Evaluating Cementum to Determine Past Reproduction in Northern Sea Otters

Abstract: Age at first reproduction (AFR) has been difficult to quantify in mammals, as the most commonly used methods require reproductive tracts or direct observations. However, work in several large mammal species suggests that the width of cementum light bands in teeth decline once females begin to reproduce, suggesting that teeth structures might provide a new tool to examine AFR. To determine if changes in cementum light band width could be used to calculate AFR for the northern sea otter (Enhydra lutris kenyoni), we measured cementum light band widths on sectioned premolar teeth and compared them to reproductive tracts. We classified otters as parous if any single light band was narrower than a threshold value, selected as the value that minimized error rates. At a threshold value of 0.32, we correctly identified otters as parous or nulliparous in 83% of cases (n = 92) as compared to reproductive tracts, and the AFR estimated from teeth samples (3.52 ± 0.032 yr) was not different from that determined by reproductive tract analysis (3.45 ± 0.031 yr; t-test, P > 0.05). These data support the use of cementum as an indicator of past reproduction in individual female otters, which can then be used to estimate average AFR. Given that declines in cementum width have been described for other mammal species, the same quantitative approach used here could be applied to other species. (JOURNAL OF WILDLIFE MANAGEMENT 72(3):618–624; 2008)     

Is there a correlation between age and <Emphasis Type="SmallCaps">D</Emphasis>-aspartic acid in human knee cartilage?

Summary. L-Aspartic acid (L-Asp) is one of the fastest racemizing amino acids such that the abnormal D-form (D-Asp) has been found in stable biological human tissues such as dentin in teeth, eye lens and brain. Earlier reports showed that there was a linear correlation between age and D-Asp in teeth. We have previously reported that significant levels of D-Asp were found in normal and osteoarthritic knee cartilage. Since cartilage is a slow regenerating tissue, we hypothesized that D-Asp should accumulate in knee cartilage and that there might be a correlation between the age of the person and the amount of D-Asp found in cartilage. Our analysis of approximately 100 samples of normal knee cartilage showed that there are detectable amounts of D-Asp (2–4% of total Asp) in knee cartilage. However, there was only a slight correlation (r = 0.35) between the age of the person and the amount of D-Asp (nmoles/g). Surprisingly, there was a better correlation between age and the amount of D-Asp in the male subjects (r = 0.57) than in the female subjects (r = 0.21).     

Skull reconstruction and ecology of <Emphasis Type="Italic">Urmiatherium polaki</Emphasis> (Bovidae,Mammalia) from the upper Miocene deposits of Maragheh,Iran

Study of a newly discovered cranial specimen of Urmiatherium polaki from Karaj Abad site in Maragheh, Iran, allows us to describe the complete skull and present a new diagnosis for this late Miocene mammal species. In the comparative study of the specimen we propose the relative position of the upper teeth as a reliable metrical method for discriminating bovids of similar size. Based on the available information, a skull reconstruction of Urmiatherium polaki is provided and its ecological profile is outlined. Both the cranial and dental features of U. polaki indicate a gregarious grazing bovid of medium size, adapted to uneven ground and exhibiting ramming as the predominant fighting behavior.     

Increased effect of harsh climate in red deer with a poor set of teeth

Teeth are vital for mammal performance and especially in ungulates relying on mechanical decomposition of plant material for effective microbial digestion and energy uptake. The main focus of the role of teeth in ungulate life histories has been on tooth wear, while no one has addressed to what extent deviation from the natural set of teeth (maldentition) causes variation in individual fitness components. Based on mandibles from 41,066 individual red deer (Cervus elaphus L.) collected from 1969 to 2001, we tested whether maldentition had an effect on individual body condition and whether this effect depended on environmental harshness. Females with maldentition (0.6% of the population) were in a poorer condition than individuals without tooth anomalies and the effect increased during unfavorable climatic conditions. The effect of maldentition in males was less clear. This study indicates that a well-functioning set of teeth is essential for mammal performance, and that selection pressure against (dental) anomalies is more pronounced when climate is unfavorable. Electronic Supplementary Material Supplementary material is available for this article at     

On the Occlusal Fit of Tribosphenic Molars: Are We Underestimating Species Diversity in the Mesozoic?

