- Email: [email protected]
Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell
Molecular and Cellular Probes 29 (2015) 254e257
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Short communication
Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell C.M. Farke a, 1, K. Olek b, W.M. Gerding c, C. Distler a, * €t Bochum, Universita €tsstr. 150, ND 7/27, 44780 Bochum, Germany Allgemeine Zoologie und Neurobiologie, Ruhr-Universita Labor für Abstammungsbegutachtungen GMBH, Marie-Curie-Str. 1, 53359 Rheinbach, Germany c €t Bochum, Universita €tsstr. 150, MA 5/39, 44780 Bochum, Germany Humangenetik, Ruhr-Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 April 2015 Received in revised form 11 May 2015 Accepted 11 May 2015 Available online 21 May 2015
We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability. © 2015 Published by Elsevier Ltd.
Keywords: Multiple paternity Sperm storage Tortoise Eggshell Testudo hermanni boettgeri Conservation
Promiscuous mating is widespread especially in vertebrates without strong social bonds, but extra-pair mating also occurs in so-called monogamous species (e.g. sharks [e.g. Refs. [1,2]], reptiles [3,4], tits [e.g. Refs. [5,6]], hedgehogs [7], man [8]). The females of polyandrous species are equipped with oviductal tubules [9] where viable sperm can be stored at least over short periods of time. As a consequence, siblings within one clutch can be sired by different males. Among chelonians, who do not form complex social structures nor do they provide parental care, multiple paternity was found in all marine turtle species except the leatherback turtle [10,11], and in all freshwater turtles tested [12e20]. In tortoises, multiple paternity occurs in the desert tortoise (Gopherus agassizii [21,22]), the Mediterranean spur-thighed tortoise (Testudo graeca [23]), the central Asian tortoise (Testudo horsfieldii [24]), and Hermann's tortoise (Testudo hermanni hermanni [25]).
* Corresponding author. E-mail addresses: [email protected] (C.M. Farke), [email protected] (K. Olek), [email protected] (W.M. Gerding), distler@ neurobiologie.rub.de (C. Distler). 1 €tenhilfe TestuDO, Rübenkamp 91, 44319 Dortmund, Present address: Schildkro Germany. http://dx.doi.org/10.1016/j.mcp.2015.05.009 0890-8508/© 2015 Published by Elsevier Ltd.
The parentage of 83 T. hermanni boettgeri from two private captive breeding colonies (group F and H, 8 adult females, 10 adult males) was analyzed during the breeding seasons 2010/2011 (see supplementary information S1, supplementary Table ST1) using a group of specific polymorphic microsatellites developed for chelonians. We successfully isolated DNA from amniotic fluid adhering to the inner surface of the eggshell of 62 live hatchlings and from 5 animals that had died during incubation thereby avoiding the difficult and potentially stressful sampling of saliva or blood in very young animals. This approach was first used in birds and alligators [26,27]. In adult potential parents (n ¼ 18; H01eH04, H7eH10, F01eF04, F07, F72, F101, F135, F201, F201a) and semi-adult animals (n ¼ 6; F102, F103, F104, F119, F120, F125) from earlier clutches, DNA was isolated from their blood, sampled from the dorsal coccygeal or femoral vein, and dried on filter paper. In addition, tissue samples of frozen and ethanol fixed animals from earlier clutches (n ¼ 10; F108, F109, F117, F118, F123, F126, F128, F129, F131, F132) were collected, and DNA was isolated. All samples were stored at 20 C until analysis. DNA was isolated and purified using the tissue or blood protocol variant of the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Twelve gene loci, that were characterized by their highly
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257
polymorphic length variation, were amplified using polymerase chain reaction (PCR). Amplification products were resolved on an ABI3730xl, and sized and scored with the GeneMapper® software. Statistical analysis was performed with CERVUS 3.0 ([28]; supplementary information S4, supplementary Tables ST5, ST6). The following polymorphic microsatellite markers developed for tortoises were used in our genotyping experiments: Test10, Test71, Test88 [29], TW161, TWR106 [30], RAD284 [31], and SK60 that was developed for T. hermanni and Testudo kleinmanni in our laboratory (supplementary information S2, supplementary Table ST2). Multiplex PCR was performed under the following conditions: initial denaturation at 94 C for 10 min, followed by 30 cycles at 94 C for 1 min, at 58 C for 1 min, at 72 C for 1 min and at 72 C for 45 min, and a break at 15 C. The final reaction volume was 20 ml: 2 ml DNA, 2.5 ml MgCl2 (25 mM), 0.2 ml Promega GoTaq (5u/ml), 3.2 ml dNTP mix (1.25 mM), 1 ml forward and 1 ml reverse primer (10 mM) for each marker, 4 ml Colorless GoTaq Flexi Buffer (Qiagen), filled with distilled water to 20 ml. We applied 3 multiplex reactions: MP1: Test10, Test71, TW161; MP2: Test88, TWR 106, RAD 284; MP 3: SK 60. Although the annealing temperature used in the multiplex reaction is a compromise and may not be optimal for the individual PCR amplification reactions, PCR products were of different sizes and could be robustly amplified. One exception was marker Test88, which was replaced by marker TW161 in further analyzes (supplementary Tables ST5, ST6). With this methodological approach we were able to analyze multiple microsatellite markers despite the limited quantity of DNA. In order to obtain additional information from monomorphic markers, PCR products were amplified and analyzed by direct DNA sequencing in three parent turtles and eight of their offspring (supplementary information S3, supplementary Table ST3, ST4) that were homozygous for one or several markers. This analysis should reveal potential single nucleotide polymorphism (SNP) within the STR-repeat or between the STR-repeat and the primer loci that could in turn provide additional information about paternity, if polymorphic. Of course, these analyses are only valuable for markers where both alleles can be amplified and are not subject of allele drop-out. Employing seven informative microsatellite markers (Test10, Test71, Test88, TWR106, RAD284, SK60, TW161) we could unequivocally determine the fathers of all juveniles at an 80% or 95% confidence level. The mothers' identity determined by DNA analysis, unequivocally confirmed our observations of the animals during oviposition. In some cases, several markers were monomorphic, e.g. only one allele could be identified. This can be caused by allele drop out, but can also be due to a problem of the STRanalysis, which is sometimes encountered in exotic