A derived ZW chromosome system in Amborella trichopoda, representing the sister lineage to all other extant flowering plants.

The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

Wide cross-species RNA-Seq comparison reveals convergent molecular mechanisms involved in nickel hyperaccumulation across dicotyledons.

The Anthropocene epoch is associated with the spreading of metals in the environment increasing oxidative and genotoxic stress on organisms. Interestingly, c. 520 plant species growing on metalliferous soils acquired the capacity to accumulate and tolerate a tremendous amount of nickel in their shoots. The wide phylogenetic distribution of these species suggests that nickel hyperaccumulation evolved multiple times independently. However, the exact nature of these mechanisms and whether they have been recruited convergently in distant species is not known. To address these questions, we have developed a cross-species RNA-Seq approach combining differential gene expression analysis and cluster of orthologous group annotation to identify genes linked to nickel hyperaccumulation in distant plant families. Our analysis reveals candidate orthologous genes encoding convergent function involved in nickel hyperaccumulation, including the biosynthesis of specialized metabolites and cell wall organization. Our data also point out that the high expression of IREG/Ferroportin transporters recurrently emerged as a mechanism involved in nickel hyperaccumulation in plants. We further provide genetic evidence in the hyperaccumulator Noccaea caerulescens for the role of the NcIREG2 transporter in nickel sequestration in vacuoles. Our results provide molecular tools to better understand the mechanisms of nickel hyperaccumulation and study their evolution in plants.

Nickel hyperaccumulation in New Caledonian Hybanthus (Violaceae) and occurrence of nickel-rich phloem in Hybanthus austrocaledonicus.

BACKGROUND AND AIMS: Hybanthus austrocaledonicus (Violaceae) is a nickel (Ni) hyperaccumulator endemic to New Caledonia. One of the specimens stored at the local herbarium had a strip of bark with a remarkably green phloem tissue attached to the sheet containing over 4 wt% Ni. This study aimed to collect field samples from the original H. austrocaledonicus locality to confirm the nature of the green 'nickel-rich phloem' in this taxon and to systematically assess the occurrence of Ni hyperaccumulation in H. austrocaledonicus and Hybanthus caledonicus populations. METHODS: X-ray fluorescence spectroscopy scanning of all collections of the genus Hybanthus (236 specimens) was undertaken at the Herbarium of New Caledonia to reveal incidences of Ni accumulation in populations of H. austrocaledonicus and H. caledonicus. In parallel, micro-analytical investigations were performed via synchrotron X-ray fluorescence microscopy (XFM) and scanning electron microscopy with X-ray microanalysis (SEM-EDS). KEY RESULTS: The extensive scanning demonstrated that Ni hyperaccumulation is not a characteristic common to all populations in the endemic Hybanthus species. Synchrotron XFM revealed that Ni was exclusively concentrated in the epidermal cells of the leaf blade and petiole, conforming with the majority of (tropical) Ni hyperaccumulator plants studied to date. SEM-EDS of freeze-dried and frozen-hydrated samples revealed the presence of dense solid deposits in the phloem bundles that contained >8 wt% nickel. CONCLUSIONS: The occurrence of extremely Ni-rich green phloem tissues appears to be a characteristic feature of tropical Ni hyperaccumulator plants.

Dioecy in Amborella trichopoda: evidence for genetically based sex determination and its consequences for inferences of the breeding system in early angiosperms.

Background and Aims: This work aimed to gain insight into the breeding system at the base of living angiosperms through both character state reconstructions and the study of sex ratios and phenotypes in the likely sister to all other living angiosperms, Amborella trichopoda . Methods: Sex phenotypes were mapped onto a phylogeny of basally diverging angiosperms using maximum parsimony. In parallel, sex ratios and phenotypes were studied over two consecutive flowering seasons in an ex situ population of A. trichopoda , while the sex ratio of an in situ population was also assessed. Key Results: Parsimony analyses failed to resolve the breeding system present at the base of living angiosperms, but indicated the importance of A. trichopoda for the future elucidation of this question. The ex situ A. trichopoda population studied showed a primary sex ratio close to 1:1, though sex ratio bias was found in the in situ population studied. Instances of sexual instability were quantified in both populations. Conclusions: Sex ratio data support the presence of genetic sex determination in A. trichopoda , whose further elucidation may guide inferences on the breeding system at the base of living angiosperms. Sexual instability in A. trichopoda suggests the operation of epigenetic mechanisms, and the evolution of dioecy via a gynodioecious intermediate.

