2003
SOUTHEASTERN
NATURALIST
2(2):159-178
A NEW SPECIES OF EURYCEA
(CAUDATA: PLETHODONTIDAE)
FROM NORTH AND SOUTH CAROLINA
JULIANR. HARRISON,111'* AND SHELDONI. GUTTMAN2
ABSTRACT - We describe a new species of hemidactyliine salamanderin the
genus Eurycea based on a study of variation in morphometric, osteologic,
reproductive, and larval characteristics and allozyme-based allele frequencies
and fixed differences in samples of two color pattern morphs known to exist in
the E. quadridigitata complex in North and South Carolina. The new species
differs from its presumptive relative Eurycea quadridigitata (Holbrook) in
having a bright yellow venter, a smaller size, relatively longer limbs, fewer
paravomerine teeth, a greater number of yolk-laden ovarian eggs, and dorsally
spotted hatchling larvae. Osteologic differences include a frontal with a welldeveloped triangular postero-lateral process, a prevomer that lacks a posteromedial bony shelf, a prevomerine tooth series that is not sharply arched, a
tetraradiateos triangulare, and fewer trunk vertebrae. The new species exhibits
between four and seven fixed allozyme differences from E. quadridigitata. The
two species are largely allopatric in distribution in the Carolinas.
INTRODUCTION
Our study of geographical variation in Eurycea quadridigitata
(Holbrook) within North and South Carolina has shown that two
color pattern morphs known to exist in this area actually correspond
to two separate, largely allopatric species. The new species differs
from E. quadridigitata in both morphological and genetic characteristics. In this paper we first discuss the literature concerning color
pattern morphs and then describe as a new species certain populations occupying the Piedmont and upper Coastal Plain of South Carolina, together with others that occupy the Piedmont and portions of
the Coastal Plain in North Carolina.
Bishop (1943) first noted the existence of two color pattern morphs
in Eurycea quadridigitata (Holbrook). One of the most striking differences between these two morphs is a completely yellow venter lacking
dark markings in what Bishop referred to as the "light form" as contrasted with a uniformly pigmented venter with small dark brown flecks
in his "dark form." The dark form or morph also possesses dark brown
sides with narrow, longitudinal or oblique yellow or white lines as
opposed to simply brownish upper sides in the light form or yellow
Department of Biology, The College of Charleston, Charleston, SC, 29424. 2
Department of Zoology, Miami University, Oxford Ohio 45058. * Corresponding author - [email protected].
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morph. Bishop provided additional details for both morphs, but did not
comment on the geographic distribution of either form. It is likely that
Bishop (1943) based the description of his "light form" on specimens
sent to him by C.S. Brimley from the Raleigh area of the lower Piedmont in North Carolina. Brimley's (1944) characterizationof Eurycea
quadridigitata fits Bishop's "light form" only, and his experience with
this species was limited primarily to the Raleigh area. Dunn's (1926)
description of Eurycea quadridigitata is a composite of both color
pattern morphs. The male described, collected in Florida, is a "dark
form" specimen, whereas the female, collected at Raleigh by Brimley, is
a "light form" specimen.
Although Mittleman (1947) studied geographic variation in Eurycea
quadridigitata throughout its then-known range, his study was based
largely on preserved material. He stated that he took notes on color
pattern and pointed out that "... various phases are found with equal
frequency in all parts of the range ..." and that he was unable "... to
distinguish specimens from one locality or another on the basis of color
and/or pattern ..." However, Mittleman did not characterize any of the
color patternshe investigated. Only 15 of the 246 specimens Mittleman
examined were collected in North or South Carolina, and 11 of the 15
were from Wake Co., NC, an area now known to harbor only populations of the yellow morph. Mittleman (1947) described two new subspecies of Eurycea quadridigitata (Holbrook) based primarily on quantitative differences involving number of prevomerine teeth, number of
costal grooves, and number of costal grooves between adpressed toes.
Later, and without comment, Mittleman (1967) relegated each of the
subspecies he described to the synonymy of Manculus quadridigitatus
(Holbrook) and regarded this species as monotypic. These allocations
were presumably based in part upon the studies of Cagle (1952) and
Neill (1949, 1954) that provided no support for subspecific differentiation within the species.
There is no furthermention of color or color patternsin the literature
concerning Eurycea quadridigitata until Folkerts (1971) pointed out the
existence of three populations of this species from Anderson and
Pickens counties in the upper Piedmont of South Carolina. All of the
specimens he examined from these three populations were referable to
Bishop's "light form." Folkerts characterizedthe Piedmont animals and
compared them with 76 "darkform" specimens from the Coastal Plain
of South Carolina. In addition to the color pattern differences, he
pointed out that the Piedmont specimens were smaller on average, but
stated there were no differences in costal groove or prevomerine tooth
counts from those given by Mittleman (1967).
Holbrook's (1842:331) description and plate of Salamandra
quadridigitata are clearly referable to Bishop's (1943) "dark form."
2003
Julian R. Harrison, III and S.I. Guttman
161
The plate (21) shows a pigmented venter and a dark-sided tail, both
characteristics of the dark morph. In his description the venter ("abdomen") is said to be bluish silvery-white, a striking feature of living
dark morph individuals. Also, the tail is described as "... compressed
only towards its tip," a characteristic of the dark morph. In yellow
morph specimens the tail is round with little or no indication of lateral
compression or a dorsal keel. Holbrook, however, provided no information concerning number of costal grooves or number of
prevomerine teeth. The provenance of Holbrook's type (ANSP 490
from South Carolina) is unknown. The type description was based on
specimens from Georgia and Florida, as well as South Carolina. Although the type is in rather poor shape (Mittleman 1967), two x-ray
photographs have revealed the number of trunk vertebrae in this specimen to be 19 (Richard Highton, pers. comm.). As discussed later in
this paper, most specimens of the yellow morph examined in the
present study had 17 trunk vertebrae, whereas most specimens of the
dark morph had 19 trunk vertebrae; these are estimates based on the
number of costal grooves (Highton 1957) and direct counts from the
study of stained and cleared material. Schmidt (1953) restricted the
type locality of E. quadridigitata to the vicinity of Charleston, SC, an
area known to harbor only the dark morph. Cope's (1871) description
of Manculus remifer also fits rather well Bishop's "darkform," as he
stated that "... color is black above, and dark brown below." On the
basis of external morphology, color pattern, and number of trunk vertebrae, we conclude that Holbrook's (1842) description of Salamandra
quadridigitata is clearly referable to the dark morph, and that the
yellow morph is an undescribed species.
