Entacmaea+quadricolor

=Bubble-tip anemone=

//Entacmaea quadricolor// (Leuckart in Rüppell & Leuckart, 1828) toc Figure 1. Rainbow bubble-tip anemone (//Entacmaea quadricolor//) (Source: Matthew Zahler, permission pending 37])

Overview
//Entacmaea quadricolor// is a sea anemone widespread throughout tropical waters, and is commonly found on Singapore's shores 21]. It is known as the bubble-tip anemone because of its distinctive bulbous tips 1]. It harbours toxins on its tentacles to catch prey 11], and is symbiotic with many species of anemonefishes, such as the common clownfish (//Amphiprion ocellaris//) 6]. This anemone also exhibits biofluorescence 10] and reproduces by releasing gametes directly into the water medium for external fertilisation 17]. This species is unfortunately threatened by the aquarium trade, owing to its wide variety of attractive colour morphs 23].

Description
// Entacmaea quadricolor // have adherent bases which conform to the shape of the substratum they attach to 1]. They have smooth flared body columns which are usually rich brown but may be greenish or reddish 1]2]. The columns of small individuals range from 50mm - 100mm in length, while that of larger individuals can reach up to 500mm 1]. At the top of the column is a flattened oral disc with an oval mouth in the middle 1]. Cylindrical tentacles fringe the oral disc and inner tentacles are 2 - 3 times longer than marginal ones (Fig. 2) 2].

Figure 2. Features of //E. quadricolor// (Adapted from: Nicola Wood, permission pending 38]) The tentacles may be bulbous at the end, with a terminal bud that usually ends in a sharp red point, or a subterminal bud which lacks a pigmented tip (Fig. 3) 1]. The bulbs may have a white ring (equator) around them (Fig. 1), or be flecked with white 2]. Some tentacles may lack a bulb and are digitiform (Fig. 3), commonly associated with the absence of symbiotic anemonefish 2]. The appearances of //E. quadricolor// are highly diverse, with sixteen colour morphs discovered in a study at North Solitary Island, Solitary Islands Marine Park, Australia 3]. The state of tentacle expansion can also differ, as tentacles often collapse when disturbed 1].

Figure 3. Bubble-tip anemone tentacle forms. Tentacle with terminal bud (left), Tentacle with subterminal bud (middle), Digitiform tentacle (right) (Adapted from: Nick Hobgood, under a CC BY-SA 3.0 license 39] (left), Chaloklum Diving, under a CC BY 3.0 license 40] (middle), Josuevg, under a CC BY-SA 3.0 license (right) 41]) Figure 4, an image taken in Singapore's waters (Terumbu Semakau), clearly shows some of the diagnostic features of //E. quadricolor.// The greenish smooth column and bulbous tentacles with the white equator are evident in the figure.

Figure 4. //Entacmaea quadricolor// in Singapore's waters (Source: Ria Tan, under a CC BY-NC-ND 2.0 license 42])

Habitats
Individuals of //E. quadricolor// occur on reef environments, from portions that are nearly exposed at low tide to as deep as 40m (although rare below 20m) 1]. They exist in two morphologies - small and colonial or large and solitary. Small clustering forms occur in shallow water, typically anchored within cracks and crevices on reefs, and are sometimes attached to branches of corals 1]. Large solitary individuals are found in deeper waters on reef slopes, anchored in holes or crevices such that only the tentacles are visible. Both forms can be found at depths in between 1].

Local
The localities of //E. quadricolor// found on Singapore's shores are Chek Jawa (Pulau Ubin), Kusu Island, Sisters Island, Pulau Biola, Pulau Semakau and Pulau Hantu 9], among others. All known localities are shown in the map below.

media type="custom" key="28798902" align="center" Figure 5 shows an individual of //E. quadricolor// at Sisters Island, with an interesting colour morph of brownish tentacles with a green sheen.

Figure 5. //Entacmaea quadricolor// at Sisters Island [Source: Ria Tan, under a CC BY-NC-SA 2.0 license 43])

Global
//Entacmaea quadricolor// inhabit the waters of the Red Sea, eastern Africa, Indian Ocean, Micronesia, Melanesia, Japan and Australia (Fig. 6) 2].

