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Home , Florida kingsnake, Kingsnake, Lampropeltis getula, Lampropeltis nigra

Journal of Heredity, 2017, 1–13 doi:10.1093/jhered/esw086 Original Article

Original Article Phylogenetics of , Complex (Serpentes: ), in Eastern North America Kenneth L. Krysko, Leroy P. Nuñez, Catherine E. Newman, and Brian W. Bowen

From the Division of Herpetology, Museum of Natural History, University of Florida, 1659 Museum Road, Gainesville, FL 32611 (Krysko and Nuñez); School of Natural Resources and Environment, University of Florida, Gainesville, FL (Nuñez); Museum of Natural Science, State University, Baton Rouge, LA (Newman); Department of Biological Sciences, Louisiana State University, Baton Rouge, LA (Newman); and Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, Hawaii (Bowen).

Address correpondence to K. L. Krysko at the address above, or e-mail: [email protected]

Received July 21, 2016; First decision September 29, 2016; Accepted December 8, 2016.

Corresponding Editor: Stephen Karl

Abstract Kingsnakes of the Lampropeltis getula complex range throughout much of temperate and subtropical North America. Studies over the last century have used morphology and color pattern to describe numerous subspecies. More recently, DNA analyses have made invaluable contributions to our understanding of their evolution and . We use genetic and ecological methods to test previous hypotheses of distinct evolutionary lineages by examining 66 total and 1) analyzing phylogeographic structure using 2 mtDNA loci and 1 nuclear locus, 2) estimating divergence dates and historical demography among lineages in a Bayesian coalescent framework, and 3) applying ecological niche modeling (ENM). Our molecular data and ENMs illustrate that 3 previously recognized subspecies in the eastern comprise well-supported monophyletic lineages that diverged during the Pleistocene. The geographic boundaries of these 3 lineages correspond closely to known biogeographic barriers (Florida peninsula, Appalachian Mountains, and Apalachicola River) previously identified for other plants and , indicating shared geographic influences on evolutionary history. We conclude that genetic, ecological, and morphological data support recognition of these 3 lineages as distinct (Lampropeltis floridana, Lampropeltis getula, and Lampropeltis meansi).

Subject areas: Molecular systematics and phylogenetics, Conservation genetics and phylogenetics Key words: biogeography, divergence dating, mtDNA, speciation

Historical biogeography and ancient barriers to gene flow are major Bowen et al. 2016; Krysko et al. 2016). In other cases, a single factors in structuring genetic divergences within and between spe- wide-ranging species actually comprises multiple evolutionary lin- cies (Avise et al. 1987). In many cases, multiple organisms share eages or, conversely, multiple recognized species comprise a single the same phylogeographic partitions, indicating similar evolution- genetic lineage (Pfenninger and Schwenk 2007; Puebla et al. 2014). ary responses to shared biogeographic history (Soltis et al. 2006; With the increasing knowledge of how ancient barriers to gene flow

© The American Genetic Association. 2017. All rights reserved. For permissions, please e-mail: [email protected] 1 2 Journal of Heredity, 2017, Vol. 00, No. 00

have partitioned regional biodiversity, Burbrink et al. (2008, p. 286) and 6 in states along the Atlantic seaboard in eastern North America. raised the question for North American , “… are there really Blaney (1977) revised the Lampropeltis getula complex using both any single transcontinental squamate species?” morphology and color pattern, and recognized only 7 subspe- The increased availability of species distribution data combined cies (Figure 1): Lampropeltis getula californiae (Blainville 1835), with fine-scale climate information allow researchers to use predic- Lampropeltis getula holbrooki Stejneger 1902, Lampropeltis getula tive modeling to test hypotheses about lineage divergence and iden- nigra (Yarrow 1882), Lampropeltis getula nigrita Zweifel and Norris tify potential areas of sympatry or hybridization (Wiens and Graham 1955, and Lampropeltis getula splendida (Baird and Girard 1853) 2005; Raxworthy et al. 2007; Kozak et al. 2008). Ecological niche west of the Appalachian Mountains; and Lampropeltis getula flori- modeling (ENM) combines occurrence data and a set of current cli- dana Blanchard 1919 and Lampropeltis getula getula (Linnaeus 1766) mate layers to generate a probability surface of species occurrence. in eastern North America. Two other taxa in eastern North America When ENMs are generated for each taxon in question and visualized are either controversial or recently named. Barbour and Engles (1942) on a map, low model overlap between taxa may indicate some degree described Lampropeltis getula sticticeps from the Outer Banks of of ecological divergence (Wiens and Graham 2005; Raxworthy et al. based on color pattern and head morphology. Lazell 2007; Rissler and Apodaca 2007), facilitating the identification of and Musick (1973) supported these findings while other authors cryptic species. remained skeptical (Hillestad et al. 1975; Blaney 1977, 1979; Gibbons Kingsnakes of the Lampropeltis getula complex (Linnaeus) range and Coker 1978; Palmer and Braswell 1995). Means (1977) hypoth- throughout much of temperate and subtropical North America; esized that an unnamed taxon occurred in the Eastern Apalachicola along the Pacific coast from southward to the Mexican Lowlands in the Florida panhandle, and Krysko (2001) and Krysko Plateau, and eastward to and southward to Florida and Judd (2006) described this form as Lampropeltis getula meansi (Figure 1; Krysko 2001). Kingsnakes of this species complex are based on analyses of mtDNA and morphology. Blaney (1977), extremely variable in color pattern, and therefore, along with their Krysko (2001), and Means and Krysko (2001) noted the occurrence mostly docile disposition, are easily recognizable and very popular in of morphological intermediates (i.e., intergrades or hybrids) in zones the pet trade (Enge 1994; Krysko 2002). between these named taxa in eastern North America. Morphological There have been at least 18 subspecies described in this species intermediates were defined as commonly found individuals that pos- complex, 12 of which occur west of the Appalachian Mountains, sess intermediate phenotypic characters between adjacent taxa.

Figure 1. Distribution of kingsnakes in the Lampropeltis getula complex in North America: (A) Lampropeltis californiae (banded); (B) Lampropeltis holbrooki; (C) ; (D) Lampropeltis getula getula; (E) Lampropeltis getula “sticticeps”; (F) Lampropeltis getula floridana; (G–I) Lampropeltis getula meansi (patternless, striped, and wide-banded, respectively); (J) Lampropeltis splendida; (K) Lampropeltis getula nigrita; (L) Lampropeltis californiae (striped). Distributions are modified after Conant and Collins (1998), Krysko (2001), Stebbins (2003), Krysko and Judd (2006), and Pyron and Burbrink (2009a, 2009b). See online color version. Journal of Heredity, 2017, Vol. 00, No. 00 3

