Molecular Phylogenetics and Evolution
Vol. 14, No. 1, January, pp. 152–164, 2000
Article ID mpev.1999.0689, available online at http://www.idealibrary.com on
Comparative Molecular Phylogeography of North American Softshell
Turtles (Apalone): Implications for Regional and Wide-Scale
Historical Evolutionary Forces
David W. Weisrock1 and Fredric J. Janzen2
Department of Zoology and Genetics, Program in Ecology and Evolutionary Biology, Iowa State University, Ames, Iowa 50011-3223
Received February 12, 1999; revised May 21, 1999
We use a comparative analysis of partial cytochrome
b sequences to evaluate the evolutionary forces shaping wide-scale phylogeographic patterns of all three
North American softshell turtles (Apalone ferox, A.
mutica, and A. spinifera). The overall phylogeographic
patterns are concordant with results from both extensive regional studies of southeastern species, implicating historical vicariant processes during the Pliocene
and Pleistocene, and investigations of more northerly
distributed species, indicating a bottleneck effect of
recent dispersal into postglacial habitat. We also resolved a novel, shared genetic break between northern–
western and southeastern populations within both A.
mutica and A. spinifera, demonstrating the value of
using widespread taxa to evaluate both regional and
wider scale phylogeographic patterns. The extensive
phylogenetic structure and sequence divergences
within both A. mutica and A. spinifera contrast sharply
with most previous studies of turtles and with the
hypothesis that turtles in general have slow rates of
mtDNA evolution. r 2000 Academic Press
Key Words: bottleneck; comparative phylogeography; cytochrome b; intraspecific phylogenetics; molecular evolution; mtDNA; softshell turtle; southeastern
United States; vicariance.
INTRODUCTION
Molecular phylogeography is a powerful concept because it affords a means for formally testing evolutionary hypotheses of the distribution of genetic variation
based on historical influences or based on morphological variation (e.g., Byun et al., 1997; Strange and Burr,
1997; Wenink et al., 1996; Zamudio et al., 1997). For
example, coupling genetic theory (e.g., Hewitt 1993,
1996; Wade et al., 1994) with the biological impact of
1 Present address: Department of Biology, Campus Box 1137,
Washington University, St. Louis, MO 63130-4899.
2 To whom correspondence should be addressed. Fax: (515) 2948457. E-mail: fjanzen@iastate.edu.
1055-7903/00 $35.00
Copyright r 2000 by Academic Press
All rights of reproduction in any form reserved.
Pleistocene glacial advances and retreats (e.g., Delcourt and Delcourt, 1991; Pielou, 1991; Holman, 1995)
provides rigorous hypotheses to address with molecular
methods (Green et al., 1996; Strange and Burr, 1997;
Avise and Walker, 1998; Avise et al., 1998). Further,
when morphological variation is lacking or biogeographic history is unknown, molecular phylogeography
can generate insights into historical evolutionary forces
affecting a species (Cracraft, 1983, 1994; Avise et al.,
1987; Moritz et al., 1987; Morrone and Crisci, 1995).
Intraspecific phylogeographic approaches have been
applied to a wide array of North American vertebrates
(for reviews see Avise, 1994; Zink, 1997). However, most
of these studies have encompassed relatively small
regions; few studies have sampled widely to understand larger phylogeographic patterns across North
America (but see Ptacek et al., 1994; Shaffer and
McKnight, 1996; Zink, 1996; Bernatchez and Wilson,
1998). This trend is especially evident in the herpetofauna and results primarily from a lack of studies on
widely distributed taxa (see below) or, when wide
ranging species were used, from relatively limited
sampling (e.g., Phillips et al., 1996). Despite this lack of
wide-scale sampling, there are excellent regional phylogeographic studies of vertebrates, primarily in southeastern North America (e.g., Lamb et al., 1989; Avise,
1992; Osentoski and Lamb, 1995; Walker et al., 1995,
1997, 1998; Baer, 1998; Walker and Avise, 1998). The
results of these studies remain to be tested with widely
distributed taxa to evaluate concordant or discordant
patterns.
North American softshell turtles (Apalone, sensu
Meylan, 1987) provide an excellent system to evaluate
comparative phylogeographic patterns on a wide scale.
Apalone is old, having been reported from the Upper
Cretaceous of Canada (Gardner et al., 1995), and
consists of three species (Meylan, 1987) broadly distributed in North America, ranging in the west from
southern Alberta (Canada) south into central Mexico
and in the east from southeastern portions of Quebec
south into Florida (Iverson, 1992; Ernst et al., 1994).
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SOFTSHELL TURTLE MOLECULAR PHYLOGEOGRAPHY
A. spinifera and A. mutica both span most of this large
distribution, whereas A. ferox is confined to Florida and
adjacent states. Of the three species, A. spinifera
exhibits the most morphological variation, resulting in
the description of seven subspecies (reviewed in Ernst
et al., 1994). In comparison, A. mutica is composed of
only two subspecies and A. ferox has none (reviewed in
Ernst et al., 1994).
