Phylogenetic Positions of Two Genera in the
Family Scrophulariaceae (Figwort Family)
Bridges to the Future Program : John Almeida working with Dr. Freeman
The Figwort Family is a very large group of plants with a lot of questions still unanswered regarding its family structure (which plants are
closely related to one another). In this study two plants will be sequenced and aligned with 38 other genera in the family using the trnL intron of the chloroplast Dna. The two plants are the Galvezia speciosa and the Scrophularia californica. Using the trnL intron the two genera were sequenced, aligned, and put into a successful family tree within the Figwort Family.
The Figwort Family is a widely diverse family of plants consisting of approximately 3000 species. Although it is a well known group of plants the Figworts have often been said to be not known very well at all. Even the limits of the family itself are not agreed upon (Thieret 1967). Many scientists have studied the group using a variety of ways such as their morphological characteristics. New technology has led scientists to study their nuclear Dna sequences, and their chloroplast Dna sequences. The morphological characteristics fail to provide enough resolution between the plants so they are not helpful in classifying the family. The nuclear Dna regions have very few differences if any at all and the Dna sequences are very difficult. The chloroplast Dna sequences have recently been utilized to provide information on plant phylogeny. This study will be using the chloroplast Dna of the genera.
Scientists have used different regions within the cpDna to try and provide resolution within the Family Scrophulariaceae. Researchers have used the trnL intron, the rbcL (e.g., Kim and Jansen 1996; Doyle et al. 1997; Morton et al. 1997; Pax, et al. 1997; Soltis and Soltis 1997; Wagstaff and Olmstead, 1997) intron, and many others. An intron is a non-coding Dna sequence which interrupts the coding Dna of the gene. Non-coding Dna sequences should change more rapidly than coding regions with time since they are constrained less by selection. As a result these sequences have the potential of resolving phylogeny at lower taxonomic levels (Clegg et al. 1994; Gielly and Taberlet 1994). The trnL intron is a highly variable region having approximately 500 bp (base pairs).
What we are trying to conclude is whether or not the trnL intron sequence is variable enough to provide resolution within the Figwort family and at what level is it most useful. The trnL intron will be used to sequence two genera in the Figwort family. The two genera are Galvezia speciosa and Scrophularia californica. The Galvezia speciosa is located in the Tribe Antirrhineae and the Scrophularia californica’s location is relatively unknown. It is either located in the Tribe Verbasceae or Cheloneae. These two genera will be aligned with 38 other plants within the Figwort Family already sequenced by Dr. Freeman. At the end of the sequencing all 40 of the plants will be aligned using the PAUP program. After they are aligned, the genera will then manufacturer a claudogram or family tree where all of the plants can be put into proper perspective.
MATERIALS AND METHODS
DNA EXTRACTION---Fresh leaves of Galvezia speciosa and Scrophularia californica were obtained by Dr. Ron Scogin from Rancho Santa Ana Botanic Garden, Claremont, California. Young leaves were selected for extraction. The
leaf samples were crushed in 300-400 ml of DNAzol ESÔ lysis buffer. Three hundred ml of lysis buffer containing crushed leaf material were transferred to a 1.5 ml microfuge tube and allowed to sit at room temperature for five minutes. An equal volume of chloroform was than added to the microfuge tube and shaken vigorously to thoroughly mix the two liquids. The mixture was allowed to sit undisturbed for 10 minutes and then centrifuged for ten minutes at 14,000 rpm to separate the two phases.
After centrifugation the chlorophyll and cell debris were found in the lower chloroform phase. Two hundred ml of the Dna-containing upper aqueous phase were removed by a wide-bore micropipet tip to another 1.5 ml microfuge tube. Dna was precipitated by the addition of 0.75 volume of absolute ethanol. The Dna precipitated at room temperature for 10 minutes and the tube centrifuged for 20 minutes at 14,000 rpm to pellet the Dna.
The pellet was washed with 80% ethanol to solublize a large portion of the proteins , polysaccharides, polyphenolics, and other contaminants in the pellet. Two ml of the total DNA extract obtained were electrophorised in a 0.8% high melting point agarose gel and the DNA visualized with ethidium bromide under UV illumination. In the case of Scrophularia californica proteins remained attached to the DNA. This total DNA solution was then treated with proteinase K and allowed to sit over night at room temperature. The total DNA solution was then reprecipitated with ammonium acetate(1/2 volume) and ethanol (2 volumes) overnight at –20C.
PCR AMPLIFICATION.---A 50ml PCR aliquot of each product was made using the c and f primers of Geilley and Taberlet (1996). This product was the trnL intron plus the trnL-trnF intergenic spacer. The temperature profile of the PCR reaction was an initial soak at 95C for five minutes followed by 35 cycles of 30 sec at 95C, 30 sec at 55C, and 1.0 min at 70C. At the end of amplification there was an additional 7.0min soak at 70C.
GEL PURIFICATION OF PCR PRODUCTS.---The PCR products were then purified by electrophoresis. To each 50ml PCR aliquot was added 15ml of glycerol-bromophenol blue loading buffer. The resulting solution was then loaded into a 0.8% high melting point agarose gel and allowed to electrophorese for approximately 30 min at 70 mV. The product was then visualized under UVA light and excised from the agarose block with a scalpel. The agarose block containing the purified PCR product was then placed in a 0.5 ml microfuge tube which contained a bed of polyester aquarium filter floss and a hole made with a 27 gauge hypodermic needle in the bottom. The 0.5 ml microfuge tube was then placed into a 1.5 ml microfuge tube and the unit was centrifuged for 20 min at 14,000 rpm. The PCR product was then ethanol precipitated from the TAE buffer liquid in the 1.5 ml microfuge tube. The PCR product pellet was then redisolved in 20 ml of water and the quantity of product estimated by electrophoresing 2ml of
this solution on a 0.8% agarose gel. The intensity of the product band was visually estimated under UVC light (265 nm).
