Journal of Heredity Advance Access published online on January 21, 2008
Journal of Heredity, doi:10.1093/jhered/esm102
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Brief Communications |
A Tetraploid Amazon Molly, Poecilia formosa
From the Department of Physiological Chemistry I, University of Wuerzburg, Biozentrum, Am Hubland, 97074 Wuerzburg, Germany (Lampert, Lamatsch, Fischer, and Schartl); and the Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK (Lamatsch)
* D.K.L. contributed equally to the work
Address correspondence to K. P. Lampert, Department of Evolutionary Ecology and Biodiversity of Animals, University of Bochum, 44803 Bochum, Germany, or e-mail: kathrin.lampert{at}ruhr-uni-bochum.de.
Polyploidization is thought to be an important driving force in evolution as it increases the genetic material on which mutation and selection can act. In the Amazon molly, Poecilia formosa, triploid genotypes can be found in the field and frequently arise from diploid breeding stocks, a tetraploid individual, however, was so far never documented. Here, we report the first tetraploid Amazon molly. Flow cytometry clearly showed the tetraploid DNA content, whereas microsatellite analysis not only confirmed the tetraploidy but also pointed to allotetraploidy. Most likely the fourth genome was received through paternal leakage, namely, by fertilization of a triploid egg with a haploid sperm. The existence of tetraploid individuals offers new explanations for the enormous clonal diversity observed in wild populations of P. formosa.
The most obvious advantage of polyploidization is the drastic increase of DNA amounts in an organism. Gene duplication leads to gene redundancy and can lead to heterosis (Comai 2005). Because redundant genes are available for the evolution of new functions or for subfunction partition (Taylor and Raes 2004), polyploidy is discussed to have a high impact on evolution (Ohno 1970). In the long term, however, loss of redundant genetic material should be advantageous. Polyploid individuals are reported for several fish species, for example, in carp (Futami et al. 2005), trout (Moghadam et al. 2005), and spiny loach (Vasil'ev et al. 1989). In fish, polyploidy is often associated with unisexual reproduction (Mogie 1986); however, it is not clear whether it is the cause or the consequence (Mable 2004). The Amazon molly, Poecilia formosa, was the first vertebrate discovered to reproduce clonally (Hubbs CL and Hubbs LC 1932). This all-female species has become a model system for studying the evolution of sex. Like all other unisexual organisms (Dawley 1989; Vrijenhoek 1989), it originated from a hybridization event of 2 distantly related species of the same genus, here most likely between a Poecilia mexicana limantouri female and a Poecilia latipinna male (Turner 1982; Avise et al. 1992; Schartl, Wilde et al. 1995). Its reproductive mode is sperm-dependent parthenogenesis (gynogenesis): Sperm from a closely related bisexual species is needed to trigger the onset of embryogenesis of the unreduced diploid eggs (Rasch and Balsano 1974; Monaco et al. 1984), but usually makes no genetic contribution to the embryo (Schartl et al. 1990; Turner et al. 1990). In very rare cases, however, the exclusion mechanism fails and either small parts of male genetic material remain inside the oocyte in the form of microchromosomes (Schartl, Nanda et al. 1995; Lamatsch et al. 2004) or the sperm nucleus fuses with the oocyte nucleus leading to gynogenetic triploid individuals (Rasch and Balsano 1974; Turner, Brett, Miller 1980; Turner, Brett, Rasch, Balsano 1980; Lamatsch, Nanda et al. 2000). In both cases, the paternal genetic material is stably transferred over at least several generations analyzed so far (Schartl, Nanda et al. 1995; Lamatsch et al. 2004). Although the majority of P. formosa populations is comprised of diploid individuals, triploids in P. formosa are most likely of monophyletic origin and show low genotypic diversity (Lampert et al. 2005). Diploid and triploid individuals reproduce gynogenetically. In the laboratory, triploidization of single offspring from diploids can be observed relatively frequently (Nanda et al. 1995) but always leads to sterile individuals. Although theoretically in a gynogenetic species an elevation from triploidy to tetraploidy seems as likely as an elevation from diploidy to triploidy, a tetraploid individual of the Amazon molly has never been reported so far neither from any of the well-studied field sites or from the laboratory. Here, we report the first tetraploid Amazon molly. We show that the tetraploid was a result of an alloploidization event and discuss the potential significance of tetraploids in the evolution of P. formosa.
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Most specimens tested were part of a study of clonal competition designed to determine whether triploid or diploid clonal lines were more successful in reproduction (Lamatsch DK, Lampert KP, Fischer P, Geiger M, Schlupp I, Schartl M, in preparation). The fish were bred in the laboratory from strains originating from fish collected from the Río Purificación. A single diploid and a single triploid line were kept in groups in large tanks in an 12:12 (light:dark) hour light cycle.
For flow cytometry, fin cells were stained with DAPI essentially as described by Lamatsch, Steinlein et al. (2000). At least 10.000 cells were measured per sample. The DNA content of the cells was determined using chicken red blood cells as a reference (Vinogradov 1998).
