Unisexual Individuals Research Articles

Cannabis sativa: Dioecious into Monoecious Plants influencing Sex Determination

This review paper highlights about sex determination and conversion of dioecious into monoecious plants by applying exogenous growth regulators or chemicals. Cannabis sativa L. (Cannabaceae) is a dioecious plant, producing male and female flowers on separate unisexual individuals. Although both male and female plants are capable of producing cannabinoids in equal concentrations, female plants produce greater floral biomass than male plants and thus are exclusively used in commercial Medical Cannabis sativa (drug or marijuana) production facilities. In commercial production, marijuana plants are all genetically unfertilized female plants and, male plants are destroyed as seed formation reduces flower quality. One male Cannabis plant can ruin the entire female plant crop due to uncontrolled pollination and crop is designated as contaminated. Moreover, after pollination, female plants alter their relative investment in phytochemicals by reducing the production of secondary metabolites like cannabinoids, flavonoids, and terpenoids. Therefore, early diagnosis of sex is very important to both breeders and farmers for Cannabis crop improvement or production purposes. Cannabis sex determination could be modified by applying exogenous growth regulators or chemicals, which can influence the ratio of endogenous hormones and hence the incidence of sex organs. Silver compounds such as silver nitrate (AgNO3) or silver thiosulfate (Ag2S2O3; STS) have been found to have masculine effects in many plant species including Cannabis. A gap in the literature highlighting Cannabis sex determination has been updated in this review paper.

Post-emergence survival and dispersal of juvenile Jefferson salamander (Ambystoma jeffersonianum) and their unisexual dependents

Abstract Understanding population demography and dispersal of species at risk is integral for evaluating population viability, identifying causes of decline, and assessing the effectiveness of recovery actions. In pond-breeding amphibians, juvenile survival and dispersal are key components linked to population and metapopulation stability but little is known about this life stage. We use mark-recapture methods to estimate juvenile daily apparent survival, dispersal distance, and initial dispersal orientation during summer and fall dispersal of endangered Ambystoma jeffersonianum and their unisexual dependents (Ambystoma laterale – jeffersonianum). Over four years (2015-2018), 1018 juveniles (612 bisexual, 406 unisexual) were marked and 192 (19%) were recaptured at least once. Total captures varied widely between years, with the highest number of captures (88% of all individuals) occurring in 2017. Cormack-Jolly-Seber estimates of daily apparent survival were low in all years (0.76-0.95) but was higher for unisexuals than bisexuals. The majority of juveniles (71%) did not disperse further than 10-40 m after which movement appeared to cease. While most juveniles remained close to their natal pond, at least 2% of juveniles in 2017 travelled further than 100 m. Dispersal orientation varied by year and there was no difference in either dispersal orientation or distance between bisexual and unisexual individuals. This work is the first to estimate and compare juvenile survival and dispersal of sympatric A. jeffersonianum and A. laterale – jeffersonianum individuals, the results of which help inform population viability assessment and increase our understanding of juvenile dispersal dynamics and habitat use.

Testing mechanisms of compensatory fitness of dioecy in a cosexual world

AbstractQuestionsAll else being equal, populations of dioecious species with a 50:50 sex ratio have only half the effective reproductive population size of bisexual species of equal abundance. Consequently, there is a need to explain how dioecious and bisexual species coexist. Increased mean individual seed mass, fecundity, and population density have all been proposed as attributes of unisexual individuals or populations that may contribute to the persistence or resilience of dioecious species. To date, no studies have compared sympatric dioecious and cosexual species with respect to all three components of fitness. In this study, we sought evidence for these compensatory advantages (higher seed mass, greater seed production per unit basal area, and higher population density) in dioecious species.LocationFive 20–25 ha forest dynamic plots spanning a latitudinal gradient in China, including two temperate, two subtropical, and one tropical forest.MethodsWe used a phylogenetically corrected generalized linear modelling approach to assess the phylogenetic dependence and joint evolution of sexual system, seed mass and production, and ecological abundances among 48–333 species and 32,568–136,237 individuals per forest.ResultsAcross all five forests, we detected no consistent advantage for dioecious relative to sympatric cosexual species with respect to mean individual seed mass, seed production or the density of stems in any size class.ConclusionsOur study suggests that seed traits may provide compensatory mechanisms in some forests, but most often the coexistence of sexual systems cannot be explained by advantages of dioecy related to seed quality and demographic parameters. Future investigations of the factors that promote coexistence may increase our understanding by expanding the search to include attributes such as lifespan and tolerance or resistance to herbivores.