The morphophysiological dormancy in Amborella trichopoda seeds is a pleisiomorphic trait in angiosperms.

Background and Aims: Recent parsimony-based reconstructions suggest that seeds of early angiosperms had either morphophysiological or physiological dormancy, with the former considered as more probable. The aim of this study was to determine the class of seed dormancy present in Amborella trichopoda , the sole living representative of the most basal angiosperm lineage Amborellales, with a view to resolving fully the class of dormancy present at the base of the angiosperm clade. Methods: Drupes of A. trichopoda without fleshy parts were germinated and dissected to observe their structure and embryo growth. Pre-treatments including acid scarification, gibberellin treatment and seed excision were tested to determine their influence on dormancy breakage and germination. Character-state mapping by maximum parsimony, incorporating data from the present work and published sources, was then used to determine the likely class of dormancy present in early angiosperms. Key Results: Germination in A. trichopoda requires a warm stratification period of at least approx. 90 d, which is followed by endosperm swelling, causing the water-permeable pericarp-mesocarp envelope to split open. The embryo then grows rapidly within the seed, to radicle emergence some 17 d later and cotyledon emergence after an additional 24 d. Gibberellin treatment, acid scarification and excision of seeds from the surrounding drupe tissues all promoted germination by shortening the initial phase of dormancy, prior to embryo growth. Conclusions: Seeds of A. trichopoda have non-deep simple morphophysiological dormancy, in which mechanical resistance of the pericarp-mesocarp envelope plays a key role in the initial physiological phase. Maximum parsimony analyses, including data obtained in the present work, indicate that morphophysiological dormancy is likely to be a pleisiomorphic trait in flowering plants. The significance of this conclusion for studies of early angiosperm evolution is discussed.

Agromining: farming for metals in the future?

Phytomining technology employs hyperaccumulator plants to take up metal in harvestable plant biomass. Harvesting, drying and incineration of the biomass generates a high-grade bio-ore. We propose that "agromining" (a variant of phytomining) could provide local communities with an alternative type of agriculture on degraded lands; farming not for food crops, but for metals such as nickel (Ni). However, two decades after its inception and numerous successful experiments, commercial phytomining has not yet become a reality. To build the case for the minerals industry, a large-scale demonstration is needed to identify operational risks and provide "real-life" evidence for profitability.

Phylogenomic resolution of order- and family-level monocot relationships using 602 single-copy nuclear genes and 1375 BUSCO genes.

We assess relationships among 192 species in all 12 monocot orders and 72 of 77 families, using 602 conserved single-copy (CSC) genes and 1375 benchmarking single-copy ortholog (BUSCO) genes extracted from genomic and transcriptomic datasets. Phylogenomic inferences based on these data, using both coalescent-based and supermatrix analyses, are largely congruent with the most comprehensive plastome-based analysis, and nuclear-gene phylogenomic analyses with less comprehensive taxon sampling. The strongest discordance between the plastome and nuclear gene analyses is the monophyly of a clade comprising Asparagales and Liliales in our nuclear gene analyses, versus the placement of Asparagales and Liliales as successive sister clades to the commelinids in the plastome tree. Within orders, around six of 72 families shifted positions relative to the recent plastome analysis, but four of these involve poorly supported inferred relationships in the plastome-based tree. In Poales, the nuclear data place a clade comprising Ecdeiocoleaceae+Joinvilleaceae as sister to the grasses (Poaceae); Typhaceae, (rather than Bromeliaceae) are resolved as sister to all other Poales. In Commelinales, nuclear data place Philydraceae sister to all other families rather than to a clade comprising Haemodoraceae+Pontederiaceae as seen in the plastome tree. In Liliales, nuclear data place Liliaceae sister to Smilacaceae, and Melanthiaceae are placed sister to all other Liliales except Campynemataceae. Finally, in Alismatales, nuclear data strongly place Tofieldiaceae, rather than Araceae, as sister to all the other families, providing an alternative resolution of what has been the most problematic node to resolve using plastid data, outside of those involving achlorophyllous mycoheterotrophs. As seen in numerous prior studies, the placement of orders Acorales and Alismatales as successive sister lineages to all other extant monocots. Only 21.2% of BUSCO genes were demonstrably single-copy, yet phylogenomic inferences based on BUSCO and CSC genes did not differ, and overall functional annotations of the two sets were very similar. Our analyses also reveal significant gene tree-species tree discordance despite high support values, as expected given incomplete lineage sorting (ILS) related to rapid diversification. Our study advances understanding of monocot relationships and the robustness of phylogenetic inferences based on large numbers of nuclear single-copy genes that can be obtained from transcriptomes and genomes.

