Genome evolution of blind subterranean mole rats: Adaptive peripatric versus sympatric speciation.

Speciation mechanisms remain controversial. Two speciation models occur in Israeli subterranean mole rats, genus Spalax: a regional speciation cline southward of four peripatric climatic chromosomal species and a local, geologic-edaphic, genic, and sympatric speciation. Here we highlight their genome evolution. The five species were separated into five genetic clusters by single nucleotide polymorphisms, copy number variations (CNVs), repeatome, and methylome in sympatry. The regional interspecific divergence correspond to Pleistocene climatic cycles. Climate warmings caused chromosomal speciation. Triple effective population size, Ne , declines match glacial cold cycles. Adaptive genes evolved under positive selection to underground stresses and to divergent climates, involving interspecies reproductive isolation. Genomic islands evolved mainly due to adaptive evolution involving ancient polymorphisms. Repeatome, including both CNV and LINE1 repetitive elements, separated the five species. Methylation in sympatry identified geologically chalk-basalt species that differentially affect thermoregulation, hypoxia, DNA repair, P53, and other pathways. Genome adaptive evolution highlights climatic and geologic-edaphic stress evolution and the two speciation models, peripatric and sympatric.

Microbiome and Longevity: High Abundance of Longevity-Linked Muribaculaceae in the Gut of the Long-Living Rodent Spalax leucodon.

With a world population living longer as well as marked disparities in life expectancy, understanding the determinants of longevity is one of the priority research agendas in 21st century life sciences. To this end, the blind mole-rat (Spalax leucodon), a subterranean mammalian, has emerged as an exceptional model organism due to its astonishing features such as remarkable longevity, hypoxia and hypercapnia tolerance, and cancer resistance. The microbiome has been found to be a vital parameter for cellular physiology and it is safe to assume that it has an impact on life expectancy. Although the unique characteristics of Spalax make it an ideal experimental model for longevity research, there is limited knowledge of the bacterial composition of Spalax microbiome, which limits its in-depth utilization. In this study, using 16S rRNA amplicon sequencing, we report the gut and skin bacterial structure of Spalax for the first time. The diversity between fecal and skin samples was manifested in the distant clustering, as revealed by beta diversity analysis. Importantly, the longevity-linked Muribaculaceae bacterial family was found to be the dominating bacterial taxa in Spalax fecal samples. These new findings contribute toward further development of Spalax as a model for longevity research and potential linkages between microbiome composition and longevity.

Convergent reduction of V1R genes in subterranean rodents.

BACKGROUND: Vomeronasal type 1 receptor genes (V1Rs) are expected to detect intraspecific pheromones. It is believed that rodents rely heavily on pheromonal communication mediated by V1Rs, but pheromonal signals are thought to be confined in subterranean rodents that live in underground burrows. Thus, subterranean rodents may show a contrasting mode of V1R evolution compared with their superterranean relatives. RESULTS: We examined the V1R evolution in subterranean rodents by analyzing currently available genomes of 24 rodents, including 19 superterranean and 5 subterranean species from three independent lineages. We identified a lower number of putatively functional V1R genes in each subterranean rodent (a range of 22-40) compared with superterranean species (a range of 63-221). After correcting phylogenetic inertia, the positive correlation remains significant between the small V1R repertoire size and the subterranean lifestyle. To test whether V1Rs have been relaxed from functional constraints in subterranean rodents, we sequenced 22 intact V1Rs in 29 individuals of one subterranean rodent (Spalax galili) from two soil populations, which have been proposed to undergo incipient speciation. We found 12 of the 22 V1Rs to show significant genetic differentiations between the two natural populations, indicative of diversifying selection. CONCLUSION: Our study demonstrates convergent reduction of V1Rs in subterranean rodents from three independent lineages. Meanwhile, it is noteworthy that most V1Rs in the two Spalax populations are under diversifying selection rather than relaxed selection, suggesting that functional constraints on these genes may have retained in some subterranean species.

Habitat and Burrow System Characteristics of the Blind Mole Rat Spalax galili in an Area of Supposed Sympatric Speciation.

A costly search for food in subterranean rodents resulted in various adaptations improving their foraging success under given ecological conditions. In Spalax ehrenbergi superspecies, adaptations to local ecological conditions can promote speciation, which was recently supposed to occur even in sympatry at sites where two soil types of contrasting characteristics abut each other. Quantitative description of ecological conditions in such a site has been, nevertheless, missing. We measured characteristics of food supply and soil within 16 home ranges of blind mole rats Spalax galili in an area subdivided into two parts formed by basaltic soil and pale rendzina. We also mapped nine complete mole rat burrow systems to compare burrowing patterns between the soil types. Basaltic soil had a higher food supply and was harder than rendzina even under higher moisture content and lower bulk density. Population density of mole rats was five-times lower in rendzina, possibly due to the lower food supply and higher cover of Sarcopoterium shrubs which seem to be avoided by mole rats. A combination of food supply and soil parameters probably influences burrowing patterns resulting in shorter and more complex burrow systems in basaltic soil.

