Indiana University - Purdue University Fort Wayne Opus: Research & Creativity at IPFW Masters' Theses Graduate Student Research 5-2016 Vocalizations of Sea Turtle Hatchlings and Embryos Lindsay N. McKenna Indiana University - Purdue University Fort Wayne Follow this and additional works at: http://opus.ipfw.edu/masters_theses Part of the Animal Sciences Commons, Behavior and Ethology Commons, and the Marine Biology Commons Recommended Citation Lindsay N. McKenna (2016). Vocalizations of Sea Turtle Hatchlings and Embryos. http://opus.ipfw.edu/masters_theses/50 This Master's Research is brought to you for free and open access by the Graduate Student Research at Opus: Research & Creativity at IPFW. It has been accepted for inclusion in Masters' Theses by an authorized administrator of Opus: Research & Creativity at IPFW. For more information, please contact admin@lib.ipfw.edu.
Graduate School Form 30 Updated PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Lindsay N. McKenna Entitled Vocalizations of Sea Turtle Hatchlings and Embryos For the degree of Master of Science Is approved by the final examining committee: Frank V. Paladino Chair Robert B. Gillespie Winfried S. Peters To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University s Policy of Integrity in Research and the use of copyright material. Approved by Major Professor(s): Frank V. Paladino Approved by: Frank V. Paladino 4/27/2016 Head of the Departmental Graduate Program Date
i VOCALIZATIONS OF SEA TURTLE HATCHLINGS AND EMBRYOS A Thesis Submitted to the Faculty of Purdue University by Lindsay N. McKenna In Partial Fulfillment of the Requirements for the Degree of Master of Science May 2016 Purdue University Fort Wayne, Indiana
For my parents, you are truly the best. I love you. ii
iii ACKNOWLEDGEMENTS First off, I would like to thank my advisor, Dr. Frank Paladino, for all the opportunities you have given me. Not only for the opportunity of letting me be your graduate student, but for allowing me to travel to Costa Rica numerous times to work with sea turtles. Without that first field trip to Costa Rica with my high school marine biology class, I would never have realized the world of marine biology. I would like to thank my committee members, Dr. Peters and Dr. Gillespie for the edits and expert advice on my thesis and presentation! I need to thank the Parque Nacional Marino Las Baulas for allowing me to do my project there as well as The Leatherback Trust for funding. One extremely important person I can t thank enough is Dr. Nathan J. Robinson. Without you, none of this would have happened, so I thank you for everything: giving me this idea, helping in every step of the way, answering all my questions, editing multiple drafts, listening to good music in Costa Rica, and being so fun! Thank you to Dr. Pilar Santidrián Tomillo, Bibi, for all the help with my questions and all the work you did for my permits. Also, thank you for all the laughs and the pizza, pizza, pizza! Thank you, Tera Dornfeld, for giving me advice about grad school when I was an undergrad, and always being the positivity I needed when things were stressful. Thank
iv you for the long walks on the beach whether they were patrolling for turtles at night or power walking in the waves. I want to thank two individuals, Jacob Hill and Jennifer Swiggs for editing, answering my questions, and the fun talks, but also thanks to all the Paladino Lab especially Jacob Bryan, Chelsey Clyde Brockway, Lauren Cruz, Jamie Price, and all the biologists I worked with in Costa Rica! I had some of the best times with all of you and will never forget them! Last, but definitely not least- my family. Thank you to my parents for all the support and encouragement along the way. You both always have the best advice. Most importantly, thank you for listening to me blab on about sea turtles and Costa Rica since my first trip in 2009. Thank you, dad, for helping me create the McKenna boxes used in my project, and mom, I got-er-done! To my sisters, Amber and Emily, thank you for all your encouragement and for accepting all my sea turtle-themed gifts I ve given to you and your kids! And lastly, I want to thank one of the best role models I ve had in my life who I lost during this grad school journey, my grandpa, Paw Paw. I will continue to mow the long grass for you.
