Site by Bennett Web & Design Co.
2007 SOUTHEASTERN NATURALIST 6(4):571–576
Multivariate Analyses of Shell Morphology of Putative
Hybrid Box Turtles
William I. Lutterschmidt1,*, Sian A. Escobar1, and Everett D. Wilson1
Abstract - Terrapene carolina triunguis (Three-toed Box Turtle) and Terrapene ornata
ornata (Ornate Box Turtle) are sympatric in areas of eastern Texas. Usually, these
species are easily distinguished by shell shape and pigmentation. However, many turtles
from areas of eastern Texas have characteristics of both Three-toed and Ornate Box
Turtles, which we presume to be a result of hybridization. We collected, marked, and
measured 103 box turtles from Walker County for morphometric analyses. Because
of our limited number of Ornate Box Turtles sampled (n = 6), an additional 68 specimens
from eastern Texas in the Texas Cooperative Wildlife Collection (TCWC) were
examined, and the carapace and plastron measured to characterize species-specific
shell morphology for Ornate Box Turtles. Multivariate analyses indicated that all morphometrics
of carapace shape (length, width, curvature length, and curvature width)
loaded heavily (r = 0.929, 0.897, 0.936, and 0.955, respectively) on the first principle
component (Factor 1) and explained 86.41% of the variation in shell morphology and
best distinguished observed differences between species and putative hybrids. Putative
hybrids demonstrated a shell morphology similar to that of Three-toed Box Turtles and
differed significantly from that of Ornate Box Turtles. Of the total 177 turtles examined,
we considered 78 to be Three-toed Box Turtles, 74 to be Ornate Box Turtles, and
25 to be hybrids between the two parent species.
Species that are closely related phylogenetically and that occur sympatrically
have a potential to hybridize. Both interspecific (e.g., Capula 1993,
2002) and intergeneric (e.g., Barber et al. 2003, Harding and Davis 1999)
hybridization have been documented in reptiles. More specifically, hybridization
between Terrapene carolina triunguis (Agassiz) (Three-toed Box
Turtle) and Terrapene ornata ornata (Agassiz) (Ornate Box Turtle) occurs
in sympatric populations (see Ernst and McBreen 1991, Ernst et al. 1994).
Shannon and Smith (1949) described an individual that displayed intermediate
characteristics of a Three-toed Box Turtle and an Ornate Box Turtle, and
Rodeck (1949) reported the mounting of a female Three-toed Box Turtle by
a male Ornate Box Turtle; copulation was interrupted. Intermediate characteristics
may include skin pigmentation and mottling that is typical of Ornate
Box Turtles, but with a keel-shaped shell and the presence of three toes on
the hind feet similar to that in Three-toed Box Turtles (Fig. 1). However,
the character of three toes is not always present in Three-toed Box Turtles
(Conant and Collins 1998), creating more difficulty in distinguishing species
based on these morphologic characters.
We collected Three-toed Box Turtles and morphologically intermediate
individuals (Fig. 1) that appear to be putative hybrids of Three-toed
1Department of Biological Sciences, Sam Houston State University, Huntsville, TX
77341. *Corresponding author - firstname.lastname@example.org.
572 Southeastern Naturalist Vol.6, No. 4
Box Turtles and Ornate Box Turtles. Natural occurrences of interspecific
hybridization are most likely when species utilize similar resources. Both
Three-toed Box Turtles and Ornate Box Turtles are reported (see Dixon
2000) from Walker County, TX, due to the county’s mosaic of grass lands,
lowland hardwoods, and pine forests. Although the Three-toed Box Turtle
prefers different habitats than does the Ornate Box Turtle (forests, meadows,
second-growth scrubs, and riparian areas versus prairies, savannas,
woodlands, and swamps, respectively; Dodd 2001), interspersion of these
preferred habitats in Walker County eliminates ecological reproductive barriers
and provide an opportunity for hybridization (Ward 1968).
We investigated the shell morphology of these morphologically intermediate
turtles suspected of hybridization. These putative hybrid turtles
collected from Walker County, TX were used to address the question: Do putative
hybrid box turtles demonstrate a shell morphology that is more similar
to that of the Three-toed Box Turtle or that of the Ornate Box Turtle?
Figure 1. Summary of morphological characters of T. c. triunguis (Three-toed Box
Turtle), T. o. ornata (Ornate Box Turtle), and putative hybrids demonstrating an
2007 W.I. Lutterschmidt, S.A. Escobar, and E.D. Wilson 573
Materials and Methods
We collected, marked, measured, and released 103 box turtles (78 Threetoed
Box Turtle; 25 putative hybrids) within Walker County, TX from April
2004 to June 2005 (sampling was under Texas Parks and Wildlife Scientific
Collectors Permit #0499-028 issued to W.I. Lutterschmidt). Turtles were
sexed, and body mass and the following shell morphology measurements
recorded: carapace length (CL), carapace width (CW), carapace curvature
Figure 2. Graphed results of Factors 1 and 2 of the PCA for shell morphology. The top
two panels represent the analysis for all individuals, middle and bottom panels are for
male and female analyses, respectively. (A) Red points represent T. c. triunguis (Threetoed
Box Turtle), blue points represent T. o. ornate (Ornate Box Turtle), and open
triangles represent putative hybrids. (B) The 95% confidence intervals for Factor 1 and
2 graphed on a smaller scale indicate that the shell morphology of putative hybrid box
turtles is similar to that of theThree-toed Box Turtle, while both putative hybrids and
Three-toed Box Turtles differ significantly from Ornate Box Turtles.
