Galkinius Perreault, 2014 or Darwiniella (Anderson, 1992)? A new coral-associated barnacle sharing characteristics of these two genera in Pacific waters (Crustacea, Cirripedia, Thoracica, Pyrgomatidae)

Abstract A new species of coral associated barnacle (Balanomorpha: Pyrgomatidae) sharing morphological features of Darwiniella (Anderson, 1992) and Galkinius Perreault, 2014 is described. It has a fused shell and opercular plates, characteristic of Darwiniella. However, the morphology of the tergum and somatic body are closer to Galkinius. Sequence divergence of mitochondrial DNA 12S rDNA and COI reveals this new species clusters with the Galkinius clade. Therefore this new form is assigned to the genus Galkinius, as G. maculosus sp. n. Concomitantly the diagnosis of Galkinius is emended to include species with fused or four- plated shells and fused opercular plates. The new species is distinct from all Galkinius species in having a fused shell. It inhabits the corals Lobophyllia spp. and is distributed from the Dongsha Atoll in the South China Sea, Orchid Island of Taiwan in the Pacific Ocean, to Madang in Papua New Guinea waters.


Introduction
Barnacles in genus Galkinius Perreault, 2014 are coral associated species of the family Pyrgomatidae. Species of Galkinius were originally grouped under the genus Creusia Leach, 1817 by Darwin (1854). Ross and Newman (1973) revised the taxonomy of pyrgomatid barnacles and redefined Creusia as having a 4-plated shell but a fused scutum and tergum. Galkin (1986) established a new genus Utinomia Galkin, 1986 to accommodate Creusia species which had a broad adductor plate and a rostral tooth in the scutum. However, the generic name Utinomia is preoccupied by Utinomia Tomlinson, 1963 for an acrothoracican barnacle (Tomlinson 1963). Ross and Newman (1995) renamed Utinomia as Galkinia, and designated G. indicum (Annandale, 1924) as the type species. Perreault (2014) pointed out the generic name Galkinia Ross & Newman, 1995 was preoccupied by a genus of fossil fish, Galkinia Ghekker, 1948 (Actinopterygii: Pholidophoriformes). He therefore renamed Galkinia as Galkinius Perreault, 2014, thereby continuing to recognize Galkin's contribution to cirripede taxonomy.
According to Ross and Newman (1973) and Ogawa (2000), there were three Galkinius species including G. decima (Ross & Newman, 1973), G. indica (Annandale, 1924), and G. supraspinulosa Ogawa, 2000. Chan et al. (2013 subsequently identified five new species of Galkinius in Taiwan waters (also see Tsang et al. 2014). Simon-Blecher et al. (2016) revealed there is geographical variation in the opercular plate morphology of Galkinius in the Indo-Pacific waters, and that there were four additional un-named cryptic species in the region suggesting there was considerably more diversity to be explored in the Pacific.
In this study, 39 specimens of a new pyrgomatid barnacle were collected in the Pacific region (Dongsha Atoll, Orchid Island in Taiwan waters and Madang in Papua New Guinea). This undescribed species has four plated shells and a fused operculum plate, which are characteristics of Darwiniella (Anderson, 1992). However, the somatic body and the shape of tergum is very similar to Galkinius. From sequence divergence in mitochondrial 12S rDNA (12S) and cytochrome c oxidase subunit I (COI) gene, this new species is closer to Galkinius than it is to Darwiniella. Therefore it was decided to classify it in the genus Galkinius. The diagnosis of Galkinius is emended to accommodate this new species of Galkinius which shares many characters with Darwiniella.

Specimen sampling and morphological analysis
The undescribed Galkinius species was sampled in Pacific waters, including the outlying islands of Taiwan waters (Dongsha Atoll in the South China Sea, Orchid Island in the Pacific Ocean) and Madang in the waters of Papua New Guinea (Fig.1). Barnacles were collected with small pieces of their coral host using hammers and chisels when SCUBA diving and then fixed in 95% EtOH. Holotype and paratype specimens are stored in the Biodiversity Museum of the Academia Sinica, Taipei, Taiwan (ASIZCR), and the National Museum of Natural History, Paris, France (NMNH). Additional specimens are stored in the Coastal Ecology Laboratory, Academia Sinica, Taiwan (CEL). After barnacle specimens were removed from the host coral with forceps, they were examined under light microscopes (LM; Zeiss Scope A1) and scanning electron microscopes (SEM; FEI Quanta 200) to further describe their morphological characters, including hard parts (shell and opercular valves) and the somatic body (cirri, penis and mouth parts). To determine the structure and articulations between individual shell parts, all the barnacle tissue, coral tissue and other organic debris adhering to the shell and the opercular valves were carefully removed by forceps, and then 1.5% bleach was used to digest the remaining tissue. After immersion in bleach for approximately three hours, the remaining organic tissue could then be torn off easily by forceps. The cleaned shells were rinsed with water for approximately 30 minutes and air-dried. The shell and opercular valves were coated with gold and then observed under SEM following the methods of Chan et al. (2013). The somatic body, including the six pairs of cirri, the penis, and the mouth parts were dissected out and observed under LM. Setal descriptions are based on Chan et al (2008).

