Wind-dispersed seeds blur phylogeographic breaks: The complex evolutionary history of Populus lasiocarpa around the Sichuan Basin.

The strength of phylogeographic breaks can vary among species in the same area despite being subject to the same geological and climate history due to differences in biological traits. Several important phylogeographic breaks exist around the Sichuan Basin in Southwest China but few studies have focused on wind-dispersed plants. Here, we investigated the phylogeographic patterns and the evolutionary history of Populus lasiocarpa, a wind-pollinated and wind-dispersed tree species with a circum-Sichuan Basin distribution in southwest China. We sequenced and analyzed three plastid DNA fragments (ptDNA) and eight nuclear microsatellites (nSSRs) of 265 individuals of P. lasiocarpa from 21 populations spanning the entire distribution range. Distribution patterns based on nSSR data revealed that there are three genetic groups in P. lasiocarpa. This is consistent with the three phylogeographic breaks (Sichuan Basin, the Kaiyong Line and the 105°E line), where the Sichuan basin acts as the main barrier to gene flow between western and eastern groups. However, the distribution pattern based on ptDNA haplotypes poorly matched the phylogeographic breaks, and wind-dispersed seeds may be one of the main contributing factors. Species distribution modelling suggested a larger potential distribution in the last glacial maximum with a severe bottleneck during the last interglacial. A DIYABC model also suggested a population contraction and expansion for both western and eastern lineages. These results indicate that biological traits are likely to affect the evolutionary history of plants, and that nuclear molecular markers, which experience higher levels of gene flow, might be better indicators of phylogeographic breaks.

De novo genome assembly of the medicinal plant Gentiana macrophylla provides insights into the genomic evolution and biosynthesis of iridoids.

Gentiana macrophylla is a perennial herb in the Gentianaceae family, whose dried roots are used in traditional Chinese medicine. Here, we assembled a chromosome-level genome of G. macrophylla using a combination of Nanopore, Illumina, and Hi-C scaffolding approaches. The final genome size was ~1.79 Gb (contig N50 = 720.804 kb), and 98.89% of the genome sequences were anchored on 13 pseudochromosomes (scaffold N50 = 122.73 Mb). The genome contained 55,337 protein-coding genes, and 73.47% of the assemblies were repetitive sequences. Genome evolution analysis indicated that G. macrophylla underwent two rounds of whole-genome duplication after the core eudicot γ genome triplication event. We further identified candidate genes related to the biosynthesis of iridoids, and the corresponding gene families mostly expanded in G. macrophylla. In addition, we found that root-specific genes are enriched in pathways involved in defense responses, which may greatly improve the biological adaptability of G. macrophylla. Phylogenomic analyses showed a sister relationship of asterids and rosids, and all Gentianales species formed a monophyletic group. Our study contributes to the understanding of genome evolution and active component biosynthesis in G. macrophylla and provides important genomic resource for the genetic improvement and breeding of G. macrophylla.

Reproduction and genetic diversity of Juniperus squamata along an elevational gradient in the Hengduan Mountains.

Elevation plays a crucial factor in the distribution of plants, as environmental conditions become increasingly harsh at higher elevations. Previous studies have mainly focused on the effects of large-scale elevational gradients on plants, with little attention on the impact of smaller-scale gradients. In this study we used 14 microsatellite loci to survey the genetic structure of 332 Juniperus squamata plants along elevation gradient from two sites in the Hengduan Mountains. We found that the genetic structure (single, clonal, mosaic) of J. squamata shrubs is affected by differences in elevational gradients of only 150 m. Shrubs in the mid-elevation plots rarely have a clonal or mosaic structure compared to shrubs in lower- or higher-elevation plots. Human activity can significantly affect genetic structure, as well as reproductive strategy and genetic diversity. Sub-populations at mid-elevations had the highest yield of seed cones, lower levels of asexual reproduction and higher levels of genetic diversity. This may be due to the trade-off between elevational stress and anthropogenic disturbance at mid-elevation since there is greater elevational stress at higher-elevations and greater intensity of anthropogenic disturbance at lower-elevations. Our findings provide new insights into the finer scale genetic structure of alpine shrubs, which may improve the conservation and management of shrublands, a major vegetation type on the Hengduan Mountains and the Qinghai-Tibet Plateau.