v TABLE OF CONTENTS Page LIST OF TABLES... vii LIST OF FIGURES... viii ABSTRACT...x INTRODUCTION...1 MATERIALS AND METHODS...5 Study Site...5 The Hatchery...6 Boxes...6 Nest Relocation...7 Olive Ridley Nest Recordings...10 Control Nest Recording...12 Olive Ridley Nest Analysis...12 Hatchlings in Buckets Recordings...15 Hatchlings in Buckets Analysis...16 Statistical Analysis...16 RESULTS...18 Olive Ridley Nest Vocalizations...18 Hatchlings in Buckets Vocalizations...24 DISCUSSION...28 Olive Ridley Nests...28 Hatchlings in Buckets...29 Conservation Implications...32 Future Study...32
vi Page CONCLUSIONS...33 LIST OF REFERENCES...34
vii LIST OF TABLES Table Page 1 Number of vocalizations, types of vocalizations, and averages of characteristics from each stage of each nest with standard error... 19 2 Number of vocalizations and averages of frequencies and duration in the incubation, hatching, and emerging stages from all three nests... 20 3 Number of vocalizations, types, and averages with standard error of the vocalizations characteristics of the three hatchlings species recorded in the bucket... 25 4 Range of averages of vocalization frequencies and durations found in olive ridley nests compared to averages found in olive ridley vocalizations in the buckets... 26 5 Comparison of leatherback hatchling vocalizations found in this experiment in the bucket to leatherback hatchling vocalizations found in nests in the experiment done by Ferrara et al., (2014)... 27
viii LIST OF FIGURES Figure Page 1 Map of study site in Costa Rica. Circle indicates location of Playa Grande and Playa Ventanas... 5 2 Dimensions of the boxes buried in sand... 7 3 Digging the egg chamber next to the plexiglass window... 8 4 Placing eggs next to the plexiglass window during the relocation process... 9 5 The nest after the relocation... 9 6 The setup of equipment while recording within the nest... 11 7 Photo of the camera placed in the box facing the plexiglass window and the cable connecting the recorder to the microphone, which is in the plastic tube. Cotton balls are plugging the plastic tube to prevent ambient noise from being recorded... 11 8 View of the eggs through the plexiglass window from inside the box... 12 9 The spectrograms of a (A) harmonic, (B) non-harmonic, and (C) pulse vocalization. Arrows point to the vocalization on the spectrogram... 14 10 Characteristics documented from a vocalization using Raven Pro software. The y-axis indicates frequency (khz) and the x-axis indicates time of recording (min:sec)... 15 11 Recording East Pacific green turtle hatchlings in a bucket... 16 12 Percent of types of vocalizations during incubation, hatching, and emerging stages of all nests... 21
ix Figure Page 13 Average highest frequency of vocalizations in each stage from all nests with standard error... 22 14 Average dominant frequency of vocalizations in each stage from all nests with standard error... 22 15 Average lowest frequency of vocalizations in each stage from all nests with standard error... 23 16 Average duration of vocalizations in each stage from all nests with standard error... 23 17 Average frequency range of vocalizations in each stage from all nests with standard error... 24
x ABSTRACT McKenna, Lindsay N. M.S., Purdue University, May 2016. Vocalizations of Sea Turtle Hatchlings and Embryos. Major Professor: Frank V. Paladino. Many animals vocalize to communicate. While this vocal communication has been studied extensively in mammals and birds, far less attention has been paid to reptile vocalizations. Sea turtles vocalize in the nest, but the purpose of these vocalizations is unknown. I aimed to characterize the vocalizations of the olive ridley turtles (Lepidochelys olivacea) in the nest during incubation, hatching, and emerging. I also aimed to characterize and compare the vocalizations of olive ridley, leatherback (Dermochelys coriacea), and East Pacific green (Chelonia mydas agassizii) turtle hatchlings. I relocated three olive ridley nests at Parque Nacional Marino Las Baulas, Costa Rica, and buried them next to a plexiglass window, which was used to obtain video recordings using a Canon digital camera. A hollow tube leading from above the eggs to the sand s surface was used to insert an Earthworks M30 microphone that was attached to a Marantz PMD61 MKII recorder to record audio. Each recording was categorized as incubation, hatching, or emerging. 60 minutes of each stage was analyzed using Raven Pro sound analysis software. The type (harmonic, non-harmonic, pulse) and
xi characteristics (highest frequency, dominant frequency, lowest frequency, duration, and frequency range) were documented. In addition, I recorded olive ridley, leatherback, and East Pacific green hatchlings in buckets. The same characteristics of vocalizations were documented from the bucket recordings as the nest recordings. For statistical purposes, the Chi-Squared Test, Friedman Test, and Kruskal-Wallis tests were used. I recorded a total of 157 vocalizations from the nests. The frequency of the vocalizations ranged from 0.05-11.4 kilohertz and the duration ranged from 0.01-0.54 seconds. I found significantly more vocalizations in the incubation stage than the other two stages (p < 0.05). The pulse vocalizations were only found in the incubation stage. Also, there was no significant difference seen in types or characteristics of vocalizations among the stages (p > 0.05, all cases). The bucket recordings revealed that the frequencies were not significantly different, but the duration of the leatherback hatchling vocalizations were significantly longer than the other two species (p < 0.05). The other characteristics of the vocalizations between species were not different. The olive ridleys hatchlings in the bucket vocalized more often than the hatchlings in the nest (p < 0.05). The frequencies in the bucket were within the same range as the frequencies in the nest, which was also seen when comparing the leatherbacks in the bucket to Ferrara et al. (2014). The results suggest that the number of vocalizations or the pulse vocalizations could possibly be used in synchronizing hatching. The significance of the frequencies of the vocalizations in the nest is unable to be determined from this study. The similar
xii frequencies of vocalizations among the three species suggest that all sea turtle species vocalize within the same frequency range both in the nest and in other situations such as a bucket. Elucidating the importance of vocalizations in hatchlings can help develop more conservation plans to make sure development and other noise pollution isn t interfering with any vocal communication.
