Microsatellite analysis supports mitochondrial phylogeography of the hellbender (Cryptobranchus alleganiensis)

We investigated genetic diversity of the hellbender (Cryptobranchus alleganiensis) throughout its range in the eastern US using nuclear markers and compared our results to a previously published mitochondrial analysis. A variety of nuclear markers, including protein-coding gene introns and microsatellites were tested but only microsatellites were variable enough for population level analysis. Microsatellite loci showed moderate among population sharing of alleles, in contrast to the reciprocal monophyly exhibited by mitochondrial DNA. However, analyses using F-statistics and Bayesian clustering algorithms showed considerable population subdivision and clustered hellbender populations into the same major groups as the mtDNA. The microsatellites combined with the mtDNA data suggest that gene flow is severely restricted or non-existent among eight major groups, and potentially among populations (rivers) within groups. The combined mtDNA and microsatellite data suggest that the currently recognized hellbender subspecies are paraphyletic. We suggest that the eight independent groups identified in our study should be managed as such, rather than basing conservation decisions on the two named subspecies of hellbender.     

Genetic relationships of hellbenders in the Ozark highlands of Missouri and conservation implications for the Ozark subspecies (<Emphasis Type="Italic">Cryptobranchus alleganiensis bishopi</Emphasis>)

The hellbender (Cryptobranchus alleganiensis) is an obligately aquatic salamander that is in decline due to habitat loss and disease. Two subspecies of hellbender have been described based on morphological characteristics: C. a. alleganiensis (eastern subspecies) and C. a. bishopi (Ozark hellbender). Current conservation strategies include captive propagation for restorative releases even though information regarding the current levels of genetic variability and structure within populations is not sufficient to effectively plan for conservation of the genetic diversity of the species. To investigate patterns of population structure in the hellbender, we genotyped 276 hellbenders from eight Missouri River drainages, representing both subspecies. Our results showed low levels of within-drainage diversity but strong population structure among rivers, and three distinct genetic clusters. F _ST values ranged from 0.00 to 0.61 and averaged 0.40. Our results confirmed previous reports that C. a. bishopi and C. a. alleganiensis are genetically distinct, but also revealed an equidistant relationship between two groups within C. a. bishopi and all populations of C. a. alleganiensis. Current subspecies delineations do not accurately incorporate genetic structure, and for conservation purposes, these three groups should be considered evolutionarily significant units.     

Characterization of 32 microsatellite loci for the Pacific red snapper, <Emphasis Type="Italic">Lutjanus peru</Emphasis>, through next generation sequencing

We developed a set of hypervariable microsatellite markers for the Pacific red snapper (Lutjanus peru), an economically important marine fish for small-scale fisheries in the west coast of Mexico. We performed shotgun genome sequencing with the 454 XL titanium chemistry and used bioinformatic tools to search for perfect microsatellite loci. We selected 66 primer pairs that were synthesized and genotyped in an ABI PRISM 3730XL DNA sequencer in 32 individuals from the Gulf of California. We estimated levels of genetic diversity, deviations from linkage and Hardy–Weinberg equilibrium, estimated the frequency of null alleles and the probability of individual identity for the new markers. We reanalyzed 16 loci in 16 individuals to estimate genotyping error rates. Eighteen loci failed to amplify, 16 loci were discarded due to unspecific amplifications and 32 loci (14 tetranucleotide and 18 dinucleotide) were successfully scored. The average number of alleles per locus was 21 (±6.87, SD) and ranged from 8 to 34. The average observed and expected heterozygosities were 0.787 (±0.144 SD, range 0.250–0.935) and 0.909 (±0.122 SD, range 0.381–0.965), respectively. No significant linkage was detected. Eight loci showed deviations from Hardy–Weinberg equilibrium, and from these, four loci showed moderate null allele frequencies (0.104–0.220). The probability of individual identity for the new loci was 1.46^?62. Genotyping error rates averaged 9.58%. The new markers will be useful to investigate patterns of larval dispersal, metapopulation dynamics, fine-scale genetic structure and diversity aimed to inform the implementation of spatially explicit fisheries management strategies in the Gulf of California.     

