Transparency tools in gene patenting for informing policy and practice

In the recent decision Association for Molecular Pathology v. Myriad Genetics1, the US Supreme Court held that naturally occurring sequences from human genomic DNA are not patentable subject matter. Only certain complementary DNAs (cDNA), modified sequences and methods to use sequences are potentially patentable. It is likely that this distinction will hold for all DNA sequences, whether animal, plant or microbial2. However, it is not clear whether this means that other naturally occurring informational molecules, such as polypeptides (proteins) or polysaccharides, will also be excluded from patents. The decision underscores a pressing need for precise analysis of patents that disclose and reference genetic sequences, especially in the claims. Similarly, data sets, standards compliance and analytical tools must be improved—in particular, data sets and analytical tools must be made openly accessible—in order to provide a basis for effective decision making and policy setting to support biological innovation. Here, we present a web-based platform that allows such data aggregation, analysis and visualization in an open, shareable facility. To demonstrate the potential for the extension of this platform to global patent jurisdictions, we discuss the results of a global survey of patent offices that shows that much progress is still needed in making these data freely available for aggregation in the first place.

Feral pig populations are structured at fine spatial scales in tropical Queensland, Australia

Feral pigs occur throughout tropical far north Queensland, Australia and are a significant threat to biodiversity and World Heritage values, agriculture and are a vector of infectious diseases. One of the constraints on long-lasting, local eradication of feral pigs is the process of reinvasion into recently controlled areas. This study examined the population genetic structure of feral pigs in far north Queensland to identify the extent of movement and the scale at which demographically independent management units exist. Genetic analysis of 328 feral pigs from the Innisfail to Tully region of tropical Queensland was undertaken. Seven microsatellite loci were screened and Bayesian clustering methods used to infer population clusters. Sequence variation at the mitochondrial DNA control region was examined to identify pig breed. Significant population structure was identified in the study area at a scale of 25 to 35 km, corresponding to three demographically independent management units (MUs). Distinct natural or anthropogenic barriers were not found, but environmental features such as topography and land use appear to influence patterns of gene flow. Despite the strong, overall pattern of structure, some feral pigs clearly exhibited ancestry from a MU outside of that from which they were sampled indicating isolated long distance dispersal or translocation events. Furthermore, our results suggest that gene flow is restricted among pigs of domestic Asian and European origin and non-random mating influences management unit boundaries. We conclude that the three MUs identified in this study should be considered as operational units for feral pig control in far north Queensland. Within a MU, coordinated and simultaneous control is required across farms, rainforest areas and National Park Estates to prevent recolonisation from adjacent localities.

A multi-genome analysis approach enables tracking of the invasion of a single Russian wheat aphid (Diuraphis noxia) clone throughout the New World

This study investigated the population genetics, demographic history and pathway of invasion of the Russian wheat aphid (RWA) from its native range in Central Asia, the Middle East and Europe to South Africa and the Americas. We screened microsatellite markers, mitochondrial DNA and endosymbiont genes in 504 RWA clones from nineteen populations worldwide. Following pathway analyses of microsatellite and endosymbiont data, we postulate that Turkey and Syria were the most likely sources of invasion to Kenya and South Africa, respectively. Furthermore, we found that one clone transferred between South Africa and the Americas was most likely responsible for the New World invasion. Finally, endosymbiont DNA was found to be a high resolution population genetic marker, extremely useful for studies of invasion over a relatively short evolutionary history time frame. This study has provided valuable insights into the factors that may have facilitated the recent global invasion by this damaging pest.

Seasonal occurrence and distribution of Bryde's whales in the Hauraki Gulf, New Zealand

