How did this snail get here? Several dispersal vectors inferred for an aquatic invasive species

1. How species reach and persist in isolated habitats remains an open question in many cases, especially for rapidly spreading invasive species. This is particularly true for temporary freshwater ponds, which can be remote and may dry out annually, but may still harbour high biodiversity. Persistence in such habitats depends on recurrent colonisation or species survival capacity, and ponds therefore provide an ideal system to investigate dispersal and connectivity. 2. Here, we test the hypothesis that the wide distributions and invasive potential of aquatic snails is due to their ability to exploit several dispersal vectors in different landscapes. We explored the population structure of Physa acuta (recent synonyms: Haitia acuta, Physella acuta, Pulmonata: Gastropoda), an invasive aquatic snail originating from North America, but established in temporary ponds in Doñana National Park, southern Spain. In this area, snails face land barriers when attempting to colonise other suitable habitat. 3. Genetic analyses using six microsatellite loci from 271 snails in 21 sites indicated that (i) geographically and hydrologically isolated snail populations in the park were genetically similar to a large snail population in rice fields more than 15 km away; (ii) these isolated ponds showed an isolation-by-distance pattern. This pattern broke down, however, for those ponds visited frequently by large mammals such as cattle, deer and wild boar; (iii) snail populations were panmictic in flooded and hydrologically connected rice fields. 4. These results support the notion that aquatic snails disperse readily by direct water connections in the flooded rice fields, can be carried by waterbirds flying between the rice fields and the park and may disperse between ponds within the park by attaching to large mammals. 5. The potential for aquatic snails such as Physa acuta to exploit several dispersal vectors may contribute to their wide distribution on various continents and their success as invasive species. We suggest that the interaction between different dispersal vectors, their relation to specific habitats and consequences at different geographic scales should be considered both when attempting to control invasive freshwater species and when protecting endangered species.

Comparison of brood size and juvenile weight between a blue strain and normal-coloured strains of the Australian freshwater crayfish Cherax Destructor Clark (Decapoda: Parastacidae)

A blue strain of the yabby Cherax destructor albidus was compared to two normal-coloured strains of C. d. destructor and C. d. albidus for brood size and juvenile weight. Reproductive performance of the blue strain was found to be significantly poorer than the two normal-coloured strains. Similarly, the weight of newly independent juveniles was also found to be significantly lower for the blue strain. No differences were detected between the two normal-coloured strains in either reproductive performance or size of newly independent juveniles. The phenotypic differences between the blue strain and normal-coloured strains are most likely genetic. However, further studies are needed to investigate whether these differences are due to pleiotropy or inbreeding effects.

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish, Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.

Allozyme evidence for a new species of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) from the south-west of Western Australia

The marron, Cherax tenuimanus (Smith), is one of the most easily recognisable members of the freshwater crayfish genus Cherax. Since its description in 1912, the taxonomy of the species has not been in dispute, but recent genetic studies have demonstrated that the species is not homogenous and consists of two genetically distinct forms. One of these forms is widespread and exploited via aquaculture and the other is restricted to a single river system, the Margaret River. This paper presents allozyme data, collected over a 19-year period, which documents the introduction of the widespread form into the Margaret River and the subsequent reproductive interactions between the two forms. These data indicate minimal interbreeding between the two forms of marron and so justify their recognition as distinct species. As the original description of the marron was based on specimens collected from the Margaret River, the form native to this river retains the name C. tenuimanus and a new species, Cherax cainii Austin is described for the common, widespread form of marron. An additional outcome of this study is that C. tenuimanus has been rapidly displaced by the introduced C. cainii within the Margaret River. Consequently, urgent conservation measures are required to protect C. tenuimanus and prevent its possible extinction.

