Download Patterns of Genetic Differentiation in Appalachian Desmognathine

Patterns of Genetic Differentiation in
Appalachian Desmognathine Salamanders
S. G. Tllley
Studies of allozymic variation In salamanders of the Desmognathus ochrophaeus
complex have revealed complex patterns of genetic fragmentation among populations and species. Populations of D. carolinensls within the Black and Great Craggy
Mountains of western North Carolina exhibit pronounced variation In allozyme frequencies at five polymorphic loci. Allozyme frequencies at one of these loci vary
with elevation. There are no obvious patterns at the remaining loci with respect to
elevation, latitude, or longitude, and only a weak suggestion of isolation by distance among populations. On a broader geographic scale, the northernmost form
(D. ochrophaeus) exhibits very little Intrademic variation, even over distances approaching 1000 km. Forms with progressively more southern distributions exhibit
progressively higher levels of Isolation by distance. Genetic distances among local
populations of the more southern forms (D. orestes, D. carolinensls, and D. ocoee)
Increase with geographic distances among populations separated by less than 100
km, but level off at greater geographic distances. Genetic distances for Interspecific
comparisons also correlate positively with geographic distances, but the relationships are not oriented toward the origins of the scatterplots, indicating additional
genetic differentiation that cannot be attributed to Isolation by distance. Patterns
of Interspecific differentiation do not relate to either geographic or cladlstlc relationships among these forms. Levels of genetic fragmentation appear higher In
southern Appalachian forms of the D. ochrophaeus complex than in two larger,
more aquatic congeners: D. montlcola and D. fuscus. Blotic Interactions with these
and other low-elevation predators and competitors may have produced genetic
fragmentation In the D. ochrophaeus complex.
From the Department of Biological Sciences, Smith College, Northampton, MA 01063. This research was supported by the Blakeslee Genetics Research Fund at
Smith College and National Science Foundation grant
BSR-8508363. Collecting permit* were provided by the
States of North Carolina and Tennessee; Plsgah, Nantahala, and Cherokee National Forests; the Great Smoky
Mountains National Park; and the Blue Ridge Parkway.
Louise Meade, Meredith Mahoney, Robert Merritt, and
David Wake provided comments on the manuscript. A
large part of the dataset was collected by Smith College
students pursuing Independent research projects supported by the Blakeslee Genetics Research Fund, the
Howard Hughes Medical Institute, and the Smith College Tomllnson Fund. I thank all these individuals, especially Nancy Karelia, Lenl-Sarah Machlnton, Meredith
Mahoney, Lada Soljan, and Emily White, for their contributions. This paper was delivered at a symposium
entitled "Genetics of Fragmented Populations" sponsored by the American Genetic Association at the University of Georgia on May 18, 1996.
Journal of Heredity 1997:88:305-315; 0022-1503/97/$5.00
Taxa that combine high species richness
with ecological diversification can teach
us much about how, to paraphrase G. Evelyn Hutchinson, the processes of genetic
fragmentation play out in various ecological theaters. Desmognathine salamanders
represent such a group. Desmognathines
are restricted to middle and eastern North
America, attaining their greatest taxonomic diversity (12 currently named species)
in the Appalachian mountains. Desmognathines combine morphological conservatism with extensive Intra- and Interdemic variation in color patterns, and some
species are extremely difficult or even impossible to distinguish in the field. Allozyme studies have greatly expanded our
understanding of species diversity in this
group, leading to the resurrection of the
cryptic species Desmognathus imitator
(Tilley et al. 1978), the description of D.
santeetlah (Tllley 1981; Tilley and
Schwerdtfeger 1981), and, most recently,
the subdivision of D. ochrophaeus into
four allopatric, genetically differentiated
forms: D. ochrophaeus, D. carolinensis, D.
ocoee, and D. orestes (Tilley and Mahoney
1996). Concommitantly our understanding
of the diversity of desmognathine life histories (Tllley and Bernardo 1993), behaviors (Arnold et al. 1993; Forester 1977),
and ecological relationships (Halrston
1986,1987) has expanded greatly in recent
years. We should now be able to at least
begin relating patterns of genetic fragmentation to the biogeographic histories and
ecological attributes of species, and to
thus begin understanding the Interplay of
ecological and genetic mechanisms that
generate taxonomic diversity in this group
of animals.
This article deals primarily with patterns of genetic differentiation in the D.
ochrophaeus complex. It summarizes and
expands on findings published recently by
Tilley and Mahoney (1996) to consider genetic differentiation at three levels: among
populations of a single species (D. caroli-
305
too complex, however, to be adequately
conveyed by any traditional taxonomic
treatment. The taxonomic statuses of D.
ocoee and D. orestes are especially problematical. Populations assigned to D.
ocoee display considerable genetic differentiation, indicating that further taxonomic subdivision may be warranted. There is
evidence of intergradation between populations assigned to D. ocoee and D. carolinensis along the Blue Ridge Divide east of
Asheville, North Carolina (Tilley and Mahoney 1996), but combining these two
forms would create an even more loosely
denned "species" than the current D.
ocoee. D. orestes consists of two distinct
Salamanders of the D.
clusters of populations, referred to by Tilochrophaeus Complex
ley and Mahoney (1996) as groups B and
C.
