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Article

Detecting the Endangered San Joaquin Kit Fox (Vulpes macrotis mutica) and Other Canine Species in Kern County, CA: Applying a Non-Invasive PCR-Based Method to Four Case Study Sites

by
Antje Lauer
*,
Sarah Alame
,
Julian A. Calvillo
,
Mario E. Gaytan
,
Jonathan R. Juarez
,
Jocelyne J. Lopez
,
Kayla Medina
,
Isaac Owens
,
Alejandro Romero
and
Jarred Sheppard
Department of Biology, California State University Bakersfield, 9001 Stockdale Highway, Bakersfield, CA 93311, USA
*
Author to whom correspondence should be addressed.
Conservation 2025, 5(1), 8; https://doi.org/10.3390/conservation5010008
Submission received: 4 December 2024 / Revised: 19 January 2025 / Accepted: 27 January 2025 / Published: 12 February 2025
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Abstract

:
The endangered San Joaquin kit fox (SJKF) (Vulpes macrotis mutica), which is endemic to the San Joaquin Valley in California, has lost most of its natural habitat due to urban sprawl and change in land use over time. Many studies have been conducted to restore and protect the remaining habitat, involving presence/absence surveys prior to urban development using camera monitoring, tracking dogs, tracking plates, spotlighting, and trapping. While these traditional methods work well, they can be invasive, expensive, labor-intensive, and require permits to perform. In our study, we used a non-invasive method based on DNA extraction from scat collected in the environment, followed by a diagnostic Polymerase Chain Reaction (PCR)-based approach on mitochondrial DNA fragments and investigated the presence of the SJKF on four case study sites that shared a high SJKF habitat suitability index but are under the threat of development. We found that the diagnostic PCR was able to accurately differentiate between different canids present at the sites, in a time- and cost-effective manner. Including this non-invasive method in the Department of Fish and Wildlife’s standardized recommendations for survey methods would help to improve future environmental assessments for SJKF populations in the Central Valley of California.

1. Introduction

The San Joaquin kit fox (SJKF) (Vulpes macrotis mutica) is the smallest canid in North America and can be differentiated from other North American fox species by their large ears, small size, long limbs, and black tipped tail [1]. The SJKF is considered a subspecies of kit fox due to its isolated geographical range within California [2]. The SJKF inhabits semi-arid areas with annual grasslands and was once common throughout the California Central Valley floor, interior basins, and adjacent foothills. The SJKF’s diet consists mainly of small mammals and occasionally arthropods [3]. One adaptation to living in arid environments is the SJKF’s ability to extract high percentages of moisture in its diet, allowing it to survive without freshwater resources for some time [4].
Much of the SJKF’s habitat has either been destroyed, damaged, or fragmented due to agricultural, industrial, and urban development, as well as human population growth [5,6,7]. In addition to habitat loss, most of the SJKF population inhabiting central and northern portions of their range have been extirpated or severely reduced due to flooding, drought, shooting, trapping, poisoning, and road kills. SJKFs have been listed as federally endangered since 1967 [8]. In 1971, this fox species was also declared endangered by the state of California under the Endangered Species Act (CESA). Even though a recovery plan was developed in 1998 with the aim of protecting the prime habitat for this key stone species, the SJKF is still in decline [9]. The exact size of kit fox populations in the Central Valley of California is not currently known but was estimated to range between approximately 7000 to 15,000 in the early 1970s with 85% of the population in six counties (Kern, Tulare, Kings, San Luis Obispo, Fresno, and Monterey). Half the population was documented in only two of those counties: Kern (41%) and San Luis Obispo (10%), mainly throughout the Temblor Mountain Range and the Carrizo Plains [10,11]. The current population size has been estimated to be likely less than 5000 range-wide, leading to concerns that their small geographic population sizes could lead to inbreeding depression when isolated, mange infestations, or other environmental or demographic stochastic events [12,13,14].
Biodiversity in California’s Central Valley has been declining for decades, as it is one of the most dense and concentrated land developments in the world [15]. Due to this urbanization, the expansion of industrial agriculture, as well as fossil fuel production, many other animal and plant species that can be found in prime SJKF habitats are also in severe decline or already extinct, among them the preferred prey of foxes, e.g., endemic kangaroo rats (Dipodomys spp.), the San Joaquin antelope squirrel (Ammospermophilus nelsoni), the San Joaquin pocket mouse (Perognathus inornatus), and the blunt-nosed leopard lizard (Gambelia sila) [16]. In the city of Bakersfield, endangered SJKFs share habitats with domestic cats (Felis catus) and dogs (Canis familiaris), as well as raccoons (Procyon lotor), striped skunks (Mephitis mephitis), and Virginia opossums (Didelphis virginiana) [17]. In the countryside, coyotes (C. latrans), bobcats (Lynx rufus), occasional mountain lions (Puma concolor), badgers (Taxidea taxus), other foxes, such as red foxes (Vulpes vulpes), gray foxes (Urocyon cinereoargenteus) and swift foxes (V. velox), as well as other animals might co-occupy SJKF habitats. Among those animals listed, red foxes and Virginia opossums are not native to the Central Valley of California [18,19].
While SJKFs have reduced in numbers in rural and exurban environments, they appear to thrive in metropolitan areas as a generalist species [20]. One area of primary interest for SJKFs is the city of Bakersfield, in which its presence has it considered as an unofficial city mascot, where a large breeding population exists, and has been studied extensively for many years. This stable population of about 200 animals is essential for the long-term recovery of the species [7,8,21].
Survey methods primarily used to detect foxes and other mammalian predators include scent/bait stations, track plating, spotlighting, and camera monitoring. Methods that are commonly used but are generally reserved for largescale projects with sufficient resources consist of dog tracking, trapping, and radio telemetry [22]. Non-traditional methods such as the non-invasive one used in this study are underused by environmental consulting groups in California and have never been included in environmental surveys in the Southern San Joaquin Valley, even though molecular biology research has resulted in a plethora of non-invasive DNA-based tools to investigate not just the presence of certain canine species but also chromosomal sex, genetics and disease burden, using DNA extraction from scat followed by Polymerase Chain Reactions (PCRs) and sequencing [2,12,23,24].
Methods of DNA collection were originally defined as ‘non-invasive’ if “the source of the DNA was left behind by the animal and could be collected without having to catch or disturb the animal” [25,26]. For example, genetic material that was left behind in scat or shed skin flakes could be collected and analyzed (i.e., sensu environmental DNA (eDNA)), implicitly avoiding any impact on animal welfare.
Several aims of this project were pursued:
  • Establish an economical and reliable non-invasive PCR-based method to detect the presence of SJKFs and other canine species in urban Bakersfield and surrounding environments in Kern County in our lab at CSUB.
  • Apply this method to selected locations in Kern County where SJKFs are suspected of roaming, including areas where a suitable habitat is threatened by development.
  • Communicate and promote this non-invasive PCR-based method to local environmental consulting companies to support efforts to identify SJKF habitats in the Central Valley of California prior to development in a timely and cost-efficient manner.
  • Contribute to SJKF protection and conservation efforts in the Central Valley of California.

