Recovery from hybrid breakdown in a marine invertebrate is faster, stronger and more repeatable under environmental stress.

Understanding how environmental stress alters the consequences of hybridization is important, because the rate of hybridization and the likelihood of hybrid speciation both appear elevated in harsh, disturbed or marginal habitats. We assessed fitness, morphometrics and molecular genetic composition over 14 generations of hybridization between two highly divergent populations of the marine copepod Tigriopus californicus. Replicated, experimental hybrid populations in both control and high-salinity conditions showed a decline in fitness, followed by a recovery. Recovery was faster in the salinity stress treatment, returning to parental levels up to two generations earlier than in the control. This recovery was stable in the high-salinity treatment, whereas in the control treatment, fitness dropped back below parental levels at the final time point. Recovery in the high-salinity treatment was also stronger in terms of competitive fitness and heat-shock tolerance. Finally, consequences of hybridization were more repeatable under salinity stress, where among-replicate variance for survivorship and molecular genetic composition was lower than in the control treatment. In a system with low effective population sizes (estimates ranged from 17 to 63), where genetic drift might be expected to be the predominate force, strong selection under harsh environmental conditions apparently promoted faster, stronger and more repeatable recovery from depressed hybrid fitness.

Sex without sex chromosomes: genetic architecture of multiple loci independently segregating to determine sex ratios in the copepod Tigriopus californicus.

Sex-determining systems are remarkably diverse and may evolve rapidly. Polygenic sex-determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male- and female-biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus.

Postzygotic isolation involves strong mitochondrial and sex-specific effects in Tigriopus californicus, a species lacking heteromorphic sex chromosomes.

Detailed studies of the genetics of speciation have focused on a few model systems, particularly Drosophila. The copepod Tigriopus californicus offers an alternative that differs from standard animal models in that it lacks heteromorphic chromosomes (instead, sex determination is polygenic) and has reduced opportunities for sexual conflict, because females mate only once. Quantitative trait loci (QTL) mapping was conducted on reciprocal F2 hybrids between two strongly differentiated populations, using a saturated linkage map spanning all 12 autosomes and the mitochondrion. By comparing sexes, a possible sex ratio distorter was found but no sex chromosomes. Although studies of standard models often find an excess of hybrid male sterility factors, we found no QTL for sterility and multiple QTL for hybrid viability (indicated by non-Mendelian adult ratios) and other characters. Viability problems were found to be stronger in males, but the usual explanations for weaker hybrid males (sex chromosomes, sensitivity of spermatogenesis, sexual selection) cannot fully account for these male viability problems. Instead, higher metabolic rates may amplify deleterious effects in males. Although many studies of standard speciation models find the strongest genetic incompatibilities to be nuclear-nuclear (specifically X chromosome-autosome), we found the strongest deleterious interaction in this system was mito-nuclear. Consistent with the snowball theory of incompatibility accumulation, we found that trigenic interactions in this highly divergent cross were substantially more frequent (>6×) than digenic interactions. This alternative system thus allows important comparisons to studies of the genetics of reproductive isolation in more standard model systems.

Fitness and morphological outcomes of many generations of hybridization in the copepod Tigriopus californicus.

Hybridization between genetically divergent populations is an important evolutionary process, with an outcome that is difficult to predict. We used controlled crosses and freely mating hybrid swarms, followed for up to 30 generations, to examine the morphological and fitness consequences of interpopulation hybridization in the copepod Tigriopus californicus. Patterns of fitness in two generations of controlled crosses were partly predictive of long-term trajectories in hybrid swarms. For one pair of populations, controlled crosses revealed neutral or beneficial effects of hybridization after the F1 generation, and hybrid swarm fitness almost always equalled or exceeded that of the midparent. For a second pair, controlled crosses showed F2 hybrid breakdown, but increased fitness in backcrosses, and hybrid swarm fitness deviated both above and below that of the parentals. Nevertheless, individual swarm replicates exhibited different fitness trajectories over time that were not related in a simple manner to their hybrid genetic composition, and fixation of fitter hybrid phenotypes was not observed. Hybridization did not increase overall morphological variation, and underlying genetic changes may have been masked by phenotypic plasticity. Nevertheless, one type of hybrid swarm exhibited a repeatable pattern of transgressively large eggsacs, indicating a positive effect of hybridization on individual fecundity. Additionally, both parental and hybrid swarms exhibited common phenotypic trends over time, indicating common selective pressures in the laboratory environment. Our results suggest that, in a system where much work has focused on F2 hybrid breakdown, the long-term fitness consequences of interpopulation hybridization are surprisingly benign.

