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.

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.

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.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda:Parastacidae) inferred from four mitochondrial gene regions

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

Population genetic studies on Australian freshwater crayfish, Cherax destructor (Crustacea: Parastacidae) using allozyme and RAPD markers

Allozyme and Random Amplified Polymorphic DNA (RAPD) variation was surveyed in the freshwater crayfish Cherax destructor Clark, an ecologically and commercially important species that is widespread throughout the freshwater systems of central Australia. At the intra-population level, allozymes revealed a similar level of variation to that found in other freshwater crayfish; RAPDs showed less diversity than allozymes, which was unexpected. At the inter-population level, both techniques revealed significant population structure, both within and between drainages. RAPD results were consistent with phylogeographic patterns previously identified using mtDNA. Although allozyme data showed little geographic pattern in relation to genetic variation based on multidimensional-scaling (MDS) plots on matrices of genetic distance, results of AMOVA and Mantel tests indicated significant population structuring. Each of the mtDNA lineages proposed in a previous study also showed significant genetic structure at similar levels as revealed by RAPDs but different levels by allozymes. These results reject hypotheses previously put forward on genetic homogenisation within the species due to wide-scale translocation. The implications of the findings for conservation and aquaculture of C. destructor are also discussed.

Differential effects of arginine, glutamate and phosphoarginine on Ca2+-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.

Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor

Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V_max of 42.0 µmol reducing sugars produced min–¹ mg protein–¹, a K_m of 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainly β-1,3-glycosidic bonds. In addition to the hydrolysis of β-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a V_max of 19.6 µmol reducing sugars produced min^–1 mg protein^–1, a K_m of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysing β-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

Sea-level changes and palaeo-ranges: reconstruction of ancient shorelines and river drainages and the phylogeography of the Australian land crayfish Engaeus sericatus Clark (Decapoda: Parastacidae )

Historical sea levels have been influential in shaping the phylogeography of freshwater-limited taxa via palaeodrainage and palaeoshoreline connections. In this study, we demonstrate an approach to phylogeographic analysis incorporating historical sea-level information in a nested clade phylogeographic analysis (NCPA) framework, using burrowing freshwater crayfish as the model organism. Our study area focuses on the Bass Strait region of southeastern Australia, which is marine region encompassing a shallow seabed that has emerged as a land bridge during glacial cycles connecting mainland Australia and Tasmania. Bathymetric data were analysed using Geographical Information Systems (GIS) to delineate a palaeodrainage model when the palaeocoastline was 150 m below present-day sea level. Such sea levels occurred at least twice in the past 500 000 years, perhaps more often or of larger magnitude within the last 10 million years, linking Victoria and Tasmania. Inter-locality distance measures confined to the palaeodrainage network were incorporated into an NCPA of crayfish (Engaeus sericatus Clark 1936) mitochondrial 16S rDNA haplotypes. The results were then compared to NCPAs using present-day river drainages and traditional great-circle distance measures. NCPA inferences were cross-examined using frequentist and Bayesian procedures in the context of geomorphological and historical sea-level data. We found distribution of present-day genetic variation in E. sericatus to be partly explained not only by connectivity through palaeodrainages but also via present-day drainages or overland (great circle) routes. We recommend that future studies consider all three of these distance measures, especially for studies of coastally distributed species.