Cytogenetics of bisexual/unisexual species of Poecilia. I. C-bands, Ag-NOR polymorphisms, and sex chromosomes in three populations of Poecilia latipinna

Three populations of the North American cyprinodont fish Poecilia latipinna, considered to be one of the progenitor species of the gynogenetic unisexual P. formosa, were analyzed by C-banding and Ag-staining. C-bands were found to be polymorphic, and Ag-staining showed a high degree of variability in both the number and location of nucleolus organizer regions. The C-banding and Ag-staining patterns allow, to a certain extent, to distinguish individual specimens from each of these populations. Females of the three populations were found to have a heteromorphic chromosome pair, which was frequently identifiable with Giemsa staining and always after C-banding. This pair could be interpreted as sex chromosomes of the ZW/ZZ type.     

Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining.

Chromosomes of Poecilia mexicana mexicana, one of the bisexual species involved in the hybrid origin of the unisexual teleost fish species P. formosa, were analyzed by several staining techniques. Sex-specific, differential heterochromatin, found in other congeneric species, was not observed in P. m. mexicana. Nucleolar organizer regions were polymorphic among individual specimens within a given population sample. A single specimen exhibiting intraindividual variability of chromosome pair 1 and a specimen with a triploid karyotype are also described.     

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.     

Monophyletic origin of multiple clonal lineages in an asexual fish (Poecilia formosa)

Despite the advantage of avoiding the costs of sexual reproduction, asexual vertebrates are very rare and often considered evolutionarily disadvantaged when compared to sexual species. Asexual species, however, may have advantages when colonizing (new) habitats or competing with sexual counterparts. They are also evolutionary older than expected, leaving the question whether asexual vertebrates are not only rare because of their 'inferior' mode of reproduction but also because of other reasons. A paradigmatic model system is the unisexual Amazon molly, Poecilia formosa, that arose by hybridization of the Atlantic molly, Poecilia mexicana, as the maternal ancestor, and the sailfin molly, Poecilia latipinna, as the paternal ancestor. Our extensive crossing experiments failed to resynthesize asexually reproducing (gynogenetic) hybrids confirming results of previous studies. However, by producing diploid eggs, female F(1) -hybrids showed apparent preadaptation to gynogenesis. In a range-wide analysis of mitochondrial sequences, we examined the origin of P. formosa. Our analyses point to very few or even a single origin(s) of its lineage, which is estimated to be approximately 120,000 years old. A monophyletic origin was supported from nuclear microsatellite data. Furthermore, a considerable degree of genetic variation, apparent by high levels of clonal microsatellite diversity, was found. Our molecular phylogenetic evidence and the failure to resynthesize the gynogenetic P. formosa together with the old age of the species indicate that some unisexual vertebrates might be rare not because they suffer the long-term consequences of clonal reproduction but because they are only very rarely formed as a result of complex genetic preconditions necessary to produce viable and fertile clonal genomes and phenotypes ('rare formation hypothesis').     

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.     

Aspects of the mating behaviour of male mollies (Poecilia spp.)

The unisexual fish Poecilia formosa (the Amazon molly) reproduces by gynogenesis, a process in which sperm from the males of the host bisexual species activates development of its eggs. Unisexuals live with one of the host species in nature and compete with bisexual females for the males. It was long thought that male discrimination and mate selection established a balance between the unisexual and bisexual populations. Thus, hierarchies of males were set up in which dominant males mated with their conspecific females and subordinates mated with the Amazon molly. Recent evidence suggested, however, that male fish do not discriminate between their own females and the Amazon molly, and that there always are more males available than sexually receptive females of both species.
Our findings indicate that male behaviour may be more complex than suggested by either hypothesis. Mate discrimination and courtship behaviour appear to increase with age, so that large males show almost complete preference for their conspecific females, but smaller males will mate with the Amazon molly. In complex groups, small males often dart in and mate with their own females whilst the large males are engaged in courtship activities and defence of territory.     

Stable inheritance of host species-derived microchromosomes in the gynogenetic fish Poecilia formosa

B chromosomes are additional, usually unstable constituents of the genome of many organisms. Their origin, however, is often unclear and their evolutionary relevance is not well understood. They may range from being deleterious to neutral or even beneficial. We have followed the genetic fate of B chromosomes in the asexual, all-female fish Poecilia formosa over eight generations. In this species, B chromosomes come in the form of one to three tiny microchromosomes derived from males of the host species that serve as sperm donors for this gynogenetic species. All microchromosomes have centromeric heterochromatin but usually only one has a telomere. Such microchromosomes are stably inherited, while the telomereless are prone to be lost in both the soma and germline. In some cases the stable microchromosome carries a functional gene lending support to the hypothesis that the B chromosomes in P. formosa could increase the genetic diversity of the clonal lineage in this ameiotic organism and to some degree counteract the genomic decay that is supposed to be connected with the lack of recombination.     