Possible incipient sympatric ecological speciation in blind mole rats (Spalax).

Sympatric speciation has been controversial since it was first proposed as a mode of speciation. Subterranean blind mole rats (Spalacidae) are considered to speciate allopatrically or peripatrically. Here, we report a possible incipient sympatric adaptive ecological speciation in Spalax galili (2n = 52). The study microsite (0.04 km(2)) is sharply subdivided geologically, edaphically, and ecologically into abutting barrier-free ecologies divergent in rock, soil, and vegetation types. The Pleistocene Alma basalt abuts the Cretaceous Senonian Kerem Ben Zimra chalk. Only 28% of 112 plant species were shared between the soils. We examined mitochondrial DNA in the control region and ATP6 in 28 mole rats from basalt and in 14 from chalk habitats. We also sequenced the complete mtDNA (16,423 bp) of four animals, two from each soil type. Remarkably, the frequency of all major haplotype clusters (HC) was highly soil-biased. HCI and HCII are chalk biased. HC-III was abundant in basalt (36%) but absent in chalk; HC-IV was prevalent in basalt (46.5%) but was low (20%) in chalk. Up to 40% of the mtDNA diversity was edaphically dependent, suggesting constrained gene flow. We identified a homologous recombinant mtDNA in the basalt/chalk studied area. Phenotypically significant divergences differentiate the two populations, inhabiting different soils, in adaptive oxygen consumption and in the amount of outside-nest activity. This identification of a possible incipient sympatric adaptive ecological speciation caused by natural selection indirectly refutes the allopatric alternative. Sympatric ecological speciation may be more prevalent in nature because of abundant and sharply abutting divergent ecologies.

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel.

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00-2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n=54→S. galili, 2n=52→S. carmeli, 2n=58→S. judaei, 2n=60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Living with stress: regulation of antioxidant defense genes in the subterranean, hypoxia-tolerant mole rat, Spalax.

Lack of oxygen is life threatening for most mammals. It is therefore of biomedical interest to investigate the adaptive mechanisms which enable mammalian species to tolerate extremely hypoxic conditions. The subterranean mole rat Spalax survives substantially longer periods of hypoxia than the laboratory rat. We hypothesized that genes of the antioxidant defense, detoxifying harmful reactive oxygen species generated during hypoxia and hyperoxia, are involved in Spalax underground adaptation. Using quantitative RT-PCR, we analyzed the mRNA expression levels of seven antioxidant defense genes (catalase, glutathione peroxidase 1, glutathione-S-transferase Pi1, heme oxygenase 1, superoxide dismutase 1 and 2) and a master regulator of this stress pathway, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in several tissues of two Israeli Spalax species, S. galili (2n=52) and S. judaei (2n=60), and rat. We also studied the differential expression of these genes after experimental hypoxia and hyperoxia as oxidative stress treatments. We found that mRNA levels and transcriptional responses are species and tissue specific. There are constitutively higher transcript levels of antioxidant genes and their transcription factor Nrf2 in Spalax tissue as compared to rat, suggesting an increased ability in the mole rat to withstand hypoxic/hyperoxic insults. In contrast to Spalax, the rat reacts to experimental oxidative stress by changes in gene regulation. In addition, Spalax Nrf2 reveals unique amino acid changes, which may be functionally important for this transcription factor and indicate positive (Darwinian) selection. Antioxidant defense genes are therefore important targets for adaptive change during evolution of hypoxia tolerance in Spalax.

Spectral and duration sensitivity to light-at-night in 'blind' and sighted rodent species.

Light-at-night (LAN) has become a defining feature of human and animal ecosystems and may possibly compromise human and animal physiology and health. Spectral and acclimation duration (AD) sensitivity were compared between social voles (Microtus socialis) and 'blind' mole rats (Spalax ehrenbergi) in four increasing ADs (0, 1, 7 and 21 days) to LAN (1×30 min, 293 µW cm(-2)) of three different monochromatic lights [blue (479 nm), yellow (586 nm) and red (697 nm)]. Animals were sampled for urine and oxygen consumption (V(O(2))) promptly after each LAN-AD. Urine samples were analyzed for production rate, urinary 6-sulfatoxymelatonin and urinary metabolites of adrenalin and cortisol. Overall, the blue light elicited the greatest effects on the biological markers of M. socialis, whereas similar effects were detected for S. ehrenbergi in response to red light. The increasing LAN-AD resulted in a dose-dependent decrement of all markers tested, except of stress hormones, which showed a direct positive correlation with LAN-AD. Our results suggest that: (1) photoperiod is an important cue for entraining physiological functions in the 'blind' S. ehrenbergi, which is essentially characterized by red-shifted sensitivity compared with the blue-shifted sensitivity detected for the sighted counterpart species, and (2) there is a strong association between LAN of the appropriate wavelength and adrenal endocrine responses, suggesting that LAN is a potential environmental stressor.

Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax.