One of us (JRH) first became interested in the status of Bishop's
two color pattern morphs during a study (Harrison 1973) of the life
history of Eurycea quadridigitata in the lower Coastal Plain of South
Carolina. All of the more than 600 specimens examined at that time
were referable to Bishop's "dark form." Later, in 1974, Harrison collected three specimens from a locality in the Sandhills region of the
upper Coastal Plain in South Carolina that proved referable to
Bishop's "light form" and distinctly different from other Coastal Plain
material. A study of this material and additional specimens from the
Clemson area in the upper Piedmont of South Carolina, the Coastal
Plain of South Carolina, and the Piedmont and Coastal Plain of North
Carolina has shown that Bishop's two color pattern forms in this
region are separate, largely allopatric species. In addition, Guttman
examined protein variation among samples from Pickens and Barnwell
counties, SC, Scotland Co., North Carolina, Osceola Parish, Louisiana,
and Beauregard Parish, Louisiana and found fixed genetic differences
as well. The new species of Eurycea is described below.
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SoutheasternNaturalist
METHODS
Measurements of adults were made with Vernier calipers to the
nearest 1.0 mm, larvae to the nearest 0.5 mm with a metric rule and a
dissecting microscope. Abbreviations used are SVL (snout to anterior
end of vent), T (tail length from anterior end of vent to tip of tail), HL
(tip of snout to posterior edge of gular fold). HW (head width), ICF
(number of intercostal folds between adpressed limbs), VT (number of
prevomerine teeth counted with a dissecting microscope after drying
roof of mouth with compressed air), HL/SVL (relative head length),
HW/SVL (relative head width), and T/SVL (relative tail length). Specimens for osteological studies were fixed in formalin, measured, sex
determined, and cleared in potassium hydroxide, stained with Alizarin
Red-S, and stored in glycerine. Ovarian egg counts in gravid females
included all obviously enlarged, yolk-laden eggs.
Specimens for genetic studies were available from the Clemson
area, Pickens Co., SC (yellow morph) Barnwell Co, SC (dark morph),
Scotland County, NC (dark morph), Osceola Parish, LA (dark morph)
and Beauregard Parish, LA (yellow morph); these were sent to
Guttman at Miami University for electrophoretic analysis of protein
variation. An organ homogenate was prepared from the kidneys,
heart, liver, tongue and rinsed digestive tract of each animal. The
tissues were homogenized together in an equal volume of chilled 2%
2-phenoxyethanol and centrifuged at 25,000 g at 4 'C for 45 min to
obtain an aqueous protein extract. The supernatant of water-soluble
proteins was decanted and frozen at -70 TCuntil used, a maximum of
72 h following preparation. Extracts were analyzed by standard techniques of horizontal starch gel electrophoresis (Selander et al. 1971,
Ayala et al. 1972). Seventeen loci were consistently resolvable (see
Table 1. Enzymes and buffer systems used in this study.
Enzyme system
Number
Buffer a
Aspartate aminotransferase(AAT)
Albumin (ALB)
Fumaratehydratase (FH)
Glucose-6-phosphate isomerase (GPI)
Glutamate dehydrogenase (GLUDH)
Isocitrate dehydrogenase (IDHP)
Lactate dehydrogenase (LDH)
Malate dehydrogenase (MDH)
Malic enzyme (MEP)
Peptidase (1-alanyl-1-leucyl; PEP)
Phosphogluconate dehydrogenase (PGDH)
Phosphoglucomutase (PGM)
Protein-2 (PT)
Superoxide dismutase (SOD)
2. 6. 1. 1
LiOH
LiOH
TC8
LiOH
TEB
TC8
TEB
TC8
TC8
LiOH
TC8
TC8
LiOH
TC8
4. 2. 1. 2
5. 3. 1. 9
1. 4. 1. 1. 1. 1. 42
1. 1. 1. 27
1. 1. 1. 37
1. 1. 1. 40
3. 4. -. 1. 1. 1. 44
5. 4. 2. 2
-.-.-.-
1. 15. 1. 1
a LiOH, Lithium hydroxide, pH 8.1 and TC8, Tris-citrate, pH 8.0 (Selander, et al. 1971; TEB, Tris-
borate-EDTA, pH 9.1 (Ayala, et al. 1972).
2003
Julian R. Harrison, III and S.I. Guttman
163
Table 1 for a list of enzyme loci, abbreviations and numbers following Shaklee et al. 1990). All gels were 12.5% starch (Electrostarch
lots #301 [LiOH] and #307 [Tris-citrate and Tris-borate-EDTA], Otto
Hiller, Madison, Wisconsin). Analysis of single individual genotypic
data was performed using the Biosys I computer program developed
by Swofford and Selander (1981).
SYSTEMATICS
EURYCEA CHAMBERLAINI SP. NOV.
CHAMBERLAIN'S DWARF SALAMANDER
Holotype. - U.S. National Museum of Natural History (USNM)
547846, an adult female collected from Sesquicentennial State Park,
Richland County, South Carolina, on 3 August 1974, by Julian R.
