A new species of Thamnophis (Serpentes, Colubridae) from Jalisco, Mexico, with a discussion on the phylogeny, taxonomy, and distribution of snakes related to Thamnophis scalaris

Garter snakes in the genus Thamnophis from Mexico have a long and convoluted taxonomic history. From 2015 to 2022, we conducted a comprehensive sampling of Mexican Thamnophis species, aiming to link molecular phylogenies with the recognized species related to T. scalaris in the highlands of Mexico. Here, we present an analysis of mitochondrial DNA to resolve the status of two enigmatic highland Thamnophis populations. Our research resulted in the identification and morphological characterization of a previously undescribed Thamnophis species from the state of Jalisco in western Mexico. We also clarify the identity and relationships of several previously enigmatic populations of Thamnophis . This work presents new data for Thamnophis phylogenetics from the Mexican highlands and offers a framework for future conservation efforts.


Introduction
The taxonomy of the Longtail Alpine Garter Snake, Thamnophis scalaris Cope, 1861, has been a subject of confusion since its description.Historically, Thamnophis scalaris was confused with both Thamnophis scaliger (Jan, 1863) and Thamnophis godmani (Günther, 1894), and these two species were considered subspecies of T. scalaris at one time (Smith 1942).The type specimen of T. scalaris was collected near Xalapa, Veracruz (Cope, 1861).Later, Cope (1887) referred additional specimens from Orizaba, Veracruz, and nearby localities in Puebla to T. scalaris.Jan (1863) described T. scaliger but did not specify a type locality, leaving the association of this name with certain Thamnophis populations unclear.However, it should be noted that Jan had access to material collected by François Sumichrast in Xalapa and other areas of Veracruz at the time he described T. scaliger (Grünwald et al. 2015, p. 402).
The first accounts of Thamnophis scalaris from Jalisco were derived from material collected by A. C. Buller in 1892, which Boulenger (1893) reported as "Tropidonotus scalaris."His concept of T. scalaris was based on seven specimens from Jalisco (which we now recognize in this study as a distinct species) and one specimen from Oaxaca, which was likely mislabeled.Later, Smith (1942) reviewed the Thamnophis of Mexico and considered T. scaliger and T. godmani to be subspecies of T. scalaris.Smith et al. (1950) also examined specimens of Thamnophis at the Natural History Museum, London, and categorized the Jalisco specimens reported by Boulenger (1893) within their concept of T. scalaris scaliger.Subsequently, Rossman and Lara-Gongora (1997) removed Thamnophis scaliger from the synonymy of T. scalaris and proposed a neotype for T. scaliger from Mexico City.This was done in order to preserve the name "T. scaliger" for the population that Smith (1943) and Smith et al. (1950), among others, had associated with it.Jan's (1863) description was extremely vague, and Smith (1942) recognized this and ignored the issue regarding the lost "T.scaliger" type and decided to apply the name to one of the morphologically distinct populations that he had at hand.At the time Rossman and Lara-Gongora (1997) were investigating the group, most authors were following Smith's (1942) concept of T. scaliger, so Rossman and Lara-Gongora (1997) wanted to stabilize the name T. scaliger, and thus they neotypified the name for populations in central Mexico.Rossman and Lara (1997) helped clarify the relationships between T. scalaris and T. scaliger, and they considered T. scalaris to be distributed along the Mexican Transverse Ranges from western Jalisco east to central Veracruz and eastern Puebla.These authors partially recognized the distinct nature of the Jalisco T. "scalaris" from typical T. scalaris in their review and grouped these individuals as part of their "western T. scalaris."However, they continued to confuse these populations with true "T.scalaris" from eastern Michoacán and the high mountains around Mexico City.Unfortunately, these authors did not publish more specific data that would facilitate comparisons between the different geographical populations of T. scalaris.
Against this historical backdrop, our research was motivated by the discovery of garter snakes in Jalisco that were morphologically distinct and could not be assigned to any known species of Thamnophis.To resolve the taxonomic status of these populations, we undertook a comprehensive molecular and morphological analysis of Mexican highland Thamnophis.Morphologically, the Jalisco population appeared to be intermediate between T. scalaris and Thamnophis errans Smith, 1942, and molecular data were needed to determine their specific identity.This study presents these molecular findings and provides a morphological description of the Jalisco population, which we recognize as a new species.In addition, we resolve some longstand-ing taxonomic conundrums around T. scalaris.This work underlines the urgent need for conservation measures since the habitats of these populations are increasingly threatened by human activities.By clarifying the taxonomy of these garter snakes, we aim to establish a foundation for future research and conservation efforts in the highlands of Jalisco.