Production of Australian freshwater crayfish in earthen-based systems using pelleted diets and forage crops as food

Juvenile Cherax destructor (commonly called theyabby) were cultured in earthen-based ponds and tanks for 70–105d, and were fed pellets and/or a forage crop of the perennialwhiteclover, Trifolium repens. Three supplementary feedingstrategies were evaluated. Yabby growth on pellets consistently exceeded (by67–159%) that obtained on clover. Base-line yields for extensiveproduction systems are around 400 kg ha^–1. Thesupplementary addition of T. repens produced yields of 635kg ha^–1 (in ponds) to 1086 kgha^–1 (in tanks). The sequential addition of cut-cloverto tanks stimulated growth to levels approaching those achieved on pellets.Yabbies stocked into ponds at 17 m^–2 and fed 33%protein pellets for 100 d, resulted in a yield of 1117 kgha^–1.Pellet inputs at a rate of 129–249 g m^–2(dry matter) and 38–83 g m^–2 (protein) over70–100 d resulted in acceptable growth and feed utilisationindices. Clover inputs of 534–682 g m–2 (asdry matter) or 84–177 g m^–2 (as protein)produced reasonable growth rates but poor feed utilisation indices. Aconsiderable quantity of the dry matter and protein content of clover waseitherinefficiently utilised or directed into other production pathways. In tanks,clover inputs from 113–296 g m^–2 (drymatter) and 24–54 g m^–2 (protein) weresufficient to maintain high growth rates for 4 weeks, while in ponds, inputs of21 g m^–2 (dry matter) and 4.3 gm^–2 (protein) were sufficient for 3 weeks. During theearly weeks of production no growth advantage was gained by providing pelletstoanimals cultured in forage-based systems. Forage depletion occurred after3–4 weeks and was probably a major growth limiting factor.

Responses of freshwater biota to rising salinity levels and implications for saline water management: a review

All of the plants and animals that make up freshwater aquatic communities are affected by salinity. Many taxa possess morphological, physiological and life-history characteristics that provide some capacity for tolerance, acclimatisation or avoidance. These characteristics impart a level of resilience to freshwater communities.     To maintain biodiversity in aquatic systems it is important to manage the rate, timing, pattern, frequency and duration of increases in salinity in terms of lethal and sublethal effects, sensitive life stages, the capacity of freshwater biota to acclimatise to salinity and long-term impacts on community structure.     We have limited understanding of the impacts of saline water management on species interactions, food-web structures and how elevated salinity levels affect the integrity of communities. Little is known about the effect of salinity on complex ecosystem processes involving microbes and microalgae, or the salinity thresholds that prevent semi-aquatic and terrestrial species from using aquatic resources. Compounding effects of salinity and other stressors are also poorly understood.    Our current understanding needs to be reinterpreted in a form that is accessible and useful for water managers. Because of their complexity, many of the remaining knowledge gaps can only be addressed through a multidisciplinary approach carried out in an adaptive management framework, utilising decision-making and ecological risk assessment tools.

Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the Genus Cherax (Decapoda: Parastacidae)

One of the most important requirements for systematic and phylogenetic studies is the identification of gene regions with the appropriate level of variation for the question of interest. Molecular phylogenetic and systematic studies of freshwater crayfish have made use of DNA sequences mainly from ribosomal genes, especially the 16S rRNA gene region. Thus, little information is available on other potentially useful mitochondrial gene regions for systematic studies in these animals. In this study, we look at nucleotide variation and phylogenetic relations within and between four species of freshwater crayfish of the genus Cherax from the southwest of Western Australia using four fragments amplified from the 16S rRNA, 12S rRNA, Cytochrome Oxidase I (COI), and Cytochrome b (Cyt b) gene regions. Samples of Engaeus strictifrons, Euastacus bispinosus, and Geocharax falcata were also sequenced for comparative purposes. The size of the fragments varied from 358 bp to 600 bp. Across all samples, the four fragments showed significant phylogenetic signal and showed similar proportions of variable sites (28.81–37.33%). Average divergence within species for the mitochondrial gene regions varied from 1.18% to 4.91%, with the 16S rRNA being the least variable and Cyt b the most variable. Average divergence between species ranged 7.63–15.53%, with 16S rRNA being the least variable and COI the most variable. At the generic level, average divergence ranged 17.21–23.82%. Phylogenetic analyses of the 16S rRNA, 12S rRNA, and COI regions generated four clades consistent with the presence of four species previously identified on the basis of allozyme and morphological studies. The relationships among samples were largely congruent across the data set, although some relationships remained unresolved. Not all samples could be amplified using the Cyt b primers, and some of those that were showed quite anomalous relationships, suggesting that one or more Cyt b pseudogenes were being amplified.