Two additional characteristics make Apalone an excellent system for phylogeographic analysis: (1) North
American softshell turtles are unusually sensitive to
desiccation (Dunson, 1986) and thus are largely restricted to aquatic environments for dispersal and
migration (e.g., Plummer et al., 1997; sensu Strange
and Burr, 1997) and (2) these turtles apparently exhibit
exceptionally large amounts of intraspecific mitochondrial DNA (mtDNA) variation. In a phylogenetic study
of the genus Apalone, extensive intraspecific variation
was detected among populations of A. spinifera and A.
mutica (Weisrock, 1997). This finding is especially
intriguing for two reasons: (1) studies of North American freshwater turtles have generally detected minimal amounts of mtDNA variation within species and
across closely related species (Avise et al., 1992; Lamb
and Avise, 1992; Lamb et al., 1989, 1994; Osentoski and
Lamb, 1995; Phillips et al., 1996; Janzen et al., 1997;
Shaffer et al., 1997; but see Walker et al., 1995, 1997,
1998; reviewed in Walker and Avise, 1998) and (2)
turtles are thought to have extremely low rates of
mtDNA evolution (Avise et al., 1992; but see Seddon et
al., 1998).
Using Apalone as a study system and mtDNA sequences for reconstructing phylogenetic relationships,
we address several important evolutionary issues: (1)
How are the three Apalone species genetically structured across both wide-scale and regional geographic
distributions? (2) What mechanisms can be invoked to
explain these patterns? (3) Given the wealth of literature dealing with phylogeographic patterns in a variety
of other species, what concordant or discordant patterns exist both within Apalone and among other
vertebrate species that might suggest either similar or
unique biogeographic histories? Systematics issues regarding acceptance or rejection of currently recognized
subspecies are treated elsewhere (Weisrock, 1997).
MATERIALS AND METHODS
DNA Extraction and Sequencing
Tissue was collected from both laboratory-raised and
field-collected turtles from across a large portion of the
distribution of all three Apalone species (Appendix 1).
In most cases, animals were released unharmed after
tissue was collected; tissue from all turtles and voucher
specimens of laboratory-raised animals are in the collection of the Janzen laboratory at Iowa State University.
Samples included carapace wedges, muscle, liver, and
153
blood stored in lysis buffer. Genomic DNA was isolated
from 49 individuals from 35 localities among the three
Apalone species and a Trionyx triunguis outgroup,
using a Proteinase K/SDS digestion and phenol/
chloroform extraction method (Hillis and Moritz, 1990).
Purified DNA was used in an initial PCR for cytochrome b under the following thermal conditions: 95°C
denature for 1 min, 50°C anneal for 1 min, and 72°C
extension for 2 min for 35 cycles. PCR was conducted in
25-µl volumes with 0.5–1.0 µg DNA, 1⫻ PCR buffer
(Tris–HCl, 1.5 mM MgCl2, and 50 mM KCl), 0.1 mM
dNTPs, 1.0 µM primers, and 1 unit Taq polymerase
(Boehringer Mannheim). Primers were developed to
amplify an ⬃800-bp fragment of the mitochondrial
cytochrome b gene. The forward primer (DW 2000; 58
ACA GGC GTA ATC CTA CTA A 38) was developed in
the Janzen laboratory. The reverse primer sequence
(DW 1594; 58 TCA TCT TCG GTT TAC AAG AC 38) was
equivalent to that of primer M of Shaffer et al. (1997).
The 38 end of DW 2000 corresponds to position 14499 in
the cytochrome b gene of the Mus musculus mtDNA
genome (Bibb et al., 1981) and the 38 end of DW1594
corresponds to position 15309 of the upstream-adjacent
threonine tRNA in Mus.
PCR product was run on a 1.5% low-melt agarose
TBE gel and the target DNA fragment was then
excised. The fragment was suspended in 1 ml deionized
H2O and heated at 95°C for 5 min. This mixture was
used as template in a second PCR to generate doublestranded DNA for sequencing. This product was run on
a 1% TBE agarose gel and the band was excised. DNA
was purified from the gel slice with a 0.22 Micropure
separator (Amicon) and concentrated in an M-100
microconcentrator (Amicon). Template was sequenced
in both directions at the Iowa State University DNA
Sequencing Facility on an ABI PRISM Model 377
automated sequencer. The region of overlap between
the pair of sequences for each individual was evaluated
to verify the integrity of the sequencing.
Phylogenetic Analysis
Forward and reverse sequences were assembled into
a contiguous fragment with Sequence Navigator vers.
1.0.1 (Applied Biosystems, 1994). All sequences were
then aligned with Clustal W for the Power PC vers. 1.5
(Thompson et al., 1994). Phylogenetic parsimony and
neighbor-joining analyses were conducted using vers.
4.0d60 of PAUP*, written by David L. Swofford. All
trees were rooted with the homologous sequence from
the African softshell turtle (T. triunguis). T. triunguis is
an Old World species that is hypothesized to be a basal
member of the tribe Trionychini to which Apalone
belongs (Meylan, 1987). GenBank Accession Nos. for
representative sequences are AF168749 to AF168767.
