Description of a new species of coral-inhabiting barnacle, Darwiniella angularis sp. n. (Cirripedia, Pyrgomatidae) from Taiwan

Abstract The present study has identified a new species from the previously monotypic genus Darwiniella Anderson, 1992. Darwiniella angularis sp. n. is similar to Darwiniella conjugatum (Darwin, 1854) in external shell morphology and arthropodal characters. Darwiniella conjugatum, however, has a sharper tergal spur and a less obvious adductor plate angle when compared to Darwiniella angularis sp. n. Molecular analyses on mitochondrial DNA 12S rDNA and COI regions also support the morphological differences. Sequence divergences in 12S rDNA and COI between Darwiniella conjugatum and Darwiniella angularis sp. n. are 5% and 13% respectively, which are equivalent to the inter-specific sequence divergences in other barnacles. Both Darwiniella species are common on Cyphastrea Milne-Edwards and Haime, 1848 corals and Darwiniella angularis sp. n. is also collected from Astreopora de Blainville, 1830 corals in Taiwan.


introduction
The coral-inhabiting barnacles of the genus Darwiniella Anderson, 1992 is a member of the family Pyrgomatidae, which has a symbiotic relationship with host corals. Darwiniella was considered to be a monotypic genus, represented by Darwiniella conjugatum (Darwin, 1854).
Darwiniella conjugatum was originally described by Darwin (1854) as Pyrgoma conjugatum. The species has a fused shell wall and a pair of fused opercular plates (scutum and tergum). Ross and Newman (1973) erected the genus Nobia to accommodate coral-inhabiting barnacles with fused shell plates and fused opercular valves, thus transferred P. conjugatum to Nobia conjugatum. However, Nobia conjugatum has a deep adductor plate and a distinct tergal spur, which is different from all Nobia species. Subsequently, Anderson (1992) erected the new genus Darwiniella to accommodate Nobia conjugatum and defined the genus as " Wall flat, fused; sheath occupying about half inner wall; scutum and tergum calcified together, without visible line of juncture; deep adductor plate and conspicuous rostral tooth; elongate spur; basis oval, deep." However, Anderson (1992) had not formally assigned the type species of Darwiniella and Ross (1999) designated the type species of Darwiniella as Darwiniella conjugatum (Darwin, 1854).
In the present study, we collected a large number of Darwiniella specimens from coral reefs in Taiwan. Based on molecular analysis of the mitochondrial 12S rDNA (12S) and DNA barcode gene fragment (COI), we revealed two species of Darwiniella, D. conjugatum and an undescribed species. We used scanning electron microscopy (SEM) to examine the shell parts and light microscopy (LM) to examine the arthropodal characters of D. conjugatum and the Darwiniella sp. n. and both species were described herein from Taiwan.

Specimen sampling and morphological analysis
Sampling of Darwiniella specimens was conducted at the main island of Taiwan (Suao and Kenting) and outlying islands (Turtle Island, Green Island and Siaoliouciou Island; see Fig. 1 and on-line Appendix 1: Table 1 for site and location details). Small pieces of coral with barnacles embedded were collected with hammers and chisels at 5-20 m depth by SCUBA diving. All barnacles and host corals were preserved in 95% EtOH. Type and paratype specimens are stored in the National Museum of Natural Science, Taichung, Taiwan (NMNS) and the Biodiversity Museum of the Academia Sinica, Taipei, Taiwan (ASIZCR), and additional specimens in the barnacle collections in the Coastal Ecology Laboratory, Academia Sinica, Taiwan (CEL). Barnacles were isolated from the host corals using forceps and the morphological characters of shell parts (shells, scutum and tergum) and somatic bodies (6 pairs of cirri, penis and oral cone) were examined. Organic debris and coral tissue on the surface of shells, scutum and tergum were removed with forceps and further cleaned ultrasonically (2-5 seconds). Cleaned shells and opercular valves (scutum and tergum) were immersed in 1.5% bleach for about five hours to completely digest organic tissue and the shells were rinsed by slow running purified water (30 minutes) and air-dried. The shells, scutum and tergum were gold coated and observed under SEM, following the methods of Achituv et al. (2009). Cirri, penis and oral cone were dissected from the somatic bodies and examined under LM (Zeiss Scope A1) with high definition lenses (Zeiss Plan APOChromat 40X/0.95 and ZEISS Plan APO Chromat 100x/1.4 oil), which allowed clear observation of setal types on cirri and mouth parts. Setal descriptions follow Chan et al. (2008).

Molecular analysis
Total genomic DNA was extracted from soft tissue of 110 Darwiniella specimens using 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 primers 12S-FB and 12S-R2 ), 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 CLUSTAL X (Thompson et al. 1997) using default settings and adjusted by eye in MacClade 4.07 (Maddison and Maddison 2005).
Scutum and tergum white, fused without junctions (consistent through 3 specimens, Fig. 2C-H). Scutum triangular, width equal to height, occludent margin slightly curved, rostral tooth basally, 15 teeth along ventral surface of occludent margin, tooth size increasing gradually from apex to base. Ventral view with oval-shaped adductor muscle scar (Fig. 2D, F, H). Dorsal view with obvious adductor plate, convex, extending below basal margin of scutum, plate about 3/5 height of scutum, basal margin with obvious angle (Fig. 2C, E, G). Dorsal surface with horizontal striations, striations with row of small pores.
Tergum trapezoid, lateral depressor muscle crests present. Spur triangular, blunt, curved, height reaching more than 1/2 height of tergum, basal margin not obvious due to curved spur. Dorsal surface with medial furrow, curving from basal margin towards the carinal margin ( Fig. 2C, E, G). Dorsal surface with horizontal striations, striations with row of small pores.
Distribution. At present only recorded from Taiwan. Etymology. The name angularis denotes the presences of the obvious adductor plate angle, which is a diagnostic character of this species.
Remarks. External shell morphology and arthropodal characters of D. angularis sp. n. are similar to D. conjugatum. However, D. angularis has an obvious adductor plate angle, whereas that of D. conjugatum is less obvious (see description below). Darwiniella conjugatum also has a sharper spur angle than D. angularis -mean ± 1 SD of the spur angle (from 10 specimens) reaching 23.6 ± 4.8 o in D. conjugatum and 32 ± 4.6 o in D. angularis, which is significantly different between the two species using the t-test (t value: -4.3, df = 18, p <0.05). (  Diagnosis. Scutum subtriangular, rostral tooth and obvious adductor plate present. Adductor plate angle not obvious. Tergum subtriangular, lateral depressor muscle crests, medial furrow and spur present. Spur triangular, long, curved and sharp.

