Biodiversity Data Journal :
Research Article
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Corresponding author: Rachel Collin (collinr@si.edu)
Academic editor: Danwei Huang
Received: 31 Oct 2018 | Accepted: 04 Feb 2019 | Published: 20 Feb 2019
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Morín J, Venera-Pontón D, Driskell A, Sánchez J, Lasker H, Collin R (2019) Reference DNA barcodes and other mitochondrial markers for identifying Caribbean Octocorals. Biodiversity Data Journal 7: e30970. https://doi.org/10.3897/BDJ.7.e30970
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DNA barcoding is a useful tool for documenting the diversity of metazoans. The most commonly used barcode markers, 16S and COI, are not considered suitable for species identification within some "basal" phyla of metazoans. Nevertheless metabarcoding studies of bulk mixed samples commonly use these markers and may obtain sequences for "basal" phyla. We sequenced mitochondrial DNA fragments of cytochrome oxidase c subunit I (COI), 16S ribosomal RNA (16S), NADH dehydrogenase subunits 2 (16S-ND2), 6 (ND6-ND3) and 4L (ND4L-MSH) for 27 species of Caribbean octocorals to create a reference barcode dataset and to compare the utility of COI and 16S to other markers more typically used for octocorals. The most common genera (Erythropodium, Ellisella, Briareum, Plexaurella, Muriceopsis and Pterogorgia) were effectively distinguished by small differences (5 or more substitutions or indels) in COI and 16S sequences. Gorgonia and Antillogorgia were effectively distinguished from each other by unique haplotypes, but the small genetic differences make distance approaches ineffective for these taxa. Plexaura, Pseudoplexaura and Eunicea were indistinguishable from each other but were generally effectively distinguished from other genera, further supporting the idea that these genera have undergone a rapid endemic radiation in the Caribbean.
cytochrome oxidase I, 16S, Panama, Bocas del Toro, gorgonian, DNA barcoding
DNA barcoding is a useful tool for documenting the diversity of most metazoan groups (
DNA barcoding has been used extensively to detect cryptic diversity within clades, with specific studies generally focusing on diversity within a single family, class or order. When COI is problematic, alternate barcodes are used, for example, 16S is commonly used as the molecular barcode in hydrozoans (
Recent assessments of biodiversity are increasingly focusing on metabarcoding analyses of bulk mixed samples, such as gut contents (
Most Caribbean gorgonian octocorals are endemic and closely related (
Here we had two main objectives: (1) to generate a reference set of DNA barcode sequences for common octocorals from the Southern Caribbean and (2) determine how the 16S and COI barcode fragments compare with other mitochondrial markers in their ability to distinguish genera and, in some cases, species in the fauna of Bocas del Toro, Panama.
A total of 180 octocoral tissue samples were collected by SCUBA-diving during the summer of 2007 from the shallow waters around the Bocas del Toro Archipelago on the Caribbean coast of Panama. Tissue samples consisted of 10-20 cm sections clipped from a distal branch for each colony. Samples were identified to species using a combination of visual identification in the field (colony morphology) and microscopic examination of spicule preparations. In some cases, individuals were intermediate in morphology or could not be identified to species and were only identified to genus. Dry tissue vouchers were deposited at the USNM and the University of Panama. Details about individual samples are provided in the BoLD project workbench (
Genomic DNA was extracted from small samples of tissue (0.5 cm3) from each specimen using the mouse tail kit on a Biosprint 96 (Qiagen). The resuspension volume was 200 µl. We used PCR to amplify fragments of the mitochondrial genes COI, 16S, ND6-ND3, 16S-ND2 and ND4L-MSH with the primers listed in Table
Primers used for amplification and sequencing of loci in this study and size of amplified fragments. *Lengths of fragments that include non-coding regions as well as ribosomal sequences can vary. 1
Locus |
Primers |
Expected Size* |
COI long |
COII-8068F1 CCATAACAGGACTAGCAGCATC COIOCTR2 ATCATAGCATAGACCATACC |
940 |
COI Folmer |
dgLCO14903 GGTCAACAAATCATAAAGAYATYGG dgHCO21983 TAAACTTCAGGGTGACCAAARAAYCA |
655 |
COI short |
GorgM13_F4 CACGACGTTGTAAAACGACGTATGTTAGGAGATGATCATCTATAT GorgM13_R4 GGATAACAATTTCACACAGGGAATGTTGTATTAAAATTYCTRTCTGT |
468 |
16S |
16Sar5 CGCCTGTTTATCAAAAACAT 16Sbr5 CCGGTCTGAACTCAGATCACGT |
668 |
ND6-ND3 |
ND6-1487F1 TTTGGTTAGTTATTGCCTTT ND3-2126R1 CACATTCATAGACCGACACTT |
611 |
16S-ND2 |
16S-647F1 TTTGGTTAGTTATTGCCTTT ND2-1418R1 ACATCGGGAGCCCACATA |
758 |
ND4L-MSH |
ND4L-2475F6 TAGTTTTACTGGCCTCTAC MUT-3458R7 TSGAGCAAAAGCCACTCC |
870 |
