Whole-genome Analyses Reveal Past Population Fluctuations and Low Genetic Diversities of the North Pacific Albatrosses.

Throughout the Plio-Pleistocene, climate change has impacted tropical marine ecosystems substantially, with even more severe impacts predicted in the Anthropocene. Although many studies have clarified demographic histories of seabirds in polar regions, the history of keystone seabirds of the tropics is unclear, despite the prominence of albatrosses (Diomedeidae, Procellariiformes) as the largest and most threatened group of oceanic seabirds. To understand the impact of climate change on tropical albatrosses, we investigated the evolutionary and demographic histories of all four North Pacific albatrosses and their prey using whole-genome analyses. We report a striking concordance in demographic histories among the four species, with a notable dip in effective population size at the beginning of the Pleistocene and a population expansion in the Last Glacial Period when sea levels were low, which resulted in increased potential coastal breeding sites. Abundance of the black-footed albatross dropped again during the Last Glacial Maximum, potentially linked to climate-driven loss of breeding sites and concordant genome-derived decreases in its major prey. We find very low genome-wide (π < 0.001) and adaptative genetic diversities across the albatrosses, with genes of the major histocompatibility complex close to monomorphic. We also identify recent selective sweeps at genes associated with hyperosmotic adaptation, longevity, and cognition and memory. Our study has shed light on the evolutionary and demographic histories of the largest tropical oceanic seabirds and provides evidence for their large population fluctuations and alarmingly low genetic diversities.

Museomics and phylogenomics of lovebirds (Psittaciformes, Psittaculidae, Agapornis) using low-coverage whole-genome sequencing.

Natural history collections contain specimens that provide important insights into studies of ecology and evolution. With the advancement of high-throughput sequencing, historical DNA (hDNA) from museum specimens has become a valuable source of genomic data to study the evolutionary history of organisms. Low-coverage whole genome sequencing (WGS) has been increasingly applied to museum specimens for analyzing organelle genomes, but is still uncommon for genotyping the nuclear DNA fraction. In this study, we applied low-coverage WGS to phylogenomic analyses of parrots in the genus Agapornis by including both modern samples and historical specimens of âˆ¼100-year-old. Agapornis are small-sized African and Malagasy parrots with diverse characters. Earlier phylogenetic studies failed to resolve the positions of some key lineages, prohibiting a robust interpretation of the biogeography and evolution of these African parrots. Here, we demonstrated the use of low-coverage WGS for generating both mitochondrial and nuclear genomic data, and evaluated data quality differences between modern and historical samples. Our resolved Agapornis phylogeny indicates the ancestor of Agapornis likely colonized Madagascar from Australasia by trans-oceanic dispersal events before dispersing to the African continent. Genome-wide SNPs also allowed us to identify the parental origins of hybrid Agapornis individuals. This study demonstrates the potential of applying low-coverage WGS to phylogenomics and population genomics analyses and illustrates how including historical museum specimens can address outstanding questions regarding the evolutionary history of contemporary lineages.

Arm Motor Recovery After Ischemic Stroke: A Focus on Clinically Distinct Trajectory Groups.

