A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian blood cells.

Understanding mitochondrial biology and pathology is key to understanding the evolution of animal form and function. However, mitochondrial measurement often involves invasive, or even terminal, sampling, which can be difficult to reconcile in wild models or longitudinal studies. Non-mammal vertebrates contain mitochondria in their red blood cells, which can be exploited for minimally invasive mitochondrial measurement. Several recent bird studies have measured mitochondrial function using isolated blood cells. Isolation adds time in the laboratory and might be associated with physiological complications. We developed and validated a protocol to measure mitochondrial respiration in bird whole blood. Endogenous respiration was comparable between isolated blood cells and whole blood. However, respiration towards oxidative phosphorylation was higher in whole blood, and whole blood mitochondria were better coupled and had higher maximum working capacity. Whole blood measurement was also more reproducible than measurement on isolated cells for all traits considered. Measurements were feasible over a 10-fold range of sample volumes, although both small and large volumes were associated with changes to respiratory traits. The protocol was compatible with long-term storage: after 24 h at 5°C without agitation, all respiration traits but maximum working capacity remained unchanged, the latter decreasing by 14%. Our study suggests that whole blood measurement provides faster, more reproducible, and more biologically and physiologically relevant (mitochondrial integrity) assessment of mitochondrial respiration. We recommend future studies to take a whole blood approach unless specific circumstances require the use of isolated blood cells.

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest-phase hypothermia.

Many animals downregulate body temperature to save energy when resting (rest-phase hypothermia). Small birds that winter at high latitudes have comparatively limited capacity for hypothermia and so pay large energy costs for thermoregulation during cold nights. Available evidence suggests this process is fueled by adenosine triphosphate (ATP)-dependent mechanisms. Most ATP is produced by oxidative phosphorylation in the mitochondria, but mitochondrial respiration may be lower during hypothermia because of the temperature dependence of biological processes. This can create conflict between increased organismal ATP demand and a lower mitochondrial capacity to provide it. We studied this in blood cell mitochondria of wild great tits (Parus major) by simulating rest-phase hypothermia via a 6°C reduction in assay temperature in vitro. The birds had spent the night preceding the experiment in thermoneutrality or in temperatures representing mild or very cold winter nights, but night temperatures never affected mitochondrial respiration. However, across temperature groups, endogenous respiration was 14% lower in hypothermia. This did not reflect general thermal suppression of mitochondrial function because phosphorylating respiration was unaffected by thermal state. Instead, hypothermia was associated with a threefold reduction of leak respiration, from 17% in normothermia to 4% in hypothermia. Thus, the coupling of total respiration to ATP production was 96% in hypothermia, compared to 83% in normothermia. Our study shows that the thermal insensitivity of phosphorylation combined with short-term plasticity of leak respiration may safeguard ATP production when endogenous respiration is suppressed. This casts new light on the process by which small birds endure harsh winter cold and warrants future tests across tissues in vivo.

Thermoregulatory consequences of growing up during a heatwave or a cold snap in Japanese quail.

Changes in environmental temperature during development can affect growth, metabolism and temperature tolerance of the offspring. We know little about whether such changes remain to adulthood, which is important to understand the links between climate change, development and fitness. We investigated whether phenotypic consequences of the thermal environment in early life remained in adulthood in two studies on Japanese quail (Coturnix japonica). Birds were raised under simulated heatwave, cold snap or control conditions, from hatching until halfway through the growth period, and then in common garden conditions until reproductively mature. We measured biometric and thermoregulatory [metabolic heat production (MHP), evaporative water and heat loss (EWL, EHL) and body temperature] responses to variation in submaximal air temperature at the end of the thermal acclimation period and in adulthood. Warm birds had lower MHP than control birds at the end of the thermal acclimation period and, in the warmest temperature studied (40°C), also had higher evaporative cooling capacity compared with controls. No analogous responses were recorded in cold birds, although they had higher EWL than controls in all but the highest test temperature. None of the effects found at the end of the heatwave or cold snap period remained until adulthood. This implies that chicks exposed to higher temperatures could be more prepared to counter heat stress as juveniles but that they do not enjoy any advantages of such developmental conditions when facing high temperatures as adults. Conversely, cold temperature does not seem to confer any priming effects in adolescence.