Krysko (2001) analyzed the 2 mitochondrial loci cytochrome b were conducted following Hillis et al. (1990) for blood and mus- (cyt b) and the nicotinamide adenine dinucleotide dehydrogenase cle tissue, Clark (1998) for air-dried shed skins, Iudica et al. (2001) subunit 4 (ND4) region; cyt b alone resulted in all samples in eastern for bone, or QIAquick PCR Purification Kit and DNeasy Blood and North America yielding one large polytomy, the ND4 region alone Tissue Kit (Qiagen Sciences, LLC) and ZR Genomic DNA™ Tissue revealed some population genetic structure, but the concatenated Microprep Kit (Zymo Research, LLC) for muscle tissue and shed sequences revealed three shallow genetic lineages that correspond to skins, following manufacturers recommendations. L. g. floridana, L. g. getula, and L. g. meansi. Subsequently, Pyron We amplified, sequenced, and analyzed a total of 2679 bp from and Burbrink (2009a, 2009b) provided a much greater sampling mtDNA cyt b; (1083 bp), mtDNA ND4 region (781 bp), and nuclear regime west of the Appalachian Mountains, and elevated 5 subspe- locus SPTBN1 (815 bp). Cytochrome b was amplified using the cies (L. g. californiae, Lampropeltis getula spendida, L. g. holbrooki, primers CYB 2 (Kessing et al. 1989), LGL765 (Bickham et al. 1995), L. g. nigra, and L. g. getula) to species status; however, based solely H15919 (Fetzner 1999), L14910 and H16064 (Burbrink et al. 2000), on analysis of cyt b they recognized a single lineage (L. getula) east and CYB 1L, CYB 2L, CYB 1H, CYB 2H (Krysko 2001). The ND4 of the Appalachian Mountains, and they did not examine samples region consisted of the ND4 gene and subsequent transfer ribonu- for L. g. nigrita from Mexico. cleic acids tRNAHis and tRNASer, and was amplified using the primers The distinct morphology and color patterns found in the ND4 and Leu (Arevalo et al. 1994; Rodríguez-Robles and de Jesús- Lampropeltis getula complex, along with its transcontinental geo- Escobar 1999), and ND4-L1 and ND4-H1 (Krysko et al. 2016). graphic distribution and occasional disjunct populations across the Because mtDNA is inherited as a single linkage unit, we expect that North American landscape make a fascinating subject for phyloge- phylogenies from cyt b and ND4 region would yield convergent gene ography. Herein, we increase our sampling effort in eastern North trees. SPTBN1 was amplified using the primers SPTBN1_F_APR and America and add a nuclear DNA locus to our data set to test previ- SPTBN1_R_APR (Ruane et al. 2014), and was chosen because it ous hypotheses of distinct genetic lineages by 1) reanalyzing phylo- represents an informative, single-copy locus that is likely evolving geographic structure, 2) estimating divergence dates and historical at a different rate than mtDNA loci (Townsend et al. 2008; Ruane demography among lineages in a Bayesian coalescent framework, et al. 2014). All 66 samples were sequenced for both mtDNA mark- and 3) analyzing niche differentiation and overlap using ENMs. ers, and a subset (n = 19) of individuals were sequenced for SPTBN1 For clarity and historic purposes, we treat named taxa in eastern (Table 1).

North America (L. g. floridana, L. g. getula, and L. g. meansi) and PCR was conducted in 25 μL reactions: 12.5 μL H2O, 9.5 μL L. g. nigrita from Mexico as subspecies prior to our species recogni- Apex™ Taq Red Master Mix 2.0X (2× buffer, 200 μM dNTPs, tion in this current study. 1.5 mM MgCl2), 1 μL each primer (10 μM), and 1 μL template DNA. PCR parameters for mtDNA included initial denaturing at 94 °C for 3 min, followed by 35 cycles of amplification: denaturing Materials and Methods at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at Laboratory Techniques 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. We used DNA sequence data from a total of 66 snakes (Table 1, PCR parameters for SPTBN1 included initial denaturing at 94 °C Figures 1 and 2); including 56 individuals from the Lampropeltis for 1.5 min, followed by 1) 5 cycles of amplification: denaturing getula complex in eastern North America and outgroups consisting at 94 °C for 30 s, annealing at 51 °C for 30 s, and extension at of 8 individuals from subspecies (1 L. g. nigrita) and recently elevated 72 °C for 1.5 min; 2) 10 cycles of amplification: denaturing at 94 °C taxa (2 L. holbrooki, 3 L. nigra, 1 L. spendida, 1 L. californiae) west for 30 s, annealing at 49 °C for 30 s, and extension at 72 °C for of the Appalachian Mountains; one of the sister taxon, short-tailed 1.5 min; and 3) 30 cycles of amplification: denaturing at 94 °C for , L. extenuata; and one from the closely related Scarlet 30 s, annealing at 48 °C for 30 s, and extension at 72 °C for 1.5 min, , Cemophora coccinea. In addition to the new DNA data followed by a final extension at 72 °C for 7 min (Sheehy 2012). PCR provided herein, this study utilizes sequences from Krysko (2001) products were electrophoresed on 1% agarose gels, visualized with and GenBank: L. spendida: spectrin beta nonerythrocytic intron 1 GelRed™ staining (Biotium, Inc., Hayward, CA), and compared (SPTBN1; Ruane et al. 2014); L. californiae: SPTBN1 (Burbrink with a DNA size standard. DNA sequences were resolved on various et al. 2012); Lampropeltis extenuata: cyt b (Burbrink and Lawson automated sequencers (Genomics Division, Interdisciplinary Center 2007), ND4 region (Rodríguez-Robles and de Jesús-Escobar 1999), for Biotechnology Research, University of Florida) and assembled SPTBN1 (Ruane et al. 2014); and C. coccinea: cyt b (Lawson et al. and edited with Geneious ver. 6.1 (http://www.geneious.com). 2005), SPTBN1 (Burbrink and Lawson 2007). To facilitate compari- son between phylogeographic findings and previous morphological Phylogenetic Analyses results, 46 of 66 (69.7%) of the individuals were used in both molec- For the combined mtDNA and nDNA analyses, a mixed-model ular and morphological analyses, including those that are considered approach was performed to infer trees and assess node support morphological intermediates (i.e., intergrades or hybrids, depending using models specific to each gene. The Bayesian information crite- on taxonomic nomenclature used; Krysko and Judd 2006). rion (BIC) in MEGA determined the best-fit nucleotide substitution DNA sequence data were obtained from blood, muscle tissue, models to be HKY (Hasegawa et al. 1985) with gamma distributed shed skins, and bone. Between 0.5 and 1.0 mL of blood was taken rate heterogeneity (HKY + Γ) for cyt b and ND4 region, and HKY from the caudal vein of live specimens and stored in lysis buffer for SPTBN1. (100 mM Tris–HCl, pH 8; 100 mM EDTA, pH 8; 10 mM NaCl; Bayesian inference analyses were conducted using BEAST ver. 1.8 1.0% sodium dodecyl sulfate) in approximately 1:10 blood to buffer (Drummond and Rambaut 2007) on the UF-HPC Galaxy instance ratio (White and Densmore 1992). Muscle tissue was taken from (http://hpc.ufl.edu; Giardine et al. 2005; Blankenberg et al. 2010; salvaged dead-on-road (DOR) specimens and stored in SED buffer Goecks et al. 2010). A relaxed clock method was used to infer line- (saturated NaCl; 250 mM EDTA, pH 7.5; 20% DMSO; Amos and age relationships without having to rely on a potentially arbitrary Hoelzel 1991, Proebstel et al. 1993) or 95% ethanol. DNA isolations molecular clock (Drummond et al. 2006). An uncorrelated lognormal 4 Journal of Heredity, 2017, Vol. 00, No. 00

