DNA extractions were performed using three different methods: the tissue and blood samples were extracted using a phenol chloroform organic extraction protocol (Sambrook et al., 1989). Additionally, many of the blood samples were washed with a silica cleanup protocol to remove excess heme and any other environmental contaminants. Hair samples were extracted using the same phenol chloroform organic extraction protocol as the tissue and blood samples (Sambrook et al., 1989) with the addition of 20µl of 1M DTT (DL-Dithiothreitol) per DNA extraction.

Every test method performed for wildlife forensic case work at the Wyoming Game and Fish Wildlife Forensics Laboratory runs a positive and negative control. The negative control moves with the case throughout the entire testing process starting with DNA extractions. The positive controls are test specific and must produce a validated result for use. In the event a control fails the results are recorded in the bench notes and the testing starts over. The lab does not report test results with failed positive or negative controls.

Three methods were used for species identification. Sanger sequencing (Sanger et al., 1977) was performed on all unknown samples as well as positive and negative controls using the mtDNA gene region CytB with universal primers ‘mcb 398’ and ‘mcb 869’ (Verma and Singh, 2002; primer sequences are listed in Supplementary Data Sheet 1 ). The samples were ran on a 3500 genetic analyzer (Applied Biosystems) and the resulting sequences were analyzed in Molecular Evolutionary Genetic Analysis (MEGA) v. 11.0.13 (Tamura et al., 2021) and then compared with sequences from NCBI using BLAST (Clark et al., 2016; Sayers et al., 2022) to determine the species of origin. DNA sequencing alone cannot differentiate between Odocoileus spp. Therefore, samples that sequenced out to be from the Odocoileus spp. had to be further tested using protein serology or STRs for species assignment.

Protein serology was used by the laboratory for taxonomic identification of blood samples (Mardini, 1984; ANSI/ASB Standard 106, 2020). Serum albumin is an excellent protein to identify species from tissue and blood samples. This protein is the most abundant plasma protein and is found throughout the extravascular spaces of the body, maintaining the interstitial fluid colloid osmotic pressure. Serum albumin is a strongly anionic monomeric protein that can be readily separated from other tissue proteins by electrophoresis (Murch and Budowle, 1986; Melsert et al., 1989; Signore et al., 2017). A key application of the Albumin Western blot analysis is the differentiation of the four closely related members of the Cervidae family (mule deer, white-tailed deer, elk, and moose). Electrophoresis was ran on a Pharmacia LKB Biotechnology PhastSystem with positive and negative controls.

The validation of OdoPlex (Hamlin et al., 2021) enabled the use of DNA STRs to differentiate species of Odocoileus spp. Multiplex PCR amplification was performed on the unknown samples that were identified as Odocoileus spp. based on sequence analysis, using the OdoPlex multiplex STR panel (Hamlin et al., 2021). STR amplification and analysis was performed using the parameters listed in Supplementary Data Sheet 2 . In order to determine the species of origin, the alleles associated with each item were compared with an in-house database of known mule deer and white-tailed deer alleles and analyzed with GenAlEx6.51b2 (Peakall and Smouse, 2006; Peakall and Smouse, 2012).

Sex identification of the Odocoileus spp, C. canadensis and U. americanas samples was amplified using multilocus STR panels. The sex identification primers are listed in Supplementary Data Sheet 1 . Samples originating from O. hemionus and O. virginianus were amplified with the OdoPlex STR panel (Hamlin et al., 2021). The sex-linked mammal-specific ZFX markers (Aasen and Medrano, 1990), which amplifies females at an amplicon size of ~443bp and an Artiodactyla-specific Y-chromosome linked SRY marker (Wilson and White, 1998) to amplify males at an amplicon size of ~190 bp were used.

Sex identification loci of C. canadensis samples was amplified, in a similar manner, with a multilocus STR panel referred to as WapitiPlex (Jones et al., 2002; Meredith et al., 2005; Meredith et al., 2007). In addition to the sex-linked mammal-specific ZFX and the Artiodactyla-specific Y-chromosome linked SRY marker the panel also includes a mammal-specific ZFY marker (Fain and LeMay, 1995) at an amplicon size of ~221bp.

Amplification of the sex identification markers for U. americanus was performed using the multilocus STR panel, UrsaPlex (Meredith et al., 2020) markers. An X chromosome linked bear-specific ZFX marker with an amplicon size of ~163bp and two Y chromosomes linked bear-specific markers (SMCY and 318.2) (Bidon et al., 2013) with amplicon sizes of ~106 bp and ~129 bp respectively were used.

