Conceived and designed the experiments: MH. Performed the experiments: MH MVM MW. Analyzed the data: MH NP MVM MW. Contributed reagents/materials/analysis tools: MH NP MW RC EB BMO. Wrote the paper: MH NP BMO. Edited the manuscript: RC.
The authors have declared that no competing interests exist.
Central to the discovery of neuroactive compounds produced by predatory marine snails of the superfamily Conoidea (cone snails, terebrids, and turrids) is identifying those species with a venom apparatus. Previous analyses of western Pacific terebrid specimens has shown that some Terebridae groups have secondarily lost their venom apparatus. In order to efficiently characterize terebrid toxins, it is essential to devise a key for identifying which species have a venom apparatus. The findings presented here integrate molecular phylogeny and the evolution of character traits to infer the presence or absence of the venom apparatus in the Terebridae. Using a combined dataset of 156 western and 33 eastern Pacific terebrid samples, a phylogenetic tree was constructed based on analyses of 16S, COI and 12S mitochondrial genes. The 33 eastern Pacific specimens analyzed represent four different species:
The auger snails (family Terebridae) are a distinctive group of carnivorous, sand-dwelling gastropods included in the superfamily Conoidea, along with cone snails and turrids
In contrast to cone snail toxins (conotoxins), terebrid toxins are largely uncharacterized and no physiological target for any terebrid venom peptide has been defined. However, the very preliminary characterization carried out to date suggests that the venoms of the Terebridae have novel components, distinct from other conoidean venoms
A significant fraction of the ∼300–400 species in the Terebridae do not have the characteristic anatomical structures that comprise the venom delivery apparatus of conoidean snails, namely a venom bulb, venom duct, and radula sac
The first molecular phylogeny of the Terebridae based on a three-gene matrix of molecular markers 12S, 16S, and cytochrome oxidase I (COI), was recently published
The original correlation between venom apparatus and molecular phylogeny was established using only western Pacific species
Panamic specimens used were dredged from the Las Perlas Archipelago in 2008, using The Smithsonian Tropical Research Institute research vessel RV-Urraca. The collected material was specifically fixed for molecular and anatomical analysis. Living specimens were anesthetized in MgCl2 isotonic with seawater for 1 or 2 hours. Samples were dissected and a piece of tissue (usually foot) was fixed in 95% ethanol.
A. The Las Perlas Archipelago, located off the west coast of Panama (see Inset), is the collection site for the terebrids analyzed. The numbers shown on the map refer to the stations for the Panamic specimens listed in
COI | 12S | 16S | VA | Station number - Coordinates/Depth | MNHNnumber | ||
x | x | x | No | 3–08°11.8′N, 078°57.1′W/21.4 m | 42093 | ||
x | x | x | No | 4–08°11.8′N, 078°57.5′W/22.4 m | 42105 | ||
x | x | x | No | 5–08°14.7′N, 079°05.55′W/17.5 m | 42136 | ||
x | x | x | No | 5–08°14.7′N, 079°05.55′W/17.5 m | 42137 | ||
x | x | No | 9–08°30.1′N, 079°06.0′W/21 m | 42159 | |||
x | x | x | Yes | 1–08°37.18′N, 079°01.12′W/25 m | 42068 | ||
x | x | x | Yes | 1–08°37.18′N, 079°01.12′W/25 m | 42069 | ||
x | Yes | 2–08°15.61′N, 078°51.57′W/24.1 m | 42071 | ||||
x | x | x | Yes | 2–08°15.61′N, 078°51.57′W/24.1 m | 42072 | ||
x | x | x | Yes | 2–08°15.61′N, 078°51.57′W/24.1 m | 42073 | ||
x | Yes | 1–08°37.18′N, 079°01.12′W/20 m | 42074 | ||||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42084 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42085 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42086 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42087 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42089 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42090 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42091 | ||
x | x | x | Yes | 3–08°11.8′N, 078°57.1′W/21.4 m | 42092 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/24 m | 42099 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/24 m | 42100 | ||
x | Yes | 4–08°11.8′N, 078°57.5′W/24 m | 42102 | ||||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42103 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42104 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42118 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42119 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42120 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42121 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42122 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42123 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42124 | ||
x | x | x | Yes | 4–08°11.8′N, 078°57.5′W/22.4 m | 42125 | ||
x | x | x | Yes | 6–08°14.94′N, 079°05.7′W/14.3 m | 42131 | ||
x | x | x | Yes | 7–08°16.86′N, 079°02.67′W/39.2 m | 42152 | ||
