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Seminars in Cell & Developmental Biology 18 (2007) 362–370 Review Evolution of the control of sexual identity in nematodes Andre Pires-daSilva ∗ UT Arlington, Department of Biology, 501 S. Nedderman, 337 LS Building, Arlington, TX 76019, United States Available online 19 January 2007 Abstract Most animals are male/female species and reproduce sexually. Variation in this pattern of reproduction has arisen many times during animal evolution, particularly in nematodes. Little is known about the evolutionary forces and constraints that influenced the origin of self-fertilization, for instance, a type of reproduction that seems to have evolved many times in the phylum Nematoda. Caenorhabditis elegans, a very well known nematode, provides the framework for comparative studies of sex determination. The relative ease with which nematodes can be studied in the laboratory and the fact that many recently developed techniques can be applied to many species make them attractive for comparative research. It is relatively poorly understood how the evolution of new types of sex determination and mode of reproduction results in changes in genome structure, ecology and population genetics. Here, I review the evolution of sex determination and mating types in the phylum Nematoda with the objective of providing a framework for future research. © 2007 Elsevier Ltd. All rights reserved. Keywords: Sex determination; Evolution; Nematodes Contents 1. 2. 3. 4. 5. 6. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Making one sex different from the other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal and environmental sex determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of the sex determination pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of self-fertilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Nematodes were one of the first animals chosen for cytological studies, which ultimately led to the discovery of the correlation between chromosomal make up of an embryo and its future sexual development—male or female [1]. The study of the mechanisms and evolution of sex determination intersects several fundamental questions in biology such as why and how sex is maintained, what forces govern the evolution of genome structure, how ecological factors constrain or favor reproductive mode, and how genetic networks evolve. ∗ Tel.: +1 817 272 1383; fax: +1 817 272 2855. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2006.11.014 362 363 364 365 366 367 367 367 It is estimated that there are about 500,000 nematode species [2], most of them uncharacterized. One species of nematode, Caenorhabditis elegans, is arguably one of the bestcharacterized metazoans. This knowledge provides a basis for comparative studies to understand the great variation found in the phylum. Now, with the numerous genome sequencing projects and the possibility of applying new technologies (e.g., FISH, microarrays and RNAi) to previously genetically intractable organisms, there is the opportunity to expand and address questions to a higher number of nematode species. Nematodes are particularly suited for comparative studies because they show a rich diversity in sex determination mechanisms, have relatively small genomes, simple body plans and live in a variety of ecological contexts. The consequences of the evolution of androdioecy in nematodes (populations consisting A. Pires-daSilva / Seminars in Cell & Developmental Biology 18 (2007) 362–370 363 of males and hermaphrodites), for example, are not well understood in terms of population genetics or evolution of the genome. Importantly, many species of nematodes are plant and animal parasites. Advances in knowledge and technology for manipulating sex ratios may open up the possibility of controlling their reproduction. In this review, I summarize recent developments in comparative analysis of evolution of sex determination and mating systems in Nematoda (for more specific reviews about this subject in C. elegans and close relatives, see Refs. [3,4]). The purpose of this review is to synthesize the variations found in this phylum and highlight potential new avenues of research. 2. Making one sex different from the other Most nematodes have conventional sexual reproduction, with morphologically distinct sexes. Males and females are defined by the size and motility of their gametes: males produce small, motile gametes (usually in profusion), whereas females produce larger, fewer and immotile gametes. Hermaphroditic nematodes produce both gametes, and reproduce mainly by self-fertilization (as opposed to the reciprocal sperm-swapping seen in other phyla). Since nematodes reproduce mainly by internal fertilization, an entire array of signals, body structures and behaviors evolved for mate recognition and efficient fertilization. In nematodes, most of the sexually dimorphic characters are restricted to the reproductive and nervous system, although differences in body size [5,6], pheromone production [7] and life span have also been noted [8]. C. elegans, a hermaphroditic species, has extensive sexual dimorphism (Fig. 1). It includes all tissue types and about one third of the adult somatic cells [9–13]. The hermaphrodites are