IDENTIFYING LOWLAND LEOPARD FROGS (LITHOBATES YAVAPAIENSIS) USING IN SITU PHOTOGRAPHY

Item Type text; Electronic Thesis

Authors THOMAS, ALYSSA SHEA

Publisher The University of Arizona.

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Abstract

Lowland leopard frogs (Lithobates yavapaiensis) are endemic to Arizona, New Mexico, Sonora, and Chihuahua. Due to conservation concerns about this species and potential risk of injury, stress, and spread of disease during research efforts, in situ photography could provide an alternative for identifying individuals without capturing and handling frogs. I administered 25 pairs of photographs to 35 participants and asked them to determine if the frogs in the photograph were the same individual. I also asked them to gauge the degree of confidence they had in each response, as well as questions about themselves, including their experience observing wildlife in general and lowland leopard frogs in particular, and whether they enjoyed puzzles. Participants identified matches (86.9% correct) and mismatches (87.4% correct) with similar accuracy, and both reflected their estimated degree of confidence. Only participants with experience observing wildlife had higher scores. Overall, in situ photography offers promise as a method to identify individual leopard frogs and perhaps other species with distinctive individual patterns.

Purpose

Lowland leopard frogs (Lithobates yavapaiensis) are endemic to areas of Arizona, New

Mexico, Sonora, and Chihuahua. The species has been extirpated from California, is considered a sensitive species in other areas, and is listed as a species of concern in the state of Arizona

(Arizona Game and Fish Department 2006; Swann and Wallace 2010). Habitat loss, wildfires, water pollution, and introduction of nonnative species has contributed to the decline of this species, in addition to concerns about the spread of pathogenic chytrid fungus (Batrachochytrium dendrobatidis) (Swann and Wallace 2010; Savage et al. 2011; Forrest and Schlaepfer 2011;

Swann et al. 2016). Consequently, it is important to study the lowland leopard frog’s ecology. To

1 meet this goal, it is crucial that researchers are able to identity individuals to understand survival rates and movements.

Along with other frog species, handling lowland leopard frogs can lead to injury, stress, or spread of disease, adversely impacting the individuals and populations being studied (USGS

National Wildlife Health Center 2001). Due to conservation concerns about this species, it is important to explore alternatives for identifying individual frogs in ways that do not involve capture. In situ photography has been used successfully to identify individuals of other species

(e.g., Meekan et al. 2006, Anderson et al. 2010) and has potential for use with leopard frogs given their complex markings. Due to the nature of in situ photographs, however, pattern recognition software is unlikely to work well given common obstructions, poor focus, poor lighting, and difficult angles, which are common and expected when photographing animals in natural settings. As a result, the reliability of identifying individual frogs manually based on their spot patterns needs to be assessed to determine if this approach is viable for field studies. In addition, we need to understand which parameters affect the reliability of identification, including degree of obstruction, focus, lighting, and orientation of individuals in photographs.

Lastly, characteristics of participants or researchers might also affect their ability to identify individuals, including their degree of experience studying wildlife as well as their affinity for puzzles and problem solving.

Relevance

Due to conservation concerns about lowland leopard frogs and its sensitivity to environmental conditions, they have been classified as an indicator of the overall health of an environment (Swann and Wallace 2010). The planet is facing its next mass extinction and amphibians are especially vulnerable (Hayes et al. 2010). As a result, it is crucial to understand

2 the ecology of the lowland leopard frog to determine how to best conserve it and the areas it inhabits.

Currently, many field methods used to mark lowland leopard frogs and other species put individuals at increased risk of injury, including toe-clipping, tagging, and radio-telemetry.

These methods can also increase rates of mortality and increase stress to the animals.

Additionally, marks can also be lost. Others have captured amphibians to take clear and consistent photographs to show the spot patterns (Bradfield 2004, Morrison et al. 2016) and high quality photographs make comparing spot patterns significantly easier and even allow for the use of pattern recognition software (Morrison et al. 2016). However, this method has potential to increase mortality, injury, and stress for the individuals because of the need to capture and manipulate frogs. As a result, it is necessary to find better methods as this species continues to be studied.

In situ photography not only has potential value for identifying lowland leopard frogs, but could serve as model for studying other species of wildlife with unique patterning. In the desert southwest, another example would be the Gila monster (Heloderma suspectum), a species that is protected in Arizona and also of conservation concern. For Gila monsters, in situ photographs also reduce risks to researchers by eliminating the need for handling. This approach could easily be adapted for many other species with unique patterning.

Methods

To test the reliability of in situ photography for identifying individual leopard frogs, I helped to develop a test with 25 pairs of photographs incorporated into a PDF. The photographs used were taken by researchers in Madrona, Wildhorse, Chiminea, and Rincon Canyons in the

Rincon Mountains east of Tucson, Arizona, between May 2013 and October 2015. When

3 selecting photo pairs, I helped to establish a set of 15 “matches” (photographs were both of the same individual frog) and 10 “mismatches” (each frog in the pair was a different individual).

