Molecular phylogeny of Gastrotricha on the basis of a comparison of the 18S rRNA genes: Rejection of the hypothesis of a relationship between Gastrotricha and Nematoda

Gastrotricha are the small meiobenthic acoelomate worms whose phylogenetic relationships between themselves and other invertebrates remain unclear, despite all attempts to clarify them on the basis of both morphological and molecular analyses. The complete sequences of the 18S rRNA genes (8 new and 7 known) were analyzed in 15 Gastrotricha species to test different hypotheses on the phylogeny of this taxon and to determine the reasons for the contradictions in earlier results. The data were analyzed using both maximum likelihood and Bayesian methods. Based on the results, it was assumed that gastrotrichs form a monophyletic group within the Spiralia clade, which also includes Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea, and Lophotrochozoa. Statistical tests rejected a phylogenetic hypotheses considering Gastrotricha to be closely related to Nematoda and other Ecdysozoa or placing them at the base of the Bilateria tree, close to Acoela or Nemertodermatida. Among gastrotrichs, species belonging to the orders Chaetonotida and Macrodasyida form two well-supported clades. The analysis confirmed monophyly of the families Chaetonotidae and Xenotrichulidae from the order Chaetonida, as well as the families Turbanellidae and Thaumastodermatidae from the order Macrodasyida. Lepidodasyidae is a polyphyletic family, because the genus Mesodasys forms a sister group for Turbanellidae; genus Cephalodasys forms a separate branch at the base of Macrodasyida; and Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To confirm these conclusions and to get an authentic view of the phylogeny of Gastrotricha, it is necessary to study more Gastrotricha species and to analyze some other genes.     

Spermatology of the genus Lepidodasys Remane, 1926 (Gastrotricha, Macrodasyida): towards a revision of the family Lepidodasyidae Remane, 1927

The spermatozoa of Lepidodasys unicarenatus and Lepidodasys sp. are filiform and composed of a cork-screw shaped acrosome, a helical nucleus surrounding a mitochondrial axis, and a 9 × 2 + 2 flagellum as in the basic structural model of the macrodasyidan sperm. The genus Lepidodasys has a debated phylogenetic position and has been linked in turn to the family Lepidodasyidae and the family Thaumastodermatidae. The sperm features of the two Lepidodasys species examined are distinct from those typical of the two families: the absence of the periaxonemal cylinder, a character shared only with Turbanellidae among Macrodasyida, could be considered as a symplesiomorphy, suggesting a basal position of the genus along the Macrodasyida clade. Moreover, a comparison of the spermatogenic process of Lepidodasys sp. with those of Acanthodasys aculeatus (Thaumastodermatidae) and Cephalodasys maximus (Lepidodasyidae) has revealed that the process of acrosome formation and nuclear morphology during spermatogenesis are peculiar in Lepidodasys sp. and differences are evident especially in the late stages of spermatogenesis. Penetrated spermatozoa were observed in the oocytes at all maturation stages in L. unicarenatus.     

A fresh look at Dinodasys mirabilis (Gastrotricha, Macrodasyida), with focus on the reproductive apparatus and sperm ultrastructure

Adults of Dinodasys mirabilis were studied for the first time. The specimens, collected from the west coast of Sweden, were investigated alive and with electron microscopy. Sexually mature specimens attain a total length of 450?C490???m; the adhesive apparatus is made up of anterior, lateral, ventrolateral, dorsal and posterior tubes; and one pair of ??cirrata?? type tubes is also present. The reproductive apparatus is hermaphroditic, paired testes extend rearward from the pharyngeo-intestinal junction to 3/4 of the trunk, and sperm ducts bend anteriorly at U52 and join together into a common, mid-ventral pore at U33. Two ovaries lie along the sides of the caudal intestine, extending anteriorly from U68. Frontal organ present on the right side of the body centered a U70; caudal organ was absent; a gland organ surrounding the terminal intestine may be present, but its homology with other organs in a similar position is uncertain. The spermatozoon is a filiform cell, formed by a long acrosome, a spring-like nucleus and a flagellum. The acrosome is divided into two regions: the anterior-most is thin and corkscrew-shaped, and the posterior one is rectilinear; both regions are delimited by a continuous external layer of thick, dense material, which in longitudinal section appears obliquely striated and surrounds a long pile of stout, electron-dense cylinders; the nucleus contains condensed chromatin and is coiled around an elongate mitochondrion; the flagellum possesses a 9?×?2?+?2 axoneme devoid of striated cylinder. Within Macrodasyida, U-bend sperm ducts and the peculiar ultrastructure of the acrosome are characteristics shared by other Turbanellidae studied so far, providing a foundation for the current systematization of Dinodasys.     

Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.     

Anatomy and ultrastructure of the reproductive organs in Dactylopodola typhle (Gastrotricha: Macrodasyida) and their possible functions in sperm transfer

Abstract. The reproductive anatomy of gastrotrichs is well known for several species, especially for the marine taxon Macrodasyida. However, there is little information on the reproductive organs and the modes of mating and sperm transfer in putative basal taxa, which is necessary for accurate reconstruction of the ground pattern of the Gastrotricha. We present the first detailed morphological investigation of the reproductive system of a putative basal gastrotrich, Dactylopodola typhle , using transmission and scanning electron microscopy, histology, and microscopic observations of living specimens. Dactylopodola typhle is a hermaphrodite that possesses paired female and male gonads, an unpaired uterus with an outlet channel that we call the cervix, and an additional accessory reproductive organ, the so-called caudal organ. We hypothesize that the hollow, secretory caudal organ serves for picking up autospermatozoa (self-sperm), for spermatophore formation, and finally for transferring the autospermatophore to a mating partner. The allospermatophore (foreign spermatophore) is stored within the uterus where fertilization occurs. We think that the mature and fertilized egg is released through the cervix and the dorsolateral female gonopore, and not by rupture of the body wall. Based on the morphology, we provide a plausible hypothesis for spermatophore formation and transfer in D. typhle . Preliminary phylogenetic considerations indicate that the stem species of Macrodasyida, perhaps that of all Gastrotricha, had paired ovaries and paired testes, an unpaired uterus, and only one accessory reproductive organ.     

Reproductive system of the genus <Emphasis Type="Italic">Crasiella</Emphasis> (Gastrotricha,Macrodasyida)

Crasiella diplura from Sweden and Crasiella sp. from Italy were studied alive and with transmission electron microscopy (TEM). The two species are simultaneous hermaphrodites and share the same reproductive system lay-out: paired ovaries extend along the posterior part of the intestine and join mid-dorsally, while bilateral, club-shaped testes lie at the sides of the anterior gut, extending as deferentia that fuse on the mid-ventral plane and open into a single pore; gametes mature in a caudocephalic and centripetal direction. The bulky, sac-like, frontal organ is lined by a simple epithelium and lies dorsolaterally to the intestine, on the left side of the body. The spindle-shaped caudal organ is musculo-glandular and is located ventrolaterally to the gut on the right side. It is characterized by the presence of a roughly Y-shaped internal channel that opens into two pores close to each other, which function for the intake and outlet of the (auto)sperm, respectively. The spermatozoa, which are peculiar and similar in the two species, are characterized by a long and complex acrosome consisting of four ultrastructurally distinct regions, three of which find equivalence in other gastrotrich species. The flagellum lacks a striated cylinder. Anatomy and ultrastructure enable us to hypothesize a modality of sperm transfer in Crasiella that is similar to that observed in Macrodasys.     

Reproductive apparatus and male accessory structures in two batrachoid species (Teleostei, Batrachoididae)

The morphology of the genital apparatus of two batrachoid species, Opsanus tau and Porichthys notatus , was studied. The anatomical organization of the female reproductive apparatus is similar in both species but differences are observed in the rhythm of gametogenesis with individual oocyte production asynchronous in O. tau and group synchronous in P. notatus. The male reproductive apparatus is similarly organized in the two species, with both showing enlongated testes with an efferent duct system, two main testicular ducts, a common sperm duct, and a pair of multi-channel accessory structures. The sperm transport system consisting of the efferent duct system, main testicular ducts, and sperm duct is more developed in P. notatus and only in this species does it secrete sialoglycoproteins. Male accessory organs also secrete sialoglycoprotein in both species, but they appear more developed in O. tau. Intraspecific variability in development of accessory structures and mucin secretion was also observed between the two male morphs of P. notatus. Type I males, which build nests and perform parental care to eggs and fry, have larger accessory organs and more abundant secretion than type II males, which adopt opportunistic spawning tactics. The possible role played by mucins as components of the seminal fluid, in both species, and their inter-and intraspecific variability are discussed in the light of the reproductive biology and the presence of alternative male mating tactics.     