Harrison (JRH 2700) and Albert E. Sanders.
Paratypes. - Fifteen specimens from South Carolina as follows:
Charleston Museum (ChM) CA4566-69 (4) [JRH 3286-89], Barnwell
Co., SavannahRiver Site, Rainbow Bay; ChM CA4570 (1) [JRH 3177],
Pickens Co., near Clemson; USNM 547847-48 (2) [JRH 2701-02],
Richland Co., Sesquicentennial State Park (topotypes, one stained and
cleared); NCSM 36021 (8), Sumter Co., N edge of Sumter. Thirty-five
specimens from North Carolina in the collection of the North Carolina
State Museum (NCSM) as follows: NCSM 14947 (5) and 17103 (3),
ChathamCo., 1.5 mi SSW Wilsonville; NCSM 36024 (1), CravenCo., ca.
3 mi E Tuscarora;NCSM 15788 (1), Johnston Co., 2 mi NE Four Oaks;
NCSM 19159 (1), Lenoir Co., 2 mi NW Deep Run; NCSM 17470 (2),
Moore Co., 8.25 mi SW Robbins; NCSM 36023 (2), Pamlico Co., 7 mi
WSW Oriental; NCSM 17556 (2), Sampson Co., 4.8 mi NNE Delway;
NCSM 14805 (3), Vance Co., 4 mi E Henderson; ChM CA4571-77 (7)
[JRH 3178-84], NCSM 17335 (1), 17579 (4), and 19783 (1), Wake Co.,
4.5 mi S Cary;NCSM 36022 (1), Wake Co., 2 mi E Holly Springs.
Description of Holotype. - After preservation, total length 81
mm, tip of snout to anterior corner of vent 27 mm, to posterior corner
of vent 29 mm; tail length from anterior corner of vent 54 mm, body
width and depth (at mid-trunk) 4.5 mm and 2.9 mm respectively,
axilla to groin length 17.2 mm, tail height and width just posterior to
vent both 2.9 mm; head length from tip of snout to gular fold 6.5 mm,
head width at widest portion 3.7 mm, head depth at angle of jaw 2.4
mm; eye diameter 1.8 mm, eye width 1.5 mm, interocular width
(shortest distance between eyes) 1.2 mm; internarial distance 1.0 mm,
tip of snout to orbit 1.2 mm. Number of costal grooves 16-16, number
of intercostal folds between adpressed limbs 4, number of costal
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grooves between adpressed limbs 5. Digits of the forelimbs 1-4-3-2
in order of length from the shortest (first digit); digits of the
hindlimbs 1-2-4-3. Number of prevomerine teeth 9-7, these widely
separated from those of the parasphenoid series.
Dorsum of head, trunk, and tail in life brownish-bronze; in preservative dorsal surfaces light brown, a consequence of abundant tiny
punctate melanophores. Dorsum of trunk between dorsolateral stripes
with a medial series of 15 dark spots about equally distributed from
gular fold to a point just posterior to hindlimbs. Dorsum of head with a
short dark occipital bar about as wide as the eyelids and a small dark
dot behind the bar. Eyelids more heavily pigmented than remainder of
head, except for a darker spot at the posteromedial corner of each eye
and a medial darker spot just posterior to the eyes. Dorsum of forelimbs light brown with scattered pigment-free areas. Dorsum of femur
with larger, mostly melanophore-free areas forming a fairly conspicuous horizontal band.
A dark dorsolateral stripe, approximately 1.0 mm in width extends
from the posterior corner of the eye to the tip of the tail. This merges
fairly abruptly on the trunk with moderately pigmented sides with
scattered pigment-free areas, which in turn are sharply demarcatedfrom
the venter by an abruptborderextending from the base of the forelimb to
the base of the hindlimb. Sides of tail moderately heavily pigmented
with relatively few pigment-free areas.
Venter of trunk in life bright yellow, venter of tail dusky-yellow; in
preservative venter of trunk and tail pale buff with yellowish tinge,
without dark pigment except scattered, very tiny punctate melanophores. Punctate melanophores somewhat more abundanton tail.
Etymology. - The name honors Edward Burnham Chamberlain,
former Curatorof VertebrateZoology, the Charleston Museum, in special recognition of his contributions to the study of amphibians and
reptiles in South Carolina.
Diagnosis. - A hemidactyliine plethodontid, probablyclosely related
to Eurycea quadridigitata, and sharing with that species the possession
of four toes on both fore and hind limbs, but differing in size, color
pattern, prevomerine and costal groove count. Eurycea chamberlaini is
smaller in size than E. quadridigitata, has a color pattern somewhat
resembling that of E. cirrigera, has relatively longer limbs than E.
quadridigitata, fewer costal grooves, and fewer prevomerine teeth. It
differs also from the nominal species in the configuration of three
skeletal elements, the prevomer, frontal and basibranchial II (os
triangulare) of the hyobranchial apparatus, in the arrangement of
prevomerine teeth, and by having fewer trunk vertebrae.
2003
Julian R. Harrison, III and S.I. Guttman
165
The new species is characterized in life by a brownish-bronze dorsum with distinct dorsolateral stripes and a bright yellow venter; snoutvent lengths of adults (SVL; measured to anteriorcorner of vent) 22-30
mm (g = 25.8 mm); modal numbers of 16 costal grooves and 17 trunk
vertebrae; relatively long limbs, 3-6 costal folds between adpressed
limbs (g = 4.2); a relatively long tail, tail to snout-vent length ratio
1.08-2.00 (g = 1.62); prevomerine teeth 5-16 (g = 10.0).
