in the D. pachea nvd coding region have caused
the loss of NVD activity with cholesterol substrate. These mutations have turned D. pachea into
an obligate specialist dependent on lathosterol, a
compound that has been found in a single plant
species in the Sonoran desert (5, 6).
Remarkably, D. melanogaster nvd RNAi flies
expressing D. pachea nvd survive significantly
better on lathosterol than on cholesterol (t test,
t10,11 = 2.029, P < 0.03) (Fig. 2), but no effect on
survival was detected with nvd RNAi flies expressing D. pachea nvd with the four ancestral
amino acid changes (Fig. 2). This suggests that
the mutations that abolished cholesterol conversion during D. pachea evolution provide a fitness
advantage on lathosterol. The underlying mechanism remains unclear. Our in vitro assay does
not uncover any benefit from the D. pachea nvd
mutations: D. pachea NVD in vitro activity with
lathosterol is not higher compared with other
species (Fig. 3), and the NVD enzymes of related
Drosophila species are already able to convert
lathosterol into 7DHC. To assess population genetic forces at play on the nvd genomic region,
we compared the 3-kb nvd locus and seven genes
on the same 100-kb scaffold with nine control
genes in 34 individuals from a single natural
population. Our analysis reveals that nvd is in a
genomic region of low nucleotide diversity, low
recombination rate, and normal divergence rate
(McDonald-Kreitman test, P > 0.85; maximum
likelihood extension of the Hudson-Kreitman-Aguadé test, P < 10−5) (Fig. 4 and tables S5 to
S11). A signature of a selective sweep is detected
[Kim and Nielsen omega (17)] over nvd and
neighboring loci (Fig. 4), but nucleotide polymorphism is too low to infer whether this recent
selection acted on the nvd mutations themselves.
Tajima’s D and Fu and Li tests are consistent with
recovery from selective sweep in the nvd region
A likely scenario is that D. pachea first evolved
a resistance toward senita cactus toxic compounds
(5) and slowly became restricted to this food
source as it escaped competition with other fly
species. Evolution of D. pachea’s resistance
most likely did not involve NVD because nvd is
not expressed in the midgut and fat body (fig.
S3), the detoxification organs in insects (16). As
lathosterol became D. pachea’s unique source of
sterols for steroid hormone synthesis, mutations
in nvd that abolished NVD activity on cholesterol
appeared and were fixed rapidly due to their
beneficial effect with lathosterol. As a result,
D. pachea became an obligate specialist on the
senita cactus. We point out that besides nvd
mutations, mutation(s) in other genes might also
have contributed to D. pachea dependence on
lathosterol. Alternatively, the identified nvd mu-
tations may have spread while D. pachea an-
cestors were still feeding on various plants and
may thus have accelerated its ecological special-
ization. Our study, which uncovered several mu-
tations underlying the obligate bond between a
specialist species and its host, illustrates how a
few mutations in a single gene can restrict the
ecological niche of a species.
References and Notes
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35, 1 (2008).
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7. J. C. Fogleman, P. B. Danielson, Am. Zool. 41, 877
8. Materials and methods are available as supplementary
materials on Science Online.
9. T. Yoshiyama, T. Namiki, K. Mita, H. Kataoka, R. Niwa,
Development 133, 2565 (2006).
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11. S. Pitnick, W. B. Heed, Ann. Entomol. Soc. Am. 87, 307
12. R. Lafont, C. Dauphin-Villemant, J. T. Warren,
H. H. Rees, in Comprehensive Molecular Insect Science,
L. I. Gilbert, K. Iatrou, S. Gill, Eds. (Oxford, 2005), vol. 3,
13. C. Blais, T. Blasco, A. Maria, C. Dauphin-Villemant,
R. Lafont, J. Chromatogr. B Analyt. Technol. Biomed.
Life Sci. 878, 925 (2010).
14. K. C. Goodnight, H. W. Kircher, Lipids 6, 166 (1971).
15. T. Yoshiyama-Yanagawa et al., J. Biol. Chem. 286, 25756
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Acknowledgments: We thank M. Joron for the linkage
disequilibrium heat map; M.-A. Félix, N. Gompel, and
C. Desplan for comments on the manuscript; C. Parada for
help with field work; T. A. Markow for D. pachea samples;
C. S. Thummel and the San Diego Drosophila Species Stock
Center for flies; Y. Hiromi for reagents; T. Blasco for liquid
chromatography tandem mass spectrometry analyses; and
M. Gho for hosting V.O. in 2009-2010. Supported by CNRS
ATIP-AVENIR (to V.O.), French Foreign Ministry postdoctoral
fellowship (to M.L.), NIH grant AI064950 (to A.G.C.),
Japan-France Bilateral Cooperating Program from the Japan
Society for the Promotion of Science (JSPS) (to H.K. and
C.D.-V.), NSF award DEB-1020009 (to L.M.M.), JSPS
postdoctoral fellowship (to T. Y.-Y.), Special Coordination
Funds for Promoting Science and Technology from the Ministry
of Education, Culture, Sports, Science and Technology in Japan
(to R.N.), and Amylin Endowment (to T.A. Markow). The nvd
sequences were deposited in GenBank under accession nos.
JF764559 to JF764595 and JX066807 to JX067384. The
authors declare no conflict of interest. V.O. conceived the
study and wrote the paper. M.L., C.B., R.L., C.D.-V., L.M.M.,
H.K., and R.N. provided technical support and conceptual
advice for designing the experiments. V.O., M.L., G.G., S.M.,
L.M.M., C.B., E.G., T. Y.-Y., C. D.-V., and R.N. performed the
experiments. A.G.C. did the population genetics analysis.
Materials and Methods
Figs. S1 to S13
Tables S1 to S11
17 May 2012; accepted 13 July 2012
Fermentation, Hydrogen, and Sulfur
Metabolism in Multiple Uncultivated
Kelly C. Wrighton,1 Brian C. Thomas,1 Itai Sharon,1 Christopher S. Miller,1 Cindy J. Castelle,2
Nathan C. VerBerkmoes,3 Michael J. Wilkins,4 Robert L. Hettich,3 Mary S. Lipton,4
Kenneth H. Williams,2 Philip E. Long,2 Jillian F. Banfield1,2*
BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but
their metabolisms remain unclear owing to lack of cultivated representatives and minimal
genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a
new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes
from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to
ferment. Three augment fermentation with archaeal-like hybrid type II/III ribulose-1,5-bisphosphate
carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO2
fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and
may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is
translated as tryptophan. All bacteria studied here may play previously unrecognized roles in
hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.
Sequencing of total DNA recovered directly from natural systems (metagenomics) often reveals previously unknown genes (1, 2)
1Department of Earth and Planetary Science, University of
California, Berkeley, Berkeley, CA 94720, USA. 2Lawrence
Berkeley National Laboratory, Berkeley, CA 94720, USA. 3Oak
Ridge National Laboratory, Oak Ridge, TN 37831, USA. 4Pacific
Northwest National Laboratory, Richland, WA 99352, USA.
*To whom correspondence should be addressed. E-mail:
and has the potential to yield near-complete genomes suitable for metabolic and phylogenetic
analyses (3–5). Numerous bacteria are known
exclusively through cultivation-independent recovery of their ribosomal RNA (rRNA) genes and
thus are important targets for this approach (6).
Here, we sequenced DNA from three microbial
communities from an acetate-amended aquifer
to reconstruct genomes of organisms that may
contribute to biogeochemical cycling in anoxic