The diet of Myotragus balearicus Bate 1909 (Artiodactyla: Caprinae), an extinct bovid from the Balearic Islands: evidence from coprolites

Myotragus balearicus Bate 1909 is an artiodactyl Caprinae endemic to the Balearic Islands which became extinct more than 4000 years ago. Coprolites produced by this species have been collected from the excavation of Holocene cave sediments in Cova Estreta (Serra de Tramuntana, Mallorca). The pollen content of several samples of coprolites has been studied in order to determine the diet of Myotragus. Myotragus balearicus from Cova Estreta was a browser, and consumed huge amounts of box, Buxus balearica , a plant known for its high content of steroidal alkaloids. The coprolites are very fine textured, probably due to the result of a very efficient digestive process.     

Molecular phylogeny and evolution of the extinct bovid Myotragus balearicus

Myotragus balearicus was a dwarf artiodactyl endemic to the Eastern Balearic Islands, where it evolved in isolation for more than 5 million years before becoming extinct between 3640 and 2135 cal BC (calibrated years BC). Numerous unusual apomorphies obscure the relationship between Myotragus and the extant Caprinae. Therefore, genetic data for this species would significantly contribute to the clarification of its taxonomic position. In this study, we amplify, sequence, and clone a 338-base pair (bp) segment of the mitochondrial cytochrome b (cyt b) gene from a >9Kyr Myotragus subfossil from la Cova des Gorgs (Mallorca). Our results confirm the phylogenetic affinity of Myotragus with the sheep (Ovis) and the takin (Budorcas). In each tree, the Myotragus branch is long in comparison with the other taxa, which may be evidence of a local change in the rate of evolution in cyt b. This rate change may be due to in part to an early age of first reproduction and short generation time in Myotragus, factors that are potentially related to the extreme reduction in size of the adult Myotragus as compared to the other Caprinae.     

Mitochondrial DNA from Myotragus balearicus, an extinct bovid from the Balearic Islands

DNA was extracted from teeth and bones of Myotragus balearicus, a bovid that evolved in isolation on the Balearic Islands (Western Mediterranean) from the end of the Miocene, becoming extinct 4,000 years BP, after the arrival of humans in the islands. The numerous morphological apomorphies of Myotragus, most strikingly its dwarfism, frontal eyes, and ever-growing incisors, obscure its phylogenetic relationships with extant bovids. Therefore, the recovery of genetic information from Myotragus is of significant interest to help clarify the taxonomic position of this species. In this study we amplified and sequenced a 95 bp (base pair) fragment of the mtDNA cytochrome b gene from 6,000-year-old specimens of Myotragus. Several experimental controls, such as amino acid analysis, independent reproduction in two different laboratories, and cloning of the PCR product, support the authenticity of the ancient DNA sequence recovered. Phylogenetic comparison with orthologous sequences from supposedly related extant genera (serow, goral, mountain goat, chamois, takin, sheep, goat, Himalayan tahr, arctic musk ox, barbary sheep, blue sheep, and saiga) from the Caprinae subfamily suggests that Myotragus is related to some of these species. However, the real phylogenetic position of Myotragus is difficult to assess, due to the lack of resolution of the present molecular study, which can be partially attributed to the short length of the genetic fragment recovered.     

First Fossil Evidence for the Advance of Replacement Teeth Coupled with Life History Evolution along an Anagenetic Mammalian Lineage

In mammals that grow up more slowly and live longer, replacement teeth tend to appear earlier in sequence than in fast growing mammals. This trend, known as ‘Schultz''s Rule’, is a useful tool for inferring life histories of fossil taxa. Deviations from this rule, however, suggest that in addition to the pace of life history, ecological factors may also drive dental ontogeny. Myotragus balearicus is an extinct insular caprine that has been proved to be an excellent test case to correlate morphological traits with life history. Here we show that Myotragus balearicus exhibits a slow signature of dental eruption sequence that is in agreement with the exceptionally slow life history of this species, thus conforming to ‘Schultz''s Rule’. However, our results also show an acceleration of the absolute pace of development of the permanent incisors in relation to that of the posterior teeth. The rodent-like incisors of Myotragus balearicus erupted early not only in relative but also in absolute terms (chronological age), suggesting that feeding characteristics also plays an important role in dental ontogeny. This is in agreement with ecological hypotheses based on primates. Our study documents a decoupling of the pace of development of teeth in mammals that is triggered by different selection pressures on dental ontogeny. Moreover, we show that Myotragus kopperi from the early Pleistocene (a direct ancestor of the late Pleistocene-Holocene M. balearicus) follows the pattern of first incisor replacement known in living bovids. Hence, the advance in the eruption sequence of the first incisors occurs along the Myotragus evolutionary lineage over a period of about 2.5 Myr. To our knowledge, this is the first fossil evidence of an advance of the emergence of the permanent first incisor along an anagenetic mammalian lineage.     