BACKGROUND AND PURPOSE: Recovery of arm function poststroke is highly variable with some people experiencing rapid recovery but many experiencing slower or limited functional improvement. Current stroke prediction models provide some guidance for clinicians regarding expected motor outcomes poststroke but do not address recovery rates, complicating discharge planning. This study developed a novel approach to defining recovery groups based on arm motor recovery trajectories poststroke. In addition, between-group differences in baseline characteristics and therapy hours were explored. METHODS: A retrospective cohort analysis was conducted where 40 participants with arm weakness were assessed 1 week, 6 weeks, 3 months, and 6 months after an ischemic stroke. Arm recovery trajectory groups were defined on the basis of timing of changes in the Fugl-Meyer Assessment Upper Extremity (FMA-UE), at least the minimal clinically important difference (MCID), 1 week to 6 weeks or 6 weeks to 6 months. Three recovery trajectory groups were defined: Fast (n = 19), Extended (n = 12), and Limited (n = 9). Between-group differences in baseline characteristics and therapy hours were assessed. Associations between baseline characteristics and group membership were also determined. RESULTS: Three baseline characteristics were associated with trajectory group membership: FMA-UE, NIH Stroke Scale, and Barthel Index. The Fast Recovery group received the least therapy hours 6 weeks to 6 months. No differences in therapy hours were observed between Extended and Limited Recovery groups at any time points. DISCUSSION AND CONCLUSIONS: Three clinically relevant recovery trajectory groups were defined using the FMA-UE MCID. Baseline impairment, overall stroke severity, and dependence in activities of daily living were associated with group membership and therapy hours differed between groups. Stratifying individuals by recovery trajectory early poststroke could offer additional guidance to clinicians in discharge planning.(See Supplemental Digital Content 1 for Video Abstract, available at: http://links.lww.com/JNPT/A337.).

Whole-Genome Analyses Resolve the Phylogeny of Flightless Birds (Palaeognathae) in the Presence of an Empirical Anomaly Zone.

Palaeognathae represent one of the two basal lineages in modern birds, and comprise the volant (flighted) tinamous and the flightless ratites. Resolving palaeognath phylogenetic relationships has historically proved difficult, and short internal branches separating major palaeognath lineages in previous molecular phylogenies suggest that extensive incomplete lineage sorting (ILS) might have accompanied a rapid ancient divergence. Here, we investigate palaeognath relationships using genome-wide data sets of three types of noncoding nuclear markers, together totaling 20,850 loci and over 41 million base pairs of aligned sequence data. We recover a fully resolved topology placing rheas as the sister to kiwi and emu + cassowary that is congruent across marker types for two species tree methods (MP-EST and ASTRAL-II). This topology is corroborated by patterns of insertions for 4274 CR1 retroelements identified from multispecies whole-genome screening, and is robustly supported by phylogenomic subsampling analyses, with MP-EST demonstrating particularly consistent performance across subsampling replicates as compared to ASTRAL. In contrast, analyses of concatenated data supermatrices recover rheas as the sister to all other nonostrich palaeognaths, an alternative that lacks retroelement support and shows inconsistent behavior under subsampling approaches. While statistically supporting the species tree topology, conflicting patterns of retroelement insertions also occur and imply high amounts of ILS across short successive internal branches, consistent with observed patterns of gene tree heterogeneity. Coalescent simulations and topology tests indicate that the majority of observed topological incongruence among gene trees is consistent with coalescent variation rather than arising from gene tree estimation error alone, and estimated branch lengths for short successive internodes in the inferred species tree fall within the theoretical range encompassing the anomaly zone. Distributions of empirical gene trees confirm that the most common gene tree topology for each marker type differs from the species tree, signifying the existence of an empirical anomaly zone in palaeognaths.

Corticospinal Tract Injury Estimated From Acute Stroke Imaging Predicts Upper Extremity Motor Recovery After Stroke.

Background and Purpose- Injury to the corticospinal tract (CST) has been shown to have a major effect on upper extremity motor recovery after stroke. This study aimed to examine how well CST injury, measured from neuroimaging acquired during the acute stroke workup, predicts upper extremity motor recovery. Methods- Patients with upper extremity weakness after ischemic stroke were assessed using the upper extremity Fugl-Meyer during the acute stroke hospitalization and again at 3-month follow-up. CST injury was quantified and compared, using 4 different methods, from images obtained as part of the stroke standard-of-care workup. Logistic and linear regression were performed using CST injury to predict ΔFugl-Meyer. Injury to primary motor and premotor cortices were included as potential modifiers of the effect of CST injury on recovery. Results- N=48 patients were enrolled 4.2±2.7 days poststroke and completed 3-month follow-up (median 90-day modified Rankin Scale score, 3; interquartile range, 1.5). CST injury distinguished patients who reached their recovery potential (as predicted from initial impairment) from those who did not, with area under the curve values ranging from 0.70 to 0.8. In addition, CST injury explained ≈20% of the variance in the magnitude of upper extremity recovery, even after controlling for the severity of initial impairment. Results were consistent when comparing 4 different methods of measuring CST injury. Extent of injury to primary motor and premotor cortices did not significantly influence the predictive value that CST injury had for recovery. Conclusions- Structural injury to the CST, as estimated from standard-of-care imaging available during the acute stroke hospitalization, is a robust way to distinguish patients who achieve their predicted recovery potential and explains a significant amount of the variance in poststroke upper extremity motor recovery.

