Behavioral Ecology and Sociobiology

, Volume 70, Issue 8, pp 1197–1208 | Cite as

Altitude underlies variation in the mating system, somatic condition, and investment in reproductive traits in male Asian grass frogs (Fejervarya limnocharis)

  • Long Jin
  • Sheng Nan Yang
  • Wen Bo Liao
  • Stefan Lüpold
Original Article


There is substantial comparative and growing experimental evidence that the competition for fertilization among sperm from different males can drive variation in male reproductive investments. However, less is known about the extent of natural variation in these investments relative to environmental variables affecting resource availability and mating system dynamics, which would allow insights into the mechanisms shaping reproductive allocation. Here, we studied interpopulation variation in male investments in testis size and sperm length across 25 populations of the Asian grass frog Fejervarya limnocharis along a 1550-km latitudinal and 1403-m altitudinal transect in China. We found relative testis mass and sperm length, male somatic condition, and the male/female sex ratio to increase with elevation but not latitude or longitude. Our results suggest that environmental variation may underlie local adaptations to reproductive investments among natural populations, mediated by differences in the availability of both resources and sexual partners (including the resulting male–male competition). These findings contrast with previous predictions that increasing latitude and/or elevation should lead to declining reproductive investments in male anurans due to shortening breeding seasons, declining resource availability, and lowering (rather than increasing) male/female sex ratios. We discuss these species differences in the context of differential resource allocation strategies, breeding ecology, and patterns of male–male competition. These differences show the need for future work on reproductive investments in anurans beyond the few model systems and for potential extension of the theoretical framework to species with different mating systems and strategies.

Significance statement

Despite a broad theoretical framework of how environmental parameters can shape mating systems and these, in turn, drive the evolution of reproductive traits, there is limited empirical evidence from natural populations. We examined differences in the size of testes and sperm between males between natural populations along latitudinal and altitudinal transects. We found that at high elevation, males were relatively heavier and had longer sperm and disproportionately large testes, and the sex ratio was more male-biased. These interpopulation differences may reflect local adaptations to variation in the resource availability and temporal patterns of female availability, which affects the breeding synchrony, sperm demand, and male–male competition for mates and paternity. However, our results differ from previous studies of frogs, possibly due to different breeding ecology, which highlights the need for further work and potential extension of theoretical predictions to non-model anurans.


Geographic variation Relative testis mass Sperm length Sperm competition Operational sex ratio Anura 



We thank Shang Ling Lou, Mao Jun Zhong, and Cheng Chen for assistance with lab work, and M. Schäfer and two anonymous reviewers for their constructive comments.

Compliance with ethical standards


Financial support was provided by the Sichuan Province Outstanding Youth Academic Technology Leaders Program (2013JQ0016) and Sichuan Province Department of Education Innovation Team Project (14TD0015; 15TD003519) to WBL and by the Swiss National Science Foundation (PZ00P3_154767) to SL.

Ethical standards

All experiments involving the sacrifice of live animals were approved by the Animal Ethics Committee at China West Normal University. We declare that all animals used in the study were treated humanely and ethically following all applicable institutional animal care guidelines in China.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

265_2016_2128_MOESM1_ESM.pdf (45 kb)
ESM 1 (PDF 45 kb)