The prevomer of Eurycea quadridigitata has a posterolateral bony
shelf and the prevomerine tooth series is sharply arched (inverted "L"
shape), an arrangement that is characteristic of hemidactyliine salamanders (Wake 1966). However, the prevomer of E. chamberlaini lacks
the posterolateral bony shelf and the prevomerine tooth series is not
sharply arched, an arrangement("plethodonine pattern")similar to that
in salamandersof the tribe Plethodontini (Wake 1966) The frontal bone
typically bears a short triangular process at its posterolateral corner.
Basibranchial II of the hyobranchial apparatusis ossified and is typically tetraradiate,i.e., 4-pointed.
The distinctive brownish-bronze dorsum and bright yellow venter
is lost in preservative, but such specimens in the Carolinas can be
identified through a combination of characteristics including especially the plethodonine prevomerine tooth pattern, narrow dark dorsolateral stripes, and an unpigmented venter, including the venter of
the head and tail. Outside the Carolinas, number of costal grooves
will not serve as a diagnostic characteristic, as some populations of
E. quadridigitata are characterized by 16 costal grooves. The venter
of the trunk may only be lightly pigmented in some E. quadridigitata,
but the venter of the head and tail are usually moderately to heavily
pigmented. Examination of the dorsal oral cavity, particularly if airdried, will reveal the plethodonine arrangement of the vomerine tooth
series, as this appears to be a fairly consistent characteristic.
Color Pattern. - Notes on color pattern in life are available for
twelve specimens (10 females, 2 males) collected in Pickens County, SC
(Fig. 1). These specimens displayed a color patternvery close to that of
the "light form" described by Bishop (1943). No sexual dimorphism in
this character was readily apparent, although only two males were
represented. The dorsum of the head and trunk was bronzy-buff or
brown in nine specimens and brownish-buff or buff in three others. The
pattern of the darker markings on the dorsum was rather variable,
ranging from none to variable patterns including faint herring bone-like
arrangements,few or numerous flecks, and a mid-dorsal line or irregular
row of dots. A deep layer of guanophores impartedthe bronzy aspect to
the dorsum; these were not evident laterally. The dorsum of the tail was
yellowish-buff or a light bronzy-buff with an orange tinge in eight
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specimens and brownish-buff, orange-buff, pale buff, or light bronzybrown in four others; darker markings present ranged from few to
numerous tiny dark flecks.
The venter of the head and limbs was flesh-colored with a suffusion
of yellow on the posterior portion of the head in some specimens. The
venter of the trunk was bright yellow in eleven specimens and yellowish-buff in one. The venter of the tail was orange-yellow or orange in
eleven specimens and yellow in one.
Seven specimens had a distinct or relatively distinct dorsolateral
dark stripe while in four the dorsolateral and lateral areas were uniformly dark in color, sharply demarcatedfrom the yellow of the venter.
In one specimen the dorsolateral stripe was replaced by a row of dots.
Laterally and ventrolaterally, eight specimens had scattered to moderately numerous white flecks or streaks but four others lacked these.
Variation. - Eurycea chamberlaini is, on average, slightly smaller in
snout-vent length than E. quadridigitata (Table 2). Forty-seven measured adults had a mean snout-vent length of 25.8 mm in comparison to
28.0 mm for 48 adults of E. quadridigitata. These results are comparable to those obtained by Folkerts (1971) for a small sample of 11
specimens of the yellow morph from Pickens and Anderson counties,
SC, and 76 specimens of the dark morphfrom the Coastal Plain of South
Carolina. The new species is also relatively longer-limbed having a
mean of 4.2 intercostal folds between adpressed limbs as opposed to a
Figure 1. Male specimen of Eurycea chamberlaini from Pickens Co., SC,
illustrating characteristic bright yellow venter.
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Julian R. Harrison, III and S.I. Guttman
167
mean of 5.5 in E. quadridigitata (Table 2). It also averages slightly
fewer prevomerine teeth, having a mean of 10.0 in comparison to a
mean of 11.8 in E. quadridigitata (Table 2). The means for each of these
three characters(snout-vent length, relative limb length, and number of
prevomerine teeth) were significantly different (Student's T) at P =
<0.01. Little sexual dimorphismin these characteristicswas noted in the
samples of either species. Males averaged slightly larger in relative head
length, head width and tail length, whereas females averaged slightly
more prevomerine teeth and had a slightly greater average relative limb
length, but these differences were non-significant.
The two species differ appreciably in number of costal grooves.
These were counted using the "maximum count," i.e., including a
groove in both the axilla and the groin but not a groove lying immediately above the hind limb (Highton 1957). Eighty-five percent of the 48
E. quadridigitata examined had 18-18 costal grooves, and 15% had 1717 or 18-17 (one specimen). By contrast, 83% of the 47 E. chamberlaini
examined had 16-16 costal grooves and 17% had 15-15 or 15-16 (one
specimen). In contrast, Folkerts (1971) found no differences in costal
groove count between specimens of the yellow morph from the South
Carolina Piedmont and the values given by Mittleman (1967) for E.
quadridigitata. However, this may simply reflect the fact that
Mittleman's data were taken from specimens collected over a broad
geographic area from North Carolina to Mississippi, and very few of
these were from the Carolinas.
Ten specimens, six Eurycea quadridigitata and four E. chamberlaini
from the Rainbow Bay complex in the Savannah River Site, Barnwell
Co., SC, illustrate differences between the two species in one of the two
known areas of sympatry. All of the E. chamberlaini have unpigmented
venters, pale buff in preservative, whereas four of the E. quadridigitata
have venters lightly to moderately pigmented with punctate melanophores. The venters of the remaining two E. quadridigitata have very
few, widely scattered punctate melanophores in one and numerous
punctate melanophores in the other. Also, the venters of the Rainbow
Bay E. quadridigitata are not as heavily pigmented as those of specimens collected elsewhere within the Carolinas. In all of the E.
