Universal DNA methylation age across mammalian tissues.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Evidence for contrasting roles for prolactin in eusocial naked mole-rats, Heterocephalus glaber and Damaraland mole-rats, Fukomys damarensis.

Elevated prolactin (PRL) has been associated with the expression of social and cooperative behaviours in a number of vertebrate species, as well as suppression of reproduction. As social mole-rats exhibit both of these traits, PRL is a prime candidate in mediating their social phenotype. While naked and Damaraland mole-rats (NMRs and DMRs) have evolved eusociality independently within their family, both species exhibit an extreme skew in lifetime reproductive success, with breeding restricted to a single female and one or two males. Non-breeding NMRs of both sexes are physiologically inhibited from reproducing, while in DMRs only the non-breeding females are physiologically suppressed. Newly emerging work has implicated the dopamine system and PRL as a component in socially induced reproductive suppression and eusociality in NMR, but the DMR remains unstudied in this context. To investigate evolutionary convergence in the role of PRL in shaping African mole-rat eusociality, we determined plasma PRL concentrations in breeders and non-breeders of both sexes, comparing DMRs with NMRs. Among samples from non-breeding NMRs 80% had detectable plasma PRL concentrations. As a benchmark, these often (37%) exceeding those considered clinically hyperprolactinaemic (25 ng ml-1) in humans: mean ± s.e.m.: 34.81 ± 5.87 ngml-1; range 0.00-330.30 ng ml-1 Conversely, 85% of non-breeding DMR samples had undetectable values and none had concentrations above 25 ng ml-1: 0.71 ± 0.38 ng ml-1; 0.00-23.87 ngml-1 Breeders in both species had the expected variance in plasma PRL concentrations as part of normal reproductive function, with lactating queens having significantly higher values. These results suggest that while elevated PRL in non-breeders is implicated in NMR eusociality, this may not be the case in DMRs, and suggests a lack of evolutionary convergence in the proximate control of the social phenotype in these mole-rats.

The Distribution of Gastrointestinal Parasites in Two Populations of Common Mole-Rats ( Cryptomys hottentotus hottentotus).

The spread of parasites through a host population is based on the variation in behavior and immune function between individuals and is rarely uniform. We studied the gastrointestinal parasites of common mole-rats ( Cryptomys hottentotus hottentotus, Lesson 1826) from 2 sites and assessed the levels of infection based on host sex, breeding status, and season. Only nematode species were found: Neoheligmonella sp. and Mammalakis macrospiculum (Ortlepp, 1939) and a single specimen of Trichuris sp., all of which have direct life cycles. Parasite burden and species richness was greater in the mesic habitat. The abundance of Neoheligmonella sp. differed significantly between seasons, and the season of peak abundance differed between sites, perhaps due to differences in host densities between sites. In addition, parasite burden did not differ between the sexes, but breeding animals had higher infections of Neoheligmonella sp. and M. macrospiculum than non-breeding animals. This and previous studies thus suggest that the subterranean environment is beneficial in reducing parasite diversity, although the restrictions on movement may lead to certain individuals suffering higher parasite burdens.

Complex evolution of X and Y autosomal translocations in the giant mole-rat, Cryptomys mechowi (Bathyergidae).

Cross-species chromosome painting was used to determine homologous chromosomal regions between two species of mole-rat, the naked mole-rat, Heterocephalus glaber (2n = 60), and the giant mole-rat, Cryptomys mechowi (2n = 40), using flow-sorted painting probes representative of all but two of the H. glaber chromosomal complement. In total 43 homologous regions were identified in the C. mechowi genome. Eight H. glaber chromosomes are retained in toto in C. mechowi, and 13 produce two or more signals in this species. The most striking difference in the karyotypes of the two taxa concerns their sex chromosomes. The H. glaber painting probes identified a complex series of translocations that involved the fractionation of four autosomes and the subsequent translocation of segments to the sex chromosomes and to autosomal partners in the C. mechowi genome. An intercalary heterochromatic block (IHB) was detected in sex chromosomes of C. mechowi at the boundary with the translocated autosomal segment. We discuss the likely sequence of evolutionary events that has led to the contemporary composition of the C. mechowi sex chromosomes, and consider these in the light of prevailing views on the genesis of sex chromosomes in mammals.

