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Several subspecies are defined within Codium fragile, including the invasive C. fragile ssp. fragile, first reported in New Zealand in 1973. An endemic subspecies, C. fragile ssp. novae-zelandiae, is also found throughout New Zealand. The two subspecies exhibit morphological and molecular variation, although these have never been evaluated together. We compared variation between subspecies at locations in Auckland, identifying subspecies using rps3- rpl16 DNA sequence data, and assessing gross morphological differences, anatomical utricle characters and morphometrics. The taxonomic utility of the morphometric data sets was assessed by linear discriminant analysis. Utricle characters and measurements varied within individual thalli and between different preservation methods. The phenotypes of both subspecies were highly variable and influenced by environment. Accurate subspecies delimitation using morphological data was not possible; the discriminant analyses performed no better than chance for all combinations of the morphological data. Specimens from New Zealand, Canada, Australia and Ireland were sequenced using both the rps3- rpl16 and tufA plastid markers. The tufA elongation factor was shown to be a good candidate for differentiating subspecies of C. fragile. This marker is twice the length of the rps3- rpl16 spacer, shows greater variation between ssp. fragile and novae-zelandiae, and is less prone to sequencing error. A simple restriction enzyme digest of the tufA amplicon can distinguish ssp. fragile and ssp. novae-zelandiae. Our study expands the known range of the ssp. fragile in New Zealand, including the first record of this subspecies from the west coast of Auckland, and points to a need to re-evaluate morphological and molecular criteria for subspecies currently defined within C. fragile. [ABSTRACT FROM AUTHOR]