Table 1. Lineage; mitochondrial haplotype; sample; locality, voucher (Lab [Lg] No.); and GenBank accession numbers (cytochrome b, ND4 region, and SPTBN1, respectively) for Kingsnakes (Lampropeltis) and outgroup taxa used in molecular analyses

Lineage Haplotype Sample Locality Voucher GenBank

Atlantic coast A GA, Banks Co.* U.S.A.: , Banks County, Yonah UF 128267 (Lg DQ360333, DQ360466, Church Rd, 9.8 km NW Homer 117, KLK 350) KU664812 Atlantic coast B GA, Walton Co.* U.S.A.: Georgia, Walton County, UF 121162 (Lg DQ360336, DQ360469, — Loganville 119) Atlantic coast C SC, Charleston Co.* U.S.A.: , Charleston UF 128282 (Lg DQ360337, DQ360470, — County, Edisto Island 120, KLK 528) Atlantic coast D SC, Greenwood Co.* U.S.A.: South Carolina, Greenwood UF 128284 (Lg DQ360338, DQ360471, — County, US 221, 2.6 km W Hwy 10 129, KLK 522) Atlantic coast E SC, McCormick Co. U.S.A.:South Carolina, McCormick (Lg 24, KLK DQ360339, DQ360472, — County, Long Cane Creek, SR 81 and 531) SR 28 Atlantic coast F SC, Jasper Co.* U.S.A.: South Carolina, Jasper County UF 128280 (Lg DQ360340, DQ360473, — 99, KLK 526) Atlantic coast G GA, Mitchell Co.* U.S.A.: Georgia, Mitchell County, (Lg 17, KME DQ360342, DQ360475, — Cotton m27) Atlantic coast G GA, Randolph Co.* U.S.A.: Georgia, Randolph County UF 128285 (Lg DQ360343, DQ360476, — 133, KLK 523) Atlantic coast G *NC, Dare Co.* U.S.A.: North Carolina, Dare County, UF 128281 (Lg DQ360341, DQ360474, — Hatteras Island 111, KLK 525) Atlantic coast H *NC, Dare Co.* U.S.A.: North Carolina, Dare County, UF 128283 (Lg DQ360346, DQ360479, — Hatteras Island 121, KLK 530) Atlantic coast H *NC, Dare Co.* U.S.A.: North Carolina, Dare County, (Lg 104, KLK DQ360347, DQ360480, — Hatteras Island 532) Atlantic coast H NC, Watauga Co.* U.S.A.: North Carolina, Watauga UF 128275 (Lg DQ360344, DQ360477, — County, Triplett 29, KLK 520) Atlantic coast H SC, Charleston Co.* U.S.A.: South Carolina, Charleston UF 128278 (Lg DQ360345, DQ360478, — County, Adams Run 37, KLK 521) Atlantic coast I NJ, Monmouth Co. U.S.A.: New Jersey, Monmouth UF 175393 (Lg KU727166, KU727165, County 141, KLK 3181 KU664825 Apalachicola J FL, Liberty Co.* U.S.A.: Florida, Liberty County, SR 67, UF 105383 (Lg DQ360325, DQ360458, — 3.7 km S SR 20 10, KLK 211) Apalachicola K FL, Liberty Co.* U.S.A.: Florida, Liberty County, SR 67, UF 128266 (Lg DQ360326, DQ36045, N Franklin County line 26, KLK 213) KU664824 Apalachicola L FL, Liberty Co.* U.S.A.: Florida, Liberty County, NFR UF 114323 (Lg DQ360327, DQ360460, — 103 and NFR 116 27, LEK 239) Apalachicola L FL, Liberty Co.* U.S.A.: Florida, Liberty County, NFR UF 114322 (Lg DQ360328, DQ360461, — 103 and NFR 116 28, KLK 240) Apalachicola M FL, Liberty Co.* U.S.A.: Florida, Liberty County UF 128276 (Lg DQ360329, DQ360462, — 30, DBM 104) Apalachicola N FL, Liberty Co.* U.S.A.: Florida, Liberty County, NFR (Lg 32, DBM 50) DQ360330, DQ360463, 110, 2.9 km S NFR 111 KU664823 Apalachicola O FL, Liberty Co.* U.S.A.: Florida, Liberty County, NFR (Lg 58, KLK DQ360331, DQ360464, 139 247) KU664822 Apalachicola P FL, Liberty Co. U.S.A.: Florida, Liberty County (Lg 31, KLK DQ360332, DQ360465, 537) KU664821 Apalachicola Q FL, Franklin Co.* U.S.A.: Florida, Franklin County, US UF 128263 (Lg DQ360323, DQ360456, — 98, 7 km E CR 30E 45, KLK 220) Apalachicola R FL, Franklin Co.* U.S.A.: Florida, Franklin County, Tates UF 128262 (Lg DQ360324, DQ360457, — Hell Swamp, New River 55, KLK 231) Apalachicola S FL, Bay Co.* U.S.A.: Florida, Bay County, SR 22, UF 109826 (Lg DQ360310, DQ360443, — 32.1 km W Wewahitchka 52, KLK 227) Apalachicola S *FL, Calhoun Co.* U.S.A.: Florida, Calhoun County, UF 114321 (Lg DQ360308, DQ360441, — Blountstown 5, KLK 31) Apalachicola S *FL, Calhoun Co. U.S.A.: Florida, Calhoun County, (Lg 6, KLK 32) DQ360309, DQ360442, — Blountstown Apalachicola S FL, Franklin Co.* U.S.A.: Florida, Franklin County, US UF 128273 (Lg DQ360312, DQ360445, — 98, 9.5 km W Carrabelle 46, KLK 221) Apalachicola S *FL, Leon Co.* U.S.A.: Florida, Leon County, Bloxham (Lg 13, KME DQ360311, DQ360444, — cutoff m3) Apalachicola T *FL, Gadsden Co.* U.S.A.: Florida, Gadsden County, US UF 128271 (Lg DQ360313, DQ360446, — 90 5.7 km W Quincy 47, KLK 222) Journal of Heredity, 2017, Vol. 00, No. 00 5