The complex occlusal fits between tribosphenic teeth are a rich source of information for taxonomic, phylogenetic, and evolutionary analysis. The degree of fit between upper and lower cheek teeth has been used to refer specimens to species-level taxa, but statistical data on occlusal fit in relation to taxonomic identity have been lacking. We used landmarks on upper and lower first molars of 20 bat populations representing 16 species to assess the degree of occlusal fit (1) between teeth from the same individual; (2) between teeth from different individuals belonging to the same populations; and (3) between teeth belonging to different populations. We found that the fit of teeth belonging to different populations was significantly worse than between those of the same population and that the degree of misfit increased linearly with time since common ancestry, albeit with substantial variance. We used our comparisons to assess the species-level diversity within Batodon, the smallest known placental mammal from the Cretaceous. Our data suggest, with caveats, that instead of belonging to a single species, the specimens assigned to Batodon represent at least two species as different as those belonging to different genera or families of living bats.     

Evolutionary Implications of Dental Eruption in Dasypus (Xenarthra)

Late eruption of the permanent dentition was recently proposed as a shared anatomical feature of endemic African mammals (Afrotheria), with anecdotal reports indicating that it is also present in dasypodids (armadillos). In order to clarify this question, and address the possiblity that late eruption is shared by afrotherians and dasypodids, we quantified the eruption of permanent teeth in Dasypus, focusing on growth series of D. hybridus and D. novemcinctus. This genus is the only known xenarthran that retains two functional generations of teeth. Its adult dentition typically consists of eight upper and eight lower ever-growing (or euhypsodont) molariforms, with no premaxillary teeth. All but the posterior-most tooth are replaced, consistent with the identification of a single molar locus in each series. Comparison of dental replacement and skull metrics reveals that most specimens reach adult size with none or few erupted permanent teeth. This pattern of growth occurring prior to the full eruption of the dentition is similar to that observed in most afrotherians. The condition observed in Dasypus and many afrotherians differs from that of most other mammals, in which the permanent dentition erupts during (not after) growth, and is complete at or near the attainment of sexual maturity and adult body size. The suture closure sequence of basicranial and postcranial epiphyses does not correlate well with dental eruption. The basal phylogenetic position of the taxon within dasypodids suggests that diphyodonty and late dental replacement represent the condition of early xenarthrans. Additionally, the inferred reduction in the number of molars to a single locus and the multiplication of premolars represent rare features for any living mammal, but may represent apomorphic characters for Dasypus.     

Vitamin D content in Alaskan Arctic zooplankton,fishes, and marine mammals

We postulated that dietary ingestion of vitamin D may be used by some Alaskan Arctic marine mammal species in addition to, or instead of, cutaneous production to meet nutritional requirements. Zooplankton (n=5) sampled near Kaktovik, Alaska, contained no measurable vitamin D₂ or D₃, but did contain provitamin D (7‐dehydrocholesterol), the cutaneous precursor for previtamin D₃ in mammals. Fillets and livers from five fish species were sampled near Barrow, Alaska, and evaluated for vitamin D₃ content (no vitamin D₂ was detected). Differences in vitamin D₃ content appeared significant (P≤0.10) among fish livers (Kruskal‐Wallis [H test]=8.25, df=4, P=0.08) and among fish fillets (H=7.80, df=4, P=0.01). We also found significant differences in several pairwise comparisons (Mann‐Whitney U‐test) of vitamin D₃ levels in fillets and livers. Blubber from six species of marine mammals had no detectable vitamin D₂. The H test results for blubber vitamin D₃ concentration were highly significant: 28.12, df=5, P<0.001. There were also significant differences in vitamin D₃ content from blubber in pairwise comparisons of primarily invertebrate feeders (bowhead whale (Balaena mysticetus) [mean=4.20 SD±1.10 ng/g], and Pacific walrus (Odobenus rosmarus divergens) [5.43±2.82 ng/g]) vs. primarily piscivorous feeders (ringed seal (Phoca hispida) [746.57±493.00 ng/g] and beluga whale (Delphinapterus leucas) [426.00±174.92 ng/g]) and a semiaquatic terrestrial carnivore (polar bear (Ursus maritimus) [406.17±311.70 ng/g]). The bearded seal (Erignathus barbatus) had intermediate blubber vitamin D₃ concentration (156.83±139.25 ng/g), which may reflect an intermediate‐type feeding strategy or an artifact of the small sample size. Zoo Biol 23:33–43, 2004. © 2004 Wiley‐Liss, Inc.     

Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

A new basal tomistomine (Crocodylia,Crocodyloidea) from Issel (Middle Eocene; France): palaeobiogeography of basal tomistomines and palaeogeographic consequences

A skull and mandible of a crocodylian from the late Lutetian of Issel, previously described as ‘Atacisaurus glareae’ is reconsidered. The holotype of ‘A. glareae’, a partial mandible, is lost, and the skull cannot be designated as a lectotype for the species. ‘Atacisaurus glareae’ is thus a nomen dubium. The skull bears a combination of characters, allowing us to assign it to the genus Kentisuchus. It differs from the, until now, only known species Kentisuchus spenceri from the Ypresian of England, in having a more robust snout, with the constriction of the snout at the level of the seventh–eighth teeth being 80% of the largest maxillary width, and not bearing anteroposterior shallow fossae along the lacrimomaxillary sutures. A new species is thus erected, K entisuchus astrei sp. nov. Phylogenetic analysis confirms that the genus Kentisuchus is one of the most primitive tomistomine. The phylogenetic and palaeogeographic distribution suggests that Kentisuchus was isolated in the Atlantic Ocean, and Ebro (Spain) and Aquitaine (France) basins, during the Ypresian, and that the south Pyrenean marine corridor between the Atlantic Ocean and the Tethys could have closed during the early Ypresian, earlier than previously supposed. This could be correlated with the first mammal migrations from the Iberian Peninsula to Southern France. The palaeogeographic distribution of early and middle Eocene tomistomines also suggests the possible presence of a marine corridor between the North Sea and the Central Tethys through the Polish Lowlands Basin during the early Lutetian. This marine corridor could be informative for studies on mammal migration, as the presence of a north–south marine corridor necessarily means there is an absence or less efficient east–west terrestrial passage. This could have consequences on the history of Asian–European mammal migrations.     

The selfie trap: A novel camera trap design for accurate small mammal identification

Camera traps are a popular tool for monitoring wildlife though they can fail to capture enough morphological detail for accurate small mammal species identification. Camera trapping small mammals is often limited by the inability of camera models to: (i) record at close distances; and (ii) provide standardised photos. This study aims to provide a camera trapping method that captures standardised images of the faces of small mammals for accurate species identification, with further potential for individual identification. A novel camera trap design coined the ‘selfie trap’ was developed. The selfie trap is a camera contained within an enclosed PVC pipe with a modified lens that produces standardised close images of small mammal species encountered in this study, including: Brown Antechinus (Antechinus stuartii), Bush Rat (Rattus fuscipes) and Sugar Glider (Petaurus breviceps). Individual identification was tested on the common arboreal Sugar Glider. Five individual Sugar Gliders were identified based on unique head stripe pelage. The selfie trap is an accurate camera trapping method for capturing detailed and standardised images of small mammal species. The design described may be useful for wildlife management as a reliable method for surveying small mammal species. However, intraspecies individual identification using the selfie trap requires further testing.     

