Diversity of intertidal, epibiotic, and fouling barnacles (Cirripedia, Thoracica) from Gujarat, northwest India

Abstract The present work studied the diversity of intertidal, epibiotic, and fouling barnacles in the state of Gujarat, northwest India. In total, eleven species belonging to eight genera and five families were recorded in the present study. The Arabian intertidal species Tetraclita ehsani Shahdadi, Chan & Sari, 2011 and Chthamalus barnesi Achituv & Safriel, 1980 are common in the high- and mid-intertidal rocky shores of Gujarat suggesting that the Gujarat barnacle assemblages are similar to the assemblages in the Gulf of Oman Ecoregion. The biogeographical boundary between the Gulf of Oman and Western Indian ecoregions for barnacles should probably extend southward towards the waters adjacent to Mumbai, where Indo-Pacific species of intertidal barnacles dominate. This study provides the first reports of the common widely distributed balanomorph barnacles Striatobalanus tenuis (Hoek, 1883), Tetraclitella karandei Ross, 1971, Amphibalanus reticulatus (Utinomi, 1967), and lepadid barnacle Lepas anatifera Linnaeus, 1758 in Gujarat, as well as of the chthamalid barnacle Chthamalus barnesi in India.


Introduction
Barnacles are marine crustaceans that inhabit a diverse range of substrates, including rocks, molluscan shells, corals, sponges, mangrove roots and leaves, turtle shells, and whale skin (Chan and Høeg 2015;Kim et al. 2020). Fossilized barnacle shells are often used to study the past environment (Bianucci et al. 2006a, b;Collareta et al. 2016aCollareta et al. , b, 2018Buckeridge et al. 2018Buckeridge et al. , 2019. Burmeister (1834) was the first to classify barnacles into cirripedes, which later attracted the attention of numerous taxonomists including Charles Darwin (Anderson 1994). Barnacles have ecological and economic importance, as some species are biofoulers and others are considered seafood in some countries (Walker 1972;Newman and Abbott 1980;Santhakumaran and Sawant 1991;Rawangkul et al. 1995;Molnar et al. 2008;Sophia Rani et al. 2010;Holm 2012). More than 1400 species of barnacles are listed globally (Chan et al. 2009), and most are abundant along the intertidal and subtidal zones of temperate and tropical regions (Frith et al. 1976;Brickner and Høeg 2010;Brick ner et al. 2010;Sophia Rani et al. 2010;Chen et al. 2012Chen et al. , 2014Hayashi 2013;Yu et al. 2016). Taxonomic study of the Indian barnacle fauna dates back to the systemic work carried out by Darwin (1854), which was followed by several important studies in the 1900s (Annandale 1906(Annandale , 1909(Annandale , 1914Nilsson-Cantell 1938;Daniel 1956Daniel , 1972Daniel , 1981. Fernando (2006) prepared a monograph on the barnacles of India in which he recorded 70 species of barnacles from Indian waters. Spalding et al. (2007) classified the world's biogeographical provinces and ecoregions within provinces. The Persian Gulf, Gulf of Oman, and Arabian Sea belong to two provinces ( Fig. 1A): the Arabian Province includes the Persian Gulf, Gulf of Oman, Western Arabian Sea, and Central Somali Coast Ecoregions. The West and South India Shelf Province covers the western and southern coastlines of India and Sri Lanka and is divided into the Western Indian Ecoregion and South India and Sri Lanka Ecoregion. Gujarat is the westernmost state of India and contains 1650 km of coastline (Fig. 1A, B). It possesses a variety of coastal habitats, including mangroves, coral reefs, rocky shores, mudflats, sandy shores, and estuaries (Fig. 1C-E;Trivedi et al. 2015). In the present work, we describe the species recorded in Gujarat and discussed the similarity in the assemblages of Gujarat between the Gulf of Oman and Western Indian Ecoregions.

