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Catastrophic wildfires impacted large areas of western Kangaroo Island (KI), South Australia in 2019–2020, burning habitat for many species, including large proportions of the distributional range of the KI micro-trapdoor spider Moggridgea rainbowi, which led to it being listed as Endangered under Australia’s Environment Protection and Biodiversity Conservation Act (EPBC Act). In order to assess population genetic structure in M. rainbowi and detect diagnosable lineages and their distributional patterns across KI, 28 individuals were genotyped for 2495 loci, sampling from all known populations of the species. Population genetic and phylogenetic analyses of nuclear and mitochondrial sequence data provided strong support for three Evolutionarily Significant Units (ESUs) within M. rainbowi; two populations on eastern KI and a heavily fire-impacted western population. High levels of divergence and fixed allelic differences between 5 and 16% indicate a lack of gene flow between ESUs and long periods of isolation. Distributional patterns of these lineages match likely locations of isolation events caused by successive changes to sea level during the Quaternary (2.58 million years ago to present), which led to KI being intermittently connected to the mainland or separated into one or more islands. Our findings have strong conservation implications for M. rainbowi and highlight the importance of inclusion of population genetic structure to inform conservation strategies and to conserve lineage biodiversity at the species level and below.

The Scirtidae Fleming, 1821 has been identified as one of the earliest diverging groups of Polyphagan beetles and is particularly speciose in Australia. However, very little is known about the origin of the Australian scirtids and there is a need for a robust, well-supported phylogeny to guide the genus and species descriptions and understand the relationships among taxa. In this study we carried out a phylogenetic analysis of the Australian Scirtinae Fleming, 1821, using DNA sequence data from ultraconserved elements (UCEs) and included representative taxa from New Zealand, New Caledonia, South America, South Africa and Eurasia in the analysis. Bayesian analyses of a concatenated dataset from 79 taxa recovered four major Southern Hemisphere groupings and two Australian–Eurasian groupings. The Veronatus group mainly consisted of genera from New Zealand, with the three Australian representatives only distantly related to each other. Relaxed molecular clock analyses, using the estimated age of the crown node of the Polyphaga for calibration, support a Gondwanan history for four of the groups of Australian Scirtinae and a northern origin for two groups. Our results highlight the value of commercially available UCEs for resolving the phylogenetic history of ancient groups of Coleoptera.

Amphipod crustaceans comprise a significant and enigmatic component of Australian groundwater ecosystems, particularly in the Pilbara region of Western Australia. Many amphipod species in the Pilbara, including species in the genus Nedsia Barnard & Williams, 1995, are considered short range endemics, poorly or contentiously defined by taxonomic treatments based on morphology alone and have uncertain distributions as a consequence of this taxonomy. A modern systematic revision of Nedsia is presented here, utilising both molecular and morphological analyses alongside distributional data to delineate species. We describe 13 new species of Nedsia, confirm three existing species and synonymise eight previously described species. Nedsia species are confirmed to be functionally morphologically cryptic, with COI divergences at the 5–20% level. We present comparatively reduced taxonomic descriptions for these cryptic amphipod species in an effort to provide an accelerated pathway for future taxonomic work. The research provides the basis for future environmental impact assessments involving Nedsia species and ongoing monitoring of the groundwater communities these form part of in the resource-rich Pilbara region.

Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. Our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to deepen our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.

Information on the extent of genetic differentiation among seabird populations is essential for conservation planning. Here we used, for the first time in little penguins (Eudyptula minor), a next generation sequencing approach to investigate population genetic structure, gene flow and inbreeding among eight colonies in South Australia. We found that Troubridge Island individuals were genetically distinct from the other sampled colonies, and that colonies on all islands were connected by moderate levels of gene flow possibly due to migration from the Kangaroo Island colonies. While further genetic sampling is needed, these data support the idea that South Australia may be a zone of subtle and rapid genetic change for little penguins.

