JRD-025** 27 G. sabrinus U.S. North Carolina Mitchell Co. LSU-M5748 28 G. sabrinus U.S. Michigan Alger Co. LSU-M5760 28 G. sabrinus U.S. Oregon Douglas Co. LSU-M5732 30 G. sabrinus U.S. Oregon Douglas Co. LSU-M5736 31 G. sabrinus U.S. Washington Pierce Co. LSU-M5727 32 G. sabrinus U.S. Washington Pierce Co. LSU-M5709 33 G. sabrinus U.S. West Virginia Webster Co. LSU-M5722 34 G. sabrinus U.S. Alaska Wrangell Island AF359212*** 35 G. sabrinus U.S. Alaska Prince of Wales Island AF271670*** 36 G. sabrinus U.S. Alaska Suemez Island AF271671*** 36 G. sabrinus U.S. Alaska Heceta Island AF271672*** 36 G. sabrinus U.S. Alaska Kosciusko Island AF359232*** 36 G. sabrinus U.S. Alaska Suemez Island AF359233*** 36 G. sabrinus U.S. Alaska Suemez Island AF359234*** 36 G. sabrinus U.S. Alaska Suemez Island AF359235*** 36 G. sabrinus U.S. Alaska Suemez Island AF359236*** 36 G. sabrinus U.S. Alaska Suemez Island AF359237*** 36 G. sabrinus U.S. Alaska Dall Island AF359238*** 36 G. sabrinus U.S. Alaska Juneau AF271668*** 37 G. sabrinus U.S. Alaska Juneau AF359220*** 37 G. sabrinus U.S. Alaska Klondike Gold Rush NP AF359211*** 38 G. sabrinus U.S. Washington Kittitas Co. AF030389*** 39 * Blood samples, no voucher specimen ** Humboldt State University specimens not yet accessioned *** GenBank Accession numbers 19

Figure 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. 20 Table 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.

Name Sequence L14724 5’-CGAAGCTTGATATGAAAAACCATCGTTG-3’ Gvo-L2 5’-TCGTTCAAATCGTCACAGGA-3’ Gvo-L3 5’-AGGCCGAGGACTCTATTATG-3’ Gvo-L4 5’-GAGGACAAATATCATTCTGAG-3’ Gvo-R1 5’-GAGGAGAAGGCGGTTATTGTG-3’ Gvo-R2 5’-ACTCCAATGTTTCAGGTTTC-3’ Gvo-R3 5’-CCTCAGATTCATTCTACAAG-3’ Gvo-R4 5’-GATGGGTTATTGGATCCTGTTT-3’ RESULTS

Thirty-nine unique haplotypes were observed among the 74 individuals examined.

These unique haplotypes were equally distributed among G. volans (n = 19) and G. sabrinus (n = 20). All internal segments of the entire 571-bp segment were obtained for

22 of 34 Mesoamerican tissue samples. Of these 22 samples, ten were unique haplotypes from eight collection localities from four major geographic areas: Sierra Madre

Occidental (n = 1), Oaxacan Highlands (n = 2), Sierra Madre de Chiapas (n = 4) and the

Sierra Madre del Sur (n = 3). Chromatograms of the sequenced data had Phred quality scores greater then 20, minimal background noise, and no double peaks. No unexpected stop codons were found and all overlapping sequences for a given individual were identical.

The best-fit model of nucleotide substitution selected by the AIC criterion for data sets that included outgroups was the GTR+G in both Modeltest and MrModeltest. Base frequencies were: A = 0.25, C = 0.28, G = 0.15 and T = 0.33, with a gamma correction value of 0.2182. A likelihood ratio test found no significant difference between phylogenetic trees with and without a molecular clock enforced (-ln likelihood clock enforced = 1639.5360, clock not enforced = 1663.2296; X2 = 47.3872, df = 37, P =

0.1178).

Both outgroup-rooted Bayesian and midpoint-rooted ML phylogenetic analyses

(Figures 7,8) recovered three major clades within Glaucomys: A Pacific coastal clade of

G. sabrinus, a widespread Continental clade of G. sabrinus, and a clade representing all

21 22 individuals of G. volans. The Pacific Coastal clade comprised two reciprocally monophyletic sister clades: one containing California populations and the other populations from Oregon and Washington. The monophyly of the Pacific Coastal clade of G. sabrinus was not supported in the outgroup-rooted ML analysis.

