An annotated checklist and integrative biodiversity discovery of barnacles (Crustacea, Cirripedia) from the Moluccas, East Indonesia

Abstract To contribute to the taxonomic knowledge of barnacles in this understudied area, the first checklist of barnacles from the Moluccas is presented, including additional information on morphology, distribution, and substrate as well as molecular data. The species of barnacles from the Moluccas have been determined using morphological analysis and DNA sequences. During 19 field trips conducted between January 2016 and September 2017, 1,513 specimens of 24 species of intertidal and one species of deep-sea barnacles were collected from 51 localities from the islands. Morphological and molecular analysis of the collected material detected members of three families of stalked barnacles and four families of acorn barnacles. In addition to sampling in the field, we also surveyed the literature on barnacles from the Moluccas. In total, our checklist comprises 97 species from the Moluccas including 23 new records, two of them yet to be described species. Results suggest that the Moluccas have a much higher diversity of barnacles than previously known, for example, from the reports of Challenger and Siboga expeditions. For further work, routine application of molecular systematics could aid the detection of cryptic species, while increased sampling of more islands and a taxonomic revision of several groups would likely lead to an even higher number of species than currently known.


Introduction
Barnacles (Crustacea, Cirripedia) are an ancient, species-rich and abundant group of crustaceans with about 1,400 extant species (Newman and Abbott 1980). They have a worldwide distribution in tropical and temperate marine environments and at different depths and are adjusted to various lifestyles, from parasites of decapod crustaceans to free-living groups. Most cirripeds usually have two free-swimming planktonic larval stages consisting of distinctive nauplii and a unique non-feeding cyprid (Darwin 1852(Darwin , 1854Pochai et al. 2017). In the most abundant group, the Thoracica, adult specimens are permanently attached to various types of substrates, other living organisms (e.g., mangroves, corals, molluscs, other barnacles, sponges), rocks, and man-made materials such as cargo ships and concrete walls (Newman and Abbott 1980;Power et al. 2010). The Thoracica comprise the orders Cyprilepadiformes, Ibliformes, Lepadiformes, Scalpelliformes, and Sessilia (Buckeridge and Newman 2006). The Indonesian Moluccas (or Spice Islands; Fig. 1) are part of the Coral Triangle, one of the most complex biogeographical and oceanographic areas on Earth. Although part of the global epicentre of marine biodiversity, knowledge of the barnacle fauna of the Moluccas is relatively sparse. The exploration of the natural history of the Moluccas dates back to the 17 th century, starting with Georg Everhard Rumphius, and later, for example, Alfred Russel Wallace (Strack 1993;Lamoureux 1990). In the 19 th and 20 th centuries, there were approximately fifty scientific expeditions passing through or specifically targeting the area, such as the British Challenger (1872-1876), the Dutch Siboga (1899)(1900) and the Snellius (1929)(1930) expeditions (Lamoureux 1990). The most recent being the French Karubar expedition in 1991 (Crosnier et al. 1997).
Rumphius provided the first record of a barnacle (the stalked Mitella Oken, 1815 (= Capitulum Gray, 1825) found on a rock near the beach at Ambon Island) in his posthumously published 'Amboinsche Rariteitkamer' (Rumphius 1705). Indonesian and Moluccan barnacles were also studied by Darwin (1854), who assigned them to one of four geographical 'provinces', the third being the East Indian Archipelago. Moluccan barnacles have not been studied since Buckeridge (1994) examined some material from the Karubar expedition.
To contribute to the taxonomic knowledge of this understudied area, we herein present the first checklist of barnacles from the Moluccas, including additional information on morphology and molecular data, as well as distribution and substrate.

Sampling
Specimens examined in this study were collected by the first author during 19 field trips between January 2016 and September 2017 to the intertidal zones of the Moluccan islands of Ambon, Saparua, Seram, Pombo, and Banda Neira (Fig. 1, Table 1, Suppl. material 1: Table S1). Deep-water barnacles (Table 1) from the Lifamatola Passage (250 m) and Halmahera Sea (250 m) were provided by Nurul Fitriya. Additional material used for the molecular analyses was collected from the island of Sulawesi in September to October 2017 (Suppl. material 1: Table S1). In total, 159 lots containing 1,513 specimens were collected from 51 Moluccan localities.

Morphological analysis
For detailed morphological analyses, all samples were studied at the Museum für Naturkunde in Berlin (ZMB), Germany. All specimens are deposited at the Museum Zoologicum Bogoriense (MZB; Suppl. material 1: Table S1), Research Center for Biology, Indonesian Institute of Sciences-LIPI, Indonesia. Barnacle species attached to other barnacle species were not separated (except for specimens dissected and measured) but were kept within the same glass container, enabling further morphological studies of different species attached to each other, e.g., with MicroCT scans. Specimens were studied by the first author. All species were determined based on external shell morphology, including the pattern of the parietes, opercular plates, mouth parts, and arthropodal characters, as described by Darwin (1852Darwin ( , 1854, Hoek (1907Hoek ( , 1913, Southward and Newman (2003), Chan et al. (2007), Pérez-Losada (2008, Chan et al. (2009a), Tsang et al. (2015), and Chan et al. (2017).
Hard body parts (parietes and opercular plates) were separated from soft body parts using a scalpel. Shell plates were separated and cleaned with a bleach solution to remove any organic material, rinsed with fresh water, dried and observed under a stereo microscope (Leica M125) and photographed with a digital camera (Leica Microsystems M205C and Leica Z16 APo-A) (Fig. 2).
The mouthparts (labrum, palps, maxilla, maxillule, and mandible) were dissected using a scalpel, each was mounted on a glass slide and examined under a light microscope (Axioskop 20). The cirri were separated into couples of cirri I-VI and the penis, before being mounted on glass slides. The anatomy of these soft body parts was studied using a light microscope.
All measurements were made using digital callipers (accurate to 0.1 mm; Suppl. material 1: Tables S2-S26) generally following the method described in Beşir and Çınar (2012): basal length of shell, basal width, orifice length, orifice width, and carinal height. For stalked cirripeds, measurement of total height, capitular height, diameter of the base of the capitulum, carina and scutum distance, scutal length, scutal width, tergal length and tergal width were taken following the method described by Igić (2007). For deep-water barnacles, capitular height, capitular width, peduncular length, orifice height, number of crests, capitular thickness, and peduncular width were taken following the method described by Chan et al. (2009b).

