Population Variation and Ecological Correlates of Tychoparthenogenesis in the Mayﬂy, Stenonema Femoratum

Biological Journal of the Linnean Society, 2002, 75, 101–123. With 10 ﬁgures

Population variation and ecological correlates of tychoparthenogenesis in the mayﬂy, Stenonema femoratum

SHELLEY L. BALL*

Division of Biological Sciences, University of Missouri, Columbia, MO, 65211–7400, USA

Received 3 January 2001; accepted for publication 28 October 2001

Species in which both sex and parthenogenesis co-occur are extremely valuable for investigating ecological conditions favouring sex. Tychoparthenogenesis is a breeding system characterized by hatching of a small proportion of unfertilized eggs (typically < 10%) from females of sexually reproducing species. With tychoparthenogenesis, both sexual and parthenogenetic reproduction co-occur within the same population. To identify ecological conditions that may favour this breeding system, I quantified population variation in females’ capacity for tychoparthenogenesis and investigated biotic and abiotic correlates of tychoparthenogenesis. I estimated tychoparthenognetic capacity (proportion of unfertilized eggs hatching) for females from 12 Missouri populations of the mayfly, Stenonema femoratum (Ephemeroptera: Heptageniidae), across three different habitat types – temporary streams, permanent streams and lakes. Tychoparthenogenetic capacity, measured as the population mean hatch success of unfertilized eggs, ranged from 3.8 to 10.7%. Tychoparthenogenetic capacity varied among habitats in 1996, but not in 1997. In 1996, temporary streams showed hatch success of unfertilized eggs twice that of permanent streams and lakes. Tychoparthenogenetic capacity also varied among sampling dates within years. Temporary streams also showed extremely low nymph densities compared to the other two habitats. However, habitats did not differ in adult density. Furthermore, in all populations nymphs showed significantly female-biased sex ratios. In contrast, adult sex ratios were equal or slightly male biased. Tychoparthenogenetic capacity was negatively correlated with nymph density in 1996, but not in 1997, suggesting possible reproductive assurance in some years. Adult densities also suggested that there may be certain times of year when tychoparthenogenesis may provide benefits of reproductive assurance. Although habitats differed significantly in their abiotic characteristics, tychoparthenogenetic capacity was correlated significantly with water temperature only. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123.

ADDITIONAL KEYWORDS: abiotic factors – breeding system – dispersal – evolution of sex – parthenogenesis – population density – sex ratio.

INTRODUCTION a more efﬁcient way of reproducing. Despite the predicted twofold advantage of asexual reproduction Since the discovery of parthenogenesis nearly due to the production of all-female offspring, paradoxi- 300 years ago, biologists have sought to explain why cally relatively few organisms reproduce asexually sexual reproduction is the predominant reproductive and asexual species appear to be evolutionarily short- mode in eukaryotes when asexual reproduction offers lived (Ghiselin 1974; Williams 1975; Maynard Smith 1978; Bell 1982; Michod & Levin 1988; Judson & Normark 1996). All else being equal, asexuals have twice the reproductive rate as sexuals, producing twice as many daughters as sexual females. Sexual *Current address: Department of Zoology, University of Guelph, Ontario, Canada N1G 2W1 females are said to pay a ‘twofold cost of sex’ due to E-mail: [email protected] production of sons. Despite this cost, the predomi-

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 101 102 S. L. BALL nance of sex indicates that it must confer large advan- ducing via meiotic parthenogenesis (Bell 1982; Suo- tages over the long-term. The primary advantage of malainen et al. 1987). Different automictic mecha- sex lies in its ability to produce genetically variable nisms can result in different genotypic consequences offspring. However, the exact way in which this advan- (White 1973). Therefore, automixis provides an oppor- tage is realized is unknown and has been the subject tunity to examine the consequences of parthenogene- of numerous hypotheses to explain the advantages of sis in different genetic backgrounds. sex (Kondrashov 1993). To understand how these Despite these advantages, tychoparthenogenesis advantages of sex are manifested, evolutionary biolo- has been largely ignored by evolutionary biologists. gists have focused their attention on the ecological Due to the low hatching success of unfertilized conditions favouring asexual reproduction. Asexual eggs, tychoparthenogenesis has often been referred reproduction is generally found in early successional to as ‘accidental’ or ‘occasional’ parthenogenesis or marginal habitats that are biotically ‘simple’, con- (White 1973) and has been dismissed as a type of taining few predators, parasites, or competitors (Bell artificial parthenogenesis that is unimportant to 1982; Bierzychudek 1985). evolution in natural populations (Maynard Smith Species in which both sex and parthenogenesis 1978; Mingo 1978; Brittain 1982). There have been co-occur are extremely valuable for investigating eco- no detailed ecological studies of tychoparthenogens logical conditions favouring sex. Tychoparthenogen- while detailed genetic studies exist for a few esis is a little studied, yet relatively common type of tychoparthenogenetic insects (Drosophila, Stalker parthenogenesis in invertebrates in which females 1951, 1952, 1954; Carson 1961, 1967, 1973; Templeton from sexually reproducing species exhibit a small et al. 1976; Templeton 1982; Bacillus stick insects, capacity for parthenogenesis (Bell 1982; Templeton Bullini 1994; Lonchoptera flies, Ochman et al. 1980; 1982; Suomalainen et al. 1987). The proportion of Locusta migratoria, Pardo et al. 1995). However, these unfertilized eggs hatching is typically less than 10% studies have been confined primarily to laboratory although enormous variation in hatch success among investigations. females has been shown (McCafferty & Huff 1974; The purpose of this study was to quantify popu- Templeton 1982; Ball 2000). With tychopartheno- lation variation in tychoparthenogenetic capacity genesis, both sex and parthenogenesis occur within of natural populations of the mayfly, Stenonema the same population. Population variation in femoratum (Ephemeroptera: Heptageniidae) and tychoparthenogenetic capacity (i.e. population mean to investigate ecological conditions correlated with proportion of unfertilized eggs hatching) offers an this breeding system. Specifically, I examined ideal opportunity to investigate ecological factors asso- whether natural populations showed evidence of ciated with different amounts of sexual reproduction. tychoparthenogenetic reproduction and the extent to Tychoparthenogenesis offers additional advantages which tychoparthenogenetic capacity varied spatially for investigating short-term ecological advantages of (among habitats and populations) and temporally sex. Because the hatching success of unfertilized eggs (between and within years). I also examined biotic and is low, relative to that of fertilized eggs, the predicted abiotic correlates of tychoparthenogenesis to elucidate twofold advantage of parthenogenesis does not exist the biological and physical conditions under which with tychoparthenogenesis. This suggests that some tychoparthenogenesis occurs and therefore, to identify other advantage must be responsible for its mainte- potential advantages of tychoparthenogenesis. nance. Tychoparthenogenesis also offers several other advantages for testing hypotheses for the maintenance of sex. Both sex and parthenogenesis occur SPECIES AND STUDY SITES not only within the same species, but also within the Parthenogenesis has been documented in 24 North same population. Thus, sexuals and parthenogens American and 35 European mayfly species (DeGrange can be compared without confounding factors related 1960; Huff & McCafferty 1974; Friesen & Flannagan to differences in habitat or phenology. Direct com- 1976; Gibbs 1977; Bergman & Hilsenhoff 1978; Mingo parisons of sexuals and parthenogens in the same 1978; Humpesch 1980; Sweeney & Vannote 1987; habitat are rare (Joekela et al. 1997). Moreover, Gillies & Knowles 1990; Harker 1997). In North tychoparthenogenesis is not confounded by hy- America, six species are known from obligately bridity. While all known unisexual vertebrates are parthenogenetic populations, while remaining species of hybrid origin (Cuellar 1974; Vrijenhoek 1989) have been classified as facultative parthenogens or tychoparthenogens are not (Templeton 1982). tychoparthenogens. Stenonema femoratum is broadly Tychoparthenogenesis also offers advantages as a distributed in North America, ranging from the north- system for studying the evolution and maintenance of eastern United States and Canada to the foothills sex because of the diversity of genotypic consequences. of the Rocky Mountains and south to Texas (Bednarik All known tychoparthenogens are automictic, repro- & McCafferty 1979). It occupies a wide range of habi-

