Tissue Samples For Genetic Analyses Research Articles

A Rapid Method to Confine and Safely Handle Bees in the Field.

Improving understanding of the basic biology and ecology of many insect pollinators, particularly specialist or rare taxa, is a priority for many researchers. As such, there is often a need to temporarily confine field-collected organisms in a non-injurious manner in order to gain information or support additional studies. This protocol represents a thoroughly tested, quick, and inexpensive field method for safely handling bees of conservation concern that can easily be tailored toward specific project needs, including organism identification, pollen removal, marking, and/or collection of non-lethal tissue samples for genetic analysis. This methodology can serve as an additional option in the researcher's toolbox to use when certain scenarios arise. It is anticipated that this methodology can be adapted for use with other insect species as well as used by individuals of varying experience and skill levels. It can be of great value to researchers studying specialist bees or conducting host-specific studies. The data collection made possible by this protocol will be invaluable to help researchers address critical data gaps for many pollinator species, plant-pollinator network structures, and pollinator conservation and management initiatives.

New records of ray-finned fishes (Actinopterygii) from Puerto Morelos Reef National Park (Mexican Caribbean)

We documented the nearshore ray-finned fishes in Puerto Morelos Reef National Park (PMRNP) by sampling 57 localities, including rocky intertidal pools, sandy bottoms, Thalassia beds, coral reefs, artificial reefs, karstic-slab bottoms, demersal-pelagic areas, and sessile-Sargassum patches. We recorded seven species new to Caribbean Mexican waters and one hundred and six species new to the PMRNP, most of which are small cryptobenthic reef fish. The resultant checklist includes 349 species, and specimens of 285 of which (81.7%) have been deposited in an ichthyological museum collection. These include both voucher organisms and with tissue samples for genetic analysis. A comparison with inventories at other localities of the Greater Caribbean indicates that more targeted sampling for cryptobenthic and deep-reef fishes is needed to provide a complete inventory. We also comment on the local use of some species as fishery resources.

Geolocator tagging links distributions in the non-breeding season to population genetic structure in a sentinel North Pacific seabird.

We tested the hypothesis that segregation in wintering areas is associated with population differentiation in a sentinel North Pacific seabird, the rhinoceros auklet (Cerorhinca monocerata). We collected tissue samples for genetic analyses on five breeding colonies in the western Pacific Ocean (Japan) and on 13 colonies in the eastern Pacific Ocean (California to Alaska), and deployed light-level geolocator tags on 12 eastern Pacific colonies to delineate wintering areas. Geolocator tags were deployed previously on one colony in Japan. There was strong genetic differentiation between populations in the eastern vs. western Pacific Ocean, likely due to two factors. First, glaciation over the North Pacific in the late Pleistocene might have forced a southward range shift that historically isolated the eastern and western populations. And second, deep-ocean habitat along the northern continental shelf appears to act as a barrier to movement; abundant on both sides of the North Pacific, the rhinoceros auklet is virtually absent as a breeder in the Aleutian Islands and Bering Sea, and no tagged birds crossed the North Pacific in the non-breeding season. While genetic differentiation was strongest between the eastern vs. western Pacific, there was also extensive differentiation within both regional groups. In pairwise comparisons among the eastern Pacific colonies, the standardized measure of genetic differentiation (FꞌST) was negatively correlated with the extent of spatial overlap in wintering areas. That result supports the hypothesis that segregation in the non-breeding season is linked to genetic structure. Philopatry and a neritic foraging habit probably also contribute to the structuring. Widely distributed, vulnerable to anthropogenic stressors, and exhibiting extensive genetic structure, the rhinoceros auklet is fully indicative of the scope of the conservation challenges posed by seabirds.

All Hands on Deck: Local Ecological Knowledge and Expert Volunteers Contribute to the First Delisting of a Marine Fish Species Under the Endangered Species Act

The listing of three species of rockfish (Sebastes spp.) under the Endangered Species Act (ESA) in 2010 motivated the development of a cooperative research program between government agencies and the local recreational fishing and SCUBA diving communities of Puget Sound (PS), WA, USA. This program has examined rockfish life history characteristics, movement behavior, young-of-year recruitment and the population responses to fishing in PS. Of most significance has been the involvement of the recreational fishing community in testing whether the three ESA-listed rockfish populations in PS were genetically discrete from populations outside of PS. There was considerable uncertainty in the answer to this question during the original ESA listing process due to limited data on these three species in PS. We used the knowledge and expert angling skills of the fishing community to locate these rare species and non-lethally collect tissue samples for genetic analyses. Results showed that yelloweye rockfish (S. ruberrimus) was genetically differentiated between PS and the outer coast, but the boundaries of the population were different than previously assumed. In contrast, canary rockfish (S. pinniger) was genetically similar across PS and the outer coast. Federal managers used these results to expand the ESA-listed boundaries of yelloweye rockfish and to delist canary rockfish – the first delisting of a marine fish species from the ESA. The knowledge and angling expertise of the Puget Sound recreational fishing community were directly responsible for the successful collection of data to test hypotheses that resulted in very specific management decisions.

