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666 NATURE | VOL 388 | 14 AUGUST 1997
the number, order and duration of copulations, and determination of
offspring paternity. Further studies of sperm competition involving
taxa at different stages of divergence are needed to determine the
frequency of conspecific sperm preference and the rapidity with
which it arises. Drosophila is an ideal genus in which to pursue genetic
and anatomic studies of conspecific sperm preference because there
are mutant markers in many species, the fate of sperm can be
followed easily within a female, and there are tools to produce males
lacking sperm or various components of the seminal fluid20. M
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods
All stocks were reared at 24 8C with a 12-h light–dark cycle. Stock information
is available from the author on request. Males and females were collected as
virgins under CO2 anaesthesia and stored in 8-dram food vials. Each female’s
first mating took place on the fourth day after eclosion, and her second mating
(if any) occurred two days later. All males were four days old at the time of
mating. Flies were transferred without anaesthesia into a food vial for
observation, and 10–60 vials were observed simultaneously. All mating
observations began within 1 h of lights coming on in the incubator, and
lasted from 45 min to 10 h depending on the ease with which mating occurred.
All copulations were observed and timed (unless otherwise noted), and males
were removed from the observation vial within 5 min of copulation ending.
Females failing to mate on the first day were discarded. Of the females that did
mate successfully, a random subset was never given the opportunity to remate.
Females that refused to remate were discarded. Females were stored individu-
ally in food vials for the two days between the first and second mating. They
were transferred to a fresh vial for the second mating, and thereafter transferred
to fresh vials every three days until they stopped laying fertile eggs. All offspring
from each of these vials were reared to adulthood and scored for paternity.
Paternity was determined by the presence of a mutant marker, except in the case
of hybrids between D. simulans and D. mauritiana, which were distinguished by
the shape of the male genital arch. F1 females were obtained by crossing D.
simulans ebony females with D. mauritiana singed males. XO males were
produced by crossing virgin D. simulans females to males from a D. simulans
attached-X, attached-XY stock. All male offspring from this cross lack only a Y
chromosome, and so should produce normal seminal fluids21. All matings were
performed as described above with the following exceptions. Copulations
between D. simulans females and D. mauritiana males are often abnormally
brief, resulting in interruptions of sperm transfer22. To control for this, all
matings known not to result in sperm transfer were excluded from the study.
Insemination was determined directly for single matings and first matings by
the presence of larvae in the female’s food vial. Insemination could not be
assessed directly for second matings, but all matings shorter than those
resulting in insemination after a single mating (,8 min) were excluded from
the study. Matings of D. mauritiana females with D. simulans males could not
be observed owing to the extreme discriminatory behaviour of the females.
Instead, 10 virgin males and 10 virgin females were crowded together on the
third day after eclosion, and left for 24 h at 24 8C. Females were transferred
under CO2 anaesthesia to individual vials on the morning of the fourth day
after eclosion. Insemination was determined by the presence of larvae in the
vials. Inseminated females were aged and re-mated to D. mauritiana males as
described above.
Received 25 February; accepted 19 May 1997.
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Acknowledgements. I thank K. C. Price, P. Rooney, K. Kyle, C. Kim and K. Dyer for technical assistance; J.
Coyne for inspiration and help; and H. A. Orr, M. Turelli, M. Noor, N. Johnson and M. Wade for
comments. This work was supported by an NSF predoctoral fellowship to the author, and by an NIH grant
to J. Coyne.
Correspondence and requests for materials should be addressed to the author (e-mail: csprice@midway.
uchicago.edu).
Molecularevidence from
retroposonsthatwhales
formacladewithin
even-toedungulates
Mitsuru Shimamura*, Hiroshi Yasue†,
Kazuhiko Ohshima*, Hideaki Abe*, Hidehiro Kato‡,
Toshiya Kishiro‡, Mutsuo Goto§, Isao Munechikak
& Norihiro Okada*
* Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Yokohama, Kanagawa 226, Japan
† Animal Genome Research Group, National Institute of Animal Industry,
2 Ikenodai, Kukisaki-machi, Inashiki-gun, Ibaraki 305, Japan
‡ Large Cetacean Section, National Research Institute of Far Seas Fisheries,
5-7-1 Orido, Shimizu, Shizuoka 424, Japan
§ Genetic Ecology Section, The Institute of Cetacean Research, 4-18 Toyomi-cho,
Chuo-ku, Tokyo 104, Japan
kChiba Zoological Park, 280 Minamoto-cho, Wakaba-ku, Chiba 264, Japan
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The origin of whales and their transition from terrestrial life to a
fully aquatic existence has been studied in depth. Palaeonto-
logical1,2, morphological3 and molecular studies4–7 suggest that
the order Cetacea (whales, dolphins and porpoises) is more closely
related to the order Artiodactyla (even-toed ungulates, including
cows, camels and pigs) than to other ungulate orders. The
traditional view that the order Artiodactyla is monophyletic has
been challenged by molecular analyses of variations in mitochon-
drial and nuclear DNA5–7. We have characterized two families of
short interspersed elements (SINEs) that were present exclusively
in the genomes of whales, ruminants and hippopotamuses, but
not in those of camels and pigs. We made an extensive survey of
retropositional events that might have occurred during the diver-
gence of whales and even-toed ungulates. We have characterized
nine retropositional events of a SINE unit, each of which provides
phylogenetic resolution of the relationships among whales, ruminants,

Nature © Macmillan Publishers Ltd 1997
letters to nature
NATURE | VOL 388 | 14 AUGUST 1997 667
hippopotamuses and pigs. Our data provide evidence that whales,
ruminants and hippopotamuses form a monophyletic group.