species or wild animals where markers are used that were originally developed for another subspecies or for a related species. In these cases the markers are often not as informative as for the original species. Furthermore, when working with degraded material it is not always clear whether the marker is really homozygous or whether a second allele cannot be amplified. Thus, for two markers (TW161 and Test71), we analyzed the flanking regions of the microsatellite for polymorphic SNP. This additional genetic variability potentially increases the informative value of the locus. As an example we show the sequencing results of the TW161 locus for two families ((H01/H07, parents; H69, H71, H73, H76, juveniles); (H01/H10, parents; H70, H72, H74, H68, juveniles)) (supplementary information S3, supplementary Table ST3, ST4). In individuals with previously identified monomorphic microsatellite markers, we could identify two polymorphic SNPs confirming the corresponding STR-based parentage. Multiple paternity was found in two of six clutches from colony F, and in seven of nine clutches from colony H. The offspring in eight
255
of these clutches was fathered by two different males, the offspring of the remaining clutch was derived from three different males. Thus, in 52.9% of the clutches analyzed offspring was fathered by more than one male. We identified three cases of sperm storage (F121, F218eF221, H28) where the fathers determined by DNA analysis were not part of the breeding groups during the season we analyzed indicating that mating had occurred during the previous season and that sperm had been stored. In the wild, tortoises are promiscuous [3,32,33]. Generally, multiple mating can have various advantages for the female ranging from decreasing harassment by aggressive males (“convenience polyandry” [2]) to direct benefits such as nuptial gifts and help during raising of the young [3,4]. In chelonians, however, indirect benefits such as “trading up” the quality of the sperm, promoting sperm competition, cryptic female choice, and “bethedging” i.e. the production of diverse geno- and phenotypes within clutches to be better equipped for environmental changes [4] seem to be more likely but these factors are mostly untested. All these benefits are invariably linked to the ability to store sperm, either within a season, e.g. mating in spring and inseminating consecutive clutches during the summer, or across seasons when sperm is stored for up to 3e4 years [3,4,34]. The mating and courtship behavior of T. hermanni has been described in detail [e.g. Refs. [25,32e38]]. In natural habitats, population density of T. hermanni can be rather low (from <3 individuals/ha [39], to ~40 individuals/ha [35,36,40]), and mating occurs during the rare encounters. Olfactory, visual, tactile, and auditory signals are important for species and mate recognition and provide honest cues for mate quality [37,41]. Thus they represent important signals for female strategies as cryptic female choice or bet-hedging [4]. Our animals produced 2-3 clutches per season, which is similar to the situation found in the wild [25,32,42]. In both colonies, the ability for sperm storage was confirmed. In colony F, multiple paternity was shown in 33%, in colony H in 78% of the clutches. Fertilization success was 85% in colony F and 95% in colony H, and thus much higher than the <60% described for permanently cohoused T. hermanni by Jasser-H€ ager and Winter [43]. Our sample is too small to deduce influences of the breeding scheme on the prevalence of multiple paternity or fertilization success. Taken together, multiple paternity in T. hermanni boettgeri was identified in 52.9% of the clutches analyzed. This rate is higher than described for T. graeca (20% [23]) and T. horsfieldii (27% [24]), and comparable to Gopherus (50e57% [21,22]) kept in captivity, or in a semi-natural environment. In a planned mating study Cutuli et al. [25] found a slightly lower rate of multiple paternity in a captive colony of T. hermanni hermanni. Multiple paternity ranges from 10% in Emys orbicularis [16] and some populations of Podocnemis expansa [15] up to 50e100% in Chelonia midas [44,45], Podocnemis expansa [13], and Podocnemis unifilis [17] with most rates lying between 20% and 80% (Lepidochelys olivacea 20% [46], Caretta 31e33% [47,48], Chrysemys picta 33% [14], Lepidochelys kempi 58% [49], Emydoidea blandingii 60e80% [18], Chelydra serpentina 66% [12], Podocnemis erythrocephala 80% [19], Elseya albagula 83% [20]). Thus, the incidence of multiple paternity in Hermann's tortoise seems similar to other tortoises and turtles even though direct comparisons are difficult due to different sample sizes and living conditions. In conclusion, the data described here extend our knowledge about multiple paternity and sperm storage to a further tortoise taxon, T. hermanni boettgeri indicating that these traits are widespread in chelonians in general. To the best of our knowledge, this is the first study in chelonians to employ the non-invasive method of collecting DNA samples from amniotic fluid adhering to the inner surface of the eggshell. The supplementary SNP analysis proved to
256
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257
be a useful method when working with wild animals and with low DNA-quantity material and can provide additional information for otherwise non-informative markers. This approach is applicable for research purposes and conservation breeding programs, i.e. if owners would store the eggshells together with genetic material of the putative parents, paternity of animals in the pet trade could be easily tracked thereby largely limiting the trade of illegally imported specimens.
[12]
[13] [14]
[15]
Funding source This study was supported by departmental funds of the faculty of Biology and Biotechnology, Ruhr-University Bochum. Author contributions CD, CMF and KO designed the experiments, KO performed the experiments, KO, CMF and WMG analyzed the data, CD, KO, WMG and CMF wrote the manuscript. Conflict of interest The authors declare no conflict of interest. All authors approve of the final article.
[16]
[17]
[18] [19]
[20]
[21] [22]
Acknowledgments We thank all owners for providing their tortoises and eggs, M. Wiegand for collecting most of the blood samples of the adult and semiadult animals, K. Krabbe for help with the PCR analysis, D.A. Akkad for help with data analysis, and the anonymous reviewers for their constructive criticism on earlier versions of the manuscript. This study was supported by departmental funds of the Faculty of Biology and Biotechnology, Ruhr-University Bochum.