Taxonomic sampling
We collected multiple specimens of garter snakes of the genus Thamnophis from the highlands of Mexico between 2015 and 2022.We photographed all live snakes, including dorsal, lateral, and ventral profiles, and euthanized them with pentobarbital.We took tissue samples from muscle or liver upon death and preserved them in 96% ethanol.We fixed specimens in 10% formalin and transferred them to 70% ethanol for permanent storage.
All collected materials were deposited at the Instituto de Investigaciones sobre los Recursos Naturales (INIRENA) of the Universidad Michoacana de San Nicolás de Hidalgo (UMSNH) in Morelia, Mexico; the Museo de Zoología, Facultad de Ciencias (MZFC) of the Universidad Nacional Autónoma de México (UNAM) in Mexico City; and the Facultad de Estudios Superiores, Zaragoza (MZFZ) of the Universidad Nacional Autónoma de México (UNAM), also in Mexico City.Museum acronyms throughout the text follow Sabaj (2020).Specimen numbers for all material examined are provided in Appendices 1, 2. We were not able to measure type specimens of previously described taxa, so we used measurements of the type specimens provided in original descriptions and other published literature (Cope 1861(Cope , 1866;;Jan 1863;Boulenger 1893;Smith 1942;Walker 1955;Rossman 1969;Rossman and Burbrink 2005).Measurements for T. errans were taken from Webb (1976), whereas measurements from Thamnophis sumichrasti Cope, 1866, andThamnophis mendax Walker, 1955, were taken from their original descriptions.
Distribution maps were generated based on the GBIF database (www.gbif.org),which includes both museum records and distribution records from the citizen scientist platform iNaturalist (inaturalist.org).iNaturalist records were curated by us to assure that no misidentifications were present before generating the maps and are current up through January 2024.Observations that could not be positively identified were removed.
The mountains of central Jalisco have numerous other species of Thamnophis that occur in sympatry or near sympatry with the species described herein.These include Thamnophis copei (Dugés, 1879), Thamnophis cyrtopsis (Kennicott, 1860), Thamnophis eques (Reuss, 1834), Thamnophis melanogaster (Peters, 1864), and Thamnophis pulchrilatus (Cope, 1885).While these species are not closely related to the species described herein, we include them in our comparatives within the species description to aid in the identification of individuals in the field.
Scale measurements were taken in the following manner: HL, distance from the tip of the snout to the posterior border of the parietal scales; HW, distance taken at the posterior edge of the jaw; RH, the distance of the rostral scale from the median point of the mouth to the vertex formed by the internasal suture; RW, distance of the rostral scale measured between each suture formed by the 1 st supralabial and prenasal; INL, distance of the internasal scale from its anterior border with the prenasal and rostral to its posterior border with the prefrontal; INW, distance from the prenasal to the medial suture between each internasal; PFL, distance from the postnasal, nasal, and internasal borders back to the border between the frontal and supraocular; PFW, distance from the postnasal to the median suture between the prefrontals; FL maximum length of the frontal shield; distance from the posterior part of the prefrontals to the medial union between the parietals; MAFW, the maximum width of the anterior portion of the frontal scale measured between each vertex formed by the prefrontal and supraocular scales; MPFW, the maximum width of the posterior portion of the frontal scale measured between each vertex formed by the parietal and supraocular scales, PW, distance from the union of the postocular and anterior temporal scales to the median suture between the parietals; PL, distance from the border between the supraocular and frontal to the posteriormost point of each parietal scale; LL, maximum length from the upper anterior border of the nasal to the lower posterior border with the preocular and supralabial; LH, distance from the supralabial to the union with the prefrontal and preocular; ML, taken from the medial point of the mouth to the posterior end of the mental scale, where the first pair of infralabials meet each other; MW, measured along the border of the mouth from one infralabial border to the other; ACSL, distance from the suture between the first and second infralabial posteriorly to the median suture between posterior chinshields; PCSL, maximum length from the union with the anterior chinshield and infralabial to the posterior border of the posterior chinshield; SC, counted on the left and right sides, with the first subcaudal scale interpreted as the first scale posterior to the cloaca that was not counted by the anal scale; Dorsal scales were counted at one head length behind the posterior edge of the parietals, at midbody, and at one head length before the anterior border of the anal scale.Muzzle length was defined as the combined length of the internasal suture (INK) and pre-frontal suture (PFK); muzzle shape was calculated by dividing INR by NR.