KINETICS OF THE INNER ENAMEL EPITHELIUM IN THE ADULT RAT INCISOR DURING ACCELERATED ERUPTION

Inner enamel epithelial (IEE) cell production was compared in accelerated and normal eruption (control). Each group consisting of thirty rats received 1 μCi/g tritiated thymidine. The animals were sacrificed at short time intervals up to 14 hr after injection. The excised incisors were cut mid-sagittally and processed autoradiographically.
The fast growing incisor produces twice as many cells as the control. Increased cell production is achieved in two ways: proliferative pool expansion (by 25.5%) and generation time ( t _c) shortening to 16 hr ( t _c= 23 hr in the control). Generation time shortening resulted mainly from a diminution in t _g1= 8.6 hr ( t _g1= 14.1 hr in the control) and t _s which equaled 5.5 hr ( t _s= 7 hr in control). Mitotic times which equaled 0.4 hr and t _g2, 1.5 hr were identical in both groups.
The eruption/IEE cell production ratio equals 1.1 in both groups.     

Photoinhibition of respiratory CO2 release in the green alga Scenedesmus

¹⁴CO₂ evolution of prelabeled Scenedesmus obliquus Kütz, has been followed in the dark and in the light. In the light, no carbon dioxide is evolved. Addition of unlabeled NaHCO, leads to ¹⁴CO₂ release attaining 20 to 30% of the dark rate. Double-reciprocal plots of NaHCO₃ concentrations vs ¹⁴CO₂ release results in a straight line, indicative of competition between exogenously supplied bicarbonate and endogenously evolved carbon dioxide. With this method, it is possible to measure CO₂ evolved by respiration in the light and to show that true photoinhibition of respiration occurs in Scenedesmus . In the light. DCMU substantially increases ¹⁴CO₂ evolution; in the presence of the uncoupler carbonyl cyanide- m -chlorophenylhydrazone. ¹⁴CO₂ evolution is comparable to that in the dark. ¹⁴CO₂ release and oxygen uptake in the dark are only slightly affected by cyanide, indicative of a cyanide-resistant respiration and/or fermentation as the essential CO₂-yielding processes in the presence of cyanide. These results, compared with concurrent ATP levels, lead us to assume that energy charge is not the only factor responsible for photoinhibition of respiration.     

A simple morphological predictor of bite force in rodents

Bite force was quantified for 13 species of North American rodents using a piezo-resistive sensor. Most of the species measured (11) formed a tight relationship between body mass and bite force (log 10(bite force)=0.43(log 10(body mass))+0.416; R ²>0.98). This high correlation exists despite the ecological (omnivores, grazers and more carnivorous) and taxonomic (Cricetidae, Heteromyidae, Sciuridae and Zapodidae) diversity of species. Two additional species, Geomys bursarius (Geomyidae) and a Sciurus niger (Sciuridae), bit much harder for their size. We found a simple index of strength based on two measurements of the incisor at the level of the alveolus ( Z_i =((anterior-posterior length)²× (medial-lateral width))/6) that is highly predictive of bite force in these rodents (R²>0.96). Z_i may be useful for prediction of bite force (log10 (Bite Force)=0.566log10 ( Z_i )+1.432) when direct measurements are not available.     

Evidence of correlated evolution of hypsodonty and exceptional longevity in endemic insular mammals

Here, we test whether the increase in tooth height in insular endemics results from the expansion of the dietary niche under resource limitation, as widely considered, or whether it represents an investment in dental durability in response to the selection for extended longevity under low levels of extrinsic mortality. We tested these hypotheses in the extremely hypsodont fossil bovid Myotragus balearicus from the Balearic Islands, an ideal model to study the evolutionary trends on islands. Dental abrasion was significantly lower in the insular bovid than in highly hypsodont continental artiodactyls, suggesting that feeding habits are not the sole driving force behind increased crown height. However, the estimated longevity for M. balearicus based on dental durability was two times that predicted from body mass. Survivorship curves confirm that an extraordinarily large number of individuals approached the longevity of the species. Our results, hence, provide evidence that hypsodonty in insular endemics is the outcome of selection for increased durability of the permanent dentition in association with an extended lifespan. In the context of insularity, our results lend additional support to the disposable soma theory of ageing confirming the dependency of somatic maintenance and repair on lifespan, and its control by resource availability and extrinsic mortality.     