Sexually Antagonistic Variation and the Evolution of Dimorphic Sexual Systems.

Multicellular Eukaryotes use a broad spectrum of sexual reproduction strategies, ranging from simultaneous hermaphroditism to complete dioecy (separate sexes). The evolutionary pathway from hermaphroditism to dioecy involves the spread of sterility alleles that eliminate female or male reproductive functions, producing unisexual individuals. Classical theory predicts that evolutionary transitions to dioecy are feasible when female and male sex functions genetically trade off with one another (allocation to sex functions is sexually antagonistic) and rates of self-fertilization and inbreeding depression are high within the ancestral hermaphrodite population. We show that genetic linkage between sterility alleles and loci under sexually antagonistic selection significantly alters these classical predictions. We identify three specific consequences of linkage for the evolution of dimorphic sexual systems. First, linkage broadens conditions for the invasion of unisexual sterility alleles, facilitating transitions to sexual systems that are intermediate between hermaphroditism and dioecy (androdioecy and gynodioecy). Second, linkage elevates the equilibrium frequencies of unisexual individuals within androdioecious and gynodioecious populations, which promotes subsequent transitions to full dioecy. Third, linkage dampens the role of inbreeding during transitions to androdioecy and gynodioecy, making these transitions feasible in outbred populations. We discuss implications of these results for the evolution of dimorphic reproductive systems and sex chromosomes.

Influence of genome and bio-ecology on the prevalence of genome exchange in unisexuals of the Ambystoma complex

BackgroundUnisexuals of the blue-spotted salamander complex are thought to reproduce by kleptogenesis. Genome exchanges associated with this sperm-dependent mode of reproduction are expected to result in a higher genetic variation and multiple ploidy levels compared to clonality. However, the existence of some populations exclusively formed of genetically identical individuals suggests that factors could prevent genome exchanges. This study aimed at assessing the prevalence of genome exchange among unisexuals of the Ambystoma laterale-jeffersonianum complex from 10 sites in the northern part of their distribution.ResultsA total of 235 individuals, including 207 unisexuals, were genotyped using microsatellite loci and AFLP. Unisexual individuals could be sorted in five genetically distinct groups, likely derived from the same paternal A. jeffersonianum haplome. One of these groups exclusively reproduced clonally, even when found in sympatry with lineages presenting signature of genome exchange. Genome exchange was site-dependent for another group. Genome exchange was detected at all sites for the three remaining groups.ConclusionPrevalence of genome exchange appears to be associated with ecological conditions such as availability of effective sperm donors. Intrinsic genomic factors may also affect this process, since different lineages in sympatry present highly variable rate of genome exchange. The coexistence of clonal and genetically diversified lineages opens the door to further research on alternatives to genetic variation.

Locomotor endurance predicts differences in realized dispersal between sympatric sexual and unisexual salamanders

Summary Dispersal is the central mechanism that determines connectivity between populations yet few studies connect the mechanisms of movement with realized dispersal in natural populations. To make such a link, we assessed how physiological variation among individuals predicted dispersal in natural populations of unisexual (all‐female) and sexual Ambystoma salamanders on the same fragmented landscape in Ohio. Specifically, we assessed variation in a trait that influences long‐distance animal movement (locomotor endurance) and determined whether variation in endurance matched patterns of realized dispersal assessed using genetic assignment tests. A possible mechanism for why unisexuals would have lower locomotor endurance than a sympatric sexual species (Ambystoma texanum) is the potential energetic cost of evolutionarily mismatched mitochondrial and nuclear genomes within polyploid unisexuals. We found that sexuals walked four times farther than unisexuals during treadmill endurance trials that mimic the locomotor endurance required for dispersal. We then applied landscape genetic methods to identify dispersed adults and quantify realized dispersal. We show that the differences in locomotor endurance between unisexual and sexual salamanders scale to realized dispersal: dispersing sexual individuals travelled approximately twice the distance between presumed natal wetlands and the site of capture compared to dispersing unisexuals. This study links variation in individual performance in terms of endurance with realized dispersal in the field and suggests a potential mechanism (physiological limitation due to mitonuclear mismatch) for the reduced endurance of unisexual individuals relative to sexual individuals although we discuss other possible explanations. The differences in dispersal between these two types of salamanders also informs our understanding of sexual/unisexual coexistence by suggesting that unisexuals are at a competitive disadvantage in terms of colonization ability under a extinction‐colonization model of coexistence. A lay summary is available for this article.

