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Many studies of sex ratio focus on the mechanism of TSD (reviewed by Wibbels 2003) or extrapolate sex ratios from various proxies (reviewed by Girondot 2010; Wyneken & Lolavar 2015) in order to understand the demographic effects that climate change will have on turtles. Sex ratios are a common method used to gauge the population’s health and plan conservation efforts. Yet, identifying sex in live turtles, particularly hatchlings, is challenging. Hatchlings and young turtles are not externally dimorphic until near puberty. In wild Atlantic loggerheads, maturation occurs at about 30 years (Frazer & Ehrhart 1985; TEWG 2009). Since marine turtles mature late in life, verifying sex in very young animals is challenging, due to a general lack of external secondary sex characteristics. The most common procedures to identify sex include histological gonadal examination of dead or sacrificed hatchlings, both of which are often limited by regulations (Yntema & Mrosovsky 1980; Mrosovsky & Provancha 1992), hatchling quality (LeBlanc et al. 2012), or ethical considerations. Laparoscopic surgery requires logistics support and special training for maintenance of hatchlings in the laboratory for months until they grow large enough (Wyneken et al. 2007). Radioimmunoassay techniques are effective only for later life stages and may only be seasonally reliable (Braun-McNeill et al. 2007). Consequently, alternative ways of non-destructively identifying hatchling sex would be a valuable tool for assessing hatchling sex ratios at nesting beaches, and for verifying sex ratios from proxies such as those extrapolated from beach sand and/or nest temperatures.

Geometric morphometrics of several characteristics of the shell, limbs and tail have been used in several previous studies of turtles to investigate if the sex of juveniles can be identified in freshwater species, including painted turtles and Arrau river turtles (Chrysemys picta, Podocnemis expansa, respectively; Valenzuela et al. 2004; Ceballos et al. 2014), common snapping turtles (Chelydra serpentina; Ceballos & Valenzuela 2011), and green turtle (Chelonia mydas; Sӧnmez et al. 2016) and (geoemydid species, Baruah & Sharma 2016). Using shell size, limb size, or shape metrics such as scute intersection landmarks to generate a morphometric grid of shell shape, these studies found that in some species sex could be identified with up to 98% accuracy as verified by histology or laparoscopy (Valenzuela et al. 2004), but required that hatchlings be raised in the laboratory for several months before measurements made were useful.

A reliable field method is needed for the time when hatchlings emerge from the nest. Geometric morphometric methods can be used to distinguish males from females in mature and maturing turtles. Do these methods also work for hatchling turtles? In this study, we applied geometric morphometric analysis to loggerhead turtle hatchling carapace features because they are accessible traits and, if shell shape differs in hatchlings, may provide an alternative way of identifying sex. 

We collected data from the photo archive of identification pictures taken during the 2014 nesting season to test if this noninvasive method would work. As part of a long-term primary sex ratio study, typically, 10 hatchlings were collected from each of 11 loggerhead turtle clutches on Boca Raton (Florida, USA) beaches. Digital pictures were taken of the hatchling carapaces on the day of collection. Individual turtles were identified using the scutes and scales in the photos and then children’s nail polish was used to mark the carapace scutes with a unique ID number of dots (Fig. 1), to ensure identification of the turtles throughout their time in the lab. Large flow-through tanks housed the hatchlings with each turtle in its own, labeled flow-through basket. Cleanings of turtles and baskets occurred weekly. Turtles were fed based on mass and spent approximately three to four months in the lab before they were ready for laparoscopy (Stokes et al. 2006). The sexes were verified by laparoscopic examination of the gonads and accessory duct characteristics, conducted when the turtles reached 120 g. (Wyneken et al. 2007). A subsample of turtles were biopsied to verify accuracy of 100%. 