Sex identification of Antilocapra americana, Alces alces and Meleagris spp. were amplified with specific sex markers and ran on a 5% agarose gel. Band separation imaging was visualized using a UV light and camera system (IGenius 3). PCR amplification of the polymorphic region of the ZFX/ZFY and SRY (P1-3EZ/P1-5EZ and Y53-3C/Y53-3D) locus was used to determine the sex of A. alces. Sex identification of A. americana was performed under the same conditions with the exception of the Y primer SRY (Y53-3E/Y53-3F) (Fain and LeMay, 1995). In Meleagris spp. the female is the heterogametic sex (ZW). The sex identification of Meleagris sp. was performed using primers Pst1 with amplification at ~177-bp and ATP amplification at ~250-bp (D’Costa and Petitte, 1998).

Multiplexed PCR amplifications were performed on the Odocoileus spp. (OdoPlex) (Hamlin et al., 2021), C. canadensis (WapitiPlex) (Jones et al., 2002; Meredith et al., 2005; Meredith et al., 2007) and U. americanas (UrsaPlex) (Meredith et al., 2020) samples with the QiagenⓇ Multiplex PCR kit using ten to sixteen STRs. Primers for the individual species are listed in the Supplementary Tables S1–S3 . Forward primers of all STR and sex identification loci were labeled with fluorescent dyes (6FAM, VIC, NED and PET; ThermoFisher Scientific) following the parameter set forth in the species specific validation studies (Hamlin et al., 2021 and Meredith et al., 2020). Multiplex PCR reactions was ran using a BioradⓇ Thermal Cycler. Fragment separation was performed on Applied Biosystems 3500 Genetic Analyzer using the Fragment Analysis50_POP7_G5 module.

Singleplex PCR amplifications were performed on Alces alces (Wilson et al., 1997; Talbot et al., 1996; Smith et al., 2002; Bishop et al., 1994; Vaiman et al., 1994), Antilocapra americana (Bishop et al., 1994; Lou, 1998; Carling et al., 2003; Stephen et al., 2010) and Meleagris (Huang et al., 1999; Reed et al., 2000). Primers for the individual species are listed in the Supplementary Tables S4–S6 . An IRDye 700 or 800 M13 tail was added to each reaction depending on the validated procedure.

The PCR reactions were ran using a BioradⓇ Thermal Cycler and fragment separation was performed on a LiCor Sequencer.

All multiplex STR analysis was performed with GeneMapper software (Applied Biosystems™). While the singleplex reactions were analyzed with Saga™ software (LiCor). The individual genotypes were visually inspected and scored by qualified analysts. The unique genetic profiles were recorded for each sample to determine individualization, minimum number of animals and sex identification.

In 2024 the laboratory received a wildlife forensic case submission from a District Wildlife Officer from Colorado Parks and Wildlife. A total of 59 items were submitted for examination. The submission form, which the submitting officer filled in, stated that items #1 and 2 originated from mule deer (O. hemionus) of unknown sex. At the time of submission, the officer did not know if other species were present in the evidentiary items and the known mule deer samples were the only species in question. The investigation aimed to address the following objectives:

A few critical decision points can streamline complex cases and save time and costs. The majority of these decision points are made during the subsampling process, but are also littered throughout the entire process. The flowchart in Figure 1 highlights the different decision points in analyzing cases, especially the more complex ones. One of the most important decision points is to determine when to stop analyses on a specific sample and report the sample as either nonreactive or inconclusive. Several factors should be considered at this step. Does the sample quality appear to be so poor that retesting may not work? How important is the sample to the case, is it one of several blood samples from the same item or is it a highly important sample? Is there enough sample to retest? How many times to retest before it becomes cost prohibitive? These factors can be considered multiple times throughout the retesting of one sample and there is always the possibility that the sample will have to be called nonreactive regardless of the importance to the case. If the item appears to have biological material and the laboratory has a presumptive test (blood) then the sample should be tested and the presumptive test should be performed prior to further testing. However, if there isn’t a presumptive test available, should the sample be tested anyway? Also, determining if there is any reason to start analyses of a specific item in the first place is a crucial decision point. For example, if the analyst does not visually identify any biological material, should the sample be tested?

In the highlighted complex case, fifty-nine samples were submitted for testing. One of the first, and sometimes the most difficult, decisions made at this juncture is determining whether or not a submitted item is suitable for downstream analyses. If a large number of samples are submitted for forensic testing, the laboratory analysts carefully examine each item to evaluate if the item contains forensically useful biological material. In this complex case, the evidentiary items ( Table 1 ) included two tissue samples from mule deer of unknown sex, eleven blood and hair samples from various clothing items, six blue shop rags with possible blood and hair, thirty-three hunting items with blood and/or hair, two coolers with blood and five miscellaneous blood and hair items. The suspect blood and the hair samples from the various clothing, shop rags, hunting items, coolers, and miscellaneous samples consist of the most difficult of the samples. Is the suspect blood actually a true blood sample? Do the hairs have any root ends? Even though the hunting items were submitted as having forensically useful material, is that material actually present?

