The tribe Bignonieae includes 393 neotropical species (classification of tribe Bignonieae), representing almost half of the species in the Bignoniaceae. The tribe is mainly composed of lianas and shrubs with widespread or highly endemic distribution patterns (Lohmann and Taylor, in press).
The first molecular phylogenetic study for the tribe (Lohmann, 2006) was mainly based on recently collected samples that did not present any problems for the amplification of large DNA fragments. However, approximately 10% of the currently recognized species of Bignonieae (37 of the 393) are highly endemic and were not encountered in the field. For those species, only five or fewer herbarium samples are available (Lohmann, unpublished data), making those specimens the only source of DNA material for phylogenetic studies.
Here, we propose new primers and protocols that allow the amplification of medium-sized DNA fragments (∼500 bp) from herbarium samples. The novel protocols here proposed are critical for the inclusion of rare and poorly known species of Bignonieae into a comprehensive phylogeny of the whole tribe.
METHODS AND RESULTS
DNA extraction–Total DNA of six herbarium samples (dating up to 53 yr old) was extracted with Invisorb Plant Mini Kit (Invitek, Berlin, Germany). The manufacturer's protocol was followed, except for the final step, in which 50 µL of elution buffer was used instead of the suggested 200 µL.
Primer development–Selected sequences of the plastid ndhF and nuclear PepC genes for Bignonieae from Lohmann (2006) were combined with newly generated sequences for the plastid rpl32-trnL intergenic spacer region following Shaw et al. (2007). Vouchers and GenBank accessions of the sequences used and/or generated in this paper are presented in Appendix 1. The data sets corresponding to the individual data partitions were aligned in Geneious 5.4 (Drummond et al., 2010) using the algorithm MUSCLE (Edgar, 2004). A thorough search for primer pairs was also conducted in Geneious, using the software Primer3 (Rozen and Skaletsky, 2000). The objective of this search was to design primers placed in highly conserved regions that would only amplify medium-sized fragments (∼500 bp) and would overlap adjacent amplicons (∼70 bp). Given that the nuclear marker PepC is present in multiple copies, with two sizes (Lohmann, 2006), we focused on the amplification of the larger fragment, which covers all of intron 4 and holds 85% of the informative sites (Lohmann, 2006). In total, 17 primers were initially developed (Table 1).
DNA amplification, cloning, and sequencing–PCR conditions were optimized using a common 25 µL master mix containing the following ingredients: 5 µL of 5× buffer, 2.5 µL of MgCl2 (25 mM), 1 µL of dNTP (10 mM), 0.5 µL of bovine serum albumin (BSA; New England Biolabs, Ipswich, Massachusetts, USA), 0.5 µL of each primer (10 µM), 1 unit of GoTaq Hot Start Polymerase (Promega Corporation, Madison, Wisconsin, USA), and 1 µL of genomic DNA. For the PepC mix, 0.25 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri, USA) was also added. A standard PCR program was implemented as follows: one initial step at 95°C for 5 min; 40 cycles at 95°C for 30 s, 48–56°C for 30 s, and 72°C for 30 s to 2 min; and a final step at 72°C for 10 min. The specific annealing temperature and elongation time for each primer pair is presented in Table 2.
The optimized PCR conditions were applied using the common mix. For ndhF and rpl32-trnL, products were purified by adding 1.5 µL of Illustra ExoStar (GE Healthcare Life Sciences, Buckinghamshire, United Kingdom) and submitting the samples to the thermal treatment as indicated by the manufacturer, with an additional step of 62°C for 15 min to renature the DNA. For PepC, PCR products were purified with the Illustra GFX purification kit (GE Healthcare Life Sciences), due to the presence of primer dimer, and then used in a ligation protocol with pGEM Easy Vector System (Promega Corporation). JM109 Competent E. coli cells (Promega Corporation) were used for the heat-shock transformation protocol. After incubation, transformant colonies were resuspended in 10 µL of 0.5× TE buffer and boiled for 10 min in a thermocycler. Up to four colonies were amplified using M13 primers and the common mix adjusted to a final volume of 10 µL. These amplifications used an initial step of 95°C for 5 min; 30 cycles of 95°C for 45 s, 53°C for 1 min, and 72°C for 90 s; and a final step of 72°C for 10 min. PCR products were purified with 0.7 µL of Illustra ExoStar (GE Healthcare Life Sciences).
Primer sequences used and/or developed to amplify and sequence selected loci for Bignonieae.
All samples were sequenced at Macrogen (Seoul, Korea), assembled in Geneious 5.4, and deposited in GenBank (Appendix 1). Annotations for ndhF and PepC follow Lohmann (2006), and those for newly generated sequences of rpl32-trnL were established using the complete plastid genomes of Nicotiana sylvestris Speg. & S. Comes (NC_007500) and Olea europaea L. (NC_013707). All cloned sequences were screened for vector contamination by comparison with the UniVec Database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html) prior to submission to GenBank. Species names follow Lohmann and Taylor (in press).
The three selected loci (ndhF, PepC, and rpl32-trnL) were successfully amplified from herbarium materials using the newly developed primer sets and proposed protocols. High-quality DNA sequences were obtained for most samples (55 of 62 sequenced fragments). In the rare cases in which low-quality sequences were generated, additional PCR optimizations were conducted, none of which led to higher-quality products. In those cases, a second PCR, using 0.5 µL of the unpurified product from the first PCR as template and the same PCR program, was adopted, leading to higher-quality products. With these optimizations, all fragments were successfully amplified and also led to high-quality sequences for ndhF and PepC. For the rpl32-trnL marker, the presence of two homopolymeric regions (polyA or polyT) was responsible for DNA polymerase slippage. As a result, low-quality sequences were seldom obtained immediately after this region (three of 13 sequences). To produce fully double-covered sequences, four primers (146R, 241F, 619R, and 682F; Table 1) were designed flanking the homopolymers. After these adjustments, high-quality sequences were produced for all samples. This protocol is already being used to reconstruct generic-level phytogenies in Bignonieae and has proved to be highly efficient in all of the genera it has been tested on (Zuntini and Lohmann, in prep.; Fonseca and Lohmann, in prep.; Medeiros and Lohmann, in prep.; Calió, Winkworth, and Lohmann, in prep.).
Optimized PCR conditions used in this study.
The 21 new primers here proposed, combined with the eight previously available primers (Fig. 1) and optimized protocols, led to high-quality sequences for the three selected molecular markers (ndhF, PepC, and rpl32-trnL). Those results demonstrate that herbarium materials can provide an excellent source of information for molecular phylogenetic studies in the plant family Bignoniaceae. These primers are now being used to obtain a comprehensive phylogeny for the whole tribe (Lohmann et al., in prep.). Given that the primers designed here were positioned in conserved regions, we believe that those primers will also yield high-quality sequences in other clades of the Bignoniaceae and other closely related families.
 The authors thank the Fundação de Amparo à Pesquisa do Estado da São Paulo (FAPESP) for scholarships to A.R.Z. (2011/09160-5) and L.H.M.F. (2009/12508-3), a regular research grant to L.G.L. (2011/50859-2), and a collaborative research grant between NSF-FAPESP (2012/50260-6). We also thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a Pq-2 grant to L.G.L. We are extremely grateful to the Missouri Botanical Garden for authorizing DNA extraction from selected specimens.