[23]
[24]
[25]
[26]
Appendix A. Supplementary data
[27]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mcp.2015.05.009.
[28]
References [1] T.S. Daly-Engel, R.D. Grubbs, K.A. Feldheim, B.W. Bowen, R.J. Toonen, Is multiple mating beneficial or unavoidable? Low multiple paternity and genetic diversity in the shortspine spurdog Squalus mitsukurii, Mar. Ecol. Prog. Ser. 403 (2010) 255e267. [2] A.M. Griffiths, D.M.P. Jacoby, D. Casane, M. McHugh, D.P. Croft, M.J. Genner, et al., First analysis of multiple paternity in an oviparous shark, the small-spotted catshark (Scyliorhinus canicula L.), J. Hered. 103 (2012) 166e173. [3] D.E. Pearse, J.C. Avise, Turtle mating systems: behaviour, sperm storage, and genetic paternity, Am. Gen. Assoc. 92 (2001) 206e211. [4] T. Uller, M. Olsson, Multiple paternity in reptiles: patterns and processes, Mol. Ecol. 17 (2008) 2566e2580. € m, H.P. Gelter, DNA fingerprinting reveals multiple [5] A. Gullberg, H. Tegelstro paternity in families of great and blue tits (Parus major and P. caeruleus), Hereditas 117 (1992) 103e108. [6] S. Strohbach, E. Curio, A. Bathen, J.T. Epplen, T. Lubjuhn, Extrapair paternity in the great tit (Parus major): a test of the “good genes” hypothesis, Behav. Ecol. 9 (1998) 388e395. [7] S. Moran, P.D. Turner, C. O'Reilly, Multiple paternity in the European hedgehog, J. Zool. 278 (2009) 349e353. [8] H.L. Lu, C.X. Wang, F.Q. Wu, J.J. Li, Paternity identification in twins with different fathers, J. Forensic. Sci. 39 (1994) 1100e1102. [9] J.D. Miller, S.A. Dinckelacker, Reproductive structures and strategies of turtles, in: J. Wynecken, M.H. Godfrey, V. Bels (Eds.), Biology of turtles, CRC press, Boca Raton, London, New York, 2008, pp. 225e278. [10] J.P. Rieder, P.G. Parker, J.R. Spotila, M.E. Irwin, The mating system of the leatherback turtle: a molecular approach, in: Fernandez Byles (Ed.), Proc 16th Ann Symp Sea Turtle Biol Conserv, Nat Ocean Atmosph Admin, Washington DC, 1998, pp. 120e121. Technical Memorandum NMFS-SESFC-412. [11] P. Dutton, E. Bixby, S.K. Davis, Tendency toward single paternity in leatherbacks detected with microsatellites, in: F.A. Abreu-Grobois, R. Briseno-Duenas,
[29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
rquez, L. Sarti (Eds.), Proc 18th Intern Symp Sea Turtle Biol Conserv, Nat R. Ma Ocean Atmosph Admin, Washington DC, 2000, p. 156. Technical Memorandum NMFS-SESFC-436. D.A. Galbraith, B.N. White, R.J. Brooks, P.T. Boag, Multiple paternity in clutches of snapping turtles (Chelydra serpentine) detected using DNA fingerprints, Can. J. Zool. 71 (1993) 318e324. N. Valenzuela, Multiple paternity in side-neck turtles Podocnemis expansa: evidence from microsatellite DNA data, Mol. Ecol. 9 (2000) 99e105. D.E. Pearse, F.J. Janzen, J.C. Avise, Multiple paternity, sperm storage, and reproductive success of female and male painted turtles (Chrysemys picta) in nature, Behav. Ecol. Sociobiol. 51 (2002) 164e171. D.E. Pearse, R.B. Dastrup, O. Hernandez, J.W. Sites Jr., Paternity in an Orinoco population of endangered Arrau river turtles, Podocnemis expansa (Pleurodira; Podocnemididae), from Venezuela, Chelonian Conserv. Biol. 5 (2006) 232e238. rez-Santigosa, J. Hidalgo-Vila, S. Roques, C. Díaz-Paniagua, A. Portheault, N. Pe Sperm storage and low incidence of multiple paternity in the European pond turtle, Emys orbicularis: a secure but costly strategy? Biol. Conserv. 129 (2006) 236e243. , I.P. Farias, Polyandry in Podocnemis unifilis C. Fantin, L.S. Viana, L.A.S. Mojelo (Pleurodira; Podocnemididae), the vulnerable yellow-spotted Amazon river turtle, Amphib. Reptil. 29 (2008) 479e486. J.M. Repsnider, High frequency of multiple paternity in Blanding's turtle (Emys blandingii), J. Herpetol. 43 (2009) 74e81. , T. Hrbek, Polyandry in the red-headed river C. Fantin, I.P. Farias, L.A.S. Mojelo turtle Podocnemis erythrocephala (Testudines; Podocnemididae), in the Brazilian amazon, Genet. Molec. Res. 9 (2010) 435e440. E.V. Todd, D. Blair, C.J. Limpus, D.J. Limpus, D.R. Jerry, High incidence of multiple paternity in an Australian snapping turtle (Elseya albagula), Austr. J. Zool. 60 (2013) 412e418. K.S. Palmer, T.A. Waite, T. Peare, Long-term sperm storage in the desert tortoise (Gopherus agassizii), Copeia 1998 (1998) 702e705. C.M. Davy, T. Edwards, A. Lathrop, M. Bratton, M. Hagan, B. Henen, et al., Polyandry and multiple paternities in the threatened Agassiz's desert tortoise, Gopherus agassizii, Conserv. Genet. 12 (2011) 1313e1322. S. Roques, C. Diaz-Paniagua, A.C. Andreu, Microsatellite markers reveal multiple paternity and sperm storage in the Mediterranean spur-thighed tortoise, Testudo graeca, Can. J. Zool. 82 (2004) 153e159. E.E. Johnston, M.S. Rand, S.G. Zweifel, Detection of multiple paternity and sperm storage in a captive colony of the central Asian tortoise, Testuda horsfieldii, Can. J. Zool. 84 (2006) 520e526. G. Cutuli, S. Cannicci, M. Vannini, S. Fratini, Influence of mating order on courtship displays and stored sperm utilization in Hermann's tortoises (Testudo hermanni hermanni), Behav. Ecol. Sociobiol. 