Casie LeeMichelle Wong

Claire GrothAIM-UP! Module: Ornithological Geographic Variation

Learning objective: Using data from natural history museum collections (accessible online), you will compare and contrast different types of geographic variation in various species of birds while investigating the possible causes of this interspecific and intraspecific variation. You will develop your own hypothesis and abbreviated observational research experiment. The use of different tools and technologies provided within this module will help you to organize and interpret relevant data to better understand how geographic variation within and between species evolves. Key Terms/Concepts:- Sensory Ecology- Founder’s Effect- Natural Selection- Sexual Selection- Phenotypic Variation- Genetic Variation- Genetic Speciation Introduction to geographic variation: Darwin’s Finches as an exemplar of geographic variation

Charles Darwin’s experiences and observations in the Galápagos Islands provided the basis for his theory of natural selection. He began to wonder why birds separated by such small distances showed notable differences in beak size and shape. After observing how beaks were adapted to handle different food sources, Darwin began to explore the geographical distribution of species across the islands. Today there at least 13 different species of finches that fill different niches across the islands. Within each niche, finches have adapted to different diets that include various seeds, insects, flowers, and leaves. As an illustrative example, the graphic below shows how finches have adapted to have different beak types because of the food sources available to them.

Source: Macmillan Publishers Ltd: Nature, Vol. 442, p. 515, 2006. Part 1: Song Variation Background information:

Bird songs and calls are vital tools both in the field and in the lab. In the field, even a cursory “glance” of the ear can unveil a bird’s identity and location. More advanced familiarity and knowledge might even reveal what the bird intends to communicate. For example, one call may be identified as an alarm for an approaching predator whereas another may be more characteristic of a territorial “marking.” A song might also be intended to attract a mate.

In the lab, spectrographs and sonograms can be modeled to visually represent the patterns of bird calls for further comparison and analysis. Songs and calls may be learned from parents or inherited “culturally” from their environment and occupy an important role in the understanding of the social dynamics of birds of a particular locality as well as the evolutionary forces leading to their existence.

For more tips on how to best listen to and analyze bird songs and calls, go to the Cornell Lab of Ornithology website and under the “Birding Basics” tab, click the subtab “Songs and Calls.” (http://www.birds.cornell.edu) Variation Example #1 - Local Regional Variation:

The White-crowned Sparrow, characterized by the bold black-and-white stripes atop its head, is a large gray sparrow found in North America. In September of 1962, The Condor published a study conducted by P. Marler and M. Tamura titled “Song Dialects of Three Populations of White-Crowned Sparrows.” In this study, differences in song were noted between sparrow populations from Inspiration Point, Contra Costa County, and Berkeley, Alameda County, areas just two miles apart in California.

Looking at tables from the study below and given that song variation can exist between local neighboring populations, do you see a difference in song between the geographic locations? What specific differences can you note about song patterns? (e.g., song duration, number of notes, maximum and minimum frequencies) What ecological factors might account for these differences?

Source: “Song ‘Dialects’ on Three Populations of White-Crowned Sparrows.” (P. Marler and M. Tamura)

For more background information, go to the Cornell Lab of Orinthology Website and conduct a search query of the White-Crowned Sparrow. Sift through the information on the “All About Birds” page and consider details such as size, migratory patterns, and other types of geographic variation (for a major clue, read through the “Cool Facts” section under the “Life History” tab). Variation Example #2 - Geographic variation on a macro (continental) scale:

Now that you have some experience with hypothesis construction and testing, let’s look at another species. The Great Tit (Parus major) is a relatively non-migratory bird commonly found throughout Europe, the Middle East, and Western Asia. They are characterized by a black head and neck, white cheeks, and a predominantly yellow body (for additional information about the species, you may read through the online resources such as that of the Cornell Lab of Ornithology). Comparing sonograms (xeno-canto.org): Look at the sonograms below, showing Great Tit songs in graphical form. Do differences in the sonograms correlate with the distance separating populations of the same species? For instance, are the songs of European Great Tit more similar than a European Great Tit’s song when compared to a Middle Eastern Great Tit’s song? Use characteristics like frequency range, song pattern, and call duration as criteria for comparison. Are certain characteristics of song the same within a species like the Great Tit? Sample sonogram: Below is a sample sonogram taken from a paper written by Hunter and Krebs. The figure demonstrates which details and factors can be expressed in a sonogram. Note that the frequency (kHz) is on the y-axis and the duration or time (s) of the song is on the x-axis. Figure 4 compares the power (dB) to the frequency (kHz) of the song.

