Genetic evidence supports polygamous mating system in a wild population of Prochilodus lineatus (Characiformes: Prochilodontidae), a Neotropical shoal spawner fish

Josiane Ribolli Carolina Isabel Miño Bianca Maria Soares Scaranto David Augusto Reynalte-Tataje Evoy Zaniboni FilhoAbout the authors

ABSTRACT

Behavioral observations made on fish have revealed remarkably diverse reproductive strategies, including polygamy by both sexes. Still, to date, most Neotropical species remain unstudied as to whether the observed reproductive behavior in natural populations correlates with their genetic mating systems. Here, we investigated the genetic mating system of a wild population of Prochilodus lineatus settled in the Middle Uruguay River basin. By using sibship reconstruction and parental inference methods based on microsatellites’ genotypes, we inferred 45 females and 47 males as potential parents of the 87 larvae analyzed. We found evidence supporting polygamous mating in both sexes: while a high percentage of males (44.7%) fertilized the eggs of one female, 55.3% of the inferred males fertilized eggs of up to four females. Likewise, while 44.5% of the inferred females had their eggs fertilized by one only male, 55.5% of females were fertilized by multiple males. The estimated proxy of the effective population size (Nb) was 126, exhibiting moderate to high levels of genetic diversity. The genetic evidence contributed in this study complements earlier behavioral observations of formation of spawning nuclei of aggregating breeders, which may be promoting a polygamous mating strategy in this long-distance migratory fish.

Keywords:
Genetic mating system; Microsatellites; Migratory fish; Parentage; Relatedness

RESUMO

Observações do comportamento de peixes neotropicais têm revelado estratégias reprodutivas marcadamente variáveis, incluindo poligamia nos dois sexos. Ainda assim, até então, a correlação entre comportamento reprodutivo observado em populações naturais e sistemas de acasalamento genético permanece pouco explorada para maioria de espécies Neotropicais. Neste estudo investigamos o sistema genético de acasalamento de Prochilodus lineatus em uma população natural estabelecida no Médio rio Uruguai. Utilizando métodos de reconstrução de grupos familiares e inferências parentais baseados em genótipos de microssatélites, inferimos 45 fêmeas e 47 machos como os possíveis parentais das 87 larvas amostradas. Encontramos evidência que permite apoiar a ocorrência de acasalamento poligâmico em ambos os sexos: enquanto uma percentagem alta de machos (44,7%) fertilizou somente uma fêmea, 55,3% dos machos inferidos fertilizaram mais de uma fêmea (até quatro por macho). Da mesma forma, enquanto que 44,5% das fêmeas inferidas tiveram seus ovos fertilizados por apenas um único macho, 55,5% das fêmeas tiveram ovos fertilizados por múltiplos machos. A estimativa do tamanho populacional efetivo (Nb) foi 126, exibindo níveis entre moderados e altos de diversidade genética. A evidência genética que apresentamos nesse estudo complementa observações iniciais da formação de núcleos de desova que podem promover estratégias de acasalamento poligâmico nessa espécie migratória de longa distância.

Palavras-Chave:
Microssatélites; Parentesco; Paternidade; Peixes migradores; Sistema genético de acasalamento

INTRODUCTION

The mating system of a given species (also known as breeding system; Reynolds, 1996Reynolds JD. Animal breeding systems. Trends Ecol Evol . 1996; 11(2):68-72. https://dx.doi.org/10.1016/0169-5347(96)81045-7
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) can be defined as a behavioral strategy for obtaining mates, and is influenced by many factors, such as life-history traits, ecology and behavior (Klug, 2011Klug H. Animal mating systems. In: eLS. Chichester: John Wiley & Sons, Ltd:; 2011. https://doi.org/10.1002/9780470015902.a0022553
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). This central biological trait of organisms can influence a number of other which, in turn, determine the effective population size, Ne (Nunney, 1993Nunney L. The influence of mating system and overlapping generations on effective population size. Evolution. 1993; 47(5):1329-41. https://doi.org/10.1111/j.1558-5646.1993.tb02158.x
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; Karageorge, Wilson, 2017Karageorge KW, Wilson RR Jr. An integrative mating system assessment of a nonmodel, economically important Pacific rockfish (Sebastes melanops) reveals nonterritorial polygamy and conservation implications for a large species flock. Ecol Evol. 2017; 7(24):11277-91. https://doi.org/10.1002/ece3.3579
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), and, thus, shape evolutionary processes. From a conservation standpoint, the mating system can accurately predict the extinction probability (Legendre et al., 1999Legendre S, Clobert J, Møller AP, Sorci G. Demographic stochasticity and social mating system in the process of extinction of small populations: the case of passerines introduced to New Zealand. Am Nat. 1999; 153(5):449-63. https://dx.doi.org/10.1086/303195
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). Mating systems can govern the population growth rate during and after periods of intense exploitation, and could be used as surrogates to assess the extinction risks and to settle conservation strategies, particularly in species exploited as economic resources (Rowe, Hutchings, 2003Rowe S, Hutchings JA. Mating systems and the conservation of commercially exploited marine fish. Trends Ecol Evol . 2003; 18(11):567-72. https://dx.doi.org/10.1016/j.tree.2003.09.004
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).

