Phillip R. England

THE POWER AND PROMISE OF POPULATION GENOMICS: FROM GENOTYPING TO GENOME TYPING

Population genomics has the potential to improve studies of evolutionary genetics, molecular ecology and conservation biology, by facilitating the identification of adaptive molecular variation and by improving the estimation of important parameters such as population size, migration rates and phylogenetic relationships. There has been much excitement in the recent literature about the identification of adaptive molecular variation using the population-genomic approach. However, the most useful contribution of the genomics model to population genetics will be improving inferences about population demography and evolutionary history.

Genetic effects of harvest on wild animal populations

Human harvest of animals in the wild occurs in terrestrial and aquatic habitats throughout the world and is often intense. Harvest has the potential to cause three types of genetic change: alteration of population subdivision, loss of genetic variation, and selective genetic changes. To sustain the productivity of harvested populations, it is crucial to incorporate genetic considerations into management. Nevertheless, it is not necessary to disentangle genetic and environmental causes of phenotypic changes to develop management plans for individual species. We recommend recognizing that some genetic change due to harvest is inevitable. Management plans should be developed by applying basic genetic principles combined with molecular genetic monitoring to minimize harmful genetic change.

Gene-culture coevolution between cattle milk protein genes and human lactase genes

Milk from domestic cows has been a valuable food source for over 8,000 years, especially in lactose-tolerant human societies that exploit dairy breeds. We studied geographic patterns of variation in genes encoding the six most important milk proteins in 70 native European cattle breeds. We found substantial geographic coincidence between high diversity in cattle milk genes, locations of the European Neolithic cattle farming sites (>5,000 years ago) and present-day lactose tolerance in Europeans. This suggests a gene-culture coevolution between cattle and humans.

Estimating Contemporary Effective Population Size on the Basis of Linkage Disequilibrium in the Face of Migration

Effective population size (Ne) is an important genetic parameter because of its relationship to loss of genetic variation, increases in inbreeding, accumulation of mutations, and effectiveness of selection. Like most other genetic approaches that estimate contemporary Ne, the method based on linkage disequilibrium (LD) assumes a closed population and (in the most common applications) randomly recombining loci. We used analytical and numerical methods to evaluate the absolute and relative consequences of two potential violations of the closed-population assumption: (1) mixture LD caused by occurrence of more than one gene pool, which would downwardly bias N^e, and (2) reductions in drift LD (and hence upward bias in N^e) caused by an increase in the number of parents responsible for local samples. The LD method is surprisingly robust to equilibrium migration. Effects of mixture LD are small for all values of migration rate (m), and effects of additional parents are also small unless m is high in genetic terms. LD estimates of Ne therefore accurately reflect local (subpopulation) Ne unless m > ∼5–10%. With higher m, N^e converges on the global (metapopulation) Ne. Two general exceptions were observed. First, equilibrium migration that is rare and hence episodic can occasionally lead to substantial mixture LD, especially when sample size is small. Second, nonequilibrium, pulse migration of strongly divergent individuals can also create strong mixture LD and depress estimates of local Ne. In both cases, assignment tests, Bayesian clustering, and other methods often will allow identification of recent immigrants that strongly influence results. In simulations involving equilibrium migration, the standard LD method performed better than a method designed to jointly estimate Ne and m. The above results assume loci are not physically linked; for tightly linked loci, the LD signal from past migration events can persist for many generations, with consequences for Ne estimates that remain to be evaluated.

Microsatellite diversity and genetic structure of fragmented populations of the rare, fire‐dependent shrub Grevillea macleayana

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self‐compatible and have limited seed dispersal. However, long‐lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self‐compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long‐lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within‐population diversity (3.2–4.2 alleles/locus; Hexp= 0.420–0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation‐drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire‐shaped distribution, may have restricted long‐distance gene flow in the past.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2010 - 31 July 2010.

