The effects of polyandry and mate preference on clutch size, hatching success and nesting location of loggerhead sea turtles (Caretta caretta) Research Proposal Fiona Dalrymple December 14, 2008 Biology 526 Dr. Robertson
1 Contents 1. Discussion 2 2. Purpose 4 3. Hypotheses 4 4. Materials & Methods 4 5. Permits and Regulations 5 6. Results 5 7. Discussion 6 8. References 7 9. Appendix A 9
1. Introduction 2 Global temperatures have warmed approximately 0.6 C in the last century according to the Intergovernmental Panel on Climate Change (2001). It is expected that, within the next 100 years, temperatures will rise a further 0.3 to 7.5 C at a rate unprecedented in the last millennium (IPCC, 2001; Zwiers, 2002, as cited in Broderick, et al., 2007). Although changes in climate have occurred in the past due to natural phenomena (IPCC, 2001), it has become evident that the rapidity of recent temperature changes is cause for concern (Hall, et al., 2003). Marine turtles are especially vulnerable to global temperature changes (Janzen, 1994). Sex ratios within these species are controlled by temperature-dependent sex determination (Mrosovsky & Yntema, 1981). The sex of the offspring is established during the middle third of embryonic development the outcome of this process being radically altered by just 1 C change in incubation temperature (Janzen, 1994). During incubation, sea turtles have a thermal tolerance range of 25-35 C (Broderick, et al., 2007). Most have a pivotal temperature of approximately 29 C at which 50% of the offspring in a clutch will be females and 50% will be males. Above this temperature only females are produced (Broderick et al., 2007). Rising annual sand temperatures will lead to the feminization of the majority of offspring within clutches and consequently, more biased operational sex ratios (Janzen, 1994). These effects are already prevalent in Southern Florida: clutches at Cape Canaveral have been found to be more than 94% female (Mrosovsky & Provancha, 1991). Although sea turtles have been facing the challenges of global temperature change for millions of years, rapid warming trends are creating insuperable problems (Janzen, 1994) as well as exacerbating the devastating impacts of improper fishing practices, pollution and predation (Heppell & Lutcavage, 2008). Unfortunately, all seven species of turtles (leatherback (Dermochelys coriacca), green (Chelonia mydas), loggerhead (Caretta caretta), hawksbill (Eretmochelys imbricata), olive ridley (Lepidochelys olivacca), Kemp s ridley (Lepidochelys kempii), flatback (Natator depressus) (Davenport, 1997)) are currently listed on the International Union of Conservation of Nature and Natural Resources Red List of Threatened Species (Heppell & Lutcavage, 2008). It has been suggested that marine turtles may be able to adapt to warmer temperatures by modifying aspects of their mating strategy. For example, pivotal temperatures could be adjusted upwards in order to moderate the effects of excessively high incubation temperatures (Janzen, 1994). However, pivotal temperatures do not vary significantly within these longliving populations and therefore this change would not be strongly selected for (Janzen, 1994). Another, more probable, adaptation to increasing land temperatures would involve the modification of spatial qualities of nesting sites. Nesting at more extreme latitudes, at different heights on the beach or at deeper levels within the substrate would provide females with some control over their offspring sex ratio (Broderick, et al., 2007). Altering nesting temporally by laying eggs earlier or later in the season could also contribute to greater overall fecundity for sea turtles (Janzen, 1994). Unfortunately, the probability this will be selected for is low because earlier or later nesting times occur infrequently in these populations (Janzen, 1994). In order to develop comprehensive and effective protection and conservation plans, it is essential that each stage of these turtles life cycles be understood, particularly in the context of changing global temperatures. Marine turtles are difficult to study (Heppell & Lutcavage, 2008); territories are inscrutable and individuals travel great distances in between nesting periods (Davenport, 1997). Research has, however, revealed that the basic life history strategy is generally similar among all species. Turtles mate near the shore of their natal beach and typically store sperm for a few weeks (although they may store sperm longer painted turtles are capable of storing sperm for up to four years) (Davenport, 1997; Avise, et al.,