Molecular phylogenetic analyses
We performed molecular phylogenetic analyses including new DNA sequences from our new samples from the Moluccas in combination with sequences of multiple barnacle specimens retrieved from GenBank. Our aim with the molecular analyses was not to provide a robust phylogeny of barnacles or to develop DNA barcodes for Moluccan barnacle taxa. Instead, our goal was to confirm the molecular taxonomic identity of the barnacles from the Moluccas with the published sequences in the GenBank, to examine whether they cluster near to congeneric or conspecific accessions. With this exercise we aimed to gain insights into the taxonomic positions of Moluccan barnacles in addition to those we may gain from morphology.
Genomic DNA was extracted from the adductor muscle tissue using CTab isolation buffer following the method of Doyle and Doyle (1987), as described at http:// www.geocities.com/ CapeCanaveral/8431/CTab.html. Tissue was ground and placed in 200 μl CTab buffer and 5 μl Proteinase K, homogenized by shaking, and incubated at 56 °C overnight. DNA was extracted from the lysate using a Qiagen BioSprint 96 using the manufacturer's protocol. The purified DNA was stored at -20 °C until required, and dilutions of 1 to 10 were used for the polymerase chain reaction (PCR).
Chromatograms were edited using CodonCode Aligner version 5.1.5 (http:// www.codoncode.com) for COI and Geneious 11 (http://www.geneious.com) for 18S. All new DNA sequences generated for this study are deposited in GenBank under the accession numbers provided in Suppl. material 1: Table S1. For comparison, 84 COI sequences and 88 18S sequences of related barnacles were downloaded from GenBank (accession numbers are provided in Figs 28, 29). Sequences were aligned using Muscle (Edgar 2004) as implemented in Geneious and later exported as nexus or fasta files.
Phylogenetic trees were reconstructed for each gene using both Maximum Likelihood (ML) and Bayesian Inference (BI). ML analyses were conducted with RAxML Black Box (Stamatakis et al. 2008) with 100 bootstrap replicates and under the GTR + I + G model of sequence evolution. Bayesian analyses were conducted in BEAST 2. As our goal was to obtain a topology and not dates for the branching events, we used a Bayesian relaxed lognormal clock with a rate of 1. Therefore, the ages obtained in the ultrametric trees emerging from this analysis are relative not absolute. Substitution model selection was performed in jModeltest (Posada 2008) using the Akaike information criterion, and GTR + I + G was identified as the best model for both genes. For each analysis, we ran two independent chains of between 10 and 40 million generations, with a birth-death tree prior. Convergence of chains and burn-ins were assessed with Tracer, runs combined using LogCombiner, and maximum clade credibility trees produced in Tree Annotator.

