Discovery of Neonrosellavitiata (Darwin) and Newmanellaspinosus Chan & Cheang (Balanomorpha, Tetraclitidae) from the Andaman Sea, eastern Indian Ocean

Abstract In this present study, distantly related acorn barnacle species in the subfamily Newmanellinae (Cirripedia, Thoracica, Tetraclitidae), including Neonrosellavitiata (Darwin, 1854) and Newmanellaspinosus Chan & Cheang, 2016, were discovered in the Andaman Sea of Thailand. Neo.vitiata can be readily distinguished from other newmanellids by shell plate and operculum morphology (external shell, tergum geometry, and pattern of parietal tube) and arthropodal characters (presence of basi-dorsal point at base of penis and triangular spines on cirri, setal type, and mouth parts). Both species were found to share overlapping territories on rocks at the rockweed zone, an area submerged under seawater most of the time throughout the year. This study highlights the first discovery of Neonrosella in the eastern Indian Ocean, whose ultrastructure compared to Newmanella is redescribed and illustrated here based on scanning electron microscopy.

The genus Newmanella was established by Ross (1969) for a group of low intertidal to subtidal tetraclitid barnacles with Balanus radiata Bruguière, 1789 as the type species (Ross, 1969: 242), later known as Newmanella radiata (Bruguière, 1789) and recently redescribed by Chan and Cheang (2016). Ross and Perreault (1999) proposed the classification for the subfamily Newmanellinae and placed Newmanella into that subfamily as well as described a new species Newmanella Kolosvaryi Ross & Perreault, 1999 from the east coast of Panama in the western Atlantic. Recently, Newmanella spinosus was described as a new species from the western Pacific (Taiwan) by Chan and Cheang (2016). Hence, the genus Newmanella is currently represented by four species: New. hentscheli Kolosvary, 1942, New. Kolosvaryi, Ross & Perreault, 1999, and New. radiata (Bruguière, 1789 from the Atlantic waters of South America (Bruguière 1789;Kolosvary 1942;Ross and Perreault 1999) and New. spinosus Chan & Cheang, 2016 from the western Pacific and the Andaman Sea, eastern Indian Ocean (Chan and Cheang 2016;Pochai et al. 2017, respectively).
A recent examination of acorn barnacle specimens from the Andaman Sea, southern Thailand, recognized two morphologically similar newmanellin species from the same area of the Na-Tai rocky shore (Phang-Nga Province); Neonrosella vitiata is new to Thailand and Newmanella spinosus is found next to Neo. vitiata at lowest low tide point. Both species are redescribed herein, based on shell plate morphology and arthropodal characters using scanning electron microscopy (SEM). This is also the first illustration of Neo. vitiata in its ultrastructure, providing clear observation of this barnacle for taxonomic identification. Both species are compared with the detailed redescription of New. radiata provided by Chan and Cheang (2016).