Figure 6. Global distribution of //E. quadricolor// (Adapted from: Global Biodiversity Information Facility, under a CC-BY 4.0 license 44])

Symbiosis
Symbiosis is a close and often long-term interaction between two different biological species, known as symbionts 5]. //Entacmaea quadricolor// is symbiotic with some species of zooxanthellae, anemonefishes and shrimps 6]7].

__Endosymbionts__
//Entacmaea quadricolor// host photosynthetic dinoflagellate algae, zooxanthellae, which supplies a rich carbon source and some essential amino acids to the host anemone 4]. The majority of the zooxanthellae (61.1%) are located in the tentacles and column walls, and belong to the genus //Symbiodinium// 4].

__Ectosymbionts__
//Entacmaea quadricolor// are also host to several species of anemonefishes including the red sea clownfish (//Amphiprion bicinctus//) 5], tomato clownfish (//Amphiprion frenatus//), cinnamon clownfish (//Amphiprion melanopus//), common clownfish (//Amphiprion ocellaris//) and red saddleblack clownfish (//Amphiprion ephippium//), (Fig. 7) 6]. They also host shrimps such as the five-spot anemone shrimp (//Periclimenes brevicarpalis//) 7]. Anemonefishes provide aggressive protection from their main predator, the chaetodontid butterflyfish, which feeds on //E. quadricolor// (Fig. 8) 8]. Chaetodontids were found to prey on //E. quadricolor// after experimental removal of symbiotic anemonefishes, causing the elimination of some anemones and retraction of others into rock crevices 8]. Besides gaining defence against predation, ammonia excreted by anemonefishes also benefit //E. quadricolor// when absorbed to increase growth rates and enrich the endosymbiotic zooxanthellae 5].

Figure 7. Anemonefishes symbiotic with //E. quadricolor// (Adapted from: Tullock in 6], permission pending 45]) Figure 8. Predator Pacific double-saddle butterflyfish (//Chaetodon ulietensis//) feeding on tentacles of //E. quadricolor// (Source: John Godwin in 8], permission pending 46])

Behaviour
//Entacmaea quadricolor// respond to predator attacks by contracting into reef holes to protect their tissues from consumption by predators, as chaetodontid butterflyfish are unable to reach into reef crevices 15]. However, remaining contracted for long periods of time may lead to to tissue degeneration as the anemone cannot feed or absorb nutrients from symbionts 15]. In some areas, //Entacmaea quadricolor// were observed to expand in light conditions only in the presence of symbiotic anemonefish, and in dark conditions when their major predators are inactive 15]. In Singapore, this behaviour is not observed as //E. quadricolor// expand in light conditions even without the presence of symbiotic anemonefish 21], possibly due to the lack of predators in local waters.

While mature //E. quadricolor// predominantly stay attached to their substratum, they are capable of movement and detach quickly from the substratum when disturbed 10]. //Entacmaea quadricolor// individuals have also been observed to escape smothering by rapid sedimentation, this was done through inflating their pedal disc and column alternatively to emerge from the sediments (shown in clip below) 10].

media type="custom" key="28798904" align="center" //Entacmaea quadricolor// escapes from being smothered by sedimentation (Adapted from: Nicola Wood, obtained and accredited under fair use 47])

Biofluorescence
Fluorescent proteins in //E. quadricolor// include Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) 10]. A study on a specimen from Heron Island, Queensland, Australia, revealed that fluorescence was inconsistent through the anemone, with varying distributions of GFP and RFP 10]. The tentacle tips were the most fluorescent for GFP, with some fluorescence present on the tentacles and oral disc 10] (Fig. 9). Fluorescence for RFP was concentrated at the tentacle ends apart from the extreme tips, with close to no fluorescence on the oral disc 10] (Fig. 9).

Figure 9. Live //E. quadricolor// specimen under GFP and RFP fluorescent channels. (A) Top view under GFP, (B) Top view under RFP, (C) Top view under GFP and RFP overlay, (D) Tentacle close-up under GFP and RFP overlay, (E) Tentacle tip close-up under GFP and RFP overlay. (Adapted from: Nicola Wood, permission pending 48]) The importance of biofluorescence and to //E. quadricolor// has not been well studied, although it has been hypothesised that fluorescent tentacle tips may attract prey for sedentary anemones to feed on 10]. Further work is required to explore the implications of GFP and RFP distribution on the ecology of //E. quadricolor//.