This article documents the addition of 205 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Bagassa guianensis, Bulweria bulwerii, Camelus bactrianus, Chaenogobius annularis, Creontiades dilutus, Diachasmimorpha tryoni, Dioscorea alata, Euhrychiopsis lecontei, Gmelina arborea, Haliotis discus hannai, Hirtella physophora, Melanaphis sacchari, Munida isos, Thaumastocoris peregrinus and Tuberolachnus salignus. These loci were cross‐tested on the following species: Halobaena caerulea, Procellaria aequinoctialis, Oceanodroma monteiroi, Camelus ferus, Creontiades pacificus, Dioscorea rotundata, Dioscorea praehensilis, Dioscorea abyssinica, Dioscorea nummularia, Dioscorea transversa, Dioscorea esculenta, Dioscorea pentaphylla, Dioscorea trifida, Hirtella bicornis, Hirtella glandulosa, Licania alba, Licania canescens, Licania membranaceae, Couepia guianensis and 7 undescribed Thaumastocoris species.

Understanding age-specific dispersal in fishes through hydrodynamic modelling, genetic simulations and microsatellite DNA analysis.

Many marine species have vastly different capacities for dispersal during larval, juvenile and adult life stages, and this has the potential to complicate the identification of population boundaries and the implementation of effective management strategies such as marine protected areas. Genetic studies of population structure and dispersal rarely disentangle these differences and usually provide only lifetime‐averaged information that can be considered by managers. We address this limitation by combining age‐specific autocorrelation analysis of microsatellite genotypes, hydrodynamic modelling and genetic simulations to reveal changes in the extent of dispersal during the lifetime of a marine fish. We focus on an exploited coral reef species, Lethrinus nebulosus, which has a circum‐tropical distribution and is a key component of a multispecies fishery in northwestern Australia. Conventional population genetic analyses revealed extensive gene flow in this species over vast distances (up to 1500 km). Yet, when realistic adult dispersal behaviours were modelled, they could not account for these observations, implying adult dispersal does not dominate gene flow. Instead, hydrodynamic modelling showed that larval L. nebulosus are likely to be transported hundreds of kilometres, easily accounting for the observed gene flow. Despite the vast scale of larval transport, juvenile L. nebulosus exhibited fine‐scale genetic autocorrelation, which declined with age. This implies both larval cohesion and extremely limited juvenile dispersal prior to maturity. The multidisciplinary approach adopted in this study provides a uniquely comprehensive insight into spatial processes in this marine fish.

Modelling wave-induced disturbance in highly biodiverse marine macroalgal communities: support for the intermediate disturbance hypothesis

As biodiversity declines globally, it is becoming increasingly important to understand the processes that create and maintain biodiverse communities. We examined whether the extraordinarily high species diversity of macroalgal communities in shallow coastal waters off south-west Western Australia is related to wave-induced physical disturbance. We used the numerical wave model SWAN to estimate the hydrodynamic forces generated by waves in bathymetrically complex coastal reefs. Oscillatory water motion at the seabed during extreme wave events was used as an index of physical disturbance in macroalgal communities. There was a significant curvilinear relationship between species diversity and disturbance index, consistent with the intermediate disturbance hypothesis (IDH). Diversity was lower at exposed offshore sites where disturbance is likely to be highest and at very sheltered sites with the least disturbance. Our results match those from some other highly diverse habitats, including rainforests, grasslands and coral reefs in which patchy, stochastic disturbance regimes have been hypothesised to prevent the development of homogeneous climax communities, promoting spatiotemporal heterogeneity and increasing total system diversity. Our results represent important evidence in support of a role for the IDH in driving diversity in marine plant communities.

Microsatellites in the Australian shrub Grevillea macleayana(Proteaceae)

