Molecular assessment of confiscated Sceloporus lizards (Squamata: Phrynosomatidae) reveals unresolved taxonomic affinities and genetic reference gaps in Honduras

Molecular tools play a crucial role in biodiversity research and wildlife forensics, yet Central America remains underrepresented in genetic reference databases, particularly for reptiles. In Honduras, species of the genus Sceloporus are poorly studied despite their ecological relevance and increasing demand in the illegal pet trade. To address this knowledge gap, we analyzed mitochondrial COI sequences from seven confiscated Sceloporus individuals assigned to three morphotypes. Morphological examination identified three distinct morphotypes: two consistent with the published diagnoses of S. schmidti and S. hondurensis, and a third displaying atypical scale arrangements that did not conform to any recognized species diagnosis. Despite this morphological variation, the sequences showed very low nucleotide divergence (mean pairwise identity 99.4%) and formed a single strongly supported COI cluster, indicating very close mitochondrial affinity among the specimens and no COI differentiation corresponding to the morphotype assignments. This discordance between morphological assignment and mitochondrial variation was the most consistent pattern recovered. Comparisons with GenBank and BOLD returned sequences labeled as S. malachiticus as the closest available references, but identity values remained low (≤93.7%), indicating that the sampled individuals are not closely matched by any currently available public COI reference sequence. These results indicate an unresolved taxonomic affinity, with the closest currently available reference sequences assigned to the S. malachiticus complex, although a single mitochondrial marker and seven individuals cannot discriminate between an unsampled lineage, misidentified reference records, and incomplete database coverage. Our findings document substantial gaps in the regional molecular reference framework for Sceloporus and, to our knowledge, provide the first forensic genetic assessment of confiscated Sceloporus in Honduras.

Molecular approaches have become an essential component in modern biodiversity research, offering powerful tools to elucidate evolutionary relationships, assess population structure, and characterize biogeographic patterns across spatial scales (Riddle et al., 2008). In Mesoamerica, such approaches are increasingly recognized as critical for guiding conservation strategies, but genetic studies across countries remain scarce (Wultsch et al., 2016). Despite the great biodiversity in Honduras, terrestrial fauna remains poorly explored genetically, limiting evidence-based conservation efforts. Consequently, significant gaps persist in understanding the evolutionary history, population structure, and taxonomic boundaries of many taxa, limiting our capacity to address threats such as habitat loss, overexploitation, and wildlife trafficking (Senior et al., 2024). Expanding genetic research at spatial scales is therefore essential to inform and strengthen conservation strategies in Honduras. Wildlife trafficking, including the illegal trade, smuggling, hunting, poisoning, poaching, and harvesting of endangered species, has directly contributed to the global decline of wild animal populations (Morton et al., 2021; Mozer and Prost, 2023). Moreover, the translocation of species from native to non-native areas can generate a wide range of collateral effects, including disruptions to local fauna, public health risks, loss of livelihoods, the introduction of invasive species, and the expansion of organized crime (Warne and Chaber, 2023; Beckmann et al., 2025).

Across the Neotropics, genetic tools have proven particularly effective for delimiting species, detecting cryptic diversity, and determining the geographic origin of individuals involved in wildlife trafficking (de Oliveira et al., 2012; Marín-Villa et al., 2025). Mitochondrial and nuclear markers have enabled researchers to delineate distinct evolutionary lineages and assess population connectivity, while also providing tools that support legal and conservation applications (Bourret et al., 2020; Kowalczyk et al., 2021). Accurate identification of confiscated specimens is essential for conservation strategies and law enforcement. Over the last few decades, molecular techniques have increasingly been incorporated into taxonomic studies due to their reliability in species identification and assessment of population structure (Chambers and Hebert, 2016; Welch et al., 2017), thereby contributing to conservation efforts, strengthening legal frameworks to combat wildlife trafficking, and helping authorities track routes prioritize conservation interventions (Alacs et al., 2010; Jiao et al., 2021). These applications highlight the growing integration of molecular systematics, conservation genetics, and wildlife forensics, an approach particularly relevant for taxa that are recurrent targets of illegal exploitation in the region (Haines et al., 2021; Meiklejohn et al., 2021).