In all analyses, populations of G. volans formed a well-supported monophyletic clade. Within this clade, populations from eastern North America and Mesoamerica formed two monophyletic sister clades. Phylogenetic relationships were identical for all analyses unless otherwise noted. In the eastern North American clade, the earliest divergence is between haplotypes from Texas and the remaining haplotypes in the clade.

Haplotypes from Missouri and Pennsylvania are nested within the multiple haplotypes found in Florida. Within Mesoamerica, haplotypes from populations from the Sierra

Madre del Sur represent the earliest divergence and are sister to a group of haplotypes from all other Mesoamerican populations. Populations from the Sierra Madre Oriental were sister to populations from the Chiapas highlands. In Bayesian analysis, populations from the Sierra Madre de Chiapas were sister to those in the Oaxaca highland (Figure 6).

This sister relationship was not recovered in the outgroup or midpoint-rooted ML analyses (Figure 7). The midpoint-rooted ML analysis resulted in a three-way polytomy between populations from the Sierra Madre de Chiapas, the Oaxaca highlands, and the clade comprised of Sierra Madre Oriental and Chiapas highlands clade.

Pair-wise sequence divergence values calculated using the Kimura two-parameter model on third position nucleotide data ranged from 2.15 – 7.92% between

Mesoamerican individuals from different mountain/highland locations. Between 23 populations of G. volans from eastern North America and Mesoamerica, the minimum pair-wise sequence divergence was 6.68%. In comparison, minimum corrected sequence divergence between the Pacific Coastal and Continental clades of G. sabrinus was 24.2%, while that between G. volans and the Pacific and Continental clades of G. sabrinus were

16.4 and 17.3% respectively. Estimated divergence times calculated using the Kimura two-parameter model and the sum of the minimum branch lengths produced similar estimates of divergence times. 24

Figure 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. 25

Figure 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. DISCUSSION

Phylogeographic relationships within Glaucomys

The phylogenetic analyses presented here agree with previous morphological studies (Braun 1988, Diersing 1980, Goodwin 1961) in supporting the hypothesis that

Mesoamerican flying squirrels are southern, disjunct populations of G. volans. The phylogenetic analyses of the cyt-b mtDNA data presented in this study also suggest that

Mesoamerican flying squirrels and those from eastern North America represent two, reciprocally monophyletic sister groups. Although this relationship was consistently recovered, it did not receive strong nodal support. Overall, these results are largely, although not entirely, consistent with relationships inferred in the most recent morphological study of these populations by Braun (1988). Braun also concluded that there are two major groups of G. volans; however, neither of these groups was composed exclusively of populations from Mesoamerica or eastern North America. Rather, one group was composed of five eastern North American populations plus one Mesoamerican population, and a second group was composed of four Mesoamerican populations plus one eastern North American population (Braun 1988).

Overall, the various methods of phylogenetic analyses used in this study produced similar relationships among the populations of Glaucomys sampled. The major exception was that the outgroup-rooted ML tree did not recover a monophyletic Pacific Coastal clade of G. sabrinus (all other methods of analyses did resolve this clade as

26 27 monophyletic). This inconsistency is most likely an artifact resulting from two factors

(1) the use of distantly related outgroups (all Asian flying squirrels are relatively divergent from Glaucomys, despite being their closest extant relatives; Arbogast 2007), and (2) the relatively large divergence between populations of G. sabrinus from

California and those from Oregon/Washington. The outgroup-rooted Bayesian analysis did recover a monophyletic Pacific Coastal clade of G. sabrinus, as did the midpoint- rooted tree and the ML analyses with a molecular clock enforced.