DNA extraction, amplification, and molecular analyses
All laboratory procedures were carried out at the UNAM FES-Zaragoza in Mexico City.We used a standard ammonium acetate protocol (Fetzner 1999) to extract genomic DNA from liver or muscle tissue.We then sequenced two mitochondrial loci: Cytochrome b (Cytb) and NADH dehydrogenase subunit 4 (ND4).For Cytb, we used Gludg-L (Palumbi 1996) as the forward primer and ATRCB3 (Harvey et al. 2000) as the reverse primer.For ND4, we employed ND4 and ND4_Leu (Arevalo et al. 1994) as the forward and reverse primers, respectively.Both loci were amplified using a standard polymerase chain reaction (PCR) protocol: an initial denaturation at 95 °C for 3 minutes and 30 seconds, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 53 °C for 30 seconds, extension at 72 °C for 1 minute, and a final extension at 72 °C for 15 minutes, with a terminal hold at 10 °C.The PCR products were purified using a polyethylene glycol method (Lis 1980) and sequenced by Macrogen Korea (Standard-Seq of Macrogen Inc.).
Raw chromatograms were trimmed and edited using Geneious v. 2023.1 (Biomatters Ltd., Auckland, NZ).To infer phylogenetic relationships from the new samples, we included additional sequences of the genus Thamnophis as well as two outgroups obtained from GenBank.All new sequences were deposited in GenBank (Appendix 2).
Maximum likelihood (ML) analysis of the concatenated dataset was performed using IQ-TREE (Nguyen et al. 2015) using the IQ-TREE web server (Trifinopoulos et al. 2016).We employed an auto-substitution model and conducted 1,000 bootstrap replicates for support assessment.Separate ML analyses were also performed for each mitochondrial gene.The resulting topologies are presented in Suppl.materials 1, 2.
For Bayesian phylogenetic inference (BI), we initially used PartitionFinder v1.1.1 (Lanfear et al. 2012) to determine the most suitable model of partitions and nucleotide evolution for each locus using the Bayesian information criterion (BIC).The identified optimal partitions were: GTR + I + gamma for the first codon position of both Cytb & ND4, HKY + I + gamma for the second codon position of Cytb, HKY + gamma for the second codon position of ND4, and GTR + gamma for the third codon position of both Cytb & ND4.Our dataset was organized accordingly by locus and codon position.We then conducted BI using Mr. Bayes v3.2.2 (Ronquist et al. 2012) on the CIPRES science gateway server (Miller et al. 2011).This analysis involved four runs, each with 10 million generations and a sampling interval of 1,000 generations, incorporating three heated chains and one cold chain.Convergence was assessed using Tracer v1.6 (Rambaut et al. 2015), focusing on likelihood and parameter estimate overlaps, effective sample sizes, and the potential scale reduction factor (PSRF).Convergence was achieved within 200,000 generations, allowing us to discard the initial 25% of each run as burn-in.The results of these runs were combined using TreeAnnotator 2.7.4. (Bouckaert et al. 2019) to create a concatenated tree and visualized with FigTree v1.4.2 (Rambaut 2014).
Considering that the topologies from both ML and BI analyses were almost identical, we have included only the maximum likelihood phylogeny in this paper, with the Bayesian Inference phylogeny provided as Suppl.material 3.