The genus Sceloporus (Wiegmann, 1828) is one of the most diverse groups of lizards, comprising more than 117 species distributed from southern Canada to western Panama (Smith, 1939; Sites et al., 1992; Leaché et al., 2016; Uetz et al., 2026), occupying a wide range of terrestrial habitats along its distribution, including lowland deserts, shrublands, grasslands, rocky and xeric landscapes, and high-elevation alpine forests (Wiens et al., 2013; Rivera et al., 2021; Hernández et al., 2024). These remarkable features make them a promising model system for ecological and evolutionary studies. Although Sceloporus lizards have been extensively studied throughout North America, particularly in the United States and Mexico (Leaché et al., 2016; López-Reyes et al., 2025), comparatively little research has been conducted in Central America. This knowledge gap likely reflects, in part, the limited availability of comprehensive taxonomic keys for regional faunas and the scarcity of molecular reference sequences in publicly accessible repositories such as GenBank, the Barcode of Life Data System (BOLD), and other genomic databases. Consequently, accurate species identification and delimitation remain challenging, hindering assessments of geographic population structure and limiting our understanding of the evolutionary history and phylogenetic relationships within this diverse group. Moreover, morphological traits, including femoral pore counts, scalation and coloration patterns, have traditionally been used for species identification and diagnosis within Sceloporus (Smith, 1939; Guillette et al., 1980; Bell et al., 2003). However, substantial uncertainty remains regarding species boundaries and the evolutionary relationships among taxa, largely due to extensive morphological variation, morphological convergence, and the presence of cryptic diversity within the genus (Campillo-García et al., 2021). Additionally, many species and subspecies remain under-sampled (Leaché, 2010; Campillo-García et al., 2021), and several lineages have been recognized but remain poorly studied (McCranie, 2018). In this context, DNA barcoding of the mitochondrial cytochrome c oxidase subunit I (COI) gene has become an essential tool for species identification, biodiversity assessment, detecting cryptic diversity, and identifying species involved in wildlife-trafficking seizures (Folmer et al., 1994; Hebert et al., 2003; Chambers and Hebert, 2016). Its resolution, however, depends entirely on the taxonomic and geographic completeness of the reference sequences available for comparison, and a single mitochondrial locus is generally insufficient on its own for evaluating gene flow, hybridization, or robust species delimitation (Yang and Rannala, 2010; Díaz-Cárdenas et al., 2017). Consequently, several studies have emphasized that robust species delimitation requires the integration of molecular, morphological, and geographic evidence rather than reliance on a single source of data (Vitecek et al., 2017; Luo et al., 2018).

In Honduras, three species of Sceloporus (S. schmidti Jones, 1927, S. squamosus Bocourt, 1874, and S. variabilis Wiegmann, 1834) have been reported, with two recently described endemic taxa within the S. malachiticus (Cope, 1864) complex (S. esperanzae and S. hondurensis McCranie, 2018). Interestingly, Stuart (1971) noted that the S. malachiticus population from La Tigra National Park (Francisco Morazán, Honduras) more closely resembled Costa Rican populations than those from the geographically closer La Esperanza (Intibucá, Honduras), highlighting the taxonomic complexity of this species complex (McCranie, 2018). Additionally, populations from western Honduras are currently considered as conspecific with those from Guatemala and El Salvador, and are identified as S. acanthinus Caballero, 1951 (McCranie, 2018). Six Honduran specimens from near the Guatemalan border support this hypothesis, and molecular data for one (FN 252207) assign it to the S. acanthinus clade with Salvadoran and Guatemalan individuals (McCranie, 2018). Despite this genetic evidence, these specimens lack the diagnostic morphological characters that define S. acanthinus. McCranie (2018) proposed the existence of three putative species within the S. malachiticus complex in Honduras. “Species A” from Joconales mountain in Santa Bárbara (northwestern Honduras), occurs between 1100–2010 m a.s.l. in wet forest habitats, but lacks clear morphological differentiation from other Honduran populations. “Species B,” from Cerro Santa Bárbara, also shows no distinct diagnostic traits but occupies an isolated mountain range at 1080–1650 m a.s.l., where individuals have been observed partially sleeping during cold nights. In contrast, “Species C,” from Ocotepeque and Celaque National Park (1400–2530 m a.s.l), is morphologically distinctive, characterized by an irregular arrangement of anterior head scales, and males exhibiting bright lime-green dorsal coloration with cobalt-blue abdominal markings.