Specimen collection and identification
Specimens were collected during low tides using a hammer and chisel from 2010-2020. Photographs of live specimens were taken in the field and then preserved in 10% formalin or 95% ethanol for further analysis in the laboratory. In the laboratory, barnacles were first identified based on their shell morphometry using a stereomicroscope. Specimens were gently dissected from their shell under a stereomicroscope with camera for specimen identification. The following barnacle parts were dissected: mouthparts (maxilla, maxillule, mandible, mandibulatory palp, and labrum), tergum, and scutum. The identification key of  was used for basic taxonomic identification as well as for general terminologies of shell morphol ogy and other important characters. All the specimens were deposited into the Zoological Reference Collection (LFSc.ZRC), Department of Life Sciences, Hemchandracharya North Gujarat University, Patan, Gujarat, India and Biodiversity Research Museum (ASIZCR), Academia Sinica, Taiwan. Rostral-carinal basal diameter of shells (BD) of sessile barnacles and capitulum length (CL, from the basal margin of scutum to apex of tergum) of stalked barnacles were measured to the nearest 0.01 mm.

Zonation pattern of rocky intertidal species at Diu, Gujarat
To examine the zonation of intertidal barnacles, stratified transect surveys were conducted in two rocky shores of Nagoa Beach in Diu (20°42.12'N,70°55.0217'E and 20°42.17'N,70° 53.94'E). The maximum tidal range at Diu is approximately 2.5 metres. At each shore, 10-m-long stretches of shoreline were selected. Sampling was conducted at the highest tidal level at which chthamalid barnacles were found (2 m above Chart Datum, C.D.). Subsequent tidal levels were sampled at 0.5 m vertical intervals, 1.5 m above C.D. and 1.0 m above C.D. At each tidal level, ten random 0.25 × 0.25 m quadrats were established and the number of individuals of each species of barnacles was scored.

Distribution in India.
This species has been reported from Gujarat (present study), Tamil Nadu (Krishnamoorthy 2007;Daniel 1956), Odisha (formerly Orissa) (Nilsson-Cantell 1938), and Karnataka (Nilsson-Cantell 1938). Remarks. The specimens examined in the present study agree with the descriptions given by Henry and McLaughlin (1975), Pochai et al. (2017). Amphibalanus amphitrite closely resembles A. reticulatus (Utinomi, 1967) but differs from the latter in the following characters: the shell plates have only vertical purple striation (shell plates have longitudinal stripes intersected with transverse striations in A. reticulatus: Pochai et al. 2017), the shape of the shell is comparatively less columnar than in A. reticulatus (Pochai et al. 2017).

Balanidae Leach, 1817
Worldwide distribution. This species has been reported from Bermuda and southeast USA to Brazil, Hawaii, California to southwest Mexico, western European waters, Mediterranean Sea, south coast of Africa, Red Sea, Black Sea, Southeast Africa, India (Trivedi et al. 2015), Australia, Indonesia, Singapore, Malaysia, Gulf of Siam in Cambodia (Jones and Hosie 2016), Vietnam (Condor Islands, Tang Trien (South Annam), Cauda Nhatrang, Hongay, Tonkin), the South China Sea, Bohai Sea (China), Taiwan, the Philippines, Japan (South Honshu, Kyushu and Ryukyu Islands) and Vladivostok (Russia) (see review in Henry and McLaughlin (1975)).
Distribution in India. This species has been reported from Gujarat (Trivedi et al. 2015;Parmar et al. 2018;present study), Maharashtra (Bhatt and Bal 1960), Goa (Desai et al. 2018), Kerala (Nilsson-Cantell 1938, Tamil Nadu (Prasanth and Sureshkumar 2020), Andhra Pradesh (Rao and Balaji 1988), Pulicat Lake (Sanjeeva 2006), Odisha (formerly Orissa) (Mitra et al. 2010), West Bengal (Ramakrishna and Talukdar 2003), and Andaman and Nicobar Islands (Mishra et al. 2010). (Utinomi, 1967 Remarks. The specimens examined in the present study agree with the descriptions and illustrations given by  and Pochai et al. (2017). Amphibalanus reticulatus is very similar to A. variegatus (Darwin, 1854), in which both shells have striated patterns. Pitriana et al. (2020) illustrated the scutum, tergum, and mandibles of A. variegatus. The gaps between the teeth in the mandibles are smaller in A. variegatus than in A. reticulatus. In the present study, we concluded the mandibles of the specimens collected from India have relatively larger gaps between the teeth compared to the illustration in Pitriana et al. (2020). In addition, the tergum of A. variegatus illustrated in Pitriana et al. (2020) has a sharp spur, while the spur of the Indian specimen is blunt. We conclude the specimens collected in the present study represent A. reticulatus.

