Resolution and evolution of the duck-billed platypus

karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex

chromosome constitution
Willem Rens*†, Frank Grützner‡, Patricia C. M. O’Brien*, Helen Fairclough*, Jennifer A. M. Graves‡,
and Malcolm A. Ferguson-Smith*
*Centre for Veterinary Science, University of Cambridge, Cambridge CB3 OES, United Kingdom; and ‡Comparative Genomics Group, Research School of
Biological Sciences, Australian National University, Canberra ACT 2601, Australia

Edited by Mary-Lou Pardue, Massachusetts Institute of Technology, Cambridge, MA, and approved October 4, 2004 (received for review August 5, 2004)

The platypus (2n ⴝ 52) has a complex karyotype that has been somes was presumed to be the X as it was present in two copies
controversial over the last three decades. The presence of unpaired in the female. Subsequently, the X was shown to carry several
chromosomes and an unknown sex-determining system especially genes that are X-linked in mammals (10–12), although human
has defied attempts at conventional analysis. This article reports on Xp genes were mapped to platypus autosomes 1 and 2 (13), and
the preparation of chromosome-specific probes from flow-sorted the mammalian SRY testis-determining gene could not be found
chromosomes and their application in the identification and clas- (14). The X chromosome clearly lay at one end of the chain (7,
sification of all platypus chromosomes. This work reveals that the 8) and a tiny ‘‘parachute-like’’ element was at the other end (9).
male karyotype has 21 pairs of chromosomes and 10 unpaired It was not possible to identify a Y chromosome unambiguously.
chromosomes (E1–E10), which are linked by short regions of The mechanism of sex determination in monotremes was a
homology to form a multivalent chain in meiosis. The female mystery.
karyotype differs in that five of these unpaired elements (E1, E3, Whether dosage compensation for the platypus X chromo-
E5, E7, and E9) are each present in duplicate, whereas the remain- some occurs has also been debated for decades. Semiquantitative
ing five unpaired elements (E2, E4, E6, E8, and E10) are absent. This PCR for three genes on the X shows that transcription from these
finding indicates that sex is determined by the alternate segrega- loci is equivalent in both sexes (2), but could not distinguish
tion of the chain of 10 during spermatogenesis so that equal between X inactivation and differential兾reduced expression
numbers of sperm bear either one of the two groups of five from both X chromosomes. Replication asynchrony between the
elements, i.e., five X and five Y chromosomes. Chromosome paint- two X chromosomes was described in echidna lymphocytes (7),
ing reveals that these X and Y chromosomes contain pairing (XY

GENETICS
but it appeared to be confined to the short arm (8), which is
shared) and differential (X- or Y-specific) segments. Y differential paired and should require no dosage compensation. No asyn-
regions must contain male-determining genes, and X differential chrony was found in fibroblasts.
regions should be dosage-compensated in the female. Two models This study reports the sorting of male platypus chromosomes
for the evolution of the sex-determining system are presented. The and production of chromosome-specific paint probes for all
resolution of the longstanding debate over the platypus karyotype but the smallest unpaired chromosome. These paint probes
is an important step toward the understanding of mechanisms of identified 10 unpaired chromosomes in the male and regions
sex determination, dosage compensation, and karyotype evolution. of homology between them that explain their involvement in
a multivalent chain. In the female five of these unpaired
monotremes 兩 multivalent chain 兩 X-inactivation 兩 sex determination chromosomes are absent and the remaining five are present in
duplicate. Thus the female karyotype does not contain any

