Morphological and molecular evidence support the intertidal barnacle Octomeris intermedia Nilsson-Cantell, 1921 (Thoracica, Chthamalidae) as a valid species in Indo-Pacific waters

Abstract Octomeris is a chthamalid intertidal barnacle with eight shell plates. There are currently two species of such barnacles: O. brunnea Darwin, 1854 (type locality in the Philippines), common in the Indo-Pacific region, and O. angulosa Sowerby, 1825, only recorded in South Africa. Octomeris intermedia Nilsson-Cantell, 1921, identified from the Mergui Archipelago in Myanmar, was considered to be conspecific with O. brunnea by Hiro (1939) based on samples collected in Taiwan. The morphological differences in shell and opercular plates between O. brunnea and O. intermedia are believed to be intra-specific variations due to different degrees of shell erosion. In the present study, the genetic and morphological differentiations of Octomeris in the Indo-Pacific region were examined. This study found two molecular clades (with inter-specific differences) based on the divergence in the COI genes, and the species also have distinct geographical distributions. The Octomeris brunnea clade covers samples collected from the Philippines and Taiwan waters and the other clade, which we argue is O. intermedia, is distributed in Phuket and Krabi, Thailand and Langkawi, Malaysia. Phuket and Krabi are located approximately 300 km south of the Mergui Archipelago, the type locality of O. intermedia. The morphology of samples collected from Thailand fits the type description of O. intermedia in Nilsson-Cantell (1921). Our study concludes that O. intermedia is a valid species based on morphological and molecular evidence.


Introduction
Octomeris is a chthamalid intertidal barnacle with eight shell plates, in contrast to most of chthamalids which have four or six shell plates. In the early 19 th century, studies on the biology and ecology of Octomeris were very rare because this species inhabits shaded habitats and its presence was often overlooked (Nilsson-Cantell 1938). A recent molecular phylogenetic analysis of the family Chthamalidae included two species of Octomeris (Pérez-Losada et al. 2012) and considered Octomeris as paraphyletic; molecular evidence does not support the hypothesis that plate number decreased from eight plates to six, then four in the chthamalid evolution (Pérez -Losada et al. 2012).
In the Indo-Pacific region, Octomeris was considered to be composed of four species: O. brunnea, O. angulosa, O. sulcata, and O. intermedia. Octomeris sulcata has a strongly fused scutum and tergum, and Poltarukha (1996) relocated O. sulcata to the monotypic genus Pseudoctomeris. Chan et al. (2017) repositioned Pseudoctomeris from Chthamalidae to Pachylasmatidae based on multiple marker molecular analyses, leaving three species in Octomeris. Octomeris brunnea was described by Darwin (1854) from the Philippine archipelago; it has a brown colored depressed shell and longitudinal furrows on its surface. The tergal and scutal margins of the opercular plates are straight. Octomeris angulosa was described by Sowerby (1825) from the Cape of Good Hope in South Africa as having a dirty white strong conical shell and coarsely crenated shell plates (see re-description in Darwin 1854). Octomeris angulosa is common in wave-exposed shores in South African waters and often interacts with Tetraclita serrata (Boland 1997). Octomeris intermedia was described by Nilsson-Cantell (1921) from Java (note the erratum on the type locality (South Atlantic Ocean in Nilsson-Cantell, 1921) stated in Nilsson-Cantell (1937); Fig. 1), having a depressed shell and sinuous tergal and scutal margin. Nilsson-Cantell (1938) further recorded O. intermedia in the Mergui Archipelago in the Malay Peninsula (Fig. 1). Hiro (1939), however, collected a different size range of O. brunnea in Taiwan and observed that there is great variation in the shape of scutum and tergum at different ages and with different degrees of erosion. Highly eroded large individuals have a smooth flattened shell and a sinuous tergal and scutal margin, which resemble the morphology of O. intermedia. Juvenile and uneroded specimens represent the morphology of O. brunnea, which has longitudinally furrowed shells and straight scutum and tergum junction. Hiro (1939) concluded that O. intermedia and O. brunnea are conspecific. The conclusion of Hiro (1939) was further supported by Pope (1965), who examined O. brunnea in Australian waters and suggested that O. intermedia is an older specimen of O. brunnea. However, Hiro (1939) did not include O. intermedia in the geographical range suggested by Nilsson-Cantell (1921, 1938 (Java and the Mergui Archipelago), nor did the former compare the latter's samples of O. brunnea collected from Taiwan. To further test the conclusion by Hiro (1939), a combined morphological and molecular approach, known as integrative taxonomy (Dayrat 2005). is needed to compare O. intermedia collected from Java, Mergui Archipelago, and their adjacent waters with O. brunnea and ascertain the taxonomic status of these two species. In the present study, we collected O. intermedia from Phuket and Krabi, Thailand (300 km south of the Mergui Archipelago) and Langkawi, Malaysia, and O. brunnea from Taiwan and the Philippines; these samples cover different sizes and degrees of erosion. The mitochondrial cytochrome C oxidase subunit I (COI) and 12S rRNA genes were used as genetic markers to test the hypothesis that O. intermedia is an eroded form of O. brunnea in the Indo-Pacific region.