We have developed nine microsatellites in Grevillea macleayana (formerly Grevillea barklyana ssp. macleayana; Makinson 1999) to study the ecology and genetics of this rare Australian shrub. G. macleayana has a very restricted and fragmented distribution near Jervis Bay, Ulladulla and in the nearby coastal ranges of New South Wales (NSW) (McGillivray 1993). It has been the focus of studies into ecological aspects of mating systems, pollination (Vaughton 1996), fire, seed banks (Vaughton 1998) and habitat fragmentation (Hogbin et al. 1998). The development of microsatellite markers in G. macleayana was necessitated by its low allozyme variability (Ayre et al. 1994). Successful transfer of microsatellite primers from G. macleayana to other species in the genus Grevillea, which comprises over 250 species, will substantially increase opportunities for ecological genetics research in the genus. Grevilleas exhibit a large range of pollinator syndromes, fire responses, seed storage and seed dormancy strategies; all of which have implications for the spatial and temporal genetic structure of plant populations. In NSW, 18 Grevillea species are listed as endangered or vulnerable under the NSW Threatened Species Conservation Act, and a further 13, including G. macleayana, are considered to be Ôrare or threatenedÕ (Briggs & Leigh 1996). To our knowledge, this study represents the first report of microsatellites in the Gondwanan family, Proteaceae, an important element in the Australian flora, being species-rich in many communities. A partial, size-selected (300Ð700 bp) G. macleayana genomic library was constructed and screened for the presence of (AC)n and (TC)n tandem repeats as outlined previously (Rassmann et al. 1991). Commercial DNA copolymers, poly(AC).poly(TG) and poly(TC).poly(AG) (Pharmacia) were used in equal proportions to probe the colony blots. Positive clones were sequenced by the dideoxy chain-termination method using a fluorescent dye-primer cycle sequencing kit (Applied Biosystems). Sequenced DNA was run on a polyacrylamide gel and the sequence read by an automated sequencer (Applied Biosystems). The program PRIMER 3 by S. Rozen and H. J. Skaletsky (code available at http://wwwgenome.wi.mit.edu/genome_software/other/primer3.html) was used to design PCR primers that flanked microsatellites identified by the screen. Where alternative primer pairs were suggested by the program, a choice was made on the basis of both maximising GC content of the primers and covering a range of PCR product sizes to facilitate multiplexing and coelectrophoresis of different loci. Primers were synthesized by Bresatec, Australia. PCR reactions were conducted in polypropylene microtitre trays (Bresatec, Australia) on an MJR thermal cycler PTC100 (MJR Research). All loci were amplified in G. macleayana with the program: 5 min at 94 ¡C then 30 cycles of 30 s at 94 ¡C, 30 s at 55 ¡C and 1 min at 72 ¡C followed by 5 min at 72 ¡C. One unit of Taq polymerase (Promega) was used in each 20 μL reaction with the buffer supplied by the manufacturer. Reactions were 2.5 mM for Mg, 200 μM for each unlabelled dNTP and 250 nM for flourescent dCTP (Perkin-Elmer). Ten pmol of each primer and ≈ 20 ng of template DNA were used per reaction. Conditions were not altered for multiplex reactions. PCR products were visualized with an ABI 377 P R I M E R N O T E S 689

SNP discovery in nonmodel organisms: strand bias and base-substitution errors reduce conversion rates.

Single nucleotide polymorphisms (SNPs) have become the marker of choice for genetic studies in organisms of conservation, commercial or biological interest. Most SNP discovery projects in nonmodel organisms apply a strategy for identifying putative SNPs based on filtering rules that account for random sequencing errors. Here, we analyse data used to develop 4723 novel SNPs for the commercially important deep‐sea fish, orange roughy (Hoplostethus atlanticus), to assess the impact of not accounting for systematic sequencing errors when filtering identified polymorphisms when discovering SNPs. We used SAMtools to identify polymorphisms in a velvet assembly of genomic DNA sequence data from seven individuals. The resulting set of polymorphisms were filtered to minimize ‘bycatch’—polymorphisms caused by sequencing or assembly error. An Illumina Infinium SNP chip was used to genotype a final set of 7714 polymorphisms across 1734 individuals. Five predictors were examined for their effect on the probability of obtaining an assayable SNP: depth of coverage, number of reads that support a variant, polymorphism type (e.g. A/C), strand‐bias and Illumina SNP probe design score. Our results indicate that filtering out systematic sequencing errors could substantially improve the efficiency of SNP discovery. We show that BLASTX can be used as an efficient tool to identify single‐copy genomic regions in the absence of a reference genome. The results have implications for research aiming to identify assayable SNPs and build SNP genotyping assays for nonmodel organisms.