As currently understood, there are two species of foxsnakes (Eastern Foxsnake,
As currently understood, the Western Foxsnake,
The approximate distributions of the two recognized lineages of foxsnakes since Conant [
In the current concept of the Eastern Foxsnake (
The disjunct distribution of the putative taxa
The subspecific status of
Both species of foxsnakes are characterized by black to chocolate blotches with a variable ground color of yellowish to light brown. The head color usually varies from a brown to reddish tint, and the belly is yellow with a black-checkered pattern. The subcloacal plate for both species is divided, and the scales are weakly keeled.
In the original description of
The objective of this study is to test the species hypotheses of foxsnakes within a phylogenetic framework. Specifically, (1) are there two species lineages or (2) is there a single lineage with a large geographic disjunction within the distribution? The cytochrome
Samples for both
The specimens collected from the Eastern Foxsnake distribution consisted of nine individuals from four populations within Ohio and the Ontario peninsula region. The Western Foxsnake specimens included individuals and populations from Nebraska, Minnesota, Iowa, Wisconsin, Illinois, and Missouri. Table
Sample localities (see Figure
Species | OTU Labels | Locality |
---|---|---|
W1 | Dakota Co., NB | |
W2a | Rock Co., MN | |
W2b | Rock Co., MN | |
W3a | Holt Co., MO | |
W3b | Holt Co., MO | |
W3c | Holt Co., MO | |
W4 | Warren Co., IA | |
W5a | Muscatine Co., IA | |
W5b | Muscatine Co., IA | |
W6 | Wabasha Co., MN | |
E7 | Vilas Co., WI | |
E8a | Pepin Co., WI | |
E8b | Pepin Co., WI | |
E9a | Buffalo Co., WI | |
E9b | Buffalo Co., WI | |
E10 | Iowa Co., WI | |
E11a | Rock Co., WI | |
E11b | Rock Co., WI | |
E12a | Green Co., WI | |
E12b | Green Co., WI | |
E13 | Putnam Co., IL | |
E14 | Dek/Lasalle Co., IL | |
E15 | Kane Co., IL | |
E16 | Dekalb Co., IL | |
E17 | Dewitt Co., IL | |
E18 | Erie Co., OH | |
E19 | Essex Co., Ontario | |
E20a | Haldimand-Norfolk Co., Ontario | |
E20b | Haldimand-Norfolk Co., Ontario | |
E20c | Haldimand-Norfolk Co., Ontario | |
E20d | Haldimand-Norfolk Co., Ontario | |
E20e | Haldimand-Norfolk Co., Ontario | |
E21 | Ottawa Co. OH |
Total DNA was isolated from tissues (muscle, liver, blood, shed skins, and scale clips), using a standard phenol-chloroform protocol, a DNeasy kit (Qiagen), or Invitrogen’s “Easy DNA” kit. All DNA isolations were tested on a 1% agarose gel to visualize the sample’s degree of yield prior to PCR amplification.
The cytochrome
PCR amplification was conducted on a Perkin-Elmer thermocycler using the standard cycle conditions for the cytochrome
PCR amplifications were visualized and tested for correct size of fragment on a 1% agarose gel. Several of the cytochrome
The PCR products were ligated into TopoTA vectors and cloned using the TopoTA cloning kit (Invitrogen). The products were transformed into TOP 10 competent
Thirty-two cytochrome
The phylogenetic placement of foxsnakes within
The data were analyzed within parsimony (MP) and likelihood (ML) frameworks with PAUP*. The MP analyses were implemented with all sites equally weighted and gaps treated as missing data. The heuristic search was conducted using 10,000 random addition searches with global branch swapping (Tree Bisection-Reconnection). Each random addition search began with a random tree and the fit of the data to the tree was evaluated with the descriptive statistics consistency index [CI; [
We used jModeltest v0.1.1 [
To estimate the rate of substitution of cytochrome
Divergence date was estimated using a strict clock model because preliminary analyses revealed clock-like evolution (ucld.stdev: 0.537; [
The cytochrome
The nucleotide composition of the region in foxsnakes is dominated primarily by adenine and thymine, with a low frequency of guanine. The base frequencies for thymine (T) ranged from 0.305 to 0.308 with a mean of 0.3065 while adenine (A) reached slightly higher frequencies of 0.327–0.331 with a mean of 0.329. The frequencies for cytosine (C) ranged from 0.258 to 0.261 at a mean of 0.2595 and guanine, the lowest, 0.104–0.106 (mean = 0.1048). The high frequencies of adenine and thymine are comparable to the outgroup taxa
The maximum parsimony and maximum likelihood analyses inferred the exact same clades so only the parsimony tree will be discussed further. The MP analysis yielded six most-parsimonious trees (strict consensus, Figure
Strict consensus tree of six equally parsimonious trees. See text for descriptive statistics of this tree. The hash marks represent unambiguous cyt-
Two distinct clades within the foxsnakes were identified; (1) an eastern clade, comprised of most foxsnakes from east of the Mississippi River, including eastern
There were a total of 11 distinct haplotypes found in the cytochrome
Haplotype distribution map. Similar haplotypes are enclosed together. Note haplotype E is distributed across the geographic disjunction. Individuals from localities 6, 7, 11, and 17 had unique haplotypes.