Do you see any overall differences between the woodland songs and the forest songs? If yes, what differences do you see? Does the song of the Great Tit in England differ from that in Germany? How? Activity: Xeno-canto is an online database of bird song recordings from around the world. Open a web browser and type in xeno-canto.org into the URL. Search “parus major” and explore the results. Play a few of the recordings and listen for differences in pitch and pattern between geographic locations. You can also access the corresponding sonogram (if available), as well as GPS coordinates, by clicking on the [sono] link under the “Remarks” column and the [options] link, respectively. (Note: The Great Tit has more than one call. Some are described as “calls of a female coaxing young to cross a clearing” and others may be identified as a hissing. Be aware of this subjective classification when comparing calls.Also, these sonograms may not be completely congruent with the article at hand). Now take a look at the differences in geographic location. Consider latitude, longitude,

ecological density, and other factors that might provide an explanation for the variation in song among different populations.

Are there any observed confounding variables? (A confounding variable is a variable irrelevant to the study that correlates with the variables taken into account for the study). Source: “Geographical Variation in the Song of the Great Tit (Parus major) in Relation to Ecological” (Malcolm L. Hunter and John R. Krebs)

Part 2: Color Variation in House Finches (Carpodacus mexicanus) Diet plays a major role in the pigmentation of male House Finches. Different types of seeds and food sources can carry organic pigments that cause color determination. A study on captive males found that birds fed a carotenoid-deficient diet grew pale yellow feathers, ones fed a beta-carotene supplemented diet grew pale orange feathers, and male birds fed a canthaxanthin (a carotenoid) diet grew bright red feathers (Hill, 1992). Thus, the coloration of an individual bird reflects its ability to attain certain food sources (Brush and Power 1976). To learn more about geographical variation in color, go tothe Ornis database (http://www.ornisnet.org/). Ornis is an online database containing information on more than 5 million bird specimens. Search for the species Carpodacus mexicanus (common name: house finch). Graph the results using the geographical mapper tool. Below is a map illustrating the geographic distribution of House Finches based on the size of the characteristic color patch on their feathers.

Source: Geographic Variation in the Carotenoid Plumage Pigmentation of Male House Finches (Geoffrey Hill) 1. Using evolutionary terms, list some possible ways that the geographic variation visualized in the map could have occurred. Next, look at pictures of two specimens of Carpodacus mexicanus (House Finch) shown below. Note their geographic origins and their distinct coloration.

Hawaiian House Finch Maryland House Finch

Carpodacus Mexicanus specimens from the U.C. Berkeley Museum of Vertebrate Zoology 2. What are some possible reasons for color variation among different geographical regions? How might different geographical environments affect pigmentation? 3. Explain how a variation in diet by location (for instance, the availability of foods high in carotenoid pigments, such as certain types of berries) might play a role in color variation. How could this be an example of sexual selection?

4. How might the existence of House Finches in Hawaii be an example of a Founder’s Effect? What does the color of Hawaiian House Finches suggest about the founding population? Food for Thought: Predict what might happen if an Eastern female (brown) were placed in a group of Western males (yellow/orange). What if a Western female (brown) were placed in a group of Eastern males (red)? Would the two scenarios exhibit different results? Explain. Part 3: Genetic Variation

In this activity, you will explore the relationship between genetic variation and the visible phenotypes with which it is associated. At one extreme, two different color morphs may differ significantly in their genetic sequences. Conversely, distinct color morphs may not differ genetically at all, instead arising from environmental influences and epigenetic factors. These two outcomes have very different evolutionary implications.

