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Background: Laurel wilt disease has caused the extensive mortality of lauraceous species in the southeastern United States. The causal agent is an invasive fungus, Raffaelea lauricola, which is a symbiont of the beetle Xyleborus glabratus and causes a rapid, fatal vascular wilt. Early diagnosis of laurel wilt is imperative for efficient disease management. The current diagnostic process, however, is slow due to the lengthy laboratory procedures required to confirm pathogen presence. Methods: We tested the robustness and field-portability of a recently developed, species-specific, loop-mediated isothermal amplification (LAMP) assay for R. lauricola, with the overall goal of eliminating the need for a laboratory confirmation of the diagnosis. We tested the robustness of the assay using benchtop equipment with naturally infected samples. We then tested the assay directly in the field using a portable device. Results: The assay successfully detected R. lauricola directly from symptomatic wood tissue using crude DNA extracts. Furthermore, the assay readily allowed users to distinguish between symptoms caused by R. lauricola infection and similar symptoms caused by other agents. In-field, we assayed wood samples from symptomatic redbay (Persea borbonia [L.] Spreng) and sassafras (Sassafras albidum [Nutt.] Nees) across the Southeast and successfully detected R. lauricola-infected trees in less than an hour. Conclusion: Results of this study confirmed that the field-deployable LAMP assay is robust and can rapidly and accurately detect R. lauricola in infected trees directly on-site. LAMP technology is well suited for in-field implementation, and these results serve as an incentive for further development and use of this technology in the field of forest pathology.

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen, Raffaelea lauricola, which, after being introduced into the xylem of trees by its vector beetle, Xyleborus glabratus, results in a fatal vascular wilt. Rapid detection and accurate diagnosis of infections is paramount to the successful implementation of disease management strategies. Current management operations to prevent the spread of laurel wilt disease are largely delayed by time-consuming laboratory procedures to confirm the diagnosis. In order to greatly speed up the operations, we developed a loop-mediated isothermal amplification (LAMP) species-specific assay that targets the β-tubulin gene region of R. lauricola, and allows for the rapid detection of the pathogen directly from host plant and beetle tissues. The assay is capable of amplifying as little as 0.5 pg of fungal DNA and as few as 50 conidia. The assay is also capable of detecting R. lauricola directly from wood tissue of artificially inoculated redbay saplings as early as 10 and 12 days postinoculation, when testing high-quality and crude DNA extracts, respectively. Finally, crude DNA extracts of individual adult female X. glabratus beetles were assayed and the pathogen was detected from all specimens. This assay greatly reduces the time required to confirm a laurel wilt diagnosis and, because LAMP technology is well suited to provide point-of-care testing, it has the potential to expedite and facilitate implementation of management operations in response to disease outbreaks.

In highly mobile philopatric species, defining the scale of natal homing is fundamental to characterizing population dynamics and effectively managing distinct populations. Genetic tools have provided evidence of regional natal philopatry in marine turtles, but extensive sharing of maternally inherited mitochondrial control region (CR) haplotypes within regions (Chelonia mydasnesting sites detected at least 2 populations, but the ubiquity of haplotype CM-A3.1 among southern rookeries decreased the power to detect differentiation. We reassessed population structure by sequencing the mitochondrial microsatellite (short tandem repeat, mtSTR) in 786 samples from 11 nesting sites spanning 700 km from Canaveral National Seashore through Dry Tortugas National Park. The mtSTR marker subdivided CM-A3.1 into 12 haplotypes that were structured among rookeries, demonstrating independent female recruitment into the Dry Tortugas and Marquesas Keys nesting populations. Combined haplotypes provided support for recognition of at least 4 management units in Florida: (1) central eastern Florida, (2) southeastern Florida, (3) Key West National Wildlife Refuge, and (4) Dry Tortugas National Park. Recapture data indicated female nesting dispersal between islands

In migratory marine species, demographic estimates are often generated from capture-mark-recapture (CMR) studies conducted at terrestrial breeding sites. However, when logistical difficulties limit the geographic area of these surveys, demographic estimates are vulnerable to biases. We compared demographic rates generated from CMR data of nesting loggerhead turtles (Caretta caretta) collected between 2010 and 2017 at one focal site (Wassaw Island, Georgia, USA; 31.89° N, 80.97° W) with estimates generated from the same group of turtles but including all other nesting events from adjacent sites in Georgia, South Carolina, and North Carolina. We found that estimates of annual recruitment at the focal site were overestimated: each year, 29–45% putative first-time nesters at the focal beach had, in fact, nested on a different beach in a previous season. Estimates of clutch frequency and breeding frequency generated at the focal site were biased low and skewed towards values of one, while estimates for remigration interval were not significantly over- or underestimated. Additionally, estimates of annual and total population productivity in terms of clutches, eggs, and hatchlings were underestimated by more than half at the focal site. Our results show how weak fidelity to a focal nesting/tagging site can affect demographic estimates in marine turtle populations and highlight the need to reconsider estimates from other populations that might be vulnerable to similar biases.