Transcriptome profiles of yellowish-white and fuchsia colored flowers in the Rheum palmatum complex reveal genes related to color polymorphism.

Flower color variation is ubiquitous in many plant species, and several studies have been conducted to elucidate the underlying molecular mechanism. There are two flower color variants (yellowish-white and fuchsia) in the Rheum palmatum complex, however, few studies have investigated this phenomenon. Here, we used transcriptome sequencing of the two color variants to shed light on the molecular and biochemical basis for these color morphs. Comparison of the two transcriptomes identified 9641 differentially expressed unigenes (DEGs), including 6477 up-regulated and 3163 down-regulated genes. Functional analyses indicated that several DEGs were related to the anthocyanin biosynthesis pathway, and the expression profiles of these DEGs were coincident with the qRT-PCR validation results, indicating that expression levels of structural genes have a profound effect on the color variation in the R. palmatum complex. Our results suggested that the interaction of transcription factors (MYB, bHLH and WRKY) also regulated the anthocyanin biosynthesis in the R. palmatum complex. Estimation of selection pressures using the dN/dS ratio showed that 1106 pairs of orthologous genes have undergone positive selection. Of these positively selected genes, 21 were involved in the anthocyanin biosynthetic pathway, indicating that they may encode the proteins for structural alteration and affect flower color in the R. palmatum complex.

A nucleotide signature for the identification of Pinelliae Rhizoma (Banxia) and its products.

BACKGROUND: Ensuring the authenticity of raw materials is a key step prior to producing Chinese patent medicines. Pinellia ternata (Thunb.) Breit. is the botanical origin of Pinelliae Rhizoma (Banxia), a traditional Chinese medicine used to treat cough, insomnia, nausea, inflammation, epilepsy, and so on. Unfortunately, authentic Pinelliae Rhizoma is often adulterated by morphologically indistinguishable plant material due to the insufficient regulatory procedures of processed medicinal plant products. Thus, it is important to develop a molecular assay based on species-specific nucleotide signatures and primers to efficiently distinguish authentic Pinelliae Rhizoma from its adulterants. METHODS AND RESULTS: The ITS2 region of 67 Pinelliae Rhizoma and its common adulterants were sequenced. Eight single nucleotide polymorphisms within a 28-43 bp stretch of ITS2 were used to develop six primer pairs to amplify these species-specific regions. We assayed 56 Pinelliae Rhizoma products sold on the Chinese market, including medicinal slices, powder and Chinese patent medicines, which revealed that about 66% of products were adulterated. The most common adulterants were Pinellia pedatisecta (found in 57% of the assayed products), Arisaema erubescens (9%), Typhonium giganteum (2%) and Typhonium flagelliforme (2%). CONCLUSIONS: A severe adulteration condition was revealed in the traditional medicine market. The species-specific nucleotide assays developed in this study can be applied to reliably identify Pinelliae Rhizoma and its adulterants, aiding in the authentication and quality control of processed products on the herbal market.

Incomplete lineage sorting and local extinction shaped the complex evolutionary history of the Paleogene relict conifer genus, Chamaecyparis (Cupressaceae).

Inferring accurate biogeographic history of plant taxa with an East Asia (EA)-North America (NA) is usually hindered by conflicting phylogenies and a poor fossil record. The current distribution of Chamaecyparis (false cypress; Cupressaceae) with four species in EA, and one each in western and eastern NA, and its relatively rich fossil record, make it an excellent model for studying the EA-NA disjunction. Here we reconstruct phylogenomic relationships within Chamaecyparis using > 1400 homologous nuclear and 61 plastid genes. Our phylogenomic analyses using concatenated and coalescent approaches revealed strong cytonuclear discordance and conflicting topologies between nuclear gene trees. Incomplete lineage sorting (ILS) and hybridization are possible explanations of conflict; however, our coalescent analyses and simulations suggest that ILS is the major contributor to the observed phylogenetic discrepancies. Based on a well-resolved species tree and four fossil calibrations, the crown lineage of Chamaecyparis is estimated to have originated in the upper Cretaceous, followed by diversification events in the early and middle Paleogene. Ancestral area reconstructions suggest that Chamaecyparis had an ancestral range spanning both EA and NA. Fossil records further indicate that this genus is a relict of the "boreotropical" flora, and that local extinctions of European species were caused by global cooling. Overall, our results unravel a complex evolutionary history of a Paleogene relict conifer genus, which may have involved ILS, hybridization and the extinction of local species.