Table 2. Morphometic characteristics of the measured samples of Euycea quadridigitata
and E. chamberlaini. See Methods and Materials for identification of abbreviations.
Eurycea chamberlaini
Eurycea quadridigitata
Character N
Mean
SD
SVL
T/SVL
HL/SVL
HW/SVL
28
1.47
0.238
0.136
2.44
0.15
0.013
0.011
48
37
48
48
Range
N
Mean
SD
Range
22-33
1.14-1.72
0.213-0.259
0.121-0.184
47
30
47
47
25.8
1.62
0.247
0.143
2.16
0.23
0.019
0.014
22-30
1.08-2.00
0.214-0.291
0.127-0.189
ICF
48
5.5
0.99
3-8
47
4.2
0.87
3-6
VT
48
11.8
2.15
8-16
47
10.0
2.99
5-16
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chamberlaini the lateral aspect of the tail is lightly pigmented, contrasting distinctly with the dorsolateral stripe. The lateral aspect of the tail is
dark-sided in all of the E. quadridigitata, not contrasting distinctly with
the dorsolateralstripe. Costal groove counts were 15 (1) or 16 (3) for the
specimens of E. chamberlaini as contrasted with 16 (1), 17 (4), or 18 (1)
for E. quadridigitata.
Osteologic Differences. - Twenty-two stained and cleared skeletons
were available for study, 10 Eurycea chamberlaini and 12 E.
quadridigitata. Some of these preparations,however, are poorly stained
and/or in poor condition, making it difficult or impossible to assess the
state of one or more of the charactersexamined. The two species differ
with respect to the structure and shape of the frontal, the prevomerine
tooth series, and basibranchialII of the hyoid apparatus.They also differ
in number of trunk vertebrae.
Eurycea is one of several plethodontid genera that possess poorly
to very well developed posterolateral frontal processes that overlap
the parietals (Wake 1966). This character is variable in both the new
and the nominal species but in E. chamberlaini the processes are
somewhat more strongly developed and typically triangular or nearly
triangular in shape, whereas they tend to be short and rounded in E.
quadridigitata (Fig. 2). Eight of the 10 specimens of E. chamberlaini
have triangular or nearly triangular processes but in the two others
they are indiscernible or short with ctenate margins, respectively. In
six specimens of E. quadridigitata in which the state of this character
could be determined, the processes are relatively short and somewhat
rounded in three specimens, relatively short and somewhat rounded
A
B
Figure 2. Conformation of the frontal processes in (A) Eurycea quadridigitata
and (B) E. chamberlaini.
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Julian R. Harrison, III and S.I. Guttman
169
but with ctenate margins in two specimens, and triangular in one
specimen.
The vomer of E. quadridigitata typically bears a sharply arched
prevomerine tooth series with a bony shelf posterolateral to the series
(Fig. 3A). This is representative of Wake's (1966) hemidactyliine pattern. No variation was noted in eight of 12 E. quadridigitata in which
the state of this character could be determined. The prevomerine tooth
series of E. chamberlaini is not sharply arched and the vomer lacks a
posterolateral bony shelf (Fig. 3B), a pattern more characteristic of
plethodonine salamanders.A similar condition was present in a stained
and cleared specimen of E. cirrigera from York Co., SC. The preorbital
process of the vomer is only slightly developed in both species. In six of
the 10 specimens of E. chamberlaini examined the tooth series is clearly
not arched, whereas it is slightly arched in the remaining four, but not to
the degree observed in specimens of E. quadridigitata. The
hyobranchial apparatus is similar in the two species, except that
basibranchial II (os triangulare) is usually tetraradiate in E.
chamberlaini and triradiatein E. quadridigitata (Fig. 4). No variation is
apparentin eight of the 10 specimens of E. chamberlaini in which this
character could be determined. Five of the 7 specimens of E.
quadridigitata in which this character could be determined have the
triradiatecondition, whereas two show the tetraradialstate. Based upon
the information presented herein, Hilton's (1945) description of the
skeleton of Manculus quadridigitatus is applicable to the new species,
E. chamberlaini, not the nominal species. Similarly, this is also the case
with regard to his (1947) illustration of the hyobranchial apparatusin
the genus Manculus.
The two species also differ in number of trunk vertebrae. According
to Highton (1957), if a costal groove located posterior to the gular fold
A
Figure 3. Conformation of the
prevomerine teeth in
(A) Eurycea
quadridigitata and
(B) E. chamberlaini.
B
'A
A
B
Figure 4. Conformation of basibranchial
II (os triangulare)in
(A) Eurycea
quadridigitata and
(B) E. chamberlaini.
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but anterior to the front limb is counted, then the number of costal
grooves in E. quadridigitata is equal to the number of trunk vertebrae.
As noted previously, seven of 47 specimens of E. chamberlaini examined have a costal groove count of 15-15, one has a count of 15-16, and
Table 3. Allele
quadridigitata.
frequencies at variable loci in five populations of purported Euycea
Missing letters represent alleles found in other species of Eurycea.