Study sites and sample collections
Samples of Octomeris intermedia were collected from Hey (or Coral) Island, Phuket (7°44'47" N; 98°22'44 E) and Ao Nang Beach, Krabi (8°02'08" N; 98°48'57 E), Thailand and Langkawi, Malaysia (Fig. 1). Octomeris in Thailand inhabits shaded rocks in the high intertidal zone, especially on vertical rock surfaces or shaded overhang surfaces in intertidal sea caves (Figs 1, 2). They can reach a percentage cover of 100% in some of the shaded rocks (Fig. 2C). Samples of O. brunnea (Fig. 2G) were collected in Kenting, Green Island, and Lanyu Island in Taiwan and Puerto Galera in the Philippines (Fig. 1). Octomeris brunnea was found on shaded rock surfaces and, occasionally, sun-exposed rocks. The abundance of O. brunnea was not high, with only a few individuals colonizing a shaded area. Representative specimens were deposited in the Biodiversity Research Museum, Academia Sinica (ASIZCR) and Collections in the first author's laboratory (CEL).

Morphological analysis
Before dissection, the shape of the junction line of scutum and tergum was recorded for all specimens with different size ranges. The morphological characters of shell parts (wall plates, scutum, and tergum) and somatic bodies (six pairs of cirri, penis, and trophi) were examined. The shells and opercular plates (scuta and terga) were immersed in 20% bleach for ca. 20 minutes to completely dissolve organic tissues, rinsed by purified water for 5 minutes, and air-dried. The cirri, penis, and trophi were dissected, mounted on glass slides, and observed under a light microscope (Olympus BX60, Japan). The morphology of the setae was described following the terminology of Chan et al. (2008).

DNA extraction, amplification, and sequencing
Total genomic DNA was extracted from the muscle tissue using Qiagen DNeasy® Blood & Tissue Kits (Qiagen, California, USA) according to the manufacturer's instructions. Partial sequences of mitochondrial DNA markers, COI, and 12S rRNA were amplified to reconstruct phylogenetic relationships. The primers used to amplify the sequences in the polymerase chain reaction (PCR) were LCO1490 and HC02198 for COI (Folmer et al. 1994) and 12S-F1 and 12S-R1 for 12S rRNA (Mokady et al. 1994). The PCR solution contained approximately 100-200 ng of template DNA, 0.4 μL each of 10 μM primer, 4 μL of Fast-Run TM Taq Master Mix with Dye (Protech Technology Enterprise, Taipei, Taiwan), and ddH 2 O to the final volume of 20 μL. PCR reactions were conducted in a DNA Engine Thermal Cycler (Bio-Rad, Richmond, California, USA). The thermal cycle began with an initial denaturation at 94°C for 4 min, then 35 cycles of denaturation at 94°C for 1 min, annealing at 49-51°C for 30 sec, and an extension at 72°C for 1 min (COI) and 30 sec (12S rRNA). The final extension step was at 72°C for 10 min. PCR products were checked by electrophoresis on 1.5 % agarose gel in 1 × TAE buffer. DNA purification and Sanger DNA sequencing were performed by Genomics BioSci & Tech Ltd. (New Taipei City, Taiwan). The sequences were assembled and edited in Geneious 7.0.6 (https://www.geneious.com).
All the sequences were aligned with ClustalW implemented in Geneious 7.0.6 (https://www.geneious.com). Neighbor-joining trees were generated on the analysis of Kimura 2-parameter (K2P) distances with bootstrap values estimated from 1,000 pseudoreplicates for two markers, separately (Felsenstein 1985;Kimura 1980;Saitou and Nei 1987). Bayesian inferences were conducted with 2 × 10 6 generations of the MCMC chain. Trees were saved every 1000 generations, and the first 500,000 trees (25%) were discarded as burn-in. Maximum likelihood was conducted with 1,000 bootstrap replicates for a Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) and ultrafast bootstrap approximation (UFB) (Guindon et al. 2010;Hoang et al. 2017). GTR+F+I+G4 and TVM+F+G4 were selected as the best-fit model under the Bayesian information criterion for COI and 12S rRNA, respectively (Kalyaanamoorthy et al. 2017). Genetic distances (K2P) between and within species were calculated by MEGA X 10.0.5 (Kumar et al. 2018).  Puerto Galera, Mindoro, Philippines