Turbidity affects association behaviour in male Poecilia latipinna

Male association preferences in a bisexual‐unisexual species complex were studied in clear and turbid environments. In south and central Texas, where the gynogenetic sexual‐parasite Poecilia formosa lives syntopically with Poecilia latipinna as its sexual host species, association times of P. latipinna males with conspecific sexual and heterospecific asexual females in clear and turbid water were measured sequentially. Turbidity had an influence on male mate association behaviour. Males spent less time with any kind of female stimulus in turbid water. There was no preference for conspecific sexual females, either in turbid water or under clear conditions. Also, origin of males and acclimatization to turbid water had no effect. How turbidity as a source of visual noise might affect communication among individuals and how this environmental factor might contribute to the stability of this sexual‐asexual mating complex in nature are discussed.     

The origin and evolution of a unisexual hybrid: Poecilia formosa

Clonal reproduction in vertebrates can always be traced back to hybridization events as all known unisexual vertebrates are hybrids between recognized species or genetically defined races. Interestingly, clonal vertebrates often also rely on interspecific matings for their reproduction because gynogenesis (sperm-dependent parthenogenesis) and hybridogenesis are common modes of propagation. While in most cases these hybridization events leave no hereditary traces in the offspring, occasionally the genome exclusion mechanism fails and either small parts of male genetic material remain inside the oocyte in the form of microchromosomes, or fusion of the sperm nucleus with the oocyte nucleus leads to polyploid individuals. In this review, we highlight the important role of hybridization for the origin and evolution of a unisexual hybrid: the Amazon molly, Poecilia formosa.     

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.     

Unusual triploid males in a microchromosome-carrying clone of the Amazon molly, Poecilia formosa

The Amazon molly, Poecilia formosa, is an all-female fish of hybrid origin which reproduces by gynogenesis, i.e. it depends on sperm of males of closely related species to trigger parthenogenetic development of the embryo. Therefore the offspring is clonal and identical to the mother. In rare cases the exclusion mechanism fails and paternal introgression occurs. This may result either in triploid offspring - if the whole haploid chromosome set of the sperm fuses with the diploid egg nucleus - or in siblings with microchromosomes - if only subgenomic amounts of paternal DNA are included. In one of our diploid, microchromosome-carrying laboratory stocks we observed eight triploid individuals which all developed into males. We investigated the mitotic and meiotic chromosomes, the synaptonemal complex (SC), and sperm production of these males, and compared them to males of the gonochoristic parental species (P. latipinna and P. mexicana) and their hybrids. This comparison revealed that P. formosa males are functional males with reduced effective fertility. They show a deviation from the typical 23 bivalents in the synaptonemal complexes as well as in diakinesis due to the triploid state. They produced offspring but only with gynogenetic Amazon molly females. This shows that the probably aneuploid sperm from P. formosa males can trigger parthenogenetic development of unreduced eggs.     

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.     

Male mate choice and sperm allocation in a sexual/asexual mating complex of Poecilia (Poeciliidae, Teleostei)

Male mate choice is critical for understanding the evolution and maintenance of sexual/asexual mating complexes involving sperm-dependent, gynogenetic species. Amazon mollies (Poecilia formosa) require sperm to trigger embryogenesis, but the males (e.g. Poecilia mexicana) do not contribute genes. Males benefit from mating with Amazon mollies, because such matings make males more attractive to conspecific females, but they might control the cost of such matings by providing less sperm to Amazon mollies. We examined this at the behavioural and sperm levels. P. mexicana males preferred to mate with, and transferred more sperm to conspecific females. However, if males mated with P. formosa, sperm was readily transferred. This underscores the importance of male choice in this system.     

Cytological evidence for temporal differences during the asynchronous ovarian maturation of bisexual and unisexual fishes of the genus Poecilia

Comparisons based upon morphological features and cytochemical staining properties of chromatin, nucleoli, and the ooplasm were used in the present analysis of ovarian maturation in wild-caught Poecilia mexicana and its related gynogenetic diploid and triploid' species'. Using an empirically derived ovarian maturation index, seasonal and collection site differences were found between the unisexual and bisexual forms. These fish seem equally suited to their environments. However, our data indicate that differences in microhabitat can have a profound influence on oocyte development. Although females of Poecilia spp. have asynchronous cycles of ovarian maturation, we could not detect any peculiar cytological events associated with the gynogenetic mode of reproduction. Sperm were found in ovaries of both unisexual and bisexual forms indicating that the unisexuals can compete effectively with females of P. mexicana for matings with males of this species. The methods employed in this study provide a sensitive measure of reproductive potentials in sympatric species.     