1 INTRODUCTION Vocalizations are used by many animals to convey information. Although much research has focused on characterizing the vocalizations made by mammals and birds (Clay et al., 2012; Wilden et al., 1998; Deecke et al., 2005), considerably less attention has been paid to reptile vocalizations. Only recently has it been discovered that freshwater turtles communicate by vocalizing. For example, Gopherus polyphemus, vocalize to attract potential mates or when another turtle invades their territory (Legler and Vogt, 2013). Similarly, sea turtle hatchlings have just been discovered to vocalize regularly, (Ferrara et al., 2014). It is hypothesized that sea turtle embryos, as well as hatchlings, could vocalize to synchronize hatching and climbing out of the nest to the sand s surface (Ferrara et al., 2013; Vergne and Mathevon, 2008). Characterizing the vocalizations made by sea turtle hatchlings and investigating whether they change during the developmental stages of incubation, hatching and emerging from the nest could provide important cues as to whether this is the case. The peak nesting season for olive ridley sea turtles in Parque Nacional Marino Las Baulas is from August to November with the peak in October (Dornfeld et al., 2015). Female sea turtles spend the majority of their lives in the ocean, but come to tropical and subtropical beaches to nest. They typically lay their eggs above the high tide line. The
2 average number of eggs in a clutch is 100.1 (da Silva et al., 2007). The eggs have an average incubation period of 49.1 + 3.6 days (Dornfeld et al., 2015). Sea turtle hatchlings often act in a coordinated fashion. When the first egg hatches, the hatchling does not immediately start climbing. Instead, it waits for others to hatch to work together and climb upward (Carr and Hirth, 1961). The hatchlings scrape sand from the ceiling of the egg chamber causing it to collapse, which raises the floor of the nest. The hatchlings gradually elevate themselves up through the sand column as a group (Carr and Hirth, 1961). There are multiple advantages for sea turtles synchronizing hatching and emerging from the nest 1) The large number of emerging hatchlings can overwhelm any potential predators and increase an individual s chance of surviving by dilution (Delm, 1990). 2) Predators might be able to smell the hatched eggs in the nest, so hatching as a group can help ensure that the majority of the hatchlings are out of the nest before the predators become attracted to it (Doody, 2011). 3) Group emergence reduces emergence time and the individual energy requirements to emerge and dig out from the nest chamber (Carr and Hirth, 1961). Yet even though synchronized hatching is advantageous, eggs may not inherently hatch at the same time. Eggs experiencing cooler temperatures, sometimes on the outer perimeter or lower parts of the nest, may result in longer incubation periods in the nest (Colbert et al., 2010; Spencer et al., 2001, Ewert, 1979). For the embryos to hatch at the same time, they must be able to adjust their rate of development in some way since they only hatch when they are fully developed. A possible explanation for this hatching behavior is the slower developing embryos are able
3 to increase their heart and metabolic rates towards the end of development by detecting heart rates of their more developed siblings in order to catch up (McGlashan et al., 2012; Spencer et al., 2001; Spencer, 2012). Another explanation for synchronized hatching is vibrations. One study suggested that feeling vibrations from sound or movement coming from more developed embryos could stimulate the less developed embryos to speed up development (Doody et al., 2012). With the recent discovery of vocalizations in sea turtle hatchlings, it has also been suggested that these vocalization could also help to synchronize hatching behavior. In this study, I aimed to characterize vocalizations of three species of sea turtle hatchlings in Costa Rica and determine if the vocalizations serve as a cue for synchronized hatching and emergence in olive ridley turtle nests. Specifically, I have two objectives. (1) To investigate whether number of vocalizations and vocalization patterns of olive ridley sea turtles change over the incubation, hatching, and emerging periods. (2) To characterize the vocalizations of olive ridley (Lepidochelys olivacea), East Pacific green (Chelonia mydas agassizii), and leatherback (Dermochelys coriacea) sea turtle hatchlings at Playa Grande, Costa Rica. With these objectives, I formulated 3 hypotheses. (1) Vocalizations will differ among the stages of development in the nest. (2) Different sea turtle species will have similar vocalization frequencies and durations since sea turtles are not known to possess a detailed vocal repertoire (Mrosovsky, 1972). (3) The number and characteristics of vocalizations of hatchlings will differ in natural situations, like the nest, and situations where they feel threatened or in fear of being predated. If evidence is found that hatchlings do vocalize as a form of communication to synchronize hatching and emergence, then a better understanding of their behavior and
4 better conservation efforts could be taken to rid or prevent any sounds from developed beaches that could conflict with the embryonic and hatchling communication.
5 MATERIALS AND METHODS Study Site The study was conducted at Parque Nacional Marino Las Baulas in the Guanacaste province of Costa Rica between October 2015 and December 2015. The national park encompasses three beaches: Playa Grande, Playa Ventanas, and Playa Langosta, which are 3.6, 1.1 and 1.3 km long respectively (10 20 N, 85 51 W). These beaches host nesting populations of olive ridley, East Pacific green, and leatherback turtles. The nesting season for olive ridley sea turtles at this location is between August and November with the peak being in October (Dornfeld et al., 2015). Figure 1. Map of study site in Costa Rica. Circle indicates location of Playa Grande and Playa Ventanas.