For this study, we used a subset of 40 hatchlings (20 males and 20 females) from the photo archives for 2014. The selection was based on clarity of the photo, the camera’s view being centered on the dorsal carapace, and completeness of view of the carapace. Digital pictures that were taken from directly overhead were used in this study to assign 30 carapacial scute intersection landmark (based on Ferreira-Júnior et al. 2011; Fig. 1) using the tps software series (tpsUtil Rohlf V1.69 2016; tpsDIG2w32 Rohlf V2.27 2016 <http://life.bio.sunysb.edu/morph/>). Plastron pictures were excluded from analysis as many hatchlings, at this stage, still had yolk sac scars making the plastral views too variable for consideration. Hindlimb and tail or cloacal characters were not available in all pictures and so were not used. The software tpsRelw32 (Rohlf V1.62 2016 <http://life.bio.sunysb.edu/morph/>) aligns the specimens and computes partial and relative warp thereby accounting for variation in hatchling size, location, and orientation so shape variables can be generated by superimposition. Shapes of each sex were modeled and visualized using thin-plate spline (Rohlf) deformation grids from the overall average shape of all specimens to the average shape of each sex. Rohlf’s software compares pairs of specimens or groups by displaying a transformation grid, which is a representation of the weighted sums of landmark shifts from the two-dimensional points assigned to each scute intersection landmark to a rotated, deformed three-dimensional grid. Procrustes ANOVA for shape data was performed in R using the geomorph package (Adams et al. 2015; geomorph R package V 2.1.x.; <http://cran.r-project.org/web/packages/geomorph/index.html>). Procrustes ANOVA uses sum of squared Procrustes distance between landmarks as a measure of sum of squares that is evaluated using permutation. Thus, it assesses relative amounts of variation among groups. Significance level was α = 0.05. Significant results for the Procrustes ANOVA analysis would indicate dimorphism between male and female hatchlings.

Figure 1. C. caretta hatchling (1 day old) photo with numbered landmarks at scute intersections. Landmarks were identified and marked using tpsUtil and tpsDIG (Rohlf V1.69 2016; Rohlf V2.27 2016).

Figure 2. Deformation grids. Left: Composite deformation grid of 20 C. caretta females. Right: Composite deformation grid of 20 C. caretta males.

Visually, the deformation grid of females seems to show more variation overall, and specifically, within the region of the supracaudal notch (Fig. 2). However, a Procrustes ANOVA analysis, F(1,38) = 1.02, p = 0.405, failed to find significant differences between the two sexes on the day of emergence. While the deformation grid (tpssplin software Rohlf V1.43 2016) seems to suggest a small degree of sexual dimorphism, at this stage it is not consistently present. 

A study of green turtle, Chelonia mydas, hatchlings appeared to show sexual dimorphism using morphometric measurements (Sӧnmez et al. 2016). Significant measurements included curved carapace width, hindlimb length, and plastron-cloaca length, but no scute intersection landmarks were used as in this study. Further, although the green turtle hatchling study found statistical differences between male and female hatchlings for these parameters, overlap in the range of these measurements was almost complete. Consequently, it is likely that very high error would occur in using such measurements for determining hatchling sex. In another study of the freshwater Arrau turtle (Podocnemis expansa), 7-day old hatchlings were not sexually dimorphic using geometric morphometrics (Ceballos et al. 2014). However, by 25 months of age the supracaudal notch measures provided a reliable sexually dimorphic characteristic. Valenzuela et al. (2004) found high accuracy using the geometric morphometric approach compared to verified sex in P. expansa (85 - 90%) and painted turtles, C. picta (98%) (2 weeks and 2 months old, respectively). Hatchling loggerheads (the present study) showed no significant sexual dimorphism in carapace characteristics. Our study population was limited to hatchlings. It was beyond the scope of our study to test this method in other sizes or life stage classes. Maintenance of turtles in the lab for many years would be necessary for turtles to reach the necessary age where sexual dimorphism could be detected grossly. Although scute patterns (and the landmarks they provide) are highly conserved as diagnostic characters, they may be too labile to reveal a sex-specific signal. In hatchlings, scutes, as well as the underlying skeleton, undergo rapid change during very high growth rates in their first few weeks of life (Stokes et al. 2006).