Table 1. Continued

Lineage Haplotype Sample Locality Voucher GenBank

Apalachicola U *FL, Gulf Co.* U.S.A.: Florida, Gulf County, Port (Lg 11, KME DQ360314, DQ360447, — St. Joe m25) Apalachicola V * FL, Gulf Co.* U.S.A.: Florida, Gulf County, Port (Lg 15, KME DQ360315, DQ360448, — St. Joe m26) Apalachicola W FL, Holmes Co.* U.S.A.: Florida, Holmes County, Rt UF 128261 (Lg DQ360316, DQ360449, — 179A, 3.8 km SW SR 2 65, KLK 257) Apalachicola X *FL, Jackson Co.* U.S.A.: Florida, Jackson County, CR (Lg 107, KLK DQ360317, DQ360450, — 271 491) Apalachicola Y FL, Jefferson Co.* U.S.A.: Florida, Jefferson County, UF 128270 (Lg DQ360318, DQ360451, — Goosepasture Rd, 1.9 km S Tram Rd 50, KLK 225) Apalachicola Z FL, Jefferson Co. U.S.A.: Florida, Jefferson County (Lg 105, KLK DQ360319, DQ360452, — 538) Apalachicola aa *FL, Leon Co.* U.S.A.: Florida, Leon County, UF 128272 (Lg DQ360320, DQ360453, — Meridian Rd, 0.2 km S Meridian 51, KLK 226) Hills Rd Apalachicola bb *FL, Wakulla Co.* U.S.A.: Florida, Wakulla County, NFR (Lg 12, KME DQ360321, DQ360454, — 312 and 313 m13) Apalachicola bb *FL, Wakulla Co.* U.S.A.: Florida, Wakulla County, (Lg 14, KME f3) DQ360322, DQ360455, — Arran Apalachicola cc GA, Echols Co.* U.S.A.: Georgia, Echols County, UF 128274 (Lg DQ360334, DQ360467, — Statenville 18, KME m10) Apalachicola dd GA, Thomas Co.* U.S.A.: Georgia, Thomas County, UF 128286 (Lg DQ360335, DQ360468, — Ochlockonee River 134, KLK 524) Florida peninsula ee FL, Charlotte Co. U.S.A.: Florida, Charlotte County, SR (Lg 34, KLK DQ360293, DQ360426, 776, S Englewood 533) KU664820 Florida peninsula ff FL, Miami-Dade Co. U.S.A.: Florida, Miami-Dade County, (Lg 3, KLK DQ360294, DQ360427, C-110 canal 94021) KU664819 Florida peninsula gg FL, Miami-Dade Co. U.S.A.: Florida, Miami-Dade County, (Lg 7, KLK 161) DQ360295, DQ360428, — Miami, 0.2 mi E SR 997 Florida peninsula hh FL, Hendry Co. U.S.A.: Florida, Hendry County, (Lg 19, KLK DQ360300, DQ360433, Hwy 80A, 18 km SE Clewiston 534) KU664818 Florida peninsula hh FL, Palm Beach Co. U.S.A.: Florida, Palm Beach County, (Lg 20, KLK DQ360299, DQ360432, — King Ranch S South Bay 535) Florida peninsula ii FL, Lee Co.* U.S.A.: Florida, Lee County, UF 128277 (Lg DQ360296, DQ360429, — Gasparilla Island, Boca Grande 35, KLK 527) Florida peninsula ii *FL, Pinellas Co.* U.S.A.: Florida, Pinellas County, UF 121121 (Lg DQ360297, DQ360430, — Pinellas Park 4, KLK 30) Florida peninsula jj FL, Monroe Co.* U.S.A.: Florida, Monroe County, UF 123777 (Lg DQ360298, DQ360431, Key Largo 36, KLK 490) KU664817 Florida peninsula kk *FL, Dixie Co.* U.S.A.: Florida, Dixie County, UF 128269 (Lg DQ360301, DQ360434, — CR 361, 5.7 km S Rocky Creek 115, KLK 316) Florida peninsula ll *FL, Duval Co.* U.S.A.: Florida, Duval County, (Lg 16, KME DQ360302, DQ360435, — Jacksonville f11) Florida peninsula mm *FL, Hernando Co.* U.S.A.: Florida, Hernando County, UF 111101 (Lg DQ360303, DQ360436, — Hernando Beach 23, KLK 230) Florida peninsula nn FL, Hillsborough Co. U.S.A.: Florida, Hillsborough County, (Lg 8, KLK 195) DQ360304, DQ360437, — Gibsonton Florida peninsula oo *FL, Levy Co. U.S.A.: Florida, Levy County, (Lg 22, KLK DQ360305, DQ360438, N Cedar Key 536) KU664816 Florida peninsula pp *FL, Levy Co. U.S.A.: Florida, Levy County, Cedar (Lg 116, KLK DQ360336, DQ360469, — Key 539) Florida peninsula qq *FL, Pinellas Co. U.S.A.: Florida, Pinellas County, (Lg 2, KLK 160) DQ360337, DQ360470, Gandy Blvd KU664815 rr Lampropeltis U.S.A.: Louisiana, Terrebonne Parish, (Lg 59, KLK DQ360348, holbrooki* Houma 519) DQ360481,KU664813 ss L. holbrooki* U.S.A.: , Perry County UF 128260 (Lg DQ360349, DQ360482, 90, KLK 259) KU664814 tt Lampropeltis nigra* U.S.A.: , Trigg County UF 128264 (Lg DQ360350, DQ360483, — 93, KLK 262) uu L. nigra U.S.A.: , Stewart County (Lg 96 KLK 529) DQ360351, DQ360484, KU664811 uu L. nigra* U.S.A.: Kentucky, Calloway County UF 128259 (Lg DQ360352, DQ360485, — 56, KLK 232) 6 Journal of Heredity, 2017, Vol. 00, No. 00

Table 1. Continued

Lineage Haplotype Sample Locality Voucher GenBank

vv Lampropeltis splen- (JF25sY), FTB AF337081, —, KF215128 dida 1554 ww Lampropeltis nigrita Mexico: Sonora (JF14sY) AF337070, KU727164, — xx Lampropeltis cali- (JF23sY), FTB AF337079, KU727163, JX648606 forniae 1530 yy Lampropeltis ex- LSUMZ 40624, DQ902131, AF138776, tenuata UF 150109 KF215118 zz Cemophora coccinea U.S.A.: Florida, Liberty County, CAS 203080 AF471091, DQ902282, Apalachicola National Forest KU727167

An asterisk before and after sample name indicates identified morphological intermediate (i.e., putative ) and/or identical sample was used in morphological analyses, respectively (Krysko and Judd 2006).

and not sampling local optima) using Tracer ver. 1.6 (http://beast. bio.ed.ac.uk/Tracer) for ESS values >200, as well as for a split stand- ard deviation less than 0.005 for −lnL tree values among chains that indicate parameter stationarity was achieved. Trees sampled prior to stationarity were discarded as burn-in, which occurred prior to 3 million generations. Trees from both independent MCMC runs were combined and burn-in was removed using LogCombiner ver. 1.8. The best statistically supported tree (i.e., maximum clade credibility tree) with mean heights was obtained using TreeAnnotator ver. 1.8 in BEAST. A phylogenetic hypothesis with posterior probabilities and estimated divergence dates was created using FigTree ver. 1.4 (http://tree.bio.ed.ac.uk/software/figtree/). The most credible infer- ences of phylogenetic relationships were confined to nodes where posterior probability was ≥95% (Felsenstein 2004).

