Explaining Science – plant genomics

Brandon Sinn performing molecular lab work

Brandon Sinn performing molecular lab work

Brandon Sinn, PhD graduate from the OSU herbarium, now a postdoctoral fellow at West Virginia University, recently published a paper on molecular work he did to better understand the evolution of genomes in Asarum (Aristolochiaceae), commonly known as wild ginger. The work was done in collaboration with Dylan Sedmak, an OSU undergraduate student, Lawrence Kelly, Associate VP of Science, New York Botanical Garden and John Freudenstein, Professor and Chair of EEOB and Brandon’s PhD advisor.

We interviewed Brandon to get a better understanding of his research findings:

Brandon: “Evolutionary relationships in the flowering plant genus Asarum served as the focus of my dissertation research, and I continue to study the group.  In this particular project we studied six Asarum species, which each represent one of the six major evolutionary lineages within the genus.

Flowers of some Asarum species found in southern Appalachians

Flowers of some Asarum species found in the southern Appalachians

Asarum is a poorly-understood genus of approximately 115 species found in temperate forests across Asia and North America. Some Asarum species are common and widespread across the continents where they are found, while the majority have highly restricted ranges – for example, one species is known only from a single gorge in North Carolina and others are found in only a few counties in the southeastern United States.

During the course of sequencing DNA for my dissertation research, I realized that the genes of some Asarum species were not in the expected order. This departure from expectation was surprising, since the clade, or evolutionary neighborhood, that Asarum belongs to is very old and had been partially characterized as having slowly-evolving and highly conserved genomes. For example, the genome of another member of the same clade has been called a “fossil” genome. It was because of this unexpected observation that we decided to sequence complete genomes from one species from each of Asarum lineage. ”

This lead to the following research questions: Note: A plastome is the genome of a plastid, the organelle responsible for photosynthesis in plants.

1) Have the plastomes of all Asarum species been destabilized and their gene order rearranged?

2) Is the plastome of Saruma henryi (commonly called upright wild ginger), the closest relative of Asarum, of typical arrangement or is it more like that of Asarum?

3) Can we understand how the ordinarily highly conserved and stable plastomes become destabilized by comparing the plastomes of many Asarum species to that of Saruma henryi?

Saruma henryi, a flowering plant in the family Aristolochiaceae, endemic to China

What should we know to understand this research?

Brandon: “Each plant cell contains at least one copy of three distinct genomes. It is easy to imagine that each cell has a copy of the plant’s genome, but many people forget that two types of plant organelles, mitochondria and plastids, also have their own genomes. Plastids, from which chloroplasts develop, have a very small genome that is relatively easy to completely sequence and the sequence of more than 2,000 are publicly available today. The sequencing of thousands of plastomes has resulted in several general trends: 1) plastomes change more slowly than the plant’s own genome; 2) the plastome is made up of three functional regions, the small single copy, large single copy, and inverted repeat regions; 3) the physical order of genes is highly conserved across even distantly related species; 4) there is very strong selective pressure on the preservation of photosynthesis, which most likely constrains the evolution of plastomes in green plants. Our knowledge of the typical layout of the plastid genome, or plastome, has long been relied upon to sequence DNA in order to study plant evolutionary relationships. Traditional DNA sequencing techniques require prior knowledge of the order of genes or regions of a genome. If this order is not as predicted, then the DNA sequencing will fail.”

What method did you use to study your research question?

Brandon: “For this study, we sequenced entire plastomes from six Asarum species and that of Saruma henryi, the closest relative of Asarum. Since traditional DNA sequencing is not useful in destabilized and dynamically rearranged genomes, and we wanted to sequence entire plastomes that we hypothesized were rearranged, we needed to use a technology called massively parallel sequencing. A major advantage of massively parallel sequencing is that a researcher can extract DNA from a tissue, break the DNA into short pieces, and simultaneously sequence all of these fragments without prior knowledge of their physical relationship to one another. The resulting millions of DNA sequences are then assembled, much like a puzzle, using specialized software. The assembled  plastomes can then be compared.”

Brandon explains one of the key figures in his manuscript:

A cruciform DNA structure that has likely destabilized a region of the plastome in Asarum species. Structure courtesy of Eric Knox.

A cruciform DNA structure that has likely destabilized a region of the plastome in Asarum species. The end of the ndhF gene is shown in red. Structure courtesy of Eric Knox.

