Research Article |
Corresponding author: David Michael Baker ( dmbaker@hku.hk ) Academic editor: Nathalie Yonow
© 2020 Adam Wang, Inga Elizabeth Conti-Jerpe, John Lawrence Richards, David Michael Baker.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wang A, Conti-Jerpe IE, Richards JL, Baker DM (2020) Phestilla subodiosus sp. nov. (Nudibranchia, Trinchesiidae), a corallivorous pest species in the aquarium trade. ZooKeys 909: 1-24. https://doi.org/10.3897/zookeys.909.35278
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Phestilla subodiosus sp. nov. (Nudibranchia: Trinchesiidae) is a novel species that feeds on corals in the genus Montipora (Scleractinia: Acroporidae) which are economically important in the aquarium industry. Nuclear-encoded H3, 28SC1-C2, and mitochondrial-encoded COI and 16S markers were sequenced. Phylogenetic analysis, Automatic Barcode Gap Discovery (ABGD), morphological data, and feeding specialization all support the designation of Phestilla subodiosus sp. nov. as a distinct species. Although new to science, Phestilla subodiosus sp. nov. had been extensively reported by aquarium hobbyists as a prolific pest over the past two decades. The species fell into a well-studied genus, which could facilitate research into its control in reef aquaria. Our phylogenetic analysis also revealed Tenellia chaetopterana formed a well-supported clade with Phestilla. Based upon a literature review, its original morphological description, and our phylogenetic hypothesis, we reclassified this species as Phestilla chaetopterana comb. nov.
Nudibranchs, aquaculture, corallivore
While many Nudibranchia species and genera have yet to be described (
Phestilla (Fionidae: Trinchesiidae) was one of the genera reinstated by
From 2017 to 2018 we observed nudibranchs feeding on Montipora spp. fragments obtained from the aquarium trade in several closed system aquaria in Hong Kong. Morphological, behavioral, and genomic analysis determined that the species was previously undescribed. Later, a single specimen was obtained from the wild in Koh Tao, Thailand that was used for morphological analysis. Here, we describe this novel species of nudibranch as Phestilla subodiosus sp. nov. and resolve inconsistencies in the systematics of its family Trinchesiidae. Phestilla subodiosus sp. nov. is a corallivorous nudibranch commonly found preying on cultured corals in the genus Montipora (Scleractinia: Acroporidae). Aquarists report that damages caused by this species can cost hundreds of dollars (USD) per outbreak (D Hui, J McNelley pers. comm. 2018). Despite the economic and environmental importance of coral aquaculture, little information is available on the eradication and control of pest species (
Sexually mature nudibranchs and their egg masses were collected from Montipora spp. fragments (N > 10) between November 2017 and March 2018 (Figs
Total genomic DNA was extracted from six specimens using the DNeasy blood and tissue extraction kit (Qiagen, Germany), following the manufacturer’s protocol. Four loci were amplified with Polymerase Chain Reaction (PCR): mitochondrial Cytochrome c oxidase subunit I (COI), mitochondrial 16S structural rRNA subunit (16S), nuclear Histone H3 (H3), and nuclear 28S structural rRNA subunit (28S). Primers used are listed in Table
LCO 1490 | 5’-GGTCAACAAATCATAAAGATATTGG-3’ | ( |
5 min at 94 °C, 35× [1min at 94 °C, 30s at 42.5 °C, 1min at 72 °C], 7 min at 72 °C |
COIH-2 | 5’-TAYACYTCRGGATGMCCAAAAATCA-3’ | ( |
|
H3AF | 5’-ATGGCTCGTACCAAGCAGACVGC-3’ | ( |
3min at 94 °C, 35× [35s at 94 °C, 1min at 50 °C, 1min at 72 °C], 7min at 72 °C |
H3AR | 5’-ATATCCTTRGGCATRATRGTGAC-3’ | ( |
|
16S arL | 5’-CGCCTGTTTAACAAAAACAT-3’ | ( |
3min at 94 °C, 39× [30s at 94 °C, 30s at 50–55 °C, 1min at 72 °C], 5min at 72 °C |
16S R | 5’-CCGRTYTGAACTCAGCTCACG-3’ | ( |
|
28SC1 | 5’-ACCCGCTGAATTTAAGCAT-3’ | ( |
5min at 94 °C, 35× [1min at 94 °C, 30s at 45 °C, 1min at 72 °C], 7min at 72 °C |
28SC2 | 5’-TGAACTCTCTCTTCAAAGTTCTTTTC-3’ | ( |
PCR products for COI, 28S, and H3 were purified with ExoSAP-IT™ PCR Product Cleanup Reagent (ThermoFisher, USA) and cycle sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher, USA), both in accordance with the manufacturer’s instructions. Sequencing was performed on an ABI 3130xl Genetic Analyzer (ThermoFisher, USA). 16S PCR products were sequenced externally by the Beijing Genomics Institute (Shenzhen, China).