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics.

Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and ∼3600-∼3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine + falcon clade, was resolved differently and with moderate (>70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis.

Genomic support for a moa-tinamou clade and adaptive morphological convergence in flightless ratites.

One of the most startling discoveries in avian molecular phylogenetics is that the volant tinamous are embedded in the flightless ratites, but this topology remains controversial because recent morphological phylogenies place tinamous as the closest relative of a monophyletic ratite clade. Here, we integrate new phylogenomic sequences from 1,448 nuclear DNA loci totaling almost 1 million bp from the extinct little bush moa, Chilean tinamou, and emu with available sequences from ostrich, elegant crested tinamou, four neognaths, and the green anole. Phylogenetic analysis using standard homogeneous models and heterogeneous models robust to common topological artifacts recovered compelling support for ratite paraphyly with the little bush moa closest to tinamous within ratites. Ratite paraphyly was further corroborated by eight independent CR1 retroposon insertions. Analysis of morphological characters reinterpreted on a 27-gene paleognath topology indicates that many characters are convergent in the ratites, probably as the result of adaptation to a cursorial life style.

Convergent evolution of parrot plumage coloration.

Parrots have remarkable plumage coloration that result in part from a unique ability to produce pigments called psittacofulvins that yield yellow to red feather colors. Little is known about the evolution of psittacofulvin-based pigmentation. Widespread color mutations of captive-bred parrots provide perfect opportunities to study the genetic basis of this trait. An earlier study on blue budgerigars, which do not possess psittacofulvins, reveals the involvement of an uncharacterized polyketide synthase (MuPKS) in yellow psittacofulvin synthesis. The blue phenotype had repeatedly appeared in different parrot species, similar to independent experimental replications allowing the study of convergent evolution and molecular mechanism of psittacofulvin-based pigmentation. Here, we investigated the genetic basis of the blue phenotypes in two species of Agapornis parrots, Fischer's lovebird (A. fischeri) and Yellow-collared lovebird (A. personatus). Using whole-genome data, we identified a single genomic region with size <2 Mb to be strongly associated with the color difference between blue and wild-type (WT) birds in both species. Surprisingly, we discovered that the mutation associated with the blue Agapornis phenotype was identical to the previously described substitution causing the functional change of MuPKS in budgerigars. Together with the evidence of shared blue-associated haplotypes and signatures of a selective sweep in this genomic region in both species, we demonstrated both de novo mutation and interspecific introgression play a role in the evolution of this trait in different Agapornis species. The convergent substitution in the same gene in both lovebirds and budgerigars also indicates a strong evolutionary constraint on psittacofulvin-based coloration.

A nuclear genome assembly of an extinct flightless bird, the little bush moa.

We present a draft genome of the little bush moa (Anomalopteryx didiformis)-one of approximately nine species of extinct flightless birds from Aotearoa, New Zealand-using ancient DNA recovered from a fossil bone from the South Island. We recover a complete mitochondrial genome at 249.9× depth of coverage and almost 900 megabases of a male moa nuclear genome at ~4 to 5× coverage, with sequence contiguity sufficient to identify more than 85% of avian universal single-copy orthologs. We describe a diverse landscape of transposable elements and satellite repeats, estimate a long-term effective population size of ~240,000, identify a diverse suite of olfactory receptor genes and an opsin repertoire with sensitivity in the ultraviolet range, show that the wingless moa phenotype is likely not attributable to gene loss or pseudogenization, and identify potential function-altering coding sequence variants in moa that could be synthesized for future functional assays. This genomic resource should support further studies of avian evolution and morphological divergence.