Feeding
//Entacmaea quadricolor// are opportunistic carnivores that capture prey using cnidocytes on their tentacles which contain many "stinging organs" known as nematocysts (or cnidae). Larger individuals generally have larger nematocysts 1]. This occurs when prey drifts or swims into the tentacles of the anemone, get immobilised and are transported to the mouth via tentacle contraction 10]. Small prey such as plankton may get trapped in the tentacles' mucus lining, and are directed to the tentacle tips followed by the mouth via ciliary action 10]. In the video below, a white //E. quadricolor// is feeding on a fish and the tentacle contractions can be seen clearly. Prey can be captured because //E. quadricolor// utilise toxins which are injected through nematocysts or toxins associated with the mucus lining of the tentacles 11].

media type="youtube" key="i0UmN01DuMw" width="560" height="315" align="center" //Entacmaea quadricolor// feeding on fish (Source: SquidMilk, obtained and accredited under fair use 49])

Toxins
Toxins ejected from nematocysts include neurotoxins which paralyse prey, and one such toxin is acetylcholinesterase inhibitor 11]. Other toxins associated with the mucus lining of tentacles are predominantly used for defence. These toxins are lethal to some fish, causing cell lysis and gill damage 12]. However, anemonefishes are not affected by the toxins as they do not elicit nematocyst discharge from //E. quadricolor// when in contact with their tentacles 13]. The anemonefishes' mucus plays an important role in protection via a blocking mechanism 14]. Anemonefishes are either innately immune to //E. quadricolor// or they acquire protection from stinging nematocysts through acclimation 13].

An interesting study published in 2014 revealed that anemones with intermediate toxicity, //E. quadricolor// included, had the most number of anemonefish symbionts (Fig. 10) 14]. This renders //E. quadricolor// a desirable host that possesses optimum toxicity for anemonefish survival, supported by the large diversity of symbiotic and highly specialised anemonefishes that inhabit //E. quadricolor// in nature 14].

Figure 10. Relationship between number of anemonefish symbionts and anemone toxicity ranking (Source: Nedosyko //et al//. in 14], under a CC BY 4.0 license 50])

__Asexual__
//Entacmaea quadricolor// can propagate asexually via longitudinal fission 1]. The anemone divides by stretching in opposite directions, starting from the base 16]. A tear is created and the tissue thins gradually, resulting in two genetically identical individuals 16] (Fig. 11). This process gives rise to individuals with irregular symmetry 1] as the individuals undergo transformations such as a change in body diameter and number of tentacles 16].

Figure 11. Longitudinal fission resulting in two genetically identical individuals (Adapted from: Nicola Wood, permission pending 51])

__Sexual__
Both sexes of //E. quadricolor// broadcast spawn their gametes into the water for external fertilisation and development, and the spawning was found to always occur after sunset between a full and waxing crescent moon 17]. Seasonal fluctuation in sea temperatures is an important environmental factor that influences reproduction in marine invertebrates 18]. //Entacmaea quadricolor// spawning events were found to occur during the warmer months of the year where sea temperatures were peaking. This can be attributed to the increased rates of embryo and larval development 19], and enhanced settlement rates associated with higher sea temperatures 20]. In Singapore, studies on //E. quadricolor// spawning events have yet to be published.

Individuals become inflated prior to and during spawning events as the process requires forceful release of gametes, facilitated by columnar muscle contractions 17]. Males commonly release sperm in milky white streams while females release eggs singly or in large assemblages 17] (Fig. 12). Males release gametes prior to females, and this synchrony of gamete release suggests that sperm or an associated product may stimulate egg release 17]. The videos at the end of this section show the spawning of a male and female //E. quadricolor//.

