Organisms have traits that are both genetically passed down and learned from one's environment. Communication is an example of a trait that is learned and developed throughout an organism's lifetime. All organisms have different ways of communication, whether that is the bioluminescence of fireflies or chemical signaling of coral (Campbell et al. 2007). Language is a significant form of communication most mammals use. Humans have many different languages amongst them, and amongst those languages are several dialects. A dialect is the form that a language takes that is specific to a region. English alone has several dialects specific to regions such as Southern United States, Northern England, and Canada. Like humans, some birds have dialects as well. Specifically, Poecile atricapillus or more commonly referred to as black-capped chickadees and Melospiza melodia or more commonly referred to as song sparrows. Black-capped chickadees use their songs and calls to attract mates, ward off predators, and establish territories (Ficken et al. 1978). Dialects vary within these vocalizations based on location of the birds as well (Miyasato et al. 1997).

The Miyasato and Baker study focuses on the gargle call, a sound used specifically by male black-capped chickadees to ward off other males when feeling threatened and in sexual contexts. These calls are comprised of syllables and frequency ranging from 2 kHz to 9 kHz (Miyasato et al. 1997). Every dialect has a distinct set of syllables and certain frequency. This is what makes each dialect unique to each geographic location. The gargle call has been observed to be shared among flocks which is the reason for the observed varying dialects (Ficken et al. 1994). Even though language is a learned trait, the development of black-capped chickadee language is due to the FoxP2 gene which is shared in humans as well (Vernes et. al 2011).

The FoxP2 gene codes for a transcription factor that inhibits the production of the forkhead boxp2 protein that blocks a neurological transmitter. Without that protein, the neurons are able to fire and create speech, but with the protein, the neurons are unable to fire (Vernes et al. 2011). When this happens, there can be speech impediments in humans and lack of vocalization in birds. FoxP2 is located on the 31st position of the seventh chromosome. The proteins FoxP2 codes for function in the Broca's area in human brains and Area X in bird brains (Arif et al. 2009). Speech is an important aspect of communication in both humans and birds, and the FoxP2 gene enables both humans and birds to have this ability.

Based on the results of the Miyasato and Baker group, it is believed that the chickadees develop different song variations based on location (Miyasato et al. 1997). Of the different gargle types found, some were present at multiple sites but most were unique to their own site. The highest degree of gargle-type sharing were in the two locations closest to each other; sites farther away showed little overlap (Miyasato et al. 1997). If chickadees are observed in two different locations in Ingham county, a variety of call types will be found due to the varying geography of the locations used. For the human study, the observation of how randome people on campus say the word "mayonnaise," as well as an anonymous survey with a set of questions, will help to determine the region unique to that dialect. Similar to the birds, if an anonymous survey is conducted that includes a variety of people around the world, there will be differences in dialect based on the region they belong.

Two different locations in Ingham County were used to create an experiment similar to the Miyasato study which used different geographic areas in Puget Sound (Baptista, 1997). The locations include the Carl G. Fenner Nature Center and Baker Woodlot Bird Sanctuary. These areas were chosen based on their diverse environments and differences in geographic locations. Besides the fact that Carl G. Fenner is a popular nature center, it provides a great environment for Black-Capped chickadees due to the open grasslands and established bird feeders. The bird feeders located next to Visitor Center were used as an observational site and included a wide variety of birds. Baker Woodlot offered an area of dense vegetation for numerous different organisms. Black Capped chickadees are seen throughout the woods and vocalizations are often heard. A personal bird feeder was placed in the woodlot and used as an observational site. Each of these will give a large number of black-capped chickadees to observe.

Data was collected with group member's smartphones, a Sony HD digital camera, and a video recorder. The smartphones were used to audio record the black-capped chickadees' calls. Along with the smartphones, the Sony HD digital camera and video recorder were used to capture the behavior of the chickadees. Every Wednesday and Friday, the group collected videos and pictures of black-capped chickadees at Baker Woodlot and Carl G. Fenner Nature Center. From October 7, 2016 until November 18, 2016, two group members went to Carl G. Fenner Nature center while the other two members went to Baker Woodlot during the same time period. The data was analyzed by listening to each sound recording and making note of the song variations. Gargle syllables, call length and pitches were important aspects of each bird song that helped with the identification and characterization in the black-capped chickadees communication. Each of the three different aspects of the bird's call were considered to be the three different variables tested. A student's t-test was used to determine the statistical difference between the data collected at Carl G. Fenner and Baker Woodlot. Then, the t-value was compared to the p-value of 0.05 to determine statistical significance.