bigger, have two gonad arms (didelphic), a pair of organs where the sperm is stored (spermathecae), a vulva and a yolk-producing intestine [10]. Didelphic gonads are probably ancestral for females, but males usually have a single testis that extends anteriorly [14]. Males are slender and have various sensory and copulatory structures (i.e., spicules, fan, and rays) in the tail that are used to locate the vulva and inseminate the hermaphrodite. Sex-specific muscles and neurons are involved in egg-laying in hermaphrodites and mating, copulation and locomotory behavior in males. In some nematodes sexual dimorphism can be very dramatic: the Trichosomoides crassicauda male is small enough to enter the female by way of the vulva, spending its life within the uterus [15,16]. Fig. 1. Schematic drawing showing extensive sexual dimorphism between C. elegans hermaphrodites and males. The rays in the male contain ciliated sensory neurons that respond to chemical and mechanical cues from the hermaphrodite. The male uses the fan to keep the male oriented with his ventral side facing the hermaphrodite. The spicules hold the vulva open for sperm transfer. Drawings based on www.wormatlas.org. About 12% of the C. elegans genes show differential transcriptional activation between males and hermaphrodites [17]. However, the differences are mostly restricted to the level of expression and tissue-specificity rather than gender-specific expression [18]. Only few genes are known to be specifically transcribed in only one sex (e.g., cwp genes [19]; lov-1 and pkd-2 [20]). It is therefore likely that the extensive differences between males and females do not arise by gender-specific genes. This conclusion is supported by genetic analyses in which gene mutations that cause defects in males also affect hermaphrodites [21–23]. Many of the mab genes (for male abnormal), for instance, which have been initially isolated because of their sexspecific defects, were found to have phenotypes in both sexes [24]. Central to sexual dimorphism is the activation of the gene transformer-1 (tra-1) that acts as a repressor of male genes in the hermaphrodite and as an activator in the male. This gene, which codes for a zinc-finger transcription factor, regulates about 364 genes in the soma of C. elegans males and hermaphrodites [18]. Most phenotypic sexual differentiation occurs postembryonically [12], and tra-1 seems to act no earlier than the first larval stage for most cell fate decisions [25,26]. To date, only two genes, egl-1 and mab-3, have been identified as direct target genes of TRA-1 in the soma (Fig. 2). egl-1 Fig. 2. Model for sex determination in C. elegans. Proteins in blue are the core components of the pathway that control sex determination in the soma and in germline, while the ones in red are involved only in the germline sex determination. Proteins upstream of HER-1 also control dosage compensation. The line connecting TRA-1 and TRA-2 represent a physical interaction. For more details, see text and reference [4]. 364 A. Pires-daSilva / Seminars in Cell & Developmental Biology 18 (2007) 362–370 and mab-3 control a subset of sex-specific events in C. elegans, such as the survival of hermaphroditic-specific neurons [27], yolk production in the intestine of the hermaphrodite [28], and expression of a male-specific cell lineage [29]. 3. Chromosomal and environmental sex determination Most nematodes have chromosomal sex determination, in which the female is XX and the male is heterogametic sex (XY) or XO (the O indicates the absence of Y). It is likely that the XX:XO sex determination system is ancestral because it is found in most of the nematode clades so far studied (Fig. 3). The few XY systems occur among parasitic nematodes of clades I, III and IV (following the nomenclature of Ref. [30]). It is thus possible that the XY system is derived from the fusion of an autosome to the old X-chromosome [31], although clear cytogenetic evidence is still lacking. Gonochoristic, i.e., male/female, nematode species with a chromosomal sex determination usually have a sex ratio of 1:1 (although exceptions are known, e.g., Ref. [32]). In hermaphroditic nematodes, however, males are rare. For example, when C. elegans is maintained in laboratory conditions, only 0.05–0.3% of the population is male [33,34]. In this nematode, the frequency of XX hermaphrodites and XO males is primarily driven by two factors: (1) the ability of male sperm to outcompete the sperm of the hermaphrodite [35]; (2) the rate of random sex chromosome non-disjunction during spermatogenesis in the hermaphrodite [36]. Normally, each hermaphrodite gamete contains one sex chromosome. However, rare chromosomal non-disjunction events lead to the production of gametes that lack an X chromosome, which then fertilize X-bearing gamete, resulting in the formation of male (XO) zygotes [37]. The molecular basis for the XX:XO sex determination system is best understood in the rhabditid C. elegans (for reviews, see Refs. [38,39]). Sex determination in C. elegans is based on a chromosome-counting mechanism that is achieved by a set of autosomal signal element genes (sea-1 and others) that oppose X signal element genes (sex-1, fox-1 and others) [40,41]. As a result, the sex-determining gene