Chromosome Research 9: 659^672, 2001.
# 2001 Kluwer Academic Publishers. Printed in the Netherlands
659
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh:
zebra¢sh, platy¢sh and guppy
Walther Traut & Heinz Winking
Institut fÏr Biologie, Medizinische UniversitÌt zu LÏbeck, D-23538 LÏbeck, Germany;
Tel: ++49/451/5004112; Fax: ++49/451/5004815; E-mail: [email protected]
Received 14 June 2001; received in revised form and accepted for publication by M. Schmid 27 July 2001
Key words: comparative genomic hybridization, ¢sh cytogenetics, sex chromosomes, sex determination,
synaptonemal complex
Abstract
We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and
meiotic chromosomes of the zebra¢sh Danio rerio, the platy¢sh Xiphophorus maculatus and the guppy
Poecilia reticulata. The three ¢sh species represent basic steps of sex chromosome differentiation:
(1) the zebra¢sh with an all-autosome karyotype; (2) the platy¢sh with genetically de¢ned sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic
chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes.
The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential
segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The
differential segment is unpaired in early pachytene but synapses later in an `adjustment' or `equalization'
process. The segment includes a postulated sex determining region and a conspicuous variable
heterochromatic region whose structure depends on the particular Y chromosome line. CGH
differentiates a large block of predominantly male-speci¢c repetitive DNA and a block of common
repetitive DNA in that region.
Introduction
The evolution of morphologically and molecularly
differentiated pairs of XY sex chromosomes from
a pair of perfectly homologous autosomes involves
three kinds of genetic changes: (1) acquisition of
the sex-determining function, either `recessive'
femaleness factors in the X or `dominant' maleness
factors in the Y chromosome; (2) evolution of a
non-recombining region between X and Y,
encompassing the sex-determining locus or loci;
and (3) accumulation of mutations, loss and gain
of sequences in that region of the Y independent
from genetic changes in the corresponding region
of the X chromosome.
Acquisition of the sex-determining function by a
chromosome pair can happen in various ways. In
plants, it has occurred repeatedly along with
transition from monoecy to dioecy (Stebbins
1971). Similar transitions from hermaphroditism
to gonochorism with chromosomal sex determination can be seen in animals, e.g. in the trematode
660
genus Schistosoma (Grossman et al. 1981). Yet,
the evolution of new sex chromosomes does not
depend on such transitions. Chromosomal sex
determination, i.e. a type in which sex determination is seemingly `monofactorial', may have
evolved from other types of sex determination,
from environmental or from polygenic sex
determination. Rather frequently in evolution,
the sex-determining function has been transferred
from one chromosome pair to a new one. This
was effected by chromosome rearrangement, e.g.
fusion or translocation (White 1973, Steinemann
et al. 1993, Charlesworth et al. 1997), or by
transposition (Traut & Willhoeft 1990, Traut
1994).
Teleost ¢shes are promising for the study of sex
chromosome evolution as the sex-determining
mechanism is not ¢xed in this group. Hermaphrodite species abound and sex-determining
mechanisms vary among gonochoristic species
and even among populations of the same species
(reviews: Morescalchi 1992, Baroiller et al. 1999).
Thus, changes must have happened frequently
and, occasionally, rather recently in the evolution
of extant species.
Sex-determining mechanisms in ¢shes include
polygenic sex determination, e.g. in the swordtail,
Xiphophorus helleri, Poeciliidae (Kosswig 1964),
and temperature-dependent environmental sex
determination, e.g. in the atherinid ¢sh Menidia
menidia (Conover & Kynard 1981). Chromosomal
sex determination of both types, XX/XY and
WZ/ZZ, occurs among ¢shes (reviewed by
Almeida-Toledo et al. 2000). The poeciliid ¢sh
Poecilia reticulata, the guppy, for example, has
an XX/XY mechanism (Winge 1934) and the
anostomid ¢sh Leporinus elongatus a WZ/ZZ type
of sex determination (Nakayama et al. 1994). But
even in species with an established chromosomal
sex determination, one or more autosomal factors
can overrule the sex chromosome constitution:
applying selection, Winge (1934) established a
strain of the guppy in which a former autosome
pair controlled sex determination. Even after
primary sex determination in ¢shes, sexual
development contrary to the genotype can be
induced by external factors. Irradiation causes
genetically male embryos of the platy¢sh,
Xiphophorus maculatus to develop into functional
females (Anders et al. 1969) and sex-hormone
W. Traut & H. Winking
treatment of embryos leads to functional males
of female genotype and/or vice versa, e.g. in the
Medaka, Oryzias latipes (Yamamoto 1958), or
in the guppy (Dzwillo 1962).
