A tetraploid Amazon molly, Poecilia formosa

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.     

A haploid-diploid-triploid mosaic of the Amazon molly, Poecilia formosa

We report the finding of the first haploid-diploid-triploid mosaic fish from the family Poeciliidae. The animal was derived from a laboratory cross of a female F1 hybrid of Poecilia mexicana and P. latipinna with a male from an ornamental strain derived from P. mexicana and P. sphenops (Black molly). It was identified because of its unusual pigmentation pattern and molecular methods (flow cytometry, NOR staining) confirmed its mosaic genotype. The mode of mosaic formation and the possible importance for poeciliid fish evolution are discussed.     

Feeding rates in the sailfin molly Poecilia latipinna and its coexisting sexual parasite,the gynogenetic Amazon molly Poecilia formosa

Feeding rates of the gynogenetic Amazon molly Poecilia formosa and one of its sexual hosts, the sailfin molly Poecilia latipinna, were measured under winter and summer temperature conditions. Food consumption of the unisexual P. formosa in winter conditions was significantly higher than that of P. latipinna, and it is hypothesized that the resulting food stress might have an important influence on the population composition of these closely related fishes via higher winter mortality in P. formosa.     

Distribution and stability of supernumerary microchromosomes in natural populations of the Amazon molly, Poecilia formosa

In animals, supernumerary chromosomes and their evolution have mostly been studied in sexual reproducing species. In the present study, for the first time, the natural distribution and stability of supernumerary microchromosomes were investigated in the unisexual fish species Poecilia formosa. Natural habitats throughout the range of P. formosa were screened for the presence of microchromosomes over several years. A high frequency of microchromosomes was found in the Río Purificación river system. Evidence points to the presence of the same microchromosome lineage over many generations. No supernumerary chromosomes were found elsewhere than in the Río Purificación representing a significant difference in the distribution of microchromosome-bearing individuals between the Río Purificación and all other collection sites.     

Penetration and distribution of carbon particles in a teleost fish, Poecilia formosa (Girard), the Amazon molly

The present paper gives data on the tissue distribution of carbon particles in Amazon mollies exposed for periods of 24–120 h to a 0.05% suspension. Very little carbon penetrated the body, either by way of the intestine or the gills. The little amount that entered appeared to pass almost exclusively through the posterior intestine. There was no anatomical specialization of the cells in this area equivalent to Peyer's patches in the intestine of mammals. We suggest that the uptake in the posterior intestine is related to its greater surface area and possibly to some enhanced mobility. After intraperitoneal (ip) injection, carbon that entered the body accumulated at three sites: the heart, the mesentery and, especially, in the head kidney. By 120 h large accumulations were seen in the intertubular areas of the kidney; there was no evidence of excretion of carbon particles from these areas.     

The role of female competition in the mating success of two clones of the Amazon molly, Poecilia formosa

Clones of the unisexual fish Poecilia formosa , the Amazon molly, live in the wild in association with their progenitor bisexual species, and compete with them for resources. These resources include food, space and also mates, since the Amazon molly reproduces gynogenetically, a process in which sperm transferred from a male host species activates their eggs. Earlier studies of the adaptive mechanisms that facilitate the coexistence of the bisexual and unisexual forms have focused upon male behaviour, but recent work with another unisexual species, Poeciliopsis , demonstrated that female aggressive behaviour may play an important part in determining the ecological standing of a clone.
We have extended our observations to female mating behaviour in two clones in the Amazon molly; clone 1 that does not thrive well under laboratory culture; and clone 2 that is hardy and survives very well. There was no aggression between the clones, nor did the clones show aggression towards females of the bisexual species. However, clone 1 consistently was more successful in the number of mating encounters made with the male; it approached the male more often than clone 2 and fled from the male less often. Males also showed some preference for mating with clone 1 females. If there are physiological differences in the wild clones similar to those seen in the laboratory, the mating advantage of the less robust clone 1 may ensure its continued survival in competition with a more vigorous clone.     