Acknowledgements. We appreciated constructive discussions with V. Erb, J. Lasala, A. Lolavar, and B. Tezak. This manuscript was improved by comments from two anonymous referees. We thank the Gumbo Limbo Nature Center’s sea turtle specialists and the FAU turtle lab independent study students who helped collect turtles and take photos. This studies’ photographic records were collected as part of a long-term study conducted under permit MTP-073 to JW.

BARUAH, C., P. DEVI & D.K. SHARMA. 2006. Comparative morphometry and biogeography of the freshwater turtles of genus Pangshura (Testudines: Geoemydidae: Pangshura). International Journal of Pure and Applied Zoology 4: 107-123.

BRAUN-MCNEILL, J., S.P. EPPERLY, D.W. OWENS, L. AVENS, E. WILLIAMS & C.A. HARMS. 2007. Seasonal reliability of testosterone radioimmunoassay (RIA) for predicting sex ratios of juvenile loggerhead (Caretta caretta) turtles. Herpetologica 63: 275-284.

CEBALLOS, C.P., O.E. HERNÁNDEZ & N. VALENZUELA. 2014. Divergent sex-specific plasticity in long-lived vertebrates with contrasting sexual dimorphism. Evolutionary Biology 41: 81-98.

CEBALLOS, C.P. & N. VALENZUELA. 2011. The role of sex-specific plasticity in shaping sexual dimorphism in a long-lived vertebrate, the snapping turtle Chelydra serpentina. Evolutionary Biology 38: 163-181.

CREWS, D. 2003. Sex determination: where environment and genetics meet. Evolution & Development 5: 50-55.

FERREIRA- JÚNIOR, P.D., R.L. TRIECHEL, T.L. SCARAMUSSA & J.T. SCALFONI. 2011. Morphometric pattern in Caretta caretta (Linnaeus, 1758) (Cheloniidae) hatchlings from nests with different embryo development rates. Brazilian Journal of Biology 71: 151-156. 

FRAZER, N.B. & L.M. EHRHART. 1985. Preliminary growth models for green, Chelonia mydas, and loggerhead, Caretta caretta, turtles in the wild. Copeia 1985: 73-79.

FUENTES, M.M.P.B., M. HAMANN & C.J. LIMPUS. 2010. Past, current and future thermal profiles of green turtle nesting grounds: implications from climate change. Journal of Experimental Marine Biology and Ecology 383: 56-64.

FUENTES, M.M.P.B., C.J. LIMPUS, & M. HAMANN. 2011. Vulnerability of sea turtle nesting grounds to climate change. Global Change Biology 17: 140-153.

GIRONDOT, M., S.B. HASSINE, C. SELLOS, M.H. GODFREY & J.M. GUILLON. 2010. Modeling thermal influence on animal growth and sex determination in reptiles: being closer to the target gives new views. Sexual Development 4: 29-38.

HAWKES, L.A., A.C. BRODERICK, M.H. GODFREY & B.J. GODLEY. 2007. Investigating the potential impacts of climate change on a marine turtle population. Global Change Biology 13: 923-923.

HAYS, G.C., A.C. BRODERICK, F. GLEN & B.J. GODLEY. 2003. Climate change and sea turtles: a 150-year reconstruction of incubation temperatures at a major marine turtle rookery. Global Change Biology 9: 642-646.

JANZEN, F.J. 1994. Climate change and temperature-dependent sex determination in reptiles. Proceedings of the National Academy of Sciences 91: 7487-7490.

JANZEN, F.J. & G.L. PAUKSTIS. 1991. Environmental sex determination in reptiles: ecology, evolution, and experimental design. Quarterly Review of Biology 66: 149-179.

JANZEN, F.J. & P.C. PHILLIPS. 2006. Exploring the evolution of environmental sex determination, especially in reptiles. Journal of Evolutionary Biology 19: 1775-1784.