Breeding systems of teleostean fishes vary widely and are extraordinarily diverse, from self-fertilization and social monogamy to promiscuous behavior and group spawning (DeWoody, Avise, 2001DeWoody JA, Avise JC. Genetic perspectives on the natural history of fish mating systems. J Hered. 2001; 92(2):167-72. https://dx.doi.org/10.1093/jhered/92.2.167
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; Haddeland et al., 2015Haddeland PJ, Junge C, Serbezov D, Vøllestad LA. Genetic parentage analysis confirms a polygynandrous breeding system in the European grayling (Thymallus thymallus). PloS One. 2015; 10(3):e0122032. https://dx.doi.org/10.1371/journal.pone.0122032
https://dx.doi.org/10.1371/journal.pone....
; Ribolli et al., 2016Ribolli J, Miño CI, Zaniboni-Filho E, Souza Guerreiro TCS, Reynalte-Tataje DA, Freitas PD, Galetti PM Junior. Preliminary insights into the genetic mating system of Neotropical Salminus brasiliensis: kinship assignment and parental reconstruction reveal polygynandry. Ichthyol Res. 2016; 63:187-91. https://dx.doi.org/10.1007/s10228-015-0487-2
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; Smith, Wootton, 2016Smith C, Wootton RJ. The remarkable reproductive diversity of teleost fishes. Fish Fish. 2016; 17(4):1208-15. https://dx.doi.org/10.1111/faf.12116
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). All possible social mating systems can be found among these fishes: monogamy by both sexes, polygyny (male’s polygamy), polyandry (females’ polygamy), polygynandry (polygamy of both sexes) and promiscuity (both sexes mate with multiple partners) (DeWoody, Avise, 2001DeWoody JA, Avise JC. Genetic perspectives on the natural history of fish mating systems. J Hered. 2001; 92(2):167-72. https://dx.doi.org/10.1093/jhered/92.2.167
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; Avise et al., 2002Avise JC, Jones AG, Walker D, DeWoody JA. Genetic mating systems and reproductive natural histories of fishes: lessons for ecology and evolution. Annu Rev Genet. 2002; 36(1):19-45. https://doi.org/10.1146/annurev.genet.36.030602.090831
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; Haddeland et al., 2015Haddeland PJ, Junge C, Serbezov D, Vøllestad LA. Genetic parentage analysis confirms a polygynandrous breeding system in the European grayling (Thymallus thymallus). PloS One. 2015; 10(3):e0122032. https://dx.doi.org/10.1371/journal.pone.0122032
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; Smith, Wootton, 2016Smith C, Wootton RJ. The remarkable reproductive diversity of teleost fishes. Fish Fish. 2016; 17(4):1208-15. https://dx.doi.org/10.1111/faf.12116
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). Furthermore, studies have demonstrated that pheromones may regulate various aspects of the reproductive behavior of teleostean fishes, such as stimulation of mating ceremonies (Liley, Stacey, 1983Liley NR, Stacey EN. Hormones, pheromones, and reproductive behavior in fish. In: Hoar WS, Randall DJ, Donaldson EM, editors. Fish Physiology. Academic Press; 1983. p.1-63.), synchronization of reproduction (Liley, Stacey, 1983Liley NR, Stacey EN. Hormones, pheromones, and reproductive behavior in fish. In: Hoar WS, Randall DJ, Donaldson EM, editors. Fish Physiology. Academic Press; 1983. p.1-63.; Kobayashi et al., 2002Kobayashi M, Sorensen PW, Stacey NE. Hormonal and pheromonal control of spawning behavior in the goldfish. Fish Physiol Biochem. 2002; 26:71-84. https://dx.doi.org/10.1023/A:1023375931734
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), spawning and increase of shoals (Chung-Davidson et al., 2010Chung-Davidson YW, Huertas M, Li W. A review of research in fish pheromones. Chemical communication in crustaceans. In: Breithaupt T, Thiel M, editors. Chemical Communication in Crustaceans. New York: Springer; 2010. p.467-82.). However, in fishes, as it occurs in other vertebrates, such as birds (Griffith et al., 2002Griffith SC, Owens IP, Thuman KA. Extra pair paternity in birds: a review of interspecific variation and adaptive function. Mol Ecol . 2002; 11(11):2195-12. https://dx.doi.org/10.1046/j.1365-294x.2002.01613.x
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), the observed reproductive behavior may not correspond to the genetic mating system: for example, both males and females may take part in extra-pair spawnings (Wootton, Smith, 2014Wootton RJ, Smith CR. Reproductive biology of teleost fishes. Oxford: Wiley-Blackwell; 2014.).

The Curimbatá Prochilodus lineatus (Valenciennes, 1837) (=Prochilodus scrofa Steindachner, 1881) is commercially exploited, and represents one of the most important freshwater resources in South America (Espinach Ros, Fuentes, 2000Espinach Ros A, Fuentes C. Recursos pesqueros y pesquerías de la cuenca del plata. In: Bezzi S, Akselman R, Boschi E, editors. Síntesis del estado de las pesquerías marítimas Argentinas y de la cuenca del Plata. Mar del Plata: INIDEP; 2000. p.1-31.; Avigliano et al., 2016Avigliano E, Fortunato RC, Biole F, Domanico A, Simone S, Neiff JJ, Volpedo AV. Identification of nurseries areas of juvenile Prochilodus lineatus (Valenciennes, 1836) (Characiformes: Prochilodontidae) by scale and otolith morphometry and microchemistry. Neotrop Ichthyol. 2016; 14(3):e160005. http://dx.doi.org/10.1590/1982-0224-20160005
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). Distributed in the Plata basin (Paraná, Paraguay, Uruguay, De La Plata River, among others), it is exploited by fisheries in Argentina, Brazil, Bolivia, Paraguay and Uruguay. Species of the genus Prochilodus Agassiz, 1829 are potamodromous, exhibit high fertility and lack of parental care (Vazzoler, 1996Vazzoler AEAM. Biologia da reprodução de peixes teleósteos: teoria e prática. Maringá: Eduem; 1996.; Winemiller, 2005Winemiller KO. Life history strategies, population regulation, and implications for fisheries management. Can J Fish Aquat Sci . 2005; 62(4):872-85. https://dx.doi.org/10.1139/f05-040
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). Prochilodus lineatus displays total spawning (Lowe-McConnell, 1999Lowe-McConnell RH. Estudos ecológicos de comunidades de peixes tropicais. São Paulo: Edusp; 1999.; Sato, Godinho, 1999Sato Y, Godinho HP. Peixes da bacia do rio São Francisco. In: Lowe-McConnel RH, editor. Estudos ecológicos de comunidades de peixes tropicais. São Paulo: Edusp ; 1999. p.401-13.): females spawn over a short time-frame and no further eggs are shed in that breeding season (Smith, Wootton, 2016Smith C, Wootton RJ. The remarkable reproductive diversity of teleost fishes. Fish Fish. 2016; 17(4):1208-15. https://dx.doi.org/10.1111/faf.12116
https://dx.doi.org/10.1111/faf.12116...
). The Curimbatá also has external fertilization which takes place under continuous water flow (Lowe-McConnell, 1999Lowe-McConnell RH. Estudos ecológicos de comunidades de peixes tropicais. São Paulo: Edusp; 1999.; Sato, Godinho, 1999Sato Y, Godinho HP. Peixes da bacia do rio São Francisco. In: Lowe-McConnel RH, editor. Estudos ecológicos de comunidades de peixes tropicais. São Paulo: Edusp ; 1999. p.401-13.). During the reproductive period, P. lineatus forms large fish aggregations, which are usually found downstream of waterfalls or dams (Godoy, 1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.). As other Neotropical migratory species, the Curimbatá performs upward reproductive migrations towards adequate spawning grounds; they release their eggs into running water, and eggs drift freely with the currents (Harvey, Carolsfeld, 2003Harvey B, Carolsfeld J. Introduction: fishes of the floods. In: Carolsfeld J, Harvey B, Ross C, Baer A, editors. Migratory fishes of South America: biology, fisheries and conservation states. Victoria; Washington (DC); Ottawa: International Development Research Centre; World Bank; World Fisheries Trust; 2003. p.1-18.). Initial reports of observations of numerous P. lineatus breeding events described large fish shoals, composed of hundreds of individuals, resembling a “mating dance”, in which fish jump out of the water in an event that can last several hours (Godoy, 1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.). Decades later, Godinho et al. (2017Godinho AL, Silva CCF, Kynard B. Spawning calls by zulega, Prochilodus argenteus, a Brazilian riverine fish. Environ Biol Fishes. 2017; 100:519-33. https://doi.org/10.1007/s10641-017-0582-5
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) described that males of a congeneric species Prochilodus argenteus Spix, Agassiz, 1829 perform characteristic spawning sounds, suggesting the occurrence of “lek choruses” (it is a call behavior performed by male, in chorus). However, despite the existence of some visual reports made on wild populations of this migratory fish species, details of its spawning and breeding system remain unclear. Therefore, investigating the genetic mating system of P. lineatus can provide useful information to support conservation and management of this species.