Isolation and characterization of 10 polymorphic microsatellite markers from striped marlin, Tetrapturus audax.

We present the isolation and characterization of 10 microsatellite loci for striped marlin, Tetrapturus audax. Thirty individuals from each of four locations revealed that all loci were polymorphic with two to 31 alleles per locus. Observed levels of heterozygosity ranged from 0.3000 to 0.9667. Significant deviations from Hardy-Weinberg equilibrium were detected in two loci, TA105 in Hawaii and New Zealand and TA155 in Hawaii, and null alleles may be present in loci TA105 and TA155 in those locations, and in locus TA193 in Mexico. No significant linkage disequilibrium was detected in any pairwise-locus comparison.

Detection of mitochondrial DNA from domestic cattle in bison on Santa Catalina Island.

In 1924, 14 American bison (Bison bison) were introduced to Santa Catalina Island, California and sporadically supplemented thereafter with additional animals. To reduce the herd and its impact on native vegetation, over 2000 animals have been exported during the past four decades. Today, the herd is estimated to contain around 250 individuals. Genetic analysis was performed on 98 animals removed from the island in 2004. Forty-four samples (45%) had domestic cattle mitochondrial DNA (mtDNA), 12 (12%) had previously reported bison haplotypes and 42 (43%) had a new haplotype differing by one base pair from a previously reported bison haplotype. A complement of five restriction enzymes was found to be useful in identifying bison with domestic cattle mtDNA.

Chromosomal basis of viability differences in Tigriopus californicus interpopulation hybrids.

Crosses between populations of Tigriopus californicus result in backcross and F2 hybrid breakdown for a variety of fitness related measures. The magnitude of this hybrid breakdown is correlated with evolutionary divergence. We assessed the chromosomal basis of viability differences in nonrecombinant backcross hybrids using markers mapped to individual chromosomes. To assess effects of evolutionary divergence we crossed one population to three different populations: two distantly related (approximately 18% mitochondrial COI sequence divergence) and one closely related (approximately 1% mitochondrial COI sequence divergence). We found that all three interpopulation crosses resulted in significant deviations from expected Mendelian ratios at a majority of the loci studied. In all but one case, deviations were due to a deficit of parental homozygotes. This pattern implies that populations of T. californicus carry a significant genetic load, and that a combination of beneficial dominance and deleterious homozygote-heterozygote interactions significantly affects hybrid viability. Pairwise tests of linkage disequilibrium detected relatively few significant interactions. For the two divergent crosses, effects of individual chromosomes were highly concordant. These two crosses also showed higher heterozygote excess in females than males across the vast majority of chromosomes.

Genetic consequences of many generations of hybridization between divergent copepod populations.

Crosses between populations of the copepod Tigriopus californicus typically result in outbreeding depression. In this study, replicate hybrid populations were initiated with first generation backcross hybrids between two genetically distinct populations from California: Royal Palms (RP) and San Diego (SD). Reciprocal F(1) were backcrossed to SD, resulting in expected starting frequencies of 25% RP/75% SD nuclear genes on either a pure RP cytoplasmic or a pure SD cytoplasmic background. After 1 year of hybridization (up to 15 generations), seven microsatellite loci were scored in two replicates on each cytoplasmic background. Frequencies of the rarer RP alleles increased significantly in all four replicates, regardless of cytoplasmic source, producing a mean hybridity of 0.97 (maximum = 1), instead of the expected 0.50. Explicit tests for heterozygote excess across loci and replicates showed significant deviations. Only the two physically linked markers showed linkage disequilibrium in all replicates. Subsequent fitness assays in parental populations and early generation hybrids revealed lower fitness in RP than SD, and significant F(2) breakdown. Computer simulations showed that selection must be invoked to explain the shift in allele frequencies. Together, these results suggest that hybrid inferiority in early generations gave way to hybrid superiority in later generations.