6 The Hatchery The hatchery is located in an area of cleared vegetation, 3m behind the beach. As the hatchery is above the high-tide line, the sand is manually dug up and rotated in the hatchery to a depth of 1m at the beginning of each season. This serves to aerate the sand. The hatchery is divided into 32 plots of size 1m 2. Relocated nests were placed into the center of these plots. The hatchery is enclosed by a fence around the perimeter to stop animals, such as raccoons or coatis, from predating the nests. Boxes Wooden boxes were constructed following the dimensions shown in Figure 1. Each box had a plexiglass window allowing for observation of the developing clutch. At the start of the nesting season, the boxes were buried to a depth of 45cm to mimic the average depth of olive ridley nests (see Dornfeld et al. 2015). When a clutch required relocation, the clutch was buried next to the clear plexiglass window allowing the eggs to be observed during development. The top of the wooden box was exposed above the sand and covered to prevent rain or animals from getting inside.
7 Figure 2. Dimensions of the boxes buried in sand. Nest Relocation Nightly patrols and morning surveys were conducted on Playa Grande and Playa Ventanas as described in Reina et al. (2002). If a female olive ridley sea turtle was found nesting at a location where the eggs were unlikely to survive, e.g. below the high tide line, the clutch was relocated to the hatchery. I used a sterile plastic bag to collect the eggs during oviposition. The eggs were taken to the hatchery where a new nest was created. The new nests were 45cm deep and were located in the center of the plot. The eggs were counted and placed next to the window of the box. The plexiglass window allowed for the eggs to be monitored visually.
8 A thermocouple was placed in the center of the clutch to record temperature every other day until the hatchlings emerged from the nest. About 3cm above the eggs, I placed a hollow plastic tube. The other end of the plastic tube was kept above the surface of the sand and was covered with plastic so nothing could pass through the tube. The covered nest was then marked with a nest number and surrounded by a metal wire cage to prevent predators from getting to the nest. The process is shown in Figures 3-5. Figure 3. Digging the egg chamber next to the plexiglass window.
9 Figure 4. Placing eggs next to the plexiglass window during the relocation process. Figure 5. The nest after the relocation.
10 Olive Ridley Nest Recordings After 45 days of incubation, I began taking video and audio recordings of the nests. I chose 45 days because the average incubation period of relocated olive ridley turtle nests is 51.0 days (da Silva et al., 2007), so I assumed that at 45 days, the eggs would still be incubating but developed enough to vocalize (Ferarra et al., 2013) and able hear the vocalizations (Miller, 1985; Crastz, 1982). A Canon A2300 16 megapixel camera was placed inside the box facing the plexiglass window in view of the eggs on the other side to record video. To record audio, I used an Earthworks M30 microphone attached to a Marantz PMD661 MKII recorder by a Mogami Gold Studio cable. The microphone was placed down the hollow tube that led to the top of the egg chamber. The top of the hollow tube was plugged with cotton balls at the time of recording to keep out as much ambient noise as possible. A Goal Zero Yeti 150 Solar Generator was used at times to keep the camera charged while recording. Each recording was at least 30 minutes in length. The recordings were taken at least four times a day between the hours of 2:00 to 5:00, 8:00 to 11:00, 14:00 to 17:00, and 20:00 to 23:00. Once recordings began, the nests were monitored every thirty minutes during daylight hours and under complete surveillance from 18:00 until 5:00. Two days after the hatchlings emerged from the nest, I stopped recording audio and video. The hatchlings were collected and released immediately onto the beach. Biologists protected the hatchlings as they crawled to the ocean to prevent crabs or other predators from getting to them before they reached the water. If the hatchlings emerged after 4:00am, they were placed in a cool dark location at the research station and released the following night when predation and dehydration pressures are reduced. The setup for recording is shown in Figures 6-8.
11 Figure 6. The setup of equipment while recording within the nest. Figure 7. Photo of the camera placed in the box facing the plexiglass window and the cable connecting the recorder to the microphone, which is in the plastic tube. Cotton balls are plugging the plastic tube to prevent ambient noise from being recorded.
12 Figure 8. View of the eggs through the plexiglass window from inside the box. Control Nest Recording A control nest was created in the same hatchery that housed the olive ridley nests. The only difference between the control nest and the olive ridley nests was that no eggs or hatchlings were present in the control nest. However, the control nest was dug to the same depth, and the hollow tube was placed in the same location as an olive ridley nest. I recorded audio in the control nest for thirty minutes. The control nest was necessary to assure that the sounds identified as vocalizations in the olive ridley nests were not also heard in the control nest. Olive Ridley Nest Analysis In order to analyze the olive ridley nests, I began by viewing the video recordings. The videos helped determine the stage of development of the eggs in the nests at the time of each recording. The development was categorized into three stages: incubation (eggs
13 only), hatching (eggs cracked), and emerging (hatchlings visible). For statistical purposes, a 60 min subset of each stage was analyzed since the recording times varied between stages and nests. I chose 60 min because that was the maximum length of time of the shortest recording. The incubation subset was taken from the last sixty minutes of recording in the incubation stage in order to get any vocalizations right before the hatching behavior. The hatching and emerging subsets were taken from the first 60 minutes of recording in each nest to record any vocalizations at the start of that behavior. This was done the same way for all three nests. The audio recordings of the subsets were then analyzed using Raven Pro software from the Cornell Lab of Ornithology. When a vocalization was encountered while observing the spectrogram of the full recording, I documented the type of vocalization it was and its characteristics. I also documented the count of vocalizations discovered in the subsets of each stage in that nest. The types of vocalizations were categorized by spectral output solely based on its appearance on the spectrogram. The three types were harmonic, non-harmonic, and pulse (a distinct type of non-harmonic). Figure 9 shows an example of the spectrogram for each of the categories. A harmonic vocalization has multiple frequencies occurring at the same time. A non-harmonic vocalization is just one frequency occurring at one time. The pulse vocalizations were a type of non-harmonic vocalization, but I separated them into their own category because the pulse vocalizations were relatively lower frequency and longer in duration than the other non-harmonic vocalizations.