67 (2013a) 273e281. J.M. Pearce, R.L. Fields, K.T. Scribner, Nest materials as a source of genetic data for avian ecological studies, J. Field Ornithol. 68 (1997) 471e481. Y. Hu, X.-B. Wu, Eggshell membranes as a noninvasive sampling for molecular studies of Chinese alligators (Alligator sinensis), Afr. J. Biotech. 7 (2008) 3022e3025. S.T. Kalinowski, M.L. Taper, T.C. Marshall, Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment, Mol. Ecol. 16 (2007) 1099e1106. A. Forlani, B. Crestanello, S. Mantovani, B. Livoreil, L. Zane, G. Bertorelle, et al., Identification and characterization of microsatellite markers in Hermann's tortoise (Testudo hermanni, Testudinidae), Mol. Ecol. Notes 5 (2005) 228e230. M. Perez, R. Bour, J. Lambourdiere, S. Samadi, M.C. Boisselier, Isolation and characterization of eight microsatellite loci for a study of gene flow between Testudo marginata and Testudo weissingeri (Testudines: Testudinidae), Mol. Ecol. Notes 6 (2006) 1096e1098. S. Rioux-Paquette, G.D. Shore, S.M. Behncke, F.J. Lapointe, E.E. Louis, Characterization of polymorphic microsatellite markers for the endangered Malagasy radiated tortoise (Geochelone radiata), Mol. Ecol. Notes 5 (2005) 527e530. I.R. Swingland, D. Stubbs, The ecology of a Mediterranean tortoise (Testudo hermanni): reproduction, J. Zool. 205 (1985) 595e610. R. Sacchi, D. Pellitteri-Rosa, M. Marchesi, P. Galeotti, M. Fasola, A comparison among sexual signals in courtship of European tortoises, J. Herpetol. 47 (2013) 215e221. cologie de la tortue d’Hermann Testudo hermanni M. Cheylan, Biologie et e ce a la connaissance des climats Gmelin, 1789, in: Contribution de l'espe m Trav EPHE, Montpellier, 1981. quaternaires de la France, Me €te, M. Cheylan, Testudo hermanni Gmelin, 1789 e Griechische Landschildkro € ten Europas, Aula, Wiebelsheim, 2012, pp. in: U. Fritz (Ed.), Die Schildkro 179e289. A. Hailey, R.E. Willemsen, Population density and adult sex ratio of the tortoise Testudo hermanni in Greece: evidence for intrinsic population regulation, J. Zool. 251 (2000) 325e338. P. Galeotti, R. Sacchi, D. Pellitteri Rosa, M. Fasola, Olfactory discrimination of species, sex, and sexual maturity by the Hermann's tortoise Testudo hermanni, Copeia 4 (2007) 980e985. A. Loy, C. Cianfrani, The ecology of Eurotestudo h. hermanni in a mesic area of southern Italy: first evidence of sperm storage, Ethol. Ecol. Evol. 22 (2010) 1e16. D. Stubbs, I.R. Swingdale, The ecology of a Mediterranean tortoise (Testudo hermanni): a declining population, Can. J. Zool. 63 (1985) 169e180.
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257 [40] G. Cutuli, M. Vannini, S. Fratini, Demographic structure and genetic variability of a population of Testudo hermanni hermanni (Reptilia: Testudines: Testudinidae) from Southern Tuscany (central Italy): a case of “happy-ending” uncontrolled reintroduction, Ital. J. Zool. 80 (2013b) 552e559. [41] P. Galeotti, R. Sacchi, M. Fasola, D. Pellitteri Rosa, M. Marchesi, D. Ballasina, Courtship displays and mounting calls are honest, condition-dependent signals that influence mounting success in Hermann's tortoises, Can. J. Zool. 83 (2005) 1306e1313. [42] A. Hailey, N.S. Loumbourdis, Egg size and shape, clutch dynamics, and reproductive effort in European tortoises, Can. J. Zool. 66 (1988) 1527e1536. €ger, A. Winter, Ergebnisse des Inkubationsprojektes für Land[43] I. Jasser-Ha €ten von 2002e2007, Radiata 16 (2007) 2e41. schildkro [44] J.S. Ireland, A.C. Broderick, F. Glen, B.J. Godley, G.C. Hays, P.L.M. Lee, D.O.F. Skibinski, Multiple paternity assessed using microsatellite markers in green turtles Chelonia mydas (Linnaeus, 1758) of Ascension island, South
257
Atlantic, J. Exp. Mar. Biol. Ecol. 291 (2003) 149e160. [45] Purnama D., Zamani N.P., Farajallah A., Microsatellite DNA analysis on the polyandry of green sea turtle Chelonia mydas. HAYATI J Biosci: 20: 182e186. glise, A.D. Schouten, S.B.J. Menken, Multiple paternity [46] W.E.J. Hoekert, H. Neufe and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea), Heredity 89 (2002) 107e113. [47] J.L. Harry, D.A. Briscoe, Multiple paternity in the loggerhead turtle (Caretta caretta), J. Hered. 79 (1988) 96e99. [48] K.M. Moore, R.M. Ball Jr., Multiple paternity in the loggerhead turtle (Caretta caretta) nests on Melbourne beach, Florida: a microsatellite analysis, Mol. Ecol. 11 (2002) 281e288. [49] K. Kichler, M.T. Holder, S.K. Davis, M.R. M arquez, D.W. Owens, Detection of multiple paternity in the Kemp's ridley sea turtle with limited sampling, Mol. Ecol. 8 (1999) 819e830.