The significance of bcr-abl molecular detection in chronic myeloid leukemia patients "late," 18 months or more after transplantation.

The bcr-abl chimeric messenger RNA is frequently detected in chronic myeloid leukemia (CML) patients after bone marrow transplantation. It was previously reported that the relapse risk of bcr-abl detection 6 to 12 months after transplantation was greater than 40%. This risk decreased as the time between transplantation and detection increased. To further define the relapse risk associated with bcr-abl molecular detection in "late" CML survivors, 379 consecutive CML patients alive at 18 months after transplantation or later were studied. Ninety of 379 patients (24%) had at least one positive bcr-abl test 18 months after transplantation or later; 13 of 90 bcr-abl-positive patients (14%) and 3 of 289 bcr-abl-negative patients (1.0%) relapsed. The median time from bcr-abl detection to relapse was 916 days (range, 251-2654 days). The hazard ratio of relapse associated with bcr-abl detection was 19.2 (P <.0001). The stage of disease, chronic graft-versus-host disease, and the donor type did not alter the association between bcr-abl and relapse. Quantification of bcr-abl was performed on 344 samples from 85 bcr-abl-positive patients by means of a real-time quantitative reverse transcriptase-polymerase chain reaction assay. The median bcr-abl change of patients who relapsed was significantly greater than those that remained in remission (P =.002). The median bcr-abl level at relapse was 40 443 bcr-abl copies per microg RNA (range, 960-299 552). Of 73 bcr-abl-positive patients who failed to relapse, 69% had only one positive test at a median of 24 copies bcr-abl per microg RNA. The detection of bcr-abl is common following transplantation. The prognostic significance of a qualitative bcr-abl can be refined by quantitative assays and thus may target patients who would benefit from early intervention.

Phylogeography of the intertidal copepod Tigriopus californicus reveals substantially reduced population differentiation at northern latitudes.

Previous studies of the intertidal copepod Tigriopus californicus revealed one of the highest levels of mitochondrial DNA differentiation ever reported among conspecific populations. The present study extends the geographical sampling northward, adding populations from northern California to south-east Alaska. The mitochondrial phylogeny for the entire species range, based on cytochrome oxidase I sequences for a total of 49 individuals from 27 populations, again shows extreme differentiation among populations (up to 23%). However, populations from Oregon northwards appear to be derived and have interpopulation divergences five times lower than those between southern populations. Furthermore, although few individuals were sequenced from each locality, populations from Puget Sound northward had significantly reduced levels of within-population variation. These patterns are hypothesized to result from the contraction and expansion of populations driven by recent ice ages.

Detection of bcr-abl transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia after marrow transplantation.

Thirty-six patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) were studied for the presence of the bcr-abl fusion mRNA transcript after an allogeneic matched related (N = 12), partially matched related (N = 4), matched unrelated (N = 14), autologous (N = 5), or syngeneic (N = 1) bone marrow transplant (BMT). Seventeen were transplanted in relapse, and 19 were transplanted in remission. Twenty-three patients had at least one positive bcr-abl polymerase chain reaction (PCR) assay after BMT either before a relapse or without subsequent relapse. Ten of these 23 relapsed after a positive assay at a median time from first positive PCR assay of 94 days (range, 28 to 416 days). By comparison, only 2 relapses occurred in the 13 patients with no prior positive PCR assays; both patients had missed at least one scheduled follow-up assay and were not tested 2 months and 26 months before their relapse. The unadjusted relative risk (RR) of relapse associated with a positive PCR assay compared with a negative assay was 5.7 (95% confidence interval 1.2 to 26.0, P = .025). In addition, the data suggest that the type of bcr-abl chimeric mRNA detected posttransplant was associated with the risk of relapse: 7 of 10 patients expressing the p190 bcr-abl relapsed, compared with 1 of 8 who expressed only the p210 bcr-abl mRNA (P = .02, log-rank test). The RR of p190 bcr-abl positivity compared to PCR-negative patients was 11.2 (confidence interval 2.3-54.8, P = 0.003), whereas a positive test for p210 bcr-abl was apparently not associated with an increased relative risk. In separate multivariable models, PCR positivity remained a statistically significant risk factor for relapse after separately adjusting for donor (unrelated and partially matched v matched, autologous, and syngeneic), remission status at the time of transplant, the presence of acute graft-versus-host disease (GVHD), and type of conditioning regimen (total body irradiation dose of < or = 1,200 cGy v > 1,200 cGy). The PCR assay appears to be a useful test for predicting patients at high risk of relapse after BMT and may identify patients who might benefit from therapeutic interventions. The finding that the expression of p190 bcr-abl may portend an especially high risk of relapse suggests a different clinical and biologic behavior between p190 and p210 bcr-abl.

