Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.     

Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicaudatus: a review

This paper assesses the present state of the art of ploidy manipulation in the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae). Diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Diploid sperm produced by artificially sex-reversed clone loaches

Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.     

Production of fertile unreduced sperm by hybrid males of the Rutilus alburnoides complex (Teleostei,cyprinidae). An alternative route to genome tetraploidization in unisexuals.

The hybrid minnow Rutilus alburnoides comprises diploid and polyploid females and males. Previous studies revealed that diploid and triploid females exhibit altered oogenesis that does not involve random segregation and recombination of the genomes of the two ancestors, constituting unisexual lineages. In the present study, we investigated the reproductive mode of hybrid males from the Tejo basin, using experimental crosses and flow cytometric analysis of blood and sperm. The results suggest that diploid hybrids produced fertile unreduced sperm, transmitting their hybrid genome intact to offspring. Triploid hybrids also produced unreduced sperm, but it was not possible to obtain data concerning their fertility. Finally, tetraploid hybrids produced fertile diploid sperm, which exhibited Mendelian segregation. Tetraploid R. alburnoides may reestablish biparental reproduction, as individuals of both sexes with the appropriate constitution for normal meiosis (two haploid genomes from each parental species) are likely to occur in natural populations. Tetraploids probably have arisen from syngamy of diploid eggs and diploid sperm produced by diploid hybrid males. Diploid hybrid males may therefore play a significant role in the dynamics of the complex, starting the evolutionary process that may ultimately lead to a new sexually reproducing species.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.     

A cryptic clonal line of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) evidenced by induced gynogenesis,interspecific hybridization,microsatellite genotyping and multilocus DNA fingerprinting

In Memanbetsu town, Hokkaido island, Japan, a high frequency of natural triploid loaches Misgurnus anguillicaudatus (7.4% on average) was detected by flow cytometry for relative DNA content. Among sympatric diploid females (n=6) from a single population, we found two unique females that laid unreduced diploid eggs. They gave normal diploid progeny even after induction of gynogenesis with genetically inert UV-irradiated sperm. When fertilized with normal loach sperm, some unreduced eggs developed into triploids, but the rest into diploids. Hybridization using goldfish Carassius auratus sperm gave both normal diploid loaches and inviable allotriploid hybrids possessing the diploid loach genome and the haploid goldfish genome. Microsatellite genotyping and DNA fingerprinting demonstrated that the diploid progeny developing from the unreduced eggs were genetically identical to the mother, while the triploids had some of the paternal DNA. These results indicate that the diploid eggs reproduced unisexually as a diploid clone and in other cases developed into triploids after accidental incorporation of sperm nucleus. The presence of at least one clonal line in this area was shown by the identical DNA fingerprint detected in five out of 17 diploid loaches examined.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Similar Performance of Diploid and Haploid Males in an Ant Species without Inbreeding Avoidance

Under haplodiploidy, a characteristic trait of all Hymenoptera, females develop from fertilised eggs, and males from unfertilised ones. Males are therefore typically haploid. Yet, inbreeding can lead to the production of diploid males that often fail in development, are sterile or are of lower fertility. In most Hymenoptera, inbreeding is avoided by dispersal flights of one or both sexes, leading to low diploid male loads. We investigated causes for the production of diploid males and their performance in a highly inbred social Hymenopteran species. In the ant Hypoponera opacior, inbreeding occurs between wingless sexuals, which mate within the mother nest, whereas winged sexuals outbreed during mating flights earlier in the season. Wingless males mate with queen pupae and guard their mating partners. We found that they mated randomly with respect to relatedness, indicating that males do not avoid mating with close kin. These frequent sib‐matings lead to the production of diploid males, which are able to sire sterile triploid offspring. We compared mating activity and lifespan of haploid and diploid wingless males. As sexual selection acts on the time of emergence and body size in this species, we also investigated these traits. Diploid males resembled haploid ones in all investigated traits. Hence, albeit diploid males cannot produce fertile offspring, they keep up with haploid males in their lifetime mating success. Moreover, by fathering viable triploid workers, they contribute to the colonies' work force. In conclusion, the lack of inbreeding avoidance led to frequent sib‐matings of wingless sexuals, which in turn resulted in the regular production of diploid males. However, in contrast to many other Hymenopteran species, diploid males exhibit normal sexual behaviour and sire viable, albeit sterile daughters.     

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

Life‐history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.     

Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark

All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.     

Hybridization studies in the planthopper genus Ribautodelphax (Homoptera,Delphacidae)

Seven known Ribautodelphax species and four recently discovered taxa were crossed. Forty-three percent of the interspecific crosses were successful. Interspecific crosses generally resulted in low numbers of hybrids. In some combinations prolonged larval development of the hybrid males was observed. Backcrosses showed that both male and female hybrids were fertile. Reproductive isolation was mainly maintained by premating barriers. Insemination tests revealed that prolonged confinement of males and females lowered mating thresholds. The taxonomic status of the known species and new taxa was shown to be in need of revision. No indications for a hybrid origin of the pseudogamous triploid Ribautodelphax females were found.     

Does triploidization produce functional sterility of triploid males of tench <Emphasis Type="Italic">Tinca tinca</Emphasis> (L.)?