Clonal and genetic diversity in foundational plant species are critical for species resiliency and ecosystem processes, both of which contribute to restoration success; however, genetic data is often lacking for common plant species used in many restoration practices. Only a few plant species dominate salt marshes, ecologically and economically valuable ecosystems targeted for restoration due to global decline. Despite being a foundational species in southeastern United States salt marshes black needlerush (Juncus roemerianus Scheele) is understudied, especially in comparison to the co-occurring smooth cordgrass (Spartina alterniflora Loisel). We used a panel of 18 microsatellite markers on 849 samples of J. roemerianus collected at 17 sites across a majority of the species range from Mississippi to South Carolina to measure clonal and genetic diversity and characterize population structure. Results are consistent with previous genetic studies on J. roemerianus and other clonal plant species in that clonal and genetic diversity were higher than expected based on life history literature, with an average genotypic diversity (GD) of 0.67 and average observed heterozygosity (HO) of 0.56. Differences in diversity between the ecologically and environmentally divergent Gulf (GD = 0.64, HO = 0.52) and Atlantic (GD = 0.80, HO = 0.45) coasts suggest that life history strategy may vary by environment. Hierarchical structure was apparent across the study area, with STRUCTURE analyses identifying three genetic clusters that further subdivided into five clusters and a broad transition zone. The scale of this genetic differentiation should be considered in sourcing plants for salt marsh restoration efforts.

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen

Population assessments conducted at reproductive sites of migratory species necessitate understanding the foraging-area origins of breeding individuals. Without this information, efforts to contextualize changes in breeding populations and develop effective management strategies are compromised. We used stable isotope analysis of tissue samples collected from loggerhead sea turtles (Caretta caretta) nesting at seven sites in the Northern Recovery Unit (NRU) of the eastern United States (North Carolina, South Carolina and Georgia) to assign females to three separate foraging areas in the Northwest Atlantic Ocean (NWA). We found that the majority of the females at NRU nesting sites (84.4%) use more northern foraging areas in the Mid-Atlantic Bight, while fewer females use more proximate foraging areas in the South Atlantic Bight (13.4%) and more southerly foraging areas in the Subtropical Northwest Atlantic (2.2%). We did not find significant latitudinal or temporal trends in the proportions of NRU females originating from different foraging areas. Combining these findings with previous data from stable isotope and satellite tracking studies across NWA nesting sites showed that variation in the proportion of adult loggerheads originating from different foraging areas is primarily related differences between recovery units: individuals in the NRU primarily use the Mid-Atlantic Bight foraging area, while individuals from the three Florida recovery units primarily use the Subtropical Northwest Atlantic and Eastern Gulf of Mexico foraging areas. Because each foraging area is associated with its own distinct ecological characteristics, environmental fluctuations and anthropogenic threats that affect the abundance and productivity of individuals at nesting sites, this information is critical for accurately evaluating population trends and developing effective region-specific management strategies.

Common species important for ecosystem restoration stand to lose as much genetic diversity from anthropogenic habitat fragmentation and climate change as rare species, but are rarely studied. Salt marshes, valuable ecosystems in widespread decline due to human development, are dominated by the foundational plant species black needlerush (Juncus roemerianus Scheele) in the northeastern Gulf of Mexico. We assessed genetic patterns in J. roemerianus by measuring genetic and genotypic diversity, and characterizing population structure. We examined population connectivity by delineating possible dispersal corridors, and identified landscape factors influencing population connectivity. A panel of 19 microsatellite markers was used to genotype 576 samples from ten sites across the northeastern Gulf of Mexico from the Grand Bay National Estuarine Research Reserve (NERR) to the Apalachicola NERR. Genetic distances (FST and Dch) were used in a least cost transect analysis (LCTA) within a hierarchical model selection framework. Genetic and genotypic diversity results were higher than expected based on life history literature, and samples structured into two large, admixed genetic clusters across the study area, indicating sexual reproduction may not be as rare as predicted in this clonal macrophyte. Digitized coastal transects buffered by 500 m may represent possible dispersal corridors, and developed land may significantly impede population connectivity in J. roemerianus. Results have important implications for coastal restoration and management that seek to preserve adaptive potential by sustaining natural levels of genetic diversity and conserving population connectivity. Our methodology could be applied to other common, widespread and understudied species.

Loblolly pine (LP; Pinus taeda L.) is an economically and ecologically important tree in the southeastern U.S. To advance understanding of the loblolly pine (LP; Pinus taeda L.) genome, we sequenced and analyzed 100 BAC clones and performed a Cot analysis. The Cot analysis indicates that the genome is composed of 57, 24, and 10% highly-repetitive, moderately-repetitive, and single/low-copy sequences, respectively (the remaining 9% of the genome is a combination of fold back and damaged DNA). Although single/low-copy DNA only accounts for 10% of the LP genome, the amount of single/low-copy DNA in LP is still 14 times the size of the Arabidopsis genome. Since gene numbers in LP are similar to those in Arabidopsis, much of the single/low-copy DNA of LP would appear to be composed of DNA that is both gene- and repeat-poor. Macroarrays prepared from a LP bacterial artificial chromosome (BAC) library were hybridized with probes designed from cell wall synthesis/wood development cDNAs, and 50 of the "targeted" clones were selected for further analysis. An additional 25 clones were selected because they contained few repeats, while 25 more clones were selected at random. The 100 BAC clones were Sanger sequenced and assembled. Of the targeted BACs, 80% contained all or part of the cDNA used to target them. One targeted BAC was found to contain fungal DNA and was eliminated from further analysis. Combinations of similarity-based and ab initio gene prediction approaches were utilized to identify and characterize potential coding regions in the 99 BACs containing LP DNA. From this analysis, we identified 154 gene models (GMs) representing both putative protein-coding genes and likely pseudogenes. Ten of the GMs (all of which were specifically targeted) had enough support to be classified as intact genes. Interestingly, the 154 GMs had statistically indistinguishable (α = 0.05) distributions in the targeted and random BAC clones (15.18 and 12.61 GM/Mb, respectively), whereas the low-repeat BACs contained significantly fewer GMs (7.08 GM/Mb). However, when GM length was considered, the targeted BACs had a significantly greater percentage of their length in GMs (3.26%) when compared to random (1.63%) and low-repeat (0.62%) BACs. The results of our study provide insight into LP evolution and inform ongoing efforts to produce a reference genome sequence for LP, while characterization of genes involved in cell wall production highlights carbon metabolism pathways that can be leveraged for increasing wood production.