Analyses of phylogenetic patterns within and among
species were conducted using all Apalone samples and
the T. triunguis outgroup. To account for possible
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WEISROCK AND JANZEN
intrapopulation variance, two or more samples from 12
populations were analyzed (Appendix 1) even though a
previous study of the same cytochrome b fragment in a
Louisiana population of A. mutica revealed a complete
lack of variation across 19 unrelated individuals (Weisrock et al., 1998). Because of the large number of
samples involved, a heuristic search option was used in
the parsimony analyses. Under this condition, the
random addition option was utilized with 10 replicates.
Also, a neighbor-joining tree with branch lengths proportional to percentage sequence divergences was constructed to illustrate the amount of genetic variation
within and among species. Sequence data were unweighted in all analyses. Bootstrapping (Felsenstein,
1985) with 500 replicates and decay analyses (Bremer,
1996) were used to test the reliability of the data in
finding the best tree and to test the robustness of
clades.
The relative rate test was used to assess variation in
evolutionary rates across different Apalone species
(Sarich and Wilson, 1973). Within each Apalone species, the average number of substitutional differences
across all populations from the outgroup was used in
the tests. T. triunguis was used as the outgroup species
and tests were conducted as outlined in Li (1997).
RESULTS
We obtained different-sized fragments in the four
softshell turtle species due to minor length variation in
the ⬃25 bp of threonine tRNA at the 38 end of the
amplified fragment; base number was consistent within
each species. The outgroup, T. triunguis, had only 806
bp, whereas A. mutica had 809 bp, A. ferox had 810 bp,
and A. spinifera had 811 bp. Among the 8 A. mutica
samples, there were 44 variable positions of which 31
were parsimony informative; among the 9 A. ferox
samples, there were 3 variable positions of which only 1
was parsimony informative; and among the 32 A.
spinifera samples, there were 67 variable positions of
which 54 were parsimony informative. Among all three
Apalone species, there were 137 variable positions of
which 121 were parsimony informative.
Phylogenetic analyses produced four most-parsimonious trees of 296 steps each, which were combined into a
strict consensus tree with a consistency index of 0.79
(Fig. 1). All three ingroup species were strongly monophyletic, as indicated by high values for both bootstrap
and Bremer decay analyses. Fairly strong support was
also indicated by both reliability analyses for a sistergroup relationship between A. mutica and an A. ferox–A. spinifera clade. These results and nearly all the
intraspecific branching patterns in the parsimony analysis were evident in the neighbor-joining analysis as
well (cf. Figs. 1 and 2).
Apalone ferox
The A. ferox subset of the phylogenetic analyses was
an unresolved clade of peninsular Florida (FL) populations plus a single South Carolina (SC) population that
was distinct from the monotypic clade of a panhandle
FL sample (Figs. 1–3). The peninsular clade was supported by a single transversion synapomorphy. Consequently, bootstrap and decay values gave low support
for this relationship (Figs. 1 and 2). Not surprisingly
then, sequence divergences were low within the A. ferox
data set (cf. lines 12 and 13 in Table 1).
Apalone mutica
In contrast to the A. ferox clade, the A. mutica portion
of the phylogenetic analyses resolved substantial
amounts of strongly supported intraspecific structure.
At the deepest level, two major clades were produced,
separating populations in southeastern North America
from northern–western populations (Figs. 1, 2, and 4).
Both clades were supported by large bootstrap values
and relatively large decay values (Figs. 1 and 2) and
were separated by sequence divergences as large as
4.0% (cf. lines 14–18 in Table 1).
Phylogenetic structure was also detected within both
major regional clades (Figs. 1, 2, and 4). Within the
southeastern clade, a population from southeastern
Louisiana (LA) grouped distinctly from a panhandle FL
population. This relationship was supported by high
bootstrap values; however, decay values were not as
high as those found in deeper nodes (Figs. 1 and 2).
Sequence divergence between LA and FL populations
was 1.0% (cf. lines 15 and 17 in Table 1). Within the
northern–western clade, a northern Texas (TX) population formed the sister clade to a clade containing
populations from Iowa (IA) and Arkansas (AR). Within
the latter clade, the AR population grouped separately
from the IA population. All nodes within this regional
clade were supported by high bootstrap values but,
again, decay values were lower than those at deeper
nodes (Figs. 1 and 2). Sequence divergences across
populations in this clade ranged from 0.2 to 1.9% (cf.
lines 14, 16, and 18 in Table 1).
Apalone spinifera
The A. spinifera component of the phylogenetic analyses resembled an amalgam of the A. ferox and A. mutica
results in that there were several large unresolved
clades as in A. ferox and a number of strongly supported
branches as in A. mutica. Two major clades were
resolved at the deepest level, corresponding to northern–
southeastern and southwestern regions of North
America (Figs. 1, 2, and 5). These two clades were
characterized by both high bootstrap and high decay
values (Figs. 1 and 2) and had sequence divergence
values as high as 5.5% (cf. lines 1–11 in Table 1).
