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T.J. Hibbitts, S.A. Wahlberg, and G. Voelker
22001155 SOUTHEASTERN NATURALIST 1V4o(2l.) :1241,3 N–2o2. 02
Resolving the Identity of Texas Desmognathus
Toby J. Hibbitts1,*, Scott A. Wahlberg2, and Gary Voelker1
Abstract - Dusky salamanders in Texas have long been identified as Desmognathus auriculatus
(Southern Dusky Salamander). However, recent taxonomic work has challenged
this notion, restricting D. auriculatus to areas east of the Apalachicola River in Florida and
Georgia, while all Texas populations are now considered D. conanti. Regrettably, Texas
specimens were not included in the studies upon which these proposed taxonomic changes
were based. Dusky salamanders have become increasingly difficult to find in Texas and have
only been observed in the state 3 times since 1980. We surveyed 18 sites of which 6 were
historical localities. We found 4 extant populations of dusky salamanders at new locations
in Texas, one of which was a county record. We were unable to find dusky salamanders at
some historically robust populations. We also used mtDNA sequence data to verify that
Texas dusky salamanders are indeed D. conanti.
Introduction
The taxonomic status of dusky salamanders (Desmognathus sp.) in East Texas
has recently come into question (Beamer and Lamb 2008). Most authors have long
considered Texas dusky salamanders to be Desmognathus auriculatus (Holbrook)
(Southern Dusky Salamander) based on morphology and habitat (Cook and Brown
1974, Dixon 2013, Petranka 1998), although some early authors considered some
Texas individuals to be D. brimleyorum (Stejneger) (Ouachita Dusky Salamander;
Livezey 1950, Sanders and Smith 1949). Doubt about the validity of East Texas
D. auriculatus arose when it was discovered that D. auriculatus from Mississippi
were nested within D. conanti Rossman (Spotted Dusky Salamander) based on
mtDNA sequence data (Kozak et al. 2005). A subsequent study also using mtDNA
to better understand the relationships of dusky salamanders from the Southeastern
Coastal Plain restricted the distribution of the Southern Dusky Salamander
to Florida and Georgia east of the Apalachicola River (Beamer and Lamb 2008).
Beamer and Lamb (2008) were unable to obtain samples from Texas, but samples
from similar habitats to those found in East Texas in central and eastern Louisiana
all were genetically identified as D. conanti. Therefore, they assigned all Texas
specimens as D. conanti.
Dusky salamanders are historically known from 18 counties in eastern Texas
(Dixon 2013). However, recent collection records are sparse despite considerable
sampling effort at historical localities (T.J. Hibbits, pers observ.). The apparent
decline in Desmognathus populations in East Texas is alarming in light of similar
1Biodiversity Research and Teaching Collection, Department of Wildlife and Fisheries Sciences,
Texas A&M University, College Station, TX 77843-2258. 2USDA Forest Service,
Southern Research Station, 506 Hayter Street, Nacogdoches, TX 75965. *Corresponding
author - thibbitts@tamu.edu.
Manuscript Editor: John Placyk
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2015 Vol. 14, No. 2
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declines documented in Florida (Means and Travis 2007) and the decline of
amphibians globally (Houlahan et al. 2000). The Texas habitats where dusky salamanders
have been most recently found were small spring-fed creeks with sandy
substrates as well as sloughs and cypress/gum swamps (T.J. Hibbits, pers observ.).