Raw reads obtained from Phestilla subodiosus sp. nov. were assembled and edited visually with Geneious 11.1.4 (
IQ-Tree (
Trees for each individual gene were computed to gain a better understanding of the systematics of the group. ML trees were estimated using IQ-Tree (
An online version of the Automatic Barcode Gap Discovery (ABGD) program (
Live adult (4 mm paratype) and juvenile individuals (1–3 mm paratypes) were photographed using a Nikon D5100 camera (Nikon, Japan) with AF-S Nikkor 18–55 mm 1:3.5–5.6G lens (Nikon, Japan). The holotype (2 mm) and a paratype (egg mass) were imaged using a Leica DFC295 microscope camera (Germany) with a 0.63X Stereo Microscope C-Mount (Leica, Germany) to examine external structures. The holotype obtained from captive Montipora spp. (2 mm) and the paratype collected from Thailand (3 mm) were dissected to isolate the buccal mass and reproductive system. Buccal mass was dissolved in dilute bleach (~ 1:30) to review radula and jaw plates. Radula, jaw plates, and reproductive system were imaged and examined under a Meiji Techno M1510 Trinocular Compound Microscope (Meiji Techno Co., Japan). Images were edited and annotated using Photoshop CC 2017 (Adobe, US). All type material was deposited at the Museum of The Swire Institute of Marine Science at The University of Hong Kong.
To elucidate the possible coral hosts of Phestilla subodiosus sp. nov., preliminary data of observed hosts were recorded. Individuals of Phestilla subodiosus sp. nov. (5 ≥ N ≥ 10) and a single fragment of one of eight species of coral (Table
Observational data of the feeding preference of Phestilla subodiosus sp. nov. Abbreviations: N indicates that this species of coral did not satisfy the two conditions needed to be counted as a host coral; Y indicates that the species did satisfy both conditions needed to be counted as a host coral.
Family | Genus | Species | Growth form | Host species |
---|---|---|---|---|
Acroporidae | Acropora | samoensis | Digitate corymbose. Thick branches. | N |
pruinosa | Digitate arborescent. Thin branches. | N | ||
Montipora | sp. 1 | Encrusting. | Y | |
sp. 2 | Digitate arborescent. Thin branches. | Y | ||
sp. 3 | Laminar scrolling. | Y | ||
sp. 4 | Laminar scrolling or encrusting. | Y | ||
Lobophylliidae | Echinophyllia | aspera | Laminar scrolling or encrusting. | N |
Poritidae | Porites | sp. 1 | Encrusting. | N |
In total, 17 of 24 sequences obtained from six sexually mature individuals were used for the final analysis: five from COI, two from 16S, four from 28S, and six from H3. GUIDANCE-2 revealed that the 16S sequence of Eubranchus rustyus was low quality and thus it was removed from the alignment. The concatenated dataset used in the phylogenetic analysis was 1255 bp (549 bp for COI, 379 bp for 16S, 327 bp for H3) long, including indels. Trees generated for each individual gene dataset (Suppl. material
The ML and BI phylogenic hypotheses (Fig.
Combined COI-16S-H3 Maximum Likelihood and Bayesian Inference phylogenetic hypotheses. Support values indicate Bootstrap (BS) and Posterior Probability (PP) rounded to two significant digits on the ML and BI trees. Phestilla subodiosus sp. nov. and P. chaetopterana comb. nov. are highlighted. Trees rooted on Eubranchus.