Divergence Dating Fossil data in the form of parametric distributions offer a high degree of flexibility in integrating a time scale into a phylogenetic analysis (Morrison 2008; Ho and Phillips 2009). For the combined mtDNA and nDNA analyses, we used two fossil calibrations with BEAST, mostly following Ruane et al. (2014). We used a minimum age of 12.35 million years ago (Ma) for the most recent common ances- tor (MRCA) between the genera Cemophora and Lampropeltis (ln mean = 2.514) ± 0.23 lognormal standard deviation (Morrison 2008; Ho and Phillips 2009) yielding a 95% prior credible interval (PCI) of 8.4–18.0 Ma, which incorporates the oldest known Lampropeltis Figure 2. Distribution and locations of samples sequenced for (Lampropeltis similis; see Holman 2000). We provided a sec- kingsnakes of the Lampropeltis getula complex in eastern North America: ond calibration point for the MRCA between the 2 closely related yellow dots = Lampropeltis getula floridana from Florida peninsula; L. extenuata and L. getula complex with a minimum age of 7.6 Ma blue dots = Lampropeltis getula getula from the Atlantic coast; red dots = Lampropeltis getula meansi from the Eastern Apalachicola Lowlands (ln mean = 2.028) ± 0.22 lognormal standard deviation yielding a in the Florida panhandle; gray dots = morphological intermediates between 95% prior credible interval (PCI) of 5.2–10.9 Ma based on the old- L. g. floridana and L. g. getula; and coral dots = morphological intermediates est known L. getula and Stilosoma vetustum (=Lampropeltis vetusta) between L. g. getula and L. g. meansi. Green and pink polygons refer to (Auffenberg 1963; Holman 2000; Hulbert 2001) from the . Lampropeltis nigra and Lampropeltis holbrooki, respectively, on the western side of the Appalachian Mountains. Distributions are modified after Conant and Collins (1998), Krysko (2001) using multi-locus phylogeny, and Krysko Historical Demography and Judd (2006) using morphology. See online color version. We used Bayesian skyline plots (BSPs) in BEAST to evaluate his- torical population demography, particularly to determine if relaxed clock with constant population size, estimated base frequen- Lampropeltis lineages in eastern North America had undergone cies, randomly generated starting tree, and exponential uncorrelated population declines, expansions, or remained stable during the lognormal relaxed clock mean (ucld.mean) priors were used. Two most recent glacial advances and retreats. BSPs evaluate the coa- independent runs were performed consisting of three heated and one lescent history of each locus and allows tracing of Ne over time cold Markov chain Monte Carlo (MCMC) run for 30 million gen- without requiring a specified demographic model (i.e., constant erations with every 3000 sample being retained. Both MCMC runs size, exponential growth, etc.). We used a mixed-model analysis were analyzed independently (to confirm chains were converging using the same substitution models as above, and partitioned cyt b, Journal of Heredity, 2017, Vol. 00, No. 00 7

ND4 region, and SPTBN1 into three linked codon positions. We used was arbitrarily set at the lowest probability value for an occurrence a strict clock with lineage-specific substitution rates (e.g., 4.3E-3 for point (Pearson et al. 2007; Raxworthy et al. 2007). The ENMs for Atlantic Coast, 5E-3 for Eastern Apalachicola Lowlands + surround- L. g. getula, L. g. floridana, and L. g. meansi were then overlaid on ing region, and 5E-3 for Florida Peninsula lineages) obtained from a map, and the Weighted Sum (Spatial Analyst) tool was used to our phylogeny, along with Bayesian Skyline Coalescent, 10 grouped sum the 3 rasters. In the resulting sum raster, cells with a value of 2 coalescent intervals, and randomly generated starting tree priors. represented areas of overlap between 2 ENMs; there was no overlap BSPs were estimated for 100 million generations with every 1 mil- between all 3 ENMs. Once the ENMs were mapped and areas of lion sample being retained. Using Tracer, we confirmed ESS values overlap were highlighted, hybrid specimens were plotted on the map, >200, discarded 10 million generations as burn-in, and conducted and concordance between location of hybrids and predicted hybrid

Bayesian Skyline Reconstructions. For comparisons of tracing of Ne zones based on ENMs was analyzed by determining the number of over time with BSPs, we also calculated the nongenealogical coa- hybrid specimens that were located in a cell where 2 ENMs overlap lescent methods of Ramos-Onsins and Rozas’s R2 (Ramos-Onsins (i.e., predicted hybrid zones). and Rozas 2002), Fu and Li’s D* (Fu and Li 1993), and Tajima’s Because the entire ENM for L. g. meansi was within the range D* (Tajima 1989) to determine the stability of population growth of L. g. getula (see Results), we further investigated whether in each lineage using DnaSP (ver. 5.10.01; Rozas 2009). Our null L. g. meansi is restricted to a subset of climatic conditions occu- hypothesis assumes a stable population size. According to Fu and pied by L. g. getula. To examine variation in climatic suitability for Li’s D* and Tajima’s D*, a stable population size would be close each subspecies in environmental space, we extracted bioclim data to zero, whereas a significant negative value would suggest a recent for each L. g. meansi and L. g. getula locality point in DIVA vers. population expansion and a significant positive value would sug- 5.4 (Hijmans et al. 2001), from the same set of bioclim layers used gest a recent population decline or bottleneck (Burbrink and Castoe to build the ENMs, and ran a principle components analysis (PCA). 2009; Shepard and Burbrink 2009). A significant positive value of R2 indicates a recent population expansion and a significant negative value indicates a recent population decline or bottleneck (Ramos- Results