Phylogenetic structure was also evident within both
major regional clades (Figs. 1, 2, and 5). The northern–
SOFTSHELL TURTLE MOLECULAR PHYLOGEOGRAPHY
155
FIG. 1. Strict consensus of four most parsimonious trees for all individuals used in this study. The tree length is 296 steps and the
consistency index is 0.79. Taxon labels are as listed in Appendix 1. Numbers above branches represent bootstrap percentages; boldface
numbers below branches represent decay values. The boldface vertical bars (right) indicate interspecific clades and the hatched vertical bars
(right) with corresponding labels detail the regional intraspecific clades described under Results.
southeastern clade split into three main groups. A clade
composed of four southeastern populations (SE1) was
characterized with a high bootstrap value and a relatively high decay value. In the neighbor-joining tree,
this group was placed as the sister clade to all northern
populations with moderate bootstrap support (Fig. 2)
but such a relationship was unresolved in the parsimony analysis (Fig. 1). Within the SE1 clade, populations from FL and Alabama (AL) grouped distinctly
from populations from LA and Mississippi (MS) but the
nodes marking these separations had lower statistical
support. Sequence divergences across these popula-
tions were low and ranged from 0.1 to 0.2% (cf. lines 1,
2, and 6 in Table 1).
Two other southeastern populations formed a distinct group (SE2) that was placed with moderate bootstrap support as the sister clade to all northern populations and SE1 in the neighbor-joining analysis (Fig. 2)
but, again, this relationship was unresolved in the
parsimony analysis (Fig. 1). Regardless, SE2, consisting of populations from northern FL and southern
Georgia (GA), had high bootstrap and decay values
(Figs. 1 and 2). Sequence divergence between these
populations was low at 0.2%. In contrast, sequence
156
WEISROCK AND JANZEN
FIG. 2. Neighbor-joining tree for all individuals used in this study. Taxon labels are as listed in Appendix 1. Numbers above branches and
arrowed numbers represent bootstrap percentages. The boldface vertical bars (right) indicate interspecific clades and the hatched vertical bars
(right) with corresponding labels detail the regional intraspecific clades described under Results.
divergences between the SE1 and the SE2 clades were
an order of magnitude higher (cf. lines 1, 2, and 6 with
line 3 in Table 1).
The third main group within the northern–southeastern clade was mostly a polytomy of all northern populations. The node delineating the northern populations as
a sister taxon to the SE1 clade had modest bootstrap
support in the neighbor-joining analysis (Fig. 2) but
was unresolved in the parsimony analysis (Fig. 1).
Within the northern clade there was minimal phylogenetic structure. A clade composed of IA, Wisconsin (WI),
and Illinois (IL) populations was resolved but statistical
support was low and the clade did not include all IL
populations examined (Figs. 1 and 2). The only other
distinct grouping that appeared in both phylogenetic analyses concerned two individuals from the Thames River
in Ontario but, surprisingly, a third individual from the
same population grouped separately (Figs. 1 and 2).
Sequence divergences among northern populations
were low and typically around 0.1% (cf. lines 4, 5, and 8 in
Table 1).
Within the southwestern regional clade, there was
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SOFTSHELL TURTLE MOLECULAR PHYLOGEOGRAPHY
FIG. 3. Overlay of the Apalone ferox parsimony tree upon its North American distribution. End points of branches correspond to sample
locations. Taxon labels are as listed in Appendix 1.
considerable phylogenetic divergence. Populations along
the Rio Grande River from New Mexico (NM) and TX
grouped together with high bootstrap and decay values
(Figs. 1 and 2) and formed the sister clade to ‘‘eastern’’
and ‘‘coastal’’ TX populations in the neighbor-joining
analysis (Fig. 2). Sequence divergence between the two
Rio Grande River populations was low at 0.1% but
divergences between these populations and the ‘‘eastern’’ and ‘‘coastal’’ TX populations were much higher at
1.1 to 1.2%, as was the divergence between the latter
two TX populations at 1.6% (cf. lines 7 and 9–11 in
Table 1).
TABLE 1
Pairwise Percentage Sequence Divergences among Selected Individuals from all Three Species of Apalone
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
ALec
FLer1-s
GAsr
ILma
ILs11
LAcr1-s
NMrg
ONtr1
TXcc
TXki
TXsc
FLca
FLco
ARwr1
FLer-m
IAcr1-m
LAcr1-m
TXbr
Trionyx
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
17.5
17.4
17.6
17.7
17.6
17.4
16.1
17.9
15.9
16.0
16.3
15.1
15.3
16.9
17.0
16.7
16.5
17.5
—
0.1
1.9
1.4
1.5
0.1
5.2
1.5
5.2
5.4
5.3
6.3
6.4
8.3
9.1
8.4
8.8
8.5
—
2.0
1.5
1.6
0.2
5.3
1.6
5.3
5.5
5.4
6.4
6.5
8.1
9.0
8.3
8.6
8.4
—
1.2
1.4
1.7
4.6
1.4
4.7
4.8
4.7
5.8
5.9
8.3
9.3
8.4
8.9
8.3
—
0.1
1.2
4.8
0.1
4.9
4.8
4.9
6.2
6.3
7.5
8.8
7.7
8.4
7.8
—
1.4
4.9
0.2
5.1
4.9
5.1
6.3
6.4
7.7
8.9
7.8
8.5
7.9
—
5.1
1.4
5.1
5.3
5.2
6.2
6.3
8.1
9.0
8.3
8.6
8.4
—
4.9
1.1
1.5
0.1
5.6
5.7
8.5
9.0
8.4
8.6
8.5
—
5.1
4.9
5.1
6.3
6.4
7.7
8.9
7.8
8.5
7.9
—
1.1
1.2
5.7
5.8
8.2
8.4
8.0
8.0
8.2
—
1.6
5.8
5.9
8.2
8.9
8.0
8.5
8.1
—
5.7
5.8
8.4
9.1
8.3
8.8
8.4
—
0.1
7.3
7.7
7.2
7.5
7.5
—
7.4
7.8
7.3
7.7
7.7
—
4.0
0.2
3.7
1.9
—
3.8
1.0
4.0
—
3.6
1.6
—
4.0
Note. Individuals illustrated are representatives from genetically similar clades. Population abbreviations are as listed in Appendix 1.