Amphibalanus reticulatus
Worldwide distribution. This species has been reported from Japan, Indo-West Pacific (the Philippines, Hawaii, Gulf of Thailand, Indonesia; Pochai et al. 2017), Australia, Persian Gulf, and India (Fernando 2006).
Distribution in India. This species has been reported from Gujarat (present study), Maharashtra (Swami et al. 2011), and Tamil Nadu (Fernando 2006).
Remarks. The specimens examined in the present study agree with the original description given by Linnaeus (1758) and the more recent one by . However, in the present specimen, the labrum does not possess teeth whereas the specimen examined by  has three sharp teeth on each side of the cutting edge.
Megabalanus tintinnabulum closely resembles M. validus Darwin, 1854, but differs from the latter in having a conical shell with a coloured external surface. The species also resembles M. volcano (Pilsbry, 1916), but differs from the latter in having the maxillule not notched.
Worldwide distribution. The species has a cosmopolitan distribution with records from Brazil, Venezuela, European waters (UK, Ireland, Belgium and Netherlands; Southward, 2008) the Mediterranean Sea, Madagascar, Cape of Good Hope, New Zealand, Australia, Singapore, Thailand, Vietnam (Jones and Hosie 2016), Hong Kong, Taiwan, Japan, and India (Trivedi et al. 2015).
Remarks. Previously, Chelonibia living on decapods were identified as C. patula and Chelonibia living on sea turtles as C. testudinaria. Cheang et al. (2013) and Zardus et al. (2014) revealed there is no significant genetic difference between C. patula and C. testudinaria, suggesting that these are the same species and their morphological differences are the result of phenotypic plasticity. We consider C. testudinaria as including two major morphs. The patula morph has a smooth white shell and lives mainly on decapods, while the testudinaria morph has oval depressions on the radii and lives mainly on surfaces of turtles. Dwarf males are often housed in these depressions on the testudinaria morph (Zardus and Hadfield 2004;Collareta 2020).
Remarks. The examined specimens in the present study agree with the description given by . Tetraclita ehsani closely resembles T. reni Chan, Hsu & Tsai, 2009, T. achituvi Ross, 1999and T. rufotincta Pilsbry, 1916, but can be differentiated from these species in the following characters: the tergum is very narrow, with the basal region slightly concave or almost straight vs. the broad tergum that has a strongly concave basal margin in T. rufotincta and T. reni, and the basi-carinal angle is larger (~ 100°) (the basi-carinal angle is smaller in T. reni (80°) and T. rufotincta (73°) .
Worldwide distribution. This species has been reported from the Gulf of Oman in Iran ) and from northwest India (Tsang et al. 2012).
Distribution in India. This species has been reported from Gujarat (Tsang et al. 2012;present study). It is not found in the region further south of Gujarat and was confirmed to be absent in Mumbai and southern India (Tsang et al. 2012). Diagnosis. Shell four-plated, surface of radii protruding with digit-like horizontal striations up to the shell apex, shell surface with fine hairs and chitin coating (Fig. 3C, D). Opercular plates white, scuta triangular, occludent margin and basal margin almost perpendicular, tergal margin straight; tergum higher than wide, scutal margin straight, spur small (Fig. 3E). Maxilla bilobed (Fig. 10A). Maxillule notched, with two cuspidate setae above notch (Fig. 10B). Mandible having four teeth, the 3 rd and 4 th of which are triple-dentated (Fig. 10C). Labrum slightly bullate, with two small teeth on each cutting edge (Fig. 10D). Mandibulatory palp elongated with dense setae on superior angle (Fig. 10E). Cirrus I: anterior ramus seven-segmented, posterior ramus longer and slender, nine-segmented. Cirrus II: rami subequal, anterior ramus six-segmented, posterior ramus seven-segmented (Fig. 10F-H). Cirrus III: both rami slender, anterior ramus 13-segmented, posterior ramus 14-segmented. Intermediate segment bears two pairs of long simple setae and three pairs of short simple setae (Fig. 10F-H).

Tetraclitella karandei Ross, 1971
Remarks. This species inhabits intertidal shore of the rocky intertidal region of Gujarat.
Worldwide distribution. This species has been recorded in India and Taiwan (Ross 1971(Ross , 1972.

