Evidence for a monophyletic origin of triploid clones of the Amazon molly, Poecilia formosa

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.     

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.     

Sperm availability in naturally occurring bisexual-unisexual breeding complexes involvingPoecilia mexicana and the gynogenetic teleost,Poecilia formosa

Synopsis Testicular maturation indices (TMI) were determined for wild-caught males ofPoecilia mexicana by quantitative analysis of the frequency of ongoing stages of spermatogenesis and spermatid differentiation in sections of testes stained with the Feulgen reaction for DNA. In nature, males maintain essentially constant levels of sperm production throughout the year and show no significant variations in mean TMI values associated with season, microhabitat, standard body length, or rank in male dominance hierarchies. Winter males or males isolated from females in the laboratory show accumulation of mature spermatophores. These findings suggest that fluctuations in the microstructure of wild populations ofPoecilia from northeastern Mexico may be due primarily to differences in the temporal scheduling of female reproductive cycles and not to limitations imposed by sperm availability or male reproductive competence. The data are discussed in terms of competitive interactions within unisexual-bisexual breeding complexes involving the Amazon mollyP. formosa, its related triploid hybrids, and the bisexual species,P. mexicana.     

Female sperm limitation in natural populations of a sexual/asexual mating complex (Poecilia latipinna, Poecilia formosa)

In sperm-dependent sexual/asexual mating systems, male mate choice is critical for understanding the mechanisms behind apparent stability observed in natural populations. The gynogenetic Amazon molly (Poecilia formosa) requires sperm from sexual males (e.g. Poecilia latipinna) to trigger embryogenesis, but inheritance is strictly maternal. Consequently, males should try to avoid or reduce the cost of mating with asexuals. We investigated male mate choice by documenting the presence of sperm in natural populations and found that a higher proportion of sexual females had sperm than asexuals. In addition, among those females that had sperm, sexuals had more sperm than asexuals. Our results hint at a role for male mate choice as a stabilizing factor in such systems.     

Cytogenetics of bisexual/unisexual species of Poecilia. III. The karyotype of Poecilia formosa, a gynogenetic species of hybrid origin.

Chromosomes of the Amazon molly, Poecilia formosa, a unisexual species of hybrid origin, were investigated by C-banding, silver staining, and fluorescent staining with DAPI, quinacrine dihydrochloride, and chromomycin A3. Analysis of heterochromatin distribution indicates that chromosomes similar to the W chromosome of P. latipinna are not present in the unisexual species. Therefore, morphologically differentiated sex chromosomes do not form the basis of the unisexuality in P. formosa. The number and location of nucleolar organizer regions vary in P. formosa and do not correlate well with those of the parental species.     

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.     

Reproductive behavior and the maintenance of all-female Poecilia

Synopsis There are four members involved in the breeding complexes of poeciliid fishes found in the freshwaters of northeastern Mexico: males and females of a bisexual species, and diploid and triploid unisexuals. Both unisexuals reproduce by gynogenesis, i.e., an asexual type of reproduction where the sperm triggers egg development but the male genome is excluded to produce clonal offspring. The three types of females are closely related, which suggests that they are potential competitors since all three require the service of the same males. The potential for competition is compounded by a highly skewed sex ratio in favor of females. On the average the unisexuals comprise about 30% of the Poecilia females. This high frequency coupled with a close genetic relatedness to their bisexual hosts, raises the question of how the unisexuals are maintained in nature.Other investigators who work with bisexual/unisexual complexes in the related genus, Poeciliopsis, have postulated that male dominance hierarchies are responsible for restricting the access of subordinate males to their conspecific females. Consequently, these subordinate males mate with unisexual females. The current report tests whether or not this hypothesis applies to bisexual/unisexual complexes of Poecilia.We have found that linear dominance hierarchies appear to function in the defense of home ranges and do not restrict access of males to females. Dominant males exhibit less mating activity than subordinate males towards females. Previous reports showed that males are reproductively competent throughout the year, whereas females show striking asynchrony in their reproductive readiness. Such asynchrony limits the proportion of receptive females at any one time. Consequently, there are more males ready to mate than there are females receptive to their mating attempts. This may lead to mating frenzies. We postulate that these indiscriminate matings maintain the fertility of both unisexuals. When the relative reproductive outputs of adult females are compared, both unisexuals appear as fit as their bisexual congeners.     