The D. ochrophaeus complex, as defined
by Tilley and Mahoney (1996) consists of
Members of the D. ochrophaeus complex
six species: apalachicolae, carolinensis, im- are all medium-size (to about 65 mm stanitator, ocoee, ochrophaeus, and orestes. D. dard length), semiterrestrial salamanders,
apalachicolae is restricted to the Gulf
whose preferred habitats are the banks of
coastal plain, and D. imitator to the Great
first-order streams, seepage areas, wet cliff
Smoky and Great Balsam Mountains of
faces, and hlgh-elevatlon forests. In the
western North Carolina and eastern Ten- forms for which Information on reproducnessee, where it is sympatrlc with D.
tive biology Is available (principally ochocoee. The work described here deals with
rophaeus, carolinensis, and ocoee), ovipothe remaining four species, which were
sition, followed by brooding by the female
until recently treated as a single form, D.
until the eggs hatch, occurs in the viciniochrophaeus. D. ochrophaeus in its new re- ties of small streams, springs, seepages,
stricted sense (Tilley and Mahoney 1996)
and wet cliff faces. Females oviposit in
ranges from Kentucky to Quebec across
brooding chambers under moss on banks
the Allegheny and Cumberland Mountains
and logs, in situations where the emerging
and Plateaus west of the Ridge and Valley
larvae can readily enter shallow surface
Physiographic Province. The remaining water. Larvae typically hatch In late sumforms (maps in Figs. 5 and 6) are para- mer and transform the following spring.
patrically distributed from north to south
Females reach sexual maturity in 4-5
in the southern Blue Ridge Physiographic
years, males in 3-4 years, with maturity
Province: D. orestes in southwest Virginia,
being delayed with increasing elevation
northwest North Carolina, and northeast
(Tilley and Bernardo 1993). Adult surviTennessee; D. carolinensis in the Unaka,
vorship is relatively high for a small verBald, Black, and Newfound Mountains
tebrate (Tilley 1980).
north of the Pigeon River in eastern TenThe lives of these salamanders are intinessee and western North Carolina; and D.
mately connected with aquatic sites asocoee in the ranges south of the Pigeon
sociated with first-order streams and their
River, including the Great Smoky, Great
headwaters. These sites provide habitats
Balsam, Cowee, Nantahala, and Unicoi
for larvae, brooding habitats for females,
Mountains and the highlands along the
and overwintering sites for all life-history
Blue Ridge Divide into northwest Georgia,
stages. At high elevations, individuals
with an isolated group of populations in
from populations centered around seepthe Cumberland Plateau of northeast Ala- age areas in the heads of coves can disbama.
perse into surrounding woodlands
Tilley and Mahoney (1996) based their
throughout even the warmest months. At
decision to taxonomically subdivide D.
low elevations, individuals seem much
ochrophaeus on an evolutionary species
more restricted to the vicinities of small
concept. Each of the four forms that they
seepage areas, and especially to wet cliff
recognized (D. carolinensis, D. ochro- faces. However, aquatic habitats at low elphaeus, D. ocoee, and D. orestes) seemed
evations usually support dense populato represent a collection of populations on
tions of D. quadramaculatus and D. montia distinct evolutionary trajectory. The pat- cola. These two more aquatic species atterns of differentiation in this group are
tain larger body sizes and prey on memnensis) within a restricted geographic region, among populations of single species
throughout their geographic ranges, and
among populations of different species of
the D. ochrophaeus complex. It also adds
comparative information on two congeners, D. fuscus and D. monticola, to show
how patterns of genetic fragmentation
may reflect the diverse ecological attributes of these species and members of the
D. ochrophaeus complex. Finally, it attempts to relate these patterns to other
attributes of the biology and biogeographic history of these animals.
3 0 6 The Journal of Heredity 1997:88(4)
bers of the D. ochrophaeus complex
(Halrston 1986, 1987). Even in habitats
where they are abundant and widely distributed, local demes may be much more
isolated than they appear, due to several
ecological and behavioral attributes that
may constrain dispersal abilities and gene
exchange among local populations.
Factors Restricting Dispersal Along
Streams
Since most of the larval stage occurs during the winter months, there is little larval
growth or opportunity for dispersal of larvae from the vicinities of the oviposition
sites. Larvae are often observed in mere
surface films at the very edges of seeps
and on wet cliff faces. Downstream dispersal is probably restricted by small size,
relatively weak swimming ability, preference for very shallow surface water, and
the short duration of the larval stage.
Downstream larval "drift" is probably
much less important in the ochrophaeus
complex than in species with larger
stream-dwelling larvae.
The streams of Appalachia are hazardous dispersal corridors for members of
the ochrophaeus complex. They are inhabited by one to four species of large plethodontids that are known to feed on smaller salamanders: D. quadramaculatus, D.
monticola, D. fuscus, and Gyrinophilus porphyriticus. The terrestrial tendencies of the
ochrophaeus complex and other small Desmognathus have been interpreted as adaptations that minimize predation by
these larger, more aquatic salamanders
(Hairston 1986, 1987; Tilley 1968). Experimental manipulations of D. monticola densities have been shown to influence densities of D. ocoee (Hairston 1986).
Factors Restricting Dispersal Across
Land
Female D. ocoee are known to exhibit phllopatry with respect to brooding sites
(Forester 1977) in successive years. Thus,
while females may disperse into the forest,
particularly in years in which they do not
brood, they may repeatedly oviposit in
their natal stream headwaters.
Mating occurs in the spring when gravid
females occur in the vicinities of oviposition sites, and in the fall when both sexes
congregate to overwinter In those same
sites. Male philopatry has not been studied, but males that disperse into terrestrial habitats during the summer may return
to and mate in their natal stream headwaters in the fall.
The manner in which the geographic
null model for such analyses is an array of
populations in which genetic differentiaLatitude
Longitude
tion occurs in response to mutation at
Min.
Sec.
Locality
Elevation (m) Deg.
Sec.
County
Deg.
Mln.
rate u and is retarded by gene exchange
1
11
42
29
1,554
35
82
at rate m. In such a system, it can be
McDowell
16
2
Yancey
933
35
44
40
82
12
50
shown that the relationship between Nei's
1,676
Yancey
3
35
43
42
82
16
49
genetic distance and geographic distance
McDowell
4
707
35
45
23
82
9
11
5
McDowell
488
35
44
11
82
7
9
eventually reaches an equilibrium, at
Yancey
1,951
6
35
44
21
82
17
8
which
that relationship is expected to be
1,987
Yancey
7
35
44
5
82
17
10
linear with zero Intercept and slope pro1,451
Yancey
8
35
42
6
82
15
56
Yancey
1,231
9
35
42
34
82
14
58
portional to V2u/m, where u and m are
Yancey
1,067
10
35
43
18
82
14
57
the
mutation and migration rates, respecYancey
11
969
35
44
2
82
15
6
tively (Nei 1972). Deviations from this simYancey
1,170
12
35
44
26
82
11
49
McDowell
1,183
13
35
44
31
82
11
24
ple relationship can illuminate the proYancey
14
829
35
52
27
82
18
40
cesses
and history of genetic fragmentaBuncombe
1,609
15
35
49
15
82
21
49
Buncombe
1,247
16
35
48
45
82
21
26
tion (Good DA, unpublished manuscript;
Buncombe
17
866
35
47
60
82
21
37
Good and Wake 1992, 1993).