The green turtle ( Chelonia mydas ) foraging aggregation along the Texas coast has increased dramatically in recent years, but the source populations for these turtles have not been adequately resolved. Previous mixed stock analysis (MSA) based on 490 base pair (bp) mitochondrial control region haplotypes suggested a large Florida contribution, but widespread sharing of common haplotypes among potential source populations and incomplete source population baseline data precluded precise assessment. To test the hypothesis that Texas turtles may represent proximal western Gulf of Mexico (GoM) nesting populations, we analyzed novel rookery samples from Rancho Nuevo, Tamaulipas, Mexico (RNMX) and conducted oceanic connectivity simulations. The RNMX samples yielded haplotypes CM-A1.1 and CM-A3.1 in frequencies not significantly different from those of the central eastern Florida nesting population. However, mitogenomic sequencing identified a diagnostic mitochondrial SNP (mtSNP) variant that is fixed in RNMX relative to the Florida CM-A1.1 lineage. Pairwise comparisons indicate that the Tamaulipas rookery represents a discrete population relative to those previously described in the northern Greater Caribbean, warranting recognition of a western GoM management unit (MU). Contrary to previous findings, the Florida populations were ruled out as major contributors to the Texas aggregation through screening of the mtSNP. Mixed stock analysis incorporating the mtSNP data suggested a western GoM origin for approximately 70% of the Texas foraging aggregation, with Quintana Roo contributing the majority of the remainder. Backtracking simulations within an ocean circulation model were broadly congruent with genetic results in indicating substantial probability of oceanic transport from Mexican rookeries to the Texas coast (68%) while also dismissing the possibility of transport from the eastern Florida rookeries (0%). The mixed stock analyses and backtracking simulations are consistent with previous hypotheses implicating oceanic dispersal followed by natal homing by neritic juveniles to explain juvenile green turtle distributions. In contrast to a pattern of stepping stone connectivity across the remaining northern Greater Caribbean, the Texas foraging aggregation was distinct from all others analyzed in the region, including one in the eastern GoM. This isolation highlights the significance of Texas as developmental habitat for the proposed western GoM MU and reiterates the importance of continued international cooperation to facilitate recovery of this stock. This study also underscores the importance of satisfying underlying assumptions of mixed stock analysis in order to make robust inferences of connectivity.

Capture–mark–recapture studies that fail to account for the frequency and dynamics of marker loss risk generating biased demographic estimates. In this study, we used permanent multilocus genotypes (i.e., “genetic tags”) and a new enhanced tag loss model to quantify the tag loss dynamics for both passive integrated transponder (PIT) and Inconel metal tags applied to loggerhead turtles (Caretta caretta) nesting on Wassaw Island, GA USA. Our results indicate that tag loss is most likely to occur within the nesting season in which tags were applied and is maximal just after tagging (maximum likelihood estimates): 0.00098 PIT tags day−1 and 0.007 Inconel tags day−1. After that, PIT tag loss was negligible and Inconel tag loss remained low but constant at 0.00028 tags day−1, such that after 5 years, the probability of losing one PIT tag was 0.06 and losing at least one Inconel tag was 0.46. The use of genetic tags in this study makes these the first truly accurate estimates of PIT and Inconel tag loss for marine turtles, and the new model of tag loss described herein represents an important advancement in the analytical methods used to estimate and compare tag loss dynamics.

The effects of predicted declines and potential loss of individual populations on species-level genetic diversity is unclear. A number of taxa, including the Canada Warbler (Cardellina canadensis), share wide-ranging geographic distributions in North American boreal forests with trailing-edge populations extending into the southern Appalachian Mountains. Trailing-edge populations in the southern portion of a species' ranges often harbor high levels of genetic diversity and unique genetic variants, and may be at risk of extinction from climate change. Climate change and other anthropogenic factors are causing declines in the Canada Warbler's southern trailing-edge populations, and with no genetic studies to date, the effect on species-level genetic diversity is uncertain. Species-specific microsatellite markers for the Canada Warbler were developed and validated using samples from three populations, including a southern trailing-edge population, to investigate their utility for intraspecific population studies. Eight of the microsatellite markers were informative for assessing genetic diversity and preliminary analysis suggests that they have potential for characterizing intraspecific neutral genetic diversity and structure among Canada Warbler populations.

Nest counts are often used as indices for nesting female abundance in marine turtle monitoring, but accurately interpreting nest count trends requires context on the scale of demographic connectivity and estimates of reproductive parameters. Weak nest site fidelity (NSF) relative to the scale of tagging effort may bias parameter estimates. The reproductive ecology of Northern Recovery Unit (NRU) loggerhead turtles (Caretta caretta) was assessed through subpopulation-scale genetic capture-recapture via clutch sampling from approximately 1000 km of coastline from Georgia to Maryland, USA (30.75–38.06°N and 75.24–81.45°W). Of 20,682 clutches recorded from 2010 to 2012, 20,222 sampled clutches were assigned to 5684 unique females through microsatellite genotyping. Approximately 73% of females detected laying multiple clutches deposited them within a 20-km span, suggesting the possibility of demographic structuring across NRU rookeries that warrants further investigation. Estimated clutch frequencies (ECF) generated from open robust design modeling were 4.28 (4.02–4.54) in 2010, 4.63 (4.45–4.80) in 2011, and 4.57 (4.28–4.77) in 2012, and were significantly higher than observed clutch frequencies. ECF generated from single-island data were biased low by 23–50% relative to those from regional genetic tagging. Among females that nested at least once on a physical tagging beach, 54% also nested elsewhere, with 81% of these “permanently” emigrating during the nesting season. This pattern of relatively strong NSF, but distributed across multiple nearby islands, confounded modeling of detection in single-island datasets and highlights the need for regional coverage for generating robust estimates of demographic parameters for marine turtles.

