Tropical Marine Science Institute, National University of Singapore, 14 Kent Ridge Road, Singapore 119223, Republic of Singapore
1. Introduction:
Barnacles of the genus Tetraclita inhabit the middle intertidal zone of temperate to tropical seas. They are also commonly found in fouling communities. Currently, there are 16 species (subspecies) distributed throughout the world, four of which are distributed in the western Pacific, namely: Tetraclita squamosa (central Japan, northern Australia to India); T. japonica (northern Japan to Hong Kong); T. formosana (central Japan to Taiwan) and T. serrata (Philippines). While species from temperate and subtropical region have been relatively well studied, those in the tropical regions are poorly known. To date, only two species, T. squamosa and T. serrata, have been reported from the tropical western Pacific, with the former widely distributed throughout the region, while the latter has only been reported from one location in the Philippines. Recent taxonomic studies by the author based on morphology found confusion in the taxonomy of the genus. Morphological data suggests there are, at least, four forms (morphs) of Tetraclita squamosa in the region. Most of these forms may well represent different species. Tetraclita serrata from the Philippines varies greatly with type material from South Africa, and probably represents an undescribed species. For East Asian species, the taxonomy is still far from satisfactory. The validity of T. formosana remains a subject of dispute (Ren & Liu, 1979; Yamaguchi, 1987; Hasegawa et al, 1996).
In the last decade, molecular data, especially mitochondrial DNA (mtDNA) has been extremely useful and extensively used in elucidating phylogenetic relationships among many crustacean groups. It has been useful in differentiating several questionable species (Dando,1987: biochemical; Dando & Southward, 1980: enzyme of Chthamalus, western Atlantic; Dando et al, 1979: enzyme of Chthamalus; Achituv & Mizrahi, 1987: allozyme, Red Sea; Pannacciulli, 1997: enzyme, NE Atlantic & Mediterranean; Wares, 2001: Chthamalus of North America; Appelbaum et al, 2002: SSCP, Red Sea; Mokady et al, 1999: coral inhabiting barnacles; 2000: Chthamalus). It has also been effective for phylogenetic and population studies of barnacles (Mizrahi et al, 1998; Harris et al, 2000; Pérez-Losada et al, 2004; Zardus & Hadfield, 2005). In the genus Tetraclita, Yamaguchi (1987) used results of enzyme electrophoresis to separate three species, T. squamosa, T. japonica and T. formosana, formerly all regarded as subspecies of T. squamosa. Hasegawa et al. (1996) demonstrated the phylogenetic relationship among these three species using mtDNA (COI), and suggested that T. squamosa represents the ancestral stock of the remaining two, which are closely related sister species. Yamaguchiˇ¦s (1987) result is, however, only based on populations from a single site in central Japan, while Hasegawa et al. (1996) examined relationships among the three based on Japanese material only.
The author has completed morphological comparisons of material collected from throughout the western Pacific. Morphological data are however not conclusive and the lack of original type material for T. squamosa makes the comparisons impossible. Thus, the study herein, to combine morphological results with molecular data, may be the only reliable way to resolve taxonomic issues in this genus. The present study compares partial sequences of the mitochondrial genes 12S rRNA, 16S rRNA, and cytochome oxidase I subunit (COI) among several species of Tetraclita, as well as among populations of T. squamosa from the western Pacific to estimate the extent of genetic diversity among these populations (or species). In combination with morphological results, the aim is to reconstruct the phylogeny of the genus in the region under study; to clarify the taxonomy of the genus Tetraclita in the region; and investigate the geographic distribution pattern of the genus in the region by examining (1) Phylogenetic relationships of populations and genetic lineages; (2) population genetic distance, and (3) morphological grouping.
2. Materials and Methods:
Collection of materials
Specimens of Tetraclita spp. were collected from 16 localities (Table 1), including two non-western Pacific areas, Australia and Panama. The specimens under study include all putative species (phenotypes) in the region, and 2 species outside the region. Individuals were collected live and muscle tissue was dissected and stored in 95% ethanol.