Seeing through the hedge: Phylogenomics of Thuja (Cupressaceae) reveals prominent incomplete lineage sorting and ancient introgression for Tertiary relict flora.

The Eastern Asia (EA) - North America (NA) disjunction is a well-known biogeographic pattern of the Tertiary relict flora; however, few studies have investigated the evolutionary history of this disjunction using a phylogenomic approach. Here, we used 2369 single copy nuclear genes and nearly full plastomes to reconstruct the evolutionary history of the small Tertiary relict genus Thuja, which consists of five disjunctly distributed species. The nuclear species tree strongly supported an EA clade Thuja standishii-Thuja sutchuenensis and a "disjunct clade", where western NA species T. plicata is sister to an EA-eastern NA disjunct Thuja occidentalis-Thuja koraiensis group. Our results suggested that the observed topological discordance among the gene trees as well as the cytonuclear discordance is mainly due to incomplete lineage sorting, probably facilitated by the fast diversification of Thuja around the Early Miocene and the large effective population sizes of ancestral lineages. Furthermore, approximately 20% of the T. sutchuenensis nuclear genome is derived from an unknown ancestral lineage of Thuja, which might explain the close resemblance of its cone morphology to that of an ancient fossil species. Overall, our study demonstrates that single genes may not resolve interspecific relationships for disjunct taxa, and that more reliable results will come from hundreds or thousands of loci, revealing a more complex evolutionary history. This will steadily improve our understanding of their origin and evolution.

The genome sequence of the European crab apple, Malus sylvestris (L.) Mill., 1768.

We present a genome assembly from an individual Malus sylvestris (the European or 'wild' crab apple; Streptophyta; Magnoliopsida; Rosales; Rosaceae). The genome sequence is 642 megabases in span. Most of the assembly (99.98%) is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial and chloroplast genomes were also assembled, with respective lengths of 396.9 kilobases and 160.0 kilobases.

Evolutionary history of two rare endemic conifer species from the eastern Qinghai-Tibet Plateau.

BACKGROUND AND AIMS: Understanding the population genetics and evolutionary history of endangered species is urgently needed in an era of accelerated biodiversity loss. This knowledge is most important for regions with high endemism that are ecologically vulnerable, such as the Qinghai-Tibet Plateau (QTP). METHODS: The genetic variation of 84 juniper trees from six populations of Juniperus microsperma and one population of Juniperus erectopatens, two narrow-endemic junipers from the QTP that are sister to each other, was surveyed using RNA-sequencing data. Coalescent-based analyses were used to test speciation, migration and demographic scenarios. Furthermore, positively selected and climate-associated genes were identified, and the genetic load was assessed for both species. KEY RESULTS: Analyses of 149 052 single nucleotide polymorphisms showed that the two species are well differentiated and monophyletic. They diverged around the late Pliocene, but interspecific gene flow continued until the Last Glacial Maximum. Demographic reconstruction by Stairway Plot detected two severe bottlenecks for J. microsperma but only one for J. erectopatens. The identified positively selected genes and climate-associated genes revealed habitat adaptation of the two species. Furthermore, although J. microsperma had a much wider geographical distribution than J. erectopatens, the former possesses lower genetic diversity and a higher genetic load than the latter. CONCLUSIONS: This study sheds light on the evolution of two endemic juniper species from the QTP and their responses to Quaternary climate fluctuations. Our findings emphasize the importance of speciation and demographic history reconstructions in understanding the current distribution pattern and genetic diversity of threatened species in mountainous regions.

Using demographic model selection to untangle allopatric divergence and diversification mechanisms in the Rheum palmatum complex in the Eastern Asiatic Region.