Materials and methods
This study is based upon material collected from the Andaman Sea at Na-Tai rocky shore, Phang-Nga Province, southern Thailand, in March 2018. Samples were collected by hand picking and were transferred into plastic containers containing 95% ethanol. In the laboratory, specimens were transferred into clean 95% ethanol for storage. Specimens were examined under a compound microscope and stereomicroscope and later selected for dissection. All taxonomically important characters, shell plate morphology, and arthropodal characters were dissected and investigated with LEO 1450 VP scanning electron microscope on gold-coated specimens at Microscopic Center, Faculty of Science, Burapha University.
Specimens are preserved in 95% ethanol and have been deposited in the Zoological Collections of Burapha University, Thailand (ZCBUU).
The general terminology of the shell morphology and arthropodal characters follows Ross (1969), Rosell (1972), Ross and Perreault (1999), and Chan and Cheang (2016). The final images were processed with Adobe Photoshop CS6 and Adobe Illustrator CS6. Abbreviations used to denote shell morphology and arthropodal characters are explained directly in figure captions.
Diagnosis. Parietes white with dark orange spots or longitudinal stripes. Tergum with broad spur. Area with lateral tergal depressor crests on basal margin long and carrying numerous and deep crests. Lateral scutal depressor crests numerous and deep. Cirrus II with equal rami. Cirrus III antenniform in both rami; lesser curvature with hook-like spines. Lesser curvature of cirrus IV without spines. Maxillule with two large spines and two smaller spines before notch; five pairs of spines and a cluster of 12 spines after notch. Mandible with five teeth; the third teeth tridentate; the fourth teeth quadridentate and the fifth teeth close to the fourth teeth; seven smaller setae on lower margin; without setae under inferior angle. Labrum with three canine-like teeth on each cutting margin. Penis with basi-dorsal point.
Description. Peduncle absent. Body length 2-3 cm. Shell white with orange longitudinal lines; low conic; composed of four shell plates including one carina, two laterals, and one rostrum (Fig. 1A, D, E). Base calcareous with parietal tubes; two rows of irregular shape and size of parietal tubes; inner laminar compartment carrying larger parietal tubes that its intraparietal septum radiating to the outer laminar; outer laminar compartment carrying three smaller and horizontal parietal tubes between larger tubes from inner laminar ( Fig. 1B, C). External shell plate ornamented with rough and white with orange longitudinal striation; some exhibited decolouration or erosion of shell plate but carina always possesses 4-5 remnants of orange spots close to the orifice (Fig. 1D). Basal margin of each shell plate irregularly undulated. Internal shell plate smooth and white; interior part close to orifice oranges and with horizontal striation (Fig. 1E). Orifice kite-shaped or pentagonal (Fig. 1F). External surface of opercular plates white with irregular orange-brown spots (Fig.  1F, G). Internal surface of opercular plates mostly white with orange ( Fig. 1F, H). Tergum smaller than scutum (Fig. 1G, H). Tergum triangular to polygonal shaped; dorsal surface with horizontal lines; longitudinal furrow on dorsal side broad. Spur of tergum broad with rounded tip. Scutal margin smooth without teeth. Basal margin of tergum longer than carina margin or area with lateral depressor muscle crests thick (ten crests). Tergal articular ridge with broad width but low ridge and thus when articulated, tergum occupies small area of scutum ( Fig. 1I-L). Scutum triangular with height similar to width. External sur-face of scutum without horizontal striation. Ventral surface of scutum with long adductor ridge. Lateral scutal depressor crests deep and numerous (five crests) ( Fig. 1M-R).
Cirrus I with unequal rami; anterior ramus (20-segmented) length longer than that of posterior ramus (10-segmented), approximately 2.5 times ( Fig. 2A); intermediate segments of posterior ramus normal or not protuberant; greater and lesser curvature of both rami  Maxilla bi-lobate; upper lobe covered with densely packed serrulated setae; lower lobe with a few serrulated setae carrying more packed setules ( Fig. 4A-C). Maxillule with Ushaped notch; two large spines and two small spines before notch; five pairs of small and slender spines after notch (Fig. 4D); cutting edge after notch carrying another 12 smaller spines followed by a cluster of serrulated setae (Fig. 4E). Mandible with five teeth; the first teeth largest; the second teeth bidentate; the third teeth tridentate; the fourth teeth quadridentate; the fifth teeth only single close to the fourth teeth; lower margin narrow with a pack of seven small setae followed by three larger setae close to inferior angle; no setae under inferior angle; simple setae scattered on surface of mandible ( Fig. 4F-H). Labrum with V-shaped notch; three canine-shaped teeth with densely packed simple setae on each side of cutting margin ( Fig. 4I-L). Mandibular palp rectangular with serrulated setae on superior margin (Fig. 4M, N). Penis long with annulation with basi-dorsal point on the dorsal side of penis base (Fig. 4O inset); a few simple setae scattered randomly along whole length; two bundles of simple and long setae found at the tip of penis ( Fig. 4O-Q).
Habitat. Neonrosella vitiata was collected only during the lowest tide (March) of the year and at the lowest littoral zone, an area submerged most of the time throughout the year. It was found on rocks covered with seaweed, densely packed green and red algae, hydroids, sponges, limpets, other acorn barnacles including Tetraclita species at the algal crust zone of the intertidal region. The barnacles were found mostly in solitary form, in connection with Newmanella spinosus, or with conspecifics as small colonies of only two or three individuals per colony.

Redescription of Newmanella spinosus Chan & Cheang, 2016
Figs 5-8 Newmanella radiata. Chan et al. 2009: 199, fig Diagnosis. Parietes and opercular plates green on external and internal surfaces. External shell plate with numerous radiating or longitudinal lines extending from apex to base. Scutal margin of tergum with serrated teeth and broad spur with cutting edges. Cirrus II with equal rami and slight curvature of both rami carrying triangular spines. Basis of cirri IV-VI without spines. Greater curvature of both anterior ramus and posterior ramus of Cirrus IV with triangular spines. Mandible with five teeth, the third teeth bidentate, the fourth teeth with serrations and small teeth along the edge, and the fifth teeth sits on the middle of lower margin surrounded by other small spines. Labrum with four teeth on each cutting margin. Penis without basi-dorsal point.
Maxilla bi-lobate; both lobes covered with serrulated setae (Fig. 8A, B, C). Maxillule with V-shaped notch; two large spines and five smaller spines before notch; six pairs of long slender spines and following seven smaller spines closed to inferior angle after notch (Fig.  8D); cutting edge after notch carrying a cluster of serrulated setae (Fig. 8E). Mandible with five teeth; the first teeth largest; the second and the third teeth bidentate; the fourth teeth serrated; the fifth teeth only single in the middle of lower margin surrounded by small and slender spines; lower margin narrow with a pack of 12 spines (irregular length); no setae under inferior angle (Fig. 8F). Labrum with V-shaped notch; four teeth with densely packed simple setae on each side of cutting margin (Fig. 8H-J). Mandibular palp rectangular carrying densely packed serrulated setae on superior margin (Fig. 8K-L). Penis long and annulated without basi-dorsal point (Fig. 8M inset); a few simple setae scattered randomly along whole length; at the tip of penis carrying two clusters of simple and long setae ( Fig. 8M-P).
Habitat. The specimens were collected only during the lowest tide (March) of the year at the lowest tide littoral zone, the same habitat as Neonrosella vitiata.
Distribution. Western Pacific from Taiwan (type locality) and Philippines (Chan and Cheang 2016) and Andaman Sea of eastern Indian Ocean (Phang-Nga Province, southern Thailand). ramus E apex of anterior ramus F serrulated setae on anterior ramus G serrulated setae on posterior ramus H plumose setae on posterior side of protopod I-P cirrus II I overview of cirrus II morphology J proximal region of cirrus II K serrulated and bidentate setae on greater curvature of posterior ramus L hook-like triangular spines on lesser curvature of posterior ramus M spines (asterisks) on lesser curvature of anterior ramus N close-up on apex on posterior ramus O serrulated setae on posterior ramus P serrulated and bidentate setae on anterior ramus Q-Y cirrus III Q overview of cirrus III morphology R slender spines on basipod S plumose setae on anterior side of basipod T hook-like triangular spines on lesser curvature of posterior ramus U hook-like triangular spines on lesser curvature of anterior ramus V greater curvature of anterior ramus with spines W serrulated setae and bidentate setae on posterior ramus X serrulated setae on anterior ramus Y bidentate setae on anterior ramus. Abbreviations: pr, protopod; bs, basipod; pos, posterior ramus; ant, anterior ramus. Scale bars in µm.