DNA is made of only four chemicals (which we abbreviate as the letters A, T, C and G) and is not entirely unlike a spiral staircase, where each handrail is a string of these letters. Holding this structure together are bonds that form between certain letters – A-T and G-C. We call these letters nucleotides. Sometimes the nucleotides making up DNA cause the molecule to form complex shapes, such as the cruciform structure shown here. Cruciform, or cross shaped, DNA structures form when the same nucleotides are repeated very close to one another, which is depicted in the vertical “stems”.


Cruciform DNA structures can be difficult for the molecular machinery in cells to work with. For example, sometimes molecules that interact with DNA get stuck on the stems, and these structures compromise the integrity of the DNA molecule. When these structures break, which you can imagine by separating the red and black halves of DNA for Saruma henryi, the cell tries to put them back together. But, repairing DNA does not always work perfectly. The results of our research suggest that faulty repairs made to this DNA structure throughout the plastomes of Asarum species have resulted in varying degrees of DNA duplication. Notice that the ndhF gene (shown in red) is typically at one end of the small single copy region, as shown on the Saruma henryi plastome. In Asarum, this gene often has a long stretch of nucleotides that can be “pasted before or after it. In other Asarum plastomes, such as Asarum canadense, we find that all of the small single copy region has been duplicated. The duplication of the formerly single copy region is most likely due to faulty repair of the cruciform DNA structure, where identical strings of nucleotides close to one another led to bonding of two identical DNA regions (as seen in the Asarum canadense cruciform structure).”

Why is this research important?

Brandon: “When you learn about DNA in high school science classes, everything sounds very concrete and well understood, but even gene function in humans is not exhaustively understood. Our basic knowledge about how genes and genomes evolve is in a constant state of improvement. This knowledge is necessary for future breakthroughs in genome engineering, evolutionary and conservation biology, and improving genome stability.  Just as it is important to understand biodiversity at the level of species, it is equally important to understand genomic diversity – the content and structure of genomes, in order to understand how mutations in particular regions of genomes can lead to genome-scale changes over deep time and how these changes affect evolutionary lineages.”

What should you take away from these findings?

1) Just because a species is a member of a very old evolutionary lineage, we should not expect that it is a living fossil and that its genome has changed little.

2) A plastome can function even when gene order is changed and more than half of its genes are present more than once.

3) Small, likely randomly generated repetitive motifs in DNA sequence that is not part of a gene can decrease genome stability, and lead to genome rearrangement and gene duplication.


Wow, we are now certainly asking questions and getting answers with new techniques that we could not have imagined decades ago. If you want to follow Brandon’s further research, click here.

About the Authors: Brandon Sinn photoBrandon Sinn earned his Ph.D. in 2015 from the Department of Evolution, Ecology and Organismal Biology, where he was a member of the Freudenstein Lab in the Museum of Biological Diversity. Brandon has held a postdoctoral research position at the Pfizer Plant Research Laboratory of the New York Botanical Garden, where he worked on the Planteome Project. He is currently a postdoctoral fellow in the Department of Biology of West Virginia University where he studies orchid genome evolution as a member of the Barrett Lab.

Angelika Nelson is the curator of the Borror Laboratory of Bioacoustics and the social media manager for the museum.

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Sinn, B. T., Sedmak, D. D., Kelly, L. M., & Freudenstein, J. V. (2018). Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in AsarumAmerican journal of botany105(1), 71-84.

Time travels with the Borror Lab of Bioacoustics

Carolina Chickadee by Dan Pancamo

Carolina Chickadee by Dan Pancamo

One thing that is unique about the sound archive of the Borror Laboratory of Bioacoustics is that it not only contains a wide diversity of animal sounds, but a great number of recordings for certain species. When I started my Ph.D. research here at OSU, I was pleasantly surprised to find that this depth of recordings also included one of my study species, the Carolina Chickadee (Poecile carolinensis).

The namesake of the Borror Laboratory of Bioacoustics (BLB), Dr. Donald J. Borror, was one of the first biologists to take recording equipment out into the field to record animal sounds. And he started in and around Columbus. Because Carolina Chickadees are rather common birds here in the central and southern portions of Ohio, Carolina Chickadees were some of the first animals Dr. Borror recorded. The oldest recording he archived in the collection dates back to 1948. Listen to a 30-second excerpt of Dr. Borror’s recording of a typical four-note whistled Carolina Chickadee song from April 1948 (note: You can listen to the entire recording (BLB21) on the BLB’s website):

You may not think that having all these chickadee recordings across a long time period is super duper exciting, but I do. See, I study chickadee song. And we know that chickadees, like other songbirds, learn their song: young chickadees must hear other individuals of their species singing and imitate those sounds in order to produce normal adult song. However, like learning in humans, song learning in birds is not always a perfect process. As young birds make imperfect copies of the songs of the adult birds they hear, variation is introduced into the songs of a population of birds. Think about how the English language has changed in the past 100 years – some words have stopped being used, new ones have come into fashion – this is analogous to what happens with song in bird populations.

