Abstract
The effects of hybridization on evolutionary processes are primarily determined by the differential between hybrid and parental species fitness. Assessing the impacts of hybridization can be challenging, however, as determining the relationship between individual fitness and the extent of introgression in wild populations is difficult. We evaluated the fitness consequences of hybridization for pure and hybrid females in a hybrid zone between two tidal marsh birds, the saltmarsh sparrow (Ammodramus caudacutus), a salt marsh obligate, and Nelson's sparrow (A. nelsoni), which has a broader ecological niche and a much younger evolutionary association with salt marshes. Biotic stressors associated with nesting in tidal environments suggest an important role for differential adaptation in shaping hybrid zone dynamics, with saltmarsh sparrows predicted to be better adapted to nesting in salt marshes. We collected DNA samples from adults (n = 394) and nestlings (n = 431) to determine the extent of introgression using 12 microsatellite loci and tested for the influence of extrinsic (nest placement) and intrinsic (genotype) factors on female reproductive success. We monitored nests (n = 228), collected data on reproductive output, and estimated daily nest survival rates using female genotype and nest characteristics as covariates. To test for reduced survival of hybrid females, we also used capture data to assess the distribution of admixed male and female individuals across age classes. Reproductive success of females varied by genotypic class, but hybrids did not have intermediate success as predicted. Instead, we found that pure Nelson's sparrows had, on average, 33% lower hatching success than any other genotype, whereas F1/F2 hybrids, backcrossed Nelson's sparrows, and backcrossed and pure saltmarsh sparrows all had similar hatching success. We found no effect of genotype or nest placement on daily nest survival probabilities. However, hybrid individuals with a higher proportion of saltmarsh sparrow alleles exhibit nesting behaviours better suited to nesting successfully in tidal marshes. Further, while the proportion of F1/F2 individuals was similar between nestling and adult males, we found that the proportion of F1/F2 individuals was 2.3 times greater in nestling females compared with adult females, indicating reduced survival of F1 females. We conclude that differences in reproductive success among pure and admixed individuals coupled with intrinsic mechanisms (reduced survival in F1 females) shape hybrid zone dynamics in this system.
Introduction
Hybridization – or the crossing of two genetically distinct species – is prevalent in a range of taxonomic groups (Mallet, 2005) and has the potential to shape species' evolutionary trajectories. Hybrid zone maintenance and the role of hybridization in reproductive isolation, however, depend on the fitness of hybrids relative to parental forms (Burke & Arnold, 2001; Lancaster et al., 2007). Fitness consequences can range from severe selection pressures against hybrids and proportionally narrow hybrid zones to hybrid vigour and hybrid swarms – a population of individuals that are all hybrids due to extensive mating among hybrids and backcrossing. Empirical studies have documented a range of fitness outcomes in both plant and animal hybrid zones, including reductions in seed viability (Cruzan et al., 1994), fecundity (Veen et al., 2001; Hurt & Hedrick, 2003; Bronson et al., 2005; Lancaster et al., 2007; Brix & Grosell, 2013), survival (Neubauer, Nowicki & Zagalska-Neubauer, 2014), hybrid vigour (Fitzpatrick & Shaffer, 2007), and no observable fitness effects (Flockhart & Wiebe, 2009). While several studies have evaluated the fitness of hybrids, the assessment of fitness consequences in wild populations is relatively underrepresented in hybrid zone research because of the logistical challenges of collecting demographic and genotypic data simultaneously. The identification of fitness consequences of hybridization provides important insight into factors that underlie reproductive isolation, and whether selection is likely to favour reductions in heterospecific-mating events (Veen et al., 2001).
Both extrinsic and intrinsic forces can alter the fitness of hybrids relative to parental taxa. Extrinsic selection pressures arise from variation in physical or social environment (Rohwer, Bermingham & Wood, 2001). Fitness reductions can occur if hybrids display intermediate morphologies (Svedin et al., 2008), mating signals (Bridle et al., 2006), or behaviours (Pearson, 2000) that make them less desirable to potential mates. Variation in habitat type and quality can also influence fitness. Hybrid zones often occur along ecological gradients, as transitional habitats may facilitate contact between species that inhabit different ecological niches (Culumber et al., 2012). In such a situation, hybrids might be less fit than the parentals in either niche (Rohwer et al., 2001). Conversely, hybrids could be favoured if they are within transitional habitats as they are better suited to these intermediate habitats compared with pure individuals – a process referred to as ‘bounded hybrid superiority’ (Moore, 1977; Moore & Price, 1993). Alternatively, intrinsic selection pressures arise from incompatibilities that result from the recombination of co-adapted genomes independent of the environment (Moore & Price, 1993; Rohwer et al., 2001). These incompatibilities are particularly evident as reductions in fertility or viability in hybrids of the heterogametic sex (Haldane's rule; Haldane, 1922; Presgraves et al., 2003; Svedin et al., 2008). The influence of extrinsic and intrinsic forces are not mutually exclusive, however, as it is usually a combination of factors that influence the relative fitness of hybrids (Price & T., 2008; Svedin et al., 2008; Arnegard et al., 2014; Neubauer et al., 2014).
In this study, we investigated extrinsic and intrinsic influences on fitness in a naturally occurring hybrid zone between two avian marsh endemics, the saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) sparrow. In the USA and Maritime Canada, these two species are restricted to a narrow ribbon of habitat along the Atlantic seaboard. The saltmarsh sparrow is a habitat specialist, exhibiting a pre-Pleistocene association with tidal salt marshes (Greenlaw & Rising, 1994; Chan et al., 2006). In contrast, the Nelson's sparrow exhibits a broader ecological niche, breeding in grassland and brackish marshes in addition to tidal marshes (Greenlaw, 1993; Nocera et al., 2007; Shriver, Hodgman & Hanson, 2011). The two sister species diverged recently (c. 600 000 years ago; Rising & Avise, 1993), as evidenced by weak genetic divergence (1.2% differentiation at the COI gene and FST of ~ 0.15 for neutral microsatellite markers: Shriver et al., 2005; Walsh et al., 2011). They are currently in secondary contact along the New England coast, where they co-inhabit salt marshes in a narrow, linear hybrid zone spanning a 210 km stretch of coastline between the Weskeag River estuary in South Thomaston, Maine and Plum Island in Newburyport, Massachusetts (Fig. 1; Hodgman, Shriver & Vickery, 2002; Shriver et al., 2005; Walsh et al., 2011). The saltmarsh–Nelson's sparrow hybrid zone corresponds geographically to a habitat discontinuity along the coastline, with a transition from smaller, isolated, and more brackish fringe marshes in the north (pure Nelson's sparrow habitat) to more expansive, continuous stretches of tidally influenced marshes in the south (pure saltmarsh sparrow habitat; Greenlaw, 1993). While there is a linear, latitudinal transition between the brackish upriver (Nelson's sparrow) and primarily coastal (saltmarsh sparrow) marsh types, the intervening habitat found within the hybrid zone is characterized by a mix of marsh types. Previous work has shown that the patchy distribution of marsh types is associated with a corresponding mosaic of genotypes and that local habitat features shape the spatial distribution of pure and admixed individuals in this system (Walsh et al., 2015b).