Describing the reproductive strategies of fishes in nature is often precluded by the complex biology of these organisms, which prevents direct behavioral observations of the individuals involved in reproductive encounters. In the wild, P. lineatus inhabits large and deep rivers, with high turbidity, and this precludes accurate observations of the spawning events and reproductive behavior of individuals (Godinho et al., 2017Godinho AL, Silva CCF, Kynard B. Spawning calls by zulega, Prochilodus argenteus, a Brazilian riverine fish. Environ Biol Fishes. 2017; 100:519-33. https://doi.org/10.1007/s10641-017-0582-5
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). In such situation, the mating system can be verified or inferred using DNA-based methods, mostly aimed at reconstructing kinship amongst individuals in an array, based on their multilocus genotypes (Jones, Ardren, 2003Jones AG, Ardren WR. Methods of parentage analysis in natural populations. Mol Ecol . 2003; 12(10):2511-23. https://doi.org/10.1046/j.1365-294X.2003.01928.x
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). When eggs are laid in masses so that the progeny may occur in family groups - as in some fishes - additional analytical power is gained by the kinship reconstruction analyses (Flanagan, Jones, 2019Flanagan SP, Jones AG. The future of parentage analysis: from microsatellites to SNPs and beyond. Mol Ecol . 2019; 28(3):544-67. https://dx.doi.org/10.1111/mec.14988
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). This approach is therefore a keystone of molecular ecology, providing important insights into behavior, ecology and evolution (Flanagan, Jones, 2019Flanagan SP, Jones AG. The future of parentage analysis: from microsatellites to SNPs and beyond. Mol Ecol . 2019; 28(3):544-67. https://dx.doi.org/10.1111/mec.14988
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), as well as surprising revelations about the behavior of species in their natural environments (Griffith et al., 2002Griffith SC, Owens IP, Thuman KA. Extra pair paternity in birds: a review of interspecific variation and adaptive function. Mol Ecol . 2002; 11(11):2195-12. https://dx.doi.org/10.1046/j.1365-294x.2002.01613.x
https://dx.doi.org/10.1046/j.1365-294x.2...
), providing cues to broaden the knowledge on evolutionary aspects of such a fundamental biological trait. Indeed, during the last two decades, molecular markers have contributed to clarify the reproductive modes and unraveled the genetic mating systems of several fish species, such as, for example, some salmonids (MacCrimmon, Gots, 1979MacCrimmon HR, Gots BL. World distribution of Atlantic salmon, Salmo solar. J Fish Res Board Can. 1979; 36(4):422-57. https://dx.doi.org/10.1139/f79-062
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; Serbezov et al., 2010Serbezov D, Bernatchez L, Olsen EM, Vøllestad LA. Mating patterns and determinants of individual reproductive success in brown trout (Salmo trutta) revealed by parentage analysis of an entire stream living population. Mol Ecol . 2010; 19(15):3193-205. https://doi.org/10.1111/j.1365-294X.2010.04744.x
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; Weir et al., 2010Weir LK, Breau C, Hutchings JA, Cunjak RA. Multiple paternity and variance in male fertilization success within Atlantic salmon Salmo salar redds in a naturally spawning population. J Fish Biol . 2010; 77(3):479-93. https://doi.org/10.1111/j.1095-8649.2010.02690.x
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; de Mestral et al., 2012de Mestral LG, Herbinger CM, O’Reilly PT. Mating structure of an endangered population of wild Atlantic salmon (Salmo salar) as determined using sibship reconstruction and a novel method of sex inference. Can J Fish Aquat Sci. 2012; 69(8):1352-61. https://dx.doi.org/10.1139/f2012-065
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), trout (Fraser et al., 2005Fraser DJ, Duchesne P, Bernatchez L. Migratory charr schools exhibit population and kin associations beyond juvenile stages. Mol Ecol . 2005; 14(10):3133-46. https://dx.doi.org/10.1111/j.1365-294X.2005.02657.x
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; Serbezov et al., 2010Serbezov D, Bernatchez L, Olsen EM, Vøllestad LA. Mating patterns and determinants of individual reproductive success in brown trout (Salmo trutta) revealed by parentage analysis of an entire stream living population. Mol Ecol . 2010; 19(15):3193-205. https://doi.org/10.1111/j.1365-294X.2010.04744.x
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; Kanno et al., 2011Kanno Y, Vokoun CJ, Letcher BH. Sibship reconstruction for inferring mating systems, dispersal and effective population size in headwater brook trout (Salvelinus fontinalis) populations. Conserv Genet. 2011; 12:619-28. https://dx.doi.org/10.1007/s10592-010-0166-9
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), and sturgeon (Duong et al., 2013Duong TY, Scribner KT, Forsythe PS, Crossman JA, Baker EA. Interannual variation in effective number of breeders and estimation of effective population size in long-lived iteroparous lake sturgeon (Acipenser fulvescens). Mol Ecol. 2013; 22(5):1282-94. https://dx.doi.org/10.1111/mec.12167
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; Jay et al., 2014Jay K, Crossman JA, Scribner KT. Estimates of effective number of breeding adults and reproductive success for white sturgeon. Trans Am Fish Soc. 2014; 143(5):1204-16. https://dx.doi.org/10.1080/00028487.2014.931301
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). To the extent of our knowledge, the molecular-parentage approach has only been applied to investigate the mating strategies of Salminus brasiliensis (Cuvier, 1816) (Ribolli et al., 2016Ribolli J, Miño CI, Zaniboni-Filho E, Souza Guerreiro TCS, Reynalte-Tataje DA, Freitas PD, Galetti PM Junior. Preliminary insights into the genetic mating system of Neotropical Salminus brasiliensis: kinship assignment and parental reconstruction reveal polygynandry. Ichthyol Res. 2016; 63:187-91. https://dx.doi.org/10.1007/s10228-015-0487-2
https://dx.doi.org/10.1007/s10228-015-04...
). Thus, in contrast to the many investigations focusing on reproductive strategies and mating behavior of such commercially important organisms, the knowledge on Neotropical migratory fishes is still incipient.