A one‐locus model of androdioecy with two homomorphic self‐incompatibility groups: expected vs. observed male frequencies

Androdioecy, the occurrence of males and hermaphrodites in a single population, is a rare breeding system because the conditions for maintenance of males are restrictive. In the androdioecious shrub Phillyrea angustifolia, high male frequencies are observed in some populations. The species has a sporophytic self‐incompatibility (SI) system with two self‐incompatibility groups, which ensures that two groups of hermaphrodites can each mate only with the other group, whereas males can fertilize hermaphrodites of both groups. Here, we analyse a population genetic model to investigate the dynamics of such an androdioecious species, assuming that self‐incompatibility and sex phenotypes are determined by a single locus. Our model confirms a previous prediction that a slight reproductive advantage of males relative to hermaphrodites allows the maintenance of males at high equilibrium frequencies. The model predicts different equilibria between hermaphrodites of the two SI groups and males, depending on the male advantage, the initial composition of the population and the population size, whose effect is studied through stochastic simulations. Although the model can generate high male frequencies, observed frequencies are considerably higher than the model predicts. We finally discuss how this model may help explain the large male frequency variation observed in other androdioecious species of Oleaceae: some species show only androdioecious populations, as P. angustifolia, whereas others show populations either completely hermaphrodite or androdioecious.     

Ultrastructural observations on the spermatozoa of a species of <Emphasis Type="Italic">Lepidodasys</Emphasis> (Gastrotricha,Macrodasyida)

The ultrastructure of the spermatozoon of a species in the marine gastrotrich genus Lepidodasys is described. The filiform cell is composed of a cork-screw acrosome, a long single mitochondrion surrounded by a helical nucleus, and a flagellum with a 9 × 2 + 2 axonemal arrangement. The structure of the sperm of this species from Denmark appears closely similar to those of the other two species of Lepidodasys studied so far from Italy and Florida (US). Peculiar features (cylindrical nucleus, absence of a periaxonemal sheath) place this genus far from the others in the family Lepidodasyidae. The absence of synapomorphies between Lepidodasys and other genera of Lepidodasyidae suggests that the family is polyphyletic. The sperm ultrastructure fully fits the species of Lepidodasys into the marine order Macrodasyida, with the sperm ground plan of which its sperm shares a number of details.     

Sperm morphology of scleractinians from the Caribbean

This study compares the sperm morphology of 2 hermaphroditic (Diploria strigosa, Montastrea annularis) and 2 gonochoric (Siderastrea siderea, Dendrogyra cylindrus) Caribbean reef corals. The spermatozoa of the gonochoric species are morphologically more specialized than those of the hermaphrodites. The cytological differences are apparent in the formation of a Spitzenkörper and pro-acrosomal vesicles, a mitochondrial mass (Nebenkern) and a large lipid vesicle in the gonochorists studied. Harrison (1985) first demonstrated that sperm morphology is correlated with sexual reproductive pattern and systematic position in scleractinian corals, and this is supported by the results of this study. The possible functional and phylogenetic bases of sperm morphology are discussed and a brief outline is given of further studies required.     

Ultrastructure of dimorphic sperm and seminal receptacle in the hermaphrodites Barrimysia siphonosomae and Pseudopythina ochetostomae (Bivalvia, Galeommatoidea)

The intertidal Bivalvia Barrimysia siphonosomae (Montacutidae) and Pseudopythina ochetostomae (Kelliidae) live commensally with Siphonosoma cumanense (Sipuncula) and Ochetostoma erythrogrammon (Echiura), respectively. Both bivalves are hermaphrodites although males and females may occur. Paired, pouch-shaped seminal receptacles are located in the suprabranchial chamber near the genital opening of both species. The interior of the receptacles of sexually mature individuals contains numerous non-epithelial cells separated by narrow spaces that serve as a depot for sperm cells. Specimens of both species produce two sperm morphotypes. Euspermatozoa are oblong with elongate conical acrosomes, and a middlepiece consisting of about eight long mitochondria spirally arranged to form a sheath surrounding the basis of the flagellum. The paraspermatozoa are vermiform, about 4 µm broad and longer (up to 240 µm) in B. siphonosomae than in P. ochetostomae (ca 150 µm). The anterior, bullet-shaped, 4.5- or 3.0-µm-long acrosome is located above an elongate, 4- to 6-µm-long subcylindrical nucleus. Adjacent to the nucleus occurs a bundle of approximately 25 (B. siphonosomae) or ca 10 (P. ochetostomae) flagella. The cytoplasm of the sperm body contains spherical lipid droplets, glycogen deposits and numerous membrane-bound spindle- to rod-shaped electron-dense granules. It is presumed that both sperm types aggregate to form spermatozeugmata. The presence of a combination of almost identical and highly specialised euspermatozoa and paraspermatozoa in species of the two genera qualifies these structures as strong synapomorphies. Pseudopythina, as presently defined, is considered paraphyletic.     

Conservation of large foci formation in arrested oocytes of <Emphasis Type="Italic">Caenorhabditis</Emphasis> nematodes

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.     