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 POPULATION VARIATION IN TYCHOPARTHENOGENESIS 103 tats from rocky shoreline of lakes to temporary and which dry in late summer forming small, isolated permanent streams. In Missouri, S. femoratum has at pools. Streams were classified as temporary if the least two generations per year (S. L. Ball, personal entire width of the stream bed was completely dry by observation). Overwintering adult nymphs emerge as late August, at a minimum of two locations along the early as March. In central Missouri, adults can be streambed, separated by at least 50m. A total of three found throughout the summer and have been observed temporary streams, four permanent streams, and five as late as November (S. L. Ball, pers. observ.). Unlike lakes were sampled. Each lake or stream was divided most insects, mayflies have retained a subadult into a series of transect points 50m apart, with up (subimago) stage. Subimagoes emerge from their to ten transect points per lake or stream. The number nymphal exuvia and spend up to 24h resting on of transect points depended on the amount of rocky vegetation near water before they molt into the final shoreline or stream reach that could be waded safely, adult (imago) stage. Imago lifespan is brief, lasting with three to eight transect points sampled in lakes ~24–48h although occasionally female imagoes have and 7–10 transect points in streams. The same tran- been kept alive in the laboratory for up to 7 days sect points were sampled each season and year. (S. L. Ball, pers. observ.). Subimagoes carry mature However, for three sites fewer transect points were gametes but, in most species, are unable to mate sampled on one occasion. because the external genitalia are not fully developed (Edmunds & McCafferty 1988). Subimagoes are easily distinguished from imagoes by their dull, grey appear- METHODS ance and translucent wings. Unlike many mayfly species, S. femoratum has asynchronous emergence EVIDENCE OF TYCHOPARTHENOGENETIC which results in relatively small mating swarms of REPRODUCTION ~5–100 males. Male imagoes form mating swarms in To determine whether natural populations showed late afternoon or early evening, flying 10–50cm above evidence of tychoparthenogenetic reproduction, I the water surface. Female imagoes join these mating measured sex ratios of nymphs, imagoes (adults) and swarms 5–15min later and copulation, which lasts subimagoes (subadults). With tychoparthenogenetic ~30s, takes place in flight. After copulating, females reproduction, populations are expected to exhibit immediately lay eggs by flying a few cm above the female biased sex ratios. To estimate nymph sex water surface and dipping the tip of their abdomen ratios, I collected nymphs from each of the 12 popu- into the water repeatedly, each time releasing a small lations in 1997 and 1998. Collections were made numbers of eggs. twice per year, in late May to July and again in late This study was conducted from 1996 to 1999 at 12 August to early November to assess seasonal varia- sites across central and southern Missouri (Table 1). tion. For each population, I collected nymphs from two Sites (hereafter referred to as populations) were clas- randomly chosen locations at each transect point. I sified into one of three habitat types: lakes, permanent used Schrader’s ‘10-rock’ method (cited in Hynes 1972) streams which flow all year, and temporary streams to estimate nymph density. At two randomly chosen

Table 1. Locations of S. femoratum populations sampled in 1996 and 1997

Population Abbreviation Habitat* County Latitude Longitude

Beaver Creek BC T Phelps 37° 52’ N 91° 48’ W Honey Creek HNC T Cole 38° 29’ N 92° 14’ W Rock Bridge Gans Creek RBG T Boone 38° 52’ N 92° 20’ W Cedar Creek CC P Callaway 38° 45’ N 92° 11’ W Grindstone Creek GC P Boone 38° 56’ N 92° 19’ W Jacks Fork River JF P Shannon 37° 09’ N 91° 27’ W Meramec River MR P Crawford 37° 48’ N 91° 26’ W Little Prairie Lake LPL L Phelps 37° 60’ N 91° 42’ W Little Dixie Lake LD L Callaway 38° 55’ N 92° 06’ W Rocky Forks Lake RF L Boone 39° 03’ N 92° 18’ W Winegar Lake WL L Cole 38° 28’ N 92° 24’ W Whetstone Lake WS L Callaway 38° 57’ N 91° 42’ W

*T = temporary stream, P = permanent streams, L = lakes.

