Populations of the "Louisiana iris" species Iris fulva, I. hexagona, and I. nelsonii were examined genetically to test for interspecific gene flow between I. fulva and I. hexagona, for pollen- versus seed-mediated introgression between these species, and for the presumed hybrid origin of I. nelsonii. Genetic markers were identified by using both a polymerase chain reaction-like method that allows the identification of random, nuclear markers and standard polymerase chain reaction experiments involving specific chloroplast DNA (cpDNA) oligonucleotides. Restriction endonuclease digestions of the cpDNA amplification products resolved diagnostic restriction site differences for I. fulva and I. hexagona. The distribution of the species-specific nuclear markers supports a hypothesis of bidirectional introgression between I. fulva and I. hexagona. Thus, individuals analyzed from a contemporary hybrid population demonstrate multilocus genotypes that are indicative of advanced-generation hybrid individuals. Furthermore, several markers from the alternate species were present in low frequency in one allopatric population each of I. fulva and I. hexagona. Data from the nuclear analysis also support the hypothesized hybrid origin of I. nelsonii from the interaction of I. fulva and I. hexagona. Finally, cpDNA data support the hypothesis that the localized and the dispersed introgression are largely due to pollen transfer. In addition to the biological implications, this study demonstrates the power of the polymerase chain reaction methodology for the rapid identification of random and specific genetic markers for testing evolutionary genetic hypotheses.
The source of nutrition of shrimp that form giant aggregations at Mid-Atlantic Ridge hydrothermal vents was explored by a combination of different molecular techniques. These animals have been hypothesized either to graze on the free-living, surface microbial community or to feed off epibiotic bacteria growing on their exoskeleton. Stable isotope compositions of potential food sources, consumer tissues, and gut contents suggest that the shrimp derive most of their carbon and nitrogen from the epibionts. However, probing of nucleic acids extracted from shrimp guts points to the existence of a gut microflora that is isotopically similar but genetically distinct from the epibionts. Carbon dioxide fixation experiments were carried out to compare the magnitude of potential primary productivity of the different nutritional sources of the shrimp. Relatively low and comparable rates were observed for both the free-living community and the epibionts but activity associated with gut preparations was surprisingly high. These high CO2 fixation rates in the gut may be indicative of a nutritional relationship between the shrimp and their gut microflora. Overall, the data indicate a nutritional symbiosis of the shrimp with their epibionts and possibly also with a separate gut microflora.
Invertebrate-chemoautotroph symbioses, in which the bacterial partner provides an internal source of autotrophically fixed carbon for its host, fall into two main groups based on previously reported stable carbon isotope ratios: those with 6l"C = -27 to -35%~1 (referred to as the -3Oo/oo group) and those with V3C = -9 to -16o/oo (referred to as the -11%~ group). The 613C values of the -31X& group resemble those of free-living chemoautotrophic bacteria, suggesting the role of the symbionts in the nutrition of the bivalves; there is presently no satisfactory explanation for the 613C values of the -1 1 ?& group. Immunological and enzyme inhibition data are presented demonstrating that the CO,-fixing enzyme expressed in representatives of the -11% group is a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), while a form I Rubisco is expressed in the -30% symbioses. Previously reported in vitro del.erminations on purified Rubisco show that the form II enzyme from free-living bacteria has a smaller kinletic isotope effect with respect to CO, than does form I. Thus, the 13C-enriched stable carbon isotope signature observed in the intact -1 lo/00 symbioses appears to be at least partially due to this diminished enzymatic discrimination.
It has been hypothesized that the ␦ 13 C values of chemoautotrophs at deep-sea hydrothermal vents, which cluster in two groups around Ϫ11‰ and Ϫ30‰, are due to variation in isotopic discrimination by different forms of RubisCO. The most enriched ␦ 13 C values are from the vestimentiferan tubeworm Riftia pachyptila, whose bacterial endosymbionts provide essentially all of its organic carbon via CO 2 fixation by a form II RubisCO. The kinetic parameters of purified R. pachyptila symbiont RubisCO were determined to assess the degree to which the ␦ 13 C values of tubeworm biomass are due to isotopic fractionation during CO 2 fixation by this enzyme. Like most form II enzymes, the K is high, at 240 mol L Ϫ1 , whereas the CO 2 /O 2 specificity factor (⍀) is low, at 8.6. The CO2 value, which is proportional to the degree of isotopic discrimination by the enzyme, was determined to be 19.5‰, lower than that for form I RubisCO. This low value supports the hypothesis that the degree of isotopic fractionation during CO 2 fixation appears to be an important influence on the ␦ 13C values of R. pachyptila biomass. Our results indicate that it is necessary to consider RubisCO values in interpreting ␦ 13 C values from autotrophs.At deep-sea hydrothermal vents, invertebrates with chemoautotrophic bacterial symbionts provide a substantial por-1 Present address: E&J Gallo Winery,
The doubly labeled water (DLW) technique for measuring CO2 production (rCO2) in free-living animals requires an assessment of the elimination of both 2H and 18O from the body over a long period of time. To calculate rCO2, it is necessary to calculate water turnover (rH2O) from the 2H flux rate. In ruminant animals, the accuracy of this calculation is affected by the loss of 2H in methane. We have quantified the effect of methane production (rCH4) on the 2H flux rate, determined in four sheep given 2H2O. The methane produced was depleted in 2H relative to the urine. A relationship between the enrichment of the methane and urine was established. The ratio of urine to methane enrichment was found on average to be 0.6536, and this value was unaffected by the level of rCH4 but showed some dependence on the absolute concentration of 2H in urine. For this reason, the ratio value obtained from four sheep not given 2H2O was different, a mean of 0.6886 was measured, this ratio was unaffected by changes in the diet supplied to the animals. Computer modeling was used to illustrate the dependence of the isotopically derived value for rCO2 on not only rCH4 but also the magnitude of rCO2 itself. The effect of rCH4 on the DLW method can be predicted from the observed ratio of rCO2 to rCH4 and the value of 0.6536 obtained for the ratio of methane to urine enrichment. With the use of data from several studies at this Institute, a limited range of 10 to 20 was found for rCO2/rCH4 in animals fed at or above maintenance.(ABSTRACT TRUNCATED AT 250 WORDS)
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