Checklist tabulation
This study provides the most comprehensive overview of barnacle species from the Moluccan islands (Table 1). The morphological analyses of the collected material revealed 24 intertidal species and one deep-water species from three families of stalked barnacles (Heteralepadidae: one genus and species; Lepadidae: two genera and species; Pollicipedidae: one genus and species) and four families of acorn barnacles (Pachylasmatidae: one genus and species; Chthamalidae: five genera and species; Tetraclitidae: five genera and eight species; Balanidae: two genera and seven species).
Including previous records from the literature, we found a total of 97 species from the Moluccan islands (Table 1) from the superorder Thoracica (free living or epizoic). Of these, 21 are new records and two (Amphibalanus sp. and Microeuraphia sp.) are currently unidentified species.
All specimens obtained from field work, except for one floating specimen, were attached to types of natural and artificial substrates (Table 1), and several smaller species, e.g., Chthamalus moro, were also attached to other larger barnacle species, e.g., Megabalanus tintinnabulum. In general, the smallest species was C. moro  Diagnosis. Capitulum rounded without hard valves and opercular plates, wall of capitulum tick with crest not more than two on the carinal region; cirrus I with filamentary appendage at the basal region; anterior rami shorter than posterior rami in cirri V-VI; caudal appendage present; maxillule strongly notched.
Description. Orifice slightly protuberant, crenulated, occupying one half to one third capitular length, parallel to or at oblique angle to capitulum; integument thick, chitinous; carinal margin sometimes with warty protuberances on slight keel; peduncle naked; colour of capitulum and peduncle yellowish (Fig. 3a-c). Cirrus I with anterior rami (19-segmented) shorter than posterior rami (25-segmented) and a filamentary appendage present at the basal region (Fig. 3d); cirri II -IV long, slender, anterior rami of cirri V and VI shorter than posterior rami; cirrus VI has a caudal appendage with 20-segmented and one fourth length of anterior ramus (Fig. 3e). Mandible with four large teeth excluding inferior angle (Fig. 3f ); maxillule strongly notched with two big teeth on upper angle and blade-shaped setae on cutting margin (Fig. 3g); labrum concave, teeth numerous. Ranges of height of capitulum 11.9-18.6 mm, width 8.7-15.3 mm, thickness 7.0-13.3 mm; length of peduncle 7.7-27.6 mm and width 5.1-10.4 mm (measurements for 25 specimens are presented in Suppl. material 1: Table S2).
Distribution. Heteralepas japonica is widely distributed in Indo-west Pacific: Indian Ocean; Australia; Singapore, Malacca Str., Indonesia; Malay Archipelago; Vietnam; Condor Island; S China Sea; E China Sea; Taiwan, Philippines; S Japan; NE New Zealand; fouling hard rock substrata, crabs, gorgonians, antipatharians, deep-sea cables; 48-500 m (Jones and Hosie 2016). In this study, Heteralepas japonica was found attached to cable moorings in Lifamatola Sea and Halmahera Sea (a map with the occurrence of Heteralepas japonica in the Moluccas is shown in Suppl. material 1: Fig. S1).
Remarks. The external appearance of this species is extremely variable (Nilsson-Cantell 1927). After an extensive study of H. japonica, Nilsson-Cantell could not dis- tinguish H. japonica and H. indica (Gruvel, 1901) and placed the latter in synonymy with H. japonica, and later authors have followed this suggestion (e.g., Broch 1931, Utinomi 1958. At the same time, Nilsson-Cantell (1927) also suggested that H. nicobarica Annandale, 1909, H. gigas Annandale, 1905and H. cygnus Pilsbry, 1907 could be invalid species and future revision may synonymize some or all of them. Zullo and Newman (1964) pointed out the uncertainty surrounding the status of several of the species assigned to Heteralepas due to a lack of zoogeographic and morphological data, since extensive collections are unavailable. Furthermore, Foster (1978) suggested that a revision of the genus was called for since the variability of H. japonica, as noted by Foster and tabulated by Nilsson-Cantell (1927), encompasses characters which have been used to distinguish several different species by other authors (e.g., H. dubia Broch, 1922, H. cornuta Darwin, 1852, H. indica Gruvel, 1901, H. lankestri Gruvel, 1900. Diagnosis. The only pelagic barnacle with its own gas-filled float; plates very thin and paper-like; carina angle bent with a prominent umbo and expanded basal disk; cirri acanthopod. Description. Five capitular plates, white, thin, delicate, wide interspaces between dark purple; base of carina almost round, not imbedded in membrane, distinct angle formed at sub-central carinal umbo peduncle short, naked (Fig. 4a); five filamentary appendages located at base of cirri on each side of body; caudal appendages small, smooth, summits rounded; mandible with five teeth (Fig. 4c); penis hirsute. Ranges of diameter of capitulum base 4.0-6.8 mm; capitular height 10.5-17.1 mm; total height 11.7-19.5 mm; scutal width 5.4-7.3 mm; scutal length 8.1-11.1 mm; tergal width 2.7-4.4 mm; tergal length 6.2-11.1 mm (measurements for six specimens are presented in Suppl. material 1: Table S3).
Distribution. Dosima fascicularis is cosmopolitan in tropical and temperate seas (Jones and Hosie 2016). While it has been reported found at New Zealand, South Africa and South America (Newman and Ross 1971). In the present study, D. fascicularis was found at Ambon Island at Tial (floating in water at the beach) (a map with the occurrence of Dosima fascicularis in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Dosima fascicularis is the only pelagic barnacle that produces its own gas-filled float enabling it to sustain itself on the sea surface (Weisbord 1979). Dosima can also be distinguished from members of the genus Lepas by the distinct angle formed at the sub-central umbo of the carina, and by very thin and brittle plates (Hinojosa et al. 2006).   Diagnosis. Capitulum with five completely calcified plates; surfaces striated with radiating lines; scuta with conspicuous growth lines; scutal margin of terga without notch, occluding margin of scutum strongly convex and swollen; carina apex extending to tergum, base of carina forked; filamentary appendages and caudal appendage present.
Distribution. Lepas anserifera is a cosmopolitan, pelagic species occurring in tropical and temperate oceans (Jones et al. 2001). In this study, Lepas anserifera was found on the islands of Ambon (at Suli, Tial, Galala, Laha), Pombo, Seram (at Lepas Pantai Kawa, Desa Murnaten, Desa Kasie, Dermaga Pelita Jaya), and Saparua (at Negeri Mahu). Lepas anserifera was found attached to mangroves, stone ship charts and ship walls, port poles, and shells of Megabalanus zebra (a map with the occurrence of Lepas anserifera in the Moluccas is shown in Suppl. material 1: Fig. S3).
Distribution. Darwin (1852) reported Capitulum mitella from the Philippine Archipelago, Ambon, East Indian Archipelago and Madagascar. Chan et al. (2009) and Jones and Hosie (2016) reported the species as widely distributed in warmer parts of the Indo-Pacific region, from Madagascar to southern Japan. In this study, C. mitella was found on the islands of Ambon (at Ureng, Alang, Dermaga Liang, Asilulu, Doc. Tawiri, Morella, Wakasihu, Laha, and Tulehu) and Saparua (at Benteng Duurstede and Teluk Saparua). Capitulum mitella attach on rocks, stone, wall of fortress, port pole and concrete wall (a map with the occurrence of Capitulum mitella in the Moluccas is shown in Suppl. material 1: Fig. S4).
Remarks. Capitulum mitella is the famous Japanese goose barnacle or 'kame-no-te' (meaning the hand of the turtle, referring to its shape). This barnacle is edible and sold as an expensive seafood in Japan, China, Taiwan, and Korea, as well as in Portugal and Spain, where it is known as 'percebes'. Diagnosis. Shell with eight plates; compound rostrum; scutum and tergum fused; mandible tridentate; multi-jointed caudal appendage present.
Distribution. Pseudoctomeris sulcata was previously recorded from southern Japan, China, and Taiwan (Jones et al. 2001;Poltarukha and Zevina 2006). In this study, P. sulcata was found on Ambon Island at Leahari and Hatu on rocks and shells of Tetraclita squamosa (a map with the occurrence of Pseudoctomeris sulcata in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Externally, the fused rostrum and rostrolaterals are six-plated, but the sutures are visible internally (Poltarukha 1996). Morphologically, Pseudoctomeris sulcata shows features of the scutum and tergum similar to those of representatives of the family Pachylasmatida. However, the species can be distinguished by its tridentate mandible and the presence of multi-jointed caudal appendages (Poltarukha 2006). A previous molecular study showed that P. sulcata clustered together with members of the family Pachylasmatidae, not with members of the Chthamalidae (Chan et al. 2017). According to Chan et al. (2017), P. sulcata is an intertidal species of the Pachylasmatidae, previously believed to be an exclusive deep-sea taxon. Description. Surface of parietes grey or light brown in colour and spotted with black; orifice rhomboidal ( Fig. 8a); basis calcareous; scutum and tergum strongly articulated, forming sinuous line; scutum elongated, triangular, tergal margin strongly articulated tergum narrow, basi-scutal angle almost 90° (Fig. 8b-c); cirrus II with multicuspid setae; mandible with three large teeth (Fig. 8f ), labrum with row of large teeth. Basal length 8.9-17.0 mm, basal width 10.3-16.4 mm, height 1.0-3.7 mm. Orifice length 4.2-6.9 mm, orifice width 3.6-5.6 mm (measurements for ten specimens are presented in Suppl. material 1: Table S7).

