Evidence for a monophyletic origin of triploid clones of the Amazon molly, Poecilia formosa.
ABSTRACT Asexual reproduction in vertebrates is rare and generally considered an evolutionary dead end. Asexuality is often associated with polyploidy, and several hypotheses have been put forward to explain this relationship. So far, it remains unclear whether polyploidization in asexual organisms is a frequent or a rare event. Here we present a field study on the gynogenetic Amazon molly, Poecilia formosa. We used multilocus fingerprints and microsatellites to investigate the genetic diversity in 339 diploid and 55 triploid individuals and in 25 P. mexicana, its sexual host. Although multilocus DNA fingerprints found high clonal diversity in triploids, microsatellites revealed only two very similar clones in the triploids. Phylogenetic analysis of microsatellite data provided evidence for a monophyletic origin of the triploid clones of P. formosa. In addition, shared alleles within the triploid clones between the triploid and diploid genotypes and between asexual and sexual lineages indicate a recent origin of triploid clones in Poecilia formosa.
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ABSTRACT: We review the different modes of meiosis and its deviations encountered in polyploid animals. Bisexual reproduction involving normal meiosis occurs in some allopolyploid frogs with variable degrees of polyploidy. Aberrant modes of bisexual reproduction include gynogenesis, where a sperm stimulates the egg to develop. The sperm may enter the egg but there is no fertilization and syngamy. In hybridogenesis, a genome is eliminated to produce haploid or diploid eggs or sperm. Ploidy can be elevated by fertilization with a haploid sperm in meiotic hybridogenesis, which elevates the ploidy of hybrid offspring such that they produce diploid gametes. Polyploids are then produced in the next generation. In kleptogenesis, females acquire full or partial genomes from their partners. In pre-equalizing hybrid meiosis, one genome is transmitted in the Mendelian fashion, while the other is transmitted clonally. Parthenogenetic animals have a very wide range of mechanisms for restoring or maintaining the mother's ploidy level, including gamete duplication, terminal fusion, central fusion, fusion of the first polar nucleus with the product of the first division, and premeiotic duplication followed by a normal meiosis. In apomictic parthenogenesis, meiosis is replaced by what is effectively mitotic cell division. The above modes have different evolutionary consequences, which are discussed. See also the sister article by Grandont et al. in this themed issue.Cytogenetic and Genome Research 06/2013; · 1.84 Impact Factor
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ABSTRACT: ▪ Abstract Most metazoans engage in recombination every generation. In theory this is associated with considerable cost, such as the production of males, so that asexual organisms, which do not pay this cost, should be able to invade populations of sexuals. Some asexuals depend on sperm of sexual males to trigger embryogenesis, a reproductive mode called gynogenesis. The genetic information of males is typically not used. Theory predicts that such mating complexes are short-lived and highly unstable. Sperm dependency is not only the defining feature of the biology of gynogenetic metazoans, it is also a major puzzle in evolutionary biology. Organisms that apparently combine disadvantages of both sexuality and asexuality are a serious challenge to theory. A number of questions about these systems are still unresolved.Annual Review of Ecology Evolution and Systematics 11/2005; 36:399-417. · 10.98 Impact Factor
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ABSTRACT: Flow cytometry, a method of rapidly characterizing optical properties of cells and cell components within individuals, populations, and communities, is advancing research in several areas of ecology, systematics, and evolutionary biology. Measuring the light emitted or scattered from cells or cell components, often in combination with specific stains, allows a multitude of physical and genetic attributes to be evaluated simultaneously and the resulting information to be rapidly processed. As a result, the technique has enabled large-scale comparative analyses of genome-size evolution, taxonomic identification and delineation, and studies of polyploids, reproductive biology, and experimental evolution. It is also being used to characterize the structure and composition of microbial communities. Here, we outline the nature of these contributions, as well as future applications, and provide an online summary of protocols and sampling methods.Annual Review of Ecology Evolution and Systematics 12/2007; 38:847-876. · 10.98 Impact Factor
? 2005 The Society for the Study of Evolution. All rights reserved.