T he duck-billed platypus (Ornithorhynchus anatinus) is one of

three extant species of the egg-laying monotremes whose
ancestors are believed to have diverged from the mammalian
unpaired elements. The sex chromosome complement in the
male is therefore heteroz ygous and interpreted as
X1Y1X2Y2X3Y3X4Y4X5Y5. This remarkable finding has impli-
lineage ⬇210 million years ago and some 30 million years before cations for sex chromosome evolution and X chromosome
the divergence of marsupials and placental mammals (1). Its dosage compensation.
unique mixture of mammalian, avian, and reptilian features (2)
has excited biologists for more than two centuries. For geneti- Materials and Methods
cists, the platypus undoubtedly provides a special outgroup Primary fibroblast cultures from the toe web of the Australian
species because it represents the earliest offshoots of the mam- platypus O. anatinus (2n ⫽ 52) were established routinely in
malian lineage. Studies in monotremes can answer questions standard medium at 32°C (Animal Experimentation Ethics
about sex determination, genomic imprinting, and dosage com- Committee Permit RCG.07.03 to F.G. and Environment Aus-
pensation in mammals. Accurate chromosome identification is tralian Capital Territory Permit LI 2002 270 to J.A.M.G.). Flow
crucial for these studies. The correct chromosome number of sorting, chromosome paint production, and fluorescence in situ
2n ⫽ 52 was determined for the platypus in 1975 (3). The hybridization were performed according to the protocol de-
karyotype was first considered to be reptilian (4), but this idea scribed (15). Chromosomes were prepared for sorting as de-
was subsequently refuted (5). However, difficulty was encoun- scribed (16), with slight modification. Colcemid (0.1 ␮g兾ml) was
tered in the analysis of the karyotype, as a number of chromo- added to flasks of male platypus fibroblasts and incubated for
somes, variably assessed as between four and eight, appeared to only 30 min instead of the usual 4–6 h. Longer incubation times
be unpaired, a feature not seen in such numbers in any other caused interphase cells to detach from the flasks. Mitotic cells
mammal except the other two monotremes, the short- and were collected by carefully tapping the culture flasks and were
long-beaked echidnas (6, 7). These unpaired chromosomes were
believed to form a multivalent chain at meiosis. However, it
remained controversial as to whether unpaired chromosomes, This paper was submitted directly (Track II) to the PNAS office.
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and therefore formation of a meiotic chain, occur in both sexes †To whom correspondence should be addressed. E-mail: [email protected].
(3, 7, 8) or males only (9). The largest of the unpaired chromo- © 2004 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0405702101 PNAS 兩 November 16, 2004 兩 vol. 101 兩 no. 46 兩 16257–16261

 Fig. 1. Flow karyotype of male O. anatinus. Almost all chromosomes are
represented by individual peaks. Only chromosomes 8 and E9, and chromo-
somes 13, 14, and E5, sort together.