Despite their ecological significance and conservation importance, the genetic diversity of Sceloporus remains poorly characterized in Honduras, with limited understanding of potential species boundaries, and geographic population structure. The undocumented lineages or misidentified populations further highlight the need for dedicated molecular research (Fernandez and Stewart, 2025). To address this knowledge gap, we analyzed genetic data from confiscated Sceloporus individuals to characterize their genetic composition and evaluate their COI-based genetic affinities within a regional framework. By comparing these samples with the reference sequences currently available for the genus, we aimed to determine their closest genetic affinities among currently available reference sequences and to evaluate the geographic distribution of the most similar reference records. These findings provide information on the taxonomic affinities of the seized specimens, document the current limitations of the regional molecular reference framework, and provide relevant information to conservation efforts and to strengthening actions against illegal wildlife trafficking.

Confiscated individuals and tissue sample collection

The analyzed specimens were obtained during the seizure of wildlife specimens by Honduran Border Police at an official border crossing to Nicaragua in May 2025. The confiscated animals, primarily reptiles (Table 1), were placed under the custody of the competent national authority (i.e., the Instituto de Conservación Forestal, Áreas Protegidas y Vida Silvestre “ICF”) and then transferred to the Colección Privada y Centro de Rescate de Fauna Silvestre El Ocotal (Sabanagrande, Francisco Morazán; 13.790961°, -87.313359° WGS84), a legally ex-situ conservation center operating under resolution GG-MP-066–2001 for preliminary taxonomic identification, health assessment, safekeeping and to promote popular scientific knowledge. To date, their geographic origin remains unknown, reflecting the clandestine nature of wildlife trafficking, highlighting the need for genetic and comparative analyses to determine taxonomic affinities and evaluate the geographic distribution of available reference records.

Only adult individuals (snout-vent length: 59.1–84 mm) were considered in this study to avoid biases in species morphological identification related to body proportions, scalation patterns and coloration. Accordingly, all individuals were preliminarily identified as Sceloporus, and each specimen was assigned to one of three morphotypes based on external morphological traits. Tissue samples were obtained from 18 adult lizards (11 males and 7 females; Table 2) authorized by the ICF, the national governing authority holding custody of the confiscated animals. Prior to sample collection, the distal tail section was cleaned with 70% alcohol to remove biological residues that could interfere with downstream molecular analyses. Approximately 1 cm of the distal tail tip was removed from each live lizard using sterilized scissors and collected for subsequent molecular processing. Then, each lizard was marked on the dorsal surface with a unique identification number using a permanent marker. Of these 18 individuals, nine were selected for molecular processing, and seven yielded usable COI sequences. The resulting molecular dataset should therefore be regarded as exploratory or pilot-scale. Although it is sufficient to document the identification problem and the lack of detectable differentiation in the COI sequences among the sampled individuals, it is not sufficient to estimate population-level genetic diversity or delimit evolutionary lineages.

Morphological characterization of Sceloporus morphotypes

Following the diagnostic characters proposed by McCranie (2018), we recognized three morphological variants based on frontal scale configuration and ventral semeion morphology. These morphotypes were initially classified according to the currently available morphological diagnoses and were used as operational categories for subsequent analyses. None of the examined specimens exhibited dorsolateral pale stripes, allowing us to exclude S. squamosus and S. variabilis, both of which possess this character. Morphotype 1 displayed a divided anterior frontal scale (transverse, longitudinal, or both) (Figure 1a) together with clearly separated ventral semeia (Figures 1b, c), corresponding to the morphology traditionally attributed to S. schmidti. Morphotype 2 exhibited an undivided anterior frontal scale (Figure 1d) and well-separated ventral semeia (Figures 1e, f), consistent with the published diagnosis of S. hondurensis. Morphotype 3 showed irregular frontal scale configurations that did not conform to the published diagnoses of either S. schmidti or S. hondurensis (Figure 1g). Nevertheless, all individuals of this morphotype possessed well-defined black abdominal bars delimiting the ventral semeia (Figures 1h, i), allowing the exclusion of S. esperanzae, which is characterized by poorly differentiated ventral semeia. Throughout the manuscript, these three units are referred to as morphotypes, defined as groups distinguished on the basis of external morphological characters. They are not considered species hypotheses unless their distinctiveness is independently supported by morphological, molecular, or other evidence.