  1. Adriaenssens B, van Damme R, Seebacher F, Wilson RS (2012) Sex cells in changing environments: can organisms adjust the physiological function of gametes to different temperatures? Glob Chang Biol 18:1797–1803CrossRefGoogle Scholar
  2. Alavi SMH, Cosson J (2005) Sperm motility in fishes. I. Effects of temperature and pH: a review. Cell Biol Int 29:101–110PubMedCrossRefGoogle Scholar
  3. Álvarez D, Viesca L, Nicieza AG (2014) Sperm competitiveness differs between two frog populations with different breeding systems. J Zool 292:202–205CrossRefGoogle Scholar
  4. Arendt JD (1997) Adaptive intrinsic growth rates: an integration across taxa. Q Rev Biol 72:149–177CrossRefGoogle Scholar
  5. Atkinson D, Sibly RM (1997) Why are organisms usually bigger in colder environments? Making sense of a life history puzzle. Trends Ecol Evol 12:235–239PubMedCrossRefGoogle Scholar
  6. Bergmann C (1847) Über die Verhältnisse der Wärmeökonomie der Thiere zu ihrer Grösse. Göttinger Stud 1:595–708Google Scholar
  7. Berry PY (1964) The breeding patterns of seven species of Singapore Anura. J Anim Ecol 33:227–243CrossRefGoogle Scholar
  8. Berven KA, Gill DE (1983) Interpreting geographic variation in life-history traits. Am Zool 23:85–97CrossRefGoogle Scholar
  9. Billard R, Christen R, Cosson MP, Gatty JL, Letellier L, Renard P, Saad A (1986) Biology of the gametes of some teleost species. Fish Physiol Biochem 2:115–120PubMedCrossRefGoogle Scholar
  10. Blanckenhorn WU, Demont M (2004) Bergmann and converse Bergmann latitudinal clines in arthropods: two ends of a continuum? Integr Comp Biol 44:413–424PubMedCrossRefGoogle Scholar
  11. Blanckenhorn WU, Fairbairn DJ (1995) Life-history adaptation along a latitudinal cline in the water strider Aquarius remigis (Heteroptera, Gerridae). J Evol Biol 8:21–41CrossRefGoogle Scholar
  12. Blanckenhorn WU, Hellriegel B (2002) Against Bergmann’s rule: fly sperm size increases with temperature. Ecol Lett 5:7–10CrossRefGoogle Scholar
  13. Bonnet X, Bradshaw D, Shine R (1998) Capital versus income breeding: an ectothermic perspective. Oikos 83:333–342CrossRefGoogle Scholar
  14. Breckels RD, Neff BD (2013) The effects of elevated temperature on the sexual traits, immunology and survivorship of a tropical ectotherm. J Exp Biol 216:2658–2664PubMedCrossRefGoogle Scholar
  15. Briskie JV (1992) Copulation patterns and sperm competition in the polygynandrous Smith’s longspur. Auk 109:563–575Google Scholar
  16. Briskie JV, Montgomerie R, Birkhead TR (1997) The evolution of sperm size in birds. Evolution 51:937–945CrossRefGoogle Scholar
  17. Burness G, Schulte-Hostedde AI, Montgomerie R (2008) Body condition influences sperm energetics in lake whitefish (Coregonus clupeaformis). Can J Fish Aquat Sci 65:615–620CrossRefGoogle Scholar
  18. Buzatto BA, Roberts JD, Simmons LW (2015) Sperm competition and the evolution of precopulatory weapons: increasing male density promotes sperm competition and reduces selection on arm strength in a chorusing frog. Evolution 69:2613–624PubMedCrossRefGoogle Scholar
  19. Byrne PG, Roberts JD, Simmons LW (2002) Sperm competition selects for increased testes mass in Australian frogs. J Evol Biol 15:347–355CrossRefGoogle Scholar
  20. Byrne PG, Simmons LW, Roberts JD (2003) Sperm competition and the evolution of gamete morphology in frogs. Proc R Soc Lond B 270:2079–2086CrossRefGoogle Scholar
  21. Chen W, Zhao L, Wang Y, Li H, He D, Ren L, Tang Z, Liu X (2013) Reproductive output of the brown frog Rana kukunoris at high altitude of the Tibetan plateau. Acta Herpetol 8:153–157Google Scholar
