DOI: 10.5061/dryad.6m905qg09
title: Survival and malformations, swimming performance and tadpole traits

description: Targeted gene flow (TGF) could bolster the adaptive potential of isolated
 populations threatened by climate change, but could also lead to
 outbreeding depression. Here, we explore these possibilities by creating
 mixed- and within-population crosses in a terrestrial-breeding frog
 species threatened by a drying climate. We reared embryos on wet and dry
 soils and quantified fitness-related traits upon hatching. TGF produced
 mixed outcomes in hybrids which depended on crossing direction (origin of
 gametes from each sex). North-south crosses led to low embryonic survival
 if eggs were of a southern origin, and high malformation rates when eggs
 were from a northern population. Conversely, east-west crosses led to one
 instance of hybrid vigour, evident by increased fitness and desiccation
 tolerance of hybrid offspring relative to offspring produced from
 within-population crosses. These contrasting results highlight the need to
 experimentally evaluate the outcomes of TGF for focal species across
 generations prior to implementing management actions.
description: We explored the potential of
 targeted gene flow (TGF) to mitigate declines in population-level fitness
 using the crawling frog, <i>Pseudophryne guentheri</i>, a
 species in which populations are threatened by habitat loss and declining
 winter rainfall. We evaluated TGF within a
 laboratory setting by creating pure and reciprocal crosses among four
 geographically distant populations. Two populations occurred in
 low-rainfall regions at the northern edge of the species’ range, and two
 other populations were from higher-rainfall regions close to the centre
 the species range. We then assessed phenotypic traits in the resulting
 offspring, comparing individuals reared on wet soils (-10 kPa, a benign
 treatment) to those reared on drier soils (-400 kPa) that significantly
 reduce survival and hatchling fitness. Detailed methods
 from manuscript: We collected adult <i>P.
 guentheri</i> from four geographically separated breeding sites,
 situated at two latitudes, in May and June 2017 (Table 1). Sites spanned a
 ~460 mm annual rainfall gradient, with site A receiving the most rain per
 year and site D receiving the least (Table 1, Fig. 1).
 <i>Pseudophryne guentheri</i> collected from breeding
 populations at each site show variation in desiccation tolerance, with
 adults and embryos from site A being the most sensitive to dry
 conditions<sup>37</sup>. Population genetic
 analysis<sup>39</sup> has demonstrated high levels of
 inbreeding in all populations (Table 1), and genetic differentiation among
 <i>P. guentheri </i>populations is high (overall
 <i>F<sub>ST</sub></i> = 0.186), which suggests low
 levels of contemporary dispersal. <i>P. guentheri </i>from
 sites A and B form distinct genetic clusters<sup>39</sup>,
 indicating low historical gene flow despite their close proximity (100
 km), whereas <i>P. guentheri</i> from sites C and D show
 admixture, but are genetically distinct from populations A and
 B<sup>39</sup>. In total, 15-16 calling males
 from each population were collected by hand and in pit-fall traps. Gravid
 females were more difficult to collect due to their cryptic behaviours,
 and so sampling was restricted to 5-13 females from each of three sites
 (A, B and C; Table 1). All frogs were temporarily housed in small (4.4 L)
 plastic terraria containing moist sphagnum moss, and transported to the
 University of Western Australia within two days of collection. There,
 frogs were fed a diet of pinhead crickets and kept in a
 controlled-temperature room at 16 °C with an 11/13 h light/dark
 photoperiod to mimic winter conditions. Breeding design and
 <i>in vitro</i> fertilisations. Egg clutches of each female
 were divided equally into four groups, and fertilised with sperm from
 males originating from each of the four populations, resulting in one pure
 and three hybrid crosses. To control for potential parental compatibility
 (i.e. specific pairwise male-by-female) effects on offspring
 fitness<sup>38,69</sup>, a sperm mixture, containing sperm
 from five random males from the appropriate population, was used to
 fertilise the eggs of each female in each
 population<sup>88</sup>. Sperm was obtained from testes
 macerates after euthanizing males via ventral immersion in &lt;0.03%
 benzocaine solution, followed by double pithing. Sperm was stored on ice
 in 25-458 μL (adjusted according to the weight of the testes) standard
 amphibian ringer (SAR; 113mM NaCl, 2mM KCl, 1.35 mM
 CaCl<sub>2</sub>). This buffer allows storage of sperm for
 extended periods (days–weeks) without substantial declines in
 motility<sup>89,90</sup>. Sperm concentrations were measured
 using an improved Neubauer haemocytometer (Hirschmann Laborgeräte,
 Eberstadt, Germany) and sperm suspensions were diluted with 1:1 SAR to 100
 sperm per μL. Upon arrival at the laboratory,
 females were gently squeezed to determine whether ovulation had occurred.
