Cnidome of Chrysaora quinquecirrha

Lastly, we examined the cnidome of multiple specimens originally identified as Chrysaora quinquecirrha to determine if species could be delineated based on nematocyst measurements (of each type) and/or nematocyst diversity (counts of nematocyst types). Nematocyst terminology followed convention used in previous studies (Weill, 1934; Calder, 1971; Calder, 1974a; Östman & Hydman, 1997; Morandini & Marques, 2010) in defining four different nematocyst types: holotrichous A-isorhiza, holotrichous a-isorhiza, holotrichous O-isorhiza, and heterotrichous microbasic rhopaloid. In agreement with Morandini & Marques (2010), we use the term heterotrichous microbasic rhopaloid, recognizing that there are likely at least two nematocysts that cannot be effectively delineated based on basic light microscopy, as shown in other previous work (Sutton & Burnett, 1969).

In all cases, formalin-preserved tentacle tissue was homogenized in distilled water in 1.5 mL microcentrifuge tubes and nematocysts were examined using differential interference contrast microscopy (DIC). A small piece of formalin-fixed tentacle tissue was homogenized in 100 μL of distilled water in a 1.5 μL tube using a plastic microcentrifuge pestle until little visible intact tissue remained. A small drop was then placed on a slide under cover slip and examined at 60× in DIC using an Olympus BX63 microscope, with photographs taken using an Olympus DP80 camera run by CellSens Dimension 1.13 (Olympus Life Science, Inc., Waltham, MA, USA).

A total of 15 individuals were examined for nematocyst size measurements (Table S1). In all cases, 10 samples of each nematocyst type were photographed and later measured using CellSens Dimension 1.13 computer program for length and width. Linear discrimination analysis (LDA) was used to determine whether species could be distinguished on the basis of nematocyst measurements using the lda routine in the R package MASS (Venables & Ripley, 2002).

A total of 10 individuals were examined for nematocyst diversity (Table S1). Since initial estimates indicated that nematocyst diversity varied by tentacle region, nematocyst counts were done from three tentacle regions for each individual: proximal (near the base of the tentacle), medial (in the middle of the tentacle), and distal (at the end of the tentacle). For each region, the first 200 nematocysts were photographed and categorized according to nematocyst type. Only lone nematocysts were enumerated, with any nematocysts still adhering to epithelial tissue ignored, since smaller nematocysts (e.g., a-isorhizas) could be obscured. In order to examine any differences in nematocyst diversity between different tentacle regions (distal, medial, proximal), a mosaic plot showing the relative proportions of nematocyst types in the various regions was made using the R package vcd version 1.4-3 (Meyer, Zeileis & Hornik, 2016). In order to visualize differences in proportions of nematocyst types (four types, three regions) between the two species we conducted non-metric multidimensional scaling of the Euclidean distance matrix using the isoMDS routine in the R package MASS (Venables & Ripley, 2002).