2002). They exhibit limited parental care except for the energy expended during egg production, choice of nesting site and nest construction. Sea turtles nests are large: 50-200 eggs depending on the species (Davenport, 1997). Females will typically lay several clutches per season at intervals of 9 to 30 days but they do not usually breed every year. Eggs incubate for a period of two months and hatchlings grow slowly, loggerheads reaching sexual maturity at 15-20 years and green turtles 20-50 years (Davenport, 1997). It is vital that research focus on the elusive mating strategies of sea turtles. Elucidating these will provide valuable insight for determining these species potential reproductive success in the future. Unfortunately, it is difficult to directly observe mating events as they occur quickly and offshore (Heppell & Lutcavage, 2008). DNA analysis is important for determining the basic biology and mating strategies of sea turtles (Ball & Moore, 2002). Sea turtles have been found to exhibit polyandry significant proportions of loggerhead clutches being fertilized by multiple males (Ball & Moore, 2002; Arlettaz, et al., 2007). Interestingly, a single copulation is usually enough to fertilize all eggs within a clutch and females receive no direct benefits, such as nuptial gifts, courtship feeding or territory access, from mating events (Avise, et al., 2002). Why, then, does polyandry occur? Most research points to indirect benefits for females (Avise, et al., 2002; Olsson & Uller, 2008; Ball & Moore, 2002). For instance, genetic variability of multiply sired broods may increase the reproductive success of polyandrous females. Multiple matings could also ensure that females find the best genotypic compatibility for their offspring. Mating with multiple males also promotes sperm competition, increasing the likelihood that the best sperm fertilizes the eggs. Because females can store their sperms for significant periods of time, multiple matings could provide a large sperm bank from which they could choose in later mating seasons (Avise, et al., 2002). It is also suggested that the trade-up hypothesis may explain polyandry in sea turtles. If the sex ratio is skewed, females may choose to mate with the first male they encounter to guarantee fertilization then later upgrade to a better male. Another explanation for the occurrence of polyandry in sea turtles is the genetic diversity hypothesis. Although females are generally philopatric, they may be forced to deviate from their natal beach and colonize new beaches. In this situation polyandry would be advantageous: polyandrous gravid females would carry more genes from their natal beach to their new colony (Ball & Moore, 2002). Multiple paternity has been found to be unevenly distributed across clutches in painted turtles: clutches fertilized by more males were found to be larger (Avise, et al., 2002). Clutch size has also been found to increase with female body size in loggerhead turtles (Broderick, et al., 2003). This could be due to male preference for large females (Avise, et al., 2002) and/or greater egg carrying capacity of those females with large body size (Hays & Speakman, 1991). However, the majority of variation in clutch size cannot be explained by the size of the female (Hays & Speakman, 1991) necessitating further research into multiple paternity. As climate changes, sex ratios will become more female biased possibly leading to a lower prevalence of polyandry (Janzen, 1994; Olsson & Uller, 2008). Now, more than ever, it is crucial that research focus on why polyandry exists in these populations. Another aspect of sea turtle mating strategy that has received little attention is that involving the relationship between mate choice and nesting location. A great deal of research supports the sexy son hypothesis which suggests that birds and other animals are able to adjust offspring sex ratios in response to cues about mate quality (Robertson & Weatherhead, 1979; Huk & Winkel, 2006). For example, female blue tits skew their offspring sex ratios towards sons when they mate with males possessing survival qualities (Avise & Pearse, 2001 maybe). Female turtles have a means of accomplishing this through temperature-dependent sex determination but the use of it in relation to mate choice is unknown (Avise & Pearse, 2001). 3
2. Purpose 4 This research will have three purposes. The first is to determine to what extent multiple paternity contributes to clutch size and hatching success when controlling for female size in loggerhead sea turtles. The second, to examine whether males prefer to mate with larger females by comparing the levels of multiple paternity of clutches in different breeding seasons. Lastly, to investigate whether females alter their nesting sites to increase the number of male offspring if their clutch s primary sire is a preferred male. 3. Hypotheses It is predicted that clutches from larger females showing multiple paternity will be significantly larger than clutches of similar sized females not exhibiting any or as much polyandry. Hatching success rates should only be weakly correlated with multiple paternity. This might be due to multiple extraneous factors that can impact embryonic growth and survivorship such as high levels of metabolic heat from within the nest, fungi, root systems, weather (excessive rain or sun) and/or predation (Caswell, et al., 1987). It is also expected that clutches from females returning in subsequent seasons will show higher levels of multiple paternity than previously. The final hypothesis: females will