Assessing sapwood depth and wood properties in Eucalyptus and Corymbia spp. using visual methods and near infrared spectroscopy (NIR)

Accurate measurement of sapwood depth (D _S) is essential for calculating volumetric water use of individual trees and stands. Various methods are available to measure D _S but their accuracy is rarely cross-validated. We sampled 15 Eucalyptus and 1 Corymbia species along a gradient of aridity and obtained reference values of D _S in fresh wood cores using light microscopy, which represents our reference method. We compared this method to the simpler and widely used macroscopic method: visual assessment of natural or induced colour change from sapwood to heartwood. In a third method, estimation of D _S was based on species-specific models that rely on wood properties measured using near infrared spectroscopy (NIR). Microscopy allowed clear identification of D _S based on the presence of blocked vessels. Measurement of D _S using microscopic methods was possible for 78 of a total of 80 cores and ranged from 3.6 mm (E. loxophleba) to 43.8 mm (E. viminalis). Macroscopic assessment clearly differentiated sapwood and heartwood in 60 cores. Results from microscopic and macroscopic methods agreed closely (<10% deviation between estimates) in 35 of 78 cores. After elimination of clearly erroneous measurements (>50% deviation between estimates), macroscopic measurement across all species agreed well with microscopic assessment of D _S (R ² = 0.92). Models developed for differentiation between sapwood and heartwood using NIR spectroscopy were very robust (high coefficient of determination) for four species, but D _S could only be predicted well for one (E. obliqua) of the four species. Even after elimination of apparent false estimates, prediction of D _S by NIR across species was not as strong as for macroscopic assessment (R ² = 0.88). D _S can accurately be measured using microscopy if vessel occlusion is clearly visible. Although slightly overestimated, D _S from macroscopic assessment was generally similar to that measured by microscopy. NIR spectroscopy was unable to predict D _S with acceptable accuracy for the majority of species. Further improvements in the prediction of D _S using NIR will require more intensive model calibration and validation, and may not be applicable to all species.     

Evaluation of vitamin D3 metabolites in Callithrix jacchus (common marmoset)

Vitamin D₃ (cholecalciferol) is endogenously produced in the skin of primates when exposed to the appropriate wavelengths of ultraviolet light (UV-B). Common marmosets (Callithrix jacchus) maintained indoors require dietary provision of vitamin D₃ due to lack of sunlight exposure. The minimum dietary vitamin D₃ requirement and the maximum amount of vitamin D₃ that can be metabolized by marmosets is unknown. Observations of metabolic bone disease and gastrointestinal malabsorption have led to wide variation in dietary vitamin D₃ provision amongst research institutions, with resulting variation in circulating 25-hydroxyvitamin D₃ (25(OH)D₃), the accepted marker for vitamin D sufficiency/deficiency. Multiple studies have reported serum 25(OH)D₃ in captive marmosets, but 25(OH)D₃ is not the final product of vitamin D₃ metabolism. In addition to serum 25(OH)D_3, we measured the most physiologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and the less well understood metabolite, 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) to characterize the marmoset's ability to metabolize dietary vitamin D₃. We present vitamin D₃ metabolite and related serum chemistry value colony reference ranges in marmosets provided diets with 26,367 (Colony A, N = 113) or 8,888 (Colony B, N = 52) international units (IU) of dietary vitamin D₃ per kilogram of dry matter. Colony A marmosets had higher serum 25(OH)D₃ (426 ng/ml [SD 200] vs. 215 ng/ml [SD 113]) and 24,25(OH)₂D₃ (53 ng/ml [SD 35] vs. 7 ng/ml [SD 5]). There was no difference in serum 1,25(OH)₂D₃ between the colonies. Serum 1,25(OH)₂D₃ increased and 25(OH)D₃ decreased with age, but the effect was weak. Marmosets tightly regulate metabolism of dietary vitamin D₃ into the active metabolite 1,25(OH)₂D₃; excess 25(OH)D₃ is metabolized into 24,25(OH)₂D₃. This ability explains the tolerance of high levels of dietary vitamin D₃ by marmosets, however, our data suggest that these high dietary levels are not required.     