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 104 S. L. BALL locations at each transect point, without looking, Frequent rainfall in late April precluded trapping. I picked up ten unimbedded rocks and collected all the Sampling resumed on May 23rd and continued S. femoratum nymphs on that rock. By collecting all until September 17th, for a total of nine samples per nymphs, I eliminated any size biases, which can bias population. nymph sex ratios as males and females may grow and develop at different rates. The 10-rock method works exceptionally well with heptageniid mayflies because SPATIAL AND TEMPORAL VARIATION IN nymphs cling tenaciously to rocks; preliminary studies TYCHOPARTHENOGENETIC CAPACITY showed that very few nymphs washed off as rocks To test for spatial variation in tychoparthenogenetic were lifted out of the water. Rocks ranged from ~3 capacity, I measured habitat and population variation to 40cm in diameter. A similar range of rock sizes in the mean proportion of unfertilized eggs that was obtained from each population. Nymphs from hatched. I did this for a sample of females in each each collection were placed in plastic Whirl Pack population and calculated population mean tych- bags containing water and transported back to the oparthenogenetic capacity. To test for temporal laboratory where the total number of nymphs per variation in tychoparthenogenetic capacity, I obtained ten rocks and the number of males and females was estimates of the population mean hatching success of counted. Males were easily identified by the presence unfertilized eggs for 1996 and 1997. Within each year, of developing genital forceps, which are visible in very I also compared the proportion of unfertilized eggs early instars. In the few instances where presence of hatching on different sampling dates. Five populations forcep could not be confidently ascertained, nymphs were sampled in 1996 and seven additional popula- were excluded from sex ratio calculations. Nymph sex tions were sampled in 1997. Late instar nymphs were ratios were calculated as percentage female by pooling collected from each population and brought to the lab- nymphs from the two 10-rock samples at each tran- oratory where they were kept in aerated glass aquaria sect point. containing water from the lake or stream in which In 1998 and 1999, I estimated imago and subimago they were collected. I also added several algae covered sex ratios from light trap collections to determine if rocks (8 ¥ 10cm) as a food source for nymphs. Water adult stages showed biased sex ratios. Light trapping temperature was maintained at 24°C. Only female was carried out repeatedly at six of the 12 populations: nymphs were placed in aquaria to ensure that all were two temporary streams (Honey Creek and Rock Bridge virgins. Mesh lids were placed on aquaria and subima- Gans Creek), two permanent streams (Cedar Creek goes collected daily as they emerged. The majority of and Grindstone Creek) and two lakes (Rocky Forks females emerged after 1–3 days in the lab but a few Lake and Little Dixie Lake). At each site, the trap spent up to 10 days in aquaria before emerging. was placed in the same location each time. I used Subimagoes were placed in small plastic vials with a BioQuip Light Trap (22V Circline UV light bulb) mesh windows in the caps. As soon as the final molt powered by a 12-V battery. The trap was filled with occurred, female imagoes were dissected and their 50mL of 70% ethanol and placed on the ground within eggs removed. Females were killed by decapitation. 2m of the water. The light was turned on automati- Each female was dissected onto a microscope slide con- cally, ~10min after sunset, using a BioQuip light taining a drop of carbon-filtered tap water. Care was sensor. The trap was run for 60min in 1998 and 90min taken to remove all eggs. Eggs were then transferred in 1999. Captured insects were taken to the laboratory to 35-mm diameter plastic Petri dishes containing and preserved in 70% ethanol. All S. femoratum adults ~10mL of carbon-filtered tap water. All pieces of were picked from these samples and stored separately exoskeleton, reproductive tissue were removed from in 20mL glass vials. Ten minutes after trapping began Petri dishes to discourage bacterial and fungal growth. and 10min before it ended, air temperature, relative Petri dishes were placed in random locations on humidity, and wind speed were measured. Tempera- shelves in the laboratory. Room temperature was ture and relative humidity were measured using a maintained at 23–25°C. Every 1–5 days, Petri dishes handheld digital thermometer-hygrometer, hung were examined for the presence of hatchlings which ~20cm from the trap. Wind speed was measured, over appeared 11–13 days after eggs were removed from 2min, by holding an MJP Student anemometer ~2m females. All hatchlings were counted and removed above the ground, near the light trap. from the Petri dishes and the cumulative number of In 1998, light trap samples were collected every hatchlings per Petri dish was recorded. I ceased check- 21–28 days except for sample 2, which was collected ing Petri dishes when no hatchlings had appeared in ~14 days after sample 1. Trapping began on July 6th a particular dish for 3 weeks. Most eggs hatched and ended on September 23rd. A total of four samples within a week of each other, but some eggs hatched per population were collected. In 1999, trapping was 10–12 days after the first eggs hatched. Once hatch- carried out every 12–16 days, beginning on April 2nd. ing had ceased, the Petri dishes were stored at 4°C in