CYCLE SEQUENCING OF ds PCR PRODUCTS---The cycle sequencing was accomplished using the Sequitherm Excel IIÔ kit (Epicentre Technologies, Madison, WI). The protocol was as specified by the manufacturer except that two ml of template were used and we used 45-50 cycles. The acrylimide gel used was Long Ranger utilizing the manufacturer’s protocol. Runs of individual reactions were 1.5 and 3.0 hours in most cases with some run for 4.5 hours for extra long reads of high quality reactions. Gels were fixed with 1.0 L of a solution containing 10% methanol, 10% glacial acetic acid, and 80% water. Gels were then dried and exposed to film for approx. 20 h. Autorads were then read and the sequences aligned using the algorithm MAP (Baylor College of Medicine, Dallas, TX) over the internet. Minor adjustments were made to that alignment as necessary.
DATA ANALYSIS---Indels were treated as presence and absence data in the database. This reduced the influence of multiple base pair insertions/deletions in the analysis. Cladograms were generated using the maximum parsimony, maximum likelihood, and neighbor joining methods (PAUP 4.0 Star).
RESULTS AND DISCUSSION
The trnL sequences of both of these species were readily alignable with the other 38 species in the Scrophulariaceae database and are shown in Table 1. Scrophularia californica is 476 bp long and this is a typical length among the taxa previously sampled. However, at 506 bp, Galvezia speciosa is the longest trnL intron sequence yet encountered within the family. This extra length is due to a long insert within a nonconserved region VI.
Table 1. The trnL sequence of Scrophularia californica (less the first 13 bp at the 5’ end and the last 17 bp at the 3’ end). This sequence is, therefore, 506 bp long ( 476 bp plus the 30 bp not included) and reads as follows.
CTTGGTATGG AACCTACTAA GTGATAACTT TCAAATTCAG AGAAACCCCG GAATTAATAA AAATGGGCAA TCCTGAGCCA AATCCTGTTT TCTCAAAACA AAGGTTCAGA AGGCGAAAAA GGATAGGTGC AGAGACTCAA TGGAAGCTGT TCTAACAAAT GGAGTTGACT GCGTTGGTAG AGAAATCTTT CCATCGAAAC TTCAGAAAGG GTGACGGATA AACGTATCTA TTGAATACTA TATCAAATGA TTAATGATGG CCCGAATCTG TATCTTTATT TTTTTATATG AAAAATAGAA AAATTGATGT GAATTTATTC CACATTGAAG AAAGAATCAA ATAAAATATT CATTCATCAA ATCATTCACT CCATAGTCTG ATAGTTCTTT TAAAGAACTG ATTAATCGGA CGAGAATAAA GATAGAGTCC CATTCTACAT GTCAATACCG
GCAACAATGA AATTTATAGT AAGAGG
Table 2. The trnL sequence of Galvezia speciosa (less the first 13 bp at the 5’end and the last 17 bp at the 3’ end). This sequence is, therefore, 542 bp long (512 bp plus the 30 bp not included) and reads as follows.
CTTGGTATGG AAACCTACTA AGTGATAACT TTCAAATTCA GAGAAACCCG GGAATTAATA AAAATGGGCA ATCCTGAGCC AAATCCTGTC TTCTCAAAAC AAAGGTTCAG AAAGCAAAAA AAGGGATAGG TGCAGAGACT CAATGGAAGC TGTTCTAACA AATGGAGTTG ACTGTATTGG TAGAGGAATC TTTCGATTAA AACTTCAGAA AAGATGAAGG ATAAATCTAT CTATTGAATA CTATAGAAAA GATGAAGGAT AAATCTATCT ATTGAATACT ATATCAAATT ATTAATGATG GCCCGAATCT GTATCTGTAT TTTTTTATAT GAAAAATGGA AGAATTGGTG TGAATTGATT CCACATTGAA GAAGAATGGA ATATTCATTC ATCAAATCAT TCACTCCATA GTCTGGTAGA TCTTTTAAAG AATCTGATTA ATCGGACGAG AATAAAGATA GAGTCCCATT CTACATGTCA ATACCGGCAA CAATGAAATT TATAGTAAGA GG
After the sequences were complete they were transferred to the Paup program. The Paup program then executed a heursitic search. The maximum parsimony analysis of the entire database, including S. californica and G. speciosa is shown in Fig. 1.
According to the claudogram the Scrophularia californica was put right beside V. thapsus and S. macranthamum. This clearly shows that according to the Paup program using the trnL intron, the S. californica is clearly located in the Tribe Verbasceae. The Galvezia speciosa is located precisely where it is suppose to be ; in the Tribe Antirrhineae.
The trnL intron was successful in putting these 40 genera in a proper family tree. Overall there were approx. 20 major sites located in the sequences between the plants which could be easily distinguished using the Paup program. There is still more research to be done and the trnL intron could be used for further study in the Figwort family.
I would just like to personally thank everybody involved in the Bridges to the Future Program, it’s been another great summer. I would especially like to thank Dr. Freeman for all of his knowledge, support, and for being a great colleague and friend for the past two summers.
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