Complete mitochondrial DNA sequence of the Australian freshwater crayfish, Cherax destructor (Crustacea: Decapoda: Parastacidae): a novel gene order revealed

The complete mitochondrial DNA sequence was determined for the Australian freshwater crayfish Cherax destructor (Crustacea: Decapoda: Parastacidae). The 15,895-bp genome is circular with the same gene composition as that found in other metazoans. However, we report a novel gene arrangement with respect to the putative arthropod ancestral gene order and all other arthropod mitochondrial genomes sequenced to date. It is apparent that 11 genes have been translocated (ND1, ND4, ND4L, Cyt b, srRNA, and tRNAs Ser(UGA), Leu(CUN), Ile, Cys, Pro, and Val), two of which have also undergone inversions (tRNAs Pro and Val). The ‘duplication/random loss’ mechanism is a plausible model for the observed translocations, while ‘intramitochondrial recombination’ may account for the gene inversions. In addition, the arrangement of rRNA genes is incompatible with current mitochondrial transcription models, and suggests that a different transcription mechanism may operate in C. destructor.

Molecular phylogeny and zoogeography of the freshwater crayfish genus Cherax Erichson (Decapoda: Parastacidae) in Australia

The evolutionary history and biogeography of freshwater-dependent taxa in Australia is of intrinsic interest given the present-day aridity of this continent. Cherax is the most widespread and one of the most species-rich of Australia's nine freshwater crayfish genera. The phylogenetic relationships amongst 19 of the 23 Australian Cherax were established from mitochondrial DNA sequences representing the 12S rRNA and 16S rRNA gene regions. The relationships among species support an initial east–west separation, followed by a north–south divergence in eastern Australia. Molecular clock estimations suggest that these divergences date back to the Miocene. The phylogenetic relationships support endemic speciation within geographical regions and indicate that long-distance dispersal has not led to recent speciation as previously hypothesized. This new evolutionary scenario is consistent with the climatic history of Australia and the evolutionary history of other similarly distributed freshwater-dependent organisms in Australia.

Phylogeography of the widespread Australian freshwater prawn, Macrobrachium australiense (Decapoda, Palaemonidae)

Aim: To investigate the phylogeographic structure of the widespread freshwater prawn, Macrobrachium australiense, within and between major Australian drainage basins using mitochondrial sequence data. This will enable the investigation of historical connections between major drainages and examination of hypotheses of biogeographic associations among Australian freshwater basins.

Location: Inland, eastern and northern Australia.

Methods: Sequencing 16S rRNA and ATPase 6 protein coding mitochondrial DNA genes from M. australiense from 19 locations from inland, eastern and northern Australia.

Results: Within drainage basins, haplotype trees are monophyletic, with the exception of the Finke River from the Lake Eyre Basin. Macrobrachium australiense from the two main inland drainages, the Murray–Darling and Lake Eyre Basin are divergent from each other and do not form a monophyletic group, instead the Murray–Darling Basin haplotypes clade with eastern coastal haplotypes. Haplotypes from neighbouring eastern coastal drainages were found to be quite divergent from each other.

Main conclusions: The phylogeographic relationships among M. australiense suggest that the two major inland drainages, the Murray–Darling Basin and the Lake Eyre Basin, are not biogeographically closely associated to each other. Instead the Murray–Darling Basin is more closely allied with the eastern coastal drainages across the Great Dividing Range. Despite their proximity the neighbouring southeast Queensland coastal Mary and Brisbane Rivers are also biogeographically divergent from each other. The results also indicate that the Finke River appears to have been isolated from the remainder of the Lake Eyre Basin catchment for a significant period of time.