In this paper, we investigate meiotic and mitotic
chromosomes with particular reference to the
differentiation of the XY chromosome pair in
the platy¢sh, X. maculatus, and the guppy,
P. reticulata (Poeciliidae). For comparison, we
include the zebra¢sh Danio rerio (Cyprinidae)
as a species that does not have sex chromosomes
(see Discussion).
Materials and methods
Animals
Danio rerio, the zebra¢sh, was from two sources.
Some animals were from a pet shop and given
to us by Dr. Thomas Becker (Hamburg,
Germany), other animals belonged to the
wild-type strain Tuebingen (Tu) and were kindly
provided by Dr. Hans Georg Frohnhoefer
(TÏbingen, Germany).We did not detect any
difference between the two sources. The platy¢sh
Xiphophorus maculatus of this study, originally
from the Rio Jamapa (Mexico), was a gift from
Professor Manfred Schartl (WÏrzburg, Germany).
The strain had sex chromosomes phenotypically
labelled by pigment markers, females were
XDr SdXDr Sd, males XDr SdYAr Sr. Some specimens of the guppy Poecilia reticulata, were caught
in Mexico and kindly provided by Reinhold Nickel
(LÏbeck-TravemÏnde, Germany), others were
bought from a pet shop. The Y chromosomes
of these specimens were phenotypically labelled
by the colour pattern caused by the Iridescens
gene.
Chromosome preparations
Meiotic chromosome spreads were obtained from
testes, and mitotic spreads from testes and ¢ns
by conventional procedures: hypotonic treatment,
¢xation in methanol^acetic acid (3 : 1) and
spreading with 60% acetic acid. Slides were stored
in the freezer until further use. The quality of ¢sh
chromosome preparations was acceptable when
stained conventionally with lactic^acetic^orcein.
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
Stained with the more sensitive £uorescence
dye, DAPI (40 6-diamidino-2-phenylindole), the
chromosomes presented a fuzzy contour even after
stabilization of the chromatin structure with
paraformaldehyde.
SCs
Visualization of synaptonemal complexes (SCs)
followed routine techniques. Brie£y, testes were
minced in cell culture medium199 (Hank's
medium). The cell suspension was centrifuged
and the cells resuspended in a small amount of
medium. A drop of the cell suspension was placed
on a coated slide and covered with sucrose
(0.15 mol/L in 0.01 mol/L borate buffer; pH
8.5^9.3; the optimum had to be determined by test
spreadings in each sample). After 3 min, the £uid
was sucked up and the slides placed in formaldehyde (4% in 0.1 mol/L sucrose) for 5 min. After
¢xation, cells were stained with 50% aqueous silver
nitrate according to Albini et al. (1984). The
plastic ¢lms with stained cells were £oated off
on the surface of distilled water and transferred
to EM grids. Images of SC complements were
recorded with an electron microscope (Philips,
EM400T).
DNA probes
DNA was isolated according to Blin & Stafford
(1976) from animals whose intestines had been
removed. DNA labelling was done by nick
translation using the Bionick Labelling system
(Life Technologies, Karlsruhe, Germany) according to the instructions of the manufacturer.
Genomic DNA from females was labelled with
FluorX-dCTP (green £uorescence), genomic
DNA from males with Cy3-dCTP (red
£uorescence) (Amersham Life Science, Arlington
Heights, IL).
661
in 70% formamide, 2 SSC. The probe cocktail
contained 400 ng FluorX-labelled DNA from
females, 400 ng Cy3-labelled DNA from males,
and 4 mg competitor DNA. The competitor
DNA was sonicated genomic DNA from the
homogametic female sex of the respective species.