The origin of Phoxinus eos-neogaeus unisexual hybrids

Phoxinus eos-neogaeus unisexual hybrids (Cyprinidae, Pisces) are among the few vertebrate taxa known to reproduce clonally by gynogenesis. These taxa have a broad distribution in North America, mostly located in regions previously covered by the last Pleistocene ice sheet. To assess whether asexual hybrids dispersed from glacial refuges at the end of the Pleistocene or they originated from current hybridization events, genetic diversity of mitochondrial DNA (mtDNA) sequences and microsatellite loci was determined in populations from 16 different sites in the Mississippi-Missouri River (Nebraska and Montana), Rainy River-Hudson Bay (Minnesota), and St Lawrence River (Quebec) drainages. The maternal species (P. neogaeus) occurred in Minnesota and Nebraska but was absent from Montana sites and was restricted to only two of 11 lakes sampled in Quebec, although hybrids were present at all sites. The genetic survey revealed a total of 49 clones, originating from 14 hybridization events. Several of the lineages were characterized by mtDNA haplotypes not detected in the maternal ancestor. Lineages as well as clones frequently displayed a large geographical distribution at a regional scale. Dating of hybridization events suggested a relatively recent origin (<50,000 years ago) from the Mississippi glacial refuge, even in regions not covered by the last Pleistocene glacier. Altogether, these results indicate P. eos-neogaeus hybrids are not the result of current hybridization events, but display a pattern predicted by postglacial dispersal. Our findings have considerable implications for the nature of selection processes affecting the diversity of these asexual taxa and their coexistence with sexual ancestors.     

Genome‐specific histories of divergence and introgression between an allopolyploid unisexual salamander lineage and two ancestral sexual species

Quantifying introgression between sexual species and polyploid lineages traditionally thought to be asexual is an important step in understanding what drives the longevity of putatively asexual groups. Here, we capitalize on three recent innovations—ultraconserved element (UCE) sequencing, bioinformatic techniques for identifying genome‐specific variation in polyploids, and model‐based methods for evaluating historical gene flow—to measure the extent and tempo of introgression over the evolutionary history of an allopolyploid lineage of all‐female salamanders and two ancestral sexual species. Our analyses support a scenario in which the genomes sampled in unisexual salamanders last shared a common ancestor with genomes in their parental species ～3.4 million years ago, followed by a period of divergence between homologous genomes. Recently, secondary introgression has occurred at different times with each sexual species during the last 500,000 years. Sustained introgression of sexual genomes into the unisexual lineage is the defining characteristic of their reproductive mode, but this study provides the first evidence that unisexual genomes have undergone long periods of divergence without introgression. Unlike other sperm‐dependent taxa in which introgression is rare, the alternating periods of divergence and introgression between unisexual salamanders and their sexual relatives could explain why these salamanders are among the oldest described unisexual animals.     

Routes of origin of two recently evolved hybrid taxa: Senecio vulgaris var. hibernicus and York radiate groundsel (Asteraceae)