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WEISROCK AND JANZEN
FIG. 4. Overlay of the Apalone mutica parsimony tree upon its North American distribution. End points of branches correspond to sample
locations. Taxon labels are as listed in Appendix 1.
DISCUSSION
This study provides important insights into phylogeography, mechanisms of genetic structuring, and
molecular evolution. These insights are due primarily
to a comparative molecular phylogeographic approach
(sensu Bermingham and Moritz, 1998) and to widespread, although by no means complete, sampling.
Such a comparative approach in turtles has been
adopted on a regional basis (Avise, 1992; Lamb et al.,
1992; Walker and Avise, 1998) but not on a wide
geographic scale (except in sea turtles; reviewed in
Bowen and Karl, 1997). We find that vicariant forces
have been of widespread importance in creating the
largely concordant, geographical genetic structure in
North American softshell turtles. However, dispersal is
likely to have been involved at least in a limited way, as
have what appear to be unusually elevated rates of
molecular evolution. We explore these themes integratively in this section.
A. ferox has the most restricted distribution of all
Apalone species. The location of this species in extreme
southeastern North America, a well-known site of
isolated Pleistocene and pre-Pleistocene refugia (discussed in Meylan, 1982), suggested the possibility of
significant amounts of intraspecific genetic differentiation. In contrast, low levels of intraspecific mtDNA
divergence could implicate a bottleneck caused by
recent radiation into the extant range (Nei et al., 1975).
Parsimony analysis of A. ferox grouped all peninsular
FL populations and a SC population distinctly from a
panhandle FL population (Fig. 1). The peninsular
FL–SC clade was supported by only a single synapomorphy and thus did not display levels of sequence divergence from the panhandle FL population (Fig. 2) expected under a model of long-term isolation. This
homogeneity in mtDNA sequence may be explained by
a vicariant divergence of these two regions followed by
extensive gene flow and fixation among the peninsular
FL and SC populations (sensu Baer, 1998). Indeed, A.
ferox is ecologically versatile and moves overland regularly in peninsular FL (Ernst et al., 1994). Explanations for the hypothesized vicariant event have been
proposed previously based upon freshwater fish that
exhibit similar intraspecific phylogeographic patterns
in southeastern North America (Bermingham and Avise,
1986; Avise, 1992). One hypothesis places divergence
between the eastern and the western assemblages
during the Pliocene 3.5–4.0 million years ago (MYA)
SOFTSHELL TURTLE MOLECULAR PHYLOGEOGRAPHY
159
FIG. 5. Overlay of the Apalone spinifera parsimony tree upon its North American distribution. End points of branches correspond to
sample locations. Taxon labels are as listed in Appendix 1.
when high sea levels pushed the Gulf and Atlantic
shorelines farther inland (Bermingham and Avise,
1986). A high-elevation area in peninsular FL may have
served as an island refuge for freshwater fauna as it
became isolated from the mainland (Meylan, 1982). If
comparable patterns among A. ferox and the sympatric
fish fauna are a result of similar vicariant forces both
spatially and temporally, then A. ferox appears to follow
the same slow molecular evolutionary trends (Table 1)
proposed for other turtle species (Avise et al., 1992).
This conclusion is tentative at best, however, because
only a single base substitution is involved.
In contrast to the low genetic variability observed in
A. ferox, we detected substantial genetic variation and
similar phylogeographic patterns in the two widely
distributed species, A. mutica and A. spinifera. Reduced sample size and geographic range in A. mutica
prevented a complete comparison across both species’
ranges (e.g., no A. mutica occur in the Rio Grande
drainage) but two major trends in softshell turtle
phylogeography were evident nonetheless.
The largest shared pattern in A. mutica and A.
spinifera is a genetic dichotomy between populations
north and west of LA (exclusive of the basal Rio Grande
drainage populations in A. spinifera) and populations
from the Gulf Coast in southeastern North America
(Figs. 4 and 5). Sequence divergences across these
assemblages in both species were large (Table 1),
although about twice as large in A. mutica as in A.
spinifera. Such large and similar patterns of intraspecific divergences in both species are suggestive of a
shared vicariant event within North American softshell
turtles.
The lack of a calibrated molecular clock for softshell
turtles imposes limitations on dating divergences and,
consequently, on forming precise hypotheses regarding
the historical vicariant forces separating the northern–
western and southeastern lineages. Still, from the size
of sequence divergences between these lineages (especially in A. mutica), the vicariant event may be very old.