Hybridization time was 3 days. A stringent wash
at 62³C in 0.1 SSC, 1% Triton X-100, was
followed by counterstaining with DAPI and
mounting in Antifade (0.233 g 1,4-diazobicyclo(2.2.2)-octane; 1 ml 0.2 mol/L Tris-HCl, pH 8.0;
9 ml glycerol).
Micrographs and image analysis
Black-and-white micrographs were taken with a
cooled CCD camera using the Pinkel ¢lter set
for measurements and ¢lters 02, 10 and 15 from
the Zeiss £uorescence ¢lter set for composite
images. False colouring and merging of images
was done with Photoshop Version 4.01 (Adobe).
Green colour was routinely adopted for the
female-derived probe, red for the male-derived
probe, and light blue for the DAPI image. We used
the histogram function together with the lasso tool
of the Adobe Photoshop program package to
compare £uorescence signals from female and
male DNA in the male-speci¢c segment of the
guppy Y chromosome. To compute the
female/male £uorescence signal ratio (Ratiofm)
of the Y chromosome segment, it was normalized
against a 1:1 signal ratio of the autosomes after
subtraction of background signal: Ratiofm ˆ
(Yf Bf)(Am Bm)/(Ym Bm)(Af Bf) where Yf
is the mean signal intensity of the bound
female-derived probe from the Y-speci¢c segment,
Af that from autosomes and Bf that from the
background, Ym is the mean signal intensity of
the bound male-derived probes from the Y-speci¢c
segment, Am that from autosomes and Bm that
from the background.
Comparative genomic hybridization
We applied the hybridization procedure of
Lapierre et al. (1998) with some modi¢cations.
After removal from the freezer, slides were
post¢xed for 1 min in cold methanol^acetic acid.
Chromatin structure was stabilized by 1 min in
1% formaldehyde. Denaturation was for 2 min
Results
Danio rerio
The diploid chromosome number is 2n ˆ 50 in the
zebra¢sh, Danio rerio; all chromosomes are
two-armed (Amores & Postlethwait 1999).
662
Centromeric and some paracentromeric regions
appear DAPI-negative (Figure 1a). CGH with
FluorX-labelled genomic DNA (green) from
females and Cy3-labelled genomic DNA (red)
from
males
stained
all
chromosomes
yellowish-brown (Figure 1a, b). This indicates
binding of equal proportions of female- and
male-derived probes to all chromosomes.
Highlighting by both probes together was
conspicuous in centromeric and paracentromeric
DAPI-negative bands and in the long arm of
one chromosome pair (inserts and arrowheads
in Figure 1c). The general pattern of these bands
corresponds to the C-band pattern described by
Pijnacker & Ferwerda (1995). Highlighting re£ects
a high concentration of bound probe and indicates
faster hybridization kinetics due to the presence of
repetitive sequences. The centromeric regions of
most chromosomes contain the type I satellite
which consists of 65% AT (Amores & Postlethwait
1999, Ecker et al. 1992). Since DAPI has a
preference for AT-rich DNA and the centromeres
are DAPI-negative, centromeric heterochromatin
should contain other sequences besides the type I
satellite. The highlighted long chromosome arm
is most probably identical to the latereplicating chromosome arm of chromosome
#3 described by Amores & Postlethwait (1999)
and the `slightly C-band positive arm' of Pijnacker
& Ferwerda (1995) and Daga et al. (1996).
In diakinesis of male meiosis, most bivalents
were terminally paired at one end; a minority
displayed cross-shaped ¢gures indicating an
intercalary chiasma and ring con¢gurations
indicating chiasmata in both chromosome arms.
No bivalent was apparently asymmetrical and
no halfbivalent was preferentially stained by
one of the two probes (Figure 1d, e).
In spreads of synaptonemal complexes (SCs)
from males, the 25 SC bivalents presented a
gradual length series (Figure 3; Table 1)
corresponding to that of mitotic chromosomes.
One to three bivalents were connected at one
end with a mass of silver-stained material
indicating terminal NORs in accord with those
desribed from mitotic chromosomes (Pijnacker
& Ferwerda 1995). Another bivalent, the third
to ¢fth in size, was regularly decorated for more
than half of its length with remnants of
chromatin, lumps of silver-stained material,
W. Traut & H. Winking
often with stalk-like extensions towards the SC
(Figures 3, H, and 4). This is most probably
chromosome #3 with its late-replicating
heterochromatic arm. None of the SCs exhibited
lateral asymmetry or regularly incomplete pairing.