Buncombe
18
878
35
43
42
82
24
33
These deviations might take two general
Buncombe
1,463
19
35
41
60
82
24
12
forms. Deviations from linearity indicate
that opposing forces that promote differentiation and cohesion have not yet
distributions of D. ochrophaeus, D. orestes, carolinensis in a topographically and eco- achieved an equilibrium. A relationship
D. carolinensis, and D. ocoee replace each logically heterogeneous area; among pop- between genetic distance and geographic
other parapatrically from north to south,
ulations from throughout the ranges of D.
distance requires two things: sufficient
across about 10 degrees of latitude,
carolinensis, D. ochrophaeus, D. ocoee, and time and geographic isolation to produce
should profoundly affect the genetic struc- D. orestes; and among those forms and two genetic fragmentation, and sufficient time
tures of these forms. We encounter D.
more aquatic desmognathines: D. monti- and gene flow to generate higher levels of
ocoee in the foothills of the Georgia Blue
cola and D. fuscus.
similarity among geographically proximal
Ridge and D. ochrophaeus in the New York
demes than among more distant ones. If
Catskills in the same sorts of habitats oc- F Statistics
levels of gene flow are sufficiently low,
cupied by the complex throughout its
Fsr, or the "fixation index" (Wright 1965) then there will be no discemable relationrange, but patterns of dispersion and gene
measures the genetic effect of population
ship between genetic and geographic disflow In Georgia and New York may be very subdivision as the proportional reduction
tance. In an array of semi-isolated demes
different. We might also expect patterns of
in overall average heterozygosity owing to
whose genetic structures are initially idengenetic differentiation in the ochrophaeus variance in allele frequencies among sub- tical, the effects of gene flow will initially
complex to be very different from those in
populations. Values of F^ were calculated
be evident at the smallest geographic disother related desmognathines, such as D.
using BIOSYS-1 version 1.7 (Swofford and
tances, and the relationship between gemonticola and D. fuscus, that inhabit larger Selander 1981). These values were aver- netic and geographic distance will initially
streams that can provide dispersal corri- aged across populations of each species
be asymptotic. Over time, genetic differdors.
(as well as D. orestes groups B and C) and
entiation raises the asymptote while gene
across loci judged polymorphic by the
flow increases the geographic distance at
95% criterion (mean frequency of the most
which the asymptote Is reached, until the
Materials and Methods
common variant averaged across popula- relationship becomes linear over the entions of a group or species ^95%).
The Data
tire range of geographic distances.
Tilley and Mahoney (1996) reported on alThe second type of deviation from the
lozymic variation across 22 loci in 52 pop- Measures of Heterozygosity
null model concerns the y intercept of the
BIOSYS-1 was employed to calculate mean
ulations of the form previously regarded
relationship, that is, the genetic distance
as D. ochrophaeus, which they subdivided heterozygoslties expected at Hardy-Weln- expected between two demes separated
berg equilibrium, using Nei's (1978) unbi- by zero geographic distance. This interinto D. carolinensis, D. ochrophaeus, D.
ocoee, and D. orestes. Information on col- ased method. Mean heterozygosities were
cept is expected to be zero within a single
averaged across the populations of each
lection sites and laboratory procedures Is
array of populations that share a simple
provided by Tilley and Mahoney (1996). In species and of D. orestes groups B and C.
history of isolation by distance. However,
addition, my students and I have accuthe array of populations might include
mulated data on microgeographic varia- Relationships Between Genetic and
multiple groups descended from ancestion at a subset of these loci among D. car- Geographic Distance
tors that underwent an initial period of difolinensis populations in the vicinity of the
ferentiation, accompanied by differentiation
Good DA (unpublished manuscript) and
Black and Great Craggy Mountains of west- Good and Wake (1992, 1993) have shown
of their constituent demes in response to
ern North Carolina (collection localities in
isolation by distance. The y intercept for
that the relationships between Nei's
Table 1), and on the entire set of loci in
comparisons between populations of two
(1978) standardized genetic distance and
several populations of D. fuscus and D.
genetically differentiated subgroups of
geographic distance among populations
monticola. We can thus examine genetic
can provide insight into patterns of differ- populations should lie above the origin.
fragmentation among local demes of D.
entiation within and among species. The The intercept's value in this case repre-
Table 1. Collecting locallUe* In the Black and Great Craggy Mountain*
Tilley • Genetic Differentiation in Appalachian Desmognaihine Salamanders
307
sents the additional genetic differentiation
accomplished by factors other than isolation by distance. These might include a
history in which populations ancestral to
the subgroups differentiated in allopatry
or current nongeographic barriers to gene
exchange.
The statistical significance of regression
parameters in these analyses cannot be
tested by traditional methods because a
given population enters into several comparisons and the data points are thus not
statistically independent (Dietz 1983; Manly 1986). Nevertheless, the broad patterns
evident In scatterplots of genetic versus
geographic distance deserve our attention. Where regression lines are calculated
in some of the analyses below, they are
provided for visual purposes only.
Mlcrogeographic Variation Among
D. carollnensis Populations in the
Vicinity of the Black and Great
Craggy Mountains
The Black and Great Craggy Mountains diverge from and then roughly parallel the
Blue Ridge (Tennessee Valley) divide east
of Asheville, North Carolina (Figure la).
The two ranges collectively form a continuous ridgeline that undulates from northeast to southwest across about 15 minutes
of latitude between Micaville (Yancey
County, North Carolina) and Swannanoa
(Buncombe County, North Carolina). For
nearly 30 km of this distance the ridgecrest never drops below 1500 m above sea
level. The highest portion of the ridgeline,
referred to as the Black Mountains, rises
above 2000 m for a distance of 20 km and
includes Mt. Mitchell (2037 m), the highest
peak in North America east of the Black
Hills.