As hypothesized for the southeastern fauna (Bermingham and Avise, 1986; Avise, 1992), high sea levels
during the warm interglacial Pliocene may have substantially affected river systems and could have pro-
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duced barriers to gene flow. Likewise, separate glacial
refugia during the Pleistocene may instead have caused
or otherwise contributed to the divergence of these
lineages (sensu Avise and Walker, 1998).
A shared vicariant history between A. mutica and A.
spinifera is also indicated by phylogeographic patterns
within southeastern North America. Both species exhibit a divergence between closely located populations
from panhandle FL and eastern LA (Figs. 4 and 5),
consistent with prior phylogeographic studies of turtles
and other vertebrate taxa in southeastern North
America (reviewed in Walker and Avise, 1998). Furthermore, magnitudes of intraspecific divergences across
these southeastern populations are again greater in A.
mutica than in A. spinifera (Table 1). This trend is
consistent with that described above for the northern–
western vs southeastern dichotomy.
The different magnitudes of sequence divergences
within each species could be considered evidence against
a shared phylogeographic history among North American softshell turtles. However, a relative rate test
(d ⫽ 7, SE ⫽ 22.8, P : 0.05) indicated that the two
species are evolving at similar rates (sensu Li, 1997).
Furthermore, strong evidence for a shared phylogeographic history derives from similar phylogenetic patterns in a variety of taxa, including mammals (e.g.,
Ellsworth et al., 1994), freshwater fish (Bermingham
and Avise, 1986; Avise, 1992; Baer, 1997), and other
species of turtles (Avise et al., 1992; Osentoski and
Lamb, 1995; Walker et al., 1995, 1997, 1998; reviewed
in Walker and Avise, 1998). These species all exhibit a
similar genetic break between western and eastern
assemblages within the southeastern regional fauna.
Discordant patterns have been detected in comparative
phylogeographic studies of other regions of North
America (Lamb et al., 1992; Zink, 1996; Strange and
Burr, 1997; Bernatchez and Wilson, 1998), making our
findings important evidence for a shared vicariant
history among many organisms in southeastern North
America (Avise, 1992; Walker and Avise, 1998).
Unlike the comparative phylogenetic results for the
southeastern region that suggest shared vicariant
events, reduced genetic variation across northern populations of softshell turtles suggests recent dispersal
into formerly glaciated habitats. For example, mtDNA
variation in A. spinifera was minimal across an area
spanning from southern IL north to WI and northeast
to Lake Champlain in Quebec (Table 1; Figs. 2 and 5).
This finding is consistent with a pattern expected
following post-Pleistocene dispersal into formerly glaciated areas (Holman, 1995) and is comparable to patterns of genetic variation across northern distributions
of other North American herpetofauna that depend on
an aquatic environment (e.g., Gray, 1995; Green, 1996;
Shaffer and McKnight, 1996; Janzen et al., 1997;
discussed in Hewitt, 1993; Wade et al., 1994).
Because turtles may exhibit slow rates of mtDNA
evolution (Avise et al., 1992), genetic variation among
recently colonized northern populations may be due to
retention of polymorphisms generated within ancestral
refugia. As glaciers receded northward, differential
fixation of haplotypes within newly founded populations could account for the small amounts of genetic
variation across the sampled northern taxa. However,
multiple haplotypes within the ONtr population of A.
spinifera is evidence of novel genetic variation within
already established northern populations. Distinct haplotypes within this extreme northern locale are unlikely to be explained as retention of ancestral polymorphism because founder events tend to genetically
homogenize newly established postglacial populations
(Nei et al., 1975; Hewitt, 1993, 1996; Green, 1996; Lair
et al., 1997; but see for example Armbruster et al.,
1998). Still, this hypothesis remains to be tested conclusively by analyzing genetic variation within and among
populations throughout the ranges of these species.
Regardless, the overall phylogeographic analyses of
A. spinifera and A. mutica raise the unexpected possibility that the rates of molecular evolution in these species
are significantly faster than those observed in most
other North American turtles (Avise et al., 1992; Osentoski and Lamb, 1995; Shaffer et al., 1997; but see
Walker et al., 1995, 1997, 1998). This scenario is
supported by substantial amounts of intraspecific
mtDNA variation in A. mutica and A. spinifera across
geographic ranges occupied by other turtle species for
which little to no mtDNA variation has been detected
(e.g., Lamb et al., 1994; Phillips et al., 1996). The reason
is unclear.
It is interesting to note in this regard that Apalone is
an ancient genus, dating from at least the Upper
Cretaceous of Canada (Gardner et al., 1995). Fossil A.
ferox are well represented in Pleistocene deposits in
Florida and fossil A. spinifera are known from a variety
of Pleistocene sites in southern and central North
America (reviewed in Holman, 1995). Surprisingly, no
fossil A. mutica have been described (reviewed in Ernst
et al., 1994). However, a cladistic analysis of osteological characters involving extinct and extant species in
the Trionychini placed A. mutica as the sister species to
the Cretaceous A. latus, with A. spinifera as the sister
species to the A. mutica–A. latus clade and A. ferox as
basal to all three of those species (Garner et al., 1995).