Phylogeographical patterns of genetic divergence and speciation in African mole-rats (Family: Bathyergidae).

African mole-rats are subterranean Hystricomorph rodents, distributed widely throughout sub-Saharan Africa, and displaying a range of social and reproductive strategies from solitary dwelling to the 'insect-like' sociality of the naked mole-rat, Heterocephalus glaber. Both molecular systematic studies of Rodentia and the fossil record of bathyergids indicate an ancient origin for the family. This study uses an extensive molecular phylogeny and mitochondrial cytochrome b and 12s rRNA molecular clocks to examine in detail the divergence times, and patterns of speciation of the five extant genera in the context of rift valley formation in Africa. Based on a value of 40-48 million years ago (Myr) for the basal divergence of the family (Heterocephalus), we estimate divergence times of 32-40 Myr for Heliophobius, 20-26 Myr for Georychus/Bathyergus and 12-17 Myr for Cryptomys, the most speciose genus. While early divergences may have been independent of rifting, patterns of distribution of later lineages may have been influenced directly by physical barriers imposed by the formation of the Kenya and Western Rift, and indirectly by accompanying climatic and vegetative changes. Rates of chromosomal evolution and speciation appear to vary markedly within the family. In particular, the genus Cryptomys appears to have undergone an extensive radiation and shows the widest geographical distribution. Of the two distinct clades within this genus, one exhibits considerable karyotypic variation while the other does not, despite comparatively high levels of sequence divergence between some taxa. These different patterns of speciation observed both within the family and within the genus Cryptomys may have been a result of environmental changes associated with rifting.

Matrilineal genetic structure within and among populations of the cooperatively breeding common marmoset, Callithrix jacchus.

Common marmosets are members of the family Callitrichidae, South American primates characterized by highly social group living and cooperative breeding. In this study we analysed 1112 base pairs (bp) of the mitochondrial control region in 59 Callithrix jacchus individuals, sampled mainly from two geographically distinct field sites in N.E. Brazil. Analysis of molecular variation revealed a highly significant genetic structuring of haplotypes between social groups and between populations. Examination of matrilineal genetic structure within social groups revealed that seven of nine recorded breeding pairs were from different maternal lineages, indicating assortative mating and outbreeding. In addition to the breeders, at least six of 10 groups contained adult individuals from different matrilines, with five haplotypes present in one social group of nine animals. Groups of mixed lineages raise questions about potential reproductive conflicts of interest, and the extent of kin-selected altruism in the evolution and maintenance of cooperative breeding in this species.

Family values: group dynamics and social control of reproduction in African mole-rats.

To exploit ecological niches where constraints have favoured selection for group living and cooperation, both vertebrates and invertebrates have evolved elaborate social systems. In mammals, numerous divergent taxa have converged at similar solutions to these ecological challenges (such as food distribution and predator avoidance), culminating in the social insect-like behaviour of the naked mole-rat. Characteristically, breeding is partitioned unequally in such groups, resulting in a 'reproductive skew'. New research linking studies of physiology, behaviour and molecular ecology in African mole-rats is helping us to elucidate why different proximate mechanisms that control groups of cooperative breeders might have evolved.

Circulating LH levels and the response to exogenous GnRH in the common mole-rat: implications for reproductive regulation in this social, seasonal breeding species.

