LAUSR

Simple Summary Pygmy grasshoppers (Orthoptera: Tetrigidae) have evaded genomic characterization thus far, impeding their evolutionary and molecular genetic analysis. Here, through high-throughput sequencing, we pioneer the first reference-grade chromosomal assembly for the Chinese endemic pygmy grasshopper, Zhengitettix transpicula. This breakthrough genome provides a foundational resource to expedite the conservation of this endangered species and further contribute to primary and comparative research across Tetrigoidea and Orthoptera in general. Our assembly advances best practices for resolving complex genomes in non-model Orthopterans. Annotation identifies an extensive catalog of protein-coding elements, availing access to the functional elements underlying Tetrigidae’s unique traits. Comparative analyses reveal lineage-specific evolutionary trajectories related to ecological specialization, unlocking questions regarding the genetic trade-offs underlying extreme adaptations. Beyond evolution, our work enables crucial genetic investigations assessing diversity and inbreeding, essential for habitat management interventions to prevent extinction. Variability in the critical defensive gene could underlie the inability of Tetrigidae to form a population outbreak. Thus, this timely and high-quality pygmy grasshopper genome is an invaluable blueprint for accelerating genotype-phenotype connections, evolutionary interpretations, and applied conservation for obscure and iconic species facing similar anthropogenic threats in increasingly fragile ecosystems. Overall, our multidimensional genomic resource promises to invigorate diverse research directions and breakthrough findings across Orthoptera biology, genomics, evolution, and conservation. Abstract The pygmy grasshopper, Zhengitettix transpicula, is a Chinese endemic species with an exceedingly limited distribution and fragile population structure, rendering it vulnerable to extinction. We present a high-continuity, chromosome-scale reference genome assembly to elucidate this species’ distinctive biology and inform conservation. Employing an integrated sequencing approach, we achieved a 970.40 Mb assembly with 96.32% coverage across seven pseudo-chromosomes and impressive continuity (N50 > 220 Mb). Genome annotation achieves identification with 99.2% BUSCO completeness, supporting quality. Comparative analyses with 14 genomes from Orthoptera-facilitated phylogenomics and revealed 549 significantly expanded gene families in Z. transpicula associated with metabolism, stress response, and development. However, genomic analysis exposed remarkably low heterozygosity (0.02%), implying a severe genetic bottleneck from small, fragmented populations, characteristic of species vulnerable to extinction from environmental disruptions. Elucidating the genetic basis of population dynamics and specialization provides an imperative guideline for habitat conservation and restoration of this rare organism. Moreover, divergent evolution analysis of the CYP305m2 gene regulating locust aggregation highlighted potential structural and hence functional variations between Acrididae and Tetrigidae. Our chromosomal genomic characterization of Z. transpicula advances Orthopteran resources, establishing a framework for evolutionary developmental explorations and applied conservation genomics, reversing the trajectory of this unique grasshopper lineage towards oblivion.