xol-1 is repressed in XX animals, thereby promoting the female fate [42–46]. xol1 repression is the first event in a cascade of genetic interactions that results in the activation of tra-1, the most downstream global regulator of sex determination (Fig. 2). It is well established that in other phyla the molecular and genetic mechanisms underlying sex determination can be completely distinct, even when comparisons of closely related species are undertaken [47,48]. The most striking differences are in the more upstream events of the sex determination pathway. Intriguingly, the ortholog of the C. elegans X signal element fox1 is also in chromosome X of the distantly related nematode P. Fig. 3. Diversity of somatic and germline sex determination in nematodes. Gonochorism and XX:XO are the most common mating types and sex determination strategies, respectively. In this phylogenetic tree, deviations from the common types of somatic and germline sex determination are highlighted; environmental sex determination and chromosomal sex determination type XX:XY have evolved independently in almost all clades. The evolution of species capable of producing oocytes and sperm by the same individual also evolved multiple times (asterisks). References: H. contortus [129]; C. elegans [97]; H. bacteriophora [130,131]; P. pacificus [132]; S. ratti [133]; R. buffonis [53]; Panagrolaimus sp. (PS1732) [134]; S. carpocapsae [135]; S. hermaphroditum [136]; A. avenae [137]; Acrobeloides sp (PS1146) [134]; S. celeris [118]; M. hapla [119]; T. alatus [138]; B. malayi [139]; H. spumosa [140]; A. lumbricoides [141]; C. incurvum [50]; P. allius [142]; T. spiralis [143]; T. muris [144]; X. attorodorum [145]; M. nigrescens [64]; C. papillatus [146]. A. Pires-daSilva / Seminars in Cell & Developmental Biology 18 (2007) 362–370 pacificus (Pires-daSilva, pers. obs.). Functional analysis of this gene and other signal element gene orthologs will be necessary to test if the counting mechanism is the same between these nematodes. Ascarids are especially rich in sex chromosome karyotype variation: spermatocytes of Toxocara canis and T. cati contain 2 X chromosomes, males of Baylisascaris transfuga have 3 X and 1 Y chromosome [49], and Contracaecum incurvum (Ascaris incurva) males have a single Y chromosome that pairs with 8 X chromosomes, while the females have 16 X chromosomes [50] (Fig. 3). The exact mechanism by which the multiple sex chromosome determination system evolved is not clear, but similar systems are also found in other groups of animals, such as insects, ostracods and the platypus [51,52]. Nothing is known about the molecular switch mechanism present in XX:XY species. One possibility is that it is based on a genic balance mechanism, which depends on a balance between female-determining genes in the X chromosome and male determining genes in autosomes, as with C. elegans. Another option is that the Y chromosome plays a dominant role in the determination of sex, similar to the situation in humans. Some nematodes alternate between different types of sex determination in their life cycle, having XX:XO sexes in one generation and XX-only animals in the other. How can XO males, which produces two kinds of sperm (half with the sex chromosome and the other half without it), generate only XX animals in the following generation? In the nematode Rhabdias bufonis (synonyms: Angiostomum nigrovenosum or Rhabdonema n.) it seems that the male X-bearing sperm have precedence for fertilization. Hence, the resulting progeny will be largely XX hermaphrodites rather than equal proportions of XX and XO [53–55]. Sperm precedence for X-bearing sperm cells has been also described for other nematode species [56]. Spermatogenesis in the ovotestis of the XX hermaphrodite follows a different course of that from males in order to generate XX females and XO males in the following generation: all the sperm contain an X at first but in half of them it is extruded and lost at the time when the developing sperm separates from its residual cytoplasm. Thus, the XX hermaphrodites produce one kind of oocyte but two kinds of sperm cells. When these sperm cells fertilize oocytes, XX and XO individuals of the sexual generation are produced once again. A more recent study reports the same kind of X-chromosome loss in a related species [57]. Oxyurids (clade III), also known as pinworms, are the only group of nematodes known to determine their sex with a haplodiploid system [58]. In this type of sex determination, males are haploid and develop from unfertilized eggs, whereas females are diploids and derive from fertilized eggs. One possible mechanism by which sex determination might be regulated is via chromosome dosage as occurs in C. elegans. Alternatively, a highly polymorphic X chromosome locus might control the sexual fate, in a similar way as occurs in honeybees [59]. In these insects the sex determination locus, when heterozygous, induces female development and when hemizygous the eggs develop into males. Additional mechanisms are possible (e.g., Ref. [60]), but testing of the different possibilities will be only possible when genetic analyses are applied. 