The pairwise genetic distances between the two (eastern versus western) major clades, as well as intraclade distances, were characterized based on individual comparisons (Table
Uncorrected (“p”) distance matrix showing estimates of genetic similarity among the different localities. Bold faced values indicate interclade distances.
(a)
(E19) | (E20a) | (E20c) | (E20d) | (E20e) | (E21) | (E13) | (E17) | (E12b) | (E12a) | (E14) | (E15) | (W5b) | (E10) | (E18) | (E9a) | (W3c) | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(E19) | — | ||||||||||||||||
(E20a) | 0.0000 | — | |||||||||||||||
(E20c) | 0.0000 | 0.0000 | — | ||||||||||||||
(E20d) | 0.0000 | 0.0000 | 0.0000 | — | |||||||||||||
(E20e) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | — | ||||||||||||
(E21) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0009 | — | |||||||||||
(E13) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0009 | — | ||||||||||
(E17) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0018 | 0.0009 | — | |||||||||
(E12b) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | — | ||||||||
(E12a) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0018 | 0.0009 | 0.0018 | 0.0009 | — | |||||||
(E14) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | — | ||||||
(E15) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | — | |||||
(W5b) | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | 0.0000 | — | ||||
(E10) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0018 | 0.0009 | 0.0018 | 0.0009 | 0.0000 | 0.0009 | 0.0009 | 0.0009 | — | |||
(E18) | 0.0018 | 0.0018 | 0.0018 | 0.0018 | 0.0018 | 0.0009 | 0.0018 | 0.0027 | 0.0018 | 0.0027 | 0.0018 | 0.0018 | 0.0018 | 0.0027 | — | ||
(E9a) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0018 | 0.0009 | 0.0018 | 0.0009 | 0.0018 | 0.0009 | 0.0009 | 0.0009 | 0.0018 | 0.0027 | — | |
(W3c) | — | ||||||||||||||||
(W1) | 0.0009 | ||||||||||||||||
(W2b) | 0.0009 | ||||||||||||||||
(W4) | 0.0009 | ||||||||||||||||
(W5a) | 0.0009 | ||||||||||||||||
(E9b) | 0.0018 | ||||||||||||||||
(W6) | 0.0027 | ||||||||||||||||
(E8a) | 0.0018 | ||||||||||||||||
(E20b) | 0.0126 | ||||||||||||||||
(E16) | 0.0126 | ||||||||||||||||
(E11b) | 0.0108 | ||||||||||||||||
(E11a) | 0.0117 | ||||||||||||||||
(W3b) | 0.0018 | ||||||||||||||||
(E8b) | 0.0018 | ||||||||||||||||
(W3a) | 0.0018 | ||||||||||||||||
(W2a) | 0.0036 | ||||||||||||||||
(E7) | 0.0117 | 0.0117 | 0.0117 | 0.0117 | 0.0117 | 0.0126 | 0.0117 | 0.0126 | 0.0117 | 0.0108 | 0.0117 | 0.0117 | 0.0117 | 0.0108 | 0.0135 | 0.0126 | 0.0027 |
(b)
(W1) | (W2b) | (W4) | (W5a) | (E9b) | (W6) | (E8a) | (E20b) | (E16) | (E11b) | (E11a) | (W3b) | (E8b) | (W3a) | (W2a) | (E7) | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(E19) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E20a) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E20c) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E20d) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E20e) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E21) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E13) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E17) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E12b) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E12a) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E14) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E15) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W5b) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E10) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E18) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E9a) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W3c) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W1) | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W2b) | 0.0018 | — | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W4) | 0.0018 | 0.0000 | — | — | — | — | — | — | — | — | — | — | — | — | — | — |