Photographs were rated on a scale of difficulty from 1 to 5 based on 4 categories: orientation of frogs in both photographs (same, 90 degrees, 180 degrees), focus (good, fair, poor), lighting

(good, fair, poor), and obstruction (none, small, big) (Figures 1 and 2). Photograph pairs were selected across the full range of difficulties. Furthermore, I noted the most difficult aspect of matching the photos, such as poor lighting, to ensure there was a variety of potential issues to assess whether certain aspects of difficulty proved more problematic. Photo pairs were then placed in 5 randomized orders, where each order was put into a separate test; this ensured that although each participant received the same photo pairs, learning or improving during the test could be assessed and bias from the order of pairs could be reduced (i.e., if participants tended to rush the end of the test, randomization would ensure that certain photo pairs would not have reduced rates of correct classification because of always being last).

Figure 1. A mismatched photo pair; same orientation; left photo (frog CH0b-3) focus is good, lighting is poor, and no obstruction; right photo (frog CH1g-2) focus is good, lighting is good, and small obstruction; overall difficulty was moderate (difficulty level 3).

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Figure 2. A matched photo pair; 90 degree orientation difference; left photo (frogCH1b-1) focus is good, lighting is good, and no obstruction; right photo (also frog CH1b-1) focus is good, lighting is good, and small obstruction; overall difficulty was slightly above moderate (difficulty level 4).

Participants were identified through word of mouth and an email server for the School of

Natural Resources and the Environment at the University of Arizona. Participants included elementary school teachers, professors, veterinarians, and students to ensure a wide spread of participants with different backgrounds, including experience with observing wildlife, observing lowland leopard frogs, and enjoyment of puzzles. Once a participant contacted the researcher, they were sent a PDF with the photograph pairs (assigned in order of when the participant contacted the researcher, ensuring all 5 test versions would be distributed in even frequency; names of participants and the version they received were recorded as the tests were sent out as to keep an accurate record for assessing responses), a response sheet, and a PDF with instructions

(Appendix 1 and 2). The test included all 25 photo pairs, in 1 of 5 orders, with 2 frogs on each page. The response sheet allowed participants to select “match” or “not a match” for each pair, their confidence for each response (low, medium, or high), a comment section, and 3 questions

5 about themselves (“Do you have experience working with wildlife? Yes or No”, “Have you seen a leopard frog in the wild? Yes many, Yes a few, or No”, and “Do you enjoy doing puzzles? Yes or No?”). The instructions gave a general overview of the study, instructions on filling out the response sheet, and suggestions on which areas of the frogs can aid identification.

As responses were returned, they were compiled into an Excel document. Each person’s response to each photo pair was scored as correct or incorrect. We summarized the percent of correct responses by characteristics of test subjects (e.g. wildlife experience) and photo pairs

(e.g. quality of lighting in each photo pair). We used generalized linear-mixed models to assess whether the probability of a correct response varied with subject- or photo-related factors. Specifically, we used a logit link and treated test subjects, nested within test version, as well as pairs of photographs, as random effects.

Literature Review

Currently, in situ photography has been used for field studies of other wild species to identify individuals. However, most of the subjects for these studies are species that have characteristics such as flat and regular tails or laterally compressed bodies, which makes the gathering of high quality photographs easier and facilitates use of pattern recognition software

(Carter et al. n.d.; Meekan et al. 2006; Anderson et al. 2010). Previous research has shown that it is possible to identify individuals based on unique pattern markings, but requires manipulation of the subject and high quality photographs. Once such example is a study of Archey’s frog

(Leiopelma Archeyi) in New Zealand (Bradfield 2004). Frogs were captured, manipulated into a standard position with a clear angle, and photographed next to a ruler as a reference. This ensured that all photographs were high quality and standardized, making individual spot patterns easy to use for identification. Other researchers have also used this method but with similar

6 manipulations, which also allows for the use of pattern recognition software due to the high quality and consistency of photographs (Goswami et al. 2011; Town, et al. 2013; Carter et al.

2014; Morrison et al. 2016). Multiple studies have shown that it is not only possible to use photography of individually distinct characteristics to identify individuals, but effective and commonly used with many species. However, the use of in situ photography has not been explored heavily.

Due to their status as a species of conservation concern and role as an indicator species, there has been a moderate amount of research on the ecology of lowland leopard frogs. Canyons that provided more water during dry summers and pools that had greater diversity of vegetation were more heavily populated, which holds great importance in determining areas and characteristics of the environment most vital for conservation of this species (Wallace et al.