DNA for polymerase chain reaction (PCR) analyses was extracted using chelex from fin-clips or tissue samples depending on animal size (Altschmied et al. 1997). For the analyses, 5 primers (Sat1, KonD15, PR39, mATG38, and mATG44) that were already shown to be variable in P. formosa were selected (Lampert et al. 2005, 2006), and PCR was performed as described in those publications. PCR products were analyzed on an ALF Express sequencer (Amersham Biosciences, Freiburg, Germany). A total number of 24 diploid and 27 triploid individuals and the tetraploid individual were analyzed.
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In a total number of 2419 diploids and 672 triploids measured in a variety of experiments in our laboratory so far, flow cytometry clearly revealed 1 tetraploid individual (0.03%) (Figure 1). The DNA content of the tetraploid nuclei was 4 pg per nucleus (N = 1; coefficient of variation = 2.56%), diploid P. formosa have a DNA content of 2.0 pg per nucleus, whereas triploids have 3.0 pg per nucleus (Lamatsch, Steinlein et al. 2000).
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Within the group of fish, the tetraploid individual originated from microsatellite analysis showed 1 diploid genotype and 1 triploid genotype (details in Table 1). For all primers analyzed diploids showed 2 alleles and triploids showed 3 alleles. The tetraploid individual showed 4 alleles in 4 of the 5 primers selected and 3 alleles for locus mATG44. In general, the tetraploid genotype resembled the triploid genotype with 1 additional allele (Table 1).
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Discussion |
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Flow cytometry and microsatellite analyses clearly revealed the tetraploidy of the individual found in the laboratory. As expected, the DNA content of the tetraploid cells was exactly twice the amount of the diploid and 1.33 times higher than in the triploids. As ploidy levels of all fish were determined before setting up the clonal competition experiment, it can be excluded that we overlooked a tetraploid individual at the beginning. Also, the tetraploid found was a juvenile, whereas all the individuals in the starting population 18 months before were adults. It can, therefore, be concluded that the tetraploid individual arose during the last phase of the experiment.
The microsatellite analyses of the tetraploid individual and the diploid and triploid individuals confirmed the tetraploidy of the individual as 4 alleles were found in 4 out of 5 loci. The tetraploid allelic pattern was concordant with the triploid genotype with the addition of 1 allele per locus. We, therefore, concluded that the tetraploid individual was an allotetraploid, which arose from the actual fertilization of a triploid egg. The additional genome in the tetraploid was then derived from P. mexicana limantouri, which was used as the host species (sperm donor) of the mixed diploid/triploid P. formosa population. In fact, the additional microsatellite alleles found in the tetraploid were well within the allelic range of P. mexicana limantouri (Table 1).
The set-up of the laboratory experiment resembles very well the genetic situation in the field: a very common triploid genotype (representing 89% of all triploids in the field [Lampert et al. 2005]) reproduces using P. mexicana limantouri males as sperm donors. It therefore seems likely that tetraploidization could also happen in the field. Even though tetraploidization might happen at a regular basis, it has so far never been reported. This might be due to the fact that it is probably a very rare event (1 in a total of 672 measured triploid individual was tetraploid) and/or that tetraploids might not be fertile, therefore, not forming a stable proportion of the population. Unfortunately, the tetraploid animal from this experiment was sacrificed before its unique genotype was known. Therefore, important life-history features such as fertility, growth rate, and life span are not known. We hypothesize that the tetraploid animal was female as P. formosa diploids as well as triploids are generally females, whereas males are extremely rare and exceptional. Its reproductive mode would then most likely have been gynogenesis with unreduced tetraploid oocytes as this is the reproductive mode in both studied levels of ploidy in P. formosa (Turner 1982). The even number of chromosome sets, however, might also allow tetraploids to form some reduced diploid eggs which could after segregation develop gynogenetically and contribute to the high clonal variability (most likely generated by mutations) found in the diploid lines of P. formosa (Lampert et al. 2005). Tetraploids in other gynogenetic fish lineages have been found to reproduce with diploid as well as tetraploid oocytes (Mada et al. 2001; Liu et al. 2004). Reproduction with diploid eggs would also explain that no tetraploid has so far been reported, other than triploids, tetraploids would not be a stable part of the population but only be present for a single generation.
Even if they are a rare phenomenon, tetraploids definitely contribute to the complexity of unisexual reproductive systems, for example, in the Rutilus alburnoides complex (Alves et al. 1999). Triploids in P. formosa show an unexpectedly low genotypic diversity and are most likely of monophyletic origin (Lampert et al. 2005); they may, however, be stepping stones on the way to creating tetraploids that might have an important role in creating genotypic variation represented by a high number of genetically distinct clones in the diploid lines of P. formosa. This may also explain why in the field the triploid clones seem to be rather stable, whereas the diploid clones have a high turnover between years (Lampert KP, unpublished data). Genotypic variation is in general thought to be advantageous as it might facilitate the adaptation to changing environmental conditions (e.g., parasites, Van Valen 1973). In addition, the acquisition of new genetic material by paternal leakage might enable P. formosa to buffer the accumulation of slightly deleterious mutations, the predicted cause for mutational meltdown in asexual species (Muller's ratchet; Muller 1964).
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Deutsche Forschungsgemeinschaft (SFB 567 Mechanismen der interspezifischen Interaktion von Organismen) and Fonds der Chemischen Industrie.
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Acknowledgments |
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We thank M. Schmid for the possibility to use the flow cytometer.
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Corresponding Editor: Lisa Seeb
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