Within-river gene flow in the hellbender (Cryptobranchus alleganiensis) and implications for restorative release

Understanding how populations are genetically and demographically connected is beneficial for species management, since gene flow and dispersal contribute to genetic diversity and population persistence. For hellbenders (Cryptobranchus alleganiensis), an aquatic salamander species experiencing dramatic declines in population size, fine-scale (i.e. within river) patterns of genetic diversity and gene flow are not well understood. Previous findings indicate that hellbenders are habitat specialists that exhibit extreme site fidelity and low vagility, suggesting that gene flow is restricted among the several, discrete habitat patches within a river. Using 15 polymorphic microsatellite loci and 497 hellbender samples from four Missouri rivers, we assessed fine-scale patterns of genetic diversity in order to infer population connectivity and aid in population management. Results indicate moderate levels of genetic variation (H_O = 0.66–0.78) with little differentiation among habitat patches (avg. F_ST = 0.002) and no evidence of isolation by distance. Our data suggest that hellbender gene flow has been extensive even among habitat patches separated by distances greater than >100 km. These results are useful for hellbender management, especially in terms of making informed decisions regarding restorative releases of captively propagated individuals.     

Asian elephant (Elephas maximus) milk composition during the first 280 days of lactation

Milk samples (n = 10) taken during the first 280 days of lactation from one Asian elephant were examined for nutrient composition including total solids, protein, fat, ash, α-tocopherol, and retinol levels. Total solids averaged 19.7 ± 2.7% SD (range 15.0–23.3). Percent protein remained fairly stable throughout this portion of lactation and averaged 3.4 ± 0.3% (range 3.0–.4.0). Ash content averaged 0.54 ± 0.03%. Milk fat and fat soluble vitamin levels varied considerably with a suggestion of a cyclic pattern. Fat content of milk averaged 7.6 ± 2.6% (range 3.9–.12.1); α-tocopheral levels averaged 0.33 ± 0.12 μg/ml; and retinol levels averaged 0.46 ± 0.1 μg/ml. © 1994 Wiley-Liss, Inc.     

Predicting Secchi disk depth from average beam attenuation in a deep,ultra-clear lake

We addressed potential sources of error in estimating the water clarity of mountain lakes by investigating the use of beam transmissometer measurements to estimate Secchi disk depth. The optical properties Secchi disk depth (SD) and beam transmissometer attenuation (BA) were measured in Crater Lake (Crater Lake National Park, Oregon, USA) at a designated sampling station near the maximum depth of the lake. A standard 20 cm black and white disk was used to measure SD. The transmissometer light source had a nearly monochromatic wavelength of 660 nm and a path length of 25 cm. We created a SD prediction model by regression of the inverse SD of 13 measurements recorded on days when environmental conditions were acceptable for disk deployment with BA averaged over the same depth range as the measured SD. The relationship between inverse SD and averaged BA was significant and the average 95% confidence interval for predicted SD relative to the measured SD was ±1.6 m (range = −4.6 to 5.5 m) or ±5.0%. Eleven additional sample dates tested the accuracy of the predictive model. The average 95% confidence interval for these sample dates was ±0.7 m (range = −3.5 to 3.8 m) or ±2.2%. The 1996–2000 time-series means for measured and predicted SD varied by 0.1 m, and the medians varied by 0.5 m. The time-series mean annual measured and predicted SD’s also varied little, with intra-annual differences between measured and predicted mean annual SD ranging from −2.1 to 0.1 m. The results demonstrated that this prediction model reliably estimated Secchi disk depths and can be used to significantly expand optical observations in an environment where the conditions for standardized SD deployments are limited.     