Evolution, 59(4), 2005, pp. 881–889
EVIDENCE FOR A MONOPHYLETIC ORIGIN OF TRIPLOID CLONES OF THE AMAZON
MOLLY, POECILIA FORMOSA
KATHRIN P. LAMPERT,1,2,*DUNJA K. LAMATSCH,1,*JO¨RG T. EPPLEN,3AND MANFRED SCHARTL1
1Department of Physiological Chemistry I, University of Wuerzburg, Biozentrum, Am Hubland, 97074 Wuerzburg, Germany
3Department of Human Genetics, University of Bochum, 44801 Bochum, Germany
is often associated with polyploidy, and several hypotheses have been put forward to explain this relationship. So far,
it remains unclear whether polyploidization in asexual organisms is a frequent or a rare event. Here we present a field
study on the gynogenetic Amazon molly, Poecilia formosa. We used multilocus fingerprints and microsatellites to
investigate the genetic diversity in 339 diploid and 55 triploid individuals and in 25 P. mexicana, its sexual host.
Although multilocus DNA fingerprints found high clonal diversity in triploids, microsatellites revealed only two very
similar clones in the triploids. Phylogenetic analysis of microsatellite data provided evidence for a monophyletic origin
of the triploid clones of P. formosa. In addition, shared alleles within the triploid clones between the triploid and
diploid genotypes and between asexual and sexual lineages indicate a recent origin of triploid clones in Poecilia
Asexual reproduction in vertebrates is rare and generally considered an evolutionary dead end. Asexuality
ternal introgression, polyploidy.
Clonal diversity, gynogenesis, microsatellite marker, Muller’s ratchet, multilocus DNA fingerprint, pa-
Received July 21, 2004.Accepted January 5, 2005.
Strictly unisexual vertebrate species are rare and an ex-
ception to the dominant mode of sexual reproduction in an-
imals. The Amazon molly, Poecilia formosa, was the first
vertebrate discovered to reproduce clonally (Hubbs and
Hubbs 1932). This all-female species has become a model
system for studying the evolution of sex. Like many unisexual
organisms it originated from a hybridization event (Dawley
1989), most likely between P. mexicana limantouri and P.
latipinna (Avise et al. 1991; Schartl et al. 1995a). Its repro-
ductive mode is gynogenesis: sperm from a closely related
bisexual species is needed to stimulate the onset of embryo-
genesis of the unreduced diploid eggs (Monaco et al. 1984),
but usually makes no genetic contribution to the embryo
(Schartl et al. 1990). In very rare cases, however, the exclu-
sion mechanism fails and either small parts of male genetic
material remain inside the oocyte in the form of microchro-
mosomes (Schartl et al. 1995b), or the sperm nucleus fuses
with the oocyte nucleus leading to gynogenetic triploid in-
dividuals (Rasch and Balsano 1974; Turner et al. 1980a,b;
Lamatsch et al. 2000a).
Many asexually reproducing organisms are polyploid even
though their ancestral closely related sexual species are dip-
loid. Three main hypotheses have been suggested to explain
this: (1) polyploidization might interrupt meiosis, therefore
creating asexual lines. (2) There might not be a high selection
against polyploids in asexual lines. (3) It might be advan-
tageous to the asexuals to be polyploid (Mogie 1986). Be-
cause P. formosa is a hybrid species, and diploid genotypes
also reproduce asexually, it can be excluded for this species
that polyploidy interrupts meiosis and therefore is the basis
for asexuality. To distinguish between the other two hypoth-
eses we considered the genetic variability of triploid indi-
viduals compared to diploids in this species. An equal or
lower level of genetic diversity in triploids would suggest
that polyploidization might simply not be selected against.
Higher genetic variability as a precondition to adapt to newly
emerging selective pressure would provide evidence that trip-
loids enjoy the advantage of additional genetic material.