Superfamily
Distribution. Previously, Hexechamaesipho pilsbryi was reported from Japan (Honshu, Shimoda, Wakayama, Okinawa); Taiwan (Turtle Island, Da Xiang Lang, Shi Ti Ping, Kenting); Philippines (Puerto Galera, Tiwi-Bicol, Boracay); Malaysia (Nexus Beach, Kota Kinnabalu, Sabah) (Tsang et al. 2013). In the present study, H. pilsbryi was collected from Hatu and Hila on Ambon Island (a map with the occurrence of Hexechamaesipho pilsbryi in the Moluccas is shown in Suppl. material 1: Fig. S2). A previous study of H. pilsbryi indicated that the distribution of this species bridges the junction of the Japan region and the Indo-Polynesian province of Briggs (Briggs 1974). Molecular results of H. pilsbryi analysed by Tsang et al. (2013) suggested that this species can be divided into two highly diverged lineages: (1) a northern lineage, predominantly distributed in Japan and Okinawa, and (2) a southern lineage, primar-ily distributed in Taiwan and Southeast Asia. Assuming that we have molecular data of H. pilsbryi in our samples, there is a probability that our samples from the Moluccas include members of the southern lineage.
Remarks. Hexechamaesipho pilsbryi was first identified from Japan as Chthamalus pilsbryi Hiro, 1936. However, due to the presence of three large teeth on the mandible, a characteristic of the subfamily Euraphiinae, the species was placed in the genus Euraphia (Nilsson-Cantell 1921). Later, due to the presence of multicuspidate setae on cirrus II, Poltarukha (1996) moved E. pilsbryi to the sub-family Notochthamalinae and determined a new genus, Hexechamaesipho, which had six parietes and a deeply interlocking scutum and tergum. Currently, H. pilsbryi is the only species in the genus.
Distribution. Nesochthamalus intertextus is known from islands in the West and Central Pacific Ocean -Indonesia, New Guinea, Malaysia to Vietnam; China; Taiwan; Philippines; Japan; Hawaii; Pitcairn I (Pope 1965;Newman and Ross 1976;Chan et al. 2009;Jones and Hosie 2016). In this study, N. intertextus was found on Ambon Island at Laha and Hila on stone (a map with the occurrence of Nesochthamalus intertextus in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Nesochthamalus intertextus can be distinguished by the conspicuous interlocking pattern exhibited by the parietal sutures and features of the basis, which is membranous in young specimens but becomes secondarily calcified with age, leaving a membranous centre only (Poltarukha 2008;Pope 1965 Diagnosis. Shell with interlocking teeth between plates; base with a true calcareous and complete secondary calcification; scutum higher than wide and interlocked but not concrescent with tergum. Description. Shell with six plates, parietes symmetrical, calcareous, solid, separable, due to coarsely serrate sutures with interlocking toothed structure (Fig. 10a-c); colour yellowish or brownish grey, inner surface of parietes smooth, white with dark brown and pale violet horizontal striations around aperture; external surface of shell irregularly ribbed around basal margin, basis calcareous; orifice rhomboidal; tergum and scutum separable; scutum triangular, occluding margin with strong teeth; tergum strongly marked with 10-12 lateral depressor crests, scutal margin strongly articulated. Measurements for one specimen are presented in Suppl. material 1: Table S9.
Distribution. Euraphia hembeli has been recorded from the Mediterranean, West Africa, Indian Ocean: Ceylon; Andaman Sea, Cocos-Keeling Islands; Malay Archipelago (Sunda Islands); Pacific Ocean (Japan; Caroline Islands; Hawaiian Islands, California (Newman and Ross 1976;Jones 2012;Barrett and Freeman 2016;Pochai et al. 2017). In this study, Euraphia hembeli was found on Ambon Island at Asilulu on rocks (a map with the occurrence of Euraphia hembeli in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Euraphia hembeli has a true calcareous basis and complete secondary calcification on its parietal wall and basis (Southward et al. 1998). It can be also distinguished from other species of the genus Euraphia by its size (up to 30 mm) and the presence of strong marked lateral depressor crests (between 10-12 in number) (Pochai et al. 2017).
Distribution. In this study, Microeuraphia sp. was found on Seram Island (at Pantai Waimeteng, Piru) (a map with the occurrence of Microeuraphia sp. in the Moluccas is shown in Suppl. material 1: Fig. S5).
Remarks. Microeuraphia sp. clustered as a unit, forming a well-supported clade in the COI tree (Fig. 29). Morphologically, one individual of this species exhibited two penises.
Remarks. Species of the genus Chthamalus are very difficult to distinguish in the field. Chthamalus moro has a stellate appearance and is smaller than C. malayensis (Southward and Newman 2003). In addition, conical spines on the dorsal side of cirrus I are absent and setae on cirrus II are without basal guards in C. moro.  Figure 13a, b, Table 1: species no. 59
Description. Shell depressed, covered by furry chitinous integument; shell plates with prominent radiating ribs; radii wide, porose, tubes running parallel to base of shell; colour of shell pale purplish; orifice diamond shaped (Fig. 13a, b); scutum triangular, tergal margin straight; tergum higher than wide, scutal margin straight, spur short; mandible with four teeth, second and third teeth bidentate; labrum with smooth cutting edge (measurements for one specimen are presented in Suppl. material 1: Table S12).
Distribution. Tetraclitella divisa was previously recorded from Western Africa, Java, Malaysia, Sumatra, Northern Australia, Singapore, South China Sea, China, Taiwan, Japan, the Pacific Ocean to Hawaii and Pitcairn (Jones and Hosie 2016). In this study, T. divisa was found on Ambon Island at Laha on a concrete wall at the port (a map with the occurrence of Tetraclitella divisa in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Tetraclita divisa exhibits a brooded phase to the cypris larval stage in the mantle cavity, whereas most other species release the first stage nauplius (Nilsson-Cantell 1921;Hiro 1939).