The end result is that not every chickadee sings exactly the same song and the acoustic traits of chickadee songs can change slightly from generation to generation. Using the BLB collection I can actually look at how Carolina Chickadee song has changed in the Columbus area over the past 65+ years.

What you are going to see below are a series of maps with representative spectrograms of chickadee songs from all over Columbus for different decade ranges. If you have never seen a spectrogram before, it is essentially a visual representation of sound, with time on the x-axis, frequency (or pitch) on the y-axis, and the darker color representing more energy (or the loudness) of the sound. Here is the spectrogram of one song of a Carolina Chickadee from the 1948 recording by Don Borror above:

Spectrogram of one song of a Carolina Chickadee recorded by Don Borror in Columbus OH in 1948

For each map below I encourage you to visually compare the different chickadee songs using the spectrograms (I have left the axes off for simplicity’s sake) and then listen to the recording containing those songs using the links below each map. Any overlapping spectrograms are from the same individual bird: Carolina Chickadees can sing up to 4 different song types each, although most only sing one or two types. If you want to listen to the original recording archived in the BLB, please click on the link for each BLB cut number.


As you can see, Carolina Chickadees usually have a four-note song of alternating high and low whistled notes, but check out the weird song at Blacklick Woods Metro Park (#3)! Dr. Borror’s 1948 recording is actually quite unique in that the notes in the chickadee’s song are not very different in pitch from one another; usually Carolina Chickadee songs sound and look more like the spectrograms seen in examples 1 and 4b.


#1 (BLB1354)

#2 (BLB2451)

#3 (BLB3909)

#4 (BLB3947)



map2The unique song type seen before persists at Blacklick Woods Metro Park through the 1960s. Also note that some Carolina Chickadee songs start with a note much lower in pitch than others (like song number 1 here, or song 4a in the 1950s map). Carolina Chickadees also sometimes add notes onto the end of their songs, resulting in five-, six-, and sometimes up to twelve-note songs, although they usually keep the alternating high-low pattern (see #4).


#1 (BLB6374)

#2 (BLB9942)

#3 (BLB5112)

#4 (BLB9026)

#5 (BLB7966)



Many of the songs in this decade are very similar, but one individual at Blendon Woods Metro Park (#2b) showed an interesting song with an additional introductory note. This song type is not seen in any other bird in any other decade, so it is possible this song type was never sung by any bird but this one.


#1 (BLB11032)

#2 (BLB14217)

#3 (BLB13312)

#4 (BLB11030)


map4As you can see, not much song variation was recorded in the 1980s, except for that three-note song up in Delware. While most of the songs follow the typical high-low-high-low pattern, there are subtle differences between individuals, like in the downward sweep of the first note.



#1 (BLB17075)

#2 (BLB17078)

Note the Black-capped Chickadee singing in the background of recording #2:

#3 (BLB15737)

#4 (BLB17063)



In Delaware a three-note song type persists in the population from the 1980s into the 1990s. Also, the song type that starts with a lower-pitched note continues to pop up in various areas of northern Columbus (e.g. 3b), but is not seen in the southern portions of the city. Interestingly, I had not heard that song type myself while living in Columbus until moving to Clintonville this past March; up near Dublin that song type is not common anymore.


#1 (BLB17435)

#2a (BLB17433)


#3 (BLB21124)

In the past 14 years none of the recordings in the BLB collection specifically target Carolina chickadee songs. It would be interesting to know if those strange songs from Blacklick Woods Metro Park are still sung in that area, or if any other unique song types have appeared in the Columbus area.

So, as you travel throughout the Columbus area, keep an ear out for some odd chickadee songs … you may even hear something that has not been recorded before.

About the author: Stephanie Wright Nelson is a graduate student in the department of EEOBiology. She studies song learning in chickadees and is particularly interested in the consequences of hybridization between Carolina and Black-capped Chickadees.