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

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:

Lampropeltis alterna (gray-banded kingsnake) was first discovered in the Davis Mountains of West Texas (Brown, 1901). It is a medium sized colubrid snake inhabiting the Trans-Pecos/Chihuahuan Desert regions of West Texas and Northern Mexico. L. alterna 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. Although the potential habitat is much larger, L. alterna are currently found in 15 counties in Texas (Brewster, Crane, Crocket, Culberson, Edwards, El Paso, Hudspeth, Jeff Davis, Kinney, Pecos, Presidio, Sutton, Terrell, Upton, and 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 the range of most colubrid snakes.

The color and patterning of L. alterna is extremely variable throughout its range. There is great variation within populations and there are characteristic differences between populations (Miller, 1979). Over their range in West Texas, small habitat isolated populations are often separated by geographic and environmental barriers with gene flow between isolated groups considered to be minimal (Tenant, 1998).

  Two distinct color morphs were described by Tanzer, 1970. L. alterna with wide red bands were called “blairi” morphs, and those with little or no red bands and the presence of some alternating bands as “alterna.” Background color in both morphs range from black to light gray. Miller, 1979, described >90% of Eastern form L. alterna as having <16 wide, solid bands, and <6 alternate broken bands (often none). Most of the Eastern form are “blairi” morphs (see Fig. 1). They are also more numerous, possibly due to the greater availability of water, with watersheds such as the Rio Grande and Devil’s rivers. <10% of Eastern form are “alterna” morphs (see Fig. 2). Miller also described >90% of Western 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. 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 southeastern Brewster County north. Though both “blairi” and “alterna” morphs are found here, typically one finds extreme variation in the color, patterning, and background color (see Fig. 6,7,8).

Lampropeltis triangulum celaenops (New Mexico milksnake), is one of two 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 alterna with its extreme variation, the color and patterning of celaenops is stable throughout the range it shares with alterna.

Lampropeltis getula splendida (desert kingsnake) is also sympatric with L. alterna. L. g. splendida is dark brown or black with yellow-centered scales located predominantly on the sides of the body and those on the middle of the back form regularly spaced narrow crosslines (see Fig. 10). L. alterna and L. t. celaenops share similar hemipenal and vertebral structures that are unlike L. g. splendida (Gartska, 1982).

The purpose of this study is to use mitochondrial DNA sequences to assess the phylogenetic relationship between L. alterna and L. t. celaenops.

Polymerase Chain Reaction (PCR): Using PCR and total cellular DNA as a template, a fragment of mtDNA was amplified. The fragment encompassed a portion of the 3’ end of the nicotinamide adenine dinucleotide dehydrogenase subunit 4 and section of three transfer ribonucleic acid (tRNA) genes (tRNAHis, tRNASer, tRNALeu). Primers used were synthesize by Synthegen and labeled ND4 and Lew (Arevalo et al, 1994). PCR reactions were carried out in 100 microL volumes consisting of 2 microL of template DNA, 5 microL of primers (1 microM), and PCR solution readily mixed with the deoxynucleotide triphosphates, Thermus aquaticus DNA polymerase, and H2O available as Supermix® by Invitrogen. DNA was denatured initially at 94C for 5 min, then 35 cycles of amplification were carried out under the following conditions: 94C denaturation for 60 sec, 55C annealing for 60 sec, and 72C for 60 sec, followed by a final 5 min extension at 72C. Two microL of the resulting PCR product was electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lone Star Labs, Houston, Texas, 77054, using an automated sequencer, directly sequenced the PCR product.

Additional sequence data were obtained from GenBank, combined with collected sequence data, and the sequences were aligned by CLUSTAL W on SDSC Biology Workbench.

RESULTS AND DISCUSSION:

A Rooted tree generated by Phylip’s Drawgram showed three clusters:

1. L. t. celaenops AY739630, AY739631, and 101 were closely related.

2. L. alterna AY739636 (Val Verde Co.), 032 (Val Verde Co.) and AY497307 (Crockett Co.) were closely related. These 3 snakes are all Eastern form alterna.

3. L. alterna AY739634 (Jeff Davis Co.), AY739635 (Jeff Davis Co.), and AY739633 (Mexico, Nuevo Leon) were closely related. These 3 snakes are the 2 Western form and the alterna from Mexico.

Current taxonomy considers L. alterna distinct from L. t. celaenops based on morphological differences (Gartska, 1982). However, a recent paper (Bryson Jr, 2007), using the sequence of the mitochondrial ND4 enzyme subunit gene, places both L. alterna and L. t. celaenops together in a “northern clade”. Further research using nuclear markers to assess gene flow among these groups will be necessary to determine if the currently recognized taxa do represent species and if the mtDNA data are indeed in error.

REFERENCES:

Arevalo E, Davis SK, Sites JW Jr (1994) Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporous grammicus complex (Phrynosomatide) in central Mexico. Systemic Biology, 43, 387-418.

Brown AE (1901) A new species of Ophibolus from western Texas. Proc. Nat Acad. Sci., 55, 543-558.

Bryson RW Jr, Pastorini J, Burbrink FT, Forstner MRJ (2007) A phylogeny of the Lampropeltis mexicanacomplex (Serpentes: Colubridae) based on mitochondrial DNA sequences suggests evidence for species-level polyphyly within Lampropeltis. Mol. Phylogen. and Evol., 43, 674-684.

Gartska WR (1982) Systematics of the mexicana complex species group of the colubrid genus Lampropeltis, with an hypothesis mimicry. Brevoria, 466, 1-35.

Miller D (1979) A life history study of the Gray-Banded kingsnake, Lampropeltis mexicana alterna in Texas. Chihuahuan Desert Res. Inst. Contrib., 87, 1-48.

 Tanzer EC (1970) Polymorphism in the mexicana complex of Kingsnakes, with notes on their natural history. Herpetologica, 26, 419-428.

Tenant A (1998) The snakes of Texas. Texas Monthly Press, Austin.

Fig. 1. .L. alterna, Eastern form, Val Verde Co, Juno Rd “blairi”

Fig. 2. L. alterna, Eastern form, Val Verde Co, Juno Rd “alterna”

MATERIALS AND METHODS:

Samples: L. alterna (031 and 032) and L. g. splendida (111) were collected from Val Verde Co - Langtry. L. t. celaenops (101) was collected from Brewster Co – Black Gap. Shed skins were taken from these snakes. DNA Isolation: Total genomic DNA was extracted from shed skin using a commercially available Kit (Invitrogen) and the protocol for isolation of DNA from mouse tails. About 10 micrograms of DNA could be extracted from about 20 milligrams of skin

 

Fig. 3. L. alterna, Western form, Jeff Davis Co, McDonald Observat.

Fig. 7.L. alterna, Intergrade, Brewster Co., Black Gap

Fig. 6. L. alterna, Intergrade, Brewster Co., Black Gap

Fig. 9. Lampropeltis triangulumcelaenops, Brewster Co., Black Gap

Fig. 10. Lampropeltis getulasplendida, Val Verde Co.

Fig. 4. L. alterna, IntergradeBrewster Co., Black Gap

Fig. 5. L. alterna, IntergradeBrewster Co., Black Gap

Fig. 8. L. alterna. Intergrade, Brewster Co., Black Gap