2. Materials and Methods

2.1. Reference Samples

To establish and verify the molecular method described below in our laboratory at CSUB, several reference scat samples from SJKFs and other mostly canine species were provided by wildlife ecologist Dr. Lucas Hall (CSUB) and by Sharon Adams, curator of the California Living Museum (CALM) in Bakersfield, CA, which is a local zoo that focuses on native and endemic species of the Central Valley. CSUB has had a viable SJKF population on campus for many years, and scat was collected from one of their latrines. These samples were used as positive controls for Polymerase Chain Reactions (PCRs) and for educational purposes. Additional scat samples were collected by CSUB students on campus and in the Bakersfield Environmental Studies Area (BESA) near campus. Some non-canine scat samples were included in this study to validify the PCR-based method prior to large scale sample collection and analyses. Supplementary Table S1 provides detailed descriptions of all reference scat samples, as well as field recognition data that are useful for scat identification and collecting efforts.

2.2. Site Selection and Description

Four sites that are at risk for the development or expansion of agriculture were selected to be investigated for the presence of SJKFs (case study sites). All these locations can be described as open grassland, with occasional shrubs and a high habitat suitability rating for SJKF between 85 and 95 out of 100 possible points, with 100 points referring to a prime habitat [26]. At some of these sites, the presence of SJKFs was confirmed by environmental consultants in previous years as part of environmental assessments or was assumed based on anecdotal knowledge. Scat samples were collected from all four case study sites. Figure 1 gives an aerial overview of these locations, and Table 1 presents information about each case study site. More specific information about each case study site has been submitted as Supplementary Materials with references [27,28,29,30,31,32,33,34]. Figure 2 shows photos of all case study sites. All case study sites received a high SJKF suitability score, which is a form of habitat suitability rating [27] (Table 2). Even in an urbanized area such as Bakersfield, a suitable SJKF habitat remained, even though it was highly fragmented (Appendix A: Figure A1).

2.3. Collection of Environmental Scat Samples

Environmental scat samples were collected from 4 different sites in Kern County between October 2023 and June 2024. In the field, we focused on collecting scats that were likely dropped by canine predators, considering the typical size and shape of the sample, and whether it contained remains of fur, berries, or insects. These scat characteristics can distinguish an actively hunting animal from a domestic dog in most cases. Canines are omnivores, compared to cats, who are strictly carnivorous, which is reflected in the scat content. Students were instructed about the typical size, shape, and consistency of different animal scats by using reference material from CALM and CSUB (Dr. Hall) (see also Supplementary Table S1). Additional visual aids and descriptions of scat were obtained from field guides [38,39]. Nitrile gloves were used to collect and handle all scat samples for safety reasons, because they likely contain parasites. The exact location of each scat sample was documented together with a short description including color, size, consistency (soft or hard), the presence of bones, fur, seeds, berries, insects, etc., as well as estimated age (fresh, older). It should be noted though that it is sometimes difficult to assess the age of a scat sample to seasonal influences, especially over the summer months when a fresh sample can dry quickly and appear older. All samples were photographed and placed in sterile Falcon Tubes or clean Ziplock bags at the time of collection and then frozen at −20 °C until processed in a biological laboratory at California State University Bakersfield (CSUB).