14 A B C Figure 9. The spectrograms of a (A) harmonic, (B) non-harmonic, and (C) pulse vocalization. Arrows point to the vocalization on the spectrogram. The characteristics of the vocalizations documented were highest frequency, lowest frequency, dominant frequency (the loudest part of the vocalization indicated by the darkest part on the spectrogram in Raven Pro), duration, and frequency range (difference between highest frequency and lowest frequency) were documented similar to the techniques in Giles et al. 2009. Figure 10 shows the characteristics of one of the vocalizations.
15 Figure 10. Characteristics documented from a vocalization using Raven Pro software. The y-axis indicates frequency (khz) and the x-axis indicates time of recording (min:sec) Hatchlings in Buckets Recordings When olive ridley, leatherback, or East Pacific green turtle hatchlings were encountered during the daytime either due to a nest hatching or during an excavation, they were placed in a cool dark location at the research station. Whenever this occurred, I recorded the turtles by placing the microphone into the bucket holding the turtles (Figure 11). A blanket was placed over the bucket and microphone to eliminate as much ambient noise as possible. Only one species of at least four individuals were in the bucket at the time of recording.
16 Figure 11. Recording East Pacific green turtle hatchlings in a bucket. Hatchlings in Buckets Analysis The recordings of the olive ridley, leatherback, and East Pacific green turtle hatchlings were analyzed using the Raven Pro software. The spectral output of each vocalization was observed and the highest frequency, dominant frequency, lowest frequency, duration, and frequency range of each vocalization was documented. Statistical Analysis IBM SPSS Statistics 23 software was used for all statistical purposes. First off, to determine if there were any differences in nest temperature, a Kuskal-Wallis test was used since the data was not normally distributed. To compare the number of vocalizations in each stage of each nest, I used a Chi Squared Test. The nest data was not normally distributed, so I used a Friedman Test, which is a non-parametric test that takes into consideration repeated measures since the data was being collected from the same nests.
17 The Friedman Test was used to compare the proportion of harmonic vocalizations and the proportion of non-harmonic vocalizations among the three stages as well as comparing the highest frequency, dominant frequency, lowest frequency, duration, and frequency range among the three stages. The data from the three species in the bucket was not normally distributed, so I used a Kruskal-Wallis test to compare the highest frequency, dominant frequency, lowest frequency, duration, and frequency range among the three species. I also used a Kruskal- Wallis test to compare the number of vocalizations per min (rate) in the olive ridley nests to the olive ridleys in the bucket. Other comparisons, which did not use a statistical test, were the highest frequency, dominant frequency, lowest frequency, duration, and frequency range of the vocalizations of the olive ridley hatchlings in the nest to the olive ridley hatchlings in the bucket. Also, I compared the vocalizations of the leatherback hatchlings in the bucket to another study done on vocalizations of leatherback hatchlings in nests in Mexico done by Ferrara et al. (2014).
18 RESULTS Olive Ridley Nest Vocalizations The three nests studied were labeled NEST 1, NEST 2, and NEST 3. All three nests were relatively successful and at least 65 (75%) turtles hatched in each nest. The Kruskal-Wallis test indicated that there was no temperature difference among the three nests (p > 0.05) or when comparing the experimental nests to non-experimental nests (p > 0.05). The subsets of each stage of each nest resulted in a total of 540 minutes, and within this time, 157 vocalizations were recorded. All vocalizations discovered had frequencies that were in the hearing range (roughly between 60-1,000 Hz) of sea turtles (Ridgway et al., 1969; Samuel et al., 2005; Mrosovsky, 1972; Cook and Forrest, 2005).The results are shown in Tables 1 and 2. No sounds like the vocalizations discovered in the nests were discovered in the 30-min recording of the control nest.
