Taxa were also assigned to species by examination of morphological characters, when formalin preserved samples were available, using descriptions from the literature11,3036,38,40,4348,6065. The 31 analyzed morphological characters are summarized as follows: arrangement of systems, position of ovaries and testes, testes morphology, number of stigmatal rows, completeness of the second stigmatal row, arrangement of stigmata, shape of intestine, location of anterior edge of intestinal loop, location of anus, number of stomach folds, appearance of the stomach folds, shape of the stomach, shape and size of the pyloric caecum, number and size orders of the oral tentacles, distribution of pigment cells in the zooid, zooid length, colony color when living and after fixation, and tunic thickness3032. If the species could not be identified with complete certainty from the literature, we used a subset of the 31 characters: arrangement of systems, position of ovaries and testes (if ovaries and testes were present), appearance of the stomach folds, shape of the stomach, and size of the pyloric caecum. At least 30 zooids were examined from each colony.

When possible, we examined the morphology of the same colony from which the mtCOI sequencing was obtained. If a formalin preserved sample of the original colony was not available, we were often able to examine the morphology of another colony with an identical mtCOI sequence. We did not assign a specimen to a specific morphologically-described species if we could not obtain a formalin sample of the original colony or of a colony with an identical mtCOI sequence.

In the course of our species assignment using morphology, several specimens did not match the descriptions of any species previously found in the geographic region in which the specimen was collected. Either these samples represent new species, or they are known species that have only recently been identified from the location where they were collected. To determine where these species fit in relation to the morphologies of the other Botryllus/Botrylloides species, we searched the literature for morphological information on the 53 described botryllid species. We compiled data on the 31 morphological characters described above. The genus names (Botrylloides or Botryllus) and the morphological data come from the type description. In a few cases, the type description is lacking data on the majority of these characters. If a re-description was available, it was used to supplement the type description. Because our examinations were more thorough than type descriptions (often type descriptions do not provide data on individual zooids or individual colonies), we averaged our quantitative data across the 30 + zooids we examined in each colony to obtain a single value for each colony. We then averaged quantitative data across multiple colonies to obtain a single value for each character for each species, to match the data available in the literature. A brief description of each considered morphological character is available in Supplementary Fig. S2, and the entire data matrix is available in Supplementary Table S1. Using the morphological data from the 53 described species and the 4 undescribed species, we then conducted a Principal Components Analysis using PCAmixdata66 as implemented in R version 3.6.1.

To accompany the PCA, we constructed a phylogenetic tree of the 57 species using the 31 morphological characters using MrBayes 3.2.267 on the CIPRES (Cyberinfrastructure for Phylogenic Research) Science Gateway68. The GTR + G model of nucleotide substitution was applied to all data sets (Nset = 6). Each analysis was run for 10 million generations, with sampling every 1000 generations. The first 2000 trees were eliminated as burn-in.

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