D. carolinensis occurs throughout this
topographically complex region, from the
lowest elevations (about 500 m) to the
summit of Mt. Mitchell (Tilley 1973). Our
sampling localities span essentially this
entire vertical range from locality 5 at 488
m at the foot of the Blue Ridge Divide
northwest of Marion (McDowell County,
North Carolina) to locality 7 on Clingman's
Peak, 1987 m, on the crest of the Black
Mountains (Table 1, Figure la). We also attempted to distribute our sampling among
the three major mountain ranges (Great
Craggy, Black, and Blue Ridge) and drainages (Ivy Creek, Cane River, South Toe River, and Catawba River) of the region. Localities 1, 2, 4, 5, and 8-13 lie along the
Blue Ridge Divide running from the point
where it joins the Black Mountains near
3 0 8 The Journal of Heredity 1997.88(4)
Pinnacle Mountain (locality 1) northeastward on opposite sides of the divide in the
drainages of the South Toe and Catawba
Rivers. Localities 3, 6, and 7 lie on the
main (eastern) ridgecrest of the J-shaped
Black Mountain range, localities 15-17 on
the lower western ridge, locality 19 on the
Great Craggy Mountains, locality 18 below
the crest of the Craggies In Dillingham
Creek drainage, and locality 14 at a low
elevation (829 m) along the Cane River
south of Burnsville (Yancey County, North
Carolina). Our two most distant sample
populations (localities 5 and 18) are separated geographically by 26 km.
D. carolinensis populations In this region
are strongly polymorphic for five of the 22
loci in our standard set: creatine Unase
(CK EC 2.7.3.2), glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8), lsocitrate
dehydrogenase-1 (IDH-1, EC 1.1.1.42),
L-lactate dehydrogenase-2 (LDH-2, EC
1.1.1.27 ), and leucyl-glycyl-glyclne peptidase (PEP, EC 3.4.-.-). All these loci show
pronounced variation In allozyme frequencies in this restricted geographic region
(Figure lb-f). F^ values average 0.219 for
the five loci (Table 2). The only regular
pattern occurs at the CK locus (Figures lb
and 2), which exhibits the highest F^ value (0.580) and where allozyme frequencies
vary strongly from west to east (Figure
lb) and with elevation (Figure 2). This pattern is most pronounced among sample
populations 1-7, between the crest of the
Black Mountains to the foot of the Blue
Ridge near Lake Tahoma. The Ckb variant
that characterizes the highest elevation
populations is otherwise a rare allozyme
in the units of the ochrophaeus complex
that we have studied (Table 3 in Tilley and
Mahoney 1996). It is known to occur In
only two other populations of D. ocoee: as
the predominant variant (P = .75) in the
Pink Beds population, Transylvania County, North Carolina, 1024 m; and as a rare
variant (P = .036) at Rough Butt Bald,
Jackson County, North Carolina, 1676 m.
Interestingly, the same allozyme (or one
with the same mobility) also distinguishes
D. santeetlah, a high-elevation form, from
low-elevation populations of D. fuscus (Tilley 1988).
None of the remaining loci exhibit any
sort of geographic pattern of variation
(Figure lc-l). The same allozymes occur
throughout the sampling regions at frequencies that vary discordantly among
the loci and that sometimes differ strongly
among sampling localities separated by
only a few kilometers.
Figure 3 shows how genetic distances
vary with respect to geographic and vertical distances for comparisons among
populations in the Black and Great Craggy
Mountains, across the five loci that are
strongly polymorphic in these populations. The dataset excludes populations 3,
9, 12, and 15, for which data are lacking
for one or more of the loci. These genetic
distances cannot be compared to those
presented below, which are based on the
entire set of 22 loci. Comparisons between
populations that are strongly differentiated at CK (represented by filled circles in
Figure 3) generally produce larger genetic
distances, which increase with elevational
distance as a consequence of the elevational cline at that locus (Figure 2). Populations 5 and 7 (separated by 15.1 km and
1500 vertical m) exhibit the greatest genetic distance (0.60) on the graphs. Geographic distance alone accounts for very
little of the variation in genetic distance.
Macrogeographlc Variation Within
and Among Forms of the D.
ochrophaeus Complex
Intraspeciflc Differentiation
The Fjy values for the four forms of the D.
ochrophaeus complex treated here are
shown in Table 2 and Figure 4. Levels of
differentiation among these forms lie well
within the range of FOT values given by Larson (1984, Table IV) and Larson et al.
(1984, Table 2), who also based their values on averages across loci judged polymorphic according to the 95% criterion. Of
the four species, D. carolinensis, D. ochrophaeus, and D. orestes exhibit similar F^
values in the middle of the distribution,
while a notably higher proportion of the
genetic variation in D. ocoee occurs at the
interdemic level. Larson et al. (1984) concluded from their analysis of fCT statistics
in species of plethodontid salamanders
that gene flow is probably too attenuated
to act as a "cohesive force" in these amphibians. The same can be said for these
members of the D. ochrophaeus complex,
in which levels of genetic differentiation
probably reflect historical patterns rather
than current levels of gene exchange.
D. ochrophaeus. Populations of this form
exhibit very low levels of average heterozygosity across the 22 loci (mean H =
0.05) and only six of these loci are polymorphic according to the 95% criterion
(Table 2). Genetic distances calculated
across all 22 loci indicate very low levels
of differentiation over distances of as
much as 1000 km (Figure 5a). However,
when only the six polymorphic loci are
BTT3CT
82°22-3Cr
82-2230"
82"15'
STTXT
. d) IDH-1
3
b)
. e) LDH-2
CK
cr
w-^it/Jg,
17
18
19
. C) G3PDH
-f)
Figure 1. Variation in allozyme frequencies at five highly polymorphic loci among populations of D carolinensis in the Black Mountains and vicinity. Buncombe. McDowell,
and Yancey Counties, North Carolina, (a) Sampling localities, numbered as in Table 1. (b)-{f) Variation in allozyme frequencies. Frequencies are proportional to shaded areas:
allelic variants are distinguished by different shading patterns In a few cases commonest and very rare variants have been pooled.