2.4. DNA Extraction, Polymerase Chain Reaction (PCR), and Agarose Gel Electrophoresis

DNA extractions were performed by using the Zymo Research Quick-DNA Fecal/Soil Microbe MiniPrep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. For scat samples that appeared older or compromised (light in color, very hard, brittle), the vortexing time was increased from 10 min to 20 min to obtain more DNA. PCRs were performed by using primer pair SIDL-forward and H3R-reverse targeting the mitochondrial DNA, resulting in a variable fragment number and size across species [40]. These primers target mitochondrial DNA fragments of different predatory species within the Canidae, Mustelidae, and Ursidae but also target some species within the Antilocapridae, Cervidae, Equidae, and Leporidae [26,41].
For our PCR process, we used 12.5 µL of Apex Taq RED DNA Polymerase Mastermix (Genesee Scientific, El Cajon, CA, USA), 1.5 µL of each primer (10 pmol/µL), 4 µL of DNA, and 6.5 µL of sterile PCR grade water in an overall 25 µL reaction. Thirty-five cycles of PCRs were performed in a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) including negative and positive controls. For scat samples that appeared older or compromised, up to 45 cycles were performed, and up to 10 µL of DNA extracts were used. The thermocycling conditions included a 15 min denaturation at 94 °C, followed by up to 35–45 cycles of 30 s at 94 °C, 90 s at 46 °C, 60 s at 72 °C, and a 30 min elongation at 60 °C. Negative and positive controls were used in each PCR to monitor contamination and accuracy of the amplification process. A subsequent 2% agarose gel electrophoresis using 4 µL of PCR products and 4 µL of a DNA marker (G316A, Promega, Madison, WI, USA) allowed the determination of amplicon lengths, intensity, and quality and was documented using the GelDocSystem 2000 (Bio-Rad, Hercules, CA, USA).

2.5. Sequencing of PCR Products

The sequencing of amplicons in the range of 300–400 bp was performed at Laragen (Culver City, CA, USA) after a purification step with ExoSapIt (Thermo Fisher Scientific, Santa Clara, CA, USA), using the Sanger sequencing method. The GenBank nucleotide database was used to identify closest matches of all sequenced amplicons with % similarity [42,43].

3. Results

Reference scat samples of known origin that were obtained from CALM and CSUB were successfully used to establish and optimize the PCR protocol originally published in 2014 [40] in our laboratory, before collecting and processing environmental scat samples from unknown species. Overall, DNA was extracted from 80 droppings collected at four individual locations named case studies near Bakersfield in Kern County, CA. Following successful amplifications via the PCR using primer pair SIDL-forward and H3R-reverse (Figure 3), products were purified, sequenced, and compared to entries in the GenBank nucleotide database, and closest matches were recorded (Table 3).
Overall, the SJKF was only detected at case study site 1 (VA). Red foxes were present only at case study site 2 (MG). The presence of coyotes was confirmed at all locations except for case study site 2. Scat from domestic dogs, which was present at site 2, was excluded from this study, and other canine species were not detected at this location. The presence of one non-canine species was confirmed for case study site 4 (BV), namely, the North American badger (Taxidea taxus) (Figure 4).
Results for the four case study sites showed the dominance of one canine species at each site. The SJKF was only detected at site 1 (VA). Red foxes were the dominant canine species at site 2 (MG), and coyotes were present at sites 3 (TS) and 4 (BV). Some droppings that were analyzed could not be associated with a canine species.
Of all environmental samples investigated, 52 (65%) were confirmed to have originated from a canine species. In addition, one sample (1.25%) belonged to a non-canine, namely, a North American badger (Taxidea taxus), a species of special concern in California. The remaining samples either resulted in no amplicons, produced multiple bands of different sizes, amplicons that were longer than 500 bp, and, when sequenced, did not result in a GenBank match, or were not associated with mammals (false positives) (Table 4).
We observed that PCRs obtained with primer pair SIDL/H3R produced a band of about 309 bp for SJKFs, which was slightly shorter compared to those obtained from other canines, which all produced longer fragments of about 333–336 bp when run on 2% agarose gels, which was confirmed by sequencing. Amplicons from T. taxus appeared shorter (291 bp) compared to those obtained from canids.
The costs to collect and analyze one scat sample at CSUB in 2024, including DNA extraction, PCR, agarose gel electrophoresis, and sequencing, were estimated to accumulate to 47 USD/sample including an estimated 10 USD/sample for fieldwork + mileage, and 37 USD/sample for lab supplies and lab work. When compared to the costs of conventional methods used for environmental assessments, this protocol is very competitive. The time needed to perform this task, including sampling (one day/location), DNA extraction and PCR and gel electrophoresis (one day), sequencing and comparing to GenBank (one day + two days for shipping and receiving), is estimated to be one week for at least 50 samples at CSUB. Currently, we are promoting this non-invasive method to two environmental consulting groups, which are known to perform environmental surveys in California.

