2007 SOUTHEASTERN NATURALIST 6(4):727–736
Life History and Color Variants in a Matriline of
Oklahoma Salamander (Eurycea tynerensis)
Mark L. McKnight1,* and Nathaniel A. Nelson1,2
Abstract - Until recently, two surface-dwelling species of multi-ribbed brook
salamanders (i.e., the metamorphic Eurycea multiplicata griseogaster [Graybelly
Salamander], and the strictly paedomorphic E. tynerensis [Oklahoma Salamander])
were recognized as living in the Missouri Ozarks. The current understanding is that
a single species (Oklahoma Salamander) is resident in the Ozarks, and that this
species is polymorphic for life-history mode from population to population. We
recently discovered that multi-ribbed salamanders at two locations in Christian and
Barry Counties, MO, show striking polymorphism in eye-coloration, with individuals
possessing either gold or black irises. To test whether the gold- and black-eyed
forms may be different species, we conducted a phylogeographic analysis of mtDNA
variation in multi-ribbed salamanders across the Missouri Ozarks. We present sequence
data from the mitochondrial cytochrome-b gene that show that transforming
and paedomorphic individuals, and the alternative eye-color morphs can all arise
from the same mitochondrially defined matriline, and thus seem to comprise a single
species. We hypothesize that color-pattern variation in multi-ribbed salamanders is
under simple genetic control.
Prior to the recent popularity of molecular phylogenetics, a number of salamander
species were named based on the (in many cases reasonable) inference
that paedomorphic individuals are intrinsically reproductively isolated from
metamorphic ones, or that consistent color-pattern variation implies independent
evolutionary lineages. For example, the paedomorphic Eurycea tynerensis
Moore and Hughes (Oklahoma Salamander) was named as a form distinct
from the metamorphic E. multiplicata Cope largely because of the difference
in life history. Similarly the several Mexican paedomorphic, caldera-lake
populations of Ambystoma retain species status despite having mitochondrial
DNA sequences very similar or identical to metamorphosing Ambystoma velasci
Dougés (Salamandra Tigre de Meseta) in the surrounding habitat (Shaffer
and McKnight 1996). Also, in the case of E. junaluska Sever, Dundee, and
Sullivan (Junaluska Salamander; Sever et al. 1976; see also Ryan 1997, 1998),
E. chamberlaini Harrison and Guttman (Chamberlain’s Dwarf Salamander;
Harrison and Guttman 2003), and E. aquatica Rose and Bush (Brownback
Salamander; Rose and Bush ), the initial impetus to investigate the
possibility of “new-species” status was the presence of modest differences in
1Department of Biology, Southwest Missouri State University, 901 South National
Avenue, Springfield, MO 65804. 2Current address - Department of Amphibians, Reptiles,
and Fishes, Sedgwick County Zoo, 5555 Zoo Boulevard, Wichita, KS 67212.
*Corresponding author - email@example.com.
728 Southeastern Naturalist Vol.6, No. 4
color pattern (though in most cases, further morphological or genetic differences
became apparent on close examination). Though some might question
the validity of E. aquatica (e.g., Jacobs 1987), a recent paper by Kozak et al.
(2006) clearly demonstrates its species status. In a recent examination of mitochondrial
DNA variation in the Eurycea multiplicata complex of the central
highlands of the United States, Bonett and Chippindale (2004) discounted the
importance of life history as a reproductive isolating mechanism when they
concluded that the 2 surface-dwelling forms in this complex from the Ozark
Plateau (i.e., the metamorphic E. multiplicata griseogaster Moore and Hughes
[Graybelly Salamander; Moore and Hughes (1941)] and the paedomorphic E.
tynerensis) are simply life-history variants of a single highly variable species,
Contemporaneous with Bonnet and Chippindale’s (2004) work on
their project, we discovered a distinctively colored form of Eurycea at
two different localities in the drainage of the White River. These salamanders
were noticeably different from the sympatric Oklahoma Salamander
and Graybelly Salamander in their possession of black irises in their
eyes, as opposed to the gold irises typical of Oklahoma Salamanders and
Graybelly Salamanders, and by their lack of gold iridophore spots along
their lateral lines. Other than these color differences, these salamanders
were morphologically clearly part of the E. multiplicata complex. Study
of individuals maintained in the laboratory for more than two years and
individuals induced to metamorphose with thyroxin showed that the color
pattern was not transient and at least persisted across metamorphosis.
