Estimating Inbreeding Rates in Natural Populations: Addressing the Problem of Incomplete Pedigrees.

Understanding and estimating inbreeding is essential for managing threatened and endangered wildlife populations. However, determination of inbreeding rates in natural populations is confounded by incomplete parentage information. We present an approach for quantifying inbreeding rates for populations with incomplete parentage information. The approach exploits knowledge of pedigree configurations that lead to inbreeding coefficients of F = 0.25 and F = 0.125, allowing for quantification of Pr(I|k): the probability of observing pedigree I given the fraction of known parents (k). We developed analytical expressions under simplifying assumptions that define properties and behavior of inbreeding rate estimators for varying values of k. We demonstrated that inbreeding is overestimated if Pr(I|k) is not taken into consideration and that bias is primarily influenced by k. By contrast, our new estimator, incorporating Pr(I|k), is unbiased over a wide range of values of k that may be observed in empirical studies. Stochastic computer simulations that allowed complex inter- and intragenerational inbreeding produced similar results. We illustrate the effects that accounting for Pr(I|k) can have in empirical data by revisiting published analyses of Arabian oryx (Oryx leucoryx) and Red deer (Cervus elaphus). Our results demonstrate that incomplete pedigrees are not barriers for quantifying inbreeding in wild populations. Application of our approach will permit a better understanding of the role that inbreeding plays in the dynamics of populations of threatened and endangered species and may help refine our understanding of inbreeding avoidance mechanisms in the wild.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Variation in migratory behavior influences regional genetic diversity and structure among American Kestrel populations (Falco sparverius) in North America.

Birds employ numerous strategies to cope with seasonal fluctuations in high-quality habitat availability. Long distance migration is a common tactic; however, partial migration is especially common among broadly distributed species. Under partial migration systems, a portion of a species migrates, whereas the remainder inhabits breeding grounds year round. In this study, we identified effects of migratory behavior variation on genetic structure and diversity of American Kestrels (Falco sparverius), a widespread partial migrant in North America. American Kestrels generally migrate; however, a resident group inhabits the southeastern United States year round. The southeastern group is designated as a separate subspecies (F. s. paulus) from the migratory group (F. s. sparverius). Using mitochondrial DNA and microsatellites from 183 and 211 individuals, respectively, we illustrate that genetic structure is stronger among nonmigratory populations, with differentiation measures ranging from 0.060 to 0.189 depending on genetic marker and analysis approach. In contrast, measures from western North American populations ranged from 0 to 0.032. These findings suggest that seasonal migratory behavior is also associated with natal and breeding dispersal tendencies. We likewise detected significantly lower genetic diversity within nonmigratory populations, reflecting the greater influence of genetic drift in small populations. We identified the signal of population expansion among nonmigratory populations, consistent with the recent establishment of higher latitude breeding locations following Pleistocene glacial retreat. Differentiation of F. s. paulus and F. s. sparverius reflected subtle differences in allele frequencies. Because migratory behavior can evolve quickly, our analyses suggest recent origins of migratory American Kestrel populations in North America.

Beware of the shrunken gallbladder - Case report of intraoperatively diagnosed gallbladder agenesis.

INTRODUCTION AND IMPORTANCE: Gallbladder Agenesis, the congenital absence of the gallbladder, is a very rare biliary anomaly found in 13 to 65 people per 100,000. Symptomatic patients usually present with abdominal pain, nausea, and emesis, and are often misdiagnosed with gallbladder pathologies leading to unnecessary operations. CASE PRESENTATION: We report the case of a 63-year-old Caucasian female patient who presented with recurrent right upper quadrant pain (RUQ) and found to have a contracted gallbladder on ultrasonography (US). A hepatobiliary iminodiacetic acid (HIDA) scan was positive, and the patient was misdiagnosed with chronic cholecystitis. No gallbladder was found on laparoscopy and patient was diagnosed intraoperatively with gallbladder agenesis. Normal biliary anatomy was confirmed intraoperatively using the Firefly technique and there was no ectopic gallbladder on computed tomography (CT) scan obtained postoperatively. Patient currently doing well on conservative pain management. CLINICAL DISCUSSION: Patients with gallbladder agenesis and normal bile ducts often have symptoms similar to that of usual gallbladder problems. Accurately diagnosing these patients preoperatively remains a challenge. CONCLUSION: Symptomatic patients with agenesis of the gallbladder are often diagnosed intraoperatively despite major advances in diagnostic imaging techniques. In these cases, minimizing the risk of injury to the biliary tree is crucial. As our case demonstrates, it is critical for surgeons to become increasingly aware of this rare and important congenital anomaly. A very high index of suspicion is warranted in patients with a shrunken or contracted gallbladder. When in doubt, obtain cross-sectional imaging.