Seasonal and environmental factors contribute to the variation in the gut microbiome: A large-scale study of a small bird.

Environmental variation can shape the gut microbiome, but broad/large-scale data on among and within-population heterogeneity in the gut microbiome and the associated environmental factors of wild populations is lacking. Furthermore, previous studies have limited taxonomical coverage, and knowledge about wild avian gut microbiomes is still scarce. We investigated large-scale environmental variation in the gut microbiome of wild adult great tits across the species' European distribution range. We collected fecal samples to represent the gut microbiome and used the 16S rRNA gene sequencing to characterize the bacterial gut microbiome. Our results show that gut microbiome diversity is higher during winter and that there are compositional differences between winter and summer gut microbiomes. During winter, individuals inhabiting mixed forest habitat show higher gut microbiome diversity, whereas there was no similar association during summer. Also, temperature was found to be a small contributor to compositional differences in the gut microbiome. We did not find significant differences in the gut microbiome among populations, nor any association between latitude, rainfall and the gut microbiome. The results suggest that there is a seasonal change in wild avian gut microbiomes, but that there are still many unknown factors that shape the gut microbiome of wild bird populations.

Measuring body temperature in birds - the effects of sensor type and placement on estimated temperature and metabolic rate.

Several methods are routinely used to measure avian body temperature, but different methods vary in invasiveness. This may cause stress-induced increases in temperature and/or metabolic rate and, hence, overestimation of both parameters. Choosing an adequate temperature measurement method is therefore key to accurately characterizing an animal's thermal and metabolic phenotype. Using great tits (Parus major) and four common methods with different levels of invasiveness (intraperitoneal, cloacal, subcutaneous, cutaneous), we evaluated the preciseness of body temperature measurements and effects on resting metabolic rate (RMR) over a 40°C range of ambient temperatures. None of the methods caused overestimation or underestimation of RMR compared with un-instrumented birds, and body or skin temperature estimates did not differ between methods in thermoneutrality. However, skin temperature was lower compared with all other methods below thermoneutrality. These results provide empirical guidance for future research that aims to measure body temperature and metabolic rate in small bird models.

Variation in breeding phenology in response to climate change in two passerine species.

Increasingly warmer springs have caused phenological shifts in both plants and animals. In birds, it is well established that mean laying date has advanced to match the earlier food peak. We know less about changes in the distribution of egg-laying dates within a population and the environmental variables that determine this variation. This could be an important component of how populations respond to climate change. We, therefore, used laying date and environmental data from 39 years (1983-2021) to determine how climate change affected laying date variation in blue tits (Cyanistes caeruleus) and marsh tits (Poecile palustris), two sympatric passerines with different life histories. Both species advanced mean laying date (0.19-0.24 days per year) and mean laying date showed a negative relationship with maximum spring temperature in both blue and marsh tits. In springs with no clear temperature increase during the critical time window (the time-window in which mean laying date was most sensitive to temperature) start of breeding in blue tits was distributed over a longer part of the season. However, there was no such pattern in marsh tits. Our findings suggest that temperature change, and not necessarily absolute temperature, can shape the variation in breeding phenology in a species-specific manner, possibly linked to variation in life-history strategies. This is an important consideration when predicting how climate change affects timing of breeding within a population.

Avian red blood cell mitochondria produce more heat in winter than in autumn.

Endotherms in cold regions improve heat-producing capacity when preparing for winter. We know comparatively little about how this change is fueled by seasonal adaptation in cellular respiration. Thus, we studied the changes of mitochondrial function in red blood cells in sympatric Coal (Periparus ater), Blue (Cyanistes caeruleus), and Great (Parus major) tits between autumn and winter. These species differ more than twofold in body mass and in several aspects of their foraging ecology and social dominance, which could require differential seasonal adaptation of energy expenditure. Coal and Great tits in particular upregulated the mitochondrial respiration rate and mitochondrial volume in winter. This was not directed toward ATP synthesis, instead reflecting increased uncoupling of electron transport from ATP production. Because uncoupling is exothermic, this increased heat-producing capacity at the sub-cellular level in winter. This previously unexplored the route of thermogenesis in birds should be addressed in future work.

Experimental warming during incubation improves cold tolerance of blue tit (Cyanistes caeruleus) chicks.

