Sexual reproduction (i.e.
meiosis and syngamy) is the predominant reproductive mode in eukaryotes, yet parthenogenesis (i.e.
asexual reproduction) is present in all major lineages. Among animals, cyclical parthenogenesis, which alternates bouts of clonal and sexual reproduction, is restricted to monogonont rotifers, digenean trematodes, and several arthropod lineages [1
]. Obligate parthenogenesis is much more common but is predicted ultimately to drive lineages to extinction due to the accumulation of deleterious mutations or inability to adapt to environmental changes [2
]. The origins of obligate parthenogens are often attributed to the loss of meiosis via
interspecific hybridization [3
] or irreversible changes in ploidy [5
], yet other mechanisms must also exist. Among animals, thousands of parthenogenetic species have been described, and volumes have been written describing the cytogenetic manifestations of many different types of parthenogenesis (e.g.
]), but little is understood about molecular determinants of these processes.
The microcrustacean Daphnia pulex
reproduces by cyclical parthenogenesis. Direct-developing eggs (also called subitaneous or summer eggs) are produced parthenogenetically and immediately develop in the female's brood chamber prior to hatching. During the sexual cycle, haploid resting eggs (also called ephippial, diapause, or winter eggs) are produced by meiosis and require fertilization and a period of extended dormancy for development to complete. Because sex determination in Daphnia
is environmentally induced [10
], males are genetic clones of their mothers. In addition, genetic and phenotypic evidence has revealed D. pulex
lineages that reproduce by obligate parthenogenesis. These obligate parthenogenetic lineages produce direct-developing eggs by parthenogenesis, which is indistinguishable from parthenogenesis in cyclical lineages. However, obligate parthenogens have lost the requirement for meiosis and fertilization to produce viable resting eggs [12
]; unfertilized resting eggs undergo a period of diapause and develop parthenogenetically to eventually hatch and produce a juvenile. Importantly, the resting egg parthenogenesis exhibited by these obligate asexual lineages is cytologically distinct from direct-developing egg parthenogenesis in both obligate and cyclical parthenogenetic lineages and from meiosis. Hence, although the terms "cyclical parthenogenesis" and "obligate parthenogenesis" may also refer to breeding systems, herein we use these terms to distinguish parthenogenetic oogenesis that takes place during direct-developing (in cyclical and obligate asexuals) and resting egg development (in obligate asexuals only), respectively. Therefore, the D. pulex
genome must contain the molecular machinery to accommodate various types of reproductive modes: meiosis (male and female) and parthenogenetic oogenesis in both cyclical and obligate parthenogenetic lineages. This feature makes D. pulex
an ideal model to investigate the genetic basis of parthenogenesis, and its consequences for gene and genome evolution.
Resting egg parthenogenesis in Daphnia
is cytologically distinct compared to direct-developing egg parthenogenesis (e.g.
with respect to chromosome morphology and egg size [13
]). However, while obligate parthenogenesis apparently involves initial meiotic pairing (but without homologous recombination) followed by a mitotic or mitotic-like division ([13
]; Tsuchiya and Zolan, pers. comm), neither obligate nor cyclical parthenogenesis seems to be strictly mitotic since a polar body is extruded during cell division, indicative of meiosis [15
]. In both cases, heterozygosity is maintained, except in rare instances of loss of heterozygosity presumably caused by mitotic crossing over [16
]. Obligate parthenogenesis in Daphnia
is limited to the D. pulex
complex (D. pulex
, D. pulicaria
and D. middendorffiana
, D. tenebrosa
] and to the D. carinata
complex (D. thomsoni, D. cephalata
], and at least in some cases, the trait is passed by male offspring of obligate asexuals into sexual backgrounds, implying a sex-limited meiosis suppressor [12
]. In D. pulex
, obligate asexuality has been migrating from northeastern to central North America, and most clonal lineages are estimated to be no more than 12,000 – 120,000 yr [18
]. Recent association mapping of obligate asexuality in Daphnia
has found markers on four different chromosomes exhibiting significant association with parthenogenetic production of resting eggs in obligate asexuals [19
]. This suggests that obligate asexuality and (by implication) the mechanistic transition from meiosis to parthenogenesis could be influenced by at least four epistatically interacting loci.
Specifically, we are interested in genes that encode components essential for meiosis in D. pulex. A cyclically parthenogenetic D. pulex lineage possesses genes required for both meiosis and parthenogenesis. To ultimately establish whether modifications to the meiotic machinery are associated with parthenogenesis, we must first determine which meiotic genes are present and expressed in cyclically parthenogenetic lineages. Then, we can compare the inventory and expression patterns of these same genes in obligate parthenogens. If obligate parthenogens have truly abandoned canonical meiosis altogether, genes required specifically for meiosis should be under reduced selective constraint and become non-functional over time. However, certain meiotic processes, perhaps in a modified form, may still be required for parthenogenesis and, thus, genes required for such processes may still be intact and expressed. Differences in the inventory, evolutionary rates and expression of meiotic genes in cyclical and obligate parthenogens may provide insight into the importance of meiotic genes for the evolution of parthenogenesis.
