Laboratory Mating Experiments Research Articles

Paternity Analyses for the Planning of SIT Projects against the Red Palm Weevil

The red palm weevil Rhynchophorus ferrugineus is an invasive pest from southeastern Asia and Melanesia that has spread widely across the Middle East and the Mediterranean Basin over the last 30 years. Its endophagous larvae cause huge amounts of damage to several palm tree species from the Arecaceae family. Many of these palms are economically important for agricultural and ornamental purposes. Therefore, a lot of attention has recently been focused on studying this species with the aim of identifying sustainable and effective eradication strategies. Sterile insect techniques are biological control strategies that are currently being investigated for their potential to eradicate this pest in selected invasion areas. Mating system features (e.g., polyandry and related features) can affect the success and suitability of these approaches. The main goal of this research was to assess the performance of a previously developed microsatellite panel in terms of the paternity assignment of progeny from laboratory mating experiments. Using a simulation approach, we evaluated the reliability of the microsatellite markers in the paternity tests both in complex laboratory experiment scenarios and on the progeny of wild-caught gravid females to help future studies on the RPW mating system. As a case study of the simulation results, we performed two double-mating experiments, genotyped the progeny and estimated the P2 values to compare to the expected progeny genotypes according to the crossing scheme of each experiment. The results of our simulations on laboratory experiments showed that it was possible to carry out paternity assignments for all progeny with reliable statistical confidence using our 13 microsatellites set. On the contrary the low genetic variability measured in red palm weevil populations in invaded areas made the resolution power of our loci too low to carry out paternity analyses on natural populations. Results of laboratory crossing were completely congruent with the expectations from the Mendelian laws.

Resource partitioning is not coupled with assortative mating in sympatrically divergent ricefish in a Wallacean ancient lake.

Sympatric speciation is considered to be difficult without the coupling between ecological traits that allow resource partitioning and reproductive traits that allow assortative mating. Such "magic traits" are known to be involved in most of the compelling examples of sympatric speciation. In this study, we report a possible case of sympatric speciation without magic traits. Three species of ricefish (genus Oryzias) are suggested to have diverged sympatrically within Lake Poso, an ancient lake in Sulawesi. An analysis of genome-wide single-nucleotide polymorphisms showed that these three species are reproductively isolated from each other throughout the lake. Stable isotope analyses revealed that the three species use different food resources, which reflect differences in their feeding morphologies (gill rakers and digestive tracts) and feeding sites. Field and laboratory observations showed that O.nebulosus and O.orthognathus share a mating habitat of cobbles, where they scatter fertilized eggs, whereas this site is never used by O.nigrimas, indicating that assortative mating is partly achieved by spatial isolation. The small, less-adhesive eggs of O.nebulosus and O.orthognathus probably reflect their adaptation to spawning on cobble beaches. Laboratory mating experiments showed strong prezygotic isolation between O.nebulosus and O.orthognathus, which is achieved by strong species recognition presumably by both sexes based on species-specific mating dances and nuptial coloration. In summary, the assortative mating of O.nebulosus and O.orthognathus is probably not coupled to resource partitioning. We discussed how sympatric speciation among these species might have been achieved even without magic traits.

Too \u201csexy\u201d for the field? Paired measures of laboratory and semi-field performance highlight variability in the apparent mating fitness of Aedes aegypti transgenic strains