Figure 12. Broadcast spawning of male (a) and female (b) //E. quadricolor// (Adapted from: Cristiana Damiano in 17], permission pending 52]) media type="youtube" key="Vrq7LEVeDdQ" width="560" height="315" align="center" Male //E. quadricolor// spawning (Source: SRKreef, obtained and accredited under fair use 53]) media type="youtube" key="i5JfNYAi_ZM" width="560" height="315" align="center" Female //E. quadricolor// spawning (Source: J Madden, obtained and accredited under fair use 54])

Status
//Entacmaea quadricolor// has not been evaluated in The IUCN Red List of Threatened Species, neither has it been listed among the threatened fauna of Singapore 21]. However, some of the obligate symbiotic anemonefishes whose essential habitats are provided by //E. quadricolor// are listed on the Red List. According to the Singapore Red Data Book, all anemonefishes, including the tomato clownfish (//Amphiprion frenatus//) and common clownfish (//Amphiprion ocellaris//) are listed as 'vulnerable' and "habitat protection and strict policing against illegal connection are required" to conserve the species 22]. Anemonefish are rarely found in Singapore anemones and thus far, only the //tomato clownfish// (//Amphiprion frenatus//) has been seen among //E. quadricolor// in the interdal areas of Singapore's shores 9]21] (Fig. 13, Fig. 14). Elsewhere in nature, it is unusual to see anemones without their symbiotic anemonefishes and it is likely that they have been removed by collectors in Singapore 21], threatening the local populations of both anemonefishes and their host sea anemones.

Figure 13. Tomato clownfish (//Amphiprion frenatus//) among //E. quadricolor// in Singapore's shores (Terumbu Raya) (Source: Ria Tan, under a CC BY-NC-ND 2.0 license 55]) Figure 14. Tomato clownfish (//Amphiprion frenatus//) among //E. quadricolor// in Singapore's shores (Pulau Semakau) (Source: Ria Tan, under a CC BY-NC-ND 2.0 license 56])

Aquarium trade
The expanding marine aquarium trade for marine ornamentals are a threat to both anemonefishes and their host sea anemones 23]. An analysis of catch from a four-month period in Cebu, Philippines, revealed that anemonefishes and anemones comprised approximately 60% of the total catch 24]. Sea anemones that host symbiotic anemonefishes are in high demand among aquarists 23] as they represent high-value species that are preferentially harvested from the wild 24]. Harvesting of marine ornamentals has led to local extinctions of some species, and poses a threat to sea anemones globally 24]. Figure 15 shows an aquarist's tank containing //E. quadricolor// and symbiotic anemonefishes. The marine aquarium trade is a global problem and large numbers of individuals of //E. quadricolor// are sold on websites online (Fig. 16). In Singapore, there is also a demand for //E. quadricolo//r as local stores have been found to be selling the anemone (Fig. 17). As these individuals sold online are most likely harvested from the wild, it poses a threat to both local and global populations of //E. quadricolor//.

Figure 15. //Entacmaea quadricolor// and symbiotic anemonefishes in an aquarist's tank (Adapted from: Melev's Reef, obtained and accredited under fair use 57]) Figure 16. Individuals of //E. quadricolor// sold online (Adapted from: LA Reefs, permission pending 58]) Figure 17. //Entacmaea quadricolor// sold by a local supplier on Facebook (Adapted from: Pinnacle Aquatics Pte Ltd, permission pending 59])

Bleaching
Rising sea temperatures can affect sea anemones through thermally-induced bleaching from the loss of symbiotic zooxanthellae, which may lead to mortality 25]. Bleaching events pose a serious threat to the viability of //E. quadricolor// populations and their symbiotic anemonefishes 25]. Following a bleaching event at the Keppel Islands, Australia, //E. quadricolor// experienced a decrease in size and abundance, and an increase in mortality rate which led to the loss of symbiotic anemonefish 25]. Bleaching is generally greatest at shallow waters where sea temperatures are highest 25], as observed in Singapore's waters where the anemones lost their endosymbionts and turned bright white 21] (Fig. 18, Fig. 19). The video below shows a pair of tomato clownfish (//Amphiprion frenatus//) among bleached //E. quadricolor// at Terumbu Raya, Singapore, in August, 2016. Figure 18. Bleached //E. quadricolor// in Singapore's shores (Sisters Island) (Source: Ria Tan, under a CC BY-NC-ND 2.0 license 60]) Figure 19. Bleached //E. quadricolor// in Singapore's shores (Sultan Shoal) (Source: Joy Wong, used with permission 61]) media type="custom" key="28798900" align="center" Bleached //E. quadricolor// at Terumbu Raya, Singapore (Source: Ria Tan, under a CC BY-NC-ND 2.0 license 62])