19 Table 1. Number of vocalizations, types of vocalizations, and averages of characteristics from each stage of each nest with standard error. INCUBATION NEST 1 NEST 2 NEST 3 (60 Minutes) (60 Minutes) (60 Minutes) # Vocalizations 66 18 15 # Harmonic 39 13 6 # Non-Harmonic 5 3 9 # Pulse 22 2 0 Highest Frequency 3.24 + 0.34 2.70 + 0.24 1.41 + 0.16 Dominant Frequency 1.16 + 0.11 1.40 + 0.18 0.74 + 0.11 Lowest Frequency 0.72 + 0.07 1.04 + 0.20 0.53 + 0.04 Duration 0.13 + 0.01 0.14 + 0.02 0.08 +0.02 Frequency Range 2.52 + 0.30 1.66 + 0.23 0.88 + 0.15 HATCHING # Vocalizations 18 14 4 # Harmonic 12 7 2 # Non-Harmonic 6 7 2 # Pulse 0 0 0 Highest Frequency 2.75 + 0.41 1.65 + 0.20 1.26 + 0.12 Dominant Frequency 1.57 + 0.30 0.99 + 0.16 0.77 + 0.03 Lowest Frequency 0.96 + 0.16 0.67 + 0.10 0.63 + 0.06 Duration 0.12 + 0.02 0.11 + 0.02 0.23 + 0.06 Frequency Range 1.79 + 0.30 0.98 + 0.17 0.63 + 0.13 EMERGING # Vocalizations 15 5 2 # Harmonic 7 5 1 # Non-Harmonic 8 0 1 # Pulse 0 0 0 Highest Frequency 3.54 + 0.28 2.53 + 0.22 3.29 + 2.67 Dominant Frequency 1.67 + 0.12 1.14 + 0.15 2.15 + 1.75 Lowest Frequency 1.32 + 0.15 0.44 + 0.04 0.98 + 0.71 Duration 0.06 + 0.01 0.07 + 0.02 0.19 + 0.004 Frequency Range 2.22 + 0.23 2.10 + 0.18 2.31 + 1.96
20 Table 2. Number of vocalizations and averages of frequencies and duration in the incubation, hatching, and emerging stages from all three nests. INCUBATION HATCHING EMERGING # Vocalizations 99 36 22 # Harmonic 58 21 13 # Non-Harmonic 17 15 9 # Pulse 24 0 0 Avg Highest Frequency 2.45 1.89 2.71 Avg Dominant Frequency 1.1 1.11 2.02 Avg Lowest Frequency 0.76 0.75 1.24 Avg Duration 0.12 0.16 0.12 Avg Frequency Range 1.69 1.13 2.15 The results of the Chi Squared Test showed that there were significantly more vocalizations in the incubation stage than the other stages (p > 0.05). Harmonic, nonharmonic, and pulse vocalizations were found in the recordings, most of the overall vocalizations were harmonic (59%). The pulse vocalizations were only found in the incubation stage. The Friedman Test showed that there was no significant difference in proportion of harmonic or non-harmonic vocalizations in any of the stages (p > 0.05, both cases). The graphs comparing the types of vocalizations in each stage of each nest are shown in Figure 12.
Percentage 21 Percentages of Types of Vocalizations in Each Stage 100 90 80 70 60 50 40 30 20 10 0 n = 24 (24%) n = 17 (17%) n = 58 (59%) n = 15 (42%) n = 21 (58%) n = 9 (41%) n = 13 (59%) Incubation Hatching Emerging Stages Pulse Non-Harmonic Harmonic Figure 12. Percent of types of vocalizations during incubation, hatching, and emerging stages of all nests. The Friedman Test showed no significant difference between the averages of the highest frequency, dominant frequency, lowest frequency, duration, and frequency range of each stage ( p > 0.05). The graphs comparing the different averages of the stages in each nest are shown below in Figures 13-17.
Frequency (khz) Frequency (khz) 22 4 Average Highest Frequency of Vocalizations in Each Stage 3 2 1 0 Incubation Hatching Emerging Stages Figure 13. Average highest frequency of vocalizations in each stage from all nests with standard error. 2.5 2 1.5 1 0.5 Average Dominant Frequency of Vocalizations in Each Stage 0 Incubation Hatching Emerging Stages Figure 14. Average dominant frequency of vocalizations in each stage from all nests with standard error.
Time (s) Frequency (khz) 23 Average Lowest Frequency of Vocalizations in Each Stage 1.5 1 0.5 0 Incubation Hatching Emerging Stages Figure 15. Average lowest frequency of vocalizations in each stage from all nests with standard error. Average Duration of Vocalizations in Each Stage 0.25 0.2 0.15 0.1 0.05 0 Incubation Hatching Emerging Stages Figure 16. Average duration of vocalizations in each stage from all nests with standard error.
Frequency (khz) 24 Average Frequency Range of Vocalizations in Each Stage 2.5 2 1.5 1 0.5 0 Incubation Hatching Emerging Stages Figure 17. Average frequency range of vocalizations in each stage from all nests with standard error. Hatchlings in Buckets Vocalizations There were no pulse vocalizations detected in the bucket recordings. The number of vocalizations, types, and averages of the characteristics of the vocalizations of the East Pacific green, leatherback, and olive ridley turtle hatchlings in the bucket are shown in Table 3.