DNA extraction, PCR amplifiication and sequencing
Genomic DNA was extracted according to the manufacturer's protocol using a QIAGENTM DNeasy nucleic acid extraction kit. DNA was eluted in deionized autoclaved water, and stored at -20oC. Polymerase chain reaction (PCR) amplification of DNA was performed and amplicons were purified with QIAquick spin columns (QIAGEN). Forward and reverse strands were cycle-sequenced using the PCR primers as listed in Table 2. DNA was extracted from 74 individual barnacles. PCR amplification and DNA sequencing of 3 genes (COI, 12S, 16S) was completed for 34 specimens. Experiments were conducted either at the Kewalo Marine Lab, University of Hawaii, or at Tropical Marine Science Institute, National University of Singapore. One sequence of the three genes from GenBank (complete genome for Tetraclita japonica mtDNA, accession numbers NC-008974) was included in the phylogenetic analysis.
Data analysis
For those species with representatives from multiple populations, one individual from each population was used in subsequent analyses. Forward and reverse sequences for each individual were edited using SequencherTM v4.5 or BioEdit v5.0.9 (Hall, 1999). COI data were confirmed and aligned by converting the sequences to their amino acid translation. Data sets for the three mtDNA genes were concatenated as all genes in the mitochondrial genome are linked and should share the same phylogenetic history as suggested by Perez-Losada et al., (2004). To test consistency of phylogenetic signal in the data, phylogenetic relationships were inferred using three different analytical approaches (neighbour- joining, maximum parsimony, and maximum likelihood), all performed with PAUP* version 4.0b10 (Swofford, 2000). Maximum parsimony (MP) (Camin and Sokal, 1965) analyses were conducted assuming equal weightings for all characters. Neighbour-joining (NJ) (Gascuel, 1997; Saitou and Nei, 1987), and maximum likelihood (ML) (Felsenstein, 1981) analyses were based on phylogenetic relationships estimated using an appropriate DNA substitution model calculated with ModelTest version 3.6 (Posada and Crandall, 1998). The program uses a hierarchical likelihood ratio test (hLRT) in order to obtain the model that best fits the data. In all cases, the heuristic search option was used with tree-bisection-reconnection (TBR) branch swapping and 50 stepwise random additions of taxa. Branch support was assessed using bootstrap resampling (Felsenstein, 1985) with 1,000 replicates, and the full heuristic search algorithm, to evaluate the reliability of the inferred topologies, for both NJ and MP. Bootstrap resampling was run with the "fast" stepwise addition algorithm and 100 replicates for ML because of the large number of taxa involved and computational time required. Sequence divergences between species pairs were calculated using the Kimura 2-parameter method (Kimura, 1980).
3. Results:
The analysis was carried out by PAUP* v4.0b10 for Macintosh (PPC/Altivec). The three analytical methods (NJ, MP and ML) produced trees with similar topologies. Hence, only the ML tree is here presented.
Box 1. Algorithm settings and parameters for phylogenetic analysis.
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Figure 1. ML consensus tree
4. Discussions:
Phylogenetic relationships
Four lineages of Tetraclita populations from the western Pacific were resolved by the phylogenetic analysis with robust bootstrap support. Two of these lineages represent a distinction between T.cf squamosa and the more basally positioned T. squamosa, divided from each other by the South American species Tetraclita panamensis. This and the fact that T. panamensis is basal to T. squamosa indicate that the there are at least two origins for the western Pacific Tetraclita.
Seven samples from various localities in Singapore form a distinct genetic lineage, separated from a sister group that includes T. japonica and the previously recognized T. formosana. T. squamosa sensus lato are separated into two lineages, one comprising samples from the Ryukyus, Taiwan , Philippines , and Australia , while the other comprises samples from Hong Kong , Malaysia and Singapore . These two clades form a polytomy together with Tetraclita serrata from South Africa .
