Laurence J. Clarke

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias

Studies of insect assemblages are suited to the simultaneous DNA‐based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR‐amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR‐amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR‐amplified the blend using five primer sets, targeting either COI or 16S, with high‐throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.

Modular tagging of amplicons using a single PCR for high-throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.

Significant population genetic structure detected for a new and highly restricted species of Atriplex (Chenopodiaceae) from Western Australia, and implications for conservation management

Atriplex sp. Yeelirrie Station (L. Trotter & A. Douglas LCH 25025) is a highly restricted, potentially new species of saltbush, known from only two sites ~30 km apart in central Western Australia. Knowledge of genetic structure within the species is required to inform conservation strategies as both populations occur within a palaeovalley that contains significant near-surface uranium mineralisation. We investigate the structure of genetic variation within populations and subpopulations of this taxon using nuclear microsatellites. Internal transcribed spacer sequence data places this new taxon within a clade of polyploid Atriplex species, and the maximum number of alleles per locus suggests it is hexaploid. The two populations possessed similar levels of genetic diversity, but exhibited a surprising level of genetic differentiation given their proximity. Significant isolation by distance over scales of less than 5 km suggests dispersal is highly restricted. In addition, the proportion of variation between the populations (12%) is similar to that among A. nummularia populations sampled at a continent-wide scale (several thousand kilometres), and only marginally less than that between distinct A. nummularia subspecies. Additional work is required to further clarify the exact taxonomic status of the two populations. We propose management recommendations for this potentially new species in light of its highly structured genetic variation.

The potential for deep groundwater use by Acacia papyrocarpa (Western myall) in a water-limited environment

Emma K. Steggles, Kate L. Holland, David J. Chittleborough, Samantha L. Doudle. Laurence J. Clarke, Jennifer R. Watling, Jose M. Facelli

Reintroduction of locally extinct vertebrates impacts arid soil fungal communities

Introduced species have contributed to extinction of native vertebrates in many parts of the world. Changes to vertebrate assemblages are also likely to alter microbial communities through coextinction of some taxa and the introduction of others. Many attempts to restore degraded habitats involve removal of exotic vertebrates (livestock and feral animals) and reintroduction of locally extinct species, but the impact of such reintroductions on microbial communities is largely unknown. We used high‐throughput DNA sequencing of the fungal internal transcribed spacer I (ITS1) region to examine whether replacing exotic vertebrates with reintroduced native vertebrates led to changes in soil fungal communities at a reserve in arid central Australia. Soil fungal diversity was significantly different between dune and swale (interdune) habitats. Fungal communities also differed significantly between sites with exotic or reintroduced native vertebrates after controlling for the effect of habitat. Several fungal operational taxonomic units (OTUs) found exclusively inside the reserve were present in scats from reintroduced native vertebrates, providing a direct link between the vertebrate assemblage and soil microbial communities. Our results show that changes to vertebrate assemblages through local extinctions and the invasion of exotic species can alter soil fungal communities. If local extinction of one or several species results in the coextinction of microbial taxa, the full complement of ecological interactions may never be restored.

Isolation of microsatellites from Baumea juncea (Cyperaceae)

We developed 13 polymorphic microsatellite loci for the rhizomatous perennial sedge Baumea juncea. The number of alleles per locus ranged from two to six, with averages of 3.0 and 3.2 at two South Australian populations. Observed and expected heterozygosity at variable loci within each population ranged from 0.034 to 1.000 and 0.034 to 0.766, respectively. These markers will allow the identification of genets, as well as the study of spatial genetic structure and genetic differentiation among populations.