Thus, there is no indication of a heteromorphic sex
chromosome pair among the SC bivalents.
Xiphophorus maculatus
The mitotic complement of the platy¢sh consists of
48 chromosomes. The XY pair of the XX/XY
strain used in this study had been identi¢ed as a
heteromorphic pair with a submetacentric X
and an acrocentric Y (Foerster & Anders 1977)
but this was not con¢rmed in a later study (Nanda
et al. 1993). All chromosomes in our study were
acrocentric with small DAPI-positive centromeric
bands (Figure 2a). After CGH with FluorXlabelled genomic DNA from females and
Cy3-labelled DNA from males, all 48 chromosomes in the male mitotic complement displayed
yellowish-brown £uorescence indicating equal
proportions of bound female- and male-derived
probes (Figure 2b). The small centromeric bands
were highlighted by both probes (Figure 2b, c)
but there was no indication of a molecularly
differentiated XY pair.
In diakinesis of male meiosis, bivalents with an
interstitial chiasma were about as frequent as
those displaying terminal pairing. All were
apparently symmetrical (Figure 2d). Likewise in
CGH, all halfbivalents bound female- and
male-derived probes equally (Figure 2e).
In SC complements from males, size
distribution among the 24 SCs was gradual
(Figure 5; Table 2), re£ecting that of mitotic
chromosomes. One to three of the SCs carried a
terminal aggregation of silver-stained material.
At least one of these aggregations was presumed
to be due to an active NOR (N in Figure 5). There
were no signs of a differentiation between paired
homologues and hence no signs of the presence
of a sex chromosome pair.
Poecilia reticulata
The mitotic complement of male guppies contains
46 acrocentric chromosomes, among them a
heteromorphic XY pair (Nanda et al. 1990).
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
663
Figure
Figure 1. CGH of zebra¢sh chromosomes with female-derived genomic
DNA (green) and male-derived DNA (red), chromosomes stained with
DAPI (blue). (a^c) male mitosis, a: DAPI-stained; b: CGH, c:
male-derived probe plus DAPI, arrowheads and inserts: chromosome
#3 with a highlighted long arm; (d, e) diakinesis of male meiosis; d:
DAPI-stained; e: CGH. Bar: 10 mm.
Figure 2. CGH of platy¢sh chromosomes from male mitosis (a^c) and diakinesis of male meiosis (d, e). Details as in Figure 1.
664
W. Traut & H. Winking
Figure 3. SC complement of male meiosis from the zebra¢sh. H: SC from chromosome #3; N: presumed remnants of a nucleolus; bar:
5 mm.
Figure 4. SCs assigned to chromosome #3 of the zebra¢sh,
decorated with stalked lumps of silver-stained material. (a^c)
pachytene; d: beginning diplotene. Bar: 5 mm.
The chromosomes displayed DAPI-negative
centromeric bands and often DAPI-negative distal
bands (Figure 6a). With CGH, these bands were
highlighted by both probes: FluorX-labelled
genomic DNA from females and Cy3-labelled
DNA from males (Figure 6b, c).
CGH identi¢ed the Y chromosome in male
mitotic complements (Figure 6b^g). While all
other chromosomes displayed yellowish-brown
£uorescence, the Y chromosome had a large
reddish segment, the colour indicating preferred
binding of male-derived probe. According to
measured signal intensities, the ratio of bound
female- vs. male-derived probe was 0.69 0.09
(mean standard deviation) in this segment
relative to a ratio of 1.0 in the remaining
chromosomes. The order of bands in the Y
chromosome varied depending on the origin of
the Y chromosome. In Y chromosomes labelled
with the Iridescens marker, four segments could
be distinguished: (1) a small DAPI-negative
centromeric band with yellow CGH £uorescence
indicating the presence of repetitive DNA
common to females and males; (2) a large
DAPI-positive presumably euchromatic segment
with brownish CGH £uorescence due to binding
of sequences common to both sexes; (3) a
DAPI-negative band with yellow CGH
£uorescence indicating chromatin with common
repetitive DNA; and (4) a large segment with
red CGH £uorescence indicating preferential
binding of male-speci¢c repetitive DNA
(Figure 6d, e). In Y chromosomes of the wild-
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
Table 1. SC lengths (mm) in male meiosis of the zebra¢sh, Danio
rerio.