 Approximately 35% of females had ovulated naturally while in transit and
 their eggs were gently stripped. For the remaining females, ovulation was
 induced via two subcutaneous injections of the hormone LHRHa over the
 course of two days<sup>37,87</sup>. Approximately 10 hours
 after the second injection, eggs were gently stripped from each female. In
 all instances, freshly stripped eggs were moistened with SAR and
 distributed equally among four small petri dishes. A standardised number
 of sperm from five random males collected at each site was pipetted onto
 one edge of each petri dish, mixed gently with the pipette tip, and then
 activated with a pre-calculated volume of 1:4 SAR
 solution<sup>38</sup>. This resulted in eggs from all females
 in the experiment being fertilised from males collected from sites A, B, C
 and D. Each dish was then manually agitated for 20 seconds to promote
 fertilisation. After 15 minutes, eggs were temporarily submerged in water,
 backlit and photographed using a digital imaging camera (Leica DFC320)
 attached to a light microscope (Leica MZ7.5) at 6.3 Χ magnification. These
 images were used to measure the ovum diameter of 50 randomly-selected eggs
 from each female, using ImageJ software<sup>91</sup>.
 Fertilisation success was initially scored one hour after mixing eggs and
 sperm by counting eggs that had rotated (Gosner<sup>79</sup>
 stage 1). However, eggs from populations A and B took substantially longer
 to show signs of fertilisation when mixed with sperm from populations C
 and D. We therefore scored fertilisation success a second time, six hours
 after sperm and eggs were mixed. Incubation treatments.
 Fertilised eggs from each cross were reared on sandy loam soil at two
 water potentials (ψ): a wet soil (ψ = -10 kPa) and dry soil (ψ = -400
 kPa). The soil was previously collected from a separate <i>P.
 guentheri </i>breeding site, and the soil water potentials
 represented a range found in natural nest sites (N. J. Mitchell, unpubl.
 data). Embryo incubation and soil preparation were performed as described
 in Rudin-Bitterli et al.<sup>92</sup>. Briefly, soil was
 oven-dried at 80°C for 24 hours, distributed into small containers and
 rewetted with an appropriate mass of deionised water using a
 water-retention curve previously determined for the soil sample (N. J.
 Mitchell, unpubl. data). The water content of the soil (g/g/ of oven dry
 soil) was approximately 50% in the wet treatment, and 21% in the dry
 treatment, and containers were sealed with a lid after wetting. Fertilised
 eggs from each cross were selected at random and distributed onto soils
 within 7 – 9 hours of fertilisation. Small plastic rings (nylon plumbing
 olives, 12 mm in diameter) were labelled and placed around eggs to
 identify individual crosses. Sealed containers were then placed in
 incubators set at 16 ± 0.5 °C, and embryos were monitored every two days.