centrifuged at 400 ⫻ g for 5 min. The cell pellet was resuspended

in 10 ml of hypotonic KCl (75 mM KCl兾0.2 mM spermine兾0.5
mM spermidine) and allowed to swell for 1 h instead of the usual
15 min at room temperature. From this point onward the
standard protocol was followed (15, 16). Fig. 2. G-banded karyotypes of O. anatinus. (Upper) The male platypus (2n ⫽
The paints produced were hybridized to male (three individ- 52) has 21 chromosome pairs and 10 unpaired chromosomes labeled E1–E10.
uals) and female (one individual) metaphase preparations. Im- The order of these chromosomes determined by their pairing regions is
ages were captured by using QFISH software (Leica Microsys- indicated by vertical bars. (Lower) G-banded female karyotype of O. anatinus.
There are no unpaired chromosomes.
tems, Cambridge, U.K.) and a cooled charge-coupled device
camera (Photometrics Sensys, Tucson, AZ) mounted on a Leica
DMRXA microscope equipped with a ⫻63, 1.3 numerical of the karyotype shows the 21 paired chromosomes that form
aperture objective. Cy3, FITC, and 4⬘,6-diamidino-2-phenylin- bivalents at meiosis. Chromosomes 1–7 can be identified easily
dole (DAPI) signals were captured separately as 16-bit black and by their size, centromere position, and G bands. Chromosomes
white images and merged to a color image. The DAPI image was 1 and 4 have large light-stained centromeric regions correspond-
enhanced with a spatial filter to obtain enhanced chromosome ing to heterochromatin; chromosomes 2 and 3 have dark-stained
bands. GTG banding was performed by using standard protocols.
centromeres. Chromosome 6 has long satellite arms, which are
All image processing was performed with Leica CW4000 software.
heteromorphic, and carry the nucleolus organizer regions (8).
Results Chromosomes 8–21 are much harder to distinguish by morphol-
ogy and G banding and remained unclassified in previous
The aim of the analysis was the identification of all platypus
chromosomes to resolve the controversial karyotype, in partic- reports. Chromosome painting classified these chromosomes
ular to identify the unpaired chromosomes and determine their and established their characteristic patterns of G banding (or
homology relationships by using chromosome-specific paint reverse 4⬘,6-diamidino-2-phenylindole banding) and centromere
probes produced from flow-sorted chromosomes. The modifi- position.
cation of the normal sort preparation protocol achieved sepa- The 10 unpaired chromosomes identified by painting and
ration of almost all chromosomes in the flow karyotype (Fig. 1), designated E1–E10 are arranged in the lower part of the
and degenerate oligonucleotide primed-PCR provided chromo- karyotype (Fig. 2 Upper). The order of all the chromosomes in
some-specific DNA free of contamination from other chromo- the multivalent was established, and the parts (some of them
somes. Only chromosomes 8 and E9, and chromosomes 13, 14, relatively small) shared by each unpaired chromosome were
and E5, sorted together in two peaks and were combined in two determined by using single- and dual-color fluorescence in situ
paint probes. However, a unique Eq paint probe was prepared hybridization. Chromosome E1 can easily be recognized by its
from a single sorted chromosome. Chromosomes 1–7, plus E1, specific size and G bands. Chromosome E2 is acrocentric.
are the larger chromosomes, and the nucleolar chromosome 6 is Chromosomes E3 and E5 are submetacentric and share a
represented by two peaks caused by polymorphism of the ‘‘stalk’’ heterochromatic region proximal on the long arm. Chromosome
of this chromosome (see Fig. 2). Numbers 8–21 represent the E4 is smaller then E3 and E5 and is metacentric. Chromosomes
medium and small chromosomes that cluster together at the E6, E7, E8, and E10 are tiny chromosomes. E7 is the biggest of
lower end of the flow karyotype. these four and is metacentric, as is E8. E6 is submetacentric. E10
Fig. 2 Upper is the G-banded karyotype of a male platypus, in is the smallest, which makes its centromere position difficult to
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which each chromosome has been identified by using the chro- determine (see also Fig. 3b). E9 is submetacentric and much
mosome-specific paints (see Fig. 3 for examples). The upper part larger than the tiny E6, E7, E8, and E10. Fig. 2 Lower is a

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 Fig. 4. Examples of chromosome painting on female metaphases of the
platypus. Chromosome paints used are indicated in each image. Chromosomes
E1, E3, E7, and E9 are present in two copies, and pairing regions are indicated
for E2 (a), E4 (b), and E6 (c) (see text). (d) Two copies of Eq in red. (Bar
represents 10 ␮m.)

flow karyotype (Fig. 1). In Fig. 3f, paint E3 (red) paints

chromosome E3 plus the short arm of E2 (see arrowhead and
Inset), and the short arm of E4 (see arrow). It also paints a band
(yellow) on E5, as E3 and E5 share a common region of
heterochromatin. E5 (green) paints E5 (the chromosome with
the red heterochromatic region) and the long arm of E4 (arrow).
As in Fig. 3e, paint E5 also paints chromosomes pairs 13 and 14
that sort together with E5 in the flow karyotype (Fig. 1). Fig. 3g

