Evolutionary Relationships Within Streptocarpus Ionanthus (Gesneriaceae) Species Complex: Insights From Population Genetics, Phylogeography And Phylogenomic Analyses

Streptocarpus ionanthus(Gesneriaceae)is an herbaceous plant species endemic to Tanzania and Kenya,distributed in Tanga,Morogoro and Kilifi regions.The species comprise nine closely related subspecies characterized by complex morphotypes and poorly understood evolutionary relationships,in which controversial and inconsistent taxonomic placements have been previously witnessed due to unclear taxa boundaries and morphological overlap.The unresolved taxon delimitation in Str.ionanthus complicates their evolutionary implication,thereby hindering design of proper conservation approaches.Therefore,Str.ionanthus is an ideal model for investigating species evolutionary dynamics over time.This work applies diverse approaches aimed at understanding Str.ionanthus evolutionary history using 23 populations sampled across the entire distribution range.Firstly,this study examined the genetic diversity,population differentiation and structure using both chloroplast and nuclear SSR markers and explored the phylogeographic structure using four chloroplast genome(cpDNA)regions.The diversification history of Str.ionanthus lineages was explored using the cpDNA haplotypes based on a range of previously calculated plastid substitution rates in plants(1.0×10~-99 substitutions/site/year to 5.6×10~-44 substitutions/site/Myrs).Ecological Niche Modeling(ENM)analysis was performed to understand the past(Last Inter-Glacial(LIG)and Last Glacial Maximum(LGM)periods),present and predict future(2070)distribution of Str.ionanthus.Further,to understand the molecular variation within Str.ionanthus,the chloroplast genomes of five representative subspecies were sequenced.Comparative analyses were done on the genomes'structural features and divergent regions identified.Finally,re-analysis of multi-locus data(nuclear:ITS and 5S-NTS;chloroplast:four cpDNA regions)generated in previous studies was done to infer phylogenetic relationships within Str.ionanthus.The obtained phylogenetic information was compared with morphological information available in literature and some comments on taxonomic aspects of Str.ionanthus were made.Despite Str.ionanthus exhibiting bottleneck events across its range,the populations maintain moderate to high genetic diversity(nSSRs:He=0.627;cpSSRs:He=0.376)attributed to historical population admixture or local adaptation arising from habitat heterogeneity.Genetic structuring revealed unclear boundaries within the Usambara populations,results supported by the Phylogeographic structure in which a high connection among the Usambara mountains haplotype lineages was witnessed.Str.ionanthus cpDNA haplotypes divergence from the outgroups date back to approx.0.21–1.5 million years ago(Mya)while the diversification within the group began approx.0.18–1.32 Mya and intensified towards the present,a conclusion of recent diversification.Niche modeling indicates that the stability of suitable areas during the Last Glacial Maximum(LGM)offered protective microhabitats which have preserved the genetic diversity of Str.ionanthus to date,thus qualify as potential climate refugia.Chloroplast phylogenomic analysis revealed a structurally conserved Str.ionanthus genome,with a narrow size range(170 base pairs(bp))and identical 115 unique genes(80 protein-coding,31 tRNAs and 4 rRNAs).Despite the high synteny among the aligned genomes,ten relatively divergent regions were identified(psbM,rps3,atpF-atpH,psbC-psbZ,psa A-ycf3,rps16,rpoC2,rpoB,ycf1 and ndhA)and these could further be investigated for phylogenetic utility in Str.ionanthus.The low polymorphism and poor phylogenetic separation could be attributed to the recent divergence reported in molecular dating above.Morphological analysis portrayed great character overlap and no single character could separate the subspecies.Similarly,phylogenetic analyses exhibited mixed signals and monophyly in only subspecies rupicola(nuclear phylogenies)and subspecies grotei,rupicola,and velutinus(chloroplast phylogeny).However,comparison of both phylogenetic and morphological data could not identify any congruent pattern and this strongly imply the existence of several lineages within Str.ionanthus which only partially satisfy the criteria for defined taxonomic concepts.In conclusion,the taxonomic arrangement of recognizing different recently diversified lineages of Str.ionanthus is more informative and convenient than recognizing non-monophyletic taxa as distinct subspecies.Therefore,Str.ionanthus appears as a complex of multiple historical refugia origin,more recently diversified and a fast-evolving group.This study provides multiple approaches,conclusions based on numerous evidence sources and avails diverse data which can be expanded for future elucidation of Str.ionanthus evolutionary relationships.