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Short communication
Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell C.M. Farke a, 1, K. Olek b, W.M. Gerding c, C. Distler a, * €t Bochum, Universita €tsstr. 150, ND 7/27, 44780 Bochum, Germany Allgemeine Zoologie und Neurobiologie, Ruhr-Universita Labor für Abstammungsbegutachtungen GMBH, Marie-Curie-Str. 1, 53359 Rheinbach, Germany c €t Bochum, Universita €tsstr. 150, MA 5/39, 44780 Bochum, Germany Humangenetik, Ruhr-Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 April 2015 Received in revised form 11 May 2015 Accepted 11 May 2015 Available online 21 May 2015
We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability. © 2015 Published by Elsevier Ltd.
Keywords: Multiple paternity Sperm storage Tortoise Eggshell Testudo hermanni boettgeri Conservation
Promiscuous mating is widespread especially in vertebrates without strong social bonds, but extra-pair mating also occurs in so-called monogamous species (e.g. sharks [e.g. Refs. [1,2]], reptiles [3,4], tits [e.g. Refs. [5,6]], hedgehogs [7], man [8]). The females of polyandrous species are equipped with oviductal tubules [9] where viable sperm can be stored at least over short periods of time. As a consequence, siblings within one clutch can be sired by different males. Among chelonians, who do not form complex social structures nor do they provide parental care, multiple paternity was found in all marine turtle species except the leatherback turtle [10,11], and in all freshwater turtles tested [12e20]. In tortoises, multiple paternity occurs in the desert tortoise (Gopherus agassizii [21,22]), the Mediterranean spur-thighed tortoise (Testudo graeca [23]), the central Asian tortoise (Testudo horsfieldii [24]), and Hermann's tortoise (Testudo hermanni hermanni [25]).
* Corresponding author. E-mail addresses: [email protected] (C.M. Farke), [email protected] (K. Olek), [email protected] (W.M. Gerding), distler@ neurobiologie.rub.de (C. Distler). 1 €tenhilfe TestuDO, Rübenkamp 91, 44319 Dortmund, Present address: Schildkro Germany. http://dx.doi.org/10.1016/j.mcp.2015.05.009 0890-8508/© 2015 Published by Elsevier Ltd.
The parentage of 83 T. hermanni boettgeri from two private captive breeding colonies (group F and H, 8 adult females, 10 adult males) was analyzed during the breeding seasons 2010/2011 (see supplementary information S1, supplementary Table ST1) using a group of specific polymorphic microsatellites developed for chelonians. We successfully isolated DNA from amniotic fluid adhering to the inner surface of the eggshell of 62 live hatchlings and from 5 animals that had died during incubation thereby avoiding the difficult and potentially stressful sampling of saliva or blood in very young animals. This approach was first used in birds and alligators [26,27]. In adult potential parents (n ¼ 18; H01eH04, H7eH10, F01eF04, F07, F72, F101, F135, F201, F201a) and semi-adult animals (n ¼ 6; F102, F103, F104, F119, F120, F125) from earlier clutches, DNA was isolated from their blood, sampled from the dorsal coccygeal or femoral vein, and dried on filter paper. In addition, tissue samples of frozen and ethanol fixed animals from earlier clutches (n ¼ 10; F108, F109, F117, F118, F123, F126, F128, F129, F131, F132) were collected, and DNA was isolated. All samples were stored at 20 C until analysis. DNA was isolated and purified using the tissue or blood protocol variant of the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Twelve gene loci, that were characterized by their highly
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257
polymorphic length variation, were amplified using polymerase chain reaction (PCR). Amplification products were resolved on an ABI3730xl, and sized and scored with the GeneMapper® software. Statistical analysis was performed with CERVUS 3.0 ([28]; supplementary information S4, supplementary Tables ST5, ST6). The following polymorphic microsatellite markers developed for tortoises were used in our genotyping experiments: Test10, Test71, Test88 [29], TW161, TWR106 [30], RAD284 [31], and SK60 that was developed for T. hermanni and Testudo kleinmanni in our laboratory (supplementary information S2, supplementary Table ST2). Multiplex PCR was performed under the following conditions: initial denaturation at 94 C for 10 min, followed by 30 cycles at 94 C for 1 min, at 58 C for 1 min, at 72 C for 1 min and at 72 C for 45 min, and a break at 15 C. The final reaction volume was 20 ml: 2 ml DNA, 2.5 ml MgCl2 (25 mM), 0.2 ml Promega GoTaq (5u/ml), 3.2 ml dNTP mix (1.25 mM), 1 ml forward and 1 ml reverse primer (10 mM) for each marker, 4 ml Colorless GoTaq Flexi Buffer (Qiagen), filled with distilled water to 20 ml. We applied 3 multiplex reactions: MP1: Test10, Test71, TW161; MP2: Test88, TWR 106, RAD 284; MP 3: SK 60. Although the annealing temperature used in the multiplex reaction is a compromise and may not be optimal for the individual PCR amplification reactions, PCR products were of different sizes and could be robustly amplified. One exception was marker Test88, which was replaced by marker TW161 in further analyzes (supplementary Tables ST5, ST6). With this methodological approach we were able to analyze multiple microsatellite markers despite the limited quantity of DNA. In order to obtain additional information from monomorphic markers, PCR products were amplified and analyzed by direct DNA sequencing in three parent turtles and eight of their offspring (supplementary information S3, supplementary Table ST3, ST4) that were homozygous for one or several markers. This analysis should reveal potential single nucleotide polymorphism (SNP) within the STR-repeat or between the STR-repeat and the primer loci that could in turn provide additional information about paternity, if polymorphic. Of course, these analyses are only valuable for markers where both alleles can be amplified and are not subject of allele drop-out. Employing seven informative microsatellite markers (Test10, Test71, Test88, TWR106, RAD284, SK60, TW161) we could unequivocally determine the fathers of all juveniles at an 80% or 95% confidence level. The mothers' identity determined by DNA analysis, unequivocally confirmed our observations of the animals during oviposition. In some cases, several markers were monomorphic, e.g. only one allele could be identified. This can be caused by allele drop out, but can also be due to a problem of the STRanalysis, which is sometimes encountered in exotic species or wild animals where markers are used that were originally developed for another subspecies or for a related species. In