The effects of breeding season and reproductive status on male and female reproduction were investigated in the common mole-rat, Cryptomys hottentotus hottentotus, a cooperatively breeding rodent which exhibits a unique combination of seasonal breeding and a reproductive division of labor. Pituitary function was examined by measuring the luteinizing hormone (LH) responses to single doses of 2 microg exogenous gonadotrophin-releasing hormone (GnRH) and physiological saline in 69 males and 58 females from 35 wild caught colonies. Neither males nor females exhibited any apparent manifestation of season on basal LH concentrations or on pituitary sensitivity to stimulation by exogenous GnRH. The continuance of reproductive function during the nonbreeding period is essential in common mole-rat males and females, as this period coincides with the period of maximal dispersal opportunity in the winter rainfall area they inhabit. Normal circulating levels of reproductive hormones in dispersing animals may aid intersexual recognition, assist pairbond formation, and thus prime animals for independent reproduction. Circulating basal concentrations of LH as well as LH levels measured in response to a single exogenous GnRH challenge were not significantly different between the reproductive and non-reproductive groups of either sex, suggest the absence of a physiologically well-defined suppression of reproduction in subordinate common mole-rats.

Kin discrimination and female mate choice in the naked mole-rat Heterocephalus glaber.

Naked mole-rats are fossorial, eusocial rodents that naturally exhibit high levels of inbreeding. Persistent inbreeding in animals often results in a substantial decline in fitness and, thus, dispersal and avoidance of kin as mates are two common inbreeding avoidance mechanisms. In the naked mole-rat evidence for the former has recently been found. Here we address the latter mechanism by investigating kin recognition and female mate choice using a series of choice tests in which the odour, social and mate preferences of females were determined. Discrimination by females appears to be dependent on their reproductive status. Reproductively active females prefer to associate with unfamiliar males, whereas reproductively inactive females do not discriminate. Females do not discriminate between kin and non-kin suggesting that the criterion for recognition is familiarity, not detection of genetic similarity per se. In the wild, naked mole-rats occupy discrete burrow systems and dispersal and mixing with non-kin is thought to be comparatively rare. Thus, recognition by familiarity may function as a highly efficient kin recognition mechanism in the naked mole-rat. A preference by reproductively active females for unfamiliar males is interpreted as inbreeding avoidance. These findings suggest that, despite an evolutionary history of close inbreeding, naked mole-rats may not be exempt from the effects of inbreeding depression and will attempt to outbreed should the opportunity arise.

Hormonal and behavioural correlates of male dominance and reproductive status in captive colonies of the naked mole-rat, Heterocephalus glaber.

Naked mole-rat colonies are societies with a high reproductive skew, breeding being restricted to one dominant female (the 'queen') and 1-3 males. Other colony members of both sexes are reproductively suppressed. Experimental removal of breeding males allowed us to investigate the relationship between urinary testosterone and cortisol, dominance rank, and male reproductive status. Dominance rank was strongly correlated with body weight, age, and urinary testosterone titres in males. No relationship between urinary cortisol levels and male reproductive status or dominance was found. Breeding males were among the highest-ranking, heaviest and oldest males in their respective colonies, and were succeeded by other high-ranking, large, old colony males. In contrast to females, no evidence of competition over breeding status was observed among males. Male-male agonism was low both before and after removal of breeders and mate guarding was not observed. The lower reproductive skew for males compared with female skew or queen control over male reproduction may explain why males compete less strongly than females over breeding status after removal of same-sexed breeders.

Ecological constraints drive social evolution in the African mole-rats.

The African mole-rats (family Bathyergidae) are subterranean hystricomorph rodents occurring in a variety of habitats and displaying levels of sociality which range from solitary to eusocial, making them a unique mammalian taxonomic group to test ecological influences on sociality. Here, we use an extensive DNA-based phylogeny and comparative analysis to investigate the relationship between ecology, sociality and evolution within the family. Mitochondrial cytochrome-b and 12s rRNA trees reveal that the solitary species are monophyletic when compared to the social species. The naked mole-rat (Heterocephalus glaber) is ancestral and divergent from the Damaraland mole-rat (Cryptomys damarensis), supporting previous findings that have suggested the multiple evolution of eusociality within the family. The Cryptomys genus is species-rich and contains taxa exhibiting different levels of sociality, which can be divided into two distinct clades. A total of seven independent comparisons were generated within the phylogeny, and three ecological variables were significantly correlated with social group size: geophyte density (p < 0.05), mean months per year of rainfall greater than 25 mm (p < 0.001), and the coefficient of rainfall variation (p = 0.001). These results support the food-aridity hypothesis for the evolution of highly social cooperative behaviour in the Bathyergidae, and are consistent with the current theoretical framework for skew theory.