  22. Chen W, Pike DA, He D, Wang Y, Ren L, Wang X, Fan X, Lu X (2014) Altitude decreases testis weight of a frog (Rana kukunoris) on the Tibetan plateau. Herpetol J 24:183–188Google Scholar
  23. Cramer ERA, Laskemoen T, Kleven O, Lifjeld JT (2013) Sperm length variation in house wrens Troglodytes aedon. J Ornithol 154:129–138CrossRefGoogle Scholar
  24. Delgado MJ, Gutiérrez P, Alonso-Bedate M (1989) Seasonal cycles in testicular activity in the frog, Rana perezi. Gen Comp Endocrinol 73:1–11PubMedCrossRefGoogle Scholar
  25. Dewsbury DA (1982) Ejaculate cost and male choice. Am Nat 119:601–610CrossRefGoogle Scholar
  26. Dziminski MA, Roberts JD, Beveridge M, Simmons LW (2010) Among-population covariation between sperm competition and ejaculate expenditure in frogs. Behav Ecol 21:322–328CrossRefGoogle Scholar
  27. Emerson SB (1997) Testis size variation in frogs: testing the alternatives. Behav Ecol Sociobiol 41:227–235CrossRefGoogle Scholar
  28. Emlen ST, Oring LW (1977) Ecology, sexual selection, and evolution of mating systems. Science 197:215–223PubMedCrossRefGoogle Scholar
  29. Endler JA (1977) Geographic variation, speciation, and clines. Princeton University Press, Princeton, NJGoogle Scholar
  30. Fei L, Ye CY (2001) The colour handbook of the amphibians of Sichuan. China Forestry Publishing House, ChengduGoogle Scholar
  31. Fitzpatrick JL, Lüpold S (2014) Sexual selection and the evolution of sperm quality. Mol Hum Reprod 20:1180–1189PubMedCrossRefGoogle Scholar
  32. Fitzpatrick JL, Montgomerie R, Desjardins JK, Stiver KA, Kolm N, Balshine S (2009) Female promiscuity promotes the evolution of faster sperm in cichlid fishes. Proc Natl Acad Sci U S A 106:1128–1132PubMedPubMedCentralCrossRefGoogle Scholar
  33. Gomendio M, Roldan ERS (2008) Implications of diversity in sperm size and function for sperm competition and fertility. Int J Dev Biol 52:439–447PubMedCrossRefGoogle Scholar
  34. Gould SJ, Johnston RF (1972) Geographic variation. Annu Rev Ecol Syst 3:457–498CrossRefGoogle Scholar
  35. Hettyey A, Roberts JD (2006) Sperm traits of the quacking frog, Crinia georgiana: intra- and interpopulation variation in a species with a high risk of sperm competition. Behav Ecol Sociobiol 59:389–396CrossRefGoogle Scholar
  36. Hettyey A, Roberts JD (2007) Sperm traits in the quacking frog (Crinia georgiana), a species with plastic alternative mating tactics. Behav Ecol Sociobiol 61:1303–1310CrossRefGoogle Scholar
  37. Hettyey A, Laurila A, Herczeg G, Jönsson KI, Kovács T, Merilä J (2005) Does testis weight decline towards the Subarctic? A case study on the common frog, Rana temporaria. Naturwissenschaften 92:188–192PubMedCrossRefGoogle Scholar
  38. Hosken DJ, Garner TWJ, Blanckenhorn WU (2003) Asymmetry, testis and sperm size in yellow dung flies. Funct Ecol 17:231–236CrossRefGoogle Scholar
  39. Hunter FM, Birkhead TR (2002) Sperm viability and sperm competition in insects. Curr Biol 12:121–123PubMedCrossRefGoogle Scholar
  40. Immler S, Pitnick S, Parker GA, Durrant KL, Lüpold S, Calhim S, Birkhead TR (2011) Resolving variation in the reproductive tradeoff between sperm size and number. Proc Natl Acad Sci U S A 108:5325–5330PubMedPubMedCentralCrossRefGoogle Scholar
  41. James FC (1970) Geographic variation in birds and its relationship to climate. Ecology 51:365–390CrossRefGoogle Scholar
  42. Katz DF, Drobnis EZ (1990) Analysis and interpretation of the forces generated by spermatozoa. In: Bavister BD, Cummis J, Roldan ERS (eds) Fertilization in mammals. Serano Symposium, Norwell, Massachusetts, pp 125–137Google Scholar
  43. Kleinbaum DG, Kupper LL, Muller KE (1998) Applied regression analysis and other multivariable methods. Duxbury Press, Pacific GroveGoogle Scholar