Climate change and increasing air temperature may alter environmental conditions for developing birds, with a range of phenotypic consequences for offspring. The thermal environment during incubation may affect the trade-off between growth and thermoregulation, but the effects of temperature on the ontogeny of endothermy are not fully understood. Therefore, we experimentally tested whether heating the nest cup of Eurasian blue tits (Cyanistes caeruleus) during incubation would influence cold tolerance of the chicks after hatching. Chicks from both heated and control nests showed a decrease in cooling rate with age as they became increasingly endothermic and homeothermic. However, chicks from previously heated nests cooled at a lower rate per unit surface area and from across the whole body. These chicks also had a greater body mass during the first 12 days of life compared with chicks from control nests. Lower cooling rates in heated chicks may reflect greater thermogenic capacity or a reduced surface area to volume ratio owing to a greater body mass. Future projections for climate change predict rising air temperature and increased likelihood of heatwaves, even in temperate regions. Our results indicate that nest microclimate can affect thermoregulation in offspring, and thus may be used to predict some of the future physiological responses of birds to climate change during breeding.

Major population splits coincide with episodes of rapid climate change in a forest-dependent bird.

Climate change influences population demography by altering patterns of gene flow and reproductive isolation. Direct mutation rates offer the possibility for accurate dating on the within-species level but are currently only available for a handful of vertebrate species. Here, we use the first directly estimated mutation rate in birds to study the evolutionary history of pied flycatchers (Ficedula hypoleuca). Using a combination of demographic inference and species distribution modelling, we show that all major population splits in this forest-dependent system occurred during periods of increased climate instability and rapid global temperature change. We show that the divergent Spanish subspecies originated during the Eemian-Weichselian transition 115-104 thousand years ago (kya), and not during the last glacial maximum (26.5-19 kya), as previously suggested. The magnitude and rates of climate change during the glacial-interglacial transitions that preceded population splits in pied flycatchers were similar to, or exceeded, those predicted to occur in the course of the current, human-induced climate crisis. As such, our results provide a timely reminder of the strong impact that episodes of climate instability and rapid temperature changes can have on species' evolutionary trajectories, with important implications for the natural world in the Anthropocene.

Low incubation temperature slows the development of cold tolerance in a precocial bird.

Incubating birds trade off self-maintenance for keeping eggs warm. This causes lower incubation temperature in more challenging conditions, with consequences for a range of offspring traits. It is not yet clear how low developmental temperature affects cold tolerance early in life. This is ecologically important because before full thermoregulatory capacity is attained, precocial chicks must switch between foraging and being brooded when their body temperature declines. Hence, we studied how cold tolerance during conditions similar to a feeding bout in the wild was affected by incubation temperature in Japanese quail (Coturnix japonica). Cold-incubated (35.5°C) chicks took the longest to develop, hatched at a smaller size, and remained smaller during their first week of life compared with chicks incubated at higher temperatures (37.0 and 38.5°C). This was reflected in increased cooling rate and reduced homeothermy, probably on account of reductions in both heat-producing capacity and insulation. Lower cold tolerance could exacerbate other temperature-linked phenotypic effects and, hence, also the trade-off between future and current reproduction from the perspective of the incubating parent.

Variation in reproductive investment increases body temperature amplitude in a temperate passerine.

Many birds and mammals show substantial circadian variation in body temperature, which has been attributed to fluctuations in ambient temperature and energy reserves. However, to fully understand the variation in body temperature over the course of the day, we also need to consider effects of variation in work rate. We made use of a dataset on body temperature during the resting and active periods in female marsh tits (Poecile palustris) that bred in a temperate area and were subjected to experimental changes in reproductive investment through brood size manipulations. Furthermore, the amplitude increased with daytime, but were unaffected by nighttime, ambient temperature. Amplitudes in females with manipulated broods were 44% above predictions based on inter-specific allometric relationships. In extreme cases, amplitudes were > 100% above predicted values. However, no individual female realised the maximum potential amplitude (8.5 °C, i.e. the difference between the highest and lowest body temperature within the population) but seemed to prioritise either a reduction in body temperature at night or an increase in body temperature in the day. This suggests that body temperature amplitude might be constrained by costs that preclude extensive use of both low nighttime and high daytime body temperatures within the same individual. Amplitudes in the range found here (0.5-6.7 °C) have previously mostly been reported from sub-tropical and/or arid habitats. We show that comparable values can also be found amongst birds in relatively cool, temperate regions, partly due to a pronounced increase in body temperature during periods with high work rate.

