Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress

We integrated field and laboratory studies in an investigation of water balance, energy use, and mechanisms of cold-hardiness in hatchling painted turtles (Chrysemys picta) indigenous to west-central Nebraska (Chrysemys picta bellii) and northern Indiana (Chrysemys picta marginata) during the winters of 1999-2000 and 2000-2001. We examined 184 nests, 80 of which provided the hatchlings (n=580) and/or samples of soil used in laboratory analyses. Whereas winter 1999-2000 was relatively dry and mild, the following winter was wet and cold; serendipitously, the contrast illuminated a marked plasticity in physiological response to environmental stress. Physiological and cold-hardiness responses of turtles also varied between study locales, largely owing to differences in precipitation and edaphics and the lower prevailing and minimum nest temperatures (to -13.2 degrees C) encountered by Nebraska turtles. In Nebraska, winter mortality occurred within 12.5% (1999-2000) and 42.3% (2000-2001) of the sampled nests; no turtles died in the Indiana nests. Laboratory studies of the mechanisms of cold-hardiness used by hatchling C. picta showed that resistance to inoculative freezing and capacity for freeze tolerance increased as winter approached. However, the level of inoculation resistance strongly depended on the physical characteristics of nest soil, as well as its moisture content, which varied seasonally. Risk of inoculative freezing (and mortality) was greatest in midwinter when nest temperatures were lowest and soil moisture and activity of constituent organic ice nuclei were highest. Water balance in overwintering hatchlings was closely linked to dynamics of precipitation and soil moisture, whereas energy use and the size of the energy reserve available to hatchlings in spring depended on the winter thermal regime. Acute chilling resulted in hyperglycemia and hyperlactemia, which persisted throughout winter; this response may be cryoprotective. Some physiological characteristics and cold-hardiness attributes varied between years, between study sites, among nests at the same site, and among siblings sharing nests. Such variation may reflect adaptive phenotypic plasticity, maternal or paternal influence on an individual's response to environmental challenge, or a combination of these factors. Some evidence suggests that life-history traits, such as clutch size and body size, have been shaped by constraints imposed by the harsh winter environment.     

Phylogenetic hypotheses for the turtle family Geoemydidae

The turtle family Geoemydidae represents the largest, most diverse, and most poorly understood family of turtles. Little is known about this group, including intrafamilial systematics. The only complete phylogenetic hypothesis for this family positions geoemydids as paraphyletic with respect to tortoises, but this arrangement has not been accepted by many workers. We compiled a 79-taxon mitochondrial and nuclear DNA data set to reconstruct phylogenetic relationships for 65 species and subspecies representing all 23 genera of the Geoemydidae. Maximum parsimony (MP) and maximum-likelihood (ML) analyses and Bayesian analysis produced similar, well-resolved trees. Our analyses identified three main clades comprising the tortoises (Testudinidae), the old-world Geoemydidae, and the South American geoemydid genus Rhinoclemmys. Within Geoemydidae, many nodes were strongly supported, particularly based on Bayesian posterior probabilities of the combined three-gene dataset. We found that adding data for a subset of taxa improved resolution of some deeper nodes in the tree. Several strongly supported groupings within the Geoemydidae demonstrate non-monophyly of some genera and possible interspecific hybrids, and we recommend several taxonomic revisions based on available evidence.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.     

Electrostatic interactions in ubiquitin: stabilization of carboxylates by lysine amino groups

To explore electrostatic interactions in ubiquitin, pK(a) values have been determined by NMR for all 12 carboxyl groups in wild-type ubiquitin and in variants where single lysines have been replaced by neutral residues. Aspartate pK(a) values in ubiquitin range from 3.1 to 3.8 and are generally less than model compound values. Most aspartate pK(a) values are within 0.2 pH unit of those predicted with a simple Tanford-Kirkwood model. Glutamate pK(a) values range from 3.8 to 4.5, close to model compound values and differing by 0.1-0.8 pH unit from calculated values. To determine the role of positive charges in modulating carboxyl pK(a) values, we mutated lysines at positions 11, 29, and 33 to glutamine and threonine. NMR studies with these six single-site mutants reveal significant interactions of Lys 11 and Lys 29 with Glu 34 and Asp 21, respectively: pK(a) values for Glu 34 and Asp 21 increase by approximately 0.5-0.8 pH unit, similar to predicted values, when the lysines are replaced by neutral residues. In contrast, the predicted interaction between Lys 33 and Glu 34 is not observed experimentally. In some instances, substitution of lysine by glutamine and threonine did not lead to the same changes in carboxyl pK(a) values. These may reflect new short-range interactions between the mutated residues and the carboxyl groups. Carboxyl pK(a) shifts > 0.5 pH unit result from mutations at groups that are <5 A from the carboxyl group. No interactions are observed at >10 A.     