The Hauraki Gulf is a large, shallow embayment located north of Auckland City (36°51′S, 174°46′E), New Zealand. Bryde's whales (Balaenoptera edeni) are the most frequently observed balaenopterid in these waters. To assess the use of the Hauraki Gulf for this species, we examined the occurrence and distribution in relation to environmental parameters. Data were collected from a platform of opportunity during 674 daily surveys between March 2003 and February 2006. A total of 760 observations of Bryde's whales were recorded throughout the study period during 371 surveys. The number of Bryde's whales sighted/day was highest in winter, coinciding with the coolest median sea-surface temperature (14.6°C). Bryde's whales were recorded throughout the Hauraki Gulf in water depths ranging from 12.1–59.8 m (mean = 42.3, SD = 5.1). Cow–calf pairs were most frequently observed during the austral autumn in water depths of 29.9–53.9 m (mean = 40.8, SD = 5.2). Data from this study suggest Bryde's whales in the Hauraki Gulf exhibit a mix of both “inshore” and “offshore” characteristics from the Bryde's whales examined off the coast of South Africa. Based on complete mitochondrial DNA sequences, Sasaki et al. (2006) recognized two sister species of Bryde's whales: Balaenoptera brydei and B. edeni, with the latter including small-type, more coastal Bryde's whales from Japan, Hong Kong, and Australia. Their samples and samples in previous analyses of small-type whales, all originated from eastern and southeastern Asia. These authors did not include the forms of Bryde's whales that occur in other regions, e.g., in the Pacific off Peru (Valdivia et al. 1981), in the Atlantic off Brazil (Best 1977) and in the western Indian Ocean off South Africa (Best 1977). Recent genetic analysis using mtDNA from the “inshore” and “offshore” forms from South Africa confirms the offshore form is B. brydei, and establishes that the inshore form is more closely related to B. brydei than to B. edeni (Penry 2010). These different forms do vary considerably in their habitat use and ecology (refer to Table 1 for a detailed comparison between the South African inshore and offshore forms, as described by Best (1967, 1977) and the Bryde's whales from New Zealand (Wiseman 2008). Recent genetic analysis on the Bryde's whales in the Hauraki Gulf suggests they are B. brydei (Wiseman 2008). However, pending resolution of the uncertainty within and between species of this genus, we follow the Society of Marine Mammal's committee on taxonomy, who state that B. edeni applies to all Bryde's whales.

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.

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.

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.

Phylogeographical relationships of Sicilian brown trout and the effects of genetic introgression on morphospace occupation

Genetic introgression of aquaculture stocks in local forms is well documented in many fish species but their evolutionary consequences for the local populations have not been thoroughly explored. Due to its wide geographical range, the existence of many locally adapted forms and the frequent occurrence of introgression of aquaculture stocks in local forms, brown trout represents the ideal system to study the effects of such introgressions. Here, we focus on a group of rivers and streams in Sicily (Italy), and, by using molecular tools, we show that autochthonous populations are probably derived from the Southern Atlantic clade, which is present in the Iberian peninsula and North Africa. Three out of the four studied rivers reveal signs of genetic introgression of domestic stocks. Finally, by using advanced geometric morphometric analyses, we show that genetic introgression produces a higher degree of morphological variability relative to that observed in non-introgressed populations.

Phylogeography of the green turtle, Chelonia mydas, in the Southwest Indian Ocean

Patterns of mitochondrial DNA (mtDNA) variation were used to analyse the population genetic structure of southwestern Indian Ocean green turtle (Chelonia mydas) populations. Analysis of sequence variation over 396 bp of the mtDNA control region revealed seven haplotypes among 288 individuals from 10 nesting sites in the Southwest Indian Ocean. This is the first time that Atlantic Ocean haplotypes have been recorded among any Indo-Pacific nesting populations. Previous studies indicated that the Cape of Good Hope was a major biogeographical barrier between the Atlantic and Indian Oceans because evidence for gene flow in the last 1.5 million years has yet to emerge. This study, by sampling localities adjacent to this barrier, demonstrates that recent gene flow has occurred from the Atlantic Ocean into the Indian Ocean via the Cape of Good Hope. We also found compelling genetic evidence that green turtles nesting at the rookeries of the South Mozambique Channel (SMC) and those nesting in the North Mozambique Channel (NMC) belong to separate genetic stocks. Furthermore, the SMC could be subdivided in two different genetic stocks, one in Europa and the other one in Juan de Nova. We suggest that this particular genetic pattern along the Mozambique Channel is attributable to a recent colonization from the Atlantic Ocean and is maintained by oceanic conditions in the northern and southern Mozambique Channel that influence early stages in the green turtle life cycle.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

The genetic structure of Australasian green turtles (Chelonia mydas): Exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Φ = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.

Genetic population structure of red snappers (Lutjanus malabaricus Bloch & Schneider, 1801 and Lutjanus erythropterus Bloch, 1790) in central and eastern Indonesia and northern Australia

