Alison J. Fitch

Rise of the machines - recommendations for ecologists when using next generation sequencing for microsatellite development

Next generation sequencing is revolutionizing molecular ecology by simplifying the development of molecular genetic markers, including microsatellites. Here, we summarize the results of the large‐scale development of microsatellites for 54 nonmodel species using next generation sequencing and show that there are clear differences amongst plants, invertebrates and vertebrates for the number and proportion of motif types recovered that are able to be utilized as markers. We highlight that the heterogeneity within each group is very large. Despite this variation, we provide an indication of what number of sequences and consequent proportion of a 454 run are required for the development of 40 designable, unique microsatellite loci for a typical molecular ecological study. Finally, to address the challenges of choosing loci from the vast array of microsatellite loci typically available from partial genome runs (average for this study, 2341 loci), we provide a microsatellite development flowchart as a procedural guide for application once the results of a partial genome run are obtained.

Metagenomic comparison of microbial communities inhabiting confined and unconfined aquifer ecosystems

A metagenomic analysis of two aquifer systems located under a dairy farming region was performed to examine to what extent the composition and function of microbial communities varies between confined and surface-influenced unconfined groundwater ecosystems. A fundamental shift in taxa was seen with an overrepresentation of Rhodospirillales, Rhodocyclales, Chlorobia and Circovirus in the unconfined aquifer, while Deltaproteobacteria and Clostridiales were overrepresented in the confined aquifer. A relative overrepresentation of metabolic processes including antibiotic resistance (β-lactamase genes), lactose and glucose utilization and DNA replication were observed in the unconfined aquifer, while flagella production, phosphate metabolism and starch uptake pathways were all overrepresented in the confined aquifer. These differences were likely driven by differences in the nutrient status and extent of exposure to contaminants of the two groundwater systems. However, when compared with freshwater, ocean, sediment and animal gut metagenomes, the unconfined and confined aquifers were taxonomically and metabolically more similar to each other than to any other environment. This suggests that intrinsic features of groundwater ecosystems, including low oxygen levels and a lack of sunlight, have provided specific niches for evolution to create unique microbial communities. Obtaining a broader understanding of the structure and function of microbial communities inhabiting different groundwater systems is particularly important given the increased need for managing groundwater reserves of potable water.

Molecular evidence for an Asian origin of monitor lizards followed by Tertiary dispersals to Africa and Australasia

Monitor lizards are emblematic reptiles that are widely distributed in the Old World. Although relatively well studied in vertebrate research, their biogeographic history is still controversial. We constructed a molecular dataset for 54 anguimorph species, including representatives of all families with detailed sampling of the Varanidae (38 species). Our results are consistent with an Asian origin of the Varanidae followed by a dispersal to Africa 41 (49–33) Ma, possibly via an Iranian route. Another major event was the dispersal of monitors to Australia in the Late Eocene–Oligocene 32 (39–26) Ma. This divergence estimate adds to the suggestion that Australia was colonized by several squamate lineages prior to the collision of the Australian plate with the Asian plate starting 25 Ma.

A molecular phylogeny of the Australian monitor lizards (Squamata : Varanidae) inferred from mitochondrial DNA sequences

To date no complete phylogeny of all of the currently recognised Indo-Australian varanid species and subspecies has been published. This paper presents a comprehensive mitochondrial gene phylogeny of these lizards. A portion of the mitochondrial genome comprising part of the ND4 gene and three adjacent tRNA genes (hereafter referred to as ND4) was analysed alone and, for a subset of the taxa, combined with previously published mitochondrial data. Similar tree topologies were produced by both datasets although combining the data helped resolve some of the unresolved or weakly supported nodes in the ND4 analyses. The monophyly of the Indo-Australian group was strongly supported in all analyses. This group comprised three major lineages: the gouldii group, the Odatria group and the varius group. Mitochondrial ND4 nucleotide sequences were successfully amplified from all of the Indo-Australian monitor species and subspecies currently recognised and, as such, is the first comprehensive phylogenetic study of the Australian monitor lizards published. Analysis of the tempo of diversification and evolution of preferred habitat use identified six episodes of increased net speciation rate, with two closely adjacent episodes showing the highest rates of diversification and correlating with the appearance of all preferred habitat types. The comprehensive molecular phylogenetic framework will also be useful for the identification of varanid species and traded products derived from monitors and, as such, has important applications for wildlife management and conservation.

Panmixia supports divergence with gene flow in Darwin's small ground finch, Geospiza fuliginosa, on Santa Cruz, Galapagos Islands

The divergence‐with‐gene‐flow model of speciation has a strong theoretical basis with a growing number of plausible examples in nature, but remains hotly debated. Darwin’s finches of the Galápagos Archipelago have played an important role in our understanding of speciation processes. Recent studies suggest that this group may also provide insights into speciation via divergence with gene flow. On the island of Santa Cruz, recent studies found evidence for adaptive divergence in Darwin’s small ground finch, Geospiza fuliginosa, between ecologically contrasting arid and humid zones. Despite the short geographical distance between these zones, strong disruptive selection during low rainfall periods is expected to generate and maintain adaptive divergence. Conversely, during high rainfall periods, when disruptive selection is predicted to be weakened, population divergence in adaptive traits is expected to break down. Because periods of low and high rainfall irregularly alternate, the geographical pattern of adaptive divergence can be assumed to break down and, importantly, regenerate in situ. Here, we use microsatellite allele frequency data to assess the genetic population structure of G. fuliginosa on Santa Cruz. We sample 21 sites and four ecological zones across the island. We reject hypotheses of population substructure linked to ecological and geographical differences among sites in favour of a single panmictic population. Panmixia implies high levels of gene flow within Santa Cruz, which favours selection over genetic drift as a valid process generating phenotypic divergence in G. fuliginosa on Santa Cruz. We discuss how our findings may support classic adaptation, phenotypic plasticity, matching habitat choice or any combination of these three processes.