During the sampling of this case, it was determined that four of the clothing items and five of the blue shop rags, when visually inspected for hair, blood, or tissue did not have any biological material. At this point, these items were not sampled for downstream testing and this was noted in the case notes.

Although only fifty-nine evidentiary items were submitted to the laboratory for the complex case and several of the submitted items did not have biological material, the number of items totaled sixty-eight ( Table 1 ). This is because multiple subsamples can be collected from the same item, these additional items are identified with a capital letter after the item number ( Table 1 ). For instance, if a shirt is submitted as one piece of evidence with multiple blood stains, several blood stains can be collected and treated as individual items. These subsampled items consisted of tissue, blood, and hair with the possibility of various environmental contaminants that may have the potential to inhibit downstream analyses. Sample types and inhibitors can dictate which of the several validated extraction protocols to use.

The genetic methods used to address the objectives in this case are described in the Methods section, with detailed methods listed in Supplementary Materials S1 .

Species identification was performed on the samples that contain biological material, as well as a positive and negative control. The Sanger sequencing method was implemented using the mitochondrial gene CytB on the unknown items. Sequencing alone cannot differentiate between Odocoileus spp. Therefore, any of these samples that sequenced out to be from the Odocoileus spp. had to be further tested using protein analysis. All other samples were identified to species and ready for downstream analyses. Species identification results for individual items are listed in Table 1 .

The fifteen blood samples that sequenced to the Odocoileus spp. had further testing using protein serology performed to determine their species of origin, either O. hemionus or O. virginianus.

At the time of this case, the Albumin Western blot test was the only test method the laboratory had validated to differentiate between mule deer and white-tailed deer. As with all analyses, positive controls were ran with the unknowns.

Species identification using DNA sequencing indicated twenty items originated from elk (Cervus canadensis), sixteen items originated from the Odocoileus spp. (O. hemionus or O virginianus), four items originated from moose (A. alces), six items originated from turkey (Meleagris), two items originated from black bear (U. americanus) and one item originated from pronghorn (A. americana). Additional species identification of the sixteen Odocoileus spp., using albumin IEF, resulted in eleven items most probably originating from mule deer (O. hemionus). Protein analysis is a reliable method for differentiating between mule deer and white-tailed deer, the method relies heavily on the analyst’s ability to score IEF gels and clean protein samples. Due to these factors the laboratory protocol states that when reporting of Odocoileus spp. using a protein test method a qualifying factor will be used, hence the most probably mule deer.

Sex identification and STR genotyping were performed on all items that produced a result for species identification. These results are listed in Table 1 .

Several items were non-reactive for species identification making this a critical decision point in the laboratory. How many times should the sample be retested prior to determining it is non-reactive? The answer to this depends on the quality of the sample, the importance of the sample, and is also based on analyst experience. If a sample is determined non-reactive at this point, further testing is not performed.

This complex case highlights several key points when making decisions to move a wildlife forensic case through the laboratory.

The results in this case demonstrated that there were five additional species outside the known mule deer items #1 and 2. It was critical that the laboratory had protocols in place to handle the sub sampling, processing, testing and reporting of the items to successfully answer the objectives in this case. The results were provided to the submitting officer to either support or refute their investigation and to provide a report in the event this case makes it to the judicial system. At this time the laboratory does not know the outcome of this case.

Early 2024 the New Mexico Department of Game and Fish submitted eight samples for forensic examination. Items #1-4 consisted of tissue samples from a gut pile and items #5-8 were tissue samples from known mule deer (O. hemionus). Using the submission request form, the Investigator requested the following ( Table 2 ):

Eight tissue samples approximately 5 mm x 5 mm in size stored in desiccant tubes were submitted for testing. Since the items submitted were tissue samples, sub sampling was straight forward and technical decision points were not required to determine if the sample type is biological material and eliminated the need for any presumptive testing. Also, four of these samples were submitted as originating from a known species, eliminating the need for species identification. In this case, approximately 100 mg of tissue from each item submitted was collected for downstream analysis. The eight tissue samples went through the laboratory’s phenol chloroform organic extraction protocol (Sambrook et al., 1989) and did not require additional DNA clean-up.

This simple case was chosen to highlight the importance of validating new test methods. Before discussing the results and conclusions of the simple wildlife forensic case, a brief overview of the validation of OdoPlex for species assignment is in order. This validation was implemented after the complex case and before the simple case was tested in the laboratory.