A primer to metabarcoding surveys of Antarctic terrestrial biodiversity

Abstract Ice-free regions of Antarctica are concentrated along the coastal margins but are scarce throughout the continental interior. Environmental changes, including the introduction of non-indigenous species, increasingly threaten these unique habitats. At the same time, the unique biotic communities subsisting in isolation across the continent are difficult to survey due to logistical constraints, sampling challenges and problems related to the identification of small and cryptic taxa. Baseline biodiversity data from remote Antarctic habitats are still missing for many parts of the continent but are critical to the detection of community changes over time, including newly introduced species. Here we review the potential of standardized (non-specialist) sampling in the field (e.g. from soil, vegetation or water) combined with high-throughput sequencing (HTS) of bulk DNA as a possible solution to overcome some of these problems. In particular, HTS metabarcoding approaches benefit from being able to process many samples in parallel, while workflow and data structure can stay highly uniform. Such approaches have quickly gained recognition and we show that HTS metabarcoding surveys are likely to play an important role in continent-wide biomonitoring of all Antarctic terrestrial habitats.

Age-related environmental gradients influence invertebrate distribution in the Prince Charles Mountains, East Antarctica

The potential impact of environmental change on terrestrial Antarctic ecosystems can be explored by inspecting biodiversity patterns across large-scale gradients. Unfortunately, morphology-based surveys of Antarctic invertebrates are time-consuming and limited by the cryptic nature of many taxa. We used biodiversity information derived from high-throughput sequencing (HTS) to elucidate the relationship between soil properties and invertebrate biodiversity in the Prince Charles Mountains, East Antarctica. Across 136 analysed soil samples collected from Mount Menzies, Mawson Escarpment and Lake Terrasovoje, we found invertebrate distribution in the Prince Charles Mountains significantly influenced by soil salinity and/or sulfur content. Phyla Tardigrada and Arachnida occurred predominantly in low-salinity substrates with abundant nutrients, whereas Bdelloidea (Rotifera) and Chromadorea (Nematoda) were more common in highly saline substrates. A significant correlation between invertebrate occurrence, soil salinity and time since deglaciation indicates that terrain age indirectly influences Antarctic terrestrial biodiversity, with more recently deglaciated areas supporting greater diversity. Our study demonstrates the value of HTS metabarcoding to investigate environmental constraints on inconspicuous soil biodiversity across large spatial scales.

Microsatellite markers for the freshwater shrimp Paratya australiensis (Atyidae)

Paratya australiensis is a common freshwater shrimp found in most catchments in south-eastern Australia. Microsatellite loci were isolated from a partial genomic library created using 454 sequencing. Of the 25 tested, 13 were found to be polymorphic, however, for pooling purposes only 12 were used for genotyping. The number of alleles per locus varied from 2 to 14 in a population from Wakefield River, South Australia, and the mean (range) observed and expected heterozygosity were 0.512 (0.136–0.909) and 0.590 (0.165–0.788), respectively across all alleles. These microsatellites will be used to study the genetic structure of populations in two catchments in South Australia.

Growth responses of Baumea juncea (Cyperaceae) plants from inland artesian spring and coastal habitats to salinity and waterlogging treatments

Artesian springs of arid inland Australia provide permanent water that supports naturally fragmented wetland communities. Some plant species that occur at springs have more extensive populations in coastal wetland areas of Australia where they may experience quite different environmental conditions. The present study investigated the growth response of one such species, Baumea juncea (R.Br.) Palla (Cyperaceae), to salinity and waterlogging. Plants from each region were subjected to combinations of salinity (freshwater or 20% seawater) and waterlogging (unsaturated or saturated soil), in a factorial design, for a period of 5 months. All plants survived and although the final aboveground biomasses did not differ significantly among the treatment combinations, for spring plants, the relative growth of roots was greater in unsaturated soil than in saturated soil. For the growth parameters of total biomass, culm biomass and rhizome biomass, spring and coastal plants showed contrasting responses to the treatment combinations; for spring plants in fresh water, these parameters were greater in the saturated treatment than in the unsaturated treatment, whereas for spring plants in saline water, these variables were lower in the saturated treatment than in the unsaturated treatment. Coastal plants displayed the reverse pattern. For the remaining parameters of root biomass, root : culm ratio and aboveground : belowground biomass ratio, plants from spring and coastal regions grown under saline conditions displayed contrasting responses to waterlogging. Our findings showed that the growth responses of plants of B. juncea to waterlogging and salinity differ for spring and coastal plants, suggesting potential differential adaptation by populations in these disjunct and distinct environments.