Molecular phylogenetic results
In our ML phylogeny (Fig. 1), we find concordance with prior evolutionary hypotheses, including those derived from mitochondrial DNA (mtDNA) studies (de Queiroz et al. 2002), those combining mtDNA and nuclear DNA in their analyses (McVay et al. 2015, Deepak et al. 2022), as well as analyses by Hallas et al. (2022), which were based on ddRADseq data.The robustness of our phylogenetic nodes is largely high, with most nodes garnering greater than 95% bootstrap support.However, several internal nodes exhibited lower support values, so we collapsed nodes below the 50% bootstrap support threshold.
Our analysis corroborates the delineation of three primary clades within Thamnophis, aligning with pre-vious studies.The first clade, "Ribbon Snakes," is composed of Thamnophis sirtalis (Linnaeus, 1758), Thamnophis proximus (Say, 1823), and Thamnophis saurita (Linnaeus, 1766), collectively forming a lineage sister to the remaining members of Thamnophis.The other sampled Thamnophis segregate into two major clades.The first of these, referenced as the "Widespread Clade" by de Queiroz et al. (2002), predominantly comprises species from the USA and northern Mexico.Our data support the close phylogenetic relationship between Thamnophis fulvus (Bocourt, 1893) and Thamnophis chrysocephalus (Cope, 1885), albeit with notable ge-netic divergence among T. chrysocephalus populations from Guerrero compared to those from Oaxaca and Veracruz.Further, the clade encompassing T. fulvus and T. chrysocephalus formed a sister lineage to a cluster of species endemic to the USA and northern Mexico, with Thamnophis.cyrtopsis forming a sister relationship to a clade comprising species from the USA and Baja California, inclusive of "T.aff.pulchrilatus" and an individual matching the original description of Thamnophis vicinus Smith, 1942 (currently a junior synonym of T. cyrtopsis), which is notable as this population has never been studied phylogenetically.In the other major clade, referenced as the "Mexican Clade," our results recover an early split between T. nigronuchalis Thompson, 1957 + T. rufipunctatus (Cope, 1875), sister to all other lineages.This split is followed by the divergence of T. copei and T. melanogaster.Remarkably, this study represents the first inclusion of T. copei in a molecular phylogeny, revealing an unexpected non-sister relationship to T. foxi, which was its sole congener in the now invalid genus Adelophis (see below).The new species reported herein formed a polytomy with T. mendax, T. sumichrasti, and T. scalaris.That group was recovered as the sister clade to a group that includes Thamnophis exsul Rossman, 1969; T. errans + T. scaliger; and a group comprising T. foxi alongside T. bogerti Rossman & Burbrink, 2005; T. conanti Rossman & Burbrink, 2005;and T. lineri Rossman & Burbrink, 2005.However, it is noteworthy that the support for this latter grouping is very low, and T. bogerti, T. conanti, and T. lineri do not form monophyletic groups.
Finally, our BI analysis exhibited a high degree of similarity to those relationships obtained through maximum likelihood, with an almost identical topology.The key distinction lies in the variation of support values for certain groups.In the BI analysis, T. ahumadai was recovered as a sister to T. mendax + T. sumichrasti + T. scalaris, but with low support (posterior probability = 0.61).The BI tree, elucidating these differences, is presented in Suppl.material 3.
Our molecular results support the hypothesis that populations formerly assigned to Thamnophis scalaris from Jalisco, Mexico, belong to an undescribed species.We analyzed molecular samples from two isolated highland populations (Sierra Cacoma, Sierra de Tapalpa; Appendix 2) of "T.scalaris" from Jalisco.Both are each other's closest relatives, with 100% bootstrap support.These populations fall within the "Mexican Clade" of our phylogenetic tree (Fig. 1).Together, these two populations are sister to a clade comprising T. scalaris, T. sumichrasti, and T. mendax, albeit with low bootstrap support (67%).The two Jalisco populations have similar genetic distances to their closest relatives.These genetic distances are 0.04-0.05(ND4) and 0.03 (Cytb) to Thamnophis bogerti (as understood herein, see below), which appears to be their closest relative according to genetic distances.The two Jalisco populations also have genetic distances of 0.04-0.06(ND4) and 0.03-0.04(Cytb) from the superficially similar T. scalaris; 0.05-0.07(ND4) and 0.04-0.05(Cytb) from geographically proximate populations of T. errans; and 0.04-0.05(ND4) and 0.04 (Cytb) from T. exsul.In comparison, the T. scalaris populations analyzed herein (Morelos, Estado de México, Querétaro, Puebla, and Veracruz) have intraspecific genetic distances of 0.00-0.02(ND4) and 0.00-0.01(Cytb).Within the T. scalaris populations analyzed, it should be noted that one specimen (AEVB 0104) from La Joya, Acajete, Veracruz had a ND4 genetic distance of 0.02-0.04from other non-Veracuz specimens of T. scalaris.Similarly, another specimen (UOGV 3932) from nearby Nogales, Veracruz, had a higher Cytb genetic distance (0.02) from all other specimens of T. scalaris.These genetic distances are still low when compared to the interspecific genetic distances that we recovered between other species, but they are nonetheless interesting and may show evidence of hybridization with nearby populations of T. bogerti (see below).
Proposed standard Spanish name.Culebra Listonada de Montaña de Ahumada.
The mountains of central Jalisco have numerous other species of Thamnophis that occur in sympatry or near sympatry with T. ahumadai.These species (except T. copei) are all distantly related to T. ahumadai and can be readily distinguished by their appearance.Thamnophis ahumadai differs from T. cyrtopsis, T. eques, and T. pulchrilatus by having 17 dorsal scale rows at mid-body (vs.19 or more) and by having a tongue that is black (vs.red with black tips).From the apparently closely related T. copei (see below), T. ahumadai differs by possessing a loreal (vs.fused with prefrontal), 17 dorsal scale rows (vs.15), and a longer head with seven supralabials (vs.5).
For comparative purposes, we include photographs of T. ahumadai and closely related species in Fig. 6 and at a higher resolution in Suppl.material 5.
Everted hemipenes are the length of seven subcaudals; they are long and narrow with no noticeable widening in the apical region.
Coloration in preservative (Fig. 2b, c).Head scales brown, but frontal, supraoculars, and parietals pale gray, apparently damaged as the snake was pre-ecdysis.Supralabials cream-white, with black lines along sutures.Dark nuchal blotch 1-3 scales long, black in coloration, complete dorsally from below the labial region on both sides.Pale mid-dorsal stripe white, starting on the fourth dorsal scale posterior to the parietals and running to the tip of the tail.One row of large dorso-lateral blotches, nine on the left, six on the right; large blotches divided into two rows of alternating dorso-lateral blotches.Lateral pale stripe bluish-gray, one second and third scale rows on the neck, and then limited to the second scale row on the rest of body and tail, slowing fading in intensity on tail.A single, lateral row of black spots present on the first dorsal scale row,  four postoculars also cream; upper one with black outline on posterior border.Anterior temporal brown above and black below; posterior temporals brown and black.A bilobed dark nuchal spot present behind head, dark brown anteriorly, and black posteriorly.Nuchal spot three scales long at mid-dorsal line, extending ventrally to the height of the jawline.This nuchal blotch partially divides the pale-yellow coloration on the posterior supralabials and the pale lateral stripe on the lower portion of the dorsum.Pale lateral stripe on first three scale rows anteriorly, pale yellow, then restricted to second scale row on the anterior four-fifths of the dorsum, only slightly involving the first and third scale rows.After the anterior fifth of the dorsum, the first scale row is cream with dark black markings on the posterior edge of each scale, giving the appearance of small black vertical blotches.Tail same color as posterior body, black above, cream below, with a pale cream mid-dorsal stripe and small black spots on the cream colored first scale row.Iris copper.INIRENA 2935).MZFZ 4593 is unique in that it presents dorsolateral blotches that are fused, similar to T. scalaris and T. scaliger.INIRENA 2934 has a dark dorsal coloration, which makes the dorsal pattern barely visible and gives the snake a dark, unpatterned appearance.Morphological and meristic variation of the all available specimens, including the holotype and all paratypes is given in Table 1.