Water, water everywhere: environmental DNA can unlock population structure in elusive marine species.

Determining management units for natural populations is critical for effective conservation and management. However, collecting the requisite tissue samples for population genetic analyses remains the primary limiting factor for a number of marine species. The harbour porpoise (Phocoena phocoena), one of the smallest cetaceans in the Northern Hemisphere, is a primary example. These elusive, highly mobile small animals confound traditional approaches of collecting tissue samples for genetic analyses, yet their nearshore habitat makes them highly vulnerable to fisheries by-catch and the effects of habitat degradation. By exploiting the naturally shed cellular material in seawater and the power of next-generation sequencing, we develop a novel approach for generating population-specific mitochondrial sequence data from environmental DNA (eDNA) using surface seawater samples. Indications of significant genetic differentiation within a currently recognized management stock highlights the need for dedicated eDNA sampling throughout the population's range in southeast Alaska. This indirect sampling tactic for characterizing stock structure of small and endangered marine mammals has the potential to revolutionize population assessment for otherwise inaccessible marine taxa.

Tonic immobility in the zebra shark Stegostoma fasciatum and its use for capture methodology

When conducting biological investigations on shark species, capture and brief restraint procedures may be required in order to collect ecological, behavioral and physiological data. Tonic immobility is an innate, reversible, coma-like stasis displayed by a large number of taxa, including sharks, and has been used for brief restraint, but is rarely used during capture itself. Here, we present a novel, non-lethal capture method of zebra sharks, Stegostoma fasciatum, using tonic immobility, in the field. Zebra sharks were caught by free diving, and on SCUBA, inducing a state of tonic immobility by quickly applying tight-fisted manual pressure to the caudal fin, instead of the dorso-ventral inversion method. Divers were then able to collect tissue samples for genetic analysis, and in some cases, maintain the animals in a state of tonic immobility during transport up to the research vessel for additional data collection. Decreased duration of anthropogenic intervention, and minimal physical struggle associated with this capture and restraint method is expected to reduce physiological markers of distress. Tonic immobility induction and other methods of non-lethal capture deserve continued investigation.

Blood gene expression studies in migraine: Potential and caveats.

Global gene expression analysis may be used to obtain insights into the functional processes underlying migraine. However, there is a shortage of high-quality post-mortem brain tissue samples for genetic analysis. One approach is to use a more accessible tissue as a surrogate, such as peripheral blood. Discuss the benefits and caveats of blood genomic profiling in migraine and its potential application in the development of biomarkers of migraine susceptibility and outcome. Demonstrate the utility of blood-based expression profiles in migraine by analysing pilot Illumina HT-12 expression data from 76 (38 case, 38 control) whole-blood samples. Current evidence suggests peripheral blood is a biologically valid substrate for genetic studies of migraine, and may be used to identify biomarkers and therapeutic pathways. Pilot blood gene expression data confirm that expression profiles significantly differ between migraine case and non-migraine control individuals.

Validation of the synonymy of the teleost blenniid fish species Salariasphantasticus Boulenger 1897 and Salarias anomalus Regan 1905 with Ecsenius pulcher (Murray 1887) based on DNA barcoding and morphology.

As currently recognized, Ecsenius pulcher includes Salarias pulcher (type material has a banded color pattern), S. anomalus (non-banded), and S. phantasticus (banded). The color patterns are not sex linked, and no other morphological features apparently distinguish the three nominal species. The recent collection of banded and non-banded specimens of Ecsenius pulcher from Iran has provided the first tissue samples for genetic analyses. Here we review the taxonomic history of E. pulcher and its included synonyms and genetically analyze tissue samples of both color patterns. Salarias anomalus is retained as a synonym of E. pulcher because DNA barcode data suggest that they represent banded and non-banded color morphs of a single species. Furthermore, the large size of the largest type specimen of S. anomalus (herein designated as the lectotype) suggests that it belongs to E. pulcher. A single non-banded specimen from Iran is genetically distinct from E. pulcher and appears to represent an undescribed species. Salarias phantasticus is retained as a synonym of E. pulcher because the primary morphological difference between the two nominal species-presence of spots on the dorsal fin in E. pulcher and absence of those spots in S. phantasticus-is not a valid taxonomic character; rather, the spots represent galls that contain the larval stages of a parasitic crustacean. As males and females of Ecsenius species have been confused in the literature, we describe and illustrate the genital regions of both and comment on possible new blenniid synapomorphies that our investigation revealed.