Plumage development and environmental factors influence surface temperature gradients and heat loss in wandering albatross chicks.

Young birds in cold environments face a range of age-specific thermal challenges. Studying the thermal biology of young birds throughout ontogeny may further our understanding of how such challenges are met. We investigated how age and environmental parameters influenced surface temperature gradients across various body regions of wandering albatross (Diomedea exulans) chicks on Bird Island, South Georgia. This study was carried out over a 200 d period during the austral winter, from the end of the brood-guard period until fledging, bridging a gap in knowledge of surface temperature variation and heat loss in developing birds with a long nestling stage in severe climatic conditions. We found that variation in surface temperature gradients (i.e. the difference between surface and environmental temperature) was strongly influenced by chick age effects for insulated body regions (trunk), with an increase in the surface temperature gradient that followed the progression of plumage development, from the second set of down (mesoptiles), to final chick feathers (teleoptiles). Environmental conditions (primarily wind speed and relative humidity) had a stronger influence on the gradients in uninsulated areas (eye, bill) than insulated regions, which we interpret as a reflection of the relative degree of homeothermy exhibited by chicks of a given age. Based on biophysical modelling, total heat loss of chicks was estimated to increase linearly with age. However, mass specific heat loss decreased during the early stages of growth and then subsequently increased. This was attributed to age-related changes in feather growth and activity that increased surface temperature and, hence, metabolic heat loss. These results provide a foundation for further work on the effects of environmental stressors on developing chicks, which are key to understanding the physiological responses of animals to changes in climate in polar regions.

Reduced immune responsiveness contributes to winter energy conservation in an Arctic bird.

Animals in seasonal environments must prudently manage energy expenditure to survive the winter. This may be achieved through reductions in the allocation of energy for various purposes (e.g. thermoregulation, locomotion, etc.). We studied whether such trade-offs also include suppression of the innate immune response, by subjecting captive male Svalbard ptarmigan (Lagopus muta hyperborea) to bacterial lipopolysaccharide (LPS) during exposure to either mild temperature (0°C) or cold snaps (acute exposure to -20°C), in constant winter darkness when birds were in energy-conserving mode, and in constant daylight in spring. The innate immune response was mostly unaffected by temperature. However, energy expenditure was below baseline when birds were immune challenged in winter, but significantly above baseline in spring. This suggests that the energetic component of the innate immune response was reduced in winter, possibly contributing to energy conservation. Immunological parameters decreased (agglutination, lysis, bacteriostatic capacity) or did not change (haptoglobin/PIT54) after the challenge, and behavioural modifications (anorexia, mass loss) were lengthy (9 days). While we did not study the mechanisms explaining these weak, or slow, responses, it is tempting to speculate they may reflect the consequences of having evolved in an environment where pathogen transmission rate is presumably low for most of the year. This is an important consideration if climate change and increased exploitation of the Arctic would alter pathogen communities at a pace outwith counter-adaption in wildlife.

Experimental facilitation of heat loss affects work rate and innate immune function in a breeding passerine bird.

The capacity to get rid of excess heat produced during hard work is a possible constraint on parental effort during reproduction [heat dissipation limit (HDL) theory]. We released hard-working blue tits (Cyanistes caeruleus) from this constraint by experimentally removing ventral plumage. We then assessed whether this changed their reproductive effort (feeding rate and nestling size) and levels of self-maintenance (change in body mass and innate immune function). Feather-clipped females reduced the number of feeding visits and increased levels of constitutive innate immunity compared with unclipped females but did not fledge smaller nestlings. Thus, they increased self-maintenance without compromising current reproductive output. In contrast, feather clipping did not affect the number of feeding visits or innate immune function in males, despite increased heat loss rate. Our results show that analyses of physiological parameters, such as constitutive innate immune function, can be important when trying to understand sources of variation in investment in self-maintenance versus reproductive effort and that risk of overheating can influence innate immune function during reproduction.

Body surface temperature responses to food restriction in wild and captive great tits.