Population
LOCUS
(N)
AAT-1
E
F
ALB
A
C
FH
A
B
C
GPI-1
B
C
GLUDH
A
B
IDHP- 1
A
B
C
D
LDH-1
C
D
E
LDH-2
A
B
MDH-1
C
D
MDH-2
A
B
PEP-1
B
C
D
PGDH
A
B
D
PGM
A
B
C
D
Yellow morph
Pickens
Beauregard
Parish, LA
Co., SC
Osceola
Parish, LA
Eurycea quadridigitata
Scotland
Co., NC
Barnwell
Co., SC
11
12
9
7
19
0.05
0.95
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.06
0.67
0.27
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.94
0.06
1.00
1.00
0.06
0.94
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.44
0.56
1.00
0.76
0.24
1.00
1.00
1.00
0.06
0.94
1.00
1.00
1.00
1.00
0.95
0.05
1.00
0.05
0.95
1.00
1.00
1.00
0.14
0.86
0.83
0.17
0.36
1.00
0.64
0.05
0.95
0.38
0.62
1.00
0.09
0.91
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.58
0.42
0.23
0.18
0.59
0.04
0.96
2003
171
JulianR. Harrison,III andS.I. Guttman
40 have a count of 16-16. Six of 48 specimens of E. quadridigitata
examined have a count of 17-17, one has a count of 18-17, and 41 have
a count of 18-18. Thus, using Highton's method, most of these E.
chamberlaini (83%) have 17 trunk vertebrae, the remainder 16 trunk
vertebrae.In contrast, most of the E. quadridigitata (85%) have 19 trunk
vertebrae, the remainder 18 trunk vertebrae. These results correspond
fairly closely with observations on 19 stained and cleared specimens in
which this character could be determined. Eight of the skeletons of E.
chamberlaini had 17 trunkvertebraeand two had 18 trunkvertebrae. Six
of the E. quadridigitata skeletons had 19 trunkvertebrae and three have
18 trunkvertebrae.
Genetic Differences. - Thirteen of the 17 protein-producing loci
surveyed were either polymorphic within populations or variable across
populations (Table 3). LAP, ME, PT2 and SOD showed no detectable
variation across all samples.
Allele frequencies (Table 3) demonstrate that extensive differentiation occurs among these five samples. Fixed differences exist at 07 loci between population pairs (Table 4). The Pickens Co., SC
sample (yellow morph) is the most strongly differentiated with seven
fixed differences between it and both the Scotland Co., NC and
Barnwell Co., SC populations (dark morphs). The Pickens Co., SC
population also maintains four fixed differences from the two Louisiana samples [Osceola Parish (dark morph) and Beauregard Parish
(yellow morph)]. In contrast, the Scotland Co., NC and Barnwell Co.,
SC populations are extremely similar with no fixed differences between them; the Osceola Parish, LA sample has one fixed difference
(MDH-1) when compared to the Scotland Co., NC and Barnwell Co.,
SC populations.
Genetic distances (unbiased, Nei 1978) between populations
(Table 4), calculated utilizing all loci, range from 0.003 (Barnwell
Co, SC and Scotland Co., NC) to 0.752 (Barnwell Co., SC and
Pickens Co., SC). When genetic distances are averaged by color
morph (Table 5), the mean genetic distance between the dark and
yellow morphs is 0.599. Genetic differentiation among the three dark
morph samples is slight (mean D = 0.053); however, the two yellow
Table 4. Number of loci showing fixed differences between the five populations examined
(above diagonal) and matrix of Nei (1978) unbiased genetic distances (below diagonal).
Populations 1 and 2 are the yellow morph; populations 3 and 5 are E. quadridigitata.
4
5
1
2
3
Population
1. Beauregard
Parish,LA.
*****
4
4
5
4
2. Pickens Co., SC
3. Osceola Co., SC
0.450
0.428
*****
0.611
4
*****
7
1
7
0
4. ScotlandCo., NC
5. BarnwellCo., SC
0.553
0.517
0.733
0.752
0.080
0.078
*****
0.003
0
*****
172
Vol. 2, No. 2
SoutheasternNaturalist
morph samples are genetically quite different from each other (D =
0.450).
The electrophoreticdata indicate that the yellow and darkmorphs are
extremely different from one another and are genetically isolated. However, the magnitude of the differentiation between the populations from
Beauregard Parish, LA and Pickens Co., SC also suggests that the
samples of Eurycea from these two sites representdistinct entities that are
not interbreeding;thus, the yellow morph genetically analyzed in this
study appears to be composed of two species. The genetic data indicate
that the populations studied belong in three, genetically distinct, groups;
there is no evidence of gene flow between the groups. Therefore, we
propose that they be recognized as three species. CarlaHass and Richard
Highton (1998; pers. comm.) are currentlyconducting analyses of genetic
variation of other populations throughoutthe range of the nominal species, and D. Bruce Means (pers. comm.) and John B. Jensen our investigating the statusof some Alabama,Florida,and Georgiapopulations.
Distribution and Habitat. - Eurycea chamberlaini is known currently from portions of the Piedmont in both North and South Carolina,
the upper Coastal Plain in South Carolina, and portions of the central
Coastal Plain in North Carolina (Fig. 5). It is sympatric with E.
quadridigitata in at least two localities, the Rainbow Bay complex
(Rainbow Bay, Bullfrog Pond, and Sun Bay) in the SavannahRiver Site,
Barnwell Co., SC (R.D. Semlitsch and J.H. Pechmann, pers. comm.),
and a site ca. 3 mi WSW Allendale, Allendale Co., SC (specimens in the
collection of the CharlestonMuseum). On the SavannahRiver Site, only
E. quadridigitata was found at Flamingo Bay and Karen's Pond (R.D.
Semlitsch, pers. comm.). Elsewhere in South Carolina the range of E.
quadridigitata appears to be parapatric with that of E. chamberlaini
along the lower edge of the sandhills region of that state. The former
species reaches into North Carolina primarily in those counties bordering the South Carolina state line from the southern edge of the sandhills
region in Scotland County eastward to Brunswick County. The pattern
of distribution of E. chamberlaini, a range encompassing the Piedmont
of both states but extending into the Coastal Plain primarily in North
Carolina, is not unique. It is duplicated in part by at least two other
species of amphibians, Hemidactylium scutatum and Bufo woodhousei
fowleri (Martof et al. 1980, Conant and Collins 1998).
Table 5. Matrix of Nei (1978) unbiased genetic distances averaged by color morph.