Various studies have tried to determine the amount of ge-
netic variability in diploid P. formosa populations. Immu-
nological techniques (tissue transplantations) were used first
(Kallman 1962), followed by allozyme studies (Turner et al.
1980a,b). DNA fingerprinting revealed very high levels of
genetic variation in natural populations (Turner et al. 1990).
Triploid clonal variability has only been estimated using al-
lozymes and was proposed to be rather high (Turner et al.
Since multilocus DNA fingerprints revealed the highest
number of clones for diploid unisexual fish, we used this
method for a study of genetic diversity in diploid and triploid
P. formosa. In addition, we used three microsatellite loci
because they allow work on a larger number of individuals.
We also wanted to validate this method for population genetic
analysis of clonally reproducing vertebrates. To investigate
whether polyploidy in clonal fish could bear the advantage
of higher genetic variability, we investigated the following
questions: Are triploids in natural populations genetically
more variable than diploid individuals? How frequently do
successful paternal introgressions lead to triploid individu-
als? Are these incorporations an old or a recent event in P.
Here we report a high degree of genetic variability in dip-
loid and less variation in triploid P. formosa indicating a
recent monophyletic origin of triploid clones in this species.
Microsatellites proved to be useful tools for the investigation
of clonal diversity in asexual organisms.
MATERIAL AND METHODS
Multilocus DNA fingerprints
and 39 diploid fish from two different years from the Rio
For multilocus DNA fingerprints, 10 triploid
KATHRIN P. LAMPERT ET AL.
The known occurrence of triploid P. formosa clones is marked with a dotted line and magnified. The magnified map inset shows the
Soto la Marina river system with the study sites (I–V). Arrows depict sampling sites; capital letters, documented catches of triploid P.
formosa (map modified from Balsano et al. 1989).
Distribution of Poecilia formosa (framed in black) and its host species P. mexicana (dark gray) and P. latipinna (light gray).
Purification (Mexico) were used. We mainly sampled one
population with especially high P. formosa abundances (I).
At this site we sampled four different habitat types: Ia, fast
flowing main river; Ib, slow flowing side arm of the river;
Ic, large, deep, cool pond (only temporarily connected to the
main river after rainy season); Id, small pond (rarely con-
nected to the main river). In addition, we investigated fish
from four more localities (II–V) to represent the full known
range of P. formosa triploids (Fig. 1). Liver, brain, eyes, and
muscles of each individual were pooled and stored at ?80?C.
DNA was extracted according to Blin
and Stafford (1976). HinfI was used for restriction digestion,
and the fragments were separated on a 0.8% agarose gel at
1 V/cm. In-gel hybridization was done essentially as de-
scribed by Nanda et al. (1988). The32P-labeled oligonucle-
otides (GATA)4, (GGAT)4, (GA)8, and (CA)8were used as
tioned above were used. Fish were captured in 1996, 1998,
In total, 450 fishes from the eight sites men-
MONOPHYLY OF POECILIA FORMOSA TRIPLOIDS
and 2002 using seine nets. One (5 mm2) piece of the fast
regenerating dorsal fin was cut from each individual and
stored in 70% alcohol. Immediately after taking the biopsy,
animals were released otherwise unharmed. In addition, fins
of 25 P. mexicana (the sexual host of P. formosa in this
region) were used. Only the subspecies P. mexicana liman-
touri lives here (hereafter P. mexicana).