Map depicting the range of pure Nelson's sparrows (dark grey), pure saltmarsh sparrows (light grey), and the hybrid zone (white) in the northeastern United States. Blue circles correspond to the four sites where fitness data were collected for this study: SC (Scarborough), EL (Eldridge), LU (Lubberland Creek), CL (Chapman's Landing).
Saltmarsh and Nelson's sparrows exhibit marked differences in behaviour, reproductive strategy (Greenlaw, 1993), and morphology (Shriver et al., 2005; Walsh et al., 2015a), as well as habitat affinities (Greenlaw, 1993; Walsh et al., 2015b), suggesting a potential role for both extrinsic and intrinsic factors in shaping fitness patterns across the hybrid zone. Introgression is widespread and ~ 50% of the individuals in the hybrid zone are admixed (Walsh et al., 2015a, b). Most admixed individuals are backcrossed by several generations, and very few F1/F2 hybrids occur. Hybrids lack an intermediate phenotype, and pure and admixed individuals cannot be distinguished by morphology alone (Walsh et al., 2015a, b). Traits associated with tidal marsh adaptations exhibit reduced introgression, suggesting an influence of natural selection on maintaining species boundaries (Walsh et al., 2016).
Differences in habitat affinity of saltmarsh and Nelson's sparrows suggest that local environmental features may also shape the fitness of hybrid individuals. Both species are ground-nesting passerines and are thus highly vulnerable to tidal flooding. In salt marshes, flooding affects nests during the highest spring tides; during this time the entire marsh is flooded and nests can be inundated with water for multiple hours (Gjerdrum et al., 2008). As a result, monthly tidal flooding is the leading cause of nest failure for both species (Greenlaw & Rising, 1994; Shriver et al., 2007; Bayard & Elphick, 2011). Previous work has shown that saltmarsh sparrows have greater nesting synchrony with tidal cycles compared with Nelson's sparrows and this synchrony is associated with increased nesting success (Shriver et al., 2007). These temporal differences in nesting may shape fitness outcomes in the hybrid zone. Further, genetic analyses have identified reduced introgression of mitochondrial and sex-linked markers across the hybrid zone (Walsh et al., 2016), indicative of reduced fitness in hybrid females (intrinsic factors). To evaluate the role of extrinsic and intrinsic selective forces as isolating mechanisms in this system, a comparison of the fitness of pure and hybrid individuals is needed.
Here, we evaluated the reproductive success and survival of admixed (recent generation hybrids and backcrossed individuals) relative to pure Nelson's and saltmarsh sparrow females. Our objective was to understand differences in reproductive success among both pure species and hybrids and how fitness consequences are shaping the saltmarsh–Nelson's sparrow hybrid zone. More specifically, we hypothesized that both extrinsic and intrinsic factors influence fitness patterns within the hybrid zone. To this end, we evaluated: (1) whether overall reproductive output varies among females as a function of genotype, (2) if there is an influence of extrinsic (nest placement) or intrinsic (genotype) mechanisms on daily probabilities of nesting success, and (3) if there is variation in nesting behaviours among females as a function of genotype. We predicted that, due to their better adaptation for nesting in tidal marshes, saltmarsh sparrows would have the highest reproductive output, hybrids intermediate, and Nelson's sparrows the lowest. Because nesting success is largely determined by nest initiation relative to tidal events, we predicted that daily nest survival probabilities would be less influenced by nest placement but still influenced by female genotype. We also tested predictions of Haldane's rule by comparing sex ratios among genotypic classes. Specifically, we predicted that: (1) female hybrids would have a lower survival probability compared with male hybrids, and (2) if female hybrids had reduced fitness, offspring sex ratios in the nests of F1 females would be male biased as a mechanism to avoid fitness reductions in the heterogametic sex.
Material and Methods
Study sites and data collection
We collected demographic data over three breeding seasons (2011–2013) in three marshes located at the southern portion of the saltmarsh-Nelson's overlap zone: (1) Eldridge in Wells, Maine (EL; 43°17.31′N, 70°34.27′W), (2) Chapman's Landing in Stratham, New Hampshire (CL; 43°02.24′N, 70°55.32′W), and (3) Lubberland Creek in Newmarket, New Hampshire (LU; 43°04.29′N, 70°54.48′W; Fig. 1). To increase sample sizes for Nelson's sparrows, we obtained nest-monitoring data from Nelson's females from a fourth site further north at Scarborough Marsh in Maine (SC; 43°33.90′N, 70°21.64′W); however, we did not include adult capture data or male/nestling genotypes from this fourth site. Previous work has shown a link between genotype and local habitat characteristics consistent with a coastal-upriver gradient (Walsh et al., 2015b). Accordingly, we classified the sites into two categories based on differences in their proximity to the coast: Lubberland Creek and Chapman's Landing, located in the Great Bay estuary, were considered upriver marshes whereas Scarborough and Eldridge were considered coastal marshes. These sites vary in terms of tidal regime with the upriver marshes exhibiting dampened average monthly high tide levels (~ 2.7 m) compared with the coastal marshes (~ 3.5 m). Due to the small size of Chapman's Landing and Lubberland Creek (11.0 and 10.5 ha, respectively), we conducted nest monitoring and banding throughout the entire marsh patches. For Eldridge, we conducted nest monitoring and banding within a 15 ha plot, which comprised only a portion of the marsh. For Scarborough, we conducted nest monitoring within three plots (averaging 10 ha in size each).