The possible pathways of origin of two recently arisen introgressant forms of Senecio vulgaris (i.e., var. hibernicus and York radiate groundsel) were investigated in experimental crosses between tetraploid S. vulgaris var. vulgaris and the normally diploid S. squalidus. Comparison of the morphology of synthesized hybrid progeny with established taxa, by discriminant function analysis, revealed that fertile hybrid offspring similar in morphology to S. vulgaris var. hibernicus and York radiate groundsel could be synthesized: (1) following formation of genomically stable diploid gametes by the triploid hybrid; (2) through the production of unreduced gametes by diploid S. squalidus; and (3) when a tetraploid form of S. squalidus acted as one of the parents. It was evident that hybrid offspring similar in morphology to the two introgressant taxa were more often produced in backcrosses to S. vulgaris than in segregating F2 or F3 generations (53% as opposed to 36%), and that fertile hybrid progeny were formed within two generations. Because hybridization between S. vulgaris and S. squalidus occurs regularly, although at very low frequency, in natural mixed populations in the British Isles, there is the potential for multiple origins to occur in the wild of both S. vulgaris var. hibernicus and York radiate groundsel.     

Reticulate evolution of the Daphnia pulex complex as revealed by nuclear markers

The study of species complexes is of particular interest to understand how evolutionary young species maintain genomic integrity. The Daphnia pulex complex has been intensively studied as it includes species that dominate freshwater environments in the Northern hemisphere and as it is the sole North American complex that shows transitions to obligate parthenogenesis. Past studies using mitochondrial markers have revealed the presence of 10 distinct lineages in the complex. This study is the first to examine genetic relationships among seven species of the complex at nuclear markers (nine microsatellite loci and one protein-coding gene). Clones belonging to the seven species of the Daphnia pulex complex were characterized at the mitochondrial NADH dehydrogenase (ND5) gene and at the Lactate dehydrogenase (LDH) locus. K-means, principal coordinate analyses and phylogenetic network analyses on the microsatellite data all separated European D. pulicaria, D. tenebrosa, North American D. pulex, D. pulicaria and their hybrids into distinct clusters. The hybrid cluster was composed of diploid and polyploid hybrids with D. pulex mitochondria and some clones with D. pulicaria mitochondria. By contrast, the phylogeny of the D. pulex complex using Rab4 was not well resolved but still showed clusters consisting mostly of D. pulex alleles and others of D. pulicaria alleles. Incomplete lineage sorting and hybridization may obscure genetic relationships at this locus. This study shows that hybridization and introgression have played an important role in the evolution of this complex.     

Using genotype simulations and Bayesian analyses to identify individuals of hybrid origin in Australian bass: lessons for fisheries management

Simulated genotypes, Bayesian analyses and molecular genetic data were used to detect individuals of hybrid origin and hybrid introgression between the Australian bass ( Macquaria novemaculeata ), a species extensively stocked in Australia, and estuary perch ( Macquaria colonorum ). Based on this analytical framework, 93% of the hybrids up to three generations later could be distinguished from the Australian bass. Individuals of hybrid origin were identified in all three rivers sampled. In addition, this study verified the fertility of hybrids between Australian bass and estuary perch as determined through genomic introgression. This study exemplifies an analytical procedure that has implications for identifying suitable individuals for use in breeding and restocking programmes for other species.     

Fitness dynamics within a poplar hybrid zone: I. Prezygotic and postzygotic barriers impacting a native poplar hybrid stand

Hybridization and introgression are pervasive evolutionary phenomena that provide insight into the selective forces that maintain species boundaries, permit gene flow, and control the direction of evolutionary change. Poplar trees (Populus L.) are well known for their ability to form viable hybrids and maintain their distinct species boundaries despite this interspecific gene flow. We sought to quantify the hybridization dynamics and postzygotic fitness within a hybrid stand of balsam poplar (Populus balsamifera L.), eastern cottonwood (P. deltoides Marsh.), and their natural hybrids to gain insight into the barriers maintaining this stable hybrid zone. We observed asymmetrical hybrid formation with P. deltoides acting as the seed parent, but with subsequent introgression biased toward P. balsamifera. Native hybrids expressed fitness traits intermediate to the parental species and were not universally unfit. That said, native hybrid seedlings were absent from the seedling population, which may indicate additional selective pressures controlling their recruitment. It is imperative that we understand the selective forces maintaining this native hybrid zone in order to quantify the impact of exotic poplar hybrids on this native system.     