Micro- and macrogeographical genetic structure of colonies of naked mole-rats Heterocephalus glaber.

Patterns of genetic structure in eusocial naked mole-rat populations were quantified within and among geographically distant populations using multilocus DNA fingerprinting and mitochondrial DNA (mtDNA) sequence analysis. Individuals within colonies were genetically almost monomorphic, sharing the same mtDNA control region haplotype and having coefficients of band sharing estimated from DNA fingerprints ranging from 0.93 to 0.99. Family analysis of a hybrid captive colony of naked mole-rats with increased levels of genetic variability using multilocus DNA fingerprinting gave results consistent with Mendelian inheritance, and has revealed for the first time that multiple paternity can occur. In a survey of wild colonies from Ethiopia, Somalia and locations in northern and southern Kenya, we have examined mtDNA control region sequence variation in 42 individuals from 15 colonies, and together with multilocus DNA fingerprinting and mtDNA cytochrome-b sequence analysis in selected individuals have shown that these populations show considerable genetic divergence. Most of the variance in sequence divergence was found to be between geographical locations (phi ct = 0.68) and there was a significant correlation between sequence divergence and geographical separation of haplotypes. Six colonies from Mtito Andei in southern Kenya shared the same control region haplotype, suggesting a recent common maternal ancestor. In contrast, out of four colonies at Lerata in north Kenya, three haplotypes were identified, and phylogenetic analysis suggests that this area may be a zone where two distinct lineages are in close proximity. Genetic distances were maximal between Ethiopian and southern Kenyan populations at 5.8% for cytochrome-b, and are approaching interspecific values seen between other Bathyergids.

Dominance and queen succession in captive colonies of the eusocial naked mole-rat, Heterocephalus glaber.

Naked mole-rat colonies exhibit a high reproductive skew, breeding being typically restricted to one female (the 'queen') and one to three males. Other colony members are reproductively suppressed, although this suppression can be reversed following the removal or death of the queen. We examined dominance and queen succession within captive colonies to investigate the relationship between urinary testosterone and cortisol, dominance rank and reproductive status; and to determine if behavioural and/or physiological parameters can be used as predictors of queen succession. Social structure was characterized by a linear dominance hierarchy before and after queen removal. Prior to queen removal, dominance rank was negatively correlated with body weight and urinary testosterone and cortisol titres in males and females. Queen removal results in social instability and aggression between high ranking individuals. Dominance rank appears to be a good predictor of reproductive status: queens are the highest ranking colony females and are succeeded by the next highest ranking females. The intense dominance-related aggression that accompanies reproductive succession in naked mole-rats provides empirical support for optimal skew theory.

LH responses to single doses of exogenous GnRH by social Mashona mole-rats: a continuum of socially induced infertility in the family Bathyergidae.

The Mashona mole-rat, Cryptomys darlingi, exhibits an extreme reproductive division of labour. Reproduction in the colony is restricted to a single breeding pair. The non-reproductive male and female colony members are restrained from sexual activity by being familiar and related to one another and the reproductive animals. Circulating basal concentrations of luteinizing hormone (LH) as well as LH levels measured in response to a single exogenous gonadotropin releasing hormone (GnRH) challenge are not significantly different between the reproductive and non-reproductive groups of either sex. Socially induced infertility in both non-reproductive males and females does not result from a reduced pituitary secretion of LH or decreased sensitivity to hypothalamic GnRH, but rather appears to result from an inhibition of reproductive behaviour in these obligate outbreeders. The African mole-rats exhibit a continuum of socially induced infertility with differing social species inhabiting regions of varying degrees of aridity. In this continuum a transition from a predominantly behavioural repression in a social mesic-adapted species through to complete physiological suppression lacking incest avoidance in an arid-adapted eusocial species occurs in this endemic African family of rodents.