  44. Kusano T, Toda M, Fukuyama K (1991) Testes size and breeding systems in Japanese anurans with special reference to large testes in the treefrog, Rhacophorus arboreus (Amphibia: Rhacophoridae). Behav Ecol Sociobiol 29:27–31CrossRefGoogle Scholar
  45. Kvarnemo C, Ahnesjö I (1996) The dynamics of operational sex ratios and competition for mates. Trends Ecol Evol 11:404–408PubMedCrossRefGoogle Scholar
  46. Laskemoen T, Albrecht T, Bonisoli-Alquati A et al (2013) Variation in sperm morphometry and sperm competition among barn swallow (Hirundo rustica) populations. Behav Ecol Sociobiol 67:301–309CrossRefGoogle Scholar
  47. Legendre P, Fortin MJ (1989) Spatial pattern and ecological analysis. Vegetatio 80:107–138CrossRefGoogle Scholar
  48. Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common mistake. Auk 104:116–121CrossRefGoogle Scholar
  49. Levitan DR (1993) The importance of sperm limitation to the evolution of egg size in marine invertebrates. Am Nat 141:517–536PubMedCrossRefGoogle Scholar
  50. Liao WB, Lu X (2011) A comparison of reproductive output of the Omei treefrog (Rhacophorus omeimontis) between high and low elevations. Anim Biol 61:263–276CrossRefGoogle Scholar
  51. Liao WB, Lu X, Shen YW, Hu JC (2011) Age structure and body size of two populations of the rice frog Rana limnocharis from different altitudes. Ital J Zool 78:215–221CrossRefGoogle Scholar
  52. Liao WB, Mi ZP, Li CL, Wei SC, Wu H (2013) Sperm traits in relation to male amplexus position in the Omei treefrog Rhacophorus omeimontis, a species with group spawning. Herpetol J 23:17–21Google Scholar
  53. Liao WB, Lu X, Jehle R (2014) Altitudinal variation in maternal investment and trade-offs between egg size and clutch size in the Andrew’s toad. J Zool 293:84–91CrossRefGoogle Scholar
  54. Liu YH, Zeng Y, Liao WB, Zhou CQ, Mi ZP, Mao M, Chen L (2012) Altitudinal variation in body size in the rice frog (Rana limnocharis) in southwestern China. Acta Herpetol 7:57–68Google Scholar
  55. Lofts B (1964) Seasonal changes in the functional activity of the interstitial and spermatogenetic tissues of green frog, Rana esculenta. Gen Comp Endocrinol 4:550–562PubMedCrossRefGoogle Scholar
  56. Lüddecke H (2002) Variation and trade-off in reproductive output of the Andean frog Hyla labialis. Oecologia 130:403–410CrossRefGoogle Scholar
  57. Lüpold S (2013) Ejaculate quality and constraints in relation to sperm competition levels among eutherian mammals. Evolution 67:3052–3060PubMedGoogle Scholar
  58. Lüpold S, Fitzpatrick JL (2015) Sperm number trumps sperm size in mammalian ejaculate evolution. Proc R Soc B 282:20152122PubMedCrossRefGoogle Scholar
  59. Lüpold S, Linz GM, Birkhead TR (2009) Sperm design and variation in the New World blackbirds (Icteridae). Behav Ecol Sociobiol 63:899–909CrossRefGoogle Scholar
  60. Lüpold S, Westneat DF, Birkhead TR (2011) Geographical variation in sperm morphology in the red-winged blackbird (Agelaius phoeniceus). Evol Ecol 25:373–390CrossRefGoogle Scholar
  61. Lüpold S, Birkhead TR, Westneat DF (2012) Seasonal variation in ejaculate traits of male red-winged blackbirds (Agelaius phoeniceus). Behav Ecol Sociobiol 66:1607–1617CrossRefGoogle Scholar
  62. Lüpold S, Tomkins JL, Simmons LW, Fitzpatrick JL (2014) Female monopolization mediates the relationship between pre- and postcopulatory sexual traits. Nat Commun 5:3184PubMedCrossRefGoogle Scholar
  63. Lüpold S, Manier MK, Puniamoorthy N, Schoff C, Starmer WT, Buckley Luepold SH, Belote JM, Pitnick S (2016) How sexual selection can drive the evolution of costly sperm ornamentation. Nature. doi: 10.1038/nature18005