Results from multiple sampling sites of the Ryukyus, Taiwan and Singapore show that genetic lineages are not location specific or homogenous. For example, one specimen from the Ryukyus groups with the four Taiwan samples, while a second groups with samples from the Philippines ; implying that gene flow between Islands is still quite active.
Taxonomy implications
In recent years, discussion has grown concerning the species, speciation and species boundaries in the marine invertebrates. However, most studies have been more interested in phylogeny than taxonomy, with the result that many taxonomic issues remain unresolved. Numerous new species have been suggested purely in the phylogenetic sense, leaving the problem to be solved by taxonomists who in many cases do not agree with the phylogenetic conclusions. Most of these new species have not been followed up with taxonomic descriptions due to the impossibility of separating them morphologically or merely due to an unwillingness to carefully examine the morphology closely. In the present study, phylogenetic trees and pairwise genetic distance measures were applied to test taxonomic results implied by prior morphological comparisons.
1. Tetraclita formosana should be recognized as a junior synonym of T. japonica.
Morphologically, T. formosana could not be separated from T. japonica, except by color pattern, which in T. formosana is pink and purple in T. japonica. Based on the phylogenetic results, T. japonica and T. formosana cluster together in a clad with 100% bootstrap support. Within the clade, samples of both species form a polytomy; indicating that both species are identical. Pair wise genetic distance values among T. japonica and T. formosana are significantly much lower than each of these samples against other species. The lowest of infra-specific value is 0.00084 between TfmTWJS1 and TjpHK58, and the highest of infra-specific value is 0.03420 between TjpCN2 and TjpHK59. While the lowest inter-specific value is 0.11746 between TjpCN2 and TsqHK4, and the highest is 0.14310 between TfmTWJS1 and TsrSAF.
2. Tetraclita sp from Singapore is genetically very distinct from all the other species. In combination with morphological data, this form should be recognized as a distinct species that is probably new to science.
Morphologically, Tetraclita sp from Singapore , Sulawesi and Lankawi ( West Malaysia ) is very different from all the known species in the form of its tergum, which has a unique and distinct "key" shape. Genetic testing results confirm that it is an undescribed species. Samples from Sulawesi and Lankawi are museum specimens, not suitable for extracting DNA, thus, comparisons were based on Singapore specimens. Seven samples from 4 localities together form a clade in the phylogenetic tree, with 100% bootstrap support. Pair wise genetic distances also show that T. sp is a legitimate species. The lowest infra-specific genetic distance value is 0.00683 between TaSGSJ56 and TaSGSJ57, and the highest infra-specific value is 0.02117 between TcSGSJ54 and TaSGRLH. While the lowest of inter-specific value is 0.10947 between TcSGFR28 andTjpJP2, and the highest is 0.13681 between TaSGRLH and TsrSAF. The morphological description of the new species is now being prepared and will be published soon in a separate paper.
3. Tetraclita cf squamosa from eastern Asian island chains is most probably a cryptic species.
Morphological comparison suggests that samples of Tetraclita squamosa from Japan , the Ryukyus, Taiwan , the Philippines and Australia share some common characters which are not present in samples from Hong Kong , southern China and Malaysia . A pit formed by the upper margin of adductor ridge and articular ridge is located distinctly lower than the adductor muscle pit (vs. higher in T. squamosa), the oblique teeth of the occludent margin are normally only distinct along the lower half (vs. distinct along 2/3 in T. squamosa) and the tergal spur is shorter than that of T. squamosa. This group also forms a clade together with 100% bootstrap support whereas with samples of T. squamosa and T. serreta it forms a polytomy. Within the clade, the Australian sample is distinctly separated from the rest and placed at the base. One of the Ryukyu samples (T1RK33) forms a clade with 4 Taiwan samples, while the remaining Ryukyu sample groups with specimens from Japan and the Philippines . Pair wise genetic comparisons within this group shows that the lowest of infra-specific distance is 0.00746 between T1RY33 and T1TWJS2, and the highest infra-specific value is 0.02236, between T1TWSM2 and T1AUS82. While the lowest inter-specific value is 0.08079 between TsqJP96 and TsqHK4, and the highest is 0.16926 between TsqJP96 and TfmTWJS1. The morphological description of this cryptic species is now being prepared and will be published soon in a separate paper.