Unusual expression of mRNA typical of Philadelphia positive acute lymphoblastic leukemia detected in chronic myeloid leukemia.

The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of "p190" CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia.

Time-dependent aspects of osmolyte changes in rat kidney, urine, blood and lens with sorbinil and galactose feeding.

Sorbitol plus myo-inositol, betaine and glycerophosphorylcholine (GPC) are cellular osmolytes in the mammalian renal medulla. Galactosemia and hyperglycemia can cause excessive levels of galactitol or sorbitol in several organs via aldose reductase (AR) catalysis. AR inhibitors can reduce these polyols. To examine osmolyte responses to polyol perturbations, male Wistar rats were fed normal diet, the AR inhibitor sorbinil (at 40 mg/kg/d), 25% galactose, or a combination, for 10, 21 and 42 days. All animals at 21 days had higher apparent renal AR activity than at 10 or 42 days, possibly providing resistance to sorbinil. Sorbinil feeding alone tended to increase urinary, plasma and renal urea levels. It reduced AR activity and sorbitol contents in renal inner medulla, though less so at 21 days; other renal osmolytes, especially betaine, were elevated. Galactose feeding caused little change in renal AR activity, and resulted in high galactose and galactitol contents in renal medulla, urine, blood and lens (and higher renal Na+ contents at 10 days). Renal sorbitol, inositol and GPC decreased, while betaine contents trended higher at all times. Sorbinilgalactose feeding reduced renal AR activities and galactitol contents (again less so at 21 days), urine, blood and lens galactitol, and further reduced renal sorbitol contents. At 10 and 21 days it tended to raise renal betaine more, and restore inositol (but not GPC) contents to control levels. At 42 days it reduced renal and urinary Na+ and galactose, and decreased renal betaine to control levels. Under most conditions, total renal (non-urea) organic osmolyte contents (presumed to be mostly intracellular) and Na+ plus galactose contents (presumed mostly extracellular) changed together such that cell volumes may have been maintained. The exception was 10 days on galactose, where total osmolytes appeared too low. In galactose-fed animals, urine/plasma ratios suggest some renal galactitol efflux, and cellular galactitol probably helps maintain osmotic balance rather than cause swelling.

Benign marrow progenitors are enriched in the CD34+/HLA-DRlo population but not in the CD34+/CD38lo population in chronic myeloid leukemia: an analysis using interphase fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was used to discriminate between benign and malignant cells in sorted populations of chronic myelogenous leukemia (CML) marrow. FISH has the advantage of allowing for a cell by cell analysis of the breakpoint cluster region (BCR) gene rearrangement immediately after flow sorting in nondividing G0/G1 cells that are potentially transcriptionally inactive. We initially selected CD34+ cells with very low expression of the activation antigen CD38 as a candidate phenotype for an immature and hypothetically more benign cell population, but found no enrichment for Ph negativity in that subtype. In five CML samples, 55% +/- 3.3% (mean +/- SE) of CD34+/CD38hi cells had the BCR gene rearrangement, similar to 57% +/- 3.7% seen in the CD34+/CD38lo population. In contrast, subsequent experiments (n = 4) determined that the CD34+/HLA-DRlo population in CML marrow does contain an increased proportion of benign cells: 15% +/- 1% of the CD34+/DRlo cells were BCR rearranged, compared with 52% +/- 5.8% of the CD34+/DRhi cells (P = .001). Our results indicate that benign progenitors in CML are enriched within the CD34+ cells with low DR antigen expression, but not low CD38 expression. One possible interpretation of these observations is that low CD38 antigen expression is not as useful as low HLA-DR expression for isolating immature cells.

Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients.

We studied 346 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML) for the presence of the bcr-abl transcript detected by the polymerase chain reaction (PCR) to understand the frequency and implication of a positive test. A total of 634 samples of BM and/or peripheral blood were obtained for PCR analysis between 3 and 192 months after BMT. A positive PCR test at 3 months post-BMT was not statistically significantly associated with an increased risk of relapse compared with PCR-negative patients. However, a positive PCR assay at 6 months and beyond was highly associated with subsequent relapse. The Kaplan-Meier estimate of relapse for patients testing PCR-positive at 6 to 12 months was 42% versus 3% for PCR-negative patients (P < .0001). The Kaplan-Meier estimate of survival at 4 years for the PCR-positive patients was 74% compared with 83% for the PCR-negative group (P = .002). Multivariable analysis indicated that a PCR-positive result at 6 to 12 months post-BMT, the type of BMT donor (allogeneic matched donor v mismatched or unrelated), and the presence of acute GVHD were independent risk factors for subsequent relapse. The relative risk (RR) for relapse for patients PCR-positive at 6 to 12 months post-BMT was 26.1 (95% confidence interval, 8.9 to 76.1, P < .0001). The outcome of long-term patients (> 36 months post-BMT) who tested PCR-positive was much better, as 15 of 59 (25%) tested positive for bcr-abl, but only one patient relapsed. There was a 91% concordance between PCR tests of simultaneously obtained BM and peripheral blood. These analyses show that the PCR assay of the bcr-abl fusion transcript 6 to 12 months post-BMT is an independent predictor of subsequent relapse which provides an opportunity for early therapeutic intervention.

Multiplex PCR of bcr-abl fusion transcripts in Philadelphia positive acute lymphoblastic leukemia.

We describe a multiplex PCR assay for the detection of bcr-abl fusion mRNA in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL). The assay provides a quick method for screening p190 (e1:a2) and p210 (b2:a2 or b3:a2) bcr-abl mRNAs simultaneously. The assay proves to be highly sensitive with detection of as little as one positive bcr-abl-expressing cell in a background of 10(5) negative bcr-abl cells. Bone marrow and peripheral blood specimens from six patients were in total accordance when run by multiplex PCR and by the single primer PCR approach. The multiplex bcr-abl assay may prove to be highly useful for screening newly diagnosed patients with ALL for the bcr-abl fusion transcript and in following the course of disease during therapy.

Rapid RT-PCR amplification from limited cell numbers.

We describe a rapid and efficient RT-PCR method particularly suited to procedures involving limited cell and target gene copy numbers. Purified leukocytes and myeloid colonies derived from patients with chronic myelogenous leukemia (CML) in chronic phase were used for direct RT-PCR. Purified cells and colonies were lysed using a small quantity of DEPC-treated water containing RNasin as an RNA inhibitor. The untreated lysate was either used immediately for RT-PCR or frozen at -70 degrees C for later use. By this method we were able to consistently amplify bcr-abl transcripts from as few as 10 cells. No noticeable difference was observed between products amplified from fresh and frozen samples.

Effects on rat renal osmolytes of extended treatment with an aldose reductase inhibitor.

1. The mammalian renal medulla uses sorbitol, myo-inositol, betaine and glycerophosphorylcholine as intracellular osmolytes. 2. Sorbitol synthesis was inhibited by feeding male Wistar rats the aldose reductase inhibitor sorbinil at 40 mg/kg/day for 71 d, and renal inner medullas were extracted for analysis. 3. Aldose reductase activities and sorbitol contents were greatly reduced in sorbinil-treated animals, while betaine contents increased significantly (with no other osmolytes changing). 4. The betaine increase compensated for the sorbitol decrease such that the total organic osmolytes maintained the same ratio to sodium contents as controls. 5. These results are identical to the pattern previously reported for sorbinil treatment of rats for 10 d, but not for 21 d.