High connectivity in rastrelliger kanagurta: Influence of historical signatures and migratory behaviour inferred from mtDNA Cytochrome b

Phylogeographic patterns and population structure of the pelagic Indian mackerel, Rastrelliger kanagurta were examined in 23 populations collected from the Indonesian-Malaysian Archipelago (IMA) and the West Indian Ocean (WIO). Despite the vast expanse of the IMA and neighbouring seas, no evidence for geographical structure was evident. An indication that R. kanagurta populations across this region are essentially panmictic. This study also revealed that historical isolation was insufficient for R. kanagurta to attain migration drift equilibrium. Two distinct subpopulations were detected between the WIO and the IMA (and adjacent populations); interpopulation genetic variation was high. A plausible explanation for the genetic differentiation observed between the IMA and WIO regions suggest historical isolation as a result of fluctuations in sea levels during the late Pleistocene. This occurrence resulted in the evolution of a phylogeographic break for this species to the north of the Andaman Sea.

Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

Molecular phylogeny supports the paraphyletic nature of the genus Trogoderma (Coleoptera: Dermestidae) collected in the Australasian ecozone

To date, a molecular phylogenetic approach has not been used to investigate the evolutionary structure of Trogoderma and closely related genera. Using two mitochondrial genes, Cytochrome Oxidase I and Cytochrome B, and the nuclear gene, 18S, the reported polyphyletic positioning of Trogoderma was examined. Paraphyly in Trogoderma was observed, with one Australian Trogoderma species reconciled as sister to all Dermestidae and the Anthrenocerus genus deeply nested within the Australian Trogoderma clade. In addition, time to most recent common ancestor for a number of Dermestidae was calculated. Based on these estimations, the Dermestidae origin exceeded 175 million years, placing the origins of this family in Pangaea.

Insights into historical drainage evolution based on the phylogeography of the chevron snakehead fish (Channa striata) in the Mekong Basin

1. The phylogeography of freshwater taxa is often integrally linked with landscape changes such as drainage re-alignments that may present the only avenue for historical dispersal for these taxa. Classical models of gene flow do not account for landscape changes and so are of little use in predicting phylogeography in geologically young freshwater landscapes. When the history of drainage formation is unknown, phylogeographical predictions can be based on current freshwater landscape structure, proposed historical drainage geomorphology, or from phylogeographical patterns of co-distributed taxa. 2. This study describes the population structure of a sedentary freshwater fish, the chevron snakehead (Channa striata), across two river drainages on the Indochinese Peninsula. The phylogeographical pattern recovered for C. striata was tested against seven hypotheses based on contemporary landscape structure, proposed history and phylogeographical patterns of codistributed taxa. 3. Consistent with the species ecology, analysis of mitochondrial and microsatellite loci revealed very high differentiation among all sampled sites. A strong signature of historical population subdivision was also revealed within the contemporary Mekong River Basin (MRB). Of the seven phylogeographical hypotheses tested, patterns of co-distributed taxa proved to be the most adequate for describing the phylogeography of C. striata. 4. Results shed new light on SE Asian drainage evolution, indicating that the Middle MRB probably evolved via amalgamation of at least three historically independent drainage sections and in particular that the Mekong River section centred around the northern Khorat Plateau in NE Thailand was probably isolated from the greater Mekong for an extensive period of evolutionary time. In contrast, C. striata populations in the Lower MRB do not show a phylogeographical signature of evolution in historically isolated drainage lines, suggesting drainage amalgamation has been less important for river landscape formation in this region.

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the β-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the β-actin and 28S markers, with the exception of Sus scrofa (pig) β-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.

