Emily D. Fountain

Effects of Diets Enriched in Omega-3 and Omega-6 Polyunsaturated Fatty Acids on Offspring Sex-Ratio and Maternal Behavior in Mice

Abstract There have been many trials describing the effects of polyunsaturated fatty acids (PUFA) on fecundity, neonatal development, and maternal behavior in humans, but few controlled studies in rodents. We examined the effects of a maternal diet high in omega 3 (N-3) or omega 6 (N-6) PUFA on NIH Swiss mice. Female mice were ad libitum fed one of three complete and balanced diets (N-3, enriched in menhaden oil; N-6, enriched in corn oil; C, control diet, Purina 5015) from age 4 wk until the end of the study. Mice were bred at ∼19 wk and 27 wk of age, providing a total of 838 pups from 129 litters in two experiments. After weaning their pups from parity 1, behavior of dams was assessed on elevated-plus and open-field mazes. Although the fraction of male pups from the N-3 and C groups was not different from 0.5, dams on the N-6 diet birthed more daughters than sons (213 vs. 133; P < 0.001). Although maternal stress has been reported to favor birth of daughters, the behavior of N-6 dams was not different from controls. By contrast, the N-3 dams displayed greater anxiety, spending less time in the open arms and more time in the closed arms of the elevated maze and traveling less distance and exhibiting less exploratory behavior in the open field (P < 0.05). N-3 dams tended to produce smaller litters than C dams, and N-3-suckled pups gained less weight (P < 0.05). In conclusion, the N-3 diet had negative effects on murine fecundity and maternal behavior, whereas the N-6 diet favored birth of daughters.

Maternal diet composition alters serum steroid and free fatty acid concentrations and vaginal pH in mice

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

Finding the right coverage: the impact of coverage and sequence quality on single nucleotide polymorphism genotyping error rates

Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction‐enzyme‐based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother–offspring dyads of Hoffmans two‐toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference‐aligned and de novo‐assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo‐assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference‐aligned and de novo‐assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference‐aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo‐assembled data sets. We observed approximately 10‐ and 13‐fold declines in the number of loci sampled in the reference‐aligned and de novo‐assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade‐off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction‐enzyme‐based SNP studies.

Arboreal Folivores Limit Their Energetic Output, All the Way to Slothfulness.

By exploiting unutilized resources, organisms expand into novel niches, which can lead to adaptive radiation. However, some groups fail to diversify despite the apparent opportunity to do so. Although arising multiple times, arboreal folivores are rare and have not radiated, presumably because of energetic constraints. To explore this hypothesis, we quantified the field metabolic rate (FMR), movement, and body temperature for syntopic two- and three-toed sloths, extreme arboreal folivores that differ in their degree of specialization. Both species expended little energy, but three-toed sloths (162 kJ/day*kg0.734) possessed the lowest FMR recorded for any mammal. Three-toed sloths were more heterothermic and moved less than two-toed sloths. We then compared FMRs and basal metabolic rates (BMRs) for 19 species of arboreal folivores along a spectrum of specialization. Overall, arboreal folivores had lower BMRs and FMRs than other mammals, and increasing specialization led to lower FMRs but not BMRs. Thus, reduced energetic expenditure in specialized species was the result of thermoregulatory and behavioral strategies, rather than simply a proportionate reduction in BMR. Altogether, our findings support the concept that arboreal folivores are tightly constrained by nutritional energetics and help to explain the lack of radiation among species exploiting a lifestyle in the trees.

Examining the uncertain origin and management role of martens on Prince of Wales Island, Alaska

Conservation biologists are generally united in efforts to curtail the spread of non-native species globally. However, the colonization history of a species is not always certain, and whether a species is considered non-native or native depends on the conservation benchmark. Such ambiguities have led to inconsistent management. Within the Tongass National Forest of Alaska, the status of American marten (Martes americana) on the largest, most biologically diverse and deforested island, Prince of Wales (POW), is unclear. Ten martens were released to POW in the early 1930s, and it was generally believed to be the founding event, although this has been questioned. The uncertainty surrounding when and how martens colonized POW complicates management, especially because martens were selected as a design species for the Tongass. To explore the history of martens of POW we reviewed other plausible routes of colonization; genetically and isotopically analyzed putative marten fossils deposited in the late Pleistocene and early Holocene to verify marten occupancy of POW; and used contemporary genetic data from martens on POW and the mainland in coalescent simulations to identify the probable source of the present-day marten population on POW. We found evidence for multiple routes of colonization by forest-associated mammals beginning in the Holocene, which were likely used by American martens to naturally colonize POW. Although we cannot rule out human-assisted movement of martens by Alaskan Natives or fur trappers, we suggest that martens be managed for persistence on POW. More generally, our findings illustrate the difficulty of labeling species as non-native or native, even when genetic and paleo-ecological data are available, and support the notion that community resilience or species invasiveness should be prioritized when making management decisions rather than more subjective and less certain conservation benchmarks.

