Abstract
Multiple paternity is widespread in nature and despite costs, it has many associated benefits like increased genetic diversity and fertilization success. It has been described in many viviparous systems, suggesting the existence of some fitness advantages counteracting the inherent costs of viviparity, such as fecundity reduction and high parental investment. Reproductively polymorphic species, like the urodele Salamandra algira, which shows two types of viviparity: larviparity (i.e., delivering aquatic larvae), and pueriparity (i.e., delivering terrestrial metamorphosed juveniles), are suitable systems to study the relationship between reproductive modes and polygamous mating. Here, multiple paternity is confirmed in a pueriparous lineage of S. algira, as previously verified for the pueriparous lineages of the reproductively polymorphic species S. salamandra, suggesting polyandry is a successful mating strategy in pueriparous systems with reduced brood sizes. We discuss the potential benefits of polyandry in the context of viviparity evolution in urodeles.
Viviparity, or live-bearing, understood as the retention of eggs and embryos within the mother’s genital tract until fully developed individuals that actively interact with their environment are delivered, has associated costs. It generally results in a reduction in clutch size and fecundity, and an increase in parental investment (Goodwin et al., 2002; Wells, 2007; Roitberg et al., 2013). Consequently, the costs of reproductive failures due to genetic incompatibilities, inbreeding, or mating with less fertile males are greater than in reproductive modes with larger broods and lower parental investment (Liu and Avise, 2011). In addition, a reduction in the number of descendants can have demographic and genetic consequences, affecting effective population size (Ne), which in turn may compromise population genetic diversity (Romiguier et al., 2014; Ellegren and Galtier, 2016). Polyandrous mating systems, that may potentially buffer the genetic consequences of the reduction in offspring number, enhance offspring genetic quality, and ensure reproduction success, will be especially valuable in viviparous species (Zeh and Zeh, 2001; Tregenza and Wedell, 2002; Avise et al., 2011; Slatyer et al., 2012). Indeed, the occurrence of multiple paternity in viviparous systems is well known and has been documented in distinct taxonomic groups (e.g., amphibians, reptiles, fishes), suggesting that multiple mating might entail fitness benefits for viviparous species (Avise and Liu, 2011; Avise et al., 2011).
Viviparity has independently evolved in all vertebrate taxa excepting birds and cyclostomes, and is an homoplastic innovation that has evolved multiple times in the tree of life (Wake, 1992; Blackburn, 2015). Within extant amphibians, viviparity is relatively common in caecilians, but rare in anurans and caudates (Duellman and Trueb, 1986; Duellman, 1989), and the origin and mechanisms underlying its acquisition vary among species (Wake, 2015). For instance, in viviparous groups that retain the typical biphasic life-cycle, we can differentiate two types of viviparity attending to stage of development at birth: larviparity, when females deliver aquatic free larvae; and pueriparity, when fully metamorphosed juveniles are born (sensu Greven, 2003). Both strategies can be found within the order Caudata, in which viviparity is restricted to two genera, Lyciasalamandra and Salamandra, of the subfamily Salamandrinae (Buckley, 2012), which are sister lineages. All species included within the genus Lyciasalamandra are pueriparous (Veith et al., 1998), while all six species in genus Salamandra display larviparity, pueriparity, or both (Buckley, 2012). While the Corsican (S. corsica) and Near Eastern (S. infraimmaculata) fire salamanders are strictly larviparous (Sparreboom, 2014), the so-called Alpine salamanders, S. atra and S. lanzai, are strictly pueriparous, showing an extreme reduction of brood size (Guex and Greven, 1994; Miaud et al., 2001). On the other hand, the sister species S. algira and S. salamandra (Burgon et al., 2021) show an unique polymorphic reproductive mode, displaying both larviparity and pueriparity (Joly, 1986; Bas and Gasser, 1994; Dopazo and Alberch, 1994; Greven and Guex, 1994; Barroso and Bogaerts, 2003; Velo-Antón et al., 2014, 2015; Dinis and Velo-Antón, 2017). In both species, larviparity is the most widespread and ancestral reproductive strategy. In contrast, pueriparity has evolved multiple times (García-París et al., 2003; Velo-Antón et al., 2007; Dinis et al., 2019), and is phylogenetically constrained to one (S. a. tingitana) or three (S. s. bernardezi; S. s. fastuosa and S. s. gallaica) subspecies, and geographically restricted to northern Moroccan and northern Iberian populations in S. algira and S. salamandra, respectively (fig. 1a) (Velo-Antón et al., 2015; Dinis and Velo-Antón, 2017).
