Pinaceae Barcodes
We evaluated seven chloroplast DNA regions for their suitability as genetic barcodes. The markers were compared across and within six families represented in Mexico: Agavaceae, Cactaceae, Cupressaceae, Crassulaceae, Orchidaceae, and Pinaceae. This page describes the results in Pinaceae.
Methods
All five Mexican Pinaceae genera were represented: Pinus, Picea, Pseudotsuga and Abies.
Table 1. Taxonomic sampling for experiment 1-1
| Taxon | Collection Number |
Locality Data |
| Abies hidalgensis |
DSG625 |
Hidalgo, Los
Mármoles |
| Pseudotsuga menziesii |
MGL1822 |
Hidalgo, El Chico |
| Picea chihuahuana |
DSG1071 |
Chihuahua,
Bocoyna |
| Pinus radiata |
DOB1045 |
Baja California,
Isla Guadalupe |
| Pinus nelsonii |
DSG1095 |
San Luis Potosi,
Guadalcazar |
Table 2. Primers used for amplifying cpDNA in Pinaceae.
| Region |
Primer Name |
Sequence (5' to 3') |
Source |
| rbcL |
rbcLA-F |
ATG TCA CCA CAA
ACA GAG ACT AAA GC |
Kress &
Erickson (2007) |
| rbcLajf634-R | GAA ACG GTC TCT
CCA ACG CAT |
Fazekas et al.
(2008) |
|
| matK | Equisetum-F |
ATA CCC CAT TTT
ATT CAT CC |
RBG Kew |
| matK2F-R | CGT ACT TTT ATG TTT ACA GGC TAA | Wang et al. (1999) | |
| trnH-psbA |
trnH |
CGC GCA TGG TGG
ATT CAC AAT CC |
Tate &
Simpson (2003) |
| psbA |
GTT ATG CAT GAA
CGT AAT GCTC |
Tate & Simpson (2003) | |
| trnL-trnF |
TabC |
CGA AAT CGG TAG
ACG CTA CG |
Taberlet et al.
(1991) |
| TabF |
ATT TGA ACT GGT
GAC ACG AG |
Taberlet et al. (1991) | |
| psbK-psbI |
psbK |
TTA GCC TTT GTT
TGG CAA G |
Lahaye et al.
(2008) |
| psbI |
AGA GTT TGA GAG
TAA GCA T |
Lahaye et al. (2008) | |
| trnS-trnG |
trnS |
AGA TAG GGA TTC
GAA CCC TCG GT |
Shaw et al.
(2005) |
| trnG |
GTA GCG GGA ATC
GAA CCC GCA TC |
Shaw et al. (2005) | |
| rpL32-trnL |
rpl32-F |
CAG TTC CAA AAA
AAC GTA CTT C |
Shaw et al. (2007) |
| trnL |
CTG CTT CCT AAG
AGC AGC GT |
Shaw et al. (2007) |
Results
Amplification and sequencing success in Pinaceae
Six of seven regions were amplified in Pinaceae. Only rpL32-trnL failed in most samples. The highest amplification success was achieved for rbcL, followed closely by psbK-psbI, trnL-trnF, and matK. Amplification problems were encountered for some taxa with trnH-psbA and trnS-trnG. Amplificaton of matK in Pinaceae required a different primer set than that used for angiosperms, while all other regions were amplified with the same primers.
High quality sequence reads of the complete amplicon were obtained most consistently for rbcL and psbK-psbI, followed by trnH-psbA. Sequence reads obtained >90% contig overlap for matK and trnS-trnG. Lower overlap was obtained for trnL-trnF. Failure of sequencing reactions was highest in rpL32-trnL, although failures also occurred in trnH-psbA. Sequence reads in trnL-trnF were typically of low quality because of a long poly-A/T simple sequence repeat.
