Reporting Complete Chloroplast Genome of Endangered Red Mulberry Useful for Understanding Hybridization and Phylogenetic Relationships

Introgressive hybridization of the North American native red mulberry ( Morus rubra ) with its invasive congener white mulberry ( Morus alba ) has severely threatened the genetic integrity of M. rubra , which is primarily found in pristine riparian forests. The major objectives of the present study were 1) to sequence, assemble, and annotate the complete chloroplast (cp) genome of M. rubra , and 2) to perform phylogenomic analyses of Morus species to assess their evolutionary history and hybridization events within the genus. We sampled 45 mulberry trees representing populations from eight US states for cp genome sequencing. We found that the cp genome of M. rubra ranged from 159,396 to 159,423 bp and contained 128 genes coding for eight rRNAs, 37 tRNAs, and 83 proteins. The cp genome of M. rubra was at least 103 bp larger than that of M. alba . The cpDNA sequence polymorphism analysis of M. rubra showed the presence of 12 haplotypes. The cp genome analysis across 12 Morus species identified trnK-UUU - rps16 , psbI - trnG-UCC , psbC - psbZ , psbZ - trnM-CAU , rps4 - trnT-UGU , trnT-UGU - trnL-UAA , ndhC - trnV-UAC , psbE - petL , clpP1 , ndhF - rpl32 , rpl32 - trnL-UAG , and ccsA - ndhD as having higher DNA polymorphism (Pi > 0.01), making them candidate species-specific markers. Phylogenomic analysis of the 12 Morus species revealed distinct clades for Asian, North American, South American, and African Morus, supporting the monophyly of the genus. Divergence time analysis showed the most common ancestor of the genus Morus diverged 38.67 million years ago (MYA), separating the African clade of M. mesozygia and M. insignis from the others, while the American and Asian clades diverged 28.63 MYA. The results from this study provide insights into the cp genome structure of M. rubra, offering foundational data for addressing complex taxonomic complexities within the genus and for developing molecular markers to study population genetics, including introgressive hybridization.