The diversity of acorn barnacles (Cirripedia, Balanomorpha) across Thailand’s coasts: The Andaman Sea and the Gulf of Thailand

The acorn barnacle is a sessile crustacean, inhabiting the intertidal areas of tropical and temperate regions worldwide. According to current practices on Cirripedia morphology, shell, opercular valves, and arthropodal characters including cirri and mouthparts are used as a tool for taxonomic classification, and using these characteristics the present study aimed to provide better resolution for the barnacle diversity and geographical distribution within coastlines of Thailand: the Andaman Sea and the Gulf of Thailand. A total of ten species belonging to three families (Chthamalidae, Tetraclitidae, and Balanidae) were identified in this study. Subsequently, five species were newly recorded for the first time from Thailand’s coasts: Newmanella spinosus Chan & Cheang, 2016, Euraphia hembeli Conrad, 1837, Euraphia depressa (Poli, 1795), Tetraclita kuroshioensis Chan, Tsang & Chu, 2007, and Tetraclita singaporensis Chan, Tsang & Chu, 2007. The others, already mentioned in previous records, include: Tetraclita squamosa (Bruguière, 1789), Chthamalus malayensis Pilsbry, 1916, Amphibalanus amphitrite (Darwin, 1854), Amphibalanus reticulatus (Utinomi, 1967), and Megabalanus tintinnabulum (Linnaeus, 1758). Interestingly, acorn barnacles along the Andaman Sea occur abundantly, and are much higher in number of species (up to 8 species) than those found in the Gulf of Thailand’s coast (up to 6 species). This biased trend of species’ preferences is possibly due to the differences in oceanographic nature between two coastlines and the history of barnacle colonization.


Introduction
Acorn barnacles, a member of marine crustaceans, inhabit a diverse array of substrates (e.g.calcareous rock or limestone, mollusk shells, corals, sponges, mangrove roots, turtle shells, and whale skins) along intertidal zones of temperate and tropical coastlines worldwide, as sessile form throughout their adulthood (Frith et al. 1976;Sophia Rani et al. 2010;Brickner and Høeg 2010;Brickner et al. 2010;Chen et al. 2012;Hayashi 2013;Chen et al. 2014;Yu et al. 2016).It is known as a marine fouling or biofouling organism and it has been considered as a problematic or invasive species for oyster farming, aquaculture, the reforestation of mangrove swamps, and for the support structures of offshore oil rig platforms and ship transport (Santhakumaran and Sawant 1991;Rawangkul et al. 1995;Molnar et al. 2008;Sophia Rani et al. 2010;Holm 2012).Although the presence of hard calcareous plates covering acorn barnacles' bodies limits abilities to search for food and new habitats, the species are still tremendously successful in occupying the coastline of tropical and temperate regions due to their free-swimming and planktonic larval stages: high-feeding nauplius and non-feeding cyprid.The nauplius larva develops in successive manner with ecdysis or molting to shed their exoskeleton and allow growth of larva, a characteristic used to classify acorn barnacles into Ecdysozoa of Protostomia clade.The metamorphosis (settlement process) alters a cyprid larva to a sessile juvenile and subsequently an adult form growing inside the ring of shell plates (4-8 in number depending on the species), homologous structure to carapace of other crustaceans (Høeg and Møller 2006;Maruzzo et al. 2012;Martin et al. 2014).The sessile body of adult barnacles has six

Sampling collection
The barnacles were collected from each station by surveying along rocky shores of an intertidal zone during both low and high tides.Whole acorn barnacle individuals were removed from the substratum and immediately preserved in ethyl alcohol (95%v/v) for further examina-tion.All work was done under certified supervision of S.K. (Certificate from Institute of Animal for Scientific Purposes Development-IAD, Royal Thai Government: U1-03104-2559).