All fins were cut in half to use one
part to determine ploidy levels via flow cytometry. For this
fins were stained with DAPI, and DNA contents were deter-
mined using chicken blood as a reference, essentially as de-
scribed (Lamatsch et al. 2000b). DNA for polymerase chain
reaction (PCR) analysis was extracted according to Altsch-
mied et al. (1997). For isolation of primers that amplify in-
formative microsatellite loci in various Poecilia species, total
genomic DNA from P. latipinna (from Key Largo, FL) and
P. catemaconis (from Laguna Catemaco, Veracruz, Mexico)
was digested to completion with MboI and cloned into p-
BSKII?. A total of 3000 recombinant clones was individu-
ally spotted on replica plates using a robotic device. The
library was screened with oligonucleotide probes (GGAT)4,
(CA)8, (TAC)8and (TAA)8TA that were end-labeled with
?32P-ATP using terminal transferase. Positive clones were
isolated and sequenced. Primers were designed from the sin-
gle copy flanking sequences of simple repeat sequences that
had a repeat number greater than eight leading to primer-pair
Sat1 (forward primer: 5?ACAGCAGCTGTTCACGGC 3?,
backwards primer: 5?TCGGAGGAAAAACAGATGAC 3?
repeat motive: (CA)CGTA(CA)10allele size range: 106–
152bp). Additionally, cross-species reactions with primers
working in Xiphophorus hellerii (Seckinger et al. 2002) and
P. reticulata (Becher et al. 2002) were used to identify mi-
crosatellites in P. formosa. A total of 35 primer pairs was
tested, three of which revealed polymorphic loci in P. for-
mosa: KonD15 (Seckinger et al. 2002); PR39 (Becher et al.
2002), and Sat1. Polymerase chain reactions were done at:
90 sec of initial denaturation, 30 sec at 94?C, 30 sec at an-
nealing temperature (52?C for KonD15, 58?C for Sat1, 54?C
for PR39) and 30 sec at 72?C for 35 cycles followed by a
300 sec elongation. PCR products were analyzed on an ALF
Express sequencer (Amersham Biosciences, Freiburg, Ger-
viations from Hardy-Weinberg equilibrium for the sexual P.
mexicana were calculated using Arlequin (Schneider et al.
2000). Genetic diversity among triploid and diploid P. for-
mosa and P. mexicana was described following Menken et
al. (1995). We calculated the proportion of distinguishable
clones (PDC: number of clones divided by sample size; a
value of 1.00 means every individual has a different geno-
type), the effective number of genotypes (ENC: 1/?
frequency of the ith clone in a population), genotypic diver-
sity (CD: 1 ? ?
) and genotypic evenness (CE: (1/?
Nc, with Nc being the number of genotypes). Genotypic even-
ness (CE) gives a relative and therefore comparable measure
for the distribution of genotypes. This was important because
sample sizes varied extensively between ploidy levels. A CE
Population differentiation (FST) and de-
value of 1.00 means all clones are equally distributed. To
estimate the effect of additional microsatellites on the ability
of this method to detect genetic differentiation, we calculated
all measurements suggested by Menken et al. (1995) not only
for the final combination of three microsatellite loci, but also
for each locus and for each combination of two loci sepa-
We used several different approaches
to analyze the phylogenetic relationships between triploid
and diploid P. formosa clones and their sexual host, P. mex-
icana. The general genetic identity between diploid and trip-
loid P. formosa and P. mexicana was estimated using the
measurement proposed by Tomiuk and Loeschke (1991). This
measure was introduced to infer genetic identity between
asexual and sexual species and populations of differentploidy
levels. For a more detailed evaluation of relationships be-
tween individual genotypes we created neighbor-joining(Sai-
tou and Nei 1987) trees using an allele sharing index (DAS;
Chakraborty and Jin 1993) and Cavalli-Sforza and Edwards
chord distance (CSE; Cavalli-Sforza and Edwards 1967).