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 POPULATION VARIATION IN TYCHOPARTHENOGENESIS 105 sealed, plastic containers. Inviable and hatched eggs subimago density (hereafter referred to as total can be maintained indefinitely under these conditions density) from light trap samples collected for the sex without disintegration or loss. ratio analyses described above. Imago and subimago To determine the proportion of unfertilized eggs densities were calculated as the number of individu- hatching for each female, I photographed each Petri als per hour of light trapping. dish using a video camera attached to a dissecting microscope. A clear, Plexiglas grid was placed under each Petri dish to aid in counting. Eggs were pho- ABIOTIC CORRELATES OF tographed at 20 ¥ magnification and digital images TYCHOPARTHENOGENESIS of each Petri dish were taken using NIH IMAGE soft- I collected data on abiotic aspects of each habitat ware. Images were printed on paper and I counted during 1997 and 1998. For each of the 12 populations, eggs by marking them with a felt tipped pen to avoid I measured stream or shoreline water depth, percent inadvertent recounting. A female’s tychopartheno- of stream bed or shoreline that was dry (habitat genetic capacity was estimated as the proportion of dryness), water current velocity, and water tempera- her unfertilized eggs hatched and was calculated by ture. Measurements were taken in summer and fall to dividing the number of hatchlings per Petri dish by test for seasonal differences in abiotic factors because the total number of eggs (hatched and unhatched) per temporary streams usually cease flowing and form Petri dish. In 1996, eggs from each female were placed small pools by late summer. For each population, mea- into a Petri dish and the proportion of unfertilized surements were taken at each transect point. For eggs hatching was calculated for each female. In 1997, streams, water depth was measured at 2-m intervals I divided a female’s clutch into two Petri dishes across the streambed, except for Beaver Creek, which to reduce losses due to bacterial or fungal infection. was measured at 1-m intervals due to the small size Proportion of unfertilized eggs hatching was calcu- of the stream. Water depth at lakeshores was mea- lated for each Petri dish separately and a females’ sured, beginning at the edge of permanent vegetation, tychoparthenogenetic capacity was calculated as the at two points, 1m apart. Only two measurements mean of the two Petri dishes. Population mean were made because water quickly became too deep tychoparthenogenetic capacity was calculated as the to measure. For lakes, these measurements were mean hatch success of all females in that population. repeated 2m to the left and right of the first mea- Mean tychoparthenogenetic capacity in each habitat surement for a total of six measurements per transect was calculated as the mean of all populations in that point. For each stream, habitat dryness (% of the habitat type. stream bed that was dry) was calculated for each tran- To test for temporal variation in tychopartheno- sect point by dividing the number of locations across genetic capacity, in 1996, hatch success was measured the stream bed that were dry, by the total number of on two sampling dates ~4 weeks apart for three of the locations measured. For lakes, measurements at the five populations tested. In 1997, six of the 12 popula- three locations per transect point were pooled so that tions were tested two, three or four times. Sampling the total number of locations without water was dates for each population differed as did the time divided by six, the total number of locations at that elapsed between repeated samples. On average, sam- transect point. In 1997, current velocity was measured pling intervals were ~3–4 weeks. once at each transect point using a neutrally buoyant weighted, Styrofoam fishing line float. The amount of time taken for the float to travel 1 m was recorded. For BIOTIC CORRELATES OF TYCHOPARTHENOGENESIS lakes, current velocity was scored as zero as the float I estimated nymph, imago, and subimago densities for usually did not move unless conditions were extremely each habitat and population. Density can affect both windy. In 1998, current velocity was measured using mate availability and the level of intraspecific compe- an MJP Student Stream Flowmeter electronic velocity tition for resources. Because S. femoratum spend meter. Water temperature was measured once at each > 95% of its lifecycle in the nymph stage and because transect point by holding a thermometer for 1min at adults do not posses functional mouthparts, competi- a known depth. tion for resources is primarily a function of nymph densities rather than imago or subimago densities. In 1997 and 1998, I estimated nymph density for each STATISTICAL ANALYSES population from ‘10-rock’ samples collected for the sex Categorical data modelling (Proc CATMOD, SAS Insti- ratio analyses described above. Nymph density per tute 1990) was used to test effects of year, season, transect point was estimated by taking the mean habitat, and population nested within habitat on of the two ‘10-rock’ samples. In 1998 and 1999, I nymph, imago, and pooled imago and subimago sex estimated imago density and combined imago and ratios (% female). This was carried out for the 12 pop-

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 106 S. L. BALL ulations sampled for nymphs in 1997 and 1998 and for in 1996 and for six populations that were sampled six populations sampled for imagoes and subimagoes repeatedly in 1997. Sampling date was considered a in 1998 and 1999. Separate analyses were carried out nested effect, instead of a repeated measure, because for nymphs and imagoes. For these analyses, year, populations were sampled a different number of times season, and habitat were considered fixed effects, and at slightly different time intervals. The popula- while population was considered a random effect and tion main effect was tested using sampling date mean was nested within habitat. The response variable in squares as the error term while sampling date was these analyses was the number of males and females. tested using the error mean squares as the error term Log-likelihood analysis was used to test whether pop- (Zar 1984). ulation sex ratios (F:M) deviated significantly from To determine if populations with higher hatch 1:1. In cases where the sample size was <25, I used success also tended to have a higher frequency of a binomial exact test (Zar 1984) to test for deviations parthenogenetic females, I tested effects of habitat, from 1:1. For each population, I pooled data for all population nested within habitat and sampling date transect points and performed G-tests on frequency of nested within population on frequency of partheno- female nymphs separately for each year. For clarity, genetic females in each population. Frequency of data are plotted as percent of females in each popula- parthenogenetic females is the number of females tion although the analysis was performed on frequen- exhibiting nonzero hatch success, divided by the total cies. Similar analyses were used to test for significant number of females tested for that population. I used deviations from 1:1 sex ratios of imagoes as well as categorical data modelling to perform log linear tests imagoes and subimagoes combined. using a nested ANOVA model. Analysis was performed I used separate nested ANOVA to test effects of separately for 1996 and 1997. To test for differences habitat and population on tychoparthenogenetic between years, I used only the five populations capacity (% hatch success of unfertilized eggs) and sampled in both years. nymph density. In all nested ANOVA tests, year and A repeated measures ANOVA was used to test effects habitat were considered fixed effects. Population was of year, habitat, population (nested within habitat) considered a random effect nested within habitat. and sampling date (the repeated measure) on density The habitat main effect was tested using population of imagoes. Separate analysis was performed for mean squares as the error term. The population effect imagoes and subimagoes combined (hereafter referred was tested using the error mean squares as the error to as total density). I performed separate analyses for term (Zar 1984). I performed analyses for each year 1999 data only, to test effects of habitat and sampling separately and then tested for a year effect by includ- date on mean density of imagoes and total density. ing only those populations sampled in both years. Because emergence and adult flight may be affected For 1996 only, I used separate one-way ANOVAS to by temperature, relative humidity and wind speed, test the effects of habitat and population on ranked I used linear regression to test for relationships hatch success because I had only one permanent between these variables and imago and total density. stream population; therefore nested ANOVA could Both imago (r2 = 0.20, N = 53, P < 0.001) and total not be used. Instead, I examined the effect of habitat density (r2 = 0.27, N = 53, P < 0.001) were significantly and used residuals to test the effect of population. related to temperature. Therefore, I used residuals of Analysis of nymph densities also included season these regressions in the repeated measures ANOVA. as a fixed main effect and transect point, which was Neither relative humidity nor wind speed was signi- considered a random effect nested within population. ficantly related to imago or total density. Fixed main effects (year, habitat, season and interac- Pearson’s correlation was used to test whether hatch tions between them) were tested using population success in a population was related to the frequency mean squares as the error term. Population was of females exhibiting parthenogenesis in 1996 and tested using transect mean squares as the error term 1997. In each year, I also used Pearson’s correlation to and transect point was tested using the error mean test for relationships between population mean hatch squares as the error term (Zar 1984). Data were success and population sex ratios, nymph density and tested for normality and homogeneity of variance imago and total density. Separate correlations were assumptions. If these assumptions were violated, performed for 1996 and 1997 hatching success. Corre- I used ranked dependent variables in the nested lations between 1996 hatch success and imago and ANOVA. When main effects or interactions were signif- combined imago and subimago sex ratios were not icant, I used Tukey’s HSD for posthoc multiple calculated due to extremely small sample sizes (N = 3). comparisons. If correlations were large, but nonsignificant, I calcu- I used a nested ANOVA to test the effect of sampling lated the power (1-b) of the correlation (Zar 1984), date, nested within population, on ranked hatch which is the probability of rejecting the null hypothe- success for three populations that were sampled twice sis when the null hypothesis is false.