Microsatellite genetic diversity and mating systems in the columnar cactus Pachycereus pringlei (Cactaceae)

Mating systems and long-term climatic fluctuations have large impact on genetic diversity and population structure of plants; however, the genetic consequences have been poorly documented in species with geographic variation in mating systems. Here we used genetic variation from microsatellite markers to investigate the biogeographic pattern of Pachycereus pringlei, a columnar cactus with exceptional assortment of mating systems and wide range of distribution in the Sonoran Desert. Bayesian and multivariate analyses distinguished five genetic populations associated with differences in mating systems. Two hermaphroditic populations restricted to Cerralvo and Catalana islands were highly differentiated (ΦPT=0.26 and 0.21, respectively). Cerralvo Island showed the lowest genetic diversity (HO=0.26). Two other populations showed wide geographical distribution: a mostly gynodioecious population in the North comprising mostly hermaphrodite and female individuals, and a trioecious population in the South comprising unisexual and hermaphrodite individuals. The latter population exhibited the lowest level of genetic differentiation (ΦPT=0.14) and highest genetic diversity (HO=0.45). Cabo San Lucas, another trioecious population restricted to the southern end of the Baja California Peninsula, exhibits prevalence of unisexual individuals (0.76) but highly differentiated (ΦPT=0.19) and relatively moderate genetic diversity (HO=0.38). Latitudinal decrease of genetic diversity and isolation by distance supports northwards range expansion and southern BCP refugia, while Ecological Niche Models supported suitable climatic conditions during the Last Glacial Maximum in this region. These results suggest that the current biogeographic pattern of P. pringlei has been driven by longstanding climatic fluctuations associated with differential colonization abilities of genders and geographic variation of selfing and outcrossing rates. In conclusion, P. pringlei is not a panmictic assemblage and the interaction of historical vicariance and dispersal processes with mating systems may well explain present-day patterns of genetic variation.

Pathways for making unisexual flowers and unisexual plants:Moving beyond the "two mutations linked on one chromosome" model.

Sex determination in higher plants is of fundamental biological interest and has great practical significance for fruit yield and high-quality seed production. Th e last decades have witnessed an explosion in the genetic-developmental understanding of a typical hermaphroditic flower; however, the genes controlling dicliny (unisexual fl owers) and dioecy (unisexual individuals) are only now being revealed ( Akagi et al., 2014 ; Boualem et al., 2015 ). These genetic-developmental insights, from distantly related groups, now need to be brought together with the large body of empirical observations showing that dicliny and dioecy each evolved independently many thousands of times, but oft en in the same genera and families suggesting related evolutionary pathways ( Darwin, 1877 ; Lloyd, 1972 , 1975a , b , 1980 ; Renner and Ricklefs, 1995 ; Renner, 2014 ). Here I highlight how the recent insights may redirect our understanding of pathways to dioecy in fl owering plants. Th e fundamental model for the evolution of dioecy in angiosperms has long been that “at least two gene mutations are necessary to transform an hermaphroditic or monoecious species into one with separate sexes; one mutation must aff ect ovule production, and the other the production of pollen” ( Charlesworth and Charlesworth, 1978 : p. 975), with the corollary that “diff erent genes determining sex [are] grouped together in one region of one chromosome in most species” (p. 976). Th is model disregards that (1) in monoecious species, mutations suppressing male and female function do not need to arise since they already exist and that (2) there is little evidence that sex-determining genes in dioecious plants are grouped together on one chromosome. Th e developmental-genetic

Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

Selfish male-determining element favors the transition from hermaphroditism to androdioecy.