Mesoamerican populations of G. volans sampled in this study can be divided into two major lineages: one composed of populations found in the Sierra Madre del Sur, and another containing all remaining Mesoamerican populations. The divergence of these populations was most likely shortly after the divergence of Mesoamerican populations from eastern North American populations (Figure 8). Minimum pair-wise sequence divergence values support the early divergence of these two Mesoamerican lineages. For example, based third position data of samples used in this study, minimum Kimura two- parameter corrected pair-wise sequence divergence values between the two

Mesoamerican lineages (7.9%) were similar to those observed between Mesoamerican and eastern North American populations (7.3%). At a finer scale, geographic locations appear to represent distinct lineages. Interestingly, genetic sorting based on geographic location appears to have happened at approximately the same time (0.14 - 0.32 x 106 years ago) throughout Mesoamerica. The synchronous diversification of Mesoamerican

G. volans populations suggests that this divergence may have been caused by widespread global climate change at this time. 28 Because of the limited specimens available, the results of this study should be interpreted as defining the minimum number of lineages of G. volans in Mesoamerica.

While sampling efforts encompassed the majority of the known populations of

Mesoamerican flying squirrel populations from the Sierra Madre Occidental, Sierra

Madre del Sur in Honduras, and the southern extreme of the Oaxaca highlands were not sampled. The large geographic distance and disjunct nature of non-sampled populations from the Sierra Madre Occidentals make them particularly likely to represent a separate major genetic lineage. Furthermore, populations of pocket gophers from the genera

Cratogeomys and Pappogeomys are represented by seven distinct clades within the

Trans-Mexican Volcanic belt (Demastes et al. 2002). This level of divergence in other

Mesoamerican mammals highlights the possibility of substantial evolutionary diversity within G. volans in the Trans-Mexican Volcanic belt.

Genetic support of Mesoamerican subspecies

Samples used in this study represented four of the six Mesoamerica subspecies proposed by Goodwin (1961) and three of the four proposed by Diersing (1980). Missing subspecies samples are of G.v. madrensis from the Sierra Madre Occidental and G.v. chontali from the southern extreme of the Oaxaca highlands. The G.v. madrensis population is known from two specimens, which were donated in sun-faded condition to the Smithsonian in 1926. Although a tissue sample was obtained from one of the G.v. madrensis specimens, repeated attempts to amplify fragments of the cyt-b gene from this specimen proved unsuccessful. The other missing sample, G.v. chontali, is known and described from a single type specimen and therefore not available for this study. 29 Genetic data do not fully support the subspecies designations proposed by

Goodwin (1961) or Diersing (1980). A conservative subspecies designation focusing only on the earliest and deepest divergence of Mesoamerican G. volans supports one of the subspecies proposed by Diersing (1980) and the synonymization of two others. Using this conservative approach the subspecies G.v. guerreroensis from the Sierra Madre del

Sur is supported, whereas G.v. oaxacensis and G.v. goldmani would be considered synonymous members of the second subspecies which includes all sampled regions except the Sierra Madre del Sur. The minimum Kimura two-parameter corrected pair- wise distance between third position data of subspecies using this approach is 7.9%.

A finer scale, analysis found genetic support for recognizing all geographically isolated populations as distinct subspecies. The minimum Kimura two-parameter corrected pair-wise genetic distance observed between any pair of geographically isolated populations is 2.15%, which was found between two pairs of populations: the Sierra

Madre de Chiapas and the Sierra Madre Oriental as well as the Sierra Madre de Chiapas and the Oaxaca highlands. This value is greater than the proposed intrasubspecific maximum for rodents (Bradley and Baker, 2001), and is therefore consistent with the classification of each disjunct population of Mesoamerican G. volans being assigned distinct subspecific status. For comparison, the minimum Kimura two-parameter corrected genetic distance between the two southern-most disjunct subspecies of G. sabrinus and the contiguous northern populations of G. sabrinus are 2.70 and 2.15%, for

G.s. fuscus from West Virginia and G.s. coloratus from North Carolina respectively. The recognition of all geographically isolated populations of Mesoamerican flying squirrels as 30 unique subspecies of G. volans closely follows the subspecies designations proposed by

Goodwin (1961), the only exception being that G.v. oaxacensis is divided into two or three subspecies. These subspecies would include populations from the Oaxacan highlands (G.v. oaxacensis) and populations from the Sierra Madre del Sur (G.v. guerreroensis). Further division of populations found in the Sierra Madre del Sur into two subspecies may be warranted. The minimum Kimura two-parameter corrected pair- wise sequence divergence between the two populations found in the Sierra Madre del Sur is 2.7%, which is above the intrasubspecific maximum proposed by Bradley and Baker

(2001).