Allopatric divergence is often initiated by geological uplift and restriction to sky-islands, climate oscillations, or river capture. However, it can be difficult to establish which mechanism was the most likely to generate the current phylogeographical structure of a species. Recently, genomic data in conjunction with a model testing framework have been applied to address this issue in animals. To test whether such an approach is also likely to be successful in plants, we used population genomic data of the Rheum palmatum complex from the Eastern Asiatic Region, in conjunction with biogeographical reconstruction and demographic model selection, to identify the potential mechanism(s) which have led to the current level of divergence. Our results indicate that the R. palmatum complex originated in the central Hengduan Mts and possibly in regions further east, and then dispersed westward and eastward resulting in genetically distinct lineages. Populations are likely to have diverged in refugia during climate oscillations followed by subsequent expansion and secondary contact. However, model simulations within the western lineage of the R. palmatum complex cannot reject a restriction to sky-islands as a possible mechanism of diversification due to the genetically ambiguous position of one population. This highlights that genetically mixed populations might introduce ambiguity regarding the best diversification model in some cases. Although it might be possible to resolve this ambiguity using other data, sometimes this could prove to be difficult in complex biogeographical areas.

Development of chloroplast microsatellite markers for Glyptostrobus pensilis (Cupressaceae).

PREMISE: Glyptostrobus pensilis (Cupressaceae) is a critically endangered conifer native to China, Laos, and Vietnam, with only a few populations remaining in the wild. METHODS AND RESULTS: Using a complete chloroplast genome sequence, we designed 70 cpSSR loci and tested them for amplification success and polymorphism in 16 samples. Ten loci were found to be polymorphic and their genetic diversity was characterized using a total of 83 individuals from three populations in China. A total of 43 haplotypes were present, the effective number of haplotypes varied from 4.55 to 13.36, and the haplotypic richness ranged from 8.04 to 16.00. Gene diversity ranged from 0.81 to 0.97 (average 0.89). The number of alleles per locus and population ranged from one to eight, and the effective number of alleles ranged from 1.00 to 3.90. All polymorphic loci were successfully amplified in the related species Cryptomeria japonica var. sinensis, Taxodium distichum, T. ascendens, and Cunninghamia lanceolata. CONCLUSIONS: These newly developed chloroplast microsatellites will be useful for population genetic and phylogeographic analyses of G. pensilis and related species.

The Complete Chloroplast Genome of Euphrasia regelii, Pseudogenization of ndh Genes and the Phylogenetic Relationships Within Orobanchaceae.

Euphrasia (Orobanchaceae) is a genus which is widely distributed in temperate regions of the southern and northern hemisphere. The taxonomy of Euphrasia is still controversial due to the similarity of morphological characters and a lack of genomic resources. Here, we present the first complete chloroplast (cp) genome of this taxonomically challenging genus. The cp genome of Euphrasia regelii consists of 153,026 bp, including a large single-copy region (83,893 bp), a small single-copy region (15,801 bp) and two inverted repeats (26,666 bp). There are 105 unique genes, including 71 protein-coding genes, 30 tRNA and 4 rRNA genes. Although the structure and gene order is comparable to the one in other angiosperm cp genomes, genes encoding the NAD(P)H dehydrogenase complex are widely pseudogenized due to mutations resulting in frameshifts, and stop codon positions. We detected 36 dispersed repeats, 7 tandem repeats and 65 simple sequence repeat loci in the E. regelii plastome. Comparative analyses indicated that the cp genome of E. regelii is more conserved compared to other hemiparasitic taxa in the Pedicularideae and Buchnereae. No structural rearrangements or loss of genes were detected. Our analyses suggested that three genes (clpP, ycf2 and rps14) were under positive selection and other genes under purifying selection. Phylogenetic analysis of monophyletic Orobanchaceae based on 45 plastomes indicated a close relationship between E. regelii and Neobartsia inaequalis. In addition, autotrophic lineages occupied the earliest diverging branches in our phylogeny, suggesting that autotrophy is the ancestral trait in this parasitic family.

A transcriptome-based resolution for a key taxonomic controversy in Cupressaceae.