Discussion
The present study represents the first discovery of Neonrosella vitiata, sharing overlapping habitat with Newmanella spinosus in the Andaman Sea, eastern Indian Ocean. We previously reported a list of new record acorn barnacles in Thailand (the Gulf of Thailand and the Andaman Sea) and New. spinosus was also observed in the low-tide intertidal zone at Na-Tai District, Phang-Nga Province, southern Thailand (Pochai et al. 2017). The collection of new batches of specimens further down the rockweed at this region uncovered the presence of two newmanellin species (clearly recognized by their low conical shell plate with four parietes): one with white-background shell plates carrying decorations of dark orange spots and one with green shell plates. The white newmanellin species were thought to be Newmanella radiata redescribed in Chan and Cheang (2016). However, based on the shell morphology characters (white shell plate with radiating orange stripes and two-layered and unequal-sized parietal tubes, tergum with broad spur and longer basal margin carrying extensive lateral depressor crests than that of New. radiata), this provides a possible clue for the occurrence of Neonrosella. By observation under the conical shell plate, New. spinosus is easily distinguished from Neonrosella in that they possess multiple layers (three or more) of parietal tubes in honeycombed pattern. Based on ex- Penis long and annulated without basi-dorsal point amination of arthropodal characters by scanning electron microscopy, Neo. vitiata carried different morphologies of cirri I-VI in the presence and absence of triangular spines on greater and/or lesser curvature of the anterior and/or posterior rami. Additionally, Neo. vitiata and New. spinosus exhibited unequal rami in cirri I and III but equal in others, as described in Table 1. Unique characters among several body parts were found in the mandible and labrum. In the mandible of Neo. vitiata, the third and fourth teeth are tridentate and quadridentate while both teeth are bidentate in New. radiata. There are three teeth on each V-shaped cutting edge of Neo. vitiata but five in New. radiata. The obvious difference between Neonrosella and Newmanella is found in their intromittent organ or penis, in that Neo. vitiata carries basi-dorsal point on the base of penis while both New. spinosus and New. radiata have smooth dorsal surface of penis base.
Neo. vitiata in this study exhibited some similarities in shell plate morphology to Tetraclita vitiata Darwin, 1854 found in Philippines and Indo-west Pacific water, as described in Rosell (1972) as following: i) white conical shell plate ii) a few layers of irregular parietal tubes iii) long basal margin of tergum with several lateral depressor crests. However, our redescription of Neo. vitiata here report more distinct feature in following terms: colouration of external and internal shell plate with dark orange spots/lines and other arthropodal characters, including less number of cirral segments, the presence of serrulated and bipinnate setal types, the presence of triangular spines on both anterior and posterior ramus in cirri III, cirri IV-VI with three pairs of unequal setae, and in particular penis carrying hair tuft-like in group of two with basi-dorsal point.
Across all regions we examined in both the Gulf of Thailand and the Andaman Sea, Neo. vitiata was found only at the rocky shore-rockweed interface of the intertidal zone during the lowest tides, and the only one site for sample collection is Na-Tai, Phang-Nga. However, further investigations of more sampling areas at deeper depths of the intertidal zone are required and they may reveal a subtidal distribution of this species. In addition, the presence of Neo. vitiata in eastern Indian Ocean provides a possible scenario that before sea levels fluctuated by glaciation during the Pleistocene (e.g., Voris 2000), Neo. vitiata was already distributed across the Pacific Ocean towards the Indian Ocean.