Tetraclitella karandei Ross, 1971
Figure 14a-g,  Diagnosis. Shell with four plates, tubiferous, not strongly articulated; radii tubiferous; summit of radii horizontal and elevated above the surface of the parietes; parietes with longitudinal ribs; scutum with nodose ornamentation.
Distribution. Tetraclitella karandei was previously recorded from India, Taiwan, the Philippine (Chan et al. 2009a). In this study, T. karandei was found on Ambon Island at Waitairi and Asilulu on stone, on the shells of Capitulum mitella and Euraphia hembeli (a map with the occurrence of Tetraclitella karandei in the Moluccas is shown in Suppl. material 1: Fig. S2). Remarks. Tetraclitella karandei can be distinguished by its radii, which are broad and have extended out and over the adjoining plates. The scutum is also unique because it has nodose ornamentation (Ross 1971).

Subfamily Tetraclitinae Gruvel, 1903 Genus Tesseropora Pilsbry, 1916
Tesseropora rosea (Krauss, 1848) Figure 15a-e, Diagnosis. Shell with four plates; wall of the parietes with a single row of parietal pore; orifice with traces of pink in colour; oral cone relatively broad; mouthparts relatively large.
Distribution. Tesseropora rosea was originally described from a specimen collected at Algoa Bay, South Africa (Krauss 1848; Darwin 1854) and has since been recorded from Australia (SW and SE); Lord Howe Island and the Kermadec Islands (Jones 1990). In this study, T. rosea was found on Ambon Island (at Rutong, Leahari, and Liang) and Saparua Island (at Ihamahu, Kulur, and Porto) on stone and mollusc shells (a map with the occurrence of Tesseroppora rosea in the Moluccas is shown in Suppl. material 1: Fig. S4).
Remarks. According to Anderson and Anderson (1985), T. rosea feeds in different ways, extending the cirral fan only in response to the fast water currents. Thus, T. rosea cannot survive in areas with a low current velocity. Tesseropora rosea exhibits a wide distribution although the species is represented by relatively few specimens. Figure 16a-g, Table 1: species no. 62  Diagnosis. Shell conical with four plates, tubiferous; radii solid; tergum broad, apex not beaked.

Tetraclita kuroshioensis Chan, Tsang & Chu, 2007
Description. Shell with four inseparable, multi-tubiferous plates, greyish black to purplish-grey or deep green to green, surfaces with mosaic scales pattern radiating randomly from base to apex, internal surface of parietes smooth, white with dark grey striations around aperture; radii solid ( Fig. 16a-c); basis membranous; scutum larger than tergum, triangular, external surface with horizontal striations, occluding margin with fine teeth; tergum broad, higher than wide, apex not produced as beak, spur sharp, basi-scutal angle smaller than that of Tetraclita squamosa (Fig. 16d, e); external surface of operculum grey and yellowish-light brown, internal surface greyish-dusky green; mandible with four large teeth; maxillule not notched with eleven setae; labrum with five small teeth on each side; cirrus I possessing serrulate setae. Basal length 12.1-21.6 mm, basal width 18.1-21.8 mm, height 7.3-10.4 mm. Orifice length 3.2-5.3 mm, orifice width 2.4-4.2 mm (measurements for five specimens are presented in Suppl. material 1: Table S15). Distribution. Tetraclita kuroshioensis was previously recorded from Japan, Taiwan, Palau, and Thailand (Chan et al. 2009a;Pochai et al. 2017). In this study, T. kuroshioensis was found on Ambon Island (at Hatu and Ureng) and Saparua Island (at Dermaga Ihamahu) on rocks and concrete wall of a port (a map with the occurrence of Tetraclita kuroshioensis in the Moluccas is shown in Suppl. material 1: Fig. S4).
Remarks. Tetraclita kuroshioensis and T. squamosa share great morphological similarity. However, DNA sequences separate the two species (Chan et al. 2007), which was confirmed in this study (Fig. 29). Morphologically, the shape of the tergum is definitive; that of T. kuroshioensis is broader and the apex blunter compared to T. squamosa (Chan et al. 2007). Figure 17a-k,   Diagnosis. Shell conical with four plates, tubiferous; radii solid; tergum narrow, concaved, apex beaked.
Remarks. Tetraclita squamosa has characteristic green parietes (Yamaguchi 1987) and a wide distribution throughout the Indo-Pacific (Newman and Ross 1976). However, the taxonomy of Tetraclita squamosa has been confusing due to a high degree of morphological variation, and it is now considered a species complex. Tetraclita squamosa and Tetraclita japonica can be separated using characters such as the shape of the parietes, scutum geometry, and mandible structures (Darwin 1854;Pilsbry 1916). In addition, a key character for T. squamosa is the tergum with a beak on its apex (Chan et al. 2009a).
Remarks. The subgenus Yamaguchiella was proposed by Ross and Perreault (1999)