Despite extensive searching, we have found no metamorphosed individuals
of this black-eyed Eurycea. This combination of distinctive color pattern
and apparent life-history mode caused us to hypothesize that these
black-eyed salamanders (as they will be referred to here) might represent
an as yet undescribed species in the Missouri Ozarks. Thus, we took the
opportunity to examine the possibility of color pattern indicating genetic
distinction in this system.
In this paper, we report results from DNA sequencing of 21 individual
Eurycea from three localities in the Ozarks. While the work of Bonett and
Chippindale (2004) was based on sequences of single individuals from each
collecting locality, by sequencing multiple salamanders per site, we extend
their results to individual variation in life history and color pattern at the collecting-
locality level. More specifically, we ask whether there is any indication
that life history or color pattern is associated with distinct evolutionary lineages
as might be expected if they represent different genealogical species.
Materials and Methods
Black-eyed individuals of the E. mutiplicata complex were found
at two localities (Fig. 1): a stream issuing from a cave in Barry County,
2007 M.L. McKnight and N.A. Nelson 729
approximately 130 km southwest of Springfield, Missouri (5 specimens, all
black-eyed; MSU 1924–28; N.B.: specimens were not found in the cave);
and a semi-permanent section of Camp Creek in Busiek State Forest and
Wildlife Area in Christian County, approximately 50 km south of Springfield
(7 specimens; MSU 1935–38, black-eyed; 1941–43, gold-eyed). Four additional
gold-eyed metamorphic specimens were collected in a tributary to
Camp Creek (MSU 1920–23). For comparison, we collected 5 salamanders
(MSU 1929–33) from the type locality of the Oklahoma Salamander (Tyner
Creek, Adair County, OK).
The salamanders used in this study were collected in two ways. Paedomorphic
and larval salamanders were collected using a shovel to scoop
gravel from the creek bottom, and then the gravel was spread over a seine
to sift for salamanders. The second method was simply to lift rocks by hand
and capture salamanders with a small dip net. Metamorphosed adults were
also caught in this second way. Animals were killed using a 10% aqueous
solution of MS–222 prior to dissection of samples of liver and muscle tissue.
The carcasses have been labeled and preserved as vouchers, and are part of
the natural history collection at Missouri State University.
DNA was extracted from the salamander liver tissue using a commercial
kit (DNAeasy, Quiagen, Valencia, CA). An approximately 1200-bp
Figure 1. Map of the western Ozark Plateau area showing localities of capture, and
730 Southeastern Naturalist Vol.6, No. 4
segment of the mitochondrial cytochrome-b gene was amplified using
touch-down polymerase chain reaction (TD-PCR), Taq-Gold DNA polymerase,
and the primer pair MVZ15 (Moritz et al. 1992) and EuryCyb9
(Hillis et al. 2001). Cycle sequencing reactions utilized the Big-Dye 3.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and were run
on an ABI 310 Automated Genetic Analyzer (Applied Biosystems, Foster
City, CA). Sequences produced from different directions were manipulated
and joined using GeneJockey (Biosoft, Cambridge, UK). Sequences have
been deposited in GenBank (Accession numbers DQ682625–DQ682645).
For comparative purposes, 74 DNA sequences of other members of the E.
multiplicata group were down-loaded from GenBank (Accession numbers
AY528330–AY528374, AY528376–AY528404; Bonett and Chippindale
2004). These down-loaded sequences were added to the sequences generated
here and aligned using Clustal-X (Thompson et al. 1997). Sequences
were analyzed phylogenetically using PAUP* 4.0b10 (Swofford 1998); a
maximum likelihood phylogeny, parameterized using Modelest (Posada
and Crandall 1998), was constructed using the 21 sequences produced here
(likelihood model was: HKY - empirical base frequencies, number of states
= 2, transition/transversion ratio = 6.7549, among-site rate variation was
equal, no invariant sites). The entire 95 “taxon” data set formed by adding
our 21 sequences to the 74 obtained from GenBank was analyzed with an
MCMC search in MrBayes (Huelsenbeck and Ronquist 2001) using starting
parameters of a HKY + I + γ model. Bayesian posterior probabilities
were generated for both topologies using MrBayes (For the 21 taxon data
set: 1,000,000 generations sampling every 100 generations, burnin of 10%;
for the 95 taxon data set: 1,558,500 generations sampling every 100 generations,
burnin of 58,500 generations).