A Sexual Preference in the Amazon Molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female species that reproduces by gynogenesis, i.e., it relies on sperm of males of closely related species to trigger embryogenesis. Sperm is supplied by males of P. latipinna and P. mexicana. Amazon mollies live in sympatry with at least one of these species, a few populations live in sympatry with two sperm-donor species. As P. formosa is sperm dependent, it needs mechanisms for species and mate recognition. To investigate the effect of rearing conditions on sexual preferences of Amazon mollies, we raised Amazon mollies in sympatry with P. latipinna and P. mexicana males. We used simultaneous choice tests to determine the effect of age on female sexual preferences. Immature Amazon mollies do not exhibit a preference if given a choice between a P. latipinna and a P. mexicana male, whereas adult P. formosa do have a preference for the P. latipinna male. We used two different stimuli in this study, live males and videotapes of males.     

Polymorphic MHC loci in an asexual fish,the amazon molly (Poecilia formosa; Poeciliidae)

Genes of the major histocompatibility complex (MHC) encode molecules that control immune recognition and are highly polymorphic in most vertebrates. The remarkable polymorphisms at MHC loci may be maintained by selection from parasites, sexual selection, or both. If asexual species show equal (or higher) levels of polymorphisms at MHC loci as sexual ones, this would mean that sexual selection is not necessary to explain the high levels of diversity at MHC loci. In this study, we surveyed the MHC diversity of the asexual amazon molly (Poecilia formosa) and one of its sexual ancestors, the sailfin molly (P. latipinna), which lives in the same habitat. We found that the asexual molly has polymorphic MHC loci despite its clonal reproduction, yet not as polymorphic as the sexual species. Although the nucleotide diversity was similar between the asexual and sexual species, the sexual species exhibited a greater genotypic diversity compared to the asexual one from the same habitats. Within‐genome diversity was similar for MHC class I loci, but for class IIB, the sexual species had higher diversity compared to the asexual — despite the hybrid origins and higher levels of heterozygosity at microsatellite loci in the asexual species. The level of positive selection appears to be similar between the two species, which suggests that these polymorphisms are maintained by selection. Thus, our findings do not allow us to rule out the sexual selection hypothesis for the evolution of MHC diversity, and although the sexual fish has higher levels of MHC‐diversity compared to the asexual species, this may be due to differences in demography, parasites, or other factors, rather than sexual selection.     

Distribution of N-methyl-N-nitrosourea in the Amazon molly, Poecilla formosa (Girard), after a single intraperitoneal injection*

The distribution and accumulation of labelled N-methyl-N nitrosourea (MNU) has been described in the Amazon molly following intraperitoneal injection. Two hours after injection the label was present throughout the body, with enhanced deposition in the macrophages of the atrium of the heart, the kidney and the spleen; about half of the liver cells were labelled. The compound was taken up by the cells of the renal excretory tubules, and certain cells in the optic tectum and corpus cerebelli of the brain. By 6 h, the distribution picture had changed radically. There was little label remaining, except in a few macrophages in the heart and kidneys, and in the nuclei of the renal tubule cells. There was a slight decrease in the numbers of cells in the brain that were labelled, but the amount of material that the individual cells had accumulated had increased. By 48 h there was further loss and only brain cells were labelled. Our findings indicate preferential accumulation of MNU in undifferentiated, pluripotential cells of the optic tectum and corpus cerebelli.     