I used Pearson’s correlation to test for relation- four abiotic variables, corrected for sample size (Sokal ships between population mean hatching success and & Rohlf 1981). Using coefficients of variation of each nymph, imago and total densities. Because nymph of the abiotic variables, I performed a MANOVA, testing densities did not vary between years, I used densities effects of year and habitat. If the multivariate effect averaged across the two years. Imago and total densi- was significant, I used Hotelling’s T2 to perform pair- ties from 1999 were used because sampling in 1999 wise comparisons for the linear combination of all four covered a greater proportion of the mayfly’s emergence abiotic variables. I also performed univariate ANOVA season (March through to September) compared to tests on each of the dependent variables separa- 1998. Population mean imago and total densities were tely to determine which of the dependent variables obtained by combining all samples collected over the contributed to the multivariate significance. I used entire emergence season. Tukey’s HSD to perform multiple comparisons on sig- To determine whether the three habitats differed nificant main effects for each univariate analysis. significantly in their abiotic characteristics, I per- I used stepwise regression to test for a relationship formed two MANOVA tests, one including depth and between tychoparthenogenetic capacity and water habitat dryness (% dry) as dependent variables and depth, habitat dryness, and current velocity. I also the other including current velocity and temperature. used stepwise regression to investigate relationships I performed separate MANOVA tests because, where between tychoparthenogenetic capacity and coeffi- percentage dry was 100%, there were no measure- cients of variation for each of the four abiotic vari- ments for current velocity or temperature. Because ables. Water temperature was not included in the current velocity was measured in different units in dif- analysis because it did not contribute to the multi- ferent years, I used standardized values (Z scores) in variate differences in habitat variability. I calculated the MANOVA. I tested main effects of year, season, partial correlation coefficients for each abiotic variable habitat and population within habitat on the linear and hatching success for 1996 and 1997, separately. combination of depth and dryness and on the linear combination of current velocity and temperature. Sig- nificant main effects were followed by univariate RESULTS analysis of those effects on each of the dependent variables. Following significant univariate main effects FIELD EVIDENCE OF TYCHOPARTHENOGENETIC and interactions, Tukey’s HSD was used to test for dif- REPRODUCTION ferences among levels of the factor. If females utilize their parthenogenetic capacity, then I compared habitat variability by estimating the populations should show female-biased sex ratios. In population mean coefficient of variation for each of the 1997 and 1998, all populations were significantly

Table 2. Tests of deviation from 1:1 sex ratios for nymphs collected from each population in 1997 and 1998. Values are ratios of Female : Male. Sample sizes are in parentheses. Signiﬁcance levels are shown with an asterisks

Year

Population Habitat† 1997 1998

Beaver Cree T 9.00:1 (20)* 2.67:1 (22)NS Honey Cree T 9.00:1 (50)*** 2.27:1 (98)*** Rock Bridge T 3.33:1 (26)** 2.09:1 (68)** Cedar Creek P 2.05:1 (119)*** 1.87:1 (198)*** Grindstone Creek P – 2.26:1 (140)*** Jacks Fork River P 2.89:1 (35)** 5.17:1 (37)*** Meramec River P 2.80:1 (38)** 2.41:1 (58)** Little Dixie Lake L 2.45:1 (494)*** 2.46:1 (384)*** Little Prairie Lake L 2.38:1 (71)*** 2.08:1 (157)*** Rocky Forks Lake L 2.55:1 (213)*** 2.93:1 (299)*** Winegar Lake L 3.41:1 (207)*** 2.34:1 (204)*** Whetstone Lake L 2.69:1 (381)*** 2.01:1 (268)***

*Significant at 0.005 < p < 0.05; **significant at 0.001 < p < 0.005; ***significant at p < 0.001; NSnon-significant. †T = temporary stream, P = permanent streams, L = lakes.

© 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 101–123 108 S. L. BALL female-biased (Table 2). Nymph sex ratio differed streams showed no difference in sex ratio across among years (c2 = 17.77, d.f. = 1, P < 0.0001) and seasons (Fig. 1B). populations (c2 = 46.76, d.f. = 10, P < 0.0001). The per- Unlike nymph sex ratios, there were no consistent centage of females per population ranged from 67% in sex biases among different habitats, populations, or Cedar Creek to 92% in Beaver Creek in 1997 and from years for imagoes and for imagoes and subimagoes 62% in Cedar Creek to 84% in the Jacks Fork River combined. Results were similar when imagoes and and in 1998. There were significant habitat by year subimagoes were combined; therefore results are (c2 = 10.59, d.f. = 2, P = 0.005) and habitat by season reported only for imagoes. In 1998, only three popula- interactions (c2 = 11.03, d.f. = 2, P = 0.004), but no sig- tions showed significant deviations from 1:1 nificant main effect of habitat (c2 = 0.63, d.f. = 2, female : male sex ratios for imagoes (Table 3). In both P = 0.73), season (c2 = 0.001, d.f. = 1, P = 0.99) or the cases, sex ratios were male biased. In 1999, only three three–way interaction (c2 = 4.99, d.f. = 2, P = 0.082). populations showed significantly male biased imago In 1996, temporary streams had a greater female bias sex ratios. There were significant effects of year compared to permanent streams and lakes while in (c2 = 8.51, d.f. = 1, P = 0.004), habitat (c2 = 7.92, d.f. = 2, 1997, all three habitats had similar sex ratios P = 0.019), year by habitat interaction (c2 = 26.82, (Fig. 1A). Lakes showed a greater female bias in fall d.f. = 2, P < 0.0001) and population nested within than in summer while both temporary and permanent habitat (c2 = 42.45, d.f. = 6, P < 0.0001) on sex ratio of imagoes. Similar results were obtained when imagoes and subimagoes were combined. Temporary streams showed a significant male bias in 1998 but not in 1999 (Fig. 2). In contrast, lakes showed a significant female A bias in 1998 but not in 1999. Permanent streams showed equal sex ratios in 1998, but in 1999 ratios were significantly male biased.