2010). The increase in fires in the Tucson area has also been linked to high mortality of lowland leopard frogs, because erosion fills pools with sediment, further adding to the considerations for management and conservation of the species (Swann et al. 2016). To protect this species, it has been recommended that pools be shielded from the effects of wildfires through preventing post- fire erosion, restoring habitat, salvaging and reintroducing frogs, and managing wildfires (Swann et al. 2016). Generally, it has also been recommended that riparian areas that frogs inhabit continue to be preserved and neighbors of these areas continue to participate in the “backyard pool project” (development of ponds as a habitat on private properties) (Swann and Wallace

2010). Chytrid fungus (Batrachochytrium dendrobatidis) is an increasing threat to this species

(Savage et al. 2011), and has affected 200 species globally and has led to dramatic declines and even extinctions of amphibian species (Forrest and Schlaepfer 2011; Savage et al. 2011). Studies have revealed that populations with warmer water conditions and larger populations were most

7 likely able to withstand chytrid fungus infection, which also holds implications for management of this species (Forrest and Schlaepfer 2011; Savage et al. 2011). These studies have yielded a promising place to start as far as understanding the ecology of this species and beginning measures to ensure its conservation.

Results and Analysis

In total, 35 of 59 (59.3%) participants returned their responses, which included 15 who had wildlife experience and 20 who did not, 14 who had observed leopard frogs in the wild and

21 who had not, and 29 who enjoyed puzzles and 6 who did not (Table 2). Overall, the percentage of correct responses between matches and mismatches was similar, with 86.9% and

87.4% correct respectively (Table 1). All combinations of photograph characteristics between pairs (i.e., good focus in both, good in one and poor in the other, etc.) were analyzed for percentage of correct responses, but sample sizes were small for some combinations (Table 1).

Participant qualities were compared with correct responses, as well as various combinations of participant qualities. Overall, those with wildlife experience had higher scores, as did those who had observed leopard frogs in the wild and those who enjoyed puzzles, although these latter two characteristics were not statistically significant (Table 2). Furthermore, participants seemed to uniformly predict their own accuracy with their confidence rating; as confidence increased, so did number of correct responses (Figure 3).

We analyzed a variety of photograph and participant based factors to see the probability of their linkage to correct responses. The only characteristic of participants that was linked with higher scores was experience with wildlife (P = 0.012). Both familiarity with lowland leopard frogs in the wild and enjoyment of puzzles did not explain higher scores (P > 0.50). There was also no evidence that participants’ scores improved as they progressed through the tests (P =

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0.43). Participants were able to accurately gauge their abilities to classify pairs correctly, as they tended to have high confidence for correct responses and low confidence for incorrect responses.

Proportions of correct responses were comparable for pairs of photographs that were matches and mismatches (86.9% correct and 87.4% correct respectively; P = 0.61). The quality of photographs did not influence the proportion of correct responses strongly, as the proportion of correct responses did not vary significantly with the following factors: lighting (P = 0.14), focus

(P = 0.30), extent of obstructions (P = 0.10), and orientation of frogs in photographs (P = 0.57).

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Table 1. Characteristics of photographs and percentage and number of individuals classifying pairs correctly.

Characteristic Level Correct (%) n Frog identities Match 86.9 15 Mismatch 87.4 10 Focus Good/Good 88.0 16 Good/Fair 85.2 6 Good/Poor 85.7 3 Fair/Fair NA 0 Fair/Poor NA 0 Poor/Poor NA 0 Lighting Good/Good 90.3 10 Good/Fair 84.1 7 Good/Poor 88.0 5 Fair/Fair 81.4 2 Fair/Poor 82.9 1 Poor/Poor NA 0 Obstruction None/None 83.8 12 None/Small 93.5 7 None/Big 85.0 4 Small/Small NA 0 Small/Big 94.3 1 Big/Big 82.9 1 Orientation Same 86.3 16 90 degrees 89.8 7 180 degrees 84.3 2

Table 2. Characteristics of participants and percentage of correct responses.

Yes No Characteristic % Correct n % Correct n Wildlife experience 93.3 15 82.4 20 Observed leopard frogs in the wild 91.1 14 84.4 21 Enjoys puzzles 87.7 29 84.0 6

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Table 3. Wildlife and leopard frog experience of participants and percentage of correct responses.

Observed frogs in the wild Yes No Wildlife experience % Correct n % Correct n Yes 93.6 10 92.8 5 No 85.0 4 81.8 16

Table 4. Wildlife experience and enjoyment of puzzles of individuals classifying and percentage of correct responses.

Enjoys puzzles Yes No Wildlife experience % Correct n % Correct n Yes 93.2 13 94.0 2 No 83.3 16 79.0 4

Table 5. Experience seeing leopard frogs and enjoyment of puzzles of individuals classifying and percentage of correct responses.