The Canada warbler (Cardellina canadensis) is a migratory neotropical warbler species of conservation concern. The 16,807 bp genome was sequenced and contains a 1224 bp control region, 37 g...

Green turtle (Chelonia mydas) nesting has increased dramatically in Florida over the past two decades, ranking the Florida nesting aggregation among the largest in the Greater Caribbean region. Individual beaches that comprise several hundred kilometers of Florida’s east coast and Keys support tens to thousands of nests annually. These beaches encompass natural to highly developed habitats, and the degree of demographic partitioning among rookeries was previously unresolved. We characterized the genetic structure of ten Florida rookeries from Cape Canaveral to the Dry Tortugas through analysis of 817 base pair mitochondrial DNA (mtDNA) control region sequences from 485 nesting turtles. Two common haplotypes, CM-A1.1 and CM-A3.1, accounted for 87 % of samples, and the haplotype frequencies were strongly partitioned by latitude along Florida’s Atlantic coast. Most genetic structure occurred between rookeries on either side of an apparent genetic break in the vicinity of the St. Lucie Inlet that separates Hutchinson Island and Jupiter Island, representing the finest scale at which mtDNA structure has been documented in marine turtle rookeries. Florida and Caribbean scale analyses of population structure support recognition of at least two management units: central eastern Florida and southern Florida. More thorough sampling and deeper sequencing are necessary to better characterize connectivity among Florida green turtle rookeries as well as between the Florida nesting aggregation and others in the Greater Caribbean region.

Investigating species' distribution and abundance over time is central to evolutionary biology, and provides important context for conservation and management. With respect to population genetic structure in green sea turtles (Chelonia mydas), certain processes such as female philopatry to natal rookeries are well understood, while others, such as male philopatry and historical changes in distribution and abundance, remain relatively understudied. Further, although inferences from mitochondrial DNA and nuclear microsatellites have both been critical in identifying management units, comparisons of these units based on both markers are still rare. Here we analyzed novel data from fifteen microsatellite markers gathered at six green turtle rookeries in the western Atlantic as well as previously published mitochondrial sequences from 13 regional rookeries. We detected low, but significant, population structure at microsatellite loci, which coincides with previous delineations of local and regional management units as well as reports of male philopatry. However, we also detected a discord between nuclear and mitochondrial data, in which two tropical rookeries (Aves Island, Venezuela; and Matapica, Surinam) clustered with the Caribbean and Mediterranean based on microsatellite data, but displayed a mitochondrial lineage characteristic of the southern Atlantic and Africa. To investigate the possible causes of this discord, we used both classical and Bayesian methods to estimate historical migration rates and the timing and magnitude of changes in population size. We detected a strong barrier to dispersal between the northern and southern Atlantic, as well as an expansion in the southern mitochondrial lineage during the Wisconsin glacial period and a later expansion in the northern lineage following the Last Glacial Maximum. We propose that the Aves and Surinam rookeries were colonized by females from a southern glacial refugium, after which they experienced male-biased gene flow from the Caribbean. This study highlights the utility of incorporating data from multiple types of molecular markers in accurately identifying conservation units and in elucidating the complex historical and contemporary processes underlying population genetic structure in marine species.

The Forest Health Initiative (FHI) was developed and implemented to test the hypothesis that a coordinated effort in biotechnology research could lead to resistant trees capable of restoring a species in a relevant time frame. As a test case, the American chestnut (Castanea dentata) was chosen for study as it is an iconic forest tree species in the eastern United States and southeastern Canada that has been nearly extirpated by chestnut blight which is caused by an introduced fungal pathogen (Cryphonectria parasitica). In addition, the species has attracted research investments over many decades, leading to some promising possibilities for effecting restoration. The FHI, now completing its third year, has integrated genomics-based candidate gene discovery with robust clonal propagation and gene transformation systems capable of producing hundreds of independent events for dozens of genes per year. A promising early leaf assay for blight reaction has been developed that will be instrumental in screening the large amount of material in production through these systems. Regulatory permits for testing some of the earliest transgene events have been obtained as small-scale field testing is beginning. High density genetic maps and various mapping populations are being analyzed to gain additional insights into the specific genes found in quantitative trait loci (QTLs) for resistances to blight and Phytophthora root rot (caused by P. cinnamomi, also known as ink disease). Nearly complete genome sequences of three of the blight resistance QTLs have been determined. These sequences are greatly assisting these analyses, as have comparisons with peach and other completed plant genomes, demonstrating the power of comparative genomics. These results show great promise for meeting the challenge proposed by the FHI hypothesis for American chestnut. In addition, other forest tree species under threat should benefit from similarly designed initiatives.