DNA extraction, mitochondrial COI gene amplification, and sequencing

Immediately upon tissue collection, samples were transferred into sterile 1.5-mL tubes containing 1 mL DNA/RNA Shield buffer (Zymo, USA), placed into a cooler with ice, transported to the Center for Genetic Research (Universidad Nacional Autónoma de Honduras) and stored at -20 °C before DNA extraction. Of the 18 adult Sceloporus individuals sampled, nine specimens were selected for molecular processing, comprising three individuals from each of the three morphotypes. This subsampling strategy was designed to provide balanced representation of the morphological variation observed among the confiscated animals while considering available laboratory and sequencing resources, and was not designed to capture population-level genetic diversity. Genomic DNA was isolated from these nine samples using the Extracta™ DNA Prep for PCR kit (QuantaBio, Beverly, MA, USA) following the manufacturer’s instructions. DNA concentration and purity were measured using a UV5Nano nano-spectrophotometer (Mettler Toledo, Canada). Extracted DNA concentrations ranged from 82.9 ng/µL to 305.96 ng/µL, providing sufficient material for PCR amplification. Prior to amplification, all samples were diluted to a standardized concentration of 40 ng/µL. Subsequently, we used the DNA barcoding primers LCO-1490 (5´-GGTCAACAAATCATAAAGATATTGG-3´) and HCO-2198 (modified) (5´-TAAACTTCAGGGTGACCAAAAATCA-3´) to amplify a fragment of approximately 650 bp of the COI gene. PCR amplifications were performed in a total volume of 50 μL of PCR mixture, containing 4 µL of DNA template, 25 µL of 2× Taq Master Mix (Promega, USA), 2 µL of each primer at a concentration of 10 µM, 2 µL of bovine serum albumin (BSA), and 15 µL of nuclease-free water. The PCR cycling settings were as follows: initial denaturation at 95 °C for 10 min, followed by 37 cycles of denaturation at 94 °C for 1 min, annealing at 48 °C for 1 min, extension at 72 °C for 1 min, and a final extension step at 72 °C for 7 min. In parallel, a PCR blank was included as a negative control, and no contamination was detected. Amplified PCR products were separated on 1% ethidium bromide-stained agarose gels, and positive samples were sequenced in both directions at Psomagen Inc. (Rockville, MD, USA). For each specimen, the forward and reverse reads were subsequently assembled into a single consensus sequence, and all downstream analyses were based on these consensus sequences.

Bioinformatic analyses

Sequencing data were processed using the Geneious ® prime software v.2024.05 (Kearse et al., 2012). Forward and reverse chromatograms were visually inspected and trimmed to remove low-quality base calls and residual primer sequence, and the paired reads of each specimen were assembled into a single consensus sequence. Pairwise identity, percentage of identical sites, and number of polymorphic sites were calculated across all consensus sequences and within morphotypes. These alignment metrics were used to assess levels of genetic variation among specimens and evaluate whether COI differentiation corresponded to morphotype assignments. To assess the genetic affinities of the sequenced individuals, each consensus sequence was queried against NCBI GenBank using the BLASTn algorithm, and against the Barcode of Life Data System (BOLD) using the barcode identification tool. For both databases, percent identity, query coverage, and associated accession or Barcode Index Number (BIN) were recorded. Geographic metadata associated with the closest reference sequences were examined to characterize the known geographic distribution of the corresponding taxa. However, given the relatively low sequence identity of the matches, these geographic data were not used to infer the geographic provenance of the confiscated specimens.