GENETICS
shows paint E3 in red and paint E4 in green. Paint E4 paints the
short arms of E3 and E5 in addition to the entire E4. Again, paint
E3 results in a red heterochromatic band on E5. Fig. 3h shows
the tiny element E6 in red and E5 in green. Fig. 3h Inset shows
that E6 paints the distal end of E5q. Fig. 3i presents E7 in red,
and the Inset shows that this probe also paints the tiny elements
Fig. 3. Examples of chromosome painting on male metaphases of the E6 and E8 (confirmed by dual-color painting, image not shown),
platypus. Chromosome paints used are indicated in each image, and unpaired
thus linking E6–E8; the unlabeled E10 is also visible in this Inset.
chromosomes E1–E9 are present in single copy. (a) Chromosome 6 painted in
red. (b) Chromosome 20 (red) and 21 (green), arrows point to tiny unpaired
Fig. 3 b–i thus links E1 to E8. Fig. 3j shows the larger element
chromosomes. (c–i) Shown is the region of homology, which links chromo- E9 without visual hybridization with E8 or E10.
somes E1–E8 (see text). (Bars represent 10 ␮m.) Fig. 4 shows the results of chromosome painting onto female
metaphases. Fig. 4a presents paint E1 in red and paint E2 in
green. Element E2 itself is not present in female metaphases,
G-banded karyotype of a female platypus in which E1, E3, E5, instead element E1 is a paired chromosome in the female
E7, and E9 are paired and E2, E4, E6, E8, and E10 are absent; platypus; its short arm is in yellow as it is painted both by paints
there are no unpaired chromosomes. E1 and E2. Fig. 4b shows the hybridization of paint E3 in red and
Fig. 3 gives examples of the fluorescence in situ hybridization E4 in green. E3 paints the entire E3 and the heterochromatic
results on male metaphases. Fig. 3a shows chromosome 6 region on E5; E3 and E5 are present in pairs. E4 is not present,
identified by its specific paint. The p-arm of this chromosome but the E4 paint probe hybridizes to its homologous region on
consists of satellite DNA and the moderately repetitive ribo-
E5p (green) and E3p (masked by the red E3 paint in this merged
somal genes. Only the tip of 6p is painted, as the rest of the short
image). Fig. 4c shows paint E6 in red and E7 in green: E7 forms
arm is composed of repetitive DNA that is blocked by compe-
tition in the hybridization procedure. Fig. 3b shows the hybrid- a pair, but E6 is not present in the female platypus and the E6
ization of chromosome paint 20 (red) and 21 (green) onto the paint reveals a tiny E6 pairing region on both homologues of E7.
two smallest paired chromosomes with arrows showing four In 70% (n ⫽ 20) of the cells, the E7 pair is heteromorphic in size.
smaller unpaired chromosomes, each of different morphology. Fig. 4d shows that E9 also forms a pair in the female platypus.
Fig. 3 c and d shows paint E1 and E2, respectively; E1 paints the The results of these hybridization experiments demonstrate that
largest unpaired chromosome plus another unpaired element in the female platypus lacks the even-numbered elements and that
the chain; the homology between them implies that it must be E2. the odd-numbered elements are all present in pairs. Although it
Paint E2 paints this second element plus the short arm of E1 and is possible that other platypus populations may have different
a region on the long arm of the next element in the chain, E3. karyotypes, we believe that the findings in the one female and
The latter region was difficult to detect but can be seen in Fig. three males that we studied are representative of the species.
3e Inset. Fig. 3e also shows the hybridization of paint E5 not only Furthermore, the diploid number of chromosomes was always
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to E5 and the heterochromatic region of E3, but also to 2n ⫽ 52 in three different reports in specimens from a total of
chromosomes pairs 13 and 14, which sort together with E5 in the 20 individuals (3, 7, 8).

Rens et al. PNAS 兩 November 16, 2004 兩 vol. 101 兩 no. 46 兩 16259
 Fig. 5. Diagram of the multivalent chain in male meiotic cells. Horizontal and
vertical lines represent differential and paired regions, respectively.

In conclusion, successive links have been demonstrated be-

tween elements E1 and E8 of the chain. We could not detect
homology between E8 and E9 or between E9 and E10 (for which
no chromosome-specific probe could be made). However,
Grützner et al. (17) have now analyzed the chain in male meiotic
preparations, using our paint probes and telomeric probes, and
have shown that E8 is linked to E9 and E10 is linked to E9 at the
end of the chain.