Frozen F1's amidst a masterpiece of nature: new insights into the rare hybrid origin of gynogenesis in the Amazon molly (Poecilia formosa)

All‐female ‘species’ of fish have been shown to be great models in ecological and evolutionary studies because of the insights they can provide into the origin and evolution of asexuality, the ecology of hybrids, associations between genotype and environment, and the maintenance of sex. Gynogenetic organisms that evolved from sexual ancestors, and combine the disadvantageous traits from sexuality and asexuality, have long baffled evolutionary biologists trying to understand their origin and persistence with their sympatric sexual counterparts. In this issue, a new study using an integrated molecular phylogenetic and classical genetic approach has uncovered compelling evidence regarding the obscure asexual origin of the Amazon molly, Poecilia formosa. By performing an extensive phylogeographic analysis, Stöck et al. (2010) provide evidence that the Amazon molly arose only once within its history, with monophyly being strongly supported by mitochondrial DNA and microsatellite analyses. This result, combined with an elaborate failed attempt to resynthesize the lineage, suggests that vertebrate gynogens such as the Amazon molly are not rare because they are at a disadvantage to their sexual counterparts, but because the genomic conditions under which they arise are rare. Organisms that apparently combine the disadvantages of both sexuality and asexuality remain difficult to understand from both an ecological and an evolutionary perspective, and Stöck et al. (2010) highlight several outstanding important questions. Nonetheless, given that we now have a better knowledge of the origin and history of this unique ‘species’, this should allow researchers to better understand how these frozen F1’s can persist amidst the masterpiece of nature.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

Diploid Amazon mollies (Poecilia formosa) show a higher fitness than triploids in clonal competition experiments

The gynogenetic livebearing Amazon molly (Poecilia formosa) is a sexual parasite that exploits males of closely related species for sperm. This is needed as physiological stimulus for embryo development; however, none of the male’s genes are normally incorporated into the genome of the gynogenetic offspring. Mostly diploid individuals were reported from the natural habitats in North-Eastern Mexico and South-Eastern Texas but stable populations of triploids have been reported from the Río Soto la Marina drainage and in the Río Guayalejo in North-Eastern Mexico. Triploidy is the result of defects in the mechanisms that normally clear the host sperm from the ameiotic diploid egg. Triploids also reproduce gynogenetically and their frequencies fluctuate markedly between years, seasons, and localities. To understand the dynamics of this mating system, it is important to understand the relative reproductive success of triploids and diploids. We hypothesize that triploids should have a selective advantage over diploids due to heterosis and/or gene redundancy based on the additional genetic material from the sexual host. However, clonal competition experiments revealed a clear reproductive advantage of diploids competing with triploids. This result contradicts not only our hypothesis but also the stable co-existence of diploids and triploids in natural habitats. Frequency dependent selection, niche partitioning and environmental heterogeneity are discussed as possible explanations.     

Chromosomal evidence for laboratory synthesis of a triploid hybrid between the gynogenetic teleost Poecilia formosa and its host species

Cytogenetic analyses were performed on a triploid hybrid individual produced in the laboratory by mating Poecilia formosa , an all-female gynogenetic diploid species, with a normal male of melanistic ornamental Poecilia sphenops (black molly). Direct chromosome preparations revealed 69 (3n) chromosomes in the somatic complement. This cytogenetic observation in conjunction with the pigmentation phenotype leads us to conclude that occasional failure of sperm exclusion may result in the presence of triploid hybrids even in the natural habitats.     

Isolation of a cancer-associated microchromosome in the sperm-dependent parthenogen Poecilia formosa

In the asexual all-female fish species Poecilia formosa, the Amazon molly, supernumerary chromosomes have frequently been found in both laboratory-reared and wild-caught individuals. While wild-caught individuals with B chromosomes are phenotypically indifferent from conspecifics, individuals carrying B chromosomes from recent introgression events in the laboratory show phenotypic changes. Former analyses showed that the expression of a pigment cell locus is associated with the presence of these B chromosomes. In addition, they contain a so far unidentified locus that confers a higher susceptibility to tumor formation in the presence of pigmentation pattern. Isolation by microdissection and hybridization to metaphase chromosomes revealed that they contain one or several sequences with similarity to a highly repetitive pericentromeric and subtelomeric sequence in A chromosomes. Isolation of one particular sequence by AFLP showed that the B chromosomes contain at least 1 copy of an A-chromosomal region which is highly conserved in the whole genus Poecilia, i.e. more than 5 million years old. We propose it to be a single copy sequence.     