Quantified reproductive isolation in Heliconius butterflies: Implications for introgression and hybrid speciation

Heliconius butterflies have become a model for the study of speciation with gene flow. For adaptive introgression to take place, there must be incomplete barriers to gene exchange that allow interspecific hybridization and multiple generations of backcrossing. The recent publication of estimates of individual components of reproductive isolation between several species of butterflies in the Heliconius melpomene–H. cydno clade allowed us to calculate total reproductive isolation estimates for these species. According to these estimates, the butterflies are not as promiscuous as has been implied. Differences between species are maintained by intrinsic mechanisms, while reproductive isolation of geographical races within species is mainly due to allopatry. We discuss the implications of this strong isolation for basic aspects of the hybrid speciation with introgression hypothesis.     

Maximum‐likelihood estimation of a hybrid index based on molecular markers

This paper elaborates on a hybrid index that utilizes information from genetic markers to quantify the genetic contribution of hybridizing species to individuals of unknown ancestry. Dominant markers will only lead to reliable and accurate estimates of hybrid index in later generation hybrids. In contrast, codominant markers can be fully resolved and their use is unproblematic. For both types of markers and allele frequencies that differ substantially between parental species (F_ST ≥ 0.17), a hybrid index based on 35–45 loci will have a nearly minimal confidence interval. Estimates of hybrid index are robust to modest errors in estimates of parental allele frequencies.     

Molecular systematics,GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)

Phylogenetic relationships in the genus Nicotiana were investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). In addition, origins of some amphidiploid taxa in Nicotiana were investigated using the techniques of genomic in situ hybridization (GISH), and the results of both sets of analyses were used to evaluate previous hypotheses about the origins of these taxa. Phylogenetic analyses of the ITS nrDNA data were performed on the entire genus (66 of 77 naturally occurring species, plus three artificial hybrids), comprising both diploid and polyploid taxa, and on the diploid taxa only (35 species) to examine the effects of amphidiploids on estimates of relationships. All taxa, regardless of ploidy, produced clean, single copies of the ITS region, even though some taxa are hybrids. Results are compared with a published plastid (matK) phylogeny using fewer, but many of the same, taxa. The patterns of relationships in Nicotiana, as seen in both analyses, are largely congruent with each other and previous evolutionary ideas based on morphology and cytology, but some important differences are apparent. None of the currently recognized subgenera of Nicotiana is monophyletic and, although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA analysis to reveal phylogenetic patterns in a complex genus such as Nicotiana is insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of natural and well-documented amphidiploids are similar or identical to one of their two parents-usually, but not always, the maternal parent-and are not in any sense themselves 'hybrid'. Knowing how ITS evolves in artificial amphidiploids gives insight into what ITS analysis might reveal about naturally occurring amphidiploids of unknown origin, and it is in this perspective that analysis of ITS sequences is highly informative.     

Molecular evidence for hybrid origin of Quercus crenata Lam. (Fagaceae) from Q. cerris L. and Q. suber L.

Abstract

Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) markers were employed to examine samples from Quercus cerris, Q. suber and Q. crenata in order to test the hypothesis of the hybrid origin of Q. crenata from Q. cerris and Q. suber in a part of its distribution area where the two putative parents do not overlap at present. Leaves from 21 Q. crenata and 37 Q. cerris individual trees were collected at natural sites in northern Italy, where Q. suber is currently lacking; 21 Q. suber and six Q. crenata plants from central Italy were also analysed. Results from Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis and principal component analysis (PCA) implied that exchange of neutral markers has been considerable between the three species, while differences in morphological characters have remained comparatively stable. The Mantel test indicated low correlation between RAPD- and ISSR-based similarity matrices, showing that the two screening techniques reveal unrelated estimates of genetic relationships. Hybrid indices computed for both markers displayed an intermediate position of Q. crenata individuals between the two putative parents shifted toward Q. cerris. Results from the present study corroborate the hypothesis of a hybrid origin for Q. crenata occurring in northern Italy, and suggest asymmetrical backcrossing with Q. cerris acting as the recurrent parent.     