BackgroundEvaluating and improving mating success and competitive ability of laboratory-reared transgenic mosquito strains will enhance the effectiveness of proposed disease-control strategies that involve deployment of transgenic strains. Two components of the mosquito rearing process, larval diet quantity and aquatic environment - which are linked to physiological and behavioural differences in adults - are both relatively easy to manipulate. In mosquitoes, as for many other arthropod species, the quality of the juvenile habitat is strongly associated with adult fitness characteristics, such as longevity and fecundity. However, the influence of larval conditioning on mating performance is poorly understood. Here, we investigated the combined effects of larval diet amount and environmental water source on adult male mating success in a genetically modified strain of Aedes aegypti mosquitoes in competition with wild-type conspecifics. Importantly, this research was conducted in a field setting using low generation laboratory and wild-type lines.ResultsBy controlling larval diet (high and low) and rearing water source (field-collected and laboratory water), we generated four treatment lines of a genetically modified strain of Ae. aegypti tagged with fluorescent sperm. Laboratory reared mosquitoes were then competed against a low generation wild-type colony in a series of laboratory and semi-field mating experiments. While neither food quantity nor larval aquatic environment were found to affect male mating fitness, the transgenic lines consistently outperformed wild-types in laboratory competition assays, an advantage that was not conferred to semi-field tests.ConclusionsUsing a model transgenic system, our results indicate that differences in the experimental conditions of laboratory- and field-based measures of mating success can lead to variation in the perceived performance ability of modified strains if they are only tested in certain environments. While there are many potential sources of variation between laboratory and field lines, laboratory adaptation - which may occur over relatively few generations in this species - may directly impact mating ability depending on the context in which it is measured. We suggest that colony-hybridization with field material can potentially be used to mitigate these effects in a field setting. Release programs utilising mass-produced modified laboratory strains should incorporate comparative assessments of quality in candidate lines.

Quantification of Reproductive Isolating Barriers Between Two Naturally Hybridizing Killifish Species

Understanding the relative importance of various reproductive barriers to the early stages of speciation is an essential question in evolutionary biology. The closely related killifishes Fundulus heteroclitus and F. grandis occasionally hybridize in a small region in coastal Northeastern Florida showing that while barriers to reproduction exist, they are incomplete. The objective of this study was to elucidate barriers to reproduction between F. heteroclitus and F. grandis in the lab, as well as to quantify their strengths and relative contributions to reproductive isolation. Pre-zygotic (mating and fertilization) and post-zygotic (hatching) barriers were investigated by performing a variety of choice and no-choice laboratory mating experiments. Under no-choice conditions, barriers to mating had the greatest influence on hybrid production in F. grandis, whereas hatching barriers contributed to the majority of reproductive isolation in F. heteroclitus. Under choice conditions, however, pre-zygotic barriers had the greatest influence on hybrid production in both species. The total reproductive isolation that was observed in females of each species was stronger in F. heteroclitus than in F. grandis, and was nearly complete in F. heteroclitus females under choice conditions and was of moderate strength in F. grandis females. These results reveal an asymmetry in the potential gene flow between these two species, with F. grandis being more likely to hybridize than F. heteroclitus in the absence of environmental influences. No-choice backcrosses were also conducted and showed that at least some F1 hybrids are fertile. The observation that pre-zygotic barriers tend to be stronger than post-zygotic barriers in the early stages of speciation is consistent with similar studies in other organisms.

Last-male sperm precedence in Rhynchophorus ferrugineus (Olivier): observations in laboratory mating experiments with irradiated males.

The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier 1790) is an invasive pest from southeastern Asia and Melanesia that in the last 30 years has spread widely in the Middle East and Mediterranean Basin. Its stem-boring larvae cause great damage to several palm species of the Arecaceae family, many of which are economically important for agricultural and ornamental purposes. Therefore, great attention has recently been focused in studying this species to identify sustainable and effective eradication strategies, such as sterile insect technique (SIT). The rapid spread of RPW is associated with its high reproductive success. To evaluate the suitability of a SIT strategy, particular physiological and behavioral aspects of RPW reproduction, such as the presence of polyandry and post-copulatory sperm selection mechanisms, were investigated. To determine paternity of progeny from multiply mated females, double-crossing experiments were carried out confining individual females with either a wild-type male or a γ-irradiated male (Co-60). Fecundity and fertility of females were scored to evaluate post-copulatory sperm selection. Results showed that progeny were almost exclusively produced by the sperm of the second male, suggesting that a last-male sperm precedence is expressed at high levels in this species, and providing interesting insights for an area-wide RPW management strategy such as the SIT.