SPATIAL AND TEMPORAL VARIATION IN TYCHOPARTHENOGENESIS In 1996, tychoparthenogenetic capacity differed sig-

niﬁcantly among habitats (F2,141 = 4.53, P = 0.012). Females from temporary streams had twice the hatch success of unfertilized eggs than those from lakes or permanent streams (Fig. 3). However, there was no signiﬁcant effect of population within habitat

(F4,139 = 0.30, P = 0.88). Within populations, females showed large variation in tychoparthenogenetic capac- B

Table 3. Log-likelihood tests of deviation from 1:1 sex ratios for imagoes for samples collected from each population in 1998 and 1999. Values are ratios of Female:Male; sample sizes are in parentheses. Bonferroni corrected probabilities are shown with an asterisks

Population 1998 1999

Honey Creek 0.63:1 (112) 1.25:1 (117) Rock Bridge 0.71:1 (758)*** 0.78:1 (673) Cedar Creek 0.93:1 (268) 0.89:1 (285) Grindstone Creek 0.52:1 (260)*** 0.51:1 (557)*** Little Dixie Lake 0.82:1 (482) 0.52:1 (285)** Rocky Forks Lake 0.75:1 (1288)*** 0.52:1 (894)*** Figure 1. Effects of (A) habitat by year and (B) habitat by season interactions on nymph sex ratio (mean percent *Significant at 0.005 < P < 0.05; **significant at female). Values are means ± SE. 0.001 < P < 0.005; ***significant at P < 0.001.

Figure 2. Effects of habitat and year on imago sex ratio (mean percent female). Values are means ± SE. Figure 3. Effects of habitat and year on tychoparthenogenetic capacity (mean % hatching success of unfertilized ity. Patterns of variation in hatch success were highly eggs). Values are means and standard errors. Means with positively skewed, with most females exhibiting small different letters are significantly different (Tukey’s HSD, tychoparthenogenetic capacities (Fig. 4). In all but one P < 0.05). population, coefficients of variation in hatch success were greater than one. For populations, which were sampled twice, there was a significant effect of sam- P = 0.038) and year by habitat interaction (F2,4 = 8.83, pling date within populations on ranked hatching P = 0.034) on ranked hatch success. In 1996, hatching success (F3,99 = 3.83, P = 0.012). Hatching success of success was greatest in temporary streams compared unfertilized eggs from females collected at Little Dixie to permanent streams and lakes while in 1997 hatch- Lake on May 29 (mean ± SE = 6.01% ± 1.64%) was sig- ing success was similar among all habitats (Fig. 3). nificantly higher than that of females collected on However, there were no main effects of habitat

June 11th (mean ± SE = 1.27% ± 0.57%). (F2,4 = 3.28, P = 0.14) or population within habitat For 12 populations tested in 1997, there was no (F4,497 = 0.25, P = 0.91) on hatch success. significant effect of habitat (F2,9 = 0.39, P = 0.61) or Hatching success of unfertilized eggs from 1996 was population (F9,377 = 1.24, P = 0.27) on ranked mean positively correlated with nymph sex ratio (r = 0.80, N hatch success. Females within populations exhibited = 5, P = 0.09), however, lack of significance may be due extremely large variation in hatch success (Table 4). to low statistical power (1 – b=0.65) resulting from In the most variable population (Rock Bridge Gans a small sample size. Correlations were not computed Creek) hatch success ranged from 0 to 78%, indicating for 1996 hatch success and imago and combined that within some populations, females showed enor- imago and subimago sex ratios because data were mous variation in hatching success. As in 1996, all available from only three populations. Hatch success populations showed significantly positively skewed for 1997 showed no significant correlations with distributions of female hatch success. For populations nymph (r = – 0.48, N = 12, P = 0.40), imago (r = 0.67, sampled repeatedly in 1997, there was a significant N = 12, P = 0.21) and combined imago plus subimago effect of sampling date nested within population (r = 0.59 N = 12, P = 0.30) sex ratios. (F13,566 = 2.16, P = 0.01; Fig. 5). Hatch success for Rock In 1996, I found no significant variation among Bridge Gans Creek females was significantly higher populations (c2 = 0.97, d.f. = 2, P = 0.62) or habitats on June 29th compared to May 20th or July 14th. Sim- (c2 = 2.93, d.f. = 2, P = 0.23) in frequency of partheno- ilarly, hatching success of Little Prairie Lake females genesis. Similarly, for populations sampled twice, no was significantly higher on July 2nd compared to April difference in frequency of parthenogenesis between 19th or June 2nd. Although Little Dixie females col- sampling dates was observed (c2 = 3.14, d.f. = 3, lected on June 10th had higher hatch success com- P = 0.37). Similarly, in 1997 I found no significant dif- pared to females collected during the three other ference in frequency of parthenogenesis among habi- sampling dates, differences were not significant. tats (c2 = 0.12, d.f. = 2, P = 0.94) or among populations For the five populations sampled in both 1996 and within habitat (c2 = 12.73, d.f. = 9, P = 0.18). However, 1997, I found significant effects of year (F1,4 = 9.28, there was a significant effect of sampling date within

Figure 4. Variation in tychoparthenogenetic capacity (% hatch success of unfertilized eggs) among females in each population sampled in 1996. Bars represent the proportion of females in the population with a given % hatch of unfertilized eggs. Medians and coefﬁcients of variation (C.V.) are also reported for each population.

