Isolation of polymorphic microsatellite loci in the New Zealand endemic sand-binder, Ficinia spiralis (Cyperaceae).

PREMISE OF THE STUDY: Ficinia spiralis (Cyperaceae) is a declining sand-binding sedge of ecological and cultural importance. Microsatellite primers were developed in F. spiralis to investigate how population genetic structure is related to the pronounced morphological, physiological, and ecological variation observed in this species. METHODS AND RESULTS: A 454 shotgun-sequencing approach was used to generate 157,274 raw sequence reads, 536 of which contained microsatellites. After initial primer testing for 40 loci, 14 polymorphic loci were isolated, containing five to 27 alleles per locus. Ten of these loci also amplified in a congener, F. nodosa. CONCLUSIONS: These loci will enable the assessment of the population genetic structure of F. spiralis, improving our understanding of the population processes underlying the observed morphological, physiological, and ecological variation in this endemic species. As the first microsatellites developed in Ficinia, these loci are a valuable resource for population genetic studies within this genus.

Building strong relationships between conservation genetics and primary industry leads to mutually beneficial genomic advances.

Several reviews in the past decade have heralded the benefits of embracing high-throughput sequencing technologies to inform conservation policy and the management of threatened species, but few have offered practical advice on how to expedite the transition from conservation genetics to conservation genomics. Here, we argue that an effective and efficient way to navigate this transition is to capitalize on emerging synergies between conservation genetics and primary industry (e.g., agriculture, fisheries, forestry and horticulture). Here, we demonstrate how building strong relationships between conservation geneticists and primary industry scientists is leading to mutually-beneficial outcomes for both disciplines. Based on our collective experience as collaborative New Zealand-based scientists, we also provide insight for forging these cross-sector relationships.

Extinct New Zealand megafauna were not in decline before human colonization.

The extinction of New Zealand's moa (Aves: Dinornithiformes) followed the arrival of humans in the late 13th century and was the final event of the prehistoric Late Quaternary megafauna extinctions. Determining the state of the moa populations in the pre-extinction period is fundamental to understanding the causes of the event. We sampled 281 moa individuals and combined radiocarbon dating with ancient DNA analyses to help resolve the extinction debate and gain insights into moa biology. The samples, which were predominantly from the last 4,000 years preceding the extinction, represent four sympatric moa species excavated from five adjacent fossil deposits. We characterized the moa assemblage using mitochondrial DNA and nuclear microsatellite markers developed specifically for moa. Although genetic diversity differed significantly among the four species, we found that the millennia preceding the extinction were characterized by a remarkable degree of genetic stability in all species, with no loss of heterozygosity and no shifts in allele frequencies over time. The extinction event itself was too rapid to be manifested in the moa gene pools. Contradicting previous claims of a decline in moa before Polynesian settlement in New Zealand, our findings indicate that the populations were large and stable before suddenly disappearing. This interpretation is supported by approximate Bayesian computation analyses. Our analyses consolidate the disappearance of moa as the most rapid, human-facilitated megafauna extinction documented to date.

Human-assisted spread of a maladaptive behavior in a critically endangered bird.

Conservation management often focuses on counteracting the adverse effects of human activities on threatened populations. However, conservation measures may unintentionally relax selection by allowing the 'survival of the not-so-fit', increasing the risk of fixation of maladaptive traits. Here, we report such a case in the critically-endangered Chatham Island black robin (Petroica traversi) which, in 1980, was reduced to a single breeding pair. Following this bottleneck, some females were observed to lay eggs on the rims of their nests. Rim eggs left in place always failed to hatch. To expedite population recovery, rim eggs were repositioned inside nests, yielding viable hatchlings. Repositioning resulted in rapid growth of the black robin population, but by 1989 over 50% of all females were laying rim eggs. We used an exceptional, species-wide pedigree to consider both recessive and dominant models of inheritance over all plausible founder genotype combinations at a biallelic and possibly sex-linked locus. The pattern of rim laying is best fitted as an autosomal dominant Mendelian trait. Using a phenotype permutation test we could also reject the null hypothesis of non-heritability for this trait in favour of our best-fitting model of heritability. Data collected after intervention ceased shows that the frequency of rim laying has strongly declined, and that this trait is maladaptive. This episode yields an important lesson for conservation biology: fixation of maladaptive traits could render small threatened populations completely dependent on humans for reproduction, irreversibly compromising the long term viability of populations humanity seeks to conserve.

The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils.

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10(-6) per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R(2) = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Sampling for microsatellite-based population genetic studies: 25 to 30 individuals per population is enough to accurately estimate allele frequencies.

One of the most common questions asked before starting a new population genetic study using microsatellite allele frequencies is "how many individuals do I need to sample from each population?" This question has previously been answered by addressing how many individuals are needed to detect all of the alleles present in a population (i.e. rarefaction based analyses). However, we argue that obtaining accurate allele frequencies and accurate estimates of diversity are much more important than detecting all of the alleles, given that very rare alleles (i.e. new mutations) are not very informative for assessing genetic diversity within a population or genetic structure among populations. Here we present a comparison of allele frequencies, expected heterozygosities and genetic distances between real and simulated populations by randomly subsampling 5-100 individuals from four empirical microsatellite genotype datasets (Formica lugubris, Sciurus vulgaris, Thalassarche melanophris, and Himantopus novaezelandia) to create 100 replicate datasets at each sample size. Despite differences in taxon (two birds, one mammal, one insect), population size, number of loci and polymorphism across loci, the degree of differences between simulated and empirical dataset allele frequencies, expected heterozygosities and pairwise F(ST) values were almost identical among the four datasets at each sample size. Variability in allele frequency and expected heterozygosity among replicates decreased with increasing sample size, but these decreases were minimal above sample sizes of 25 to 30. Therefore, there appears to be little benefit in sampling more than 25 to 30 individuals per population for population genetic studies based on microsatellite allele frequencies.