Noninvasive determination of genome size and ploidy level in fishes by flow cytometry: detection of triploid Poecilia formosa

BACKGROUND: In order to understand the evolutionary significance of single triploids among the mostly diploid Poecilia formosa we have developed a simple, noninvasive technique for DNA content and ploidy determination. METHODS: From dorsal fin clips of 14 different fish species single cell suspensions were obtained by chopping the material in 2.1% citric acid/0.5% Tween20, passing it through a 0. 6-gauge needle and incubating it for 20 min at room temperature (RT) with gentle agitation. After overnight fixation in 70% ethanol, the cells were treated with 1ml 0.5% pepsin/0.1 M HCl for 15 min at RT before adding DAPI to a final volume of 2 ml. The cells were stained for 1-3 h and then analyzed by flow cytometry. RESULTS: We obtained good measurements with CVs ranging from 1.23% to 3.36%. The poeciliid species measured contain from 1.6 to 2.0 pg/nucleus, Oryzias latipes (Medaka) exhibits a nuclear DNA content of 2.2 pg, Danio rerio (zebrafish) 4.6 pg, Tetraodon fluviatilis (freshwater fugu) 0.70 pg. All values except zebrafish are in good agreement with the literature. CONCLUSIONS: The identification of living specimens of different ploidy for breeding experiments, behavioral studies and tissue transplantations is now made possible. With slight modifications the method can be extended to a field technique, providing therefore a useful tool for a variety of researchers.     

Male mating behaviour of a molly, <Emphasis Type="Italic">Poecilia latipunctata</Emphasis>: a third host for the sperm-dependent Amazon molly, <Emphasis Type="Italic">Poecilia formosa</Emphasis>

The Tamesí molly, Poecilia latipunctata, has a very limited biogeographical range in northeast Mexico. This area is nested within the ranges of the Atlantic molly, Poecilia mexicana, and the unisexual Amazon molly, Poecilia formosa. Based on morphology, especially fin shape, the Tamesí molly has been considered to be a "short-fin" molly. We describe the courtship sequence of P. latipunctata. The courtship clearly places the species into the clade of "long-fin" mollies, a finding that corroborates earlier studies based on nuclear DNA and mitochondrial DNA. All three species live together in certain habitats. This renders P. latipunctata a potential host species for the sperm-dependent, unisexual Amazon molly. Using behavioural tests, we demonstrate that P. latipunctata males actually copulate with Amazon mollies, despite a pronounced preference for conspecific females. In laboratory experiments P. latipunctata males are capable of triggering embryogenesis in P. formosa females. Field observations support the hypothesis that P. latipunctata is a third host species for P. formosa, indicating that the Amazon molly effectively exploits all available host species for its gynogenetic mode of reproduction. Electronic Publication     

Influence of male competition on male mating behaviour in the cave molly, Poecilia mexicana

In many species, male mating behaviour is correlated with male body size, with large males often being preferred by females. Small surface-dwelling Poecilia mexicana males compensate for this disadvantage by being more sexually active and using sneaky copulations. In a cave-dwelling population, however, small males do not show this behaviour. Do small males alter their behaviour in the presence of a large rival? Here, we investigated the influence of male competition on male mating behaviour in the cave form. Two males of different sizes were mated with a female either alone or together with the other male. No aggressive interactions were observed between either fish. There was no statistically significant difference in the frequency of sexual behaviours between the two treatments. In both treatments, large males were more sexually active than small males. Thus, small cave molly males do not switch to an alternative mating behaviour in the presence of a larger rival. Possibly, the extreme environmental conditions in the cave (e.g. low oxygen content and high levels of hydrogen sulphide) favour saving energetic costs, resulting in the absence of alternative mating behaviour in small males.