This study investigates two parasitic reptile ticks — Bothriocroton hydrosauri and Amblyomma limbatum — of the sleepy lizard (Tiliqua rugosa) that abut at a 1–2 km wide parapatric boundary in South Australia. Long‐term research has investigated potential mechanisms to explain the maintenance of this boundary but has not uncovered why the distribution of A. limbatum does not extend further south. It has been previously hypothesised that pathogens may be responsible for maintaining parapatric boundaries. Rickettsia spp. has previously been reported in B. hydrosauri ticks. This study explored whether Rickettsia spp. occurs in co‐occurring A. limbatum. We observed that Rickettsia spp. was absent from all A. limbatum ticks and that 83% of examined B. hydrosauri were found to be positive with a spotted fever group Rickettsia strain. This study puts forward the hypothesis that Rickettsia spp. could contribute to the maintenance of the Mt Mary parapatric boundary between these two tick species. Further work is required to determine whether Rickettsia spp. can be transmitted from B. hydrosauri to A. limbatum and — if transmission can occur — to explore whether Rickettsia is lethal to A. limbatum ticks.

In the framework of neutral theory of molecular evolution, genes specific to the development and function of eyes in subterranean animals living in permanent darkness are expected to evolve by relaxed selection, ultimately becoming pseudogenes. However, definitive empirical evidence for the role of neutral processes in the loss of vision over evolutionary time remains controversial. In previous studies, we characterized an assemblage of independently-evolved water beetle (Dytiscidae) species from a subterranean archipelago in Western Australia, where parallel vision and eye loss have occurred. Using a combination of transcriptomics and exon capture, we present evidence of parallel coding sequence decay, resulting from the accumulation of frameshift mutations and premature stop codons, in eight phototransduction genes (arrestins, opsins, ninaC and transient receptor potential channel genes) in 32 subterranean species in contrast to surface species, where these genes have open reading frames. Our results provide strong evidence to support neutral evolutionary processes as a major contributing factor to the loss of phototransduction genes in subterranean animals, with the ultimate fate being the irreversible loss of a light detection system.

Amphipod crustaceans comprise a significant and enigmatic component of Australian groundwater ecosystems, particularly in the Pilbara region of Western Australia. Many amphipod species in the Pilbara, including species in the genus Nedsia Barnard & Williams, 1995, are considered short range endemics, poorly or contentiously defined by taxonomic treatments based on morphology alone and have uncertain distributions as a consequence of this taxonomy. A modern systematic revision of Nedsia is presented here, utilising both molecular and morphological analyses alongside distributional data to delineate species. We describe 13 new species of Nedsia, confirm three existing species and synonymise eight previously described species. Nedsia species are confirmed to be functionally morphologically cryptic, with COI divergences at the 5–20% level. We present comparatively reduced taxonomic descriptions for these cryptic amphipod species in an effort to provide an accelerated pathway for future taxonomic work. The research provides the basis for future environmental impact assessments involving Nedsia species and ongoing monitoring of the groundwater communities these form part of in the resource-rich Pilbara region.

Mitochondrial heteroplasmy is the occurrence of more than one type of mitochondrial DNA within a single individual. Although generally reported to occur in a small subset of individuals within a species, there are some instances of widespread heteroplasmy across entire populations.

ContextIn the era of the Anthropocene, habitat loss and environmental change threaten the persistence of many species. Genotyping-By-Sequencing (GBS) is a useful molecular tool for understanding how patterns of gene flow are associated with contemporary habitat distributions that may be affected by environmental change. Two parapatric subspecies of the threatened thick-billed grasswren (TBGW; Amytornis modestus) more frequently occur in different plant communities. As such, a preference for plant community type could reduce subspecific introgression and increase genetic diversity at the parapatric boundary.AimsWe aimed to measure gene flow within and among two TBGW subspecies and tested whether divergent genomic markers were associated with plant community type.MethodsWe sequenced 118 individuals from either of the two TBGW subspecies or in the region of parapatry and identified 7583 SNPs through ddRADseq.Key resultsWe found evidence of asymmetric gene flow and a genomic pattern of isolation-by-distance. There were sixteen genomic outliers correlated with plant community type (regardless of location).ConclusionsThese findings show that plant community type does not prevent introgression in one subspecies (A. m. raglessi), but low dispersal and habitat heterogeneity could contribute to the maintenance of distinct subspecific morphotypes. Local adaptation in different plant community types could also provide a mechanism for future divergence.ImplicationsWe suggest subspecific introgression could increase genetic variation and the adaptive potential of the species, facilitating species persistence under conditions of climate change.Introgression between grasswren subspeciesCharacterising gene flow facilitates conservation management. This study used genomic markers to measure gene flow between thick-billed grasswren subspecies and found results that support taxonomic identification of the two subspecies and suggests grasswrens have low dispersal and may benefit from increased genetic diversity. Recognition of models of divergence with gene flow will be necessary for future conservation management.