Home-Range Use and Activity Patterns of the Red Langur (Presbytis rubicunda) in Sabangau Tropical Peat-Swamp Forest, Central Kalimantan, Indonesian Borneo

Knowledge of a species’ ranging patterns is vital for understanding its behavioral ecology and vulnerability to extinction. Given the abundance and even distribution of leaves in forested habitats, folivorous primates generally spend less time feeding; more time resting; have shorter day ranges; and require smaller home ranges than frugivorous primates. To test the influence of frugivory on ranging behavior, we established the activity budget and home-range size and use in a highly frugivorous population of the Borneo-endemic colobine, Presbytis rubicunda, within Sabangau tropical peat-swamp forest, Central Kalimantan, and examined relationships between fruit availability and ranging patterns. We collected 6848 GPS locations and 10,702 instantaneous focal behavioral scans on a single group between January and December 2011. The group had the largest home-range size recorded in genus Presbytis (kernel density estimates: mean = 108.3 ± SD 3.8 ha, N = 4 bandwidths). The annual activity budget comprised 48 ± SD 4.0% resting; 29.3 ± SD 3.9% feeding, 14.2 ± SD 2.5% traveling, and 0.4 ± SD 0.4% social behaviors. Mean monthly day-range length was the highest recorded for any folivorous primate (1645 ± SD 220.5 m/d). No significant relationships existed between ranging variables and fruit availability, and ranging behaviors did not vary significantly across seasons, potentially owing to low fluctuations in fruit availability. Our results suggest that colobine monkeys maintain larger than average ranges when high-quality food resources are available. Their extensive range requirements imply that protecting large, contiguous tracts of habitat is crucial in future conservation planning for Presbytis rubicunda.     

Space use and site fidelity of wintering whooping cranes on the Texas Gulf Coast

The Aransas-Wood Buffalo population (the only non-reintroduced, migratory population) of endangered whooping cranes (Grus americana) overwinters along the Texas Gulf Coast, USA. Understanding whooping crane space use on the wintering grounds reveals essential aspects of this species' ecology, which subsequently assists with conservation. Using global positioning system telemetry data from marked whooping cranes during 2009–2017, we fit continuous-time stochastic process models to describe movement and home range using autocorrelated kernel density estimation (AKDE) and explored variation in home range size in relation to age, sex, reproductive status, and drought conditions. We used the Bhattacharyya coefficient of overlap and distance between home range centroids to quantify site fidelity. We examined the effects of time between winter home ranges and the sex of the crane on site fidelity using Bayesian mixed-effects beta regression. Winter whooping crane 95% AKDE home range size averaged 30.1 ± 45.2 (SD) km² (median = 14.3, range = 1.1–308.6). Home ranges of sub-adult females were approximately 2 times larger than those of sub-adult males or families. As drought worsened, home ranges typically expanded. Between consecutive years, the home ranges of an adult crane exhibited 68 ± 31% overlap (site fidelity), but fidelity to winter sites declined in subsequent winters. The overlap of adult home ranges with the nearest unrelated family averaged 33 ± 28%. As a whooping crane aged, overlap with its winter home range as a juvenile declined, regardless of sex. By 4 years of age, a whooping crane had approximately 14 ± 28% overlap with its juvenile winter home range. Limited evidence suggested male whooping cranes return to within 2 km of their juvenile home range by their fifth winter. Previous data obtained from aerial surveys led ecologists to assume that whooping crane families normally used small areas (~2 km²) and expressed persistent site fidelity. Our analyses showed <8% of families had home ranges ≤2 km², with the average area 15 times greater, and waning site fidelity over time. Our work represents an analysis of whooping crane home ranges for this population, identifying past misconceptions of winter space use and resulting in better estimates of space requirements for future conservation efforts.     

Water velocity dependence of phosphate uptake on a coral-dominated fringing reef flat,La Réunion Island,Indian Ocean