Finding the right coverage : The impact of coverage and sequence quality on SNP genotyping error rates

Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction‐enzyme‐based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother–offspring dyads of Hoffmans two‐toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference‐aligned and de novo‐assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo‐assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference‐aligned and de novo‐assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference‐aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo‐assembled data sets. We observed approximately 10‐ and 13‐fold declines in the number of loci sampled in the reference‐aligned and de novo‐assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade‐off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction‐enzyme‐based SNP studies.

The effects of island forest restoration on open habitat specialists: the endangered weevil Hadramphus spinipennis Broun and its host-plant Aciphylla dieffenbachii Kirk

Human alteration of islands has made restoration a key part of conservation management. As islands are restored to their original state, species interactions change and some populations may be impacted. In this study we examine the coxella weevil, (Hadramphus spinipennis Broun) and its host-plant Dieffenbach’s speargrass (Aciphylla dieffenbachii Kirk), which are both open habitat specialists with populations on Mangere and Rangatira Islands, Chathams, New Zealand. Both of these islands were heavily impacted by the introduction of livestock; the majority of the forest was removed and the weevil populations declined due to the palatability of their host-plant to livestock. An intensive reforestation program was established on both islands over 50 years ago but the potential impacts of this restoration project on the already endangered H. spinipennis are poorly understood. We combined genetic and population data from 1995 and 2010–2011 to determine the health and status of these species on both islands. There was some genetic variation between the weevil populations on each island but little variation within the species as a whole. The interactions between the weevil and its host-plant populations appear to remain intact on Mangere, despite forest regeneration. A decline in weevils and host-plant on Rangatira does not appear to be caused by canopy regrowth. We recommend that (1) these populations be monitored for ongoing effects of long-term reforestation, (2) the cause of the decline on Rangatira be investigated, and (3) the two populations of weevils be conserved as separate evolutionarily significant units.

Genomics meets applied ecology: Characterizing habitat quality for sloths in a tropical agro-ecosystem

Understanding how habitat quality in heterogeneous landscapes governs the distribution and fitness of individuals is a fundamental aspect of ecology. While mean individual fitness is generally considered a key to assessing habitat quality, a comprehensive understanding of habitat quality in heterogeneous landscapes requires estimates of dispersal rates among habitat types. The increasing accessibility of genomic approaches, combined with field‐based demographic methods, provides novel opportunities for incorporating dispersal estimation into assessments of habitat quality. In this study, we integrated genomic kinship approaches with field‐based estimates of fitness components and approximate Bayesian computation (ABC) procedures to estimate habitat‐specific dispersal rates and characterize habitat quality in two‐toed sloths (Choloepus hoffmanni) occurring in a Costa Rican agricultural ecosystem. Field‐based observations indicated that birth and survival rates were similar in a sparsely shaded cacao farm and adjacent cattle pasture–forest mosaic. Sloth density was threefold higher in pasture compared with cacao, whereas home range size and overlap were greater in cacao compared with pasture. Dispersal rates were similar between the two habitats, as estimated using ABC procedures applied to the spatial distribution of pairs of related individuals identified using 3,431 single nucleotide polymorphism and 11 microsatellite locus genotypes. Our results indicate that crops produced under a sparse overstorey can, in some cases, constitute lower‐quality habitat than pasture–forest mosaics for sloths, perhaps because of differences in food resources or predator communities. Finally, our study demonstrates that integrating field‐based demographic approaches with genomic methods can provide a powerful means for characterizing habitat quality for animal populations occurring in heterogeneous landscapes.

Cophylogenetics and biogeography reveal a coevolved relationship between sloths and their symbiont algae

Specialized species, like arboreal folivores, often develop beneficial relationships with symbionts to exploit ecologically constrained lifestyles. Although coevolution can drive speciation by specialization of a symbiont to a host, a symbiotic relationship is not indicative of coevolution between host and symbiont. We tested for coevolved relationships between highly specialized two- and three-toed sloths (Choloepus spp. and Bradypus spp., respectively) and their symbiotic algae using cophylogenies and phylogeography. Our phylogeographic analysis showed a biogeographic pattern for the sloth distribution that was not found in the algal phylogeny. We found support for congruence between the sloth and algae phylogenies, implying cospeciation, only in the Bradypus lineage. Algae host-switching occurred from Bradypus spp. to Choloepus spp. Our results support a previously hypothesized symbiotic relationship between sloths and the algae in their fur and indicate that coevolution may have played a role in algae diversification. More broadly, convergent evolution may facilitate host switching between deeply diverged host lineages.

Finding the right coverage

Restriction‐enzyme‐based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction‐enzyme‐based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother–offspring dyads of Hoffmans two‐toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference‐aligned and de novo‐assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo‐assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference‐aligned and de novo‐assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference‐aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo‐assembled data sets. We observed approximately 10‐ and 13‐fold declines in the number of loci sampled in the reference‐aligned and de novo‐assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade‐off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction‐enzyme‐based SNP studies.