The impact of digging on the evolution of the rodent mandible

There are two main (but not mutually exclusive) methods by which subterranean rodents construct burrows: chisel-tooth digging, where large incisors are used to dig through soil; and scratch digging, where forelimbs and claws are used to dig instead of incisors. A previous study by the authors showed that upper incisors of chisel-tooth diggers were better adapted to dig but the overall cranial morphology within the rodent sample was not significantly different. This study analyzed the lower incisors and mandibles of the specimens used in the previous study to show the impact of chisel-tooth digging on the rodent mandible. We compared lower incisors and mandibular shape of chisel-tooth digging rodents with nonchisel-tooth digging rodents to see if there were morphological differences between the two groups. The shape of incisors was quantified using incisor radius of curvature and second moment of area (SMA). Mandibular shape was quantified using landmark based geometric morphometrics. We found that lower incisor shape was strongly influenced by digging group using a Generalized Phylogenetic ancova (analysis of covariance). A phylogenetic Procrustes anova (analysis of variance) showed that mandibular shape of chisel-tooth digging rodents was also significantly different from nonchisel-tooth digging rodents. The phylogenetic signal of incisor radius of curvature was weak, whereas that of incisor SMA and mandibular shape was significant. This is despite the analyses revealing significant differences in the shape of both mandibles and incisors between digging groups. In conclusion, we showed that although the mandible and incisor of rodents are influenced by function, there is also a degree of phylogenetic affinity that shapes the rodent mandibular apparatus.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Disproportionate eruption of maxillary and mandibular incisors in the long-tailed ground squirrel

The surface of the maxillary and mandibular incisors of Spermophilus undulatus long-tailed ground squirrels, including those born in the current year and those that have hibernated (trapped one month or later after hibernation) is studied. The presence of daily growth increments on the incisors’ surface allows the evaluation of the eruption rate of the incisors; a specific change in the character of the growth increments corresponds to winter hibernation (hibernation zone), which serves as the time mark. Ratio between the eruption rates of the maxillary and mandibular incisors typical for rodents is found in young-of-the-year and some animals after hibernation. In these animals the eruption rate of the mandibular incisors is higher than the eruption rate of the maxillary incisors and can be taken as proportional to their length. In individuals that have hibernated and show proportional eruption of the incisors, the proportions of the total length of the incisor formed before hibernation zone are equal for the maxillary and mandibular incisors. In the individuals that also have hibernated and show the ratio between the total length of the maxillary and mandibular incisors typical for rodents, the eruption rate of the mandibular incisor is equal to or less than the eruption rate of the maxillary incisor and the proportion of the incisor formed before hibernation is greater in the mandibular incisor than in the maxillary. This disproportionate pattern of incisor eruption is not typical for rodents and is a result of inequal attrition of the maxillary and mandibular incisors, which ultimately results in the normal ratio of the total length of the maxillary and mandibular incisors.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

C2H2 and Ar induce rapid declines in nitrogenase activity and CO2 evolution in nodules of Datisca glomerata

Preliminary studies have indicated that after addition of C₂H₂ there is a rapid decline in nitrogenase activity in the nodules of Datisca glomerata . The present work was undertaken to determine whether (1) there is also a decline in respiration and (2) the decline is associated with the cessation of ammonia production. The rates of C₂H₄ and CO₂ evolution by nodulated root systems of Datisca were measured as a function of time after exposure to C₂H₂. The peak rate of C₂H₄ evolution occurred at 30 s after C₂H₂ exposure, while the rate of CO₂ evolution started to decline at 60 s after exposure to C₂H₂. Incubation of nodules in a gas mixture containing Ar also caused a decline in CO₂ evolution. Further, pretreatment with Ar eliminated most of the C₂H₂-induced decline in nitrogenase activity and CO₂ evolution. These C₂H₂- and Ar-induced declines in Datisca nodules are more rapid than those reported in any other nodules. They are evidence that continued ammonia formation is essential for maintenance of normal nitrogenase activity in Datisca nodules.     

Degradation activity of adhered and suspended Pseudomonas cells cultured on 2,4,6-trichlorophenol, measured by indirect conductimetry

The degradation activity (expressed as specific CO₂ production rates) of adhered and suspended Pseudomonas cells, strains SP1 and SP2, during the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), was compared using indirect conductimetry technique. This technique is defined as the measurement of CO₂ ionization in an alkaline solution and expressed as the negative conductance change values of such solution. The attachment surfaces were porous glass and silicone rubber. The 2,4,6-TCP concentrations ranged from 10 to 500 mg 1⁻¹. Specific respiration rates were determined from CO₂ evolution rates and biomass yields of both suspended and adhered cell cultures. CO₂ evolution rates were determined after conversion of conductance change values into CO₂ produced values. Results indicate that glass-adhered cells reached a higher maximum specific CO₂ evolution rate ( Q _CO2max) than both suspended and silicone rubber-adhered cells. However, suspended cells showed a lower saturation constant ( K_s ) than the adhered cells. These results suggest that depending on support nature the respiration activity of adhered cells could be higher than of suspended cells. Moreover, the indirect conductimetry technique could efficiently be used by measurements of respiration activities of both attached or suspended xenobiotic-degrading micro-organisms.     