these cases the markers are often not as informative as for the original species. Furthermore, when working with degraded material it is not always clear whether the marker is really homozygous or whether a second allele cannot be amplified. Thus, for two markers (TW161 and Test71), we analyzed the flanking regions of the microsatellite for polymorphic SNP. This additional genetic variability potentially increases the informative value of the locus. As an example we show the sequencing results of the TW161 locus for two families ((H01/H07, parents; H69, H71, H73, H76, juveniles); (H01/H10, parents; H70, H72, H74, H68, juveniles)) (supplementary information S3, supplementary Table ST3, ST4). In individuals with previously identified monomorphic microsatellite markers, we could identify two polymorphic SNPs confirming the corresponding STR-based parentage. Multiple paternity was found in two of six clutches from colony F, and in seven of nine clutches from colony H. The offspring in eight
255
of these clutches was fathered by two different males, the offspring of the remaining clutch was derived from three different males. Thus, in 52.9% of the clutches analyzed offspring was fathered by more than one male. We identified three cases of sperm storage (F121, F218eF221, H28) where the fathers determined by DNA analysis were not part of the breeding groups during the season we analyzed indicating that mating had occurred during the previous season and that sperm had been stored. In the wild, tortoises are promiscuous [3,32,33]. Generally, multiple mating can have various advantages for the female ranging from decreasing harassment by aggressive males (“convenience polyandry” [2]) to direct benefits such as nuptial gifts and help during raising of the young [3,4]. In chelonians, however, indirect benefits such as “trading up” the quality of the sperm, promoting sperm competition, cryptic female choice, and “bethedging” i.e. the production of diverse geno- and phenotypes within clutches to be better equipped for environmental changes [4] seem to be more likely but these factors are mostly untested. All these benefits are invariably linked to the ability to store sperm, either within a season, e.g. mating in spring and inseminating consecutive clutches during the summer, or across seasons when sperm is stored for up to 3e4 years [3,4,34]. The mating and courtship behavior of T. hermanni has been described in detail [e.g. Refs. [25,32e38]]. In natural habitats, population density of T. hermanni can be rather low (from <3 individuals/ha [39], to ~40 individuals/ha [35,36,40]), and mating occurs during the rare encounters. Olfactory, visual, tactile, and auditory signals are important for species and mate recognition and provide honest cues for mate quality [37,41]. Thus they represent important signals for female strategies as cryptic female choice or bet-hedging [4]. Our animals produced 2-3 clutches per season, which is similar to the situation found in the wild [25,32,42]. In both colonies, the ability for sperm storage was confirmed. In colony F, multiple paternity was shown in 33%, in colony H in 78% of the clutches. Fertilization success was 85% in colony F and 95% in colony H, and thus much higher than the <60% described for permanently cohoused T. hermanni by Jasser-H€ ager and Winter [43]. Our sample is too small to deduce influences of the breeding scheme on the prevalence of multiple paternity or fertilization success. Taken together, multiple paternity in T. hermanni boettgeri was identified in 52.9% of the clutches analyzed. This rate is higher than described for T. graeca (20% [23]) and T. horsfieldii (27% [24]), and comparable to Gopherus (50e57% [21,22]) kept in captivity, or in a semi-natural environment. In a planned mating study Cutuli et al. [25] found a slightly lower rate of multiple paternity in a captive colony of T. hermanni hermanni. Multiple paternity ranges from 10% in Emys orbicularis [16] and some populations of Podocnemis expansa [15] up to 50e100% in Chelonia midas [44,45], Podocnemis expansa [13], and Podocnemis unifilis [17] with most rates lying between 20% and 80% (Lepidochelys olivacea 20% [46], Caretta 31e33% [47,48], Chrysemys picta 33% [14], Lepidochelys kempi 58% [49], Emydoidea blandingii 60e80% [18], Chelydra serpentina 66% [12], Podocnemis erythrocephala 80% [19], Elseya albagula 83% [20]). Thus, the incidence of multiple paternity in Hermann's tortoise seems similar to other tortoises and turtles even though direct comparisons are difficult due to different sample sizes and living conditions. In conclusion, the data described here extend our knowledge about multiple paternity and sperm storage to a further tortoise taxon, T. hermanni boettgeri indicating that these traits are widespread in chelonians in general. To the best of our knowledge, this is the first study in chelonians to employ the non-invasive method of collecting DNA samples from amniotic fluid adhering to the inner surface of the eggshell. The supplementary SNP analysis proved to
256
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257
be a useful method when working with wild animals and with low DNA-quantity material and can provide additional information for otherwise non-informative markers. This approach is applicable for research purposes and conservation breeding programs, i.e. if owners would store the eggshells together with genetic material of the putative parents, paternity of animals in the pet trade could be easily tracked thereby largely limiting the trade of illegally imported specimens.
[12]
[13] [14]
[15]
Funding source This study was supported by departmental funds of the faculty of Biology and Biotechnology, Ruhr-University Bochum. Author contributions CD, CMF and KO designed the experiments, KO performed the experiments, KO, CMF and WMG analyzed the data, CD, KO, WMG and CMF wrote the manuscript. Conflict of interest The authors declare no conflict of interest. All authors approve of the final article.
[16]
[17]
[18] [19]
[20]
[21] [22]
Acknowledgments We thank all owners for providing their tortoises and eggs, M. Wiegand for collecting most of the blood samples of the adult and semiadult animals, K. Krabbe for help with the PCR analysis, D.A. Akkad for help with data analysis, and the anonymous reviewers for their constructive criticism on earlier versions of the manuscript. This study was supported by departmental funds of the Faculty of Biology and Biotechnology, Ruhr-University Bochum.
[23]
[24]
[25]
[26]
Appendix A. Supplementary data
[27]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mcp.2015.05.009.
