Reproductive timing and reliance on hoarded capital resources by lactating red squirrels

Successful reproduction in a seasonal environment can be accomplished with resources that are stored before use (“capital resources”) or resources that are used immediately (“income resources”). Research examining capital versus income resource usage during reproduction has primarily focused on assigning species to positions along a capital–income gradient. Here, we examine the causes and reproductive consequences of among and within-year variation in hoarded capital versus income resource usage by female North American red squirrels (Tamiasciurus hudsonicus) during mid-lactation in a highly seasonal environment. Among years, the proportion of feeding events that were on capital resources (PROPCAP) averaged 39 % during the yearly median mid-lactation periods, but ranged widely between 2 and 100 %. In years with earlier parturition dates, females primarily used hoarded capital resources during mid-lactation, whereas in years with later parturition dates, females primarily used income resources during mid-lactation. Within years, PROPCAP during mid-lactation tended to be greater in early-breeding females than in late-breeding females. Rates of water flux in females during mid-lactation provided further evidence that late-breeding females used more water-rich income resources. The proportion of litters that were partially or completely lost, and the litter mass that lactating females supported, was not influenced by the large among-year differences in hoarded capital resource usage. Red squirrels appear to delay reproduction following years with low cone production to time peak reproductive demands to be late enough to be supported by income resources that only become available later in the season. In conclusion, our results offer a rare example of the capacity of a food-hoarding mammal to support reproduction exploiting a wide range of capital and income resources.     

Optimisation of energetic and reproductive gains explains behavioural responses to environmental variation across seasons and years

Animals switch between inactive and active states, simultaneously impacting their energy intake, energy expenditure and predation risk, and collectively defining how they engage with environmental variation and trophic interactions. We assess daily activity responses to long‐term variation in temperature, resources and mating opportunities to examine whether individuals choose to be active or inactive according to an optimisation of the relative energetic and reproductive gains each state offers. We show that this simplified behavioural decision approach predicts most activity variation (R² = 0.83) expressed by free‐ranging red squirrels over 4 years, as quantified through accelerometer recordings (489 deployments; 5066 squirrel‐days). Recognising activity as a determinant of energetic status, the predictability of activity variation aggregated at a daily scale, and the clear signal that behaviour is environmentally forced through optimisation of gain, provides an integrated approach to examine behavioural variation as an intermediary between environmental variation and energetic, life‐history and ecological outcomes.     

Indirect effects on fitness between individuals that have never met via an extended phenotype

Interactions between organisms are ubiquitous and have important consequences for phenotypes and fitness. Individuals can even influence those they never meet, if they have extended phenotypes that alter the environments others experience. North American red squirrels (Tamiasciurus hudsonicus) guard food hoards, an extended phenotype that typically outlives the individual and is usually subsequently acquired by non‐relatives. Hoarding by previous owners can, therefore, influence subsequent owners. We found that red squirrels breed earlier and had higher lifetime fitness if the previous hoard owner was a male. This was driven by hoarding behaviour, as males and mid‐aged squirrels had the largest hoards, and these effects persisted across owners, such that if the previous owner was male or died in mid‐age, subsequent occupants had larger hoards. Individuals can, therefore, influence each other's resource‐dependent traits and fitness without ever meeting, such that the past can influence contemporary population dynamics through extended phenotypes.     

Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.The chlorinated form of auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), is a highly active hormone that is thought to play a key role in early pericarp growth (Reinecke et al., 1995, 1999; Ozga et al., 2009). Exogenous 4-Cl-IAA, for example, has been shown to promote the pericarp elongation of deseeded pea (Pisum sativum) pods (Reinecke et al., 1999). Johnstone et al. (2005) reported that 4-Cl-IAA and bioactive GA (GA₃ or GA₁) act synergistically on pericarp growth when applied simultaneously, and a growth regulatory role has been proposed for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. In other species, e.g. tomato (Solanum lycopersicum), the nonchlorinated form of auxin, indole-3-acetic acid (IAA), also stimulates fruit growth via GAs (Serrani et al., 2008; Tang et al., 2015). The chlorinated auxin is mainly found in reproductive structures (Katayama et al., 1988), in which its levels often exceed those of the more widespread IAA (Tivendale et al., 2012). The chlorinated form is thought to be restricted to members of the leguminous tribe Fabeae (Reinecke 1999), which includes the genera Vicia, Pisum, Lathyrus, Lens, and Vavilovia (Schaefer et al., 2012). However, there is a curious exception: 4-Cl-IAA has been reported also from Scots pine (Pinus sylvestris; Ernstsen and Sandberg, 1986).We previously published evidence that most 4-Cl-IAA in maturing pea seeds is synthesized from 4-Cl-tryptophan (4-Cl-Trp) via 4-Cl-indole-3-pyruvic acid (Tivendale et al., 2012, 2014). 4-Cl-Trp has been identified in extracts from pea and broad bean (Vicia faba) seeds (Kettner et al., 1992; Manabe et al., 1999), but whether the precursors of Trp can be chlorinated is unknown.Virtually nothing is known about the enzymes that catalyze halogenation reactions in plants. In bacteria, fungi, and marine algae, there are six types of enzymes responsible for the addition of halogen atoms to organic molecules. These include heme haloperoxidases, vanadium-dependent haloperoxidases, mononuclear nonheme iron halogenases, flavin-dependent halogenases, S-adenosyl-l-Met-dependent chlorinases and fluorinases, and methyl halide transferases (Butler and Sandy, 2009; Wagner et al., 2009). However, in the genomes of angiosperms, the only type of halogenating enzyme that has been annotated are haloperoxidases, but very little is known about these enzymes. To further understand the activity and action of halogenating enzymes in plants, a comparative system is required.In this study, we investigated the distribution of 4-Cl-IAA and 4-Cl-Trp in the Fabaceae by monitoring these compounds in the seeds of representative species spanning the phylogeny of this family. Most of these species have not been previously tested for the presence of the chlorinated compounds. In addition, we reexamined the reported occurrence of 4-Cl-IAA outside the Fabaceae, namely in Scots pine; several other Pinus species were investigated here as well. We also examined the endogenous levels of 4-Cl-IAA in both vegetative tissues and seeds of broad bean to address the question of whether 4-Cl-IAA is largely restricted to seeds (Pless et al., 1984; Katayama et al., 1988).     