For the three marshes from which adult capture data were obtained (EL, CL, LU), we subdivided each site into subplots and we systematically trapped adults using mist nets; a minimum of three netting sessions were conducted per subplot each season. Once captured, we banded adults and collected standard morphological measurements. Over the 3 years of the study, we captured 394 adult sparrows. We sexed adults by presence or absence of a cloacal protuberance or brood patch. For adults sampled in 2012 and 2013, we drew 10–20 μL of blood from the brachial vein and transferred samples to Nobuto blood filter strips (Sterlitech, Kent, Washington). For adults sampled in 2011, we pulled two tail feathers (R1/R6) and stored feathers for later genetic analyses.
We found nests through systematic plot searches; once found, we marked nests and visited them every 1–5 days to record overall fate. We estimated nest initiation based on clutch completion, hatching dates, or chick age, assuming a 24-day nesting period for both species (1 day to lay each egg for a modal clutch size of 4, incubation begins with the last egg and follows for 10 more days, and 10 days to fledge; Greenlaw & Rising, 1994; Shriver et al., 2007). We captured females at their associated nests to confirm identity. We banded all chicks at age 6–7 days and collected morphological measurements, including weight, tarsus length, and bill length; pinfeathers were collected from each nestling for genetic analyses. We banded 431 nestlings at the three demographic sites (EL, CL, LU); 36 additional nestlings were associated with females with nests monitored on Scarborough (SC). When chicks died during nest flooding or predation events, we obtained tissue samples when possible. We determined nest failure or success based on evidence at the nest site (Ruskin et al., 2015). Briefly, we considered a nest successful if it was found empty when at least one nestling would have been 10 days old; we counted a nest successful if at least one chick fledged. We classified nests as flooded if they contained drowned chicks or if eggs were found outside of the nests (or a nest with young chicks or eggs was empty after a high tide), and we considered nests depredated if nests were torn, or contained broken or punctured eggs (or a nest with young chicks or eggs was empty and dry).
We also collected data on nest placement characteristics, which may differ between saltmarsh sparrows, Nelson's sparrows, and their hybrids or between successful and failed nests. Once a nest became inactive, we estimated the percent cover of the dominant vegetation types within a 1-m2 area around the nest and recorded the presence or absence of a woven vegetation canopy over the nest. We also measured nest height (cm) as the distance from the lip of the nest cup to the ground.
Genetic analyses and hybrid identification
For the adults, we extracted DNA from blood samples using a DNeasy blood Kit (Qiagen, Valencia, CA, USA) according to manufacturer protocol. We extracted DNA from pinfeathers collected from banded nestlings using a DNeasy Tissue Kit, with a minimum of 24-h incubations for the lysis stage. For adult tail feathers, we isolated the calamus and followed the same protocol as with the pinfeathers, except with the addition of 10 μL of DTT (dithiothreitol) to the lysis buffer and a 48-h incubation. To genotype individuals, we selected a subset of informative markers used previously to identify patterns of introgression in saltmarsh and Nelson's sparrows (Walsh et al., 2015a, b). These included a combination of six polymorphic, putatively neutral anonymous microsatellite loci and six diagnostic microsatellites (Ammo markers; Kovach et al., 2015) that were developed specifically to differentiate saltmarsh sparrows, Nelson's sparrows, and hybrids. We amplified DNA using fluorescent dye-labelled primers for 12 microsatellite loci in two multiplexes: Ammo001, Ammo006, Ammo008, Ammo015, Ammo017, Ammo027, Ammo028 (Kovach et al., 2015), Escμ1 (Hanotte et al., 1994), Asμ18 (Bulgin et al., 2003), Aca01, Aca08, and Aca12 (Hill et al., 2008), following the conditions of Walsh et al. (2012, 2015a). We conducted two replicates for adult feather samples to reduce genotyping error associated with lower quality samples. For nestlings, we included dye-labelled primers P2 and P8 for the CHD gene (Griffiths et al., 1998) in one of the multiplexes for sex determination. Because of a difference in the size of an intron on the CHD1-W and CHD1-Z genes, this method results in two fragments of different sizes in females and one fragment in males. We validated sexing primers with 16 samples of adult saltmarsh sparrows of known sex (8 males and 8 females). In all 16 tests, the genetic identification corresponded with field identification. Sex ratios for nestlings were calculated as the proportion of males in a clutch. Amplified products were electrophoresed on an automated DNA sequencer (ABI 3130 Genetic Analyzer: Applied Biosystems, Foster City, CA, USA) and individual genotypes were scored manually using PEAKSCANNER software (Applied Biosystems).
To define genotypic classes, we first calculated a hybrid index for each individual using the R package introgress (Gompert & Buerkle, 2009, 2010), defined as the proportion of alleles inherited from the saltmarsh sparrow (0 = pure Nelson's sparrow and 1 = pure saltmarsh sparrow). We then estimated interspecific heterozygosity, defined as the proportion of genotypes that are heterozygous for the parental alleles (0 = all homozygous genotypes and 1 = all heterozygous genotypes). Using the combination of hybrid index and interspecific heterozygosity, we assigned sparrows to five genotypic classes (pure Nelson's sparrow, backcrossed in the direction of Nelson's sparrow, F1/F2 hybrids, backcrossed in the direction of saltmarsh sparrow, and pure saltmarsh sparrow) following the methods of Milne & Abbott (2008). Individuals with intermediate hybrid indices (0.25–0.75) and high heterozygosity (> 0.3) were considered recent generation hybrids (F1, F2), and individuals with low hybrid index (0.05–0.25 or 0.75–0.95) and low heterozygosity (< 0.3) were considered backcrossed in the direction of Nelson's or saltmarsh sparrows, respectively. We considered individuals to be pure Nelson's sparrows if they had a hybrid index of 0–0.05 and pure saltmarsh sparrows if they had a hybrid index of 0.95–1.
Variation in reproductive output as a function of genotype
We first evaluated variation in overall reproductive output among females as a function of their genotypic class. To do this, we included seven reproductive parameters as indicators of female fitness. These included clutch size, hatching success (proportion of eggs per nest to hatch), fledging success (proportion of nestlings per nest to fledge), average chick weight per nest, the range of chick weights per nest, the maximum chick weight per nest, and average daily nest survival rates. We included average chick weight, range in chick weights, and maximum chick weights in our analyses because field observations suggest that larger nestlings are better able to climb out of the nest during flooding events than smaller nestlings (Gjerdrum et al., 2008). We used ANOVA and a Tukey's test to compare the seven reproductive parameters among individuals to evaluate fitness differences among the five genotypic classes. We conducted these analyses in the base package in R (R core team, 2014).