Our study had 2 main objectives. First, we wanted to locate populations of Desmognathus
in Texas. We chose to search in counties that historically had the largest
number of specimens and collection sites to increase our chances of finding extant
populations. Second, we wanted to determine the relationships of Texas dusky
salamanders to other dusky salamanders of the Coastal Plains of the southeastern
United States. We chose to use mtDNA to investigate these relationships
Methods
We searched for dusky salamanders at historic localities and other habitats that
were suitable based on habitat descriptions for D. conanti and D. auriculatus (Petranka
1998). Historic localities were located by querying the VerpNet database
(http://www.vertnet.org) and its 192 museum collections as well as the Biodiversity
Research and Teaching Collection (TCWC), the Texas Natural History Collection
(TNHC), and the Amphibian and Reptile Diversity Research Center (UTAA). Surveys
for salamanders consisted of walking along spring runs, streams, and sloughs
overturning logs and other debris. We also dip-netted the leaf litter and vegetation
in these aquatic habitats and sifted through surface leaf litter, especially around
springs and seeps. Each survey site was searched for at least 2 person hours with at
least 2 observers. Dusky salamanders in Texas have been found year round based
on museum records; therefore we did not focus surveys on any specific time of year,
although most surveys were done in the spring months.
When encountered, at least 2 individual Dusky Salamanders were collected
per locality. We used the E.Z.N.A. tissue DNA kit (Omega) to extract whole
genomic DNA from liver samples and the polymerase chain reaction (PCR) to
amplify the mitochondrial cytochrome oxidase subunit c (cox1; 550 bp segment);
primers and reaction conditions followed those in Beamer and Lamb (2008).
We selected this gene because it allowed us to enhance our overall dataset by
including extensive Desmognathus sequences available from GenBank (i.e.,
from Beamer and Lamb 2008). We first did an analysis with the complete dataset
from Beamer and Lamb (2008), which included D. auriculatus, D. conanti,
and our new material. We then pruned the clade using a subset of Desmognathus
species that were most closely related to D. conanti based on the results of our
initial analysis of the complete dataset, including: D. apalachicolae Means and
Karlin (Apalachicola Dusky Salamander), D. brimleyorum, D. carolinensis Dunn
(Carolina Mountain Dusky Salamander), D. fuscus, D. monticola Dunn (Seal
Salamander), D. ocoee Nicholls (Ocoee Salamander), and D. santeetlah Tilley
(Santeetlah Dusky Salamander) (Table 1).
Automated sequencing was performed using BigDye chemistry (Applied Biosystems)
and both forward and reverse sequencing products were run on an ABI 377
or 3730 sequencer at the University of Florida sequencing facility (Interdisciplinary
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Table 1. Dusky salamander (Desmognathus ssp.) samples and their collection localities. These
specimens were used in our phylogenetic analysis to determine the taxonomic placement of the Texas
dusky salamander samples.
Specimen number Species State County
EU311666 D. apalachicolae Florida Liberty
EU311708 D. apalachicolae Florida Leon
BLowe57 D. apalachicolae Florida Liberty
FC11578 D. brimleyorum Oklahoma LeFlore
RMB2327 D. brimleyorum Arkansas Nevada
RMB2201 D. brimleyorum Arkansas Polk
EU311713 D. carolinensis Tennessee Yancey
EU311651 D. conanti Georgia Effingham
EU311668 D. conanti South Carolina Barnwell
EU311709 D. conanti Georgia Wayne
EU311710 D. conanti Georgia Wayne
TJR2470 D. conanti Georgia Richmond
EU311698 D. conanti North Carolina Henderson
EU311667 D. conanti Kentucky Livingston
EU311712 D. conanti Alabama Lawrence
EU311672 D. conanti Mississippi Jasper
EU311685 D. conanti Mississippi Jasper
ASU23806 D. conanti Mississippi Jasper
EU311673 D. conanti Louisiana Washington
EU311677 D. conanti Alabama Butler
EU311679 D. conanti Florida Santa Rosa
EU311678 D. conanti Alabama Baldwin
EU311684 D. conanti Florida Washington