Octomeris brunnea Darwin, 1854
Shihtiping, Hualien, Taiwan Ao Diagnosis. Shell eight plated, conically depressed. Shell brown, surface with longitudinal furrows and tergo-scutal junction straight in young and uneroded specimens. Shell gray, surface smooth and tergo-scutal junction sinuous in old and eroded specimens. Maxillule with deep notch at upper 1/3 of cutting edge, lower 1/3 strongly protruded, cutting edge clearly divided into upper, middle, and lower region by the clear notch and protrusion of lower margin.
Cirrus I rami unequal (Fig. 5A). Posterior ramus short, six-segmented. Anterior ramus seven-segmented. All segments height greater than width (Fig. 5A). Bidentate serrate setae and simple setae present. Bidentate serrate setae appear up to seven segments in anterior ramus and present up to first three distal segment in posterior ramus (Fig. 5B-D). Cirrus II, posterior ramus seven-segmented, anterior ramus eight-segmented. Bidentate serrate setae present up to seven segments in anterior ramus and up to first four distal segments in posterior ramus (Fig. 5E-H). Cirri III to VI similar in morphology, long and slender (Figs 6, 7). Cirrus III, posterior and anterior rami 12 segmented ( Fig. 6A-D). Cirri IV and V, posterior and anterior rami 15-segmented (Figs 6E-H, 7A-C). Dorsal surface of cirri IV-VI has small spines (Figs 6G, 7F). Cirrus VI with 16 segmented rami ( Fig. 7D-E). Intermediate segments of cirri III and VI with three pairs of long and one pair of short simple setae (Figs 6B, F, 7B, E). Distal segments of cirrus III bear two pairs of long and one pair of short setae (Figs 6C, D, H,  7C). Caudal appendage absent. Penis short, tip with a few simple setae (Fig. 7G, H).
Maxilla subtriangular, distal lobe prominent and proximal lobe flat, shallow notch present in inner margin between the two lobes (Fig. 8A), inner and outer margin with serrulate setae (Fig. 8B-D). Maxillule with a deep notch on upper 1/3 and lower 1/3 of cutting edge. Cutting edge obviously divided into three distinct portions. Cutting edge above upper notch with two large and a few setae (length of setae ranges from 80-100 μm); middle portion of cutting edge has six setae; 1/3 of lower portion of cutting edge has eight short setae (Fig. 8E-H). Mandibles with three teeth, cutting edge of first tooth smooth, second tooth with one or two spines, third tooth with a few spines on cutting edge (Fig. 9A-D). Mandibular palp elongated, with serrulate setae on outer margin (Fig. 9E, F). Cutting margin of labrum concave, with small fine teeth (Fig. 9G, H).      Distribution. Southern Japan, Taiwan, Philippines, east coast of Queensland in Australia, Santa Cruz and New Hebrides (based on specimens in Australian Museum stated in Pope, 1965). Figures 2A-F, 10-14 Diagnosis. Shell eight plated, very depressed, surface brown with longitudinal furrows on uneroded specimens, tergo-scutal junction sinuous, except for young specimens. Maxillule with very shallow notch at upper 1/3 of cutting edge, lower 1/3 slightly protruded.
Maxilla subtriangular, inner margin with an inconspicuous notch, inner and outer margins with serrulate setae (Fig. 13A-D). Maxillule with two very shallow notches on upper 1/3 and lower 1/3 of cutting edge. Cutting edge more or less straight but the region above notch with one large and a few setae; middle margin has nine setae; cutting edge below upper notch has > 20 short setae (Fig. 13E-H). Mandibles with three teeth, first tooth with smooth edge and second teeth with slightly serrated edge, third tooth with smooth edge but occasionally with some spine on edge region (Figs 14A-D, 15A-C). Mandibular palp elongated, with serrulate setae on outer margin (Fig. 14E, F). Cutting margin of labrum concave, with small fine teeth (Fig. 14G-H).
Distribution. Java in Indonesia, Mergui Archipelago in Myanmar, Phuket and Krabi in Thailand.
Remarks. O. intermedia collected in the present study represents the specimens described by Nilsson-Cantell (1921, 1938 who collected O. intermedia from Java and the Mergui Archipelago; Phuket and Krabi are approximately 300 km south of the Mergui Archipelago. The morphology of our specimens fits the description in Nilsson-Cantell (1921, 1938: the shell is depressed and has a sinuous junction between the tergum and scutum. The external shell morphology of O. brunnea and O. intermedia is very similar. Old and eroded specimens of O. brunnea have a sinuous junction line between scutum and tergum, which is one of the characteristics of O. intermedia described by Nilsson-Cantell (1921). There are, however, several consistent diagnostic features between O. intermedia and O. brunnea. The shell of O. intermedia is much depressed in comparison to that of O. brunnea. The junction of the tergum and scutum in O. intermedia is sinuous, even at the young stage (except for very small individuals, RC-diameter < 10 mm; Fig.  3). In contrast, young individuals of O. brunnea have a straight junction between the scutum and tergum, and this junction line becomes sinuous when the barnacles get older (Fig. 3). There are some variations in the number of spines (-2-4) on the region between the second and third teeth of mandibles in O. brunnea and O. intermedia (Fig. 15). But both species have similar range of variations and there are no diagnostic differences between the two species (Fig. 15). The maxillule of O. brunnea has very deep notches on the upper and lower 1/3 portions of the cutting edge dividing it into three distinct portions. While the maxillule of O. intermedia has shallow notches on the upper and lower 1/3 portions of the cutting margin looking more or less straight without being dividing into three distinct regions as in O. brunnea. Such differences are considered consistent based on observations of the additional three specimens from both species (Fig. 16). Nilsson-Cantell (1921) has not state any deposition nor specimen number of type or paratype specimens of O. intermedia. The foreword section of Nilsson-Cantell (1921) stated majority of specimens in Nilsson-Cantell (1921) were obtained from collections in Swedish Imperial Museum in Stockholm, Sweden and Zoological Museum in Uppsala, Sweden. The specimens of O. intermedia are possibly housed in either one of the two museums above. Before checking the presences or absences of O. intermedia in museum collections in Sweden, the present study did not attempt to establish any neotypes of O. intermedia to avoid taxonomic confusion. The information of the COI gene in GenBank for O. intermedia is currently adequate for future studies to confirm identification of specimens collected.