Intrinsic and extrinsic influences on nesting survival
In addition to evaluating variation in reproductive output among females belonging to different genotypic classes, we also assessed the influence of intrinsic and extrinsic factors on daily nest survival probabilities. We evaluated reproductive output (number of fledglings produced) and daily nest survival probabilities separately. We tested for the influence of intrinsic and extrinsic factors on female reproductive success, by modelling nesting survival in relation to genotype and nesting behaviour. We used the program MCestimate (Etterson, Nagy & Robinson, 2007; Etterson, Greenberg & Hollenhorst, 2014) to generate daily nest survival probabilities using nest-monitoring data. MCestimate employs a Markov chain algorithm to estimate daily nest survival probabilities via a generalization of the Mayfield method (Mayfield, 1975). We developed two sets of models to independently estimate the influence of: (1) female genotype (intrinsic), and (2) nest characteristics (extrinsic) on daily nest survival probabilities. For the intrinsic – genotypes – models, we estimated daily nest survival probabilities as a function of the genotype of an individual female. We used five different models to test for the influence of genotype. We evaluated the influence of genotype on daily survival probabilities by modelling females using: (1) hybrid index as a continuous variable, and (2) categorical values corresponding to the five genotypic classes. To evaluate basic differences between pure females and admixed females, we also modelled genotype by grouping females as follows: (3) pure vs. admixed, (4) F1/F2 hybrids vs. all other females, and (5) pure Nelson's females vs. all other females. Due to known differences in site (tidal regime) and likely differences between years, we included a covariate for upriver (Chapman's Landing and Lubberland Creek) and coastal sites (Scarborough marsh and Eldridge marsh) and year in all models. With the inclusion of an intercept-only (null) model, we ran a total of eight candidate models for the intrinsic – genotypes – model set.
For the extrinsic – nesting behaviours – models, we estimated daily nest survival probabilities as a function of nest placement by an individual female, to evaluate the influence of local vegetation features on nest success, independent of female genotype. Because we predict that the ability to nest successfully in tidal marshes will be reduced in Nelson's sparrows, intermediate in admixed individuals, and greatest in saltmarsh sparrows, these models included nesting behaviours that may be influential in avoiding or alleviating the impacts of flooding events: (1) height of the nest off of the ground, (2) proportion of high marsh vegetation surrounding the nest, (3) the construction of a canopy over the nest (which may prevent eggs from washing out of the nest during flooding events; Humphreys et al., 2007), and (4) timing of nest initiation relative to most recent spring high tide. Again, to disentangle any potential effect of temporal and local variation, we ran the models with and without site and year as covariates. We included single and additive models for each variable, with a total of 32 candidate models for the extrinsic – nesting behaviours – model set. We used Akaike's Information Criterion to compare candidate models in each set and determine which models most parsimoniously described the daily nest survival probabilities (Burnham & Anderson, 2002).
Variation in nesting behaviours as a function of genotype
In addition to assessing the influence of genotype (intrinsic) and nest placement (extrinsic) factors on reproductive output and daily nest survival probabilities, we also evaluated the relationship of these two factors to each other. To evaluate whether potential differences in nest placement were driven by marsh type (i.e. differential availability of high marsh/low marsh in tidal vs. costal marshes) or genotype (innate behavioural differences among females), we ran mixed effect models to test the relative influence of marsh type (coastal or upriver) and hybrid index on nest placement. All mixed models were run using the nlme package in R, with female as a random effect to control for multiple nesting attempts of the same individuals.
Testing predictions of Haldane's rule
We tested two a priori predictions about the sex of hybrid offspring and adults based on expectations of Haldane's rule. First, if female hybrids had reduced fitness, we predicted that offspring of F1/F2 females should be male biased either due to reduced viability of female embryos or due to direct manipulation of nestling sex ratios by female hybrids in order to increase life-time fitness. To test this prediction, we compared the proportion of males produced by F1/F2 females compared with pure females using a chi-squared test. Second, if female hybrids exhibited reduced survival we predicted a reduction in the proportion of F1 hybrids in adult females compared with hatch-year females. To test this prediction, we used multilocus genotypes from both adult and nestlings to compare the proportion of recent generation hybrids (hybrid index of 0.25–0.75) among four different groups: hatch-year females, hatch-year males, after-hatch-year females, and after-hatch-year males using a chi-squared test to evaluate the survival of hybrids from nestling to adult.
Results
We sampled 228 nests associated with 147 females; 65 of the females (44%) were associated with more than one nest over the three breeding seasons. Across the four sites, we monitored 125 nests at Chapman's Landing, 57 nests at Eldridge marsh, 24 nests at Lubberland Creek, and 22 nests at Scarborough marsh. We obtained multilocus genotypes for 784 individuals, including 147 adult females, 247 adult males, and 390 nestlings; with the exception of 14 Nelson's sparrow females from Scarborough marsh, these individuals were sampled from three sites (EL, CL, LU). Of the 390 nestlings, we were able to ascertain the sex of 355 individuals (91%); the rest were dropped from the data set due to poor amplification success or ambiguous sex IDs, likely due to degraded samples.
Of the 784 individuals (adults and nestlings combined), we classified 27 (3.4%) as pure Nelson's sparrows and 212 (27.0%) as pure saltmarsh sparrows. The remaining 69.5% of the individuals exhibited some degree of admixture: we classified 63 (8%) as backcrossed Nelson's, 387 (49.4%) as backcrossed saltmarsh, and 95 (12.1%) as F1/F2 hybrids. Across the three sites where we captured adult males and females (EL, CL, LU), on average, based on morphological field identification, saltmarsh sparrows (pure and backcrossed individuals) outnumbered Nelson's sparrows (pure and backcrossed) 5.5: 1. In addition, adult Nelson's sparrow males (pure and backcrossed) outnumbered Nelson's sparrow females (pure and backcrossed), on average 3.5: 1, with similar imbalance at all three of the sites: 12 males: 4 females at Chapman's Landing, 21 males: 6 females at Eldridge, and four males: 0 females at Lubberland Creek. Sex ratios for adult saltmarsh sparrows (pure and backcrossed) were also skewed toward males, but less so, averaging 1.6: 1 on the three sites (183 males: 115 females).