Rank and taxa names
26]
 * Kingdom || Animalia ||
 * Phylum || Cnidaria ||
 * Class || Anthozoa ||
 * Subclass || Hexacorallia ||
 * Order || Actiniaria ||
 * Suborder || Enthemonae ||
 * Superfamily || Actinioidea ||
 * Family || Actiniidae ||
 * Genus || //Entacmaea// ||
 * Species || //Entacmaea quadricolor// ||

Original description
The basionym of //E. quadricolor// is //Actinia quadricolor//, originally described by Friedrich Sigismund Leuckart, a german naturalist 1]9]. It was in a publication titled "Atlas zu der Reise im nördlichen Afrika" by Rüppell & Leuckart in 1828, with its description on page 4 of the publication 1] (Fig. 20). An illustration labelled Fig. 3 was also provided 1]27] (Fig. 21).

Figure 20. Original description of //E. quadricolor// (basionym: //Actinia quadricolor//) (Adapted from: Biodiversity Heritage Library. Digitized by Field Museum of Natural History Library | www.biodiversitylibrary.org) Figure 21. Illustration of //E. quadricolor// (basionym: //Actinia quadricolor//) NB: Mislabelled as Fig. 2 in description, correctly labelled as Fig. 3 in caption 1]27] (Adapted from: Biodiversity Heritage Library. Digitized by Field Museum of Natural History Library | www.biodiversitylibrary.org)

Type information
The holotype of //E. quadricolor//, which the original description was based on, is shown in Figure 22. It was recorded on Jan 1, 1827 29], and the type locality is near Suez, the Red Sea, Egypt 1]2]. The specimen is now found in Senckenberg Museum, with catalog number 34 (SMF-34) 28]. It was labelled "//Actinia quadricolor//" and found to be a male 1].

Notes on holotype: Whole animal. Oral, pedal disc diameters 40mm, length 35mm; tentacles poorly preserved. Wedges removed from margins, mid-column for microscope slides by D. G. Fautin. Labeled from Rotes Meer, attributed to Rüppell 1827 29].

Figure 22. Holotype of //E. quadricolor// in Senckenberg Museum (Source: Daphne Fautin, permission pending 63]) Although there is a holotype for //E. quadricolor//, multiple syntypes and lectotypes exist due to nomenclatural reorganisations of previously different species that are now synonymous with //E. quadricolor//. The list of other type specimens can be found here.

Nomenclature
The majority of sea anemone species described in the eighteenth century or early nineteenth century were originally placed in genus //Actinia// created by Linnaeus in 1767 1]30]. This included //E. quadricolor//, which was originally named //Actinia quadricolor// 1]. There was much uncertainty in the assignment of species and definition of the genus //Actinia// 1]. The definition was later narrowed, and the subgenus //Entacmaea// was created by Ehrenberg in 1834 to encompass fifteen species of predominantly Red Sea and Mediterranean anemones, in which the inner tentacles are longer than marginal ones 1]. //Entacmaea// later became a genus of its own and currently includes two species, //Entacmaea medusivora// and //Entacmaea quadricolor// 31].

Synonyms
Many synonyms exist for this species and the full inventory can be found here. The large number of synonyms could be due to the variable appearance and wide geographical range of //E. quadricolor// 9], together with nomenclatural reorganisations over the years. For example, the 13 species that were previously placed in //Gyrostoma// Kwietniewski, 1897 were moved to //Entacmaea//, rendering //Gyrostoma// a junior synonym 30].

Phylogeny
//Entacmaea// species have yet to be included in phylogenetic studies done within the family Actiniidae, possibly due to the small number of species present. However, the nucleotide sequence of //E. quadricolor// has been used in a study to investigate the systematic relationships among the orders Scleractinia (hard corals), Actiniaria (sea anemones) and Corallimorpharia (corallimorphs) within the subclass Hexacorallia, using nucleotide sequences from the 5'-end of the 28S rDNA 32]. In this study, the DNA sequences were aligned using CLUSTAL V, followed by manual editing. Both parsimony and distance methods (neighbour-joining) were used in the phylogenetic analysis 32]. Scleractinia was found to form a monophyletic group while Actiniaria and Corallimorpharia did not (Fig. 23) 32], suggesting that Actiniaria and Corallimorpharia are more closely related to one another than either one is to Scleractinia.