Biogeographic history of G. volans in Mesoamerica

Dispersal of G. volans into Mesoamerica likely occurred via a forested corridor between northeastern Mexico and Texas during a Pleistocene glacial maximum when cooler, wetter climatic conditions prevailed in the region (Martian and Harrell 1957).

Assuming a molecular clock and a rate of sequence divergence for third positions of the cyt-b gene for Glaucomys of 10 - 15% / 106 years (Arbogast 1999), it is possible to estimate approximate dates of population divergence among the lineages sampled in this study. Populations of G. volans from eastern North America appear to have diverged from those in Mesoamerica approximately 0.49 - 0.73 x 106 years ago. This estimate is consistent with the hypothesis that G. volans recently invaded Mesoamerica (Diersing

1980, Martian and Harrell 1957) and places the divergence of Mesoamerican and eastern

North American G. volans populations in the mid-Pleistocene. Although nodal support only weakly supported the reciprocal monophyly of Mesoamerican and eastern North 31 American G. volans population, all methods of phylogenetic analysis recovered this relationship. As such, there is no clear evidence for multiple invasions of G. volans from eastern North America into Mesoamerica, or vice versa. Rather, a single divergence of populations from Mesoamerica and eastern North America is the most parsimonious conclusion based on the mtDNA data at hand. Divergence of G. volans populations in eastern North America from those in Mesoamerica likely occurred during the Aftonian interglacial period (Cox and Moore 2005). Warming and drying conditions during this interglacial period may have severed a forested corridor connecting eastern North

American and Mexican populations of G. volans.

The directionality of movement between eastern North American and

Mesoamerican populations of G. volans remains ambiguous. A southern migration of G. volans during the Pleistocene is likely responsible for the colonization of Mesoamerica.

However, it is unclear whether current populations in eastern North America are remnants of populations that colonized Mesoamerica, a result of a recolonization of eastern North America by Mesoamerican populations, or a combination of both.

The first divergence of lineages of G. volans within Mesoamerica most likely occurred shortly after the divergence from eastern North American populations, possibly in the Aftonian interglacial period (Figure 8). Further diversification of G. volans likely happened synchronously throughout Mesoamerica. I estimate the timing of this divergence to be between 0.14 – 0.32 x 106 years ago, roughly the time the of Yarmouth interglacial period. At this time, warmer and dryer conditions could to have forced oak and pine-oak forests to higher elevations, breaking forest continuity and stimulating the 32 divergence observed in Mesoamerican populations of G. volans. Further evidence that this diversification was linked to a global change in climate is the contemporaneous divergence within the Continental clade of G. sabrinus populations from the southern

Appalachian mountains and those found further north (Figure 8).

It appears that the biogeographic patterns in Mesoamerica were shaped largely by the complex topology of the region combined with climatic changes associated with

Quarternary glacial and interglacial cycles (Luna et al. 1999, Dawson 2005). Several previous studies have used phylogenetic relationships of small mammals to elucidate biogeographic patterns in the mountain and highland regions of Mesoamerica (e.g.

Arellano et al. 2005, Carleton et al. 2002, León-Paniagua et al. 2006, Sullivan 1997,

2000). The widespread and disjunct, high-elevation distribution of Mesoamerican flying squirrels makes them an excellent species to facilitate our biogeographic understanding of the region.

One of the most notable biogeographic patterns seen in several Mesoamerican small mammals is a deep phylogenetic divergence across the Isthmus de Tehuantepec.