choose shadier, more protected sites for nesting if their clutch is fathered primarily by a preferred male. 4. Materials & Methods All field research will be conducted on Bald Head Island, North Carolina, USA (Broderick, et al., 2005) from June 1st to August 1st over an eight year period. This beach is patrolled nightly by volunteers with the Bald Head Island Conservancy (Broderick, et al., 2005). It is hoped that these volunteers will collaborate with the research team in identifying, counting and measuring the turtles. The curved carapace length will be recorded for all female turtles coming ashore and each will be marked with stainless steel tags on the trailing edges of their front flippers (Frazer & Richardson, 1985). It is hoped that data from at least 90 females will be obtained. If a turtle has not been tagged before, skin punched out during this process will be used as a biopsy (Arlettaz, et al., 2007). Otherwise, skin biopsies will be taken with a 6 mm biopunch and stored in ethanol. Nests will be marked and descriptions of locations will be recorded based on the vegetation cover, height on beach and depth of nest (once eggs have hatched). 50 days after eggs have been laid, nests will be caged (Ball & Moore, 2002) and checked at intervals of 30 minutes or less. This time interval is essential (especially during the day) as hatchlings are extremely vulnerable to desiccation (Arlettaz, et al., 2007). Many volunteers will be required in order to maintain necessary vigilance. 20 hatchlings will be sampled from each nest. Non-destructive blood samples will be taken from the dorsal cervical sinus according to Owens & Ruiz, (1980, as cited in Hays & Lee, 2004) and stored in buffered solution (50 mm EDTA/2% SDS/10 mm NaCl/50 mm TrisHCl at ph 8) at room temperature (Hays & Lee, 2004). All hatchlings will be released immediately after counting and sampling. Nests will be excavated 48 hours after the last hatchling emerges (Broderick, et al., 2003) 1. Dead hatchlings and embryos will be counted, biopsied and preserved in absolute ethanol (Arlettaz, et al., 2007, Hays & Lee, 2004). Females will be grouped according to curved carapace length and clutches will be analyzed for multiple paternity, number of eggs and hatching success. Paternity analysis will be carried out according to Ball & Moore (2002) at the Center for Marine Science at the University of North Carolina, Wilmington. DNA will be isolated from biopsies and blood samples using the standard phenolchloroform extraction protocol and ethanol precipitation. A
microsatellite-enriched DNA library will be established and primers will be designed using PRIMER3 (Rozen & Skaletsky, 1998, as cited in Ball & Moore, 2002) and the Operon oligo toolkit webpage (http://www.operon.com/toolkit). Primers will then be synthesized by Operon Technologies, Inc. Samples will be amplified through polymerase chain reaction and run on 6% acrylamide gels. The DNA from each mother will be run alongside the DNA from her offspring. It will be assumed that each new allele that appears indicates a different father. If this occurs, DNA will be re-amplified and re-run for confirmation. Because females will be marked, the prevalence of multiple paternity and number of eggs could be compared among clutches from females in earlier and later seasons. It is assumed that females, because of sea turtles indeterminate growth, will be significantly larger in subsequent nesting seasons. This assumption must be tested in order for this comparison to be valid. Females will also be compared in order to determine whether a better nesting site for producing males will be chosen if their clutch is primarily sired by a preferred male. Preferred males will be identified as those fathering the most offspring with the most females. 5. Permits and Regulations The North Carolina Wildlife Resources Commission (NCWRC) has established the Sea Turtle Protection Program through a Cooperative Agreement with United States Fish and Wildlife Service under Section 6 of the Endangered Species Act. All research will be conducted according to the regulations set out by this program. A permit will be required from NCWRC s Nongame and Endangered Wildlife Program as well as from the North Carolina Department of Marine Fisheries. A permit must also be obtained from United States Fish and Wildlife Services. Permission and cooperation will also be required from the Bald Head Island Conservancy. Procedures will also comply with regulations of the Coastal Area Management Act and Endangered Species Act. 6. Results It is predicted that one-way analysis of variance will indicate a significant difference between groups of females based on curved carapace length. Figure 1 provides an example of the potential number of females falling into each size category during the first nesting season. Working with data obtained from females in the same size category, it is predicted that the number of eggs per clutch will increase significantly with the number of sires. By controlling for female size, the correlation between clutch size and multiple paternity