365 Environmental sex determination evolved independently in various nematode clades [61,62] (Fig. 3). For the parasitic mermithid nematodes (e.g., Mermis nigrescens), the degree of infestation, the size of the host and the sex of the parasite which first penetrated the host seem to be important factors [63,64]. For the tylenchid Meloydogine incognita sex determination depends on conditions of feeding: when favorable the eggs develop into females and when not favorable they become males [65]. In Meloidodera floridensis, larvae subjected to starvation by keeping them in tap water for four months resulted in large proportion of males [66]. In Diplenteron colobocercus (syn. Mononchoides potohikus) the induction of males seems to be mediated by a pheromone-like substance [67]. Nematodes with environmental sex determination have a high rate of intersexes [65]. Intersexes are usually functional females (i.e., individuals with well developed reproductive system that produces oocytes) with some male somatic characters such as spicules [68]. In M. javanica and M. incognita, female larvae can undergo sex reversal relatively late in development, which causes the appearance of males with two gonadal arms (typical of females) instead of one [65]. Sex reversal and intersexes can be induced experimentally in crosses between two different species, such as in the rhabditids C. remanei and C. briggsae [69]. In this case, the effect has been attributed to dysgenic interactions among fast-evolving sex determination genes. The existence of all this diversity in sex determination systems raises many questions about what selective forces underlie their evolution and how one system evolves into another. Hodgkin [70] demonstrated that by using combinations of different C. elegans sex determination mutant strains it is possible to mimic many types of sex determination systems, including environmental sex determination and multiple X and Y chromosomes. These results suggest that a few mutational events can radically change the sex determination system. 4. Evolution of the sex determination pathway The sex determination pathway that leads to the regulation of TRA-1 has been very well characterized at the genetic and molecular level in C. elegans [38]. Chromosomal signals are translated into a set of genetic interactions that result in the sexspecific activation of a signal transducing pathway mediated by HER-1 (Fig. 2). HER-1, a male-specific protein [71], is a small secreted ligand that binds to the TRA-2 membrane receptor on the cell that secretes it, or on neighboring cells [72,73]. In a final step, TRA-2 regulates TRA-1 via three FEM proteins by unknown mechanisms. Some components and dynamics of the sex determination pathway show resemblance in at least three aspects when compared to hedgehog (hh) signalling, a pathway involved in various developmental processes in vertebrates and invertebrates [74]. First, the membrane receptor of Hh, Patched, is structurally similar to TRA-2 [75,76]. It is important to note, however, that Hh and HER-1 show no sequence or protein fold similarity and thus are not likely to be related by descent. Second, TRA-1 is a homolog of Drosophila cubitus interrruptus (Ci), the transcription factor of the hh signalling pathway [77]. Third, TRA-1 is 366 A. Pires-daSilva / Seminars in Cell & Developmental Biology 18 (2007) 362–370 post-trancriptionally regulated by proteolytic cleavage, like Ci [78,79]. These similarities suggest that the hh signalling pathway has been co-opted for sex determination in C. elegans [80]. As an alternative hypothesis, it has been suggested that the sex determination pathway arose in sequential steps, rather than being recruited from an already existent pathway. According to Wilkins [81], mutations in sex determination genes that cause a detrimental sex ratio are countered by the acquisition of modifiers into the pathway. These modifier genes would then reestablish the optimal sex ratio for a given population. Mutations that cause a production of excess of females in the population, for example, would be countered by the recruitment of malepromoting genes that can inhibit the female promoting factor TRA-1. The prediction for such a scenario is that the global sexual regulator TRA-1 is the most ancient part of the sex determination cascade, and the genes upstream of it are sequentially younger acquisitions of the pathway. These hypotheses can be tested by comparing the sex determination between different nematode species. So far, no additional components of the hh signalling pathway have been found in nematodes, such as Hh, Hedgehog-interacting protein (Hip) and Smoothened (Smo) [80,82]. It is therefore not clear whether the entire pathway was present in the lineage that gave rise to the nematodes or in which nematode clade this pathway might has been recruited for sex determination. With the various genome projects underway, it is possible that these homologs will still be identified in nematodes. Although the sex determination gene sequences are known to evolve rapidly [83–85], functional tests suggest that the pathway is essentially conserved within the Caenorhabditis genus [86,87,3]. In the diplogasterid Pristionchus pacificus, a more distantly related nematode, many of the C. elegans sex determination gene homologs have been found (A. Pires da Silva, unpublished data). Their