Distribution in India.
This species has been reported from Gujarat (present study) and Mumbai (Ross 1971;Fernando 2006).
Remarks. The examined specimen in the present study agree with the description given by Achituv and Safriel (1980) and . Chthamalus barnesi forms part of the challengeri group and closely resembles C. moro Pilsbry, 1916, C. neglectus Yan &Chan, 2004, andC. challengeri Hoek, 1883, but can be differentiated based on the following characters: a depression towards the tergo-occludent corner of the scutum (C. moro, lacks this depression, Southward and Newman 2003), the tergal margin is not straight (tergal margin straight in C. moro, Southward & Newman, 2003), the scutal margin of the tergum shows a deep articular furrow (scutal margin of tergum almost straight in C. neglectus, Yan & Chan, 2004), and the maxillule possess a very shallow notch (maxillule possesses a distinct notch in C. challengeri, . Worldwide distribution. The species has been reported from the Red Sea, Gulf of Aden, and Gulf of Oman including Yemen, Oman, Iran, Saudi Arabia , and northwest India (present study).
Distribution in India. This species is reported for the first time in India from the coastal regions of Gujarat.
Diagnosis (modified from Pilsbry 1916). Specimens depressed, cinnamonbrown with smooth surface, with a large, wide aperture; alae broad with arched, subhorizontal summits (Fig. 3I). Scutum thin, triangular, conical, almost twice as long as wide, lower part with fine growth-lines (Fig. 3J). Articular ridge feebly developed with median lobe, not extending beyond the scutal border. Articular furrow shallow and sharply notched. Tergum narrow, club-shaped, very thick (Fig. 3J). Cirrus I: anterior ramus (with seven or eight segments) longer than posterior (usually with six or seven segments). Cirrus II: anterior ramus (with seven or eight segments) longer than posterior (usually with six segments). Setae of terminal segment non-pectinated. The carinal lobe narrow, situated high. Maxilla bilobed (Fig. 12A), group of short spines on the lower edge. Maxillule not notched (Fig. 12B). Mandible with three large teeth and pectinated lower point with eight spines (Fig. 12C-E). Mandibulatory palp rectangular (Fig. 12F). Labrum with broad, nearly straight edge, the middle fold having a series of strong teeth (Fig. 12G, H).
Remarks. The specimens examined in the present study agree with the description by Pilsbry (1916). Microeuraphia withersi closely resembles M. depressa and M. permitini, but can be distinguished from the latter based on the following characters: the scutum is comparatively narrow in (scutum is comparatively wide in M. depressa, Poltarukha, 1997), the width to height ratio fluctuates from 0.8 to 1.4 (width to height ratio commonly > 1.5 in M. depressa, Poltarukha, 1997), the basal comb of mandible with eight equally distanced slender spines (1-3 stout spines after third tooth, and a row of small and 2-4 long spines in M. permitini, , and both the rami of cirri II without finely pectinate setae on terminal segments (both rami of cirri II with finely pectinate setae on terminal segments in M. permitini; . Worldwide distribution. The species has been reported from the Philippines (Pilsbry 1916), the west coast of Sumatra (Nilsson-Cantell 1921), Indonesia, Singapore, Java, Vietnam, Hong Kong, the South China Sea (Jones and Hosie 2016), the East China Sea (Zevina and Tarasov 1963), Australia, Madagascar (Utinomi 1968), andIndia (Nilsson-Cantell 1938).
Remarks. The specimens examined in the present study agree with the description given by . Lepas anatifera closely resembles L. anserifera Linnaeus, 1767, but can be differentiated by the following characters: maxillule notched into four distinct regions (maxillule not clearly notched in L. anserifera, ), upper portion of tergum blunt (upper portion of tergum pointed in L. anserifera, ), scutum sometimes with dark marking or spots (no such markings or spots on scutum in L. anserifera, ).
Worldwide distribution. The species has a cosmopolitan distribution Schiffer and Herbig 2016) that includes India (Krishnamoorthy 2007).
Remarks. The specimens examined in the present study agree with the descriptions given by Fernando (2006) and .
Worldwide distribution. This species has a cosmopolitan distribution in tropical and temperate seas Jones and Hosie 2016;Schiffer and Herbig 2016) and in India (Annandale 1909).