Reproductive phenology of a food-hoarding mast-seed consumer: resource- and density-dependent benefits of early breeding in red squirrels

The production of offspring by vertebrates is often timed to coincide with the annual peak in resource availability. However, capital breeders can extend the energetic benefits of a resource pulse by storing food or fat, thus relaxing the need for synchrony between energy supply and demand. Food-hoarding red squirrels (Tamiasciurus hudsonicus) breeding in the boreal forest are reliant on cones from a masting conifer for their nutrition, yet lactation is typically completed before the annual crop of cones is available for consumption such that peaks in energy supply and demand are not synchronized. We investigated the phenological response of red squirrels to annual variation in environmental conditions over a 20-year span and examined how intra- and inter-annual variation in the timing of reproduction affected offspring recruitment. Reproductive phenology was strongly affected by past resource availability with offspring born earlier in years following large cone crops, presumably because this affected the amount of capital available for reproduction. Early breeders had higher offspring survival and were more likely to renest following early litter loss when population density was high, perhaps because late-born offspring are less competitive in obtaining a territory when vacancies are limited. Early breeders were also more likely to renest after successfully weaning their first litter, but renesting predominantly occurred during mast years. Because of their increased propensity to renest and the higher survival rates of their offspring, early breeders contribute more recruits to the population but the advantage of early breeding depends on population density and resource availability.     

Very low levels of direct additive genetic variance in fitness and fitness components in a red squirrel population

A trait must genetically correlate with fitness in order to evolve in response to natural selection, but theory suggests that strong directional selection should erode additive genetic variance in fitness and limit future evolutionary potential. Balancing selection has been proposed as a mechanism that could maintain genetic variance if fitness components trade off with one another and has been invoked to account for empirical observations of higher levels of additive genetic variance in fitness components than would be expected from mutation–selection balance. Here, we used a long‐term study of an individually marked population of North American red squirrels (Tamiasciurus hudsonicus) to look for evidence of (1) additive genetic variance in lifetime reproductive success and (2) fitness trade‐offs between fitness components, such as male and female fitness or fitness in high‐ and low‐resource environments. “Animal model” analyses of a multigenerational pedigree revealed modest maternal effects on fitness, but very low levels of additive genetic variance in lifetime reproductive success overall as well as fitness measures within each sex and environment. It therefore appears that there are very low levels of direct genetic variance in fitness and fitness components in red squirrels to facilitate contemporary adaptation in this population.     

Within-season synchrony of a masting conifer enhances seed escape

Predator satiation resulting from interannual reproductive synchrony has been widely documented in masting plants, but how reproductive synchrony within a year influences seed escape is poorly understood. We evaluated whether the intra-annual reproductive synchrony of individual white spruce trees (Picea glauca) increased seed escape from their primary predispersal seed predator, North American red squirrels (Tamiasciurus hudsonicus). Trees with cones that matured synchronously relative to those of other trees within red squirrel territories were significantly more likely to escape squirrel predation in years with both low and superabundant levels of cone production, generating a significantly positive linear selection differential for increasing intra-annual reproductive synchrony. Thus, this masting plant escapes seed predation in numbers through interannual synchrony in seed production and in time through intra-annual synchrony of seed availability.     

Cohort effects in red squirrels: the influence of density, food abundance and temperature on future survival and reproductive success

1. Environmental conditions experienced early in life may have long-lasting effects on individual performance, thereby creating 'silver-spoon effects'. 2. We used 15 years of data from a North American red squirrel (Tamiasciurus hudsonicus Erxleben) population to investigate influences of food availability, density and spring temperature experienced early in life on reproduction and survival of female squirrels during adulthood. 3. We found that spring temperature and food availability did not affect female survival after 1 year of age, whereas higher squirrel densities led to lower survival, thereby affecting longevity and lifetime fitness. 4. In addition, both food availability experienced between birth and weaning, and spring temperature in the year of birth, had long-lasting positive effects on female reproductive success. These results emphasize the critical effect environmental conditions during the early life stages can have on the lifetime performance of small mammals. 5. These long-term effects of early food and temperature were apparent only once we controlled for conditions experienced during adulthood. This suggests that silver-spoon effects can be masked when conditions experienced early in life are correlated to some environmental conditions experienced later in life. 6. The general importance of silver-spoon effects for adult demographic performance might therefore be underestimated, and taking adult environment into account appears to be necessary when studying long-term cohort effects.     

Distinct Recognition of Non-Clade B Human Immunodeficiency Virus Type 1 Epitopes by Cytotoxic T Lymphocytes Generated from Donors Infected in Africa

We present detailed studies of human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) responses to clade A or C HIV type 1 in three donors infected in East Africa. We define several novel non-clade B CTL epitopes, including some restricted by HLA alleles common in Africans. Although cross-clade CTL recognition of these epitopes does occur, recognition can also be highly clade specific.