MtDNA diversity of the critically endangered Mekong giant catfish (Pangasianodon gigas Chevey, 1913) and closely related species : implications for conservation

Catfishes of the family Pangasiidae are an important group that contributes significantly to the fisheries of the Mekong River basin. In recent times the populations of several catfish species have declined, thought to be due to overfishing and habitat changes brought about by anthropogenic influences. The Mekong giant catfish Pangasianodon gigas Chevey, 1913 is listed as Critically Endangered on the IUCN Red List. In the present study, we assessed the level of genetic diversity of nine catfish species using sequences of the large subunit of mitochondrial DNA (16S rRNA). Approximately 570 base pairs (bp) were sequenced from 672 individuals of nine species. In all species studied, haplotype diversity and nucleotide diversity ranged from 0.118±0.101 to 0.667±0.141 and from 0.0002±0.0003 to 0.0016±0.0013, respectively. Four haplotypes were detected among 16 samples from natural populations of the critically endangered Mekong giant catfish. The results, in spite of the limited sample size for some species investigated, indicated that the level of genetic variation observed in wild populations of the Mekong giant catfish (haplotype diversity=0.350±0.148, nucleotide diversity=0.0009±0.0008) is commensurate with that of some other related species. This finding indicates that (1) wild populations of the Mekong giant catfish might be more robust than currently thought or (2) present wild populations of this species carry a genetic signature of the historically larger population(s). Findings from this study also have important implications for conservation of the Mekong giant catfish, especially in designing and implementing artificial breeding programme for restocking purposes.

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.

Lack of genetic divergence found with microsatellite DNA markers in the tarakihi Nemadactylus macropterus

Three classes of molecular markers are commonly employed during population genetic studies of marine taxa: allozymes, mitochondrial DNA (mtDNA), and microsatellite DNA. These markers differ in their levels of polymorphism, and the ease and cost of their application. Nemadactylus macropterus is a commercially important marine fish from New Zealand and southern Australia that has been the subject of genetic (allozyme, mtDNA) and non-genetic (otolith microchemistry, larval advection) studies of stock structure. We collected microsatellite DNA data from this species to compare the utility of these molecular markers with those genetic methods previously applied to N. macropterus. Microsatellites did not indicate significant divergence among Australian samples, or between Australian and New Zealand samples. The latter is incongruent with the allozyme and mtDNA studies, and it is suggested that allelic homoplasy has hindered the resolution of population structure when using microsatellites.

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.

Using mitochondrial nucleotide sequences to investigate diversity and genealogical relationships within common carp (Cyprinus carpio L.)

Direct sequencing of mitochondrial DNA (mtDNA) D-loop (745 bp) and MTATPase6/MTATPase8 (857 bp) regions was used to investigate genetic variation within common carp and develop a global genealogy of common carp strains. The D-loop region was more variable than the MTATPase6/MTATPase8 region, but given the wide distribution of carp the overall levels of sequence divergence were low. Levels of haplotype diversity varied widely among countries with Chinese, Indonesian and Vietnamese carp showing the greatest diversity whereas Japanese Koi and European carp had undetectable nucleotide variation. A genealogical analysis supports a close relationship between Vietnamese, Koi and Chinese Color carp strains and to a lesser extent, European carp. Chinese and Indonesian carp strains were the most divergent, and their relationships do not support the evolution of independent Asian and European lineages and current taxonomic treatments.

Mitochondrial DNA sequence and gene organization in the Australian Blacklip Abalone Haliotis rubra (Leach)

The complete mitochondrial DNA of the blacklip abalone Haliotis rubra (Gastropoda: Mollusca) was cloned and 16,907 base pairs were sequenced. The sequence represents an estimated 99.85% of the mitochondrial genome, and contains 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes found in other metazoan mtDNA. An AT tandem repeat and a possible C-rich domain within the putative control region could not be fully sequenced. The H. rubra mtDNA gene order is novel for mollusks, separated from the black chiton Katharina tunicata by the individual translocations of 3 tRNAs. Compared with other mtDNA regions, sequences from the ATP8, NAD2, NAD4L, NAD6, and 12S rRNA genes, as well as the control region, are the most variable among representatives from Mollusca, Arthropoda, and Rhynchonelliformea, with similar mtDNA arrangements to H. rubra. These sequences are being evaluated as genetic markers within commercially important Haliotis species, and some applications and considerations for their use are discussed.

Complete mitochondrial DNA sequences of the decapod crustaceans pseudocarcinus gigas (Menippidae) and macrobrachium rosenbergii (Palaemonidae)

The complete mitochondrial DNA sequence was determined for the Australian giant crab Pseudocarcinns gigas (Crustacea: Decapoda: Menippidae) and the giant freshwater shrimp Macrobrachium rosenbergii (Crustacea: Decapoda: Palaemonidae). The Pse gigas and Mrosenbergii mitochondrial genomes are circular molecules, 15,515 and 15,772 bp in length, respectively, and have the same gene composition as found in other metazoans. The gene arrangement of M. rosenbergii corresponds with that of the presumed ancestral arthropod gene order, represented by Limulus polyphemus, except for the position of the tRNA^Leu(UUR) gene. The Pse. gigas gene arrangement corresponds exactly with that reported for another brachyuran, Portunus trituberculatus, and differs from the M. rosenbergii gene order by only the position of the tRNA^His gene. Given the relative positions of intergenic nonoding nucleotides, the “duplication/random loss” model appears to be the most plausible mechanism for the translocation of this gene. These data represent the first caridean and only the second brachyuran complete mtDNA sequences, and a source of information that will facilitate surveys of intraspecific variation within these commercially important decapod species.