Phosphate uptake (P-uptake) into coral reef communities has been hypothesized to be mass-transfer limited. One method of demonstrating mass-transfer limitation of P-uptake is to show dependence of P-uptake on water velocity. Water velocity across reef flats varies with tides and swell; thus, we measured P-uptake over the entire reef flat on eight different days, representing a range in water velocities. P-uptake was calculated from changes in P concentration of the water column. Changes in P concentration were measured by water sampling at six sites along a 300-m cross-reef transect while simultaneously measuring water velocity. To smooth the variability in phosphate concentrations, peristaltic pumps were used to get time-integrated water samples for 4–6 h at each site. Water velocities were measured in the middle of the transect using an acoustic Doppler current profiler and were averaged to match the time-integrated water sampling. Depth-averaged cross-reef water velocities were 0.031 ± 0.013 m s⁻¹ (mean ± SD), while the root-mean-square water velocities, accounting for oscillatory flow, averaged 3.3 times higher, 0.101 ± 0.021 m s⁻¹ (mean ± SD). Phosphate decreased along all transects. The first-order rate constant for P-uptake (S) was 8.5 ± 2.4 m d⁻¹ (mean ± SD) and increased linearly with root-mean-square water velocity. The Stanton number derived from oscillatory flow, the ratio of the first-order rate constant for P-uptake to the root-mean-square water velocity (S/U _rms), was (9.4 ± 1.2) × 10⁻⁴ (mean ± SD). P-uptake ranged from 0.2 to 1.1 mmol P m⁻² d⁻¹, demonstrating that P-uptake is variable on short time scales and is directly related to P concentration and water velocity.

     

Highly Polymorphic Microsatellite Markers for the Assessment of Male Reproductive Skew and Genetic Variation in Critically Endangered Crested Macaques (<Emphasis Type="Italic">Macaca nigra</Emphasis>)

Genetic analyses based on noninvasively collected samples have become an important tool for evolutionary biology and conservation. Crested macaques (Macaca nigra), endemic to Sulawesi, Indonesia, are important for our understanding of primate evolution as Sulawesi macaques represent an exceptional example of primate adaptive radiation. Crested macaques are also Critically Endangered. However, to date we know very little about their genetics. The aim of our study was to find and validate microsatellite markers useful for evolutionary, conservation, and other genetic studies on wild crested macaques. Using fecal samples of 176 wild macaques living in the Tangkoko Reserve, Sulawesi, we identified 12 polymorphic microsatellite loci through cross-species polymerase chain reaction amplification with later modification of some of these primers. We tested their suitability by investigating and exploring patterns of paternity, observed heterozygosity, and evidence for inbreeding. We assigned paternity to 63 of 65 infants with high confidence. Among cases with solved paternity, we found no evidence of extragroup paternity and natal breeding. We found a relatively steep male reproductive skew B index of 0.330 ± 0.267; mean ± SD) and mean alpha paternity of 65% per year with large variation across groups and years (29–100%). Finally, we detected an excess in observed heterozygosity and no evidence of inbreeding across our three study groups, with an observed heterozygosity of 0.766 ± 0.059 and expected heterozygosity of 0.708 ± 0.059, and an inbreeding coefficient of ?0.082 ± 0.035. Our results indicate that the selected markers are useful for genetic studies on wild crested macaques, and possibly also on other Sulawesi and closely related macaques. They further suggest that the Tangkoko population of crested macaques is still genetically variable despite its small size, isolation, and the species’ reproductive patterns. This gives us hope that other endangered primate species living in small, isolated populations may also retain a healthy gene pool, at least in the short term.     

Isolation and characterization of microsatellite loci in Palinurus elephas

The European spiny lobster (Palinurus elephas) mean annual catches have decreased alarmingly during recent decades along its entire distribution area due to stock over‐exploitation, which makes it a primary target for conservation plans. A total of 164 microsatellite loci were isolated from a genomic library of P. elephas enriched for CA, GA, CAA and GATA repeats. A total of 15 polymorphic loci have been screened in 48 individuals. High numbers of alleles per locus (averaging 20 ± 10.5) and observed heterozygosity (averaging 0.789 ± 0.197) have been detected. None of the pairs of loci showed significant linkage disequilibrium. Two of the loci (Pael1 and Pael2) showed significant departure from Hardy–Weinberg equilibrium in Sagres, while Pael38 showed significant departure in Tunis. These highly polymorphic markers will be useful in determining the spatial patterns of genetic diversity between and within populations of Palinurus elephas.     