GenBank accession number. 18S (MK981384).
Diagnosis. Parietes low with wall spreading; peritreme slightly toothed; base calcareous with two rows of irregular shape and size of parietal tubes; tergum with broad spur; lateral scutal depressor crests numerous and deep; five toothed mandibles; segments of posterior cirri with four pairs spines.
Distribution. Neonrosella vitiata was previously recorded from the Indo-west Pacific, Indian Ocean, Nicobar Island to Australia, Indonesia, Malay Archipelago, Sulu Archipelago, Philippines, and the Pacific Ocean (Jones and Hosie 2016). Recently, Neonrosella vitiata also was discovered in the Andaman Sea of Thailand (Sukparangsi et al. 2019). In this study, N. vitiata was found on Ambon Island (at Liang), Banda Island and Saparua Island (at Tuhaha) on port poles, reef and stones (a map with the occurrence of Neonrosella vitiata in the Moluccas is shown in Suppl. material 1: Fig. S7).

Remarks.
Neonrosella vitiata can be distinguished by its irregular parietal tubes, the shape of the terga, the five toothed mandibles and four pairs of spines on the segments of the posterior cirri (Darwin 1854). Figure 20a-e, Table 1: species no. 66
Description. Shell low conical, four plates externally greyish in colour, parietes with deep longitudinal, radiating lines from base to apex, internally with multiple rows of irregular parietal tubes (Fig. 20a, b); radii wide with horizontal striations, summits oblique (Fig. 20c); scutum triangular, external surface with horizontal striations, adductor ridge conspicuous; tergum high, narrow, basal margin with well-developed depressor muscle crests projecting beyond border; orifice pentagonal ( Fig. 20d, e); basis calcareous, tubiferous, tubes in single layer; mandible with five teeth, the first tooth is the largest and separated from the rest, while the fifth tooth is the smallest and located at the middle of lower margin; labrum with V-shaped notch, two large teeth on the right side, five teeth on the left side of cutting margin; penis without basidorsal point, with few bundles of setae distally. Basal length 17.4-20.9 mm, basal width 15.9-20.5 mm, height 6.8-8.9 mm. Orifice length 5.3-7.3 mm, orifice width 5.0-6.7 mm (measurements for five specimens are presented in Suppl. material 1: Table S19).
Distribution. Newmanella spinosus was previously recorded from Japan, Taiwan, Philippines, and Thailand (Chan and Cheang 2016;Pochai et al. 2017). In the current study, this range is extended to Rutong (on stones and reef surface), Ambon Island (a map with the occurrence of Newmanella spinosus in the Moluccas is shown in Suppl. material 1: Fig. S2).
Remarks. Newmanella spinosus is morphologically close to Newmanella radiata but it can be distinguished by the morphology of the scutum, tergum, cirrus II, mandible and maxillule. N. spinosus also has numerous spines on its cirri, especially on cirrus II, which is different from N. radiata (Chan and Cheang 2016).  Balanus amphitrite denticulata Henry, 1959: 192, pl  Diagnosis. Primary parietal tubes with transverse septa; exterior of shell with longitudinal purple striations, horizontal striations absent; tergum short with wide spur; cirri III-VI with erect teeth below posterior angles of distal; cirrus III without complex setae.
Distribution. Amphibalanus amphitrite is commonly found on beaches and in estuaries, lives attached to harsh natural substrate, such as bedrock, rocks, shells of molluscs, as well as the roots and trunks of mangrove trees. Many specimens also stick to artificial substrates, such as ship hulls and the walls and pillars of docks. Amphibalanus amphitrite is spread globally tropical and subtropical waters (Zullo et al. 1972;Henry and McLaughlin 1975;Chen et al. 2014). In this study, A. amphitrite was found on the islands of Ambon (at Galala) and Saparua (at Desa Mahu, Negeri Mahu and Tuhaha) on stone, mollusc shells and the capitulum of Lepas anserifera (a map with the occurrence of Amphibalanus amphitrite in the Moluccas is shown in Suppl. material 1: Fig. S4).
Distribution. Amphibalanus reticulatus is native to the Indo-Pacific region and has been introduced by shipping to tropical-subtropical waters of the Eastern Pacific (Coles et al. 1999;Carlton et al. 2011). A. reticulatus can be found from Japan to the Malay Archipelago, east Asia from the Yellow Sea to Gulf of Siam, from Malaysia to southeast Africa, in the Mediterranean Sea, West Africa, the Southeast United States to the West Indies (Henry and McLaughlin 1975).
In this study, A. reticulatus was found on the islands of Ambon (at the port of Yos Sudarso) on stone and concrete wall of the port (a map with the occurrence of Amphibalanus reticulatus in the Moluccas is shown in Suppl. material 1: Fig. S2).
Distribution. Amphibalanus variegatus has been reported from the Indo-west Pacific: Bay of Bengal; Sumatra; New Zealand, Australia; Indonesia; Singapore; Vietnam; Gulf of Siam; Hong Kong; W Kyushu; Vladivostok; and is a common fouling species (Henry and McLaughlin 1975;Jones and Hosie 2016). In this study, A. variegatus was found on the islands of Ambon (at Waitatiri) and Saparua (at Teluk Saparua) on stones and a plastic bag (a map with the occurrence of Amphibalanus variegatus in the Moluccas is shown in Suppl. material 1: Fig. S4).
Remarks. Amphibalanus variegatus is a member of the Balanus amphitrite complex, whose members can be difficult to distinguish morphologically. Amphibalanus variegatus can be differentiated by its vesicular sheath, and from A. reticulatus by features of the tergum, armature of cirrus II and the lack of erect teeth below the posterior distal angles of cirri III-VI (Henry and McLaughlin 1975). Figure 24a-j,  fig. 2 (1-14). Diagnosis. Primary parietal tubes without transverse septa; exterior of shell with longitudinal striations; scutum without adductor ridge, external surface scutum with row of pits; anterior margin of cirri III with conical denticles, erect hooks below posterior angles of distal articles of rami present; cirrus IV with erect hooks on posterodistal angles of articles. Description. Shell six-plated, conic, purplish-white with longitudinal stripes of purple, not cross-hatched by transverse striations; parietes externally smooth, parietal tubes lacking transverse septa and subsidiary tubes; radii wide with slightly oblique summits; orifice rhomboidal, toothed (Fig. 24a-c); scutum trigonal, exterior of scutum with single row of pits extending down centre of valve, occluding margin toothed, inner surface smooth, adductor ridge lacking; tergum with carinal margin convex, spur furrow open, basal margin straight on both sides of spur (Fig. 24d, e); cirrus III without complex setae; cirri III-VI with erect hooks around posterior angle; first maxilla without notch, mandible with five teeth (Fig. 24h). Basal length 5.8-21.6 mm; basal width 4.8-19.2 mm; height 2.1-16.5 mm; orifice length 3.0-10.0 mm; orifice width 2.6-7.6 mm (measurements for eleven specimens are presented in Suppl. material 1: Table S23).

