The coral crab Fungicola fagei (Decapoda: Brachyura: Cryptochiridae) is recorded for the first time from the Hawaiian Islands, where it was discovered in a previously unknown association with the solitary, free-living mushroom coral Lobactis scutaria (Anthozoa: Scleractinia: Fungiidae). The associated crab species was discovered off Hilo on the island of Hawai‘i, where it appeared to be relatively common. It could have been previously overlooked because of its small size (max. ca. 1 cm long) and its hidden life style inside the host coral. Species identification is based on the morphology of the carapace and use of the cytochrome oxidase subunit I (COI) barcode gene as molecular marker. Fungicola fagei is known from other localities in the Indo-West Pacific region, where it is only hosted by mushroom coral species of the genera Podabacia and Sandalolitha. The record of F. fagei from the Hawaiian Islands represents a northeastward extension of its known geographical distribution range, 6,000 km away from its nearest locality in Vanuatu, despite the availability of host species as eastward as the Cook Islands and French Polynesia. Because these other host species do not occur in the Hawaiian Islands, F. fagei's occurrence here is only possible because of its association with L. scutaria, uniquely recorded from Hawai‘i.
Lobactis scutaria (Lamarck, 1801) is a solitary, free-living scleractinian coral of the family Fungiidae that shows a wide distribution range in the Indo-West Pacific region, from the Red Sea to the Hawaiian Islands, and French Polynesia (Hoeksema 1989, Gittenberger et al. 2011). It is the largest and most common mushroom coral in the Hawaiian archipelago, where it was traditionally known as Fungia scutaria (Edmondson 1946, Maragos 1977, Hoover 1999, Fenner 2005). This species has been used in coral reef research in Kane‘ohe Bay and Waikiki Reef (O‘ahu Island) or in laboratory experiments with specimens taken from those localities, which has resulted in numerous publications on its ecology and physiology over a long period of time (e.g., Edmondson 1929, Stephens 1962, Bosch 1967, Yamazato 1970, Pearse 1971, Jokiel and Cowdin 1976, Krupp 1982, 1983, 1984, 1985, Chadwick 1988, Romano 1988, Krupp et al. 1993, 2006, Jokiel and Bigger 1994, Schwarz et al. 1999, Weis et al. 2001, Hagedorn et al. 2006, Concepcion et al. 2010, Schnitzler et al. 2012). Additional references can be found in the work by Coles et al. (2002).
Despite all the research on Lobactis scutaria in the Hawaiian Islands, limited attention has been given to its associated fauna. For example, Bosch (1965) and Bell (1985) reported on epitoniid snails and their egg capsules found on the undersurface of several L. scutaria corals in Kane‘ohe Bay, O‘ahu, which probably feeds on its host's tissue and can be considered parasitic. They were described as Epitonium ulu Pilsbry, 1921, with Hilo at Hawai‘i as type locality (Pilsbry 1921, Edmondson 1946). Now, this gastropod is classified as Epifungium ulu and considered widespread in the Indo-West Pacific region, where it has been recorded from 11 mushroom coral species (Gittenberger and Gittenberger 2005, Gittenberger and Hoeksema 2013).
Furthermore, Maragos (1977) mentioned that small xanthid crabs find shelter underneath mushroom corals in the Hawaiian Islands. Finally, van der Meij, Fransen, et al. (2015) reported on empty galls of cryptochirids found in museum specimens of L. scutaria from Hawai‘i, which were assigned to Fungicola sp. In an earlier review paper, Hoeksema et al. (2012) reported on two gall crab species, Fungicola fagei (Fize & Serene, 1956) and F. utinomi (Fize & Serene, 1956), in association with L. scutaria, but they did not give specific locality data or references. In a successive, more elaborate study on cryptochirids associated with mushroom corals, van der Meij, Fransen, et al. (2015) mentioned that F. fagei crabs were known to dwell only in Podabacia and Sandalolitha corals and that F. utinomi crabs were hosted by Ctenactis, Danafungia, Fungia fungites (Linnaeus, 1758), Halomitra, Lithophyllon, and Sandalolitha corals, whereas F. syzygia van der Meij, 2015, was predominantly associated with Cycloseris and Pleuractis. Hence, there was no certainty about which gall crab species lived in association with Lobactis in the Hawaiian Islands, and no occurrence records of this association are known from elsewhere.