Table 4. Population variation in hatch success (mean percent) of unfertilized eggs for females from populations sampled in 1997. N = number of females tested

Population Region* Mean (%) SD Range N

Beaver Creek T 7.31 13.95 0-53.16 15 Honey Creek T 4.33 7.83 0-25.47 11 Rock Bridge Gans Creek T 5.66 13.93 0-77.86 38 Cedar Creek P 6.56 9.64 0-46.52 64 Grindstone Creek P 8.56 14.77 0-57.32 19 Jacks Fork River P 5.28 8.28 0-30.96 28 Meramec River P 5.49 10.25 0-45.76 45 Little Dixie Lake L 7.96 14.54 0-67.66 71 Little Prairie Lake L 6.86 7.84 0-32.40 36 Rocky Forks Lake L 3.31 4.12 0-16.14 43 Winegar Lake L 10.73 17.58 0-41.64 5 Whetstone Lake L 7.36 12.93 0-63.38 57

*T = temporary stream, P = permanent streams, L = lakes.

Figure 5. Temporal variation in tychoparthenogenetic capacity among populations sampled in 1997. Values are means ± SE. Sampling dates are given above each bar. population (c2 = 26.13, d.f. = 13, P = 0.016; Fig. 6). The Dixie and Rocky Forks also showed large difference in greatest variation in frequency of parthenogenesis frequency of parthenogenesis among sampling dates. occurred in Whetstone Lake. On May 4th, 65% of For populations sampled in both 1996 and 1997, I females tested produced offspring parthenogenetically found no signiﬁcant effect of year (c2 = 1.15, d.f. = 1, while 100% of females sampled on July 1st produced P = 0.28), habitat (c2 = 3.60, d.f. = 2, P = 0.16) and no offspring parthenogenetically. Similarly, both Little signiﬁcant year by habitat interaction (c2 = 4.54,

Figure 6. Temporal variation in the percentage of females exhibiting tychoparthenogenetic capacity (non-zero % hatch of unfertilized eggs) for six populations sampled repeatedly in 1997. See Table 1 for population names and habitat designations.

d.f. = 2, P = 0.10) on the frequency of parthenogenesis. nymphs were patchily distributed along streambeds There was a nearly significant effect of population and lakeshores. None of the two or three-way interac- within habitat (c2 = 8.04, d.f. = 3, P = 0.09). tions between year, habitat, or season were significant. Correlation of population mean hatch success in Repeated measures ANOVA showed a significant 1996 with mean percent of females producing off- effect of population nested within habitat on total spring parthenogenetically was not significant (r = density, but no significant effects of year, habitat or 0.76, N = 5, P = 0.13). However small sample size any two-way interaction (Table 6). Similar results resulted in low power to detect a statistically signifi- were obtained for imagoes only. In contrast to nymph cant relationship (1-b=0.55). Similarly in 1997, vari- densities, Rock Bridge Gans Creek, a temporary ation in hatch success was unrelated to variation in stream, showed relatively high total densities while percent of females producing offspring parthenogenet- Little Dixie Lake showed low densities that were ically (r = –0.09, N = 12, P = 0.78). comparable to those of the permanent streams (Fig. 8A). Total density varied over time, declining significantly in early September. A similar pattern was BIOTIC CORRELATES OF TYCHOPARTHENOGENESIS observed for imago density, although the effect of time Nymph densities did not differ significantly between was not significant (P = 0.066). A separate analysis for years, but there were significant differences among 1999 that included all nine sampling dates, from April habitats (Table 5). Lake densities were an order of to September, showed a significant effect of sampling magnitude higher than those in either temporary or date (F8,24 = 2.67, P = 0.029) on total density, but no permanent streams (Fig. 7). Nymph densities also effect of habitat (F2,24 = 0.19, P = 0.83) or habitat by varied significantly among populations. There was a sampling date interaction (F16,24 = 1.08, P = 0.42). significant effect of transect point, indicating that Similar results were obtained for imago density. Light

Table 5. Nested ANOVA of effects of year, season, habitat, population (nested within habitat) and transect point (nested within population) for mean nymph density (number of nymphs per ten rocks)

Source df MS FP

Year 1 52147.48 0.63 0.43 Season 1 25403.17 0.31 0.58 Habitat 2 3566697.98 43.10 0.0001 Year x Season 1 182168.70 2.20 0.15 Year x Habitat 2 159664.69 1.93 0.16 Season x Habitat 2 41265.15 0.50 0.61 Year x Season x Habitat 2 24106.47 0.29 0.75 Population (Habitat) 33 82761.51 2.57 0.0001 Transect point (Population) 288 32213.92 3.54 0.0001 Error 333 3029017.00

Figure 7. Variation in nymph density among populations and habitats. Populations are grouped by habitats (TS = temporary streams, PS = permanent streams, LK = lakes). Values are means ± SE, averaged across two years (1997 and 1998).

trap samples collected early in April and late May and relations between 1996 hatching success and imago toward the end of the emergence season in mid Sep- and total density were not performed due to small tember showed extremely low total densities relative sample sizes (N = 3). Hatch success in 1997 showed no to samples collected in June through August (Fig. 8B). relationship with any of the density measures; in all Hatch success in 1996 was negatively correlated cases, correlation coefﬁcients were relatively small with nymph density (r = –0.80, P = 0.034; Fig. 9). Cor- and nonsigniﬁcant (P > 0.50 for all correlations).

Figure 8. Population variation (A) and temporal variation (B) in mean total density (imagoes plus subimagoes) from light trap samples. Population means in (panel a) are averaged across 2 years (1998 and 1999). Temporal variation (panel b) is shown for 1999 only, across nine different sampling dates. Values are means ± SE.

Table 6. Repeated measures ANOVA for effects of year, habitat, population (nested within habitat) and time (repeated measure) on mean total density . Density is the number of individuals caught per hour of light trapping on four sampling dates: July 10, July 26, August 24, September 9, in 1998 and 1999

Source df MS FP

Year 1 96690.40 2.63 0.21 Habitat 2 23649.05 0.64 0.59 Time 3 32854.69 3.78 0.050 Year x Habitat 2 33302.06 0.90 0.49 Habitat x Time 6 14448.14 1.66 0.24 Population (Habitat) 3 36821.88 4.24 0.039 Time x Population 9 8681.92 0.87 0.57 (Habitat) Error 21 9997.92 Figure 9. Relationship between tychoparthenogenetic capacity (% hatch of unfertilized eggs) measured in 1996 and nymph density.