Discussion
Chromosome-specific paint probes from the male platypus were
used to identify and classify the platypus karyotype and resolve
the decades-long debate about the nature of the unpaired
chromosomes and their participation in the multivalent chain in
male meiosis. We show that the male has 21 pairs of homologous
chromosomes and 10 unpaired chromosomes, designated E1–
E10 (Figs. 1 and 2). In the female, the odd-numbered elements
(E1, E3, E5, E7, and E9) are present in two copies, and the
even-numbered elements (E2, E4, E6, E8, and E10) are absent.
Thus females have no unpaired chromosomes and are not Fig. 6. A model for the evolution of the platypus sex chromosome comple-
expected to produce a multivalent chain in meiosis. As sex in ment. Evolution starts with an ancestral pair of differential chromosomes, one
platypus is associated with particular combinations of these of them is repeatedly involved in exchanges with an autosome. Two pathways
elements, either homozygous E1, E3, E5, E7, and E9 in the are shown. (Left) The ancestral pair has homology to the mammalian X
female or heterozygous E1, E3, E5, E7, E9兾E2, E4, E6, E8, and chromosome (indicated by the androgen receptor gene mapped to E1).
E10 in the male, by convention the odd elements are considered (Right) The homology is to the avian Z based on the mapping of DMRT1 on E9.
as X chromosomes and the even elements as Y chromosomes. The model can be explained step by step. On the left there are two differential
chromosomes (ancestral X-Y chromosomes). A break (indicated by a horizon-
The X1Y1X2Y2X3Y3X4Y4X5Y5 sex chromosome constitution
tal line) occurs within one of these and within a homolog of an autosomal pair
that we describe in the male platypus seems unique among (a). These breaks lead to a rearrangement and a chain of four elements.
mammals in the number of chromosomes involved in the mul- Element E1 and E3 are the derivative chromosomes, and E2 is the nonderiva-
tivalent chain at meiosis. Meiotic chains have been described in tive homolog of the original autosomal pair (b). The color change from red to
certain plants (18, 19), termites, and spiders (20–23). In mam- pink indicates the pairing regions of the element E2 with its ‘‘old’’ autosomal
mals a meiotic chain has been reported in a primate with an homolog within the chain of four elements (c). A new break and rearrange-
X1X2Y1Y2 quadrivalent at male meiosis (24). It is unclear how ment with another autosomal pair (blue) result in a chain of six elements (c and
the complex platypus sex chromosome system can achieve d). The derivative blue homolog is rearranged and can be found on elements
E3 and E5, whereas the nonderivative blue homolog forms element E4.
alternate segregation of X and Y chromosomes into individual
Similarly, a color change from blue to yellow indicates the pairing regions with
sperm with high probability (17) and apparently without repro- its old autosomal homolog (e). Note that the new element E3 has a region
ductive wastage. derived from the original X, whereas the new element E4 does not. Two
Homologous regions of variable size, demonstrated here by additional rearrangements result in the chain of 10 elements as detected by
chromosome painting, link the 10 unpaired elements in the male chromosome painting. The right pathway, which starts with ancestral Z-W
platypus karyotype. These homologous regions allow chiasmata chromosomes, can be explained similarly.
to form during prophase of male meiosis I, thus forming a
multivalent chain as shown in Fig. 5. The differential (unpaired)
regions in the multivalent cannot form chiasmata and the region that we now confirm to be the differential region of the
horizontal lines in Fig. 5 represent these regions. In somatic cells, platypus X, should require dosage compensation. The other four
the female has a double dose of genes carried by these differ- X chromosomes (E3, E5, E7, and E9) also have regions that are
ential segments of the five X chromosomes. not represented on the Y chromosomes and are thus present in
Because the E1 short arm pairs almost entirely with E2, it single dose in the male. It will be of interest to discover whether
follows that genes known to map to the short arm of E1 [the dosage compensation occurs for these X chromosomes as it does
human Xq genes GLA, PLP, F8, F9, and RCP and the human Xp apparently for the differential region of E1 (2). Ohno (26)
genes ALAS2 and GATA1 (11, 12)] all are present in the platypus explained the stability of the X chromosome across mammalian
in double doses in both males and females. This region therefore species by the inactivation mechanism. This stability would be
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has no need for dosage compensation. However, four human Xq disrupted if parts of the X chromosome were translocated to
genes (AR, GDX, P3, and G6PD) (11, 25) that map to E1q, a autosomes. As discussed below, exchanges between the ancestral

16260 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0405702101 Rens et al.