Gallbladder volvulus with gangrenous cholecystitis - A case report.

INTRODUCTION AND IMPORTANCE: Gallbladder torsion (volvulus) is a very rare pathology that occurs when the gallbladder rotates on the cystic duct/cystic artery axis, resulting in blockage of bile drainage and blood flow. CASE PRESENTATION: We present the case of an elderly 87-year-old female who experienced acute gallbladder torsion. The patient presented with acute left chest pain that radiated to the patient's back and nausea but no emesis. A thorough physical examination, serologic studies, abdominal ultrasound, and computed tomography scan revealed gallbladder dilation, a thickened wall, enlarged common bile duct (approximately 1 cm), and the presence of pericholecystic fluid, all of which were consistent with acute cholecystitis. Laparoscopic cholecystectomy led to an intraoperative diagnosis of completely gangrenous gallbladder volvulus (GBV) with a 360-degree counter clockwise rotation. The pathology led to a final diagnosis of acute necrotizing cholecystitis without evidence of malignancy. CLINICAL DISCUSSION: GBV (gallbladder torsion) is typically observed in elderly patients at a female-to-male ratio of 4:1 and a median age at presentation of 77 years. Few advancements have been made in accurate diagnosis of GBV using clinical findings or the results of radiographic imaging, leading to accurate preoperative diagnoses in only 25 % of patients. CONCLUSION: Though GBV usually presents with right upper quadrant pain, our patient had the rare presentation of left sided chest pain mimicking acute coronary syndrome. GBV is an uncommon condition that occurs frequently in the elderly, particularly in women. Accurate preoperative diagnosis remains daunting, since clinical, laboratory, and radiographic findings often lead to an incorrect diagnosis of acute cholecystitis. Prompt diagnosis necessitates a high level of suspicion, and laparoscopic cholecystectomy is the recommended treatment/management.

Comparison of Bayesian clustering and edge detection methods for inferring boundaries in landscape genetics.

Recently, techniques available for identifying clusters of individuals or boundaries between clusters using genetic data from natural populations have expanded rapidly. Consequently, there is a need to evaluate these different techniques. We used spatially-explicit simulation models to compare three spatial Bayesian clustering programs and two edge detection methods. Spatially-structured populations were simulated where a continuous population was subdivided by barriers. We evaluated the ability of each method to correctly identify boundary locations while varying: (i) time after divergence, (ii) strength of isolation by distance, (iii) level of genetic diversity, and (iv) amount of gene flow across barriers. To further evaluate the methods' effectiveness to detect genetic clusters in natural populations, we used previously published data on North American pumas and a European shrub. Our results show that with simulated and empirical data, the Bayesian spatial clustering algorithms outperformed direct edge detection methods. All methods incorrectly detected boundaries in the presence of strong patterns of isolation by distance. Based on this finding, we support the application of Bayesian spatial clustering algorithms for boundary detection in empirical datasets, with necessary tests for the influence of isolation by distance.

Changes in genetic diversity and differentiation in Red-cockaded woodpeckers (Dryobates borealis) over the past century.

Red-cockaded woodpeckers (RCW; Dryobates borealis) declined after human activities reduced their fire-maintained pine ecosystem to <3% of its historical range in the southeastern United States and degraded remaining habitat. An estimated 1.6 million RCW cooperative breeding groups declined to about 3,500 groups with no more than 10,000 birds by 1978. Management has increased RCW population abundances since they were at their lowest in the 1990s. However, no range-wide study has been undertaken since then to investigate the impacts of this massive bottleneck or infer the effects of conservation management and recent demographic recoveries. We used mitochondrial DNA sequences (mtDNA) and nine nuclear microsatellite loci to determine if range-wide demographic declines resulted in changes to genetic structure and diversity in RCW by comparing samples collected before 1970 (mtDNA data only), between 1992 and 1995 (mtDNA and microsatellites), and between 2010 and 2014 (mtDNA and microsatellites). We show that genetic diversity has been lost as detected by a reduction in the number of mitochondrial haplotypes. This reduction was apparent in comparisons of pre-1970 mtDNA data with data from the 1992-1995 and 2010-2014 time points, with no change between the latter two time points in mtDNA and microsatellite analyses. The mtDNA data also revealed increases in range-wide genetic differentiation, with a genetically panmictic population present throughout the southeastern United States in the pre-1970s data and subsequent development of genetic structure that has remained unchanged since the 1990s. Genetic structure was also uncovered with the microsatellite data, which like the mtDNA data showed little change between the 1992-1995 and 2010-2014 data sets. Temporal haplotype networks revealed a consistent, star-like phylogeny, suggesting that despite the overall loss of haplotypes, no phylogenetically distinct mtDNA lineages were lost when the population declined. Our results may suggest that management during the last two decades has prevented additional losses of genetic diversity.