(W5a) | 0.0018 | 0.0000 | 0.0000 | — | — | — | — | — | — | — | — | — | — | — | — | — |
(E9b) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | — | — | — | — | — | — | — | — | — | — | — | — |
(W6) | 0.0018 | 0.0018 | 0.0018 | 0.0018 | 0.0009 | — | — | — | — | — | — | — | — | — | — | — |
(E8a) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0000 | 0.0009 | — | — | — | — | — | — | — | — | — | — |
(E20b) | — | — | — | — | — | — | — | — | — | |||||||
(E16) | 0.0000 | — | — | — | — | — | — | — | — | |||||||
(E11b) | 0.0099 | 0.0117 | 0.0117 | 0.0117 | 0.0108 | 0.0117 | 0.0108 | — | — | — | — | — | — | — | ||
(E11a) | 0.0108 | 0.0108 | 0.0108 | 0.0108 | 0.0099 | 0.0108 | 0.0099 | 0.0027 | — | — | — | — | — | — | ||
(W3b) | 0.0009 | 0.0027 | 0.0027 | 0.0027 | 0.0018 | 0.0027 | 0.0018 | 0.0108 | 0.0117 | — | — | — | — | — | ||
(E8b) | 0.0009 | 0.0009 | 0.0009 | 0.0009 | 0.0000 | 0.0009 | 0.0000 | 0.0108 | 0.0099 | 0.0018 | — | — | — | — | ||
(W3a) | 0.0027 | 0.0027 | 0.0027 | 0.0036 | 0.0045 | 0.0036 | 0.0144 | 0.0126 | 0.0135 | 0.0036 | 0.0036 | — | — | — | ||
(W2a) | 0.0027 | 0.0027 | 0.0027 | 0.0027 | 0.0018 | 0.0027 | 0.0018 | 0.0126 | 0.0117 | 0.0036 | 0.0018 | 0.0054 | — | — | ||
(E7) | 0.0135 | 0.0135 | — |
The results for sequence divergence at first, second, and third codon positions indicated an expected pattern of substitution rate. At first codon positions there were two informative, 15 variable, and 353 constant characters. Second condon positions included two informative, 12 variable, and 355 constant characters. Third positions contained 11 of the 15 informative characters with 93 variable and 265 constant. In the interclade comparison of the most diverged sequences 16 sites of the 1108 were different with a transition-transversion ratio of 3.0 (12/4). Ten of the divergent characters were in third codon postions with a ts/tv ratio of 4.0 (8/2).
The best-fit model of evolution estimated by jModeltest was GTR + I. In all BEAST analyses, ESSs were high (>300), indicating increased reliability estimates. Calibration of substitution rate resulted in a mean substitution rate of 0.58% per million years (95% highest posterior density range: 0.28%–0.93%). The universal cytochrome
The results of the phylogenetic analyses indicate the presence of two distinct clades. The Eastern Clade is diagnosed by four unambiguous synapomorphies and contains most of the individuals of
Diagnostic cyt-
EAST CLADE | WEST CLADE | ||||
---|---|---|---|---|---|
CHAR | TRANS | CI | CHAR | TRANS | CI |
317 | T | 1.00 | 121 | C | 1.00 |
728 | T | 1.00 | 191 | A | 1.00 |
770 | A | 1.00 | 284 | T | 1.00 |
830 | T | 1.00 | 679 | C | 1.00 |
689 | A | 1.00 | |||
707 | T | 1.00 | |||
830 | T | 1.00 |
The two clades diagnosed in the analyses do not correspond to the currently accepted concepts of foxsnake species nor with the currently recognized distributions. The hypothesis of two widely disjunct taxa is rejected. In addition, there is no evidence that foxsnakes form a single species represented by a single lineage. In contrast, the results indicate two genetically distinct lineages (species) and reveal historical relationships previously unrecognized.