LEBLANC, A.M., K.K. DRAKE, K.L. WILLIAMS, M.G. FRICK, T. WIBBELS & D.C. ROSTAL. 2012. Nest temperatures and hatchling sex ratios from loggerhead turtle nests incubated under natural field conditions in Georgia, United States. Chelonian Conservation & Biology 11: 108-116.

LEWISON, R.L., S.A. FREEMAN & L.B. CROWDER. 2004. Quantifying the effects of fisheries on threatened species: the impact of pelagic longlines on loggerhead and leatherback sea turtles. Ecology Letters 7: 221-231.

MAZARIS, A.D., G. MATSINOS & J.D. PANTIS. 2009. Evaluating the impacts of coastal squeeze on sea turtle nesting. Ocean & Coastal Management 52: 139-145.

MITCHELL, N.J. & F.J. JANZEN. 2010. Temperature-dependent sex determination and contemporary climate change. Sexual Development 4: 129-140.

MROSOVSKY, N. & J. PROVANCHA. 1992. Sex ratio of hatchling loggerhead sea turtles: data and estimates from a 5-year study. Canadian Journal of Zoology 70: 330-338.

ORÓS, J., A. TORRENT, P. CALABUIG & S. DÉNIZ. 2005. Diseases and causes of mortality among sea turtles stranded in the Canary Islands, Spain (1998-2001). Diseases of Aquatic Organisms 63: 13-24.

PIEAU, C., M. DORIZZI & N. RICHARD-MERCIER. 1999. Temperature-dependent sex determination and gonadal differentiation in reptiles. Cellular and Molecular Life Sciences 55: 887-900.

ROHLF, F.J. (2016). tpsRelw32 Version 1.62 <http://life.bio.sunysb.edu/morph/> [Computer software]. 

ROHLF, F.J. (2016). tpsSplin Version 1.22 [Computer software]. 

SӦNMEZ, B., C. TURAN, Ş. ӦZDILEK & F. TURAN. 2016. Sex determination of green sea turtle (Chelonia mydas) hatchlings on the basis of morphological characters. Journal of Black Sea/ Mediterranean Environment 22: 93-102.

STOKES, L., J. WYNEKEN, L.B. CROWDER & J. MARSH. 2006. The influence of temporal and spatial origin on size and early growth rates in captive Loggerhead sea turtles (Caretta caretta) in the United States. Herpetological Conservation and Biology 1: 71-80.

TEWG (Turtle Expert Working Group). 2009. An assessment of the loggerhead turtle population in the western northern Atlantic Ocean. NOAA Tech Memo NMFS-SEFSC-575, Miami, Florida. 131p.

VALENZUELA, N., D.C. ADAMS, R.M. BOWDEN & A.C. GAUGER. 2004. Geometric morphometric sex estimation for hatchling turtles: a powerful alternative for detecting subtle sexual shape dimorphism. Copeia 2004: 735-742.

WIBBELS, T. 2003. Critical approaches to sex determination in sea turtles. In: Lutz, P.L., J.A. Musick & J. Wyneken (Eds.) The Biology of Sea Turtles. Volume 2. CRC Press, Inc. Boca Raton, Florida. pp. 103-134.

WIBBELS, T., J.J. BULL & D. CREWS. 1991. Chronology and morphology of temperature-dependent sex determination. The Journal of Experimental Zoology 260: 371-381.

WITT, M.J., L.A. HAWKES, M.H. GODFREY, B.J. GODLEY & A.C. BRODERICK. 2010. Predicting the impacts of climate change on a globally distributed species: the case of the loggerhead turtle. The Journal of Experimental Biology 213: 901-911.

WYNEKEN, J., S.P. EPPERLY, L.B. CROWDER, J. VAUGHAN & K.B. ESPER. 2007. Determining sex in posthatchling loggerhead sea turtles using multiple gonadal and accessory duct characteristics. Herpetologica 63: 19-30.

YNTEMA, C.L. & N. MROSOVSKY. 1980. Sexual differentiation in hatchling loggerheads (Caretta caretta) incubated at different controlled temperatures. Herpetologica 36: 33-36.