Sperm morphology in the Pterastericolidae (Platyhelminthes, Rhabdocoela): phylogenetic implications

The sperm morphology of five species of the Pterastericolidae was studied with transmission electron microscopy. The spermatozoa of all species have two axoncmes, which are incorporated in the sperm cell body for most of their length. The axonemes arc of the'9 + 1'pattern characteristic of the flatworm taxon Trepaxonemata. Outer dynein arms are absent from the microtubule doublets in the axonemes. Dense bodies are few, and occur only in the distal part of the spermatozoa, but small electron dense granules are numerous. A sister group of the Neodermata consisting of the Pterastericolidae and the Fecampidae is proposed. The monophyly of the taxon Dalyellioida is discussed     

The artificial insemination of bumblebee queens

Summary: Artificial insemination (AI) is a technique to transfer instrumentally sperm from the male into the female's reproductive system. AI is widespread in vertebrates for economical animal breeding and for conservation biology. However, in invertebrates only a few cases of successful AI have been reported. In this paper we describe a new technique to artificially inseminate bumblebee queens (Bombus spp.). Males were dissected and the accessory testes were removed and washed in insect ringer. They were then opened and the outflowing sperm was picked up with a glass capillary mounted on a syringe. For the sperm transfer into the queen we adapted a standard apparatus used for honeybee inseminations. The queen was anaesthetized with CO₂, held in place by a queen holder and the sting chamber was opened using two hooks. The sperm containing glass capillary was introduced into the queen's sexual tract. The sperm was released into the bursa copulatrix very near the opening of the spermathecal duct of the queen. Inseminated queens were hibernated for 2 weeks and produced a normal colony under field conditions. Multiple inseminations proved to be successful since different patrilines could be detected in the worker offspring. Successful inseminations were performed for B. terrestris, B. lucorum and B. hypnorum.     

Intense Sperm-Mediated Sexual Conflict Promotes Reproductive Isolation in Caenorhabditis Nematodes

Conflict between the sexes over reproductive interests can drive rapid evolution of reproductive traits and promote speciation. Here we show that inter-species mating between Caenorhabditis nematodes sterilizes maternal individuals. The principal effectors of male-induced harm are sperm cells, which induce sterility and shorten lifespan by displacing conspecific sperm, invading the ovary, and sometimes breaching the gonad to infiltrate other tissues. This sperm-mediated harm is pervasive across species, but idiosyncrasies in its magnitude implicate both independent histories of sexually antagonistic coevolution within species and differences in reproductive mode (self-fertilizing hermaphrodites versus females) in determining its severity. Consistent with this conclusion, in androdioecious species the hermaphrodites are more vulnerable, the males more benign, or both. Patterns of assortative mating and a low incidence of invasive sperm occurring with conspecific mating are indicative of ongoing intra-specific sexual conflict that results in inter-species reproductive incompatibility.     

Differences in sperm morphology in foam‐nesting leptodactyline frogs (Anura,Leptodactylidae)

Sperm morphology is diverse among vertebrates and is influenced by the reproductive strategies adopted by species. In anurans, sperm morphology is associated with reproductive modes and mating systems. Here, we describe the sperm morphology of 11 frog species in the genus Leptodactylus and that of Lithodytes lineatus and discuss the relationship between sperm morphology and species' mating systems. We observed two distinct sperm morphotypes among the leptodactyline species, which differed mostly in head morphology. Type I sperm had triangular head, discrete acrosome vesicle with posterior margin not clearly visible; type II sperm had elongated head, clear acrosomal vesicle with posterior margin clearly visible. These sperm types do not seem to be associated with phylogeny; instead, type II sperm was observed in all polyandrous species analysed and in species with evidences of polyandry. Moreover, sperm of all species presented tail with undulating membrane connected to the axial fibre. We suggest that differences in sperm morphology might be associated with sperm competition to what polyandrous species are subjected. However, natural history observations on polyandrous mating in some species presenting type II sperm and phylogenetic comparative studies are need to elucidate the role of mating systems in the evolution of sperm morphology in leptodactylines.     