 X and autosomes may have occurred during the evolution of the regions (Fig. 6a). A reciprocal exchange between the X and an
multivalent in platypus, which would imply that a mechanism for autosome leads to a meiotic chain of four (Fig. 6b). Element 2
dosage compensation based on X inactivation must accommo- in this chain is homologous with the short arm of E1 (Fig. 6c).
date multiple X chromosomes. On the other hand, dosage The next event is an exchange between the derivative X and
compensation may have been achieved in this species without X another autosome (Fig. 6c). The new element E3 has a region
inactivation. derived from the original X, whereas the new element E4 does
The five Y chromosomes share homologies with the X chro- not. Two similar events lead to the final 10 elements in which
mosomes only at their pairing segments. The differential seg- the derivative X is involved on each occasion. Note that in the
ments of E2, E4, E6, E8, and E10, although not detectable by our diagram after each rearrangement we have given one of the
methods, are likely to contain male-determining and other exchanged segments and its homologous segment a different
male-specific sequences. In the absence of a detectable homo- color to emphasize the regions that subsequently pair with one
logue of SRY (14), the molecular basis for testis determination another in the multivalent. It is evident that E1, E3, E5, E7, and
in the platypus is unknown. It will be important to explore E9 have X regions and the other elements do not. Four succes-
male-specific sequences on the Y chromosomes for a novel sive X-autosome exchanges can provide a explanation for the
sex-determining gene. Nevertheless, our results show that sex in homologies within the 10 elements of the chain. An alternative
the platypus is determined by alternate segregation of X and Y possibility (Fig. 6 Right) is that E9 and E10 originated from an
chromosomes during male meiosis. This finding is confirmed by ancestral sex chromosome pair and that E1 and E2, with
Grützner et al. (17) using the chromosome-specific paint probes homology to the eutherian X chromosome, represent the latest
described here on platypus sperm, which reveals that male addition to the chain. Either pathway might be an early stage in
haploid germ cells contain either X or Y chromosomes but not the evolution of sex-determination systems from birds to mam-
both. mals. The recent observation that DMRT1, the putative sex-
Our findings have consequences for the postulated model (8) determining gene carried by the Z chromosome in birds, maps
of evolution ⬎210 million years of the platypus sex chromosome to chromosome E9 in platypus (17) supports the second possi-
system. Most models of sex-chromosome evolution assume an bility. In this model the ZW pair would be the ancestors of
origin from an ancestral pair of sex chromosome in which one chromosomes E9 and E10 and the starting point in the evolution
member acquires a male-determining role in a differential of the monotreme multivalent. Comparative mapping studies
segment. It is possible that the system in platypus originated from with birds and reptile species may provide answers to the most
such an ancestral pair. We propose a model (alternative to ref. likely mechanism of evolution of the sex chromosome system in
8) in which part of an original sex chromosome is repeatedly monotremes and may help to identify the key sex-determining
involved in exchanges with autosomes. In the absence of infor- genes.
mation on the gene content of any element except E1 and E9, it
cannot be certain which end of the chain represents the initial sex This work was performed at the Cambridge Resource Centre for

GENETICS
pair. One possibility (Fig. 6 Left) is that the ancestral therian X Comparative Genomics at the University of Cambridge, which was
and Y initiated the chain from a large X and an attenuated Y supported by a generous grant from the Wellcome Trust (to M.A.F.-S.).
chromosome with small pairing regions and large differential The Australian Research Council supports F.G. and J.A.M.G.

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