Amphibalanus zhujiangensis (Ren, 1989)
Distribution. Amphibalanus zhujiangensis was first recorded from the estuary of the Zhujiang River, South China Sea (Puspasari et al. 2002). Afterwards found on Okinawa Island, Japan and Taiwan (Chan et al. 2009a). In this study, A. zhujiangensis was found on the islands of Ambon Island (at Galala, Laha, and Talake), Saparua Island (at Dusun Pia, Negeri Mahu, Desa Mahu), and Seram Island (at Desa Kasie, Lepas Pantai Kawa) on stone and capitulum of Lepas anserifera (a map with the occurrence of Amphibalanus zhujiangensis in the Moluccas is shown in Suppl. material 1: Fig. S8).
Remarks. The presence of a row of pits on the external surface of the scutum and the absence of an adductor ridge on the scutum are diagnostic for A. zhujiangensis. The species can be distinguished from A. variegatus by characters of the shell, cirri III and cirri IV; on A. reticulatus by characters of the shell and first maxilla. Amphibalanus zhujiangensis is distinct from A. thailandicus in lacking transverse septa in the longitudinal tubes and a notch on the first maxilla (Puspasari et al. 2002). Diagnosis. Primary parietal tubes with transverse septa, exterior of shell with longitudinal striations; orifice toothed; scutum without adductor ridge; anterior margin of cirri III with conical denticles, erect hooks below posterior angles of distal articles of rami present; cirrus IV with erect hooks on posterodistal angles of articles; basidorsal of penis absent.
Distribution. In this study, Amphibalanus sp. was found on Ambon (at Talake and Waitatiri) and Seram islands (at Dermaga Pelita Jaya) (a map with the occurrence of Amphibalanus sp. in the Moluccas is shown in Suppl. material 1: Fig. S9).
Remarks. In the molecular phylogeny, Amphibalanus sp. forms a well-supported clade in both, the COI and the 18S tree (Figs 28, 29). This species also has different maxilla than those of A. amphitrite, A. reticulatus and A. variegatus, which have setae on its upper and lower margins.
Distribution. Megabalanus tintinnabulum is a cosmopolitan species and widely distributed worldwide (Pochai et al. 2017). In this study, M. tintinnabulum was found on Ambon Island (at Laha) and in Saparua Island (at Desa Pia) on concrete bridge at the port, stones and reef surface (a map with the occurrence of Megabalanus tintinnabulum in the Moluccas is shown in Suppl. material 1: Fig. S4).
Remarks. The name Megabalanus was given by Hoek (1913), referring to the largest form of existing Balani. With the exception of Balanus amphitrite, Darwin (1854) considered Balanus tintinnabulum as the most difficult and variable species in the genus Balanus (Henry and McLaughlin 1986). Megabalanus tintinnabulum can be distinguished by its large shell plates and purple surface with irregular, unclear longitudinal stripes (Pochai et al. 2017). Figure 27a-c, Diagnosis. Parietes reddish purple with strong longitudinal white ribs; radii and sheath dark purple to reddish brown; scutum with narrow tergal segment slightly inflected; tergum approximately as wide as scutum, crest for depressor muscle prominent.
Distribution. Megabalanus zebra is a well-known fouling species of ship hulls, floating structures, moveable oil platforms, etc., and has been widely recorded from the Atlantic Ocean; W Africa; Indo-west Pacific: Indian Ocean; Australia; Thailand; China; Philippines; Taiwan (Pilsbry 1896(Pilsbry , 1916Stubbings 1961;Karande and Pakelar 1966;Foster and Willan 1979;Chan et al. 2009a;Jones and Hosie 2016;Pitombo et al. 2017). In this study, Megabalanus zebra was found on Ambon Island (at Galala and Laha) on stones and the capitulum of Lepas anserifera (a map with the occurrence of Megabalanus zebra in the Moluccas is shown in Suppl. material 1: Fig. S1).
Remarks. Megabalanus zebra can be distinguished from other species in the Megabalanus group by, for example, the inflection of the tergal segment of the scutum and the position of the spur (Henry and McLaughlin 1986). M. zebra can also be differentiated by an intermediate shape of the tergum and scutum compared to M. tintinnabulum and M. coccopoma (Pitombo et al. 2017).
In general, support values (bootstrap and posterior probability) were low for both markers, with the majority of internal nodes receiving support values below 50% bootstrap or 0.5 posterior probability. However, there are several highly supported nodes throughout (> 70% bootstrap; > 0.85 posterior probability), which allow us to gain insights into the evolutionary history of the group. In general, and as expected, COI ( Fig. 29) provided higher resolution at terminal nodes but low resolution at deeper nodes (rapidly evolving marker), whereas 18S (Fig. 28) provided higher resolution than COI at basal nodes (slowly evolving marker). The trees resulting from the BEAST and RaxML analyses were fully congruent, with no highly supported relationships being favoured in one analysis but not the other. The main purpose of our phylogenetic analyses was to find out where the new accessions from the Moluccas sequenced for this study are retrieved on the barnacle tree, and to see whether putative species are retrieved as monophyletic. We therefore show multiple accessions per species on the trees. The trees resulting from RaxML analyses and the concatenated analyses are given in the Suppl. material (Suppl. material 1: Figs S10-S13).