Pairwise distances (Table
Uncorrected COI p-distances (%) among all species of described Phestilla with available sequences. Percentages all rounded to one decimal place. Standard error (%) estimates rounded to one decimal place generated from bootstrapping (N = 10,000) are shown above the diagonal.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | ||
1 | Phestilla minor | 1.6 | 1.4 | 1.4 | 1.4 | 1.4 | 1.4 | 1.4 | 1.6 | 1.6 | 1.4 | 1.4 | 1.4 | 1.3 | 1.4 | |
2 | P. sibogae | 20 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.7 | 0.6 | 1.6 | 1.6 | 1.5 | 1.5 | 1.5 | |
3 | P. sp. 2 | 15.2 | 18.9 | 1.4 | 1.4 | 1.4 | 1.4 | 1.4 | 1.6 | 1.5 | 1.3 | 1.5 | 1.4 | 1.4 | 1.3 | |
4 | P. subodiosus sp. nov. PS1 | 14.6 | 17.5 | 13.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.5 | 1.4 | 1.4 | 1.4 | 0.4 | 1.3 | 0.4 | |
5 | P. subodiosus sp. nov. PS3 | 14.6 | 17.5 | 13.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.5 | 1.4 | 1.4 | 1.4 | 0.4 | 1.3 | 0.4 | |
6 | P. subodiosus sp. nov. PS4 | 14.6 | 17.5 | 13.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.5 | 1.4 | 1.4 | 1.4 | 0.4 | 1.3 | 0.4 | |
7 | P. subodiosus sp. nov. PS5 | 14.6 | 17.5 | 13.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.5 | 1.4 | 1.4 | 1.4 | 0.4 | 1.3 | 0.4 | |
8 | P. subodiosus sp. nov. PS6 | 14.6 | 17.5 | 13.7 | 0.0 | 0.0 | 0.0 | 0.0 | 1.5 | 1.4 | 1.4 | 1.4 | 0.4 | 1.3 | 0.4 | |
9 | P. chaetopterana comb. nov. | 17.1 | 18.4 | 17.1 | 14.9 | 14.9 | 14.9 | 14.9 | 14.9 | 1.6 | 1.6 | 1.5 | 1.5 | 1.5 | 1.5 | |
10 | P. lugubris | 19.9 | 2.2 | 18.7 | 16.7 | 16.7 | 16.7 | 16.7 | 16.7 | 17.8 | 1.6 | 1.5 | 1.4 | 1.5 | 1.4 | |
11 | P. melanobrachia | 14.6 | 19.7 | 12.6 | 15.9 | 15.9 | 15.9 | 15.9 | 15.9 | 19.3 | 19.6 | 1.5 | 1.4 | 1.4 | 1.4 | |
12 | P. poritophages | 14.2 | 18.5 | 16.7 | 14.2 | 14.2 | 14.2 | 14.2 | 14.2 | 15.8 | 17.7 | 17.6 | 1.4 | 1.4 | 1.4 | |
13 | Phestilla sp. L | 14.1 | 16.9 | 13.6 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 14.7 | 16.1 | 15.9 | 14.1 | 1.3 | 0.5 | |
14 | Phestilla sp. 1 | 12.9 | 18 | 14.6 | 11.8 | 11.8 | 11.8 | 11.8 | 11.8 | 15.5 | 17.7 | 14.9 | 15.1 | 11.8 | 1.3 | |
15 | Phestilla sp. 3 | 13.7 | 17.4 | 13.2 | 1.2 | 1.2 | 1.2 | 1.2 | 1.2 | 14.5 | 16.9 | 15.8 | 14.1 | 1.5 | 11.6 |
All three ABGD models elucidated ten partitions: simple distance found ten partitions with eight groups; while JC69 and K80 found five partitions with eight groups and five partitions with ten groups. In the partitions with eight groups, Phestilla subodiosus sp. nov., P. sp. L, and P. sp. 3 as well as P. lugubris and P. sibogae were grouped together. This provides additional evidence that Phestilla subodiosus sp. nov., P. sp. L, and P. sp. 3 are the same species and that P. lugubris and P. sibogae are synonymous. However, in the partitions with ten groups, while P. lugubris and P. sibogae were grouped together, Phestilla subodiosus sp. nov. was distinct to P. sp. L and P. sp. 3. These partitions are likely statistical anomalies due to the oversampling of virtually identical Phestilla subodiosus sp. nov. sequences.