The genetic mating system of fishes showing external fertilization in the freshwater courses, as the Curimbatá, can be approached by sibship reconstruction based on genotypic information. Here, we assessed aspects of the breeding system of wild P. lineatus by means of molecular markers and kinship reconstruction of larvae sampled at the Uruguay River, South Brazil. Our aims were to: i) estimate the number of breeding males and females in the population; ii) assess the most prevalent genetic mating system in both sexes; and iii) estimate the reproductive contribution of males or females during spawning.

MATERIAL AND METHODS

Field sampling and laboratory procedures. We sampled P. lineatus eggs at a site located in the Middle Uruguay River basin (27°15’50.16”S; 54°03’16.97” W), located between Turvo State Park (Brazil) and Yabotí Biosphere Reserve (Argentina), which are both National protected areas. This area represents the largest dam-free stretch in the Middle Uruguay River (ca. over 800 km), and is of paramount importance for the reproduction of long-distance migratory fishes (Reynalte-Tataje et al., 2012Reynalte-Tataje DA, Zaniboni-Filho E, Bialetzki A, Agostinho AA. Temporal variability of fish larvae assemblages: influence of natural and anthropogenic disturbances. Neotrop Ichthyol . 2012; 10(4):837-46. http://dx.doi.org/10.1590/S1679-62252012000400017
http://dx.doi.org/10.1590/S1679-62252012...
; Ziober et al., 2015Ziober SR, Reynalte-Tataje DA, Zaniboni-Filho E. The importance of a conservation unit in a subtropical basin for fish spawning and growth. Environ Biol Fish. 2015; 98:725-37. https://doi.org/10.1007/s10641-014-0307-y
https://doi.org/10.1007/s10641-014-0307-...
; Reynalte-Tataje et al., 2017Reynalte-Tataje DA, Barcellos RP, Hartmann PB, Scherer JB, Martine G, DeVlieger IT, Zaniboni-Filho E, Hermes-Silva S, Pelicice FM. O médio rio Uruguai como importante área de reprodução do surubim-pintado Pseudoplatystoma corruscans (Siluriformes: Pimelodidae). Boletim Sociedade Brasileira de Ictiologia. 2017; (122):10-15.; Ribolli et al., 2018Ribolli J, Zaniboni-Filho E, Freitas PD, Galetti Junior PM. Genetic evidences of non-reproductive shoaling in the freshwater fish Salminus brasiliensis. Hydrobiologia . 2018; 815:65-72. https://dx.doi.org/10.1007/s10750-018-3550-y
https://dx.doi.org/10.1007/s10750-018-35...
). Sampling was conducted in March 2014 (summer season in the southern hemisphere), at night time (around 9:00 PM), using conical-cylindrical plankton nets (0.5 mm mesh and mouth area of 0.11 m2) following methods described in Hermes-Silva et al. (2009Hermes-Silva S, Reynalte-Tataje D, Zaniboni-Filho E. Spatial and temporal distribution of ichthyoplankton in the upper Uruguay river, Brazil. Braz Arch Biol Technol. 2009; 52(4):933-44. http://dx.doi.org/10.1590/S1516-89132009000400017
http://dx.doi.org/10.1590/S1516-89132009...
). All eggs were sampled simultaneously and using the same plankton net, and were immediately incubated in plastic bags supplied with water flow and oxygen input. Prochilodus lineatus eggs were captured at the initial segmentation stage (about 8-10 hours after fertilization), thus confirming that all eggs derived from the same spawning event, and the larvae were cultivated in the laboratory until juvenile stage (3-month-old) according to the live ichthyoplankton methodology (Reynalte-Tataje, Zaniboni-Filho, 2008Reynalte-Tataje DA, Zaniboni-Filho E. Biologia e identificação de ovos e larvas de peixes do alto rio Uruguai, Brasil. In: Zaniboni-Filho E, Nuñer APO, editors. Reservatório de Itá: estudos ambientais, desenvolvimento de tecnologias de cultivo e conservação da ictiofauna. Florianópolis: Editora UFSC; 2008. p.157-76.). A fin fragment of each 87 juveniles were cut using sterilized surgical scissors and preserved in ethanol 96% at -20 °C until processed. Voucher of P. lineatus from Uruguay River is deposited in the Museum of the State University of Londrina (MZUEL11729).