EU311671 D. conanti Louisiana West Feliciana
EU311674 D. conanti Mississippe Amite
TJH2696 D. conanti Texas Tyler
94726 D. conanti Texas Tyler
TJH2756 D. conanti Texas Newton
TJH3263 D. conanti Texas Newton
TJH3264 D. conanti Texas Newton
TJH3265 D. conanti Texas Newton
TJH3266 D. conanti Texas Sabine
TJH3270 D. conanti Texas Sabine
TJH3262 D. conanti Texas Newton
TJH2757 D. conanti Texas Newton
TJH2758 D. conanti Texas Newton
TJH3269 D. conanti Texas Sabine
CSA330 D. conanti Texas Newton
EU311699 D. conanti Louisiana Grant
EU311702 D. fuscus North Carolina Caldwell
EU311662 D. fuscus North Carolina Wilkes
EU311688 D. monticola North Carolina Watauga
EU311690 D. monticola North Carolina Watauga
EU311647 D. ocoee Georgia Lumpkin
EU311652 D. ocoee Georgia Douglas
EU311715 D. ocoee Georgia Union
EU311676 D. santeetlah North Carolina Graham
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Center for Biotechnology Research, Gainesville, FL). Sequences were aligned with
Sequencher® version 5.0.1 sequence analysis software (Gene Codes Corp., Ann Arbor,
MI), and we used MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001) to generate
a Bayesian tree. In MrBayes, we incorporated codon-position–specific models, as
determined in MrModeltest 2.3 (Nylander 2004), and initiated 2 runs of 4 Markovchain
Monte Carlo (MCMC) chains for 5,000,000 generations, each starting from
a random tree and sampling every 100 generations. Default temperature-profile
settings were used. Each run resulted in 50,000 trees and converged on the same
topology. The first 5000 trees from each analysis were removed as “burn-in”; visualization
of –ln scores versus generations confirmed that chain stationarity had been
reached within 5000 trees. We used the remaining 90,000 trees to generate a 50%
majority-rule consensus tree.
Results
Our query of HerpNet and the major Texas herpetological collections revealed
68 localities where 361 specimen records of dusky salamanders have been found
in eastern Texas since 1940. The most recent collection date was 1995, but only 3
specimens from 2 localities were found after 1975. We searched 18 localities in 9
counties of East Texas. Six of these localities historically supported populations of
dusky salamanders (111 of the 361 specimen records for the state). We found 4 localities
that contained populations of dusky salamanders (TCWC 94274, 94726–27,
95134–36, 97079–85, 98122; Fig. 1). All of these localities were in clear, springfed,
first-order streams, and none of these localities were historical collection sites.
Our initial Bayesian analysis of Desmognathus (not shown) included all of the
samples used by Beamer and Lamb (2008) in addition to our new samples from
Texas. This analysis resulted in a very similar phylogeny to that of Beamer and
Lamb (2008), and showed that our new Texas specimens were sister to D. conanti,
and not closely related to D. auriculatus. Therefore we pruned the dataset to include
just those species that were most closely related to D. conanti, and this analysis
produced a generally well-supported tree (Fig. 2). Most importantly, the Texas
Desmognathus populations formed a monophyletic, albeit moderately supported
(0.86 posterior probability) group. This clade falls inside the larger D. conanti
clade. Within the D. conanti clade, 3 general clades can be identified (Fig. 2). First
is a well-supported clade distributed along the Atlantic Coastal Plain of Georgia
and the Carolinas. The second is another well-supported clade from Kentucky, Alabama,
eastern Mississippi and the panhandle of Florida. The third clade included
our samples from Texas and samples collected just east of the Mississippi River in
Louisiana and Mississippi.
However, if the moderate to weakly supported clades are collapsed, then the
third clade is better recognized as a polytomy comprising 3 strongly supported
clades: 1) eastern Louisiana and Mississippi, 2) Newton County, TX, and 3) Tyler
County, TX. Geographically, these 3 areas are separated by potentially strong river
barriers: the Mississippi River, the Sabine River, and the Neches River.