Morphology analysis
Samples were primarily identified based on their shell morphology using an Olympus SZ51 stereomicroscope and was photographed with digital camera.For better species identification in some families, arthropodal characters were observed.Soft bodies were removed from the shells and dissected.Cirri and mouthparts were mounted onto slides for light microscopy observation and imaging using digital camera.Taxonomic identification was performed using keys of Newman and Ross (1976) and Chan et al. (2009).The general terminology of shell morphology and the important characters used in this paper follow Chan et al. (2009).All voucher specimens from each station were deposited in the collection of Laboratory of Zoology, Department of Biology, Faculty of Science, Burapha University, Thailand.

Results
Based on shell morphology, total ten species (6 genera) of acorn barnacles along the coastlines of Thailand in both the Andaman Sea and the Gulf of Thailand were identified and are categorized into three families: Chthamalidae (2 subfamilies: Chthamalinae and Euraphiinae), Tetraclitidae (2 subfamilies: Newmanellinae and Tetraclitinae), and Balanidae (2 subfamilies: Amphibalaninae and Megabalaninae).The descriptions of the identified barnacles are as follows:
In this study, the presence of E. depressa in Khao Sam Muk station (Chon Buri) in Thailand was unexpected as it was previously unrecorded along Thailand's coastal areas.They were found along rocky shores exposed to heavy wave action inhabiting sheltered crevices of the rocky platform and high shore.The abundance of E. depressa is much less than that of the cosmopolitan barnacle Amphibalanus amphitrite in the same area of observation.
Remarks.Euraphia depressa (Poli, 1795) was the reassigned name from Chthamalus depressus (Poli, 1795).According to Southward (1964), Euraphia depressa can be distinguished from Chthamalus stellatus, based on the shell morphology showing smooth unribbed shell on the external surface from younger specimens to more adult stage and the operculum characters, showing joints between tergum and scutum without sinous or slightly sinous, and smaller tergum.In addition, a barnacle of the genus Euraphia is distinguished from the genus Chthamalus based on the number of teeth on mandible and as described in Southward (1964) our specimens have mandible with three large teeth and three large setae on the lower edge (Figure 3F & G) and lacking of caudal appendages, leading to species identification of our specimens as Euraphia depressa.However, the number of setae at the pectinated margin of mandible in our specimens is different.Only small 5 setae after larger three setae were found in our specimens while up to 12 setae were mentioned in Southward (1964).
Distribution.Barnacles in the genus Euraphia were recorded in several regions including West Africa, the Mediterranean, Hawaii and Southern Japan (Newman and Ross 1976).Euraphia hembeli was previously recorded in California around San Diego (Barrett and Freeman 2016).In this study, we report the presence of Euraphia hembeli distributing along low and middle shore of the intertidal zone, which was only found at Na Tai station, the Andaman Sea (Tables 2 and 3).In addition, this is the first report of its presence in Thailand.