4. Discussion

Direct observations of SJKF and other small foxes in their natural environments are rare because of their mostly secretive nature and nocturnal activity patterns, such as avoiding larger predators, especially coyotes, which are common in the Southern San Joaquin Valley. Exceptions are some urban areas, such as the CSUB campus, where these foxes have become acquainted with the presence of humans and can be observed during the day. In the natural environment, the presence of a canine species might be overlooked when traditional methods alone are pursued or require many field biologists, time, and equipment, such as traps and cameras, and, thus, might be costly. If the purpose of a project is to solely determine the presence of SJKFs or other canines on a property (e.g., for environmental surveys), and no additional data such as age, weight, chromosomal sex, health status, etc., are needed, a non-invasive method, like the one described in this study, can provide reliable results in a week at moderate costs. Therefore, to determine if a specific location can be considered as suitable habitat for this endangered species, either as hunting ground, as wildlife corridor, or for mating and denning, a non-invasive method based on scat analysis can be considered as an alternative method of choice.
Detecting scat from canines and other territorial animals is a relatively simple process. SJKFs, like other canines, leave scat easily visible at dens, trail intersections, along unpaved roads, and fence lines [44,45], as well as near conspicuous objects. Examples include trash cans, skeletal remains [46,47], lids of sewage canals (personal observation, A. Lauer) or landmarks, such as large rocks, berms, poles, or concrete, where other individuals have left their markings, creating so-called latrines. It is, therefore, very likely to find at least a few scat samples from many, if not all, predatory mammal species that are present in an area by simply following jogging and hiking trails and small roads or by following animal trails through the vegetation. Non-canines mark their territory as well, and it is not always easy to distinguish scat from foxes from those of other predators. When visiting a location to collect scat, potential dens and burrows of animal tracks or other signs of the presence of canine predators can be documented, which are all non-invasive methods.
Collectors of scat should be trained to be able to distinguish potential canine scat from droppings of other animals. Descriptions of all collected samples, including potential age, will aid in explaining potential false negative results when using DNA-based methods. We noticed that old scat samples that appeared whitish and very hard often fail to provide enough DNA for PCRs, even though, based on their description, they were likely of canine origin. Increasing bead beating time in the DNA extraction from 10 min to up to 40 min, increasing the amount of DNA in the PCRs, as well as adding additional PCR cycles can improve the outcome for older scat samples. We also noticed that many scat samples that were identified to belong to a certain canine species varied to some degree in shape, size, and content, which made it difficult to assess the likely origin of the scat when collecting them in the field. Therefore, we recommend also collecting scats that, at first sight, seem unlikely to originate from a canine species to avoid missing the detection of some species in the target area.
Based on the habitat suitability score, all four case study locations are suitable habitats for SJKFs, either being characterized as grassland/ruderal or as scrub with a slightly higher score [27]. Ongoing urban development in Kern County is destroying or fragmenting some of the remaining larger grassland areas within the city limits that could support SJKF populations. This has resulted in an increase in habitat fragments that are less suitable for these endangered animals, affecting population genetics, animal health, and, ultimately, survival of the species. The Metropolitan Bakersfield Habitat Conservation Plan (MBHCP) included an incidental take permit (CESA ITP 2081-2013- 025-04) for the SJKF and other endangered species but excluded special status species, such as the American badger. However, the MBHCP expired on 1 June 2023 and has not been renewed. This has implications for developers who must apply for their own permits through the California Department of Fish and Wildlife if they have not paid a mitigation fee by 1 January 2023 [48]. In most urban development cases, including those highlighted in this paper, impacts on the environment including endangered species are considered less than significant; therefore, mitigation measures are not required at all.
Results of our analyses did not confirm the presence of SJKFs at the case study site 2 (MG), in contrast to a survey conducted by an environmental consulting group in 2021 [32]. Instead, all scat that was detected confirmed the presence of red foxes (Vulpes vulpes) on that property. It is known that different carnivores, including fox species, often avoid sharing a habitat due to competition for prey and dens [49,50], and it can therefore be speculated that the larger red fox might have outcompeted the smaller SJKF at this location. Related to that, coyotes (Canis latrans) were the only carnivores detected on study sites 3 (TS) and 4 (BV), even though the habitat type was also indicated as highly suitable for SJKFs. Coyotes are known to often kill the smaller canines, which was noted as the primary cause of mortality in a study conducted in the Carrizo Plains, likely due to exploitative competition [4]. The only case study site that showed the dominance of SJKFs was site 1 (VA), where many large dens of California ground squirrels (Spermophilus beecheyi) were observed when students sampled the area in addition to potential SJKF dens. Evidence of the presence of this endangered fox species was documented earlier [29]. This location is close to several restaurants, which tend to attract urban foxes as they have learned to scavenge [20]. Prey was also plentiful at this site in the form of a large population of California ground squirrels (A. Lauer, personal observation). Developing this area can therefore be detrimental to the SJKF population.
