Microsatellite development for Theridion evexum (Araneae: Theridiidae) using low-coverage genome sequencing and the MiMi script.

Habitat fragmentation and unplanned urban expansion increasingly threatens biodiversity, yet the genetic impacts on arthropods, and particularly spiders, remains understudied due to the lack of suitable molecular makers. Here, we used low-coverage genome sequencing and a bioinformatics pipeline to develop polymorphic microsatellite markers for Theridion evexum, a tropical spider with urban and natural populations. To increase the yield of DNA extracted from small spiders like T. evexum, we also optimized a Cetyltrimethylammonium bromide (CTAB) DNA extraction protocol. We sequenced eight individuals at 4X using paired-end sequencing on an Illumina Novaseq 6000. Reads were cleaned and processed using the Multi-individual Microsatellite Identification (MiMi) python pipeline. MiMi produced a total of 3999 putative microsatellites. After filtering for polymorphic loci with an allelic richness greater than three and present in at least 5 of the 8 sequenced individuals, 34 final markers were identified. An experimental validation of 13 of these 34 markers showed that 10 loci were polymorphic with at least three detectable alleles, one locus was monomorphic, and two loci did not produce PCR products. These markers will allow a better assessment of the effects of urban fragmentation and isolation across populations of this spider species. Furthermore, developing markers using low-coverage next-generation sequencing (NGS) and bioinformatic protocols provide a valuable approach for uncovering microsatellite markers at a reduced cost for other tropical species, thereby broadening the scope of molecular ecology research in the tropics.