 Any dead eggs were removed and discarded. Response variables. Putative
 fitness from within- and between-population crosses, reared in dry and wet
 rearing environments, was assessed at hatching. At 33 days after
 fertilisation (when embryos were approximately at
 Gosner<sup>79</sup> Stage 26), hatching was induced by placing
 embryos individually in small test tubes containing 2 ml of deionised
 water<sup>38</sup>. Embryos were then monitored at least every
 30 min until hatching, defined as when an individual completely escaped
 their egg capsule. Embryonic survival was recorded for each family as the
 percentage of fertilised eggs that hatched. Swimming performance was recorded
 6 - 12 hours after hatching on a subset of hatchlings
 (<i>N</i> = 633 across all within and between-population
 crosses). For this purpose, individual hatchlings were placed in a petri
 dish (diameter = 150 mm) containing water 10 mm deep. After an initial
 acclimation period of 1 min, the tail of each hatchling was nudged with a
 glass cannula to elicit a burst swimming response. A video camera (Canon
 PowerShot G16, recording at 60 fps) installed 300 mm above the petri dish
 was used to film three burst swimming responses for each hatchling, and
 their movement was later tracked and analysed using EthoVision v8.5
 software<sup>93</sup>. EthoVision enabled the quantification
 of the following swimming parameters: maximum velocity (cm
 s<sup>-1</sup>), mean velocity (cm s<sup>-1</sup>)
 and total distance moved (cm). We also recorded mean meander (deg
 cm<sup>-1</sup>), a measure of the straightness of the
 swimming response, as dry rearing environments can lead to asymmetrically
 shaped hatchlings<sup>37,38</sup> that swim in a more circular
 motion. A hatchling was considered to be moving when it exceeded 0.45 cm
 s<sup>-1</sup>. As each video recording contained three burst
 swimming responses with periods of no movement in between them, EthoVision
 only analysed frames in which a hatchling moved faster than 0.45 cm
 s<sup>-1</sup> (consequently merging the three swimming
 responses for each hatchling). Immediately following the swimming
 performance trials, hatchlings were euthanized in &lt;0.03% benzocaine and
 preserved in 10% neutral buffered formalin. Wet masses of preserved
 hatchlings were recorded to the nearest 0.001 g after blotting on tissue.
 Hatchlings were then photographed in lateral view (while submerged in
 water to minimize refraction) using a digital imaging camera
 (Leica DFC320) attached to a light microscope (Leica MZ7.5) at X 6.3
 magnification. These images were used to score malformations for each
 hatchling and to determine their developmental
 stage<sup>79</sup> by examining the hind limb
 buds. Statistical
 analysis.<i> </i>All analyses were performed in R version
 3.4.3 (R Development Core Team 2017). Linear mixed-effects models (with
 restricted maximum-likelihood methods; REML) were run using the lme4
 package<sup>94</sup> to compare offspring traits from pure and
 hybrid crosses. In these models, treatment, female (population) origin,
 male (population) origin and all interactions (female origin-by-male
 origin, female origin-by-treatment, male origin-by-treatment, female
 origin-by-male origin-by-treatment) were considered as fixed factors.
 We
 checked for overdispersion in our models using the ‘overdisp_fun’ function
 proposed by Bolker et al <sup>95</sup>. Only one trait,
 embryonic survival, was overdispersed; we added observation level as an
 extra random factor to account for overdispersion when analyzing this
 trait (Harrison 2014). Because the eggs from each female were
 subjected to a split-clutch design, the term for dam (i.e. individual
 female ID) was added as a random effect to account for the use of
 individual females across multiple fertilisation events. We also included
 ovum size as a covariate in all analyses to control for possible maternal
 effects arising from different patterns of egg provisioning among
 females<sup>38</sup>. The significance of the fixed effects
 was evaluated using Wald chi-squared tests. Embryonic survival and hatchling
 malformation data were binomial variables and thus a generalized-linear
 mixed-effects model (GLMM) with a logit-link function was used for the
 analysis of these traits. In these models we included treatment, female
 (population) origin, male (population) origin and all interactions (see
 above) as fixed effects. As above, dam ID was treated as a random effect
 and ovum size was added as a covariate to control for possible maternal
 effects<sup>38</sup>. The significance of the
 fixed effects was evaluated using Wald Z tests.