Darwiniella conjugatum
Description. Shell (8 mm in maximum basal diameter, rostro-carinal diameter to 6 mm) plates fully fused, purple, oval, externally surface with about 25 strip-like projections differing in length and radiating from nearly subcentral orifice to plate margin (Fig. 10A). Bases of shell with about 27 internal ribs radiating from the rim of the inner operculum to the basal margin of the shells (Fig. 10B). Orifice oval, long and narrow, about 3/8 length of rostro-carinal diameter.
Scutum and tergum white, fused without any junctions (Fig. 10C, D). Scutum subtriangular, width 1.5 times length of height, occludent margin slightly curved, with a rostral tooth on bottom (Fig. 10F) and continuous teeth along the ventral surface of occludent margin, tooth size increasing gradually from apex to base. Ventral view with an apparent oval-shaped adductor muscle scar (Fig. 10D). Dorsal view with obvious adductor plate, extending below basal margin, plate more than 1/2 height of scutum. Basal margin of adductor plate without obvious adductor plate angle (Fig. 10C). Dorsal surface of scutum with horizontal striations, striations with row of small pores (Fig. 10E).
Tergum subtriangular, lateral depressor muscle crests present. Spur triangular, long, curved and sharp, height reaching more than 1/2 height of tergum, basal margin not obvious due to the curved spur. Dorsal surface with medial furrow, curving from basal margin towards the carinal margin of tergum, width of furrow increased gradually from apex to base (Fig. 10C). Dorsal surface with horizontal striations, striations with row of small pores.
Distribution Remarks. D. conjugatum is widely reported in the Indo-Pacific Ocean. Note Ogawa and Matsuzaki (1992) and Asami and Yamaguchi (1997) misspelled the species name Darwiniella conjugatum as 'conjugata'.

Molecular analysis
After trimming and aligning the sequences, 472bp of 12S rDNA and 642bp of COI were obtained from 107 and 92 Darwiniella specimens without indels, respectively (see on-line Appendix 2: Table 2 for GenBank accession numbers, on-line Appendix 3: alignment Figure). The NJ inferred genealogical relationships based on 12S and COI were congruent to each other (Fig. 18). Both datasets showed the presence of two wellsupported distinct lineages, corresponding to D. conjugatum and D. angularis sp. n.. In 12S, 37 out of the 69 variable nucleotide sites were parsimony informative. Evolutionary distances based on p-distance/K2P-distance were 0.004/0.004 and 0.006/0.006 within D. conjugatum and D. angularis sp. n., respectively, and 0.056/0.058 between the two species. In COI, 100 out of the 127 variable nucleotide sites were parsimony informative. Evolutionary distances based on p-distance/K2P-distance were

Discussion
In the present study, a new species is identified from the previously monotypic genus Darwiniella that exhibit diagnostic morphological and molecular differences from D. conjugatum. These two species are difficult to distinguish from external shell morphology. The diagnostic characters are of the adductor plate angle of the scutum and the spur angle of the tergum.
Based on molecular analyses, the sequence distance of mitochondrial DNA markers 12S and COI within the two Darwiniella species (12S: 0. 4-0.6%, COI: 0.6-1.0%) is much smaller than between species (12S: 5.6-5.8%, COI: 12.0-13.2%). The sequence divergence of these two species has reached the level of congeneric species in another coral barnacle genus, Cantellius Ross & Newman, 1973 (12S: 5-7%, COI: 10-11 %) (Achituv et al. 2009), therefore further supporting D. angularis sp. n. as a separate species from D. conjugatum. Although the application of mitochondrial DNA in delimitating species can be limited by its natural history, e.g. reduced effective population size and introgression, and maternal inheritance (Rubinoff et al. 2006), the species boundary is evident based on the differentiation of 12S, COI and morphology of abundant samples from various localities.
Both Darwiniella species show significant host preference for the massive-form coral Cyphastrea. In the present study, 21 of 23 coral pieces with D. conjugatum embedded are Cyphastrea and only one is Astreopora (Appendix 2: Table 2). A similar pattern is observed in D. angularis where six of eight coral pieces are Cyphastrea, and only two are Astreopora. In previous studies, D. conjugatum has been recorded in Cyphastrea corals by Soong and Chang 1983 (Taiwan), Ren 1986 (Mainland China), Ogawa et al. 1998 (Mauritius) and Jones et al. 2000 (South China Sea), agreeing with the current study. However, Ogawa and Matsuzaki (1992) also recorded Darwiniella conjugatum from Cyphastrea, Goniopora de Blainville, 1830 and Favites Link, 1807 species in Japan. Explanation note: Study sites and sampling locations for Darwiniella in the present study.

Appendix 1
Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.  Explanation note: GenBank accession numbers for the barnacle species used in the present study.
Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.