All new test methods routinely used at the Wyoming Game and Fish Forensics Laboratory are validated via a full validation process following the laboratory’s Quality Manual section on developmental and/or internal validation. The validations also follow the validation guidelines for DNA analysis methods recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM, 2016).

Shortly after the complex case was completed the laboratory performed an internal validation using OdoPlex (Hamlin et al., 2021) to differentiate between O. hemionus and O. virginianus species. This STR multiplex panel was previously validated for use for individualization, leading the way for the internal validation for species identification. Hamlin et al., 2021. demonstrated that the OdoPlex STR multiplex panel can be used as a diagnostic tool to determine species identity of the two closely related deer species and several subspecies. The laboratory’s internal validation focused on the identification of O. hemionus and O. virginianus.

Tissue samples from 313 deer, including 143 known mule deer (O. hemionus), 150 known white-tailed deer (O. virginianus) and 20 deer of unknown species (selected by a third party for species assignment) from Wyoming, Montana, and Colorado were selected. These samples were collected by law enforcement officers and agency biologists, primarily from hunter harvested deer and are stored in the laboratory’s in-house database. DNA was extracted using a phenol chloroform organic extraction protocol (Sambrook et al., 1989). PCR optimization, amplification, and capillary electrophoresis were performed following the process listed in the Methods section for OdoPlex.

Sixty-one individuals were analyzed using STRUCTURE (v2.3.4) (Pritchard et al., 2000; Evanno et al., 2005). The data set was analyzed with two populations assumed, with a 100,000 Burn-in period, and 100,000 Markov chain Monte Carlo repetitions for 10 iterations (K = 1-18). Results were analyzed using Evanno’s Best K (Evanno et al., 2005) and plotted with a bar graph to determine if species separation was possible. Results from STRUCTURE clearly demonstrated that the OdoPlex STR panel (Hamlin et al., 2021) can differentiate between the two Odocoileus spp. It is important to note that all sixty-one individuals produce a complete profile. The bar plot ( Figure 2 ) demonstrates that both species clearly separate when a full genetic profile is produced using the Odoplex STR panel.

The same sixty-one individuals were analyzed using GenAlEx (Peakall and Smouse, 2006; Peakall and Smouse, 2012). Although both programs correctly identified two species, GenAlEx was determined to be the best fit for the laboratory’s data formatting. Therefore, only GenExAl was used to analyze the larger data set of 313 deer to assign the unknown deer samples. GenAlEx demonstrated that the OdoPlex STR multiplex (Hamlin et al., 2021) plan can be used to determine species assignment using microsatellites to differentiate between mule deer and white-tailed deer with 100% accuracy of species assignment of unknowns. OdoPlex (Hamlin et al., 2021) has a number of species-specific alleles that can be used in conjunction with population assignment software to determine the species of unknown evidentiary items. Figure 3 demonstrates species assignment of ten unknown deer samples using GenAlEx. The green unknown triangles plotted in the figure show the correct assignment of all ten unknown deer samples.

Additionally, as demonstrated in the original developmental validation for species determination using OdoPlex (Hamlin et al., 2021), OarFCB193 has a fixed allele at “10” when ran with O. hemionus. The observation of a fixed allele at “10” was seen at 100% during the internal validation with OarFCB 193 in all Montana, Wyoming, and Colorado mule deer that have been tested on OdoPlex. A small number of O. virginianus samples that were heterozygous at the “10” locus at OarFCB193 were detected. This was seen 1% of the time. With this observation and the fact that the Wyoming Game and Fish work with both deer species on a regular basis, it was determined that the entire OdoPlex panel (Hamlin et al., 2021) would be used for species assignment.

Key points to consider when using OdoPlex for species assignment:

Species identification was performed on the first four items using Sanger sequencing of the CytB gene. DNA sequencing indicated all four items originated from the Odocoileus spp. meaning additional testing was required to determine species of origin. Previously, the laboratory used proteins to determine O. hemionus and O. virginianus. However, with the new validation of OdoPlex (Hamlin et al., 2021) to differentiate species of Odocoileus DNA STRs were used to determine species of origin. Species identification using OdoPlex for population assignment indicated the four items originated from mule deer (O. hemionus).

Sex identification and STR genotyping was performed on all eight samples using the parameters listed in the Methods section. The results, using Odoplex for genotyping, sex identification, and species identification are in Table 2 .

This simple case highlights a couple of key points when making decisions to move a simple wildlife forensic case through the laboratory.

The results in this case demonstrated that there were two male mule deer. The implementation of OdoPlex for species assignment played a key role in species identification of this case. The results were provided to the submitting officer and they supported their investigation. The results in this case led to the defendant pleading guilty to taking of big game animals without the required license. Their plea resulted in several thousand dollars in fines and restitution and the loss of their hunting privileges for one year.