Airlift bioreactors (ALBs) were constructed and tested for their potential to enhance chestnut embryogenic tissue proliferation for genetic transformation and mass propagation. Multiple genotypes of American chestnut (Castanea dentata) and backcross hybrids of American chestnut and Chinese chestnut (C. dentata × Castanea mollissima) were cultured in ALBs. Medium for tissue proliferation in ALBs was woody plant medium supplemented with 3 g l−1 sucrose, 0.5 g l−1 l-glutamine and 2 mg l−1 2,4-dichlorophenoxyacetic acid. Optimum culture conditions for 1-l ALBs included 2 % (w/v) tissue inoculation density for culture initiation, 200 ml min−1 airflow rate, and weekly feeding with fresh medium (80–85 %, v/v). Compared with flasks, ALBs generated higher yields of tissue mass and larger fractions of small cell clumps (

Diet items and habitat constitute some of the environmental resources that may be used differently by individuals within a population. Long-term fidelity by individuals to particular resources exemplifies individual specialization, a phenomenon that is becoming increasingly recognized across a wide range of species. Less is understood about the consequences of such specialization. Here, we investigate the effects of differential foraging ground use on reproductive output in 183 loggerhead sea turtles (Caretta caretta) nesting at Wassaw Island, Georgia (31.89°N, 80.97°W), between 2004 and 2011 with resulting possible fitness effects. Stable isotope analysis was used to assign the adult female loggerheads to one of three foraging areas in the Northwest Atlantic Ocean. Our data indicate that foraging area preference influences the size, fecundity, and breeding periodicity of adult female loggerhead turtles. We also found that the proportion of turtles originating from each foraging area varied significantly among the years examined. The change in the number of nesting females across the years of the study was not a result of uniform change from all foraging areas. We develop a novel approach to assess differential contributions of various foraging aggregations to changes in abundance of a sea turtle nesting aggregation using stable isotopes. Our approach can provide an improved understanding of the influences on the causes of increasing or decreasing population trends and allow more effective monitoring for these threatened species and other highly migratory species.

The southeastern USA hosts the largest nesting concentration of loggerhead turtles Caretta caretta in the Atlantic. Regionally significant nesting also occurs along the Caribbean coast of Mexico, in Cuba, and in the Bahamas. Previous studies of North Atlantic loggerhead turtle rookeries based on a 380 bp fragment of the mitochondrial control region supported recognition of 8 demographically independent nesting populations (management units) in the Northwest Atlantic in addition to Cape Verde in the eastern Atlantic. Recent analysis of expanded mitochon- drial control region sequences revealed additional genetic diversity and increased population structure between western and eastern Atlantic loggerhead turtle rookeries. We se quenced an 817 bp mitochondrial DNA fragment in 2427 samples from nesting beaches in the southeastern USA, Cay Sal Bank, Bahamas, and Quintana Roo, Mexico. Pairwise FST comparisons, pairwise exact tests of population differentiation, and analysis of molecular variance support previously proposed management unit designations and additionally indicate that southeastern and south- western Florida rookeries should be recognized as distinct management units. Therefore, North- west Atlantic loggerhead turtle rookeries can be subdivided into 10 management units, corre- sponding to the beaches from (1) Virginia through northeastern Florida, (2) central eastern Florida, (3) southeastern Florida, (4) Dry Tortugas, Florida, (5) Cay Sal, Bahamas, (6) southwestern Cuba, (7) Quintana Roo, Mexico, (8) southwestern Florida, (9) central western Florida, and (10) northwestern Florida. We confirmed increased resolution of stock structure between many North- west Atlantic management units and the Cape Verde rookery with the expanded control region haplotypes.

We describe isolation and characterization of 14 polymorphic tetranucleotide loci from the hawksbill turtle (Eretmochelys imbricata). We identified an average of 14.5 alleles per locus based on screening of 36 individuals captured on foraging grounds in Bocas del Toro Province, Panama, and 9.6 alleles in 13 individuals captured on foraging grounds in Bermuda. Observed heterozygosity ranged from 0.67 to 1.00, with a mean of 0.85 for the Panama foraging aggregation and 0.83 for the Bermuda foraging aggregation. This microsatellite suite has a combined non-exclusion probability of identity of 8.26 × 10–23. These markers should be informative in individual and population-focused analyses.

The seaside sparrow (Ammodramus maritimus; SESP) includes seven extant subspecies ranging from the northeastern coast of the United States to the Gulf of Mexico. Genetic diversity of SESP may be impacted since coastal marsh habitats are highly fragmented, thereby limiting gene flow among adjacent populations. Genetic analysis of SESP populations will provide insight for development of conservation and management strategies for the species. We describe development and characterization of 11 novel microsatellite markers for SESP. These loci will be useful for individual identification as well as studies of fine-scale genetic relatedness within populations and subspecies of SESP.

Analyses of mitochondrial control region polymorphisms have supported the presence of several demographically independent green turtle (Chelonia mydas) rookeries in the Greater Caribbean region. However, extensive sharing of common haplotypes based on 490-bp control region sequences confounds assessment of the scale of natal homing and population structure among regional rookeries. We screened the majority of the mitochondrial genomes of 20 green turtles carrying the common haplotype CM-A5 and representing the rookeries of Buck Island, St. Croix, United States Virgin Islands (USVI); Aves Island, Venezuela; Galibi, Suriname; and Tortuguero, Costa Rica. Five singlenucleotide polymorphisms (SNPs) were identified that subdivided CM-A5 among regions. Mitogenomic pairwise /ST values of eastern Caribbean rookery comparisons were markedly lower than the respective pairwise FST values. This discrepancy results from the presence of haplotypes representing two divergent lineages in each rookery, highlighting the importance of choosing the appropriate test statistic for addressing the study question. Haplotype frequency differentiation supports demographic independence of Aves Island and Suriname, emphasizing the need to recognize the smaller Aves rookery as a distinct management unit. Aves Island and Buck Island rookeries shared mitogenomic haplotypes; however, frequency divergence suggests that the Buck Island rookery is sufficiently demographically isolated to warrant management unit status for the USVI rookeries. Given that haplotype sharing among rookeries is common in marine turtles with cosmopolitan distributions, mitogenomic sequencing may enhance inferences of population structure and phylogeography, as well as improve the resolution of mixed stock analyses aimed at estimating natal origins of foraging turtles.