Combined olfactory contact with the parent colony and direct contact with nonbreeding animals does not maintain suppression of ovulation in female naked mole-rats (Heterocephalus glaber).

The study investigated the role of odor cues from two naked mole-rat colonies, in conjunction with behavioral cues from nonbreeding colony members, in maintaining suppression of ovulation in subordinate female naked mole-rats isolated from the two parent colonies. Four high ranking nonbreeding female naked mole-rats were removed from their respective parent colonies and singly housed in separate burrow systems. For a 64-day period, the removed females were maintained in daily odor contact with their parent colony by daily rotating soiled bedding material between the parent colony and the burrow systems of removed females. In addition, subsets of nonbreeding animals from the respective parent colony were regularly moved into the burrow systems of removed females for 2-day periods during this 64-day period. Removed females were therefore in continual social contact with subsets of parent colony animals except for the breeding pair. All four removed females exhibited raised levels of urinary progesterone (< 2 ng/mg Cr) indicative of the onset of ovarian function within 3 days of being separated from the parent colony. Removed females exhibited a normal ovulatory cycle with levels of progesterone remaining elevated for 25-35 days (mean concentration of progesterone +/- SEM; 16.2 +/- 2 ng/mg Cr). Initiation of aggression and sexual behavior by removed females increased significantly when they were isolated from the parent colony. The results demonstrated that odor cues from the complete colony in conjunction with behavioral/tactile/vocal cues from the nonbreeding colony members were not the major cues maintaining reproductive suppression in nonbreeding female naked mole-rats. Instead, our results suggest that female reproductive suppression in naked mole-rats is caused by a dominance-related behavioral mechanism requiring direct contact with the breeding female.

Reproductive suppression in subordinate, non-breeding female Damaraland mole-rats: two components to a lifetime of socially induced infertility.

The Damaraland mole-rat Cryptomys damarensis exhibits an extreme reproductive division of labour. Reproduction in the colony is restricted to a single breeding pair, resulting from a two-fold control: (i) a reduced pituitary synthesis and/or secretion of luteinizing hormone leading to a block to ovulation in non-reproductive females; and (ii) a strong inhibition to breeding with familiar kin. Circulating basal concentrations of luteinizing hormone as well as luteinizing hormone levels measured in response to a single exogenous gonadotrophin releasing hormone challenge, were significantly lower in non-reproductive females in the presence of the reproductive female than those in colonies lacking a reproductive female. Urinary progesterone concentrations before the removal of the reproductive female were significantly higher in non-reproductives than the post removal values. Behavioural studies from sib-sib and non-sib pairings provide evidence for a strong incest avoidance, probably resulting from an inhibition of breeding with familiar colony members. A total of four pairings of non-sibs resulted in copulatory activity and eventual conception. In contrast, four couples of sib-sib combinations failed to produce any sexual activity or offspring. Thus, suppression of reproduction in these non-reproductive, subterranean bathyergids is complicated by the masking effect of familiarity that prevents incest, in addition to the physiological inhibition of fertility in the presence of the reproductive female.

A dispersive morph in the naked mole-rat.

Close inbreeding is known for a variety of small mammal species for which a high probability of mortality during dispersal makes helping and delayed maturation a relatively secure fitness option. Prolonged inbreeding, however, is usually associated with lowered fitness, and it has been shown that most highly inbred small mammals and social insects have inbreeding-avoidance mechanisms that promote some degree of outbreeding. However, previous field and laboratory research on the naked mole-rat (Heterocephalus glaber) suggested that this cooperatively breeding rodent is highly inbred, with new colonies forming by fission. Here we report the discovery of a dispersal phenotype that may occasionally promote outbreeding in naked mole-rats. These dispersers are morphologically, physiologically and behaviourally distinct from other colony members. They are laden with fat, exhibit elevated levels of luteinizing hormone, have a strong urge to disperse, and only solicit matings with non-colony members. These findings suggest that, although rare, a dispersive morph exists within naked mole-rat colonies.