Because morphology-based identification within the S. malachiticus complex can be challenging, and because public reference sequences may include misidentified or taxonomically unresolved records, GenBank and BOLD matches were interpreted cautiously. These databases were used to assess genetic affinity with the closest available reference sequences, rather than to provide definitive species-level identifications. Accordingly, low identity values were interpreted as evidence that the confiscated specimens are not confidently assignable to any currently represented reference sequence, highlighting the need for voucher-based regional sampling and integrative taxonomic revision. All newly generated consensus sequences were deposited in NCBI GenBank. The correspondence among specimen codes, consensus sequences, and accession numbers is provided in Table 2.

To assess whether the seven consensus sequences obtained in this study formed distinct COI-based genetic clusters, a barcode clustering analysis was conducted using the Tamura-Nei genetic distance model and the Neighbor-Joining method with 1,000 bootstrap replicates to assess nodal support. These dendrograms are used here as an exploratory barcode-clustering tool and are not intended as phylogenetic reconstructions or as a formal species-delimitation analysis. To provide a comparative framework across the genus, we conducted an exhaustive search of the GenBank Nucleotide database for all publicly available Sceloporus COI sequences (search terms: Sceloporus [Organism] AND (COI[Gene] OR COX1[Gene] OR “cytochrome oxidase subunit I”[Title] OR “cytochrome c oxidase subunit 1”[Title]). After excluding duplicate and redundant records, a final dataset of 69 reference sequences representing 16 named Sceloporus species was retained and aligned together with the seven newly generated consensus sequences (Supplementary Table 1). This dataset included all Sceloporus COI sequences recovered from the public database search and therefore represents the broadest currently available COI reference framework for comparison. Nonetheless, it encompasses only 16 of the more than 117 currently recognized species in the genus, meaning that unsampled congeners or populations may exhibit closer mitochondrial affinities to the confiscated specimens.

Sequencing quality and length

DNA extraction and COI PCR amplification were performed for nine selected specimens, representing the three morphotypes. Seven of these nine specimens yielded high-quality bidirectional COI reads suitable for downstream analyses, with HQ percentages above 92% and final consensus lengths between 658 and 678 bp. Alignment of the seven consensus sequences resulted in a mean aligned length of 657 bp, of which 643 sites (97.9%) were identical across all seven sequences simultaneously, whereas identity averaged over all pairwise comparisons was 99.3%. The two values differ because the first counts only those sites conserved in every sequence, while the second averages identity across sequence pairs; together they indicate very limited COI divergence among the sampled individuals.

Genetic variation

Within morphotypes, only minor polymorphisms were detected. Morphotype 1 (AJT46, AJT47) showed four polymorphic sites and 99.7% pairwise identity. Morphotype 2 (AJT40, AJT41, AJT45) exhibited nine polymorphic sites and 99.3% identity. Morphotype 3 (AJT53, AJT54) displayed five polymorphic sites and 99.5% identity. The GenBank accession number of the consensus sequence of each specimen is given in Table 2. Overall, despite the morphological criteria used for morphotype assignment, no COI differentiation corresponding to the predefined morphotypes was detected.

Database comparisons and genetic affinity assessment

BLAST searches in GenBank recovered sequences labeled as S. malachiticus as the closest available matches for all seven sequences. However, percent identities were comparatively low (93.26–93.7%), with no alignment exceeding 94%. The top GenBank match in all cases corresponded to accession MH274629.1, a record reported from Colón, Honduras. At this level of similarity, however, the locality associated with the closest reference record cannot be taken as an indication of the geographic origin of the confiscated specimens. Similarly, searches in the BOLD Barcode Identification System recovered sequences labeled as S. malachiticus as the nearest available references, but identity values remained low, ranging from 92.58% to 93.42%. The closest BIN-associated records were BOLD: ADI4162, corresponding to a specimen from Colón, Honduras; BOLD: ADD7876, derived from a record from Panama; and BOLD: ACB7946, associated with a record without detailed geographic information. These BINs were interpreted as the nearest available BOLD reference clusters, rather than as definitive BIN assignments for the newly generated sequences. The low identity values and incomplete metadata associated with some records further support a cautious interpretation of BOLD matches as indicators of broad mitochondrial affinity, not definitive species-level identification.