Tilley • QenetJC Differentiation in Appalachian Desmognathine Salamanders 3 0 9
Table 2. Levels of genetic variation and average
ocoee, and D. orestes
Species
D. carolinensis:
Throughout range
Black and Great
Craggy Mountains
No. of
populations
O
0.8
o
Mean no. of
polymorphic Mean
loci
heterozygoslty
02
9
13
0.131
0.469
19
9
16
14
4
10
6
16
16
10
15
0.055
0.097
0.139
0.121
0.146
0.420
0.644
0.437
0.170
0.352
Numbers of polymorphic loci are averaged across populations. Heterozygotles were calculated using Nel's (1978)
unbiased method and averaged across loci and populations. Fn values are averaged across populations for loci In
which the mean frequency of the most common variant was ^95%.
considered, the mean F^ value for this
form (0.420, Table 2) is similar to those for
D. carolinensis and D. orestes (0.469 and
0.437, respectively; Table 2 and Figure 4).
Genetic differentiation in these forms nevertheless involves more loci and has occurred over much more restricted geographic areas.
D. orestes. Patterns of differentiation in
this, the next form to the south (Figure
5b), are complicated by the fact that D.
orestes consists of two distinct groups of
populations (groups B and C, respectively,
of Tilley and Mahoney 1996). Group B is
apparently restricted to the Roan and Iron
Mountain highlands along the North Carolina-Tennessee state line, and lowland
populations occur in the vicinity of Johnson City, Tennessee. In that region it forms
a wedge between group C D. orestes to the
north and D. carolinensis to the south.
Group B constituted the most highly differentiated form studied by Tilley and Mahoney (1996), appearing as the most basal
group of populations on their neighborjoining and parsimony trees. Nevertheless,
Tilley and Mahoney (1996) presented evidence of intergradation between group B
and the remaining D. orestes populations
(their group C). Differentiation between
groups B and C contributes to the magnitude of the overall F^ for D. orestes populations (0.437), as indicated by the lower
values for groups B (0.170) and C (0.352;
Table 2).
Population comparisons within the
group C D. orestes (Figure 5b, filled circles) all yield genetic distances below 0.2,
with a very modest tendency for genetic
3 1 0 The Journal of Heredity 1997.88(4)
0.0
400
0580
0.170
0.152
0.079
0.114
0.219
distance to increase with geographic distance. Adding the four group B populations to the analysis steepens this relationship by superimposing a series of
points across which the relationship between geographic and genetic distance rises much more steeply, to genetic distances of about 0.4. Six of these additional
points near the origin of the graph represent comparisons among the group B populations; the remainder all represent comparisons between groups B and C. The
overall relationship between genetic and
geographic distance trends toward the origin of the graph in Figure 5b. Interdemic
comparisons within D. orestes as a whole
thus reflect, in striking contrast to the
much more wide-ranging D. ochrophaeus,
isolation by distance. There is, however,
an interesting substructure to this general
pattern. Much of the trend owes to a
strong tendency for group B and group C
D. orestes populations to become more genetically differentiated as they grow more
geographically distant. Group C D. orestes
populations are genetically quite homogeneous with only a weak tendency for
levels of differentiation to increase with
geographic distance.
D. carolinensis. The distribution of this
form extends only about 100 km from its
contact with D. orestes to the Newfound
Mountains southwest of Asheville, North
Carolina. Nevertheless, comparisons among
populations of this form yield a mean F^
of 0.469 and suggest Isolation by distance
(Figure 5c), with genetic distance rising
from 0 to about 0.3 as populations grow
more geographically separated.
O
o
O
0.6
0.4
CK
G3PDH
1DH-1
LDH-2
PEP
Mean
D. ochrophaeus
D. ocoee
D. orestes
Group B
Group C
1.0
values among D. carolinensis, D, ochrophaeas, D.
O
O
o
<a o
°o o
-K3
900
1400
KD
1900
Elevation (m)
Figure 2. Frequency of the Oallele (Figure lb) plotted against elevation among populations of D. carolinensis In the Black Mountains and vicinity.
D. ocoee. This form occupies the most
southerly and topographically complex region of any of the species treated here: the
southwestern Blue Ridge Physiographic
Province from the Great Smoky and Great
Balsam Mountains southward. D. ocoee
populations formed the loosest cluster In
the study by Tilley and Mahoney (1996),
with some pairs of populations in different
mountain ranges displaying genetic distances as great as those between different
species of Desmognathus across the same
set of 22 loci. This form displays the greatest fjT value (0.644) of the four species
(Table 2). Populations of D. ocoee show a
tendency to become more differentiated
over the first 100 km of geographic separation, but at higher geographic distances
genetic distance appears to level off at
about 0.3-0.4 (Figure 5d). All but two of
the points beyond geographic distances of
more than 150 km involve comparisons
with the Alabama populations, but suggestion of an asymptotic relationship persists
even with these points omitted.
Figure 5e summarizes the patterns suggested by the scatterplots in Figure 5a-d.
Levels of genetic fragmentation appear to
increase from north to south. Patterns
suggesting Isolation by distance characterize differentiation within each of the
southern Appalachian forms, in very distinct contrast to the situation in the most
northern form, D. ochrophaeus. Furthermore, in the northernmost member of the
southern Appalachian complex, group C
D. orestes, the isolation-by-distance pattern appears distinctly weaker than In the
two more southerly forms. In D. carolinensis and D. ocoee, and in comparisons involving the most southerly (form "B") D.
orestes populations, genetic distance rises
with geographic distance so that populations separated by 100 km typically differ
by a Nei distance of about 0.3. There is no
evidence, in any species of the complex,
of isolation by distance among populations separated by more than 100 km.
a)
0.7
0.6
0.5
t
0.4
0.3
o °» o o
0.2
rt.