The composition and evolutionary relationships of the subclass Hexacorallia remains unresolved because analyses have yet to provide a consistent framework for understanding the phylogeny of higher-level groups within Hexacorallia 33]. The three aforementioned orders are particularly problematic because they are morphologically very similar 32]33]34], and the relationships among the three orders are still controversial 33].

Figure 23. Relationships of orders (Scleractinia, Actiniaria and Corallimorpharia) within the anthozoan subclass Hexacorallia (Adapted from: Springer-Verlag in 34], permission pending 64]) Zooming into the order Actiniaria, they are amongst the most diverse members of the subclass Hexacorallia. Relationships within Actiniaria are also unresolved due to insufficient taxon sampling conducted in previous studies to derive consistent relationships 35]. A phylogenetic reconstruction of the order Actiniaria was published in 2014 using three mitochondrial (partial 12S rDNA, 16S rDNA and cox3) and two nuclear (18S rDNA and partial 28S rDNA) markers 36]. The DNA sequences for each marker were separately aligned using MAFFT. Multiple tree optimality criteria were used, namely maximum parsimony, Maximum Likelihood (ML) and Bayesian 36]. In the maximum parsimony analysis, trees of minimum length were found at least five times, data was subjected to 1000 rounds of bootstrap sampling to assess clade support, and gaps were treated as missing data 36] - one advantage of this gap treatment is that trees can be identified solely from the pattern of gaps in an alignment, so treating gaps as missing data may decrease the input of erroneous characters while still producing a reliable phylogenetic tree. However, missing data also has the potential to result in trees that are biologically meaningless, where gaps were treated as missing data when insertion events were more likely to have occured.

In the ML analysis, clade support was conducted using bootstrapping 36]. At the internal nodes of the phylogenetic tree derived from the ML analysis, maximum parsimony ancestral state reconstructions were performed using Mesquite for seven morphological characters - acontia, basilar muscles, deciduous tentacles, endosymbionts, longitudinal ectodermal muscles in the column, marginal sphincter muscle, and nematocysts with apical flaps 36].

Although some nodes had low bootstrap support which allows for ambiguity, the resultant phylogenetic tree derived from the ML analysis of molecular data was found to be consistent with the aforementioned morphological features (Fig. 24) 36]. It was mentioned that the analysis of members in the superfamily Actinioidea, within which //E. quadricolor// is placed, was not extensive relative to the clade diversity 36]. Hence, more phylogenetic work needs to be done to resolve the ambiguity and increase the understanding of higher-level groups, to reveal more information about the phylogeny of //Entacmaea// species within the family Actiniidae.

Figure 24. Construction of morphological characters within Hexacorallia, with red arrow showing position of //E. quadricolor// (Adapted from: Rodríguez //et al//. in 36], under a CC BY 4.0 license 65]). A higher resolution image can be found here.

DNA barcode
The National Center for Biotechnology Information contains nucleotide and protein sequences for //E. quadricolor//, which can be found here.

Glossary
**Nomenclature** - A system or procedure of assigning names to groups of organisms as part of a taxonomic classification. **Phylogeny** - The phylogeny of an organism is the evolutionary history of the organism.
 * Digitiform** - Shaped like a finger.
 * Endosymbionts** - Smaller symbiotic partners living inside a host organism, establishing endosymbiosis.
 * Ectosymbionts** - Partners in a symbiotic relationship that remain on the surface of its host of occupy a body cavity.
 * Chaetodontid** - Tropical marine fish in the family Chaetodontidae (butterflyfish, bannerfish, coralfish).
 * Cnidocyte** - Also known as a cnidoblast or nematocyte, is an explosive cell containing a secretory organelle or cnida/nematocyst.
 * Nematocyst** - Produced by cnidocyte for capturing or paralysing prey or for defense. Each nematocyst contains a coiled, hollow thread that can have barbs or spines and often contains poison.
 * Acetylcholinesterase inhibitor** - A chemical that inhibits acetylcholinesterase enzyme from breaking down neurotransmitter acetylcholine.
 * Broadcast spawn** - Animals release their eggs and sperm into the water for external fertilisation.
 * Waxing crescent moon** - A crescent moon expanding in illumination.
 * Basionym** - The original name on which a new name is based.