Five of the 33 individuals were found in clades that did not support an east-west separation at the Mississippi River. Individuals (E9b, E8a, E8b, and E7) of
Some individuals of
Harding [
The divergence time estimates between the Eastern and Western Clades yielded a range of 369,100–1,183,000 ya (mean = 742,800 ya) which nearly encompasses the entire set of Pleistocene glaciation events in North America [
Given estimated divergence times, the two clades as distinct lineages, and the patterns of genetic divergence relative to geographical space, we infer foxsnakes underwent allopatric speciation with the Pleistocene glacial lobes and the Mississippi River acting as vicariant barriers. There is evidence indicating that the Mississippi River has played an important role in the evolutionary history and speciation of several closely related species in North America (e.g., [
The presence of four western haplotypes east of the Mississippi River indicates dispersal across or around the river in Minnesota and northern Wisconsin. The presence of a single eastern haplotype in Iowa west of the Mississippi River indicates that dispersal also has occurred across the river in the opposite direction. Again, these data suggest that the Mississippi River may be no longer an absolute barrier to dispersal. The extent of dispersal affecting gene flow has yet to be investigated, but it is assumed that some genetic exchange is occurring (based on specimens from Buffalo Co., Wisconsin that occur in opposite clades). It is worth noting that it is well documented that mtDNA haplotypes cross what appear to be established species boundaries [
An alternative hypothesis to leakage (dispersal) across the barrier is that the river is a functioning barrier and the distribution of western haplotypes east of the river represents an old capture event. Rivers change course over time, and the meander could have captured some individuals on the “wrong” side. Without recombination of the mtDNA, these haplotypes would persist regardless of hybridization with the eastern populations.
The DNA and the geography indicate two lineages, but what about the morphology
The recognition of new species boundaries (Figure
Map depicting the approximate distributions of the two foxsnake mtDNA lineages as hypothesized from this study. The light shaded area represents the range of
(Western Foxsnake)
National Museum of Natural History, USNM 578514 (original number JRP 1395), adult female gravid with seven eggs collected in Warren Co. (R24W, T76N, Sec. 30), Iowa, 21 June 2002 by Jeff Parmelee.
National Museum of Natural History, USNM 578515 (original number JRP 1449), Marion Co. (R21W, T75N, Sec. 25), Iowa, 5 June 05; USNM 578516 (original number JRP 1447), Warren Co. (R22W, T76N, Sec. 23), Iowa, 5 June 05; USNM 578517 (original number JRP 1448), Warren Co. (R22W, T76N, Sec. 21), Iowa, 5 June 05; USNM 578518 (original number JRP 1450), Warren Co. (R24W, T76N, Sec. 25), Iowa, 18 May 05; SLU 1036 (original number JRP 1558), Madison Co. (R27W, T77N, Sec. 36), Iowa, 15 September 02; USNM 578519 (original number JRP 1555), Warren Co. (R24W, T77N, Sec. 33), Iowa, 27 May 03.
A relatively large
Rostral wider than high, notched below. Nasal divided with nasal opening approximately centered between the two parts. Loreal wider than high in a somewhat trapezoidal shape. One preocular, twice a high as wide. Two post oculars. Temporals right: 2-3-3, left 2-3-4 with the most dorsal scale in the second row fused with the corresponding scale in the third row and in the third row a scale is divided making the third row with 4 scales but in a 1-2-1 dorsal to ventral pattern. Internasals two, one-half to one-third the size of the two prefrontals. Frontal higher than wide, broadening anteriorly and forming a pointed tip posteriorly where it contacts the parietals. Supraoculars longer than wide and at their widest less than twice as long as wide. Paired parietals smooth, less than one and a half times longer than the frontal, regular, and symmetrical posteriorly. Eight supralabials, 4+5 in contact with the orbit, and the seventh the largest. Infralabials 11 on each side, with 5 the largest on the right and 6 the largest on the left. One pair anterior chin shields in contact and larger than posterior pair which are separated by pair of smaller scales anteriorly and a row of 4 smaller scales posteriorly. Dorsal scale rows 27-25-21. First 9 rows smooth (opposite seventh ventral) grading from weakly keeled to moderately keeled beyond the cloaca. Dorsal scales with 2 apical pits at tips. Ventrals 206. Anal plate divided. Subcaudals 56 in two rows terminated by a long pointed claw-like tip. Dorsal ground color of specimen (in alcohol, six years old) light olive brown anteriorly grading darker towards cloaca and becoming lighter again towards tail tip. Color also grades to lighter from dorsal midline laterally towards ventrals with the two most ventral lateral scales distinctly lighter. The first six cm behind the head gives the appearance of narrow light stripping laterally. Scales variably stippled in black with perimeter appearing cream over anterior third of body then becoming brown. Some scales along dorsal midline appear discolored white. Dorsal blotches dark brown with perimeter often black. Dorsal blotches 44, with two irregular, one smaller than normal, and three smallish blotches separated by 1-2 scales counted as one. Tail blotches 13. Top of head hazel-olive brown with dark band across posterior half of prefrontals reaching to the dorsal anterior portion of the eyes. Another band extends from posterior of the eyes at an angle terminating at the two rear supralabials. On the right side a dark band extends from below the eye on supralabials 4+5, on the left the band extends across 2-5. Parietals and frontal with distinct dark markings. Looking from the anterior, the frontal has a broad triangle with the point anterior. The parietals have a broad “M” shape and two spots in contact with the frontal at the two posterior tips of the triangle. There is also a short dark stripe along the medial line of the parietal scale contact posteriorly. The upper and lower labials are yellow-cream with the sutures tinged with dusky gray. Chin and throat yellow-cream. Temporals same as basic head color but becoming darker posteriorly with color extending into a dorsal blotch outlined in black on each side. Venter yellow-cream with 4 rows of black blotches, with most lateral blotches extending into the first 2-3 scale rows and either alternate with dorsolateral blotches or connect with them (on the right side 31 do not and 28 connect, on the left side 2 do not and 31 connect). SVL = 74.5 cm, TL = 13.4.