  64. Mallick PK (1986) Mating behaviour of Rana limnocharis. Biol Bull India 8:89–94Google Scholar
  65. Mallick PK (1987) The reproductive behaviour and the chronology of meiosis and spermiogenesis in Rana limnocharis, Rana verrucosa and Rana cyanophlyctis. PhD thesis, University of BurdwanGoogle Scholar
  66. Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220PubMedGoogle Scholar
  67. Mi ZP, Liao WB, Jin L, Lou SL, Cheng J, Wu H (2012) Testis asymmetry and sperm length in Rhacophorus omeimontis. Zool Sci 29:368–372PubMedCrossRefGoogle Scholar
  68. Minoretti N, Stoll P, Baur B (2013) Heritability of sperm length and adult shell size in the land snail Arianta arbustorum (Linnaeus, 1758). J Molluscan Stud 79:218–224CrossRefGoogle Scholar
  69. Moran PAP (1950) Notes on continuous stochastic phenomena. Biometrika 37:17–23PubMedCrossRefGoogle Scholar
  70. Morisawa M, Suzuki K, Shimizu H, Morisawa S, Yasuda K (1983) Effects of osmolality and potassium on motility of spermatozoa from freshwater cyprinid fishes. J Exp Biol 107:95–103PubMedGoogle Scholar
  71. Morrison C, Hero JM (2003) Geographic variation in life-history characteristics of amphibians: a review. J Anim Ecol 72:270–279CrossRefGoogle Scholar
  72. Olsson M, Madsen T, Shine R (1997) Is sperm really so cheap? Costs of reproduction in male adders, Vipera berus. Proc R Soc Lond B 264:455–459CrossRefGoogle Scholar
  73. Othman MS, Khonsue W, Kitana J, Thirakhupt K, Robson MG, Kitana N (2011) Reproductive mode of Fejervarya limnocharis (Anura: Ranidae) caught from Mae Sot, Thailand based on its gonadosomatic indices. Asian Herpetol Res 2:41–45PubMedPubMedCentralCrossRefGoogle Scholar
  74. Parker GA, Ball GF (2005) Sperm competition, mating rate and the evolution of testis and ejaculate sizes: a population model. Biol Lett 1:235–238PubMedPubMedCentralCrossRefGoogle Scholar
  75. Parker GA, Pizzari T (2010) Sperm competition and ejaculate economics. Biol Rev 85:897–934PubMedCrossRefGoogle Scholar
  76. Pinheiro JC, Bates DM (2000) Mixed-effects models in S and S-PLUS. Springer, New YorkCrossRefGoogle Scholar
  77. Pitcher TE, Stutchbury BJM (1998) Latitudinal variation in testis size in six species of North American songbirds. Can J Zool 76:618–622CrossRefGoogle Scholar
  78. Pitnick SS, Markow TA, Spicer GS (1999) Evolution of multiple kinds of female sperm-storage organs in Drosophila. Evolution 53:1804–1822CrossRefGoogle Scholar
  79. Preston BT, Stevenson IR, Pemberton JM, Coltman DW, Wilson K (2003) Overt and covert competition in a promiscuous mammal: the importance of weaponry and testes size to male reproductive success. Proc R Soc Lond B 270:633–640CrossRefGoogle Scholar
  80. Ramm SA, Schärer L (2014) The evolutionary ecology of testicular function: size isn’t everything. Biol Rev 89:874–888PubMedCrossRefGoogle Scholar
  81. Roberts JD, Byrne PG (2011) Polyandry, sperm competition, and the evolution of anuran amphibians. Adv Study Behav 43:1–53CrossRefGoogle Scholar
  82. Ryser J (1996) Comparative life histories of a low- and high-elevation population of the common frog Rana temporaria. Amphibia-Reptilia 17:183–195CrossRefGoogle Scholar
  83. Sasso-Cerri E, de Faria FP, Freymüller E, Miraglia SM (2004) Testicular morphological changes during the seasonal reproductive cycle in the bullfrog Rana catesbeiana. J Exp Zool 301:249–260CrossRefGoogle Scholar
  84. Schulte-Hostedde AI, Millar JS (2004) Intraspecific variation of testis size and sperm length in the yellow-pine chipmunk (Tamias amoenus): implications for sperm competition and reproductive success. Behav Ecol Sociobiol 55:272–277CrossRefGoogle Scholar