Non-invasive methods based on DNA extraction from scat might be very useful in areas where large surveys are planned and where camera/track monitoring would be prohibitively expensive. For example, large parcels of land must be surveyed before they can be considered as suitable land for mitigation efforts or before the construction of a project can commence. Results from surveys based on scat analyses via PCR can be performed prior to in-depth traditional survey methods that may be required by state and federal agencies [22], increasing accuracy of the study, while also saving money and time. For a more general focus on determining habitat suitability based on proof that animals used a specific location, non-invasive methods like scat analysis might be sufficient, provide results quicker, and are more affordable. Surprisingly, non-invasive methods are poorly implemented in wildlife studies and environmental surveys. A recent review showed that only 22% of studies on the genetics of animals used non-invasive methods, even though non-invasive genetic analyses have been shown to be reliable tools in wildlife research when compared with other approaches [51,52]. Also, among conservationists, wildlife biologists, and environmental consultants in Kern County, the use of non-invasive methods to detect animals in the environment is not common (A. Lauer, personal observation) and are not included in survey and monitoring protocols and guidelines provided by the California Department of Fish and Wildlife. The standard recommendation for monitoring SJKFs has not been updated since 2011 and includes only traditional survey methods [22], which may explain why non-invasive methods are rarely taken up by professional environmental consultants. It can be criticized that detecting scat from an animal species might not confirm its current occupation of a habitat, because the scat can be several months old, and it will take some time until scat is completely decomposed in dry environments such as the San Joaquin Valley. However, finding scat from an SJKF is certainly an indicator that a particular habitat was suitable at least for hunting or traveling. We like to emphasize that older scat samples that were deposited several months prior to collection still provide valuable information about habitat suitability for a certain canine species, although it might not reflect the status of the species’ presence at the time of sampling.
With Bakersfield predicted to grow by 6% over the next 6 years reaching almost 450,000 inhabitants [53], more land will be needed for housing, schools, and other infrastructure. Land that is considered vacant often includes a suitable habitat for SJKF populations, but, to allow development, the city is pursuing rezoning efforts, especially for large scale warehouses and shopping malls, such as those proposed for case study site 2 [54]. Only a few locations where SJKF populations thrive within the city of Bakersfield have remained. Two of the locations include the CSUB campus and the Bakersfield Community College campus located on the bluffs above the Panorama Vista Preserve, near the Kern River and its floodplains. Small populations of SJKFs are also observed on other school campuses, churches, and medical centers throughout the city [6,55]. Among those sites that support a viable SJKF population within the city of Bakersfield is also case study site 1 that was investigated in this study.
The diagnostic PCR method based on the mitochondrial DNA used in this study allowed the successful differentiation between several different canine species that can be encountered in Kern County, CA. We confirmed that this method accurately indicated the presence of SJKFs in all controls (reference samples from CALM and CSUB) and was reliable for environmental samples of known origin (CSUB campus). We further confirmed that the amplicon obtained via PCR from V. macrotis mutica scat samples was shorter compared to those from other canine species, which allowed it to be easily distinguishable from other foxes, coyotes, or feral dogs that are likely to share the habitat [40]. We also confirmed that the North American badger (T. taxus), which belongs to the family Mustelidae, can be detected with this method and was not included as target animal in the original publication [40].
About 35% of the collected scat samples could not be identified and might have originated from members of the Felidae family (Felis catus, Lynx rufus, Puma concolor), raccoons (Procyon lotor), the Virginia opossum (Didelphis virginiana), or from larger rodents (e.g., the California ground squirrel [Otospermophilus beecheyi]) that are common in Kern County. Animal tracks from various non-canine species were common at all case study sites that were investigated in this study. However, we do not know how many individual non-canine species were present at each site, which may have affected the variations in identified and non-identified scat between the case study sites. Other factors to consider for the variation in PCR success between sites are likely, for example, different ages of samples and the unequal number of samples collected at each study site.
Even though SJKF scat is generally smaller in size compared to feces from coyotes, dogs, or larger cat species, and generally larger than rodent or skunk droppings, for example, it is sometimes difficult to determine the origin of a scat sample, considering the age, health, and diet of an individual animal, which affect size, color, consistency and content of the sample. In addition, some scat samples collected in the field might have been too old to extract enough DNA suitable for downstream applications, such as PCR. An incomplete removal of PCR inhibitors during DNA extraction could explain some of the failed amplifications and variations in PCR success among sites. Furthermore, we sometimes observed unspecific amplifications, resulting in amplicons that had no match in the GenBank database or were related to microorganisms abundant in the scat. PCR products that resulted in amplicons larger than 330 bp were not of canine origin, as observed in this study, and generally had no match in the GenBank nucleotide database. However, most of our samples (65%) were either negative or resulted in an amplicon that was related to a canine species. Additional efforts could be considered to improve the DNA extraction procedure from older or compromised samples by adding a second DNA purification step. Furthermore, the use of primers that amplify shorter mitochondrial fragments of mitochondrial DNA could increase PCR success rates for degraded samples, as suggested by [56], could be explored.
Next steps to continue and expand this work include the following:
  • Continuing data collection (potentially in collaboration with environmental consulting companies) in areas of Kern County where the presence of SJKFs is unknown or anecdotal (e.g., potential mitigation sites).
  • Developing primer pairs that are more specific to canines, or, alternatively, allow the identification of all non-canine species that are common in Kern County, thereby reducing the number of unidentified scat samples.
  • Amplifying the DNA extracts that failed to produce an amplicon using an additional primer pair [54] that amplifies a shorter fragment of the mitochondrial DNA, which might help with slightly compromised DNA from older scat samples and exclude the amplification of non-target species.
  • Performing a phylogenetic analysis of SJKF sequences to investigate if differences in populations can be detected based on the mitochondrial DNA indicating potential mixing of the gene pool from different populations.
  • Expanding this non-invasive method to include endangered rodents, such as Dipodomys spp. using primers based on the mitochondrial DNA or the cytochrome oxidase DNA [57,58].
  • Alerting Kern County officials and the city of Bakersfield about results from this work and future work prior to certifying developmental projects that could destroy SJKF habitat.
  • Communicating this method to local environmental consultants to improve future environmental surveys for SJKF populations for EIRs in a cost and time-effective way.