Measurements (mm
Distribution and habitat.This species appears to be restricted to grasslands and meadows in pine-oak woodland and pine forest above 2100 m asl.Only known from two mountain ranges in Jalisco, the Cumbre de Guadalupe region of the Sierra Cacoma, and in the vicinities of the towns of Atemajac de Brizuela and Juanacatlán in the Sierra de Tapalpa.This species has been collected at elevations ranging from 2140 to 2450 m asl.We have included a range map with known localities of this species and closely related species in Fig. 7. Etymology.A patronym honoring Iván Trinidad Ahumada-Carrillo (1984-), who has made many contributions to diverse areas in herpetology, including extensive studies of the herpetofauna of Jalisco and Zacatecas.Iván collected the first specimen of this new species in the Sierra Cacoma (MZFZ 4593) and pointed out its distinctiveness from typical T. scalaris and T. errans.
Conservation.This species of garter snake is only known from two relatively small high-elevation areas in the highlands of Jalisco, which fall within the "Jaliscan Transverse Range Pine-Oak Woodland (42)" and "Jaliscan Sierra Madre del Sur Mixed Temperate Woodland ( 46)" biogeographical formations as mapped by Grünwald et al. (2015).Due to its small distribution, we recommend that this species be rewarded with the highest level of protection possible from the Mexican government.As habitat destruction such as logging and farming is ongoing in both localities where it occurs, this species may qualify as Endangered under the IUCN criterion.However, it can also be considered to be in the DD (data deficient) category due to uncertainties about whether or not this species occurs in Michoacán (see below).More fieldwork should be done to determine the full extent of the distribution of T. ahumadai and whether or not it is present in other highland regions of Jalisco or adjacent Michoacán.