Dense, small and male-biased cages exacerbate male–male competition and reduce female choosiness in Bicyclus anynana

Sexual selection is increasingly recognized to depend upon, and to fluctuate with, major ecological factors in natural environments. The operational sex ratio (OSR) and population density can affect the opportunity for, and strength of, sexual selection but their effects are rarely taken into account in laboratory behavioural studies. In Bicyclus anynana, a model butterfly for mate choice studies, the experimental set-up widely used in laboratories often involves very high densities compared to the field, malebiased sex ratios and small cage volumes. We hypothesized that these conditions impede the proper expression of female mating preference by promoting male competition and sexual harassment of females. Using various cage volumes, we separately manipulated OSR and density to cover the range of values used in B. anynana laboratory mating experiments and to approach field values. Male competition, quantified by the number and duration of courtships aborted by males, became stronger with increasing densities, specifically under more male-biased sex ratios, and decreasing cage volumes. Thus, male eagerness to mate was essentially due to the use of small cage volumes and very high experimental densities. Concomitantly, female choosiness, quantified by the proportion of rejections of male mating attempts, decreased with increasing densities under a male-biased sex ratio. Females also accepted more matings with decreasing cage volume, and mated more rapidly with increasing density. We conclude that the laboratory social environments frequently used to test mate choice in B. anynana, with unnaturally high densities and male-biased sex ratios, exacerbate maleemale competition and strongly hinder female choice, biasing the estimates of the strength and direction of sexual selection that were shaped under very different natural environments in the wild. We propose a set-up for B. anynana mate choice studies that allows the assessment of female choosiness in statistically robust mating experiments. © 2015 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. Understanding the processes of pair formation is a necessary, yet neglected, step to fully grasp how secondary sexual traits and mating preferences evolve under sexual selection. It requires taking into account the fact that sexual selection, like natural selection, takes place in ecologically complex environments and examining the two major selective agents of sexual selection, mate choice and within-sex competition, in an integrative way (Miller & Svensson, 2014). It is increasingly acknowledged that mating patterns depend on ecological and social constraints, which determine the

Latitudinal Variation in Male Competitiveness and Female Choosiness in a Fish: Are Sexual Selection Pressures Stronger at Lower Latitudes?

Tropical animals are characterized by their showy ornaments and conspicuous colors, and numerous studies on sexual selection have been conducted using tropical species as a model system. In this study, hypothesizing that sexual selection pressures are stronger at lower latitudes, we examined the latitudinal variation in the degree of (1) male aggressiveness in male–male competitions, (2) male eagerness in courtship, and (3) female choosiness to mates in the medaka fish (Oryzias latipes complex). Laboratory mating experiments revealed that males from lower-latitude populations fight each other and court females more frequently than males from higher-latitude populations, supporting the hypothesis that both intra- and intersexual selection pressures are stronger at lower latitudes. In addition, females from lower-latitude populations, where males are more conspicuous, did not accept males easily, especially when mated with higher-latitude males, suggesting that female choosiness has also coevolved across latitudes through Fisherian runaway process. We suggest that latitudinal cline in reproductive seasonality, which is the case not only in the medaka but also among a variety of taxa, leads to latitudinal cline in operational sex ratios of local populations, which ultimately results in latitudinal cline in sexual selection pressures.

Impacts of an endoparasitic copepod, Ismaila belciki, on the reproduction, growth and survivorship of its nudibranch host, Janolus fuscus