(a) Distribution of both species displaying reproductive polymorphism, Salamandra salamandra (in blue) and Salamandra algira (in green). In red, the area of occurrence of pueriparity within each species. Values next to each pueriparous nuclei denote the frequency of multiple paternity. (b) Detail of the western-most distribution area of S. algira where the pueriparous lineage occurs (in red). The box shows the number of fathers and the proportion of the offspring sired by each male for all four studied females. Numbers in brackets are clutch size in each female.
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10075
The evolution, occurrence and main reproductive traits of pueriparity have been thoroughly studied in S. salamandra (Greven and Guex, 1994; García-París et al., 2003; Buckley et al., 2007; Velo-Antón et al., 2007, 2012, 2015; Alarcón-Ríos et al., 2020). Differences in brood size between viviparous modes (Velo-Antón et al., 2015) and multiple paternity within both viviparous strategies (Steinfartz et al., 2006; Caspers et al., 2014; Alarcón-Ríos et al., 2020) have been documented for S. salamandra. Moreover, those studies show that multiple paternity (i.e., polyandry) is a common phenomenon within S. salamandra irrespective of the reproductive strategy and differences in brood sizes (Alarcón-Ríos et al., 2020). However, knowledge about pueriparity in the North African salamander is scarce (Donaire-Barroso and Bogaerts, 2000; Donaire-Barroso et al., 2001; Beukema et al., 2010; Dinis and Velo-Antón, 2017). Pueriparity in this species has been only described for a few populations from the north western Rif mountains belonging to S. a. tingitana (fig. 1) (Donaire-Barroso and Bogaerts, 2000; Donaire-Barroso et al., 2001; Dinis and Velo-Antón, 2017), and most reproductive traits of this polymorphic species, such as brood size, mating system or the existence of multiple paternity, remain unknown or largely unexplored.
The reproductive polymorphism displayed by sister species S. salamandra and S. algira and the multiple origins of pueriparity that occurred within this clade provide a unique system to investigate the evolution of terrestrial reproduction in salamanders. It also allows testing hypotheses linking both viviparous strategies to other reproductive traits (e.g., mating systems and patterns of paternity), and evaluate the evolutionary relationship between pueriparity and multiple paternity. Here, we provide novel data on patterns of paternity in pueriparous S. a. tingitana and discuss the relationship between multiple paternity and pueriparity in salamanders in terms of potential fitness benefits.
Molecular data for S. a. tingitana was obtained from the offspring (N = 35) of four females (table 1, fig. 1b) captured in a single population from Amsa (Morocco) in 2015 (fig. 1b). Females were kept in captivity using boxes filled with moss, bark, and small containers of water until parturition (see Dinis and Velo-Antón, 2017). After sample collection, consisting of tissue samples from a toe-clip in the case of females and a tail-clip from the offspring, both females and offspring were released at their place of capture.
Frequency of multiple paternity (Incidence) in each pueriparous group of the species Salamandra salamandra (S. s. bernardezi and S. s. gallaica) and Salamandra algira tingitana, indicating the number of studied females (Nfemales), mean clutch size (Clutch), and mean number of fathers siring a clutch (Nfathers).
Citation: Amphibia-Reptilia 43, 1 (2022) ; 10.1163/15685381-bja10075
We used a protein precipitation protocol for DNA extraction followed by the elution of the extraction product with 50 μl of Elution Buffer (Omega BIO-TEK). We quantified DNA and diluted each sample to a concentration of 20 ng/μl. We amplified 12 microsatellites (see supplementary table S1; Steinfartz et al., 2004; Hendrix et al., 2010) distributed in two optimized duplexes and two multiplexes (supplementary table S1), through polymerase chain reaction (PCR). Each duplex/multiplex mix contained distilled H2O, fluorescently labelled forward (6-FAM, VIC, NED or PET; supplementary table S1 for sequence details) and reverse primers. Each PCR reaction contained a total volume of 10 μl: 5 μl of Multiplex PCR Kit Master Mix (QIAGEN), 3 μl of distilled H2O, 1 μl of primer multiplex mix and 1 μl of DNA extract (∼20 ng/μl). To identify possible contaminations, we used a negative control. PCR touchdown cycling conditions were equal in all multiplex reactions: the reaction started with an initial step at 95°C for 15 min, 19 cycles