Table 1. Collection localities, description of eye color, and life-history of salamanders used in
Field collection Eye accession
numbers color Locality numbers Notes
MSU 1920–1923 Gold Trib. Camp Creek, DQ682625– Metamorphic
Christian County, MO DQ682628
MSU 1924–1928 Black Galena Spring, DQ682629– Paedomorphic
Barry County, MO DQ682633
MSU 1929–1933 Gold Tyner Creek, DQ682634– Type locality of tynerensis
Adair County, OK DQ682638
MSU 1935–1938 Black Camp Creek, DQ682639– Paedomorphic - sympatric
Christian County, MO DQ682642 with 1941–1943
MSU 1941–1943 Gold Camp Creek, DQ682643– Paedomorphic - sympatric
Christian County, MO DQ682645 with 1935–1938
2007 M.L. McKnight and N.A. Nelson 731
The 21 individual salamanders (Table 1) used in this study were fully
adult, based on values of snout–vent length (Dundee 1958, Tumlison et
al. 1990), or the presence of yolked eggs in females or developed hedonic
glands in males. From these specimens, 817 bp of unambiguous sequence
were obtained. The phylogeny depicting the relationships among the 21
sequences (Fig. 2) shows the gold-eyed Oklahoma Salamander topotypes
(GT: MSU1929–MSU1933) to be the most divergent of the samples in our
analysis (uncorrected p distances to the rest of the samples range from 4%
to 5%). This is not surprising, given the geographic distances separating
Tyner Creek from the remaining collecting localities. The black-eyed paedomorphic
salamanders from Barry County (BB: MSU1924–MSU1928)
Figure 2. Maximum
phylogeny of the
in this paper. Eurycea
are used as the
lengths are proportional
amount of genetic
is no separation
of black-eyed (B)
(G) and paedomorphic
m e t a m o r p h i c
State Forest and
(Camp Creek tributary:
MSU1938, MSU1941–MSU1943). BB are black-eyed salamanders from Barry
County, and GT are gold-eyed topotypes of Oklahoma Salamander. Numbers above
branches are Bayesian posterior probabilities (1,000,000 generations sampling every
100 generations, burnin of 10%).
732 Southeastern Naturalist Vol.6, No. 4
form a tight monophyletic group (1.1% to 5% uncorrected p distance
to the remaining salamanders). It is the placement of the remaining
eleven specimens on this phylogeny that is most interesting. These
specimens, from the localities within Busiek State Forest and Wildlife
Area in Christian County (MSU1920–MSU1923, MSU1935–MSU1938,
MSU1941–MSU1943), form two rather divergent monophyletic groups
(differing from each other by 1.5% to 1.7% uncorrected p distances).
Each group, however, contains a mixture of gold-eyed metamorphic
(GM), gold-eyed paedomorphic (GP) and black-eyed paedomorphic (BP)
salamanders. In addition, each group contains individuals from each
of the localities within Busiek State Forest and Wildlife Area in Christian
County. In fact, specimens MSU1920, MSU1935, MSU1941, and
MSU1943 all have identical DNA sequences spanning the 817 bp studied
here, despite their identification as gold-eyed metamorphic, black-eyed
paedomorphic, and 2 gold-eyed paedomorphic salamanders, respectively.
A similar set of identical sequences exists in the other group from Christian
County (MSU1921–3, MSU1942). Genetic variation within these two
groups from Christian County is quite small, ranging from 0% sequence
difference to an uncorrected p distance of 0.25%. The cause of the deeper
divergence (ca. 1.6% p) between the two groups of haplotypes from
Christian County is unknown, but it probably represents the degree of
within-lineage variation that only becomes evident with the sequencing
of multiple individuals from a locality.