DAS was recommended for microsatellites by Goldstein et
al. (1995) as a useful tool for clustering related individuals,
and CSE by Takezaki and Nei (1996) as the most reliable
tool to generate tree topologies. Both methods have recently
been used to investigate phylogenetic relationships between
sexual and asexual populations of animals (Simon et al. 1999;
Delmotte et al. 2003). Calculations for the Tomiuk and Loes-
chke (1991) genetic identity matrix were done using the pro-
gram Popdist 1.1.1 (Guldbrandtsen et al. 2000). All other
distances and the neighbor-joining trees were calculated us-
ing the program Populations 1.2.28 (Langella 1999). Boot-
strapping values for the DAS and CSE trees were derived
from 1000 iterations. Because the reduction of discrete mor-
phologies to genetic distances might result in a loss of in-
formation, we additionally calculated a Wagner maximum
parsimony tree (Felsenstein 1989). This program uses binary
data; we therefore coded our microsatellite size data as n
discrete characters (n, number of alleles; Schalkwyk et al.
1999). To circumvent the problem of diploid and triploid
genotypes in the same dataset, we first calculated a tree cod-
ing every genotype as triploid. We added the same allele to
all diploid P. formosa and P. mexicana. Because this approach
might overestimate the degree of relatedness within the dip-
loids, we performed another analysis in which all triploid
individuals were treated as diploid by randomly leaving out
one of their three alleles in each locus. Bootstrapping values
were obtained from 1000 (all-triploid approach) and 1350
(all-diploid approach—50 per allele combination) replicates.
We used the programs Seqboot to generate the jackknife rep-
licates, MIX to generate the Wagner parsimony trees, and
Consense to generate a consensus tree from all the replicate
trees (Felsenstein 1989).
Ploidy level and genetic variability between years and
For all individuals of P. formosa, ploidy was determined
by flow cytometry. Ploidy levels varied between years: one
triploid and 20 diploids in the samples from 1996, 11 triploids
KATHRIN P. LAMPERT ET AL.
and 15 diploids in 1998, and 43 triploid and 316 diploid P.
formosa in 2002. In two of the eight populations no triploids
were found. Therefore, these sampling sites were excluded
from further analysis. For the remaining six sites sample sizes
varied greatly (n: Ia ? 85, Ib ? 105, Ic ? 60; Id ? 21; II
? 3; III ? 29). Because we could not find a significant level
of genetic diversification between the sampling sites (FST:
0.00–0.07; P ? 0.05), all data were pooled.
Clonal variability detected by multilocus DNA fingerprints
Multilocus DNA fingerprints of 10 triploid and 39 diploid
P. formosa revealed five genotypes in the triploids and 12
different diploid genotypes (Table 1). Statistical analysis re-
vealed a higher proportion of distinguishable clones in trip-
loids. The effective number of clones was clearly higher in
diploids than in triploids, whereas clonal diversity was only
slightly higher in diploids. Clonal evenness was higher in
triploids (Table 2A).
Low clonal variability of triploids detected by microsatellite
We found three polymorphic microsatellite loci for P. for-
mosa: Sat1, KonD15, and PR39. Analysis of their alleles
(Tables 1, 2B) led to the definition of 10 genotypes for primer
Sat1, seven for KonD15, and five in PR39 in diploid fish
(Table 1A). In triploids only two very similar genotypes were
found for each microsatellite (Table 1B). All diploids were
heterozygous at all three loci. Triploids always showed three
alleles. Genotypes varied highly in abundance. Intriguingly,
we found the same two triploid clones in all years and at all
sites. Triploid and diploid individuals shared a high number
of alleles: 71% (Table 1). An overall higher clonal variety
was observed in diploids (26 clones in 339 individuals) than
in triploids (two clones in 55 individuals). Consequently, a
higher proportion of distinguishable clones in diploids than
in triploids was found, as well as a higher number of effective
clones in diploids but lower clonal evenness than in triploids
Twenty-three genotypes were found for the 25 P. mexicana
investigated (Table 1C). They had an effective number of
21.6 different genotypes and a proportion of distinguishable
genotypes of 0.93. Genotypic diversity was very high and
different genotypes almost equally distributed (Table 2B). A
slight heterozygote deficiency in the Sat1 and KonD15 loci
was found in P. mexicana, probably due to the low number
of individuals scored. Increasing the number of microsatellite
loci revealed more genotypes in sexual (P. mexicana) and
asexual (P. formosa) diploids. Consequently, the proportion
of distinguishable genotypes increased. However, triploids
did not show any difference in their genotypic pattern wheth-
er one, two, or three loci were included in the analysis.