The leatherback turtle (Dermochelys coriacea) is a globally endangered marine species. Numerous questions regarding life history and demographics that are of conservation interest remain and many of these can be addressed through the use of highly polymorphic nuclear markers. We describe primers and polymerase chain reaction conditions to amplify 19 tetranucleotide microsatellite loci from the leatherback turtle. The primers were tested on samples from 22 females that nested at Archie Carr National Wildlife Refuge, Melbourne Beach, Florida, USA. The primers developed in this study yielded an average of 9.4 alleles per locus (range of 5–19) and an average observed heterozygosity of 0.84 (range 0.36–1.00). These markers should prove useful in supplementing existing markers for individual and population level analyses.

Green turtle nesting has been recorded in North Carolina, since 1980, but how these nesting females fit into the broader regional context genetically has not been determined. Genetic tagging through microsatellite genotyping of clutches laid in northern South Carolina, North Carolina, and Delaware from 2010 through 2014 identified at least 52 individual nesting females. The mitochondrial control region haplotype frequencies of these individuals were significantly different from all northern Greater Caribbean subpopulations, including those in Florida, suggesting that these northern US females represent an incipient subpopulation that warrants distinct management unit status.

The southeastern United States supports one of two large loggerhead turtle (Caretta caretta) nesting aggregations worldwide and is therefore critical to global conservation and recovery efforts for the species. Previous studies have established the presence of four demographically distinct nesting populations (management units) corresponding to beaches from (1) North Carolina through northeastern Florida, (2) peninsular Florida, (3) the Dry Tortugas, and (4) northwest Florida. Temporal and geographic genetic structure of the nesting aggregation was examined utilizing partial mitochondrial control region haplotype frequencies from 834 samples collected over the 2002 through 2008 nesting seasons from 19 beaches as well as previously published haplotype data. Most rookeries did not exhibit interannual genetic variation. However, the interannual variation detected did significantly impact the interpretation of spatial genetic structure in northeastern Florida. Based on pairwise F ST comparisons, exact tests of population differentiation, and analysis of molecular variance, the present study upholds the distinctiveness of the four currently recognized management units and further supports recognition of discrete central eastern, southern (southeastern and southwestern), and central western Florida management units. Further subdivision may be warranted, but more intensive genetic sampling is required. In addition, tools such as telemetry and mark-recapture are needed to complement genetic data and overcome limitations of genetic markers in resolving loggerhead turtle rookery connectivity in the southeastern USA.

The eastern indigo snake (Drymarchon couperi) is the longest snake in North America. Population declines due to extensive loss of habitat led to federal listing as a threatened species in 1978. Knowledge deficiencies regarding the biology of the eastern indigo snake, including population connectivity and gene flow, are impediments to the development of effective conservation and restoration strategies. We describe primers and polymerase chain reaction (PCR) conditions to amplify 22 tetranucleotide and pentanucleotide nuclear microsatellite loci isolated from the eastern indigo snake. We tested primers using 28 shed skins representing 24 individuals from Fort Stewart, Georgia. Primers yielded an average of 4.6 alleles per locus (range 3–7) and an average observed heterozygosity of 0.63 (range 0.46–0.79). These loci should prove useful for individual identification as well as population level analyses of this federally threatened species.

Tagging studies on nesting beaches are commonly used to estimate nesting frequency, remigration interval and nesting population size for marine turtle rookeries. Estimates of these demographic parameters from tagging projects may be biased because of the small scale of tagging efforts relative to female nest site fidelity and the logistical difficulty of intercepting all nesting females. Therefore, alternative and supplemental means of individual identification of nesting females are required. We demonstrate that maternal nuclear microsatellite DNA can be isolated from unincubated eggshells of the loggerhead sea turtle (Caretta caretta) through comparison of DNA extracted from 59 eggs collected within 15 h of oviposition and DNA derived from skin samples from respective nesting females. Scorable microsatellite genotypes were produced in 897 of 994 (90.2%) single-locus egg amplifications attempted. Among eggs from known females, 730 of 748 (97.6%) single-locus, egg-derived genotypes matched the respective skin-derived genotypes. Allelic dropout was the most common type of error, followed by the presence of nonmaternal, presumably paternal, alleles. Genotypes derived from unincubated eggshells permit individual assignment of nests and therefore demographic parameter estimates for loggerhead turtle nesting populations, despite genotyping errors that require further optimization. Although sampling unincubated eggs is destructive, this technique is noninvasive to nesting females and is applicable in marine turtle population genetics studies when individual resolution is required but direct interception of nesting females is undesirable or logistically infeasible.

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).

Some of the first applications of transgenic trees in North America may be for the conservation or restoration of threatened forest trees that have been devastated by fungal pathogens or insect pests. In some cases, where resistance has yet to be found in the natural population of a tree species, incorporating genes from other organisms may offer the only hope for restoration. In others, transgenics may play a role as part of an integrated approach, along with conventional breeding or biocontrol agents. American chestnut (Castanea dentata) was wiped out as a canopy species by a fungal disease accidentally introduced into the United States around 1900. Similarly, American elm (Ulmus americana) virtually disappeared as a favored street tree from Northeastern U.S. cities after the introduction of the Dutch elm disease fungus in the 1940s. In both cases, progress has been made toward restoration via conventional techniques such as selection and propagation of tolerant cultivars (American elm) or breeding with a related resistant species (American chestnut). Recently, progress has also been made with development of systems for engineering antifungal candidate genes into these “heritage trees.” An Agrobacterium-leaf disk system has been used to produce transgenic American elm trees engineered with an antimicrobial peptide gene that may enhance resistance to Dutch elm disease. Two gene transfer systems have been developed for American chestnut using Agrobacterium-mediated transformation of embryogenic cultures, setting the stage for the first tests of potential antifungal genes for their ability to confer resistance to the chestnut blight fungus. Despite the promise of transgenic approaches for restoration of these heritage trees, a number of technical, environmental, economic, and ethical questions remain to be addressed before such trees can be deployed, and the debate around these questions may be quite different from that associated with transgenic trees developed for other purposes.