Isolation and characterization of 18 polymorphic microsatellite markers for the “Female Ginseng” Angelica sinensis (Apiaceae) and cross-species amplification

Angelica sinensis (Apiaceae) is an endangered alpine herb that is widely used as a medicinal plant in traditional Chinese medicine (TCM). Wild populations of A. sinensis have become quite rare in China. Thus, population genetics studies of this species are urgently needed for its effective conservation and sustainable use. However, to date, no microsatellite loci have been isolated in A. sinensis. To address this issue, we isolated 18 polymorphic loci and genotyped 120 individuals collected from 6 populations. The number of alleles per locus ranged from 1.2 to 5.5, and the average was 2.4. The observed and expected heterozygosity per locus for a population varied, respectively, from 0.000 to 0.983 (averaged at 0.198) and from 0.066 to 0.661 (averaged at 0.333). Deviation from the Hardy–Weinberg equilibrium (p < 0.01) was observed for 4 to 14 loci in various populations. These microsatellite markers were cross-amplified in 10 species affinis, and 7 loci were successfully amplified in all species. These microsatellite markers are useful for genetic studies, the conservation management of A. sinensis, and identification of A. sinensis.     

Cross-amplification and characterization of 13 tetranucleotide microsatellites in multiple species of Neotropical canids

We tested 47 tetranucleotide microsatellite loci developed for the domestic dog in four species of Neotropical canids, aiming to produce a standardized set that could be successfully used even in noninvasive samples across this group. We identified 13 suitable loci, nine of which constitute a standardized set for all species. Considering only the ideal panel of nine loci, the mean expected heterozygosity (averaged across species) per locus ranged from 0.58 to 0.92 (overall mean 0.76), and the maximum probability of identity value was 1.3 × 10^?9. This set of loci has a great potential for application in evolutionary, ecological and conservation studies.     

Predator-recognition training: a conservation strategy to increase postrelease survival of hellbenders in head-starting programs

For species with declining populations, captive rearing with subsequent release into natural habitats ("head-starting") is often used as part of a conservation strategy. One challenge to head-starting programs is that head-started individuals can suffer high rates of postrelease predation. Head-starting programs are currently being established for hellbenders (Cryptobrancus alleganeinsis), large aquatic salamanders that are experiencing population declines throughout much of the species' range. Although hellbenders have innate recognition of many predators, inexperienced juveniles show only weak recognition of introduced trout. We used a classical conditioning protocol to train captive-reared hellbender larvae to show fright responses to the scent of trout. We exposed hellbender larvae to trout-scented water plus a hellbender distress secretion during training trials. In a subsequent test, these larvae responded to trout cues alone with a fright response; control larvae that were trained with the trout scent plus a blank control did not show a fright response to the trout cues. Learning was specific to trout because trained larvae did not respond to water that had been scented by a suckermouth catfish. Although a number of details remain to be addressed concerning standardized procedures, we recommend that head-starting programs for hellbenders include trout-recognition training.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Using environmental DNA and occupancy modelling to identify drivers of eastern hellbender (Cryptobranchus alleganiensis alleganiensis) extirpation

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Association between genome-wide association studies reported SNPs and pediatric-onset Crohn’s disease in Canadian children

A recent pediatric-focused genome-wide association study has implicated three novel susceptibility loci for Crohn’ disease (CD).We aimed to investigate whether the three recently reported and other previously reported genes/loci were also associated with CD in Canadian children. A case–control design was implemented at three pediatric gastroenterology clinics in Canada. Children <19 years of age with a confirmed diagnosis of CD were recruited along with controls. Single nucleotide polymorphisms (SNPs) in 19 reported genes/loci were genotyped. Associations between individual SNPs and CD were examined. A total of 563 cases and 553 controls were studied. The mean (±SD) age of the cases was 12.3 (±3.2) years. Most cases were male (56.0%), had ileo-colonic disease (L3 ± L4, 48.8%) and inflammatory behavior (B1 ± p, 87.9%) at diagnosis. Allelic association analysis (two-tailed) showed that 8 of the 19 targeted SNPs were significantly associated with overall susceptibility for CD. Associations with one additional SNP was borderline non-significant. Significantly associated SNPs included SNPs rs1250550 (p = 0.026) and rs8049439 (p = 0.04), recently reported to be specifically associated with pediatric-onset CD.Based on the results, we confirmed associations between two of the three novel pediatric-CD loci and other regions reported for associations with either pediatric and/or adult-onset CD.     