5. Conclusions

The non-invasive PCR-based method used in this study was efficient and produced reliable results and thus has potential to be adopted in the portfolio of survey methods to detect endangered SJKFs and other canines in the field. Using this method on a large-scale project with an adequate population size of canines will help to establish trust in this method. Regardless of the timeframe in which DNA analysis on scat could become an accepted practice for environmental assessments, it is still an effective tool for other researchers and citizen scientists to monitor the range and movement of SJKFs and other animals of interest. With additional research, the factors for accurate identification (e.g., preventing false negative results) can be determined, and a standardized protocol can be developed and included in the list of accepted survey methods of wildlife in California.
Communicating the benefits of the non-invasive method used in this study as shown here to local environmental consulting companies may help to improve future environmental surveys for SJKF populations and other animals in a cost and time-effective way, with the goal to identify and preserve suitable habitats for this endangered species, in urban and rural areas. Strategies for mitigating the impact of urban development on SJKFs have been proposed many years ago [21] and should be considered by urban planners and developers for sustainable SJKF populations in Kern County and elsewhere in the future.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/conservation5010008/s1, Supplementary Information for case study sites 1–4, as well as Supplementary Table S1. Description of reference scat samples used in this study, as well as field recognition criteria. It should be considered that scat content from omnivores like foxes and coyotes varies seasonally, often including insects in the spring, seeds and berries in the summer/fall, and more bones and fur or feathers from animals in the winter.

Author Contributions

Conceptualization, A.L.; methodology, A.L., K.M. and A.R.; validation, A.L.; formal analysis, K.M., A.R., S.A., J.A.C., M.E.G., J.R.J., J.J.L., I.O. and J.S.; investigation, S.A., J.A.C., M.E.G., J.R.J., J.J.L., K.M., I.O., A.R. and J.S.; resources, A.L.; data curation, A.L.; writing—original draft preparation, A.L., M.E.G. and J.S.; writing—review and editing, S.A., J.A.C., M.E.G., J.R.J., J.J.L., I.O. and J.S.; visualization, A.L., J.R.J. and J.S.; supervision, A.L.; project administration, A.L. funding acquisition, A.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Office of the Provost at CSU Bakersfield, Grant MX117-PJ0176-2023/24.

Data Availability Statement

The authors declare that the data collected for this project can be shared with other scientists.

Acknowledgments

We thank Gordon Nipp for providing detailed information on the case study sites. Furthermore, we would like to thank Lucas Hall (CSUB) and Sharon Adams (Curator, CALM) for providing fecal samples from known animals. Some of the data were collected as part of the Advanced Topics in Biology course (BIOL 5710) in the fall semester of 2023. Lastly, we would like to thank S. Del Rio, A. Mateiro, C. Gude, and J. Lo for performing some of the DNA extractions and PCRs.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Appendix A

