Survival and malformations, swimming performance and tadpole traits

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.,We explored the potential of targeted gene flow (TGF) to mitigate declines in population-level fitness using the crawling frog, Pseudophryne guentheri, 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 P. guentheri 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). Pseudophryne guentheri 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 conditions37. Population genetic analysis39 has demonstrated high levels of inbreeding in all populations (Table 1), and genetic differentiation among P. guentheri populations is high (overall FST = 0.186), which suggests low levels of contemporary dispersal. P. guentheri from sites A and B form distinct genetic clusters39, indicating low historical gene flow despite their close proximity (100 km), whereas P. guentheri from sites C and D show admixture, but are genetically distinct from populations A and B39. 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 in vitro 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 fitness38,69, a sperm mixture, containing sperm from five random males from the appropriate population, was used to fertilise the eggs of each female in each population88. Sperm was obtained from testes macerates after euthanizing males via ventral immersion in

靶向基因流（Targeted Gene Flow, TGF）能够提升受气候变化威胁的孤立种群的适应潜力，但也可能引发远交衰退。本研究通过对一种因气候干旱化而受到威胁的陆栖繁殖蛙类开展种群内和种群间杂交实验，探究了上述两种可能性。我们将胚胎分别置于湿润和干燥土壤环境中饲养，并量化了孵化后与适合度相关的性状。研究结果显示，靶向基因流对杂交后代的影响存在混合效应，且取决于杂交方向（即两性配子的来源群体）。当卵来自南方种群时，南北向杂交的胚胎存活率较低；而当卵来自北方种群时，杂交后代的畸形率显著升高。与之相反，东西向杂交则出现了一例杂种优势现象：相较于种群内杂交产生的后代，杂交子代的适合度与耐脱水性均有所提升。这些截然相反的研究结果表明，在实施管理措施前，需通过实验评估靶向基因流对目标物种跨代的影响效果。

本研究以冈瑟氏澳小蛙（Pseudophryne guentheri）为研究对象，探讨了靶向基因流（TGF）在缓解种群水平适合度下降方面的潜力，该物种种群正受到栖息地丧失与冬季降雨减少的威胁。我们在实验室环境下开展了靶向基因流评估实验，对四个地理距离较远的种群进行了纯系杂交与正反交实验。其中两个种群分布于该物种分布范围北缘的低降雨区域，另外两个种群则来自接近该物种分布中心的高降雨区域。随后我们对杂交后代的表型性状进行了评估，并将饲养于湿润土壤（-10 kPa，良性处理组）的个体与饲养于干燥土壤（-400 kPa，该环境会显著降低存活率与幼体适合度）的个体进行了比较。

论文详细方法如下：我们于2017年5月至6月间，从四个地理隔离的繁殖位点采集了成年冈瑟氏澳小蛙（表1）。这些位点的年降雨量跨度约为460毫米，其中A位点年降雨量最高，D位点最低（表1、图1）。从各繁殖位点采集的冈瑟氏澳小蛙在耐脱水性上存在差异：来自A位点的成体与胚胎对干燥环境最为敏感37。种群遗传学分析39显示，所有种群均存在较高程度的近交，且种群间遗传分化显著（整体FST=0.186），这表明当代扩散水平较低。A、B位点的冈瑟氏澳小蛙形成了独立的遗传簇39，尽管二者地理距离仅100公里，但历史基因交流水平较低；而C、D位点的种群存在遗传混合，但与A、B种群遗传分化显著39。

我们通过手捕与陷阱诱捕的方式，共采集了每个种群15-16只鸣叫雄性个体。由于雌性成体具有隐蔽行为，较难采集，因此仅从A、B、C三个种群中分别采集了5-13只雌性个体（表1）。所有蛙类被暂时饲养于容积为4.4升的塑料饲养盒中，盒内放置湿润的泥炭藓，采集后两天内被转运至西澳大学。在实验室中，我们以针头蟋蟀作为饲料，将蛙类饲养于温度控制为16℃、光周期为11小时光照/13小时黑暗的恒温室内，以模拟冬季环境。

杂交设计与体外受精：将每只雌性的卵团均分为四组，分别用来自四个种群的雄性的精子进行受精，从而获得一组纯系杂交与三组远缘杂交组合。为了控制潜在的亲本兼容性效应（即特定的雌雄配对对子代适合度的影响38,69），我们使用混合精子进行受精：将来自对应种群的5只随机雄性的精子混合，用于受精每只雌性的卵88。精子通过将雄性个体经腹浸法安乐死后，研磨睾丸获取。