Sequences were screened for quality and contigs of forward and reverse sequences were produced using Sequencher 5.4.6 (Gene Codes). Only sequences with a Phred quality score of at least 30 for more than 85% of the bases were combined into contigs and used for analysis. Sequences were compared internally across our dataset and BLASTned against GenBank sequences to check for contamination or mislabelling. After this step, through an abundance of caution, all 27 ND4L-MSH sequences from a single plate were excluded from the analyses because a subset of them showed unexplained divergences from previously published GenBank sequences. No other plate of sequences in our analysis showed similar problems. Sequences of each marker were aligned with ClustalX (gap-opening penalty: 15, gap-extension penalty: 6.66, transition weight: 0.5, delay divergent cutoff: 30%) and used to generate a matrix of pairwise differences [including the number of substitutions and indels (insertions/deletions)] between all the sequenced specimens. This matrix was then used to build heatmaps displaying the average pairwise differences between species. The patterns observed in heatmaps were contrasted with those observed in maximum parsimony and neighbour-joining trees (BIONJ,
Most markers showed two groups within which the pairwise differences amongst species (and genera) were consistently small. For these groups, we constructed haplotype networks for all the markers using Haplotype Viewer (Center for Integrative Bioinformatics Vienna; http://www.cibiv.at/) to determine if haplotypes were unique or related to particular species or genera.
The data underpinning the analysis reported in this paper are deposited in the Barcode of Life Database (dx.doi.org/10.5883/DS-OCTOCORA) (
We were able to collect and successfully sequence 28 species represented by 180 individuals of octocorals from Bocas del Toro (Table
Number of individuals collected and successfully sequenced for each genetic marker for samples identified to species. The colour of the cells indicates the availability of sequences for the same marker and species in GenBank. Dark grey: A new contribution; no available sequences overlap our fragment by ≥100 bp. Light grey: Partial sequence is already available; our sequences overlap existing sequences by >350 bp; our data extends this by >50 bp. White: Sequence already available; sequences available in GenBank overlap ours completely with <50bp additional data. X mark indicates species that failed to amplify or resulted in unusable or suspicious sequences. *Sequences were also generated for an additional 4 Antillogrogia, 3 Erythopodium, 44 Eunicea, 3 Muricea, 1 Plexaura, 5 Plexaurella, 16 Pseudoplexaura and 9 Pterogrogia that were not identified to species.
Species |
Individuals collected* |
COI |
16S |
16S-ND2 |
ND6-ND3 |
ND4L-MSH |
Antillogorgia acerosa |
4 |
3 |
3 |
3 |
3 |
3 |
Antillogorgia americana |
4 |
4 |
4 |
4 |
4 |
3 |
Antillogorgia bipinnata |
8 |
5 |
5 |
5 |
5 |
4 |
Antillogorgia rigida |
2 |
1 |
1 |
1 |
1 |
X |
Briareum asbestinum |
9 |
8 |
7 |
7 |
3 |
7 |
Ellisella schmitti |
2 |
2 |
2 |
1 |
2 |
2 |
Erythropodium caribaeorum |
3 |
3 |
1 |
X |
3 |
X |
Eunicea calyculata |
3 |
3 |
2 |
3 |
3 |
3 |
Eunicea clavigera |
1 |
1 |
1 |
1 |
1 |
1 |
Eunicea flexuosa |
9 |
8 |
8 |
8 |
8 |
8 |
Eunicea fusca |
5 |
4 |
4 |
4 |
4 |
4 |
Eunicea laxispica |
1 |
1 |
1 |
1 |
1 |
1 |
Eunicea succinea |
1 |
1 |
1 |
1 |
1 |
X |
Eunicea tourneforti |
6 |
6 |
4 |
6 |
6 |
3 |
Gorgonia mariae |
8 |
8 |
8 |
8 |
8 |
X |
Gorgonia ventalina |
4 |
4 |
4 |
4 |
4 |
X |
Muricea laxa |
7 |
7 |
3 |
6 |
7 |
6 |
Muricea muricata |
3 |
3 |
3 |
3 |
3 |
3 |
Muricea pinnata |
5 |
5 |
3 |
5 |
5 |
5 |
Muriceopsis bayeriana |
6 |
6 |
6 |
6 |
6 |
X |
Muriceopsis flavida |
4 |
3 |
4 |
3 |
3 |
X |
Plexaura homomalla |
11 |
8 |
6 |
8 |
8 |
7 |
Plexaura kuna |
4 |
4 |
4 |
4 |
4 |
2 |
Plexaurella dichotoma |
3 |
3 |
3 |
3 |
3 |
3 |
Plexaurella nutans |
4 |
2 |
2 |
2 |
2 |
2 |
Pseudoplexaura porosa |
2 |
2 |
1 |
2 |
2 |
2 |
Pseudoplexaura wagenaari |
1 |
1 |
X |
1 |
X |
1 |
Pterogorgia anceps |
4 |
X | X |
1 |
X | X |
Pterogorgia citrina |
2 |
2 |
2 |
2 |
2 |
X |
New additions to GenBank from this study include COI for 19 species, 16S for 20 species, 16S-ND2 and ND4L-MSH for 3 species and 16S-ND2 and ND6-ND3 for 13 species (Table
As expected for octocorals, none of our 5 mitochondrial markers was suitable for distinguishing amongst congeneric species across the entire dataset, but they were generally useful to distinguish amongst most genera except those in the Plexaura-Pseudoplexaura-Eunicea group and the Antillogorgia-Gorgonia group (Figs
Heatmap of the mean pairwise differences between species of Caribbean octocorals, based on nucleotide substitutions in their COI sequences. Colours on the diagonal indicate the mean intra-species differences. Species with a dash in the diagonal were represented by only one individual and thus their intra-species differences could not be calculated.