Echolocation calls, wing shape, diet and phylogenetic diagnosis of the endemic Chinese bat Myotis pequinius

We describe the echolocation calls, flight morphology and diet of the endemic Chinese bat Myotis pequinius Thomas, 1908. Orientation calls are broadband, and reach low terminal frequencies. Diet comprised 80% beetles by volume. Wing shape and call design suggest that the bats fly in cluttered habitats, and the possession of moderately long ears and the dietary composition imply they forage at least sometimes by gleaning. Myotis pequinius resembles a larger Oriental version of the western Palaearctic species M. nattereri. Phylogenetic analysis based on sequences of the cytochrome b gene of mitochondrial DNA (1,140 base pairs) from a range of Palaearctic Myotis species confirmed that M. pequinius is close to the nattereri group, and is a sister-species to the eastern Palaearctic M. bombinus. One bat sequenced from China could not be identified from available species descriptions. It was smaller than M. pequinius, and also differed from it in sequence divergence by 6.7%, suggesting the existence of additional, cryptic taxonomic diversity in this group. Our phylogenetic analysis also supports the recognition of M. schaubi as a species distinct from M. nattereri in Transcaucasia and south-western Asia. Myotis nattereri tschuliensis is more closely related to M. schaubi than to M. nattereri, and is best considered either as a subspecies of M. schaubi, or possibly as a distinct species.

Phylogenetics of small horseshoe bats from east Asia based on mitochondrial DNA sequence variation

We undertook analyses of mitochondrial DNA gene sequences and echolocation calls to resolve phylogenetic relationships among the related bat taxa Rhinolophus pusillus (sampled across China), R. monoceros (Taiwan), R. cornutus (main islands of Japan), and R. c. pumilus (Okinawa, Japan), Phylogenetic trees and genetic divergence analyses were constructed by combining new complete mitochondrial cytochrome-b gene sequences and partial mitochondrial control region sequences with published sequences. Our work showed that these 4 taxa formed monophyletic groups in the phylogenetic tree. However, low levels of sequence divergence among the taxa, together with similarities in body size and overlapping echolocation call frequencies, point to a lack of taxonomic distinctiveness. We therefore suggest that these taxa are better considered as geographical subspecies rather than distinct species, although this should not diminish the conservation importance of these island populations, which are important evolutionarily significant units. Based on our findings, we suggest that the similarities in body size and echolocation call frequency in these rhinolophids result from their recent common ancestry, whereas similarities in body size and call frequency with R. hipposideros of Europe are the result of convergent evolution.

The long and short of it: branch lengths and the problem of placing the New Zealand short-tailed bat.

The taxonomic position of the endemic New Zealand bat genus Mystacina has vexed systematists ever since its erection in 1843. Over the years the genus has been linked with many microchiropteran families and superfamilies. Most recent classifications place it in the Vespertilionoidea, although some immunological evidence links it with the Noctilionoidea (=Phyllostomoidea). We have sequenced 402 bp of the mitochondrial cytochrome b gene for M. tuberculata (Gray in Dieffenbach, 1843), and using both our own and published DNA sequences for taxa in both superfamilies, we applied different tree reconstruction methods to find the appropriate phylogeny and different methods of estimating confidence in the parts of the tree. All methods strongly support the classification of Mystacina in the Noctilionoidea. Spectral analysis suggests that parsimony analysis may be misleading for Mystacina's precise placement within the Noctilionoidea because of its long terminal branch. Analyses not susceptible to long-branch attraction suggest that the Mystacinidae is a sister family to the Phyllostomidae. Dating the divergence times between the different taxa suggests that the extant chiropteran families radiated around and shortly after the Cretaceous–Tertiary boundary. We discuss the biogeographical implications of classifying Mystacina within the Noctilionoidea and contrast our result with those classifications placing Mystacina in the Vespertilionoidea, concluding that evidence for the latter is weak.