Remarks.
Based on the shell and opercular valve morphology (Newman and Ross 1976;Kim and Yamaguchi 1996), two candidates: Euraphia hembeli Conrad, 1837 and Euraphia pilsbryi Hiro, 1936 (reassigned as Hexechamaesipho pilsbryi (Hiro, 1936)) show similar patterns of opercular plates to our collected specimens.Based on Newman and Ross (1976)  Description.Peduncle absent; base calcareous.Shell greyish green, shell with 4 plates (1 carina, 2 latus, 1 rostrum); parietes low conical, 3-4 rows of irregular parietal tubes (parietes multiple tubiferous), radii board with horizontal striation and summit oblique.External surface with deep longitudinal/radiating lines from base to apex, internal surface of parietes smooth and white with greyish green striations close to operculum.Orifice pentagonal, diamond-shaped.External surface of operculum brownish grey, internal surface of operculum white.Scutum triangular, external surface of scutum with horizontal striations; tergum high and narrow, tergum with numerous depressor crests.
Distribution.Newmanella spinosus was previously recorded from low intertidal to subtidal levels on rock shores along the coastlines of Taiwan and the Philippines and they were also collected from the surfaces of buoys used in fishing cages in the open sea (Chan and Cheang 2016).In this present study, N. spinosus specifically distributes along low shores, and the intertidal zones of Na Tai beach, Takua Thung District, Phang-nga (the Andaman Sea).
Remarks.N. spinosus is morphologically similar to N. radiata, based on shell and scutum.The shell of N. spinosus is green while those of N. radiata is white.In addition, lateral scutal depressor muscle crest is shallow in the scutum of N. radiata, but deep in N. spinosus.The distribution of N. spinosus is around the North Pacific Ocean, from Okinawan Japan to Taiwan and the Philippines (Chan and Cheang 2016).The presence of N. spinosus in Thailand is surprising in our study, and creates the first record of this species distributing specifically in Na Tai beach, Takua Thung district, Phang-nga province.
Distribution.Tetraclita kuroshioensis is reassigned the name from Tetraclita squamosa which were collected from Taiwan, and Okinawa and Honsu of Japan, and Tetraclita pacifica.The distribution of this species occurs in broad area along north-west Pacific region (Chan et al. 2007a, b;Chan 2009).In this present study, the species distribution occurs along littoral intertidal zones in both the Andaman Sea (Ao Yon, Ao Khoei, Na Tai, and Kalim) and the Gulf of Thailand (Ban Krut and Ko Kham Yai).
Remark.Tetraclita kuroshioensis is quite similar to Tetraclita singaporensis in following characteristics: tergum without beak and with wide spur, scutum with short articular ridge-basal margin.However, angle between tergal margin and basal margin of T. kuroshioensis is more perpendicular (90°) or shaper while that of T. singaporensis is curved.
Description.Peduncle absent; base membranous; shell purplish-dusky green with 4 plates (1 carina, 2 latus, 1 rostrum), parietes conical, plates inseparable, 5-6 rows of parietal tubes (parietes multiple tubiferous), external surface with deep and irregular longitudinal striations from apex to base and small radiating lines, internal surface of parietes smooth and white with greyish-green horizontal striations around aperture.External surface of operculum yellowish brown mixed with dusky green, internal surface of operculum dusky green-purplish and white around spur of the tergum.Scutum bigger than tergum, scutum triangular, short articular ridge-basal margin, external surface of scutum with horizontal striations, occludent margin of scutum with rough teeth.Tergum higher than wide, tergum with broad spur and not beaked, spur angle 30-35°, basi-scutal margin 148-150°.Mandible with 4 big teeth, 2 nd and 3 rd teeth consisting double teeth, 1 st tooth with small spines, lower margin pectinate with 8 small teeth and obvious double bigger teeth at the edge; maxillule notched, two large setae above notch, 13-17 setae below notch; labrum with 4-5 large teeth on each side; cirri I possessing bidenate serrulate setae.
Distribution.Tetracliata singaporensis has been reassigned the name from previously known as Tetraclita squamosa, which were collected from Singapore.Hence, the distribution of this species is firstly marked at Singapore, Indo-West Pacific region (Chan et al. 2007a).In this present work, the specimens were collected from Na Tai, Andaman Sea and it distributes in the mid shore.