Microsatellites as tools to detect genetic variation even in
Since microsatellites have so far not been used extensively
to estimate genetic differentiation in clonal vertebrates, we
compared the microsatellite data with the results of the mul-
tilocus DNA fingerprints. A direct comparison of methods
was possible because 36 of the 49 individuals used for the
DNA fingerprint study were also used in the microsatellite
analysis. Microsatellites revealed fewer clones (six) than did
multilocus fingerprints (12). Some microsatellite clones di-
rectly represented a distinct clone (also found using multi-
locus fingerprints), though in some cases multilocus finger-
prints differentiated more than one clone in a microsatellite
line (Table 1).
The genetic identities calculated after the method of Tom-
iuk and Loeschke (1991) revealed low to medium levels of
relatedness between the sexual P. mexicana and the asexual
P. formosa genotypes, whereas the asexual groups (diploid
and triploids) showed a much higher genetic similarity (ge-
netic similarity: diploid/triploid ? 0.590; mexicana/diploid
? 0.088; mexicana/triploid ? 0.232).
The neighbor-joining trees created from the DAS and the
CSE genetic distances (data not shown) were very similar
and revealed three clearly distinct groups: P. mexicana ge-
notypes were separated from all asexual P. formosa geno-
types, while within the P. formosa clones, triploids were sep-
arated from diploids (bootstrapping values: 90%). The mi-
crosatellites, however, did not further resolve genotype re-
latedness within these major groups (all bootstrapping values
?70%). The results from the Wagner parsimony analysis
supported these findings. A consensus tree calculated from
three alleles per individual revealed strong support for a
monophyletic origin of the triploids (76%; data not shown).
The consensus tree calculated from two alleles per genotype
also found a monophyletic origin of the two triploid geno-
types to be the most likely solution, although bootstrapping
support for this solution was only 35% (Fig. 2).
A detailed look at allele sizes revealed that the diploid
HBB genotype and the triploid genotypes YYY shared a high
number of alleles. The triploid clone ZZZ still had one allele
in common with YYY and HBB in the Sat1 and KonD15
locus. In the PR39 locus it even shared two alleles with both
other genotypes. Although ZZZ and YYY shared only one
allele in the Sat1 locus, their allelic pattern was very similar.
YYY alleles that were not found in the HBB genotype were
very close (Sat1) or even similar in size (KonD15, PR39) to
alleles found in P. mexicana (Table 1).
Are microsatellites adequate tools to describe clonal
Since microsatellites have so far not been used to determine
genetic variability in asexual vertebrate species, we estimated
their value by comparing microsatellite results with the re-
sults of the multilocus DNA fingerprints. The three micro-
satellites developed for this study proved to be excellent tools
to investigate clonal diversity in P. formosa. Even though in
a direct comparison they identified fewer clones than did
multilocus fingerprints in the asexuals, their resolution was
good enough to differentiate between almost every sexual
individual. In addition, they allowed analysis of a large group
MONOPHYLY OF POECILIA FORMOSA TRIPLOIDS
mexicana (C). n, number of animals. Shared alleles between triploid and diploid genotypes are bold; shared alleles between P. formosa
and P. mexicana (with either diploids or triploids or both) are italic. In the last column multilocus DNA fingerprint genotypes (letters)
for 36 P. formosa that were analyzed with both methods are given (numbers of individuals).
Number of microsatellite genotypes and allele sizes found in 337 diploid (A) and 55 triploid (B) Poecilia formosa and in P.
Allele sizes at locus
24f(7); h(1); i(1)
A(1); C(4); C?(1)
1Significant heterozygote deficiency.