COI-based clustering and genetic affinity to reference sequences

The COI-based Neighbor-Joining clustering analysis of the seven newly generated sequences (Figure 2A) showed that all individuals grouped together with 100% bootstrap support, regardless of their assigned morphotype. Although minor sub-grouping was observed, no cluster corresponded uniquely to a specific morphotype, indicating that no COI differentiation corresponding to the three morphotype assignments was detected. When the seven confiscated sequences were analyzed together with the expanded reference dataset of 69 sequences spanning 16 Sceloporus species (Figure 2B), the confiscated specimens again formed a strongly supported COI cluster (100% bootstrap support). Among the available reference sequences, this cluster showed its closest mitochondrial affinity to sequences labeled as S. malachiticus, and no closer match was recovered among the currently available public COI sequences included in the analysis. Because the reference dataset represents only 16 of the more than 117 recognized species in the genus, this result should be read as the absence of a closer match within the currently available reference framework, rather than as evidence that incomplete reference sampling can be excluded.

The illegal wildlife trade poses serious risks to local populations and can disrupt trophic dynamics. Accurate morphological and molecular identification of seized specimens is essential for enforcing wildlife protection laws and preventing further wild population declines. Here, we applied molecular analyses to assess the genetic affinities between a group of confiscated Sceloporus individuals and the reference sequences currently available for the genus. The frontal morphological variation observed among the three morphotypes falls within the range of diagnostic criteria proposed for Honduran S. malachiticus sensu lato (McCranie, 2018). Moreover, the presence of black abdominal semeion bars across all male morphotypes was useful here for excluding S. esperanzae, although the diagnostic value of this character across the complex remains to be tested with broader sampling.

However, the presence of individuals exhibiting irregular frontal scalation suggests additional morphological variation not captured by current diagnostic descriptions, a pattern that has also been documented in other Sceloporus spiny lizards (Buckley et al., 2010; García-Rosales et al., 2017). Importantly, these assignments represent morphological hypotheses based solely on external characters. As shown by the molecular analyses presented here, the three morphotypes were not differentiated by the COI sequences, despite their differences in frontal scale morphology. This discordance between morphology-based assignments and mitochondrial data suggests that frontal scale configuration, traditionally used as a diagnostic character within the S. malachiticus complex, may exhibit greater intraspecific variation than currently assumed, or may be of limited diagnostic value within this group. Similar intraindividual phenotypic variation has been reported in other lizard species, potentially arising from developmental instability, environmental influences, or other sources of phenotypic variation (Sites et al., 1992; Leaché et al., 2016). It should be emphasized, however, that intraspecific variation represents only one of several plausible explanations. Incomplete morphological diagnoses, misinterpretation or misassignment of diagnostic characters, and recent divergence between taxa retaining similar or shared mitochondrial haplotypes cannot be excluded based on the present dataset. While morphological analyses are useful for taxonomic delimitation, the limited availability of comprehensive taxonomic keys for Honduran Sceloporus species can hinder accurate species-level identification, as exemplified by the three morphotypes analyzed here, which may represent intraspecific morphological variation. Therefore, integrative studies combining morphological and molecular methods are essential for taxonomic surveys, evolutionary analyses, and the detection of potential cryptic species.