4. Tetraclita squamosa is restricted to continental East and Southeast Asia .
Pilsbry (1916) discussed the disposition of the type specimen of Tetraclita squamosa Bruguière. He suggested that " The type-specimen was probably lost, ... it has seemed best to consider the common form of China and the Philippine Islands as typical T. squamosa". As such, in the present study, I would like to assign a specimen from Hong Kong as the neotype for T. squamosa to settle the taxonomy. From the phylogenetic tree, two samples from Singapore and one from Malaysia are joined together with the two from Hong Kong , and form a clade with 100% bootstrap support. Interestingly, this seems to imply that the species is restricted to continental East and South East Asia . Pairwise genetic distances also suggest that it is a unique and distinct phylogenetic group. The lowest infra-specific distance is 0.00497 between TdMA and TsqHK4, and the highest is 0.02735 between TsqHK4 and TbSGSJ10. While the lowest inter-specific value is 0.07893 between TspSGSJ and T1RK33, and the highest is 0.12957 between TbSGSJ and TfmTWJS1.
5. Tetraclita serreta is not presented in West Pacific.
Rosell (1972, 1986) reported the occurrence of T. serrata in the Philippines. Comparisons of his drawing with type material in the British Museum and other freshly collected specimens from South Africa , the type locality of the species, show that the Philippine specimen is not T. serrata. This form of "philippine T. serrata" is also found in Singapore . A molecular comparison between the Singapore (TbSGSJ) and South African specimens shows that the two are very distinct genetically. TbSGSJ forms a clade with other samples of T. squamosa, and should be assigned to T. squamosa. Thus, T. serrata is excluded from the western Pacific cirripedian fauna.
Biogeography
Two origins in the region
It is premature to speculate on the evolution of the Tetraclita in the region as many other related species from the region are not presently available for study. However, some interesting geographic patterns are worth noting. By comparing species from the Indian Ocean ( South Africa ) and eastern Pacific ( Panama ), we can at least conclude that species from the western Pacific are not monophyletic. At least two independent origins can be surmised: one giving rise to the new Singapore species and Tetraclita japonica (including T. formosana); the other leading to T. squamosa and its sibling (cryptic) species T. cf. squamosa.
Distribution patterns:
According to the distribution of species in this study, three biogeographic regions can be recognized:
1. The chain of East Asian islands including the main Island of Japan , the Ryukyu Islands , Taiwan , the Philippines form a distinct region which hosts a cryptic species T. cf squamosa. This distribution is probably linked to ocean currents which are a major force responsible for the dispersal of barnacle larvae.
2. In the present study, the continental coast of the South China Sea possesses T. squamosa, indicating that it differs faunistically from oceanic islands in the region. However, as no specimens of T. squamosa from temperate areas of the Chinese mainland were available in the present study, it cannot be confirmed at this time.
3. The discovery of an undescribed species in Singapore , Malaysia and Sulawesi implies that it is probably a tropical species. Thus, a tropical component to geographic patterns in the region is suggested.
Acknowledgements
This study was supported by a Pacific Institutes of Marine Science (PIMS) Fellowship. Molecular genetic work was conducted mostly at the host institution, the Kewalo Marine Laboratory, University of Hawaii at Manoa, during my two-month visit there from 21 April to 19 June 2005, and continued at the Tropical Marine Science Institute, National University of Singapore (funding support from ASTAR No. 012-105-0037). I am most grateful to Professor Michael Hadfield, for his great encouragement, and generous support during the course of the study. I am also indebted to Dr. John Zardus, who provided training and assistance in molecular techniques and analysis for the current project. Many graduate and postgraduate students at the Kewalo Lab were very helpful in the lab and provided valuable discussion. I am also very grateful to Dr. Serena L. M. Teo for her encouragement concerning the PIMS fellowship application and collaboration with the Kewalo Lab, and for her generous support during the course of study. Thanks are also due to Drs. Li Xinzheng, Shih Shi-Te, Rachel Collin, Toshi Yamaguchi, Tohru Naruse, Hiroshi Suzuki, Benny K. K. Chan, Yair Achituv, John Zardus, Serena Teo, Tan Koh Siang, Sin Tsai Min, and Mr Lim Swee Cheng for help in collecting specimens for the present study.