The genetic population structure of red snapper Lutjanus malabaricus and Lutjanus erythropterus in eastern Indonesia and northern Australia was investigated by allozyme electrophoresis and sequence variation in the control region of mtDNA. Samples were collected from eight sites in Indonesia and four sites in northern Australia for both species. A total of 13 allozyme loci were scored. More variable loci were observed in L. malabaricus than in L. erythropterus. Sequence variation in the control region (left domain) of the mitochondrial genome was assessed by RFLP and direct sequencing. MtDNA haplotype diversity was high (L. erythropterus, 0.95 and L. malabaricus, 0.97), as was intraspecific sequence divergence, (L. erythropterus, 0.0-12.5% and L. malabaricus, 0.0-9.5%). The pattern of mtDNA haplotype frequencies grouped both species into two broad fisheries stocks with a genetic boundary either between Kupang and Sape (L. malabaricus) or between Kupang and Australian Timor Sea (L. erythropertus). The allozyme analyses revealed similar boundaries for L. erythropterus. Seven allozymes stocks compared to two mtDNA stocks of L. malabaricus including Ambon, which was not sampled with mtDNA, however, were reported. Possible reasons for differences in discrimination between the methods include: i) increased power of multiple allozyme loci over the single mtDNA locus, ii) insufficient gene sampling in the mtDNA control region and iii) relative evolutionary dynamics of nuclear (allozyme loci) and mitochondrial DNA in these taxa. Allozyme and haplotype data did not distinguish separate stocks among the four Australian locations nor the central Indonesian (Bali and Sape locations) for both L. malabaricus and L. erythropterus.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Trapping to monitor tephritid movement: Results, best practice, and assessment of alternatives

Movement of tephritid flies underpins their survival, reproduction, and ability to establish in new areas and is thus of importance when designing effective management strategies. Much of the knowledge currently available on tephritid movement throughout landscapes comes from the use of direct or indirect methods that rely on the trapping of individuals. Here, we review published experimental designs and methods from mark-release-recapture (MRR) studies, as well as other methods, that have been used to estimate movement of the four major tephritid pest genera (Bactrocera, Ceratitis, Anastrepha, and Rhagoletis). In doing so, we aim to illustrate the theoretical and practical considerations needed to study tephritid movement. MRR studies make use of traps to directly estimate the distance that tephritid species can move within a generation and to evaluate the ecological and physiological factors that influence dispersal patterns. MRR studies, however, require careful planning to ensure that the results obtained are not biased by the methods employed, including marking methods, trap properties, trap spacing, and spatial extent of the trapping array. Despite these obstacles, MRR remains a powerful tool for determining tephritid movement, with data particularly required for understudied species that affect developing countries. To ensure that future MRR studies are successful, we suggest that site selection be carefully considered and sufficient resources be allocated to achieve optimal spacing and placement of traps in line with the stated aims of each study. An alternative to MRR is to make use of indirect methods for determining movement, or more correctly, gene flow, which have become widely available with the development of molecular tools. Key to these methods is the trapping and sequencing of a suitable number of individuals to represent the genetic diversity of the sampled population and investigate population structuring using nuclear genomic markers or non-recombinant mitochondrial DNA markers. Microsatellites are currently the preferred marker for detecting recent population displacement and provide genetic information that may be used in assignment tests for the direct determination of contemporary movement. Neither MRR nor molecular methods, however, are able to monitor fine-scale movements of individual flies. Recent developments in the miniaturization of electronics offer the tantalising possibility to track individual movements of insects using harmonic radar. Computer vision and radio frequency identification tags may also permit the tracking of fine-scale movements by tephritid flies by automated resampling, although these methods come with the same problems as traditional traps used in MRR studies. Although all methods described in this chapter have limitations, a better understanding of tephritid movement far outweighs the drawbacks of the individual methods because of the need for this information to manage tephritid populations.

Grey Mackerel (Scomberomorus semifasciatus) microsatellite genotypes (nine loci) representing twelve regions along Australia's northern coastline from the eastern coast of Queensland to the western coast of Western Australia.

Scomberomorus semifasciatus is an Australian endemic found in tropical, coastal waters from eastern to western Australia. Commercial and recreational exploitation is common and regulated by state-based authorities. This study used mitochondrial DNA sequence and microsatellite markers to elucidate the population structure of Scomberomorus semifasciatus collected from twelve, equidistant sampling locations. Samples (n=544) were genotyped with nine microsatellite loci, and 353 were sequenced for d-loop (384 bp) and ATP (800bp) mitochondrial DNA gene regions. Combined interpretation of microsatellite and mtDNA data identified four genetic stocks of S. semifasciatus: Western Australia, northwest coast of the Northern Territory, Gulf of Carpentaria and the east coast of Queensland. Connectivity among stocks across northern Australia from the Northern Territory to the east coast of Queensland was high, but in contrast, there was a clear genetic break between populations in Western Australia compared to the rest of northern Australia. This indicates a restriction to gene flow possibly associated with suboptimal habitat along the Kimberley coast (northwestern Australia). The appropriate scale of management for this species corresponds to the jurisdictions of the three Australian states, except that the Gulf of Carpentaria stock should be co-managed by authorities in Queensland and Northern Territory.