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.     

Cytogenetic studies of Poecilia (Pisces)V. Cytophotometric evidence for the production of fertile offspring by triploids related to Poecilia formosa

To monitor the perpetuation of triploid genomes in laboratory-reared progeny of wild-caught females of Poecilia, amounts of DNA-Feulgen staining in scale epithelial nuclei were determined for 58 different fish. Based on estimates of average DNA content, these specimens were readily separated into two groups: (a) diploids, with an average of 1–57 × 10⁻¹² g DNA per cell and (b) triploids, with an average of 2.38 × 10⁻¹² g DNA per cell. These values yield a ratio of 1: 1.5, as would be expected from the presence of an extra chromosome set in putative triploid females. Ploidy status of the latter, based on DNA cytophotometry, was independently confirmed for several specimens by direct cytological examination of chromosome number, by electrophoresis of plasma proteins and by morpho-metric analysis. These findings unequivocally document one case involving the production of triploid offspring from a typical, diploid P. formosa and several instances in which broods of all-female triploid offspring were derived in the laboratory from both wild-caught and laboratory-reared triploid mothers. The relationship and significance of these triploid forms to the breeding complex of P. formosa and P. mexicana in nature are discussed.     

Spectral sensitivity of mollies: comparing surface- and cave-dwelling Atlantic mollies, Poecilia mexicana

The visual pigments of cones and rods in three species of mollies, Poecilia mexicana , Poecilia latipinna and their asexual hybrid Poecilia formosa , were examined using microspectrophotometry. In P. mexicana , populations from extreme photic habitats were used: one population originated from a clear water habitat, one from a milky water habitat and another from a completely dark cave. Ultraviolet-sensitive cones were found in all species. Differences in the λ_max values of the visual pigments were small between species and among the three P. mexicana populations, but dark-reared cave fishes showed appreciably higher variance. The hybrid species P. formosa showed a highly variable long wavelength cone absorbance, ranging from 528·9 to 598·5 nm, suggesting multiple opsin expression or chromophore mixing.     

Geographical variation in reproductive character displacement in mate choice by male sailfin mollies

Female Amazon mollies, Poecilia formosa, are a unisexual species that reproduce by gynogenesis. They must coexist and mate with males of other species (usually the mollies Poecilia latipinna or Poecilia mexicana) to induce embryogenesis, but inheritance is strictly maternal. We examined the mating preference of the male sailfin molly, P. latipinna, for female sailfin mollies versus Amazon mollies, P. formosa. We compared the mating preferences of sympatric and allopatric populations collected throughout the Gulf Coast of North America. Male P. latipinna from six populations sympatric with Amazon mollies showed a significantly greater strength of preference for conspecific sailfin females than males from five populations that were allopatric with Amazon mollies. These results provide strong evidence for reproductive character displacement of male mate choice in sympatry. Furthermore, the large geographical range of populations that we tested revealed variation among populations within sympatry and allopatry, indicating that it is important to evaluate a large number of populations when examining reproductive character displacement.     

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.     

PEERING THROUGH THE LOOKING GLASS AT A SEXUAL PARASITE: ARE AMAZON MOLLIES RED QUEENS?

Abstract.— The gynogenetic Amazon molly ( Poecilia formosa ) is a clonal, all-female lineage of livebearing fish that faces an unusual obstacle to evolutionary persistence. Sperm from heterospecific males (either sailfin, P. latipinna , or Atlantic, P. mexicana , mollies) is necessary to trigger embryogenesis. However, none of the male's genes are incorporated into the genome of the gynogenetic offspring. Some investigators have proposed that the evolution of male mate discrimination is a result of this cost, leading to a coevolutionary arms race between male avoidance of P. formosa and P. formosa attractiveness. Given that P. formosa successfully reproduces and has not yet gone extinct, it is clear there are mechanisms by which they attract the sexual attention of males. Although a Red Queen coevolutionary process in typical host/parasite systems has been shown to favor the persistence of sexual species, in this system an arms race has been invoked to explain the reverse. Here I present behavioral data supporting a more parsimonious scenario: that mechanisms of attraction in P. formosa are simply a consequence of its hybrid origin. Poecilia latipinna and P. mexicana males do not discriminate between gynogenetic P. formosa females and first generation sexual hybrid females, and females do not differ in agonistic behaviors associated with competition for mates. Both results contradict predictions from the Red Queen hypothesis. Therefore, coevolution is not necessary to explain the apparent evolutionary persistence of P. formosa.