DNA extraction and marker amplification. Genomic DNA was isolated from fin samples following an universal salt extraction protocol (Aljanabi, Martinez, 1997Aljanabi SM, Martinez I. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 1997; 25(22):4692-93. https://dx.doi.org/10.1093/nar/25.22.4692
https://dx.doi.org/10.1093/nar/25.22.469...
). Samples were amplified at 11 microsatellite loci, using fluorescently-labeled primers (see details in Additional File S1). Polymerase Chain Reactions were carried out in 10 µL mixes containing: 50 ng of template DNA, 15 mM of MgCl2, 1U Taq DNA polymerase (Uniscience, Brasil), 1X PCR buffer, 2 mM of each forward and reverse primer, and 1.1 mM of dNTPs (Invitrogen, Brasil). Standard cycling parameters were: initial denaturation at 95 °C for 1 min, followed by 35 cycles of 95 °C for 30 sec, annealing temperature of each primer (File S1) for 45 s, extension at 72 °C for 30 sec, followed by a final elongation step at 72 °C for 20 min. Allele sizing was carried out by capillary electrophoresis using 1 µL of diluted PCR product in ultrapure water (2:15 µL), added to 0.25 µL of GS600 LIZ® and 8.75 µL of formamide HIDI™ and run in an ABI 3500XL Sequencer (Applied Biosystems). Genotyping of each sample was carried out using automatic bins in Gene Mapper v.3.2 software (Applied Biosystems) and eye-checked.

Statistical analyses. The genotypic dataset was analyzed for the presence of null alleles, allelic dropout and stutter peaks using MICRO-CHECKER v2.2.3 (van Oosterhout et al., 2004Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P. MICRO‐CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes. 2004; 4(3):535-38. https://doi.org/10.1111/j.1471-8286.2004.00684.x
https://doi.org/10.1111/j.1471-8286.2004...
). Departures from Linkage Equilibrium or Hardy-Weinberg Equilibrium (HWE) were investigated using GENEPOP 1.2 (Raymond, Rousset, 1995Raymond M, Rousset F. An exact test for population differentiation. Evolution. 1995; 49(6):1280-83. http://dx.doi.org/10.2307/2410454
http://dx.doi.org/10.2307/2410454...
) and GenAlEx v6.4 (Peakall, Smouse, 2006Peakall ROD, Smouse PE. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes. 2006; 6(1):288-95. https://doi.org/10.1111/j.1471-8286.2005.01155.x
https://doi.org/10.1111/j.1471-8286.2005...
), respectively. Mean number of alleles per locus, and mean observed (Ho) and expected (He) heterozygosity values were calculated using GenAlEx. Allelic richness (Ar; Leberg, 2002Leberg PL. Estimating allelic richness: effects of sample size and bottlenecks. Mol Evol. 2002; 11(11):2445-49. https://dx.doi.org/10.1046/j.1365-294x.2002.01612.x
https://dx.doi.org/10.1046/j.1365-294x.2...
), inbreeding coefficients (F IS ; Weir, Cockerham, 1984Weir BS, Cockerham CC. Estimating F-statistics for the analysis of population structure. Evolution, 1984; 38(6):1358-70. https://dx.doi.org/10.2307/2408641
https://dx.doi.org/10.2307/2408641...
) and tests for heterozygotes’ excess (pL) or deficit (pS) were calculated using FSTAT 2.9.3.2 (Goudet, 2001Goudet J. FSTAT: a computer program to calculate F-statistics. J Hered . 2001; 86(6):485-86. Release 2.9. 3.2. https://dx.doi.org/10.1093/oxfordjournals.jhered.a111627
https://dx.doi.org/10.1093/oxfordjournal...
).

Given that our aim was to maximize our ability to correctly reconstruct sibship arrays, in order to accurately infer the genetic mating system of P. lineatus, we first conducted an analysis in KinInfor v.1 (Wang, 2006Wang J. Informativeness of genetic markers for pairwise relationship and relatedness inference. Theor Popul Biol. 2006; 70(3):300-21. https://doi.org/10.1016/j.tpb.2005.11.003
https://doi.org/10.1016/j.tpb.2005.11.00...
), to determine the effect of the number of loci used on the power of relationship analysis (PWR). For this, we simulated 1,000,000 genotypes of full-siblings as primary hypothesis and unrelated individuals as null hypothesis, used empirical allele frequencies, a genotypic error of 0.01 for each marker, and 0.05 alpha level.

We then used COLONY v2 (Jones, Wang, 2010Jones OR, Wang J. COLONY: a program for parentage and sibship inference from multilocus genotype data. Mol Ecol Resour. 2010; 10(3):551-55. https://dx.doi.org/10.1111/j.1755-0998.2009.02787.x
https://dx.doi.org/10.1111/j.1755-0998.2...
) to infer potential parentage and full- and half-sibship arrays based on larvae genotypes, by running a full-likelihood method and assuming, separately: i) a polygamous mating system without inbreeding for both sexes, ii) a polygamous mating system for males and monogamy for females, iii) a polygamy for females and monogamy for males and iv) monogamy for both sexes. COLONY v2 allows for deviations from Hardy-Weinberg equilibrium. We run five replicate runs for each of the different mating system assumptions and only recorded the resulting full- and half-sibling assignments with a probability >0.80 which were consistently recovered in all runs (i.e., the five replicates of each of the four types of mating system). Finally, we used the Sibship Assignment method implemented in COLONY to estimate the effective number of breeders, Nb (and its corresponding 95% Confidence Interval) as proxy of the population effective size (Ne), assuming an unknown number of parents, very high likelihood precision algorithm and polygamy in both-sexes.