Onsins and Rozas 2002). BSPs may be more sensitive at tracing Ne Sequence comparisons including outgroup taxa from the 66 speci- over time than nongenealogical methods that do not consider phy- mens revealed 369 polymorphic sites, and 171 parsimony-inform- logenetic structure (Felsenstein 1992; Pybus et al. 2000; Pilkington ative characters within the analyzed 2679 bp of combined mtDNA et al. 2008; Shepard and Burbrink 2009). and nDNA. The full coverage among all samples for only the Summary statistics were calculated using DnaSP. Sequence mtDNA loci revealed 52 unique haplotypes with haplotype diversity comparisons for the number of informative characters and unique h = 0.988 (A–zz; Table 1). Genbank accession numbers are listed in haplotypes were obtained. Using haploid data, we estimated within Table 1. lineage and overall genetic variation with differentiation (χ2) and Phylogenetic Analyses levels of population genetic divergence and migration rate (FST and Nm, respectively; Hudson et al. 1992) with 10 000 permutation test Our multi-locus Bayesian phylogeny illustrates three genetic lineages replicates (Librado and Rozas 2009). in eastern North America, which correspond to the named taxa, Lampropeltis getula floridana, L. g. getula, and L. g. meansi. All Ecological Niche Modeling lineages are well supported with posterior probabilities at or above Based on the mtDNA phylogeny (see Results), tissue sample localities 95% (Figure 3), with the exception of 3 individuals (haplotype G) were classified into 3 clades (L. g. getula, L. g. floridana, L. g. meansi) that cluster with the appropriate Atlantic Coast Lineage (L. g. get- and plotted on a map in ArcGIS ver. 10.1 (Environmental Systems ula) but lack statistical support. The monophyly of L. g. nigrita from Research Institute, Redlands, CA). We generated a minimum con- Mexico along with its sister taxon, L. californiae, is also well sup- vex polygon (MCP) around the points for each lineage and used ported, with posterior probabilities of 99%. these MCPs as a conservative estimate of lineage distributions. All collection localities (latitude, longitude) were downloaded from the Divergence Dating online databases VertNET (vertnet.org) and GBIF (gbif.org). Only Divergence dating estimates (Figure 3) indicate that the MRCA those collection localities falling within one of the MCPs were used between Cemophora and Lampropeltis occurred ca. 11.9 Ma to create the models. Because there is no evidence that hybrid zones (95% highest posterior density [HPD] = 16.5–7.8 Ma; during the are inhabited exclusively by hybrids, identified morphological inter- Miocene). The MRCA between L. extenuata and the L. getula mediate specimens used in the genetic analyses were included when complex occurred ca. 6.7 Ma (95% HPD = 9.4–4.3 Ma; during generating the MCPs, but to be conservative, all designated morpho- the Miocene through the Pliocene). The MRCA between L. califor- logical intermediates were excluded from the ENMs. niae and L. g. nigrita occurred ca. 1.5 Ma (95% HPD = 2.4–0.6 To generate the ENMs, we used the 19 WorldClim bioclimatic Ma; during the Pleistocene). The MRCA between L. californiae + layers (http://www.worldclim.org) representing various trends in L. g. nigrita and L. splendida occurred ca. 2.5 Ma (95% HPD = 3.8– precipitation and temperature from 1950–2000 (Hijmans et al. 1.3 Ma; during the Pliocene through Pleistocene). The MRCA 2005). Spatial resolution of the layers was 1 km2. ENMs were gener- between L. californiae + L. g. nigrita + L. splendida and all remain- ated in Maxent vers. 3.2.19 (Phillips et al. 2006) using default set- ing taxa of the L. getula complex eastward occurred ca. 4.7 Ma tings, the 19 climate layers, and the NHC species occurrence data. (95% HPD = 6.8–2.7 Ma; during the Miocene through Pliocene). ENMs were converted from ASCII format to raster in ArcGIS and The MRCA between L. nigra and L. holbrooki occurred ca. 0.4 visualized on a map. Ma (95% HPD = 0.7–0.1 Ma; during the Pleistocene). The MRCA To analyze niche overlap, each ENM was converted from a con- between L. nigra + L. holbrooki and the L. getula complex in eastern tinuous distribution to binary (presence/absence), with presence North America occurred ca. 0.4 Ma (95% HPD = 3.1–1 Ma; during cells coded as 1 and absence cells coded as 0. Presence threshold the Pliocene through the Pleistocene). 8 Journal of Heredity, 2017, Vol. 00, No. 00

Figure 3. Bayesian inference phylogeny using 2679 base pairs of combined mtDNA (cytochrome b and ND4 region) and nDNA (SPTBN1) for Kingsnakes (Lampropeltis getula complex) in eastern North America. Note that values above nodes represent posterior probabilities (≥95%); values below major nodes represent mean divergence time estimates of the most recent common ancestor (MRCA); bars at major nodes represent 95% highest posterior density (HPD); letters at end of sample names indicate haplotype (Table 1); and an asterisk before or after sample name indicates a morphological intermediate/hybrid or identical sample was used in morphological analyses, respectively (Krysko and Judd 2006). See online color version.

In eastern North America, the MRCA between cohorts in the Florida peninsula (L. g. floridana) and Atlantic coast (L. g. getula) + Eastern Apalachicola Lowlands (L. g. meansi) occurred ca. 0.54 Ma (95% HPD = 0.8–0.2 Ma; during the Pleistocene), and the MRCA between L. g. getula and L. g. meansi occurred ca. 0.45 Ma (95% HPD = 0.7–0.2 Ma; during the Pleistocene). Extant hap- lotypes within the Florida peninsula, Atlantic coast, and Eastern Apalachicola Lowlands + surrounding region lineages, each coalesce to a MRCA ca. 0.3 Ma (95% HPD = 0.5–0.1 Ma).

Historical Demography Using genealogical coalescent methods, BSPs for the Atlantic coast and Eastern Apalachicola Lowlands + surrounding region show a stable population over the last 60 000 and 100 000 years, respectively (Figure 4). The Florida peninsula Lineage shows an overall gradual increase in historical population size beginning 150 000 years ago before becoming stable. Using nongenealogical methods, we fail to reject our null hypothesis of a stable popula- tion over time for the Atlantic coast (Fu and Li’s D* = −1.937, P = 0.057; Tajima’s D* = −1.417, P = 0.076) and Florida peninsula lineages (Fu and Li’s D* = −1.031, P = 0.167; Tajima’s D* = −1.466, P = 0.066). However, we reject our null hypothesis of a stable popu- Figure 4. Bayesian skyline plots (BSPs; left) and mismatch distributions lation over time for the Eastern Apalachicola Lowlands + surround- with tests against expectation of growth (right) of 3 genetic lineages of ing region lineage (Fu and Li’s D* = −2.944, P = 0.006; Tajima’s Lampropeltis in eastern North America. Note that the central line in each BSP D* = −2.201, P = 0.003), which indicates a recently increasing pop- represents the median value for the log of the population size (Ne* t) and ulation. R2 statistics reject our null hypothesis of a stable population the shaded area represents 95% highest posterior density (HPD). See online over time for all 3 lineages (Atlantic coast: R2 = 0.0845, P = 0.008; color version. Eastern Apalachicola Lowlands + surrounding region: R2 = 0.046, P < 0.001; Florida peninsula: R2 = 0.0752, P = 0.003), indicating Ecological Niche Modeling recently increasing populations. Mismatch distributions support The ENMs for L. g. getula, L. g. floridana, and L. g. meansi closely the results of the R2 statistic and show an expanding population corresponded to each of their geographic ranges based on morphol- (Figure 4). ogy (Figures 1 and 5). Some overprediction was observed in the Journal of Heredity, 2017, Vol. 00, No. 00 9

ENM for L. g. getula, with species occurrence predicted westward between L. g. meansi and L. g. getula is generally warmer, wetter, of the range limit into coastal Louisiana and . The ENM of and has lower seasonality than the rest of the L. g. getula range L. g. meansi overlaps entirely with that of L. g. getula and overpre- northward along the Atlantic coast. dicts into regions westward of the Apalachicola River, eastward of the Ochlockonee River, and northward of Telogia Creek. The ENM Species Recognition for L. g. floridana is consistent with the hypothesized range (Krysko We follow the General Lineage Concept of Species (de Queiroz 2001) and is restricted to peninsular Florida. All of the L. g. meansi × 1998, 2007) for recognizing lineages at the species level; each are L. g. getula morphological intermediates fell within their respective made up of connected subpopulations in an ancestor-descendant region of ENM overlap, and all of the L. g. floridana × L. g. get- series (those in eastern North America) and exhibits diagnosability ula morphological intermediates fell within their respective region using morphology and color pattern. Because all 3 lineages in east- of ENM overlap (Figure 5). There were no areas of ENM overlap ern North America, as well as L. g nigrita in Mexico, can also be between all 3 morphotypes. A scatterplot of the PCA factor scores diagnosed using morphology (secondary operational criterion; de shows that L. g. meansi is restricted to a small subset of the cli- Queiroz 1998, 2007), we recognize each as a full species. matic space occupied by L. g. getula (Figure 6). The area of overlap