574 Southeastern Naturalist Vol.6, No. 4
length (CCL), carapace curvature width (CCW), and plastron length (PL).
Blood from the femoral vein of each box turtle was drawn and immediately
frozen and stored at -70 °C for future molecular analyses. Female
turtles were x-rayed to determine reproductive condition. Because only six
Ornate Box Turtles were collected, an additional 68 specimens from the
Texas Cooperative Wildlife Collection (TCWC) at Texas A&M University
were measured for shell morphology. Only adult specimens from eastern
Texas were measured and used in the analysis to avoid potential influences
of geographic variation in shell morphology. Statistical analyses of shell
morphology were conducted using SPSS® 8.0 statistical software, and all
analyses were considered to be significant at P < 0.05.
Multivariate analyses were used to investigate possible morphometric
differences in shell morphology among Three-toed Box Turtles, Ornate
Box Turtles, and putative hybrids. Preliminary analyses indicated that body
mass and plastron length did not distinguish differences between Three-toed
Box Turtles, Ornate Box Turtles, and putative hybrids (P > 0.05) and were
therefore removed from the final analysis. Secondly, due to preservation
techniques, the museum specimens of Ornate Box Turtles cannot offer accurate
measures of body mass, thereby making comparisons of body mass with
living specimens invalid. Thus, morphometric data consisted of measures
characterizing carapace shape (i.e., CL, CW, CCL, and CCW) for each turtle
species and individuals identified as putative hybrids.
The principle components analysis (PCA) indicated that there was a
significant effect of species and sex on shell morphology (Table 1). Eigen
values indicated that all morphometrics of carapace shape (CL, CW, CCL,
and CCW) loaded heavily (r = 0.929, 0.897, 0.936, and 0.955, respectively)
on the first principle component (Factor 1) and explained 86.41% of the variation
in shell morphology among species groups (Table 2). Carapace width
(CW) was the only morphometric that correlated slightly (r = 0.438) with
the second principle component (Factor 2), which explained only 6.97% of
Table 1. Reported results (SPSS® 8.0, 1997) of the analysis of variance within the principle
components analysis (PCA) of). Factor 1 and Factor 2 explain 86.41% and 6.97% of the variation
in shell morphology, respectively.
Source variable df SS MS F P
Species Factor 1 2 342.88 171.44 62.39 less than 0.001
Factor 2 2 28.84 14.42 22.51 less than 0.001
Sex Factor 1 1 39.40 39.40 14.34 less than 0.001
Factor 2 1 4.08 4.08 6.37 0.012
Species*sex Factor 1 2 10.64 5.32 1.94 0.147
Factor 2 2 6.74 3.37 5.26 0.006
Within (error) Factor 1 171 469.90 2.75
Factor 2 171 109.56 0.641
Corrected total Factor 1 176 841.79
Factor 2 176 145.64
2007 W.I. Lutterschmidt, S.A. Escobar, and E.D. Wilson 575
the variation in shell morphology (Fig. 2a). The first principle component
(Factor 1) best distinguished species groups and shows that putative hybrids
have a shell morphology most similar to that of the Three-toed Box Turtle.
The 95% confidence intervals for Factor 1 and Factor 2 overlap for the
Three-toed Box Turtle and putative hybrids; the Ornate Box Turtle does not
overlap and differs significantly in shell morphology (Fig. 2b).
Pigmentation and basic shell morphology are commonly used to distinguish
Three-toed Box Turtles and Ornate Box Turtles. A solid yellow plastron
and a narrow and keeled carapace with flaring on the posterior portion of the
shell identify the Three-toed Box Turtle. The carapace of the Ornate Box
Turtle is dorsally compressed, unkeeled, and strongly pigmented with five
to nine light stripes on the second costal scute. The plastron is also heavily
pigmented with dark mottling. Individuals with a morphology intermediate
between two closely related species are assumed to occur from hybridization
between those species (e.g., Azevedo et al. 2003, Kasahara et al. 1998,
Sullivan 1986). Our morphometric analysis indicates that individuals with
intermediate characteristics are more similar to the shell morphology of the
Three-toed Box Turtle than that of the Ornate Box Turtle. Putative hybrids
also retain the presence of three toes on the hind feet as in Three-toed Box
Turtles, but demonstrate shell pigmentation patterns that closely resemble
Ornate Box Turtles. Although, museum (TCWC) and county records (Dixon
2000) document the occurrence of Ornate Box Turtles within Walker County,
our sampling provided no Ornate Box Turtles while providing 78 individuals
of Three-toed Box Turtles. Second, based upon putative hybrid shell morphology
being more similar to that of the Three-toed Box Turtle, we believe
that these hybrids are most likely Three-toed Box Turtles that have obtained
shell pigmentation from occasional hybridization events with Ornate Box
Turtles (Ernst and McBreen 1991).