The Bayesian consensus tree from the larger data set (Fig. 3) including
the sequences of Bonett and Chippindale (2004) helps to place the variation
among our specimens into the context of a more geographically extensive
sampling of multi-ribbed salamanders from the central highlands of North
America. Though Bonett and Chippindale (2004) did not mention any blackeyed
salamanders among their collections, they did recognize two forms
based on life history: those that fully metamorphose (Graybelly Salamander:
Emg on the phylogeny) and those that remain paedomorphic (Oklahoma Salamander:
Ety on the phylogeny). The salamanders from our study again fall
into the same 4 groups, often with 1 specimen from Bonett and Chippindale
(2004; Ety 56) embedded within. What is clear from this larger sampling is
that, with one possible exception of a clade of Graybelly Salamanders from
the southern Ozarks, all Ozark salamanders are closely related genetically,
and that there is no genetic distinction among metamorphosing and nonmetamorphosing
salamanders, nor is there a genetic distinction between
gold-eyed or black-eyed salamanders.
Our hypothesis of a new black-eyed species of Eurycea was not supported
by our data. Instead the metamorphosing gold-eyed form (Graybelly
2007 M.L. McKnight and N.A. Nelson 733
Figure 3. Bayesian consensus phylogeny of the full data set comprised of the 21
sequences presented here and 74 sequences from Bonett and Chippindale (2004).
The phylogeny is rooted using several species of plethodontid salamanders. Note
again the lack of discrimination between paedomorphic and metamorphic, and goldeyed
and black-eyed forms of Ozark multi-ribbed salamanders. Codes for the specimens
from this paper are the same as in Figure 2, codes for specimens from Bonett
and Chippindale (2004) are: Emm = E. multiplicata multiplicata (Many-Ribbed
Salamander), Emg = E. m. griseogaster (Graybelly Salamander), Ety = E. tynerensis
(Oklahoma Salamander), Tsp = Typhlotriton spelaeus (Grotto Salamander). Numbers
above branches are Bayesian posterior probabilities (1,558,500 generations sampling
every 100 generations, burnin of 58,500 generations).
734 Southeastern Naturalist Vol.6, No. 4
Salamander), the paedomorphic gold-eyed form (Oklahoma Salamander),
and the paedomorphic black-eyed salamander share very similar or identical
mitochondrial cytochrome-b sequences, and can apparently arise from within
the same line of maternal descent. The lack of genetic difference between
the different life-history and color types of multi-ribbed salamanders in the
Ozarks is in complete agreement with the results of Bonett and Chippindale
(2004). They, in fact, concluded that it is probably best to recognize only
a single species of surface dwelling multi-ribbed salamander on the Ozark
Plateau north of the Arkansas River, namely the Oklahoma Salamander. We
agree with their taxonomic conclusion, and with their characterization of this
species as a highly variable and plastic form in life-history mode. We found
that transforming and paedomorphic salamanders collected from geographically
proximate locations within a single watershed (Camp Creek, Christian
County, MO) have identical cytochrome-b DNA sequences for the 816-bp
fragment we examined.
The highly variable nature of Oklahoma Salamanders is also seen in
the presence of the black-eyed morph that was the impetus for this study.
The presence of identical cytochrome-b sequences in “typical” Oklahoma
Salamanders and in the black-eyed salamanders suggests either that the
color pattern is evolving extremely rapidly, or that the black-eyed phenotype
is segregating within a matriline. An interesting parallel situation
exists in the Axolotl. An allele (axanthic; Ambystoma Genetic Stock
Center 2007) at a single color locus produces a very similar phenotype
to that seen in this study. We propose, as a working hypothesis, that the
black-eyed form (also lacking gold/silver iridophores along their sides)
results from homozygosity for a similar allele. If this hypothesis is correct,
the coexistence of gold-eyed and black-eyed salamanders at Busiek
State Forest and Wildlife Area would be explained by simple Mendelian
segregation at this locus, and the absence of gold-eyed salamanders at the
Barry County location would, according to this hypothesis, suggest the
fixation of the hypothesized allele in this population. With time and skill
at captive breeding, it should be possible to do the requisite crosses to test
We thank Lynn Robbins for allowing collection under his permit, and Jeff Briggler
and the Missouri Department of Conservation for help with gaining access to
Busiek State Forest and Wildlife Area. While ectothermic vertebrates were not covered
as part of our institution’s animal care and use document at the time this work
was done, our procedures followed recommendations of all herpetological societies.