  85. Schulte-Hostedde AI, Millar JS, Hickling GJ (2005) Condition dependence of testis size in small mammals. Evol Ecol Res 7:143–149Google Scholar
  86. Shuster SM, Wade MJ (2003) Mating systems and strategies. Princeton University Press, Princeton, NJGoogle Scholar
  87. Simmons LW (2001) Sperm competition and its evolutionary consequences in insects. Princeton University Press, Princeton, New JerseyGoogle Scholar
  88. Simmons LW, Fitzpatrick JL (2012) Sperm wars and the evolution of male fertility. Reproduction 144:519–534PubMedCrossRefGoogle Scholar
  89. Simmons LW, Kotiaho JS (2002) Evolution of ejaculates: patterns of phenotypic and genotypic variation and condition dependence in sperm competition traits. Evolution 56:1622–1631PubMedCrossRefGoogle Scholar
  90. Simmons LW, Moore AJ (2009) Evolutionary quantitative genetics of sperm. In: Birkhead TR, Hosken DJ, Pitnick S (eds) Sperm biology: an evolutionary perspective. Academic, San Diego, pp 405–434CrossRefGoogle Scholar
  91. Snook RR (2001) Absence of latitudinal clines in sperm characters in North American populations of Drosophila subobscura (Diptera: Drosophilidae). Pan Pac Entomol 77:261–271Google Scholar
  92. Stearns SC (1992) The evolution of life histories. Oxford University Press, OxfordGoogle Scholar
  93. Stutchbury BJ, Morton ES (1995) The effect of breeding synchrony on extra-pair mating systems in songbirds. Behaviour 132:675–690CrossRefGoogle Scholar
  94. Thomsen R, Soltis J, Matsubara M, Matsubayashi K, Onuma M, Takenaka O (2006) How costly are ejaculates for Japanese macaques? Primates 47:272–274PubMedCrossRefGoogle Scholar
  95. Tourmente M, Gomendio M, Roldan ERS (2011) Sperm competition and the evolution of sperm design in mammals. BMC Evol Biol 11:12PubMedPubMedCentralCrossRefGoogle Scholar
  96. Vahed K, Parker DJ (2012) The evolution of large testes: sperm competition or male mating rate? Ethology 118:107–117CrossRefGoogle Scholar
  97. van Noordwijk AJ, de Jong G (1986) Acquisition and allocation of resources: their influence on variation in life history tactics. Am Nat 128:137–142CrossRefGoogle Scholar
  98. Vasudeva R, Deeming DC, Eady PE (2014) Developmental temperature affects the expression of ejaculatory traits and the outcome of sperm competition in Callosobruchus maculatus. J Evol Biol 27:1811–1818PubMedCrossRefGoogle Scholar
  99. Vieites DR, Nieto-Román S, Barluenga M, Palanca A, Vences M, Meyer A (2004) Post-mating clutch piracy in an amphibian. Nature 431:305–308PubMedCrossRefGoogle Scholar
  100. Wang J, Xue W, Li N, Wang X, Jiang H, Xu H (2006) Hibernation of Rana limnocharis in Shanghai farmland. Chin J Ecol 25:1289–1291Google Scholar
  101. Wells KD (2010) The ecology and behavior of amphibians. University of Chicago Press, Chicago, ILGoogle Scholar
  102. Xu F, Adler GH, Li Y (2013) Covariation in insular life-history traits of the rice frog (Fejervarya limnocharis) in eastern China. Asian Herpetol Res 4:28–35CrossRefGoogle Scholar
  103. Zeng Y, Lou SL, Liao WB, Jehle R (2014) Evolution of sperm morphology in anurans: insights into the roles of mating system and spawning location. BMC Evol Biol 14:104PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Long Jin
    • 1
  • Sheng Nan Yang
    • 1
  • Wen Bo Liao
    • 1
  • Stefan Lüpold
    • 2
  1. 1.Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education)China West Normal UniversityNanchongPeople’s Republic of China
  2. 2.Department of Evolutionary Biology and Environmental StudiesUniversity of Zurich-IrchelZurichSwitzerland

Personalised recommendations