Future work will analyze the collected blood samples with molecular
techniques to investigate the degree of hybridization for individual box turtles
with characteristics intermediate between Three-toed Box Turtles and Ornate
Box Turtles. Although such techniques, including the use of allozymes and
microsatellites, are helpful in evaluating hybridization, morphometric analyses
are used often and are adequate to identify and investigate hybridization in
turtles (e.g., Seidel and Palmer 1991, Seidel et al. 1999) and other amphibians
and reptiles (e.g., Arevalo et al. 1993, Gergus et al. 1999).
Table 2. Reported Eigen values and % variance for each of the four principle components of
the PCA .
Component Eigen values % of variance
1 (CL, CW, CCL, CCW) 3.456 86.409
2 (CW) 0.279 6.971
3 0.165 4.125
4 0.099 2.494
576 Southeastern Naturalist Vol.6, No. 4
We thank the curators and staff of the Texas Cooperative Wildlife Collection at
Texas A&M University as well as J. Kelly McCoy, Diane L. Neudorf, Monte L. Thies,
and anonymous reviewers for helpful comments that have improved this manuscript.
Arevalo, E., G. Casas, S.K. Davis, G. Lara, and J.W. Sites, Jr. 1993. Parapatric hybridization
between chromosome races of the Sceloporus grammicus complex
(Phrynosomatidae): Structure of the Ajusco transect. Copeia 1993:352–372.
Azevedo, M.F.C., F. Foresti, P.R.R. Ramos, and J. Jim. 2003. Comparative cytogenetic
studies of Bufo ictericus, B. paracnemis (Amphibia, Anura), and an intermediate
form in sympatry. Genetics and Molecular Biology 26:289–294.
Barber, R.C., C.T. Fontaine, J.P. Flanagan, and E.E. Louis, Jr. 2003. Natural hybridization
between a Kemp’s Ridley (Lepidochelys kempii) and Loggerhead Sea
Turtle (Caretta caretta) confirmed by molecular analysis. Chelonian Conservation
and Biology 4:701–704.
Capula, M. 1993. Natural hybridization in Podarcis sicula and P. wagleriana (Reptilia:
Lacertidae). Biochemical Systematics and Ecology 21:373–380.
Capula, M. 2002. Genetic evidence of natural hybridization between Podarcis sicula
and Podarcis tiliguerta (Reptilia: Lacertidae). Amphibia-Reptilia 23:313–321.
Conant, R., and J.T. Collins. 1998. Reptiles and Amphibians of Eastern/Central
North America. Houghton Mifflin Co., New York, NY. 616 pp.
Dixon, J.R. 2000. Amphibians and Reptiles of Texas. Texas A&M University Press,
College Station, TX. 421 pp.
Dodd, K. 2001. North American Box Turtles: a Natural History. University of Oklahoma
Press, Norman, OK. 231 pp.
Ernst, C.H., and J.F. McBreen. 1991. Terrapene carolina. Catalog of American Amphibians
and Reptiles 512:1–13.
Ernst, C.H., R.W. Barbour, and J.E. Lovich. 1994. Turtles of the United States and
Canada. Smithsonian Institution Press, Washington, DC. 578 pp.
Gergus, E.W.A., D.B. Malmos, and B.K. Sullivan. 1999. Natural hybridization
among distantly related toads (Bufo alvarius, Bufo cognatus, Bufo woodhousii)
in central Arizona. Copeia 1999:281–286.
Harding, J.H., and S.K. Davis. 1999. Clemmmys insculpta (Wood Turtle) and Emydoidea
blandingii (Blanding’s Turtle) hybridization. Herpetological Review 30:225–226.
Kasahara, S., A.P.Z. Silva, and S.L. Gruber. 1998. Use of lymphocyte cultures for
BrdU replication banding patterns in anuran species (Amphibia). Genetics and
Molecular Biology 21:471–476.
Rodeck, H.G. 1949. Notes on box turtles in Colorado. Copeia 1949:32–34.
Seidel, M.E., and W.M. Palmer. 1991. Morphological variation in turtles of the genus
Pseudemys (Testudines: Emydidae) from central Atlantic drainages. Brimleyana
Seidel, M.E., J.N. Stuart, and W.G. Degenhardt. 1999. Variation and species status
of slider turtles (Emydidae: Trachemys) in the southwestern United States and
adjacent Mexico. Herpetologica 55:470–487.
Shannon, F.A., and H. Smith. 1949. Herpetological results of the University of Illinois
field expedition, spring 1949. I. Introduction, Testudines, Serpentes. Transactions
of the Kansas Academy of Sciences 52:494–509.
Sullivan, B.K. 1986. Hybridization between the toads Bufo microscaphus and Bufo
woodhousii in Arizona: Morphological variation. Journal of Herpetology 20:11–21.
Ward, J.P. 1968. Presumed hybridization of two species of box turtle. Copeia