RESULTS

Population genetic parameters, larvae sibship arrays and inference on parentage patterns. The 11 microsatellites were polymorphic (total of 178 alleles, 4-33 allele/locus) (Tab. 1). Loci Pli34 and Plin139 deviated significantly from Linkage Equilibrium (P = 0.00), therefore, locus Pli34 was excluded from sibship reconstruction analyses and estimates of Nb, which were then performed with a set of 10 unlinked microsatellites. Six loci deviated significantly from HWE (Tab. 1). The mean He ranged from 0.13 to 0.95 and Ho ranged from 0.13 to 0.93 (Tab. 1). The multilocus PWR to discriminate between full-siblings and unrelated individuals was 0.99. COLONY analyses recovered the number of sires producing each full-sib array with a high probability (minimum = 0.95, the value obtained for the two broods sired by four males, see below). Likewise, the number of mothers producing each progeny array was recovered with a high probability (minimum = 0.95).

TABLE 1
| Summary population genetic diversity for eleven microsatellite loci genotyped in Prochilodus lineatus larvae collected in the Middle Uruguay River basin, Southern Brazil. Number of individuals genotyped (N), Number of alleles (A), observed heterozygosity (Ho), expected (He) heterozygosity, inbreeding coefficient (F IS ) and probabilities of tests for deviation from Hardy-Weinberg equilibrium (PHWE) are shown (the asterisks denote significant values, P < 0.05).

Our analyses inferred 47 males as sires of the larvae, 21 of which (44.7%) sired one larva, while 14 (29.8%) sired two larvae, 10 males (21.3%) sired three and two of males (4.2%) sired four larvae (Fig. 1A). Forty-five females were inferred as dams, 19 of which (42.2%) mothered only one larva, while 14 (31.1%) produced two, nine (20.0%) sired three larvae, two females (4.4%) produced four larvae, and one (2.3%) mothered five larvae (Fig. 1A). While 21 males fertilized the eggs from only one female, 16 males (34.0%) fertilized eggs of two females each, nine males (19.2%) fertilized eggs of three females each, and a single male (2.1%) fertilized eggs from up to four females (Fig. 1B). Likewise, while females (44.5% of the inferred females) had their eggs fertilized by a single male, 13 females (28.8%) had their eggs fertilized by two males, 10 females (22.2%) by three, and two (4.5%) by four males (Fig. 1B). From these figures, the number of males siring a brood varied from one to four and the population frequency of multiple paternity was 55.5% (sum of the sibship arrays sired by two or more males).

Under polygamy by both sexes, and assuming random mating, the Nb estimated in the studied population was 126 fishes (95% CI: 94-171).

FIGURE 1
| Inferred parentage and mating patterns derived from analysis of 87 Prochilodus lineatus larvae sampled at the Middle Uruguay River in Brazil. A. Number of adults identified as parents of the sampled larvae; B. Number of different mates inferred for males and females.

DISCUSSION

We conducted kinship reconstruction in Prochilodus lineatus based on microsatellites’ genotypes of larvae, and found genetic evidence suggesting that polygamy is the most prevalent mating system in both sexes, in agreement with previous field observations of shoals during P. lineatus spawning events in nature (Godoy, 1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.). Kinship analyses showed that 45.98% of the 87 analyzed larvae were not attributed to any sibship array, i.e. they were unrelated to each other. This result was further supported by the fact that a high percentage of males (44.7%) and females (42.2%) contributed with only one descendant each. These results seem to suggest that the analyzed breeding population of P. lineatus exhibits a percentage of monogamous mating.

It is worth noting that 47 larvae were produced by two or more females and two or more males (57.8% and 55.3%, respectively) with multiple breeders participating in the same reproductive event. Theoretically, reproductive aggregations in species that release pelagic eggs imply little opportunity to choose mates (Stockley et al., 1997Stockley P, Gage MJG, Parker GA, Møller AP. Sperm competition in fishes: the evolution of testis size and ejaculate characteristics. Am Nat . 1997; 149(5):933-54. https://dx.doi.org/10.1086/286031
https://dx.doi.org/10.1086/286031...
). Despite the scarce information about spawning of Neotropical fishes in nature, parentage analyses conducted on P. lineatus in semi-natural conditions (reproduction in the laboratory) identified unequal contribution of parents to the progeny, suggesting reproductive advantage (Souza et al., 2018Souza FPD, Castro PLD, Goes ESDR, Ribeiro RP, Santos SCAD, Lima ECSD, Murari PJF, Lopera-Barrero NM. Genetic variability of Prochilodus lineatus in artificial and semi-natural reproduction. Ital J Anim Sci. 2018; 17(2):321-25. https://doi.org/10.1080/1828051X.2017.1365312
https://doi.org/10.1080/1828051X.2017.13...
). Additionally, some gametes (or breeders) may have dominance during fertilization, as observed in pooled-milt fertilizations of Rhamdia quelen (Quoy, Gaimard, 1824) in laboratory conditions (Ribolli, Zaniboni-Filho, 2009Ribolli J, Zaniboni-Filho E. Individual contributions to pooled-milt fertilizations of silver catfish Rhamdia quelen. Neotrop Ichthyol . 2009; 7(4):629-34. https://doi.org/10.1590/S1679-62252009000400011
https://doi.org/10.1590/S1679-6225200900...
). It is important to emphasize here that the observed pattern of skewed parental contribution, instead of suggesting male or female dominance, could be reflecting the sampling strategy. For example: considering that each P. lineatus female can produce on average 160,000 oocytes per kg of weight (Vazzoler, 1996Vazzoler AEAM. Biologia da reprodução de peixes teleósteos: teoria e prática. Maringá: Eduem; 1996.), the 45 potential females inferred as participating in the spawning event would have produced at least 7,200,000 eggs, of which we randomly sampled 87. Therefore, the single-larvae arrays can be explained as the result of the mixture of eggs from distinct spawning nuclei (i.e., sired by different parents) carried out by the river flow.