We describe isolation and characterization of 20 polymorphic tetranucleotide loci from the green turtle (Chelonia mydas). We identified an average of 12.5 alleles per locus based on screening of 31 individuals foraging in the Indian River Lagoon, Florida, USA. Observed heterozygosity ranged from 0.53 to 1.00, with a mean of 0.85. This microsatellite suite has a combined non-exclusion probability of identity of 2.45 × 10–28. These markers will complement those already available for green turtles in individual-based as well as population level genetic analyses.

Premise of the study: Juncus roemerianus (Juncaceae) is a foundational species and ecosystem engineer of salt marshes in the Gulf of Mexico. These ecosystems provide coastal flood attenuation, nurseries for important species, and other ecosystem services, but are experiencing significant decline. Nuclear microsatellite markers were developed for J. roemerianus to study genetic diversity and population structure for conservation and restoration efforts. Methods and Results: Illumina NextSeq high-throughput sequencing was used to develop a panel of 19 polymorphic microsatellite markers that were tested across individuals from three populations on the Gulf Coast. All markers were polymorphic, with observed and expected heterozygosities ranging from 0.212 to 0.828 and from 0.362 to 0.873, respectively. Allelic richness ranged from two to 13 alleles per locus with an average of 5.737. Conclusions: The 19 microsatellite markers are useful for population studies throughout the range of J. roemerianus. Three loci...

In order to provide information to better inform management decisions and direct further research, vessel-based visual transects, snorkel transects, and in-water capture techniques were used to characterize hawksbill sea turtles in the shallow marine habitats of a Marine Protected Area (MPA), the Key West National Wildlife Refuge in the Florida Keys. Hawksbills were found in hardbottom and seagrass dominated habitats throughout the Refuge, and on man-made rubble structures in the Northwest Channel near Cottrell Key. Hawksbills captured (N = 82) were exclusively juveniles and subadults with a straight standard carapace length (SSCL) ranging from 21.4 to 69.0cm with a mean of 44.1 cm (SD = 10.8). Somatic growth rates were calculated from 15 recaptured turtles with periods at large ranging from 51 to 1188 days. Mean SSCL growth rate was 7.7 cm/year (SD = 4.6). Juvenile hawksbills (

We describe 12 polymorphic tetranucleotide and pentanucleotide loci in the red-cockaded woodpecker (Picoides borealis). An average of 6.25 alleles per locus was identified based on a screening of 21 individuals from the Joseph W. Jones Ecological Center in southwestern Georgia. Observed heterozygosity ranged from 0.048 to 0.952. These markers could be used for both population level studies and individual identification.

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.

Previous genetic studies have demonstrated that natal homing shapes the stock structure of marine turtle nesting populations. However, widespread sharing of common haplotypes based on short segments of the mitochondrial control region often limits resolution of the demographic connectivity of populations. Recent studies employing longer control region sequences to resolve haplotype sharing have focused on regional assessments of genetic structure and phylogeography. Here we synthesize available control region sequences for loggerhead turtles from the Mediterranean Sea, Atlantic, and western Indian Ocean basins. These data represent six of the nine globally significant regional management units (RMUs) for the species and include novel sequence data from Brazil, Cape Verde, South Africa and Oman. Genetic tests of differentiation among 42 rookeries represented by short sequences (380 bp haplotypes from 3,486 samples) and 40 rookeries represented by long sequences (~800 bp haplotypes from 3,434 samples) supported the distinction of the six RMUs analyzed as well as recognition of at least 18 demographically independent management units (MUs) with respect to female natal homing. A total of 59 haplotypes were resolved. These haplotypes belonged to two highly divergent global lineages, with haplogroup I represented primarily by CC-A1, CC-A4, and CC-A11 variants and haplogroup II represented by CC-A2 and derived variants. Geographic distribution patterns of haplogroup II haplotypes and the nested position of CC-A11.6 from Oman among the Atlantic haplotypes invoke recent colonization of the Indian Ocean from the Atlantic for both global lineages. The haplotypes we confirmed for western Indian Ocean RMUs allow reinterpretation of previous mixed stock analysis and further suggest that contemporary migratory connectivity between the Indian and Atlantic Oceans occurs on a broader scale than previously hypothesized. This study represents a valuable model for conducting comprehensive international cooperative data management and research in marine ecology.

The major protein found in the leaves, bark and hardened roots of trifoliate orange ( Poncirus trifoliata L. Raf.) is a glycoprotein ( M r 24 000). Characteristics of trifoliate orange protein, now called trifoliatin, are consistent with those reported for other plant storage proteins, the low sequence similarity with other leaf proteins indicates that trifoliatin may represent a new class of vegetative proteins. Levels of glycoprotein vary inversely with leaf age and temperature [6] and can reach levels up to 65% of total leaf protein. The purification of this protein was accomplished by a combination of free zone isoelectric focusing and chromatography techniques. The N-terminal sequence determined by Edman (P. Edman and G. Begg, Eur. J. Biochem., 1 (1967) 80) degradation shares little to no sequence similarity with other known leaf proteins. The partial sequence of trifoliatin has the highest sequence similarity with sporamin, the major tuberous root storage protein in sweet potato. Trifoliatin has no protease activity to casein hydrolysate or inhibitory activity to trypsin or α-chymotrypsin.