can be more clearly established. Figure 2 demonstrates the estimated linear relationship between the number of eggs per clutch and the minimum number of sires in nests of females within the 81-85 cm curved carapace length category. The minimum number of sires will be used as the operational variable for multiple paternity, as the absolute number cannot be determined due to sample size and limitations of the microsatellite library (Arlettaz, et al., 2007). A two-tailed t-test will be used to verify whether the relationship between these variables is significant. It is expected that only a weak positive correlation will be found between hatching success and the minimum number of sires. This data will be statistically analyzed by using a two-tailed t-test the correlation between these two variables may not be significant. Figures 3 and 4 display predicted data points. Figure 3 represents the relationship between hatching success and minimum number of sires of one clutch per female in the first nesting season. Figure 4 shows the same variables but because females can lay more than one nest per season, the data is pooled; all data from nests of each female are pooled and averaged for the first nesting season. The turtles at Bald Head Beach will be studied for a period of eight years ensuring that the minimum number of sires of each female s clutches can be compared between seasons. 5
Figure 5 provides an example of predicted results from 20 females. It is expected that most females will, on average, lay clutches with a higher minimum number of sires in later seasons relative to those laid in earlier seasons. Analysis of variance will be used to determine whether there is a significant positive increase in the mean number of sires between seasons. Results should also indicate that females laying clutches fathered primarily by a preferred male will choose sites with more ground cover (vegetation, dunes, trees, etc.) and will bury their eggs deeper in the substrate. Location descriptions will be recorded using a likert scale. Figure 6 is taken from Arlettaz, et al. (2007) to illustrate potential results. This graph displays proportions of clutches fathered by primary, secondary, tertiary and quaternary males. This, as well as paternity analysis, will be used to determine which males father the most offspring with the most females. A two-tailed t-test will be used to determine the significance of the relationship between nest location and paternity. 7. Discussion The degree of multiple paternity should be positively correlated with the probability of mate encounters (Olsson & Uller, 2008). Due to highly female biased sex ratios resulting from global warming, aggregations and competition for females may be reduced (Ball & Moore, 2002) leading to lower frequencies of multiple mating. If polyandry can be directly linked to the number of eggs per clutch, and turtles always want to maximize clutch size (Hays & Speakman, 1991), studying this mating strategy would be incredibly important to the future success of loggerhead sea turtles. This research will provide insight into male mate choice. By establishing whether females lay clutches fathered by more males in their second nesting season, will help determine factors influencing male preference. If males are found to prefer larger, older females, further research should focus on whether smaller, younger females will experience less reproductive success as the sex ratio becomes more female biased. Information obtained from analysis of female nesting site in relation to paternity will be valuable for conservation efforts. No previous study has investigated whether females choose shadier, deeper nesting sites if their clutch is primary fathered by a preferred male. These results would provide greater depth of understanding of this species as well as valuable insight into possible focuses for beach improvement and conservation. Most importantly, this research study will provide a wide range of valuable data accumulated over an eight-year period. It will serve as a departure from many other studies results not being hindered by small sample sizes and ambiguous DNA markers (Ball & Moore, 2002). All data would contribute greatly to the development of conservation and protection programs for loggerhead sea turtles around the world. Future research should concentrate on whether smaller females make up for their size by having shorter inter-nesting periods. Small females may have smaller clutches but nest more frequently within season and take smaller breaks between seasons (Broderick, et al., 2003). It is also suggested that polyandry occurs because female turtles are making the best of a bad job. Male harassment can be intense and perhaps larger, preferred females succumb to more males, therefore yielding clutches with higher levels of multiple paternity. More research is necessary to determine whether female choice should be excluded from factors affecting polyandry (Hays & Lee, 2004). It also remains unclear whether males are philopatric. If females continue producing fewer males, decreasing numbers will return to their natal beach (assuming they are philopatric) contributing to major population declines. 6