Molecular analysis
All the phylogenetic results suggested that both Octomeris brunnea and O. intermedia were clustered their own clades with high bootstrap values and posterior probabilities. The sequences from Fisher et al. (2004) and Pérez-Losada et al. (2012), which were designated as O. brunnea, were clustered with O. intermedia collected from Malaysia and Thailand by the NJ method (Fig. 17A, B). The phylogeny reconstructed by ML and BI suggested that O. brunnea and O. intermedia were sister groups (Fig. 18).
The K2P distances within O. brunnea and O. intermedia were 0.007±0.001 and 0.005±0.001 for the COI sequences, and 0.004±0.001 and 0.004±0.001 for the 12S rRNA sequences, respectively. The K2P distances between O. brunnea and O. intermedia were 0.098±0.013 and 0.043±0.001 for the COI and 12S rRNA sequences, respectively. The K2P distances between these two species and other species ranged from 0.207 to 0.251 for the COI sequences and 0.167 to 0.303 for the 12S rRNA sequences (Table 2).

Discussion
In the present study, we conclude that Octomeris intermedia is a valid species using integrative taxonomy. There are consistent morphological differences in the shell and maxillule of O. intermedia and O. brunnea, suggesting they are two distinct species. intermedia and O. brunnea in the phylogenetic analysis suggests that these two species may have formed when the Sunda Shelf was exposed during the Pleistocene glaciations, separating the Indian and Pacific Oceans (Voris 2000). Many sister taxa or distinct population genetic divergences in other marine species, including Tetraclita and Chthamalus barnacles and coral reef fishes, also formed when the Indian and Pacific Oceans separated during the last glacial maxima (Bowen et al. 2001;Tsang et al. 2011Tsang et al. , 2012.     Fisher et al. 2004). Pérez-Losada et al. (2012) included Octomeris angulosa collected in Sydney, Australia in their phylogenetic analysis. According to Pope (1965) and Jones (2012), only Octomeris brunnea has been recorded in Australian waters. Only the 12S rRNA sequences of this O. angulosa from Pérez-Losada et al. (2004)  Future studies should focus on the diversity and taxonomy of Octomeris in Australia.
Intertidal barnacle diversity in Thailand received very little attention until the recent studies of Pochai et al. (2017) and Sukparangsi et al. (2019), who conducted detailed surveys of Thai intertidal barnacles and recorded a total of eleven species from the Thai coastline. The distribution of intertidal barnacles is different between the coastline in the Gulf of Thailand and the Andaman Sea (Pochai et al. 2017): the Andaman side has a higher species diversity (nine total species in Andaman side and six in the Gulf of Thailand). Octomeris was not reported by Pochai et al. (2017) or Sukparangsi et al. (2019). The record of O. intermedia in the present study brings the number of Thai intertidal barnacle species to 12. No Octomeris were found during sampling trips by the first author to Si-Chang Island and Chumporn in the Gulf of Thailand. This suggests that the coastlines in Thai waters that O. intermedia is located is probably the Andaman Sea. Therefore, there are ten species of intertidal barnacles on the Andaman side, and six in the Gulf of Thailand.