[28]
References [1] T.S. Daly-Engel, R.D. Grubbs, K.A. Feldheim, B.W. Bowen, R.J. Toonen, Is multiple mating beneficial or unavoidable? Low multiple paternity and genetic diversity in the shortspine spurdog Squalus mitsukurii, Mar. Ecol. Prog. Ser. 403 (2010) 255e267. [2] A.M. Griffiths, D.M.P. Jacoby, D. Casane, M. McHugh, D.P. Croft, M.J. Genner, et al., First analysis of multiple paternity in an oviparous shark, the small-spotted catshark (Scyliorhinus canicula L.), J. Hered. 103 (2012) 166e173. [3] D.E. Pearse, J.C. Avise, Turtle mating systems: behaviour, sperm storage, and genetic paternity, Am. Gen. Assoc. 92 (2001) 206e211. [4] T. Uller, M. Olsson, Multiple paternity in reptiles: patterns and processes, Mol. Ecol. 17 (2008) 2566e2580. € m, H.P. Gelter, DNA fingerprinting reveals multiple [5] A. Gullberg, H. Tegelstro paternity in families of great and blue tits (Parus major and P. caeruleus), Hereditas 117 (1992) 103e108. [6] S. Strohbach, E. Curio, A. Bathen, J.T. Epplen, T. Lubjuhn, Extrapair paternity in the great tit (Parus major): a test of the “good genes” hypothesis, Behav. Ecol. 9 (1998) 388e395. [7] S. Moran, P.D. Turner, C. O'Reilly, Multiple paternity in the European hedgehog, J. Zool. 278 (2009) 349e353. [8] H.L. Lu, C.X. Wang, F.Q. Wu, J.J. Li, Paternity identification in twins with different fathers, J. Forensic. Sci. 39 (1994) 1100e1102. [9] J.D. Miller, S.A. Dinckelacker, Reproductive structures and strategies of turtles, in: J. Wynecken, M.H. Godfrey, V. Bels (Eds.), Biology of turtles, CRC press, Boca Raton, London, New York, 2008, pp. 225e278. [10] J.P. Rieder, P.G. Parker, J.R. Spotila, M.E. Irwin, The mating system of the leatherback turtle: a molecular approach, in: Fernandez Byles (Ed.), Proc 16th Ann Symp Sea Turtle Biol Conserv, Nat Ocean Atmosph Admin, Washington DC, 1998, pp. 120e121. Technical Memorandum NMFS-SESFC-412. [11] P. Dutton, E. Bixby, S.K. Davis, Tendency toward single paternity in leatherbacks detected with microsatellites, in: F.A. Abreu-Grobois, R. Briseno-Duenas,
[29]
[30]
[31]
[32] [33]
[34]
[35]
[36]
[37]
[38]
[39]
rquez, L. Sarti (Eds.), Proc 18th Intern Symp Sea Turtle Biol Conserv, Nat R. Ma Ocean Atmosph Admin, Washington DC, 2000, p. 156. Technical Memorandum NMFS-SESFC-436. D.A. Galbraith, B.N. White, R.J. Brooks, P.T. Boag, Multiple paternity in clutches of snapping turtles (Chelydra serpentine) detected using DNA fingerprints, Can. J. Zool. 71 (1993) 318e324. N. Valenzuela, Multiple paternity in side-neck turtles Podocnemis expansa: evidence from microsatellite DNA data, Mol. Ecol. 9 (2000) 99e105. D.E. Pearse, F.J. Janzen, J.C. Avise, Multiple paternity, sperm storage, and reproductive success of female and male painted turtles (Chrysemys picta) in nature, Behav. Ecol. Sociobiol. 51 (2002) 164e171. D.E. Pearse, R.B. Dastrup, O. Hernandez, J.W. Sites Jr., Paternity in an Orinoco population of endangered Arrau river turtles, Podocnemis expansa (Pleurodira; Podocnemididae), from Venezuela, Chelonian Conserv. Biol. 5 (2006) 232e238. rez-Santigosa, J. Hidalgo-Vila, S. Roques, C. Díaz-Paniagua, A. Portheault, N. Pe Sperm storage and low incidence of multiple paternity in the European pond turtle, Emys orbicularis: a secure but costly strategy? Biol. Conserv. 129 (2006) 236e243. , I.P. Farias, Polyandry in Podocnemis unifilis C. Fantin, L.S. Viana, L.A.S. Mojelo (Pleurodira; Podocnemididae), the vulnerable yellow-spotted Amazon river turtle, Amphib. Reptil. 29 (2008) 479e486. J.M. Repsnider, High frequency of multiple paternity in Blanding's turtle (Emys blandingii), J. Herpetol. 43 (2009) 74e81. , T. Hrbek, Polyandry in the red-headed river C. Fantin, I.P. Farias, L.A.S. Mojelo turtle Podocnemis erythrocephala (Testudines; Podocnemididae), in the Brazilian amazon, Genet. Molec. Res. 9 (2010) 435e440. E.V. Todd, D. Blair, C.J. Limpus, D.J. Limpus, D.R. Jerry, High incidence of multiple paternity in an Australian snapping turtle (Elseya albagula), Austr. J. Zool. 60 (2013) 412e418. K.S. Palmer, T.A. Waite, T. Peare, Long-term sperm storage in the desert tortoise (Gopherus agassizii), Copeia 1998 (1998) 702e705. C.M. Davy, T. Edwards, A. Lathrop, M. Bratton, M. Hagan, B. Henen, et al., Polyandry and multiple paternities in the threatened Agassiz's desert tortoise, Gopherus agassizii, Conserv. Genet. 12 (2011) 1313e1322. S. Roques, C. Diaz-Paniagua, A.C. Andreu, Microsatellite markers reveal multiple paternity and sperm storage in the Mediterranean spur-thighed tortoise, Testudo graeca, Can. J. Zool. 82 (2004) 153e159. E.E. Johnston, M.S. Rand, S.G. Zweifel, Detection of multiple paternity and sperm storage in a captive colony of the central Asian tortoise, Testuda horsfieldii, Can. J. Zool. 84 (2006) 520e526. G. Cutuli, S. Cannicci, M. Vannini, S. Fratini, Influence of mating order on courtship displays and stored sperm utilization in Hermann's tortoises (Testudo hermanni hermanni), Behav. Ecol. Sociobiol. 