Investigation of numbers and motility of spermatozoa in reproductively active and socially suppressed males of two eusocial African mole-rats, the naked mole-rat (Heterocephalus glaber) and the Damaraland mole-rat (Cryptomys damarensis).

Reproductive tracts and spermatozoa from reproductively active and reproductively suppressed non-breeding males from two species of eusocial African mole-rats Cryptomys damarensis and Heterocephalus glaber were examined. In two captive colonies of Heterocephalus glaber, reproductive tracts from seven non-breeding males removed from their colonies, and housed singly for 5-6 weeks to cause reproductive activation, were compared with reproductive tracts from seven non-breeding males. The body weight of the separated, reproductively active males increased significantly (P < 0.01), and the mean testis weights relative to body weight of the reproductively active males were significantly larger (P < 0.05) than those of non-breeding males. The number of spermatozoa, in one half of the reproductive tract, was higher in active males than in non-breeding males (mean +/- SEM: 8.59 x 10(6) +/- 2.69 x 10(6) versus 1.78 x 10(6) +/- 1.43 x 10(6), respectively; P < 0.05). In addition, six of the seven reproductively active males, but only two of seven non-breeding males, had motile spermatozoa. A total of 28 wild Cryptomys damarensis from two colonies were examined in the field. The testis weights relative to body weight of breeding males (n = 7) were higher than those of non-breeding males (n = 19; P < 0.01), but the number of spermatozoa did not differ significantly between the two groups (0.13 x 10(6) +/- 0.06 x 10(6), n = 7 versus 0.29 x 10(6) +/- 0.14 x 10(6), n = 21, respectively). Breeding and non-breeding males produced similar numbers of motile spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermoregulation in three species of Afrotropical subterranean mole-rats (Rodentia: Bathyergidae) from Zambia and Angola and scaling within the genus Cryptomys.

The thermoregulatory characteristics of three species of Cryptomys from Zambia and Angola are examined and, together with published data on four other species of Cryptomys from southern Africa, used to determine whether scaling occurs in this genus of subterranean rodents. The thermoregulatory properties of acclimated giant Zambian mole-rats, Cryptomys mechowi ([Formula: see text]=267 g), Angolan mole-rats, Cryptomys bocagei ([Formula: see text]=94 g) and Zambian common mole-rats Cryptomys hottentotus amatus ([Formula: see text]=77 g) are as follows. Mean resting metabolic rates (RMRs) within the respective thermoneutral zones were 0.60±0.08 cm3 O2 g-1 h-1 (n=12) for C. mechowi; 0.74±0.06 cm3 O2 g-1 h-1 (n=8) for C. bocagei and 0.63±0.06 cm3O2 g-1 h-1 (n=21) for C. h. amatus. The thermoneutral zones (TNZs) of all three species are narrow: 29-30°C for C. mechowi; 31.5-32.5°C for C. bocagei and 28-32° C for C. h. amatus. The increase in mean RMR at the lowest temperatures tested (15° C for C. mechowi, 18° C for C. bocagei and C. h. amatus) was 2.35, 2.2 and 3.82 times their RMR in the TNZ respectively. Body temperatures are low, 34±0.53° C (n=24) for C. mechowi, 33.7±0.32° C (n=20) for C. bocagei and 33.8±0.43° C (n=40) for C. h amatus. At the lower limit of thermoneutrality, conductances are 0.09±0.01 cm3 O2 g-1 h-1 °C-1 (n=30) in C. mechowi; 0.12±0.01 cm3 O2 g-1 h-1 °C-1 (n=20) in C. bocagei and 0.12±0.03 cm3 O2 g-1 h-1 °C-1 (n=32) in C. h. amatus. The range in mean body mass among the seven species of Cryptomys examined for scaling was 60 g (C. darlingi) to 267 g (C. mechowi). There is no clear relationship between RMR within the TNZ and body mass. The resultant relationship is represented by the power curve RMR=2.45 mass-0.259.