at 9 °C for 30 s, 90 s of annealing at 65°C (decreasing 0.5°C each cycle), 72°C for 40 s, followed by 25 cycles of 95°C for 30 s, 56°C for 60 s, 72°C for 40 s, and ended with a final extension of 30 min at 60°C. We verified the quality of PCR products on a 2% agarose gel. To determine the relative size of fragments, we employed the DNA Size Standard LIZ 500 DSMO-100 (NIMAGEN) on an ABI3130XL capillary sequencer (Applied Biosystems). We then scored alleles in GENEIOUS v. 2020.2.4. (Biomatters Ltd.), only considering those alleles exhibiting clear fluorescence peaks higher than 100 relative fluorescent units. To increase the likelihood of amplification, we amplified in duplex or uniplex reactions those female (mothers) samples in which any microsatellite marker failed to amplify or exhibited dubious allelic profiles (e.g., peak artefacts) and when any mother-offspring genotype incongruence existed (e.g., any descendant did not present any allele from the mother at any loci). If incongruences persisted, we reamplified incongruent loci in duplexes or uniplexes. Cycling conditions were the same as those described for multiplexes. Some of the incongruences persisted after regenotyping attempts. Specifically, two markers, SalE8 and SalG9, did not amplify in our dataset and were excluded from further analyses. We assessed the entire dataset for null alleles, allelic dropout and false alleles (genotyping errors) using the program MicroErrorAnalyzer v. 1 (Wang, 2010). This software estimates the mistyping rates of a set of markers given the observed numbers of mismatches among a number of known parent-offspring dyads. Maximum likelihood estimates of error rates were <0.0001 for null alleles and allelic dropout and 0.065 for genotyping error when assessing all 10 markers combined. Although this error applies to all loci, it mostly resulted from loci SST-A6-II, SalE14, SalE6 and Sal3, as revealed when analysed separately (data not shown).
To infer the number of sires in each family, we estimated the most likely number of fathers in COLONY 2.0.6.4. (Jones and Wang, 2010), a software which implements a maximum likelihood method to infer parentage based on individual multilocus genotypes. We set the species as dioecious and diploid, assumed polygamy for both sexes, no inbreeding, provided the maternal genotype and maternal sibship with no candidate father genotypes, and considered a genotyping error of 0.065 for all loci. We applied the maximum likelihood approach with high likelihood precision and two very long runs with different random number seeds.
We found evidence of multiple paternity in two out of four analysed females, with the number of fathers ranging from 1 to 3 (see fig. 1b, table 1). When there is more than one male siring a single clutch, each father sired on average 40% (±32%) of the offspring, with males siring between 17% and 59% of the clutch (fig. 1, table 1).
The identification and characterization of the mating strategy in viviparous species constitutes a preliminary step for testing further hypotheses about the evolutionary relationship between reproductive modes and multiple mating. Here, we unveil for the first time the occurrence of multiple paternity in pueriparous S. a. tingitana, as previously documented for both pueriparous (Alarcón-Ríos et al., 2020) and larviparous (Caspers et al., 2014) groups of its sister species, S. salamandra. The small sample size prevents us from reaching a robust estimate of multiple paternity frequency in pueriparous S. a. tingitana populations. However, the high incidence of multiple paternity in pueriparous Salamandra lineages (S. s. tingitana, 50%; S. s. gallaica, 88%; and S. s. bernardezi, 60%), in comparison with the more fecund larviparous lineages (54% or 37.5% according to Caspers et al., 2014), suggests that multiple paternity is a successful strategy in pueriparous systems (Alarcón-Ríos et al., 2020). Yet, the scarcity of pueriparous amphibian species, with the exception of the understudied caecilians, hinder comparisons with other groups. To the best of our knowledge, patterns of paternity in viviparous amphibians have only been documented in S. salamandra and one pueriparous anuran, the Nimba toad (Nimbaphrynoide occidentalis, Sandberger-Loua et al., 2016). Regarding the remaining pueriparous species within family Salamandridae, the alpine salamander S. atra has been described as polyandrous (Trochet et al., 2014), but the prolonged gestation period and the extremely reduced brood sizes of one or two offspring (Häfeli, 1971) challenge the study of multiple paternity.