According to the current, widely accepted paradigm, the evolutionary transition from hermaphroditism toward separate sexes occurs in two successive steps: an initial, intermediate step in which unisexual individuals, male or female, sterility mutants coexist with hermaphrodites and a final step that definitively establishes dioecy. Two nonexclusive processes can drive this transition: inbreeding avoidance and reallocation of resources from one sexual function to the other. Here, we report results of controlled crosses between males and hermaphrodites in Phillyrea angustifolia, an androdioecious species with two mutually intercompatible, but intraincompatible groups of hermaphrodites. We observed different segregation patterns that can be explained by: (1) epistatic interactions between two unlinked diallelic loci, determining sex and mating compatibility, and (2) a mutation with pleiotropic effects: female sterility, full compatibility of males with both hermaphrodite incompatibility groups, and complete male-biased sex-ratio distortion in one of the two groups. Modeling shows that these mechanisms can explain the high frequency of males in populations of P. angustifolia and can promote the maintenance of androdioecy without requiring inbreeding depression or resource reallocation. We thus argue that segregation distortion establishes the right conditions for the evolution of cryptic dioecy and potentially initiates the evolution toward separate sexes.

Sexual system of Garcinia indica Choisy: geographic variation in trioecy and sexual dimorphism in floral traits

Most flowering plants are hermaphroditic with both stamens and carpels in the same flower. Other sexual systems are derived from ancestral hermaphroditism through one of the following pathways namely via monoecy, directly from hermaprhoditism, via gynodioecy, via distyly or via androdioecy. Sexual system in the genus Garcinia is highly diverse and includes dioecious, gynodioecious, androdioecious, polygamodioecious, monoecious and andromonoecious species. Garcinia indica Choisy (kokum tree) is reported to be polygamodioecious or gynodioecious. In the present study, sexual variation was estimated in seven populations of Garcinia indica of Western Ghats to determine the extent of variation among the populations and 14 floral traits were studied to assess the sexual dimorphism in floral traits. Four basic kinds of flowers namely, male flowers, male flowers with pistillode, female flowers with staminodes and bisexual flowers, were found in G. indica. These basic kinds of flowers occurred individually on a particular tree, producing unisexual individuals, or in combination of two, forming cosexual individuals. Thus, this species is trioecious, although the percentage of cosexual trees varied in different geographical locations. All the populations included cosexuals except one whereas pure hermaphroditic trees were present only in one. Significant differences were observed in 11 out of the 14 floral traits studied in the seven populations resulting in the diversity of sex forms. Male flowers had larger petal and female flowers were in general smaller than the other kinds. Interestingly, bisexual flowers were not always intermediate in size between male and female flowers.

Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae)

The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described. The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD. PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption. Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.

Sexual stability in the nearly dioeciousPinus johannis(Pinaceae)

Even though dioecy is a dominant sexual system among gymnosperms, little is known about its evolutionary history. Pinus johannis may represent a model system because unisexual and monoecious individuals compose its populations. The presence of unisexual individuals in other Pinus species is a consequence of sexual lability. Here we determined whether P. johannis represents the first example of a dioecious or nearly dioecious reproductive system in conifers by evaluating its sexual stability. • To assess the stability of sexual expression, we quantified the proportion of male vs. female reproductive structures produced by trees over multiple years and tested for the presence of sexual dimorphism. Sexual lability hypotheses were also examined by looking at the relationship between environmental factors and sexual expression and by comparing the reproductive behavior of P. johannis with its closest labile relative, P. edulis. • Pinus johannis is nearly dioecious: ~99% of individuals are unisexual or express a low proportion of the opposite gender with few changes in sexual expression through time. We found sexual dimorphism consistent with sexual stability. Sexual expression did not vary with tree size/age, abiotic environment, or herbivore removal, providing evidence against sexual lability. Individuals of P. johannis tended to produce only male or female strobili, whereas those of P. edulis were mainly monoecious with a gradient in the female to male strobili ratio. • This study represents the first report of a nearly stable dioecious Pinus species. The variety of sexual morphs coexisting in the same population makes P. johannis a model for studying the evolution of dioecy in gymnosperms.