role, however, has been uncovered for only one of these genes, tra-1. P. pacificus tra-1 (Ppa-tra-1) lossof-function mutants show the same sex reversal phenotype as Cel-tra-1, suggesting that the function of this gene is conserved between C. elegans and P. pacificus [88]. It is still not clear whether the direct regulation of C. elegans tra-1 targets, mab-3 and egl-1, is conserved in P. pacificus. mab-3 is member of a family of transcription factors that contain a DM domain [89]. This domain is also present in a male-promoting transcription factor, Doublesex (DSXM ), the most downstream regulator of sex determination in the fruitfly Drosophila melanogaster. The similarity in biological functions between DSXM and mab-3 suggests that a component of the sex-determination mechanism is shared between different phyla. Additional DM family members have been implicated in various roles in sexual development for C. elegans [90], mouse [91] and fish [92]. However, neither the regulation of these genes, nor the targets show significant conservation across phyla [93]. It is therefore possible that the recruitment of DM-domain factors for male-specific function in development occurred multiple times as result of convergent evolution. In summary, more comparative studies between nematodes may shed light on to the evolution of sex determination. With the recent development of sophisticated genetic and molecular tools for P. pacificus, traditional forward genetics approaches can be applied to this nematode [94,95,88]. Together with the availability of the genome sequence, forward genetic screens can be complemented with reverse genetic approaches to test whether C. elegans homologs have conserved functions in other species. 5. Evolution of self-fertilization Many nematodes are capable of autotokous reproduction, which is the production of progeny by a single parent. This mode of reproduction includes hermaphroditism and parthenogenesis, in which progeny develop from fertilized and unfertilized eggs, respectively. Hermaphroditism in nematodes is characterized by the production of sperm and oocytes by the same individual. Only rarely will the male – as characterized by the presence of spicules and absence of vulva – produce both types of germ cells [96]. Typically, the external morphology of hermaphrodites is morphologically identical to females of closely related gonochoristic species [97]. Hermaphroditic nematodes probably evolved from gonochoristic animals and therefore represent a derived condition [98]. In most hermaphroditic nematodes a single organ, the ovotestis, produces both oocytes and sperm. There are about 17/460 rhabditid species with an ovotestis, scattered in 11 out of a total of 44 genera [2,99,100]. The presence of an ovotestis has been also found in parthenogenetic species, where the sperm serve as an activator of embryogenesis but do not contribute genetic material to the next generation [101–104]. At least 5/17 species with ovotestis have been confirmed to self-fertilize and produce zygotes by pronuclei fusion, which meet the criteria for being hermaphroditic: C. elegans, C. briggsae, Oscheius tipulae CEW1, P. pacificus and H. bacteriophora. Phylogenetic analysis indicates that the most parsimonious interpretation for the evolution of hermaphroditism is that it evolved several times within the eurhabditids [99] and diplogasterids [100]. General features of germline development are conserved between different groups of nematodes. However, the role of the somatic gonad in the regulation of spermatogenesis and the timing of spermatogenesis seems to vary [35,105]. In C. elegans, spermatogenesis and oogenesis do not occur simultaneously: the gonad produces several hundred sperm during the last larval stage and then switches to oocyte production as adults. At the distal part of the ovotestis, the germ cells are mitotic and serve as stem cells. Although germline and somatic sex determination proceed by a similar genetic pathway [38], they differ in two aspects. First, the brief production of sperm in the last larval stage is independent of the X chromosome to autosome ratio. Second, there are additional genes in germline sex determination that have no role in somatic sex determination (i.e., the fog and mog genes). The genetic basis for germline sex determination has been well characterized in C. elegans [106]. fog-2, the most upstream germline sex-specific regulator, codes for a cytoplasmic protein of the F-box family [107], and is required for spermatogenesis only in XX hermaphrodites but not in XO males. Together with the RNA-binding protein GLD-1, FOG-2 down-regulates A. Pires-daSilva / Seminars in Cell & Developmental Biology 18 (2007) 362–370 tra-2 mRNA translation, resulting in the production of sperm [107]. Phylogenetic analyses suggest that hermaphroditism in C. elegans and C. briggsae has occurred due to an evolutionary convergence rather than being homologous. This conclusion is supported by genetic data, which indicate that there are fundamental differences in spermatogenesis between these two species. fog-2, the most upstream germline-specific regulator of spermatogenesis in C. elegans hermaphrodites [108], is not present in the C. briggsae genome [109]. Furthermore, other genes involved