To understand the evolution of polyandry in pueriparous systems in general, and in pueriparous salamanders in particular, it is important to consider jointly the potential benefits of polyandry, and some exclusive reproductive traits of viviparous species. Salamander females do not receive any direct benefit from polyandry (e.g., nuptial gifts, parental contribution to egg production, or extra parental care by males), and thus the evolutionary success of multiple mating across pueriparous species is expected to be related to the genetic benefits associated to multiple paternity (Yasui, 1998; Jennions and Petrie, 2000; Wolff and Macdonald, 2004). One of such genetic benefits might be enhancing reproduction success. Although female fertility is ultimately limited by the number of ova available to be fertilized, mating with multiple mates might ensure fertilization success, avoiding fertilization failures or infertile mates. Indeed, a positive correlation between number of fathers and clutch size was detected in both larviparous S. s. terrestris and pueriparous S. s. gallaica populations, but not in S. s. bernardezi (Caspers et al., 2014; Alarcón-Ríos et al., 2020). It is important to note that in some pueriparous salamander species/lineages there is also a high production of unfertilized eggs. Although they can be seen as fertilization failures, this is considered a selective strategy to ensure the provision of additional nutrients to developing embryos within uteri beyond egg yolk (i.e., matrotrophy) in the form of oophagy, and in some cases, adelphophagy (i.e., intrauterine cannibalism over aborted eggs or siblings, respectively) (Dopazo and Korenblum, 2000; Buckley et al., 2007).
Another fitness benefit usually associated with multiple mating is the increase of offspring genetic diversity (Jennions and Petrie, 2000), which can thus constitute a compensation mechanism to maintain offspring genetic diversity despite the reduction in fecundity (Yasui, 2001). However, the relationship between multiple paternity and offspring genetic diversity is not clear in any of the two S. salamandra viviparous modes (Caspers et al., 2014; Alarcón-Ríos et al., 2020). In addition, Caspers et al. (2014) showed that mates of polyandrous females were genetically more similar between them, and to the female, suggesting active mate selection of females as a mechanism to avoid outbreeding in an incipient speciation system between stream- and pond breeding populations (Steinfartz et al., 2007; Caspers et al., 2014). However, the reproductive mode itself might also play an important role on mate selection and population differentiation. According to the Viviparity Driven Conflict hypothesis (Zeh and Zeh, 2000, 2008), and due to the intense genomic conflicts derived from viviparity (Furness et al., 2015), the evolution of this reproductive mode may increase reproductive isolation among populations (Schrader and Travis, 2009) and, in turn, favour polyandry to reduce the risk of mating with incompatible mates (Zeh and Zeh, 2000; Schrader and Travis, 2008; Coleman et al., 2009). If we assume that pueriparity in salamanders implies a closer mother-offspring relationship than larviparity because of associated developmental changes and matrotrophy, polyandry might be favoured in pueriparous systems.
Other ecological and reproductive traits can also influence the evolution of polyandry in viviparous species. First, species of Salamandra are considered philopatric, presenting high site-fidelity and relatively low effective dispersal (Helfer et al., 2012; Hendrix et al., 2017; Lourenço et al., 2018; Dinis et al., 2019). This may increase the risks of mating with close relatives, and thus multiple mating can be a potential strategy to avoid the negative impacts of inbreeding on females fitness (Michalczyk et al., 2011). Indeed, this strategy has been suggested to explain the relatively high levels of genetic diversity unveiled in isolated urban (Lourenço et al., 2017), woodland (Lourenço et al., 2019), and insular (Velo-Antón et al., 2012; Alarcón-Ríos et al., 2020) pueriparous populations of S. salamandra. Furthermore, the existence of internal fertilization and oviductal retention of developing offspring allows processes acting beyond copulation, such as sperm storage and competition, post mating cryptic female choice (i.e., female-driven bias in sperm use), and post-fertilization processes (e.g., nutrient reallocation and intrauterine cannibalism). Those processes can bias patterns of paternity and, consequently, enhance the selective pressures over multiple mating behaviour in both sexes (Parker, 1970; Birkhead and Pizzari, 2002; Gilmore et al., 2005; Simmons, 2005). Long term sperm storage has been documented in fire salamanders, although it is rare under natural conditions (Greven and Guex, 1994; Steinfartz et al., 2006). In larviparous S. salamandra, sperm from multiple males mixes in the spermatheca following a topping-off pattern with first male precedence (Jones et al., 2002; Caspers et al., 2014), facilitating the occurrence of sperm competition and cryptic female choice. Taken together, the reduction in pueriparous females’ fecundity compared to larviparous ones, and the occurrence of intra and intersexual competition beyond copulation might result in strong selection on mating multiply in pueriparous salamanders (Avise and Liu, 2010). This also raises the possibility that the observed higher incidence of multiple paternity in pueriparous salamanders compared to larviparous ones results from the combined action of an intense selection on polygamous behaviour, and local factors such as population density, rate of mate encounters, and mate accessibility, that facilitate mating behaviours such as female harassment (Fitze et al., 2005; Avise et al., 2011; Marino et al., 2015).