ABIOTIC HABITAT CHARACTERISTICS Multivariate ANOVA revealed that water depth and was a significant interaction between year, season, habitat dryness varied significantly among habitats and habitat as well as a significant season by habitat (Table 7A). Neither year, season, nor any of the two- interaction (Table 7C; Figs 10C,D). way or three-way interactions were significant with Hatch success in 1996 was negatively related to respect to these two variables. Populations differed water temperature (r = –0.95, N = 5, P = 0.012) but significantly, indicating spatial variation for depth and showed no correlation with percentage dryness dryness within the different habitats. Univariate (r = 0.74, N = 5, P = 0.26), water depth (r = –0.41, N = 5, analyses showed that significant differences in depth P = 0.56) or current velocity (r = 0.60, N = 5, P = 0.40). among habitats and among populations within habi- In 1997, hatch success showed no correlations with tats contributed to the multivariate significance water depth (r = 0.17, N = 12, P = 0.59),% dryness (Table 7B). Temporary streams were significantly (r = –0.32, N = 12, P = 0.31), current velocity (r = –0.18, shallower than either permanent streams or lakes N = 12, P = 0.57), or water temperature (r = 0.07, (Fig. 10A). All other main effects were not significant. N = 12, P = 0.84). Multivariate analysis of current velocity and tem- Multivariate ANOVA on coefficients of variation perature showed significant differences among habi- for water depth, dryness, current velocity, and tem- tats and seasons, but no significant differences perature showed significant differences in variability between years and no significant two- or three-way among habitat (Wilk’s l=0.086, F8,30 = 9.06, interactions (Table 8A). Populations within habitats P = 0.0001) and a trend toward differences in vari- also varied significantly in current velocity and tem- ability between years (Wilk’s l=0.55, F4,15 = 3.04, perature. Univariate analysis of current velocity P = 0.052) but no multivariate interaction between showed significant variation among habitats and pop- year and habitat (Wilk’s l=0.56, F8,30 = 1.25, P = 0.30). ulations within habitats (Table 8B). Permanent Pairwise multivariate comparisons using Hotelling’s streams showed the highest (Fig. 10B) current veloc- T2 indicated that all three habitats differed signifi- ity; temporary streams were intermediate and lakes cantly in variability (P < 0.02 in all cases). Univariate showed the lowest current velocity. For temperature, analyses showed that habitats differed significantly in univariate tests showed significant differences variability of water depth, with temporary streams between seasons and among populations within habi- significantly more variable compared to permanent tats, but no differences among years, habitats, or streams and lakes (F2,18 = 16.81, P = 0.0001; Table 9). with any of the two-way or three-way interactions Similarly, habitats differed in their variability in (Table 8C). Summer water temperatures were signifi- dryness, with lakes being significantly more variable cantly higher (mean ± SD = 24.7 ± 4.7°C) than fall compared to temporary or permanent streams temperatures (mean ± SD = 17.3 ± 6.5°C). (F2,18 = 6.46, P = 0.008; Table 9). There were no sig- Univariate analysis showed that habitat dryness nificant year or year by habitat interactions for also contributed to multivariate significance. There either variability in water depth or dryness (all

P values >0.50). There were also signiﬁcant habitat 1996; Cloeon simile, Harker 1997), including several differences in variability of current velocity obligately parthenogenetic species in which males are

(F2,18 = 42.34, P = 0.0001) as well as differences exceedingly rare or absent (Sweeney & Vannote 1987). between years (F2,18 = 11.63, P = 0.003). Current veloc- In contrast, equal sex ratios were reported for Dolania ity was most variable in permanent streams and americana nymphs, a species which showed no slightly less variable in temporary streams while lakes parthenogenetic capability (Peters & Peters 1977). showed no variation in current velocity (Table 9). The Several factors, other than tychoparthenogenesis, interaction between habitat and year was nearly may lead to female biased sex ratios. However, it is significant (F2,18 = 3.44, P = 0.054). Variability in tem- unlikely that these factors explain entirely the female perature did not contribute significantly to the biased nymph sex ratios in S. femoratum populations. multivariate effect (F2,9 = 1.20, P = 0.35; Table 9). Female bias can also result from sampling biases, dif- Hatch success in 1996 was positively related to vari- ferential mortality, environmental sex determination, ability in water depth (r = 0.98, N = 5, P = 0.02) but or cytoplasmic symbionts. In addition, mating systems showed no correlation with variability in current where mating occurs exclusively between sibs are typ- velocity (r = 0.92, N = 5, P = 0.17) or with variability ically characterized by strong female biases (Thornhill in dryness (r = –0.43, N = 5, P = 0.98). There were no & Alcock 1983). It is unlikely that female biases are significant correlations between hatch success in 1997 an artifact of sampling because nymphs were collected and variability in depth, dryness or current velocity randomly without bias toward certain size classes or (P = 0.90 in all cases). developmental stages. Like many mayfly species, S. femoratum shows protandrous emergence. Collect- ing nymphs immediately after male emergence begins, DISCUSSION but before female emergence begins, could result in female bias. However, female biases were consistent FIELD EVIDENCE OF TYCHOPARTHENOGENETIC across sampling dates between years and seasons, REPRODUCTION suggesting that they were not simply a sampling In both years sampled, all S. femoratum populations artifact. Sib mating is unlikely to occur frequently en- showed significantly female biased nymph sex ratios, ough in S. femoratum populations to lead to female with populations ranging from moderately (62%) to biases because of asynchronous adult emergence. The strongly female biased (92%). These data are consis- likelihood of sib mating should be less than for tent with the hypothesis that tychoparthenogenetic synchronously emerging mayfly species. Mating is reproduction does occur in these natural populations promiscuous, with swarming males intercepting and that hatching of unfertilized eggs was not simply females as they fly into the swarm, which suggests a case of artificial parthe