Although this arrangement contradicts our phylogenetic reconstruction based on molecular data (Figs. 1
and 2), both analyses nonetheless suggest that A.
mutica is old (⬎65 MYA based on Gardner et al., 1995
and basal to A. spinifera and A. ferox in this study). The
age of this genus could explain the substantial genetic
structure not detected in codistributed turtles of other
genera, assuming that those genera are younger than
Apalone (which is almost certainly true; see summaries
of fossil occurrences in Ernst et al., 1994), without
resort to invoking faster rates of molecular evolution. A
161
SOFTSHELL TURTLE MOLECULAR PHYLOGEOGRAPHY
APPENDIX 1
APPENDIX 1—Continued
Taxa and Locale Information for all Softshell Turtle
Samples Included in the Analyses
Sample
Taxa
Locale information
Trionyx
FLca
Trionyx triunguis
A. ferox
FLco
A. ferox
Unknown; (J51896)
Apalachicola River east of
Blountstown, Calhoun Co.,
FL; (J52188)
US Highway 41, ⬃5 km east
of junction with state road
29, Collier Co., FL;
(J53743)
Suwanee River, Lafayette
Co., FL; (J52186)
Rainbow Run near Dunnellon, Marion Co., FL;
(J53741)
De Leon Springs, Spring
Garden Lake, Volusia Co.,
FL; (J52187)
Palm Beach Co., FL;
(J51895)
Palm Beach Co., FL;
(J52183)
Palm Beach Co., FL;
(J52184)
Edisto River, Edisto Island,
Colleton Co., SC; (J20046)
White River near
Georgetown, White Co.,
AR; (J51264)
White River near
Georgetown, White Co.,
AR; (J51268)
Escambia River just north of
state road 4, Escambia Co.,
FL; (J51894)
Near Wiese Slough on Cedar
River, Muscatine Co., IA;
(J51486)
Near Wiese Slough on Cedar
River, Muscatine Co., IA;
(J51487)
Comite River at Highway 64,
Comite Drive, and Dyer
Road, 30°308N, 91°048W,
East Baton Rouge Parish,
Baker, LA; (J51306)
Comite River at Highway 64,
Comite Drive, and Dyer
Road, 30°308N, 91°048W,
East Baton Rouge Parish,
Baker, LA; (J51309)
Brazos River bridge on Route
209 near Graham, Young
Co., TX; (J20040)
Euphapee Creek, 1.5 km east
of Route 49 bridge, Macon
Co., AL; (J20044)
Escambia River just east of
Century, Escambia Co.,
FL; (J52172)
Escambia River near FL-AL
state line, Escambia Co.,
FL; (J51892)
FLlf
A. ferox
FLma
A. ferox
FLvo
A. ferox
FLpb1
A. ferox
FLpb2
A. ferox
FLpb3
A. ferox
SCer
A. ferox
ARwr1
A. mutica
ARwr2
A. mutica
FLer-m
A. mutica
IAcr1-m
A. mutica
IAcr2-m
A. mutica
LAcr1-m
A. mutica
LAcr2-m
A. mutica
TXbr
A. mutica
ALec
A. spinifera
FLer1-s
A. spinifera
FLer2-s
A. spinifera
Sample
Taxa
Locale information
FLor
A. spinifera
GAsr
A. spinifera
IAcr1-s
A. spinifera
IAcr2-s
A. spinifera
ILac1
ILac2
ILma
A. spinifera
A. spinifera
A. spinifera
ILsl1
A. spinifera
ILsl2
A. spinifera
INsr
A. spinifera
LAcr1-s
A. spinifera
LAcr2-s
A. spinifera
MIcc
A. spinifera
MOsc
A. spinifera
MSmc
A. spinifera
NMrg
A. spinifera
ONlp
A. spinifera
ONrp
A. spinifera
ONsr
A. spinifera
ONtr1
A. spinifera
ONtr2
A. spinifera
Ochlockonee River, Whitehead Landing in Apalachicola National Forest, Liberty Co., FL; (J51893)
Suwanee River, Lanier Co.,
GA; (J20034)
Near Wiese Slough on Cedar
River, Muscatine Co., IA;
(J51573)
Near Wiese Slough on Cedar
River, Muscatine Co., IA;
(J51574)
Alexander Co., IL; (J20035)
Alexander Co., IL; (J20036)
Piasa Island, Mississippi
River mile 210, Madison
Co., IL; (J53755)
Stump Lake near Alton,
Madison Co., IL; (J52084)
Stump Lake near Alton,
Madison Co., IL; (J52085)
Sugar River east of Crawfordsville, Montgomery
Co., IN; (J20041)
Comite River at Highway 64,
Comite Drive, and Dyer
Road, 30°308N, 91°048W,
East Baton Rouge Parish,
Baker, LA; (J51588)
Comite River at Highway 64,
Comite Drive, and Dyer
Road, 30°308N, 91°048W,
East Baton Rouge Parish,
Baker, LA; (J51600)
Muskegon River, Clare Co.,
MI; (J20037)
Airport Slough, Mississippi
River mile 220, St. Charles
Co., MO; (J53756)
Mill Creek, Pearl River Co.,
MS; (J20043)
North Elephant Butte Reservoir near Nogal Canyon,
⬃40 air km North of Truth
or Consequences, Socorro
Co., NM; (J20013)
Long Point Provincial Park,
Lake Erie, Ontario,
Canada; (J53767)
Rondeau Provincial Park,
Lake Erie, Ontario,
Canada; (J53785)
Sydenham River south of
Alvinston at border of
Lambert and Middlesex
Co. Mun., Ontario,
Canada; (J53774)
Thames River north of
London, Ontario, Canada;
(J53773)
Thames River north of
London, Ontario, Canada;
(J53776)
162
WEISROCK AND JANZEN
APPENDIX 1—Continued
Sample
Taxa
Locale information
ONtr3
A. spinifera
QBlc1
A. spinifera
QBlc2
A. spinifera
TXcc
A. spinifera