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.

Invasion history affects genetic structure in island rat populations

Invasive species usually go through a period of reduced genetic variability due to a bottleneck. The genetic constitution of an invading population should therefore reflect the time since invasion and the number of introduction events. We studied genetic population structure of three rat species occurring sympatrically on the island of Futuna (46 km2), where they were introduced recently (Rattus rattus), 80–170 years ago (R. norvegicus) and about 3000 years ago (R. exulans). From 2007 to 2012, we trapped 3900 rats during a rodent regulation programme over 20 000 trap nights and took 565 rat tissue samples for genetic analyses. By examining genetic variation and spatial distribution of genetic resemblance, we evaluated the consequences of the time after introduction for genetic population structure. Observed heterozygosity, expected heterozygosity, number of alleles and allelic richness generally increased with increasing length of time a species was introduced to the island. The species with longer invasion history had also higher spatial admixture. However, in R. norvegicus, the observed heterozygosity was lower and inbreeding higher than expected by the invasion history. We relate this with the assumption that this species was introduced once or a few times and in low numbers, while R. exulans was intentionally introduced many times by Polynesian navigators and R. rattus had a high chance of being introduced on multiple occasions in recent times.

A novel conservation approach provides insights into the management of rare cetaceans

The conservation of rare or cryptic species in inaccessible habitats represents a particular challenge to biologists. Since 1991, a collaborative research program involving members of the public, indigenous communities, the Museum of New Zealand Te Papa Tongarewa (NMNZ) and the Department of Conservation has provided tissue samples for genetic analysis of stranded, or beach-cast cetaceans. The New Zealand Cetacean Tissue Archive (NZCeTA), initiated and maintained by the University of Auckland, is now one of the largest archives of its kind in the world, with tissue samples or extracted DNA from 1982 individuals. Species identity has been confirmed by DNA barcoding, using mtDNA control region sequences for 65% of the samples representing 35 species, 11 of which are from the poorly known beaked whale family, Ziphiidae. Although these animals regularly strand around the coastline of New Zealand there are no known areas at sea where they can be reliably found and very few reported live sightings. Samples collected from strandings of three species: Gray’s (Mesoplodon grayi); straptoothed (Mesoplodon layardii) and Cuvier’s beaked whale (Ziphius cavirostris), represent 83% (n=225) of all ziphiids in the NZCeTA (n=272). As an example of the archives utility, we used the spatial and temporal distribution of these records to provide new evidence for key habitat of these cryptic species and for seasonal and sex-biased patterns of stranding mortality. As beaked whales are known to be threatened by anthropogenic activity in other parts of the world, these records provide a critical baseline for understanding the future impacts of planned development in New Zealand’s near and offshore waters.

Got It Clipped? The Effect of Tail Clipping on Tail Gripping Performance in Chameleons

Toe and tail clipping are commonly used methods for permanent marking of animals and for obtaining tissue samples for genetic analyses. Although it has been tested whether toe clipping affects locomotor performance (and thus potentially the fitness of an individual), little is known about the effect of tail clipping. Tails are important organs in many amphibians and reptiles and are used for balance or stability during locomotion or as prehensile organs. Effects of tail autotomy or the removal of large parts of the tail have previously been demonstrated. Here, we test whether the removal of a small part (,5 mm) of the distal tail in chameleons affects their ability to cling to branches of different diameters by measuring gripping strength using of a force platform. Our data show no significant or directional effect of tail clipping on the maximal forces that can be generated by the tail and, thus, suggest that tail clipping can be used as a method for tissue collection. Toe and tail clipping are commonly used methods for permanent marking of animals and to obtain tissue samples for genetic analyses (Ferner, 1979; Gosner and Collura, 1996). In lizards, tail clipping is predominantly used to obtain tissue for genetic analysis. Toe clipping is, in addition, used to permanently mark individuals. Given the possibility of clipping several toes on each foot, this gives the researcher access to a nearly unlimited number of individual codes. Despite being minimally invasive, previous studies have documented how toe clipping can affect locomotor perfor-