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.

The molecular systematics of Leiopotherapon unicolor (Günther, 1859): testing for cryptic speciation in Australia’s most widespread freshwater fish

Leiopotherapon unicolor is the most widespread freshwater fish species in Australia. A comprehensive allozyme and mitochondrial DNA 16S rRNA data set was assembled from 141 specimens of L. unicolor collected Australia-wide in order to test for cryptic speciation in this far-ranging species. Surprisingly, little genetic diversity was observed within L. unicolor and provided no evidence for the existence of cryptic species within this lineage. In contrast, a small sample set of L. aheneus used as the outgroup showed two highly divergent haplotypes strongly suggestive of cryptic speciation. L. unicolor has a number of ecological and life history attributes that may explain the lack of significant genetic divergence over substantial geographical distances. The occurrence of other widespread fish and crustacean species that also display only limited genetic diversity indicate that climate conditions more favourable to dispersal across central and northern Australia than is suggested by the extent of present-day aridity have occurred in the relatively recent geological past.

Cultured muscle cells display defects of mitochondrial myopathy ameliorated by anti-oxidants

The mitochondrial DNA A3243G mutation causes neuromuscular disease. To investigate the muscle-specific pathophysiology of mitochondrial disease, rhabdomyosarcoma transmitochondrial hybrid cells (cybrids) were generated that retain the capacity to differentiate to myotubes. In some cases, striated muscle-like fibres were formed after innervation with rat embryonic spinal cord. Myotubes carrying A3243G mtDNA produced more reactive oxygen species than controls, and had altered glutathione homeostasis. Moreover, A3243G mutant myotubes showed evidence of abnormal mitochondrial distribution, which was associated with down-regulation of three genes involved in mitochondrial morphology, Mfn1, Mfn2 and DRP1. Electron microscopy revealed mitochondria with ultrastructural abnormalities and paracrystalline inclusions. All these features were ameliorated by anti-oxidant treatment, with the exception of the paracrystalline inclusions. These data suggest that rhabdomyosarcoma cybrids are a valid cellular model for studying muscle-specific features of mitochondrial disease and that excess reactive oxygen species production is a significant contributor to mitochondrial dysfunction, which is amenable to anti-oxidant therapy.

Mitochondrial DNA diversity of broodstock of two indigenous mahseer species, Tor tambroides and T. douronensis (Cyprinidae) cultured in Sarawak, Malaysia

Tor tambroides and T. douronensis, locally referred to as empurau and semah, respectively, are high valued mahseer species, indigenous to Sarawak, East Malaysia, with an aquaculture potential and of conservational value. Direct sequencing of mitochondrial DNA (mtDNA) 16S rRNA gene region (542 bp) was used to investigate genetic variation of T. tambroides and T. douronensis broodstock collected from different geographic locations in Sarawak and maintained at the Indigenous Fish Research and Production Center (IFRPC), Tarat, Sarawak, Malaysia. A total of 11 unique haplotypes were identified, of which six were detected in T. tambroides, and five in T. douronensis. Overall, nucleotide diversity (π) was low, ranging from 0.000 to 0.006, and haplotype diversity (h) ranged from 0.000 to 0.599. Although the analysis failed to detect genetic variation amongst populations of T. tambroides (significant pairwise FST was found for only one test, but pairwise haplotype frequencies were not statistically significant), substantial inter-population divergence among T. douronensis was recognised, especially those originating from different river systems (pairwise F_ST = 0.754 to 1.000, P < 0.05). Fixed haplotype differences were found in one population of T. douronensis. Average nucleotide divergence between T. tambroides and T. douronensis was 0.018, similar to the amount recognised between T. tambroides and the outgroup T. khudree (0.017). In addition, phylogenetic analysis revealed that the T. douronensis mtDNA consisted of two highly divergent clusters (0.020), one of which is more closely related to T. tambroides rather than with the other group of haplotypes of the conspecifics. The findings from the present study have important implications for aquaculture, management and conservation of these two species. The data also raise some concerns regarding the taxonomic status of T. douronensis, which needs to be addressed.

Foot mucus is a good source for non-destructive genetic sampling in polyplacophora

A non-destructive method for collecting samples for DNA analysis from the mucus of molluscs was successfully adapted for use with the genus Ischnochiton. DNA was extracted using a Chelex-based method and the COI subunit of the mtDNA was amplified and sequenced. Sequences from the mucus were crosschecked against sequences from the foot tissue of the same animal and were found to be identical. This method provides a non-destructive way of carrying out larger studies of the genetics of rare organisms and may be of general use for genetic-based field studies of molluscs.