Variation in reproductive output as a function of genotype
Comparison among the five genotypic classes revealed differences in reproductive output, however, F1/F2 hybrids were not intermediate with respect to the two parental species. Instead, Nelson's sparrows had reduced reproductive output compared with both saltmarsh sparrows (pure and backcrossed) and F1/F2 individuals. Significant differences were found in hatching success (F4,216 = 3.43, P = 0.009) and fledging success (F4,220 = 2.88, P = 0.023; Fig. 2). Pure Nelson's sparrows had 39%, 32%, and 28% lower hatching success (percentage of laid eggs to hatch) compared with F1/F2 hybrids (P = 0.01), backcrossed saltmarsh sparrows (P = 0.006), and pure saltmarsh sparrows (P = 0.02), respectively. Pure Nelson's sparrows also had 42, 30, and 32% lower fledging success (percentage of hatched chicks to fledge), compared with F1/F2 hybrids (P = 0.04), backcrossed saltmarsh sparrows (P = 0.07), and pure saltmarsh sparrows (0.07), respectively. Backcrossed Nelson's sparrows exhibited a trend for moderately higher frequencies of nests with at least one hatched egg (P = 0.10) compared with pure Nelson's sparrows but no differences in the frequency of nests with at least one fledged offspring (P = 0.95). Clutch size (P = 0.70), average chick weight (P = 0.60), maximum chick weight (P = 0.56), and range of chick weights (P = 0.26) did not differ for the five genotypic classes.
Patterns in average daily survival rates, fledging success, and hatching success – compared among female sparrows assigned to one of five genotypic classes NESP (pure Nelson's; n = 19), BCNESP (backcrossed Nelson's; n = 18), F1/F2 (first/second generation hybrids; n = 17), BCSALS (backcrossed saltmarsh; n = 110), and SALS (pure saltmarsh sparrow; n = 62). Mean ± SE for each of the five groups shown. Letter comparisons are included only for the reproductive parameters that differed among genotypic classes.
Intrinsic and extrinsic influences on nest survival
For intrinsic (genotypic) effects on nest survival, models including hybrid index while controlling for marsh type (coastal or inland) and year predicted daily nest survival probabilities better than the null model (Table 1). The model comparing F1/F2 females to the remainder of the genotypic classes out-performed all of the other categorical genotype models and the continuous hybrid index. Despite this, there were no differences among nest survival rates based on marsh type, year, or genotype. The top-ranked model included only marsh type and year (null model), and the addition of genotype to this model was equivalent (∆AICc for F1/F2 females vs. all other genotypic classes of females = 0.57; ∆AICc for continuous hybrid index = 1.98). Although the daily nest survival probabilities did not vary significantly with either the continuous hybrid index or the categorical genotypic classes, there was a non-significant trend for higher probabilities of daily nest survival in F1/F2 hybrids (estimate ± SE = 0.97 ± 0.01), backcrossed saltmarsh sparrows (0.96 ± 0.005), and pure saltmarsh sparrows (0.96 ± 0.006) compared with pure (0.93 ± 0.01) and backcrossed (0.94 ± 0.01) Nelson's sparrows (Fig. 2).
Intrinsic (genotypic) nest survival model selection results from MCestimate with estimated daily nest survival probabilities as a function of female genotype
| Model name | Negative log likelihood | AICc | Delta AICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year | 362.66 | 733.33 | 0 | 0.32 | 4 |
| Marsh type + Year + F1/F2 | 361.94 | 733.9 | 0.57 | 0.24 | 5 |
| Marsh type + Year + NESP | 362.63 | 735.28 | 1.95 | 0.12 | 5 |
| Marsh type + Year + PURE | 362.64 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index | 362.65 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index + HybridIndex^2 | 362.43 | 736.89 | 3.56 | 0.05 | 6 |
| Marsh type + Year + Genotypic class | 361.78 | 739.61 | 6.28 | 0.01 | 8 |
| Intercept only | 377.7 | 757.41 | 24.08 | 0 | 1 |
| Model name | Negative log likelihood | AICc | Delta AICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year | 362.66 | 733.33 | 0 | 0.32 | 4 |
| Marsh type + Year + F1/F2 | 361.94 | 733.9 | 0.57 | 0.24 | 5 |
| Marsh type + Year + NESP | 362.63 | 735.28 | 1.95 | 0.12 | 5 |
| Marsh type + Year + PURE | 362.64 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index | 362.65 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index + HybridIndex^2 | 362.43 | 736.89 | 3.56 | 0.05 | 6 |
| Marsh type + Year + Genotypic class | 361.78 | 739.61 | 6.28 | 0.01 | 8 |
| Intercept only | 377.7 | 757.41 | 24.08 | 0 | 1 |
Female genotype was modelled five different ways: continuous hybrid index, genotypic class, pure females vs. admixed females, pure Nelson's sparrow females vs. all other females, and F1/F2 females vs. all other females. Marsh type (coastal vs. river) and year were included in all models. AICc, delta AICc, model weight, and the number of parameters (K) are included for all models.
Intrinsic (genotypic) nest survival model selection results from MCestimate with estimated daily nest survival probabilities as a function of female genotype
| Model name | Negative log likelihood | AICc | Delta AICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year | 362.66 | 733.33 | 0 | 0.32 | 4 |
| Marsh type + Year + F1/F2 | 361.94 | 733.9 | 0.57 | 0.24 | 5 |
| Marsh type + Year + NESP | 362.63 | 735.28 | 1.95 | 0.12 | 5 |
| Marsh type + Year + PURE | 362.64 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index | 362.65 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index + HybridIndex^2 | 362.43 | 736.89 | 3.56 | 0.05 | 6 |
| Marsh type + Year + Genotypic class | 361.78 | 739.61 | 6.28 | 0.01 | 8 |
| Intercept only | 377.7 | 757.41 | 24.08 | 0 | 1 |
| Model name | Negative log likelihood | AICc | Delta AICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year | 362.66 | 733.33 | 0 | 0.32 | 4 |
| Marsh type + Year + F1/F2 | 361.94 | 733.9 | 0.57 | 0.24 | 5 |
| Marsh type + Year + NESP | 362.63 | 735.28 | 1.95 | 0.12 | 5 |
| Marsh type + Year + PURE | 362.64 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index | 362.65 | 735.31 | 1.98 | 0.12 | 5 |
| Marsh type + Year + Hybrid index + HybridIndex^2 | 362.43 | 736.89 | 3.56 | 0.05 | 6 |
| Marsh type + Year + Genotypic class | 361.78 | 739.61 | 6.28 | 0.01 | 8 |
| Intercept only | 377.7 | 757.41 | 24.08 | 0 | 1 |
Female genotype was modelled five different ways: continuous hybrid index, genotypic class, pure females vs. admixed females, pure Nelson's sparrow females vs. all other females, and F1/F2 females vs. all other females. Marsh type (coastal vs. river) and year were included in all models. AICc, delta AICc, model weight, and the number of parameters (K) are included for all models.