Background and Aims: Rapid evolutionary divergence and reticulate evolution may result in phylogenetic relationships that are difficult to resolve using small nucleotide sequence data sets. Next-generation sequencing methods can generate larger data sets that are better suited to solving these puzzles. One major and long-standing controversy in conifers concerns generic relationships within the subfamily Cupressoideae (105 species, approx. 1/6 of all conifers) of Cupressaceae, in particular the relationship between Juniperus, Cupressus and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) clade. Here we attempt to resolve this question using transcriptome-derived data. Methods: Transcriptome sequences of 20 species from Cupressoideae were collected. Using MarkerMiner, single-copy nuclear (SCN) genes were extracted. These were applied to estimate phylogenies based on concatenated data, species trees and a phylogenetic network. We further examined the effect of alternative backbone topologies on downstream analyses, including biogeographic inference and dating analysis. Results: Based on the 73 SCN genes (>200 000 bp total alignment length) we considered, all tree-building methods lent strong support for the relationship (HCX, (Juniperus, Cupressus)); however, strongly supported conflicts among individual gene trees were also detected. Molecular dating suggests that these three lineages shared a most recent common ancestor approx. 60 million years ago (Mya), and that Juniperus and Cupressus diverged about 56 Mya. Ancestral area reconstructions (AARs) suggest an Asian origin for the entire clade, with subsequent dispersal to North America, Europe and Africa. Conclusions: Our analysis of SCN genes resolves a controversial phylogenetic relationship in the Cupressoideae, a major clade of conifers, and suggests that rapid evolutionary divergence and incomplete lineage sorting probably acted together as the source for conflicting phylogenetic inferences between gene trees and between our robust results and recently published studies. Our updated backbone topology has not substantially altered molecular dating estimates relative to previous studies; however, application of the latest AAR approaches has yielded a clearer picture of the biogeographic history of Cupressoideae.

Genetic and chemical differentiation characterizes top-geoherb and non-top-geoherb areas in the TCM herb rhubarb.

Medicinal herbs of high quality and with significant clinical effects have been designated as top-geoherbs in traditional Chinese medicine (TCM). However, the validity of this concept using genetic markers has not been widely tested. In this study, we investigated the genetic variation within the Rheum palmatum complex (rhubarb), an important herbal remedy in TCM, using a phylogeographic (six chloroplast DNA regions, five nuclear DNA regions, and 14 nuclear microsatellite loci) and a chemical approach (anthraquinone content). Genetic and chemical data identified two distinct groups in the 38 analysed populations from the R. palmatum complex which geographically coincide with the traditional top-geoherb and non-top-geoherb areas of rhubarb. Molecular dating suggests that the two groups diverged in the Quaternary c. 2.0 million years ago, a time of repeated climate changes and uplift of the Qinghai-Tibetan Plateau. Our results show that the ancient TCM concept of top-geoherb and non-top-geoherb areas corresponds to genetically and chemically differentiated groups in rhubarb.

DNA barcoding a taxonomically complex hemiparasitic genus reveals deep divergence between ploidy levels but lack of species-level resolution.

DNA barcoding is emerging as a useful tool not only for species identification but also for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode data sets can provide insights into the mechanisms underlying the generation of species diversity. Here, we study evolutionary processes in taxonomically complex British Euphrasia (Orobanchaceae), a group with multiple ploidy levels, frequent self-fertilization, young species divergence and widespread hybridization. We use a phylogenetic approach to investigate the colonization history of British Euphrasia, followed by a DNA barcoding survey and population genetic analyses to reveal the causes of shared sequence variation. Phylogenetic analysis shows Euphrasia have colonized Britain from mainland Europe on multiple occasions. DNA barcoding reveals that no British Euphrasia species has a consistent diagnostic sequence profile, and instead, plastid haplotypes are either widespread across species, or are population specific. The partitioning of nuclear genetic variation suggests differences in ploidy act as a barrier to gene exchange, while the divergence between diploid and tetraploid ITS sequences supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, analyses of DNA barcoding data can reveal evolutionary patterns in taxonomically complex genera.

Authentication of Eleutherococcus and Rhodiola herbal supplement products in the United Kingdom.