x | x | Yes | 8–08°24.50′N, 079°04.66′W/18.4 m | 42153 | |||
x | x | x | No | 9°37.4′N, 123°46.9′E, 3–20 m | 30370 | ||
x | x | x | No | 15°32.5′S, 167°10.5′E, 5–10 m | 30372 | ||
x | x | x | No | 15°36.9′S, 167°10.5′E, 6–33 m | 30373 | ||
x | x | x | No | 15°34.4′S, 167°13.1′E, 9 m | 30377 | ||
x | x | x | No | 15°32.5′S, 167°10.5′E, 5–10 m | 30379 | ||
x | x | x | No | 15°35.4′S, 166°59.7′E, 3–37 m | 30381 | ||
x | x | x | No | 15°28.7′S, 167°15.2′E, 19 m | 30389 | ||
x | x | x | No | 15°38.1′S, 167°05.9′E, intertidal | 30428 | ||
x | x | x | No | 9°37.4′N, 123°54.5E, 6–8 m | 30443 | ||
x | x | x | No | 9°37.4′N, 123°54.5E, 6–8 m | 30445 | ||
x | x | x | No | 15°22.6′S, 167°11.6′E, intertidal | 30490 | ||
x | x | x | No | 15°34.4′S, 167°13.1′E, 9 m | 30494 | ||
x | x | x | No | 9°37.4′N, 123°46.9′E, 3–20 m | 30587 | ||
x | x | x | Yes | 9°27.4′N, 123°49.4′E, 273–356 m | 15724 | ||
x | x | x | Yes | 9°36.2′N, 123°43.8′E, 382–434 m | 16735 | ||
x | x | x | Yes | 9°29.4′N, 123°44.4′E, 271–318 m | 30390 | ||
x | x | x | Yes | 9°35.3′N, 123°52.2′E, 84–87 m | 30404 | ||
x | x | x | Yes | 9°39.2′N, 123°47.5′E, 255–268 m | 30410 | ||
x | x | x | Yes | 9°39.2′N, 123°47.5′E, 255–268 m | 30418 | ||
x | x | x | Yes | 15°32.5′S, 167°10.5′E, 5–10 m | 30485 | ||
x | x | x | Yes | 8°39.5′ S, 157°23.0′ E, 214–243 m | 30487 | ||
x | x | x | Yes | 15°28.6′S, 167°15.1′E, 3–31 m | 30544 | ||
x | x | x | Yes | 15°35.2′S, 167°59.4′E, intertidal | 30552 | ||
x | x | x | Yes | 15°35.2′S, 167°59.4′E, intertidal | 30420 | ||
x | x | x | No | 9°37.4′N, 123°54.5′E, 6–8 m | 30430 | ||
x | x | x | Yes | 15°33.1′S, 167°12.2′E, 3–40 m | 30376 | ||
x | x | x | Yes | 15°31.1′S, 167°10.5′E, 7 m | 30380 | ||
x | x | x | Yes | 15°36.6′S, 167°10.1′E, 8–20 m | 30386 | ||
x | x | x | Yes | 15°33.1′S, 167°12.2′E, 3–40 m | 30387 | ||
x | x | x | Yes | 15°36.9′S, 167°10.5′E, 6–33 m | 30394 | ||
x | x | x | Yes | 15°33.1′S, 167°17.8′E, 15–25 m | 30409 | ||
x | x | x | Yes | 9°36.8′N, 123°52.2′E, intertidal | 30431 | ||
x | x | x | Yes | 9°37.4′N, 123°54.5E, 6–8 m | 30444 | ||
x | x | x | Yes | 9°32.8′N, 123°42.1′E, 3–35 m | 30483 | ||
x | x | x | Yes | 15°38.5′S, 167°15.1′E, 13 m | 30493 | ||
x | x | x | Yes | 15°26.6′S, 167°15.2′E, intertidal | 30597 | ||
x | x | x | Yes | 15°43.4′S, 167°15.0′E, 6 m | 30603 | ||
x | x | x | Yes | 15°31′S, 167°09′E, intertidal | 30613 | ||
x | x | x | Yes | 15°31′S, 167°09′E, intertidal | 30632 | ||
x | x | x | Yes | 9°37.5′N, 123°40.2′E, 606–631 m | 30395 | ||
x | x | x | 21°10′S, 158°39′E, 650–723 m | 40568 | |||
x | x | x | Yes | 9°32.5′N, 123°41.8′E, 111–115 m | 17922 | ||
x | x | x | 9°32.5′N, 123°41.8′E, 111–115 m | 40569 | |||
x | x | x | 15°33.6′S, 167°16.6′E, 8–9 m | 17685 |
DNA was extracted from foot or other tissue using Qiagen QIAamp Dneasy Tissue kit. Fragments of mitochondrial genes 12S, 16S and COI were amplified using universal primers 12S1/12S3
COI sequences were manually aligned and 12S and 16S were automatically aligned using ClustalW multiple alignment implemented in BioEdit version 7.0.5.3
Phylogenetic analyses were based on reconstructions using two approaches: (i) Maximum Likelihood (ML) using PhyML 2.4.4
The 33 Panamic specimens analyzed were assigned to four different terebrid species:
After alignment, DNA fragments of 658, 534, and 455 bp were obtained for COI, 12S, and 16S genes, respectively. No contradictions were observed when independently constructed gene trees for COI, 12S, and 16S genes were analyzed (results not shown). These Panamic sequences were combined with sequences from western Pacific terebrid specimens to reconstruct the phylogeny illustrated in
Shown is a consensus tree (BA) using COI, 16S, and 12S data sets. Posterior probabilities and bootstrap values are specified for each node. Shaded clades were collected in Panama. The bar on the right shows which taxa have venom glands (black bars) and which do not (white bars). Clade A refers to the sister group that includes
Of the 5 distinct terebrid clades previously identified, Clade A (
The phylogenetic analysis strongly indicates that the Panamic
All Panamic specimens collected were dissected and the presence or absence of a venom apparatus was noted (
Predatory marine snails of the superfamily Conoidea produce several neurotoxins in their venom that are used to capture and subdue prey
The three Panamic species
The species-level taxonomy of
The figure shows the diversity of the venomous eastern Pacific forms tentatively assigned to Clade C,
In this instance the molecular characters used in the phylogenetic analyses confirmed the shell-based morphological characters used to identify different terebrid species. The specimens of
The Panamic
The authors thank Felix Rodriquez, Edwin Diaz, Trinidad Pardo, Moises Bernal, and the crew of the RV-Urraca with collection efforts. We also acknowledge James Ernest for input on the various forms of Panamic Terebridae. Authors also thank Carey Matz for shell images.