BackgroundThe repeatome, the collection of repetitive DNA sequences represented by transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), is found in high proportion in organisms across the tree of life. Grasshoppers have large genomes (average 9 Gb), containing large amounts of repetitive DNA which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of the repeatome and its contribution to genome evolution, in four chromosomal races of the morabine grasshopperVandiemenella viaticaspecies complex.ResultsWe obtained linked-read genome assemblies of 2.73-3.27 Gb from estimated genome sizes of 4.26-5.07 Gb DNA per haploid genome of the four chromosomal races ofV. viatica. These constitute the third largest insect genomes assembled so far (the largest being two locust grasshoppers). Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs constituting 66 to 75 % per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314-463 Mb per assembly), indicating that their large genome size is likely due to similar rates of TE accumulation across the four races. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a repertoire of satDNA families in the ancestor of theV. viaticachromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of theV. viaticaspecies complex.ConclusionIn-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entireDrosophilagenomes, which likely explains the large genome sizes in grasshoppers. Although the TE and satDNA repertoires were rather similar between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.

Most subterranean animals are assumed to have evolved from surface ancestors following colonisation of a cave system, however very few studies have raised the possibility of 'subterranean speciation' in underground habitats (i.e. obligate cave-dwelling organisms (troglobionts) descended from troglobiotic ancestors). Numerous endemic subterranean diving beetle species from spatially-discrete calcrete aquifers in Western Australia (stygobionts) have evolved independently from surface ancestors; however, several cases of sympatric sister species raises the possibility of subterranean speciation. We tested this hypothesis using vision (phototransduction) genes that are evolving under neutral processes in subterranean species and purifying selection in surface species. Using sequence data from 32 subterranean and five surface species in the genus Paroster (Dytiscidae), we identified deleterious mutations in: long wavelength opsin (lwop), arrestin 1 (arr1), and arrestin 2 (arr2) shared by a sympatric sister-species triplet, arr1 shared by a sympatric sister-species pair, and lwop and arr2 shared among closely related species in adjacent calcrete aquifers. In all cases, a common ancestor possessed the function-altering mutations, implying they were already adapted to aphotic environments. Our study represents one of the first confirmed cases of subterranean speciation in cave insects. The assessment of genes undergoing pseudogenisation provides a novel way of testing modes of speciation and the history of diversification in blind cave animals. This article is protected by copyright. All rights reserved.

BackgroundRepetitive DNA sequences, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), collectively called the “repeatome”, are found in high proportion in organisms across the Tree of Life. Grasshoppers have large genomes, averaging 9 Gb, that contain a high proportion of repetitive DNA, which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of repetitive DNA sequences in four chromosomal races of the morabine grasshopperVandiemenella viaticaspecies complex and determine their contribution to genome evolution.ResultsWe obtained linked-read genome assemblies of 2.73–3.27 Gb from estimated genome sizes of 4.26–5.07 Gb DNA per haploid genome of the four chromosomal races ofV. viatica. These constitute the third largest insect genomes assembled so far. Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs, constituting 66 to 75% per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314–463 Mb per assembly), indicating that their large genome sizes are likely due to similar rates of TE accumulation. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a diversity of satDNA families in the ancestor of theV. viaticachromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of theV. viaticaspecies complex.ConclusionThis in-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entireDrosophilagenomes, which likely explains the large genome sizes in grasshoppers. Despite an overall high similarity of the TE and satDNA diversity between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.

The major histocompatibility complex (MHC) is a hypervariable gene family that plays an essential role in the recognition of pathogens and immune response. Research on the reptilian MHC has lagged behind other vertebrate groups. Here, we genotyped individuals of an Australian agamid lizard species at MHC class I loci using a recently developed clustering method and family group data. Our method allowed identification of low amplification efficiency alleles and estimation of both type I and II genotyping error rates. The number of MHC class I alleles per individual varied within populations and together with allele segregation patterns, suggests either natural copy number variation or allele dropout. Genotypes from individuals across five populations revealed shared alleles among populations and low allelic diversity in an island population. Finally, we identified sites under selection and designated them putative peptide binding regions. Our results provide a foundation for future work on the MHC class I region of agamid lizards.