The markers were compared in five phylogenetically divergent species. The spacers had the highest number of variable sites; trnS-trnG (233 sites) and trnH-psbA (167 sites) had the most. The least variable spacer was psbK-psbI (104 sites). The least variable region was the rbcL gene fragment (39 sites)
Table 3. Results of Experiment 1. Variation in six regions as measured in five Pinaceae taxa.
| region |
length (b.p.) |
aligned length (b.p.) | variable sites |
| rbcL
|
553 |
553 |
39 |
| matK | 837 |
837 |
109 |
| trnH-psbA |
505-636 |
650 |
167 |
| trnL-trnF |
864-916 |
953 |
152 |
| psbI-psbK |
506-512 |
525 |
104 |
| trnS-trnG |
735-910 |
1021 |
233 |
Experiment 1-2
An issue of great practical importance is the ability of DNA barcodes to distinguish closely related species of the same genus. Closely related species not only present low levels of sequence divergence, they are also potentially able to hybridize, or to share ancestral alleles (ones with coalescence times that predate speciation). Many studies to date have not chosen species pairs within genera that are hypothesized to be either sister species or to be more closely related (within the genus) than expected by chance. Here we used pre-existing morphological and often molecular information to chose closely related species. For experiment 1-2 in Pinaceae, two pairs of closely related species of Pinus subsection Cembroides, as inferred from morphology (Malusa 1991), a prior phylogenetic study with rbcL, matK and rpL16 (Gernandt et al. 2003; the former two also candidate barcode loci), and also consistent with phylogenies inferred with nuclear loci (Gernandt et al. 2001; Syring et al. 2007). Other species complexes in pines with lower cpDNA divergences are known in pines (Gernandt et al. 2005), but these have not yet been evaluated with candidate barcode regions.
Table 4. Results of Experiment 1-2. Variation in six regions as measured in two closely related species pairs in Pinus subsection Cembroides.
| region |
length (b.p.) | aligned length (b.p.) | variable maximartinezii versus
pinceana |
variable cembroides versus
johannis |
| rbcL |
553 |
553 |
0 |
1 |
| matK |
837 |
837 |
2 |
2 |
| trnH-psbA |
596 |
596 |
8 (inversion=6) |
1 |
| trnL-trnF |
872-876 |
877 |
2 |
0 |
| psbI-psbK |
501 |
501 |
2 |
2 |
| trnS-trnG |
663-870 |
870 |
2 |
1 |
Literature cited
CBOL Plant Working Group. 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences 106: 12794-12797.
Fazekas, A.J., K.S. Burgess, P.R. Kesanakurti, S.W. Graham, S.G. Newmaster, B.C. Husband, D.M. Percy, M. Hajibabaei, and S.C.H. Barrett. 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. 2008. PLoS ONE 3(7): e2802.
Gernandt, D.S., A. Liston, and D. Piñero. 2001. Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes. Molecular Phylogenetics and Evolution 21: 449-467.
Gernandt, D.S., A. Liston, and D. Piñero. 2003. Phylogenetic of Pinus subsection Cembroides: and Nelsoniae inferred from cpDNA sequences. Systematic Botany 28: 657-673.
Gernandt, D. S., G. Gaeda Lopez, S. Ortiz Garcia, and A. Liston. 2005. Phylogeny and classification of Pinus. Taxon 54: 29–42.
Lahaye, R., V. Savolainen, S. Duthoit, O. Maurin, and M. van der Bank. A test of psbK-psbI and atpF-atpH as potential plant DNA barcodes using the flora of the Kruger National Park as a model system (South Africa). Nature Precedings: hdl:10101/npre.2008.1896.1
Malusa, J. 1992. Phylogeny and biogeography of the pinyon pines (Pinus subsection Cembroides). Systematic Botany 17: 42-66.
Syring, J., K. Farrell, R. Businsky, R. Cronn, and A. Liston. 2007. Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus. Systematic Biology 56: 163-181.
Taberlet, P., L. Gielly, G. Pautou, and J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105–1109.