Antiplasmodial activity of New Caledonia and Vanuatu traditional medicines.

CONTEXT: With the emergence of strains multiresistant to antimalarial drugs, the search for new active molecules remains a priority. Ethnopharmacology appears to be a good method of selection in such investigations. OBJECTIVE: The aim of this research work is to select plants used in Melanesian traditional medicine, in New Caledonia and Vanuatu, which should be a promising source for the isolation of new antimalarial drugs. MATERIALS AND METHODS: Forty-seven plant extracts belonging to 12 families, traditionally used by the Melanesian people or belonging to an antimalarial known genus, were screened in vitro for antimalarial activity on Plasmodium falciparum chloroquine (CQ)-resistant (FcB1) and CQ-sensitive (HB3) strains. They were also tested for their inhibitory effects on a protein kinase (Pfnek) and their cytotoxicity on human breast adenocarcinoma (MCF7) cells. RESULTS: Among all extracts, four displayed strong in vitro activities against P. falciparum: Gardenia urvillei Montrouzier, Scleria polycarpa Boeckeler, Terminalia catappa L. and Acronychia laevis J.R. & J.G. Forster, the latter being also toxic on MCF7 cells. Except for the extracts of S. polycarpa, all others that were active on P. falciparum, also possess an inhibitory effect on Pfnek. DISCUSSION AND CONCLUSION: These results confirm that ethnopharmacology is an excellent approach for such investigations. The two countries considered clearly present advantages in the field. Indeed, local populations keep their traditional knowledge alive, and their flora is exceptionally rich. In New Caledonia, the high endemicity rate (74%) ranks the island as one of the world's biodiversity hotspots. As a consequence, chances to discover new active natural compounds are also high.

The Common Fruit-Piercing Moth in the Pacific Region: A Survey of the Current State of a Significant Worldwide Economic Pest, Eudocima phalonia (Lepidoptera: Erebidae), with a Focus on New Caledonia.

When referring to fruit-piercing moths, the genus Eudocima, and more specifically Eudocima phalonia (Linneaus), is cited as a worldwide crop pest. Damages associated with this pest are substantial on more than 100 fruit species, wherever it is encountered. In New Caledonia, the once occasional pest has become a serious threat to the current fruit arboriculture. Particularly devastating during outbreak periods, it has become an urgent need to find a suitable solution able to support farmers in the ecological transition of our agricultural models. This review proposes a synthesis of the existing data and publications on E. phalonia, worldwide and especially in New Caledonia, with recent observations. The assessment of this knowledge and the dynamics of the species in the territory of New Caledonia provide key information for a better prospect of adapted solutions.

Novel Insights Into the Hyperaccumulation Syndrome in Pycnandra (Sapotaceae).