0.1
o
8
10
to
Q
o
(D
CD
n O
20
30
Geographic distance (km)
b)
0.7
0.6
0.5 \
0.4
0.3 \
0.2
0.1
%%
o . .
o
&_
8
o
OCP6)'
500
1000
1500
Elevational distance (m)
Figure 3. Genetic distance plotted against (a) geographic distance and (b) elevatlonal distance for comparisons
among populations of D. carolmensis In the Black Mountains and vicinity. Genetic distances are calculated across
five loci polymorphic In these populations, and are not comparable to those shown in Figures 5-7, which are based
on 22 loci.
Differentiation Among Forms of the
D. ochrophaeus Complex
Figure 6 shows the relationships between
genetic and geographic distances for comparisons involving populations of different
species. Comparisons involving D. ochrophaeus populations and those of the three
other species (Figure 6a-c) yield genetic
distances of about 0.3, on average. Since
the most northern (New York) and southern (Kentucky) D. ochrophaeus popula-
tions are so genetically similar to one another (Figure 5a), there is no relationship
between genetic and geographic distance
for either of the three categories of Interspecies comparisons.
The remaining three relationships between genetic and geographic distance for
interspecies comparisons (Figure 6d-f)
are all strongly positive. For all the interspecific comparisons Involving D. orestes,
D. carolinensis, and D. ocoee, positive re-
lationships between genetic and geographic distance are evident even at geographic distances exceeding 100 km In
contrast to the pattern of intraspecific differentiation within D. ocoee (Figure 5d).
The relationship between genetic and
geographic distance for comparisons of D.
orestes to D. carolinensis (Figure 6d) is
particularly revealing. The sample includes populations of these two parapatric forms that are separated by only a few
kilometers, and even these geographically
adjacent populations differ by genetic distances of about 0.3. This is approximately
the same genetic distance that separates
D. ochrophaeus from the other three species. The relationship between genetic and
geographic distance for comparisons of D.
orestes to D. carolinensis thus does not
trend toward the origin of the graph. Its
upward displacement indicates that populations of these two forms are more genetically differentiated than we would predict from geographic distances alone, and
that isolation by distance does not entirely account for the pattern of genetic fragmentation.
In the remaining two classes of interspecific comparisons, D. orestes versus D.
ocoee (Figure 6e) and D. carolinensis versus D. ocoee (Figure 6f), it is less clear
whether isolation by distance alone falls
to account for the patterns of differentiation. D. orestes and D. ocoee are allopatric,
and the contact zone between D. carolinensis and D. ocoee is currently under
study. Thus we lack pairs of geographically adjacent populations for either of these
two interspecific comparisons. A further
complication is that the Alabama D. ocoee
populations are consistently less differentiated from either D. orestes or D. carolinensis than would be predicted from geographic distances. If comparisons involving the Alabama populations are excluded,
the least-squares regression lines fit to the
remaining comparisons (involving only
the D. ocoee populations in the southern
Blue Ridge Physiographic Province) intersect the genetic distance axes between 0.1
and 0.2. Since the points in the scatterplots are not statistically independent, we
cannot determine whether these y intercepts differ significantly from zero. It does
appear that D. orestes and D. carolinensis
are more genetically dissimilar than D. orestes and D. ocoee or D. carolinensis and
D. ocoee, when genetic distances are "corrected" for geographic distances among
populations.
Tiltey • Genetic Differentiation in Appalachian Desmognathine Salamanders 3 1 1
4 •
ft 3req
3
Itl
00.10
0.20
o
o
Q>
o
Q
O
•• •
^m
0.00
CO
IK?
!•
2 •
O O
cara //n
If
0.30
0.40
050
^^^^B
;
1IH|
• • I
• • • •
060
0.70
0.80
FST
Figure 4. Histogram showing distributions of F^ value* for plethodontld salamanders. Shaded squares represent
values given by Larson (1984, Table IV). Squares with filled circles indicate species Inhabiting middle and eastern
North America; empty circles Indicate the species studied here.
Comparisons With Other
Desmognathines
D. monticola and D. fuscus are abundant
species along low-elevation streams in the
Appalachians. Both species are more
aquatic than members of the D. ochrophaeus complex, seldom venturing more
than a few meters from flowing water. The
low-elevation regions that evidently act as
barriers to dispersal in the D. ochrophaeus
complex may actually function as dispersal corridors for D. monticola and D. fuscus.
It is thus of interest to compare patterns
of genetic variation in these forms to
those in the D. ochrophaeus complex.
We have accumulated allozyme data for
four populations of D. monticola (Cocke
County, Tennessee, n = 29; Polk County,
Tennessee, n = 28; Washington County,
Tennessee, n = 16; Macon County, North
Carolina, n = 20) and three populations of
D. fuscus (Grayson County, Virginia, n = 7;
Scioto County, Ohio, n = 16; Franklin
County, Massachusetts, n = 18). These
and the D. ochrophaeus complex data are
based on the same 22 loci. The relationships between genetic and geographic distance for intraspecific comparisons in our
samples of D. monticola and D. fuscus are
shown in Figure 7. When adjusted for geographic distances among populations, levels of intraspecific differentiation in D. htscus and D. monticola are quite low compared to those in D. carolinensis, D. ocoee,
or D. orestes, but not to those in D. ochrophaeus. Our four D. monticola populations are all from the southwestern Blue
Ridge Physiographic Province. These samples are nearly genetically identical to one
another, in contrast to samples of D. carolinensis, D. ocoee, or D. orestes in the
same geographic region and separated by
3 1 2 The Journal of Heredity 1997.88(4)
similar geographic distances (Figure 5be). The highest genetic distances for intraspecific comparisons in D. fuscus or D.
monticola correspond to the greatest geographic distances: at about 1000 km of
geographic separation, Massachusetts D.
fuscus differs from Ohio and Virginia D. fuscus by genetic distances of 0.14 and 0.25,
respectively. Karlln and Guttman (1986)
reported a very similar range of genetic
distances (0.0-0.2 versus our 0.09-0.22)
for comparisons among northern populations of D. fuscus over a similar latitudinal
range, employing a set of 21 loci that had
14 allozyme systems in common with
ours. Thus D. fuscus populations apparently require 1000 km of geographic separation to achieve the same degree of genetic differentiation (D « 0.2) that is typically exhibited by D. carolinensis, D.
ocoee, or D. orestes populations separated
by less than 100 km. In contrast, this level
of differentiation is approximately twice
the average genetic distance between D.
ochrophaeus populations separated by the
same geographic distance (Figure 5a).