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Figure A1. Information on habitat suitability for SJKF can be obtained from this interactive map: Kit Fox Habitat Suitability Model|Data Basin based on the work by [27]. The figure shows SJKF habitat suitability score for the Veteran’s Clinic (VA, case study site 1) and Major Gateway Project site (MG, case study site 2) indicated as 85, class 1: Grassland/ruderal (light turquoise color). The Beardsley canal, connecting different locations suitable for SJKFs, is seen in the brown east of site 1. The SJKF habitat suitability score for the Travel Center site and the location at Cole’s Levee Road (BV) (case study sites 3 and 4) is indicated as 95, class1: Scrub, undeveloped.
Figure A1. Information on habitat suitability for SJKF can be obtained from this interactive map: Kit Fox Habitat Suitability Model|Data Basin based on the work by [27]. The figure shows SJKF habitat suitability score for the Veteran’s Clinic (VA, case study site 1) and Major Gateway Project site (MG, case study site 2) indicated as 85, class 1: Grassland/ruderal (light turquoise color). The Beardsley canal, connecting different locations suitable for SJKFs, is seen in the brown east of site 1. The SJKF habitat suitability score for the Travel Center site and the location at Cole’s Levee Road (BV) (case study sites 3 and 4) is indicated as 95, class1: Scrub, undeveloped.
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Figure 1. Aerial overview of four sites (case studies) that were investigated for the presence of SJKFs using a non-invasive PCR-based method. The sites are all located in the Southern San Joaquin Valley near Bakersfield, CA, and are indicated with red dots and white labels (scale bar: 20,000 feet).
Figure 1. Aerial overview of four sites (case studies) that were investigated for the presence of SJKFs using a non-invasive PCR-based method. The sites are all located in the Southern San Joaquin Valley near Bakersfield, CA, and are indicated with red dots and white labels (scale bar: 20,000 feet).
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Figure 2. Case study sites: (A). Overview of site 1 (VA) (Veteran’s Affairs Community Clinic Project). Note that the soil has been disked. (B). Undisturbed grassland to the east of the project area with visible burrows and dens. (C). Site 2 (MG) (Majestic Gateway Project) showing the disturbance and illegal dumping of trash. (D). Larger den on project area (near site MG-9). (E). Overview of Travel Center site 3 (TS) showing grassland and playa-like soils with occasional salt bushes (Atriplex spp.). (F). Animal den and plenty of small mammal tracks (near TS-2). (G). Overview of the sampling area at site 4 (BV) Cole’s Levee Road (CLR) on Green Acres Farm showing grassland and playa soil (site BV—Buena Vista Lake Area). (H). Animal den near site BV (CLR-7).
Figure 2. Case study sites: (A). Overview of site 1 (VA) (Veteran’s Affairs Community Clinic Project). Note that the soil has been disked. (B). Undisturbed grassland to the east of the project area with visible burrows and dens. (C). Site 2 (MG) (Majestic Gateway Project) showing the disturbance and illegal dumping of trash. (D). Larger den on project area (near site MG-9). (E). Overview of Travel Center site 3 (TS) showing grassland and playa-like soils with occasional salt bushes (Atriplex spp.). (F). Animal den and plenty of small mammal tracks (near TS-2). (G). Overview of the sampling area at site 4 (BV) Cole’s Levee Road (CLR) on Green Acres Farm showing grassland and playa soil (site BV—Buena Vista Lake Area). (H). Animal den near site BV (CLR-7).
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Figure 3. Example of PCR results obtained with primer pair SIDL/H3R: (A). PCR products obtained from reference samples and from four environmental samples (SJKF 1–4, and unknown 5 [V. macrotis mutica], 6 [neg], 7 [C. lupus familiaris], 8 [neg], 9 [V. m. mutica], NC = negative control) collected near CSUB and at the Bakersfield Environmental Studies Area (BESA) in preparation of this project. (B). PCR products obtained from environmental samples only. Note the slight difference in amplicon size between samples KF-56 and KF-60, which were identified as SJKFs compared to samples KF-52 (C. latrans) and samples KF-57 and KF-58 (C. lupus familiaris). Samples KF-53 and KF-54 cannot be identified, and KF-59 (present twice) is negative.
Figure 3. Example of PCR results obtained with primer pair SIDL/H3R: (A). PCR products obtained from reference samples and from four environmental samples (SJKF 1–4, and unknown 5 [V. macrotis mutica], 6 [neg], 7 [C. lupus familiaris], 8 [neg], 9 [V. m. mutica], NC = negative control) collected near CSUB and at the Bakersfield Environmental Studies Area (BESA) in preparation of this project. (B). PCR products obtained from environmental samples only. Note the slight difference in amplicon size between samples KF-56 and KF-60, which were identified as SJKFs compared to samples KF-52 (C. latrans) and samples KF-57 and KF-58 (C. lupus familiaris). Samples KF-53 and KF-54 cannot be identified, and KF-59 (present twice) is negative.
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Figure 4. Detection of canine predators at 4 study sites: A. corner of Knudsen and Olive Drive (site 1 [VA]), B. South H Street and Berkshire Dr. (site 2 [MG]), C. intersection of Stockdale Hwy. and I-5 (site 3 [TS]), and D. farmland on Cole’s Levee Road (site 4 [BV]).
Figure 4. Detection of canine predators at 4 study sites: A. corner of Knudsen and Olive Drive (site 1 [VA]), B. South H Street and Berkshire Dr. (site 2 [MG]), C. intersection of Stockdale Hwy. and I-5 (site 3 [TS]), and D. farmland on Cole’s Levee Road (site 4 [BV]).
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Table 1. Location of all sampling sites, number of samples collected, habitat type and rating [27], as well as sampling dates.
Table 1. Location of all sampling sites, number of samples collected, habitat type and rating [27], as well as sampling dates.
Sampling Locations and SitesSamples (n)Habitat Type and Rating (Class 1) [27]Sampling Date
Central Bakersfield:
Knudsen Dr. (VA) (case study 1)22Grassland/ruderal, 8530 October 2023
South Bakersfield:
South H Str. (MG) (case study 2)31Grassland/ruderal, 8531 December 2023 and 15 June 2024
West Bakersfield:
I5-Stockdale Hwy. (TS) (case study 3)18Scrub, 9513 + 17 December 2023
Southwest Bakersfield:
Cole’s Levee Rd. (BV) (case study 4)9Scrub, 951 April 2024
Table 2. Overview of sites that served as case studies for the detection of canine predators via a non-invasive PCR-based method.
Table 2. Overview of sites that served as case studies for the detection of canine predators via a non-invasive PCR-based method.
Case Study SiteSize (sf)Description of SiteAdditional Information
Case study 1 (VA) Knudsen Dr.±39,648Grassland, mostly invasive species, disked at least once, partially trashed, presence of SJKF confirmed by previous environmental surveysConstruction of Veteran’s Affairs Community-Based Outpatient Medical Clinic [35].
Case study 2 (MG) South H Street.187,500 + 1,012,185Grassland, mostly invasive species, disked at least once, partially trashed, presence of SJKF suspected by previous environmental surveysMajestic Gateway Project, 12 commercial buildings and warehouses planned [36].
Case study 3 (TS) I-5/Stockdale Hwy.1,141,272Natural valley grassland with occasional shrubs, presence of SJKF not knownTravel center with 4 fast food restaurants, 28 fuel pumps and a nine-bay, island-like diesel truck fueling station associated with truck scales and parking [33].
Case study 4 (BV) Cole’s Levee Rd. 3450Unused agricultural plot with semi-natural grassland vegetation, invasive species, and occasional shrubs, presence of SJKF suspectedLos Angeles Bureau of Engineering, Green Acres Biosolids Land Application Project [37].
Table 3. Overview of PCR amplification success from all environmental scat samples collected at four case study sites and amplified with primer pair SIDL-F/H3R-R (id. = identified; unid. = unidentified).
Table 3. Overview of PCR amplification success from all environmental scat samples collected at four case study sites and amplified with primer pair SIDL-F/H3R-R (id. = identified; unid. = unidentified).
Samples Species
Sampling Location# of Samples% of id. Samples% of unid. SamplesV. m. muticaV. vulpesC. latransT. taxus
City of Bakersfield
Case study 1 (VA) Knudsen Dr.2245.554.556040
South of Bakersfield
Case study 2 (MG) South H Str.3180.6519.3502500
West of Bakersfield
Case study 3 (TS) I-5/Stockdale Hwy.1866.6733.3300120
Buena Vista Lake Area
Case study 4 (BV) Cole’s Levee Rd.955.5644.440041
Table 4. Summary of PCR results obtained with primer pair SIDL/H3R for all environmental scat samples collected.
Table 4. Summary of PCR results obtained with primer pair SIDL/H3R for all environmental scat samples collected.
nPos PCR *Neg PCRMultiple AmpliconsAmplicon > 500 bp **
805211125
100%65.00%13.75%15.00%6.00%
* Sequencing successful. ** No match in GenBank.
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Lauer, A.; Alame, S.; Calvillo, J.A.; Gaytan, M.E.; Juarez, J.R.; Lopez, J.J.; Medina, K.; Owens, I.; Romero, A.; Sheppard, J. Detecting the Endangered San Joaquin Kit Fox (Vulpes macrotis mutica) and Other Canine Species in Kern County, CA: Applying a Non-Invasive PCR-Based Method to Four Case Study Sites. Conservation 2025, 5, 8. https://doi.org/10.3390/conservation5010008

