Phylogeny-Guided Barcoding of Marine Mollusks

We sequenced 2,026 cowries for 614 bp of COI mtDNA, the traditional Folmer primer region proposed for barcoding most metazoans. Two or more individuals were sequenced from 82% (216) of ESUs, ≥5 from 54% (143), and ≥10 from 23% (60). To maximize recovery of the greatest intraspecific variation and test for geographical structuring, sequences were generated from the most geographically distant populations available. Molecular methods followed standard procedures and are reviewed in Meyer [25 ,26 ], Kirkendale and Meyer [27 (link)], and Meyer et al. [28 (link)].
We used standard, tree-based methods to address accuracy of identification in a thoroughly sampled phylogeny using both a species-level and ESU approach. One exemplar from each recognized species (the nominal subspecies if the species included multiple subspecies) or each identified ESU was used as the reference “barcode” exemplar in topological comparisons. We randomly selected 1,000 sequences from the cowrie COI dataset, excluding barcode exemplars, and limiting representation of each species or ESU to 15 or ten sequences, respectively, to minimize bias toward well-sampled taxa. Hybrid individuals (see above) were excluded. These 1,000 sequences were tested one at a time, and their placement relative to the barcoding exemplars evaluated in both neighbor-joining (K2P) and parsimony phylogenies. Identification was considered correct if the sister taxon of the test sequence was the exemplar sequence of its corresponding species or ESU. Identification was considered incorrect if the sister taxon was wrong. If the random sequence fell below a node linking two recognized sister taxa including the corresponding species, the identification was considered ambiguous, as assignment to one or the other is equivocal, as the unknown could also represent a novel taxon. Similar analyses were performed with the turbinid (n = 200 from 278) and limpet (n = 100 from 125) datasets.
Pairwise K2P distances, theta, and coalescent depth were used to characterize intraspecific variation. Genetic distance between terminal taxa and their closest sister was used to characterize interspecific divergence. While the phylogenies used are based upon sequence data from two mtDNA markers (16S and COI: [26 –28 (link)]), only COI was used for these analyses. The two most genetically distant individuals within each ESU (based on pairwise comparisons) were chosen to bookend genetic diversity and recover coalescent depth (maximum intra-ESU variability). These two individuals replaced the exemplar taxon used to construct the overall phylogeny (Figure 3). A likelihood ratio test (GTR + G with and without a clock enforced) was used to test for clock-like behavior (using only COI) in the resulting tree. A clock could not be falsified for turbinids and limpets (p > 0.05); but was falsified (p = 0.007) for cowries. Coalescent depths and interspecific divergence estimates throughout are based on topologies with a molecular clock enforced, although the overall cowrie data marginally rejected rate constancy. We estimated theta by calculating the average intraspecific difference using K2P distances. All analyses were conducted using PAUP* version 4.0b10 [43 ]. A listing of ESUs, number of individuals examined, interspecific divergence, and intraspecific metrics can be found in the supporting information for cowries (Table S1), turbinids (Table S2), and limpets (Table S3).