Weaning mass affects changes in body composition and food intake in harbour seal pups during the first month of independence

In phocid seals, the transition to nutritional independence is abrupt, with females abandoning their offspring after weaning and returning to sea. We hypothesized that body size at weaning may play an important role in the nature of this transition. We studied the changes in body composition and water flux of newly weaning harbour seals over the first 4-6 wk postweaning. Thirty-three pups were dosed with deuterium oxide to estimate total body water (TBW) and a subset of 24 was dosed twice to estimate changes in body composition and water flux. All pups lost body mass over the study period, but TBW increased during the period of mass loss, indicating continued lean tissue growth. Combined data from this and our early study indicated that heavy (>median mass) pups were relatively fatter (41.0% vs. 37.1%) and had significantly greater total body energy at weaning than did light (< or = median mass) pups. Percentage TBW declined linearly over time in light pups but was constant in heavy pups for the first 19 d postweaning and then declined linearly. Both the temporal pattern and composition of mass loss differed between light and heavy pups. Estimated food intake increased in the second 2 wk of study compared to the first 2 wk, in both light and heavy pups, reflecting increased foraging success but at levels still insufficient to meet daily expenditures of most individuals.     

ESTIMATION OF TOTAL BODY WATER IN HARBOR SEALS: HOW USEFUL IS BIOELECTRICAL IMPEDANCE ANALYSIS?

We evaluated bioelectrical impedance analysis (BIA) as a means of rapidly and inexpensively estimating total body water (TBW) of harbor seals ( Phoca vitulina ). Deuterium oxide dilution was used to estimate TBW in 17 adult females and 16 of their pups between birth and late lactation. Isotope dilution was also used to determine TBW in 12 adult males early and 10 of these males late in the breeding season. At the same time, resistance ( Rs ) and reactance ( Xc ) measurements were taken using a tetrapolar, impedance plethysmograph (Model 101 A, RJL Systems). Seals were sedated with diazepam prior to taking BIA measurements. Within-day duplicate Rs measurements on pups and adults, taken 2-240 min apart, differed by an average of 3.0%± 1.4% ( n = 42, CV = 102%). Movement of the seal during BIA measurements caused variability in both Rs and Xc values. BIA measurements were generally poor predictors of TBW. Rs was significantly correlated with TBW in pups only ( Rs = 0.93, P = 0.001, n = 11). Bioelectrical conductor volume (length2/ Rs ) was significantly correlated with TBW only in adult females ( Rs = 0.63, P = 0.02, n = 14). We conclude that BIA is not a reliable method of estimating TBW in wild harbor seals.     

A new fourier transform approach for protein coding measure based on the format of the Z curve

MOTIVATION: At the core of most protein gene-finding algorithms are the coding measures used to make a decision on coding/non-coding. Of the protein coding measures, the Fourier measure is one of the most important. However, due to the limited length of the windows usually used, the accuracy of the measure is not satisfactory. This paper is devoted to improving the accuracy by lengthening the sequence to amplify the periodicity of 3 in the coding regions. RESULTS: A new algorithm is presented called the lengthen-shuffle Fourier transform algorithm. For the same window length, the percentage accuracy of the new algorithm is 6-7% higher than that of the ordinary Fourier transform algorithm. The resulting percentage accuracy (average of specificity and sensitivity) of the new measure is 84.9% for the window length 162 bp. AVAILABILITY: The program is available on request fromC.- T. Zhang. Contact: ctzhang@tju.edu.cn      

RAPID EVOLUTION OF REPRODUCTIVE ISOLATION BETWEEN INCIPIENT OUTCROSSING AND SELFING CLARKIA SPECIES

A major goal of speciation research is to understand the processes involved in the earliest stages of the evolution of reproductive isolation (RI). One important challenge has been to identify systems where lineages have very recently diverged and opportunities for hybridization are present. We conducted a comprehensive examination of the components of RI across the life cycle of two subspecies of Clarkia xantiana, which diverged recently (ca. 65,000 bp). One subspecies is primarily outcrossing, but self‐compatible, whereas the other is primarily selfing. The subspecies co‐occur in a zone of sympatry but hybrids are rarely observed. Premating barriers resulted in nearly complete isolation in both subspecies with flowering time and pollinator preference (for the outcrosser over the selfer) as the strongest barriers. We found that the outcrosser had consistently more competitive pollen, facilitating hybridization in one direction, but no evidence for pollen–pistil interactions as an isolating barrier. Surprisingly, postzygotic isolation was detected at the stage of hybrid seed development, but in no subsequent life stages. This crossing barrier was asymmetric with crosses from the selfer to outcrosser most frequently failing. Collectively, the results provide evidence for rapid evolution of multiple premating and postzygotic barriers despite a very recent divergence time.