1. Identification and Evolution of Brunfelsia australis
Carlos Olivaris
Raffia Ahmed
Lab Group 8
May 5, 2014

2. Purpose of Study We had two goals:
1) DNA Barcoding – This was used to confirm our plant’s identity
2) Phylogenetic Analysis – This allowed us to better understand our plant’s genetic background and evolutionary relationships

3. Our Plant Specimen
We chose Brunfelsia australis, commonly known as Yesterday-Today-and-Tomorrow.
We thought this plant was interesting because of the tri-coloured flowers. When we did research, we found out that the flowers start purple and slowly fade to lavender and then white over a few days.

4. Our Plant Specimen Family Solanaceae Tribe Petunieae
Originated in light woodlands and thickets of Brazil
Popular garden plant but no other economic uses
The Solanaceae family contains some of the most economically important food crops, including potatoes, tomatoes, and tobacco. There are 2900 species spread over 76 genera. Our genus – Brunfelsia – contains 40 species and like most other solenoids contains toxic compounds. Since Brunfelsia australis is a cultivated crop, it looks very much like some of the other species in its genus like Brunfelsia pauciflora. However, Brunfelsia australis seems to be promoted as more drought-resistant.

5. Methods Our protocol can be broken down into three major steps:
Obtaining and preparing our sample
DNA Extraction, PCR, and electrophoresis
Aligning and analyzing the DNA sequences
Done once for each sequence (ITS2 and rbcL)
Data was analyzed for phylogeny and compared to GenBank and BOLD using BLAST
Our sample size was 7mm x 3mm and we were able to extract 340 microlitres of lysate. We used electrophoresis to check our results after PCR, and like almost everyone else, our first electrophoresis attempt at both ITS2 and rbcL was unsuccessful. Note: be careful that you put them in the correct wells, because we did not! Our sequences were much shorter than the ones we obtained from GenBank, suggesting that we were only partially successful at extraction and sequencing. Luckily, we were still able to obtain good data. Aligning our sequences worked alright, but our bootstrapping analysis for rbcL took a very long time. Luckily, we were able to use 500 replications for both and had good results.

6. Results
Our results from the ITS2 Bootstrapping analysis confirms Brunfelsia australis’s place within the Solanaceae family. Note that no other Brunfelsia species are represented – that’s because only partial ITS2 data was sequenced for all of the species available. It’s disappointing that we couldn’t find ITS2 data for another species within the Brunfelsia genus, but hopefully more data will be available in the future or another group can replicate our study and include the partial data.

7. Results Comparing our ITS2 data to GenBank showed interesting results:
Our top 5 species were all within the Brunfelsia genus (and included Brunfelsia australis)
However, number 1 was Brunfelsia pauciflora
This is not unexpected; the species look similar and the ident percentage was 97 as opposed to 96 for Brunfelsia australis
rbcL data was also queried in BOLD

8. Results
This is the result of the rbcL bootstrapping analysis. Note that the Nicotiana species are listed near the bottom, away from our sample species. This differs from our results in GenBank and BOLD.

9. Results
Most of our rbcL results in GenBank and BOLD were in the Nicotiana genus!
BOLD did not match our sequence to any species in the Brunfelsia family – the top species was Nicotiana tomentosiformis
N. tomentosiformis was number 2 on GenBank, but number 1 there was Brunfelsia americana
Why did this happen? We suspect that many more closely related species to B. australis have not yet had their rbcL data sequences. Since rbcL is also more well-preserved between species, it is not surprising that more distantly related species that are still within the same family come up as close matches – the ident values for all of these species except Brunfelsia americana were no more than 98%, and Brunfelsia americana was 99. What is N. tomentosiformis? It’s a wild flowering species within the tobacco genus that originated in South America.

10. Conclusions
Building phylogeny trees from sequenced data is successful at clarifying evolutionary relationships, but requires lots of processing power
DNA Barcoding works with some caveats
Species need to be previously sequenced to be matched properly in GenBank and BOLD
Accuracy should improve over time as more species are sequenced