Characterization of 12 polymorphic microsatellite markers in the liverwort Marchantia inflexa (Marchantiaceae)

Microsatellite markers were developed in Marchantia inflexa, a haploid liverwort with unisexual individuals, to identify clonal genotypes and measure population genetic variability. Twelve polymorphic primer sets were developed from three enriched genomic libraries. Primers were fluorescently labeled, and alleles were identified by fragment analysis. These primers were tested in four natural populations and revealed a moderate level of genetic variation within four populations, as indicated by the number of alleles per locus (range = 1-5). Development of polymorphic markers is crucial to the identification of individuals and will allow additional research into this species, particularly on its population genetics and metapopulation dynamics.

Sex in unisexual salamanders: discovery of a new sperm donor with ancient affinities

Although bisexual reproduction has considerable evolutionary benefits, several all-female vertebrates exist. Unisexual salamanders in the genus Ambystoma are common around the Great Lakes region in eastern North America. They originated from a hybridization event that involved a female that shared a common ancestor with Ambystoma barbouri 2.4 to 3.9 million years ago but, unexpectedly, A. barbouri nuclear genomes were unknown in unisexuals. Unisexual salamanders steal sperm from donors of normally bisexual species, so their reproductive mode is described as kleptogenesis. Most known unisexuals are polyploid and they all possess at least one A. laterale genome. One or more other genomes are taken from sperm donors that may include A. jeffersonianum, A. laterale, A. texanum and A. tigrinum. We examined unisexual adults and larvae in a southern Ohio pond where unisexual individuals coexist with male A. barbouri. This population provided an opportunity to test hypotheses pertaining to the role of A. barbouri in the evolution of the disparate cytoplasmic and nuclear genomes in unisexual salamanders. Microsatellite DNA loci, mitochondrial DNA sequences and genomic in situ hybridization were used to identify the genomic constitution of individuals. A. barbouri was found to be an acceptable sperm donor for unisexuals but only contributed genomes in ploidy-elevated individuals. In the absence of A. jeffersonianum, this Ohio population is likely experiencing a recent switch in sperm donors from A. jeffersonianum to A. barbouri and demonstrates the evolutionary flexibility and dynamics of kleptogenesis.

Phylogenetic analysis of sexual systems in Inuleae (Asteraceae)

From an ancestor with bisexual flowers, plants with unisexual flowers, or even unisexual individuals have evolved in different lineages of angiosperms. The Asteraceae tribe Inuleae includes hermaphroditic, monoecious, dioecious, and gynomonoecious species. Gynomonoecy, the sexual system in which female and bisexual flowers occur on the same plant, is prevalent in the Asteraceae. We inferred one large gene phylogeny (ndhF) and two supertrees to investigate whether gynomonoecy was a stage in the evolution from hermaphroditism to monoecy. We identified transitions in sexual system evolution using the stochastic character mapping method. From gynomonoecious ancestors, both hermaphroditic and monoecious descendants have evolved. Gynomonoecy was not restricted to a stage in the evolution toward monoecy because the number of transitions and the rate of change from monoecy to gynomonoecy were much higher than the opposite. We also investigated one hypothesized association among female flowers and the development of a petaloid ray as an explanation of gynomonoecy maintenance in Asteraceae. We found that peripheral female flowers and petaloid rays were phylogenetically correlated. However, empirical evidence shows that a causal relationship between these traits is not clear.

Genealogical relationships of southern Ontario polyploid unisexual salamanders (genus Ambystoma) inferred from intergenomic exchanges and major rDNA cytotypes