History, Biogeography, and
Genetic Fragmentation
Patterns of genetic differentiation within
and among Appalachian units of the D.
ochrophaeus complex are quite intricate. It
may be premature to attempt to develop
a historical hypothesis that attempts to
account for all of them, but we can identify patterns that such a hypothesis must
eventually explain:
1. Evidence for genetic differentiation
over very short distances among populations of D. carolinensis.
2. The lack of differentiation among pop-
ulations of the most widely distributed
form, D. ochrophaeus.
3. A general tendency for levels of genetic differentiation to increase from
north to south within the D. ochrophaeus
complex.
4. The lack of evidence for a relationship
between genetic and geographic distance
among D. ocoee populations separated by
more than 100 km.
5. The positive relationships between
genetic and geographic distance for interspecific comparisons Involving D. orestes,
D. carolinensis, and D. ocoee, even for populations separated by more than 100 km.
6. Lower degrees of differentiation,
when corrected for geographic distances
among populations, for D. ocoee versus D.
orestes and D. ocoee versus D. carolinensis
than for any of the other interspecific
comparisons.
7. Higher degrees of Intraspecific differentiation, when corrected for geographic
distances, in D. carolinensis, D. ocoee, and
D. orestes than in D. monticola or D. fuscus.
Taken as a whole, the data for D. carolinensis populations in the Black and Great
Craggy Mountains indicate that within this
restricted, topographically diverse region,
these populations exhibit considerable local differentiation in allozyme frequencies.
The tendency for allozyme frequencies to
vary with elevation at CK suggests that selection may account for some of this differentiation, as Tilley (1973) and Bernardo
(1993) suggested that it did for elevational
variation in life-history characteristics of
D. carolinensis and D. ocoee populations.
It must also be true that levels of gene
flow among these populations are extremely low. Any model of genetic differentiation within this complex must accommodate the contrast between D. carolinensis demes that exhibit differentiation within a single high-elevation region that
contains no evident barriers to dispersal
and D. ochrophaeus, which exhibits exceedingly low levels of differentiation
among local populations as distant as
1000 km from one another. D. ochrophaeus
has clearly expanded its range northward
since the last (Wisconsin) episode of continental glaciation. Its northernmost populations lie about 500 km north of the
southernmost extent of Wisconsin glaciation, which was attained about 21,000 BP
(Peltier 1994). Salamanders of the genus
Plethodon that have invaded formerly glaciated territory also exhibit high levels of
genetic uniformity (Highton et al. 1989;
Highton and Webster 1976; Larson 1984;
ochrophaeus
carolinensis
CO O
0
200
400
600
800
1000
orestes
0
_i\
100
ocoee
•within "form C"
200
300
• comparisons involving
Alabama populatons
o
100
200
300
0
100
200
300
O
"55
e)
0)
o
ocoee
0.3-
0.1ochrophaeus
100
Geographic distance (km)
Figure 5. Relationships between geographic and genetic distances for comparisons among populations of (a) D.
ochrophaeus, (b) D. orestes, (c) D. carolinensis, and (d) D. ocoee. (e) Curves, fit by eye, summarizing the trends In
(aM<0-
Larson et al. 1984). They and members of
the D. ochrophaeus complex confront us
with a biogeographic paradox: their genetic fragmentation in the south, particularly
the southern Appalachians, requires very
low levels of gene exchange, yet they have
colonized vast areas of formerly glaciated
terrain since the retreat of Wisconsin ice
sheets.
The curves in Figure 5e resemble the series that would represent the relationships
between genetic and geographic distance
in populations that have either been undergoing isolation by distance for increasing lengths of time or have been differentiating for the same period but under different levels of gene flow. It may be impossible to disentangle these two
mechanisms In this complex, because
both levels of gene flow and time available
for differentiation probably vary with latitude in this complex of species. Group C
D. orestes may represent a complex of populations that has been differentiating longer than D. ochrophaeus, or which are
more isolated from one another in the topographically complex southwestern Blue
Ridge Physiographic Province, or both.
The still greater tendency for genetic distance to accumulate with geographic distance in D. carolinensis and D. ocoee
would be expected in these still more
southerly complexes of populations. The
roughly asymptotic relationship between
geographic and genetic distance for comparisons among D. ocoee populations is to
be expected in an array of populations
that has not had time to achieve the equilibrial linear relationship predicted under
simple isolation by distance. Viewing the
D. ocoee pattern as a stage in progression
toward an equilibrium may be misleading.
There can presently be no gene exchange
at all between the Alabama populations
and the remainder of D. ocoee. Even
among more continuously distributed
populations, at geographic distances beyond 100 km, the time required for the
equilibria! relationship to be attained may
exceed the time scales of major climatic
and biogeographic shifts.
Turning to the interspecific comparisons, the model proposed by Good DA
(unpublished manuscript) accounts for
the positive relationships between genetic
and geographic distance seen In Figure
6d-f as manifestations of past gene exchange between these forms, and the upward displacements of the relationships
as the consequences of additional differentiation during subsequent periods of genetic isolation. If these positive, nona-
Tffley • Genetic Differentiation in Appalachian Desmognathine Salamanders 3 1 3
i
l0.3
3 0.2
CD
S
• Desmognathuifuscia
O Desmognathus monttcota
01
o.o
200
400
600
800
1000
Geographic distance (km)
Figure 7. Relationships between geographic and genetic distances In D. monticola and D fuscus.
a)
ochrophaeus vs. orestes
r\\ orestes vs. carolinensis
1.0 i
0.8
0.6
0.4
0.2
0.0
500
8
CO
w
b)
1.00.8
06
0.4
02
0.0
0
c)
1.0 T
08
0.6
0.4
0.2
00
0
1000
1500
0
ochrophaeus vs. carolinensis ^\
e)
500
1000
1500
ochrophaeus vs. ocoee
0
r\
200
600
400
orestes vs. ocoee
I comparisons involving
Alabama populations
200
400
600
carolinensis vs. ocoee
S"i*a
V * i
• comparisons involving
Alabama populations
CDo
500
1000
1500
0
200
400
600
Geographic distance (km)
Figure 6. Relationships between geographic and genetic distances lor between-specles population comparisons,
between (a) D. ochrophaeus and D orestes, (b) D carolwensis, and (c) D ocoee; between (d) D. orestes and D.
carolinensis and (e) D. ocoee; and between (f) D. carolinensis and D ocoee. Least-squares regression lines are
shown for visual comparisons only. Regression calculations for (e) and (0 exclude the Alabama populations.