The discovery of nickel hyperaccumulation, in Pycnandra acuminata, was the start of a global quest in this fascinating phenomenon. Despite recent advances in the physiology and molecular genetics of hyperaccumulation, the mechanisms and tolerance of Ni accumulation in the most extreme example reported to date, P. acuminata, remains enigmatic. We conducted a hydroponic experiment to establish Ni tolerance levels and translocation patterns in roots and shoots of P. acuminata, and analyzed elemental partitioning to gain insights into Ni regulation. We combined a phylogeny and foliar Ni concentrations to assess the incidence of hyperaccumulation within the genus Pycnandra. Hydroponic dosing experiments revealed that P. acuminata can resist extreme Ni concentrations in solution (up to 3,000 µM), and dosing at 100 µM Ni was beneficial to growth. All plant parts were highly enriched in Ni, but the latex had extreme Ni concentrations (124,000 µg g-1). Hyperaccumulation evolved independently in only two subgenera and five species of the genus Pycnandra. The extremely high level of Ni tolerance is posited to derive from the unique properties of laticifers. The evolutionary and ecological significance of Ni hyperaccumulation in Pycnandra is discussed in light of these findings. We suggest that Ni-rich laticifers might be more widespread in the plant kingdom and that more investigation is warranted.

Evidence for the Extensive Conservation of Mechanisms of Ovule Integument Development Since the Most Recent Common Ancestor of Living Angiosperms.

The ovules and seeds of most angiosperm groups are enclosed by two integuments, whose evolutionary origins are considerably separated in time, as the inner integument arose over 300 million years ago (MYA) in an ancestor of all living seed plants, while the outer integument arose, perhaps as recently as 164 MYA, in an ancestor of all living angiosperms. Studies of the model angiosperm Arabidopsis thaliana indicate that the mechanisms of development of the inner and outer integuments depend on largely different sets of molecular players. However, it was not known, in most cases, whether these differences were already present in early flowering plants, or arose later in the Arabidopsis lineage. Here, we analyze the expression patterns of integument regulators in Amborella trichopoda, the likely sister to all other living angiosperms. The data obtained indicate that regulators of the YABBY, KANADI, and homeodomain-leucine zipper class III transcription factor families have largely conserved their integument-specific expression profiles in the Amborella and Arabidopsis lineages since the most recent common ancestor (MRCA) of living angiosperms. We identified only one case, involving the paralogous genes ETTIN and AUXIN RESPONSE FACTOR4, in which integument-specific expression patterns had clearly diverged between Amborella and Arabidopsis. We use the data obtained to partially reconstruct molecular mechanisms of integument development in the MRCA of living angiosperms and discuss our findings in the context of alternative hypotheses for the origin of the angiosperm outer integument.

"Liaisons dangereuses": The invasive red-vented bulbul (Pycnonotus cafer), a disperser of exotic plant species in New Caledonia.

The biodiversity hotspot of New Caledonia hosts high levels of endemism (74% of flora) that is threatened increasingly by climate change, habitat reduction, and invasive species. The fruit-eating red-vented bulbul (Pycnonotus cafer) is currently invading the main island of the archipelago, and its recent dispersal out of urbanized habitats raises questions about its potential to disperse noxious plant seeds along urban corridors and beyond. Indeed, the red-vented bulbul is considered a vector of several introduced plant species in its alien range including Miconia calvescens, Lantana camara, and Schinus terebinthifolius. We conducted a quantitative assessment of the bulbul's fruits consumption by analyzing the gut contents of shot birds. We estimated gut passage times for four species of fruit found in gut contents (S. terebinthifolius, Myrtastrum rufopunctatum, Passiflora suberosa, and Ficus prolixa) and tested the effects of bird digestion on seed germination rates for two species. Finally, we monitored the movements of individual VHF radio-tagged red-vented bulbuls. All of the consumed fruit species we identified here have red fleshy diaspore, including fruit of the shrub M. rufopunctatum that occurred frequently (9.6%) in bulbul gut samples. Median gut passage times were short (15-41 min), corresponding to short-distance seed transportation (77-92 m). The effect of gut passage was positive for the germination of the invasive S. terebinthifolius and negative for the endemic M. rufopunctatum, suggesting a potential bias in the contribution to the dispersal toward alien species. This study provides the first integrated assessment of mechanisms involved in the seed dispersal effectiveness of this high-concern invasive bird species that is expected to face similar plant communities in most of its alien range in tropical islands. More generally, our results enhance knowledge of synergies between non-native frugivores and plant species dispersal.