Similar to the avian study, human communication methods were recorded through an online survey to determine how location alters dialect. The group used SurveyMonkey to create a ten question survey which was distributed using email, Facebook, and Twitter. The questions included place of origin and generic questions regarding words that vary between locations. An example of a question used was, "What do you call a carbonated beverage?" and the participants were given both specific options to choose from and an area to type in their own response. In addition to the survey, the group observed how humans pronounced the word "mayonnaise." This was done by having a mutual friend of the group hold up a card with the word written on it and asking random people on Michigan State University campus to read it. The audio of the answers were recorded on smartphones and analyzed by the group members with the same program as the bird calls. They were analyzed to determine which syllable of the word was stressed based on its length and pitch. This data was grouped based on which continent the specimen originated from. Five of the continents were analyzed (Europe, Asia, North America, South America, and Australia). The average syllable number stressed for each continent were compared using a one way anova test and the Tukey post hoc test using the astatsa calculating tool.

In order to fully understand the connection between black-capped chickadees and humans, DNA was collected from both to be analyzed. The group contacted the Department of Integrative Biology at Michigan State University to obtain a DNA sample of a black-capped chickadee. The group also contacted Dr. Ayako Yamaguchi, an associate professor of biology at the University of Utah who specifies in neurobiology, behavior, and physiology and functional behavior. Her knowledge of how the brain is connected to communication helped the group to further understand the FoxP2 gene and how it is connected to language in both birds and humans. PCR was used to show the presence of FoxP2 in both organisms, Poecile atricapillus and Homo sapiens. The PCR procedure was modeled after the Soejima Group's methods (2012). First, the samples were denatured at 95°C for 5 seconds. Then, they were annealed at 55°C for 30 seconds and extended at 75°C. This was done 20 times using the primers created by the group. These primers were used to target the gene. Forward primer 5'-GCGTCAGGGACTCATCTCC-3' and reverse primer 3'-CTCCAGATCGGGAGGTACAAAT-5'. Then, using a BioRad T100 Thermo-Cycler, samples were run through a 1% agarose gel and measured with gel electrophoresis.

It was found that the dialects of Homo sapiens varied based on different geographic areas around the world. This correlates with the research done by Bent and her colleagues that found different syllables in a word altered in stress depending on the organism's place of origin (Bent et al., 2016). Through analysis of data, it was found that humans in Australia (with an average stress syllable of 1.63) and North America (with an average stress syllable of 1.53) stressed the first syllable more often as compared to the dialects of humans in Europe (with an average stress syllable of 2.47) and South America (with an average stress syllable of 2.32) who stressed the third syllable (Figure 1). This data was found to be significance through the use of the one way anova test and the Tukey post hoc test by comparing the Tukey p-values of 0.011 between Europe and Australia, 0.0034 between Europe and North America, and 0.019 between North and South America to the interfence value of 0.05. This was found to happen because language patterns are learned through social interactions (Roach, 1982).

It was found that pitch, duration of call and number of syllables in the black-capped chickadee's call differed based on the geographic environment. Each chickadee call is composed of different units called syllables, which were found to have a different average (mean=4.6) at Carl G. Fenner as compared to calls at Baker Woodlot (mean=3.75) (Figure 2). The study found that generally the difference in syllables were slight, for instance, the addition or deletion of a few syllables, and most occurred at the beginning of the gargle (Ficken et al. 1978). Furthermore, the frequency of the "dees" obtained from the chickadee call were compared and revealed that the pitch was higher among the population at Baker Woodlot (mean=7.325 kHz) (Figure 2). The final variable, duration of call, showed that the average song lengths among the two sites being studied had a slight variation, where Carl G. Fenner was longer (mean=1.169s) than that of Baker Woodlot (mean=0.9775s). After the completion of the student's t-test, the p-value for each of the three variables were found to be insignificant (pitch: p= 0.132, length: p= 0.486, syllables: p= 0.390) because the found p-value is greater than the alpha value 0.05. With more evidence we predict that there will be statistical significance between the sites of interest because in the Miyasato study it was found that the gargle calls among each of the populations were site unique (Miyasato et al. 1997).