Discussion
With the description of T. ahumadai, we remove T. scalaris from the herpetofauna of Jalisco.We also suggest that all specimens of "T.scalaris" collected from Jalisco (NHMUK 92.9.5.39,UTA 4040,4949, are assignable to T. ahumadai.A detailed morphological examination of these specimens will undoubtedly expand the known variation in this newly described species.Boulenger (1893) gave a detailed description of specimens of "T.scalaris" from Jalisco.The combination of 7 supralabial scales and low ventral and subcaudal scale counts that he reported precludes their assignment to any other species of Thamnophis known from Jalisco except for T. scaliger.While we cannot definitely exclude T. scaliger from the description given by Boulenger, it is important to note that despite intensive recent collecting at both localities (Cumbre de Guadalupe and Atemajac de Brizuela), no T. scaliger has been collected from either of these mountain ranges.We tentatively suggest that these specimens are referrable to T. ahumadai.Smith et al. (1950) reviewed these same specimens, but they confused them with their concept of "T.scalaris scaliger." Apparently, T. ahumadai and T. scalaris are widely isolated along the Mexican Transverse Ranges, with no known records between the Sierra Tapalpa in central Jalisco and the closest populations occurring near the vicinity of Nahuatzen in central Michoacán (Rossman et al. 1996;Rossman and Lara-Gongora 1997).The Nahuatzen population is of special interest itself.The Nahuatzen area is a particularly high elevation (ca.3000 m) upland situated in the middle of a continuous swath of moderate mountains (ca.2000 m) in central Michoacán.While this region is not particularly isolated, the nearest collecting localities of highland Thamnophis are T. ahumadai, 190 km to the west at Atemajac de Brizuela, Jalisco, and T. scalaris, 170 km to the east near Zitacuaro, Michoacán.Based on color pattern alone, these snakes seem very similar to and may be conspecific with T. ahumadai.We were unable to acquire molecular data from this population as all specimens in Mexican collections are fixed in formalin.Field work should be done around Nahuatzen to determine whether this population represents an isolated population of T. ahumadai, an isolated population of T. scalaris, or another yet undescribed species of Thamnophis.

Monophyly of Thamnophis scaliger and Thamnophis scalaris
Based on eleven genetic samples of Thamnophis scalaris collected across its range and an additional five samples of Thamnophis scaliger from several localities in central Mexico, we found that both T. scalaris and T. scaliger are monophyletic and do not represent sister species (Fig. 1).De Queiroz et al. ( 2002) had confusing results for the relationship between T. scalaris and T. scaliger based on the placement of their "T.scaliger 2" within T. scalaris.Their "T.scaliger 2" was based on LSUMZ 42638 (Accession Number: AF420189), which is supposedly a T. scaliger from the vicinity of Villa Victoria, Estado de México.We compared this sample to five T. scaliger samples and nine T. scalaris samples, all identified in the field by us.This sample was consistently recovered in a clade containing morphologically verified T. scalaris in all of our analyses.While we were not able to examine the specimen, we hy-  (2002), thus dispelling the possibility of PCR contamination.Nonetheless, these authors also refrained from making any taxonomical changes because the sequenced specimen was the same individual used earlier by de Queiroz et al. (2002) and was thus subject to the same caveats as the prior study.Later, Hallas et al. (2022) found Adelophis to be placed within Thamnophis across three molecular datasets (mtDNA, nDNA, and ddRADseq) and formally sank the genus Adelophis into Thamnophis.They cautioned that "some might refrain from formal changes to A. copei until that taxon can be suitably evaluated in a phylogenetic analysis."However, Hallas et al. (2022) considered the putative sister relationship between "A.copei" and "A.foxi" (Rossman and Blaney 1968;Rossman and Wallach 1987) to be sufficient evidence refuting the argument that "A.copei" could be nested outside of Thamnophis.In this study, we sequenced samples from a specimen of Thamnophis copei for the first time and confirmed that T. copei falls within the "southern clade" of Thamnophis like T. foxi (de Queiroz et al. 2002;McVay et al. 2015;Hallas et al. 2022).This confirms that the placement of Thamnophis copei is correct and that Adelophis should be subsumed into Thamnophis.Surprisingly, however, our results suggest that the morphologically similar T. copei and T. foxi are not sisters to one another, though both are consistently nested within the "Mexican clade" of Thamnophis.More extensive sequence data is needed to determine the exact phylogenetic relationship between these two species and closely related species.It is noteworthy that T. foxi has not been collected since the 1970s, despite several recent collection attempts.