Conservation genetics of the water mouse, Xeromys myoides Thomas, 1889

The water mouse, Xeromys myoides, is currently recognised as a vulnerable species in Australia, inhabiting a small number of distinct and isolated coastal regions of Queensland and the Northern Territory. An examination of the evolutionary history and contemporary influences shaping the genetic structure of this species is required to make informed conservation management decisions. Here, we report the first analysis undertaken on the phylogeography and population genetics of the water mouse across its mainland Australian distribution. Genetic diversity was assessed at two mitochondrial DNA (Cytochrome b, 1000 bp; D-loop, 400 bp) and eight microsatellite DNA loci. Very low genetic diversity was found, indicating that water mice underwent a recent expansion throughout their Australian range and constitute a single evolutionarily significant unit. Microsatellite analyses revealed that the highest genetic diversity was found in the Mackay region of central Queensland; population substructure was also identified, suggesting that local populations may be isolated in this region. Conversely, genetic diversity in the Coomera region of south-east Queensland was very low and the population in this region has experienced a significant genetic bottleneck. These results have significant implications for future management, particularly in terms of augmenting populations through translocations or reintroducing water mice in areas where they have gone extinct.

The Long and Short of It: Branch Lengths and the Problem of Placing the New Zealand Short-Tailed Bat, Mystacina

The taxonomic position of the endemic New Zealand bat genus Mystacina has vexed systematists ever since its erection in 1843. Over the years the genus has been linked with many microchiropteran families and superfamilies. Most recent classifications place it in the Vespertilionoidea, although some immunological evidence links it with the Noctilionoidea (=Phyllostomoidea). We have sequenced 402 bp of the mitochondrial cytochrome b gene for M. tuberculata (Gray in Dieffenbach, 1843), and using both our own and published DNA sequences for taxa in both superfamilies, we applied different tree reconstruction methods to find the appropriate phylogeny and different methods of estimating confidence in the parts of the tree. All methods strongly support the classification of Mystacina in the Noctilionoidea. Spectral analysis suggests that parsimony analysis may be misleading for Mystacina's precise placement within the Noctilionoidea because of its long terminal branch. Analyses not susceptible to long-branch attraction suggest that the Mystacinidae is a sister family to the Phyllostomidae. Dating the divergence times between the different taxa suggests that the extant chiropteran families radiated around and shortly after the Cretaceous-Tertiary boundary. We discuss the biogeographical implications of classifying Mystacina within the Noctilionoidea and contrast our result with those classifications placing Mystacina in the Vespertilionoidea, concluding that evidence for the latter is weak.

Mitochondrial DNA sequence analysis from multiple gene fragments reveals genetic heterogeneity of Crassostrea ariakensis in East Asia

The native Asian oyster, Crassostrea ariakensis is one of the most common and important Crassostrea species that occur naturally along the coast of East Asia. Molecular species diagnosis is a prerequisite for population genetic analysis of wild oyster populations because oyster species cannot be discriminated reliably using external morphological characters alone due to character ambiguity. To date there have been few phylogeographic studies of natural edible oyster populations in East Asia, in particular this is true of the common species in Korea C. ariakensis. We therefore assessed the levels and patterns of molecular genetic variation in East Asian wild populations of C. ariakensis from Korea, Japan, and China using DNA sequence analysis of five concatenated mtDNA regions namely; 16S rRNA, cytochrome oxidase I, cytochrome oxidase II, cytochrome oxidase III, and cytochrome b. Two divergent C. ariakensis clades were identified between southern China and remaining sites from the northern region. In addition, hierarchical AMOVA and pairwise UST analyses showed that genetic diversity was discontinuous among wild populations of C. ariakensis in East Asia. Biogeographical and historical sea level changes are discussed as potential factors that may have influenced the genetic heterogeneity of wild C. ariakensis stocks across this region.

Cytochrome p450 isozyme protein verified in the skin of southern hemisphere humpback whales (megaptera novaeangliae) : implications for biochemical biomarker assessment

Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress.

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wingshape data

Background Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. Results Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P < 0.0001) as compared to a ‘Euclidean’ scenario for which direct geographic distances between sample sites was used (r2 = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. BEAST analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the ‘non-Euclidean’ scenario (r2 = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. Conclusions Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

Delivery of nitric oxide for analysis of the function of cytochrome c'

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.