Siberian ginseng (Eleutherococcus senticosus, Araliaceae) and roseroot (Rhodiola rosea, Rosaceae) are popular herbal supplements which have been shown to improve resilience to conditions such as stress and exhaustion. Using DNA barcoding methods we tested 25 Siberian ginseng and 14 roseroot products which are widely available to UK customers to test whether the herbal ingredient stated on the label is also in the product. All Siberian ginseng supplements contained E. senticosus, however, 36% also contained an Eleutherococcus species other than E. senticosus. In three out of the 13 roseroot products which produced amplifiable DNA, we could only retrieve sequences matching alfalfa (declared on the product label) and fenugreek (not declared). In the other 10 supplements Rhodiola was detected but only five matched the target species R. rosea. As DNA can get severely degraded during the manufacturing process we did not take the absence of Rhodiola DNA as proof for a compromised product. Contamination could explain the presence of non-target species such as fenugreek but is unlikely to be account for the detection of congeneric Rhodiola species in roseroot preparations. Our results therefore suggest that the substitution or mixing of the target medicinal ingredient in these two popular supplements with other species is common.

Hidden in plain view: Cryptic diversity in the emblematic Araucaria of New Caledonia.

PREMISE OF THE STUDY: Cryptic species represent a conservation challenge, because distributions and threats cannot be accurately assessed until species are recognized and defined. Cryptic species are common in diminutive and morphologically simple organisms, but are rare in charismatic and/or highly visible groups such as conifers. New Caledonia, a small island in the southern Pacific is a hotspot of diversity for the emblematic conifer genus Araucaria (Araucariaceae, Monkey Puzzle trees) where 13 of the 19 recognized species are endemic. METHODS: We sampled across the entire geographical distribution of two closely related species (Araucaria rulei and A. muelleri) and screened them for genetic variation at 12 nuclear and 14 plastid microsatellites and one plastid minisatellite; a subset of the samples was also examined using leaf morphometrics. KEY RESULTS: The genetic data show that populations of the endangered A. muelleri fall into two clearly distinct genetic groups: one corresponding to montane populations, the other corresponding to trees from lower elevation populations from around the Goro plateau. These Goro plateau populations are more closely related to A. rulei, but are sufficiently genetically and morphological distinct to warrant recognition as a new species. CONCLUSIONS: Our study shows the presence of a previously unrecognized species in this flagship group, and that A. muelleri has 30% fewer individuals than previously thought. Combined, this clarification of species diversity and distributions provides important information to aid conservation planning for New Caledonian Araucaria.

The evolutionary history of ferns inferred from 25 low-copy nuclear genes.

UNLABELLED: • PREMISE OF THE STUDY: Understanding fern (monilophyte) phylogeny and its evolutionary timescale is critical for broad investigations of the evolution of land plants, and for providing the point of comparison necessary for studying the evolution of the fern sister group, seed plants. Molecular phylogenetic investigations have revolutionized our understanding of fern phylogeny, however, to date, these studies have relied almost exclusively on plastid data.• METHODS: Here we take a curated phylogenomics approach to infer the first broad fern phylogeny from multiple nuclear loci, by combining broad taxon sampling (73 ferns and 12 outgroup species) with focused character sampling (25 loci comprising 35877 bp), along with rigorous alignment, orthology inference and model selection.• KEY RESULTS: Our phylogeny corroborates some earlier inferences and provides novel insights; in particular, we find strong support for Equisetales as sister to the rest of ferns, Marattiales as sister to leptosporangiate ferns, and Dennstaedtiaceae as sister to the eupolypods. Our divergence-time analyses reveal that divergences among the extant fern orders all occurred prior to ∼200 MYA. Finally, our species-tree inferences are congruent with analyses of concatenated data, but generally with lower support. Those cases where species-tree support values are higher than expected involve relationships that have been supported by smaller plastid datasets, suggesting that deep coalescence may be reducing support from the concatenated nuclear data.• CONCLUSIONS: Our study demonstrates the utility of a curated phylogenomics approach to inferring fern phylogeny, and highlights the need to consider underlying data characteristics, along with data quantity, in phylogenetic studies.

Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy-to-access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.