The sonograms depicted show a visual representation of the comparison between the two sites studied, Carl G. Fenner and Baker Woodlot. Based on studies, songs vary from flock to flock since they are unique to each population of birds (Ficken et al. 1994). In Figure 3a and Figure 3b, it can be seen that the more complex call of the black-capped chickadee at Carl G. Fenner contained multiple dee syllables (seven) as compared to the call documented at Baker Woodlot (four). At Baker Woodlot, the frequency of the sonogram was determined to be 6.5kHz (Figure 3a) when compared to the call from Carl G. Fenner that reached a frequency of 7.46kHz (Figure 3b). These were only two of the 15 calls that were analyzed to perform statistical tests.

We predict that through DNA purification we will be able to amplify the FoxP2 gene taken through secretions of both the organisms of study, Poecile atricapillus and Homo sapiens. With this intensified DNA, a forward and reverse primer will be selected to be used to bind to the target DNA. The forward primer will bind to the base pairs ranging from 711 to 729 and the reverse primer will attach to the sense DNA strand located at base pairs 852 through 873, which will both occur during the polymerase chain reaction (PCR) that takes place during the annealing process (Soejima, 2012). The DNA will undergo denaturation, annealing, and elongation during PCR amplification (Abdel-Rahman, 1996). We predict to see the presence of the FoxP2 gene when compared to the ladder in gel electrophoresis shown in Figure 4 because Soejima and their colleagues found the presence of the FoxP2 gene in both the wild-type human and the black-capped chickadee DNA samples (2012).

1. Abdel-Rahmana, Sherif A., Randa A. El-Zeinb, Wagida A. Anwa, and William W. Au. "A multiplex PCR procedure for polymorphic analysis of GSTMI and GSTTI fenes in population studies." Cancer Letters 107.2 (1996): 229-233.
   

2. Arif, Hakim. "FOXP 2: A gene for language and speech." Dhaka University Journal of Linguistics, 2.3 (2009): 173-184.

3. Baptista, Luis F. Geographic variation in song and dialects of the Puget Sound white-crowned sparrow. The Condor (1977): 356-370.
   

4. Bent, Tessa, Eriko Atagi, Amal Akbik, and Emma Bonifield. "Classification of Regional Dialects, International Dialects, and Nonnative Accents." Journal of Phonetics 58 (2016): 104-17. Web. 7 Oct. 2016.
   

5. Campbell NA. and Reese JB. 2007. Biology - 8th ed., Chapter 13 "Mendel and the Gene". Benjamin Cummings, CA.
   

6. Ficken, Millicent Sigler, Robert W. Ficken, and Steve R. Witkin. "Vocal Repertoire of the Black-Capped The Auk 95.1 (1978): 34-48.
   

7. Ficken, Millicent Sigler, and James W. Popp. "Long-term Persistence of a Culturally Transmitted Vocalization of the Black-capped Chickadee." Animal Behaviour 50.3 (1995): 683-93.
   

8. Miyasato, Lori E., and Myron C. Baker. "Black-capped Chickadee Call Dialects along a Continuous Habitat Corridor." Animal Behaviour 57.6 (1997): 1311-318.
   

9. Roach, Peter. 1982. On the distinction between 'stress-timed' and syllable timed languages. Linguistic controversies, ed. David Crystal, 73-79.
   

10. Soejima, Mikiko, Kenichi Hiroshige, Joji Yoshimoto, and Yoshiro Koda. "Selective Quantification of Human DNA by Real-time PCR of FOXP2." Forensic Science International: Genetics 6.4 (2012): 447-51.
    

11. Soha, J. A., D. Nelson A., and P. Parker G. Genetic Analysis of Song Dialect Populations in PugetSound White-crowned Sparrows. Behavioral Ecology 15.4 (2004): 636-46.
    

12. Vernes, Sonja C., Peter L. Oliver, Elizabeth Spiteri, Helen E. Lockstone, Rathi Puliyadi, Jennifer M. Taylor, Joses Ho, Cedric Mombereau, Ariel Brewer, Ernesto Lowy, Jerome Nicod, Matthias Groszer, Dilair Baban, Natasha Sahgal, Jean-Baptiste Cazier, Jiannis Ragoussis, Kay E. Davies, Daniel H. Geschwind, and Simon E. Fisher. "Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain." PLoS Genetics PLoS Genet 7.7 (2011).
    

