DNA barcode identification of Podocarpaceae--the second largest conifer family

Abstract

We have generated matK, rbcL, and nrITS2 DNA barcodes for 320 specimens representing all 18 extant genera of the conifer family Podocarpaceae. The sample includes 145 of the 198 recognized species. Comparative analyses of sequence quality and species discrimination were conducted on the 159 individuals from which all three markers were recovered (representing 15 genera and 97 species). The vast majority of sequences were of high quality (B 30 = 0.596-0.989). Even the lowest quality sequences exceeded the minimum requirements of the BARCODE data standard. In the few instances that low quality sequences were generated, the responsible mechanism could not be discerned. There were no statistically significant differences in the discriminatory power of markers or marker combinations (p = 0.05). The discriminatory power of the barcode markers individually and in combination is low (56.7% of species at maximum). In some instances, species discrimination failed in spite of ostensibly useful variation being present (genotypes were shared among species), but in many cases there was simply an absence of sequence variation. Barcode gaps (maximum intraspecific p-distance > minimum interspecific p-distance) were observed in 50.5% of species when all three markers were considered simultaneously. The presence of a barcode gap was not predictive of discrimination success (p = 0.02) and there was no statistically significant difference in the frequency of barcode gaps among markers (p = 0.05). In addition, there was no correlation between number of individuals sampled per species and the presence of a barcode gap (p = 0.27).

Figure 1. Phylogenetic relationships among complete samples.

Strict consensus of 3600 most parsimonious trees (L = 1205; CI = 0.59; RI = 0.93; all tree statistics exclude uninformative nucleotide positions) obtained from the simultaneous analysis of matK, rbcL, and nrITS2 sequence data. Numbers at nodes indicated jackknife support above 50%. Species that can be distinguished from all other species using the ‘least inclusive clade’ method are in boldface (the least inclusive clade method cannot be applied to species with only one sample). Genera have been abbreviated: Ac.  =  Acmopyle, Af.  =  Afrocarpus, Dc.  =  Dacrycarpus, Dd.  =  Dacrydium, F.  =  Falcatifolium, La.  =  Lagarostrobos, Le.  =  Lepidothamnus, Ma.  =  Manoao, Mi.  =  Microcachrys, N.  =  Nageia, Ph.  =  Pherosphaera, Po.  =  Podocarpus, Pr.  =  Prumnopitys, R.  =  Retrophyllum, and S.  =  Saxegothaea. Sample codes correspond to those used in Dataset S1.

Figure 2. Barcode sequence quality (B₃₀) versus linguistic complexity (LC) for complete samples.

Circles represent individual matK (blue; m), rbcL (red; r), and nrITS2 (yellow; i) sequences. Black squares indicate marker means. Error bars span three standard deviations.

Figure 3. Species discrimination by barcode marker for complete samples.

Squares indicate means for matK (blue; m), rbcL (red; r), nrITS2 (yellow; i), matK combined with rbcL (purple; mr), matK combined with nrITS2 (green; mi), rbcL combined with nrITS2 (orange, ri), and all markers combined (black; mri). Error bars indicate 95% confidence intervals.

Figure 4. Barcode variation within and among species for complete samples.

Circles represent the set of matK (blue), rbcL (red), and nrITS2 (yellow) sequences for each species. Opaque filled circles denote diagnostic sequence sets. Non–diagnostic sequence sets are indicated with semi–transparent filled circles. Equal intra– and inter–specific variation is marked by the gray line. Points above the gray line indicate species with ‘barcode gaps’.