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Escherichia coli  not only inhabit the large intestines of human and warm-blooded animals but could also persist in the external environment. However, current knowledge was largely based on host-associated strains. Moreover, cryptic  Escherichia  clades that were often misidentified as  E. coli  by conventional diagnostic methods were discovered. Failure to distinguish them from  E. coli sensu stricto  could lead to inaccurate conclusions about the population genetics of  E. coli . Based on seven housekeeping genes, we determine the genetic and ecological diversity of  E. coli  and cryptic clades as they occupy aquatic habitats with different characteristics and human impact levels in subtropical Hong Kong. Contrary to previous reports, clade II was the most abundant cryptic lineage co-isolated with  E. coli , being especially abundant in relatively pristine subtropical aquatic environments. The phylogenetically distinct cryptic clades and  E. coli  showed limited recombination and significant genetic divergence. Analyses indicated that these clade II strains were ecologically differentiated from typical  E. coli ; some may even represent novel environmental  Escherichia  clades that were closely related to the original clade II strains of fecal origins.  E. coli  of diverse origins exhibited clonality amidst divergent genotypes STs, echoing other studies in that recombination in housekeeping genes was insufficient to disrupt phylogenetic signals of the largely clonal  E. coli . Notably, environmental  E. coli  were less diverse than fecal isolates despite contributing many new alleles and STs. Finally, we demonstrated that human activities influenced the distribution of  E. coli  and clade II in a small aquatic continuum. Moving from relatively pristine sites toward areas with higher human disturbance, the abundance of clade II isolates and new  E. coli  genotypes reduces, while  E. coli  bearing class I integrons and belonging to CCs of public health concern accumulates. Altogether, this work revealed the new genetic diversity of  E. coli  and cryptic clades embedded in selected subtropical aquatic habitats, especially relatively pristine sites, which will aid a more thorough understanding of the extent of their genetic and functional variations in relation to diverse habitats with varied conditions.

Genetic and Ecological Diversity of Escherichia coli and Cryptic Escherichia Clades in Subtropical Aquatic Environments