Of all the coral species examined (Table
Family Trinchesiidae Nordsieck, 1972
“Physical form quite depressed. An edge anterior to the head, wing-like, attached to [...]; oral tentacles short, rhinophores simple. Cerata arranged on singular slanting rows, lacking cnidosacs. [...] Masticatory edge contains mandibles behind teeth (round, with irregular serration). Radula uniserial.” – Bergh, 1874: 1, partially translated.
Phestilla chaetopterana (Ekimova, Deart & Schepetov, 2017), comb. nov., Phestilla lugubris (Bergh, 1870), Phestilla melanobrachia (Bergh, 1874), Phestilla minor (Rudman, 1981), Phestilla panamica (Rudman, 1982), Phestilla poritophages (Rudman, 1979), Phestilla subodiosus sp. nov.
Historically, Phestilla was placed in the family Tergipedidae. This family contained a large “unnecessary and unnatural” number of genera (
At the same time that
Tenellia
sp. L:
Tenellia
sp.:
Holotype
: [SWIMS-MOL-17-001]. 1 specimen 2 mm long in 95% ethanol, dissected, Hong Kong SAR: Montipora spp., cultured in aquaria, coll. A. Wang, 19 Nov. 2017 (Figs
Paratypes
: [SWIMS-MOL-17-002]. 1 egg case 1 mm long in 95% ethanol. Hong Kong SAR: Montipora spp., cultured in aquaria, coll. A. Wang, 25 Nov. 2017 (Figs
The specific epithet, subodiosus, Latin for odious and vexatious, is symbolic of its status as a pest in the aquarium trade, and also a homage to the time and prized Montipora colonies the first author lost to in an outbreak of this species.
Specimen collected from Koh Tao, Thailand (this paper). Reported from Jeju Island, Korea (
External morphology
(Figs
Internal morphology
(Fig.
Internal morphology of Phestilla subodiosus sp. nov.: A schematic of rachidian tooth. Abbreviations: B, base; D, denticles; CC, central cusp B schematic of jaw plates overlaid onto microscope imagery C microscope imagery of reproductive system. Abbreviations: GO, genital opening; Pr, prostate; FGM, female gland mass; Am, ampulla D schematic of reproductive system. Abbreviations: PG, penile gland; Pr, prostate; VD, vas deferens; Va, vagina; FGM, female gland mass; Am, ampulla; HS, hermaphrodite system.
Color. Two ontogenetic color forms. Juvenile animals with white epidermal pigment throughout entire body. Adults with white epidermal pigment and translucent ceratal epidermis. Cerata speckled with brown clots, possibly from internal fluids or dinoflagellates of Symbiodiniaceae from coral hosts. Swollen regions on cerata lack speckles. Speckle density decreases towards the posterior of the cerata.
Defense mechanisms. Cerata observed to autotomize and secrete viscous adhesive mucus, usually encapsulating abscised ceras, when animal is disturbed tacitly.
Observed prey items.