SC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
S
Cell 1
9.5
9.2
7.9
7.6H
7.5
7.4
7.2
7.2
7.1
6.8
6.8
6.6
6.5
6.5
6.4
6.2
6.1
6.0
5.7
5.6
5.6
5.5
5.2
4.9
4.7
165.7
Cell 2
10.6
10.5
10.0
9.7H
9.2
9.0
8.9
8.7
8.6
8.3
8.1
7.8
7.6
7.6
7.4
7.4
7.2
7.0
6.9
6.6
6.6
6.5
6.4
6.3
5.5
198.4
Cell 3
12.9
10.9
10.5
10.3
10.2H
9.4
9.3
8.8
8.7
8.4
8.1
8.0
8.0
7.7
7.6
7.5
7.4
7.4
7.0
7.0
6.8
6.7
6.7
6.1
5.7
207.1
Cell 4
12.9
12.9
12.0H
10.7
10.4
10.4
9.2
9.0
8.8
8.6
8.3
8.2
7.7
7.7
7.6
7.5
7.4
7.3
7.0
7.0
6.9
6.5
6.3
6.2
4.6
211.1
Cell 5
16.3
13.8
10.3H
10.3
9.9
9.8
9.7
9.4
9.2
9.2
9.1
9.0
8.9
7.8
7.8
7.7
7.7
7.6
7.3
7.1
7.0
7.0
6.8
6.7
6.7
222.1
H
, bivalent with stalked lumps of silver-stained material, presumably decorating the long heterochromatic arm of chromosome
#3.
caught Mexican guppy, the order of the two distal
segments was reversed and the two segments were
separated by a small dull band (Figure 6f, g).
In DAPI-stained diakineses of male meiosis,
most bivalents appeared to be paired endto-end and cross-shaped bivalents were rare
(Figure 6h, k). DAPI-staining, however, is
deceptive when terminal chromosome segments
are DAPI-negative. CGH revealed some more
bivalents with subterminal chiasmata: in several
bivalents, the DAPI-negative yellow terminal
bands were split, indicating underlying crossshaped ¢gures invisible in the DAPI picture
(compare Figure 6h with j, k with l). With two
exceptions (n ˆ 49), the XY bivalent was paired
end to end. The two exceptions were a ring
con¢guration with both ends paired and a crossshaped bivalent. Autosomal and sex chromosomal
univalents were not rare in the guppy. In a few
665
complements, sex chromosomes were the only
univalents. This allowed us to identify, besides
the Y chromosome, the X chromosome which
had a conspicuous (though smaller than the Y)
terminal yellow-£uorescing segment (Figure 6k, l).
Segregation of X and Y happens predominantly
in anaphase I of meiosis. This was deduced from
the presence or absence of the conspicuous
Y-speci¢c segment in metaphase II plates. In 10
of 22 metaphase II plates, both Y chromosomal
chromatids were present, 11 had no Y, the remaining plate may have had X and Y but was not clear
enough for a safe determination (not shown).
The 23 synaptonemal complexes presented a
gradual length series (Figure 7, Table 3). The
smallest SC was regularly decorated with lumps
of silver-stained material at a site about a quarter
of the length from one end (Figure 7, arrowhead).
Another SC, the second- to seventh-longest,
displayed lateral asymmetry and represented the
XY pair (Table 3, Figure 7, XY). The structure
of the XY SCs varied. In presumably early
pachytene when all autosomal SCs were completely paired, X and Y displayed a paired region
and unpaired axial elements of unequal length,
the longer Y and the shorter X (Figure 8a^c, f^h).
Up to 57% of the Y and 28% of the X axis
remained unpaired in such SCs. In presumably
later stages, the unpaired parts of X and Y became
smaller and ¢nally formed completely paired SCs,
but had lateral elements of unequal thickness
(Figure 8e, i, j). Solari (1992) described a similar
phenomenon from chicken and quail WZ chromosomes and termed it `equalization'. The pairing
pattern of XY bivalents from Iridescens guppies
(Figure 8a^e) was not different from that of
XYs from the Mexican guppy (Figure 8f^j)
although the order of distal Y chromosome bands
was different.