Isolation by distance versus landscape resistance: Understanding dominant patterns of genetic structure in Northern Spotted Owls (Strix occidentalis caurina).

Landscape genetics investigations examine how the availability and configuration of habitat influence genetic structure of plants and animals. We used landscape genetics to evaluate the role that forest connectivity plays in determining genetic structure of the federally-threatened Northern Spotted Owl (Strix occidentalis caurina) using genotypes of 339 Northern Spotted Owls obtained for 10 microsatellite loci. Spatial clustering analyses identified a distinct genetic cluster at the southern extent of the region examined. This cluster could not be linked to landscape connectivity patterns and suggested that post-Pleistocene processes were involved with its development rather than contemporary landscape configuration. We also compared matrices of pairwise inter-individual genetic distances with resistance distances derived from a circuit-theory based framework. Resistance distances were obtained for an idealized raster map that reflected continuous unimpeded dispersal habitat across the landscape along with five empirically-derived raster maps reflecting the 1870's, 1940's, 1986, 1994, and 2012. Resistance distances from the idealized map served as surrogates for linear geographic distances. Relative to idealized conditions, resistance distances were ~250% higher in the 1940's and ~200% higher from 1986 onward. Resistance distances from the 1870's were ~40% higher than idealized conditions. Inter-individual genetic distances were most highly correlated with resistance distances from the idealized map rather than any of the empirical maps. Two hypotheses explain our results. First, our results may reflect temporal lags between the onset of large-scale habitat alterations and their novel effects on genetic structure in long-lived species such as Northern Spotted Owls. Second, because Northern Spotted Owls disperse over long distances, our results may indicate that forest habitat has never been sufficiently fragmented to the point where connectivity was disrupted. The second hypothesis could indicate that forest management practices mandated by the Northwest Forest Plan succeeded with one of its primary goals. However, our results do not represent a complete portrayal of the status of Northern Spotted Owls given detection of significant population declines and bottlenecks in other studies. Future investigations based on computer simulations may help distinguish between hypotheses.

Allele frequencies of 15 autosomal STR loci in the Iraq population with comparisons to other populations from the middle-eastern region.

Allele frequencies for the 15 autosomal STR loci included in the AmpFlSTR((R)) IdentifilerTM PCR Amplification Kit panel from Applied Biosystems (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, CSF1PO, D19S433, D2S1338, D16S539) and several statistical parameters were estimated from a sample of 103 unrelated individuals, mostly Shia and Sunni Arabs, living in most of central and southern Iraq provinces. We compared the allele frequency spectrum detected in the Iraqi population to allele frequencies from 11 other data sets from published studies of individuals from Turkey, Iraqi-Kurdistan, Saudi Arabia, Arab Emarates, Oman, Iran, Syria, and Jordan. Significant global differences in allele frequencies were detected in 9 of the 11 comparisons following sequential Bonferroni corrections. Comparisons with the two independent panels from Saudi Arabia were not significant after applying Bonferroni corrections, however, low P-values (P<0.05) associated with these two contrasts nonetheless suggested that at least slight genetic differences between populations may exist.

Genetic differentiation and inferred dynamics of a hybrid zone between Northern Spotted Owls (Strix occidentalis caurina) and California Spotted Owls (S. o. occidentalis) in northern California.