Molecular analysis
Total genomic DNA was extracted from soft tissue of individual specimens using a Qiagen (Chatsworth, CA) QIAquick Tissue Kit following the manufacturer's instructions.
Partial sequences of mitochondrial genes 12S rDNA (12S) and cytochrome c oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) with primer 12S-FB and 12S-R2 (Tsang et al. 2009), and COI-F5 5' AAACCTATAGCCTTCAAAGCT 3' and COI-R4 5' GTATCHACRTCYATWCCTACHG 3', respectively. The PCR solution contained 40 ng of template DNA, 5 μl Taq DNA Polymerase Master Mix (1.5 mM MgCl 2 ; Ampliqon, Denmark), 1 μM of each primer, and ddH 2 O with a final volume of 10 μl. The PCR reaction was conducted under the following conditions: 2 min at 95 °C for initial denaturing, 35 cycles of 30 sec at 95 °C, 1 min at 48 °C, 1 min at 72 °C with a final extension for 5 min at 72 °C. The PCR products were then purified using the DNA Gel purification kit (Tri-I Biotech, Taipei, Taiwan). Direct sequencing of the purified PCR products was performed on an ABI 3730XL Genetic Analyzer with BigDye terminator cycle sequencing reagents (Applied Biosystems, Foster City, California, USA). Sequences were then aligned with BioEdit Sequence Alignment Editor V7.2.5 (Hall et al. 2013) using default settings and adjusted by eye.
The genealogical relationships of specimens based on 12S were inferred using both Maximum Composite Likelihood model, 1000-replicate Neighbor-Joining (NJ) method and T92 model, 1000-replicate Maximum Likelihood (ML) method implemented in MEGA v7.0.14 (Kumar et al. 2016). We reconstructed the relationship between three species of Darwiniella (Darwiniella angularis, D. conjugatum, and D. maculosus sp. n.) and eight Galkinius Perreault, 2014 species (Galkinius adamanteus Chan, Chen & Lin, 2013, G. equus Chan, Chen & Lin, 2013 (Ross & Newman, 1973), G. tabulatus Chan, Chen & Lin, 2013, G. depressa Chan, Chen & Lin, 2013, G. altiapiculus Chan, Chen & Lin, 2013, G. trimegadonta Chan, Chen & Lin, 2013, and G. indica (Annandale, 1924). Additionally, five specimens of the coral barnacle Nobia grandis Sowerby, 1839 were used as the outgroup. Additionally, three sequences of Darwiniella spp. and four sequences of Galkinius species form Malay and Michonneau 2014 were downloaded from EMBL and added into the analysis. The evolutionary distance (number of base differences per site) between sequence pairs was calculated with uncorrected p-distance and Tamura 3-parameter model (T92) models by MEGA. Diagnosis (emended). Shell wall fused or four plated, flat, with high radial ridges at the junction with coral skeleton. Scutum and tergum fused, the two parts being approximately subequal. Adductor ridge and lateral depressor muscle scars absent, adductor plate and rostral tooth present. Tergal spur well developed and wide. Apertural frill coloured and spotted. Maxilla and cirri with numerous dark spots and bands.