Confined aquifers as viral reservoirs

Knowledge about viral diversity and abundance in deep groundwater reserves is limited. We found that the viral community inhabiting a deep confined aquifer in South Australia was more similar to reclaimed water communities than to the viral communities in the overlying unconfined aquifer community. This similarity was driven by high relative occurrence of the single-stranded DNA viral groups Circoviridae, Geminiviridae and Microviridae, which include many known plant and animal pathogens. These groups were present in a 1500-year-old water situated 80 m below the surface, which suggests the potential for long-term survival and spread of potentially pathogenic viruses in deep, confined groundwater. Obtaining a broader understanding of potentially pathogenic viral communities within aquifers is particularly important given the ability of viruses to spread within groundwater ecosystems.

A direct benefit of native saltbush revegetation for an endemic lizard (Tiliqua rugosa) in southern Australia

Abstract. Land alteration for intensive agriculture has been a major cause of species decline and extinction globally. In marginal grazing regions of southern Australia, native perennial shrubs are increasingly being planted to supplement pasture feeding of stock. Such revegetation has the benefits of reducing erosion and salinity, and importantly, the potential provision of habitat for native fauna. We explored the use of revegetated native saltbush by the sleepy lizard (Tiliqua rugosa) an endemic Australian species common in the region. We repeatedly sampled revegetated saltbush throughout 2010 and 2011 for adults (n = 55) and juveniles (n = 26). Using genotypes from eight microsatellite loci, parents were assigned to half of all juveniles with high statistical confidence. Parents were sampled in the same patch of revegetated saltbush as their offspring, thus supporting the observation that juvenile sleepy lizards remain within the home range of their parents before dispersal. Most importantly, our findings indicate that revegetated saltbush provides important habitat for T. rugosa at significant life stages – before and during breeding for adults, and before dispersal for juveniles. We conclude that revegetation using simple, monoculture plantations provides beneficial habitat for T. rugosa and may also be beneficial habitat for other native species in human-altered agricultural landscapes.

Isolation and characterisation of eighteen microsatellite markers from the sea cucumber Holothuria scabra (Echinodermata : Holothuriidae)

Abstract. We isolated 18 new, polymorphic, microsatellite markers from Holothuria scabra, a commercially important species of sea cucumber found throughout the Asia-Pacific region. Next-generation sequencing was used to identify 206 unique loci for which primers were designed. Of these unique loci, we trialled 65 primer pairs in the target species, and 48 (74%) amplified a product of the expected size. Eighteen loci were found to be polymorphic and reliable, and were screened for variation in 50 individuals, from a single population from Croker Island, Northern Territory, Australia. Observed heterozygosity ranged from 0.00 to 0.96 (mean = 0.46) and the number of alleles per locus from 2 to 28 (mean = 9.61). These loci will be useful for the investigation of population structure and mating systems in H. scabra and may also be of use in other holothurian species.

Plasmid Maintenance and Localisation of Vibrio sp. S141(p519ngfp) Cells within Monoculture and Mixed-species Biofilms

This study examined cell localisation and plasmid maintenance of Vibrio sp. S141(p519ngfp) cells which grew in, and detached from, monoculture and mixed-species marine biofilms under continuous flow conditions. Over the 48 h time course of the experiments, the broad host range IncQ RSF1010 derivative plasmid, p519 ngfp , was maintained in S141(p519ngfp) cells detaching from the biofilms irrespective of selection for the plasmid, the presence of another bacterial species, or the order of substratum colonisation. S141(p519ngfp) cell localisation within mixed-species biofilms was affected by the order, and length of time, of colonisation. When S141(p519ngfp) was the initial coloniser of the biofilm, the localisation of the majority of the plasmid-bearing cells near the substratum surface was not affected. When S141(p519ngfp) cells colonised a pre-existing Psychrobacter sp. SW5HR biofilm, or the two species simultaneously colonised the substratum, non plasmid-bearing cells initially dominated the substratum surface; as the time of S141(p519ngfp) colonisation increased, the plasmid-bearing cells appeared to displace the SW5HR cells from the surface. Since the p519ngfp plasmid was stably maintained in the Vibrio sp. S141 host forming biofilms over a 48 h period, GFP-producing S141 cells were able to be localised in mixed-species biofilms and were found to dominate the substratum surface by 48 h.

Isolation, via 454 sequencing, and characterisation of microsatellites for Phalacrocorax fuscescens, the black-faced cormorant (Aves:Phalacrocoracidae)

Abstract. The black-faced cormorant, Phalacrocorax fuscescens, is a brood-reducing seabird endemic to the southern waters of Australia. Microsatellite loci were isolated from genomic DNA using 454 shotgun sequencing. Thirty-one loci were tested and, of these, 16 were found to be polymorphic. Further characterisation was conducted on seven loci that were genotyped in 42 adult individuals from a single breeding colony in South Australia. The number of alleles per locus ranged from three to eight (s.d. ± 2.16), and the mean observed and expected heterozygosity was 0.66 (s.d. ± 0.249) and 0.62 (s.d. ± 0.178) respectively. We confirm that four loci conformed to Hardy–Weinberg expectations. Four other Phalacrocorax species were trialled for amplification of these four polymorphic loci. Amplification success varied between loci and species. These loci will be useful in determining genetic family structure and exploring nestling relatedness to further understand how relatedness influences competitive behaviours in brood-reducing species.