Color Morph
No. of populations
1
1. Dark Morph
3
0.053
(0.003-0.080)
2. Yellow Morph
2
0.599
(0.428-0.752)
2
0.450
(0.450-0.450)
2003
JulianR. Harrison,IIIandS.I. Guttman
173
In the Carolinas Eurycea quadridigitata occurs in a variety of habitats, but is especially associated with pine litter or other debris at the
margins of pine flatwood or pine savanna ponds (Harrison 1973). It also
occurs in the vicinity of Carolinabays, in tupelo-cypress swamps, and in
bottomland hardwood forests. Eurycea chamberlaini also occurs in a
variety of habitats but seems to prefer seepage areas near streams or
ponds, particularly in Piedmont and upper Coastal Plain areas. Thirtythree records of the new species in files of the NCSM provide fairly
specific habitat data. Twenty-three (ca. 70%) of these collections were
associated with streams and/or seepages from springs, and ten (ca. 30%)
were associated with floodplain sites or a pond. At the Savannah River
Site in Barnwell Co., SC, both species have been caught in pitfall traps
at Carolina bays, presumably entering these habitats to breed. However,
E. chamberlaini tends to migrate to Rainbow Bay later in the fall than E.
quadridigitata and there are seepage or wet areas in the vicinity of
Rainbow Bay and Bullfrog Pond (J. H. Pechmann, pers. comm.).
Whether or not E. chamberlaini oviposits in these sites and/or in the
seepage or wet areas remains undetermined.
Differences in Fecundity. - Data relative to the fecundity of
Eurycea chamberlaini are available from 18 gravid females in two
samples, one from Wake Co., NC, the other from Barnwell Co., SC.
000o0
0o
0,'D0
0~
o o
oD
o
ro
0
0o
00
o
0
0
00
o
0
00k
50 km
0
Figure 5. Distribution of Eurycea chamberlaini (filled circles) and E.
quadridigitata (open circles) in the Carolinas. The type locality of E.
chamberlaini is indicated by a filled triangle. Known sites occupied by both
species are shown by half-filled circles. Map is based on selected records in the
ChM and NCSM collections; it includes only those records of Eurycea
chamberlainifromNorthCarolinathathavebeenverified.
174
SoutheasternNaturalist
Vol. 2, No. 2
Data for E. quadridigitata are available from 14 gravid females in the
collections of the Charleston Museum (ChM) and the senior author
(JRH) from the South Carolina Coastal Plain. The number of enlarged,
yolk-laden ovarian eggs was rather variable in both species but in E.
chamberlaini (gt SVL = 25.6 mm) the mean was 45.2 (35-64), whereas
the mean in E. quadridigitata was 39.8 (23-62). Although the female
E. quadridigitata were larger (gi SVL = 30.8 mm), they bore on average the smallest number of enlarged yolk-laden ovarian eggs. In contrast, Semlitsch and McMillan (1980) found that females (dark morph;
R. D. Semlitsch, pers. comm.) from Rainbow Bay and Ellenton Bay on
the Savannah River Site differed significantly in number of ovarian
eggs (.t = 32.7 and 21.4 eggs, respectively), and that the larger females
from Rainbow Bay produced more eggs. The mean number of eggs in
12 clutches oviposited by E. quadridigitata in the Charleston, SC, area
was 21.9 (13-36) (Harrison 1973). Most of these were clutches deposited in the laboratory, so it is possible that some females did not
deposit all of their eggs. There are only two other reports in the
literature concerning the fecundity and/or field clutches of females
from populations herein assigned to E. chamberlaini and only one of
these gives the number of eggs oviposited. The mode of egg deposition
is similar in the two species. Bishop (1943) states that a female sent to
him from the Raleigh area by Brimley deposited 48 eggs in the laboratory. These were deposited in groups of 5-8 on the undersurfaces of
damp leaves. Brimley (1923) reports finding field-deposited eggs in
leafy trickles attached to leaves and debris singly or in groups of 3-6.
These results are comparable to those reported by Harrison (1973) for
E. quadridigitata. He observed laboratory-deposited eggs attached singly and ratherloosely to pine needles.
Larval Differences. - Larvae (n = 102) of the new species were
available for examination. These were compared with Goin's (1951)
description of early Eurycea quadridigitata larvae, 21 specimens of
hatchling larvae of the latter species from Berkeley Co., SC, and 50
Figure 6. Hatchling larva of Eurycea chamberlaini from Vance Co., NC. Scale
represents1.0 mm.
2003
Julian R. Harrison, III and S.I. Guttman
175
older larvae from Bladen Co., NC. The Berkeley County larvae agree
closely with the description provided by Goin (1951). As he notes, one
of the most striking features of the hatchling larvae is the presence of a
type of dorsal fin normally associated with ponds. A similar fin is
present in hatchling larvae of the new species (Fig. 6). Hatchling E.
quadridigitata are more or less uniformly grayish-brown dorsally but
have pigment-free venters. There is a tendency for lateral rows of small
rounded, pigment-free areas, especially ventrolaterally. There are no
dorsal spots. Older larvae have lateral pigment-free areas in the form of
longitudinal dashes or streaks, a characteristic absent in larvae of the
new species. In hatchlings the ventral portion of the caudal fin is wider
than that of the new species, especially near the vent. In older larvae of
E. quadridigitata dark head markings of the postorbital and preorbital
areas are more sharply delineated than those in larvae of the new
species. Also, in older larvae of that species, the venter of the tail is
moderately pigmented whereas in larvae of the new species the venter of
the tail is only lightly pigmented or unpigmented.
An average of 74% of the larvae of E. chamberlaini have symmetrically or asymmetrically paired dorsal spots (Fig. 4). The number of
specimens with spots declines over the larval period. When spots are
present these average 4.5 (1-7) on the right and 4.4 (1-8) on the left.