Remarks.
In the original diagnosis of Galkinius, the shell consisted of four separated plates and the fused scutum and tergum, which differs from Darwiniella which has a fused shell as well as a fused scutum and tergum. In the present study, a new species of Galkinius was identified as having a fused shell wall. Therefore it is necessary to emend the diagnosis of Galkiniusto accommodate this species (see discussion below). Galkinius differs from Darwiniella in having much wider tergal spur and tergal furrow. Height of the adductor ridge of the scutum in Darwiniella is much greater than in species of Galkinius. In Darwiniella, the height of adductor ridge is approximately 2/3 to 1/2 total height of scutum. In Galkinius, height of adductor plate is often approximately 1/3 of the total height of scutum. Maxilla of Galkinius and cirri with large number of coloured spots and bands, when compared to Darwiniella. The apertural frills of Darwniella angularis and D. conjugatum are white, while Galkinius has a coloured or spotted aperture frill. Diagnosis. Galkinius with fused shell wall, spotted aperture frill; cirri, maxilla, and penis with dark spots, scutum with relatively narrow adductor plate, tergum with wide spur.
Scutum and tergum white, plates fused without junctions ( Fig. 3D-G). Width of scutum similar to width of tergum. Scutum triangular, transversely elongated, width two times longer than height. Occludent margin straight, with 6-8 rostral teeth basally along ventral surface of occludent margin, teeth gradually increasing in size from apex to base (Fig. 3D-G). Ventral view with oval-shaped adductor muscle scar. Dorsal view with horizontal striations, each bearing rows of small pores (Fig. 3H). Adductor plate convex, extending below basal margin half height of scutum (Fig. 3D, F). Tergum trapezoid, three times higher than scutum. Tergum apex pronounced, lateral depressor muscle crests not apparent. Spur wide, reaching one third width of basal margin of tergum, base convex, height of scutal side of spur three times longer than carinal side, height of spur about one third height of tergum. Dorsal surface with middle spur furrow, curving slightly from the basal margin towards carinal margin (Fig. 3D). Dorsal surface with horizontal striations, each bearing rows of small pores (Fig. 3I).
Cirrus I with rami unequal. Dark spots and stripes on each segment of anterior and posterior rami (Fig. 7A). Posterior ramus short (nine segments), bearing serrate setae (Fig.  7B), the anterior edges of the rami carry simple and serrulate setae (Fig. 7C). Anterior ramus long (17 segments), slender, anterior edges of the segments bearing simple and bi-        dentate serrulate setae (Fig. 7D). Cirrus II rami sub-equal. Dark spots and stripes on each segment of anterior and posterior rami (Fig. 7E) Anterior ramus (nine segments) and posterior ramus (seven segments), bearing serrulate setae. Anterior edges of both anterior and posterior rami with both simple and bidentate serrulate setae (Fig. 7F). Fan-shaped denticles present at the margins of middle segments (Fig. 7G) and conical spines present at the margin of distal two to three segments (Fig. 7H). Cirrus III rami subequal (Fig.  8A), dark spots and stripes exist on each segment of anterior and posterior rami. Anterior ramus (12 segments) and posterior ramus (10 segments), with simple and serrulate setae. Fan-shaped denticles (Fig. 8B) present at the surface of basal segments of posterior ramus Conical spines present at the margin of the distal three up to eight segments at both anterior and posterior rami (Fig. 8C). Anterior sides of both anterior and posterior rami with bidentate serrulate setae (Fig. 8D). Cirrus IV-VI long, slender, with equal rami length. Number of segments on Cirrus IV (22, 20) (Fig. 8E), Cirrus V (24, 24) (Fig. 9A), Cirrus VI (23, 23) (Fig. 9D). Stripes exist on each segment of the ramus (Figs 8E, 9A, 9D). Intermediate segments of Cirrus IV-VI has four pairs of serrulate setae (Figs 8F, 9B, C, E, F), distal pair longest, proximal pair shortest. Penis long (about one and a half times length of Cirrus VI), annulated, with scattered irregular dark spots (Fig. 9G). Pedicel with basidorsal point (Fig. 9G, H), apex of penis with short, simple setae (Fig. 9I).
Etymology. The name maculosus means dappled or mottled, and therefore denotes the spots scattered around the aperture frill, maxilla, palp, Cirrus I-VI, and penis of this species.
Distribution. Taiwan waters (Dongsha Atoll in the South China Sea, Orchid Island in the Pacific Ocean), Madang, Papua New Guinea.

Discussion
Galkinius maculosus sp. n. has shared similarities between Galkinius and Darwiniella. There are two possible genera for Galkinius maculosus sp. n. Based on the fused shell and opercular plates, Galkinius maculosus sp. n. can be placed under Darwiniella. Subsequently, the molecular phylogenetic pattern of Dawiniella will become diphyletic, with D. conjugatum and D. angularis in one molecular clade, and Galkinius maculosus sp. n. (if identified as Darwiniella) will be located in the other molecular clade with Galkinius species together. Identification of Galkinius maculosus sp. n. under the genus Darwiniella, based only on its fused shell character, probably trumps in characters of somatic body, tergum shape and molecular data.
Apart from the character of fused shell, there are many morphological characters of Galkinius maculosus sp. n. which fit well to Galkinius rather than Darwiniella. The shape of the opercular plates, especially the wide spur in the tergum of Galkinius maculosus sp. n., is similar to species of Galkinius ( Fig. 12; also see Chan et al. 2013, Simon-Blecher et al. 2016. The adductor plate of Galkinius maculosus sp. n. is narrow, which is similar to other Galkinius species, rather than the wide adductor plate in Darwiniella (Fig. 12). The aperture frill, maxilla, mandibular palp, and cirrus of Galkinius maculosus sp. n. are spotted, similar to those of Galkinius, in contrast to those of species of Darwiniella which have very few spots. The size of the Galkinius maculosus sp. n. is comparable to Galkinius (see Chan et al. 2013) and much larger than Darwiniella (see Chen et al. 2012). Adults of Galkinius maculosus sp. n. are approximately twice as large as D. angularis and one and a half times larger than D. conjugatum. Based on the morphological similarities of Galkinius maculosus sp. n. to Galkinius, this species is classified under Galkinius and, in this case, the monophyly of Darwiniella and Galkinius in the molecular phylogeny tree is preserved.
The sequences divergence of the two Darwiniella species (UF8661 and UF7460) from Malay and Michonneau (2014) clustering into the clades with the Darwiniella species further supports the monophyly of Darwiniella. These two Darwiniella sequences from Malay and Michonneau (2014) were collected in the Oman and the Philippines, indicating that there is further diversity within Darwiniella waiting to be explored in the Pacific and Indian oceans.