13. White, Stephanie A., S. Fisher, D. Geschwind, C. Scharff, and T. Holy. Singing Mice, Songbirds, and More: Models for FOXP2 Function and Dysfunction in Human Speech and Language. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience. U.S. National Library of Medicine, 11 Oct. 2006.

    Figures
    
    Finalized by: Bailey Borycki
    
    
    
    Figure 1. Average syllable number stressed in the word "mayonnaise" in five observed continents. We found that different syllables in the word "mayonnaise" was stressed based on certain geographic regions because the Bent study observed that different syllables in a word altered in stress depending on the organism's place of origin (Bent et al. 2016). Responses from humans in certain locations were grouped together based on their location in the world. The person was asked to say "mayonnaise" when shown a picture of the word and was recorded using a microphone. The data was inserted into a sonogram to determine which syllable was stressed. The syllable emphasis was then averaged for each continent which is shown on the X and Y axis. Standard error is represented by error bars using a Tukey post hoc test using the astatsa calculating tool. The results showed that p>0.05 when comparing Europe to Australia, Europe to North America, and North America to South America. All other p-values were below .05 or insignificant. For each data population n=0.

    
    
    

    
    Figure 2. Averages of focused variables (pitch, length, and number of "dees") in black-capped chickadee calls. We found that the mean call length and pitch for black-capped chickadees varied between the two locations because the Miyasato study observed that black-capped chickadees from different habitats have distinct songs and therefore song lengths and pitches (Miyasato et al. 1997). The student's t-test was used to analyze the recordings in each location. As seen in the figure, our data did not show significance because p>0.05, shown by the overlapping error bars . Bird calls were recorded at each site (Carl G. Fenner Nature Center and Baker Woodlot) and were measured to determine the average pitch and length of the bird call unique to each location. The calls were then inserted into both garageband and a sonogram application to be analyzed. The pitch was measured in kHz and the length was measured in seconds. We found that the average number of "dees" at the end of the each chickadee call differed slightly based on location. However, the student's t-test test justified that the data was insignificant and therefore the calls were not site unique. Carl G. Fenner n=10, Baker Woodlot n=5.

    
    
    

    
    A.
    B.
    Figure 3. Sonogram comparing syllables of chickadee calls in Black-Capped Chickadees at Carl G. Fenner nature center. We found that the syllables of each chickadee call differed based on location because in the Miyasato study the majority of chickadee call types were found to be site unique rather than shared between sites (Miyasato et al. 1997). This figure illustrates an example of two different chickadee calls, A and B. A represents a call from Carl G. Fenner Nature center and B represents a call from Baker Woodlot bird sanctuary. Each individual chickadee component is called a syllable. The frequency of the call is measured in kHz. The space between each syllable represents the quiet period. This graph analyzes the structure of the calls so we can clearly scrutinize the differences. We found that the chickadee call from Carl G. Fenner was higher in frequency and had a greater amount of "dees" at the end of the call.

    
    
    

    
    
    Figure 4. Predicted results from Homo sapien and Poecile atricapillus DNA viewed on gel electrophoresis. DNA samples from black-capped chickadees (denoted as BCC in the figure) and humans (denoted as HS in the figure) will be gathered. The non-discriminating reverse primer is 3'-CTCCAGATCGGGAGGTACAAAT - 5'. The discriminating forward primer is 5'- GAGGTCTAGCCCTCCATGTTTA- 3'. The predicted binding site is on chromosome 7 p.31. The denaturing set will be set to 95°C for 5 seconds and then annealed at 55°C for 30 seconds and elongated at 75°C. This will be repeated 20 times. Then they will run through gel electrophoresis and be compared to the DNA templates of Homo sapiens and Poecile atricapillus. Blue dye will be added to the gel which will tell us when gel electrophoresis is complete. This PCR procedure was modeled after the Soejima Group's methods (2012). The FoxP2 gene codes for the forkhead box P2 protein, which is a transcription factor, meaning it controls the activity of other genes. If this gene is mutated in either of the organisms, then it will not be present in the PCR gel electrophoresis which then leads to the development of speech and language disorders which can be seen in Area X of songbirds and the Broca's area in humans.
    
    
    
    
    Figure 5. Group's Video Project. iMovie was used to create a documentary to discuss the research and findings of the group's experiment. The film includes real footage of the black-capped chickadees at Carl G. Fenner Nature Center and Baker Woodlot. It also includes video and audio of humans on Michigan State University's campus. Voice overs were added to explain the group's research and any additional information needed to understand.