The establishment of a hybrid zone between red and sika deer (genus Cervus)

Japanese sika deer ( Cervus nippon nippon ) were introduced to Scotland around 80 years (20 generations) ago. The sika phenotype is expanding its range and hybridizing extensively with native red deer ( Cervus elaphus ) leading to the establishment of a hybrid zone. This zone is currently moving and cannot be considered to be at equilibrium. Cervid genotypes and mitochondrial haplotypes were mapped across the sika phenotype range, using diagnostic protein isozymes, microsatellite nuclear DNA markers and RFLPs in mtDNA. These were analysed to estimate heterozygote deficits and nuclear linkage disequilibria and cytonuclear disequilibria in relation to gene frequencies and time since contact. Introgression was found in both taxa and strong linkage disequilibria and heterozygote deficits characterize the populations longest exposed to hybridization. Populations further from the introduction site, where hybridization is facilitated by the dispersal of sika-like stags, show low values for linkage disequilibria and heterozygote deficit. The observed patterns in genotype are explained in terms of assortative mating and a selective advantage of the sika genotype. The genetic integrity of the Scottish mainland red deer is shown to be at risk from the invasion of sika.     

Assessment of gene flow across a hybrid zone in red-tailed chipmunks (Tamias ruficaudus)

The role of hybridization in animal speciation is controversial and recent research has challenged the long-standing criterion of complete reproductive isolation to attain species status. The speciation-with-gene-flow model posits that the genome is semi-permeable and hybridization may be a phase in the process of divergence. Here, we apply these concepts to a previously identified zone of mtDNA introgression between the two strongly morphologically differentiated subspecies of red-tailed chipmunk ( Tamias ruficaudus ) in the US Inland Northwest. Using multilocus genotype data from the southern, older contact zone, we demonstrate that neutral gene flow is unusually low between the subspecies across the Lochsa River. This is geographically congruent with the discontinuity in bacular morphology, indicating that the cline of mitochondrial DNA (mtDNA) haplotypes is displaced. Furthermore, we elucidate the evolutionary forces responsible by testing hypotheses of lineage sorting and hybridization. We determined that introgressive hybridization is the cause of mtDNA/morphology incongruence because there are non-zero levels of migration and gene flow. Although our estimate of the age of the hybrid zone has wide credibility intervals, the hybridization events occurred in the Late Pleistocene and the divergence occurred in the Middle Pleistocene. Finally, we assessed substructure within and adjacent to the hybrid zone and found that the hybrid zone constitutes a set of populations that are genetically differentiated from parental sets of populations; therefore, hybridization in this system is not likely an evolutionary sink, but has generated novel combinations of genotypes.     

Introgressive hybridization and evolution of a novel protein phenotype: Glue protein profiles in thenasuta-albomicans complex ofDrosophila

Glue proteins are tissue-specific proteins synthesized by larval salivary gland cells ofDrosophila. InDrosophila nasuta nasuta andD. n. albomicans of thenasuta subgroup, the genes that encode the major glue protein fractions are X-linked. In the present study, these X-linked markers have been employed to trace the pattern of introgression ofD. n. nasuta andD. n. albomicans genomes with respect to the major glue protein fractions in their interracial hybrids, called cytoraces. These cytoraces have inherited the chromosomes of both parents and have been maintained in the laboratory for over 400–550 generations. The analysis has revealed that cytoraces withD. n. albomicans X chromosome show eitherD. n. nasuta pattern or a completely novel pattern of glue protein fractions. Further, quantitative analysis also shows lack of correlation between the chromosomal pattern of inheritance and overall quantity of the major glue protein fractions in the cytoraces. Thus, in cytoraces the parental chromosomes are not just differentially represented but there is evidence for introgression even at the gene level.