Morphological and molecular characterization of a new species of leech (Glossiphoniidae,Hirudinida): Implications for the health of its imperiled amphibian host (Cryptobranchus alleganiensis)

The hellbender (Cryptobranchus alleganiensis) is among the most intriguing and imperiled amphibians in North America. Since the 1970s and 80s, western populations of the Ozark and eastern subspecies in Missouri have declined by nearly 80%. As a result of population declines, the Ozark hellbender was recently federally protected as an endangered species, and the eastern subspecies was granted protection under CITES. Although habitat degradation is probably the biggest threat to hellbender populations, recent evidence suggests that pathogens including chytrid fungus and “flesh-eating” bacteria may also contribute to declines in Ozark hellbenders. Leeches, which are very common on Ozark hellbenders, have recently been implicated as possible vectors of disease among Ozark hellbenders but have not been described in eastern hellbenders or outside of Missouri and Arkansas. We discovered a population of leeches on eastern hellbenders in southwest Virginia and confirmed that the species of leech is within the genus Placobdella, but is morphologically and genetically distinct from all previously described leech species. We named the new species Placobdella appalachiensis sp. n. Moser and Hopkins, based on the mountainous region in which it was discovered. Our surveys over a three consecutive year period suggested that this leech species may be patchily distributed and/or have a narrow geographic range. We consistently detected leeches at one site (mean prevalence in 80 hellbenders = 27.5%; median intensity = 3.0 leeches per parasitized hellbender [range 1 – >250 leeches]) during three years of surveys, but we never found leeches in any of our other seven study sites in two streams (mean prevalence in 139 hellbenders = 0%). We found a significant positive relationship between hellbender body size and the intensity of parasitism, and we suggest the possibility that the behavioral ecology of adults leading up to reproduction may increase their encounter rates with parasites. We discuss the potential conservation implications of discovery of leeches in this stream, and make recommendations for future mitigation and monitoring efforts.     

Selected aspects of female white-faced saki (Pithecia pithecia) reproductive biology in captivity

Little is known regarding the reproductive biology of the white-faced saki. Although sakis have been in zoos since the mid-1970s, little attention has been paid to the reproductive biology of this threatened species. This study describes the results of a 3-year study of the reproduction of the white-faced saki in captivity. Using noninvasive methods to determine levels of estrone conjugates (E₁C) and pregnanediol-3-glucuronide (PdG) in daily urine samples, we have characterized the female reproductive cycle of this species. Nonconceptive ovarian cycles were 16.95 ± 1.57 (SD) days, gestational length was 146.1 ± 5.2 (SD) days, and the length of lactationally induced infertility varied among females but averaged 163 ± 40.6 (SD) days. Attempts to accelerate puberty by pairing a young female with an adult male was without effect. Puberty occurred at 32 months of age in one female. This study provides information that enhances our understanding of the reproductive biology of this species. © 1995 Wiley-Liss, Inc.     

Microsatellites for the Neotropical ant,Camponotus leydigi (Hymenoptera: Formicidae)

Ants (Hymenoptera: Formicidae) are dominant social insects that play important ecological roles in terrestrial ecosystems. Camponotus leydigi (Forel) is widely distributed in the Neotropical region and is frequently found in the Brazilian cerrado savanna interacting with plants and other insects. Field observations indicate that C. leydigi has a polydomous nesting habit, but little is known about the genetic relationship among workers. In this study, we identify the first nine microsatellite loci for C. leydigi that will allow further investigation on its genetic diversity. We used a microsatellite-enriched library method. According to this method, repetitive sequences are captured with (CT)₈ and (GT)₈ biotin-linked probes, with subsequent recovery by streptavidin magnetic-coated beads. We observed that eight loci were polymorphic. The mean (± standard error) observed and expected heterozygosities were 0.55 ± 0.23 and 0.73 ± 0.28, respectively. The rarified allelic richness ranged from 1 to 5.32. The polymorphism contents were similar to diversity estimates found in markers previously developed for other Camponotus ants. These markers will be useful for future studies on population genetics and ecology of Camponotus ants in cerrado, including nesting ecology, colony structure, dispersal and conservation.