It is important to highlight the scarcity of information about pueriparous reproduction in S. algira. Thus, most of the exposed ideas rely on information collected in S. salamandra, although the close phylogenetic relationship between these two sister species, and other reproductive traits such as the occurrence of siblings at different developmental stages within a single clutch (Dinis and Velo-Antón, 2017), suggest similar processes underlying pueriparity in the North African fire salamander. The occurrence of multiple paternity in all pueriparous nuclei within S. salamandra and S. algira, despite their general smaller brood sizes, points to the existence of a specific fitness benefit of multiple mating for pueriparous populations. In addition, considering traits that characterize pueriparous systems such as reduced fecundity, the high production of aborted eggs (Häfeli, 1971; Guex and Greven, 1994; Dopazo and Korenblum, 2000), the existence of matrotrophy through oophagy and adelphophagy (Guex and Chen, 1986; Buckley et al., 2007), and the potential for sperm storage and cryptic female choice, may constitute important pressures for the selection of multiple mating in pueriparous salamanders.
Corresponding author; e-mails: alarconrios@cibio.up.pt; guillermo.velo@uvigo.es; guillermo.velo@cibio.up.pt
Acknowledgements
We thank M. Dinis, F. Martínez-Freiría, S. Fahd, F. Giménez and L. García-Cardenete for help during field work, and Adam Marques for assistance during laboratory work and allele calling. Sebastian Steinfartz, the handling editor and one anonymous reviewer provided edits and helpful comments on earlier drafts of the manuscript. This work was supported by National Funds through FCT – Foundation for Science and Technology (SALOMICS: PTDC/BIA-EVL/28475/2017); the authors also acknowledge funding from other FCT projects and FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE (EVOVIV: PTDC/BIA-EVF/3036/2012; FCOMP-01-0124-FEDER-028325 and UIDB/500027/2020); and by a grant from Instituto de Estudios Ceutíes (IEC 2015). GVA was supported by FCT (IF/01425/2014 and CEECIND/00937/2018). Fieldwork for obtaining tissue samples was done with the corresponding permits from the Moroccan administration (n° 19-2015052).
Supplementary material
Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.16722601
References
Alarcón-Ríos, L., Nicieza, A.G., Lourenço, A., Velo-Antón, G. (2020): The evolution of pueriparity maintains multiple paternity in a polymorphic viviparous salamander. Sci. Rep. 10: 14744.
Avise, J.C., Liu, J.-X. (2010): Multiple mating and its relationship to alternative modes of gestation in male-pregnant versus female-pregnant fish species. Proc. Natl. Acad. Sci. 107: 18915-18920.
Avise, J.C., Liu, J.-X. (2011): Multiple mating and its relationship to brood size in pregnant fishes versus pregnant mammals and other viviparous vertebrates. Proc. Natl. Acad. Sci. 108: 7091-7095.
Avise, J.C., Tatarenkov, A., Liu, J.-X. (2011): Multiple mating and clutch size in invertebrate brooders versus pregnant vertebrates. Proc. Natl. Acad. Sci. 108: 11512-11517.
Barroso, D.D., Bogaerts, S. (2003): A new subspecies of Salamandra algira Bedriaga, 1883 from northern Morocco. Pod@rcis 4: 84-100.
Bas, S., Gasser, F. (1994): Polytypism of Salamandra salamandra (L.) in north-western Iberia. Mertensiella 4: 41-74.
Beukema, W., De Pous, P., Donaire, D., Escoriza, D., Bogaerts, S., Toxopeus, A.G., De Bie, C.A.J.M., Roca, J., Carranza, S. (2010): Biogeography and contemporary climatic differentiation among Moroccan Salamandra algira. Biol. J. Linn. Soc. 101: 626-641.
Birkhead, T.R., Pizzari, T. (2002): Evolution of sex: postcopulatory sexual selection. Nat. Rev. Genet. 3: 262.
Blackburn, D.G. (2015): Evolution of vertebrate viviparity and specializations for fetal nutrition: a quantitative and qualitative analysis. J. Morphol. 276: 961-990.
Buckley, D. (2012): Evolution of Viviparity in Salamanders (Amphibia, Caudata). In: eLS. Wiley, Chichester.
Buckley, D., Alcobendas, M., García-París, M., Wake, M.H. (2007): Heterochrony, cannibalism, and the evolution of viviparity in Salamandra salamandra. Evol. Dev. 9: 105-115.
Burgon, J.D., Vences, M., Steinfartz, S., Bogaerts, S., Bonato, L., Donaire-Barroso, D., Martínez-Solano, I., Velo-Antón, G., Vieites, D.R., Mable, B.K. (2021): Phylogenomic inference of species and subspecies diversity in the Palearctic salamander genus Salamandra. Mol. Phylogenet. Evol. 157: 107063.