During winter at temperate and high latitudes, the low ambient temperatures, limited food supplies and short foraging periods mean small passerines show behavioural, morphological and physiological adaptations to reduce the risk of facing energy shortages. Peripheral tissues vasoconstrict in low ambient temperatures to reduce heat loss and cold injury. Peripheral vasoconstriction has been observed with food restriction in captivity but has yet to be explored in free-ranging animals. We experimentally food restricted both wild and captive great tits (Parus major) during winter months and measured surface temperatures of the bill and eye region using thermal imaging, to investigate whether birds show rapid local heterothermic responses, which may reduce their thermoregulatory costs when facing a perceived imminent food shortage. Our results of a continuously filmed wild population showed that bill temperature was immediately reduced in response to food restriction compared with when food was available ad libitum, an apparent autonomic response. Such immediacy implies a 'pre-emptive' response before the bird experiences any shortfalls in energy reserves. We also demonstrate temporal variation in vasoconstriction of the bill, with bill temperature gradually rising throughout the food restriction after the initial drop. Eye-region temperature in the wild birds remained at similar levels throughout food restriction compared with unrestricted birds, possibly reflecting the need to maintain steady circulation to the central nervous and visual systems. Our findings provide evidence that birds selectively allow the bill to cool when a predictable food supply is suddenly disrupted, probably as a means of minimising depletion of body reserves for a perceived future shortage in energy.

The metabolic cost of subcutaneous and abdominal rewarming in king penguins after long-term immersion in cold water.

Marine endotherms in the polar regions face a formidable thermal challenge when swimming in cold water. Hence, they use morphological (fat, blubber) adjustment and peripheral vasoconstriction to reduce demands for heat production in water. The animals then regain normothermia when resting ashore. In the king penguin (Aptenodytes patagonicus) metabolic rate is lower in fed than in fasted individuals during subsequent rewarming on land. This has been suggested to be a consequence of diversion of blood flow to the splanchnic region in fed birds, which reduces peripheral temperatures. However, peripheral temperatures during recovery have never been investigated in birds with different nutritional status. The aim of this study was, therefore, to measure subcutaneous and abdominal temperatures during the rewarming phase on land in fasted and fed king penguins, and investigate to which extent any different rewarming were reflected in recovery metabolic rate (MRR) after long term immersion in cold water. We hypothesized that fed individuals would have a slower increase of subcutaneous temperatures compared to fasted penguins, and a correspondingly lower MRR. Subcutaneous tissues reached normothermia after 24.15 (back) and 21.36 min (flank), which was twice as fast as in the abdomen (46.82 min). However, recovery time was not affected by nutritional condition. MRR during global rewarming (4.56 ± 0.42 W kg-1) was twice as high as resting metabolic rate (RMR; 2.16 ± 0.59 W kg-1). However, MRR was not dependent on feeding status and was significantly elevated above RMR only until subcutaneous temperature had recovered. Contrary to our prediction, fed individuals did not reduce the subcutaneous circulation compared to fasted penguins and did not show any changes in MRR during subsequent recovery. It seems likely that lower metabolic rate in fed king penguins on land reported in other studies might not have been caused primarily by increased circulation to the visceral organs.

Deep body and surface temperature responses to hot and cold environments in the zebra finch.

Global warming increasingly challenges thermoregulation in endothermic animals, particularly in hot and dry environments where low water availability and high temperature increase the risk of hyperthermia. In birds, un-feathered body parts such as the head and bill work as 'thermal windows', because heat flux is higher compared to more insulated body regions. We studied how such structures were used in different thermal environments, and if heat flux properties change with time in a given temperature. We acclimated zebra finches (Taeniopygia guttata) to two different ambient temperatures, 'cold' (5 °C) and 'hot' (35 °C), and measured the response in core body temperature using a thermometer, and head surface temperature using thermal imaging. Birds in the hot treatment had 10.3 °C higher head temperature than those in the cold treatment. Thermal acclimation also resulted in heat storage in the hot group: core body temperature was 1.1 °C higher in the 35 °C group compared to the 5 °C group. Hence, the thermal gradient from core to shell was 9.03 °C smaller in the hot treatment. Dry heat transfer rate from the head was significantly lower in the hot compared to the cold treatment after four weeks of thermal acclimation. This reflects constraints on changes to peripheral circulation and maximum body temperature. Heat dissipation capacity from the head region increased with acclimation time in the hot treatment, perhaps because angiogenesis was required to reach peak heat transfer rate. We have shown that zebra finches meet high environmental temperature by heat storage, which saves water and energy, and by peripheral vasodilation in the head, which facilitates dry heat loss. These responses will not exclude the need for evaporative cooling, but will lessen the amount of energy expend on body temperature reduction in hot environments.

