1. Given that natural populations contain so many
genotypes, how can this organism be considered truly clonal?
Ans. The definition of clonality used is that of G. Bell: "Clones are assemblages of organisms that are genetically identical by descent." Genetic identity is the key concept, regardless of the level of variation of natural populations. Genetic identity is most rigorously established by demonstrating that parent and progeny are isogenic, and this, in turn, is most efficiently done by progeny testing field-caught individuals. This has now been accomplished for some 29 field-caught hermaphrodites collected from throughout the range of the species (except for Twin Cays, Belize), using DNA fingerprinting with a variety of multilocus microsatellite and minisatellite probes. These data do not rule out small amounts of outcrossing in these populations, but they are convincing evidence of clonality as a general rule.2. What is the evidence that these clones are, in fact, homozygous? Granted that clonal reproduction has been demonstrated, the data could also be compatible with reproduction by some apomictic mode rather than self-fertilization. This has actually happened in some invertebrates which were initially thought to be self-fertilizing but turned out to be parthenogenetic instead. How, for example, has gynogenesis (pseudogamy), a common apomictic pattern in fishes, been ruled out?
Ans. Syngamy and mendelian segregation have been demonstrated in R. marmoratus, and both are incompatible with gynogenesis. Harrington & Kallman (1968) were able to cross an aged, male-sterile hermaphrodite from one clonal lineage with a male from another clone. The F1 progeny accepted grafts from both parental strains, but progeny-to-parent grafts failed, i.e., the F1 were heterozygotes that could only have been generated by syngamy between the two strains. Progeny testing of (heterozygous) hermaphrodites from Twin Cays, Belize showed obvious segregation of components of parental DNA fingerprint patterns (Lubinski et al., 1995) .3. It would be quite interesting if clones could be routinely crossed in the laboratory, and fitness-related traits compared in heterozygous vs homozygous clonal progeny. Why has this not been done?
Ans. Outcrossing has thus far been achieved in the laboratory only under special circumstances (see above). Adult hermaphrodites are extremely aggressive, particularly when held in small containers (which facilitate egg collection). In our hands, they regularly and promptly kill males (or other hermaphrodites) placed with them., even if the male is considerably larger. Young hermphrodites, which have little testicular tissue, are less aggressive, but are frequently killed by males. Individuals get along better in larger containers, and when held in larger numbers, but then egg collection becomes problematic because of predation. Artificial techniques, including in vitro fertilization, hormone-induced gamete maturation , etc., seem more promising than further attempts at actual matings , but these apparently have not yet been tried.4. What is the evidence of inbreeding depression in this species?
Ans. There is thus far no definitive evidence of inbreeding depression in R. marmoratus, and this must be regarded as a serious challenge to the "overdominace" school of inbreeding depression/heterosis. The developmental anomalies induced by low temperature in some clones (Fisher, 1997) may be evidence of an "I.M. Lerner effect"---i.e., environmentally-induced failure of developmental canalization because of homozygosity--- but this is far from clear. Pilot scale studies of "fluctuating asymmetry" of paired body components, an approach frequently suggested in the literature to detect inbreeding depression, have thus far proven uniformly disappointing.5. Is there any evidence that heterozygotes have lower fitness than homozygotes, i.e., is there any obvious outbreeding depression?
Ans. At the present time there is no such evidence. Inability to make heterozygotes at will by laboratory crossing has hampered work in this area as well as the detection of inbreeding depression.6. Bifunctional gonads such as the ovotestis of R. marmoratus are extremely rare, and the processes of spermatogenesis and oogenesis (including vitellogenesis), which can occur in adjacent tubules in this species, are generally thought to be hormonally mutually incompatible. How does this species solve the potential problems that this poses?
Ans. There are apparently no data relevant to this question. This area is entirely open for investigation.
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