The validity of the species related to
Thamnophis godmani in the Sierra Madre del Sur Thamnophis godmani was described from Omiltemi and "Amula" in Guerrero (Günther 1886) and was later considered to be distributed in the Sierra Madre del Sur and Sierra Madre Oriental from central Guerrero and central Veracruz (respectively) to the Isthmus of Tehuantepec (Rossman et al. 1996).Thamnophis godmani has historically been recognized as a close relative of T. scalaris, and Smith (1942) even considered T. godmani to be a subspecies of the former taxon.The specific identity of T. godmani was reaffirmed when more specimens and morphological data became available (Rossman in Varkey 1979).Later, Rossman and Burbrink (2005) conducted a multivariate study of morphological characters in various populations of T. godmani and described three species: T. bogerti, T. conanti, and T. lineri.Rossman and Burbrink (2005) provided subtle morphological and mensural characters to justify the description of T. bogerti, T. conanti, and T. lineri as species distinct from T. godmani.While the morphological data provided does suggest that T. godmani may be specifically distinct from the other three, the morphological differences are trivial once data from T. bogerti, T. conanti, and T. lineri are compared to one another.When compared to the intraspecies variation of other widespread highland Thamnophis such as T. scaliger, T. scalaris, T. errans, T. chrysocephalus, and T. sumichrasti, the subtle geographic variation found across T. bogerti and its relatives over a relatively broad range suggests that these taxa likely represent a single evolutionary species unit.Morphological and mensural differences for the three species were presented in the description only in text; however, here we compare them in Table 2.
We evaluated the status of these species (T.bogerti, T. conanti, and T. lineri) in our phylogeny, and our results indicate that T. bogerti is paraphyletic with respect to T. conanti and T. lineri (Fig. 1, Suppl.materials 1-3).Additionally, we found very low levels of genetic divergence between these species.Genetic distances in the mitochondrial gene ND4 were as low as 0.002 between T. bogerti and T. conanti, whereas both of these species show a genetic distance of 0.04-0.05with their closest relative, T. scalaris.Genetic distances in the Cytb between T. bogerti, T. conanti, and T. lineri ranged from 0.00-0.01,whereas all three of these "species" had distances between 0.03-0.04from their closest relative, T. ahumadai.(Suppl.material 4).These results support the tree topologies of McVay et al. (2015) and Hallas et al. (2022).Unfortunately, we did not have any genetic material of T. godmani to compare with, and recent analyses that did include T. godmani (Hallas et al. 2022) are based on a sample of T. bogerti from Oaxaca (J.Campbell, pers.comm).Thamnophis godmani is not known to occur in Oaxaca, and all samples of "T.godmani" found on Genbank are based on specimens that were collected before T. bogerti was described (Rossman and Burbrink 2005).Consequently, our phylogenetic results indicate that T. bogerti, T. conanti, and T. lineri belong to the same evolutionary unit.As these three species were described in the same paper, we invoke Article 24.2 of the International Code of Zoological Nomenclature (ICZN 1999) and suggest that T. lineri and T. conanti are junior synonyms of T. bogerti, the latter of which was alphabetically and sequentially described first in Rossman and Burbrink (2005).The specific relationship between T. godmani and T. bogerti remains to be tested; however, the isolated nature of the Guerrero populations of this species may indeed prove to be of a specific nature.Rossman and Burbrink (2005), in their review of the populations formerly assigned to T. godmani, stated that "T.godmani occurs in at least four discrete geographic areas that are effectively separated at the present time by habitat disjunctions unsuitable for these residents of montane pineoak forests (1768-3048 m)."However, these sky islands inhabited by the snakes formerly assigned to T. godmani have not been proven to act as biogeographic barriers for other reptiles (Bryson et al. 2011;Palacios-Aguilar et al. 2021).Furthermore, T. bogerti is frequently collected as low as 1100-1200 m in humid environs in Oaxaca, and oak woodland in the Sierra Juárez and Sierra Zongolica ranges down to at least 1500 m.Thus, these perceived "isolated mountain ranges" are not barrier-isolated for an elevation-adapted snake like T. bogerti.