The subterranean mole rat Spalax is an excellent model for studying adaptation of a mammal toward chronic environmental hypoxia. Neuroglobin (Ngb) and cytoglobin (Cygb) are O(2)-binding respiratory proteins and thus candidates for being involved in molecular hypoxia adaptations of Spalax. Ngb is expressed primarily in vertebrate nerves, whereas Cygb is found in extracellular matrix-producing cells and in some neurons. The physiological functions of both proteins are not fully understood but discussed with regard to O(2) supply, the detoxification of reactive oxygen or nitrogen species, and apoptosis protection. Spalax Ngb and Cygb coding sequences are strongly conserved. However, mRNA and protein levels of Ngb in Spalax brain are 3-fold higher than in Rattus norvegicus under normoxia. Importantly, Spalax expresses Ngb in neurons and additionally in glia, whereas in hypoxia-sensitive rodents Ngb expression is limited to neurons. Hypoxia causes an approximately 2-fold down-regulation of Ngb mRNA in brain of rat and mole rat. A parallel regulatory response was found for myoglobin (Mb) in Spalax and rat muscle, suggesting similar functions of Mb and Ngb. Cygb also revealed an augmented normoxic expression in Spalax vs. rat brain, but not in heart or liver, indicating distinct tissue-specific functions. Hypoxia induced Cygb transcription in heart and liver of both mammals, with the most prominent mRNA up-regulation (12-fold) in Spalax heart. Our data suggest that tissue globins contribute to the remarkable tolerance of Spalax toward environmental hypoxia. This is consistent with the proposed cytoprotective effect of Ngb and Cygb under pathological hypoxic/ischemic conditions in mammals.

Photoentrainment in blind and sighted rodent species: responses to photophase light with different wavelengths.

Our study examined the impact of daylight (photophase) wavelength on the photoentrainment sensitivity of two species with vastly different visual systems. Social voles (Microtus socialis) and 'blind' mole rats (Spalax ehrenbergi) were exposed to short-wavelength (479 nm) or long-wavelength (697 nm) light at an intensity of 293 µW cm(-2). Rhythms of urine production, urinary 6-sulfatoxymelatonin (6-SMT), urinary metabolites of adrenaline and cortisol, and oxygen consumption (VO(2)) were used as markers for the sensitivity of the photoentrainment system. Significant 24-h rhythms were detected in all variables for both species under short-wavelength light, whereas ultradian rhythms of 12- or 8-h were detected under long-wavelength light. Wavelength inversely affected 6-SMT levels in M. socialis (negative correlation) and S. ehrenbergi (positive correlation). Increased levels of stress hormone metabolites were detected in M. socialis under the long-wavelength light whereas, in S. ehrenbergi elevated levels were secreted under short-wavelength light. Long-wavelength light increased VO(2) in M. socialis and decreased it in S. ehrenbergi; short-wavelength light elicited the opposite effects. Our results indicate that photophase wavelength is an integral light property for modulating photoperiodic responses in mammals, including visually challenged species. Finally, the spectral-induced differential responses between the two species potentially represent adaptive physiological flexibility in species with contrasting visual and habitat challenges.

Photosensitivity to different light intensities in blind and sighted rodents.

Photoperiod is an important cue regulating biological rhythms in mammals, including 'blind' subterranean and sighted fossorial rodent species. These species may respond differentially to changes in light quality according to their retinal complexity. The effects of increasing light intensity on daily rhythms of urine excretion and urinary output of 6-sulfatoxymelatonin levels were compared in 'blind' mole rats Spalax ehrenbergi and sighted social voles, Microtus socialis. Our results show that the threshold irradiance required to entrain rhythms of voles is three magnitudes greater than that for mole rats. The results suggest that mole rats have an operational photoreceptive pathway with a lower threshold irradiance than voles. Such a low threshold reflects the remarkable capability of this 'blind' species to utilize light signals even under challenging light conditions.

Exploration and navigation in the blind mole rat (Spalax ehrenbergi): global calibration as a primer of spatial representation.

The aim of this study was to uncover the process of initial spatial mapping of the environment. For this, blind mole rats (Spalax ehrenbergi), were tested in an unfamiliar square arena, in order to reveal how they construct a spatial representation. The mole rats first displayed a build-up phase, in which they gradually formed a path along the perimeter while travelling slowly, frequently pausing and repeating previously travelled segments of the path. This behaviour was followed by a free-travel phase, in which the mole rats appeared to locomote smoothly along the perimeter and through the centre of the arena while travelling faster with fewer stops or repetitions of path segments. Familiarity with the environment was reflected in local shortcuts at the arena corners and global shortcuts (crosscuts) through the arena centre. We suggest that scanning the perimeter throughout the build-up phase constitute a process of calibration, i.e. forming an initial representation of the size and perhaps the shape of the environment--a sort of basic global map. We further suggest that this calibration is later used for navigation, as indicated by the emergence of global crosscuts in the subsequent phase. Further investigation of the build-up phase, e.g. by manipulating environment size, might provide additional insight into the course of establishment of global environment representation (mapping).