Copepods from the genus Ismaila are large endoparasites that inhabit the main body cavity and/or cerata of opisthobranch molluscs. These parasites exhibit many life history characteristics typically found in parasitic castrators, yet the actual impact of infection on reproduction, growth or survivorship of the hosts are unknown. On the Oregon (USA) coast, Ismaila belciki can infect over 80% of their hermaphroditic hosts, Janolus fuscus. In laboratory mating experiments, we compared the reproductive output (egg mass weight, number of egg capsules, number of viable embryos) and the gonadal somatic index of infected versus uninfected J. fuscus. Infected J. fuscus could produce viable sperm and copulate. Mating with an infected individual did not limit a sea slug’s reproductive output. However, infected J. fuscus had significantly lower reproductive output (by 34–54%), producing smaller egg masses with fewer capsules and viable embryos. Infected hosts had significantly lower gonadal somatic index than their uninfected counterparts, although there was no significant difference in gonadal somatic index between hosts with single and double infections. By collecting the egg sacs produced by the copepod parasite during experiments, we estimated that 25–34% of the host’s reproductive output is usurped by the parasite and re-directed to the parasite’s own reproduction. In the laboratory, infection did not alter growth in J. fuscus. However, infection significantly decreased survivorship in mature (but not immature) nudibranch hosts. These results suggest that I. belciki is not a true castrator, but it does reduce the reproductive output of its host and may therefore limit the natural population size of J. fuscus.

Two new species of Chaco Tullgren from the Atlantic coast of Uruguay (Araneae, Mygalomorphae, Nemesiidae).

We describe two new species of the nemesiid spider genus Chaco from Rocha Province, Uruguay. These new species are diagnosed based on genital morphology, male tibial apophysis spination, and burrow entrance. We test cospecificity of one species, Chaco costai,via laboratory mating experiments. The new species are diagnosed and illustrated and habitat characteristics, and capture behavior are described. We conduct a cladistic analysis based on a previously published morphological character matrix that now includes the newly described species.

A comparative analysis of sperm storage and mating strategies in five species of brachyuran crabs

Variation in female sperm storage is explained, in part, by the amount of sperm transferred at mating. Laboratory mating experiments were conducted on Eurypanopeus depressus and Rhithropanopeus harrisii from the Chesapeake Bay and Pachygrapsus transversus from Florida, while mated pairs of Uca beebei and U. terpsichores were collected from mudflats in Panama. All experiments and collections were conducted during the summer of 2006 and 2007. More sperm was transferred to larger than smaller females, and by species with long copulation durations (R. harrisii and E. depressus). These two species live in cryptic habitats, have high sperm/egg ratios, and likely store sperm across multiple broods. In contrast, P. transversus and U. beebei mate conspicuously, have short copulations, transfer fewer sperm, and have low sperm/egg ratios. Comparisons of sperm transfer across different mating strategies and habitats provide a better understanding of female sperm storage in the Brachyura.

SPECIES HYBRIDS IN THE LABORATORY BUT NOT IN NATURE: A REANALYSIS OF PREMATING ISOLATION BETWEENDROSOPHILA ARIZONAEANDD. MOJAVENSIS

Understanding speciation relies critically on the identification of mechanisms responsible for maintaining species integrity (i.e., reproductive isolation) especially when closely related species are sympatric in nature. Studies of reproductive isolation in Drosophila often involve laboratory mating experiments that assume that patterns of mate choice in the laboratory are similar to those in the wild. Two sibling species, Drosophila arizonae and D. mojavensis, known to exhibit low levels of interspecific hybridization in the laboratory, but not in nature, were used in multiple-choice mating trials using various mating chamber designs as well as synthetic and natural media for developing larvae and courting adults. Sympatric populations were more sexually isolated than allopatric ones, consistent with past studies, and all experimental variables tested (chamber size, host plant presence and rearing substrates) had significant effects on levels of premating isolation between these species. Flies reared on cactus showed increased premating isolation versus those reared on synthetic laboratory food as did providing fermenting host plant tissue during mating trials. Also, surprisingly, smaller mating chambers led to an increase in premating isolation versus larger containers. The design of these types of mating trials is thus critical to understanding how mating behaviors in the laboratory are related to those in natural populations.