This paper was improved tremendously by the helpful comments of T. Uzzell, S.R.
Voss, H.B. Shaffer, two anonymous reviewers, and our editor, K.H. Kozak. Financial
support for this work was provided by the Department of Biology and the Graduate
College of Southwest Missouri State University to N.A. Nelson, and a grant from the
Missouri Department of Conservation to M.L. McKnight.
2007 M.L. McKnight and N.A. Nelson 735
Ambystoma Genetic Stock Center. 2007. Available online at http://bigapple.uky.
edu/~axolotl/mutantslist.htm#ax. Accessed 9 April 2007.
Bonett, R.M., and P.T. Chippindale. 2004. Speciation, phylogeography, and evolution
of life history and morphology in plethodontid salamanders of the Eurycea
multiplicata complex. Molecular Ecology 13:1189–1203.
Cope, E.D. 1869. A review of the species of the Plethodontidae and Desmognathidae.
Proceedings of the National Academy of Sciences Philadelphia 21:106.
Dundee, H.A. 1958. Habitat selection by aquatic Plethodontid salamanders of the
Ozarks, with studies on their life histories. Ph.D. Dissertation. University of
Michigan, Ann Arbor, MI.
Harrison III, J.R., and S.I. Guttman. 2003. A new species of Eurycea (Caudata:
Plethodontidae) from North and South Carolina. Southeastern Naturalist 2:159–
Hillis, D.M., D.A. Chamberlain, T.P. Wilcox, and P.T. Chippindale. 2001. A new species
of subterranean blind salamander (Plethodontidae: Hemidactyliini: Eurycea:
Typhlomolge) from Austin, Texas, and a systematic revision of central Texas
paedomorphic salamanders. Herpetologica 57:266–280.
Huelsenbeck, J.P., and F. Ronquist. 2001. MrBayes: Bayesian inference of phylogeny.
Bioinformatics 17: 754–755.
Jacobs, J.F. 1987. A preliminary investigation of geographic genetic variation and
systematics of the Two-lined Salamander, Eurycea bislineata (Green). Herpetologica
Kozak, K.H., R.A. Blaine, and A. Larson. 2006. Gene lineages and eastern
North American palaeodrainage basins: Phylogeography and speciation in
salamanders of the Eurycea bislineata species complex. Molecular Ecology
Moore, G.A., and R.C. Hughes. 1939. A new plethodontid from eastern Oklahoma.
American Midland Naturalist 22:696–699.
Moore, G.A., and R.C. Hughes. 1941. A new plethodont salamander from Oklahoma.
Moritz C., C.J. Schneider, and D.B. Wake. 1992. Evolutionary relationships within
the Ensatina eschscholtzii complex confirm the ring-species interpretation. Systematic
Posada, D., and K.A. Crandall. 1998. Modeltest: Testing the model of DNA substitution.
Rose, F.L., and F.M. Bush. 1963. A new species of Eurycea (Amphibia: Caudata)
from the southeastern United States. Tulane Studies in Zoology 10:121–128.
Ryan, T.J. 1997. Larva of Eurycea junaluska (Amphibia: Caudata: Plethodontidae),
with comments on distribution. Copeia 1997:210–215.
Ryan, T.J. 1998. Eurycea junaluska (Junaluska Salamander). Morphology. Herpetological
Sever, D.M., H.A. Dundee, and C.D. Sullivan. 1976. A new Eurycea (Amphibia:
Plethodontidae) from southwestern North Carolina. Herpetologica 32:26–29.
736 Southeastern Naturalist Vol.6, No. 4
Shaffer, H.B., and M.L. McKnight. 1996. The polytypic species revisited: Genetic
differentiation and molecular phylogenetics of the Tiger Salamander (Ambystoma
tigrinum) (Amphibia: Caudata) complex. Evolution 50:417–433.
Swofford, D.L. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Version 4. Sinauer Associates, Sunderland, MA.
Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D.G. Higgins. 1997.
The ClustalX windows interface: Flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Research 24:4876–4882.
Tumlison R., G.R. Cline, and P. Zwank.1990. Morphological discrimination between
the Oklahoma Salamander (Eurycea tynerensis) and the Graybelly Salamander
Eurycea multiplicata griseogaster. Copeia 1990:242–246.