Profiling the dead: generating microsatellite data from fossil bones of extinct megafauna--protocols, problems, and prospects.

We present the first set of microsatellite markers developed exclusively for an extinct taxon. Microsatellite data have been analysed in thousands of genetic studies on extant species but the technology can be problematic when applied to low copy number (LCN) DNA. It is therefore rarely used on substrates more than a few decades old. Now, with the primers and protocols presented here, microsatellite markers are available to study the extinct New Zealand moa (Aves: Dinornithiformes) and, as with single nucleotide polymorphism (SNP) technology, the markers represent a means by which the field of ancient DNA can (preservation allowing) move on from its reliance on mitochondrial DNA. Candidate markers were identified using high throughput sequencing technology (GS-FLX) on DNA extracted from fossil moa bone and eggshell. From the 'shotgun' reads, >60 primer pairs were designed and tested on DNA from bones of the South Island giant moa (Dinornis robustus). Six polymorphic loci were characterised and used to assess measures of genetic diversity. Because of low template numbers, typical of ancient DNA, allelic dropout was observed in 36-70% of the PCR reactions at each microsatellite marker. However, a comprehensive survey of allelic dropout, combined with supporting quantitative PCR data, allowed us to establish a set of criteria that maximised data fidelity. Finally, we demonstrated the viability of the primers and the protocols, by compiling a full Dinornis microsatellite dataset representing fossils of c. 600-5000 years of age. A multi-locus genotype was obtained from 74 individuals (84% success rate), and the data showed no signs of being compromised by allelic dropout. The methodology presented here provides a framework by which to generate and evaluate microsatellite data from samples of much greater antiquity than attempted before, and opens new opportunities for ancient DNA research.

Genetic analyses reveal hybridization but no hybrid swarm in one of the world's rarest birds.

Hybridization facilitated by human activities has dramatically altered the evolutionary trajectories of threatened taxa around the globe. Whereas introduced mammalian predators and widespread habitat loss and degradation clearly imperil the recovery and survival of the New Zealand endemic black stilt or kaki (Himantopus novaezelandiae), the risk associated with hybridization between this critically endangered endemic and its self-introduced congener, the pied stilt or poaka (Himantopus himantopus leucocephalus) is less clear. Here, we combine Bayesian admixture analyses of microsatellite data with mitochondrial DNA sequence data to assess the levels of hybridization and introgression between kaki and poaka. We show that birds classified as hybrids on the basis of adult plumage are indeed of hybrid origin and that hybridization between kaki and poaka is both extensive and bidirectional. Despite this, we found almost no evidence for introgression from poaka to kaki, thus negating the popular belief that kaki represent a hybrid swarm. To our knowledge, ours represents the first comprehensive study to document a lack of widespread introgression for a species at risk despite a recent history of extensive bidirectional human-induced hybridization. We attribute this rather surprising result, in part, to reduced reproductive success in female hybrids combined with a transient male-biased kaki sex ratio. To maximize the evolutionary potential of kaki, we use these data to recommend conservation management activities aimed to maintain the genetic integrity and to maximize the genetic diversity of this iconic rare bird.

Merging ancient and modern DNA: extinct seabird taxon rediscovered in the North Tasman Sea.

Ancient DNA has revolutionized the way in which evolutionary biologists research both extinct and extant taxa, from the inference of evolutionary history to the resolution of taxonomy. Here, we present, to our knowledge, the first study to report the rediscovery of an 'extinct' avian taxon, the Tasman booby (Sula tasmani), using classical palaeontological data combined with ancient and modern DNA data. Contrary to earlier work, we show an overlap in size between fossil and modern birds in the North Tasman Sea (classified currently as S. tasmani and Sula dactylatra fullagari, respectively). In addition, we show that Holocene fossil birds have mitochondrial control region sequences that are identical to those found in modern birds. These results indicate that the Tasman booby is not an extinct taxon: S. dactylatra fullagari O'Brien & Davies, 1990 is therefore a junior synonym of Sula tasmani van Tets, Meredith, Fullagar & Davidson, 1988 and all North Tasman Sea boobies should be known as S. d. tasmani. In addition to reporting the rediscovery of an extinct avian taxon, our study highlights the need for researchers to be cognizant of multidisciplinary approaches to understanding taxonomy and past biodiversity.

Characterization of microsatellite markers for Sycoscapter nonpollinating fig wasps.

We isolated 18 microsatellites from Sycoscapter australis, a nonpollinating fig wasp that develops in figs of Ficus macrophylla, and assessed their variability in 20 wasps. We further optimized nine of these loci for use in three other Sycoscapter species that develop in Ficus rubiginosa figs and assessed their variability in 47-140 wasps per species. These are the first microsatellites developed for nonpollinating fig wasps and show sufficient polymorphism to become important tools in evolutionary and genetical studies of Sycoscapter wasps.

Development of polymorphic microsatellite markers for the New Zealand black stilt (Himantopus novaezelandiae) and cross-amplification in the pied stilt (Himantopus himantopus leucocephalus).

Eight polymorphic microsatellite primer pairs were developed for the critically endangered New Zealand black stilt, Himantopus novaezelandiae, representing the first microsatellite markers available for birds in the family Recurvirostridae. The number of alleles ranged from two to four per locus. Observed and expected heterozygosities ranged from 0.30 to 0.80 and from 0.37 to 0.70, respectively. All eight loci were polymorphic in the related species Himantopus himantopus leucocephalus, indicating these primer pairs may be useful for additional taxa in the globally distributed genus Himantopus.

Causes of size homoplasy among chloroplast microsatellites in closely related Clusia species.

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.