Genetic differentiation among Spotted Owl (Strix occidentalis) subspecies has been established in prior studies. These investigations also provided evidence for introgression and hybridization among taxa but were limited by a lack of samples from geographic regions where subspecies came into close contact. We analyzed new sets of samples from Northern Spotted Owls (NSO: S. o. caurina) and California Spotted Owls (CSO: S. o. occidentalis) in northern California using mitochondrial DNA sequences (mtDNA) and 10 nuclear microsatellite loci to obtain a clearer depiction of genetic differentiation and hybridization in the region. Our analyses revealed that a NSO population close to the northern edge of the CSO range in northern California (the NSO Contact Zone population) is highly differentiated relative to other NSO populations throughout the remainder of their range. Phylogenetic analyses identified a unique lineage of mtDNA in the NSO Contact Zone, and Bayesian clustering analyses of the microsatellite data identified the Contact Zone as a third distinct population that is differentiated from CSO and NSO found in the remainder of the subspecies' range. Hybridization between NSO and CSO was readily detected in the NSO Contact Zone, with over 50% of individuals showing evidence of hybrid ancestry. Hybridization was also identified among 14% of CSO samples, which were dispersed across the subspecies' range in the Sierra Nevada Mountains. The asymmetry of hybridization suggested that the hybrid zone may be dynamic and moving. Although evidence of hybridization existed, we identified no F1 generation hybrid individuals. We instead found evidence for F2 or backcrossed individuals among our samples. The absence of F1 hybrids may indicate that (1) our 10 microsatellites were unable to distinguish hybrid types, (2) primary interactions between subspecies are occurring elsewhere on the landscape, or (3) dispersal between the subspecies' ranges is reduced relative to historical levels, potentially as a consequence of recent regional fires.

The conservation genetics juggling act: integrating genetics and ecology, science and policy.

The field of conservation genetics, when properly implemented, is a constant juggling act integrating molecular genetics, ecology, and demography with applied aspects concerning managing declining species or implementing conservation laws and policies. This young field has grown substantially since the 1980s following the development of polymerase chain reaction and now into the genomics era. Our laboratory has 'grown up' with the field, having worked on these issues for over three decades. Our multidisciplinary approach entails understanding the behavior and ecology of species as well as the underlying processes that contribute to genetic viability. Taking this holistic approach provides a comprehensive understanding of factors that influence species persistence and evolutionary potential while considering annual challenges that occur throughout their life cycle. As a federal laboratory, we are often addressing the needs of the U.S. Fish and Wildlife Service in their efforts to list, de-list, or recover species. Nevertheless, there remains an overall communication gap between research geneticists and biologists who are charged with implementing their results. Therefore, we outline the need for a National Center for Small Population Biology to ameliorate this problem and provide organizations charged with making status decisions firmer ground from which to make their critical decisions.

Intercontinental genetic structure and gene flow in Dunlin (Calidris alpina), a potential vector of avian influenza.

Waterfowl (Anseriformes) and shorebirds (Charadriiformes) are the most common wild vectors of influenza A viruses. Due to their migratory behavior, some may transmit disease over long distances. Migratory connectivity studies can link breeding and nonbreeding grounds while illustrating potential interactions among populations that may spread diseases. We investigated Dunlin (Calidris alpina), a shorebird with a subspecies (C. a. arcticola) that migrates from nonbreeding areas endemic to avian influenza in eastern Asia to breeding grounds in northern Alaska. Using microsatellites and mitochondrial DNA, we illustrate genetic structure among six subspecies: C. a. arcticola,C. a. pacifica,C. a. hudsonia,C. a. sakhalina,C. a. kistchinski, and C. a. actites. We demonstrate that mitochondrial DNA can help distinguish C. a. arcticola on the Asian nonbreeding grounds with >70% accuracy depending on their relative abundance, indicating that genetics can help determine whether C. a. arcticola occurs where they may be exposed to highly pathogenic avian influenza (HPAI) during outbreaks. Our data reveal asymmetric intercontinental gene flow, with some C. a. arcticola short-stopping migration to breed with C. a. pacifica in western Alaska. Because C. a. pacifica migrates along the Pacific Coast of North America, interactions between these subspecies and other taxa provide route for transmission of HPAI into other parts of North America.

Identifying shared genetic structure patterns among Pacific Northwest forest taxa: insights from use of visualization tools and computer simulations.

BACKGROUND: Identifying causal relationships in phylogeographic and landscape genetic investigations is notoriously difficult, but can be facilitated by use of multispecies comparisons. METHODOLOGY/PRINCIPAL FINDINGS: We used data visualizations to identify common spatial patterns within single lineages of four taxa inhabiting Pacific Northwest forests (northern spotted owl: Strix occidentalis caurina; red tree vole: Arborimus longicaudus; southern torrent salamander: Rhyacotriton variegatus; and western white pine: Pinus monticola). Visualizations suggested that, despite occupying the same geographical region and habitats, species responded differently to prevailing historical processes. S. o. caurina and P. monticola demonstrated directional patterns of spatial genetic structure where genetic distances and diversity were greater in southern versus northern locales. A. longicaudus and R. variegatus displayed opposite patterns where genetic distances were greater in northern versus southern regions. Statistical analyses of directional patterns subsequently confirmed observations from visualizations. Based upon regional climatological history, we hypothesized that observed latitudinal patterns may have been produced by range expansions. Subsequent computer simulations confirmed that directional patterns can be produced by expansion events. CONCLUSIONS/SIGNIFICANCE: We discuss phylogeographic hypotheses regarding historical processes that may have produced observed patterns. Inferential methods used here may become increasingly powerful as detailed simulations of organisms and historical scenarios become plausible. We further suggest that inter-specific comparisons of historical patterns take place prior to drawing conclusions regarding effects of current anthropogenic change within landscapes.

