This study compiles evidence that reproductive traits favor the resilience of the annual fish Leptopanchax opalescens in habitats with extreme cyclical hydrological variations, such as temporary wetlands. Spawn type, stages of gonadal development, and reproductive characteristics (fecundity) have not previously been reported for species of the genus Leptopanchax. In addition, we compile evidence that the eggs of annual fishes inhabiting temporary wetlands are relatively larger than those of perennial freshwater species.
DNA barcoding
The genus Leptopanchax currently comprise six valid species (Costa 2019), but only a few genes of L. aureoguttatus, and L. citrinipinnis have been sequenced to date for evolutionary studies of Rivulidae (Murphy and Collier 1997; Murphy et al. 1999; Costa et al. 2016). In addition to their use in phylogenetic studies, these genes may be useful to delimit Leptopanchax species through DNA barcoding methods (Hebert et al 2003a), as an alternative to the use of coloration of living males as the main diagnostic feature (Costa, 2019). To reduce the molecular information gap in Leptopanchax, here we sequenced samples of L. opalescens from Guandu River drainage.
Specimens with COI genetic distances less than 2% are considered conspecific following DNA barcoding standards for fish (Hebert et al. 2003a, 2003b, Pereira et al. 2013). Following this criterion, the comparison of sequences available so far supports the morphological hypothesis that L. aureoguttatus, L. citrinipinnis, and L. opalescens are distinct species, despite the coloration similarity and geographic proximity of L. opalescens and L. citrinippinis (Costa 2019). The specimens of L. opalescens used in the present study are from the Sepetiba Bay drainage, while previous samples associated to this taxon are from the Guanabara Bay drainage. Morphometric and meristic data from the L. opalescens populations are overlapped between these different basins (Guedes et al. 2020), but the populations show small color variations difficult to analyze by photographs taken with different parameters (Costa 2019; Souto-Santos et al. 2021). Therefore, it is desirable that specimens from the type locality of L. opalescens (or at least from some locality associated with Guanabara Bay) be sequenced and compared with the Sepetiba sequences made available here.
Spawn type and phases of maturation
The reproductive cycle in females of L. opalescens is discontinuous and spawning in batches, indicated by the presence of oocytes at different stages of development and post-ovulatory follicles in specimes capable of spawning. Similar histological results were observed for other rivulids, such as the annuals Austrolebias charrua (Arezo et al. 2007), Millerichthys robustus (Domínguez-Castanedo et al. 2017), and non-annuals Melanorivulus aff. punctatus (Cassel et al. 2013) and Atlantirivulus riograndensis (Cavalheiro and Fialho 2015). According to the scale of gonad development proposed by Brown-Peterson et al. (2011), L. opalescens has four stages of the reproductive cycle: immature, developing, spawning capable, and regressing. The regressing phase showed disorganized structure with presence of postovulatory follicles and atresia, characterized by the disappearance of the zona radiata in degenerating and resorbing oocytes. However, the regressing phase did not represent the interruption of the reproductive cycle due to the simultaneous presence of oocytes capable of spawning.
In males, the histological organization of the testes corresponds to the restricted lobular pattern because spermatogonia are confined to the distal end of the lobules (Uribe et al. 2015). Restricted spermatogonial testis type is characteristic of all Atherinomorpha (which includes Cyprinodontiformes) and is considered a feature of more derived taxa (Parenti and Grier 2004). Reproduction of L. opalescences was characteristic of species with a continuous cycle and splitted spermiation, due to the consistent presence of sperm capable of being ejaculated in the mature phases. A continued production of sperm has also been reported for other rivulids, such as Millerichthys robustus (Domínguez-Castanedo and Uribe 2019), Melanorivulus aff. punctatus (Cassel et al. 2013) e Austrolebias charrua (Arezo et al. 2007). These characteristics are important in the ecological context, as males will be able to readily release sperm for fertilization whenever females produce new batches of eggs.
Leptopanchax opalescens inhabit temporary wetlands that experience abrupt variations in their areas due to fluctuations in water level (Guedes et al. 2020; Souto-Santos et al. 2021). In these variable and unpredictable habitats, spawning in batches means that during the beginning of the flooding period and at the end of the dry season, egg deposition must occur closer to the center of the swamps, while in the period of maximum flooding, egg deposition may occur in areas more away from the center, i.e., in the water-fluctuating ecotones. Therefore, spawning in batches in habitats in continuous expansion and retraction may determine an asynchrony in time under embryonic diapause within the population, consequently causing an asynchrony in the hatching of eggs of the next generation of annual fish. According to Lowe-McConnell (1987), asynchrony in offspring development increases the chance of survival by reducing potential predation on offspring and intraspecific competition for food and shelter.
