Contributions to Zoology, 86 (2) – 2017Gerrit Potkamp; Mark J.A. Vermeij; Bert W. Hoeksema: Genetic and morphological variation in corallivorous snails (Coralliophila spp.) living on different host corals at Curaçao, southern Caribbean
Results

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Genetics

The three Caribbean Coralliophila species clustered into three well-supported, separate clades on the phylogenetic tree (posterior probability (PP) = 0.997-1.000; Fig. 15). Phylogenetic trees constructed for both markers separately showed the same pattern (Online Supplementary Material 5). No monophyletic Caribbean cluster was found: Coralliophila curacaoensis sp. nov. is the sister species of a group consisting of both C. fontanangioyae Smriglio and Mariottini, 2000 and C. meyendorffii (Calcara, 1845) from the Eastern Atlantic and the Mediterranean; C. galea is sister of C. mira (Cotton and Godfrey, 1932) from the Indo-Pacific, and C. caribaea is sister of the Indo-Pacific Leptoconchus sp. Hence, the genus Coralliophila is also not monophyletic.

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Fig. 15. Phylogenetic tree based on the 12S rRNA (12S) and cytochrome c oxidase subunit I (COI) markers of a selection of the collected Coralliophila spp. specimens. The three shaded clades represent the three species of Coralliophila found in the present study. Within the shaded clusters, tip labels represent the host order and host species with which the snail was associated. Specimens outside the shaded cluster are previously published sequences. Branch labels are posterior probabilities (PP). Intraspecific PP-values are not shown (except for the main clade within C. caribaea) and were all lower than 0.90. Scale bar: 0.01 substitutions per site.

No host-associated genetic divergence was found within Coralliophila galea (p = 0.458 and p = 0.342 for 12S and COI, respectively). In C. caribaea on the other hand, a small divergence (mean uncorrected distance of 0.6% for 12S and 2.9% for COI) was found between snails associated with Alcyonacea and those associated with Scleractinia (p < 0.0001 for both 12S and COI). This genetic structuring within C. caribaea was also visible on the phylogenetic tree, as C. caribaea collected from Scleractinia cluster all on a single branch (with one exception), although the support value for this branch was low (PP = 0.803). There was no host-associated divergence within C. caribaea from scleractinian corals (p = 0.213 and p = 0.971 for 12S and COI, respectively) or alcyonacean hosts (p = 0.941 and p = 0.945 for 12S and COI, respectively).

The small divergence among C. caribaea individuals associated with either scleractinians or alcyonaceans was also observed in the haplotype networks constructed for both markers (Fig. 16). With one exception for COI, haplotypes were unique to snails associated with hosts from either Scleractinia or Alcyonacea. In addition, haplotypes of snails associated with alcyonaceans mostly (with one exception for both markers) clustered together on a single branch in the haplotype network. Snails associated with scleractinians formed (again, with one exception for both markers) the other branches in the networks. The two clusters were separated by two mutations for 12S and eight mutations for COI. No clear correlation between host species and haplotype was present within C. galea.

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Fig. 16. Haplotype networks based on an infinite site model (using simple, uncorrected distances) of the sequenced specimens C. caribaea, both for the markers 12S rRNA (12S) (a) and cytochrome c oxidase subunit I (COI) (b). Haplotype are coloured based on host species with which the haplotypes were associated, size represents the frequency of haplotypes. Length between haplotypes is based on the number of mutations between haplotypes. Circles and lines in the bottom left of each figure represent a frequency of one and one mutation between haplotypes respectively.

The ABGD analysis based on COI, using a more extensive dataset of coralliophiline snails, proposed nine different groupings of specimens into MOTUs, depending on the a priori boundary between intra- and interspecific divergence. A histogram of the frequencies of pairwise Kimura two-parameter (K2P) distances revealed a multimodal distribution, with the lowest minimum frequency around a K2P distance of 0.05 (Fig. 17). Using this value as a boundary between intra- and interspecific divergence, the coralliophiline dataset can be subdivided into 16 MOTUs. At this value, the specimens of C. galea, C. caribaea and C. curacaoensis sp. nov. are clustered into three different MOTUs, in agreement with the phylogenetic tree. The divergence within C. caribaea was confirmed to be intraspecific, not revealing any cryptic species, using this boundary value of 0.05.

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Fig. 17. Automatic Barcode Gap Discovery analysis based on Kimura two-parameter distances of a dataset of coralliophiline cytochrome c oxidase subunit I (COI) sequences, including the Coralliophila spp. specimens sequenced in the present study.