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Discussion
It appears that populations of Desmognathus in Texas may have suffered from
local extirpations. Recent searches for the species in the state previous to our efforts
Figure 1. Survey sites for dusky salamanders (Desmognathus spp.) in East Texas. Open
circles are sites where dusky salamanders were not observed and filled circles are sites
where they were collected.
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2015 Vol. 14, No. 2
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have been unsuccessful at a number of historical localities (Beamer and Lamb
2008). After the initial rediscovery in the Big Thicket National Preserve Canyonlands
Unit (P. Crump, Houston Zoo, Houston, TX, pers comm.), we concentrated
our efforts on first-order, spring-fed streams with sandy substrates, and all populations
were found in these sorts of habitats. Undoubtedly more populations exist in
similar conditions, but access on private property is not readily available and not all
18 counties in which they were historically found (or surrounding counties) were
surveyed for this study. Thus, additional data is needed before we can say with
absolute certainty that there has been a range reduction or population declines. We
did, however, survey without any success the sites that had the most known specimen
records.
It is clear from our results that Texas populations of Desmognathus should be
referred to as D. conanti, as proposed by Beamer and Lamb (2008). Also the habitat
associations of the specimens that we found fit those of D. conanti in other areas.
Figure 2. Bayesian phylogram of the mtDNA cox1 gene for Desmognathus in east Texas.
Species included in this analysis were: Desmognathus conanti, D. apalachicolae, D. brimleyorum,
D. carolinensis, D. fuscus, D. monticola, D. santeetlah, and D. ocoee. Asterisks
at nodes denote Bayesian posterior probabilities ≥ 0.95, and numbers indicate posterior
probabilities ≥ 50%. Numbers appended to non-Texas samples indicate sequences obtained
from GenBank.
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Desmognathus have also been found in cypress sloughs and swamps in Texas, but
we found no specimens in any of these habitats. It is possible that old specimens
from cypress swamps represent D. auriculatus or another undescribed species, but
we believe that is unlikely. Desmognathus auriculatus, sensu Beamer and Lamb
(2008), is restricted to east of the Apalachicola River in Georgia and Florida. No
populations sampled to date west of the Apalachicola River are genetically more
similar to D. auriculatus than to D. conanti, even with fairly thorough sampling
(Beamer and Lamb 2008, this study).
As in several other amphibian species, it appears that major rivers are dispersal
barriers (Vieites et al 2006, Zhao et al 2009). Within Texas, the salamanders to the
east of the Neches River are all more closely related to one another than to those
west of the Neches, although our specimens from central Newton County are closer
in straight line distance to those salamanders from Tyler County than the ones from
southern Sabine County. Specimens are known from west of the Trinity River, but
we were unable to find an extant population. Our results would suggest that if these
populations are indeed extirpated, then we have already lost genetically distinct
population segments. Our results further highlight the importance of the population
to the west of the Neches River and the need to locate additional populations in this
area, if any in fact exist. Fortunately, this area is under the protection of the national
park system.
In conclusion, our data support the assertion made by Beamer and Lamb (2008)
that the Texas dusky salamanders are D. conanti. We documented 4 extant populations
in eastern Texas, and Texas populations can be genetically distinguished from
other populations of D. conanti; however, at some Texas localities that seemed to
contain robust populations in the past (judged by specimen collection numbers at
localities), D. conanti are apparently now either extirpated or in very low numbers.
Acknowledgments
We would like to thank Don Shepard for tissues of several species of Desmognathus as
well as discussions about the project. The following people helped with field surveys: Rich
Adams, Jarred Melvin, Matthijs Hollanders, Matt Buckingham, Taylor Hall, and Bob Dink.
The Texas A&M University Institutional Animal Care and Use Committee approved this
research under AUP 2013-0081, and this project was also completed under the authority
of a permit issued by the Texas Parks and Wildlife Department (SPR-0506-662). This is
publication number 1489 of the Biodiversity Research and Teaching Collections at Texas
A&M University.
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