Another avenue of evidence which could support our findings comes from observations of a peculiar reproductive display in some Prochilodus species: in the spawning arenas, some males made ‘calls’ to attract partners (Godinho et al., 2017Godinho AL, Silva CCF, Kynard B. Spawning calls by zulega, Prochilodus argenteus, a Brazilian riverine fish. Environ Biol Fishes. 2017; 100:519-33. https://doi.org/10.1007/s10641-017-0582-5
https://doi.org/10.1007/s10641-017-0582-...
), similar to the spawning description by Godoy (1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.). According to the last author, during the reproduction of P. lineatus, a group with thousands of individuals was observed, where males called females by characteristic sounds (“males concentrate in the center of the river and females remain on the margins, waiting for the moment to attack the males”; details in Fig. 2A; Godoy, 1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.). Decades after Godoy’s (1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.) report, Godinho et al. (2017Godinho AL, Silva CCF, Kynard B. Spawning calls by zulega, Prochilodus argenteus, a Brazilian riverine fish. Environ Biol Fishes. 2017; 100:519-33. https://doi.org/10.1007/s10641-017-0582-5
https://doi.org/10.1007/s10641-017-0582-...
) reported a similar reproductive behavior for P. argenteus suggesting a “lek mating system”, stimulated by male calls. On the other hand, direct observation of natural spawning of P. lineatus indicated that fishes aggregate in several nuclei composed by many shoals of fishes moving the water simultaneously. Likewise, in laboratory conditions, males touch female genitalia and compress the body of females, thus stimulating the release of oocytes (Evoy Zaniboni-Filho, 2019, pers. comm.). In these cases, several males simultaneously stimulated a mature female, which, when exhausted, would move away, allowing males to start the same process with another mature female, and over again (for details see Fig. 2B). The sexual pheromones released by mature females would stimulate reproductive synchrony (Stacey, 2003Stacey N, Chojnacki A, Narayanan A, Cole T, Murphy C. Hormonally derived sex pheromones in fish: exogenous cues and signals from gonad to brain. Can J Physiol Pharmacol. 2003; 81(4):329-41. https://dx.doi.org/10.1139/y03-024
https://dx.doi.org/10.1139/y03-024...
), and could promote the reproductive aggregation described above. Fonseca et al. (2010Fonseca FAL, Ituassu DR, Cavero BAS, Bordinhon AM. Cultivo de curimatã (Prochilodus spp.). In: Baldisserotto B, Gomes LC, organizers. Espécies nativas para piscicultura no Brasil. Santa Maria: Editora da Universidade Federal de Santa Maria; 2010. p.57-83.) also report that artificially induced females of Prochilodus sp. do not spawn alone; they naturally spawn when stored in tanks together with males. The sexual pheromones released by mature females in this case would be responsible for attracting males and synchronize the reproduction, as described above. In teleost fish, the pre-spawning release of sex pheromones causes a rapid increase of sexual activity and attraction and excitation of partners to mate (Hoar et al., 1983Hoar WS, Randall DJ, Donaldson EM. Fish Physiology Reproduction: behavior and fertility control. Academic Press; 1983.; Chung-Davidson et al., 2010Chung-Davidson YW, Huertas M, Li W. A review of research in fish pheromones. Chemical communication in crustaceans. In: Breithaupt T, Thiel M, editors. Chemical Communication in Crustaceans. New York: Springer; 2010. p.467-82.). Females’ pheromones have been found to modulate males’ reproductive behavior in several fish species, including Salmo trutta Linnaeus, 1758 (Laberge, Hara, 2003Laberge F, Hara TJ. Behavioural and electrophysiological responses to F-prostaglandins, putative spawning pheromones, in three salmonid fishes. J Fish Biol. 2003; 62(1):206-21. https://dx.doi.org/10.1046/j.1095-8649.2003.00020.x
https://dx.doi.org/10.1046/j.1095-8649.2...
), Carassius auratus (Linnaeus, 1758) (Kobayashi et al., 2002Kobayashi M, Sorensen PW, Stacey NE. Hormonal and pheromonal control of spawning behavior in the goldfish. Fish Physiol Biochem. 2002; 26:71-84. https://dx.doi.org/10.1023/A:1023375931734
https://dx.doi.org/10.1023/A:10233759317...
) and Misgurnus anguillicaudatus (Cantor, 1842) (Ogata et al., 1994Ogata H, Kitamura S, Takashima F. Release of 13, 14-dihydro-15-ketoprostaglandin F2a, a sex pheromone, to water by cobitid loach following ovulatory stimulation. Fish Sci. 1994; 60(2):143-48.), but have not yet been reported in P. lineatus. The descriptions and hypotheses aforementioned of the spawning behavior of P. lineatus (Evoy Zaniboni-Filho, 2019, pers. comm.) could explain the polygamous mating by males and females inferred in our study, and complement our understanding of the reproductive behavior of P. lineatus in nature.

FIGURE 2
| Alternative schematic representations of spawning in Prochilodus lineatus. A. Schematic drawing of spawning moment of Prochilodus lineatus (=Prochilodus scrofa), according to Godoy (1975Godoy MP. Peixes do Brasil, subordem Characoidei: bacia do rio Mogí Guassú. Piracicaba: Editora Franciscana; 1975.), whom described that males, positioned in the center of the river channel, performed distinctive sounds, possibly attracting females (figure drawn by Josiane Ribolli and Dennis Fernando Moreno, inspired from the original by Godoy, M. P, 1975, fig. 153, p.684); B. Hypothetical schematic representation of reproduction and spawning of P. lineatus in the wild according to direct observations made by Evoy Zaniboni-Filho (personal communication). The exhausted mature female is marked with an asterisk. Figure drawn by Josiane Ribolli and Dennis Fernando Moreno.

Our genetic data inferring many males and females as sires of the collected eggs support previous evidence from experimental conditions and field observations in suggesting that reproduction of P. lineatus occur in different spawning nuclei. Although not investigated in the present study, the formation of such nuclei may result of orchestrated calls made by mature males or by attraction and stimulation mediated by sexual pheromones, or both factors acting complementarily. Yet, hormone-behavior studies were not carried out in P. lineatus to date, so that this hypothesis remains to be thoroughly investigated.