Inheritance of molecular markers and sex in the Australian freshwater crayfish, Cherax destructor Clark

Inheritance of three kinds of molecular genetic markers (mtDNA, random-amplified polymorphic DNAs (RAPDs) and allozymes) and sex were investigated in crossbreeding experiments between three populations of the Australian freshwater crayfish Cherax destructor. Crossbreeding did not disrupt the ively maternally inherited, and allozyme and RAPD markers were transmitted following expected Mendelian principles for co-dominant and dominant traits respectively. Unlike these three markers, sex ratios were found to be distorted by crossbreeding in some families. Two crossbred families produced only females. The implications of these findings for freshwater crayfish population genetics, taxonomy and aquaculture are discussed.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.

Mitochondrial 12S rRNA sequences support the existence of a third species of freshwater blackfish (Percicthyidae: Gadopsis) from south-eastern Australia

Fish of the genus Gadopsis are a distinctive component of the freshwater fish fauna of south-eastern Australia. Gadopsis marmoratus and G. bispinosus are the only two species recognised within the genus, with the former of uncertain taxonomic status, as it is thought to be composed of at least two distinct geographical forms based on morphological and allozyme data. The objective of this study was to investigate DNA sequence divergence in Gadopsis, especially in the western portion of its distribution, using an approximately 400 base pair fragment of the mitochondrial small subunit 12S rRNA gene region in order to reassess the taxonomy of the genus. Individuals from 11 locations were sequenced and confirm that G. marmoratus and G. bispinosus are genetically distinct, and further that the G. marmoratus complex consists of two divergent clades representing the previously identified northern and southern forms. The degree of divergence between the three Gadopsis clades was similar (5–6% nucleotide substitutions), suggesting that they diverged from a common ancestor at approximately the same period in geological time. These results are consistent with previous allozyme studies and highlight the usefulness of mitochondrial DNA data coupled with allozyme information for clarifying taxonomic boundaries in morphologically conservative aquatic organisms.

Freshwater reserves in Australia : directions and challenges for the development of a comprehensive, adequate and representative system of protected areas

The establishment of a system of protected areas that samples all ecosystems, including freshwater environments, in a comprehensive, adequate and representative (CAR) manner is regarded as a cornerstone for the conservation of biodiversity. There have been few quantitative assessments of the comprehensiveness, adequacy and representativeness of freshwater reserves in Australia. This paper reviews and quantifies the effect of classification of freshwater ecosystems for conservation planning, the importance of reservation status and protection measures for developing a CAR reserve system, and aspects of reserve design for freshwater ecosystems. We propose a strategic and iterative process that incorporates these measures to assist in the efficient and effective development of freshwater reserve systems worldwide. However, the provision of suitable water regimes for freshwater reserves presents further ecological and political challenges, and even adequate reservation of freshwater ecosystems may not conserve constituent biodiversity without effective management.

The effects of various binders and moisture content on pellet stability of research diets for freshwater crayfish

Two experiments were conducted to assess the water stability of a practical research diet manufactured with various binders and differing levels of moisture. In the first experiment the binders – agar, gelatine, carrageenan, and carboxymethylcellulose (CMC) were included at both 3 and 5% of total ingredient weight. All binders were tested with equal ingredient weight to water volume, and additionally carrageenan was tested in a diet with double the water volume. The dry matter remaining following immersion for up to 180 min was calculated and the rate of pellet decay was modelled using the Weibull distribution. The analysis revealed that the rate of dry matter loss decreased with time, and that carrageenan and CMC binders were significantly better (P < 0.001) binders than the agar and gelatine. The 5% binder concentration slowed the decay rate by as much as 62% as compared with the 3% binder concentration. The second experiment compared the binding performance of carrageenan and sodium alginate in both 50% moisture and 10% moisture pellets. The same analysis revealed that 10% moisture alginate-bound pellets were more water stable than the others. A discussion of the use of moist diets for crayfish research is included.