Heatmap of the mean pairwise differences between species of Caribbean octocorals, based on nucleotide (substitutions) and indel (insertions/deletions/gaps) differences of their 16S sequences. Colours on the diagonal indicate the mean intra-species differences. Species with a dash in the diagonal were represented by only one individual and thus their intra-species differences could not be calculated.
Heatmap of the mean pairwise differences between species of Caribbean octocorals, based on nucleotide (substitutions) and indel (insertions/deletions/gaps) differences of their 16S-ND2 sequences. Colours on the diagonal indicate the mean intra-species differences. Species with a dash in the diagonal were represented by only one individual and thus their intra-species differences could not be calculated.
Heatmap of the mean pairwise differences between species of Caribbean octocorals, based on nucleotide (substitutions) and indel (insertions/deletions/gaps) differences of their ND6-ND3 sequences. Colours on the diagonal indicate the mean intra-species differences. Species with a dash in the diagonal were represented by only one individual and thus their intra-species differences could not be calculated.
Heatmap of the mean pairwise differences between species of Caribbean octocorals, based on nucleotide (substitutions) and indel (insertions/deletions/gaps) differences of their ND4L-MSH sequences. Colours on the diagonal indicate the mean intra-species differences. Species with a dash in the diagonal were represented by only one individual and thus their intra-species differences could not be calculated.
Haplotype network analyses of the Plexaura-Pseudoplexaura-Eunicea group and the Antillogorgia and Gorgonia group, were able to distinguish some genera and species which were not easily distinguished by distance analysis as visualised in the heatmaps. All of the markers except for 16S show that Gorgonia and Antillogorgia can be separated, as each is comprised of unique haplotypes Fig.
Haplotype networks involving Gorgonia and Antillogorgia DNA sequences. Each haplotype is represented by a circle. The number of individuals within each haplotype is indicated within the corresponding circle. The circle's sizes are proportional to the number of individuals. The white areas within some circles represent individuals identified only to the genus level (species unknown), whereas other colours are species-specific. The length between haplotypes represents the number of nucleotide differences between them: 1 length unit = 1 substitution or indel (insertion/deletion).
In contrast, network analysis of the Plexaura-Pseudoplexaura-Eunicea group further reinforces the results of the heatmaps, indicating that these genera cannot be distinguished using any of the markers Fig.
Haplotype networks of DNA sequences belonging to genera that could not be clearly separated by their nucleotide differences (heatmaps). For each gene, only the genera that were not clearly different in the heatmaps were included. Each haplotype is represented by a circle and each genus is represented by a unique colour. The number of individuals belonging to each haplotype are indicated within the corresponding circle. The circle's sizes are proportional to the number of individuals. The length between haplotypes represents the number of nucleotide differences (substitutions or indels) between them. Haplotypes present in multiple species, all belonging to the same genus, are indicated with a black star and haplotypes with all individuals belonging to the same species are indicated with underlined numbers. To be conservative, where samples only identified to genus were included in a circle, we did not consider them conspecific with samples identified to species, although they could have been.
As has been previously demonstrated for octocorals from other regions (
We thank the Autoridad de los Recursos Acuáticos de Panamá for issuing permits for this work, the participants in the 2007 workshop on Gorgonian Biology and Systematics in Bocas del Toro and C. McFadden whose suggestions significantly improved this manuscript. This research was supported by a Grand Challenges Level 1 grant from the Smithsonian Institution. All or portions of the laboratory and computer work were conducted in and with the support of the L.A.B. facilities of the National Museum of Natural History.
JM– contributed to the analysis and manuscript preparation; RC– contributed to the analysis and manuscript preparation; DVP– contributed to the analysis and manuscript preparation; ACD– generated the sequences and contributed to the analysis; JAS– Collected and identified the samples; HRL– Collected and identified the samples