KATHRIN P. LAMPERT ET AL.
investigated, numbers of clones, and statistical analysis of data following Menken et al. (1995) are given. (B) Allele numbers, genotypes,
and statistical description of microsatellite data on genotypic variation in diploid and triploid P. formosa and P. mexicana. Numbers of
alleles represent the total number of different alleles found in the always heterozygous diploids and in the triploids that also differed in
all three alleles. All results for the three variable microsatellites and for their combined genotypes are given.
(A) Results from the multilocus DNA fingerprint analysis for diploid and triploid Poecilia formosa. Total number of fish
A. Multilocus DNA fingerprintsDiploidTriploid
No. of genotypes
Effective no. of genotypes (ENC)1
Proportion distinguishable (PDC)2
No. of unique genotypes3
Genotypic diversity (CD)4
Genotypic evenness (CE)5
B. MicrosatellitesDiploid Triploid
number of alleles
number of genotypes
number of alleles
number of genotypes
number of alleles
number of genotypes
no. of genotypes
effective no. of genotypes (ENC)1
proportion distinguishable (PDC)2
no. of unique genotypes3
genotypic diversity (CD)4
genotypic evenness (CE)5
All loci 26
1Calculated as 1/? p , pi being the frequency of the ith genotype.
2No. of genotypes divided by sample size.
3A unique genotype is represented by one individual.
4Calculated as 1 ? ? p , pi being the frequency of the ith genotype.
5Effective number of genotypes divided by number of genotypes.
of several hundred individuals and gave valuable additional
information by identifying all alleles of an individual.
Clonal variability of triploids
From the multilocus fingerprint data we had expected high
levels of clonal variability in triploids. However, microsat-
ellites revealed very low levels of genetic differentiation in
triploid P. formosa. These findings contradict a number of
earlier studies investigating clonal diversity in the asexual
fish Poeciliopsis (e.g., Vrijenhoek et al. 1978). Also, Turner
et al. (1983), using allozymes, found no difference in genetic
variability between diploid and triploid P. formosa. We pur-
posefully sampled all habitat types (see Ia–d) to exclude a
possible sampling bias due to differences in ecological niche
adaptations in different clones as proposed by Vrijenhoek
(1998). However, a more recent study of the Poeciliopsis
complex (Quattro et al. 1992) revealed low diversity in trip-
loids compared to diploids and therefore supports our finding.
How frequent are successful introgressions?
The neighbor-joining trees and the Wagner parsimony
analysis clearly point toward a single triploidization event.
All asexual lineages were separated from the sexual lineages,
and within the asexuals triploids formed their very own clade.
Overall bootstrap values were relatively low, most likely due
to the low number of informative microsatellite loci that are
available in this parthenogenetic species. However, the boot-
strap values for the branches separating the sexuals from the
nonsexuals, and the triploids from the diploids, were rea-
sonably high to be considered valid (90%). Constructing the
Wagner parsimony tree was somewhat difficult as diploid
and triploid genotypes could not be analyzed in the same
dataset. The result of the diploid-only consensus tree was
therefore very conservative, but still revealed the monophy-
letic origin as the most parsimonious solution, even though
the bootstrapping support was not overwhelming. Since both
ways of creating phylogenetic trees showed the same tree,
which was additionally supported by the Tomiuk and Loesch-
ke (1991) genetic identity, we are confident that those trees
give a reliable picture of the actual relatedness of clones.
More support for a monophyletic origin of the triploid
clones came from the allelic patterns observed in the two
triploid clones. Both triploid clones shared at least one allele
at every locus. Alleles that were not identical showed a very
similar pattern that could be explained by subsequent mu-
tations. It seems highly unlikely that two independent intro-
gression events converged by mutation to such a similar al-
lelic pattern. Furthermore, in the whole geographic range of
P. formosa, triploid clones are found in only one river system
(Schlupp et al. 2002) and new triploidizations in laboratory
strains are always sterile (Nanda et al. 1995). A random com-
bination of genotypes is obviously not sufficient to produce
fertile new clones. Considering the rarity of hybridizations
leading to successful unisexual lineages (Dawley and Bogart
1989), most likely due to the genetic (Moritz et al. 1989)
and ecological (Vrijenhoek 1989) constraints on asexuality,
it seems likely that the origin of triploids in P. formosa was
restricted to a single introgression event. Similar arguments
have been used by Quattro et al. (1992) to explain the origin
MONOPHYLY OF POECILIA FORMOSA TRIPLOIDS
allele combination; see text).