Lampropeltis floridana, Blanchard (1919)

Coluber getulus (part) Linnaeus (1766) Ophibolus getulus (part) Baird and Girard (1853) getulus (part) Duméril et al. (1854) Lampropeltis getulus (part) Cope (1860) Lampropeltis getulus floridana Blanchard (1919) Lampropeltis getulus brooksi Barbour (1919) in Blaney (1977) Lampropeltis getulus sticticeps Barbour and Engels (1942) in Blaney (1977) Lampropeltis getulus goini (part) Neill and Allen (1949) in Blaney (1977) Lampropeltis getula (part) Frost and Collins (1988) Lampropeltis getula floridana Conant and Collins (1991) Lampropeltis getula brooksi Krysko and Judd (2006) Lampropeltis floridana (this study)

Holotype: USNM 22368; female collected by William Palmer in Figure 5. Ecological niche models for Lampropeltis getula getula, March 1895. Lampropeltis getula floridana, and hybrids. Overlap of >1 ENM is shown in Type Locality: Orange Hammock, De Soto County, Florida. dark gray. Note that the ENM for Lampropeltis getula meansi is not depicted, Distribution: A lineage incorporating individuals only from the as it entirely overlaps with that for L. g. getula. See online color version. Florida peninsula and upper Florida Keys; from Key Largo, Monroe County, northward to Citrus, Alachua, and Volusia counties (Figure 2). This species is speckled, has ≥34 narrow (1.5 dorsal scale rows wide) and light-colored crossbands, and a degenerate lateral chain pattern (Blaney 1977; Krysko 2001; Krysko and Judd 2006). Neonates are mostly black with light-colored crossbands, may pos- sess reddish coloration on the light-colored crossband scales, and the dark dorsal interband scales undergo various degrees of ontogenetic lightening, giving it a yellowish speckled appearance in the adult stage (Blaney 1977; Krysko 2001; Krysko and Judd 2006). The light pig- ment within each crossband scale is situated on the anterior portion of the scale (Krysko and Judd 2006). The venter consists of a tight checkered pattern with alternating squares of dark and light colora- tion (Means and Krysko 2001; Krysko and Judd 2006). Individuals from the hypothesized hybrid zone (Figure 2) can possess phenotypes typical of L. floridana or L. getula (in the more northern region of this zone), or a combination of both taxa (Krysko and Judd 2006).

Lampropeltis getula, Linnaeus (1766) Eastern Kingsnake

Coluber getulus (part) Linnaeus (1766) Figure 6. PCA of climate data for Lampropeltis getula getula and Lampropeltis Ophibolus getulus (part) Baird & Girard (1853) getula meansi. For each subspecies, climate data were extracted from all sampling localities used to generate the ENMs. Gray: L. g. getula, red: Coronella getulus (part) Duméril et al. (1854) L. g. meansi. See online color version. Lampropeltis getulus (part) Cope (1860) 10 Journal of Heredity, 2017, Vol. 00, No. 00

Lampropeltis getulus sticticeps (part) Barbour & Engels (1942) in 2001), and 2 individuals (UF-herpetology 55450 and 115959) from Blaney (1977) Wakulla County on the eastern side of the Ochlockonee River also Lampropeltis getulus goini (part) Neill & Allen (1949) in Blaney possess the phenotype of L. meansi. This species is speckled and has (1977) ≤25 wide (>2.5 dorsal scale rows wide) and light-colored crossbands; Lampropeltis getula (part) Frost & Collins (1988) some individuals are striped or completely patternless (i.e., non- Lampropeltis getula getula (part) Conant & Collins (1991) banded) (Means and Krysko 2001; Krysko and Judd 2006). Neonates Lampropeltis getula sticticeps (part) Conant & Collins (1991) are mostly black (banded individuals) with light-colored crossbands, Lampropeltis getula goini (part) Krysko & Judd (2006) may possess reddish coloration on their light colored crossband scales, and the dark dorsal interband scales undergo a high degree Holotype: N/A, collected by D. Garden. of ontogenetic lightening, giving it a yellowish speckled appearance Type Locality: “Carolina” (Linnaeus (1766:382), but later desig- in the adult stage (Means and Krysko 2001; Krysko and Judd 2006). nated as Charleston, South Carolina (Klauber 1948). Nonbanded striped and patternless neonates do not necessarily have Distribution: A lineage incorporating individuals from the northern light-colored crossbands, but rather are made up mostly of already Florida peninsula and panhandle, east of the Appalachian Mountains lightened interband scales that continue to undergo ontogenetic to southern New Jersey (Figure 2). This species is typically solid black lightening (Means and Krysko 2001; Krysko and Judd 2006). The to chocolate brown with 19–32 narrow (1.5–2.5 dorsal scale rows light pigment within each crossband scale is situated on the anterior wide) and light-colored crossbands that widen or divide laterally portion of the scale (Krysko and Judd 2006). The venter consists of giving it a chain-like pattern (Krysko and Judd 2006; Jensen et al. either a loose checkered pattern with alternating squares of dark and 2008). Neonates may possess reddish coloration on the light-colored light coloration with interspersed bicolor scales, or entirely bicolored crossband scales, and the dark dorsal interband scales do not typi- scales (Means and Krysko 2001; Krysko and Judd 2006). Individuals cally lighten with age (Jensen et al. 2008). However, interband scales from the hypothesized hybrid zone (Figure 2) in the Florida panhan- may lighten ontogenetically in some adult specimens from south- dle can possess phenotypes typical of L. getula (in the far peripheries eastern Georgia and the Outer Banks of North Carolina (Barbour of this zone) or a combination of L. getula and L. meansi (Means and and Engels 1942; Lazell and Musick 1973; Krysko and Judd 2006; Krysko 2001; Krysko and Judd 2006). Jensen et al. 2008). The light pigment within each crossband scale is situated on the anterior portion of the scale. The venter consists Lampropeltis nigrita, Zweifel and Norris (1955) of a checkered pattern with alternating squares of dark and light Mexican Black Kingsnake coloration (Means and Krysko 2001; Jensen et al. 2008). Individuals from the hypothesized hybrid zone (Figure 2) in the Florida panhan- Coluber californiae (part) Blainville (1835) dle can possess phenotypes typical of L. getula (in the far peripher- Coronella californiae (part) Duméril et al. (1854) ies of this zone), or a combination of both L. getula and L. meansi Lampropeltis getulus yumensis (part) Blanchard (1919) (Means and Krysko 2001; Krysko and Judd 2006). Individuals from Lampropeltis getula yumensis (part) Klauber (1938) the hypothesized hybrid zone in the Florida peninsula can possess Lampropeltis getulus californiae (part) Stebbins (1954) phenotypes typical of L. floridana, or L. getula (in the more northern Lampropeltis getulus nigritus Zweifel and Norris (1955) region of this zone), or a combination of both taxa (Krysko and Judd Lampropeltis getulus nigrita (part) Smith and Taylor (1966) 2006). Another hybrid zone between L. getula and L. nigra is known Lampropeltis getulus splendida (part) Hardy and McDiarmid (1969) from northwestern Georgia on the southern edge of the Appalachian Lampropeltis getula nigrita (part) Crother (2000) Mountains (Jensen et al. 2008). Lampropeltis californiae (part) Pyron and Burbrink (2009) Lampropeltis nigrita (this study) Lampropeltis meansi, Krysko and Judd (2006) Holotype: MVZ 50814, male collected by Kenneth S. Norris and Eastern Apalachicola Lowlands Kingsnake Richard G. Zweifel on 3 August 1950. Lampropeltis getulus goini (part) Neill & Allen (1949) in Blaney Type Locality: 49.2 km south of Hermosillo, Sonora, Mexico. (1977) Distribution: A lineage incorporating individuals from northern Lampropeltis getula (part) Frost & Collins (1988) Sinaloa northward to Sonora, Mexico, and extreme southeastern Lampropeltis getula goini (part) Krysko & Judd (2006) , USA. This species is solid black or brown in the adult stage Lampropeltis getula meansi Krysko & Judd (2006) (Zweifel and Norris 1955; Blaney 1977; Stebbins 2003; Krysko and Lampropeltis meansi (this study) Judd 2006). There may be considerable variation within a single clutch of , where some neonates might exhibit a nearly solid Holotype: UF-Herpetology 73433, male collected by D. Bruce dark dorsum and venter, while other siblings might exhibit a narrow Means on 9 June 1970. banded dorsum and tight checkerboard ventral pattern with alternat- Type Locality: Apalachicola National Forest, FH-13 ca. 3.2 km W ing squares of dark and light coloration like those of adjacent popu- SR 67, Liberty County, Florida. lations of L. splendida (Krysko and Judd 2006). It might hybridize Etymology: Named for Dr D. Bruce Means in recognition of his dis- where it comes into contact with adjacent species (i.e., L. californiae covery of the first known Eastern Apalachicola Lowlands kingsnake, and L. splendida) (Blanchard 1921; Blaney 1977; Stebbins 2003). as well as his contributions to our knowledge of the flora and fauna of the Coastal Plains. Distribution: A lineage incorporating individuals mostly from Franklin Discussion and Liberty counties, between the Apalachicola and Ochlockonee riv- Nearly all of the subspecies within the Lampropeltis getula complex ers and south of Telogia Creek in the Florida panhandle (Figure 2). were named solely using morphology and color pattern characters. We note that some individuals are found in Franklin County on the More recently, DNA analyses have improved our understanding of southwestern side of the Apalachicola River (Means and Krysko evolutionary relationships and corresponding taxonomy. Journal of Heredity, 2017, Vol. 00, No. 00 11