25 Table 3. Number of vocalizations, types, and averages with standard error of the vocalizations characteristics of the three hatchlings species recorded in the bucket. East Pacific Green Leatherback Olive Ridley # Vocalizations 12 132 20 # Harmonic 7 78 10 # Non-Harmonic 5 54 10 # Pulse 0 0 0 Average Highest Frequency (khz) Average Dominant Frequency (khz) Average Lowest Frequency (khz) 5.52 + 2.91 3.74 + 3.07 3.48 + 2.32 2.58 + 1.04 1.9 + 1.07 1.83 + 0.63 1.58 + 0.72 1.33 + 1.06 1.27 + 0.53 Average Duration (s) 0.08 + 0.06 0.14 + 0.13 0.09 + 0.16 Average Frequency Range (khz) 3.94 + 2.83 2.41 + 3.02 2.21 + 2.23 The results of the Kruskal-Wallis test determined that the duration of the leatherback vocalizations were significantly different than the other two species (p < 0.05). None of the other characteristics among the three species were significantly different (p > 0.05, all cases). The averages of the highest frequency, dominant frequency, lowest frequency, duration, and frequency range from the olive ridley vocalizations in the bucket compared to the ranges of averages of the olive ridleys in the nests are shown in Table 4.
26 Table 4. Range of averages of vocalization frequencies and durations found in olive ridley nests compared to averages found in olive ridley vocalizations in the buckets. Range of Averages Found in All Stages and Nests Averages Found in Buckets Lowest-Highest Highest Frequency (khz) 1.26-3.69 3.48 Dominant Frequency (khz) 0.74-2.76 1.83 Lowest Frequency (khz) 0.44-2.30 1.27 Duration (s) 0.06-0.24 0.09 Frequency Range (khz) 0.63-2.52 2.21 The averages of highest frequency, dominant frequency, lowest frequency, duration, and frequency range of the olive ridley vocalizations in the bucket were in the range of the vocalizations in the nests. The Kruskal-Wallis test showed that the number of vocalizations per min (rate) from the olive ridley hatchlings in the bucket was significantly higher than the olive ridley hatchlings in the nest (p < 0.05). An observation was made between this study and the Ferrara et al. (2014) study. The range of frequencies of the leatherback hatchlings in the bucket from this study was found in the range of frequencies from the Ferrara et al. (2014) study of leatherback hatchlings in the nests in Mexico. Also, harmonic and non-harmonic vocalizations were found in both studies, and the duration of the leatherback hatchling vocalizations were similar (Table 5).
27 Table 5. Comparison of leatherback hatchling vocalizations found in this experiment in the buckets to leatherback hatchling vocalizations found in nests in the experiment done by Ferrara et al., (2014). Lowest Frequency- Highest Frequency (khz) This Experiment Ferrara, 2014 0.59-21.62 0.12-24.00 Harmonics Present Yes Yes Non-Harmonics Present Yes Yes Average Duration (s) 0.14 0.10
28 DISCUSSION In my study I aimed to characterize the vocalization patterns of sea turtle hatchlings in order to uncover insights into their function. First, I characterized the vocalization patterns of olive ridley turtles during the stages of incubation, hatching, and emerging from the nest. Secondly, I compared the vocalizations of olive ridley turtles in buckets to the vocal repertoire of East Pacific green turtles and leatherback turtles in a bucket. Thirdly, I compared the vocalizations of the olive ridleys in the nest to the olive ridleys in the bucket. Lastly, I compared the leatherback vocalizations discovered in this study to those found in Ferrara et al. 2014. I provide the first-ever report of vocalization in olive ridley and East Pacific green turtle hatchlings, and further confirm the occurrence of vocalizing in leatherback turtle hatchlings. Considering that all sea turtle species investigated so far vocalize as hatchlings, I consider this a potentially universal trait of Cheloniids. Olive Ridley Nests Sea turtles may be communicating in the nest using vocalizations as well as the catch up method (McGlashan et al., 2012; Spencer et al., 2001; Spencer, 2012) and vibrations (Doody et al., 2012). Multiple cues to stimulate hatching behavior aren t uncommon and have been found in other species (Gomez-Mestre et al., 2008; Warkentin
29 et al., 2001). We know sea turtle hatchlings vocalize in the nest (Ferrara et al., 2014; Vogt, 2014). This study found the same result, but with the use of the camera I was able to observe the stage of development of the hatchlings while the vocalizations were heard. Vocalizations were discovered in all stages, but the highest number of vocalizations occurred in the incubation stage, which was while the hatchlings were still in the egg but right before hatching. This result supports my hypothesis that the vocalizations differ among the three stages of development. Perhaps the hatchlings are vocalizing more in the incubation stage when they are still in the egg and can t be heard as well by predators. Harmonic vocalizations are thought to be used when animals are in nonthreatening situations (Seyfarth and Cheney, 2003). Although there were more harmonic vocalizations overall than non-harmonic vocalizations, There was not a statistically significant different between the percentage of non-harmonic and harmonic vocalizations among the three stages. Therefore, it is not possible to determine the biological significance of the use of harmonic and non-harmonic vocalizations. Finding the pulse vocalizations only in the incubation stage and never in the bucket experiments suggest that these could possibly have something to do with the vocalizations responsible for coordinating hatching. If this was the case, then this finding would be the first vocalization that has been discovered to aid in the hatching process. Hatchlings in Buckets Mrosovsky (1972) suggests that turtles do not have a very detailed vocal repertoire. My results showed that the three species vocalize within the same frequencies, which supports my second hypothesis. This discovery suggests that not only