Identity of Thamnophis similar to T. scalaris in the Sierra de Pinal de Amoles, Querétaro
An unidentified population of Thamnophis occurs in the vicinity of Pinal de Amoles in the Sierra Gorda region of Querétaro (Rossman and Lara-Gongora, 1997).These specimens (UMMZ 105415-416) are cataloged as T. scalaris but originate from well outside of the known range of this species.Rossman and Lara-Gongora (1997) discussed these specimens in detail, comparing them to T. scalaris, T. scaliger, and T. exsul, and concluded that "until fresh material becomes available, it would seem prudent to defer judgment of the identity of these Querétaro specimens."We collected two specimens (INI-RENA 2937-38, Fig. 8) of this population in 2020 and included them in our analyses.
Our phylogenetic analysis suggests that these specimens are nested with T. scalaris.Genetic distances for ND4 between this population and nearby Veracruz T. scalaris range from 0.00-0.01.In comparison, the genetic distances of ND4 between the Queretaro specimens and T. scaliger range from 0.02-0.06,between T. exsul 0.04 and T. errans 0.05.The genetic distance of Cytb in these specimens to nearby T. scalaris in Puebla and northern Veracruz ranges from 0.00-0.01.Comparatively, the genetic distances of Cytb in the Queretaro specimen to specimens of T. scaliger are 0.04-0.07,and for T. exsul, they are 0.04.Based on these results, we formally assign the Pinal de Amoles (Sierra Gorda) population to T. scalaris and confirm the presence of T. scalaris in Querétaro.

Figure 7 .
Figure 7. Distribution map of snakes similar to Thamnophis scalaris in Mexico.Circles represent museum records; squares represent verified iNaturalist observations, or field observations made by us but without a specimen deposited in a collection.The diamond represents the type locality of Thamnophis ahumadai sp.nov.We have included the different populations of Thamnophis bogerti in the key with their former names in quotation marks, and we used different shades of green to depict their individual ranges.
bar along posterior suture of SL 5 equal to or greater than bar along SL 6 and SL 7 suture prominence of black bar along posterior suture of SL 5 equal to or less than bar along SL 6 and SL 7 suture prominence of black bar along posterior suture of SL 5 equal to or less than bar along SL 6 and SL 7 suture black bar along posterior suture of SL 5 reduced or absent

Table 1 .
Morphometric and meristic variation of Thamnophis ahumadai sp.nov.Measurements of the holotype are shaded gray.

The positioning of the former genus Adelophis within Thamnophis
De Queiroz et al. (2002)suggested that their sample of Adelophis may have been mixed up with another sample; however, they assessed that the genetic distances of the analyzed sequences were too far removed from any of the other analyzed species to represent any of them.These authors had samples from all valid Mexican McVay et al. (2015)is at that time except for two (Thamnophis postremusSmith, 1942, and Thamnophis rossmani  Conant, 2000)and cautioned against making any taxonomical changes pending further sampling of "A.foxi" and the similar "A.copei."McVayetal. (2015)obtained additional sequences from the same specimen of "A.foxi" and confirmed the results obtained by de Queiroz et al.

Table A1 .
Specimens examined.Two samples are from shed skins where specimens were not collected, but genetic material was included in the analysis and these are included in bold print.

Table A2 .
Genbank accession numbers used in this study.New sequences generated by us are indicated in bold.Maximum Likelihood phylogenetic inference of members of the genus Thamnophis and closely related genera, based on the mitochondrial gene Cytb.Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0/).The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Link: https://doi.org/10.3897/herpetozoa.37.e122213.suppl1Author: Jacobo Reyes-Velasco Data type: pdf Explanation note: Maximum Likelihood phylogenetic inference of members of the genus Thamnophis and closely related genera, based on the mitochondrial gene ND4.Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0/).The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Link: https://doi.org/10.3897/herpetozoa.37.e122213.suppl2