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Table 1. Species, Collection sites and code used in the present study
| species | Locality | Code used in the study | Collection site |
| T. squamosa | Hong Kong 1 | TsqHK4 | Hong Kong |
| Hong Kong 2 | TsqHK42 | Hong Kong | |
| Malaysia | TdMA | Patu Palat, Peninsular Malaysia | |
| Singapore | TspSGSJ | St John's Island, Singapore | |
| TbSGSJ10 | St John's Island, Singapore> | ||
| T. cf squamosa | Japan | TsqJP96 | Kagoshima, Japan |
| Ryukyus | T1RK22 | Okinawa, Ryukyus | |
| T1RK33 | South of Kolenzoko, Okinawa, Ryukyu Islands | ||
| Taiwan | T1TWSM2 | I-Lan, Northeast coast of Taiwan | |
| T1TWSM4 | I-Lan, Northeast coast of Taiwan | ||
| T1TWSM5 | I-Lan, Northeast coast of Taiwan | ||
| T1TWJS2 | I-Lan, Northeast coast of Taiwan | ||
| Philippines | TsqPHL212 | Panay Island, Central Philippines | |
| Australia | T1AUS82 | Dampier, Australia | |
| T. japonica | Japan | TjpJP96 | Kagoshima Island, Japan |
| TjpJP2 | Kagoshima Island, Japan | ||
| TjpGB | Data from Gene Bank (NC-008974) | ||
| China | TjpCN2 | Nan'ao Island, Guangdong Province, China | |
| TjpCN29 | Nan'ao Island, Guangdong Province, China | ||
| Hong Kong | TjpHK58 | Hong Kong | |
| TjpHK59 | Hong Kong | ||
| T. formosana | Ryukyus | TfmRK052 | Okinawa, Ryukyu Islands |
| Taiwan | TfmTWJS1 I-Lan, | Northeast coast of Taiwan | |
| T. sp | Singapore | Tc SGSJ54 | St John's Island, Singapore |
| TaSGSJ55 | St John's Island, Singapore | ||
| TaSGRLH15 | Raffles Light House, Singapore | ||
| TcSGFR28 | Fort Road End at East Coast, Singapore | ||
| TaSGMR19 | Navigational Buoy in Marina Bay, Singapore | ||
| TaSGSJ56 | St John's Island, Singapore | ||
| TaSGSJ57 | St John's Island, Singapore | ||
| T. panamensis | Panama | TpnmPNM | Panama |
| T. serrata | South Africa | TsrSAF6 | Cap Town, South Africa |
| Newmanella sp | Singapore | NspSGSJ | St John's Island, Singapore |
Table 2. Primers used in the present study
| Gene | Primer | Sequence | Reference |
| 12S | 12S F | GAA ACC AGG ATT AGA TAC CC | Mokady, O., Loya, Y., Achituv, Y., Geffen, E., Graur, D., Rozenblatt, S. and Brickner, I. (1999). |
| 12S R | TTT CCC GCG AGC GAC GGG CG | ||
| 16S | 16S 1471 | CCT GTT TAN CAA AAA CAT | Crandall, K.A. and Fitzpatrick, J.F., Jr. (1996). |
| 16S 1472 | AGA TAG AAA CCA ACC TGG | ||
| COI | LCO 1490 | GGT CAA CAA ATC ATA AAG ATA TTG G | Folmer, O., Black, M., Hoeh, W.R., Lutz, R.A. and Vrijenhoek, R.C. (1994). |
| HCO 2198 | TAA ACT TCA GGG TGA CCA AAA AAT CA |