The studied larvae showed moderate to high levels of neutral genetic diversity, which could be indicative of a stable and large population, as observed in other Neotropical migratory fishes, such as, for example, Prochilodus costatus Valenciennes, 1850 (Carvalho-Costa et al., 2008Carvalho-Costa LF, Hatanaka T, Galetti Júnior PM. Evidence of lack of population substructuring in the Brazilian freshwater fish Prochilodus costatus. Genet Mol Biol . 2008; 31(S1):377-80. https://doi.org/10.1590/S1415-47572008000200036
https://doi.org/10.1590/S1415-4757200800...
; Braga-Silva, Galetti Jr., 2016Braga-Silva A, Galetti Júnior PM. Evidence of isolation by time in freshwater migratory fish Prochilodus costatus (Characiformes, Prochilodontidae). Hydrobiologia. 2016; 765:159-67. https://dx.doi.org/10.1007/s10750-015-2409-8
https://dx.doi.org/10.1007/s10750-015-24...
) and P. lineatus (Rueda et al., 2013Rueda EC, Carriquiriborde P, Monzón AM, Somoza GM, Ortí G. Seasonal variation in genetic population structure of sábalo (Prochilodus lineatus) in the Lower Uruguay River. Genetica. 2013; 141:401-07. https://doi.org/10.1007/s10709-013-9739-0
https://doi.org/10.1007/s10709-013-9739-...
). It is worth mentioning that the studied larvae were sampled at an area with low disturbance lying between two areas of high conservation concern, officially protected by Argentina and Brazilian laws. In such preserved habitats, it is possible that P. lineatus populations remain large and stable, thus maintaining moderate-high levels of genetic diversity. Retention of genetic diversity is important for the conservation of P. lineatus which is vulnerable to habitat fragmentation, degradation, pollution, overexploitation, and cascade effects imposed on population dynamics by reduced dispersal, restricted by dams (Smolders et al., 2002Smolders AJP, Guerrero Hiza MA, van der Velde G, Roelofs JGM. Dynamics of discharge, sediment transport, heavy metal pollution and sábalo (Prochilodus lineatus) catches in the Lower Pilcomayo River (Bolivia). River Res Appl. 2002; 18(5):415-27. https://dx.doi.org/10.1002/rra.690
https://dx.doi.org/10.1002/rra.690...
; Agostinho et al., 2004Agostinho AA, Gomes LC, Veríssimo S, Okada EK. Flood regime, dam regulation and fish in the Upper Paraná River: effects on assemblage attributes, reproduction and recruitment. Rev Fish Biol Fish. 2004; 14:11-19. https://dx.doi.org/10.1007/s11160-004-3551-y
https://dx.doi.org/10.1007/s11160-004-35...
; Pesoa, Schulz, 2010Pesoa NA, Schulz UH. Diel and seasonal movements of grumatã Prochilodus lineatus (Valenciennes 1836) (Characiformes: Prochilodontidae) in the Sinos River, southern Brazil. Braz J Biol. 2010; 70(4):1169-77. http://dx.doi.org/10.1590/S1519-69842010000600006
http://dx.doi.org/10.1590/S1519-69842010...
; Rueda et al., 2013Rueda EC, Carriquiriborde P, Monzón AM, Somoza GM, Ortí G. Seasonal variation in genetic population structure of sábalo (Prochilodus lineatus) in the Lower Uruguay River. Genetica. 2013; 141:401-07. https://doi.org/10.1007/s10709-013-9739-0
https://doi.org/10.1007/s10709-013-9739-...
; Costa et al., 2018Costa RS, Okada EK, Agostinho AA, Gomes LC. Variação temporal no rendimento e composição específica da pesca artesanal do alto rio Paraná, PR-Brasil: os efeitos crônicos dos barramentos. Bol Inst Pesca. 2018; 38(3):199-213.). The analyzed population could be thus seen as a source of genetic diversity which can enable adaptation to new environmental pressures.

The set of polymorphic microsatellite markers was powerful enough to perform kinship reconstruction of family groups, and the significant deviations from Hardy-Weinberg equilibrium possibly reflect a non-closed population with migration. The Ne estimated for the studied population was higher than the number of potential inferred parents; such an increased Ne may be reflecting a balanced individual reproductive success during spawning, a low variation in family size, or the reproductive strategy of polygamy by both sexes. In addition, the dispersion of eggs by current-drifting allows the admixture of progeny from several families and different spawning nuclei, and might concurrently be contributing to the high genetic diversity of the examined sample, maximizing its effective size.

In sum, this study investigated, for the first time, the genetic mating system in of a wild population of P. lineatus, a spawner migratory fish which aggregates in shoals during reproduction. We contribute novel evidence suggesting that this species shows a monogamous genetic mating system in combination with polygamy by males and females, supporting earlier behavioral observations. We suggest that spawning happens with the formation of several nuclei, in which the eggs released by a female could be fertilized by several males, which in turn can accompany the spawning of other females and also take part in other fertilizations. The result of this reproductive behavior associated with the environmental characteristics of the site used for spawning (large river flow, which guarantees the dispersal and mixture of the eggs) has allowed the maintenance of a similar sexual proportion of the parents. This study expands the knowledge about the mating strategies and reproductive behavior of Neotropical migratory fishes in nature through the use of molecular approaches. Our results can also support the management and conservation of such an important fishery resource.

ACKNOWLEDGMENTS

JR thanks Dennis Fernando Moreno for the preparation and edition of the figures. JR thanks PNPD/CAPES; EZF thanks CNPq (Grant 304949/2017-5). We acknowledge the Laboratory of Developmental Physiology and Plant Genetics (LFDGV-UFSC) for laboratory facilities. This study was partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Brasil, Finance Code 001). CIM is grateful to Indio and Sergio Quintana for their love, time and patience.

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ADDITIONAL NOTES

  • HOW TO CITE THIS ARTICLE

    Ribolli J, Miño CI, Scaranto BMS, Reynalte-Tataje DA, Zaniboni Filho E. Genetic evidence supports polygamous mating system in a wild population of Prochilodus lineatus, a Neotropical shoal spawner fish. Neotrop Ichthyol. 2020; 18(2):e190123. https://doi.org/10.1590/1982-0224-2019-0123

Publication Dates

  • Publication in this collection
    26 June 2020
  • Date of issue
    2020

History

  • Received
    06 Nov 2019
  • Accepted
    22 Apr 2020
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E-mail: neoichth@nupelia.uem.br