The effect of host resistance on the metabolic rate of engorged females of Rhipicephalus evertsi evertsi

Abstract. This study investigated the effect of acquired resistance in guinea-pigs on the metabolic rate of adult females of the tick Rhipicephalus evertsi evertsi. Guinea-pigs were subjected to three successive infestations of ticks and the rate of CO₂ production (Vco₂) measured in first and third infestation engorged females. Ticks which fed on resistant hosts showed a 52% decrease in mass compared to ticks that fed on naive animals. Reduction in mass was accompanied by a decrease in Vco₂ (mlh^-1) per tick but an increase in mass specific Vco₂ (mlg^_1h^_1). However, both groups shared a single allometric relationship between body mass and metabolic rate (Vco₂). We suggest that the differences in size rather than any factor directly relating to the mechanism of acquired resistance account for the differences in metabolic rate between ticks fed on naive and resistant guinea-pigs.     

Modifications of the leaf surface structures of Quercus ilex L. in open, naturally CO2-enriched environments

Two Italian CO₂ springs allowed us to study the long-term effect of a 350–2600 μ mol mol^–1 increase in CO₂ concentrations on the surface structures of leaves of Quercus ilex L. Carbon dioxide increased the quantity of cuticular waxes, above an apparent threshold of 750 μ mol mol^–1 CO₂. Leaf wettability was not modified by CO₂ concentrations. Reduction in stomatal frequency was observable up to 750 μ mol mol^–1 CO₂, the slope being almost the same as that estimated for the increase in CO₂ concentration from pre-industrial times to the present. At higher concentrations, CO₂ seemed to exert no more impact on stomatal frequency.     

Reversal by light of deleterious effects of chilling on oxygen evolution, manganese and free fatty acid content in tomato thylakoids is not accompanied by restoration of the original membrane conformation

Free fatty acids (FFA) generated in thylakoids upon chilling of tomato leaves at 0°C for a few days result in release of functionally active Mn and inactivation of O₂ evolution. Chilling does not lead to a decrease in the extrinsic 16, 23 and 33 kDa polypeptides. Upon illumination of chilled leaves both Mn content and O₂ evolution in thylakoids are restored and FFA content is reduced to the level of the control. Photoactivation of O₂ evolution in chilled leaves does not change the ratio of unsaturated/saturated FFA. Constant Arrhenius activation energy (Ea) for O₂ evolution by thylakoids isolated from control leaves was found, whereas it increased at temperatures below 8.0 and 10.5°C in thylakoids from cold-treated and photoactivated leaves, respectively. This indicates that restoration of O₂ evolution as well as of FFA and Mn contents is not accompanied by a complete reversal of membrance conformation.     

Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

Stomatal limitation of photosynthesis in winter production of greenhouse tomato plants

In May, greenhouse tomato ( Lycopersicon esculentum Mill.) plants near the end of their winter production cycle were shown to exhibit a diurnal photosynthetic decrease. In order to identify the physiological causes of this decline, we compared in May the photosynthetic characteristics of the fifth youngest leaves from tomato plants of different ages corresponding to a winter production (11-month-old plants) and to a spring production (5-month-old plants). Although the leaves were developed simultaneously under the same environmental conditions, only the ones from the winter production showed a diurnal decline of the in situ CO₂ assimilation rate (A _{CO 2}). This was accompanied by a decline of internal CO₂ and stomatal conductance and by large accumulations of hexoses. When stomatal closure was relieved under saturated CO₂ concentration (5%) using a leaf-disc electrode system, the fifth leaves of both tomato cultures had similar maximum quantum efficiency of O₂ evolution (Φ_max), light-saturated rate of O₂ evolution (P_max) and quantum efficiency of photosystem II (PSII) photochemistry (ΔF/F'_m, q _P and q _N ). We concluded that the diurnal decline of A _{CO 2} observed in winter tomato production during May originates from a stomatal limitation that is not dependent on environmental conditions but rather related to the developmental stage of the plants.     

31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain

Abstract: Ischemia-induced changes in ³¹P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T ₁ were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P₁ levels had reached a plateau. The T ₁ for P₁ did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T ₁ relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P₁ during ischemia, implying that the T *₂ values remain constant. Our results suggest that the ³¹P T ₁ and T *₂ for phosphocreatine, P_i, and ATP do not change during ischemia, and therefore changes in ³¹P NMR peak intensity accurately reflect changes in metabolite concentrations.