Enjoys puzzles Yes No Observed frogs % Correct n % Correct n Yes 91.3 12 90.0 2 No 85.2 17 81.0 4

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Figure 3. Correct responses increased with confidence and participants with wildlife experience had higher scores. Error bars represent 95% confidence intervals.

Conclusions

We found that only wildlife experience was linked with higher scores, whereas previous experience with lowland leopard frogs and enjoyment of puzzles had little effect. I found these results surprising as initially I expected that all of the characteristics we considered might explain variation in the rate of correct responses, as experience with wildlife and with frogs likely helped participants look for certain features and puzzles might indicate that participants would spend more time on this challenging task. My results suggest that experience with lowland leopard frogs does not familiarize participants with what to look for (though this might explain why those with wildlife experience did better) and enjoyment of puzzles did not condition participants

12 to have a greater determination and methodology to identify matches more accurately. We also found that participants did not improve as the test progressed and they became more familiar with the task. Because this is only one short assignment, I did not find the lack of learning surprising; over time and through more photo pairs, however, I would be surprised if participants did not learn and become increasingly more accurate. Across the board, participants could gauge their own accuracy and their confidences related to more correct responses as confidence was high and fewer correct responses as confidence was low. I expect participants to be able to gauge their accuracy, or have a confidence that corresponds to correctness; the results from this study support these expectations.

The proportion of correct responses was similar for pairs of photographs that were matches and mismatches, meaning participants can just as effectively identify a match as they can a mismatch. Although I had no expectations about whether participants would be better at correctly identifying one or the other, these results are assuring in that researchers in another scenario could just as accurately differentiate between two frogs as they could match a single frog. Contrary to what I expected, the quality of photographs did not influence the proportion of correct responses strongly, as the proportion of correct responses did not vary with lighting, focus, extent of obstructions, or orientation of frogs in photographs. After spending many months analyzing photographs of leopard frogs, these issues often made it challenging for me to distinguish characteristics on a frog, so much so that other plans for this study were discarded because there was often a great discrepancy in how a single person would characterize the same frog from photograph to photograph. Initially, differences in orientation and lighting were regarded as extremely difficult obstacles when comparing frogs.

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Overall, with high accuracy of correct responses (86.9% and 87.4% for true matches and mismatches respectively) it seems that in situ photography and pattern identification for individuals could be a helpful alternative to current physical handling procedures. However, when using this method two things should be considered: confidence and experience of the researcher. Because participants could predict their own accuracy, this could be used as a cautionary measure for researchers; simply, if someone does not feel confident in their classification, it may be best to skip that photograph to avoid the risk of an incorrect classification. As for the experience of the researcher, this could indicate that those with wildlife experience may be more capable at making correct identifications; this could help a research team select who would be the most competent at looking at photographs to make correct identifications. These results also lead to the conclusion that photographs should not initially be discarded due to issues inherit to the method (i.e., terrible focus or lighting), as they tended to have a much smaller effect than expected.

Although this method has legitimacy, it may not be perfect. One issue is that this method takes time; a researcher would have to go through countless photographs before potentially finding a match, and as more frogs are documented, this method will become increasingly more time consuming. This issue could be reduced through recruiting more researchers, but this is not always an option. To reduce the time required by this process, a database was created to allow participants to enter values for a characteristic (i.e., “spots on the head-yes or no”, “number of spots on the eyelid”, etc.) and the database would have retrieved frogs with matching characteristics. This was eventually abandoned because even the same frog would receive different responses depending on the photograph. If further research was done on a more effective way to use this method from the perspective of time, this could greatly increase the

14 practicality of using in situ photography to identify individuals over time. Additionally, this method should be studied in other species; while it may work well in lowland leopard frogs, are the patterns on Gila monsters as easily discernible? This requires more in-depth study of the method. It is also worth noting that many of the samples sizes for the characteristics of photo pairs were small, at times only 0 or 1. To further assess the reliability of this method and explore the influence of these parameters, more photographs with these characteristics should be selected and tested to ensure that they do not limit reliability, as tentatively shown with this study. This method does have its merits, including reducing potential adverse effects on subjects, reducing time spent by the researcher capturing a subject, as well as promising accuracy, but it should still be explored more in depth before being adopted.

Acknowledgements

I thank Dr. Robert Steidl and Erin Zylstra of the School of Natural Resources and the

Environment of the University of Arizona for their incredible guidance through this process.

Additionally, this project would not have been possible without the photographs from Erin’s fieldwork, her assistance with excel coding, and her help analyzing the data.

I also thank Dr. James Jarchow and Dr. Walter Merker of Orange Grove Animal Hospital for answering general questions about amphibian morphology, behavior, and physiology.

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Appendices

Appendix 1. Instructions issued to each participant in the study.

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Appendix 2. Response sheet provided to each participant to record their answers.

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