Shading patterns as In Figure 5.
D. orestes and D. carolinensis are less differentiated from the Alabama populations
of D. ocoee than from less distant populations of that form. Tilley and Mahoney
(1996) suggested that the geographic distributions of these species may have been
quite different in the past. Any historical
blogeographic scenario that explains the
current patterns of genetic differentiation
would be at odds with the evolutionary relationships suggested by parsimony analyses of the allozyme data. The shortest
tree obtained by Tilley and Mahoney
(1996), rooted with respect to D. imitator,
rendered D. orestes paraphyletic, with
group B populations basal and group C
populations terminal. D. carolinensis
(group D in Tilley and Mahoney 1996) was
likewise paraphyletic, with respect to
group C D. orestes, on this tree. A tree that
portrayed D. carolinensis as monophyletic
was only two steps longer. The phylogenetic relationships suggested by this tree
are shown in Figure 8. Of the relationships
suggested by this phylogeny, only the relatively basal position of D. ochrophaeus
agrees with the patterns shown in Figure
6, which portray that form as about equally differentiated from D. carolinensis, D. orestes, and D. ocoee. It seems prudent to
refrain from further speculation on the
evolutionary and biogeographic history of
this complex until additional data, such as
nucleotide sequences and information on
imitator
Group B orestes
ochrophaeus
ocoee
symptotic relationships were established
at some time in the past when the three
species were genetically contiguous, levels of gene flow at that time were evidently
higher than they presently are among populations of D. ocoee, which show no evidence of isolation by distance among populations separated by more than 100 km.
3 1 4 The Journal of Heredity 1997:88(4)
Levels of differentiation among the
three species in the southern Blue Ridge
Physiographic Province do not agree with
the current geographic relationships
among these forms. D. orestes is clearly
more differentiated from D. carolinensis,
its neighbor to the south, than from the
more distant D. ocoee. Furthermore, both
carolinensis
Group C orestes
Figure 8. Phyolgenetlc relationships suggested by
cladlstlc analysis of allozyme data, rooted with D. imitator designated as the outgroup and constrained to
portray all of the population groups recognized by Tilley and Mahoney (1996) as monophyletic-
morphological characters, are brought to
bear on the issue of phylogenetic relationships.
Patterns of intraspecific genetic differentiation in the southern Blue Ridge desmognathines seem consistent with the
ecological attributes of these species. The
genetically homogeneous D. monticola is
an abundant, stream-bank dwelling, lowland form for which there are probably
few barriers to dispersal. The genetically
much more heterogeneous D. carolinensis,
D. ocoee, and D. orestes are montane forms
whose lives seem centered on the headwaters of first-order streams. These habitat requirements may very well reflect
competition with and predation by D.
monticola and D. quadramaculatus, another low-elevation, streambank form (Hairston 1986, 1987).
Interspecific interactions may, in fact,
have a long history of involvement in genetic differentiation in the D. ochrophaeus
complex. If the forms of this complex are
simply adapted to cooler, moister, environments, their present geographic fragmentation could be interpreted as a postPleistocene event. Highton (1995) proposed that speciation events in the P. glutinosus complex predate the Pleistocene,
attributing them to vicariance events during Pliocene episodes of glaciation and
aridity. His criterion for species recognition was a Net genetic distance of at least
0.15, which is considerably less than the
genetic distances we have obtained
among units of the D. ochrophaeus complex. Furthermore, our set of loci excludes
"fast-evolving" transferrin, albumin, and
esterase loci that Highton employed. Had
we employed these loci, we probably
would have obtained even greater genetic
distances. Unless rates of molecular evolution are unusually high in these salamanders, levels of differentiation within and
among D. carolinensis, D. ocoee, and D. orestes appear far too high to have accumulated only since the last episode of continental glaciation. The beginnings of genetic fragmentation in the D. ochrophaeus
complex appear to be at least as ancient
as the fragmentation of the P. glutinosus
complex, and I suggest that they may have
different origins. Plethodon are completely
terrestrial animals that require relatively
mesic conditions for reproduction and foraging. Highton's (1995) proposal that arid
climates have imposed geographic isolation among populations in cool, moist refugia seems perfectly reasonable. Mem-
bers of the D. ochrophaeus complex, however, require stream headwaters and seepage areas for opposition, brooding, and
larval development. Their dispersal
across lowland areas may be constrained
by biotic interactions with the larger, more
aquatic species of desmognathines that inhabit higher-order streams. These interactions have apparently generated associations between small body size and terrestriality (Halrston 1986, 1987; Tilley
1968; Tilley and Bernardo 1993) in
desmognathines. Tendencies toward genetic fragmentation may be an additional
consequence of adaptation to terrestrial
environments in these salamanders. D.
monticola is the most abundant, ubiquitous occupant of low-elevation streams in
the southern Appalachians. It may be significant that the genetic distances between D. monticola and members of the D.
ochrophaeus complex average about 0.41,
while those among units of the complex
average 0.33 (Tilley and Mahoney 1996,
Table 7). These genetic distances suggest
that diversification of the D. ochrophaeus
complex followed, perhaps not too distantly, the appearance of a large-bodied,
stream-dwelling desmognathine that came
to be exceedingly abundant in low-elevation habitats in the southern Appalachians. The occurrence of large predatory
salamanders in low elevation habitats may
have been the main barrier to gene exchange among populations of the D. ochrophaeus complex, while ice caps and
spruce-fir forests have expanded and contracted, for as long as these species have
coexisted In the southern Appalachians.
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