Generation-dependent female choice: behavioral polyphenism in a bivoltine butterfly

Climatic and biotic circumstances vary as seasons shift, and different cohorts of multivoltine species are likely subjected to different selection regimes. The bivoltine butterfly Leptidea reali (Réal's wood white; Lepidoptera: Pieridae) appears during May and June in central Sweden and has a partial second generation in late July. We manipulated both generations to appear simultaneously and performed laboratory mating experiments that showed the presence of a behavioral polyphenism in mating propensity, which is induced during the developmental stages. Females of the summer generation expressed higher mating propensities than spring generation females. Spring females showed an increase in mating propensity with increasing age, whereas summer females accepted most matings already when they were only 1 or 2 days old. It is likely that larval time constraints, a lower abundance of males and a lower risk of accepting a male of their univoltine sister species Leptidea sinapis (wood white), have relaxed selection on mate discrimination among summer generation females. A major challenge for future research is to further investigate the developmental pathways causing the polyphenism and the adaptive implications of cohort-dependent behaviors.

Reproductive interference, priority effects and the maintenance of parapatry inSkistodiaptomuscopepods

Reproductive interactions among species, such as attempts to mate with heterospecifics, can have profound impacts on patterns of geographic range and co‐occurrence. For example, several theoretical models demonstrate that reproductive interference‐the negative influence of heterospecifics on the abilities of conspecifics to successfully mate‐can lead to parapatry, even across homogeneous landscapes, when the parapatric species are initially allopatric. However, the potential consequences of reproductive interactions on patterns of range limits and co‐occurrence have been largely ignored by ecologists. Here, I use a combination of laboratory mating experiments and a genetic survey for two parapatric species ofSkistodiaptomuscopepods to evaluate the potential importance of interspecific reproductive interactions on the maintenance of parapatry. The genetic survey demonstrates phylogenetic exclusivity of these species, suggesting that gene flow has not occurred between them. Moreover, laboratory crosses between both species demonstrate that their parapatric boundary is not maintained as a hybrid zone because the two species cannot form viable hybrids. However,Skistodiaptomus oregonensisandS. pygmaeusmales mate indiscriminately and interfere with the ability of heterospecific females to successfully reproduce. I suggest that reproductive interference results in priority effects that strongly influence the maintenance of this parapatric boundary and stress that reproductive traits are important and often‐overlooked in ecological models for species range limits.

Estimates of natural allosperm storage capacity and self-fertilization rate in the hermaphroditic freshwater pulmonate snail, Physa acuta

Summary Previous estimates of allosperm storage capacities and self-fertilization rates in pulmonate snails have been derived almost exclusively from laboratory mating experiments. Here we report observations on a single adult albino Physa acuta collected from a natural pond in Charleston, South Carolina. Cultured in isolation from August 2002 until its death in February, this individual laid an average of 78.3 eggs per week (s.e.m. = 6.2), with a hatching success of 35.1% and an overall frequency of albino (putatively selfed) offspring of 6.4%. We found no evidence of reduced fecundity, reduced offspring viability, or increased self-fertilization over 20 weeks of observation, suggesting that stores of allosperm at no time approached exhaustion. We suggest that self-fertilization in primarily outcrossing populations of pulmonate snails may not be an adaptation, but rather may be a consequence of inefficiency in the mechanism sorting autosperm from allosperm.

Factors influencing paternity success in Antechinus agilis: last-male sperm precedence, timing of mating and genetic compatibility

Abstract We describe the patterns of paternity success from laboratory mating experiments conducted in Antechinus agilis, a small size dimorphic carnivorous marsupial (males are larger than females). A previous study found last-male sperm precedence in this species, but they were unable to sample complete litters, and did not take male size and relatedness into account. We tested whether last-male sperm precedence regardless of male size still holds for complete litters. We explored the relationship between male mating order, male size, timing of mating and relatedness on paternity success. Females were mated with two males of different size with either the large or the small male first, with 1 day rest between the matings. Matings continued for 6 h. In these controlled conditions male size did not have a strong effect on paternity success, but mating order did. Males mating second sired 69.5% of the offspring. Within first mated males, males that mated closer to ovulation sired more offspring. To a lesser degree, variation appeared also to be caused by differences in genetic compatibility of the female and the male, where high levels of allele-sharing resulted in lower paternity success.