During a typical animal meiosis (Fig. ), a germline stem-cell (GSC) divides asymmetrically producing a daughter GSC and either a cystoblast (females) or gonialblast (males) [9
]. During both meiosis and parthenogenesis in Daphnia
females, incomplete mitoses create a 4-cell cystoblast which matures into an oocyte cluster of three nurse cells and the presumptive oocyte [15
]. Only later in vitellogenesis can parthenogenetically-produced oocytes be distinguished visually from meiotically-produced oocytes [15
]. As the oocyte cluster matures, pre-meiotic S-phase DNA replication occurs in the oocyte, followed by heterochromatin and centromere specification and, in most animals, appearance of the synaptonemal complex (SC) [20
]. In most organisms studied, cohesin complexes are recruited during S-phase to promote cohesion between sister chromatids [21
]. Several mechanisms have been reported to initiate chiasmata formation and recombination between homologous chromosomes, including double-strand break (DSB) formation and DSB-independent pathways [22
]. As recombination progresses, syntelic attachment of sister kinetochores (i.e.
both attached to the same spindle pole) generates monopolar tension towards the spindle poles, leading to segregation of homologous chromosome pairs at anaphase and cytokinesis resulting in two diploid cells [23
]. In the second meiotic division, amphitelic attachment of kinetochores (i.e
. associated with microtubules from opposite spindle poles) and the complete removal of cohesin allow sister chromatids to segregate to opposite poles [23
]. As a result, one haploid cell is formed; it becomes the ovum while two polar bodies are produced and eventually degenerate.
Figure 1 Meiotic genes annotated in the D. pulex genome (shown in boxes) and a schematic of a possible model for parthenogenesis. Arrows indicate their roles in meiosis, and potentially in parthenogenesis. Proteins in bold are encoded by multiple gene copies in (more ...)
While parthenogenesis in Daphnia
shares some features with meiosis (e.g.
oocyte cluster formation, extrusion of polar bodies), there are important differences. First, during parthenogenesis sister chromatids segregate in a mitosis-like fashion, suggesting that sister chromatid cohesion must be different. This could be a result of parthenogenesis-specific cohesin complexes or altered timing of cohesin removal. Second, parthenogenetic kinetochore orientation should be amphitelic (bi-oriented, as in mitosis and meiosis II), again to allow pairs of sister chromatids to segregate towards opposite poles. Lastly, recombination likely differs compared to meiosis because heterozygosity is maintained during parthenogenetic reproduction and chiasmata are not observed [15
]. These changes likely involve a modification of recombination bias away from reciprocal and homologous exchange to between sisters or to no recombination at all [16
The major stages of meiosis and the genes which are the targets of our inventory in D. pulex are indicated in Fig. . The genes were chosen with a focus on female meiosis and their potential role(s) in parthenogenesis. In this study, we report an inventory of genes in the genome of a cyclically parthenogenetic strain of D. pulex (strain TCO) that encode proteins with roles throughout meiosis. This represents an initial step in identifying and characterizing the genes that are central to reproduction in D. pulex. We have divided these meiotic genes intro two broad categories. First, we investigate "meiosis-related genes": these are genes that encode proteins involved in meiosis but whose functions and expression are not specific to meiosis. These include genes encoding Argonaute proteins (PIWI and AGO subfamilies), cell cycle regulation proteins (cyclins, cyclin dependent kinases (CDKs) and polo kinases) and several proteins involved in DNA replication, cohesion and meiotic recombination (minichromosome maintenance (MCM), TIMELESS (TIM) and RecQ proteins). Second, we investigate several meiosis-specific genes in our inventory: these are genes for which homologs in most model organisms function are expressed only during meiosis and mutants containing null alleles are defective only in meiosis. These genes include SPO11, MND1, HOP2, DMC1, REC8, MSH4, and MSH5, which encode proteins that together generally affect the initiation and progression of meiotic recombination and sister chromatid cohesion. We also examine gene families that are closely involved in the above processes: these include structural maintenance of chromosome (SMC) and stromal antigen (SA) gene families, RAD54 and RAD54B paralogs, and eukaryotic homologs of bacterial mutL and mutS genes. Database homology searches and rigorous phylogenetic analyses are employed to identify orthologs and distinguish paralogs. For 42 gene copies, we use RT-PCR to compare expression levels in ovaries of females undergoing meiosis or obligate (resting egg) parthenogenesis, in males (i.e. undergoing meiosis) and in female somatic tissue. We interpret our results from these experiments in light of a model of the genetic underpinnings of parthenogenesis we have developed for D. pulex.
The gene inventory and the expression patterns of these genes during meiosis and parthenogenesis will help us address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved. While thelytokous parthenogenesis may occur via various cytological mechanisms [9
], parthenogenesis in Daphnia
appears to be apomictic and does not involve gametic fusion as would be observed with automictic reproduction [15
]. The transition from meiosis to parthenogenesis in Daphnia
requires at least three modifications: altered spindle attachment of the kinetochore, modified sister chromatid cohesion and abrogation of homologous recombination (cf. [24
]). It is not clear whether any one of these changes is necessary or sufficient for the origin of thelytokous parthenogenesis in D. pulex
, or whether they are pertinent for other types of parthenogenesis such as arrhenotoky. However, these modifications must involve characterized pathways in mitosis and meiosis, for which mutant phenotypes closely resemble the cytogenetic manifestations characteristic of parthenogenotes [20
]. Therefore, our inventory includes genes required for these and other meiotic processes.