During exploratory dives at the reef off Leleiwi Beach, Hilo, Hawai‘i, in July 2016, J.E.G.-H. came across numerous mushroom corals of Lobactis scutaria and of another solitary, free-living mushroom coral species, Pleuractis granulosa (Klunzinger, 1879). Individuals of L. scutaria were common, and many of them contained visible injuries (Figure 1). Closer inspection revealed that these injuries were gall crab dwellings and that most of them contained living crabs. This finding offered the opportunity to investigate the identity of the gall crab species associated with L. scutaria, which so far has remained unknown.
materials and methods
Mushroom corals off Leleiwi Beach, Hilo, island of Hawai‘i (19° 44′ 05.6″ N, 155° 01′ 00.1″ W), were observed and photographed in situ at ca. 10–15 m depth (4 July 2016). Individuals containing galls of cryptochirids were investigated more closely. A total of six crabs (two females and four males) was extracted from the corals with the help of forceps and stored in 96% ethanol. The crabs were brought to the laboratory for identification by morphological examination and molecular analyses based on the cytochrome oxidase subunit I (COI) barcoding gene as marker. Close-up photographs of the crabs were made with a stereo microscope (Carl Zeiss Discovery V20) equipped with a camera (Axiocam Mrc5) for stacking photography.
Genomic DNA was extracted using the Macherey-Nagel NucleoMag 96 Tissue Kit and the KingFisher Flex (Thermo Scientific). One leg of a gall crab was used for the DNA extraction and was lysed overnight in Lysis buffer T1 and Proteinase K at 56°C. A polymerase chain reaction (PCR) was carried out by using a M13-LepFol primer mix (M13FLepFolF mix and M13R-LepFolR mix) with an amplification product between the primers of 658 base pairs (bp). M13F-LepFolF primer mix was a mix of M13F-LepF1 and M13FLCO1490, and M13R-LepFolR primer mix was a mix of M13R-LepR1 and M13RHCO2198 (Folmer et al. 1994, Herbert et al. 2004). A PCR amplification of the mitochondrial COI was performed in a volume of 25 μl containing 2 μl DNA template, 2.5 μl of 10x PCR Coralload buffer (Qiagen), 0.5 μl of 10 μM of each primer, 0.5 μl of 2.5 mM dNTP, and 0.25 μl of 5 U/μl Taq DNA polymerase (Qiagen) under standard PCR conditions (initial denaturation at 94°C for 3 min, 40 cycles of: denaturation at 94°C for 15 sec, annealing at 50°C for 30 sec, elongation at 72°C for 40 sec, and a final elongation step at 72°C for 5 min). PCR products were visualized by agarose gel electrophoresis (2% agarose E-Gel, Invitrogen), and suitable amplicons were sequenced by Baseclear Leiden using the M13 primers (forward and reverse sequencing). Four out of six sequences were successful. The raw sequences were edited by using Geneious software (version 8.1.8). For the purpose of molecular identification, a Neighbor-Joining tree was constructed to show the phylogenetic affinities of the crabs from Hawai‘i, along with all three Fungicola species recognized so far (van der Meij, Fransen, et al. 2015). Data are derived from GenBank (NCBI) based on specimens from Southeast Asia and the West Pacific (van der Meij 2015a). Because Fungicola fagei and F. syzygia together form a sister group of F. utinomi (van der Meij 2015a), the latter was selected as outgroup.
The six specimens have been deposited in the collections of the Bernice B. Bishop Museum in Honolulu (bpbm) and Naturalis Biodiversity Center (previously known as Rijksmuseum van Natuurlijke Historie) in Leiden (rmnh). The following catalog numbers were assigned: bpbm-S17057 (one female and one male), rmnh.CRUS.D.57237 (one female), rmnh.CRUS.D.57238 (one male), rmnh.CRUS.D.57239 (one male), rmnh. CRUS.D.57240 (one male). The latter four had sequences that were deposited in Genbank (NCBI) under accession numbers MF973150–MF973153.
