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. Introduction 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 [1963]), 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 - markmcknight@missouristate.edu. 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, E. tynerensis. 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 sample sizes. 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 this study. GenBank 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 Results 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 likelihood phylogeny of the 21 salamander cytochrome-b sequences produced in this paper. Eurycea tynerensis (Oklahoma Salamander) samples are used as the outgroup. Branch lengths are proportional to the amount of genetic divergence between taxa. There is no separation of black-eyed (B) from gold-eyed (G) and paedomorphic (P) from m e t a m o r p h i c (M) salamanders from Busiek State Forest and Wildlife Area (Camp Creek tributary: MSU1920– MSU1923; Camp Creek: MSU1935– 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. Discussion 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 this hypothesis. Acknowledgments 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. 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