Caspers, B.A., Krause, E.T., Hendrix, R., Kopp, M., Rupp, O., Rosentreter, K., Steinfartz, S. (2014): The more the better-polyandry and genetic similarity are positively linked to reproductive success in a natural population of terrestrial salamanders (Salamandra salamandra). Mol. Ecol. 23: 239-250.
Coleman, S.W., Harlin-Cognato, A., Jones, A.G. (2009): Reproductive isolation, reproductive mode, and sexual selection: empirical tests of the viviparity-driven conflict hypothesis. Am. Nat. 173: 291-303.
Dinis, M., Merabet, K., Martínez-Freiría, F., Steinfartz, S., Vences, M., Burgon, J.D., Elmer, K.R., Donaire, D., Hinckley, A., Fahd, S., Joger, U., Fawzi, A., Slimani, T., Velo-Antón, G. (2019): Allopatric diversification and evolutionary melting pot in a North African Palearctic relict: the biogeographic history of Salamandra algira. Mol. Phylogenet. Evol. 130: 81-91.
Dinis, M., Velo-Antón, G. (2017): How little do we know about the reproductive mode in the north African salamander, Salamandra algira? Pueriparity in divergent mitochondrial lineages of S. a. tingitana. Amphibia-Reptilia 38: 540-546.
Donaire-Barroso, D., Bogaerts, S. (2000): Observations on viviparity of Salamandra algira in north Morocco. In: Herpetologia Candiana, p. 147-151. SEH, Irakleio.
Donaire-Barroso, D., Bogaerts, S., Herbert, D. (2001): Confirmación de desarrollo larvario completo intrauterino en Salamandra algira (Bedriaga, 1883) del Noroeste de Marruecos. Butll. Soc. Cat. Herp. 5: 107-110.
Dopazo, H., Alberch, P. (1994): Preliminary results on optional viviparity and intrauterine siblicide in Salamandra salamandra populations from Northern Spain. Mertensiella 4: 125-137.
Dopazo, H.J., Korenblum, M. (2000): Viviparity in Salamandra salamandra (Amphibia: Salamandridae): adaptation or exaptation? Herpetologica 56: 144-152.
Duellman, W.E. (1989): Alternative life-history styles in anuran amphibians: evolutionary and ecological implications. In: Alternative Life-History Styles of Animals, p. 101-126. Bruton, M.N., Ed., Springer.
Duellman, W.E., Trueb, L. (1986): Biology of Amphibians. JHU Press, New York.
Ellegren, H., Galtier, N. (2016): Determinants of genetic diversity. Nat. Rev. Genet. 17: 422.
Fitze, P.S., Le Galliard, J.F., Federici, P., Richard, M., Clobert, J. (2005): Conflict over multiple-partner mating between males and females of the polygynandrous common lizards. Evolution 59: 2451-2459.
Furness, A.I., Morrison, K.R., Orr, T.J., Arendt, J.D., Reznick, D.N. (2015): Reproductive mode and the shifting arenas of evolutionary conflict. Ann. N. Y. Acad. Sci. 1360: 75-100.
García-París, M., Alcobendas, M., Buckley, D., Wake, D.B. (2003): Dispersal of viviparity across contact zones in Iberian populations of fire salamanders (Salamandra) inferred from discordance of genetic and morphological traits. Evolution 57: 129-143.
Gilmore, R.G., Putz, O., Dodrill, J.W. (2005): Oophagy, intrauterine cannibalism and reproductive strategy in lamnoid sharks. In: Reproductive Biology and Phylogeny of Chondrichthyes: Sharks, Batoids and Chimaeras, p. 435-462. Hamlett, W.C., Ed., Science Publishers, Enfield, New Hampshire, USA.
Goodwin, N.B., Dulvy, N.K., Reynolds, J.D. (2002): Life-history correlates of the evolution of live bearing in fishes. Philos. Trans. R. Soc. London. Ser. B Biol. Sci. 357: 259-267.
Greven, H. (2003): Larviparity and pueriparity. In: Reproductive Biology and Phylogeny of Urodela (Amphibia), p. 447-475. Jamieson, B.G.M., Sever, D.M., Eds, Science Publishers, Enfield, NH.
Greven, H., Guex, G.D. (1994): Structural and physiological aspects of viviparity in Salamandra salamandra. Mertensiella 4: 139-160.