For extrinsic (nest placement) effects on nesting survival, top models included combinations of the four nest placement characteristics of interest (Table 2). Most of these models were equivalent based on differences in AICc values < 2.0, and the only covariate consistently included in the top models was the timing of nest initiation relative to flood tides. Thus, daily nest survival probabilities are linked strongly to timing of nest initiation (daily survival probability decreased with increasing number of days nests were initiated post flood tide), and do not appear to be as strongly influenced by other nest placement characteristics.
Extrinsic (nest placement) nest survival model selection results from MCestimate with estimated daily nest survival probabilities as a function of nesting characteristics
| Model name | Negative log likelihood | AICc | DeltaAICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year + Nest height + Nest initiation post flood tide | 190.93 | 393.91 | 0 | 0.29 | 6 |
| Marsh type + Year + Nest initiation post flood tide | 192.16 | 394.35 | 0.44 | 0.23 | 5 |
| Marsh type + Year + Nest height + Nest initiation post flood Tide + Proportion of high marsh | 190.63 | 395.33 | 1.42 | 0.14 | 7 |
| Marsh Type + Year + Nest initiation post flood tide + Proportion of high marsh | 191.69 | 395.44 | 1.53 | 0.14 | 6 |
| Marsh type + Year + Nest initiation post flood tide + nest canopy | 191.59 | 397.25 | 3.34 | 0.05 | 7 |
| Marsh type + Year + Nest height + Nest initiation post flood tide + Nest canopy | 190.64 | 397.37 | 3.46 | 0.05 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh | 191.18 | 398.44 | 4.53 | 0.03 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh + Nest height | 190.35 | 398.8 | 4.89 | 0.03 | 9 |
| Nest height + Nest initiation post flood tide | 197.56 | 401.14 | 7.22 | 0.01 | 3 |
| Nest height + Nest initiation post flood tide + Proportion of high marsh | 196.81 | 401.64 | 7.72 | 0.01 | 4 |
| Model name | Negative log likelihood | AICc | DeltaAICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year + Nest height + Nest initiation post flood tide | 190.93 | 393.91 | 0 | 0.29 | 6 |
| Marsh type + Year + Nest initiation post flood tide | 192.16 | 394.35 | 0.44 | 0.23 | 5 |
| Marsh type + Year + Nest height + Nest initiation post flood Tide + Proportion of high marsh | 190.63 | 395.33 | 1.42 | 0.14 | 7 |
| Marsh Type + Year + Nest initiation post flood tide + Proportion of high marsh | 191.69 | 395.44 | 1.53 | 0.14 | 6 |
| Marsh type + Year + Nest initiation post flood tide + nest canopy | 191.59 | 397.25 | 3.34 | 0.05 | 7 |
| Marsh type + Year + Nest height + Nest initiation post flood tide + Nest canopy | 190.64 | 397.37 | 3.46 | 0.05 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh | 191.18 | 398.44 | 4.53 | 0.03 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh + Nest height | 190.35 | 398.8 | 4.89 | 0.03 | 9 |
| Nest height + Nest initiation post flood tide | 197.56 | 401.14 | 7.22 | 0.01 | 3 |
| Nest height + Nest initiation post flood tide + Proportion of high marsh | 196.81 | 401.64 | 7.72 | 0.01 | 4 |
The top ten models (out of 32) are shown here. AICc, delta AICc, model weight, and the number of parameters are included for all models.
Extrinsic (nest placement) nest survival model selection results from MCestimate with estimated daily nest survival probabilities as a function of nesting characteristics
| Model name | Negative log likelihood | AICc | DeltaAICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year + Nest height + Nest initiation post flood tide | 190.93 | 393.91 | 0 | 0.29 | 6 |
| Marsh type + Year + Nest initiation post flood tide | 192.16 | 394.35 | 0.44 | 0.23 | 5 |
| Marsh type + Year + Nest height + Nest initiation post flood Tide + Proportion of high marsh | 190.63 | 395.33 | 1.42 | 0.14 | 7 |
| Marsh Type + Year + Nest initiation post flood tide + Proportion of high marsh | 191.69 | 395.44 | 1.53 | 0.14 | 6 |
| Marsh type + Year + Nest initiation post flood tide + nest canopy | 191.59 | 397.25 | 3.34 | 0.05 | 7 |
| Marsh type + Year + Nest height + Nest initiation post flood tide + Nest canopy | 190.64 | 397.37 | 3.46 | 0.05 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh | 191.18 | 398.44 | 4.53 | 0.03 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh + Nest height | 190.35 | 398.8 | 4.89 | 0.03 | 9 |
| Nest height + Nest initiation post flood tide | 197.56 | 401.14 | 7.22 | 0.01 | 3 |
| Nest height + Nest initiation post flood tide + Proportion of high marsh | 196.81 | 401.64 | 7.72 | 0.01 | 4 |
| Model name | Negative log likelihood | AICc | DeltaAICc | Weight | K |
|---|---|---|---|---|---|
| Marsh type + Year + Nest height + Nest initiation post flood tide | 190.93 | 393.91 | 0 | 0.29 | 6 |
| Marsh type + Year + Nest initiation post flood tide | 192.16 | 394.35 | 0.44 | 0.23 | 5 |
| Marsh type + Year + Nest height + Nest initiation post flood Tide + Proportion of high marsh | 190.63 | 395.33 | 1.42 | 0.14 | 7 |
| Marsh Type + Year + Nest initiation post flood tide + Proportion of high marsh | 191.69 | 395.44 | 1.53 | 0.14 | 6 |
| Marsh type + Year + Nest initiation post flood tide + nest canopy | 191.59 | 397.25 | 3.34 | 0.05 | 7 |
| Marsh type + Year + Nest height + Nest initiation post flood tide + Nest canopy | 190.64 | 397.37 | 3.46 | 0.05 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh | 191.18 | 398.44 | 4.53 | 0.03 | 8 |
| Marsh type + Year + Nest initiation post flood tide + Nest canopy + Proportion of high marsh + Nest height | 190.35 | 398.8 | 4.89 | 0.03 | 9 |
| Nest height + Nest initiation post flood tide | 197.56 | 401.14 | 7.22 | 0.01 | 3 |
| Nest height + Nest initiation post flood tide + Proportion of high marsh | 196.81 | 401.64 | 7.72 | 0.01 | 4 |
The top ten models (out of 32) are shown here. AICc, delta AICc, model weight, and the number of parameters are included for all models.