The endangered subspecies of Southern Emu-Wren (Stipiturus malachurus intermedius) in the Mount Lofty Ranges (MLR), South Australia has a fragmented distribution and poor dispersal capabili...

A recent DNA barcoding study of Australian microgastrines (Hymenoptera: Braconidae) sought to use next-generation sequencing of the cytochrome c oxidase subunit 1 (COI) barcoding gene region, the wingless (WG) gene and the internal transcribed spacer 2 (ITS2) to delimit molecular species in a highly diverse group of parasitic wasps. Large intragenomic distances between ITS2 variants, often larger than the average interspecific variation, caused difficulties in using ITS2 for species delimitation in both threshold and tree-based approaches, and the gene was not included in the reported results of the previous DNA barcoding study. We here report on the intragenomic, and the intra- and interspecies, variation in ITS2in the microgastrine genus Diolcogasterto further investigate the value of ITS2as a marker for species delimitation and phylogenetics of the Microgastrinae. Distinctive intragenomic variant patterns were found in different species of Diolcogaster, with some species possessing a single major variant, and others possessing many divergent variants. Characterizing intragenomic variation of ITS2is critical as it is a widely used marker in hymenopteran phylogenetics and species delimitation, and large intragenomic distances such as those found in this study may obscure phylogenetic signal.

Next-generation sequencing has revolutionised molecular ecology. Its key advantages are a more accurate representation of genetic variation made possible by the generation of large volumes of data, more quickly and at a lower price per sequence than traditional sequencing methods. Yet these benefits come with a cost. For example, next-generation sequencing is error prone and requires increased quality control compared with traditional methods. Problems associated with next-generation sequencing may be exacerbated when sequencing gene complexes such as the major histocompatibility complex (MHC). Although not eliminated, significant progress has been made in addressing some of those problems and there is an increasing literature utilising this technology for studies of the MHC. However, what is generally lacking is detailed documentation of the methods used, and clear reasoning, for each step. Here we document detailed methodology, using an Australian lizard, Egernia stokesii, as a case study, with ...

The Microgastrinae are a hugely diverse subfamily of endoparasitoid wasps of lepidopteran caterpillars. They are important in agriculture as biological control agents and play a significant ecological role in the regulation of caterpillar populations. Whilst the group has been the focus of intensive rearing and DNA barcoding studies in the Northern Hemisphere, the Australian fauna has received little attention. In total, 99 species have been described from or have been introduced into Australia, but the real species diversity for the region is clearly much larger than this. In this study, museum ethanol samples and recent field collections were mined for hundreds of specimens of microgastrine wasps, which were then barcoded for the COI region, ITS2 ribosomal spacer and the wingless nuclear genes, using a pooled sequencing approach on an Illumina Miseq system. Full COI sequences were obtained for 525 individuals which, when combined with 162 publicly available sequences, represented 417 haplotypes, and a total of 236 species were delimited using a consensus approach. By more than doubling the number of known microgastrine wasp species in Australia, our study highlights the value of DNA barcoding in the context of employing high-throughput sequencing methods of bulk ethanol museum collections for biodiversity assessment.