A Combination of Histological, Physiological, and Proteomic Approaches Shed Light on Seed Desiccation Tolerance of the Basal Angiosperm Amborella trichopoda.

Desiccation tolerance allows plant seeds to remain viable in a dry state for years and even centuries. To reveal potential evolutionary processes of this trait, we have conducted a shotgun proteomic analysis of isolated embryo and endosperm from mature seeds of Amborella trichopoda, an understory shrub endemic to New Caledonia that is considered to be the basal extant angiosperm. The present analysis led to the characterization of 415 and 69 proteins from the isolated embryo and endosperm tissues, respectively. The role of these proteins is discussed in terms of protein evolution and physiological properties of the rudimentary, underdeveloped, Amborella embryos, notably considering that the acquisition of desiccation tolerance corresponds to the final developmental stage of mature seeds possessing large embryos.

Application of a simplified method of chloroplast enrichment to small amounts of tissue for chloroplast genome sequencing.

PREMISE OF THE STUDY: High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. METHODS: Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. RESULTS: Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. DISCUSSION: This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species).

Pre-Gondwanan-breakup origin of Beauprea (Proteaceae) explains its historical presence in New Caledonia and New Zealand.

New Caledonia and New Zealand belong to the now largely submerged continent Zealandia. Their high levels of endemism and species richness are usually considered the result of transoceanic dispersal events followed by diversification after they re-emerged from the Pacific Ocean in the mid-Cenozoic. We explore the origin and evolutionary history of Beauprea (Proteaceae), which is now endemic to New Caledonia but was once spread throughout eastern Gondwana, including New Zealand. We review the extensive Beauprea-type pollen data in the fossil records and analyze the relationship of these fossil taxa to extant genera within Proteaceae. We further reconstruct the phylogenetic relations among nine extant species of Beauprea and estimate the age of the Beauprea clade. By incorporating extinct taxa into the Beauprea phylogenetic tree, we reconstruct the ancient distribution of this genus. Our analysis shows that Beauprea originated c. 88 Ma (million years ago) in Antarctica-Southeastern Australia and spread throughout Gondwana before its complete breakup. We propose that Beauprea, already existing as two lineages, was carried with Zealandia when it separated from the rest of Gondwana c. 82 Ma, thus supporting an autochthonous origin for Beauprea species now in New Caledonia and historically in New Zealand up to 1 Ma. We show that the presence of Beauprea through transoceanic dispersal is implausible. This means that neither New Caledonia nor New Zealand has been entirely submerged since the Upper Cretaceous; thus, possible vicariance and allopatry must be taken into account when considering the high levels of endemism and species richness of these island groups.

Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia.

Chemical study of Cunonia macrophylla, a New Caledonian Cunoniaceae, based on bioactive effects of a crude methanol extract of the leaves, detected bioactive tannins for the first time in this plant family. These ellagitannins have been identified as ellagic acid-4-O-beta-D-xylopyranoside (6), mallorepanin (3), mallotinic acid (1) along with corilagin (2), chebulagic acid (4), ellagic acid (5) and gallic acid (7) and have been shown to possess antimicrobial activity and to inhibit xanthine oxidase. Antimicrobial effects on bacterial human pathogens (Staphylococcus aureus, Corynebacterium accolans) and on a plant pathogen (Erwinia carotovora) as well as on a human pathogenic yeast (Candida albicans) were investigated. Activity is reported here for the first time for compounds 1, 3, 4 and 6. The inhibitory effects of all molecules against xanthine oxidase in relation to their structure was evaluated and compared. Compound 6 presented the best activity and seems to be of considerable interest for further studies.

Mining in New Caledonia: environmental stakes and restoration opportunities.