TXki
A. spinifera
TXsc
A. spinifera
WImr1
A. spinifera
WImr2
A. spinifera
Thames River north of
London, Ontario, Canada;
(J53777)
Chapman Bay, Lake Champlain, Quebec, Canada;
(J53779)
Chapman Bay, Lake Champlain, Quebec, Canada;
(J53780)
Coleto Creek intersection
with Camp Coleto Road,
⬃5 km from Schroeder,
Goliad Co., TX; (J20047)
Kingsville, Kleberg Co., TX;
(J20042)
Sycamore Creek, Route 277
bridge, Del Rio, Valverde
Co., Texas; (J20045)
Pool 8 of the Mississippi
River near Stoddard,
Vernon Co., WI; (J51496)
Pool 8 of the Mississippi
River near Stoddard,
Vernon Co., WI; (J51501)
Note. Numbers in parentheses are F. J. Janzen tissue collection
numbers.
similar argument could explain the differences observed between A. mutica and A. spinifera if the former
is older than the latter. Still, softshell turtles are
remarkably distinct from other turtles morphologically
(Meylan, 1987), karyotypically (Bickham and Carr,
1983), and in other traits (e.g., Janzen and Paukstis,
1991), which could conceivably reflect a unique underlying genetic architecture and atypical molecular processes in this family.
Comparative biogeography and, more recently, comparative phylogeography emphasizes shared patterns
among similarly distributed organisms. Although inferring process from pattern is an inexact science, the
abundance of concordant phylogeographic patterns in
regional assemblages of North American vertebrates
comprises strong evidence of shared historical processes. Patterns detected within the three species of
Apalone strengthen evolutionary conclusions drawn
from regional studies in southeastern North America
regarding vicariant forces (Avise, 1992; Walker and
Avise, 1998) and in northern latitudes of North America
regarding processes of post-Pleistocene dispersal (e.g.,
Hewitt, 1993, 1996; Green, 1996; Shaffer and McKnight, 1996; Janzen et al., 1997). These patterns are
unlikely to be shared by all species though, due to
ecological and historical differences; thus, further studies are needed to address different processes (Avise,
1998).
Phylogeographic studies of widespread species, as
exemplified by two of the three Apalone species exam-
ined herein, are also beneficial for detecting patterns
not evident in smaller regional studies. The large
genetic break between northern–western and southeastern populations in both A. mutica and A. spinifera
suggests a shared history of vicariance that predates
vicariant processes thought to have occurred in southeastern North America (e.g., Avise, 1992). The sequence
of vicariant events for A. spinifera is, however, somewhat more complex than that for A. mutica (cf. Figs. 4
and 5); additional strategic sampling from throughout
the range of both species would resolve many such
phylogeographic issues raised in this study. Still, the
large genetic break in A. spinifera separating southwestern populations from all others attests to the potential
for ancient, extensive phylogenetic structure within
other widespread North American species (e.g., Zamudio et al., 1997). Detection of these heretofore unrecognized patterns sets the stage for future phylogeographic studies of widespread, sympatric aquatic
species.
ACKNOWLEDGMENTS
Tissue and egg samples were kindly provided by K. Darnell, S.
Doody, M. Fletcher, J. Harding, C. Lanthier, P. Magwene, P. Moler, C.
Painter, B. Shaffer, and J. Tucker. Eggs collected by F.J.J. were
obtained with Iowa Department of Natural Resources Scientific
Collecting Permit SC14 9501 and Wisconsin Department of Natural
Resources Scientific Collecting Permit SCP-WD-82-C-95. Special
thanks go to T. Haselkorn for assistance with the molecular work; P.
Meylan, G. Orti, E. Routman, B. Shaffer, and an anonymous reviewer
for insightful comments on various drafts of the manuscript; R.
Sterner for the use of his maps; K. Darnell for going out of his way
simply for the love of softshell turtles; and P. Meylan for encouraging
the project and for advice in getting started. This work was conducted
by D.W.W. in partial fulfillment of his M.S. requirements at ISU.
Partial funding was provided by the Department of Zoology and
Genetics and NSF Grant DEB 96-29529 to F.J.J. This is Journal
paper No. J-18433 of the Iowa Agriculture and Home Economics
Experiment Station, Ames, IA, Project No. 3369, and was supported
by Hatch Act and State of Iowa funds.
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