The origin of the mammalian order Eulipotyphla has been debated intensively with arguments around whether they began diversifying before or after the Cretaceous-Palaeogene (K-Pg) boundary at 66 Ma. Here, we used an in-solution nucleotide capture method and next generation DNA sequencing to determine the sequence of hundreds of ultra-conserved elements (UCEs), and conducted phylogenomic and molecular dating analyses for the four extant eulipotyphlan lineages—Erinaceidae, Solenodontidae, Soricidae, and Talpidae. Concatenated maximum-likelihood analyses with single or partitioned models and a coalescent species-tree analysis showed that divergences among the four major eulipotyphlan lineages occurred within a short period of evolutionary time, but did not resolve the interrelationships among them. Alternative suboptimal phylogenetic hypotheses received consistently the same amount of support from different UCE loci, and were not significantly different from the maximum likelihood tree topology, suggesting the prevalence of stochastic lineage sorting. Molecular dating analyses that incorporated among-lineage evolutionary rate differences supported a scenario where the four eulipotyphlan families diversified between 57.8 and 63.2 Ma. Given short branch lengths with low support values, traces of rampant genome-wide stochastic lineage sorting, and post K-Pg diversification, we concluded that the crown eulipotyphlan lineages arose through a rapid diversification after the K-Pg boundary when novel niches were created by the mass extinction of species. We are grateful to Rafael Borroto-Páez, Jorge L. Delgado-Labañino,José Alvarez-Lemus for their help in sampling of solenodons in Cuba.We thank Kieren Mitchell for his advice on our next generation se-quencing experiments. We also appreciate Junji Moribe and AkioShinohara for providing the animal photos ofCrocidura dsinezumiandMogera wogura, respectively. This study was supported by theLeadership Fund of Hokkaido University (no grant number to SDO) andJSPS, Grants-in-Aid for Scientific Research (16H05655 to MK). Scopus

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km(2) region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag–Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

Three new species of Koonunga were discovered in surface and subterranean waters in southern Australia, and were defined using mtDNA analyses and morphology. The new species are: Koonunga hornei Leijs & King; K. tatiaraensis Leijs & King and K. allambiensis Leijs & King. Molecular clock analyses indicate that the divergence times of the species are older than the landscape that they currently inhabit. Different scenarios explaining this apparent discrepancy are discussed in the context of the palaeography of the area. A freshwater epigean origin for Koonunga is considered the most likely hypothesis, whereby some lineages made the transition to the subterranean environment within the last few million years influenced by significant climatic cooling/drying. We discuss the possibility that one stygobitic lineage secondarily regained some of its body pigmentation as adaptation to increased photic conditions after cave collapse and forming of cenotes during the last glacial maximum.

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to β-strands and α-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

A monoclonal antibody designated MBR 39 has been generated against a membrane associated protein found selectively on lysosomes. MBR 39 reacts with the cytosolic face of the lysosome and was used to develop an organelle binding assay which reacted with high density organelles characteristic of lysosomes. These organelles contained lysosomal enzyme markers which included the integral membrane protein acetyl-CoA:α-glucosaminide N-acetyltransferase and the soluble lysosomal enzyme markers acid phosphatase (mature form), β-hexosaminidase, arylsulfatase, and α- l -iduronidase. Under conditions which disrupt lysosomes the release of the latter soluble lysosomal enzymes was demonstrated from MBR 39 bound organelles. Immunoblots of MBR 39 with purified fibroblast lysosomal membrane, demonstrated reactivity with polypeptides of molecular mass 63 kDa (major species) and 73 kDa (minor species).

Summary The importance of microeukaryotes (protists, ciliates, fungi, algae and small invertebrates) in riverine food webs is well recognised, but the difficulty in surveying these taxa has meant that monitoring of biodiversity in rivers has focused on larger organisms. Microeukaryote biodiversity was assessed by pyrosequencing of the conserved 18S rRNA gene during summer and autumn at two river sites and a weir pool site that form a flow gradient in the Murray River, Australia. Concurrent measurements were made of phytoplankton concentrations, irradiance conditions, river metabolism and associated water quality attributes. Based on operational taxonomic units derived from the molecular analyses, significant differences were identified in eukaryotes between the river and the weir pool. Taxonomic assignment was possible for 66% of the operational taxonomic units, reflecting the large amount of unsurveyed diversity present. Visual identification of algae corroborated the orders identified in the molecular data set, and more orders were detected using pyrosequencing. Day length, depth of light penetration and phytoplankton biomass and primary productivity were strongly linked to eukaryote community composition suggesting the importance of algae as an energy source. Our finding that microeukaryote biodiversity is linked with environmental characteristics demonstrates the potential of 18S rRNA pyrosequencing for more comprehensive assessment of the condition of an aquatic ecosystem, but further data are required to confirm this potential.

A deficiency of the enzyme arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type arylsulfatase B.