Molecular study of Moluccan barnacles
The vast majority of new samples from the Moluccas produced in this study matched sequences from the same species that are available on GenBank. For example, DNA sequence of our Heteralepas japonica matched the sequence of H. japonica EU884146.1 and EU884169.1 from Chan, et al. (2009c); and our Nesochathamalus intertextus matched the sequence of N. intertexus JX083869.1 from Perez-Losada, et al. (2012). This applies to all species for which we have new sequences. The only exception is Chthamalus moro, for which one of our samples in the 18S tree does not match the GenBank samples of that species. However, for this particular case, the support values of that clade in the tree are very low, therefore the odd positioning is not strongly supported (that clade is essentially a polytomy).
Two taxa for which we sequenced multiple accessions, but for which we could not assign a species name, were retrieved in positions on the tree that lead us to propose these may constitute new unidentified species. The first one is Amphibalanus sp., clustering as a unit in both COI and 18S trees (Figs 28, 29; Suppl. material 1: Figs S10-S13). The other was Microeuraphia sp., which formed well supported and separated clades in the COI tree, and was clustered in the same unresolved clade in 18S (Figs 28, 29; Suppl. material 1: Figs S10-S13).  The K2P distances within Microeuraphia sp. were 1.74%±0.51% for the COI sequences. The K2P distances between Microeuraphia sp. and other species ranged from 10.90% to 22.70%; and overall averaged distances between the species and other species were 13.82% (Suppl. material 1: Table S27). Whilst for Amphibalanus sp. the K2P distances within the species were 0.22%±0.13% for the COI sequences. The K2P distances between Amphibalanus sp. and other species ranged from 13.34% to 18.33%; and overall averaged distances between the species and other species were 14.37% (Suppl. material 1: Table S28).

Discussion
This checklist lists 97 species, including 23 new records the Moluccas and two of which still await their species descriptions. The past record on barnacles from these islands dates back to the Challenger (1872-1876) and Siboga (1899-1900) expeditions (Hoek 1913). Later, Kolosváry (1950) only mentioned some balanids living in corals collected during Snellius Expedition (1929)(1930), which contrasts reports from other groups numerously collected during the same expedition, such as in decapod crustaceans and Foraminifera.
Hoek (1913) listed a total of 210 species from the Malay Archipelago that were collected during the Challenger and Siboga expeditions. Among these, 45 species were found in the Moluccas. However, the majority of the Moluccan species listed by Hoek (32 species) were deep-water barnacles found at depths of 204-2,798 m, while 10 species barnacles were found at depths of 9-90 m, and only three inshore species were recorded: Temnaspis fissum (Darwin, 1851) from Ternate; Yamaguchiella coerulescent (Spengler, 1790) and Tetraclitella costata (Darwin, 1854) from Banda Island. In contrast, sampling for this study focused on inshore habitats with only two deep-sea locations. In consequence to the different sampling approaches, we found 24 inshore species and only one deep-sea species among the new samples.
A comparison of the number of species previously recorded from Ambon, Seram, and Banda by Hoek (1913) and Jones (2001Jones ( , 2016 with those recorded in this study indicates that species diversity for each island has been heavily underestimated. On Ambon, for example, seven species were previously known compared to the 24 species listed here. For other smaller islands such as Saparua and Pombo, no barnacle species was previously recorded. Given the size of the Moluccan Archipelago, with ca. 1,000 islands, many of which have never been sampled despite including relatively large islands such as Haruku, Buru, Yamdena or Wetar, a much higher number of species can be expected in the Moluccas. The molecular results also indicate that the barnacle fauna of the region is understudied. In addition to evidence for two potentially new species (see above), the generic assignment of some described species is also challenged. For example, Amphibalanus zhujiangensis was found to be more closely related to Megabalanus than to other Amphibalanus species, suggesting the need to conduct in-depth research on this species to clarify its taxonomy. However, we must caution against over interpretation of our phylogenetic trees, because the markers we used revealed low node support overall.
The molecular phylogeny failed to reveal any biogeographic pattern of barnacles from the Moluccas, which is not surprising given the limited scope of sampling. These points all underline again the necessity of a more comprehensive approach to sampling in the region as well as the need to explore more molecular markers for a truly integrative taxonomy of barnacles, not just in the Moluccas.
Yamaguchi T (1987) Changes in the barnacle fauna since the Miocene and the infraspecific structure of Tetraclita in Japan (Cirripedia: Balanomorpha  Values next to nodes are support values; Table S1.