Although Barbour and Engels (1942) and Lazell and Musick Acknowledgments (1973) believed that L. g. sticticeps from the Outer Banks of North We thank Frank T. Burbrink, David L. Reed, and Julie Allen for suggestions Carolina deserved taxonomic recognition, this name was rejected by on analyses; Richard C. Hulbert for information on geological names and fos- Blaney (1977, 1979) and Krysko (2001), and our molecular results sil taxonomy; Chris McMartin for photographs; and Stephen A. Karl and 2 herein support this conclusion. Additionally, ontogenetically light- reviewers for constructive comments. This study was conducted under the fol- ened interbands occur in Florida populations, as well as individu- lowing permits and grants: Florida Fish and Wildlife Conservation Commission als from the Outer Banks and coastal Georgia, and therefore is not (Project NG00-002), Everglades National Park (Permit 940015), South Florida a diagnosable character (Lazell and Musick 1973; Blaney 1977; Water Management District (Permit 1069), United States Forest Service (Permit Palmer and Braswell 1995; Krysko 2001). 2670), UF Institutional Care and Use Committee (IACUC) (Permit Our mtDNA and nDNA sequence data illustrate that previously A187), Central Florida, Volusia County, and Suncoast herpetological societies. described subspecies in eastern North America represent natural groups corresponding to L. floridana, L. getula, and L. meansi, distinct Data Availability lineages as proposed by Blaney (1977), Krysko (2001), and Krysko and Judd (2006). The elevation of these forms to full taxonomic rec- We have deposited the primary data underlying these analyses as follows DNA sequences: for Genbank accessions see Table 1. ognition follows a trend established for western kingsnakes based on a fuller knowledge of morphology, genetics, and distributions (Pyron and Burbrink 2009a). The presence of morphological intermediates References at the fringes of each species range indicates suture (hybridization) Amos B, Hoelzel AR. 1991. Long-term preservation of whale skin for DNA zones, and to a lesser extent (regarding to L. getula) incomplete line- analysis. Rep Int Whal Comm. 13:99–103. age sorting because of a recent evolutionary divergence. Arevalo E, Davis SK, Sites JW Jr. 1994. Mitochondrial DNA sequence diver- ENM results show that Lampropeltis meansi overlaps entirely gence and phylogenetic relationships among eight chromosome races of with L. getula but is restricted to a small subset of the climatic the Sceloporus grammicus complex (Phrynosomatidae) in central Mexico. environment occupied by L. getula. This is not surprising, given the Syst Biol. 43:387–418. very short tree branch between these 2 lineages (Figure 3). Taken Auffenberg W. 1963. The fossil snakes of Florida. Tulane Stud Zool. 10:131– together, the short branch length and lack of ENM differentiation 216. Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE, Reeb CA, suggest very recent divergence between these taxa, as sufficient Saunders NC. 1987. Intraspecific phylogeography: the mitochondrial time may not have passed for complete ecological divergence (de DNA bridge between population genetics and systematics. Ann Rev Ecol Queiroz 2007). These results also indicate that L. meansi has a Syst. 18:489–522. more restricted set of environmental requirements than L. getula, Baird SF, Girard C. 1853. Catalogue of North American reptiles in the museum which is predicted to occur across a much broader range. In con- of the Smithsonian Institution. Part 1. Serpents. Washington, DC: Smithso- trast, ENMs for L. getula and L. floridana were distinct with a clear nian Institute. p. 172. area of overlap in northern peninsular Florida (Figure 5), support- Barbour T. 1919. Another new race of the kingsnake. Proc New Engl ZoClub. ing recognition of the 2 lineages as parapatric species separated by 7:1–3. a hybrid zone. Barbour T, Engels WL. 1942. Two interesting new snakes. Proc New Engl The distributions of these genetic lineages are concordant with ZoClub. 20:101–104. Bickham JW, Wood CC, Patton JC. 1995. 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