Polyandry in field‐collected Spodoptera littoralis moths and laboratory assessment of the effects of male mating history

AbstractTo estimate multiple mating in females in the field, moths of S. littoralis were light‐trapped in June, July, August, and September in 1997 and 1998 in the edge of farming land in Assiut (Egypt). Females were dissected for the spermatophores in their bursae. Virgin females represented 8.04%, whereas once‐mated and multiply‐mated females were 37.2% and 54.76% of the trapped females, respectively. The average number of matings per trapped female (1.68±0.05) was significantly lower than that of laboratory‐reared females (2.48±0.11). As the number of captured males declined from June to September, there was a significant shift towards remating in the captured females. Laboratory mating experiments were performed with reared females that were daily supplied with males of different mating histories, including virgin males (V) or males that had previously mated once (PM1), twice (PM2), or three times (PM3). Females supplied with virgin males exhibited significantly lower numbers of matings acquired during a 5‐day experiment than females supplied with previously mated males. There was also a highly significant association between the frequency of mating among females and the mating history of males involved. Females supplied with previously mated males showed a higher propensity to remate than those supplied with virgin males. Significant increase of the reproductive capacity of females was associated with virginity (versus non‐virginity) of males or multiple mating (versus single mating) by females. Females paired with V or PM1 males exhibited shorter longevity and higher rates of egg laying than those paired with PM2 and PM3 males. The results suggest that females are able to assess the male mating status and, accordingly, alter their mating behavior to achieve higher reproductive capacity.

Size-assortative mating, male choice and female choice in the curculionid beetle Diaprepes abbreviatus

In the beetle Diaprepes abbreviatus (L.) females are larger on average than males, as indicated by elytra length. Size-assortative matings were observed in wild populations in Florida and in laboratory mating experiments. We tested three mechanisms for this size-assortative mating: (1) mate availability; (2) mating constraints; and (3) mate choice. We found that mate choice influenced size-assortative mating by: (1) large and small males preferring to mate with large females; (2) large males successfully competing for large females, leaving small males to mate with small females; and (3) females accepting large males as mates more readily than small males. Males increased their reproductive success by mating with larger, more fecund females. They transferred protein to females during mating.

The effect of male size, age, and mating behavior on sexual selection in the seed beetle Callosobruchus maculatus

We use laboratory mating experiments to examine the effect of male size, age, and mating behavior on fecundity selection and sexual selection in the seed beetle, Callosobruchus maculatus (Coleoptera Bruchidae), a species in which females are larger than males. Female C. maculatus gain a fitness advantage, in the form of increased lifetime fecundity, from mating with large males (which contribute larger ejaculates), but the partial correlation between male size and fecundity is weaker than the partial correlation between female size and her fecundity. Large males had a mating advantage relative to small males, both when a single male was presented to a female and when two males were present. However, this did not appear to be due to females rejecting male courtship attempts, but instead may be due to male-male competition. When females were mated to two males sequentially, neither the size of the first male nor the size of the second male influenced whether or how quickly a female remated. None of the other potential bases for sexual selection — male age, male mating experience, and male courtship persistence — appeared to influence male mating success. We discuss how patterns of sexual selection on body size and sexual size dimorphism in C. maculatus differ from patterns of sexual selection and dimorphism in another seed beetle, S. limbatus.

New Potential Inhibitors of Pheromonal Attractionin the Oriental Fruit Moth, Cydia molesta

New analogues of (Z)-dodec-8-en-1-yl acetate (Z8-12:OAc, 1), the main sex pheromone component of the Oriental fruit moth, Cydia molesta, were designed by formally transferring the terminal propyl group from the C-9 to the C-7 position to form vinyl-branched (2, 3) or, after isomerization, ethylidene-branched (4) structures and by replacing the -CH=CH- grouping by the -S-CH2- moiety (5, 6). Their biological activities were studied both electrophysiologically and behaviourally (laboratory mating and wind tunnel experiments). All the structural modifications resulted in analogues whose electroantennographic activities were lower than that of 1 following the order 1 >> 6 ≈ 5 >> 2 ≈ 4 ≈ 3. The single sensillum recording activities indicated that all the analogues stimulate the same Z8-12:OAc receptor neurone. In behavioural experiments, the analogues were generally found to reduce the ability of males to find a pheromone source, however, to different degrees. The highest inhibitory effect (90%) was observed for the thia analogues 5 and 6. The results support the view that the inhibitory properties of the analogues should not be entirely associated with their pheromone-mimicking capabilities.