30 these three sea turtle species, but all species, vocalize within the same frequencies. This would make sense because the hearing range of sea turtles is similar among species (Ridgway et al., 1969; Samuel et al., 2005; Mrosovsky, 1972; Cook and Forrest, 2005), so their vocalizations would have to be within those frequencies if they were used for communication. The significantly longer duration of leatherback hatchlings vocalizations could have to do with their body size. The leatherback hatchlings are larger in size than the other two species. Price et al., (2007), and Ey et al. (2006) suggest that larger lung capacity can provide longer vocalizations because the air being exhaled can be more easily controlled. These comparisons of the three species allow us to better characterize the range of the vocal ability of sea turtle hatchlings. The recordings of the olive ridley hatchlings in the bucket compared to the olive ridley hatchlings in the nest show that the hatchlings increase the rate of vocalizing in the bucket. When the hatchlings were in the bucket, they were handled and moved around, which I assumed would be when they would portray any behavior associated with feeling threatened. However, when the hatchlings were in the nest, they were protected from any predators so they would not feel threatened. Like stated before, harmonic vocalizations are thought to be used more in situations when there is no threat, whereas non-harmonic vocalizations are thought to be used when the animal is in danger, and the individual is in more of a panicked state (Seyfarth, and Cheney, 2003). With this information, I assumed that the vocalizations in the nest would be mostly harmonic, and the vocalizations in the bucket would be mostly non-harmonic. However, my results did not support this hypothesis. So, the importance of using harmonic and non-harmonic
31 vocalizations, if any, is not able to be determined in this study. However, discovering the use of both harmonic and non-harmonic vocalizations in sea turtle hatchlings give us more evidence that turtle species are using these different types of vocalizations (Giles et al., 2009; Ferrara et al., 2013; Ferrara et al., 2014). Perhaps it is not the type of vocalizations used, but rather the rate of vocalizing that signals different meanings. The hatchlings are vocalizing more when they are in the bucket than in the nest, which shows support for my third hypothesis. Possibly, the hatchlings are feeling more in danger in the buckets than the nest and are vocalizing more because they feel panicked. This has been seen in other animals including some bird species and mammals change their calls depending on the size closeness of the predator (Leavesley and Magrath, 2005; Templeton et al., 2005) and speed up the rate when they feel threatened (Seyfarth and Cheney, 2003). Similar to the olive ridley comparison of vocalizing in the nest and in the bucket, the comparison of the leatherbacks in the bucket in this study to the leatherbacks in the nest done by Ferrara et al. (2014), suggest that the characteristics of vocalizations do not change between environments. While I was not able to obtain the rate of leatherback vocalization in the nest from the Ferrara et al. (2014) study, I calculated the rate of vocalizing in the bucket, and discovered that the rate of the leatherback hatchlings in the bucket was even higher than the olive ridley turtles in the bucket. So, possibly there is more vocalizing in leatherback hatchlings in general. Also, perhaps the same behavior as in the olive ridley hatchlings will be seen, and the leatherback hatchlings will vocalize more when they feel threatened as well.
32 Conservation Implications This study and Ferrara et al. (2014) confirm that three species of sea turtles vocalize, but suggest that all sea turtle species vocalize. An increase in ambient noise on developing beaches could hinder the communication between hatchlings (Dumyahn and Pijanowski, 2011). If the noises from the developing beaches are in the frequency range that sea turtle hatchlings are vocalizing in, this could possibly interfere with any communication. If this is the case, then the noises from construction, which usually takes place during the day, could stimulate hatching at inopportune times for sea turtle hatchlings. Conservation efforts have been made on Playa Grande (this study) (Tomillo, et al., 2007) and other beaches (Sarti-Martínez et al., 2007) in relation to other aspects of the lives of sea turtles and physicality of their nesting beaches, i.e. light pollution. This study gives us the insight into the range of the vocalizations of three sea turtle species and suggests that possibly all species vocalize within this same range. With this knowledge, better plans can be generated to take noise from developing beaches that may be within this range into consideration just like studies have done in the water (Samuel et al., 2005). Future Study With the trends seen in this study, we can assume that vocalizations are a part of the hatching process. Further studies on the hatching process and vocalizations could help us further confirm the use of vocalizations during the hatching process. Also, studying more vocalizations could help us come up with a better way to characterize vocalizations that could be used universally.
33 CONCLUSIONS This study gives insight into the function of the vocalizations heard in the nests. While there is still more to discover, I found that three species of sea turtles are vocalizing as hatchlings. By studying the behavior of olive ridley hatchlings in the nest, I discovered that the turtles inside the egg (embryos) and hatchlings are vocalizing while still in the nest and during their emergence to the surface of the sand. Most vocalizing is occurring while the turtle is still in the egg and right before hatching begins. The pulse vocalizations could be used in stimulating hatching and the relative lower frequency of those vocalizations could be because lower frequencies travel through solids better. The types and characteristics of vocalizations in terms of harmonic, non-harmonic, pulse, frequency, and duration are unable to be associated with a specific behavior in this study. Sea turtle hatchlings vocalize in similar vocal frequency and durations in the nest and in environments where they feel threatened. With all this new knowledge of sea turtle vocalization, we can predict better conservation plans in terms of avoiding interference of sounds in the frequency ranges of sea turtle hatchling vocalizations found in this study.
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