The total number of spots averaged 8.9 (2-15). Spots are symmetrically
arranged as pairs in 45% of the sample, asymmetrically in 55%.
Hatchling larvae of E. quadridigitata are slightly larger than those of the
new species averaging 6.8 mm SVL (6.5-7.0) for 10 measured specimens as compared to 6.3 mm SVL (5.5-7.0) for 10 measured specimens
of E. chamberlaini. Gill rami of E. quadridigitata are longer and
stockier, with the second and third pairs reaching the elbow. Those of E.
chamberlaini reach just beyond the brachial mid-point. E.
quadridigitata larvae are darker, more heavily pigmented. There are
dense patches of melanophores with more or less uniformly dispersed
pigment-free areas that come into contact but do not anastomose. Patch
edges are relatively discrete. In E. chamberlaini there are anastomosing
patches of melanophores enclosing pigment-free areas; patch edges are
more dendritic.
Concluding Remarks. - In Barnwell County, SC at the Savannah
River Site, both Eurycea chamberlaini and E. quadridigitata appear to
occur in similar habitats but apparently breed at somewhat different
times, with the former species entering bucket traps at drift fences later
in the fall. This pattern merits furtherinvestigation, including the question of whether or not different egg deposition sites are chosen. The
geographic range and habitat requirements of E. chamberlaini in the
Carolinas are incompletely known, but the hiatus between the geo-
176
Naturalist
Southeastern
Vol.2, No. 2
graphically isolated populations of E. chamberlaini in Pickens Co., SC
and populations of this species in the upper Coastal Plain is perhaps an
artifact of collecting as the intervening Piedmont is the least explored
region of this state. With the exception of papers by Brimley (1923,
1944) the life history and ecology of E. chamberlaini is relatively
unknown and should prove to be fruitful areas of research, especially at
sites occupied by both the new species and E. quadridigitata.
The electrophoretic data indicate that the yellow and dark morphs
are extremely different from one another and are genetically isolated.
However, the magnitude of the differentiation between the populations
from Beauregard Parish, LA and Pickens Co., SC also suggests that the
samples of Eurycea from these two sites represent distinct entities that
are not interbreeding; thus, the yellow morph genetically analyzed in
this study appears to be composed of two species. It is important to
conduct genetic analyses of sympatric populations of the two morphs,
e.g., Barnwell Co., SC. In addition, genetic studies of additional yellow
morph populations are needed to determine the number and range of
taxa included in this morph.
ACKNOWLEDGMENTS
Permission to collect specimens in South Carolina State Parks was granted
to Harrison by the South Carolina Departmentof Parks and Tourism. We thank
Alvin L. Braswell (North Carolina State Museum of Natural Sciences - NCSM),
Albert E. Sanders (The Charleston Museum - ChM) and the late Joseph R.
Bailey (Duke University - DU) for permission to study specimens housed in
their respective institutions. Specimens in the DU collection have been transferred to NCSM. Specimens for genetic studies from the upper Piedmont of
South Carolina were supplied by Richard R. Montanucci (Clemson University),
from the Savannah River Site by R.D. Semlitsch, S. J. Morreale, and J.H.
Pechmann (Savannah River Ecology Laboratory), and from North Carolina by
Alvin L. Braswell (NCSM). We appreciate also the effort of others in collecting
the Louisiana specimens used in genetic analyses. Alvin L. Braswell (NCSM)
furnished information concerning the larvae, habitats, and distribution of the
two species in North Carolina, and otherwise assisted our study in significant
ways. R. D. Semlitsch (University of Missouri), J.H. Pechmann (University of
New Orleans) and J. W. Gibbons (Savannah River Ecology Laboratory) provided information concerning the distribution and ecology of the two species on
the SavannahRiver Site. RichardHighton (University of Maryland)reviewed an
earlier version of this paper and has provided valuable information and encouragement to the senior author over the years. We also thank Paul Chippindale
(University of Texas at Arlington) and Raymond D. Semlitsch (University of
Missouri) for providing many helpful comments and suggestions that significantly improved the manuscript. This is contribution number 223 of the Grice
Marine Biological Laboratory,College of Charleston.
2003
JulianR. Harrison,IIIandS.I. Guttman
177
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Appendix I.
Specimens examined. In addition to the holotype and paratypes of Eurycea
chamberlaini, the study included measurements taken from 37 specimens of E.
quadridigitata from North Carolina as follows: Scotland County, NCSM
15401 (4), 1.25 mi SSE Wagram, NCSM 17017 (1), 2 mi SSE Wagram, NCSM
17334 (1), 3.25 mi NW Wagram, NCSM 17016 (1), 3.5 mi S Wagram; NCSM
16984 (13), 5.4 mi NE Laurinburg,NCSM 19872 (1), 5.5 mi NE Laurinburg,
NCSM 18615 (2), 6 mi NE Laurinburg,NCSM 16526 (1), NCSM 19230 (2), and
NCSM 19873 (1), 6.5 mi NE Laurinburg, NCSM 17015 (1), 7 mi ENE
Laurinburg;Robeson County, NCSM 35995 (1) [DU A1103], Shoeheel Creek
on Route 501, NCSM 35978 (2) [DU Al 104], 2 mi NE Fairmont; Bladen
County, NCSM 35976 (4) [DU A2131], N.C. State Game Club, 0.25 mi toward
Council from club house, NCSM 36000 (1) [DU A2172], 3 mi N Bladinboro on
NC Highway 131; Columbus County, NCSM 35996 (1) [DU A8566], Cerro
Gordo, and 11 specimens from South Carolina as follows: Clarendon County,
JRH 3163-3166 (4) and 3193-99 (7), Santee National Wildlife Refuge, Dingle
Pond.