This divergence has been documented in the genera Neotoma, Peromyscus, Habromys and Reithrodontomys (2000, Carleton et al. 2002, Edwards and Bradley 2002, Sullivan et al. 1997). In a thorough review, Carleton et al. (2002) suggested that divergence at the

Isthmus should be considered the null model for Mesoamerican mammals restricted to middle and upper montane humid forests. Interestingly, Mesoamerican populations of G. volans do not exhibit the predicted deep phylogenetic divergence at the isthmus. I found the level of divergence between populations of G. volans on either side of the isthmus to 33 be similar to those observed between other geographically isolated populations in

Mesoamerica (Figure 8). The most likely hypothesis to explain this phenomenon is that

G. volans was not present in Mesoamerica, or at least not south of the isthmus, before it became a major biogeographic barrier for many small mammals. Sullivan et al. (2000) proposed that vicariance at the isthmus may have been during the last marine transgression in the late Pliocene / early Pleistocene. If so, this would likely pre-date the presence of G. volans in Mesoamerica based on the divergence dates estimated in this study.

Two small mammal species, Neotoma picta and Peromyscus winkelmann, are known only to occur in the Sierra Madre del Sur. Habitat expansion-contraction cycles of the Pleistocene are likely responsible for the isolation and subsequent speciation of these taxa (Edwards and Bradley 2002, Sullivan et al. 1997). The deepest divergence between populations of G. volans in Mesoamerica is between those found in the western portion of the Sierra Madre del Sur and all other Mesoamerican populations. Similarly, more recent Pleistocene expansion-contraction cycles could be responsible for the isolation and subsequent divergence of G. volans populations in the Sierra Madre del Sur from those found elsewhere in Mesoamerica.

Another common biogeographic pattern seen in Mesoamerica is that isolated mountain and highland regions often reveal monophyletic assemblages based on phylogenetic analysis of genetic data. This biogeographic pattern is observed in birds and mammals (Garcia-Moreno 2004, Sullivan et al. 2000). The small sample size of

Mesoamerican G. volans specimens prevents the rigorous testing of this biogeographic 34 pattern. However, the data presented here suggest that mtDNA lineages of G. volans in

Mesoamerica are geographically sorting amongt distinct montane regions.

Conclusions

This is the first molecular phylogenetic study of Mesoamerican flying squirrels.

The data presented here provide new evidence relevant to several important aspects of the systematics and biogeographic history of this enigmatic group of mammals. First, phylogenetic analyses of the cyt-b mtDNA data support the hypothesis that flying squirrels found in Mesoamerica are conspecific with populations of G. volans from eastern North America. Mesoamerican flying squirrels appear to represent a monophyletic clade that is sister to the eastern G. volans clade, and levels of sequence divergence between the two are considerably less than those observed between G. volans and G. sabrinus. The directionality of G. volans migrations with respect to these two clades is ambiguous. Second, unlike the pattern observed in many other small mammals, the Isthmus de Tehuantepec does not appear to have played a major role in the biogeographic diversification of Mesoamerica flying squirrels. This lack of biogeographic influence from the isthmus likely results from a relatively recent colonization of the region south of the isthmus by flying squirrels (i.e., after the isthmus played an important vicariant role in structuring evolutionary lineages of other small- mammal taxa). Third, the earliest divergence recovered among populations of G. volans in Mesoamerica was between those in the Sierra Madre del Sur and all others. Finally, populations found in each isolated highland region are genetically distinct, consistent with their recognition as distinct subspecies. 35 Future work should focus on clarifying the distributions, phylogenetic relationships, and conservation status of Mesoamerican flying squirrels. This work includes further testing of the reciprocal monophyly of eastern North American and

Mesoamerican populations of G. volans. Obtaining more Mesoamerican G. volans samples would allow the use of coalescent approaches to test the directionality of population movement as well as likely reveal genetically and/or morphologically unique populations. In concurrence with this the most recent collections of G. volans from

Mesoamerica are from previously unknown areas, illustrating the limitations of described distributions and the high probability of unknown, unique populations in the

Mesoamerican highlands (Ceballos and Galindo 1983, Ceballos and Miranda 1985).

Further phylogenetic analysis will help elucidate Mesoamerican biogeographic patterns, phylogenetic relationships within G. volans, and provide a genetic basis for conservation planning. With high levels of deforestation taking place in Mesoamerica (>1% / year in

Mexico, Sanchez-Cordero et al. 2005), developing a comprehensive conservation strategy is paramount. 36

Figure 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. LITERATURE CITED

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