Olfactory receptor subgenome and expression in a highly olfactory procellariiform seabird.

Procellariiform seabirds rely on their sense of smell for foraging and homing. Both genomes and transcriptomes yield important clues about how olfactory receptor (OR) subgenomes are shaped by natural and sexual selection, yet no transcriptomes have been made of any olfactory epithelium of any bird species thus far. Here, we assembled a high-quality genome and nasal epithelium transcriptome of the Leach's storm-petrel (Oceanodroma leucorhoa) to extensively characterize their OR repertoire. Using a depth-of-coverage-assisted counting method, we estimated over 160 intact OR genes (∼500 including OR fragments). This method reveals the highest number of intact OR genes and the lowest proportion of pseudogenes compared to other waterbirds studied, and suggests that rates of OR gene duplication vary between major clades of birds, with particularly high rates in passerines. OR expression patterns reveal two OR genes (OR6-6 and OR5-11) highly expressed in adults, and four OR genes (OR14-14, OR14-12, OR10-2, and OR14-9) differentially expressed between age classes of storm-petrels. All four genes differentially expressed between age classes were more highly expressed in chicks compared to adults, suggesting that OR genes may exhibit ontogenetic specializations. Three highly differentially expressed OR genes also had high copy number ratios, suggesting that expression variation may be linked to copy number in the genome. We provide better estimates of OR gene number by using a copy number-assisted counting method, and document ontogenetic changes in OR gene expression that may be linked to olfactory specialization. These results provide valuable insight into the expression, development, and macroevolution of olfaction in seabirds.

Characterization and locus-specific typing of MHC class I genes in the red-billed gull (Larus scopulinus) provides evidence for major, minor, and nonclassical loci.

A major challenge facing studies of major histocompatibility complex (MHC) evolution in birds is the difficulty in genotyping alleles at individual loci, and the consequent inability to investigate sequence variation and selection pressures for each gene. In this study, four MHC class I loci were isolated from the red-billed gull (Larus scopulinus), representing both the first characterized MHCI genes within Charadriiformes (shorebirds, gulls, and allies) and the first full-length MHCI sequences described outside Galloanserae (gamebirds + waterfowl). Complete multilocus genotypes were obtained for 470 individuals using a combination of reference-strand conformation analysis and direct sequencing of gene-specific amplification products, and variation of peptide-binding region (PBR) exons was surveyed for all loci. Each gene is transcribed and has conserved sequence features characteristic of antigen-presenting MHCI molecules. However, higher allelic variation, a more even allele frequency distribution, and evidence of positive selection acting on a larger number of PBR residues suggest that only one locus (Lasc-UAA) functions as a major classical MHCI gene. Lasc-UBA, with more limited variation and PBR motifs that encompass a subset of Lasc-UAA diversity, was assigned a putative minor classical function, whereas the divergent and largely invariant binding-groove motifs of Lasc-UCA and -UDA are suggestive of nonclassical loci with specialized ligand-binding roles.

Convergent regulatory evolution and loss of flight in paleognathous birds.

A core question in evolutionary biology is whether convergent phenotypic evolution is driven by convergent molecular changes in proteins or regulatory regions. We combined phylogenomic, developmental, and epigenomic analysis of 11 new genomes of paleognathous birds, including an extinct moa, to show that convergent evolution of regulatory regions, more so than protein-coding genes, is prevalent among developmental pathways associated with independent losses of flight. A Bayesian analysis of 284,001 conserved noncoding elements, 60,665 of which are corroborated as enhancers by open chromatin states during development, identified 2355 independent accelerations along lineages of flightless paleognaths, with functional consequences for driving gene expression in the developing forelimb. Our results suggest that the genomic landscape associated with morphological convergence in ratites has a substantial shared regulatory component.

Interdisciplinary Stroke Recovery Research: The Perspective of Occupational Therapists in Acute Care.

