Tropical rainforests, harboring over half the world's species, are crucial for ecosystem services but face significant threats from anthropogenic climate change. Understanding the forces driving and maintaining biodiversity in these hotspots is essential for conservation efforts. While both adaptive and non-adaptive processes contribute to biodiversity, their relative roles remain unclear, particularly in tropical rainforests where the processes generating and sustaining diversity are debated. Earlier research focused on vicariant influences like refugial isolation and landscape breaks, but these views have been increasingly challenged by evidence for parapatric and sympatric divergence. The challenge lies in disentangling the coexisting signatures of neutral and adaptive processes in these complex ecosystems. Landscape genomics offers powerful tools to identify ecological adaptations, using genotype-environment associations (GEAs) and phenotype-environment associations (PEAs). However, distinguishing between adaptive evolution and phenotypic plasticity remains crucial. Integrative genotype-phenotype-environment (GxPxE) associations offer a promising approach to separate these mechanisms. Freshwater ecosystems, particularly those within rainforests, present unique constraints due to restricted niches and dendritic habitat structures. This study leverages previous research on Australian rainbowfishes, which showed the importance of hydroclimate and the effectiveness of integrative methods in assessing aquatic adaptation. Focusing on *Melanotaenia splendida splendida*, abundant in the Wet Tropics of Queensland, Australia, this study aims to understand the interplay between adaptive and neutral influences on variation in tropical rainforest freshwater ecosystems. The study asks: (1) To what extent do genetic and morphological diversities correlate with hydroclimate beyond expectations based on neutral genetic structure? (2) If ecological associations exist, can they be linked to a genetic (heritable) component to morphology? (3) What is the contribution of catchment structure to divergence?

The literature review highlights the historical emphasis on vicariant drivers of tropical rainforest diversity and the increasing recognition of contemporary environmental factors in shaping biodiversity. Studies on both terrestrial and aquatic species support the role of temperature and precipitation as significant drivers. However, a nuanced understanding of the interplay between neutral and adaptive processes is needed. Previous research on Australian rainbowfishes (*Melanotaenia* spp.) has established the importance of hydroclimate and the utility of integrative methods in detecting adaptation. Work on various *Melanotaenia* species has demonstrated heritable body shape variation associated with streamflow and selection for plasticity in thermal response mechanisms. However, genome-wide studies on tropical representatives of the genus have been lacking. This study bridges this gap by using *M. s. splendida* as a model species.

This study employed a landscape genomics approach, integrating genomic and morphometric data with environmental variables. Wild *M. s. splendida* were sampled from nine rainforest creek sites across five drainages in the Wet Tropics of Queensland. DNA was extracted from fin clips, and double-digest restriction-site-associated DNA (ddRAD) libraries were prepared and sequenced using Illumina HiSeq2500 and HiSeq4000. Bioinformatics analysis involved read trimming, alignment to a reference genome (*M. duboulayi*), variant calling, and filtering to obtain high-quality SNPs. Putatively neutral loci were identified using Bayescan, and neutral genomic diversity was estimated using Arlequin. Population structure was analyzed using FastStructure and DAPC. Environmental variables (stream segment aspect, river disturbance index, summer mean runoff, annual mean rainfall, annual mean temperature, and upstream segment length) were obtained from the National Environmental Stream Attributes. Genotype-environment associations (GEAs) were assessed using BAYPASS and RDA, controlling for neutral genetic variation. Geometric morphometrics were used to analyze body shape variation based on 18 landmarks. Phenotype-environment associations (PEAs) were conducted using RDA, controlling for allometric effects and neutral genetic structure. Finally, genotype-phenotype-environment (GxPxE) analyses were performed using RDA to investigate links between adaptive genetic variation and morphology. BLAST annotation was used to identify functional information for candidate genes.

The study found high neutral genetic diversity within *M. s. splendida* populations, but substantial population subdivision was observed, largely explained by restricted gene flow among drainages. However, a significant proportion of genetic variation (12-17%) was associated with environmental variables, particularly annual rainfall and annual temperature, even after controlling for neutral genetic structure. Furthermore, a substantial portion of body shape variation (11.6-14%) was also linked to environmental variables, primarily annual temperature and stream density. GxPxE analysis revealed a strong association (6.5%) between environmentally associated genetic loci and body shape, suggesting a heritable component to the observed morphological variation. Several candidate loci were identified through this analysis, some with known functional relevance in other species (e.g., *cdh2*, *SPTAN1*). Drainage structure was also found to influence demographic divergence, emphasizing the interaction between gene flow and environmental selection.

The findings demonstrate that both neutral and adaptive processes have shaped the regional diversity of *M. s. splendida*. The strong environmental associations, particularly with hydroclimate variables, highlight the role of local adaptation in driving morphological and genomic divergence. The GxPxE results suggest that heritable, climate-adaptive shape variation has evolved. These findings challenge historical views emphasizing vicariance and offer support for contemporary environmental factors as major drivers of tropical rainforest biodiversity, similar to findings in other tropical systems. The relatively greater influence of environmental factors on morphology compared to genotype is attributed to the functional constraints on morphology. The specific morphological changes observed, especially in fin position and head shape, are consistent with adaptations to varied streamflow and resource availability. The study acknowledges the difficulty in fully separating direct and indirect environmental effects.

This study provides strong evidence for the role of hydroclimatic variation and drainage connectivity in shaping the diversity of *M. s. splendida*. The integration of genomic, morphometric, and environmental data revealed significant associations supporting local adaptation, highlighting the importance of heritable, climate-adaptive traits. The findings emphasize the need for proactive conservation strategies focused on maintaining habitat connectivity and promoting evolutionary resilience in response to climate change. Future studies should continue to integrate multiple data types to further unravel evolutionary processes in tropical rainforests.

The study's interpretation of environmental associations is limited by the difficulty in fully disentangling direct and indirect effects of environmental variables. While the reference genome used was closely related, using a *M. splendida splendida* genome would strengthen the analysis. The functional annotation was limited by the availability of protein sequence data for the reference genome. Further research is needed to confirm the functional roles of the identified candidate genes. Finally, while the study offers a comprehensive approach, potential confounders in natural environments may influence the results.