Martin S. Elphinstone

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817)

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species’ range. Eighteen different haplotypes were defined in the ≈ 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within‐population haplotypic diversity of 0.180 ± 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour‐joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA‐haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short‐term management unit (MU) for koalas is the local population.

Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol ‐ Outgroup Heteroduplex Analysis (OHA). We assess this techniques sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433‐base‐pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.

Phylogeographical population structure of tiger quolls Dasyurus maculatus (Dasyuridae: Marsupialia), an endangered carnivorous marsupial

Tiger quolls, Dasyurus maculatus, are the largest carnivorous marsupials still extant on the mainland of Australia, and occupy an important ecological niche as top predators and scavengers. Two allopatric subspecies are recognized, D.m. gracilis in north Queensland, and D.m. maculatus in the southeast of the mainland and Tasmania. D.m. gracilis is considered endangered while D.m. maculatus is listed as vulnerable to extinction; both subspecies are still in decline. Phylogeographical subdivision was examined to determine evolutionarily significant units (ESUs) and management units (MUs) among populations of tiger quolls to assist in the conservation of these taxa. Ninety‐three tiger quolls from nine representative populations were sampled from throughout the species range. Six nuclear microsatellite loci and the mitochondrial DNA (mtDNA) control region (471 bp) were used to examine ESUs and MUs in this species. We demonstrated that Tasmanian tiger quolls are reciprocally monophyletic to those from the mainland using mtDNA analysis, but D.m. gracilis was not monophyletic with respect to mainland D.m. maculatus. Analysis of microsatellite loci also revealed significant differences between the Tasmanian and mainland tiger quolls, and between D.m. gracilis and mainland D.m. maculatus. These results indicate that Tasmanian and mainland tiger quolls form two distinct evolutionary units but that D.m. gracilis and mainland D.m. maculatus are different MUs within the same ESU. The two marker types used in this study revealed different male and female dispersal patterns and indicate that the most appropriate units for short‐term management are local populations. A revised classification and management plan are needed for tiger quolls, particularly in relation to conservation of the Tasmanian and Queensland populations.

Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species-specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species-specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species-specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species-specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36-base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.

Phylogenetics and revised taxonomy of the Australian freshwater cod genus, Maccullochella (Percichthyidae)

Determining the phylogenetic and taxonomic relationships among allopatric populations can be difficult, especially when divergence is recent and morphology is conserved. We used mitochondrial sequence data from the control region and three protein-coding genes (1253 bp in total) and genotypes determined at 13 microsatellite loci to examine the evolutionary relationships among Australia’s largest freshwater fish, the Murray cod, Maccullochella peelii peelii, from the inland Murray–Darling Basin, and its allopatric sister taxa from coastal drainages, the eastern freshwater cod, M. ikei, and Mary River cod, M. peelii mariensis. Phylogenetic analyses provided strong support for taxon-specific clades, with a clade containing both of the eastern taxa reciprocally monophyletic to M. peelii peelii, suggesting a more recent common ancestry between M. ikei and M. peelii mariensis than between the M. peelii subspecies. This finding conflicts with the existing taxonomy and suggests that ancestral Maccullochella crossed the Great Dividing Range in the Pleistocene and subsequently diverged in eastern coastal drainages. Evidence from the present study, in combination with previous morphological and allozymatic data, demonstrates that all extant taxa are genetically and morphologically distinct. The taxonomy of Maccullochella is revised, with Mary River cod now recognised as a species, Maccullochella mariensis, a sister species to eastern freshwater cod, M. ikei. As a result of the taxonomic revision, Murray cod is M. peelii.

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae)

Premise of the study: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow. Methods and Results: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species—M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii—and in commercial macadamia cultivars including hybrids. Conclusions: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.

Population structure, timing of divergence and contact between lineages in the endangered Hastings River mouse (Pseudomys oralis)

Management of threatened species requires understanding their genetic structure, particularly when the potential for cryptic lineages exists for species with a broad geographic range. The Hastings River mouse (Pseudomys oralis) is an endangered species found along the eastern coast of Australia. Previous genetic investigation revealed two mitochondrial lineages, separated by an unsampled gap in northern New South Wales. Using new samples from within this gap and from throughout the species’ range, we recovered two evolutionary lineages in agreement with previous studies. Importantly, we identified Washpool National Park as the area of lineage overlap, located at the northern limit of the Macleay–McPherson Overlap Zone. We confirmed limited haplotype sharing between localities, suggesting low levels of gene flow. Historical demography suggested recent population expansion and decline for Lineages I and II, respectively. Our dating estimates placed lineage divergence at 300 000–900 000 years ago, at the lower limit of percentage divergence between other sister species in Pseudomys. While these results support separate management consideration for each lineage, nuclear markers are needed to evaluate whether these lineages represent separate species. In addition, comparative phylogeographic analyses of divergence times among lineages of other species distributed across the Macleay–McPherson Overlap Zone are needed to determine the significance of this biogeographic contact zone for ecological communities in the region generally.

Correction to “Whole Genome Shotgun Sequences for Microsatellite Discovery and Application in Cultivated and Wild Macadamia (Proteaceae)”

Vol. 2 No. 4: 1300089, April 2014, doi: 10.3732/apps.1300089 The authors discovered an error in a primer sequence reported in Table 1. The corrected primer sequence (locus Mac0010, reverse primer sequence) is listed below in boldface. The authors apologize for the error. The published article (full-text and PDF) has been corrected.