Remarks. Tetraclita singaporensis differs from
Tetraclita squamosa in that it has tergum without beak and broader spur, and scutum with short articular ridge-basal margin.
Remark.As described in Chan et al. 2007a, b, T. squamosa (southern China) has unique tergum characteristics including tergum with beak and long spur, and scutum with long articular ridge-basal margin.Our specimens  from Hin Ngam beach have all of these characteristics; hence, it is more fitted into T. squamosa (Southern China) rather than T. squamosa (Singapore), which is reassigned as T. singaporensis.
Distribution.Amphibalanus amphitrite is a common fouling barnacle and cosmopolitan species distributed along intertidal zones of coastlines in both the Gulf of Thailand and the Andaman Sea.It was found in all stations examined.The settlement patterns are various (e.g.rocks, shells of oyster and green mussels, concrete walls of bridges and harbors, offshore vessels, dock pilling, and mooring robes).In previous records, this species distributes worldwide in both tropical and temperate regions including the Indo-West Pacific, and Western Australia (Jones 2004;Chen et al. 2014) and it has been suggested that this wide range of distribution was due to human-mediated activities during global trade expansion (Chen et al. 2014).
Remark.The morphology of Amphibalanus amphitrite is variable from diverse habitats worldwide.Shells exposed and eroded by heavy wave action showed no purple stripes on the external surface.The molecular analysis has confirmed its genetic differentiation which might be due to local adaptation and geographical isolation (Chen et al. 2014).Due to hypothesis on human-mediated activities as the main cause of A. amphitrite's distribution across the globe, this species is considered as non-native or introduced species in these examined regions: Hawaii, California, North Carolina, and the Atlantic coast (Carlton et al. 2011), whereas it is considered as native in tropical waters (e.g.Hong Kong, Thailand, Malaysia) supported by molecular study (Chen et al. 2014).Despite the diverse morphology of A. amphitrite, another species in the same genus Amphibalanus reticulatus exhibits clear patterns of shell carrying both vertical and longitudinal striations on the external surface.The separation of settlement type is distinct between these two species; one is found mostly on rocky shores exposed to waves and the other one is found on some mollusk shells.
Distribution.Amphibalanus reticulatus is widely distributed from Japan, the Indo-West Pacific to Australia, of which the latter is considered as an introduced species carried by ship transport (Jones 2004).In this study, A. reticulatus occurred in the intertidal zone along the Andaman Sea and the Gulf of Thailand.These specimens were found at Si Racha, Khao Sam Muk, Ko Kham Yai (Chon Buri) and Na Tai (Phang-nga).
Remark.Amphibalanus reticulatus exhibits clear vertical and horizontal striations while Amphibalanus amphitrite shows only vertical purple striation in all shell plates.In addition, the shapes of shell of A. reticulatus is more columnar than that of A. amphitrite, which might be due to elongation of parietes in response to crowding when growing as colonies.On all examined stations, distinct distribution and settlement between A. amphitrite and A. reticulatus can be noticed, in that A. amphitrite were found in almost all kinds of substrates but A. reticulatus preferred its attachment on shells which obviously did not live along the rocky shores and it might probably inhabit the deeper areas of the sea and were occasionally carried away into the shores by wave action.
Distribution.Megabalanus tintinnabulum is widely distributed across almost all continents and is a well-known cosmopolitan fouling species.It was previously found in French Guiana, the United States, Australia, Mexico, Ecuador, Kuwait, Saudi Arabia, Sweden, France, Netherlands, Singapore, Indonesia and India (Henry and Mclaughlin 1986;Thiyagarajan et al. 1997;Jones et al. 2000;Jones 2004).Similar to Amphibalanus, it is considered as an introduced species in several regions and its distribution has been facilitated via shipping (Jones 2004).In Thailand, M. tintinnabulum specifically occurs in the low shores at Na Tai beach, Phang-nga province (the Andaman Sea).However, it does not appear to be a common fouling species as seen in some regions.M. tintinnabulum might have been introduced to Phang-nga beaches via ship transport, and the competition for habitat niche is compromised, compared to previously occupying cosmopolitan A. amphitrite.
Remarks.Megabalanus tintinnabulum has relatively larger shell plates than those of Amphibalanus.All three examined species (M.tintinnabulum, A. amphitrite and A. reticulatus) in family Balanidae have opercular valves with prominent growth ridges horizontally, and tergum with a clear spur.The coloration among these three species is easily distinguishable, in that purplish longitudinal striations presenting A. amphitrite, vertical and longitudinal red-orange striations with orange-pale pink background presenting A. reticulatus and brownish red surface with some irregular and unclear longitudinal stripes presenting M. tintinnabulum.
Identification key