As acute stroke treatments advance, more people survive the initial stroke event and live with long-term neurological impairments that impact functional outcomes and quality of life. In accordance with International Classification of Functioning (ICF), living with long-term neurological impairments can limit survivors' activity performance and restrict participation in valued life roles and routines. Research focused on longitudinal analysis of functional measures and outcomes after stroke are critical for determining early indicators of long-term participation and quality of life and guiding rehabilitation resource allocation. As core members of the interdisciplinary stroke recovery treatment team throughout the post-acute care continuum, occupational therapists (OTs) directly address stroke survivors' ability to participate in meaningful daily activities to promote function and quality of life. Just as in clinical care in which multidisciplinary, team-based perspectives are vital, OTs provide invaluable perspectives for stroke recovery research. Here we describe OTs' role in a collaborative, interdisciplinary research study aimed at comprehensively understanding upper extremity motor recovery after stroke and its impact on individuals across the post-acute care continuum. This article discusses the importance of the OTs' perspectives in conducting interdisciplinary, longitudinal stroke recovery research. The challenges, strategies and recommendations for future directions of advancing the role of OTs in multidisciplinary stroke recovery research are highlighted. We use this perspective as a call to action to the stroke recovery field to incorporate OTs as members of the research team and for OTs to provide their perspectives on ongoing stroke recovery research.

Pervasive Correlated Evolution in Gene Expression Shapes Cell and Tissue Type Transcriptomes.

The evolution and diversification of cell types is a key means by which animal complexity evolves. Recently, hierarchical clustering and phylogenetic methods have been applied to RNA-seq data to infer cell type evolutionary history and homology. A major challenge for interpreting this data is that cell type transcriptomes may not evolve independently due to correlated changes in gene expression. This nonindependence can arise for several reasons, such as common regulatory sequences for genes expressed in multiple tissues, that is, pleiotropic effects of mutations. We develop a model to estimate the level of correlated transcriptome evolution (LCE) and apply it to different data sets. The results reveal pervasive correlated transcriptome evolution among different cell and tissue types. In general, tissues related by morphology or developmental lineage exhibit higher LCE than more distantly related tissues. Analyzing new data collected from bird skin appendages suggests that LCE decreases with the phylogenetic age of tissues compared, with recently evolved tissues exhibiting the highest LCE. Furthermore, we show correlated evolution can alter patterns of hierarchical clustering, causing different tissue types from the same species to cluster together. To identify genes that most strongly contribute to the correlated evolution signal, we performed a gene-wise estimation of LCE on a data set with ten species. Removing genes with high LCE allows for accurate reconstruction of evolutionary relationships among tissue types. Our study provides a statistical method to measure and account for correlated gene expression evolution when interpreting comparative transcriptome data.

A set of BAC clones spanning the human genome.

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.

Molecular sexing of prey remains permits a test of sex-biased predation in a wintering population of western sandpipers.

Population sex ratios in monogamous birds are often male biased. One factor that can affect population sex ratios is sex-biased predation. However, most estimates of sex-biased predation in birds have focused on species with obvious sexual colour dimorphism or body size dimorphism. Data on sexually monomorphic birds are generally lacking. In the present study, we adopt a PCR-based sexing procedure to help test for sex-biased predation in a wintering population of western sandpipers (Calidris mauri), a shorebird that shows only subtle sexual size dimorphism. Specifically, by comparing the a priori determined sex ratio of live birds wintering at a site in western Mexico to the molecular estimate obtained from depredated birds at this same site, we were able to perform a population-specific test for sex bias in predator-induced mortality. The proportion of females estimated from living (ca. 25%) versus dead (ca. 24%) individuals was in fact not significantly different, indicating that the strong male bias in this population is not due to differential predation. However, molecular sexing of prey remains is a hitherto unexploited test of sex-biased predation in birds, and is potentially applicable to any species for which prey remains can be gathered. We discuss our results in the context of alternate ecological hypotheses for population sex biases.

Partial reductions in mechanical loading yield proportional changes in bone density, bone architecture, and muscle mass.