Preys on coral species in the genus Montipora. Does not feed on corals of genera Porites, Acropora, and Echinophyllia. Reports of feeding on corals in genus Anacropora (
Based solely on the morphological key given in
Morphologically, Phestilla subodiosus sp. nov. is most similar to P. minor and P. poritophages in color forms and swollen cerata, but is distinguished by several characters: firstly, adult Phestilla subodiosis sp. nov. we observed averaged 3.5 mm in length, approximately half of the size P. minor (
One species, P. panamica, did not have any sequences available online so a molecular comparison was infeasible. However, it is clear that P. panamica and Phestilla subodiosus sp. nov. are not the same species. P. panamica grows up to 24 mm, has 18 cerata per row, five precardiac rows, and eight postcardiac rows (
There were considerable differences in the reproductive system and radula of Phestilla subodiosus sp. nov. and the rest of the genus, notably in the presence of a female gland mass. This arrangement is surprisingly similar to the reproductive system of the Chromodorididae. While it possible that the “female gland mass” is a bursa copulatrix, this would be extremely large for the genus, with a diameter 1.5 times the penile bulb’s length, and directly attached to the female genital opening. In all other species of Phestilla, with the exception of P. chaetopterana comb. nov., the bursa copulatrix is much smaller than the penile bulb and attached to the oviduct. In P. chaetopterana comb. nov., the bursa copulatrix is small, but attached directly to the female genital opening. As the function of the bursa copulatrix’ is to store sperm and/or digest it when needed, a larger one would allow a nudibranch to store more sperm longer thus explaining the phenomena reported by aquarists where the introduction of a single nudibranch can result in an outbreak and their ability to survive long periods without food (D Hui pers. comm. 2018). However, while the specimens dissected for the internal morphology analysis were sexually mature, they were only 2 mm and 3 mm in length. As previously shown, internal morphology has high ontogenetic plasticity throughout development (
While several molecular studies have investigated the phylogenetic relationships within Fionoidae, taxonomic assignment of groups has resulted in debate, including the placement and composition of some genera in Trinchesiidae such as Phestilla. Several genera were combined due to their close relationships recovered in a molecular phylogenetic analysis (
In recent decades, the introduction of coral aquaculture has reshaped both the aquarium industry and coral reef conservation efforts (
Despite being a prolific pest in aquaria, we were only able to find two reports of nudibranchs that resemble Phestilla subodiosus sp. nov. in situ (Roberston 1987:3;
Phestilla subodiosus sp. nov. displayed prey selectivity in our preliminary tests; however, the underlying mechanism is unclear. It has been established that other Phestilla species rely on chemical cues to differentiate host corals (
The description of Phestilla subodiosus sp. nov. is a key step that will allow for research to be conducted on its ecology and biology, and eventual control within reef aquaria. Given the wide number of common names in use to describe nudibranchs that feed on Montipora spp. (D Hui, J McNelley pers. comm. 2018), it is unclear whether several species exist or whether these names all refer to Phestilla subodiosus sp. nov. By formally describing Phestilla subodiosus sp. nov., further research can be conducted with confidence in the identity of the species being examined, allowing for clear collaboration and communication while a basic biological and ecological understanding of this species is developed. Furthermore, Phestilla subodiosus sp. nov. has been placed on the taxonomic tree of life within a well-understood genus containing several model organisms. Previous studies have described the proteins involved in Phestilla metamorphosis and drugs have been discovered that inhibit this vital process (
Despite the scientific advances enabled by the aquarium industry (
We thank the reviewers for their time and effort to review this paper and provide insightful comments and suggestions. The authors gratefully acknowledge Dr. Moriaki Yasuhara and Ms. Maria Lo Gar Yee of the University of Hong Kong for allowing us to use their microscope and microscopy assistance, Kerry Samantha Hsu from the University of Pennsylvania and Dr. Han Guo Hong from the Fourth Military Medical University for manuscript comments, Jeffrey Chan for the design of feeding experiments, and Vriko Yu of the University of Hong Kong for aiding the sequencing and other slug-related work. We are also extremely thankful to Marcus Hibbins from Indiana University Bloomington for his help on phylogenetic analysis, as well as James Townsend and Alejandro Damián Serrano from the University of Pennsylvania, and everybody else from our science meme page admin group chat for their advice on Latin translations, stylistic elements, and providing moral support. Last but not least, the first author is also extremely grateful and indebted to Dr. Theodore Faunce for helping him receive this opportunity to conduct research for the first time. This project was funded by the Environment and Conservation Fund (# 67/2016) and is manuscript number 48 of MarineGEO-Hong Kong.
Table S1: List of sequences used for molecular analysis
Data type: GenBank Accession Numbers
Explanation note: The species name and GenBank accessions of sequences we obtained and used for phylogenetic inference, ABGD analysis, p-distance are included within this table.
S2: Phylogenetic hypotheses based upon individual COI, 16s, and H3 partial gene sequences
Data type: Newick Trees
Explanation note: The compressed file contains 6 newick tree files and are named based upon the partial gene and phylogenetic inference method that were used to generate that tree. BI = Bayesian Inference, ML = Maximum Likelihood.