You will use GenBank, an online genetic database, to compare the genetic sequences taken from the DNA of Carpodacus mexicanus specimens with different geographical origins in North America. Specifically, you will be comparing the sequences of the CO1 (cytochrome oxidase 1 gene) which is a specific gene sequence commonly used for constructing molecular-based phylogenetic trees. Though it is a specific gene sequence that controls the production of certain proteins in the electron transport chain, when comparing between multiple species, it is a good indicator of overall genetic similarity. 1) To begin, go to http://www.ncbi.nlm.nih.gov/genbank/ and make sure that “taxonomy” is selected as the search field at the top of the page. 2) Enter “Carpodacus mexicanus” in the search field at the top, and select “nucleotide” underneath the link that shows up. This will take you to a page listing all of the different DNA nucleotide sequences that have been sequenced from House Finch specimens and uploaded to GenBank. 3) Look at the different markers that have been used to study House Finches.These include nuclear and mitochondrial markers, which have different modes of inheritance and thus capture somewhat different information about genetic variation. One of the best studied loci is CO1 (cytochrome oxidase 1), which serves as the blueprint for proteins involved in the mitochondrial electron transport chain. You will specifically compare the nucleotide sequences of the CO1 gene. In the search field, enter “carpodacus mexicanus cytochrome oxidase hawaii,” which should lead you to three specific sequences taken from specimens collected in Hawaii (accession numbers JF498839.1, JF498840.1, and JF498841.1). 4) Click on the nucleotide sequence with the accession number JF498841.1. Notice that the actual nucleotide sequence is displayed towards the bottom of the page and information such as the institution that submitted it and the number of base pairs in the sequence are included. 5) On the right hand side of the page under the heading “analyze this sequence,” click on the “Run BLAST” link. BLAST (Basic Local Alignment Search Tool) allows you to determine the similarity of multiple nucleotide sequences based on the number of fragments of successive nucleotides they share. This method is often used in creating phylogenies based on molecular data.

6) In the “Enter accession number(s), gi(s), or FASTA sequence(s)” box under the “Enter Query Sequence” heading, the accession number that you selected (JF498841.1) should already be entered. Click the “BLAST” button at the bottom of the page. The resulting red bars are a visual depiction of the extent to which certain sequences in GenBank locally align with your selected sequence. The greater the alignment score at a locus, the more similar a locus from a specimen in the GenBank databse is to that part of the BLASTed sequence. 7) Below the alignment bars, you will see a list of other sequences compared to the one you blasted. The accession number you entered in the box will appear first on the list, and all of the sequences listed below are compared to it. The “Max ident” value tells you how similar a given sequence is to the first accession number that you entered. The higher the percent value, the more similar it is to the first sequence.

The second and third specimens/accession numbers listed are for another House Finch specimen from Hawaii (JF498839.1), and a House Finch from Ontario (AY666266.1). How does the genetic similarity of the two Hawaii specimens compare to the genetic similarity between the Hawaii and Ontario specimens? Use the alignment scores and “Max ident” values as the grounds for your answer. Like the “Max ident” value, the higher the alignment score, the more similar the two sequences are. What does this say about the effect of geographic separation on genetic similarity between members of the same species?

8) Ontario House Finches tend to be red while Hawaiian House Finches tend to be yellow. Based on this fact and on the amount of genetic similarity you found between the two Hawaiian House Finches and between one of the Hawaiian House Finches and the Ontario House Finch, what can you conclude about phenotypic variation in its relation to genetic variation? In answering this question, it is important to consider the sample size of the specimens you are comparing. How does this affect the validity of your analysis of the genetic sequences you compared? 9) Look at the “Max ident” and alignment scores for the sequence taken from a Tachyphonus coronatus (common name: Ruby-crowned Tanager) specimen (FJ028384.1). Here is a photograph of a female Ruby-crowned Tanager:

Source: http://en.wikipedia.org/wiki/File:Tachyphonus_coronatus_female.jpg

Given the physical appearance of this species, are you surprised by its BLAST scores when compared with the Hawaiian House Finch? Why or why not? Part 4: Design your own exploration of geographic variation Choose a species that exists in multiple geographic regions, and compare the different regions. Use tools from the previous exercises to compare the morphology and genome of the two populations. 1) Suggest reasons that could account for the geographic variation you find based on what you have learned from the previous activities (e.g. speciation, drift, habitat). Explain why these reasons are logical. Potential bird species to investigate: - Cardinal (Richmondena cardinalis)- Eastern Meadowlark (Sturnella magna) vs. Western Meadowlark (Sturnella neglecta)- Western Bluebird (Sialia mexicanus)