Heritability: the link between development and the microevolution of molar tooth form

Abstract

The developmental gene expression, morphogenesis, and population variation in mammalian molar teeth has become increasingly well understood, providing a model system for synthesizing evolution and developmental genetics. In this study, we estimated additive genetic covariances in molar shape (G) using parent-offspring regression in Cryptotis parva, the Least Shrew. We found that crown shape had an overall h² value of 0.34 (±0.08), with higher heritabilities in molar cusps than notches. We compared the genetic covariances to phenotypic (P) and environmental (E) covariances, and to the covariances in crown features expected from the enamel knot developmental cascade (D). We found that G and D were not strongly correlated and that major axes of G (evolutionary lines of least resistance) are better predictors of evolutionary divergences in soricines than is D. We conclude that the enamel knot cascade does impose constraints on the evolution of molar shape, but that it is so permissive that the divergences among soricines (whose last common ancestor lived about 14 million years ago) do not fully explore its confines. Over tens of millions of years, G will be a better predictor of the major axes of evolution in molar shape than D.     

Late Jurassic Mammals from Tendaguru, Tanzania, East Africa

Records of Mesozoic mammals are extremely rare in Africa. The only previous record from the Upper Jurassic of Africa is a fragmentary mandible without teeth of Brancatherulum tendagurense. Here I report the discovery of two new mammals from the Upper Jurassic of Tendaguru, Tanzania. The fossils were recovered from the Middle Saurian Bed of the Tendaguru Series. A lower molar of a triconodontid mammal is described as Tendagurodon janenschi gen. et sp. nov., and a fragmentary dentary of a eupantothere as Tendagurutherium dietrichi gen. et sp. nov. The eupantothere in particular contributes to documenting the evolution of mammals during the Mesozoic. The posterior portion of the mandible of Tendagurutherium dietrichi gen. et sp. nov. shows that the angular (tympanic) bone was not yet completely separated from the dentary, a previously undocumented stage of eupantotherian middle ear evolution.     

Fractals and the analysis of growth paths

A simple practical method exists for classifying and comparing planar curves composed of connected line segments. This method assigns, a single numberD, the fractal dimension, to each curve.D=log(n)/[log(n)+log(d/L)], where:n is the number of line segments,L is the total length of the line segments, andd is the planar diameter of the curve (the greatest distance between any two endpoints). At one end of the spectrum, for straight line curves,D=1; at the other end of the spectrum, for random walk curves,D→2. Standard statistics are done on the logarithms of the fractal dimension [log(D)]. With this measure, trails of biological movement, such as the growth paths of the cells and the paths of wandering organisms, can be analyzed to determine the likelihood that these trails are random walks and also to compare the straightness of the trails before and after experimental interventions.     

MEASURING THE HYDRAULIC DIAMETER OF A PORE OR CONDUIT

A method for estimating the hydraulic diameter of a pore or conduit having a noncircular opening is presented with special reference to plant anatomy. An ellipse or a rectangle is inscribed within the opening, and the length of the short axis (a) is measured. The hydraulic diameter (D_h) is estimated for the ellipse (D_h = 1.4a) or rectangle (D_h = 2a). Use of these equations often gives a more accurate estimate of the hydraulic diameter of a pore or conduit than does averaging the lengths of the short and long axes (D_h = [a + b]/2, b is the long axis) or assuming that the opening is circular (D_h = a). A table of the error in each method is included, and the errors inherent in the use of D_h are discussed. Because fewer measurements are required, estimation based on the measurement of one axis is much faster than calculation using both the short and long axes. The equations and table should permit anatomists and physiologists to rapidly determine the best method for estimating the hydraulic diameter of a pore or conduit, and to more accurately and quickly estimate the hydraulic diameters of large numbers of openings. However, because of potential pitfalls in applying theoretical fluid dynamics equations to real-world functional anatomy, botanists must ensure that their applications of hydraulic diameter are appropriate in each case.     