Discussion
In the zebra¢sh, sex ratios vary among broods.
Even gynogenesis produces mixed progeny of
variable, mostly male-biased sex ratios independent of whether diploidization is induced by
suppression of the second meiotic or the ¢rst
mitotic division (Pelegri & Schulte-Merker 1999).
Hence, sex determination is not chromosomal.
666
W. Traut & H. Winking
Figure 5. SC complement of male meiosis from the platy¢sh. N: presumed remnants of a nucleolus; bar: 5 mm.
Table 2. SC lengths (mm) in male meiosis of the platy¢sh Xiphophorus maculatus.
SC
Cell 1
Cell 2
Cell 3
Cell 4
Cell 5
Cell 6
Cell 7
Cell 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5.3
5.3
5.3
5.1
5.0
4.9
4.8
4.8
4.7
4.5
4.5
4.5
4.4
4.3
4.2
4.1
4.0
3.9
3.8
3.8
3.8
3.3
3.3
2.8
5.7
5.5
5.4
5.2
5.1
5.1
5.1
4.9
4.9
4.9
4.9
4.9
4.8
4.7
4.6
4.5
4.5
4.2
4.2
3.9
3.8
3.7
3.6
2.5
6.1
5.7
5.4
5.2
5.1
5.0
5.0
5.0
4.9
4.9
4.8
4.8
4.7
4.7
4.5
4.4
4.3
4.3
4.3
4.0
3.9
3.9
3.6
2.4
6.0
5.5
5.4
5.3
5.3
5.2
5.2
5.1
5.1
5.1
5.0
5.0
4.9
4.8
4.8
4.5
4.5
4.4
4.4
4.3
4.1
4.0
3.9
2.5
7.0
6.9
6.8
6.7
6.6
6.4
6.4
6.3
6.2
6.1
6.0
6.0
5.9
5.7
5.6
5.6
5.2
5.1
5.0
4.7
4.4
4.4
4.0
2.9
7.4
7.3
7.1
7.0
6.9
6.9
6.8
6.8
6.7
6.7
6.7
6.6
6.3
6.0
5.9
5.9
5.6
5.6
5.4
5.4
5.3
5.2
5.0
3.1
7.9
7.9
7.5
7.5
7.4
7.2
7.0
7.0
7.0
6.8
6.8
6.6
6.5
6.3
6.3
6.2
6.1
5.7
5.7
5.7
5.7
5.6
5.2
3.4
8.9
8.6
8.2
8.2
7.7
7.6
7.5
7.3
7.1
6.7
6.7
6.4
6.4
6.4
6.3
6.3
6.2
6.2
6.0
5.7
5.3
5.0
4.8
3.2
104.4
110.6
110.9
114.3
135.9
147.6
155.0
158.7
S
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
667
Figure 6. CGH of guppy Poecilia reticulata chromosomes with female-derived genomic DNA (green) and male-derived DNA (red),
chromosomes stained with DAPI (blue). (a^e) male mitosis from Iridescens guppies; a: DAPI-stained; b: CGH; c: CGH plus DAPI;
d, e: Y chromosomes from Iridescens guppies; (f, g): Y chromosomes from the Mexican guppy; (h^l): diakinesis of male meiosis
from an Iridescens guppy; h: DAPI-stained; i: CGH; j: CGH plus DAPI; (k, l): X and Y unpaired in an Iridescens guppy; k:
DAPI-stained; l: CGH plus DAPI. Bar: 10 mm.
668
W. Traut & H. Winking
Figure 7. SC complement of male meiosis from the Mexican guppy. Arrowhead: the shortest SC, decorated with lumps of
silver-stained material at about a quarter of its length; XY : XY bivalent; bar: 5 mm.
