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.

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.

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.