67 (2013a) 273e281. J.M. Pearce, R.L. Fields, K.T. Scribner, Nest materials as a source of genetic data for avian ecological studies, J. Field Ornithol. 68 (1997) 471e481. Y. Hu, X.-B. Wu, Eggshell membranes as a noninvasive sampling for molecular studies of Chinese alligators (Alligator sinensis), Afr. J. Biotech. 7 (2008) 3022e3025. S.T. Kalinowski, M.L. Taper, T.C. Marshall, Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment, Mol. Ecol. 16 (2007) 1099e1106. A. Forlani, B. Crestanello, S. Mantovani, B. Livoreil, L. Zane, G. Bertorelle, et al., Identification and characterization of microsatellite markers in Hermann's tortoise (Testudo hermanni, Testudinidae), Mol. Ecol. Notes 5 (2005) 228e230. M. Perez, R. Bour, J. Lambourdiere, S. Samadi, M.C. Boisselier, Isolation and characterization of eight microsatellite loci for a study of gene flow between Testudo marginata and Testudo weissingeri (Testudines: Testudinidae), Mol. Ecol. Notes 6 (2006) 1096e1098. S. Rioux-Paquette, G.D. Shore, S.M. Behncke, F.J. Lapointe, E.E. Louis, Characterization of polymorphic microsatellite markers for the endangered Malagasy radiated tortoise (Geochelone radiata), Mol. Ecol. Notes 5 (2005) 527e530. I.R. Swingland, D. Stubbs, The ecology of a Mediterranean tortoise (Testudo hermanni): reproduction, J. Zool. 205 (1985) 595e610. R. Sacchi, D. Pellitteri-Rosa, M. Marchesi, P. Galeotti, M. Fasola, A comparison among sexual signals in courtship of European tortoises, J. Herpetol. 47 (2013) 215e221. cologie de la tortue d’Hermann Testudo hermanni M. Cheylan, Biologie et e ce a la connaissance des climats Gmelin, 1789, in: Contribution de l'espe m Trav EPHE, Montpellier, 1981. quaternaires de la France, Me €te, M. Cheylan, Testudo hermanni Gmelin, 1789 e Griechische Landschildkro € ten Europas, Aula, Wiebelsheim, 2012, pp. in: U. Fritz (Ed.), Die Schildkro 179e289. A. Hailey, R.E. Willemsen, Population density and adult sex ratio of the tortoise Testudo hermanni in Greece: evidence for intrinsic population regulation, J. Zool. 251 (2000) 325e338. P. Galeotti, R. Sacchi, D. Pellitteri Rosa, M. Fasola, Olfactory discrimination of species, sex, and sexual maturity by the Hermann's tortoise Testudo hermanni, Copeia 4 (2007) 980e985. A. Loy, C. Cianfrani, The ecology of Eurotestudo h. hermanni in a mesic area of southern Italy: first evidence of sperm storage, Ethol. Ecol. Evol. 22 (2010) 1e16. D. Stubbs, I.R. Swingdale, The ecology of a Mediterranean tortoise (Testudo hermanni): a declining population, Can. J. Zool. 63 (1985) 169e180.
C.M. Farke et al. / Molecular and Cellular Probes 29 (2015) 254e257 [40] G. Cutuli, M. Vannini, S. Fratini, Demographic structure and genetic variability of a population of Testudo hermanni hermanni (Reptilia: Testudines: Testudinidae) from Southern Tuscany (central Italy): a case of “happy-ending” uncontrolled reintroduction, Ital. J. Zool. 80 (2013b) 552e559. [41] P. Galeotti, R. Sacchi, M. Fasola, D. Pellitteri Rosa, M. Marchesi, D. Ballasina, Courtship displays and mounting calls are honest, condition-dependent signals that influence mounting success in Hermann's tortoises, Can. J. Zool. 83 (2005) 1306e1313. [42] A. Hailey, N.S. Loumbourdis, Egg size and shape, clutch dynamics, and reproductive effort in European tortoises, Can. J. Zool. 66 (1988) 1527e1536. €ger, A. Winter, Ergebnisse des Inkubationsprojektes für Land[43] I. Jasser-Ha €ten von 2002e2007, Radiata 16 (2007) 2e41. schildkro [44] J.S. Ireland, A.C. Broderick, F. Glen, B.J. Godley, G.C. Hays, P.L.M. Lee, D.O.F. Skibinski, Multiple paternity assessed using microsatellite markers in green turtles Chelonia mydas (Linnaeus, 1758) of Ascension island, South
257
Atlantic, J. Exp. Mar. Biol. Ecol. 291 (2003) 149e160. [45] Purnama D., Zamani N.P., Farajallah A., Microsatellite DNA analysis on the polyandry of green sea turtle Chelonia mydas. HAYATI J Biosci: 20: 182e186. glise, A.D. Schouten, S.B.J. Menken, Multiple paternity [46] W.E.J. Hoekert, H. Neufe and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea), Heredity 89 (2002) 107e113. [47] J.L. Harry, D.A. Briscoe, Multiple paternity in the loggerhead turtle (Caretta caretta), J. Hered. 79 (1988) 96e99. [48] K.M. Moore, R.M. Ball Jr., Multiple paternity in the loggerhead turtle (Caretta caretta) nests on Melbourne beach, Florida: a microsatellite analysis, Mol. Ecol. 11 (2002) 281e288. [49] K. Kichler, M.T. Holder, S.K. Davis, M.R. M arquez, D.W. Owens, Detection of multiple paternity in the Kemp's ridley sea turtle with limited sampling, Mol. Ecol. 8 (1999) 819e830.