8. References 7 Arlettaz, R., Largiader, C.R, Leippert, F., Margaritoulis, D., Zbinden, J.A. (2007). High frequency of multiple paternity in the largest rookery of Mediterranean loggerhead sea turtle. Molecular Ecology 16: 3703-3711. Avise, J.C., Janzen, F.J. & Pearse, D.E. (2002). Multiple paternity, sperm storage, and reproductive success of females and male painted turtles (Chrusemys picta) in nature. Behav. Ecol. Sociobiol. 51: 164-171. Avise, J.C. & Pearse, D.E. (2001). Turtle mating systems: behaviour, sperm, storage, and genetic paternity. The Journal of Heredity 92(2): 206-211. Ball, R.M. Jr. & Moore, M.K. (2002). Multiple paternity in loggerhead turtle (Caretta caretta) nests on Melbourne Beach, Florida: a microsatellite analysis. Molecular Ecology 11: 281-288. Broderick, A.C., Glen, F., Godley, B.J., Hays, G.C. (2003). Variation in reproductive output of marine turtles. Journal of Experimental Marine Biology and Ecology 288: 95-109. 1 Broderick, A.C., Glen, F., Godley, B.J. & Hays, G.C. (2003). Incubation environment affects phenotype of naturally incubated green turtle hatchlings. J. Mar. Biol. Ass. U.K. 83: 1183-1186. Broderick, A.C., Godfrey, M.H., Godley, B.J., Hawkes, L.A. (2007). Investigating the potential impact of climate change on marine turtle population. Global Change Biology 13: 923-932. Broderick, A.C., Godfrey, M.H., Godley, B.J., Hawkes, L.A. (2005). Status of nesting loggerhead turtles Caretta caretta at Bald Head Island (North Carolina, USA) after 24 years of intensive monitoring and conservation. Oryx 39(1): 65-72. Caswell, H., Crouse, D.T., Crowder, L.B. (1987). A stage-based population model for loggerhead sea turtles and implications for conservation. Ecology 68(5): 1412-1423. Davenport, J. (1997). Temperature and the life-history strategies of sea turtles. J. therm. Biol. 22(6): 479-488. Frazer, N.B. & Richardson, J.I. (1985). Annual variation in clutch size and frequency for loggerhead turtles, Caretta caretta, nesting at Little Cumberland Island, Georgia, USA. Herpetologica 41(3): 246-251. Intergovernmental Panel on Climate Change. (2001). Climate change 2001: the scientific basis. New York: Cambridge University Press. Hall, K.R., Pounds, J.A., Price, J.T., Root, T.L., Rosenzweig, C., Schnelder, S.H. (2003). Fingerprints of global warming on wild animals and plants. Nature 42: 57-60. Hays, G.C. & Lee, P.L.M. (2004). Polyandry in a marine turtle: Females make the best of a bad job. Proceedings of the National Academy of Sciences of the United States of America 101(17): 6530-6535. Hays, G.C. & Speakman, J.R. (1991). Reproductive investment and optimum clutch size of loggerhead sea turtles (Caretta caratta). Journal of Animal Ecology 60: 455-462.
Heppell, S. & Lutcavage, M. (2008). The Pacific-Atlantic Sea Turtle Assessment Project (PASTA): Bringing disciplines together to evaluate the causes of sea turtle decline. Pelagic Fisheries Research Program 13(1): 1-8. 8 Huk, T & Winkel, W. (2006). Polygyny and its fitness consequences for primary and secondary female pied flycatchers. Proceedings for the Royal Society B-Biological Sciences 273(1594): 1681-1688. Janzen, F.J. (1994). Climate change and temperature-dependent sex determination in reptiles. Proc. Natl. Acad. Sci. USA: 91: 7487-7490. Mrosovsky, N. & Provancha, J. (1992). Sex ratio of hatchling loggerhead sea turtles: data and estimates from a 5-year study. Can. J. Zool. 70: 530-538. Mrosovsky, N. & Yntema, C.L. (1981). Critical periods and pivotal temperatures for sexual differentiation in loggerhead sea turtles. Can. J. Zool. 60: 1012-1016. Olsson, M. & Uller, T. (2008). Multiple paternity in reptiles: patterns and processes. Molecular Ecology 17: 2566-2580. Robertson, RJ & Weatherhead, PJ. (1979). Offspring quality and the polygyny threshold: the sexy son hypothesis. The American Naturalist 113(2): 201-208.
9. Appendix A 9 Figure 1. An example of the number of females expected to fall within each category of curved carapace length (CCL) (cm) for the first season. 200 Females with CCL of 81-85cm 180 160 140 120 100 80 60 40 20 0 0 1 2 3 4 5 6 Minimum number of sires Figure 2. The predicted relationship between clutch size (number of eggs per clutch) and minimum number of sires. 100% Graph 3: (one clutch/female) Hatching 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 1 2 3 4 5 6 Minimum number of Sires Figure 3. The expected weak positive correlation between hatching success (%) and minimum number of sires of one nest per female in season one.
10 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 1 2 3 4 5 6 Minimum number of Sires Figure 4. The expected weak positive correlation between hatching success (%) and minimum number of sires for pooled data in season one. Figure 5. The predicted difference in minimum number of sires between seasons for 20 females clutches. Figure 6. An example of potential data presentation for the proportions of each clutch being fathered by particular males (Arlettaz, et al., 2007).