Chilean Blue Whales as a Case Study to Illustrate Methods to Estimate Abundance and Evaluate Conservation Status of Rare Species

Often abundance of rare species cannot be estimated with conventional design-based methods, so we illustrate with a population of blue whales (Balaenoptera musculus) a spatial model-based method to estimate abundance. We analyzed data from line-transect surveys of blue whales off the coast of Chile, where the population was hunted to low levels. Field protocols allowed deviation from planned track lines to collect identification photographs and tissue samples for genetic analyses, which resulted in an ad hoc sampling design with increased effort in areas of higher densities. Thus, we used spatial modeling methods to estimate abundance. Spatial models are increasingly being used to analyze data from surveys of marine, aquatic, and terrestrial species, but estimation of uncertainty from such models is often problematic. We developed a new, broadly applicable variance estimator that showed there were likely 303 whales (95% CI 176-625) in the study area. The survey did not span the whales' entire range, so this is a minimum estimate. We estimated current minimum abundance relative to pre-exploitation abundance (i.e., status) with a population dynamics model that incorporated our minimum abundance estimate, likely population growth rates from a meta-analysis of rates of increase in large baleen whales, and two alternative assumptions about historic catches. From this model, we estimated that the population was at a minimum of 16.5% (95% CI 7.3-34.4%) of pre-exploitation levels in 1998 under one catch assumption and 12.4% (CI 5.4-26.3%) of pre-exploitation levels under the other. Thus, although Chilean blue whales are probably still at a small fraction of pre-exploitation abundance, even these minimum abundance estimates demonstrate that their status is better than that of Antarctic blue whales, which are still <1% of pre-exploitation population size. We anticipate our methods will be broadly applicable in aquatic and terrestrial surveys for rarely encountered species, especially when the surveys are intended to maximize encounter rates and estimate abundance.

Comparative mitochondrial and nuclear quantitative PCR of historical marine mammal tissue, bone, baleen, and tooth samples

AbstractThe use of historical and ancient tissue samples for genetic analysis is increasing, with ever greater numbers of samples proving to contain sufficient mitochondrial and even nuclear DNA for multilocus analysis. DNA yield, however, remains highly variable and largely unpredictable based solely on sample morphology or age. Quantification of DNA from historical and degraded samples can greatly improve efficiency of screening DNA extracts prior to attempting sequencing or genotyping, but requires sequence‐specific quantitative polymerase chain reaction (qPCR) based assays to detect such minute quantities of degraded DNA. We present two qPCR assays for marine mammal DNA quantification, and results from analysis of DNA extracted from preserved soft tissues, bone, baleen, and tooth from several cetacean species. These two assays have been shown to amplify DNA from 26 marine mammal species representing 12 families of pinnipeds and cetaceans. Our results indicate that different tissues retain different ratios of mitochondrial to nuclear DNA, and may be more or less suitable for analysis of nuclear loci. Specifically, historical bone and tooth samples average 60‐fold higher ratio of mitochondrial DNA to nuclear DNA than preserved fresh soft tissue, and the ratio is almost 8000‐fold higher in baleen.

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci: a conservation alert?

Genetic variation at allozyme and mitochondrial DNA loci was investigated in the Australian lungfish, Neoceratodus forsteri Krefft 1870. Tissue samples for genetic analysis were taken non-lethally from 278 individuals representing two spatially distinct endemic populations (Mary and Burnett rivers), as well as one population thought to be derived from an anthropogenic translocation in the 1890's (Brisbane river). Two of 24 allozyme loci resolved from muscle tissue were polymorphic. Mitochondrial DNA nucleotide sequence diversity estimated across 2,235 base pairs in each of 40 individuals ranged between 0.000423 and 0.001470 per river. Low genetic variation at allozyme and mitochondrial loci could be attributed to population bottlenecks, possibly induced by Pleistocene aridity. Limited genetic differentiation was detected among rivers using nuclear and mitochondrial markers suggesting that admixture may have occurred between the endemic Mary and Burnett populations during periods of low sea level when the drainages may have converged before reaching the ocean. Genetic data was consistent with the explanation that lungfish were introduced to the Brisbane river from the Mary river. Further research using more variable genetic loci is needed before the conservation status of populations can be determined, particularly as anthropogenic demands on lungfish habitat are increasing. In the interim we recommend a management strategy aimed at conserving existing genetic variation within and between rivers.