in C. elegans hermaphroditic spermatogenesis (e.g., gld-1, fem-2 and fem-3) do not have a function in sperm formation in C. briggase hermaphrodites [109,86,110]. Traditional forward genetic approaches will be necessary to unravel the genes involved in germline sex determination in C. briggsae [4,111]. Sperm size and spermiogenesis in hermaphrodites show at least two differences relative to males of the same species. First, male-derived sperm are substantially bigger than hermaphrodite-derived sperm [112,113]. Second, spermiogenesis seems to be sex-specific: mutant screens for C. elegans spermatogenesis defects have uncovered genes with hermaphroditespecific [114–116] or male-specific defects [117]. This raises the question of how the convergent evolution of spermiogenesis in hermaphrodites has been reflected at the molecular level. Hermaphroditism outside of clade V is based solely on morphological criteria (Fig. 3). Studies of the aphelenchids Seinura oxura and S. steineri showed that the gonads of these species can produce oocytes and sperm (syngonic hermaphroditism), and that egg production stops when the supply of sperm cells is exhausted [118]. This mode of reproduction suggests that it is similar to C. elegans, where the gonad produces first sperm and then switches to oogenesis. In other nematode species, however, spermatogenesis and oogenesis seem to occur simultaneously [57,119], or in alternation [53,120]. The production of sperm and oocytes in two different gonads of the same animal (digonic hermaphroditism) has been suggested for Clarkus (Mononchulus) ventralis and Helicothylenchus digonicus [121]. For C. ventralis, Cobb [122] describes females with one anterior gonad that produces oocytes and a small posterior testis that produces sperm. More detailed analysis of H. digonicus, however, failed to confirm hermaphroditism in this species: the large spherical structure that projects dorsally from the oviduct female gonad that has been regarded as sperm-producing organ does not contain sperm [123]. It is clear that more detailed cytological and genetic studies are necessary to evaluate to confirm that there are hermaphroditic species in many nematode clades. 367 other animals, nematodes that reproduce mainly asexually are one of the most ubiquitous plant parasites in the world [124]. To understand the factors that are driving the evolution of sex determination and asexual reproduction, integration of many diverse fields such as ecology, genetics and cell biology will be required. Furthermore, basic questions such as whether there is the occurrence of hermaphroditism in non-rhabditid nematodes are still unanswered. The evolution of self-fertile hermaphroditism appears to be associated with an adaptation to low effective population density, which can be caused either by low absolute numbers or low mobility [125,126]. This mode of reproduction is of advantage for organisms that pioneer new environments, because it assures the production of progeny in the absence of a mating partner. This advantage, however, comes at the cost of an increase in the level of inbreeding. The discussion of the interplay between factors that favor or hinder self-fertilization is beyond the scope of this review, but much research is needed in this area. No significant correlation has been found between the ecological niche of autotokous nematodes and their gonochoristic relatives [2]; sometimes sister species with different reproductive modes are found in the same habitat [127]. More details about life history characteristics are needed to better understand the variables that influence fitness, and therefore the selective factors acting on the reproductive mode. Sex determination is known to evolve fast in various phyla. However, it is not clear how exactly the sex determination pathway evolves: does it evolve from bottom up [81]? Was the C. elegans pathway recruited from hh signaling pathway? Were DM-domain containing genes recruited independently in various phyla for a role in sex determination? With the development of unbiased, forward genetic methods in various nematodes species, the comparison of the evolution of sex determination will be addressed at various phylogenetic scales [86,88]. Furthermore, with the various nematode EST and genome sequence projects (e.g., http://www.sanger.ac.uk/Projects/Helminths/, http://www.nematodes.org/nematodeESTs/nembase.html), candidate genes can be readily identified and investigated using reverse genetics methods, e.g., RNAi, in non-rhabditid species [128]. Acknowledgments I want to thank Ellen Pritham, Lenore Price and Ralf Sommer for critically reading the manuscript, and Zach Jacquays for drawing Fig. 1. Andre Pires da Silva is funded by NSF grant IOB#0615996. 6. Perspectives References Nematodes are very diverse in reproductive modes and in sex determination mechanisms [62]. It has been suggested that gonochoristic nematodes evolved into hermaphrodites, and that some of those became parthenogenetic [101]. Autotokous reproduction seems to have evolved relatively often in nematodes, which make an interesting case for studying which factors lead to the evolution of this reproductive mode. Although rare in [1] McClung CE. 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