Although the musculoskeletal system is known to be sensitive to changes in its mechanical environment, the relationship between functional adaptation and below-normal mechanical stimuli is not well defined. We investigated bone and muscle adaptation to a range of reduced loading using the partial weight suspension (PWS) system, in which a two-point harness is used to offload a tunable amount of body weight while maintaining quadrupedal locomotion. Skeletally mature female C57Bl/6 mice were exposed to partial weight bearing at 20%, 40%, 70%, or 100% of body weight for 21 days. A hindlimb unloaded (HLU) group was included for comparison in addition to age-matched controls in normal housing. Gait kinematics was measured across the full range of weight bearing, and some minor alterations in gait from PWS were identified. With PWS, bone and muscle changes were generally proportional to the degree of unloading. Specifically, total body and hindlimb bone mineral density, calf muscle mass, trabecular bone volume of the distal femur, and cortical area of the femur midshaft were all linearly related to the degree of unloading. Even a load reduction to 70% of normal weight bearing was associated with significant bone deterioration and muscle atrophy. Weight bearing at 20% did not lead to better bone outcomes than HLU despite less muscle atrophy and presumably greater mechanical stimulus, requiring further investigation. These data confirm that the PWS model is highly effective in applying controllable, reduced, long-term loading that produces predictable, discrete adaptive changes in muscle and bone of the hindlimb.

Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading.

Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11-17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased -9.2% ± 1.0% in HLU-VEH, whereas it increased 4.2% ± 0.7%, 13.1% ± 1.0%, and 30.6% ± 3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p < 0.0001). Trabecular bone volume, assessed by micro-computed tomography (µCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p < 0.05), and was 2- to 3-fold higher in SclAbII groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by three-point bending, and distal femoral strength, assessed by micro-finite element analysis (µFEA), were significantly higher in SclAbII versus VEH-groups in both loading conditions. Serum sclerostin was higher in HLU-VEH (134 ± 5 pg/mL) compared to CON-VEH (116 ± 6 pg/mL, p < 0.05). Serum osteocalcin was decreased by hindlimb suspension and increased by SclAbII treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.

Caloric restriction leads to high marrow adiposity and low bone mass in growing mice.

The effects of caloric restriction (CR) on the skeleton are well studied in adult rodents and include lower cortical bone mass but higher trabecular bone volume. Much less is known about how CR affects bone mass in young, rapidly growing animals. This is an important problem because low caloric intake during skeletal acquisition in humans, as in anorexia nervosa, is associated with low bone mass, increased fracture risk, and osteoporosis in adulthood. To explore this question, we tested the effect of caloric restriction on bone mass and microarchitecture during rapid skeletal growth in young mice. At 3 weeks of age, we weaned male C57Bl/6J mice onto 30% caloric restriction (10% kcal/fat) or normal diet (10% kcal/fat). Outcomes at 6 (n = 4/group) and 12 weeks of age (n = 8/group) included body mass, femur length, serum leptin and insulin-like growth factor 1 (IGF-1) values, whole-body bone mineral density (WBBMD, g/cm(2)), cortical and trabecular bone architecture at the midshaft and distal femur, bone formation and cellularity, and marrow fat measurement. Compared with the normal diet, CR mice had 52% and 88% lower serum leptin and 33% and 39% lower serum IGF-1 at 6 and 12 weeks of age (p < .05 for all). CR mice were smaller, with lower bone mineral density, trabecular, and cortical bone properties. Bone-formation indices were lower, whereas bone-resorption indices were higher (p < .01 for all) in CR versus normal diet mice. Despite having lower percent of body fat, bone marrow adiposity was elevated dramatically in CR versus normal diet mice (p < .05). Thus we conclude that caloric restriction in young, growing mice is associated with impaired skeletal acquisition, low leptin and IGF-1 levels, and high marrow adiposity. These results support the hypothesis that caloric restriction during rapid skeletal growth is deleterious to cortical and trabecular bone mass and architecture, in contrast to potential skeletal benefits of CR in aging animals.

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.