North American unisexual salamanders in the genus Ambystoma are common around the Great Lakes region of North America. They contain an almost identical mitochondrial genome across their distribution that is unlike that of any of the four species whose genomes may be included in their nuclei. Thus, sequence-based phylogenies of unisexual populations are confusing. We used chromosomal intergenomic exchanges and major rDNA cytotypes as combined cytogenetic markers to tentatively construct a genealogy of unisexual Ambystoma in southern Ontario. We employed GISH and sequential/simultaneous GISH/FISH-rDNA to reveal intergenomic exchanges and rDNA cytotypes in unisexual A. laterale--2 jeffersonianum (LJJ) triploids and their tetraploid derivative A. laterale--3 jeffersonianum (LJJJ). We identified 10 different patterns of intergenomic exchanges from 18 isolated populations and used them as primary cytogenetic markers. Major rDNA cytotypes served as independent and supplementary markers. Our results suggest that current LJJ and LJJJ populations in southern Ontario are likely derived from a few unisexual individuals. Intergenomic exchanges are common phenomena and widely distributed in the salamanders of the A. laterale--A. jeffersonianum unisexual complex. Integration of GISH and FISH can exhibit multiple unrelated chromosomal markers on the same chromosome spread and demonstrate lineage relationships in unisexual populations. Similar methods may be applied for studying the molecular cytogenetics of other unisexuals to improve our understanding of their genealogical relationships and historical dispersal.

Additional Distributional Records of Ambystoma Laterale, A. Jeffersonianum (Amphibia: Caudata) and Their Unisexual Kleptogens in Northeastern North America

Abstract Several species of mole salamanders in the genus Ambystoma are targeted by various state, provincial, and federal agencies for conservation. These salamanders have specific wetland and forested upland habitat requirements that render them vulnerable to environmental alteration. The blue-spotted salamander, Ambystoma laterale (LL) and the Jefferson salamander, A. jeffersonianum (JJ) have both been listed for protection in various parts of their ranges, but the identification of these salamanders is confusing because they often coexist with unisexual individuals that are mostly polyploid and use the sexual species as sperm donors. We used isozyme electrophoresis, blood erythrocytes, and chromosome counts in a continued effort to identify sexual and unisexual individuals in eastern North America. We examined 1377 salamanders from 118 sites in Connecticut, Massachusetts, New Jersey, New York, Pennsylvania, and Virginia. Most Pennsylvania salamanders were A. jeffersonianum (JJ) but A. laterale (LL), p...

Unisexual salamanders (genus Ambystoma) present a new reproductive mode for eukaryotes

To persist, unisexual and asexual eukaryotes must have reproductive modes that circumvent normal bisexual reproduction. Parthenogenesis, gynogenesis, and hybridogenesis are the modes that have generally been ascribed to various unisexuals. Unisexual Ambystoma are abundant around the Great Lakes region of North America, and have variously been described as having all 3 reproductive modes. Diploid and polyploid unisexuals have nuclear genomes that combine the haploid genomes of 2 to 4 distinct sexual species, but the mtDNA is unlike any of those 4 species and is similar to another species, Ambystoma barbouri. To obtain better resolution of the reproductive mode used by unisexual Ambystoma and to explore the relationship of A. barbouri to the unisexuals, we sequenced the mitochondrial control and highly variable intergenic spacer region of 48 ambystomatids, which included 28 unisexuals, representatives of the 4 sexual species and A. barbouri. The unisexuals have similar sequences over most of their range, and form a close sister group to A. barbouri, with an estimated time of divergence of 2.4-3.9 million years ago. Individuals from the Lake Erie Islands (Kelleys, Pelee, North Bass) have a haplotype that demonstrates an isolation event. We examined highly variable microsatellite loci, and found that the genetic makeup of the unisexuals is highly variable and that unisexual individuals share microsatellite alleles with sexual individuals within populations. Although many progeny from the same female had the same genotype for 5 microsatellite DNA loci, there was no indication that any particular genome is consistently inherited in a clonal fashion in a population. The reproductive mode used by unisexual Ambystoma appears to be unique; we suggest kleptogenesis as a new unisexual reproductive mode that is used by these salamanders.

A nonlethal method of identification of Ambystoma laterale, A. jeffersonianum and sympatric unisexuals

AbstractUnisexual salamanders in the genus Ambystoma live in obligate sympatry with the sexual species Ambystoma laterale or Ambystoma jeffersonianum, from which they are not easily distinguished. Because the sexual species are protected in many parts of their range, accurate identification of sexual and unisexual individuals is required for conservation purposes. Unisexual individuals are currently identified using isozyme electrophoresis, which requires sacrificing the animal. Here we present a nonlethal method of identification for members of the A. laterale–jeffersonianum part of the Ambystoma complex utilizing genome‐specific microsatellites with or without the addition of flow cytometry.