Age-dependent effects of predation risk on night-time hypothermia in two wintering passerine species.

Small animals that winter at northern latitudes need to maximize energy intake and minimize energy loss. Many passerine birds use night-time hypothermia to conserve energy. A potential cost of night-time hypothermia with much theoretical (but little empirical) support is increased risk of night-time predation, due to reduced vigilance and lower escape speed in hypothermic birds. This idea has never been tested in the wild. We, therefore, increased perceived predation risk in great tits (Parus major) and blue tits (Cyanistes caeruleus) roosting in nest boxes during cold winter nights to measure any resultant effect on their use of night-time hypothermia. Roosting birds of both species that experienced their first winter were less prone to use hypothermia as an energy-saving strategy at low ambient temperatures when exposed to increased perceived predation risk either via handling (great tits) or via predator scent manipulation (blue tits). However, we did not record such effects in birds that were in their second winter or beyond. Our results suggest that effects of increased predation risk are age- and temperature specific. This could be caused by age-related differences in experience and subsequent risk assessment, or by dominance-related variation in habitat quality between young and old birds. Predation risk could, through its effect on use and depth of night-time hypothermia, be important for total energy management and winter survival for resident birds at northern latitudes.

Testing the heat dissipation limit theory in a breeding passerine.

The maximum work rate of animals has recently been suggested to be determined by the rate at which excess metabolic heat generated during work can be dissipated (heat dissipation limitation (HDL) theory). As a first step towards testing this theory in wild animals, we experimentally manipulated brood size in breeding marsh tits (Poecile palustris) to change their work rate. Parents feeding nestlings generally operated at above-normal body temperatures. Body temperature in both males and females increased with maximum ambient temperature and with manipulated work rate, sometimes even exceeding 45°C, which is close to suggested lethal levels for birds. Such high body temperatures have previously only been described for birds living in hot and arid regions. Thus, reproductive effort in marsh tits may potentially be limited by the rate of heat dissipation. Females had lower body temperatures, a possible consequence of their brood patch serving as a thermal window facilitating heat dissipation. Because increasing body temperatures are connected to somatic costs, we suggest that the HDL theory may constitute a possible mediator of the trade-off between current and future reproduction. It follows that globally increasing, more stochastic, ambient temperatures may restrict the capacity for sustained work of animals in the future.

Seasonal variation in the thermal responses to changing environmental temperature in the world's northernmost land bird.

Arctic homeotherms counter challenges at high latitudes using a combination of seasonal adjustments in pelage/plumage, fat deposition and intricate thermoregulatory adaptations. However, there are still gaps in our understanding of their thermal responses to cold, particularly in Arctic birds. Here, we have studied the potential use of local heterothermy (i.e. tissue cooling that can contribute to significantly lower heat loss rate) in Svalbard ptarmigan (Lagopus muta hyperborea) - the world's northernmost land bird. We exposed birds kept under simulated Svalbard photoperiod to low ambient temperatures (Ta; between 0 and -30°C) during three seasons (early winter, late winter, summer), whilst recording resting metabolic rate (RMR), core temperature (Tc) and several cutaneous temperatures. Leg skin temperature varied the most, but still only by up to ∼15°C, whereas body trunk skin temperature changed <1°C when Ta decreased from 0 to -30°C. At the same time, Tc increased by 0.9°C, concomitant with increased RMR. This was probably driven by the triggering of cerebral thermosensors in response to cooling of the poorly insulated head, the skin of which was 5.4°C colder at -30°C than at 0°C. Thermal conductance in winter was higher in yearlings, probably because they were time/resource constrained from acquiring a high-quality plumage and sufficient fat reserves as a result of concomitant body growth. In conclusion, Svalbard ptarmigan do not employ extensive local heterothermy for cold protection but instead rely on efficient thermogenesis combined with excellent body insulation. Hence, cold defence in the world's northernmost land bird is not mechanistically much different from that of its lower latitude relatives.