Temporals range from 2-3-4 to 2-4-5. One specimen with left temporal arrangement of 2-4-1-5 (SLU 1036). Infralabials sometimes 10. Chin shields variably divided by 1-3 rows of scales. Dorsal scale rows 25-25-21 and 24-25-21 dorsal keeling variably starts 8–11 scales behind head. Ventrals range from 191 to 208 and subcaudals 60 to 65. Dorsal blotches 39-46 and tail blotches 17–19.
West of the Mississippi River in southeastern Minnesota, most of Iowa, southeastern corner of South Dakota, eastern Nebraska, northwestern and northeastern corners of east-central Missouri.
The specific epithet
In the course of the present study, the issue arose as to which specimen should serve as the name bearing type of
“Ledger has “Type B&G Cat. Serp. P. 75” in remarks but it is crossed out, and “topotype” has been added. All are original or old annotations”.
We examined USNM 7269 and compared it to Baird and Girard’s original type description and found that it did not match. Two other topotypic specimens from the original series from Racine were sent one each to the Natural History Museum, London (USNM 1625) and the Hungarian Natural History Museum, Budapest (USNM 1626), and one was possibly the missing holotype. The Hungary collection was destroyed in the revolt of 1956 but after examination the specimen in London (BMNH 1861.8.15.41) was determined not to be the missing holotype. Therefore the name bearing specimen for
(Eastern Foxsnake)
As described in [
All populations east of the Mississippi River including Wisconsin, Illinois, eastern Missouri, Indiana, Michigan, Ohio, and Ontario, Canada.
As noted above, with the new understanding of the distribution of these two forms of foxsnakes, the holotype for the name
Although there is some debate (as previously noted), much of the current distribution of vertebrates in North America, especially the herpetofauna, has been in some way shaped by the glaciation events during the Pleistocene [ We concur with Holman [ After the last ice age at the end of the Wisconsinan, approximately 20,000 ybp, foxsnakes were divided by the Mississippi River. Presumably, speciation was well underway by then because the eastern and western distributions were separated by the ice sheets and the Mississippi River. The estimated dates and the low genetic divergence (0.903–1.444%) between the clades east and west of the river support the recent separation. The postglacial distribution of foxsnakes expanded north, east, and west into the current northern distributions. Holman [ A rapid northward expansion by foxsnakes is suggested by the low intraclade genetic variability. This pattern of low genetic diversity with increasing latitude has been observed in plants [
The furthest extents of Pleistocene glaciation superimposed on the current and partial paleodistribution of foxsnakes. Notice that the current distribution (shaded area) is completely within the Pleistocene ice sheet coverage. The different colored lines represent the furthest extents of the four classical subdivisions of the Quaternary glaciations in North America. Nebraskan 1 mya – 950 kya, Kansan 750 kya – 650 kya, Illinoian 350 kya – 250 kya, Wisconsin 125 kya – 10 kya.
The molecular data employed in this study diagnose two clades and indicate that the previously accepted taxonomy is inconsistent with the geographic distribution of the clades. One clade,
Numerous people contributed to this study. F. Burbrink, F. Durbian, B. Hay, R. King, J. LeClere, E. McCumber, J. Parmelee, C. Phillips, and R. Siegel all kindly contributed specimens/tissues. I. Ineich (MNHN), L. Ford, and D. Kizirian (AMNH) are thanked for generously answering museum queries. Roy McDiarmid, Steve Gotte and James Poindexter (USNM), and Colin McCarthy (BMNH) are thanked for helping with specimen loans. J. Parmelee kindly contributed specimens for the type series of