Discussion
In the present study, we examine geographical distribution of sessile acorn barnacles along Thai Peninsular coastal areas including the Gulf of Thailand and the Andaman Sea.So far, there has been a lack of information regarding the diversity of sessilian Thoracican barnacles in Thailand.Hence, we attempt to generate a checklist to understand the species diversification and how they distribute on intertidal rocky shores and sandy shores along the coast of Thailand.At least ten different forms of acorn barnacles were diagnosed so far that are classified into 6 genera and 3 families (Chthamalidae, Tetraclitidae and Balanidae), which can be distinguished based on their external shell morphology, including pattern of parietes, opercular plates, and arthropodal characters as described in previous literatures (Ross and Perreault 1999;Chan 2001;Chan et al. 2007a, b;Chan et al. 2009;Lozano-Cortés and Londoño-Cruz 2013;Chen et al. 2014;Hayashi and Chan 2015;Chan and Cheang 2016).
Our study also shows that the numbers of species found in the Andaman Sea (8 species) are more than those found in the Gulf of Thailand (6 species).At Na Tai station located in the Andaman Sea, up to 8 species (6 genera and 3 families) were recorded.Four of these 8 species were found only at this station including Newmanella spinosus, Euraphia hembeli, Megabalanus tintinnabulum and Tetraclita singaporensis.In other examined stations, only 2-3 species could be found, and most of them were of the genus Amphibalanus, Tetraclita, and Chthamalus.The differences in species abundance between two coastlines might probably due to the past history of the barnacle colonization.It has been shown in Voris (2000) that sea level was fluctuated during the Pleistocene, caused by glaciation.The spread of acorn barnacle seen in present day is possibly due to successful colonization when there were the connections between the eastern part of Indian Ocean and the Gulf of Thailand.The nature of local habitats such as the incoming oceanic current and freshwater discharge might also be another factor promoting or limiting the boundary of barnacle distribution.In addition, the spread of the barnacles found in the Gulf of Thailand was probably facilitated by the influence of the South China Sea Warm Current (SCSWC) as shown in the case of Chthamalus malayensis (Tsang et al. 2012).However, at this present work, we cannot clearly conclude that all of these species found in this work successfully distributed before the glaciation or influenced by nature of specific local habitats as further extensive works need to be done to include more stations along both coastlines with proper oceanographic data.
In addition, we found five new records identified as Newmanella spinosus, Euraphia depressa and Euraphia hembeli, Tetraclita singaporensis, and Tetraclita kuroshioensis on which the presence of these species in Thailand has not been mentioned in any literatures.N. spinosus, E. hembeli and T. singaporensis can only be seen at Na Tai station, Phang-nga province while E. depressa is specific to Khao Sam Muk, Chon Buri province.However, we cannot rule out the possibility of their presences in other places and more intensive field surveys covering all provinces along Thailand's coasts are required.
Recently, there are 26 species in the genus Chthamalus (Chan et al. 2009).In this study, one of them is clearly diagnosed as Chthalamus malayensis based on distinct shell, operculum morphology and arthropodal characters.However, we also found another Chthamalid which has shallow articulation of tergum and scutum; suggesting the possibility of a different species.Surprisingly, this Chthamalids is similar to Chthamalus depressus (reassigned as Euraphia depressa), described in Southward (1964).The presence in Thailand was not mentioned as they were thought to be found around the Mediterranean.In addition, the Chthamalids we found exhibit great variation and this has previously been reported that Chthamalids have high intraspecific variation in external morphology (Helmuth et al. 2006;Hawkins et al. 2008) and thus using shell morphology is not ideal for taxonomic identification; thus several studies have used other measures for species diagnosis, including opercular plate geometry (Tsang et al. 2012), light microscopy and SEM of arthropodal characters (e.g. the number of conical spines and the number of setules of the basal guard setae on cirri and pattern of oral cones) (Miller and Blower 1989;Southward and Newman 2003;Yan and Chan 2004;Tsang et al. 2012) and molecular approaches (Tsang et al. 2012).In any future studies, we will use all of these measures, particularly the examination of mitochondrial COI, 12s rDNA, 16s rDNA sequences or performing DNA barcoding in order to get accurate identifications of chthamalid barnacles.
According to a field survey on water quality and metal contamination of both coastal regions of Thailand, the Andaman Sea is still in a good condition compared to the Gulf of Thailand.On the other hand, habitat degradation along the Gulf of Thailand is much more severe and the number of species of these sessile arthropods has been declining dramatically over the last 20 years due to high amount of water pollution.For example, along Chon Buri's coast around 20 years ago, at least five species were commonly seen along rocky shores of the now developing centrum area.Recently, however, only Amphibalanus amphitrite have been able to tolerate severe human activities and even in some sites there are no more barnacles on rocky shores.This might be because the local communities have been releasing non-treated waste water directly into the sea (personal communication and unpublished report (1996): Department of Biology, Faculty of Science, Burapha University).Hence, the richness of barnacle species can also be used to indirectly monitor the conditions of sea water.
Taken together, we demonstrate a clearer view of diversity for acorn barnacles from various localities in Thailand.This study shows at least 10 species of barnacles, in total, exist along Thai coast regions.Future works with more sampling sites and further in-depth investigations using SEM and molecular approaches with the help of phylogenetic analysis will provide a much better view especially of the history of barnacles and intraspecific variation between sessile crustaceans and that may reveal new barnacle species inhabiting Thailand.