AMA Style

Lauer A, Alame S, Calvillo JA, Gaytan ME, Juarez JR, Lopez JJ, Medina K, Owens I, Romero A, Sheppard J. Detecting the Endangered San Joaquin Kit Fox (Vulpes macrotis mutica) and Other Canine Species in Kern County, CA: Applying a Non-Invasive PCR-Based Method to Four Case Study Sites. Conservation. 2025; 5(1):8. https://doi.org/10.3390/conservation5010008

Chicago/Turabian Style

Lauer, Antje, Sarah Alame, Julian A. Calvillo, Mario E. Gaytan, Jonathan R. Juarez, Jocelyne J. Lopez, Kayla Medina, Isaac Owens, Alejandro Romero, and Jarred Sheppard. 2025. "Detecting the Endangered San Joaquin Kit Fox (Vulpes macrotis mutica) and Other Canine Species in Kern County, CA: Applying a Non-Invasive PCR-Based Method to Four Case Study Sites" Conservation 5, no. 1: 8. https://doi.org/10.3390/conservation5010008

APA Style

Lauer, A., Alame, S., Calvillo, J. A., Gaytan, M. E., Juarez, J. R., Lopez, J. J., Medina, K., Owens, I., Romero, A., & Sheppard, J. (2025). Detecting the Endangered San Joaquin Kit Fox (Vulpes macrotis mutica) and Other Canine Species in Kern County, CA: Applying a Non-Invasive PCR-Based Method to Four Case Study Sites. Conservation, 5(1), 8. https://doi.org/10.3390/conservation5010008

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