New Caledonia is a widely recognised marine and terrestrial biodiversity hot spot. However, this unique environment is under increasing anthropogenic pressure. Major threats are related to land cover change and include fire, urban sprawling and mining. Resulting habitat loss and fragmentation end up in serious erosion of the local biodiversity. Mining is of particular concern due to its economic significance for the island. Open cast mines were exploited there since 1873, and scraping out soil to access ores wipes out flora. Resulting perturbations on water flows and dramatic soil erosion lead to metal-rich sediment transport downstream into rivers and the lagoon. Conflicting environmental and economic aspects of mining are discussed in this paper. However, mining practices are also improving, and where impacts are inescapable ecological restoration is now considered. Past and ongoing experiences in the restoration of New Caledonian terrestrial ecosystems are presented and discussed here. Economic use of the local floristic diversity could also promote conservation and restoration, while providing alternative incomes. In this regard, Ecocatalysis, an innovative approach to make use of metal hyperaccumulating plants, is of particular interest.

Phytoextraction from mine spoils: insights from New Caledonia.

Increasing pressure on mineral resources has drawn research efforts into innovative supply and recycling. Metal-rich biomass produced in phytoextraction recently proved an interesting starting material for green chemistry. It allows the production of new catalysts, referred to as ecocatalysts. Ecocatalysts provide increased yields in chemical production and increased regio- and chemo-selectivity, which result in high added value. This new approach to using metal-rich biomass could spur the development of phytoextraction, a technique considered promising for long, yet without credible economic outlets. In this regard, metallophyte biodiversity hotspots, such as New Caledonia, are of particular interest for biomass supply. Potential phytoextraction from mine spoils using two species endemic to New Caledonia is discussed here. Geissois pruinosa, a hypernickelophore, and Grevillea exul, a Mn accumulator, were selected for these original experiments. The results presented here 20 months after plantation of young trees from a nursery show the interest of the approach. Mean Ni concentrations of up to 1513 mg kg(-1) are reported in G. pruinosa, as well as 2000 mg kg(-1) Mn in G. exul. Concentrations of Ni and Mn in the leaves of each species appear to be correlated with leaf age. Plantation of these species may also ensure mine reclamation, and experiments were conducted with the principles of ecological restoration in mind adding a further dimension to the approach.

Leaf-age and soil-plant relationships: key factors for reporting trace-elements hyperaccumulation by plants and design applications.

Relationships between the trace-elements (TE) content of plants and associated soil have been widely investigated especially to understand the ecology of TE hyperaccumulating species to develop applications using TE phytoextraction. Many studies have focused on the possibility of quantifying the soil TE fraction available to plants, and used bioconcentration (BC) as a measure of the plants ability to absorb TE. However, BC only offers a static view of the dynamic phenomenon of TE accumulation. Accumulation kinetics are required to fully account for TE distributions in plants. They are also crucial to design applications where maximum TE concentrations in plant leaves are needed. This paper provides a review of studies of BC (i.e. soil-plant relationships) and leaf-age in relation to TE hyperaccumulation. The paper focuses of Ni and Mn accumulators and hyperaccumulators from New Caledonia who were previously overlooked until recent Ecocatalysis applications emerged for such species. Updated data on Mn hyperaccumulators and accumulators from New Caledonia are also presented and advocate further investigation of the hyperaccumulation of this element. Results show that leaf-age should be considered in the design of sample collection and allowed the reclassification of Grevillea meisneri known previously as a Mn accumulator to a Mn hyperaccumulator.

Development of nuclear and chloroplast microsatellite markers for the endangered conifer Callitris sulcata (Cupressaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Callitris sulcata (Cupressaceae), an endangered conifer species in New Caledonia. METHODS AND RESULTS: Using sequencing by synthesis (SBS) of an RNA-Seq library, 15 polymorphic nuclear and chloroplast microsatellite markers were developed. When evaluated with 48 individuals, these markers showed genetic variations ranging from two to 15 alleles and expected heterozygosity ranging from 0 to 0.881. CONCLUSIONS: These markers will be useful for examining the genetic diversity and structure of remaining wild populations and improving the genetic status of ex situ populations.