The arid Yilgarn region of Western Australia contains numerous subterranean calcrete aquifers with unique assemblages of obligate groundwater invertebrates (stygofauna). We aimed to establish a DNA barcoding framework for the macro-invertebrates present in a single calcrete, as a basis for future assessment of biodiversity of the Yilgarn calcretes and for investigating food webs. Intense sampling of a bore field grid in the Sturt Meadows calcrete was undertaken to obtain representatives of the entire macro-invertebrate ecosystem. A 623-bp fragment of the mitochondrial cytochrome c oxidase 1 (COI) gene was used to provide DNA barcodes for stygobiont macro-invertebrates plus terrestrial organisms that are found in the calcrete. Phylogenetic analyses revealed the existence of 12 divergent monophyletic groups of haplotypes. Subterranean amphipods (Chiltoniidae) showed three groups of COI haplotypes with sequence divergences between them of >11%. Allozyme analyses found a large number of fixed allelic differences between these three amphipod groups, indicating that there are three morphologically cryptic species within the Sturt Meadows calcrete. Unlike the sister triplet of dytiscid beetles present, the amphipods are not sister clades and are more closely related to other Yilgarn and non-Yilgarn amphipods than to each other. Our results show that the aquifer contains at least 12 macro-invertebrate species and DNA barcoding provides a useful means for discriminating species in this system.

A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.

The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcγRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcγRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcγRIIa may be an essential feature of the FcγRIIa signaling cascade.

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.

The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.

The Yilgarn calcrete aquifers in Western Australia are an interesting system for investigating the process of speciation within subterranean habitats, because of the limited opportunities for dispersal between isolated calcretes. The presence of different-sized diving beetles (Dytiscidae) in separate calcretes, including sympatric sister-species pairs, suggests that species may have evolved within calcretes by an adaptive shift as a result of ecological-niche differentiation. We have studied the potential for trophic niche partitioning in a sister triplet of diving beetles, of distinctly different sizes, from a single aquifer. Fragments of the mitochondrial COI gene, specific to known species of amphipods and copepods, were polymerase chain reaction-amplified from each of the three beetle species, indicating that there is an overlap in their prey items. Significant differences were found in the detected diets of the three species, and results showed a propensity for prey preferences of amphipods by the large beetles and one species of copepod for the small beetles. A terrestrial source of carbon to the calcrete was suggested by stable isotope analyses. The combined approach of molecular, stable isotope and behavioural studies have provided insight into the trophic ecology of this difficult-to-access environment, providing a framework for more fine-scale analyses of the diet of different-sized species to examine speciation underground.

Australia was historically considered a poor prospect for subterranean fauna but, in reality, the continent holds a great variety of subterranean habitats, with associated faunas, found both in karst and non-karst environments. This paper critically examines the diversity of subterranean fauna in several key regions for the mostly arid western half of Australia. We aimed to document levels of species richness for major taxon groups and examine the degree of uniqueness of the fauna. We also wanted to compare the composition of these ecosystems, and their origins, with other regions of subterranean diversity world-wide. Using information on the number of ‘described’ and ‘known’ invertebrate species (recognised based on morphological and/or molecular data), we predict that the total subterranean fauna for the western half of the continent is 4140 species, of which ~10% is described and 9% is ‘known’ but not yet described. The stygofauna, water beetles, ostracods and copepods have the largest number of described species, while arachnids dominate the described troglofauna. Conversely, copepods, water beetles and isopods are the poorest known groups with less than 20% described species, while hexapods (comprising mostly Collembola, Coleoptera, Blattodea and Hemiptera) are the least known of the troglofauna. Compared with other regions of the world, we consider the Australian subterranean fauna to be unique in its diversity compared with the northern hemisphere for three key reasons: the range and diversity of subterranean habitats is both extensive and novel; direct faunal links to ancient Pangaea and Gondwana are evident, emphasising their early biogeographic history; and Miocene aridification, rather than Pleistocene post-ice age driven diversification events (as is predicted in the northern hemisphere), are likely to have dominated Australia’s subterranean speciation explosion. Finally, we predict that the geologically younger, although more poorly studied, eastern half of the Australian continent is unlikely to be as diverse as the western half, except for stygofauna in porous media. Furthermore, based on similar geology, palaeogeography and tectonic history to that seen in the western parts of Australia, southern Africa, parts of South America and India may also yield similar subterranean biodiversity to that described here.