Variation in reproductive success and nest placement among genotypic classes
We found a positive relationship between nest height and hybrid index (nest height increased with increasing hybrid index, suggesting that pure saltmarsh sparrows build higher nests; β ± SE = 2.53 ± 1.17, t = 2.17, P = 0.03). We attribute differences in nest heights to female genotype, as we detected no differences in nest height between coastal and upriver marshes (i.e., this difference in nesting behaviour can not be attributed to variation in vegetation height in different habitat types; β ± SE = 0.91 ± 0.80, t = 1.15, P = 0.25). Similarly, the proportion of high marsh surrounding a nest did not differ between coastal and upriver sites (β ± SE = 1.35 ± 3.78, t = 0.35, P = 0.73) but did show a negative relationship with hybrid index. We observed increases in vegetation structure with increasing hybrid index, suggesting that pure saltmarsh sparrows build in a mix of low and high marsh, while Nelson's sparrows build primarily in high marsh (β ± SE = −11.54 ± 5.27, t = −2.20, P = 0.03). We did not find a significant relationship between canopy cover (none, partial, or complete; β ± SE = −0.20 ± 0.22, t = −0.88, P = 0.37) or nest initiation timing post flood tide (β ± SE = −0.51 ± 1.02, t = −0.5, P = 0.62) with hybrid index.
Testing predictions of Haldane's rule
Using molecular sexing, we identified 174 female and 181 male offspring. Due to loss of eggs and nestlings during flooding events, we had sex ratio information from full clutches for 78 of the 228 nests. The proportion of males in a nest ranged from 0.25 to 0.75 for a majority (67%) of the nests; ten nests (13%) had 0 male nestlings and 16 nests (20%) had all male nestlings. We did not detect a difference in the proportion of males in a clutch among genotypic classes (P = 0.63) or in hybrid index between male and female nestlings (2 tailed student's t-test: t = 0.89, P = 0.37). The distribution of F1/F2 hybrids varied among sexes and age classes, however. While the proportion of F1/F2 individuals was similar between hatch-year males (n = 22; 12%) and adult males (n = 33; 13%), the proportion of F1/F2 individuals was significantly higher (2.3 times higher; χ2 = 8.59, d.f. = 1, P = 0.003) in hatch-year females (n = 24; 14%) compared with adult females (n = 8; 6%; Fig. 3).
Bar plot showing distribution of pure and hybrid genotypes by sex and age for 735 sparrows sampled from three marshes: Chapman's Landing, Lubberland Creek, and Eldridge marsh. The five panels show proportions of each group: hatch-year (nestling) males (HY males; n = 181), after-hatch-year (adult) males (AHY males; n = 247), hatch-year (nestling) females (HY females; n = 174), and after-hatch-year (adult) females (AHY females; n = 133) for each of the five genotypic classes (NESP, BCNESP, F1/F2, BCSALS, and SALS), across sample sites.
Discussion
We found differences in reproductive output (hatching and fledging success) and nesting behaviours among females as a function of their genotype. Contrary to predictions of intrinsic reproductive isolation, we found no reduction in reproductive success in recent generation (F1/F2) hybrid females compared with pure saltmarsh sparrows. When comparing both fledging and hatching success, F1/F2 females exhibited success rates comparable with pure and backcrossed saltmarsh sparrows but higher than pure and backcrossed Nelson's sparrows. This result suggests that adaptations for breeding in tidal marshes contribute to reproductive isolation or dominance of alleles favored in salt marsh environments. Consistent with previous work in this system (Shriver et al., 2007), we documented lower hatching and fledging success in pure Nelson's sparrows compared with pure saltmarsh sparrows. Given their exclusive use of salt marsh habitat, greater hatching and fledging success in saltmarsh sparrows compared with Nelson's sparrows is not surprising. However, the similar reproductive success of F1/F2 hybrids compared with pure saltmarsh sparrows in tidal marshes was not predicted. As such, it is possible that nesting behaviours inherent to pure saltmarsh sparrows may benefit admixed individuals nesting in salt marshes, resulting in a trend for increased reproductive success with increasing hybrid index (higher proportion of saltmarsh sparrow alleles in an individual).
Our results suggest a strong role for extrinsic mechanisms in shaping reproductive success. Decreased fledging success in all individuals was predominantly attributed to tidal flooding, which was the leading cause of nest failure. To this end, the observed differences in nesting behaviour between saltmarsh sparrows, Nelson's sparrows, and hybrids may afford differential capacities for withstanding flooding events. Saltmarsh sparrow eggs are known to remain viable after some flooding, and even successful nests can experience up to ten flooding events (Bayard & Elphick, 2011). We hypothesize, however, that more frequent flooding reduces hatching success by increasing the likelihood that any given egg becomes inviable (damaged from cumulative effects of multiple flooding events) or washed out of the nest. Furthermore, flooding is most likely to influence hatching success rather than fledging success, as nestlings that are partially mobile but still too young to fledge have been shown to climb out of nests during flooding events and return afterward (Gjerdrum et al., 2008; Bayard & Elphick, 2011). Small differences in nest height might thus make the difference between complete nest failure and the ability of partial fledging success. Our results are consistent with such a mechanism. Saltmarsh sparrows, with their higher nests, exhibited higher egg viability rates (manifest in higher hatching success) and a greater proportion of fledged young. Across all females, individuals with a higher hybrid index (i.e. pure saltmarsh sparrows) exhibited nesting behaviours more likely to result in offspring production (i.e., nests built higher off of the ground and a greater diversity of high and low marsh surrounding the nest) by decreasing the extent of nest flooding during high tide events. Nests that are built higher intuitively will be less impacted by flood tides than nests that are built closer to the ground. Alternatively, increased nest structure provided by weaving nests with a mixture of Spartina patens (high marsh) and Spartina alterniflora (low marsh) may allow nests to withstand flooding better than nests built primarily in the softer, less rigid Spartina patens.