Based on the association with mushroom corals (Figure 1), the crabs could be identified at family level as belonging to the Cryptochiridae (Decapoda: Brachyura). The elongated pits were wedged in between the host's septa, with the crabs themselves in perpendicular position (Figure 1f). These dwellings were similar to those made by Fungicola crabs in corals of Cycloseris and Pleuractis (van der Meij, Fransen, et al. 2015: fig. 1). Most crabs lived as a pair consisting of a large female and a small male (Figure 1a, c, d, f), whereas others lived alone (Figure 1b, e). The females were about 8–11 mm long and the males 3–4 mm, a ratio of ca. 3:1 (Figure 2). The upper surface of the female carapace shows a keyhole-shaped sculpture (Figure 2a, b), which is also seen in Fungicola fagei (van der Meij 2015a: figs. 1A, 4A) but not in its congeners.
In the COI-based phylogenetic tree, all four crabs from Hawai‘i clustered with specimens of F. fagei found in corals of the fungiid genera Podabacia and Sandalolitha, which were collected from Indonesia, Malaysia, and New Caledonia (Figure 3). Hence, results of the molecular analysis are consistent with those of the morphological examination, confirming the crab's identity as F. fagei.
Gall crabs belonging to the genus Fungicola were previously unknown from the Hawaiian Islands, except for the listing by Hoeksema et al. (2012). Until now, only four gall crab species (family Cryptochiridae) were previously recognized from the Hawaiian Islands (Kropp 1989, 1990, Castro 2011): (1) Hapalocarcinus marsupialis Stimpson, 1859, associated with corals of the scleractinian family Pocilloporidae; (2) Opecarcinus crescentus (Edmondson, 1925), known from corals of the genus Pavona (Agariciidae); (3) Pelycomaia minuta (Edmondson, 1933), found in corals of the scleractinian genera Cyphastrea (family Merulinidae) and Leptastrea (family incertae sedis); and (4) Utinomiella dimorpha (Henderson, 1906) in association with pocilloporid corals. Hence, F. fagei is the fifth cryptochirid species recorded from the Hawaiian Islands.
The new record is also an addition to the known associated fauna of L. scutaria. Besides the parasitic snail Epifungium ulu, L. scutaria is known to host various other invertebrates. It has been observed to host three barnacle species, an excavating mytilid bivalve, a coralliophiliid snail, and a hydroid (Gittenberger and Gittenberger 2011, Hoeksema et al. 2012, Montano et al. 2015).
Until now, the easternmost record of F. fagei is from New Caledonia and Vanuatu (Figure 4), where it was reported from the corals Podabacia motuporensis Veron, 1990, and Sandalolitha robusta (Quelch, 1886), respectively (van der Meij 2015a; van der Meij, Fransen, et al. 2015). Hawai‘i is about 6,000 km away from the nearest locality of F. fagei at Vanuatu (Figure 4). Other F. fagei host records concern Podabacia crustacea (Pallas, 1766) from eastern Indonesia and northeastern Borneo (Malaysia); Podabacia sinai Veron, 2000, from Palau; and Sandalolitha dentata Quelch, 1884, also from northeastern Borneo. Some of the coral species themselves have more eastward distribution localities, such as P. crustacea in French Polynesia (Hoeksema 1989); S. dentata in the Marshall Islands, Caroline Islands, and French Polynesia (Hoeksema 1989, Hoeksema and Benzoni 2013); and S. robusta in the Cook Islands (Hoeksema 1989). Fungicola fagei may occur at these localities, in which case it has been overlooked, as previously in Hawai‘i. Other easternmost records for Fungicola concern F. syzygia from French Polynesia (van der Meij, Fransen, et al. 2015) and F. utinomi from Guam and Palau (Kropp 1990, Paulay et al. 2003).