Egg size and zona pellucida morphology
Leptopanchax opalescens eggs reached a maximum diameter of 1050 µm, similar to that observed for other Leptopanchax species (maximum 1005 µm; Costa and Leal 2009), and within the diameter range (688–2104 µm) observed for another 60 annual or non-annual killifishes (Thompson et al. 2017). Determining why annual species have eggs with different sizes is not a trivial task. Congeneric annual species of the family Nothobranchiidae can coexist in the same pools, but may present eggs with significantly different sizes (Reichard 2016). Fish can exhibit great variability in egg size among species, and between and within populations. Parental care, environmental quality, fecundity, temperature, latitude, predation, resource availability, size and age of females are some factors that, isolated or in interaction, have been identified as influencing egg size in fish (e.g., Duarte and Alcaraz 1989; Einum et al. 2002; Vrtílek and Reichard 2015; Feiner et al. 2016; Barneche et al. 2018b; Iglesias-Rios et al. 2022).
The zona pellucida of mature eggs of L. opalescens featured mushroom-like projections similar to other species in the genera Leptopanchax and Notholebias (Costa and Leal 2009; Thompson et al. 2015). Leptopanchax opalescens presented a single mushroom-like projection in the centre of each polygonal groove, identical to L. citrinipinnis (Costa and Leal 2009). This corroborates the genetic similarity (DNA barcoding) between these two species in this study. Wourms and Sheldon (1976) hypothesized that these mushroom-like projections on the zona pellucida of Notholebias constitutes a chorionic respiratory system, since there is a network of channels leading to hollow spikes that may function as egg-like aeropiles, similar to insect eggs. This may be an adaptation for annual fishes since a thick, hard and consequently poorly oxygen-permeable zona pellucida may be necessary to prevent desiccation (Thompson et al. 2015).
Fecundity
Leptopanchax opalescens showed reduced batch fecundity (27 ± 7.0 eggs), like other annual species, such as Cynopoecilus melanotaenia (19 ± 26 eggs; Gonçalves et al. 2011) and Austrolebias nigrofasciatus (21.5 ± 12 eggs; Volcan et al. 2011). Fecundity is a currently known reproductive parameter for less than 10 species (e.g., Volcan et al. 2011; Gonçalves et al. 2011; Schalk et al. 2014; Cavalheiro and Fialho 2015) among the 471 species of Rivulidae that occur in the Neotropical Region. Optimal egg size theory suggests that populations evolve a particular egg size that balances the trade-off between egg size and fecundity to maximize reproductive yield (Smith and Fretwell 1974). In other words, larger eggs come at a cost of reducing the number of eggs, which is supported by the findings of this study. In addition to batch fecundity, it is necessary to consider the number of spawning events throughout the breeding season (Wotton and Smith 2015). We do not have information on the number of reproductive events of L. opalescens, but the African Nothobranchiidae and Neotropical Rivulidae annual fish may have daily or weekly spawning events, lasting until senescence (e.g., Polačik et al. 2016; Volcan et al. 2011).
Trade-off between egg size and maximum body length
Analyzes based on absolute diameter of fish eggs demonstrated the occurrence of smaller sizes in more seasonal and less predictable environments (Winemiller and Rose 1992; Morrongiello et al. 2012). However, when the diameter of the eggs is weighted by the species' maximum body size (egg size/TLmax ratio) different perspectives arise. We found that fish with an annual life cycle inhabiting unpredictable habitats (i.e, temporary wetlands) have relatively larger eggs (p < 0.001) than other freshwater perennial species that occur in more hydrologically stable environments (i.e., rivers, lakes and reservoirs). This means that annual fish have a greater reproductive investment in offspring fitness disproportionate to the maternal reserve allocation capacity. Females with smaller reserves draw more heavily on exogenous resources, closer to the income breeding strategy (McBride et al. 2013). This implies in a rapid conversion in the acquired energy from feeding to use to spawn (Aristizabal 2007).
Eggs of annual fish undergo embryonic diapause, a process that requires a larger size to storage of nutrients, lipids and water for long periods buried in the substrate (Riddle and Hu 2021). Large eggs increase survival during the pre- and post-hatch stages especially in environments with low dissolved oxygen (Einum et al. 2002) typical of temporary wetlands. Larger eggs contain more yolk, produce larger larvae with reduced susceptibility to starvation, lower risk of predation, and greater food capture abilities (Wotton and Smith 2015). The energy cost of bath spawning of larger eggs is disproportionately expensive for annual fishes due to their reduced body size (56 mm on average). Preparation for reproduction can cause oxidative stress and affect maternal self-maintenance (Godoy et al. 2020) and consequently somatic growth. However, this seems to be an effective strategy of annual fishes to (1) increase embryo survival in unpredictable habitats that can remain without water for months, and (2) increase offspring fitness in larval stages, as free-living time is limited by the time constraints imposed by the hydrological cycle.