Analyses of mitochondrial control region polymorphisms have supported the presence of several demographically independent green turtle (Chelonia mydas) rookeries in the Greater Caribbean region. However, extensive sharing of common haplotypes based on 490-bp control region sequences confounds assessment of the scale of natal homing and population structure among regional rookeries. We screened the majority of the mitochondrial genomes of 20 green turtles carrying the common haplotype CM-A5 and representing the rookeries of Buck Island, St. Croix, United States Virgin Islands (USVI); Aves Island, Venezuela; Galibi, Suriname; and Tortuguero, Costa Rica. Five single-nucleotide polymorphisms (SNPs) were identified that subdivided CM-A5 among regions. Mitogenomic pairwise φ(ST) values of eastern Caribbean rookery comparisons were markedly lower than the respective pairwise F(ST) values. This discrepancy results from the presence of haplotypes representing two divergent lineages in each rookery, highlighting the importance of choosing the appropriate test statistic for addressing the study question. Haplotype frequency differentiation supports demographic independence of Aves Island and Suriname, emphasizing the need to recognize the smaller Aves rookery as a distinct management unit. Aves Island and Buck Island rookeries shared mitogenomic haplotypes; however, frequency divergence suggests that the Buck Island rookery is sufficiently demographically isolated to warrant management unit status for the USVI rookeries. Given that haplotype sharing among rookeries is common in marine turtles with cosmopolitan distributions, mitogenomic sequencing may enhance inferences of population structure and phylogeography, as well as improve the resolution of mixed stock analyses aimed at estimating natal origins of foraging turtles.

Acidic chitinases (EC 3.2.1.14) were isolated and characterized from 4-week-old nonembryogenic Citrus sinensis L. Osbeck cv 'Valencia' callus tissue. The enzymes were purified using size exclusion, anion exchange, and chromatofocusing HPLC techniques. Eleven isoforms were isolated with M(r)s between 26,000 and 37,400. Eight of the isoforms were purified to homogeneity, and all but one cross-reacted with a polyclonal antibody raised against a basic class I potato leaf chitinase. The isoelectric points (determined by chromatofocusing) were from pH 4.5 to 5.4. All hydrolases degraded chitin and four were capable of hydrolyzing solubilized shrimp shell chitosan suggesting they may be chitosanases (EC 3.2.1.99). Apparent chitosanase activity generally decreased with decreasing acetylation of the chitosan (i.e. from 20% to 0% acetylation). The chitinases and chitinases/chitosanases are predominantly endochitinases. Chitosanase activity was optimal at pH 5 while the pH optimum for chitinase activity ranged between pH 3.5 and 5.5. The chitinases and chitinases/chitosanases were stable up to 60 degrees C and showed their highest enzyme activity at that temperature. N-terminal sequences were obtained on three of the isoforms. One of the isoforms was identified as a class II chitinase and the other two as class III chitinases.

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.

Genetic markers are often used to designate population units for management and conservation, but widespread sharing of mitochondrial DNA control-region haplotypes defined from short (

The DNA sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila L. was determined. The Zamia small-subunit rRNA was found to be 1813 nucleotides in length and approximately 92% identical to published angiosperm small-subunit rRNA sequences. Conserved regions interspersed with variable regions are observed corresponding to those found in other eukaryotic small-subunit sequences. Using representatives from protist, fungal, plant, and animal groups, a distance matrix was constructed of average nucleotide substitution rates for pairs of organisms. Phylogenetic trees were inferred from similarities between sequences. The sequence of Zamia represents the earliest divergence from the higher plant lineage reported to date for small-subunit rRNA data. Inferred phylogenies also support a monophyletic origin for the angiosperms consistent with studies citing phenotypic characters.

An 1830-bp genomic DNA segment containing an Arabidopsis thaliana actin gene, AAc1, has been cloned and sequenced. The AAc1 gene is present as a single-copy gene, but at least two other actin-like genes have been detected. Comparison of the nucleotide sequence of AAc1 with other cloned plant actin genes reveals four exons separated by three introns conservatively located in all plant actin genes. The deduced amino acid sequence has also been compared with actin protein sequences from other plants.

Capture–mark–recapture (CMR) studies on marine turtle nesting beaches provide data on reproductive periodicity that inform population trends and models. Annual survival is estimated from observations of remigration, the return of females in subsequent nesting seasons. However, a significant proportion of tagged females are never encountered remigrating in many studies, presumably due to weak nest site fidelity (NSF). We employed a genetic CMR approach based on subpopulation-scale clutch sampling to conduct a 5-year evaluation of inter-seasonal recapture rates and NSF for Northern Recovery Unit loggerhead turtles (Caretta caretta). Of 1770 females genetically tagged from Georgia through Maryland in 2010, 1156 (65%) remigrated between 2011 and 2015. Inter-seasonal NSF, measured as shifts in median latitude nesting locations between years, was highly variable among individuals but strong overall (mean: 15.08 (± 44.61) km, median: 1.84 km). Among three focal beaches with nocturnal tagging projects, 69 of 173 females (40%) remigrated onsite whereas 115 (66%) were detected overall. Regional genetic sampling therefore yielded significantly higher inter-seasonal recapture rates, which may improve precision in future survival analyses. However, despite sampling ~ 1000 km with high annual detection probabilities (p* ≥ 0.94), 35% of 2010-females were not detected remigrating. Several non-exclusive hypotheses to explain these remaining “missing” remigrants should be considered: longer remigration intervals, imperfect detection within the study area, emigration to Florida, anthropogenic mortality, and natural mortality or senescence. This genetic tagging approach can be applied over large spatial scales where nesting densities permit, better characterizing inter-seasonal dispersal.