Environmental sex determination has been
suggested for the zebra¢sh (Pelegri &
Schulte-Merker 1999) but we cannot rule out
polygenic sex determination with or without an
environmental component. On the cytogenetic
level, all 25 chromosome pairs have been identi¢ed
with replication banding (Amores & Postlethwait
1999) and none of the 25 linkage groups displays
sex-linkage (http://z¢sh.uoregon.edu). Thus,
none of the 25 chromosome pairs plays a decisive
role in sex determination. It was no surprise,
therefore, to see the 2n ˆ 50 chromosomes of
the female and male diploid complement without
any sign of sex-speci¢c molecular differentiation
after CGH. Sex chromosomes had not been
identi¢ed with C-banding, chromomycin A3 or
with replication banding (Pijnacker & Ferwerda
1995, Daga et al. 1996, Gornung et al. 1997,
Amores & Postlethwait 1999). In pachytene and
in diakinesis, none of the 25 bivalents displayed
signs of morphological or molecular sex chromosome differentiation.
The platy¢sh has a clearly de¢ned sex
chromosome system but with local variation. A
population from the Rio Jamapa in Mexico has
an XX/XY mechanism while several other
populations have W chromosomes besides X
and Y (Gordon 1954, Kallman 1984). The strain
used in this study, was originally derived from
the Rio Jamapa. X and Y chromosomes were
labelled by phenotypically visible genetic markers.
Chromosome morphology does not show obvious
differences between X and Y chromosomes. A
molecular marker, XIR, however, was located
by FISH at the distal end of one of the acrocentric
chromosomes, thereby identifying it as the Y
chromosome (Nanda et al. 2000). This maps the
non-recombining
region
including
the
sex-determining region to the distal end of the
Y chromosome but it is not clear yet how far it
extends proximally as no chiasma distribution
data exist for the sex chromosomes. The distal
region was neither conspicuous in the SC
complement nor with CGH on meiotic and
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
669
Table 3. SC lengths (mm) in male meiosis of the guppy Poecilia reticulata from Mexico.
SC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Cell 1
7.7
6.4XY
5.9
5.5
5.5
5.5
5.4
5.3
5.3
5.2
5.1
5.1
5.1
5.1
5.0
4.8
4.8
4.5
4.5
4.5
4.3
3.9
3.4
S
117.8
Cell 2
7.1
6.0
5.8
5.7XY
5.6
5.6
5.6
5.5
5.4
5.3
5.3
5.2
5.2
5.2
5.2
5.0
4.9
4.6
4.5
4.3
4.3
4.1
3.4
118.8
Cell 3
7.1
6.1
6.1
6.0
5.9
5.7XY
5.7
5.6
5.5
5.2
5.1
4.9
4.9
4.9
4.9
4.7
4.7
4.8
4.6
4.5
4.5
4.1
3.5
119.0
Cell 4
7.7
5.7XY
5.7
5.7
5.7
5.6
5.5
5.4
5.3
5.3
5.3
5.2
5.2
5.1
5.0
5.0
4.9
4.8
4.6
4.6
4.2
3.9
3.7
119.1
Cell 5
7.3
6.5
6.4
6.0XY
5.9
5.8
5.7
5.7
5.5
5.5
5.4
5.2
5.2
5.1
5.0
4.9
4.8
4.6
4.6
4.4
4.4
4.5
3.0
121.4
Cell 6
7.7
7.0XY
6.6
6.0
6.0
6.0
5.9
5.8
5.8
5.6
5.6
5.6
5.3
5.3
5.1
5.1
5.1
5.0
4.6
4.4
4.3
4.2
3.4
125.4
Cell 7
8.5
7.9
6.8
6.8
6.7
6.6
6.3XY
6.1
6.1
6.1
5.8
5.8
5.7
5.7
5.6
5.5
5.4
5.1
5.1
4.9
4.8
4.8
3.7
135.8
Cell 8
9.1
7.0
6.9
6.6
6.5
6.3XY
6.3
6.3
6.2
6.1
6.0
5.9
5.8
5.8
5.7
5.5
5.3
5.2
5.1
4.9
4.9
4.6
3.9
135.9
XY
, XY bivalent.
mitotic chromosomes. Thus sex chromosome
differentiation in the platy¢sh is in an early stage.
Molecular differentiation of X and Y is either
not far enough advanced or restricted to too small
a chromosome segment to be recognizable in
SCs or by CGH.
In contrast, there is clear XY differentiation on
the genetic and the cytogenetic level in the guppy.
A variety of known sex-linked genes control the
form of ¢ns, the colour and pigmentation pattern.