Guex, G.-D., Chen, P.S. (1986): Epitheliophagy: intrauterine cell nourishment in the viviparous Alpine salamander, Salamandra atra (Laur.). Experientia 42: 1205-1218.
Guex, G.-D., Greven, H. (1994): Structural and physiological aspects of viviparity in Salamandra atra. Mertensiella 4: 161-208.
Häfeli, H.P. (1971): Reproductive biology of the Alpine salamander (Salamandra atra Laur). Rev. Suisse Zool. 78: 235-293.
Helfer, V., Broquet, T., Fumagalli, L. (2012): Sex-specific estimates of dispersal show female philopatry and male dispersal in a promiscuous amphibian, the Alpine salamander (Salamandra atra). Mol. Ecol. 21: 4706-4720.
Hendrix, R., Schmidt, B.R., Schaub, M., Krause, E.T., Steinfartz, S. (2017): Differentiation of movement behaviour in an adaptively diverging salamander population. Mol. Ecol. 26: 6400-6413.
Jennions, M.D., Petrie, M. (2000): Why do females mate multiply? A review of the genetic benefits. Biol. Rev. 75: 21-64.
Joly, J. (1986): La reproduction de la salamandre terrestre (Salamandra salamandra L.). Trait. Zool. Amphib. 14: 471-495.
Jones, A.G., Adams, E.M., Arnold, S.J. (2002): Topping off: a mechanism of first-male sperm precedence in a vertebrate. Proc. Natl. Acad. Sci. 99: 2078-2081.
Jones, O.R., Wang, J. (2010): COLONY: a program for parentage and sibship inference from multilocus genotype data. Mol. Ecol. Resour. 10: 551-555.
Liu, J.-X., Avise, J.C. (2011): High degree of multiple paternity in the viviparous Shiner Perch, Cymatogaster aggregata, a fish with long-term female sperm storage. Mar. Biol. 158: 893-901.
Lourenço, A., Álvarez, D., Wang, I.J., Velo-Antón, G. (2017): Trapped within the city: integrating demography, time since isolation and population-specific traits to assess the genetic effects of urbanization. Mol. Ecol. 26: 1498-1514.
Lourenço, A., Antunes, B., Wang, I.J., Velo-Antón, G. (2018): Fine-scale genetic structure in a salamander with two reproductive modes: does reproductive mode affect dispersal?. Evol. Ecol. 32: 699-732.
Lourenço, A., Gonçalves, J., Carvalho, F., Wang, I.J., Velo-Antón, G. (2019): Comparative landscape genetics reveals the evolution of viviparity reduces genetic connectivity in fire salamanders. Mol. Ecol. 28: 4573-4591.
Marino, I.A.M., Riginella, E., Gristina, M., Rasotto, M.B., Zane, L., Mazzoldi, C. (2015): Multiple paternity and hybridization in two smooth-hound sharks. Sci. Rep. 5: 1-11.
Miaud, C., Andreone, F., Ribéron, A., De Michelis, S., Clima, V., Castanet, J., Francillon-Vieillot, H., Guyétant, R. (2001): Variations in age, size at maturity and gestation duration among two neighbouring populations of the Alpine salamander (Salamandra lanzai). J. Zool. 254: 251-260.
Michalczyk, Ł., Millard, A.L., Martin, O.Y., Lumley, A.J., Emerson, B.C., Chapman, T., Gage, M.J.G. (2011): Inbreeding promotes female promiscuity. Science 333: 1739-1742.
Parker, G.A. (1970): Sperm competition and its evolutionary consequences in the insects. Biol. Rev. 45: 525-567.
Roitberg, E.S., Kuranova, V.N., Bulakhova, N.A., Orlova, V.F., Eplanova, G.V., Zinenko, O.I., Shamgunova, R.R., Hofmann, S., Yakovlev, V.A. (2013): Variation of reproductive traits and female body size in the most widely-ranging terrestrial reptile: testing the effects of reproductive mode, lineage, and climate. Evol. Biol. 40: 420-438.
Romiguier, J., Gayral, P., Ballenghien, M., Bernard, A., Cahais, V., Chenuil, A., Chiari, Y., Dernat, R., Duret, L., Faivre, N. (2014): Comparative population genomics in animals uncovers the determinants of genetic diversity. Nature 515: 261-263.
Sandberger-Loua, L., Feldhaar, H., Jehle, R., Rödel, M.-O. (2016): Multiple paternity in a viviparous toad with internal fertilisation. Sci. Nat. 103: 51.