Zonation patterns of rocky intertidal species
The high shores (2 m above C.D.) of the sandstone rocky shores at Diu are filled with Chthamalus barnesi, reaching a mean abundance of 20-50 individuals per 0.25 × 0.25 m 2 quadrat. In the mid-shores (1.5 m above C.D.), C. barnesi and T. ehsani occur together, with similar abundances of 40-90 individuals per 0.25 × 0.25 m 2 . In the low shores, C. barnesi is absent, and T. ehsani has a low abundance and co-exists with Megabalanus tintinnabulum (Fig. 15).

Discussion
The present study reported a total of eleven species from Gujarat, northwest India and is the first record of the rocky intertidal barnacle Chthamalus barnesi in India. Tetraclita ehsani was previously recorded from the Gulf of Oman, Iran, and northwest India. Tetraclita ehsani is absent from the Persian Gulf and Red Sea, where T. rufotincta is a common species (Tsang et al. 2012). Northwest India is probably the southern limit of T. ehsani, as this species is absent from Mumbai and Tamil Nadu (based on personal sampling trips by BKKC). Chthamalus barnesi was first identified along the coastline of the inner Red Sea (Achituv and Safriel 1980) and was subsequently reported in the Persian Gulf and Gulf of Oman . Northwest India appears to be the eastern biogeographical limit of C. barnesi, as it is absent from Mumbai and further south. From Mumbai and along the southern and eastern coastlines of India, C. malayensis becomes dominant (Tsang et al. 2012). Based on the classification of the world's biogeographical provinces and ecoregions by Spalding et al. (2007), Gujarat is located in the Western India Ecoregion of the West and South India Shelf Province (Fig. 1). The Gulf of Oman and Persian Gulf are two separate ecoregions located in the Arabian Province. Based on rocky intertidal barnacles, the Gulf of Oman Ecoregion should include Gujarat, while the boundary to the Western Indian Ecoregion appears to be adjacent to waters around Mumbai. Similar patterns may emerge from other groups of marine species. Extensive studies on the biogeography of different groups of organisms across these two ecoregions should be conducted. There are nine species with a very wide geographical distribution in the Indo-Pacific, all of which are recorded in Gujarat. Lepas anatifera and L. anserifera are pelagic species that attach to floating objects and get carried by ocean currents (Schiffer and Herbig 2016). Chelonibia testudinaria is epibiotic on turtle and decapod hosts. Population genetics studies revealed that there are genetic differences among Western Pacific, Eastern Pacific and Western Atlantic populations of C. testudinaria (Rawson et al. 2003).
Amphibalanus amphitrite, A. reticulatus, and Megabalanus tintinnabulum are common fouling species that disperse via ballast water or shipping industries. Chen et al. (2014) examined the world-wide genetic differentiation of A. amphitrite and identified three molecular clades, which include a worldwide clade (present in most of the world's oceans); a second clade common in tropical regions; and a third clade that is only found in the Eastern Atlantic waters. The genetic differentiation among fouling barnacles could be a result of the combined effects of historical events such as Pleistocene sea level changes and human-mediated dispersals (Chen et al. 2014).
Some Indo-Pacific species were recorded in the present study. The intertidal barnacle T. karandei was first identified in Mumbai, India (Ross 1971); Ross (1972) subsequently recorded it in Taiwan. The present study is the third report of this species in northwest India. Striatobalanus tenuis is a widely reported epibiotic species that often attaches to deep-water crustaceans and mollusc shells. Microeuraphia withersi is a high shore chthamalid barnacle common on shaded regions of the Indo-Pacific rocky shores (Poltarukha 1997). There are currently no genetic studies on the diversity or population genetics of these species. It is possible that cryptic species are present among these nominal species across the Indo-Pacific region, like for many other barnacles, as well as for hermit crabs and other decapods (Chan et al. 2007;Tsang et al. 2012;Jung et al. 2018;Shih and Poupin 2020) or that they are homogeneous populations across large geographical expanses (see example of intertidal blennies in Hongjamrassilp et al. 2020). Future research should also focus on the diversity and biogeography of rhizocephalan species in India, as this superorder of barnacles remains extremely understudied in India. It is possible that Indian rhizocephalan species are present in decapods and hermit crabs and exhibit distinct biogeographical distributions similar to the patterns recognised in the Northwest Pacific (Jung et al. 2019).