This study represents, to our knowledge, the first genetic characterization of confiscated Sceloporus individuals in Honduras. Although the individuals were initially assigned to three distinct morphotypes, their COI sequences showed minimal nucleotide variation (pairwise identity: 99.3–99.7%), indicating that all sampled specimens showed very limited COI divergence and clustered together in the barcode analysis. However, comparison with GenBank and BOLD revealed unexpectedly low similarity to sequences labeled as S. malachiticus, the closest available reference taxon, with identity values not exceeding 93.7%. Given these low values, the possibility of uncertain or outdated taxonomic assignments in public databases, the limited taxonomic and geographic coverage of the available reference sequences, and the fact that our comparative dataset included representative rather than all available locality-specific Sceloporus sequences, our results do not support a definitive species-level identification or inferences regarding cryptic diversity. Instead, they indicate that the confiscated specimens are not closely matched by the currently available COI reference sequences. Because the analyses relied on a single mitochondrial marker and on seven individuals, the Neighbor-Joining dendrograms presented here should be interpreted as barcode-based clustering analyses rather than definitive phylogenetic reconstructions, and no formal lineage delimitation is attempted. Resolving the taxonomic status of these animals will require additional nuclear and mitochondrial markers, broader geographic sampling, more specimens, voucher-based reference sequences, and integrative morphological analyses. These findings highlight important gaps in the molecular reference framework currently available for Central American Sceloporus.

Following McCranie (2018), S. hondurensis was primarily diagnosed using morphological traits in combination with geographic distribution, particularly locality and elevation. Although some of the morphotypes recognized in this study exhibit character combinations traditionally associated with S. hondurensis and S. schmidti, the molecular evidence recovered here does not allow a definitive assignment to any recognized species within the S. malachiticus complex. The morphological assessment identified three morphotypes, but all sampled individuals fell within a single COI cluster. This result suggests that the observed variation in frontal scale configuration may represent intraspecific variation or limitations of the morphological characters currently used for species diagnosis. Similar discrepancies between morphology and mitochondrial data have been documented previously within the genus (Wiens and Penkrot, 2002).

The taxonomic uncertainty surrounding the S. malachiticus complex is particularly relevant in western Honduras, a region recognized for high levels of endemism and the presence of poorly studied reptile assemblages (Wilson and McCranie, 2003; Wilson and Townsend, 2006). McCranie (2018) further suggested the possible existence of additional undescribed diversity within northwestern and western Honduras (and even eastern Honduras in La Mosquitia). From a wildlife forensic perspective, the inability to confidently assign confiscated specimens to a described taxon underscores the current limitations of regional reference databases. Illegal wildlife trade continues to represent a major conservation challenge in Central America, where weaknesses in enforcement capacity and legal frameworks may facilitate the movement of wild-caught animals through commercial networks (Reuter and O’Regan, 2017; Richards et al., 2003; Gluszek et al., 2021; Wilson, 2023). However, it is essential to distinguish empirical documentation from inference. While the confiscation of these lizards during a wildlife trafficking seizure is documented, their geographic provenance and trafficking route remain unknown. Our molecular data do not resolve the source population, geographic origin, or transboundary movement of the specimens, nor do they establish whether the animals are native to Honduras or were imported from elsewhere. Accordingly, the findings support only the conclusion that specimens with unresolved taxonomic affinities were recovered during a wildlife trafficking seizure, which is itself relevant to conservation forensics. Expanding validated, voucher-based genetic reference datasets will be essential for improving species identification and may ultimately facilitate more reliable assessments of geographic origin, while strengthening the capacity of conservation and law-enforcement authorities to investigate and address wildlife trafficking across the region.

The confiscated specimens formed a strongly supported COI cluster that was genetically distinct from the currently available S. malachiticus reference sequences, and from S. virgatus (Valenciennes, 1835) and S. orcutti (Stejneger, 1893), the two next closest taxa included in our comparative dataset. This pattern indicates that the mitochondrial variation recovered from the confiscated lizards is not adequately represented in the COI reference sequences available in public databases. Several non-mutually exclusive explanations may account for this result, including erroneous taxonomic assignments of reference sequences, low-quality or incomplete reference data, unsampled variation within the S. malachiticus complex, or the possibility that the confiscated specimens belong to a taxonomically distinct lineage not currently represented in public repositories. The present data cannot discriminate among these alternatives. The contribution of this study is therefore best understood as a wildlife forensic case study that documents an unresolved taxonomic affinity, a discordance between morphotype assignment and mitochondrial variation, and substantial gaps in the molecular reference framework available for Central American Sceloporus. Resolving the taxonomic identity of these lizards will require broader geographic sampling, voucher-based reference datasets, morphological analyses, and multilocus approaches combining nuclear and mitochondrial markers with integrative morphological analysis.