There was no difference, however, in overall nest survival (i.e., probability of a nest producing at least a single fledged offspring) by genotype, suggesting that both species and their hybrids were at similar risk of complete nest loss due to tidal flooding. Although we found no differences in overall nest success between F1/F2 individuals and pure and backcrossed saltmarsh sparrows, we did detect differences in other indices between pure and backcrossed Nelson's sparrows and pure Nelson's sparrows and F1/F2 hybrids that may suggest benefits of hybridization for this species. Backcrossed Nelson's sparrows had moderately higher hatching success than pure Nelson's sparrows, and F1/F2 individuals exhibited significantly higher hatching success than pure Nelson's sparrows. Additionally, although the differences in daily nest survival probabilities were not significant, backcrossed Nelson's sparrows and F1/F2 individuals exhibited a trend of higher survival probabilities (0.97 and 0.94, respectively) compared with pure Nelson's sparrows (0.93), suggesting a potential benefit to introgression of saltmarsh sparrow alleles. This factor may be an important driver of ongoing hybridization and introgression between saltmarsh and Nelson's sparrows. Specifically, increased genetic diversity resulting from ongoing hybridization and gene flow between these species may increase the adaptive capacity of Nelson's sparrows as they move into more coastal environments (Nicotra et al., 2015).
While there may be a potential benefit to hybridization for Nelson's sparrows, trends in behavioural differences in relation to hybrid index may be less positive for saltmarsh sparrows. Although F1/F2 hybrids did not have significantly reduced nesting success compared with pure saltmarsh sparrows, we found a decrease in nest height with decreasing hybrid index and a decrease in nest vegetation diversity with decreasing hybrid index. Based on these patterns, admixed individuals appear to exhibit lower nest heights and higher proportion of high marsh surrounding the nest compared with pure saltmarsh sparrows. If Nelson's and saltmarsh sparrows represent ends to the nest placement spectrum, arguably, intermediate nesting behaviours observed in F1/F2 individuals may place them at a disadvantage compared with pure saltmarsh sparrows. While this hypothesis would require further research with larger sample sizes of F1/F2 hybrids, fitness reductions resulting from intermediate behaviours have been documented in a number of natural hybrid zones. Specifically, hybrid intermediaries have been documented in song and mating displays (Svedin et al., 2008), aggression and territorial behaviours (Pearson & Rohwer, 2000), and migratory behaviour (Delmore & Irwin, 2014).
We also observed notable differences in the distribution of the species and sexes that may play an important role in structuring hybrid zone dynamics. Substantial differences in abundance of saltmarsh and Nelson's sparrows (5.5:1, respectively) and, in particular, differences in sex ratios within Nelson's sparrows may suggest an additional mechanism for ongoing hybridization and asymmetrical introgression. Previous work in this system has offered multiple lines of support for asymmetrical introgression toward saltmarsh sparrows (Shriver et al., 2005; Walsh et al., 2011, 2016). While these patterns could be driven by differences in mating strategy (Shriver et al., 2005), hybridization between populations differing in abundance can result in asymmetrical backcrossing of F1 hybrids to the more abundant parent by chance alone (Anderson, 1948; Haygood, Ives & Andow, 2003; Burgess et al., 2005). On Lubberland Creek, we captured Nelson's males but not Nelson's females, despite intensive nest searching and banding efforts. Unsurprisingly, 65% of the nestlings banded on Lubberland Creek were F1/F2 hybrids. Sites that display these extreme differences in the proportions of Nelson's males and females are clearly important in facilitating ongoing hybridization and gene flow between the two species. It is possible that decreased fecundity of Nelson's females is partially responsible for the observed differences in sex ratios. Decreased reproductive success can drive females out of marginal habitats (Switzer, 1993, 1997; Doligez et al., 1999) or an increase in reproductive effort as a result of continuous nest loss may reduce overall survival of females (Nur, 1984; Daan, Deerenberg & Dijkstra, 1996; Nordling et al., 1998). Alternatively, males may disproportionately settle in marginal habitats due to social queues, however it should be noted that this behaviour has not been observed in Nelson's sparrows, which are thought to be a solitary species (Nocera, Forbes & Giraldeau, 2006).
Our findings also support a role for intrinsic selection in shaping hybridization outcomes between saltmarsh and Nelson's sparrows, through reductions in hybrid female survival. Comparison of F1/F2 individuals across age groups and sexes revealed a reduction in F1/F2 females from hatch-year (14%) to adult (6%); the same comparison in males showed no reduction in hybrids between age classes. This evidence for reduced survival in F1/F2 females aligns with predictions of Haldane's rule (Haldane, 1922). Even in the absence of immediate fitness consequences, the strength of post-zygotic isolation is often more evident over the long term (Wiley et al., 2009; Neubauer et al., 2014). Fitness reductions in the heterogametic sex, as predicted by Haldane's rule, appear to play an important role in speciation in a variety of organisms (Coyne & Orr, 2004). Reduced survival in F1/F2 females translates into reduced reproductive output of hybrid females compared with parental species and can lead to less gene flow between hybridizing species over time (Neubauer et al., 2014). Our finding of reduced survival of hybrid females is consistent with findings of reduced introgression of sex-linked and mitochondrial genes across the saltmarsh–Nelson's sparrow hybrid zone (Walsh et al., 2016). Extensive genetic data collected from 34 marshes spanning the entire hybrid zone further indicated an overall deficit of F1/F2 individuals, with most individuals being pure or backcrossed (Walsh et al., 2015a). Although F1/F2 offspring are viable and fertile, the deficit of hybrid adults supports an important role for post-zygotic isolation in maintaining species boundaries in the face of ongoing gene flow.
ACKNOWLEDGEMENTS
We thank the Nature Conservancy and the Maine Department of Inland Fisheries and Wildlife for allowing sample collection in protected marshes. We also thank M.B Hunt, K.E. Papanastassiou, B. Flemer, and L. Kordonowy for help in the field. We thank C. Benkman and one anonymous reviewer for their helpful comments. Funding for this project was provided by the United States Fish and Wildlife Service Region 5, Division of Natural Resources, National Wildlife Refuge System, the Northeast Regional Conservation Needs Grant Program, the New Hampshire Agricultural Experiment Station, through a USDA National Institute of Food and Agriculture McIntire-Stennis Project #225575, the University of Maine, a Competitive State Wildlife Grant (U2-5-R-1) via the United States Fish and Wildlife Service, Federal Aid in Sportfish and Wildlife Restoration to the states of Delaware, Maryland, Connecticut, and Maine, and the Maine Association of Wetland Scientists. This is Scientific Contribution Number 2676 of the New Hampshire Agricultural Experiment Station. Sampling was conducted in accordance with the Institutional Animal Care and Use Committee of the University of New Hampshire (100605, 130604) and of the University of Maine (A2011-04-02). The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the US Fish and Wildlife Service.