Triploidy interferes with gametogenesis in all fish species tested so far. In fish it results in complete female sterility however, males are still able to develop testis. The reason why sterility levels in triploid fishes differ among species and between sexes is unclear. In the present study the reproductive capacity of triploid males of tench was studied. Flow cytometry revealed sperm cells of triploids to be largely aneuploid with high mosaic DNA, oscillating from haploid DNA to diploid DNA content. Analysis of variance showed an insignificant influence of ploidy level on the percentage of motile spermatozoa, as well as on spermatozoa velocity. Experimental crosses between normal diploid female and triploid males resulted in the appearance of triploid progeny, which exhibited genotypes composed of microsatellite alleles inherited from the founder female and additional allele derived from the donor male. We can conclude that the triploid males analysed in the present study were capable to fertilize eggs derived from diploid females.     

Fertile triploid males: an uncommon case among hybrid vertebrates

The endemic Iberian minnow Squalius alburnoides is a complex of fishes of hybrid origin including both males and females with distinct ploidy levels and varying proportions of the parental genomes. In this paper we demonstrated that in contrast to many vertebrate hybrid lineages the sperm of triploid hybrid males of S. alburnoides is viable and fully functional. Flow cytometry and analysis of sequences of a fragment of the beta-actin nuclear gene applied to progenitors and offspring evidenced that these males produced their sperm clonally, as already described for diploid hybrids. The presence of different types of fertile males (nonhybrid diploids with normal meiosis and both diploid and triploid hybrids) coupled with hybridogenetic meiosis in females endows this vertebrate complex with a high level of independence from other species and contributes to maintain its genetic variability.     

Reproductive behaviour and sexual production in the first‐generation hybrids of roach Rutilus rutilus L. × common bream Abramis brama L

The aim of this study was to further test the viability of the roach Rutilus rutilus × common bream Abramis brama hybrid in terms of reproductive behaviour and sexual production. Egg release, mating and aggressive acts in reproductive behaviour, as well as absolute fecundity and sperm density in milt for sexual production were examined in the first generation of these hybrids at their first sexual maturity. The F2 and backcrosses of hybrids were also studied. The results revealed that these hybrids expressed a normal and typical mating behaviour, producing viable gametes. Under experimental reproduction between hybrids (hybrid reproduction), the number of egg‐release acts (range, 21–66) was nearer (χ² test, P > 0.05) the number of mating acts (11–65). Moreover, hybrid males exhibited territorial and aggressive behaviours. However, in experimental reproduction of female and male hybrids mixed with parental males (mixed reproduction), the egg‐release act and the mating act were inhibited by the intense territorial and aggressive activities of the common bream male. Absolute fecundity values (median, <2.2 × 10³ eggs) and sperm density (<7 × 10⁹ spermatozoa ml^?1) of hybrids showed a greater decrease (U test, P < 0.05) than in parental species (median, >6.0 × 10³ eggs and >14 × 10⁹ spermatozoa ml^?1, respectively). Nevertheless, these hybrids were fertile. F2 and backcross generations were produced, although with a significantly lower viable hatching rate (FEP test, P < 0.05) in F2 individuals from the female and its corresponding hybrid male (<6%), indicating a very low chance of survival in rivers.     

Durum wheat as a candidate for the unknown female progenitor of bread wheat: an empirical study with a highly fertile F<Subscript>1</Subscript> hybrid with <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss.

Hexaploid bread wheat was derived from a hybrid cross between a cultivated form of tetraploid Triticum wheat (female progenitor) and a wild diploid species, Aegilops tauschii Coss. (male progenitor). This cross produced a fertile triploid F₁ hybrid that set hexaploid seeds. The identity of the female progenitor is unknown, but various cultivated tetraploid Triticum wheats exist today. Genetic and archaeological evidence suggests that durum wheat (T. turgidum ssp. durum) may be the female progenitor. In previous studies, however, F₁ hybrids of durum wheat crossed with Ae. tauschii consistently had low levels of fertility. To establish an empirical basis for the theory of durum wheat being the female progenitor of bread wheat, we crossed a durum wheat cultivar that carries a gene for meiotic restitution with a line of Ae. tauschii. F₁ hybrids were produced without using embryo rescue techniques. These triploid F₁ hybrids were highly fertile and spontaneously set hexaploid F₂ seeds at the average selfed seedset rate of 51.5%. To the best of our knowledge, this is the first example of the production of highly fertile F₁ hybrids between durum wheat and Ae. tauschii. The F₁ and F₂ hybrids are both similar morphologically to bread wheat and have vigorous growth habits. Cytological analyses of F₁ male gametogenesis showed that meiotic restitution is responsible for the high fertility of the triploid F₁ hybrids. The implications of these findings for the origin of bread wheat are discussed.     

Reproductivity of early males of the temperate paper wasp Polistes rothneyi iwatai

In Polistes paper wasps, haploid early males can mate with early emerging females and leave viable offspring. In contrast, diploid early males are eventually sterile because they contribute triploid offspring via diploid sperm. Clarifying the ploidy of early males is important for determining whether early male production is a reproductive strategy for the species. We examined the mating behavior and the ploidy of early males in the Japanese paper wasp, Polistes rothneyi iwatai van der Vecht. Thirteen early males from four colonies were all diploid. Two of the nine early males (22.2%) attempted to mate with females, but only one individual (11.1%) was successful (the female's spermatheca contained spermatozoa). These results suggest that although most early males of P. rothneyi iwatai do not produce offspring, their mating may be linked to the occasional production of triploid females.