A Phylogeny of  Three Sympatric Species of Lampropeltis: L. alterna, L. triangulum celaenops, and L. getula splendida (Serpentes: Colubridae) Jenny Hernandez, Miguel Quirch, and E. L. NordykeDepartment of Biology, University of St. Thomas, Houston TX, 77006

1. A Phylogeny of Three Sympatric Species of Lampropeltis:
L. alterna, L. triangulum celaenops, and L. getula splendida (Serpentes:
Colubridae)
Jenny Hernandez, Miguel Quirch, and E. L. Nordyke
Department of Biology, University of St. Thomas, Houston TX, 77006
ABSTRACT:
The gray-banded kingsnake, Lampropeltis alterna, is a medium sized colubrid snake found in the Trans-Pecos/Chihuahuan Desert regions of west Texas and northern Mexico. It inhabits the dry hillsides, canyons, and mountain slopes of the
northern Chihuahuan Desert along the Rio Grande, eastward into the Edwards Plateau, and westward into the mountains of the western Trans-Pecos. The color and patterning of L. alterna is extremely variable throughout its range with
local population groups often-times separated by geographic barriers and availability of water. Gene flow between isolated population groups is thought to be minimal. The New Mexico milksnake, Lampropeltis triangulum celaenops, and
the desert kingsnake, Lampropeltis getula splendida, whose range extends across the southwestern United States, are both sympatric with L. alterna all throughout its very limited range, were selected as outgroup taxa. The phylogeny of
these species was studied using DNA sequences of the mitochondrial gene ND4. Sequences were aligned manually by eye and then analyzed using maximum parsimony and/or Bayesian inference.
INTRODUCTION:
MATERIALS AND METHODS:
Lampropeltis alterna (gray-banded kingsnake) was first discovered in the
Davis Mountains of West Texas (Brown, 1901). It is a medium sized colubrid Samples: L. alterna (031 and 032) and L. g. splendida (111) were collected from Val
snake inhabiting the Trans-Pecos/Chihuahuan Desert regions of West Texas and Verde Co - Langtry. L. t. celaenops (101) was collected from Brewster Co – Black
Northern Mexico. L. alterna inhabits the dry hillsides, canyons, and mountain Gap. Shed skins were taken from these snakes.
slopes of the northern Chihuahuan Desert along the Rio Grande, eastward into
the Edwards Plateau, and westward into the mountains of the western Trans- DNA Isolation: Total genomic DNA was extracted from shed skin using a
Pecos. Although the potential habitat is much larger, L. alterna are currently commercially available Kit (Invitrogen) and the protocol for isolation of DNA from
Fig. 3. L. alterna, Western form,
found in 15 counties in Texas (Brewster, Crane, Crocket, Culberson, Edwards, El Fig. 1. .L. alterna, Eastern form, Fig. 2. L. alterna, Eastern form,
Jeff Davis Co, McDonald Observat. mouse tails. About 10 micrograms of DNA could be extracted from about 20
Val Verde Co, Juno Rd “blairi” Val Verde Co, Juno Rd “alterna”
Paso, Hudspeth, Jeff Davis, Kinney, Pecos, Presidio, Sutton, Terrell, Upton, and milligrams of skin
Val Verde), Eddy County in New Mexico, and in the Mexican states of Coahuila,
Durango, and Nuevo Leon. Area-wise, this is a small range when compared to Polymerase Chain Reaction (PCR): Using PCR and total cellular DNA as a
the range of most colubrid snakes. template, a fragment of mtDNA was amplified. The fragment encompassed a
portion of the 3’ end of the nicotinamide adenine dinucleotide dehydrogenase
The color and patterning of L. alterna is extremely variable throughout its subunit 4 and section of three transfer ribonucleic acid (tRNA) genes (tRNA His,
range. There is great variation within populations and there are characteristic tRNASer, tRNALeu). Primers used were synthesize by Synthegen and labeled ND4
differences between populations (Miller, 1979). Over their range in West Texas, and Lew (Arevalo et al, 1994). PCR reactions were carried out in 100 microL
small habitat isolated populations are often separated by geographic and volumes consisting of 2 microL of template DNA, 5 microL of primers (1 microM),
environmental barriers with gene flow between isolated groups considered to be Fig. 4. L. alterna, Intergrade Fig. 5. L. alterna, Intergrade Fig. 6. L. alterna, Intergrade, and PCR solution readily mixed with the deoxynucleotide triphosphates, Thermus
minimal (Tenant, 1998). Brewster Co., Black Gap Brewster Co., Black Gap Brewster Co., Black Gap aquaticus DNA polymerase, and H2O available as Supermix® by Invitrogen. DNA
Two distinct color morphs were described by Tanzer, 1970. L. alterna with was denatured initially at 94°C for 5 min, then 35 cycles of amplification were
wide red bands were called “blairi” morphs, and those with little or no red bands carried out under the following conditions: 94°C denaturation for 60 sec, 55°C
and the presence of some alternating bands as “alterna.” Background color in annealing for 60 sec, and 72°C for 60 sec, followed by a final 5 min extension at
both morphs range from black to light gray. Miller, 1979, described >90% of 72°C. Two microL of the resulting PCR product was electrophoresed on a 1%
Eastern form L. alterna as having <16 wide, solid bands, and <6 alternate broken agarose gel and stained with ethidium bromide. Lone Star Labs, Houston, Texas,
bands (often none). Most of the Eastern form are “blairi” morphs (see Fig. 1). 77054, using an automated sequencer, directly sequenced the PCR product.
They are also more numerous, possibly due to the greater availability of water, Additional sequence data were obtained from GenBank, combined with collected
with watersheds such as the Rio Grande and Devil’s rivers. <10% of Eastern sequence data, and the sequences were aligned by CLUSTAL W on SDSC Biology
Fig. 7.L. alterna, Intergrade, Fig. 8. L. alterna. Intergrade,
form are “alterna” morphs (see Fig. 2). Miller also described >90% of Western Brewster Co., Black Gap Brewster Co., Black Gap Workbench.
form L. alterna as having >16 narrow, solid bands, >6 alternate broken bands,
and speckling on the body, usually around the head and neck. Though Western
form are highly variable, most are recognized as “alterna” morphs (see Fig. RESULTS AND DISCUSSION:
3,4,5). The Western form inhabits a dryer region (Chihuahua Desert) and are far
fewer in number. An area of intergradation between the two forms runs through A Rooted tree generated by Phylip’s Drawgram showed three clusters:
southeastern Brewster County north. Though both “blairi” and “alterna” 1. L. t. celaenops AY739630, AY739631, and 101 were closely related.
morphs are found here, typically one finds extreme variation in the color,
patterning, and background color (see Fig. 6,7,8). 2. L. alterna AY739636 (Val Verde Co.), 032 (Val Verde Co.) and AY497307
Fig. 9. Lampropeltis triangulum
(Crockett Co.) were closely related. These 3 snakes are all Eastern form
Fig. 10. Lampropeltis getula
Lampropeltis triangulum celaenops (New Mexico milksnake), is one of two celaenops, Brewster Co., Black Gap splendida, Val Verde Co. alterna.
Lampropeltis species that is sympatric (shares the same range) with L. alterna.
Fig. 9 shows a typical celaenops with its triads of red, black, and white. Unlike 3. L. alterna AY739634 (Jeff Davis Co.), AY739635 (Jeff Davis Co.), and
alterna with its extreme variation, the color and patterning of celaenops is stable AY739633 (Mexico, Nuevo Leon) were closely related. These 3 snakes are the 2
throughout the range it shares with alterna. Western form and the alterna from Mexico.
REFERENCES:
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