Understanding the potential for organisms to tolerate thermal stress through physiological or evolutionary responses is crucial given rapid climate change. Although climate models predict increases in both temperature mean and variance, such tolerances are typically assessed under constant conditions. We tested the effects of temperature variability during development on male fitness in the rainforest fly Drosophila birchii, by simulating thermal variation typical of the warm and cool margins of its elevational distribution, and estimated heritabilities and genetic correlations of fitness traits. Reproductive success was reduced for males reared in warm (mean 24 °C) fluctuating (±3 °C) vs. constant conditions but not in cool fluctuating conditions (mean 17 °C), although fluctuations reduced body size at both temperatures. Male reproductive success under warm fluctuating conditions was similar to that at constant 27 °C, indicating that briefly exceeding critical thermal limits has similar fitness costs to continuously stressful conditions. There was substantial heritable variation in all traits. However, reproductive success traits showed no genetic correlation between treatments reflecting temperature variation at elevational extremes, which may constrain evolutionary responses at these ecological margins. Our data suggest that even small increases in temperature variability will threaten tropical ectotherms living close to their upper thermal limits, both through direct effects on fitness and by limiting their adaptive potential.

Rapid ecological speciation along depth gradients has taken place repeatedly in freshwater fishes, yet molecular mechanisms facilitating such diversification are typically unclear. In Lake Masoko, an African crater lake, the cichlid Astatotilapia calliptera has diverged into shallow littoral and deep benthic ecomorphs with strikingly different jaw structures within the last 1,000 years. Using genome-wide transcriptome data, we explore two major regulatory transcriptional mechanisms, expression and splicing QTL variants, and examine their contributions to differential gene expression underpinning functional phenotypes. We identified 7,550 genes with significant differential expression between ecomorphs, of which 5.4% were regulated by cis-regulatory expression QTLs, and 9.2% were regulated by cis-regulatory splicing QTLs. We also found strong signals of divergent selection on differentially expressed genes associated with craniofacial development. These results suggest that large-scale transcriptome modification plays an important role during early-stage speciation. We conclude that regulatory-variants are important targets of selection driving ecologically-relevant divergence in gene expression during adaptive diversification.

Evolutionary responses to environmental change require heritable variation in traits under selection. Both heritability and selection vary with the environment, and may also covary, meaning that environmental variation can be an important source of evolutionary constraint. However, estimates of heritability and selection along environmental gradients in the field are rare. We estimated environmental variation in selection on three traits (cold tolerance, heat tolerance and wing size) of the rainforest fly Drosophila birchii by transplanting flies in cages along two elevational gradients in north-east Queensland, Australia, and calculating the genetic covariance of trait values with cage productivity at each elevation. We estimated heritability of each trait from laboratory crosses, and environmental variation in heritability of wing size from the correlation of mothers and daughters in cages at each elevation. We then used estimates of selection and heritability to predict selection responses along the elevation gradients. Laboratory assays revealed low-moderate genetic variation in all traits and low covariation among traits, suggesting the potential for a strong response to selection. Estimated selection responses predicted divergence of cold tolerance with elevation at one gradient. However, this was not observed at either gradient, with no difference between high and low elevation populations for this trait. Despite substantial variation in heritability (and predicted selection response) of wing size, this appeared random with respect to elevation, preventing overall divergence and suggesting that local environmental variation constrains evolutionary responses along natural ecological gradients. Such an effect, if widespread, may significantly slow evolutionary responses to environmental change.

Mating success is the main source of fitness variation in males, meaning that males should capitalise on all opportunities for mating. Strong selection on male mating success should also reduce genetic variation in male mating traits relative to other traits. We quantified mating latency, mating duration and productivity in males of the tropical fruitfly, Drosophila birchii, from 30 isofemale lines collected from across two elevational gradients, when they were given opportunities to mate with up to four females consecutively. Male remating rates were low compared to other Drosophila (only 14 – 27% of males achieved a fourth mating), with mean mating durations approximately doubling across successive copulations. However, although successive remating produced progressively fewer offspring, it consistently increased overall male reproductive success, with males that mated four times more than doubling offspring number compared to males mating only once. We also found no reduction in the productivity of sons emerging from later matings, indicating a sustained cumulative fitness benefit to remating. Heritable variation was observed for most traits (H2 = 0.035 – 0.292) except mating latency, but there was no divergence in trait means with elevation. The observed restricted remating ability of male D. birchii, despite the clear benefits of remating, may be due to a low encounter rate with females in the field, leading to high investment per gamete (or ejaculate). However, it remains unclear why genetic variation in these traits is high, given we observe no variation in these traits across elevational gradients known to affect local population density.

Molecular phylogenetic evidence clearly resolves the African cichlid fish genus Ctenochromis, as defined by Greenwood (1979), as paraphyletic. Here, we redefine the genus Ctenochromis and assign Ctenochromis horei, a member of the Tropheini from Lake Tanganyika, to a new genus Shuja gen. nov. We restrict Ctenochromis to Ctenochromis pectoralis and Ctenochromis scatebra sp. nov., both of which are endemic to the Pangani River catchment in northern Tanzania, and are resolved as sister taxa in a phylogenetic analysis using genome-wide data. Ctenochromis pectoralis is the type species of the genus and described from specimens collected near Korogwe, Tanzania. The species was declared extinct in a 2016 IUCN Red List Assessment. We confirm the continued presence of a population of C. pectoralis within the Ruvu tributary linking Lake Jipe to Nyumba ya Mungu Reservoir. The new taxon Ctenochromis scatebra sp. nov. is described from Chemka Springs, and recognised on the basis of differences from C. pectoralis in tooth and jaw morphology.

Rapid ecological speciation along depth gradients has taken place independently and repeatedly in freshwater fishes. While the extent of genomic divergence between ecomorphs is often well understood, the molecular mechanisms facilitating such rapid diversification are typically unclear. In Lake Masoko, an East African crater lake, the cichlid Astatotilapia calliptera has diverged into shallow littoral and deep benthic ecomorphs with strikingly different jaw structures within the last 1,000 years. Using genome-wide transcriptome data from jaw tissue, we explore two major regulatory transcriptional mechanisms, expression and splicing QTL variants and examine their contribution to differential gene expression underpinning functional phenotypes. We identified 7,550 genes with significant differential expression between ecomorphs, of which 4.2% were regulated by cis-regulatory expression QTLs, and 6.4% were regulated by cis-regulatory splicing QTLs. There were also strong signals of divergent selection of differentially expressed genes that showed divergent regulation from expression, splicing or both QTL variants, including genes associated with major jaw plasticity and adaptation networks, adaptive immune system response, and oxidoreductase processes. These results suggest that transcriptome plasticity and modification have important roles during early-stage ecological speciation and demonstrate the role of regulatory-variants as important targets of selection driving ecologically-relevant divergence in gene expression that is associated with adaptive diversification.