The new record presented here for F. fagei is not only unique because of the eastward extension of its known distribution range, but also because L. scutaria is documented as a newly recorded host coral (Figure 1). Extensive surveys of the mushroom coral fauna have been performed in various localities in eastern Indonesia and eastern Malaysia, where L. scutaria is most common on offshore reefs: the Spermonde archipelago, South Sulawesi (Hoeksema 2012a,b); Ternate, Moluccas (Gittenberger et al. 2015); and Kudat and Semporna, Sabah (Waheed and Hoeksema 2013, Waheed et al. 2015). Corals at the same localities were also searched for cryptochirids, but this did not result in host records for L. scutaria (van der Meij and Hoeksema 2013; van der Meij, Fransen, et al. 2015). Unfortunately, we have no occurrence data on F. fagei from localities between Palau / Vanuatu and Hawai‘i. Nevertheless, the Hawaiian crabs appear closely related to those from remote localities (Figure 3), which indicates that they may have been introduced relatively recently.
It is important to note that the population of F. fagei would not have been able to establish and maintain itself in Hawai‘i without a host switch to L. scutaria from Podabacia / Sandalolitha spp., which are absent in the Hawaiian Islands. The reverse is unlikely because then L. scutaria would also have been found as a host in other investigated areas. Because Lobactis is not directly related to Podabacia /Sandalolitha, and elsewhere it is unknown to serve as a host for F. fagei, it is also not likely that this association originated in a shared ancestral host taxon. A host switch is therefore more probable, but it is unclear when and where this could have happened, either in the Hawaiian Islands or in more westward localities. Host switches have also been reported from other crustacean taxa, such as pea crabs (Becker and Türkey 2017), xanthid crabs (Garcia-Hernandez et al. 2016), and palaemonid shrimps (Brinkmann and Fransen 2016, Horka et al. 2016, Hoeksema and Fransen 2017).
Because F. fagei appears to be common in Hawai‘i, it is likely that this association also occurs at other islands throughout the Hawaiian archipelago. The absence of field records despite a long research history concerning its host off O‘ahu is probably due to a lack of focus on coral-associated fauna. Cryptochirid crabs are small and live concealed inside their pits, and therefore they are usually not easily noticed (Hoeksema 2017), except perhaps when they appear in extremely high densities (Hoeksema and van der Meij 2013). Recent research targeting coral gall crabs resulted in the discovery of a number of new species (Badaro et al. 2012, Zayasu et al. 2013, van der Meij 2014a, 2015b, 2017, van der Meij, Berumen, and Pauley 2015) and new host records (van der Meij 2012, 2014b, Hoeksema et al. 2017). Likewise, recent surveys on mushroom coral hosts have also led to new discoveries of associated fauna, including some undescribed species, such as a benthic ctenophore (Hoeksema et al. 2013, Alamaru et al. 2016), a serpulid worm (Hoeksema and ten Hove 2014), a hydroid (Montano et al. 2015), and cryptobenthic fishes (Bos and Hoeksema 2015, 2017). The discovery reported here will undoubtedly be succeeded by new observation records of F. fagei and perhaps of other coral-associated fauna within the Hawaiian archipelago. Because of its common occurrence in Hawai‘i, the abundance of the association between F. fagei and L. scutaria could become the subject of in-depth ecological studies.
J.E.G.-H. is grateful to SeaGrant Puerto Rico for providing travel funds to attend and present at the 13th International Coral Reef Symposium in Honolulu, which also allowed for field work to take place on the Island of Hawai‘i. J.E.G.-H. thanks Anna Baker Mikkelsen, Jean Devine, Nick Vanderzyl, and Zac Higgins for assisting in scuba diving. Last, and wholeheartedly, J.E.G.-H. thanks the Ogata family (Crystal, Skyler, Shani, and Royce) for hosting him at their home and would like to dedicate this work to Skyler Ogata, a true Hawaiian warrior who left us unexpectedly too soon. Holly Bollick (bpbm) and Karen van Dorp (rmnh) assigned museum catalog numbers. Charles Fransen constructed the phylogenetic tree. Kees van den Berg assisted with the microscopic photography. Erik-Jan Bosch made the final version of the distribution map. Three anonymous reviewers provided constructive comments, which helped to improve the article.