Figure 3 .
Figure 3. Euraphia depressa collected from Khao Sam Muk beach, Chon Buri (BUU16.CH.ED01).A. Dorsal and ventral view of external shell, B. External (left panel) and internal (right panel) view of tergum (upper panel) and scutum (lower panel), C. External (upper panel) and internal (lower panel) view of shell plates, D-G.Light microscopy on mouthparts, D. Labrum, E. Close up on the teeth of the labrum, F. Mandible with three large teeth, G. Close up on the pectinated lower margin of mandible.D-G.Scale bars in µm.Abbreviations: c, carina; cl, carinal latus; l, latus; r, rostrum.

Figure 6 .
Figure 6.Tetraclita kuroshioensis collected from (BUU16.TC.TK01) from Na Tai beach, Phang-nga.A. Dorsal and ventral view of external shell, B. External (left panel) and internal (right panel) view of tergum (upper panel) and scutum (lower panel), C-F.Light microscopy on mouthparts, C. Labrum, D. Close up on the teeth of the labrum, E. Mandible, F. Close up on the lower margin of mandible.C-F.Scale bars in µm.

Figure 7 .
Figure 7. Tetraclita singaporensis collected from (BUU16.TC.TSG02) from Na Tai beach, Phang-nga.A. Dorsal and ventral view of external shell, B. External (left panel) and internal (right panel) view of tergum (upper panel) and scutum (lower panel), C. Lateral side showing external surface of shell, D. Close up on the external surface of shell, E-H.Light microscopy on mouthparts, E. Labrum, E. Close up on the teeth of the labrum, G. Mandible, G. Close up on the pectinated lower margin of mandible.E-H.Scale bars in µm.

Figure 8 .
Figure 8. Tetraclita squamosa collected from Hin Ngam beach, Nakhon Si Thammarat (BUU16.TC.TS01).A. Dorsal and ventral view of external shell, B. External (left panel) and internal (right panel) view of tergum (upper panel) and scutum (lower panel), C. External (upper panel) and internal (lower panel) view of shell plates, D. Close up on external surface of shell, E-H.Light microscopy on mouthparts, E. Labrum, E. Close up on the teeth of the labrum, G. Mandible, G. Close up on the pectinated lower margin of mandible.E-H.Scale bars in µm.Abbreviations: c, carina; l, latus; r, rostrum.

Table 1 .
Sampling locations, arranged from north to south.

Table 2 .
Species list and distribution of acorn barnacles found in ten sampling sites along the coastlines of the Andaman Sea and the Gulf of Thailand.Abbreviations: +, presence; abs, absence.See Table1for acronyms of sampling sites.

name Habitat type Settlement pattern on habitats LS MS HS Family Chthamalidae
large setae at the edge; labrum with obvious teeth; caudal appendage absent.