Schrader, M., Travis, J. (2008): Testing the viviparity-driven-conflict hypothesis: parent-offspring conflict and the evolution of reproductive isolation in a poeciliid fish. Am. Nat. 172: 806-817.
Schrader, M., Travis, J. (2009): Do embryos influence maternal investment? Evaluating maternal-fetal coadaptation and the potential for parent-offspring conflict in a placental fish. Evol. Int. J. Org. Evol. 63: 2805-2815.
Simmons, L.W. (2005): The evolution of polyandry: sperm competition, sperm selection, and offspring viability. Annu. Rev. Ecol. Evol. Syst. 36: 125-146.
Slatyer, R.A., Mautz, B.S., Backwell, P.R.Y., Jennions, M.D. (2012): Estimating genetic benefits of polyandry from experimental studies: a meta-analysis. Biol. Rev. 87: 1-33.
Sparreboom, M. (2014): Salamanders of the Old World: the Salamanders of Europe, Asia and Northern Africa. Brill.
Steinfartz, S., Stemshorn, K., Kuesters, D., Tautz, D. (2006): Patterns of multiple paternity within and between annual reproduction cycles of the fire salamander (Salamandra salamandra) under natural conditions. J. Zool. 268: 1-8.
Steinfartz, S., Weitere, M., Tautz, D. (2007): Tracing the first step to speciation: ecological and genetic differentiation of a salamander population in a small forest. Mol. Ecol. 16: 4550-4561.
Tregenza, T., Wedell, N. (2002): Polyandrous females avoid costs of inbreeding. Nature 415: 71.
Trochet, A., Moulherat, S., Calvez, O., Stevens, V.M., Clobert, J., Schmeller, D.S. (2014): A database of life-history traits of European amphibians. Biodivers. Data J. e4123.
Veith, M., Steinfartz, S., Zardoya, R., Seitz, A., Meyer, A. (1998): A molecular phylogeny of ‘true’salamanders (family Salamandridae) and the evolution of terrestriality of reproductive modes. J. Zool. Syst. Evol. Res. 36: 7-16.
Velo-Antón, G., García-Cardenete, L., Cazalla, F.J., Martínez-Freiría, F. (2014): New record of Salamandra algira isolated on the north-western Tingitana peninsula, with some notes on the reproductive modes within the species. Bol. Asoc. Herpetol. Esp. 25: 46-50.
Velo-Antón, G., García-París, M., Galán, P., Cordero-Rivera, A. (2007): The evolution of viviparity in Holocene islands: ecological adaptation versus phylogenetic descent along the transition from aquatic to terrestrial environments. J. Zool. Syst. Evol. Res. 45: 345-352.
Velo-Antón, G., Santos, X., Sanmartín-Villar, I., Cordero-Rivera, A., Buckley, D. (2015): Intraspecific variation in clutch size and maternal investment in pueriparous and larviparous Salamandra salamandra females. Evol. Ecol. 29: 185-204.
Velo-Antón, G., Zamudio, K.R., Cordero-Rivera, A. (2012): Genetic drift and rapid evolution of viviparity in insular fire salamanders (Salamandra salamandra). Heredity 108: 410-418.
Wake, M.H. (1992): Evolutionary scenarios, homology and convergence of structural specializations for vertebrate viviparity. Am. Zool. 32: 256-263.
Wake, M.H. (2015): Fetal adaptations for viviparity in amphibians. J. Morphol. 276: 941-960.
Wang, J. (2010): Effects of genotyping errors on parentage exclusion analysis. Mol. Ecol. 19: 5061-5078.
Wells, K.D. (2007): The Ecology and Behavior of Amphibians. The University of Chicago Press, Chicago.
Wolff, J.O., Macdonald, D.W. (2004): Promiscuous females protect their offspring. Trends Ecol. Evol. 19: 127-134.
Yasui, Y. (1998): The genetic benefits’ of female multiple mating reconsidered. Trends Ecol. Evol. 13: 246-250.
Yasui, Y. (2001): Female multiple mating as a genetic bet-hedging strategy when mate choice criteria are unreliable. Ecol. Res. 16: 605-616.
Zeh, D.W., Zeh, J.A. (2000): Reproductive mode and speciation: the viviparity-driven conflict hypothesis. Bioessays 22: 938-946.
Zeh, J.A., Zeh, D.W. (2001): Reproductive mode and the genetic benefits of polyandry. Anim. Behav. 61: 1051-1063.
Zeh, J.A., Zeh, D.W. (2008): Viviparity-driven conflict. Ann. N. Y. Acad. Sci. 1133: 126-148.
Footnotes
Associate Editor: Iñigo Martínez-Solano