Functional morphology of the sperm-containing chambers of the sea slug Okenia polycerelloides in the context of sexual selection

Sea slugs are interesting models to study post-copulatory sexual selection in simultaneous hermaphrodites due to the enormous variation of their reproductive systems. However, the knowledge of the functional morphology of their reproductive system is limited to few species, and it is rarely discussed in the context of sexual selection theory. In this study, we investigated the functional morphology of the sperm-containing chambers (i.e., ampulla, seminal receptacle, and bursa copulatrix) of the reproductive system of Okenia polycerelloides (Ortea & Bouchet, 1983), based on light, confocal, and electron microscopy. Although the morphology of the ampulla is similar to other species, indicating that it is a site for autosperm storage, we found some sperm facing the ampullar epithelium, a feature commonly regarded as characteristic of the seminal receptacle of sea slugs. The seminal receptacle of O. polycerelloides showed secretory activity and contained sperm with distribution and orientation suggestive of stratification of allosperm from distinct mating events, a feature that would affect sperm competition. The bursa copulatrix had epithelial cells with secretory and absorptive characteristics, and contained degraded sperm and yolk granules within its lumen. Comparative analyses of the contents of each organ demonstrated that sperm digestion occurs in the bursa copulatrix and affects sperm heads first, changing their morphology from slender and curved to shorter and ellipsoid before complete lysis. Although digestion and absorption of surplus sperm are currently the main hypothesized functions for the bursa copulatrix, its role in cryptic female choice should not be ruled out. The close structural connection between the seminal receptacle and bursa copulatrix, as well as their muscular walls, would enable control over the fate of the sperm received in each mating event, that is, storage or digestion.     

The Reproductive System of Gastrotrichs. I Introduction with morphological data for two new Dolichodasys species1

This paper begins a series aimed at clarifying the structure and function of reproductive organs in Gastrotricha Macrodasyida. Morphological data are presented for Dolichodasys carolinensis sp.n. and D. delicatus sp.n. Both are elongate, have reduced adhesive organs and complex accessory reproductive organs, termed the frontal-caudal organ system. The system consists of a band of cells behind the gonads in the central body chamber. The anterior portion, the frointal organ, stores foreign spermatozoa; the posterior portion, the caudal organ, may function as a copulatory organ. The caudal organ has two canals forming a loop opening at a single ventral pore. One canal contains a spiralled secretion, the other elongated filaments that appear to be muscle cell derivatives. Systematic groupings are made for other long-bodied Gastrotricha Macrodasyida.     

Reproduction in the genus Limacina (Opisthobranchia: Thecosomata)

Reproductive mechanisms in the seven species of the thecosomatous pteropod genus Limacina are described and compared. All species are protandrous hermaphrodites. Five species– L. bulimoides, L. helicina, L. lesueuri, L. retroversa and L. trochiformis –have a similar reproductive anatomy in which the gonoduct leading from the gonad to the common genital pore functions as a seminal vesicle in the male and is elaborated into mucous and albumen glands in the female. The male system consists of a prostate gland and penis connected to the common genital pore by an external ciliary tract. All five species have a free-swimming veliger stage which hatches from free-floating egg masses. Limacina helicoides has the same reproductive anatomy but is ovoviviparous, with embryos retained in capsules in the mucous gland until they are juveniles of 50 mm in shell diameter. Limacina inflata lacks mucous and albumen glands and a penis; a spermatophore formed by the prostate gland is used in aphallic sperm transfer. This species exhibits brood protection with un-encapsulated embryos retained in the mantle cavity until they are released as veligers measuring 0067 mm in diameter. L. inflata is the most abundant of the seven species despite lowered fecundity; reasons for its ecological success are discussed.     

Androdioecy and hermaphroditism in five species of clam shrimps (Crustacea: Branchiopoda: Spinicaudata) from India and Thailand

Crustaceans in the order Spinicaudata display a broad range of reproductive strategies, ranging from pure hermaphroditism to pure dioecy (separate males and females), and intermediate combinations. One particularly interesting genus of these “clam shrimps” is Eulimnadia. Based on offspring sex ratios, it has been suggested that all members of the genus are androdioecious: populations consist of mixtures of males and hermaphrodites. However, only two of the ~40 species in this genus have been examined histologically to confirm the presence of ovotestes in the purported hermaphrodites of this group. Here, we report both sex ratio and histological evidence showing that populations of five additional Eulimnadia species from India and Thailand are indeed mixes of males and hermaphrodites (four species) or hermaphrodite only (one species). Sex ratios of adults and offspring from isolated hermaphrodites are in accordance with those previously reported for 15 Eulimnadia species, and histological assays of four of the five species show the presence of both testicular and ovarian tissue in these hermaphrodites. As has been previously reported, the testicular tissue in members of these Eulimnadia spp. is located in a small section at the distal end of the gonad. In addition, the sperm produced in these hermaphrodites forms distinct plaques of compacted chromatin. Overall, these data are consistent with a single origin of hermaphroditism in Eulimnadia, and support the notion that all members of the genus are either androdioecious or all‐hermaphroditic.