We attempted to resolve the issue of whether the order Artio-
dactyla is monophyletic or paraphyletic by basing our analysis on
the presence or absence of SINEs at particular orthologous loci of
certain groups of species. SINEs are retroposons that have been
amplified and integrated into genomes by retroposition8–11, that is,
by the integration of a reverse-transcribed copy of RNA. As a
consequence of the nature of retroposons, SINEs can be found
specifically within members of a particular clade10–13. It is generally
believed that SINEs are not excised precisely and, moreover, that
SINEs have not been inserted independently at orthologous loci
within different evolutionary lineages. These features mean that
SINEs are very useful for the reconstruction of phylogenetic
relationships among closely related species12,13.
We have characterized two new and different families of SINEs,
designated the CHR-1 (for Cetacea, hippopotamus and Ruminan-
tia) and CHR-2 family of repeats, from the genomes of several
species of whales. The consensus sequences of these two families of
SINEs are shown in Fig. 1a. The order Artiodactyla is traditionally
divided into three suborders: Ruminantia (chevrotains, deer, cows,
sheep), Tylopoda (camels) and Suiformes (pigs, peccaries and
hippopotamuses). Dot-hybridization studies showed that these
two families of SINEs are distributed extensively in the genomes
of Cetacea, Ruminantia and hippopotamus, but were not detected
in those of Tylopoda or of Suiformes other than the hippopotamus
(Fig. 1b). These results suggest that whales, ruminants and hippo-
potamuses form a monophyletic group. This possibility prompted
us to isolate specific genomic loci at which SINEs had been inserted.
The first approach to this involved random screening, to identify
loci that contained a CHR-1 or CHR-2 SINE unit, followed by
cloning and sequencing. Polymerase chain reactions (PCRs) were
performed with genomic DNA from various cetacean and artio-
dactyl species to determine whether or not the locus might be
informative from a phylogenetic perspective. Second, we performed
a comprehensive survey of the protein-coding genes, in standard
databases, in which an intron contained one unit of CHR-1 or CHR-
2. When the length of the intron was short enough for generation of
a PCR product from the entire intron, we designed one set of
primers by reference to the sequences of exons. We used these two
approaches to characterize seven different loci with a CHR-1 or
CHR-2 SINE unit, as described below.
Our analysis indicates that a CHR-2 SINE had been integrated at
the locus Pm52 in a common ancestor of cetaceans (Fig. 2A). The
patterns of PCR products are shown in Fig. 2A, a. We performed
hybridization experiments with the SINE sequence to confirm that
the SINE unit had been integrated in a common ancestor of all
cetaceans (Fig. 2A, b), and with the flanking sequence to confirm
that the orthologous locus of each species had been amplified
accurately (Fig. 2A, c). The presence of the SINE unit in longer
fragments (about 620 base pairs in length) in cetaceans (lanes 1–7)
and the absence of the SINE unit in shorter fragments (about
230 base pairs (bp) in length) in artiodactyls (lanes 8–15) were
confirmed by sequencing. The small fluctuations in fragment length
were due to insertions and deletions of several nucleotides (data not
shown). The Pm72 locus yielded similar results (Fig. 2B). The
presence of these two loci indicates that the order Cetacea forms a
monophyletic group.
The locus pgha3, at which a CHR-1 SINE was integrated in intron
C of the gene for the a-subunit of a pituitary glycoprotein hormone
(Fig. 2C), and locus c21-352, at which a CHR-1 SINE was integrated
in intron C of the gene for steroid 21-hydroxylase (Fig. 2D),
demonstrate the monophyly of ruminants.
Locus Gm5 was isolated by random screening by using CHR-1
SINE as probe. The SINE unit seems to have been integrated in a
common ancestor of cetaceans, ruminants and hippopotamuses,
suggesting that these three evolutionary lineages are monophyletic
(Fig. 2E). The sequences of the fragments from the short-finned
pilot whale, cow, hippopotamus and Bactrian camel confirmed the
presence of the SINE unit in the longer fragment, and its absence in
the shorter fragment, respectively. In lane 15 for the pig, a longer
band was detected, but sequencing showed that this was due to
insertion of another SINE unit, PRE-1 (ref. 14) in another site of this
locus (data not shown).
The loci aaa228 and aaa792 are both derived from the gene for the
a-subunit of the F(0)F(1) ATP synthase (designated the atpA1 gene)
in the bovine genome. At locus 228, a CHR-1 SINE is present in the
intron between exons 2 and 3, suggesting monophyly of cetaceans,
ruminants and hippopotamuses (Fig. 2F, a). Hybridization experi-
ments using two different kinds of probe (Fig. 2F, b and c)
confirmed this conclusion.
Locus aaa792, between exons 10 and 11, is more complex. Three
different families of SINEs (CHR-1, CHR-3 and Bov-tA15) became
associated independently with this locus during the evolution of
Figure 1 The two newly isolated families of SINEs, CHR-1 and CHR-2, were
present exclusively in the genomes of whales, ruminants and hippopotamuses.
a, The consensus sequences of CHR-1 and CHR-2. The tRNA-derived region is
underlined in each case. These sequences will appear in the DDBJ, EMBL and
GenBank databases under the accession numbers: AB005033 and AB005034. b,
Dot hybridization experiment.