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 four 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.