Rapid natural selection for resistance to an introduced parasite of rainbow trout.

Introduced species and infectious diseases both independently pose challenges for the preservation of existing biodiversity. However, native species or disease hosts are by no means 'unarmed' when faced with novel environmental challenges, provided that adequate adaptive genetic variation exists to mount effective evolutionary responses. In this study, we examined the consequences of the recently introduced parasite and causative agent of whirling disease (Myxobolus cerebralis) in a wild rainbow trout (Oncorhynchus mykiss) population from Harrison Lake, Montana (USA). Consistent with the parasite's age-specific effects, juvenile rainbow trout recruitment into Harrison Lake was substantially reduced following parasite detection in 1995. However, experimental data suggest that natural selection has rapidly reduced whirling disease susceptibility within the population over time. The rapid observed temporal change in resistance patterns argues that the standing genetic variation for parasite resistance facilitated this process. Our findings ultimately underscore the importance of preserving genetic diversity to ensure that species of economic importance or of conservation concern have maximal chances for persistence in future changing environments.

Phylogeography and spatial genetic structure of the Southern torrent salamander: implications for conservation and management.

The Southern torrent salamander (Rhyacotriton variegatus) was recently found not warranted for listing under the US Endangered Species Act due to lack of information regarding population fragmentation and gene flow. Found in small-order streams associated with late-successional coniferous forests of the US Pacific Northwest, threats to their persistence include disturbance related to timber harvest activities. We conducted a study of genetic diversity throughout this species' range to 1) identify major phylogenetic lineages and phylogeographic barriers and 2) elucidate regional patterns of population genetic and spatial phylogeographic structure. Cytochrome b sequence variation was examined for 189 individuals from 72 localities. We identified 3 major lineages corresponding to nonoverlapping geographic regions: a northern California clade, a central Oregon clade, and a northern Oregon clade. The Yaquina River may be a phylogeographic barrier between the northern Oregon and central Oregon clades, whereas the Smith River in northern California appears to correspond to the discontinuity between the central Oregon and northern California clades. Spatial analyses of genetic variation within regions encompassing major clades indicated that the extent of genetic structure is comparable among regions. We discuss our results in the context of conservation efforts for Southern torrent salamanders.

Effects of historical climate change, habitat connectivity, and vicariance on genetic structure and diversity across the range of the red tree vole (Phenacomys longicaudus) in the Pacific Northwestern United States.

Phylogeographical analyses conducted in the Pacific Northwestern United States have often revealed concordant patterns of genetic diversity among taxa. These studies demonstrate distinct North/South genetic discontinuities that have been attributed to Pleistocene glaciation. We examined phylogeographical patterns of red tree voles (Phenacomys longicaudus) in western Oregon by analysing mitochondrial control region sequences for 169 individuals from 18 areas across the species' range. Cytochrome b sequences were also analysed from a subset of our samples to confirm the presence of major haplotype groups. Phylogenetic network analyses suggested the presence of two haplotype groups corresponding to northern and southern regions of P. longicaudus' range. Spatial genetic analyses (samova and Genetic Landscape Shapes) of control region sequences demonstrated a primary genetic discontinuity separating northern and southern sampling areas, while a secondary discontinuity separated northern sampling areas into eastern and western groups divided by the Willamette Valley. The North/South discontinuity likely corresponds to a region of secondary contact between lineages rather than an overt barrier. Although the Cordilleran ice sheet (maximum approximately 12,000 years ago) did not move southward to directly affect the region occupied by P. longicaudus, climate change during glaciation fragmented the forest landscape that it inhabits. Signatures of historical fragmentation were reflected by positive associations between latitude and variables such as Tajima's D and patterns associated with location-specific alleles. Genetic distances between southern sampling areas were smaller, suggesting that forest fragmentation was reduced in southern vs. northern regions.