Wagner parsimony tree for all genotypes. Bootstrapping values are derived from 1350 randomizations (50 trees per triploid
of triploids in the Poeciliopsis complex. Turner et al. (1990)
interpreted the extraordinarily high level of clonal diversity
found in a DNA fingerprint study as evidence of mutation as
a driving force in variation processes in P. formosa. Other
authors also proposed that mutations are more likely to be
the source of variation rather than multiple hybridizations
(Kallman 1962; Schartl et al. 1995a). The rarity of successful
introgressions and the lack of tetraploids or higher ploids
makes the hypothesis of low selective pressure against po-
lyploidization in asexuals rather unlikely.
Is triploidization an ancient or recent event in Poecilia
The high proportion of shared alleles in triploids and dip-
loids (71%) points to a rather recent origin of the triploid
clones in P. formosa (Birky 1996). Diploids, however,
showed a high number of allelic divergences and are therefore
likely to be more ancient. We identified HBB as a clone
closely similar to the likely diploid ancestor with the triploid
clone YYY. This assumption was based on the striking sim-
ilarity of alleles. Also, the close proximity of the ‘‘third’’
alleles in triploid P. formosa to alleles found in P. mexicana
lends support to the assumption of a rather recent introgres-
sion of a P. mexicana genome. Triploids seem to be old
enough to be slightly different in one of the three loci but
still rather young considering the close proximity to each
other and the diploid HBB clone.
Triploidization is often interpreted as being advantageous
for asexually reproducing organisms, as it might increase the
ecological and evolutionary potential by providing additional
genetic variability. It was generally assumed that diploid P.
formosa arose from a single or very few hybridization events
whereas subsequent triploidization was a more common event
(Turner et al. 1990). Our data, however, point in a different
direction: the low amount of variation in all of the micro-
satellite loci in triploids provides evidence for a relatively
recent monophyletic origin. Thus, it is reasonable to assume
that mutations rather than multiple hybridization events are
the major source of genetic variation and divergence in the
triploid clones of P. formosa. Triploidization seems unlikely
to be P. formosa’s main ‘‘strategy’’ for overcoming the mu-
tational meltdown predicted for its species (Lynch et al.
1993). However, triploidization definitely contributes to ge-
netic diversification and might, therefore, have a key function
by creating multiple starting points for mutations. On an in-
dividual basis, being triploid might still be advantageous,
even though on a population scale diploids seem to be more
KATHRIN P. LAMPERT ET AL.
diverse than triploids. Even rare diversification events may
be sufficient to slow down Muller’s ratchet (Muller 1964).
We are grateful to the Mexican government for issuing
permit no. 210696-213-03 to collect P. formosa, and to J.
Parzefall, M. Do ¨bler, I. Schlupp, U. Hornung, and A. Fros-
chauer for help in the field. For help with the laboratory work
we thank S. Schories. B. Wilde developed the microsatellite
Sat1 used in this study. We thank H. Schwind, G. Schneider,
and P. Weber for breeding the fish in the laboratory, and C.
West Wheat and N. Pongratz for critically reading the man-
uscript. Financial support for this study was granted by the
Deutsche Forschungsgemeinschaft (SFB 567 Mechanismen
der interspezifischen Interaktion von Organismen) and Fonds
der Chemischen Industrie. We thank M. Schmid for the pos-
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Corresponding Editor: G. Ortı ´