Sixteen of these markers exchange by crossingover between X and Y, seventeen of them are
restricted to the Y chromosome (reviewed by
Kirpic­nikov 1987). The latter class de¢nes a
non-recombining segment including the hypothetical sex locus, the former one a recombining part of
the Y. The segment with male-speci¢c repetitive
DNA which was visualized by CGH, maps the
non-recombining segment to the distal end of
the Y chromosome. The region probably overlaps
with a distal C-band detected by Nanda et al.
(1990) in Y chromosomes of ornamental strains
of the guppy. With in-situ hybridization, this band
bound a (GACA)4 simple repeat probe.
Another aspect of the recombining and
non-recombining segments of the guppy Y
chromosome is provided by SCs. Nearly half of
the Y chromosomal axis is always paired in
pachytene, presumably the homologous and
recombining part, while the other half, the
non-recombining part, is unpaired in presumably
early pachytene stages but engages in
non-homologous pairing in an `equalization'
(Solari 1992) or `adjustment' (Moses & Poorman
1981) process during later pachytene stages.
Similar observations were made of the XY
bivalent from the Nile Tilapia, Oreochromis
niloticus (Carrasco et al. 1999).
The predominant end-to-end pairing con¢guration of the sex chromosomes in diakinesis may
be non-chiasmatic since the non-recombining ends
are involved. In both types of XY bivalents, those
from Iridescens guppies and those from the
Mexican guppy, red and yellow CGH bands
670
W. Traut & H. Winking
Figure 8. SCs of XY pachytene bivalents from the guppy in different stages of adjustment. (a^e) Iridescens guppy; (f^j) Mexican
guppy. Bar: 5 mm.
show up in an order as if mitotic X and Y chromosomes were placed end to end. This excludes
recombination in the homologous part with
subsequent terminalisation of the chiasma.
Dzwillo (1959) determined 8.4% genetic exchange
between the Y-speci¢c marker Ds and the X-Y
marker Cp. If the total recombination frequency
between X and Y is not much higher, then the
frequency of chiasmata (2 in 49) seen in the
homologous segment is not signi¢cantly different
from the expectation (16.8%) on the basis of the
recombination data but this point needs further
investigation.
The three ¢sh species of this study represent a
series of different stages of sex chromosome
evolution. The zebra¢sh is a species with only
homologous chromosome pairs, a stage from
which all sex chromosome evolution is thought
to have started. Acquisition of the sex-determining
function at one end of a chromosome pair would
lead to the form of sex chromosomes found in
the platy¢sh: an XY pair with little differentiation,
terminal molecular differences between X and Y
but no visible effects on the cytogenetic level or
by CGH. Addition of large amounts of genetic
material to the distal chromosome ends of
platy¢sh-like sex chromosomes enlarges the
non-recombining region and results in a situation
as seen in the guppy: conspicuous molecular
and cytogenetic differentiation at the distal
chromosome ends. Interestingly, X and Y
chromosomes are de¢ned by the distal segments
only while the centromeres and roughly the
proximal half of the guppy Y chromosomes
(probably even more in the platy¢sh Y) are not
Y-speci¢c but shared by X and Y chromosomes.
Molecular changes in this segment, e.g. the
different order of bands, are transmitted in the
respective male lines of offspring. In Southern
hybridization, (GACA)4 and (GATA)4 probes
detected male-speci¢c variation among outbred
guppies and various inbred strains (Nanda et al.
Meiotic chromosomes and stages of sex chromosome evolution in ¢sh
1990). Thus, there is presumably more variation
among guppies in the structure of the distal Y
chromosome region than described here.
Acknowledgements
We thank Ulrike Eickhoff, Hans-GÏnther Mertl
and Conny Reuter for technical assistance. Dr.
Thomas Becker (Hamburg, Germany), Dr. Hans
Georg Frohnhoefer (TÏbingen, Germany),
Professor Manfred Schartl (WÏrzburg, Germany)
and Reinhold Nickel (LÏbeck-TravemÏnde,
Germany) have kindly provided us with ¢sh
specimens. Dr. Annerose Anders (Giessen,
Germany), Professor Michael Dzwillo (Hamburg,
Germany), Dr. Indrajit Nanda (WÏrzburg,
Germany) and Dr. Petr Räb (Libechov, Czech
Republic) shared their expertise of ¢sh genetics
with us.
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