HYBRIDIZATION FREQUENCY IS NEGATIVELY CORRELATED WITH DIVERGENCE TIME OF MITOCHONDRIAL DNA HAPLOTYPES IN A SEA STAR (LEPTASTERIAS SPP.) SPECIES COMPLEX.

If interspecific reproductive isolation is caused by cumulative genetic change within species, then the degree of isolation of recently diverged species should increase with the amount of time since divergence from the common ancestral population (Coyne and Orr 1989). This prediction is fairly robust, being independent of whether or not control of reproductive isolation is polygenic, and excluding obvious exceptions like polyploidization. However, tests of this prediction have so far been restricted to laboratory mating experiments in Drosophila (references in Coyne and Orr 1989). In some cases, laboratory experiments may not directly reflect the extent of natural hybridization between sympatric species, particularly for those that are externally fertilizing (see discussion and references in McClary 1992). In such situations, reproductive isolation between sympatric species may be more appropriately assessed by natural hybridization frequency. However, analysis of natural hybrids is obviously limited to sympatric species, so some important evolutionary issues relating to reproductive isolation and hybridization (such as the existence of reinforcement) cannot be addressed. Recent genetic analyses of a sea star cryptic species complex (Leptasterias spp.) offer the possibility of testing the correlation between estimated degree of reproductive isolation in nature and divergence time for various pairs of sympatric mtDNA PCR-RFLP haplotypes. Occasional hybridization among members of this species complex had long been suspected (Fisher 1930; Kwast et al. 1990), but detailed analysis had to await the use of molecular polymorphisms as phylogenetic characters. Recent studies of this complex have uncovered at least 11 mtDNA PCR-RFLP haplotypes (denoted A-K in order of discovery), with many pairs that are broadly sympatric in Alaska, British Columbia, and the northwestern Pacific coast of the continental United States. Joint analysis of mtDNA and allozyme data suggests that the haplotypes constitute a species complex, similar to the sibling species complexes previously described in many other marine invertebrate taxa (Knowlton 1993). Genetic cohesiveness of each of the five most common and broadly distributed haplotypes (A, B, C, F, and G) was demonstrated in three ways: (1) principal components analysis (PCA) of multilocus allozyme genotypes within each haplotype revealed few outliers and no evidence for multimodal distributions of PCA scores (Foltz et al. 1996a); (2) Nei's (1978) unbiased genetic distances based on 14 allozyme loci were consistently smaller among population samples of haplotypes (range 0.001-0.045) than between haplotypes (range 0.086-0.198), leading to discrete clusters (Foltz et al. 1996a); and (3) direct sequencing of PCR products revealed negligible amounts of intrahaplotypic sequence polymorphism, compared to the level of interhaplotypic divergence (Hrincevich and Foltz 1996). Morphological differences between many pairs of mtDNA haplotypes were limited (especially for sympatric pairs), with statistically significant differences in canonical discriminant scores based on 55 characters but few diagnostic morphological characters (Foltz et al. 1996b), a pattern similar to those described for other marine invertebrate sibling species complexes (Knowlton 1993). The existence of numerous sympatric and closely related haplotypes within the Leptasterias cryptic species complex offered the possibility of studying the occurrence of hybridization between haplotypes in more detail. To test whether the extent of reproductive isolation increases with increased divergence time in this species complex, the frequency of putative F1 hybrids (as judged from the relative frequency of two-locus heterozygotes for informative allozyme alleles distinguishing the haplotypes) for sympatric pairs of haplotypes was compared to the amount of divergence estimated from mtDNA sequence data.