Classification of ancient mammal individuals using dental pulp MALDI-TOF MS peptide profiling

Background

The classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets.

Methodology/Principal Findings

We present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279–modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively.

Conclusions/Significance

Mass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals.     

Temporal shifts of DNA‐microsatellite allele profiles in roe deer (Capreolus capreolus L.) within three decades

DNA‐microsatellite polymorphism (four loci) was studied in 56 male roe deer (Capreolus capreolus) from a 900‐ha hunting territory in the Vosges du Nord Mountains (France), culled over 34 years (1956–1990). Changed allele frequencies at two loci within this period, and increased allelic diversity, were traced to a phase of reduced population density and subsequent immigration. Decadic population samples collected within 900‐ha were distinguished by higher genetic variability measures than were certain geographical samples across Central Europe (4–900 km). On average, the decadic cohorts were distinguished by a gene diversity index of G_ST = 0.0286, and a genetic distance of D = 0.0938, which reflect 54% (G_ST) and 69% (D) of the respective geographic (350 km) differentiation indices of roe deer in Central Europe. The importance of demography and population ecology effects for microevolution in a large mammal is demonstrated, as is the risk of artefact by composing population samples of deer over several years. Population genetic screening should cover various demes of roe deer from the same general region, and be based on many unlinked polymorphic loci, to minimize the distorting effects of genetic dynamics at the small spatial scale.     

Estimation of the D period from residual division after exposure of exponential phase bacteria to chloramphenicol

Summary Values of the D period, between termination of chromosome replication and cell division, were determined from measurements of residual cell division after exposure of exponential phase cultures of Escherichia coli B/r and K12 and of Salmonella typhimurium to chloramphenicol. The results obtained by this method were compared with earlier results for E. coli B/r obtained from measurements of DNA content per cell and were found to be almost identical. For each, values of the D period were independent of growth rate, and the average value of D=26.1±1.2 min obtained by residual division is in good agreement with the value of 25 min obtained earlier. These results indicate that the method of residual division provides a good measure of the duration of the D period. Values of D were also independent of growth rate for each of the other strains.This work supported by the U.S. Atomic Energy Commission.     

Plasticity in hydraulic architecture of Scots pine across Eurasia

Widespread tree species must show physiological and structural plasticity to deal with contrasting water balance conditions. To investigate these plasticity mechanisms, a meta-analysis of Pinus sylvestris L. sap flow and its response to environmental variables was conducted using datasets from across its whole geographical range. For each site, a Jarvis-type, multiplicative model was used to fit the relationship between sap flow and photosynthetically active radiation, vapour pressure deficit (D) and soil moisture deficit (SMD); and a logarithmic function was used to characterize the response of stomatal conductance (G _s) to D. The fitted parameters of those models were regressed against climatic variables to study the acclimation of Scots pine to dry/warm conditions. The absolute value of sap flow and its sensitivity to D and SMD increased with the average summer evaporative demand. However, relative sensitivity of G _s to D (m/G _s,ref, where m is the slope and G _s,ref is reference G _s at D = 1 kPa) did not increase with evaporative demand across populations, and transpiration per unit leaf area at a given D increased accordingly in drier/warmer climates. This physiological plasticity was linked to the previously reported climate- and size-related structural acclimation of leaf to sapwood area ratios. G _s,ref, and its absolute sensitivity to D (m), tended to decrease with age/height of the trees as previously reported for other pine species. It is unclear why Scots pines have higher transpiration rates at drier/warmer sites, at the expense of lower water-use efficiency. In any case, our results suggest that these structural adjustments may not be enough to prevent lower xylem tensions at the driest sites.